On-line version ISSN 1678-992X
Sci. agric. vol.57 n.1 Piracicaba Jan./Mar. 2000
Parasitism of Rhizoctonia solani by strains of Trichoderma spp.
Itamar Soares de Melo1*; Jane L. Faull2
1Embrapa Meio Ambiente, C.P. 69 - CEP: 13820-000 - Jaguariúna, SP.
2Birkbeck College, University of London, Malet Street, London, WC1E 7HX, UK.
*Corresponding author <firstname.lastname@example.org>
ABSTRACT: Rhizoctonia solani causes serious diseases in a wide range of plant species. The fungus Trichoderma has been shown to be particularly effective in the control of the pathogen. Thus, this research was carried out to screen fourteen Trichoderma strains against R. solani in vitro. All strains tested inhibited the growth of R. solani. Three T. koningii strains produced toxic metabolites with strong activity against R. solani, inhibiting the mycelial growth by 79%. T. harzianum, Th-9 reduced the viability of sclerotia of R. solani by 81.8% and T. koningii, TK-5 reduced by 53%. Electron microscopic observations revealed that all T. harzianum strains interacted with R. solani. Th-9 grew toward and coiled around the host cells, penetrating and destroying the hyphae. Penetration of host cells was apparently accomplished by mechanical activity.
Key words: Rhizoctonia, Trichoderma, biological control
Parasitismo de Rhizoctonia solani por linhagens de Trichoderma spp.
RESUMO: Rhizoctonia solani é um dos mais destrutivos patógenos de plantas cultivadas. Métodos alternativos de controle têm sido empregados com sucesso, particularmente, utilizando-se o fungo Trichoderma. Este trabalho visou, portanto, selecionar linhagens efetivas desse micoparasita contra o patógeno. Onze linhagens de T. harzianum e três de T. koningii foram testadas in vitro com relação ao parasitismo de hifas e de escleródios e produção de metabólitos tóxicos. Todas as linhagens de Trichoderma spp. inibiram o crescimento miceliano de R. solani e as três linhagens de T. koningii produziram potentes antibióticos, que inibiram mais de 79% o crescimento do patógeno. Uma linhagem de T. harzianum, Th-9, reduziu a viabilidade dos escleródios em 81,8% e uma de T. koningii em 53%. Microscopia eletrônica de varredura revelou que todas as linhagens de T. harzianum parasitaram R. solani enquanto nenhuma linhagem de T. koningii interagiu com R. solani, possivelmente, devido à forte inibição causada pelos metabólitos que impediu o contato entre os dois fungos. T. harzianum, Th-9, cresceu ao redor, penetrou e destruiu as hifas de R. solani. A penetração das células hospedeiras parece ser acompanhada por atividade mecânica.
Palavras-chave: Rhizoctonia, Trichoderma, controle biológico
Rhizoctonia solani Kühn is the major fungus responsible for damping-off and root rot diseases. With most vegetables, no effective fungicides are available against Rhizoctonia diseases, although chlorothalonil, thyophanate methyl, iprodione and some other chemicals are sometimes recomended (Agrios, 1988). Moreover, the use of chemicals for pest control is a growing concern to environmentalists. In this context, the major task would be to develop a biocontrol program. The literature on biological control of soilborne pathogens with fungal mycoparasites is huge and several fungi have been reported to be good antagonists of R. solani (Roy, 1989; Velvis & Jager, 1983; Poromarto et al., 1998; Benyagorub et al., 1994).
The most prominent are the fungi Trichoderma spp. (Papavizas, 1985; Harman et al., 1980; Melo, 1996). Various mechanisms have been proposed to explain biocontrol of R. solani, namely production of antibiotics (Dennis & Webster, 1971; Claydon et al., 1987; Faull et al., 1994) and hydrolytic enzymes (Cruz et al., 1992; Lorito et al., 1993; Sivan & Chet, 1989) and mycoparasitism and hyphal disruption (Elad et al., 1983; Elad et al., 1987).
Trichoderma spp. may affect the viability of sclerotia of R. solani (Naiki, 1986; Velvis & Jager, 1983).
Biocontrol mechanisms are likely to be specific for particular antagonists and plant pathogens and several mechanisms could operate independently or synergistically in any microbial interaction.
The objective of this research was to evaluate the use of Trichoderma spp. in the biocontrol of R. solani in vitro. This approach involved the introduction of selected Trichoderma strains, isolated from Sclerotinia sclerotiorum sclerotia.
