Control biológico de estados edáficos de tefrítidos (Diptera: Tephritidae) mediante tratamientos de suelo con ascomicetos mitospóricos entomopatógenos (Ascomycota: Hypocreales)

Background: The entomopathogenic anamorphic fungus Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spainwere characterized, integrating group I intron insertion patterns at the 3’-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana. Results: Group I intron genotype analysis was based on the four highly conserved insertion sites of the LSU (Ec2653, Ec2449, Ec2066, Ec1921). Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1- phylogeny served to classify most of the strains in the B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin (host, geographic location and climatic conditions) were also examined but in general no association could be found. Conclusion: Most Spanish B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with wordwide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled. BMC Microbiology 11 (2011) 84-95 51 2003), universally primed (UP) PCR (Meyling and Eilenberg, 2006), amplified fragment length polymorphism (AFLP) (Aquino de Muro et al., 2003), isoenzyme analyses (St Leger et al., 1992), or combinations of these methods (Fernandes et al., 2009) have provided useful polymorphisms to access genetic diversity among B. bassiana isolates. Although some molecular studies have correlated B. bassiana genetic groups and host affiliation (Berreta et al., 1998; Gaitan et al., 2002), more recent evidence indicates that this is not the case since B. bassiana contains generalist enthomopathogens with no particular phylogenetic association with their insect host (Fernandes et al., 2009; Meyling et al., 2009), environmental factors being the prime selective forces for genotypic evolution in B. bassiana (Meyling et al., 2009). In this sense, several studies have demonstrated the association between B. bassiana genetic groups and Canadian (Bidochka et al., 2002), Brazilian (Fernandes et al., 2009) and worldwide (Ghikas et al., 2010) climatic zones. Entomopathogenic species displayed a high degree of variability-mainly attributed to the presence of group I intronsat specific sites of the coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA). Group I introns in entomopathogenic fungi were initially reported in Beauveria brongniartii LSU genes (Neuvéglise and Brygoo, 1994). Work addressing the presence and usefulness of these non-coding elements has been reported for Beauveria. For example, Neuvéglise et al. (1997) found 14 form variants of introns, differing in size and restriction patterns, at four different LSU positions from among a panel of 47 isolates of B. brongniartii, two of B. bassiana, and one of Metarhizium anisopliae from several geographic origins. Coates et al. (2002) found 12 intron forms in the SSU from 35 Beauveria isolates. Wang et al. (2003) analyzed the presence of group I introns in the four LSU insertion positions, designated Bb1 (also known as Ec2563), Bb2 (Ec2449), Bb3 (Ec2066) and Bb4 (Ec1921), and distributed a collection of 125 B. bassiana isolates in 13 different genotypes. In that study, their sequence analyses confirmed that the introns were invariably inserted in specific target sequences, and a strong correlation between specific insertion sites and intron subgroups was also observed. In addition, the features and behaviour of these group I intron were also detected in related genera such as Cordyceps (Nikoh and Fukatsu, 2001) and Metarhizium (Pantou et al., 2003). The present study was undertaken to investigate the genetic variability existing in a collection of 53 Spanish isolates of B. bassiana, obtained from different substrates or insect hosts, and 4 isolates from other European countries. The insertion patterns of group I introns at the 3’end of the LSU rDNA genes and EF1-a phylogenetic distribution were integrated in order to explore any possible correlation between genetic groups and geographical/climate origin, and habitat or insect host.