VIEW ARTICLE

Molecular Plant Pathology

Detection of the Bean Common Blight Bacteria, Xanthomonas campestris pv. phaseoli and X. c. phaseoli var. fuscans, Using the Polymerase Chain Reaction. Patrice Audy, Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta T1J 4B1; André Laroche(2), Gilles Saindon(3), Henry C. Huang(4), and Robert L. Gilbertson(5). (2)(3)(4)Research Centre, Agriculture and Agri-Food Canada, Lethbridge, Alberta T1J 4B1; (5)Department of Plant Pathology, University of California, Davis 95616. Phytopathology 84:1185-1192. Accepted for publication 4 August 1994. Copyright 1994 Department of Agriculture, Government of Canada. DOI: 10.1094/Phyto-84-1185.

A 3.4-kb plasmid DNA fragment (p7) of Xanthomonas campestris pv. phaseoli that hybridizes specifically to X. c. phaseoli was subcloned and partially sequenced to design primers for a polymerase chain reaction (PCR) assay for the detection of bacteria causing common blight in bean. In Southern hybridization experiments, p7X3 and p7X4 subfragments showed high specificity for total genomic DNA of pathogenic X. c. phaseoli, whereas the p7X2 segment also weakly hybridized to DNA extracted from some strains belonging to other X. campestris pathovars. For each of these p7 fragments, a set of G+C-rich oligonucleotide primers was devised and tested under high-stringency conditions in PCR assays. The p7X4-derived primers specifically directed the amplification of a 730-bp fragment from DNA of 27 pathogenic X. c. phaseoli isolates from various geographic regions, whereas DNA from 61 other strains of X. campestris, Pseudomonas syringae pv. phaseolicola (the cause of bean halo blight), and plant-pathogenic species of Agrobacterium, Clavibacter, and Erwinia did not produce any discrete bands upon amplification on ethidium bromide-stained agarose gel. An additional fragment of 550 bp was occasionally amplified from the DNA of fuscans strains of X. c. phaseoli. The p7X4 primers successfully mediated the amplification of PCR products of the expected size from DNA extracted from common blight lesions on bean leaves. The high melting temperature of these primers allowed us to shorten the PCR assay time considerably by using a two-step repetitive cycle—95 C for denaturation and 72 C for annealing and extension. As little as 100 fg of X. c. phaseoli DNA (approximately 10 cfu genomic equivalent) could be detected on ethidium bromide-stained agarose gel after a 35-cycle PCR-amplification assay using p7X4-derived primers, and the threshold of detection could be lowered to 10 fg of DNA (approximately 1 cfu genomic equivalent) with two successive amplifications of 20 and 35 cycles.