Whole-Genome Re-Alignment Facilitates Development of Specific Molecular Markers for Races 1 and 4 of Xanthomonas campestris pv. campestris, the Cause of Black Rot Disease in Brassica oleracea

Abstract

Black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is a seed borne disease of Brassicaceae. Eleven pathogenic races have been identified based on the phenotype interaction pattern of differential brassica cultivars inoculated with different strains. Race 1 and 4 are the two most frequent races found in Brassica oleracea crops. In this study, a PCR molecular diagnostic tool was developed for the identification of Xcc races 1 and 4 of this pathogen. Whole genomic sequences of races 1, 3, 4 and 9 and sequences of three other Xanthomonas pathovars/species (X. campestris pv. incanae (Xci), X. campestris pv. raphani (Xcr) and X. euvesicatoria (Xev) were aligned to identify variable regions among races. To develop specific markers for races 1 and 4, primers were developed from a region where sequences were dissimilar in other races. Sequence-characterized amplified regions (SCAR) and insertion or deletion of bases (InDel) were used to develop each specific set of primers. The specificity of the selected primers was confirmed by PCR tests using genomic DNA of seven different Xcc races, two strains of X. campestris pathovars and other species of bacteria. Bacterial samples of the races 1 and 4 isolates were collected from artificially inoculated cabbage leaves to conduct bio-PCR. Bio-PCR successfully detected the two Xcc isolates. By using our race-specific markers, a potential race 1 strain from the existing Korean Xcc collection was identified. The Xcc race 1 and 4-specific markers developed in this study are novel and can potentially be used for rapid detection of Xcc races through PCR.

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 molecular marker-based pathogen detection technique has successfully been used to identify plant-infecting bacterial and fungal pathogens. Rubel et al [22] reported molecular markers (InDel and SCAR) for detection of Xcc race 1 and race 4, the pathogen of black rot disease. Later, Afrin et al [23] also reported molecular markers (InDel and SCAR) for detection of Xcc race 3.…”

“…S2). Rubel et al [22] developed 'Xcc_47R1' and 'Xcc_85R1' markers with a similar technique and demonstrated that those markers can distinguish race 1 from race-unknown Xcc strains.…”

“…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.…”