An Aspergillus terreus strain showed in vitro antagonistic activity against tha plant pathogens Sclerotinia sclerotiorum (Lib.) de Bary.The interacton between A. terreus and sclerotia revealed that the mycoparasite sporulated abundantly on the sclerotial surface. Cell breakdown due to host cell wall disruption was observed in inner rind cells, by a scanning electron microscopy

FAULL, J. L.

MELO, I. S. de

NASCIMENTO, R. dos S.

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Brazilian Journal of Microbiology (2006) 37:417-419ISSN 1517-8382417*Corresponding Author. Mailing address: Centro Nacional de Pesquisa de Monitoramento e Avaliação de Impacto Ambiental, Empresa Brasileira dePesquisa Agropecuária, Rodovia SP 340, Km 127.5. 13820-000, Jaguariúna, SP, Brasil. E-mail: itamar@cnpma.embrapa.brANTAGONISM OF ASPERGILLUS TERREUS TO SCLEROTINIA SCLEROTIORUMItamar S. Melo1*; Jane L. Faull2; Rosely S. Nascimento11Centro Nacional de Pesquisa de Monitoramento e Avaliação de Impacto Ambiental, Empresa Brasileira de PesquisaAgropecuária, Jaguariúna, SP, Brasil; 2School of Biological and Chemical Sciences, Birkbeck College, LondonSubmitted: May 17, 2004; Approved: July 18, 2006ABSTRACTAn Aspergillus terreus strain showed in vitro antagonistic activity against the plant pathogen Sclerotiniasclerotiorum (Lib.) de Bary. The interaction between A. terreus and sclerotia revealed that the mycoparasitesporulated abundantly on the sclerotial surface. Cell breakdown due to host cell wall disruption was observedin inner rind cells, by a scanning electron microscopy.Key words: biological control, mycoparasitism, morphologyINTRODUCTIONIn Brazil, sclerotinia wilt is a serious disease of manyimportant crops, including tomato, dry beans, soybeans,sunflower, letucce and many others, caused by infection ofroots by hyphae from myceliogenic germination of Sclerotiniasclerotiorum.Several microorganisms including bacteria and fungi havebeen reported to be good antagonists to Sclerotinia sclerotiorum.The most prominent and extensively studied antagonists areseveral species of Trichoderma (11,13), Coniothyrium minitans(15), Sporidesmium sclerotivorum (1) and Talaromyces flavus(10). The success of these antagonists is based, in general, ontheir ability to parasitize sclerotia, the primary source of inoculum.Because they prove to be effective agents of biolocical controlfor pathogenic fungi, mycoparasites have attracted widespreadattention in recent years with many commercial biofungicidesavailable worldwide (3,5,8,12).During a survey of soils for antagonists of S. sclerotiorum,many isolates belonging to the genus Aspergillus wereobtained besides others antagonistic organisms, such asTrichoderma and Penicillium. This mould produces substanceswhich are able to inhibit growth of different bacteria and ofTrichophyton mentagrophytes and Sporothrix schenckii (4).Antifungal activity of A. terreus was observed by Slagg andFellows (14), who reported complete inhibition of Ophiobolusgraminis, the causal organism of take-all of wheat. A similareffect on Fusarium udum, the cause of wilt of pigeon peas, wasnoted by Vasudeva and Roy (16). Inhibition of nematode-trapping fungi by A. terreus was reported by Mankau (9).Therefore, the present study was conducted to investigate,using scanning electron microscopy, the mode ofhyperparasitism of S. sclerotiorum by an isolate of  A. terreus,isolated from soil, using the sclerotial bait technique.S. sclerotiorum sclerotia were produced on oat-agar (50grolled oat, 18g agar, 1000 mL water esterilized at 121ºC for 30min.) by growing at 26ºC for three weeks in the dark. Soilsamples, obtained from rhizosphere of tomato, were air-driedand placed on the sterile petri dishes. Twenty 2-3 mm diametersclerotia were placed on the soil surface. The soil wasmoistened with 10 mL of sterile distilled water. After 