Antagonist potential of Trichoderma indigenous isolates for biological control of Phytophthora palmivora the causative agent of black pod disease on cocoa (Theobroma cacao L.) in Côte d’Ivoire

The biodiversity of Trichoderma isolates from cocoa rhizosphere in cocoa production areas of Côte d’Ivoire, and their antagonist potential with Phytophthora palmivora using in vitro assays and bioassays, were investigated and screened for field trials. A total of 135 isolates were analysed at the species level by using sequence analysis of ITS1 and 2 of the rRNA region and a fragment of translation elongation factor 1a (tef1) gene. Sixty-four isolates were identified as T. virens, 60 as T. harzianum, 7 as T. spirale, two as T. asperellum and two unidentified. Forty-three Trichoderma isolates were in vitro confronted with P. palmivora on agar plates, and their antagonist activity was further evaluated by the damages on leaf discs and detached pods. Twenty-five isolates reduced the mycelial growth of P. Palmivora more than 50%. The isolate T17 assigned to T .virens was the best to reduce mycelium growth upto 97.9%. All of Trichoderma isolates with the exception of isolate T39 reduced foliar sensitivity to P. palmivora. Tweenty-six Trichoderma isolates reduced the pod sensitivity to P. palmivora more than 50%. Based on the combined analysis, T. virens T7, T. harzianum T40, T. asperellum T54 and T. spirale T4 isolates were selected for field trials

Relevance of the light signaling machinery for cellulase expression in trichoderma reesei (hypocrea jecorina)

<p>Abstract</p> <p>Background</p> <p>In nature, light is one of the most important environmental cues that fungi perceive and interpret. It is known not only to influence growth and conidiation, but also cellulase gene expression. We therefore studied the relevance of the main components of the light perception machinery of <it>Trichoderma reesei </it>(<it>Hypocrea jecorina</it>), ENV1, BLR1 and BLR2, for production of plant cell wall degrading enzymes in fermentations aimed at efficient biosynthesis of enzyme mixtures for biofuel production.</p> <p>Findings</p> <p>Our results indicate that despite cultivation in mostly dark conditions, all three components show an influence on cellulase expression. While we found the performance of the enzyme mixture secreted by a deletion mutant in <it>env1 </it>to be enhanced, the higher cellulolytic activity observed for <it>Δblr2 </it>is mainly due to an increased secretion capacity of this strain. <it>Δblr1 </it>showed enhanced biomass accumulation, but due to its obviously lower secretion capacity still was the least efficient strain in this study.</p> <p>Conclusions</p> <p>We conclude that with respect to regulation of plant cell wall degrading enzymes, the blue light regulator proteins are unlikely to act as a complex. Their regulatory influence on cellulase biosynthesis involves an alteration of protein secretion, which may be due to adjustment of transcription or posttranscriptional regulation of upstream factors. In contrast, the regulatory function of ENV1 seems to involve adjustment of enzyme proportions to environmental conditions.</p

Species diversity of Trichoderma in Poland

In the present study, we reinvestigate the diversity of Trichoderma in Poland utilizing a combination of morphological and molecular/phylogenetic methods. A total of 170 isolates were collected from six different substrata at 49 sites in Poland. These were divided among 14 taxa as follows: 110 of 170 Trichoderma isolates were identified to the species level by the analysis of their ITS1, ITS2 rDNA sequences as: T. harzianum (43 isolates), T. aggressivum (35), T. citrinoviride (11), T. hamatum (9), T. virens (6), T. longibrachiatum (4), T. polysporum (1), and T. tomentosum (1); 60 isolates belonging to the Viride clade were identified based on a fragment of the translation-elongation factor 1-alpha (tef1) gene as: T. atroviride (20 isolates), T. gamsii (2), T. koningii (17), T. viridescens (13), T. viride (7), and T. koningiopsis (1). Identifications were made using the BLAST interface in TrichOKEY and TrichoBLAST (http://www.isth.info). The most diverse substrata were soil (nine species per 22 isolates) and decaying wood (nine species per 75 isolates). The most abundant species (25%) isolated from all substrata was T. harzianum

Disruption of Trichoderma reesei cre2, encoding an ubiquitin C-terminal hydrolase, results in increased cellulase activity

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/1472-6750/11/103Background: The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is an important source of cellulases for use in the textile and alternative fuel industries. To fully understand the regulation of cellulase production in T. reesei, the role of a gene known to be involved in carbon regulation in Aspergillus nidulans, but unstudied in T. reesei, was investigated. Results: The T. reesei orthologue of the A. nidulans creB gene, designated cre2, was identified and shown to be functional through heterologous complementation of a creB mutation in A. nidulans. A T. reesei strain was constructed using gene disruption techniques that contained a disrupted cre2 gene. This strain, JKTR2-6, exhibited phenotypes similar to the A. nidulans creB mutant strain both in carbon catabolite repressing, and in carbon catabolite derepressing conditions. Importantly, the disruption also led to elevated cellulase levels. Conclusions: These results demonstrate that cre2 is involved in cellulase expression. Since the disruption of cre2 increases the amount of cellulase activity, without severe morphological affects, targeting creB orthologues for disruption in other industrially useful filamentous fungi, such as Aspergillus oryzae, Trichoderma harzianum or Aspergillus niger may also lead to elevated hydrolytic enzyme activity in these species.Jai A Denton and Joan M Kell

