枯草芽胞杆菌HMB19198在番茄叶片上定殖能力的分子检测

枯草芽胞杆菌HMB19198能有效防治番茄灰霉病，为快速、准确检测HMB19198在叶面的定殖能力，本研究通过对HMB19198全基因组序列比对分析，获得该菌株102 bp功能未知的独有基因序列，设计出针对HMB19198的特异性引物和探针。荧光定量PCR结果表明，该引物和探针对HMB19198具有较高的特异性，在有番茄叶片DNA干扰下，体系检测阈值为10²拷贝/μL。利用荧光定量PCR技术和菌落计数法检测了HMB19198在叶片上定殖动态。叶片喷施1×10⁸ cfu/mL的菌体悬浮液，0 d后菌体数量分别为1.7×10⁸拷贝/g叶片和8.9×10⁷ cfu/g叶片，2、4、6和8 d后菌株HMB19198在叶面的定殖数量逐渐降低，8 d后定殖数量分别为1.0×10⁷拷贝/g叶片和1.2×10⁷cfu/g叶片。防效试验结果表明，喷施菌株HMB19198悬浮液2 d后防效在80%以上，8 d后防效降为37.9%。

Quantitative Detection of Bacillus subtilis HMB19198 on Tomato Leaf by Real-time PCR

The biocontrol agent Bacillus subtilis HMB19198 is proved to be effective in control of tomato gray mold in the field.To evaluate the colonization ability of strain HMB19198 on tomato leaf surface，a specific gene of strain HMB19198 was obtained by BLAST of all genes in HMB19198, followed by designing a pair of the strain-specific primers and DNA probe. Real-time PCR (qPCR) experiments showed that the primers and probe were efficient in specific detection of strain HMB19198 in laboratory and the detection sensitivity was 10²copies/μL when using HMB19198 DNA spiked with tomato leaf DNA. Combing the qPCR technique and the colony-counting method (CCM), the colonization dynamics of strain HMB19198 on tomato leaves were monitored. Results showed that 0 day after inoculation (DAI), the population of strain HMB19198 on the leaf was detected as 1.0×10⁷ copies/g leaf by qPCR and 8.9×10⁷ cfu/g leaf by CCM. The population of strain HMB19198 on the leaf was gradually decreased at 4, 6 and 8 DAI, and the population at 8 DAI was detected as 1.0×10⁷ copies/g leaf by qPCR and 1.2×10⁷ cfu/g by CCM. The control efficacy was above 80% againsr tomato gray mold after application of strain HMB19198 for 2 days, while the efficacy decreased to 37.9% after 8 days.