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ABSTRACTS

63rd International Symposium

on Crop Protection

May 24, 2011 Ghent

Belgium

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Tel. no. + 32 9 264.61.45 (B. Vandekerkhove) Fax. no : + 32 9 264 62 23

E-mail : [email protected] Website: http://www.iscp.ugent.be/

HONORARY CHAIRMEN

R. BULCKE, D. DEGHEELE ( ), W. DEJONCKHEERE( ), A. GILLARD( ), R.H. KIPS ( ), C. PELERENTS, J. POPPE, J. STRYCKERS( ), J. VAN DEN BRANDE ( ), W. WELVAERT

ORGANIZING COMMITTEE

G. SMAGGHE (Chair), B. DE CAUWER, P. DE CLERCQ, M. HÖFTE, M. MOENS, W. STEURBAUT, L. TIRRY P. SPANOGHE & B. VANDEKERKHOVE (Secretaries) L. GOETEYN, L. GOSSYE & H. VAN BOST (Assistant‐secretaries)

ADVISORY COMMITTEE

A. CALUS, J. COOSEMANS, P. CORNELIS, P. CREEMERS, W. DE COEN, R. DE VIS, B. GOBIN, E. PRINSEN, D. REHEUL, E. VAN BOCKSTAELE, Els VAN DAMME, J. VANDEN BROECK, G. VAN HUYLENBROECK, M.C. VAN LABEKE, W. VERSTRAETE

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THE SYMPOSIUM VENUE

Section Topic Blok (Building) Floor Room

No

Session

PS Plenary Session E first 1.002

SP Biopesticides and Green Adjuvants A ground 0.024

1 Agricultural Entomology and Acarology E first 1.002

2 Insect and Plant Nematology E second 2.009

3 Exploiting genomics to understand plant-

nematode Interactions – Part 1 (COST action 872)

E second 2.010

4 IPM E first 1.015

5 Phytopathology

6 Phytopathology: Integrated Control Strat-egies

E first 1.012 7 Phytopathology: Plant-pathogen interac-

tions and Resistance Mechanisms

8 Application Technology A first 1.015

9 Pesticide Residues, Toxicology and Ecotox-icology

10 Herbology A second 2.025

25/05/11 Satellite meeting: Exploiting genomics to understand Plant-nematode interactions –

Part 2 (COST action 872) E first 1.012

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Posters

Posters S01-S03 Special Session: Biopesticides and Green Adjuvants

A ground 0.041

Posters E01-E46 Agricultural Entomology and Acarology

Posters N01-N10 Insect and Plant Nematology

Poster C01-C18

Exploiting genomics to understand plant-nematode Interactions (COST Action 872) – part 1

Posters P01-P22 Phytopathology

Posters P23-P39 Phytopathology: Biocontrol

Posters P40-P48 Phytopathology: Fungicides

Posters P49-P55 Phytopathology: Integrated Control

Posters R01-R02 Application Technology

Posters R03-R15 Pesticides Residues, Toxicology and Ecotoxicology

Posters H01-H16 Herbology

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CONTENT Plenary 9

Special Session - Biopesticides and green adjuvants 13

Session 1 – Agricultural Entomology and Acarology 29

Session 2 – Insect and Plant Nematology 45

Session 3 – Exploiting genomics to understand plant-nematode interactions -

Part 1 (COST Action 872) 59

Session 4 – IPM 73

Session 5 – Phytopathology 81

Session 6 – Phytopathology: Integrated Control Strategies 91

Session 7 – Phytopathology: Plant-pathogen Interactions and Resistance Mechanisms 99

Session 8 – Application Technology 109

Session 9 – Pesticide Residues, Toxicology and Ecotoxicology 119

Session 10 – Herbology 129

Wednesday 25 May 2011: Satellite meeting: Exploiting genomics to understand plant-nematode Interactions - Part 2 (COST Action 872) 137 POSTERS 149 S01-S03 Special Session – Biopesticides and green adjuvants 151

E01-E46 Agricultural Entomology and Acarology 155

N01-N10 Insect and Plant Nematology 203

C01-C18 Exploiting genomics to understand plant-nematode interactions (COST Action 872) – Part 1 215

P01-P22 Phytopathology 235

P23-P39 Phytopathology: Biocontrol 259

P40-P48 Phytopathology: Fungicides 279

P49-P55 Phytopathology: Integrated Control 291

R01-R02 Application Technology 301

R03-R15 Pesticide Residues, Toxicology and Ecotoxicology 305

H01-H16 Herbology 321

Plenary

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PLENARY

Plenary

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Plenary

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9:40 RNA INTERFERENCE AS A NEW BIOTECHNOLOGICAL

DEVELOPMENT IN CROP PROTECTION

S.R. PALLI & Fang ZHU Department of Entomology, College of Agriculture University of Kentucky, Lexington,KY 40546, USA

Since its initial description in the nematode Caenorhabditis elegans RNA interference (RNAi) mediatedby double‐stranded RNA (dsRNA) inducing gene‐specific silencinghas become one of the most promising methods for studying thefunction of genes. This technology has also become a potentialrobusttool for cropprotectionagainst insect pests. Forefficientpest con‐trol methods using RNAi in the field, the major challengeis the development of easy and reliable methods for productionand delivery of dsRNA. Delivering dsRNA by spraying on the cropplants fits well with current insecticide delivery methods. Severalresearch groups have explored the possibility of delivering dsRNAto insects throughfeeding.However,most of these studies used in vitro synthesized dsRNA, which isnot cost effective. Two recent papers described studies that usedtransgenic plants for synthesis and delivery of dsRNA. The major limitation of the transgenic plant approach for RNAi pest management is public hesitancy in accepting transgenic food. Inaddition, technologies for making transgenic plants are availa‐ble for only a few commercial crops.RNAi works very well in beetles and is systemic. Recent studies in our laboratories showed that feeding dsRNA is effective in down regulating the expression of genes in the Colorado potato beetle (CPB), a major pest of potatoes, tomatoes and eggplants. Feeding RNAi successfully triggered the silencing of all five target genes test‐ed and caused significantmortality and reduced body weight gain in the treated beetles. The Colorado potato beetle RNAi is the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. These studies suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible methodfor management of CPB. Feed‐ing RNA could be also a promising bioassay approach for genome‐wide screens to identify effective targetgenes for use as novel RNAi‐based insecticides. In addition, feeding RNAi could be also used to prolong the life of currently used insecticides. The mechanism and potential applications of this exiting technology in pest management will be discussed. Key words: feeding RNAi; bacteria-expressed dsRNA; Colorado potato beetle; Leptinotar-sadecemlineata; insecticide resistance

Plenary

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10:10 IMPORTANCE AND IMPACT OF THE NEW SUSTAINABLE

DIRECTIVE BY EUROPE

Anne ALIX Office on Regulation on Plant Protection Products and Inputs

Deputy Head Ministry of Food, Agriculture, Fisheries, Rural affairs and Spatial Planning

General Directorate on Food 251 rue de Vaugirard, FR-75732 Paris cedex 15, France

Directive 2009/128/EC (SUD) establishes a framework for action to achieve sustainable use of pesticides within the European Union.While Directive 91/414/EEC and EC Regulation 1107/2009 condition the placing of individual products on the market on the demonstration of uses being safe for human health and the environment, Directive 2009/128/EC lists a series of measures and action aiming at minimizing further the overall pressure exerted by pesticides in conventional agriculture.Measures and action are targeted on each of the lever that may contribute, from the decision to turn to chemical pest control, to global use poli‐tics. The main measures are i) the implementation of National Action Plans aiming at reduc‐ing the use of products containing substances of particular concern, ii) the professionaliza‐tion of users through training programs along the chain from distribution to farmers, and through certification systems, both aiming at reinforcing the safety of users and of the envi‐ronment through a better knowledge of the conditions of uses, iv) regular technical inspec‐tion of application equipments, v) implementation of protection measures for aquatic sys‐tems (e.g. buffer zones), vi) restrictions concerning the uses leading to the most important transfers to public areas/aquatic systems and the environment, vii) limitation of the access of pesticides to non professional users and viii) promotion of alternative methods, where possible. Once Member States have evaluated the level of compliance of their agricultural systems with the principles of SUD, a proper implementation of the directive implies to identify and develop the technical, scientific and economical tools that will allow to comply with the reduction objectives. The efficacy of these tools in then assessed through appropri‐ate indicators that will also help to monitor the efficacy of the adopted strategy over time. This presentation aims at depicting the implementation of SUD in a Member State (France) through a selection of measures to illustrate, beside regulatory aspects, the level of technical and scientific progress that is needed to reach the SUD objectives. A state of the art of the implementation will also be presented with the aim to inform about the organizations in‐volved into the process andthe time scales. Finally, a review of areas for improvement and research needs will be proposed.

Session Biopesticides and green adjuvants

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Special Session Biopesticides and green adjuvants


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11:00 HOW TO DEFINE GREEN ADJUVANTS?

P. SPANOGHE, B. BECK & W. STEURBAUT

Laboratory of Crop Protection Chemistry, Faculty of Bio-Science Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium

The concept ‘green adjuvants’ is difficult to interpret. Discussion on how to define the green accent has been ongoing for many years. In this presentation, an answer is looked for from different angles. A first approach looks at the production source. Green adjuvants might be adjuvants derived from natural/organic resources. They can be extracted and refined out of a natural source (e.g. vegetable oil) or they might be produced by microbiological systems (e.g. biosurfac‐tants). Depending on chemical processing afterwards, they might be natural or semi‐synthetic compounds. In the same framework one might define an adjuvant as green, when it is produced following the principles of ‘green chemistry’ (use of renewable sources, less toxic solvents etc.) The second approach focuses on the environmental impact of the compounds. The common view on adjuvants is that they are inert and/or very biodegradable. Work at the UGent dept. of Crop Protection Chemistry showed that this is not the case for all adjuvants. Even natural compounds may be very persistent and cause (eco‐)toxic harm. This calls for another ap‐proach on green adjuvants. Using this definition of less harmful, fast degradable compounds, no distinction is made between synthetic, semi‐synthetic or naturally produced substances. A third approach is to identify the word ‘green’ with the word ‘sustainable’. One can score the benefits of adjuvants for People, Profit and Planet. Economical benefit is obtained from enhanced crop protection, resulting in a higher production value. The benefits for humans and the environment are replacement of old adjuvants with ‘green’ ones having a lower impact. Next to this, green adjuvants might reduce drift potential and enhance a more effi‐cient application of active ingredients, which reduces the need for these compounds. Considering all of the above, this presentation concludes with a tentative definition of “Green adjuvants.” Key words: green adjuvants, pesticides, definition


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11:20 EVALUATION OF THE IMPACT OF ADJUVANTS

ON THE ENVIRONMENT: THE USE OF A BIOSENSOR METHOD

Ingrid NOBELS1, Freddy DARDENNE1, Ronny BLUST1,

Pieter SPANOGHE2 & Geert HAESAERT3 1University of Antwerp; Laboratory for ecophysiology, biochemistry and toxicology

Dept. Biology, Groenenborgerlaan 171, BE2020 Antwerpen, Belgium 2 Department of Crop Protection, Ghent University, Ghent, Belgium

3 Faculty of Biosciences and Landscape Architecture, Hogeschool Ghent, Ghent, Belgium The omnipresent group of pesticide adjuvants are often referred to as “inert” ingredients, a rather misleading term since consumers associate this term with “safe”. The upcoming new EU regulation concerning the introduction of plant protection products on the market (EC1107/2009) includes for the first time the demand for information on the possible nega‐tive effects of not only the active ingredients but also the used adjuvants. This new regula‐tion requires basic toxicological information that allows to decide on the use/ban or prefer‐ence of use of available adjuvants. In this study, we obtained toxicological relevant infor‐mation for a broad selection of commonly used adjuvants (e.g. ethoxylated alcohols, isopho‐rone,…) through a multiple endpoint reporter assay. The used assay allows the toxicity screening in a mechanistic way, with direct measurement of specific toxicological responses (e.g. oxidative stress, DNA damage, membrane damage and general cell lesions). The results show that the selected solvents are less toxic than the surfactants, therefore solvents should have a preference of use. The gene expression profiles of the selected sur‐factants reveal that a phenol (ethoxylatedtristyrylphenol) and an organosilicone surfactant (ethoxylatedtrisiloxane) show little or no inductions at EC20 concentrations, making them preferred surfactants for use in different applications. The organosilicone surfactant is a fairly new compound that looks very promising, shows little or no toxicity and good adjuvant properties. Although the number of compounds that were evaluated is rather limited (13), the results show that the used reporter assay is a promising tool to rank commonly used agricultural adjuvants based on toxicity and toxic mode of action data.


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11:40 NATURAL PLANT EXTRACTS AS PHOTOPROTECTING ADJUVANTS FOR PHYTOSANITARY FORMULATIONS

A. TER HALLE1,2, B. EYHERAGUIBEL1,2 C. RICHARD1,2 & G. LEDOIGT3

1 Clermont Université, Université Blaise Pascal Laboratoire de Photochimie Moléculaire et Macromoléculaire

BP 10448, FR-63000 Clermont-Ferrand, France 2 CNRS, UMR 6505, LPMM, FR-63177 Aubiere, France

3EA 3296 “ERTAC”, Physiologie et Génétique Végétales, Université Blaise Pascal 6505 FR-63177 Aubière Cedex, France

Concerns about the environmental impact of pesticides has prompted research on their transport from treated fields to the air, other land and water bodies, together with ecotoxi‐cological studies about effects on ecosystems. As a consequence, pesticides are already some of the most thoroughly evaluated molecules on the market. The effect of sunlight on pesticide degradation is systematically studied in water and on soil. However, photodissipa‐tion on leaves, first recipients of sprayed phytochemicals, is not considered. We designed a protocol to assess photostability of pesticides on leaves. To mimic the hydrophobic surface of plants a test based on thin wax films was developed. The thin film model has proven valid by field monitoring. In the case of two triketone herbicides (mesotrione and sulcotrione), DT50 of photolysis on leaves are between 6 to 8 hours when used pure. With commercial formulation DT50 drastically drops to 1‐2 hours. The half‐life's of photolysis on leaves are very low compared to the ones reported in water (over 100 days*) and soils (between 30 to 90 days*). In total ten pesticides from very distinct chemical families were studied. The increase in photoreactivity on leaves is systematic. A mechanistic investigation is under work in order to explain this phenomenon. The results indicate that pesticide photolysis on crops fater application is a significant phenomenon and this evaluation should be systematic. If pesticides are photolabile, the use of photoprotecting agents is a promising technique. The molecules proposed until now do not meet nowadays requirements: good efficacy and no toxicity. We have developed a new class of natural photoprotecting agents. They are not harmful for crops or the environment and significantly decrease the rate of photolysis. Key words: photodissipation, photoproducts, leaves, plant extracts


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12:00 INCREASED EFFECTIVENESS OF FOLIAR-APPLIED

ENTOMOPATHOGENIC NEMATODES WITH ADJUVANTS AND ATTRACTANTS?

B. BECK1, E. BRUSSELMAN2, D. NUYTTENS2, S. POLLET3, F. TEMMERMAN3,

W. STEURBAUT1 & P. SPANOGHE1 1 Laboratory of Crop Protection Chemistry, Faculty of Bio-Science Engineering,

Ghent University, Coupure Links 653, BE-9000 Ghent, Belgium 2 Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit,

Agricultural Engineering Burgemeester Van Gansberghelaan 115-1, BE-9820 Merelbeke, Belgium

3 Provincial Research and Advisory Centre for Agriculture and Horticulture (POVLT) Ieperseweg 87, BE-8800 Rumbeke, Belgium

This work explores the influence of a selection of adjuvants on the foliar application of en‐tomopathogenic nematodes (EPN). The goal of this study was to increase the deposition and infectivity of EPN on leaves by adding adjuvants to the spray liquid. The first test presented in this work focuses on the effect of adjuvants on the deposition of EPN. This test showed that a good choice of nozzle size and adjuvants is essential to increase the deposition of EPN on leaves. The highest depositions were obtained with an extended range standard flat fan XR 110 04 nozzle, with 0.5 g/l of a modified heptamethyltrisiloxane superwetter added to the tank. Similar, but less variable results were obtained with the same nozzle, with a com‐bination of an adjuvant consisting of a surfactant and a vegetable oil (2.5 ml/l) and xanthan gum (0.3 g/l) added to the spray suspension. The second test presented in this work handles about the effect of two attractants on the infectivity of EPN against the cabbage moth (Mamestra brassicae). Both yeast extract and bee feed, consisting of various types of sugar, were tested. Results of this test were not yet available while writing this abstract. Key words: spray application, surfactant, sedimentation, deposition, nozzle


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12:20 IN SITU PROTEOME STUDY OF PICHIA ANOMALA STRAIN K, AN ANTAGONIST OF APPLE PATHOGEN

BOTRYTIS CINEREA

A. KWASIBORSKI1, J.RENAUT2,P. LEPOIVRE3 & H. JIJAKLI4 1,3,4 Unity of Phytopathology, Gembloux Agro-Biotech, University of Liège

Passage des Déportés 2, BE-5030, Gembloux, Belgium 2 Department of Environment and Agro-Biotechnology, CRP Gabriel Lippmann

41 rue du Brill, L-4422 Belvaux, Luxemburg Postharvest fungal pathogens, mainly Botrytis cinerea, Penicillum expansum and Gloeospori‐um spp., annually cause 5‐20% losses of fruit. Control measures against these diseases in‐clude chemical fungicide applications, but the development of resistant fungal explains the growing interest for biological control. Pichia anomala strain K was previously identified as an efficient antagonist of pathogens on apples. Indeed, the percentage of protection of P.anomala against B.cinerea reached from 90 to 100% on apple wounds according to the experimental conditions. Microbiological, biochemical and molecular approaches demon‐strated the implication of exo‐β‐1,3‐glucanases in the mechanism of action of P.anomala. However, study of these mechanisms could be improved by observations under natural infection conditions in order to take into account the tripartite interactions host/antagonist/pathogen. The proteomic tool allowed an overview of process implicated in the antagonism against B.cinerea in such conditions. One 50mm wound per apple were covered by a membrane and inoculated by a P.anomala suspension then by B.cinerea or not. Samples were collected during the exponential and stationary phase to identify the early and later responses to the presence of B.cinerea. After extraction, proteins were separated on 2DE gels. Spots influenced by the presence of B.cinerea in exponential and stationary phases were identified by MALDI‐ToF. One hundred five and sixty spots of proteins were influenced by the presence of B.cinerea in exponential and stationary phase respectively. In exponential phase, influenced proteins were implicated in the different steps of the proteins biosynthe‐sis: amino acid synthesis, translation or mRNA maturation and in energy synthesis. On the other hand, in stationary phase, influenced proteins were mainly implicated in energy meta‐bolic pathway: glycolysis, alcoholic fermentation or gluconeogenesis. The presence of B.cinerea seemed to induce a slowdown in the metabolism of P.anomala without influenced its growth. Further studies have to be realized to understand the complexity of these modes of action. Key words: biocontrol, Pichia anomala, Botrytis cinerea, in situ, proteome


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12:40 ENTOMOVECTORED BIOCONTROL IS SUPERIOR TO SPRAYING AGAINST STRAWBERRY GREY MOULD

I. MENZLER-HOKKANEN & H.M.T. HOKKANEN

Department of Agricultural Sciences University of Helsinki, Finland Botrytis cinerea is the most important strawberry disease. Several fungicide treatments annually are needed in commercial crops to control it. Numerous antagonists occur naturally in the fields, but they seldom provide adequate protection on their own. One widespread natural antagonist, Gliocladium catenulatum, isolated from Finnish agricultural soil, has been developed into a commercial product (Prestop, VerderaOy, Finland). It is used to protect plants from fungal attack usually as a soil mix, but also as spray. We have studied its poten‐tial in protecting strawberry from grey mould. A continuous precision delivery system for the product was developed with honeybees as the delivery agent, because earlier experiences of spraying it failed to protect the crop. We report here on successful application and rapid adoption of this ‘entomovector’ technology in Finland during the past 5 years. Studies were carried out on commercial strawberry and raspberry farms in Finland during four seasons (2006‐2009). In total 16 strawberry‐farm‐years and 5 raspberry‐farm‐years were involved in intensive assays; in addition several dozen other farms have used this technology, freely available for Finnish growers since 2008. Organic growers and many conventional growers have been involved in the experiments. At the conventional farms 3‐4 fungicide sprays were applied annually, in addition to the bee‐vectored biocontrol. Two strong beehives/ha with dispensers (BeeTreat, Aasatek Ltd, Finland) were placed close to the berry fields, and 5 g of Prestop Mix was filled into the dispenser daily during flowering. Bees visited each strawberry flower 10 times every day during flowering, carrying a load of the biocontrol fungus to the flower each time. Four treatments were included in our study: untreated control, chemical control alone, bee‐vectored biocontrol alone, and chemical + biocontrol combined. Consist‐ently, all control treatments were highly effective regardless the weather conditions during the past 5 years: overall, bee‐vectored biocontrol alone decreased disease incidence by 50%, chemical control by 65%, and both methods together by 80%. However, these differences between the control methods disappeared when the marketable yield was measured: bio‐control was at least as effective as the two other methods. Furthermore, biocontrol in‐creased the storage durability of the berries more than chemical control. Overall the bee‐vectored biocontrol was reliable, easy to operate, and it saved the berry growers time, work, equipment, environment, and money: the cost of chemical control is 500‐1000 €/ha, biocon‐trol about 300 €/ha. Use of honeybees also provides additional 20‐35% yield benefit to growers via improved pollination. Key words: Biocontrol, entomovector, application, strawberry, Botrytis cinerea


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15:00 SYSTEMIC RESISTANCE INDUCED BY PSEUDOMONAS

PUTIDA BTP1 IN TOMATO IS ASSOCIATED WITH CHANGES IN THE OXYLIPIN PATHWAY

Martin MARIUTTO1, Marie-Laure FAUCONNIER2, Marc ONGENA3,

Philippe THONART3, Patrick DU JARDIN2 & Jacques DOMMES1 1 Laboratory of Plant Molecular Biology and Biotechnology, University of Liège, Belgium

2 Plant Biology Unit, Gembloux Agro-Biotech, University of Liège, Belgium 3CWBI, Gembloux Agro-Biotech, University of Liège, Belgium

Some rhizobacteria, called Plant Growth Promoting Rhizobacteria, are able to induce in their plant host defence mechanisms effective against pathogens. This resistance is called the induced systemic resistance (ISR) is molecularly different of the systemic acquired resistance (SAR), induced by pathogen attack. We studied the effect of the PGPR Pseudomonas putida BTP1 on tomato, at the transcriptional, enzymatic and metabolic levels on tomato leaves, before and after inoculation of the pathogen fungus Botrytis cinerea. Precedent study showed that these bacteria protect tomato against this fungus. The activity of the first en‐zyme of the pathway, the lipoxygenase (LOX), was stimulated in treated plants before and two days after the infection, and was caused by the increase of the expression level of two genes, LoxD and LoxF. One product of Lox, the 13‐HPOT, was more abundant in treated plants under esterified forms before infection, but free forms, that did not show difference of accumulation between controls and treated before infection, accumulated more rapidly after pathogen inoculation in treated plants. Its reduced product, the 13‐HOT, showed a similar profile. Downstream the Lox, the transcription level of allene oxide synthase, leading to the formation of jasmonic acid, and divinyl ether synthase, leading to the production of fungitoxic compounds, was similar: the transcription level was stimulated by the infection, but the increase was more important in control plants. The differential stimulation of the divinyl ether synthase was confirmed by the quantification of its products, the colneleic and colnelenic acids, which accumulated faster in control plants than in bacterised plants. For another gene of the pathway, the hydroperoxidelyase (HL), leading to the production of defensive volatiles, no differences were detected between control and bacterized plants. This result was confirmed with the analysis of the enzymatic activity of HL which was not simulated by the infection nor by the treatment. These results strongly suggest that the resistance conferred by P. putida BTP1 is associated in tomato with the stimulation of the Lox pathway, and that the bacterial treatment leads to a faster accumulation of fungitoxic 13‐HPOT in tomato leaves after infection by B. cinerea, by stimulating the LOX and inhibiting the 13‐HPOT‐consuming enzymes. Key words: Biocontrol, Induced systemic resistance, Plant Growth Promoting Rhizobacteria, Oxylipin


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15:20 INDUCTION OF RESISTANCE IN WHEAT AGAINST

POWDERY MILDEW BY BACTERIAL CYCLIC LIPOPEPTIDES

B. RANDOUX, Ch. TAYEH, N. BOURDON, B. TISSERANT, F. LARUELLE

&Ph. REIGNAULT Université du Littoral Côte d’Opale

Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), GIS PhyNoPi, B.P. 699, FR-62228, Calais cedex, France.

Our contribution to the PhytoBioINTERREG IV project aims at characterizing fengycin, myco‐subtilin and surfactin, three bacterial cyclic non‐ribosomal lipopeptides provided by the ProbioGEM laboratory, for their ability to induce resistance in wheat against powdery mil‐dew (Blumeria graminis f.sp. tritici). We will also investigate biological activities induced on leaves subsequently infected by B. graminis in order to detect possible priming activities. We are currently measuring the reduction of fungal infection induced by these 3 lipopeptides and will investigate different wheat defence responses potentially induced by them: PR‐proteins synthesis, reactive oxygen species (ROS) metabolism, lipid peroxidation, phenylpro‐panoid and octadecanoid pathways. Level expression of several genes involved in these pathways will be measured using real‐time qPCR in time course experiments. In a first step of our study, treatments consisted of spraying of first wheat leaves with solutions at three different concentrations and distinct resistance levels have been obtained depending on the tested lipopeptide. In order to evaluate both plant‐mediated resistance and direct fungistat‐ic activities, we also measured in vitro the impact of fengycin, mycosubtilin and surfactin solutions on germination of B. graminis spores. Prior to gene expression analysis, two refer‐ence genes showing constant expression levels in our different experimental conditions have been found out and will be used in our RT‐qPCR experiments: β‐tubuline and Ef1‐α�elongation factor‐encoding genes. Moreover, a set a sequences corresponding to genes involved in the different defence responses pathways have been designed and expression of genes encoding the following activities are going to be measured in treated/untreated and infected/uninfected conditions: chitinase, lipoxygenase, gluthatione‐S‐transferase, oxalate oxidase. Key words: Blumeria graminis f.sp. tritici, Triticum aestivum, cyclic lipopeptides, NRPs, Real-Time q-PCR, gene


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15:40 RHIZOBACTERIA-GENERATED CYCLIC LIPOPEPTIDES

AS INDUCERS OF PLANT SYSTEMIC IMMUNITY

G. HENRY, P. THONART & M. ONGENA Walloon Center for Industrial Biology, University of Liège/Gembloux Agro-Bio Tech

Gembloux BE-5030, Belgium Cyclic lipopeptides (cLPs) constitute a structurally diverse group of metabolites produced by various fungal and bacterial genera. It appears that the panel of natural functions of these cLPs in the ecological fitness of the producing microorganism is larger than previously sus‐pected. Recent findings on cLPs isolated from plant beneficial Pseudomonas and Bacillus strains have evidenced the crucial role played by these compounds in biocontrol of plant diseases notably by stimulating host immunity. More specifically, the rhizobacterial cLPs surfactin and massetolide tightly interact with plant cells to trigger systemic resistance at a micromolar range of concentration. The macroscopic disease reduction is associated at the molecular level with the stimulation of metabolic pathways known to be tightly involved in pathogen restriction. As biosurfactants and together with rhamnolipids, these molecules thus constitute a novel class of microbial elicitors with a possibly specific mechanism of action that we wanted to further investigate. Surfactin induces in cultured plants cells, a whole panel of early defense events also typically observed upon recognition of pathogen‐derived signals. However, the surfactin‐induced plant response also displays some specific aspects suggesting that, rather than a receptor‐mediated recognition process, this cLP pref‐erably interact with the lipid fraction of the plant plasma membrane. Some structural traits have been shown to alter the activity potential, opening the door to structure/activity inves‐tigations. As these cLPs do not create irreversible pore formation, they may act in a way sufficient to induce some disturbance or transient channeling in the phospholipid bilayer that can in turn activate a biochemical cascade of molecular events leading to defensive responses. Such an original lipid‐based mode of action may explain why some structurally close cLPs are more efficient than others. Key words: Cyclic lipopeptides, systemic immunity, elicitors


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16:00 OPTIMIZATION OF AN ENUMERATION METHOD TO STUDY THE EFFICIENCY OF TOMATO ROOTS

COLONIZATION BY SEVERAL MUTANTS OF BACILLUS SUBTILIS HAVING A POTENTIAL AS BIOPESTICIDE

J. DERAVEL1, E. JOURDAN2, F. KRIER1, M. BIGAN1, F. COUTTE1,

M. ONGENA2&P. JACQUES1 1 Laboratoire ProBioGEM, IUT A/Polytech-Lille, Université Lille1, Boulevard Paul Langevin, FR-59655 Villeneuve d’Ascq, France

2 Centre Wallon de Biologie Industrielle, Unité de Bio-Industries Gembloux-AgroBio-Tech- Université de Liège, BE-5030 Gembloux, Belgium

Owing to ecological concerns, there is an increasing interest in finding alternatives to the chemical pesticides. Biocontrol, a concept using a living organism or the product of this organism to suppress or to limit a pathogen is one of these alternatives. Some rhizobacteria, coded as PGPR (Plant Growth Promoting Rhizobacteria), are able to colonize plant roots and to develop a biofilm‐like on this surface. Studies reveal that biofilm‐like formed on roots by some PGPR initiates biocontrol against plant diseases. Bacillus subtilis is one of these PGPR. It produces surfactins, iturins or fengycins/plipastatins which are three families of cyclic lipopeptides well known for their importance in the biocontrol effect of the strain. The aim of our work is to analyse the precise role played by these lipopeptides in the roots coloniza‐tion by the strain. Several mutants derivated from strains ATCC6633 and 168 were con‐structed to get a panel of non‐producing and overproducing strains of these different lipopeptidic compounds. The strain Bacillus subtilis BBG111 (a derivative from strain 168, which produces both surfac‐tin and fengycin) was used to statistically optimize an enumeration method of roots film‐like. The experimental design constructed as a matrix functional complete allows the comparison of two methods (sonication or abrasion with glass beads) used with or without detergent and applied during 5 or 10 minutes. The glass beads method without detergent applied during 5 minutes allows the unhooking of 4.4x106 CFU/g of root while, in the same condi‐tions, sonication allows to unhook 1.4x106 CFU/g of root. The glass beads method was cho‐sen to compare the efficiency of tomato roots colonization by the mutants previously de‐scribed. Preliminary results indicate that the efficiency of root colonization is partially de‐pending on the lipopeptide production by the mutant strains. To our knowledge, it is the first time that such a comparison is carried out. Key words: enumeration method, root-colonization, Bacillus, lipopeptides


25

16:20 CYCLIC LIPOPEPTIDES OF PSEUDOMONAS SP.

CMR12A PLAY CONTRASTING ROLES IN SWARMING MOTILITY AND BIOFILM FORMATION

Jolien D’AES1, Marc ONGENA2 & Monica HÖFTE1

1 Laboratory of Phytopathology, Ghent University, Ghent, Belgium 2 Gembloux Agro-Bio Tech, University of Liege, Gembloux, Belgium

Biocontrol strain Pseudomonas sp. CMR12a was isolated from cocoyam roots in Cameroon and produces several interesting metabolites, including phenazine‐type antibiotics and two different types of cyclic lipopeptides (CLPs). CLPs are composed of a partially cyclised pep‐tide linked to a fatty acid of variable length, and can exhibit powerful toxicity towards a wide range of organisms. Their synthesis is governed by non‐ribosomal peptide synthetases, which are encoded on large gene clusters. In plant pathogenic Pseudomonas spp., CLPs often play an important role in virulence. Many plant commensal or beneficial pseudomonads also produce CLPs, in which case they are involved in a wide range of natural functions, such as motility, biofilm formation, and even defence against predation. So far, simultaneous pro‐duction of two CLPs by one strain has only been demonstrated for plant pathogenic pseu‐domonads. In this study, we elucidated the structure of both cyclic lipopeptides by combin‐ing two approaches. Firstly, the biosynthesis gene clusters for the CLPs were identified and analysed, which provided an in silico structure prediction. Secondly, chemical structure identification was performed, involving purification of the compounds by liquid chromatog‐raphy followed by mass spectrometry, and amino acid hydrolysis and analysis by HPLC. It appeared that CMR12a produces two distinct CLPs, which were designated sessilin and motilin, respectively. Sessilin is structurally related to the toxin tolaasin, produced by the bacterial mushroom pathogen Pseudomonas tolaasii. The structure of motilin on the other hand is very similar to that of orfamide, which was recently described in biocontrol strain Pseudomonas fluorescens Pf‐5. Thus, both compounds are related to other known CLPs, but have never before been reported to occur together in one strain. Site‐specific biosynthesis mutants in both sessilin and motilin were generated to test for a possible role of these CLPs in swarming motility and biofilm formation by CMR12a. Swarming motility was assessed on soft agar plates, while biofilm formation was tested in polystyrene 96 well plates. This showed that a sessilin mutation drastically reduced biofilm forming capacity, while motilin seems indispensable for swarming motility of CMR12a. Hence, both CLPs appear to play contrasting roles in biofilm formation and swarming motility, which may at least partly ex‐plain their co‐production by CMR12a. A previous study already demonstrated that sessilin is an important determinant of the biocontrol capacity of CMR12a towards R. solani root rot on bean (Phaseolus vulgaris). This was attributed to a direct antagonistic activity of sessilin, since sessilin showed antifungal effects in vitro, and the colonization capacity of a sessilin mutant was not significantly reduced. A possible role for motilin in the colonization capacity and/or biocontrol activity of CMR12a is currently under investigation. Key words: cyclic lipopeptides, Pseudomonas, biofilm, swarming motility


26

16:40 LIPOPEPTIDE PRODUCTION OF PSEUDOMONAS

CICHORII SF1-54 CAUSING MIDRIB ROT ON LETTUCE

E. PAUWELYN1, M. ONGENA2 & M. HÖFTE1 1 Laboratory of Phytopathology, Ghent University, Ghent, Belgium

2 Walloon Centre for Industrial Biology, University of Liège Gembloux Agro-Bio Tech, Gembloux, Belgium

During the last decade, bacterial midrib rot has caused severe losses of greenhouse butter‐head lettuce (Lactuca sativa L. var. capitata) in Flanders (Belgium).This disease, character‐ized by a dark‐brown rot of the midrib of one or more inner head leaves, is caused by Pseu‐domonas cichorii. The P. cichorii strains, isolated from symptomatic butterhead leaves, can be differentiated into three subgroups, that differ in virulence. P. cichorii is introduced in Belgian greenhouses by contaminated irrigation water. The type III secretion system is un‐important for midrib rot development and it is not clear which pathogenicity or virulence factors are involved in the infection process of P. cichorii. Several plant pathogenic bacteria produce cyclic lipopeptides, which may contribute to virulence.An extract of P. cichorii strain SF1‐54 showed biosurfactant activity, antibiotic activity against Rhodotorula pilimanae and Galactomyces geotrichum and phytotoxicity, which are hallmarks of cyclic lipopeptides. HPLC‐analysis of this extract showed the production of three CLPs with different biological activity. The structure of these CLPs has been characterized partially and mining of the P. cichorii strain S1‐54 genome revealed the non‐ribosomal peptide synthethases, encoding for these CLPs. Mutants in these NRPS‐genes will elucidate their role in pathogenicity for P. cichorii SF1‐54.


27

17:00 ELICITOR-INDUCED WHEAT DEFENCE GENES

AGAINST POWDERY MILDEW

C. TAYEH, B. RANDOUX, N. BOURDON & Ph. REIGNAULT Université du Littoral Côte d’Opale

Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), GIS PhyNoPi B.P. 699, FR-62228, Calais cedex, France.

Our work aims at characterizing the mode of action in wheat (Triticum aestivum) of treha‐lose, salicylic acid (SA) and heptanoyl salicylic acid (HSA), three previously characterized inducers of resistance against wheat powdery mildew (Blumeria graminis f.sp. tritici). We focuse on different wheat defence responses: PR‐proteins synthesis, reactive oxygen species (ROS) metabolism, lipid peroxidation, phenylpropanoid and octadecanoid pathways, and level expressions of several genes involved in these pathways are being measured using real‐time qPCR in time course experiments. In a first step of our study, treatments of first wheat leaves consisted of infiltration with trehalose and SA solutions. The wheat chitinase‐encoding chi gene was up‐regulated by treatments with both resistance inducers during the early period of the time course (0‐36 hours post‐infiltration, hai), but only SA‐infiltrated leaves exhibited a higher additional late up‐regulation (48 hai). Moreover, two other wheat chitinase‐encoding genes, chi1 and chi4, have been shown to be up‐regulated within the earliest hai with SA and to a greater extent after trehalose treatment. On the other hand, no effect on the lipoxygenase (lox), oxalate oxidase (oxo) and glutathione‐S‐transferase (gstf) genes expression was observed in trehalose‐infiltrated leaves, while their expression was up‐regulated 15 hai following SA infiltration. The expression of lox gene was also highly up‐regulated by SA treatment from 48 hai. Common and distinct features of trehalose and SA‐induced gene expression regulations will be discussed, as well as putative defence pathways induced by these resistance inducers. We will extend our work to biological activities in‐duced on leaves treated with sprayings of resistance inducers (including HSA), as well as those induced in leaves subsequently infected by B. graminis in order to investigate possible priming activities. Key words: Blumeria graminis f.sp. tritici, Triticum aestivum, elicitors, Real-Time PCR, gene expression.

Session 1 Agricultural Entomology and Acarology

29



30


31

11:00 IMPACT OF ARTIFICIAL ILLUMINATION ON THE

DIAPAUSE INDUCTION OF MOTHS

Eva HÄFFNER1, Sibylle SCHROER2, Barbara JÄCKEL3& Franz HÖLKER2 1Freie Universität Berlin, Institute Applied Zoologie / Animal Ecology

Haderslebener Str. 9, DE-12163 Berlin, Germany 2Leibniz-Institut of Freshwater Ecology and Inland Fisheries

Müggelseedamm 310, DE-12587 Berlin, Germany 3Pflanzenschutzamt Berlin, Department of Crop Protection

Mohriner Allee 137, DE-12347 Berlin, Germany The vanishing night due to artificial illumination in conurbations has unknown implications on the natural environment. For insects light controls not only the rhythm of night and day, but has an impact on the timing of egg laying, hatching, pupation, mating and the induction of the diapause. For the present research the horse chestnut leaf miner (Cameraria ohridella) was used as a test organism, because it is highly adaptive to changing environmental condition and moths are known to be light‐sensitive. The horse chestnut (Aesculus hippocastanum) is among the most common and favored trees in Berlin. The city alone has over 48.000 specimens. The invasive leave miner has major impact on both the aesthetics and the plant health. Infested leaves wilt in early summer and appear prematurely like autumn foliage.Noble trees appear sere and other impairments are amplified. Cameraria ohridella develops three generations in Berlin and up to five in warmer regions. In field trials, in October 2010, infested leaves were collected from seven sites illuminated by street lights versus nine sites with natural nightly darkness. Densities of cocoons, larvae, mines and the degree of discoloration did not differ significantly at illuminated trees versus trees in natural darkness. However, the number of mines was significantly increased on illuminated trees. For the greenhouse trials horse chestnut seedlings were infected with C. ohridella larvae at 20°C under long‐day conditions (16 h). At 8‐15°C 12 seedlings were treated with artificial illumination for 24 h (Megaman dimmable energy‐saving lamps, ~100 lux). Another 12 seedlings were kept under short‐day light regime (8 h).After only two weeks the size of the mines of treated leaves was significantly increased compared to the trees kept in natural darkness. Non‐diapausing pupae, which develop another generation, were mainly observed at leaves treated with artificial illumination. The number of diapausing cocoons was significantly higher on leaves kept in natural darkness. The larval feeding behavior might have changed, presumably due to changes in leaf metabolism. Artificial illumination combined with higher temperature due to climate change might lead to a fourth generation of C. ohridella in Berlin. The leaf miner, like other invasive species, has a high potential to adapt to changes in climate and other conditions. Decline of food supply can induce diapause for larvae of the first and second generation. Consequently parts of each generation annually go dormant for six months. The population is therefore not threatened by the event of early frost. However, other moth species, less adapted to climate conditions, might also be impeded in dormancy by artificial light. The effect on moths as pollinators due to artificial light is today unknown Key words: Cameraria ohridella, Aesculus hippocastanum, light, vanishing night


32

11:20 APHID POPULATION DENSITY IN WARE POTATOES

INFLUENCED BY FOLIAGE COLOR?

J.P. JANSEN Walloon Agricultural Research Centre Life Science, Plant Protection and Ecotoxicology unit

Chemin de Liroux 2,BE-5030 Gembloux, Belgium Between 2006 and 2009, aphid population density were compared in two groups of ware potato: a “light green” foliage variety (cv Exempla) and a “dark green” variety group (cv Bintje, Celina, Nicola, …) planted side by side in the same fields and cultivated under the same conditions (date of plantation, fertilizers and pesticide treatments). Aphid population were estimated by weekly visual counts on 200 leaves between mid June to end of July, period corresponding to the arrival, outbreak and decline of aphids in potato in Belgium. Even if the aphid population remained relatively low between 2006 and 2009, with no aphid outbreaks and no insecticide application required in the fields followed, statistical analysis of the results was indicating a highly significant difference between the two variety groups, with a mean of 3.4x more aphids in the dark green group than in the Exempla group. In fields with the highest aphid density, the difference was even greater, with up to 5‐10x more aphids in the “dark green” group than in the “light green”. Specific counts of alate aphids in aphids with the highest populations were indicating that similar differences were observed between “light green” and “dark green” groups, with more alate aphids in the “dark green” group than in the “light green” group, especially during the first weeks of observation, corresponding to the arrival of aphids in the crops and beginning of infestation. These results suggests that a darker green foliage could be more attractive for alate aphids than lighter green foliage and therefore could explain, at least partly, why aphid populations were lower in fields of the Exempla variety than in darker green variety planted side by side. The interest of this possible higher attractiveness of dark green foliages is discussed in terms of integrated aphid management. Key words: aphid, potato, foliage color, attractiveness


33

11:40 ATTRACTION OF WIREWORMS TO ROOT-EMITTED

VOLATILE ORGANIC COMPOUNDS OF BARLEY

F. BARSICS, E. HAUBRUGE & F.J. VERHEGGEN Unit of Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech

University of Liège, Belgium Wireworms, the belowground larvae of click beetles (Coleoptera: Elateridae), represent a threat for various crops, including cereals, potatoes and small fruits, in many countries. In order to promote the development of bio‐pesticides or the selection of resistant varieties, studies concerning the close relationship between wireworms and the roots they attack have to be conducted. Indeed, lacks of knowledge in the chemical ecology of these small but consequent pests baffle such achievements. Here, we studied the reactions of wireworms (Agriotes sordidus Illiger) facing root‐emitted volatile organic compounds (VOCs) of barley. We first tested the ability of wireworms to orientate towards a blend of volatile compounds emitted by seven day’s old mashed roots of barley grown in vermiculite. We used a Y‐shaped horizontal olfactometre, which inferior half was filled with compressed and humidified vermiculite. Wireworms were able to detect blends of volatiles emitted by mashed roots, and to orientate their foraging behaviour toward the emission source. Volatile compounds were identified from barley roots using SPME‐GC‐MS and several chemicals were identified. This study is part of a larger programme which purpose is the identification of VOCs emitted by the roots of barley and different actors of the rhizosphere, and to assess their semiochemical functions. Key words: Wireworms, Agriotes sordidus, volatile organic compounds, rhizosphere, barley.


34

12:00 ALTERNATIVE HOST PLANTS AS POTENTIAL FOOD

SOURCES FOR DIABROTICA VIRGIFERA VIRGIFERA LECONTE AS EXEMPLIFIED FOR SEVERAL LOCATIONS

IN SLOVENIA AND ROMANIA

T. NEDELEV1, S. DINNESEN1, C. ULRICHS1, Hans E. HUMMEL2,3, R. ZEHAN4, I. GROZEA4 & A. CĂRĂBEŢ4 1Urban Ecophysiology, Humboldt University Berlin

Lentzeallee 75, DE-14195 Berlin, Germany. [email protected] 2Chair of Organic Agriculture, Justus-Liebig-University Giessen

Karl-Gloeckner-Str. 21 C, DE-35394 Giessen, Germany 3Illinois Natural History Survey, University of Illinois Urbana-Champaign, Institute of Natural

Resources Sustainability, 1816 S.Oak Street, Champaign , Il. 61820, USA 4Banat’s University of Agricultural Sciences and Veterinary Medicine, Timişoara, Romania

INTRODUCTION. Since 2006, three annual study projects of the first and second authors have been conducted with experiments under the direction of Profs. C. Ulrichs, Hans E. Hummel, and M. Gossmann. These projects included the population and feeding ecology of the Western Corn Rootworm Diabrotica virgifera virgifera LeConte (Dvv) and potential alternative host plants in Southeastern Europe. In all, three study projects have been carried out: (1) August 2006 in Gaberje, Slovenia; (2) July and August 2006 in Timisoara and Sag, Romania; (3) July, August and September 2009 in Timisoara, Sag, Gottlob, Varias and Sistarovat, all Romania. One objective of our research was to capture and quantify Dvv and to compare the kinds of traps used for this purpose with regard to their efficiency. Additionally, the significance of Dvv as potential vector of fungal spores was analyzed. MATERIALS AND METHODS. Flight curves describing Dvv populations have been recorded for the years 2006, 2008 and 2009. The following traps were used: (1) the Hungarian Csalomon® sex‐pheromone trap with 8‐methyl‐2‐decyl propanoate as bait, (2) the Temmen company’s mass capacity trap with sex pheromones (after Kovalev or Mori) or kairomones; synthetic or natural diatomaceous earth, as developed by Prof. Ulrichs and colleagues, is used to kill the sampled beetles, and (3) sticky cup trap of the Metcalf type with kairomone and pheromone bait and a sticky polyisobuten surface. Captured beetles were frozen in liquid nitrogen to analyze the gut content of Dvv for determining the identity of the pollen consumed. Detailed methodology is described in the bachelor’s thesis of the first and in the master‘s thesis of the second author. RESULTS. We did not find significant differences in the effectiveness of the three trap types used in our study. Identification was easier when the beetles were captured with mass capacity traps than with sticky traps, especially when there were long periods between collections. We showed that there is a significant influence of the lure used and of weather conditions on the effectiveness of the traps. In our research on corn (Zea mays), sorghum (Sorghum halepense), pumpkins (Cucurbita spp.) as well as water melons, honeydew melons and unspecified weeds (Order: Poaceae) growing at field margins we found that the adult Dvv can be regarded as a potential vector of fungal spores of at least five potential producers of mycotoxines and may potentially be spreading Ustilago maydis. CONCLUSIONS & OUTLOOK. Gut analyses will be used to draw conclusions regarding details of the rather complicated nutritional ecology of Dvv beetles.


35

12:20 COMPARISON OF TWO DIFFERENT SEX PHEROMONE

PREPARATIONS FOR THE WESTERN CORN ROOTWORM IN MAIZE FIELDS OF ROMANIA, THE CANTON TICINO,

AND THE STATE OF ILLINOIS, USA

Hans E. HUMMEL1,2, K. MORI3, B. KOVALEV4, M. BERTOSSA5

& S. DINNESEN6 1Justus-Liebig-University Giessen, Organic Agriculture Karl-Gloeckner-Str. 21 C, DE-35394 Giessen, Germany

2Illinois Natural History Survey, University of Illionois, Urbana-Champaign, Illinois 6l820, USA 3The University of Tokyo, 1-20-6-1309 Mukogaoka, Bunkyo-ku, 113-0023 Japan

4Scientific Productive Company “Biochemtech” 2060, Kishinev, Bd. Dachia 58, Republic of Moldova

5Agroscope-Changings-Wädenswil ACW, Centro di Cadenazzo, CH-6594 Contone, Switzerland 6Urban Ecophysiology, Humboldt University Berlin, Lentzeallee 75, DE-14195 Berlin, Germany.

E-mail: [email protected] INTRODUCTION: The Western corn rootworm Diabrotica v. virgifera LeConte (Dvv) (Cole‐optera: Chrysomelidae), a quarantine pest ever since introduction into Europe in 1992, is being monitored by a number of different trapping systems. The Metcalf sticky cup trap is the most sensitive and certainly the least expensive design imaginable. It costs 15 cent per piece including lure after its identification and synthesis. Little attention has been paid to the stereoisomeric mixture of sex pheromone lure 8‐methyl‐decane‐2‐ol propanoate itself and to its standardization. MATERIALS AND RESULTS: Data from a systematic comparison of two different synthetic pheromone sources (with origin Mori and Kovalev) at three locations in Europe (Romania, Ticino/Switzerland) and Illinois/USA, indicate no consistent, statistically significant prefe‐rence of the beetles to either source. DISCUSSION. Thus, as long as the identity and purity of the synthetic pheromone sources are well characterised by chromatographic and spectrometric analysis and as long as the Dvv populations are genetically uniform, the considerable variability observed in the trapping systems are probably more a consequence of the trap design and, first and foremost, of the ecological and weather factors prevalent at individual field locations. CONCLUSIONS. For all practical intents and purposes, a stereoisomeric mixture of synthetic sex pheromone from either one of the two sources is usable as a reliable monitoring tool for Dvv males.


36

12:40 POPULATION DYNAMICS OF THE ENSIGN SCALE,

INSIGNORTHEZIA INSIGNIS BROWNE (ORTHEZIIDAE) ON LANTANA CAMARA SHRUBS, IN REFERENCE TO

SOME ABIOTIC FACTORS AFFECTING ITS SEASONAL ABUNDANCE IN ALEXANDRIA, EGYPT

H.A. MESBAH1, K. MOURSI, A.K. MOURAD1, O.A., ZAGHLOUL1

& S. ABDEL-FATAH, RASHA 1 Faculty of Agriculture, Saba- Basha, Department of Plant Protection

Alexandria University, Alexandria, Egypt Plant Protection Research Institute, Agricultural Research Center, Alexandria, Egypt

Investigation throughout the subsequent years of 2005‐2006 and 2006‐2007 indicated that the main pest species prevailing on Lantana camara Linn. was ensign insect, Insignorthezia insignis. It has been recorded in relatively high rates all over the year on the host species in El‐ Nozha public gardens, Alexandria, Egypt. Generally, the calculated rates of the inspected insects were relatively low in Autumn months and January in the first year. Where it lasted till March in the second year. Considering the adult females, their highest population incidence occurred in September and October in both years. More or less low numbers occurred in Spring, Summer and late Winter months in both years, were as in the second year the rate of the inspected adults reincreased during January. Results showed that there was a significant weak positive correlation between daily mean temperature and total counted individuals of I. insignis, while it was highly significant positive with dew point. On the other hand, the correlations with the factors of relative humidity , wind speed and total counted individuals of I. insignis showed strong negative significant relationship. Key words: Insignorthezia insignis;Lantana camara;humidity, wind speed and total counted individuals .


37

15:00 AGROECOLOGICAL CROP PROTECTION IN REUNION:

FIRST RESULTS UNDER COMMERCIAL FARM CONDITIONS

J-P. DEGUINE1, P. ROUSSE2, K. LE ROUX3 & X. AUGUSSEAU1

1 Cirad, UMR PVBMT, Cirad/Université de La Réunion 7 chemin de l’Irat, FR-97410 Saint-Pierre, La Réunion, France

2 Chambre d’Agriculture de La Réunion 24 rue de la Source B.P 134,FR-97463 Saint-Denis Cedex, La Réunion, France

3 FARRE Réunion, 7 chemin de l’Irat,FR-97410 Saint-Pierre, La Réunion, France Cucurbit fruit flies (Diptera: Tephritidae) are major pests of vegetable crops in Reunion. Up to now, the protection of these crops relied mainly on agrochemistry. Broad spectrum insecticides are, however, considered as ineffective by producers, and are largely harmful to public health and to the environment. We thus conceived a project of research, development and education, in order to manage these pests using sustainable methodology. This innovative project, GAMOUR (a French abbreviation for agroecological management of cucurbit flies in Reunion), is one of the first such large‐scale initiatives in the European Union. The project includes a wide spectrum of stakeholders, involved in research, training, development and the commercial sector. It further involves numerous producers, who are the principle beneficiaries of the project. GAMOUR relies on the three pillars of agroecological crop management: sanitation, habitat management and conservation biological control. Our action has started since early 2009 in three intensive agricultural pilot areas totalling about 50 hectares of crops and 30 farms. The technical package is further compatible with organic farming specifications, and the project also includes four pilot organic farms.The objective of the study was to examine the project’s performance on both intensive and organic pilot farms following two years of agroecological crop protection practices. Four kinds of method were used for this assessment: (i) field experimentation of method effectiveness, (ii) monitoring of fly populations in pilot areas, (iii) socio‐economic monitoring of crop production, (iv) farmer satisfaction surveys. The results are highly encouraging: chemical spraying has been stopped while vegetable yields are higher or, at least, as high as before. As a consequence, farmers’ incomes are increasing. Our field experimentation confirms the effectiveness of techniques first assessed under controlled research station conditions. In particular, sanitation is highly efficient, as it is applied using the locally designed “augmentorium” (a tent‐like structure which sequesters adult flies emerging from infested fruit and allow the parasitoids to escape, via a net placed at the top of the structure). In addition, the trap plant (maize) concentrates more than 95% of fly populations, which are thereafter suppressed by food baits. Finally, mass trapping, without insecticides, is efficient for two of the three vegetable fly species present. As a result of these successes, farmers have quickly appropriated the project package. These results confirm that agroecology is a suitable alternative to agrochemistry for crop protection purposes. We now plan to extend this methodology beyond the pilot project area, both within Reunion, and to other islands in the Indian Ocean. Moreover, this agroecological dynamic may now be adapted to other agricultural systems such as tomatoes, citrus and mangoes. Key words: Agroecology, Crop protection, Tephritidae, Reunion


38

15:20 BIOEFFICACY OF THYMOL AND 1,8-CINEOLE AGAINST DIAMONDBACK MOTH LARVAE, PLUTELLA XYLOSTELLA

L. (LEPIDOPTERA: YPONOMEUTIDAE)- A POSSIBLE ENZYMATIC INTERACTION

Nutchaya KHUMRUNGSEE1, Wanchai PLUEMPANUPAT2,

Vasakorn BULLANGPOTI1* & Opender KOUL3 1 Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2 Department of Chemistry and Center of Excellence for Innovation in Chemistry Faculty of

Science, Kasetsart University, Bangkok 10900, Thailand 3 Insect Biopesticide Research Centre, 30 Parkash Nagar, Jalandhar 144 003, India

Essential oils are now well known to effect insects in various ways. We selected Thymol and 1,8‐cineole, well known natural essential oil allelochemicals to effect various insects mi‐crobes and fungi, to investigate the efficacy against an economically important pest dia‐mondback moth (DBM), Plutella xylostella larvae in Thailand using topical bioassays. After 24 h Thymol was more toxic (LC50 = 289 mg/larva) than 1,8‐cineole (LC50 = 1395 mg/larva). In order to demonstrate, the interaction with enzyme system of the candidate insect, car‐boxylesterase and glutathione‐s‐transferase activities were determined in the treated larvae that survived beyond 24 h. The present initial studies suggest that both compounds have potential for use in crop protection but the increase in both detoxification enzyme activities after treatment also suggests the risk of resistance development in DBM in the future. Key words: thymol, cineole, carboxyl esterase, glutathione‐s‐transferase, Plutella xylostella


39

15:40 INSECTICIDAL ACTION OF ALLYL ESTERS

M. GINER1, J. AVILLA1, M. BALCELLS1 & G. SMAGGHE2

1Department of Crop Protection, UdL – IRTA, Lleida, Spain Department of Chemistry University of Lleida, Lleida, Spain

2Department of Crop Protection, Ghent University, Ghent, Belgium The insecticidal action of five allyl esters from aromatic acids and middle‐chain fatty acids was assessed “in vivo” on three arthropod pests [Cydia pomonella (L.) (Lepidoptera: Tortricidae), Acyrthosiphon pisum (Harris) (Hemiptera: Aphididae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)], and “in vitro” on four insect cell lines [epithelial cells from Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), ovarian cells from Bombyx mori (L.) (Lepidoptera: Bombycidae), embryonic cells from Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) and fat body cells from Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)]. Allyl esters of fatty acids were more active than allyl esters of aromatic acids by topical application on C. pomonella eggs and on T. castaneum adults. By contrast, C. pomonella neonate larvae that feed on topically treated diet were more susceptible to allyl esters of aromatic acids than fatty acids and no significant effect or low mortality was observed when A. pisum fed on diet containing allyl esters. In all cases, mortality was produced in a short time from application (did not increase after 24h from application) and no deferred effects were observed. Acute cytotoxicity was also observed and, in general, few differences in cell response to tested allyl esters were recorded. Results from “in vitro” assays revealed that all allyl esters disrupted cell membranes and this fact explained most of the recorded cell mortality. Tested allyl esters may be promising alternative insecticides acting by contact, although their mode of action, fate and effects on non‐target organisms have to be more precisely discerned. Key words: allyl esters, codling moth, pea aphid, red flour beetle, insect cell lines


40

16:00 GROWTH-INHIBITION EFFECTS OF PACIFASTIN-LIKE

INHIBITORS ON A PEST INSECT: THE DESERT LOCUST, SCHISTOCERCA GREGARIA

Bert BREUGELMANS, Jornt SPIT, Vincent VAN HOEF, Gert SIMONET

Sven ZELS & Jozef VANDEN BROECK Department of Animal Physiology and Neurobiology, Zoological Institute K.U.Leuven,

Naamsestraat 59, BE-3000 Leuven, Belgium The main reason for the varying degrees of success of peptidase inhibitors (PI) as biological insecticides is the existence of a poorly understood mechanism, which allows pest insects to adapt to PI present in their diet. To challenge this highly flexible adaptation mechanism and to prolong the inhibitory effect of PI on insect growth, a number of measures were taken into account before and during feeding experiments on a notorious pest insect, the desert locust, Schistocerca gregaria: (i) non‐plant PI (pacifastin‐related inhibitors) were used to eliminate the risk of co‐evolutionary adaptation of the pest insect, (ii) based on the main types of digestive enzymes present in the midgut, mixtures of multiple PI with different enzyme specificity were selected, ensuring maximal inhibition and (iii) digestive peptidase samples were taken during the feeding experiments to study adaptation responses. Contrary to larvae fed on a diet containing plant‐derived PI, a significant growth impediment was observed in larvae that were fed a mixture of different pacifastin‐like PI, illustrating the strength of our strategy to use non‐plant PI. Nevertheless, the insects adapted to the PI after a few days, attenuating the growth inhibition effect. Moreover, a comprehensive study of the observed adaptation responses that occurred during the feeding experiments revealed that S. gregaria larvae can respond to PI in their diet in two distinct ways depending on the composition/concentration of the PI‐mixture. Key words: peptidase inhibitor, growth inhibition, pacifastin, adaptation response


41

16:20 FILM DOCUMENTATION OF THE BEHAVIOUR AND DEVELOPMENT OF THE EUROPEAN CORN BORER

OSTRINIA NUBILALIS

Urs WYSS Institute of Phytopathology, University of Kiel

Hermann-Rodewald-Str. 9, DE-24118 Kiel, Germany The video film (15 min. duration) presents the univoltine cycle of Ostrinia nubilalis, a serious pest of corn (Zea mays L). Male moths can be distinguished from female moths by the darker colour of the wings and by their protruding abdominal hind end. Egg clusters on the underside of corn leaves, embryonic development and hatching of the 1st instar larvae (L1) larvae from the eggs are shown. Nearly all L1 hatch simultaneously: most of them clean themselves intensively after hatching, some feed for a while on the egg shells and the few latecomers are occasionally killed by cannibalism. The neonate caterpillars then disperse over the leaves without any attempts to feed. Some use silk threads on which they hang for wind dispersal. Feeding is initiated on the underside of the leaves without affecting the upper epidermis. In this way characteristic ‘window panes’ are formed. All leaf feeding larvae (1st and 2nd instars) protect themselves under a loose web of silk threads on which faeces are suspended. Feeding on young leaves when still rolled up inside the corn whorl causes so‐called ‘shot holes’. The moulting process of a 2nd (L2) to the 3rd instar larva (L3) is shown in detail. L3 enter the stem at the level of the leaf axil. Extensive feeding inside the young stem, shown with a special illumination technique, leads to heaps of moist expelled faeces around the entrance hole. The 4th and 5th instar larvae (L4 and L5) move progressively deeper towards the base of the stalk. All instars that feed inside the stem or stalk show the same protective behaviour when in danger, i.e. when stems or stalks are cut to reveal the larva, the larva immediately stops feeding, starts to spin a loose web and covers it with frass. On only one occasion was it possible to demonstrate the fierce and powerful feeding activities of a L5 larva near the base of the lignified stalk. When fully grown L5 have reached the base of the stalk, they start to construct a dense protective pupation web. The L5 overwinters under this web as a prepupa. It is shown how the young pupa emerges from this diapausing stage and how it hardens rapidly. Female and male pupae can be distinguished by characteristic features at their rear end. Final sequences show how the adult moth emerges from the pupa and how it expands its wings soon thereafter. Apart from behaviour studies, the film demonstrates typical damage symptoms, including the cob, on which the feeding activities of the larvae have also been recorded. Key words: Ostrinia nubilalis, Behaviour, Development


42

16:40 BEHAVIOUR AND DEVELOPMENT OF THE PARASITOID BRACON BREVICORNIS, AN ENEMY OF THE EUROPEAN

CORN BORER OSTRINIA NUBILALIS

Urs WYSS Institute of Phytopathology, University of Kiel

Hermann-Rodewald-Str. 9, DE-24118 Kiel, Germany In recent years, in Germany, the ectoparasitic wasp Bracon brevicornis (Hym. Braconidae) was often detected parasitising Ostrinia nubilalis larvae in infested maize fields. Therefore, a video film (duration 14 ½ min.) was produced that documents the behaviour and development of the braconid parasitoid. Infested maize stalks were partially cut open to reveal the host larvae (L4‐L5) and were then offered to mated B. brevicornis females under a stereomicroscope. As a typical response, the Ostrinia larvae first tried to protect themselves within the cut stalk by spinning a web that was then partially covered by frass and faeces. The wasp, standing nearby, continuously observed this mending process with great care, awaiting the right moment for the first sting in order to paralyse the mighty larva with an injected venom. Several attempts with the partially protruded ovipositor preceded the first very quick successful sting, upon which the host larva responded violently. Several minutes elapsed until the affected larva gradually calmed down. This process was continuously observed by the parasitoid. Then the host was stung again. The paralysed Ostrinia larva was then removed from the stalk to document host feeding and oviposition. First host feeding stings again evoked a strong defensive response. Finally, after the larva had calmed down completely, host feeding was initiated by forceful deep ovipositor stings. Then the exuding haemolymph was immediately imbibed. Host feeding was followed by oviposition. The female protruded its ovipositor nearly full length along the body of the host in order to release the flexible egg through a lateral slit of the ovipositor, some distance proximal to its tip. Subsequent sequences show that many (up to 30) eggs are deposited at different locations onto the paralysed, still pulsating host. The development of the parasitoid from egg deposition until adult emergence was recorded at 27‐28°C. Emphasis was placed on the following features: embryonic development, hatch of the L1 larvae that immediately start feeding, food ingestion at high magnification, and the rapid growth of the larvae until they are fully developed after about three days. These larvae start to spin a dense silken pupation cocoon, the process of which is presented in detail. The final sequences show the praepupa and pupa within the cocoon and how, five days after the onset of pupation, an adult male and female emerge from the cocoon. Key words: parasitoid,Bracon brevicornis, enemy, Ostrinia


43

17:00 FILM DOCUMENTATION OF THE BEHAVIOUR AND

DEVELOPMENT OF THE HAZELNUT WEEVIL CURCULIO NUCUM

Urs WYSS

Institute of Phytopathology, University of Kiel Hermann-Rodewald-Str. 9, DE-24118 Kiel, Germany

Although Curculio nucum is the most important pest of hazelnut in many European countries, especially Turkey, little is yet known about its biology. This video film (16 min. duration) was produced to close some of the gaps. Sequences show that adult females can easily be distinguished from each other by the size of the rostrum, which is considerably smaller in males. The mating behaviour is filmed, showing details of sperm transfer. After mating, the female finally gets rid of the male by shaking it off from its body. During maturation feeding on young nuts, the long rostrum is first drilled through the nut’s soft involucre and then deeper and deeper. Drilling is achieved by a characteristic twisting action of the sharp mandibles at the tip of the rostrum as well as by turning the head to and fro around the pivot point. Drilling for egg deposition is similar to that of maturation feeding but not as deep. As soon as the female has withdrawn its rostrum from the drilled oviposition canal, it turns round, locates the hole with the tip of its ovipositor which is then inserted deep into the canal. It takes a few seconds until the flexible egg is forcefully squeezed at high speed through the narrow ovipositor tube. After tube retraction, the female turns round, locates the hole with its antennae and plugs it with its mouthparts. Plugging the oviposition hole is a characteristic behaviour pattern for all females that have deposited an egg. Subsequent sequences show the penetration depth of the fully extended ovipositor and how setae at the tip of the ovipositor are used to discriminate between suitable deep and unsuitable shallow oviposition canals. In all cases examined, the deposited egg was seen to be located in the still soft nut shell. However, by the time the L1 larvae had hatched, they had to gnaw their way through a harder nut shell and through the fluffy endocarp in order to reach the small young kernel at the top of the nut. Kernel penetration was achieved with ease by the L1. Two days later they moulted for the first time inside the kernel. The moulting process of a L2 to the L3 is shown in detail. L3 and L4 were most destructive by excavating large parts of the kernel that by this time has grown considerably. When fully developed, the L4 had to bite its way out through the hard nut shell. In spite of prominent strong mandibles, this was a tough task that could last up to 3 hours until the L4 was able to squeeze its massive apodous body through the small exit hole. The L4 then burrowed into the soil, where it constructed a solid earth cocoon (hibernaculum) for hibernation. Key words: Hazelnut,Curculio nucum, Behaviour, Development

Session 2 Insect and Plant Nematology

45



46

11:00 MOLECULAR VARIABILITY AND SENSITIVITY TO

ROOT EXUDATES WITHIN THE GLOBODERA TABACUM SPECIES COMPLEX

C. ALENDA1,2, D. FOUVILLE2, A. GALLOT2 & E. GRENIER2

1 ANITTA, Domaine de la Tour, FR-24100 Bergerac, France 2 INRA, Agrocampus Rennes, Université Rennes 1,

UMR1099 BiO3P, FR-35653 Le Rheu, France The tobacco cyst nematode (TCN) is a highly specialized and sedentary pathogen composed of at least 3 subspecies: Globodera tabacum tabacum, Globodera tabacum solanacearum and, Globodera tabacum virginiae. These organisms are morphologically difficult to distinguish one from another but display different developmental abilities on Nicotiana plants. Here, we have further investigated the intra‐ and inter‐subspecies variability by sequencing nuclear coding genes in a panel of 12 TCN populations. Using a candidate gene approach, we sequenced three parasitism genes involved in different stages of the interaction, one coding for an expansin protein (ExpB3) and the two others for CLAVATA like ESR (CLE) peptides. Study of the sequence polymorphism of these genes has allowed the development of specific subspecies molecular markers. In the light of this molecular variability, we looked further at the phenotypic variability among the subgroups of TCN and investigated the variability of two life‐history traits: hatching and attractiveness. In this prospect, responses of each TCN subspecies to strigolactones and root exudates of different Solanaceae (tomato, potato, and tobacco) were assessed and compared through in‐vitro bioassays. Key words: cyst nematode, parasitism gene, molecular marker, hatching, attractiveness.


47

11:20 DESSICATION TOLERANCE AMONG DIFFERENT

ISOLATES OF THE ENTOMOPATHOGENIC NEMATODE STEINERNEMA FELTIAE (FILIPJEV)

P. NIMKINGRAT & R.U. EHLERS

Institute for Phytopathology, Department for Biotechnology and Biological Control Christian-Albrechts-University Kiel

Hermann-Rodewald Str. 9, DE-24118, Kiel, Germany E-mail: [email protected]

Poor storage capacity is a major constraint limiting further expansion of the use of entomopathogenic nematodes.In order to prolong shelf life, a quiescent state of the dauer juveniles (DJs) should be induced. This can be achieved by means of desiccation of DJs. In this study, 24 natural isolates of Steinernema feltiae were exposed to desiccation stress in non‐ionic polyethylene glycol 600. The dehydrating conditions were measured as water activity, aw‐value. Non‐adapted and adapted DJs were tested separately under a series of dehydrating conditions. The mean tolerated aw‐value (MW50) ranged from 0.85 for the isolate NEP1 to 0.95 for FIN1, ISR5 and PAL4 when not adapted to desiccation stress and from MW50 of 0.822 for CR1 to 0.98 for ISR6 when adapted to the stress conditions. CR1 tolerated the lowest desiccation stress at an aw‐value for the most tolerant 10% of the population (MW10) at 0.65 when DJs had been adapted to stress. No significant differences were recorded between all isolates in non‐adapted DJs populations MW10 was compared. No correlation between tolerance under non‐adapted and adapted conditions were found. Most tolerant isolates will now be used for cross‐breeding and subsequent genetic selection to enhance desiccation tolerance.


48

11:40 CONTROLLING THE SUGAR BEET FLY

PEGOMIYA MIXTA

M.M.E. SALEH*, K.A.A. DRAZ**, M.A. MANSOUR**, Mona A. HUSSEIN* & M.F.M. ZAWRAH**

* Pests and Plant Protection Department, National Research Centre Elbehooth St., Dokki, Giza, Egypt

** Pest Control and Environmental Protection Department Faculty of Agriculture Damanhour University

Sugar beet, Beta vulgaris L. is a strategic crop of sugar industry in Egypt. It is threatened by several insect pests among most important of them is the beet fly Pegomyia mixta. This work deals with the biological control of this insect using four entomopathogenic nematodes (EPNs). The nematodes included Steinernema carpocapsae S2, Steinernema feltiae, Hete‐rorhabditis bacteriophora (HB1‐3) and Heterorhabditis bacteriophora S1. Daily mortality of larvae and pupae of P. mixta were recorded after treatment with serial concentrations (500, 1000, 2000 and 4000 infective juveniles (IJs)/ml) of each of four studied EPNs. In the laboratory all tested nematodes killed the larvae inside their mines in the sugar beet leaves and developed in their bodies in different extends. They also killed the insect pupae in the soil and developed in their bodies. Young larvae were more susceptible than old ones. New pupae were more susceptible than old ones. In the field a single spray of S. feltiae or H. bacteriophora caused 81.3 or 75.9% reduction in the larval population of the in sugar beet leaves. Key words: Pegomyia mixta , Heterorhabditis, Steinernema, Beta vulgaris


49

12:00 THE PINEWOOD NEMATODE AND PINE WILT DISEASE:

A SERIOUS FOREST PROTECTION ISSUE

M. MOTA Universidade de Evora, Evora, Portugal

Bursaphelenchus xylophilus, the pinewood nematode (PWN), and the causal agent of pine wilt disease (PWD), was detected for the first time in 1999, in Europe, in Portugal. The PWN has been detected in new forest areas in the center of the country, in 2008, despite efforts developed by the national forestry and quarantine authorities to control the nematode and its insect vector (Monochamus galloprovincialis). The nematode has also recently been reported to be present from Spain. Circulation of non‐treated wood and wood products may explain the spread of the nematode. Control strategies have been focused on the vector by using chemical traps, by cutting down symptomatic trees, heat‐treatment of lumber, and monitoring of main roads and ports through which lumber and wood products are transported, by the Portuguese authorities. The nematode constitutes a threat to the rest of Europe, if proper measures are not taken European governments. Study topics on this issue include nematode and insect bioecology, pathogenicity, use of molecular biology in diagnostics and detection, histopathology, etc. Many gaps in the knowledge of this complex biological system persist. The involvement of bacteria, associated with the PWN in causing pine wilt, has been claimed. New quick detection methods and the understanding of the nematode population dynamics are being developed. Nematode genomics may provide some insight to better understand the pathogenic effects caused inside the plant. Pathogenicity testing of susceptible pine species is imperative. A review of the progress is hereby presented. Key words: Pinewood nematode, Bursaphelenchus xylophilus


50

12:20 DUAL ROLE OF BACTERIA IN PINE WILT DISEASE

Cláudia VICENTE1, Francisco NASCIMENTO1, Margarida ESPADA1,

Solange OLIVEIRA2 & Manuel MOTA1 1NemaLab, Universidade de Évora

Instituto de Ciências Agrárias e Ambientais Mediterrânicas (UE/ICAAM) 2Laboratório de Microbiologia do Solo, Universidade de Évora

Instituto de Ciências Agrárias Mediterrânicas (UE/ICAAM) Pine wilt disease (PWD) is one of the most serious problems in pine forests, and also one of the most serious biological invasion issues worldwide. The causal agent, the pinewood nematode (PWN), Bursaphelenchus xylophilus, is considered to have its center of origin in North America, where it has co‐evolved for millions of years, and is in balance with the autochthonous pine species. The PWN was firstly described as the casual organism of PWD in 1971. Later, in 1980, a possible involvement of toxin producing‐bacteria in association with B. xylophilus in PWD was hypothesized. Ever since, efforts have been made to unravel the role(s) of bacteria in PWD. Preliminary results show that bacteria associated with B. xylophilus can induce PWD, although the symptoms shown by the plant host are slightly different from those observed when the nematode is inoculated alone. These observations suggest that bacteria can act in a different pathway than the pinewood nematode. It has been hypothesized that toxin producing‐bacteria can lower plant defense systems, thus helping nematode infection and proliferation in the plant host. If so, how, where and when do bacteria act? Which mechanisms are utilized? These questions remain unanswered. Still the role of bacteria in PWD seems to be evident.