MATERIAL AND METHODS
Fourteen Trichoderma strains were isolated from Sclerotinia slcerotiorum sclerotia by selective isolation employing the sclerotial bait technique (Melo et al., 1996). Identification of Trichoderma to species level was performed by light microscopy and the comparison to type species, was confirmed by the International Mycological Institute, Egham , UK.
One isolate of Rhizoctonia solani, isolated from hypocotyl of a diseased tomato plant was tested as host to Trichoderma.
R. solani and Trichoderma spp. were maintained and subcultured routinely at 26°C on potato dextrose agar (PDA). Sclerotia of R. solani were produced on PDA at 24°C after 4 weeks.
Dual culture testes
Plates of PDA were inoculated with a 5mm disc of R. solani (R.s.) 10mm from the edge of the plate. A 5mm disc of the tested Trichoderma isolate was placed 60mm from the R. solani disc 24 hours after the original inoculation. Paired cultures were incubated at 26°C in the light for 3 days. The growth of the fungi was recorded and interaction between mycelia scored for degree of antagonism using a scale of 1-5 (Bell et al., 1982), where 1=Trichoderma overgrowing R. solani and 5=R. solani overgrowing Trichoderma.
Survival of R. solani mycelium when paired with Trichoderma
Survival of R. solani mycelium when paired with T. harzianum (Th-9) and T. koningii (Tk-5) conidia was determined using the technique of Sivan & Chet (1989). Triplicate mycelial discs (5mm) were removed from 7 day old cultures of R. solani. The discs were soaked in a conidial suspension of Trichoderma strains (108 conidia ml-1 from 8 day old culture) and then placed in Petri dishes on moistened whatman 1 filter paper for 12 hours. Discs were then removed to PDA supplemented with benomyl (1%) to suppress the growth of Trichoderma. Control discs were washed with sterile distilled water. Survival was expressed as the percentage of the mycelial discs from which R. solani grew.
Extraction of metabolites with antibiotic activity
Trichoderma strains were grown in Erlenmeyer flasks (1L) containing 250ml PD broth (PDB). Inoculum consisted of 1ml of spore suspension (1 x 107 conidia ml-1). The cultures were incubated at 28°C on an orbital shaker at 140 rpm in the dark for 7 days. Culture filtrates were extracted twice by shaking in a separatory funnel with two third of its volume of ethyl acetate (EtOAc). The EtOAc phases were then pooled and concentrated to give a final volume equivalent to a 250-fold concentration of the original volume of the filtrate. Antimicrobial activity was tested in vitro against R. solani and inhibition of growth was calculated as the percentage of reduction in diameter of colonies compared with control colonies of the same age that were treated with EtAOc.
Effect of T. harzianum (Th-9) and T. koningii (Tk-5) on the viability of sclerotia
Sclerotia of R. solani were placed on the surface of PDA which was overgrown with mycelium and spores of a 4 days-old colony of T. harzianum strain, Th-9 and T. koningii strain, Tk-5. The cultures were incubated at 26°C in the darkness for up to 30 days. Untreated sclerotia served as the control. The viability of R. solani sclerotia were estimated by placing them on water-agar for 24 hours at 26°C and the germination was detected with a stereo microscope.
Preparation of specimens for scanning electron microscopy
The parasitism of hyphal cells of R. solani by T. harzianum was studied in detail by scanning electron microscopy (SEM). To obtain interaction sites of hyphae, 10% PDA was inoculated at a constant distance from the edge of the Petri dish with a mycelial disc (5mm) cut from the leading edge of a colony of Trichoderma and the pathogen. R. solani was inoculated 24 hours before Trichoderma. The mycoparasite and its host grew toward each other and their hyphae intermingled. After 48 hours of incubation, the plate cultures were observed under a light microscope to verify the early stage of interaction. The interaction sites were marked and an agar block of 1cm2was removed for SEM preparation. Mycelial samples from the interaction region were fixed for 24 hours with vapors of glutaraldehyde and osmium tetroxide (3:1), air-dried for 48 hours and sputter coated with gold.
RESULTS AND DISCUSSION
Assessment of the antagonism of Trichoderma strains against R. solani
All Trichoderma strains tested inhibited the growth of R. solani (TABLE 1). The strain Th-9 showed a high degree of antagonism overgrowing R. solani and reducing the growth by over 60% relative to that of the control and also infecting the pathogen (Figure 1). This strain has been shown to produce high levels of cellulase (Melo et al., 1997), an enzyme that hydrolyzes the cell walls of other fungi (Ridout et al., 1988; Elad et al., 1982; Sivan & Chet, 1989; Köhl & Schösser, 1991).
