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The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression.
PLoS ONE
PUBLISHED: 01-01-2014
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Trichoderma reesei is the industrial producer of cellulases and hemicellulases for biorefinery processes. Their expression is obligatorily dependent on the function of the protein methyltransferase LAE1. The Aspergillus nidulans orthologue of LAE1 - LaeA - is part of the VELVET protein complex consisting of LaeA, VeA and VelB that regulates secondary metabolism and sexual as well as asexual reproduction. Here we have therefore investigated the function of VEL1, the T. reesei orthologue of A. nidulans VeA. Deletion of the T. reesei vel1 locus causes a complete and light-independent loss of conidiation, and impairs formation of perithecia. Deletion of vel1 also alters hyphal morphology towards hyperbranching and formation of thicker filaments, and with consequently reduced growth rates. Growth on lactose as a sole carbon source, however, is even more strongly reduced and growth on cellulose as a sole carbon source eliminated. Consistent with these findings, deletion of vel1 completely impaired the expression of cellulases, xylanases and the cellulase regulator XYR1 on lactose as a cellulase inducing carbon source, but also in resting mycelia with sophorose as inducer. Our data show that in T. reesei VEL1 controls sexual and asexual development, and this effect is independent of light. VEL1 is also essential for cellulase gene expression, which is consistent with the assumption that their regulation by LAE1 occurs by the VELVET complex.
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The polyketide synthase gene pks4 of Trichoderma reesei provides pigmentation and stress resistance.
Eukaryotic Cell
PUBLISHED: 09-13-2013
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Species of the fungal genus Trichoderma (Hypocreales, Ascomycota) are well-known for their production of various secondary metabolites. Nonribosomal peptides and polyketides represent a major portion of these products. In a recent phylogenomic investigation of Trichoderma polyketide synthase (PKS)-encoding genes, the pks4 from T. reesei was shown to be an orthologue of pigment-forming PKSs involved in synthesis of aurofusarin and bikaverin in Fusarium spp. In this study, we show that deletion of this gene in T. reesei results in loss of green conidial pigmentation and in pigmentation alteration of teleomorph structures. It also has an impact on conidial cell wall stability and the antagonistic abilities of T. reesei against other fungi, including formation of inhibitory metabolites. In addition, deletion of pks4 significantly influences the expression of other PKS-encoding genes of T. reesei. To our knowledge, this is the first indication that a low-molecular-weight pigment-forming PKS is involved in defense, mechanical stability, and stress resistance in fungi.
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Two novel class II hydrophobins from Trichoderma spp. stimulate enzymatic hydrolysis of poly(ethylene terephthalate) when expressed as fusion proteins.
Appl. Environ. Microbiol.
PUBLISHED: 05-03-2013
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Poly(ethylene terephthalate) (PET) can be functionalized and/or recycled via hydrolysis by microbial cutinases. The rate of hydrolysis is however low. Here, we tested whether hydrophobins (HFBs), small secreted fungal proteins containing eight positionally conserved cysteine residues, are able to enhance the rate of enzymatic hydrolysis of PET. Species of the fungal genus Trichoderma have the most proliferated arsenal of class II hydrophobin-encoding genes among fungi. To this end, we studied two novel class II HFBs (HFB4 and HFB7) of Trichoderma. HFB4 and HFB7, produced in Escherichia coli as fusions to the C terminus of glutathione S-transferase, exhibited subtle structural differences reflected in hydrophobicity plots that correlated with unequal hydrophobicity and hydrophily, respectively, of particular amino acid residues. Both proteins exhibited a dosage-dependent stimulation effect on PET hydrolysis by cutinase from Humicola insolens, with HFB4 displaying an adsorption isotherm-like behavior, whereas HFB7 was active only at very low concentrations and was inhibitory at higher concentrations. We conclude that class II HFBs can stimulate the activity of cutinases on PET, but individual HFBs can display different properties. The present findings suggest that hydrophobins can be used in the enzymatic hydrolysis of aromatic-aliphatic polyesters such as PET.
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Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism.
BMC Genomics
PUBLISHED: 02-19-2013
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Trichoderma is a genus of mycotrophic filamentous fungi (teleomorph Hypocrea) which possess a bright variety of biotrophic and saprotrophic lifestyles. The ability to parasitize and/or kill other fungi (mycoparasitism) is used in plant protection against soil-borne fungal diseases (biological control, or biocontrol). To investigate mechanisms of mycoparasitism, we compared the transcriptional responses of cosmopolitan opportunistic species and powerful biocontrol agents Trichoderma atroviride and T. virens with tropical ecologically restricted species T. reesei during confrontations with a plant pathogenic fungus Rhizoctonia solani.
