Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas (original) (raw)
Related papers
Horticulture, Environment, and Biotechnology, 2019
Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of cruciferous crops that causes black rot disease throughout the world. At present, based on the host-pathogen interactions among differential cultivars of Brassica crops, 11 pathogenic Xcc races have been identified, but the race identification method based on host-pathogen interactions is time-consuming. However, early and rapid detection of the pathogen could reduce economic loss by allowing appropriate control measures against black rot disease to be taken more quickly. In this study, a PCR-based molecular marker has been developed for identifying the Xcc race 1 and Xcc race 2 bacterial strains together. The specificity of the marker was tested by PCR using 8 available Xcc races, X. campestris strains, and other bacteria. Upon amplification, a polymorphic band was observed in the PCR amplicon with a size of 1523 bp and 929 bp in Xcc races 1 and 2, respectively. A deletion of 594 bp conferred the specificity in Xcc race 2 compared to race 1. The identified PCR-based molecular marker clearly discriminated the Xcc race 1 and race 2 from other races when tested in artificially infected cabbage leaves. Thus, PCR-based development of an Xcc race 1-and 2-specific marker could be a valuable tool for the accurate detection of Xcc race 1 and 2 together for implementing control measures more quickly. Keywords Molecular marker • PCR assay • Xcc • Races 1 and 2 • Bio-PCR • Black rot disease • Cabbage Communicated by Sung-Chur Sim.
International journal of molecular sciences, 2017
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...
The Plant Pathology Journal
Xanthomonas campestris pv. campestris (Xcc) is a plant pathogen of Brassica crops that causes black rot disease throughout the world. At present, 11 physiological races of Xcc (races 1-11) have been reported. The conventional method of using differential cultivars for Xcc race detection is not accurate and it is laborious and time-consuming. Therefore, the development of specific molecular markers has been used as a substitute tool because it offers an accurate and reliable result, particularly a quick diagnosis of Xcc races. Previously, our laboratory has successfully developed race-specific molecular markers for Xcc races 1-6. In this study, specific molecular markers to identify Xcc race 7 have been developed. In the course of study, whole genome sequences of several Xcc races, X. campestris pv. incanae, X. campestris pv. raphani, and X. campestris pv. vesicatoria were aligned to identify variable regions like sequence-characterized amplified regions and insertions and deletions ...
Plant Disease
Xanthomonas campestris pv. campestris is a causative agent of black rot disease of cruciferous crops. A whole-genome sequence of any race of X. campestris pv. campestris has not been reported from India. The isolate Xcc-C7, race 4, of X. campestris pv. campestris was isolated from cabbage (Brassica oleracea var. capitata) from Bengaluru, in the southern parts of India. Whole-genome sequence data were generated by the next-generation sequencingbased single-molecule real-time sequencing (SMRT) techniques. This study will improve our knowledge of genomic diversity in X. campestris pv. campestris and pave the way for research on host-pathogen interactions (crucifer crops-X. campestris pv. campestris) to develop resistance in cultivated Brassicaceae crops. X. campestris pv. campestris (Pammel) Dowson is a Gram-negative, rod-shaped pathogenic bacterium that causes black rot disease in many cruciferous crops, worldwide. It is a vascular disease developing 'V'-shaped yellow lesions accompanied by blackened veins at margin of the leaf (Fig. 1A). It is genetically diversified by the specific host range of crucifer crops, including B. oleracea vegetables (cole crops such as cabbage, cauliflower, brussels sprout, broccoli, knoll khol, and kale), oilseed crops, ornamentals, and weeds (Singh et al. 2016; Vicente et al. 2001). The annual worldwide spread of black rot disease limits the yield of cole crops under favorable conditions (Singh and Dhar 2011; Singh et al. 2014). The diversity within X. campestris pv. campestris is reported by several researchers (Jensen et al. 2010; Singh and Dhar 2011; Singh et al. 2016). In fact, the pathovar is subdivided into nine races based on host-pathogen interactions (Vicente et al. 2001). According to Singh et al. (2016), only races 1, 4, and 6 of X. campestris pv. campestris of crucifer crops have been identified in India, with races 1 and 4 occurring predominately worldwide. Races 2, 3, and 5 seem to be rare (
Development of a PCR test for detection of Xanthomonas campestris pv. raphani
Australasian Plant Pathology, 2019
Leaf spot of Brassica is an important bacterial seed borne disease caused by X. campestris pv. raphani (Xcr), one of the three pathovars of Xanthomonas campestris species which makes significant economic loss throughout the world. A rapid and reliable detection method will be helpful for early diagnosis of the disease. To do that, we designed Xcr specific sequence-characterized amplified region (SCAR) markers from the Xcr specific genomic fragments, identified by aligning the whole genome sequences of several closely related bacterial pathovars and other bacterial species. Our primer set Xcr_59 produced Xcr specific amplicon of 679 bp in PCR assays. This primer pair was also capable of detecting Xcr strains directly from the artificially infected leaf samples prepared by suspending the diseased leaf area in sterile water without extracting bacterial DNA. This rapid, sensitive and reliable detection technique will be useful for adopting any firsthand plant protection measures against the disease. Keywords X. campestris pv. raphani. campestris pv. Raphani. Leaf spot. PCR marker. Brassica
Plant Disease, 2022
