禾谷镰孢荧光定量检测体系的建立及其在玉米苗枯病上的应用
Development of fluorescent quantitative detection system for fungal pathogen Fusarium graminearum and its application in maize seedling blight

为实现对田间土壤中禾谷镰孢Fusarium graminearum的定量检测，本研究构建了土壤含菌量与玉米苗枯病病情指数的回归模型。基于禾谷镰孢甾醇14α-去甲基化酶基因CYP51C序列，设计特异性引物HQ1-F/HQ1-R，利用引物建立实时荧光定量PCR （RT-qPCR）体系，选取4个玉米自交系品种进行室内苗枯病接种试验，调查其病情指数，利用RT-qPCR体系检测土壤禾谷镰孢含菌量，并对病情指数和土壤禾谷镰孢含菌量进行回归。结果表明，仅禾谷镰孢扩增出目的条带并且可从多种病原菌土壤中检测出。RT-qPCR的熔解曲线具有单一吸收峰，扩增曲线的循环阈值与模板浓度呈良好的线性关系，扩增效率为104.7%，标准曲线为y=-3.2137x+34.9560（R2=0.9968），最低可检测到1 pg/μL的DNA。随着土壤禾谷镰孢接菌量的增加，单位土壤禾谷镰孢含菌量呈线性增加，即y=13.603x-85.370（R2=0.9998）。4个玉米品种的病情指数与土壤禾谷镰孢含菌量的回归曲线分别为y=0.0789x+22.0590（R2=0.7949）、y=0.0304x+7.8686（R2=0.9579）、y=0.0458x+23.7540（R2=0.5420）、y=0.0471x+32.0760（R2=0.6753）。
In order to quantify fungal pathogen Fusarium graminearum in field soil, the regression model between the number of soil fungi and the disease index of maize seedling blight was established. The specific primers HQ1-F and HQ1-R were designed based on the sequence of the Sterol 14α-de-methylase gene CYP51C. The real-time quantitative PCR (RT-qPCR) system was established by using the serial dilution of F. graminearum DNA as the standard. Indoor pot experiments with soil inoculation of maize seedling blight pathogen were conducted to detect fungal content in the soil and investigate the disease index. The results showed that the target band was amplified only from F. graminearum and detectable from various pathogenic soils. The melt curve had a single absorption peak, and a fine linear relationship existed between threshold cycle and template concentration. The standard curve based on the formula y=-3.2137x+34.9560, was established with the correlation coefficient of 0.9968 and with high amplification efficiency (1.047), and as low as 1 pg/μL of F. graminearum DNA could be detected. Meanwhile, with the increase of the inoculation concentration of F. graminearum spores, the pathogen number in the soil also linearly increased, and the equation was:y=13.603x-85.370 (R2=0.9998). In addition, the regression equations between disease index of different resistant inbred maize lines and the F. graminearum content in soil were constructed and they were y=0.0789x+22.0590 (R2=0.7949), y=0.0304x+7.8686 (R2=0.9579), y=0.0458x+23.7540 (R2=0.5420), y=0.0471x+32.0760 (R2=0.6753).