Identification of Trichoderma strains by image analysis of HPLC chromatograms.

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.

Endochitinase gene-based phylogenetic analysis of Trichoderma.

DNA sequences of the single-copy gene coding for the 42 kDa endochitinase enzyme (EC 3.2.1.14) were used for phylogenetic analysis in Trichoderma. A set of 12 primers was developed and the entire gene was sequenced for 16 strains, and nucleotide and deduced amino acid sequences were compared to data from GenBank for additional Trichoderma strains. Analysis of the sequences revealed parsimony informative variation from 2.4 to 43.6% depending on the part of the gene (exons/introns) and the taxonomic level considered. Results are discussed in comparison to previous data from ITS-1 and ITS-2 rDNA sequencing and suggest the 42 kDa endochitinase gene as a potential molecular marker for reconstructing phylogenetic relationships in the genus Trichoderma at species level.

A morphological and molecular perspective of Trichoderma viride: is it one or two species?

Trichoderma (Ascomycetes, Hypocreales) strains that have warted conidia are traditionally identified as T. viride, the type species of Trichoderma. However, two morphologically distinct types of conidial warts (I and II) have been found. Because each type corresponds to a unique mitochondrial DNA pattern, it has been questioned whether T. viride comprises more than one species. Combined molecular data (sequences of the internal transcribed spacer 1 [ITS-1] and ITS-2 regions and of part of the 28S rRNA gene along with results of restriction fragment length polymorphism analysis of the endochitinase gene and PCR fingerprinting), morphology, physiology, and colony characteristics distinguish type I and type II as different species. Type I corresponds to "true" T. viride, the anamorph of Hypocrea rufa. Type II represents a new species, T. asperellum, which is, in terms of molecular characteristics, close to the neotype of T. hamatum.

Molecular reidentification of human pathogenic Trichoderma isolates as Trichoderma longibrachiatum and Trichoderma citrinoviride.

Several species of the well-known saprophytic genus Trichoderma have been identified as the cause of infections in immunosuppressed humans. Because the differentiation and identification of Trichoderma species based on morphological characters only, is very difficult, two molecular approaches were applied for species identification. Six human pathogenic Trichoderma isolates were investigated by PCR-fingerprinting and analysis of ribosomal DNA internal transcribed spacer (ITS) sequences and compared with the corresponding data sets established for described species of the genus. Five of these strains were identified as T. longibrachiatum, whereas one single strain turned out to be T. citrinoviride. Both species are very closely related and belong to Trichoderma section Longibrachiatum. These data indicate that the occurrence of pathogenic Trichoderma strains may be restricted to species of section Longibrachiatum.

Molecular evidence that the asexual industrial fungus Trichoderma reesei is a clonal derivative of the ascomycete Hypocrea jecorina.

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.

Cellulase formation by species of Trichoderma sect. Longibrachiatum and of Hypocrea spp. with anamorphs referable to Trichoderma sect. Longibrachiatum.

The cellulolytic potential of the wild-type strain of Trichoderma reesei was compared to other members of Trichoderma sect. Longibrachiatum and Hypocrea spp. that have anamorphs referable to that section. There was high diversity even within the same species (as defined by morphological and macromolecular characters). Differences, where notable, were more pronounced for carboxymethyl-cellulase activity than for filter paper activity. High cellulase activities were observed for several strains of T. longibrachiatum and T. citrinoviride, whereas T. parceramosum formed only low levels of activity. Among the corresponding teleomorphs, most strains of H. schweinitzii were comparatively poor producers, whereas the highest percentage of high producers was found among H. jecorina isolates, and many strains were even more active than the parent T. reesei QM 6a. Immunoblot analysis of corresponding culture filtrates of various H. jecorina strains showed that the three major cellulase proteins (cellobiohydrolase I, cellobiohydrolase II, and endoglucanase I) were present in culture filtrates and their M(r) was identical to that of the respective T. reesei proteins. ELISA analysis demonstrated that these enzymes were also present in the same relative proportions in culture filtrates from H. jecorina and T. reesei. With the aid of primers, corresponding to conserved sequences in the cellobiohydrolase I-encoding gene cbh1, a fragment of this gene was amplified from selected strains of H. jecorina, T. reesei, T. longibrachiatum, T. citrinoviride, and H. schweinitzii. The fragments had the same size in all fungi. Cleavage of this fragment with Hhal produced a RFLP pattern which was identical in H. jecorina and T. reesei, but different in the other species. In the latter, the RFLP pattern was also species specific. These results provide support for a close genetic similarity of T. reesei and H. jecorina cellulases. In the latter, an ascomycetous model system for cellulase biosynthesis is now available. The results further indicate that other anamorphs of Trichoderma section Longibrachiatum are promising sources of high cellulase production.

