from september 25th to 28th, 2018 perpignan, france · the key role of chitooligosaccharides in...

from September 25th to 28th, 2018 Perpignan, FRANCE

Comité d’organisation / Organizing Committee : Cédric BERTRAND – UPVD /PO²N (Président) Marie-Virginie SALVIA – UPVD /PO²N Dimitri DILES – CCI Occitanie & Enterprise Europe Network Denis LONGEVIALLE – IBMA France Laurent AUGIER – Agri Sud-Ouest Innovation Elsa BALLINI – Supagro, Montpellier Régis BERTHELOT – Arvalis, Elicitra Marie TURNER – Vegenov, Elicitra

Comité scientifique / Scientific Committee : Dr. Claire PRIGENT-COMBARET, University Lyon 1, France (Présidente) Dr. Emilio MONTESINOS, University of Girona, Institute of Agriculture and Food Technology, Spain Dr. Christoph KEEL, University of Lausanne, Switzerland Dr. Bernard DUMAS, University Paul Sabatier, Toulouse, France Dr. Philippe REIGNAULT, University of Littoral, Elicitra, France Dr. Olivier LEGALL, INRA Bordeaux, Elicitra, France Dr. Fabienne BAILLIEUL, University of Reims, Elicitra, France Dr. Anne-Emmanuelle HAY, University Lyon 1, France Dr. Azucena GONZALEZ-COLOMA, ICA, CSIC Madrid, Spain Pr. Cédric BERTRAND, University of Perpignan France Dr. Marc BARDIN, INRA Avignon, France

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Contents

SESSION 1: REGULATION AND INTEGRATED PEST MANAGEMENT .................................................................................................... 4

PLANT RESISTANCE INDUCERS: A LONG BUT PROMISING WAY FROM THE RESEARCH LABORATORY TO INTEGRATED PROTECTION SCHEMES IN THE FIELD .............. 5

SUBSTANCE ACTIVES DE BIOCONTROLE: EVOLUTIONS DEPUIS L’ENTREE EN VIGUEUR DU RCE 1107/2009/ BIOCONTROL AGENT ACTIVE SUBSTANCES:

EVOLUTION SINCE THE ENTRY IN VIGOUR OF REG. 1107/2009 .............................................................................................................................................................. 6

RELEASE AND MONITORING OF TWO SPIDERS (CHEIRACANTHIUM MILDEI AND CLUBIONA SP.) IN AN APPLE ORCHARD AND ITS EFFECTS ON CODLING

MOTH ABUNDANCE ................................................................................................................................................................................................................................ 7

MILDIOU DE LA POMME DE TERRE : RECHERCHE ET MAITRISE DES PRODUITS DE BIOCONTROLE DANS UN SCHEMA DE PROTECTION INTEGREE DES CULTURES

(PROJET MILPOMBIO) ............................................................................................................................................................................................................................. 8

MALADIE DES PROTEAGINEUX : VERS UNE REDUCTION DES INTRANTS ET UNE SECURISATION DES RENDEMENTS

(PROJET PROGRAILIVE DU PROGRAMME SOS-PROTEIN) ........................................................................................................................................................................ 9

EVALUATION DE PRODUITS DE BIOCONTROLE POUR LUTTER CONTRE LA POURRITURE GRISE DE LA VIGNE ...................................................................................... 10

INVESTING THE MODE OF ACTION OF RESISTANCE INDUCERS DURING PROTECTION OF WHEAT AGAINST POWDERY MILDEW ........................................................ 11

SESSION 2 : ELICITORS OF PLANT DEFENSES AND PLANT DEFENSES ............................................................................................... 12

PRIMING PLANTS FOR ENHANCED DEFENSE ......................................................................................................................................................................................... 13

STIMULATION OF PLANT IMMUNITY BY BENEFICIAL BACILLI : MECHANISTICS OF SURFACTIN PERCEPTION ....................................................................................... 14

STUDY OF A STREPTOMYCES SP. DERIVED SIGNALS AND THEIR PLANT PERCEPTION MECHANISMS ................................................................................................... 15

NITROGEN FERTILIZATION: A DETERMINING FACTOR FOR EFFICIENCY OF PLANT DEFENSE ELICITORS ? ............................................................................................. 16

PHAEODACTYLUM TRICORNUTUM : NEW SOURCE OF ELICITING MOLECULES FOR PLANT DEFENSE AND HEALTH............................................................................. 17

MECHANISMS UNDERLYING ASM-INDUCED RESISTANCE TO THE APPLE ROSY APHID, DYSAPHIS PLANTAGINEA ................................................................................ 18

BRASSICA NAPUS DEFENSE RESPONSES TRIGGERED BY BACTERIAL RHAMNOLIPIDS - FROM PERCEPTION TO EFFECTIVE PROTECTION ............................................. 19

EVALUATION DES PROPRIETES DE STIMULATION DES DEFENSES DES PLANTES D’UN EXTRAIT NATUREL DE CHAMPIGNON DE CULTURE ......................................... 20

ALKYL GALLATES INDUCE PLANT DEFENCE REACTIONS ON TOBACCO .................................................................................................................................................. 21

THE KEY ROLE OF CHITOOLIGOSACCHARIDES IN PLANT-MICROBE INTERACTIONS .............................................................................................................................. 22

RNA-SEQ ANALYSIS OF PLANT INNATE IMMUNITY AFTER ELICITATION WITH COS-OGA ...................................................................................................................... 23

SESSION 3: BIOCONTROL MICROORGANISMS AND MACROORGANISMS AND THEIR MECHANISMS OF ACTIONS .......................... 24

PATHOGENICITY OF RHIZOSPHERE PSEUDOMONADS TOWARDS PLANT PEST INSECTS ....................................................................................................................... 25

ANTIMICROBIAL PEPTIDES IN BIOCONTROL MICROORGANISMS. POTENTIAL AND LIMITATIONS AS BIOPESTICIDES FOR PLANT DISEASE CONTROL ......................... 26

PATHOGEN SUPPRESSION BY RHIZOSPHERE MICROORGANISMS BUILDS UP DURING PLANT DEVELOPMENT .................................................................................... 27

PLANT-ASSOCIATED LACTIC ACID BACTERIA AS EFFICIENT BIOCONTROL AGENTS OF BACTERIAL PLANT DISEASES ............................................................................. 28

PARABURKHOLDERIA PHYTOFIRMANS STRAIN PSJN A PROMISING TOOL TO HELP GRAPEVINE FACING BOTH BIOTC AND ABIOTIC STRESSES .................................. 29

BIOLOGICAL CONTROL OF QUARANTINE BACTERIAL DISEASES WITH SELECTED STRAINS OF BACILLUS AMYLOLIQUEFACIENS .......................................................... 30

CYTOLOGICAL AND MOLECULAR MECHANISMS OF PYTHIUM OLIGANDRUM MYCOPARASITIC INTERACTIONS WITH FUSARIUM GRAMINEARUM ........................... 31

LEGUME NODULES ARE SITTING DUCKS FOR PATHOGENS ................................................................................................................................................................... 32

HOW DOES A FLAVOR LACTONE TRIGGER BACTERIAL BIOCONTROL OF BLACKLEG AND SOFT-ROT DISEASES ? ................................................................................ 33

IDENTIFICATION OF POTENTIAL MYCOHERBICIDES USING A METABARCODING APPROACH ................................................................................................................. 34

SEED COATING WITH NATURAL BIOSTIMULANTS : A PRACTICAL TOOL FOR IMPROVING TOLERANCE TO BIOTIC AND ABIOTIC STRESSES IN TUNISIA ....................... 35

ALD1202, A NEW BIOFUNGICIDE BASED ON YEAST TO CONTROL BOTRYTIS AND MONILIA ROTS IN GRAPEVINE, VEGETABLE CROPS AND STONE FRUITS ................... 36

BIOCONTROL OF GREY MOLD OF TOMATO USING ENDOPHYTIC BACTERIA PRODUCING VOLATILE ORGANIC COMPOUNDS ............................................................. 37

AN ENDOPHYTIC BACILLUS SUBTILIS STRAIN PROTECTS GRAPEVINE AGAINST DOWNY MILDEW BY DIRECT EFFECT AND DEFENSE STIMULATION ............................ 38

AGROINDUSTRIAL AND FORESTRY WASTES POTENTIAL AS BIOCONTROL PRODUCTS : CHEMICAL CHARACTERIZATION AND EVALUATION ....................................... 39

BIODÉSINFECTION DES SOLS EN CULTURE DE TOMATE ........................................................................................................................................................................ 40

SCREENING STRATEGIES FOR DISCOVERY AND CHARACTERIZATION OF ANTIFUNGAL LIPOPEPTIDES USABLE AS BIOCONTROL AGENTS ........................................... 41

POTENTIALITIES OF ACTINOMYCETES FROM SOILS FOR THE DEVELOPMENT OF NOVEL BIOCONTROL PRODUCTS TO MANAGE CEREALS FUNGAL DISEASES

AND MYCOTOXINS RISK ........................................................................................................................................................................................................................ 42

OF THE INTEREST TO LISTEN AND DISRUPT THE DIALOGUE OF PATHOGENS: DETAILS OF SOFT-ROT BIOCONTROL BY THE RHODOCOCCAL QUORUM-QUENCHING 43

EVALUATION OF HIGHLY EFFICIENT LOCAL STRAINS OF 3 ENTOMOPATHOGENIC FUNGI SPECIES AGAINST 3 APHID PEST SPECIES .................................................... 44

STUDY OF RHIZOBACTERIA ISOLATED FROM ARGANIA SPINOZA L. OF TINDOUF REGION ................................................................................................................... 45

BACILLUS METHYLOTROPHICUS STRAIN B25, A NEW BACTERIAL BIOSTIMULANT MADE ITS WAY TO THE MARKET UNDER THE NAME HÉLÈS .................................. 46

DECRYPTAGE DES MECANISMES BIOLOGIQUES IMPLIQUES DANS LE BIOCONTROLE DES CHAMPIGNONS MYCOTOXINOGENES DES CEREALES .................................. 47

PSEUDOMONAS VRANOVENSIS COMME BACTERIE ANTAGONISTE : ANALYSE PAR TN-SEQ DE GENES ESSENTIELS A LA COLONISATION DE LA RHIZOSPHERE

DE POMME DE TERRE ........................................................................................................................................................................................................................... 48

CYCLIC LIPOPEPTIDE BIOSYNTHETIC GENES AND PRODUCTS AND ANTIFUNGAL ACTIVITY OF PLANT-ASSOCIATED BACTERIA ............................................................ 49

RELATIONSHIP BETWEEN THE NUMBER OF CO-OCCURRING PLANT PROTECTION PROPERTIES OF FLUORESCENT PSEUDOMONAS AND THEIR ABILITY

TO INHIBIT THE GROWTH OF PHYTOPATOGENIC MICROORGANISMS ................................................................................................................................................. 50

EVALUATION OF NEW BACTERIAL SPECIES AS POTENTIAL BIOSTIMULANTS FOR AGRICULTURE. ........................................................................................................ 51

LABORATORY EVALUATION OF ENTOMOPATHOGENIC NEMATODES TO CONTROL AGRIOTES WIREWORMS IN FRANCE ................................................................... 52

LA COLLECTION DE NEMATODES ENTOMOPATHOGENES DE L’INRA, RESSOURCES BIOLOGIQUES UTILISABLES COMME AGENTS DE BIO-CONTROLE ............................. 53

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SESSION 4: PLANT AND NATURAL PRODUCTS USED AS BIOCONTROL PRODUCTS .......................................................................... 54

LES ACIDES AMINES POUR LUTTER CONTRE LES ADVENTICES .............................................................................................................................................................. 55

EFFECTS OF 3 FORMULATIONS BASED ON OYSTER SHELL AND NEEM ON GROWTH AND RESISTANCE OF COCOA SEEDLINGS IN NURSERIES ........................................... 56

OXYLIPINS ARE INVOLVED IN PLANT PROTECTIONS PROCESSES AND ARE POTENTIAL BIOCONTROL AGENTS ........................................................................................ 57

ARE ESSENTIAL OILS EFFICIENT AS BIOPESTICIDES AGAINST SAP-SUCKING PESTS? A BIOASSAY SCREENING AT DIFFERENT SCALES .................................................. 58

POTENTIALITES BIOINSECTIDES DES EXTRAITS BRUTS DE DEUX PLANTES SAHARIENNES ENVERS LE PUCERON NOIR DE LA FEVE APHIS FABAE ................................. 59

ESSENTIAL OILS FROM THREE ALGERIAN SPECIES OF APIACEAE AS NEW BIO-HERBICIDE .................................................................................................................... 60

EVALUATION OF MICROALGAE EXTRACTS EFFICIENCY AGAINST FUSARIUM ROOT ROT IN WHEAT ..................................................................................................... 61

SCREENING FOR NATURAL UV ABSORBERS : RESIDUAL BIOLOGICAL ACTIVITY .................................................................................................................................... 62

AGRONOMY OF A SPANISH POPULATION OF SATUREJA MONTANA L. AND EESSENTIAL OIL/HYDROLATE VALORIZATION ................................................................. 63

HOW COULD THE HERBICIDAL EFFECTS OF SELECTED ESSENTIAL OIL COMPOUNDS BE RELATED TO THEIR MEMBRANE ACTIVITY ? ................................................. 64

ENCAPSULATION OF CYMBOPOGON WINTERIANUS JOWITT AND CINNAMOMUM VERUM J. PRESL ESSENTIAL OILS WITH GLYCERODENDRIMERS IN ORDER

TO CREATE A BIO HERBICIDE ................................................................................................................................................................................................................ 65

EFFET BIOCIDE DES HUILES ESSENTIELLES ET DE LA MENTHE POIVREE (MENTHA PEPERITA) ET DU PIN MARITIME (PINUS PINASTER) SUR LA LONGEVITE DES

ADULTES MALES DIAPAUSANTS DE LA BRUCHE DE LA FEVE (BRUCHUS RUFIMANUS) ......................................................................................................................... 66

STUDY OF THE LARVICIDAL ACTIVITY OF HYDRO-ALCOHOLIC EXTRACTS OF NERIUM OLEANDER L. AND RICINUS COMMUNIS L. ON TUTA ABSOLUTA (MEYRICK) .... 67

LES HUILES ESSENTIELLES COMME ALTERNATIVES DE CONTROLE D’INSECTES DE DENREES STOCKEES ET VECTEURS DE MALADIES AU BURKINA FASO ................... 68

BIOLOGICAL ACTIVITIES OF ESSENTIAL OIL AND HYDROSOL EXTRACT OF CENTAUREA ACAULIS L ....................................................................................................... 69

POTENTIAL OF PLANT EXTRACTS AS CONTROL TOOL FOR THE SOUTH AMERICAN TOMATO PINWORM (TUTA ABSOLUTA) ............................................................... 70

CHEMICAL CHARACTERIZATION OF THE NEMATICIDAL AGENTS FROM MENTHA SUAVEOLENS HYDROLATE. ..................................................................................... 71

DEVELOPPEMENT DE SOLUTION DE BIOCONTROLE « SUBSTANCE NATURELLE » ............................................................................................................................... 72

EFFECT OF DIFFERENT CONCENTRATIONS OF M2ITM PHEROMONE DISPENSERS AND THE IMPACT OF WATER AND PARAFFIN IN PHEROMONE TRAPS

FOR RHYNCHOPHORUS FERRUGINEUS (COLEOPTERA: CURCULIONIDAE) ............................................................................................................................................ 73

ETUDE IN VITRO DU POTENTIEL NEMATICIDE DE QUELQUES HUILES ESSENTIELLES ET L’EXTRAIT D’AIL SUR LES NEMATODES A GALLES

(MELOIDOGYNE INCOGNITA) ................................................................................................................................................................................................................ 74

CASTANEA AND SCHINOPSIS SP. TANNINS, FOR CROP PROTECTION .................................................................................................................................................... 75

EFFET DU POTENTIEL INSECTICIDE DE L’HUILE ESSENTIELLE DE THYM ET DE L’EXTRAIT DE L’AIL SUR LA MINEUSE DE LA TOMATE .................................................... 76

EFFICACITE D’UN INSECTICIDE A BASE DE NEEM DANS LA LUTTE CONTRE QUELQUES RAVAGEURS DU COTONNIER AU BENIN ......................................................... 77

USAGES DES PLANTES À EFFET PESTICIDE AU CAMEROUN ................................................................................................................................................................... 78

INSECTICIDAL AND ANTIMICROBIAL ACTIVITY OF THREE APIACEAE PLANTS ESSENTIAL OILS AGAINST MICROORGANISM PATHOGEN .............................................. 79

COMPARATIVE PHYTOCHEMICAL INVESTIGATION BETWEEN ESSENTIAL OILS AND HYDROSOL OF ALGERIAN JUGLANS REGIA.L LEAVES OBTAINED

USING TWO DIFFERENT EXTRACTION METHODS .................................................................................................................................................................................. 80

Β-AMINOBUTYRIC ACID (BABA) TREATMENT INDUCES A TEMPORAL DISRUPTION OF EXPANSIVE AND STRUCTURAL GROWTH ASSOCIATED WITH

A METABOLIC REORGANISATION IN FLAX (LINUM USITATISSIMUM) ................................................................................................................................................... 81

MICROALGAL COMPOUNDS FOR BIOCONTROL PRODUCT DEVELOPMENT: AN INNOVATIVE METABOLOMIC APPROACH BY MOLECULAR NETWORKING ................ 82

SESSION 5 : EVALUATION AND METHODS DEDICATED TO THE ANALYSIS OF BIOCONTROL NATURAL PRODUCTS AND OF THEIR

ENVIRONMENTAL IMPACT. ............................................................................................................................................................ 83

LATEST ADVANCES IN METABOLITE ANNOTATION FOR METABOLOMICS – MOLECULAR NETWORKING FOR DECIPHERING KEY DEFENCE INDUCTION PHENOMENA

.............................................................................................................................................................................................................................................................. 84

PLANT ROOT MODULATION OF PSEUDOMONAS SECONDARY METABOLISM INVOLVED IN BIOLOGICAL CONTROL OF PLANT DISEASES ........................................... 85

METABOLIC FOOT-PRINTING APPROACH TO ASSESS THE ENVIRONMENTAL FATE AND IMPACT OF BIOINSECTICIDES........................................................................ 86

INNOVATIVE BIOINFORMATICS TOOLS TO DECIPHER THE STRUCTURE OF NEW MICROBIAL LIPOPEPTIDES ........................................................................................ 87

« OLIGOGALACTURONOMICS » OF PLANT-FUNGUS INTERACTIONS .................................................................................................................................................... 88

ENVIRONMENTAL METABOLIC FOOTPRINTING (EMF) APPROACH TO STUDY THE QUALITY AND THE ENVIRONMENTAL IMPACT OF BIOCONTROL PRODUCTS ON

MEDITERRANEAN CROPS ...................................................................................................................................................................................................................... 89

INTRODUCING HIGH RESOLUTION MASS SPECTROMETRY IN THE ENVIRONMENTAL METABOLIC FOOTPRINTING METABOLOMICS APPROACH; APPLICATION ON

EXTRACTED NATURAL HERBICIDE “MYRIGALONE A” ............................................................................................................................................................................ 90

A NEW TOOL FOR ASSESSMENT OF BIOSTIMULANTS EFFECTS ON GRAPEVINE ................................................................................................................................... 92

RECHERCHE DE BIOMARQUEURS DE TOLERANCE A LA VERTICILLIOSE CHEZ LE LIN PAR UNE APPROCHE DE METABOLOMIQUE PAR RMN ....................................... 93

SCREENING STRATEGY TO SELECT NEW FUNGAL BIOCONTROL PRODUCTS AGAINST PLANT DISEASES ............................................................................................... 94

A FIELD SAMPLING KIT TO EVALUATE AND CONFIRM PLANT DEFENSE INDUCTION OF ELICITORS IN FIELD CONDITIONS ................................................................... 95

DEVELOPMENT OF LOCAL BIOCONTROL PRODUCTS ADAPTED TO MEDITERRANEAN CROPS .............................................................................................................. 96

"GRAPEVINE BIOCONTROL" PLATFORM (INRA / IFV - BORDEAUX) FOR EVALUATION OF BIOCONTROL PRODUCTS AND GRAPEVINE PROTECTION ........................... 97

UTILISATION DES MOYENS BIOLOGIQUES (BACTERIES LACTIQUES) POUR LA CONSERVATION DES ALIMENTS .................................................................................... 98

PRESENTATION OF THE FRENCH PUBLIC-PRIVATE CONSORTIUM FOR RESEARCH, DEVELOPMENT AND INNOVATION ON BIOCONTROL ........................................... 99

ANIMATION OF EXPERIMENTATION NETWORKS DEDICATED TO THE BIOCONTROL .......................................................................................................................... 100

EFFICACITE DU KAOLIN (KALABA) DANS LA GESTION INTEGREE DES CHENILLES ENDOCARPIQUES DU COTONNIER DANS LA ZONE CENTRE DU BENIN ................... 101

EVALUATION DE PRODUITS DE BIOCONTRÔLE POUR LUTTER CONTRE LA POURRITURE GRISE DE LA VIGNE .................................................................................... 102

USE OF MICROBIAL ANTAGONISTS OF THE CHESNUT ROT AND CANKER AGENT GNOMONIOPSIS SMITHOGILVYI TO REDUCE THE MORTALITY

OF CHESTNUT TREES IN NURSERY ....................................................................................................................................................................................................... 103

Natural Products & Biocontrol 2018

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Session 1:

Regulation and Integrated Pest management

Keynote speaker: Philippe Nicot


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PLANT RESISTANCE INDUCERS: A LONG BUT PROMISING WAY FROM THE RESEARCH LABORATORY TO INTEGRATED PROTECTION SCHEMES

IN THE FIELD

Philippe Nicot

Unité de Pathologie Végétale, INRA, 84140 Montfavet, France

Pioneering studies in the late 1800's and early 1900's already demonstrated the induction of resistance

to infectious agents in plants by abiotic stress and microorganisms. Starting in the 1990's, this topic

generated increasingly strong interest for its potential in crop protection and it developed into a very

dynamic field of research which produced more than 20000 scientific publications in the last 20 years.

A working group of the International Organisation for Biological Control (www.iobc-wprs.org) has been

fully dedicated to scientific exchanges on this topic since 1998.

Over the years, commercial preparations with plant defense-inducing properties have been developed

and they are increasingly registered as plant protection products in many parts of the world for use both

in organic and conventional agriculture. Plant defense inducers rarely provide 100% protection against

pests or infectious agents, and the consistency of their efficacy may be a challenge in field conditions.

However, plant defense inducers present unique advantages that make them particularly useful tools

for Integrated Pest Management. These features will be presented and remaining challenges for their

practical inclusion in integrated protection schemes will be evoked, highlighting needs for future

research.

Keywords: biocontrol, resistance induction, IPM

http://www.iobc-wprs.org/


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Oral communications - O1

SUBSTANCE ACTIVES DE BIOCONTROLE: EVOLUTIONS DEPUIS L’ENTREE EN VIGUEUR DU RCE 1107/2009/ BIOCONTROL AGENT

ACTIVE SUBSTANCES: EVOLUTION SINCE THE ENTRY IN VIGOUR OF REG. 1107/2009

Diane ROBIN, Patrice MARCHAND*

[email protected] | ITAB, 149 rue de BERCY, F-75595 PARIS CEDEX 12

Actuellement, le Biocontrôle est en forte progression avec un marché de 1,6 milliards d’euros dans le

monde et 550 millions d’euros en Europe. La croissance annuelle est estimée entre 15 et 20% et les

fabricants espèrent atteindre 15% du marché de la protection des plantes en France d’ici 2025.

Les Produits de Biocontrôle sont des agents utilisant des mécanismes naturels dans le cadre de la lutte

intégrée contre les ennemis des cultures. A l’exception des macroorganismes, les substances ou produits

comprenant des micro-organismes, des médiateurs chimiques (comme les phéromones et les

kairomones) et des substances naturelles (d'origine végétale, animale, microbienne ou minérale)

dépendent de la réglementation phytopharmaceutique sur la protection des cultures.

Dès l’entrée en vigueur du règlement d’exécution (UE) No 540/2011 (Partie A), les substances actives

contenues dans ces Produits de Biocontrôle ont été présentes. Leur nombre a évolué en fonction des

approbations et des renouvellements (Partie B) ainsi que des retraits et des non-approbations, mais a

également été impacté par l’arrivée de nouvelles substances :

Apport des substances de base, dont 17 ont été approuvées depuis 2014, qui sont compatibles

avec la définition des substances naturelles, des micro-organismes ou des médiateurs chimiques

de Biocontrôle et qui sont directement utilisables sans AMM (Partie C, Art. 23 du RCE 1107).

Apport des substances à faibles risques (Partie D, Art. 22 du RCE 1107), dont 12 ont été

approuvées depuis 2015 et qui sont entièrement reconnues comme substances actives de

Biocontrôle.

Des contributions négatives existent également, comme le retrait, le non renouvèlement ou la non-

approbation initiale de substances actives compatibles avec les catégories « phytosanitaires » de

Biocontrôle.

Nous avons quantifié l’évolution du Biocontrôle « phytosanitaire » au niveau des substances actives

depuis la mise en place de la nouvelle réglementation (règlement (CE) No 1107/2009 en 2011), en

partant des substances initiales et en comptabilisant annuellement les substances approuvées,

renouvelées, non renouvelées ou abandonnées par les pétitionnaires dans tous les registres :

médiateurs chimiques (phéromones, allomones, kairomones, attractants…), microorganismes, et

substances naturelles (d’origine végétale, animale, minérale ou microbienne). Nous avons tenté de

vérifier l’hypothèse que le Biocontrôle progresse globalement et quelle a été son évolution depuis la

mise en place du règlement 1107/2009. Mots-clés / Key words: biocontrôle – substance active – Règlement 1107/2009– Règlement 540/2011

mailto:[email protected]


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Oral communications – O2

RELEASE AND MONITORING OF TWO SPIDERS (CHEIRACANTHIUM MILDEI AND CLUBIONA SP.) IN AN APPLE ORCHARD AND ITS EFFECTS

ON CODLING MOTH ABUNDANCE

Sadeq Alzubik (1,2), Christophe Mazzia (2), Alain Pasquet (3), Yvan Capowiez (1)

(1) INRA, UR 1114, Domaine Saint Paul, 84914 Avignon cedex 09

(2) UAPV, UMR 7263, IMBE, 301 rue Baruch de Spinoza, BP 21239, 84916 Avignon cedex 9

(3) CNRS, UR AFPA, Université de Lorraine, BP 239, 54504 Vandoeuvre les Nancy

Some spiders such as Cheiracanthiium mildei or Clubiona sp. are known to be able to eat codling moth

eggs and larvae, the main pest in apple orchard. However, their abundance is very variable in orchards,

ranging from a few individuals per tree to several tens and their efficiency as biological agents remains

largely unknown.

To fill this gap in knowledge, we caught spiders in an organic orchard where their abundances was very

high (17 C. mildei and 12 Clubiona sp per tree) using cardboard traps where the spiders generally

hibernate. In December, the traps were transferred in an experimental and « spider-poor » orchard (less

than one C. mildei or Clubiona sp. per tree) with one trap every two trees.

The abundance of both released spider were then monitored using artificial transparent shelters located

in each apple tree during two years from April 2016 to December 2017 with one or two sampling per

month. Additionally we monitored codling moth abundances in June and October using cardboard traps

to determine whether the density of spiders could decrease the density of this pest on each apple tree.

The release of both spider was successful: the number of Cheiracanthium mildei and Clubiona sp.

pertress was increased by a factor 3 and 4. 5 respectively the year following the realease. However the

effects of the release was not significant two years after the release with abundance close to those

before the release for both species. Interestingly, the abundance of Cheiracanthium mildei was

negatively and significantly related to the number of codling moth larvae on apple trees the year after

the release. No such trend could be observed anymore two year after the release.

If the role of spiders in biocontrol has gained interest in the recent years, especially in orchards, this

study based on a release of targeted spiders is to our knowledge the first. We concluded that this kind

of approach, i.e. the transfer of natural enemies, sometimes applied for earwigs between peach and

apple orchards, is feasible at a limited cost and may increase the control of codling moth, albeit not

sufficiently regarding the thresholds generally used in orchards.

Keywords: biocontrol, habitat manipulation, generalist predator


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MILDIOU DE LA POMME DE TERRE : RECHERCHE ET MAITRISE DES PRODUITS DE BIOCONTROLE DANS UN SCHEMA DE PROTECTION

INTEGREE DES CULTURES (PROJET MILPOMBIO)

Antoine MENIL (1), Jean-Eric CHAUVIN (2), Roland PELLE (2), Marie BOUSSEAU (2), Denis GAUCHER (3), Guillaume BEAUVALLET (3), Claude MAUMENE (3), Didier ANDRIVON (4),

Claudine PASCO (4), Florence VAL (4), Arnaud BARBARY (5), Marie TURNER (1)

(4) Protection et Nutrition des Plantes, VEGENOV, SAINT-POL-DE-LEON (5) UMR IGEPP, INRA, PLOUDANIEL (6) SPIC, ARVALIS-Institut du végétal, BOIGNEVILLE (7) UMR IGEPP, INRA, LE RHEU (8) Bretagne Plants Innovation, HANVEC

Le mildiou (causé par Phytophthora infestans) est le principal bio-agresseur des cultures de pommes de

terre en France. Face à ce pathogène, la lutte chimique reste largement employée conduisant à un IFT

important sur cette culture (IFT fongicide > 10). En revanche, aucune solution de biocontrôle n’est à

l’heure actuelle disponible. Le projet MilPomBio financé par l’ONEMA (2015-2018) vise ainsi à identifier

des produits de biocontrôle efficaces contre le mildiou pour les proposer aux producteurs. Ces produits

seront accompagnés de leur mode d’utilisation et intégré dans des itinéraires techniques, par exemple,

en association avec d’autres produits phytosanitaires et/ou avec l’usage de variétés plus résistantes.

Cela permettra ainsi d’atteindre des niveaux de symptômes compatibles avec des rendements

satisfaisants et de répondre aux attentes de la société d’une agriculture plus économe en intrants et

plus respectueuse de l’environnement.

La première phase du projet MilPomBio a consisté à évaluer l’efficacité de protection d’une quarantaine

de produits de biocontrôle (extraits d’algues, extraits de plantes, microorganismes, éléments minéraux

et composés organiques) vis-à-vis du mildiou de la pomme de terre en conditions contrôlées. Une

quinzaine de produit se sont montrés efficaces pour faire diminuer les symptômes.

Les produits les plus intéressants ont été sélectionnés pour 1/ mieux comprendre leur mode d’action :

une étude de la stimulation des défenses des plantes et des tests d’effet direct ont été réalisés 2/ étudier

l’interaction avec la génétique et l’architecture de la plante aux champs 3/ évaluer l’efficacité de

protection aux champs en association avec une dose réduite de fongicide sur une variété. Plusieurs

produits contenant des phosphites ont montré une efficacité de protection intéressante en plein champ

contre le mildiou de la pomme de terre. L’étude de leur mode d’action a révélé une stimulation des

défenses des plantes et un faible effet in vitro sur la croissance mycélienne ou la germination des

sporanges.

La dernière étape du projet est en cours et consiste à évaluer, en conditions de production, plusieurs

stratégies de protection intégrée, associant variétés présentant des niveaux de résistance variables, OAD

Miléos, produit de biocontôle et fongicides susceptibles d’apporter un niveau de maladie acceptable. Ce

travail est réalisé sur deux sites différents en termes de conditions pédoclimatiques. Un produit est

également évalué sur une dizaine de variétés afin d’approfondir la question de l’interaction avec la

génétique de la plante. Des résultats seront disponibles en aout.

Mots-clés : MilPomBio, Phytophthora infestans, Pomme de terre, Biocontrôle, Résistance variétale


9

Poster communications – P1

MALADIE DES PROTEAGINEUX : VERS UNE REDUCTION DES INTRANTS ET UNE SECURISATION DES RENDEMENTS

(PROJET PROGRAILIVE DU PROGRAMME SOS-PROTEIN)

Antoine MENIL (1), Marie TURNER (1)

(9) Protection et Nutrition des Plantes, VEGENOV, SAINT-POL-DE-LEON

L'objectif du projet Prograilive du programme SOS-Protein est de sécuriser et d'augmenter la production

de protéagineux grains pour les élevages de l'ouest de la France (Bretagne et Pays de la Loire). Ce projet

de recherche et d'expérimentation est centré sur les attentes des agriculteurs du territoire. Les

protéagineux ciblés dans ce projet sont le pois, le lupin et la fèverole utilisés en grains pour augmenter

l'autonomie protéique des élevages de l'ouest de la France. Un observatoire et des travaux de

caractérisation, notamment en termes de diversité génétiques, des maladies ont été réalisés pour mieux

appréhender leur contrôle.

Afin d’envisager une réduction de l’utilisation d’intrants chimiques tout en maintenant une bonne

performance des cultures, des travaux d’étude de résistances variétales et/ou d’efficacités de produits

de biocontrôle ont été réalisés sur l’anthracnose et le botrytis du lupin, ainsi que la rouille de la fèverole.

Le niveau de résistance à la rouille (Uromyces fabae) a été étudié sur 10 variétés de féverole. Deux

variétés présentaient des sensibilités réduites, les autres présentaient une forte sensibilité. Le niveau de

résistance à l’anthracnose (Colletotrichum lupini) a été étudié sur 15 variétés de lupin. Trois variétés

présentaient des sensibilités réduites, les autres présentaient une forte sensibilité.

