假臭草丛枝病植原体鉴定与多样性分析

Identification and diversity analysis of phytoplasma associated with Praxelis clematidea witch’s broom

研究假臭草丛枝病植原体的多样性,并确定其分类地位,对于利用假臭草丛枝病植原体对假臭草进行生物防治具有重要的意义。本研究采集了海南省8个地区的假臭草丛枝病样品,采用PCR以及巢式PCR方法扩增了假臭草丛枝病植原体的16S rDNA序列片段,进一步选用AfaI、AluI、EcoRI、HaeⅢ、HpaⅡ、HhaI、HinfI、KpnI、Sau3AI、TaqI和XspI等11种限制性内切酶对巢式PCR产物进行酶切分析(RFLP),并对16SrDNA序列进行测序,确定假臭草丛枝病植原体多样性和生物学地位。结果发现:假臭草丛枝病的病原确为植原体;8个地区的假臭草丛枝病植原体的酶切图谱基本一致,与已知植原体的相似度为0.26～0.97;8个地区假臭草丛枝病植原体的序列同源性均在99.4%以上,应为同种植原体;假臭草丛枝病植原体与16S rRNA Ⅱ-A组的花生丛枝病(PnWB)的同源性最高达到99.1%,说明假臭草丛枝病植原体在分类学地位上应归属于植原体16Sr RNA Ⅱ-A组。

To study the diversity and classification of phytoplasma associated with Praxelis clema tidea witch＇ s broom is of significance for the bio-control of the pathogen. In this paper, the P. clematidea plants with the presence of the phytoplasma were collected from eight counties in Hainan Province of South China. The samples were tested by PCR amplification using phytoplas- ma-specific 16S rDNA primers. Furthermore, the diversity of the phytoplasma associated with P. clematidea witch＇ s broom was investigated with restricted fragment length polymorphisms （RFLP） by using the restriction enzymes Afa Ⅰ, Alu Ⅰ, EcoR Ⅰ, HaeⅢ, Hpa Ⅱ, Hha Ⅰ, Hinf Ⅰ, Kpn Ⅰ, Sau3A Ⅰ, Taq Ⅰ, and Xsp Ⅰ. The PCR products were cloned and sequenced, and the se- quences were compared with the members of 16SrⅡ group of phytoplasma. The results showed that the causal agent of P. clematidea witch＇ s broom was the phytoplasma. The patterns of RFLP analysis of 16S rDNA sequence primed from the phytoplasma collected from the eight counties were basically the same, and the similarity between the 16S rDNA and that of other known phyto- plasmas ranged from 0.26 to 0.97. There was a high similarity （ 〉99.4% ） within the sequences from the eight counties, and the highest similarity （99.1% ） with the phytoplasma from peanut witch＇ s broom. It was suggested that the phytoplasma of P. clematidea witch＇ s broom could be categorized as a branch of 16S rRNAⅡ -A group.