Trichoderma Biocontrol: Signal Transduction Pathways Involved in Host Sensing and Mycoparasitism

Fungi of the genus Trichoderma are used as biocontrol agents against several plant pathogenic fungi like Rhizoctonia spp., Pythium spp., Botrytis cinerea and Fusarium spp. which cause both soil-borne and leaf- or flower-borne diseases of agricultural plants. Plant disease control by Trichoderma is based on complex interactions between Trichoderma, the plant pathogen and the plant. Until now, two main components of biocontrol have been identified: direct activity of Trichoderma against the plant pathogen by mycoparasitism and induced systemic resistance in plants. As the mycoparasitic interaction is host-specific and not merely a contact response, it is likely that signals from the host fungus are recognised by Trichoderma and provoke transcription of mycoparasitism-related genes

Secondary Metabolites of Mycoparasitic Fungi

Mycoparasitic fungi, fungi preying on other fungal species, are prolific producers of volatile and non-volatile secondary metabolites. Several secondary metabolites are produced during mycoparasitism to weaken the host and support attack and parasitism. Further, evidence accumulated that some secondary metabolites also act as communication molecules. Besides their antagonistic activity, several fungal mycoparasites exhibit beneficial effects on plants and some of their secondary metabolites have plant growth-promoting and defense stimulating activities. As many secondary metabolism-associated gene clusters remain silent under standard laboratory conditions, the full variety as well as the underlying biosynthetic pathways employed by fungal mycoparasites for secondary metabolite production still await clarification. Nonetheless, the variety of currently known secondary metabolites and their range of activities is impressive already and they exhibit a great potential for agriculture, pharmacology and other industrial applications

Monitoring the volatile language of fungi using gas chromatography-ion mobility spectrometry

Fusarium oxysporum is a plant pathogenic fungus leading to severe crop losses in agriculture every year. A sustainable way of combating this pathogen is the application of mycoparasites—fungi parasitizing other fungi. The filamentous fungus Trichoderma atroviride is such a mycoparasite that is able to antagonize phytopathogenic fungi. It is therefore frequently applied as a biological pest control agent in agriculture. Given that volatile metabolites play a crucial role in organismic interactions, the major aim of this study was to establish a method for on-line analysis of headspace microbial volatile organic compounds (MVOCs) during cultivation of different fungi. An ion mobility spectrometer with gas chromatographic pre-separation (GC-IMS) enables almost real-time information of volatile emissions with good selectivity. Here we illustrate the successful use of GC-IMS for monitoring the time- and light-dependent release of MVOCs by F. oxysporum and T. atroviride during axenic and co-cultivation. More than 50 spectral peaks were detected, which could be assigned to 14 volatile compounds with the help of parallel gas chromatography-mass spectrometric (GC-MS) measurements. The majority of identified compounds are alcohols, such as ethanol, 1-propanol, 2-methyl propanol, 2-methyl butanol, 3-methyl-1-butanol and 1-octen-3-ol. In addition to four ketones, namely acetone, 2-pentanone, 2-heptanone, 3-octanone, and 2-octanone; two esters, ethyl acetate and 1-butanol-3-methylacetate; and one aldehyde, 3-methyl butanal, showed characteristic profiles during cultivation depending on axenic or co-cultivation, exposure to light, and fungal species. Interestingly, 2-octanone was produced only in co-cultures of F. oxysporum and T. atroviride, but it was not detected in the headspace of their axenic cultures. The concentrations of the measured volatiles were predominantly in the low ppbv range; however, values above 100 ppbv were detected for several alcohols, including ethanol, 2-methylpropanol, 2-methyl butanol, 1- and 3-methyl butanol, and for the ketone 2-heptanone, depending on the cultivation conditions. Our results highlight that GC-IMS analysis can be used as a valuable analytical tool for identifying specific metabolite patterns for chemotaxonomic and metabolomic applications in near-to-real time and hence easily monitor temporal changes in volatile concentrations that take place in minutes

Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride

Gα subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus

Transcriptomic response of the mycoparasitic fungus Trichoderma atroviride to the presence of a fungal prey

