Group-I intron based strain-specific diagnosis of entomopathogenic Metarhizium spp. fungi from Cuba

Abstract: Mitosporic entomopathogenic ascomycetes from the genus Metarhizium (Ascomycota: Hypocreales) are of particular interest as biological control agents of numerous pest insects. Bioprospection for these fungi in Cuba has given rise to a set of strains from a wide range of hosts. Internal transcribed spacer (ITS) based molecular taxonomic analysis locates the strains within the Metarhizium anisopliae species complex. In order to facilitate the development of strain LBM-41 into a myco-insecticide, a PCR-based diagnostic tool allowing the reliable and fast differentiation of this strain from the other Cuban isolates was needed. Screening of this set of fungal strains for the presence or absence of self-splicing group-I introns disrupting the 18S and 28S rRNA encoding genes at previously identified intron insertion hot-spots revealed a unique intron constellation comprising two group-I introns in insertion positions 1 and 4, for the 28S rRNA gene of strain LBM-41. These findings permit to develop an identification assay for this strain. Primer pairs hybridizing against the 28S rRNA intron sequences were designed and used to amplify partial rRNA gene sequences in a strain specific manner. The approach was shown to unambiguously identify strain LBM-41 among all strains investigated. In conclusion, the feasibility of strain-specific identification based on group-I intron sequences has been demonstrated for a potential Metarhizium biocontrol strain from Cuba.