Sm1, a proteinaceous elicitor secreted by the biocontrol fungus Trichoderma virens induces plant defense responses and systemic resistance

The soilborne filamentous fungus Trichoderma virens is a biocontrol agent with a well-known ability to produce antibiotics, parasitize pathogenic fungi, and induce systemic resistance in plants. Even though a plant-mediated response has been confirmed as a component of bioprotection by Trichoderma spp., the molecular mechanisms involved remain largely unknown. Here, we report the identification, purification, and characterization of an elicitor secreted by T. virens, a small protein designated Sm1 (small protein 1). Sm1 lacks toxic activity against plants and microbes. Instead, native, purified Sm1 triggers production of reactive oxygen species in monocot and dicot seedlings, rice, and cotton, and induces the expression of defense-related genes both locally and systemically in cotton. Gene expression analysis revealed that SM1 is expressed throughout fungal development under different nutrient conditions and in the presence of a host plant. Using an axenic hydroponic system, we show that SM1 expression and secretion of the protein is significantly higher in the presence of the plant. Pretreatment of cotton cotyledons with Sm1 provided high levels of protection to the foliar pathogen Colletotrichum sp. These results indicate that Sm1 is involved in the induction of resistance by Trichoderma spp. through the activation of plant defense mechanisms.     

Tvbgn3, a beta-1,6-glucanase from the biocontrol fungus Trichoderma virens, is involved in mycoparasitism and control of Pythium ultimum

Even though beta-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a beta-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of beta-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.     

Enhanced biocontrol activity of Trichoderma virens transformants constitutively coexpressing β-1,3- and β-1,6-glucanase genes

Evidence for the role of chitinases, proteases and β-1,3- and β-1,6-glucanases in mycoparasitism by Trichoderma species has been well documented. Moreover, constitutive over-expression of genes encoding individual cell-wall-degrading enzymes (CWDEs) has been shown to improve the potential of biological agents. In this study, we generated transformants of T. virens in which β-1,3- and β-1,6-glucanase genes, TvBgn2 and TvBgn3 , respectively, were constitutively coexpressed in the same genetic T. virens Gv29.8 wild-type background. The double over-expression transformants (dOEs) grow and sporulate slower than the wild-type (WT). However, the reduction in growth did not seem to affect their mycoparasitic and biocontrol capabilities, as dOEs displayed much higher levels of total β-1,3- and β-1,6-glucanase activity than the WT. This higher enzymatic activity of dOEs positively correlated with observed in vitro inhibition of Pythium ultimum and Rhizoctonia solani mycelia, and with enhanced bioprotection of cotton seedlings against P. ultimum , R. solani and Rhizopus oryzae . Besides effective biocontrol of all pathogens at an original inoculum level, the performance of dOEs was highly enhanced (up to 312% of WT performance) when pathogen pressure was greater (i.e. concentration of inoculum was higher or pathogens applied in combination). These results demonstrate that the strategy of introducing multiple lytic enzyme-encoding genes through transformation of a given biocontrol strain can be successfully used to achieve better biocontrol.     

A proteinaceous elicitor Sm1 from the beneficial fungus Trichoderma virens is required for induced systemic resistance in maize

