Stolbur and "bois noir" phytoplasma strains represent a distinct novel species, ‘Candidatus Phytoplasma solani'

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae (Lee et al., 2000). Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including ‘Candidatus Phytoplasma australiense’, ‘Ca. Phytoplasma japonicum’, ‘Ca. Phytoplasma fragariae’ (IRPCM, 2004; Valiunas et al., 2006). Other group 16SrXII phytoplasma strains, associated with stolbur and bois noir diseases, have been informally proposed to represent a separate species, ‘Ca. Phytoplasma solani’, but a formal description of this taxon has not previously been published. In the present communication, on the basis of nucleotide sequence and phylogenetic analyses of multiple genes and assessment of biological properties, stolbur phytoplasma strains are described as representing a distinct species level lineage and novel taxon, ‘Ca. Phytoplasma solani’. DNA fragments of the tuf, secY, and rplV-rpsC genes were PCR-amplified from strain STOL11 (STOL), originally from a diseased plant of Capsicum annuum in Serbia (Seemüller et al., 1994), which had been transmitted and maintained by grafting in plants of Madagascar periwinkle. Nucleotide sequences of 16S rRNA, tuf, secY, and rplV-rpsC genes from diverse group 16SrXII strains, and from reference strains of formally described ‘Ca. Phytoplasma’ species, were retrieved from the GenBank database, compiled in FASTA format, and aligned using BioEdit version 7.0.5. Phylogenetic analyses were done using the software MEGA4. Moreover, deduced amino acid sequences were aligned for phylogenetic analyses. Data on plant hosts, insect vectors, and geographic distribution of 16SrXII phytoplasmas were compared in order to determine whether stolbur and bois noir phytoplasmas formed an ecologically distinct population. Our data provide evidence that ‘Ca. Phytoplasma solani’ strains clearly represent a distinct gene pool, based on 16S rDNA and variable gene sequence analyses, and occupy a distinct ecological niche. According to IRPCM guidelines, phytoplasmas sharing >97.5% 16S rDNA nucleotide sequence similarity can be described as separate species if they are clearly distinguished by evident molecular diversity and ecological niche. Strain STOL11, selected as the potential 'Ca. Phytoplasma solani’ reference strain, shares 97.6% 16S rDNA sequence similarity with ‘Ca. Phytoplasma australiense’. We found that 'Ca. Phytoplasma solani’ intra-species sequence similarity was significantly and consistently greater (>99%) than inter-species similarity (<98.2% vs ‘Ca. Phytoplasma australiense’). Moreover, 16S rDNA sequence alignments identified a STOL11-unique signature sequence and two distinguishing sequence blocks (DSBs), whose combined use facilitates assignment of strains to the species. In previous work, 16S rDNA signature sequences have already been employed for separating biologically distinct ‘Ca. Phytoplasma’ species that shared >97.5% 16S rDNA sequence similarity (Seemüller et al., 2004). In our view, a strain of ‘Ca. Phytoplasma solani’ should (i) share >99% sequence similarity with a minimum of 1.2kb within the 16S rRNA gene of reference strain STOL11, and (ii) contain the identical STOL11-unique signature 16S rDNA sequence, and (iii) contain both DSBs of reference strain STOL11, with a tolerance of a single nucleotide difference in no more than one of the sequences. Strains that do not fulfill either criterion (ii) or criterion (iii) are considered ‘Ca. Phytoplasma solani’-related strains, even if they fulfill criterion (i). Since additional genes were proposed for future delineation of species (Davis et al., 2012), further work was carried out that distinguished ‘Ca. Phytoplasma solani’ on the basis of sequence analyses of tuf, secY, and rplV-rpsC gene loci. Nucleotide sequence alignments revealed unique single nucleotide polymorphisms (SNPs) in STOL11 and other ‘Ca. Phytoplasma solani’ strains and reinforced the evidence that intra-species sequence similarity was remarkably and consistently greater than inter-species similarity between ‘Ca. Phytoplasma solani’ and ‘Ca. Phytoplasma australiense’. Moreover, phylogenetic analyses of 16S rRNA, tuf, secY, and rplV-rpsC gene sequences yielded congruent trees on 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. In addition, we found that ‘Ca. Phytoplasma’ species within group 16SrXII possess distinct biological properties. Focus was on comparisons with ‘Ca. Phytoplasma australiense’, the only described species sharing >97.5% sequence similarity of 16S rDNA. In Australia and New Zealand, ‘Ca. Phytoplasma australiense’ strains are associated with Australian grapevine yellows and other diseases (Streten et al., 2005), and Oliarius atkinsoni has been described as its insect vector in New Zealand (Liefting et al., 1997). In Europe and in the Mediterranean basin, ‘Ca. Phytoplasma solani’ strains are associated with bois noir disease of grapevine, with stolbur disease in wild and cultivated herbaceous and woody plants, and with yellowing, reddening, decline, dwarfism, leaf malformation and degeneration diseases of other plants. Hyalesthes obsoletus, its most common vector (Maixner, 1994), is not known to transmit any other phytoplasma, possibly indicating a long and intimate co-evolution of phytoplasma and vector, and a unique phytoplasma-vector association distinguishing ‘Ca. Phytoplasma solani’ from other species. In summary, the distinct molecular characteristics and unique vectorship support recognition of ‘Ca. Phytoplasma solani’ as a new species in the genus ‘Ca. Phytoplasma’ (Quaglino et al., 2013).