Identification of Trichoderma spp. by DNA Barcode and Screening for Cellulolytic Activity

摘要

Species identification of isolates of Trichoderma from different locations of Nile delta of Egypt was performed and their cellulolytic activities were analyzed. On the basis of morphological characteristics, 75% of isolates were identified to species level and they were divided into four aggregate groups. Morphological characterization alone was insufficient to precisely identify Trichoderma species because they have relatively few morphological characters and limited variation that cause overlapping and misidentification of the isolates. Therefore, there was a necessity to use molecular technique to compensate for the limitations of morphological characterization. DNA sequencing of 5.8S-ITS region was carried out using specific primers ITS1 and ITS4. By comparing the sequences of the 5.8S-ITS region to the sequences deposited in GenBank using BLAST program all isolates can be identified to species level with homology percentage of at least 99%. In addition, TrichOKEY search tool, was used to assess the reliability of Genbank and results were in 92% agreement with the BLAST results. Data indicated a narrow species diversity and there were two main species predominated namely; T. longibarchiatum and T. harzianum. Distribution of nucleotides as well as the (G+C) content in ITS region of isolates indicated a wide range of interspecies variation. Finally, isolates were assessed for their total cellulase activities using a cellulose-azur method, for exoglucanases activity using Avicel method and for endoglucanases activity using carboxymethyl cellulose (CMC) and acid swollen cellulose methods. Consequently, eleven isolates were selected to be the best isolates among the 28 isolates used for cellulolytic ability.