山东蚕区桑黄化型萎缩病病原物的分子鉴定

Molecular Identification of Pathogen Causing Mulberry Yellow Dwarf Disease in Shandong Sericultural Areas

以采自山东省宁阳市桑园的桑黄化型萎缩病发病植株叶脉组织为材料,通过PCR扩增植原体16S rRNA基因及延伸因子基因(tuf)和核糖体蛋白基因(rp),分别得到大小约为1.4、0.8和1.2 kb的目的片段并测定序列。以该病原物的16S rRNA基因与GanBank中相关的植原体16S rRNA基因序列构建系统发育树并进行RFLP分析,结果显示该病原物属于翠菊黄化组的16SⅠr-B亚组,与翠菊黄化组16SⅠr-B亚组典型成员的同源性为99.9%。进一步对延伸因子基因和核糖体蛋白基因构建系统发育树并做RFLP分析,结果显示该病原物与翠菊黄化组的tuⅠf-B亚组典型成员和rpⅠ-B亚组典型成员的同源性分别达到99.6%、99.9%。由此在3个基因水平确定该植原体的分类地位属于16SrⅠ-B、tuⅠf-B和rpⅠ-B亚组。

Infected tissues of mulberry yellow dwarf disease were collected from Ningyang of Shandong Province for PCR amplification of a 1.5 kb fragment of 16S rRNA gene,a 0.8 kb fragment of elongation factor gene and a 1.2 kb fragment of ribosomal protein gene.All three amplified fragments were sequenced.The sequence of mulberry dwarf phytoplasma of 16S rRNA gene was compared with the reported related sequences of phytoplasma in GenBank.Phylogenetic analysis and virtual RFLP pattern of 16S rRNA gene revealed that the phytoplasma belonged to 16SrⅠ-B subgroup of aster yellow dwarf phytoplasma group.This phytoplasma shared the highest sequence identity（99.9%） with typical phytoplasma strains in subgroup 16SrⅠ-B.Further phylogenetic analysis in combination with analytical results of virtual RFLP pattern of tuf gene and rp gene showed that the phytoplasma shared 99.6% and 99.9% identities with typical phytoplasma strains in subgroups tufⅠ-B and rpⅠ-B,respectively.Based on analyses of the above three aspects,we therefore classified mulberry dwarf phytoplasma into subgroup 16SrI-B,tufⅠ-B and rpⅠ-B.