Search

Background: The proteins Sm1 and Sm2 from the biocontrol fungus Trichoderma virens belong to the cerato-platanin protein family. Members of this family are small, secreted proteins that are abundantly produced by filamentous fungi with all types of life-styles. Some species of the fungal genus Trichoderma are considered as biocontrol fungi because they are mycoparasites and are also able to directly interact with plants, thereby stimulating plant defense responses. It was previously shown that the cerato-platanin protein Sm1 from T. virens - and to a lesser extent its homologue Epl1 from Trichoderma atroviride - induce plant defense responses. The plant protection potential of other members of the cerato-platanin protein family in Trichoderma, however, has not yet been investigated. Results: In order to analyze the function of the cerato-platanin protein Sm2, sm 1 and sm2 knockout strains were generated and characterized. The effect of the lack of Sm1 and Sm2 in T. virens on inducing systemic resistance in maize seedlings, challenged with the plant pathogen Cochliobolus heterostrophus, was tested. These plant experiments were also performed with T. atroviride epl1 and epl2 knockout strains. In our plant-pathogen system T. virens was a more effective plant protectant than T. atroviride and the results with both Trichoderma species showed concordantly that the level of plant protection was more strongly reduced in plants treated with the sm2/epl2 knockout strains than with sm1/epl1 knockout strains. Conclusions: Although the cerato-platanin genes sm1/epl1 are more abundantly expressed than sm2/epl2 during fungal growth, Sm2/Epl2 are, interestingly, more important than Sm1/Epl1 for the promotion of plant protection conferred by Trichoderma in the maize-C. heterostrophus pathosystem. [ABSTRACT FROM AUTHOR]

Screening and obtaining a novel high activity cellulase and its producing microbe strain is the most important and essential way to improve the utilization of crop straw. In this paper, we devoted our efforts to isolating a novel microbe strain which could produce high activity cellulase. A novel strain Trichoderma virens ZY-01 was isolated from a cropland where straw is rich and decomposed, by using the soil dilution plate method with cellulose and Congo red. The strain has been licensed with a patent numbered ZL 201210295819.6. The cellulase activity in the cultivation broth could reach up to 7.4 IU/mL at a non-optimized fermentation condition with the newly isolated T. virens ZY-01. The cellulase was separated and purified from the T. virens culture broth through (NH4)2SO4 fractional precipitation, anion-exchange chromatography and gel filtration chromatography. With the separation process, the CMC specific activity increased from 0.88 IU/mg to 31.5 IU/mg with 35.8 purification fold and 47.04% yield. Furthermore, the enzymatic properties of the cellulase were investigated. The optimum temperature and pH is 50 °C and pH 5.0 and it has good thermal stability. Zn2+, Ca2+ and Mn2+ could remarkably promote the enzyme activity. Conversely, Cu2+ and Co2+ could inhibit the enzymatic activity. This work provides a new highly efficient T. virens strain for cellulase production and shows good prospects in practical application.

Nine fungal isolates [Clonostachys rosea(1),Coniothyrium minitans(1),Trichoderma crassum(1),T. hamatum(4),T. rossicum(1) andT. virens(1)] were tested in two bioassays for their ability to degrade sclerotia and reduce apothecial production and carpogenic infection of cabbage seedlings.C. minitansLU112 reduced apothecial production in both bioassays, withT. virensLU556 significantly reducing apothecial production in the sclerotial parasitism assay. Both isolates also reduced sclerotial viability in this assay to 5% forC. minitansand 22% forT. virens.C. minitansLU112 andT. virensLU556 reduced the infection of cabbage seedlings in the pot bioassay 126 days after sowing but not after 147 days, partly due to ascospore cross-infection between treatments.C. minitansLU112,T. virensLU556 andT. hamatumLU593 as maizemeal-perlite (MP) soil incorporation and transplant potting mix incorporation were evaluated for their ability to controlSclerotinia sclerotiorumdisease of cabbage in field experiments.S. sclerotioruminfection of cabbage was reduced by 46–52% and 31–57% by bothC. minitansLU112 andT. hamatumLU593 as MP soil incorporations, respectively, in the two field experiments.T. virensLU556 MP soil incorporation andC. minitansLU112 andT. hamatumLU593 transplant potting mix incorporations reducedS. sclerotiorumdisease in the first experiment but not in the second experiment. A commercialC. minitansLU112 formulation, C. Mins LU112 WG, also significantly reducedS. sclerotiorumdisease by 59%. Soil incorporation ofC. minitansandT. hamatumwas shown to have potential to controlS. sclerotiorumdisease in cabbage. [ABSTRACT FROM PUBLISHER]

