Aster yellows (AY) group (16SrI) phytoplasmas are associated with over 100 economically important diseases worldwide and represent the most diverse and widespread phytoplasma group. Strains that belong to the AY group form a phylogenetically discrete subclade within the phytoplasma clade and are related most closely to the stolbur phytoplasma subclade, based on analysis of 16S rRNA gene sequences. AY subclade strains are related more closely to their culturable relatives, Acholeplasma spp., than any other phytoplasmas known. Within the AY subclade, six distinct phylogenetic lineages were revealed. Congruent phylogenies obtained by analyses of tuf gene and ribosomal protein (rp) operon gene sequences further resolved the diversity among AY group phytoplasmas. Distinct phylogenetic lineages were identified by RFLP analysis of 16S rRNA, tuf or rp gene sequences. Ten subgroups were differentiated, based on analysis of rp gene sequences. It is proposed that AY group phytoplasmas represent at least one novel taxon. Strain OAY, which is a member of subgroups 16SrI-B, rpI-B and tufI-B and is associated with evening primrose (Oenothera hookeri ) virescence in Michigan, USA, was selected as the reference strain for the novel taxon 'Candidatus Phytoplasma asteris'. A comprehensive database of diverse AY phytoplasma strains and their geographical distribution is presented.

Extensive phylogenetic analyses were performed based on sequences of the 16S rRNA gene and two ribosomal protein (rp) genes, rplV (rpl22) and rpsC (rps3), from 46 phytoplasma strains representing 12 phytoplasma 16Sr groups, 16 other mollicutes and 28 Gram-positive walled bacteria. The phylogenetic tree inferred from rp genes had a similar overall topology to that inferred from the 16S rRNA gene. However, the rp gene-based tree gave a more defined phylogenetic interrelationship among mollicutes and Gram-positive walled bacteria. Both phylogenies indicated that mollicutes formed a monophyletic group. Phytoplasmas clustered with Acholeplasma species and formed one clade paraphyletic with a clade consisting of the remaining mollicutes. The closest relatives of mollicutes were low-G+C-content Gram-positive bacteria. Comparative phylogenetic analyses using the 16S rRNA gene and rp genes were performed to evaluate their efficacy in resolving distinct phytoplasma strains. A phylogenetic tree was constructed based on analysis of rp gene sequences from 87 phytoplasma strains belonging to 12 16Sr phytoplasma groups. The phylogenetic relationships among phytoplasmas were generally in agreement with those obtained on the basis of the 16S rRNA gene in the present and previous works. However, the rp gene-based phylogeny allowed for finer resolution of distinct lineages within the phytoplasma 16Sr groups. RFLP analysis of rp gene sequences permitted finer differentiation of phytoplasma strains in a given 16Sr group. In this study, we also designed several semi-universal and 16Sr group-specific rp gene-based primers that allow for the amplification of 11 16Sr group phytoplasmas.

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In the spring of 2000, an aster yellows (AY) epidemic occurred in carrot crops in the Winter Garden region of southwestern Texas. A survey revealed that vegetable crops, including cabbage, onion, parsley, and dill, and some weeds also were infected by AY phytoplasmas. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis of PCR-amplified phytoplasma 16S rDNA were employed for the detection and identification of phytoplasmas associated with these crops and weeds. Phytoplasmas belonging to two subgroups, 16SrI-A and 16SrI-B, in the AY group (16SrI), were predominantly detected in infected plants. Carrot, parsley, and dill were infected with both subgroups. Onion and three species of weeds (prickly lettuce, lazy daisy, and false ragweed) were predominantly or exclusively infected by subgroup 16SrI-A phytoplasma strains, while cabbage was infected by subgroup 16SrI-B phytoplasmas. Both types of phytoplasmas were detected in three leafhopper species, Macrosteles fascifrons, Scaphytopius irroratus, and Ceratagallia abrupta, commonly present in this region during the period of the epidemic. Mixed infections were very common in individual carrot, parsley, and dill plants and in individual leafhoppers. Sequence and phylogenetic analyses of 16S rDNA and ribosomal protein (rp) gene sequences indicated that phytoplasma strains within subgroup 16SrI-A or subgroup 16SrI-B, detected in various plant species and putative insect vectors, were highly homogeneous. However, based on rp sequences, two rpI subgroups were identified within the subgroup 16SrI-A strain cluster. The majority of subgroup 16SrI-A phytoplasma strains were classified as rp subgroup rpI-A, but phytoplasma strains detected in one onion sample and two leafhoppers (M. fascifrons and C. abrupta) were different and classified as a new rp subgroup, rpI-N. The degree of genetic homogeneity of the phytoplasmas involved in the epidemic suggested that the phytoplasmas came from the same pool and that all three leafhopper species may have been involved in the epidemic. The different phytoplasma population profiles present in various crops may be attributed to the ecological constraints as a result of the vector-phytoplasma-plant three-way interaction.

During the growing seasons of 2003 and 2004, a disease occurred in several carrot crops in south central Washington with symptoms suggestive of infection by phytopathogenic mollicutes (phytoplasmas and spiroplasmas). In the fall, many affected carrot plants exhibited extensive purple or yellow-purple leaf discoloration, general stunting of shoots and taproots, and formation of bunchy, fibrous secondary roots. For detection of the putative causal agents, polymerase chain reaction (PCR) assays were performed using primers specific to phytoplasmas as well as primers specific to plant-pathogenic spiroplasmas. Restriction fragment length polymorphism (RFLP) analyses of PCR-amplified 16S rDNA sequences revealed that about 81% of affected plants showing dark purple or yellow-purple leaf symptoms tested positive for Spiroplasma citri. Of affected plants showing mild purple discoloration of leaf margins, 18% tested positive for a phytoplasma strain belonging to the clover proliferation group (16SrVI), subgroup 16SrVI-A, and 11% for another phytoplasma strain belonging to the aster yellows group (16SrI), subgroup 16SrI-A. Nucleotide sequence analysis of cloned 16S rDNA confirmed the phytoplasma group affiliations. Some symptomatic plants were co-infected with S. citri and either aster yellows phytoplasma or clover proliferation group phytoplasma. To our knowledge, this is the first documentation of spiroplasma infection of carrot in the United States.