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^δ-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.     

In vitro Antagonism of Phytophthora cinnamomi and P. citricola by Isolates of Trichoderma spp. and Gliocladium virens

Three fungi, isolated from soil from which Phytophthora was not obtained, were evaluated for antagonism of Phytophthora spp. shown to cause root rot of chestnut in South Australia. Trichoderma hamatum and T. pseudokoningii appeared to inhibit P. cinnamomi by mycoparasitism. with evidence of parallel growth and coiling, and both Trichoderma spp. and Gliocladium virens grew over P. cinnamomi in vitro, preventing further growth of this pathogen. Antibiotics produced by young T. hamatum cultures and G. virens in culture filtrate experiments inhibited growth of P. cinnamomi and P. citricola. with filtrate from 4-day-old cultures of G. virens showing the greatest potential for biocontrol. All three antagonists prevented P. cinnamomi and P. citricola from causing infection symptoms on micropropagated shoots of chestnut cvs Goldsworthy and Buffalo Queen in an in vitro excised shoot bioassay for biocontrol.     

Production of 6-kestose by the filamentous fungus Gliocladium virens as affected by sucrose concentration

The filamentous fungus Gliocladium virens is able to produce fructooligosaccharides (FOS), fructose-containing sugars, used as functional ingredients to improve nutritional and technological properties of foods. In this work we evaluated FOS production by G. virens when grown in a wide range of sucrose concentrations (10–400 g l^?1). High sucrose concentrations increased both biomass and FOS production, including 6-kestose, a trisaccharide comprising β (2 → 6) linked fructosyl units, with enhanced stability and prebiotic activity when compared to the typical FOS β (2 → 1) linked. The highest 6-kestose yield (3 g l^?1) was achieved in media containing 150 g l^?1 sucrose after 4–5 days of culture, production being 90% greater than in media containing 10, 30, or 50 g l^?1 sucrose. After 5 days, FOS production declined markedly, following complete sucrose depletion in the medium. Although most of the β-fructofuranosidases preferentially catalyze sucrose hydrolysis, FOS production in G. virens grown in high sucrose concentration, might be attributed to a reverse hydrolysis by these enzymes. In conclusion, high sucrose concentrations increase growth of G. virens whilst 6-kestose accumulation in the medium seems to be controlled both by specific properties of β-fructofuranosidases and on the sucrose concentration.     

HdaA, a class 2 histone deacetylase of Aspergillus fumigatus, affects germination and secondary metabolite production

Histone deacetylases (HDACs) play an important role in regulation of gene expression through histone modifications. Here we show that the Aspergillus fumigatus HDAC HdaA is involved in regulation of secondary metabolite production and is required for normal germination and vegetative growth. Deletion of the hdaA gene increased the production of several secondary metabolites but decreased production of gliotoxin whereas over-expression hdaA increased production of gliotoxin. RT-PCR analysis of 14 nonribosomal peptide synthases indicated HdaA regulation of up to nine of them. A mammalian cell toxicity assay indicated increased activity in the over-expression strain. Neither mutant affected virulence of the fungus as measured by macrophage engulfment of conidia or virulence in a neutropenic mouse model.     

Inoculum dynamics ofGliocladium virens associated with roots of cotton seedlings

A system was developed to evaluate the effects of root growth of cotton seedlings on the inoculum dynamics ofGliocladium virens in nonsterile soil. In soil infested withG. virens, inoculum densities of the fungus increased when plants remained alive. After 30 days, shoots were excised and the roots allowed to deteriorate. During this portion of the experiment (30–60 days) soil inoculum densities ofG. virens declined. In infested soil without a seedling, inoculum densities remained constant throughout the duration of the experiments. Colonization of roots byG. virens was found to increase throughout the duration of the experiments. At 60 daysG. virens was recovered from approximately 60% of the root pieces (1-cm) sampled. The percentage of primary, secondary, or tertiary roots colonized was different (P = 0.01), but the total colonization of roots at three depths (0–10, 10–20, and 20–30 cm) was not different (P = 0.64). In noninfested soil, colonization of roots by indigenous propagules ofG. virens was never greater than 3%. Offprint requests to: C. M. Kenerley.     

Tvbgn3, a β-1,6-Glucanase from the Biocontrol Fungus Trichoderma virens, Is Involved in Mycoparasitism and Control of Pythium ultimum

Even though β-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 β-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 β-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.     

Antibiotic production by strains of Gliocladium virens and its relation to the biocontrol of cotton seedling diseases

Strains of the fungal antagonist Gliocladium virens were separated into two distinct groups on the basis of secondary metabolite production in vitro. Strains of the ‘P’ group produced the antibiotics gliovirin and heptelidic acid but not the antibiotic gliotoxin and its companion, dimethylgliotoxin. Strains of the ‘Q’ group produced gliotoxin and dimethylgliotoxin but not gliovirin or heptelidic acid. Strains from both groups produced the antibiotic viridin and phytotoxin viridiol. Gliovirin was very inhibitory to Pythium ultimum but had no activity against Rhizoctonia solani, and strains that produce it were more effective seed treatment biocontrol agents of disease incited by P. ultimum. Conversely, gliotoxin was more active against R. solani than against P. ultimum, and strains that produced it were more effective seed treatments for controlling disease incited by R. solani. These results indicate that the antibiotic profiles of strains should be considered when screening strains for biocontrol efficacy, and that it may be necessary to treat seeds with a combination of strains in order to broaden the disease control spectrum.     

Production of Extracellular Proteins by the Biocontrol Fungus Gliocladium virens

Gliocladium virens is a common saprophytic fungus that is mycoparasitic on a large number of fungi. Responses of G. virens toward its environment were examined by monitoring the presence of extracellular proteins in culture fluid during time course experiments. Culture fluid of G. virens grown on glucose, washed cell walls of Rhizoctonia solani (one of its hosts), olive oil, or chitin contained β-glucanase, N-acetylglucosaminidase, lipase, and proteinase activities. There were relatively minor amounts of other enzymatic activities tested. Levels of extracellular enzyme activity varied with the age of the culture and the substrate used as the carbon source. Substrate-associated differences in enzyme activities were detected as early as 8 h after transfer of mycelia from stationary-phase cultures to fresh media. When G. virens was grown on host cell wall material, β-glucanase had the greatest specific activity of any enzyme tested at 8 h. This result suggests that β-glucanase may be the first enzyme important in the G. virens-R. solani interaction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that some of the polypeptides were present in the culture fluid at relatively constant amounts and others accumulated early, at intermediate times, or late in the 8-day incubation test period. Several of the polypeptides present in the culture fluid during the first 24 h disappeared completely by 48 h. Consequently, it appears that extracellular proteins in cultures of G. virens are regulated by a combination of gene regulation and protein degradation.     

Genetic Transformation of the Biocontrol Fungus Gliocladium virens to Benomyl Resistance

Methodology was developed to isolate and regenerate protoplasts from the biocontrol fungus Gliocladium virens and to transform them to benomyl resistance with a Neurospora crassa β-tubulin gene. Southern blots demonstrated that multiple copies of the vector integrated into the chromosomal DNA of stable biotypes but not of abortive transformants. Analysis of nuclear condition in vegetative and asexual structures demonstrated that no structure of G. virens is dependably uninucleate and thus preferentially suitable for transformation.     