The effects of the interaction between Suillus luteus (L.) Roussel and Trichoderma virens (J.H. Mill., Giddens & A.A. Foster) Arx on Pinus sylvestris var. mongolica Litv. were studied using plant physiology, mycorrhizal science, forest pathology, and biochemistry. Seedling growth and physiological parameters were determined, including the colonization rate of mycorrhizal fungi, biomass, root activity, photosynthetic pigment content, soluble protein content, antioxidant enzyme activities, rhizosphere soil enzyme activities, and protective enzyme activities. In addition, an optimal resistance system involving T. virens, mycorrhizal fungus ( S. luteus), and P. sylvestris var . mongolica seedlings was constructed. Synergies between S. luteus and T. virens were observed, and most of the parameters of P. sylvestris var. mongolica seedlings inoculated with S. luteus 30 days + T. virens were higher than other treatments. After three months, when compared the control, the S. luteus 30 days + T. virens treatment gave increases in height (42.3 %); collar diameter (66.7 %); fresh weight (54 %); dry weight (50 %); soluble protein content (69.86 %); root activity (150 %); chlorophyll a (77.6 %); chlorophyll b (70.5 %); carotenoids (144 %); CAT activity (876.9 %); POD activity (268.3 %); SOD activity (66.18 %); β-1,3-glucanase activity (125.8 %); chitinase activity (40 %); rhizosphere soil catalase activity (97.8 %); and phosphatase activity (266.7 %). These results indicate that there may be a stimulating factor between S. luteus and T. virens when they are inoculated together ( S. luteus 30 days + T. virens). [ABSTRACT FROM AUTHOR]

A Total of 13 isolates of Trichoderma virens were isolated from soil samples collected from 9 different sites. All isolates inhibited the mycelial growth of Alternaria brassicicola that causes Chinese kale leaf spot. There were two strains, T-ST-01 and T-NST-01, that gave high values of inhibition, 72.5 and 67.0%, respectively. Strains T-ST-01 and T-NST-01 were used to evaluate antifungal properties using dialysis membrane technique testing with A. brassicicola. The results showed that mycelial growth of A. brassicicola was completely inhibited. Antifungal metabolite was extracted and tested against germination of A. brassicicola spores. The results showed that spores of A. brassicicola germinated 9.4% compared with 98.5% for the control. Use of antifungal metabolite was effective both in laboratory and glasshouse conditions. Severity of the disease was significantly (P

The present study is an attempt to isolate and optimize the fermentation conditions of three fungus, namely Trichoderma virens, Alternaria alternata and Curvularia lunata to get pigments for the purpose of textile dyeing. Highest optical density has been achieved in potato dextrose broth at 28°C in 25 days under static conditions. Wool and silk are dyed unmordanted with very good wash and rub fastness. Trichoderma virens has demonstrated antifungal property. There is no adverse effect on the tensile strength of the fabric and on the human skin. Paper and thin layer chromatography has revealed that the pigments are multi-component in nature. [ABSTRACT FROM AUTHOR]

Abstract: Biological control of Botrytis gray mold (BGM, Botrytis cinerea) of chickpea by foliar, seeds or soil treatments of seven biocontrol agents (BCA) isolated from commercial biopesticides was evaluated. Foliar inoculation with BGM fungus caused disease of greater severity (82%) in comparison to soil (38%) or seed inoculation (42%) and reduced the plant dry weight and yield of chickpea cv. BG-256 by 23% and 29%, respectively. Soil or seed inoculation with the fungus reduced the plant dry weight (10–12%) and yield (13–16%). Greatest control of the disease was observed in the plants which were inoculated with BGM fungus and BCA through same method i.e. seed–seed, soil–soil, etc. Soil treatment of Trichoderma harzianum (PBAT-1 and Biowilt-X) significantly improved the yield of soil inoculated plants (P ⩽0.05). Similarly seed treatment with T. harzianum decreased the disease severity and increased the yield by 8–13% over seed inoculated control (P ⩽0.05). Foliar treatment of T. harzianum effectively checked the disease (39–41%) and improved the yield (19–22%) of foliar inoculated plants over the respective controls (P ⩽0.05). T. harzianum PBAT-1, Trichoderma virens Sanjeevni and Pseudomonas fluorescens Biocomp-X were found to be the most effective biopesticide strains. Treatment of biopesticides resulted in substantial decreases in the population of BGM fungus, whereas the population of biocontrol agents increased in the presence of B. cinerea. [Copyright &y& Elsevier]

