Three new species of Hypocrea/Trichoderma sect. Trichoderma (Hypocreaceae, Hypocreales, Ascomycota, Fungi) are described from recent collections in southern Europe and the Canary Islands. They have been characterized by morphological and molecular methods, including microscopic examination of the teleomorph in thin sections, the anamorph, growth rate experiments and phylogenetic analyses based on a part of the translation elongation factor 1-alpha encoding gene (tef1) containing the two last introns and a part of the rpb2 gene, encoding the second largest RNA polymerase subunit. Analyses involving tef1 did not unequivocally resolve the sister clade relationship of Hypocrea caerulescens relative to the Koningii and Viride clades, while analyses based on rpb2 clearly suggest a close relationship with the former, although the phenotype of H. caerulescens is similar to H. viridescens, particularly by its warted conidia and a coconut-like odor in CMD culture. Hypocrea hispanica and T. samuelsii however are clearly related to the Viride clade by both phylogenetic markers, despite their morphological similarity to H. koningii and its relatives. An apparently specific blue pigment is formed in CMD cultures by Hypocrea caerulescens but could not be obtained by extraction with organic solvents.

Trichoderma spp. are free-living fungi that are common in soil and root ecosystems. Recent discoveries show that they are opportunistic, avirulent plant symbionts, as well as being parasites of other fungi. At least some strains establish robust and long-lasting colonizations of root surfaces and penetrate into the epidermis and a few cells below this level. They produce or release a variety of compounds that induce localized or systemic resistance responses, and this explains their lack of pathogenicity to plants. These root-microorganism associations cause substantial changes to the plant proteome and metabolism. Plants are protected from numerous classes of plant pathogen by responses that are similar to systemic acquired resistance and rhizobacteria-induced systemic resistance. Root colonization by Trichoderma spp. also frequently enhances root growth and development, crop productivity, resistance to abiotic stresses and the uptake and use of nutrients.

Trichoderma reesei is the main industrial source of cellulases and hemicellulases used to depolymerize biomass to simple sugars that are converted to chemical intermediates and biofuels, such as ethanol. We assembled 89 scaffolds (sets of ordered and oriented contigs) to generate 34 Mbp of nearly contiguous T. reesei genome sequence comprising 9,129 predicted gene models. Unexpectedly, considering the industrial utility and effectiveness of the carbohydrate-active enzymes of T. reesei, its genome encodes fewer cellulases and hemicellulases than any other sequenced fungus able to hydrolyze plant cell wall polysaccharides. Many T. reesei genes encoding carbohydrate-active enzymes are distributed nonrandomly in clusters that lie between regions of synteny with other Sordariomycetes. Numerous genes encoding biosynthetic pathways for secondary metabolites may promote survival of T. reesei in its competitive soil habitat, but genome analysis provided little mechanistic insight into its extraordinary capacity for protein secretion. Our analysis, coupled with the genome sequence data, provides a roadmap for constructing enhanced T. reesei strains for industrial applications such as biofuel production.

One of the biggest obstructions to studies on Trichoderma has been the incorrect and confused application of species names to isolates used in industry, biocontrol of plant pathogens and ecological surveys, thereby making the comparison of results questionable. Here we provide a convenient, on-line method for the quick molecular identification of Hypocrea/Trichoderma at the genus and species levels based on an oligonucleotide barcode: a diagnostic combination of several oligonucleotides (hallmarks) specifically allocated within the internal transcribed spacer 1 and 2 (ITS1 and 2) sequences of the rDNA repeat. The barcode was developed on the basis of 979 sequences of 88 vouchered species which displayed in total 135 ITS1 and 2 haplotypes. Oligonucleotide sequences which are constant in all known ITS1 and 2 of Hypocrea/Trichoderma but different in closely related fungal genera, were used to define genus-specific hallmarks. The library of species-, clade- and genus-specific hallmarks is stored in the MySQL database and integrated in the TrichOKey v. 1.0 - barcode sequence identification program with the web interface located on . TrichOKey v. 1.0 identifies 75 single species, 5 species pairs and 1 species triplet. Verification of the DNA-barcode was done by a blind test on 53 unknown isolates of Trichoderma, collected in Central and South America. The obtained results were in a total agreement with phylogenetic identification based on tef1 (large intron), NCBI BLAST of vouchered records and postum morphological analysis. We conclude that oligonucleotide barcode is a powerful tool for the routine identification of Hypocrea/Trichoderma species and should be useful as a complement to traditional methods.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)8,(GTG)5 and (GACA)4, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)4 produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.

Trichoderma harzianum is a ubiquitous species in the environment and is effective in the biological control of plant-pathogenic fungi. T. harzianum has not been linked unequivocally to its sexual state nor has its phylogeny been studied in detail. It has been suggested that T. harzianum is a species complex based on the phenotypic and genotypic variability encountered. On the basis of morphological and cultural characters and DNA sequence data analysis of four genes (ITS rDNA, translation elongation factor 1-alpha, calmodulin, and alpha-actin), Hypocrea lixii was found to be the sexual state of T. harzianum. Both the asexual and sexual states of this species have wide geographic distributions. Phylogenetic analysis of four genes showed that T. harzianum/H. lixii is a cohesive group that is supported by bootstrap values higher than 95%. Principles of genealogical concordance indicated that T. harzianum/H. lixii is a complex of independent monophyletic lineages, but no diagnostic morphological distinctions were identified that justify formal taxonomic recognition for the different lineages.

Toxic-metabolite-emitting microbes were isolated from the indoor environment of a building where the occupant was suffering serious building-related ill-health symptoms. Toxic substances soluble in methanol and inhibitory to spermatozoa at <10 microg (dry weight) ml(-1) were found from six bacterial isolates and one fungus. The substances from isolates of Bacillus simplex and from isolates belonging to the actinobacterial genera Streptomyces and Nocardiopsis were mitochondriotoxic. These substances dissipated the mitochondrial membrane potential (Deltapsi) of boar spermatozoa. The substances from the Streptomyces isolates also swelled the mitochondria. The substances from isolates of Trichoderma harzianum Rifai and Bacillus pumilus damaged the cell membrane barrier function of sperm cells.

For efficient fungal strain selection in microbial screening, we applied the random amplified polymorphic DNA (RAPD) method using the polymerase chain reaction (PCR). In order to evaluate this system, the genus Trichoderma was employed, because its species are difficult to distinguish from each other. We selected an appropriate oligonucleotide decamer, R28 (5'-ATGGATCCGC), determined the optimal cycles of PCR as 30 cycles, simplified the template preparation method, and determined optimal concentrations of the template and Taq DNA polymerase. We then examined 74 closely related strains of Trichoderma. The electrophoretic band patterns of the PCR products were compared. According to the statistical analysis with the phylogenetic analysis using parsimony (PAUP), the results were consistent with the morphological, physiological and ecological data on these strains. Therefore, we conclude that RAPD is a simple, efficient and reliable method for the selection of fungal strains employed in screening.