Classification of plant-pathogenic mycoplasma-like organisms using restriction-site analysis of PCR-amplified 16S rDNA

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.

Phylogenetic positions of phytoplasmas associated with dieback, yellow crinkle and mosaic diseases of papaya, and their proposed inclusion in ' Candidatus Phytoplasma australiense' and a new taxon, 'Candidatus Phytoplasma australasia'

“…To date, the most reliable classifications of phytoplasmas have been based on DNA restriction analysis and phylogenetic sequence analysis, usually of the PCR-amplified 16s rRNA gene plus 16s-23s rRNA intergenic spacer region (R. E. ; R. I. Gundersen et al, 1994Gundersen et al, , 1996Lee et al, 1993;Namba et al, 1993;Schneider et al, 1993Schneider et al, , 1995aSeemuller et al, 1994). These analyses have allowed the provisional classification of phytoplasmas from Europe, North America, Asia and Australia.…”

Polymerase Chain Reaction for Phytoplasmas Detection

“…However, serological methods weren't always sensitive enough to detect various phytoplasmas (13,47). Finally, in the early 1990's PCR coupled with RFLP analysis allowed the accurate identification of different strains and species of phytoplasma (127,91,145). Nowadays, diagnosis of phytoplasmas is routinely done by PCR and can be divided into three phases: total DNA extraction from symptomatic tissue or insects; PCR amplification of phytoplasma-specific DNA; characterization of the amplified DNA by sequencing, RFLP analysis or nested PCR with group-specific primers (117).…”

“…More specific detection methods involve using phytoplasma-specific primers or differentiation on the basis of phylogenetic RFLP analysis of PCR amplified sequences (91,145). RFLP analysis of PCR amplified DNA sequences using a number of endonuclease restriction enzymes (93).…”

Polymerase Chain Reaction Detection and Phylogenetic Characterization of an Agent Associated with Yellow Vine Disease of Cucurbits

“…The DNA amplified using protocol II was digested with Bcll and EcoRI to determine if the amplified DNA corresponded to the 16S rDNA of prokaryotes or chloroplasts. The chloroplast 16S rDNA has one or more restriction sites for BclI, while the prokaryotic 16S rDNA has none (27). The latter also has a centrally located restriction site for EcoRI (27) absent from chloroplast 16S rDNA (pea, EMBL accession no.…”

“…The chloroplast 16S rDNA has one or more restriction sites for BclI, while the prokaryotic 16S rDNA has none (27). The latter also has a centrally located restriction site for EcoRI (27) absent from chloroplast 16S rDNA (pea, EMBL accession no. X55033).…”