Concernant l’évaluation de produits de protection, 18 produits de biocontrôle ont été évalués en

conditions contrôlées. Ces produits ont été choisis pour la diversité de leur composition : extraits de

plantes, extraits d’algues, micro-organismes, minéraux, acides aminées et produits stimulant la défense

des plantes (SDP). Parmi les 18 produits testés, et dans nos conditions, 4 se sont révélés efficaces (à plus

de 50%) pour lutter contre le botrytis du lupin, 6 contre l’anthracnose du lupin et 9 contre la rouille de

la féverole. Les produits minéraux semblent les plus efficaces et polyvalent, un même produit s’est

révélé capable de contrôler plusieurs maladies.

Des essais sur le terrain sont en cours pour valider des itinéraires techniques intégrant ces résultats et

pouvoir ainsi proposer des solutions concrètes aux producteurs

Mots-clés : lupin, fèverole, Biocontrôle, Résistance variétale

Ce projet est co-financé par le fonds européen agricole pour le développement rural.

http://europe.bzh/jcms/wcrb_223387/fr/accueil

http://www.paysdelaloire.fr/politiques-regionales/europe/les-fonds-europeens-en-pays-de-la-loire-2014-2020/feader-en-pays-de-la-loire/


10


EVALUATION DE PRODUITS DE BIOCONTROLE POUR LUTTER CONTRE LA POURRITURE GRISE DE LA VIGNE

Aveline N. (1), Dupin S. (2), Calvo Garrido C. (3), Davidou L. (2), Roudet J. (3), Fermaud M. (3)

(1) Institut Français de la Vigne et du Vin– 39 rue Michel Montaigne 33290 Blanquefort- France (2) Chambre d’Agriculture de la Gironde- 39 rue Michel Montaigne 33290 Blanquefort- France (3) INRA – UMR SAVE, 71 avenue Edouard Bourlaux CS 20032, 33882 Villenave-d'Ornon cedex- France

La pourriture grise (B.cinerea) est une maladie de la vigne qui affecte la vendange de façon quantitative

et qualitative. La lutte fongicide classique utilise des traitements à stades préalablement fixés en saison

(floraison, fermeture de la grappe et/ou début véraison) pour limiter l’apparition des symptômes lors

des dernières semaines, voire jours avant récolte. L’efficacité de cette lutte n’est pas toujours optimale

et peut générer notamment des résidus phytosanitaires dans les vins. Depuis quelques années, le statut

« Biocontrôle » a fait émerger quelques produits de protection alternatifs avec un profil à faible risque,

homologués contre la pourriture grise. Ces produits présentent des origines et des modes d’action

divers : i) micro-organismes vivants (bactéries, champignons) agissant par compétition spatiale /

nutritive et/ou antibiose; ii) des produits d’origine naturelle à action physique (choc osmotique) ou

fongicide directe. Malgré ce panel disponible, des comparatifs d'efficacité et des exemples d’emploi

optimisés au vignoble sont peu répandus.

En Aquitaine, des projets collaboratifs menés de 2014 à 2017 ont fait intervenir différents

dispositifs (réseaux ou des plateformes d’essai) pour obtenir plus d’informations sur ces produits et

proposer aux viticulteurs des stratégies efficaces contre B.cinerea. Les résultats montrent que les

produits à base de micro-organismes vivants présentent des efficacités très variables et irrégulières

selon les millésimes et les sites d’essai. Pour les substances naturelles, l’Armicarb (Bicarbonate de

potassium) se détache comme le meilleur candidat, avec une attaque moindre comparée au témoin non

traité. Néanmoins les taux d’efficacité sont faibles à moyens (40-45% maximum).

Cependant le biocontrôle du Botrytis au vignoble pose de nombreuses questions, en lien notamment

avec son épidémiologie complexe et de nombreux facteurs influençant son développement en saison.

Un indice de développement du Botrytis (INRA) basé sur la climatologie à partir de la véraison a été

évalué pour positionner certains produits de biocontrôle. De plus, la nature même des produits de

biocontrôle testés et leur mode d’action restent trop mal connus. L’implantation de micro-organismes

au vignoble soulève également les problématiques de survie en lien aux conditions climatiques,

d’interaction avec les autres traitements de l’itinéraire et de cinétique de développement face à B.

cinerea.

Mots-clés : vigne, biocontrôle, pourriture grise, pilotage


11


INVESTING THE MODE OF ACTION OF RESISTANCE INDUCERS DURING PROTECTION OF WHEAT AGAINST POWDERY MILDEW

Alice Fourquez (1), Maryline Magnin-Robert (1), Béatrice Randoux (1), Ali Siah (2),

Patrice Halama (2), Philippe Reignault (1) et les membres du consortium Iris+

(4) Univ. Littoral Côte d’Opale, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV-EA 4492), SFR Condorcet FR CNRS 3417, CS 80699, F-62228, Calais cedex, France

(5) Equipe Biotechnologie et Gestion des Agents Pathogènes en agriculture, Laboratoire Charles Viollette, SFR Condorcet FR CNRS 3417, Institut Supérieur d’Agriculture, Université Lille Nord de France, 48 Bd Vauban, – F-59046 Lille cedex, France

In the context of the Ecophyto French National Action Plan, France is committed to dramatically reduce

by 2025 the use of pesticides on agricultural crops and to remove from the market the most hazardous

molecules, while maintaining a high production rate and good food quality. As an alternative to

fungicides, known to act directly on pathogens, an increasing number of elicitors, or Resistance Inducers

(RIs), are being developed to protect plants via the stimulation of plant immunity. The efficacy of RIs to

protect plants is now well established in controlled and greenhouse conditions, but may substantially

decrease when applied to crops in field conditions. In the IRIS+ project, we focus on one major crop of

great economic interest: bread wheat (Triticum aestivum L.) to screen and evaluate the impact of a set

of five new molecules considered as potential RIs against a foliar cryptogamic disease: powdery mildew

(PM) caused by the biotrophic agent Blumeria graminis f sp. tritici.

Among the five RIs tested to protect wheat against PM, SDN2, SDN3 and SDN5 were efficient, with

protective effects of 20-30%, 20-40% and 65% respectively. The direct activity of RIs on the fungus was

also tested in vitro: SDN2 weakly inhibited B. graminis spores germination whereas SDN5 totally

inhibited it and SDN2 has no effect. The defense marker peroxidase activity was also measured: SDN2,

SDN3 and SDN5 increase the enzyme activity in non-inoculated and inoculated conditions compared to

the control, but SDN5 at higher level.

We also aim at determining how SDN3 and SDN5, which are the two most efficient treatments to protect

wheat, affect the content of secondary metabolites in leaf tissues under non-inoculated and inoculated

conditions using a global method of metabolomics based on Ultra-High Performance Liquid

Chromatography (UHPLC).

Keywords: Bread wheat, Powdery mildew, Resistance inducer, Protection


12

Session 2 :

Elicitors of plant defenses and plant defenses

Keynote speaker: Dr Uwe Conrath


13

PRIMING PLANTS FOR ENHANCED DEFENSE

Uwe Conrath

Department of Plant Physiology, RWTH Aachen University, Aachen, NRW 52056, Germany

When locally infected by pathogens, plants activate a systemic immune response, called systemic

acquired resistance (SAR). In this process, distal leaves become primed to activate a more robust defense

response upon further infection.

Defense priming is associated with an elevated level of receptors for microbial patterns (e.g. flagellin-

sensing [FLS] 2), accumulation of dormant signaling enzymes (e.g. mitogen-activated protein kinases

[MPKs] 3 and 6), and with modification to chromatin. The latter comprises the covalent modification of

histones (e.g. histone H3 and H4 acetylation and/or methylation) and the formation of open chromatin

indicative of regulatory DNA sites with a role in defense priming.

Together, these events provide a memory to the initial infection and enable the boosted recall defense

response of plants. I will disclose the impact of these fascinating discoveries on sustainable agriculture

by introducing smart tools and approaches for the identification of priming-inducing chemistry. I will

also elaborate on the so-called Green Release technology that allows the controlled release of

agrochemicals from plant surface-binding micro-gels, thus allowing the smart management of plant

performance.

References: Schillheim et al. (2018) Plant Physiol. 176:2395-2405; Martinez-Medina et al. (2016) Trends Plant Sci.

21:818-822; Conrath and Reimer-Michalski (2016) Sem. Immunol. 28:319-327; Conrath et al. (2015) Annu. Rev.

Phytopathol. 53:97-119; Schilling et al. (2015) BMC Plant Biol. 15:282; Jaskiewicz et al. (2011) EMBO rep. 12:50-55;

Conrath (2011) Trends Plant Sci. 16:524-531; Beckers et al. (2009) Plant Cell 21:944-953; Beckers and Conrath

(2007) Curr. Opin. Plant Biol. 10:425-431; Conrath et al. (2006) Molec. Plant-Microbe Interact. 19:1062-1071;

Conrath et al. (2002) Trends Plant Sci. 7:210-216.


14


STIMULATION OF PLANT IMMUNITY BY BENEFICIAL BACILLI : MECHANISTICS OF SURFACTIN PERCEPTION

Magali Deleu (1), Laurence Lins (1), Marc Ongena (2)

(6) Laboratory of Molecular Biophysics at Interfaces, Gembloux Agro-Bio Tech, University of Liege (7) Microbial Processes and Interactions Research Unit, Gembloux Agro-Bio Tech, University of Liege

Some strains of the plant-associated Bacillus velezensis (formerly B. amyloliquefaciens subsp. plantarum

clade) are of particular interest regarding their potential to combat a range of phytopathogenic microbes

for the sake of crop health.

Like other groups of the Bacillales, these bacteria are excellent producers of a wide variety of

antimicrobials either ribosomally synthesized such as bacteriocins and lantibiotics or enzymatically

formed via multi-modular mega-enzymes like polyketides (PKs) and non-ribosomally synthesized

peptides (NRPs). Among these last compounds, cyclic lipopeptides (CLPs) are of particular interest since

they retain crucial functions for biocontrol activity of the producing strains i.e. rhizosphere fitness, direct

antagonism of pathogens and host resistance stimulation. In this last context, the CLP surfactin (SF) has

been identified as main Bacillus product triggering the so-called Induced Systemic Resistance (ISR), a

layered innate immunity process that first involves the perception of such elicitors referred as Microbe-

Associated Molecular Patterns (MAMPs) at the plant cell membrane level.

MAMPs from pathogens are typically perceived by dedicated pattern recognition receptors (PRRs)

functioning as Receptor-Like Kinases. By contrast, the mechanistics underlying perception of ISR elicitors

typically secreted by beneficial bacteria still remain much more obscure.

In this work, we combined functional and biochemical assays together with in silico and experimental

biophysics to deeply investigate the molecular mechanisms of surfactin perception by tobacco cells. Our

results indicate that SF recognition does obviously not involve dedicated PRRs but primarily rely on an

unsuspected process at the lipid phase of root cell plasma membrane. We hypothesize that SF insertion

into or at the border of specific nanodomains induces some re-organization of this lipid bilayer, which

could in turn affect the localization/recruitment, and thereby activate key proteins involved in early

signaling processes. Such a peculiar PRR-independent mechanism of recognition may explain why this

CLP acts as a real priming agent of plant immunity with no impact on fitness upon elicitation but with

clear stimulation of antifungal phytochemicals upon infection.

Key words: B. velezensis - lipopeptide – priming - lipid bilayer – proteomics - lipid binding – lipid specificity -

molecular interactions


15


STUDY OF A STREPTOMYCES SP. DERIVED SIGNALS AND THEIR PLANT PERCEPTION MECHANISMS

Damien Gayrard (1) (2), Thomas Rey (1), Bernard Dumas (2)

(8) De Sangosse, Bonnel, 47480 Pont-Du-Casse, France (9) Laboratoire de Recherche en Sciences Végétales, UMR 5546 CNRS- Université Paul Sabatier, Pôle de Biotechnologie

Végétale, Castanet Tolosan, FRANCE

A Streptomyces strain inducing plant defence responses against foliar pathogens was characterized.

Multi-loci Sequence Typing and morphological characteristics identified this strain, as a novel

Streptomyces strain belonging to the Streptomyces violaceusniger subclade of the S. hygroscopicus

clade.

Arabidopsis thaliana plants treated with the Streptomyces strain cell suspensions or culture filtrates

showed intense defence responses resulting in significant reduction of symptoms when plants were

challenged with fungal pathogens. Microscopic examination of treated plant tissues revealed that

Streptomyces strain mycelia efficiently colonized leaf surface leading to the production of spores. The

effect of foliar spray of Streptomyces strain or culture filtrate on the plant transcriptome was examined

by RNA-Seq. Multiple markers of salicylic acid and jasmonate/ethylene pathways were regulated by the

treatment. Partial protection of immune-deficient Arabidopsis mutants inoculated A.brassicicola

conidies suggests that induction of defense responses is required to obtain high protection levels.

Together, this work shows that Streptomyces bacteria produce compounds displaying strong elicitor

activity and could be used as foliar treatment to efficiently protect plants against pathogen attacks.

Mots-clés / Key words: Streptomyces – plant defence stimulation – transcriptomics


16


NITROGEN FERTILIZATION: A DETERMINING FACTOR FOR EFFICIENCY OF PLANT DEFENSE ELICITORS ?

Camille Verly (1,2), Alia Dellagi (1), Frédéric Giraud (2), Loïc Rajjou (1), Martine Rigault (1),

Chloe Vanniere (4), Marie Emmanuelle Saint Macary (4)

(10) Institut Jean-Pierre Bourgin (IJPB), UMR 1318 INRA-AgroParisTech, ERL CNRS 3559 - Université Paris-Saclay, LabEx SPS, RD10, F-78026 Versailles,

(11) Staphyt-Service L&G / BIOTEAM, Site de Montesquieu, Av François Magendie, 33650 Martillac, France (12) Staphyt - BIOTEAM, La Paluzette, Route des Mas 34590 Marsillargues, France

In the context of crop protection, the development of elicitors is a promising strategy to reduce the use

of conventional products. However, the effectiveness of elicitors in the field remains uncertain and

depends on different agronomic parameters, such as the cultivated variety, the agricultural practices

and the climate conditions of production. Among these factors, nitrogen (N) nutrition has been shown

to play an important role in establishing defenses after infection by a pathogen. What about the impact

of N fertilization on the efficiency of elicitors?

Should this parameter be included in the strategy for using and optimizing the effectiveness of these

elicitors?

In order to address these issues, 2 complementary approaches have been considered: a classic one in

the laboratory and another in field conditions.

In the laboratory, the model plant Arabidopsis thaliana was chosen. Plants were grown in soil under N

deficiency (2 mM of nitrate), optimal N nutritional status (10 mM of nitrate) or N over-fertilization (20

mM of nitrate). They were then treated with elicitors activating either the salicylic acid- dependent

pathway or the jasmonic acid-dependent pathway in order to quantify the impact of nitrogen

fertilization level on the activation of the two major defense systems described in plants. Activation of

defenses in response to elicitor treatments on A. thaliana was measured by both quantifying the

expression of marker genes specific to each of the 2 defense pathways and the level of protection against

a pathogenic bacterium, Dickeya dadantii.

In parallel, field trials were also undertaken on wheat and grapevine. In the same way as in the

laboratory experiments, 3 levels of nitrogen fertilization were applied (deficiency, optimal and over-

fertilization) depending on the target crop and the production system (agro- climatic conditions). The

effectiveness of elicitors in the field was assessed by following-up of diseases and the quantification of

symptoms over time (frequency and intensity).

Our first results have already demonstrated the importance of controlling the level of fertilization in the

context of the use of elicitors, both at the molecular level by efficiency of the induction of defense genes

and at the level of effective protection against a pathogen. All these results will be presented during this

seminar. Keywords: SDP - nitrogen - fertilization - plant protection - systemic defenses


17


PHAEODACTYLUM TRICORNUTUM : NEW SOURCE OF ELICITING MOLECULES FOR PLANT DEFENSE AND HEALTH

Coralie Chuberre (1), Muriel Bardor (1,2), Driouich Azzedine (1),

Marie-Christine Kiefer-Meyer (1), Bruno Gügi (1)*, Maité Vicré (1)*

(13) Université de Rouen Normandie, Laboratoire Glyco-MEV, EA4358Centre Universitaire de Recherche et d’Innovation

en Biologie (CURIB), 25 rue Lucien Tesnière,76821 Mont-Saint Aignan Cedex (14) Institut Universitaire de France (I.U.F.) 1, rue Descartes, 75231 Paris Cedex 05, France

*Equal contribution of the last authors

Recently, the use of effective and eco-friendly substitutes to traditional agrochemicals has aroused great

interest in agriculture. The development of these new products is based on the characterization of

eliciting molecules, also called elicitors that mimic pathogen aggression to trigger plant defense.

Different storage and parietal polysaccharides such as β-glucan, chitin and chitosan oligomers,

oligogalacturonides have been reported to induce plant immunity1. Among them, the laminarin which

is extracted from the brown macroalga Laminaria digitata is a famous elicitor of plant defense2. Defense

reactions induced by laminarin include cell wall reinforcement, specific phytoalexin/enzymes

production2,3 and a reduction of disease infection against botrytis cinerea and plasmopara viticola3.

However, macroalgae culture requires large natural culture areas and take a long time to develop. In

contrast, microalgae grow fast and in control restricted areas. Furthermore, they represent a highly

diverse group with a wide range of metabolites which represent potential source of eliciting molecules.

Thus, we decided to investigate the diatom Phaeodactylum tricornutum, as a potential source of plant

defense elicitor.

This diatom had the peculiarity to be pleiomorphic according to the environmental conditions. In this

study, our aim is to evaluate the protective activities of the three P. tricornutum morphotypes on

Arabidopsis thaliana roots after 7 days of growth. Our data shown that a crude extract, from one

morphotype, induces parietal remodelling and defense polysaccharide deposition in roots, some well-

known markers of plant defense4,5. Activation of plant defense pathways is being further explored by

cellular and molecular investigations.

Key words: Phaeodactylum tricornutum, eliciting molecules, plant immunity, microalgae

Regional funding from the GRR VASI and NORVEG

Citations

1. Trouvelot, S. et al. Carbohydrates in plant immunity and plant protection: roles and potential application as foliar

sprays. Frontiers in Plant Science 5, (2014).

2. Klarzynski, O. et al. Linear beta-1,3 glucans are elicitors of defense responses in tobacco. Plant Physiol. 124, 1027–

1038 (2000).

3. Aziz, A. et al. Laminarin elicits defense responses in grapevine and induces protection against botrytis cinerea and

plasmopara viticola. Molecular Plant-Microbe Interactions 16, 1118–1128 (2003).

4. Millet, Y. A. et al. Innate Immune Responses Activated in Arabidopsis Roots by Microbe-Associated Molecular

Patterns. The Plant Cell 22, 973–990 (2010).

5. Plancot, B. et al. Deciphering the Responses of Root Border-Like Cells of Arabidopsis and Flax to Pathogen-Derived

Elicitors. PLANT PHYSIOLOGY 163, 1584–1597 (2013).


18


MECHANISMS UNDERLYING ASM-INDUCED RESISTANCE TO THE APPLE ROSY APHID, DYSAPHIS PLANTAGINEA

Romain Warneys (1), Sylvia Anton (2), Christophe Gadenne (2), Matthieu Gaucher (1),

Christelle Heintz (1), Sébastien Aubourg (1), Nicolas Barthes (3), Marie Noëlle Brisset (1) and Alexandre Degrave (1)

(15) Institut de Recherche en Horticulture et Semences, IRHS, UMR1345 (INRA / Agrocampus-ouest / Université d'Angers) Centre Angers-Nantes, 42 rue Georges Morel – CS 60057 ; 49071 Beaucouzé cedex - France

(16) Institut de Génétique, Environnement et Protection des Plantes, IGEPP, UMR 1349 (INRA/Agrocampus Ouest/Université Rennes), 2, rue Le Nôtre, 49045 Angers cedex 01 - France

(17) Centre d’Ecologie Fonctionnelle & Evolutive, CEFE, UMR 5175 (Centre National Recherche Scientifique / Université Montpellier / Université Paul Valery Montpellier 3 / Ecole Pratique des Hautes Etudes / Institut Recherche pour le Développement), 1919, route de Mende, 34293 Montpellier cedex 05 - France

Apple, one of the most heavily treated culture, can be protected against various pest and disease when

previously sprayed with a salicylate analog (acibenzolar-S-methyl – ASM). However, the mechanisms

underlying ASM-induced resistance remain elusive, especially in apple. The project presented here

aimed at characterizing the molecular and biochemical events activated in ASM-treated apple plants

leading to resistance against the apple rosy aphid (Dysaphis plantaginea).

By analyzing the differences in fitness and behavior of aphids between mock and ASM plants, we

identified that ASM was acting by antibiosis and antixenosis. We undertook the characterization of these

mechanisms by performing first a transcriptomic study, complemented by proteic and metabolic

analysis. This strategy allowed us to identify putative factors and molecular processes leading to

enhanced resistance upon ASM treatment. We undertook functional validation assays in order to show

that the antibiosis effect is due to the strong production of a specific lectin with putative toxical

properties, and that the antixenosis effect relies on the release of repellent sesquiterpenes.

Finally, we will discuss the possible development or enhancement of existing protection strategies,

illustrating how basic research can pave the way for environmental friendly practices.

Keywords: apple - plant resistance inducers – Dysaphis plantaginea – lectin - sesquiterpene


19


BRASSICA NAPUS DEFENSE RESPONSES TRIGGERED BY BACTERIAL RHAMNOLIPIDS - FROM PERCEPTION TO EFFECTIVE PROTECTION

Noadya Monnier (1) (2), Aurélien Furlan (1), Sébastien Buchoux (1), Stéphanie Guénin (3),

Gaëlle Mongelard (3), Sylvain Cordelier (4), Stéphan Dorey (4), Christophe Clément (4), Sonia Rippa (2), Catherine Sarazin (1)

(18) Université de Picardie Jules Verne, UFR des Sciences, Unité de Génie Enzymatique et Cellulaire- FRE 7025 CNRS 33 rue Saint-Leu, 80039 Amiens Cedex 1, France

(19) Université Technologique de Compiègne, Unité de Génie Enzymatique et Cellulaire - FRE 7025 CNRS - Rue Roger Couttolenc CS60319, 60203 Compiègne Cedex, France

(20) Université de Picardie Jules Verne, UFR des Sciences, Centre Régional de Ressources en Biologie Moléculaire - 33 rue Saint-Leu, 80039 Amiens Cedex 1, France

(21) Université de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Resistance Induite et Bioprotection des plantes - EA 4707, Laboratoire de Stress, Défenses et Reproduction des Plantes, Moulin de la Housse - Bâtiment 18, BP 1039, 51687 REIMS Cedex 2, France

Pseudomonas aeruginosa rhamnolipids are glycolipids with both antimicrobial and eliciting activities.

They trigger defense responses in grapevine and Arabidopsis thaliana but their mode of perception and

their efficiency toward crops are still unknown.

The potential use of rhamnolipids to protect the agronomical plant Brassica napus against the

opportunistic pathogenic fungus Botrytis cinerea was studied. The direct impact of rhamnolipids on

spore germination and mycelium growth was characterized. Independently, the response of B. napus to

rhamnolipids was investigated, following canonical defense markers of physical and chemical defenses.

The effects of rhamnolipids on plant physiology were also measured. The obtained results were

considered in the light of data from a transcriptomic study on the model brassicaceae A. thaliana treated

with rhamnolipids.

As amphiphilic compounds, rhamnolipids have been proposed to interact directly with plasma

membrane lipids. We explored the possibility of an elicitation triggered by a direct interaction of

rhamnolipids with lipid membranes. A biophysical approach combining solid state NMR and molecular

dynamic simulations allowed to localized rhamnolipids and their dynamic within plant plasma lipid

membrane models.

We gratefully acknowledge the financial support of the Hauts de France region council and the European

Union (ERDF). We also thank the SFR Condorcet FR 3417 CNRS.

Keywords: rhamnolipids, Brassicaceae, rapeseed, MAMP, transcriptomics, biophysics, NMR, molecular dynamics


20


EVALUATION DES PROPRIETES DE STIMULATION DES DEFENSES DES PLANTES D’UN EXTRAIT NATUREL DE CHAMPIGNON DE CULTURE

Koçi Rromir (1), Raynaud Anaïs (2), Célérier Julien(2), Moine Charlotte (2),

Gloaguen Vincent (1), Faugeron-Girard Céline (1)

(1) Laboratoire Peirene (EA 7500), Université de Limoges, Faculté des Sciences et Techniques, 123 avenue Albert Thomas, 87060 Limoges cedex

(2) COVERTIS, Ester Technopole, 1 avenue d'Ester, 87069 Limoges Cedex

Parmi les produits de biocontrôle, figurent les éliciteurs définis comme des molécules capables de

stimuler les réactions de défense des plantes, ceci indépendamment de l’attaque par un pathogène. Ces

molécules permettent ainsi de mettre la plante « en alerte » afin qu’elle résiste mieux aux éventuelles

agressions ultérieures qu’elle pourrait subir. Ainsi, la société COVERTIS a développé un extrait de

champignon de culture (COV 17-01) dont les propriétés élicitrices ont été évaluées en conditions

contrôlées.

Des essais ont été menés sur des cultures de tomate réalisées sous serre. Le produit COV 17-01 est

appliqué sous forme de pulvérisation foliaire. Puis, les plantes ont été inoculées par un champignon

pathogène (Botrytis cinerea). Après récolte, les plantes ont été analysées pour quantifier d’une part

certains marqueurs des réactions de défense, notamment d’enzymes, telles que la PAL (Phénylalanine

Ammonia Lyase) ou la peroxydase, ainsi que les teneurs en callose ou en composés phénoliques. D’autre

part, l’expression des gènes codant pour des PR protéines (Pathogenesis Related proteins) a été évaluée

via l’outil qPFD® développé par Vegepolys Innovation.

Plusieurs marqueurs des réactions de défense présentent des activités supérieures chez les plantes

ayant subi le pré-traitement avec le COV 17-01 comparé aux plantes témoins sur lesquelles a été

appliqué un blanc de formulation. Cette élicitation a été confirmée par des analyses moléculaires

puisque plusieurs gènes codant pour des PR protéines sont exprimés à un niveau plus élevé chez les

plantes traitées par COV17-01 comparé au témoin, notamment PR1 et PR2 et, en moindre mesure, la

peroxydase.

Des prélèvements sur des plantes issues d’essais plein champ seront prochainement menés pour

confirmer l’activité SDN du produit COV 17-01 sur différents modèles de plantes cultivées. Il s’agira alors

de confronter l’efficacité du produit COV 17-01, déterminée à partir de notations réalisées sur le terrain,

des données issues des analyses moléculaires faites sur ces prélèvements pour proposer des hypothèses

sur le mécanisme d’action du produit.

Mots-clés : stimulateur des défenses - éliciteur - extrait naturel - PR protéines


21


ALKYL GALLATES INDUCE PLANT DEFENCE REACTIONS ON TOBACCO

Goupil Pascale (1,2), Benouaret Razik (1,2), Galien Antonin (1,2), Richard Claire (3,4)

(1) Université Clermont Auvergne, UMR 547-UBP/INRA PIAF, Campus universitaire des Cézeaux, 8 avenue Blaise Pascal, 63178 Aubière cedex, France,

(2) INRA, UMR 547 PIAF, Clermont-Ferrand, France (3) Université Clermont Auvergne, CNRS, UMR 6296, Campus universitaire des Cézeaux, Institut de Chimie de Clermont-

Ferrand (ICCF), 24 avenue Blaise Pascal, 63178 Aubière cedex, France (4) Equipe Photochimie CNRS, UMR 6296, ICCF, 63178 Aubière, France

In the context of sustainable development, agriculture is incorporating more eco-friendly plant

technology and limiting the use of chemical pesticides. Increasing plants natural resistance is one

favoured line of research, notably using biological substances that can stimulate defence mechanisms

in plants. Plants synthesise a large range of phenolics, known to be an integral part of their non-host

resistance strategy as efficient chemical barriers to pathogens. The elicitation properties of alkyl gallates

are newly investigated.

The elicitor activities of alkyl gallates were measured on mature tobacco plants (Nicotiana tabacum L.)

in controlled conditions. Tobacco cell suspensions of Nicotiana tabacum L. Bright Yellow (BY-2) were

used to assess early defence reactions. Alkyl gallates showed elicitor activities on both tobacco whole

plants and cell suspensions. Methyl gallate (MG), ethyl gallate (EG) and n-propyl gallate (PG) infiltration

into tobacco leaves induced hypersensitive reaction-like lesions and topical production of

autofluorescent compounds revealed under UV light. When sprayed on tobacco plants at 5 mM, EG

promoted upregulation of defence-related genes such as the antimicrobial PR1, b-1,3-glucanase PR2,

chitinase PR3 and osmotin PR5 target genes. Tobacco BY-2 cells challenged with EG underwent cell death

in 48 h, significantly reduced in the presence of the protease inhibitor aprotinin. The three alkyl gallates

all caused alkalinisation of the BY-2 extracellular medium, whereas gallic acid did not trigger any pH

variation. Using EGTA or LaCl3, we showed that Ca2+ mobilisation occurred in BY-2 cells elicited with EG.

Our findings evidence the alkyl gallate elicitor properties with early perception events on plasma

membrane, potential hypersensitive reactions and PR-related downstream defence responses in

tobacco.

The research addresses the question of the potential use of naturally-occurring elicitors for plant

defence against plant pathogens, a challenging issue in sustainable agriculture and green environmental

approaches.

Keywords: Alkyl gallates, BY-2, Elicitors, Ethyl gallate, Pathogenesis-related protein, Plant defence reactions, Tobacco


22


THE KEY ROLE OF CHITOOLIGOSACCHARIDES IN PLANT-MICROBE INTERACTIONS

Laurent KAMEL (1), Etienne DARINOT (1), Adeline BASCAULES (1),

Virginie PUECH-PAGES (1), Fabienne MAILLET (2), Sylvain COTTAZ (3), Sébastien FORT (3), Sandra BENSMIHEN (2), Christophe JACQUET (1), Clare GOUGH (2) et Guillaume BECARD (1)

(1) Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 31326 Castanet Tolosan, France (2) Laboratoire des Interactions Plantes Micro-organismes, Université de Toulouse, INRA, 31326 Castanet Tolosan, France (3) Centre de recherches sur les macromolécules végétales, CNRS, 38041 Grenoble

In the rhizosphere, plant roots must continuously interact with a multitude of microorganisms.

Plants have developed intricate mechanisms based of the recognition of some signals released by

both beneficial and pathogenic microorganisms, so they can activate appropriate responses leading to

symbiotic relationship and defense, respectively. These signals include chitin- related molecules already

used in sustainable agriculture to enhance plant nutrition and growth, or to stimulate plant defense.

Here, we focus on two types of molecules produced by microorganisms interacting with plants:

Chitooligosaccharides (COs) and Lipochitooligosaccharides (LCOs).

LCOs were already described as symbiotic signals produced by the rhizobia (Nod factors) to activate the

nodulation process in leguminous plants, and by the arbuscular mycorrhizal (AM) fungi to promote

mycorrhization1,2. In the rhizobium-legume interaction, they also participate in the mechanism of host

specificity3. It was recently discovered that short-chain COs (consisting of 3 to 5 N-acetyl-glucosamine

residues, CO3-5), produced by AM fungi, can also be important for AM symbiosis, while long-chain of

COs (CO6-8) have been originally described as elicitors of plant defenses4,5. All these described

molecules are active at extremely low concentrations, down to the nano- or even to the picomolar

range.

The aim of this study is to investigate how plants can discriminate and integrate simultaneous

stimulation by these different molecules. For this, we study with the legume model plant Medicago

truncatula the impact of each of these closely-related molecules on the regulation of important genes

involved either in defense or in symbiosis. Based on the obtained results, we will test various

combinations of chitin-related molecules to try enhancing plant resistance against pathogens while

favoring symbiotic associations.

Keywords: Lipochitooligosaccharides, Chitooligosaccharides, Symbiosis, Defense, Medicago truncatula, Fluidigm

References

1. Lerouge, P. et al. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344, 781–784 (1990).

2. Maillet, F. et al. Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza. Nature 469, 58–63 (2011).

3. Wang, D., Yang, S., Tang, F. & Zhu, H. Symbiosis specificity in the legume - rhizobial mutualism. Cell. Microbiol. 14, 334–342 (2012).

4. Genre, A. et al. Short-chain chitin oligomers from arbuscular mycorrhizal fungi trigger nuclear Ca2+ spiking in Medicago truncatula roots and their production is enhanced by strigolactone. New Phytol. 198, 179–189 (2013).

5. Sanchez-Vallet, A., Mesters, J. R. & Thomma, B. P. H. J. The battle for chitin recognition in plant-microbe interactions. FEMS Microbiol. Rev. 39, 171–183 (2015).


23


RNA-SEQ ANALYSIS OF PLANT INNATE IMMUNITY AFTER ELICITATION WITH COS-OGA

Moreau Sophie (1), Van Aubel Géraldine (2), Janky Rekin’s (3)

and Van Cutsem Pierre (1)

(1) University of Namur, 61 rue de Bruxelles, 5000 Namur - Belgium (2) Fytofend SA Rue Phocas Lejeune, 25-6 B-5032 Isnes - Belgium (3) VIB Nucleomics Core, Campus Gasthuisberg Herestraat 49, 3000 Leuven - Belgium

This study focuses on an elicitor called FytoSave® whose physicochemical characteristics mimic the

natural plant-pathogen interactions. The so-called “COS-OGA” active substance of this product

combines plant non self-molecules, i.e. chitooligosaccharides (COS), and plant self-compounds called

oligogalacturonides (OGA). This elicitor is used as a preventive treatment which leads to a strengthening

of the plant natural physical and chemical barriers before the arrival of the threat.

