Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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Direct PCR Detection of the Causal Agents, Soybean Bacterial Pustule, Xanthomonas axonopodis pv. glycines in Soybean Seeds

콩 종자에서 Xanthomonas axonopodis pv. glycines의 검출을 위한 Direct PCR 방법 개발

  • Lee, Yong-Ju (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.) ;
  • Kang, Mi-Hyung (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.) ;
  • Noh, Tae-Hwan (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.) ;
  • Lee, Du-Ku (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.) ;
  • Lee, Geon-Hwi (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.) ;
  • Kim, Si-Ju (Reclaimed Land Agriculture Division, Department of Rice and Winter Cereal Crop, National Institute Crop Science, R.D.A.)
  • 이용주 (국립식량과학원 벼맥류부 간척지농업과) ;
  • 장미형 (국립식량과학원 벼맥류부 간척지농업과) ;
  • 노태환 (국립식량과학원 벼맥류부 간척지농업과) ;
  • 이두구 (국립식량과학원 벼맥류부 간척지농업과) ;
  • 이건휘 (국립식량과학원 벼맥류부 간척지농업과) ;
  • 김시주 (국립식량과학원 벼맥류부 간척지농업과)
  • Published : 2009.08.01
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Abstract

Direct Polymerase Chain Reaction (PCR) method that combines biological and enzymatic amplification of PCR targets was developed for the detection of Xanthomonas axonopodis pv. glycines on soybeen seeds without DNA isolation. Primers Xag F1 and Xag R1 were designed to specifically amplify a 401 bp fragment of the glycinecin A gene of X axonopodis pv. glycines. Xag F1 and Xag R1 were used to carry out the PCR analysis with genomic DNA from 45 different bacterial strains including phylogenetically related bacteria with X axonopodis pv. glycines, and other bacterial strains of different genus and species. The PCR assay using this set of primers were able to detect X axonopodis pv. glycines with DNA concentration as low as 200 fg and $1.8{\times}10^3$ cfu/ml. The Xag was detected from the seed samples incubated for 2 hrs with shaking and the intensity of the band was increase with the incubation time of seeds. The Direct PCR assay method without DNA isolation makes detection of X. axonopodis pv. glycines on soybean seeds easier and more sensitive than other conventional methods. The developed seed assay using direct PCR method will be useful for the specific detection of X. axonopodis pv. glycines in soybean seed samples.

콩 불마름병을 일으키는 Xanthomonas axonopodis pv. glycines를 종자에서 DNA 추출없이 바로 검출하는 방법에 대하여 연구하였다. 콩 종자에서 X. axonopodis pv. glycines를 특이적으로 검출하기 위해 특이적인 유전자로 알려져 있는 glycinecin A로부터 증폭 size가 401 bp인 primer Xag F1 & Xag R1을 고안하였다. Xag Fl과 Xag R1 primer는 콩 종자와 잎에서 분리한 균주와 KACC에서 분양받은 X. axonopoids pv. glycines 균주를 증폭시켰으나 근연종인 X. axonopodis pv. citri, X. axonopodis pv. vesicatoria 등은 증폭되지 않았다. 콩 종자에 존재하는 것으로 알려진 다른 세균들 역시 증폭되지 않았다. 고안된 Xag F1 & Xag R1 primer를 이용한 X. axonopodis pv. glycines의 검출 한계 농도 측정은 genomic DNA와 세포 현탁액을 이용하였다. X. axonopodis pv. glycines의 genomic DNA는 200 fg까지 증폭이 되었으며, 세포현탁액은 $OD_{600nm}$ 0.1로 농도를 조정한 뒤, $10^{-8}$까지 희석하여 측정한 결과 $10^{-6}$인 $1.8{\times}10^3$ cfu/ml까지 증폭이 확인되었다. 자연 감염된 종자에서 병원균을 검출하기 위해 종자를 육안상 건전종자와 불건전한 종자(변색립, 피해립)로 구분하여 direct PCR 방법으로 실험 한 결과 육안상 건전종자에서는 병원균이 검출되지 않았으나 불건전한 종자에서는 병원균의 검출이 확인되었다. 또한 진탕 배양 시간에 따른 병원균의 검출여부를 조사한 결과 진탕 배양 2시간부터 병원균의 검출이 확인되었으며 시간이 지날수록 증폭 밴드가 더욱 선명해 지는 것을 확인할 수 있었다. 그러므로 고안된 Xag Fl & Xag R1 primer를 이용한 direct PCR 방법은 다른 많은 미생물들로 오염되어진 콩종자에 있는 X. axonopodis pv. glycines를 신속하고 민감하게 검정할 수 있는 효과적인 방법으로 활용될 수 있을 것이다.

Keywords

References

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