柿树带化病相关植原体的形态及分子鉴定

Morphological and molecular detection of phytoplasma associated with persimmon fasciation disease

柿树为我国重要的经济林果树,柿果、柿叶和柿霜等包含丰富的生物活性成分,具有重要的医学价值。从山东省邹城市采集到表现带化并伴随节间缩短和叶片变小的柿树枝条样品,利用透射电子显微镜进行植原体细胞检测,结果显示染病的柿枝条韧皮部筛管细胞内存在直径为200~600 nm的圆形或者椭圆形植原体粒子。提取染病柿树枝条和健康枝条的韧皮部总DNA,利用植原体16S rRNA基因及延伸因子tuf基因通用引物进行PCR扩增,从染病组织DNA中得到了长度分别约为1.2和0.8 kb的片段,健康柿树枝条没有扩增条带。序列一致率分析表明,柿树带化植原体16S rRNA基因和tuf基因与枣疯植原体相应基因的核苷酸一致率分别高达99.8%和99.9%;i Phy Classifi er在线分析表明,柿树带化植原体与16Sr V-B亚组代表株系的16S rRNA基因具有相同的酶切图谱,相似系数为1.0;基于两个基因构建的系统进化树均表明,柿树带化植原体与16Sr V-B亚组成员聚为一簇。生物学和分子生物学鉴定结果表明,柿树带化病存在植原体,且该植原体属于榆树黄化组(16Sr V)B亚组,与候选种‘Candidatus Phytoplasma ziziphi’相关。

Persimmon （Diospyros kaki） is an important economic forest fruit tree species in China. Fruit, leaves and powder on the surface of a dried persimmon contain several biologically active compounds, used traditionally for many medicinal purposes. Persimmon plants with stems fasciation, internodes shorten, and little leaf were sampled in Zoucheng, Shandong, China. Transmission electron microscope technology was used to detect phytoplasmal cells from the tissue of symptomatic plants and a great quantity of structures resembling phytoplasmas, about 200-600 nm, circular or elliptical, were observed in phloem sieve elements. Total DNA was extracted from symptomatic plants as well as asymptomatic plants via the modified CTAB method. PCR was conducted to detect putative phytoplasma using universal primers for phytoplasmal 16S rRNA gene and tufgene, and specific fragments of circa 1.2 kb and 0.8 kb in length were amplified from diseased persimmon tissue, respectively. Sequence analysis indicated that 16S rRNA gene and tuffragrnents shared 99.8% and 99.9% identity with that of jujube witches＇-broom phytoplasma, respectively, iPhyClassifier analysis for 16S rRNA gene showed that persimmon fasciation phytoplasma has the same restriction enzyme pattern with the typical isolate of subgroup 16SrV-B, and the similarity coefficient was 1.0. Phylogenetic tree based on 16S rRNA or tufgene revealed that persimmon fasciation phytoplasma strain formed a sublineage with members of subgroup 16SrV-B. In conclusion, phytoplasma associated with persimmon fasciation in Zoucheng, Shandong, China was classified to the subgroup B of the elm yellows group （16SrV） and related to ＂Candidatus Phytoplasma ziziphi＂.