Sucrose exuded by plants into the rhizosphere is a crucial component for the symbiotic association between the beneficial fungus Trichoderma and plant roots. In this article we sought to identify and characterize the molecular basis of sucrose uptake into the fungal cells. Several bioinformatics tools enabled us to identify a plant-like sucrose transporter in the genome of Trichoderma virens Gv29-8 (TvSut). Gene expression profiles in the fungal cells were analyzed by Northern blotting and quantitative real-time PCR (qRT-PCR). Biochemical and physiological studies were conducted on Gv29-8 and fungal strains impaired in the expression of TvSut. TvSut exhibits biochemical properties similar to those described for sucrose symporters from plants. The null expression of tvsut caused a detrimental effect on fungal growth when sucrose was the sole source of carbon in the medium, and also affected the expression of genes involved in the symbiotic association. Similar to plants, T. virens contains a highly specific sucrose /H+ symporter that is induced in the early stages of root colonization. Our results suggest an active sucrose transference from the plant to the fungal cells during the beneficial associations. In addition, our expression experiments suggest the existence of a sucrosedependent network in the fungal cells that regulates the symbiotic association. [ABSTRACT FROM AUTHOR]

The aim of this study was to apply a generated Δ tku70 strain with increased homologous recombination efficiency from the mycoparasitic fungus Trichoderma virens for studying the involvement of laccases in the degradation of sclerotia of plant pathogenic fungi. Inactivation of the non-homologous end-joining pathway has become a successful tool in filamentous fungi to overcome poor targeting efficiencies for genetic engineering. Here, we applied this principle to the biocontrol fungus T. virens, strain I10, by deleting its tku70 gene. This strain was subsequently used to delete the laccase gene lcc1, which we found to be expressed after interaction of T. virens with sclerotia of the plant pathogenic fungi Botrytis cinerea and Sclerotinia sclerotiorum. Lcc1 was strongly upregulated at early colonization of B. cinerea sclerotia and steadily induced during colonization of S. sclerotiorum sclerotia. The Δ tku70Δ lcc1 mutant was altered in its ability to degrade the sclerotia of B. cinerea and S. sclerotiorum. Interestingly, while the decaying ability for B. cinerea sclerotia was significantly decreased, that to degrade S. sclerotiorum sclerotia was even enhanced, suggesting the operation of different mechanisms in the mycoparasitism of these two types of sclerotia by the laccase LCC1. [ABSTRACT FROM AUTHOR]

Sheath blight caused by Rhizoctonia solani is one of the most serious rice diseases worldwide. The disease is currently managed only by the excessive application of chemical fungicides which are toxic and not environmentally friendly. Therefore, greater emphasis should be given to biological control as being both safe and effective. Trichoderma species are ubiquitous fungi in the soil and have an antagonistic activity against several soil-borne plant pathogens including R. solani. The present study was undertaken to evaluate the potential of indigenous Trichoderma strains from Mazandaran province, Northern Iran (a Mediterranean region on the southern coast of the Caspian Sea) against R. solani AG1-IA in vitro, and against sheath blight disease in the glasshouse, in order to find biocontrol isolates for application in the field. More than 200 Trichoderma strains were isolated from the soil, plant debris and the phyllosphere in rice fields. Strains were first screened for their antagonism to R. solani by in vitro antagonism tests including dual culture, antibiosis, the effect of Trichoderma strains on the production and viability of R. solani sclerotia, and hyperparasitism on microscopic slides. According to the in vitro experiments, several strains belonging to T. harzianum, T. virens and T. atroviride showed excellent biocontrol. These potential antagonist strains were further evaluated for their effectiveness in controlling sheath blight under glasshouse conditions. Among the 55 selected strains, seven significantly controlled the disease. T. harzianum AS12-2 was the most effective strain in controlling rice sheath blight, better even than propiconazole, the most commonly used fungicide in Iran. [ABSTRACT FROM AUTHOR]

