Multilocus genotyping based species identification of entomopathogenic fungi of the genus Lecanicillium (=Verticillium lecanii s.l.).

Mitochondrial gene NADH dehydrogenase subunit 1 (nad1), β-tubulin gene, and elongation factor 1-alpha (tef) were used to characterize and to identify 42 Lecanicillum spp. isolates (former complex species Verticillium lecanii Zimm. Viegas) and to study the phylogenetic relationships in this group. Within the isolates under investigation, Lecanicillium muscarium was the most common species (about 70% of all isolates, collected on the different hosts, predominantly on the insects from the order Hemiptera). Based on nad1 sequencing four main molecular haplotypes were revealed. All four haplotypes have Holarctic origin. Most of them were isolated in the Central part of Russia. One haplotype showed a specific association with the certain geographical area, limited to southwest Georgia and the Krasnodar Territory. For most strains their affiliation to species L. muscarium, L. longisporum, L. psalliotae, L. pissodes were confirmed by the phylogenetic tree, based on the combined sequences of nad1, β-tub, and tef genes. Only five strains of haplotype C and strain F-2643 could not be identified to any present Lecanicillium species and their position remains ambiguous. Thus, the use of multilocus molecular approach based on these genes was useful to identify the Lecanicillium species. Inter-simple sequence repeat (ISSR) study evaluated a high diversity among the L. muscarium strains. The topology of the NJ-tree based on the ISSR-PCR markers has shown the genetic relationships with the support values 62-91% between L. muscarium isolates.