51

12:40 WILL MELOIDOGYNE MINOR BECOMES A

THREAT TO OUR AGRICULTURE?

G.W. KORTHALS, W. VAN GASTEL, J. VISSER, T. THODEN, A. VAN DER SOMMEN & L. DEN NIJS Nematology, PPO-AGV, Lelystad, Netherlands

M. minor is a recently described nematode (Karssen et al, 2004) that has been found on sport fields, golf courses, and more recently also in potato growing fields in the UK and the Netherlands. A Pest Risk Analyses (PRA) was carried out in 2007, showing a lack of existing information on the damage thresholds on arable crops. Therefore, a project was started in 2008 aimed to resolve this lack of knowledge. Thus, a naturally M. minor‐infested field was subjected to a variety of crops (maize, rye, annual ryegrass, potato, sugar beet, black fallow) to asses their host status and, to achieve various initial population densities (Pi) of M. minor in the soil. Subsequently, in spring 2009, two potato cultivars were planted in these plots. From these studies it is obvious that nematode were able to multiply on potatoes whereby reproduction took place within roots as well as potato tubers, respectively. This, of course, increases the risk of spreading this nematode species by seed potatoes. No substantial reproduction was present on other host plants. Key words: nematodes, pest risk analysis, root‐knot nematodes, host study


52

15:00 RECENT FINDINGS ON THE TROPICAL ROOT-KNOT

NEMATODE MELOIDOGYNE ETHIOPICA

Sasa ŠIRCA, Polona STRAJNAR, Matej KNAPIČ, Barbara GERIC STARE & Gregor UREK

Agricultural Institute of Slovenia Hacquetova ulica 17, 1000 Ljubljana, Slovenia

E-mail: [email protected] Meloidogyne ethiopica is a tropical root‐knot nematode species which has recently been found in Europe. M. ethiopica was reported to be the major root knot nematode pest of grapevine, kiwi and other crops in Chile and Brazil causing serious crop damage and economic losses. The species was recently listed on the EPPO Alert list of harmful organisms. M. ethiopica is typical polyphagous pest which can parasitize at least 80 different woody and herbaceous host plant species belonging to dicotyledons as well as monocotyledons. We examined its ability to survive in open fields located in regions with sub‐Mediterranean and continental European climates. Several tests were performed to determine the influence of temperature on the duration of reproduction cycle. The species required 67, 48 and 36 days to complete reproduction cycle at mean daily temperatures of 18.3, 22.7 and 26.3°C, respectively. The entry of the host roots and further juvenile development was observed at 14.0°C. The data obtained from these experiments were used to develop a correlation between temperature and duration using a mathematical equation which can be used for modelling. In addition, the relationship between the morphological changes of roots and physiological plant responses of M. ethiopica infested tomatoes was studied. Infested plants had a very limited ability to mitigate the effects of root infection by altering root growth, and root knots have a major influence on the hydraulic conductivity of the root system. Low leaf water potential of infested plants was indicative of decreased stomatal conductivity, transpiration and photosynthesis.


53

15:20 INFLUENCE OF TEMPERATURE ON THE

LIFE-CYCLE OF GLOBODERA SPP.

V. BLOK, A. KACZMAREK & J.E. PALOMARES-RIUS Plant Pathology Programme

Scottish Crop Research Institute Invergowrie, Dundee DD2 5DA, UK

The potato cyst nematodes (PCN) G. rostochiensis (Woll.) and G. pallida (Stone) are the most economically important pests of potato (Solanum tuberosum L.) in the UK and are widespread in ware potato growing regions in Europe.The new EU directive 2007/33/EC which came into effect July 1, 2010 aims to control their spread and limit further increases in populations.We are investigating the role of temperature in the life cycle of PCN to assess how this effects population multiplication in relation to managing PCN.Hatching and adult maturation are stages in the life cycle that are affected by temperature and these have implications for the length of time required for one generation to be completed and the potential for final populations to increase.In some conditions a partial or complete second generation has also been observed within the growing season.Females have been observed on the surface of tubers and “pecking” skin damage can occur which may be a result of a second generation.We are investigating the influence of temperature on the potential for a second generation or the induction of diapause.


54

15:40 A METHOD FOR ESTIMATING THE CONTRIBUTION OF SEED POTATOES, MACHINERY AND SOIL TARE

IN FIELD INFESTATIONS WITH POTATO CYST NEMATODES ON A NATIONAL SCALE

M. GOEMINNE, N. VIAENE & K. DEMEULEMEESTER*

Institute for Agricultural and Fisheries Research (ILVO), Nematology Burg. van Gansberghelaan 96 bus 2, BE-9820 Merelbeke, Belgium

E-mail: [email protected] * Interprovinciaal Proefcentrum voor de Aardapppelteelt (PCA)

Ieperseweg 87, 8800 Roeselare, Belgium E-mail: [email protected]

In order to make a cost benefit analysis for the management of the potato cyst nematodes Globodera rostochiensis and G. pallida we developed a method to estimate the relative importance of three basic distribution channels of potato cyst nematodes: seed potatoes, machinery and soil tare. This quantification is a baseline for the evaluation of the effects of control measures for potato cyst nematode (PCN) on a national scale. The baseline is determined by the surface planted with potatoes, the area infested with PCN, the proportion of resistant potato cultivars and the distribution of cysts trough different channels. The relative importance of the potato cyst distribution channels is determined by the frequency with which these channels are used, as well as the agricultural and other practices applied. We calculated the numbers of parcels per crop type planted with seed potatoes of a certain origin and their degree of infestation. We estimated all machinery operations in each type of crop and their characteristics: number of parcels, number of infested parcels, amount of soil distributed. Per evacuation scenario and per product transaction of potatoes we estimated the volume of soil tare, the incidence and the level of the infestation in the soil tare, and the destination of the soil tare. Combination of all these data lead to an estimation of the number of infested fields and soil removed, the total amount of cysts moved, the amount of cysts per infestation, the number of infestations in the same field, in another field of the same farmer and in a field of another farmer per year. We concluded that within a region, the main causes of spread are machinery and soil tare. The most important spread occurs within the field itself and in the fields of the same farmer. Soil tare causes heavier infestations in one spot. Machinery disperses less cysts, but the infestation is in multiple spots. Fields of other farmers can be infested by seed (little importance), machinery (subcontractors) and soil tare. For long distance dispersion of cysts, soil tare appears to be the most important vector. Soil tare is easily transported over longer distances via trade and processing industry when soil tare is returned to farmers. It seems practically impossible to exclude infestation of soil tare with soil of other farmers. Therefore, it is recommended to investigate the cost/benefit of disinfection techniques , e.g. inundation of soil, at the level of the processors. Machinery of long distance subcontractors, as well as seed potatoes, are also possible vectors of cysts between regions, but the number of cysts is smaller than with soil tare. Key words: Globodera spp, contamination, control measures, cost benefit analysis, distribution


55

16:00 A NEW BIOASSY FOR ROOT-KNOT NEMATODE

DETECTION IN POTATO

G.W. KORTHALS, J. VISSER & W. VAN GASTEL Nematology, PPO-AGV, Lelystad, Netherlands

Some of the temperate root‐knot nematode species (Meloidogyne chitwoodi and M. fallax) do have a quarantine status and can form high risks for growing planting material, such as seed potatoes. At the moment there are several methods to detect both species in soil and crops, in order to assess their presence and the consequences upon the potential risks of introductions of M. chitwoodi or M. fallax via infected planting material (in this case potatoes). Many factors of thelife‐strategy of these nematodes species are not well known, in comparison with the better studied PCN‐nematodes, but some characteristics such as a broad host‐plant‐range, several generations a year, and a relatively quick decline during periods without a host‐plant, may have a high impact on population dynamics, detection methods and their potential risks of new introductions.Therefore new detection methods to detect M. chitwoodi or M. fallax should be investigated. In the present study results on the development of a new bioassay’s for the early detection of M. chitwoodi with the use of natural infested soils and different crops will be presented and discussed.


56

16:20 STATISTICAL ANALYSIS OF NEMATODE COUNTS FROM INTERLABORATORY PROFICIENCY TESTS

W. VAN DEN BERG1, O. HARSTEMA1& L.J.M.F. DEN NIJS2

1 Applied Plant Research, Wageningen UR Edelhertweg 1, NL-8219 PH, Lelystad, The Netherlands

2 Plant Protection Service Geertjesweg 15, NL-6706 AE, Wageningen, The Netherlands

Participation in proficiency tests is a requirement for laboratories to obtain accreditation for performing nematological analyses. Therefore, in The Netherlands data from these tests are summarized each year and reported to the participants. In this study the accuracy was investigated of the results of proficiency tests on Globodera rostochiensis and G. pallida since 1992 in The Netherlands. Particularly, changes in time, differences between measuring institutes, effects of the methods used and soil type are established. The same procedure is applied to counts of the endoparasite Meloidogyne spp. and the migratory endoparasite Pratylenchus spp., both originating from yearly proficiency tests on free‐living nematodes since 2000. For potato cyst nematode, the mean number of cysts and living larvae, as well as the precision of these estimates are presented. Further, the number of living larvae per cyst, the percentage of living larvae and the percentage of cysts other than Globodera spp. are relevant. Cysts and larvae are assumed to be Poisson and gamma distributed. The percentage other cysts and percentage living larvae are considered to be binomially distributed. For Pratylenchus spp. and Meloidogyne spp. the number of nematodes is counted before and after incubation. The total number counted and the percentage gathered before incubation are usually reported. The variances and precision of these figures are studied. The total counts are assumed to be Poisson distributed and the number before incubations is assumed to be binomial distributed. In the yearly proficiency test, each participant receives 10 soil samples. The variance between the counts from these samples and the variance of the mean of the 10 counts relative to the overall mean of all the counts in the yearly proficiency test are estimated per participant. Therefore, the data from all years are used. Hence these estimates of the two sources of variation are used to evaluate the precision of a measurement from each laboratory. The quality of a proficiency test is determined by the quality of the distributed samples, the statistics used and the communication with participants about the results. This study provides insight into the accuracy and precision per laboratory in recent years and the trend over the years in which the ring tests were conducted. The results of the statistical analysis will be presented. Key words: proficiency test, laboratory, nematode, counts, statistical analysis


57

16:40 HOST STATUS OF FODDER RADISH

(RAPHANUS SATIVUS VAR. OLEIFORMIS) TOWARDS MELOIDOGYNE CHITWOODI

Misghina G. TEKLU1, Thomas H. BEEN2 & Corrie H. SCHOMAKER2

1 Nematology Section, Department of Biology, Ghent University K.L. Ledeganckstraat 35, BE-9000 Ghent Belgium

2 Plant Research International, Wageningen University and Research Center Plant science group, P.O.Box 16, NL-6700 Wageningen, the Netherlands.

Five resistant varieties of fodder radish; Anaconda, Contra, Defender, Doublet, and Terranova, were compared to the susceptible variety, Radical, in order to estimate their degree of resistance towards Meloidogyne chitwoodi. All varieties were grown in 6 liter pots in a greenhouse under controlled conditions and subjected to initial population densities (Pi) ranging from 0, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64 to 128 J2/g dry soil in 5 replications (3 replications for Pi = 128). Five pots with one susceptible tomato plant, cv. Moneymaker, at Pi = 4 J2/g dry soil were used to check the quality of the inoculums. The fodder radish plants, 5 per pot, were allowed to grow for a period of 11 weeks until their flowering stage. Water uses, fresh and dry weight, were measured to estimate the total yield. The root system of each plant was examined for the presence of galls. The final population density (Pf) was measured by adding nematode counts from both the soil and the roots. The relationship obtained between Pi and Pf was fitted to the Seinhorst model for free living nematodes using R. (version). Plants of all varieties, germinating at the highest density (Pi = 128) died within a week after their germination. The replanted seedlings developed into plants. The relative fresh weight of all varieties was the same at all densities. Therefore, yield, expressed as total fresh weight was not affected by the nematodes. The total fresh weight of Radical was 5 till 12% higher than weights observed in Anaconda, Contra, Defender (P<0.05). The mean total fresh weight of Doublet and Terranova were not different from Radical (P>0.05). A higher percentage of galled roots were observed on the susceptible variety (40%) compared to the resistant varieties (<12%). Galling increased with nematode density. For the susceptible variety, Radical, a maximum multiplication rate (a) of 0.39 and maximum population density (M) of 6.43 was estimated. Radical proved to be a bad host for M. chitwoodi with all final population densities below the equilibrium line and effectively reducing population densities of M. chitwoodi by 60% at the smallest and 95% at the highest density. The resistant varieties performed even better. The maximum population densities (M) for Anaconda, Contra, Defender, Doublet and Terranova were 0.021, 0.170, 0.373, 0.033, and 0.018. With Radical taken to be 100% susceptible, this resulted in relative susceptibilities (r.s.) of these varieties in to: 0.34, 2.73, 5.97, 0.53 and 0.29% respectively, reducing high levels of M. chitwoodi with more than 99%. Key words: Resistance testing, maximum‐multiplication rate, maximum‐population density.


58

17:00 THE USE OF SEAWEED-BASED PRODUCTS FROM

ECKLONIA MAXIMA AND ASCOPHYLUM NODOSUM AS CONTROL AGENTS FOR MELOIDOGYNE

CHITWOODI AND M. HAPLA ON TOMATO PLANTS

Bruno M. NGALA1, Wim M.L. WESEMAEL2 & Roland N. PERRY1,3 1 Faculty of Science, Department of Biology, Ghent University, Ghent, Belgium

2 Institute for Agricultural and Fisheries Research, Merelbeke, Belgium 1,3 Plant Pathology and Microbiology Department

Rothamsted Research, Harpenden, United Kingdom Two commercially available seaweed products, derived from Ascophylum nodosum (An) and Ecklonia maxima (Em), were evaluated for their potential as control agents for the temperate root‐knot nematodes, Meloidogyne chitwoodi and M. hapla. The effects of both products on hatching, host location and penetration by second‐stage juveniles (J2) were examined. The effect on nematode populations and plant growth were assessed in a pot experiment with tomato (Solanum lycopersicum cv. Moneymaker). For M. chitwoodi, continuous exposure off egg masses to 100% An significantly reduced the final percentage hatch. For M. hapla the effect was less pronounced but a reduction in hatching was also observed after immersion in a 50% An solution. Em did not affect hatching of either nematode species. In a bioassay in pluronic gel on Petri dishes, significantly more J2 of M. chitwoodi were found in the 0.5 cm vicinity of the roots of a tomato seedling after 24h exposure to Em. The same effect was observed for M. hapla after 6h exposure but there was no difference with the control (distilled water) after 24h exposure of J2. Treatments with An did not differ from the control for both exposure time and species. There was a significant reduction in penetration of tomato plants after 24h exposure of M. chitwoodi J2 to Em and An. The highest reduction was observed after treatments with An. For M. hapla only 24h exposure to An induced a reduction in penetration. In the pot experiment, treatments with the seaweed based products had no effect on the final nematode populations. Shoot length and biomass of the tomato plants did not differ between treatments. The root biomass increased when plants were treated with An and Em but this was only significant in the treatments with M. chitwoodi. Based on our results An had a clear effect on hatching and infectivity of M. chitwoodi. However, the level of control might be limited under normal agricultural conditions. Key words: attraction, biological control, hatching, infectivity, Meloidogyne

Session 3 Exploiting genomics to understand plant-

Nematode interactions – Part 1 (COST Action 872)

59

Session 3 Exploiting genomics to understand plant‐nematode interactions Part 1 (COST Action 872)

Session 3 Exploiting genomics to understand plant- Nematode interactions – Part 1 (COST Action 872)

60



61

11:00 DE NOVO ANALYSIS OF 454 GSFLX

TRANSCRIPTOME OF PRATYLENCHUS THORNEI

Michael G.K. JONES, Gill REETINDER, Paul NICHOL & John FOSU-NYARKO

WA State Agricultural Biotechnology Centre, Murdoch University Perth WA 6150, Australia

Root lesion nematodes, Pratylenchus spp. are economically important migratory endoparasites.They are the most serious nematode pathogens of cereals (wheat, barley) and sugarcane in Australia, where they cause losses of about 10% for wheat, and 10‐15% for sugarcane.Different populations of a range of Pratylenchus spp (P. thornei, P. neglectus, P. penetrans, P. zeae) have been obtained and maintained on carrot tissues in vitro. We are undertaking a de novo analysis of the P. thornei transcriptome using 454 GSFlx technology to facilitate genomic studies to underpin knowledge that might lead to new approaches to their control. In this study, 6,989 contigs common to MIRA and CAP3 assemblers were assembled from 393,637 reads, with six fold average sequence coverage. The contigs were annotated using the Kyoto KEGG biochemical pathway database and Gene Ontology (GO) hierarchy. In functional analysis of these transcripts we have focussed on four subjects: wall degrading enzymes, sterol metabolism, neuropeptides and parasitism genes. This enables a compari‐son of such genes in P. thornei with those already studied in the sedentary endoparasites root‐knot and cyst‐nematodes.


62

11:20 ANALYSIS OF THE TRANSCRIPTOME OF

HIRSCHMANNIELLA ORYZAE TO GAIN FURTHER INSIGHT INTO THE PLANT-NEMATODE INTERACTION

Lander BAUTERS, Annelies HAEGEMAN & Godelieve GHEYSEN

Ghent University, dpt. Molecular Biotechnology Coupure links 653, BE-9000 Ghent, Belgium

The rice‐root nematode Hirschmanniella oryzae is the most abundant nematode in flooded rice fields all over the world. It can cause severe yield reductions and economic losses depending on the environmental circumstances. Due to the fact that H. oryzae is a migratory nematode, it is economically less important compared to the root‐knot and cyst nematodes, although yield losses up to more than 30% have been reported. Little is known about the arsenal of secreted proteins used by this nematode to colonize the host plant. We have recently performed 454 sequencing on a mixed population to generate expressed sequence tags (EST), hereby obtaining 48347 contigs and 58564 singletons after assembly. Sequences where analyzed and compared with other freely available EST‐data of nematodes to screen for putative effector genes and candidate cell wall modifying proteins. The data was also compared with recently obtained EST‐data from another migratory nematode, Pratylenchus coffeae. Several putative plant cell wall degrading enzymes where identified and some of them could be specifically assigned to migratory species. Some interesting putative effector genes were also detected during the screen. By means of in situ hybridization and functional studies these putative parasitism genes are currently further characterized



63

11:40 NEW CANDIDATE EFFECTORS SPECIFICALLY

EXPRESSED IN EARLY PARASITIC STAGES OF ROOT KNOT NEMATODE

Maëlle JAOUANNET, Emeline DELEURY, Laetitia PERFUS-BARBEOCH,

Marc MAGLIANO, Nathalie MARTHEU, Pierre ABAD & Marie-Noëlle ROSSO INRA, UMR 1064, Université de Nice Sophia Antipolis, CNRS, UMR 6243

Interactions Biotiques et Santé Végétale 400, route des Chappes, BP 167, FR-06903 Sophia Antipolis, France

Root‐knot nematodes are obligate root parasites that establish a unique relationship with their hosts as they induce the re‐differentiation of root cells into specialized feeding cells called giant‐cells essential to the development of the nematode into adult stages and offspring production. Five to seven giant‐cells are formed in the root vascular cylinder after the second stage juveniles have invaded the root tissue and injected effector molecules in the selected cells. Nematode effectors are the compounds secreted during the interaction that manipulate host functions to promote parasitism. Of particular importance are the effectors produced by oesophageal gland cells and secreted through the stylet in the plant tissue during parasitism. Genome sequencing of two RKN species, M. incognita and M. hapla provided the first genome data available for plant parasitic nematodes and a fabulous opportunity to further explore the key effectors required for the success of infection. In order to optimally benefit from these genome sequences we generated additional EST dataset from various M. incognita developmental stages and added 38,769 EST sequences to the 20,312 publicly available sequences. We searched for new candidate effectors by comparative analyses of EST datasets and identified genes specifically expressed during the early steps of infection. A series of sequence selection criteria allowed for the identification of three RKN genes that encode predicted secreted proteins expressed in the oesophageal glands of parasitic stages. These effector candidates are specific to RKN species. Two of them have unknown function and one encodes a putative protease. Interestingly, two candidates are localized in duplicated regions of the genome in M. incognita. Transient expression in N. benthamiana leaf cells indicated that one candidate effector is targeted to the nucleus. Further functional characterizations addressed the roles of these candidate effectors in the development of the disease.


64

12:00 SMALL SUBUNIT RIBOSOMAL DNA SEQUENCES OF A

WIDE RANGE OF TYLENCHIDAE SUGGEST FOR A DEEP SPLIT IN THE ORDER TYLENCHIDA

Akbar KAREGAR1, Hanny VAN MEGEN2, Sven VAN DEN ELSEN2,

Paul MOOIJMAN2, Gerrit KARSSEN3 & Johannes HELDER2 1Department of Plant Protection, College of Agriculture, Shiraz University

Shiraz, 71441-65186 Iran 2Laboratory of Nematology, Department of Plant Sciences, Wageningen University,

Droevendaalsesteeg 1, NL-6708 PB Wageningen, The Netherlands 3Plant Protection Service, Nematology Section

Geertjesweg 15, NL-6706 EA Wageningen, The Netherlands Most of the high‐impact plant parasitic nematodes such as root knot (RKN), cyst (CN) and lesion (LN) nematodes belong to the order Tylenchida. Small subunit ribosomal DNA (SSU rDNA) sequences suggest for a distal positioning of these advanced plant parasites in Clade 12 (e.g. Holterman et al. 2006); a robustly supported node gives rise to CNs (and Hoplolaimidae) on the one hand, and to LNs and RKNs on the other. Insight in the evolution of these advanced plant parasites requires a neutral molecular framework for the order Tylenchida. Siddiqi (2000) distinguished four Tylenchida suborders: Hoplolaimina (including RKN, CN and LN), Criconematina, Hexatylina (mostly insect parasites) and Tylenchina. Ribosomal DNA data are available from a fair number of representatives of the first three suborders. However, the Tylenchina are highly underrepresented. This is understandable as these algal, moss, lichen and fungal feeding nematodes have hardly any, or no economical impact, and as expertise on the identification of these small, long‐tailed Tylenchida is scarce. Recently we’ve identified and characterized a substantial number of representatives of the diverse family Tylenchidae. The resulting data suggest for a deep split in the order Tylenchida; one branch harbors the Hoplolaimina, Criconematima and a part of the Tylenchina, whereas the other branch includes the Hexatylina and the remaining part of the Tylenchina (including all Anguinoidea). Implications of these results for the reconstruction of the evolution from fungivorous lifestyles towards advanced obligatory plant parasites will be discussed. Holterman, M., van der Wurff, A., van den Elsen, S., van Megen, H., Bongers, T., Holovachov,

O., Bakker, J., and Helder, J. 2006. Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Mol. Biol. Evol. 23:1792-1800.

Siddiqi, M. R. 2000. Tylenchida: Parasites of Plants and Insects. CABI Publishing, Wallingford, UK.



65

12:20 ORIGINS AND EVOLUTION OF SATELLITE DNAS:

THE NEMATODE VIEW

Nevenka MEŠTROVIĆ1, Philippe CASTAGNONE-SERENO2 & Miroslav PLOHL1

1Ruđer Bošković Institute, Bijenička 54, HR-10000 Zagreb, Croatia 2INRA UMR1301/UNSA/CNRS UMR6243, FR-06903 Sophia Antipolis, France

The noncoding DNA sequences repeated in tandem, satellite DNAs, compose significant portion of every eukaryotic genome. Despite tremendous advances in sequencing and assembly of genomic DNA, satellite repeats are underrepresented in outputs of genome projects, which are still focused on non‐repetitive, gene‐rich euchromatic segments. However, more and more attention is given to the noncoding genomic fraction, because it gives an organisational framework to every eukaryotic genome, underlies pivotal genomic functions and may represent a major trigger in speciation. Unlike coding DNA, satellite DNA vary greatly in size and sequence even in closely related species and this extreme diversity raised difficulties in deriving general conclusions about the origin, evolution and function of satellite DNA in higher organisms. The study of satellite DNAs in closely related species contributes to the understanding of satellite DNA evolution and dynamic processes that structure eukaryotic genomes. Moderate abundance of different satellite repeats, parthenogenetic mode of reproduction, defined phylogenetic relationships and available source of genome sequencing data make RKN species a good model system to study satellite DNAs. The overview of our present studies on a subset of Meloidogyne species will be presented. In particular, the results obtained relate stochastic processes and selective pressure driving the evolution of satellite DNAs, explain the origins of different forms of satellite DNAs coexisting in genomes in the form of a common library, and establish genomic composition of satellite DNAs as a phylogenetic marker capable to help in resolving some questions in RKN species evolution. Although obtained on a limited sample of RKN species, we suggest that such information describes a general behavior characteristic of all satellite DNAs.


66

12:40 THE MITOCHONDRIA GENOME OF

GLOBODERA PALLIDA

V. BLOK1, P. COCK1, P. HEDLEY1, J. MORRIS1, N. GRUJIC1, M. HUNT2, L. PYLYPENKO3, A. HOOLAHAN4, M. DOWTON4 & J. JONES1

1Plant Pathology Programme, SCRI, Invergowrie, Dundee, DD2 5DA, UK 2Pathogen Genomics, The Wellcome Trust Sanger Institute

Genome Campus, Hinxton, Cambridge, UK 3Plant Quarantine Department, Institute of Plant Protection

33 Vasilkovskaya Str., Kiev 22, 03022, Ukraine 4Institute of Biomolecular Sciences, School of Biology

University of Wollongong, Wollongong, NSW, 2522, Australia Animal mitochondrial genomes are typically small circular molecules of between 14 and 20 Kb containing 37 genes, including 13 protein encoding genes that produce various subunits of the enzyme complexes forming the respiratory chain, 2 rRNA genes, and 22 tRNA genes. However, there are deviations from this model amongst them plant‐parasitic nematodes: Globodera pallida has a multipartite mitochondrial genome, the genome of Xiphinema americanum is compact with overlapping genes and the translation termination codons differ for Radopholus similis. The multipartite genome of G. pallida consists of several small circular molecules and sequencing has revealed gene duplication, the presence of apparent pseudogenes as well as a substantial noncoding region in each scmtDNA.In addition there is considerable diversity in the scmtDNAs amongst G. pallida populations and this intraspecific diversity in the mtDNA of G. pallida is being explored as a means of differentiating populations.The multipartite structure appears to be a general feature for the species and is also found in G. rostochiensis suggesting that this unusual structure predates their evolutionary separation, however, the biological significance and mechanisms of inheritance of the unusual mitochondrial genome of PCN are unclear. The G. pallida genome sequencing project has confirmed the existence of 7scmtDNAs and provided transcription information at different life stages. The relative representation of each of the scmtDNAs apparently differs in the population of G. pallida used in the genome sequence analysis with most hits for scmtDNA IV and VII. Other populations of G. pallida differ in scmtDNA composition with some for example having limited representation of scmtDNA I. The transcriptome has been mapped onto the scmtDNAs to investigate whether differential expression occurs during different life stages and to assess whether pseudogenes are expressed and the large noncoding regions.



67

15:00 MOLECULAR AND HISTOLOGICAL ANALYSIS OF RICE (ORYZA SATIVA) ROOTS RESPONSES TO

MELOIDOGYNE SPP. INFECTION

S. BELLAFIORE1, A. EL ASHRY2, M. BELLIS4, A. BAK1, P. NGUYEN1,7, A-S. PETITOT1, H. ADAM5, J. ESCOUTE6, S. BALZERGUES3 & D. FERNANDEZ1

1 UMR 186 RPB (IRD-UM2-Cirad) 911, avenue Agropolis BP 64501, FR-34394 Montpellier Cedex 5, France

2 Institute of Crop Science and Resource Conservation, Department Molecular Phytomedicine University Bonn, Karlrobert-Kreiten-Str. 13, DE-53115 Bonn, Germany

3 UMR INRA 1165 - CNRS 8114 – UEVE, 2, rue Gaston Crémieux, CP5708, 91057 Evry cedex, France

4 CRBM - CNRS - UMR5237 1919, Route de Mende,FR-34293 Montpellier Cedex 5, France 5UMR DIADE (IRD-INRA-Cirad) 911

avenue Agropolis BP 64501,FR-34394 Montpellier Cedex 5, France 6 Plate-forme d'Histocytologie et d'Imagerie Cellulaire Végétale, Biochimie et Physiologie Moléculaire des Plantes-Développement et Amélioration des Plantes, INRA-CNRS-Cirad,

TA96/02 Avenue Agropolis, FR-34398 Montpellier, France 7Département de Biotechnologie, Université d’Agriculture et de Foresterie

de Hồ Chí Minh ville (UAF),Việt Nam Meloidogyne graminicola and M. incognita are responsible for rice (Oryza sativa) production losses in Asia, Latin America and Africa. All currently grown rice varieties are highly susceptible and resistance to root‐knot nematode species is only found in two rice species from Africa: Oryza glaberrima and O. longistaminata. Identification of nematode virulence effectors and their plant targets may provide new insights for developing control strategies towards Meloidogyne spp. Currently, little is known about the monocotyledonous plants reactions to Meloidogyne infection as most studies were performed on dicotyledonous hosts. We are looking to see if specific answers can be observed in rice‐Meloidogyne interactions. The M. graminicola and M. incognita life cycles in rice were investigated by microscopic observations of infected roots and histological analyses. In parallel, the molecular plant responses to M. graminicola and M. incognita infection were assessed by whole genome transcriptomic analyses (Affymetrix GeneChip® rice genome array) at early stages of the rice‐nematode interaction (migration and feeding site formation). These analyses revealed convergent and specific rice root answers to Meloidogyne infections. Among the differentially regulated rice genes due to the infection by both nematode species, we identified genes that are involved in metabolic pathways, thus providing new insights for possible targets of nematodes effectors.


68

15:20 FUNCTIONAL ANALYSIS OF THE ROLE OF ETHYLENE

IN INDUCED RICE DEFENSE TO INFECTION BY MELOIDOGYNE GRAMINICOLA

Kamrun NAHAR, Tina KYNDT & Godelieve GHEYSEN

Department of Molecular Biotechnology, Faculty of Bioscience Engineering Ghent University, Belgium

The gaseous hormone ethylene (ET) is known to differentially affect the defense mechanism of plants against pathogens and plays an important role in induced defense. Our previous work shows that young rice plants sprayed with Ethephon (an ethylene generator) 24 hrs before M. graminicola infection had a considerable reduction of galls per plant and per gram of root. The goal of this study was to functionally analyse the role of ET‐dependent defenses in rice (Oryza sativa) to infection with the root knot nematode M. graminicola. In order to verify the effectiveness of chemical sprays as an inducer of either or both EIN2 genes (OSEIN2a and OsEIN2b) that could be functional in ET‐treatment, we analyzed transcript levels of these two genes in ET‐treated and non‐treated Nipponbare plant roots. Both OsEIN2‐homologues showed an elevated expression pattern in ET treated plants indicating ET as the signal for enhanced OsEIN2 transcription in rice roots. The antisense ein2b transgenic line was more susceptible than the wild type to infection by M. graminicola. The ein2a silenced plants showed a slightly higher (but this was not significant) susceptibility compared to the wild type rice. The foliar treatment with ethephon did not protect the ethylene insensitive antisense line ein2b suggesting that OsEIN2b plays a role in basal defense against M. graminicola. In addition, in planta ethylene production was inhibited by applying the ET biosynthesis inhibitor aminooxyacetic acid (AOA), one day before nematode inoculation. ET biosynthesis inhibition resulted in a higher sensitivity towards the root knot nematodes, confirming that the ethylene pathway plays an important role in basal defense against M. graminicola. Our results collectively suggest the importance of ET signalling as well as ET biosynthesis in plant defence to root knot nematodes in young rice plants. The contribution of the ET signal transduction component EIN2b is required in this response.



69

15:40 THE TRANSCRIPTOME OF RICE UPON

NEMATODE ATTACK

Tina KYNDT, Simon DENIL, Geert TROOSKENS, Tim DE MEYER, Hongli JI*, Annelies HAEGEMAN, Wim VAN CRIEKINGE

& Godelieve GHEYSEN Department of Molecular Biotechnology, Ghent University (UGent)

Coupure Links 653, BE-9000, Ghent, Belgium * NXTGNT, Ghent University, Medical Research Building

Ghent University Hospital, De Pintelaan 185, BE-9000 Ghent, Belgium Rice is one of the most important crops worldwide and an excellent model system for monocotyledonous plants. Two agronomically very important nematodes attacking rice are the rice root knot nematode Meloidogyne graminicola and the migratory root rot nematode Hirschmanniella oryzae. Since these two nematodes have very different lifestyles, comparing the transcriptome in rice upon infection with these pathogens will provide important insights into general and specific defence strategies of the rice plant. Recently, Next Generation Sequencing methodologies have been introduced into transcriptome research, with mRNAseq currently being the most innovative and high‐throughput method for expression profiling. The goal of our research project is to apply mRNAseq to study the transcriptome of rice roots when challenged by nematode infection. In 2010 an Illumina GAII mRNAseq‐run was performed on roots infected with H. oryzae and root galls caused by M. graminicola, and the corresponding control tissue. This analysis was done at two different time points after infection: 3 and 7 days. For each treatment 3 biological replicates were investigated. On average 7 million reads (76 nt, single end) per sample were generated, they were mapped onto the rice genome and differential expression was evaluated. At each timepoint more than 400 genes were found to be differentially expressed in the root galls, while about 100 genes were differentially expressed in rice roots infected with migratory nematodes (at an FDR cut‐off of 0.05). A subset of these differentially expressed genes were independently validated using qRT‐PCR analysis, all of which confirmed the expression pattern obtained by sequencing. To further analyse the transcriptome changes specifically induced by root knot nematodes, we have now isolated transcripts from nematode induced giant cells, by applying Lasure Capture Microdissection, and mRNAseq on these samples is currently in progress.


70

16:00 ROS AND NO: THEIR ROLE IN TOMATO ROOTS CHALLENGED BY MELOIDOGYNE INCOGNITA

Maria Teresa MELILLO, Paola LEONETTI,Pasqua VERONICO

& Teresa BLEVE-ZACHEO Institute for Plant Protection - CNR, Bari, Italy

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. To investigate the key components involved in oxidative and nitrosative metabolism, experiments were carried out at different infection times by using a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root‐knot nematode Meloidogyne incognita. We analyzed the superoxide radical (O2‐) production, hydrogen peroxide (H2O2) content, and nitric oxide synthase (NOS)‐like, and nitrate reductase (NR) activities, as potential sources of NO. A rapid NO accumulation and ROS production were differently linked to incompatible and compatible tomato‐nematode interactions. NOS‐like arginine‐dependent rather than NR was the main source of NO production, and NOS‐like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H2O2 cooperating in triggering hypersensitive cell death. The results obtained from this studies reveal a defence mechanism that has not been previously described in tomato‐nematode interaction and provide new insight into the complex regulation of ROS and NO metabolism by avr‐ and vir‐ RKN pathotypes in their hosts roots.



71

16:20 ROLE OF THE PLANT CELL WALL AND

CELL WALL SIGNALLING IN THE PLANT-NEMATODE INTERACTION

Krzysztof WIECZOREK

Institute of Plant Protection, University of Natural Resources and Life Sciences Peter Jordan-Straße 82, AT-1190 Vienna, Austria

Most of the plant pathogens encounter the cell wall at the onset of their infection. Plant‐parasitic nematodes have to deal with this structure throughout the parasitic process. They have to enter the root tissue, they migrate through it in order to find the suitable initial cell, they pierce the wall with their stylet and finally they massively change the architecture of the cell wall in the feeding structures they induce. To facilitate all this steps nematodes are armed with a set of different cell wall modifying and degrading proteins and enzymes that are secreted either during the migration or released through the stylet into the feeding site during the stationary phase. Moreover, these secretions are thought to contain factors that might change expression of plant genes, among others those ones that are involved in the cell wall dynamics. During last years we extensively analyzed proteins and enzymes of plant‐origin that are specifically activated in nematode‐feeding structures. We found expansins and glucanases to be differentially expressed during the development of syncytia induced by cyst nematode Heterodera schachii. More recently, we found two upregulated pectate lyases that might play a crucial role in the establishment of syncytia and development of the nematode.


72

16:40 DOES SYNTHETIC BIOLOGY HAVE A

ROLE IN DEVELOPING STRATEGIES TO CONTROL NEMATODES?

Keith G. DAVIES

Plant Pathology and Microbiology, Rothamsted Research, Harpenden Hertfordshire, AL5 2JQ, United Kingdom

Plant‐parasitic nematodes are important crop pests that until recently have been controlled by the application of nematicides. Since 2003 European regulations (91/414) have resulted in the loss of 600 active substances, including nematicides, and new regulations (1107/2009) that come into force in June 2011 will continue this trend; alternative methods of control are therefore necessary. Crop rotation and the use of resistance genes have been deployed as a method of control for many years and several laboratories are investigating the use of genetic modification. Biological control has always been in the shadow of these other approaches, notwithstanding the observation that nematode suppressive soils are a well established phenomenon that is known to develop in mono‐cropping systems. Biological control scientists have isolated numerous strains of bacteria and fungi that are pathogenic to plant‐parasitic nematodes and, although inundation of soil with individual organisms can produce control in glasshouse situations, this approach has generally not proved suitable to transfer into field situations. New approaches and thinking are now required. Arguably, the concept of synthetic biology dates back to the 1974 when it was stated that, “…the real challenge will start when we enter the synthetic biology phase of research in our field. We will then devise new control elements and add these new modules to the existing genomes or build up wholly new genomes.” (Szybalski, 1974). Last year saw the growth of the first microorganism from a synthetically constructed genome (Gibson et al., 2010) heralding that synthetic biology has become a reality. The literature contains a vast store of knowledge regarding the mechanisms and modes of actions of potential microbial pathogens and parasites and the challenge is whether or not synthetic biology can combine the key elements to produce a designer biological control organism that can be applied in the field to produce robust and consistent nematode control. Gibson, DG; Glass, JI; Lartigue, C, et al. (2010) Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome Science 329:52‐56 Szybalski, W. !974) In Vivo and in Vitro Initiation of Transcription, Page 405. In: A. Kohn and A. Shatkay (Eds.), Control of Gene Expression, pp. 23–4, and Discussion pp. 404–5 (Szybalski's concept of Synthetic Biology), 411–2, 415–7. New York: Plenum Press Key words: Key words: biological control, plant‐parasitic nematodes, synthetic biology

Session 4 IPM

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Session 4 IPM

Session 4 IPM

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Session 4 IPM

75

11:00 PREDOMINANT EFFECTS OF HOST PLANT SPECIES ON APHIS GOSSYPII APHID AND

EPISYRPHUS BALTEATUS HOVERFLY IN A TRITROPHIC APPROACH

René-Noel POLIGUI, Frédéric FRANCIS & Eric HAUBRUGE

University of Liege, Gembloux Agro-Bio Tech Passage de Deportes 2, BE-5030 Gembloux, Belgium

The aphid Aphis gossypii Glover (Homoptera: Aphididae) is known as worldwide important pest. It causes serious injuries to crops, but is also able to feed on various cultivated and wild plants. Some programs are developped to control this aphid. In many studies, hoverfy Episyrphus balteatus De Geer (Diptera : Syrphidae) is used as an efficient and aphid specific predator in biological control systems. In this work, we study cotton aphid choice plant discrimination, the effect of host plants on its developpment (fecondity and mortality), and the E. balteatus foraging and oviposition behavior according aphid host plants. Biologic parameters were observed using various plant combinations. E. balteatus behavioral observations were conducted using the Noldus Observer v.5.0. A. gossypii makes discriminant choice of his hosts, with better preference on Cucurbita pepo L. (Cucurbitaceae) and Capsicum annuum L. (Solanaceae). Dacryodes edulis (G. Don) H.J. Lam. (Burseraceae) and Vicia faba L. (Fabaceae) are the midle choice while Hibiscus sabdariffa L. (Malvaceae) is less chosen. A. gossypii develops easily on C. pepo, and weakly on C. anuum, H.sabdariffa,and V. faba. D. edulis induces very high mortality to A. gossypii. According to E. balteatus behavior, A. gossypii is more attractive when infesting C. pepo and V. faba, less on H. sabdariffa. While non infested, the hoverfly more attractive plant is V. faba. Our results highlited the importance of interactions between aphids and their host plants and the prey‐host influence on a specific aphid predatory hoverfy. Suitable ways in correlation with this work are to investigated to complete this study and perform aphids biological control. Key words: Aphids, Hoverfly, host plants, prey‐host influence, biological control

Session 4 IPM

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11:20 OPTIMAL PROFIT OF THE PARASITATION

BY APHELINUS MALI IN AN IPM COMPLEMENTARY STRATEGY FOR THE CONTROL

OF ERIOSOMA LANIGERUM

D. GOOSSENS2, E. BANGELS1, T. BELIEN1, C. SCHOEVAERTS2 & L. DE MAEYER2

1pcfruit, Zoology Department Fruittuinweg 1, BE-3800 Sint-Truiden, Belgium

2Bayer CropScience J.E. Mommaertslaan 14, BE-1831 Diegem (Machelen), Belgium

During summer the parasitoid Aphelinus mali may certainly impact woolly apple aphid (Eriosoma lanigerum), but studies on the single interaction rarely indicate sufficient biocontrol in the period May‐June. In this period, on a high infestation of woolly apple aphid, chemical control by spirotetramat or pirimicarb remains indispensable in order to anticipate on the dense migration waves and subsequent colonization of extension shoots. The insufficient contribution of the early parasitation by A. mali to avoid woolly apple aphid damage is linked with a later emergence from diapauze and slower reproduction than its host. In 2010, modeling for A. mali based on activity threshold set at at 8.4°C and T° sums, corresponded perfectly with the first adult flights detection monitored on yellow sticky traps in 3 orchards. Further accurate monitoring of adult flights all along the season enabled also to determine an identical period of the endoparasitic stages of A. mali in these 3 orchards. During this endoparasitic phase A. mali larvae reside inside their host and compounds with known acute toxicity on A. mali adults, like CNI’s, are preferably positioned here to claim selectivity in the time. Respecting this principle, the further population development and parasitation potential of A. mali in summer is influenced negligibly. The preservation of the first peak of flights of A. mali in the preflowering period is essential for the exponential flight increase and subconsequent culmination of the parasitation ratio in summer, which constitutes a valuable complement in the integrated E. lanigerum control strategy. A. mali is not favored by humid conditions nor by frequent mowing of the grass lanes where adults roam for nectar collection on herbs. Studies on alternated mowing of grass lanes in the period from half of June till mid July to promote the high expansion of A. mali in summer are ongoing. Optimal management of parasitoids through simple practices can significantly contribute to the complementary IPM strategy for woolly apple aphid control can be realized. Key words: monitoring parasitoids, woolly apple aphid, selectivity in the time, IPM

Session 4 IPM

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11:40 THE EFFECTS OF SCALE INSECT PREDATOR CHILOCORUS NIGRITUS ON GLASSHOUSE

PEST CONTROL

J.P.J. BURMAN & D.J. PONSONBY Geographical Life Sciences Dept, Canterbury Christ Church University

North Holmes Road, Canterbury, Kent, England, CT1 1QU Chilocorus nigritus is a commercially important ladybird considered to have been one of the most successful biocontrol agents for scale insect management in recent history. It has provided vital pest control in various classical biocontrol releases in South Africa and South East Asia where successfully established populations still remain to this day. The ability of these insects to suppress pests in glasshouses still remains largely un‐tested, despite their common usage in glasshouse control since the 1980s. This experiment aimed to establish the efficacy of Chilocorus nigritus larvae in augmentative release program to control scale insects in heterogeneous glasshouse environments. Chilocorus nigritus larvae were released on a monthly basis for 6 months at Royal Botanical Gardens, Kew. Their subsequent establishment in both the Palm House and Temperate House was monitored. The impact on various scale insect populations was also monitored over a period of 18 months before and during the release schedule, whilst climatic and pesticide spray data were also collected. Data on scale insect populations were obtained via a fully randomised sampling method devised specifically for heterogeneous plant communities. Differences in the abundance of several species of soft and armoured scale insects before and after the release program were tested. There were no signs of long term establishment of Chilocorus nigritus in either glasshouse, but the augmentative release appeared to reduce the host population on some plants. The potential effects of pesticide usage and varying climate are also discussed in with mind to their potential impacts on the insect populations. Key words: Chilocorus nigritus, Biocontrol, Glasshouse, Scale insect, Pesticide, Release.

Session 4 IPM

78

12:00 LABORATORY ASSAYS ON THE COLORADO

POTATO BEETLE PREDATION BY LACEWING LARVA

L. SABLON, E. HAUBRUGE & F.J. VERHEGGEN Unit of Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech

University of Liège, Belgium The Colorado Potato beetle (CPB), Leptinotarsa decemlineata, is the most efficient defoliator of potatoes in Europe and North America. It was introduced in Europe in 1922 and quickly colonized Europe except the British Isles and Scandinavia, before reaching Belgium 1935. Many control techniques have been implemented to control this pest, from which the application of insecticides remains the mostly used. Meet future European norms on pesticide and resurgence of CPB in our fields, we evaluated the ability of the lacewing Chrysoperla carnea, a generalist predator, to consume CPB individuals. During laboratory experiments, we have placed in Petri dishes a lacewing larva in presence of a determined CPB larvae instar, in excess. 24 hours later, we have observed the surviving individuals. These laboratory results clearly indicate a high predation rate of CPB larvae and eggs by lacewing larvae. Our results also highlight the different predation strategies and food consumption for each larval instar of CPB. These initial laboratory results suggest a possible biological control approach against the Colorado potato beetle using lacewing larvae. Key words: Leptinotarsa decemlineata,Chrysoperla carnea, predation, larvae, eggs

Session 4 IPM

79

12:20 SIDE EFFECTS OF MODERN PESTICIDES ON

THE PREDATORY BUG ORIUS LAEVIGATUS (FIEBER) (HEMIPTERA: ANTHOCORIDAE)

UNDER LABORATORY CONDITIONS F. AMOR, P. BENGOCHEA, O. CHRISTIAENS*, E. VELÁZQUEZ, I. MORALES,

Mª. M. FERNÁNDEZ, G. SMAGGHE* & E. VIÑUELA Department of Crop Protection, Escuela Técnica Superior de Ingenieros Agrónomos, Technical

University of Madrid (UPM), ES-28040, Madrid (Spain) * Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University

Coupure Links 653, BE-9000 Ghent (Belgium) The initial contact toxicity and persistence of 7 modern insecticides, that are included or with requested inclusion in annex I of the directive 91/414/EEC, were studied on O. laevigatus. Pesticides were applied on glass surfaces using the Potter Precision Spray Tower (standard deposit of 1.5‐2 mg spray fluid per cm2; 1 ml, 50 kPa) at their respective maximum field recommended rate for practical use. Adults of O. laevigatus were exposed to fresh spray deposits for 72 hours. Mortality at 72 hours as well as fecundity and fertility of the surviving bugs were recorded and pesticides classified according to IOBC toxicity ratings. Flubendiamide (ryanodine receptor modulator), methoxyfenozide (moulting accelerator) and spiromesifen (lipid synthesis inhibitor) were harmless (1), emamectin benzoate (chloride channel agonist) was slightly toxic (2), and abamectin (chloride channel agonist), deltamethrin (neurotoxic pyrethroid) and spinosad (neurotoxic naturalyte) were harmful. The duration of harmful activity was only tested for those compounds which proved to be not harmless at the initial contact tests. Persistence studies showed that emamectin benzoate was slightly persistent (Class B: toxicity 5‐15 days), while abamectin, deltamethrin and spinosad were very persistent (Class D: toxicity >30 days). Further studies under semi‐field conditions are needed to completely ascertain their effects on the predatory bug. Key words: Orius laevigatus, pesticides side‐effects, laboratory method.

Session 4 IPM

80

12:40 EFFECTS OF INSECTICIDE SUBLETHAL

CONCENTRATIONS ON THE RINGLEGGED EARWIG, EUBORELLIA ANNULIPES (LUCAS)

R. PORAMARCOM

Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520, Thailand

Integrated pest control of sugarcane stem borers, Chilo infuscatellus, recommends the use of a voracious predator as the ringlegged earwig, Euborellia annulipes (Lucas) and insecticide application. This study intended to evaluate in the laboratory the sublethal effects of one recommended insecticide, cypermethrin, on E. annulipes adults related to their ability to continue to predate and reproduce offspring. Sublethal concentrations of 0.025 and 0.15 % active ingredients of cypermethrin (10% EC) were topically applied in two different ways to tested individuals. They were applied directly to adults of E. annulipes and indirectly to the 3rd instar larvae of C. infuscatellus that were then fed to E. annulipes adults to test for sublethal effects. E. annulipes adults treated directly with 0.025 and 0.15% concentrations showed slower onset to prey averaged 14.20 and 18.35 h, lower consumption of preys averaged 0.022 and 0.018 gm/d, lower number of eggs/batch averaged 55.9 and 31.8 eggs and lower hatchability averaged 65.0 and 27.6%, respectively, as compared to those of untreated ones. Similar results were obtained on E. annulipes adults treated indirectly with the same concentrations of cypermethrin. The only different result was recorded on the consumption of preys by treated adults at 0.025% averaged 0.035 gm/d which was not significantly different as compared to that of untreated ones. Developmental period of the offspring from adults treated directly and indirectly at 0.15% was significantly prolonged to 91.8 and 95.6 d as compared to 54.9 and 52.8 d, respectively, from untreated ones. This study concluded that sublethal concentrations of cypermethrin which did not kill E. annulipes adults reduced their continued ability to predate, caused them to produce fewer offspring, and prolonged the developmental period of their offspring. It is suggested that E. annulipes cannot be used safely together with cypermethrin to control C. infuscatellus larvae. Key words: Euborellia annulipes, ringlegged earwig, predator, sublethal concentrations, Chilo infuscatellus

Session 5 Phytopathology

81



82


83

15:00 RAPID DETECTION AND QUANTIFICATION FOR THE CAUSAL AGENT OF CERCOSPORA

LEAF SPOT IN SUGAR BEET

Abdallah ALI, Peter WOLF & Joseph-Alexander VERREET Department of Phytopathology, Christian-Albrechts-Universität Kiel

Hermann Rodewald Strasse 9, DE-24118 Kiel, Germany Cercospora leaf spot (CLS) is considered to be the most common and destructive foliar disease in sugar beet worldwide. It causes reductions in sugar yield and financial returns. It is caused by the fungus Cercospora beticola which survives as stromata in sugar beet leaf residues in soil. It germinates under optimal conditions and produces conidia which are dispersed as primary inoculum to initiate infection in sugar beet. The objective of this research was to develop a method for qualitative detection of the fungus in soil and to quantify the amount of its inoculum in order to improve predictions of disease onset within an integrated disease management system. In this study two diagnostic methods, polymerase chain reaction (PCR) and ELISA technique were successfully applied for qualitative and quantitative detection of C. beticola in soil. Several fields in Germany and USA were surveyed for C. beticola. Soils were sampled from several sugar beet growing fields, from three layers of soil; 0‐5 cm depth, 5‐15 cm depth, 15‐30 cm depth. The fields were either under sugar beet cultivation or in rotation with other crops. The DNA was purified from soil using Power Soil DNA Kit (MO BIO Lab.) according to manufacturer’s instructions. The purified DNA was collected and subjected to PCR reaction with CBACTIN based primers. Amplification was carried out over 40 cycles at 94°C for 1 min denaturation, 65°C for 30sec annealing, 72°C for 1 min extension and 5 min final extension at 72°C. The quantification of Cercospora DNA in soil was analyzed by real time PCR. QPCR sensitivity assessed by10‐fold serial dilutions of genomic DNA of C. beticola from pure culture (100, 10, 1ng/µ1, 100, 10,1pg/µ1, 100fg/µ1) as a standard DNA for real time PCR reactions. Two replicates were used to quantify the amount of DNA for every sample. The second method of detection was ELISA technique using monoclonal antibodies which were developed using pre‐ absorbed C. beticola serum and it achieved in biochemistry institute, Christian‐Albrechts‐University of Kiel, Germany. The monoclonal antibodies were tested against fungal organisms from different classes, for potential cross reactivity. Key words: C. beticola, sugar beet, QPCR, ELISA technique


84

15:20 DETECTION AND CLASSIFICATION OF

FOLIAR SUGAR BEET DISEASES BY MEANS OF LEAF REFLECTANCE

T. RUMPF1, A-K. MAHLEIN2, H-W. DEHNE2

, U. STEINER2 & E-C. OERKE2 1 Institute of Geodesy and Geoinformation, Department of Geoinformation, University of Bonn,

Meckenheimer Allee 172, DE-53115 Bonn, Germany 2 Institute of Crop Science and Resource Conversation (INRES – Phytomedicine)

University of Bonn, Nussallee 9, DE-53115 Bonn, Germany Precision crop protection offers a high potential to reduce the costs and environmental impact of fungicide use. However, a precise, reproducible and time‐saving disease monitoring method is essential for a site specific management of variable diseased field sites. Hence the suitability of hyperspectral reflectance and two different analysis methods from machine learning were proven in this work. Hyperspectral data was recorded from healthy sugar beet leaves and leaves inoculated with the pathogens Cercospora beticola, Uromyces betae or Erysiphe betae causing Cercospora leaf spot, sugar beet rust and powdery mildew, respectively for a period of 21 days after inoculation. Nine spectral vegetation indices from remote sensing science, related to biochemical and biophysical plant parameters were calculated on reflectance data. These spectral vegetation indices were used as features for machine learning by Support Vector Machines. With this kernel‐based statistical approach an early differentiation between healthy and inoculated plants and among the different diseases was achieved with classification accuracy up to 96%. In a second attempt spectral vegetation indices, specific for the different diseases were evaluated to simplify the detection and identification procedure. Therefore, significant wavelengths for each disease were analyzed by reflectance difference, standard deviation and the Wilcoxon rank‐sum test. Furthermore band combinations, highly correlated to the diseases were assessed testing all possible two band differences and ratios. For the generation of hyperspectral vegetation indices, specific for the detection of sugar beet diseases the best weighted combination of a single wavelength and wavelengths difference were searched systematically using a correlation‐based filter algorithm according to Hall. The final, optimized, disease specific vegetation indices were proven for their ability to detect and to classify healthy and diseased sugar beet leaves. With high accuracies up to 93% healthy sugar beet leaves and leaves, diseased with Cercospora leaf spot, powdery mildew and sugar beet rust were classified. Both methods, based on common vegetation indices and the developed disease specific vegetation indices will fulfill non‐invasive and automatic disease detection and monitoring to improve precision agricultural applications. Key words: hyperspectral reflectance, spectral vegetation indices, automatic classification, sugar beet


85

15:40 DISTRIBUTION OF CLAVIBACTER MICHIGANENSIS SUBSP. MICHIGANENSIS IN PRIMARILY INFECTED

TOMATO PLANTS

J.M. VAN DER WOLF1, D. LUDEKING2, R. HAMELINK2, M. SCHENK2 & P.S. VAN DER ZOUWEN1

1 Plant Research International, Biointeraction and Plant Health, Wageningen, Netherlands 2 Wageningen UR Greenhouse Horticulture, Bleiswijk, Netherlands

Clavibacter michiganensis subsp. michiganensis (Cmm), the causative agent of bacterial canker of tomato, is a regulated seed transmitted disease (EPPO A2). The disease of great importance for the tomato producing industry, as outbreaks often result in high economic losses. For seeds, validated methods for testing exist, but not for testing of plants, because knowledge on the distribution of Cmm in plant material is limited and hence effective sampling strategies are lacking. Testing protocols for plants will be important in case of disease outbreaks. We studied the distribution of Cmm in plants grown from infected seed as a first step in the development of a sampling protocol. Seeds were used, 100% infected with a spontaneous rifampicin resistant strain of Cmm. Cmm was detected by plating of extracts on the semi‐selective medium SCMFast supplemented with rifampicin. The identity of suspected colonies was confirmed with PCR. Seed contamination levels ranged from 5 to 2200 cfu per seed. Seeds were grown in rockwool blocks placed in plastic containers (2 plants per container). Sixty days after sowing, 94% of the plants were infected, from which 73% showed symptoms. The first symptoms were visible at 25 days after sowing. Fourteen days after sowing, from 60 plants a cotyledon was sampled of which 37 were infected. After 35 days the percentages of infected plant parts were 77, 67, 63 and 53% for roots, leaves stems and cotyledons, respectively (n=30). After 60 days, the percentages of infected plant parts were 69, 63, 63 and 44% for stems, young leaves, roots and old leaves respectively (n=16). The population densities in the different plant parts ranged from ca. 30 to higher than 100.000 cells per gram of tissue. The distribution of the density levels were similar for the different plant parts at the different sampling times. Sixteen days after sowing, one plant grown from Cmm‐free seed was placed in 30 containers with plants grown from Cmm‐infected seeds. After 60 days, 2 plants (6.7%) showed wilting. The percentages of infected plant parts were 25, 10, 10 and 15% for stems, old leaves, young leaves and roots, respectively. In conclusion, Cmm is efficiently transmitted from seed to seedling, even at low densities of ca. 20 cfu per seed. Plants grown from infected seeds can be sampled in different plant stages. All plant parts can be sampled because only small differences were found in the infection incidence for the different plant parts. In containers, Cmm is efficiently transmitted from infected to non‐infected plants. Key words: sampling, spreading, infected tomato seed,


86

16:00 STUDYING XANTHOMONAS FRAGARIAE

PATHOGENICITY THROUGH WHOLE GENOME ANALYSIS

J. VANDROEMME1, P. DE VOS4, T. DECKERS3, P. MELIS2,

M. THOELEN3, W. VERJANS3 & M. MAES1 1 Plant Sciences Unit – Crop Protection, Institute for Agricultural and Fisheries Research,

Merelbeke, Belgium 2 Research Station Hoogstraten, Hoogstraten, Belgium

3 Research Station Fruit, Sint-Truiden, Belgium 4 Laboratory of Microbiology, Ghent University, Ghent, Belgium

The introduction of next generation sequencing techniques has created new possibilities in the study of plant pathogenic bacteria. Now labs have access to genomic data of own iso‐lates through commercial sequencing services and online gene annotation. Four Xanthomonas genomes have been sequenced in ILVO, and they are comparatively analysed for genes involved in pathogenicity and host range delineation. One was a Xan‐thomonas fragariae (Xf) strain. Xf has a host range restricted to strawberry and causes angu‐lar leaf spot. In earlier ILVO research Xf manifested some exceptional traits such as fastidious growth, repellence to genomic transformation and apparent lack of expressed pathogenicity functions. Two other sequenced strains were representatives of Xanthomonas arboricola pv. fragariae (Xaf), also a pathogen on strawberry. Although both strains have been identified as Xaf, one was reported to be deficient in pathogenicity. The fourth sequenced strain was a member of Xanthomonas arboricola pv. pruni (Xap), taxonomically closely related to Xaf, but with Prunus as host. The genomic sequence of the non‐pathogenic Xaf strain confirmed it missed all genes relat‐ed to the type three secretion system (T3SS) that is pivotal in Xanthomonas pathogenicity. The second Xaf strain, although closely related to the former one, represented a different genomic lineage within Xanthomonas arboricola. The genome sequence of Xf revealed a history of intense contact with external DNA. The genome has a high presence of insertion sequence elements, comparable to Xanthomonas oryzae pv. oryzae (Xoo) (1). The presence of a large CRISPR (clustered regularly interspersed short palindromic repeats) element ‐ believed to be a bacterial immune system against invading viral and plasmidal DNA (2) ‐ could be an evolutionary answer of Xf to this external DNA pressure. The Xf genome evolved away from that of other Xanthomonads, as is reflect‐ed in a lower presence of genes for cell wall degrading enzymes, a deletion in a key patho‐genicity region and the presence of several new putative T3SS effectors. 1. Genome sequence and rapid evolution of the rice pathogen Xanthomonas oryzae pv. oryzae

PXO99 Salzberg et al., BMC Genomics 2008, 9:204 2. Analysis of CRISPR system function in plant pathogen Xanthomonas oryzae, Semenova et

al., FEMS Microbiol Lett (2009), 296:110–116


87

16:20 BACTERIAL CANKER OF PLUM TREES, CAUSED BY

PSEUDOMONAS SYRINGAE PATHOVARS, AS A SERIOUS THREAT FOR PLUM

PRODUCTION IN THE NETHERLANDS'

M. WENNEKER1, J.D. JANSE2 & A. DE BRUINE1 1Applied Plant Research, Research Unit Fruit, Wageningen UR

P.O. Box 200, 6670 AE Zetten, the Netherlands E-mail: [email protected]

2Dutch General Inspection Service PO Box 1115, 8300 BC Emmeloord, TheNetherlands

E-mail: [email protected] Bacterial canker in plum trees (Prunus domestica) is a serious and recent problem in plum production. It is caused by Pseudomonas syringae pathovars syringae and morsprunorum. The trunks of the affected plum trees are girdled by bacterial cankers resulting in sudden death of infected trees. A rapid death was observed in several orchards 3‐4 years after planting. Disease incidences can be very high, and sometimes complete orchards have to be removed. Similar observations have been described in Switzerland (Vignutelli & Hasler, 2001) and Germany (Hinrichs‐Berger, 2004). Recently, plum cultivation in the Netherlands has changed from a relatively extensive into an intensive cultivation. This was realized by the use of weak rootstocks (e.g. VVA‐1 and St‐Julien A). Also, some new plum varieties have been developed (e.g. ‘Lazoet’). However, due to the risks of losses of trees due to bacterial canker, growers are reluctant to plant new plum orchards.Although several control measures are advised in the Netherlands, e.g. cultivation measures at the planting site, careful pruning with disinfection of pruning tools, and removal of heavily infected trees, in practice no control measures are taken. In general nurseries and fruit growers are not familiar with bacterial diseases and lack knowledge in order to prevent infections. Therefore, control strategies to manage plum decline have to be developed. In 2010 a project is started to study the epidemiology and possible control of plum decline. The project will focus on plum decline in commercial plum orchards and plum nurseries. Factors such as root stocks and cultural practices will be evaluated. References: Vignutelli, A, and Hasler, T., 2001. Zwetschgensterben: eine Gefahr für den schweizerischen

Zwetgenanbau? Schweiz. Z. Obst-Weinbau 20/01: 550- 552. Hinrichs-Berger, J., 2004. Epidemiology of Pseudomonas syringae pathovars associated with

decline of plum trees in the Southwest of Germany. J. Phytopathology 152: 153-160. Key words: Bacterial canker, Pseudomonas, Control


88

16:40 TWIG SCAB: AN IMPORTANT FACTOR

DURING THE EPIDEMIOLOGICAL CYCLE OF SCAB ON PEARS

W. VAN HEMELRIJCK1 , E. CROES1, W. KEULEMANS2 & P. CREEMERS1

1 Proefcentrum Fruitteelt vzw, Fruittuinweg 1, BE-3800 Sint-Truiden, Belgium 2 Laboratory of fruit breeding and -biotechnology, KULeuven Department of Biosystems

W. De Croylaan 42, BE-3001 Heverlee, Belgium Scab is a common disease on apple and pear worldwide. The disease is caused by the ascomycete Venturia inaequalis and V. pirina on apple and pear, respectively. Although they are two distinct species, their biological cycle is mainly the same. Two distinctions can be made, i.e. the appearance of twig scab and the more important contribution of secondary scab infections on pears. Until now, most research concerning scab disease is performed on apples and as such, the disease management of pear scab is based on that of apple. Based on the two discrepancies in their biological cycle, we believe that the timing of the fungicides to control pear scab can be better positioned and that less fungicide sprayings may be needed to control pear scab. For that reason an IWT (Agency for Innovation by Science and Technology)‐funded research project was started to study pear scab in more detail. In this project the etiology and the importance of twig scab in the epidemiological cycle of pear scab and its contribution to scab infections during the primary and secondary scab season is investigated. Furthermore, the diversity of V. pirina biotypes and their pathogenicity on different plant parts or cultivars will be determined. The first results indicate that twig scab on pear plays an important role in the biological cycle of pear scab and that plant part or type of cultivar do not set up a barrier for cross infections with scab conidia. Key words: pear, twig scab, epidemiology, cross inoculations


89

17:00 EFFECT OF CLIMATE CHANGE ON INFECTION OF GRAPEVINE BY DOWNY AND POWDERY

MILDEWS UNDER CONTROLLED ENVIRONMENT

M. PUGLIESE, M.L. GULLINO & A. GARIBALDI Centre of Competence for the Innovation in the agro-environmental field (AGROINNOVA) -

University of Torino Via L. da Vinci, 44, IT-10095 Grugliasco (TO)

Plant responses to elevated CO2 and temperature have been much studied in recent years, but effects of climate change on pathological responses are largerly unknown. The pathosystems grapevine (Vitis vinifera) – downy mildew (Plasmopara viticola) and powdery mildew (Eysiphe necatrix) were chosen as models to assess the potential impact of increased CO2 and temperature on disease incidence and severity under controlled environment. Grapevine potted plants were grown in phytotrons under 4 different simulated climatic conditions: (1) standard temperature (ranging from 18° to 22°C) and standard CO2 concentration (450 ppm); (2) standard temperature and elevated CO2 concentration (800 ppm); (3) elevated temperature (ranging from 22° to 26°C, 4°C higher than standard) and standard CO2 concentration; (4) elevated temperature and CO2 concentration. Each plant was inoculated with a spore suspension containing 5x105 cfu/ml. Disease index and physiological parameters (chlorophyll content, fluorescence, assimilation rate) were assessed. Results showed an increase of the chlorophyll content with higher temperatures and CO2 concentration, to which consequently corresponded an higher fluorescence index. Disease incidence of downy mildew increased when both CO2 and temperatures were higher, while an increase in CO2 did not influenced powdery mildew incidence, probably due to the increased photosynthetic activity of plants under such conditions. Considering that the rising concentrations of CO2 and other greenhouse gases will lead to an increase in global temperature and longer seasons, we can assume that this will allow more time for pathogens evolution and could increase pathogen survival, indirectly affecting downy and powdery mildews of grapevine. Key words: climate change, Vitis vinifera, Plasmopara viticola, Eysiphe necatrix, phytotrons

Session 6 Phytopathology: Integrated Control Stategies

91

Session 6 Phytopathology: Integrated Control Strategies


92


93

11:00 COMPARATIVE EFFECTS OF DIFFERENT

FUNGICIDES ON THE PHYSIOLOGY AND YIELD OF WHEAT PLANTS

Carlos A. BERDUGO, Ulrike STEINER, E-C. OERKE & H-W. DEHNE

Institute of Crop Science and Resource Conservation (INRES) – Phytomedicine University of Bonn, Nussallee 9, DE-53115 Bonn, Germany

Several strategies have been used to control foliar pathogens in wheat crops, among these the use of fungicides remains the most implemented method. In addition to fungicidal effects, application of some groups of fungicides such as strobilurins has often resulted in higher grain yields; they were associated with positive physiological effects on the crop like delayed leaf senescence and modifications in the balance of plant growth regulatory substances. Bixafen, a pyrazole carboxamide inhibiting fungal succinate dehydrogenase in the respiratory chain, is a new broad‐spectrum fungicide from Bayer CropScience developed for the control of pathogens in cereals. The objective of these studies was to investigate in a disease‐free environment the effects of bixafen on the physiology and yield of wheat plants and to compare them to those caused by triazoles and strobilurins. Fungicides were applied at two growth stages (GS), first when the flag leaf ligule was visible (GS 39) and again at the end of heading, when the inflorescence was fully emerged (GS 59). Green leaf area duration (GLAD) was assessed weekly after the second fungicide application as percentage of green area of the blades of the top leaves. Digital IR‐thermal images were taken at four growth stages: GS 75, GS 80, GS 85 and GS 90. The following parameters were analyzed as well: senescence of leaves and maturation of ears, photosynthetic activity and yield parameters such as grain yield, thousand kernels mass and numbers of kernels per ear. Effects of bixafen on morphogenesis of wheat was more pronounced than those produced by the other fungicides, it increased the length and width of flag leaf. Bixafen and fluoxastrobin increased GLAD of the flag leaf compared to the other treatments. Differences in the senescence of leaves and ears between treatments were confirmed by the temperature of plant surfaces representing transpiration activity of leaves and ears. Images from a digital infrared camera revealed significant differences between untreated and fungicide‐treated plants at different growth stages. The combination of the positive effects produced by bixafen application on morphogenesis and physiology of wheat resulted in a yield advantage of bixafen treated plants. Key words: Green leaf area, morphogenesis, plant discrimination, senescence, thermal imagines.