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DNA barcoding survey of Trichoderma diversity in soil and litter of the Colombian lowland Amazonian rainforest reveals Trichoderma strigosellum sp. nov. and other species.
Antonie Van Leeuwenhoek
PUBLISHED: 01-09-2013
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The diversity of Trichoderma (Hypocreales, Ascomycota) colonizing leaf litter as well as the rhizosphere of Garcinia macrophylla (Clusiaceae) was investigated in primary and secondary rain forests in Colombian Amazonia. DNA barcoding of 107 strains based on the internal transcribed spacers 1 and 2 (ITS1 and 2) of the ribosomal RNA gene cluster and the partial sequence of the translation elongation factor 1 alpha (tef1) gene revealed that the diversity of Trichoderma was dominated (71 %) by three common cosmopolitan species, namely Trichoderma harzianum sensu lato (41 %), Trichoderma spirale (17 %) and Trichoderma koningiopsis (13 %). Four ITS 1 and 2 phylotypes (13 strains) could not be identified with certainty. Multigene phylogenetic analysis and phenotype profiling of four strains with an ITS1 and 2 phylotype similar to Trichoderma strigosum revealed a new sister species of the latter that is described here as Trichoderma strigosellum sp. nov. Sequence similarity searches revealed that this species also occurs in soils of Malaysia and Cameroon, suggesting a pantropical distribution.
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The intracellular galactoglycome in Trichoderma reesei during growth on lactose.
Appl. Microbiol. Biotechnol.
PUBLISHED: 01-09-2013
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Lactose (1,4-0-?-D-galactopyranosyl-D-glucose) is used as a soluble carbon source for the production of cellulases and hemicellulases for-among other purposes-use in biofuel and biorefinery industries. The mechanism how lactose induces cellulase formation in T. reesei is enigmatic, however. Previous results from our laboratory raised the hypothesis that intermediates from the two galactose catabolic pathway may give rise to the accumulation of intracellular oligogalactosides that could act as inducer. Here we have therefore used high-performance anion-exchange chromatography-mass spectrometry to study the intracellular galactoglycome of T. reesei during growth on lactose, in T. reesei mutants impaired in galactose catabolism, and in strains with different cellulase productivities. Lactose, allo-lactose, and lactulose were detected in the highest amounts in all strains, and two trisaccharides (Gal-?-1,6-Gal-?-1,4-Glc/Fru and Gal-?-1,4-Gal-?-1,4-Glc/Fru) also accumulated to significant levels. Glucose and galactose, as well as four further oligosaccharides (Gal-?-1,3/1,4/1,6-Gal; Gal-?-1,2-Glc) were only detected in minor amounts. In addition, one unknown disaccharide (Hex-?-1,1-Hex) and four trisaccharides were also detected. The accumulation of the unknown hexose disaccharide was shown to correlate with cellulase formation in the improved mutant strains as well as the galactose pathway mutants, and Gal-?-1,4-Gal-?-1,4-Glc/Fru and two other unknown hexose trisaccharides correlated with cellulase production only in the pathway mutants, suggesting that these compounds could be involved in cellulase induction by lactose. The nature of these oligosaccharides, however, suggests their formation by transglycosylation rather than by glycosyltransferases. Based on our results, the obligate nature of both galactose catabolic pathways for this induction must have another biochemical basis than providing substrates for inducer formation.
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The putative protein methyltransferase LAE1 of Trichoderma atroviride is a key regulator of asexual development and mycoparasitism.
