Browse > Article
http://dx.doi.org/10.5423/RPD.2011.17.1.052

One-step Multiplex RT-PCR Method for Simultaneous Detection of Seed Transmissible Bacterium and Virus Occurring on Brassicaceae Crop Seeds  

Jeong, Kyu-Sik (Variety Testing Division, Korea Seed and Variety Service)
Soh, Eun-Hee (Variety Testing Division, Korea Seed and Variety Service)
Publication Information
Research in Plant Disease / v.17, no.1, 2011 , pp. 52-58 More about this Journal
Abstract
The aim of this research was to develop specific and sensitive PCR-based procedures for simultaneous detection of economically important plant pathogenic bacteria and seed borne virus in commercial Brassicaceae crop seeds, Xanthomonns campestris pv. campestris (Xcc) and Lettuce Mosaic Virus (LMV). Bacterial and virus diseases of Brassicaceae leaves are responsible for heavy losses. PCR with arbitral primers: selection of specific primers, performance of PCR with specific primers and determination of the threshold level for pathogens detection. To detect simultaneously the Xcc and LMV in commercial Brassicaceae crop seeds (lettuce, kohlrabi, radish, chinese cabbage and cabbage), two pairs of specific primer (LMV-F/R, Xcc-F/R) were synthesized by using primer-blast program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The multiplex PCR for the two pathogens in Brassicaceae crop seeds could detect specifically without interference among primers and/or cDNA of other plant pathogens. The pathogen detection limit was determined at 1 ng of RNA extracted from pathogens. In the total PCR results for pathogen detection using commercial kohlrabi (10 varieties), lettuce (50 varieties), radish (20 varieties), chinese cabbage (20 varieties) and cabbage (20 varieties), LMV and Xcc were detected from 39 and 2 varieties, respectively. In the PCR result of lettuce, LMV and Xcc were simultaneously detected in 8 varieties.
Keywords
Brassicaceae crop; Mutliplex RT-PCR; Seed pathogens;
Citations & Related Records
Times Cited By KSCI : 3  (Citation Analysis)
연도 인용수 순위
1 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.   DOI
2 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. J. Plant Pathol. 109: 775-789.   DOI
3 Park, D. S., Shim, J. K., Kim, J. S., Lim, C. K., Shrestha, R.,Hahn, J. H., Parrella, G., Verdin, E., Gognalons, P. andMarchoux, G. 2006. Detection and characterization of tobacco mild green mosaic virus (TMGMV) large type isolate from trailing petunia in france. Commun. Agric Appl. Biol. Sci. 71: 1237-1244.
4 Park, Y. J., Lee, B. M., Hahn, J. H., Lee, G. B. and Park, D. S.2004. Sensitive and specific detection of Xanthomonas campestis pv. campestris by PCR using species-specific primers based on hrpF gene sequence. Microbiological Research, 159: 419-423.   DOI
5 Ro, H. S., Lee, N. J., Lee, C. W. and Lee, H. S. 2006. Isolation of a novel mycovirus OMIV in Pleurotus ostreatus and its detection using a triple antibody sandwich-ELISA. J. Virol. Methods 138: 24-29.   DOI   ScienceOn
6 Schaad, N. W. 1979. Serological identification of plant pathogenic bacteria. Annu. Rev. Phytopathol. 17: 123-147.   DOI
7 Schaad, N. W. ed. 1980. Laboratory Guide of Identification of Plant Pathogenic Bacteria. APS Press, St. Paul, MN.
8 Sharman, M., Thomas, J. E. and Dietzgen, R. G. 2000. Development of a multiplex immunocapture PCR with colourimetric detection for viruses of banana. J. Virol. Methods 89: 75-88.   DOI   ScienceOn
9 Suehiro, N., Matsuda, K., Okuda, S. and Natsuaki, T. 2005. A simplified method for obtaining plant viral RNA for RT-PCR. J. Virol. Methods 125: 67-73.   DOI
10 De Leon, L., Siverio, F. and Rodriguez, A. 2006. Detection of Clavibacter michiganensis subsp. michiganensis in tomato seeds using immunomagnetic separation. J. Microbiol. Methods 67: 141-149.   DOI
11 Franken, A. A. J. M. 1992. Application of polyclonal and monoclonal antibodies for the detection of Xanthomonas campestris pv. campestris in crucifer seeds using immunofluorescence microscopy. Eur. J. Plant Pathol. 98: 95-106.
12 Ghabrial, S. A., Li, D. and Shepherd, R. J. 1982. Radioimmunosorbent Assay for Detection of Lettuce Mosaic Virus in Lettuce Seed. Plant Disease 66: 1037-1040.   DOI
13 Jin, K. S., Kang, I. B., Ko, K. I., Lee, E. S., Heo, J. Y., Kang, Y. K. and Kim, B. K. 2001. Detection of Xanthomonas axonopodis pv. citri an Citrus Fruits Using Enzyme-Linked Immunosorbent Assay. Plant Pathol. J. 17: 62-66.   과학기술학회마을
14 Kim, J. H., Choi, G. S., Kim, J. S., Lee, S. H., Choi, J. K. and Ryu, K. H. 2006. Development of single-tube multiplex immunocapture RT-PCR Assay for simultaneous detection of two pepper Tobamoviruses. Plant Pathol. J. 22: 164-167.   과학기술학회마을   DOI
15 Kim, S. W., Kim, M. G., Kim, J., Lee, H. S. and Ro, H. S. 2007.Detection of a mycovirus OMSV in edible mushroom Pleurotus ostreatus using SPR biosensor chip. J. Virol. Methods.
16 Lee, S. H. 1981. Studies on virus disease occurring in various crops in Korea. Res. Report RDA 23: 62-74.
17 Lee, Y. A., Sung, A. N., Liu, T. F. and Lee, Y. S. 2009. Combination of chromogenic differential medium and estAspecific PCR for isolation and detection of phytopathogenic Xanthomonas spp. Appl. Environ. Microbiol. 75: 6831-6838.   DOI
18 Berg, T., Tesoriero, L. and Hailstones, D. L. 2006. A multiplex real-time PCR assay for detection of Xanthomonas campestris from brassicas. Lett. Appl. Microbiol. 42: 624-630.
19 식물병리학 교재연구모임. 2006. 식물병리학(제5판). 월드싸이언스. 653-654, 764-769 pp.
20 홍성준, 홍연규, 이봉춘, 임미정, 윤영남, 황재복, 송석보, 박성태. 2007. PCR assay 이용 콩 종자에서 Xanthomonas axonopodis pv. glycines 검출 및 종자오염 조사. 식물병연구 13: 145-151.   과학기술학회마을   DOI
21 Cho, J. D., Kim, J. S., Lee, S. H. and Chung, B. N. 2007. Triplex Virion Capture (VC)/RT-PCR for Three Seed Transmissible Tobamoviruses of CGMMV, ZGMMV and KGMMV Occurring on Cucurbitaceae. Res. Plant Dis. 13: 82-87.   DOI