Diversity of Beauveria spp. isolates from pollen beetles Meligethes aeneus in Switzerland Nicolai V. Meyling a,⇑ , Christina Pilz b , Siegfried Keller b , Franco Widmer b , Jürg Enkerli b a Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Denmark b Molecular Ecology, Agroscope Reckenholz-Tänikon Research Station ART, Reckenholzstrasse 191, Zurich, Switzerland article info Article history: Received 13 May 2011 Accepted 3 October 2011 Available online 10 October 2011 Keywords: Entomopathogenic fungi Phylogeny Microsatellite markers Ecological host range Genetic diversity abstract Pollen beetles Meligethes aeneus were collected in oilseed rape fields at different sites in Switzerland in spring 2004–2005 and 32 isolates of the fungal genus Beauveria occurring as latent infections in the bee- tles were obtained and molecularly characterized. Three major clades, Beauveria bassiana sensu stricto (Clade A: n = 13), Beauveria brongniartii (Clade B: n = 1) and Beauveria Clade C (n = 18) were identified among the isolates based on sequences of the ITS region and the 5 0 end of EF1-a. B. bassiana s.s. was fur- ther separated in the two clades, Eu_1 (n = 10) and Eu_4 (n = 3). The intergenic region Bloc provided best resolution of the individual clades B. bassiana s.s. Eu_1, Eu_4 and B. brongniartii. No specific clade of Beau- veria appeared to be associated with adult M. aeneus populations. However, data suggested high relative abundance of Beauveria Clade C among the fungal entomopathogens infecting M. aeneus. Characterization of the isolates by simple sequence repeats (SSR) revealed further genotypic diversity within the clades except B. bassiana s.s. Eu_4 which appeared to be clonal. However, the individual SSR markers were dif- ferentially amplifiable from isolates of the different clades. It is therefore important to identify the under- lying phylogenetic affinity of Beauveria isolates to interpret results based on SSR markers. The data suggest that not all available SSR markers are suitable for reliable characterization of diversity within Beauveria Clade C. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction The pollen beetle Meligethes aeneus F. (Coleoptera: Nitidulidae) is a major pest in oilseed rape cultures and other cruciferous crops in Central and Northern Europe (Nilsson, 1987; Alford et al., 2003; Warner et al., 2008). Adult M. aeneus beetles hibernate in hedge- rows and forested areas and migrate to oilseed rape fields in the following spring (e.g., Alford et al., 2003). There they feed on flower buds, flowers as well as pollen and oviposit into flower buds caus- ing a severe reduction in seed production (e.g. Alford et al., 2003). M. aeneus is attacked by different natural enemies, including pre- dators (e.g., Büchs and Alford, 2003; Warner et al., 2008), parasit- oids (e.g. Nilsson, 2003) and microsporidian parasites (Hokkanen et al., 1988; Lipa and Hokkanen, 1992). Fungal entomopathogens are known to infect a wide range of in- sect species from many different orders and are readily isolated from soils in various ecosystems (e.g., Roy et al., 2010). However, little is known about fungal entomopathogens infecting M. aeneus under natural conditions and only few field surveys have been performed to elucidate this interaction. In Finland a study has revealed no natural infections by fungal entomopathogens (Hokkanen et al., 1988, 2003), while in Switzerland 1.8% of 2139 M. aeneus adults collected in oilseed rape fields in different regions were infected by fungi (Pilz, 2005; Pilz and Keller, 2006) identified as Beauveria spp. (Ascomycota: Hypocreales). Treatments of oilseed rape field soil with Beauveria bassiana as a biological control agent against M. aeneus have resulted in a 50% decrease in winter survival of adult beetles compared to non-treated controls (Hokkanen, 1993). However, the mechanisms leading to the observed effect of the fungal application have not become clear as no epizootics have been observed in M. aeneus in the treated fields (Hokkanen, 1993). Traditional identification of species of the genus Beauveria is principally based on conidial morphology. However, molecular phylogenies have revealed that the genus includes cryptic species (Rehner and Buckley, 2005). The globally distributed Beauveria bassiana sensu lato (s.l.) (Balsamo) Vuillemin morphospecies is not monophyletic and consists of members placed in separate clades which have been proposed to be considered separate spe- cies (Rehner and Buckley, 2005; Rehner et al., 2006; Ghikas et al., 2010; S.A. Rehner pers. comm.). One clade, Clade A, corresponds to B. bassiana sensu stricto (s.s.) and is the sister clade of Clade B which comprises Beauveria brongniartii (Saccardo) Petch (Rehner and Buckley, 2005). A third and distantly related clade, Clade C, 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.10.001 ⇑ Corresponding author. Address: Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Freder- iksberg C, Denmark. Fax: +45 3533 2670. E-mail address: nvm@life.ku.dk (N.V. Meyling). Journal of Invertebrate Pathology 109 (2012) 76–82 Contents lists available at SciVerse ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip