DOI QR코드

DOI QR Code

Race- and Isolate-specific Molecular Marker Development through Genome-Realignment Enables Detection of Korean Plasmodiophora brassicae Isolates, Causal agents of Clubroot Disease

  • Received : 2017.12.20
  • Accepted : 2018.08.30
  • Published : 2018.12.01

Abstract

Clubroot is one of the most economically important diseases of the Brassicaceae family. Clubroot disease is caused by the obligate parasite Plasmodiophora brassicae, which is difficult to study because it is nonculturable in the laboratory and its races are genetically variable worldwide. In Korea, there are at least five races that belongs to four pathotype groups. A recent study conducted in Korea attempted to develop molecular markers based on ribosomal DNA polymorphism to detect P. brassicae isolates, but none of those markers was either race-specific or pathotype-specific. Our current study aimed to develop race- and isolate-specific markers by exploiting genomic sequence variations. A total of 119 markers were developed based on unique variation exists in genomic sequences of each of the races. Only 12 markers were able to detect P. brassicae strains of each isolate or race. Ycheon14 markers was specific to isolates of race 2, Yeoncheon and Hoengseong. Ycheon9 and Ycheon10 markers were specific to Yeoncheon isolate (race 2, pathotype 3), ZJ1-3, ZJ1-4 and ZJ1-5 markers were specific to Haenam2 (race 4) isolate, ZJ1-35, ZJ1-40, ZJ1-41 and ZJ1-49 markers were specific to Hoengseong isolate and ZJ1-56 and ZJ1-64 markers were specific to Pyeongchang isolate (race 4, pathotype 3). The PCR-based sequence characterized amplified region (SCAR) markers developed in this study are able to detect five Korean isolates of P. brassicae. These markers can be utilized in identifying four Korean P. brassicae isolates from different regions. Additional effort is required to develop race- and isolate-specific markers for the remaining Korean isolates.

E1PPBG_2018_v34n6_506_f0001.png 이미지

Fig. 1. Alignment of whole genome sequences of Plasmodiophora brassicae. (A) Alignment of five isolates: the dotted rectangles in Yeoncheon and ZL1 indicate the regions where isolate-specific markers were targeted. (B) Identified Yeoncheon- and ZJ1-specific markers regions are highlighted by black arrows.

E1PPBG_2018_v34n6_506_f0002.png 이미지

Fig. 2. Three markers specific to the Yeocheon (Ycheon 9 and Ycheon 10), Yeocheon and Hoengseong (Ycheon14) isolates of race 2. Akimeki is a control host. R, race; P, pathotype.

E1PPBG_2018_v34n6_506_f0003.png 이미지

Fig. 3. Three markers showing specificity to the Haenam2 isolate of race 4 and pathotype 4. Akimeki, a Japanese Chinese cabbage cultivar, was a control host. R, race; P, pathotype.

E1PPBG_2018_v34n6_506_f0004.png 이미지

Fig. 4. Four markers showing specificity to the Hoengseong isolate of race 2 and pathotype 1. Akimeki, a Japanese Chinese cabbage cultivar, was a control host. R, race; P, pathotype.

E1PPBG_2018_v34n6_506_f0005.png 이미지

Fig. 5. Four markers showing specificity to the Pyeongchang isolate of race 4 and pathotype 3. Akimeki, a Japanese Chinese cabbage cultivar, was a control host. R, race; P, pathotype.

Table 1. Plasmodiophora brassicae isolates used for comparing genome sequences. Sequences of e3 and ZJ-1 isolates were compared to four Korean isolates

E1PPBG_2018_v34n6_506_t0001.png 이미지

Table 2. Forward and reverse primer sequences used to identify Korean Plasmodiophra brassicae isolates

E1PPBG_2018_v34n6_506_t0002.png 이미지

Acknowledgement

Supported by : Ministry of Agriculture, Food and Rural Affairs in the Republic of Korea (MAFRA)