We have previously shown that the beneficial filamentous fungus Trichoderma virens secretes the highly effective hydrophobin-like elicitor Sm1 that induces systemic disease resistance in the dicot cotton (Gossypium hirsutum). In this study we tested whether colonization of roots by T. virens can induce systemic protection against a foliar pathogen in the monocot maize (Zea mays), and we further demonstrated the importance of Sm1 during maize-fungal interactions using a functional genomics approach. Maize seedlings were inoculated with T. virens Gv29-8 wild type and transformants in which SM1 was disrupted or constitutively overexpressed in a hydroponic system or in soil-grown maize seedlings challenged with the pathogen Colletotrichum graminicola. We show that similar to dicot plants, colonization of maize roots by T. virens induces systemic protection of the leaves inoculated with C. graminicola. This protection was associated with notable induction of jasmonic acid- and green leaf volatile-biosynthetic genes. Neither deletion nor overexpression of SM1 affected normal growth or development of T. virens, conidial germination, production of gliotoxin, hyphal coiling, hydrophobicity, or the ability to colonize maize roots. Plant bioassays showed that maize grown with SM1-deletion strains exhibited the same levels of systemic protection as non-Trichoderma-treated plants. Moreover, deletion and overexpression of SM1 resulted in significantly reduced and enhanced levels of disease protection, respectively, compared to the wild type. These data together indicate that T. virens is able to effectively activate systemic disease protection in maize and that the functional Sm1 elicitor is required for this activity.     

Role of the 4-phosphopantetheinyl transferase of Trichoderma virens in secondary metabolism and induction of plant defense responses

Trichoderma virens is a ubiquitous soil fungus successfully used in biological control due to its efficient colonization of plant roots. In fungi, 4-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary and secondary metabolism. Therefore, we cloned the PPTase gene ppt1 from T. virens and generated PPTase-deficient (?ppt1) and overexpressing strains to investigate the role of this enzyme in biocontrol and induction of plant defense responses. The ?ppt1 mutants were auxotrophic for lysine, produced nonpigmented conidia, and were unable to synthesize nonribosomal peptides. Although spore germination was severely compromised under both low and high iron availability, mycelial growth occurred faster than the wild type, and the mutants were able to efficiently colonize plant roots. The ?ppt1 mutants were unable of inhibiting growth of phytopathogenic fungi in vitro. Arabidopsis thaliana seedlings co-cultivated with wild-type T. virens showed increased expression of pPr1a:uidA and pLox2:uidA markers, which correlated with enhanced accumulation of salicylic acid (SA), jasmonic acid, camalexin, and resistance to Botrytis cinerea. Co-cultivation of A. thaliana seedlings with ?ppt1 mutants compromised the SA and camalexin responses, resulting in decreased protection against the pathogen. Our data reveal an important role of T. virens PPT1 in antibiosis and induction of SA and camalexin-dependent plant defense responses.     

Identification of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase

The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.     

绿木霉Gv29-8丝氨酸蛋白酶S8/S53超家族基因特性及功能

【背景】丝氨酸蛋白酶在木霉菌生物防治过程中发挥重要作用。【目的】研究绿木霉丝氨酸蛋白酶S8/S53超家族基因信息及其生物学功能,进而为该蛋白酶生防制剂的开发及基因改造提供理论支持。【方法】通过生物信息学分析方法,从绿木霉Gv29-8基因组中鉴定出23个丝氨酸蛋白酶基因,以少孢节丛孢菌ATCC 24927基因组中鉴定的4个丝氨酸蛋白酶基因作为对照,对这27个丝氨酸蛋白酶基因的特性、蛋白结构、进化地位、功能等进行预测分析。【结果】27个基因结构差异较大,编码的蛋白具有典型的丝氨酸蛋白酶催化三联体结构,属于S8/S53超家族,分为6个亚家族,同一亚家族的蛋白酶保守区长度相近,相似性较高,催化残基附近序列比较保守。系统进化分析显示,同一亚家族丝氨酸蛋白酶聚为一类。【结论】绿木霉和少孢节丛孢菌的部分丝氨酸蛋白酶基因在结构和蛋白性质上相似性强,亲缘关系较近,均属于S8_PCSK9_ProteinaseK_like亚家族,推测绿木霉与少孢节丛孢菌该亚家族的丝氨酸蛋白酶具有相似的功能,可抑制植物病原真菌和降解线虫体壁。     

Peptide synthetase gene in Trichoderma virens.

