Diversity of glycosil hydrolase family 18 genes in environment isolates of the entomopathogenic fungus Metarhizium anisopliae

Abstract

Backgound Metarhizium anisopliae is a model for host-pathogen studies due to its ability to infect several different arthropods. The first barrier to accomplish successful host-infection is transverse the host cuticle, which is a rigid chitin-rich structure. To surpass this barrier, the fungus produces several hydrolytic enzymes, among which are chitinases and endo-b-N-acetylglucosaminidases, glycosil hydrolase 18 (GH18) members [1,2]. In fungi, GH18 enzymes have nutritional importance and exhibit morphogenic and autolytic functions, acting at different processes of fungal development and life cycle maintenance. Assigning functional role for these genes in each process is one of the goals in entomopathogenic fungi study [3]. A genomic analysis performed in our laboratory, in M. anisopliae E6 strain, identified twentythree GH18 putative genes [3-5]. Considering this variety, this study aims to evaluate the diversity of these genes amongst M. anisopliae strains, to access their distribution in environmental isolates of the fungus. DNA samples from 23 M. anisopliae strains (CG291, NORDESTE, CARO7, CG125, CG343, CG374, CG46, CARO12, CARO14, CARO19, CG30, CG97, CG320, AL, MT, M5, CARO11, CARO15, CARO16, CG47, PL57, CG87 e CG491) isolates from different arthropods and places were subjected to PCR analysis to evaluate the presence of each of the 23 GH18 putative genes found in the genome of strain E6. Methods All strains were grown on Cove’s Complete Medium (MCc) agar plates at 28°C until sporulation. Spores were harvested with 0.01% Tween solution and inoculated into liquid MCc cultured in a rotatory shaker at 28oC, 180 rpm for 48 hours. After growth, DNA was extracted using lysis solution and phenol/chloroform method. The presence of GH18 genes was detected by PCR, using primers designed for M. anisopliae strain E6 genes.