从浙江蚕区桑园发生桑黄化型萎缩病植株分离植原体的分子鉴定

Molecular Identification of Phytoplasma Strain Isolated from Mulberry Trees Infected by Mulberry Mosaic Leaf Roll Disease in Zhejiang Province

从浙江省蚕区的桑园内采集表现桑黄化型萎缩病症状的植株叶片,并提取叶脉总DNA,通过巢式PCR和常规PCR方法对桑黄化型萎缩病植原体分离物(MYD-ZJ)的16S r DNA、延伸因子基因tuf和核糖体蛋白基因rp进行扩增及克隆,分别得到大小为1 239 bp、842 bp和1 240 bp的序列。对这些基因片段序列进行系统进化分析,结果表明,MYD-ZJ的16S r DNA序列与来自山东、安徽等省感病桑树分离桑黄化型萎缩病植原体的同源序列的相似度为98%,在进化树中位于同一进化支;MYD-ZJ的tuf基因序列与来自山东省的桑黄化型萎缩病植原体的同源序列的相似度为89%~100%,并与该同源序列以及同样来自山东省的枣疯病植原体、苦楝丛枝病植原体等聚为一支;MYD-ZJ的rp基因序列与来自山东省、安徽省的桑黄化型萎缩病植原体的同源序列的相似度为100%,在进化树中位于同一进化支。研究结果明确了MYD-ZJ属于翠菊黄化植原体组的16SrⅠ-B亚组、tufⅠ-B亚组及rpⅠ-B亚组。

In this study, we collected mulberry leaves infected by yellow dwarf disease from Lin＇an, Zhejiang Province. Using total DNA from diseased mulberry leaf vein as template, 16S rDNA, elongation factor gene tuf and ribosomal protein gene rp of the isolated mulberry yellow dwarf phytoplasma （MYD-ZJ） were amplified and cloned by nested PCR and con- ventional PCR. The amplified product length of 16S rDNA, tufgene and rp gene were 1 239 bp, 842 bp and 1 240 bp, re- spectively. Phylogenetic analysis showed that 16S rDNA sequence of MYD-ZJ had 98% similaritv with mulberrv vellnw dwarf phytoplasmas isolated from infected mulberry trees in Shandong Province, Anhui Province and so on, and these sequences were located on the same phyletic clade, tuf gene of MYD-ZJ had 89% to 100% similarity with mulberry yellow dwarf phytoplasma from Shandong Province, and was grouped into the same clade with its homologous se- quences of mulberry yellow dwarf phytoplasma, jujube wit- ches＇ broom disease phytoplasma and Chinaberry witches＇ broom disease phytoplasma from Shandong Province. Rp gene of MYD-ZJ had 100% similarity with mulberry yellow dwarf phytoplasmas from Shandong and Anhui Province, and they were grouped into the same clade. The above results con- firmed that MYD-ZJ belongs to subgroup 16Sr I-B, tuf I-B and rp I-B of aster yellow phtoplasma.