ORIGINAL ARTICLE PCR-based assay for the detection of Xanthomonas euvesicatoria causing pepper and tomato bacterial spot C. Moretti, M.T. Amatulli and R. Buonaurio Dipartimento di Scienze Agrarie e Ambientali, University of Perugia, Perugia, Italy Introduction Bacterial spot of pepper and tomato plants, incited by bacterial spot-causing xanthomonads (BSX): Xanthomonas euvesicatoria, Xanthomonas vesicatoria, Xanthomonas perforans and Xanthomonas gardneri (Jones et al. 2004), is one of the most economically important disease; espe- cially, in tropical and subtropical countries. For many years, the disease was attributed to Xanthomonas campes- tris pv. vesicatoria, which was considered to be a relatively homogeneous organism (Jones et al. 1998). In the 1990s, the bacterium was found to be composed of two geneti- cally and phenotypically distinct groups (A and B), which Vauterin et al. (1995) placed into Xanthomonas axonopo- dis pv. vesicatoria (A) and X. vesicatoria (B). Two other BSX pathogenic on tomato plants, assigned to groups C and D, were successively described (Jones et al. 2000). Jones et al. (2004) demonstrated that groups A, C and D have <70% DNA relatedness with each other, with the type strain of Xanthomonas axonopodis and with the cur- rently classified species within the Xanthomonas genus. For these reasons, they renamed A, C and D groups as X. euvesicatoria, X. perforans and X. gardneri, respectively. They also stated that B group can be retained in X. vesicatoria. BSX are quarantine organisms in the European Union (EPPO A2 list) and are subjected to stringent interna- tional phytosanitary measures. Because of the economic and legal importance of the disease, the accurate detection of BSX in pepper and tomato seeds where they can survive for a long period, as well as in asymptomatic greenhouse-grown transplants, is critical and is also the best means to prevent further bacterial dissemination (Stall 1993). Although diagnostic protocols based on the use of semi-selective media and serological tests were developed to detect BSX (McGuire et al. 1986; Tsuchiya et al. 2003), they present many inconvenience (O’Brien et al. 1967; Gitaitis et al. 1987) Keywords bacterial spot disease, diagnosis, pepper, phytobacteria, tomato. Correspondence Chiaraluce Moretti, Dipartimento di Scienze Agrarie e Ambientali, Universita` degli Studi di Perugia, Via Borgo XX Giugno 74, 06121 Perugia, Italy. E-mail: chiaraluce.moretti@unipg.it 2009 ⁄ 1127: received 23 June 2009, revised and accepted 30 June 2009 doi:10.1111/j.1472-765X.2009.02690.x Abstract Aims: To develop a PCR-based assay for Xanthomonas euvesicatoria detection in culture and in planta. Methods and Results: A fragment of 1600 bp specific for X. euvesicatoria was found by repetitive extragenic palindromic sequence-PCR. Among the primers designed on the basis of the partially sequenced fragment, the primers Xeu2.4 and Xeu2.5 direct amplification of the expected product (208 bp) for all the X. euvesicatoria strains and not for other related and unrelated phytopathogenic bacteria or saprophytic bacteria isolated from pepper and tomato phyllosphere. The assay permits the detection of X. euvesicatoria in pure culture, with a limit of detection of two bacterial cells and 1 pg of DNA per PCR, and in extracts obtained from asymptomatic inoculated tomato and pepper plants. Conclusions: Primers Xeu2.4 and Xeu2.5 provide a specific, sensitive and rapid assay for the detection of X. euvesicatoria in culture and in pepper and tomato plants. Significance and Impact of the Study: Because X. euvesicatoria is a quarantine organism in the European Union, and it is subjected to stringent international phytosanitary measures, this highly sensitivity PCR-based assay is suitable for its detection in pepper and tomato plant materials to avoid the introduction and spread of the bacterium. Letters in Applied Microbiology ISSN 0266-8254 466 Journal compilation ª 2009 The Society for Applied Microbiology, Letters in Applied Microbiology 49 (2009) 466–471 ª 2009 The Authors