comparison of cross inoculation potential of south african avocado and mango isolates of...
Post on 21-Sep-2016
Embed Size (px)
Three hundred and eighteen Colletotrichum gloeosporioidesisolates from stem-end rot and anthracnose infected avocadosas well as from stem-end rot/anthracnose and soft brown rot onmango, were compared using fruit inoculations. Isolates couldbe categorised according to lesion size and both avocado andmango isolates produced larger lesions when inoculated ontheir own hosts. Cross-inoculation potential of these isolateswas also compared on strawberries, peppers, guavas, papayasand citrus. All isolates produced lesions on all hosts exceptcitrus. Factors such as area of origin and symptom type fromwhich original isolations were made, could not be correlatedwith lesion development on these hosts.
Key words: avocado mango Colletotrichum gloeosporio-ides inoculation study
Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.in Penz. is a ubiquitous, proliferate and economicallyimportant pathogen causing substantial yield losses due to fruit decay and damage to vegetative parts in avariety of plant species (Freeman and Shabi 1996).Diseases caused by C. gloeosporioides include anthrac-nose, dieback, root rot, leaf spot, blossom rot and seed-ling blight on a wide range of crops including avocado,almond, peach (Freeman et al. 1998), peppers (Manand-har et al. 1995), papaya (Dickman 1994) mango (Ploetz1994), Stylosanthes spp. (Chakraborty and Jones 1993),
citrus (Timmer et al. 1994), rubber trees (Brown andSoepena 1994), passion fruit (Jeffries et al. 1990) andstrawberry (Denoyes and Baudry 1995).Isolates of C. gloeosporioides from almond, apple,
mango and avocado inoculated into detached apple,avocado, almond, mango and nectarine were shown tosuccessfully cross-infect (Freeman and Shabi 1996), butwere more virulent on the same host from which theywere originally isolated (Hayden et al. 1994). Cross-inoculation studies with C. gloeosporioides on a widerange of hosts have been reported (Maas and Howard1985; Alahakoon et al. 1992, 1994; Freeman et al.1996). Differential virulence of C. gloeosporioides iso-lated from several hosts was shown when inoculatedinto other hosts (Quimio and Quimio 1975; Freemanand Shabi 1996). Furthermore, the pathogen was shownto genetically adapt to the new host, eventually inducingthe same level of disease as isolates from the host inquestion (Alahakoon et al. 1992).Many hosts susceptible to C. gloeosporioides are
cultivated world-wide and losses where multiple hostssuch as mango, avocado, coffee, papaya and citrus aregrown in close proximity, could be enormous (Freemanet al. 1998). This is especially true in South Africa,where there are small climatic zones suited for cultiva-tion of subtropical crops, and avocados and mangoes areoften cultivated in adjacent blocks or orchards. Isolationfrequency of C. gloeosporioides from stem-end rot,anthracnose and soft brown rot was variable in avocadosand mangoes, although generally higher on mangoes(Sanders et al. 2000). Higher levels of C. gloeosporioi-des on mango trees could therefore serve as an inoculumsource for adjacently cultivated crops.
0944-5013/03/158/02-143 $15.00/0 Microbiol. Res. 158 (2003) 2 143
Microbiol. Res. (2003) 158, 143150http://www.urbanfischer.de/journals/microbiolres
Comparison of cross inoculation potential of South Africanavocado and mango isolates of Colletotrichum gloeosporioides
G. M. Sanders*, L. Korsten
Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute, University of Pretoria,Pretoria 0002, South Africa
Accepted: December 23, 2002
Corresponding author: Dr. G. M. Sanders (Swart)e-mail : firstname.lastname@example.org
Because of the close proximity in which crops such asavocados, mangoes, papayas and citrus are often culti-vated, it is important to determine whether the differenthosts can serve as inoculum sources for the others. Thepurpose of this study was therefore to determine thevirulence of C. gloeosporioides isolates collected dur-ing a market survey of post-harvest disease incidence onavocados and mangoes (Sanders et al. 2000) and theircross-inoculation potential each other as well as onpapayas, strawberries, peppers, guavas and citrus.
Materials and methods
Culture collection. Three hundred and eighteen mono-conidial C. gloeosporioides isolates were randomlyselected from the market survey collection described bySanders et al. (2000). Details of all isolates includinghost, symptom and stage of ripeness from which isola-tions were made were also recorded (data not shown;Swart 1999). Of these, 207 were from avocado and 111from mango (Table 1). Cultivation and maintenance ofisolates was done on oatmeal agar (20 g oatmeal, 20 gagar (Biolab), 1 l distilled water) (OA). Plates wereincubated at ambient temperature under constant mixedirradiation from near ultraviolet and daylight typefluorescent tubes (Phillips TL 40W/08RS, F40 B43 andTL 40W/33RS respectively) until sporulation occurred.Identities of isolates were confirmed using physiologi-cal and inoculation studies, molecular analysis (Swart1999) and morphology (Baxter et al. 1983). Cultureswere preserved for further use by freezing in 50% gly-cerol at 78C, as well as on potato-dextrose agar (Bio-lab) (PDA) slants and under sterile water.
