The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
권호 표지
DOI QR코드

DOI QR Code

Development of PCR-Based Molecular Marker for Detection of Xanthomonas campestris pv. campestris Race 6, the Causative Agent of Black Rot of Brassicas

  • Received : 2020.06.14
  • Accepted : 2020.08.11
  • Published : 2020.10.01
Download PDF
⟨ Previous Next ⟩

Abstract

Xanthomonas campestris pv. campestris (Xcc), the pathogen of black rot which is the most destructive disease of Brassica vegetables throughout the world. Here, we reported two novel sequence-characterized amplified region (SCAR) markers (i.e., XccR6-60 and XccR6-67) for the detection of Xcc race 6 via re-alignment of the complete genome sequences of Xcc races/strains/pathovars. The specificity of SCAR primer sets was verified by mean of PCR amplification using the genomic DNA template of Xcc races/strains/pathovars and two other plant infecting bacterial strains. The PCR result revealed that the XccR6-60 and XccR6-67 primer sets amplified 692-bp and 917-bp DNA fragments, respectively, specifically from race 6, while no visible amplification was detected in other samples. In addition, the SCAR primers were highly sensitive and can detect from a very low concentration of genomic DNA of Xcc race 6. However, the complete genome sequence of Xcc race 6 is not yet publicly available. Therefore, the cloning and sequencing of XccR6-60 and XccR6-67 fragments from race 6 provide more evidence of the specificity of these markers. These results indicated that the newly developed SCAR markers can successfully, effectively and rapidly detect Xcc race 6 from other Xcc races/strains/pathovars as well as other plant pathogenic bacteria. This is the first report for race-specific molecular markers for Xcc race 6.

Keywords

References

  1. Afrin, K. S., Rahim, M. A., Jung, H.-J., Park, J.-I., Kim, H.-T. and Nou, I.-S. 2019. Development of molecular marker through genome realignment for specific detection of Xanthomonas campestris pv. campestris race 5, the pathogen of black rot disease. J. Microbiol. Biotechnol. 29:785-793. https://doi.org/10.4014/jmb.1901.01050
  2. Afrin, K. S., Rahim, M. A., Rubel, M. H., Natarajan, S., Song, J.-Y., Kim, H.-T., Park, J.-I. and Nou, I.-S. 2018. Development of race-specific molecular marker for Xanthomonas campestris pv. campestris race 3, the causal agent of black rot of crucifers. Can. J. Plant Sci. 98:1119-1125. https://doi.org/10.1139/cjps-2018-0035
  3. An, S.-Q., Lu, G.-T., Su, H.-Z., Li, R.-F., He, Y.-Q., Jiang, B.-L., Tang, D.-J. and Tang, J.-L. 2011. Systematic mutagenesis of all predicted gntR genes in Xanthomonas campestris pv. campestris reveals a GntR family transcriptional regulator controlling hypersensitive response and virulence. Mol. Plant.-Microbe. Interact. 24:1027-1039. https://doi.org/10.1094/MPMI-08-10-0180
  4. Arias, R. S., Nelson, S. C. and Alvarez, A. M. 2000. Effect of soil-matric potential and phylloplanes of rotation-crops on the survival of a bioluminescent Xanthomonas campestris pv. campestris. Eur. J. Plant Pathol. 106:109-116. https://doi.org/10.1023/A:1008760609372
  5. Berg, T., Tesoriero, L. and Hailstones, D. L. 2005. PCR-based detection of Xanthomonas campestris pathovars in Brassica seed. Plant Pathol. 54:416-427. https://doi.org/10.1111/j.1365-3059.2005.01186.x
