Integration of soil application and seed treatment formulations of Trichoderma species for management of wet root rot of mungbean caused by Rhizoctonia solani

BACKGROUND: The efficacy of seed dressing and soil application formulations from the isolates of Trichoderma viride (IARI P1; MTCC 5369), T. virens (IARI P3; MTCC 5370) and T. harzianum (IARI P4; MTCC 5371) were evaluated individually and in combination in pot and field experiments during the rainy seasons of 2005, 2006 and 2007 for the management of wet root rot (Rhizoctonia solani) and improvement in the yield of mungbean. RESULTS: A seed dressing formulation, Pusa 5SD, and soil application formulations, Pusa Biogranule 6 (PBG 6) and Pusa Biopellet 16G (PBP 16G), based on Trichoderma virens, were found to be superior to other formulations in reducing disease incidence and increasing seed germination and shoot and root lengths in mungbean. In field experiments, a combination of soil application with PBP 16G (T. virens) and seed treatment with Pusa 5SD (T. virens) + carboxin was superior to any of these formulations individually in increasing seed germination, shoot and root lengths and grain yield and reducing wet root rot incidence in mungbean. Seed treatment was more effective than soil application for all the evaluated parameters. The combined application of Pusa 5SD and carboxin was also superior to individual treatment. CONCLUSION: The efficacy of the evaluated formulations against wet root rot of mungbean proved that the integration of soil application of PBP 16G and seed treatment with Pusa 5SD + carboxin is highly effective for the management of wet root rot, increasing plant growth and grain yield of mungbean Copyright © 2011 Society of Chemical Industry     

木霉分生孢子和厚垣孢子对黄瓜叶片抗氧化系统及枯萎病防效的影响

采用前期筛选出的对黄瓜枯萎病菌有较好拮抗作用的3株木霉菌,即哈茨木霉菌(Trichoderma harzianum)809、拟康氏木霉菌(Trichoderma pseudokoningii)886和棘孢木霉菌(Trichoderma asperellum)525,利用盆栽试验,测定了木霉菌分生孢子和厚垣孢子对黄瓜幼苗...     

Selective accumulation of Trichoderma species in soils suppressive to radish damping-off disease after repeated inoculations with Rhizoctonia solani, binucleate Rhizoctonia and Sclerotium rolfsii

Artificial suppression of radish damping-off disease was induced by repeated soil inoculations with Rhizoctonia solani, binucleate Rhizoctonia (BNR) and Sclerotium rolfsii in pot systems. Soils repeatedly inoculated with R. solani and BNR showed suppressive to disease caused by R. solani and S. rolfsii, while soils repeatedly inoculated with S. rolfsii were suppressive to disease caused by S. rolfsii but not by R. solani. Species of Trichoderma were consistently isolated from soils repeatedly inoculated with R. solani, BNR and S. rolfsii. These Trichoderma spp. accumulated selectively in relation to the fungal species that was repeatedly added to the soils. The ratios of the frequencies of T. viride, T. harzianum and T. hamatum were 5:2:2 and 8:5:2 in soils repeatedly inoculated with R. solani and BNR, respectively. In S. rolfsii- inoculated soils, T. koningii was predominantly isolated. T. viride, T. harzianum and T. hamatum isolates obtained from either R. solani or BNR after repeated additions to the soils suppressed radish damping-off disease caused after challenge inoculations with R. solani or S. rolfsii. Among the Trichoderma species, T. viride consistently yielded high levels of suppression. However, isolates of T. koningii obtained from S. rolfsii-infested soils suppressed disease caused by S. rolfsii but failed to suppress disease caused by R. solani. Generally, the species of Trichoderma accumulated in a selective pattern that was closely related to the species of fungal pathogen used to induce the suppressive soil.     

Survival in the phylloplane of an introduced biocontrol agent (Trichoderma harzianum) and populations of the plant pathogenBotrytis cinerea as modified by abiotic conditions

Leaf populations ofTrichoderma were studied on tomato, pepper and geranium plants incubated under various conditions. Treatments involved high (>90%) or lower (75–85%) relative humidity (r.h.), temperatures of 15±3°C or 25±3°C, and soil fertilization with formulations of 2,2,5%, 3,3,8% or 5,3,8% NPK. The size of populations on leaves treated with the fungusTrichoderma harzianum differed according to plant species, leaf age, length of incubation, atmospheric conditions, and plant nutrition.T. harzianum populations were promoted in many cases by high r.h. and by 3,3,8% NPK. Interactions of introduced populations ofBotrytis cinerea with populations ofT. harzianum on tomato leaves under combinations of the above conditions showed that the population ofB. cinerea wasca tenfold lower in the presence ofT. harzianum than in the absence of this fungus.     

