Evolution and Ecophysiology of the Industrial Producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a New Sympatric Agamospecies Related to It

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov. CONCLUSIONS: Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour--T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies

Comparative transcriptomics reveals different strategies of Trichoderma mycoparasitism

BACKGROUND: 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. RESULTS: The three Trichoderma spp. exhibited a strikingly different transcriptomic response already before physical contact with alien hyphae. T. atroviride expressed an array of genes involved in production of secondary metabolites, GH16 ß-glucanases, various proteases and small secreted cysteine rich proteins. T. virens, on the other hand, expressed mainly the genes for biosynthesis of gliotoxin, respective precursors and also glutathione, which is necessary for gliotoxin biosynthesis. In contrast, T. reesei increased the expression of genes encoding cellulases and hemicellulases, and of the genes involved in solute transport. The majority of differentially regulated genes were orthologues present in all three species or both in T. atroviride and T. virens, indicating that the regulation of expression of these genes is different in the three Trichoderma spp. The genes expressed in all three fungi exhibited a nonrandom genomic distribution, indicating a possibility for their regulation via chromatin modification. CONCLUSION: This genome-wide expression study demonstrates that the initial Trichoderma mycotrophy has differentiated into several alternative ecological strategies ranging from parasitism to predation and saprotrophy. It provides first insights into the mechanisms of interactions between Trichoderma and other fungi that may be exploited for further development of biofungicides

The VELVET A Orthologue VEL1 of Trichoderma reesei Regulates Fungal Development and Is Essential for Cellulase Gene Expression

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

Generacija novih genotipskih i fenotipskih svojstava prirodnih i umjetnih hibrida kvasaca

Evolution and genome stabilization have mostly been studied on the Saccharomyces hybrids isolated from natural and alcoholic fermentation environments. Genetic and phenotypic properties have usually been compared to the laboratory and reference strains, as the true ancestors of the natural hybrid yeasts are unknown. In this way the exact impact of different parental fractions on the genome organization or metabolic activity of the hybrid yeasts is difficult to resolve completely. In the present work the evolution of geno- and phenotypic properties is studied in the interspecies hybrids created by the cross-breeding of S. cerevisiae with S. uvarum or S. kudriavzevii auxotrophic mutants. We hypothesized that the extent of genomic alterations in S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii should affect the physiology of their F1 offspring in different ways. Our results, obtained by amplified fragment length polymorphism (AFLP) genotyping and karyotyping analyses, showed that both subgenomes of the S. cerevisiae x S. uvarum and of S. cerevisiae × S. kudriavzevii hybrids experienced various modifications. However, the S. cerevisiae × S. kudriavzevii F1 hybrids underwent more severe genomic alterations than the S. cerevisiae × S. uvarum ones. Generation of the new genotypes also influenced the physiological performances of the hybrids and the occurrence of novel phenotypes. Significant differences in carbohydrate utilization and distinct growth dynamics at increasing