The aim of this study is to deeper investigate the mode of action and the signaling pathways triggered

by COS-OGA. A better comprehension of its mode of action could improve field application strategies.

In this view, transcriptomic-based approaches provide a powerful tool to address the questions of how

FytoSave® acts on the plant defensive mechanisms. More precisely, a RNA-Seq analysis was performed

to bring to light and analyze the large changes undergone by tomato plant (Solanum lycopersicum) after

three applications of the elicitor.

Mots-clés / Key words: COS-OGA – Elicitation – RNA-Seq – Mode of Action


24

Session 3:

Biocontrol microorganisms and macroorganisms and

their mechanisms of actions

Keynote speakers: Pr Christoph Keel

and Pr Emilio Montesinos


25

PATHOGENICITY OF RHIZOSPHERE PSEUDOMONADS TOWARDS PLANT PEST INSECTS

Keel Christoph

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

Root-associated bacteria of the Pseudomonas protegens and Pseudomonas chlororaphis groups are well

known for their plant-beneficial activities, which include the suppression of plant pathogenic fungi and

protists, the promotion of plant growth and nutrition, and priming plants for enhanced defense. We

discovered that these bacteria, in addition, can exhibit potent insecticidal activities towards plant-

feeding larvae of certain Lepidopteran and Dipteran pest insects. Following their ingestion by the larvae,

the insecticidal pseudomonads colonize the gut, breach the intestinal barrier, invade the hemocoel,

proliferate and ultimately kill the insects. Some of the interactions of the pseudomonads with the pest

insects are less virulent, yet still result in morphological defects, others cause no apparent damage.

Recent experiments indicate that the pseudomonads can persist inside several pest insect species

throughout different life stages and can use adult stages to travel to new host plants. Depending on the

nature, the fitness and the development stage of the insect, these bacteria appear to exploit the insect

they infect either as food source or as vector for dispersal.

The arsenal of insect pathogenicity determinants of P. protegens and P. chlororaphis includes a large

insecticidal protein, which we termed the Fit toxin. The bacteria activate the costly production of the

insecticidal toxin only in the insect but not on plant roots and a sophisticated regulatory system

guarantees tight host-dependent control. Several lytic and toxic extracellular products including a

chitinase, a phospholipase, cyclic lipopeptides and hydrogen cyanide further support the insecticidal

activity of the pseudomonads. Specific cell surface constituents, notably the lipopolysaccharide O-

antigen and probably certain exopolysaccharides, protect the bacteria from the immune defenses of the

insect, in particular from antimicrobial peptides. Insect pathogenic function of these bacteria requires

efficient colonization of the insect gut that is densely populated by bacteria. We obtained evidence that

in P. protegens the type VI secretion system, a contractile phage tail-like apparatus that functions like a

deadly syringe to inject toxic and lytic effector proteins into attacked cells, contributes to colonization

of the insect gut, invasion of the gut microbiome, competition with dominant microbiome members,

and consequently to insect pathogenicity.

The in-depth understanding of the molecular basis of the insect pathogenic activities of these fascinating

bacteria and of their role in the microbial ecology of plants and insects are a prerequisite to generate

knowledge for the potential future implementation of such bacteria in agricultural pest management

strategies. Within the Swiss National Research Program 68 on the sustainable use of soil, we investigated

the potential of the entomopathogenic pseudomonads as biocontrol agents of soil-dwelling insect pests

in Swiss and U.S. greenhouse and field experiments. Targeted pests included the cabbage root fly Delia

radicum on vegetables and the corn rootworm Diabrotica virgifera.

Key words: Pseudomonas – rhizosphere – insecticidal – plant pest insects – insect pathogenicity determinants


26

ANTIMICROBIAL PEPTIDES IN BIOCONTROL MICROORGANISMS.

POTENTIAL AND LIMITATIONS AS BIOPESTICIDES FOR PLANT DISEASE CONTROL

Emilio Montesinos

Institute of Food and Agricultural Technology-CIDSAV- University of Girona (Spain)

Antimicrobial peptides (AMPs) are interesting compounds in plant health because there is a need for

new products in plant protection that fit into the new regulations. Living organisms secrete a wide range

of AMPs. Microorganisms produce a wide range of AMPs involved in competition processes, that include

small bacteriocins and fungal defensins synthesized through ribosomal synthesis, and peptaibols,

cyclopeptides and pseudopeptides that are secondary metabolites produced by non-ribosomal

synthesis.

Bacteriocins are a type of protein and peptide secreted by major groups of bacteria that kill closely

related species like nisin, mesentericin, and plantaricins, produced by lactic acid bacteria, typically

produced by species of Lactobacillus. Several filamentous fungi secrete fungal defensins similar to

defensins from animals and plants with a compact structure of antiparallel strands stabilized by disulfide

bridges. Peptaibols are linear peptides usually composed of a C-terminal amino alcohol and an acyl

Nterminus, that are rich in dialkylated aminoacids such as diaminobutiric acid, like harzianins,

trichorzins, trichokonins, typically produced by Trichoderma spp. Antimicrobial cyclopeptides are

secondary metabolites reported from bacteria, fungi and cyanobacteria that are composed of amino

acid residues including D- and L-forms as well as allo and diamino derivatives, arranged in a cyclic ring,

usually without disulphide bridges. Examples are iturins, fengycins, surfactins, agrastatins, polymixins,

syringopeptides and amphisins, produced by several species of Bacillus and Pseudomonas.

Pseudopeptides of interest in plant disease control have few peptide bonds and complex aminoacid

modifications (e.g. nucleosides) and are mainly produced by bacteria, like agrocins produced by

Agrobacterium spp. (Rhizobium), or pantocins produced by Pantoea spp.

These AMPs are secreted in situ by most microorganisms that are active ingredients of commercial

biopesticides, or form part of the formulated product, and they play generally an important role in their

mechanisms of action against plant pathogenic fungi and bacteria.

The potential for production of AMPs from natural sources and their limitations at the time of

registration for commercialization, and their toxicological profiles will be discussed.

Mots-clés / Key words: Keywords: bacteriocins, peptaibols, cyclolipopeptides, pseudopeptides,activity, toxicology.


27


PATHOGEN SUPPRESSION BY RHIZOSPHERE MICROORGANISMS BUILDS UP DURING PLANT DEVELOPMENT

Jie Hu (1,2), Zhong Wei (2), Ville-Petri Friman (3), Yang-chun Xu (2), Qi-rong Shen (2),

GeorgeKowalchuk (1,2), Alexandre Jousset (1,2)

(1) Utrecht University, Institute for Environmental Biology, Ecology & Biodiversity, Padualaan 8, 3584CH Utrecht, the

Netherlands; (2) Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, National Engineering Research Center for Organic-

based Fertilizers, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Weigang 1, Nanjing, 210095, PR China;

(3) University of York, Department of Biology, Wentworth Way, York, YO10 5DD, United Kingdom

Bacterial communities associated with plant roots play an important role in the suppression of soil-borne

pathogens. However, we still have limited information about how rhizosphere community assembly

impacts disease suppression.

Here, we assessed the inhibition of the phytopathogen Ralstonia solanacearum by bacteria isolated from

tomato roots at different growth stage. Functional diversity increased during growth, suggesting that

late microbial communities can better compete with the pathogen. Further, late stage bacteria were

more inhibitory against the pathogen.

Together, the results indicate a rapid ecological succession in the rhizosphere and that young plants may

be more at risk and may benefit from targeted inoculation speeding up the build- up of disease

suppressive species.

This study provides the base for informed bio- engineering strategies to optimize the functionality of the

rhizosphere microbiome for plant health and sustainable agriculture.

Keywords : microbiome assembly; pathogen suppression; sustainable agriculture


28


PLANT-ASSOCIATED LACTIC ACID BACTERIA AS EFFICIENT BIOCONTROL AGENTS OF BACTERIAL PLANT DISEASES

Núria Daranas, Gemma Roselló, Esther Badosa, Jesús Francés, Jordi Cabrefiga,

Emilio Montesinos, Anna Bonaterra

Plant Pathology-CIDSAV-CeRTA, Institute of Food and Agricultural Technology, University of Girona (Spain)

Strains of lactic acid bacteria are good candidates for development of biocontrol agents. They are

antagonists of bacterial and fungal plant pathogens and benefit of the lack of biosafety concerns of most

lactic acid bacteria. A set of 55 isolates of lactic acid bacteria obtained from a wide range of plant sources

and environments were characterized in order to select lead candidates.

The Lactobacillus plantarum strains PM411 and TC92 have been selected because their wide spectrum

of in vitro antagonism against several bacterial plant pathogens, including quarantine bacteria in the EU.

They showed differential MLST profiles and shared the same plantaritype. More in detail, these strains

showed a strong inhibitory activity against Erwinia amylovora (fire blight of apple and pear),

Pseudomonas syringae pv. actinidiae (bacterial canker of kiwifruit trees), Xanthomonas arboricola pv.

pruni (bacterial spot of Prunus spp.), and Xanthomonas fragariae (angular leaf spot of strawberry)(in

vitro antagonism of cells and culture supernatants).

Moreover, their efficacy in preventing infections by these pathogens was confirmed in experiments with

the corresponding plant hosts under greenhouse and field conditions. To increase its tolerance to water

stress and their fitness on the plant surface, and counteract the adverse effects upon field application,

an adaptive strategy consisting of hyperosmotic and acidic conditions during growth was used.

Remarkably, the physiological adaptation of strain PM411 increased its performance under stress and

field conditions. The beneficial effect was demonstrated by the expression of several stress-related

genes using RT- qPCR (groEL, dnaK, ftsH, ctsR, clpB, clpC, hsp1, hsp2, hsp3, cspP, cspL, efTU, trxB1) that

was studied in preadapted cells, and during cell survival under low relative humidity conditions.

In addition, to study the colonization and survival in field conditions, a viability qPCR (v-qPCR) assay for

L. plantarum PM411 was developed and validated for the specific detection and quantification of their

viable cells in aerial plant surfaces.

Funding was provided by Spain MINECO (AGL-2012-39880-C02-01 and AGL2015-69876-C2-1-R), FEDER,

and project FP7-KBBE.2013.1.2-04 613678 DROPSA of the European Union.

Keywords : Lactic acid bacteria, Lactobacillus plantarum


29


PARABURKHOLDERIA PHYTOFIRMANS STRAIN PSJN A PROMISING TOOL TO HELP GRAPEVINE FACING BOTH

BIOTC AND ABIOTIC STRESSES

Essaid AIT BARKA, Stéphane COMPANT, Andreas THEOCHARIS, Fan SU, Lidiane MIOTTO-VILLANOVA, Qassim ESMAEEL, Sandrine DHONDT-CORDELIER, Lisa SANCHEZ,

Nathalie VAILLANT-GAVEAU, Christophe CLÉMENT

University of Reims Champagne Ardenne, Lab of Induced Resistance and Plant Bioprotection, CNRS Research Federation Condorcet (FR CNRS 3417), UFR Sciences Exactes et Naturelles, BP 1039, 51687 Reims cedex 2, France

In the current context, plant/crop protection is a delicate issue since it is imperative to preserve the

environment and to face the climate change. Viticulture is a special case owing to the number of

fungicide treatments required to get healthy berries at vintage.

Using the soil bacterium Paraburkholderia phytofirmans strain PsJN as an endophyte of grapevine

results in plant protection against both biotic and abiotic stresses. The bacterium perceives the plant

root in the soil, then colonizes its surface and finally penetrates the roots and reaches the vascular

bundles. Afterwards, the bacterium diffuses through the xylem in the whole plant organs, including

flowers and berries.

The presence of the bacterium in the whole plant provides some protection against biotic and abiotic

stresses. Plant response was followed after Botrytis cinerea inoculation. In bacterized plants, the

stimulation of plant defence genes was faster and stronger than in the control plants, which leads to

plant protection against grey mould, without using any pesticide nor the GMO technology. Similarly,

when the plant is confronted to abiotic stresses such as temperature variations, the presence of

Paraburkholderia phytofirmans strain PsJN results in the maintenance of plant physiology (ie

photosynthesis) at the same level when compared to plant grown at standard temperature. Here again,

plant reaction is faster and stronger in bacterized plants than in control plants.

Altogether, these results strongly suggest that endophytic micro-organisms may be used to develop

sustainable technologies of plant/crop protection in the future. It is now necessary to transfer these

results from the lab to the field.

Keywords : biocontrol, biotic and abiotic stresses, Botrytis cinerea, cold temperatures, Paraburkholderia

phytofirmans PsJN


30


BIOLOGICAL CONTROL OF QUARANTINE BACTERIAL DISEASES WITH SELECTED STRAINS OF BACILLUS AMYLOLIQUEFACIENS

J. Cabrefiga, I. Mora and E. Montesinos

Laboratory of Plant Pathology, Institute of Food and Agricultural Technology-XaRTA-CIDSAV, University of Girona, Campus Montilivi, 17071, Girona, Spain

Quarantine plant bacterial diseases are important problems in crop production in the European Union

and also in other countries. Some of the more relevant diseases are caused by Erwinia amylovora,

Pseudomonas syringae pv. actinidiae, Xanthomonas arboricola pv. pruni and Xanthomonas fragariae.

There are strong difficulties to control these diseases and fruit production losses are very relevant

depending on years. In addition, chemical control in the EU is limited to copper compounds, because

antibiotics are not authorized. Therefore, there is an urgent need for alternative or complementary

control methods.

Our group has focused research in selecting strains of Bacillus spp. for candidates as biocontrol agents

of bacterial diseases, in contrast with the great efforts done by others targetting fungal diseases. For

this purpose, a large collection of strains of Bacillus subtilis and B. amyloliquefaciens was build-up from

plant environments from Mediterranean crops and forests. The isolation and selection process was

based on PCR-assisted molecular tools using biosynthetic genes related with the production of

antimicrobial compounds, especially cyclo-lipopeptides (cLPs), directly in the samples prior to the

isolation. The isolates obtained were characterized by the presence of cLPs biosynthetic genes, the

production of cLPs and the range of antagonistic activity in vitro against several plant pathogenic

bacteria. The best strains were tested for the control of infections in the corresponding plant hosts (pear,

peach, strawberry, kiwifruit) under controlled environment conditions in the quarantine greenhouse,

and finally under semi-field assays.

Some of the strains pertaining to B. amyloliquefaciens showed significant and consistent disease control

with promising results compared to reference commercial products.

Key words: bacillus, quarantine diseases, biocontrol, bacterial pathogens


31


CYTOLOGICAL AND MOLECULAR MECHANISMS OF PYTHIUM OLIGANDRUM MYCOPARASITIC INTERACTIONS

WITH FUSARIUM GRAMINEARUM

Charlène FAURE (1), Thomas REY (1,2), Elodie GAULIN (1), Bernard DUMAS (1)

(1) Laboratoire de Recherche en Sciences Végétales, Université de Toulouse, CNRS, UPS, 24 Chemin de Borde Rouge, Auzeville, BP42617, 31326 Castanet Tolosan, France

(2) De Sangosse, Bonnel, 47480 Pont-Du-Casse, France

Mycoparasitism is a common but understudied mechanism which has a substantial ecological

significance as well as translational research potential to control fungal and oomycete phytopathogens.

Pythium oligandrum is a mycoparasite oomycete with a large host range ranging from fungi to

oomycetes. However, the molecular bases of P. oligandrum pathogenic mechanisms underlying prey

colonization are still poorly described.

We devised and implemented a P. oligandrum strain M1 mycoparasitism assay based on a dual-culture

system using a nutrient poor medium. We observed that Fusarium graminearum growth was quickly

halted following the first contact with P. oligandrum. At the end of the co-culture kinetic at 9 days, all

remaining fungal structures were dead whilst oospores of the oomycete were produced, proving the

transfer of nutrients crucial for P. oligandrum sexual lifestyle from the fungus to the oomycete. We

performed follow up experiments suggesting that mycoparasitism is instrumental to provide fungal

sterols to P. oligandrum which in turn allow sexual hormone production in this sterol auxotroph

oomycete. Further electronic and confocal microscopy analyses using several subcellular markers

illustrate the necrotrophic behavior of P. oligandrum strain M1 and cellular biology shifts related to

mycoparasitism. These approaches highlighted the development of specialized secondary mycoparasitic

hyphae of P. oligandrum which penetrate into F. graminearum.

To identify the genetic determinants involved in P. oligandrum fungal preys colonization, we performed

sequencing of the M1 strain genome and RNAseq analysis. RNAseq analysis revealed 1380 genes induced

in mycoparasitic conditions with F. graminearum. Combined with Pythiale genomic comparative

analyses, it led to the identification of genes, notably coding secreted proteins, specifically found in

mycoparasitic Pythium species but no in phytopathogens nor in zoopathogens and highly expressed

during pathogenesis.

Together, our results show that P. oligandrum engages a specific cellular and genetic program upon

contact its host, leading to the penetration and the rapid killing of the host. Further studies will aim to

functionally characterized genes specifically involved in mycoparasitism and molecular signals inducing

mycoparasitic development.

Keywords: Pythium oligandrum, Fusarium graminearum, mycoparasitism, genome sequencing, RNAseq


32


LEGUME NODULES ARE SITTING DUCKS FOR PATHOGENS

Claire BENEZECH (1), Alexandre LE SCORNET (1), Jeoffrey GEORGE (2), Fathi BERRABAH (2), Thomas BADET (1), Gaofei JIANG (1), Alain JAUNEAU (3), Yves MARTINEZ (3),

Marie-Christine AURIAC (3), Marie- Françoise JARDINAUD (1), Fabienne VAILLEAU (1), Sylvain RAFFAELE (1), Pascal RATET (2), Benjamin GOURION (1)

(1) LIPM, Université de Toulouse, INRA, CNRS-Université Toulouse, Castanet-Tolosan, France (2) Institute of Plant Sciences Paris-Saclay, Centre National de la Recherche Scientifique, Institut National de la Recherche

Agronomique, Université Paris-Sud, Université Evry, Université Paris- Diderot, Université Paris-Saclay, Orsay, France (3) CNRS, Plateforme Imagerie-Microscopie, Fédération de Recherche FR3450, Castanet-Tolosan, France

Legume nodules are symbiotic root organs where in the range of billion(s) of bacteria reside without

triggering plant defenses. This state rises the questions of how the plants tolerate such colonization and

how sensitive to pathogens are these organs.

To better understand the benefits and drawbacks of using symbiosis for the agriculture, we initiated

experiments to assess the influence of nodulation and of nitrogen fixation on the sensitivity to

phytopathogenic microorganisms. To do this, we set up two tripartite interaction systems. These

systems involve the model plant Medicago truncatula nodulated in vitro by Sinorhizobium medicae and

two distinct phytopathogenic organisms: either the bacterium Ralstonia solanacaearum or the fungus

Sclerotinia sclerotiorum. With these tripartite systems we are also characterizing the immune responses

within the nodules notably by using transcriptomic analysis, plant, rhizobia and pathogen mutants,

histology and molecular biology.

Data obtained with these innovative experimental set ups show that nodules represent an opportunity

for phytopathogenic organisms to infect the plant and to proliferate. For instance, S. sclerotiorum

development is more important on nodules than on root segments. Data obtained with R. solanacearum

also indicate that the bacterium can penetrate in the plant via the nodule. However, we observed that

dissemination of this bacterium from a nodule to the rest of the plant is delayed as compared to an

infection on a root segment. We are currently trying to understand at the molecular and cytological

basis of these observations.


33


HOW DOES A FLAVOR LACTONE TRIGGER BACTERIAL BIOCONTROL OF BLACKLEG AND SOFT-ROT DISEASES ?

Chane Andrea (1), Barbey Corinne (1,2), Amélie Beury-Cirou (2,3), Bouteiller Mathilde (1), Merieau Annabelle (1), Konto-Ghiorghi Yoan (1), Virginie Gobert (2,3), Latour Xavier (1)*

(1) Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM EA 4312) - Normandie Université - LMSM, 55 rue Saint-Germain, 27000 Evreux, France & Structure Fédérative de Recherche Normandie Végétale 4277 (NORVEGE).

(2) Seeds Innovation Protection Research and Environment (SIPRE), Rue des Champs Potez, 62217 Achicourt, France (3) French Federation of seed potato growers (FN3PT/RD3PT), 43-45 rue de Naples, 75008 Paris, France

*Corresponding author: [email protected]

During the last decade, the means of communication used by numerous Gram-negative pathogenic

bacteria have emerged as a target for the development of novel biocontrol strategies (Diallo et al.,

2011, FEMS Microbiol. Ecol.). These therapies aim at reducing the expression of virulence systems rather

than eradicating the pathogen, using bacteria or enzymes degrading signaling molecules (for a

recent review see Grandclément et al. 2016, FEMS Microbiol Rev). Unfortunately, these anti-virulence

strategies will likely suffer a lack of consistency when they will be submitted to various soils and climatic

conditions as observed for most other biocontrol methods. Indeed, the effectiveness of biocontrol

treatments greatly depends on the fitness of the protecting agent and its sustainable activity in the

vicinity of plant-host.

In the fight against the economically important blackleg and soft-rot phytopathogens, we have

previously isolated from potato rhizosphere the Rhodococcus erythropolis strain R138, a bacterium able

to degrade N-acyl homoserine lactone signaling molecules used by numerous pathogens (Cirou et al.,

2007, Environ Microbiol). The plant protection provided by R138 strain mainly involves the quorum-

sensing signal degradation (qsd) pathway, the regulation of which has just been elucidated (Barbey et

al., submitted). Using the qsd pathway, and only when the γ-caprolactone (GCL) biostimulant was

spread, the R138 biocontrol agent was able to suppress the disease during field trials (Cirou et

al., 2012, Appl Environ Microbiol). GCL is poorly present in the environment and is rather known as a

synthetic additive conferring a nutty and malty odor to products intended for human consumption (e.g.

beverages, ice cream, candy and tobacco).

In this work, we unveil and explain at a molecular level how GCL promotes the elective growth of

biocontrol agent and acts as a powerful inducer of the qsd operon. The findings presented here shows

that preventive or curative anti-virulence therapies may be triggered and boosted by the addition

of cheap biostimulating lactone molecules. Maybe the potato fields will soon smell nutty?

Keywords: microbiological control, bacterial communication, quorum-sensing, quorum-quenching, Solanum tuberosum



34


IDENTIFICATION OF POTENTIAL MYCOHERBICIDES USING A METABARCODING APPROACH

Triolet Marion (1,2), Véronique Edel-Hermann (1), Nadine Gautheron (1)

André Olivier (2), Guillemin Jean-Philippe (1), Steinberg Christian (1)

(1) UMR Agroécologie, AgroSup Dijon, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon (2) Société DE SANGOSSE, Lieu-dit Rue Bonnel, 47480 Pont-du-Casse

Using chemical herbicides to control weeds is very common but some are human unhealthy,

environmentally detrimental and they stimulate herbicide resistance. Therefore, using microorganisms

as natural herbicides appears as a promising alternative. A project aiming at identifying new

mycoherbicides was initiated between the INRA Research Unit Agroecology (Dijon, France) and the

international crop protection and pest control products supplyer DE SANGOSSE® (Agen, France). Part of

this project is based on comparing the endophytic mycoflora of different species of symptomatic and

asymptomatic weeds by metabarcoding to characterize i) the fungal component associated with

symptoms and likely to harbor potential mycoherbicidal candidates, and ii) the component associated

with non-symptomatic plants that may be a hindrance to mycoherbicidal activity.

Symptomatic and asymptomatic weeds were collected in different fields in France. The sampling

includes 475 plant samples representing 24 plant species and 29 plots. Aerial parts of the plants were

disinfected, frozen and crushed. Fungal diversity was analyzed using a metabarcoding analysis based on

PCR amplification of internal transcribed spacer (ITS) of the ribosomal DNA and high throughput

sequencing. Two pairs of primers (ITS1F/ITS2 and ITS86F/ITS4) were used, targeting the ITS1 and ITS2

regions, respectively.

In total more than 40 million reads were obtained and deciphered through bioinformatics analyzes using

appropriate pipelines. Taxonomic groups specific to each component have been identified and provide

candidates that a Pasteurian approach coupled with access to fungal collections should test for their

mycoherbicidal activity or their triggering of plant defense reactions.

Keywords: biocontrol, bioherbicide, genetic diversity, weeds, ITS1, ITS2


35


SEED COATING WITH NATURAL BIOSTIMULANTS : A PRACTICAL TOOL FOR IMPROVING TOLERANCE

TO BIOTIC AND ABIOTIC STRESSES IN TUNISIA

Maissa Ben Jabeur (1), Zayneb Kthiri (1), Kalthoum Harbaoui (2), Cedric Jacquard (3), Essaid Ait barka (3) and Walid Hamada (1)

(1) Laboratory of Genetics and plant breeding, National institute of Agronomy of Tunis, Tunisia (2) Laboratory of Agronomy, Regional Field Crops Research Center (CRRGC) Béja-Tunisia (3) Laboratory of Stress, Defenses and Reproduction of Plants, University of Reims Champagne-Ardenne, France

Due to climate change in the area, emerging problems due to foliar and soil diseases as well as drought

stress are causing severe losses in durum wheat. Coating seeds with beneficial micro-organisms and

plant extracts appears to be a promising approach to maintain the productivity of plants under stress

condition. In this study, we evaluated the endophytic bacterium, Burkholderia phytofirmans strain PsJN,

thyme essential oil, the antagonist Trichoderma harzianum, the yeast Meyerozyma guilliermondii, and

their different associations as well as a reference product (Panoramix, Koppert) for their ability to control

the diseases mentioned both under controlled conditions and in field. Seeds of a sensitive Tunisian

cultivar of durum wheat “Karim” were coated with B. phytofirmans (10⁸ CFU/ml) and T. harzianum (10⁶

spores/ml), thyme essential oil (5 ppm), M. guilliermondii (108 spores/ml) and Panoramix at 4ml/g.

Under controlled conditions, Septoria leaf blotch was monitored in pots while Fusarium head blight was

assessed in hydroponic system.

Treatments reduced pycnidial coverage of septoria to 10% compared to control (40%).

Cytology and enzymatic analysis showed that these treatments enhance plant resistance with increased

catalases activity, reduced peroxidases activity and H2O2 levels and reduced fungal colonization and

development in leaf cells. In field, coated seedlings showed a reduced septoria leaf blotch attack to an

average 10-20% compared to control (43%).

As for Fusarium head blight, under controlled conditions, severity was reduced of about 30%, with

reduced peroxidases activity in roots, enhanced phenolic compounds content in leaves and roots and

reduced colonization and macroconidia abundance in root cells. Moreover, molecular analysis on leaves

sampled after 21 days of growth showed clearly by QRT-PCR overexpression of genes involved in several

defense response pathways.

Keywords: abiotic and biotic, stress, Burkholderia, Trichoderma, thyme, Meyerozyma, Panoramix, defense

response.


36


ALD1202, A NEW BIOFUNGICIDE BASED ON YEAST TO CONTROL BOTRYTIS AND MONILIA ROTS IN GRAPEVINE, VEGETABLE CROPS

AND STONE FRUITS

P. Letousey, B. Albert, F. Boulisset, M. Cadiou, J. Detoc, H. Houdbine, R. Kempf, F. Manceau, A. Martin, M.-C. Reveillaud, A. Theriez and C. Profizi

AGRAUXINE LESAFFRE PLANT CARE, 2&4 Rue Henri Becquerel, 49070 BEAUCOUZE, France

AGRAUXINE Lesaffre Plant Care, a business unit of the LESAFFRE group, develops, produces and markets

biocontrol products derived from microorganisms to improve crop protection. Based on this expertise,

AGRAUXINE Lesaffre Plant Care developed ALD1202 product containing a living yeast Saccharomyces

cerevisiae strain LAS02. Agrauxine selected this specific strain for its good potential efficacy against

Botrytis and Monilia crop pathogens. The mode of action of ALD1202 consists in competition for space

and nutrients so that ALD1202 yeasts form a natural barrier against pathogens on treated leaves and

fruits.

In viticulture, vegetable crops and stone fruit production, ALD1202 demonstrated a significant efficacy

against rots like Botrytis and Monilia, quite similar to conventional chemical references. We also

demonstrated an efficacy post-harvest on peach and strawberry. ALD1202 product has several

interesting advantages: low-risk substance, compatibility in tank-mix with anti-Botrytis chemical

products, absence of MLRs, compatibility with organic farming. Moreover, we ensured that ALD1202

has no effect on vinification (wine processes, analytical quality of wine, sensory quality after one year of

storage).

Keywords: antagonism, biocontrol, crop protection, low-risk substance, yeast


37

Poster communications - P10

BIOCONTROL OF GREY MOLD OF TOMATO USING ENDOPHYTIC BACTERIA PRODUCING VOLATILE ORGANIC COMPOUNDS

Manel CHAOUACHI (1,2,3), Takwa MARZOUK (1,2), Sabrine HAJRI (1), Wafa FRIDHI (1), Feriel WERGHIMI (2), Imen BEN SLIMENE (1), Selim JALLOULI (1), Salem ELKAHOUI (1),

Cécile BEN (3), Laurent GENTZBITTEL (3) and Naceur DJEBALI (1)

(1) Laboratory of Bioactive Substances, Center of Biotechnology of Borj Cedria, BP 901, Hammam-Lif 2050,Tunisi (2) University of Tunis El Manar, Faculty of Mathematical, Physical and Natural Sciences of Tunis, 2092, Tunis, Tunisia (3) Laboratoire d’Ecologie Fonctionelle et Environnement, Université de Toulouse, CNRS, INPT, UPS, Toulouse, France *Correspondence to [email protected]; [email protected];

Grey Mold, caused by the fungus Botrytis cinerea, is one of the major airborne diseases that attack more

than 270 plant species. Several methods have been developed to control B. cinerea, particularly

biological control based on the use of living organisms to prevent or to reduce the damage caused by

this pathogen. This study is interested in using the volatile organic compounds (VOCs) produced by

tomato endophytic bacteria to reduce the damage of grey mold on tomato fruits.

A collection of bacterial strains was established from the different organs of several Solanaceae species.

The effect of the bacterial VOCs on the growth of two B. cinerea strains (S2 and S5) was studied using

the double culture method avoiding any physical contact between the two microorganisms.

The results showed that 28% of the bacterial strains specifically inhibit the S2 strain, 30% inhibit the S5

strain, 33% inhibit both fungal strains and 9 % of the strains have no activity. The activity of bacterial

VOCs on the germination of tomato seeds showed that 42.5% of the strains improved the percentage of

germination, 38.3% of the strains reduced it and 12.8 % have no effect on this parameter.

The later experiments were the basis for the choice of three bacterial strainsDHT2, H1F1, H2FL2

producing VOCs that inhibit the growth B. cinerea and enhance tomato seed germination. Some

biochemical tests (production of HCN and certain enzymes) were carried out on the selected strains.

The results showed that the bacterial strains have different levels of cellulosic, pectinolytic activities but

do not produce HCN. The test of protection of tomato fruit from B. cinerea infection by the bacterial

VOCs showed that those strains was able to reduce the maceration of the fruit tissues 48 h after

inoculation at different levels. The biochemical identification of the three bacterial strain showed that

they belong to Bacillusgenus.

The characterization of bacterial VOCs was also carried out by GC-MS methods and showed different

types of volatile compounds produced by the three strains. The results of this study show the potential

use of the bacterial VOCs for post-harvest disease management.

Keywords: Endophytic bacteria, Botrytis cinerea, Biological control, Tomato, Volatile organic compounds.




38


AN ENDOPHYTIC BACILLUS SUBTILIS STRAIN PROTECTS GRAPEVINE AGAINST DOWNY MILDEW BY DIRECT EFFECT

AND DEFENSE STIMULATION

Yan Li (1), Marie-Claire Héloir (2), Xun Zhang(1), Mareen Geissler(3), Marius Henkel(3), Qi Wang (1), Marielle Adrian(2)

(1) Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing, China (2) Agroécologie, AgroSup Dijon, CNRS, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France Adresse/Adress (3) Institute of Food Science and Biotechnology, Department of Bioprocess Engineering, University of Hohenheim,

Stuttgart, Germany

Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape

pathogen. Phytochemicals are generally used to control infections, but the appearance of resistant

strains and the concern for possible adverse effects on environment and human health are increasing

the search for alternative strategies. Biological control has received a great deal of attention as an

alternative and promising measure to control different plant diseases. Many antagonistic

microorganisms, including Bacillus spp., have been exploited against different pathogens. Bacillus spp.

activity results from multiple modes of action including antibiosis, competition, and induction of the

host systemic resistance. Bacillus production of the antibiotics lipopeptides, especially surfactin, iturin,

and fengycin, is known as an efficient way to suppress diseases.

We have isolated the Bacillus subtilis strain GLB191 and demonstrated it has a strong activity against

grapevine downy mildew. Assays performed in greenhouses showed a reduction of more than 95 % of

the leaf sporulating area. The bacterial activity results from both direct effect against the pathogen, and

indirect effect via stimulation of the plant defenses. This strain indeed induces defense gene expression

and defense reactions such as callose production. Mutants affected in lipopeptide production were

obtained to further investigate the mode of action of this strain. The absence of production of the

targeted lipopeptides was confirmed using high-performance thin-layer chromatography (HPTLC).

