Molecular techniques are necessary for reliable identification of Trichoderma species. For this, sequencing of several genomic loci, such as ITS, TEF1 and RPB2, are advised, but this is costly when a lot of isolates are to be analyzed. RFLP of the ITS region, on the other hand, is a simple and cost-effective method for differentiation between large numbers of Trichoderma species. In the current work, 77 Trichoderma isolates were classified using the ITS-RFLP technique by combination of three restriction enzymes (MspI, NlaIII and NlaIV). Regarding the results, the isolates were divided into 8 groups. Out of these, 14 representative isolates were selected for sequencing of ITS, TEF1 and RPB2. In order to evaluate the enzymes used in this study, 227 ITS sequences of Trichoderma species from the RefSeq database of NCBI were analyzed. Based on these, about 65% of the tested Trichoderma sequences can be categorized into small groups including less than 10 species and thus allowing a low-cost pre-classification. Our results showed that the ITS-RFLP method and the enzymes used in this study can make easy the differentiation of a large numerous of Trichoderma species. We present the ITS-RFLP method using the combination of three restriction enzymes as an efficient and reliable method for the identification of Trichoderma isolates.

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