Lee, I.-M.

ABSTRACT “ Candidatus Phytoplasma pruni” strain CX, belonging to subgroup 16SrIII-A, is a plant-pathogenic bacterium causing economically important diseases in many fruit crops. Here, we report the draft genome sequence, which consists of 598,508 bases, with a G+C content of 27.21 mol%.

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the class Mollicutes . Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, ‘Candidatus Phytoplasma’, and the genus was embraced within the order Acholeplasmatales . However, phytoplasmas differ significantly from species of the genus Acholeplasma in their habitat specificities, modes of life, metabolic capabilities, genomic architectures, and phylogenetic positions. This communication describes the unique ecological, nutritional, biochemical, genomic and phylogenetic properties that distinguish phytoplasmas from species of the genus Acholeplasma and all other taxa in the class Mollicutes . Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the order Acholeplasmatales and of all other existing orders, namely Mycoplasmatales , Entomoplasmatales and Anaeroplasmatales , this communication raises the question of whether ‘ Candidatus Phytoplasma ’ should be retained in the order Acholeplasmatales or whether a novel provisional order and family should be created to accommodate the genus ‘ Ca. Phytoplasma ’.

X-disease is one of the most serious diseases known in peach (Prunus persica). Based on RFLP analysis of 16S rRNA gene sequences, peach X-disease phytoplasma strains from eastern and western United States and eastern Canada were classified in 16S rRNA gene RFLP group 16SrIII, subgroup A. Phylogenetic analyses of 16S rRNA gene sequences revealed that the X-disease phytoplasma strains formed a distinct subclade within the phytoplasma clade, supporting the hypothesis that they represented a lineage distinct from those of previously described ‘Candidatus Phytoplasma ’ species. Nucleotide sequence alignments revealed that all studied X-disease phytoplasma strains shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Candidatus Phytoplasma ’ species. Based on unique properties of the DNA, we propose recognition of X-disease phytoplasma strain PX11CT1R as representative of a novel taxon, ‘Candidatus Phytoplasma pruni’. Results from nucleotide and phylogenetic analyses of secY and ribosomal protein (rp) gene sequences provided additional molecular markers of the ‘Ca. Phytoplasma pruni’ lineage. We propose that the term ‘Ca. Phytoplasma pruni’ be applied to phytoplasma strains whose 16S rRNA gene sequences contain the oligonucleotide sequences of unique regions that are designated in the formally published description of the taxon. Such strains include X-disease phytoplasma and - within the tolerance of a single base difference in one unique sequence - peach rosette, peach red suture, and little peach phytoplasmas. Although not employed for taxon delineation in this work, we further propose that secY, rp, and other genetic loci from the reference strain of a taxon, and where possible oligonucleotide sequences of unique regions of those genes that distinguish taxa within a given 16Sr group, be incorporated in emended descriptions and as part of future descriptions of ‘Candidatus Phytoplasma ’ taxa.

Symptoms of abnormal proliferation of shoots resulting in formation of witches’-broom growths were observed on diseased plants of passion fruit (Passiflora edulis f. flavicarpa Deg.) in Brazil. RFLP analysis of 16S rRNA gene sequences amplified in PCRs containing template DNAs extracted from diseased plants collected in Bonito (Pernambuco) and Viçosa (Minas Gerais) Brazil, indicated that such symptoms were associated with infections by two mutually distinct phytoplasmas. One phytoplasma, PassWB-Br4 from Bonito, represents a new subgroup, 16SrIII-V, in the X-disease phytoplasma group (‘Candidatus Phytoplasma pruni’-related strains). The second phytoplasma, PassWB-Br3 from Viçosa, represents a previously undescribed subgroup in group 16SrVI. Phylogenetic analyses of 16S rRNA gene sequences were consistent with the hypothesis that strain PassWB-Br3 is distinct from previously described ‘Ca. Phytoplasma ’ species. Nucleotide sequence alignments revealed that strain PassWB-Br3 shared less than 97.5 % 16S rRNA gene sequence similarity with previously described ‘Ca. Phytoplasma ’ species. The unique properties of its DNA, in addition to natural host and geographical occurrence, support the recognition of strain PassWB-Br3 as a representative of a novel taxon, ‘Candidatus Phytoplasma sudamericanum’.

A novel phytoplasma, designated strain SoyST1c1, associated with a newly emerging disease in soybean (Glycine max), known as soybean stunt (SoyST), was found in 2002 in a soybean plantation in Alajuela Province, Costa Rica. The same phytoplasma, or a very closely related strain, also infected sweet pepper (Capsicum annuum) with purple vein syndrome (SwPPV) and passion fruit vine (Passiflora edulis) with bud proliferation disease (PasFBP) in the same region. Sequence analysis of cloned 16S rRNA gene sequences (GenBank accession nos FJ226068–FJ226073 and HQ225624–HQ225635) indicated that all three affected plants were infected by phytoplasmas that shared <97.5 % sequence similarity with previously described phytoplasmas. The SoyST-causing phytoplasma represents a new taxon, most closely related to phytoplasma group 16SrI and 16SrXII strains. Virtual RFLP analysis indicated that the SoyST-causing phytoplasma and its closely related strains represent a novel 16Sr group, designated 16SrXXXI. Phylogenetic analysis of 16S rRNA gene sequences from the new phytoplasma strains, those previously described as ‘Candidatus Phytoplasma spp.’ and other distinct, as yet unnamed, phytoplasmas indicated that the SoyST-causing phytoplasma represents a distinct lineage within the aster yellows/stolbur branch on the phylogenetic tree. On the basis of its unique 16S rRNA gene sequence and biological properties, strain SoyST1c1 represents a novel taxon, for which the name ‘Candidatus Phytoplasma costaricanum’ is proposed with SoyST1c1 as the reference strain.