Activity against downy mildew was lost for double mutants affected in both fengycin and surfactin,

suggesting a key role of these two lipopeptides in the direct mode of action of Bacillus subtilis strain

GLB191.

This study shows that Bacillus subtilis strain GLB191 could be a potential biological agent to control

grapevine downy mildew. It would be interesting to check activity against other grapevine diseases,

especially Grapevine Trunk Diseases, and also against other crop pathogens.

Keywords: grapevine, downy mildew, Plasmopara viticola, biocontrol, Bacillus subtilis, lipopeptides


39


AGROINDUSTRIAL AND FORESTRY WASTES POTENTIAL AS BIOCONTROL PRODUCTS : CHEMICAL CHARACTERIZATION

AND EVALUATION

Andreu Vanessa (1), Bertrand Cédric (1,2), Merad Nadjiya (2), Chaïb Slimane (2), Levert Annabel (1)

(1) AkiNaO SAS, University of Perpignan Via Domitia, Perpignan, France (2) EPHE, PSL Research University, UPVD-CNRS, USR3278 CRIOBE, Perpignan, France

According to the FAO, the processing of raw materials in agri-food industry generates a large quantity of waste in various forms that is usually disposed of in landfills or incinerators with economical and environmental negative effects. Preventing the overproduction of waste is one of the wagers of European environmental policies, to move towards an efficient Europe in the use of resources according to the EU 2020 Strategy, or the Framework Directive on Residues. Within this context, the REDVALUE collaborative project (Technological consortium to complete the cycle of agroindustrial and forestry production) was initiated in 2017. Its purpose is to strengthen the establishment of agro-industrial and forestry waste recycling sectors in the South-West countries which are particularly involved in these sectors and impacted by the economic crisis. This project is bringing together Research centers (Institute of Emerging Chemical Technologies of La Rioja), Universities and higher education centers (Polytechnic Institute of Viana do Castelo, University of Lleida, University of Zaragoza, University of Minho, University of Perpignan) and private companies (AkiNaO, Indulleida) from France, Spain and Portugal. By-products were selected according to their importance as agroindustrial and forestry wastes in the countries involved in the project: pine bark, olive pomace, peach, apple, cucumber and red pepper wastes. Those raw material were extracted under different conditions (different solvent, maceration extraction, ohmic extraction…). They were then analyzed and their biological activities (antioxidative, anticancer, biocontrol potential…) were assessed by different project partners. AkiNaO analyzed the chemical composition (LC-DAD-MS analysis) and evaluated the generated extracts as biopesticides (herbicidal and antifungal activity).

Keywords: Agroindustrial and forestry waste extracts – Biopesticide potential – Chemical characterization - Antifungal in vitro activity – Herbicidal in vitro activity


40


BIODÉSINFECTION DES SOLS EN CULTURE DE TOMATE

I. Arnault (1), P. Deberdt (2,4), G. Dubreuil (3), Y. Prin (4), P. Fernandes (2,4), H. Védie (5), P. Sunder (6), T. Fouillet (7), G. Dufretay (7), C. Souty-Grosset (8), N. Pourtau (8),

M. Zimmermann (3), D. Giron (3)

(1) CETU Innophyt, Université de Toursavenue Monge, 37200 Tours (2) CIRAD, UPR HORTSYS, Campus Agro-environnemental Caraïbe, F-97285 Le Lamentin, Martinique, France (3) IRBI - UMR 7261 CNRS / Université François-Rabelais de Tours, UFR Sciences et Techniques- Parc Grandmont - Avenue

Monge 37200 Tours (4) Hortsys, Université de Montpellier, CIRAD, Montpellier, France (5) Groupe de Recherche en Agriculture Biologique, 84 911 Avignon Cedex 9 (6) Certis Europe, 5 rue Galilée 78280 Guyancourt, France ; Université de Poitiers (7) ARKEMA France 420, Rue d’Estienne d’Orves, 92705 Colombes Cedex (8) Ecologie et Biologie des Interactions, 3, rue Jacques Fort, 86073 Poitiers cedex 9

Le projet SERUM (PSPE 2 2015-2019) propose de concevoir une stratégie de biocontrôle socialement et

économiquement acceptable pour lutter contre deux bioagresseurs telluriques de la tomate en région

tempérée et en région tropicale humide (départements et régions d’outre-mer DOM) : les nématodes à

galles du genre Meloidogyne et l’agent du flétrissement bactérien Ralstonia solanacearum (organisme

de quarantaine A2 de l'OEPP). L’idée du projet est d’utiliser des plantes connues pour leur potentiel

assainissant contre Meloidogyne sp et R. solanacearum, adaptées aux conditions agro-

environnementales des régions ciblées. Les données acquisent suggèrent que les Alliacées (oignons,

cives…) et les crotalaires peuvent être utilisées (I) en association ou (II) en précédent cultural suivi d’un

mulch ou (III) en enfouissement dans le sol selon le concept de biofumigation, pour produire leur effet

assainissant.

A mi-chemin du projet, les deux espèces de crotalaires choisies sont C. spectabilis et C. juncea et, pour

les Allium : Allium fistulosum en Martinique (oignon-pays) et Allium cepa, Allium schoenaprasum

en métropole. Les premiers résultats montrent des résultats confortant le potentiel de biodésinfection

des sols et d’allélopathie des Allium, crotalaires et du disulfure de diméthyle (DMDS). Contre les

nématodes en conditions contrôlées, la poudre de crotalaire et le broyat frais de ciboulette montrent un

effet de réduction du nombre de galles sur tomate. Contre l’agent du flétrissement bactérien,

l’incorporation au sol des broyats de crotalaires et d’oignon-pays permettent de réduire l’incidence du

flétrissement bactérien de la tomate. Par ailleurs, seuls les extraits d’oignon- pays et le DMDS montrent

un effet biocide direct sur les populations de R. solanacearum dans le sol. Des effets stimulants des

faibles doses d’oignon-pays et de crotalaires ont été observés au niveau des racines de tomates et, du

DMDS au niveau des feuilles. Les extraits d’oignons-pays et des deux espèces de crotalaires montrent

effet de stimulation des populations microbiennes du sol. Les effets non intentionnels de la méthode de

biocontrole sur les arthropodes sont en cours d’évaluation grâce à l’application mobile Insectfinder

(projet CaRamba, financement conseil régional du Centre-Val de loire).

Mots-clés : Ralstonia solanacearum, nématodes à galles, crotalaires, alliacées, biofumigation, allélopathie


41


SCREENING STRATEGIES FOR DISCOVERY AND CHARACTERIZATION OF ANTIFUNGAL LIPOPEPTIDES USABLE AS BIOCONTROL AGENTS

Bricout Alexandre (1, 2), Morris Cindy (3), Bardin Marc (3), Nicot Philippe (3),

Chataigné Gabrielle (1), Jacques Philippe (4), Leclère Valérie (1), Rochex Alice (1)

(1) Univ. Lille, INRA, ISA, Univ. Artois, Univ. Littoral Côte dOpale, EA 7394 - ICV - Institut Charles Viollette, F-59000 Lille, France

(2) Agence de l’Environnement et de la maîtrise de l’Energie, 20 avenue du Grésillé BP 90406, 49004 Angers Cedex 01, France

(3) Pathologie Végétale, INRA, 84140 Montfavet, France (4) TERRA Research Centre, Microbial Processes and Interactions (MiPI), Gembloux Agro-Bio Tech University of Liege,

B-5030 Gembloux, Belgium

Field crops fungal diseases such as wheat Septoria tritici blotch or rape and sunflower sclerotinioses lead

to serious economic consequences. Apart from chemical pesticides whose widespread use is known to

generate dire environmental and health hazards, few products are available to handle the fungi

responsible for these diseases. Thus, it becomes crucial to establish new alternatives, more environment

respectful, harmless and meeting the criteria of producers and consumers. The use of biocontrol agents

and more particularly naturally occurring substances, less toxic and more biodegradable than

chemically synthetized products is one of the undertaken solutions. In this way, bacterial lipopeptides,

molecules made of a peptide chain linked to a fatty acid and produced by Non-Ribosomal Peptide

Synthetase (NRPS) are promising candidates. Thanks to their antifungal and surfactant activities,

lipopeptides produced by Bacillus species have shown, for instance, a great ability to combat the

oomycete Bremia lactucae (lettuce) or the ascomycete Fusarium culmorum (leek).

This project aims at developing various screening strategies allowing the identification of new antifungal

lipopeptides usable as biocontrol agents. Currently the strategy is based on a combined approach of

microbiology, bioinformatics and biochemistry and applied to a collection of 754 Pseudomonas

strains. Hence, in vitro tests have been carried out to study the antagonist effect against the

pathogens Botrytis cinerea, Zymoseptoria tritici and Sclerotinia sclerotiorum, using either bacterial

colonies or cell free supernatants. 161 strains show an antifungal activity on solid media. For 90 of

the 100 most active, this activity is maintained in liquid cultures. Bioinformatics tools are used to analyze

the genomic sequences of the active strains to predict the potential production of active compounds.

Mass spectrometry analyses (MALDI-ToF, LC-MS) are also performed on bacterial colonies and cell free

supernatants to detect the produced molecules. New lipopeptides and particularly syringopeptin

variants have already been highlighted. An in-depth characterization of these molecules using

purification methods and in vivo tests on plants is in progress.

Keywords: lipopeptides, antifungal activity, biocontrol, Pseudomonas


42


POTENTIALITIES OF ACTINOMYCETES FROM SOILS FOR THE DEVELOPMENT OF NOVEL BIOCONTROL PRODUCTS TO MANAGE

CEREALS FUNGAL DISEASES AND MYCOTOXINS RISK

Campos Avelar Ixchel, Colas de la Noue Alexandre, Fontana Angélique,

Strub Caroline, Schorr-Galindo Sabine

UMR QualiSud, University of Montpellier 2, Place E. Bataillon, 34095, Montpellier, FRANCE

The fungal pathogens of cereals represent a major risk and lead to various issues such as yield loss and

the apparition of mycotoxins. Until now, the main solution for the control of molds is the application of

fungicides. However, excessive use of these fungicides can lead to environmental pollution, pesticide

resistance, and public health concerns. In order to avoid these issues, alternative strategies are being

developed, such as the biocontrol of plant pathogens by microorganisms naturally present in the

soil. Actinomycetes show some of the most promising features for the biocontrol applications. Carrying

a large toolbox of molecules of interests (e.g. enzymes, active metabolites such as antibiotics) that allow

them to settle in the soil environment, they are also able to interact directly with the plant (endophytic

habitat, plant growth promoting traits). The main objective of this project is to propose a global

approach to evaluate the potential of actinomycetes isolates from the QualiSud collection for the control

of wheat fungal diseases, from in vitro testing till in planta efficacy, but also regarding their

technological potential for a future industrial production.

Fifty-nine strains of actinomycetes bacteria have been tested by in vitro confrontations against

five strains of phytopathogenic (Magnaporthe oryzae, Zymoseptoria tritici and Parastagonospora

nodorum) and mycotoxinogenic fungi (Fusarium graminearum, Fusarium verticillioides). The objective

of this first approach is to measure the potential capacity of the actinomycetes strains to reduce the

growth of the fungi and/or to inhibit the production of mycotoxins. In the preliminary results, pathogen

growing inhibition rates until 85% have been observed. In vitro complementary tests allowed to

determinate those of the studied strains which present enzymatic activities that could explain their

inhibitory capacity, together with the production of other molecules that have fungicide properties.

Asides these in vitro tests, the extraction and purification of secondary metabolites from the most

promising strains will be carrying out to verify the presence of well-known molecules, but mostly to

identify new ones. Plant Growing Promoting traits will be evaluated in vitro and in planta in order to

predict some positive effects of the actinomycetes on the plant that could have a positive impact on

plant defenses against fungal pathogens. Finally, in planta assay in greenhouse conditions will be

performed on wheat to verify the real efficacy of actinomycete strains to inhibit the fungi proliferation

and to reduce the mycotoxin content on the plant, a verification that is often put aside.

The results of the precedent tests will allow the selection of the most performant strains concerning

disease control efficiency. The last step will be to study the technological properties of selected strains

concerning their production yield on various media originated mostly from agro industrial wastes, with

a particular attention regarding their sporulation capacity and their resistance to conservation processes

such as drying. Keywords: Actinomycetes, biocontrol, fungal pathogens, mycotoxins, cereals


43


OF THE INTEREST TO LISTEN AND DISRUPT THE DIALOGUE OF PATHOGENS: DETAILS OF SOFT-ROT BIOCONTROL BY THE

RHODOCOCCAL QUORUM-QUENCHING

Chane Andrea (1), Gallique Mathias (1) Beury-Cirou Amélie (2,3), Gobert Virginie (2,3), Merieau Annabelle (1), Barbey Corinne (1,2), Latour Xavier (1)*

(5) Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM EA 4312) - Normandie Université

LMSM, 55 rue Saint-Germain, 27000 Evreux, France & Structure Fédérative de Recherche Normandie Végétale 4277 (NORVEGE).

(6) Seeds Innovation Protection Research and Environment (SIPRE), Rue des Champs Potez, 62217 Achicourt, France (7) French Federation of seed potato growers (FN3PT/RD3PT), 43-45 rue de Naples, 75008 Paris, France


The virulence of soft-rot bacteria due to Pectobacterium and Dickeya is under the control of a quorum

sensing process based on the synthesis and perception of signaling molecules belonging to N-acyl

homoserine lactone (NAHSL) family. We are therefore developing a biocontrol approach for the Solanum

tuberosum model based on the selective stimulation of NAHSL-degrading bacteria. This approach

involves the use of the Rhodococcus erythropolis strain R138 as a biocontrol agent, which is able both

to degrade signaling molecules effectively in vitro and to suppress the maceration of tubers in laboratory

and in controlled greenhouse culture conditions. The objective of this study consists in analyzing the

involvement of the NAHSL catabolic pathway, in the control of tuber soft-rot by R. erythropolis R138.

The functions of the key enzyme, the QsdA lactonase, were investigated by making molecular tools

(∆qsdA; qsdA::gfp transcriptional fusion; QsdA expressing E. coli strain) used to inoculate potato tubers

infected with the soft-rot Pectobacterium atrosepticum. The qsdA gene transcription was induced in

potato tubers in the presence of NAHSL producing pathogens only. The biocontrol and NAHSL-

breakdown activities of the QsdA expressing E. coli strain were similar with those of the R. erythropolis

wild-type strain. The deletion of the qsdA gene partially abolished the biocontrol activity of the R.

erythropolis parental strain.

These results demonstrate the involvement of the γ-lactone catabolic pathway both in NAHSL-

breakdown and in the control of the disease. This is a novel and unusual biocontrol pathway, based on

an anti-virulence strategy instead of the pathogen eradication, differing from those previously described

as protecting plants. The regulation mechanism of this pathway by pathogen signals (NAHSL) and diverse

other lactone molecules is being studied.

Keywords: microbiological control, bacterial communication, quorum-sensing, quorum-quenching, Solanum tuberosum


44


EVALUATION OF HIGHLY EFFICIENT LOCAL STRAINS OF 3 ENTOMOPATHOGENIC FUNGI SPECIES AGAINST 3 APHID PEST SPECIES

Eckert François (1), Crovadore Julien (1), Cochard Bastien (1),

Chablais Romain (1), Lefort François (1)

(1) Plants and Pathogens Group, Research Institute Land Nature and Environment, hepia, HES-SO University of Applied

Sciences and Arts Western Switzerland, Jussy, Geneva, Switzerland

To fight the numerous pests and pathogens causing heavy production losses, the agriculture mainly

relies on the use of chemical pesticides, with the disadvantages of a low sustainability, since they

provoke contaminations of soils and waters and can be recovered as chemical residues in raw vegetal

products and food, raising public concern about health. Aphids represent very important pests

worldwide and in Europe, about 50 aphid species may generate heavy losses in crops. Very few biological

control solutions are yet available against such pests. Some fungi, known as entomopathogenic fungi,

may cause lethal pathologies in arthropods and could be the alternative to chemical pesticides.

We present here the evaluation of the pathogenicity of 26 strains of 3 fungal species, Beauveria

bassiana, Metharhizum anisopliae and Paecilomyces fumosoroseus on 3 aphid pests: Myzus persicae,

Chaetosiphon fragaefolii and Acyrthosiphon pisum. These strains have been isolated from organic

orchards and meadows in Canton Geneva. All aphids were reared in climatic chambers on their host

plants: A. pisum on faba beans, C. fragaefolii on strawberry plants and M. persicae on Chinese cabbage.

Fungi were grown on PGA, Sabouraud and diluted Sabouraud media, for optimal sporulation. Repetitive

experimental units consisted in five aphids placed in a Petri dish on leaf disks previously soaked for 15 s

in fungal spore suspensions standardised at 107 spores per milliliter. Mortality was recorded at 3 and 6

days. Myzus persicae expressed a mortality rate between 30 and 50% after 6 days for 12 strains and

showed to be very sensitive to two strains, B. bassiana 11.6 and P. fumosoroseus 28.2, with a mortality

rate of 86% and 80%, respectively. Some insects were quickly covered with mycelium and conidiophores.

Six strains were very infectious to Acyrthosiphon pisum causing 100% mortality: B. bassiana 1.1, B.

bassiana 4.1, B. bassiana 11.6, B. bassiana 13.1, P. fumosoroseus 28.2 and M. anisopliae 28.4. The isolate

B. bassiana 13.1 caused 100% mortality after 3 days. Nine other strains induced mortality rates higher

than 80% after 6 days. Chaetosiphon fragaefolii was killed at 80% or more by the strains B. bassiana 2.1,

M. anisopliae 2.6, M. anisopliae 10.1, B. bassiana 11.4, B. bassiana 12.1, B. bassiana 28.1 et P.

fumosoroseus 28.2. The most interesting strains were B. bassiana 2.1, B. bassiana 11.4, B. bassiana 12.1,

B. bassiana 11.6 and P. fumosoroseus 28.2., all of them provoked a mortality higher than 80% on 2 aphid

species The strains 11.4 and 28.2, being highly lethal to the 3 aphid species, must be in priority

considered for further development as a biological pesticide against aphids.

Keywords: Beauveria bassiana, Metarhizium anisopliae, Paecilomyces fumosoroseus, Myzus persicae,

Acyrthosiphon pisi, Chaetosiphon fragaefolii


45


STUDY OF RHIZOBACTERIA ISOLATED FROM ARGANIA SPINOZA L. OF TINDOUF REGION

Housseyn Medjahed (1, 2), Meriem Medjkane (3), Mazari Ait Kaci (1), Kamel Zirar (1),

Abdellah Noui (3), Abdelazize Merouane (3), Kamel Nedjari Ben Hadj Ali (3), Mohamed Cheurfa (3), Abdelkader Saadi (3)

(1) University Center Nour Bachir, El Bayadh, BP 900 32000, Algeria (2) Laboratory of Natural Bio-Resources, Hassiba Benbouali University, BP 151 Chlef 02000, Algeria (3) Faculty of Science of Nature and Life, Hassiba Benbouali University, BP 151 Chlef 02000, Algeria

The use microorganisms as inoculants in agriculture is one of the most promising approach to improve

cultivated plants production and yield. The first step of our study was the isolation of the rhizobacteria

from the rhizosphere of the argan tree of Tindouf regions in Algeria, then the investigation of their power

to boost the plants growth was done by the determination of IAA after purifying our isolates on King B

medium. This step allowed us to select 41 bacteria approving concentrations of AIA higher than 13.5

μg/ml among 78 isolates. The highest rate of IAA was 56μg/ml. The productions of HCN and NH3 were

also measured. In the second step, the isolated PGPRs were applied on seeds planting and their effects

on plant growth by the germination test on the varieties SIMITO and ARZ representing durum and soft

wheat respectively were examined.

The germination rates were 47%, 55%, 25%, 56%, 68%, 64%, and 36% varying with bacteria. The finale

step was a statistical study which confirm the power of our PGPRs on wheat growth with very

satisfactory results. All the results were higher than the control however one of the bacteria gave the

most interesting result with an average of dry root weight of 125 ± 4.08 mg leaves length of 110 ± 29.15

cm and rods length of 100 ± 33.02 cm.

Keywords: IAA, Germination test, Growth test, PGPR (Plant Growth Promoting Rhizobacteria), The Argan


46


BACILLUS METHYLOTROPHICUS STRAIN B25, A NEW BACTERIAL BIOSTIMULANT MADE ITS WAY

TO THE MARKET UNDER THE NAME HÉLÈS

Lefort François (1), Gerbore Jonathan (2), Cochard Bastien (1), Crovadore Julien (1), Aline Brutel (2), Pierre Joly (2), Chablais Romain (1), Berthon Jean-Yves (2)

(1) Plants and Pathogens Group, Research Institute Land Nature and Environment, hepia, HES-SO University of Applied Sciences and Arts Western Switzerland, Jussy, Geneva, Switzerland

(2) Biovitis, Le Bourg, 15400 St-Etienne-de-Chomeil, France

Isolated from a decaying Platanus x acerifolia in Geneva in 2012, Bacillus methylotrophicus B25 has

found its way to the European biostimulant market early 2018, under the commercial name Hélès,

through a collaboration between the French company Biovitis and hepia HES-SO. We detail here the

results of in vitro assays showing the capacities of the strain B25 to produce indole acetic acid (IAA) and

siderophores, to solubilize phosphate. The effects of applications of Bacillus methylotrophicus B25 on

crop yields are also reported for open field experiments on potato, carrot and onions cultures, carried

out in 4 conventional farms in different locations in France. The capacity of solubilizing the inorganic

phosphate Ca3(PO4)2 was measured in liquid cultures in NBRIP-BPB (National Botanical Institute’s

phophore- Bromophenol Blue) medium. The qualitative assay showed the bacterium was able to secrete

an organic acid while the solubilized phosphorus was quantitatively assessed by ICP-Radial after

centrifugation of the culture medium which measured an increase of 830% of solubilized phosphorus.

The production of indole acetic acid was measured on TSA medium supplemented with L-tryptophane

and reached 13 µg/ml after 32h incubation.

The production of siderophores of bacterial origin was revealed on a ChromeAzurol S (CAS) culture

medium and showed the capacity of the B25 strain to produce such siderophores. Open field

experiments in potato, carrots and onions cultures were carried out in 2016, and showed statistically

significant yield increases for all three cultures with gains of 7.6-36%, 42% et 20%, when compared to

control, for potato, carrot and onions, respectively. Concerning potatoes, the number and size of tubers

were significantly higher compared to the control and the part of marketable tubers was increased by

17.7%. Similarly, the number of onions was 36% higher in the treatment. Besides that, the biocontrol

potential of this strain was assessed by in vitro challenge tests, which showed an antibiosis effect against

10 pathogenic fungi out of 13 tested species. These promising results, among others paved the way of

the successful homologation of Bacillus methylotrophicus B25 as a commercial biostimulant traded

under the name of Hélès by Biovitis.

Keywords: Bacillus methylotropicum, biostimulant, crops, agriculture


47


DECRYPTAGE DES MECANISMES BIOLOGIQUES IMPLIQUES DANS LE BIOCONTROLE DES CHAMPIGNONS MYCOTOXINOGENES

DES CEREALES

PELLAN Lucile (1), Caroline STRUB (1), Noël DURAND (2), Angélique FONTANA (1), Sabine SCHORR-GALINDO (1)

(1) UMR Qualisud (Université Montpellier), Place Eugene Bataillon, 34095 Montpellier Cedex 5, France (2) UMR Qualisud (Cirad), 73 Rue Jean-François Breton, 34398 Montpellier Cedex 5, France

Les mycotoxines sont des métabolites secondaires toxiques synthétisés par des moisissures qui

contaminent de nombreuses denrées d’origine végétale, notamment les céréales qui sont le premier

facteur d’exposition des consommateurs à ces molécules présentant un réel risque pour la santé

humaine et animale. En particulier, les espèces Fusarium graminearum et Fusarium verticillioides

sont responsables de la production de mycotoxines de type trichothécènes et fumonisines

respectivement, et leurs incidences sur les productions de blé et de maïs en pré- et post-récolte sont

bien connus. Pour lutter contre ces champignons, une stratégie alternative aux produits phytosanitaires

est l’utilisation de microorganismes antagonistes qui par différents mécanismes vont réduire la

progression de ces champignons et limiter l’accumulation de mycotoxines.

Cependant, la mise en place de telles méthodes de protection nécessite une bonne connaissance des

mécanismes biologiques qui régissent l’interaction entre les moisissure mycotoxinogènes et les

agents de biocontrôle (BCA), afin de favoriser les phénomènes d’antagonismes mais aussi pour faciliter

la sélection de nouveaux candidats de biocontrôle en fonctions des processus les plus prometteurs. La

finalité de cette étude est donc d’utiliser les technologies méta-omiques, notamment la

transcriptomique et la métabolomique afin de : (i) décrypter les mécanismes impliqués dans le

biocontrôle des moisissures toxinogènes pour comprendre comment les BCA potentiels interviennent,

(ii) identifier des biomarqueurs (gène clé ou métabolite) inhibiteurs de la mycotoxinogenèse pour

optimiser le screening de BCA, (iii) optimiser leur usage et évaluer les possibilités de régulation des

populations microbiennes pour favoriser les phénomènes d’antagonisme par des itinéraires

techniques optimisés.

En vue de ces objectifs, des BCA commerciaux et de la collection de l’UMR QualiSud ont été

sélectionnés, caractérisés et étudiés en confrontation in vitro avec ces pathogènes du genre

Fusarium et ont permis de proposer une ou plusieurs hypothèses concernant leur mode d’action et sur

la manière dont la physiologie des Fusaria est impactée. L’étude de la confrontation des couples BCA-

pathogène a été complétée sur épillets grâce à une méthode innovante de screening, puis la question

de la transposition vers la plante entière sera investiguée. L’analyse du transcriptome et du

métabolome des différents systèmes plantes-pathogènes-BCAs permettra d’explorer quels gènes

sont différentiellement exprimés en confrontation, quelles voies biologiques sont impliquées lors de

cette réponse, et quelles sont les signatures de ces mécanismes antagonistes (signaux VIP issus des

meta-omic : gènes ou métabolites). L’identification de ces biomarqueurs servira à faire émerger de

nouveaux BCAs antimycotoxinogènes pour de futures études de développement ou autorisations de

mise sur le marché (AMM). D’une manière globale, les résultats de cette étude pourront contribuer à

limiter l’accumulation des mycotoxines dans les céréales ainsi que les résidus de produits

phytosanitaires, ce qui est un double objectif de sécurité sanitaire.


48


PSEUDOMONAS VRANOVENSIS COMME BACTERIE ANTAGONISTE : ANALYSE PAR TN-SEQ DE GENES ESSENTIELS A LA COLONISATION DE

LA RHIZOSPHERE DE POMME DE TERRE

Kévin Robic1, 2, Jérémy Cigna1, 2, Valérie Hélias2, Amélie Beury-Cirou2, Denis Faure1

(1) Institut de Biologie Intégrative de la Cellule (I2BC), CNRS, Gif-sur-Yvette, France (2) Recherche Développement Promotion du Plant de Pomme de Terre (RD3PT), FN3PT, Paris, France

La jambe noire est une maladie de la pomme de terre provoquée par des populations bactériennes

associant une ou plusieurs espèces ou sous-espèces telles que Pectobacterium carotovorum subsp.

carotovorum, P. carotovorum brasiliense, P. atrosepticum, P. parmentieri, Dickeya dianthicola et plus

récemment D. solani. Actuellement, les principales méthodes de lutte contre le développement de la

maladie reposent sur des mesures prophylactiques. Une nouvelle approche en cours de développement

et d’évaluation est l’utilisation d’agents de biocontrôle pour lutter contre ces pathogènes.

Plusieurs souches bactériennes montrant des activités d’antibiose envers ces pathogènes isolées du sol

ont été évaluées (Raoul Des Essarts et al, 2016). Ces agents de biocontrôle montrent des efficacités

d’antibiose différentes selon les pathogènes testés. Une des souches ayant une action d’antibiose contre

le plus large spectre de pathogènes est une souche de Pseudomonas putida.

La recherche, chez cette souche, de gènes essentiels à la colonisation de la rhizosphère via une approche

de Transposon sequencing (Tn-seq) est actuellement en cours. Les premières expériences de

colonisation de la rhizosphère de plants de pomme de terre par P. vranovensis ont déjà été réalisées.

Les résultats de séquençage des mutants Tn-seq seront présentés.

Raoul des Essarts Y, Cigna J, Quêtu-Laurent A, et al. Biocontrol of the Potato Blackleg and Soft Rot Diseases Caused by Dickeya dianthicola. Schottel JL, ed. Applied and Environmental Microbiology. 2016;82(1):268-278. doi:10.1128/AEM.02525-15.


49


CYCLIC LIPOPEPTIDE BIOSYNTHETIC GENES AND PRODUCTS AND ANTIFUNGAL ACTIVITY OF PLANT-ASSOCIATED BACTERIA

Imen HADDOUDI (1,2,3), Isabel MORA (3), Jordi CABREFIGA (3),

Emilio MONTESINOS (3) and Moncef MRABET (2)

(1) University of Tunis El Manar 2092, Tunis, Tunisia, Faculty of sciences Tunis El Manar, (2) Laboratory of Legyme, Centre of Biotechnology of Borj-Cédria (CBBC) BP. 901, Hammam-Lif 2050, Tunisia, (3) Laboratory of Plant Pathology, Institute of Food and Agricultural Technology-XaRTA-CIDSAV, University of Girona,

Campus Montilivi, 17071, Girona, Spain

The new approach in biocontrol research to obtain a candidate strain based on the relationship between

the antifungal activity and presence of cLPs biosynthetic genes and lipopeptide products was used in

this work.

The antifungal activity is the aim in this study, which 85% of plants diseases are caused by fungi and the

fungal infection causes more plant disease that other group of plants agent with over 8,000 species

inducing fungal disease. A collection of 33 bacterial strains was selected from 500 bacterial isolates

based on their high antifungal activity against Fusarium oxysporum as first screening, then they show

variables levels of antifungal activities against 11 different phytopathogenic fungi. The detection of cLPs

genes shows that FEND and SRFA were the most frequently genes detected (50% and 70%, respectively),

then BMYB (40%), when the SPAS and dfnM were the less frequent genes (10% and 20%, respectively).

A 78% of stains shows at least one of the cLPs genes, although 22% of isolates did not had any

biosynthetic genes. The analysis of the production of the cLPs shows that 30% of strains produced Iturin,

Fengycin and Surfactin, when 12% only Iturin and Surfactin, 6% Iturin and Fengycin, when others strains

were able to produce only one type of lipopeptide Fengycin and Iturin (18% and 6%, respectively).

Hierarchical and correspondence analysis confirmed the presence of three clearly differentiated groups,

mainly composed of Bacillus strains, which produces the group with higher antifungal activity, presented

several biosynthetic genes and several isoforms of cLPs. Whereas, the second group of strains presents

moderate antifungal activity with some cLPs genes and products when the last group was characterized

by strains with very low or none antifungal activity, that showed one or none of the cLPs genes and

products.

Keywords: Antifungal activity, cLPs biosynthetic genes, cLPs products, Bacillus


50


RELATIONSHIP BETWEEN THE NUMBER OF CO-OCCURRING PLANT PROTECTION PROPERTIES OF FLUORESCENT PSEUDOMONAS AND

THEIR ABILITY TO INHIBIT THE GROWTH OF PHYTOPATOGENIC MICROORGANISMS

Tiffany Garin (1), Jordan Vacheron (2), Beatriz Manriquez (1), Daniel Muller (1), Claire Prigent-Combaret (1)

(1) UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Villeurbanne, France (2) Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

Plants must face a wide diversity of diseases that strongly affect crop yields. The means to efficiently

protect plants against diseases remain so far based on the use of chemical pesticides. But they induce

adverse effects on the health of ecosystems and humans. Alternative solutions that respect the

environment must be sought. The use of biocontrol bacteria, able to inhibit the growth of plant

pathogens by mechanisms of antagonism and / or induction of plant defenses is one of them.

We hypothesize that rhizobacteria harboring a high number of known plant-protecting properties

(production of secondary metabolites with antimicrobial properties such as 2,4-diacetylphloroglucinol,

pyoluteorines, pyrrolnitrines, phenazines, proteases, chitinases,…) co-occurring in their genome might

be more effective to protect plants against pathogens than rhizobacteria harboring a low number of

those functions.

To test this hypothesis, we compare the ability of 61 strains of fluorescent Pseudomonas, with low or

high numbers of plant protective functions, to inhibit or not the growth of plant pathogen

microorganisms (Fusarium graminearum, the fungal causal agent of fusariosis on many cereals, and

Dickeya dadantii 3937, the bacterial causal agent of soft-rot diseases on many crops).

A relationship between the number of co-occurring plant protection functions and the ability to inhibit

mycelial growth has been observed but this relationship was not established for the growth inhibition

of the bacterial plant pathogen. This suggests that other mechanisms of antagonism than those

considered in this study might be involved in the biocontrol activity of Pseudomonas strains against the

bacterial plant pathogen. On-going screening of genomes of several fluorescent Pseudomonas strains

will help us to identify other potential gene candidates involved in plant-protecting activities of

fluorescent Pseudomonas strains against fungal and bacterial pathogens.

