Entomology Publications Entomology 1-2002 Beauveria bassiana haplotype determination based on nuclear rDNA internal transcribed spacer PCR±RFLP Brad S. Coates Iowa State University, [email protected] Richard L. Hellmich Iowa State University, [email protected] Leslie C. Lewis Iowa State University Follow this and additional works at: http://lib.dr.iastate.edu/ent_pubs Part of the Entomology Commons, and the Plant Breeding and Genetics Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ ent_pubs/87. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Entomology at Iowa State University Digital Repository. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Beauveria bassiana haplotype determination based on nuclear rDNA internal transcribed spacer PCR±RFLP Abstract DNA sequence alignment of the nuclear 5.8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6.62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8.39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR-RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I,Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR-RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived. Disciplines Entomology | Plant Breeding and Genetics Comments This article is from Mycological Research; 106 (2002); 40-50; doi: 10.1017/S0953756201005305 Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted. This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/ent_pubs/87 Mycol. Res. 106 (1): 40–50 (January 2002). # The British Mycological Society 40 DOI: 10.1017\S0953756201005305 Printed in the United Kingdom. Beauveria bassiana haplotype determination based on nuclear rDNA internal transcribed spacer PCR–RFLP Brad S. COATES1,2,3, Richard L. HELLMICH1,3 and Leslie C. LEWIS1,3 " USDA-ARS, Corn Insect and Crop Genetics Research Unit, Genetics Laboratory, c\o Insectary, Iowa State University, Ames, IA 50011, USA. # Interdepartmental Genetics Program, Iowa State University, Ames, IA 50011, USA. $ Department of Entomology, Iowa State University, Ames IA 50011, USA. E-mail: anthesia!iastate.edu Received 21 December 2000; accepted 20 August 2001. DNA sequence alignment of the nuclear 5n8S ribosomal RNA (rRNA) gene and internal transcribed spacers (ITS) from Beauveria bassiana demonstrated that 6n62% sequence variation existed between nine isolates. A higher level of mutation was observed within the ITS regions, where 8n39% divergence occurred. Polymerase chain reaction restriction fragment length polymorphism, PCR–RFLP, and DNA sequence alignment of the ITS1 and ITS2 regions identified seven polymorphic restriction endonuclease sites, Alu I, Hha I, Hinf I, Sin I, Tru 9AI and two Tha I restriction sites. The allelic frequency of each genetic marker was determined from 96 isolates. PCR–RFLP defined 24 B. bassiana genotypes within the sample set, from which eight phylogenetic clusters were predicted to exist. AMOVA and Fst (θ) indicated that no significant correlation existed between B. bassiana haplotype and insect host range as defined by insect order from which each isolate was derived. regions within the initial transcripts have demonstrated INTRODUCTION differential rates of change at the nucleotide level, and The entomopathogenic fungus Beauveria bassiana (As- intraspecies comparison of fungi has shown a pre- comycota, Hypocreales) has shown use within insect ponderance of sequence polymorphism within two biocontrol regimes for suppression of the crop pests internal transcribed spacer (ITS) regions (Neuveglise et Ostrinia nubilalis (Bing & Lewis 1991, 1992), Diabrotica al. 1994, Buscot et al. 1996, Chew, Strongman & unidecimpunctata (Krueger & Roberts 1997) and Dia- MacKay 1997). A moderate level of mutation was brotica virgifera virgifera (Mulock & Chandler 2000). reported in the first internal transcribed spacers (ITS1) Success within biological control practices has spear- of B. bassiana, where two DNA sequences showed 91% headed an interest in the documentation of molecular similarity (Shih et al. 1995). The second B. bassiana differences, and the genetic identification (‘finger- internal transcribed spacer (ITS2) sequence was de- printing’) of particular strains. Variable morphological termined to be invariant, and the 5n8S rRNA gene characteristics, such as conidia size and shape, have showed 98% similarity (Shih et al. 1995). Additional been defined between B. bassiana (Brady 1979a), and nucleotide data have been provided from B. bassiana Beauveria brongniartii (Brady 1979b). The presence of rRNA coding