Identification of Xanthomonas campestris pv. mangiferaeindicae using the polymerase chain reaction.
Huang H. C., Chang J. A., Lin Y. C., Chu M. K., Lin H. C., Hsu S. T.
Author Affiliation: Agricultural Biotechnology Laboratories, Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan.
Plant Pathology Bulletin 6 : 1-9
Abstract : A genomic library of the mango pathogen X. c. pv. mangiferaeindicae (XCM) strain HI5 was constructed in the pBluescript SK, and was transformed into Escherichia coli DH10B. Three recombinant clones, designated as pXCM58, pXCM24 and pXCM21, respectively, were randomly selected and labelled by the nonradioactive system with Digoxigenin-11-dUTP (DIG) using the random priming method. In Southern-blot hybridization and dot-blot hybridization analyses, the 3 probes specifically hybridized to XCM strains but not to other microorganisms including Erwinia carotovora, E. chrysanthemi, Pseudomonas solanacearum [Ralstonia solanacearum], X. c. pv. begoniae [X. axonopodis pv. begoniae], X. c. pv. campestris, X. c. pv. citri [X. axonopodis pv. citri], X. c. pv. diffenbachiae [X. axonopodis pv. dieffenbachiae], X. c. pv. pruni [X. arboricola pv. pruni], X. c. pv. vesicatoria [X. vesicatoria] and Colletotrichum gloeosporioides [Glomerella cingulata] chromosomes. Four sets of primers, P21-3/P21-7, P24-3/P24-7, P24-3/P24-1-7 and P58-P1-3/P58-P1-7, were generated from the nucleotide sequences of insert DNAs in pXCM21, pXCM24 pXCM24-1 (subcloned from pXCM24) and pXCM58-P1 (subcloned from pXCM58), respectively. The 4 primer sets specifically amplify 1.9, 2.2, 0.9 and 1 kb DNA fragments, respectively, using chromosomal DNAs of XCM strains as templates in PCR; the rates of detection for the 4 primer sets using 35 strains of XCM were 82.9%, 100%, 94.3% and 100%, respectively. In sensitivity test, P24-3/P24-7 and P58-P1-3/P58-P1-7 detected the lowest level of DNA, 10-100 fg, and lowest number of cells, 100-500. It is concluded that 2 primer sets, P24-3/P24-7 and P58-P1-3/P58-P1-7, can be potentially developed to diagnose fruits naturally infected with bacterial black spot using PCR.