The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.     

Carbon Requirements of Some Nematophagous, Entomopathogenic and Mycoparasitic Hyphomycetes as Fungal Biocontrol Agents

Thirty-three carbon sources were evaluated for their effects on spore germination, hyphal growth and sporulation of 11 fungal biocontrol agents, i.e. the nematophagous fungi Paecilomyces lilacinus, Pochonia chlamydosporia, Hirsutella rhossiliensis, H. minnesotensis and Arkansas Fungus 18, the entomopathogenic fungi Lecanicillium lecanii, Beauveria bassiana and Metarhizium anisopliae, and the mycoparasitic fungus Trichoderma viride. Variations in carbon requirements were found among the fungal species or strains tested. All strains studied except for T. viride grew on most carbon sources, although B. bassiana had more fastidious requirements for spore germination. Monosaccharides and disaccharides were suitable for fungal growth. For most isolates, d-glucose, d-mannose, sucrose and trehalose were superior to pectin and soluble starch among the polysaccharides and lactic acid among the organic acids. Both ethanol and methanol could accelerate growth of most isolates but not biomass. d-mannose, d-fructose and d-xylose were excellent carbon sources for sporulation, while d-glucose, sucrose, cellobiose, trehalose, chitin, dextrin, gelatin and lactic acid were better for some isolates. Neither sorbic acid nor linoleic acid could be utilized as a single carbon source. These findings provided a better understanding of the nutritional requirements of different fungal biocontrol agents that can benefit the mass production process.     

SCP production by Chaetomium cellulolyticum,a new thermotolerant cellulolytic fungus

Chaetomium cellulolyticum, a newly isolated cellulolytic fungus, showed 50–100% faster growth rates and over 80% more final biomass-protein formation than Trichoderma viride, a well-known high cellulase-producing cellulolytic organism, when cultivated on Solka-floc (a purified, predominantly amorphorous form of cellulose) or partially delignified sawdust (consisting of a mixture of hardwoods) as the sole-carbon source in the fermentation media. However, in both cases, T. viride produced much higher quantities of free cellulases at faster rates and also degraded more substrate than C. cellulolyticum. It is concluded that the synthesis mechanisms and/or the nature of the cellulase complexes of the two types of organisms are quite different such that C. cellulolyticum is more optimal for single-cell protein (SCP) production, while T. viride is more optimal for the production of extracellular cellulases. It was also found that the amino acid composition of C. cellulolyticum is generally better than that of T. viride and compares favorably with those of the FAO reference protein, alfalfa, and soya meal. In addition, preliminary feeding trials on rats have shown no adverse effects of the SCP produced by C. cellulolyticum fermentations.     

Enzyme mechanisms involved in cellulose hydrolysis by the rot fungus Sporotrichum pulverulentum

This article presents a review of the enzyme mechanisms involved in degradation of cellulose by the white-rot fungus Sporotrichum poulverulentum. The hydrolytic enzymes involved include: (1) five endo-1,4-β-glucanases; (2) one exo-1,4-β-glucanase, and (3) one or several 1,4-β-glucosidases. A recently discovered oxidative enzyme of importance in in vitro cellulose degradation seems to be a cellobiose oxidase. An oxidoreductase, cellobiose:quinone oxidoreductase, is of importance both in cellulose and in lignin degradation. Regulatory mechanisms of the extracellular enzyme activities, such as monosugar levels causing catabolite repression of the endoglucanases, have also been investigated. The enzymes used by S. pulverulentum in cellulose hydrolysis are compared to those used by Trichoderma viride. Very similar types of enzymes are used in both cases. However, no oxidative enzyme has so far been found to be involved in extracellular cellulose degradation in the case of T. viride. Recommendations for further research are given.     