3 weeksof incubation, sclerotia was recovered, washed in steriledistilled water, surface sterilized by immersion in 10% sodiumhypochlorite for 10 minutes, transferred to potato, dextrose,agar medium (PDA) + aureomycin (0.003%) and incubated at26ºC. Unsterilized sclerotia were also incubated as above. After48 hours, fungal colonies were purified and transferred to(PDA) slopes.Survival of S. sclerotiorum sclerotia when paired with conidiaof A. terreus was tested by immersing sclerotia in a conidial418Melo, I.S. et al.suspensions (108 conidia mL-1) from 14 - day-old Aspergilluscultures for 10 minutes. Sclerotia were blotted on sterile filterpaper, placed on to sterile sand in Petri dishes, and incubated at28ºC for 30 days. Sclerotia, washed in sterile distilled water weretreated similarly to act as controls. After this period, sclerotiawere recovered, washed, surface sterilized and placed on PDAcontaining aureomycin (0.003% w/v). Viability was determinedafter 7 days of incubation, when sclerotia were score forgermination, when the antagonist could be observed growingon non-viable sclerotia.For scanning electron microscopy SEM, parasitized sclerotiawere fixed in 2% glutaraldehyde. Following buffer – rinsing, thespecimens were post-fixed in 2% osmium tetroxide at 8ºC for 2h,rinsed in buffer, and dehydrated in an ethanol series. Followingethanol dehydration, the material was critical – point dried,mounted on SEM stubs, sputter–coated with gold and examinedon an ultra – high performance SEM (Leo 982-Zeiss and Leica)at Embrapa Environment.The sclerotial bait technique was successful in isolatingdifferent fungal strains. In dual culture, an Aspergillus isolateshowed a high degree of antagonism by overgrowing S.sclerotiorum and was readily distinguishable from its host byfine hyphae it produced in contrast to the coarse hyphae ofS. sclerotiorum. This antagonistic fungus was identified by itsmorphological characteristics as A. terreus (Fig. 1A). Conidiaare globose smooth and connected each other.A. terreus occurs in tropical and subtropical zones and hasa worldwide distribution on different soil. One very commonhabitat is the rhizosphere of plants (4). This species was foundby White et al., (17) to be the most strongly cellulolytic speciesof the genus. This fungus produces a large number of specificmetabolites, including the nephrotoxin citrinin, the neurotoxinscitroviridin, patulin, terrain, terreic acid and geodin. (4) andseveral other compounds.A. terreus (EQ) was found to affect sclerotial viability whensclerotia of the pathogen were treated with conidia of thehyperparasite. The fungus caused 100% mortality of sclerotia(Data not shown). This strain of A. terreus was able to colonizesclerotia of S. sclerotiorum and produce small hyphal branchesthat penetrated host cell wall without forming appressoria.Plasmolysis was evident in some of the host cells infectedby A. terreus. A close examination of these parasitized sclerotiarevealed that hyphae of the antagonist grew abundantly onthe sclerotial surface, forming dense forest of conidiophores(Fig. 1 E). At a more advanced stage of parasitism, A. terreuscompletely destroyed the sclerotial cells and penetrated andcollapsed the medulary tissue (Fig. 1 C, D), with internal growth,and subsequent sporulation outside the host. This mode ofparasitism is similar to previously described necrotrophicmycoparasites of S. sclerotiorum, such as Trichodermaharzianum (7,13), Talaromyces flavus (10) and Coniothyriumminitans (6).A. terreus has proved to be a destructive necrotrophic parasiteof S. sclerotiorum, where this agent destroys the host cell afteror slightly before invasion.For a mycoparasite to be considered a successful biocontroleagent, it should be effective against resistant survival structuresof plant pathogens (2). These results herein obtained showedthat A. terreus