Objectives: Trichoderma spp. are known as cosmopolitan soil inhabiting filamentous fungi. Certain members of the genus are emerging as causative agents of opportunistic infections in humans. Here we present the discriminatory power of different phenetic and phylogenetic approaches applied for the taxonomic characterization of clinical Trichoderma isolates. Methods: Twelve clinical Trichoderma isolates were involved in the experiments. Molecular phylogenetic analysis was performed for the sequences of the internal transcribed spacer 1 and 2 (ITS1 and 2) regions of the rDNA cluster and for the 4th large intron of the gene encoding translation elongation factor 1-alpha (tef1). RFLP patterns of mtDNA were generated by BsuRI and Hin6I. Phenotype profiles were examined by isoenzyme analysis of 7 enzyme systems with cellulose-acetate electrophoresis (CAE) and by carbon source utilization arrays performed on BIOLOG FF microplates. Results: Based on morphological characters, the 12 clinical Trichoderma isolates were originally identified as members of 3 species from section Longibrachiatum: T. longibrachiatum (5), T. pseudokoningii (3), T. citrinoviride (1); and 2 species from section Trichoderma: T. viride (2) and T. koningii (1). However, the ITS barcode identification by TrichOKEY 1.0 (www.isth.info) revealed that all of them belong to the triplet of species T. longibrachiatum/Hypocrea orientalis/H. cerebriformis. Phylogenetic analysis of tef1 sequences shows that 11 strains belong to the clade of T. longibrachiatum, while one is attributed to H. orientalis. The examination of further, non-clinical isolates indicated that the tef1 marker clearly separates these two species. RFLP of mtDNA revealed 7 and 10 different patterns with BsuRI and Hin6I, respectively, resulting in 4 groups on the dendrogram, while CAE separated the strains into 4 distinct electrophoretic types. BIOLOG Phenotype Microarrays were performed for all clinical and a series of non-clinical isolates from several closely related species. Comparisons were done at 9 time points and at 3 temperatures in order to detect possible physiological shifts specific for clinical isolates. Conclusions: Our results support that fungal opportunists belonging to the genus Trichoderma are restricted almost exclusively to section Longibrachiatum. Besides sequence analysis, the methods of CAE, mtDNA RFLP and BIOLOG Phenotype Microarrays proved also appropriate for the characterization of clinical Trichoderma strains.
Full conference title:
16th European Congress of Clinical Microbiology and Infectious Diseases
- ECCMID 16th (2006)