concentrations of sodium chloride, urea and miconazole were observed within and between the S. cerevisiae × S. uvarum and S. cerevisiae × S. kudriavzevii hybrids. Parental strains also demonstrated different contributions to the final metabolic outcomes of the hybrid yeasts. A comparison of the genotypic properties of the artificial hybrids with several hybrid isolates from the wine-related environments and wastewater demonstrated a greater genetic variability of the S. cerevisiae × S. kudriavzevii hybrids. Saccharomyces cerevisiae × S. uvarum artificial and natural hybrids showed considerable differences in osmolyte tolerance and sensitivity to miconazole, whereas the S. cerevisiae × S. kudriavzevii hybrids exhibited differences also in maltotriose utilization. The results of this study suggest that chromosomal rearrangements and genomic reorganizations as post-hybridization processes may affect the phenotypic properties of the hybrid progeny substantially. Relative to their ancestors, the F1 segregants may generate different phenotypes, indicating novel routes of evolution in response to environmental growth conditions.Evolucija i stabilizacija genoma kvasca uglavnom se proučavaju s pomoću interspecijskih hibrida roda Saccharomyces, izoliranih iz prirodnih staništa ili tijekom alkoholnih fermentacija. Njihova genetska i fenotipska svojstva obično se uspoređuju sa svojstvima laboratorijskih i referentnih sojeva, budući da su izvorni roditeljski sojevi prirodnih hibrida kvasaca nepoznati. Na ovaj je način teško u potpunosti razumjeti utjecaj različitih roditeljskih frakcija na organizaciju genoma ili metaboličku aktivnost hibrida kvasaca. U ovom je radu proučena evolucija genotipskih i fenotipskih svojstava interspecijskih hibrida, nastalih križanjem kvasca S. cerevisiae s auksotrofnim mutantima kvasaca S. uvarum i S. kudriavzevii. Naša je hipoteza bila da bi genomske promjene nastale u hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii trebale na različite načine utjecati na fiziologiju njihovih F1 segreganata. Rezultati dobiveni genotipizacijom, tj. analizom polimorfizma duljine umnoženih fragmenata (engl. amplified fragment length polymorphism - AFLP) i kariotipizacijom pokazuju da su oba subgenoma hibrida S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii izmjenjena. Međutim, promjene genoma segreganata F1 hibrida S. cerevisiae × S. kudriavzevii bile su znatnije od onih segreganata hibrida S. cerevisiae × S. uvarum. Novi su genotipovi utjecali na fiziološke značajke hibrida te nastanak novih fenotipova. Bitna je razlika među hibridima S. cerevisiae × S. uvarum i S. cerevisiae × S. kudriavzevii opažena u potrošnji šećera i različitoj dinamici rasta kod povećanih koncentracija natrijevog klorida, uree i mikonazola. Roditeljski su sojevi različito utjecali na konačnu metaboličku sliku hibrida kvasaca. Usporedbom genotipskih svojstava umjetnih hibrida s nekoliko hibrida izoliranih iz prirodnih staništa (vinograda i otpadnih voda) utvrđena je veća genetska raznolikost hibrida S. cerevisiae × S. kudriavzevii. Umjetni i prirodni hibridi Saccharomyces cerevisiae × S. uvarum bili su različito osjetljivi prema osmolitima i mikonazolu, dok su se hibridi S. cerevisiae × S. kudriavzevii razlikovali i u potrošnji maltotrioze. Iz dobivenih se rezultata može zaključiti da kromosomalna rekombinacija i genomska reorganizacija kao post-hibridizacijski procesi mogu značajno utjecati na fenotipska svojstva hibridnih potomaka. U usporedbi s roditeljskim sojevima, segreganti F1 mogu generirati različite fenotipove, što upućuje na zaključak da su specifični uvjeti rasta kvasaca uzrokovali nastanak novih evolucijskih tokova

