小麦印度腥黑穗病菌的分子检测

根据GenBank中登录Tilletia属的20个种的核糖体转录间隔区(ITS)序列差异设计1对引物T1／T2，可以特异地从T.indica和T.wallceri菌株中扩增到一条450bp左右的条带，而从其他供试菌株中不能扩增出条带，表明该对引物可以将这二个种与Tilletia其他种区分开。进一步根据T．indica和T．walked的线粒体DNA序列的差异设计一对引物M1／M2，只能特异地从T．indica DNA中扩增到一条250bp左右的条带，表明该对引物可以将T．indica与T．walked区分开。利用上述2对引物分别与ITS区通用引物ITS1/ITS4．和线粒体引物Ti-1／Ti4组合建立套式PCR检测体系，能够直接将T．indica与其他相似或相关种区分开，且灵敏度可达1个冬孢子。此外，还观察到在反应体系中加入适量的(NH4)2SO4对PCR反应具有增效作用，可以提高检测灵敏度。上述结果提示建立的套式PCR反应体系可望能应用于口岸对T．indica的检疫。

Molecular Detection of Tilletia indica,Causal Agent of Karnal Bunt of Wheat

The oligonucleotide primers T1/T2 (5'-CAG AGT TGC TGG TAC TTC G-3'/5'-AGC TAT CGA TGA TTC CGA AG-3'), derived from the differentiation of internal transcribed space (ITS) regions within Tilletia spp., amplified a single 450 bp product from T.indica and T.walkeri. The 20 ITS DNA sequences of Tilletia spp. were gained from GenBank. Oligonucleotide primers M1/M2 (5'-GCG TGA AGG TTG CCG AAA C-3'/5'-GTC ATA GCA TGG CTC ACA CG-3'), derived from the mitochondrial DNA, amplified a product of 250 bp which was unique to T.indica. The nested PCR using primers ITS1/ITS4 and T1/T2 identified T.indica and T.walkeri from other Tilletia spp., and using primers Ti-1/Ti-4 and M1/M2 identified T.indica from T.walkeri. The product of PCR could be increased by adding appropriate concentration of (NH 4) 2SO 4 into the reaction system. Using the method we demonstrated that T.indica can be reliably detected at the level of one single teliospore.