Submitted to: International Journal of Systematic and Evolutionary Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/9/1998
Publication Date: N/A
Citation: Interpretive Summary: Phytoplasmas, formerly called mycoplasmalike organisms, are associated with diseases in several hundred plant species. Thus far, none have been cultured in vitro. Until the last decade, differentiation and classification of uncultured phytoplasmas primarily relied on their biological properties, such as plant and insect vector host specificities s and symptomatology of affected plants. The determination of biological properties is often time consuming, laborious, and is sometimes unreliable. The development of molecular-based tools, especially polymerase chain reaction (PCR) assays using universal primers designed from conserved gene sequences, has made it possible for us to detect and identify a wide array of phytoplasmas associated with infected plants or insects. Numerous new phytoplasma strains have been reported within the last four years. A comprehensive classification scheme is needed for identification of these new strains. In this study we have constructed the most comprehensive classification scheme (with 14 phytoplasma groups and 41 subgroups) thus far for differentiation and classification of phytoplasmas based on RFLP analyses (DNA finger printing) of conserved 16S rRNA and ribosomal protein gene sequences. The classification by this approach is phylogenetically valid. The information and pathogen- identification technology will be of benefit to diagnosticians and to APHIS for implementation of new quarantine regulations.
Technical Abstract: RFLP analyses of 34 phytoplasma 16S rDNA (R16F2n/R16R2 nested PCR products) sequences with 17 restriction enzymes delineated distinct pattern types. Based on similarity coefficients derived from RFLP analyses, the 34 representative phytoplasma strains were differentiated into 14 major groups (termed 16S rRNA groups) and 32 subgroups. The similarity coefficients of RFLP patterns between two distinct groups wer 90 percent or below. By including the additional groups and subgroups identified based on phylogeny and putative restriction site analysis of 16S rDNA sequences from the phytoplasmas of which RFLP analyses were not performed, a total of 14 groups and 41 subgroups were proposed. By combined RFLP analyses of 16S RNA (rr) and subgroups (rr-rp) were recognized. The phytoplasma 16S rRNA groups delineated were consistent with phylogenetic groups (subclades) delineated based on phylogenetic analysis of near full length 16S rRNA gene sequences, indicating that th RFLP-based groups are phylogenetically valid. The approach using RFLP analyses of PCR-amplified 16S rDNA (and ribosomal protein gene sequences) provides a simple and reliable means that can afford differentiation and classification of many unknown phytoplasmas.