Abstract: The beneficial fungus Trichoderma virens secretes a small cysteine-rich protein (Sm1) that induces defense responses in dicot and monocot plants and is a member of the cerato-platanin family. Purification of Sm1 from T. virens results in low protein yield limiting the application of this protein for crop disease protection to small-scale assays. To increase the yield of Sm1, we cloned the sm1 gene in the pPIC9K vector for transformation into the AOX1 locus of Pichia pastoris strain GS115. Transformants of P. pastoris were selected based on the presence of the vector insert as indicated by PCR analysis and the ability to secrete high levels of the rSm1 protein. The optimal incubation period and methanol concentrations for induction were determined for production of rSm1 in shake flasks. One Pichia transformant was estimated to express approximately 55mg/l of rSm1 after 4days culture in a 1% final concentration of methanol. The secreted rSm1 was purified by ammonium sulfate precipitation, ion exchange chromatography and gel column chromatography. SDS–PAGE and Western blot analysis revealed that the purified rSm1 expressed in Pichia was recognized by anti-Sm1 polyclonal antibody. The protein sequence was verified by ESI/MS/MS analysis of a tryptic digest of the rSm1. Greater than 90% peptide coverage was obtained and determined to be identical to the predicted sequence. The MALDI/TOF/MS analysis revealed the molecular mass of rSm1 to be 13.1kDa, which is higher than native Sm1 (12.6kDa). Edman sequencing of the purified protein revealed an N-terminal extension of six amino acids (EAEAYV). The extension is the result of insufficient activity of the Ste13 protease preventing efficient cleavage of the spacer (EAEA) downstream of the Kex2 cleavage site. Maize (cv. Silver Queen) treated with rSm1 or native Sm1 demonstrated the induction of two defense genes. Enhanced production of this elicitor has implications for the treatment of specialty crops to promote disease resistance. [Copyright &y& Elsevier]

Copyright of Journal of Plant Protection Research is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Development of transgenic Nicotiana tabacum and Lycopersicon esculentum expressing an endochitinase ( ech42) gene from biocontrol fungus Trichoderma virens using Agrobacterium-mediated genetic transformation is reported in this paper. Integration of transgene in the genome of transgenic plants was demonstrated using polymerase chain reaction and Southern-blot hybridization, while expression was ascertained by reverse transcription polymerase chain reaction. Histochemical analysis confirmed the expression of GUS enzyme in transformed shoots. Levels of endochitinase enzyme in transgenic plants were found to be up to 10 fold higher compared to control plants. Endochitinase enzyme of 42 kDa was also visualized on SDS-PAGE gel using fluorimetric zymogram in transgenic plants. Endochitinase activity was found to be higher in leaf and stem than the root tissue in transgenic tomato plants. Transgenic lines of both plants showed enhanced resistance to fungal pathogens and a strong negative correlation was found between expression level of endochitinase enzyme and size of disease lesions. Inheritance of transgene, expression and resistance to fungal pathogens of T1 transgenic tobacco lines was also analysed. The results of the present studies show that ech42 is a promising candidate gene for developing fungal disease resistance in tomato plants. [ABSTRACT FROM AUTHOR]

There are many reports on obtaining disease-resistance trait in plants by overexpressing genes from diverse organisms that encode chitinolytic enzymes. Current study represents an attempt to dissect the mechanism underlying the resistance to Rhizoctonia solani in cotton plants expressing an endochitinase gene from Trichoderma virens. Several assays were developed that provided a powerful demonstration of the disease protection obtained in the transgenic cotton plants. Transgene-dependent endochitinase activity was confirmed in various tissues and in the medium surrounding the roots of transformants. Biochemical and molecular analyses conducted on the transgenic plants showed rapid/greater induction of ROS, expression of several defense-related genes, and activation of some PR enzymes and the terpenoid pathway. Interestingly, even in the absence of a challenge from the pathogen, the basal activities of some of the defense-related genes and enzymes were higher in the endochitinase-expressing cotton plants. This elevated defensive state of the transformants may act synergistically with the potent, transgene-encoded endochitinase activity to confer a strong resistance to R. solani infection. [ABSTRACT FROM AUTHOR]