94

11:20 NEW CEREAL FUNGICIDES WITH NEW

STICK & STAY FORMULATION TECHNOLOGY

C. OOSTLAND BASF Belgium, Waterloo, Belgium

When we speak about "innovation" in the plant protection world, in general we think about "new active ingredient", "new family" or "new mode of action". The new STICK & STAY formulation technology developed by BASF demonstrates that high investments and researches in the formulation domain can lead to an important improvement in efficacy of existing active ingredients. PALAZZO and OSIRIS, two new fungicides with this STICK & STAY technology, are offering to the Belgian cereals growers a new level of protection against Septoria tritici one of the most important desease in wheat and an important yield increase, clearly higher than the current azoles standard,. With the recent and perhaps future ban of products, the formulation will play for sure an important role to express all the potential of the current active ingredients. Key words: Fongicides, STICK & STAY, PALAZZO, OSIRIS, septoria


95

11:40 INFLUENCE OF FUNGICIDE SEED TREATMENT

ON THE INFECTION AND DISEASE DEVELOPMENT OF CROWN ROT FUSARIUM CULMORUM ON WHEAT WITH DIFFERENT LEVELS OF GENETIC RESISTANCE

S.R. PARIYAR1, A.A. DABABAT2, J.M. NICOL2, G. ERGINBAS2, M. KLIX3,

C. WARTIN3, N. BOLAT4 & R.A. SIKORA1 1Soil- Ecosystem Phytopathology & Nematology, University of Bonn, Germany.

2CIMMYT (International Maize and Wheat Improvement Centre) K. 39 Emek, TR-06511 Ankara – Turkey

3Syngenta Crop Protection AG, WRO 1004.6.65 Schwarzwaldallee 215, P.O. Box, CH-4002 Basel, Switzerland 4Anatolian Agricultural Research Institute, Eskisehir, Turkey

This greenhouse experiment was carried out to investigate the influence of seed treatment with the fungicide FO EZA 14510 on the infection of the crown rot disease caused by Fusarium culmorum as well as on possible interactions with wheat germplasm having different genetic sources of resistance. The three different susceptible (S) wheat germplasm (Seri 82, Kiziltan 91 & Demir 2000) and the three moderately resistant (MR) wheat germplasm (2‐49, Altay 2000 & Burbot 6) were tested at increasing fungicide concentrations of 25 g ai/100 kg seed, 50 g ai/100 kg and 100 g ai/100 kg seeds in greenhouses at ATAEM, Eskisehir, Turkey. The fungicide was applied as a standard seed coating used to control damping‐off fungi. The increasing concentrations of fungicide caused a significant reduction in the F. culmorum crown score (CS) on 4 out of the 6 wheat germplasms. The highest CS reduction was recorded on two MR germplasms Altay 2000 (2.97±2.6) and Burbot‐6 (3.35±1.64). The S germplasm Seri (2.93±1.86) and Demir 2000 (3.98±3.32) also showed significant reductions in CS. However, all fungicide concentrations caused a significant reduction in CS; the concentration of 100 g ai/100 kg seeds gave the highest CS reduction in combination with both S and MR wheat germplasm. Neither shoot height, shoot weight; root length nor root weight were significantly increased by the seed treatment when compared with the control. However, the greatest increase in shoot height was detected on the fungicide treated moderately resistant germplasm. No phytotoxicity symptoms were observed on the plants. The results demonstrated the presence of an interaction between fungicide and specific types of germplasm. The mechanisms involved in the interrelationship still need to be identified. Key words: Wheat germplasm, fungicide, seed treatment, host‐plant interactions


96

12:00 CHARACTERIZING INSENSITIVITY TOWARDS

TRIFLOXYSTROBIN IN FUSARIUM GRAMINEARUM

T. DUBOS, M. PASQUALI, F. POGODA, L. HOFFMANN & M. BEYER Centre de Recherche Public – Gabriel Lippmann

Département Environnement et Agro-biotechnologies 41, rue du Brill, LU-4422 Belvaux, Luxembourg

Fusarium graminearum is the main causal agent of Fusarium head blight (FHB), one of the most devastating diseases of wheat worldwide, in terms of quality as well as quantity. Spikes infected by the fungus are subjected to mycotoxin contaminations, which are harmful to both humans and animals. Fusarium graminearum strains can be categorized into three chemotypes according to the primary mycotoxin they are able to synthesize: 3‐acetylated deoxynivalenol, 15‐acetylated deoxynivalenol and nivalenol. In this study, the sensitivity of a collection of strains of Fusarium graminearum towards the fungicide trifloxystrobin was investigated. Eight concentrations ranging from 0 to 1.24g/L of trifloxystrobin were tested on each isolate. More than 50 strains isolated in Europe and North America within the last 4 decades were tested, so far. It appeared that all isolates were able to grow in presence of trifloxystrobin, even at the highest concentration tested. A control assay was also produced, by using prothioconazole on a reference strain. This isolate was totally inhibited by a concentration of only 2.4mg/L. The maximum level of inhibition obtained using trifloxystrobin averaged 42 ± 8 %. The chemotype, as well as the country of origin did not have a significant influence on the maximum level of inhibition. This information, coupled with the fact that even the strains isolated before the year of market introduction of the strobilurins showed to be resistant to the substance, goes in favor of a natural resistance towards trifloxystrobin occurring among Fusarium graminearum strains. Key words: Chemotype,Gibberella zeae, fungicide, respiration inhibition


97

12:20 AN ANALYSIS OF THE EXPLANATORY VARIABLES

AFFECTING FUSARIUM INFECTION AND DON CONTENT IN WHEAT

Sofie LANDSCHOOT, Willem WAEGEMAN, Kris AUDENAERT,

Jan VANDEPITTE, Jan BAETEN, Bernard DE BAETS & Geert HAESAERT Department of Biological Sciences and Landscape Architecture, University College Ghent,

Schoonmeersstraat 52, BE-9000 Ghent, Belgium Models for predicting Fusarium Head Blight (FHB) and deoxynivalenol (DON) contamination in wheat provide farmers with a handy tool for preventing yield loss and mycotoxin contamination. Nevertheless, these predictive models suffer from a moderate accuracy and limited applicability. Most models are location‐specific and are only reliable in the geographic area of development or in other very similar places. Due to the complex nature of FHB, these models typically take only a subset of explanatory variables as inputs. Moreover, they generally consider only weather conditions (mainly temperature and rainfall) during anthesis and to a lesser extent field‐specific variables and crop charac‐teristics. Yet a complete picture of all factors influencing FHB and DON contamination remains to be revealed. More research is needed, taking into account all abiotic and biotic factors, as well as fungal interactions. We present a thorough statistical analysis of a broad spectrum of variables that affect FHB and DON contamination, using an extensive and unique database of field observations covering eight different years. This study clearly unveils new variables that play a more important role in FHB and DON contamination. Evidence is brought forward demonstrating the effect of species interactions in wheat ears on the DON content and the effect of weather conditions during the vegetative growth stage during winter. Indeed, monthly temperature from November till May was positively correlated with FHB and DON content, whereas monthly relative humidity and rainfall were negatively correlated with FHB and DON content in the same period. Moreover, our findings demonstrate that maize in crop rotation systems is conducive for the production of DON. Finally, our data reveal that a high relative humidity during anthesis is positively correlated with FHB and DON content. Moreover, these correlations were even higher than was the case for rainfall. In a broader view of the divergent results obtained regarding FHB and DON contamination in wheat crops, this study underscores that the prediction of FHB and DON contamination should be seen as two distinct goals. Key words: crop rotation, deoxynivalenol, disease index, Fusarium head blight, weather


98

12:40 POSTHARVEST USE OF ULTRASOUNDS TO

CONTROL PENICILLIUM EXPANSUM DECAY: PROMISING RESULTS IN VITRO AND DURING LONG STORAGE OF APPLES

A. DORE, M.G. MOLINU, T. VENDITTI, G. PANI, A. PETRETTO

& G. D’HALLEWIN CNR – ISPA

Traversa La Crucca, 3 - Loc. Baldinca, IT-07040 Sassari, Italy The potential of several physical treatments to control postharvest pathogens on horticul‐tural products has been investigated, and some have provided good results. Despite this, the diffusion, on a commercial scale, of these treatments is still limited. This may be in part related to the difficulty to integrate these treatments into an existing packing line (e.g. tim‐ing, space, costs), or eventually to the hazard of any physical damage to the commodity. Among the potential physical means, little attention has been given to ultrasounds (US). Preliminary experiments carried out with Citrus fruits highlight that, low‐intermediate levels, did not produce damage to the fruit (unpublished data), while decay was reduced. Here, we report the results obtained with apples (Malus domestica Borkh. cv. Miali) harvested when commercially mature, sonicated and stored at 2 °C and 98% RH for 90 days. Sonication was performed in 10 L de‐ionized water (DW) with a ultrasonic processor (1500 W, 20Khz) con‐nected to a 25 mm Ø probe. Two levels of continuous sonication (20 and 40% of max. ampli‐tude) were adopted during a 1 or 2 min immersion. Each set of fruit was made out of 18 apples with an average weight of 2 Kg, and fruit was completely immersed and moved dur‐ing the treatment. Control fruit was only immersed, for 1 and 2 min, in DW. Following the treatment, fruit was left to dry, put into boxes and cold‐stored. Fruit appearance (index) and moulds (%) were monitored monthly during storage and at the end of a simulated marketing period (6 d at 20 °C and 75% RH). In addition, the effect on P. expansum conidia and hyphae was checked. For this assay, conidia or infected apples were put in 10 L DW and sonication was performed as aforementioned. After each sonication, to valuate the effect of US on suspended conidia, 250 mL of water was collected and 100 μl were spread onto three PDA plates. To cheque the effect on hyphae, tissue of the infection area was removed from the apple and placed onto PDA plates. All plates were kept at 25 °C in the dark, the growth was monitored after 3 days. A clear delay of conidia germination (3 days after the control) and hyphae growth was observed when sonication was performed for 2 min, either at 20 or 40% of the max. amplitude. No complete inhibition was found with the sonication levels em‐ployed. Key words: physical treatments, Malus domestica, apple blue mold

Session 7 Phytopathology: Plant-pathogen Interactions

and Resistance Mechanisms

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Session 7 Phytopathology: Plant‐pathogen Interactions and Resistance Mechanisms

Session 7 Phytopathology: Plant-pathogen Interactions and Resistance Mechanisms

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101

15:00 CONSTRUCTION OF A BIO-TEST FOR INFECTION

OF RED CLOVER PLANTS WITH SCLEROTINIA TRIFOLIORUM

T. VLEUGELS1, J. BAERT1 & E. VAN BOCKSTAELE2

1 Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit Caritasstraat 21, BE-9090 Melle, Belgium

2 Institute for Agricultural and Fisheries Research, Plant Sciences Unit Burg. Van Gansbergenlaan 96, BE-9820 Merelbeke, Belgium

Sclerotinia trifoliorum is an ascomycete fungus that infects red clover crops and other leguminous crops during the winter. Ascospores infect plants in autumn and the disease develops during the winter and early spring, with a high plant mortality by mid‐spring, depending on the weather conditions. In some years entire fields are destroyed. In red clover crops the disease is called clover cancer, Sclerotinia crown and stem rot, or just clover rot. This is a major disease in Europe. It is difficult to control, and no red clover varieties are completely resistant. Breeding for resistant red clover varieties is being slowed down because little is known about the diversity of European S. trifoliorum populations and because of the lack of bio‐tests that are useable in breeding programs. The first objective of this research was to develop a reliable high‐throughput bio‐test. Such a test would be useable in breeding programs. The second objective was to develop another bio‐test to more precisely assess the disease intensity. We developed such a test based on infecting isolated leaves from red clover plants. This bio‐test can be used to determine the susceptibility level of a plant genotype or the aggressiveness of different Sclerotinia isolates. First, we optimized a method for ascospore production of S. trifoliorum. Once produced, the ascospores were used to evaluate the effect of climate conditions, ascospore concentration and plant age on disease development. For the bio‐test on isolated leaves, the effects of infection method, incubation conditions, incubation period, ascospore concentration and leaf growth stage were evaluated. Finally, the correlation between both bio‐tests was determined by first applying the leaf bio‐test then the whole plant bio‐test. In the whole plant bio‐test, disease levels rose with increasing ascospore concen‐tration up to 20,000 spores/ml. Concentrations higher than 20,000 spores/ml did not cause a higher disease level. The plant age did not have a significant effect on the disease level. For the isolated leaf bio‐test, the most effective and most re‐peatable infection method was spraying of an ascospore suspension. Six ascospore concentrations between 10,000 and 80,000 spores/ml were tested, and disease levels continued to increase with rising concentrations. Incubation time did not interact with plant susceptibility levels. Finally, the leaf growth stage was evaluated: using the youngest completely opened leaf yielded the most repeatable results. These new bio‐tests could be valuable instruments for breeding programs and for studying plant‐pathogen interactions. Key words: Sclerotinia trifoliorum, Trifolium pratense, infection, ascospores, bio‐test


102

15:20 EXPLORING PLANT DEFENSE PATHWAYS

IN THE CARROT-ALTERNARIA DAUCI PATHOSYSTEM, A NON-MODEL INTERACTION.

M. LECOMTE1, C. ALLENDA1, F. SEMENT1, M. BERTHET1, M. BRIARD1,

P. POUPARD2 & R. BERRUYER3 1 Agrocampus Ouest - Institut National d’Horticulture et de Paysage- Centre d’Angers,

IFR Quasav 149, UMR GenHort 1259, 2 rue Le Nôtre, FR-49045 Angers Cedex 01, France

2 Université d’Angers, IFR Quasav 149, UMR PaVé A77, UFR Sciences 3 Université d’Angers, IFR Quasav 149, UMR GenHort 1259, UFR Sciences

2 boulevard Lavoisier, FR-49045 Angers Cedex 01, France Most of the molecular mechanisms underlying plant partial resistance QTLs are still unknown. Two competing hypothesis are generally invoked to link observed field resistance with actual molecular gene function. An older hypothesis links partial resistance QTLs with overcome typical R genes encoding classical NBS‐LRR or LRR‐TM type proteins. In recent papers (Hu et al., 2008), partial plant resistance was linked with defense mechanisms. These results led us to develop a candidate gene approach to study partial plant resistance of carrot (Daucus carota) towards its main foliar fungal pathogen, Alternaria dauci. Since carrot is a non‐model plant, little sequence data is available on public databases. We thus chose to develop a homology‐based cloning strategy in order to detect and sequence defense‐related genes in carrot. Since A. dauci is a necrotrophic pathogen, we focused this strategy on Jasmonic acid (JA) signaling pathway and JA controlled defense genes (such as JAZ3 or PR4). Since the degenerate primer strategy is not known to be effective on each and every gene, we chose to apply it in a parallel fashion on a rather large set of genes. Alignments of sequence data from eight already sequenced dicotyledonous plant species were performed for 15 genes. Degenerate primers were defined for 10 genes involved in this JA pathway. Additionally, we defined degenerate primers for two defense genes that are not mainly JA‐ regulated: the SA‐regulated defense gene PR1, and the non host defense gene PAL1. Five out of 12 genes were partially cloned and sequenced. Two strategies are currently deployed to link these defense related genes with partial resistance QTLs observed in the carrot‐A. dauci interaction (Leclerc et al., 2009). SNPs are being found between the resistant and susceptible parents of our mapping populations. They will help us to find potential QTL‐candidate co‐localizations. Absence of such a co‐localization does not mean that a potential candidate is not involved in defense. It is also possible that the QTL influences defense‐related genes activation rather than the efficiency of the cognate PR proteins. For this reason, we also plan to study the induction of these genes by A. dauci in both susceptible and resistant backgrounds. Le Clerc, V., Pawelec, A., Birolleau‐Touchard, C., Suel, A. & Briard, M. (2009). Genetic architecture of factors underlying partial resistance to Alternaria leaf blight in carrot. Theor. Appl. Gen. 118: 1251‐9. Hu, K.‐M., Qiu, D.‐Y., Shen, X.‐L., Li, Y.‐H. & Wang, S.‐P. (2008). Isolation and Manipulation of Quantitative Trait Loci for Disease Resistance in Rice Using a Candidate Gene Approach.(Review) Mol. Plant 1: 786‐793. Key words: Degenerate oligonucleotides PCR, homology cloning, PR proteins, partial resistance, QTLs.



103

15:40 MULTIFACETED DEFENSE MECHANISM IN SITIENS,AN

ABSCISIC ACID DEFICIENT TOMATO MUTANT, AGAINST THE NECROTROPHIC FUNGUS BOTRYTIS CINEREA

Hamed SEIFI & Monica HÖFTE

Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University Coupure Links 653, BE-9000, Ghent, Belgium

The role of amino acid metabolism during a necrotrophic fungal/plant interaction has been poorly studied. We have previously demonstrated that the ABA deficient sitiens mutant of tomato is highly resistant to the necrotrophic fungus Botrytis cinerea via a timely and localized production of H2O2 followed by cell wall fortifications in the epidermis, which effectively arrest spreading of the pathogen. To further investigate how downstream defense responses are regulated in sitiens, the relative transcript level of several candidate genes was analysed using qRT‐PCR. Phenylalanine ammonia lyase was markedly upregulated in sitiens 8hpi, while the enzyme activity inhibition led to extreme susceptibility. Epi‐fluorescence microscopy confirmed the attenuating effect of PAL inhibition on the deposition of phenolic compounds in the epidermal cell wall during the infection progress. Increased PAL expression level was paralleled with an early increase in expression of the cytosolic glutamine synthetase (GS1), which intimately functions with glutamate synthase via GS‐GOGAT cycle. In spite of the early (8hpi) increase in GS1 expression level, no significant change was detected in total protein content of sitiens leaves after 72hpi, indicating that upregulation in cytosolic GS could not be associated with the putative role of the enzyme in natural and stress‐induced senescence. Interestingly, inhibition of GS activity resulted in promoted cell death and ultimately subdued the effective resistance in sitiens, while the early cell wall fortification in the epidermis of the mutant remained intact. Glutamate is a key precursor for various compounds with possible roles in plant‐pathogen interaction such as polyamines and gamma amino butyric acid (GABA). It has been reported that GABA, via GABA‐shunt, has a regulatory role in central C/N metabolism and provides a major link between amino acid metabolism and TCA cycle. Further qRT‐PCR analyses in sitiens, showed increases in genes encoding for polyamines and GABA biosynthesis; suggesting a link between GS‐GOGAT cycle and GABA‐shunt. Furthermore, it was revealed that GABA application could significantly reduce susceptibility in wild type. According to these results we propose that a successful defensive response in sitiens consists of two crucially linked components: firstly, the rapid epidermal ROS accumulation and cell wall fortification and consequently arresting the pathogen; and secondly, a durable maintenance of the basic metabolism in the challenged tissue to retard the necrotroph‐induced senescence. It seems that the ammonium produced by PAL activity in the rapid phase, is re‐assimilated by GS1 and fed into the GS‐GOGAT cycle, providing sufficient fuel for the second phase. In the maintenance part, GABA‐shunt regulates the central C/N metabolism by injecting the scavenged supply into the TCA cycle to enable the mutant to resist the lethal strategy of the necrotrophic pathogen. Key words: Abscisic acid, sitiens, tomato, Botrytis, gaba, amino acid metabolism, pathogen‐induced senescence


104

16:00 CATECHOLAMINE BIOSYNTHESIS PATHWAY

POTENTIALLY INVOLVED IN BANANA DEFENSE MECHANISMS TO CROWN ROT DISEASE

Ludivine LASSOIS1, Patrick FRETTINGER2, Luc DE LAPEYRE DE

BELLAIRE3,4, Philippe LEPOIVRE1 & M. HAÏSSAM JIJAKLI1 1 Plant Pathology unit, University of Liège, Gembloux Agro-Bio Tech

2 Passage des Déportés, BE-5030 Gembloux, Belgium 2 Unité Mixte de Recherches INRA-CNRS-Université de Bourgogne, Plante-Microbe

Environnement, INRA, 17 Rue Sully, BP 86510, FR-21065 Dijon cedex, France 3 CARBAP, African Center for Research on Bananas and plantains

Phytopathology Unit, BO Pox, 832. Douala, Cameroon 4 CIRAD, Research Unit, banana, plantain and pineapple cropping systems

FR-34398 Montpellier, France Variations in Cavendish bananas susceptibility to crown rot disease have been observed (Lassois et al., 2010), but the molecular mechanisms underlying these quantitative host‐pathogen relationships were still unknown. The present study was designed to compare gene expression between bananas (Musa acuminata, AAA, ‘Grande‐Naine’) showing a high post‐harvest susceptibility (S+) and bananas showing a low post‐harvest susceptibility (S‐) to crown rot disease. This comparison was performed between crowns (S+ and S‐) collected one hour before standardized artificial inoculations with Colletotrichum musae. Fruit susceptibility was evaluated through lesion size on the crown 13 days later. Gene expression comparisons were performed with the cDNA‐AFLP technique (Lassois et al., 2009). This revealed that a gene showing a strong homology with a dopamine‐β‐monooxygenase (DoH) combined with a cytochrome B561 (CB) is differently expressed between S+ and S‐ (Lassois et al., 2011). Furthermore, semi‐quantitative real‐time RT‐PCR analyses between S+ and S‐ were applied to confirm the differential expression results for DoH‐CB obtained by cDNA‐AFLP. Two biological replicates were tested. These semi‐quantitative analyses were performed not only on tissues collected one hour before C. musae inoculation but also on crown tissues collected 13 days after inoculation. The real‐time RT‐PCR confirmed that DoH‐CB was upregulated in the S‐ tissues collected at harvest, just before C. musae inoculation. This gene was also highly upregulated in the S‐ tissues collected 13 days after crown inoculation. Similar results were obtained for both biological replicates. Our results suggest that cathecolamines could play a role in banana defense mechanisms to crown rot disease. The roles of catecholamine pathways in plant are discussed. Key words: post‐harvest disease, susceptibility, Banana, cDNA‐AFLP, real time RT‐PCR



105

16:20 SILICON-INDUCED BROWN SPOT

RESISTANCE IN RICE (ORYZA SATIVA L.)

J. VAN BOCKHAVEN, D. DE VLEESSCHAUWER & M. HÖFTE Ghent University, Phytopathology lab

Coupure-Links 653, BE-9000 Ghent, Belgium E-mail: [email protected]

In a world where half of the population depends on rice as their main staple food the need of a consistent manner to protect rice plants against a wide range of pathogens is of high importance. Silicon (Si) enables rice plants to fend off these pathogens. The resulting silicon–induced broad spectrum disease resistance in rice is the result of a combination of passive and active defense. The passive defense originates from the silicon deposition in the epidermal rice cells which renders a certain rigour to these cells. However, relatively little is known about the role of silicon in active defense. In our research several intruiging results in the field of Si‐induced defense against Cochliobolus miyabeanus, the causal agent of brown spot, have been found. Extensive microscopic analysis on brown spot–infected rice plants, revealed that fungal growth is hampered in the mesophyll of Si‐amended plants. Different stainings also suggested that the Si‐induced defense responses against brown spot mainly consist of an enhanced production of phenolic compounds, whereas active oxygen species and cell wall reinforcements do not seem to be part of Si‐induced brown spot resistance. The mechanisms underlying the Si‐induced defense responses against brown spot appear to be regulated by the abscisic acid (ABA), cytokinin (CK) and especially ethylene (ET) pathway and also seem to be SA independent which is confirmed by previously conducted experiments with mutant and transgenic lines. Moreover, pharmacological experiments suggested that especially the ET pathway is a major regulator of Si‐induced brown spot resistance. These results along with several microarray studies strongly suggest that the mode of action of silicon can be found in the priming of the defense mechanisms. Dependent on the type of pathogen Si‐treated plants can activate specific defense responses much quicker and stronger upon pathogen infection granting the plant with a strategy to fend off a broad spectrum of pathogens. To validate this hypothesis the role of various signaling components thought to play a key function in the Si‐mediated priming for enhanced defense will be studied thoroughly. These future studies not only will shed new light on how plants cope with pathogen assault, but may also guide novel strategies for biologically based, environmentally friendly and durable disease control in various agricultural settings. Key words: silicon, Cochliobolus miyabeanus, rice, induced disease resistance


106

16:40 NEW INSIGHTS ON THE MOLECULAR

RELATIONSHIPS BETWEEN PEACH LATENT MOSAIC VIROIDS AND PEACH TREES

Olivier PARISI, Philippe LEPOIVRE & M. Haissam JIJAKLI

Plant Pathology Unit, University of Liège, Gembloux Agro-Bio Tech 2 passage des Déportés, BE-5030 Gembloux Belgium

Viroids are circular single‐stranded RNA molecule that do not encode protein. From them, the Peach latent mosaic viroid ranges in size from 335 to 351 nucleotides and folds in a highly branched secondary structure consisting in stems‐loop and pseudoknots. It mainly infects peach. The mechanisms involved in viroids pathogenicity are poorly understood. However several hypothesizes were postulated: (1) the pathogenic effect of viroids can be induced by an interaction with host proteins, (2)the gene silencing which protect the plant genome against viruses and viroids could be responsible of the symptoms expression. In this latter case, two ways can be borrowed: in the first one, the degradation of the viroid by this defence mechanism produces small RNA which can silence host genes; in the second, the silencing machinery of the host can be affected during the viroid infection by an unknown mechanism interfering with the normal host gene regulation. Our study aimed to characterize the molecular relationship between peach trees and the PLMVd. To explore the mechanisms involved by the infection a cDNA‐AFLP analysis on total RNA peach extract was performed. This technique offers an open strategy which does not need any knowledge of the host genome to determine the transcriptional modifications induced by the infection. Previously studied pathogenic variants of the PLMVd were isolated and induced different intensities of mosaic and chlorosis but also a latent infection. These variants have permitted to compare (1) the effect of different symptoms as well as the (2) effect of a latent variant on the transcription profile of the infected plants. We have obtained differentially expressed bands corresponding to genes whose expression is modified by the infection. From them, we found genes implicated in photosynthesis and photosystem protection, mRNA translation, protein metabolism and plant defence. All of our results will be discussed during the meeting. Key words: cDNA‐AFLP, viroid, molecular relationships, photosynthesis, defence



107

17:00 TRANSMISSION STUDIES AND CONTROL OF

POTATO SPINDLE TUBER VIROID IN ORNAMENTALS AND TOMATO

K. DE JONGHE1, S. MORIO1, M. MAES1, I. HANSSEN2,

T. OLIVIER3, J. GOVERS3 & S. STEYER3 1 Instituut voor Landbouw- en Visserijonderzoek, Eenheid Plant

Burg. Van Gansberghelaan 96, BE-9820 Merelbeke, Belgium E-mail: [email protected]

2 Scientia Terrae vzw Fortsesteenweg 30A, BE-2860 St.-Katelijne-Waver, Belgium

3 Centre Wallon de Recherches Agronomiques, Département Science du Vivant, Unité Biologie des Nuisibles et Biovigilance

4 rue de Liroux, BE-5030 Gembloux, Belgium

Potato spindle tuber viroid (PSTVd) is infectious un‐encapsidated, small, circular, single‐stranded RNA, capable of autonomous replication when inoculated into a host plant. The host range of this pathogen is thought to be relatively narrow. The primary hosts are potato and tomato, yet a number of ornamental Solanaceae can also be infected by PSTVd. A major problem is that the presence of the viroid in those ornamentals remains unnoticed, since no symptom development occurs. Recent surveys show that PSTVd and other related (pospi)viroids are quite commonly found in Solanum jasminoides, Solanum rantonetti and Brugmansia suaveolens. The viroid has been given a quarantine status because of a realistic risk for tomato and potato on which PSTVd does cause important economic damage due to serious growth reductions and malformation of plants and tubers. The main objectives of the current study is to provide new knowledge of the epidemiology, the transmission risk from ornamental plants to tomato and potato cultures, and the control of PSTVd and other pospiviroids. To address these challenges the following activities were carried out: evaluation of the pathogenicity of a series of Belgian pospiviroid isolates, evaluation of the sensitivity of the most common tomato and potato cultivars against PSTVd, assessment of several controlled transmission experiments (pollen, insects, transmission through root and leaf contact and via irrigation waters, the importance of solanaceous weeds in PSTVd transmission), and chemical and physical inactivation tests. Together with the data of parallel studies on origin, status and molecular characterization of the most important pospiviroids in solanaceous crops (PSTVd, CEVd, CLVd and TCDVd), control strategies can be proposed to avoid further spreading of this quarantine pathogen.

Session 8 Application Technology

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111

11:00 COMPARISON OF THE PERFORMANCE

BETWEEN A SPRAY GUN AND A SPRAY BOOM IN ORNAMENTALS

D. FOQUÉ & D. NUYTTENS

Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science Unit, Agricultural Engineering

Burgemeester van Gansberghelaan 115, bus 1, BE-9820 Merelbeke, Belgium E-mail: [email protected]; [email protected]

Flemish greenhouse growers predominantly use handheld spray guns and spray lances for their crop protection purposes although these techniques are known for their heavy workload and their high operator exposure risks. Moreover, when these techniques are compared with spray boom equipment, they are often found to be less effective. On the other hand, handheld spraying techniques are less expensive and more flexible to use. The aim of this work was to evaluate and compare the spray deposition, penetration and uniformity between a manually pulled horizontal spray boom and a spray gun under controlled laboratory conditions. In total, six different spray application techniques were evaluated. With the spray boom, three different nozzle types were tested at an application rate of 1000 L/ha i.e. an XR 80 03 extended range standard flat fan nozzle at 3.0 bar, a TXA 80 03 hollow cone nozzle at 3.0 bar and an ID 90 02 air inclusion nozzle at 6.0 bar. Besides the boom application, three different spray lances were tested at a higher spray pressure (7‐15 bar) by two experienced and one non‐experienced sprayer. Spray deposits were measured in an Ivy crop by sampling different plants at three plant levels on the top and on the bottom side of the leaves using mineral chelates as a tracer and filter paper collectors. Two plant densities were compared i.e. 21 and 32 plant/m². Spray distribution was assessed using wooden frames with filter papers and water sensitive papers. In general, the total deposition results were comparable between the spray boom and the spray gun applications but the boom applications resulted in a more uniform spray distribution over the crop. On a plant level, the spray distribution was not uniform for all the different techniques with highest deposits on the upper side of the top leaves and lowest deposits on the bottom side of the leaves in the crop. Using spray guns at a higher spray pressure and higher spray volumes did not improve spray penetration and deposition on the bottom side of the leaves. Plant density clearly affected crop penetration and deposition on the bottom side of the leaves. From the different nozzle types, the XR 80 03 gave the best results. The spray boom technique may increase productivity, reduce labour costs and decrease operator exposure. Key words: Spray lance, spray boom, ornamentals, nozzle, deposition


112

11:20 ORGANIC NANOFIBERS CONTAINING INSECT

PHEROMONE DISRUPTANTS: A NOVEL TECHNICAL APPROACH TO CONTROLLED RELEASE WITH POTENTIAL FOR PROCESS MECHANIZATION

Hans E. HUMMEL1,2, D.F. HEIN1, M. BREUER3, I. LINDNER1,

A. GREINER4, J.H. WENDORFF4, C. HELLMANN4, R. DERSCH4, A. KRATT5, H. KLEEBERG5 & G. LEITHOLD1

1Chair of Organic Agriculture, Justus-Liebig-University Giessen Karl-Gloeckner-Str. 21 C, DE-35394 Giessen, Germany

E-mail: [email protected] 2Illinois Natural History Survey, University of Illinois Urbana-Champaign

Institute of Natural Resources Sustainability 1816 S.Oak Street, Champaign, Il. 61820, USA

3State Institute for Viticulture and Enology, Freiburg, Department of Ecology Merzhauser Str. 119, DE-79100 Freiburg, Germany

4Phillips-University Marburg, Department of Chemistry, Hans-Meerwein-Strasse DE-35032 Marburg, Germany

5Trifolio-M GmbH Dr.-Hans-Wilhelm-Weg 1, DE-35633 Lahnau, Germany

INTRODUCTION. The search for suitable dispensers containing insect pheromones grew with the availability of these synthetic biotechnical tools beginning fifty years ago. Many economic entomologists and application engineers dearly wish they had the "smart, intelligent and ideal dispenser". More or less suitable approximations are available commercially, but none so far meets all demands. METHODS. Under economic strictures novel inexpensive systems would be advantageous with release characteristics taylored to the specific life histories of pest insects and the numerous requirements of growers alike. Simultaneously, their field distribution should be mechanizable and be accomplished by one (or very few) application runs. The dispensers should be biodegradable, biocompatible, sustainably applicable, and they should be based on renewable resources. RESULTS. In this report, we present first results of a novel organic, electrospun nanofiber dispenser with dimensions in the upper nanometer range. Its load of pheromone can be adjusted to be sufficient for 7 weeks of constant disruptive action in vineyards and can be directed against the European Grape Vine Moth Lobesia botrana (Lepidoptera: Tortricidae) which here serves as a readily available model. Mating disruption in Lobesia botrana and the related Eupoecilia ambiguella is a well studied and developed engineering process. Equally, nanofiber production by electrospinning (for a comprehensive review see Greiner & Wendorff, 2007) is well known and already has numerous applications in filtration technology, air conditioning, and medical wound dressing. Our goal was to bring together and successfully mate these (partly incompatible) technologies via technical tricks of a proprietary nature. DISCUSSION. Even though we still must double the lifetime and effectiveness of currently available nanofibers, we know the rather complicated system of their production and analysis well enough to identify the parameters that need future adjustment. Another


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challenge is the mechanical distribution of the fibers in the vineyards by suitable machinery. Also, here we have available certain technical leads for following up during 2011. Technical details of fibers and their production by electrospinning are protected by patent applications via TransMIT GMBH Giessen‐Marburg. We are very grateful to Schwarz Foundation for a starting grant and for support by the Innovation Program of the Federal Ministry of Agriculture and Nutrition. JKI Berlin as a federal independent authority kindly monitors in numerous test systems the compatibility of organic nanofibers for their effects on human and environmental health. So far no contraindications have been identified.


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11:40 ORGANIC ELECTROSPUN NANOFIBERS AS

SEMI-INTELLIGENT PHEROMONE DISPENSERS: CHARACTERIZATION BY LABORATORY

INVESTIGATIONS

I. LINDNER1, D.F. HEIN1, M. BREUER2, Hans E. HUMMEL1,3, A. DEUKER1, A. VILCINSKAS4, G. LEITHOLD1, Ch. HELLMANN5, A. GREINER5,

J.H. WENDORFF5, R. DERSCH5 & A. KRATT6 1Chair of Organic Agriculture, Justus-Liebig-University Giessen, DE-35394 Giessen, Germany

2State Institute for Viticulture and Enology, Freiburg Department of Ecology, DE-79100 Freiburg, Germany

3Illinois Natural History Survey, University of Illinois Urbana, Institute of Natural Resources Sustainability, Champaign, 61820, USA

4Phytopathology and Applied Zoology, Justus-Liebig-University, DE-35392 Giessen, Germany 5Philipps-University Marburg, Department of Chemistry, DE-35032 Marburg, Germany

6Trifolio-M GmbH, DE-35633 Lahnau, Germany INTRODUCTION Organic nanofibers have a history of technical application in various independent fields, including medical technology, filtration technology, and applications of pharmaca via inhalation. Very recently, in a joint effort with polymer chemists, agricultural applications have been added to this list of options with the aim of finding novel approaches to insect control. Pheromones, dispensed in a quantifiable way, are being used here in disrupting the mating communication between male and female pest insects, e.g. the European grape vine moth Lobesia botrana (Lepidoptera: Tortricidae), where current dispenser technology does not fully meet the exact expectations of growers and environmentalists with respect to release longevity, self decomposition of dispensers, mechanical distribution, renewability as well as sustainability of resources. MATERIALS & METHODS The methodology of electrospinning is exhaustively covered by Greiner and Wendorff (2007), with technical details reported by Hellmann et al. (2009), Hein et al. (2010), and Hummel et al. (2010). ‐ Wind tunnel studies were run within a self built tunnel with adjustable laminar flow and 0.5 m/sec air velocity. Mass losses of the nano fiber bundels were determined with a sensitive analytical balance 2‐3 times per week and recorded as time vs. mass change. ‐ CLSA experiments were performed with a self‐developed glass apparatus (Lindner 2010) partly based on suggestions of previous authors. Microgram quantities of volatile pheromone (E,Z)‐7,9‐Dodecadienylacetate were absorbed on a filter of rigorously purified charcoal and desorbed by repeated microextraction with a suitable solvent mixture. Aliquots of the solution were subjected to temperature programmed capillary GLC. Retention times were used for identification, while the area covered by the pheromone peak originating from a FID detector signal was integrated and compared with a carefully calibrated standard peak in the low nanogram range. Several replications were averaged. ‐ Thermogravimetric analysis between ambient temperature and 500 degrees centigrade provided a series of degradation curves where the diagram contained information on the evaporation of pheromone alone, polymer fiber alone and pheromone included in the fiber. ‐ Scanning electron microscopy resulted in pictures of nanofibers from which the overall morphology and the fiber dimensions could by quantified.


115

RESULTS & DISCUSSION Organic nanofibers loaded with the grape vine moth pheromone have been well characterized by 4 different lab methods, followed by field bioassays reported elsewhere in these abstracts (Hummel et al. 2011). This comprehensive analytical approach to fiber characterization is new and will be further refined. The federal agency JKI Berlin subjected the pheromone loaded organic fibers to various independent toxicological and ecotoxicological tests and found no adverse side effects that would prevent their future development and distributio


116

12:00 RECENT STUDIES OF MEASURES TO IMPROVE

BASAMID SOIL DISINFESTATION

E.J.G.L. VAN WAMBEKE Certis Europe B.V.

Woluwedal 60, BE-1200 Brussels Belgium Basamid granule is used worldwide as a broad spectrum soil fumigant generator and has replaced methyl bromide for many applications. A lot is known for decades regarding the factors determining the success of the application from soil preparation and conditions to the application and soil sealing or soil tarping, as well as the operations and hygienic measures after the fumigant contact time. This paper explains last 6 years studies regarding the improvement of application methods, both from the viewpoint of homogenous incorporation of the granule over the soil profile to become treated as well as from possible premature loss of the gaseous active methyl isothiocyanate (MITC) by using improved tarping materials. Both result in lower environmental exposure and better biological performance of the application. In that respect, product incorporation in soil was studied in France and in Italy with more recent commercially available Basamid application machinery, and about 24 plastic films have been compared for their MITC barrier properties with an ‘in house’ developed method. Film testing allowed clear categorizing in standard (monolayer) films, V.I.F. (Virtually Impermeable Film) and T.I.F. (Totally Impermeable Film). The paper presents the methodology for granule incorporation study and results from trials with two specific Basamid application machines compared with a classic rotovator, the methodology and comparison of plastic film barrier properties testing, and directives to minimize exposure and to maximize performance. Key words: Basamid, methyl isothiocyanate, soil incorporation, barrier properties, tarpaulin


117

12:20 TOPPS-PROWADIS A NEW EUROPEAN

MULTISTAKEHOLDER PROJECT TO REDUCE LOSSES OF PLANT PROTECTION PRODUCTS TO WATER

Manfred ROETTELE1& Inge MESTDAGH²

1Better Decisions – DE-48249 Dülmen, Haverlandhöhe 21 a (Germany) Projectmanager of ECPA TOPPS – Prowadis project

E. v.Nieuwenhuyselaan 6, BE-1060 Brussels, Belgium ²Provincial Research centre for Agri- and Horticulture (POVLT)

Ieperseweg 87, BE-8800 Rumbeke, Belgium The new TOPPS‐PROWADIS project builds on the success of TOPPS (co‐funded by ECPA and EU ‐ www.TOPPS‐life.org) which was focused on reducing point sources. TOPPS‐PROWADIS will focus on reducing losses of Plant Protection Products (PPP) from field run‐off and drift. Run‐off is considered the most important entry route of PPP into water from diffuse sources. Mitigation measures are complex as field run‐off is predominantly influenced by weather conditions, soil properties and specific landscape characteristics, which require field specific approaches. Currently consistent and adequate Best Management Practices (BMPs) to mitigate run off hardly exist across the EU. Drift is not a big source for PPP water contamination but it receives a lot of public attention due to its visibility and it offers the opportunity for effective mitigation. Anti‐drift technology has been developed, but has not yet received wide enough acceptances by operators. TOPPS‐PROWADIS will contribute to the implementation of the Water Framework Directive and the Directive on Sustainable use of Pesticides by developing and offering practical tools and recommendations. Currently recommendations referring to water protection across the EU show very little harmonization. It is hoped that with TOPPS and TOPPS‐PROWADIS a rather comprehensive set of consistent BMPs on water protection will help to harmonize approaches to ensure a more equal playing field for farmers and to gain more credibility and acceptance. Implementation of BMPs need a structured approach including its development, transfer to advice and stakeholders and the implementation through information, training, demonstrations and advice to operators. Methodology The TOPPS‐PROWADIS project will work in a multi skilled team of experts to bring in the understanding of the local situations, which is needed to develop common “EU core” recommendations with the flexibility for some needed local adoptions. The project will be executed in two main steps: a) Development of diagnosis tools (run‐off and drift) and BMPs recommendations b) Training, demonstration on the application of the diagnosis tools and the dissemination of the BMPs, which will include the diffuse and the point source mitigation. Expected results TOPPS and PROWADIS if implemented can avoid most of the PPP losses to water. The success of BMPs implementation and acceptance depend largely on the translation of research results into practice. This means recommendations need to be understandable and practical. Mitigation measures should be focused on those working processes, techniques or infrastructure where results can be achieved most effective. This requires a wider understanding of the different measures and their implications at various stakeholder levels. BMPs based on a “common EU core” will create common understanding and acceptance and will therefore be a prerequisite for the successful implementation of the respective EU directives. Key words: TOPPS‐PROWADIS; run‐off; drift; BMPs, diagnosis tools; training


118

12:40 NEW PERSPECTIVES IN THE CONTROL OF LODGING IN CEREALS WITH MEDAX TOP

C. OOSTLAND

BASF Belgium, Waterloo, Belgium Lodging can have serious consequences in cereals, such as in 2010. Yield losses upto 40% are not unlikely. Since the end of the 60’s until now, only 6 active ingredients have been developed for cereals in Belgium. As a specialist in cereal growth regulation with 5 a.i. in its portfolio, BASF is launching now Medax Top. This new growth regulator combines prohexadione calcium, a new a.i. in cereals, with the mepiquat chloride. Thanks to this unique combination of two different modes of action, Medax Top provides a better control of lodging, is safer for the cereal crop (can be used for seed production) and is more flexible in use than the current competitors. Cereals gowers will benefit of a interesting and highly performing product. Key words: Lodging, growth regulator, cereals, Medax Top

Session 9 Pesticide Residues,

Toxicology and Ecotoxicology

119

Session 9 Pesticide Residues, Toxicology and Ecotoxicology


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121

15:00 EFFECTS OF FOOD PROCESSING ON PESTICIDE

RESIDUES IN FRUITS AND VEGETABLES

Boitshepo M. KEIKOTLHAILE, Pieter SPANOGHE & Walter STEURBAUT Laboratory of Crop Protection Chemistry

Faculty of Bioscience Engineering, Ghent University Coupure Links 653, BE-9000 Ghent, Belgium

Pesticides are being used during crop production to sustain the food supply for the increasing world population. As a result, their residues are found in raw agriculture commodities and some processed foods. Due to potential health risks of acute and chronic poisoning, researchers are continuously investigating ways to reduce the use of pesticides in crops and their residues in food products. Food processing has been found to help in reducing the pesticide residues in agricultural commodities except in processes like oil production and drying where concentration can occur. Various researchers have come up with many processing factors concerning fruits and vegetables of importance in their geographic regions. These processing factors are important in the risk assessment of pesticide residues in food. In this paper, the state of the art concerning effects of food processing in fruits and vegetables is investigated using meta‐analysis, distributions and influence of pesticide physico‐chemical properties during processing using public market samples are investigated and the impact of food processing in relation to consumer health. Key words: pesticide residues, food processing, fruits, vegetables, risk assessment


122

15:20 DESIGNING AN INDICATOR SET FOR

SUSTAINABLE CROP PROTECTION AT FARM LEVEL

Hilde WUSTENBERGHS1, Ilse DELCOUR2, Karoline D’HAENE1, Pieter SPANOGHE2, Walter STEURBAUT2 & Ludwig LAUWERS1 1 Institute for Agriculture and Fisheries Research (ILVO), Social Sciences Unit

Burg. Van Gansberghelaan 115, box 2, BE-9820 Merelbeke, Belgium 2 Ghent University, Faculty of Bioscience Engineering, Department of Crop Protection

Coupure Links 653, BE-9000 Ghent, Belgium The objective is to develop an indicator set for sustainable crop protection, that can be used in integrated farm sustainability assessment, such as the Monitoring Tool for Integrated Farm Sustainability MOTIFS (Meul et al., 2008). The indicator is built using a participatory process. In the design phase crop protection experts and environmental stakeholders are consulted in every stage. To draw up the framework for crop protection, a definition was formulated following Directive 2009/128/EC on sustainable pesticide use and stakeholder consultations: “A sustainable crop protection should not harm the actors in fundamental natural processes, i.e. it should not have any intolerable impact on humans nor the environment”. Translating this to the farm level, essential elements of sustainable crop protection were defined: (1) Prevention of diseases, pests and weeds and of unnecessary treatment; (2) If chemical treatment is inevitable, choose the least environmentally harmful product; (3) Comply with pesticide legislation; (4) Avoid resistance to pesticides; (5) Take precautions for operator safety; (6) Avoid point and diffuse pollution For monitoring these elements, we propose a set of indicators consisting of • POCER, the Pesticide Occupational and Environmental Risk indicator (Vercruysse & Steurbaut, 2002); • an inquiry with farmers concerning their crop protection management practices. POCER assesses risk to humans and different environmental compartments: persons applying pesticides or re‐entering treated fields, bystanders exposed to spray drift, persistence in the soil, groundwater contamination, aquatic organisms, birds, bees, earthworms and beneficial arthropods. The inquiry sounds out farmers’ attitude towards and management of crop protection: information acquirement; awareness and attitude to environmental effects and pollution paths; disease, pest and weed prevention; monitoring and risk evaluation; alternative protection strategies; resistance management; operator exposure; spraying equipment and pollution prevention. These themes were defined through literature and stakeholder consultation. The final stage in the participatory design is benchmarking and weighting the indicators in the set against each other by experts and stakeholders. By attributing weights to the sub‐indicators, they can be added up to a single crop protection sustainability indicator at the farm level. A later phase is the implementation in discussion groups with farmer and farm advisors. While discussing the indicators farmers become the main stakeholders. Not only do they learn about the sustainability of their practices and means for improvement, the indicator designers also learn about refining it and about appropriately bringing it into practice (De Mey et al., 2011). De Mey et al. (2011) INT J AGRIC SUSTAIN, in press; Meul et al. (2008) AGRON SUSTAIN DEV 28: 321‐332; Vercruysse & Steurbaut (2002) CROP PROT 21: 307‐315 Key words: indicator design, participatory approach, crop protection, POCER, farm inquiry



123

15:40 (CROP) PROTECTION PRODUCTS:

QUANTIFICATION AND GEOGRAPHIC DISTRIBUTION OFEMISSIONS TOWARDS SURFACE

WATERWITHIN THE FRAMEWORK OF WEISS

Kim DOAN NGOC1, Karen CALLEBAUT2, Stefaan HERMANS3, Greta VOS3 & Pieter SPANOGHE1

1 Dept of Crop Protection Chemistry, Ghent University Coupure links 653, BE-9000 Ghent, Belgium

2 ARCADIS Belgium nv Posthofbrug 12 blok 2,BE-2600 Antwerpen-Berchem, Belgium

3 Vlaamse Milieumaatschappij, Afdeling Rapportering Water, Dienst Meetnet Afvalwater, Verantwoordelijke Emissie Inventaris Water

Dr. De Moorstraat 24-26, BE-9300 Aalst, Belgium To assess the effectiveness of the policy measures it is crucial to have a clear view of the emission situation of pesticides in Flanders. WEISS is a LIFE+ project that will create a geo‐graphically explicit, transparent inventory of emissions of all kinds of pollutants towards water for the Flemish region of Belgium, with focus on priority substances. This study has two goals: quantification and localization of the source (use) and the emissions. Operating at a high spatial resolution (1 ha grid) the WEISS system integrates all relevant emission sources (both diffuse and point), all transport routes, and a planning support mod‐ule. This enables calculation of pollutant loads in distinctive nodes of the pathways, as re‐quired for monitoring and reporting. For each source and pollutant WEISS will proceed in three consecutive steps: spatial distribu‐tion of the source, computation of the pathways and accounting in distinctive nodes of the pathways. For every step, specific algorithms are developed that can easily be chained to perform the required calculations. This allows the system to be extended with new sources and pollutants. A new case study on pesticides was initiated in 2010 within WEISS. Both agricultural and non‐agriculture use of pesticides are taken into consideration. Data on agricultural use (lim‐ited to corn and winter wheat) and non‐agricultural use (limited to the municipalities) are provided by the ‘Landbouwmonitoringsnetwerk’ and the database ‘Zonder is Gezonder’, respectively. A prioritization of the list of used active ingredients is made based on the SEQ value and measurement data (monitoring).Direct losses, drift, drainage and runoff were identified as the major transport processes towards surface water. An emission factor is calculated for each relevant substance and transport process. These calculated emission factors are validated by comparing them with emission factors found in the open literature. Finally, the gross emissions towards surface water are calculated. From the established shape files, the use, the gross emissions as well as the contribution of each transport process can be plotted on a map.


124

16:00 CONTRIBUTION BY NEIGHBOURING COUNTRIES TO

PESTICIDES IN THE DUTCH SURFACE WATERS

M. VAN 'T ZELFDE, W.L.M. TAMIS, M.G. VIJVER & G.R. DE SNOO Department of Conservation Biology Institute of Environmental Sciences (CML)

Leiden University P.O. Box 9518, NL-2300 RA Leiden, The Netherlands

In the Netherlands pesticides concentrations in surface water are monitored by the regional water boards and the Ministry of Water‐management to assess water quality. These monitoring data were processed, compared with several environmental quality standards and spatially analyzed (see: www.pesticidesatlas.nl). These data show that many different pesticides are measured in high concentrations. At a national level, the percentage of pesticides concentrations that exceed the maximum tolerable risk (MTR) has declined between 1998 and 2009 with 50‐70%, but there are still locations where the measured concentrations exceed the environmental quality standards. The Dutch government tries to improve the quality of the surface waters by specific policy on admission of pesticides in specific crops. This policy assumes that the contamination of the surface water with pesticides have occurred in the Netherlands. The Netherlands however is the delta for the rivers Rhine, Meuse and Schelde which pass through many countries. The aim of the current study is to determine the extent in which these pesticides concentrations have their origin abroad. Therefore we have used the data in the Dutch pesticidesatlas. Monitoring locations differ in water‐type and catchment area. Water‐types vary from very small ditches, which are located besides the arable fields, to big rivers. This kind of location‐ and concentration information is used in this study. For some water‐types (like stagnant) the pesticides concentrations very probably come from the adjacent fields, for other running water‐types like rivers and small running waters like brooks they may originate from areas which are located further away. Water at monitoring locations in running water‐types near the Dutch border is coming from abroad. We analyse the number of monitoring locations in border zones to Belgium and Germany of different sizes. The measured pesticide concentrations in these ‘border‐zone’‐locations are compared to the other measuring locations in the Netherlands in magnitude. Furthermore the list of most exceeding pesticides is compared between the border‐area and the remainder of the Netherlands. This study will answer the question if the neighbouring countries contribute to the pesticides in the Dutch surface water. Key words: pesticides neighbouring countries surface waters environmental standards atlas netherlands



125

16:20 SIMILARITIES AND DIFFERENCES BETWEEN

MEASURED AND PREDICTED CONCENTRATIONS OF PESTICIDES IN DUTCH SURFACE WATERS

R. KRUIJNE1, M.G. VIJVER2, M. VAN’T ZELFDE2, W.L.M. TAMIS2

& G.R. DE SNOO2 1 Wageningen University and Research Centre Team

Environmental Risk Assessment Centre for Water and Climate P.O. Box 47 6700 AC Wageningen, The Netherlands

E-mail: [email protected] 2 Leiden University Institute of Environmental Sciences (CML)

Department of Conservation Biology P.O. Box 9518, NL-2300 RA Leiden, The Netherlands

E-mail: [email protected] For the assessment of the environmental quality of surface water and to evaluate pesticide policy measures and regulations two approaches are used. First, the actual pesticides concentration in surface waters can be measured. Second, the potential pesticide concentration in the water can be predicted by using models taken into account pesticide use data, spray drift data etc. The aim of the current study is to identify similarities and dissimilarities in measured and predicted pesticides concentrations in Dutch surface waters at national scale. To this purpose, monitoring data obtained from regional water authorities and other monitoring institutes were processed. Pesticide data were aggregated over a two years time period and over space (see the Dutch Pesticides Atlas (www.pesticideatlas.nl). Information can be found on where a pesticide is being measured, observed and possibly constitutes an exceedance of the environmental quality standards. The monitoring data were used to compare with predicted emissions of pesticides to surface water. Information such as aggregation steps over time and space, grid sizes, information on crop areas was geared to one another. The model used for the predictions was the Dutch National Environmental Indicator (NMI version 2) (www.nmi.alterra.nl) that has input data regarding average agricultural use, crop interception, spray drift ‐ and point source emissions of pesticides. In our comparison we systematically look to the outcome of both approaches with respect to the exceedance of environmental criteria, and with respect to pesticide concentrations at national scale. For this study, 10 different combinations of pesticide and exposure concentration were selected covering a large range of pesticides characteristics and pesticides use. Six out of these 10 cases gave similar results between measurements and predictions with respect to exceedance of the environmental quality criteria. Comparison of the concentrations measured and predicted showed higher variability. Predicted 21‐time‐weighted average concentrations of pesticides gave higher agreement with pesticides measurements compared to predictions based on the maximum peak concentration. Dissimilarities could be explained by many factors, in which information at a regional scale was highly important. An overview of the dominant factors is discussed. Key words: emissions, measurements, pesticides, surface water, quality standard exceedance


126

16:40 SIDE EFFECTS OF THE SBI FUNGICIDE,

PROPICONAZOLE, ON A BENEFICIAL ARBUSCULAR MYCORRHIZAL FUNGUS

Maryline CALONNE, Joël FONTAINE, Djouher DEBIANE, Frédéric LARUELLE,

Anne GRANDMOUGIN & Anissa LOUNÈS-HADJ SAHRAOUI Univ Lille Nord de France, Lille Université du Littoral Côte d’Opale,

Unité de Chimie Environnementale et Interactions sur le Vivant FR-62228 Calais, France

Corresponding author E-mail: [email protected] Nowadays, the use of fungicides is generalized in modern agriculture for the control of fungal diseases. Among them, the Sterol Biosynthesis Inhibitor (SBI) family, including propiconazole, is one of the most used. Unfortunately, little is known about their potential side effects on non‐target plant‐beneficial soil organisms such as arbuscular mycorrhizal fungi (AMF). Thanks to axenic cultures, the direct impact of increasing propiconazole concentrations (0.02; 0.2 and 2 mg.L‐1) on the lipid metabolism of the AMF Glomus irregulare in relation with its development, was studied. The propiconazole impact on Glomus irregulare was investigated, firstly, through sterol (the target‐metabolism of SBI fungicides), phospholipids (PL) and their associated fatty acids (PLFA) analysis (the main membrane components) and secondly by measuring malondialdehyde (MDA) (a biomarker of lipid peroxidation) formation. Finally, the activity of peroxidase (POD: an antioxidant enzyme) was assayed. Our results demonstrated that the drastic reduction of Glomus irregulare development (germination, germ tube elongation, colonization, extraradical hyphae growth and sporulation) could be explained not only by the decreases of the total sterol end‐products (24‐methylcholesterol and 24‐ethylcholesterol) and by eburicol (a sterol precursor) accumulation, suggesting an inhibition of a key enzyme in sterol biosynthesis pathway (14a‐demethylase), but also by the increases in phosphatidylcholine (PC) and PLFA (C16:0; C18:0 and C19:0) quantities and MDA production. Moreover, POD activity was found to be inhibited in the presence of propiconazole. Taken together, the findings of the current study highlighted a relationship between the IBS fungicide toxicity against the beneficial AMF Glomus irregulare and (1) a disturbance in the sterol metabolism, (2) a lipid peroxidation, (3) a membrane alteration and (4) an inhibition of the antioxidant enzyme. In conclusion, this work contributed to investigate the toxicity of agricultural chemicals on AMF and underlined the emergency of using sustainable alternative method to control plant diseases. Furthermore, these data can provide a useful approach in soil ecotoxicology studies and risk assessment. Key words: Glomus irregulare, arbuscular mycorrhizal fungi, fungicides, malondialdehyde, sterols, phospholipids



127

17:00 CYANOBACTERIA FROM LAB TO FIELD:

SCREENING FOR PESTICIDE RESISTANCE IN CYANOBACTERIA

R. GYANA PRASUNA, G.V.N.S. DEVIRAM, SAIDANI & GAURAV PANT

Department of mcirobiology, GITAM Institue of Science, GITAM University, India Cyanobacteria are known to have potent applications as a biofertilizer. in addition to nitrogen fixation they also bring about alteration in soil profile leading to better water retention and aeration. pesticide build up in soils is one of the problems of paramount importance in agriculture. To alleviate this problem, it is required to develop pesticide resistant biofertilizers. This paper presents experimental studies with several strains of cyanobacteria in conjunction with pesticides Key words: Cyanobacteria, biofertilizer, pesticide resistance

Session 10 Herbology

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130


131

15:00 LONG-TERM EFFECTS OF REDUCED HERBICIDE

DOSAGES AND NITROGEN FERTILISATION INPUTS ON APERA SPICA-VENTI POPULATION DYNAMICS

Andrea SCHULZ, Bernhard PALLUTT & Bärbel GEROWITT

University of Rostock Faculty for Agricultural and Environmental Sciences Group Crop Health Satower Str. 48,DE-18051 Rostock, Germany

Apera spica‐venti is one of the most abundant grass weeds on light‐textured soils in Central and Eastern Europe. Matured plants produce up to 10,000 seeds, which are able to survive for 2 to 7 years in the soil. As a winter annual grass weed it is well adapted to the development of winter sowing crops. It became one of the most serious annual grass weeds in Germany and may cause considerably yield losses in untreated fields. Very limited crop rotations with high percentage of winter wheat combined with reduced/no tillage practices and early sowing dates support a high infestation and spreading with A. spica‐venti. These problems may be avoided, if selected preventive and cultural weed management practices are adopted. A long‐time field experiment was conducted investigating the effects of crop rotation, weather conditions and limited herbicide and nitrogen fertiliser input on A. spica‐venti population dynamics and crop yield. The effects of situation related herbicide doses and application rates reduced by 50% were investigated in a six‐field cash‐crop and a six‐field fodder crop over two rotation periods. Cash‐crop rotation contained 75% winter cereals whereas the fodder crop rotation 50%. Due to soil heterogeneity on experimental site six replications were investigated. In the first six years the occurrence of A. spica‐venti increased. Fertilisation reduced by 50% lead to a significant increase in weed density. Appropriate nitrogen fertilisation improved cereal competitive power, which reduced weed‐caused yield losses and the increase in weed infestation after non‐application of herbicides. There was also observed a significant influence of weather factors on A. spica‐venti emergence. The improved understanding gained from this study on the behaviour of this important grass weed with limited chemical weed control and nitrogen mineral fertiliser inputs, provides guidelines for practical agriculture and future research. Key words: Apera spica‐venti, herbicide use, crop yield, weather


132

15:20 DYNAMIC OF BLACK-GRASS POPULATIONS

DEPENDING ON THE SOWING TIME OF WINTER WHEAT

J. VANDERSTEEN1, G. MAHY2, J-P BIZOUX2, A. MONTY2, F. HENRIET3,

F. VANCUTSEM1 & B. BODSON1 1 Unit of Crop Management, Gembloux Agro Bio-Tech, University of Liège, Gembloux, Belgium

2 Biodiversity and Landscape Unit, Gembloux Agro Bio-Tech University of Liège, Gembloux, Belgium

3Plant Protection and Ecotoxicology Unit, Walloon Agricultural Research Center Gembloux, Belgium

Herbicides are used from decades in agriculture but currently, there is a significant interest of reducing their use. The development of appropriate weed management strategies can help to reduce the amount of applied herbicide. The delay of sowing date can be a technique to reduce weed populations through the impact on their life cycle. A better understanding of these potential changes is necessary for a good adaptation of agricultural practices. The main goal of this research is to study dynamic of wheat and weed populations ‐ including a cereals specialized species: black‐grass (Alopecurus myosuroides Huds.) ‐ in relation to sowing date. The winter wheat crop was sown at four different dates at two weeks interval, from 15 October to 26 November. The sowing densities increased with sowing date from 220 to 375 seeds per square meter. The experiment was carried out in Gembloux, on a Luvisol (FAO classification) in Hesbaye (Belgium) during the growing season 2009 ‐ 2010. A split plot design with four replications was used. The main plot was the use of chemical weeding and the subplot was the sowing date. Weeds and wheat were assessed every two weeks during all the vegetation season in all plots. Reproduction rate evaluation was done on the non weeding plots. Total black‐grass density ranged, in May, between 0 and 32 plants per square meter. It was shown that this density like the number of ears and seeds produced per black‐grass plant decreased significantly with the sowing date. Consequently, we conclude that the sowing date delay appears a promising way to reduce black‐grass populations and their reproduction rate under our conditions. In order to confirm these results, similar experiments are repeated this year in similar soil conditions. Key words: Black‐grass, sowing date, winter wheat, non chemical management, Wallonia


133

15:40 LOCAL SPREAD OF METAMITRON RESISTANT

CHENOPODIUM ALBUM L. PATCHES USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS

J. APER, J. DE RIEK, E. MECHANT, R. BULCKE & D. REHEUL

Weed Science Unit Department of Plant Production, Ghent University

Coupure links 653, BE-9000 Ghent, Belgium Molecular markers can provide valuable information on the spread of resistant weed biotypes. In particular, tracing local spread of resistant weed patches will give details on the importance of seed migration with machinery, manure, wind or birds. This study investigated the local spread of Chenopodium album patches in the southwest region of the province West‐Flanders (Belgium). During the summer of 2009, leaf and seed samples were harvested in 27 patches, distributed over 10 sugar beet fields and 1 maize field. The fields were grouped in four local clusters. Each cluster corresponded with the farmer who cultivated these fields. A PCR‐RFLP procedure identified the Ser264 to Gly mutation in the D1 protein, endowing resistance to metamitron, a key herbicide applied in sugar beet. The majority of the sampled plants within a patch (97% on average) carried this mutation. Analysis of molecular variance revealed that a significant part of the genetic variability was attributed to variation among the farmer locations (12%) and variation among Chenopodium album patches within the farmer locations (14%). In addition, Mantel tests revealed a positive correlation between genetic distances (linearised Ð¤pt between pairs of patches) and geographic distances (Mantel‐coefficient significant at p=0.002), suggesting isolation‐by‐distance. In one field, a decreased genetic diversity and strong genetic relationships between all the patches in this field supported the hypothesis of a recent introduction of resistant biotypes. Furthermore, genetic similarity between patches from different fields from the same farmer indicated that seed transport between neighbouring fields is likely to have an important impact on the spread of metamitron resistant biotypes. Key words: genetic differentiation, herbicide resistance, Chenopodium album L., sugar beet, AFLP


134

16:00 CROSSING ABILITIES OF ALS TARGET-SITE RESISTANT BLACK-GRASS (ALOPECURUS

MYOSUROIDES HUDS.)

Maréchal PIERRE-YVES*1, François HENRIET2, Françoise VANCUTSEM1 & Bernard BODSON1

1 Université de Liège – Gembloux Agro Bio Tech Unité de Phytotechnie des Régions Tempérées 2, Passage des Déportés, BE-5030 Gembloux Belgique

*E-mail: [email protected] 2 Centre Wallon de Recherches Agronomiques Département Sciences du Vivant (D1)

Unité Protection des plantes et écotoxicologie (U4) 11, rue du Bordia, BE-5030 Gembloux Belgique E-mail: [email protected] *Boursier FRIA

Black‐grass crossings were conducted outdoor for two successive years in a way to determine the percentage of resistant black‐grass that can be engendered in one generation, after the introduction of one Target‐Site resistant individual. After germination, plants were genotyped and couples of resistant and susceptible individuals were formed, then planted outdoor and enclosed with a pollen‐proof cloth to avoid any external pollen contamination. Seeds were collected and tested in glasshouses with herbicide spraying. DNA analyses were also performed on surviving plants to distinguish hetero‐ from homozygotes. The resistance level of the progeny seems to be fairly high and Mendelean distribution is generally confirmed. The offspring of self‐crossings was also observed to assess the facultative autogamy ability, according to the genotype and the presence of mutant ALS alleles (position W‐574). Key words: Black‐grass, Target‐Site Resistance (TSR), AcetoLactateSynthase (ALS), Crossing, Autogamy


135

16:20 EFFICACY OF ECHINOCHLOA MURICATA AND ECHINOCHLOA CRUS-GALLI TO HPPD- AND

ALS-INHIBITING HERBICIDES IN CORN

B. DE CAUWER, R. ROMBAUT, R. BULCKE & D. REHEUL Department of Plant Production, Ghent University, Ghent, Belgium

Untill recently, Echinochloa muricata var. microstachya Wiegand (rough barnyardgrass), an alien species native to North America, was completely overlooked in Belgium. Since 1887, E. muricata var. microstachya has gradually spread and is now locally naturalized and abundant in and along maize fields, particularly on moister and heavier soils. Unfortunately, E. muricata is hardly known and distinguishable morphologically from Echinochloa crus‐galli (L.) P. Beauv. (barnyardgrass), particularly at early growth stages. One of the possible raisons for its expansion in maize fields, besides e.g. the lack of crop rotation, might be a lower sensitivity to postemergence (POST) herbicides acting against panicoid grasses, in particular 4‐hydroxyphenyl pyruvate dioxygenase (HPPD)‐inhibiting herbicides and acetolactate synthase (ALS) inhibiting herbicides. Dose‐response pot experiments were conducted in the greenhouse to evaluate the effectiveness of four HPPD‐inhibitor herbicides [topramezone (ARIETTA®), mesotrione (CALLISTO®), tembotrione (LAUDIS®), sulcotrione (MIKADO®) and the ALS‐inhibitor herbicide nicosulfuron (KELVIN®) for controlling E. crus‐galli and E. muricata populations collected in Flanders, Belgium. Pots were planted with 25 seeds , thinned afterwards to 5 plants (one week after sowing) and irrigated overhead. Herbicides were applied at the3‐4 leaf stage (BBCH stage 13‐14). Fresh biomass was harvested 28 d after treatment (DAT). Echinochloa muricata populations were less sensitive to HPPD‐inhibitor herbicides than E. crus‐galli populations. The 90% effective doses (ED90) of sulcotrione and topramezone were fourfold higher for E. muricata than for E. crus‐galli. However, nicosulfuron sensitivity of both species was similar. Due to the intragenus variability in sensitivity to HPPD‐inhibitor herbicides, higher awareness is required for the presence of E. muricata plants in maize fields in order to avoid insufficient “barnyardgrass” control. Key words: Rough barnyardgrass, barnyardgrass, maize herbicide, panicoid grass, sensitivity


136

16:40 WEED INHIBITORY EFFECT OF DIFFERENT

PAVING CONSTRUCTIONS

M. FAGOT1, B. DE CAUWER1, D. REHEUL1, R. BULCKE1, E. BOONEN2 & A. BEELDENS2

1Department of Plant Production, Ghent University, Coupure Links 653, BE-9000 Ghent, Belgium

2Belgian Road Research Centre Woluwedal 42, BE-1200 Brussels, Belgium

The recent phase out of herbicidal use on public pavements by the Flemish government triggers the development of new alternative weed control strategies. Besides the search for effective non‐chemical curative methods, there is also a need for weed preventive strategies on pavements. In this study, effects of type of joint filling material, joint width, pollution and type of street layer material were assessed for their impact on weed germination and coverage. In 2009, a paving experiment with 96 block paving plots was set up under a rain shelter. Block paving plots were arranged in a randomized complete block with 3 replications. Each block paving plot consisted of a conventional (non permeable) concrete block paving on top of a street layer, supported by a base layer. Paving plots comprised factorial combinations of five joint filling materials, two joint widths, two pollution levels and two street layer materials. Tested joint filling materials were: fine white sand, sea sand, crushed porphyry, crushed limestone and Dansand® (a sodium silicate enriched sand). Joint filling materials were evaluated in pure and organically polluted (10% fine compost by volume) state. Street layer materials tested comprised crushed porphyry (a good water permeable aggregate) and crushed limestone (a less water permeable aggregate). After establishment of the block paving plots, each paving plot was oversown with a mixture comprising Taraxacum officinale, Poa annua, Plantago major and Trifolium repens, all dominant, hard to control weed species on pavements. Weed inhibitory effect of paving plots was determined by examining weed germination and weed coverage. Weed germination was recorded weekly during the first five weeks after sowing. Weed coverage was recorded monthly over a two‐year period by using Image J Software. Pavings were watered three times a day by automatic sprinkler irrigation (2.1 mm/day). Weed germination and weed coverage was highest in pavings with wide joints and polluted joint filling material. In pavings with polluted filling material, the type of street layer material significantly influenced weed germination and weed coverage. Weed preventive ability of joint fillers was highest for sodium silicate enriched sand (Dansand®) irrespective of pollution level, joint width or type of base layer. The weed inhibitory effect of the coarser filling materials porphyry and limestone completely dropped once organically polluted. Within standard filling materials, unpolluted sea sand and porphyry reduced seedling emergence of all species tested. Shifts in weed composition occurred due to observed species‐specific inhibitory effects of joint filling materials. Overall, these results show an opportunity for weed prevention by using suitable paving materials in the construction of pavements. Key words: Weed prevention, pavements, joint fillers

Wednesday 25 May 2011 Satellite meeting: Exploiting genomics to understand

Plant-nematode interactions – (COST Action 872) – Part 2

137

Wednesday 25 May 2011 Satellite meeting: Exploiting genomics to understand plant‐nematode interactions – (COST Action 872) – Part 2

Wednesday 25 May 2011 Satellite meeting: Exploiting genomics to understand Plant-nematode interactions – (COST Action 872) – Part 2

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139

09:00 MINING THE MELOIDOGYNE HAPLA GENOME

David MCK. BIRD1,2, Charles H. OPPERMAN1, Elizabeth SCHOLL1,

Peter DIGENNARO1, Valerie WILLIAMSON3 & Axel ELLING4 1Plant Nematode Genomes Group

2Bioinformatics Research Center, NC State University, Raleigh, NC 27608, USA 3Department of Nematology, University of California, Davis, CA 95616, USA

4Department of Plant Pathology, Washington State University Pullman, WA 99164, USA We have established Meloidogyne hapla, a diploid root‐knot nematode (RKN) with a compact (54 Mbp) sequenced genome, as a tractable model to study the genetic and biochemical basis for plant parasitism (Opperman et al., 2008). The current annotation freeze (HapPep4) predicts 14,207 proteins; experimental confirmation by LC‐MSE is ongoing (Mbeunkui et al., 2010). Comparative genomics, particularly with the sympatric RKN species M. chitwoodi and the migratory species Pratylenchus coffeae, is proving to be a powerful approach to deduce the evolution and mechanisms of parasitic ability by these nematodes. A current target of functional analysis is genes encoding small proteins that exhibit sequence similarity to members of at least three distinct classes of plant peptide hormones. Unlike the canonical plant proteins which are expressed as pre‐pro‐proteins, the RKN genes encode simple pre‐proteins, likely reflecting direct secretion by the nematode into the apoplast. We have developed bioassays to assess the functional role of these apparent ligand mimics in the host‐parasite interaction. Based on initial computational analyses, it appears that these mimics may have evolved by convergent evolution. Like genes acquired horizontally, ligand mimic genes in RKN appear to be expanding and diversifying into families. Collectively, RKN genes with strong inter‐kingdom analogues appear to lie at the core of the parasitic armory. Mbeunkui F, Scholl EH, Opperman CH, Goshe MB, DMcK Bird. 2010. J Proteome Res, 9: 5370–5381 Opperman CH, Bird DMcK, Williamson VM, et al. 2008. Proc Natl Acad Sci (USA), 105: 14802‐14807.


140

10:00 IDENTIFICATION OF PUTATIVE PARASITISM GENES FROM WHOLE GENOMES AND TRANSCRIPTOMES OF

PLANT-PARASITIC NEMATODES

Etienne G.J. DANCHIN1, Amandine CAMPAN-FOURNIER1, Laetitia PERFUS-BARBEOCH1, Marie-Noëlle ROSSO1,

Marie-Jeanne ARGUEL1, Corinne DA SILVA2, Celine VENS3,Jérôme GOUZY4, Martine DA ROCHA1, Marc MAGLIANO1,Nathalie MARTEU1, Karine LABADIE2, François ARTIGUENAVE2 & Pierre ABAD1

1INRA, UNS, CNRS, UMR 1301 Biotic Interactions and Plant Health 400 route de Chappes, Sophia-Antipolis, 06903, France 2CEA, Institut de Génomique, Genoscope, Evry, France

3Katholieke Universiteit Leuven, Department of Computer Science Celestijnenlaan, 3001 Leuven, Belgium

4INRA, CNRS, UMR 0441 LIPM Plants - Micro-organisms Interaction, Castanet-Tolosan, France Initial analysis of the genome of Meloidogyne incognita has allowed the identification of thousands of genes that apparently did not have homologs in model animal and fungal species. A proportion of these M. incognita‐restricted gene models may be involved in functions linked to plant‐parasitism. To identify genes putatively involved in adaptation to parasitism in root‐knot nematode genomes, we have undertaken a comparative analysis of the sets of predicted genes in M. incognita and M. hapla against a large dataset of whole genomes and transcriptomes. As our objective is to identify druggable parasitism genes we have searched for genes conserved in other parasitic or plant‐associated species (including bacteria, fungi and oomycetes) but absent from the genomes of species that could be negatively affected by newly developed drugs or control means (e.g. plants, vertebrates, annelids..). The rationale of our analysis is that the more a gene is conserved in plant‐parastic or plant‐pathogenic species while absent from model non‐parasitic species, the more likely it is to be involved in key parasitism processes. To partially overcome the scarcity of omics data available for plant‐parasitic nematodes, we have initiated RNA‐seq transcriptome sequencing of four plant‐parasitic nematode species presenting diverse parasitic strategies (Pratylenchus coffeae, Ditylenchus dipsaci, Bursaphelenchus xylophilus and Xiphinema index). We have also generated the RNA‐seq of different developmental stages of M. incognita in order to bring additional and dynamic transcription support to the identified genes. We have performed bioinformatics annotation of genes associated with plant parasitism, including but not limited to: detection of signal peptide and Pfam domains, assignment of gene ontology terms, and identification of specific motifs. Using RNAi assays, we will test the effect of gene silencing for a series of the most promising targets identified during bioinformatics screens.



