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.     

Trichoderma harzianum might impact phosphorus transport by arbuscular mycorrhizal fungi

Trichoderma sp. is a biocontrol agent active against plant pathogens via mechanisms such as mycoparasitism. Recently, it was demonstrated that Trichoderma harzianum was able to parasitize the mycelium of an arbuscular mycorrhizal (AM) fungus, thus affecting its viability. Here, we question whether this mycoparasitism may reduce the capacity of Glomus sp. to transport phosphorus ((33)P) to its host plant in an in vitro culture system. (33)P was measured in the plant and in the fungal mycelium in the presence/absence of T. harzianum. The viability and metabolic activity of the extraradical mycelium was measured via succinate dehydrogenase and alkaline phosphatase staining. Our study demonstrated an increased uptake of (33)P by the AM fungus in the presence of T. harzianum, possibly related to a stress reaction caused by mycoparasitism. In addition, the disruption of AM extraradical hyphae in the presence of T. harzianum affected the (33)P translocation within the AM fungal mycelium and consequently the transfer of (33)P to the host plant. The effects of T. harzianum on Glomus sp. may thus impact the growth and function of AM fungi and also indirectly plant performance by influencing the source-sink relationship between the two partners of the symbiosis.     

Co-expression of two genes, a chitinase (chit42) and proteinase (prb1), implicated in mycoparasitism by Trichoderma hamatum

Mycoparasitism of fungal plant pathogens by Trichoderma species is a complex process that involves the production and coordinated secretion of cell-wall degrading enzymes. Genes implicated in mycoparasitism by Trichoderma atroviride contain motifs in the promoter region, designated MYRE1-MYRE4, that are proposed to act as binding sites for a global inducer of the mycoparasitic response. The aim of our study was to establish whether these motifs also were present in Trichoderma hamatum and whether the presence of these motifs could predict co-expression when T. hamatum was confronted by a pathogen. Using a combination of targeted, degenerate and inverse PCR, homologues of the mycoparasitism-related genes ech42 (chit42), prb1 and lam1.3 (xbg1.3-110), which encode an endochitinase, proteinase, and β-1,3-glucanase, respectively, were cloned and sequenced from T. hamatum. Alignment of the promoter regions of the three genes revealed identical regions in the chit42 and prb1 promoters, which were 6-9 base pairs in length and conserved in position. Specifically, the regulator y motifs MYRE1-MYRE4 were fully conserved, together with a fifth motif, identified by this research. A substrate assay designed to investigate the response of these genes from T. harzianum and T. hamatum to a simple carbon source (glycerol) showed that, in contrast to chit42 and prb1, xbg1.3-110 was not expressed. Further comparison of the expression patterns of these three genes between T. harzianum and T. hamatum using the glycerol substrate assay showed that no chit42 or prb1 expression could be detected in T. harzianum when it was grown under the same conditions as T. hamatum. This showed that the response of these genes to glycerol was species specific and that a single expression pattern for these genes was not common to all Trichoderma species. Confrontation assays were used to investigate the response of the three T. hamatum genes to the more complex substrate posed by the fungal pathogen Sclerotinia sclerotiorum. Once again gene expression analysis showed that both chit42 and prb1 were co-expressed and moderately induced during confrontation against Sclerotinia sclerotiorum. Although xbg1.3-110 previously had been implicated in mycoparasitism by T. harzianum, this study detected no xbg1.3-110 expression during confrontation between T. hamatum and S. sclerotiorum. These findings show that the MYRE1-MYRE4 together with MYRE5 are present in two species of Trichoderma, T. atroviride and T. hamatum and that the presence of these motifs could predict co-expression in response to two carbon sources.     