Keywords: Biocontrol, fluorescent Pseudomonas, plant-protecting functions, comparative genomics


51


EVALUATION OF NEW BACTERIAL SPECIES AS POTENTIAL BIOSTIMULANTS FOR AGRICULTURE.

Bastien Cochard, François Raffini, Nicolas Vuille, Julien Crovadore,

Romain Chablais, François Lefort (1)


Facing the mundialization of an intensive agricultural model, heavily dependent of chemical entrants,

with documented effects on the ecosystems and human health, researching alternative methods for an

environmentally friendly and sustainable agriculture is a major issue.

Among these alternatives, the use of microorganisms promoting plant growth and resistance to biotic

and abiotic stresses is a promising possibility to convert the chemically-assisted agriculture to a more

sustainable and productive agriculture. While the search for such microorganisms is blooming, the

selection of efficient candidate microorganisms for the development of commercial biostimulants

remains a tedious and time-consuming task. A key step resides in setting rapid methods for precisely

identifying microorganisms and characterizing their biostimulant potential as well as their harmlessness

for animals and plants. We describe here the methodology that we routinely use for the in vitro

characterization of several strains of the following species Bacillus methyltrophicus, Bacillus aryabhattai,

Paenibacilium sp., Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas koreensis,

Pseudomonas graminis and Pseudomanas xanthomarina, in regard to their biostimulant potential.

Microorganisms are genetically identified by ribosomal genes sequencing. Their capacity to metabolize

carbohydrates, as well as their sensitivity to 23 chemicals are assessed through adapted cultures on

Biolog Ecoplate and Gen III microplates. Minimum inhibitory concentrations (MICs) are measured for

the 7 antibiotic classes in order to evaluate their resistance spectrum to antibiotics. The biostimulant

potential is evaluated by measuring the capacity of all strains to solubilize inorganic phosphate, to

synthesize indole acetic acid and to produce siderophores. The biocontrol capacities of the strains are

also evaluated by in vitro challenge against 14 fungal plant pathogens. In planta small scale experiments

in climatic chambers aim to confirm a potential biostimulant properties on different plant species.

Finally, the genomes of the best candidates are sequenced with an Illumina MiniSeq DNA sequencer.

Keywords: biostimulant, crops, agriculture, bacteria


52


LABORATORY EVALUATION OF ENTOMOPATHOGENIC NEMATODES TO CONTROL AGRIOTES WIREWORMS IN FRANCE

Frayssinet Marie (1), Pagès Sylvie (1), Lawac Floriane (1), Duvic Bernard (1),

Givaudan Alain (1) and Ogier Jean-Claude (1)

(1) Laboratory DGIMI (Diversité, Génomique et Interactions Microorganismes-Hôtes), INRA, Université de Montpellier, 34095 Montpellier, France Contact : [email protected]

Wireworms, the larvae of click beetles (Coleoptera: Elateridae), are considered an economically

important pest of a wide variety of crops in many parts of the world. In France, the main agricultural

pests are Agriotes sordidus, A. lineatus and A. sputator. The use of chemical pesticides is more and more

controversial due to their detrimental effect to both environment and human health. The development

of effective biological control agents is therefore particularly needed to control this pest.

Our study1 aims to examine the virulence of different strains of entomopathogenic nematodes (EPNs)

against A. sordidus larvae in laboratory conditions.

The behaviour of 12 strains of EPNs belonging to 5 different species (Steinernema feltiae, S. carpocapsae,

S. affine, S. boemarei & Heterorhabditis bacteriophora) was analyzed. Ten Agriotes larvae were used

and each larva was exposed to 100 IJs in macroplates. The infestations were performed at two different

temperatures 18°C and 23°C, and the susceptibility of wireworm larvae to EPNs was measured after 72

h of contact. The wireworm larvae susceptibility was mainly dependent of the EPN strains and species,

but insecticidal performances are generally low. Only two strains of S. carpocapsae caused 50% mortality

of wireworm larvae at 18°C, whereas only one strain caused 70% mortality of wireworm larvae at 23°C.

Other bio-assays were performed in the same conditions for the 6 most efficient EPNs strains, but the

susceptibility of wireworm larvae after 72 h of contact was lower than 10% for all tested strains.

In order to facilitate the entrance of the EPNs in their host, we optimized the conditions of the bio-assays

by selecting wireworm larvae that are actively feeding. Three EPNs strains, two laboratory strains (S.

carpocapsae and H. bacteriophora) and one field strain of S. feltiae collected from an Agriotes cadaver,

were selected. Mortality rates of the wireworm larvae after 72 h of contact were low for the three EPNs

strains (respectively 16.7%, 6.7% and 16.7%), and no mortality was observed in the control.

Overall, our results are in agreement with previous studies which state that wireworms are generally

considered to be resistant to EPN infection. Further investigations at laboratory scale are needed to

better understand the interactions between EPNs and insect larvae, in order to improve their efficacy

to control wireworm pests. Furthermore, we are currently investigating the pathogenicity of the

bacterial symbionts of EPNs.

1 Thibord et al., 2017. Prévision des risques et élaboration de nouvelles techniques de lutte pour la

protection des cultures contre les attaques de taupins. Innovations Agronomiques 55 (2017), 215-233

Keywords: Biological control, wireworms, Agriotes sordidus, entomopathogenic nematodes, Steinernema,

Heterorhabditis


53


LA COLLECTION DE NEMATODES ENTOMOPATHOGENES DE L’INRA, RESSOURCES BIOLOGIQUES UTILISABLES COMME AGENTS

DE BIO-CONTROLE

Pagès Sylvie (1), Ogier Jean-Claude (1), Hellequin Manaël (1), Gaudriault Sophie (1), Givaudan Alain (1)

(1) Laboratoire DGIMI (Diversité, Génomique et Interactions Microorganismes-Hôtes), INRA, Université de

Montpellier, 34095 Montpellier, France

Les nématodes entomopathogènes (NEPs) sont utilisés depuis les années 70 comme auxiliaires pour la

défense des productions agricoles contre les insectes ravageurs et leur utilisation représente aujourd’hui

environ 20 % du marché des bio-pesticides. Ils sont surtout efficaces contre les Lépidoptères et les

Coléoptères. Ces NEPs appartenant aux deux genres Steinernema et Heterorhabditis véhiculent

respectivement Xenorhabdus et Photorhabdus (Enterobacteriacae) dans leur tube digestif. Après

infestation des larves d’insectes présentes dans les sols, la bactérie se multiplie et tue ses hôtes par

septicémie en 24 à 48 heures.

Depuis 1999, l’unité DGIMI héberge une collection unique de nématodes entomopathogènes vivants.

Aussi 26 espèces du genre Steinernema sont représentées comprenant 100 souches stockées à 8°C

et 3 espèces du genre Heterorhabditis comprenant 40 souches conservées à 15°C. Cette ressource est

unique et riche en diversité biologique, elle provient de sols d’origine géographique variée et

internationale. Nos missions sont de pérenniser dans le temps la viabilité et par conséquent le pouvoir

pathogène des souches par infestation naturelle sur insectes et de valoriser leurs utilisations comme

bio-pesticides. La collection est associée depuis 2016 au pilier environnement du centre de Ressources

Agronomiques pour la Recherche :

https://www.brc4env.fr/BRCs-and-collections/Invertebrates/Nematodes/Entomopathogenic-nematodes

La collection est régulièrement enrichie de nouveaux isolats à caractériser suite aux prospections

réalisées dans des réserves naturelles (forêt de Comté, réserve d’Eynes des Pyrénées) ou dans des sites

sauvages (bord de l’Hérault) et via notre réseau de collègues nématologistes internationaux (République

Tchèque, Liban, Afrique du Sud, USA, Irlande, Italie, Belgique, ...). Nous avons développé des outils

standardisés de caractérisation phylogénétique au niveau de l’espèce pour les NEPs (marqueurs

moléculaires ITS et 28S) et pour sa bactérie symbiotiquement associée (5 gènes ubiquitaires dnaN, recA,

gyrB, gltX, infB).

Suite à des difficultés de renouvellement et des pertes de viabilité de certaines souches de NEPs, nous

avons engagé un programme d’évaluation des températures de stockage et de production sur Galleria

mellonella (Lepidoptera). Nous évaluons l’efficacité du cycle parasitaire selon l’espèce en fonction de

trois températures 15°C, 18°C et 23°C en mesurant après infestation de l’insecte Galleria le % de

mortalité, le succès reproducteur (qualité et la quantité des stades infestants de nématodes sortant du

cadavre) et la survie des symbiotes.

In fine, ces bio-essais permettront d’optimiser la qualité de la production et de transmettre nos

expériences aux industriels producteurs de nématodes entomopathogènes.

Mots-clés : nématodes entomopathogènes, Steinernema, Heterorhabditis, bio-contrôle, température

https://www.brc4env.fr/BRCs-and-collections/Invertebrates/Nematodes/Entomopathogenic-nematodes


54

Session 4:

Plant and Natural products used as biocontrol

products


55


LES ACIDES AMINES POUR LUTTER CONTRE LES ADVENTICES

Claude Alabouvette (1), Cindy E. Morris (2), David C. Sands (3)

(1) Agrene, 47 rue Constant Pierrot F 21000 Dijon, (2) INRA PACA Pathologie végétale, 67 allée des chênes, CS 60094, F-84143 Montfavet, (3) Plant Sciences and Plant Pathology, Montana State University, Bozeman, MT 59717, USA

La lutte contre les adventices repose essentiellement sur des méthodes chimiques ou mécaniques. Bien que des chercheurs aient depuis longtemps proposé d’utiliser des microorganismes ou insectes attaquant spécifiquement la plante à détruire, la lutte biologique ne s’est jamais réellement développée. Une approche différente, mais complémentaire à l’utilisation de microorganismes est proposée (Sands et al. 2001) ; elle consiste à utiliser des acides aminés toxiques pour les plantes. Ces acides aminés peuvent être appliqués directement sur les plantes ou bien livrés par des microorganismes qui sont sélectionnés pour les excréter en abondance. Il est peu connu que certains acides aminés peuvent être toxiques pour les prototrophes comme les plantes. Cette toxicité est la cause d’une maladie du tabac « Frenching disease ». Celle-ci est due à la présence, dans la rhizosphère du tabac, de bactéries saprophytes hyperproductrices de leucine. L’ hyper-production de leucine inhibe l’activité d’une enzyme (acetolactate synthase) qui régule la production d’acides aminés indispensables au développement de la plante. L’étude des voies de biosynthèse des acides aminés a permis de montrer qu’il est possible d’inhiber le développement des plantes en favorisant l’installation, dans leur rhizosphère, de microorganismes hyper-producteurs d’acides aminés toxiques pour la plante. La mise en œuvre d’une telle stratégie de lutte consiste donc, dans une première étape à identifier les acides aminés toxiques pour la plante à détruire et dans une seconde étape à identifier des microorganismes capables de produire ces acides aminés dans la rhizosphère de la plante. Parmi différents exemples illustrant cette approche, la lutte contre une plante parasite de la famille des orobanches, à savoir Striga hermonthica mérite d’être présentée. S hermonthica provoque des dégâts considérables en Afrique sur de nombreuses cultures vivrières. Le challenge est ici de détruire la plante parasite sans affecter la plante hôte. Ayant identifié des acides aminés toxiques pour Striga, mais sans effet néfaste sur le développement de la plante-hôte, ce sont des variants de Fusarium oxysporum f.sp strigae, hyper-producteurs d’acides aminés, qui ont été sélectionnés pour excréter ces acides aminés dans la rhizosphère. Après avoir validé ce concept de lutte qui associe deux modes d’action complémentaires, le parasitisme de Fo strigae et la toxicité d’acides aminés vis-à-vis de Striga, une expérimentation portant sur 500 fermes a été conduite au Kenya, pendant deux années consécutives. Elle a permis de montrer l’efficacité de la méthode; par rapport au témoin et selon la période de culture, l’augmentation des rendements varie de 42 à 56%. Une autre innovation caractérise ce modèle, elle concerne la production in situ de l’agent de biocontrôle. Un inoculum « primaire » préparé dans un laboratoire est cultivé sur des cure-dents puis expédié sous cette forme aux utilisateurs qui le multiplient à la ferme, sur grains de riz bouillis puis l’appliquent à raison de 2g par trou de plantation (Nzioki et al. 2016). Longtemps négligée en Europe et particulièrement en France, la lutte biologique contre les adventices mérite d’être prise en considération et l’utilisation d’acides aminés ouvre des perspectives encore largement inexplorées. Seul un article récent rend compte des résultats d’une expérimentation réalisée au champ, pour lutter contre une orobanche du trèfle violet (Fernández-Aparicio, 2017). Mais l’exemple africain appelle également à réfléchir à l’approche européenne du biocontrôle. N’y aurait-il pas, à côté de l’approche industrielle, place pour une production décentralisée des agents de biocontrôle autorisés au niveau des exploitations qui vont les mettre en œuvre ? L’objectif de cette présentation est de communiquer sur cette stratégie de lutte biologique contre des adventices et d’ouvrir le débat sur son potentiel en Europe.

Mots clé : Acides aminés, Fusarium oxysporum, Striga


56


EFFECTS OF 3 FORMULATIONS BASED ON OYSTER SHELL AND NEEM ON GROWTH AND RESISTANCE OF COCOA SEEDLINGS IN NURSERIES

Dzelamonyuy A. (1,2), Magne P. T. F. (1,2), Foka T.E. (1,2), Tene Tayo P.M. (1,2),

Boudjeko T.* (1,2)

(1) Laboratory of Phytoprotection and Plant Valorization, Biotechnology Center, University of Yaounde 1. P.O BOX 3851 Messa Yaounde,

(2) Department of Biochemistry, Faculty of Science, University of Yaounde 1, P.O BOX 812, Yaounde, Cameroon. *Corresponding author: [email protected]

Cocoa (Theobroma cacao L.) culture in Cameroon represents nearly 30% of non-oil exports and

generates a revenue of over 100 billion CFA francs per year to more than 600,000 producers. This

has however been affected negatively by parasitic attacks (fungi; Phytophtora megakarya and insects;

Mirids and cochineals), poor quality of cocoa seeds and consequently falling prices of cocoa in the world

market. To counter the parasitic attacks, chemical pesticides have been used but the latter have proven

to be risky environmentally and health wise. Biocontrol methods have emerged as potential alternatives.

The aim of our study was developed a series of bio-agents capable of protecting plants against parasitic

attacks while maintaining a balance on quality of cocoa produced (organic) and being environmentally

friendly. Oyster shell was therefore used to formulate a bio-fungicide for substrate (soil) amendment

while Neem, citronella, and sunflower (Biopesticide 1) and Neem, corossol and calcium (Biopesticide 2)

were used to formulate two biopesticides with anti-insectidal and anti-fungal properties. The effects of

the oyster shell bio-fungicide were evaluated by treating the soil with cocoa seedlings in the nursery at

a concentration 10 % W/W every two weeks for three months. The two bio-pesticides formulated were

tested for stability and effects against insects in vitro and their effects evaluated in the nursery on cocoa

seedlings for 3 months. In the nursery the agro-morphological parameters of growth (height of stem,

number of leaves, leaf surface area, fresh and dry weight of roots) were evaluated for three consecutive

months every month. With the oyster shell based bio-fungicide, the load of P. megakarya in the soil was

equally determined after 12 weeks by measuring the speed of development of necrosis after inoculation

of cocoa pods with samples of soil suspensions from both treated and non-treated batches while the

two biopesticides were applied on the leaves every two weeks. For the three formulations, the level of

resistance was evaluated through quantification of the biochemical markers of resistance (total

polyphenols, flavonoids, tannins, total proteins, PR-proteins such as Polyphenoloxidases, peroxidases,

polyamine ammoniac lyase, β-1,3-glucanases and chitinases). Our biopesticides 1 and 2 showed strong

anti- insectidal effects in vitro as they demonstrated 100 % and 75 % ability to kill insects respectively.

We observed a slight increase in all the agro-morphological parameters of growth for plant treated with

any out three formulations. From a sanitary point of view, the soil samples amended with the organic

matter showed a weak level of the P. megakarya inoculum which was demonstrated by a low speed of

development of necrosis on cocoa pods. Plants treated with our formulations showed a weak disease

severity index on leaves which was correlated by significant increases in the level of biomarkers

synthesized by various plants from samples treated with our formulations. These significant increases

both in defense and growth markers demonstrate that our formulations are effective against various

pathogens affecting cocoa and constitute a potential bio-control agent against fungal and insect attacks

on plants.

Keywords: Cocoa, Phytophtora megakarya, insects, biopesticide, biocontrol, oyster shell, Neem.



57


OXYLIPINS ARE INVOLVED IN PLANT PROTECTIONS PROCESSES AND ARE POTENTIAL BIOCONTROL AGENTS

Estelle DEBOEVER (1,2), Manon GENVA (1,2), Mats X. ANDERSSON (3), Magali DELEU (2), Laurence LINS (2) and Marie-Laure FAUCONNIER (1)

(1) Laboratoire de Chimie Générale et Organique, Gembloux Agro-Bio Tech, Université de Liège, 2, Passage des Déportés, B-5030 Gembloux, Belgique

(2) Laboratoire de Biophysique Moléculaire aux Interfaces, Gembloux Agro-Bio Tech, Université de Liège, 2, Passage des Déportés, B-5030 Gembloux, Belgique

(3) Department of Biological and Environmental sciences, University of Gothenburg, Box 461, SE-405 30, Göteborg, Sweden

E-mails: [email protected], [email protected]

Nowadays, biopesticides have emerged as a main alternative to conventional agriculture. In this context,

plant oxylipins, a vast and diverse family of secondary metabolites originated from polyunsaturated fatty

acids (PUFAs), appear to be crucial agents in plant defence mechanisms. Among plant oxylipins, the 13-

hydroperoxy oxylipins (13-HPO) constitute key intermediate oxylipins (KIOs) as they can be converted

into jasmonic acid, OPDA, dn-OPDA or traumatic acid, well-characterized components involved in plant

resistance mechanisms [1][2]. Their presumed functions include direct antimicrobial effect, stimulation

of plant defence gene expression, and/or regulation of plant cell death [3]. Otherwise, OPDA and dn-

OPDA were also found esterified in more complex structures such as galactolipids. Those compounds

are called arabidopsides. However, the precise contribution of each of those molecules in plant defence

remains unknown.

The first part of this study aims to understand the oxylipins action mechanisms and especially their

membrane activities. As arabidopsides are produced under stress and localized at the chloroplast

membranes, their interactions with those were studied using biomimetic membranes via a

complementary in silico informatics and in vitro biophysical approaches. On the other hand, as KIOs are

found in the literature to be potential biocontrol agents, there effect on different pathogens of

agronomic interest were studied in vitro, by the same approach.

As far as arabidopsides are concerned, results show that they possess different interfacial properties

compared to major chloroplast lipids, which they are produced from. Arabidopsides modify the fluidity

and permeabilize chloroplast membranes. As chloroplast membrane lipid composition is essential to its

photosynthetic ability, such changes in its composition under stress will affect its function. Concerning

KIOs, they seem to interact with pathogens plasma membranes. Indeed, in vitro assays show that KIOs

can hinder growth of some plant microbial pathogens, with differences between strains and KIOs forms.

Keywords: oxylipins, arabidopsides, fatty acid hydroperoxides, antimicrobial activities, biomimetic membranes

Acknowledgment: Estelle Deboever is supported by a grant from the Formation à la recherche dans l’Industrie et l’Agriculture (FRIA).

References :

[1] Jones and Dangl 2006. Nature 444, 323-329.

[2] Mosblech et al. 2009. Plant Physiol Biochem 47, 511-517.

[3] Prost et al. 2005. Plant Physiol.139, 1902–1913.




58


ARE ESSENTIAL OILS EFFICIENT AS BIOPESTICIDES AGAINST SAP-SUCKING PESTS? A BIOASSAY SCREENING AT DIFFERENT SCALES

S. Benhamou (1), T. Malanga (1), B. Gard (3), T. Michel (2), M. Laigle (2), N. Desneux (1), AV. Lavoir (1)*

(1) Université Côte d'Azur, UNS, INRA, CNRS, ISA, France. 400 Rte des Chappes, 06903 Sophia-Antipolis Cedex FRANCE (2) Université Côte d'Azur, CNRS, Institut de Chimie de Nice, UMR 7272, Nice, France (3) CTIFL Operational Center of Balandran, 751 chemin de Balandran, 30127 Bellegarde, France


In the last decades, aromatic plant essential oils (EOs) have received growing attention as a low-risk

alternative to pesticides in integrated pest management owing to their low mammalian toxicity and

environmental impact.

The toxicity of various Mediterranean EOs experimental formulations was evaluated to assess their

insecticidal activity against two world-wide distributed sap-sucking pests, the silverleaf whitefly Bemisia

tabaci (Gennadius) and the potato aphid Macrosiphum euphorbiae (Thomas).

Two EOs ways of exposure - by contact or by fumigation - were tested in bioassays at leaf scale. All six

oils showed dose-dependent insecticidal activity against both insects in contact experiments, but high

mortality rates were always associated with severe phytotoxicity on tomato leaflets. This effect might

have been accentuated as the polysorbate surfactant (Tween®) alone exhibited severe chlorosis in

recommended concentration on tomato leaves, questioning its routine use in EOs bioactivity

assessment. Fumigation seems to be a good alternative to reduce phytotoxicity. This way of exposure

allowed Rosemary and Mugwort oils to be highly effective against B. tabaci, as well as green anise and

fennel oils against M. euphorbiae, all inducing more than 90% of mortality due to a suspected neurotoxic

effect at non-phytotoxic doses.

This effect was partly found again at plant scale but also at greenhouse scale. In order to identify the

blend of active compounds, the volatile constituents of EOs were analyzed by a combination of GC-

FID/MS. This study provides a new potential resource to develop more eco-friendly means of sap-sucking

pest control in greenhouse crops through essential oil vapour-phase exposure.

The experiments received financial support from the ERA-NET ARIMNet2, project STomP and from the

European Union, EUCLID project: H2020-SFS-2014-2 Sustainable Food Security, Research and Innovation

Action

Keywords: entomotoxicity, phytotoxicity, ecotoxicology Bemisia tabaci, Macrosiphum euphorbiae, tomato crop, biocontrol, IPM strategies,


59


POTENTIALITES BIOINSECTIDES DES EXTRAITS BRUTS DE DEUX PLANTES SAHARIENNES ENVERS LE PUCERON NOIR

DE LA FEVE APHIS FABAE

Acheuk Fatma (1), Abdellaoui Khemais (2), Belaid Messaouda (1), Lakhdari Wassima (3), Dehliz Abderrahmène (3), Baba Khouya Noura (1) et Rezig Fadhila (1)

(1) Laboratoire Valorisation et Conservation des Ressources Biologiques, Faculté des Sciences, Université de Boumerdes,

Algérie. (2) Département des Sciences Biologiques et de la Protection des Végétaux, Institut Supérieur Agronomique de Chott-

Mariem, Université de Sousse, Tunisia (3) Institut National de Rercherche Agronomique, Station de Sidi Mehdi, Touggourt, Algeria

Le recours au traitement par les métabolites secondaires des plantes à activités insecticides constitue

une des plus grandes préoccupations scientifiques des chercheurs s’intéressant au domaine de la lutte

contre les insectes ravageurs des cultures. La richesse et la diversification de la flore algérienne,

notamment celle du Sahara, en espèces d’intérêt biologique a ouvert des axes de recherches en lutte

biologique par l’utilisation des molécules bioactives des plantes. Deux espèces sahariennes, Euphorbia

guyoniana et Cotula cinerea ont été explorées sur la plan phytochimique et insecticide. La présente

étude s’intéresse à l’évaluation de l’activité insectidicide des extraits éthanoliques de ces deux plantes

sahariennes sur les adultes du puceron noir de la fève Aphis fabae. L’étude a été ménée en testant une

gamme de cinq doses par contact : 1,16 ; 3,12 ; 6,25 ; 12,5 et 25 mg/mL. Les analyses phytochimiques

effectuées ont permis d’identifier les différents groupes chimiques présents dans les extraits des deux

plantes étudiées. Ces tests phytochimiques ont mis en évidence la présence massive des flavonoïdes,

des tanins notamment les tanins galliques, des alcaloïdes, des saponosides et des glucosides. Les deux

plantes sont dépourvues d’anthocyanes, des leuco-anthocyanes, des tanins cathéchiques, des

coumarines et des iridoides.

Les résultats des essais insecticides des extraits bruts des deux plantes montrent qu’à la plus forte dose

(25 mg/mL), les 100 % de mortalité sont obtenus 72 h après traitement. Alors qu’à la plus faible (1,16

mg/mL), les 100 % de mortalité sont enregistrés 144 h et 168 h après traitement respectivement pour

l’extrait de E. guyoniana et C. cinerea. La valeur de la DL50 obtenue au temps le plus court (6 heures

après traitement) est de l’ordre de 6,76 mg/mL pour l’extrait d’E. guyoniana tandis que pour le deuxième

extrait, elle est de 10 mg/mL. Ce résultat explique l’effet insecticide puissant de la première plante E.

guyoniana.

Au vue de ces résultats, il apparait que les extraits de ces deux plantes sont dotés d’activité insecticide

et qu’après un complément d’étude les extraits de ces deux plantes peuvent être impliqués dans des

programmes de lutte intégrée contre cet insecte nuisible.


60


ESSENTIAL OILS FROM THREE ALGERIAN SPECIES OF APIACEAE AS NEW BIO-HERBICIDE

N.Merad (1), V.Andreu (2), S.Chaib (2), C. Bertrand (2,3), N.Djabou (1)

(1) COSNA, Université de Tlemcen, BP 119, Tlemcen 13000, Algérie, [email protected]. (2) Akinao, 8 AV Paul Alduy, 66000 Perpignan. (3) CRIOBE USR3278, Université de Perpignan Via Domitia, F66860, France.

Potential damage to human health and to the environment from herbicides is regarded as a real problem

today. It has resulted in an increased interest in alternative strategies leading to the development of

biodegradable compounds. Inhibition of growth of plants by other plants in their vicinity has been known

for a long time. The chemical interaction between plants, which can cause enhancement or inhibition of

growth, has been named allelopathy [1]. The latter refers to beneficial or harmful effects on a plant by

another plant through the release of plant chemicals by leaching, root exudation, volatilization and

decomposition, among other processes. The main effects that are commonly cited include reduced seed

germination and seedling growth.

As synthetic herbicides, there is a common mode of action or a physiological destination for all

allelochemicals. However, the actions of some allelochemicals include changes in cell division, pollen

germination, nutrient uptake, photosynthesis and a specific enzymatic function [2]. As natural inhibitors

of plant germination and growth, essential oils could be used as alternative bioherbicide.

The essential oils were extracted from 3 different species of the family Apiaceas; Smyrnium Olusatrum,

Daucus Crinitus, Eryngium Triquetrum, collected in western Algeria. The objectives of this study were to

characterize the chemical composition and evaluate the allelopathic effect of these three essential oils

that are rich in oxygenated terpenic and sesquiterpene compounds. The phytotoxic activity was

evaluated on the basis of inhibition rate of i) growth the model watercress (Lepidium sativum) ii)

thylakoids activity. The results obtained with the essential oil of S.Olusatrum reveal a phytotoxic activity

on the growth of Cresson seeds with a 65% inhibition at 82 mg / ml and a photosynthesis inhibition of

74% at 0.01 mg / ml. The possible use of essential oils as herbicides is discussed.

References:

[1] n. Dudai, a.poljakoff-mayber, a.m. Mayer, “essential oils as allelochemicals and their potential use as bioherbicides,”

Chemical Ecology., 1999, 25, 5, 1079–1089.

[2] Th. N. Füllera, C. Bertrand, B. Antonelloa, “Chemical diversity of wild populations of Elionurus muticus (Spreng.) and the

allelopathic effect of its essential oil,”Essential Oil Research,2017,1-6.

Keywords: Bio-control; Essential oils, bio-herbicide, Smyrnium Olusatrum, Daucus Crinitus, Eryngium Triquetrum


61


EVALUATION OF MICROALGAE EXTRACTS EFFICIENCY AGAINST FUSARIUM ROOT ROT IN WHEAT

Aline Brutel (1), Wafa Achouak (2), Jean-Paul Cadoret (3), Sonia Hallier (4), Thierry Heulin (2),

Pierre Joly (1), Pierre Ruaud (5), Carole Vialleix (3), Aurélie Wauquier (1), Jonathan Gerbore (1)

(1) Biovitis, Le Bourg, 15400 Saint-Etienne-De-Chomeil, France (2) CEA, Lab Ecol Microb Rhizosphere & Environ Extrem (LEMiRE), 13108, Saint Paul-Lez-Durance, France (3) CNRS, BVME UMR 7265, ECCOREV FR 3098, 13108, Saint Paul-Lez-Durance, France (4) Greensea, Prom. Sergent Jean Louis Navarro, 34140 Mèze, France (5) Bio-Eva, Kermorvan, 29410 Guiclan, France (6) Limagrain, 15 route d’Ennezat, 63720 Chappes, France

The objective of the MOPAD project (Micro-Organisms for a Sustainable Agriculture) is to develop

biocontrol solutions using micro-organisms or micro-organism extracts to control fusarium in bread

wheat, a fungal disease causing yield losses and a decrease of use value of grains. The MOPAD

consortium involves the SME Biovitis (project coordinator), Limagrain group and two research institutes

of CEA’s Life Sciences Division.

Among the tested solutions (including bacteria, fungi…), a part of the project focused on the potential

of microalgae to control fusarium disease. Due to their nature, these extracts of microalgae were more

susceptible to induce systemic resistance in wheat than fighting directly the pathogens with antagonistic

behavior. Thus, we designed a specific protocol to evaluate their efficiency against two Fusarium species.

A previous screening on 50 extracts was made on Fusarium culmorum and the 11 best candidates were

kept for a second screening on Fusarium graminearum.

The second screening phase of this protocol, (i.e. the evaluation of extracts phytotoxicity, effects on

spores germination and roots & aerial parts notations) will be presented. Promising extracts with high

in vitro efficiency have been identified and selected for further screening experiments in realistic in vivo

conditions.

At the end of the project, competitive advantages are foreseen. First, in terms of performance,

developed products will be at least as efficient as chemical products to reduce pest potential of

Fusarium. Second, the project meets strong societal expectations and will give results at a good time

given European regulation changes, and the integration of constraints faced by seed producers and the

agri-food sector.

Keywords: fusarium, microalgae, biocontrol, MOPAD


62


SCREENING FOR NATURAL UV ABSORBERS : RESIDUAL BIOLOGICAL ACTIVITY

Midthassel Audun (1), Oger Julien (1), Crevel Francois-Xavier (2)

(1) Certis Europe BV, 5 rue Galilée, 78280 Guyancourt, France (2) Japan Agro Services SA, Lieu-dit Cabreirolles, Route de Beaucaire, 30320 Marguerittes, France

Exposure to natural light can lead to photolysis, or photodegradation, of plant protection products

shortening their residual activity, which can reduce their overall efficacy. Biorational plant protection

products, examples of which include microorganisms (and their metabolites), viruses, plant extracts and

pheromones are often photolabile. It is primarily the UV wavelengths that are responsible for photolysis

and the level of photolability can be studied under artificial UV light or a UV-sun lamp emitting the

wavelength distribution of natural light.

Natural pyrethrum is a botanically derived insecticide (extracted from the seeds of Chrysanthemum

cinerariifolium) containing a mixture of 6 natural pyrethrins. These are biologically active with rapidly-

acting ‘knock-down’ activity on a broad spectrum of insect pests. The relatively short persistence of

natural pyrethrum on plant surfaces is primarily due to the photolability of the pyrethrins. Improving

the photostability of the pyrethrins can yield a prolonged residual activity in the field and therefore more

efficacious pest control.

This study focused on the screening of natural UV absorbers and antioxidants in order to reduce the rate

of photodegradation of natural pyrethrum. Through accelerated exposure to simulated natural light

using an OSRAM UV-sun radiation lamp, samples were exposed to light 16 times stronger than natural

light at midday. Natural pyrethrum in combination with UV absorbers were exposed to UV-light as dry

residue on leaf discs and the residual biological activity of the pyrethrum evaluated by inoculating aphids

(Myzus persicae) onto the UV-treated residues and assessing subsequent rates of mortality. Pyrethrum

in combination with an oligosaccharide, an ethoxylated plant oil and a phenolic phytochemical resulted

in significantly improved residual activity.

Mots-clés : Photodegradation, natural UV absorbers, natural pyrethrum, pyrethrins, aphids


63


AGRONOMY OF A SPANISH POPULATION OF SATUREJA MONTANA L. AND EESSENTIAL OIL/HYDROLATE VALORIZATION

J. Navarro-Rocha (1,6), J. Burillo1, M. Bailén (2), M. Fe Andrés (3), C. Elisa Díaz (4), A.M.R. Pino-Otín (5,6), D. Ballestero (5,6) , J. Val (5) and A. González-Coloma (3)

(1) Centro de Investigación y Tecnología Agroalimentaria de Aragón, CITA, Spain. Email: [email protected] (2) Universidad Europea de Madrid, UEM, Spain (3) Instituto de Ciencias Agrarias, ICA, CSIC, Spain. (4) Instituto de Productos Naturales y Agrobiología IPNA CSIC, Spain (5) Universidad San Jorge, Campus Universitario Villanueva de Gállego, Zaragoza, Spain. (6) Grupo GATHERS, Universidad de Zaragoza, c/ Mariano Esquillor s/n, 50018-Zaragoza, Spain.

