Department of Biotechnology, Guru Jambheshwar University, Hisar, Haryana, India Characterization of Xanthomonas axonopodis pv. cyamopsidis, the Bacterial Blight Pathogen of Cluster Bean Using PCR-based Molecular Markers B. B. Kaur Kaur, S. S. Purkayastha Purkayastha, N. N. Dilbaghi Dilbaghi and and A. A. Chaudhury Chaudhury AuthorsÕ address: Department of Biotechnology, Guru Jambheshwar University, Hisar 125001, Haryana, India (correspondence to A. Chaudhury. E-mail: ashokchaudhury@hotmail.com) Received March 4, 2005; accepted June 1, 2005 Keywords: bacterial blight, cluster bean, genetic diversity, IS1112-polymerase chain reaction, pathogenicity, random amplified polymorphic DNA, repetitive polymerase chain reaction Abstract Xanthomonas axonopodis pv. cyamopsidis causes bacterial blight of cluster bean, an industrially import- ant legume. A total of 76 isolates of X. axonopodis pv. cyamopsidis obtained from cluster bean fields of north and north-west India were identified at the pathovar level using virulence trials on the susceptible cluster bean genotype, PNB which confirmed their phytopath- ogenic specialization on cluster bean. Different polym- erase chain reaction (PCR)-based techniques, such as, random amplified polymorphic DNA (RAPD)–PCR, repetitive (rep)-PCR and IS1112-PCR were success- fully employed to elucidate the pathogen population structure and intrapathovar relationship among a set of 25 X. axonopodis pv. cyamopsidis isolates. There was a significant variation in their pathogenicity and genetic structure. The pathogenicity data were found to be in line with the molecular data to some extent. RAPD markers revealed a high level of genetic diver- sity across the isolates employed in comparison with the other molecular markers employed. A weak corre- lation was observed among the various molecular markers employed as each technique explores genetic variation differently. There was no correlation between DNA amplification product patterns and geographic sites of isolation, suggesting that this bacterium has spread largely through exchange of infected plant germplasm. This is the first report on the molecular characterization of X. axonopodis pv. cyamopsidis iso- lates from India. Introduction Bacterial blight of cluster bean or guar, first repor- ted by Patel et al. (1953) as bacterial leaf spot and later as bacterial blight by Patel and Patel (1958), caused by Xanthomonas axonopodis pv. cyamopsidis is a major impediment to cluster bean production in rain-fed and irrigated environments in India. Yield losses of as high as 68% have been reported (Gandhi and Chand, 1985). This seed-borne disease (on the seed coat or in the embryo) causes infesta- tion of plants from the seedling stage until maturity (Srivastava and Rao, 1963). Principal symptoms include large angular necrotic lesions at the tips of leaves, yellow leaf spots, blighting, wilting which causes defoliation and in severe cases vascular necro- sis, dieback and black streaking of the stem along with continuous oozing of exudates. Infected/contam- inated seed or the propagating planting material is a major source of inoculum for most of the phytobac- terial disease manifestation. Furthermore, the bacter- ium survives in tissue without causing discernable symptoms. Cluster bean [Cyamopsis tetragonoloba (L.) Taub] is grown mainly in arid and semiarid regions of the Indian subcontinent and to a small extent in Australia, Brazil, South Africa, Oklahoma and Texas in North America. In India, it is grown in the states of Rajasthan, Haryana, Gujarat and parts of Punjab and Uttar Pradesh under rain-fed conditions. Cluster bean endosperm contains signifi- cant amounts of galactomannan gum, which has tre- mendous industrial application, and bacterial blight infestation adversely affects the quality and quantity of gum production, which earns considerable foreign exchange to the country. This entails the need for designing effective disease management programme in order to save this economically important crop and regimens are, therefore, required to monitor persist- ence and distribution of this important pathogen. Detailed characterization of the genetic variability among strains of X. axonopodis pv. cyamopsidis has not been reported despite the prevalence and increas- ing economic importance of this bacterium. In sev- eral other crop species, the use of DNA-based molecular markers is a novel strategy in undertaking genetic diversity studies for gaining an insight into the intricate population structure of plant pathogenic bacteria. Analysis of genomic DNA using polymerase www.blackwell-synergy.com J. Phytopathology 153, 470–479 (2005) Ó 2005 Blackwell Verlag, Berlin