Apparent endogenous circadian rhythm inent-kaurene biosynthesis

Net synthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid was assayed in cell-free enzyme extracts prepared from Alaska pea (Pisum sativum L.) seedlings throughout 44 h of a regimen consisting of a 16-h day and an 8-h night. Activities generally followed an upward trend during the dark period and a downward trend during the photoperiod. Activity was also assayed in enzyme extracts prepared at intervals during a 12-h photoperiod and a following, continuous 36-h dark period after entrainment of plants to a regimen of 12-h days and 12-h nights.Ent-kaurene synthesis activity again followed an upward trend in enzyme extracts prepared during what would have been the entrainment dark period, and a downward trend during the entrainment photoperiod. The apparent endogenous rhythm ofent-kaurene biosynthesis may have implications for the regulation of gibberellin biosynthesis.     

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-ß-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-ß-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-ß-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.     

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.     

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans downstream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.     

Cloning and sequencing of endochitinase genes from gliocladium virens and trichoderma species

Nucleotide sequences containing the genetic determinants of the endochitinase of Gliocladiurn virences as well as those of Trichoderma harzianum, T. hamatum, T. viride and T. pseudokoningii were determined. The ORFs of 1287 or 1293 nucleotides were interrupted by three introns. The deduced amino acid sequences of endochitinase of G. virens were highly homologous to those of Trichoderma species (82–97% identity), although the culture filtrates from G. virens showed a much stronger inhibitory effect on growth of Helicobasidium mompa than those from Trichoderma species. Phylogenic analysis showed that these sequences could be divided into three clusters regardless of genus or species.     

Lethal effects of Gliocladium virens,Beauveria bassiana and Metarhizium anisopliae on the malaria vector Anopheles albimanus (Diptera: Culicidae)

Control of Anopheles albimanus, the main vector of malaria on the coast of the State of Chiapas, is based mainly on application of chemical insecticides, which has resulted in resistance to most registered insecticides. Strategies for biological control may provide sustainable alternatives. We report on the lethal effects of a native isolate of Gliocladium virens on An. albimanus larvae and adults, compared to that of strains of Beauveria bassiana and Metarhizium anisopliae. Conidial suspensions of G. virens, B. bassiana and M. anisopliae cultured on Sabouraud agar were tested in bioassays with An. albimanus larvae and adults. Mosquito larvae were more susceptible to all fungi, compared to adults. On early and late instar larvae, M. anisopliae showed the most pathogenic effect (LC₅₀ of 1.4×10⁵ conidia/mL in early instars; 1.1×10⁵ conidia/mL in late instars), followed by G. virens (LC₅₀ of 3.3×10⁵ conidia/mL in early instars and 3.5×10⁶ conidia/mL in late instars). Metarhizium anisopliae sensu lato and the native G. virens could be considered good choices for An. albimanus control in southern Mexico.     

Towards understanding the biosynthetic pathway for ustilaginoidin mycotoxins in Ustilaginoidea virens

Ustilaginoidins, toxic to plants, animals and human, are one of major types of mycotoxins produced by Ustilaginoidea virens. In this study, a gene cluster containing the polyketide synthase gene UvPKS1 was analysed via gene replacement and biochemical studies to determine ustilaginoidin biosynthetic pathway in U. virens. UvPKS1 was first proven to be responsible for the first step of ustilaginoidin biosynthesis, since neither ustilaginoidin derivatives nor intermediates were produced when UvPKS1 was deleted. Replacement of ugsO greatly reduced ustilaginoidin production but increased the ratios of dehydrogenated/hydrogenated ustilagioidin derivatives. The enhanced growth rate of the ΔugsO mutant indicates that accumulation of certain ustilaginoidin derivatives may adversely affect mycelial growth in U. virens. Deletion of ugsT encoding a putative MFS transporter disrupted the ability to generate ustilaginoidins. The ustilaginoidin derivatives produced in the ΔugsJ mutant all lack C3-methyl, indicating that UgsJ is responsible for C3-methylation. Only monomeric intermediates, such as 3-methyl-dihydro-nor-rubrofusarin, but no ustilaginoidin derivatives were generated in the ΔugsL mutant, indicating that UgsL is responsible for the dimerization of nor-rubrofusarin derivatives to produce ustilaginoidins. However, ugsR2 deletion had no dramatic effect on ustilaginoidin biosynthesis. Together, biochemical analyses with bioinformatics and chemoinformatics uncover a multiple-step enzyme-catalysed pathway for ustilaginoidin biosynthesis in U. virens.     

