Zhao, Yan

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’.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including ‘CandidatusPhytoplasma australiense’, ‘CandidatusPhytoplasma japonicum’ and ‘CandidatusPhytoplasma fragariae’. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis viniferaL.). Such latter strains have been informally proposed to represent a separate species, ‘CandidatusPhytoplasma solani’, but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the ‘CandidatusPhytoplasma’ genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated (‘Ca.Phytoplasma solani’) strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. ‘Ca.Phytoplasma solani’ is the only phytoplasma known to be transmitted byHyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse ‘Ca.Phytoplasma solani’ strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA,tuf,secYandrplV–rpsCgene sequences supported this view and yielded congruent trees in which ‘Ca.Phytoplasma solani’ strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of ‘Ca.Phytoplasma australiense’-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, ‘Ca.Phytoplasma solani’; STOL11 is designated the reference strain.

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.