PCR-fingerprinting used for comparison of ex type strains of Trichoderma species deposited in different culture collections.

PCR-fingerprinting with primers (GACA)4, (GTG)5, M13 (core sequence of phage M13), and OPB-05 was used to compare ex type strains of various species of Trichoderma. The ex type strain of each species was obtained from different culture collections. PCR-fingerprints of each ex type culture, despite of separate cultivation over a long period of time (sometimes decades) by different culture collections, were identical. Ex type strains could be discriminated from a number of other strains belonging to the same species, since every strain was characterized by its individual PCR-fingerprint. PCR-fingerprinting is recommended as a basic tool for proving the identity of strains, especially with regard to comprehensive culture collections. Investigations of Trichoderma todica, a species of undefined systematic position within the genus Trichoderma, suggest a close relationship to Trichoderma paraceramosum. Morphological and molecular data indicate that Trichoderma todica and Trichoderma parceramosum are conspecific.

Fingerprinting reveals gamma-ray induced mutations in fungal DNA: implications for identification of patent strains of Trichoderma harzianum.

We have analyzed different patent strains and gamma-ray induced mutants of Trichoderma harzianum by DNA fingerprinting and PCR fingerprinting (RAPD). Applying wild-type phage M13 DNA, with the oligonucleotides (CT)8 and (GTG)5 as probes for hybridization, as well as the oligonucleotides GGCATCGGCC, (GTG)5, (CAC)5 and the M13 sequence GAGGGTGGCGGTTCT as primers in PCR, we were able to obtain different and discriminative fingerprint patterns for all strains and mutants investigated. Irradiation of fungi led to mutations which resulted in new fingerprint patterns. Consequently, irradiation-induced mutants can be clearly distinguished from the original wild-type isolates by genomic fingerprinting which is of importance for the patent protection of fungal strains. Sequencing of the ITS-1 and ITS-2 regions of the rDNA gene complex revealed the same sequence for all mutant strains and the original wild-type strain.

Rapid identification and differentiation of yeasts by DNA and PCR fingerprinting.

We have used the techniques of DNA fingerprinting and polymerase chain reaction (PCR) with probes specific for hypervariable repetitive DNA sequences (mini- and microsatellite DNAs) to analyze 36 yeast strains belonging to 10 species and 2 genera. Using (GTG)5, (GACA)4, phage M13 DNA and the M13 sequence GAGGGTGGCGGTTCT as probes and primers, respectively, we obtained DNA polymorphisms which allowed us to discriminate 23 biotechnologically important strains of the yeast Saccaromyces cerevisiae and to distinguish them from strains of S. pastorianus, S. bayanus and S. willianus. Our results demonstrate that both DNA and PCR fingerprinting are suitable tools for an easy, fast and reliable molecular typing of yeasts. The DNA fingerprinting method seems to be more sensitive than PCR fingerprinting with respect to the individualization of strains. Nevertheless, using the PCR fingerprinting technique we were able to unambigously discriminate between genotypes of different species. Therefore, PCR fingerprinting might become a useful tool in the classification of yeasts on the basis of phylogenetic relatedness.

DNA- and PCR-fingerprinting in fungi.

DNA-fingerprinting has been successfully used to detect hypervariable, repetitive DNA sequences (minisatellites and microsatellites) in fungi. Combined with methods used to identify random amplified polymorphic DNA (RAPD), conventional DNA-fingerprinting hybridization probes can also be used as single primers to detect DNA polymorphisms among fungal species and strains. The oligonucleotides (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, as well as the phage M13 and its core sequence, have been used as specific probes in hybridization experiments and as primers for PCR analysis. Both methods have enabled the differentiation of all the fungal species and strains that were examined, including species of Penicillium, Trichoderma, Leptosphaeria, Saccharomyces, Candida and Cryptococcus. These methods have been used 1) to clarify the taxonomic relationships among relevant species of the Trichoderma aggregate, 2) to discriminate between aggressive and non-aggressive isolates of the rape seed phytopathogen, Leptosphaeria maculans, and 3) to identify strains of the pathogenic yeasts, Cryptococcus neoformans and Candida albicans. PCR-fingerprinting allowed serotypes of C. neoformans to be distinguished. The application of DNA- and PCR-fingerprinting to fungal DNA should aid in clarification of their taxonomy and improved diagnosis of mycotic disease.