Bioefficacy of Trichoderma Species Against Javanese Root-Knot Nematode,Meloidogyne javanica,in Green Gram

Root-knot nematodes are mainly controlled by using synthetic nematicides, but their excessive use is prohibited due to associated health hazards which demand for suitable alternatives. The overreliance on nematicides can be curtailed by using biological control agents possessing nematicidal or nematostatic properties. Therefore, in the present study, effectiveness of seven indigenous species of Trichoderma were tested for their ability to suppress the population of Javanese root-knot nematode, Meloidogyne javanica, and improve growth variables of green gram. All the Trichoderma species resulted in an increase in shoot and root lengths and shoot weight while a decrease was observed in root weight. Maximum increase in shoot length (45.5%) was found in case of T. harzianum followed by T. hamatum and T. viride whereas the increase was the minimum where T. pseudokoningii and T. koningii were applied. Similarly, maximum increase in shoot weight was recorded with T. viride (56.1%) followed by T. harzianum (55%) and the minimum with T. pseudokoningii. As regards root length, it was the maximum in treatments with T. hamatum (46.2%) and T. harzianum (45.1%) and minimum with those where T. koningii and T. pseudokoningii were applied. Contrarily, maximum reduction in root weight was observed in treatments where T. harzianum (37.8%) and T. viride (35.8%) were applied while T. koningii and T. pseudokoningii resulted in minimum decrease. All the Trichoderma species significantly caused reductions in the number of galls and eggs and reproductive factor of the nematode over control. Maximum reduction in numbers of galls and eggs were observed with T. viride (49 and 53%) followed by T. harzianum (46 and 53%) while the minimum reduction was recorded with T. pseudokoningii followed by T. atroviride. Likewise, T. viride caused the maximum reduction in reproductive factor of M. javanica (81%) followed by T. harzianum (78%) and T. asperellum (75%). On the other hand, the minimum reductions in reproductive factor were observed with T. pseudokoningii and T. koningii.

     

Effect of label and sublabel rates of metam sodium in combination with<Emphasis Type="Italic">Trichoderma hamatum,T. harzianum,T. virens,T. viride</Emphasis> on survival and growth of<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

This work was undertaken to determine the effects ofTrichoderma spp. combined with label and sublabel rates of metam sodium on survival ofRhizoctonia solani in soil. Soils were infested with wheat bran preparations ofTrichoderma hamatum Tri-4,T. harzianum Th-58,T. virens Gl-3, andT. viride Ts-1-R3. Soil was also infested with sterile beet seeds that were colonized withR. solani. Beet seeds were later recovered, plated onto water agar plus antibiotics, and the growth ofR. solani was recorded. Preliminary experiments showed thatT. hamatum andT. virens reduced survival and saprophytic activity ofR. solani when the biocontrol fungi were incorporated into soil at 1.5% (w:w) or greater. Based on these data, biocontrol fungi in subsequent experiments were incorporated into soil at 2%. Metam sodium at label rate killed all biocontrol fungi andR. solani. At 1:2 and 1:5 dilutions, metam sodium reduced survival ofR. solani and allTrichoderma spp. When biocontrol fungi plus the label rate of metam sodium and 1:5, 1:10, 1:50 or 1:100 dilutions of the label rate were tested together, there were no interactions between any biocontrol agent and the fumigant with respect to colony diameter, reflecting that allTrichoderma isolates tested reacted similarly to increasing concentrations of metam sodium. At the label rate of metam sodium, allTrichoderma spp. significantly reduced colony diameter, but not growth rate, ofR. solani from beet seed. For the levels of metam sodium tested in combination withTrichoderma, it does not appear feasible to use a reduced rate of metam sodium to controlR. solani. However, the combination ofTrichoderma with metam sodium does reduce growth ofR. solani in comparison with that provided by metam sodium at the label rate. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Application of Trichoderma harzianum Strain KABOFT4 for Management of Tomato Bacterial Wilt Under Greenhouse Conditions