141

10:20 FUNCTIONAL CHARACTERIZATION OF EFFECTORS FROM THE POTATO CYST

NEMATODE GLOBODERA PALLIDA

Sophie MANTELIN1, Peter J. COCK1, Peter THORPE1,3, Alisa SMITH1, Martin HUNT2, Matt BERRIMAN2, P.E. URWIN3, C.J. LILLEY3,

Mirela COKE3, Vivian C. BLOK1 & John T. JONES1 1PPI Programme, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK

2Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB101SA 3Centre for Plant Sciences, Faculty of Biological Sciences

University of Leeds, Leeds, LS2 9JT, UK Globodera pallida induces complex changes in its host and effectors secreted from the pharyngeal gland cells are thought to be important in this process.The genome sequencing project for G. pallida is now well underway.The current assembly consists of just over 112Mb in 17,000 supercontigs.Long‐range information is now being generated in order to reduce the fragmentation of the sequence.Candidate effectors have been identified from the G. pallida genome sequence using a variety of bioinformatic approaches coupled to in situ hybridization.Little overlap exists between the effectors present in G. pallida and in root knot nematodes, with the exception of the cell wall degrading enzymes and chorismate mutaseIllumina transcriptome sequencing for several life stages has been carried out, allowing analysis of expression profiles of all target effectors.We have developed a high‐throughput system for analyzing localization of effector proteins in plant cells and used this to demonstrate that effectors are localized to a variety of plant subcellular structures including the nucleus.In addition, we have developed a system for assaying suppression of basal defences (pathogen triggered immunity – PTI) and effector triggered immunity (ETI).Using these assays we have identified a G. pallida effector that suppresses PTI and are currently investigating the mechanism by which this suppression is achieved.


142

11:10 A NOVEL CYST NEMATODE EFFECTOR PROTEIN

PROMOTES NEMATODE INFECTION AND INTERACTS WITH A HOST PLANT BETA-1,3-ENDOGLUCANASE

Noureddine HAMAMOUCH1, Chunying LI1,Thomas J. BAUM2, Melissa G. MITCHUM3, Richard S. HUSSEY4 & Eric L. DAVIS1

1North Carolina State University, Department of Plant Pathology, Raleigh, NC 27607, USA 2Iowa State University, Department of Plant Pathology, Ames, IA 50011, USA 3University of Missouri, Division of Plant Sciences, Columbia, MO 65211, USA

4University of Georgia, Department of Plant Pathology, Athens, GA 30602, USA Phytoparasitic nematodes secret an array of effector proteins into host plant cells to successfully infect host roots. The biological function of the soybean cyst nematode, Heterodera glycines 30C02 effector protein was studied using Arabidopsis thaliana as host and the beet cyst nematode, Heterodera schachti that contains a homolog of the 30C02 effector. Expression of Hg30C02 in Arabidopsis did not affect plant growth and development but augmented plant susceptibility to infection by H. schachtii. The 30C02 protein interacted specifically with a host beta‐1,3‐endoglucanase in both yeast and plant cells, possibly to interfere with its role as a pathogenesis‐related protein. In addition, Arabidopsis T‐DNA plants that are impaired in beta‐1,3‐endoglucanase activity showed hyper‐susceptibility to nematode infection, suggesting a potential role of host beta‐1,3‐endoglucanase in defense response against H. schachii infection. Interestingly, real‐time PCR analysis of beta‐1,3‐endoglucanase showed that this gene is down‐regulated in expression at the nematode infection site, indicating that, in addition to interacting with the host bet‐1,3‐endoglucanase, secretions of H. schachtii may also act to down‐regulate the expression of this gene at the nematode‐formed feeding site. Moreover, Arabidopsis plants expressing double‐stranded RNA complementary to 30C02 sequence were not phenotypically different from non‐transformed plants but exhibited a strong RNAi‐mediated tolerance to infection by H. schachtii. Taken together, these results indicate that 30C02 is a nematode effector protein that interacts specifically with host plant beta‐1,3‐endoglucanase and is required for plant parasitism by cyst nematodes. Key words: Heterodera schachtii, cyst nematode, Meloidogyne incognita, root‐knot nematode, RNAinterference



143

11:30 A POSSIBLE ROLE FOR TREHALOSE 6-PHOSPHATE

SYNTHASE (TPS) IN THE PARASITISM OF THE PLANT-PARASITIC NEMATODES

ABD EL NASER EL ASHRY1, Etienne G.J. DANCHIN2, Julia HOFMANN3,

Pierre ABAD2 & Florian M.W. GRUNDLER1 1INRES-Molecular Phytomedicine, Rheinische-Friedrich-Wilhelms University of Bonn, Germany

2INRA National Institute for Agricultural Research, Plant-Microbe Interactions and Plant Health, Sophia Antipolis, France

3 Institute of Plant Protection, University of Natural Resources and Life Sciences Vienna, Austria

The establishment and progress of a successful plant‐nematode interaction depends on directed changes in the gene expression of plant and nematode. A. thaliana GeneChip analyses showed that genes involved in carbohydrates metabolism and regulation were differentially expressed after H. schachtii infection. Furthermore, metabolomic studies proved a significant difference in the level of enrichment of certain sugars in A. thaliana after H. schachtii infection. One of the sugars that were both locally and systemically strongly enriched after nematode infection is trehalose. A. thaliana trehalose 6‐phosphate synthase genes (AtTPS), which play a role in catalyzing trehalose, were significantly up regulated in H. schachtii infected plants. On the plant parasitic nematode (PPN) side, there is only little information about the TPS genes. Analysis of TPS genes of M. incognita (MINC) and M. hapla (Mh) showed an over‐representation of TPS in comparison with the TPS from free‐living nematodes (FLN) and animal‐parasitic nematodes (APN).We identified 6 MINC‐TPS and their Mh‐TPS homologues while FLN‐TPS have typically two TPSs, TPS‐1 and TPS‐2.TPS phylogenetic tree, which was formed using the available nematode TPS showed that there is only one of MINC‐TPS and its M. hapla homologue that clustered with FLN‐ and APN‐TPS‐2. All other MINC‐ and Mh‐TPS clustered in a separate branch.We conclude that the latter TPS genes have evolved in Meloidogyne spp. or generally in PPN species to fulfil functions related to pathogenicity. Meanwhile, the gene that clustered with TPS‐2 might still represent the ancestral TPS functions in FLN and APN. Furthermore, our analysis showed that two MINC‐TPS have a predicted signal peptide. Interestingly, these MINC‐TPS genes were included under the PPN specific branch. We expect that further studies on the PPN‐TPS genes can help us gaining more understanding about evolutionary processes in PPN.


144

11:50 UDP-GLUCOSE DEHYDROGENASE (UGD) GENES ARE INVOLVED IN CELL WALL MODIFICATIONS DURING

DEVELOPMENT OF SYNCYTIUM IN ROOTS OF ARABIDOPSIS INDUCED BY HETERODERA SCHACHTII

Shahid SIDDIQUE1*, Rebecca REBOUL2, Miroslaw SOBCZAK3,

Raimund TENHAKEN2, Florian GRUNDLER1*, Holger BOHLMANN1 1Institute of Plant Protection, University of Natural Resources and Life Sciences,

Vienna, Austria 2 Department of Plant Physiology, University of Salzburg, Austria

3Department of Botany, Warsaw University of Life Sciences (SGGW), Warsaw, Poland *Current address, INRES-Molecular Phytomedicine,

Rheinische-Friedrich-Wilhelms University of Bonn, Germany Second stage juveniles (J2) of beet cyst nematode Heterodera schachtii invade the roots of the host plants and induce a feeding site called syncytium. During development, cell walls of syncytia undergo modifications to support its function as sole source of nutrients for the nematodes. These modifications include thickening as well as local dissolution of cell wall. Moreover, cell wall ingrowths are formed at the interface between xylem and syncytium. In order to undergo these modifications, syncytium requires the de novo synthesis of cell wall polysaccharides. In plants, UDP‐glucuronic acid (UDP‐GlcA) is a precursor for cell wall polysaccharides and is synthesized by the oxidation of UDP‐glucose by UDP‐glucosedehydrogenase (UGD). The enzyme UGD is encoded by four genes in Arabidopsis (UGD1, UGD2, UGD3, UGD4). In this study, we analysed the importance of these genes in syncytium development and maintenance in Arabidopsis plants. Histological assays were performed with the help of promoter::GUS lines, which showed a syncytium specific expression of all four UGD genes during different stages of nematode development. At least two of these genes (UGD2 and UGD3) were expressed in syncytium as early as 1 days post inoculation (dpi). Nematode infection assays with T‐DNA inserted knockout mutants showed a significantly reduced number of females in two single (∆ugd2, ∆ugd3) and a double UGD mutant (∆ugd23). A time course ultrastructural investigation revealed degeneration of syncytia as early as 3 dpi in roots of ∆ugd23 mutant. Moreover, syncytia contain electron translucent cytoplasm in which cell wall ingrowths were either absent or collapsed. As these genes are involved in synthesis of cell wall matrix, we analysed the hemi‐cellulose content of roots and syncytia of ∆ugd23 plants. The resulting data showed syncytium specific changes in cell wall composition in these mutants.We, therefore conclude that UGD2 and UGD3 play an important role in cell wall modifications during development of syncytium.



145

12:10 LIPID SIGNALS GOVERNING PLANT

INTERACTIONS WITH ROOT KNOT NEMATODE

Sigal BROWN HOROWITZ1, Ionit IBERKLEID1,2 & Rachel OZELVO1 1Departments of Nematology; Plant Protection Institute, ARO, The Volcani Center

2Department of Microbiology; The Hebrew University of Jerusalem, The Robert H. Smith Faculty of Agriculture, Food and Environment, Israel

Root‐knot nematodes, Meloidogyne spp., are extremely destructive pathogens with a cosmopolitan distribution and a host range that spans most crops. Safety and environmental concerns of toxic nematicides along with lack of natural resistance sources for the four major Meloidogyne spp. threatening most crops worldwide emphasize the need to identify the precise mechanisms that determine whether the plant host is susceptible or resistant to nematodes. A group of novel but little understood chemical signals that have been implicated for either successful defense or colonization are small lipid derivatives called oxylipins. In plants, oxylipins are synthesized by lipoxygenases (LOX). Recent findings in Arabidopsis suggesting that oxylipins from the 9‐LOX pathway function in cell wall modifications required for lateral root development and pathogen arrest. To understand the function of 9‐ and 13‐ lipoxygenases in response to nematodes infection the expression profile of these genes will be monitored and functional analyzing will determine which are important in host‐parasite interactions. We will report the temporal regulation of transcription of these genes encoding 13‐LOX, 9‐LOX, α‐DOX1 and α‐DOX2 through the early parasitic life cycle of the root knot nematode M. javanica. To function analyzing their role in the parasitic interaction Arabidopsis thaliana mutants disrupted in 13‐LOX, 9‐LOX, α‐DOX1 and α‐DOX2 encoding genes were inoculated with M. javanica eggs and are being studied to analyze parasitic development during mutant parasitism. Results obtained here will provide evidence in terms of a possible strategy of M. javanica to manipulate early plant defense mechanisms governed by LOX signaling cascades.


146

14:00 ROLE OF THE PLANT SIGNALLING PEPTIDES

PHYTOSULFOKINES AND THEIR RECEPTOR FOR PLANT NEMATODE COMPATIBLE INTERACTION

Natalia RODIUC, Laetitia ZURLETTO, Pierre ABAD, Harald KELLER

& Bruno FAVERY INRA-Université de Nice-Sophia Antipolis-CNRS

UMR 1301-6243 Interactions Biotiques et Santé Végétale 400 route des Chappes FR-06903 Sophia Antipolis France,

During a compatible interaction, root knot nematodes Meloidogyne spp. induce an elaborate permanent feeding site characterized by the redifferentiation of vascular root cells into multinucleate and hypertrophied cells. These giant cells result from repeated nuclear divisions without cell division. In addition, hyperplasia of the surrounding cells lead to the formation of the typical root gall. Genome wide expression profiling experiments of the plant response to root‐knot nematode Meloidogyne incognita infection identified many genes differentially expressed during giant cell formation in Arabidopsis. Among them, we found those coding for phytosulfokines (PSKs) to be downregulated. PSKs are sulfated penta‐ or tetra‐peptides, and are unique mitogens regulating cellular dedifferentiation and redifferentiation in plants. In Arabidopsis, five paralogous genes have previously been identified, which code for PSK precursors of approximately 80 amino acids. From these pre‐pro‐proteins, PSKs are produced by proteolytic processing and post‐translational tyrosine sulfation. To study the role of PSK signalling in the plant‐nematode interaction, we subjected the five PSK genes, as well as the gene coding for the PSK putative receptor, PSKR1, to a functional analysis. The first results obtained in Arabidopsis will be presented.



147

14:20 IMMUNOLOCALISATION OF VACUOLE SPECIFIC V-ATPASE AND γ-TIP PROTEINS IN SYNCYTIA

INDUCED BY CYST NEMATODES IN ARABIDOPSIS AND TOMATO ROOTS

Lukasz BARANOWSKI, Miroslaw SOBCZAK &Wladyslaw GOLINOWSKI

Department of Botany, Warsaw University of Life Sciences (SGGW) Nowoursynowska 159, 02-787 Warsaw, Poland

Plant parasitic cyst forming nematodes are biotrofic pathogens that induce in host plant root a specific feeding site called a syncytium. Characteristic features of the syncytium are condensation and proliferation of its cytoplasm, hypertrophy of syncytial nuclei and general increase of organelles numbers. Concomitantly, central vacuoles typical for differentiated plant cells become replaced by numerous small vacuoles and vesicles. Very little is known about their origin and functions in syncytia. Thus using specific antibodies directed against V‐ATPase and γ‐TIP proteins, being molecular markers of vacuole and tonoplast, respectively, immunolocalisation studies were performed on sections of syncytia. V‐ATPase protein is a general marker of all types of vacuole. In contrast, γ‐TIP protein is a marker specific for only lytic vacuole compartment. Both proteins were localised in syncytia induced in roots of Arabidopsis thaliana and tomato (Solanum lycopersicum) by Heterodera schachtii or Globodera rostochiensis, respectively. Two‐step immunolocalisation in conjunction with fluorochrome (FITC) or colloidal gold detection was performed. Both proteins were detected in membranes surrounding vesicles in syncytia induced by both nematodes. In uninfected roots, central vacuoles labelled strongly for the presence of V‐ATPase protein only. Labelling with anti‐γ‐TIP antibody indicated the presence of lytic vacuoles only in differentiating tracheary elements.


148

14:40 ANALYSIS OF THE ARGININE METABOLISM IN PLANT

NEMATODE INTERACTIONS

Shahbaz ANWAR1, Erich INSELSBACHER2, Florian M.W. GRUNDLER3 & Julia HOFMANN1

1Institute of Plant Protection, University of Natural Resources and Life Sciences, Peter Jordan-Straße 82, AT-1190 Vienna, Austria

2Department of Forest Ecology and Management, Swedish University of Agricultural Sciences, SE-901 83 Umeå, Sweden

3INRES Molekulare Phytomedicine University Bonn Karlrobert-Kreiten-Str.13, DE-53115 Bonn, Germany

The plant parasitic cyst nematode Heterodera schachtii infects next to a variety of plant species also the roots of Arabidopsis where it induces specialized feeding structures called syncytia. A previous transcriptome analysis of nematode‐induced syncytia showed a significant up‐regulation of genes involved in the arginine metabolism (Szakasits et al., 2009). Arginine is an essential and proteinogenic amino acid and it is an important medium for transport and storage of nitrogen in plants. Several arginine‐derived metabolites such as polyamines have been recognized as cell differentiation regulators. In order to study the role of arginine metabolism in nematode‐induced syncytia, gene expression analyses were performed. We selected genes coding for argininosuccinate synthase and argininosuccinate lyase playing a key role in arginine biosynthesis. Further, the expression of ARGAH1 and ARGAH2 were studied that code for arginase, which is hydrolyzing arginine into ornithine and urea. Relative mRNA expression levels of these genes were quantified using qRT‐PCR in 5, 10 and 15 day old H. schachtii induced syncytia. All the analyzed genes were up‐regulated as compared to the non‐infected control roots. Promoter::gus lines, when infected with H. schachtii, showed high promoter activity in syncytia, 5 and 10 day post‐inoculation. Gene silencing lines of argininosuccinate synthase and argininosuccinate lyase under the control of the PDF 2.1 promoter were produced. These lines showed a higher susceptibility towards Heterodera schachtii compared to the wild type, whereas infection tests with the T‐DNA mutants argah1‐1 and argah2‐1 did not show any significant difference. These results suggest that arginine cycling plays an important role for nematode development.

Posters S01-S03

149

Posters Special Session: Biopesticides and green adjuvants

Posters S01-S03

150

Posters S01-S03

151

S01 LETHAL EFFECTS PLANT EXTRACTS FUMARIA

PARVIFLORA LAM. AND EUCALYPTUS CAMALDULENSIS AND INSECTICIDES, AMITRAZ ON AGONOSCENA PISTACIAE BURCKHARDT AND LAUTERER UNDER

LABORATORY CONDITIONS

F. MIRZAEI, M.A. SAMIH, M.K. IRANNEJAD, Z. SHABANI & A. ALIZADEH Department of Plant Protection, Faculty of Agriculture, Valieasr University, Rafsanjan-Iran

The common pistachio psylla Agonoscena pistaciae Burckhardt and Lauterer (Hem.: Aphalaridae) was know as the most important in pistachio orchards Iran. Different methods like pesticides had been used for controlling the pest till now, but due to environment hazard and prevention of natural resources especially in water and soil systems management and chains food uses of other method is necessary. In recent years, plant extracts have received much attention as pest's control because of their insecticidal properties In this research, the effect of two ethanolic extracts Fumaria parviflora Lam. (Fumariaceae) and Eucalyptus camaldulensis (Myrtaceae) using a modified sukselh apparatus and insecticides, amitraz on the5nd instar nymph of Agonoscena pistaciae Burckhardt and Lauterer was assessed at 26±2°C , 65±5 % RH and 16: 8 (L: D) photoperiod. In bioassay experiments was used by dipping in pistachio leaf disk at different concentrations plant extracts and exposed insecticides reated for the5nd instar nymph. mortality was determined 24 hours after treatment. Experiments were computation under CRD procedures,with three replicates were performed per treatments. Estimates of LC50 value for amitraz was 26.8 (17.1‐36.2) mg/l, this value for extracts Fumaria parviflora Lam. 77900 (45600‐109500), and was determined for Eucalyptus camaldulensis 283600 (227100 – 332700) mg/l , and dosage‐response gradient was1.528±0.271, 3.28±0.507, 2.837±0.714,1.552±0.226. Based on the results, the 5nd instar nymph are more susceptible as compared with insecticides amitraz. between extracts, plant extracts Fumaria parviflora Lam.has comparatively effective on pest and It can be as a less effective compound and agreeable with environment in controlling this pest. Key words: Agonoscena pistaciae, amitraz, Eucalyptus camaldulensis, Fumaria parviflora

Posters S01-S03

152

S02 POWDERY MILDEW INTEGRATED CONTROL ON

ZUCCHINI WITH 'SOFT CHEMISTRY' MATERIALS AND QUINOXYFEN

T. ANNESI, V. POMPI, E. CONTE, C. CIOFFI & L. DONNARUMMA

CRA- Research Center on Plant Pathology via C.G. Bertero, 22- Rome, Italy

Powdery mildew (Podosphaera xanthii) is obligate biotroph that rapidly colonize green tissues of zucchini and can negatively affect host physiology. The aim of this trial was to evaluate the efficacy of one strengthener and one essential oil used alternating with quinoxyfen in order to suggest an integrated control management able to reduce the use of synthetic fungicides. Experimental trial, carried out in plastic greenhouse on zucchini crop (‘Romanesco’ cultivar), consisted in treatments with ‘soft chemistry’ materials alone or in alternation with synthesis fungicide. Each thesis has been composed of six plants, according to a randomized blocks design, with 3 replications. Four alternative application strategies were carried out: theses 1 and 2 consisted respectively in applications of strengthener NTI B based on a mix of edible oils alone and in rotation with quinoxyfen; while in theses 3 and 4 Tea Tree essential oil (leaf) emulsified with Tween 20 were applied alone and in rotation with quinoxyfen. The first treatment was carried out when natural fungi infection appeared and, thereafter, treatments were repeated in seven day intervals for five weeks, plants were sprayed until runoff. Theses treated weekly with water, Tween and quinoxyfen were also performed. Disease incidence (percentage of infected leaves) and disease severity (colony density on leaf) were detected and the effect of the treatments was analyzed by analysis of variance (ANOVA) by Tukey test for p≤ 0.05 and p ≤ 0.01. After 6 weeks it has been observed a general reduction of disease incidence in all treated theses. As regards disease severity only the strengthener alternated with quinoxyfen has been statistically different from the untreated control. Preliminary results seem to confirm that the alternation of ‘soft chemistry’ materials with effective synthetic fungicide can maintain a good disease control and may also assist with resistance management. Key words: Powdery mildew, zucchini, strengthener, quinoxyfen, integrated management

Posters S01-S03

153

S03 BIOCONTROL ABILITY OF FLUORESCENT

PSEUDOMONADS ISOLATED FROM WHEAT RHIZOSPHERE AGAINST SOIL BORNE PHYTOPATOGENS

S. MEZAACHE-AICHOUR, A. GUECHI & M.M. ZERROUG Département de Microbiologie/Faculté des Sciences de la Nature et de la Vie

Université Ferhat Abbas, Sétif, Algérie E-mail: [email protected]

There is increasing public concern regarding the continued use of agrochemicals that are damaging our ecosystems. So, It is necessary nowday to search and to develop alternative methods for the control of plant diseases that will contribute to the goal of sustainability development of environment. Within the last decade intense efforts and scientific research were focused on the biocontrol agents particularly Pseudomonads (biocontrol agents: BCAs) which attracted particular interest. This group became the model of studiyng and understanding the mechanisms and the regulation within Gram negative rhizospheric bacteria. Soil fluorescent Pseudomonads, are exellent biocontrol agents colonizing plant rhizosphere and producing active secondary metabolites, because of their ability to reduce the incidence of the root soil borne phytopatogens and presenting a realistic alternative to chemical pesticides. The aim of this work was to isolate, test biocontrol ability of local strains of fluorescent Pseudomonads (Sétif, Algeria), and their exploitation. Form soil samples(30 g in 75 ml water), seraial dilution series were made, aliquots (100 µl) from dilutions 10‐3and 10‐4 were plated on PDA in dual culture for two weeks at room temperature with phythopathogenic soil borne fungi. In vitro, screening revealed an antifungal activity estimated from 10% to 75%. The dual culture test showed an effective action which reaches for some strains a rate of 75% for Phytophtora infestans, Fusarium oxysporum f.sp. albedinis, 40% for Fusarium solani var. coeruleum, and fungistase estimated at 10% for Fusarium solani spp. These results are very promising for biological prospects by using fluorescent Pseudomonads as biocontrol agents. Key words: Alternative pesticides, Biocontrol agents, fluorescent Pseudomonads, Environment.

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Posters Agricultural Entomology and Acarology

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E01 HALF A CENTURY OF PHEROMONE SCIENCE:

A WELCOME GOLD-MINE FOR BASIC BIOLOGY, SENSORY PHYSIOLOGY, AND CROP PROTECTION

Hans E. HUMMEL1,2, K-E. KAISSLING3 & E. HECKER4

1 Chair of Organic Agriculture, Justus-Liebig-University Karl-Gloeckner-Strasse 21C, DE-35394 Giessen, Germany

2 Illinois Natural History Survey, University of Illinois, Champaign, Ill. 61820, USA 3 Max-Planck-Institute of Ornithology, DE-82346 Seewiesen-Andechs, Germany

4 German Centre of Cancer Research, Im Neuenheimer Feld, DE-69120 Heidelberg, Germany INTRODUCTION. Contemporary biologists take the term as well as the concept of "pheromones" for granted. Yet, it was a sensational and controversial event when the January 1959 issue of Nature magazine published a two page communication by Karlson and Lüscher on "Pheromones, a new term for a class of biologically active substances". This paper appeared in the same year with the seminal identification of the chemical structure of bombykol by Butenandt, Hecker and co‐workers (1959). Bombyx mori the silkworm moth, with its main pheromone component (E,Z)‐10,12‐hexadecadien‐l‐ol served as a prototype for an entirely new class of exogenously active natural signal compounds. Two years later, the total synthesis via several independent routes by Butenandt, Hecker et al. 1961 concluded 20 years of pioneering work into unknown territory. Immediately, the impact of these discoveries for chemical communication, chemical ecology, sensory physiology and practical plant protection was recognized. SIGNIFICANCE. Nothing documents the significance of pheromones better than several thousand original research articles and dozens of monographs. Today, pheromones are known from pest insects in all major food and fiber crops and from stored products. Most prominent are insect sex attractants. But chemical signals for territorial marking, trail following, general chemical orientation to food plants and oviposition sites, hive cohesion, and caste determination in social insects are also known. In insect pest management, pheromones and related kairomones play a key role as lures in traps for monitoring, mass trapping and for mating disruption. In a number of favourable cases, pheromones can compete with non sustainable pesticides which provoke events of poisoning, ecotoxicity and resistance. Today, pheromones of other arthropods including spiders are also known. CONCLUSIONS: The progress in vertebrate pheromone research is noteworthy, but slower. If exploited, its long term impact on human society, however, may be enormous. Pheromone research influenced other fields like nanogram analytics, electrophysiological methods including electroantennography and single cell recording, and biochemical genetics. Without pheromones, entomology would be like a soup without spices.

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E02 POPULATION DYNAMICS 2010 OF DIABROTICA V.VIRGIFERA AT VARIOUS LOCATIONS OF THE SWISS CANTON TICINO UNDER MANDATORY

CROP ROTATION MANAGEMENT

Hans E. HUMMEL1,2 & M. BERTOSSA3 1 Chair of Organic Agriculture, Justus-Liebig-University Giessen

Karl-Gloeckner-Str. 21 C, DE-35394 Giessen, Germany E-mail: [email protected]

2 Illinois Natural History Survey, University of Illinois Urbana-Champaign, Institute of Natural Resources Sustainability, 1816 S.Oak Street, Champaign, Il. 61820, USA

3 Agroscope-Changings-Wädenswil ACW, Centro di Cadenazzo, CH-6594 Contone, Switzerland INTRODUCTION: Populations of Diabrotica v. virgifera LeConte (Coleoptera: Chrysomelidae) (Dvv) suddenly increased in Ticino maize fields sharply after the turn of the century. In response, federal authorities enforced a production system based on mandatory crop rotation without insecticide applications. For long term management and efficacy control, Dvv populations were systematically quantified by monitoring with pheromone and kairomone traps. MATERIAL AND METHODS: Inexpensive, reliable and highly specific traps of the Metcalf sticky cup type were baited with the sex pheromone 8‐methyl‐decane‐2‐ol propanoate and the kairomone 4‐methoxy‐cinnamaldehyde (MCA). Start, maximum and end of flights have been exactly monitored. During the summer of 2010, 13 locations in the Magadino plain, adjacent valleys and hills were monitored twice a week. RESULTS: (1) Dvv flights occurred between July 12 to Oct. 3, with a maximum from July 19 to Aug. 15, 2010. (2) The location Lostallo in the Misox side valley with N= 530 Dvv under otherwise comparable conditions showed a population maximum and is responsible for more than 25 % of all Dvv captured in the region. Not by design but in actuality Lostallo thus serves as a standard control for the "maize after maize" growing strategy without crop rotation. Populations sampled in 12 other locations throughout the Magadino plain are much less numerous and collectively represent only 58% of the Dvv captured. (3) MCA attracts both sexes but with N=75 is with a factor 16 less active than the synthetic female sex pheromone which lures males only. (4) As previously seen, Dvv have been present at Mt. Ceneri pass at 530 m elevation in the absence of any maize fields. This indicates a trans‐cantonal flight path of Dvv from S to N. (5) Mandatory canton‐wide crop rotation results in a considerable decrease and eventual stabilization of Dvv at a significantly lower density. Yearly variations result from changes in weather and influx activity from Northern Italy. CONCLUSION: The well proven crop rotation is characterized by considerably lower cost, resource saving sustainability and lowest possible environmental burden, if any. Adoption of this old and proven strategy by other countries is urgently advocated.

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E03 POPULATION FLUCTUATIONS OF TWO ICERYA SPECIES,

ICERYA PURCHASI AND ICERYA SEYCHELLARUM (MONOPHLEBIDAE, COCCOIDEA) ALONG WITH

ECOLOGICAL STUDIES ON ACALYPHA WILKESIANA IN ALEXANDRIA, EGYPT

K. MOURSI, H.A. MESBAH, A.K. MOURAD, A.S. ABO-SHANAB,

Nagda AHMED EL-SAYED & S. ABDEL-FATAH, RASHA Faculty of Agriculture, Saba- Basha, Department of Plant Protection

Alexandria University, Alexandria, Egypt Plant Protection Research Institute, Agricultural Research Center, Alexandria, Egypt

Two monophlebid species were recorded infesting A. wilkesiana in Montazah gardens in Alexandria, Egypt during the period of study. They were identified as; Icerya purchasi (Maskell) and I. seychellarum ( Westwood). The estimated values of total counted numbers of the two monophlebid species per five shrubs in the successive years (2005 – 2006 & 2006 – 2007) exhibited the highest occurrence rates of mealy bugs population during October & November. These highest rates were decreased during December, January and February. But, While, the lowest population density was observed during the Spring months (March, April and May) and early Summer (June and July) in the two subsequent years. The highest values of Quotient of increase during August occurred at 26°C and 27°C, R.H. 73 and 68%, Dew point 22°C and 22°C and Wind speed 6 and 11m/sec for the first and second year, respectively. The high population density of I. seychellarum gradually decreased during Summer and Autumn months reaching a comparative and somewhat low calculated percentages of the total count/month during the following Winter months: December, January and February. On the other hand, the other inspected species, I. purchasi, the data showed its complete disappearance during Spring months on Acalypha wilkesiana leaves. Key words: Icerya purchasi (Maskell) and I. seychellarum ( Westwood);Acalypha wilkesiana

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E04 POPULATION DYNAMICS OF PSYLLIDS IN AN

APPLE ORCHARD AND THEIR INFECTIVITY WITH APPLE PROLIFERATION

G. PEUSENS1, C. DUCHÊNE3, P. LEPOIVRE2 & T. BELIËN1

1 Proefcentrum Fruitteelt vzw, Fruittuinweg 1, BE-3800 Sint-Truiden 2 Unité de Phytopathologie, Faculté des sciences agronomiques, Université de Liège

Passage des Déportés 2,BE-5030 Gembloux 3 Université de Liège, Gembloux Agro-Bio Tech, Unité de Phytopathologie

Avenue Marechal Juin, 13 BE-5030 Gembloux In apple trees an infection of apple proliferation can cause severe damage and economic loss to the crop. As some psyllid species are able to transmit this phytoplasma from infected trees to healthy trees within and between orchards, their population was investigated in 4 apple orchards from March till August. Marked trees where sampled on a 2‐weekly basis by the beating tray method and sticky yellow plates. After collection of the insects they were identified, numbered and their infectivity was checked by transmission trials and/or PCR analysis. Transmission was tested through exposure of individual insects to an artificial feeding source at different intervals. In addition hedges of hawthorn nearby apple orchards were also monitored to reveal the presence of psyllids and their dispersal behaviour. Preliminary results illustrated the presence of 3 important vectors Cacopsylla mali, Cacopsylla melanoneura and Cacopsylla picta in apple trees or/and hawthorn and of Candidatus Phytoplasma mali in psyllids. Key words: psylla, vector, apple proliferation

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E05 CYDIA POMONELLA L., THE CODLING MOTH

IN BELGIUM: SITUATION 2010

E. BANGELS & T. BELIËN Department of Zoology pcfruit vzw

Fruittuinweg 1, BE-3800 Sint-Truiden, Belgium The codling moth, Cydia pomonella L. (Tortricidae), is a key pest in Belgian apple and pear orchards. Analysis of more than 50 years of historical life cycle data revealed that the active part of the life cycle has elongated since the late eighties until 2005. The flights are starting earlier in spring and last longer in summer, thus requiring more insecticide applications to cover the full flight period. This analysis is now updated until 2010, the year of a general and subsidized introduction of mating disruption techniques in the Flemish part of Belgium. Recently, new insecticides have been introduced for the control of the codling moth. On top, mating disruption camouflages our monitoring trap catches and does not only disrupt codling moths but also growers in the field. An overview of catches in more than 70 codling moth pheromone traps under diverse conditions is presented. Pcfruit, department of Zoology has a long tradition of executing insecticide field trials according to EPPO guidelines. An overview of recent efficacy values out of field trials with currently registered products against codling moth is shown. The choice of products is determined by their true efficacy in the field, legal restrictions and the potential development of resistance or cross‐resistance to active ingredients with similar modes‐of‐action. Recently also residue risks and side effects on beneficial arthropods more and more reach the centre of attention. Key words: Life Cycle, Cydia pomonella, pheromone traps, mating disruption, biological efficacy

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E06 A NEW PEST OF SUNFLOWERS IN EUROPE –

DISCOVERY OF STRAUZIA LONGIPENNIS IN BERLIN

S. LERCHE1, P. BAUFELD2, B. KUMMER3, T. SCHOBER4 & C. BÜTTNER1 1 Humboldt-Universität Berlin, Faculty of Agriculture and Horticulture

Dept. Phytomedicine, Germany 2 Julius Kühn-Institut, Federal Research Centre for Cultivated Plants

Institute for National and International Plant Health, Germany 3 Plant Protection Office Berlin, Germany

4 Humboldt-University Berlin, Faculty of Agriculture and Horticulture Dept. Urban Ecophysiology of Plants, Germany

Brückner & Kornveyev (2010) reported on the first appearance of Strauzia longipennis Wiedemann 1830 (Diptera: Tephritidae) in the palearctic region. This species is native in North America (Foote et al. 1993). The catch of flies from Brückner & Kornveyev (2010) in Berlin‐Johannisthal is not a single finding. Four additional adults of the species were caught in Berlin‐Wartenberg in the same month. Subsequently, the existence of another specimen was known, found in Berlin‐Lankwitz in the year 2009. The locations of the findings are between 11 and 21 km apart from each other. To determine the actual infestation situation of the pest species, a specific monitoring is necessary within the city as well as within the adjacent federal state Brandenburg. With the oviposition by female adults on sunflowers being observed as well as the successful development of the larvae within the plant and their pupation under ambient conditions in the field, an establishment of the species in Berlin seems possible. Brueckner & Korneyev (2010): Strauzia longipennis (Diptera: Tephritidae), an important pest of sunflowers recorded for the first time in the Palaearctic Region. Ukrainska Entomofaunistyka 2010, 1(1): 55‐57 Foote, R.H., Blane, F.L. & A.L. Norrbom (1993): Handbook of the fruit flies (Diptera: Tephritidae) of America North of Mexico. Ithaca, Comstock Publishing Associates, USA: 571 Key words: Diptera, Tephritidae, Strauzia longipennis, sunflower maggot, Germany

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E07 FIRST REPORT OF CYDALIMA PERSPECTALIS

(LEPIDOPTERA:CRAMBIDAE) IN BELGIUM

H. CASTEELS1, J. WITTERS1, S. VANDIERENDONCK1

& L. VAN REMOORTERE2 1Institute for Agricultural and Fisheries Research (ILVO)

Burg. Van Gansberghelaan 96, BE-9820 Merelbeke, Belgium 2Research Centre for Ornamental Plants

Schaessestraat 18, BE-9070 Destelbergen, Belgium The box tree caterpillar Cydalima perspectalis (Walker, 1859) is one of the most destructive insect pests of box in Asia, with reports in China, South Korea and Japan. The larvae feed on leaves and shoots and severe infestations can lead to almost complete defoliation of the plants. This invasive alien species was first detected in Europe in Weil‐am‐Rhein in Baden‐Würtemberg (Germany) in 2007. Since its introduction, the insect of the EPPO Alert List, has spread at a rather rapid pace in Europe. Box is widely planted as shrub in gardens and parks, where it is commonly used in hedges, for topiary work and as terrace plant. The introduction of this new pest in Belgium could not only present a threat to private gardens and public green, but also to nurseries. Due to the occurrence of the box tree pyralid in some European countries, phytosanitary authorities ask for data concerning the presence of C. perspectalis in Belgium through a monitoring project called FYQUARSTAT (financed by the Federal Public Service for Health, Food Chain Safety and the Environment). The monitoring program started in October 2009. Monthly observations were done in several box nurseries. During these observations we looked for batches of eggs, larvae, pupae and damage on the plants. Adults were monitored using pheromone traps. A technical sheet was also distributed to more than 700 members of the “Observation and Warning System for Nursery Stock and Landscape Gardening” of the Research Centre for Ornamental Plants (Destelbergen). This technical sheet both gave information on the insect and requested information from the sector. The first occurrence of the box tree caterpillar was registered in a private garden in Kapellen (Antwerp) in May 2010; in August we detected caterpillars in five private gardens in the surroundings of Ghent. A total of three adults were caught in amateur light traps, one in Stokkem (Limburg) and two in Nieuwrode (Brabant). Despite the limited number of locations where larvae and adults have been found and the chemical eradication of those caterpillars, the box tree pyralid is expected to be established in Belgium in the near future. Key words: Cydalima perspectalis, box, box tree caterpillar

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E08 MONOPHAGOUS ARTHROPODAL PESTS ON REED

(PHRAGMITES AUSTRALIS (CAVAN) TRIN EX STEUDEL)

Edit HORVÁTH, Zsolt MARCZALI & Éva LEHOCZKY University of Pannonia Georgikon Faculty, Institute of Plant Protection

Department of Applied Zoology HU-8360 Keszthely, Deák Ferenc st. 57., Hungary

Continuous decay of reedy areas is a worldwide known problem nowadays. The European Union has just declared reed‐beds ecologically sensitive areas. Reed‐beds play a considerable role in stabilizing the sediment and providing nutrients for a number of heterotrophic species. In addition to it, it has significance from a human point of view as well. There are 80000 hectares covered by reed in Hungary, which means 0.8% of the total Hungarian countryside. Our homeland has Europe's second largest reed‐bed which is located in the district of Lake Fertő. The most protected reeds are on the Small‐Balaton region on 2000 hectares. In order to prevent the eutrophisation processes at Lake Balaton a Small‐Balaton Water Quality Protection System was established and the Small‐Balaton landscape protection area was founded in 1986. The aim was to restore the former marsh in this protected zone. Several examinations were carried out in Hungary studying the sediment of the reeds, its plant communities and plant chemistry, its ecophysiology and morphology of. Just a few researches were aimed at studying the arthropod fauna of reeds. Researches with pests (especially with the legless reed mealy bug (Chaetococcus phragmitis) were accomplished within the framework of the Institute of Plant Protection in Keszthely. In Hungary there are 39 monophagous reed consumer species. These are 3 Acari, 7 Auchenorrhyncha, 1 Coccinea, 3 Coleoptera, 12 Lepidoptera and 15 Diptera species. One of my aims is the description of the gall gnet which was found by our researchers furthermore the better cognition of insects with a sticking‐sucking mouth organ on the reed. Primarily the phytophagous bugs occurring on Small ‐ Balaton area which I wish to examine. In the course of my watch I examine the swarming the nourishment and the injury of the species. In the growing season we are looking for the harmful species by netting with the collection of the plant samples in a whole year and we examine their biological cycle. The insects which can be found on the other parts of the plant we can collect with knocking net and with bugsucking appliance too. This study has been generously supported by TÁMOP‐4.2.2‐08/1/2008‐0018 ‐ entitled as “Liveable environment and healthier people ‐ Bio innovation and Green Technology research atthe University of Pannonia. The project is co‐financed by the European Social Fund with the support of the European Union. Key words: reed, monophagous arthropods

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E09 BUSSEOLA SPECIES COMPLEX (LEPIDOPTERA:

NOCTUIDAE) IN ETHIOPIAN SUGARCANE FARMS: DISTRIBUTION, HOST PLANTS AND NATURAL ENEMIES

Y. ASSEFA ARMIDE Department of Crop Science and Production, Botswana College of Agriculture

University of Botswana Private Bag 0027, Gaborone, Botswana

Surveys sugarcane Estates and in peasant farmer fields in three regional states of Ethiopia conducted in 2003, 2004 and 2007 indicated that Busseola fusca Fuller (Lepidoptera: Noctuidae) and a species of the Busseola phaia Bowden species complex (Lepidoptera: Noctuidae) are the major and most widely distributed sugarcane stem borers in the country. Over 70% of the peasant sugarcane fields visited and two of the three sugarcane Estates were infested by these borers, with the highest levels of infestation (35% and 50%) being in the northern and western part of the country, respectively. The natural enemy complex observed in sugarcane fields was completely different from that previously reported in cereal grain fields of the country. An unknown solitary hymenopteran larval parasitoid, a solitary dipteran parasitoid, Linnaemya sp. (a solitary dipteran pupal parasitoid), an Entomophthora fungus and Bacillus thuringiensis were recovered from the two Busseola species in sugarcane and Pennisetum purpureum (Napier grass). Agronomic practices that may contribute to the colonization of sugarcane by the borers are discussed. Key words: Busseola fusca, Busseola phaia, sugarcane, natural enemy, stem borer

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E10 DATA ON THE DIAPAUSE OF COTTON

BOLLWORM (HELICOVERPA ARMIGERA HBN.)

Edit HORVÁTH, Péter BALOGH, Lynette ECHESSA & Zsolt MARCZALI 1University of Pannonia Georgikon Faculty, Institute of Plant Protection

Department of Applied Zoology HU-8360 Keszthely, Deák Ferenc st. 57., Hungary

Cotton bollworm (Helicoverpa armigera Hbn.) is one of the most highly polyphagous pests of many economically important crops in the world. It is spread in the Mediterranean regions of Africa, Asia, Australia but it has already occupied the temperate zones of Europe. It was a pending question whether it is able to overwinter in Hungary or not. Therefore, our study was aimed at studying the minimum temperature at which the overwintering pupae of the Helicoverpa armigera can survive. Helicoverpa armigera larvae were collected from a hybrid cornfield and were reared in the laboratory on pepper fruit at the University of Pannonia Georgikon Faculty, Institute of Plant Protection in Keszthely until their pupation. The developed pupae where then kept at a temperature of 20°C and the temperature was gradually reduced to 8°C before subjecting them to cold treatments. The pupae were then transferred to a special laboratory in and placed into a thermostat, where they were subjected to varying low temperatures for different duration of time. They were divided into 5 groups with 16 specimens per group and there was a control group, which was kept at the constant temperature of 8°C. After the cold treatments the pupae where transferred back to Keszthely, where under 25°C degrees in a climate chamber the mortality was examined. For the statistical evaluation, two replications were produced from each treatment with random choosing and then 2way ANOVA analyses were carried out to find out whether the treatments caused statistically significant mortality in the diapausing pupae. The highest mortality was observed at 7days at ‐9°C though it indicated that about 30% of the pupae could be able to survive for over one week at the very low temperature of‐ 9°C. No mortality however was observed in the control group. The lowest mortality percentage was measured at ‐6°C for three days showing that at this temperature H. armigera is able to survive longer and the same was observed in the next treatment where the duration was 7 days. This study has been generously supported by TÁMOP‐4.2.2‐08/1/2008‐0018 ‐ entitled as “Liveable environment and healthier people ‐ Bio innovation and Green Technology research at the University of Pannonia". The project is co‐financed by the European Social Fund with the support of the European Union. Key words: cotton bollworm, Helicoverpa armigera, diapause, cold treatment

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E11 FIELD EXPERIMENTS ON MANAGEMENT OF THE TOMATO BORER TUTA ABSOLUTA (MEYRICK) IN

SARDINIAN TOMATO GREENHOUSES

M. NANNINI, F. ATZORI, F. CHESSA, F. FODDI, G. MURGIA, R. PISCI & F. SANNA

DIRVE, Agris Sardegna, Cagliari, Italy Since its introduction into Sardinia, the tomato borer Tuta absoluta (Meyrick) has become the most serious threat to the island’s open field and greenhouse tomato crops. Several insecticides have been shown to be successful against the pest, but those regarded as the most effective, e.g. spinosad, are frequently overused by growers, increasing the risk of selecting insecticide‐resistant populations. To evaluate the efficacy of products having different modes of action to be used in insecticide rotation programmes, we tested selected biorational and conventional insecticides authorized for use on greenhouse tomatoes in Italy. A few products not yet registered for this crop, as well as a vegetable oil adjuvant, were also tested. Moreover, we investigated the correlation between pheromone trap catches of T. absoluta adults and larval infestation in commercial tomato greenhouses, and evaluated the reliability of action thresholds for tomato borer management based on the number of adults caught per week in pheromone traps. Two to four foliar applications of neem‐based products reduced pest infestation by 65‐72% compared to the untreated controls, whereas a single spinosad application showed 85% efficacy. The efficacy of insecticides based on different subspecies of Bacillus thuringiensis Berliner, sprayed four times at weekly intervals, varied widely from around 35 to 70%. Two foliar treatments of abamectin, indoxacarb and metaflumizone reduced infestation by approximately 70%, significantly less than two spinosad applications (96%). The addition of a vegetable oil adjuvant to some standard products for tomato borer control enhanced their efficacy. Finally, a double application of a commercial formulation of chlorantraniliprole, not authorized for tomatoes, proved similar in efficacy to spinosad. The use of a decision support tool based on pheromone trap catches of T. absoluta adults was successful in controlling the pest. In the two crops surveyed density did not exceed 1.7 live larvae per plant. However, strict application of the proposed thresholds resulted in a very intense, and hence far from economically sustainable, use of insecticides. The weak correlation observed between the number of adults caught in pheromone traps and larval infestation raises questions about the suitability of trap catches for reliably estimating tomato borer density and determining action thresholds for control of the pest. Key words: action thresholds, insecticide efficacy assessment, integrated pest management , pheromone traps

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E12 POTENTIAL OF MACROLOPHUS PYGMAEUS (RAMBUR)

(HETEROPTERA: MIRIDAE) FOR TUTA ABSOLUTA (MEYRICK) CONTROL IN GREENHOUSE TOMATOES

M. NANNINI, F. ATZORI, G. MURGIA, R. PISCI & F. SANNA

DIRVE, Agris Sardegna, Cagliari, Italy The introduction into Mediterranean countries of the tomato borer Tuta absoluta (Meyrick) has led to a considerable increase in recent years in the use of insecticides in greenhouse tomato production, raising concern about the economic and environmental sustainability of chemical pest control. The search for alternatives to insecticide treatments has prompted studies into the role of native predators and parasitoids for management of this new invasive pest. Among the species surveyed, the mirid Macrolophus pygmaeus (Rambur) has been shown to feed on immature stages of the tomato borer, and is presently recommended for T. absoluta control. To gather more detailed information about the potential of this biocontrol agent for tomato borer management, we evaluated the impact of different densities of the mirid bug on pest infestation under semi‐field conditions, as well as the effect on T. absoluta control of the occurrence on plants of another prey. Furthermore, we tested the efficacy of releasing 2 M. pygmaeus/m2 into 10 commercial tomato greenhouses located in southern Sardinia (Italy). The release of 10 mirids per plant significantly affected tomato borer infestation regardless of other experimental conditions. When lower predator densities were tested, pest control results seemed to be influenced by the density and age composition of T. absoluta infestation and seasonal climatic trends. On the other hand, the occurrence of the sweetpotato whitefly Bemisia tabaci (Genn.), a natural prey of M. pygmaeus, proved to adversely affect tomato borer control, resulting in higher levels of infestation. In commercial protected tomato crops predatory mirids reached a maximum density of 1.4 individuals per plant. Furthermore, most of the specimens detected were identified as Nesidiocoris tenuis (Reuter), another zoophytophagous mirid which frequently colonizes tomato crops in this area. Notwithstanding the low mirid population levels achieved in the greenhouses where the beneficials were released, at the end of the growing period significantly lower levels of T. absoluta infestation were detected in these crops than in the controls where no predators were artificially introduced. Therefore it cannot be ruled out that this result was due to a certain extent to factors other than the use of biocontrol agents, e.g. differences in the use of insecticides. While the poor establishment of M. pygmaeus in some crops was caused by treatments with abamectin, in most cases the delay observed in mirid population build‐up was likely determined by biological factors (e.g. scarce prey) and growing practices (e.g. intense plant deleafing) known to adversely affect crop colonization by the predator. Key words: biological control, Dicyphinae, natural enemies, predators, zoophytophagous mirids

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E13 WITHIN-PLANT DISTRIBUTION OF MACROLOPHUS

PYGMAEUS (RAMBUR) (HETEROPTERA: MIRIDAE) AND ITS EFFECTS ON PREDATOR ESTABLISHMENT IN

TOMATO CROPS

M. NANNINI, F. FODDI, R. PISCI &F. SANNA DIRVE, Agris Sardegna, Cagliari, Italy

Macrolophus pygmaeus (Rambur) (Heteroptera: Miridae) is a zoophytophagous species native to the Mediterranean area, used on greenhouse crops for the biological control of whitefly and other pests, including the tomato borer Tuta absoluta (Meyrich). Since the cost of this beneficial limits its rate of introduction to a few individuals per m2, e.g. 1‐2 mirids/m2 in Sardinian tomato greenhouses, a good establishment of the predator in the crop and a rapid population build‐up are generally necessary to achieve effective pest control. However several factors, such as poor fitness of the beneficials released, low prey availability, occurrence of pesticide residues on crops, and also severe leaf removal from tomato plants, are known to adversely affect mirid population growth. To investigate the relation between within‐plant distribution of M. pygmaeus and the effects of plant deleafing on predator establishment in tomato crops, observations were carried out under semi‐field and field conditions. The proportion of recently emerged M. pygmaeus nymphs (< 3‐day‐old) located on the 4 oldest leaves of tomato plants was found to be markedly influenced by plant size during oviposition, duration of oviposition and within‐plant location of prey. The time of year the predator was released was not found to have any direct effect on the distribution of young stages. However, in winter egg eclosion took approximately 3 times longer than in autumn (at least 41‐49 against 14‐16 days, respectively), increasing the risk of removing mirid eggs and neonate nymphs along with the basal leaves during deleafing operations. In fact, the removal of cotyledons and the 4 oldest leaves carried out one month after M. pygmaeus release (i.e. about 2 weeks after and 2 weeks before the beginning of egg eclosion, respectively in autumn and winter) resulted in a reduction of predator progeny, compared to the controls, of 29% when the mirid was released in October and 71% when released in December. Observations carried out in commercial greenhouses showed a non‐negligible proportion of young nymphs of the predator to be located in the lower leaf layer of tomato plants, suggesting that also under field conditions deleafing may have an impact on the abundance of the mirid bug. Key words: biological control, greenhouse tomatoes, natural enemies, plant deleafing, predators

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170

E14 ALTERNATIVE FOOD SOURCES FOR ENHANCING THE

ESTABLISHMENT OF MACROLOPHUS PYGMAEUS (RAMBUR) (HETEROPTERA: MIRIDAE) ON

GREENHOUSE TOMATOES

M. NANNINI, F. ATZORI, F. FODDI, G. MURGIA, R. PISCI & F. SANNA DIRVE, Agris Sardegna, Cagliari, Italy

Over the last two decades, the use of Miridae Dicyphinae for the biological control of pests in greenhouse crops has become common practice in several European countries. Notwithstanding their zoophytophagous feeding habits, which play a role in sustaining mirid populations when prey abundance is low, adequate animal food sources need to be available to ensure good biological fitness for these predatory bugs. In fact, low prey density is regarded as a factor determining the poor establishment and delayed population build‐up of Macrolophus pygmaeus (Rambur) on greenhouse tomatoes. Similar constraints are encountered in the use of the mirid bugs in protected tomato crops grown in Sardinia (Italy), where inoculative releases of the predator frequently result in inadequate population growth and poor control of target pests. An investigation has been conducted to explore the possibility of enhancing the establishment of M. pygmaeus in tomato greenhouses by supplying the predator with Ephestia kuehniella (Zeller) eggs as alternative food source. Experiments were conducted under controlled, semi‐field and field conditions. Since in some horticultural districts of the island the use of floating row covers has become fairly common practice for tomato pest and disease control, the effects of releasing the mirid bug beneath non‐woven fabrics were also studied. The mirids, kept in a climate cabinet at 23°C and fed with 8 mg of flour moth eggs per mirid per week, showed increased fertility and lower mortality rates than unfed controls. Similarly, on tomato plants grown in a glasshouse the number of nymphs produced by predators supplied with the fictitious prey was greater than that of non‐supplementary‐fed colonies. This was especially true of commercially mass‐reared bugs, whose reproductive performances were significantly enhanced when fed with Ephestia kuehniella eggs. However, when in commercial greenhouses the same amounts of eggs were supplied for 4‐6 weeks at the mirid bugs’ release points, no significant improvement was noted. The experiments for testing M. pygmaeus release under a non‐woven fabric showed that when the predator was confined to the same plants on which supplementary food was supplied, it generated a greater number of progeny than when it was allowed to move from release points. Further studies are needed to evaluate the impact of the supply of alternative food sources during early establishment of the predator on the overall build‐up of mirid populations and on pest control. Key words: biological control, Ephestia kuehniella, fictitious preys, natural enemies, predators

Posters E01-E46

171

E15 A SURVEY OF NATURAL ENEMIES OF WHITEFLY

(HOMOPTERA: ALEYRODIDAE) ON SELECTED WEEDS IN A MEDITERRANEAN TOMATO PRODUCTION AREA

M. NANNINI, F. CORDA, F. FODDI, L. MANCA, G. MURGIA,

R. PISCI & F. SANNA DIRVE, Agris Sardegna, Cagliari, Italy

A three‐year survey of natural enemies of the whiteflies Trialeurodes vaporariorum (Westw.) and Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) has been carried out on uncultivated host plants in a major greenhouse tomato production area in southern Sardinia (Italy). The occurrence of the two whitefly species and their indigenous enemies on selected weeds (5 plant species in 2005, and 3 in 2006/7), were evaluated in two adjacent farms and the impact assessed of beneficial insects on pest populations, by both insecticide interference and exclusion experiments. While the population trends of whitefly and predators were estimated monthly by vacuum samplings, the occurrence of parasitoids, as well as whitefly species composition and mortality rates, were assessed on leaf samples. The most frequently collected predators during the survey were mirid bugs of the genus Macrolophus, probably M. melanotoma (Costa), which were found to be generally associated with Dittrichia viscosa (L.). Actually, whereas on this plant the predator reached relatively high densities in spring (up to approximately 20 individuals per plant for a suction time of 10 seconds), it was only detected sporadically on the other host plants examined, even if heavily infested by whitefly. Nesidiocoris tenuis (Reuter), another mirid bug of the subfamily Dicyphinae, was found mainly in summer on Solanum nigrum L. (up to 6 individuals per plant), but also on D. viscosa, Conyza and Sonchus spp.. Coccinellid beetles were collected in non negligible numbers on the above‐mentioned hosts: among these, Clitostethus arcuatus (Rossi) is known to be a predator of whitefly eggs and nymphs. Also anthocorid bugs and lacewings were caught occasionally. As concerns whitefly parasitoids, Encarsia pergandiella Howard was found to be the predominant species throughout the entire survey period, along with Encarsia mineoi Viggiani and Eretmocerus mundus Marcet. The experiments carried out on Conyza bonariensis (L.) Cronquist, a common weed in the study area, to estimate the impact of biological control by natural enemies on whitefly abundance, showed significantly higher densities of the pests on plants in exclusion cages, or treated with bifenthrin, than on control plants. However, these outcomes did not appear to be fully supported by data on whitefly mortality and beneficials abundance, and it cannot be ruled out that factors other than natural enemy suppression (e.g. hormoligosis) may have contributed to a certain extent to the resurgence of whitefly populations observed on plants sprayed with bifenthrin. Key words: Bemisia tabaci, biological control, parasitoids, predators, Trialeurodes vaporariorum

Posters E01-E46

172

E16 FIRST REPORT OF THE PREDATOR FLY

COENOSIA ATTENUATA IN LETTUCE AND CELERY CROPS IN BELGIUM

J. WITTERS1 , I. LEENKNEGT2, H. CASTEELS1& R. DE VIS2

1 Institute for Agricultural and Fisheries Research (ILVO) Burg. Van Gansberghelaan 96, BE-9820 Merelbeke, Belgium

2 Research Station for Vegetable Production Duffelsesteenweg 101, BE-2860 St-K-Waver, Belgium

Coenosia attenuata (Diptera:Muscidae), a paleotropical insect, was recently found by the authors in a lettuce nursery in Belgium. According to literature this predator fly could play an important role in controlling pests like whitefly, leaf miners (Diptera), shore flies and fungus gnats. In 2010 a monitoring program was carried out to study the occurrence of C. attenuata in the production areas of lettuce and celery. By using yellow sticky plates the presence of C. attenuata, leaf miners, fungus gnats and shore flies were examined. In every monitored greenhouse C. attenuata was detected which indicates that this hunter fly seems to be established and widespread in the production areas in Belgium. In addition to the monitoring results, preliminary data of the population dynamics of C. attenuata, fungus gnats and shore flies in celery and lettuce crops will be given. Key words: Coenosia attenuata, population dynamics, hunter fly, fungus gnats, leaf vegetables

Posters E01-E46

173

E17 DIVERSITY OF INSECT PARASITOIDS AND

PREDATORS OF THE BAGWORM, PTEROMA PENDULA (LEPIDOPTERA: PSYCHIDAE) IN RELATION TO

THE ENVIRONMENT, WITHIN AN OIL PALM PLANTATION IN PERAK, MALAYSIA

Norman KAMARUDIN & Othman ARSHAD

Biology Division, Malaysian Palm Oil Board, No 6, Persiaran Institusi, Bandar Baru Bangi 43000 Kajang Selangor, Malaysia

Bagworms are one of the important leaf‐eating pests of oil palm in Malaysia and Indonesia. Crop losses, due to the extent of defoliation by a serious bagworm attack is inevitable. A moderate defoliation of about 10‐13% may cause a crop loss of about 33‐40%. Parasitoids play an important role in regulating bagworm numbers. The propagation of the insect natural enemies depend on species of flowering plants as sources of nectar, which prolong the life span of the adults parasitoids. A leguminous nectar procucing shrub, Cassia cobanensis, was deemed suitable for propagation of natural enemies due to its attractiveness to most bagworm parasitoids. Environmental parameters such as light intensity, temperature and relative humidity were measured during the sampling of insects via sticky traps and sweep net. Sticky traps tend to measure activities of flying insects whereas sweep net tends to capture the resident population of insects within a certain habitat. Samplings were conducted on the C. cobanensis plant, within the planting block where C. cobanensis was planted at the roadside and a control block, devoid of C. cobanensis within its vicinity. The overall diversity of insect natural enemies occurring on the C. cobanensis plant was quite high (Shannon‐Wiener diversity index: 2.32). However, within the harvesting paths of the C. cobanensis block, the diversity index was reduced to 1.09. In the control plot, the diversity index was only slightly higher (1.12) than the C. cobanensis block (1.09) however, much lower than on the C. cobanensis plant (2.32). Similarly, the captures of insect parasitoids were also low (less than 5 individuals per species) within the control block, in the absence of C. cobanensis plant. The activities of insect natural enemies were seen highest at medium light intensities of between 2000‐4999 fc, with the highest diversity index (2.38) between 4000‐4999 fc. In contrast, at low range light intensity (1000‐1999 fc), natural enemies were mostly inactive, indicated by high species richness (9.0) and high diversity index (2.10) in the sweep net captures. Activities of natural enemies were observed higher at humidities of between 60‐79% RH, indicated by higher species richness (16.0‐19.0) and high diversity index (2.51) in the sticky traps samples. Between 70‐89% RH, natural enemies seem to be less active, indicated by higher species richness (7.0) and diversity index (1.84) in the sweep net samples. Activities of the natural enemies were highest between 30‐34 C, shown by the high species richness (20.00) and diversity index (2.68) in the sticky trap samples. In contrast, at lower temperatures of between 25‐30 C, most natural enemies became less active, as indicated by high species richness (9.00) and high diversity index (2.06) in the sweep net samples. This study provides better understanding on the conducive environment for the diversity and activities of the bagworm parasitoids in the oil palm environment. Key words: Oil Palm, Bagworm, Cassia cobanensis, natural enemies, species richness, diversity index.

Posters E01-E46

174

E18 FIELD EVALUATION OF SURVIVAL RATES OF

HARMONIA AXYRIDIS (COLEOPTERA: COCCINELLIDAE) AND OTHER APHIDOPHAGOUS SPECIES

Axel VANDEREYCKEN, Delphine DURIEUX, Emilie JOIE,

Eric HAUBRUGE & François J. VERHEGGEN Entomologie fonctionnelle et évolutive Liege University

Gembloux Agro-Bio Tech Passage des Déportés 2, BE-5030 Gembloux, Belgium

The multicoloured Asian ladybird Harmonia axyridis Pallas (Coleoptera: Coccinellidae), previously introduced as a biological control agent against aphids, is now frequently considered as an intraguild predator, consuming other aphids natural enemies. Interactions between this exotic ladybird and the other aphidophagous species present in Belgian agro‐ecosystems such as Coccinella septempunctata, Epysirphus balteatus, Chrysoperla carnea are asymmetric to the benefit of H. axyridis. Many studies have already shown its predatory behaviour. This study focuses on the survival rate of three aphidophagous species Harmonia axyridis (Ha), Coccinella septempunctata (C7) and Epysirphus balteatus (Eb) in biological fields of potato and sugar beet. During June and July, hermetic cages were disposed on the crops with 10 larvae (second stage) of each aphidophagous. There were 4 combinations of insects placed inside the cages (1) Ha+Eb+C7+ aphids, (2) Ha+Eb+C7, (3) Eb+C7+ aphids and (4) Eb+C7. Each combination was repeated three times. Aphids (Aphis fabae) were added in excess when needed. In sugar beet fields, E. balteatus was the species with the highest mortality rate. The two coccinellid species had a better survival rate than E. balteatus. All aphidophagous species had higher mortality rates in absence of aphids. In potato, the survival rate of all aphidophagous was lower than sugar beet when aphids were present in excess probably because A. fabae reproduced less in potato. Nevertheless, we have observed in potato no mortality of C. 7‐punctata during all its development cycle. This suggests that C. 7‐punctata could use more efficiently aphids as food sources than H. axyridis. In potato and sugar beet, H. axyridis had the longest development cycle, being still at the larval stage when C. 7‐punctata and E. balteatus reached the pupae stage. We can suppose that pupae of E. balteatus and C. 7‐punctata were an alternative food (intraguild prey) to H. axyridis when aphids were lacking. Pupae are immobile and therefore more sensitive to the attack of predators. Key words: Interspecific interaction, multicoloured ladybird, Coccinella septempunctata, Epysirphus balteatus

Posters E01-E46

175

E19 PREDOMINANT EFFECTS OF HOST PLANT SPECIES ON

APHIS GOSSYPII APHID AND EPISYRPHUS BALTEATUS HOVERFLY IN A TRITROPHIC APPROACH

René-Noel POLIGUI, Frédéric FRANCIS & Eric HAUBRUGE

University of Liege, Gembloux Agro-Bio Tech, Passage de Deportes 2, BE-5030 Gembloux, Belgium

The aphid Aphis gossypii Glover (Homoptera: Aphididae) is known as worldwide important pest. It causes serious injuries to crops, but is also able to feed on various cultivated and wild plants. Some programs are developped to control this aphid. In many studies, hoverfy Episyrphus balteatus De Geer (Diptera: Syrphidae) is used as an efficient and aphid specific predator in biological control systems. In this work, we study cotton aphid choice plant discrimination, the effect of host plants on its developpment (fecondity and mortality), and the E.balteatus foraging and oviposition behavior according aphid host plants. Biologic parameters were observed using various plant combinations. E.balteatus behavioral observations were conducted using the Noldus Observer v.5.0. A.gossypii makes discriminant choice of his hosts, with better preference on Cucurbita pepo L. (Cucurbitaceae) and Capsicum annuum L. (Solanaceae). Dacryodes edulis (G. Don) H.J. Lam. (Burseraceae) and Vicia faba L. (Fabaceae) are the midle choice while Hibiscus sabdariffa L. (Malvaceae) is less chosen. A.gossypii develops easily on C.pepo, and weakly on C.anuum, H.sabdariffa,and V.faba. D. edulis induces very high mortality to A.gossypii. According to E. balteatus behavior, A. gossypii is more attractive when infesting C.pepo and V.faba, less on H. sabdariffa. While non infested, the hoverfly more attractive plant is V.faba. Our results highlited the importance of interactions between aphids and their host plants and the prey‐host influence on a specific aphid predatory hoverfy. Suitable ways in correlation with this work are to investigated to complete this study and perform aphids biological control. Key words: Aphids, Hoverfly, host plants, prey‐host influence, biological control

Posters E01-E46

176

E20 STUDY OF THE INTERACTION BETWEEN PLANT

AND GREEN PEACH APHID (MYZUS PERSICAE SULTZER) BY A PROTEOMIC APPROACH

William LUWAERT1, Marc ONGENA2, Jacky DOMMÈS3, Edwin DE PAUW4,

Eric HAUBRUGE1 & Frédéric FRANCIS1 Unit of Functional and Evolutionary Entomology

Gembloux Agro-Bio Tech, University of Liège, Belgium Interactions between plants and insects are numerous, complex and varied. Indeed, feeding behaviour of many insects is directly linked with defensive compounds in host plants. Until now, most studies are based on plant defence mechanisms associated with chewing insects. According to a theory, each species of herbivore insects produces its own particular molecular signature, especially concerning saliva. The role of insect saliva is crucial concerning establishment of defence mechanisms in plants because composition of saliva allows plants to recognize insects. Up to now, only few studies focused on the identification of elicitors in aphid saliva. Moreover, aphids are considered phenotypically closest to plant pathogenic fungi and bacteria to other chewing insects. For this study, we used a proteomic approach to analyse differentially expressed proteins in plants after different infestation periods by green peach aphid Myzus persicae. We investigated the proteome of two different plant models: Arabidopsis thaliana and Nicotiana tabacum. We first considered effect of aphid saliva on cell suspension and also effect of aphids on entire plants. Proteomes of healthy and infested plants are compared with 2D‐DIGE, a 2 dimensions electrophoresis method that allows quantitative comparison of protein samples. Following this work, we observed a differential protein expression in A. thaliana and in N. tabacum after M. persicae infection. Identification of proteins is in progress and other experiments will be carry out to compare responses and proteomes between different species and different supports. Key words: Protein expression, Defense mechanisms, Particular molecular signature

Posters E01-E46

177

E21 EFFECTS OF SPIRODICLOFEN AND HEXAFLUMURON ON SOME PHYSIOLOGICAL ASPECTS OF LADYBIRD,

HIPPODAMIA VARIEGATA (GOEZ) (COL: COCCINELLIDAE) LARVAE UNDER LABORATORY

CONDITION

N. ALIMOHAMMADI, M.A. SAMIH & H. IZADI Department of Plant Protection, Faculty of Agriculture

Vali-e-Asr University, Rafsanjan, Iran The Ladybird, Hippodamia variegata is one of the most important natural enemies of common pistachio psylla, Agonoscena pistaciae Burckhardt and Lauterer (Hem: Psyllidae). Pesticides application has indirectly indirect effects on physiology and behaviors of predators. In this research, the side effects of insecticide hexaflumuron and acaricide spirodiclofen were investigated on some physiological aspects (glycogen, glucose and lipids contents) of the 4th instars larvae of H. variegata (Goeze) under controlled condition (26±2ºC, 60‐70٪ RH, 16: 8 L: D). Carbohydrates (glycogen and glucose) and whole body lipids were measured by a method described by Warburg and Yuval (1997). No significant difference in glucose was observed between the larvae treated with these two pesticides and control (F2,12 = 1.53, P = 0.25), but glycogen content in the larvae treated with spirodiclofen and hexaflumuron with 17.42±1.6 and 16.07±2.4 mg/g fresh body weight, respectively was significantly different from the larvae treated with ethanol as control (29.02±2.1 mg/g fresh body weight) (F2,15 = 11.37, P = 0.01). There was no significant difference in total body protein (F2,10 = 2.06, P = 0.175) in the larvae treated with spirodiclofen and hexaflumuron and ethanol as control. There was significant difference between lipids content of spirodiclofen treated larvae (5.29±0.2 mg/g fresh body weight) and control (7.89±0.4 mg/g fresh body weight) whereas there was no significant difference between hexaflumuron and control (F 2,11 = 6. 19, P = 0.016). Key words: Agonoscena pistaciae, Carbohydrates, hexaflumuron, Hippodamia variegata, lipid, spirodiclofen

Posters E01-E46

178

E22 INVESTIGATION ON EFFECT OF FUMARIA PARVIFLORA

AND TEUCRIUM POLIUM EXTRACTS ON BIOLOGICAL PARAMETERS OF BEMISIA TABACI (GENN.) ON TOMATO

UNDER CONTROLLED CONDITIONS

F. JAFARBEIGI1, M.A. SAMIH1, M. ZARABI2 & S. ESMAEILY1 1Department of Plant Protection, Faculty of Agriculture

Vali-e-Asr University, Rafsanjan, Iran 2Department of Plant Protection, College of Plant and Animal Sciences

Abureihan campus of Agriculture, University of Tehran, Iran Cotton whitefly, Bemisia tabaci (Genn.) (Hem.: Aleyrodidae) is a major pest of field crops, vegetables and ornamentals. Development of alternative methods except the chemicals application is necessary for pest management because of human health and safety of environment. Usage of some plant extracts which are toxic for insects is a good way in pest control. In this research, the effect of two plant extracts fineleaf fumitory Fumaria parviflora and germander Teucrium polium studied on biological parameter Cotton whitefly. Each unit consists of two clear plastic glasses (10 cm diameter, 15 cm height). The leaflets treated by plant extracts for 20s and allowed to air dry. The same ages adults released in the upper glass (n=30). Adults gather after 24 hours then life duration of egg, nymph and pupa collected daily. The experiments carried out in a CRD with six replications. All experiments done in controlled condition (27±20°C, 50±5 RH and 16L:8D). Results showed that the effect of plant extracts, was significant on life duration egg (F2, 218= 5.5, P= 0.004), pupa (F2, 114= 5.01, P= 0.008) and egg‐adult (F2, 98= 4.5, P= 0.013) and there was no significant difference on nymphs (F2, 131= 0.55, P= 0.57) in 5% level. Mean of longevity egg and nymph in Teucrium polium with 7.35±0.12, 34.12±0.03 were highest respectively and control with 6.86±0.09, 11.82±0.38 days were least respectively. The mean of longevity pupa and egg‐adult in control and Teucrium polium were highest and the least with 5.78±0.43, 24.92±0.98 and 4.27±0.27, 24.24±0.44 days respectively. Results indicated that germander extract have most number of casualties on egg and nymph stages cotton whitefly. Key words: Biological parameters, plant extract, cotton whitefly, Bemisia tabaci

Posters E01-E46

179

E23 TESTING GARLIC EXTRACT AS A SEMIOCHEMICAL FOR APHIDOPHAGOUS PREDATORS AND APHIDS

H-C. XIE1, P.D. LEROY2, A. VANDEREYCKEN2, J. FARMAKIDIS2,

J. CHEN1, Y. LIU3 & F. FRANCIS2 1 Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS),

West Yuan-ming-yuan Road 2, 100193 Beijing, China 2 Department of Functional and Evolutionary Entomology, University of Liege,

Gembloux Agro-Bio Tech, Passage des Déportés 2, BE-5030 Gembloux, Belgium 3 College of Plant Protection, Shandong Agricultural University

Taian, 271018 Shandong, China Semiochemicals are involved in tritrophic interactions, affecting the behaviours of both the phytophagous and the beneficial insects. Garlic extract is known to act as a repellent for many pests like aphids and so could be used in pest control management. Here, through four arm‐olfactometer and wind‐tunnel (L × W × H = 2.4 × 0.8 × 0.6 m) experiments, we investigated the impact of garlic extract at different doses (0.1, 1 and 10 µg) on two major aphid predators encountered in field crops, the hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) and the Asian lady beetle Harmonia axyridis (Pallas). To determine the effect of garlic extract on a phytophagous insect, the same doses were tested on the pea aphid Sitobion avenae (Fabricus), an important pest in several crops. No repellent effect was noted nor for H. axyridis neither for the aphid A. pisum since individuals were observed to be randomly dispersed into the four arm‐olfactometer. Hoverflies were not repelled and responded to the garlic extract at the 1 µg dose since a higher number of individuals were located on the plants in comparison with the negative control (plant without odour) during the wind tunnel experiments. This study showed that garlic extract did not repel H. axyridis and A. pisum but also that E. balteatus was attracted at a 1 µg garlic extract dose. Key words: Garlic extract, semiochemical, aphid, Syrphidae, Coccinellidae, wind‐tunnel, four arm‐olfactometer

Posters E01-E46

180

E24 A SEMIOCHEMICAL ENHANCING THE

ATTRACTIVENESS OF APHIDOPHAGOUS PREDATORS IN POTATO CROPS

P.D. LEROY1, A. VANDEREYCKEN1, A. SABRI2, S. HEUSKIN3,

F.J. VERHEGGEN1, Q. CAPELLA1, J. FARMAKIDIS1, P. THONART2, G. LOGNAY3, J-P. WATHELET4, Y. BROSTAUX5 & E. HAUBRUGE1

1 Department of Functional and Evolutionary Entomology, University of Liege, Gembloux Agro-Bio Tech, Passage des Déportés 2, BE-5030 Gembloux, Belgium