Inoculation studies on Fuerte avocado fruit. Freshlyharvested untreated, unwaxed, physiologically mature,but unripe Fuerte fruit from Westfalia Estates (LimpopoProvince, South Africa) were used for plug inoculations.Prior to inoculation, all fruit were swabbed with 70%(v/v) ethanol to reduce surface contamination and left toair-dry in the laboratory. Starter cultures were preparedby incubating each C. gloeosporioides isolate for fivedays on OA as described previously. Plugs (4 mm dia-meter) were cut from actively-sporulating areas near the edge of each colony. Four 10 mm deep holes wereaseptically punched around the broadest side part ofeach fruit with a 4 mm diameter stainless steel corkborer. Three plugs, each from a different isolate, wereplaced into a hole in three replicate fruit with an OAmedium plug (control) into the fourth hole. Fruit plugsremoved from punched holes were replaced and coveredwith parafilm. Fruit were incubated upright at ambienttemperature (approximately 25C). After five days,lesion diameters were measured in two opposite direc-tions. Lesions 59 mm in size were rated as category 1,1014 mm as 2, 15 20 mm as 3, 2124 mm as 4,2529 mm as 5, 3034 mm as 6, 3539 mm as 7 and4044 mm as 8. Means were determined for all isolatesand all data were analysed with the SAS system usinganalysis of variance based on unequal subclass numbers.Pearsons product moment correlation coefficient (r)was used to correlate various factors under investiga-tion. Treatment means and ranks were compared usingDuncans multiple range test. Categories were compa-red using the chi-square test for equal proportions. Inoculation studies on Sensation mango fruit. Freshlyharvested, untreated, unwaxed, physiologically matureSensation fruit from Moria Mango Estate (LimpopoProvince, South Africa) were used for plug inoculations.Preparation of inoculum, inoculation, evaluation anddata analyses was carried out as described for avocado.Inoculation potential on different hosts. Cultures wereprepared in the same way as for avocado and mangoinoculation studies. After five days incubation, plateswere flooded with sterile distilled water (SDW) andconidia aseptically harvested using a glass rod. Conidialconcentrations were determined using a haemocyto-meter and adjusted with SDW to 1 104 conidia ml1.Strawberries (Fragaria ananassa Duchesne), guavas(Psidium guajava L.), peppers (Capsicum annuum L.),papayas (Carica papaya L.) and citrus (Citrus sinensisL. Osbeck) were selected for this study since they areoften cultivated in close proximity to avocados andmangoes. Fruit were collected from the National FreshProduce Market in Pretoria, South Africa. All fruit werethoroughly washed under running water, swabbed with70% (v/v) ethanol and left to air-dry in the laboratory.Inoculum droplets of 10 l containing ca. 104 conidia
144 Microbiol. Res. 158 (2003) 2
Table 1. Distribution and origin of Colletotrichum gloeospo-rioides isolates used in this study
Host Geographical origin Number of isolates
Mango Levubu 5Tzaneen 4Hoedspruit 6Kaapmuiden 7Letsitele Valley 26Hazyview 15Malelane 11
Total 111Avocado Louis Trichardt 39
Kwa-Zulu Natal 6Tzaneen 98Nelspruit 41Levubu 21Hazyview 2
ml1 SDW were placed onto 2 mm deep prick woundson the surfaces of the various fruits Three isolates perfruit with three to five fruit replicates per isolate wereused (three replicates: papayas, guavas and citrus; fivereplicates: strawberries and peppers). A single prickwound receiving SDW was included as control on eachfruit replicate. Fruit were incubated at 25C in cartonslined with moist laboratory wipes and covered withplastic bags. Lesion development was monitored at five,seven and 11 days after inoculation. Isolations weremade from fruit to confirm that lesions were due toinfection by C. gloeosporioides. Sequence analysis. DNA extractions were conductedout on all isolates using the procedure described by Rae-der and Broda (1985). Representative isolates wereselected (Table 2) and compared by means of ribosomalDNA internal transcribed spacer sequence analysisusing primers ITS1 and ITS4. These primers describedby White et al. (1990) were used to amplify the inter-nally transcribed spacer region. Primers were synthe-sised by MWG Biotech (Germany). PCR reactions wereperformed in 50 l volumes, each reaction containing0.5 l template DNA, 0.6 M of each primer, 5 lrecommended 10 buffer (supplied with Taq poly-merase), 2 mM MgCl2, 100 M each of dCTP, dGTP,dATP and dTTP (Promega) and 0.5 U Taq polymerase(Promega). Twenty-five PCR cycles were p