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<p>The woody oil crop industry has become a national strategy for China to solve the grain and oil crisis. As one of the woody oil crops, the development of oil peony has received ever-increasing attention. Pests and diseases have gradually become the factors that must be considered in the development of oil peony industry, especially the red spot caused by <i>Cladosporium paeoniae</i>, which poses the greatest threat to the production of oil peony. Accurate and timely diagnosis of disease plays an important role in disease prevention and control. Loop-mediated isothermal PCR (LAMP) has been more and increasingly widely used in plant disease detection. In the paper, based on the low homology ITS sequence of <i>C. paeoniae</i>, LAMP primers were designed, the components and conditions were optimized, and the optimal LAMP reaction system was determined: 10 × ThermoPol Buffer 2.5&#xa0;μL, Bst DNA polymerase 0.16&#xa0;U/μL, F3/B3 0.2&#xa0;μmol/L, dNTP 1.2&#xa0;mmol/L, MgSO<sub>4</sub> 8&#xa0;mmol/L, FIP/BIP 1.6&#xa0;μmol/L, HNB 0.15&#xa0;mmol/L, betaine 0.6&#xa0;mol/L, DNA 1&#xa0;μL, ddH<sub>2</sub>O added to 25&#xa0;μL; the reaction temperature was set at 64&#xa0;°C, and the minimum reaction time was 60&#xa0;min. The LAMP detection system has high sensitivity, which is 100 times that of conventiaoal PCR detection; and it is specific, which means it only responds positively to <i>C. paeoniae</i> and does not respond to other leaf pathogenic fungi of oil peony. According to the color change of PCR tubues and the bands on the 2% agarose gel electrophoresis diagram after the LAMP reaction, determine whether it was the red spot. The results of LAMP detection were consistent with the results of pathogen isolation, indicating that LAMP can be applied to the detection of oil peony red spot in the field. The LAMP reaction system has been further verified in four oil peony planting bases, which means it can be used to diagnose oil peony red spot, and provide a practical guarantee for the precise prevention and control of the disease in production.</p>

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Development and application of loop-mediated isothermal amplification (LAMP) for detection of Cladosporium paeoniae, the causal agent of oil peony red spot

  • 期刊论文

  • 发表日期：

    2025年5月9日

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  • Delong Kong,
  • Pu Miao,
  • Qiuyuan Chai,
  • Zejun Cheng,
  • Wei Zheng,
  • Ying Hou,
  • Xiaogai Hou,
  • Jianqiang Xu

摘要

The woody oil crop industry has become a national strategy for China to solve the grain and oil crisis. As one of the woody oil crops, the development of oil peony has received ever-increasing attention. Pests and diseases have gradually become the factors that must be considered in the development of oil peony industry, especially the red spot caused by Cladosporium paeoniae, which poses the greatest threat to the production of oil peony. Accurate and timely diagnosis of disease plays an important role in disease prevention and control. Loop-mediated isothermal PCR (LAMP) has been more and increasingly widely used in plant disease detection. In the paper, based on the low homology ITS sequence of C. paeoniae, LAMP primers were designed, the components and conditions were optimized, and the optimal LAMP reaction system was determined: 10 × ThermoPol Buffer 2.5 μL, Bst DNA polymerase 0.16 U/μL, F3/B3 0.2 μmol/L, dNTP 1.2 mmol/L, MgSO4 8 mmol/L, FIP/BIP 1.6 μmol/L, HNB 0.15 mmol/L, betaine 0.6 mol/L, DNA 1 μL, ddH2O added to 25 μL; the reaction temperature was set at 64 °C, and the minimum reaction time was 60 min. The LAMP detection system has high sensitivity, which is 100 times that of conventiaoal PCR detection; and it is specific, which means it only responds positively to C. paeoniae and does not respond to other leaf pathogenic fungi of oil peony. According to the color change of PCR tubues and the bands on the 2% agarose gel electrophoresis diagram after the LAMP reaction, determine whether it was the red spot. The results of LAMP detection were consistent with the results of pathogen isolation, indicating that LAMP can be applied to the detection of oil peony red spot in the field. The LAMP reaction system has been further verified in four oil peony planting bases, which means it can be used to diagnose oil peony red spot, and provide a practical guarantee for the precise prevention and control of the disease in production.