References

  1. Bryngelsson, T., Gustafsson, M., Green, B. and Lind, C. 1988. Uptake of host DNA by the parasitic fungus Plasmodiophora brassicae. Physiol. Mol. Plant Pathol. 33:163-171. https://doi.org/10.1016/0885-5765(88)90017-3
  2. Buczacki, S. T., Toxopeus, H., Mattusch, P., Johnston, T. D., Dixon, G. R. and Hobolth, L. A. 1975. Study of physiologic specialization in Plasmodiophora brassicae: Proposals for attempted rationalization through an international approach. Trans. Br. Mycol. Soc. 65:295-303. https://doi.org/10.1016/S0007-1536(75)80013-1
  3. Buhariwalla, H. and Mithen, R. 1995. Cloning of a Brassica repetitive DNA element from resting spores of Plasmodiophora brassicae. Physiol. Mol. Plant Pathol. 47:95-101. https://doi.org/10.1006/pmpp.1995.1045
  4. Buhariwalla, H., Greaves, S., Magrath, R. and Mithen, R. 1995. Development of specific PCR primers for the amplification of polymorphic DNA from the obligate root pathogen Plasmodiophora brassicae. Physiol. Mol. Plant Pathol. 47:83-94. https://doi.org/10.1006/pmpp.1995.1044
  5. Darling, A. E., Mau, B. and Perna, N. T. 2010. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PloS One 5:e11147. https://doi.org/10.1371/journal.pone.0011147
  6. Dixon, G. R. 2009. The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease. J. Plant Growth Regul. 28:194-202. https://doi.org/10.1007/s00344-009-9090-y
  7. Faggian, R. and Strelkov, S. E. 2009. Detection and measurement of Plasmodiophora brassicae. J. Plant Growth Regul. 28:282-288. https://doi.org/10.1007/s00344-009-9092-9
  8. Hatakeyama, K., Fujimura, M., Ishida, M. and Suzuki, T. 2004. New classification method for Plasmodiophora brassicae field isolates in Japan based on resistance of F1 cultivars of Chinese cabbage (Brassica rapa L.) to clubroot. Breed. Sci. 54:197-201. https://doi.org/10.1270/jsbbs.54.197
  9. Ito, S., Maehara, T., Tanaka, S., Kameyaiwaki, M., Yano, S. and Kishi, F. 1997. Cloning of a single-copy DNA sequence unique to Plasmodiophora brassicae. Physiol. Mol. Plant Pathol. 50:289-300. https://doi.org/10.1006/pmpp.1997.0087
  10. Ito, S., Maehara, T., Maruno, E., Tanaka, S., Kameya-Iwaki, M. and Kishi, F. 2008. Development of a PCR-based assay for the detection of Plasmodiophora brassicae in soil. J. Phytopathol. 147:83-88.
  11. Jo, E., Jang, K., Choi, Y., Ahn, K. and Choi, G. 2016. Resistance of cabbage plants to isolates of Plasmodiophora brassicae. Korean J. Hortic. Sci. Technol. 34:442-452.
  12. Jo, S. J., Shim, S. A., Jang, K. S., Choi, Y. H., Kim, J. C. and Choi, G. J. 2011. Resistance of cultivars of Chinese cabbage to Plasmodiophora brassicae isolates of several races collected in Korea. Korean J. Hortic. Sci. Technol. 29:610-616.
  13. Karling, J. S. 1968. The Plasmodiophorales. 2nd ed, Hafner Publishing Company, New York, USA. 256 pp.
  14. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Mentjies, P. and Drummond, A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647-1649. https://doi.org/10.1093/bioinformatics/bts199
  15. Koressaar, T. and Remm, M. 2007. Enhancements and modifications of primer design program Primer3. Bioinformatics 23:1289-1291. https://doi.org/10.1093/bioinformatics/btm091
  16. Kim, H., Jo, E. J., Choi, Y. H., Jang, K. S. and Choi, G. J. 2016. Pathotype classification of Plasmodiophora brassicae isolates using clubroot-resistant cultivars of Chinese cabbage. Plant Pathol. J. 32:423-430. https://doi.org/10.5423/PPJ.OA.04.2016.0081
  17. Kim, W. G., Moon, M. H., Kim, J. H., Choi, H. W. and Hong, S. K. 2009. Occurrence of clubroot on pak-choi caused by Plasmodiophora brassicae. Mycobiology 37:69-71. https://doi.org/10.4489/MYCO.2009.37.1.069
  18. Kuginuki, Y., Yoshikawa, H. and Hirai, M. 1999. Variation in virulence of Plasmodiophora brassicae in Japan tested with club root resistant cultivars of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Eur. J. Plant Pathol. 105:327-332. https://doi.org/10.1023/A:1008705413127
