Determination of Genetic variation among Beauveria bassiana and Metarhizium anisopliae Isolates from Palestine and other Geographic regions using ITS and β-tubulin Sequences, RFLP and ISSR Data

Abstract

In this work we report the genetic variation of ten isolates of entomopathogenic fungi, four of which were isolated from Palestine while the remaining six were from other geographic regions. The variation was determined using molecular-based techniques including; sequence analysis of β-tubulin (BT) gene, the Internal Transcribed Spacers (ITS) 1 & 2 of the rDNA, Restriction Fragment Length Polymorphism (RFLP), and Inter-Simple Sequence Repeat (ISSR) analysis. The sequencing data of ITS regions confirmed the taxonomic identity of six isolates as Metarhizium anisopliae while the identity of the other four as Beauveria bassiana. These data also revealed that the two Palestinian Beauveria b. isolates are probably new strains since theyshowed 99% homology with the best match sequences reported in the gene bank. The restriction digestion of the ITS2 sequences produced a fingerprint for the PAL-M01 isolate. The ITS and BT sequences were able to distinguish the B-Bot genotype from the other Beauveria b. isolates. In contrast, the ITS could discriminate the three Metarhizium isolates: Ma-7, PAL-M01, and PPRC. Moreover, the ISSR analysis detected high levels of polymorphism among the Beauveria b. (80%) and the Metarhizium a. (90%) isolates. The fact that the PPRC and B-Bot isolates areobviously genetically different from their Metarhizium a. and Beauveria b. counterparts could be attributed to their distant geographic origins. The molecular data obtained in this work can be utilized for specific identification of each ofthese isolates which, would facilitate the local use of these fungi as biocontrol agents. To the best of our knowledge, this is the first study, in Palestine, attempting to discriminate between EPF isolates using advanced molecular techniques.