《植物生理学报》 2017, 53(1): 96-102

黄蒿丛枝病植原体的鉴定及其16S rDNA序列分析

余贤美, 王洁, 付丽, 翟浩, 孙清荣^*

山东省果树研究所, 山东泰安271000

通信作者：孙清荣；E-mail: sdipss@163.com

摘　要：

黄蒿为菊科蒿属一、二年生草本植物, 在医学和植物病害生物防治方面具有重要价值。植原体是一类重要的病原物, 常引起丛枝、小叶、黄化、簇生等症状。从山东泰山山涧采集到表现丛枝症状的感病黄蒿植株, 以叶片总DNA为模板, 利用植原体通用引物对R16F2n/R2进行PCR扩增, 获得了大小为1 244 bp的片段, 与葡萄黄化(Stolbur)植原体(AF248959)和纸花黄化植原体(JX128698)一致性分别为99.4%和99.8%。系统进化树显示, 黄蒿丛枝病植原体(登录号: KT899994)与葡萄黄化(Stolbur)植原体(AF248959)和纸花黄化植原体(JX128698)处于同一分支。植原体分类鉴定在线工具iPhyClassifier分析结果显示, 所获得的片段与16SrXII-A亚组代表性植原体(AF248959)相似系数为1.0, 表明黄蒿丛枝病植原体归属于16SrXII-A亚组。利用葡萄黄化(Stolbur)植原体特异性引物对STOL4f/r进行PCR扩增, 获得大小为1 754 bp的片段, 与葡萄黄化(Stolbur)植原体(AF248959)一致性为99.5%, iPhyClassifier分析结果显示, 该片段(登录号: KU759570)与AF248959相似系数为1.0, 验证了植原体通用引物对R16F2n/R16R2分析结果的可靠性。综上, 将黄蒿丛枝病植原体的分类地位确定为16SrXII-A亚组。

关键词：黄蒿丛枝病; 葡萄黄化植原体; 分子鉴定; 16S rDNA序列分析

收稿：2016-11-14   修定：2016-12-30

资助：山东省农业良种工程项目(鲁科字[2014]96)。

Molecular identification and 16S rDNA sequence analysis of phytoplasma associated with Artemisia scoparia witches’ broom

YU Xian-Mei, WANG Jie, FU Li, ZHAI Hao, SUN Qing-Rong^*

Shandong Institute of Pomology, Tai’an, Shandong 271000, China

Corresponding author: SUN Qing-Rong; E-mail: sdipss@163.com

Abstract:

Artemisia scoparia is a faintly scented annual or biennial herb widespread and found to be used in traditional medicine and biological control of plant disease. Phytoplasma is an important pathogen associated with the symptoms of witches’ broom, leaflet, yellow, fasciation, et al., and causes great economical loss. Artemisia scoparia plants with witches’ broom symptoms suggestive of a phytoplasma disease were sampled in Mount Tai, Shandong, China. Total DNA was extracted from symptomatic plants as well as asymptomatic plants via the modified CTAB method. Analyses by PCR using universal primer pairs R16F2n/R2 was performed to detect putative phytoplasma, and a fragment of 1 244 bp was amplified from symptomatic plants. The Blastn analysis revealed that the obtained fragment shared 99.4% and 99.8% identity with that of stolbur phytoplasma (AF248959) and the paper flower yellows phytoplasma (JX128698), respectively. The phylogenetic tree constructed based on the 16S rDNA sequences of related phytoplasmas showed that the obtained fragment (GenBank accession No.: KT899994) was located at the same branch with stolbur phytoplasma (AF248959) and paper flower yellows phytoplasma (JX128698). Then the iPhyClassifier software was used to perform sequence comparison and generate the virtual restriction fragment length polymorphism (RFLP) profile, the result showed that the obtained 16S rDNA R16F2n/R2 fragment was identical (similarity coefficient 1.0) to the reference pattern of 16Sr group XII, subgroup A (AF248959). The above results indicated that the phytoplasmas associated with Artemisia scoparia witches’ broom belongs to Ca. Phytoplasma solani. Finally, A fragment of 1 754 bp was amplified from the total DNA of symptomatic Artemisia scoparia plants by using the primer pairs of STOL4f/r which were specific for stolbur group phytoplasmas (16SrXII subgroup A), while no fragment from asymptomatic plants, the obtained fragment (GenBank accession No.: KU759570) shared 99.5% identity with AF248959. RFLP profile by iPhyClassifier software showed that the fragment was also identical (similarity coefficient 1.0) to AF248959, which further verified the amplifying result by universal primer pairs R16F2n/R2. Taken together, these results indicated that the phytoplasma accociated with Artemisia scoparia witches’ broom in Mount Tai, Shandong, China is a member of 16SrXII-A subgroup.

Key words: Artemisia scoparia witches’ broom; stolbur phytoplasma; molecular identification; 16S rDNA sequence analysis