PLoS ONE
PUBLISHED: 01-01-2013
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In Ascomycota the protein methyltransferase LaeA is a global regulator that affects the expression of secondary metabolite gene clusters, and controls sexual and asexual development. The common mycoparasitic fungus Trichoderma atroviride is one of the most widely studied agents of biological control of plant-pathogenic fungi that also serves as a model for the research on regulation of asexual sporulation (conidiation) by environmental stimuli such as light and/or mechanical injury. In order to learn the possible involvement of LAE1 in these two traits, we assessed the effect of deletion and overexpression of lae1 gene on conidiation and mycoparasitic interaction. In the presence of light, conidiation was 50% decreased in a ? lae1 and 30-50% increased in lae1-overexpressing (OElae1) strains. In darkness, ? lae1 strains did not sporulate, and the OElae1 strains produced as much spores as the parent strain. Loss-of-function of lae1 also abolished sporulation triggered by mechanical injury of the mycelia. Deletion of lae1 also increased the sensitivity of T. atroviride to oxidative stress, abolished its ability to defend against other fungi and led to a loss of mycoparasitic behaviour, whereas the OElae1 strains displayed enhanced mycoparasitic vigor. The loss of mycoparasitic activity in the ? lae1 strain correlated with a significant underexpressionn of several genes normally upregulated during mycoparasitic interaction (proteases, GH16 ß-glucanases, polyketide synthases and small cystein-rich secreted proteins), which in turn was reflected in the partial reduction of formation of fungicidal water soluble metabolites and volatile compounds. Our study shows T. atroviride LAE1 is essential for asexual reproduction in the dark and for defense and parasitism on other fungi.
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Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize.
Microbiology (Reading, Engl.)
PUBLISHED: 11-10-2011
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Trichoderma virens genome harbours genes encoding 22 non-ribosomal peptide synthetases (NRPSs) with at least one complete module (containing adenylation, thiolation and condensation domains) and four PKS/NRPS (polyketide synthase/NRPS) hybrid enzymes. After a primary screen for expression of these 26 genes when mycelia of T. virens are in contact with maize roots, seven genes that are upregulated were selected for further study. Using homologous recombination, loss-of-function mutants in six of these were obtained (the seventh, tex2, was acquired from our previous studies). Plant assays in a hydroponics system revealed that all seven mutants retained the ability to internally colonize maize roots. However, a mutation in one of the PKS/NRPS hybrid genes impaired the ability of T. virens to induce the defence response gene pal (phenylalanine ammonia lyase), suggesting a putative role for the associated metabolite product in induced systemic resistance. Interestingly, the mutant retained its ability to induce another defence response gene aos (allene oxide synthase). We thus provide evidence that a PKS/NRPS hybrid enzyme is involved in Trichoderma-plant interactions resulting in induction of defence responses.
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Taxon-specific metagenomics of Trichoderma reveals a narrow community of opportunistic species that regulate each others development.
Microbiology (Reading, Engl.)
PUBLISHED: 11-10-2011
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In this paper, we report on the in situ diversity of the mycotrophic fungus Trichoderma (teleomorph Hypocrea, Ascomycota, Dikarya) revealed by a taxon-specific metagenomic approach. We designed a set of genus-specific internal transcribed spacer (ITS)1 and ITS2 rRNA primers and constructed a clone library containing 411 molecular operational taxonomic units (MOTUs). The overall species composition in the soil of the two distinct ecosystems in the Danube floodplain consisted of 15 known species and two potentially novel taxa. The latter taxa accounted for only 1.5?% of all MOTUs, suggesting that almost no hidden or uncultivable Hypocrea/Trichoderma species are present at least in these temperate forest soils. The species were unevenly distributed in vertical soil profiles although no universal factors controlling the distribution of all of them (chemical soil properties, vegetation type and affinity to rhizosphere) were revealed. In vitro experiments simulating infrageneric interactions between the pairs of species that were detected in the same soil horizon showed a broad spectrum of reactions from very strong competition over neutral coexistence to the pronounced synergism. Our data suggest that only a relatively small portion of Hypocrea/Trichoderma species is adapted to soil as a habitat and that the interaction between these species should be considered in a screening for Hypocrea/Trichoderma as an agent(s) of biological control of pests.
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Trichoderma: the genomics of opportunistic success.
Nat. Rev. Microbiol.
PUBLISHED: 09-16-2011
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Trichoderma is a genus of common filamentous fungi that display a remarkable range of lifestyles and interactions with other fungi, animals and plants. Because of their ability to antagonize plant-pathogenic fungi and to stimulate plant growth and defence responses, some Trichoderma strains are used for biological control of plant diseases. In this Review, we discuss recent advances in molecular ecology and genomics which indicate that the interactions of Trichoderma spp. with animals and plants may have evolved as a result of saprotrophy on fungal biomass (mycotrophy) and various forms of parasitism on other fungi (mycoparasitism), combined with broad environmental opportunism.