A quantitative bioassay for extracellular metabolites that antagonize growth of filamentous fungi,and its use with biocontrol fungi

A bioassay and an empirically derived formula were developed to quantify fungitoxic effects. This bioassay can be easily performed and objectively read, and it is suitable for low-volume samples originating from aqueous or organic solvents. The formula defines the Inhibition Index (I), a single value that incorporates both the response to concentrations of the inhibitory compound and the persistence of inhibition. Antagonistic efficacy of metabolites produced by biocontrol strains of Trichoderma spp. were measured based on inhibition of growth of Rhizoctonia solani. Although the bioassay itself was not influenced by pH or light conditions, these factors affected metabolite production or activity. Aqueous extracts from light-grown germlings of Trichoderma virens inhibited R. solani more than extracts from germlings grown in the dark. Low pH increased the inhibitory activity of extracts from T. virens. Tests of fungal strains developed for biocontrol demonstrated that the bioassay reflected their activity both in the field and in other in vitro tests. The bioassay and formula are readily adapted for use with other fungi.This revised version was published online in October 2005 with corrections to the Cover Date.     

Regulation of Morphogenesis and Biocontrol Properties in Trichoderma virens by a VELVET Protein,Vel1

Mycoparasitic strains of Trichoderma are applied as commercial biofungicides for control of soilborne plant pathogens. Although the majority of commercial biofungicides are Trichoderma based, chemical pesticides, which are ecological and environmental hazards, still dominate the market. This is because biofungicides are not as effective or consistent as chemical fungicides. Efforts to improve these products have been limited by a lack of understanding of the genetic regulation of biocontrol activities. In this study, using gene knockout and complementation, we identified the VELVET protein Vel1 as a key regulator of biocontrol, as well as morphogenetic traits, in Trichoderma virens, a commercial biocontrol agent. Mutants with mutations in vel1 were defective in secondary metabolism (antibiosis), mycoparasitism, and biocontrol efficacy. In nutrient-rich media they also lacked two types of spores important for survival and development of formulation products: conidia (on agar) and chlamydospores (in liquid shake cultures). These findings provide an opportunity for genetic enhancement of biocontrol and industrial strains of Trichoderma, since Vel1 is very highly conserved across three Trichoderma species.Trichoderma-based formulation products account for about 60% of the biofungicide market (35). Despite the use of Trichoderma-based biofungicides as an alternative and additive to chemical fungicides, the applications of these preparations are limited because their efficacy is lower than that of fungicides. A lack of understanding of the regulation of biocontrol has limited progress in enhancing the competitiveness of these fungi through genetic manipulation of desired traits. The success of a biocontrol agent also depends on the ability of researchers to develop an effective formulation based on active propagules that survive under the conditions that occur in nature and are effective against the target pathogens. Trichoderma spp. produce two types of propagules, conidia during solid-state fermentation and chlamydospores during liquid fermentation. Both types are used in commercial formulations depending on the growth conditions (17, 35). Thus, understanding how the two sporulation pathways are controlled is critical for obtaining an improved, balanced formulation product. Identification of a global regulator of morphogenesis and biocontrol properties (such as antibiosis and mycoparasitism) would provide an opportunity to manipulate the morphogenetic and antagonistic traits, leading to wider commercial acceptance of Trichoderma spp. in the long run.Trichoderma virens is a commercially formulated biocontrol agent that is effective against soilborne plant pathogens, such as Rhizoctonia solani, Sclerotium rolfsii, and Pythium spp.; its major direct mode of action is