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中国农学通报

中国农学通报 ›› 2024, Vol. 40 ›› Issue (19): 126-132.doi: 10.11924/j.issn.1000-6850.casb2023-0470

• 植物保护·农药 • 上一篇    下一篇

3种葡萄座腔菌的检测方法比较

王丽1,2(), 侯珲1, 涂洪涛1,2, 黄天祥1, 柳友亮1, 袁洪波1,2()   

  1. 1 中国农业科学院郑州果树研究所，郑州 450009
    2 中国农业科学院中原研究中心，河南新乡 453003
  • 收稿日期:2023-06-28 修回日期:2024-01-10 出版日期:2024-07-05 发布日期:2024-07-02
  • 通讯作者:
    袁洪波，男，1987年出生，河南安阳人，助理研究员，博士，主要从事果树病害研究。通信地址：450009 河南省郑州市未来路南首郑州果树所，Tel：0371-65330953，E-mail：。
  • 作者简介:

    王丽，女，1981年出生，河南正阳人，副研究员，博士，主要从事果树病害研究。通信地址：450009 河南省郑州市未来路南首郑州果树所，Tel：0371-65330953，E-mail：。

  • 基金资助:
    河南省重点研发专项“智慧果园生产管理技术体系研发与示范”(221111111800); 中国农业科学院科技创新工程“猕猴桃资源与育种科研团队”(CAAS-ASTIP-2023-ZFRI); 长江猕猴桃产业技术研究中心“猕猴桃关键虫害绿色防控技术研究集成”(CJZX20210109)

Comparison of Three Detection Methods for Botryosphaeria dothidea

WANG Li1,2(), HOU Hui1, TU Hongtao1,2, HUANG Tianxiang1, LIU Youliang1, YUAN Hongbo1,2()   

  1. 1 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009
    2 Zhongyuan Research Center, Chinese Academy of Agricultural Sciences, Xinxiang, Henan 453003
  • Received:2023-06-28 Revised:2024-01-10 Published:2024-07-05 Online:2024-07-02

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摘要：

为精准检测葡萄座腔菌(Botryosphaeria dothidea)引起的果树病害，基于B. dothidea的ITS序列设计常规PCR、巢式PCR和LAMP引物，对3种检测方法的灵敏度进行比较分析，依据研究结果选用巢式PCR和LAMP反应检测田间病样。结果表明，常规PCR、巢式PCR和LAMP检测方法均能特异性检测出B. dothidea。普通PCR检测到的DNA最低浓度为10-1 ng/μL，巢式PCR的检测限为10-4 ng/μL，LAMP方法的检测限为10-5 ng/μL。灵敏度较高的巢式PCR和LAMP方法都可用于检测B. dothidea引起的田间病害样品。LAMP方法检测田间疑似病害样品的检出率为65.0%、常规PCR为58.8%、巢式PCR为62.5%，而常规分离鉴定方法的检出率仅为27.5%。

关键词: 果树病害, 葡萄座腔菌, 聚合酶链式反应, 环介导等温扩增技术, 检测

Abstract:

Being able to rapidly and reliably detect Botryosphaeria dothidea will enable better disease monitoring and control efforts. In the present study, we developed conventional PCR, nested PCR, and LAMP assays in order to specifically differentiate between B. dothidea and other fungal species. Then, the sensitivity of the three detection methods was compared and analyzed. Finally, these assays were used to detect diseased samples collected from fields. The results showed that the conventional PCR, nested PCR and LAMP assays were able to detect and diagnose fruit diseases caused by B. dothidea. Sensitivity analyses revealed that the detection limit for conventional PCR assay was 10-1 ng/μL of B. dothidea DNA, whereas the nested PCR assay had a 10-4 ng/μL detection limit, and the LAMP assay was sensitive to as little as 10-5 ng/μL of fungal DNA. The nested PCR and LAMP assays can be reliably utilized to diagnose fruit diseased samples from field caused by B. dothidea. The positive rates of the LAMP detection, nested PCR, conventional PCR and traditional identification method were 65.0%, 62.5%, 58.8% and 27.5% respectively.

Key words: fruit diseases, Botryosphaeria dothidea, PCR, LAMP, detection