Conserved gene sequences, including 16S rRNA and ribosomal protein gene sequences, were used to evaluate genetic variations in phytoplasma strains belonging to 16S rRNA groups I (aster yellows and related phytoplasmas) and III (X-disease and related phytoplasmas). We used PCR to amplify the sequences of the 16S ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rps19, all of rpl22, and rps3) from diverse phytoplasma group I and III strains. Additional chromosomal gene sequences of group I strains were also amplified. The PCR products amplified from members of each group of phytoplasmas were compared by performing restriction fragment length polymorphism (RFLP) analyses. On the basis of the RFLP patterns observed and similarity coefficients derived from combined RFLP analyses, the phytoplasma strains belonging to groups I and III were placed in distinct 16S rRNA, ribosomal protein, and 16S rRNA-ribosomal protein subgroups. Analyses of two or more conserved gene sequences revealed that members of the two groups were more diverse than previously thought. Subgroup differentiation on the basis of our combined analyses of 16S rRNA and ribosomal protein gene sequences seemed to adequately reflect the levels of chromosomal homology determined by DNA-DNA hybridization assays. On the basis of unique RFLP profiles, we identified new, previously unclassified group I phytoplasma strains, including the organisms that are associated with witches'-broom [subgroup 16SrI-F(rr-rp)], maize bushy stunt [subgroup 16SrI-I(rr-rp)], and Mexican periwinkle virescence [subgroup 16SrI-J(rr-rp)], and new, previously unclassified group III phytoplasma strains, including the organism that is associated with pecan bunch [subgroup 16SrIII-H(rr-rp)]. On the basis of the results of our analyses of 16S rRNA and ribosomal protein conserved gene sequences, we recognized 9 group I subgroups and eight group III subgroups. We propose that phytoplasma strains belonging to each group I and III subgroup should be distinguished taxonomically at a level equivalent to the subspecies level.


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