三种猕猴桃根腐病致病菌多重实时定量PCR检测技术的建立及应用

Development and application of a multiplex real-time PCR assay for quantitative detection of three pathogens related to kiwifruit root rot disease

[背景]近年来,随着猕猴桃种植面积的不断扩大,病害的频繁发生已逐渐影响猕猴桃的产量和品质。恶疫霉(Phytophthora cactorum)、樟疫霉(P.cinnamomi)和雪松疫霉(P.lateralis)是一类可以引起猕猴桃根腐病的致病疫霉菌。[目的]建立并优化可以同时检测3种致病疫霉的多重实时定量检测技术,并调查猕猴桃主要产区的致病菌分布情况。[方法]基于Ypt1 (ras-related protein gene)基因设计恶疫霉、樟疫霉和雪松疫霉的特异性TaqMan探针和引物,建立并优化多重实时荧光定量PCR检测体系。利用近缘种检验检测体系特异性并进行灵敏度测试,应用该检测体系分析猕猴桃主要产区根际土壤中3种致病疫霉的Yt1基因含量。[结果]供测试的11个恶疫霉近缘种、11个樟疫霉近缘种、13个雪松疫霉近缘种及非目标菌种DNA样品中均无荧光信号,反应结果为阴性,而在恶疫霉、樟疫霉和雪松疫霉DNA样品中分别检测出HEX、FAM和ROX荧光信号,反应结果为阳性。三种疫霉的检测灵敏度均达到100fg。此外,通过对猕猴桃主产区陕西省周至县和眉县果园共166份土壤样品的检测发现,恶疫霉的分布最广泛且Ypt1基因含量最高,樟疫霉和雪松疫霉则相对较少。[结论]建立的猕猴桃根腐病致病疫霉多重实时定量检测体系特异性强、灵敏度高,适合于恶疫霉、樟疫霉和雪松疫霉的检测及定量分析。该技术可为猕猴桃疫霉病害的早期诊断、监测及预防提供指导。

[Background] In recent years, with the expansion of kiwifruit cultivation area, frequent occurrences of diseases have increasingly affected the yield and quality of kiwifruit. Phytophthora cactorum, P. cinnamomi and P. lateralis are a group of pathogens that cause the kiwifruit root rot disease. [Objective] The present study aimed to establish and optimize a multiplex quantitative real-time PCR assay for simultaneously detecting the three pathogenic Phytophthora species, and to investigate the distribution of these pathogens in the main production areas of kiwifruit. [Methods] The Ypt1(ras-related protein gene) sequences were aligned to develop species-specific TaqMan probes and primers for P. cactorum, P. cinnamomi and P. lateralis, respectively. A multiplex quantitative real-time PCR assay was established and optimized, and the specificity and sensitivity were tested. Finally, the detection system was used to analyze the Ypt1 gene content of three pathogens from the rhizosphere soils in the main production areas of kiwifruit. [Results] HEX, FAM and ROX fluorescence signals were detected in the DNA samples of P. cactorum, P. cinnamomi and P. lateralis, respectively, but no fluorescence signals in those of their closely related and other soil-borne pathogens. The sensitivity was 100 fg for each pathogen. By assaying 166 rhizosphere soil samples of kiwifruit plants from Zhouzhi and Meixian Prefecture of Shaanxi Province, P. cactorum was found to be the most widely distributed along with the highest Ypt1 gene content, while P. cinnamomi and P. lateralis were relatively less frequent. [Conclusion] The established multiplex quantitative detection method was specific and sensitive, and was suitable for the detection and quantification of P. cactorum, P. cinnamomi and P. lateralis. This technique would be useful in early diagnosis, monitoring and prevention of Phytophthora disease in kiwifruits.