香蕉枯萎病菌1号和4号生理小种的快速检测与鉴定

Rapid Detection and Identification of Fusarium oxysporum f. sp. cubense Race 1 and Race 4

【目的】从香蕉枯萎病菌1号和4号生理小种特有的基因序列入手,建立一种快速可靠的分子检测技术,为防止香蕉枯萎病的传播蔓延、尽早采取防治对策、指导香蕉生产进行品种配置提供理论依据。【方法】根据研究室已经筛选到的4号生理小种候选致病相关基因序列设计引物,分别以来自海南、广东和广西的6个香蕉枯萎病菌1号生理小种菌株、7个4号生理小种菌株、7个尖镰孢其它专化型菌株以及2个外围菌株DNA为模板进行PCR扩增,筛选香蕉枯萎病菌1号、4号生理小种特异性引物及尖镰孢菌的通用引物。【结果】筛选到的特异性引物不仅可用于香蕉枯萎病菌DNA的检测,还可直接用于对罹病香蕉组织和土壤中的香蕉枯萎病菌的检测;筛选到的尖镰孢菌通用引物,可作为内参照以检测DNA的质量,以避免假阴性情况的出现。【结论】所建立的三重PCR检测方法实现了在一次PCR反应中快速、准确地同步检测香蕉枯萎病菌1号和4号生理小种,对检测香蕉苗是否感染枯萎病及蕉园土壤是否受到香蕉枯萎病菌的污染具有重要意义。

[ Objective ] The objective of this study is to establish a method of rapid detection and identification of Fusarium oxysporum f. sp. cubense （FOC） race 1 and race 4 based on their respective specific sequence, avoid spreading and control banana Fusarium wilt, and further to provide a scientific basis for production and banana cultivar configuration. [Method] DNAs of 6 FOC race 1 isolates, 7 FOC race 4 isolates, 7 F. oxysporum isolates and 2 outgrouped isolates were used as templates to screen the specific primers designed on the basis of candidate pathogenicity-relative gene sequences of FOC race 4 by PCR. [Result] Two pairs of primers specific to FOC race 1 and race 4 respectively and one pair of universal primers ofE oxysporum were screened. Using the specific primers, pathogenic fungai DNA and diseased banana plant DNA and even contaminated soil DNA with the pathogen could be detected and identified. Meanwhile, the universal primers of E oxysporum could also be used as control to test the DNA quality and avoid producing false negative and further to ensure the detected result. [Conclusion] A rapid and accurate triple PCR detection method was established to identify FOC race 1 and race 4 in one PCR reaction, which is very useful to detect FOC races from banana tissue cultured lalantlets and filed soil.