Clavibacter michiganensis subsp. michiganensis is a very important pathogen that causes bacterial wilt of tomato (BWT). Biological control of plant diseases is a critical tool for protecting the environment from chemical pollution. Twenty-five isolates of the genus Trichoderma were obtained from a healthy tomato root. Of the 25 isolates, KABOFT4 showed highly antagonistic activity that controlled the growth of C. michiganensis subsp. michiganensis (Cmm7) under in vitro conditions. The 5.8S ribosomal RNA gene and internal transcribed spacer identified the isolate as Trichoderma harzianum KABOFT4. The effect of this isolate as a soil drench and/or foliar application on bacterial wilt under greenhouse conditions was studied. The germination percentage of tomato seed treated with KABOFT4 increased by 36.7% compared to infected seed treated with only the pathogen Cmm7. Under greenhouse conditions, tomato seedlings treated with KABOFT4 as a soil drench, foliar and soil treatment, and foliar treatment had a 61.3, 26.7, and 40% reduced disease severity relative to the infected control, respectively. All treatments had a positive effect on tomato plants that presented as greater vegetative growth and accumulation of dry matter. The best fresh and dry weight was recorded when plants were treated with KABOFT4 as a soil and foliar application. Tomato plants treated with KABOFT4 also had increased total phenol and flavonoid contents in inoculated and non-inoculated plants compared to untreated plants. Under greenhouse conditions, T. harzianum strains can be used as an environmentally friendly way to manage the most economically important tomato disease. The results showed that a native endophytic strain of T. harzianum was a potent biocontrol agent against C. michiganensis subsp. michiganensis. Application of this strain to tomatoes in the greenhouse resulted in a decrease in disease severity and an increase in crop biomass.

     

Effect of coffee pulp on Trichoderma spp. in Kenyan tea soils

Abstract

Six organic amendments and one soil sterilant (Basamid) were screened at different rates to a given amount of soil, for their efficiency to enhance the population of Trichoderma spp. Coffee pulp significantly (P = 0.01) enhanced the population of Trichoderma spp. in the soil compared to other amendments. The enhancement was rate‐dependent with 16 and 22 g carbon giving the optimum population. The Trichoderma species enhanced included T. koningii, T. harzianum and T. longibrachiatum. The implications of these results in relation to biological control of soil‐borne plant pathogens are discussed.     

Effect of antagonistic<Emphasis Type="Italic">Fusarium</Emphasis> spp. and of different commercial biofungicide formulations on Fusarium wilt of lettuce

Five experimental trials were carried out to test different biological control agents against Fusarium wilt of lettuce, cause byFusarium oxysporum f.sp.lactucae. In the presence of a very high disease incidence, the best results in terms of disease control as well as increased growth response were shown byTrichoderma harzianum T 22 (RootShield), which, at 3 gl ⁻¹ of substrate, provided very consistent results.F. oxysporum IF 23 gave good disease control but in two out of five trials reduced the biomass produced. Less consistent, but still significant results were provided byF oxysporium MSA 25, at 3 gl ⁻¹ of substrate, and byTrichoderma viride TV 1. The twoF. oxysporum agents Fo 251/2 and Fo 47 and the mixture ofT. harzianum + T. viride (Remedier) partially reduced disease incidence but were less effective than the above mentioned. Less interesting results were offered byStreptomyces griseoviridis (Mycostop). The results obtained show that biological control can play a role in the management of Fusarium wilt of lettuce.     

Integrated management of Meloidogyne incognita‐Fusarium oxysporum f. sp. ciceri wilt disease complex in chickpea

A field experiment was conducted to study the management of a root‐knot nematode, Meloidogyne incognita (Kofoid and White) Chitw. (Tylenchida: Heteroderidae)‐wilt inducing fungi, Fusarium oxysporum Schlecht. emend. Snyd & Hans. f. sp. ciceri (Padwick) Snyd. & Hans. (Moniliales: Tuberculariaceae) wilt disease complex in chickpea (Cicer arietinum L.). cv. Annegiri. The results indicated that integration of soil solarization (for 6 weeks), VA mycorrhizal fungus (VAM), Glomus fasciculatum inoculation (12g/hill) and seed treatment with carbosulfan (3% w/w) was highly effective in reducing population levels of both pathogens, root‐knotdisease and wilt incidence and in increasing chickpea grain yield significantly. However, seed treatment with carbendazim (0.25% w/w) together with carbosulfan (3% w/w) was not only effective in reducing the wilt disease complex but also economic with an incremental cost: benefit ratio of 1: 2.4.     

