Letters in Applied Microbiology 1999, 28, 258–262 Presence of a 20 bp insertion/deletion in the ITS1 region of Verticillium lecanii R. Zare, V.N. Kouvelis 1 , M.A. Typas 1 and P.D. Bridge CABI Bioscience, Egham, Surrey, UK, and 1 Division of Genetics and Biotechnology, Department of Biology, University of Athens, Greece 2055/98: received 27 January 1999 and accepted 28 January 1999 R. ZARE, V.N. KOUVELIS, M.A. TYPAS AND P.D. BRIDGE. 1999. Polymerase chain reaction (PCR) amplification of the complete ribosomal RNA internally transcribed spacer (ITS) region of 36 isolates of Verticillium lecanii and related species gave a single 620 bp product in 31 isolates. Five isolates received as V. lecanii, however, gave a single product of 600 bp. Restriction fragment analysis of the PCR products from all isolates gave consistent patterns for the 31 isolates with a 620 bp product. The five isolates with the 600 bp product showed only minor discrepancies to these, generally related to the size of only one restriction fragment. The total ITS region was sequenced from 10 typical 620 bp isolates and one 600 bp isolate. Sequence variation between the isolates varied from 0 to 14·5%, and the 20 bp size discrepancy was found to relate to an insertion or deletion in the centre of the ITS1 region. INTRODUCTION The sequence of the internally transcribed spacer (ITS) regions of the ribosomal RNA (rRNA) gene cluster has become established as an important feature of fungal sys- tematics, and numerous studies have shown how conservation and mild sequence variation in these spacers can be used to determine phylogenetic relationships, define species and generate species-specific probes and primers (e.g. Bruns et al. 1991; Boysen et al. 1996; Kuninaga et al. 1997; Mugnier 1998). Sequence variation between members of an individual species will vary according to the species considered and the spacer studied (see Sherriff et al. 1994; Seifert et al. 1995), and typically, the ITS1 region exhibits higher variability than the ITS2 region (Boysen et al. 1996; Kuninaga et al. 1997). Intraspecific variation in ITS sequences of filamentous fungi was reviewed by Seifert et al. (1995) who reported values of generally between 0 and 5% sequence divergence within species. Values of greater than 10% divergence within species have been reported for some fungi (e.g. Viljoen et al. 1993; Neuve ´glise et al. 1994) and extreme cases of over 50% vari- ation have also been reported (e.g. Kuninaga et al. 1997). Apart from transitions, transversions, deletions and dupli- cations, small group-I introns of around 100–400 bp have been reported in both the small and large subunit genes of some filamentous fungi (e.g. Neuve ´glise et al. 1994; Crespo Correspondence to: Dr P.D. Bridge, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK (e-mail: p.bridge@cabi.org). © 1999 The Society for Applied Microbiology et al. 1998). Work on these gene regions in entomopathogenic fungi is rather limited; ITS sequence divergence within spec- ies of Metarhizium has been reported to be between 0 and 5% (Curran et al. 1994; Mavridou and Typas 1998; Driver and Milner 1998), while Beauveria brongniartii and B. bassiana both show greater than 5% sequence divergence (Neuve ´glise et al. 1994; Shih et al. 1995). In the present study, the ITS1 and ITS2 regions of isolates of the V. lecanii species complex are examined. MATERIALS AND METHODS Growth conditions and DNA extraction The 36 isolates of the V. lecanii species complex examined are listed in Table 1. The fungi were grown on either Czapek- Dox with the addition (2 g l -1 ) of casein hydrolysate, malt extract, yeast extract and mycological peptone (Typas et al. 1992), or GYM medium (Mugnai et al. 1989). Liquid cultures were grown in 250 ml flasks containing 60 ml of medium on an orbital shaker at 150 rev min -1 , 25 °C, for 3–6 d. Mycelium was harvested by vacuum filtration, washed with distilled sterile H 2 O and lyophilized. Small scale (50 mg dry weight) extractions of total genomic DNA were made using the pro- tocol of Raeder and Broda (1985) and re-resuspended in 30 ml TE buffer, pH 8·0 (10 mmol l -1 Tris-HCl, 1 mmol l -1 EDTA pH 8·0).