and ITS regions (Rakotonirainy et al. phenotypic diversity with respect to host specificity 1991, 1994, Neuveglise et al. 1994), but no intraspecies implies that genetic differences are present within the comparisons were made. species (St Leger et al. 1992). Mutation detection Mutation detection within DNA sequences can be techniques have proven useful in the differentiation of facilitated by several means. Restriction endonuclease species of Beauveria (Neuveglise et al. 1994, Glare & cleavage of PCR-amplified DNA fragments, PCR– Inwood 1998), yet, insufficient data exists regarding RFLP, has proven useful in the intraspecies differen- molecular differences among B. bassiana isolates. tiation of B. bassiana (Glare & Inwood 1998) and Nuclear ribosomal RNA gene sequences are well B. brongniartii (Neuveglise et al. 1994). Based on a described, with degrees of conservation observed DNA fragment that contained the ITS1, 5n8S rRNA throughout evolution (White et al. 1990) that have gene, and ITS2, Glare & Inwood (1998) reported five allowed for phylogenetic comparison among species RFLPs that separated Beauveria species into two (Guadet et al. 1989, Rakotonirainy et al. 1994). Defined groups. The first contained B. bassiana and B. brong- B. S. Coates, R. L. Hellmich and L. C. Lewis 41 Table 1. Origin, subpopulation designation, and haplotype of 96 Beauveria bassiana isolates as determined by PCR–RFLP of the rRNA ITS regions 1 and 2. Four subpopulations, Lepidoptera (Lep.), Coleoptera (Col.), Hymenoptera\Diptera (HD), Hemiptera\Homoptera and others (HHO), based on insect order(s) from which the isolate was originally recovered (Humber 1992), are separated by horizontal spaces. Restriction fragments are labeled 1 or 0, as described in Table 2. Hi l Hinf I, Tr l Tru 9AI, Th l Tha I, Al l Alu I, Hh l Hha I, and Si l Sin I. ITS1 ITS2 Isolate Host Geographic origin Subpop. Hi Tr Th Al Hh Si Th Haplotype Bb 151 Cydia pomonella France Lep. 1 0 1 1 0 0 1 G (Lepidoptera; Torticidae) Bb 501 Ostrinia nubilalis PR China Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Crambidae) Bb 502 Ostrinia nubilalis PR China Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Crambidae) Bb 533 Ostrinia nubilalis PR China Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Crambidae) Bb 652 O. nubilalis PR China, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Pyralidae) Beijing Bb 959 Spordoptera frugiperda Brazil, Lep. 1 0 1 1 1 0 1 C (Lepidoptera; Noctuidae) Londrina, Parana Bb 1001 Dioryctria sylvestrella Japan, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Pyralidae) Hachiojo, Tokyo Bb 1022 Maize: O. nubilalis infective USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Crambidae) Champaign, IL Bb 1038 Ostrinia nubilalis USA, Lep. 1 0 0 0 1 1 1 N (Lepidoptera; Crambidae) Tulley, NY Bb 1113 Galleria mellonella Italy, Lep. 1 0 0 1 0 0 1 L (Lepidoptera; Pyralidae) Bologna Bb 1121 Ostrinia nubilalis Italy, Lep. 1 0 0 1 0 0 1 L (Lepidoptera; Crambidae) Padus, Venetia Bb 1149 Heliothis aramigera Spain, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Noctuidae) Cordoba Bb 1314 Heliothis virescens France, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Noctuidae) La Miniere Bb 1564 Hyphantria cunea Italy, Lep. 1 0 1 1 1 1 1 L (Lepidoptera; Actiidiae) Cade (Reggio) Bb 2297 Brassolis larva Columbia, Lep. 1 0 1 1 1 0 1 C (Lepidoptera; Brassolidae) Cumaral, Meta Bb 2570 Lymantria dispar USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Lymantriidae) Dover, DE Bb 2619 Brassolis saphorae Brazil, Lep. 1 0 0 1 0 0 1 L (Lepidoptera; Brassolidae) Moju, Para Bb 2629 Diatraea saccharalis Brazil, Lep. 1 0 0 1 0 0 1 L (Lepidoptera; Pyralidae) Usina Salgado, Upojuca Bb 2737 Bombyx mori Phillipines, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Bombycidae) La Trinidad, Benguet Bb 2976 Ostrinia nubilalis USA, Lep. 1 0 0 0 0 0 1 P (Lepidoptera; Crambidae) Centre Co., PA Bb 3113 Soil: O. nubilalis infective USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Crambidae) Ames, IA Bb 3543 Galleria mellonella USA, Lep. 1 0 1 0 0 0 0 I (Lepidoptera; Pyrallidae) Warren, VT EL03 Ostrinia nubilalis USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Cramblidae) Ames, IA EL12 Ostrinia nubilalis USA, Lep. 0 1 0 0 0 0 0 X (Lepidoptera; Cramblidae) Ames, IA EL13 Ostrinia nubilalis USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Cramblidae) Ames, IA EL14 Ostrinia nubilalis USA, Lep. 1 0 1 1 1 1 1 A (Lepidoptera; Cramblidae) Ames, IA EL15 Ostrinia nubilalis USA, Lep.