DHINGRA, O. D.

SANTOS, A. F. dos

English

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FITOPATOLOGIA BRASILEIRAv.14, n.3/4, OUT 1989.I 1\\II\l\II\l\111 \\11\\\111 \\111 \1\1\ 11\1\ 11\111\\11 \\1\1 11\11\\1\1 1\111\\1422 - 108. h-';;-YI~ IH Ul"'l\#1RL UA :suCIEDADE BRASfLEiFiA DE FITOPATOLOGIAI of, "'J 7, "i ', ' .ARTIGOSPAlHOGENICIlY OF GLIOCLADIUM ON lHE SCLEROTIA OFSCLEROTINIA SCLEROTIORUMAF. DOS SANTOSl & O.D. DIDNGRA2lCNPSD/EMBRAPA, Divisão de Fitopatologia do CEPEC Cx. Postal 7, 45600 - Itabuna-BA, and2CENTREINAR, Universidade Federal de Viçosa 36570 - Viçosa-MG - Brasil.(Accepted for publication on 27/07/89)ABSTRACfSANTOS, A.F. & DHINGRA, O.D. Pathogenicity of Gliocladium on the scJerotia of Sclerotinia sclerotiorum. Fitopatol. bras. (14):198-200.1989.Seventeen isolates of Gliocladium spp. obtained from21 vegetable field soils by baiting with the sclerotia of Sclero-tinia sclerotiorum were tested for pathogenicity and aggres-siveness 'in vitro' and in field soil against the latter. Twoisolates of G. penicilloides, one of G. roseum and two of anunidentified species killed over 80% ofthe sclerotia in 30 days.In the aggressiveness test, one isolate of G. penicilloides andone of the unidentified species killed 100% of the sclerotia in15 days when inoculated with inoculum suspension of 106 or108 conidia/ml. The same isolates killed 80 to 90% of thesclerotia in 60 days in field soil; thus showing their pontentialfor use in bio-control of the pathogen. Of ali the isolates, G.roseum was the least aggressive.RESUMOPatogenicidade de gliocladium spp no escleródio de sclerotinia sclerotiorum.Dezessete isolados de Gliocladium spp., obtidos de 21 G. penicilloides e um de espécie não identificada mataramtipos de solo de campo utilizando-se escleródios de Scleroti- 100% dos escleródios em 15 dias quando os escleródios foramnia sclerotiorum como isca, foram testados quanto à patogeni- inoculados com suspensão de inóculo de 106 ou 108 coní-cidade e agressividade, "in vitro" e no campo, a S. sclerotio- dios/rnl. Os mesmos isolados mataram 80 a 90% de escleró-rum. Dois isolados de G. penicilloides, um de G. roseum e dois di os em solos de campo, dentro de 60 dias, assim mostrandode espécie não identificada, mataram mais de 80% dos es- seu potencial para uso em bio-controle do patógeno. De todoscleródios em 30 dias. No teste de agressividade, um isolado de os isolados o menos agressivo foi G. roseum.INTRODUCTIONSclerotinia s clero tiorum (Lib.). De Bary, a fungal patho-gen ofvegetable crops, causes heavy losses in certain vegetableseed production farms in the state of Minas Gerais, Brasil. Inmany of these farms, inoculum density of 8 to 10 sclerotia/kgsoil and yield losses of 85 to 100%, have been observed. Thesoils in these fields are sandy, acidic (pH 4 to 5.5), low in or-ganic matter, and with poor water retention capacity. Furni-gation and application of soil fungicides did not control thedisease to satisfaction; therefore, a search for bio-controlagents was initiated. Considering the properties of these soils,we focused our attention on the fast growing fungal my-coparasites that could resist water deficit and grow well underacidic conditions. Besides Trichoderma spp., Gliocladiumspp. ais o satisfy these requirements and are reported to beparasites of many sclerotial and nonsclerotial fungal patho-gens. Gliocladium virens has been most studied as a possible198bio-control agentfor Rhizoctonia solani (Levis & Papavizas,1985), Pythium and Fusarium spp. (Teyers & Dirks, 1985)and S. sclerotiorum (Tu, 1980; Lee & Wu, 1984). The íollow-ing study was done to determine the potential of Gliocladiumspp., occurring in the soils of Minas Gerais, in reducing theinoculum density of S. sclerotiorum.MATERIALS AND METHODSIsolates of Gliocladium spp. were obtained from 21 soilscollected from vegetable fields in different agro-climaticregions, with or without the ocorrence ofthe disease caused byS. clerotiorum. Isolations were made using the following bait-ing technique. After adjusting the soil moisture to about 30%ofits moisture holdingcapacity, 25 sclerotia, freshly harvestedfrom corn-meal culture and without drying, were mixed with500 g soil and incubated in polyethylene bags at room tem-perature. Afterone month, thesclerotia wererecovered bywetFitopatol. bras. 14(3/4), Outubro/Dezembro 89sieving, washed under running tap water for 15 ot 20 minutes,surface-sterilized in 1.7% sodium hypoehlorite for 30 seeondsand in 70% ethanol for one minute, and then rinsed in steriledistilled water. Sclerotia were then plaeed on autoclavedmoistsand in clearplastieboxes (20x 20x3 em) and ineubatedat 25°C for 15 days. The fungi growing on the sclerotia wereisolated and multiplied on potato-dextrose agar (PDA).For initial pathogenieity tests, eonidial suspension ofisolates of Gliocladium spp. was prepared by washing theeonidia from five-day old PDA eultures with sterile distilledwater. Conidial eounts were adjusted to 106/m!. Freshly pre-pared non-dried sclerotia were inoeulated by immersion inthe eonidial suspension foroneminute. Control sclerotia wereimmersed in sterile distilled water. Sclerotia were then plaeedon autoclaved moist sand as deseribed earlier and ineubated at25°C. After 30 days, they were removed, washed in running tapwater, surfaee-sterilized and plated on 1.5% water agar, andineubated at 20°C for 15 days to eheek their viability.The isolates that killed over 80% of the sclerotia in theabove experiment were studied for aggressiveness 'in vitro'and in a field soi!. 'In vitro' test was done as deseribed earlier,exeept that the sclerotia were inoeulated with suspensions of104, 106 or 108 eonidia/ml and the ineubation period wasreduced to seven or 15 days. The fie\d test was done in avegetable seed produetion farm where the pathogen was wellestablished. Holes of 15 em diameter and 10 em deep were dug2 meters apart. The soil was infested with 10 sclerotia of S.sclerotiorum and with respeetive isolate of Gliocladium at theeoneentrationsof 104 ,106 or l O" eonidia/gofsoi!. Thesoilwas then plaeed in a nylon mesh (1 mm) eylindrieal bag andplaeed in the respeetive holes. After 20, 40 or 60 days the bagswith soil were removed. Sclerotia reeovered by wet sievingwere tested for viability and infeetion by Gliocladium sp.Ali studies had three replieations and were repeatedtwiee. The field experiment was repeated after one month at adifferent site of the same field.RESULTSOfthe 17 isolates of Gliocladium isolated from sclerotiaand tested for pathogenieity 'in vitro', two isolates of G. peni-cilloides, one of G. roseum and two of an unidentified speeieskilled 94 to 98% of the sclerotia. The other isolates either didnotaffect the germination or reduetion in viabilitywas too lowto demand further testing. The isolates that killed the sclerotiagrew profusely on them, eausing a dry rot.In the aggressiveness test, during the first seven days ofineubation, 57 to 68% and 67 to 92% of sclerotia were killedwhen inoeulated with 104 and 108 eonidia/ml, respeetively(Table 1). Isolates G-l and G-2 of Gliocladium eausedmaximum decline in viability. The