  6. Bogdanove, A. J., Koebnik, R., Lu, H., Furutani, A., Angiuoli, S. V, Patil, P. B., Van Sluys, M.-A., Ryan, R. P., Meyer, D. F., Han, S.-W., Aparna, G., Rajaram, M., Delcher, A. L., Phillippy, A. M., Puiu, D., Schatz, M. C., Shumway, M., Sommer, D. D., Trapnell, C., Benahmed, F., Dimitrov, G., Madupu, R., Radune, D., Sullivan, S., Jha, G., Ishihara, H., Lee, S.-W., Pandey, A., Sharma, V., Sriariyanun, M., Szurek, B., Vera-Cruz, C. M., Dorman, K. S., Ronald, P. C., Verdier, V., Dow, J. M., Sonti, R. V., Tsuge, S., Brendel, V. P., Rabinowicz, P. D., Leach, J. E., White, F. F. and Salzberg, S. L. 2011. Two new complete genome sequences offer insight into host and tissue specificity of plant pathogenic Xanthomonas spp. J. Bacteriol. 193:5450-5464. https://doi.org/10.1128/JB.05262-11
  7. Bolot, S., Cerutti, A., Carrere, S., Arlat, M., Fischer-Le Saux, M., Portier, P., Poussier, S., Jacques, M.-A. and Noel, L. D. 2015. Genome sequences of the Race 1 and Race 4 Xanthomonas campestris pv. campestris strains CFBP 1869 and CFBP 5817. Genome Announc. 3:e01023-15.
  8. Bolot, S., Guy, E., Carrere, S., Barbe, V., Arlat, M. and Noel, L. D. 2013a. Genome sequence of Xanthomonas campestris pv. campestris strain Xca5. Genome Announc. 1:e00032-12.
  9. Bolot, S., Roux, B., Carrere, S., Jiang, B.-L., Tang, J.-L., Arlat, M. and Noel, L. D. 2013b. Genome sequences of three atypical Xanthomonas campestris pv. campestris strains, CN14, CN15, and CN16. Genome Announc. 1:e00465-13.
  10. Chidamba, L. and Bezuidenhout, C. C. 2012. Characterisation of Xanthomonas campestris pv. campestris isolates from South Africa using genomic DNA fingerprinting and pathogenicity tests. Eur. J. Plant Pathol. 133:811-818. https://doi.org/10.1007/s10658-012-0002-9
  11. Cook, A. A., Walker, J. C. and Larson, R. H. 1952. Studies on the disease cycle of black rot of crucifers. Phytopathology 42:162-167.
  12. Cruz, J., Tenreiro, R. and Cruz, L. 2017. Assessment of diversity of Xanthomonas campestris pathovars affecting cruciferous plants in Portugal and disclosure of two novel X. campestris pv. campestris races. J. Plant Pathol. 99:403-414.
  13. da Silva, A. C. R., Ferro, J. A., Reinach, F. C., Farah, C. S., Furlan, L. R., Quaggio, R. B., Monteiro-Vitorello, C. B., Van Sluys, M. A., Almeida, N. F., Alves, L. M. C., do Amaral, A. M., Bertolini, M. C., Camargo, L. E. A., Camarotte, G., Cannavan, F., Cardozo, J., Chambergo, F., Ciapina, L. P., Cicarelli, R. M. B., Coutinho, L. L., Cursino-Santos, J. R., El-Dorry, H., Faria, J. B., Ferreira, A. J. S., Ferreira, R. C. C., Ferro, M. I. T., Formighieri, E. F., Franco, M. C., Greggio, C. C., Gruber, A., Katsuyama, A. M., Kishi, L. T., Leite, R. P., Lemos, E. G. M., Lemos, M. V. F., Locali, E. C., Machado, M. A., Madeira, A. M. B. N., Martinez-Rossi, N. M., Martins, E. C., Meidanis, J., Menck, C. F. M., Miyaki, C. Y., Moon, D. H., Moreira, L. M., Novo, M. T. M., Okura, V. K., Oliveira, M. C., Oliveira, V. R., Pereira, H. A., Rossi, A., Sena, J. A. D., Silva, C., de Souza, R. F., Spinola, L. A. F., Takita, M. A., Tamura, R. E., Teixeira, E. C., Tezza, R. I. D., Trindade dos Santos, M., Truffi, D., Tsai, S. M., White, F. F., Setubal, J. C. and Kitajima, J. P. 2002. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417:459-463. https://doi.org/10.1038/417459a
  14. Darling, A. C. E., Mau, B., Blattner, F. R. and Perna, N. T. 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14:1394-1403. https://doi.org/10.1101/gr.2289704
  15. Desai, D., Li, J.-H., van Zijll de Jong, E., Braun, R., Pitman, A., Visnovsky, S., Hampton, J. and Christey, M. 2015. Draft genome sequences of two New Zealand Xanthomonas campestris pv. campestris isolates, ICMP 4013 and ICMP 21080. Genome Announc. 3:e01247-15.