2 Walloon Center of Industrial Biology, University of Liege, B40, BE-4000 Sart Tilman, Belgium 3 Department of Analytical Chemistry, University of Liege, Gembloux Agro-Bio Tech,

Passage des Déportés 2, BE-5030 Gembloux, Belgium 4 Department of General and Organic Chemistry, University of Liege, Gembloux Agro-Bio Tech,

Passage des Déportés 2, BE-5030 Gembloux, Belgium 5 Department of Applied Statistics, Computer Science and Mathematics, University of Liege,

Gembloux Agro-Bio Tech, Avenue de la Faculté 8, BE-5030 Gembloux, Belgium Aphids are major pests of crops worldwide and the use of pesticides has led to resistant populations. The integration of aphid natural enemies in integrated management programs could be an option, but their efficacy is often limited by their quick dispersal from the ecosystem where they are released. Here, using wind‐tunnel and field experiments, we have demonstrated that 3‐methyl‐2‐butenal acts as an efficient attractant and ovipositional stimulant for the hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae), enhancing its efficiency as biological control agent in crop fields. Wind‐tunnel assays allowed determining that a minimal dose of 250 µg of 3‐methyl‐2‐butenal is required to attract the hoverflies over a distance of 2.5 m while a minimal dose of 500 µg is needed to induce the oviposition. Both attraction and oviposition increased proportionally to the tested doses showing that hoverflies are able to regulate their oviposition according to the chemical stimuli from their environment. In field experiments, both Syrphidae and Chrysopidae were strongly attracted by the 3‐methyl‐2‐butenal at a dose of 10 mg which also induces the oviposition of these two aphid predators in potato crops. This study also highlighted that hoverflies are the most abundant aphid predators found in natural environment. Against aphids, the use of this semiochemical could certainly enhance the efficiency of natural enemies. Furthermore, the use of the chemical cue 3‐methyl‐2‐butenal could provide a novel approach to control aphids in field and greenhouse systems. Key words: biological control, aphids, predators, 3‐methyl‐2‐butenal, attractant, oviposition

Posters E01-E46

181

E25 TESTING Z,E-NEPETALACTONE AS A

POTENTIAL KAIROMONE FOR APHIDOPHAGOUS PREDATORS AND APHIDS

L-L CUI1, P.D. LEROY2, A. VANDEREYCKEN2, J. FARMAKIDIS2,

Y. LIU1, J. CHEN3 & F. FRANCIS2 1 College of Plant Protection, Shandong Agricultural University, Taian, 271018 Shandong, China

2 Department of Functional and Evolutionary Entomology, University of Liege Gembloux Agro-Bio Tech, Passage des Déportés 2, BE-5030 Gembloux, Belgium 3 Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS),

West Yuan-ming-yuan Road 2, 100193 Beijing, China Semiochemicals from aphids are known to act as kairomones for many predatory and parasitic insects, inducing specific behaviours like the active search for prey or the eggs laying. Here, through wind‐tunnel experiments, we investigated the impact of the Z,E‐nepetalactone, the main aphid sex pheromone component, on two aphidophagous predators but also on one aphid specie. In a small glass wind‐tunnel (L x W x H = 80 x 9.0 x 9.0 cm), if the Asian lady beetle Harmonia axyridis Pallas was significantly attracted by this semiochemical at a dose of 1 mg, the aphid Myzus persicae (Sulzer) did not show any behavioural response to the Z,E‐nepetalactone tested at the same dose. Behaviours (attraction and oviposition) of the hoverfly Episyrphus balteatus (De Geer) (Diptera: Syrphidae) were recorded in response to 10 ng, 100 ng, 1 µg and 1 mg doses of Z,E‐nepetalactone in another wind‐tunnel (L × W × H = 2.4 × 0.8 × 0.6 m) highlighting that Z,E‐nepetalactone had no effect on this syrphid. This study showed that two aphidophagous predators respond differently to the major component of the aphid sex pheromone but also that this semiochemical does not attract the aphid M. persicae when tested alone. Key words: Z,E‐nepetalactone, semiochemical, aphid, Syrphidae, Coccinellidae, attractant, wind‐tunnel

Posters E01-E46

182

E26 TRANSMISSION AND DISPERSAL OF PANDORA

NEOAPHIDIS BY EPISYRPHUS BALTEATUS LARVAE

B.M. DIAZ1,3, D. CALVO1,3, M. PLAZA1,3, M.A. MARCOS-GARCÍA2,3

& A. FERERES1,3 1 Departamento de Protección Vegetal. Instituto de Ciencias Agrarias. Consejo Superior de

Investigaciones Científicas. C/Serrano 115 dpdo., ES-28006 Madrid, Spain, 2 Centro Iberoamericano de la Biodiversidad (CIBIO), Universidad de Alicante,

Campus San Vicente del Raspeig, ES-03080, Alicante, Spain 3 Unidad Asociada Universidad de Alicante-CSIC. Grupo de Interrelaciones

Insecto-Patógeno -Planta y sus Agentes de Biocontrol The successful of biological control, to achieve an effective pest supression, is dependent of positive relationships between natural enemies. This is essential when consider specific natural enemies, which share a common and unique extraguild prey, as occurr with the entomopathogenic fungus Pandora neoaphidis (Remaudière & Hennebert) Humber and the aphidophagous syrphid Episyrphus balteatus (De Geer) on aphids. Pandora neoaphidis is a highly specific parasite of aphids, which impede causing direct infection on natural enemies; whereas E. balteatus, is one of the most common syrphid in Europe and its larvae are effective predators of aphids. The aim of this study was to access the role of E. balteatus in the transmission and a passive dispersion of P. neoaphidis within and between colonies of the lettuce aphid, Nasonovia ribisnigri (Mosley). To carry out the experiments, 15 apterous adults of N. ribisnigri were placed on each of 40 four‐leaf lettuce plants. After 1h of aphid settlement, plants were divided in two groups. On twenty plants, five sporulating aphid cadavers were distributed on the inner leaves of the lettuce plants (Treatment), whereas the control plants (20 plants) did not receive sporulating aphid cadavers. Then were selected a half of plants of both, treatment and control, and one E. balteatus larvae (L3) were allowed to forage for 4h. The remaining half of plants did not receive any syrphid larvae. After 4 h, larvae of E. balteatus were removed from all plants and were transferred on a new plant infested with 40 healthy apterous of N. ribinigri for 20 h. The experiment was repeated three times, under the same experimental conditions (T=18ºC, HR= 90±10% and LD 16:8h photoperiod). The consumption of aphids by syrphid larvae was recorded after 4 h of exposition; whereas mortality of aphids due P. neoaphidis, after 4h and 20h of exposition to syrphid larvae, was recorded daily for 7 days. Results indicated that the foraging capacity of E. balteatus larvae did not decreased significantly with the presence of P. neoaphidis cadavers (4.4 ± 0.22 aphids) compared to healthy colonies of N. ribisnigri (5.2 ± 0.17 aphids). N. ribisnigri infection due P. neoaphidis increased 10% with the presence of a syrphid larvae within the aphid colony. Syrphid larvae were able to vector conidia of P. neoaphidis and to begin fungal infection on healthy colonies of N. ribisnigri. The results suggest that the presence of E. balteatus larvae enhance fungal infections of P. neoaphidis, encouraging the promotion of both natural enemies in a conservation biological control strategy, to control N. ribisnigri in a sustainable context. Key words: Biological Control, aphids, Entomophthorales, aphidophagous hoverflies, intraguild relationships

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183

E27 BIOLOGICAL ACTIVITY OF ENTOMOPATHOGENS

AND MUTUALISTIC FUNGAL ENDOPHYTES ON AGROTIS SEGETUM

J. AKELLO, G. SICHTERMANN & R. SIKORA

INRES-Phytomedicine, Soil-Ecosystem, Phytopathology and Nematology University of Bonn, Nussallee 9, DE-53115 Bonn, Germany

The Turnip Moth, Agrotis segetum, (Denis and Schiffermüller) (Lepidoptera: Noctuidae) is an economically important pest of seedlings in greenhouses cultivation and of field crops. The larvae reside in soil while feeding on the roots and stems of several species of important crops, in particular, solanaceae and brassicaceae vegetables and cereal crops like maize and sorghum. Our research program is geared towards the treatment of seed and seedling with entomopathogenic and mutualistic endophytic fungi to improve pest management in IPM programs. The effect of such endophytic fungi on fecundity, development and survival of A. segetum is poorly understood. The current study examined the influence of entomopathogenic and non‐entomopathogenic fungi, isolated from the stem tissue of tomato on survival, oviposition, and development of A. segetum. In the first assay, mortality of second larval instars (L2) were monitored for 15 days after being sprayed with fungal suspensions of Metarhizium anisopliae (Ma96), Beauveria bassiana (Bba149), Paecilomyces farinosus (Pfr10), Verticillium lecanii (V25) or Fusarium oxysporum (Fo162) at increasing concentration of 103 – 109 conidia/ml. In the second assay, maize seeds were surface sterilized and sown in sterile soil treated with fungal suspension of the above fungi. Two weeks after planting, the seedlings were infested with adult moths of mixed sexes and the number of eggs laid on each plant counted after 48 h. For the last bioassay, L2 were fed on 10‐14 day old treated maize seedlings and weight gain assessed daily for ten days. The results revealed that all entomopathogenic fungi, except strain V25, were more pathogenic to A. segetum larvae than the non‐entomopathogenic mutualistic endophyte strain Fo162. Seed treatment with different fungi had no effect on oviposition but negatively affected larval weight gain. The current findings demonstrate that entomopathogenic fungi, especially B. bassiana strain Bba149, have potential biological control activity as an endophyte toward A. segetum when administered at planting stage or later during plant development. The results of these experiments will be presented and discussed in detail. Key words: Entomopathogenic fungi, endophytes, pathogenicity, development, Agrotis segetum

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184

E28 DEFENSIVE RESPONSE OF TRITRICUM AESTIVUM

O THE ATTACK OF SITOBION AVENAE: APHID GENOTYPE, CULTIVAR AND IRRIGATION

SYSTEM EFFECTS

M.E. RUBIO-MELÉNDEZ1, J.A. VERDUGO1,2 & C.C. RAMÍREZ1 1 Laboratorio Interacciones Insecto-Planta, Instituto de Biología Vegetal y Biotecnología,

Universidad de Talca. Casilla 747, Talca, Chile 2 Gembloux Agro-Bio Tech - Université de Liège, Unité d’Entomologie fonctionnelle et évolutive,

Passage des Déportés 2, BE-5030 Gembloux, Belgium Resistance and tolerance are defensive responses in plants, which may be affected by plant genotype, type of insect and conditions of plant development. In this study, resistance and tolerance in two cultivars of wheat Tritricum aestivum (resistant and susceptible cultivars) were evaluated against to the attack of two genotypes of the aphid Sitobion avenae (specialist and generalist genotypes) with two irrigation conditions. Essays were conducted under controlled laboratory conditions. Resistance was evaluated by the reciprocal aphid population growth rate, while tolerance was assessed as the difference in growth (height, number of leaves produced and cumulative length of leaves) between damaged and undamaged plants by aphids. Trade‐off between resistance and tolerance was evaluated. The results show: resistance was significantly affected by wheat cultivar and aphid genotype and also, it was higher to generalist genotype attack, particularly in the susceptible cultivar with reduced irrigation. Respect tolerance: susceptible cultivar showed higher tolerance than resistant cultivar, and plants with normal irrigation have better tolerance. A negative correlation was found between resistance and tolerance, suggesting that exist trade‐off between them. Key words: resistance, tolerance, trade‐off

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185

E29 RESISTANCE OF PRUNUS PERSICA (L.) BATSCH

TO THE ATTACK OF THE APHID MYZUS PERSICAE (SULZER) IN CHILE

J.A. VERDUGO1,2, M.E. RUBIO-MELÉNDEZ1, C.C. RAMÍREZ1

& F. FRANCIS2 1Laboratorio Interacciones Insecto-Planta, Instituto de Biología Vegetal y Biotecnología,

Universidad de Talca, Casilla 747, Talca, Chile. 2Gembloux Agro-Bio Tech - Université de Liège, Unité d’Entomologie fonctionnelle et évolutive,

Passage des Déportés 2, BE-5030 Gembloux, Belgium. Resistance is a mechanism of defense that plants use to deal with insect attack. In Chile, there are no records of cultivars of P. persica resistant to the attack by M. persicae, generating a problem for organic production of this fruit. In the present study we evaluate the genetic variability of resistance in commercial 14 cultivars of P. persica in persica and nectarine varieties to the attack of the aphid M. persicae. We recorded the number of infected shoots (100 shoots sampled). The study was conducted in three seasons: 2007‐08, 2008‐09 and 2009‐10. Subsequently, as the degree of infestation of cultivars showed large variation, ten cultivars were selected to evaluate the resistance cultivars through estimates of the performance of M. persicae during the 2009 and 2010. In addition, the electrical penetration graph (EPG) technique was used to determine the feeding behavior of aphids on this cultivars. Infestation levels in the three seasons show significant difference among cultivars. The resistance had a differential response among the cultivars studied, whose differences were explained by the following variables of feeding behavior of M. persicae: 1) pre‐phloematic variables: total length of F, mean duration of pd, 2) phloematic variables: number of E1, total duration of E1 and 3) all tissue variables: total duration of pd. The more resistant was the nectarine NS92, but this is no correlated with any EPG variable, but the cultivars peaches DU23 and White Lady their response is correlated with total duration of pd and mean total of pd. Thus, we can infer that there are different levels of resistance among the cultivars tested in Chile against the attack of M. persicae. Key words: performance, EPG, defensive mechanism

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186

E30 EVALUATION FOR RESISTANCE TO APHIDS

OF WHEAT GERMPLASM RESOURCES IN CHINA

Haibo ZHOU1,2, Julian CHEN1, Frédéric FRANCIS2, Dengfa CHENG1,Yong LIU3 & Jingrui SUN1

1State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, PR China

2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liege, Gembloux,Belgium

3 Shandong Agricultural University, Taian, PR China Host plant resistance plays important roles in controlling pests and protecting of natural enemies in an agroecosystem, and the effect of insect resistant varieties in reducing damage is conspicuous. Fuzzy recognition technique evaluation were used to determine sources of resistance in wheat to populations of the wheat aphids in five field experiments over 2 years in wheat‐producing areas of China. Results showed that the majority of wheat varieties identified were susceptible to aphids, and no immune and highly resistant varieties was observed. The mean percentage from the wheat varieties with resistant, lowly susceptible, moderately susceptible and highly susceptible to aphid were 9.30, 23.15, 42.32, and 25.23% in whole wheat varieties respectively. 5 wheat varieties (Lantian18, Lantian20, Lantian22, Lantian00‐30 and Shanmai175) were moderately resistant in Jiangyou in 2009, other resistant sources were low levels for aphid‐resisant. And more importantly, 2 wheat varieties (Lantian20, Lantian22) with the continuous resistance to wheat aphid in the five experimental fields over 2 years were found. Although resistance of wheat varieties had a close relation to their genetics and inheritance, we also found that the evaluation result of the same wheat varieties were varied in different place. The results indicated that the resistance to aphid could be affected by geographical environment.These evaluations on reaction to aphid metapopulation that could be crucial when germplasm is used in crosses to improve wheat cultivars. This research is part of program to evaluate germplasm for useful traits and make this information available to the germplasm system. Key words: wheat line; aphid; resistance identification Key words: wheat line; aphid; resistance identification

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187

E31 TOXICITY AND EFFECT ON DETOXIFICATION

ENZYME ACTIVITIES FROM JATROPHA GOSSYPIFOLIA (EUPHORBIACEAE) CRUDE EXTRACT ON

SPODOPTERA LITURA (LEPIDOPTERA: NOCTUIDAE)

Thritsaporn BAWORNNANTHADEJ & Vasakorn BULLANGPOTI Zoology Department, Faculty of Science, Kasetsart University

Phahonyothin Rd., Bangkok, Thailand This research was aimed to study the effectiveness of Thai botanical insecticides, Jatropha gossypifolia ethanol crude extracts which extract by Soxhlet apparatus using ethanol as solvent then was compared it toxicity to cypermethrin and piperonylbutoxide. All treatments were diluted by 70%ethanol and were tested against 2 instars Spodoptera litura by dipping method for 1s. The J. gossypifolia showed its insecticidal activity (24‐hours LC50 = 1647ppm); however, it still very less toxicity than cypermethrin and PBO (24‐hours LC50 = 0.22 and 914 ppm, respectively). In vitro for mode of action study found both cypermethrin and J. gossypifolia treatment induced CYP450, CarE and GST activities of treated –survival larvae whereas PBO treatment inhibit CYP450 but induced CarE and GST. The result shows although J. gossypifolia can control S.litura; however, this insect may use detoxification enzymes for become resistant in the future. Key words: Jatropha gossypifolia, Spodoptera litura, detoxification enzyme

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188

E32 THE COMPARISON OF TOXICITY FROM ALPINIA

GALANGAL AND EURYCOMA LONGIFOLIA EXTRACTS ON MORTALITY OF SPODOPTERA LITURA

(LEPIDOPTERA: NOCTUIDAE)

Rungsimalin TEPATIP, Thritsaporn BAWORNNANTHADEJ & Vasakorn BULLANGPOTI

Zoology Department, Faculty of Science, Kasetsart University Phahonyothin Rd., Bangkok, Thailand

The objective of this study was observed the best solvent extraction for insecticidal effect from the Alpinia galanga and Eurycoma longifolia on second instars Spodoptera litura. Four solvents, ethyl acetate, hexane, ethanol and dichloromethane were used for extract using Soxhlet apparatus. Each crude extract were treated to second instars of S. litura in ranges of 0.4–20 mg/ml by dipping method. The results revealed that Alpinia galangal crude extract is the better control effect than Eurycoma longifolia crude extract followed by 24‐hour LC50 value. The Alpinia galangal ethyl acetate crude extract shows the most effective than other extract as LC50 value as 3.82 mg/ml for ethyl acetate crude extract. For Eurycoma longifolia, hexane crude extract shows the best toxicity compare with other solvent (LC50 = 14 mg/ml); however, it control efficiency still less than all treatment of Alpinia galangal. The toxicity is not significant different by longer expose (p<0.05, DMRT). These results provide the preliminary information that could be further studied in order to apply for this insect pest control in the future. Key words: Alpinia galangal, Eurycoma longifolia , Spodoptera litura, biopesticide

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189

E33 INSECTICIDAL ACTIVITY OF BAUHINA SCANDENS

CRUDE EXTRACT ON MORTALITY AND DETOXIFICATION ENZYMES ACTIVITY OF PLUTELLA XYLOSTELLA

(LEPIDOPTERA: YPONOMEUTIDAE) Waraporn POONSRI,Wanchai PLUEMPANUPAT2 & Vasakorn BULLANGPOTI1

1 Department of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand 2 Department of Chemistry and Center of Excellence for Innovation in Chemistry

Faculty of Science, Kasetsart University, Bangkok 10900, Thailand Bauhina scandens was extracted by Soxhlet apparatus using sequencing solvents as hexane, dichloromethane, ethyl acetate and ethyl alcohol. The toxicities of each crude extract to the 2nd instars Plutella xylostella (DBM), reared on cabbage were tested by topical applicator. The 24‐hours LC50 values of dichloromethane crude extract shows the highest toxicity to DBM (LC50 = 4621ppm) which was significant difference among them (P > 0.05). The activity of carboxylesterase and glutathione‐s‐transferase in the survival treated larvae from all extracts were induced. Although Bauhina scandens could shows insecticidal effect on DBM which could serve as a new way to control DBM, however, the increase in both detoxification enzyme activities after treatment in the tested insects could partly explain the risk of resistance of DBM. In addition, other mode of action and the active ingredient needs further investigation. Key words: Bauhina scandens, Plutella xylostella, carboxylesteras, glutathione‐s‐transferase

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190

E34 EFFECT OF LACTONE GLYCOSIDE, A NOVEL PLANT

ACTIVE PRINCIPLE ON GANGLIONIC ACETYL CHOLINESTERASE AND NA+ - K+ ATPASE ENZYMES OF

SPODOPTERA LITURA F.

T. SELVAMUTHUKUMARAN & S. ARIVUDAINAMBI Department of Entomology, Faculty of Agriculture, Annamalai University,

Annamalainagar, Tamilnadu, India Challenges such as resistance development, resurgence and residues in the present day pest management scenario necessitated the on going search for newer molecules and formulations. The plant active molecules with their novel modes of action are important leads to development of such newer insecticides. In our earlier studies, Lactone glycoside, a lignin lactone glycoside was identified as the active molecule responsible for the significant anti insect activity of Cleistanthus collinus (Roxb.) Benth., a small deciduous wild tree. Sub lethal (LC30& LC50)and lethal doses (LC95) of Lactone glycoside were bioassayed on third, fourth and fifth instar Spodoptera litura Fab. larva through leaf disc method along with a solvent (Acetone) control. Ganglionic acetyl cholinesterase and Na+ ‐ K+ ATPase enzymes were carefully extracted. Their activity measured by Ellman and Fiske and Subbarow methods respectively and compared with an absolute control. The results revealed that acetyl cholinesterase and Na+ ‐ K+ ATPase activities were reduced at all the concentrations among all the instars tested. The efffect was more pronunced in third instar. At 30, 50 and 95 per cent lethal concentrations (LC30, LC50 and LC95) the acetyl cholinesterase activity was measured as 16.36, 13.28 and 11.43 n mol mg‐1 min‐1 as against 20.16 n mol mg‐1 min‐1 in control, a reduction of 19, 34 and 43 per cent when compared with control. Similarly, the ATPase activity at 30, 50 and 95 per cent lethal concentrations (LC30, LC50 and LC95) against third instar was determined to be as 42.87, 37.15 and 24.36 µmol mg‐1 h‐1 a reduction of 36, 45 and 64 per cent respectively. Against fourth and fifth instars the reduction of acetyl cholinesterase activity was 28 and 23 per cent and ATPase activity was 23 per cent only even at 95 per cent lethal concentration (LC95). These results clearly exhibits concentration dependent and age dependent effect of Lactone glycoside. Lactones efficiently alkylate thiols and amines in all systems and inhibited thiol dependent enzymes like acetyl cholinesterase and ATPase in nervous system. This underlines the possibility of development of a newer insecticidal molecule with a possible derailment of nerve conduction as mode of action. Key words: Lactone glycoside, Cleistanthus collinus, Acetyl cholinesterase, Na+‐ K+ ATPase, Mode of action

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191

E35 INTERACTIONS OF RHIZOCTONIA SOLANI

AGGLUTININ WITH INSECT MIDGUT CF-203 CELLS

Mohamad HAMSHOU1,2, Guy SMAGGHE1 & Els J.M. VAN DAMME2 ¹Laboratory of Agrozoology, Department of Crop Protection

²Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology Faculty of Bioscience Engineering, Ghent University

Coupure links 653, BE-9000 Ghent, Belgium. Recently, more attention has been given to the use particular proteins as biocontrol agents for the control of insect pests. Some of these proteins belonging to class of lectins which are defined as proteins possessing at least one non‐catalytic domain that binds reversibly to specific mono‐ or oligosaccharides. Several plant lectins have been reported to possess insecticidal activity against different insects while very little is known about the insecticidal activity of fungal lectins. In previous work we reported the insecticidal activity of Rhizoctonia solani agglutinin (RSA) towards cotton leafworm Spodoptera littoralis (Hamshou et al. 2010) and demonstrated that midgut cells are the primary target for RSA (on unpublished data). In the present study we focused on the interaction of RSA with insect midgut CF‐203 cells. First, the toxic effect of RSA was investigated by incubation of CF‐203 cells with different RSA concentrations. The EC50 value was calculated to be 0.3 µM, demonstrating high toxicity of RSA towards these midgut cells. Interestingly, the toxicity of RSA was reduced to 50% by preincubation of the lectin with N‐acetyl galactosamine. Analyses using the confocal fluorescence microscope demonstrated that FITC‐labeled RSA was not internalized CF‐203 cells, but was bound to the cell surface. Furthermore, the RSA cytotoxicity in CF203 cells was accompanied by DNA fragmentation and the effect was caspase‐3 independent but caspase‐7, ‐8 and ‐9 dependent. Further studies are required to determine the mode of action of RSA. Reference Hamshou, M., Smagghe, G., Van Damme, E.J.M., 2010. Entomotoxic effects of fungal lectin

from Rhizoctonia solani towards Spodoptera littoralis. Fungal Biol.114, 34-40.

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192

E36 THE EFFECT OF KAOLIN ON THE MITE ASSEMBLAGES

OF AN ORGANIC APPLE ORCHARD IN HUNGARY

Peter SIPOS1, Arpad SZABÓ2 & Viktor MARKÓ2 1Pannon University, Georgikon Faculty, Plant Protection Institute, Keszthely

Deák Ferenc u. 57. Hungary E-mail: [email protected]

2Corvinus University of Budapest, Faculty of Horticulture, Department of Entomology, Budapest, 1118 Ménesi út 44. Hungary

E-mails: [email protected], [email protected] The impact of Surround WP, a kaolin particle film product, on abundance of phytophagous and predacious mites was evaluated in an organic apple orchard in Hungary. In the orchard planted in 2000, six plots were separated, each consisting of 250 trees. In two plots 10 kaolin applications per year, in two plots six applications per year were performed and two plots served as untreated control. The abundance of mites was observed on two apple cultivars, Florina (M9) and Prima (M9), from May to October in 2009 and 2010. From each plot, seven leaves of 10 different apple trees were examined monthly under microscope. The kaolin treatments dramatically reduced the number of predatory mites, including phytoseiid mites (Phytoseiidae) and the stigmaeid mite, Zetzellia mali in 2009, however in 2010 the number of Z. mali increased in the treated plots. The occurrence of the herbivorous apple rust mite, Aculus schlechtendali slightly decreased responding to the kaolin treatments, however the abundance of the two‐spotted spider mite (Tetranychus urticae) increased in the kaolin treated plots in both years. The mite abundances in the plots with intermediate kaolin regime were between the control plots, and the plots with high kaolin pressure. The density of tydeid mites did not show clear differences between the three treatments. In the control plots the dominant phytoseiid mite species the same as in the whole region was Euseius finlandicus, and the subdominant was Amblyseius andersoni. Kaolin treatments changed the rate of this two species and in the treated plots A. andersoni became the dominant, which seemed to tolerate better the kaolin applications. This study demonstrated, that multiple applications of kaolin particle films can suppress the apple rust mite however, by disrupting beneficial mites they promote spider mite populations. Key words: kaolin particle film, mite assembleges, organic apple

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193

E37 EFFICACY OF INDOXACARB AGAINST MELIGETHES

AENEUS IN OILSEED RAPE IN HUNGARY

I. FARKAS1, V. LIPOSITS1, M. VESZELKA1, I. MOLNÁR2, I. SOMLYAY2 & E. TÓTH

1 Agricultural Administration Office, Plant, Soil and Agroenvironmental Protection Directorate, HU-1118 Budapest, Hungary

2 DuPont Hungary Ltd. HU-2040 Budaörs, Hungary Indoxacarb a globally registered insecticide and biocide active substance which is mostly applied to control Lepidoptera species in different crops. Oilseed rape (OSR) area has been increased rapidly in the past years in Hungary reaching in 2010 close to 300 000 ha. The increased acreage of oilseed rape was not followed by the enlargement of the available insecticide portfolio from crop protection chemical producers. The aim of this registration expansion of indoxacarb in oilseed rape was to widen the insecticide offer in oilseed rape and supply the farmers with a new low dose and selective insecticide. Also important aim was to give a new mode of action product, that has no cross resistance with the main chemical class affected: Pyrethroïds. The common pollen beetle (Melighetes aeneus, Fabricius 1775.) is an important pest of oilseed rape. It is unknown if it contributes to the pollination of the crop. Eggs are laid in the flower buds of the host plant the larvae develop within the flowers. Oviposition damage to the buds of oilseed rape can cause the flowers to drop off. Both adults and larvae feed on the pollen and in the flowers. Three different dosages of AVAUNT® 150 EC (containing the single enantiomer active) were tested to obtain efficacy data on this important OSR pest ‐ the common pollen beetle (Meligethes aeneus, Fabricius 1775. ) ‐ applied once at green/yellow bud phenology (BBCH 51‐59) of the crop using conventional ground sprayer 250‐400 liter spray water/ha.

Year Pest Test site scale Dose (l/ha) Efficacy Phenology 2009 M. aeneus Vas county small 0,3 Medium

(67-75 %) BBCH 51-59

2009 M. aeneus Zala county small 0,3 Good (76-90 %)

BBCH 51-59

2010 M. aeneus Nógrád county large 0,17 0,25

good. good

BBCH 51-59

2010 M. aeneus Zala county large 0,17 0,25

medium good

BBCH 51-59

2010 M. aeneus Vas county large 0,17 0,25

medium. good

BBCH 51-59

Based on the evaluation of the large scale (1 ha) trial data 0,17‐0,25 l/ha Avaunt® 150 EC was suggested as effective dose range against common pollen beetle in oilseed rape which also can be a new tool in insecticide resistance management program of OSR farmers. The detailed efficacy data will be discussed in the poster. Key words: indoxacarb, oilseed rape, common pollen beetle

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194

E38 ENZYME ACTIVITY AND FITNESS COST OF

NEONICOTINOID RESISTANT AND SUSCEPTIBLE POPULATIONS OF THE APHIS GOSSYPII GLOVER

(HEM: APHIDIDAE)

Shadieh GERAMI Department of Plant Protection, Faculty of Agricultural Sciences and Engineering,

College of Agriculture and Natural Sciences, University of Tehran, Karaj, Iran Populations of Aphis gossypii of distinct susceptibility to neonicotinoids show differences in the accumulation and mobilization of energy reserves, what may allow the production of their defensive tools against such compounds without impairing their reproductive performance. Biochemical properties with energy metabolism, including sugar, lipid, glycogen and protein, to test this hypothesis. Levels of these characteristics were determined in the MUMV for resistant population, and susceptible population, MUMM population and semi‐ sensitive poplation, MUMD. Bioassays were also carried out for these aphid populations. According to the results obtained, the MUMV population showed resistant rate significantly higher than the other two populations. Among the enzymes studied, CaE showed significant difference among the three populations with higher activity in the MUMV population. The results obtained in the assays with biochemical parameters also indicate significant difference in activity among the populations, with higher activity in using glycogen in the MUMV population. The inverse activity trends of T (Generation time) and DT (Doubling time) in both resistant and susceptible populations, one showing fitness disadvantage without insecticide exposure and the other not showing it, may underlay the mitigation of insecticide resistance physiological costs observed in the MUMV population. Key words: Insecticide resistance, fitness cost, cotton aphid, carboxyl esterse

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195

E39 DETOXIFYING ENZYMES OF COTTON APHID IN

RESPONSE TO HOST-PLANTS AND NEONECOTINOID

Shadieh GERAMI, Khalil TALEBI & Vahid HOSSEININAVEH Department of Plant Protection, Collage of Agriculture and Natural Sciences

University of Tehran, Karaj, Iran, Postal code 3158711157 In the present investigation an effort was made to realize the role of feeding host plants including of Cucurmis sativusvar Sultan and var Turkey and Cucurbita zucchini on some enzymatic characteristics such as mono oxygenase(MFO), carboxyl esterase (CaE) and Glutathione S‐transferase (GST) in cotton aphid, Aphis gossypii Glover(Hemiptera: Aphididae). Esterase assays were performed on cotton aphids according to Grant. et al. method with slight modifications. Glutathion S‐Transferase activity was determined using CDNB as the substrate (Kao et al., 1989). MFO activity was determined by measuring the production of p‐nitrophenol photometric as described by Kinoshita et al. (1969) by some modification. The results showed that the enzymes’ activities were changed in the aphids feeding from different host plants assist in detoxification of their host metabolites. It is important when the aphids exposured to insecticides. Results indicated that the measured enzyme activity have significant changes depending on the host plant. It is showed in this study that there is not any significant differences between different host‐ plants on esterase activity (p=0.446), however there is a significant differences between GSH activity (p=0.047) but this relationship is not significant on MFO activity (p=0.417). Among three strains of Ag‐PP, Ag‐MO and Ag‐FA, strain Ag‐PP was the most resistant strain with neonicotinoids and the resistance mechanism was related to metabolic increasing of carboxyl esterase activity. Results showed that strain of Ag‐MO was the most susceptible strain against neonicotinoids, imidacloprid and thiametoxam. The result of this investigation also showed the general esterases might play an important role in conferring or contributing to neonicotinoid resistance in the cotton aphids. Key words: neonicotinoid, detoxifying enzymes, cotton aphid, host plant

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196

E40 SUBLETHAL EFFECTS OF TEBUFENOZIDE AND

METHOXYFENOZIDE ON REPRODUCTIVE EVENTS AND TESTES IN THE MEDITERRANEAN FLOUR MOTH

H. BOUZERAA1, R. MESKACHE1, G. SMAGGHE2& N. SOLTANI-MAZOUNI1,*

1 Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences, University Badji Mokhtar of Annaba, DZ-23000 Annaba, Algeria

2 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, BE-9000 Ghent, Belgium *E-mail: [email protected]

The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: Pyralidae), is an important pest in stored products worldwide, and is one of the major pests in flour mills in Algeria. Due to environmental concerns, biorational control products such as insect growth regulators (IGRs) are an alternative to methyl bromide, the most widely used fumigant. Tebufenozide (RH‐5992) and methoxyfenozide (RH‐2485) are lepidopteran‐specific insec‐ticides that belong to a new class of IGRs, the non‐steroidal ecdysteroid agonists. In this project, methoxyfenozide and tebufenozide were applied topically at their respective LD50 value (0.01 and 0.005 µg/pupa) on newly ecdysed male pupae. Subsequently, the surviving males were coupled with untreated female adults and the sublethal effects on different reproductive parameters as the pre‐oviposition and oviposition period, fecundity and egg viability were determined. In an attempt to better understand the effects by the IGR compounds, we dissected the testes from the newly emerged males from treated pupae and determined the amounts of carbohydrates, total proteins, lipids and nucleic acids. Key words: Insecta, Ephestia kuehniella, methoxyfenozide, tebufenozide, toxicity, reproduction, testes

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197

E41 TOXICITY OF BORIC ACID BY INJECTION AGAINST

ADULTS OF THE GERMAN COCKROACH AND THE ANALYSIS OF RESIDUES IN SEVERAL ORGANS

D. HABES, K. BOUAZDIA, R. MESSIAD & N. SOLTANI*

Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences, University Badji Mokhtar of Annaba, DZ-23000 Annaba, Algeria

*E-mail: [email protected] In Algeria, Blattella germanica L. (Dictyoptera: Blattellidae) is a serious pest in the urban environment and their infestations were controlled by conventional insecticides such as organophosphate, carbamate, or pyrethroid compounds. However, the intensive utilisation of these conventional insecticides caused secondary side‐effects on the environment and subsequently alternative methods are developed. Thus, interest has again centred on lesser‐used compounds such as boric acid. This inorganic insecticide has been used for many years, especially against cockroaches and its mode of action remains understood. In order to obtain more information on its mode of action, we first evaluated the toxicity of boric acid administered by injection on male adults of B. germanica. Then, boric acid residues were determined in several organs (fat body, testes, hemolymph and gut) by a colorimetric method. The insecticide was injected at different doses ranging over 25, 50, 100 and 200 µg/insect to newly emerged male adults. Mortality was measured at different times following treatment during the adult life (1, 2, 3 and 6 days). Treatment resulted in a dose‐dependent mortality, since the respective LD50 and LD90 (µg/insect) were 282 and 603 at day 1, 209 and 417 at day 2, 158 and 295 at days 3, 78 and 194 at day 6, while the respective LT50 and LT90 (days) were 44 and 126 for 25 µg/insect, 21 and 41 for 50 µg/insect, 4.7 and 16 for 100 µg/insect, 3.0 and 7.6 for 200 µg/insect. This insecticidal activity depends on the dose and the duration of treatment. The quantity of boric acid accumulated in several organs (hemolymph, foregut, midgut, hindgut, testes, fat body) was determined as a function of the dose (LD50 and LD90) and the time following treatment (1 to 6 days). Results revealed that the amount of residues in these organs increased as a function of the time following treatment. In addition, the amount of residues was relatively important in fat body followed by testes, midgut, hindgut and foregut and hemolymph. Data from the present injection experiment are discussed in relation to previous bioassays made by ingestion Key words: Blattella germanica, boric acid, toxicity, injection, residues, organs

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E42 EFFECTS OF HALOFENOZIDE (RH-0345) ON

CUTICULAR HYDROCARBONS OF MOSQUITO LARVAE OF CULEX PIPIENS

H. BOUDJELIDA1,*, J.P. FARINE2, G. SMAGGHE3 & N. SOLTANI1

1 Laboratory of Applied Animal Biology, Department of Biology, Faculty of Sciences, University Badji Mokhtar of Annaba, DZ-23000 Annaba, Algeria

2 Université de Bourgogne, CSGA, CNRS UMR626, Faculté des Sciences, FR-21000 Dijon, France 3 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Biosciene Engineering,

Ghent University, BE-9000 Ghent, Belgium *E-mail: [email protected]

Halofenozide (RH‐0345) is an insect growth regulator belonging to the class of non‐steroidal ecdysone agonists that are developed as insecticides particularly against Coleoptera. In a previous study halofenozide was found to be effective against larvae of Culex pipiens L. (Diptera: Culicidae) and exerted its toxicity primarily by the induction of a precocious lethal molting (Boudjelida et al., 2005). In this study, newly molted 4th‐instar larvae of C. pipiens were exposed to halofenozide (23% EC) for 24 h at its LC50 (12.58 µg/l) and LC90 (28.58µg/l) under standard laboratory conditions in accord with WHO recommendations. Then to better understand the mode of action of halofenozide, the profiles of the cuticular hydrocarbons in treated and control larvae were evaluated by use of gas chromatography (GC) and gas chromatography‐electron impact coupled to mass spectrometry (GC‐MS). In essence, the chromatographic profiles of the purified hexane extracts mainly consisted of n‐alkanes (C21‐C31). Typically, treatment with halofenozide reduced the amounts of different mean peaks, and shifted the cuticular hydrocarbon profile towards longer‐chain alkanes compared to controls. The current data can help in further studies on the potential role of mosquito cuticular hydrocarbons in chitin synthesis and mating behavior. Key words: Culex pipiens, halofenozide, cuticle, cuticular hydrocarbons, n‐alkanes

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E43 STAND STRUCTURE OF MOSQUITOES

(DIPTERA CULICIDAE) IN FARMS AND ESTABLISHMENT OF A NEW POTENTIAL VECTOR

OF WEST NILE VIRUS FOR BELGIUM

Slimane BOUKRAA, Jean-Yves ZIMMER, Grégory SIMONON, Eric HAUBRUGE & Frédéric FRANCIS

Department of functional and evolutionary Entomology, Gembloux Agro-Bio Tech (University of Liege), Gembloux, Belgium

Many Diptera, spread across the globe are likely to play a role in the transmission of various human and animal diseases. Mosquitoes in particular, give rise to various countries on research increasingly expanded and deepened. To better understand the structure of the Culicidae at cattle farms in Belgium and identify habitats favorable to the development of each species, a taxonomic inventory was conducted in 2008 and 2009 in ten different sites. They fall into five natural regions: Condroz, Ardenne, Fagne‐Famenne, Lorraine and Compine. The collection of mosquitoes is based on sampling of larvae in 13 biotopes such as water troughs, tires, abandoned utensils and temporary puddles or not. The results of the morphotaxonomic study of the mosquitoes collected in the different study sites show the presence of five species of Culicidae divided into three genera, Anopheles, Culex and Culiseta. Of the 1843 individuals examined in 2009, Culex pipiens represents 79.98% of the total harvest. A new species is reported for Belgium, Culex modestus Ficalbi, 1890. This species is one of the most important from an epidemiological and medical‐veterinary entomology, given its role in the transmission of West Nile and myxomatosis viruses. Key words: mosquitoes, culicidae, wnv, farm, belgium, transmission, systematic

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E44 ANALYSIS OF THE EGYPTIAN PROPOLIS COLLECTED

BY DIFFERENT STRAIN HYBRIDS OF HONEY BEE APIS MELLIFERA L. (HYMENOPTERA: APIDAE)

OSMAN A. ZAGHLOUL, A.K. MOURAD, O. EL-ANSARY1 & F.A. KASSEM2

Faculty of Agriculture, Saba-Basha, Department of Plant Protection, Alexandria University, Alexandria, Egypt

1Faculty of Agriculture University of Alexandria, Egypt 2Faculty of Agriculture Omar El Mokhtar, University of Sabha, Libya

The current study was carried out in a private apiary located in Kafr‐El Dawar Province, Egypt. Langstroth hives containing three different hybrids of the strains i.e, Carniolan (Apis mellifera carnica), Italian (A. mellifera ligustica) and Cucasian (A. mellifera caucasica). Equal number of hives for each hybrid was assigned in this study. For each hybrid, quantities of propolis were harvested in each season of the year (Spring, Summer, Autumn and Winter) The analytical analysis of the alcoholic extracts of the compiled propolis for each hybrid in each season, based on the percentage of the total ion current (%TIC) showed twelve fatty acids; six sugar derivatives; nine organic acids; twelve aromatic compounds; nineteen esters; eight phenolic compounds; nine flavonoids and eleven terpenes. The average percentages of "TIC" of the aforementioned compounds were 22.7; 4.45; 6.40;18.49; 12.43; 21.56; 8.10 and 5.79, respectively. In general, it was noticed that the %TIC for the detected compounds were high in Spring and Summer and low in Autumn and Winter. As to the quantitative analysis detected flavonoids, it was found that the mean quantities were 7.00; 6.98 ; 4.42; 3.65; 1.03 and 1.02 mg/g. for Chrysin, Galangin, Pinocembrin, Pinobanksin O‐acetate, Pinostrobin chalcone and Pinostrobin, respectively. Regarding the determination of lead (Pb)and Arsine (As), the concentration of (Pb) ranged from 11.30 to 17.00 mg/kg. of propolis whereas (As) has not been detected (0.00mg/kg.). These two levels did not exceed the world upper limits for both the heavy metals. Therefore, propolis under this study is not in need for purification; thus the Egyptian propolis is considered of good quality. Key words: propolis, Apis mellifera hybrid strains, heavy metals, flavonoids.

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201

E45 RNA INTERFERENCE OF NUCLEAR RECEPTOR

GENES IN THE PEA APHID (ACYRTHOSIPHON PISUM)

Olivier CHRISTIAENS, Hanneke HUVENNE & Guy SMAGGHE

Laboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, BE-9000 Ghent, Belgium

RNA interference (RNAi) is a relatively novel technique that is widely used in functional genomics, e.g. of newly found genes in genome projects. By bringing sequence‐specific double stranded RNA (dsRNA) into a cell, the RNAi machinery will cause down‐regulation of the expression of the gene of interest. Through monitoring of possible gene‐expression related effects indications about possible functions of the gene can be determined. The possibility to use RNAi in crop protection is also being investigated. In this strategy insects would have to take up dsRNA orally. The main advantage of RNAi compared to most other pest control strategies is the species‐specificity of this approach. However, one of the biggest challenges is to find easy and reliable dsRNA uptake strategies. Here, we report the use of RNAi in the pea aphid (Acyrthosiphon pisum) by administering double stranded RNA of some of the nuclear receptors. These were recently identified in this hemipteran insect (Christiaens et al., 2010). In this research the dsRNA is delivered to the aphids by injection or orally, by adding the dsRNA to the artificial diet. The delivery methods were optimized and the efficacy of gene silencing through RNAi depending on method of delivery and during different stages of the insect was assessed. Christiaens O., Iga M., Velarde R., Rougé P. and Smagghe G. (2010). Halloween genes and

nuclear receptors in ecdysteroid biosynthesis and signaling in the pea aphid. Insect Mol Biol, 19 (suppl. 2), 187-200.

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202

E46 PRELIMINARY STUDY ON PVY TRANSMISSION

EFFICIENCY IN APHID CLONES

Saman BAHRAMI-KAMANGAR1,2, Kris DE JONGHE2, Martine MAES2

& Guy SMAGGHE1 1 Lab Agrozoology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium

2 Plant Sciences Unit, Institute for Agricultural and Fisheries Research, Merelbeke, Belgium Potato virus Y (PVY) is the type representative of the Potyviridae family with a host range that comprises more than 60 species including several solanaceous crops such as potato, tobacco and pepper. PVY isolates have historically been classified into PVYO, PVYN and PVYC groups based on symptomatology and transmission efficiency. Besides mechanical transmission, PVY is transmitted by aphids in a non persistent matter. The mechanism of vector transmission is not yet fully understood. The different PVY strains are transmitted by many aphid species. The transmission efficiency can strongly vary and not only depends on the PVY strain, but also on the aphid species and the host plants. Additionally, the number of virus particles and presence of virus in efficient sites in the vector could be crucial. In this project the transmission efficiency in different clones of Myzus persica (11), Myzus nicotiana (1) and Acyrthosiphon pisum (1) was recorded by transmission tests in order to evaluate correlation between transmissibility and number of virus particles in aphids. To evaluate the number of virions in aphids, PVY detection in single aphids by means of a one step qRT‐PCR protocol was optimized using PVY transcript dilution series to standardize the virus particle quantification. Results of the PVY transmission tests revealed differences among the clones. Evaluation of the transmission efficiency showed significant differences that can be linked to the virion quantity in the aphid clones

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Posters Insect and Plant Nematology

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N01 FACTORS AFFECTING THE EFFICACY OF

BRASSICA SPP. AND LOLIUM PERENNE L. ON ROOT-KNOT NEMATODE INFESTATION OF TOMATO

Ioannis ANASTASIADIS1 & Eirini KARANASTASI2

1 Laboratory of Nematology, Department of Agricultural Entomology & Zoology,Benaki Phytopathological Institute, 8, St. Delta str., GR-14561, Kifissia, Greece

2 Laboratory of Plant Protection, Department Of Greenhouse Crops and Floriculture, Technological Educational Institute of Messolonghi, Nea Ktiria, GR-30200, Messolonghi, Greece The effect of metham‐sodium and finely chopped broccoli, cabbage, cauliflower, and ryegrass residues incorporated in soil against Meloidogyne incognita and M. javanica, over increasing periods of time and a range of dose rates, with or without plastic covering were studied in growth chamber experiments. The aim of this work was to compare the effect of treatment duration, tarping and application rate of fresh organic amendments originating from four plant species of the family Brassicaceae on Meloidogyne spp. eggs. Also, to evaluate and compare the efficacy of volatiles deriving from fresh broccoli or ryegrass tissues with the fumigant nematicide metham‐sodium against Meloidogyne spp. egg masses, in relation to treatment duration. The amendments were applied at 5 or 10 kg/m2 dose rates. The most effective treatment in maintaining low numbers of nematodes was pre‐treatment with metham‐sodium volatiles, which resulted in zero invasion of tomato roots. Application of fresh‐chopped broccoli, ryegrass and, occasionally, cauliflower, also caused a significant reduction in the root‐knot nematode population. Nematode reduction induced by the broccoli amendment was slightly better than ryegrass. On the other hand, cabbage incorporation did not suppress Meloidogyne spp. to the same extent, though a 19% reduction was observed, even at the lowest dose rate. However, increased doses of all treatments were further effective in reducing nematode populations. Additionally, nematode suppression was greater in pots amended and covered with plastic tarp, as compared to the non‐covered, and these results demonstrated that retention of these gases through the use of an impermeable film improved the efficacy of all green manures against root‐knot nematodes. The failure of non‐covering, even though frequent watering maintained a thin crust of clay on the surface, suggests that application of a plastic cover is crucial in biofumigation. Also, a more significant effect of biofumigants may be achieved by developing methods that increase plant cell disruption and thereby the degree of volatile release from plant tissues. Key words: biofumigation, biological control, Brassica oleracea, broccoli, cabbage, cauliflower, grass, management, Meloidogyne incognita, Meloidogyne javanica, metham‐sodium, ryegrass, soil amendment, Solanum lycopersicum.

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206

N02 EFFECT OF PLANTING DENSITY OF POTATO

USED AS A TRAP CROP AGAINST GLOBODERA ROSTOCHIENSIS

Milan RADIVOJEVIĆ & Nikola GRUJIĆ

Institute for Phytomedicine, Faculty of Agriculture, Beograd, Serbia Potato cyst nematodes require an integrated approach of control, where various ways of using plants in order to reduce nematode field populations are available. Trap crops are among them, offering itself a range of approaches. Growing resistant potato cultivars with H1 gene, highly resistant to the first pathotype (Ro 1) of Globodera rostochiensis is widely practiced. In Serbia, Ro 1 is still prevailing in several areas and sustainable strategies to control it from the quarantine side is being investigated. Herein we provide preliminary results in estimating effect of planting density of trap crop plants on reducing population density of G. rostochiensis, pathotype Ro 1. A small plot trial was set in 2010 in a field part know to be moderately infested with G. rostochiensis, pathotype Ro 1. The trial involved 3 plant densities , with the plants of potato variety Agria distributed in a rectangular grid pattern with distances of 1, 0.5 and 0.25m between the plants in 1 m2 small field plots, with 6 replicas. Soil samples of c.a. 1 kg soil (80 cores) per plot were collected at planting and harvest time (late May and October 2010, respectively). Cysts were extracted by flotation and living content counted to estimate initial (Pi) and final (Pf) population density. In all three treatments the density of cysts was reduced by c.a. 20 %, from c.a. 250 to 200 cysts per 100 g of soil. There was no significant decleane in the cyst viability in plots with 1 m distance between the plants. However, in both the plots with plants 0.5 m and 0.25 m apart the population decline was high and similar. In plots with 0.5 m between the plants viability of cysts at harvest was reduced by 64.2 % vs. 57.8 % in plots with plants 0.25 cm apart. Density of encysted larvae in soil at harvest was lower by 68 % (Pi 142.6, Pf 46.3 L2/g soil) in 0.5 m plots vs. 70.8 % (Pi 124.0, Pf 33.6 L2/g soil) in 0.25 m plots. The trial will be continued in 2011. The present data suggest that optimal distance between trap crop potato plants is around 0.5 m, providing c.a. 70 % annual population decline of G. rostochiensis, pathotype Ro 1 in moderately infested soil. Key words: Potato cyst nematodes, resistant cultivars, trap crops

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N03 SCREENING ENTOMOPATHOGENIC NEMATODE SPECIES FOR MANAGEMENT OF THE WOOLLY

APPLE APHID (ERIOSOMA LANIGERUM)

Nick BERKVENS1, Tim BELIËN2 & Nicole VIAENE1 1 Plant – Crop Protection – Nematology, ILVO, Merelbeke, Belgium

2 Zoology Department, pcfruit, Sint-Truiden, Belgium The withdrawal and restriction of certain effective insecticides has led to the woolly apple aphid Eriosoma lanigerum becoming a major pest in apple growing areas across Western Europe. Since a part of the aphid’s life cycle occurs in the soil, application of entompathogenic nematodes (EPN) is considered to be a possible alternative strategy for management of this serious pest. The aim of this study was to evaluate the efficacy of commercially available EPN species (Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema carpocapsae, Steinernema feltiae, Steinernema glaseri and Steinernema kraussei). Woolly apple aphids were held individually in a multiwell cell lined with filter paper. Each aphid was exposed to a nematode suspension of 40µl containing approximately 150 infective juveniles of one of the EPN species. Mortality of each aphid was assessed after 3 days. Aphids were dissected and the number of dead and living nematodes inside the insect bodies were counted. The following mortality rates (with Abbott’s correction) were determined for E. lanigerum: 1% with H. bacteriophora, 5% with H. megidis, 43% with S. carpocapsae, ‐2% with S. feltiae, ‐4% with S. glaseri and 17% with S. kraussei. Only S. carpocapsae was able to cause significant mortality to the aphids. Addition of the non‐ionic surfactant Trend 90 (isodecyl alcohol ethoxylate) to the nematode suspension significantly increased parasitization rates for H. bacteriophora (p < 0.001), S. carpocapsae (p = 0.025), S. feltiae (p < 0.001) and S. kraussei (p < 0.001). Furthermore, the number of EPN individuals found in the aphid bodies was significantly higher for H. bacteriophora (p < 0.001), S. carpocapsae (p = 0.041) and S. kraussei (p < 0.001) when adding Trend 90. We will further examine the influence of several biotic and abiotic factors on the efficacy of the EPN application. In addition, field studies will be performed to monitor the phenology of the woolly apple aphid in Belgian orchards and to examine the control capacity of selected EPN species under field conditions. Key words: aphid, biocontrol, entomopathogenic nematode, Heterorhabditidae, Steinernematida

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208

N04 INFLUENCE OF DIFFERENT FERTILIZER TYPES

OF ZUCCHINI (CUCURBITA PEPO) ON THE STRUCTURE OF NEMATODE COMMUNITES

Dimka HAYTOVA & Tatyana BILEVA

Department of Horticulture and Department of Ecology and Environmental Protection Agricultural university 12 Mendeleev str., BG-4000 Plovdiv, Bulgaria

Increasing efficiency of production of vegetable crops is directly related to search for appropriate solution to increase their productivity. Organic amendments have been used for centuries to improve soil fertility and crop yield. Our study suggests that organic amendments can also be used as nematicidal agents. The survey was conducted on Experimental field of Department Horticulture at Agricultural University of Plovdiv, Bulgaria in 2009 in nematodes infested sites. The experiment was laid out in a randomized block design with four replicated thrice along with the control. Sizes of experimental plots were 6.4 m2 with zucchini cv. “Black Beauty”. Combination with two types of fertilizers was used to investigate their effects on the community of soil nematodes. Characterization and comparative analysis among treatments of soil nematode community structure based on different ecological measures such as total nematode abundance, number of genera, trophic diversity and etc., was made. Changes in the composition and structure of nematode community as result of different fertilizer types were assessed. Maximum yield of zucchini was obtained from test plants in infested plots with soil and foliar fertilization. Key words: Cucurbita pepo, organic amendments, nematode community, zucchini, fertilizers,

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N05 PATHOGENICITY, DEVELOPMENT AND REPRODUCTION

OF THE ENTOMOPATHOGENIC NEMATODE STEINERNEMA SP., IN MEALWORM,TENEBRIO

MOLITOR

Yuliantoro BALIADI1, Ika ROCHDJATUN SASTRAHIDAYAT2, Syamsuddin DJAUHARI2 & Bambang TRI RAHARDJO2

1 Indonesian Legume and Tuber Crops Research Institute, Kendalpayak-Malang, Indonesia 2 Faculty of Agriculture, Brawidjaya University Malang-Indonesia

The pathogenicity, development and reproduction of Steinernema sp., isolate Skpr‐20/Str, an entomopathogenic nematode (EPN) isolated from soil samples in Sukapura (Indonesia isolate) were studied in vivo using larvae of the Tenebrio molitor, as host. Results revealed that pathogenicity, development and reproduction was significantly influenced by the nematode doses, particularly due to the penetraion rate of infective juvenile (IJ) into T. molitor larvae. Although the number of invading nematodes increased with increasing dose, percentage penetration declined. The invading IJs resumed development, moulted to the J4 stage and reached adulthood within three days after nematode inoculation. In more detail, immature females appeared likewise within two days after inoculation and adult female developed three days after inoculation. The females laid eggs from day 4th until 7th. All eggs which remain inside the uterus after the period of egg‐laying develop inside the maternal body. The first female bearing endotokia matricida were observed at day 5th. In a sand‐based assay, nematode being more pathogenic at lower dose than at higher ones. The optimum dose for nematode penetration and establishment in T. molitor larvae was 80 nematodes/larvae which average number of progeny per female is 5438. Under crowded conditions development proceeds to IJ stage instead of the 3rd juvenile stage (J3). The average length (Y = ‐ 16.714x + 602; R2 = 0.8474) and width (Y = ‐ 1.1857x + 30.857; R2 = 0.967) decreased with increasing of nematode doses. The IJ produced in cadavers infested with 640 nematodes/larvae were significantly smaller (492 ± 6.4 µm) than offspring from other doses. The number of days which nematodes first emerged from the cadavers decreased with increasing dose (Y = ‐ 1.2286x + 18.943; R2 = 0.9904). IJ first emerged on average 10‐13 days after infestation at high IJ densities, while in larvae infested with 10‐40 IJ/larva the emergence period began 15‐17 days after infection, respectively. It is conclude that the wide experimental characteristic of EPNs, also hold true for Steinernema sp., isolate Skpr‐20/Str. Key words: endotokia matricida, entomopathogenic nematode, Steinernema sp., isolate Skpr‐20/Str.

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N06 A STUDY ON THE GROWTH OF OLIVE CULTIVARS SEEDLINGS IN THE PRESENCE OF VERTICILLIUM WILT, VERTICILLIUM DAHLIAE, AND ROOT-KNOT

NEMATODE, MELOIDOGYNE JAVANICA

Ayatollah SAEEDIZADEH1, Ahmad KHEIRI2, Javad ZAD2, Hasan REZA ETEBARIAN3, Ali REZA BANDANI2 & Fahimeh NIASTI1

1 Dep. of Plant Protection, Shahed University, Tehran, Iran 2 Dep. of Plant Protection, Agriculture Campus, University of Tehran, Karaj, Iran

3 Dep. of Plant Protection, Aboureyhan Campus, University of Tehran, Iran In this study the non‐defoliant strain of the fungus (SS‐4) was isolated from olive groves showing symptom of the disease from Toshan area in southern Gorgan city. Root‐knot nematode, M. javanica, was recovered from infested olive seedling and after identification it was propagated on tomato seedlings cv. Rutgers. In this study one‐year‐old seedlings of olive cultivars, Zard, Roghani, Koroneiki and Manzanilla, were transplanted to pots containing 2000g of sterilized sandy loam soil. Experiment was conducted in completely randomized design with 32 treatments and five replications. Treatments were as fallows: control, nematode alone, fungus alone and fungus+nematode. Pots were inoculated with (2000, 3000, 4000) J2 of nematode and/or (10 no/g soil) microsclerotia of fungus according to the treatments. Pots were arranged in glasshouse benches with the temperature of 25‐27°C and natural light. Experiment was terminated after 10 months and fallowing parameters were determined i.e., fresh weight of root and stem, no. of galls and egg masses per root system, and percentage of incidence of symptom on aerial part, browning of vascular tissue, decrease of seedling height and stem/root tissue colonization by fungus. Results showed that presence of nematode caused reduction on colonization of the fungus in the root and stem and vice versa i.e. presence of fungus caused reduction on no. of galls and egg masses produced by the nematode. Whereas maximum of percentage of disease symptoms on aerial part was observed in fungus+nematode treatments. Severe fungal wilt on aerial part of Zard cultivare was observed in fungus+nematode treatments and mild fungus wilt was observed in fungus alone treatments of Koroneiki cultivar. Galling and egg mass production in root system were reduced in cvs. Manzanilla, Zard, Roghani and Koroneiki, respectively (p≤0/05). Key words: root‐knot nematode, verticillium wilt, seedling growth, olive

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N07 IDENTIFICATION OF FUSARIUM SPP. ISOLATED

FROM EGG MASSES AND FEMALES OF MELOIDOGYNE JAVANICA IN GREENHOUSES OF KERMANSHAH

PROVINCE, IRAN

R.KARKHANEH1, A.A. HOJAT-JALALI1 & M. SHEIKHOLESLAMI2 1 Dep. of Plant Protection, College of Agriculture, University of Kordestan, Iran

2 Dep. of Plant Protection, Agricultural Research Center of Kermanshah, Iran Meloidogyne spp. especially M.Javanica are among the most important agents to limit growing greenhouse crops in Kermanshah province, Iran. In order to study on natural contamination of egg masses and females of M.Javanica to soil‐borne pathogenic fungi these organs were initially disinfected with NaOCl 0.5% and after drying placed on various culture media containing antibiotic streptomycin sulfate. Grown fungi were purified through single spore or hyphal swelling methods. Many isolates belong to Fusarium spp. including F.chlamydosporium, F.oxysporum, F.solani, F.tricinctum and F.verticilloides were identified. This is the first report of these fungi isolated from M.javanica in Iran and 4 species including F. chlamydosporium, F.proliferatum, F.tricinctum and F. verticilloides are reported for the first time on M.javanica in the world. F.solani specimens had the highest number of isolates among these 6 species. Key words: Fusarium, Meloidogyne, biological control, fungi

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N08 NEW RECORD OF SOME MITOSPORIC FUNGI

ISOLATED FROM EGG MASSES AND FEMALES OF MELOIDOGYNE JAVANICA IN COMMERCIAL

GREENHOUSES OF KERMANSHAH PROVINCE, IRAN

R. KARKHANEH1, A.A. HOJAT-JALALI1 & M. SHEIKHOLESLAMI2 1 Dep. of Plant Protection, College of Agriculture, University of Kordestan. Iran

2 Dep. of Plant Protection, Agricultural Research Center of Kermanshah, Iran Meloidogyne javanica is the most widespread species of Meloidogyne spp. to damage greenhouse crops in Kermanshah province, Iran. In order to study on natural contamination of M.javanica to pathogenic fungi and then application of these fungi for biological controlling actions some experiments were performed. Females and egg masses of M.javanica were isolated from root knots of greenhouse cucumber and tomato plants. After disinfection of these organs with NaOCl 0.5% and then drying, they were placed on various culture media containing antibiotic streptomycin sulfate. 160 specimens of fungi were taxonomically identified based on morphometric specifications. These fungi belonged to 24 species in 15 Genera. Among these species Cladosporium lucidum, C. macrocarpum, Penicillium coprophilum, Peyronellaea glomerata, Plectosporium tabacinum, Sporothrix schenckii, Ulocladium chladosporium and Beniowskia sp. Are reported for the first time on M. javanica in the world. Key words: Meloidogyne, biological conrol, mitosporic fungi

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N09 MAPPING OF NEMATODE DISTRIBUTION AND

ASSESSMENT OF ITS ECOLOGICAL STATUS USING GIS TECHNIQUES IN PLOVDIV REGION, BULGARIA

Tatyana BILEVA & Zhulieta ARNAUDOVA

Dep. Melioration and land surveying; Dep. Ecology and Environmental Protection The current investigation was carried out in the vineyards in Plovdiv region, South Bulgaria. The plant‐parasitic nematodes from family Longidoridae associated with grapevine of the region were described using GIS application. The impact of longidorids as virus vectors and some ecological aspects of their occurrence in vineyards in Plovdiv region, Bulgaria are discussed. This paper focuses on mapping of nematode distribution assessment using GIS technique. Key words: GIS techniques, plant‐parasitic nematodes, Longidoridae, soil type, grapevine

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N10 THE USE OF GIS TO SUPPORT SUSTAINABLE MANAGEMENT OF VINEYARDS IN PLOVDIV

REGION, BULGARIA

Zhulieta ARNAUDOVA & Tatyana BILEVA Dep. Melioration and land surveying

Dep. Ecology and Enveronmental protection Agricultural University, Plovdiv, Bulgaria Vine is a traditional branch of plant growing in Bulgaria. The exact location of the vine culture is a specific complex of environmental factors influencing its development, such as climate, soil, landscape and traditions of the region. GIS is a platform to better manage, evaluate and present spatial data in a useful visual form. It’s improves the decision‐making by combining data with accurate location and management the vineyards. In the present survey were studied and analyzed the factors in choosing an appropriate location for the cultivation of vine varieties in selected regions of Plovdiv. Climatic and soil characteristics, topography and environmental factors as well as presence of virus vector nematodes from family Longidoridae in creating the vines through GIS spatial analysis are taken into account. Key words: GIS, Longidoridae, sustainable management, vine, environmental factors

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Posters Exploiting genomics to understand plant‐nematode interactions (COST Action 872) – part 1

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217

C01 ANALYSIS OF ARBUSCULAR MYCORRHIZA-

INDUCED DEFENSE AGAINST MELOIDOGYNE INCOGNITA EARLY INFECTION IN TOMATO

Christine VOS1, Annemie ELSEN2, Bart PANIS1, Odile CHATAGNIER3,

Diederik VAN TUINEN3, Vivienne GIANINAZZI-PEARSON3

& Dirk DE WAELE1 1 Laboratory of Tropical Crop Improvement, Katholieke Universiteit Leuven

Kasteelpark Arenberg 13, BE-3001 Heverlee, Belgium 2 Bodemkundige dienst van België

Willem de Croylaan 48, BE-3001 Heverlee, Belgium 3 UMR INRA/CNRS/Université Bourgogne Plante-Microbe-Environnement

BP 86510, FR-21065 Dijon Cedex, France Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts colonizing the majority of vascular plants on earth. These fungi receive photosynthetic carbon from their host, while the plant benefits from improved water and nutrient uptake, resulting in enhanced growth and survival. In addition, AMF have been shown to reduce the occurrence and impact of several soil pathogens, including plant‐parasitic nematodes, on a wide range of agricultural crops. A well‐established AMF‐colonization is a prerequisite for AMF‐induced biocontrol against nematodes, both locally and systemically. Apart from these observations however, the defense mechanisms involved have rarely been investigated for AMF‐nematode interactions. Our previous experiments indicated that the presence of AMF already influences the root‐knot nematode in its early stage of plant infection. In order to understand the underlying mechanisms of this effect, we analyzed the AMF‐plant‐nematode interaction during early nematode infection at transcriptome level. A suppressive subtractive hybridization (SSH) experiment was conducted, using the Clontech PCR‐select cDNA subtraction kit. Briefly, in this experiment the tomato mRNA population of the bioprotection treatment was compared with the nematode‐infected and the mycorrhizal treatments in two subsequent subtrac‐tions, to significantly enrich the differentially expressed sequences linked to bioprotection. The resulting cDNA fragments were then amplified and cloned into the pCR4‐TOPO vector, yielding 467 clones. Of these obtained clones, 190 were selected for sequencing. Finally, 50 tomato genes could be identified, mainly involved in signal transduction and secondary metabolism. Transcript abundance of a selection of the genes will be further examined by qPCR. This work was supported by an STSM grant from the EU COST action 872 ‘Exploiting genomics to understand plant‐nematode interactions’.

Posters C01-C18

218

C02 USE OF DSRNA EXPRESSING PLANTS TO

KNOCK DOWN GPCR GENES IN MELOIDOGYNE INCOGNITA

S. ARCIELLO1, A. TORTORA1, M.T. MELILLO2, M. BIMONTE1,

G. COLUCCI1 & F. APONE1 1 Arterra Bioscience srl, via Brin 69, Napoli, Italy

2 IPP, CNR, via Amendola 165/A, Bari, Italy G protein coupled receptors (GPCR) represent key targets for novel strategies to fight plant parasitic nematodes, since these receptors are involved in many vital functions of the worm, such as reproduction, feeding and movement. According to some studies carried out by using C. elegans as model system, receptors involved in the recognition of biogenic amines and neuropeptides represent the most useful target to reduce nematode attacks to plants. After cloning 7 different GPCR genes from Meloidogyne incognita cDNA, we chose two of these genes to be expressed as double strand RNA (dsRNA) in tabacco plants. The transgenic plants expressing the dsRNA were used in infection experiments by using J2 larvas of Meloidogyne incognita. The results showed that the worms fed on transgenic plants, which expressed the dsRNA corresponding to the receptor MiNPR1‐like, had serious developmental and reproductive defects, compared to worms fed on wt untransformed plants. Moreover, J2 larvas derived from a first infection cycle were less aggressive in infecting new plants and produced a lower number of egg‐containing galls, suggesting that GPCR genes represent excellent targets to control nematode infection.

Posters C01-C18

219

C03 A NOVEL ARABIDOPSIS PP2C-TYPE PHOSPHATASE

IS INVOLVED IN NEMATODE INDUCED PLANT SIGNALING CASCADES

Ekaterina SIDONSKAYA1, Irute MESKIENE2, Krzysztof WIECZOREK1

& Julia HOFMANN1 1Institute of Plant Protection, University of Natural Resources and Applied Life Sciences

AT-1190 Vienna, Austria 2Max F. Perutz Laboratories of the University of Vienna, AT-1030 Vienna, Austria

Plant‐parasitic nematodes infect roots of host plants and thereby activate signalling mechanisms that lead to the significant reorganization of the expression and regulation of relevant plant genes. These changes trigger the formation of the sophisticated feeding structures, such as syncytia of cyst nematodes. MAPK cascades may be employed activating the host defence‐related gene expression. Protein phosphatases of type 2C (PP2C) are implicated in the regulation of MAPK signalling pathways. By use of the GUS reporter gene system we showed a specific and early activation of the phosphatase promoter in early stages of syncytium development. To determine the response of Arabidopsis to the cyst nematode Heterodera schachtii mutant line of the phosphatase has been examined. Further, the pertinence of phosphatase during nematode infection has been studied by kinase assay using mutant and overexpressing lines. Our results suggest that the PP2C‐type phosphatase might play an essential role in the early signalling events during plant‐nematode interaction.

Posters C01-C18

220

C04 AAA+ ATPASE FAMILY GENE AT1G64110 IS

INVOLVED IN THE DEVELOPMENT OF SYNCYTIA INDUCED BY HETERODERA SCHACHTII

Muhammad AMJAD ALI1, Stephan PLATTNER1, ABD-EL NASER EL ASHRY1,2,

Krzysztof WIECZOREK1, Shahid SIDDIQUE1,2, Moritz AMMELBURG3

& Holger BOHLMANN1 1Institute of Plant Protection, Department of Plant Sciences and Plant Biotechnology, University

of Natural Resources and Life Sciences, Peter Jordan-Straße 82, AT-1190 Vienna, Austria. 2Institute of Phytomedicine, University of Bonn, Bonn, Germany

3Department of Protein Evolution, Max-Planck-Institute for Developmental Biology DE-72076 Tübingen, Germany

The plant parasitic cyst nematode Heterodera schachtii induces specialized feeding sites in the roots of Arabidopsis called syncytia. A transcriptome study of nematode‐induced syncytia by Heterodera schachtii in Arabidopsis roots showed a significant upregulation of various genes including At1g64110. At1g64110 is a member of a small gene family within the large group of AAA+ ATPases. The results from RT‐PCR and qRT‐PCR and in situ hybridization of 5 and 15 dpi syncytia confirmed the gene chip data and showed that the gene At5g52882, which is not covered by the gene chip, is also expressed in syncytia. Promoter::GUS fusion lines were developed using different lengths of promoter of At1g64110. All the promoter lengths showed GUS staining in syncytia at 5, 10 and 15 dpi. Moreover, histochemical GUS assays displayed GUS staining especially in imbibed seeds and sperm cells. The gene expression was modified by developing artificial microRNA (amiRNA) constructs using the constitutive CaMV promoter and promoters like PDF2.1 and MIOX5 with strong expression in syncytia. The knock down lines from PDF and MIOX promoters showed silencing of the gene which resulted in a reduced number of female nematode per unit root length. The T‐DNA knock out mutants of gene At1g64110 also showed a reduced number of females as compared to wild type plants. The results indicate that the AAA+ ATPase family gene At1g64110 is important for the function of syncytia in Arabidopsis roots.

Posters C01-C18

221

C05 THE ROLE OF THE SUGAR-BREAKDOWN GENES

IN THE INTERACTION BETWEEN THE BEET CYST NEMATODE HETERODERA SCHACHTII AND

ITS HOST ARABIDOPSIS THALIANA

Susana CABELLO & Julia HOFMANN University of Natural Resources and Applied Life Sciences, Institute of Plant Protection,

Department of Applied Plant Sciences and Plant Biotechnology, Peter-Jordan-Strasse 82, AT-1190 Vienna, Austria

The obligate plant‐parasitic cyst nematode Heterodera schachtii induces highly specific feeding cell system in the central cylinder of plant root, which serves as a source of nutrients and thus energy. Among different sugars, sucrose is the major transport molecule in the phloem of Arabidopsis thaliana, and is therefore expected to be the major sugar imported into nematode‐induced feeding structure. In plants it needs to be degraded by a sucrose synthase (SUS) or an invertase (INV) in order to make it available for energy gaining or the synthesis of macro‐molecules like cellulose or starch. Two simple sugar residues, fructose and glucose, are the products of this reaction. In order to elucidate whether the sucrose‐converting enzymes involved in this process play a role in nematode feeding structure, we analyzed the development of H. schachtii in different sus and inv Arabidopsis T‐DNA lines (Barratt et al., 2009). In this study we found that most of the mutants showed an increase in the infection rate. In addition, the double mutant of cytosolic invertases cinv1/cinv2 showed also an increase in size of the females as well as in egg production. These results suggest that the lack of sucrose cleavage, what results in an elevated concentration of sucrose, might be beneficial for the nematode development.

