Molecular Identification of Local Isolates Aspergillus nidulans from Erbil Province Using Internal Transcribed Spacers

Abstract

Aspergillus nidulans recognized as unique cell factory to manufacture several of useful natural products for human beings. Also it considered as a rich reservoir for wide range of the unknown secondary metabolites. Due to the great capacity of Aspergillus nidulans for fabrication of valuable natural products and in order to have deeper insight for biosynthesis of these metabolites, precise identification is required. Accurate identification of Aspergillus nidulans from local isolates at the species level based on the multi-copies of internal transcribed regions of the rDNA unit (ITS1-5.8S-ITS2 rDNA), was the aim of current study. Thus, in this study, morphological features, molecular approach, sequence alignment, phylogenetic analysis and detection of the matrix diversification for amplified ITS regions, in combination with highest BLAST bit score, utilized for accurate identification of Aspergillus nidulans. Therefore, soil samples of Erbil province from different agricultural fields subjected for this study. Out of 106 samples only 12 of them successfully identified as Aspergillus nidulans. Similarity and uniformity of isolates determined by aligning sequencing results of amplified ITS1-5.8S-ITS2 regions with available sequences of close related species in NCBI GenBank database. Sequence alignment results demonstrate that, all isolates displayed (%100) similarity with sequence of at least 14 species previously identified as Aspergillus nidulans. Concurrently, they displayed (%100) similarity with other species in the same genus as well, such as with Aspergillus rugulosus, Aspergillus variecolor, Aspergillus rugulovalvus and Emericella dentate. Furthermore, Maximum likelihood method used for construction of phylogenetic tree for all isolates with referenced strain Aspergillus nidulans with accession number (AF138289) to ascertain phylogenetic position of isolates. Interpretation for outcome of phylogenetic analysis of isolates, determine that, isolates (MW228089, MW228090, MW228094, MW228095, MW228096, MW228097, MW228098, and MW228099) belonged to an ITS genotype corresponding to the reference strain Aspergillus nidulans.While due to the interspecies variation, profound changes, single and multiple substitutions in ITS1 region of isolates (MW228088, MW228091, MW228092 and MW228093), they displayed divergent phylogenetic position in dendrogram in compare with reference strain. In spite of that, taxonomic analysis results revealed that, all isolates be affiliated with class of Aspergillaceae (124 hits). Overall in present study we confirmed that, colony morphology, molecular approche, sequence alignment analysis for amplified regions ITS1 and ITS2 in combination with highest aligned bit score of species addressed in listed of BLAST search required for accurate identification of isolates at the species level and distinguishing them from other close related species.