香蕉种苗中枯萎病菌和细菌性软腐病菌实时荧光PCR检测

Detection of Fusarium Wilt and Bacterial Soft Rot of Banana Seedlings by Real-Time Fluorescence Quantitative PCR

香蕉枯萎病菌和细菌性软腐病菌复合侵染且潜伏期较长,建立香蕉种苗病害早期监测的分子检测方法非常重要。本文基于尖孢镰刀菌古巴专化型1号生理小种(Fusarium oxysporum f.sp.cubense race 1,FOC1)基因组Contig 438区间(35631~37693 bp)(GenBank:AMGP01000438.1)设计特异扩增引物qFOC1-F/-R、4号生理小种(Fusarium oxysporum f.sp.cubenserace4,FOC4)基因组Contig195区间(4028~6126bp)(GenBank:AMGQ01000195.1)设计特异扩增引物qFOC4-F/-R,同时以香蕉细菌性软腐病菌Dickeya zeae的促旋酶B亚单位基因(Subunit B of gyrase gene)(GenBank:JQ284039)序列设计特异扩增引物qgyrB-F/-R。qPCR检测结果显示:实时荧光检测引物扩增特异性强。通过拟合曲线方程分析得到:检测香蕉枯萎病菌的DNA浓度最低限为3 pg·μL^(-1),细菌性软腐病菌的菌液浓度最低限灵敏度为70 cfu·mL^(-1),检测灵敏度较高,结果稳定可靠。因此,本研究建立的实时荧光PCR检测方法可有效应用于检测香蕉种苗中的香蕉枯萎病菌和细菌性软腐病菌,确保种苗的安全生产。

Establishing a molecular detection method for early monitoring of banana seedling diseases is important because of mixed infection and long incubation of banana Fusarium wilt and bacterial soft rot pathogens.The specific amplification primers of qFOC1-F/-R were designed based on the sequences of the contig 438(35631-37693 bp)(AMGP01000438.1)in the DNA of Fusarium oxysporum f.sp.cubense race 1(FOC1)and qFOC4-F/-R based on the contig 195(4028-6126 bp)(AMGQ01000195.1)in the DNA of F.oxysporum f.sp.cubense race 4(FOC4).Based on sequence of subunit B of gyrase gene of banana Fusarium wilt pathogen(GenBank:JQ284039),Dickeya zeae,the primers of qgyrB-F/-R were designed.The results of qPCR showed that the specificity of primers amplification was very strong.Curve-fitting equation analysis showed that:the minimum limit of DNA concentration for detection of F.oxysporum f.sp.cubense was 3 pg·μL^(-1),and the sensitivity de‐tection of D.zeae was 70 cfu·mL^(-1);with high sensitivity if the detection,the results were stable and reliable.Therefore,the qPCR detection method developed in this study is effective to detect banana Fusarium wilt and bacterial soft rot pathogens of banana seedlings to ensure the safe production of seedlings.