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, fourof which were isolated from Palestine while the remaining six were from other geographicregions. The variation was determined using molecular-based techniques including; sequenceanalysis 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 asMetarhizium anisopliae while the identity of the other four as Beauveria bassiana. These dataalso 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 restrictiondigestion of the ITS2 sequences produced a fingerprint for the PAL-M01 isolate. The ITS andBT 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, andPPRC. Moreover, the ISSR analysis detected high levels of polymorphism among the Beauveriab. (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 beattributed 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 bestof our knowledge, this is the first study, in Palestine, attempting to discriminate between EPFisolates using advanced molecular techniques.