Morphology and molecular characterization of an entomopathogenic fungus, Metarhizium anisopliae isolate TS6 from north-west of Iran
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عنوان دوره: سومین کنگره بین المللی حشره شناسی ایران
نویسندگان
Department of Plant Protection, Faculty of Agriculture, Azarbaijan Shahid Madani University, Tabriz, Iran,
چکیده
An entomopathogenic fungus, Metarhizium anisopliae, is extensively used as a soil born ubiquitous insect biocontrol agent in integrated pest management programs. The purpose of the study was to understand the entomopathogenic fungi distribution across cultivated and non-cultivated areas in north-west of Iran and characterization of M. anisopliae. In total, more than 100 soil samples were collected randomly. Insect baiting technique using Galleria mellonella larva was also used to isolate fungi from the soil. The larvae were checked daily and dead insects with the symptoms of entomopathogenic fungi infection were subjected to further studies. After washing the cadavers with sodium hypochlorite solution 1%, they were washed three times with distilled water. The cadavers then were transferred to Petri dishes and incubated for two weeks at 25 ºC. Monoxenic cadavers were cultured in Sabouraud dextrose agar supplemented with 0.5% yeast extract (SDAY) medium. The isolated fungus was distinguished based on their morphology and phylogenetic analysis of ITS-rDNA sequence using maximum parsimony, maximum likelihood and neighbor joining methods. After amplification of ITS-rDNA using universal primers, PCR products were subjected to direct sequencing. Five replications of query were used to construct consensus sequence. DNA sequences were edited with Chromas 2.01 and aligned using Clustal X 1.64 with the homologous sequences of other species / isolate of the genus obtained from NCBI. The evolutionary history was inferred using the Maximum Parsimony (MP), Maximum Likelihood (ML) and Neighbor-Joining (NJ) methods. MEGA6 with the following settings was used for evolutionary analysis. The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. The MP tree was obtained using the SPR algorithm in which the initial trees were obtained by the random addition of sequences with ten replicates. For ML analysis, initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the MCL approach, and then selecting the topology with superior log likelihood value. In the case of NJ, the evolutionary distances were computed using the Maximum Composite Likelihood method. Morphological study revealed that the isolate has one-celled, cylindrical and slightly constricted and smooth-walled conidia with branched conidiophore. Reconstruction of phylogenetic trees based on ITS-rDNA sequence of isolate TS6 and homologous ones approved accuracy of identification as Metarhizium anisopliae by placing it in a monophyletic clade containing well-defined members of the species.
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