2种桑树病原真菌多重PCR检测方法的建立

罗龙辉; 张兴楠; 孟芳; 黄裕鑫; 刘吉平

doi:10.7671/j.issn.1001-411X.202201019

2种桑树病原真菌多重PCR检测方法的建立

Establishment of multiplex PCR detection method for two mulberry pathogenic fungi

摘要

摘要:
目的褐斑病与轮纹病是桑树Morus alba 的2种常见真菌性病害，危害严重，给生产上带来极大损失。本研究旨在快速准确地诊断2种病害的主要病原菌新褐斑壳丰孢Neophloeospora maculans与膝节霉Gonatophragmium triuniae。

方法基于多重PCR原理，针对2种病原菌的核糖体内转录间隔区(Internal transcribed spacer, ITS)序列设计多重PCR的特异性引物，优化多重PCR反应的条件，并通过对45份不同地区桑树褐斑病与轮纹病样本进行检测以验证所建立的多重PCR的可行性。

结果建立的多重PCR检测体系具有良好的可操作性，特异性良好，2种病原菌的DNA检测灵敏度分别达0.1 和1.0 pg/μL，通过对不同地区的田间收集的多份桑树病样进行检测，可以明显区分出不同地区2种病害病原菌的种类。

结论所建立的多重PCR技术可用于桑树褐斑病与轮纹病病原菌的快速检测，可为桑树真菌性病害的防控建立基础。

Abstract:
Objective Mulberry brown spot disease and mulberry ring leaf spot disease are two common fungal diseases of mulberry trees (Morus alba), which are serious and cause great losses in production. This study was aimed to quickly and accurately diagnose the main pathogens of the two diseases, Neophloeospora maculans and Gonatophragmium triuniae.

Method Based on the principle of multiplex PCR, specific primers for multiplex PCR were designed for the ribosome internal transcribed spacer (ITS) sequences of two pathogenic fungi, and the conditions of multiplex PCR reaction were optimized. Tests were performed with 45 samples to verify the feasibility of the established multiplex PCR.

Result The established multiplex PCR detection system had good operability and good specificity, and the sensitivities for simultaneously detecting DNA of two pathogens were 0.1 and 1.0 pg/μL, respectively. The detection of mulberry disease samples could clearly distinguish the two types of pathogenic fungi in different regions.

Conclusion The established multiplex PCR technology can be used for rapid detection of the pathogens of mulberry brown spot and ring leaf spot diseases, and provides a foundation for the prevention and control of mulberry fungal disease.

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