Antonie van Leeuwenhoek 80: 1–10, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 1 Development of a PCR test for the detection of Curtobacterium flaccumfaciens pv. flaccumfaciens Patricia Messenberg Guimara ¯ es 1 , Sabrina Palmano 2 , Julian J. Smith 3 , Maria F. Grossi de S´ a 1 & Gerry S. Saddler 4,∗ 1 National Research Center for Genetic Resources and Biotechnology (CENARGEN/EMBRAPA), SAIN Parque Rural, Bras´ ılia-DF, Brazil; 2 Dipartimento di Biologia Applicata alla Difesa delle Piante, Universit` a di Udine, via delle Scienze 208, 3100 Udine, Italy; 3 CABI Bioscience, UK Centre, Bakeham Lane, Egham, Surrey TW20 9TY, UK; 4 Scottish Agricultural Science Agency, 82 Craigs Road, East Craigs, Edinburgh, EH12 8NJ, Scotland ( ∗ Author for correspondence; E-mail: gerry.saddler@sasa.gov.uk) Received 26 June 2000; accepted 9 January 2001 Key words: Curtobacterium flaccumfaciens pv. flaccumfaciens, PCR detection Abstract A chromosomal DNA library of the bacterial pathogen of bean, Curtobacterium flaccumfaciens pv. flaccumfaciens NCPPB 559 was constructed in the plasmid pGEM-7Zf(+). Several clones were identified that hybridised to all Curtobacterium flaccumfaciens pathovars including: C. f. betae, C. f. flaccumfaciens, C. f. oortii, C. f. poinsettiae and, in addition, to some strains of Clavibacter michiganensis subsp. insidiosus and Clavibacter michiganensis subsp. One of these clones (pPMP-26), after subsequent digestion with restriction endonucleases EcoRI/SacI, yielded a fragment of approximately 0.2 Kb (pPMP-26D) that hybridised specifically to C. f. flaccumfaciens and not to any of the other plant pathogenic members of the order Actinomycetales or any of the other prokaryotic bean pathogens tested. This fragment was subcloned and sequenced, analysis of the resultant 198 bp sequence showed that no significant homology existed with any other sequence currently deposited in public databases. Further analysis of these data facilitated the design of PCR primers which were subsequently tested against a wide range of plant pathogenic actinomycetes and other prokaryotic bean pathogens. Results show that these primers are highly specific for all strains of C. f. flaccumfaciens with no cross-reaction to strains from any other bacterial taxa tested. Introduction Curtobacterium flaccumfaciens pv. flaccumfaciens (Hedges) Collins & Jones is the causal agent of bac- terial wilt of beans. The pathogen is seed-borne and of phytosanitary significance (Calzolari et al. 1987; Smith et al. 1997). The bacterium has been reported to cause serious disease outbreaks in the United States (Thomas & Graham 1952; Dowson 1957; Coyne & Schuster 1979; Venette et al. 1995) and its presence has been recorded in diverse geographical areas within Europe, Australia, Asia, North and South America, and Africa (Bradbury 1986; Smith et al. 1997). The long latency period prior to the development of the disease, the relatively slow growth of the bacterium on complex media, its endophytic nature and occur- rence in low numbers (Van Vuurde et al. 1983) have made disease diagnosis and pathogen detection dif- ficult, especially in seed certification programmes or quarantine inspection of imports. To date, a variety of different methods have been employed to circumscribe, identify and detect Cur- tobacterium spp., in particular C. f. flaccumfaciens. These include: numerical taxonomy (Jones 1975; Locci et al. 1989), identification kits (e.g. BIO- LOG; Zhao et al. 1997), analysis of cell wall amino acids (Yamada & Komagata 1972), whole cell pro- teins (Carlson & Vidaver 1982), lipids (Collins et al. 1980; Collins & Jones 1980, 1983; Henningson & Gudmestad 1991) and polyamines (Altenburger et al. 1997). In addition, serology (Lazar 1968; Calzolari et al. 1987; Diatlof et al. 1993; Mizuno et al. 1995;