Trichoderma virens (synonym, Gliocladium virens), a deuteromycete fungus, suppresses soilborne plant diseases caused by a number of fungi and is used as a biocontrol agent. Several traits that may contribute to the antagonistic interactions of T. virens with disease-causing fungi involve the production of peptide metabolites (e.g., the antibiotic gliotoxin and siderophores used for iron acquisition). We cloned a 5,056-bp partial cDNA encoding a putative peptide synthetase (Psy1) from T. virens using conserved motifs found within the adenylate domain of peptide synthetases. Sequence similarities with conserved motifs of the adenylation domain, acyl transfer, and two condensation domains support identification of the Psy1 gene as a gene that encodes a peptide synthetase. Disruption of the native Psy1 gene through gene replacement was used to identify the function of this gene. Psy1 disruptants produced normal amounts of gliotoxin but grew poorly under low-iron conditions, suggesting that Psy1 plays a role in siderophore production. Psy1 disruptants cannot produce the major T. virens siderophore dimerum acid, a dipetide of acylated N(delta)-hydroxyornithine. Biocontrol activity against damping-off diseases caused by Pythium ultimum and Rhizoctonia solani was not reduced by the Psy1 disruption, suggesting that iron competition through dimerum acid production does not contribute significantly to disease suppression activity under the conditions used.     

Multiple non-ribosomal peptide synthetase genes determine peptaibol synthesis in Trichoderma virens

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.     

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.     

Suppression of Fusarium Colonization of Cotton Roots and Fusarium Wilt by Seed Treatments with Gliocladium virens and Bacillus subtilis

In a growth chamber at 25 C, the fungal antagonist Gliocladium virens colonized tap roots and secondary roots of cotton in non-sterile soil after seed treatment with preparations of G. virens. Colonization of tap roots by G. virens increased over time, and decreased with root depth. Seed treatments with G. virens strains G-4 and G-6 and with Bacillus subtilis strains GB03 and GB07 reduced the colonization of tap roots and secondary roots of cotton seedlings by Fusarium spp. Under greenhouse conditions, the same seed treatments suppressed the incidence and severity of fusarium wilt of cotton in soil infested with Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita. Gliotoxin, produced by 'Q-group' strains of G. virens, inhibited F. oxysporum f. sp. vasinfectum in vitro. The seed treatments with G. virens strain G-6 and B. subtilis strains GB03 and GB07 did not affect the reproduction of root-knot nematodes. The results of this study may help to explain why the treatment of cotton seed with biocontrol agents often results in more vigorous and higher yielding plants, and indicate that there is potential for using G. virens and B. subtilis as seed treatments to control fusarium wilt of cotton.     

Isolation and characterization of protease overproducing mutants of Trichoderma harzianum

Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes is considered as the main mechanism involved in the antagonistic process. Strain Trichoderma harzianum T334 is a potential biocontrol agent against plant pathogenic fungi with the ability to produce low levels of proteases constitutively. To improve its fungal antagonistic capacity, mutagenetic program was undertaken for the construction of protease overproducing derivates. The mutant strains were obtained by means of UV-irradiation and were selected for p-fluorophenyl-alanine resistance or altered colony morphology. It was revealed by means of specific chromogenic protease substrates that both trypsin-like and chymotrypsin-like protease secretion was elevated in most of the mutant strains. The profiles of isoenzymes were different between the mutants and the wild-type strain, when examined by gel filtration chromatography. Certain mutants proved to be better antagonists against plant pathogens in in vitro antagonism experiments. This study suggests the possibility of using mutants with improved constitutive extracellular protease secretion against plant pathogenic fungi.     