Biological control of root-rot of Coleus forskohlii Briq. using microbial inoculants

The root-rot or wilt of Coleus forskohlii is a very serious soil-borne disease caused by Fusarium chlamydosporum. A field study was undertaken to study the possibility of controlling the disease using three biocontrol agents viz., Glomus mosseae, Pseudomonas fluorescens, Trichoderma viride, singly and in combination. Planting of coleus cutting was done in wilt sick soil. Inoculation with Trichoderma viride + Glomus mosseae gave the best result in controlling the disease. The same treatment also resulted in maximum growth, yield and root forskolin concentration of coleus. Plants treated with T. viride + G. mosseae showed a disease severity index of 33.28% compared to uninoculated control plants, which had a maximum disease severity index of 85.5%. The fungicide Emisan (0.2%) was not as effective as the biocontrol agents in controlling the pathogen.     

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.     

In vitro and in vivo antagonism of biocontrol agents against Colletotrichum lindemuthianum causing bean anthracnose

Bean anthracnose caused by Colletotrichum lindemuthianum is a serious seed borne disease. For devising an effective management strategy, the efficacy of different bioagents, viz. Trichoderma viride, Trichoderma harzianum, Trichoderma hamatum and Gliocladium virens conducted under in vitro and in vivo conditions revealed maximum inhibition of mycelial growth in dual culture (59.48%) and inverted plate (55.98%) with T. viride. All the bioagents overgrew the pathogen and the principal mechanism of mycoparisitism observed was coiling, brusting and disintegration of pathogen hyphae. Culture filtrate from T. viride was found best as it completely inhibited radial growth at 25 and 50% concentration and reduced the spore germination of test fungus significantly. However, lower concentrations of culture filtrate from all bioagents showed little effect on spore germination. Seed application of bioagents was found better as compared to soil application. A maximum increase in seed germination and inhibition of seed borne infection was observed with T. viride followed by T. harzianum under pot culture conditions. T. viride has the maximum potentiality to suppress the spore germination, mycelial growth, seed borne infection of C. lindemuthianum and increased seed germination when compared with the other biocontrol agents.     

An Antifungal Protein Purified from Pearl Millet Seeds Shows Sequence Homology to Lipid Transfer Proteins

In the course of a search for antifungal proteins from plant seeds, we observed inhibition of mycelial growth of Trichoderma viride with extracts of pearl millet. We have identified several proteins with antifungal properties in the seeds of pearl millet. One of these proteins has been purified to homogeneity and characterized. The purified protein has a molecular mass of 25 kDa. The N-terminal sequence of the protein (25 residues) shows homology to non-specific lipid transfer proteins (LTPs) of cotton, wheat and barley. The purified LTP inhibited mycelial growth of T. viride and the rice sheath blight fungus, Rhizoctonia solani in vitro.     

Secretome analysis of the fungus Trichoderma harzianum grown on cellulose

Trichoderma harzianum is a mycoparasitic filamentous fungus that produces and secretes a wide range of extracellular hydrolytic enzymes used in cell wall degradation. Due to its potential in biomass conversion, T. harzianum draws great attention from biofuel and biocontrol industries and research. Here, we report an extensive secretome analysis of T. harzianum. The fungus was grown on cellulose medium, and its secretome was analyzed by a combination of enzymology, 2DE, MALDI-MS and -MS/MS (Autoflex II), and LC-MS/MS (LTQ-Orbitrap XL). A total of 56 proteins were identified using high-resolution MS. Interestingly, although cellulases were found, the major hydrolytic enzymes secreted in the cellulose medium were chitinases and endochitinases, which may reflect the biocontrol feature of T. harzianum. The glycoside hydrolase family, including chitinases (EC 3.2.1.14), endo-N-acetylglucosaminidases (EC 3.2.1.96), hexosaminidases (EC 3.2.1.52), galactosidases (EC 3.2.1.23), xylanases (EC 3.2.1.8), exo-1,3-glucanases (EC 3.2.1.58), endoglucanases (EC 3.2.1.4), xylosidases (EC 3.2.1.37), α-L-arabinofuranosidase (EC 3.2.1.55), N-acetylhexosaminidases (EC 3.2.1.52), and other enzymes represented 51.36% of the total secretome. Few representatives were classified in the protease family (8.90%). Others (17.60%) are mostly intracellular proteins. A considerable part of the secretome was composed of hypothetical proteins (22.14%), probably because of the absence of an annotated T. harzianum genome. The T. harzianum secretome composition highlights the importance of this fungus as a rich source of hydrolytic enzymes for bioconversion and biocontrol applications.     