The CRE1 carbon catabolite repressor of the fungus Trichoderma reesei: a master regulator of carbon assimilation

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Here, we deciphered the CRE1 regulatory range in the model cellulose and hemicellulose-degrading fungus <it>Trichoderma reesei </it>(anamorph of <it>Hypocrea jecorina</it>) by profiling transcription in a wild-type and a delta-<it>cre1 </it>mutant strain on glucose at constant growth rates known to repress and de-repress CCR-affected genes. Analysis of genome-wide microarrays reveals 2.8% of transcripts whose expression was regulated in at least one of the four experimental conditions: 47.3% of which were repressed by CRE1, whereas 29.0% were actually induced by CRE1, and 17.2% only affected by the growth rate but CRE1 independent. Among CRE1 repressed transcripts, genes encoding unknown proteins and transport proteins were overrepresented. In addition, we found CRE1-repression of nitrogenous substances uptake, components of chromatin remodeling and the transcriptional mediator complex, as well as developmental processes.</p> <p>Conclusions</p> <p>Our study provides the first global insight into the molecular physiological response of a multicellular fungus to carbon catabolite regulation and identifies several not yet known targets in a growth-controlled environment.</p

Ecological genomics of the opportunistic fungus Trichoderma

Zsfassung in dt. SpracheTrichoderma (Hypocreales, Ascomycota) ist eine Gattung der filamentösen Pilze, die durch ihr hohes opportunistisches Potenzial und ihre Anpassungsfähigkeit an verschiedene ökologische Bedingungen in zahlreichen natürlichen und künstlichen Habitaten verbreitet ist.Die Fruchtkörper der Trichoderma spp. (Teleomorph; geschlechtliche Form) wurden hauptsächlich auf Basidiomyceten gefunden. Viele Trichoderma Anamorphe (asexuelles Entwicklungsstadium) haben die Fähigkeit biotrophe Interaktionen (z.B. Parasitismus) mit anderen Pilzen einzugehen.Aufgrund dieser Eigenschaft sind Trichoderma spp. für Forschung und Industrie von großer Bedeutung und werden zur Bekämpfung von phytopathogenen Pilzen als Biofungizide eingesetzt. Trichoderma Mykoparasitimus steht daher im Fokus zahlreicher wissenschaftlicher Studien, was dazu führte, dass mittlerweile sieben Genome (T.asperellum, T. atroviride, T. citrinoviride, T. harzianum sensu stricto, T. longibrachiatum, T. reesei und T. virens) sequenziert und für die Öffentlichkeit freigegeben wurden.In der vorliegenden Dissertation wird die Ökologie der Gattung Trichoderma anhand von genomischen, transcriptomischen und ökophysiologischen Studien diskutiert. Die komparative Transkriptomik zeigte unterschiedliche Mykoparasitismus-Strategien der T. atroviride, T. reesei und T. virens. Die phylogenomische Studie der drei genannten Spezies zeigte, dass Mykoparasitismus eine Ureigenschaft dieser Gattung ist. Dies wurde auch in der Fallstudie über die evolutionär bedingten Unterschiede des mykoparasitischen Potenzials der eng verwandten, jedoch genetisch getrennten Spezies T. reesei und T. parareesei, unterstrichen.Die Studie des Polyketid-Synthase Gens pks4 in T. reesei brachte hervor, dass dieses Gen für die Pigmentbildung verantwortlich ist und auch Einfluss auf andere wichtige ökophysiologische Funktionen, inklusive Mykoparasitismus dieser Spezies hat. Diese Dissertation bietet erste Einblicke in den Mykoparasitismus von Trichoderma aus genomischer und ökophysiologischer Perspektive.Trichoderma (Hypocreales, Ascomycota) is a genus of common filamentous fungi that display a remarkable range of lifestyles. They have been isolated from numeral natural and artificial habitats demonstrating their high opportunistic potential and adaptability to variety of ecological conditions. Fruiting bodies of Trichoderma spp.are mainly found associated with basidiomycetes and many of Trichoderma anamorphs can form biotrophic associations (in a broad sense parasites) with different fungi. This property is widely used by researchers and industry for the antagonization and eventual killing of fungal pathogens of plants (biological control or biocontrol). The exploitation of the mycotrophic properties of Trichoderma in biotechnology and agriculture made the genus well studied and brought it in focus of numerous -omic studies including the seven complete genomes (T. asperellum, T.atroviride, T. citrinoviride, T. harzianum sensu stricto, T.longibrachiatum, T. reesei and T. virens) recently released for public access. This doctoral thesis focuses on the ecology of the genus Trichoderma, discussing it by our ecophysiological, genomic and transcriptomic studies. The comparative transcriptomic analysis of T. atroviride, T.reesei and T. virens revealed different strategies of Trichoderma mycoparasitism. The phylogenomic study of the three mentioned species showed that mycoparasitism is an innate property of the genus. This was further underlined by the case study on the versatility of antagonistic potentials in closely evolutionary related but genetically delimited species T. reesei and T. parareesei. The study of a polyketide synthase gene (pks4) in T. reesei revealed the involvement of a pigment producing pks in such ecophysiological functions as stress resistance, defense against competitive microorganisms and in mycoparasitism. This thesis provides the first insight into ecological genomics of Trichoderma through the genome-wide integrated analysis of mechanism, evolution and ecophysiology.14

Novel Endophytic Trichoderma spp. Isolated from Healthy Coffea arabica Roots are Capable of Controlling Coffee Tracheomycosis

One of the biggest threats to coffee growers in East Africa are emerging vascular wilt diseases (tracheomycosis) caused by Fusarium spp. Many Trichoderma species are known to be natural antagonists of these pathogens and are widely used in biological control of fungal plant diseases. More recently, several Trichoderma spp., which exhibited high antifungal activity have been isolated as endophytes. Consequently, we have investigated the presence and the antagonistic activity of endophytic Trichoderma isolated from roots of healthy coffee plants (Coffea arabica) from the major coffee growing regions of Ethiopia. Our results showed that community of Trichoderma spp. in roots of C. arabica contains fungi from coffee rhizosphere, as well as putatively obligate endophytic fungi. The putatively “true” endophytic species, until now, isolated only from coffee plant ecosystems in Ethiopia and recently described as T. flagellatum and novel T. sp. C.P.K. 1812 were able to antagonize Fusarium spp., which cause coffee tracheomycosis. Moreover, we found that strains of these species are also highly antagonistic against other phytopathogenic fungi, such as Alternaria alternata, Botryotinia fuckeliana (anamorph: Botrytis cinerea), and Sclerotinia sclerotiorum