Research in Plant Disease (식물병연구)

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

DOI QR Code

Detection of Fusarium verticillioides Contaminated in Corn Using a New Species-specific Primer

종 특이 primer를 이용한 옥수수 오염 Fusarium verticillioides의 PCR 검출

  • Kang, Mi-Ran (Department of Medical Biotechnology, Soonchunhyang University) ;
  • Kim, Ji-Hye (Department of Medical Biotechnology, Soonchunhyang University) ;
  • Lee, Seung-Ho (Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Ryu, Jae-Gee (Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Lee, Theresa (Microbial Safety Division, National Academy of Agricultural Science, Rural Development Administration) ;
  • Yun, Sung-Hwan (Department of Medical Biotechnology, Soonchunhyang University)
  • 강미란 (순천향대학교 의료생명공학과) ;
  • 김지혜 (순천향대학교 의료생명공학과) ;
  • 이승호 (농촌진흥청 국립농업과학원 유해생물과) ;
  • 류재기 (농촌진흥청 국립농업과학원 유해생물과) ;
  • 이데레사 (농촌진흥청 국립농업과학원 유해생물과) ;
  • 윤성환 (순천향대학교 의료생명공학과)
  • Received : 2011.10.27
  • Accepted : 2011.11.22
  • Published : 2011.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Fusarium verticillioides (teleomorph: Gibberella moniliformis), a member of the Gibberellea fujikuroi species complex, causes rots of corn stalks and ears, and produces a group of mycotoxins known as fumonisins that are harmful to animals and humans. Here, we focus on the development of a species-specific PCR primer set for differentiating F. verticillioides from other fumonisin-producing Fusarium species belonging to the species complex, such as F. proliferatum, F. fujikuroi, and F. subglutinans that are frequently associated with corn. The specific primers (RVERT1 and RVERT2) derived from the nucleotide sequences of RNA polymerase II beta subunit (RPB2) gene amplified a 208 bp-DNA fragment from only F. verticillioides isolates among the potential fumonisin-producing species examined; all of these isolates were shown to carry FUM1 required for fumonisin biosynthesis. The PCR detection limit using this specific primer set was approximately 0.125 pg/${\mu}l$ genomic DNA of F. verticillioides. In addition, the F. verticillioides-specfic fragment was successfully amplified from genomic DNAs of corn samples contaminated with Fusarium spp. This primer set would provide a useful tool for the detection and differentiation of potential fumonisin-producing F. verticillioides strains in cereal samples.

Fusarium verticillioides(완전세대: Gibberella moniliformis)는 Gibberellea fujikuroi 종 복합체에 속하는 식물병원균으로서 옥수수의 줄기와 이삭에 썩음병을 일으킬 뿐 아니라 인축에 중독증을 일으키는 fumonisin 곰팡이 독소를 생산한다. 본 연구의 목적은 옥수수에 주로 발생하는 fumonisin 생성가능 G. fujikuori 종 복합체 소속 Fusarium 곰팡이 중 F. verticillioides와 그 외 F. proliferatum, F. fujikuori 등을 서로 구별할 수 있는 종 특이적 PCR primer 조합을 개발하는 것이다. RNA polymerase II beta subunit 유전자(RPB2)의 염기서열로부터 제작된 특이 primer 조합(RVERT1와 RVERT2)은 우리나라 옥수수에서 분리한 잠재적인 fumonisin 생성 G. fujikuori 종 복합체 균주 중 오직 F. verticillioides로부터 208 bp 크기의 단일 DNA 절편을 증폭하였다. 한편 F. verticillioides를 포함한 모든 조사균주는 fumonisin 생합성에 필수적인 FUM1 유전자를 포함하고 있었다. 개발된 특이 primer 조합의 검출한계는 분석 곰팡이 DNA 0.125 pg/${\mu}l$ 수준이었다. 한편, 같은 primer 조합으로 Fusarium spp.에 오염된 옥수수 시료의 게놈 DNA로부터 F. verticillioides 특이 DNA 절편이 증폭되었다. 이와 같은 결과를 종합할 때, 본 연구에서 개발된 primer 조합은 여러 곡물 시료에 오염되어 있는 F. verticillioides 균주의 검출과 종 동정에 유용하게 사용될 것이다.

