广州桑树植原体分子检测及多样性初探

Molecular Detection of Mulberry Phytoplasma in Guangzhou and Primary Study of its 16S rDNA Diversity

采用植原体16S-23SrDNA区段的通用引物对P1／P7和巢式引物对Rml6F2／Rml6R1，建立了快速准确的桑树植原体巢式PCR检测技术。对广州的两个桑树品种资源圃中的部分桑树品种进行了植原体分子检测，结果在两个资源圃中均发现有植原体存在。对巢式PCR的扩增产物（16SrDNA片段）进行了限制性片断长度多态性（RFLP）分析，显示出3种RFLP带型，暗示桑树植原体存在多样性。对所得植原体16SrDNA片段进行序列测定，并与其它植物植原体作亲缘关系分析，结果表明该植原体的16SrDNA序列与其它植物病原植原体之间的同源性为83．3％-99．9％，并初步判断所检测到的桑树植原体属于16SrI组。

Based on the 16S-23S rDNA sequence of phytoplasma, a set of universal primers P1/P7 and a set of nested primers Rml6F2/Rml6R1 were employed to perform nested-PCR detection for pathogenic phytoplasma in mulberry leaf tissues sampled from two variety-collecting bases in Guangzhou, China. It was confirmed that some mulberry varieties in these bases were phytoplasma-infected. The products of nested-PCR, 16S rDNA fragments, were then analyzed by restricted fragment length polymorphism with Kpn Ⅰ and Msp Ⅰand by 6% polyacylamide gel electrophoresis. The result showed that there were three types of RFLP bands which indicated the diversity of mulberry phytoplasma. The obtained 16S rDNA fragments were sequenced and then compared with the reported phytoplasma 16S rDNA. The nucleotide identities were from 83.3% to 99.9% between the detected phytoplasma and others. According to 16S rDNA sequence the phylogenetic tree was constructed. Based on the above results, we deduced that the phytoplasma causes mulberry dwarf disease in Guangzhou belongs to group 16S r Ⅰ.