Xanthomonas campestris pv. campestris is a causative agent of black rot disease of cruciferous crops. A whole-genome sequence of any race of X. campestris pv. campestris has not been reported from India. The isolate Xcc-C7, race 4, of X. campestris pv. campestris was isolated from cabbage (Brassica oleracea var. capitata) from Bengaluru, in the southern parts of India. Whole-genome sequence data were generated by the next-generation sequencingbased single-molecule real-time sequencing (SMRT) techniques. This study will improve our knowledge of genomic diversity in X. campestris pv. campestris and pave the way for research on host-pathogen interactions (crucifer crops-X. campestris pv. campestris) to develop resistance in cultivated Brassicaceae crops. X. campestris pv. campestris (Pammel) Dowson is a Gram-negative, rod-shaped pathogenic bacterium that causes black rot disease in many cruciferous crops, worldwide. It is a vascular disease developing 'V'-shaped yellow lesions accompanied by blackened veins at margin of the leaf (Fig. 1A). It is genetically diversified by the specific host range of crucifer crops, including B. oleracea vegetables (cole crops such as cabbage, cauliflower, brussels sprout, broccoli, knoll khol, and kale), oilseed crops, ornamentals, and weeds (Singh et al. 2016; Vicente et al. 2001). The annual worldwide spread of black rot disease limits the yield of cole crops under favorable conditions (Singh and Dhar 2011; Singh et al. 2014). The diversity within X. campestris pv. campestris is reported by several researchers (Jensen et al. 2010; Singh and Dhar 2011; Singh et al. 2016). In fact, the pathovar is subdivided into nine races based on host-pathogen interactions (Vicente et al. 2001). According to Singh et al. (2016), only races 1, 4, and 6 of X. campestris pv. campestris of crucifer crops have been identified in India, with races 1 and 4 occurring predominately worldwide. Races 2, 3, and 5 seem to be rare (
PCR-based detection of Xanthomonas campestris pathovars in Brassica seed
Plant Pathology, 2005
Xanthomonas campestris is a seedborne bacterium that causes black rot of crucifers. Substantial crop losses may result from the rapid spread of the bacteria under favourable conditions, especially those occurring during seedling production. A PCR-based method has been developed for the rapid and sensitive detection of the pathovars of X. campestris that affect crucifers. Primers were designed to specifically amplify a 619 bp fragment of the hrpF gene from X. campestris . Amplification products were not detected from other Xanthomonas species, or from other pathogenic or epiphytic bacteria occurring on these plants. To avoid false-negative results arising from the presence of amplification inhibitors in plant extracts, primers targeting a 360 bp section of the internal transcribed spacer (ITS) region from Brassica spp. were included in a multiplex PCR. The assay readily detected X. campestris pv. campestris infections in diseased plants and from bacterial colonies isolated on growth media, and was more sensitive and specific than traditional plating methods and a commercially available ELISA. A seed-washing protocol was optimized to allow the detection of a single artificially infected seed among 10 000 healthy seeds using the multiplex PCR.
European Journal of Plant Pathology, 2019
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.
A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas
Letters in Applied Microbiology, 2006
Aims: To develop a sensitive real-time PCR-based protocol for the detection of Xanthomonas campestris pathovars from Brassica seed. Methods and Results: A 5¢ nuclease real-time PCR assay was developed to screen Brassica spp. seed for the presence of X. campestris pathovars that cause black rot. The assay amplifies a 78-bp segment of the X. campestris hrpF gene and a 100-bp segment of the Brassica spp. 18S-25S internal transcribed spacer region. The Brassica spp. target provides an internal control for the amplification process to prevent false negatives that may arise from inhibitors that are often present in extracts from plant material. Whilst the primers were compatible with SYBRÒ Green I assays, the use of fluorescently labelled probes in a 5¢ nuclease assay afforded greatest sensitivity and specificity. Seed batches carrying one artificially infected seed among 10 000 were readily detected using the assay. The multiplex real-time PCR assay permitted the rapid detection of pathogenic strains of X. campestris from bacterial colonies, Brassica seed and plants.
Journal of Plant Pathology & Microbiology, 2015
Xanthomonas campestris pv. campestris (Pammel) Dowson (Xcc) is the causal agent of black rot disease of crucifers worldwide. Seventy five isolates of Xcc were collected from 12 agro-climatic regions of India to determine the distribution pattern of races and diversity of the population. Based on pathogenic reaction on seven standard differential crucifers, race 1, 4 & 6 were found to be prevalent. For assessing the pathogenic diversity, forty one cultivars of crucifers comprising seven Brassica and coeno species were inoculated artificially under field conditions. Brassica juncea cultivars (Pusa Bold, Varuna, Pusa Vijay, Pusa Mustard 21 and Pusa Mustard 25) showed resistance against all the strains of Xcc, whereas the Brassica olerecea cultivar Pusa Ageti was found to be resistant to races 1 and 4. Genetic characterization of these 75 strains of Xcc was carried out using rep-PCR (ERIC, REP and BOX-PCRs) followed by phylogenetic analysis. The strains of Xcc clustered into 6 groups at 50% similarity coefficient and among these groups, 28 strains of Xcc belonging to races 1, 4 & 6 were clustered together under Group 5. Sequences of the 16S rRNA, hrpF and efP genes of five strains representing the races 1, 4 and 6 were used for multilocus sequence analysis. Based on sequence analysis of 16S rRNA and hrpF genes, the Indian strains were found to be very closely related to the strain Xcc ATCC 33913 (race 3, UK), whereas based on efP sequences, they were found to be closely related to strains race 1 Xcc B100 (Italy) and race 9 Xcc 8004 (UK).