Therefore, the potential

usefulness of mt intergenic sequence variation for intra- and inter- species discrimination and phylogenetic studies of Beauveria was examined following an in silico analysis based on criteria of size, complexity and suitability (for designing primers) of all Beauveria mt intergenic regions. More specifically, smaller than 200 bp interenic regions Ferroptosis inhibitor were excluded due to the few informative characters they contained, whereas ideal regions were considered those with sizes between 200-800 bp click here because they can be easily cloned and/or obtained by PCR. Regions containing trn genes -due to their cloverleaf structures- and regions with dispersed repetitive elements were avoided because their structures make them unsuitable for designing primers for PCR amplification (for details of all intergenic regions see Additional File 1, Table S1). Thus, the most suitable intergenic regions following the above criteria for the population analyses were nad3-atp9 and atp6-rns. Population and phylogenetic studies based on ITS1-5.8S-ITS2 and intergenic

mt region sequences PCR amplicons for the ITS1-5.8S-ITS2 region showed little variation in size, being almost identical ITF2357 price for all B. bassiana (480-482 bp) and B. brongniartii (478-481 bp) isolates, but with sizeable differences for the other Beauveria species (471-512 bp). On the contrary, the intergenic nad3-atp9 and atp6-rns amplicons exhibited a much greater variability in sizes even within B. PIK3C2G bassiana isolates, ranging from 259-332 bp for the former and 283-483 bp for the latter (Additional File 2, Table

S2 and Additional File 3, Table S3), thus providing excellent tools for species or species-group identification. For example, using high-resolution agarose electrophoresis (data not shown), nad3-atp9 B. bassiana amplicons can be easily differentiated from the other Beauveria species and at the same time can be grouped into Clades A and C according to their sizes and in congruence to the classification proposed earlier [1] (Additional File 3, Table S3). Variability for the other Beauveria species was even greater, ranging from 84-302 bp and 249-441 bp for the nad3-atp9 and atp6-rns, respectively. When analyzed, these differences were found to be mainly due to deletions and/or additions of 3-5 nucleotides for nad3-atp9, scattered throughout this region, and rarely due to single point mutations. The atp6-rns sequence differences were primarily due to a 4-bp repeat (GCTT) inserted in the corresponding sequence up to 13 times (e.g., R184-483bp), thus providing in many cases excellent tools for isolate identification. Amplicon sequences from all isolates listed in Additional File 2, Table S2 were used to draw phylogenetic trees deduced from NJ analyses (Fig. 2, 3, 4 and 5), and parsimony and Bayesian methods were applied to examine the sensitivity of the resulting trees and tree topologies.