Natural Distribution And Entomopathogenicity Of Insect-associated Fungal Pathogens From Forest In West China

This study sought to determine the distribution and diversity of Entomopathogenic Fungi(EFs)in soil habitats of forest in west China.The study is composed of two parts: first section is to determine the natural distribution of EFs in Foping Natural Nature Reserve(FNNR)in the Qinling Mountains and second part is to show the diversity of EFs in the forest of Ba Mountains and Ganzi hignland in Sichuan province.The insect-associated fungal pathogen species such as Metarhizium and Beauveria were isolated with semi-selective agar from Shaanxi and Metarhizium,Beauveria,Isaria,Purperocillium and Clonostachys were isolated by insect susceptible bait method with Tenebrio molitor(Coleoptera: Tenebrionidae)from the soil diverse of above forests in Sichuan province.Morphological observation and analysis of DNA sequence data from nuclear gene regions were used to identify taxa and examine phylogenetic relationship.Molecular characterization of Shaanxi EFs isolates using the Mz_IGS3 intergenic region revealed that Metarhizium isolates phylogenetically clustered in the PARB clade with four different distinguishable species,but the 5′-TEF gene allowed only ambiguous delimitation of Metarhizium species.Beauveria isolates were characterized by sequence analyses of the translation elongation factor 1 alpha and the Bloc region.The richness of Metarhizium species decreased with increasing elevation,with Metarhizium robertsii sensu lato being the most abundant species along the elevational gradient in FNNR.Morphological and molecular characterizations of EFs from Sichuan province verified four main genera in insect fungal pathogens,Metarhizium(n= 173 isolates),Beauveria(n= 15 isolates),Isaria(n= 10 isolates),Purpureocillium(n= 1 isolate)and Clonostachys(n= 1 isolate).Maximum likelihood of the both intergenic spacers,Mz_IGS3 and Mz_FG543 sequences of the 173 Metarhizium strains were performed separately.The result revealed that M.pingshaense was predominant species and phylogenetic reconstructions verified one independent Metarhizium cluster in M.anisopliae species complex.We conducted GCPSR-based on multi-loci(a 6796 bp segments composed of whole translation Elongnation Factor-1 alpha 5’ and 3’-TEF),β-tubulin,ITS,with two intergenic spacers(Mz_IGS3 and Mz_FG543)phylogenetic analysis to illustrate this unexplained Metarhizium taxon.We found this cluster is the sister cluster with the M.anisopliae species complex.The relationship between two Metarhizium IGS markers and geographic distances was performed by Mantel test and this test yielded no congruence between geographic isolation positions with phylogenetic species entities suggesting that the natural distribution of Metarhizium species might be independent from geographic locations.