Trichoderma harzianum metabolites pre-adapt mushrooms to Trichoderma aggressivum antagonism

Trichoderma spp. is the cause of green mold, a disorder that affects cultivated mushrooms. The aims of the study were to establish whether improvement of mushroom resistance to Trichoderma aggressivum could be obtained by inducing reaction mechanisms before contact with the pathogen and whether this ability was species or strain dependent. Twenty nine isolates of Agaricus bisporus, 29 isolates of Lentinula edodes and 18 isolates of Pleurotus spp. were studied. The effect of T. harzianum metabolites on mycelial growth of these isolates was evaluated on YMEA (yeast, malt extract and agar), supplemented or not with Lysing Enzymes from T. harzianum (Sigma?, L1412). Mycelial growth generally was affected by Lysing Enzymes, but some L. edodes and Pleurotus spp. adapted to Lysing Enzymes. When mycelium was taken from a first culture with Lysing Enzymes and placed on YMEA with Lysing Enzymes for a second culture, their growth rate was not different from those of the controls. In the case of A. bisporus, only partial adaptation was obtained with a few isolates. The effect of adaptation to Lysing Enzymes on resistance to T. aggressivum was assayed for one strain of each group. Trichoderma aggressivum was exposed to the margin of 5- to 9-day-old mushroom colonies. Agaricus bisporus produced brown droplets, and T. aggressivum overgrew its mycelium. Lentinula edodes and P. ostreatus produced brown lines blocking the progression of T. harzianum, both on YMEA and YMEA plus Lysing Enzymes. The line was visible after 3 d on YMEA and after only 2 d on YMEA plus Lysing Enzymes. Improvement in the resistance to antagonists by introduction of some of their metabolites to the culture medium is a method for mushroom protection.     

Resource allocation ability of wild isolates of Agaricus bisporus on conventional mushroom compost

Abstract: Twenty-one wild isolates from two distinct sites and six cultivated strains of Agaricus bisporus were cultivated on a conventional mushroom compost. Their degradative abilities were studied by measuring 12 extracellular enzyme activities produced during mycelial growth. Differences in production of enzyme activities and in compost colonisation were observed between the three groups of strains and within each group. They were used to define the mechanisms of resource allocation in mushroom compost. The ability to grow and produce sporophores on mushroom compost appeared to be linked with the production of a balanced pool of enzymes including moderate levels of polysaccharidases active on straw cell walls and of enzymes able to degrade microbial biomass and microbial products.     

PCR-based genotyping of epidemic and preepidemic Trichoderma isolates associated with green mold of Agaricus bisporus.

We used randomly amplified polymorphic DNA (RAPD)-PCR to estimate genetic variation among isolates of Trichoderma associated with green mold on the cultivated mushroom Agaricus bisporus. Of 83 isolates examined, 66 were sampled during the recent green mold epidemic, while the remaining 17 isolates were collected just prior to the epidemic and date back to the 1950s. Trichoderma harzianum biotype 4 was identified by RAPD analysis as the cause of almost 90% of the epidemic-related episodes of green mold occurring in the major commercial mushroom-growing region in North America. Biotype 4 was more closely allied to T. harzianum biotype 2, the predominant pathogenic genotype in Europe, than to the less pathogenic biotype 1 and Trichoderma atroviride (formerly T. harzianum biotype 3). No variation in the RAPD patterns was observed among the isolates within biotype 2 or 4, suggesting that the two pathogenic biotypes were populations containing single clones. Considerable genetic variation, however, was noted among isolates of biotype 1 and T. atroviride from Europe. Biotype 4 was not represented by the preepidemic isolates of Trichoderma as determined by RAPD markers and PCR amplification of an arbitrary DNA sequence unique to the genomes of biotypes 2 and 4. Our findings suggest that the onset of the green mold epidemic in North America resulted from the recent introduction of a highly virulent genotype of the pathogen into cultivated mushrooms.     

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.     

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.     