  16. Fargier, E. and Manceau, C. 2007. Pathogenicity assays restrict the species Xanthomonas campestris into three pathovars and reveal nine races within X. campestris pv. campestris. Plant Pathol. 56:805-818. https://doi.org/10.1111/j.1365-3059.2007.01648.x
  17. Grimm, R. and Vogelsanger, J. 1989. Black rot disease on cabbage, irrigation and spreading. In: Proceeding of the 7th International Conference on Plant Pathogenic Bacteria, ed. by Z. Klement, pp. 225-229. Akademiai Kiado, Budapest, Hungary.
  18. Jakubowski, S. J., Krishnamoorthy, V., Cascales, E. and Christie, P. J. 2004. Agrobacterium tumefaciens VirB6 domains direct the ordered export of a DNA substrate through a type IV secretion system. J. Mol. Biol. 341:961-977. https://doi.org/10.1016/j.jmb.2004.06.052
  19. Jensen, B. D., Vicente, J. G., Manandhar, H. K. and Roberts, S. J. 2010. Occurrence and diversity of Xanthomonas campestris pv. campestris in vegetable Brassica fields in Nepal. Plant Dis. 94:298-305. https://doi.org/10.1094/PDIS-94-3-0298
  20. Kamoun, S., Kamdar, H. V., Tola, E. and Kado, C. I. 1992. Incompatible interactions between crucifers and Xanthomonas campestris involve a vascular hypersensitive response: role of the hrpK locus. Mol. Plant-Microbe Interact. 5:22-33. https://doi.org/10.1094/MPMI-5-022
  21. Kim, B. S. 1986. Testing for detection of Xanthomonas campestris pv. campestris in crucifer seeds and seed disinfection. Korean J. Plant Pathol. 2:96-101.
  22. King, E. O., Ward, M. K. and Raney, D. E. 1954. Two simple media for the demonstration of pyocyanin and fluorescin. J. Lab. Clin. Med. 44:301-307.
  23. Leite, R. P. Jr., Minsavage, G. V., Bonas, U. and Stall, R. E. 1994. Detection and identification of phytopathogenic Xanthomonas strains by amplification of DNA sequences related to the hrp genes of Xanthomonas campestris pv. vesicatoria. Appl. Environ. Microbiol. 60:1068-1077. https://doi.org/10.1128/AEM.60.4.1068-1077.1994
  24. Lema, M., Cartea, M. E., Sotelo, T., Velasco, P. and Soengas, P. 2012. Discrimination of Xanthomonas campestris pv. campestris races among strains from northwestern Spain by Brassica spp. genotypes and rep-PCR. Eur. J. Plant Pathol. 133:159-169. https://doi.org/10.1007/s10658-011-9929-5
  25. Lin, Y.-H., Chang, J.-Y., Liu, E.-T., Chao, C.-P., Huang, J.-W. and Chang, P.-F. L. 2009. Development of a molecular marker for specific detection of Fusarium oxysporum f. sp. cubense race 4. Eur. J. Plant Pathol. 123:353-365. https://doi.org/10.1007/s10658-008-9372-4
  26. Liu, Y.-C., Wang, S.-C., Yu, Y.-J., Fung, K.-M., Yang, M.-T., Tseng, Y.-H., Tsai, S.-F., Sun, H. S., Lyu, P.-C. and Chou, S.-H. 2015. Complete genome sequence of Xanthomonas campestris pv. campestris strain 17 from Taiwan. Genome Announc. 3:e01466-15.