Analysis of expressed sequence tags from Chaetomium cupreum grown under conditions associated with mycoparasitism

Aims:  To elucidate the molecular mechanisms associated with mycoparasitism from Chaetomium cupreum , an effective biocontrol agent with ability against plant pathogenic fungi.
Methods and Results:  One cDNA library was constructed from conditions predicted to resemble mycoparasitic process. A total of 1876 ESTs were generated and assembled into 1035 unigenes. B last X search revealed that 585 unigenes had similarities with sequences available from public databases. Based on the ESTs abundance, MFS monosaccharide transporter was found as the gene expressed at the highest level. A KEGG analysis allowed mapping of 60 metabolic pathways well represented by the glycolysis/gluconeogenesis, d -arginine and ornithine metabolism, and tryptophan metabolism. The genes related to mycoparasitism were detected.
Conclusions:  The results revealed that the cell walls of the fungal pathogen can simulate some aspects of the mycoparasitic interaction between C. cupreum and its targets.
Significance and Impact of the Study:  This is the first report to study genes expression under conditions associated with the mycoparasitic process. The findings contribute to elucidate the molecular mechanisms involved in mycoparasitism and will help to advance our efforts in developing novel strategies for biocontrol of plant fungal diseases.     

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.     

Expressed sequence tags-based identification of genes in the biocontrol agent <Emphasis Type="Italic">Chaetomium cupreum</Emphasis>

Chaetomium cupreum has a potential as biocontrol agent against a range of plant pathogens on the basis of production of antifungal metabolites, mycoparasitism, competition for space and nutrients, or various combinations of these. To explore genes expressed in C. cupreum, a cDNA library was constructed from mycelium and 3,066 expressed sequence tags (ESTs) were generated. Clusters analysis enabled the identification of 1,471 unigenes with 392 contigs and 1,079 singleton sequences. Putative functions were assigned to 874 unigenes that exhibited strong similarity to genes/ESTs in public databases putatively containing genes involved in cellular component, molecular function, and biological process. Other 597 ESTs representing novel genes showed no significant similarity to public database resource of NCBI. A proportion of genes was identified related to degradation of pathogen cell wall, antifungal metabolite production, as was estimated in the biocontrol fungus. The paper described is a first step towards the knowledge of the C. cupreum genome. The results present the useful application of EST analysis on C. cupreum and provide a preliminary indication of gene expression putatively involved in biocontrol.     

Transcriptional profiling of Zea mays roots reveals roles for jasmonic acid and terpenoids in resistance against Phytophthora cinnamomi

Phytophthora cinnamomi is a soil-borne plant pathogen that has caused widespread damage to vulnerable native ecosystems and agriculture systems across the world and shows no sign of abating. Management of the pathogen in the natural environment is difficult and the options are limited. In order to discover more about how resistant plants are able to defend themselves against this generalist pathogen, a microarray study of plant gene expression following root inoculation with P. cinnamomi was undertaken. Zea mays was used as a resistant model plant, and microarray analysis was conducted using the Affymetrix GeneChip Maize Genome Array on root samples collected at 6- and 24-h post-inoculation. Over 300 genes were differentially expressed in inoculated roots compared with controls across the two time points. Following Gene Ontology enrichment analysis and REVIGO visualisation of the up-regulated genes, many were implicated in plant defence responses to biotic stress. Genes that were up-regulated included those involved in phytoalexin biosynthesis and jasmonic acid/ethylene biosynthesis and other defence-related genes including those encoding glutathione S-transferases and serine-protease inhibitors. Of particular interest was the identification of the two most highly up-regulated genes, terpene synthase11 (Tps11) and kaurene synthase2 (An2), which are both involved in production of terpenoid phytoalexins. This is the first study that has investigated gene expression at a global level in roots in response to P. cinnamomi in a model plant species and provides valuable insights into the mechanisms involved in defence.     

