Variation in morphology and ribosomal DNA among isolates of Metarhizium anisopliae from Thailand.

Abstract

Metarhizium anisopliae is an important entomopathogenic fungus that mainl y used for the biological control. The objectives of this work wer e to isolate and identify varieties of Metarhizium anisopliae mainly based on the morphological and molecular characteristics. We investigated 24 Metarhizium isolates which collected from different insect host s and soil from different sites in Thailand and one isolate was col lected from the Philippines. These isolates have been partially characterized using morphologic al traits such as features of colony morphology, size and shape of conidia that were gro wn on Potato-dextrose-agar (PDA) at room temperature. Colony features could be separated int o five groups. The length/width ratio of the conidia were calculated and generated into three gr oups: ≤ 2.18 is low ratio group, between 2.33 and 2.87 is medium ratio group and ≥ 2.91 is high ratio group. Internal transcribed spa cer (ITS) and 5.8S DNA sequences analysis was employed to identify genotypes. The ITS regions were amplified using the ITS1 and ITS4 primers that was a unique fragment of approximately 550 bp. The sequences were aligned using the Clusta lX program and compared with 19 isolates of Metarhizium available in the GenBank/NCBI database and 2 isolates Beauveria bassiana sequences as outgroup. Phylogenetic trees construct ed using the Neighbor-joining algorithm in the Phylip package. Our study showed that M. anisopliae , M. album , M. anisopliae var. acridum and M. flavoviride can be clearly differentiated and generated the 25 isolates into four main groups (Clade B, C, E and F). However, it is n ot clearly difference between M. anisopliae var. anisopliae and M. anisopliae var. majus and most of them are related to M. anisopliae var . anisopliae . This study indicated that M. anisopliae are highly divergent. Nevertheless, most of M. anisopliae isolates from Thailand distantly related to M. anisopliae from other countries. It would be interesting to compare them using other molecular technique in the future.