Abstract
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.
Highlights
Trichoderma species are cosmopolitan fungi, frequently present in all types of soil, manure and decaying plant tissues
Four isolates were given from Tanta university collection and three isolates were from our laboratory collection
The morphological characterization is not reliable for identification of the isolates and oligonucleotide barcode is a powerful tool for the identification of Trichoderma species and should be useful as an alternative or as a complement to morphological methods
Summary
Trichoderma species are cosmopolitan fungi, frequently present in all types of soil, manure and decaying plant tissues. Their dominance in soil may be attributed to their diverse metabolic capability and aggressive competitive nature [1]. Degradation of cellulose results in the release of a bound dye, the vertical migration of which can be observed and the intensity of blue dye indicates the activity of cellulase [6]. Another assay was developed for screening of highly producing exoglucanase isolates using microcrystalline cellulose (Avicel) as substrate crystalline pure cellulose [7]. Quantitative assay of endoglucanase activity can be detected using carboxymethyl cellulose (CMC) by detection of clear zone around the colony using the Congo red stain [9]
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