Composting of a crop residue through treatment with microorganisms and subsequent vermicomposting

Preliminary studies were conducted on wheat straw to test the technical viability of an integrated system of composting, with bioinoculants and subsequent vermicomposting, to overcome the problem of lignocellulosic waste degradation, especially during the winter season. Wheat straw was pre-decomposed for 40 days by inoculating it with Pleurotus sajor-caju, Trichoderma harzianum, Aspergillus niger and Azotobacter chroococcum in different combinations. This was followed by vermicomposting for 30 days. Chemical analysis of the samples showed a significant decrease in cellulose, hemicellulose and lignin contents during pre-decomposition and vermicomposting. The N, P, K content increased significantly during pre-decomposition with bioinoculants. The best quality compost, based on chemical analysis, was prepared where the substrate was treated with all the four bioinoculants together followed by vermicomposting. Results indicated that the combination of both the systems reduced the overall time required for composting and accelerated the composting of ligno-cellulosic waste during the winter season besides producing a nutrient-enriched compost product.     

Einfluß der Gerbsäure in Kombination mit Strohmehl auf die Verdaulichkeit der Rohnährstoffe und Aminosäuren bei Legehennen der Mastrichtung

Four fungi varieties (Wild variety, Chaetomium cellulolyticum, Trichoderma harzianum and Penicillium colony 10) were cultivated in a solid‐state fermentation on a wheat straw‐wheat bran‐sugar beet pulp mixture (20 : 40: 40%).

The fermented substrates contained xylanase (22.1 to 78.6 Units g^‐1 DM) and cellulase (0.47 to 14.0 Units g^‐1 DM). Fermented substrate was homogeneously mixed with ground wheat straw (1: 2; 1: 5 and 1 : 10), moistened to 30 or 50% DM, airtightly stored for 24 h at 50°C and deep frozen. All samples were given in nylon bags and incubated for 48 h in the rumen of 4 sheep.

In sacco DMD decreased with higher straw proportions in the mixture (1: 2; 1 : 5 and 1: 10: 52.0; 46.8 and 44.2% resp.), the DM‐content did not significantly influence the in sacco DMD. The Wild variety and Chaetomium cellulolyticum did not significantly influence the in sacco DMD, Trichoderma harzianum and Penicillium colony 10 decreased DMD in some cases.

In general added fungi did not improve the rumen degradability of fibre.     

Scanning electron microscopy observations of the interaction between Trichoderma harzianum and perithecia of Gibberella zeae

Chronological events associated with the interaction between a strain of Trichoderma harzianum, T472, with known biological control activity against perithecial production of G. zeae, were studied with scanning electron microscopy to investigate the mechanisms of control. Large clusters of perithecia consisting of 5-15 perithecia formed on the autoclaved, mulched wheat straw inoculated with G. zeae alone (control) with an average of 157 perithecia per plate. Small clusters consisting of 3-6 and an average of 15 perithecia per plate perithecia formed on straw that was treated with T. harzianum. The mature perithecia from straw treated with T. harzianum produced less pigment and were lighter in color than those from the control plates. Furthermore the cells of the outer wall of these perithecia were abnormal in appearance and unevenly distributed across the surface. Immature perithecia were colonized by T. harzianum approximately 15 d after inoculation (dai) with the biocontrol agent and pathogen. Few perithecia were colonized at later stages. The affected perithecia collapsed 21 dai, compared to the perithecia in the control samples that began to collapse 28 dai. Abundant mycelium of T. harzianum was seen on the perithecia of treated samples. Perithecial structures may be resistant to penetration by the mycelium because direct penetration was not observed. Trichoderma harzianum colonized the substrate quickly and out-competed the pathogen, G. zeae.     

Purification of a chitinase from Trichoderma sp. and its action on Sclerotium rolfsii and Rhizoctonia solani cell walls

Trichoderma harzianum is an effective biocontrol agent of several important plant pathogenic fungi. This Trichoderma species attacks other fungi by secreting lytic enzymes, including beta-1,3-glucanase and chitinolytic enzymes. Superior biocontrol potential may then be found in strains having a high capacity to produce these enzymes. We have therefore evaluated the capacity of six unidentified Trichoderma spp. isolates to produce chitinolytic enzymes and beta-1,3-glucanases in comparison with T. harzianum 39.1. All six isolates demonstrated substantial enzyme activity. However, while the isolates hereafter called T2, T3, T5, and T7 produced lower amounts of enzymes, the activity of isolates T4 and T6 were 2-3 fold higher than that produced by T. harzianum 39.1. A chitinase produced by the T6 isolate was purified by a single ion-exchange chromatography step and had a molecular mass of 46 kDa. The N-terminal amino-acid sequence showed very high homology with other fungal chitinases. Its true chitinase activity was demonstrated by its action on chitin and the failure to hydrolyze laminarin and p-nitrophenyl-beta-N-acetylglucosaminide. The hydrolytic action of the purified chitinase on the cell wall of Sclerotium rolfsii was convincingly shown by electron microscopy studies. However, the purified enzyme had no effect on the cell wall of Rhizoctonia solani.     