When R. solani mycelium was treated with conidia of T. harzianum (Th-9) and T. koningii (TK-5), these strains caused total inhibition of mycelial growth. Light microscope observation of R. solani mycelium on the inoculum discs showed it to be vacuolated and disrupted.
Effects of antibiotic of Trichoderma on the growth of R. solani
The percent of reduction of colony size of R. solani by antifungal activity of Trichoderma was observed in vitro. The T. koningii strains (TK-3, TK-5 and TK-12) showed strong inhibition of mycelial growth (Figure 2) whilst only two T. harzianum strains (Th-13 and Th-14) reduced about 10% of the colony size of R. solani (TABLE 2). Other T. harzianum strains tested did not produce any antibiotic activity.
Viability of R. solani sclerotia
The viability of sclerotia was decreased after 30d of incubation (TABLE 3). T. harzianum, Th-9 was able to reduce the germination of sclerotia in 72% and T. koningii in 43%. In fact, in this investigation T. harzianum, Th-9 has shown to be an efficient mycoparasite of R. solani. On the other hand, T. koningii has proved to be a good antibiotic producer. To be considered a successful biocontrol agent a mycoparasite should be effective against resistant survival structures of plant pathogens (Baker & Cook, 1974).
SEM observation on the mycoparasitic nature of Trichoderma
It was observed hyphae coiling of R. solani with all the T. harzianum strains studied. In this case only one of them (Th-9) was chosen for visualization via SEM. Strains TK-3,5 and 12 could not be studied as strong antibiosis zones persisted between them and R. solani. Examination of the interaction after 3 days of contact showed complete colonization of R. solani with all Trichoderma strains. The parasitic hyphae reached the host hyphae and grew on the surface always with coiling (Figure 3) and later they penetrated the cell wall directly without formation of appressorium-like structures (Figure 4). The R. solani invaded hyphae looked disintegrated. A collapse of the parasitized hyphae was common. Furthermore, total disappearance of the host hyphae after 7 days of interaction was observed. Lytic enzymes seem to be capable of degrading the cell walls of R. solani (Figure 5), as is the strain (Th-9), which produces high amounts of b-glucosidase (Melo et al., 1997). A variety of extracellular lytic enzymes may play an important role for the parasite. High chitinase and b-(1,3)-glucanase activities have been reported to be produced by T. harzianum (Sivan & Chet, 1989; Harman et al., 1981) and there may be a relationship between the production of these enzymes and the ability to control plant diseases (Ridout et al., 1988; Sivan & Chet, 1989; Elad et al., 1982).
In this paper, the fixation of the specimens with OsO4 vapor was not observed with the cellophane membrane to obtain interaction sites. These were observed directly in the culture medium.
Trichoderma harzianum and T. koningii, isolated from S. sclerotiorum sclerotia, were effective in inhibiting the mycelium growth of R. solani. Ultrastructural studies demonstrated that T. harzianum (Th-9) was capable of parasitizing and destroying R. solani mycelium. T. koningii strains produced large quantities of antibiotics, causing between 79-82% inhibition of R. solani growth. By the methods employed in this work little or no antibiotic was detected from T. harzianum strains.
AGRIOS, G.N. Plant pathology. New York: Academic Press, 1988. 803p.
BAKER, K.F.; COOK, R.J. Biological control of plant pathogens. San Francisco: Freeman, 1974. 433p.
BELL, D.K.; WELLS, H.D.; MARKHAN, C.R. In vitro antagonism of Trichoderma species against six fungal pathogens. Phytopathology, v.72, p.379-382, 1982.
BENYAGORUB, M.; JABAJI-TARE, H.; BANVILLE, G.J.; CHAREST, P.M. Stachybotrys elegans: a destructive mycoparasite of Rhizoctonia solani. Mycological Research, v.98, p.493-505, 1994.
CLAYDON, N.; ALLAN, M.; ITANSON, J.R.; AVENT, A. G. Antifungal alkyl pyrones of Trichoderma harzianum. Transactions of the British Mycological Society, v.88, p.503-513, 1987.
CRUZ, J.; HILDALGO-GALLEGO, A; LORA, J.M.; BENITEZ, T.; PINTOR-TORO, J.A.; LLOBEL, A. Isolation and characterization of three chitinases from Trichoderma harzianum. European Journal of Biochemical, v.206, p.859-867, 1992.
DENNIS, C.; WEBSTER, J. Antagonistic properties of species-groups of Trichoderma: II. Production of volatile antibiotics. Transactions of the British Mycological Society, v.57, p.41-48, 1971.