  27. Luongo, L., Vitale, S., Haegi, A. and Belisario, A. 2012. Development of SCAR markers and PCR assays for Fusarium oxysporum f. sp. melonis race 2-specific detection. J. Plant Pathol. 94:193-199.
  28. Massomo, S. M. S., Nielsen, H., Mabagala, R. B., Mansfeld-Giese, K., Hockenhull, J. and Mortensen, C. N. 2003. Identification and characterisation of Xanthomonas campestris pv. campestris strains from Tanzania by pathogenicity tests, Biolog, rep-PCR and fatty acid methyl ester analysis. Eur. J. Plant Pathol. 109:775-789. https://doi.org/10.1023/A:1026194402701
  29. Park, H. G. 2006. Genetical improvement of Brassica in Korea. Acta Hortic. 706:31-48. https://doi.org/10.17660/ActaHortic.2006.706.1
  30. Qian, W., Jia, Y., Ren, S.-X., He, Y.-Q., Feng, J.-X., Lu, L.-F., Sun, Q., Ying, G., Tang, D.-J., Tang, H., Wu, W., Hao, P., Wang, L., Jiang, B.-L., Zeng, S., Gu, W.-Y., Lu, G., Rong, L., Tian, Y., Yao, Z., Fu, G., Chen, B., Fang, R., Qiang, B., Chen, Z., Zhao, G.-P., Tang, J.-L. and He, C. 2005. Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris. Genome Res. 15:757-767. https://doi.org/10.1101/gr.3378705
  31. Roux, B., Bolot, S., Guy, E., Denance, N., Lautier, M., Jardinaud, M.-F., Fischer-Le Saux, M., Portier, P., Jacques, M.-A., Gagnevin, L., Pruvost, O., Lauber, E., Arlat, M., Carrere, S., Koebnik, R. and Noel, L. D. 2015. Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome. BMC Genomics 16:975. https://doi.org/10.1186/s12864-015-2190-0
  32. Rubel, M. H., Robin, A. H. K., Natarajan, S., Vicente, J. G., Kim, H.-T., Park, J.-I. and Nou, I.-S. 2017. Whole-genome realignment 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. Int. J. Mol. Sci. 18:2523. https://doi.org/10.3390/ijms18122523
  33. Saha, P., Kalia, P., Sharma, M. and Singh, D. 2016. New source of black rot disease resistance in Brassica oleracea and genetic analysis of resistance. Euphytica 207:35-48. https://doi.org/10.1007/s10681-015-1524-y
  34. Schaad, N. W. and Alvarez, A. 1993. Xanthomonas campestris pv. campestris: cause of black rot of crucifers. In: Xanthomonas, eds. by J. G. Swings and E. L. Civerolo, pp. 51-55. Chapman and Hall, London, UK.
