巢式PCR检测油茶根腐病菌的研究

为建立油茶Camellia oleifera根腐病菌层生镰刀菌Fusarium proliferatum的巢式聚合酶链式反应（PCR）快速检测体系．扩增层生镰刀菌核糖体DNA基因间隔序列（ITS）区并测定其序列，比较该序列与GenBank中近似种的ITS序列差异．设计了特异性引物GF1和GR2，利用该对引物与ITS1和ITS4进行巢式PCR扩增检测层生镰刀菌。结果显示，巢式PCR对层生镰刀菌的检测灵敏度可达100ag基因组DNA，比常规扩增方法提高了1万倍。利用设计的GF1／GR2特异性引物与ITS区通用引物进行巢式PCR扩增．可灵敏地扩增出油茶根腐病菌层生镰刀菌DNA。

Detection of Camellia oleifera root rot pathogen with nested-PCR

A species-specific polymerase chain reaction(PCR)assay for rapid and accurate detection of the pathogenic root rot of diseased Camellia oleifera plant tissues was developed. Based on differences in inter-nal transcribed spacer(ITS)sequences of Fusarium proliferatum, a pair of species-specific primers, GF1/GR2. was designed. Other species of F proliferatum were used to test the specificity of the primers. The GF1/GR2 primer only amplified a unique 400 bp band from the CSUFT070109 strain, and its detection sensitivity was 1 Pg of genomic DNA in 25 μL reaction solution. A nested PCR procedure using ITS1/ITS4 as the first-round primers followed by GF1/GR2 increased detection sensitivity 10 000-fold to 100 ag. This assay detected the pathogen rapidly and accurately meaning methods developed here could simplify both plant disease diagnosis and pathogen monitoring as well as guide plant disease management. Ch, 4 fig. 22ref. ]