Posters C01-C18

222

C06 RAPID MICROSCOPIC AND MOLECULAR METHOD

OF IDENTIFICATION OF LEAF PARASITISING APHELENCHOIDES SPECIES

Aneta CHALANSKA1, Tadeusz MALEWSKI2 & Gabriel LABANOWSKI1

1 Institute of Horticulture PL-96-100 Skierniewice, Konstytucji 3 Maja 1/3, Poland

2 Museum and Institute of Zoology, Polish Academy of Sciences PL-00-979 Warsaw, Wilcza 64, Poland

Aphelenchoides sp. (Nematoda) feeding on plant leaves are increasing problem in ornamental plant nurseries. Leaves of some ornamental shrubs and many perennials can be injured by: A. ritzemabosi, A. fragariae or A. besseyi. However, only A. besseyi is the quarantine organism being subjected to the obligatory control, but has not yet detected in ornamental plant nurseries in Poland. Occurrence of A. ritzemabosi and A. fragariae does not entail legal sanctions, but they cause economic loses, thus it is necessary to evaluate their occurrence and control their distribution and dispersal. To achieve this, we have developed a quick and easy procedure distinguishing these species on the basis of the morphometric and molecular features. A microscopic examination of ten basic taxonomic features was conducted on 10 individuals of each species. The range of value of individual features as well as essential differences between these features among compared species were statistically quantified. Additionally, a molecular test based on Real‐Time PCR was developed to discriminate A. besseyi, A. ritzemabosi and A. fragariae. Comparing sequences of cytochrome c oxidase subunit I (COI) gene from three Aphelenchoides sp. species‐specific primers were designed. These primers demonstrated the sensitivity and specificity in qPCR detection assay carried out on field samples.

Posters C01-C18

223

C07 DURABILITY OF THE FIRST FRENCH POTATO

CULTIVAR FOR HUMAN CONSUMPTION RESISTANT TO GLOBODERA PALLIDA

Sylvain FOURNET2, Marie-Claire KERLAN1 & Eric GRENIER2

1 INRA - Agrocampus Ouest - Univ Rennes 1, UMR118 Amélioration des Plantes et Biotechnologies Végétales

FR-29260 Ploudaniel, France 2 INRA, Agrocampus Rennes, Université Rennes 1

UMR1099 BiO3P, FR-35653 Le Rheu, France Different oligogenic resistances to the potato cyst nematode Globodera pallida were introduced from wild species (Solanum sparsipilum, S. spegazzinii and S. vernei) in cultivated potato. At INRA we have focused most on these 20 last years on the S. vernei resistance and the first cultivar carrying this highly effective resistance was registered in 2009 in the French potato catalog. Its durability was estimated by studying the adaptation of French G. pallida populations to 4 potato genotypes carrying the S. vernei resistance during eight years of continuous selection pressure. After five years of continuous selection pressure, some circumvention of the resistance was already observed in some potato genotypes and not with others. It may be inferred that the resistance from S. vernei is durable in some genotypes and not in others even when using the same resistant source. These results strengthen the need to manage the use of these resistances to nematodes and to increase our knowledge of the processes that govern pathogen evolution.

Posters C01-C18

224

C08 USE OF RNA POLYMERASE II SEQUENCES TO

ELUCIDATE THE EVOLUTIONARY ORIGIN OF ROOT-KNOT NEMATODES

K.D. RYBARCZYK-MYDŁOWSKA, H.H.B. VAN MEGEN, S.J.J. VAN DEN ELSEN,

P.J.W. MOOIJMAN & J. HELDER Laboratory of Nematology, Department of Plant Sciences, Wageningen University,

Droevendaalsesteeg 1, NL-6708 PB Wageningen, The Netherlands. A Small Subunit ribosomal DNA tree of the order Tylenchida containing most of the economically important plant parasitic species suggests that root‐knot nematodes (Meloidogyne spp.) are nested in, and derived from representatives of the genus Pratylenchus (lesion nematodes) (Holterman et al., 2009). However, a precise origin of root‐knot nematodes and their relationship with the Pratylenchidae remained unresolved. A new phylogenetic analysis of pathogenicity‐related gene, namely β‐1,4‐endoglucanase (a cellulase from glycosyl hydrolase family 5), confirms the overall topology of the SSU rDNA tree (manuscript in preparation). Moreover, it shows stronger support for the Pratylenchus origin of root‐knot nematodes. The basal positioning of M. ichinochei within the Meloidogyne clade, is confirmed by cellulase data, and our results suggest Pratylenchus thornei to be sister to all Meloidogyne species. Currently, we are testing RNA polymerase II gene, a single copy gene in most eukaryotes, to further confirm this hypothesis. We designed and tested semi‐universal rbp1‐primers for the amplification of the largest subunit of the RNA polymerase II gene (rbp1), and this allowed us to generate rbp1 sequences from numerous root‐knot and lesion nematodes. The phylogenetic tree base on those sequences will be presented during the COST 872 – 2011 meeting.

Posters C01-C18

225

C09 SPRYSECS SECRETED BY POTATO CYST

NEMATODES ACT AS ELICITORS AND SUPPRESSORS OF PLANT INNATE IMMUNITY

Wiebe POSTMA, Sajid REHMAN, Kamila KOROPACKA, Tom TYTGAT,

Hein OVERMARS, Casper VAN SCHAIK, Aska GOVERSE, Jaap BAKKER & Geert SMANT

Laboratory of Nematology, Wageningen University Droevendaalsesteeg 1, NL-6708PB Wageningen, The Netherlands.

Plant parasitic nematodes secrete effectors from their esophageal glands to invade the host, modify plant cells into feeding sites, and to suppress plant innate immunity. The sedentary biotrophic nematodes feed from their feeding site for several weeks, and it is thus extremely important to protect this feeding site from adverse responses by the plant. Recent transcriptome analysis of plant parasitic nematodes has revealed that these plant microbes released several hundred effectors in the plant to establish a long‐lasting infection. It is our objective to identify and functionally characterize nematode effectors that are modulating plant innate immunity. We have previously shown that potato cyst nematodes (Globodera rostochiensis) secrete SPRY‐domain containing effectors (SPRYSECs). We hypothesise that SPRYSECs are able to elicit and suppress immune responses via interactions with CC‐NB‐LRR type resistance proteins.

Posters C01-C18

226

C10 REPROGRAMMING ROOTS FOR GALL

DEVELOPMENT: THE FUNCTION OF TRANSCRIPTION FACTORS LBD16 AND HSFB4

Alejandra GARCÍA1, Javier CABRERA1, María SÁNCHEZ1, Fernando E. DÍAZ1, Marta BARCALA2, Julia HOFFMAN,

Carmen FENOLL1 & Carolina ESCOBAR1 1Facultad de Ciencias del Medio Ambiente y Bioquímica, Universidad de Castilla-La Mancha,

Avenida de Carlos III s/n, ES-45071 Toledo, Spain. 2Centro de Biología Molecular 'Severo Ochoa'-CSIC, Universidad Autónoma de Madrid

ES-28049 Madrid, Spain How root‐knot nematodes manipulate the plant metabolism to induce giant cells (GCs) into the vascular cylinder remains still unclear. Our previous transcriptomic analysis showed that a high number of genes encoding transcription factors (TFs) were differentially expressed in micro‐dissected GCs (Barcala et al., 2010., Portillo et al., 2009). TFs are key regulators of signalling cascades and can control the expression of groups of genes. Thus, we are studying the putative role of some of these TFs during nematode infection. LBD16is induced specifically in the galls formed by Meloidogyne javanica as soon as the nematode is established in the vascular cylinder, its expression is maintained until 12‐15 days after infection (dai). The activity was centred in GCs and in a few layers of newly forming cells inside the vascular cylinder surrounding them. This pattern correlated with the GUS activity of an auxin synthetic promoter DR5, also induced very early in the infection. However, activity of DR5 was maintained during the whole gall development, till the completion of nematodes cycle. Conversion of LBD16 in a dominant repressor by fusing the protein to the suppression domain SRDX, caused a pronounced decrease of galling by Meloidogyne javanica. However, infection by Heterodera schachtii was not affected. This suggest that LBD16 could play a crucial role in galls but not syncitia development. Another TF early induced in GCs was the HSFB4. Its transcripts were significantly induced in the transcriptome of laser‐micro‐dissected GCs with a fold change of 2.2 and p<0.04. Promoter::GUS fusions confirmed its activation pattern from early infection stages (2‐3 dai) in galls. In addition, mutants in both the Col‐o and Ler backgrounds confirmed that the gall formation was compromised. HSFB4, has recently been described as a crucial factor for cell fate determination in roots and it is suggested that HSFB4 might be integrated into signaling pathways different to the heat‐shock response. These results suggest that both transcription factor tested, LBD16 and HSFB4 play crucial roles in galls development.

Posters C01-C18

227

C11 MOLECULAR VARIABILITY AND PHYLOGENETIC RELATIONSHIPS OF PRATYLENCHUS SPECIES

BY USING THE ITS rDNA

Francesca DE LUCA1, Aurelio REYES2, Alberto TROCCOLI1, Elena FANELLI & Pablo CASTILLO3

1 Institute of Plant Protection, CNR, Bari, Italy 2 MRC-Dunn Human Nutrition Unit, Cambridge, UK

3 Institute for Sustainable Agriculture (IAS), CSIC, Cordoba, Spain Root‐lesion nematodes of the genus Pratylenchus are migratory endoparasites distributed worldwide and regarded as severe constraints of economic crops. Crop damage is often aggravated by their interactions with soilborne fungi and bacteria, resulting in complex diseases which are biological and physiological rather than physical in nature. Morphological identification and species delimitation of these nematodes is still problematic due to their high morphological plasticity, the small number of diagnostic features available at species level, the intraspecific variability of some of these characters and many incomplete descriptions published in the literature. Thus, molecular approach, by using the ribosomal DNA, plays a key role within the taxonomy of this genus. The ITS containing region of eighteen species of Pratylenchus was sequenced and the variation within and between species was assessed. Phylogenetic analyses were conducted by using different tree reconstruction approaches: Bayesian inference (BI), neighbour‐joining Log‐Det (NJ), maximum parsimony (MP) and maximum likelihood (ML). The alignment revealed small species‐specific DNA sequences suitable for the construction of potentially useful species‐specific primers or for a more promising approach for DNA barcoding of root‐lesion nematodes.

Posters C01-C18

228

C12 SEQUENCE VARIABILITY OF EXPANSIN B LIKE GENES IN THE GLOBODERA PLANT-PARASITIC

NEMATODES

Barbara GERIČ STARE1, Janja GORŠEK1, Sasa ŠIRCA1, Gregor UREK1 & Eric GRENIER2

1 Agricultural Institute of Slovenia, Plant Protection Department Hacquetova 17, SI-1001 Ljubljana, Slovenia

2 INRA, UMR1099 BiO3P (Biology of Organisms and Populations applied to Plant Protection), Domaine de la Motte, BP 35327, FR-35653 Le Rheu, France

When plant parasitic nematodes invade the plant, they excrete the whole arsenal of effectors including a variety of plant cell wall degrading enzymes. We have evaluated molecular variability of expansin like genes in different Globodera nematodes. Our results revealed that there are at least three copies of expB genes present in the potato cyst nematode G. rostochiensis. ExpB3 gene sequences of the closely related species G. tabacum and G. pallida were analysed to confirm whether the evolution of expansins was similar in the genus Globodera as that we have previously observed for the pectate lyases (pel1 and pel2); ie: a topology of the phylogenetic trees different from the admitted species topology. Our results suggest more members of the expansin B like genes family.

Posters C01-C18

229

C13 BARCODING QUARANTINE NEMATODES AND

THEIR CLOSE RELATIVES: AN UPDATE ON THE STATUS OF THE QBOL-PROJECT

Martijn H.M. HOLTERMAN1, Jürg E. FREY1, Hans HELDER2,

Paul J.W. MOOYMAN2 & Sebastion KIEWNICK1 1 Agroscope Changins-Wädenswil

Schloss, CH-8820 Wädenswil, Switzerland 2 Laboratory of Nematology, Wageningen University

Droevendaalsesteeg 1, NL-6708 PB Wageningen, The Netherlands Identification of plant pests, in particular quarantine species, needs to be fast and accurate to enable timely plant protection measures. In addition, a false diagnosis can cause serious financial losses for trade and producers. It is now well established that genetically based diagnosis is a reliable alternative to the classical identification procedures generally based on morphological features, which usually require expert taxonomic skills. On the other hand, genetic diagnosis through the use of so‐called DNA‐barcodes, stretches of DNA that contain taxon‐specific information, can be performed by any skilled lab‐worker. The European Union 7th framework program QBOL aims to establish DNA‐barcodes for all European quarantine organisms as well as their close relatives and disseminate the results and protocols in a publicly available and curated database. To enable genetically based identification requires knowledge of the genetic variation both within and between the species of interest as well as their close relatives. Several gene regions (the COI, COII, SSU, LSU and RNA polymerase subunit II) are being evaluated for their barcoding potential. An update on the current status of the nematode part of QBOL will be provided as well as an example case of the genus Meloidogyne.

Posters C01-C18

230

C14 RNAi-MEDIATED CROP IMPROVEMENT FOR

SUSTAINABLE RESISTANCE TO GLOBODERA PALLIDA

Richard M. COLLINS, Howard J. ATKINSON & Peter E. URWIN Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK

The potato cyst nematode, Globodera pallida, is one of the most economically important nematodes to UK arable agriculture. Current control measures are largely dependent on chemical nematicides and there is increasing demand for alternative control methods.One potential method is the use of transgenic plants that express dsRNA with homology to nematode or plant genes that are essential for maintenance of the parasitic interaction. The dsRNAs trigger RNA interference (RNAi) leading to transcript depletion and gene silencing. RNAi of nematode digestive genes was initially demonstrated through in vitro soaking experiments and subsequent infection assays and by using transgenic potato hairy root lines transformed with hairpin constructs. Both approaches resulted in a 50‐60% reduction of established nematodes. Transgenic potato plants expressing hairpin constructs have since been produced and screened for resistance in both containment and field trials. RNAi of plant genes has focused on those specifically expressed in the nematode feeding cell. A potato MIOX gene has been identified that is expressed within the syncytium and anther tissues. Potato plants expressing dsRNA to silence this gene have also been screened for nematode resistance. In addition, we are exploring approaches to increase the efficacy of resistance.

Posters C01-C18

231

C15 FROM WORM TO DATABANK, AN INTEGRATED

APPROACH TO COMBINE MORPHOLOGY, MOLECULES AND OTHER RELEVANT NEMATODE DATA

Wim BERT, Pamela FONDERIE & Hanne STEEL Nematology Unit, Department of Biology, Ghent University

Ledeganckstraat 35, BE-9000 Ghent, Belgium Exciting new molecular directions in nematology are vastly growing, including sequence‐only studies that rely on single sequences as a proxy for biological data. On the other hand, plant‐parasitic nematode surveys and descriptions are still often based on morphological and morphometrical criteria without any connection to informative molecules. The latter approach relies on a taxonomic expertise that is strongly declining.We want to reduce the gap between “traditional” taxonomic expertise and emerging next‐generation molecular methods, using efficient digital dissemination of a combination of multifocal images, a morphometrical dataset and molecular data. This should be stored in an open database and connected with other associated information (e.g. biographical‐, ultrastructural‐, ecological‐, physiological‐ data and taxonomic literature). The advantages of an open access of vouchers and metadata are numerous: (1) global access to specimen‐based information would allow taxonomists in the developing world to far more efficiently contribute to the global taxonomic effort (2) more insight in the value of single sequences as a proxy to infer comprehensive information associated with organism based biology (3) improved quality control in species delimitation (4) promotion of reciprocal illumination (morphology vs. molecules) in phylogenetic studies.

Posters C01-C18

232

C16 CHARACTERIZATION OF EFFECTORS FROM THE POTATO CYST NEMATODE GLOBODERA PALLIDA

Peter THORPE1,2, John T. JONES1, Sophie MANTELIN1, Mirela COKE2, Catherine J. LILLEY2 & Peter URWIN2

1Plant Pathology Programme, SCRI, Invergowrie, Dundee, DD2 5DA, UK 2IICB, University of Leeds, Leeds, LS2 9JT, UK

The potato cyst nematode Globodera pallida is an economically important pathogen of potatoes. During a parasitic infection the nematode induces complex changes to host gene expression to induce a specialised feeding site termed a syncytium, from which the animal feeds throughout the rest of it’s development. During parasitism the nematode injects secretory products made in the oesophageal glands into the plants via a stylet. These secretions are important in establishing and maintaining the syncytium, and are often termed effector proteins. A putative pool of effector genes has been identified and constructs have been developed to allow constitutive over‐expression in Arabidopsisthaliana and Solanum tuberosum cv. Desiree. Preliminary results have shown differential growth between transgenic lines. The transgenic plant lines will be challenged with nematodes and other pathogens in order to identify lines that are compromised in their ability to respond to mount a defence response. This may allow candidate genes that are essential for suppression of the host defences to be identified. Such genes will be taken forward for further characterisation.

Posters C01-C18

233

C17 IDENTIFICATION OF CEREAL CYST NEMATODE

SPECIES WITH MORPHOMETRIC AND MOLECULAR METHODS IN TURKEY

I. HALIL ELEKCIOĞLU1, HALIL TOKTAY2, MUSTAFA İMREN2, E. SAHIN3,

A.A. DABABAT4 & J.M. NICOL4 1 Çukurova Üniversitesi, Ziraat Fakültesi, Bitki Koruma Bölümü, 01360, Sarıçam, Adana

2 Zirai Mücadele Araştırma Enstitüsü, 01321, Adana, Turkey 3 Karamanoğlu Mehmetbey University, Karaman, Turkey

4 CIMMYT (International Maize and Wheat Improvement Centre), Turkey Office, Ankara, Turkey Turkey is one of the world’s larger wheat producers with more than 20Mt of wheat produced per year and average yields around 3 t/ha. The Turkish Central Anatolian Plateau (CAP) is the ‘bread basket’ of Turkey with over 5Mha rain fed wheat area. Southeast Anatolia (SEA) region is the second important wheat production area in Turkey. Diploid and durum wheat were firstly cultivated historically in this region especially Karacadag/Diyarbakır province. This region is very rich for wild genotypes of wheat such as Triticum dicoccoides, T. urartu, T. boeoticum. T. dicoccoides has an important role in the wheat evolution. It is therefore very important to determine nematode fauna in these regions for possible genetic control strategies.Cereal Cyst nematodes (Heterodera spp.) are very important pest of wheat especially rainfed monoculture wheat system. Intensive survey studies carried out in wheat areas in both regions to determine Heterodera species. Nematodes were identified morphologically and molecularly using PCR‐RFLP techniques. The obtained results show the predominant species on the rain‐fed winter wheat production system of the Central Anatolian Plateau is H. filipjevi. While, in the semi‐arid wheat growing area of Turkey, Southeast Anatolia region, where spring wheat mostly grown, the main species are H. avenae and H. latipons. The presence of the different species in the different regions mainly contributed to the different climatic conditions.

Posters C01-C18

234

C18 IDENTIFICATION OF PUTATIVE PARASITISM

GENES FROM WHOLE GENOMES AND TRANSCRIPTOMES OF PLANT-PARASITIC NEMATODES

Amandine CAMPAN-FOURNIER1, Marie-Jeanne ARGUEL1,

Laetitia PERFUS-BARBEOCH1, Marie-Noëlle ROSSO1, Corinne DA SILVA2, Celine VENS3, Jérôme GOUZY4, Martine DA ROCHA1, Marc MAGLIANO1,

Nathalie MARTEU1, Karine LABADIE2, François ARTIGUENAVE2, Pierre ABAD1 & Etienne G.J. DANCHIN1

1 INRA, UNS, CNRS, UMR 1301 Biotic Interactions and Plant Health 400 route de Chappes, Sophia-Antipolis, FR-06903, France

2 CEA, Institut de Génomique, Genoscope, Evry, France 3 Katholieke Universiteit Leuven, Department of Computer Science

Celestijnenlaan, BE-3001 Leuven, Belgium 4 INRA, CNRS, UMR 0441 LIPM Plants - Micro-organisms Interaction, Castanet-Tolosan, France Initial analysis of the genome of Meloidogyne incognita has allowed the identification of thousands of genes that apparently did not have homologs in model animal and fungal species. A proportion of these M. incognita‐restricted gene models may be involved in functions linked to plant‐parasitism. To identify genes putatively involved in adaptation to parasitism in root‐knot nematode genomes, we have undertaken a comparative analysis of the sets of predicted genes in M. incognita and M. hapla against a large dataset of whole genomes and transcriptomes. As our objective is to identify druggable parasitism genes we have searched for genes conserved in other parasitic or plant‐associated species (including bacteria, fungi and oomycetes) but absent from the genomes of species that could be negatively affected by newly developed drugs or control means (e.g. plants, vertebrates, annelids..). The rationale of our analysis is that the more a gene is conserved in plant‐parastic or plant‐pathogenic species while absent from model non‐parasitic species, the more likely it is to be involved in key parasitism processes. To partially overcome the scarcity of omics data available for plant‐parasitic nematodes, we have initiated RNA‐seq transcriptome sequencing of four plant‐parasitic nematode species presenting diverse parasitic strategies (Pratylenchus coffeae, Ditylenchus dipsaci, Bursaphelenchus xylophilus and Xiphinema index). We have also generated the RNA‐seq of different developmental stages of M. incognita in order to bring additional and dynamic transcription support to the identified genes. We have performed bioinformatics annotation of genes associated with plant parasitism, including but not limited to: detection of signal peptide and Pfam domains, assignment of gene ontology terms, and identification of specific motifs. Using RNAi assays, we will test the effect of gene silencing for a series of the most promising targets identified during bioinformatics screens.

Posters P01-P22

235

Posters Phytopathology

Posters P01-P22

236

Posters P01-P22

237

P01 HARMFULNESS OF STAGONOSPORA NODORUM

LEAF BLOTCH IN SPRING BARLEY IN LATVIA

O. TREIKALE, B. JAVOISHA & J. PUGACHEVA Plant Pathology, Latvian Plant Protection Research Centre, Riga, Latvia

In Latvia, spring barley is one of the most important agricultural crops: cultivation area is 94.6 thsd. ha or 20% of total area under cereals. The average yield of spring barley in 2009 was 2.2 t ha‐1. The yield and grain quality depends on many factors included preventive and curative measures to control harmful organisms. The most widespread diseases of barley in Latvia are net blotch (Pyrenophora teres) and powdery mildew (Blumeria graminis). In last years the leaf blotch caused by Stagonospora nodorum has a leading position in barley sowings. The rapid increase of incidence of Stagonospora nodorum was recorded over 2008‐2010. In order to reduce yield losses in intensive spring barley cultivation, effective management practices are needed. The field experiments with general aiming to develop the strategy of application of fungicides in different cultivars of spring barley were carried out at the LPPRC. Recent observations of Stagonospora nodorum leaf blotch in barley have demonstrated that distribution and severity of the disease depend on the humidity in crop stands, air temperature and crop growth stage (GS). The best conditions for development of an infection are: the air temperature 20‐25°С, high humidity of air and moisture of the leaves for a longer period of time (after frequent rain). It was possible to derive the relationships between severity of disease and yield losses in different meteorological conditions. In favourable conditions for disease development a strong correlation between yield and severity of disease on leaf was obtained. In spring barley cultivated by intensive technology was demonstrated that infestation of diseases, included Stagonospora nodorum leaf blotch, at the GS 70‐71 (BBCH) had the most influence on yield losses. The severity of Stagonospora nodorum leaf blotch in both varieties ‘Pasadena’ and ‘Tocado’ under favourable meteo‐rological conditions was increased from 2‐4% up to 12‐14% during the two weeks after the first appearance of symptoms at the GS 37‐41. There were high significant differences between yield in treated and untreated variants. The application of fungicides gave increase of yield up to 1.03 t ha‐1 for ‘Pasadena’ and 1.23 t ha‐1 for ‘Tocado’. The relationship between yield (y) in t ha‐1 and percentage severity (x) of Stagonospora nodorum leaf blotch in spring barley at the GS 72 was: y = ‐0.0919 x + 5.1961, R2 = 0.64 (P‐value=0.05). For variety ‘Tocado’ a significant correlation between yield losses and the severity of disease at the GS 70‐75 was clearly illustrated. In field experiments the application of fungicides showed the significant efficacy to control most widespread diseases during the trials, including Stagonospora nodorum leaf blotch. The highest effect gave new fungicide Acanto Prima (picoxystrobin 80 g, cyprodinil 300 g kg‐1) by application on spring barley at the GS 37‐41 in dosage 1.0 kg ha‐1. Key words: barley, Stagonospora nodorum, harmfulness, fungicides

Posters P01-P22

238

P02 DIAGNOSIS OF EARLY BLIGHT ON POTATO

CROPS IN THE NETHERLANDS IN 2009 AND 2010: NOT AS EASY AS EXPECTED!

J. SPOELDER & L.J. TURKENSTEEN

Hilbrands Laboratory for Soildiseases Kamspweg 27, NL-9418 PD Wijster, The Netherlands

From June till September in 2009 and 2010, a survey was made on early blight leaf spots on potato leaflets in The Netherlands. Early blight leaf spots are marked by concentric rings and a dark brown colour. Leaflets with lesions were sampled and sent to HLB. These lesions were examined microscopically and through culturing on the presence of the three fungi Alternaria solani, A. alternata and Cladosporium cladosporioides; organisms which are considered as pathogenic, weakly pathogenic and non‐pathogenic to potato foliage, respectively. In addition, other sporulating fungi were scored as well. In total, 1900 lesions were examined, many of which were void of either Alternaria, implying that a correct diagnosis of Early Blight can be difficult to make. Further it was found that A. solani showed up relatively late during the growing season. The first sample carrying A. solani was found mid‐late July and more than 50% of all samples with A. solani were collected in the period September 4 till 18. From these results it is to be concluded that throughout the growing season, A. solani was not the main cause of Alternaria‐like lesions, creating confusion among growers in regard to crop protection. It is shown that several nutrient deficiencies and excesses can cause the formation of lesions which are mistaken for early blight. A proper nutrient supply and structure of the soil before planting will take away most of the problems with Alternaria‐like lesions. The authors suggest that ozone damage, possibly linked to nutrient deficiencies, is also likely to be confused with Early Blight symptoms. Key words: early blight, Alternaria, Nutrient deficiency, potato, ozone

Posters P01-P22

239

P03 SCREENING ELISA METHOD TO ASSESS IN-VITRO EFFICACY OF NEW AGENTS

AGAINST PHYTOPATHOGEN

Françoise BAFORT1, Jean-PauL PERRAUDIN2 & M. Haïssam JIJAKLI1 1 Gembloux Agro-Bio Tech, Université de Liège, Unité de Phytopathologie

Passage des Déportés 2, BE-5030 Gembloux, Belgium 2 Taradon Laboratory, Avenue Léon Champagne 2, BE-1480 Tubize, Belgium

The lactoperoxydase system (LPS) is a natural biological protection system in human saliva, milk and tears. Lactoperoxydase catalyzes the oxidation of iodide (I‐) and thiocyanate (SCN‐) in presence of peroxide hydrogen in hypoiodite (OI‐) and hypothiocyanite (OSCN‐) ions. These ions have bacteriostatic or bactericide action against pathogenic bacteria, fungi and viruses. Until now, the ions were not tested against plant pathogen. Testing efficacy of new molecules against plant pathogenic bacteria and/or fungi needs long‐term trying. Gembloux Agro‐Bio Tech has developed a simple and reliable method which permits to test together 10 molecules on ELISA plate. The pathogen is incubated on a 96‐well ELISA microplate in growing medium with or without inhibitory molecule. After growing period, optical density is measured to determine the inhibitory activity of the molecule. The percentage of pathogen inhibition is calculated by comparing the development of the pathogen in presence or in absence of the inhibitory agent. Seventeen different I‐/SCN‐ ratios with 3 dilutions for each one were screened against Botrytis cinerea. From the seventeen different I‐/SCN‐ ratios screened against Botrytis cinerea, 4 ratios were selected and tested, at various dilutions, against 6 phytopathogenic fungi. Results showed that LPS give 87, 88 and 100 % inhibition against Penicillium expansum, Botrytis cinerea and Verticillium dahlia respectively. The inhibitory activity of the LPS was in a range of 70 to 79 % regarding Phytophthora infestans, Colletotrichum lindemuthianum and Fusarium avenaceum. The LPS showed an insufficient inhibitory activity against Septoria tritici. The screening method highlighted that high I‐/SCN‐ ratios and low dilution were more efficient against phytopathogens. The efficient ratios have to be tested in‐vivo. Key words: Lactoperoxydase system, screening elisa method, phytopathogenic fungi

Posters P01-P22

240

P04 SCREENING OF JATROPHA CURCAS GENOTYPES

TO ANTHRACNOSE CAUSED BY COLLETOTRICHUM GLOEOSPORIOIDES

M.M. FERREIRA-PINTO, M.J. SILVA & M.R. SANTOS

ECOBIO, Instituto de Investigação Científica Tropical (IICT) Apartado 3014, PT-1301-901 Lisboa, Portugal

Anthracnose caused by different species of Colletotrichum is an important physic nut (Jatropha curcas) disease causing damages on leaves, stems and fruits and consequently a decrease in seed quantity and quality. Physic nut is a native plant of Mexico and Central America that has gained international attention due to its potential as biodiesel crop. However despite its highest relevance, studies concerning the response of different Jatropha genotypes to infection with this disease are very limited. The main objective of this study is the evaluation of susceptibility of 38 Jatropha curcas accessions from a worldwide germplasm collection to Colletotrichum gloeosporioides Penz. A simple method using wounded detached leaves inoculated with a 5‐mm mycelial plug was used and observations (lesion diameter and % of infection) were made after 3 and 7 days under controlled conditions (25°C and 85% RH). Results showed significantly differences (P≤0.001) in both percentage of infection and lesion diameter among the tested accessions. Two accessions originally from Brazil and Cape Verde, presented no lesions after 7 days of treatment, while the others presented between 60 and 100% of infection. Lesion diameter showed an increase with time of incubation and proved to be a useful tool for evaluation of plant susceptibility. Key words: anthracnose, Colletotrichum gloeosporioides, physic nut, germplasm evaluation,

Posters P01-P22

241

P05 FUSARIUM SPP. THAT CAUSE ROOT AND CROWN

ROT OF WHEAT IN KERMANSHAH PROVINCE

D. SAFAEE, M. SHEIKHOLESLAMI & H. YOUNESI Plant protection department of agricultural and natural resources

Research Center of Kermanshah, Iran In order to identify fungi involving root and crown rot of wheat and to determine their distribution and frequency, wheat fields in different regions of Kermanshah province were sampled at seedling, tillering and heading stages during 2004‐9. For isolation of fungi, some samples of infected root, subcrown internode, crown and foot were surface sterilized and then placed on potato dextrose agar containing streptomycin sulfate. Pathogenecity test was performed by mixing of pasteurized soil with inoculum (colonized wheat seeds with an isolate) under greenhouse condition. Disease severity (D.S.) was evaluated by scoring from 1 to 5 (1= no symptoms, 5= extensive necrosis of the crown and stem base resulting in plant death). Besides other genera, 231 isolates of Fusarium spp. were isolated. Also the results revealed that Fusarium spp. exist in most parts of the province and their D.S. are from 1 to 2.9. Among Fusarium species Fusarium culmorum and F. pseudograminearum had the highest D.S., F. acuminatum, F.avenaceum, F. crookwellense, F. proliferatum and F. udum had medium D.S. and that of F. nygama, F. reticulatumand, F. merismoides was the least amount. The species that could not cause disease were F.equiseti, F. lateritium, F. moniliforme, F. oxysporum, F. sambucinun, F. semitectum, F. solani and F. trisinctum. This is the first report of pathogenicity of F. udum and F. merismoides on wheat root in the world. Key words: fusarium , wheat , root and crown rot , kermanshah, iran

Posters P01-P22

242

P06 USE OF MULTIPLE DNA BARCODES FOR

PATHOVAR-LEVEL DIAGNOSIS OF PLANT QUARANTINE XANTHOMONADS

L. DETEMMERMAN1,2, B. COTTYN1, P. DE VOS2 & M. MAES1

1 Institute for Agricultural and Fisheries Research – ILVO, Unit Plant-Crop Protection Burg. van Gansberghelaan 96, BE-9820 Merelbeke, Belgium

2 Laboratory of Microbiology, Ghent University K.L. Ledeganckstraat 35, BE-9000 Ghent, Belgium

Plant pathogens and pests can have a significant economic impact; therefore rapid and accurate identification is crucial. The EU‐FP7 program QBOL aims to make DNA barcoding available for plant health diagnostics, as an accurate identification tool for quarantine organisms on the EU Directive 2000/29/EC and EPPO list. DNA barcoding uses short genomic DNA sequences from key gene regions for species identification. In the framework of QBOL, we focus on the genetic barcoding of a set of quarantine and Q‐alert bacteria within the genus Xanthomonas. Target pathogens are X. fragariae, X. translucens, X. oryzae pathovars oryzae and oryzicola, the group of pathovars on citrus and multiple pathovars of X. axonopodis. For each target organism, a working collection was assembled, consisting of the pathotype, other voucher strains and taxonomically related bacteria. As a single gene region is not suitable for reliable identification of all target bacteria, the strategy is to use a combination of several barcodes in a step‐by‐step approach. We adopted a 530‐bp sequence of the gyrB gene as a first barcode region, which was adequate for identifying the species X. oryzae, X. fragariae, X. vesicatoria and X. translucens. However for identification at the pathovar level, the gyrB sequence does not provide sufficient resolution and has to be complemented with a second barcode region in order to obtain reliable identification. Other gene sequences were investigated, with a focus on genes coding for effector proteins and proteins involved in the type III secretion system. Sequence divergence in the AvrBs2 gene, a type III effector protein, provides enough resolution to discriminate the target pathovars of X. axonopodis and the pathovars that cause diseases on citrus. For these target organisms, a two‐step approach will be used for identification. This research also provides new insights into the current classification of Xanthomonas pathovars

Posters P01-P22

243

P07 OVERWINTERING FORM OF THE CAUSAL

AGENT OF SHOT HOLE DISEASE IN KHORASAN RAZAVI, IRAN

Naser PANJEHKEH1, Azam YOUSEFI1 & Mohammad HAJIAN SHAHRI2

1Department of Plant Protection, Faculty of Agriculture, University of Zabol, Iran 2Agricultural and Natural Resources Centre of Khorasan Razavi, Iran

Shot hole disease of stone fruit trees caused by some plant pathogenic fungi is a major constraint to stone fruit production worldwide where the trees are grown. Identification of the causal agents of the disease and their overwintering forms in stone fruit trees of Khorasan Razavi was necessary for disease management programs. Buds, twigs, fallen leaves and fruits were collected from the infected peach, apricot, nectarine and almond trees in winter 2007. The samples were superficially disinfested in 1% sodium hypochlorite for 2‐3 min and then in 70% ethanol for 45 sec. Two to three fragments of 4 ×4 mm from each tissue were separately cultured on 2% water agar and potato dextrose agar (PDA) and purified on PDA. Just a fungal species, Wilsonomyces carpophilus was isolated from the infected buds and twigs. No microorganism was isolated from the fallen leaves and fruits collected from the infested stone fruit trees. Pathogenicity of the fungus was examined on detached shoots of current year of four varieties of stone fruit trees. Fungal discs were placed under the bark of the bud base. Control shoots were similarly treated with sterile PDA discs. Inoculated shoots were placed in a humid growth chamber at 25 ˚C. Fungal hyphae appeared at 30 days post inoculation. Control shoots were asymptomatic. Pathogenicity intensities or lesion lengths were significantly different among the four varieties tested. A completely rando‐mised design with five replicates was employed to measure the number of spores in infested buds and twigs of each variety of stone fruit trees. The samples were sliced and placed into a glass tube of centrifuge containing 3 ml of sterile distilled water. They were mixed on a vortex mixer for 30‐40 min and centrifuged at 3000 rpm for 5 min. Pelleted material from each sample was suspended in 500 μl of sterile distilled water and the spores were counted using a hemocytometre. Results revealed that the fungus overwinters as hyphae and conidia in the infected buds, and as hyphae and globular chlamydospores in twig lesions. Key words: Shot hole, overwinter, pathogenicity

Posters P01-P22

244

P08 PRE-HARVEST CONDITIONS AFFECT THE BANANA

SUSCEPTIBILITY TO POST-HARVEST DISEASE

Ludivine LASSOIS1, Cécile ANNIE EWANE1,2, Marie FORRET1, M. Haïssam JIJAKLI1 & Luc DE LAPEYRE DE BELLAIRE2,3

1 Plant Pathology unit, University of Liège, Gembloux Agro-Bio Tech, 2 Passage des Déportés, BE-5030 Gembloux, Belgium

2 CARBAP, African Center for Research on Bananas and plantains, Phytopathology Unit, PO Pox 832, Douala, Cameroon.

3 CIRAD, Research Unit, banana, plantain and pineapple cropping systems FR-34398 Montpellier, France

Crown rot disease affects export bananas in all producing countries and is considered as one of the main post‐harvest diseases of bananas. The symptoms appear on the crown, i.e. the tissue joining the fruit pedicels with each other. The disease develops during shipping, ripening, and storage and has a negative impact on the market value of bananas. Crown rot is caused by a parasitic complex. However, Colletotrichum musae is often regarded as one of the most prevalent fungi contributing to the crown rot complex. Variations in the susceptibility to this disease have been noted between bananas but the factors involved are still unknown. The aim of this study was to evaluate the influence of some pre‐harvest factors on fruit susceptibility to crown rot post‐harvest disease. The fruit susceptibility was estimated through lesion size on the crown after standardized artificial inoculations with C. musae. Four pre‐harvest factors that influence the fruit susceptibility to crown rot, were identified: (i) Banana hand position on the bunch: It was shown that within a bunch, there is a gradient of susceptibility to crown rot (r= ‐0.95), the hands initiated first (the upper ones) being more susceptible than those initiated last (the lower ones); (ii) Source‐sink ratio of the banana plant (hand considered as sink and leaves as source): When the sink is decreased by artificial removal of many hands, the level of fruit susceptibility to crown rot decreases. These results also confirmed that source‐sink ratio changes have a significant effect on fruit morphology; (iii) Fruit physiological age at harvest: a linear relationship between fruit age (expressed in degree day) and the susceptibility of the fruit to crown rot was reported, the oldest fruit being most susceptible to this disease; (iiii) Level of black leaf streak disease (BLSD) caused by Mycosphaerella fijiensis during plant growth: the level of BLSD severity significantly influenced fruit sensitivity of crown rot. All these results highlight that the field environmental conditions during plant growth greatly influence the fruit susceptibility and thus potential post‐harvest disease development. Key words: post‐harvest disease, susceptibility, source sink ratio, physiological age, banana

Posters P01-P22

245

P09 NEW MSC PROGRAM CROP PROTECTION AT

GÖTTINGEN UNIVERSITY, GERMANY

Susanne WEIGAND & Andreas VON TIEDEMANN Institute for Plant Pathology and Plant Protection University of Goettingen

Grisebachstrasse 6, DE-37077 Goettingen, Germany Email: [email protected]

Why MSc crop protection? At a time of population growth and concerns over global food security the investments made in crop production need to be protected from losses due to plant pathogens, insects and weeds. Crop protection is one of the main components to improve crop production efficiency. Science, research and innovation offer the only prospect to develop and apply modern crop protection techniques while observing the challenges of natural resources conservation. In view of the growing food demand there is an increasing need for qualified employees. Therefore the MSc program "Crop Protection" was newly established at the Department of Crop Sciences, University of Göttingen, which has long successful experience in research and training. Course description The two years master program is an international, job and research oriented program and follows an interdisciplinary approach of crop protection within the larger framework of crop production systems. In addition to the advanced study courses the capability for interdisciplinary research, improvement of oral and written communication and presentation skills are focused on. The program consists of four semesters during which 120 ECTS have to be completed. The courses of advanced study contribute 81 credits, an internship of 6 weeks including a written report and oral presentations 9 credits and the master thesis including the colloquium 30 credits. The training in concepts and practical skills is achieved through lectures, seminars, laboratory classes, field courses and project work. The language of instruction is English. The crop protection study program will cooperate with research institutions and the agrochemical industry at different levels (Internship, lectures and practical courses) and provide the opportunity to focus on the topics and tools applicable and on demand for research in national and international crop protection. Career perspectives Graduated students will have broad research and analytical skills related to crop protection and be able to select and combine suitable technologies to solve the problems of crop protection and increase crop production efficiency. Possible employment areas are agrochemical industry, agricultural services and research institutions, international organizations and institutes, public administration and advisory boards, public and private research centers. Best performing students are eligible for a scholarship in the subsequent PhD Program on Crop Protection. This international Master program opens attractive perspectives for an international career in the multidisciplinary fields of crop protection. Key words: MSc crop protection, Göttingen university, job and research oriented, international

Posters P01-P22

246

P10 THE REACTION OF EIGHT BEAN CULTIVARS TO

IRANIAN ISOLATES OF CUCUMBER MOSAIC VIRUS AND BEAN YELLOW MOSAIC VIRUS

A. TAHMASEBI, A. DIZADJI & M. KOOHI HABIBI

Department of plant protection, College of agriculture, University of Tehran Cucumber mosaic virus (CMV) and Bean yellow mosaic virus (BYMV) infect naturally bean (Phaseolus vulgaris) plants worldwide. During 2009, fields of three bean cultivating provinces of Iran were surveyed and totally 180 symptomatic plants tested by DAS‐ELISA using CMV and BYMV polyclonal antibodies (DSMZ, AS‐0475, AS‐0471). The results showed 11.5% and 10.6% of symptomatic bean plants were infected with CMV and BYMV respectively. Also the reaction of eight bean cultivars, including Darakhshan, Akhtar, Talash (three Iranian cultivars), Jules, Karacasehiro 90, Goynuk, Cos16 and Bountiful was evaluated to CMV and BYMV isolates in greenhouse. Thirteen plants of each cultivar were mechanically inoculated with biologically pure isolates of CMV and BYMV and 24 days post inoculation were tested by DAS‐ELISA. Bountiful with 77.8% and Akhtar, Karacasehiro 90 with 13.3% infection were the maximum and minimum infection rate to CMV, while 34.8% of Bountiful (maximum) and 3.3% of Cos16 (minimum) were infected by BYMV. Relative concentration of each virus in infected plants of cultivars was compared based on the absorbance mean of infected plants at 405 nm. Maximum and minimum relative concentration of CMV was estimated in Bountiful and Talash with 0.661 and 0.141 OD405 value and for BYMV in Goynuk and Cos16 with 0.352 and 0.114, respectively. The low value of some cultivars in both infection rate and relative concentration can be considered in breeding programs for producing resistant plants. On the basis of these results, Bountiful with respect to other cultivars had the most sensitivity to CMV and BYMV. Key words: Cucumber mosaic virus, Bean yellow mosaic virus, infection status, cultivars reaction

Posters P01-P22

247

P11 EFFECTS OF INDOLE-3-ACETIC ACID ON

BOTRYTIS CINEREA ISOLATES OBTAINED FROM POTTED PLANTS

Juan Antonio MARTÍNEZ1, RaqueL VALDÉS1, María José GÓMEZ BELLOT2

& Sebastian BAÑÓN1,3 1 Departamento de Producción Vegetal, Universidad Politécnica de Cartagena, Spain

2 Departamento de Riego, Centro de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, Murcia, Spain

3 Horticultura Sostenible en Zonas Áridas, Unidad Asociada al CEBAS-CSIC, Murcia, Spain We study the growth of different isolates of Botrytis cinerea collected from potted plants which were affected by Botrytis blight in southern Spain during recent years. These isolates, which show widely phenotypic differences when grown in vitro, are differentially affected by growth temperature, gibberellic acid applications and paclobutrazol, an efficient plant growth retardant and fungicide at the same time. In this work, we have evaluated the effect of the auxin indole‐3‐acetic acid (IAA) dose (0, 1, 10, and 100 mg/plate) on the growth of the collection of B. cinerea isolates obtained from the following potted plants: Cyclamen persicum, Hydrangea macrophylla, Lantana camara, and Lonicera japonica. B. cinerea produces indolacetic acid, but so far the precise biosynthetic pathway and some effects on this fungal species are still unclear, although recent studies have revealed an antifungal activity of IAA on several fungi, including B. cinerea isolated from harvested fruits. Mycelial growth curves and growth rates assessed from difference in colony areas during the both linear and deceleration phase, conidiation (measured as time of appearance), conidia length (µm), and sclerotia production (number/plate) were evaluated in the isolates, which were grown at 26ºC on Petri dishes containing potato dextrose agar for up to 35 days. Mycelial growth curves fitted a typical kinetic equation of fungi grown on solid media. B. cinerea isolates showed a high degree of variability in their growth kinetics, depending on the isolate and auxin dose. This plant growth substance delayed mycelial growth during the linear phase in an isolate‐dependent manner, thus isolates from C. persicum, H. macrophylla and L. camara were more affected by IAA than L. japonica. On the other hand, 100 mg of IAA was the critical dose to significantly reduce the growth rate in all isolates and to promote brown‐striped hyphae development, especially in isolate from C. persicum. 10 and 100 mg IAA inhibited conidiation in isolates from H. macrophylla but no effect was found in the conidia length. The sclerotia production process was blocked at IAA doses of 100 mg in isolates from L. camara and L. japonica, and was reduced in isolate from H. macrophylla. However, dose of 100 mg IAA had no effect on sclerotia production in isolate from C. persicum. It was concluded that the effect of IAA on B. cinerea growth depends on the isolate, thus isolates from H. macrophylla and L. camara were the most affected by IAA. B. cinerea reduced its development under IAA applications, depending on the isolate and dose. These results confirm those recently published on the inhibitory effect of IAA on Botrytris species growth. Key words: Botrytis blight, mycelial growth, auxins, plant growth substances, antifungal activity

Posters P01-P22

248

P12 ARBUSCULAR MYCORRHIZAL FUNGI IN TERMS

OF SYMBIOSIS-PARASITISM CONTINUUM

B. SCHMIDT, S. GAŞPAR, D. CAMEN, I. CIOBANU (POPESCU) & R. ŞUMĂLAN

Department of Plant physiology, Banat’s University of Agricultural Sciences and Veterinary Medicine, Timişoara, Romania

Arbuscular mycorrhizal fungi are forming the most wide‐spread mycorrhizal relationships on Earth. Mycorrhiza contributes to phosphorous acquisition, water absorption and resistance to diseases. The fungus promotes the absorption of mineral nutrients and water from soil, meanwhile the host plant offers photosynthetic assimilates in exchange, like carbohydrates, as energy source. The plant benefits from the contribution of symbiotic partner only when nutrients are in low concentrations in soil and the root system would not be able to absorb sufficiently the minerals. When the help of mycorrhizal fungi is not necessarily needed, the host plant is making an economy of energy, suppressing the development of fungi in the internal radicular space. In this moment, the nature of relationship turns from symbiotic to parasitic, triggering a series of defensive reactions from the plant. Also, there were several cases reported when the presence of arbuscular mycorrhizal fungi negatively influenced the host plant. For example, in adverse environmental conditions, like very high temperatures, instead of determining a higher plant biomass and flowering, the mycorrhiza reduces the growth of the host plants. We conducted a pot experiment with hydroponic culture to examine the effect of arbuscular mycorrhiza on development of French marigold as a host plant. As experimental variants, the phosphorous content in nutrient medium and temperature varied. Plants were artificially infected with arbuscular mycorrhizal fungi using a commercial inoculum containing three fungal species, as following: Glomus intraradices, Glomus etunicatum and Glomus claroideum. Colonization intensity and arbuscular richness were checked using root staining with aniline blue and estimation with the Trouvelot method. To observe the differences between plants from the experimental variants, we examined the number of leaves, side shoots, flower buds and fully developed flowers, fresh biomass, plant height and total leaf area. Results show that adverse climatic conditions, like temperature shock at the beginning of growing period modified the nature of symbiosis. In this case, the physiological parameters were reduced at colonized plants, while usual, constant growing conditions permitted the normal, efficient and beneficial development of symbiosis. Key words: symbiosis, arbuscular mycorrhiza, physiological parameters

Posters P01-P22

249

P13 PHYLOGENETIC STUDY AND IDENTIFICATION

OF PLANT PATHOGENIC CLAVIBACTER

Joanna ZALUGA1, Kim HEYLEN1, Martine MAES3 & Paul DE VOS1,2 1 Department of Biochemistry and Microbiology, Gent University, Gent, Belgium

2 BCCM/LMG Bacteria Collection, Gent, Belgium 3 Institute for Agricultural and Fisheries Research-ILVO, Merelbeke, Belgium

The genus Clavibacter includes five subspecies, most of them belong to quarantine or q‐alert organisms and cause a variety of plant diseases and serious crop losses. To protect plants rapid and reliable identification is required. The development of accurate DNA‐based diagnostic tools is essential for quick and effective identification and in consequence for preventing the distribution of the diseases in pathogen‐free areas. In the frame of the QBOL project (Quarantine Barcoding of Life) we focus on the fine taxonomic resolution of Clavibacter and close relatives using a polyphasic approach, with the development of barcodes as the final goal. To reevaluate taxonomic/phylogenetic position in the genus Clavibacter, a total number of 173 strains of five Clavibacter michiganensis subspecies (C. michiganensis subsp. michiganensis (Cmm), C. michiganensis subsp. sepedonicus (Cms), C. michiganensis subsp. nebraskensis (Cmn), C. michiganensis subsp. insidiosus (Cmi), C. michiganensis subsp. tesselarius (Cmt)) and some outgroups were included for sequence analysis of the 16S rRNA gene and housekeeping gene gyrB. A new method introduced into bacterial taxonomy, matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spec‐trometry (MALDI‐TOF MS) was evaluated for chemotaxonomic typing. Cell extracts from strains were measured to establish proteins pick profiles. Closely related Clavibacter subspecies could be then grouped using UPGMA together with Pearson's product moment correlation coefficient and correctly assigned by defining unique biomarker ions. To find suitable barcode region the two available Clavibacter genome sequences, namely from a Cmm and a Cms strain, together with the genome of the outgroup Leifsonia xyli subsp. xyli were searched for shared single copy genes. The whole genome comparison resulted in a high number of potentially suitable genes for further research. Thus, the literature was search to find other possible good markers. Already described gyrB primer sets gave a good amplification of a 550‐bp fragment in all subspecies and outgroups. Gene sequence analysis in BioNumerics 5.1 and Mega 4.1 software showed that the gyrB gene fragment is a good taxonomic marker. The gyrB sequences showed enough resolution and specificity to identify the subspecies based on sequence relatedness in a neighbor‐joining tree or on specific characters within the sequence. Results with both methods were in concordance and distinguished the five Clavibacter subspecies from each other and from other closely related bacterial genera like Rathayibacter, Leifsonia or Curtobacterium. Our study suggests that the proteomic analysis using MALDI‐TOF mass spectrometry and the gyrB sequence barcode are powerful diagnostic tools for reliable identification of the Clavibacter plant pathogens. Key words: Clavibacter, gyrB, identification, quarantine organism, Maldi‐Tof

Posters P01-P22

250

P14 SYMPTOM DEVELOPMENT OF DOWNY MILDEW ON ROSES CAUSED BY PERONOSPORA SPARSA

UNDER GREENHOUSE CONDITIONS AT THE BOGOTÁ PLATEAU, COLOMBIA

Sandra GÓMEZ & German ARBELÁEZ

Faculty of Agronomy, Universidad Nacional, Bogotá, Colombia Downy mildew is considered one of the most important diseases affecting roses in the Bogota Plateau (Cundinamarca, Colombia). Under proper environmental conditions, rapid and hard to control epidemics of this pathogen may cause direct losses of a susceptible rose cultivar plantation under greenhouses conditions in the tropics due to the affection of flowers and indirect losses by defoliation of plants. In addition, plants recovering following a severe attack by this disease takes several months. In spite of this, no detailed information of the development of the disease is available. Thus, the aim of this study was to investigate the development of the symptoms caused by P. sparsa under greenhouse conditions. For this purpose healthy rose plants of cultivars “Charlotte”, “Classy” and “First Red” grafted on rootstocks Natal Briar and Manetti and plants rooted directly were inoculated with a sporangia suspension of the pathogen collected from samples of commercial crops. After the inoculation, the plants were kept under moisture chamber conditions and darkness for 12 hours. To register the presence of downy mildew symptoms, the plants were divided in three levels according to the predominant stage of the development of the foliage and were evaluated daily starting two days after inoculation. The percentage of affected leaves per stratum per variety was established. Furthermore, the degree of the disease in apical leaflets was registered according to the scale proposed in this study. Symptoms of downy mildew were first observed on leaves seven days post inoculation (dpi). The symptoms on stems occurred 17 and 18 dpi in cv. “Charlotte” and between 21 and 28 dpi in cv. “Classy” and cv. “First Red”. The symptoms on flower buds appeared 14 and 16 dpi in “Charlotte”, 14 days after inoculation in cv. “Classy” and between 16 and 26 days in cv. “First Red”. Defoliation of affected leaves occurred first in cv. “Classy” 12 dpi. In cv. “Charlotte” this event was observed 18 and 28 days and in “First Red” plants in a period of 15 and 28 days. No interactions were observed between the rose variety and the levels of the plants on the incidence of the disease. Key words: cultivars, rootstocks, incubation period.

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251

P15 SEASONAL DIFFERENCES IN ENDOPHYTIC

FUNGI IN CHERRY TREES GRAFTED ON DIFFERENT ROOTSTOCKS

N. HADDADDERAFSHI, K. HALÁSZ, T. PÓSA, G. PÉTER,

K. HROTKÓ & N. LUKÁCS Department of Plant Physiology and Plant Biochemistry, Budapest, Hungary

Endophytic fungi are currently considered as symbionts which can colonize a wide range of hosts and do not induce any pathological signs. The main aim of this study was to identify the endophytic fungi colonizing different organs (root, twig and leaf) of cherry (Prunus avium) in spring and summer and to find out whether rootstocks have an effect on the frequency and species composition of the endophytes. Samples were collected two times from eight‐ year old trees of the Hungarian cultivar ‘Peter’ grafted on 11 different rootstocks. After surface sterilisation samples were put on potato dextrose agar and outgrowing fungal colonies were visually classified after 1 and 2 weeks. Then monosporation and monohyphation technique was applied to generate single colonies of each individual fungus, Isolated colonies were classified morphologically. Molecular identification at species level was performed by applying the polymerase chain reaction (PCR) to amplify a nuclear ribosomal internal transcribed spacer region (ITS) and by sequence comparison of the PCR products. We observed a significant difference between the frequency of endophytic fungi in different plant organs. The main influence of the rootstock seemed be on the diversity of fungi. Endomycota showed the highest diversity in root, while Alternaria spp was predominantly found in twigs and leaves. Our data indicate that fungal endophytic infection may show rootstock‐ as well as tissue‐specific differences. Further analyses are being carried out to verify these results and to find out and which endophytes can be used to improve the physiological functions and tolerance threshold of the composite trees against environ‐mental stresses. Key words: Keywords: Endophytic fungi, Symbiosis, Prunus avium, Rootstock .

Posters P01-P22

252

P16 FUSARIUM SPECIES AND FUSARIUM MYCOTOXINS

IN CEREALS FROM WEST ROMANIA

A. ALEXA, G. POP, R. SUMALAN, I. RADULOV, M. POIANA & C. TULCAN Faculty of Agro-Food Technologies, Banat`s Unvirsity of Agricultural Sciences

Timisoara, Romania Fungal contamination of plant products is an important risk factor for health, because of the high mycotoxin potential deriving from these contaminations with multiple effects: hepatic toxicity, teratogenic, mutagenic and carcinogenic. The contamination of cereals with mycotoxins has been a serious problem in Balkan communities. Several studies implicated mycotoxins, in endemic kidney disease geographically limited to Balkan region (Balkan endemic nephropathy). The trichothecenes are of particular concern because they are ubiquitous found in wheat, corn and barley throughout the world. Fumonisins have been isolated from certain Fusarium species of which FB1, FB2 and FB3 are the major ones produced in naturally contaminated foods.These mycotoxins are produced on cereal grains infected by Fusarium while being grown in‐the‐field. The aim of this study is to evaluate the presence of the Fusarium species in cereals from West side of Romania and to determinate the concentrations of deoxynivalenol (DON) and fumonisine (F1+F2). Furthermore, the level of contamination has been correlated to the agro‐climatic, agro‐technical and storage conditions from West Romania. Identification of Fusarium species was done using the total number of fungal species determination method. The level of mycotoxins was determined with the immune‐enzymatic method ELISA. 27 cereal samples from rural households in three counties in West Romania were analysed. The experimental results indicated that the most frequent fungal species in cereal samples prelevated from the West side of Romania were Fusarium graminearum and Fusarium culmorum.The DON level registered in the cereal samples ranged between 0,22‐53,92 ppm, the maximum level being found in barley used in beer manufacturing. In 22,22% from all samples the maximum concentration limit of DON was exceeded. The highest incidence of DON was registered in barley samples (100% contaminated samples), folowed by wheat for human consumption (42,85% contaminated samples). Corn samples, both for human and animal consumption, did not exceed the maximum admited limit. Concerning the analysed area, the largest number of positive samples were registered in the Arad county (25%), followed by Timis county (23,59%) and Satu Mare county, which did not display any contamination.The concentration of fumonisins in the analysed samples ranged between 0,22‐56,48 ppm, the maximum level being found in feed corn. The study has shown the fact that the incidence of Fusarium species and mycotoxins in the samples taken from West Romania is high and it is the result of agro‐climatic conditions typical for Romania, as well as of the storage conditions which favour fungus development and mycotoxins formation. Acknowledgements: This research was supported by SEE-ERA.NET PLUS no. 139/01:Systems to reduce mycotoxins contamination of cereals and medicinal plants in order to preservation native species and traditional products in Romania-Serbia-Croatia area Key words: Fusarium, cereals, deoxynivalenol (DON), fumonisins (F1+F2)

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253

P17 FUSARIUM WILT OF CHICKPEA

(CICER ARIETINUM L.) IN NORTH-WESTERN ALGERIA

F. BENFREHA1,J.E. HENNI2,A. MERZOUG1 & M. BENYELESS1

1 University of Mascara, Algeria 2 University of Oran, Algeria

Fusarium oxysporum f.sp. ciceri (FOC) is a soil fungus that constitutes a permanent threat to chickpea (Cicer arietinum L.) causing wilting. In Algeria the studies on pathogens of chickpea, have focused on anthracnose caused by Ascochyta rabiei, a real obstacle to culture plus the currently complex wilt and root rot, wilt syndrome called (Rhrib, 1990). The aim of this study is to estimate the incidence and severity of Fusarium wilt caused by Fusarium oxysporum (Schletend: Fr) f.sp ciceri (Padwick) (Foc) during surveys at the 3 agro‐climatic zones of north‐western Algeria, made on 50 chickpea fields, at different phenological stages of the plant and during five campaigns (from 2005 to 2009). The special shape ciceri is determined by inoculating a variety of chickpea ILC482, sensitive to the FOC. The isolation is carried from the plant (stems and roots) and from the seed of chickpea plants affected by the methods of incubating plant fragments deposited on nutrient agar and the test method respectively. The presence of the disease is found in all the fields visited and the pathogen was isolated from all infected plants harvested. For the only region of Mascara over 5 rounds, the incidence (ID) is estimated (it is expressed as a percentage of the number of sick units on the total number of plants considered ID% = number of diseased plants/number total plant X100) between 5% and 31% for stage 2 branching, 10% and 60.2% for stage pre‐bloom, 54% and 98.6% for stage maturation. It is higher in dry years. For each plot the values of the impact have been grouped into classes of similar extent to that seen by Traperos‐Casas (1980) [0% (zero), 0.1 to 0.5% (low), 1 20% (moderately high), 20.1 to 50% (high)> 50% (very high)]. The indice of severity of the disease (ISD) was estimated based on visual assessment of the proportion of plant parts affected by wilting or yellowing. affected plants are ISD ascribed depending on the severity of symptoms [0: no symptoms 1: yellowing or wilting of 1/3 of the plant, 2: yellowing or wilting of 2/3, 3: yellow or the whole plant wilts 4: dead plant]. 0 <ISD>1 a minor illness, 1 <ISD <2 disease moderately severe, 2 <ISM> 3 illness; 3 <ISD > 4 very severe disease The severity varies from 2 to 3.56 for the three stages. The index of disease severity (Disease Intencity index) (DII) DII gives the relationship between DI and ISD. ( DII = ID x ISD x X / 4: X = 1) ,It varies between 2.5 and 68.77. The average estimate is made between the regions of Mascara, Sidi Bel Abbes, Ain Temouchent, Mostaganem, Relizane, Tiaret and Saida, the incidence varies between 50 and 100%, the severity score between 1 and 2.79 and index between 15 and 68.77. Key words: Cicer arietinum, Fusarium oxysporum f.sp. ciceri, Incidence.

Posters P01-P22

254

P18 SERIOUS OUTBREAK OF CLAVIBACTER

MICHIGANENSIS SUBS. MICHIGANENSIS ON TOMATO IN SOUTHERN ITALY

Angela FANIGLIULO1, Antonella VIGGIANO1, Guiseppe PIEGARI1

& Aniello CRESCENZI2 1 Bioagritest Test Facility, Bioagritest Srl.

Zona PIP lotto E2. IT-85010 Pignola (PZ, Italy) E-mail: [email protected]; www.bioagritest.it.

2 Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali Università degli Studi della Basilicata.

Viale dell’Ateneo Lucano Campus Macchia Romana 3A310, IT-85100 Potenza (PZ) Italy E-mail: [email protected]

An outbreak of bacterial cancer was observed in tomato fields in the main tomato growing area of Apulia and Basilicata regions, respectively in the campaigns of Foggia and Matera province, in 2010 summer, starting 15 – 20 days from transplant. Symptoms were observed in open field in the period between the fruit set and the beginning of ripening. They consisted in systemic wilt of the plant, often starting unilaterally on a lower leaf, a creamy‐white, yellow or reddish‐brown discoloration of vascular tissue in stems, petioles and peduncles of wilted plants, necrosis of the leaf margin and upward curling of the leaf edges, irregular watery spots between leaf veins which become necrotic, adventitious roots on the stems, dark streaks on stems and stem cankers, “Bird’s eyes” on fruits: spots with raised brown centers surrounded by an opaque white halo, external marbling on fruits, yellow vascular areas in fruits leading to the seed. The disease reached, in some instances, above all at the beginning of ripening, a 100% incidence, with the complete loss of the crop. Detection assays for C. michiganensis subs. michiganensis were performed in order to ascertain the etiologic agent, in any case of symptom observation. The diagnostic procedure comprised isolation from infected tissue, presumptive diagnosis with a rapid test, identification of presumptive isolates and determination of pathogenicity (2005 OEPP/EPPO, Bullettin OEPP/EPPO Bullettin 35, 271‐273. PM 7/42). Unfortunately, all symptomatic plants resulted infected with the bacterium under investigation. They belonged always to the same cultivar of tomato, cv. Uno Rosso, and to the same seed batch. This let us to believe that bacterial infection was associated with tomato seed. The suspected tomato seed batch cv. Uno Rosso was analyzed using the method for the detection of C. michiganensis subsp. michiganensis on tomato seed (International Seed Federation), working on a slight modification because the seeds under investigation were pills. The result was that the seeds were positive to C. michiganensis subsp. michiganensis. About 40 milion of these tomato pills (100Kg tomato seeds) were distributed, with imaginable consequences on plant health of the industrial tomato crops. This event allow us to reflect on how important is the use of health seed for the preservation of our agro‐systems.

Posters P01-P22

255

P19 SPORE TRAPS NETWORK:

A NEW TOOL TO PREDICT THE EPIDEMICS OF YELLOW RUST ON WHEAT

G. DEDEURWAERDER1, S-M. MVUYENKURE1, M. DUVIVIER2,

J-M. MOREAU2 & A. LEGRÈVE1 1 Applied Microbiology - Phytopathology Group, Earth and Life Institute

Université Catholique de Louvain, Louvain-la-Neuve, Belgium 2 Unité de Protection des plantes et d' écotoxicologie, CRA-W,

Centre wallon de Recherches agronomiques, Gembloux, Belgium A network of Burkard 7‐day spore‐recording traps was set up in the Walloon Region in Belgium to monitor the airborne inoculum of wheat pathogens. The relationship between the airborne inoculum of Puccinia striiformis, the causal agent of wheat yellow rust, and the disease incidence on plants in untreated plots located near each spore traps was studied during the season 2008‐2009. The presence of airborne inoculum was tested on tapes collected from 4 Burkard spore traps from 1 April to the 14 June 2009. Total DNA from each fragment of spore trap tape corresponding to 1 day sampling was extracted and the quantity of P. striiformis was assessed by real‐time polymerase chain reaction (PCR) assay using specific primers and SYBRGreen. The first detection of airborne inoculum of P. striiformis occurred between 7 and 13 April 2009 depending on the location in the Walloon Region. The first symptoms of the yellow rust were observed in the fields between 15 May and 2 June 2009. In each location, the onset of the disease in the 4 monitored fields was always preceded by a higher peak of airborne inoculum about 10 days before. When P. striiformis was detected, the daily quantities of spores, collected from a volume of 14.4 m³, fluctuated between 0.23 and 154.66 spores. This study confirms that spore traps coupled with real‐time PCR could be used to assess the airborne inoculum of P. striiformis, to understand and to predict the outbreak of the yellow rust. Key words: Puccinia striiformis, wheat, spore trap, real‐time PCR, airborne inoculum

Posters P01-P22

256

P20 VALIDATION OF THE SEROLOGICAL SCREENING

FOR TOMATO YELLOW LEAF CURL VIRUS (TYLCV) ON TOMATO

R. TAHZIMA, D. SCHEERLINCK, M. MAES & K. DE JONGHE

Instituut voor Landbouw- en Visserijonderzoek, Eenheid Plant Burg. Van Gansberghelaan 96, BE-9820 Merelbeke, Belgium

Tomato yellow leaf curl virus (TYLCV) disease is one of the most devastating virus diseases of tomato (Solanum lycopersicon) crops worldwide. The whitefly‐transmitted geminivirus causes serious economic losses to tomato crops worldwide. All strains cause indistinguishable symptoms on tomatoes, including severe reduction of plant growth (stunting), small, yellow and misshaped leaves which are usually curled upward and yellow erect shoots. When young plants are infected they produce few ‐ if any ‐ marketable fruits reducing severely the total production. Since its first description in Israel during the 1960s, the disease has expanded progressively causing significant crop damages in many tropical, subtropical and temperate regions. TYLCV outbreaks of different strains (including Tomato yellow leaf curl Sardinia virus) and other closely related geminiviruses are being reported in new areasand affecting new crops in the EPPO region, but so far, the virus has never been reported in Belgium. To prepare for a possible future outbreak we optimized a triple antibody sandwich–enzyme‐linked immunosorbent assay (TAS‐ELISA) to screen for the presence of TYLC(S)V in different tomato tissues, starting from a DSMZ (German Collection of Microorganisms and Cell Cultures) antibody set detecting all known European strains of TYLC(S)V. The TAS‐ELISA method employed in our diagnostic procedure was evaluated, optimized and validated for monitoring viral presence and accumulation in different plant tissues. In the first part of our study, the influence of different sampling places on a TYLCV infected tomato plant on the ELISA test results was assessed. In a second part, a series of critical test parameters for the validation of serological test methods in plant pest diagnostic labs such as analytical specificity, analytical sensitivity, selectivity and repeatability and reproducibility have been evaluated. Additionally, we report some important results on robustness of the test method with respect to pre‐analysis sample treatment, storage time and temperature and the influence of some analysis parameters such as buffer and incubation temperatures. The results of the optimization and validation tests which are presented here are compiled in a standard protocol maximizing the expected OD/background ratio of the ELISA reading results, thus minimizing the risk on false positive and negative results.

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257

P21 SURVEY OF MICROFUNGI IN THE

LANDSCHAFTSPARK DUISBURG-NORD (GERMANY) PART I

Manfred JENSEN**, Nadine NERAT & Nosratollah ALE-AGHA*

Universität Duisburg-Essen (Standort Essen), FB Biologie/Geographie – Allgemeine Botanik, Universitätsstr. 5, DE-45117 Essen, Germany

* Corresponding author E-mail: [email protected] ** Corresponding author E-mail: [email protected]

During an excursion in the Landschaftspark Duisburg‐Nord in 2009 and 2010 we were able to collect and identify more than 100 specimens of microfungi on different parts of cultivated and wild plant species. We found parasitic and saprophytic microfungi on trees, bushes and herbaceous plants. Some of them have been observed only rarely until now. Most of the collected microfungi species belong to the classes of Ascomycetes, Basidiomycetes and Deuteromycetes – for example Leptosphaeria modesta (Desm.) Rabenh. on Knautia cf. arvensis (L.) Coult., Ramularia urticae Ces. on Urtica dioica L., Stigmina glomerulosa (Sacc.) S. Hughes on Juniperus communis L., Pseudomassaria corni (Sowerby) Arx on Cornus alba L., Mollisia discolor (Mont.) W. Phillips on Cornus alba L., Botryosphaeria quercuum (Schwein.) Sacc. on Quercus robur L., Peronospora cytisi Rostr. on Laburnum anagyroides Med., Microsphaera guarinonii Briosi & Cavara on Laburnum anagyroides Med., Brachysporium dingleyae S. Hughes on Fraxinus angustifolia Vahl and Rhododendron spec., Mamiania fimbriata (Pers.) Ces. & De Not. on Carpinus betulus L., Atopospora betulina (Fr.) Petr. on Betula pendula Roth, Septoria robiniae (Lib.) Desm. (=Phloeospora robiniae (Lib.) Höhn.) on Robinia pseudoacacia L., Chalara hughesii Nag Raj & W.B. Kendr. on Quercus robur L.. All specimens are located in the Herbarium ESS, Mycotheca Parva collection G.B. Feige & N. Ale‐Agha. Key words: fungi, microfungi, biodiversity, parasitism

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258

P22 EVOLUTION OF THE PHYTOPHTHORA PORRI COMPLEX

BY MEANS OF HOST ADAPTATION, INTERSPECIFIC HYBRIDIZATION AND POLYPLOIDIZATION

L. BERTIER1, L. D’HONDT2, H. BROUWER3, A.W.A.M. DE COCK3,

L. LEUS2 & M. HÖFTE1 1 Ghent University, Department of Crop Protection

Coupure links 653, BE-9000 Gent, Belgium 2 Institute for Agricultural and Fisheries Research (ILVO)

Plant Sciences Unit, Applied Genetics and Breeding Caritasstraat 21, BE-9090 Melle, Belgium 3 CBS-KNAW, Fungal Biodiversity Centre

Uppsalalaan 8, NL-3584 CT Utrecht, the Netherlands

Since the description of Phytophthora infestans, the causal agent of the Irish potato famine in the 1850’s, the number of Phytophthora species has grown exponentially. Nowadays more than 100 different Phytophthora species are known to exist, and nearly all of them are plant pathogens. An extensive phylogenetic study has divided the genus into 10 well supported clades. Our research objectives were to have a closer look at the occurrence, genetic diversity and host adaptation of Phytophthora porri and other Phytophthora clade 8b species (with the exception of Phytophthora syringae). Clade 8b contains a group of slow growing, homothallic and cold tolerant species that are found on a range of winter grown crops cultivated in Belgium and in regions with similar climatic conditions. We have built a collection of 91 cultures isolated from 11 different hosts. The cultures were either freshly isolated from diseased plants or derived from culture collections around the world. Sequencing of the rDNA ITS region and the mtDNA Cox1 gene led us to hypothesize that next to the three officially described species (P. porri, P. primulae and P. brassicae); clade 8b contains several other distinct species that are still undescribed. The phylogenetic data together with pathogenicity information also reveal a clear host‐driven speciation in this clade. Interestingly, some isolates showed dimorphic peaks in their ITS sequences, a feature that is typical for interspecific hybrids. Cloning of the ITS region, Cox1 sequencing and genome size determination by use of flow cytometry further supports the hybrid origin of these isolates. Our data suggests that interspecific hybridization occurred at least twice in this clade, with different parental species involved. Since all hybrids were isolated from different hosts than the parental isolates, we also suggest that interspecific hybridization can be a means of adaptation to new hosts in Phytophthora. Moreover, by use of flow cytometry, we found that polyploidy is a widespread feature among these species. The ecological significance of this polyploidy is now under research. In conclusion, we want to stress the fact that evolution of Phytophthora clade 8b species is an ongoing process and that host shifts can occur quickly, especially when interspecific hybridization and polyploidization are in play.