Identification of<Emphasis Type="Italic">Trichoderma</Emphasis> biocontrol isolates to clades according to ap-PCR and ITS sequence analyses

A collection ofTrichoderma isolates, with different biocontrol capabilities, were identified by molecular methods. Arbitrarily-primed PCR (ap-PCR) using repeat motif primers was performed on DNA from aTrichoderma spp. collection of 76 isolates, and representative isolates were further characterized into three main clades by internal transcribed spacer (ITS) sequence analysis. Consequently, a reliable phylogenetic tree was constructed containing isolates belonging to theT. harzianum clade (comprisingT. aureoviride, T. inhamatum, andT. virens), theT. longibrachiatum andT. saturnisporum cluster, and that including the speciesT. asperellum, T. atroviride, T. koningii andT. viride. http://www.phytoparasitica.org posting July 14, 2004.     

木霉分生孢子和厚垣孢子对黄瓜幼苗生理特性及枯萎病防效的影响

采用前期筛选出的对黄瓜枯萎病菌有较好拮抗作用的3株木霉菌,即哈茨木霉Trichoderma harzianum 809、拟康氏木霉Trichoderma pseudokoningii 886和棘孢木霉Trichoderma asperellum 525,利用盆栽试验,测定了木霉菌孢子不同类型施用对黄瓜幼苗生长、生理特性...     

Changes induced by Trichoderma harzianum in suppressive compost controlling Fusarium wilt

The addition of species of Trichoderma to compost is a widespread technique used to control different plant diseases. The biological control activity of these species is mainly attributable to a combination of several mechanisms of action, which may affect the microbiota involved in the suppressiveness of compost. This study was therefore performed to determine the effect of inoculation of Trichoderma harzianum (T. harzianum) on compost, focusing on bacterial community structure (16S rRNA) and chitinase gene diversity. In addition, the ability of vineyard pruning waste compost, amended (GCTh) or not (GC) with T. harzianum, to suppress Fusarium wilt was evaluated. The addition of T. harzianum resulted in a high relative abundance of certain chitinolytic bacteria as well as in remarkable protection against Fusarium oxysporum comparable to that induced by compost GC. Moreover, variations in the abiotic characteristics of the media, such as pH, C, N and iron levels, were observed. Despite the lower diversity of chitinolytic bacteria found in GCTh, the high relative abundance of Streptomyces spp. may be involved in the suppressiveness of this growing media. The higher degree of compost suppressiveness achieved after the addition of T. harzianum may be due not only to its biocontrol ability, but also to changes promoted in both abiotic and biotic characteristics of the growing media.     

Trichoderma Biocontrol of Colletotrichum acutatum and Botrytis cinerea and Survival in Strawberry

Trichoderma isolates are known for their ability to control plant pathogens. It has been shown that various isolates of Trichoderma, including T. harzianum isolate T-39 from the commercial biological control product TRICHODEX, were effective in controlling anthracnose (Colletotrichum acutatum) and grey mould (Botrytis cinerea) in strawberry, under controlled and greenhouse conditions. Three selected Trichoderma strains, namely T-39, T-161 and T-166, were evaluated in large-scale experiments using different timing application and dosage rates for reduction of strawberry anthracnose and grey mould. All possible combinations of single, double or triple mixtures of Trichoderma strains, applied at 0.4% and 0.8% concentrations, and at 7 or 10 day intervals, resulted in reduction of anthracnose severity; the higher concentration (0.8%) was superior in control whether used with single isolates or as a result of combined application of two isolates, each at 0.4%. Only a few treatments resulted in significant control of grey mould. Isolates T-39 applied at 0.4% at 2 day intervals, T-166 at 0.4%, or T-161 combined with T-39 at 0.4% were as effective as the chemical fungicide fenhexamide. The survival dynamics of populations of the Trichoderma isolates (T-39, T-105, T-161 and T-166) applied separately was determined by dilution plating and isolates in the mixtures calculated according to the polymerase chain reaction (PCR) using repeat motif primers. The biocontrol isolates were identified to the respective species T. harzianum (T-39), T. hamatum (T-105), T. atroviride (T-161) and T. longibrachiatum (T-166), according to internal transcribed spacer sequence analysis.     