绿色木霉对生物降解和生物防治的影响机理与应用研究进展

绿色木霉(Trichoderma viride)是一种广泛分布于自然界的有益微生物,具有高效生物降解和生物防治功能。研究表明,绿色木霉在代谢过程中会产生纤维素酶、几丁质酶、木质素过氧化物酶等一系列水解酶类,能够高效降解环境中的有机物质,同时,作为农业上常见的生防菌,具有抑制病原菌、促进植物生长、提高土壤肥力等有益功能。基于此,围绕绿色木霉生物功能最新研究进展,从绿色木霉生物降解和生物防治角度,探究绿色木霉实际应用和作用机理,以期为充分利用绿色木霉资源提供依据,并对本领域未来的发展方向和应用前景进行展望。     

Evaluation of cellulases produced from four fungi cultured on furfural residues and microcrystalline cellulose

Four fungal strains—Trichoderma viride, Aspergillus niger, Trichoderma koningii, and Trichoderma reesei—were selected for cellulase production using furfural residues and microcrystalline cellulose (MCC) as the substrates. The filter paper activity (FPA) of the supernatant from each fungus was measured, and the performance of the enzymes from different fungal strains was compared. Moreover, the individual activities of the three components of the cellulase system, i.e., β-glucosidase, endoglucanase, and exoglucanase were evaluated. T. koningii showed the highest activity (27.81 FPU/ml) on furfural residues, while T. viride showed an activity of 21.61 FPU/ml on MCC. The FPA of the crude enzyme supernatant from T. koningii was 30% higher on furfural residues than on MCC. T. koningii and T. viride exhibited high stability and productivity and were chosen for cellulases production. The crystallinity index (CrI) of the furfural residues varied after digested by the fungi. The results indicated differences in the functioning of the cellulase system from each fungus. In the case of T. koningii, T. reesei and T. viride, furfural residues supported a better environment for cellulase production than MCC. Moreover, the CrI of the furfural residues decreased, indicating that this material was largely digested by the fungi. Thus, our results suggest that it may be possible to use the cellulases produced from these fungi for the simultaneous saccharification and fermentation of lignocellulosic materials in ethanol production.     

Cellulase and protein production from mixed cultures of Trichoderma viride and a yeast

Fermentations with mixed cultures of the cellulolytic fungus Trichoderma viride and the yeast Saccharomyces cerevisiae or Candida utiliswere examined. The fermentations were carried out in an aerated 5 liter fermentor with NaOH treated barley straw as the cellulose source (2–4%). Yeast was inoculated 24–32 hr after the fungus and the growth of the two organisms was followed through the production of CO₂ and cell protein. In comparison with fermentations with T. viridealone, the production time for maximum yields of cellulases and cell protein was reduced by several days, depending on the straw concentrations. The protein content of the growth product was 21–22% and the amino acid composition of the product resembled that of T. viride alone.     

Two‐dimensional proteome reference maps for the human pathogenic filamentous fungus Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus has become the most important airborne fungal pathogen causing life‐threatening infections in immunosuppressed patients. We established a 2‐D reference map for A. fumigatus. Using MALDI‐TOF‐MS/MS, we identified 381 spots representing 334 proteins. Proteins involved in cellular metabolism, protein synthesis, transport processes and cell cycle were most abundant. Furthermore, we established a protocol for the isolation of mitochondria of A. fumigatus and developed a mitochondrial proteome reference map. 147 proteins represented by 234 spots were identified.     