ELAD, Y.; CHET, I.; HENIS, Y. Degradation of plant pathogenic fungi by Trichoderma harzianum. Canadian Journal of Microbiology, v.28, p.719-725, 1982.
ELAD, Y.; BARAK, R.; HENIS, Y. Ultrastructural studies of the interaction between Trichoderma spp. and plant pathogenic fungi. Phytopathologische Zeitschrift, v.107, p.168-175, 1983.
ELAD, Y.; SADWOSKY, Z.; CHET, I. Scanning electron microscopical observations of early stages of interaction of Trichoderma harzianum and Rhizoctonia solani. Transactions of the British Mycological Society, v.88, p.259-263, 1987.
FAULL, J.L.; GRAEME-COOK, K.A.; PILKINGTON, B.L. Production of an isonitrille antibiotic by an UV-induced mutant of Trichoderma harzianum. Phytochemistry, v.36, p.1273-1276, 1994.
HARMAN, G.E.; CHET, I.; BAKER, R. Trichoderma hamatum effects on seed and seedling disease induced in radish and pea by Pythium spp. or Rhizoctonua solani. Phytopathology, v.70, p.1167-1172, 1980.
HARMAN, G.E.; CHET, I.; BAKER, R. Factors affecting Trichoderma hamatum applied to seeds as a biocontrol agent. Phytopathology, v.71, p.569-572, 1981.
KÖHL, J.; SCHÖSSER, E. Antagonism against Rhizoctonia solani and cellulolytic activity of strains of Trichoderma. In: BEEMSTER A. B.R.; BOLLEN G. J.; GERLAGH, M.; RUISSEN, M.A.; SCHIPPERS, B.; TEMPEL, A. Biotic interactions and soil-borne diseases. Amsterdam: Elsevier, 1991. p.160-164
LORITO, M.; HAYES, C.K.; DI PIETRO, A.; WOO, S.L.; HARMAN, G.E. Purification, characterization and synergistic activity of a glucan-b-1,3-glucosidase and a N-acetyl-B-glucosaminidase from Trichoderma harzianum. Phytopathology, v.84, p.398-405, 1993.
MELO, I.S. Trichoderma e Gliocladium como bioprotetores de plantas. In: LUZ, W.C. (Ed.) Revisão anual de patologia de plantas. Porto Alegre, 1996. v.4, p.261-296.
MELO, I.S.; FAULL, J.L.; GRAEME-COOK, K.A. Trichoderma koningii and Trichoderma harzianum as destructive mycoparasites of Sclerotinia sclerotiorum. In: INTERNATIONAL CONGRESS OF MYCOLOGY DIVISION, 8., Jerusalém, 1996. Proceedings. Jerusálem, 1996. p.183.
MELO, I.S.; FAULL, J.L.; GRAEME-COOK, K.A. Relationship between in vitro cellulase production of uv-induced mutants of Trichoderma harzianum and their bean rhizosphere competence. Mycological Research, v.101, n.11, p.1389-1392, 1997.
NAIKI, T. Differences in susceptibility of sclerotia of Rhizoctonia solani to Trichoderma sp. Soil Biology and Biochemistry, v.18, p.551-553, 1986.
PAPAVIZAS, G.C. Trichoderma and Gliocladium: biology, ecology and the potential for biocontrol. Annual Review of Phytopathology, v.23, p.23-54, 1985.
POROMARTO, S.H.; NELSON, B.D.; FREEMAN, T.P. Association of binucleate Rhizoctonia with soybean and mechanism of biocontrol of Rhizoctonia solani. Phytopathology, v.88, p.1056-1067, 1998.
RIDOUT, C.J.; COLEY-SMITH, J.R.; LYNCH, J.M. Fractionation of extracellular enzymes from a mycoparasitic strain of Trichoderma harzianum. Enzyme Microbiology and Technology, v.10, p.180-187, 1988.
ROY, A.K. Biological control of Rhizoctonia solani. In: AGNIHORTI, V.P.; SINGH, N.; CAIDE, H.S; SINGH, U.S.; DWNEDI, T.S. (Ed.) Perspectives in plant pathology. New Delhi: Today and Tomorrows Printers and Publishers, 1989. p.391-407.
SIVAN, A.; CHET, I. Degradation of fungal cell walls by lytic enzymes of Trichoderma harzianum. Journal of General Microbiology, v.135, p.675-682, 1989.
VELVIS, H.; JAGER, G. Biological control of Rhizoctonia solani on potatoes by antagonists: 1. Preliminary experiments with Verticillium biguttatum, a sclerotium inhibiting fungus. Netherlands Journal of Plant Pathology, v.89, p.113-123, 1983.
Received June 16, 1999