  35. Singh, D., Raghavendra, B. T., Rathaur Singh, P., Singh, H., Raghuwanshi, R. and Singh, R. P. 2014. Detection of black rot disease causing pathogen Xanthomonas campestris pv. campestris by bio-PCR from seeds and plant parts of cole crops. Seed Sci. Technol. 42:36-46. https://doi.org/10.15258/sst.2014.42.1.04
  36. Soengas, P., Hand, P., Vicente, J. G., Pole, J. M. and Pink, D. A. C. 2007. Identification of quantitative trait loci for resistance to Xanthomonas campestris pv. campestris in Brassica rapa. Theor. Appl. Genet. 114:637-645. https://doi.org/10.1007/s00122-006-0464-2
  37. Song, E.-S., Kim, S.-Y., Noh, T.-H., Cho, H., Chae, S.-C. and Lee, B.-M. 2014. PCR-based assay for rapid and specific detection of the new Xanthomonas oryzae pv. oryzae K3a race using an AFLP-derived marker. J. Microbiol. Biotechnol. 24:732-739. https://doi.org/10.4014/jmb.1311.11005
  38. Thieme, F., Koebnik, R., Bekel, T., Berger, C., Boch, J., Buttner, D., Caldana, C., Gaigalat, L., Goesmann, A., Kay, S., Kirchner, O., Lanz, C., Linke, B., McHardy, A. C., Meyer, F., Mittenhuber, G., Nies, D. H., Niesbach-Klosgen, U., Patschkowski, T., Ruckert, C., Rupp, O., Schneiker, S., Schuster, S. C., Vorholter, F.-J., Weber, E., Puhler, A., Bonas, U., Bartels, D. and Kaiser, O. 2005. Insights into genome plasticity and pathogenicity of the plant pathogenic bacterium Xanthomonas campestris pv. vesicatoria revealed by the complete genome sequence. J. Bacteriol. 187:7254-7266. https://doi.org/10.1128/JB.187.21.7254-7266.2005
  39. Vicente, J. G., Conway, J., Roberts, S. J. and Taylor, J. D. 2001. Identification and origin of Xanthomonas campestris pv. campestris races and related pathovars. Phytopathology 91:492-499. https://doi.org/10.1094/PHYTO.2001.91.5.492
  40. Vicente, J. G. and Holub, E. B. 2013. Xanthomonas campestris pv. campestris (cause of black rot of crucifers) in the genomic era is still a worldwide threat to brassica crops. Mol. Plant Pathol. 14:2-18. https://doi.org/10.1111/j.1364-3703.2012.00833.x
  41. Villamil Giraldo, A. M., Sivanesan, D., Carle, A., Paschos, A., Smith, M. A., Plesa, M., Coulton, J. and Baron, C. 2012. Type IV secretion system core component VirB8 from Brucella binds to the globular domain of VirB5 and to a periplasmic domain of VirB6. Biochemistry 51:3881-3890. https://doi.org/10.1021/bi300298v
  42. Vorholter, F.-J., Schneiker, S., Goesmann, A., Krause, L., Bekel, T., Kaiser, O., Linke, B., Patschkowski, T., Ruckert, C., Schmid, J., Sidhu, V. K., Sieber, V., Tauch, A., Watt, S. A., Weisshaar, B., Becker, A., Niehaus, K. and Puhler, A. 2008. The genome of Xanthomonas campestris pv. campestris B100 and its use for the reconstruction of metabolic pathways involved in xanthan biosynthsis. J. Biotechnol. 134:33-45. https://doi.org/10.1016/j.jbiotec.2007.12.013
  43. Wang, B., Hu, X., Li, Q., Hao, B., Zhang, B., Li, G. and Kang, Z. 2010. Development of race-specific SCAR markers for detection of Chinese races CYR32 and CYR33 of Puccinia striiformis f . sp. tritici. Plant Dis. 94:221-228. https://doi.org/10.1094/PDIS-94-2-0221
  44. Williams, P. H. 1980. Black rot: a continuing threat to world crucifers. Plant Dis. 64:736-742. https://doi.org/10.1094/PD-64-736
  45. Yuen, G. Y., Alvarez, A. M., Benedict, A. A. and Trotter, K. J. 1987. Use of monoclonal antibodies to monitor the dissemination of Xanthomonas campestris pv. campestris. Phytopathology 77:366-370. https://doi.org/10.1094/Phyto-77-366
  46. Zaccardelli, M., Campanile, F., Spasiano, A. and Merighi, M. 2007. Detection and identification of the crucifer pathogen, Xanthomonas campestris pv. campestris, by PCR amplification of the conserved Hrp/type III secretion system gene hrcC. Eur. J. Plant Pathol. 118:299-306. https://doi.org/10.1007/s10658-007-9115-y