Diversity of Xanthomonas spp

Bacterial spot has become a major problem in Brazilian processing tomato fields due to favorable environmental conditions, lack of resistant cultivars or lack of efficient chemical control. The occurrence of genetic groups/species of Xanthomonas associated with bacterial spot in tomato in Brazil was studied from 1995 to 2000, upon phenotypic and genotypic characterization of 447 strains collected in commercial fields in the Central-West and Northeast. The four recently described Xanthomonas species associated with bacterial spot of tomato were detected. The populations from the Central-West were mainly composed of X. gardneri (group D/race T2). Xanthomonas vesicatoria (group B/race T2) and X. euvesicatoria (group A/race T1) were also present in some fields but at low rates. In the Northeast, only X. euvesicatoria (group A/race T1) and X. perforans (group C/race T3) were found. Genetic variability was analysed using pulsed-field electrophoresis. Strains of X. euvesicatoria, which were collected mostly from an open pollinated variety, were more variable in contrast with those of X. vesicatoria, X. perforans, and X. gardneri. Further collection and characterization of 53 strains from 2002 and 2003 revealed that race T3 is already present in the Central-West associated with imported hybrid cultivars. Surveys on bacterial spot occurrence should be continuous in order to detect eventual changes in population composition. INTRODUCTION Previously distributed to southeast and northeastern states of Brazil, processing tomatoes are now concentrated in the Central-West, which accounts for approximately 80% of cultivated area. High technological cropping systems, which make use of hybrid cultivars and usually imported seeds, sprinkle irrigation, and mechanized sowing, transplanting and harvest are typical in the region, where yields range from 70 to 100 t/ha (tomato processing companies/Embrapa Hortaliças, 2001, unpublished). Among the 101 Proc. 1st IS on Tomato Diseases Eds. M.T. Momol, P. Ji and J.B. Jones Acta Hort 695, ISHS 2005 tomato diseases, bacterial spot has become a major problem due to favorable environmental conditions ensured by sprinkle irrigation, lack of resistant cultivars or lack of efficient chemical control (Lopes et al., 2000). Bacterial spot was until recently thought to be caused by a homogeneous group of strains of Xanthomonas campestris pv. vesicatoria. At present, however, it is accepted that this disease is caused by four different species comprising four genetic/phenotypic groups and three races: X. euvesicatoria (group A/race T1), X. vesicatoria (group B/race T2), X. perforans (group C/race T3) and X. gardneri (group D/race T2) (Jones et al., 2004). These species/groups/races can be reliably distinguished by genomic fingerprinting using different molecular techniques such as rep-PCR, restriction profiles of hrp genes, and rRNA sequence homology (Bouzar et al., 1999; Jones et al., 2000; Obradovic, 2004). Some phenotypic features are also related to the groups, such as the use of cis-aconitic acid and maltose, whole-cell protein profiles, reaction to monoclonal antibodies and hypersensitive reaction on tomato differential varieties. The latter is used for race determination (Jones et al., 1998a). Whereas groups A and B have a worldwide distribution, groups C and D are thought to be restricted to Mexico, Thailand and the USA and to Costa Rica and Yugoslavia, respectively (Jones et al., 2000). The occurrence of these species/groups/races in Brazil was not well known, but ten years ago, Brazilian strains of group A and B from producing regions of fresh market tomatoes were reported by Bouzar et al. (1994). This study was carried out with a group of strains collected in processing tomato fields from 1995 to 2003. Preliminary results of this study have been published elsewhere (Quezado-Duval and Camargo, 2004; Quezado-Duval et al., 2003; Quezado-Duval et al., 2004). In this report, an overview of the population composition of xanthomonads associated with bacterial spot in Brazilian processing tomato and its dynamics during this period is presented. MATERIALS AND METHODS Strain Collection Samplings were done in the contrasting edaphoclimatic regions represented by the Northeast (states of Pernambuco and Bahia) and the Central-West (states of Goiás and Minas Gerais) (Fig. 1). The first region was characterized by a warmer climate, fields of about 2 to 5 ha, of furrow or conventional sprinkler irrigation, and was mostly cultivated with the national open pollinated variety IPA-5. In the second region in high plateaux, the fields ranged from 20 to 50 ha, most of them irrigated by central pivot systems. Diseased leaves and fruits were collected randomly throughout the fields. Nutrient agar medium (NA) was used for isolation. Strains derived from single colonies were stored in phosphate buffer (pH 7.0) at room temperature. From 1995 to 2000, 447 strains obtained from both regions were characterized by pulsed-field electrophoresis (PFGE) and by amylolytic and pectolytic activity. Selected representive strains were further tested for the presence of α or β proteins; utilization of maltose; in vitro sensitivity to copper, streptomycin and oxitetracycline; race determination in relation to tomato and pepper hosts; and presence of avirulence genes avrRxv and avrXv3 by PCR with specific primers (Bouzar et al., 1993; Astua-Monge et al., 2000). Additionally, 53 strains from the CentralWest collected in 2002 and 2003 were partially analysed (races, amylolitic and pectolytic activities, and maltose utilization) and incorporated in the overall analysis. Strains 75-3, XV56, and XG101 were included as reference of X. euvesicatoria, X. vesicatoria, and X. gardneri, respectively (Jones et al., 2000; Jones et al., 2004). PFGE of Digested Genomic DNA PFGE analysis was performed as described by Egel et al. (1991) with minor modifications. Genomic DNA from lysed agar-embedded cells of bacterial strains was digested with Xba I and the DNA fragments resolved in 1.2% agarose gels prepared with 0.5X TBE running buffer. Electrophoresis was carried out in a CHEF-DR II apparatus