A new disease of potatoes, tentatively named zebra chip (ZC) because of the intermittent dark and light symptom pattern in affected tubers which is enhanced by frying, was first found in Mexico in 1994 and in the southwestern United States in 2000. The disease can cause severe economic losses in all market classes of potatoes. The cause of ZC has been elusive, and only recently has been associated with ‘Candidatus Liberibacter’ sp. Field samples of potato plants were collected from several locations in the United States, Mexico, and Guatemala to determine transmission to potato and tomato by grafting of ZC-infected scions and psyllid feeding. The disease was successfully transmitted, through up to three generations, by sequential top- and side-grafting ZC-infection scions to several potato cultivars and to tomato. The disease was also successfully transmitted to potato and tomato plants in greenhouse experiments by potato psyllids collected from potato plants naturally affected with ZC. Transmission electron microscopic observation of ZC-affected tissues revealed the presence of bacteria-like organisms (BLOs) in the phloem of potato and tomato plants inoculated by grafting and psyllid feeding. The BLOs were morphologically similar in appearance to BLOs associated with other plant diseases. Polymerase chain reaction (PCR) amplified 16S rDNA sequences from samples representing different geographic areas, including the United States, Mexico, and Guatemala, were almost identical to the 16S rDNA of ‘Ca. L. solanacearum’ previously reported from solanaceous plants in New Zealand and the United States. Two subclades were identified that differed in two single base-pair substitutions. New specific primers along with an innovative rapid PCR were developed. This test allows the detection of the bacteria in less than 90 min. These data confirm the association of ‘Ca. L. solanacearum’ with potatoes affected by ZC in the United States, Mexico, and Guatemala.

Potato purple top wilt (PPT) is a devastating disease that occurs in various regions of North America and Mexico. At least three distinct phytoplasma strains belonging to three different phytoplasma groups (16SrI, 16SrII and 16SrVI) have been associated with this disease. A new disease with symptoms similar to PPT was recently observed in Texas and Nebraska, USA. Two distinct phytoplasma strain clusters were identified. One belongs to the 16SrI phytoplasma group, subgroup A, and the other is a novel phytoplasma that is most closely related to, and shares 96.6 % 16S rRNA gene sequence similarity with, a member of group 16SrXII. Phylogenetic analysis of 16S rRNA gene sequences of the novel PPT-associated phytoplasma strains, previously described ‘Candidatus Phytoplasma’ organisms and other distinct unnamed phytoplasmas indicated that the novel phytoplasma, termed American potato purple top wilt (APPTW) phytoplasma, represents a distinct lineage and shares a common ancestor with stolbur phytoplasma, ‘Candidatus Phytoplasma australiense’, ‘Candidatus Phytoplasma japonicum’, ‘Candidatus Phytoplasma fragariae’, bindweed yellows phytoplasma (IBS), ‘Candidatus Phytoplasma caricae’ and ‘Candidatus Phytoplasma graminis’. On the basis of unique 16S rRNA gene sequences and biological properties, it is proposed that the APPTW phytoplasma represents ‘Candidatus Phytoplasma americanum’, with APPTW12-NE as the reference strain.

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.

Elm yellows group (16SrV) phytoplasmas, which are associated with devastating diseases in elm, grapevine, blackberry, cherry, peach and several other plant species in America, Europe and Asia, represent one of the most diverse phytoplasma clusters. On the basis of phylogenetic analysis of 16S rDNA sequences, elm yellows group phytoplasmas form a discrete subclade within the phytoplasma clade. Three phylogenetic parameters, namely 16S rRNA, ribosomal protein and secY genes, have been evaluated for their usefulness in differentiating elm yellows group phytoplasmas. RFLP analysis of 16S rRNA sequences differentiated the elm yellows group phytoplasmas into five subgroups. Twelve RFLP subgroups were differentiated on the basis of ribosomal protein and 13 were differentiated using secY gene sequences. Phylogenetic analysis of the ribosomal protein genes and secY gene alone or in combination indicated that the subgroups constitute 12 genetically distinct lineages, each of which appears to have evolved under different ecological constraints such as specific vector or plant hosts. On the basis of unique DNA and biological properties, it is proposed that the elm yellows phytoplasma EY1T represents a novel taxon, ‘Candidatus Phytoplasma ulmi’.