A multiplex-PCR assay for identification of the quarantine plant pathogen Xanthomonas axonopodis pv. phaseoli

• Published in: Journal of microbiological methods 2013-01, Vol.92 (1), p.42-50
• Main Authors: Boureau, T., Kerkoud, M., Chhel, F., Hunault, G., Darrasse, A., Brin, C., Durand, K., Hajri, A., Poussier, S., Manceau, C., Lardeux, F., Saubion, F., Jacques, M.-A.
• Format: Article
• Language: English
• Subjects: algorithms; Bacterial plant pathogens; Bacteriological methods and techniques used in bacteriology; Bacteriological Techniques - methods; Bacteriology; beans; Biological and medical sciences; dipping; Fundamental and applied biological sciences. Psychology; genes; Identification; inoculum; leaves; Life Sciences; Microbiology; Multiplex PCR; Multiplex Polymerase Chain Reaction - methods; pathogenicity; Pathogenicity assay; Phytopathology. Animal pests. Plant and forest protection; Plant Diseases - microbiology; plant pathogens; Plants - microbiology; polymerase chain reaction; Quarantine; Vegetal Biology; Virulence Factors - genetics; Xanthomonas; Xanthomonas axonopodis; Xanthomonas axonopodis - genetics; Xanthomonas axonopodis - isolation & purification; Xanthomonas axonopodis pv. phaseoli
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.10.012

In this study we developed an algorithm to screen for all exact molecular signatures of the quarantine pathogen Xanthomonas axonopodis pv. phaseoli (Xap), based on available data of the presence or absence of virulence-associated genes. The simultaneous presence of genes avrBsT and xopL is specific to Xap. Therefore we developed a multiplex PCR assay targeting avrBsT and xopL for the molecular identification of Xap. The specificity of this multiplex was validated by comparison to that of other molecular identification assays aimed at Xap, on a wide collection of reference strains. This multiplex was further validated on a blind collection of Xanthomonas isolates for which pathogenicity was assayed by stem wounding and by dipping leaves into calibrated inocula. This multiplex was combined to the previously described X4c/X4e molecular identification assay for Xap. Such a combination enables the molecular identification of all strains of Xanthomonas pathogenic on bean. Results also show that assay by stem wounding does not give reliable results in the case of Xap, and that pathogenicity assays by dipping should be preferred. ► An algorithm finds signatures of pathovars from matrices of characters of strains. ► We design a multiplex PCR assay for detection of X. axonopodis pv. phaseoli (Xap). ► For Xap, pathogenicity assays by dipping are more reliable than by wounding.