Diversidad y control biológico de insectos - CyberTesis UACh ...

and put it in a phylogeographical perspective, relating any potential pattern with
geographical origin and other relevant traits of the isolates. In addition, the second aim was
to assess the potential use of nuclear gene sequencing for strain fingerprinting.
Materials and methods.
We adopted a three-step sequential approach to examine the genetic variation of Chilean B.
bassiana populations. The rationale and background of this approach are given in detail by
Bernatchez (2001) and Althoff and Pellmyr (2002). The first step was to examine the
potential patterns of relatedness among haplotypes by standard phylogenetic analyses
(phylogenetic trees and haplotype network). Then, we incorporated analyses of
demographic history such as mismatch distributions and surveys of haplotype and
nucleotide diversity. Finally, we performed population genetic analyses to deal with recent
population structure: distance pairwise comparisons and AMOVA seem particularly
suitable to provide insights into genetic structure at this level.
Sample collection.
Over three hundred B. bassiana accessions are hold in the Entomopathogenic Organisms
Collection at Instituto de Investigaciones Agropecuarias (INIA), Ministry of Agriculture,
Chillán, Chile. This collection has been built since 1997, collecting soil samples across
Chile. The preferred isolation method was Galleria mellonella baiting, described by Alves
et al., (1998). Some other accessions have been obtained directly from parasitized
individuals. Strains were preserved under –196 ºC.
Isolate selection and populations.
A total of 97 isolates, morphologically identified as Beauveria bassiana (Balsamo)
Vuillemin, were selected to be included in the study. They were selected with the aim to
represent contrasting ecological conditions, natural and anthropogenic habitats, insect host
and pathogenicity, when they were available. Based on geographic and ecological
characteristics, seven hypothetical populations were defined. Names, number of isolates
and climatic data for each population are shown in Table 1. Monosporic cultures were
obtained through dilution plating method, inoculating Petri dishes with diluted PDY
medium (one-quarter strength). A diluted suspension of spores was spread on a thin layer of
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