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans, and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens, Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens. Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Plant Protection Research is the property of Institute of Plant Protection and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A novel furanosteroid named 9-epi-viridiol (1), along with viridiol (2) and mevalonic acid (3), was isolated from Trichoderma virens. The structure of 1 was verified by combined spectroscopic data (COSY, HSQC, HMBC and NOESY) to be a C-9 epimer of viridiol. 9-epi-Viridiol exhibited cytotoxicity towards HeLa and KB cells with IC50 values of 19 and 50 µg mL-1, respectively. [ABSTRACT FROM AUTHOR]

Abstract: A novel gene, MRSP1 (MAP kinase repressed secreted protein 1) is strongly overexpressed in the tmkA MAPK mutant of the biocontrol fungus Trichoderma virens. Transcriptional regulation of MRSP1 is determined by presence or absence of TmkA, rather than by light or sporulation, making it a molecular marker for the unusual, negative, regulation by TmkA. The predicted protein is 15.9kDa, has a secretory signal, and the four-cysteine pattern, C-X29-CP(G)C-X31-C, may define a new cysteine-rich motif. This is a novel protein with functions not known from any other organism. Conservation in ascomycete, basidiomycete, and Dictyostelium homologs, as well as tight MAPK regulation, might indicate important cellular functions. [Copyright &y& Elsevier]

A transcriptional comparison of wild type and a secondary metabolite deficient Trichoderma virens mutant resulted in the identification of six genes similar to those involved in secondary metabolism in other fungi, including four cytochrome P450 genes, one O-methyl transferase and one terpene cylase. Four of the genes (three cytochrome P450s and the cyclase) are located as a cluster. Transcript levels of three of the P450 genes, the O-methyl transferase and the terpene cyclase were measured. These genes are underexpressed in the mutant, which lacks the major secondary metabolites produced by this strain, viridin and viridiol. Expression levels of clones from the differential library with similarity to fungal trehalose synthase and a hydrophobin were also underexpressed in the mutant, while a heat shock protein hsp98 homolog was not. Based on the gene expression pattern and associated secondary metabolite profile, along with similarity to other secondary metabolism pathways in related fungi, we predict that the cluster is associated with the production of a terpene. The terpene could be viridin. This is the first report on cloning of secondary metabolism related genes from T. virens, and of their organization in a cluster, in this biocontrol fungus. [ABSTRACT FROM AUTHOR]

Classical models of population dynamics predict that with increasing initial population densities the per capita growth will diminish. Observations over a broad range of initial densities with a wild-type and a genetically engineered strain of the filamentous fungus , Trichoderma virens (Arx), in soil and autoclaved soil differed from these predictions. The per capita growth response of T. virens in vitro was found to be density dependent on potato dextrose agar, but density independent on water agar. Further experiments with a defined, carbon-free medium (Vogel's medium) and, with the same medium containing sucrose, indicated that density dependent per capita growth occurred in the nutrient-rich medium but not the oligotrophic medium. This hypothesis was tested and supported experimentally through observation of density dependent per capita growth after adding nutrients to autoclaved soil. Development of better models of population dynamics will be important to predict successfully the likelihood and extent of establishment after field release of microorganims. [ABSTRACT FROM AUTHOR]

Trichoderma virens (Gliocladium virens) (Miller et al.) von Arx is a soilborne fungus with a high degree of rhizosphere competence that produces a potent herbicidal compound, viridiol, and therefore has potential for development as a bioherbicide. We investigated the possibility of using composted chicken manure (CCM) as a medium for the production and deployment of T. virens. We chose CCM since the safe disposal of chicken manure presents significant logistic problems, and composted manures, as well as serving as an organic source of nitrogen, have been shown to support the activity of other biological control agents. Composted chicken manure supported the growth of T. virens and the rapid production of high concentrations of viridiol, but only when it was supplemented with large quantities of nutrients, including sucrose (16% w/w). Viridiol was not stable when stored in CCM, with a rapid decline in viridiol concentrations observed in T. virens-inoculated CCM cultures. Clearly, a cheaper alternative to sucrose is required as a carbon source for T. virens in CCM or similar media, and effective storage methods would need to be found for a T. virens-based bioherbicide product. Importantly, CCM did not need to be sterilized to support the growth of T. virens and its concomitant production of viridiol, suggesting that on-farm production systems may be feasible. Trichoderma virens-colonized CCM reduced the emergence and seedling growth of redroot pigweed (Amaranthus retroflexus L.) in a greenhouse experiment and dramatically reduced the emergence of a mixed community of broadleaf weeds in the field. [ABSTRACT FROM AUTHOR]

