Development of species-specific diagnostic primers for Zoophthora radicans and Pandora blunckii; two co-occurring fungal pathogens of the diamondback moth, Plutella xylostella Ariel W. GUZMA ´ N-FRANCO a,c, *, Simon D. ATKINS b , Peter G. ALDERSON c,y , Judith K. PELL a a Department of Plant and Invertebrate Ecology, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK b Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK c School of Biosciences, University of Nottingham, Loughborough, Leicestershire LE12 5RD, UK article info Article history: Received 31 January 2007 Received in revised form 25 March 2008 Accepted 1 April 2008 Corresponding Editor: Richard A. Humber Keywords: Bicontrol Entomogenous fungi Entomophthorales Genetic variation Species-specific primers abstract Species-specific primers for Zoophthora radicans and Pandora bluckii were developed. To achieve this, partial sequences of DNA that encode for rRNA, more specifically, the ITS region (rDNA-ITS) were obtained from different isolates and analysed. Seven Z. radicans isolates (four from P. xylostella, and three from other lepidopteran hosts) and one P. blunckii isolate (from P. xylostella) were used. These isolates were selected based on PCR-RFLP patterns obtained from 22 isolates of P. blunckii and 39 isolates of Z. radicans. All P. blunckii isolates were from the same host (P. xylostella); 20 isolates were from Mexico, one from the Philippines, and one from Germany. The Z. radicans isolates were more diverse in geo- graphical origin (Mexico, Kenya, Japan, New Zealand, Australia, Taiwan, Philippines, Malaysia, Uruguay, France, USA, Poland, Indonesia, Switzerland, Israel, China, and Denmark) and host origin (Lepidoptera, Hemiptera, Hymentoptera, and Diptera). Using conven- tional PCR, each pair of species-specific primers successfully detected each species of fun- gus from DNA extracted from infected host larvae either single- or dual-inoculated with both fungal species. The PCR-RFLP analysis also showed that Z. radicans was genetically more diverse than P. blunckii, although only a limited number of P. blunckii isolates from one country were considered. There was no direct relationship between genetic diversity and host or geographical origin. The relationship between genetic variation within both fungal species and host specificity or ecological adaptation is discussed. ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. Introduction The entomopathogenic fungi Zoophthora radicans and Pandora blunckii (Glomeromycetes: Entomophthorales) are amongst the most important natural enemies of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) (Pell et al. 2001). Zoophthora radicans, Pandora blunckii, and their host, Plutella xylostella, co-occur in space and time (Riethmacher & Kranz 1994; Velasco-Silva et al. 2000), and the fungi may, therefore, interact with each other directly within individuals and * Corresponding author. Current Address: Instituto de Fitosanidad, Colegio de Postgraduados, Km 36.5, Carretera Mexico-Texcoco, Montecillo, Texcoco, Estado de Mexico, 56230, Mexico. Tel.: þ52 595 9520200x1741; fax: þ52 595 9520267. E-mail address: gariel@colpos.mx y Present address: The University of Nottingham Malaysia Campus, Jalan Broga, 43500 Semenyih, Selangor Darul Ehsan, Malaysia. journal homepage: www.elsevier.com/locate/mycres mycological research 112 (2008) 1227–1240 0953-7562/$ – see front matter ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.mycres.2008.04.006