Satureja montana L., is a bushy perennial subshrub with woody stems at the base, small linear leaves,

pale pink and white flowers [1]. This plant is abundant in the Mediterranean basin and in Spain grows in

mountain areas as a ruderal species. It colonizes arid, stony and lime-filled soils, especially in high

altitudes [2, 3]. S. montana has developed several morphological and physiological adaptations,

affecting oil yields and composition [4]. The aim of this study was the pre-domestication of S. montana

to develop a chemically stable population and the valorization of its essential oil and hydrolate

byproduct for the production of biopesticides.

A selected population (MSAMO-7) was cultivated for four growing seasons (2011-2014) to give a pre-

domesticated one (SAMO-0). The plants were harvested yearly to evaluate production (fresh and dry

biomass), essential oil yield from steam and hydro-distilled biomass (% and l/ha) and plant mortality.

The population SAMO-0 maintained a stable yield of dry material (44.5%) and essential oil (0.45%) per

year.

The essential oils from the different crops of the pre-domesticated population SAMO-0 showed

carvacrol as the major component, little variation in their chemical composition and strong biocidal

effects (antifungal, nematicidal and insect antifeedant effects). The essential oils and hydrolyte

presented strong phytotoxic effects. [1] A. Wesolowska, M. Grzeszczuk, D. Jadczak. Notulae Botanicae Horti Agrobotanici Cluj-Napoca. 2014, 42: 392-397. [2] J. Burillo. Investigación y experimentación de plantas aromáticas y medicinales en Aragón: cultivo, transformación y analítica, 262 p. 2003. [3] AP. Cunha, JA. Ribeiro, OR. Roque. Plantas Aromaticas em Portugal Caracterizac ̧̃ao e Utilizacoes. Fundacao Calouste Gulbenkian. Lisboa. 2007. [4] S. Mirjana, B. Nada. Journal of Essential Oil Research. 2004, 16: 387-391.

Financial Support: CTQ2015-64049-C3-1-R (MINECO/FEDER), Gobierno de Aragón FSE (Grupo GATHERS E39_17R).

Keywords: Satureja montana L., pre-domestication, essential oil, hydrolate, biopesticide, production and valorization


64


HOW COULD THE HERBICIDAL EFFECTS OF SELECTED ESSENTIAL OIL COMPOUNDS BE RELATED TO THEIR MEMBRANE ACTIVITY ?

Lins L. (1), Bettaieb I. (2), Dal Maso S. (3), Kenné T. (2), De Clerck C. (3),

Deleu M. (1), Jijakli H. (3), Fauconnier ML. (2)

(1) Laboratoire de Biophysique Moléculaire aux Interfaces, Gembloux Agro Biotech, University of Liege, Belgium (2) Laboratoire de Chimie Générale Organique, Gembloux Agro Biotech, University of Liege, Belgium (3) Laboratoire de Phytopathologie, Gembloux Agro Biotech, University of Liege, Belgium

The European legislation on plant protection products (PPP) is about to undergo important changes in

the coming years. The chemical PPP destined to be removed from the European market are responsible

for the appearance of resistance phenomena to plants pests. It is therefore necessary to explore new

alternatives, one of them being the search for natural herbicides. Essential oils (EO) could provide a

welcome alternative due to their proven activity as PPP. Even if these compounds seem to have a bright

future as PPP, their activity is mainly approached through empirical observations. EO are also a complex

mix of of different molecules that could act individually or in synergy. Particularly, very little is known

when it comes to molecular mechanisms of action and the relations between structure and activity of

the active compounds. This study aims to investigate the structure/activity relationships of some EO

molecules, among which cinnamaldehyde (CIN) from cinnamon and citronellal and citronellol from

lemongrass.

Due to their lipophilic properties, EO tend to interact with one or more of the layers found in the outer

plant tissues, among which the cell membranes. For cinnalmaldehyde (and other EO compounds), it has

been shown that they are able to interact with bacterial phospholipids and induce change in lipid

organization (fluidity, packing, ...) on model lipid monolayers (Nowotarska et al, 2014). Citronnellol was

notably shown to displace cholesterol from its phospholipid complexes (Lange, Y et al, 2009). However,

nothing is known about any interaction with lipids specific to plant plasma membrane (PPM). In this

study, we analyzed the effects of the above three EO compounds on model PPM by complementary in

vitro and in silico biophysical approaches. We showed that the three compounds have differential effects

on plant lipids and different herbicidal properties on plantae. While part of the herbicidal activity could

be related to membrane perturbation, some clues remain to be elucidated.

Future studies at a molecular point of view would help to better decipher the herbicidal action involving

the membrane, other outer plant tissues such as the cuticule and/or and a potential effect on EO

compounds on proteins or genomic DNA, as it was shown for CIN on E. Coli (He, TF et al, 2018).

References: Nowotarska et al, Molecules, 2014, 19, 7497 He et al, J. Photochem and Photobiol, 2018, 178, 623

Keywords: essential oils, plasma membrane interaction, structure/activity relationships, herbicidal effect


65


ENCAPSULATION OF CYMBOPOGON WINTERIANUS JOWITT. AND CINNAMOMUM VERUM J. PRESL ESSENTIAL OILS WITH

GLYCERODENDRIMERS IN ORDER TO CREATE A BIO HERBICIDE

Chloé Maes (1,2), Sandrine Bouquillon (1), Marie-Laure Fauconnier (2)

(1) Institut de Chimie Moléculaire de Reims, UMR CNRS 7312, Université de Reims-Champagne-Ardenne, UFR Sciences, BP 1039 boîte 44, 51687 Reims Cedex 2, France

(2) Gembloux Agro-Bio Tech, Université de Liège, 2 Passage des Déportés, 5030 Gembloux, Belgique

Essential oils have already proven their ability for biocontrol such as herbicide or fungicide properties,

but they are characterized by a high volatility, which beget a challenge: increase the time during the

essential oil act efficiently at a given place. In this work, an innovative matrix for essential oils

encapsulation is proposed. Indeed, glycerol carbonate surface-modified dendrimers have shown their

capacity to encapsulate some metallic complexes and organic compounds. As a consequence, the final

goal is to produce an efficient bioherbicide based on a glycerodendrimer - essential oil combination.

More precisely, two types of dendrimers (glycero poly(propylenimine) and glycero poly(aminoamine))

and two essential oils (Cymbopogon winterianus J. and Cinnamomum zeylanicum Blume) have been

used. Both essential oils have been chosen because of their herbicide properties. Dynamic headspace

gas chromatography with mass spectrometry have allowed to measure the total encapsulation rate and

then to quantify the main compounds encapsulated. Results show that dendrimers encapsulate

essential oils with some efficiency and that there is an influence of the dendrimer size, the

concentration, the agitation duration on the encapsulation rate.

Furthermore, the interaction between glycerodendrimers and essential oils could be studied by nuclear

magnetic spectrometry. During this period, efficiency of created products will be controlled by an

analysis of the inhibition of mono- and dicotyledonous seeds germination in petri dishes and the

inhibition of the growing of new leaves of Arabidopsis Thaliania (L.) Heynh.

The poster will summarize the optimization results and some information about the possible

relationship between dendrimers’s structure and biocontrol.

Figure 1. Encaspulation of Cymbopogon winterianus J. major coumpounds (citronellal, geraniol and limonene)

in the second generation of glycerodendrimer poly(prolynimine) (GD-PPI-2).

Keywords : dendrimer, essential oil, bio herbicide, encapsulation


66


EFFET BIOCIDE DES HUILES ESSENTIELLES ET DE LA MENTHE POIVREE (MENTHA PEPERITA) ET DU PIN MARITIME (PINUS PINASTER) SUR LA LONGEVITE DES ADULTES MALES DIAPAUSANTS DE LA BRUCHE DE LA

FEVE (BRUCHUS RUFIMANUS)

Medjdoub-bensaad F. (1), Hamani-Aoudjit S. (2) et Goucem-Khelfane K. (3)

1, 2 et 3: laboratoire de production, sauvegarde des espèces menaces et des récoltes. Département de

Biologie Animale et végétale. Faculté des sciences biologiques et des sciences agronomiques. Université

Mouloud Mammeri de Tizi-Ouzou. [email protected]

La bruche de la fève, Bruchus rufimanus (Coleoptera : Chrysomelidae) est insecte dont les adultes se

nourrissent de nectar et de pollen de la plantes hôte Vicia faba. Les larves se développent aux dépens

des réserves contenues dans les cotylédons des graines de fève qui perdent leur qualité gustative et leur

pouvoir germinatif. L’utilisation des plantes dotées de propriétés insecticides représente une solution

alternative à la lutte chimique pour la protection des récoltes et préserver l’environnement.

Ainsi, la toxicité des huiles essentielles de la menthe poivrée et du pin maritime a été testée vis-à-vis des

adultes mâles de B.rufimanus durant la période de diapause.

Le traitement pat inhalation par les deux huiles essentielles de la M.peperita et de P.pinaster présentent

un effet toxique sur les bruches où la mortalité est de 100% après 24 heures d’exposition à la plus faible

dose de 0,5µl.

Le test par contact montre une efficacité au bout de 8h d’exposition de l’huile essentielle de M.peperita

à la dose 6µl où 35% des mâles sont morts et 100% au bout de 24 heures d’exposition.

Pour l’huile de P.pinaster la mortalité de 100% est observée au bout de 24h pour la dose de 8µl.

Mots clés : Bruchus rufimanus, menthe poivrée, pin maritime, huiles essentielles, toxicité



67


STUDY OF THE LARVICIDAL ACTIVITY OF HYDRO-ALCOHOLIC EXTRACTS OF NERIUM OLEANDER L. AND RICINUS COMMUNIS L.

ON TUTA ABSOLUTA (MEYRICK)

Osmane Badiaa (1), Boualem Malika (2), Saiah Farida (3)

Laboratory of Plant Protection, Laboratory of Plant Biochemistry. Department of Biotechnology, Abdelhamid Ibn Badis-Mostaganem University, Algeria [email protected] +(213)556796877

The purpose of the current study is to suggest alternative solutions based on the use of natural products

“bioinsecticides of plant origin”, to fight against the tomato leafminer, Tuta absoluta (Meyrick), which is

considered as a serious threat of tomato production in Algeria. To meet this objective, we evaluated the

toxicity of hydro-methanoic, hydro-ethanoic and synergistic extracts of tow toxic plants, including

Nerium oleander; Ricinus communis growing spontaneously in the province of Mostaganem, on larvae

of T. absoluta.

The extraction yields (Method of Soxhlet) obtained by the two hydro-methanoic and hydro-ethanoic

extracts are: (26%, 18%) for N. oleander and (35%, 29%) for R. communis respectively; However, the

hydro-methanoic extract remains the most effective. These extracts were tested adopting the method

of toxicity by direct contact or spray.

The biological tests on the larvae of T. absoluta have shown that there is a difference between the

toxicity of hydro-methanoic and hydro-ethanoic extracts within the same plant. The synergistic extract

(mixture of hydro-methanoic extracts of the two plants) caused a remarkable mortality rate that

exceeded 90% mortality. While N. oleander and R. communis presented a low toxicity not exceeding

30% for the hydro-ethanoic extracts and 40% for the hydro- methanoic extracts. After 24 hours of

exposure to the test, the LD50 values were very high (above 50%) for hydro-ethanoic and hydro-

methanoic extracts of the two plants used. However, the LD50 values were relatively low for the

synergistic extract (19%). The results showed that L2 and L1 larvae are most sensitive to all extracts used.

While, the L3 and L4 are the most resistant, except for the synergistic extract.

Keywords: Bioinsecticides, Tuta absoluta, Nerium oleander, Ricinus communis, Toxicity, Hydro-alcoholic extracts



68


LES HUILES ESSENTIELLES COMME ALTERNATIVES DE CONTROLE D’INSECTES DE DENREES STOCKEES ET VECTEURS DE MALADIES AU

BURKINA FASO

Sanon Antoine, Wangrawa Dimitri, Ilboudo Zakaria, Badolo Athanase

Laboratoire d’Entomologie Fondamentale et Appliquée, UFR Sciences de la Vie et de la Terre. Université Ouaga I Pr Joseph KI-ZERBO. 03 BP 7021 Ouagadougou 03. Burkina Faso. Email : [email protected]

L'utilisation d’insecticides de synthèse pour contrôler les insectes nuisibles est une pratique courante

qui représente cependant un risque pour les utilisateurs, l'environnement et pourrait également

exacerber le contrôle des insectes cibles suite au développement de la résistance. Des études réalisées

au Burkina Faso ont permis de montrer l’existence d’un potentiel insecticide dans plusieurs huiles

essentielles issues de la flore locale. Ainsi, dans le but de trouver des alternatives efficaces aux

insecticides de synthèse, une évaluation plus approfondie de ces huiles a été conduite en ciblant d’une

part, des insectes ravageurs des stocks de graines de niébé et, d’autre part, des vecteurs de maladies.

Les huiles essentielles provenant de plantes aromatiques, Ocimum canum, Hyptis suaveolens, Hyptis

spicigera, Lippia multiflora et Lantana camara, ont ainsi fait l’objet de recherches.

Les effets comparés de ces huiles essentielles sur Callosobruchus maculatus, le principal ravageur du

niébé, Vigna unguiculata Walp, en stockage, mettent en évidence une activité insecticide marquée des

HE sur les adultes et, dans une moindre mesure, sur les stades immatures de cet insecte. L’HE de

O. canum est dans tous les cas la plus efficace et la plus rémanente en conditions ambiantes.

Les recherches sur les effets des mêmes huiles essentielles sur Anopheles gambiae s.l. ont mis en

évidence de fortes activités larvicide, adulticide, excito-répulsive et d’inhibition de la ponte des femelles

gravides de moustiques. L’HE de L. camara s’est illustrée avec une plus grande toxicité vis-à-vis des œufs

(CL50 =70,70 ppm), des larves (CL50 =102,7 ppm pour An. coluzzii et CL50 = 92,2 ppm pour An. gambiae)

et des adultes (CL50 = 0,84 %). L’inhibition de la ponte a été supérieure à 95 % avec toutes les HE testées

à partir de 500ppm.

Il ressort des études réalisées que les huiles essentielles ont une activité biologique variable en fonction

des nuisibles ciblés. Ainsi, pendant que l’HE de O canum était la plus efficace sur C. maculatus, c’est celle

de L. camara qui s’est révélée la plus prometteuse sur les moustiques. Ces résultats appellent à la prise

en compte de la spécificité des HE et des espèces d’insectes nuisibles pour une utilisation optimale de

ces alternatives prometteuses.

Mots-clés : Alternatives de lutte, Insectes de stocks, vecteurs de maladies, huiles essentielles, Afrique de l’Ouest



69


BIOLOGICAL ACTIVITIES OF ESSENTIAL OIL AND HYDROSOL EXTRACT OF CENTAUREA ACAULIS L

Mohammed Semaouia, Nabila Ain Sebaa*(1), Mohammed El Amine Dib (1),

Boufeldja Tabtia (1), Jean Costa (2) and Alain Muselli (2)

(1) Laboratoire des Substances Naturelles et Bioactives (LASNABIO), Université de Tlemcen, BP 119, 13000, Algérie (2) Laboratoire Chimie des Produits Naturels, Université de Corse, UMR CNRS 6134, 7 Campus Grimaldi,

BP 52, 20250 Corte, France

Objectives: This study aimed to chemically characterize the essential oils of Centaurea acaulis and to

evaluate their antioxidant and antimicrobial activities.

Methods: Essential oils were obtained by a Clevenger-type apparatus and analyzed by using Gas

Chromatography (GC) and Gas Chromatography Mass Spectroscopy (GC/MS). Antioxidant activity was

evaluated by free radical-scavenging capacity, β-carotene bleaching and FRAPS methods. The

antimicrobial activities were tested to four phytopathogenic stains. Results: Altogether, forty-two

compounds were identified in the essential oils of aerial part with sixteen compounds in roots parts,

representing 0.16% and 0.07% of the total oil composition respectively.

The essential oil of roots was dominated by oxygenated sesquiterpenes (33,0%) and sesquiterpeniques

hydrocarbons with 3 derivatives (10.5%). While, aerial part was constituted by oxygenated

sesquiterpenes (66.2-68,4%) and oxygenated diterpenes (5.3-6.4 %). The main components in both

aerial and roots parts were caryophyllene oxyde (27.6-29.6 %), verridiflorol (11.5-12.8 %), β-eudesmol

(6.7-7.2 %), (E)-phytol (5.2-6.3%), α-pinene (1.7-3.1 %), myrcene (0.9-1.7 %) and limonene (2.7-4.8 %).

The best antioxidant activity was exhibited by aerial part essential oil at low concentrations (IC50 of

73.01 μg/mL and 114.20 μg/mL) for DPPH essay and (IC50 40.60 μg/mL; 44.32 μg/mL) for β-carotene

bleaching of aerial and roots parts respectively. However, a high result of ferric reducing antioxidant

power for essential oil tested was observed. Antibacterial tests showed antibacterial activity of roots

part against only gram-positive bacteria with inhibition diameter of 10 and 12 mm against

Staphylococcus aureus and Bacillus cereus respectively.

Conclusion: The essential oil can be used as a potential source of sustainable eco-friendly botanical to a

variety of food commodities.

Keywords: Centaurea acaulis, essential oils composition, antioxidant activities, antibacterial activity


70


POTENTIAL OF PLANT EXTRACTS AS CONTROL TOOL FOR THE SOUTH AMERICAN TOMATO PINWORM (TUTA ABSOLUTA)

Orlando Campolo (1), Michele Ricupero (2), Antonio Biondi(2), Gaetano Siscaro(2), Vincenzo Palmeri(1), Thomas Michel (3), Lucia Zappalà (2)

(1) University of Reggio Calabria, Department of Agriculture, loc Feo di Vito, 89122 Reggio Calabria, Italy. (2) University of Catania, Department of Agriculture, Food and Environment, via Santa Sofia 100, 95123, Catania, Italy. (3) Université Côte d’Azur, CNRS, Institut de Chimie de Nice UMR7272, Nice, France.

The implementation of environmental friendly methods for the management of key crop pests is

strongly required both by public opinion and European regulation. Among sustainable control tools

botanical insecticides, and in particular plant essential oils (EOs) have shown a good potential in

controlling insect pests, as well as managing plant pathogens. Tomato has a very high social and

economic relevance in Europe and the whole Mediterranean area. Several key insect pests (e.g. Tuta

absoluta, aphids and whiteflies) affect the tomato industry in the Mediterranean. Their control often

relies on agrochemicals which can disrupt the existing integrated pest management programs (IPM) and

have unwanted side effects.

In this context, laboratory trials were conducted to assess the insecticidal activity, through contact and

ingestion exposure route of seven commercial essential oils (EOs), against T. absoluta. Toxicity to various

instars and repellency towards adults, as well as the potential toxic effects on tomato plants, were

evaluated. Chemical characterization of the tested EOs was carried out by GC-MS and GC/FID. Both the

zeta potential (ζ) and the particle size distribution of the formulations developed were assessed.

Fennel and anise nanoemulsions caused significant mortality of T. absoluta eggs. In the ingestion toxicity

trials, all the tested EOs caused significant mortality. Fennel, artemisia, sage, lavender and peppermint

EOs significantly reduced the number of laid eggs on sprayed plants. The formulations developed had

nanometric scale dimensions (113-146 nm) and good stability over time (average ζ: -15.4 mV). Chemical

characterization of the tested EOs highlighted some simpler [e.g. anise and clove EOs were mainly

constituted of anethol (87%) and eugenol (91%), respectively] and some more complex phytochemical

profiles.

These results provide the scientific bases for further studies aimed at integrating these EOs into IPM

packages in tomato.

Keywords: Essential oil, IPM, biopesticide, Tuta absoluta, gas chromatography


71


CHEMICAL CHARACTERIZATION OF THE NEMATICIDAL AGENTS FROM MENTHA SUAVEOLENS HYDROLATE.

Carmen E. Díaz (1), Luis F. Julio (2), L. Quesada (2), M. Fe Andrés (2), Juliana Rocha (3),

Azucena González-Coloma (2)

(1) Instituto de Productos Naturales y Agrobiología, CSIC, Avda. Astrofísico F. Sánchez 3, 38206 La Laguna, Tenerife, Spain. (2) Instituto de Ciencias Agrarias, CSIC, Serrano 115-dpdo, 28006 Madrid, Spain. (3) Centro de Investigación y Tecnología Agroalimentaria de Aragón, Avda. Montañana, 930. 50059 Zaragoza, Spain.

Mentha suaveolens also known as apple mint, is an aromatic and medicinal plant of

ethnopharmacological interest due to its antiemetic, antidiarrhaea, antihemorroidal, carminative and

analgesic effects. Several studies 1, 2 have demonstrated the antimicrobial, antifungal, antioxidant,

insecticide and nematicidal3 activity of its essential oil.

The essential oil (EO) of Spanish M. suaveolens with piperitone oxide as major component4 has been

been described as being nematicidal. Following a domestication process, a chemically stable population

has been developed for further valorization. In this context, the pilot-plant vapor-pressure essential oil

extraction of this population of M. suaveolens generated EO and hydrolate as byproduct, both showing

strong in vitro nematicidal effects against the root knot nematode Meloidogyne javanica.

The aim of this study was to determine the compounds responsible for the nematicidal activity of the

hydrolate byproduct. The dichloromethane liquid-liquid extraction of the hydrolate gave a nematicidal

extract and an inactive aqueous residue. The bio-guided study of the chemical composition of the

organic extract by flash chromatography and HPLC resulted in the isolation of monoterpene compounds,

including piperitenone also present in the EO, along with several new monoterpene alcohols. These

compounds were identified by spectroscopic techniques (1D and 2D NMR) and mass spectrometry (GS-

MS). The potential of M. suaveolens hydrolate as a potential root-nematode control agent will be

discussed.

Keywords: hydrolate, Mentha suaveolens, organic fraction, nematicidal effect

References: 1 Kusari S., Hertweck C., Spiteller M., Chem. & Biol., 2012, 19, 792–798. 1 Brahmi F., Hauchard D., Guendouze N., Madani K., Kiendrebeogo M., Kamagaju L., Stévigny C., Chibane M., Duez

P., Ind. Crops Prod., 2016, 88, 96–105 1 OkaY., Nacar S., Putievsky E., Ravid U., Yaniv Z., Spiegel Y., Phytopathology, 2000, 90, 710–715 1 Andrés M.F., González-Coloma A., Sanz J., Burillo J., Sainz P., Phytochem. Rev., 2012, 11, 371–390

Acknowledgements: This work has been supported by grant CTQ2015-64049-C3-1-R (MINECO/FEDER).


72


DEVELOPPEMENT DE SOLUTION DE BIOCONTROLE « SUBSTANCE NATURELLE »

Taïeb BEN-NEJMA (1), Caroline CAYZAC (1), Fanny ROLET (1)

(1) Antofénol, Place Eugène Bataillon, Bâtiment 15, 34 095 Montpellier

Ces dernières années, la protection de l’environnement représente un enjeu majeur. En agriculture,

l’obligation de réduire les intrants phytosanitaires pousse les laboratoires à développer des solutions

naturelles alternatives aux solutions chimiques. L’autre enjeu majeur pour l’agriculture est de produire

suffisamment pour nourrir une population mondiale croissante. Afin de répondre à ces enjeux Antofénol

propose de réduire les pertes post-récolte. En effet, sur les 1,7 milliards de tonnes produites

annuellement, il y a entre 30 et 50% de pertes post-récolte. 80% de ces pertes sont dus à des maladies

fongiques.

La société Antofénol dans une logique éco-responsable, oriente ses travaux sur la valorisation de «

déchets » végétaux en développant des solutions de biocontrôle pour la conservation naturelle de fruits

et légumes. Pour cela, elle a développé un procédé d’éco-extraction reposant sur une technologie

innovante combinant micro-ondes, ultrasons, vide et brassage et utilisant un agrosolvant afin de garantir

des éco-extraits les plus naturels possibles.

Ainsi est née l’Antoferine, extrait végétal issu de co-produits viticoles riches en polyphénols, présentant

in vitro une activité antifongique sur ne quinzaine de pathogènes fongiques. Aujourd’hui, l’efficacité

antifongique de l’Antoférine est démontrée à l’échelle laboratoire sur un modèle pomme / Botrytis

cinerea, phytopathogène largement répandu, responsable de la pourriture grise sur de nombreuses

cultures.

Grâce à sa solution de biocontrôle issue de co-produits viticoles et dans une logique d'économie

circulaire, l’entreprise propose donc des solutions aux problématiques du traitement naturel post-

récolte de fruits et légumes, ainsi qu’à la revalorisation de déchets.

Mots-clés : biocontrôle, conservation, substance naturelle, antifongique, valorisation


73


EFFECT OF DIFFERENT CONCENTRATIONS OF M2ITM PHEROMONE DISPENSERS AND THE IMPACT OF WATER AND PARAFFIN IN PHEROMONE TRAPS FOR RHYNCHOPHORUS FERRUGINEUS

(COLEOPTERA: CURCULIONIDAE)

Mohamed Habib DHOUIBI (1), Wiem HAOUARI (1), Ilhem KHRISSI (1), Olivier GUERRET (2), Hatem CHAAR (1) and Kevin DE COZAR (2)

(1) National Institute of Agronomy of Tunisia, 43 avenue Charles Nicole 1082 Tunis mahrajene; Email : [email protected]

(2) Groupe M2I, 112 Bureaux de la Colline, 92213 St Cloud, France

Red palm weevil is considered nowadays one of the major threats to palm trees worldwide.

Due to the current issue towards the use of environmentally friendly control measures, dealing with this

pest by mass trapping was used in this study. In order to enhance the current management practices to

control the pest, field studies were conducted to know the attractiveness of red palm weevil to 3

concentrations of polymers in M2ITM dispensers in comparison to ISCA capsules, and to test the

effectiveness of putting water inside the traps in the adults’ attraction.

Besides we tested the impact of three solutions: bare paraffin, paraffin mixed with water and bare water

in order to evaluate the best solution that attract more adults of Red palm weevil.

Studies indicated that the concentration 0.5 % and 1% (of polymers) attracted significantly more number

of weevils.

Studies revealed also that there wasn’t significance in putting water inside traps.

The solutions tested inside the traps proved that paraffin is the best substance that bans more adults to

escape from trap. This study indicated the existence of red palm weevil throughout the year but with

lower adults capture in the study period (October-December).

We presented climatic data in details mainly temperature and Relative humidity. These parameters were

main explication for the lower rate of RPW adults captured in baited traps.

Pheromone traps baited with M2I dispensers can be placed in fields for better IPM for RPW.

Keywords: Rhynchophorus Ferrugineus, Capsules M2ITM, Water, Traps


74


ETUDE IN VITRO DU POTENTIEL NEMATICIDE DE QUELQUES HUILES ESSENTIELLES ET L’EXTRAIT D’AIL SUR LES NEMATODES A GALLES

(MELOIDOGYNE INCOGNITA)

Mokrini (1), Hormatallah (2), Janati (3), Houari (3), Sbaghi (4)

(1) Institut National de la Recherche Agronomique (INRA), UR-Production intégrée des cultures, Laboratoire de

Nématologie, CRRA-Agadir-Maroc (2) Département de Protection des Plantes, Institut Agronomique et Vétérinaire Hassan II, Complexe Horticole d’Agadir,

Maroc (3) Faculté Polydisciplinaire de Taroudant, Laboratoire des Biotechnologies, Valorisation et Environnement (4) Institut National de la Recherche Agronomique (INRA), Division Scientifique, Rabat-Maroc

De par sa contribution à la sécurité alimentaire du pays, à la création de l’emploi et de l’équilibre de la

balance commerciale, la filière de la tomate constitue l’un des secteurs clés pour l’économie marocaine.

Néanmoins, la culture de tomate est sujette à diverses attaques de bioagresseurs dont les nématodes

à galles causé par le genre Meloidogyne. Si la lutte chimique demeure la méthode la plus usitée par les

producteurs pour contrôler le développement des nématodes à galles, elle est actuellement confrontée

à divers enjeux réglementaires, sanitaires, environnementaux et sociétaux. C’est ainsi le recours à des

nouvelles méthodes alternatives basées sur la recherche de produits « bio-némacticides » d’origine

végétale sont testées.

Dans ce cadre, une étude a été menée afin d’évaluer l’efficacité in vitro des différentes concentrations

de (2, 4, 8, 16 et 32 µl/ml) de l’huile essentielle d’Origan (Origanum compactum), l’huile essentielle de

Cannelle (Cinnamomum zeylanicum) et l’extrait d’ail (Allium sativum) sur la mortalité des juvéniles après

une période de 24h, 48 et 72h d’exposition, et sur l’éclosion des œufs de Meloidogyne incognita.

Les résultats ont montré que les traitements testés tuent les larves du deuxième stade et inhibent

l’éclosion des oeufs de M. incognita dont l’effet est en fonction de la nature de traitement, le temps

d’exposition et la concentration.

Concernant l'effet des traitements testés sur la mortalité des juvéniles, on constate une augmentation

de taux mortalité ́en fonction du temps d'exposition et de la concentration. Il atteint 82,4% et 81,6%

respectivement pour les huiles essentielles de Cannelle et d’Origan et 84,6% pour l’extrait d’ail. Par

d’ailleurs, le pourcentage d’inhibition de l’éclosion des œufs de M. incognita est respectivement 78,4%,

73,9%, et 67,3% pour l’extrait d’ail et les huiles essentielles d’Origan et Cannelle à la concentration 32

ppm après 8 jours d’incubation.

Mots clés : huile essentielle d’Origan, huile essentielle de Cannelle, extrait de l’ail Meloidogyne incognita, éclosion, mortalité des juvéniles.


75


CASTANEA AND SCHINOPSIS SP. TANNINS, FOR CROP PROTECTION

Gabriele Monguisti (1), Renzo Paoletti (1), Christophe Zambaux (2)

(1) Adresse SILVATEAM via Torre, 7 12080 San Michele Mondovì CN – Italy (2) IAZ DEVELOPPEMENT 4 Bis Avenue De Louvois 92370 Chaville

The substance Castanea and Schinopsis sp. Tannins was applied as a basic substance under the

Regulation (EC) No 1107/2009 Article 23.

Tannins are natural components of wood, synthesized by plants as a natural defence coponent. These

substances correspond to large polymers soluble in water, of different natures according to the

botanical origin. They all have a high reactivity; an acid function and the ability to interact with cations

and proteins. These products have been used for many years in the leather industry or for animal and

human food.

SILVATEAM Company extracts and sells Tannins. Since the beginning of the 2010’s many works have

been set up to identify and measure the interest of Tannins for the crop protection. In partnership with

IAZ DEVELOPPEMENT after a selection and combination of tannins, a preparation of interest for

Agriculture and crop protection has been developed.

This preparation, which contains 40% of Castanea sp and Shinopsis sp tannins in liquid form, is of interest

in nematodes control, soil diseases like Rhizoctonia, Sclerotinia and also plant disesases like downy

mildew or bacterial control in many crops.

The presentation specifies the nature of the extracts, details the development of the product, its

characteristics and illustrates the different crops and possible uses, also showing the interest of this new

complementary mode of action for bio-control strategies in crop protection.

Keywords: Tannins, Bio-contrôle, nematods, Soil diseasesl, Downy mildew, Bactérioses


76


EFFET DU POTENTIEL INSECTICIDE DE L’HUILE ESSENTIELLE DE THYM ET DE L’EXTRAIT DE L’AIL SUR LA MINEUSE DE LA TOMATE

Hormatallah A. (1), Chtaina N. (2), Antour H. (1)

(1) Département de Protection des Plantes, Institut Agronomique et Vétérinaire Hassan II, Complexe Horticole d’Agadir, Maroc

(2) Département de Production Protection et Biotechnologie Végétales. Unité de Protection intégrée des végétaux. Institut Agronomique et Vétérinaire Hassan II, Rabat, Maroc

Au Maroc, la culture de tomate occupe une place très importante dans le contexte socio-économique

national. Néanmoins, la culture de tomate est sujette à diverses attaques de bio agresseurs, dont la

mineuse connue scientifiquement sous le nom de Tuta absoluta (Meyrick). En dépit des efforts déployés

par tous les professionnels du secteur pour la gestion de Tuta absoluta, la campagne 2016/2017 a connu

une recrudescence de ce redoutable ravageur. Afin de respecter les exigences sanitaires, les cahiers de

charges imposées par les marchés importateurs (zéro fruit infesté, respect des LMRs…) et éviter les

phénomènes de résistance, des nouvelles méthodes alternatives basées sur la recherche de produits «

bio-insecticides » d’origine végétale sont testées. C’est dans ce cadre que l’huile essentielle du thym et

l’extrait de l’ail utilisé comme biostimulant, ont été choisies pour évaluer leur efficacité contre Tuta

absoluta dans les conditions in vitro au laboratoire et in vivo en conditions contrôlées de serre. Les tests

in vitro de l’efficacité larvicide, par la méthode « Leaf dip bioassay », et ovicide de l’huile de thym préparé

aux concentrations de 1000 ppm, 1500 ppm et 2000 ppm et de l’extrait de l’ail à 60 ppm ont été

comparés à deux insecticides de référence à savoir le Spinétoram (Radiant) et le Chlorantraniliprole +

Lambda-Cyhalothrine ( Ampligo) préparés respectivement aux 30 ppm et aux 30 ppm + 15 ppm.

