Molecular biodiversity of phytoplasmas

A new, more efficient procedure based on heteroduplex mobility assay (HMA) was developed for studies of molecular biodiversity of phytoplasmas. By analyzing the artificially introduced substitutions and insertion/deletions into the 16S rRNA gene of aster yellows phytoplasma, HMA was shown to be capable of detecting a single base-pair deletion/insertion or a single two-base-pair substitution in a 520 base-pair DNA fragment. A single base-pair substitution was differentiated by HMA when a suitable reference was used. Heteroduplex mobilities were affected by base number and base composition in mismatches or gaps and were proportional to the degree of DNA divergence. Gaps caused greater retardation in heteroduplex mobility than did mismatches. HMA was also highly sensitive in detecting mixed infections of phytoplasmas.; The partial and full 16S rRNA, the 16/23S spacer region, and the large DNA fragment comprising the full 16S rRNA and 16/23S spacer region amplified from 24 representative phytoplasmas were subjected to HMA analysis. Different groups were clearly discerned in HMA profiles and fully agreed with those derived from DNA sequencing analysis. The 16/23S spacer region was found to be a suitable sequence for using HMA in the classification of phytoplasmas with aster yellows phytoplasma isolate 27 (AY27) as a reference.; A comparative study was conducted to evaluate RFLP and HMA for differentiating closely-related phytoplasmas by analyzing both the 16S rRNA gene and the 16/23S spacer regions amplified from phytoplasma strains of clover proliferation (CP), alfalfa witches'-broom (AWB), and potato witches'-broom (PWB). Two subgroups among each of the phytoplasmas CP, AWB, and PWB were identified by HMA but not by RFLP. A total of 62 phytoplasma isolates were identified and classified into five major groups by HMA analysis of the 16/23S spacer region. More genetic diversity of phytoplasmas was observed than in previous studies. Twenty-five phytoplasma diseases were reported and identified for the first time in Canada. Phytoplasmas in subgroups I-A and I-B were prevalent in Alberta and more phytoplasmas were found in subgroup I-A than in subgroup I-B. These studies have indicated that HMA is a reliable, rapid, and accurate method for the detection and estimation of the genetic divergence of phytoplasmas when other methods such as RFLP analysis are not readily applicable.; The secondary structure model of the 16S rRNA gene of AY phytoplasma was constructed and the highly variable sections were identified among phytoplasmas in ten major groups. The tRNAIle located in the 16/23S spacer region was found to evolve slowly. Inferences about the molecular evolution of phytoplasmas were made from a phylogenetic tree with a high confidence level constructed by analyzing the nucleotide sequences of the 16S rRNA gene and the 16/23S spacer region of 24 phytoplasmas. This study suggested that phytoplasmas might have evolved into two branches with different genome sizes from their ancestor and that phytoplasmas in the CP group likely originated from the AP group, with the WX group acting as a possible bridge.