Integrated control of soilborne and bulbborne pathogens in Iris

Coating iris bulbs with a preparation ofTrichoderma harzianum was highly effective under greenhouse conditions in reducing incidence of diseases caused byRhizoctonia solani andSclerotium rolfsii. In field experiments with irises for bulb production, the incidence ofR. solani in plants and bulbs was effectively reduced (up to 93%), and the yield increased (by 35–41%), by applyingT. harzianum either as a bulb coating or broadcast application (biological treatment), treating soil with pentachloronitrobenzene (PCNB; quintozene) (chemical treatment), or solarizing the soil by mulching it with transparent polyethylene sheets (physical treatment) prior to planting. Combined treatments,i.e., chemical-biological or physicalbiological, were the most effective.T. harzianum bulb treatment and broadcast application in field plots increasedTrichoderma population density in the soil by 4–27 times.     

Potential of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. and <Emphasis Type="Italic">Talaromyces flavus</Emphasis> for biological control of potato stem rot caused by <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Sixteen isolates belonging to 11 species of Trichoderma (T. asperellum, T. ceramicum, T. andinensis, T. orientalis, T. atroviride, T. viridescens, T. brevicompactum, T. harzianum, T. virens, T. koningii and T. koningiopsis) were evaluated for biological control of potato (Solanum tuberosum) stem rot caused by Sclerotinia sclerotiorum. In dual culture tests, all antagonists significantly reduced sclerotia formation, and were able to inhibit radial growth of the pathogen. Growth inhibition by production of volatile and non-volatile inhibitors was also measured in in vitro tests. In screening the most efficient species of Trichoderma, establishment of mycelium on sclerotia and sclerotia lysis were also considered as important biocontrol qualities. Excluding T. asperellum, T. brevicompactum, T. andinensis and T. harzianum, all tested Trichoderma species were able to lyse sclerotia. The sclerotia-destroying species of Trichoderma and one isolate of Talaromyces flavus were tested in greenhouse tests and during 2 years of field experimentation during the 2007 and 2008 cropping seasons. After one aerial application of spore suspension in greenhouse trials, T. koningii, T. virens, T. ceramicum and T. viridescens were the most effective bio-agents and reduced significantly disease severity, and the least biocontrol efficacy was observed in T. flavus. Under field conditions and after five soil and foliar applications of spore suspension, all tested antagonists reduced significantly disease incidence. T. viridescens followed by T. ceramicum showed the best results. T. flavus and T. orientalis were less effective than other tested antagonists in both field trials.     

黄淮海夏玉米主产区穗腐病病原菌的分离鉴定

为明确我国黄淮海夏玉米主产区玉米穗腐病的病原菌种类、优势种群及虫害、年度、省份对病原菌的影响,以形态学为基础,结合分子生物学方法对2013、2015年随机采自河南、河北、山东3省的155份玉米穗腐病样品进行分离鉴定。结果表明,引起黄淮海夏玉米主产区玉米穗腐病的主要致病菌为镰孢菌Fusarium spp.,包括拟轮枝镰孢F.verticillioides、禾谷镰孢F.graminearum、层出镰孢F.proliferatum、木贼镰孢F.equiseti及藤仓镰孢F.fujikuroi,分离频率分别为49.7%、28.4%、12.3%、3.9%和1.3%;其次为木霉菌Trichoderma spp.,包括哈茨木霉T.harzianum、绿色木霉T.viride和棘孢木霉T.asperellum,分离频率分别为8.4%、3.2%和5.2%;青霉菌Penicillium spp.分离频率较低,为14.2%;曲霉菌Aspergillus spp.包括黑曲霉A.niger和黄曲霉A.flavus,分离频率分别为2.6%和1.9%。研究表明,黄淮海主产区玉米穗腐病优势病原菌为拟轮枝镰孢、禾谷镰孢和木霉菌,不同省份不同年度间病原菌种类及优势病原菌存在差异,虫害能加重玉米穗腐病的发生。     

Parasitism of <Emphasis Type="Italic">Trichoderma</Emphasis> on <Emphasis Type="Italic">Meloidogyne javanica</Emphasis> and role of the gelatinous matrix