death rate of sclerotia ino-eulated with suspension of 106/ml was similar to that of108/ml. However, after 15 days of ineubation 94 to 100%sclerotia were killed by ali isolates exeept G. roseum, with noTABLE 1 - Pereent germination of sclerotia of Sc/erotinia sclero-tiorum inoeulated by immersion in a eonidial suspen-sion ofGlioc/adium spp. and ineubated on sterile moistsand at 25"CI,2 •Ineubation InoeulumIsolate period Coneentration/ml Mean(days) 104 106 IOSG. penicilloides 7 43 3S 33 37(Gp-I) 15 5 5 2 4G. penicilloides 7 35 34 30 33(Gp-2) 15 O O O OG. roseum 7 38 16 13 2115 13 12 10 12Gliocladium sp. 7 26 23 13 21(G-I) 15 6 O O 2Gliocladium sp, 7 22 10 8 13(G-2) 15 5 5 2 4Control 7 89 89 89 8915 91 91 91 911. Average of two experiments and three replications. The numbers arerounded to the nearest whole number.2. Considering large differences among treatments and small differencesamong replications, statistical analysis was discarded.TABLE2 - Pereentage germination ofsclerotia ofSclerotina sclerotiorum reeovered fromfield soil infested with eonidia ofGliocladium to adepth of 10 em 1,2.Ineubation G. penicilloides Gliocladium sp.Conidia/g period Control G. roseum(days) Gp-l Gp-2 G-I G-220 89 67 63 67 60 63104 40 63 47 57 57 27 2060 63 43 43 33 27 10Mean 72 52 54 52 38 3120 89 57 67 66 53 57106 40 63 43 40 67 50 5360 63 33 30 47 40 20Mean 72 44 46 60 48 4320 89 57 57 57 53 63108 40 63 30 37 47 27 4360 63 17 40 27 17 10Mean 72 35 45 44 32 39Mean of isolate 72 44 48 52 39 38I. Average of three replications and two experiments. The numbers are rounded to the nearest whole number.2. Tukey for comparison of treatments with contraI: 14.Fitopatol. bras. 14(3/4), Outubro/Dezembro 89 199significant difference among concentration of inoculum andisolates (Table 1).In the field test viability of sclerotia in soil infested withGliocladium declined significantly and constantly with time.The effect of inoculum concentration was more pronounced at60-day incubation period than earlier. However, averagingtheeffect of isolates and incubation period, did not show signifi-cant difference among inoculum concentrations in reducingthe sclerotial viability. On the other hand, averaging the effectof isolates and inoculum concentrations, showed that thesclerotial death increased with time, with maximum declineoccuring between 40 and 60 days (Table 2).Tne rate of sclerotial death in the soil was more rapidwith isolates G-1 and G-2, even at lower inoculum concen-trations. On an average, these isolates were more aggressivethan Gp-1 and Gp-2. Gliocladium roseum was the leastaggressive. Respective Gliocladium spp. were recovered fromali non-germinating and majority of germinating sclerotia.DISCUSSIONThis study shows that some species and strains of Glio-cladium parasitize the sclerotia of S. sclerotiorum. This con-firms ar adds to the list of mycoparasites that show potentialfor controlling this and some other soil-borne pathogens(Santos & Dhingra, 1982; Huang, 1978, 1980; Huang& Hoes,1976; Tu, 1980; Uecker et aI., 1978). Gliocladium catenu-latum and G. uirens have been reported to be aggressive my-co parasites of S. sclerotiorum (Zazzerini & Tosi, 1985; Lee &Wu, 1984). This study reports, for the first time, G. penicil-loides as an aggresive pathogen of S. sclerotiorum. Glio-cladi um catenulatum and G. uirens were notfound in the soilssampled. From the small sample of isolates tested, it wasobvious that species ar strains of Gliocladi um nonpathogenicto sclerotia of S. sclerotiorum are more common than thepathogenic ones in the soils ofMinas Gerais. However, withinthe pathogenic strains, there is a great difference in the aggres-siveness. The isolates G-1 and G-2, that showed high aggres-siveness, appear to be promising for bio-control of S. sclero-tiorum.LITERATURE CITEDHUANG, H.C. Gliocladium catenulatum: hyperparasite ofSclerotinia sclerotiorum and Fusarium species. Cano J.Bot 56:2243-2246.1978.HUANG, H.C. Contrai of Sclerotinia wilt of sunflower by hy-perparasites. Cano J. Plant Pathol. 