Abstract: A number of techniques exist that have potential to be integrated into low-external-input weed management systems but it is not the norm for these different techniques to be investigated in combination. Here, we evaluate the weed management potential of two cultural weed management techniques, cover cropping and fertility management, with two allelochemical-releasing organisms compared to herbicides. Trichoderma virens (=Gliocladium virens), which releases the herbicidal molecule viridiol, is applied incorporated in composted chicken manure (CCM), and rye, which is known to release the herbicidal molecules (3H)-benzoxazolinone (BOA) and 2,4-dihydroxy-1,4-(2H)benzoxazine-3-one (DIBOA), is used as the cover crop. Both T. virens-inoculated CCM and rye cover crops provided significant weed control in some experiments without a significant loss of crop vigor and yield. Yield was comparable to those obtained with chemical herbicides, although the results were not consistent. Clear weed suppressive activity exists with these nonherbicidal treatments, and effective weed management in transplanted vegetables seems feasible with these approaches. However, further refinements in these treatments is needed to ensure reliable performance prior to grower acceptance. [Copyright &y& Elsevier]

Abstract: The genetic stability and ecological persistence of a strain of the filamentous fungus, Trichoderma virens, genetically modified (GM) with a hygromycin resistance gene and a gene encoding an organophosphohydrolase was evaluated over 243 days in soil mesocosms. The GM populations declined over time, a trend similar to the wild-type, parental strain (WT), but was still present at the end of the incubation. Similar population estimates were obtained by plating serial dilutions on selective media or by quantitative polymerase chain reaction (QC-PCR) after 243 days’ incubation. However, the absolute population size differed between the two methods by more than a log order of magnitude with QC-PCR being more sensitive. The presence of the WT strain did not adversely affect the persistence of the GM strain. The GM strain stably maintained the transgene and still expressed antibiotic resistance and the ability to degrade organophosphate xenobiotics. Even after the long-term incubation the GM and WT strains rapidly and efficiently colonized freshly added substrates, indicating a high level of responsiveness. [Copyright &y& Elsevier]

Trichoderma virens, an imperfect fungus, is used as a biocontrol agent to suppress plant disease caused by soilborne fungal pathogens. Antimicrobial peptides it produces include peptaibols of 11, 14, and 18 amino acids in length. These peptaibols were previously reported to be synthesized by a non-ribosomal peptide synthetase (NRPS) encoded by the Tex1 gene in strain Tv29-8. The present study examined the Tex1 homolog in a commercially relevant T. virens strain, G20. Although the gene in G20 was 99% identical in DNA sequence to Tex1 in the 15.8 kb compared, gene disruption results indicate that it is only responsible for the production of an 18-mer peptaibol, and not 11-mer and 14-mer peptaibols. Additional NRPS adenylate domains were identified in T. virens and one was found to be part of a 5-module NRPS gene. Although the multimodule gene is not needed for peptaibol synthesis, sequence comparisons suggest that two of the individual adenylate domain clones might be part of a separate peptaibol synthesis NRPS gene. The results indicate a significant diversity of NRPS genes in T. virens that is unexpected from the literature. [ABSTRACT FROM AUTHOR]

Abstract: Strains of Trichoderma virens belonging to the “P” group are ineffective as biocontrol agents of seedling disease in cotton, and they are pathogenic to susceptible seed lots. Those strains belonging to the “Q” group are effective biocontrol agents of cotton seedling disease, and they are not pathogenic to cotton. To account for these behavioral differences, comparative assays were made of “P” and “Q” strains for production of phytotoxin, for cellulase, polygalacturonase and protease activity, for induction of phytoalexin synthesis in cotton roots, and for metabolism of pathogen germination stimulants. The results showed little difference in phyotoxin production or enzyme activity between the two groups, and that “P” strain mutants deficient for viridiol production were still pathogenic to cotton. There was also no difference between strains in their ability to metabolize pathogen germination stimulants. HPLC analyses of extracts from roots treated with “P” or “Q” strains, however, showed that “Q” strains induced high levels of phytoalexin synthesis, while “P” strains did not. Treatment of seeds or seedling radicles with combination “P”+“Q” seed coat preparations or cultures filtrates, respectively, ameliorated seedling kill, and increased phytoalexin production in treated roots. These results indicate that an inability by “P” strains to induce high levels of phytoalexins in cotton, not only makes them ineffective as biocontrol agents, but renders them pathogenic to susceptible cultivar seed lots. Induction by “Q” strains of high levels of phytoalexin synthesis in cotton makes them effective biocontrol agents, and it inhibits their development in cotton roots which might lead to pathogenesis. [Copyright &y& Elsevier]

