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.     

Cloning and characterization of bgn16.3, coding for a beta-1,6-glucanase expressed during Trichoderma harzianum mycoparasitism

AIMS: To clone and characterize the gene coding for BGN16.3, a beta-1,6-glucanase putatively implicated in mycoparasitism by Trichoderma harzianum, a biocontrol agent used against plant pathogenic fungi. METHODS AND RESULTS: Using degenerate primed PCR and cDNA library screening, we have cloned the cDNA coding BGN16.3. bgn16.3 showed a significant sequence identity (50%) to bgn16.1; however, they both have low identity to the previously cloned bgn16.2, allowing the identification of amino acid sequences putatively involved in the common catalytic activity of the three proteins. bgn16.3 is a single-copy gene and highly homologous sequences are present in all tested Trichoderma species. bgn16.3 expression pattern is analysed by Northern blot, finding that it is expressed during the interaction of T. harzianum CECT 2413 with Botrytis cinerea, supporting the implication of the enzyme in the mycoparasitic process. CONCLUSIONS: The cloned bgn16.3 completes the knowledge on the beta-1,6-glucanase isozyme system from T. harzianum CECT 2413. A highly homologous gene is present in all analysed Trichoderma strains. bgn16.3 is expressed under few specific conditions, including the mycoparasitic process. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to the knowledge of beta-1,6-glucanases. It implicates this group of enzymes in the mycoparasitism by some biocontrol agents such as T. harzianum.     

BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

A new component of the beta-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.     

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.     

A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum.

The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.     

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.     

An antifungal exo-alpha-1,3-glucanase (AGN13.1) from the biocontrol fungus Trichoderma harzianum.

Trichoderma harzianum secretes alpha-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type alpha-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.     

Purification and properties of a beta-1,6-glucanase from Penicillium brefeldianum.

An inducible endo-beta-1,6-glucanase was purified from Penicillium brefeldianum by DEAE-cellulose, Bio-Gel P-150 and high-pressure liquid chromatography. The final preparation was essentially free from beta-1,3-glucanase and beta-glucosidase activities. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed one protein band with an Mr of 44000. The Vmax. and Km values were calculated to be 624 units (mumol/min)/mg and 2.78 mg/ml respectively. The glucanase had lytic activity against mycelial cells of the yeast Candida albicans. The yield of purified beta-1,6-glucanase from 100 mg dry weight of freeze-dried culture filtrate varied from 60 to 180 units.     

Characterization of a 29-kDa beta-1,3-glucanase from Trichoderma harzianum

A beta-1,3-glucanase, from culture filtrates of Trichoderma harzianum, was purified in sequential steps by gel filtration, hydrophobic interaction and ion exchange chromatography. A typical procedure provided 69-fold purification with 0.32% yield. The molecular mass of the protein was found to be approximately 29 kDa, as estimated by SDS-PAGE on a 10% slab gel. The K(M) and V(max) values for beta-1,3-glucanase, using laminarin as substrate, were 1. 72 mg ml(-1) and 3.10 U ml(-1), respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 50 degrees C. The enzyme was strongly inhibited by HgCl(2) and SDS. These results suggest that each beta-1,3-glucanase produced by T. harzianum is different and is probably encoded by different genes.     

Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities

Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.     

Involvement of G-alpha protein GNA3 in production of cell wall-degrading enzymes by Trichoderma reesei (Hypocrea jecorina) during mycoparasitism against Pythium ultimum

The involvement of the G-alpha protein GNA3 in the production of cell wall-degrading enzymes (CWDEs) by Trichoderma reesei during antagonism against Pythium ultimum was investigated. cAMP content was 2.8-fold higher in the T. reesei mutant gna3QL than in the parental TU-6. The gna3QL, like TU-6, inhibited the growth of P. ultimum in dual culture assays. Scanning electron microscopy showed that the gna3QL promoted more morphological alterations of P. ultimum cell wall than TU-6. In general, gna3QL produced higher activities of CWDEs than TU-6. We therefore suggest that CWDEs production during mycoparasitism by T. reesei against P. ultimum may be associated with the level of GNA3 activity.     

Detection of beta-1,6-glucanase isozymes from Trichoderma strains in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and isoelectrofocusing gels.

The filamentous fungus Trichoderma produces, under specific growth conditions, several extracellular fungal cell wall degrading enzymes, amongst them beta-1,6-glucanases. These enzymes seem to play an important role in the antagonistic action of Trichoderma against a wide range of fungal plant pathogens. In this report we describe two different methods for the specific detection of the activity of beta-1,6-glucanase isozymes in gels. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, beta-1,6-glucanase activity can be assayed in the gel by renaturation of the enzyme, incubation with an overlay agarose gel containing solubilized pustulan (a commercially available beta-1,6-glucan), followed by the staining of the agarose gel with Congo Red. In native isoelectrofocusing gels, as little as 1 mU can be detected after incubation with solubilized pustulan followed by a detection reaction of the released reducing sugars with 2,3,5-triphenyltetrazolium chloride. The latter technique has been successfully applied to the screening of beta-1,6-glucanase isozymes from different Trichoderma strains under different growth conditions.     

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.     