2: 26-32. 1980.HUANG, H. C. and HOES, J.A Penetration and infection ofSclerotinia sclerotiorum by Coniothyrium minitans.Cano J. Bot. 54: 406-410.1976.LEE, Y.A & WU, W.S. The antagonisms of Trichoderma spp.and Gliocladium virens against Sclerotinia sclero-tiorum. PI. Prot. BulI. 26: 293-304. 1984.LEW1S, J.A. & PAPAVIZAS, G.C. Effect of mycelial pre-paration of Trichoderma and Gliocladium on popu-lation of Rhizoctonia solani and the incidence of dam-ping off. Phytopathology, 75: 812-817. 1985.SANTOS, AF. dos and DHINGRA, O.D. Pathogenicity ofTrichoderma spp. on the sclerotia of Sclerotinia sclero-tiorum. Cano J. Bot. 60: 472-475. 1982.TEYERS, A.A & DIRKS, V.A Suppression of Fusarium andPythium pea root rot by antagonistic microorganisms.Phytoprotection. 66: 23-29. 1985.TU, J.c. Gliocladium virens, a destructive mycoparasite ofSclerotinia sclerotiorum. Phytopathology, 70: 670-674.1980.UECKER, F.A.; AYRES, W.A & ADAMS, P.B. A newhyphomycete on sclerotia of Sclerotinia sclerotiorum.Mycotaxon, 6: 275-282.1978.ZAZZERINI, A. & TOSI, L. Antagonistic activity of fungiisolated from sclerotia of Sclerotinia sclerotiorum. PlantPathology, 34: 415-421. 1985.EFEITO DE REGI9ES E ÉPOCAS DE PRODUÇÃO NAQUALIDADE SANITARIA DE S:t:MENTESDE FEI}OEIRO NOESTADO DE SAO PAULO~~SILVAMA H. FURLANl & }.a.M. MENTEN2-Seção de Doenças das Pls. Alim. Bás. e Olerícolas Inst. Biológico. C. Postal 70, 13001 - Campinas-SP.2Prof~ Assistente Dr. ESALQ - Depto. Fitopatologia Caixa Postal 9 - 13400 - Piracicaba-SP.(Aceito para publicação em 22/09/89)RESUMOFURLAN, S.H. & MENTEN, J .O.M. Efeito de regiões e épocas de produção na qualidade sanitária de sementes de feijoeiro no Estado de São Pau-lo. Fitopatol. bras. (14):200-205.1989.Com o objetivo de conhecer regiões e épocas de cultivomais adeauadas à produção de sementes de feijoeiro no Esta-ao de São Paulo, utilizou -se o parâmetro sanidade comoprincipal, seguido da germinação e envelhecimento aceleradocomo indicativos da qualidade das sementes. Sementes gené-ticas, básicas e certificadas produzidas nas épocas da seca de1984, das águas de 1984/85, e do inverno de 1985 provenien-tes de 46 municípios foram avaliadas. Os testes de sanidadeenvolveram o método de papel de filtro comum e o método depapel de filtro com congelamento. De maneira geral, a sanida-de das sementes foi boa, considerando-se a baixa freqüênciade fungos potencialmente importantes à cultura como Colle-totrichum lindemuthianum, havendo, no entanto, efeito de ••regiões dentro de cada época de produção. Houve, também, oefeito de regiões e épocas de produção na germinação e enve-lhecimento acelerado das sementes. As melhores áreas para• Parte da Dissertação apresentada pelo autor à E.S.A. "Luiz de Queiroz" para obtenção do título de Mestre em Fitopatologia.** Bolsista da FAPESP.200 Fitopatol. bras. 14(3/4), Outubro/Dezembro 89

Pathogenicity of Gliocladium on the Sclerotia of Sclerotinia sclerotiorum.

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Pathogenicity of Gliocladium on the Sclerotia of Sclerotinia sclerotiorum.

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