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甘蔗宿根矮化病是由Leifsonia xyli subsp.xyli(Lxx)引起的一种世界性甘蔗细菌病害。根据Lxx的Pat1基因保守序列,设计并合成了一对特异性引物Pat1F(5’-GGTTCCATTGCTTACCGATT-3’)/Pat1R(5’-CAAGTTTCGACAGGAACAGC-3’),和一条Taq M an探针(FAM-5’-CCACGGCTACGTCAATTCGGG-3’-TAM RA),建立了一种特异性强、灵敏度高的甘蔗宿根矮化病菌实时荧光定量PCR检测方法。结果表明,本研究建立的实时荧光定量PCR方法,对Lxx的检测最低下限为102copies·μL-1。应用实时荧光定量PCR与常规PCR方法对14个甘蔗品种进行Lxx检测,阳性检出率分别为86%和43%,表明实时荧光定量PCR比常规PCR检测方法具有更高的灵敏度。研究结果为甘蔗宿根矮化病的诊断、田间发生动态监测、脱毒健康种苗检测及品种/材料交换检疫检测提供了新技术支撑。
Sugarcane root blight is a worldwide cane bacterial disease caused by Leifsonia xyli subsp. Xyli (Lxx). A pair of specific primers Pat1F (5’-GGTTCCATTGCTTACCGATT-3 ’) / Pat1R (5’-CAAGTTTCGACAGGAACAGC-3’) and a TaqMan probe (FAM-5) were designed and synthesized based on the consensus sequence of Lxx’s Pat1 gene ’-CCACGGCTACGTCAATTCGGG-3’-TAM RA), a highly specific and sensitive method for real-time PCR detection of sugarcane-resistant root-dwarfing pathogen was established. The results showed that the lowest limit of detection of Lxx was 102copies · μL-1 by real-time fluorescence quantitative PCR method. Real-time PCR and conventional PCR methods were used to detect Lxx in 14 sugarcane varieties. The positive detection rates were 86% and 43%, respectively, indicating that real-time PCR has higher sensitivity than conventional PCR. The results provided a new technical support for the diagnosis of sugarcane root-dwarfing disease, dynamic monitoring in the field, the detection of healthy detoxification seedlings and the quarantine inspection of variety / material exchange.