The expression of extracellular fungal cell wall hydrolytic enzymes in different Trichoderma harzianum isolates correlates with their ability to control Pyrenochaeta lycopersici

Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16) were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of beta-1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici.     

Colonisation of phase II compost by biotypes of Trichoderma harzianum and their effect on mushroom yield and quality

Colonisation assessments confirmed that Trichoderma harzianum biotypes Th1, Th2a, Th2b and Th3 inoculated into two distinct compost types at spawning became established by first flush assessment; the extension rate of two Th2 isolates was over 1000 times that of Th1 and Th3. Results subsequently confirmed that while Th1 and Th3 did not significantly affect yield, Th2 could reduce mushroom quality and productivity by as much as 80%. Analysis of compost type also indicated that the speed and magnitude of T. harzianum colonisation was influenced by key compost characteristics, most notably, moisture, ash content and degree of fermentation. This study has shown that compost parameters which have a positive influence on Agaricus growth and productivity also resulted in increased compost colonisation by T. harzianum. Commercially acceptable yields obtained from uninoculated compost confirmed that production of a high quality, productive substrate does not confer inherent immunity to colonisation by T. harzianum. Received: 25 September 1998 / Received revision: 30 November 1998 / Accepted: 5 December 1998     

The antagonistic action of Trichoderma sp. hyphae to Lentinula edodes hyphae changes lignocellulotytic activities during cultivation in wheat straw

Lentinula edodes (Berk.) Pegler was cultivated in sterilized or pasteurized wheat straw both with and without inoculation with Trichoderma sp. Enhancements of -mannosidase and laccase activities and lowering of Mn-dependent peroxidase activity were observed seven days after inoculation in substrates inoculated with Trichoderma sp. These enzymes were not produced by Trichoderma sp. Most of the polysaccharidase activities were higher in substrates with Trichoderma sp. than in absence of Trichoderma sp. The area of the substrate contaminated with T. harzianum significantly correlated with cellulase, laccase and Mn-dependent peroxidase activities measured in the substrate. The increase of cellulase activity was due to enzymes produced by Trichoderma sp. and the decrease of Mn-dependent peroxidase activity was due to diminished growth of L. edodes. The stimulation of laccase activity was linked with the formation of brown lines (oxidation of polyphenols) at the contact between the mycelia of the two antagonists.     

Effect of fungal consortium and animal manure amendments on phosphorus fractions of paddy-straw compost

The study was conducted to reveal the type of phosphorus (P) fractions present in mature compost prepared by co-composting paddy straw (P.S) with cattle manure (CM), farm yard manure (FYM) and poultry manure (PM), each added separately as nitrogen (N) and P source. A consortium of phytate mineralizing fungi developed by including Aspergillus niger ITCC 6719, Aspergillus flavus ITCC 6720 and Trichoderma harzianum ITCC 6721 was applied for recovery of P from plant and animal residues. Chemical evaluation of compost after 4 months of aerobic decomposition revealed that inoculation improved the sodium bicarbonate-extractable P content of CM and FYM supplemented P.S compost by 32.3% and 23.5% respectively compared with their respective un-inoculated control. However, the peak values for water soluble-P fractions were recorded in CM–straw compost followed by PM–straw compost. Fungal inoculation also improved the agronomic quality of PM–straw compost as the latter had the highest total P content and lowest C:N and E4/E6 ratio of 18:1 and 5.36:1 respectively. The recovery of organic P from agricultural residue has the potential to reduce the application of synthetic P fertilizer. P-enriched organic manure can offer potential environment and economic benefits to farmers under sustainable agriculture.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