Abstract: The soilborne pathogens Rhizoctonia solani, Pythium ultimum, and Meloidogyne incognita can cause severe economic losses to field- and greenhouse-grown cucumber. A collection of bacterial isolates and isolates GL3 and GL21 of Trichoderma virens were screened for suppression of diseases caused by these pathogens. T. virens isolates GL3 and GL21 provided the most effective suppression of damping-off caused by R. solani in greenhouse bioassays. Burkholderia ambifaria BC-F, B. cepacia BC-1, and Serratia marcescens N1-14 also provided significant suppression of R. solani relative to the pathogen check in some experiments. T. virens isolates GL3 and GL21 and S. marcescens isolates N1-6, N1-14, and N2-4 provided the most consistent and effective suppression of damping-off of cucumber caused by P. ultimum in growth chamber experiments. No microbial treatment containing individual or combined microbes significantly suppressed populations of M. incognita on cucumber or improved plant vigor in greenhouse bioassays. T. virens GL21 applied as a granular formulation, in combination with B. cepacia BC-1 or B. ambifaria BC-F applied as a seed treatment, significantly improved suppression of damping-off caused by R. solani over individual applications of these microbes in at least one experiment. Treatments combining B. cepacia BC-1, B. ambifaria BC-F, or S. marcescens isolates N1-14 or N2-4 with T. virens GL21 in R. solani biocontrol assays always resulted in plant stands that were similar or greater than treatments containing individual applications of these microbes. B. ambifaria BC-F combined with T. virens GL21 in seed treatments resulted in significantly improved suppression of damping-off caused by P. ultimum in two of three experiments. Populations of T. virens GL3 and GL21 were both substantially reduced after coincubation with B. cepacia BC-1, or S. marcescens isolates N1-14 or N2-4 for 10 to 12 d in cucumber rhizospheres. Populations of T. virens GL21 were slightly reduced after coincubation with B. ambifaria BC-F. Results presented here substantiate other studies reporting enhanced biocontrol performance with certain combinations of biocontrol agents. These results also indicate that antagonism among combinations of biocontrol agents can vary with the assay system employed. [Copyright &y& Elsevier]

This work was undertaken to determine the effects of Trichoderma spp. combined with label and sublabel rates of metam sodium on survival of Rhizoctonia solani in soil. Soils were infested with wheat bran preparations of Trichoderma hamatum Tri-4, T. harzianum Th-58, T. virens Gl-3, and T. viride Ts-1-R3. Soil was also infested with sterile beet seeds that were colonized with R. solani. Beet seeds were later recovered, plated onto water agar plus antibiotics, and the growth of R. solani was recorded. Preliminary experiments showed that T. hamatum and T. virens reduced survival and saprophytic activity of R. 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 and R. solani. At 1:2 and 1:5 dilutions, metam sodium reduced survival of R. solani and all Trichoderma 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 all Trichoderma isolates tested reacted similarly to increasing concentrations of metam sodium. At the label rate of metam sodium, all Trichoderma spp. significantly reduced colony diameter, but not growth rate, of R. solani from beet seed. For the levels of metam sodium tested in combination with Trichoderma, it does not appear feasible to use a reduced rate of metam sodium to control R. solani. However, the combination of Trichoderma with metam sodium does reduce growth of R. solani in comparison with that provided by metam sodium at the label rate. [ABSTRACT FROM AUTHOR]

Summary Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens . Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T[sub 2] plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata . Transgenic cotton plants showed significant resistance to both pathogens. [ABSTRACT FROM AUTHOR]