<p>Abstract</p> <p>Background</p> <p>Combating the action of plant pathogenic microorganisms by mycoparasitic fungi has been announced as an attractive biological alternative to the use of chemical fungicides since two decades. The fungal genus <it>Trichoderma </it>includes a high number of taxa which are able to recognize, combat and finally besiege and kill their prey. Only fragments of the biochemical processes related to this ability have been uncovered so far, however.</p> <p>Results</p> <p>We analyzed genome-wide gene expression changes during the begin of physical contact between <it>Trichoderma atroviride </it>and two plant pathogens <it>Botrytis cinerea </it>and <it>Rhizoctonia solani</it>, and compared with gene expression patterns of mycelial and conidiating cultures, respectively. About 3000 ESTs, representing about 900 genes, were obtained from each of these three growth conditions. 66 genes, represented by 442 ESTs, were specifically and significantly overexpressed during onset of mycoparasitism, and the expression of a subset thereof was verified by expression analysis. The upregulated genes comprised 18 KOG groups, but were most abundant from the groups representing posttranslational processing, and amino acid metabolism, and included components of the stress response, reaction to nitrogen shortage, signal transduction and lipid catabolism. Metabolic network analysis confirmed the upregulation of the genes for amino acid biosynthesis and of those involved in the catabolism of lipids and aminosugars.</p> <p>Conclusion</p> <p>The analysis of the genes overexpressed during the onset of mycoparasitism in <it>T. atroviride </it>has revealed that the fungus reacts to this condition with several previously undetected physiological reactions. These data enable a new and more comprehensive interpretation of the physiology of mycoparasitism, and will aid in the selection of traits for improvement of biocontrol strains by recombinant techniques.</p

The Lipoxygenase Lox1 Is Involved in Light‐ and Injury-Response, Conidiation, and Volatile Organic Compound Biosynthesis in the Mycoparasitic Fungus Trichoderma atroviride

18 páginas, 10 figuras. -- The first publication by Frontiers Media is avalilable at https://www.frontiersin.org/articles/10.3389/fmicb.2020.02004/fullThe necrotrophic mycoparasite Trichoderma atroviride is a biological pest control agent frequently applied in agriculture for the protection of plants against fungal phytopathogens. One of the main secondary metabolites produced by this fungus is 6-pentyl-α-pyrone (6-PP). 6-PP is an organic compound with antifungal and plant growth-promoting activities, whose biosynthesis was previously proposed to involve a lipoxygenase (Lox). In this study, we investigated the role of the single lipoxygenase-encoding gene lox1 encoded in the T. atroviride genome by targeted gene deletion. We found that light inhibits 6-PP biosynthesis but lox1 is dispensable for 6-PP production as well as for the ability of T. atroviride to parasitize and antagonize host fungi. However, we found Lox1 to be involved in T. atroviride conidiation in darkness, in injury-response, in the production of several metabolites, including oxylipins and volatile organic compounds, as well as in the induction of systemic resistance against the plant-pathogenic fungus Botrytis cinerea in Arabidopsis thaliana plants. Our findings give novel insights into the roles of a fungal Ile-group lipoxygenase and expand the understanding of a light-dependent role of these enzymes.This research was supported by the Austrian Science Fund (FWF; grant P32179-B), Tyrolean Science Fund (TWF; grant number AP718021) and the doctoral program BioApp from the University of InnsbruckPeer reviewe

Functional Analyses of Trichoderma reesei LAE1 Reveal Conserved and Contrasting Roles of This Regulator

The putative methyltransferase LaeA is a global regulator that affects the expression of multiple secondary metabolite gene clusters in several fungi, and it can modify heterochromatin structure in Aspergillus nidulans. We have recently shown that the LaeA ortholog of Trichoderma reesei (LAE1), a fungus that is an industrial producer of cellulase and hemicellulase enzymes, regulates the expression of cellulases and polysaccharide hydrolases. To learn more about the function of LAE1 in T. reesei, we assessed the effect of deletion and overexpression of lae1 on genome-wide gene expression. We found that in addition to positively regulating 7 of 17 polyketide or nonribosomal peptide synthases, genes encoding ankyrin-proteins, iron uptake, heterokaryon incompatibility proteins, PTH11-receptors, and oxidases/monoxygenases are major gene categories also regulated by LAE1. chromatin immunoprecipitation sequencing with antibodies against histone modifications known to be associated with transcriptionally active (H3K4me2 and -me3) or silent (H3K9me3) chromatin detected 4089 genes bearing one or more of these methylation marks, of which 75 exhibited a correlation between either H3K4me2 or H3K4me3 and regulation by LAE1. Transformation of a laeA-null mutant of A. nidulans with the T. reesei lae1 gene did not rescue sterigmatocystin formation and further impaired sexual development. LAE1 did not interact with A. nidulans VeA in yeast two-hybrid assays, whereas it interacted with the T. reesei VeA ortholog, VEL1. LAE1 was shown to be required for the expression of vel1, whereas the orthologs of velB and VosA are unaffected by lae1 deletion. Our data show that the biological roles of A. nidulans LaeA and T. reesei LAE1 are much less conserved than hitherto thought. In T. reesei, LAE1 appears predominantly to regulate genes increasing relative fitness in its environment.Keywords: Flavus, Secondary metabolism, Aspergillus nidulans, Gene expression, Hypocrea jecorina, DNA methylation, Histone H3, Protein kinase, Neurospora crassa, Penicillin biosynthesi