  19. Laila, R. 2018. Molecular investigations towards clubroot resistance and expression of wax biosynthesis genes in Brassicaceae family. Ph. D. thesis. Sunchon National University, Sunchon, Korea.
  20. Laila, R., Robin, A. H., Yang, K., Choi, G. J., Park, J. I. and Nou, I. S. 2017. Detection of ribosomal DNA sequence polymorphisms in the protist Plasmodiophora brassicae for the identification of geographical isolates. Int. J. Mol. Sci. 18:84. https://doi.org/10.3390/ijms18010084
  21. Li, Y., Yuan, Y., Zhao, Y., Wei, X., Yao, Q., Jiang, W., Wang, Z., Yang, S., Zhang, X. and Tian, B. 2017. Pathogen identification of clubroot disease in Chinese cabbage from Yuanyang county, Henan province. Mol. Plant Breed. 8:45-51.
  22. Li, Z., Niu, Y. Z., Wu, Y. C. and Guo, S. X. 2013. Research advances in clubroot disease. Southwest China J. Agri. Sci. 26:1733-1737 (in Chinese).
  23. Manzanares-Dauleux, M. J., Divaret, I., Baron, F. and Thomas, G. 2001. Assessment of biological and molecular variability between and within field isolates of Plasmodiophora brassicae. Plant Pathol. 50:165-173. https://doi.org/10.1046/j.1365-3059.2001.00557.x
  24. Nikolaev, S. I., Berney, C., Fahrni, J. F., Bolivar, I., Polet, S., Mylnikov, A. P., Aleshin, V. V., Petrov, N. B. and Pawlowski, J. 2004. The twilight of heliozoa and rise of Rhizaria, an emerging supergroup of amoeboid eukaryotes. Proc. Natl. Acad. Sci. U.S.A. 101:8066-8071. https://doi.org/10.1073/pnas.0308602101
  25. Robin, A. H., Yi, G. E., Laila, R., Yang, K., Park, J. I., Kim, H. R. and Nou, I. S. 2016. Expression profiling of glucosinolate biosynthetic genes in Brassica oleracea L. var. capitata inbred lines reveals their association with glucosinolate content. Molecules 21:E787. https://doi.org/10.3390/molecules21060787
  26. Schwelm, A., Fogelqvist, J., Knaust, A., Julke, S., Lilja, T., Bonilla-Rosso, G., Karlsson, M., Shevchenko, A., Dhandapani, V., Choi, S. R., Kim, H. G., Park, J. Y., Lim, Y. P., Ludwig- Muller, J. and Dixelius, C. 2015. The Plasmodiophora brassicae genome reveals insights in its life cycle and ancestry of chitin synthases. Sci. Rep. 5:11153. https://doi.org/10.1038/srep11153
  27. Some, A., Manzanares, M. J., Laurens, F., Baron, F., Thomas, G. and Rouxel, F. 1996. Variation for virulence on Brassica napus L. amongst Plasmodiophora brassicae collections from France and derived single-spore isolates. Plant Pathol. 45:432-439. https://doi.org/10.1046/j.1365-3059.1996.d01-155.x
  28. Tsushima, S. 2000. Perspective of integrated pest management-a case study: clubroot disease of crucifers. J. Pest. Sci. 25:296-299 (in Japanese). https://doi.org/10.1584/jpestics.25.296
  29. Untergasser, A., Cutcutache, I., Koressaar, T., Ye, J., Faircloth, B. C., Remm, M. and Rozen, S. G. 2012. Primer3-new capabilities and interfaces. Nucleic Acids Res. 40:e115. https://doi.org/10.1093/nar/gks596
  30. Voorrips, R. E. 1995. Plasmodiophora brassicae: aspects of pathogenesis and resistance in Brassica oleracea. Euphytica 83:139-146. https://doi.org/10.1007/BF01678041
  31. Wallenhammar, A. C. 1996. Prevalence of Plasmodiophora brassicae in a spring oilseed rape growing area in central Sweden and factors influencing soil infestation levels. Plant Pathol. 45:710-719. https://doi.org/10.1046/j.1365-3059.1996.d01-173.x
  32. Wallenhammar, A. C. and Arwidsson, O. 2001. Detection of Plasmodiophora Brassicae by PCR in naturally infested soils. Eur. J. Plant Pathol. 107:313-321. https://doi.org/10.1023/A:1011224503200
  33. Wang, J., Huang, Y., Hu, X. L., Niu, Y. Z., Li, X. L. and Liang, Y. 2008. Study on symptom, yield loss of clubroot and modality of Plasmodiophora brassicae in rape. Chinese J. Oil Crop Sci. 30:112-115 (in Chinese).
  34. Williams, P. H. 1966. A system for determination of races of Plasmodiophora brassicae that infect Cabbage and Rutabaga. Phytopathology 56:624-626.
  35. Yang, P. W., Yang, Q. Z., Wang, Q., Li, J. R. and Zeng, L. 2002. PCR detection of Plasmodiophora brassicae causing cruciferae clubroot. J. Yunnan Agric. Univ. 17:137-139.