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Marine isolates of Trichoderma spp. as potential halotolerant agents of biological control for arid-zone agriculture.
Appl. Environ. Microbiol.
PUBLISHED: 06-10-2011
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The scarcity of fresh water in the Mediterranean region necessitates the search for halotolerant agents of biological control of plant diseases that can be applied in arid-zone agriculture irrigated with saline water. Among 29 Trichoderma strains previously isolated from Mediterranean Psammocinia sp. sponges, the greatest number of isolates belong to the Trichoderma longibrachiatum-Hypocrea orientalis species pair (9), H. atroviridis/T. atroviride (9), and T. harzianum species complex (7), all of which are known for high mycoparasitic potential. In addition, one isolate of T. asperelloides and two putative new species, Trichoderma sp. O.Y. 14707 and O.Y. 2407, from Longibrachiatum and Strictipilosa clades, respectively, have been identified. In vitro salinity assays showed that the ability to tolerate increasing osmotic pressure (halotolerance) is a strain- or clade-specific property rather than a feature of a species. Only a few isolates were found to be sensitive to increased salinity, while others either were halotolerant or even demonstrated improved growth in increasingly saline conditions. In vitro antibiosis assays revealed strong antagonistic activity toward phytopathogens due to the production of both soluble and volatile metabolites. Two marine-derived Trichoderma isolates, identified as T. atroviride and T. asperelloides, respectively, effectively reduced Rhizoctonia solani damping-off disease on beans and also induced defense responses in cucumber seedlings against Pseudomonas syringae pv. lachrimans. This is the first inclusive evaluation of marine fungi as potential biocontrol agents.
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The amsterdam declaration on fungal nomenclature.
IMA Fungus
PUBLISHED: 05-17-2011
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The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19-20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented.
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Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma.
Genome Biol.
PUBLISHED: 03-28-2011
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Mycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.
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The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation.
BMC Genomics
PUBLISHED: 01-17-2011
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The identification and characterization of the transcriptional regulatory networks governing the physiology and adaptation of microbial cells is a key step in understanding their behaviour. One such wide-domain regulatory circuit, essential to all cells, is carbon catabolite repression (CCR): it allows the cell to prefer some carbon sources, whose assimilation is of high nutritional value, over less profitable ones. In lower multicellular fungi, the C2H2 zinc finger CreA/CRE1 protein has been shown to act as the transcriptional repressor in this process. However, the complete list of its gene targets is not known.
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Clonal species Trichoderma parareesei sp. nov. likely resembles the ancestor of the cellulase producer Hypocrea jecorina/T. reesei.
Appl. Environ. Microbiol.
PUBLISHED: 09-03-2010
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We have previously reported that the prominent industrial enzyme producer Trichoderma reesei (teleomorph Hypocrea jecorina; Hypocreales, Ascomycota, Dikarya) has a genetically isolated, sympatric sister species devoid of sexual reproduction and which is constituted by the majority of anamorphic strains previously attributed to H. jecorina/T. reesei. In this paper we present the formal taxonomic description of this new species, T. parareesei, complemented by multivariate phenotype profiling and molecular evolutionary examination. A phylogenetic analysis of relatively conserved loci, such as coding fragments of the RNA polymerase B subunit II (rpb2) and GH18 chitinase (chi18-5), showed that T. parareesei is genetically invariable and likely resembles the ancestor which gave raise to H. jecorina. This and the fact that at least one mating type gene of T. parareesei has previously been found to be essentially altered compared to the sequence of H. jecorina/T. reesei indicate that divergence probably occurred due to the impaired functionality of the mating system in the hypothetical ancestor of both species. In contrast, we show that the sexually reproducing and correspondingly more polymorphic H. jecorina/T. reesei is essentially evolutionarily derived. Phenotype microarray analyses performed at seven temperature regimens support our previous speculations that T. parareesei possesses a relatively high opportunistic potential, which probably ensured the survival of this species in ancient and sustainable environment such as tropical forests.
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Muscodor fengyangensis sp. nov. from southeast China: morphology, physiology and production of volatile compounds.
