435 Penny R. HIRSCH, Tim H. MAUCHLINE, Tom A. MENDUM and Brian R. KERRY 1 Soil Science Department and Entomology and Nematology Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK. Accepted 18 June 1999. PCR-based methods to detect Verticillium chlamydosporium on infected plant roots were developed. Arbitrary ERIC primers and those based on rRNA genes, to identify fungi grown in pure culture, were unsuitable for DNA extracted from nematode-infested roots, because of interference by plant and nematode DNA. A novel method utilizing specific primers designed from an amplified and cloned fragment of the V. chlamydosporium β-tubulin gene was developed. Although it could not discriminate between different isolates of V. chlamydosporium, one primer set could identify the fungus on tomato roots infested with root-knot nematodes. The V. chlamydosporium β-tubulin sequence data showed close homology to sequences from plant endophytic Acremonium and Epichloe  species and the saprotrophic Trichoderma viride. INTRODUCTION Biological control and other management methods will become more important when the use of methyl bromide as a soil fumigant for the control of nematodes, soil-borne diseases and weeds in agriculture and horticulture, is banned in 2001 because of its adverse environmental effects (Thomas, 1996). Certain isolates of the nematophagous Verticillium chlamydo- sporium have considerable potential as agents for control of root-knot nematodes (Bourne, Kerry & de Leij, 1996). The fungus colonizes the surface of roots and invades the nematode egg masses which protrude into the rhizosphere. Destruction of eggs and the subsequent control of nematode populations depend on the effectiveness of this fungal invasion, which differs with plant species, nematode density and V. chlamydosporium isolate. Laboratory, pot and microplot experiments to compare different isolates have identified those with potential for biological control (de Leij, Kerry & Dennehy, 1993 ; Bourne, Kerry & de Leij, 1994). Ecological studies and monitoring the fungus in soils after its application are, however, hampered by difficulties in differentiating between the inoculant and native V. chlamydosporium isolates, and other root-colonizing fungi. There is great variation between isolates of V. chlamydosporium in soils which are suppressive to the cereal cyst nematode (Irving & Kerry, 1986) but the importance of such variation for the regulation of nematode populations is unclear. Different V. chlamydo- sporium isolates grown in pure culture can be discriminated using PCR-based DNA fingerprinting (Arora, Hirsch & Kerry, 1996), but approaches which avoid the need for culture will be more practical for analysing large numbers of infected plants from field trials. PCR-based methods to detect mycorrhizal and pathogenic fungi in plant roots have been reported (e.g. Gardes et al., 1991 ; Lovic et al., 1995). These have relied on primers designed to recognize specific sequences within the mito- chondrial genome, or the transcribed (ITS) and non-transcribed (IGS) spacers within the ribosomal RNA genes (White et al., 1990). In this paper, we describe a method that relies on direct DNA extraction from fungi in planta. Our approach involved primers designed to amplify a section of the β-tubulin gene. MATERIALS AND METHODS Cultures of several isolates of V. chlamydosporium and other fungi (Table 1) were maintained on corn-meal agar (Oxoid) at 28 C ; plates were stored at 4. Liquid cultures were grown in Table 1. Fungal isolates Source Verticillium chlamydosporium 10 Meloidogyne incognita eggs, Rothamsted, UK V. chlamydosporium 66 Heterodera avenae eggs, New Zealand V. chlamydosporium 69 H. avenae eggs, New Zealand V. chlamydosporium 77 H. avenae eggs, Hampshire, UK V. chlamydosporium TH6 H. schachtii-infested soil, Germany V. chlamydosporium P H. incognita-infested soil, Pakistan V. dahliae Strawberry, East Malling, UK – D. J. Barbera V. albo-atrum Hop, East Malling, UK – D. J. Barbera V. tricorpus Tomato, East Malling, UK – D. J. Barbera Acremonium sp. Globodera rostochiensis, UK – D. Crump Beauveria bassiana ARSEF2879 Diuraphis noxia USDA, USA – M. Feng Fusarium avenaceum Soil, Rothamsted, UK – D. K. Arora Paecilomyces lilacinus Soil, Rothamsted, UK – P. Williamson P. fumosoroseus ARSEF348 Myzus persicae USDA, USA – A. D. Ali Mycol. Res. 104 (4) : 435–439 (April 2000). Printed in the United Kingdom. Detection of the nematophagous fungus Verticillium chlamydosporium in nematode-infested plant roots using PCR