Role of two G-protein alpha subunits, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens

G-protein alpha subunits are involved in transmission of signals for development, pathogenicity, and secondary metabolism in plant pathogenic and saprophytic fungi. We cloned two G-protein alpha subunit genes, tgaA and tgaB, from the biocontrol fungus Trichoderma virens. tgaA belongs to the fungal Galphai class, while tgaB belongs to the class defined by gna-2 of Neurospora crassa. We compared loss-of-function mutants of tgaA and tgaB with the wild type for radial growth, conidiation, germination of conidia, the ability to overgrow colonies of Rhizoctonia solani and Sclerotium rolfsii in confrontation assays, and the ability to colonize the sclerotia of these pathogens in soil. Both mutants grew as well as the wild type, sporulated normally, did not sporulate in the dark, and responded to blue light by forming a conidial ring. The tgaA mutants germinated by straight unbranched germ tubes, while tgaB mutants, like the wild type, germinated by wavy and highly branched germ tubes. In confrontation assays, both tgaA and tgaB mutants and the wild type overgrew, coiled, and lysed the mycelia of R. solani, but tgaA mutants had reduced ability to colonize S. rolfsii colonies. In the soil plate assay, both mutants parasitized the sclerotia of R. solani, but tgaA mutants were unable to parasitize the sclerotia of S. rolfsii. Thus, tgaA is involved in antagonism against S. rolfsii, but neither G protein subunit is involved in antagonism against R. solani. T. virens, which has a wide host range, thus employs a G-protein pathway in a host-specific manner.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Trichoderma spp. Antagonism to the Dermatophyte Trichophyton rubrum: Implications in Treatment of Onychomycosis

Onychomycosis--the dermatophytic invasion of the nail--is difficult to eradicate with drug treatment. The hyphae of the main invading pathogen, Trichophyton rubrum, are often interwoven with the nail plate, preventing effective anti-mycotic agents from reaching its growing tips. An alternative approach to treat onychomycosis may possibly be the application of a biological control agent against the pathogen. In analogy with the success of biocontrol of phytopathogenic fungi, we screened a series of commercially available Trichoderma strains for potential antagonism between Trichoderma and Trichophyton spp. A wide spectrum of antagonism capacity, ranging from effective overgrowth to no interaction was found, with Trichoderma virens NRRL 26672 being the most effective against the Trichophyton strains tested e.g. T. rubrum NCPF118. Furthermore, T. virens NRRL 26672 grown with T. rubrum NCPF118 hyphae as a carbon source, exhibited enhanced induced secretion of active extracellular chitinases and beta-glucosidases, affecting lysis and sporulation on T. rubrum NCPF118 hyphae. Growth of Trichod. virens NRRL 26672 in poor medium also resulted in secretion of antibiotics active in arresting the growth of T. rubrum NCPF118 inoculum. Our findings may open new directions for the treatment of onychomycosis, either in combination with known medications or as a new "natural" route.     

Histopathological studies of sclerotia of Rhizoctonia solani parasitized by the EGFP transformant of Trichoderma virens

Aims:  The aim of the study was to investigate the antagonistic interactions of Trichoderma species against Rhizoctonia solani sclerotia by enhanced green fluorescence protein (EGFP)-tagged transformant of Trichoderma virens TY009.
Methods and Results:  An EGFP was used as a report gene for transforming T. virens strain, and a stable EGFP transformant GF5 was obtained with the mycoparasitic activity against sclerotia of R. solani . Observation of parasitized sclerotia by fluorescence microscopy showed hyphae of transformant GF5 was able to invade into sclerotia and its colonization was mainly intercellar with uniformly distributed mycelium in sclerotia. The host cells were colonized, penetrated, and then the whole cells were replaced by transformant GF5 hyphae. Chlamydospores were seen after 10 days but mature ones after 20 days. Sclerotia became soft and decayed after 40 days but a few cells seemed not to be colonized completely.
Conclusions:  Trichoderma virens was able to parasitize sclerotia to make sclerotia soft and decayed, and its colonization was mainly intercellar in sclerotial tissues.
Significance and Impact of the Study:  This is first report of parasitism of sclerotia of R. solani by EGFP-tagged transformant, providing useful information for using T. virens as effective biocontrol agent.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

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.