Fungal Biol
PUBLISHED: 04-24-2010
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The fungal genus Muscodor was erected on the basis of Muscodor albus, an endophytic fungus originally isolated from Cinnamomum zeylanicum. It produces a mixture of volatile organic compounds (VOCs) with antimicrobial activity that can be used as mycofumigants. The genus currently comprises five species. Here we describe the isolation and characterization of a new species of Muscodor on the basis of five endophytic fungal strains from leaves of Actinidia chinensis, Pseudotaxus chienii and an unidentified broad leaf tree in the Fengyangshan Nature Reserve, Zhejiang Province, Southeast of China. They exhibit white colonies on potato dextrose agar (PDA) media, rope-like mycelial strands, but did not sporulate. The optimum growth temperature is 25°C. The results of a phylogenetic analysis based on four loci (ITS1-5.8S-ITS2, 28S rRNA, rpb2 and tub1) are consistent with the hypothesis that these five strains belong to a single taxon. All five strains also produce volatile chemical components with antimicrobial activity in vitro, which were different from those previously described for other Muscodor species.
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The Trichoderma harzianum demon: complex speciation history resulting in coexistence of hypothetical biological species, recent agamospecies and numerous relict lineages.
BMC Evol. Biol.
PUBLISHED: 04-01-2010
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The mitosporic fungus Trichoderma harzianum (Hypocrea, Ascomycota, Hypocreales, Hypocreaceae) is an ubiquitous species in the environment with some strains commercially exploited for the biological control of plant pathogenic fungi. Although T. harzianum is asexual (or anamorphic), its sexual stage (or teleomorph) has been described as Hypocrea lixii. Since recombination would be an important issue for the efficacy of an agent of the biological control in the field, we investigated the phylogenetic structure of the species.
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Review: Global nutrient profiling by Phenotype MicroArrays: a tool complementing genomic and proteomic studies in conidial fungi.
J Zhejiang Univ Sci B
PUBLISHED: 03-06-2010
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Conidial fungi or molds and mildews are widely used in modern biotechnology as producers of antibiotics and other secondary metabolites, industrially important enzymes, chemicals and food. They are also important pathogens of animals including humans and agricultural crops. These various applications and extremely versatile natural phenotypes have led to the constantly growing list of complete genomes which are now available. Functional genomics and proteomics widely exploit the genomic information to study the cell-wide impact of altered genes on the phenotype of an organism and its function. This allows for global analysis of the information flow from DNA to RNA to protein, but it is usually not sufficient for the description of the global phenotype of an organism. More recently, Phenotype MicroArray (PM) technology has been introduced as a tool to characterize the metabolism of a (wild) fungal strain or a mutant. In this article, we review the background of PM applications for fungi and the methodic requirements to obtain reliable results. We also report examples of the versatility of this tool.
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Evolution and ecophysiology of the industrial producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a new sympatric agamospecies related to it.
PLoS ONE
PUBLISHED: 01-18-2010
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Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina.
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Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 09-02-2009
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Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.
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Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom.
FEMS Microbiol. Lett.
PUBLISHED: 08-21-2009
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Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1alpha gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.
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Differential regulation and posttranslational processing of the class II hydrophobin genes from the biocontrol fungus Hypocrea atroviridis.
Appl. Environ. Microbiol.
PUBLISHED: 03-27-2009
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Hydrophobins are small extracellular proteins, unique to and ubiquitous in filamentous fungi, which mediate interactions between the fungus and environment. The mycoparasitic fungus Hypocrea atroviridis has recently been shown to possess 10 different class II hydrophobin genes, which is a much higher number than that of any other ascomycete investigated so far. In order to learn the potential advantage of this hydrophobin multiplicity for the fungus, we have investigated their expression patterns under different physiological conditions (e.g., vegetative growth), various conditions inducing sporulation (light, carbon starvation, and mechanical injury-induced stress), and confrontation with potential hosts for mycoparasitism. The results show that the 10 hydrophobins display different patterns of response to these conditions: one hydrophobin (encoded by hfb-2b) is constitutively induced under all conditions, whereas other hydrophobins were formed only under conditions of carbon starvation (encoded by hfb-1c and hfb-6c) or light plus carbon starvation (encoded by hfb-2c, hfb-6a, and hfb-6b). The hydrophobins encoded by hfb-1b and hfb-5a were primarily formed during vegetative growth and under mechanical injury-provoked stress. hfb-22a was not expressed under any conditions and is likely a pseudogene. None of the 10 genes showed a specific expression pattern during mycoparasitic interaction. Most, but not all, of the expression patterns under the three different conditions of sporulation were dependent on one or both of the two blue-light regulator proteins BLR1 and BLR2, as shown by the use of respective loss-of-function mutants. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of mycelial solvent extracts provided sets of molecular ions corresponding to HFB-1b, HFB-2a, HFB-2b, and HFB-5a in their oxidized and processed forms. These in silico-deduced sequences of the hydrophobins indicate cleavages at known signal peptide sites as well as additional N- and C-terminal processing. Mass peaks observed during confrontation with plant-pathogenic fungi indicate further proteolytic attack on the hydrophobins. Our study illustrates both divergent and redundant functions of the 10 hydrophobins of H. atroviridis.