Three native and two commercial biocontrol agents (Clonostachys rosea and Trichoderma spp.) were evaluated against the cocoa diseases moniliasis, witches'' broom, and black pod. Antagonists were applied either separately or as mixed inoculum in comparison with a copper fungicide and a nontreated control. Cultural control (weekly removal of diseased pods) was practiced in all treatments. Field trials were conducted on neglected cocoa farms in eastern Peru from 1998 to 2000. The fungicide treatment and C. rosea strain G-4 did not reduce disease. The other single-strain antagonists reduced moniliasis. Additionally, Trichoderma longibrachiatum and Trichoderma stromaticum reduced witches'' broom and Trichoderma virens reduced black pod. With strain mixtures, yield increases of up to 15% were obtained. A mixture of four antagonists was superior to mixtures of two or three antagonists with respect to multiple disease control and yield. Biocontrol in combination with cultural control was more economical than cultural control alone; chemical control was least economical. Fertilization improved yields by 11% independent of the disease control measure and compensated for the additional costs with net returns improving by 9%. Increasing application frequencies of a mixed biocontrol inoculum improved moniliasis and witches'' broom control linearly. Moniliasis exhibited a stronger response. Witches'' broom was significantly lower than the nontreated control only if ten applications were administered in two-week intervals. This application frequency increased yields by 15%. It was followed by three applications adjusted to the production cycle (yield increase: 12%). Three adjusted applications were the most economical biocontrol strategy under the conditions of eastern Peru. Net returns were increased by 12%. Recommendations for technology transfer are presented. [Copyright &y& Elsevier]

Trichoderma virens is a widely distributed soil fungus that is parasitic on other soil fungi. The mycoparasitic activity of T. virens is correlated with the production of numerous antifungal activities, including the secretion of a considerable repertoire of fungal cell wall-degrading enzymes. Here, we report the characterization of a diverse set of chitinase and glucanase genes from T. virens. In each case, full-length genomic clones were isolated and characterized, while sequencing of the corresponding cDNA clones and manual annotation provided a basis for establishing gene structure. Based on homology of the deduced amino acid sequences, we have identified three members of the 42Kd endochitinase gene family, two 33Kd exochitinases, two exochitinases with homology to N-acetylglucosaminidases, and three glucanase genes predicted to encode β-1,3- and β-1,6-proteins. The majority of these genes appear to encode signal peptides, suggesting an extracellular location for the corresponding proteins. Despite their overall similarity, the 42Kd class of chitinases can be subdivided, based on the presence of distinct N-terminal domains, suggesting that the proteins may have distinct cellular roles, while Northern blot analysis confirms that these genes possess distinct patterns of gene regulation. Similarly, one of the 33Kd chitinase genes is unique, because it is predicted to encode a protein C-terminus with high homology to the conserved family I cellulose-binding domain. The expression patterns of the chitinase genes were analyzed in both a wild-type strain and a strain disrupted for the major 42Kd chitinase gene of T. virens. The results of these transcript analyses, together with enzymatic assay of the extracellular proteins, suggest interdependent regulation of this important gene family in T. virens. [ABSTRACT FROM AUTHOR]

The nematicidal properties of Trichoderma species have potential for developing safer biocontrol agents. In the present study, 13 native Trichoderma strains from T. citrinoviride, T. ghanense (2 strains), T. harzianum (4), T. koningiopsis, T. simmonsii, and T. virens (4) with nematicidal activity were selected and cultured in potato dextrose broth to obtain a culture filtrate (CF) for each. Each CF was partitioned with ethyl acetate to obtain organic (EA) and residual filtrate (RF) fractions, which were then tested on second-stage juveniles (J2s) of the nematodes Meloidogyne javanica and M. incognita in a microdilution assay. The most lethal strains were T. harzianum Th43-14, T. koningiopsis Th41-11, T. ghanense Th02-04, and T. virens Th32-09, which caused 51–100% mortality (%M) of J2s of both nematodes, mainly due to their RF fractions. Liquid chromatography–diode array detector-electrospray-high resolution mass spectrometry analysis of the most-active fractions revealed sesquiterpene and polyketide-like metabolites produced by the four active strains. These native Trichoderma strains have a high potential to develop safer natural products for the biocontrol of Meloidogyne species. [ABSTRACT FROM AUTHOR]