Davis, Robert E.

Phy.to.plas'ma. Gr. masc. n.phytosa plant; Gr. neut. n.plasmasomething formed or molded, a form.Tenericutes / Mollicutes / Acholeplasmatales / incertae Sedis ‐ Family II / “Candidatus Phytoplasma”Phytoplasmas (Sears and Kirkpatrick, 1994) are wall‐less, nutritionally fastidious, phytopathogenic prokaryotes 0.2–0.8 µm in diameter that morphologically resemble members of theMollicutes. Sequencing of nearly full‐length PCR‐amplified 16S rRNA genes (Gundersen et al., 1994; Namba et al., 1993; Seemüller et al., 1994), combined with earlier studies (Kuske and Kirkpatrick, 1992b; Lim and Sears, 1989), provided the first comprehensive phylogeny of the organisms and showed that they constitute a unique, monophyletic clade within theMollicutes. These organisms are most closely related to members of the genusAcholeplasmawithin theAnaeroplasmaclade as defined by Weisburg et al. (1989). Sustained culture in cell‐free media has not yet been demonstrated for any phytoplasma. Their genome sizes have been estimated to range from 530 to 1350 kb, and the G+C content of phytoplasma DNA is about 23–30 mol%. The presence of a characteristic oligonucleotide sequence in the 16S rRNA gene, CAA GAY BAT KAT GTK TAG CYG GDC T, and standard codon usage indicate that phytoplasmas represent a distinct taxon for which the name “CandidatusPhytoplasma” has been adopted by specialists in the molecular biology and pathogenicity of these and similar phytopathogenic organisms (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma Taxonomy Group, 2004). At present, the designation “Candidatus” must still be used for new types.

North American grapevine yellows (NAGY) disease has sometimes been attributed to infection of Vitis vinifera L. by Prunus X-disease phytoplasma (‘Candidatus Phytoplasma pruni’) but this attribution may not be fully adequate. In this study, phytoplasma strains related to ‘Ca. Phytoplasma pruni’ were found in NAGY-diseased grapevines in Maryland, Pennsylvania, Virginia, Ohio, Missouri, and New York State. Based on restriction fragment length polymorphism analysis of 16S ribosomal RNA gene (16S rDNA) sequences, the strains (termed NAGYIII strains) were classified in group 16SrIII (X-disease group) but they contained a recognition site for the restriction endonuclease MseI that is not present in the 16S rDNA of ‘Ca. Phytoplasma pruni’. The 16S rDNA of the strains differed by three or four nucleotides from that of ‘Ca. Phytoplasma pruni’, indicating that they belonged to two novel 16S rDNA sequevars, designated NAGYIIIα and NAGYIIIβ. Both sequevars differed from ‘Ca. Phytoplasma pruni’ by a single base in each of three regions corresponding to species-unique (signature) sequences described for ‘Ca. Phytoplasma pruni’. Phylogenetic analyses of 16S rRNA genes and SecY proteins, and single-nucleotide polymorphism analyses of secY and ribosomal protein genes, further distinguished the two grapevine sequevar lineages from one another and from ‘Ca. Phytoplasma pruni’. The NAGYIIIα and NAGYIIIβ sequevars also differed from ‘Ca. Phytoplasma pruni’ in regions of the folded SecY protein that are predicted to be near or exposed at the outer surface of the phytoplasma membrane. No evidence indicated that diseased grapevines contained any phytoplasma strain conforming to ‘Ca. Phytoplasma pruni’ sensu stricto. Because the NAGYIII sequevars have not been reported in X-disease, a question is raised as to whether NAGYIII and Prunus X-disease are caused by different phytoplasma genotypes.

Phytoplasmas are a diverse but phylogenetically coherent group of cell-wall-less bacteria affiliated with the classMollicutes. Due to difficulties in establishing axenic culture, phytoplasmas were assigned to a provisional genus, ‘CandidatusPhytoplasma’, and the genus was embraced within the orderAcholeplasmatales. However, phytoplasmas differ significantly from species of the genusAcholeplasmain 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 genusAcholeplasmaand all other taxa in the classMollicutes. Since such distinguishing properties of the phytoplasmas are not referable to the descriptions of the orderAcholeplasmatalesand of all other existing orders, namelyMycoplasmatales,EntomoplasmatalesandAnaeroplasmatales, this communication raises the question of whether ‘CandidatusPhytoplasma’ should be retained in the orderAcholeplasmatalesor whether a novel provisional order and family should be created to accommodate the genus ‘Ca.Phytoplasma’.