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Evaluation of matrix-assisted laser desorption/ionization (MALDI) preparation techniques for surface characterization of intact Fusarium spores by MALDI linear time-of-flight mass spectrometry.
Rapid Commun. Mass Spectrom.
PUBLISHED: 02-19-2009
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Unambiguous identification of mycotoxin-producing fungal species as Fusarium is of great relevance to agriculture and the food-producing industry as well as in medicine. Protein profiles of intact fungal spores, such as Penicillium, Aspergillus and Trichoderma, derived from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were shown to provide a rapid and straightforward method for species identification and characterization. In this study, we applied this approach to five different Fusarium spp. strains which are known to affect the growth of different grain plants. To obtain a suitable MALDI matrix system and sample preparation method, thin-layer, dried-droplet and sandwich methods and several MALDI matrices, namely CHCA, DHB, FA, SA and THAP dissolved in various solvent mixtures (organic solvents such as ACN, MeOH, EtOH and iPrOH and for the aqueous phase water and 0.1% TFA), were evaluated in terms of mass spectrometric pattern and signal intensities. The most significant peptide/protein profiles were obtained with 10 mg ferulic acid (FA) in 1 mL ACN/0.1% TFA (7:3, v/v) used as matrix system. Mixing the spores with the matrix solution directly on the MALDI target (dried-droplet technique) resulted in an evenly distributed spores/matrix crystal layer, yielding highly reproducible peptide/protein profiles from the spore surfaces. Numerous abundant ions throughout the investigated m/z range (m/z 1500-15 000) could be detected. Differences in the obtained mass spectral patterns allowed the differentiation of spores of various Fusarium species.
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Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan-European, invasive species of Hypocrea/Trichoderma.
Environ. Microbiol.
PUBLISHED: 02-07-2009
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We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.
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Fungal diversity in the rhizosphere of endemic plant species of Tenerife (Canary Islands): relationship to vegetation zones and environmental factors.
ISME J
PUBLISHED: 01-24-2009
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Knowledge about fungal diversity scaling relationships relative to that of plants is important to understand ecosystem functioning. Tenerife Island, a natural laboratory to study terrestrial biodiversity, is represented by six different vegetation zones characterized by specific abiotic conditions and plant communities with a high proportion of endemic plants. Little is known about the biodiversity of associated fungi. To understand the relationship between plant and fungal communities, we analysed soil/rhizosphere fungi from all vegetation zones. From 12 sampling points dispersed on the whole island, molecular analysis of fungal communities was determined by single-strand conformation polymorphism (SSCP) analysis using universal and specific primers for Trichoderma. The highly diverse fungal communities were mainly characterized by ectomycorrhiza-forming Basidiomycota and a high proportion of yet-unidentified species. Besides, Trichoderma-specific SSCP resulted in low diversity of mainly cosmopolitan species, for example Hypocrea lixii/T. harzianum. The dominance of T. harzianum was confirmed by cultivation. All Trichoderma isolates show an extraordinarily high antagonistic potential towards different groups of plant pathogens, supporting the hypothesis of extensive colonization by highly competitive Trichoderma species from the continent. In contrast, biodiversity patterns of the whole fungal and plant communities follow the same ecological rules. Furthermore, a high statistical correlation between fungal communities and the main environmental factors, temperature and precipitation, was found.
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Isotope-assisted screening for iron-containing metabolites reveals a high degree of diversity among known and unknown siderophores produced by Trichoderma spp.
Appl. Environ. Microbiol.
