Development of race-specific molecular marker forXanthomonas campestrispv.campestrisrace 3, the causal agent of black rot of crucifers

Abstract

Race-specific molecular markers were established to distinguish Xanthomonas campestris pv. campestris (Xcc) race 3, the causal agent of black rot disease of crucifers. The available genome sequences of Xcc races were aligned and identified three DNA fragments specific to Xcc race 3. The identified race-specific DNA fragments namely XccR3-49, XccR3-52, and XccR3-55 were used for designing the race-specific primers to detect and identify Xcc race 3. The specificity of race-specific primers was tested against the genomic DNA extracted from Xcc (races 1–7), Xcc strains, Xc pathovars, and other bacterial species. XccR3-49, a specific sequence characterized amplified region (SCAR) primer set, gave a single band with 867 bp length for Xcc race 3 only. The remaining two markers XccR3-52 and XccR3-55 showed polymorphic amplification with amplicon sizes of 1889 and 2109 bp for Xcc race 3, respectively. Additionally, the SCAR primer set detected Xcc race 3 rapidly and efficiently in artificially infected cabbage leaves with bio-PCR. This result showed that the newly developed race-specific markers can successfully and efficiently detect and identify Xcc race 3 from Xanthomonas campestris pv. campestris races, Xanthomonas species/pathovars, as well as other plant pathogenic bacteria (Pseudomonas syringae pv. maculicola and Erwinia carotovora subsp. carotovora). Up to now, this is the first report describing the race-specific marker for the detection of Xcc race 3.

Genetic diversity and virulence of Xanthomonas campestris pv. campestris isolates from Brassica napus and six Brassica oleracea crops in Serbia

“…In view of the importance of identifying this plant pathogenic bacterium at the species, pathovar or race level, many genotyping methods have lately been proposed that are superior to classical methods of identification. Investigators have developed Xcc‐specific primer sets (Park et al , ; Berg et al , ; Singh et al , ) and race 3‐specific molecular markers (Afrin et al , ) for fast molecular identification. The most commonly used techniques for accurate identification and typing of Xanthomonas isolates at species and subspecies level include DNA fingerprinting techniques based on the amplification of repetitive elements, such as REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (Rademaker et al , ; Singh et al , ), as well as multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) using two schemes, both using different housekeeping genes: fusA (elongation factor 4), gap‐1 (glyceraldehyde‐3‐phosphate dehydrogenase A), gltA (citrate synthase), gyrB1 (DNA gyrase B), lacF (PTS system lactose‐specific EIIA component) and lepA (elongation factor 4) (Almeida et al , ), or rpoD (RNA polymerase sigma factor RpoD), dnaK (chaperone protein DnaK), fyuA (TonB dependent receptor) and gyrB2 (Young et al , ).…”

Development of Molecular Marker through Genome Realignment for Specific Detection of Xanthomonas campestris pv. campestris Race 5, a Pathogen of Black Rot Disease

“…Thus, proper detection of Xcc races is crucial to manage this disease and to understand its occurrence and evolution. Recently, race-specific molecular markers for detection of Xcc race 1, race 3 and race 4 have been reported [22,23]. The complete genome sequence of some Xcc races/strains and subspecies opened the avenue for comparative genome analysis which facilitates us in finding out highly specific and variable genomic regions ( Table 2 and Fig.…”

“…The leaves of 3-week-old cabbage plants of a black rot susceptible inbred line (SCNU-C-3328) were inoculated with Xcc races (races 1-8) following the method as previously described by Afrin et al [23] by clipping secondary veins followed by dipping into a bacterial suspension (10 9 CFU/ml) and maintaining high humidity [1]. About 1-cm infected leaves were collected when black rot symptoms were visible and cut into small pieces and soaked in sterile water (250 µl) for 40 min at room temperature.…”

“…The PCR-based race detection technique has been proven as a very powerful, rapid and reliable method for Xanthomonas oryzae pv. oryzae (Xoo) and Xcc [21][22][23]. Thus, one of our research goals is to identify and determine the predominant Xcc races in Korea.…”

“…Thus, one of our research goals is to identify and determine the predominant Xcc races in Korea. Our research group has already developed race-specific molecular markers for Xcc race 1, 3, and 4 [22,23] and some others are under development. Therefore, the objective of this study was to also develop a marker for rapid detection of Xcc race 5.…”