A selective medium for quantitative reisolation of Trichoderma harzianum from Agaricus bisporus compost

We adapted a selective medium, previously developed for reisolation of Trichoderma spp. from soil, for quantitative determination of growth of T. harzianum from commercial Agaricus bisporus composts. This medium enables comparisons of aggressive (sensu inhibition of A. bisporus yield) with nonaggressive T. harzianum groups. The resulting medium contains the antimicrobials chloramphenicol, streptomycin, quintozene, and propamocarb and was highly selective, allowing the recovery of T. harzianum, as viable conidia and hyphal fragments, in compact colonies with the absence of visible microbial contaminants.     

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures

Aims:  To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures.
Methods and Results:  Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus–Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus–Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms.
Conclusions:  The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma -mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes.
Significance and Impact of the Study:  Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.     

Biological control of phytopathogenic fungi through lytic action of Trichoderma species

Abstract The antagonistic effect of Trichoderma reesei and Trichoderma harzianum was studied towards a range of phytopathogenic fungi; Alternia solani, Botrytis fabae, Cladosporium cucumerinum, Fusarium oxysporum, Fusarium tricintum . Coculture of the phytopathogens and Trichodema under laboratory conditions clearly showed dominance of the Trichoderma species. In all cases, Trichoderma overgrew the phytopathogens and subsequently developed a conidial lawn over the surface. In these studies the lytic action of the pathogen was clearly apparent and the inhibition of growth appears directly related to its ability to hydrolyze the cell walls of the tested microorganisms rather than through the inhibitory action of antibiotics or toxins. Proteinase, mannanase, laminarinase and chitinase activities were determined in the extracellular fluid of Trichoderma . Glucose and laminaribiose were detected after the in vitro hydrolysis of the cell walls of the phytopathogens. The data imply that the nature of T. reesei and T. harzianum antagonism is based on mycoparasitism (lysis) and appears to optimalize with contact between the mycelia.     

Production and detection of muramidase and acetylglucosaminidase from Agaricus bisporus

The production and regulation of extracellular bacteriolytic enzymes of Agaricus bisporus are being studied to understand better the nutrition of this fungus and to identify factors that regulate the selectivity of mushroom compost as a growth medium. Both muramidase (EC.3.2.1.17) and N -acetyl-β- D -glucosaminidase (β-GlcNAcase, EC.3.2.1.30) have been detected in liquid cultures of A. bisporus , and in cultures fruiting in sterile and non-sterile compost. A turbidometric assay, based on the decrease in optical density of suspended Bacillus subtilis bacterial cell walls, was used to measure muramidase production by A. bisporus . A colorimetric assay was used to measure β-GlcNAcase. Both bacteriolytic enzyme activities were produced on a range of sole carbon sources, including killed freeze-dried B. subtilis cells. Muramidase activity was highest in axenic compost cultures. Bacteriolytic enzyme activity peaked as the first group of fruit bodies was harvested in both sterile and non-sterile compost.     

Investigating the role of a Verticillium fungicola beta-1,6-glucanase during infection of Agaricus bisporus using targeted gene disruption

Studies on the mycopathogen Verticillium fungicola have shown the up-regulation of beta-1,6-glucanases when grown in the presence of host cell walls and host cell wall components including chitin. These cell-wall-degrading enzymes are hypothesized to contribute to the pathogenic ability of mycopathogens. A beta-1,6-glucanase gene, VfGlu1, showing high similarity to beta-1,6-glucanase genes from Hypocrea virens, Neotyphodium sp., and Trichoderma harzianum, was isolated using degenerate PCR from V. fungicola, a serious mycopathogen of the cultivated mushroom Agaricus bisporus. Agrobacterium-mediated transformation of V. fungicola using homologous DNA from VfGlu1 resulted in homologous integration at the VfGlu1 locus in 75% of transformants, generating mutants disrupted in the VfGlu1 gene. VfGlu1 mutants displayed reduced virulence and diminished ability to utilize chitin as a carbon source, implicating VfGlu1 in the disease process. Agrobacterium-mediated transformation affords an efficient technique for the disruption of genes associated with disease symptom development in the complex V. fungicola-A. bisporus interaction.