The gene task1 is involved in morphological development,mycoparasitism and antibiosis of Trichoderma asperellum

Competitive activity, mycoparasitism and antibiosis of Trichoderma asperellum are considered essential mechanisms in its suppressive activity against soil-borne plant pathogens. The role of the mitogen-activated protein kinase encoding gene task1 on morphological development, mycoparasitic interaction and the production of cell wall degrading enzymes and secondary metabolites were examined in T. asperellum. The Δtask1 mutant had altered growth morphology, lost its ability to parasitise plant pathogens and showed increased expression of several cell wall degrading enzymes during confrontation with Rhizoctonia solani. T. asperellum task1 expression was negatively correlated with cell wall degrading enzyme activities during inducing experiments using pathogen cell wall compounds. In antibiosis assays, task1 deletion caused increased output of 6-pentyl-α-pyrone and inhibition of pathogen growth.     

Interactions of Gliocladium virens with Rhizoctonia solani and Pythium ultimum in Non-sterile Potting Medium

Interactions of Gliocladium virens with Pythium ultimum and Rhizoctonia solani under simulated in vivo conditions were observed microscopically. Different types of propagules of the three fungi were paired on nitrocellulose membranes and incubated at 25°C in non-sterile potting medium in Petri dishes for 1-5 days. Alginate-wheat bran prill were used as carriers for G. virens. Prill inoculated with G. virens and pre-incubated in potting medium for 3-5 days before placement on membranes did not inhibit the germination of Pythium sporangia, but subsequent Pythium growth was markedly stunted and distorted, with some hyphal collapse and cytoplasmic leakage. G. virens had no visible effect on older Pythium mycelium. Two to 5 days' growth of G. virens caused cytoplasmic leakage of Rhizoctonia mycelium, prevented secondary branching of hyphae and occasionally coiled around Rhizoctonia hyphae. Prill that were newly colonized by G. virens, but not prill pre-incubated for 3 or 5 days, stimulated the growth of Pythium mycelium and sporangia, Rhizoctonia mycelium and unprimed monilioid cells, probably by supplying nutrients. The timing of the interactions and their specificity for the different pathogen propagules were consistent with the production of gliotoxin by G. virens. This view was supported by in vitro experiments, in which pathogen propagules were incubated in a range of concentrations of gliotoxin in potato dextrose broth. Pythium sporangia and mycelium were inhibited by 1 or 2 μmg ml^-1, but Rhizoctonia monilioid cells and mycelium required 3-5 μmg ml^-1 for inhibition. At the lowest effective concentrations the inhibition was sometimes reversible, but propagules were killed at high concentrations of gliotoxin.     

Biocontrol of Damping-off Diseases Caused by Rhizoctonia solani and Pythium ultimum with Alginate Prills of Gliocladium virens, Trichoderma hamatum and Various Food Bases

Alginate prills were formulated with the biomass of isolates of Gliocladium virens and Trichoderma spp. and various food bases (wheat bran, corn cobs, peanut hulls, soy fiber, castor pomace, cocoa hulls and chitin). Alginate prills with G. virens (Gl-21) biomass and all food bases except cocoa hull meal significantly reduced the damping-off of zinnia in a soil-less mix caused by Rhizoctonia solani and Pythium ultimum. The prills with bran, soy fiber, castor pomace or chitin resulted in stands similar to those in the non-infested control. In soil, prills with all the food bases and Thrichoderma hamatum (TRI-4) biomass controlled the damping-off of cotton caused by R. solani and gave stands comparable to, or better than, those in the non-infested control soil. Prills with all the food bases resulted in a proliferation of Gl-21 in a soil-less mix and of Gl-21 and TRI-4 in soil. Prills with food bases and TRI-4 biomass reduced the survival of R. solani in infested beet seed to less than 30%, with bran and chitin being the most effective food bases; prills with Gl-21 biomass and all food bases also reduced the survival of R. solani in beet seed, but not as much as did prills with TRI-4 biomass. In prills containing wheat bran, soy fiber or chitin, the biocontrol isolate Th-58 (T. harzianum) was almost as effective as TRI-4, but isolate Gl-3 (G. virens) was less effective. There was no significant interaction between the biocontrol fungus and the food base. The results suggest that the intrinsic properties of a selected fungus isolate are more important than some formulation variables in biocontrol.     

Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.     

Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to pathogenesis or biocontrol processes. A gene (tvsp1) encoding an extracellular serine protease was cloned from Trichoderma virens, a biocontrol agent effective against soilborne fungal pathogens. The gene was expressed in Escherichia coli and a polyclonal antibody was raised against the recombinant protein. The expression pattern of tvsp1 was determined and its physiological role was addressed by mutational analysis. Strains of T. virens in which tvsp1 was deleted (PKO) or constitutively overexpressed (POE) were not affected in growth rate, conidiation, extracellular protein accumulation, antibiotic profiles nor in their ability to induce phytoalexins in cotton seedlings. Tvsp1 overexpression, however, significantly increased the ability of some strains to protect cotton seedlings against Rhizoctonia solani. Our data show that Tvsp1 is not necessary for the normal growth or development of T. virens, but plays a role in the biocontrol process.