Detection of potentially valuable polymorphisms in four group I intron insertion sites at the 3'-end of the LSU rDNA genes in biocontrol isolates of Metarhizium anisopliae

Abstract

Background: The entomopathogenic anamorphic fungus Metarhizum anisopliae is currently used as a biocontrol agent (BCA) of insects. In the present work, we analyzed the sequence data obtained from group I introns in the large subunit (LSU) of rDNA genes with a view to determining the genetic diversity present in an autochthonous collection of twenty-six M. anisopliae isolates selected as BCAs.

Results: DNA fragments corresponding to the 3'-end of the nuclear LSU rDNA genes of 26 M. anisopliae isolates were amplified by PCR. The amplicon sizes ranged from 0.8 to 3.4-kb. Four intron insertion sites, according to Escherichia coli J01695 numbering, were detected--Ec1921, Ec2066, Ec2449 and Ec2563--after sequencing and analysis of the PCR products. The presence/absence of introns allowed the 26 isolates to be distributed into seven genotypes. Nine of the isolates tested showed no introns, 4 had only one, 3 two, and 10 displayed three introns. The most frequent insertion sites were Ec1921 and Ec2449. Of the 26 isolates, 11 showed insertions at Ec2563 and a 1754-bp sequence was observed in ten of them. The most-parsimonious (MP) tree obtained from parsimony analysis of the introns revealed a main set containing four-groups that corresponded to the four insertion sites.

Conclusion: Four insertion sites of group I introns in the LSU rDNA genes allowed the establishment of seven genotypes among the twenty-six biocontrol isolates of M. anisopliae. Intron insertions at the Ec2563 site were observed for first time in this species.

Figure 1

Phylogenetic analysis of group I introns of the LSU rDNA genes from entomopathogenic fungi. The MP tree was generated by parsimony analysis after heuristic searching (TBR option). Bootstrap full heuristic analysis with bootstrap intervals from 1000 replications and nodes supported in >50% of bootstrap replicates was generated using the PAUP 4.0 program. The branch lengths are proportional to the number of changes. Eleven different intron sequence types identified from 26 Metarhizium anisopliae isolates were aligned with 21 representative intron sequences from Metarhizium anisopliae (Ma), Beauveria bassiana (Bb), Cordyceps profilica (Cp) and Cordyceps sp. (Csp) and, and an intron sequence from Naegleria sp. (Nsp) was used as an outgroup.