利用基于 DNA 的 qRT-PCR 检测法定量分析茶花中的真菌病原 Didymella segeticola。

IF 4.7 2区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Plant Methods Pub Date : 2024-10-08 DOI:10.1186/s13007-024-01284-2

You Zhang, Yiyi Tu, Yijia Chen, Jialu Fang, Fan'anni Chen, Lian Liu, Xiaoman Zhang, Yuchun Wang, Wuyun Lv

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引用次数: 0

摘要

真菌病原体半知菌（Didymella segeticola）会导致茶树（Camellia sinensis）叶斑病和叶枯病，造成生产损失，并影响茶叶的品质和风味。准确检测和量化茶树叶片中的半知菌（D. segeticola）生长情况对于诊断病害严重程度或评估寄主抗性至关重要。在这项研究中，我们监测了接种了 GFP 表达菌株的茶树叶片的病害进展和 D. segeticola 的生长情况。相比之下，我们采用了基于 DNA 的 qRT-PCR 分析方法，以更方便、更易操作地检测茶叶中 D. segeticola 的生长情况。这种方法是通过比较一种编码 Cys2His2-锌指蛋白（NCBI登录号：OR987684）的 D. segeticola 特异性 DNA 与茶树 Cs18S rDNA1 的关系。与 ITS 和 TUB2 序列不同的是，这种特异性 DNA 只在 D. segeticola 分离物中扩增，而不在其他茶树病原体中扩增。这种检测方法也适用于检测与不同茶树品种交互作用过程中的 D. segeticola。与'龙井43'（LJ43）、'中茶108'（ZC108）和'中茶302'（ZC302）相比，'中茶102'（ZC102）和'福鼎大白茶'（FDDB）对半知菌更易感。不同的 D. segeticola 分离物对 LJ43 也表现出不同程度的侵染性。总之，基于 DNA 的 qRT-PCR 分析是一种高灵敏度、简便而有效的方法，可用于定量分析茶叶中 D. segeticola 的生长情况。该技术可用于诊断茶叶叶斑病和枯萎病的严重程度，或评估茶树对该病原体的抗性。

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Quantification of the fungal pathogen Didymella segeticola in Camellia sinensis using a DNA-based qRT-PCR assay.

The fungal pathogen Didymella segeticola causes leaf spot and leaf blight on tea plant (Camellia sinensis), leading to production losses and affecting tea quality and flavor. Accurate detection and quantification of D. segeticola growth in tea plant leaves are crucial for diagnosing disease severity or evaluating host resistance. In this study, we monitored disease progression and D. segeticola development in tea plant leaves inoculated with a GFP-expressing strain. By contrast, a DNA-based qRT-PCR analysis was employed for a more convenient and maneuverable detection of D. segeticola growth in tea leaves. This method was based on the comparison of D. segeticola-specific DNA encoding a Cys2His2-zinc-finger protein (NCBI accession number: OR987684) in relation to tea plant Cs18S rDNA1. Unlike ITS and TUB2 sequences, this specific DNA was only amplified in D. segeticola isolates, not in other tea plant pathogens. This assay is also applicable for detecting D. segeticola during interactions with various tea cultivars. Among the five cultivars tested, 'Zhongcha102' (ZC102) and 'Fuding-dabaicha' (FDDB) were more susceptible to D. segeticola compared with 'Longjing43' (LJ43), 'Zhongcha108' (ZC108), and 'Zhongcha302' (ZC302). Different D. segeticola isolates also exhibited varying levels of aggressiveness towards LJ43. In conclusion, the DNA-based qRT-PCR analysis is highly sensitive, convenient, and effective method for quantifying D. segeticola growth in tea plant. This technique can be used to diagnose the severity of tea leaf spot and blight or to evaluate tea plant resistance to this pathogen.

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来源期刊

Plant Methods 生物-植物科学

CiteScore

9.20

自引率

3.90%

发文量

121

审稿时长

2 months

期刊介绍： Plant Methods is an open access, peer-reviewed, online journal for the plant research community that encompasses all aspects of technological innovation in the plant sciences. There is no doubt that we have entered an exciting new era in plant biology. The completion of the Arabidopsis genome sequence, and the rapid progress being made in other plant genomics projects are providing unparalleled opportunities for progress in all areas of plant science. Nevertheless, enormous challenges lie ahead if we are to understand the function of every gene in the genome, and how the individual parts work together to make the whole organism. Achieving these goals will require an unprecedented collaborative effort, combining high-throughput, system-wide technologies with more focused approaches that integrate traditional disciplines such as cell biology, biochemistry and molecular genetics. Technological innovation is probably the most important catalyst for progress in any scientific discipline. Plant Methods’ goal is to stimulate the development and adoption of new and improved techniques and research tools and, where appropriate, to promote consistency of methodologies for better integration of data from different laboratories.