桑椹菌核病菌实时荧光定量PCR检测体系的建立和应用

Establishment and application of real-time PCR for detecting pathogens causing mulberry fruit sclerotiniosis

为及时高效地鉴别桑椹菌核病的病原菌桑实杯盘菌Ciboria shiraiana和肉阜状杯盘菌C.carunculoides,根据RPB2基因序列设计引物并验证其特异性,建立这2种病原菌的实时荧光定量PCR(real-time fluorescent quantitative PCR,qPCR)检测体系,运用该体系对抗、感果桑品种不同时间点芽(果)内的2种病原菌进行检测。结果表明,设计的2对引物特异性强,qPCR检测灵敏度均为10^(-4) ng/μL,均为常规PCR灵敏度的10^(5)倍,建立的桑实杯盘菌和肉阜状杯盘菌qPCR标准曲线的扩增效率分别为104.89%和95.30%。利用所建方法成功从采集的果桑样品中检测出病原菌主要为肉阜状杯盘菌,且感病品种中肉阜状杯盘菌的含量随着时间不断增加,在将要显症时激增。表明所建qPCR体系适用于桑椹菌核病早期无症状芽(果)的检测,可通过监测其病原菌类型和含量为该病害的预测预报及早期防治提供依据。

In order to timely and efficiently identify Ciboria shiraiana and C.carunculoides,the pathogens associated with mulberry fruit sclerotiniosis,primers were designed based on RPB2 gene sequences and their specificity were verified,and the real-time quantitative polymerase chain reaction(qPCR)detection systems were established for the two pathogens.The constructed qPCR systems were used to detect the two pathogens in buds(fruits)of resistant and susceptible mulberry cultivars at different time points.The results showed that the two pairs of primers were highly specific,and the sensitivities of qPCR detection were both 10^(-4) ng/μL,which were both 105 times higher than conventional PCR.The amplification efficiency of the constructed qPCR standard curves for C.shiraiana and C.carunculoides were 104.89%and 95.30%,respectively.C.carunculoides was successfully detected by these methods as the main pathogen in the samples,and its amount in the susceptible cultivar has increased with time and increased sharply at the onset of disease symptoms.The results indicated that the qPCR systems are suitable for early detection of the two pathogens from asymptomatic buds(fruits)of mulberry.Timely identification and quantification of causal pathogens can provide a basis for the prediction and early control of the disease.