Keywords

References

  1. Amoah, B. K., Rezanoor, H. N., Nicholson, P. and McDonald, M. V. 1995. Variation in the Fusarium section Lesiola: pathogencity and genetic studies of Fusarium moniliforme Sheldon from different hosts in Ghana. Plant Pathol. 44: 563-572. https://doi.org/10.1111/j.1365-3059.1995.tb01678.x
  2. Baird, R., Abbas, H. K., Windham, G., Williams, P., Baird, S., Ma, P., Kelley, R., Hawkins, L. and Scruggs, M. 2008. Identification of select fumonisin forming Fusarium species using PCR applications of the polyketide synthase gene and its relationship to fumonisin production in vitro. Int. J. Mol. Sci. 9: 554-570. https://doi.org/10.3390/ijms9040554
  3. Bezuidenhout, C. C., Prinsloo, M. and Van der Walt, A. M. 2006. Multiplex PCR-based detection of potential fumonisinproducing Fusarium in traditional African vegetables. Environ. Toxicol. 21: 360-366. https://doi.org/10.1002/tox.20193
  4. Chi, M. H., Park, S. Y. and Lee, Y. H. 2009. A quick and safe method for fungal DNA extraction. Plant Pathology J. 25: 108-111. https://doi.org/10.5423/PPJ.2009.25.1.108
  5. Desjardin, A. E., Manandhar, H. K., Plattner, R. D., Manandhar, G. G. Poling, S. M. and Maragos, C. M. 2000. Fusarium species from Nepalese rice and production of mycotoxins and gibberellic acid by selected species. Appl. Environ. Microbiol. 66: 1020-1025. https://doi.org/10.1128/AEM.66.3.1020-1025.2000
  6. Hong, S.-Y., Kang, M.-R., Cho, E.-J., Kim, H.-K. and Yun, S.-H. 2010. Specific PCR detection of four quarantine Fusarium species in Korea. Plant Pathology J. 26: 409-416. https://doi.org/10.5423/PPJ.2010.26.4.409
  7. Jurjevic, Z., Wilson, D. M., Wilson, J. P., Geiser, D. M., Juba, J. H., Mubatanhema, W., Widstrom, N. W. and Rains, G. C. 2005. Fusarium species of the Gibberella fujikuroi complex and fumonisin contamination of pearl millet and corn in Georgia, USA. Mycopathologia 159: 401-406. https://doi.org/10.1007/s11046-004-1050-2
  8. Leslie, J. F., Anderson, L. L., Bowden, R. L. and Lee, Y. W. 2007. Inter- and intra-specific genetic variation in Fusarium. Int. J. Food. Microbiol. 119: 25-32. https://doi.org/10.1016/j.ijfoodmicro.2007.07.059
  9. Leslie, J. F., Pearson, C. A. S., Nelson, P. E. and Toussoun, T. A. 1990. Fusarium spp. from corn, sorghum, and soybean fields in the central and eastern United States. Phytopathology 80: 343-350. https://doi.org/10.1094/Phyto-80-343
  10. Leslie, J. F. and Summerell, B. A. 2006. The Fusarium lab manual, Blackwell, Ames, Iowa, USA.
  11. Liu, Y. L., Whelen, S. and Hall, B. D. 1999. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol. Biol. Evol. 16: 1799-1808. https://doi.org/10.1093/oxfordjournals.molbev.a026092
  12. Marasas, W. F. O. 2001. Discovery and occurrence of the fumonisins: a historical perspective. Environ. Health Perspect. 109: 239-243. https://doi.org/10.1289/ehp.01109s2239
  13. Marasas, W. F. O., Kellerman, T. S., Gelderblom, W. C. A., Coetzer, J. A. W., Thiel, P. G. and Vanderlugt, J. J. 1988. Leukoencephalomalacia in a horse induced by fumonisin-B1 isolated from Fusarium moniliforme. J. Vet. Res. 55: 197-203.
  14. Mule, G., Susca, A., Stea, G. and Moretti, A. 2004. A speciesspecific PCR assay based on the calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. Eur. J. Plant Pathol. 110: 495-502. https://doi.org/10.1023/B:EJPP.0000032389.84048.71
  15. Munkvold, G. P. and Desjardins, A. E. 1997. Fumonisins in maize: can we reduce their occurrence? Plant Dis. 81: 556-565. https://doi.org/10.1094/PDIS.1997.81.6.556
  16. Nelson, P. E., Desjardins, A. E. and Plattner, R. D. 1993. Fumonisins, mycotoxins produced by Fusarium species: biology, chemistry, and significance. Annu. Rev. Phytopathol. 31: 233-252. https://doi.org/10.1146/annurev.py.31.090193.001313
  17. O'Donnell, K., Cigelnik, E. and Nirenberg, H. I. 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90: 465-493. https://doi.org/10.2307/3761407
  18. Patino, B., Mirete, S., González-Jaen, M., Mule, G., Rodríguez, M. T. and Vazquez, C. 2004. PCR detection assay of fumonisin-producing Fusarium verticillioides strains. J. Food Prot. 67: 1278-1283.
  19. Proctor, R. H., Desjardins, A. E., Plattner, R. D. and Hohn, T. H. 1999. A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A. Fungal Genet. Biol. 27: 100-112. https://doi.org/10.1006/fgbi.1999.1141
  20. Proctor, R. H., Platter, R. D., Brown, D. W., Seo, J.-A. and Lee. Y.-W. 2004. Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex. Mycol. Res. 108: 815-822. https://doi.org/10.1017/S0953756204000577
  21. Sreenivasa, M. Y., Gonzalez-Jaen, M. T., Dass, R. S., Raj, A. P. C. and Janardhana, G. R. 2008. A PCR-based assay for the detection and differentiation of potential fumonisin-producing Fusarium verticillioides isolated from Indian maize kernels. Food Biotechnol. 22: 160-170. https://doi.org/10.1080/08905430802043206
  22. Thompson, J. D., Higgins, D. G. and Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  23. Yazeed, H. A. E., Hassan, A., Moghaieb, R. E. A., Hamed, M. and Refai, M. 2011. Molecular detection of fumonisin-producing Fusarium species in animal feeds using polymerase chain reaction (PCR). J. Appl. Sci. Res. 7: 420-427.
  24. Yoshizawa, T., Yamashita, A. and Luo, Y. 1994. Fumonisin occurrence in corn from high- and low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol. 60: 1626-1629.

Cited by

  1. Fumonisin Production by Field Isolates of the Gibberella fujikuroi Species Complex and Fusarium commune Obtained from Rice and Corn in Korea vol.18, pp.4, 2012, https://doi.org/10.5423/RPD.2012.18.4.310
  2. Population Structure of the Gibberella fujikuroi Species Complex Associated with Rice and Corn in Korea vol.28, pp.4, 2012, https://doi.org/10.5423/PPJ.OA.09.2012.0134