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A global phylogenetic analysis using parsimony of 16S rRNA gene sequences from 46 mollicutes, 19 mycoplasmalike organisms (MLOs) (new trivial name, phytoplasmas), and several related bacteria placed the MLOs definitively among the members of the class Mollicutes and revealed that MLOs form a large discrete monophyletic clade, paraphyletic to the Achokplasma species, within the Anaeroplasma clade. Within the MLO clade resolved in the global mollicutes phylogeny and a comprehensive MLO phylogeny derived by parsimony analyses of 16S rRNA gene sequences from 30 diverse MLOs representative of nearly all known distinct MLO groups, five major phylogenetic groups with a total of 11 distinct subclades (monophyletic groups or taxa) could be recognized. These MLO subclades (roman numerals) and designated type strains were as follows: i, Maryland aster yellows AY1; ii, apple proliferation AP-A; iii, peanut witches'-broom PnWB; iv, Canada peach X CX; v, rice yellow dwarf RYD; vi, pigeon pea witches'-broom PPWB; vii, palm lethal yellowing LY; viii, ash yellows AshY; ix, clover proliferation CP; x, elm yellows EY; and xi, loofah witches'-broom LfWB. The designations of subclades and their phylogenetic positions within the MLO clade were supported by a congruent phylogeny derived by parsimony analyses of ribosomal protein L22 gene sequences from most representative MLOs. On the basis of the phylogenies inferred in the present study, we propose that MLOs should be represented taxonomically at the minimal level of genus and that each phylogenetically distinct MLO subclade identified should represent at least a distinct species under this new genus.Mycoplasmalike organisms (MLOs) have been found to be associated with diseases in several hundred plant species since 1967, when Doi et al. (7) first discovered the presence of cell wall-less prokaryotes within the sieve cells of plants exhibiting "yellows" symptoms. These unique plant pathogenic prokaryotes have been termed MLOs because of their resemblance in morphology and ultrastructure to animal mycoplasmas (members of the class Mollicutes). For nearly three decades, attempts to isolate MLOs in pure culture have failed.The inability to culture MLOs has made it difficult to characterize these pathogens and to determine their taxonomic status by traditional methods. Consequently, it has long remained uncertain whether all MLOs are members of the class Mollicutes.The members of the class Mollicutes, commonly referred to as mollicutes (13) (50) and Maniloff (28) indicated that the mollicutes arose from a gram-positive clostridium-like ancestor of the lactobacillus lineage. The phylogenetic relationships among mollicutes established by these studies revealed that the previous taxonomic scheme based on phenotypic (biological and biochemical) characteristics was not always consistent with phylogeny, underscoring a necessity to modify the traditional concept of taxonomy (38). On the basis of the study by Weisburg et al. (50), the taxonomy of the class Mollicutes was recently revised to be ...

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 I11 (X-disease and related phytoplasmas). We used PCR to amplifjr the sequences of the 16s ribosomal DNA and a segment of the ribosomal protein gene operon (encoding the 3' region of rpsl9, all of rp122, and rps3) from diverse phytoplasma group I and I11 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 I11 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 Ipomoea obscura 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 I11 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 I11 subgroups. We propose that phytoplasma strains belonging to each group I and I11 subgroup should be distinguished taxonomically at a level equivalent to the subspecies level. Phytoplasmas (26), which previously have been called mycoplasmalike organisms, are unculturable prokaryotes that are associated with diseases in a wide variety of economically important crop plants (44). In nature, phytoplasmas are carried by different homopterous insect vectors (both monophagous and oligophagous) and are transmitted to different host plants (7-10, 31, 44). Traditionally, phytoplasma-induced diseases have been named and the identities of the associated phytoplasmas have been determined on the basis of the plant hosts and the symptoms induced in the plant hosts. In general, it has been assumed that each disease is caused by a single distinct phytoplasma. These biological properties and vector specificity have been used by workers in some laboratories to identify major phytoplasma groups (10). This system is complicated and laborious and often has resulted in misleading conclusions because the phytopl...