ORIGINAL ARTICLE Rapid and reliable DNA extraction and PCR fingerprinting methods to discriminate multiple biotypes of the nematophagous fungus Pochonia chlamydosporia isolated from plant rhizospheres R.H. Manzanilla-Lo ´ pez, I.M. Clark, S.D. Atkins, P.R. Hirsch and B.R. Kerry Nematode Interactions Unit, Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire, UK Introduction The fungus Pochonia chlamydosporia (syn. Verticillium chlamydosporium) (Goddard) Gams and Zare (2001) is a facultative parasite of eggs of sedentary endoparasitic nematodes such as cyst (Heterodera spp. and Globodera spp.), root-knot (Meloidogyne spp.) and false root-knot nematodes (Nacobbus spp.). A range of laboratory and field trials has confirmed its potential as a biological con- trol agent (Atkins et al. 2003; Verdejo-Lucas et al. 2003). Analysis of genetic variation in the fungus at the sub- species level demonstrates that it is related to host nema- tode preference (Morton et al. 2003; Mauchline et al. 2004). These studies relied on single-step polymerase chain reaction (PCR) fingerprinting of DNA extracted from cultured isolates. Many approaches have been used successfully to provide PCR fingerprints of fungi using primers that recognize universal consensus, repeated or arbitrary sequences, including in fungi with biological control potential such as Trichoderma spp. (Arisan-Atac et al. 1995). The most appropriate primers depend on the fungal species under investigation (Bridge et al. 1998). Primers designed originally to amplify the enterobacterial repetitive intragenic consensus (ERIC) sequence Keywords alkaline lysis, DNAeasy Ò, DNA extraction, ERIC-PCR fingerprinting, FTA Ò cards, fungal mycelium, microLYSIS Ò -PLUS, Pochonia chlamydosporia. Correspondence Rosa H. Manzanilla-Lo ´ pez, Department of Plant Pathology and Microbiology, Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK. E-mail: rosa.manzanilla-lopez@bbsrc.ac.uk 2007 ⁄ 2026: received 17 December 2007, revised and accepted 21 July 2008 doi:10.1111/j.1472-765X.2008.02489.x Abstract Aims: To develop a simple, rapid, reliable protocol producing consistent poly- merase chain reaction (PCR) fingerprints of Pochonia chlamydosporia var. chl- amydosporia biotypes for analysing different fungal isolates during co-infection of plants and nematodes. Methods and Results: DNA extracted from different P. chlamydosporia biotypes was fingerprinted using enterobacterial repetitive intragenic consensus (ERIC)- PCR. Four extraction methods (rapid alkaline lysis; microLYSIS Ò -PLUS; DNeasy Ò ; FTA Ò cards) gave consistent results within each protocol but these varied between protocols. Reproducible fingerprints were obtained only if DNA was extracted from fresh fungal cultures that were free of agar. Some DNA degradation occurred during storage, except with the FTA Ò cards, used with this fungus for the first time, which provide a method for long-term archiving. Rapid alkaline lysis and ERIC-PCR identified fungal isolates from root and nematode egg surfaces when plants were treated with different combinations of fungal biotypes; the dominant biotype isolated from the rhizosphere was not always the most abundant in eggs. Conclusions: ERIC-PCR fingerprinting can reliably detect and identify different P. chlamydosporia biotypes. It is important to use fresh mycelium and the same DNA isolation method throughout each study. Significance and Impact of the Study: This evaluation of methods to assess genetic diversity and identify specific P. chlamydosporia biotypes is relevant to other mycelial fungi. Letters in Applied Microbiology ISSN 0266-8254 ª 2008 The Authors Journal compilation ª 2008 The Society for Applied Microbiology, Letters in Applied Microbiology 48 (2009) 71–76 71