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Due to low iron availability under environmental conditions, many microorganisms excrete iron-chelating agents (siderophores) to cover their iron demands. A novel screening approach for the detection of siderophores using liquid chromatography coupled to high-resolution tandem mass spectrometry was developed to study the production of extracellular siderophores of 10 wild-type Trichoderma strains. For annotation of siderophores, an in-house library comprising 422 known microbial siderophores was established. After 96 h of cultivation, 18 different iron chelators were detected. Four of those (dimerum acid, fusigen, coprogen, and ferricrocin) were identified by measuring authentic standards. cis-Fusarinine, fusarinine A and B, and des-diserylglycylferrirhodin were annotated based on high-accuracy mass spectral analysis. In total, at least 10 novel iron-containing metabolites of the hydroxamate type were found. On average Trichoderma spp. produced 12 to 14 siderophores, with 6 common to all species tested. The highest number (15) of siderophores was detected for the most common environmental opportunistic and strongly fungicidic species, Trichoderma harzianum, which, however, did not have any unique compounds. The tropical species T. reesei had the most distinctive pattern, producing one unique siderophore (cis-fusarinine) and three others that were present only in T. harzianum and not in other species. The diversity of siderophores did not directly correlate with the antifungal potential of the species tested. Our data suggest that the high diversity of siderophores produced by Trichoderma spp. might be the result of further modifications of the nonribosomal peptide synthetase (NRPS) products and not due to diverse NRPS-encoding genes.
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The Longibrachiatum Clade of Trichoderma: a revision with new species.
Fungal Divers.
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The Longibrachiatum Clade of Trichoderma is revised. Eight new species are described (T. aethiopicum, T. capillare, T. flagellatum, T. gillesii, T. gracile, T. pinnatum, T. saturnisporopsis, T. solani). The twenty-one species known to belong to the Longibrachiatum Clade are included in a synoptic key. Trichoderma parareesei and T. effusum are redescribed based on new collections or additional observations. Hypocrea teleomorphs are reported for T. gillesii and T. pinnatum. Previously described species are annotated.
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Novel traits of Trichoderma predicted through the analysis of its secretome.
FEMS Microbiol. Lett.
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Mycotrophic species of Trichoderma are among the most common fungi isolated from free soil, dead wood and as parasites on sporocarps of other fungi (mycoparasites). In addition, they undergo various other biotrophic associations ranging from rhizosphere colonization and endophytism up to facultative pathogenesis on such animals as roundworms and humans. Together with occurrence on a variety of less common substrata (marine invertebrates, artificial materials, indoor habitats), these lifestyles illustrate a wealthy opportunistic potential of the fungus. One tropical species, Trichoderma reesei, has become a prominent producer of cellulases and hemicellulases, whereas several other species are applied in agriculture for the biological control of phytopathogenic fungi. The sequencing of the complete genomes of the three species (T. reesei, T. virens, and T. atroviride) has led to a deepened understanding of Trichoderma lifestyle and its molecular physiology. In this review, we present the in silico predicted secretome of Trichoderma, and - in addition to the unique features of carbohydrate active enzymes - demonstrate the importance of such protein families as proteases, oxidative enzymes, and small cysteine-rich proteins, all of that received little attention in Trichoderma genetics so far. We also discuss the link between Trichoderma secretome and biology of the fungus.
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The production of multiple small peptaibol families by single 14-module Peptide synthetases in Trichoderma/Hypocrea.
Chem. Biodivers.
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The most common sequences of peptaibiotics are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid-adding modules. Here, we provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11- and 14-residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid-activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.
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Molecular phylogeny and species delimitation in the section Longibrachiatum of Trichoderma.
Fungal Genet. Biol.
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The phylogenetically most derived group of the genus Trichoderma - section Longibrachiatum, includes some of the most intensively studied species, such as the industrial cellulase producer T. reesei (teleomorph Hypocrea jecorina), or the facultative opportunistic human pathogens T. longibrachiatum and H. orientalis. At the same time, the phylogeny of this clade is only poorly understood. Here we used a collection of 112 strains representing all currently recognized species and isolates that were tentatively identified as members of the group, to analyze species diversity and molecular evolution. Bayesian phylogenetic analyses based on several unlinked loci in individual and concatenated datasets confirmed 13 previously described species and 3 previously recognized phylogenetic species all of which were not yet described formally. When the genealogical concordance criterion, the K/? method and comparison of frequencies of pairwise nucleotide differences were applied to the data sample, 10 additional new phylogenetic species were recognized, seven of which consisted only of a single lineage. Our analysis thus identifies 26 putative species in section Longibrachiatum, what doubles the currently estimated taxonomic diversity of the group, and illustrates the power of combining genealogical concordance and population genetic analysis for dissecting species in a recently diverged group of fungal species.
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