Posters P23-P39

259

Posters Phytopathology: Biocontrol

Posters P23-P39

260

Posters P23-P39

261

P23 EFFICACY OF SOME PLANT EXTRACTS

AGAINST ALTERNARIA SOLANI THE CAUSAL AGENT OF EARLY BLIGHT OF TOMATO

K. ABO-ELYOUSR & Sallam NASHWA Plant Pathology Dept. Faculty of Agriculture

Assiut Univ. 71526 Assiut, Egypt The antimicrobial activity of six plant extracts, Ocimum basilicum Sweat Basil, Azadirachta indica Margosa tree, Eucalyptus chamadulonsis eucalyptus, Datura stramonium Jimsonweed, Nerium oleander oleander and Allium sativum garlic, were tested in controlling Alternaria solani in vitro and in vivo. In in vitro study the leaf extracts of D. stramonium, A. indica and A. sativum at 5% concentration caused highest reduction of mycelial growth of A. solani (44.4, 43.3 and 42.2% respectively), while O. basilicum at 1 and 5% and N. oleander at 5% caused the lowest inhibition of mycelia growth of the pathogen. In greenhouse experiments the highest reduction of diseases severity was achieved by fungicides (Ridomil Plus at 2 g/l) 82.8% followed by the extracts of A. sativum at 5% and D. stramonium at 1 and 5% concentration. All treatments, plants extracts and fungicides (Ridomil Plus), significantly reduced the early blight disease as well as increased the yield of tomato compared to infected control under field condition. The greatest reduction of diseases severity was achieved by Ridomil Plus 74.2 % followed by A. sativum at 5% and the smallest reduction was obtained when tomato plant was treated with O. basilicum at 1 and 5% (46.1 and 45.2 % respectively). Fungicide, D. stramonium and A. sativum at 5% increased the fruit yield 85.7, 76.2 and 66.7% compared to infected control. Key words: Alternaria solani, plant extracts, antimicrobial activity, early blight, tomato.

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262

P24 ANTAGONISTIC ACTIVITY IN VITRO AND IN VIVO OF CUBAN RHIZOBACTERIA AGAINST

PHYTOPHTHORA PALMIVORA AND PHYTOPHTHORA TROPICALIS IN CACAO

Y. ACEBO-GUERRERO1, M. HEYDRICH-PÉREZ1, Y. MATOS CUETO3,

M. EL JAZIRI2 & A. HERNÁNDEZ-RODRÍGUEZ1 1 Laboratory of Microbial Ecology, Faculty of Biology, University of Havana, Cuba

2 Laboratory of Plant Biotechnology, Université Libre de Bruxelles, Belgium 3 Experimental Station for Coffee and Cacao Research, Baracoa, Cuba

In Cuba, black pod has become one of the most prominent diseases of cacao. Once black pod has infected a cacao field, its eradication is very difficult. As the application of chemical inputs for disease control and prevention of black pod is not only frequently ineffective, but also hazardous to environment, there is an increasing tendency to consider Plant Growth Promoting Bacteria (PGPB) as an alternative for plant disease management and therefore to protect crops and the environment, as well as to cut down costs in agricultural production. There is also little knowledge of the molecular dialog involving genes and metabolites that is established during the interactions between the antagonists, the plant and the pathogen. This work was aimed to isolate and select Cuban autochthonous bacterial strains associated to cacao rhizosphere with antagonistic activity against 9 strains of Phytophthora palmivora and two strains of Phytophthora tropicalis, all isolated from the same ecosystem in Baracoa, Cuba. Samples from rhizospheric soil from different cacao cultivars were taken and 400 bacterial isolates were obtained. Antagonistic activity was determined in vitro in V8 Juice Agar. The best performing strains were selected and in vivo antagonism assays against two strains of P. palmivora and P. tropicalis were carried out in leaves and pods. The selected strains from the in vivo assays were identified by micromorphological and biochemical tests and the partial sequencing of 16s rDNA, rpoD and gyrB genes. The results showed that 9 of the isolates had antagonistic activity in vitro against at least two fungal strains and four of them were antagonists of the 11 fungal strains. The in vivo antagonism assays demonstrated that four of the strains that showed in vitro activity retained such activity in vivo, diminishing the disease symptoms in leaves and pods. In this study, the responses in vitro corresponded to the in vivo assays, although further studies must be carried out to ensure that the effect is extended to all of the Cuban Phytophthora strains. The isolates were identified as Pseudomonas chlororaphis, a nonpathogenic species with high potentialities to be used as a biocontrol agent. The feasibility of the use of microorganisms as biological control agents is highly attractive to producers, since the use of antagonistic microorganisms is frequently considered as one of the safest and most affordable control strategies. Key words: black pod, rhizobacteria, antagonistic activity

Posters P23-P39

263

P25 THE POTENTIAL OF SOME LOCAL ISOLATESOF STREPTOMYCES TO CONTROL DAMPING-OFF

DISEASE IN SOYBEAN CAUSED BY SCLEROTIUM ROLFSII

Ika ROCHDJATUN SASTRAHIDAYAT1, Syamsuddin DJAUHARI1

& Nasir SALEH3 1 Plant Protection Department, Agriculture Faculty, Universitas Brawijaya , Malang, Indonesia

2 Indonesia Legume and Tuber Crops Research Institute, Malang, Indonesia Biocontrol on plant pathogen has attractive extension by phytophathologies, recently due to reduction of environmental pollution by over using pesticide. The aim of this research was to determine the effectiveness of Streptomyces isolates alone or combined with VAM fungus to suppress damping‐off disease caused by S. rolfsii. Laboratory test using the opposing isolated in agar plates, the paper disc‐gelatin method and an over lay of Cellophane to control Sclerotium rolfsii by several isolates of Streptomyces showed that all Streptomyces isolates were able to inhibit S. rolfsii growth. On Cellophane test, Wajak isolate showed better growth than Pare and Cangar isolates to pressure S. rolfsii. The same effect was also shown by direct opposite test, in which all isolates inhibited S. rolfsii growth. The antibiotic test with paper disc method showed thickness of S. rolfsii colony; tested by the Wajak isolate surface S. rolfsii colony have a flat and thin, while the other isolates have a wavy surface. Field test showed that application of Streptomyces Wajak isolate combined with VAM pressed the attack of S.rolfsii. Combination of Streptomyces and VAM on soybean seedling to control this pathogen in endemic area was able to press seedling death until 50 %. Streptomyces can be used in liquid form for soaking seed up to 24 hours, and VAM can be introduced either in pellet or tablet form. The growth and yield were also influenced by the different inoculations applied to the combination of both Streptomyces and VAM. The research result shows that the combination of Streptomyces and VAM increases the yield up to 20% ‐ 28 %. Key words: Key words : Sclerotium rolfsii, Streptomyces antagonist, VAM, Soybean,

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264

P26 A JUDICOUS CHOICE OF SUBSTRATE

FORMULATION: THE KEY TO DEVELOPMENT OF AN EFFECTIVE BIOPESTICIDE

P. LALITHA1 & K. ARUNALAKSHMI2

1 Department of Mcrobiology 2 Department of Biotechnology GITAM Institute of Technology

GITAM University, Visakhapatnam 530045 India The world’s increasing population necessitates the development of new strategies for higher agricultural produce. Technology in one way has led to the synthesis of chemicals that enhance the growth of plants and some which reduce the crop loss due to pests. But the resistance in pests demands higher doses of the same chemicals that are hazardous to aquatic and terrestrial life. Scientists, since few decades have realised the serious problems with regard to the recalcitrant nature of the chemicals and have turned to biofertilizers and biopesticides. Trichoderma viride is one of the fugal species which has pesticidal properties. Almost the entire world makes use of this fungal species as a biopesticide. There is ample data regarding mass production of Trichoderma viride in submerged fermentation and relatively scanty data pertaining to solid state fermentation. Furthermore, a number of countries are working on developing strategies to increase the biomass. We have formulated four different media using agricultural wastes viz corn cob residue, sugarcane baggasse, corn leaf and peel of Citrus limette keeping, the nitrogen source same in all, and we have found that the biomass produced and the amount of cellulase produced by the culture in all formulations has no direct relationship. Higher biomass may not necessarily lead to higher production of chitinases as well. Hence, special formulation of medium, at farmer’s disposal has been made and tested in our lab for growth enhancement of plants as well as antagonising pathogens. The details shall be discussed. Key words: Substrate, Trichoderma viride, cellulose, biomass

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265

P27 EPICOCCUM NIGRUM FOR BIOCONTROL

AGENTS IN VITRO OF PLANT FUNGAL PATHOGENS

R. OGÓREK & E. PLĄSKOWSKA Department of Plant Protection, Section of Plant Pathology and

Mycology Wrocław University of Environmental and Life Sciences pl. Grunwaldzki 24a, PL-53-363 Wrocław, Poland

Epicoccum nigrum strains were evaluated in vitro as potential biological agents for control of the growth of Fusarium spp. and Botrytis cinerea. Epicoccum nigrum is an ubiquitous hyphomycete which grows saprophytically on many substrates. It has been isolated as well from a variety of soils and plants. It produces pigments (ß‐, y‐carotene) and antibiotics (epicorazine A, B and flavipin). It has been described as an organism antagonistic to several fungal pathogens, such as Sclerotinia sclerotiorum, Colletotrichum spp., Botrytis cinerea, Monilinia laxa and to some bacteria, such as Pseudomonas spp., Bacillus subtilis and Enterobacter cloacae Fusarium species is among the most important group of plant pathogens and it is also reported as etiological agents of opportunistic infections in humans. Mycotoxins produced by Fusarium spp. pose a significant hazard to food safety and human health. Botrytis cinerea, the cause of grey mould, is a necrotrophic pathogen causing pre‐harvest and post‐harvest diseases in at least 235 plant species. The five strains of E. nigrum used in the experiment were isolated from rye grain and from wheat grain, whereas the pathogens: B. cinerea and double isolates of F. avenaceum, F. graminearum and F. oxysporum were obtained only from wheat grain. The interaction of the fungi was studied using the biotic series method, that was elaborated for fungi on PDA medium. The inocula of 4 mm diameter were taken from ten‐day‐old cultures, one of E. nigrum and the other one of a pathogenic fungus. They were placed mycelium down and 2 cm apart in the centre of a Petri dish, on the solidified PDA medium. Petri plates with mycelium of single fungal species were used as reference. Each one of the experimental variants was prepared in 4 replicates (dishes), except for the reference ‐ two replicates. After inoculation the dishes were incubated for 10 days in the darkness at 24°C. While evaluating the biotic effect, the surrounding of one colony by another fungal species was observed and the occurrence of the inhibition zone between the colonies, as well as the reduction of the colony size were both taken into account. The individual biotic effect (IBE) may be positive or negative, as well as may also receive the value of 0. The results of IBE were analyzed by ANOVA, using Statistica 9.0 package. Means were compared using Fisher’s least significant difference (LSD) test at α 0.01. All strains of E. nigrum limited the growth of all isolates of Fusarium spp. after 10 days of dual growth, which was shown by positive values of IBE. However, Botrytis cinerea limited the growth all isolates of E. nigrum. CONCLUSIONS 1. The tested strains of E. nigrum have shown positive IBE and proved to be effective in vitro antagonists of F. avenaceum, F. graminearum and F. oxysporum, but they were not effective for B. cinerea. 2. Epicoccum nigrum strains should be therefore examined in vivo as potential biocontrol agents against Fusarium spp. Key words: Epicoccum nigrum, biotic effect, Fusarium spp., Botrytis cinerea

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266

P28 LACK OF NIFH GENE IN SOME ENDOPHYTIC BACTERIA ISOLATED ON RC SOLID MEDIUM

K. PISARSKA & M. ADAMSKI

Department of Agriculture Microbiology, Wroclaw University of Environmental and Life Science, Grunwaldzka 53, PL-50-357 Wroclaw, Poland

Presence of endophytic bacteria has been proved in many crops including maize (Zea mays L.). Endophytes play a significant role in plant nutrient and pesticide uptake. Application of endophytic bacteria is a goal of environmentally friendly agriculture. Occurrence of Azospirillum strains is often reported as tissue inhabiting bacteria of maize. They were reported as a potential plant growth promoting bacteria as well as biocontrol agent of several phytopathogenic microbes. The biological N2‐fixation is one of most important processes assigned to this bacteria. The objective of this study was to examine the biodiversity of Azospirillum spp. isolated from the leaves of 6 cultivates Zea mays L. growth on two experimental fields (Smolice and Kobierzyce, Poland). Isolation of Azospirillum spp. were done on the solid RC medium, based on typical appearance of colony. Forty four isolates grown as a small intensive small colonies were selected. Occurrence of Azospirillum‐like strains were not noticeably different among tested cultivars and at both locations. To verify ability to N2‐fixation isolates were analyzed based on nifH gene presence. Several publications describe nifH gene as good marker of nitrogen fixing microorganisms isolated from different habitats. This gene encodes Fe protein of nitrogenase complex (nitrogenase reductase). Presence of nifH gene was tested using PCR method with PolF and PolR universal degenerate primers. Positive results gave 360bp product size, same as reference Azospirillum brasiliense strain 36S and A. brasiliense strain 35Bb. The presence of nifH gene was found in 5 tested strains isolated from leaves of 3 cultivars (Cyrkon, KB1903, KB1902) from Smolice location, only. Our results suggest that selection of Azospirillum‐like strains on RC solid medium based on appearance of colony is not correlated with theirs ability to nitrogen fixation or used degenerated primers (PolF, PolR) are not universal enough. Key words: enpdohytic diazotrophs, nifH gene, N2‐fixation

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267

P29 CHARACTERIZATION OF BACILLUS STRAINS

ASSOCIATED TO RICE (ORYZA SATIVA L.) FOR THE GROWTH PROMOTION AND BIOLOGICAL CONTROL

Berto TEJERA1, Marcia M. ROJAS1, Yanelis ACEBO GUERRERO1,

Mayra HEYDRICH1 & Jacques MAHILLON2 1 Dpto de Microbiología y Virología, Facultad de Biología

Universidad de la Habana, La Habana, Cuba 2 Laboratory of Food and Environmental Microbiology

Université catholique de Louvain, Louvain-la-Neuve, Belgium The rice is one of the main cereals crop in the world, feeding more than 50% of the world population. Thus, rice fields demand managerial care to obtain the best quality and quantity of production with less environmental impact. The study of plant growth promoting bacteria (PGPB), constitute an important tool, they can stimulate the growth of their host, and represent a way to improve the crop. Among them, the genus Bacillus and their relatives have a great potential uses in the agriculture. This work shows the isolation and characterization of Bacillus strains from rhizosphere of rice crop (Oryza sativa L.). Two isolation methods were used, microcosm model to isolates bacteria from variety J‐104 and direct isolation from soil cultivated with INCA LP‐5 variety. Physiological characterization of 13 isolates was carried out, considering indole compounds production, phosphate solubilization, qualitative nitrogen fixing ability and antagonistic effect against phytophatogenic fungi of rice (Alternaria solani, Pyricularia grisea, Fusarium sp. and Curvularia sp.). All isolated strains have antagonistic effect in vitro against the four fungi and showed high inhibitions percents (50‐90%). Also, all strains produce indole compounds auxine type measured by colorimetric method until value of 16 µg.mL‐1. Also, 9 strains solubilize inorganic phosphate in solid NBRIP medium, showing haloes between 2 and 4 cm. On the other hand, 11 strains fix nitrogen in semisolid medium without any source of nitrogen. A conglomerated cluster analysis was performed to select the best isolates to be identified by biochemical test and 16SrDNA partial sequencing. The four selected strains belonged to the genus Bacillus and were classified as Bacillus subtilis, Bacillus cereus and two as Bacillus sp. This work shows the potential capacities of Bacillus strains associated with rice to be use in the biological control and growth promotion in order to obtain better results in the yield of this important crop in a sustainable agriculture. Key words: Bacillus, rice, biological control, plant growth promotion

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268

P30 STUDY OF INHIBITORY EFFECT OF BACTERIA

ISOLATED FROM PISTACHIO TREES ON ASPERGILLUS FLAVUS LINK, PRODUCING AFLATOXIN

S. AHMADI TAJ ABADI, H. ALAEI, A. MAREFAT & Gh. BARADARAN

Department of Plant Protection, Faculty of Agriculture, Zanjan University E-mail: [email protected]

Department of Plant Protection, Faculty of Agriculture, Vali-e-Asr University of Rafsanjan Plant Pests and Diseases Department of Agricultural and

Natural Resources Research Center of Kerman Aflatoxin contamination of pistachio, is the most important problem in production and export. Biological control is one of the more promising to reduce and eliminate aflatoxin contamination particularly for the near‐term. For this purpose, numerous organisms including bacteria, yeasts and nontoxigenic strains of A. flavus and A. parasiticus have been tested. Due to beginning of pistachio contamination to A. flavus on trees, before harvest and increased infection delaying the harvest, likely by using the active bacterial isolates with antifungal activity could be reduced aflatoxin contamination to acceptable limits. Therefore, during September and October 2009 (harvest time) as well as April and May 2010 (growing time), fruits and foliage samples of pistachio trees collected from different areas of Rafsanjan. In preliminary work, the number of 877 bacterial isolates was purified. Inhibitory effect of these isolates was tested on mycelial growth and sporulation of A. flavus on Potato Dextrose Agar and a medium prepared from pistachio kernel extract. Preliminary results showed that 125 isolates were able to prevent A. flavus sporulation and mycelial growth significantly. The results of volatile antifungal activity test showed that 17 out of 145 tested isolates were able to control mycelia growth significantly (P<0.01). 13 out of 17 selected isolates were able to produce antibiotics and had significant differences (P<0.01) to prevent mycelial growth. Based on physiology and biochemistry test the selected isolates were identified as Bacillus spp. (gram positive) and Pseudomonas spp. (gram negative). To determination of antagonistic effects of the selected isolates on pistachio fruit, further tests are running in our lab. Key words: Aspergillus, Aflatoxin, Biological control, Pistachio, Rafsanjan

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269

P31 INHIBITORY EFFECTS OF ESSENTIAL OILS

OF MEDICINAL PLANTS FROM GROWTH OF PLANT PATHOGENIC FUNGI

N. PANJEHKEH1, Z. JAHANI HOSSEIN-ABADI1 & M. FLAHATI RASTGAR2

1 Department of Plant Protection, Faculty of Agriculture University of Zabol, Zabol, Iran

2 Department of Plant Protection, Faculty of Agriculture Ferdowsi University of Mashhad, Mashhad, Iran

Plant cells produce a vast amount of secondary metabolites. Production of some compounds is restricted to a single species. Some compounds are nearly always found only in certain specific plant organs and during a specific developmental period of the plant. Some secondary metabolites of plants serve as defence agents against invading microorganisms. Nowadays, it is attempted to substitute the biological and natural agents with chemically synthesized fungicides. In the present research, the antifungal activities of essential oils of seven medicinal plants on mycelial growth of three soilborne plant pathogenic fungi were investigated. The plants consisted of Zataria multiflora, Thymus carmanicus, Mentha pieperata, Satureja hortensis, Lavandual officinalis, Cuminum cyminum and Azadirachta indica. The first five plants are from the family Labiatae. Examined fungi, Fusarium oxysporum f.sp. lycopersici, Fusarium solani and Rhizoctonia solani are the causal agents of tomato root rot. Essential oils of Z. multiflora, T. carmanicus, M. pieperata, S. hortensis and C. cyminum were extracted by hydro‐distillation method. Essential oils of L. officinalis and A. indica were extracted by vapor‐distillation method. A completely randomized design with five replicates was used to examine the inhibitory impact of each concentration (300, 600 and 900 ppm) of each essential oil. Poisoned food assay using potato dextrose agar (PDA) medium was employed. Results showed that essential oils of A. indica, Z. multiflora, T. carmanicus and S. hortensis in 900 ppm at 12 days post‐inoculation, when the control fungi completely covered the plates, prevented about 90% from mycelial growth of each of the fungi. While, the essential oils of M. pieperata, C. cyminum and L. officinalis in the same concentration and time prevented 54.86, 52.77 and 48.84%, respectively, from F. solani growth. These substances did not prevent from F. oxysporum f.sp. lycopersici and R. solani growth. Minimum inhibitory concentration (MIC) of essential oils of T. carmanicus, Z. multiflora and A. indica from R. solani and F. solani growth was 900 and 600 ppm, respectively. In addition, the MIC of essential oils of these plants and essential oil of S. hortensis from F. oxysporum f.sp. lycopersici growth was 900 ppm. The MIC of essential oils of M. pieperata, C. cyminum and L. officinalis from F. solani growth was 900 ppm. Key words: essential oil, inhibitory, soilborne fungi

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270

P32 EFFECTS OF VOLATILE ORGANIC COMPOUNDS

OF THE ROOTS OF THE BARLEY IN THE RHIZOSPHERE M. FIERS1, F. BARSICS2, M. CAMERMAN3, P. DELAPLACE3, N. DE LA VALLÉE POUSSIN4, M-F. DESTAIN5, P. DU JARDIN3, A. GFELLER3, E. HAUBRUGE2,

L. HIRTT5, D.E. KATI4, L. LASSOIS1, P. LEPOIVRE1, G. LOGNAY6, F. OOMS5, F. VERHEGGEN2, J-P WATHELET4 & M-L. FAUCONNIER3 1 Plant Pathology Unit, 2 Functional and Evolutionary Entomology Unit

3 Plant Biology Unit, 4 General and Organic Chemistry Unit 5 Mechanics and Construction Unit, 6 Analytical Chemistry Unit

Gembloux Agro-Bio Tech, University of Liège Passage des Déportés 2, BE-5030 Gembloux, Belgium

Volatile organic compounds emitted by plants are known to intervene with various biotic environmental factors. Up to now, most of the studies have been focused on aerial volatiles and root liquid exudates. Very few researches have been completed concerning belowground volatiles released into the rhizosphere despite their potential capacity to carry information between organisms. The Rhizovol project involves 5 different units of Gembloux Agro‐Bio Tech collectively studying the production of belowground volatiles by barley roots underlying various biotic interactions in the rhizosphere. To achieve this goal, analytical methods allowing the sampling, separation, identification and quantification of belowground volatile compounds have to be developed. They enable the subsequent characterization and study of the interactions between barley and its rhizospheric partners chosen for this study. These interactions imply beneficial organisms, pathogenic agents and plant and insect pests. Beneficial organisms can promote the growth of barley by the emission of volatiles; on the other hand barley can support their growth and metabolism. These phenomenons will be assessed by the study of 19 strains of plant growth‐promoting bacteria (PGPR). Three pathogenic agents ‐ two fungi (Fusarium culmorum and Cochliobolus sativus) and one virus (Barley yellow dwarf virus) ‐ were chosen as they are known to cause various diseases on barley, especially on roots. The attractive or repellent effects of barley root volatiles on the pathogenic agents or their vectors, as well as the effect of volatiles on the diseases evolution will be evaluated. The project also includes several types of pests such as plants and insects. Plants can compete with barley for space and nutrients through volatile interactions. This will be assessed by the study of allelopathy with 8 weeds and a hemiparasitic plant (Rhinanthus spp.). The effects of barley volatiles can also impact the severity of the attacks by insects. This part will be conducted with wireworms as they represent worldwide known pests, and aphids, through their viral vector role. Eventually, as soil characteristics can strongly influence the diffusion of volatile compounds, the diffusion behaviour of the identified volatile biomolecules through the soil will be modelled. Tritrophic interactions (e.g. insect‐plant‐pathogenic fungi) will be studied based on each bitrophic interaction results. Over‐all, the Rhizovol project aims at improving the knowledge of interactions mediated by volatile compounds in the rhizosphere and at establishing new biocontrol methods that could contribute to integrated disease and pest management systems. Key words: volatile compound, Hordeum vulgare, multitrophic interaction, beneficial organism, pathogen, pests

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271

P33 IN VITRO INDUCTION OF BIOFILM IN THE BIOCONTROL YEAST CANDIDA OLEOPHILA

(ISOLATE 13L) INHIBITS P. DIGITATUM CONIDIA GERMINATION

G. D’HALLEWIN1, G. PANI1, T. VENDITTI1, M.G. MOLINU1,

A. DORE1, S. MARCEDDU1 & G. LADU2 1 CNR - ISPA, Traversa La Crucca, 3 - Loc. Baldinca, IT-07040 Sassari, Italy

2 DISAABA - Università degli Studi Sassari, viale Italia 39, IT-07100 Sassari, Italy Even if the modes of action of several biocontrol agents have been investigated, evidenced and the efficacy for the control of postharvest pathogens implemented, several aspects of the antagonist‐pathogen‐host interaction are still undefined. Indeed, little is known about the involvement and role, if any, of biofilm formed during the antagonist‐pathogen interaction. This aspect is well documented for those micro‐organisms involved in human diseases, such as Candida spp., in wood‐degradation (e.g. Pseudomonas aeruginosa), in brewery and winery (e.g. Saccharomyces spp.) and for some filamentous fungi (e.g. Aspergillus niger, Penicillium spp.). In order to gain knowledge on the role of the biofilm in the antagonist‐pathogen interaction we studied in vitro the behaviour of 4 yeasts, with similar colonization ability but with different antagonistic activity, against Penicillium digitatum Sacc., the agent cause of citrus green mold. The antagonists employed were isolates of Candida oleophila, Pichia guilliermondii, Candida saitoana and Rhodotorula glutinis labelled 13L, 5A, 8C and 21A, respectively. Each isolate (25 µl of a 105cfu.mL‐1) was plated on potato dextrose agar (PDA) or yeast peptone dextrose (YPD with 2 or 0.3% agar) in Petri dishes (9 cm Ø) and incubated at 25 °C in the dark. After 12 h, half of each Petri dish with the antagonists in a planktonic form were covered with a portion of a 47 mm sterile membrane filter (pore size 0.45 µm) containing or not conidia of P. digitatum (~100 conidia), and dishes were returned to the incubation chamber. After that, the behaviour of the antagonists, exposed or not to the conidia or to the sterile membrane, was investigated by performing the invasive growth assay (after 3d), by checking the mat formation ability (at 10 d) and by scanning electron microscopy (SEM) observations. Three isolates, 5A 8C and 21A, delayed conidia germination compared to the control, while isolate 13L inhibited completely conidia germination up to the end of the experiment (15 d). SEM observations evidenced that isolate 13L formed a biofilm after application of conidia, and that under these condition no germination occurred. When conidia were removed and put on PDA, germination occurred regularly. This indicating that conidia were not killed but only germination was inhibited. According to previous experiments in vivo, with the 4 reported antagonists, the most effective in controlling citrus green mould was isolate 13L. Indeed, a complete control was obtained when this isolate was applied to wounds 24 h before conidia of P. digitatum. Based on this result and on the ones achieved in vitro, it is likely that the biofilm, formed upon the contact with the conidia, is a stress reaction of this isolate that inhibits the germination of conidia, controlling in this way the infection process in vivo. Key words: Citrus fruits, biological control, postharvest

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272

P34 INDUCTION OF DEFENCE IN CHICKPEA PLANTS BY RHIZOBIA IN RESPONSE TO INFECTION BY

RHIZOCTONIA SOLANI

Y. MABROUK2, I. HEMISSI1,2,3, S. MEJRI2, M. SAIDI2 & G. SIFI1 1 Institut National de Recherche Agronomique de Tunisie (INRAT)

2 Unité d’Utilisation Médicale et Agricole des Techniques Nucléaires, Centre National des Sciences et Technologies Nucléaires, Sidi Thabet, 2020 Tunis

3 Institut National d’Agronomie de Tunis (INAT) Nodulation effects on chickpea–Rhizoctonia solani relationships were studied using the commercial chickpea variety Béja1 and different Rhizobium strains using Petri dish experiments. First, the benefit of bacterial inoculation on plant growth was demonstrated for two isolates, Pch Azm and Pch S.Nsir2. Moreover, these isolates led to reduced chickpea infection by R. solani, resulting from the effect direct of rhizobia on pathogens and possible induced resistance in chickpea. Concomitantly, reduction in infection was accompanied by enhanced level of defence‐related enzymes, L‐phenylalanine ammonia lyase (PAL) and peroxidase (POX). An increased level of phenol content was recorded in the roots of bacterized plants grown in the presence of pathogen. The results promise the use of rhizobia for protection of chickpea against R. solani. Key words: Rhizobium, Rhizoctonia solani, mechanisms of defence, PAL, Pox

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273

P35 FLUORESCENT PSEUDOMONADS ISOLATED FROM

RICE RHIZOSPHERE HAVE IN VITRO ANTAGONISTIC ACTIVITY AND INDUCE IN VIVO PLANT RESISTANCE

AGAINST PYRICULARIA GRISEA (SACC.)

Annia HERNÁNDEZ-RODRÍGUEZ1, Yanelis ACEBO-GUERRERO1, Narovis RIVES-RODRIGUEZ1, Michel ALMAGUER CHAVEZ1,

Mayra HEYDRICH-PÉREZ1, Mondher EL JAZIRI2 & Olivier M. VANDEPUTTE2 1 Departamento de Microbiología y Virología, Facultad de Biología, Universidad de la Habana,

Calle 25 # 455. Plaza, Ciudad Habana, Cuba. 10347 E-mail: [email protected]

2 Laboratoire de Biotechnologie Végétale. Université Libre de Bruxelles (ULB) 8 rue Adrienne Bolland, BE-6041, Gosselies, Belgium

Rice blast is widely spread worldwide, being the most important disease for this crop. It is frequently managed through chemical inputs, however, this strategy has proven to be often ineffective and pollutant for the environment. Fluorescent pseudomonads are an excellent example of the combination of multiple mechanisms to exert an effective biocontrol The aim of this work was to isolate Cuban native fluorescent pseudomonads from rice rhizosphere, to select thoses that show in vitro antagonistic activity against Pyricularia grisea and to evaluate the in vivo efficiency of selected Pseudomonas strains as a biocontrol agents in P. grisea infected plants. The results showed that 40 isolates that showed the typical growth and cultural characteristics of fluorescent pseudomonads in King B plates. Antagonistic activity against 5 strains of Pyricularia grisea was assessed, selecting 12 isolates for their higher antagonistic activity, being identified as P. putida (9 strains) and P. fluorescens (3 strains). These strains were capable of producing siderophores under limiting‐iron conditions. Subsequently, in vivo experiments for the biocontrol of P. grisea AAM‐275 were carried out and the results showed that only 8 of the strains showed efficient control in vivo, showing that the shoot length, shoot and root fresh weight were increased in all the plants treated with bacteria, compared to the nonfungus‐inoculated and fungus‐inoculated controls. All the strains exerted biocontrol over P. grisea, showing a decrease of the disease symptoms in the rhizobacteria‐treated plants (28‐43 %), in comparison with fungus‐inoculated control. These results showed that P. fluorescens AI05 and AI08 and P. putida AI03, AI02, AJ01, AJ29 and AJ30 promote plant growth and biocontrol P. grisea in rice, which could lower the application of pesticides and chemical fertilizers, hence reducing the hazardous effects on the environment Key words: Fluorescent pseudomonads, rice, antagonistic activity, plant resistance, Pyricularia grisea

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274

P36 CONTROL OF PHYTOPHTHORA CINNAMOMI

ONHEDERA USING FUNGAL BIOLOGICAL CONTROL AGENTS

S. INGHELBRECHT1, E. VAN WAMBEKE2, B. GOBIN3, K. HEUNGENS1

& M. VISSERS3 1 Institute for Agricultural and Fisheries Research (ILVO), Plant Sciences Unit

Burg. Van Gansberghelaan 96 bus 2, BE-9820 Merelbeke, Belgium 2 Certis Europe, Woluwedal 60, BE-1200 Brussel, Belgium

3 Research Centre for Ornamental Plants (PCS) Schaessestraat 18, BE-9070 Destelbergen, Belgium

Hedera, a popular potted ornamental plant product, is often grown on ebb‐and‐flow irrigation benches. This increases the risk of disease from root infecting oomycetes such as Pythium spp. and Phytophthora spp. Due to increased regulation and a negative public opinion towards the use of chemical pesticides, growers welcome non‐chemical control options that offer adequate pathogen control at acceptable prices. We developed a bioassay to test the control capacity of three fungal biocontrol agents and then conducted a GEP‐level efficacy test. The biocontrol treatments were Trianum P (Trichoderma harzianum T‐22), Tusal (Trichoderma atroviride T‐11 and T. asperellum T‐25) and Prestop (Gliocladium catenulatum). These treatments were applied in suspension to the potting soil at the manufacturers’ recommended dose 1 day after planting the cuttings and again 6 weeks after planting. A chemical control reference treatment (Fenomenal) and a water reference (no control of pathogen) treatment were also included. Ten cuttings were placed per 1 L pot. One replicate consisted of 10 pots, and 4 replicates were used for each treatment in a randomized complete block design. The Phytophthora cinnamomi isolate used for the artificial inoculation originated from diseased Hedera roots. Zoospores were generated using a modification of the technique described by Byrt and Grant (1979). The pathogen was inoculated twice, 1 week and 7 weeks after planting, using 15 ml of 1x104 and 5x104 zoospores per ml per pot, respectively. Root and rhizosphere colonization with Trichoderma spp. was verified in the water reference, Trianum, and Tusal treatments at 1 month after planting using a semi‐selective medium based on Askew and Laing (1993). Disease progression was monitored weekly during 3 months. Final average disease incidence in the water reference treatment was moderate (8.75%) but representative of a commercial situation and still allowed discrimination between the treatments. The best control was observed with the Fenomenal (0.75% disease incidence) and Tusal (3.00% disease incidence) treatments, which were both significantly different (p<0.05) from the Trianum (6.75% disease incidence), water, and Prestop (9.25 % disease incidence) treatments. Rhizosphere colonization with Trichoderma spp. was 5.0x102 cfu/g for the water treatment, 7.05x103 cfu/g for the Tusal treatment, and 3.66x104 cfu/g for the Trianum treatment. The difference in root colonization level was similar to the difference in the number of originally inoculated cfu per pot. This experiment revealed that one of the biocontrol treatments offered a level of disease control that was not significantly different from the chemical control treatment. References: Byrt P. and Grant B.R. (1979). Australian Journal of Botany 27(2): 103‐115Askew D.J. and Laing M.D. (1993). Plant Pathology 42 (5): 686‐690 Key words: bioassay, biocontrol, oomycete, colonization, Trichoderma, Gliocladium

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275

P37 VERTICILLIUM TRICORPUS: A POTENTIAL

INDIGENOUS BIOLOGICAL CONTROL AGENT OF VERTICILLIUM WILT IN BELGIUM

Soraya C. FRANÇA1, Lien TYVAERT1, Jane DEBODE2, Katrijn SPIESSENS3, Luc DE ROOSTER3, Sabien POLLET4, Danny CALLENS4 & Monica HÖFTE1

1 Laboratory of Phytopathology, Faculty of Bioscience Engineering Ghent University, Gent, Belgium

2 Crop Protection, Institute for Agricultural and Fisheries Research, Merelbeke, Belgium 3 Proefstation voor de Groenteteelt, Sint-Katelijne-Waver, Belgium

4Provinciaal Onderzoeks- en Voorlichtingscentrum voor Land- en Tuinbouw, Beitem, Belgium Verticillium wilt is an important soil‐borne disease, affecting many crops worldwide. In Belgium, Verticillium wilt causes losses to cauliflower, strawberry, chrysanthemum and tree nurseries. Three species of Verticillium can cause wilt: V. albo‐atrum, V. dahliae and V. longisporum. The control of these pathogens is difficult because they produce melanized structures that are very long‐lasting in soil. V. dahliae and V. longisporum form microsclerotia and V. albo‐atrum, dark resting mycelium. Specially microsclerotia are very persistent in soil. Our research focuses on cauliflower as a model crop. The disease in cauliflower is caused by V. longisporum. Soil chemical disinfestation is not rentable for this crop and, additionally, not ecologically sound. Field experiments in Oppuurs‐Antwerpen and Ardooie ‐West‐Vlaanderen have shown that 1 microsclerotium/g soil is enough to cause 100% of Verticillium wilt incidence in cauliflower. Therefore to control the disease, microsclerotia in soil have to be reduced to very low levels. However, in some plots in Ardooie, besides microsclerotia of V. longisporum, a high number of microsclerotia of Verticillium tricorpus was found. Interestingly, the disease incidence in these plots was around 50% lower than in neighboring plots, although more than 1 V. longisporum microsclerotium/g soil was present. The ability of V. tricorpus to protect potato and lettuce against virulent isolates of V. dahliae has been reported in the literature. To investigate the interaction of the indigenous V. tricorpus with V. longisporum in cauliflower, a time course experiment was carried out. Cauliflower seedlings were inoculated with V. tricorpus by using the root‐dip method. Seven days later, V. longisporum was applied in the same way to the seedlings. Root and stem samples were taken 28, 49, 63 and 77 days post inoculation of the pathogen and fungal DNA of both Verticillium species was quantified with real‐time PCR. Disease symptoms and plant growth were also recorded. V. tricorpus behaved as an endophyte, i.e. it colonized the plants without causing symptoms and even stimulated the growth of small seedlings. Reduced root and stem colonization by V. longisporum occurred when plants were previously inoculated with V. tricorpus. Consequently, disease incidence and severity were clearly lower in plants associated with V. tricorpus than in non‐ associated ones. We demonstrated that the indigenous V. tricorpus found in our soils is a potential biocontrol agent of Verticillium wilt. Key words: Verticillium wilt, Verticillium tricorpus, biocontrol, endophyte

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276

P38 ABILITY OF SERRATIA PLYMUTHICA STRAINS TO

CONTROL BOTRYTIS CINEREA IN TOMATO AND BEAN BY DIRECT ANTAGONISM AND INDUCED RESISTANCE

Zabihollah AZAMI-SARDOOEI1,2 & Monica HÖFTE1

1 Laboratory of Phytopathology, Faculty of Bioscience Engineering, Ghent University Coupure Links 653, BE-9000 Ghent, Belgium

2 Crop Productions Department, Faculty of Agriculture, University of Jiroft, Jiroft, Iran The main plan of biological control is to find alternative agents to replace fungicides. Grey mould disease (Botrytis cinerea) seriously hampers vegetables and other crops. The soil strains Serratia plymuthica IC14 and IC1270, are produced the antibiotic pyrrolnitrin (Prn), as well as chitinolytic enzymes (chitinases). We investigated the potential of both strains to protect tomato and bean from the leaf pathogen B. cinerea. In addition, chitinolytic activity and antibiosis were studied as possible biocontrol mechanisms. Tomato (5‐week‐old) and bean (2‐week‐old) plants were sprayed with bacterial suspensions 108 cfu ml‐1 or tap water as control. Tertiary leaves of tomato and primary leaves of bean plants were inoculated with B. cinerea at 1, 5 and 7 days post treatment (dpt) using the detached leaf method. Ten 5µl and 10µl droplets of the conidial suspension (105 spores ml‐1) were applied on each tomato and bean leaf, respectively. Disease development was evaluated 3 and 4 days later by recording number of spreading and non‐spreading lesions. Foliar application of strains IC14 and IC1270 on tomato and bean resulted in an enhanced resistance to the grey mould pathogen in a detached leaf assay. Both strains were able to significantly reduce the number of spreading B. cinerea lesions and the bacteria protected plants up to 7 dpt, despite the fact that the bacteria survived poorly on the leaves and could no longer be detected 5 or 7 dpt. Foliar spraying of a chitinase‐deficient (IC14#18) mutant of strain IC14 decreased the severity of disease to a level comparable to that achieved with the parental strain in both tomato and bean, indicating that the 58 kDa chitinase is not important in biocontrol. Mutant IC1270‐C7 deficient in chitinase production and mutant IC1270‐P1 lacking Prn production still had a residual protective effect against B. cinerea on tomato but not on bean. Mutant IC1270‐RpoS deficient in Prn production was nearly as effective as the parental strain to suppress grey mould on both plants. Mutant IC1270‐grrA, impaired in chitinase and Prn, retained some activity in tomato but not in bean. These data indicate that chitinases and pyrrolnitrin are both involved in biocontrol by IC1270 on bean. Both S. plymuthica strains could play a key role in crop protection and are promising candidates for application in sustainable agriculture, especially in vegetable production. Key words: Biocontrol, Serratia plymuthica, Botrytis cinerea, Solanum lycopersicon, Phaseolus vulgaris, Direct antagonism,Induced resistance

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277

P39 SCREENING OF DIFFERENT BIOFUNGICIDES TO

CONTROL GREY MOULD IN TOMATO

Sarah VAN BENEDEN1,2, Johan DE KOKER2, Jasper CARRETTE2, Monica HÖFTE1 & Geert HAESAERT2

1 Laboratory of Phytopathology, Department of Crop Protection, Ghent University Ghent, Belgium.

2 Faculty Biosciences and Landscape Architecture, University College Ghent Ghent, Belgium

E-mail: [email protected] Botrytis cinerea,a saprotrophicfungus with a wide host range, is the causal agent of grey mould in tomato. The infection often is initiated on wounds where leaves and side shoots are removed.When the disease spreads to the healthy stem it results in stem rot, wilting and sometimes even in plant death. Currently a strict chemical fungicide scheme is used to control B. cinerea in tomato greenhouses in Belgium. In this study the efficacy of different biofungicides (plant extracts and antagonists) was evaluated in a detached leaf assay. Briefly, 8 week old tomato plants were treated with a biofungicide; 1 and 7 days after treatment the 6th, 7th and 8th leaf were detached and placed in a moisture chamber. Subsequently, each leaflet was drop‐inoculated with 10 µl (105B. cinerea spores ml‐1) spore suspension. Ten days post infection, lesions were evaluated using a score 0‐4. The experiment was repeated in time. Promising results were obtained with 5 biofungicides, (3 productscontaining plant extracts and 2 products containing antagonists).Soybean lecithin resulted in a very good control of B. cinerea.Products based on citrus oil + cinnamon and red thyme oil significantly reduced thesymptoms. In addition, two products containing Gliocladium sp. had a moderate but consistent control effect on B. cinerea. Currently we are evaluating the control efficacy of the biofungicides when applied directly on woundsand we are investigating the underlying mechanism.

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279

Posters Phytopathology: Fungicides

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280

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281

P40 EFFECT OF FUNGICIDES ON EPIPHYTIC

MICROORGANISMS ASSOCIATED WITH STRAWBERRY

J. DEBODE1, W. VAN HEMELRIJCK2, S. BAEYEN1, C. VAN MALDERGHEM1, M-A. VAN BOST1, P. CREEMERS2 & M. MAES1

1 Plant Sciences Unit – Crop Protection, Institute for Agricultural and Fisheries Research (ILVO) Burg. van Gansberghelaan 96 bus 2, BE-9820 Merelbeke, Belgium.

2 Research centre for fruit cultivation (PCF) Fruittuinweg 1, BE-3800 Sint-Truiden, Belgium.

Epiphytic microorganisms could provide an important natural buffer against foliar plant pathogens. Under laboratory conditions, certain isolates have shown potential for use as biological control agents. But before these epiphytic microorganisms can be exploited as protective and biological control organisms, their behaviour under field conditions must be studied, including the effects of the applications of fungicides. In the present study, the effect of two commonly used fungicides on the density and diversity of epiphytic microorganisms of field‐grown strawberry was investigated, with special attention to the epiphytic yeast community. In the summer of 2010, Switch® (cyprodinil plus fludioxonil) and Signum® (boscalid plus pyraclostrobin) were applied weekly over a 1‐month period to strawberry plants. Epiphytic populations on leaves and (im)mature fruits were assessed in treated and untreated plots at several time points by plating and denaturing gradient gel electrophoresis (DGGE) analysis. The treated strawberry plants had similar epiphytic counts to the control plants. Immature fruits showed significantly larger counts than mature fruits or leaves. Similarly, DGGE analysis showed that the tested fungicides had only a minor impact on the composition of the epiphytic communities, in contrast to strawberry tissue (fruit or leaves) and sampling time. Among yeast species identified, Cryptococcus, Rhodosporidium and Sporidiobulus dominated on the plates and in the DGGE analysis. These results suggest that fungicide resistance exists among epiphytic microorganisms of strawberry. This shows potential for the development of integrated control of the important fungal diseases of strawberry. Key words: DGGE, Fragaria × ananassa, plating, phylloplane, yeast

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282

P41 SCREENING OF FUNGICIDE EFFICACY AND FUNGICIDE

RESISTANCE AS PART OF INTEGRATED CONTROL OF CYLINDROCLADIUM BUXICOLA ON BUXUS

B. GEHESQUIERE1-2, F. RYS2, F. GOOSSENS3, K. VAN POUCKE1

B. GOBIN2, M. MAES1 & K. HEUNGENS1 1 Plant Sciences Unit – Crop Protection, Institute for Agricultural and Fisheries Research (ILVO)

Burg. Van Gansberghelaan 96/2, BE-9820 Merelbeke, Belgium 2 Ornamental Plant Research (PCS), Schaessestraat 18, BE-9070 Destelbergen, Belgium

3 Agricultural Policy Department (ADLO) Burg. Van Gansberghelaan 115A, BE-9820 Merelbeke, Belgium

Cylindrocladium buxicola is an ascomycete fungus that causes “box blight” in Buxus spp. First detected in nurseries in the United Kingdom in the mid‐1990s, the disease has spread throughout Western Europe. Under warm and humid conditions, disease symptoms can increase rapidly, causing substantial economic losses to boxwood growers. During two experiments in 2007, 11 fungicides were tested in vivo for their preventive and curative efficacy against C. buxicola. As an imitation of preventive and curative treatments, B. sempervirens plants were treated with fungicides either 1 day before or 2 days after artificial inoculation. The pathogen was inoculated as a conidiospore suspension (2 x 104 spores/ml ; 400 ml/m²) using a pressurized spray bottle. To maximize disease development, the plants were incubated under humid conditions. Preventive and curative efficacy of the different fungicides was assessed using a percentage disease severity scale at 1, 2 and 3 weeks after inoculation. Five of the most effective fungicides were selected and retested using the Good Experimental Practice (GEP) standard adopted by the EU: Eminent 125 ME (tetraconazole), Candit 50 WG (kresoxim‐methyl), Bravo 500 SC (chloorthalonil), Topsin M 70 WG (thiofanaat‐methyl) and Ortiva Top 200/125 SC (azoxystrobin + difenoconazole). As a result of this GEP test, these products are currently registered in Belgium for use against box blight. In 2010, in vitro mycelium growth tests (PDA, 20°C) were conducted to screen isolates for resistance against the fungicides Eminent 125 ME, Candit 50 WG and Topsin M 70 WG. Three fungicide‐sensitive isolates were first tested on a range of concentrations of each fungicide. Once the discriminatory dose (i.e., the lowest dose for maximum inhibition) for each product was determined, all isolates could be tested at a single concentration to classify them as sensitive or resistant. One hundred isolates originating from Belgium, United Kingdom, the Netherlands, France, Germany, New Zealand and Switzerland were tested. Five isolates (5%) were at least 10 times less sensitive to tetraconazole (Eminent 125 ME) and kresoxim‐methyl (Candit 50 WG) than the other isolates. Conidiospores of two resistant and two sensitive isolates were artificially inoculated on plants (Buxus sempervirens ‘Suffruticosa’) that were preventively treated with the standard dose of either Eminent 125 ME or Candit 50 WG. These standard treatments did provide adequate control using the sensitive isolates. However, they both failed to control disease development using the two resistant isolates, demonstrating that the level of fungicide resistance in these isolates is sufficient for failure of these products under practical conditions. This research demonstrates that several fungicides can provide adequate control of box blight but that special care is needed to prevent or manage fungicide resistance. Key words: Buxus ‐ Cylindrocladium buxicola ‐ box blight ‐ fungicide efficacy ‐ fungicide resistance

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283

P42 EFFICACY OF DIFFERENT FUNGICIDES FOR THE

CONTROL OF EARLY BLIGHT

B. HEREMANS, L. DE LANGE, J. CARRETTE & G. HAESAERT University College Ghent, Faculty of Biosciences and Landscape Architecture, Schoonmeersstraat 52 – Valentin Vaerwyckweg 1, BE-9000 Ghent, Belgium

Early blight caused by two species of the genus Alternaria occurs commonly worldwide on potato crops and other Solanaceae. A. solani and A. alternata are destructive pathogens, particularly in regions with high temperature and alternating dry and high humidity periods. Early blight results in premature dying of foliage and yield losses. Early blight was normally controlled by fungicide treatments against Phytophthora infestans, but in the last years the disease gained in importance. This change is due to several reasons: reduction of nitrogen supply to the crop, climatic change, the growing of more susceptible potato varieties and the use of new fungicides against late blight with less efficacy against early blight. In recent years some specific fungicides against early blight were developped. In this study the efficacy of several fungicides was tested against A. solani and A. alternata in in vitro tests. The tested fungicides showed differences of efficacy in controlling the two Alternaria species. Azoxystrobin (Amistar) and boscalid plus pyraclostrobin (Terminett) were developped for the control of Alternaria species in potatoes. Azoxystrobin (Amistar) and boscalid plus pyraclostrobin (Terminett) completely inhibited the mycelium growth of the tested A. alternata isolates. Nevertheless, an efficiency of 70 % and 50 % was observed for respectively the field dose of boscalid plus pyraclostrobin (Terminett) and azoxystrobin (Amistar) on the German isolate of A. alternata. For azoxystrobin (Amistar) a mean inhibition of 32 % was observed for the A. solani isolates. Boscalid plus pyraclostrobin (Terminett) completely controlled the growth of the tested A. solani isolates even at reduced dose. The other fungicides tested are fungicides used to control late blight in potatoes. These fungicides controlled very well A. alternata. The field dose of these fungicides completely inhibited the growth of all the A. solani isolates tested. The Belgian isolates were less sensitive to the lower doses of these fungicides then the German isolate. In vitro the fungicides based on mancozeb were very efficient in controlling early blight. The disease control was better then the control by the strobilurin azoxystrobin, which act by inhibiting mitochondrial respiration in fungi. Furthermore, fungicides may be less efficient on different isolates in an Alternaria population and Alternaria isolates may develop resistance to some azoxystrobin. Key words: A. solani ‐ A. alternata ‐ early blight ‐ fungicides

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284

P43 THE CONTROL OF WHEAT AND MAIZE DISEASES IN

SOIL CONSERVATIVE AGRICULTURE SYSTEM

E. NAGY, K. NAGY, M. IGNEA, V. HAS, F. MURESANU & L. TICUDEAN Plant Protection,Agricultural Research-Development Station, Turda, Romania

Conservative agriculture system is large used in the world for many crops, but in Romania have been lately adapted, even though the advantages of soil conservation by reduced erosion and the reduced energy, time and labor needed are widely recognized. This study was undertaken to investigate the control of wheat diseases in conservative agriculture system. The experiments were carried out at Agricultural Research Development Station Turda. In a polyfactorial experiments with two fertilizers levels having two graduated: N50

P50K50 kg/ha at sowing,and N50 P50K50 kg/ha at sowing + N50 P30 kg/ha at the renewal of vegetation in the spring. The wheat cultivars are four: Ariesan, Turda 2000, Appulum, Dumbrava, created in our station. Treatments variants eight were constituted by applying the foliar fertilizers, insecticides and fungicides, concomitant or consecutive in four wheat vegetation faze: early in the spring, end of tiller, blossom and flowering. For maize, it has been studied, the influence of main technological factors: three plant density, two fertilizer levels on the main diseases at five hybrids in conservative agriculture system. The dominant wheat diseases were tan spot (Pyrenophora tritici repentis)(PTR), leaf and glume blotch (Septoria tritici and Stagonospora nodorum) and Fusarium head blight (Fusarium spp.) (FHB). The others diseases such as powdery mildew (Blumeria graminis f.sp. tritici), rusts (Pucciniastriiformis, Puccinia recondita) appeared later and presented a reduced attack degrees, above 2 %. Analyzing of obtained data in this experimental system, resulted that fertilizer levels have not influenced significantly the increase of the attack degree cause by PTR and FHB. Referring at the behavior of wheat cultivars, there is a differential types, Apullum and Dumbrava cultivars being significantly more resistant to the PTR and FHB, Turda 2000 is the most susceptible to both diseases, respectively. Analyzing of 8 treatment variants, resulted that, one foliar fungicidal treatment against the foliar and ear diseases is not sufficiently under a strong infection pressure. By applying two fungicidal treatments, at least, at blossom and flowering, it was assured a good protection of leaf flag and ear, obtaining the higher and improving qualitative wheat yields. In maize, only stalk rot (Fusarium spp.) and logged plants were influenced significantly by technological factors in soil conservative agriculture system. In soil conservative agriculture system, wheat and maize diseases had a increased tendency, but not significantly in almost cases. Keywords: conservative agriculture system, wheat and maize diseases, attack degree, fungicidal treatments, technological factors. Poster presentation Phytopathology

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285

P44 INHIBITION OF ASPERGILLUS FLAVUS GROWTH BY

CHEMICAL TREATMENTS ON CANOLA STRAW

M. GHIASVAND1, A. TAHMASEBI2, B. GHODS ALAVI2, S. GHASEMI2, M. DEHGHAN BANADAKI1 & K. REZA YAZDI1

1 Department of Animal Science 2 Department of Plant Protection University of Tehran, Karaj, Iran

Various fungi, including Aspergillus flavus increase mold on Canola straw during processing practice. Canola straw is used to feed animals. It should be processed for enhancing straw digestion in animals. In this study, effects of chemical treatments including urea 3.5 percent of dry matter, urea 3.5 percent plus molasses 2 percent of dry matter, ammoniac 3.5 percent of dry matter, sodium hydroxide 5 percent of dry matter, sodium hydroxide 5 percent plus hydrogen peroxide 2 percent of dry matter and hydrogen peroxide 2 percent of dry matter were measured against Aspergillus flavus growth in small 8 kg silos and PDA culture. sodium hydroxide 5 percent plus hydrogen peroxide 2 percent of dry matter and urea 3.5 percent plus molasses 2 percent of dry matter treatments with 2.5 cm and 5.75 cm showed the most and least effect on longitudinal growth of mold in silos. While control treatment without chemical compounds was calculated 5.5 cm. Also Inhibition of Aspergillus growth was carried out by disc diffusion method on potato dextrose agar (PDA) culture in Petri dishes with 9 cm diameter by mentioned treatments at concentrations of 0, 20, 30, 60, 80 and 100 microliter. Hydrogen peroxide with 27.8 percent and ammoniac with 4.4 percent had the highest and lowest effect on inhibition of Aspergillus growth at concentration of 100 microliter on PDA culture. With regard to the harmful effects of Aspergillus growth and mycotoxin production by different species of Aspergillus, the use of hydrogen peroxide plus sodium hydroxide treatment is recommended to increase the ability of straw digestion and reduction of Aspergillus growth. Key words: Aspergillus flavus, canola straw processing, chemical treatments, inhibitory effect

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286

P45 EFFECT OF RESISTANCE TO THIOPHANATE-METHYL

ON FITNESS OF BOTRYTIS CINEREA

J. DERPMANN1, U. STEINER1,H. BUSCHHAUS2 & H-W. DEHNE1 1 University of Bonn, Institute for Crop Sciences and Resource Conservation (INRES) –

Department of Phytomedicine, Bonn, Germany 2 Nisso Chemical Europe GmbH, Duesseldorf, Germany

Gray mold disease caused by Botrytis cinerea is a severe disease, which occurs on many fruit, vegetable and ornamental crops. In grapevine, B. cinerea reduces quantity of yield as well as quality of grapes at harvest. The pathogen has a high tendency to become resistant to frequently applied systemic fungicides. Only a few years after introduction of benzimidazoles like carbendazim (MBC) and the related active compound thiophanate‐methyl strains resistant to benzimidazoles appeared frequently. The development of fungicide resistance in a population is largely dependent on the fitness of the resistant portion of the population. Usually fitness parameters are tested at favourable growth conditions. But fitness costs might be more costly under conditions that are suboptimal for the fungus. Therefore two fitness parameters of MBC‐sensitive and MBC‐resistant isolates were compared at favourable and unfavourable development conditions for the fungus. Mycelial growth and sporulation of twenty isolates of B. cinerea were tested at an optimal temperature of 21 °C and on rich nutrition medium (PDA). Additionally both fitness parameters of all isolates were tested at a low temperature of 4 °C and on a medium with low nutrition availability (CZA 10 % and autoclaved leaf discs). The isolates were divided into a group of ten MBC‐sensitive and a group of ten MBC‐resistant isolates, which showed a substitution of glutamic acid by alanine at position 198 of the beta‐tubulin gene. The presence of this point mutation was confirmed by an allele specific PCR. Radial growth was measured after an incubation time of 3 days at favourable and 10 days at unfavourable conditions. Spore production at favourable conditions was induced by continuous incubation for 11 days at 21°C with a 14 h photo‐period. At favourable conditions mycelial growth and spore production of MBC‐sensitive isolates were not significantly different (p>0.05) compared to that of MBC‐resistant isolates. However at unfavourable conditions the two sensitivity groups showed different results. The mycelial growth of MBC‐sensitive isolates was significantly higher (p < 0.05) than that of MBC‐resistant isolates. Also spore production of MBC‐sensitive isolates was significantly higher (p < 0.05) with about twice as much in average produced spores as the MBC‐resistant isolates. Without selection pressure a fitness penalty of MBC‐resistant isolates at unfavourable conditions might lead to a decrease of the resistant portion within the population at beginning of the epidemic. This might explain the observed decrease of the portion of MBC‐resistant isolates in Germany since the use of benzimidazoles was discontinued in viticulture thirty years ago. Key words: Botrytis, fitness, resistance

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287

P46 DETECTION OF STROBILURIN-RESISTANT

ISOLATES OF MYCOSPHAERELLA GRAMINICOLA IN MOROCCO

A-Y. ELBEKALI1, A. RAMDANI1, A. SIAH2*, B. TISSERANT3*, C. DEWEER2*,

M. HAFIDI4, PH. REIGNAULT3* & P. HALAMA2* 1 Institut National de la Recherche Agronomique, BP 578, Meknès, Maroc

2 Laboratoire Biotechnologie et Gestion des Agents Pathogènes en agriculture, GIS PhyNoPi, Institut Supérieur d’Agriculture, 48 Boulevard Vauban, F-59046 Lille cedex, France

3 Unité Chimie Environnementale et Interactions sur le Vivant, GIS PhyNoPi, Université du Littoral Côte d’Opale, BP 699, F-62228 Calais cedex, France

4 Université My Ismail, Faculté des Sciences, Département de Biologie, Meknès * Univ. Lille-Nord de France

Septoria tritici blotch caused by Mycosphaerella graminicola (anamorph: Septoria tritici) is nowadays one of the most frequently occurring diseases on both bread and durum wheat crops. Two hundred and thirty isolates of the fungus were sampled from six distinct wheat‐producing regions of Morocco in order to investigate the resistance of M. graminicola to strobilurins in this country, where this fungicide class is increasingly used in wheat‐pest management. A subset of 134 isolates was first collected in 2008 from Meknès‐Tafilalet, Tadla‐Azilal, Gharb and Chaouia. Furthermore, 96 additional isolates were sampled in 2010 from the fourth regions investigated in 2008 plus Fes‐Boulmane and Doukkala‐Abda. Sensitivity or resistance within the isolates were determined by screening the G143A cytochrome b substitution conferring resistance. We used a mismatch amplification mutation assay allowing the amplification of either G143 (sensitive) or A143 (resistant) allele. All the 2008 isolates were found to be sensitive since they carry the wild‐type allele G143. However, 9 (9 %) out of the 2010 isolates were found to contain the resistant allele A143 and therefore to be resistant. Four of them were from Gharb and five from Fes‐Boulmane. This study highlighted for the first time the occurrence of strobilurin‐resistant isolates of M. graminicola in Morocco. Further genetic investigations should determine if the resistant isolates emerged independently in Morocco or traveled by wind‐migration from Europe. Key words: Septoria tritici blotch, strobilurin fungicides, G143A substitution

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288

P47 IN VITRO STUDIES ON THE EFFECT OF METHANOL AND DIMETHYL SULFOXIDE ON THE GROWTH AND SPORIFICATION OF PENICILLIUM EXPANSUM AND

BOTRITIS CINEREA

G. PANI, M.G. MOLINU, A. DORE, T. VENDITTI, A. PETRETTO & G. D’HALLEWIN

CNR – ISPA Traversa La Crucca, 3 - Loc. Baldinca, IT-07040 Sassari, Italy

Penicillium expansum and Botritys cinerea are, among the pathogens, most frequently affecting apples and grapes, respectively, after harvest. We studied the behaviour of theses moulds when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO) as an alternative method to fungicides in controlling postharvest decay of horticultural products. The experiments were performed with 5 cm Petri dishes containing on potato dextrose agar) (PDA) amended with 0, 5, 10, 20, 30, 40 and 50 μL/mL of each of the two tested chemicals. Freshly prepared conidia (~ 50 conidia/plate) of P. expansum and B. cinerea were sown onto the media and incubated at 21 °C (then kept into an incubation chamber at 21 °C) up to 6 and 3 days, respectively. Daily, the formed colonies (cfu), the colony diameter and the degree of sporification were monitored. Moreover, Scanning Electron Microscopy (SEM) observations were performed in order to shed light on the effect of the chemicals on P. expansum and B. cinerea growth. Compared to the control MeOH and DMSO affected the growth rate of the two pathogens. The cfu value of P. expansum and B. cinerea was not inhibited significantly, instead the colony diameter and the sporification degree decreased as concentration was raised. B. cinerea cultured on DMSO showed a significant drop of sporification up to the tested concentration of 10 μL/mL, and a complete inhibition of cfu when the concentrations was higher than 20 μL/mL. SEM observations evidenced changes in the pathogen growth behaviour and structure. The importance to fasten or inhibit the sporification will be discussed and data of preliminary in vivo experiments will be presented. Key words: dimethyl sulfoxide, methanol, postharvest.

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289

P48 CORRELATION BETWEEN TRIAZOLE SENSITIVITY

AND MUTATIONS IN THE CYP51 GENE OF MYCOSPHAERELLA GRAMINICOLA IN FLANDERS

Katrien CURVERS1, Bart PYCKE2, Tina KYNDT1, Geert HAESAERT2

& Godelieve GHEYSEN1 1 Department Molecular Biology, University of Ghent

Coupure Links 653, BE 9000 Ghent, Belgium 2 Department of Biotechnological Sciences, University Academy Gent

Schoonmeersstraat 52, BE-9000 Gent, Belgium Mycosphaerella graminicola (anamorph Septoria tritici) is a fungal pathogen that causes leaf blotch in wheat. Worldwide, Septoria tritici blotch is one of the most important wheat diseases, with yield losses up to 50%. No Septoria‐resistant wheat cultivars are available and due to resistance to multiple fungicides, this economically important plant pathogen is difficult to control. The only fungicides still effective against M. graminicola belong to the group of the sterol 14Î±‐demethylation inhibitors (DMIs) of which triazole derivatives are most commonly used. The use of azole fungicides may however be jeopardized by the emergence of resistance, since a slow but constant shift towards reduced sensitivity of M. graminicola has been observed in Europe. This shift is caused mainly by mutations (single nucleotide polymorphisms or SNPs) in the CYP51 gene encoding the demethylase target protein for these fungicides, whereby some specific mutations could be linked to the use of certain triazoles. To date, 19 SNPs have been reported. In this work, about 100 M. graminicola field isolates were sampled at 23 sampling plots spread over wheat‐growing areas in Flanders and their sensitivity towards different triazoles was analysed. Furthermore, we sequenced the CYP51 gene of these isolates and performed a SNP analysis on them. The results showed that there is a large variety in triazole sensitivity between the isolates, even within one field, which is reflected in a high SNP diversity within the CYP51 gene of the M. graminicola population in Flanders. We also found new haplotypes that haven't been described in literature before, indicating cross‐country variation in the M. graminicola populations. Key words: Mycosphaerella graminicola, fungicide resistance, SNP

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290

Posters P49-P55

291

Posters Phytopathology: Integrated control

Posters P49-P55

292

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293

P49 INTERACTION OF VARIOUS SUGAR BEET

GENOTYPES WITH THE ROOT AND CROWN ROT PATHOGEN, RHIZOCTONIA SOLANI

Parissa TAHERI

Department of Crop Protection, Faculty of Agriculture, Ferdowsi University of Mashhad P.O.Box 91775-1163, Mashhad, Iran.

Root and crown rot of sugar beet, caused by the soil borne fungus Rhizoctonia solani AG2‐2, is one of the most important diseases of this crop causing significant yield losses every year worldwide. We investigated the reaction of four sugar beet genotypes, including Dorothea, Ramona, Sonja and Paulina, to infection with the pathogen in greenhouse conditions. The Plants of each cultivar were infected at 9 weeks after seedling emergence by placing a toothpick, which was colonized by the pathogen, next to the crown of each plant. Disease evaluation was carried out at 7 days after inoculation by estimating the lesion length. Data were analyzed statistically, using the Kruskall‐Wallis multiple comparison tests completed by a Mann‐Whitney comparison test at P = 0.05 using SPSS 12.0 for Windows. Our results revealed that Ramona was the most resistant, whereas Dorothea was the most susceptible cultivar to the pathogen. Using a pharmacological approach and thioglycolic‐acid assay, the host defense mechanisms associated with basal resistance of Ramona genotype was investigated. Accumulation of lignin in the cell walls was found to be one of the defense responses of sugar beet plants against R. solani. Higher level of lignification in the resistant Ramona cultivar was correlated with lower level of infection structure formation on plant surface by the pathogen. Lignin forms a structural barrier that limits the spread of pathogen and prevents the diffusion of extracellular enzymes and RS‐toxin produced by this necrotrophic fungus. Key words: Beta vulgaris, cultivars, lignification, resistance, Thanatephorus cucumeris.

Posters P49-P55

294

P50 GRAPEVINE DOWNY MILDEW CONTROL USING

REDUCED COPPER AMOUNT IN ORGANIC VITICULTURE

A. LA TORRE, V. POMPI, C. MANDALÀ & C. CIOFFI C.R.A. - Plant Pathology Research Centre Via C.G. Bertero 22, IT-00156 Rome, Italy

Copper occurs in nature and is an essential micronutrient; however, copper used as plant protection product may have long‐term consequences due to its accumulation in the soil. Therefore, the limitations on copper use have been defined in organic farming (Regulation EC 889/2008). In the light of new developments and evidence, European Commission will review copper authorisation for further possible restriction of the allowed copper quantities. Taking into account this situation, two‐year field trial was set up with new copper formulations to evaluate the possibility to reduce copper quantities to be applied with the treatments and consequently to reduce copper soil residues. Plots were prepared, each containing 12 plants and repeated 4 times in randomized blocks. The test organism was Plasmopara viticola (Berk. et Curt.) Berl. et De Toni. Cupric formulations characterised by low metallic content (Glutex CU 90 and Labicuper) have been tested in comparison with reference product and untreated control. The assessments to evaluate the effectiveness of treatments have been carried out periodically on 100 leaves and 100 bunches for each replicate. Data were analysed statistically with ANOVA and Tukey's test (p ≤ 0.05). The chemical analyses have been performed to determine copper residues in soil, leaves and grapes. Samplings of leaves and grapes have been carried out for every plot. With regard to soil, each single sampling was considered at two depths (0‐20; 20‐40 cm). The total copper has been determined by means of spectrophotometry in atomic absorption by acetylene‐air flame (FAAS at λ= 324.8 µm). The obtained results showed that the tested products gave a good control of downy mildew with a lower copper dosage in comparison with cupric formulations used as reference product. Glutex CU 90 formulation gave an annual input of copper that was a little more than a third in comparison with the reference product; Labicuper formulation gave an annual input of copper that was about a fifth or a sixth in comparison with the reference product. At harvest, copper levels determined in grapes have been much smaller than RML (fixed at 50 mg/kg). With regard to cupric product treatments impact in organic vineyard soil, no statistically significant differential increase of Cu residue has been observed in soil between tested products versus untreated control plots. In conclusion, the new investigated cupric formulations, by reducing copper input in organic viticulture, are able to minimise its environmental impact. Key words: copper formulations, grapevine downy mildew, organic farming, copper residues

Posters P49-P55

295

P51 RESPONSES OF BARLEY VARIETIES AND LINES TO

ALGERIAN ISOLATES OF PYRENOPHORA TERES

K. BOUNGAB1, L. BELABID1, Z. FORTAS2 & F. LAZRAG1 1 Laboratoire de recherche sur les systèmes biologiques et la géomatique

Eniversité de Mascara. BP 763, Mascara, Algérie 2 Laboratoire de biologie des microorganismes et de biotechnologie

Département de biotechnologie. Université d’Oran .Algérie. The prevalence of net blotch in barley (Hordeum vulgare L.), caused by Pyrenophora teres Drechs., conidial state Drechslera teres (Sacc.) Shoemaker, has increased in recent years, and net blotch has become one of the most important foliar diseases in Algeria. it causes important crop losses to barley nearly every year. The transmission of the pathogen by seeds is an effective way for its spread. Two forms of the disease, the net form, caused by P. teres f. teres, and the spot form, caused by P. teres f. maculata, are differentiated by the type of symptoms on leaves. The two forms are present in Algeria, since the teres form is more common. A knowledge of the reaction of different cultivars, putative resistant lines, and variability in the net blotch pathogen is necessary to develop a successful resistance breeding program. The present study aims to evaluate the reaction of 17 barley varieties and lines from diverse origins (Algeria and Syria) against some virulent isolates of Pyrenophora teres Drechs collected from different barley‐growing areas of Algeria in order to select the best genotypes resistant to this disease. Our test is led in vitro and in the field. A rating scale from 1 to 9 based on the type and size of lesions was used to characterize the infection response observed on the second leaves of barley seedlings. The results obtained revealed a variability of interaction between the varieties of barley and the isolates of P. teres, with reactions ranging from moderate resistance to high susceptibility. Cultivars of six‐row barley were more sensitive to net blotch, on the contrary, two‐row barleys demonstrated less sensitivity to isolates of P. teres f. teres. Barley varieties and Lines had different susceptibility spectra and could be divided into four homogeneous groups, the most susceptible group was composed of two varieties each susceptible to all isolates. The results demonstrated that all Algerian cultivars were susceptible to P.teres, Those from Syria were less sensitive, where two lines were moderately resistant to the disease. These lines can be used in the future in the crossing program. They may be used in combination with other sources of resistance. Deployment of resistant cultivars is the most economic and environmentally sound means of preventing net blotch epidemics. Key words: Algeria, barley, net blotch, cultivars, resistance

Posters P49-P55

296

P52 SCREENING OF MAIZE VARIETIES FOR RESISTANCE

TO RHIZOCTONIA SOLANI AG 2-2 IIIB

S. INGHELBRECHT, K. HEUNGENS, M. MAES, J. VAN WAES & J. PANNECOUCQUE

Institute for Agricultural and Fisheries Research (ILVO) Plant Sciences Unit, Merelbeke, Belgium

Rhizoctonia solani AG2‐2IIIB has caused increasing problems in the culture of maize in Western Europe, especially when the crop is grown in rotation with beet, which is also highly susceptible to isolates of this anastomosis group. Fungicide treatments are either uneconomical or undesired and soil moisture content is not easily managed for field crops. In cases where crop rotations cannot be altered, disease resistance becomes the principal strategy to mitigate the problem. But only limited knowledge exists on the resistance level of the different commercial maize varieties. Our aim was to develop a practical and robust bioassay and apply it to test a set of varieties from the Belgian variety list. To develop the bioassay, different doses of ground and non‐ground bran‐based inoculum were evaluated together with different seeding depths and soil moisture levels. Consistent symptom development was obtained by using a controlled mixture of sand and potting soil, standardized inoculum preparation and application, seeding at a constant depth, controlled climate conditions and regimented watering during germination and seedling growth. A disease index was developed on a scale of 0 to 5. Root volume and the level of necrosis to the main and the adventitious roots were accounted for. Using the very disease‐conducive conditions of the bioassay and two to three replicates consisting of up to 30 seeds each, 21 varieties from the Belgian variety list were tested for their level of resistance. The average disease index was 3.4 and varied between 2.3 and 4.0, with significant differences being present between the varieties. Shoot weight varied between 10.9% and 44.3% of the non‐inoculated controls. A strong negative correlation (R2=0.76) was observed between disease index and shoot weight, indicating that the latter can also be used as an objective evaluation tool. The disease resistance data will be made directly available to the growers via the national variety list. The bioassay is available to the commercial seed producers and can assist them when breeding for more resistant varieties. Key words: bioassay, disease resistance, corn, fungi

Posters P49-P55

297

P53 INFLUENCE OF CURING TIMES ON THE

EFFECTIVENESS OF TREATMENTS WITH ACETIC ACID ON THE CONTROL OF P. DIGITATUM ON LEMONS

T. VENDITTI, A. DORE, M.G. MOLINU & G. D’HALLEWIN

CNR – ISPA Traversa La Crucca, 3 - Loc. Baldinca, IT-07040 Sassari, Italy

The restricted number of postharvest fungicides used in packing houses is leading to the selection of resistant strains of Penicillium digitatum (citrus green mould), one of the most common and serious pathogens during storage and marketing of lemons. Furthermore a growing concern for human health and a greater awareness for environmental conservation have multiplied the studies on new ecological technologies. Among the alternatives to synthetic postharvest fungicides, the use of acetic acid (classified as GRAS) followed by a physical method such as curing, have led to encouraging results. In the present study is reported the combined use of curing, performed at reduced times compared to those reported to be effective, followed by acetic acid (AAC) treatments. Lemons of the variety “Limone di Massa” artificially inoculated with P. digitatum at a concentration of 104 spores per milliliter were cured for 0, 3, 6, 12 and 24 hours and then treated with three different concentrations of AAC (25, 50 and 75 µL/L) for 15 min. Fruit was then stored at 20 °C and 80% relative humidity (RH) for 9 days, when the number of decayed fruits was monitored. The same combined treatments were also carried out on naturally infected lemons, stored for 6 or 8 weeks at 5° C and 90% RH. After 9 days of storage, in artificially inoculated fruit, the lowest percentage of infected wounds was 0% after 6 hours of curing followed by AAC fumigation performed at 50 µL/L, while untreated or cured for three hours lemons showed the worst results with 71.4 and 61.9 % of rotted fruit respectively. In naturally infected lemons the best results were achieved, after 6 or 8 weeks of storage, with curing performed for 24 hours followed by AAC fumigation at 50 µL/L. In this cases the combined treatment reduced the decayed fruit by the 91.0 and 66.5% respectively, if compared to untreated fruit. The weight loss was not affected by any of the treatments. These results show that a good control of green mould during storage could be achieved, on lemon fruit, by combining a reduced curing time of 24 hours to the effect of AAC. The best results were obtained after 6 week of storage even if a satisfactory control was observed after 8 weeks of storage. Key words: curing, acetic acid, postharvest, lemons

Posters P49-P55

298

P54 SEQUENTIAL APPLICATION OF NaHCO3, CaCL2 AND

CANDIDA OLEOPHILA (ISOLATE 13L) AFFECTS SIGNIFICANTLY PENICILLUM EXPANSUM GROWTH AND

THE INFECTION DEGREE IN APPLES

M.G. MOLINU1, G. PANI1, T. VENDITTI1, A. DORE1, G. LADU2

& G. D’HALLEWIN1 1CNR - ISPA, Traversa La Crucca 3, Loc. Baldinca, IT-07040 Sassari, Italy

2 DISAABA - Università degli Studi Sassari, viale Italia 39,IT-07100 Sassari, Italy The employment of biocontrol agents to restrain postharvest pathogens is a promising approach, although, efficacy and consistency are still below those of synthetic pesticides. Up to date, the ‘integrated control strategy’ seems to be the most promising way to overcome this gap. Here, we report the results attained by applying, separately or sequentially, NaHCO3, CaCl2 with the yeast Candida oleophila (isolate ‘13L’) to wounded or wound‐inoculated apples. Fruits were harvested at commercial maturity, selected, part were disinfected and then each fruit was wounded (3x3mm) at two sites in the equatorial area. The surface‐disinfected fruit (‘exp. 1’, 840 apples) was divided into two groups of 420 fruits (A and B), wounds in fruits of group ‘A’ were inoculated with P. expansum (20 µL of a 104 conidia�mL‐1) 24 h before treatment, while those in fruits of group ‘B’ were 24 h after treatment. Within the groups, 7 sets (for 7 different experiments) of 60 fruit were obtained and treated as follows: set 1) H2O (control); set 2) isolate ‘13L’ (20 µL of a 10

7 cfu�mL‐1); set 3) NaHCO3 (SBC 2%, w:v, in water); set 4) CaCl2 (Ca 1%, w:v, in water); set 5) SBC‐isolate ‘13L’; set 6) SBC – Ca; set 7) SBC – Ca – isolate ’13L’. Fruits of ‘exp. 1’ were stored for 7 d in a thermo‐regulated room, at 20±1°C and 98% RH, during which the growth rate and the degree of infected wounds by P. expansum were recorded. Again, for the remaining un‐sterilized wounded, but not inoculated fruits, 7 sets (each of 100 fruits) were prepared and treated as in ‘exp. 1’. Then, fruits were stored at 2±1°C for 45 d. Decay was monitored after 14 and 45 d of storage. Following storage, 40 apples from each set were selected and half was inoculated with a suspension of P. expansum (20 µL of a 104 conidia�mL‐1), after that all fruits were kept, at 20 °C and 75% RH for 7 d, and the infection degree was monitored. As a general role, the best results were attained with treatment ‘6’ and ‘7’. Compared to the sole yeast, the efficacy of treatment ‘7’ in ‘exp. 1’ resulted three times more effective when inoculation was performed 24 h before treatment and one time when performed 24 h after the treatment. Interestingly, SBC treated apples had the highest percentage of decay during both storage and following the inoculation after storage. Key words: postharvest decay, biological control, integrated pest control.