antibiosis and mycoparasitism (20, 36). This species has also been used as a model system for studies of biocontrol mechanisms, and the genome has recently been sequenced (http://genome.jgi-psf.org/Trive1). The role of beta-glucanases, chitinases, and proteases in biocontrol has been reported previously (2, 8, 29). Some strains of T. virens (designated Q strains) produce copious amounts of the antibiotic gliotoxin that is involved in biocontrol (10, 12, 39). In an attempt to identify regulators of biocontrol properties, the role of a mitogen-activated protein kinase (MAPK) pathway was studied previously (22, 24). Deletion of the TmkA/Tvk1 MAPK gene resulted in derepressed conidiation and different biocontrol behavior for two strains of T. virens; Mukherjee et al. (24) noted the reduced ability of these mutants to parasitize the sclerotia of S. rolfsii and R. solani, while Mendoza-Mendoza et al. (22) found that deletion of this MAPK gene improved the biocontrol activity of T. virens against R solani and P. ultimum. The production of secondary metabolites was not affected by deletion of this gene. To date, no gene that regulates the balance between conidiation or chlamydospore formation, secondary metabolism, and antagonistic or biocontrol properties has been identified in any Trichoderma sp.The Vel1 VELVET protein has been shown to be a regulator of morphogenesis and secondary metabolism in some filamentous fungi (6). In Aspergillus nidulans, VeA physically interacts with VelB and the regulator of secondary metabolism LaeA to form a complex that regulates secondary metabolism and sexual reproduction (3). Deletion of the VeA gene leads to an increase in asexual development (conidiation in the dark) and reduced biosynthesis of sterigmatocystin (the product of a polyketide synthetase [PKS]) and penicillin (the product of a nonribosomal peptide synthetase [NRPS]), while it reduces and delays sexual reproduction (15, 16). VeA is also required for the production of sclerotia and for aflatoxin biosynthesis in Aspergillus parasiticus (7). Deletion of the VeA gene in Neurospora crassa, like deletion of the VeA gene in A. nidulans, results in deregulated conidiation, while in Acremonium chrysogenum, loss of VeA leads to increased hyphal fragmentation and reduced cephalosporin production (4, 9). Deletion of the VeA gene in Fusarium verticilliodes resulted in a loss of hydrophobicity and an increased macroconidium-to-microconidium ratio; these defects could be restored by growing the organism on osmotically stabilized media (18). The mutants were also defective in production of the mycotoxins fumonisin and fusarin (25).To test the hypothesis that Vel1 is a global regulator of gene expression in T. virens, we examined the functions of Vel1 in this organism by using gene knockout and complementation. Here we report that in addition to a role in conidiation and secondary metabolism, Vel1 also regulates conidiophore aggregation, chlamydosporogenesis, mycoparasitism, and biocontrol efficacy in T. virens. Thus, we identified the first master regulator of morphogenesis and antagonistic properties in this economically important fungus.     

Comparative Antagonistic Properties of Gliocladium virens and Trichoderma harzianum on Sclerotium rolfsii and Rhizoctonia solani—its Relevance to Understanding the Mechanisms of Biocontrol

An isolate of Trichoderma harzianum which is less effective than G. virens in suppressing S. rolfsii and R. solani was compared with G. virens for various mechanisms of antagonism in vitro, viz., antagonism in dual culture/hyphal parasitism, parasitism of sclerotia and antibiosis. G. virens and T. harzianum were equally effective in parasitizing the hyphae of R. solani. Only T. harzianum parasitized the hyphae of S. rolfsii, and the two antagonists were comparable with respect to antibiosis on the test pathogens. However, G. virens readily parasitized the sclerotia of the test pathogens and was found to be more effective than T. harzianum in destroying the sclerotia. Under SEM, G. virens was found to colonize, penetrate, and sporulate inside the sclerotia of the test pathogens.Parasitism of sclerotia is suggested as the principal mechanism of biological control of S. rolfsii and R. solani by G. virens.