Trichoderma (T. asperellum-203, 44 and GH11; T. atroviride-IMI 206040 and T. harzianum-248) parasitism on Meloidogyne javanica life stages was examined in vitro. Conidium attachment and parasitism differed beween the fungi. Egg masses, their derived eggs and second-stage juveniles (J2) were parasitized by Trichoderma asperellum-203, 44, and T. atroviride following conidium attachment. Trichoderma asperellum-GH11 attached to the nematodes but exhibited reduced penetration, whereas growth of T. harzianum-248 attached to egg masses was inhibited. Only a few conidia of the different fungi were attached to eggs and J2s without gelatinous matrix; the eggs were penetrated and parasitized by few hyphae, while J2s were rarely parasitized by the fungi. The gelatinous matrix specifically induced J2 immobilization by T. asperellum-203, 44 and T. atroviride metabolites that immobilized the J2s. A constitutive-GFP-expressing T. asperellum-203 construct was used to visualize fungal penetration of the nematodes. Scanning electron microscopy revealed the formation of coiling and appressorium-like structures upon attachment and parasitism by T. asperellum-203 and T. atroviride. Gelatinous matrix agglutinated T. asperellum-203 and T. atroviride conidia, a process that was Ca²⁺-dependent. Conidium agglutination was inhibited by carbohydrates, including fucose, as was conidium attachment to the nematodes. All but T. harzianum could grow on the gelatinous matrix, which enhanced conidium germination. A biomimetic system based on gelatinous-matrix-coated nylon fibers demonstrated the role of the matrix in parasitism: T. asperellum-203 and T. atroviride conidia attached specifically to the gelatinous-matrix-coated fibers and parasitic growth patterns, such as coiling, branching and appressoria-like structures, were induced in both fungi, similarly to those observed during nematode parasitism. All Trichoderma isolates exhibited nematode biocontrol activity in pot experiments with tomato plants. Parasitic interactions were demonstrated in planta: females and egg masses dissected from tomato roots grown in T. asperellum-203-treated soil were examined and found to be parasitized by the fungus. This study demonstrates biocontrol activities of Trichoderma isolates and their parasitic capabilities on M. javanica, elucidating the importance of the gelatinous matrix in the fungal parasitism.     

Growth of Coniothyrium minitans,Gliocladium roseum,Trichoderma harzianum and T. viride from alginate pellets and interaction with water availability1

Conidia, chlamydospores and mycelia of Coniothyrium minitans, Gliocladium roseum, Trichoderma harzianum and T. viride were obtained from liquid or solid culture and formulated within alginate pellets. Quantitative assessment of these pellets over a 12-week period showed a decrease in the number of colony-forming units (cfu) from about 10⁷ to 10³-10⁴ g^-1 air-dried pellets. Qualitatively, growth was assessed by direct plating the alginate pellets on rich (potato dextrose agar, PDA) and poor (water agar, WA) substrates. Rate of growth (mm day^-1) from individual pellets was decreased by water potential in the range -0.25 to-2.8 MPa, with growth significantly less on WA than PDA for all species except G. roseum. All species grew from 95 to 100% of direct-plated pellets on PDA over the water-potential range tested. However, on WA, interaction between water stress (-2.8 MPa) and lack of nutrients resulted in a reduction, with less than 30% of pellets of some formulations showing growth. In contrast to the quantitative results, there was little change in the growth rate of test species from pellets over a 12-week period, particularly on PDA. Growth from pellets could not be conclusively demonstrated in unsterile soil. The shelf-life and viability of alginate pellets in soil is assessed.     

Diversity of soil-dwelling Trichoderma in Colombia and their potential as biocontrol agents against the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary

Twenty-one isolates of Trichoderma spp. were collected from eight states in Colombia and characterized based on the 5′ end of the translation elongation factor-1α (EF1-α1) gene and RNA polymerase II gene encoding the second largest protein subunit (RPB2) by using mixed primers. Seven species of soil-dwelling Trichoderma were found: T. atroviride, T. koningiopsis, T. asperellum, T. spirale, T. harzianum, T. brevicompactum and T. longibrachiatum. Species identifications based on the EF1-α1 gene were consistent with those obtained from the RPB2 gene. Phylogenetic analyses with high bootstrap values supported the validity of the identification of all isolates. These results suggest that using the combination of the genes EF1-α1 and RPB2 is highly reliable for molecular characterization of Trichoderma species. Trichoderma asperellum Th034, T. atroviride Th002 and T. harzianum Th203 prevented germination of more than 70 % of sclerotia of Sclerotinia sclerotiorum in bioassay tests and are promising biological control agents. No relationship between mycelium growth rate and parasitism level was found.