Biology and biotechnology of Trichoderma

Fungi of the genus Trichoderma are soilborne, green-spored ascomycetes that can be found all over the world. They have been studied with respect to various characteristics and applications and are known as successful colonizers of their habitats, efficiently fighting their competitors. Once established, they launch their potent degradative machinery for decomposition of the often heterogeneous substrate at hand. Therefore, distribution and phylogeny, defense mechanisms, beneficial as well as deleterious interaction with hosts, enzyme production and secretion, sexual development, and response to environmental conditions such as nutrients and light have been studied in great detail with many species of this genus, thus rendering Trichoderma one of the best studied fungi with the genome of three species currently available. Efficient biocontrol strains of the genus are being developed as promising biological fungicides, and their weaponry for this function also includes secondary metabolites with potential applications as novel antibiotics. The cellulases produced by Trichoderma reesei, the biotechnological workhorse of the genus, are important industrial products, especially with respect to production of second generation biofuels from cellulosic waste. Genetic engineering not only led to significant improvements in industrial processes but also to intriguing insights into the biology of these fungi and is now complemented by the availability of a sexual cycle in T. reesei/Hypocrea jecorina, which significantly facilitates both industrial and basic research. This review aims to give a broad overview on the qualities and versatility of the best studied Trichoderma species and to highlight intriguing findings as well as promising applications

A homologous production system for Trichoderma reesei secreted proteins in a cellulase-free background

Recent demands for the production of biofuels from lignocellulose led to an increased interest in engineered cellulases from Trichoderma reesei or other fungal sources. While the methods to generate such mutant cellulases on DNA level are straightforward, there is often a bottleneck in their production since a correct posttranslational processing of these enzymes is needed to obtain highly active enzymes. Their production and subsequent enzymatic analysis in the homologous host T. reesei is, however, often disturbed by the concomitant production of other endogenous cellulases. As a useful alternative, we tested the production of cellulases in T. reesei in a genetic background where cellulase formation has been impaired by deletion of the major cellulase transcriptional activator gene xyr1. Three cellulase genes (cel7a, cel7b, and cel12a) were expressed under the promoter regions of the two highly expressed genes tef1 (encoding translation elongation factor 1-alpha) or cdna1 (encoding the hypothetical protein Trire2:110879). When cultivated on d-glucose as carbon source, the Δxyr1 strain secreted all three cellulases into the medium. Related to the introduced gene copy number, the cdna1 promoter appeared to be superior to the tef1 promoter. No signs of proteolysis were detected, and the individual cellulases could be assayed over a background essentially free of other cellulases. Hence this system can be used as a vehicle for rapid and high-throughput testing of cellulase muteins in a homologous background

Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4- methylumbelliferyl-β-D-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.Fundação para a Ciência e a Tecnologia (FCT

Towards barcode markers in Fungi: an intron map of Ascomycota mitochondria

<p>Abstract</p> <p>Background</p> <p>A standardized and cost-effective molecular identification system is now an urgent need for Fungi owing to their wide involvement in human life quality. In particular the potential use of mitochondrial DNA species markers has been taken in account. Unfortunately, a serious difficulty in the PCR and bioinformatic surveys is due to the presence of mobile introns in almost all the fungal mitochondrial genes. The aim of this work is to verify the incidence of this phenomenon in Ascomycota, testing, at the same time, a new bioinformatic tool for extracting and managing sequence databases annotations, in order to identify the mitochondrial gene regions where introns are missing so as to propose them as species markers.</p> <p>Methods</p> <p>The general trend towards a large occurrence of introns in the mitochondrial genome of Fungi has been confirmed in Ascomycota by an extensive bioinformatic analysis, performed on all the entries concerning 11 mitochondrial protein coding genes and 2 mitochondrial rRNA (ribosomal RNA) specifying genes, belonging to this phylum, available in public nucleotide sequence databases. A new query approach has been developed to retrieve effectively introns information included in these entries.</p> <p>Results</p> <p>After comparing the new query-based approach with a blast-based procedure, with the aim of designing a faithful Ascomycota mitochondrial intron map, the first method appeared clearly the most accurate. Within this map, despite the large pervasiveness of introns, it is possible to distinguish specific regions comprised in several genes, including the full NADH dehydrogenase subunit 6 (ND6) gene, which could be considered as barcode candidates for Ascomycota due to their paucity of introns and to their length, above 400 bp, comparable to the lower end size of the length range of barcodes successfully used in animals.</p> <p>Conclusion</p> <p>The development of the new query system described here would answer the pressing requirement to improve drastically the bioinformatics support to the DNA Barcode Initiative. The large scale investigation of Ascomycota mitochondrial introns performed through this tool, allowing to exclude the introns-rich sequences from the barcode candidates exploration, could be the first step towards a mitochondrial barcoding strategy for these organisms, similar to the standard approach employed in metazoans.</p

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