In this study, the taxonomic position and group classification of the phytoplasma associated with a lethal yellowing-type disease (LYD) of coconut (Cocos nucifera L.) in Mozambique were addressed. Pairwise similarity values based on alignment of nearly full-length 16S rRNA gene sequences (1530 bp) revealed that the Mozambique coconut phytoplasma (LYDM) shared 100 % identity with a comparable sequence derived from a phytoplasma strain (LDN) responsible for Awka wilt disease of coconut in Nigeria, and shared 99.0–99.6 % identity with 16S rRNA gene sequences from strains associated with Cape St Paul wilt (CSPW) disease of coconut in Ghana and Côte d’Ivoire. Similarity scores further determined that the 16S rRNA gene of the LYDM phytoplasma shared <97.5 % sequence identity with all previously described members of ‘Candidatus Phytoplasma ’. The presence of unique regions in the 16S rRNA gene sequence distinguished the LYDM phytoplasma from all currently described members of ‘Candidatus Phytoplasma ’, justifying its recognition as the reference strain of a novel taxon, ‘Candidatus Phytoplasma palmicola’. Virtual RFLP profiles of the F2n/R2 portion (1251 bp) of the 16S rRNA gene and pattern similarity coefficients delineated coconut LYDM phytoplasma strains from Mozambique as novel members of established group 16SrXXII, subgroup A (16SrXXII-A). Similarity coefficients of 0.97 were obtained for comparisons between subgroup 16SrXXII-A strains and CSPW phytoplasmas from Ghana and Côte d’Ivoire. On this basis, the CSPW phytoplasma strains were designated members of a novel subgroup, 16SrXXII-B.

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.

This study addressed the taxonomic position and group classification of a phytoplasma responsible for virescence and phyllody symptoms in naturally diseased Madagascar periwinkle plants in western Malaysia. Unique regions in the 16S rRNA gene from the Malaysian periwinkle virescence (MaPV) phytoplasma distinguished the phytoplasma from all previously described ‘ Candidatus Phytoplasma ’ species. Pairwise sequence similarity scores, calculated through alignment of full-length 16S rRNA gene sequences, revealed that the MaPV phytoplasma 16S rRNA gene shared 96.5 % or less sequence similarity with that of previously described ‘ Ca. Phytoplasma ’ species, justifying the recognition of the MaPV phytoplasma as a reference strain of a novel taxon, ‘Candidatus Phytoplasma malaysianum’. The 16S rRNA gene F2nR2 fragment from the MaPV phytoplasma exhibited a distinct restriction fragment length polymorphism (RFLP) profile and the pattern similarity coefficient values were lower than 0.85 with representative phytoplasmas classified in any of the 31 previously delineated 16Sr groups; therefore, the MaPV phytoplasma was designated a member of a new 16Sr group, 16SrXXXII. Phytoplasmas affiliated with this novel taxon and the new group included diverse strains infecting periwinkle, coconut palm and oil palm in Malaysia. Three phytoplasmas were characterized as representatives of three distinct subgroups, 16SrXXXII-A, 16SrXXXII-B and 16SrXXXII-C, respectively.

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’.

Phytopathogenic mollicutes, which include spiroplasmas and phytoplasmas, are cell wall-less bacteria that parasitize plant hosts and insect vectors. Knowledge of the evolution of these agents is important in understanding their biology. The availability of the first complete phytoplasma and several partial spiroplasma and phytoplasma genome sequences made possible an investigation of evolutionary relationships between phytopathogenic mollicutes and other micro-organisms, especially Gram-positive bacteria, using a comparative genomics approach. Genome data from a total of 41 bacterial species were used in the analysis. Sixty-one conserved proteins were selected from each species for the construction of a hypothetical phylogenetic tree. The genes encoding these selected proteins are among a core of genetic elements that constitute a hypothetical minimal genome. The proteins were concatenated into five superproteins according to their functional categories, and phylogenetic trees were reconstructed using distance, parsimony and likelihood methods. Phylogenetic trees based on the five sets of concatenated proteins were congruent in both clade topology and relative branching length.Spiroplasma kunkeliiand phytoplasmas clustered together with other mollicutes, forming a monophyletic group. Phytoplasmas diverged from spiroplasmas and mycoplasmas at early stages in the evolution of mollicutes. Branch lengths on the phylogenetic trees were noticeably longer in theMollicutesclade, suggesting that the genes encoding the five sets of proteins evolved at a greater rate in this clade than in other clades. This observation reinforces the concept that mollicutes have rapidly evolving genomes.