No abstract

In order to develop a diagnostic tool to identify phytoplasmas and classify them according to their phylogenetic group, we took advantage of the sequence diversity of the 16S-23S intergenic spacer regions (SRs) of phytoplasmas. Ten PCR primers were developed from the SR sequences and were shown to amplify in a group-specific fashion. For some groups of phytoplasmas, such as elm yellows, ash yellows, and pear decline, the SR primer was paired with a specific primer from within the 16S rRNA gene. Each of these primer pairs was specific for a specific phytoplasma group, and they did not produce PCR products of the correct size from any other phytoplasma group. One primer was designed to anneal within the conserved tRNA Ile and, when paired with a universal primer, amplified all phytoplasmas tested. None of the primers produced PCR amplification products of the correct size from healthy plant DNA. These primers can serve as effective tools for identifying particular phytoplasmas in field samples.

No abstract

A method has been developed to amplify the 16s rRNA gene of plant-pathogenic mycoplasma-like organisms (MLOs) from infected plant material using the polymerase chain reaction (PCR). The procedure is dependent on the presence of a BcZI restriction site in the 16s rDNA of chloroplasts but not in that of the MLOs. This difference permits the specific amplification of the 16s rDNA of the MLOs from BcZI-digested total DNA from infected plants using primers from conserved regions of this gene. In this study 16s rDNA was obtained from 52 MLO isolates from herbaceous dicots and monocots as well as woody plants. Digestion of the 16s rRNA genes using A M endonuclease revealed seven restriction patterns, which were used to group the isolates examined. Group I, which is also characterized by the presence of two KpnI sites, consisted of 31 isolates, most of which are from herbaceous dicots. Isolates assigned to groups I1 to VI were mostly from woody plants, while the isolates of group VII were from monocots or obtained from a leafhopper. The restriction patterns varied little within groups; however, four group I isolates and one group IV isolate differed slightly from the typical patterns of these groups as a result of a deletion or a slight shift of one restriction site. The groupings uncovered by Ah1 restriction were also obtained by digesting the 16s rDNA with RsaI endonuclease. However, some atypical patterns were observed within group V isolates. The groups described on the basis of restriction digest data were supported by sequence analysis. With one exception, the 16s rDNA of isolates within the same group exhibited 97.8 to 99-5 YO homology while those of different groups showed 89.6 to 92.0% homology.