Griffiths H M, Sinclair W A, Smart C D, Davis R E
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.
Int J Syst Bacteriol. 1999 Oct;49 Pt 4:1605-14. doi: 10.1099/00207713-49-4-1605.
Phytoplasmas associated with the plant diseases ash yellows (AshY, occurring in Fraxinus) and lilac witches'-broom (LWB, occurring in Syringa) represent a putative species-level taxon. Phytoplasmal DNA from 19 ash or lilac sources across the known geographic range of AshY (71-113 degrees W) was examined to determine if AshY and LWB phytoplasmas are a coherent group, if variability exists in both conserved and anonymous DNA, and if variability in 16S rDNA is related to host or geographic origin. The 16S rRNA gene and the 16S-23S spacer were amplified using primer pair P1/P7 and analysed using 15 restriction enzymes. RFLPs were detected in digests obtained with Alul, Hhal or Taql, for a total of four RFLP profile types. Sequencing of the amplimers from strains AshY1T, AshY3, AshY5 and LWB3 (which represent the four 16S rDNA RFLP profile types) revealed only three positions in the 16S rRNA gene and one position in the 16S-23S spacer at which differences occurred; these were single nucleotide substitutions. Sequence homology between any two strains was > 99.8%. A portion of a ribosomal protein operon, amplified with primer pair rpF1/R1 from each of the four strains noted above, was analysed with six restriction enzymes, resulting in the detection of two RFLP profiles with Msel. Southern analysis, utilizing two non-specific probes from other phytoplasma groups, revealed three RFLP profile types in anonymous chromosomal DNA of strains representing the four 16S rDNA genotypes. Two strains, AshY3 and LWB3, had unique combinations of characters in the various assays. On the basis of RFLP profiles, the strains from the other plants sampled comprised two groups. The grouping was not clearly related to host or geographic origin. The genome size of strain AshY3 was estimated from PFGE data to be 645 kbp. Phylogenetic analysis of a 1423 bp 16S rDNA sequence from strains AshY1T, AshY3, AshY5 and LWB3, together with sequences from 14 other mollicutes archived in GenBank, produced a tree on which the AshY and LWB strains clustered as a discrete group, consistent with previous analyses utilizing only type strain AshY1T. Thus, the AshY phytoplasma group is coherent but heterogeneous. The name 'Candidatus Phytoplasma fraxini' is proposed for this group.
与植物病害白蜡黄化病（AshY，发生在白蜡属植物上）和丁香丛枝病（LWB，发生在丁香属植物上）相关的植原体代表了一个假定的物种级分类单元。对来自AshY已知地理范围（西经71 - 113度）内19个白蜡或丁香来源的植原体DNA进行了检测，以确定AshY和LWB植原体是否为一个连贯的群体，保守DNA和非编码DNA中是否存在变异性，以及16S rDNA中的变异性是否与宿主或地理起源相关。使用引物对P1/P7扩增16S rRNA基因和16S - 23S间隔区，并使用15种限制酶进行分析。在用Alul、Hhal或TaqI酶切获得的消化产物中检测到了RFLP，总共四种RFLP图谱类型。对代表四种16S rDNA RFLP图谱类型的菌株AshY1T、AshY3、AshY5和LWB3的扩增子进行测序，结果显示16S rRNA基因中仅三个位置和16S - 23S间隔区中一个位置存在差异；这些差异均为单核苷酸替换。任意两个菌株之间的序列同源性> 99.8%。用引物对rpF1/R1从上述四个菌株中扩增核糖体蛋白操纵子的一部分，并用六种限制酶进行分析，用Msel酶切检测到两种RFLP图谱。利用来自其他植原体群体的两种非特异性探针进行Southern分析，在代表四种16S rDNA基因型的菌株的非编码染色体DNA中检测到三种RFLP图谱类型。两个菌株AshY3和LWB3在各种检测中具有独特的特征组合。根据RFLP图谱，从其他采样植物中获得的菌株分为两组。这种分组与宿主或地理起源没有明显关系。根据PFGE数据估计菌株AshY3的基因组大小为645 kbp。对菌株AshY1T、AshY3、AshY5和LWB3的1423 bp 16S rDNA序列以及GenBank中存档的其他14种柔膜菌的序列进行系统发育分析，得到一棵树，其中AshY和LWB菌株聚为一个离散的群体，这与之前仅使用模式菌株AshY1T的分析结果一致。因此，AshY植原体群体是连贯但异质的。建议将这个群体命名为“Candidatus Phytoplasma fraxini”。