Trichoderma atroviride LU132 promotes plant growth but not induced systemic resistance to Plutella xylostella in oilseed rape

Several species of the fungus Trichoderma can promote plant health and are widely used as commercial biopesticides. Beneficial effects of this fungus are attributed to various mechanisms such as mycoparasitism, plant-growth promotion, increased stress tolerance and elicitation of induced systemic resistance against pathogens via jasmonic acid/ethylene-dependent pathways. Despite such well-established effects on pathogens, surprisingly little is known about the influence of Trichoderma on plant defences against herbivorous insects. This study investigated whether soil-supplementation of the established biocontrol agent Trichoderma atroviride LU132 affected the performance of oilseed rape (Brassica napus) and the development of Plutella xylostella caterpillars. Furthermore, induction and priming of defence-related phytohormones, genes and secondary metabolites by fungus and herbivore were assessed. Plants colonized by T. atroviride LU132 had significantly larger root and shoot biomass than controls. No effects of fungal inoculation were found on herbivore development. Leaf feeding of the herbivore induced higher jasmonic acid levels, but this was not influenced by fungal treatment. Similarly, the defence-related genes MYC2 and TPI were induced by herbivory but not primed or induced by T. atroviride. Expression of the gene PDF1.2 was repressed by herbivore feeding while no effects on the gene ACO and glucosinolates were observed. We conclude that T. atroviride LU132 has positive effects on the growth of oilseed but it does not enhance above-ground insect defences.     

Genomic Analysis of the Mycoparasite Pestalotiopsis sp. PG52

Pestalotiopsis sp. is a mycoparasite of the plant pathogen Aecidium wenshanense. To further understand the mycoparasitism mechanism of Pestalotiopsis sp., we assembled and analyzed its genome. The genome of Pestalotiopsis sp. strain PG52 was assembled into 335 scaffolds and had a size of 58.01 Mb. A total of 20,023 predicted genes and proteins were annotated. This study compared PG52 with the mycoparasites Trichoderma harzianum, Trichoderma atroviride, and Trichoderma virens. This study reveals the entirely different mycoparasitism mechanism of Pestalotiopsis compared to Trichoderma and reveals this mycoparasite’s strong ability to produce secondary metabolites.     

Expressed sequence tags-based identification of genes in a biocontrol strain Trichoderma asperellum

Trichoderma asperellum, a filamentous soil fungus, is an effective biocontrol agent against many fungal plant pathogenic species. In the present study, we investigated the biological control properties of the strain T. asperellum T4. T. asperellum fermentation products significantly decreased the ability of Rhizoctonia solani and Sclerotinia sclerotiorum to infect rice and soybean, respectively. To further elucidate the biocontrol mechanisms of T. asperellum at the molecular level, a cDNA library was constructed from its mycelium. In total, 3114 expressed sequence tags (ESTs) were generated, which represented 1,554 unigenes, including 354 contigs and 1,200 singletons. Among these unigenes, 731 represented known genes while 823 were novel genes. Forty-six unigenes potentially involved in biocontrol processes were identified from the EST collection. Among them, the expressions of 16 genes were studied, and 15 genes were highly differentially regulated during confrontation with 2 phytopathogens, suggesting that they play roles in the T. asperellum response to phytopathogens. Our study may provide helpful insight in the mechanism of biocontrol by T. asperellum T4 against plant pathogens.     

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.     

The expression of genes involved in parasitism by Trichoderma harzianum is triggered by a diffusible factor

The mycoparasite Trichoderma harzianum has been extensively used in the biocontrol of a wide range of phytopathogenic fungi. Hydrolytic enzymes secreted by the parasite have been directly implicated in the lysis of the host. Dual cultures of Trichoderma and a host, with and without contact, were used as means to study the mycoparasitic response in Trichoderma. Northern analysis showed high-level expression of genes encoding a proteinase (prb1) and an endochitinase (ech42) in dual cultures even if contact with the host was prevented by using cellophane membranes. Neither gene was induced during the interaction of Trichoderma with lectin-coated nylon fibres, which are known to induce hyphal coiling and appressorium formation. Thus, the signal involved in triggering the production of these hydrolytic enzymes by T. harzianum during the parasitic response is independent of the recognition mediated by this lectin-carbohydrate interaction. The results showed that induction of prb1 and ech42 is contact-independent, and a diffusible molecule produced by the host is the signal that triggers expression of both genes in vivo. Furthermore, a molecule that is resistant to heat and protease treatment, obtained from Rhizoctonia solani cell walls induces expression of both genes. Thus, this molecule is involved in the regulation of the expression of hydrolytic enzymes during mycoparasitism by T. harzianum.