Studies on Biological Control of Postharvest Rot in Yams (Dioscorea spp.) using Trichoderma viride

From in vitro and in vivo screening tests for antagonism by isolates of Trichoderma against postharvest pathogens of yams (Dioscorea spp.), an isolate of Trichoderma viride Pers. ex S.F. Gray was selected as the most promising candidate for the biocontrol of postharvest rot of yams. Inoculation of white yam (Dioscorea rotundata Poir.) with conidiaspores of T. viride and subsequent storage of the tubers under the ambient environment conditions of a traditional yam barn, resulted in a drastic reduction in the frequency of occurrence of the normal tuber surface mycoflora over a 4‐month (December‐April) storage period. Trichoderma viride on the other hand, maintained a high frequency of occurrence during the same period. Furthermore, whereas up to 52.0%) rot was found among groups of tubers that were artificially inoculated with the postharvest pathogens of yams, Aspergillus niger Van Tiegh., Botryodiplodia theobromae Pat. or Penicillium oxalicum Currie and Thom, and also the group that was not inoculated with any organism (control), among groups of tubers that were inoculated with T. viride the rot was either totally suppressed or only a low percentage of rot occurred. The significance of the findings is discussed in relation to yam storage especially by farmers with limited resources.     

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.     

Biocontrol of onion white rot by application of Trichoderma species formulated on wheat bran powder

In vitro, Trichoderma album, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride and Trichoderma virens showed antagonistic effect against the most pathogenic isolate (Sc2) of Sclerotium cepivorum, the cause of onion white rot disease. Five Trichoderma preparations of each Trichoderma sp. were prepared on wheat bran powder to be used for controlling white rot disease of onion. Greenhouse and field experiments followed the same trend where T. harzianum and T. koningii were the most effective in reducing the incidence and severity of white rot disease compared with the control. Trichoderma species preparations caused promotion to vegetative parameters of onion plants in pots and increase bulb productivity in filed. In this regard, T. harzianum and T. koningii were the most effective. A positive correlation was found between the biocontrol activity of Trichoderma species preparations and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in onion plants to resist infection with S. cepivorum.     

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

Background

Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.

Results

Here we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.

Conclusions

The data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.     

Signal transduction by Tga3, a novel G protein alpha subunit of Trichoderma atroviride

Trichoderma species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when Trichoderma specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the tga3 gene of Trichoderma atroviride, which encodes a novel G protein alpha subunit that belongs to subgroup III of fungal Galpha proteins. Deltatga3 mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with Rhizoctonia solani or Botrytis cinerea, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or N-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes ech42 and nag1 were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus, T. atroviride Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.     

Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain

The gfp gene from the jellyfish Aequorea victoria, coding for the Green Fluorescent Protein (GFP), was used as a reporter gene to transform a Trichoderma virens strain I10, characterized as having a promising biocontrol activity against a large number of phytopathogenic fungi. On the basis of molecular and biological results, a stable GFP transformant was selected for further experiments. In order to evaluate the effects of GFP transformation on mycoparasitic ability of T. virens I10, sclerotia of Sclerotium rolfsii, Sclerotinia sclerotiorum and S. minor were inoculated with the T. virens strain I10 GFP transformant or the wild type strain. Statistical analysis of percentages of decayed sclerotia showed that the transformation of the antagonistic isolate with the GFP reporter gene did not modify mycoparasitic activity against sclerotia. Sclerotium colonization was followed by fluorescent microscopy revealing intracellular growth of the antagonist in the cortex (S. rolfsii) and inter-cellular growth in the medulla (S. rolfsii, and S. sclerotiorum). The uniformly distributed mycelium of T. virens just beneath the rind of sclerotia of both S. rolfsii and S. sclerotiorum suggests that the sclerotia became infected at numerous randomly distributed locations without any preferential point of entry.