一种快速检测和定量小麦叶片中潜伏侵染白粉病菌的方法

doi:10.1016/j.jia.2024.06.004

摘要/Abstract

摘要：

由禾布氏白粉菌小麦专化型（Blumeria graminis f. sp. tritici）引起的小麦白粉病在世界范围内广泛发生，对小麦安全生产构成严重威胁。明确小麦白粉病菌的秋苗菌源量对于预测和评估其春季病害流行风险具有重要意义。然而，在晚秋或冬季进行秋苗调查时，如遇温度较低情况，小麦白粉病菌常以潜伏侵染的形式寄生在叶片组织内，而不显示肉眼可见的症状。本研究开发了一种快速检测和定量小麦叶片中潜伏侵染白粉病菌的新方法。该方法基于重组酶聚合酶扩增反应（RPA）并通过侧向流试纸条（LFD）进行终点检测。小麦白粉病菌DNA的检出下限为100 ag μL^-1，且不受其他常见小麦病原真菌或其寄主小麦（Triticum aestivum）DNA的干扰。在小麦幼苗上对该法检测小麦白粉病菌潜伏侵染进行了试验验证，结果显示其准确性高、灵敏性强。在此基础上，将RPA-LFD检测技术与图像分析技术相结合，开发了用于定量估计小麦白粉病菌潜伏侵染量的分析方法。试验结果显示，该分析方法和实时荧光定量PCR技术估计的小麦白粉病菌DNA量之间存在极显著的相关性（P ≤ 0.01），说明所建立的新方法能够快速准确地定量测定小麦叶片中潜伏侵染小麦白粉病菌的含量，并有望进一步发展成为能够直接用于田间现场监测小麦白粉病菌潜伏侵染水平的工具。本研究建立的方法还可为小麦其他叶部病原真菌（如小麦锈菌）潜伏侵染的检测和定量提供借鉴，并有潜力结合空气中的病原菌孢子捕捉技术提供孢子浓度的定量信息。

Abstract:

Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is an important disease worldwide. Detection of latent infection of leaves by the pathogen in late autumn is valuable for estimating the inoculum potential to assess disease risks in the spring. We developed a new tool for rapid detection and quantification of latent infection of seedlings by the pathogen. The method was based on recombinase polymerase amplification (RPA) coupled with an end-point detection via lateral flow device (LFD). The limit of detection is 100 ag μL^-1 of Bgt DNA, without noticeable interference from either other common wheat pathogens or wheat material (Triticum aestivum). It was evaluated on wheat seedlings for this accuracy and sensitivity in detecting latent infection of Bgt. We further extended this RPA-LFD assay to estimate the level of latent infection by Bgt based on imaging analysis. There was a strong correlation between the image-based and real-time PCR assay estimates of Bgt DNA. The present results suggested that this new tool can provide rapid and accurate quantification of Bgt in latently infected leaves and can be further development as an on-site monitoring tool.

Key words: Blumeria graminis f. sp. tritici , recombinase polymerase amplification (RPA) , , lateral flow device (LFD) , , image-based quantification , , disease monitoring

. 一种快速检测和定量小麦叶片中潜伏侵染白粉病菌的方法[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.06.004.

Aolin Wang, Ru Jiang, Meihui Zhang, Hudie Shao, Fei Xu, Kouhan Liu, Haifeng Gao, Jieru Fan, Wei Liu, Xiaoping Hu, Yilin Zhou, Xiangming Xu. A rapid tool for quantification of latent infection of wheat leaves by powdery mildew[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.06.004.

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