Posters P49-P55

299

P55 CHANGES IN MICROORGANISMS POPULATIONS

IN THE SOIL AFTER FUMIGATION

Beata MESZKA, Aneta CHALANSKA, Piotr SOBICZEWSKI, Hanna BRYK, Eligio MALUSA

Institute of Horticulture PL-96-100 Skierniewice, Konstytucji 3 Maja 1/3, Poland

Soil‐borne pests and diseases management is the most important factor deciding crop success in intensive horticulture. Beside positive effect, pesticides have negative influence on human health and environment. It encourages to apply low‐input of pesticides in crop farming by applying IPM principles, alternative methods and biofumigants. This approach supports the EU policy for achieving the sustainable use of pesticides. In 2010, in a typical system of Central Europe, the sustainable use of chemical fumigants of soil for the control of soil‐borne pathogens and nematodes were studied in Poland. Experiments were performed on four plantations in three localities: pepper and tomatoes in the Radom, cucumber in the Kozienice and strawberry in the Skierniewice. The owners of all plantations indicated on phytosanitary problems in soil. Soil fumigation was conducted in autumn (October 12) 2010 with application dazomet, metham sodium, chloropicrin and chloropicrin + 1.3 D. It was found that after fumigation the total number of bacteria significantly increased in all combinations. On plots treated with dazomet and chloropicrin +1.3D almost twofold increase of bacteria number was noted. A particular atention should be pay on populational changes within fluorescent Pseudomonads. In soil of all disinfected plots their number increased 2‐36 times. It indicates great increase of soil biological potential including soil suppressiveness, because biocontrol agents are often selected from Pseudomonas genus. On the other hand the population number of Bacillus spp. bacteria, playing also significant role in soil suppressiveness, did not changed and some cases even decreased. Microorganisms communities in soil before fumigation consisted of fungi from genera: Trichoderma, Penicillium, Cladosporium, Mucor, Rhizopus, Zygorynhus, Gymnoascus, Alternaria. Plant parasitic nematodes from Pratylenchus, Rotylenchus, Trichodorus and Paratylenchus species was also observed. The presence of yeasts and pathogenic fungi Fusarium (F. culmorum i F. oxysporum), Verticillium dahliae and Phytophthora cactorum and P. megasperma was also recorded.Total number of fungi in the soil before fumigation was 11.4 x 104 cfu (colony forming units) in 1 g of soil. Plant‐parasitic nematodes was present at samples from 0,78 all nematodespercentage in tomatoes to 54,17 all neamtodes percentage in the strawberry. All tested products reduced the number of fungi populations to 0.04 ‐ 3,87 x 104 cfu depending on compund used. The highest efficacy showed mixture of chloropicrin and 1.3 D. Key words: fumigation, soil‐borne pathogens, plant parasitic nematodes

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300

Posters R01-R02

301

Posters Application Technology

Posters R01-R02

302

Posters R01-R02

303

R01 POTENTIAL USE OF A MONO-DROPLET GENERATOR

AS AN ALTERNATIVE APPLICATION DEVICE FOR A PRECISE WEED CONTROL

Sabin BASI1, Mauricio HUNSCHE1, Georg NOGA1, Lutz DAMEROW2

& Peter SCHULZE LAMMERS2 1 University of Bonn, INRES-Horticultural Science Auf dem Huegel 6, DE-53121 Bonn, Germanry

2 University of Bonn, Institute of Agricultural Engineering Nussallee 5, DE-53115, Bonn, Germany

In the context of minimizing the herbicide use contrasting to the conventional application technology, while increasing the required biological efficacy, a pneumatic droplet generator has been constructed to produce mono‐droplets of herbicide solution aiming a target‐oriented weed control. Principally, the device works by pneumatic pressure generated with nitrogen gas. A solenoid valve controlled by impulse generator regulates the pressure, breaking the surface tension of the liquid jet exiting from the orifice, thus discharging a single droplet. Excess gas is exhausted through the vent, dampening the pressure so that no more liquid discharges. As shown in our experiments, the application device can be used accurately to deliver the required amount of herbicide consistently. As integrative part or the tests the effect of impulse width for controlling the gas flow through solenoid valve, vent opening, the pressure, and the composition of liquid affecting surface tension of the herbicide has been evaluated. Increase in the impulse width, vent closer or orifice size raise the droplet volume. While, decreasing the surface tension of the liquid with surfactants, had an effect of reducing the volume and the pressure required for droplet formation. With an optimum adjustment, the parameters, impulse width, exhaust vent opening, and the pressure, the droplet generator can successfully be used to deliver the droplets of volume 0.05 µl to 2.5 µl with our model, though the range could be varied easily by changing the parameters. Delivery efficiency of the device for herbicide (glyphosate) application has been tested with model weed species having hydrophobic (Chenopodium album and Setaria viridis) or hydrophilic leaf surfaces (Viola arvensis). The effect of height of application (5, 10 and 30 cm), volume (~0.5 and 2 µl) and solution (Glyphosate ± Silwet‐L77, Tween‐20, RSO 10; and Roundup Ultra®) has been studied with regard to droplet retention on horizontal leaves. In hydrophilic surface, retention was 100% independent of height, volume or surfactant, while, in hydrophobic surfaces, retention was highly dependent on those factors. In general, surfactants had a positive effect on retention capacity, while volume and the height of application had a negative effect. As a conclusion, droplet generator could be used in herbicidal application, with selection of proper solution at low application volume and height. Key words: Monodroplet, herbicide, adjuvant, weed, retention

Posters R01-R02

304

R02 B-VARAN, A NEW SPRAY DEVICE FOR REGISTRATION

TRIALWORK IN POMEFRUIT ENABLING CORRECT TESTING OF ANTIDRIFT NOZZLES RESPECTING

PRACTICALLY RELEVANT ADJUSTMENTS

C. SCHOEVAERTS, D. GOOSSENS, N. HENDRICKS1, D. BAETS, H. VAN DYCK & L. DE MAEYER

Bayer CropScience J.E. Mommaertslaan 14, BE-1831 Diegem, Belgium 1PCFRUIT, Fruittuinweg 1, BE-3800, Sint-truiden, Belgium

The B‐Varan (Belgian Variable Application Nozzle) is a prototype sprayer developed by Bayer CropScience Belgium. It is especially designed for complete randomized block trials in pomefruit aiming at a maximal alignment with growers’ standard practices in terms of watervolume, pressure, turbulence and deposit. The B‐Varan device was calibrated for airflow characteristics and homogeneity of nozzle flow rate to allow its use for Good Experimental Practice (GEP) registration trials. The biological efficacy on two pests was proven to be at least as effective as the results obtained with the previously used knapsacksprayer Solo Port 423. The B‐Varan spray device is appropriate for spray technology testing and offers multifunctional testing of nozzle type and watervolume directly linked with droplet size. Furthermore it allows testing of primary and secondary deposit and the accurate assessement of biological efficacy of driftreducing air induction nozzles. Field trials in 2010 have indicated that the biological efficacy of a systemic insecticide (spirotetramat) obtained with antidrift nozzles is in line with the biological efficacy on Eriosoma lanigerum and Psylla pyri obtained via holocone nozzles. Improved spray technology contributes significantly to the sustainability goal in pome fruit. The B‐Varan device can bring evidence to support the successful use of drift‐reducing nozzles and can, as such play a key role in increasing the credibility of improved spray technology at growers’ level. Key words: trialsprayer, pomefruit, antidrift nozzles

Posters R03-R15

305

Posters Pesticide Residues, Toxicology and Ecotoxicology

Posters R03-R15

306

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307

R03 LEACHING OF OXADYXIL AND TEBUCONAZOLE

IN COLOMBIAN SOIL

Martha Isabel ALDANA-CASTAÑEDA1, Rafael DE PRADO AMIAN2 & María José MARTÍNEZ-CORDÓN1

1Departamento de Química. Universidad Nacional de Colombia. Sede Bogotá. Colombia 2 Departamento de Química Agrícola y Edafología

Campus Universitario de Rabanales Córdoba. España Lake Tota (Boyaca, Colombia) supplies water for human consumption, agriculture and industry for more than 500.000 people. Oxadixyl and tebuconazole are fungicides used in onion crops in the lake catchment area. The mobility of pesticides in soil, bioavailability and transfer to other environmental compartments depend on sorption and desorption kinetics and mechanisms. An understanding of these processes is essential for transport modeling and the rational design of corrective measures against pollution. A displacement study was performed on a hand packed soil column in laboratory conditions. A pulse of 0,01 M CaCl2 solution, containing a tracer (bromide) and the fungicides oxadixyl y tebuconazole, was injected. Column experiment was performed at 0.78 mm h‐1 flow rate under unsaturated conditions. Eluates were collected in flasks at constant intervals. After rainfall simulation, the soil from the column was sliced into six successive sections (5 cm). Methanol extraction was used to determine the fungicides in each soil section. Water and methanolic samples were analyzed by HPLC. Only bromide and oxadixyl were recovered at the bottom of the column. Unlike bromide breakthrough curve, oxadixyl was asymmetrical, with early breakthrough and increased tailing. The oxadixyl recovery was 96.7 % after ten pore volumes. Tebuconazole showed the highest retention in the fist five cm of soil layer. The results suggest that oxadyxil presents highs risk to leachate through the soil profile and that tebuconazole is strongly absorbed in Colombian soil. Key words: Tebuconazole, oxadixyl, soil column, leaching

Posters R03-R15

308

R04 EVALUATION OF THE TOXICITY OF CYPERMETHRIN PESTICIDE ON ORGANS WEIGHT LOSS AND SOME BIOCHEMICAL AND HISTOLOGICAL PARAMETERS

SALIHA DAHAMNA1*, Assia BELGUET1, DalilaBOUAMRA2, Ali GUENDOUZ1,

Mounira MERGHAM1 & Daoud HARZALLAH2 1 Laboratory of Phytotherapy Applied to Chronic Diseases

2 Laboratory of Applied Microbiology, Faculty of Natural Sciences and Life University Ferhat Abbas, 19000, Algeria

An increase in global food demand has resulted in a significant increase in the use of pesticides in agriculture. Synthetic pyrethroid pesticides account for over 30% of the global pesticide use; Pyrethroid pesticides were used preferably over organochlorines and organophosphates due to their high effectiveness, low toxicity to non‐target organisms and easy biodegrability. It has widespread applications in agriculture through the world and as well in Algeria. Cypermethrin is one of the most insecticidal pyrethroids widely used in agriculture regions of Setif. to control wide range of insect pests in a variety of crops. The aim of this study is to investigate the effects of cypermethrin (Cyper‐Ac 271 g/l from the active substance of the cypermethrin) on hematological, biochemical parameters, body weight loss, and histopathological study of some organs. Male mice weighing 30‐40g were used, separated in 5 groups, n=6, two groups controls given vehicle (oil vegetable) and three experimental groups (Cypermetherin and vegetable oil). The animals were gavaged by 1/5 LD50 (LD50 = 485 mg/kg b/w) for 2 and 4 weeks respectively, and with 1/20 LD50 for 12 weeks, then the animals sacrificed at the end of the experiment.. Blood was collected. Enzyme activities were assayed in the plasma samples obtained. Glutamate oxaloacetate transaminase (GOT), Glutamate pyruvate transaminase (GPT), Alkaline phosphatase (ALPH) and Glucose. Red blood cells, (RBC), and white blood cells (WBC) were calculated too. The samples of liver and kidney were processed for histology. The results indicated a significant increase in transaminases GOT, GPT, and AlP. The decrease in Hb, RBC and WBC which are related to the immunity, this is probably due to cell lyses explain the effect of Cypermetherin on erythropoeisis. cypermethrin treatment exhibited severe histopathological changes, especially in the liver and kideney accompanied by weight loss of some organs. We conclude that cypermethrin induces oxidative stress and modifies biochemical parameters and histological aspects of liver and kidney. Key words: Cypermetherin, biochemical and histological alterations, mice, kidney, liver

Posters R03-R15

309

R05 EFFECT OF GLYPHOSATE-BASED HERBICIDE ON

ACETYLCHOLINESTERASE ACTIVITY IN TADPOLES, HOPLOBATRACHUS RUGULOSUS

Watcharaporn RUAMTHUM1*, Suraphon VISETSON1, John R. MILNE2

& Vasakorn BULLANGPOTI1 1 Department of Zoology, Faculty of Science, Kasetsart University 50 Phahon Yothin Road,Cha-tuchak, Bangkok 10900, Thailand 2 Department of Biology, Faculty of Science, Mahidol University

272 Rama VI Road, Bangkok 10400, Thailand This study focused on toxicity and its effect on neuron enzyme, acetylcholinesterase (AchE) activities from glyphosate‐based herbicide on tadpole stage (stage 35‐39) of Hoplobatrachus rugulosus. Each 20 tadpoles were tested by adding glyphosate‐based herbicide at 21, 24, 27 and 30 µl in 1L de‐chlorinated water at glass container (4x6x8 inches). The toxicity results were compared with distilled water as control treatment. After 24, 48, 72 and 96 hours exposure of glyphosate‐based herbicide concentrations, an LC50 showed as 25.21, 24.66, 24.16 and 23.63 µl/L, respectively. The acetylcholinesterase activities were observed in treated tadpoles which the activities were induced following increasing concentration of treatment group. This result is for the base knowledge of toxicity and its effect on neuron enzyme, acetylcholinesterase activities from glyphosate‐based herbicide on tadpole stage of Hoplobatrachus rugulosus. Key words: Glyphosate‐based herbicide, Herbicide, Hoplobatrachus rugulosus, Tadpole, Acetylcho‐linesterase

Posters R03-R15

310

R06 TOXICITY OF S-METOLACHLOR CONTAINING

FORMULATION AND HEAVY METALS TO CHICKEN EMBRYOS

Rita SZABÓ1, Péter BUDAI1, József LEHEL2 & Éva KORMOS1

1University of Pannonia, Georgikon Faculty, Institute of Plant Protection, Keszthely, Hungary 2Szent István University, Faculty of Veterinary Science, Budapest, Hungary

Nowadays, besides food production, agriculture must pay close attention to the economical use of non‐recyclable raw materials and the decrease or complete elimination of the damage to the environment, which affects wildlife. The traditional mass production with its huge need of energy and chemicals is not suitable to solve these multiple tasks at the same time. This kind of production spoils the environment continuously by polluting the water and the soil and by endangering wildlife, and the consumption of the polluted products largely contributes to the health impairment of the population. Environmental pollution of metal modelled by copper‐sulphate, cadmium‐sulphate and a 960 g/l S‐metolachlor containing herbicide formulation (Dual Gold 960 EC) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air‐chamber in a volume of 0.1 ml/egg on day 0 of incubation. The concentration of copper‐, and cadmium‐sulphate was 0.01%. The applied concentration of Dual Gold 960 EC was 0.375%. Evaluation was done on day 19 of the hatching period. In comparison with the values of the control group, the rate of embryomortality also increased significantly in the groups which were treated by herbicide and heavy metals individually. When the herbicide (Dual Gold 960 EC) was used individually, one quarter of the treated animals died in comparison with the values measured in the control group. In the groups which were treated with cadmium‐sulphate and Dual Gold 960 EC embryomortality increased significantly in comparison with both the control and individually treated groups. In the groups, which were treated together, the occurrence of development disorders remained as low level as in the case individual treatments. Looking at the types of development disorders, the most frequent problems were the oedema, the shortening of the beak mandible, and the incorrect posture of the feet and the neck. As the collective result of the treatment with cadmium‐sulphate and Dual Gold 960 EC, the body mass of the treated embryos decreased significantly in comparison with the control and individually treated groups, therefore we can conclude that the combined treatment resulted in increased embryo toxic effect in comparison with the individual embryo damaging effect of the used components. This study has been generously supported by TÁMOP‐4.2.2‐08/1/2008‐0018 ‐ entitled as “Liveable environment and healthier people ‐ Bio innovation and Green Technology research at the University of Pannonia". The project is co‐financed by the European Social Fund with the support of the European Union. Key words: cadmium, copper, chicken embryo, embryotoxicity, S‐metolachlor

Posters R03-R15

311

R07 THE EFFECTS OF HERBICIDE NICOSULFURON

ON SOME BIOCHEMICAL ACTIVITY OF CLAY LOAM SOIL

Ljiljana RADIVOJEVIC1, Ljiljana SANTRIC1, Dragana MARISAVLJEVIC2,

Slavica GASIC1 & Jelena GAJIC UMILJENDIC1 1 Institute of Pesticides and Enviromental Protection, Belgrade, Serbia

2 Institute of Plant Protection and Enviroment, Belgrade, Serbia Nicosulfuron is a postemergence herbicide that provides selective grass and broadleaf weed control in corn. This sulfonylurea herbicide inhibits acetolactate synthase to effectively block synthesis of the branched‐shain amino acids valine, leucine and isoleucine. Much research concerning the toxicity of nicosulfuron on weed and crops has been reported, there are few reports about its effects on biochemical activity on soil. The effect of nicosulfuron on biochemical activity in soil was investigated. Trials were set up in laboratory on clay loam soil (pH 7.10, organic matter 3.32 %, sand 21 %, silt 49 %, clay 30 %) at Zemun Polje, Serbia. Soil samples were collected from the upper layer (0‐10 cm), carefully dried, sieved to pass 5 mm mesh, and stored at 4oC. Nicosulfuron was added at rates 0.3, 1.5 and 3.0 mg per kilogram. Untreated soil served as control. Samples were collected for analysis 1, 7, 14, 21, 30 and 60 days after nicosulfuron application. The activity of the enzyme dehydrogenase was determined according to Tabatabai (1982). Fumigation‐extraction was employed to determine microbiological biomass carbon (Vance at al, 1987). Walter’s method (1952) was employed to determine soil respiration. The microbiological metabolic coefficient (qCO2) was computed from the ratio of soil respiration intensity and microbiological biomass (Andreson and Domsch, 1990). Nicosulfuron was found to cause different effects on the biochemical activity in soil and its influence depended on the rate of application and duration of activity. Nicosulfuron inhibited dehydrogenase. Nicosulfuron decreased microbial biomass carbon over the initial seven days, and increased it from the seventh day onward and increased the microbiological metabolic coefficient (qCO2) from the first to 30th day. The changes detected were found to be transient, and there is no real risk of the compound disrupting the balance of biochemical processes in soil. Key words: nicosulfuron,dehydrogenase,biomass carbon,respiration, microbiological metabolic coefficient, (qCO2)

Posters R03-R15

312

R08 EFFECTS OF WASHING AND SUNLIGHT ON LONG

LASTING INSECTICIDAL MOSQUITO NETS'

Jean Pierre NABLENI OUATTARA1, Pieter SPANOGHE1, Olivier PIGEON2 & Walter STEURBAUT1

1 Laboratory of Crop Protection Chemistry, Department of Crop Protection, Ghent University, Coupure links 653, BE-9000 Gent, Belgium

2 Walloon Agricultural Research Centre (CRA-W), Agriculture and Natural Environment Department, Rue du Bordia 11, BE-5030 Gembloux, Belgium

Malaria is one of the public health problems for most of developing countries in the world. It kills a child in the world every 30 seconds, infects more than 250 million people each year and 1 million of them die. \nThe use of long lasting insecticidal mosquito Nets (LNs) remains the best recommended method to prevent malaria in the world. Therefore manufacturers are constantly working to improve technologies in order to make LNs easier, safer and more efficient for households.The efficacy of LNs depends a lot on their use in field condition. It is observed that although they received a recommendation from WHOPES (World Health Organization Pesticides Evaluation Scheme) before being provided to population, questions still arise. The aim of this study is to use 4 recommended LNs in order to assess the impact of 2 ways of washing followed by 3 ways of drying.For this purpose, 4 brands of LN will be used. Two types of washing (laboratory and field washing) will be applied 20 times to each brand of LN followed by 3 types of drying (Hanging sample in room to dry without direct sunlight ‐ Hanging sample in room to dry with ventilator and without direct sunlight ‐ exposing sample at a specific UV light for drying). The time between each wash step will take the regeneration time into account. Just before each next washing step the median knock‐down time (MKDT) will be determined by a bioassay and the active ingredient content by chromatographic analysis. This poster summarizes a methodology to evaluate the effect of washing and drying on the active ingredient content for 4 LNs, to assess the effect of UV light on the active ingredient content of LNs (kinetic of UV light degradation) and to compare the laboratory washing using the WHO/CIPAC method with the field washing. Key words: Malaria, Long Lasting Insecticidal mosquito Nets, Washing, sunlight effect

Posters R03-R15

313

R09 INFLUENCE OF SURFACTANTS ON THE SORPTION OF TWO CHLOROACETANILIDE IN AN ROMANIAN

CHERNOZEM SOIL

I. COROI1, T. DE WILDE2, M.S. CARA1, G. JITAREANU1 & W. STEURBAUT2 1 Department of Soil Management

''Ion Ionescu de la Brad'' University of Agricultural Sciences and Veterinary Medicine Faculty of Agriculture, Iasi - 700490, Romania

2 Department of Crop Protection, Faculty of Bioscience Engineering Chemistry, Ghent University Coupure Links 653, BE-9000 Gent, Belgium

Pesticides have been extensively used in modern agriculture. Due to the prevalent use, there have been serious problems generated by pesticides wastes which could eventually endanger water resources and human health. The development of technologies for the decontamination of soils and waters polluted by hydrophobic organic compounds has encouraged research into the use of non‐ionic surfactants as potential agents for the enhanced solubilization and removal of contaminants from soils and sediments. Surfactants are a class of amphipathic chemicals possessing hydrophobic moieties and hydrophilic groups which might have an influence on the sorption and mobility properties of pesticides. Sorption of two chloroacetanilide herbicides, acetochlor and metolachlor was studied on a representative chernozem soil of the Main Agricultural Research Station Ezareni belonging to the ’’Ion Ionescu de la Brad’’ University of Agriculture and Veterinary Medicine Iasi, Romania, in the presence and absence of surfactants. Three different non‐ionic surfactants were selected: Tween‐20, Synperonic 91/5 and Silwet L‐77, to verify the influence of their presence on herbicide sorption at different concentrations. Our results showed that the sorption of the studied herbicides within the soil‐water‐non‐ionic surfactant system was influenced by the presence of non‐ionic surfactants. The n values obtained were lower than 1 for all pesticide‐surfactant combinations, which indicates that the amount of acetochor and metolachlor sorbed decreased with an increase in pesticide concentration. The sorption of acetochlor increased in the following order: Acetochlor+Synperonic 91/5 < Acetochlor < Acetochlor+Tween‐20 < Acetochlor+Silwet L‐77. In the case of metolachlor+Synperonic and metolachlor+Silwet L‐77, the Kf values were significantly higher than the Kf value of metolachlor+Tween‐20 on soil, where a lower Kf value could be observed with however a higher n value which indicate a higher sorption capacity at higher concentrations. Key words: sorption; soil; acetochlor; metolachlor; non‐ionic surfactants.

Posters R03-R15

314

R10 METAL ORGANIC FRAMEWORKS AN ALTERNATIVE

SORBENT FOR THE TREATMENT OF PESTICIDE CONTAMINATED WATER?

T. DE WILDE

Laboratory of Crop Protection Chemistry, Ghent University Coupure Links 653, BE-9000, Gent, Belgium

The use of various solid‐phase sorbents, such as carbon black, polymeric resins, organoclays, etc. have been explored for the removal of pesticides from water. But they display generally poor adsorption specificity and often low uptake affinity toward organic molecules. Very polar pesticide molecules are hardly retained in the soil. The design of advanced pesticide adsorbents, capable of selectively capturing high amounts of targeted organic compounds, is an area of research that is still in its infancy. A relatively new potential sorbent are crystalline microporous coordination polymers, also called metal‐organic frameworks (MOFs). MOFs are formed by coordinate bonds from multidentate ligands to mono‐ or polynuclear metal centers and may have extensive open‐framework structures. MOFs are porous and have a large surface area exceeding those of the best activated carbons. Guest molecules can be sorbed into this porous structure. These materials have potential applications in many fields including separation, heterogeneous catalysis, and gas storage. Sorption of pesticides on these materials from aqueous media was never studied before. Therefore the aim of the presented study is to optimize the sorption of polar pesticides on currently existing or newly developed MOFs. Optimization consists of different aspects, namely, maximizing sorption capacity, selective sorption, competitive sorption with dissolved organic matter (DOM), and finally the regeneration capacity of the MOFs. Key words: sorption, metal organic framework, anionic pesticides, porous

Posters R03-R15

315

R11 PLANT PROTECTION PRODUCTS - ADHESION TO

AND DISTRIBUTION ON TREATED SEEDS

P. DE VOS & O. PIGEON CRA-W - Agriculture and Natural Environment Department (D3)

Plant Protection Products and Biocides Physico-chemistry and Residues Unit (U10) Rue du Bordia 11BE-5030 GemblouxBelgium

Seed treatment with fungicides and insecticides are used to protect crops against pests and pathogens. To assure a good efficacy, each seed and so each plant has to be correctly treated. Good quality seed treatment with plant protection products means that the average concentration of active substance on seeds has to be as close as possible to the target rate and that the distribution of the active substance on individual seeds has to be uniform. To test this quality of treatment, several official methods are now available and recommended and other additional methods can be proposed. Treatment of some seeds (barley, wheat, maize, peas...) can be performed at the laboratory scale with the laboratory seed treatment machinery Hege 11 according to the CIPAC method MT 175. This CIPAC method is also used to determine the seed‐to‐seed uniformity of distribution of the pesticide. The CIPAC method MT 194 is used to verify the adhesion to the treated seeds after a controlled dropping stress. Our laboratory recommends for both CIPAC methods the analysis of the active substance content using a chromatographic determination instead of the colorimetric measurement (dye concentration on each seed). To determine the particulate matter ("dust") of seeds treated with insecticides and fungicides, a Heubach‐dustmeter or an equivalent apparatus can be preconized. This range of tests permits to ensure the quality of the treated seeds and so the protection of each plant. Key words: plant protection products, treated seeds, adhesion, distribution

Posters R03-R15

316

R12 BYSTANDERS, RESIDENTS, OPERATORS AND WORKERS EXPOSURE MODELS FOR PLANT

PROTECTION PRODUCTS (BROWSE)

Kim DOAN NGOC, Walter STEURBAUT & Pieter SPANOGHE Department of Crop Protection Chemistry

Ghent University, Ghent, Belgium It is well‐known that the application of plant protection products (PPPs) poses a potential risk for operators, re‐entry workers, residents and bystanders.Different models have already been developed to evaluate the exposure to PPPs. BROWSE is a project within the Seventh Framework Programme (FP7) and has as main goal the development of exposure models beyond the state‐of‐the‐art.One work package (WP) of BROWSE deals with the exposure of workers. Workers are persons who, as part of their employment, enter an area or handle a crop that has been treated previously with a PPP. The main objective of this WP is to review, improve and extend the currently used exposure models for workers. The existing models are based on limited data for a limited range of crop/task combinations. Recently‐generated, high‐quality exposure data should be incorporated in the model to expand the range of scenarios. Key factors and mechanisms will be identified to develop a new mechanistic model that goes beyond the state‐of‐the‐art. Regional and gender differences in worker activities will be investigated and incorporated. Key words: worker exposure, exposure model, plant protection products, BROWSE

Posters R03-R15

317

R13 THE INFLUENCE OF CLIMATE CHANGE ON THE

INCIDENCE OF PLAGUES AND DISEASES Ilse DELCOUR1, Michael HOUBRAKEN, Anne GOBIN² & Pieter SPANOGHE1

1Crop Protection Department, Ghent University Coupure Links 653, BE-9000 Ghent, Belgium

²VITO, Industriezone Vlasmeer 7, BE-2400Mol, Belgium It is generally known that our climate evolves and that this will have many consequences in the future. Several models have already been developed to show future trends in temperature and precipitation. Veg‐i‐trade (FP7 project) goes further by studying the effect of climate change on food safety and focusing on fresh produce for which some case studies will be elaborated. One facet of Veg‐i‐trade considers the residues of crop protection products or pesticides. The concentration of residues found on fresh produce is mainly linked to the amount of pesticides applied on the crop, which goes hand in hand with the occurrence of pests and plagues on the plants. In a first step we evaluate if a trend in the occurrence (frequency and intensity) of some important pests and plagues can be discovered. Our approach is based on several Belgian “decision models” that are used as part of the integrated pest management strategy (IPM). Those models predict when a pest or plague will occur and based on this, farmers are informed on when to treat their crops. At present some important models and their underlying parameters have been evaluated and analyzed, which revealed that temperature and humidity are important parameters. In a next step, weather data predictions (delivered by VITO) will be put into the models. In this way a trend analysis will be conducted to represent the changing pest and plague incidence as a result of climate change.

Posters R03-R15

318

R14 ACROPOLIS: CUMULATIVE AND AGGREGATE RISK

ASSESSMENT OF PESTICIDES IN EUROPE

I. DELCOUR1, F. VANHONACKER2, F. ROSSENEU3, W. VERBEKE2, W. STEURBAUT1, P. SPANOGHE1 & J. VAN KLAVEREN4 1 Department of Crop Protection, Ghent University, Ghent, Belgium

2 Department of Agricultural Economics 3 General Europatat, Av. De Broqueville 272 box 4, BE-1200 Brussels, Belgium

4 Natl Inst Publ Hlth & Environm RIVM, Bilthoven, the Netherlands www.acropolis‐eu.com

Current risk assessment of pesticides, and MRL setting does not sufficiently account for cumulative and aggregate exposure (EU Directive 396/2005). The central aim of the project is to improve risk assessment strategies in Europe. The project will develop a framework for cumulative and aggregate risk assessment of pesticides that is scientifically sound and accessible for all actors involved in the European risk assessment and risk management. This work consists of: 1. studying the data needs, data availability and organization including uncertainties for

cumulative exposure and effect assessment in a probabilistic risk assessment framework; 2. integrating models describing various routes of exposure into an aggregate exposure

model; 3. setting up new toxicological testing for identifying possible additive or synergistic effects

and developing a strategy for refinement of cumulative assessment groups; 4. integrating cumulative and aggregate risk models including uncertainty analyses in a

web‐based tool, including accessible data for all stakeholders; 5. improving risk assessment strategies in Europe by analysing stakeholders attitudes, by

training and by discussing the new methodology in several stakeholder conferences. The project brings together the key experts and key risk models in Europe such as dietary exposure (MCRA), dermal and inhalation exposure (ConsExpo, EUROPOEM and UK POEM) related to professional and non‐professional use. The new risk model will be validated and testedin different countries to ensure its usability at the international level. Cumulative pesticide risk assessment is a multi‐stakeholder issue. European consumer groups have raised their concern and the pesticide industry has an urgent need for clear criteria in order to justify the enormous investments in producing new pesticides. The ACROPOLIS project will therefore organise stakeholder conferences to address the complex issue of cumulative and aggregate risk assessment and the usability and user‐friendliness of the models will be tested.

Posters R03-R15

319

R15 EFFECTS OF CLIMATE CHANGE ON INSECTICIDES:

THE TOXICITY OF BINARY MIXTURES OF HARMFULCYANOBACTERIA AND INSECTICIDES

ON DAPHNIA PULEX

Jana ASSELMAN1, Guy SMAGGHE2, Colin R. JANSSEN1 & Karel A.C. DE SCHAMPHELAERE1

1Laboratory of Environmental Toxicology and Aquatic Ecology, Ghent University BE-9000 Gent, Belgium

2Laboratory of Agrozoology, Ghent University, BE-9000 Gent, Belgium We investigated the mixture toxicity of harmful cyanobacteria (i.e. Micorcystis aeruginosa)and insecticides on Daphnia pulex. Cyanobacterial blooms of this species, which are predicted to become increasingly prominent under climate change conditions, areknown for their production of toxins such as microcystin.We therefore exposed Daphnia pulex to binary mixture combinations of cyanobacteria and insecticides for a period of 21 days based on the central composite design. Results of these life tables were statistically analyzed with both concentration addition and independent action reference models to reveal additive, synergistic or antagonistic mixture effects. This analysis resulted in additive effects for the combination carbaryl and Microcystis aeruginosa, synergistic effects for the combination chlorpyrifos and Microcystis aeruginosa. These results suggest complicated interaction patterns between insecticides and harmful cyanobacteria with a potential impact on future risk assessment.

Posters H01-H16

321

Posters Herbology

Posters H01-H16

322

Posters H01-H16

323

H01 GERMINATION ECOLOGY OF

RHYNCHOSIA CAPITATA (ROTH) DC: AN EMERGING ANNUAL SUMMER SEASON WEED

HAFIZ HAIDER ALI, ASIF TANVEER, MUHAMMAD ATHER NADEEM

& HAFIZ NAEEM ASGHAR Department of Agronomy, University of Agriculture, Faisalabad-38040, Pakistan

The genus Rhynchosia is widely distributed with several species occurring in mountainous regions in the tropics. Rhynchosia capitata (Roth) DC, a member of Fabaceae family, is indigenous to Pakistan, India, and Sri Lanka. It invaded much cultivated areas of Pakistan, and become difficult to manage. Experiments were conducted to study the seed germination patterns of Rhynchosia capitata (Roth) DC. We investigated whether the diverse ecological factors such as temperature, salinity, drought stress, pH, soil depth and darkness affect the germination of R. capitata. Germination decreased as the temperature increased from 25 oC and completely inhibited at 45 °C. Presence or absence of light did not influence germi‐nation. Germination of R. capitata was sensitive to increasing salt and drought stress as well as seed burial depth. Only 47.50 % seeds germinated at a NaCl concentration of 150 mM as compared to 100% in control (distilled water). Similarly, 15% seeds germinated at drought stress level of –0.8 MPa as compared to 87.5% at –0.2 MPa level. In burial depth trial, maximum seedling emergence of 92.50 % was at 2cm depth.The optimal pH for seed germination of R. capitata was 7 (97.5 %), but the seeds also germinated at lower level of pH 5 (85%) and at higher level of pH 10 (75 %). The high germination ability of R. capitata under a wide range of ecological factors suggests that this species is likely to be the most problematic in near future if not managed appropriately.It can be concluded that a better understanding of the way in which environmental factors affect weed seed germination could be used to develop more accurate weed emergence predictions and improve weed control strategies. This will require leading weed scientists to focus their research on the development of more sustainable weed control strategies based on better knowledge on weed biology and ecology to meet the challenge of reduction of pesticide inputs in agricultural systems. Key words: Rhynchosia capitata, germination, drought stress, pH, salinity, seedling emergence.

Posters H01-H16

324

H02 STUDY METHODS OF DORMANCY BREAKING AND

GERMINATION OF RHYNCHOSIA CAPITATA (ROTH) DC. AN ANNUAL SUMMER SEASON WEED.

HAFIZ HAIDER ALI, ASIF TANVEER, MUHAMMAD ATHER NADEEM

& HAFIZ NAEEM ASGHAR Department of Agronomy, Institute of soil and environmental Sciences

University of Agriculture, Faisalabad-38040, Pakistan Rhynchosia capitata (Roth) DC, a member of Fabaceae family, is indigenous to Pakistan, India and Sri Lanka. It invaded the cultivated areas of Pakistan and become difficult to manage. Weed control is an integral part of efficient crop production that has benefited from developing new methods of dormancy release. Experiments were conducted to investigate the effect of different dormancy breaking treatments on the germination of Rhynchosia capitata (Roth) DC, an emerging weed in Pakistan. The seeds were soaked in thiourea (2500, 5000, 7500 and 10,000 ppm) and KNO3 (10000, 20000, 30000, 40000, 50000 and 60000 ppm) for 24 hours; HCl for 20, 40, 60, 80, 100 and 120 min; HNO3 for 20, 40, 60, 80, 100 and 120 min; HCl for 20, 40, 60, 80, 100 and 120 min + H2SO4 for 30 min; HNO3 for 20, 40, 60, 80, 100 and 120 min; + H2SO4 for 30 min and H2SO4 alone for 20, 40, 60, 80, 100 and 120 min. Maximum germination (100%) was recorded when seeds were treated with H2SO4 for 60 and 80 min. Germination of R. capitata did not differ statistically when seeds were treated with HCl for different durations + H2SO4 for 30 min. Seeds soaking in HNO3 for 60, 80, 100 and 120 min and then for 30 min in H2SO4 also showed non significant difference in respect of germination. While various concentrations of HCl, HNO3 alone, thiourea and KNO3 were ineffective for breaking hard seed coat dormancy of R. capitata. Results of these experiments showed that the R. capitata seeds were in a dormant state and concentrated sulphuric acid scarification significantly increased seed germination. Seed coat was the major barrier to Rhynchosia capitata seed germination, since the treatments that induce germination were those that can rupture the seed coat. These studies indicated that the success of this species is largely attributed to the occurrence of seed dormancy, which allows the seed to persist for long periods in soil and thus escape the effects of post germination weed control measures. Key words: Rhynchosia capitata, Seed dormancy, HCl, HNO3, H2SO4, Germination

Posters H01-H16

325

H03 EFFECT OF FERTILIZERS ON THE GERMINATION

AND GROWTH OF VELVETLEAF (ABUTILON THEOPHRASTI MEDIC.)

Viktor NAGY & Erzsébet NÁDASY

University of Pannonia Georgikon Faculty, Institute of Plant Protection H-8360, Keszthely Deák F. Str. 57.

Velvetleaf (Abutilon theophrasti Medic.) is one of the most important and invasive weed species in Hungary and in Europe, too. The dangerousness of this species derives from the biological characteristics of the germination and its strong competitive and allelopathic ability. As a weed of cultivated crops with wide line space such as corn, sunflower and soybean, it can reduce the yield significantly. Some authors established, that the yield reducing ability is lower under higher nutrient supply. Our experiment was carried out in Hungary, in Keszthely (46o45’35.53’’; 17o14’26.9’’) at the Univerity of Pannonia, Georgikon Faculty, Institute of Plant Protection in 2009 and 2010. We studied the single and collective effect of different fertilizers on the germination and growth of velvetleaf in greenhouse pot experiments. The applied fertilizers were Linzer NAC (27% N) in doses of 200kg N ha‐1 (2,325g Linzer NAC / pot), Patent Káli (30% K2O) in 100kg K2O ha‐1 (1,05g Patent Káli / pot) and DC Szuperfoszfát (20,5% P2O5) in 100kg P2O5 ha‐1 (1,05g DC Szuperfoszfát / pot). We made our study in 1,5litre pots with Ramman brown‐forest soil in four replications, with 25 seeds of velvetleaf per pot. Five velvetleaf plants were removed four and six weeks after planting for measurement of plant length, fresh‐ and air dried weight, leaf area and nitrogen, K2O, P2O5 and CaO content of the plants. The data sets were analyzed by ANOVA. We established, that nitrogen applied at a rate of 200kg N ha‐1 (alone or with other nutrients) can reduce the germination and growth of Abutilon . The potassium and phosphorus stimulated the germination and the growth. Potassium applied alone showed the biggest stimulating effect . We could not find differences in the nutrient content among the treatments. The research was funded by: "Livable Environment and Healthier People – Bioinnovation and Green Technology Research at the University of Pannonia" Key words: Abutilon theophrasti, fertilizer, potassium, nitrogen, phosphorus

Posters H01-H16

326

H04 STUDY OF THE PEST COMMUNITY OF VELVETLEAF

(ABUTILON THEOPHRASTI MEDIC.)

Viktor NAGY, Balázs KERESZTES & Erzsébet NÁDASY University of Pannonia Georgikon Faculty, Institute of Plant Protection

H-8360, Keszthely Deák F. Str. 57 Velvetleaf (Abutilon theophrasti Medicus 1787) is one of the most economically threatening weed species in Hungary. Researching biological control against it, and identifying a possible and effective biocontrol agent is an important challenge, as chemical control is difficult and expensive, and there is an increasing claim to practice minimal plant protection. Entomological studies were made in several parts of the world, for evaluating the species, occuring in velvetleaf, but none of these kind of experiments were assessed in Hungary. Our observations were made in field and plastic boxes, both under open field circumstances in 2008 and 2009 by visually assessing pests, netting and damage based identifying. Meanwhile 8 pest species were identified, including (Helix pomatia Linnaeus 1758 – roman snale; Trialeurodes vaporariorum (Westwood 1856) – greenhouse whitefly; Oxycarenus lavaterae (Fabricius 1787) – lime seed bug; Pyrrhocoris apterus (Linnaeus 1758) – fire bug; Rhopalus parumpunctatus Schilling 1829 – common hyaline bug; Liorhyssus hyalinus ‐ hyaline grass bug (Fabricius 1794); Mamestra brassicae (Linnaeus 1758) – cabbage moth; Helicoverpa armigera (Hübner 1808) – corn earworm). On the whole the literature data was enlarged with four new velvetleaf pests (roman scale, lime seed bug, common hyaline bug, cabbage moth). Considering the earlier literature and our results, Liorhyssus hyalinus may play an important role on biological management of velvetleaf. However this pest, considered as polyphagous, was discovered to occur in great numbers on velvetleaf. This points to the fact that it can be its main host plant. By sucking on the plant, it can cause decreased germination rate. We suggest the „hyaline velvetleaf bug” name instead of „hyaline grass bug”. Of course, additional experiments are needed to determine whether this pest might be used savely and effectively in the future. The research was subsidized by: "Livable Environment and Healthier People – Bioinnovation and Green Technology Research at the University of Pannonia" (TÁMOP‐4.2.2.‐08/1/2008‐0018). Key words: Abutilon theophrasti, biological weed control, Liorhyssus hyalinus

Posters H01-H16

327

H05 ALLELOPATHIC EFFECT OF BROMUS SPP. AND

LOLIUM SPP. SHOOT EXTRACTS ON SOME CROPS

Éva LEHOCZKY1, Martha OKUMU NELIMA1, Rita SZABÓ1, Attila SZALAI1 & Péter NAGY2

1University of Pannonia, Georgikon Faculty, Institute of Plant Protection, Keszthely, Hungary 2Dow AgroSciences Export S.A.S., Mougins, France

Allelopathy is an untapped resource for weed control in crops that could give good possibilities for environmentally sound, integrated crop production. Allelopathy is defined as the direct or indirect harmful or beneficial effects of one plant on another through the production of chemical compounds, called allelochemicals, which escape into the environment. Allelochemicals can be produced by weeds and affect crops, and the reverse is also true. Allelopathic interactions include weed‐weed, weed‐crop, and crop‐crop. Allelopathy offers potential for selective biological weed control for instance weed‐suppressing crops and the use of plant residues in cropping systems, allelopathic rotational crops, or companion plants with allelopathic potential. Bromus species occur in many habitats in temperate regions of the world, including America, Eurasia, Australia, and Africa. The genus Lolium is one of the most important forage grasses. The weed species usually grow in the same production zones as wheat and are considered weeds since they parasitize wheat fields. Some of the weed species in these two genus have been reported to have allelopathic effect. One of the methods that has been successful in studying allelopathic activity are bioassays. Laboratory experiments were conducted to determine allelopathic effect of watery shoot extracts of four weed species of the Poaceae family, namely Bromus rigidus, Bromus diandrus, Lolium multiflorum and Lolium temulentum on germination and growth of winter wheat (Triticum aestivum L.), spring barley (Hordeum vulgare L.), corn (Zea mays L.), perennial ryegrass (Lolium perenne L.), bean (Phaseolus sp.) and sunflower (Helianthus annuus L.) and on each other. The experiment was carried out during the period March 2010 to October 2010. Twenty five seeds were put into one Petri‐dish on filter paper, adding 15ml of extract to each in four repeats. The germination took place in a Binder‐type thermostat in the dark. The timing of germination was checked in every two days and the rate of growth was estimated after a week, by counting the number of germinated seeds and measuring the length of the radicle and plumule. The measured data was statistically analyzed and the effect of the extracts on germination percentage and seedling length was evaluated. This study has been generously supported by TÁMOP‐4.2.2‐08/1/2008‐0018 ‐ entitled as “Liveable environment and healthier people ‐ Bio innovation and Green Technology research at the University of Pannonia". The project is co‐financed by the European Social Fund with the support of the European Union. Key words: allelopathy, Bromus spp., crops, germination, Lolium spp.

Posters H01-H16

328

H06 ALLELOPATHIC EFFECTS OF RAGWEED (AMBROSIA ARTEMISIIFOLIA L.) ON

CULTIVATED PLANTS

Éva LEHOCZKY, Gellért GÓLYA, Rita SZABÓ & Attila SZALAI University of Pannonia, Georgikon Faculty, Institute of Plant Protection

Keszthely, Hungaria During the past years ragweed has been coming to the forefront of interest in Hungary and in other European countries as well because its serious health risk. Results of the 5th National Weed Survey has proven that ragweed is the most important weed species on Hungarian field lands, its coverage shows a rising tendency in cereals moreover it not only occurs in cultivated plants. Allelopathic effects of aqueous extracts derived from different parts of ragweed plants (air dried leafy stems, seeds) on the germination and growth of other cultivated plants [corn (Zea mays L.), soy‐bean (Glycine max L.), sunflower (Helianthus annuus L.), bean (Phaseolus vulgaris L.), winter wheat (Triticum aestivum L.), and rapeseed (Brassica napus oleifera L.)] were studied. The extracts made for the trials were prepared with distilled water. Petri dishes were used for the germination experiments and distilled water was used as a control treatment. The seven days long experiment was carried out within a Binder‐type thermostat under dark conditions. The germination percentage was checked in every two days and the growth of sprouts was evaluated after a week counting the germinated seeds and measuring the length of the radicle and plumule. The measured data were statistically analysed and the effect of extracts on germinating and length of sprouts were assessed. This study was supported by TÁMOP‐4.2.2‐08/1/2008‐0018 ‐ entitled as “Liveable environment and healthier people ‐ Bio innovation and Green Technology research at the University of Pannonia". The project is co‐financed by the European Social Fund with the support of the European Union. Key words: allelopathy, Ambrosia artemisiifolia L., cultivated plants, germination

Posters H01-H16

329

H07 ALLELOPATHIC ACTIVITY OF FRACTIONS OF

IPOMOEA BATATAS EXTRACT ON SORGHUM BICOLOR

Amed PUPO RODRÍGUEZ, Ramón CRISTOBAL RIOS ALBUERNE, Rafael SOSA MARTÍNEZ, Sinesio TORRRES GARCÍA, Maykel HERNÁNDEZ

& Ray ESPINOSA RUIZ Carretera a Camajuaní, km 5 1/2 Universidad Central

"Marta Abreu" de Las Villas. Santa Clara, Cuba It was studied different fractions obtained by gel filtration chromatography processes and dilution of the aqueous extract (sweet potato) Ipomoea batatas (L.) Lam, identifying the qualitative and quantitative presence of phenolic compounds, specifically flavonoids. The allelopathic effects of different fractions of aqueous extract of sweet potato (I. batatas), were tested on seedlings of Sorghum bicolor (L.) Moench (sorghum) in vitro condition. The variables measured were: length of the hypocotyl, radicle length, dry weight and fresh weight. The different treatment (aqueous extract 8100, 162, 1 mg L‐1, 10 ‐30 mg L‐1, F1‐F9 fractions) were administrated based on the value of total dissolved solids using destilled and deionized water as control. The hypocotyl and radicle were stimulated by diluting 162 mg L‐1 more than the extract that stimulated it, too. The treatment (1 mg L‐1) stimulated the growth of radicle meanwhile there were no statistical differences with control in the case of hypocotyl. The dilution of 10‐30 mg L‐1 did not show differences. No fractions stimulated hypocotyl S. bicolor, although F4 and F5 stimulated the growth of the radicle, the fraction 9 inhibited the growth of it. With the exception of fractions 2 and 4, weight loss occurred with respect to cool control, and there were no variations in the dry weigh. Key words: Allelopathy, Ipomoea batatas, Sorghum bicolor

Posters H01-H16

330

H08 EFFECT OF PRE-PLANTING IRRIGATION,

MAIZE PLANTING PATTERN AND NITROGEN ON WEED SEED BANK POPULATION

Ehsan HEMMATI, Saeed VAZAN & Mostafa OVEISI

Department of Agronomy,Islamic Azad University,Karaj-Branch,Iran Department of Agronomy,Islamic Azad University,Karaj-Branch,Iran Department of Agronomy and Plant

breeding, University of Tehran,Iran Pre‐planting irrigation and planting patterns are important factors in weed management and seed bank dynamics. Additionally, the nitrogen is the most important factor in plant growth that affects weed‐crop competition and ultimately, seed rain into the soil. A field experiment was conducted to study the effect of nitrogen application rates, pre‐planting irrigation and maize planting patterns on weed seed bank population. Experimental factors were nitrogen rates at 4 levels (200,300,400 and 500 kg per hectare) as main plot; and pre‐planting irrigation at 2 levels (irrigation before planting plus weeding emerged seedlings and, irrigation after sowing), and maize planting patterns (one‐row and two‐row planting of maize with same density per square of row length) that were assigned in a factorial arrangement to the sub plots. Soil samples were taken at the beginning of the season (before planting of maize) and at the end of the season (after harvest) at depth of 0‐5 cm in the fixed quadrates (60 cm × 60 cm). The weed seeds were extracted from the soil samples and were identified using standard methods. The majority of weed seed bank populations included 6 weed species: Portulaca oleracea, Chenopodium album, Amaranthus retroflexus, Sorghum halepense, Datura stramonium, Xanthium strumarium. Results showed that population of weed seed bank increased significantly with increasing nitrogen rate. The increasing rate was different between one‐row and two‐row planting patterns. The parameters indicated that seed bank population was much higher in a one row planting pattern of maize. With two‐row planting, seed bank was decreased by 34, 26, 20 and 5% at 200,300,400 and 500 kg N/ha, respectively. Pre‐planting irrigation was also found an effective implement to reduce the weed seed bank. When pre‐planting irrigation was applied, seed bank was decreased by 57, 43, 34 and 9% at 200,300,400 and 500 kg N/ha. Increasing nitrogen because of weed’s better growth and higher seed production neutralized the decreasing effect of pre‐planting irrigation and two‐row planting of maize on weed seed bank population. Key words: seed bank, nitrogen rate, weed management, maize

Posters H01-H16

331

H09 EFFECT OF MUNGBEAN LIVING MULCH ON DENSITY

AND DRY WEIGHT OF WEEDS IN CORN FIELD

M. BAKHTIARI MOGHADAM, S. VAZAN, F. GOLZARDI, S. AZIZKHANI & M. ESFINI FARAHANI

Departement of Agronomy, Islamic Azad University-Karaj Branch, Iran Departement of Agronomy, Islamic Azad University-Karaj Branch, Iran Departement of Agronomy, Islamic Azad University-Karaj Branch, Iran Departement of Agronomy, Islamic Azad University-Karaj Branch,

Iran Departement of Agronomy, Islamic Azad University-Karaj Branch, Iran Living mulch is a suitable solution for ecological weed management and it is considered an effective method to decrease weed density and dry weight. In order to evaluate the effect of mungbean living mulch on density and dry weight of weeds in corn, an experiment was set up in 2010 at the agricultural faculty of Islamic Azad University Karaj branch. The experimental design was a split plot based on randomized complete block design with four replications. Experimental factors included time of mungbean suppression with 2‐4,D herbicide (four levels: 4, 6, 8 and 10 corn leave stage) and control without weeding as main plots and densities of mungbean (three levels: 50 %, 100% and 150% more than optimum density) as sub plots. Per plot, density and dry weight of weeds were measured after tasseling of corn in 60 x 100 cm quadrates, . In total, 9 species of weeds were identified in the field, which included 4 broadleaf weeds that were present in all plots. The results showed that the best time for suppression of mungbean is the 8th leaf stage of corn, which decreased density and dry weight of weeds by 53% and 51% compared to the control, respectively. Increase of density of mungbean, decreased the density and dry weight of weeds: optimum weed suppression was achieved by the 150% mungbean density which decreased density and dry weight of the weeds by 69% and 63.5% respectively compared to the control. Key words: living mulch, weeds ecological management, corn, mungbean

Posters H01-H16

332

H10 ROLE OF RICE VARIETIES AND MINERAL FERTILIZER

IN THE INTEGRATED CONTROL OF THE HEMI PARASITIC WEED RHAMPHICARPA FISTULOSA IN THE

INFESTED INLAND OF BENIN

S.H.N. ZOSSOU, J. RODENBURG, A. AHANCHEDE & G. GBEHOUNOU Vegetables Production, FSA/UAC, Benin Weeds management, Africarice

Tanzania Vegetables Production, FSA/UAC, Benin FAO, Rhum, Italie Inlands of Benin are today enjoying a resurgence of interest with the increasing degradation of soil trays. However, the development of new parasitic weed Rhamphicarpa fistulosa in some of these inland valleys constitutes a major constraint for the development of rice cultivation. This sets out to improve rice production in Africa inland valleys and has been initiated to find an integrated management of the parasite. Experimentation has been carried out in order to evaluate the potential role of some varieties of NERICAs and other popular rice varieties grown in the lowland combined with the supply of mineral fertilizer. It comes out from the results that NERICA‐L‐32 and IR64 are in order the most tolerant and the most resistant varieties of all those tested. The contribution of mineral fertilizers at a dose of 200 kg / ha of NPK (15‐15‐15) and 75 kg / ha of Urea allows better control of the parasite. In conclusion, we can say that Rhamphicarpa fistulosa is a new pest of rice that deserves the attention of the entire international scientific community. Key words: Rhamphicarpa, rice, weed, inland, parasite, fertilizer

Posters H01-H16

333

H11 INTEGRATED WEED MANAGEMENT IN MAIZE

ASIM RAZA CHADHAR, MUHAMMAD ATHER NADEEM, MUHAMMAD AWAIS,

ASIF TANVEER& MUHAMMADTAHIR Department of Agronomy, University of Agriculture, Faisalabad, Pakistan

Maize is an important cereal crop of Pakistan, demanding a proper weed management approach. A field trial was accomplished to appraise the integrated effect of weed control practices on weed density and biomass, and yield of autumn planted maize (Zea mays L.), under agro‐ecological conditions of Faisalabad. Treatments comprised manual hoeing and earthing up alone and in combination with metolachlor + atrazine at1110+740 g a.i.ha‐1, manual hoeing + earthing up, metolachlor + atrazine at1110+740 g a.i.ha‐1 alone and acetochlor at 618 g a.i.ha‐1 alone along with weedy check as control. All the weed control treatments significantly decreased weed density compared with weedy check after 30 and 60 days of sowing and at harvesting. Manual hoeing integrated with metolachlor at1110 g a.i.ha‐1 + atrazine at 740 g a.i. ha‐1 provided the maximum control of narrow and broad leaved weeds followed by manual hoeing. All the weed control treatments significantly decreased weed fresh and dry weight compared with weedy check and minimum fresh and dry weight was recorded in manual hoeing integrated with metolachlor at1110 g a.i.ha‐1 + atrazine at 740 g a.i.ha‐1, followed by manual hoeing. The maximum grain and biological yield was obtained with manual hoeing integrated with metolachlor at1110 g a.i.ha‐1 + atrazine at740 g a.i.ha‐1. Key words: integrated, weed management, maize

Posters H01-H16

334

H12 INTEGRATED APPROACH ON WEED CONTROL

IN SWEET NARROW-LEAFED LUPINS (LUPINUS ANGUSTIFOLIUS)

T. COUSSENS1, K. DEWITTE1,2, K. AUDENAERT1,2,

J. LATRÉ1 & G. HAESAERT1,2 1 Faculty of Biosciences and Landscape architecture, University College Ghent,

Schoonmeersstraat 52, BE-9000 Gent, Belgium 2 Faculty of Bioscience Engineering, Ghent University

Coupure Links 653, BE-9000 Gent, Belgium Narrow‐leafed lupins are a protein rich, nitrogen fixing crop which can be of importance for sustainable agriculture in general and organic farming in particular. Since lupins have a slow development of the canopy, they are bad competitors of weeds. Therefore a field trial with 5 different weed management systems applied on L. angustifolius var. SNS1 with a 30 cm row spacing has been set up during the growing season 2010. The weed control systems varied from intensive harrowing and hoeing on the one hand to extensive harrowing and hoeing in combination with burning or a herbicide application at a low dose on the other hand. Weed control: Extensive harrowing and hoeing resulted in a reduction of 60 % of the Stellaria media population, but a higher germination of Chenopodium album (220 %) and of Polygonum convolvulus (120 %) was observed. Intensive mechanical weed control caused the highest reduction (40 %) of the Chenopodium album population and resulted also in a reduction of the Stellaria media and Polygonum convolvulus population, with respectively 80 % and 67 %. Previous weed populations were measured in the row, between the rows weed control was almost complete. Extensive mechanical weed control in combination with burning or with a herbicide application resulted in an overall weed reduction. The Chenopodium album population was less under control if extensive mechanical weed control was combined with a pre‐ and post‐emergence herbicide mix of respectively chlorotoluron (750 g/ha) with isoxaben (37.5 g/ha) and diflufenican (50 g/ha). Yield: Carrying out no weed control at all resulted in the significantly lowest yield (1963 kg/ha). This was not significantly lower than the extensive mechanical weed control (2350 kg/ha) but was significantly lower than all other objects. Intensive mechanical weed control resulted in the highest yield (3058 kg/ha).This was not significantly higher than the objects in which an extensive weed management was combined with a herbicide application at low dose or burning but was significantly higher than the other objects. The lowest population of Chenopodium album probably resulted in a lower competition for light and nutrients and in a higher yield. An integrated weed management in sweet narrow‐leafed lupins is possible, especially an intensive combination of harrowing and hoeing seems interesting. In case of unstable weather conditions, some safety can be built in e.g. with an application of herbicides at a low dose. Key words: integrated weed control, sweet lupins, Lupinus angustifolius

Posters H01-H16

335

H13 POST-EMERGENT WEED CONTROL IN

NARROW-LEAFED LUPINS (LUPINUS ANGUSTIFOLIUS)

T. COUSSENS1, K. DEWITTE1,2, K. AUDENAERT1,2

J. LATRÉ1 & G. HAESAERT1,2 1 Faculty of Biosciences and Landscape architecture, University College Ghent,

Schoonmeersstraat 52, BE-9000 Gent, Belgium 2 Faculty of Bioscience Engineering, Ghent University

Coupure Links 653, BE-9000 Gent, Belgium In Australia, the world leader in lupin production, new lupin genotypes contain tolerance for metribuzin. On the contrary, in Europe are no metribuzin resistant varieties available and post‐emergence weed control in narrow‐leafed sweet lupins remains difficult. There are only few studies on the impact of herbicides sprayed on sweet lupins in post‐emergence. Therefore a field trial with 5 different herbicides or combinations of herbicides applied in post emergence has been set up during the growing season 2010. The herbicides were chosen based on previous screening results and were compared with an untreated control. A pre‐emergence weed control ‐ at low dose ‐ with chlorotoluron (750 g/ha) in combination with isoxaben (37.5 g/ha) was sprayed on all objects to enhance the weed control. Phytotoxicity: During the growing season no growth inhibition or necrosis could be observed, only chlorosis was visible. The combination of bromoxynil (600 g/ha) and carfentrazone‐ethyl (6.4 g/ha) on the one hand and the combination of tepraloxydim (50 g/ha) with carfentrazone‐ethyl (6.4 g/ha) on the other hand caused respectively 20.0 % and 18.3 % chlorosis. This was higher than noticed on the other objects. Ten days later, this severe crop injury had almost disappeared. Yield: The application of tepraloxydim (50 g/ha) in combination with carfentrazone‐ethyl (6.4 g/ha) resulted in the highest yield (3382 kg/ha). This was not significantly different from the other treatments and only slightly higher than the control (3354 kg/ha). Spraying bromoxynil (600 g/ha) resulted in the lowest yield (3071 kg/ha) but was not significantly lower than the yield of the other treatments. Weed control: The applied overall pre‐emergence herbicides in combination with the different herbicides or combinations of herbicides succeeded well and resulted in an almost clean crop. Differences could only be observed for Stellaria media. The combinations of diflufenican (100 g/ha) with bromoxynil (600 g/ha) and of diflufenican (100 g/ha) with carfentrazone‐ethyl (6.4 g/ha) resulted in a complete destruction of the present S. media plants. Spraying one of the other herbicides or herbicide combinations resulted in a reduction of at least 66 % of the S. media population. The results show some possibilities, but weed control in post‐emergence remains difficult. Key words: chemical weed control, sweet lupins, Lupinus angustifolius

Posters H01-H16

336

H14 TO GENERATE EFFICACY DATA IN ITALY AND TO

EVALUATE THE EFFECT OF APPLICATION TIMING OF THE HERBICIDE SIT90 ON TRITICUM DURUM

Angela FANIGLIULO1, Vittorio FILÌ1 & Aniello CRESCENZI2

1 Bioagritest Test Facility, Bioagritest Srl. Zona PIP lotto E2. 85010 Pignola (PZ, Italy) E-mail: [email protected]; www.bioagritest.it.

2 Dipartimento di Biologia, Difesa e Biotecnologie Agro-Forestali – Università degli Studi della Basilicata. Viale dell’Ateneo Lucano Campus Macchia Romana 3A310

IT-85100 Potenza (PZ) Italy E-mail: [email protected]

A study was performed in the period February to May 2010 by Bioagritest test facility according to EPPO guidelines and Principles of Good Experimental Practice (GEP), in the land of Altamura (BA), in the core of Murgia. The purpose of the study was to generate efficacy data in Italy and to evaluate the effect of application timing of the herbicide SIT90 (a.i. propoxycarbazone + iodosulfuron + mefenpyr) on Triticum durum. Two different dosages of the herbicide SIT90 – 0,250 and 0,333 kg/ha ‐ were applied in a T. durum field at two different application timings, i.e. in early post‐emergence (25th February) and late post – emergence (27th March). In early post‐emergence it was also used, at the lowest dosage 0,250 kg/ha, in combination with the post emergence herbicide Dicuran (a.i. chlorotoluron) 3,2 L/ha. In late post‐emergence it was applied at dosage 0,333 kg/ha, both alone and in combination with the adjuvant Biopower 1 L/ha. At a lower dosage, 0,150 kg/ha, it was also used combined with both Biopower and the commercial formulate Atlantis WG (mesosulfuron+ iodosulfuron+ mefenpyr) 0,250 kg/ha. T. durum cultivar was “Iride”. Three assessments were performed starting from 21 days after the 2nd application, exactly on 22nd April, 3rd May and 18th May 2010. They consisted in the evaluation of control activity (expressed as %) on every single weed checked (Phalaris spp., Fumaria officinalis, Papaver rhoeas, Alopecurus myosuroides, Avena ludoviciana), where 0% = same vegetative situation of the check; 100% = total weed control. Also, possible phytotoxic effects on T. durum, identifiable as necrosis, chlorosis and burns were evaluated, using a 0‐100 scale with 0 = no damage and 100 = death of the plant. The study has given sufficient results on the use of SIT90, in the conditions of use foreseen by the protocol and in consideration of the weeds present, which represented the most species present in the Murgia. Treatments with the SIT90 alone (even in combination with chlorotoluron), applied in early or in late post‐emergence, were very ineffective on grasses and dicotyledonous weeds, highlighting the importance of the adjuvant Biopower to enhance the expression of the herbicide. By contrast, the presence of the adjuvant Biopower allowed the expression of a clear and good overall herbicide activity of SIT90, for the control of P. rhoeas and other dicotyledonous weeds, but also a discrete activity against grasses (although less than that exerted by the standard Atlantis). More reliable was the treatment with SIT90 mixed with Atlantis, against the entire community of grass weeds and on F. officinalis. The performance of all the treatments was insufficient on P. rhoeas, because of the evident resistance shown by the weed. No expression of phytotoxicity, induced by the treatments being tested, was observed.

Posters H01-H16

337

H15 USING HERBICIDES IN SPRING RAPESEED AND EFFECT ON QUANTITY AND QUALITY

PARAMETERS OF YIELD

Dragana MARISAVLJEVIC2 , Petar MITROVIC1, Radovan MARINKOVIC1, Danijela PAVLOVIC2, Erika PFAF DOLOVAC2, Igor ELEZOVIC2

& Ljiljana RADIVOJEVIC3 1 Institute of Field and Vegetable Crops, Novi Sad

2 Institute for plant protection and enviroment, Belgrade 3 Institute of Pesticides and Enviromental Protection, Belgrade, Serbia

Possibility to chemically control weeds in spring rapeseed has been tested in two locations (Novi Sad and Kragujevac) and following herbicides (a.i.) were tested: trifluralin, clomazone, quizalofop‐P‐ethyl and clopyralid. Simultaneously we tested the effect of the herbicides on yield and hectoliter weight of seed and oil and protein contents in seed. Chemical weed control in spring rapeseed crop was carried out in 2009, using the standard EPPO method for testing the efficiency of herbicides in rapeseed crops. In addition to the tested herbicides, the experiment included also two controls ‐ one with and another without hoeing. In the trial in Kragujevac, a large number of weed species were present, with somewhat increased density and uneven distribution of weed plants. This was particularly evident with grassy weeds and with the species Rubus caesius in several plots. Rapeseed yield and quality were determined by measuring and analyzing the following parameters: grain yield (kg/plot (30 m2), hectoliter weight, oil content (%) and protein content (%) in seed.Basic statistical calculations of rapessed yield and quality were done by the t‐test. Neither herbicide tested in the location of Kragujevac was sufficiently efficient in controlling the dominant weed species in the experiment (Hibiscus trionum, Cynodon dactylon, Elytrigia repens, Cirsium arvense and Rubus caesius). The tested herbicides exhibited a certain measure of efficiency in the control of annual grasses and broadleaf weeds at this site. In plots dominated by these weeds, best results were obtained with the combinations trifluralin + clomazone, trifluralin + clopyralid and clopyralid + quizalofop‐P‐ethyl. The tested herbicides showed no adverse effect on the yield and hectoliter weight of seed in either location, with the exception of quizalofop‐P‐ethyl in Kragujevac, which affected the control variants. Oil content was negatively affected by the combination, trifluralin + clopyralid in the location of Novi Sad and by quizalofop‐P‐ethyl in the other location. Trifluralin and quizalofop‐P‐ethyl exhibited a negative effect on protein content in the location of Novi Sad, while there were no statistically significant negative effects in the other location. Key words: spring rapeseed, herbicides, weeds, yield, hectoliter weight, oil and protein contents

Posters H01-H16

338

H16 PHYLLOSPHERIC STUDY OF SOME

DOMINANT CROP WEEDS

Irum MUKHTAR*, Ibatsam KHOKHAR, Amna ALIA & Sobia MUSHTAQ Institute of Plant Pathology, University of the Punjab, Lahore Pakistan

* E-mail: [email protected] Diversity in epi‐ and endophytic microorganisms of some dominant agricultural weeds has been thoroughly discussed in this study. In the search of diversity and relationship of epi and edophytic microorganism, 46 fungal strains and 19 bacterial strains were isolated from the surface and the inner tissue of four weeds.Leaf wash and homogenized leaf mixture solution respectively were used for the isolations from healthy leaves of four weeds viz. Chenopodium album, Euphorbia helioscopia, Parthenium hysterophorus, Convolvulus arvensis. Current study indicated that complex interactions existed between the host and their epi and endophytic microflora. Each weed has specific bacterial community with the reference of epi and endo phyllospere. The number and species of bacterial strains varied not only with their host weed plants but also in epi and endo phyllospere. Sørensen’s QS of all tested weeds for the endophytic and epiphytic bacterial assemblages was 0.00 that indicated no species overlap/similarity between the communities. Five fungal genera were common as epi and endophytes in all weeds samples: Aspergillus (56% of all isolates), Drechslera (10%), Alternaria (10%) Penicillium (6%) and Cladosporium (4%). Frequency of all five common genera differed significantly among weeds. It was also noted that entophytic fungal communities were not noticeably less speciose than epiphyte communities. Sørensen’s QS of Euphorbia sp. (0.23), Chenopodium sp. (0.37) and Convolvulus sp. (0.46) for the endophytic and epiphytic fungal assemblages was intermediate in the range (0.12–0.79) of previous studies. Although in case of P. hysterophorus, the value for Sørensen’s QS was 0.00 which means no species similarity. The other identified genera were rare, such as Absidia, Cuvularia, Phoma and Trichoderma. However, if some strain of any of these fungal or bacterial species are developed which meet the requirements of using them as biocontrol agents i.e. host specificity and strong virulence, they can be considered as potential bioherbicides for biocontrol of these weeds. Key words: phyllospher, weeds, aspergillus, alternaria, epiphytic, endophytic

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