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

Abstract

Black rot caused by <i>Xanthomonas campestris</i> pv. <i>campestris</i> (Xcc) is the most damaging disease in <i>Brassica</i> crops around the world. In this study, we developed a molecular marker specific to <i>Xcc</i> race 5. To do this, the available whole genome sequences of <i>Xcc</i> races/strains and <i>Xc</i> subspecies were aligned and identified a highly variable genomic region (XccR5-89.2). Subsequently, a primer set covering the 'XccR5-89.2' region was designed and tested against the genomic DNA of <i>Xcc</i> races/strains, <i>Xc</i> subspecies and other plant-infecting bacterial strains (<i>Pseudomonas syringae</i> pv. <i>maculicola</i> and <i>Erwinia carotovora</i> subsp. <i>carotovora</i>). The results showed that the 'XccR5-89.2' primer pair amplified a 2,172-bp fragment specific to <i>Xcc</i> race 5. Moreover, they also amplified a 1,515-bp fragment for <i>Xcc</i> race 1 and an over 3,000-bp fragment for <i>Xcc</i> race 3. However, they did not amplify any fragments from the remaining <i>Xcc</i> races/strains, subspecies or other bacterial strains. The 'XccR5-89.2' primer pair was further PCR amplified from race-unknown <i>Xcc</i> strains and ICMP8 was identified as race 5 among nine race-unknown <i>Xcc</i> strains. Further cloning and sequencing of the bands amplified from race 5 and ICMP8 with 'XccR5-89.2' primers revealed both carrying identical sequences. The results showed that the 'XccR5-89.2' marker can effectively and proficiently detect, and identify <i>Xcc</i> race 5 from <i>Xcc</i> races/strains, subspecies and other plant-infecting bacteria. To our knowledge, this is the first report for an <i>Xcc</i> race 5-specific molecular marker.