was able to exploit sclerotial tissue of S.sclerotiorum.Figure 1. Scanning electron micrograph of conida A. terreusand interactions with sclerotia of S. sclerotiorum. (A) Conidialheads long, compactly colummar; (B) Note the presence of aconidiophore and a mass of conidia on the surface of thesclerotia; (C) The cell wall has been disintegrated and hyphaeof the microparasite has penetrated and colonized the modu-lar tissue. The breakage of the host cell wall is disintegrated;(D) A conidium of A. terreus germination towards to medullartissue of a disintegrated sclerotia; (E) A forest of conidiophoreswith conidia of A. terreus on the surface of a S. sclerotiorumsclerotia.Antagonism of A. terreus to  S. sclerotiorum419RESUMOAntagonismo de Aspergillus terreus contraSclerotinia sclerotiorumUma linhagem de Aspergillus terreus mostrou forte atividadeparasítica contra Sclerotinia sclerotiorum. Interações entre opatógeno e o antagonista revelaram que A. terreus esporulouprofusamente sobre os escleródios. Quando visto em microscopiaeletrônica de varredura, o antagonista mostra-se rompendo elisando a parede celular e penetrando o interior do escleródio,onde se estabelece no tecido medular.Palavras-chaves: controle biológico, micoparasitismo,morfologiaREFERENCES1. Adams, P.B.; Fravel, D.R. Economical biological control ofSclerotinia lettuce drop by Sporidesmium sclerotivorum. Phytopathol.,80, 1120-1124, 1990.2. Baker, K.F.; Cook, R.J. Biological Control of plant Pathogens. SanFrancisco: Freeman, 1974, 433p.3. Cook, R.J.; Baker, K.F. The nature and practice of biological controlof plant pathogens. St. Paul. American Phytopathological Society,1983, p.539.4. Gravesen, S.; Frisvad, J.C.; Samson, R.A. Descriptions of somecommon fungi. In Microfungi. Munksgaard Copenhagen, 1994, p.41.5. Harman, G.E.; Taylor, A.G. Improved seedling performance byintegration of biological control agents at favorable pH levels withsolid matrix priming. Phytopathology, 78, 520-525. 1988.6. Huang, H.C. Control of sclerotinia wilt of sunflower by hyperparasites.Canadian J. Plant Pathol., 2, 26-32, 19807. Jones, D.; Watson, D. Parasitism and lysis by soil fungi of Sclerotiniasclerotiorum (Lib.) de Bary a phytopathogenic fungus. Nature, 224,287-288, 1969.8. Lewis, J.A.; Papavizas, G.C. Effect of mycelial preparations ofTrichoderma and Gliocladium on populations of Rhizoctonia solaniand the incidence of damping-off. Phytopathology, 75, 812-817.1985.9. Mankau, R. Antagonism to nematode trapping fungi in soil.Phytopathology, 51, 66. 1961.10. McLaren, D.L.; Huang, H.C.; Rimmer, S.R. Hyperparasitism ofSclerotinia sclerotiorum by Talaromyces flavus. Canadian J. PlantPathol., 8, 43-48, 1986.11. Melo, I.S. Trichoderma e Gliocladium como bioprotetores de plantas.Revisão Anual de Patologia de Plantas, 4, 415, 199612. Ricard, J.J.L. Method of using immunizing commensals. U.S. Patent4,678, 669. 1987.13. Santos, A.F.; Dhingra, D.D. Pathogenicity of Trichoderma spp. onthe sclerotia of Sclerotinia sclerotiorum. Canadian J. Bot., 60, 472-475, 1982.14. Slagg, C.M.; Fellows, H. Effects of certain soil fungi and their by-products on Ophiobolus graminis. J. Agr. Res., 75, 279-293, 2 figs.,1947.15. Trutman, P.; Keane, P.J.; Merriman, P.R. Reduction of sclerotialinoculum of Sclerotinia sclerotiorum with Coniothyrium minitans.Soil Biol. Biochem., 12, 461-465, 1980.16. Vasudeva, R.S.; Roy, T.C. Effects of associated soil microflora onFusarium udum Butl., the fungus causing wilt of pigeon-pea (Cajanuscajan (L.) Millisp.). Ann. Appl. Biol., 37, 169-178. 1950.17. White, W.L.; Darby, R.T.; Stechert, G.M.; Sandersen, K. Assays ofcellulolytic activity of molds isolated from fabrics and related itemsexposed in the tropics. Mycology, 40, 34-48, 1984.

Antagonism of Aspergillus terreus to Sclerotinia sclerotiorum.

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