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

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

Glucosinolate Profile and Glucosinolate Biosynthesis and Breakdown Gene Expression Manifested by Black Rot Disease Infection in Cabbage

Cabbage (Brassica oleracea var. capitata) is an economically important crop in the family Brassicaceae. Black rot disease is a top ranked cabbage disease, which is caused by Xanthomonas campestris pv. campestris (Xcc) and may reduce 50% crop loss. Therefore, we need a clear understanding of black rot disease resistance for sustainable disease management. The secondary metabolites, like Glucosinolate (GSL) presents in Brassica species, which plays a potential role in the defense mechanism against pathogens. However, there is little known about GSL-regulated resistance mechanisms and GSL biosynthesis and the breakdown related gene expression after black rot disease infection in cabbage. In this study, relative expression of 43 biosynthetic and breakdown related GSLs were estimated in the black rot resistant and susceptible cabbage lines after Xcc inoculation. Ten different types of GSL from both aliphatic and indolic groups were identified in the contrasting cabbage lines by HPLC analysis, which included six aliphatic and four indolic compounds. In the resistant line, nine genes (MYB122-Bol026204, MYB34-Bol017062, AOP2-Bo9g006240, ST5c-Bol030757, CYP81F1-Bol017376, CYP81F2-Bol012237, CYP81F4-Bol032712, CYP81F4-Bol032714 and PEN2-Bol030092) showed consistent expression patterns. Pearson’s correlation coefficient showed positive and significant association between aliphatic GSL compounds and expression values of ST5c-Bol030757 and AOP2-Bo9g006240 genes as well as between indolic GSL compounds and the expression of MYB34-Bol017062, MYB122-Bol026204, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714 genes. This study helps in understanding the role of GSL biosynthesis and breakdown related genes for resistance against black rot pathogen in cabbage, which could be further confirmed through functional characterization either by overexpression or knock-out mutation

Development of race-specific molecular marker for Xanthomonas campestris pv. campestris race 3, the causal agent of black rot of crucifers

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 the 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. The 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 such as XccR3-52 and XccR3-55 showed polymorphic amplification with amplicon size of 1889 bp 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.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author