Detection of Xanthomonas campestris pv. campestris through a real-time PCR assay targeting the Zur gene and comparison with detection targeting the hrpF gene

Abstract

Xanthomonas campestris pv. campestris (Xcc) is a seedborne bacterium that causes black rot of crucifers. A real-time PCR assay based on a dual-labeled hydrolysis TaqMan® probe has been developed for the rapid and sensitive detection of Xcc and related pathovars that affect mainly Brassicaceae crops and ornamentals. Primers were designed to specifically amplify a 152 bp fragment of the Zur gene from X. campestris. To confirm the specificity of the detection, primers targeting the Zur and hrpF genes were used for standard and real-time PCR with DNA samples from 13 Xcc strains, seven Xanthomonas species and pathovars and five different bacterial endophytes including Bacillus, Erwinia, Klebsiella, Pantoea and Pseudomonas, previously isolated from tissues of crucifers. PCR products amplified with Zur and hrpF primers were sequenced to assess the genetic diversity of these genes in the tested isolates. The real-time PCR protocol was optimized to allow the detection at the level of ten copies of Zur PCR fragment per one microliter of DNA. Although the real-time based on detection of Zur also detected X. campestris pvs raphani, armoraciae, incanae and a strain of X. hortorum pv. carotae, it improved the specificity in relation to the previously published hrpF based real-time method. A multiplex assay for Zur and hrpF genes further improved the specificity by excluding X. hortorum pv. carotae. Tests of brassica tissues and seeds artificially inoculated with Xcc showed that the real-time PCR based on detection of Zur is an efficient and robust assay.