Les observations ont été réalisées 24h, 48h et 72h après traitement. L’effet répulsif de l’extrait de l’ail

aux concentrations de 30, 45 et 60 ppm à l’égard des larves de Tuta absoluta a été évalué en utilisant la

méthode de la zone préférentielle sur papier filtre décrite par McDonald et al (1970). Les résultats

obtenus ont montré que l’huile de thym a pu engendrer un taux de mortalité des larves dépassant les

60%, à partir de la concentration 1500 ppm. L’analyse statistique a révélé que l’effet larvicide de l’huile

de thym à 2000 ppm était similaire à celui du Spinétoram à 30 ppm puisque les deux produits ont

provoqué la mortalité la plus élevé de larves allant jusqu’à 95% après 24h, 48h et 72 h. Quoi que l’extrait

de l’ail ait montré une efficacité larvicide par rapport au témoin, le taux de mortalité n’a pas pu dépasser

les 10%. Les résultats de l‘effet ovicide ont montré que l’huile de thym à la concentration 2000 ppm a

engendré un taux d’éclosion de 67%. Le Spinétoram a montré une efficacité ovicide par rapport à l’huile

de thym puisque uniquement 13,9% des œufs ont pu éclore. Le taux d’éclosion des œufs était de 100%

pour le témoin non traité. L’extrait de l’ail et l’insecticide «Chlorantraniliprole+Lambda-Cyhalothrine»

n’ont pas montré d’effet sur les oeufs de Tuta absoluta. L’extrait de l’ail présente une forte activité

insectifuge à l’égard des larves de Tuta absoluta puisqu’il présente un taux de répulsion de 78% et

appartient à la classe répulsive IV (répulsif) selon le classement de McDonald. Si l’application de l’huile

de thym sur des plants de tomate sous serre à des concentrations entre 100 à 1500 ppm, a pu engendrer

une phytotoxicité, l’extrait de l’ail a permis de réduire le nombre de mines causées par Tuta absoluta

jusqu’à 31% en traitement curatif.

Mots clés : Tomate, Tuta absoluta, Huile de Thym, Extrait de l’Ail, Spinétoram, Chlorantraniliprole + Lambda-Cyhalothrine, effet larvicide, effet ovicide


77


EFFICACITE D’UN INSECTICIDE A BASE DE NEEM DANS LA LUTTE CONTRE QUELQUES RAVAGEURS DU COTONNIER AU BENIN

G. Bonni (1), M. Adegnika (1), A. Paraïso (2)

(1) Centre de Recherche Agricole Coton et Fibres (CRA-CF), Institut National des Recherches Agricoles du Bénin (INRAB)

Abomey-Calavi, Bénin (2) Université de Parakou, Faculté d’Agronomie, Laboratoire de Protection des Végétaux, de Pathologie et de

Parasitologie des abeilles (LAPPAB)

L’effet d’un insecticide à base de neem, Azadirachta indica A. Juss (Meliaceae) a été évalué et comparé

à des produits insecticides de synthèse dans la lutte contre les chenilles du cotonnier : Haritalodes

derogata, Helicoverpa armigera, Earias. spp., Pectinophora gossypiella et Thaumatotibia leucotreta.

L’expérimentation a été conduite dans deux localités incluses dans les aires de distribution des espèces,

Kandi et Savè situées respectivement dans la zone septentrionale et Centrale du Bénin. La formulation

de neem contenait 0,5 % d’azadirachtine et a été utilisée aux doses de 2, 3 et 4 l/ha.

Les formulations insecticides de synthèse contenaient de l’emamectine 24 g/l + acetamiprid 32 g/l ; de

la cyperméthrine 35g/l + chlorpyrifos 300 g/l et du betacyfluthrine 45 g/l + imidachloprid 100 g/l.

Les différentes applications ont été réalisées selon le programme calendaire de protection du cotonnier

recommandé dans chacune des deux régions. Au total, dix traitements ont été réalisés avec l’insecticide

botanique et six traitements avec les trois insecticides de synthèse.

Les résultats ont montré que l’insecticide à base de neem s’était montré autant efficace que le témoin

de référence (cypermethrine 35g/l+chlorpyrifos 300 g/l) dans le contrôle des chenilles endocarpiques

(Pectinophora gossypiella et Thaumatotibia leucotreta). L’insecticide botanique a réduit le pourcentage

de plants attaqués par le phyllophage, Haritalodes derogata de 25 % contre 100 % de réduction pour le

produit chimique de synthèse. Un effet dose de l’insecticide botanique a été observé avec 36,1 %, 29,8

et 6,1 % de plants attaqués, pour les doses de 2, 3 et 4 l/ha respectivement.

Les rendements de coton graine obtenus des parcelles traitées à l’extrait aqueux de neem ont été

supérieurs à ceux des parcelles non traitées. En zone endocarpique, ces rendements issus des parcelles

traitées à l’extrait de neem ne sont pas différents statistiquement de ceux des parcelles traitées au

produit de synthèse utilisé en référence.

Cet extrait à base de neem pourrait être utilisé dans la zone de pullulation des chenilles à régime

endocarpique pour une gestion intégrée de ces nuisibles. Mots clés : Cotonnier, ravageurs, Azadirachta indica, insecticides, Efficacité, rendement, Bénin


78


USAGES DES PLANTES À EFFET PESTICIDE AU CAMEROUN

Tagne Appolinaire (1), Silvie Pierre (2,3), Martin Pierre (2,3), Ndzana Francois Xavier (1), Tindo Maurice (4), Fekam Boyom Fabrice (5), Bokogo Claudine (1), Foka Ebenezer (5) Ntaffeu

Edmond (6), Chimi Laure (1), Marnotte Pascal (2,3)

(1) Institut de la Recherche Agricole pour le Développement (IRAD), Yaoundé, Cameroun (2) CIRAD, UPR AIDA, F-24398 Montpellier, France (3) AÏDA, Université Montpellier, CIRAD, Montpellier, France (4) Université de Douala, Cameroun (5) Université de Yaoundé 1, Cameroun (6) Université Agricole et de Management des Métiers de Productions, Yaoundé, Cameroun

Les pesticides d’origine végétale constituent une des alternatives à la lutte chimique avec des produits

de synthèse et un moyen complémentaire aux autres méthodes de lutte. Dans le cadre du projet

Knomana (Knowledge management), portant sur la gestion des connaissances et la conservation des

données relatives aux plantes pesticides en Afrique (http://urlz.fr/6P4a), un état des lieux des usages

expérimentaux (laboratoire et champs) et des utilisations par les producteurs a été réalisé.

La recherche des articles publiés, leurs saisies pour alimenter une base de connaissances et l’analyse

préliminaire effectuée ont permis de recenser 48 espèces végétales utilisées au Cameroun pour

protéger 14 espèces végétales : Apium graveolens, Citrus sinensis, Monodora myristica, Oryza sativa,

Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Sorghum bicolor, Triticum sp., Vigna mungo,

Vigna radiata, Vigna subterranea, Vigna unguiculata, Zea mays. Les travaux ont notamment concerné la

composition chimique des extraits de plantes, leurs propriétés insecticide et antimicrobienne. Les

usages relevés concernent la protection des semences contre des maladies telles que la pourriture des

racines et des épis du maïs causé par le champignon Fusarium verticiloïdes, la maladie des taches brunes

du riz (due au pathogène Bipolaris oryza) et le flétrissement de la tomate (Ralstonia solanacearum). Les

connaissances enregistrées relevant de la protection contre les insectes des cultures concernent

essentiellement ceux des denrées stockées comme Sitophilus zeamais ou Callosobruchus maculatus et

d’autres espèces du genre Sitophilus. À ce stade de la collecte des informations les usages en protection

foliaire semblent donc faibles dans ce pays.

Les résultats obtenus indiquent clairement qu’il existe plusieurs plantes à effet pesticide dans la flore

végétale du Cameroun. En dehors du neem (Azadirachta indica) déjà bien connu en Afrique, des espèces

apparaissent intéressantes à exploiter de façon extemporanée par les agriculteurs ou à développer de

façon commerciale à l’exemple de Ocimum gratissimum, Callistemon citrinus, Cymbopogon citratus,

Ageratum conyzoides, Tephrosia vogelii, Bidens pilosa, Commelina benghalensis, Emilia coccinea,

Erigeron floribundus, Eucalyptus tereticornis, Euphorbia hirta, Oxalis barrelieri, Podocarpus milanjianus.

Dans certains cas, l’adoption nécessitera des études complémentaires sur les risques de toxicité vis-à-

vis des organismes non cibles (dont l’homme) et de l’environnement.

Keywords: pesticide plants, natural extracts, essential oils, Africa


79


INSECTICIDAL AND ANTIMICROBIAL ACTIVITY OF THREE APIACEAE PLANTS ESSENTIAL OILS AGAINST MICROORGANISM PATHOGEN

N. Merad (1), V. Andreu (2), D. Raviglione (2), C. Bertrand (2,3), N. Djabou (1)

(1) COSNA, Université de Tlemcen, BP 119, Tlemcen 13000, Algérie, [email protected] (2) SAS AkiNaO, Perpignan, F66860, France (3) CRIOBE USR3278, Université de Perpignan Via Domitia, F66860, France, [email protected]

In recent years, crop loss remains a problem due to plant diseases caused by insects, plant pathogen

fungi, bacteria, and viruses. Microorganisms have also unfavorable effects on the quality, safety, and

shelf life of foods. Therefore, the postharvest diseases caused by microorganisms are still a most

important problem. Synthetic chemicals are widely used in the control of plant diseases.

However, these chemicals are associated with undesirable effects and some toxic residues in the

products. Therefore, there has been a growing interest in research concerning alternative pesticides and

antimicrobial active compounds, including the plant extracts and essential oils that are relatively less

damaging to the health and environment. The essential oils and volatile constituents extracted from the

aromatic plants are widely used as new bio-control alternative agents against microbial strains and

insect pests, because of their specificity of action, biodegradable nature, and potential for commercial

application.

The essential oils were extracted from 3 different species of the family Apiaceas; Smyrnium Olusatrum,

Daucus Crinitus, Eryngium Triquetrum, collected in western Algeria. The objectives of this study were to

characterize the chemical composition and evaluate the antimicrobial and insecticide activity of three

essential oils witch are rich in oxygenated terpenic and sesquiterpene compounds. Essential oils

activities were evaluated against two bacterial strains (Pectobacterium and Pseudomonas) and against

Artemia Salina as a biological model for preliminary insecticide evaluation.

The essential oil of Eryngium Triquetrum contains Falcarinol as a major compound which is present 93%

of the total oil. It revealed an insecticidal activity with IC50 = 0.0007 mg / ml and 100% and 85% of

antimicrobial activity against Pseudomonas and Pectobacterium respectively. Thus, the essential oil of

Smyrnium olusatrum contains Curzerene and furanoermophilone as a major compounds at 19%, 31%

respectively of the total oil. It revealed an antimicrobial activity against both strains with a rate of

inhibition 63% and 58% for Pectobacterium and Pseudomonas respectively.

In conclusion, the two tested essential oils could be considered as a good bio-control agent to protect

plants against bacterial diseases and as an alternative to chemical pesticides and insecticides.

Keywords: essential oils-bacterial strain-insecticide-biocidal




80


COMPARATIVE PHYTOCHEMICAL INVESTIGATION BETWEEN ESSENTIAL OILS AND HYDROSOL OF ALGERIAN JUGLANS REGIA.L

LEAVES OBTAINED USING TWO DIFFERENT EXTRACTION METHODS

Fayçal Boukhari (1), Nacéra Tigrine-Kordjani (1), Hamza Aliboudhar (1), and Fatma Zohra Geuzil (1)

(1) USTHB, Laboratoire d’Analyse Organique Fonctionnelle, Faculté de Chimie, Université des Sciences et de la Technologie

Houari Boumediene, BP 32, El Alia, Bab Ezzouar, 16111 Alger, Algérie

Juglans regia L. commonly called ‘Al-Jouz’ in Algeria, is an important deciduous tree. This species is well

known medicinally for its powerful anti-diabetic, anti-diarrhea and depurative properties and frequently

it is used as cosmetic dyes agent for hair. In this work, a comparative phytochemical investigation is

carried out between essential oils and hydrosol of Algerian Juglans regia.L leaves obtained using two

different extraction methods. An advanced hydrodistillation; microwave-assisted hydrodistillation

(MAHD) and conventional hydrodistillation (HD), were conducted to extract the essential oil from walnut

leaves. In addition, the hydrolat or hydrosol; a sub-product of microwave-assisted hydrodistillation

essential oil obtained from leaves collected in May, was reported for the first time, and a chemical

comparison content variation with its corresponding oil was realized.

All samples were analyzed using GC–FID and GC/MS. Quantitative and qualitative differences were

established between the chemical composition of liquid-liquid extraction of hydrolat and its

corresponding essential oil. Five new oxygenated compounds were detected and identified in hydrolat;

α-terpineol (9.92%), eugenol (8.15%), linalol (2.32%), borneol (1.10%), Menth-2-en-I-ol (trans-ҏ)

(0.65%), while they were not found in its correspondent essential oil.

Conversely, a large part of sesquiterpene compounds identified in essential oil were also absents in

hydrolat, such as; α-cubebene, copaene, β-elemene, α-muurolene, α-farnesene , δ-cadinene.

Keywords : Juglans regia L, Leaves, Essential oil, Hydrosol


81


Β-AMINOBUTYRIC ACID (BABA) TREATMENT INDUCES A TEMPORAL DISRUPTION OF EXPANSIVE AND STRUCTURAL GROWTH ASSOCIATED

WITH A METABOLIC REORGANISATION IN FLAX (LINUM USITATISSIMUM)

Nicole Pontarin (1,2), Anthony Quéro (1), Job Tchoumtchoua (1), Maja Gredelj (1),

Roland Molinié (1), David Mathiron (3), Anne-Isaline Van Bohemen (1), Jean-Xavier Fontaine (1), Julien Boccard (4), Vivien Sarazin (2), François Mesnard (1)

(1) Biologie des Plantes & Innovation (BIOPI EA3900), UPJV, Amiens, France Adresse (2) Laboulet Semences, Airaines, France (3) PFA, UPJV, Amiens, France (4) School of Pharmaceutical Sciences, University of Geneva, Switzerland

β-aminobutyric acid (BABA) is a non-protein amino acid synthesised by plants in a hormone-like

response to stress1. When exogenously applied, BABA is a powerful priming agent of plant natural

defences, active against 80 different pathogens and pests, and a variety of abiotic stresses 2,3. Though,

at the treatment doses, BABA engenders a growth delay. Within this context, this study aims to

characterise BABA-induced response on flax growth in a spatio-temporal fashion.

For leaves, the stage of development is generally assigned on the basis of their photosynthetic and

metabolic independence, so we can distinguish three populations of leaves: source, transition and sink

leaves.

Firstly, flax source, transition and sink leaves were differentiated based on their relative growth rate and

their metabolic composition via multivariate data analysis. Secondarily, BABA-induced response on the

growth of those leaf populations was evaluated during a time frame of ten days. The contribution of

expansive and structural growth to the overall leaf growth was assessed by measuring fresh mass, dry

mass, leaf surface, water content and transpiration. For further understanding, metabolic changes were

investigated by metabolic profiling through GC-MS and LC-MS, and osmotic potential was measured. In

addition, the structure elucidation of purified compounds potentially involved in the osmotic potential

was unambiguously performed using NMR experiments.

Multivariate data analysis revealed that BABA-induced response was characterised by early expansive

growth retardation, followed by the recovery of a new homeostatic state, characterised by a lower

deposition of biomass. The metabolic reorganisation played a central role in growth regulation, revealing

how the various phases of the response were driven by the leaf populations, in different but coordinated

fashions. 1Thevenet D, Pastor V, Baccelli I, Balmer A, Vallat A, Neier R, Glauser G, Mauch-Mani B. The priming molecule β-aminobutyric acid is naturally present in plants and is induced by stress. New Phytologist 2017; 213:552-9; PMID:27782340; http://dx.doi.org/10.1111/nph.14298.

2Cohen Y, Vaknin M, Mauch-Mani B. BABA-induced resistance: milestones along a 55-year journey. Phytoparasitica 2016; 44:513-38; http://dx.doi.org/10.1007/s12600-016-0546-x. 3Quéro A, Fliniaux O, Elboutachfaiti R, Petit E, Guillot X, Hawkins S, Courtois J, Mesnard F. β-Aminobutyric acid

increases drought tolerance and reorganizes solute content and water


82


MICROALGAL COMPOUNDS FOR BIOCONTROL PRODUCT DEVELOPMENT: AN INNOVATIVE METABOLOMIC APPROACH BY

MOLECULAR NETWORKING

CHAÏB Slimane (1), RAVIGLIONE Delphine (1), ANDREU Vanessa (2), VIALLEIX Carole (3), LEVERT Annabel (2), CADORET Jean-Paul (3), BERTRAND Cédric (1), BONNARD Isabelle (1)

(1) PSL Research University EPHE-UPVD- CNRS, USR 3278 CRIOBE, LabEx-CORAIL, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

(2) AKINAO, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France (3) GREENSEA, Promenade du Sergent Navarro, 34140 Mèze, France

Uses of synthetic pesticide may have negative ecological consequences due to direct soil and water

contamination. In this context, the use of products from natural origin seems a sustainable option due

to their perceived eco-friendly profile over environmentally harmful chemical pesticides [1]. In this

context, micro-algae constitute, within the marine environment, a source of bioactive molecules [2].

Micro-algae metabolites are active on a wide range of biological targets and can be recycled in various

ways.

This project is aimed to develop an innovative tool adapted to the analysis of the chemical diversity from

the microalgae and evaluation of their biocide potential. This project proposes to combine, on the one

hand, differential metabolic fingerprints and, on the other hand, analysis of the molecular diversity maps

drawn by the study of “molecular networks”. The “molecular network” is an innovative bio-informatic

approach that allows to characterize classes of metabolites based on their spectral data and study their

degrees of similarity [3].

Through the use of molecular networks, data and complex mixtures can be compared in silico and

annotated. This approach facilitates the study of the metabolome of living organisms and the

characterization by dereplication of active metabolites.

In the context of biocidal products research, an herbicidal assay targeted on thylakoids as well as an

antioxidant assay [4] were selected. This poster will present therefore the methodology for samples

preparation and metabolic fingerprints acquisition, as well as, the first results on metabolic

characterization coupled with the biological activity of extracts from micro-algae model.

References

[1] Dayan et al. Natural products in crop protection. Bioorganic & Medicinal Chemistry. (2009),17(12), 4022-4034. [2] John Berry. Marine and Freshwater Microalgae as a Potential Source of Novel Herbicides, Herbicides and Environment, Dr Andreas Kortekamp (Ed.), (2011). 705-734. [3] Justine Chervin et al. Dereplication of natural products from complex extracts by regression analysis and molecular networking : case study of redox-active compounds from Viola alba subsp. Dehnhardtii. Metabolomics (2017) 13: 96. [4] Idaira Jerez-Martel et al. (2017). Phenolic Profile and Antioxidant Activity of Crude Extracts from Microalgae and Cyanobacteria Strains, Journal of Food Quality. (2017) ID 2924508, 8 p.

Keywords: Metabolomic, Microalgae, Molecular networks, Bio-pesticides


83

Session 5 :

Evaluation and methods dedicated to the analysis of biocontrol natural products and of their environmental

impact.

Keynote speaker: Pr Jean-Luc Wolfender


84

LATEST ADVANCES IN METABOLITE ANNOTATION FOR METABOLOMICS – MOLECULAR NETWORKING FOR DECIPHERING KEY

DEFENCE INDUCTION PHENOMENA

Jean-Luc Wolfender(1), Hicham Chahtane(2), Luis Lopez-Molina(2), Emerson Ferreira Queiroz(1),

Pierre-Marie Allard (1)

(1) School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU – Rue Michel Servet

1, 1211 Geneva 11, Switzerland

(2) Department of Plant Biology, University of Geneva, Geneva, Switzerland;

The rapid innovations in metabolite profiling, bioassays and chemometrics lead to a paradigm shift in

natural product (NP) research. Indeed, having at hand partial/full structure information of possibly all

secondary metabolites and an estimation of their levels in plants and microorganisms, provides a way

to perform deep metabolome investigations from a new and holistic perspective (1,2). The increasing

amount of accurate metabolome data that can be acquired on massive sample sets, notably through

data dependent HRMS/MS, allows mapping natural extracts at an unprecedented precision level (3). In

this context, data contextualization is however still a lagging process.

For this, we investigated methods that could provide enhanced annotation confidence level through

multiple scores integrating taxonomy information and molecular network (MN) structural consistency

as well as other orthogonal analytical data. Benchmarking of such approaches is currently assessed by

profiling mixtures of complex extracts with well-studied composition. We also investigate the best way

to integrate extracts bioactivity data in MN and shortcut bioactivity guided isolation for an efficient

targeted identification of bioactive NPs (4). To this end, accurate chromatography gradient methods at

various scales have been developed for MS-targeted purification of biomarkers and their full de novo

structure identification by NMR.

Different recent applications of our metabolomics/phytochemical investigations will illustrate these

aspects and also highlight their efficiency for the study of the dynamic production of biomarkers in plant

and microorganism in ecologically relevant problematics. We also will show how this led to the

identification of a key biomarker released by P. aeruginosa, as a biotic compound triggering germination

arrest in Arabidopsis (5). Evaluation of what is readily implemented and is still required in NP research

will be made.

Key words: metabolomics Dereplication, metabolite profiling, MS-targeted isolation, bioactive natural products

References: (1) Allard, P.-M., et al. Curr. Opin. Biotechnol. 2018, 54, 57-64 ; (2) Allard, P. M., et al. Anal. Chem. 2016, 88, 3317-3323 ; (3) Wolfender, J.-L., et al. J Chromatogr A 2015, 1382, 136-164. (4) Olivon, F., et al. ACS Chem Biol 2017, 12, 2644-2651 ; (5) Chahtane, H., et al. eLife 2018, 7, e37082.


85


PLANT ROOT MODULATION OF PSEUDOMONAS SECONDARY METABOLISM INVOLVED IN BIOLOGICAL CONTROL

OF PLANT DISEASES

Laura RIEUSSET (1), Gilles COMTE (1), Claire PRIGENT-COMBARET (1)

(1) UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Villeurbanne, France

The plant root adherent soil houses an important microbial community. Roots exudate a wide variety of

secondary metabolites able to attract and/or manage a large diversity of microbial species. In return,

among microbiota, some bacteria are able to promote plant development, using plant growth-

promotion and plant protection properties. These bacteria are named PGPR for Plant Growth-Promoting

Rhizobacteria. Some PGPR belong to the Pseudomonas genus. Pseudomonas are known to produce an

important diversity of secondary metabolites that could have antimicrobial activity. They play a major

role in microbial biocontrol strategies and multiple Pseudomonas strains are used for crop protection.

But yet, the impact of host plant on antimicrobial metabolite production and biocontrol activity is still

poorly understood.

The aim of our project is to better understand the impact of the host plant on secondary metabolite

production by fluorescent Pseudomonas strains. To respond to this objective a metabolomic approach

was developed in order to decipher which plant signal mediators may modulate antimicrobial secondary

metabolite production in Pseudomonas. Three different Pseudomonas strains were thus cultivated in

the presence of root extracts of three wheat genotypes, at low concentration. This experimentation

allows us to evaluate the impact of root metabolites on Pseudomonas secondary metabolism, and

especially on the production of antimicrobial compounds.

Analysis of our metabolomics workflow revealed that the production of several Pseudomonas secondary

metabolites are significantly up or down regulated when bacteria were cultivated with root extract at

low concentration. This shows that root wheat metabolites may act as signaling compounds and show

capacity to modulate Pseudomonas secondary metabolism, including antimicrobial metabolites

involved in biocontrol properties. Interestingly, root extract modulation seems to be different according

to wheat genotypes and Pseudomonas strains. That suggests specific bacterial strain / plant genotype

interactions.

Keywords: PGPR, Pseudomonas, metabolomics, wheat genotype, antimicrobial secondary metabolites, biocontrol, signaling


86


METABOLIC FOOT-PRINTING APPROACH TO ASSESS THE ENVIRONMENTAL FATE AND IMPACT OF BIOINSECTICIDES.

Chandrashekhar PATIL (1), Amani BEN JRAD (1), Hikmat GHOSSON (1), Delphine RAVIGLIONE

(1), Marie-Virginie SALVIA (1,2), Cédric BERTRAND (1,2,3)

(1) CRIOBE USR3278, Université de Perpignan Via Domitia, F66860, France. (2) Laboratoire d’excellence “CORAIL”, France (3) AkiNaO, Perpignan F66860, France

Presence and establishment of invasive mosquito species such as Aedes aegypti and Aedes albopictus is

rapidly increasing in the European environment. Mosquitoes can carry infectious diseases from person

to person and from place to place. The most efficient means to fight against mosquitos is the use of

insecticides. A lot of chemical insecticides were developed. First of all, organochlorine compounds such

as DDT were used. However, most of these substances were then removed from the market as they

showed severe risks for the human and animal health as well as for the ecosystem.

The European Directive in 1998 led to the increasing use of biological insecticides such as cry proteins

produced by the bacterium Bacillus thuringiensis israelensis (Bti) that kill mosquito larvae after being

ingested. Considering the interest in Bti as more environmentally sustainable bioinsecticide, it is

important to examine in detail environmental fate and impact of Bti. The available tool, the half-life,

t1/2 has a limitation as it does not consider the biodegradation and biotransformation phenomenon as

well as the impact on biodiversity. Furthermore, it cannot be used for complex mixtures. To address this

challenge, 'Environmental Metabolic Footprinting' (EMF), giving an idea of the resilience time, a new

integrative proxy, was recently developed in the laboratory (Patil et al. 2016; Salvia et al, 2017) to

evaluate the impact of synthetic, botanical and microbial insecticides on soil and sediment matrices.

The project ‘EnvFate’ aims to employ an EMF approach in order to assess the Bti environmental impact.

A chemical biocide will be used for comparison, the α-cypermethrin. First, the resilience time, will be

evaluated. It corresponds to the time required for the compound dissipation and its effect on the matrix.

On longer-term, the goal is to dynamically characterize environmental markers of Bti pollution found

among the sediment matrix meta-metabolome. The whole study will require to develop and optimize

extraction and detection protocols using LC-MS platform. In addition, metabarcoding approach will

allow to understand microbial community responses to the Bti pollution. Emphasis will be placed on

better standardisation, data interpretation and evaluation that will build confidence in the value of

“omics” technologies – this being essential to increase their (regulatory) use. These activities will

advance our understanding of environmental risks associated with Bti, and pave the way for the

development and adaptation of new environmental monitoring tools.

We will present sensible preliminary findings from metabolomics data. The changes in the sediment

meta-metabolome after Bti and α-cypermethrin treatment were investigated at various time intervals

as compared to non-treated controls in order to evaluate the resilience time of the sediment. Keywords: Natural products, biopesticides, Bti, metabolomics, environment, fate, LC-MS


87


INNOVATIVE BIOINFORMATICS TOOLS TO DECIPHER THE STRUCTURE OF NEW MICROBIAL LIPOPEPTIDES

Mickaël CHEVALIER (1), Emma RICART (2), Maude PUPIN (3,4), Areski FLISSI (3,4),

Frédérique LISACEK (2), Philippe JACQUES (5), Christophe FLAHAUT (1) and Valérie LECLERE (1)

(1) Univ. Artois, INRA, ISA, Univ. Lille, Univ. Littoral Côte d'Opale, EA 7394 - ICV – Institut Charles Viollette, F-59000 Lille, France.

(2) Proteome informatics Group, Swiss Institute of bioinformatics (SIB), Geneva (3) Univ Lille, CNRS, Centrale Lille, UMR9189 – CRIStAL- Centre de Recherche en Informatique Signal et Automatique de

Lille, F-59000 Lille, France (4) Inria-Lille Nord Europe, Bonsai team, F-59655 Villeneuve d’Ascq Cedex, France (5) TERRA Research Centre, Microbial Processes and Interactions (MiPI), Gembloux Agro-Bio Tech University of Liege, B-

5030 Gembloux, Belgium

The nonribosomal peptides (NRPs) are microbial secondary metabolites, displaying a broad range of

biological activities. Especially, lipopeptides have been show to display antifungal and surfactant

activities leading to potential biocontrol applications.

Moreover, some of them are also inducing plant defence. Despite their peptidic nature, NRPs are not

produced through ribosome machinery and therefore their structure cannot be directly deduced from

genomic DNA sequences. Indeed, there are synthesized by microorganism through modular

multienzymatic complexes working as assembly lines named NonRibosomal Peptide Synthetases (NRPS).

This mode of biosynthesis leads to the production of NRPs presenting a structural biodiversity due to

the presence of nonproteogenic aminoacids, original monomers, and D-isomers. Moreover, most of the

NRPs are not linear as classical peptides but include at least one cycle and/or branching.

Considering the specific features related to the NRPSs and their products, we developed innovative

bioinformatics tools allowing the screening for new lipopeptides.

These tools mostly include a database named NORINE gathering more than 1200 annotated NRPs (freely

available at http://bioinfo.lifl.fr/NRP/). We also propose a workflow allowing the lipopeptide structure

prediction from genome sequencing data. We are currently developing a calculation method, based on

Kendrick mass defect allowing to assign a molecular formula from high resolution mass spectra (HRMS).

The smart easy-to-use tool, combining Kendrick calculation and molecular formula corresponding to all

NRPs annotated in Norine database, was very useful to decipher the structure of cyclic lipopeptides

produced by several bacterial strains displaying antifungal activities.

Keywords: lipopeptide, microorganism, bioinformatics, mass spectrometry


88


« OLIGOGALACTURONOMICS » OF PLANT-FUNGUS INTERACTIONS

Aline Voxeur (1), Fabien Miart (1), Marie-Christine Soulié (1,2), Christophe Rihouey (3), Corinne Pau-Roblot (4), Jean-Marc Domon (4), Jérôme Pelloux (4), Grégory Mouille (1),

Mathilde Fagard (1), Herman Höfte (1) and Samantha Vernhettes (1)

(1) Institut Jean-Pierre Bourgin, INRA, Centre National pour la Recherche Scientifique, AgroParisTech, Université. Paris-Saclay, RD10, 78026 Versailles Cedex France

(2) Sorbonne Universités, Université Pierre et Marie Curie Univ Paris 06, UFR 927, Paris, France (3) Normandie Univ, UNIROUEN, INSA Rouen, CNRS, UMR6270, Polymères Biopolymères Surfaces, 76000 Rouen, France (4) EA3900-BIOPI Biologie des Plantes et Innovation, FR CNRS 3417, Université de Picardie Jules Verne, 80039, Amiens,

France

Despite considerable interest for pectin-derived oligogalacturonide (OG) signaling in plant immunity,

very little is known about the OGs that accumulate during pathogen infection, in part due to technical

limitations.

Here we developed a sensitive analytical method for the analysis of the OG profile or OGome. This

showed, that, despite the presence of polygalacturonases (PG) in the fungus Botrytis cinerea, 80% of the

OGs are produced by pectin lyases and are surprisingly highly acetylated and methyl-esterified.

The remaining PG products accumulate as, previously unnoticed, oxidized GalA dimers.

Finally, the comparison of the OGomes of leaves infected with B. cinerea mutants with reduced

pectinolytic activity but with decreased or increased virulence respectively, led to the identification of a

novel OG candidate elicitor.

In conclusion, this study shows that OGome analysis provides unprecedented insights into the

evolutionary enzymatic arms race between plant and fungus and facilitates the identification of novel

defense elicitors.


89


ENVIRONMENTAL METABOLIC FOOTPRINTING (EMF) APPROACH TO STUDY THE QUALITY AND THE ENVIRONMENTAL IMPACT OF

BIOCONTROL PRODUCTS ON MEDITERRANEAN CROPS

Ramos Mélina (1,2,3), Salvia Marie-Virginie (1), Llugany Mercè (2), Badosa Esther (3), Montesinos Emilio (3), Bertrand Cédric (1,4)

(1) PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Université de Perpignan, 52 Avenue Paul Alduy, 66860

Perpignan Cedex, France (2) Unitat de Fisiologia vegetal, Universitat Autonoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain (3) Center for Innovation and Development in Plant Health (CIDSAV), University of Girona, Maria Aurèlia Capmany, 61,

17003 Girona, Spain (4) AKINAO, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

To meet both farmers and consumers’ expectations, French and Spanish governments want less use of

conventional pesticides and more use of products from natural sources. Although biocontrol market recorded a

growth rate of around +15% in a year, we lack technical efficacy and ecotoxical profile references. Based in

Catalogne and Roussillon, the PALVIP project (local Mediterranean crops’ alternative protection) associates

universities and technical structures to evaluate new biocontrol products developed by the small local businesses

partners. The purpose is 1) to characterize biocontrol products adapted to local Mediterranean crops (wine,

fruits and vegetables growing), 2) to give advices to the farmers about the use of these products, and 3) to

enhance the development of biocontrol industry and sustainable agriculture.

To reach that goal, the biopesticides selected in the project are studying according to their efficiency through

field experimentation, their effect on plants and their environmental impact.

The UPVD (University of Perpignan Via Domitia) will contribute to the part of the project regarding the evaluation

of biocontrol products’ environmental impact. To date, the half-life, t1/2, was often used to study the fate of

pesticides in environmental matrices. However, this value gives restricted information as it doesn’t give any

information regarding the formation of by-products and the effect on biodiversity. Moreover, biopesticides are

usually really complex mixtures and yield chromatograms that are extremely difficult to interpret which

precludes the t1/2 from being considered as a viable tool. Consequently, an innovative approach based on

metabolomics (LC-MS), the Environmental Metabolic Footprinting (EMF), was recently developed in the

laboratory (Patil et al, 2016; Salvia et al, 2017).

On one hand, the EMF gives rise to a new integrative proxy, the resilience that corresponds to the time needed for

the compound dissipation and its effects on the matrix. It has the potential to evaluate all the po st-

application effects of the biopesticide.

On the other hand, the EMF can be used to determine the preharvest interval (PHI) that corresponds to the

time needed to have no residue difference between the treated sample and the control.

Different and complex matrices from fields (fruits, leaves and soils) will be studied. Optimizations will be done in

completely controlled environment (climatic chamber). Then, on the basis of these optimizations, samples from

the field experimentations will be analyzed in order to determine the effects of the different treatments

through the evaluation of the resilience time and the PHI in particularly.

Keywords : biocontrol compounds, environmental impact, LC-MS, metabolomics, resilience time Patil C et al (2016). Environmental Metabolic Footprinting : A novel application to study the impact of a natural and a synthetic β-triketone herbicide in soil. Science of the Total Environment 566-567 (552-558). Salvia M-V et al (2017). Environmental Metabolic Footprinting (EMF) vs. half-life: a new and integrative proxy foe the discrimination between control and pesticides exposed sediments in order to further characterize pesticides’ environmental impact. Environnemental Science and Pollution Research.


90


INTRODUCING HIGH RESOLUTION MASS SPECTROMETRY IN THE ENVIRONMENTAL METABOLIC FOOTPRINTING METABOLOMICS APPROACH; APPLICATION ON EXTRACTED NATURAL HERBICIDE

“MYRIGALONE A”

Hikmat Ghosson (1,2), Rahul Suryawanshi (1), Delphine Raviglione (1,2,3), Marie-Virginie Salvia (1,2,3), Cédric Bertrand (1,2,3,4)


Perpignan Cedex, France (2) UPVD, UFR Sciences Exactes et Expérimentales, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860

Perpignan Cedex, France (3) Laboratoire d’Excellence « CORAIL », Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France (4) S.A.S AkiNaO, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

Herbicide consumption is yearly increasing in order to enhance crop yield and its quality. However, these

chemical compounds are threatening environment, and for instance, soil. Thus, tendency for natural

bioherbicide use is increasing. In fact, these “bio-compounds” are supposed less harmful for the

environment and its biodiversity. However, the evaluation of the environmental impact for these

compounds is still not widely studied. This is because of multiple limitations in classical evaluation

protocols (e.g. half-life). Thus, new omics-based methods are being developed in order to adapt new

suitable protocols and assess the environmental risk of such biocontrol products.

Recently, an innovative metabolomics-based approach was developed in the laboratory as an alternative

tool for the evaluation of the environmental impact. This approach named “Environmental Metabolic

Footprinting” (EMF), was applied on pure chemical pesticides in soil1, and then on a complex bio-

insecticide in sediments2, using Liquid Chromatography-Mass Spectrometry (LC-MS). The aim of this

approach is to determine the “resilience time” as a new integrative evaluation concept. It corresponds

to the time required for the dissipation of the compound and its effect on the matrix microbiome.

In fact, this approach takes in consideration the analysis of the whole “meta-metabolome” of the

environmental matrix. This meta-metabolome contains the pesticide and its by-products

(xenometabolome), but also the microbial metabolome. Thus, as EMF approach proved efficiency and

several advantages, improving this approach was considered by introducing High Resolution Mass

Spectrometry (HRMS), using a Quadrupole/Time-of- Flight Mass Spectrometer (QToF MS). This

improvement allowed higher selectivity by exact mass measuring. Hence, potential bias issued from

interferences were eliminated. In addition, necessary information for biomarkers discovery and

identification were provided.

These advantages are necessary to study complex natural biopesticides containing several unknown

metabolites. Thus, this work was applied to evaluate the environmental impact of a natural complex

bioherbicide; the Myrica gale extract, containing the “Myrigalone A”. The impact of this natural

bioherbicide will be compared to the pure synthetized Myrigalone A compound. A reference product,

“Sulcotrione”, a well-known β-triketone herbicide, will be included in the study.


91

This bioherbicide presenting high affinity to the soil, makes the kinetics monitoring insufficient for low

concentration abundance. Thus, the interest of applying EMF is to analyze its by-products and the impact

on microbial metabolome. For that, a 150 days kinetic experiment was performed with 10 time points

in soil microcosms.

Five replicates were done for each treatment and time condition. After soil extraction, the extracts

were analyzed after optimizing an exhaustive analytical method by LC-QToF system. Acquired data were

processed on “Workflow4metabolomics” online platform after critical review and rigorous optimization

of preprocessing parameters.

Processed data were used for multivariate analysis, and resilience time was explored by comparison of

herbicide-spiked microcosms with controlled microcosms. Results showed high discrimination between

controlled batch and the Myrica gale extract batch. Hence, biomarkers “fishing” and discovery is planned

in order to identify discriminant metabolites, so we can explain better the environmental impact of this

bioherbicide.

Keywords: HRMS, Environmental Metabolic Footprinting, Myrica gale, Myrigalone A, Soil metabolomics


92


A NEW TOOL FOR ASSESSMENT OF BIOSTIMULANTS EFFECTS ON GRAPEVINE

Sophie Trouvelot (1), Marielle Adrian (1), Marie-Claire Héloir (1), Lucile Jacquens (1),

Yuko Krzyzaniak (1), Anthony Laybros (1), Frédéric Cointault (1), Jean-Claude Simon (1), Estelle Moreau (1), and other members of the FUI Iris + consortium

(1) Agroécologie, AgroSup Dijon, CNRS, INRA, Univ. Bourgogne Franche-Comté, F-21000 Dijon, France (2) Arysta Goëmar Parc Technopolitain Atalante, CS41908, 35435 Saint Malo cedex

Despite an increasing interest for the use of biostimulants (BS) in agriculture, methods allowing a precise

description of their effects on plants remain rather limited. In the IRIS+ FUI project, two major and highly

different worldwide crops, wheat (annual, monocotyledon) and grapevine (perennial, broadleaf) were

chosen to deepen our knowledge of such compounds and explore their potential additional interest.

One objective was to develop a tool adapted to the screening and study of the impact of a series of BS

on the development and physiology of these crops in controlled conditions.

We managed to develop such a tool adapted to grapevine herbaceous cuttings. It allows a quite rapid

and non-destructive phenotyping of BS effects on the aerial and root parts of plantlets over time. At the

end of the experiment, plant material can be sampled for biomass measurements (fresh and dry weight)

and analysis of gene expression or metabolites. This tool was validated with some commercial BS prior

being used to study new putative ones.

Using this tool, we have identified a new BS provided by Arysta-Goëmar. The positive effects of this BS

was dose dependent. We have observed that it transiently accelerates the shoot development and

increases the number of roots, and also shoot and root biomass. Moreover, analysis of the root and

shoot metabolomes highlighted clear fingerprints associated to BS effects.

This tool will be useful to screen new BS potentially active in grapevine. Moreover, it will be useful to

study and compare the effects of diverse BS on grapevine development, metabolism and physiology ;

and to decipher their mode of action. In this context, work is still in progress to further develop this tool

to make it suitable to the highlighting of diverse effects related to BS and other crops, such as wheat.

Keywords : grapevine, biostimulants, phenotyping


93


RECHERCHE DE BIOMARQUEURS DE TOLERANCE A LA VERTICILLIOSE CHEZ LE LIN PAR UNE APPROCHE DE METABOLOMIQUE PAR RMN

Lecomte Sylvain (1, 2, 3), Fliniaux Ophélie (1), Fontaine Jean-Xavier (1), Molinie Roland (1), Van Wuystwinkel Olivier (1), Gutierrez Laurent (2),

Pineau Christophe (3), Mesnard François (1)

(1) BIOPI, EA3900, UFR de Pharmacie, Université de Picardie Jules Verne, 80000 Amiens (2) CRRBM, Université de Picardie Jules Verne, 80000 Amiens (3) Linea, Semences de Lin, 60210 Grandvilliers

Le lin, Linum usitatissimum, est une plante cultivée en Europe pour sa fibre, dans les régions allant de la

Normandie au Nord de la France et s’étendant jusqu’en Belgique et aux Pays-Bas, pour de nombreuses

applications dans les domaines du textile, des matériaux composites… Lors de sa culture, le lin peut être

soumis à l’attaque de différents agents pathogènes, comme des insectes (altises, thrips…), comme des

champignons (Fusarium oxysporum f. sp. lini, Septoria linicola, Oidium lini, Verticillium dahliae...). Ces

attaques pathogènes entrainent des pertes de rendements et/ou des problèmes de qualité de la fibre*.

Pour limiter les risques liés à ces agents pathogènes, différentes approches sont envisagées : le respect

de bonnes pratiques culturales, la certification de graines exemptes d’agents pathogènes, l’utilisation

de produits phytosanitaires ou encore la création/sélection variétale.

Pour lutter contre la verticilliose**, aucun traitement phytosanitaire n’est efficace et aucune variété de

lin ne présente de résistance totale. Dans ce contexte, la recherche de biomarqueurs de résistance à cet

agent causal nécessite d’être envisagée. Une approche par métabolomique par RMN est proposée pour

étudier les variations métaboliques dans différents organes (tiges, feuilles, racines) de deux variétés de

lin, Evéa (variété partiellement résistante à la verticilliose) et Violin (variété sensible à la verticilliose),

infectées artificiellement. L’analyse multivariée réalisée à partir des données de RMN

monodimensionnelle du proton montre qu’en conditions témoins, ces deux variétés de lin révèlent des

profils métaboliques distincts au niveau des organes étudiés. D’une manière générale, la teneur en

acides aminés, tels la proline, le glutamate sont en concentration plus élevée chez Evéa que chez Violin,

alors que la teneur en monosaccharides est plus élevée chez Violin que chez Evéa, aussi bien dans les

racines que dans les tiges. Il en est de même pour les extraits obtenus avec les deux variétés de lin

cultivées en présence de V. dahliae aux différents points de cinétique de l’infection. Certaines variations

métaboliques engendrées par l’infection par V. dahliae sont communes aux différents organes et aux

deux variétés. D’autres sont organe-spécifiques et/ou variété-spécifiques et/ou cinétique spécifique. Par

exemple, en conditions de stress, les cyanogènes ont leur concentration dans les feuilles et les racines

augmentée à J7, puis diminuée à J20 chez Violin alors que chez Evéa, la concentration en ces composés

n‘est augmentée qu’à J7 au niveau des racines. La putrescine a sa concentration augmentée pour Evéa

à J7 et J20 alors que pour Violin sa concentration augmente à J7 et diminue à J20. L’alanine a sa

concentration augmentée pour Evéa à J7 et à J20 alors que ce phénomène n’est observé qu’à J20 pour

Violin.

Mots-clés : Linum usitatissimum - Verticillium dahliae - métabolomique - RMN

* Muir AD, Wescott ND. 2003. Flax – the genus Linum. CRC press, Boca Raton. ** Klosterman SJ, Atallah ZK, Vallad

GE, Subbarao KV. 2009. Diversity, Pathogenicity and Management of species. Annual Review of Phytopathology,

47, 39-62.


94


SCREENING STRATEGY TO SELECT NEW FUNGAL BIOCONTROL PRODUCTS AGAINST PLANT DISEASES

Marchi Muriel, Franck Bastide, Daniel Sochard, Sophie Aligon, Bruno Hamon, Thomas Guillemette

Institut de Recherche en Horticulture et Semences, UMR1345, INRA, Université d’Angers, Agrocampus-Ouest, SFR QUASAV, F-49000 Angers (France)

Seeds are the staple of human and animal diets but also efficient microorganism vectors. Seed sanitary

quality and vigor are crucial for seedling development and crop productions in general (horticulture,

cereal and vegetable productions). The seed microbiota structure and dynamic alter plant heath and

development. In this context characterization of microorganism interactions between themselves and

with the plant contribute to plant adaptation to biotic and abiotic stresses and represent new sources

for biocontrol and biostimulation innovations.

This work displays the set of fungal phenotyping tools developed in our laboratory to screen fungal

strains in order quantify biocontrol and biostimulation activities during seedling development.

This screening strategy considers the antagonistic property (in vitro and in planta antagonism assays,

effect on the plant defenses) and the biological properties of each isolate (spore production, growth

rate, mycotoxin risks, germination and growth at 37°C, cold tolerance, compatibility with fungicides…).

To screen the strains, parasitism and competition between them are also observed by cultural approach

and using filamentous fungi – tailored procedure based on liquid culture and automated nephelometric

recording of growth in 96 wells microplate. This growth monitoring technique is applicable for the

evaluation of antifungal activity and for large-scale phenotypic profiling. A molecular diagnostic tool

called qPFD ® (quantitative low-density microarray: microplate quantitative RT-PCR / low-density DNA

microarray) was used to provide information on the level of stimulation of the plant defenses in contact

with antagonist strains or metabolites.

Some results will be exposed as proof of concept concerning this phenotyping strategy.


95


A FIELD SAMPLING KIT TO EVALUATE AND CONFIRM PLANT DEFENSE INDUCTION OF ELICITORS IN FIELD CONDITIONS

Bryone F. (1), Roelofsen C. (2), Bonneau C. (1)

(1) VEGEPOLYS Innovation, 26 rue Dixmeras, 49066 Angers, France (2) NSure B.V., PO Box 14, 6700 AA Wageningen, Netherlands

The qPFD® is a molecular tool patented by INRA for the screening in laboratory conditions of plant

defense inducers (PDIs) also called elicitors.

We combined the qPFD with the FTA® technology using the sampling and extraction method developed

by NSure B.V., to evaluate the expression of plant defense genes in field conditions. FTA cards were used

to collect and transport nucleic acids from field to laboratory. Extraction methods were optimized and

adapted to tomato and grapevine, to increase RNA yield and quality, and the transcription levels of PR

genes were evaluated in different field trials.

Our results demonstrated, using this field kit, defense induction in field trials, after treatment with

elicitors. The methodology developed in this work is now proposed to agribusiness and more generally

to the agricultural sector to accelerate the use of PDIs.

Keywords: plant defense inducers, field, elicitors


96


DEVELOPMENT OF LOCAL BIOCONTROL PRODUCTS ADAPTED TO MEDITERRANEAN CROPS

Ramos Mélina (1,2,3), Salvia Marie-Virginie (1), Llugany Mercè (2), Badosa Esther (3),

Gabolde Sophie (4), Andreu Vanessa (5), Elorduy Vidal Xoán (6), Fernández Carolina (7), Montesinos Emilio (3), Bertrand Cédric (1,5)


Perpignan Cedex, France (2) Unitat de Fisiologia vegetal, Universitat Autonoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Spain (3) Center for Innovation and Development in Plant Health (CIDSAV), University of Girona, Maria Aurèlia Capmany, 61,

17003 Girona, Spain (4) Chambre d'Agriculture des Pyrénées-Orientales, 19 avenue de Grande Bretagne, 66025 Perpignan Cedex, France (5) AKINAO, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France (6) INCAVI, Pl. Agora 2, 08720 Vilafranca del Penedes, Spain (7) Futureco Bioscience, Pol. Ind. St. Pere Molanta, Avda Cadì 19-23, 08799 Olèrdola, Spain

Consumers as well as farmers are supporting the decrease of conventional pesticides use in favor of the employment of biocontrol products. To meet those expectations, French and Spanish governments promote the biocontrol products market: it recorded a growth rate of around +15% in a year. Nevertheless, references regarding technical efficacy and ecotoxic data are lacking. To fill that gap, the PALVIP project (local Mediterranean crops’ alternative protection) associates universities and technical structures to evaluate new biocontrol products BP developed by the local SMEs. The purpose is 1) to characterize biocontrol products adapted to Mediterranean crops (wine, fruits and vegetables growing), 2) to give advices to the farmers about the use of these products, and 3) to enhance the development of biocontrol industry and sustainable agriculture. To reach that goal, the biopesticides selected in the project will be studied according to their efficiency through field experimentations (Chambre d’Agriculture 66, INCAVI), their environmental impact (Université de Perpignan Via Domitia, Universitat de Girona, Futureco Bioscience) and their effect on plants (Universitat de Girona, Universitat Autonoma de Barcelona). Field experimentations are done on i) vine against mildew, powdery mildew and botrytis; ii) on peach tree against brown rot; iii) on apricot tree against flowers’ drying up caused by Monilia laxa; iv) on cherry tree against flies; and v) on salad against weeds. This experimentation design includes pesticide commercial references; selected biocontrol products from SMEs partners (AkiNaO, Futureco Bioscience) and controls (untreated plants). Tested biocontrol products are microbials and botanicals. Impact of all treatment on wine quality will be investigated Environmental impact of biocontrol products will be studied using an innovative approach based on metabolomics (LC-MS), the Environmental Metabolic Footprinting (EMF). This technique allows the study of really complex mixtures like soils, leaves and fruits. It gives information regarding the products’ residue, the formation of by-products and the effect on biodiversity. After applying the different biocontrol treatments, plants’ response is studied using a transcriptomic approach based on ARN sequencing technique together with quantitative PCR. It will give information about the different gene expression following treatments with biocontrol products and other compounds compared with the non - treated controls. Furthermore, qPCR will enable a quantification of the plant response. That information will be completed with metabolic analysis of phytoalexins (phenols, resveratrol and defensive hormones).

Keywords: biocontrol compounds, environmental impact, LC-MS, metabolomics, resilience time,

phytochemistry, transcriptomic


97


"GRAPEVINE BIOCONTROL" PLATFORM (INRA / IFV - BORDEAUX) FOR EVALUATION OF BIOCONTROL PRODUCTS AND GRAPEVINE

PROTECTION

Dufour Marie-Cécile, Pierre Sauris, François Delmotte

INRA, UMR Santé et Agroécologie du vignoble (SAVE-1065), CS 20032, ISVV, 33882 Villenave d’Ornon CEDEX, France.

The "GRAPEVINE BIOCONTROLE" platform is an instrumented experimental device deployed at different

spatial scales to assess new biocontrol products with appropriate methodologies and propose optimal

use strategies for the vineyard (characterization of the mode of action; product efficacy assessment

under controlled conditions and the vineyard and integration into protection strategies to maximize

their effectiveness).

The platform focuses on three major grapevine diseases such as powdery mildew (Erysiphe necator),

downy mildew (Plasmopara viticola) and gray mold (Botrytis cinerea). It is also able to respond to

inquiries about the diseases responsible for dieback (Eutypa lata, Esca) or secondary aerial diseases such

as black rot (Guignardia bidwellii).

DEVICE DESCRIPTIONS :

VINEYARDS :

1. A first device aims to evaluate products at intermediate scales coupled with epidemic control. It is

composed of large parcels (total of 0.9 ha, sensitive grape varieties) which are equipped with a misting

system for the realization of artificial inoculations. This makes it possible to envisage tests with different

aggressiveness strains as well as multi-pathogenic tests. Currently used for the evaluation of

phytosanitary products, it will be fully remobilized as part of the "GRAPEVINE BIOCONTROLE" platform.

2. A second device is in progress (funding INSTITUT CARNOT-PLANT2PRO). It will be dedicated to the fine

exploration of the conditions of biocontrol products effectiveness. This will be an instrumented parcel

of 0.4 ha which is on the site of INRA Bordeaux planted with sensitive and resistant grape varieties and

equipped with instruments for measuring microclimatic and soil parameters. The molecular tools for

characterizing the physiological state of the vine (NeoViGen96 chip) can be mobilized in a relevant way

on this device.

3. A third device is entirely dedicated to the evaluation of solutions in production conditions on natural

epidemics based on access to plots in private vineyards.

LABS :

Equipment for carrying out bioassays in controlled conditions: in vitro biotests, evaluation of

competition between fungi by antibiosis, measurement of defense gene expression, etc...

Keywords : efficacy and mode of action, biocontrol products, grapevine diseases


98


UTILISATION DES MOYENS BIOLOGIQUES (BACTERIES LACTIQUES) POUR LA CONSERVATION DES ALIMENTS

Dr Bendimerad Nahida, Boumedienne Karima et Pr Moussa Boudjemaa Boumedienne

Laboratoire LAMAABE, Département de Biologie, Faculté SNV, Université de Tlemcen Algérie Mail : [email protected]

La préservation de l’innocuité des aliments qui est un combat constant des organismes agro-

alimentaires, et la demande croissante des consommateurs de pouvoir bénéficier des produits frais,

poussent les professionnels de l’agro-alimentaire à se tourner vers l’utilisation des moyens biologiques

comme les bactéries lactiques. En effet dans les industries agro-alimentaires, les bactéries lactiques sont

utilisées pour accomplir plusieurs rôle, comme celui de fermenter les aliments, de produire des

composés aromatiques et le rôle de conserver les aliments et les protéger contre les microorganismes.

Tel est le but de ce travail.

Cette étude consiste à montrer l’effet antagoniste d’une souche lactique « S94 » appartenant à l’espèce

et sous espèce Lactococcus lactis subsp lactis biovar diacétylactis vis –à-vis des bactéries pathogènes et

d’altération comme Staphylococcus aureus (ATCC25923), Listeria monocytogenes (ATCC19145),

Escherichia coli (ATCCP25922), Enterococcus faecalis (ATCC29212). L’étude des interactions entre

bactérie lactique et bactéries contaminantes a nécessité l’application de la technique de Fleming et al,

(1975), la méthode de Barefoot (1983), et la mesure de la densité optique. Les trois techniques ont

montrés que les bactéries contaminantes des produits alimentaires sont inhibées par la souche lactique

S94, cette inhibition est dû probablement à la production par cette souche d’une substance de nature

protéique appelée bactériocine. Cette substance est sensible à deux enzymes, la protéinase K et la

pepsine, de nature protéique aussi.

Ainsi la souche lactique S94 est recommandée pour être sélectionner comme « Starter » afin d’être

utiliser dans les industries agro-alimentaire comme conservatrice.

Mots clés : bactéries lactique, aliment, conservateur, bactéries contaminantes



99


PRESENTATION OF THE FRENCH PUBLIC-PRIVATE CONSORTIUM FOR RESEARCH, DEVELOPMENT AND INNOVATION ON BIOCONTROL

MULTEAU Cécilia (1), MALAUSA Thibaut (2)

(1) INRA, centre Montpellier – Département SPE – 2 place Pierre Viala 34060 MONTPELLIER (2) INRA, centre PACA – UMR ISA – 400 route des Chappes BP167 06903 Sophia Antipolis

The French public-private Consortium for Research, Development and Innovation on Biocontrol has for

ambition to foster the use of biocontrol and to support the deployment of an industrial biocontrol

activity in France.

Launched in 2016, the 48 private and public members of the consortium are implementing a scientific

and technical program of collective interest in order to facilitate the development of the biocontrol by

research and innovation.

Consortium strategy aims to increase the expertise and know-how about biocontrol by catalyzing

initiatives of its members through precompetitive research actions.

By relying on joint public-private proposals, the consortium is also a contact point of choice for

stakeholders at both national and international levels to work on the orientation of R&D&I strategies.

To get more information : www.consortium-biocontrole.fr

Keywords: Consortium Biocontrol; Biocontrol

http://www.consortium-biocontrole.fr/


100


ANIMATION OF EXPERIMENTATION NETWORKS DEDICATED TO THE BIOCONTROL

Berthelot Régis, Besnier Thomas

(1) ARVALIS INSTITUT DU VEGETAL - Station Expérimentale - 91720 Boigneville (2) VEGENOV - Pen ar Prat, 29250 St Pol de Léon

Today, factors leading to the successful use of biocontrol products remain difficult to identify due to the

complexity of parameters to be considered while testing them.

Moving from lab to fields, with well-established technical routes, is often a delicate step, because

biocontrol strategies, based on natural regulations, consist of complex interactions between organisms.

Standards available to evaluate phytoprotection products are not suitable for living organisms compared

to inert materials such as chemicals.

To facilitate the transition of biocontrol products toward fields, the Consortium Biocontrôle, composed

of public and private members, granted a first research project (named XP-BC) aiming to implement

experimental networks dedicated to biocontrol. Three networks were built focusing on: 1) airborne

diseases on field crops, 2) mildews and 3) insect and mite pests.

This project gathers together 12 Agricultural Technical Institutes or Applied Research Institutes and the

French National Institute for Agricultural Research (INRA) for 3 years (2017-2019).

Keywords: experimentation, new biocontrol products, methodology


101


EFFICACITE DU KAOLIN (KALABA) DANS LA GESTION INTEGREE DES CHENILLES ENDOCARPIQUES DU COTONNIER

DANS LA ZONE CENTRE DU BENIN

G. Bonni (1,2), A. Hougni (1), A. Paraiso (2), T. Houndete (1)

(1) Centre de Recherche Agricole Coton et Fibres (CRA-CF), Institut National des Recherches Agricoles du Bénin (INRAB) Abomey-Calavi, Bénin

(2) Université de Parakou, Ecole Doctorale Sciences Agronomiques et Eau, Laboratoire de Protection des Végétaux, de Pathologie et de Parasitologie des abeilles (LAPPAB)

Les travaux sur l’efficacité du Kaolin dans la gestion intégrée des chenilles endocarpiques du cotonnier

ont été réalisés dans le Centre du Bénin. L’objectif visé par les travaux était d’évaluer la potentialité du

Kaolin dans la lutte contre quelques chenilles endocarpiques du cotonnier.

Pour atteindre cet objectif, un dispositif en Bloc de Fisher ayant 4 traitements et 4 répétitions a été mis

sur pied sur deux sites.

Les traitements mis en comparaison étaient constitués d’une parcelle non traitée ; d’une parcelle traitée

à Lambdacyalothrine 15 g/l - chlorpyrifos éthyl 300 g/l; d’une parcelle traitée au kaolin 5 % et d’une

autre traitée au mélange kaolin + Lambdacyalothrine 15 g/l - chlorpyrifos éthyl 300 g/l. Le traitement au

kaolin a été réalisé avec un pulvérisateur à dos, à pression entretenue. Dix applications ont été réalisées

au kaolin utilisé seul et 6 sur les autres objets.

Les résultats ont montré qu’il n’y a pas de différence significative entre le nombre moyen de chenilles

répertoriées dans les parcelles traitées avec le kaolin et les parcelles qui ont servi de témoin de référence

(Lambdacyalothrine 15 g/l - chlorpyrifos éthyl 300 g/l, à 1 l/ha). Il en est de même pour le pourcentage

de capsules vertes attaquées et la production de coton graine. L’effet du kaolin dans le contrôle du

phyllophage Haritalodes derogata a été faible, (11,6 % de plants attaqués contre 0,1% pour le témoin

chimique de référence et 22,9 % pour la parcelle non traitée).

Les résultats du Kaolin associé au binaire acaricide, ont montré que le nombre de chenilles

endocarpiques a été significativement réduit par rapport à celui du témoin de référence. Cette étude

indique que le kaolin peut, à l’instar des autres pesticides biologiques, être utilisé comme alternative à

la lutte chimique contre les ravageurs du cotonnier et constitué une composante importante dans la

gestion intégrée des ravageurs du cotonnier.

Mots clés : Cotonnier, Pectinophora gossypiella, Thaumatotibia leucotreta, kaolin, gestion intégrée


102


EVALUATION DE PRODUITS DE BIOCONTROLE POUR LUTTER CONTRE LA POURRITURE GRISE DE LA VIGNE

Aveline N (1), Dupin S (2), Calvo Garrido C (3), Davidou L (2), Roudet J (3), Fermaud M (3)

(1) Institut Français de la Vigne et du Vin– 39 rue Michel Montaigne 33290 Blanquefort- France (2) Chambre d’Agriculture de la Gironde- 39 rue Michel Montaigne 33290 Blanquefort- France (3) INRA – UMR SAVE, 71 avenue Edouard Bourlaux CS 20032, 33882 Villenave-d'Ornon cedex- France

La pourriture grise (B.cinerea) est une maladie de la vigne qui affecte la vendange de façon quantitative

et qualitative. La lutte fongicide classique utilise des traitements à stades préalablement fixés en saison

(floraison, fermeture de la grappe et/ou début véraison) pour limiter l’apparition des symptômes lors

des dernières semaines, voire jours avant récolte. L’efficacité de cette lutte n’est pas toujours optimale

et peut générer notamment des résidus phytosanitaires dans les vins. Depuis quelques années, le statut

« Biocontrôle » a fait émerger quelques produits de protection alternatifs avec un profil à faible risque,

homologués contre la pourriture grise. Ces produits présentent des origines et des modes d’action

divers : i) micro-organismes vivants (bactéries, champignons) agissant par compétition spatiale /

nutritive et/ou antibiose; ii) des produits d’origine naturelle à action physique (choc osmotique) ou

fongicide directe. Malgré ce panel disponible, des comparatifs d'efficacité et des exemples d’emploi

optimisés au vignoble sont peu répandus.

En Aquitaine, des projets collaboratifs menés de 2014 à 2017 ont fait intervenir différents

dispositifs (réseaux ou des plateformes d’essai) pour obtenir plus d’informations sur ces produits et

proposer aux viticulteurs des stratégies efficaces contre B.cinerea. Les résultats montrent que les

produits à base de micro-organismes vivants présentent des efficacités très variables et irrégulières

selon les millésimes et les sites d’essai. Pour les substances naturelles, l’Armicarb (Bicarbonate de

potassium) se détache comme le meilleur candidat, avec une attaque moindre comparée au témoin non

traité. Néanmoins les taux d’efficacité sont faibles à moyens (40-45% maximum).

Cependant le biocontrôle du Botrytis au vignoble pose de nombreuses questions, en lien notamment

avec son épidémiologie complexe et de nombreux facteurs influençant son développement en saison.

Un indice de développement du Botrytis (INRA) basé sur la climatologie à partir de la véraison a été

évalué pour positionner certains produits de biocontrôle. De plus, la nature même des produits de

biocontrôle testés et leur mode d’action restent trop mal connus. L’implantation de micro-organismes

au vignoble soulève également les problématiques de survie en lien aux conditions climatiques,

d’interaction avec les autres traitements de l’itinéraire et de cinétique de développement face à B.

cinerea.

Mots-clés : vigne, biocontrôle, pourriture grise- pilotage


103


USE OF MICROBIAL ANTAGONISTS OF THE CHESNUT ROT AND CANKER AGENT GNOMONIOPSIS SMITHOGILVYI TO REDUCE THE

MORTALITY OF CHESTNUT TREES IN NURSERY

Jonathan Bourquin (1), Julien Crovadore (1), Francesco Bonavia (2), Bastien Cochard (1), Romain

Chablais (1), Paolo Piattini (3), François Lefort (1)


(2) Vivaio forestale cantonale, Dipartimento del territorio, Divisione dell’ambiente, Sezione forestale Repubblica e Cantone Ticino, 6835 Morbio Superiore, Switzerland

(3) Associazione dei castanicoltori della svizzera italiana, Casella postale 112, 6947 Vaglio

Gnomoniopsis smithogilvyi is an endophytic fungus, recently identified in Europe and Switzerland, as the

main agent of chestnut brown rot but also as a chestnut canker agent. It seems to be the main cause of

the high mortality occurring in chestnut nurseries and orchards. Previous works showed that it was

possible to detect the presence of G. smithogilvyi presence as an as an endophyte in chestnut material

(roots, fruits, leaves) of rootstocks and grafts, endophyte but that C. parasitica, the main agent of

chestnut canker, was never detected. Rootstock material showed to be slightly contaminated while

grating material of all varieties tested was highly contaminated by the endophytic fungus G. smithogilvyi.

As two antagonists, Trichoderma hamatum UASWS1405 and Pseudomonas putida UASWS0946 had

showed a high level of protection against G. smithogilvyi in experiments run in climatic chambers, it was

decided to test these organisms in planta at the State Nursery of Canton Ticino. Several experiments

were carried out from September 2017 to August 2018 to evaluate the biocontrol efficacy of these

organisms with the goal to develop a practical solution to reduce chestnut mortality in nurseries and

orchards. In parallel, a metagenomic experiment was conducted to understand how these two

organisms may influence the microbiota of chestnut trees. Concerning the two field experiments carried

out in Ticino, the first experiment aimed to evaluate the biostimulating and curing effects of these

antagonists on trees infected by G. smithogilvyi. The presence of the pathogen had been previously

detected by a specific molecular diagnostic test. Commercial formulations of the two antagonists were

inoculated in the culture substrate by humectation, under several modalities. As we observed that the

contamination mostly occurs at grafting, a second experiment aimed to evaluate the use of these

antagonists as a preventive treatment applied at grafting by soaking grafts in suspension of

microorganisms and to observe the survival rate of grafted trees in nursery. Until now, molecular

diagnostic showed the presence of G. smithogilvyi in almost all the mother trees (89 samples analyzed)

used for grafting in the nursery. Old varieties and local varieties show the higher percentage of G.

smithogilvyi presence, while infection by C. parasitica remains globally very low in all the mother trees

samples analyzed. Concerning the 200 young trees from the biostimulant experiment, the same analysis

showed the presence of G. smithogilvyi in about 10% of the trees all modalities combined. Compared to

controls, soaking grafts in aqueous suspensions 18h hours before grafting seems to have no effects on

trees survival, while the first experiment by humection of potting soil showed promising results on short

term trees survival in nursery, with a reduction of mortality in all modalities.

Keywords: Gnomoniopsis smithogilvyi, chestnut canker, biocontrol, Trichoderma hamatum, Pseudomonas

putida


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