Ribosomal protein gene-based phylogeny for finer differentiation and classification of phytoplasmas

Abstract

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.

‘Candidatus Phytoplasma pruni’, a novel taxon associated with X-disease of stone fruits, Prunus spp.: multilocus characterization based on 16S rRNA, secY, and ribosomal protein genes

“…To confirm the presence of sequence-heterogeneous rRNA operons, products of PCR primed by P1/16S-SR were subjected to restriction endonuclease digestion, and the resulting digest was analysed by 1 % agarose gel electrophoresis. For analysis of ribosomal protein (rp) genomic regions, DNA segments were amplified in hf-PCRs primed by primer pair rpL2F3/rp(I)R1A in direct PCR (for strain CX-95), and in nested PCRs primed by rpL2F3/rp(I)R1A (Martini et al, 2007) followed by rp(III)-FN/rp(I)R1A (for the remaining strains) under conditions after Martini et al (2007). The nucleotide sequence of primer rp(III)-FN was 59-GGTGAATTTTCTCCAACTCG-39 (this study).…”

Molecular Characterization and Phylogeny of a Phytoplasma Associated with Phyllody Disease of toria (Brassica rapa L. subsp. dichotoma (Roxb.)) in India

“…The thermal conditions were as follows: denaturation at 94°C for 1 min (5 min for the first cycle), annealing at 60°C for 2 min and extension at 72°C for 3.5 min. The last cycle was extended for an additional 10 min at 72°C [19].…”

“…16S rRNA, 23S rRNA, rp and secA gene sequences were aligned with phytoplasma group/subgroup representatives available in GenBank using Clustal W [26]. Phylogenetic relationship of four isolates of TP phytoplasma with other group representative phytoplasma sequences available in GenBank were assessed based on sequences of 16S rRNA gene between primers R16F2n and R16R2, secA gene between primers SecAfor2 and SecArev3 [12], 23S rRNA gene between primers P7 and 23Srev [12], rp gene between primers rp(IX)F2 and rp(IX)R2 [19] and 16S-23S rRNA intergenic spacer region between primers P3 and P7 [12]. Sequence alignments were performed using CLUSTAL W [26].…”

“…SecY, map and uvrB-degV genes were used to analyse strains in the 16SrV cluster [1]. As five subgroups in 16SrIX phytoplasma group have been identified [8,19,31], it was important to identify the phytoplasma subgroup associated with toria phyllody (TP) disease. Therefore, in silico virtual RFLP and phylogenetic analysis of 16S rRNA gene, actual RFLP of 16S rRNA and 23S rRNA genes, sequencing and phylogenetic analysis of ribosomal protein gene, 16S-23S rRNA intergenic spacer region, 23S rRNA and secA genes were attempted for characterization and phylogeny of a phytoplasma associated with TP disease using samples from four different locations in India.…”

Molecular identification of phytoplasmas in fasciated cacti and succulent species and associated hormonal perturbation

“…Phytoplasmas can be detected by PCR assays using specific and universal primers designed on the basis of 16S rRNA gene (Deng & Hiruki 1990;Ahrens & Seemüller 1992;). The extremely conserved 16S rRNA gene is the most widely used for phylogenetic studies of prokaryotes (Martini et al 2007). Phylogenetic analysis of 16S rRNA gene sequence revealed the phylogenetic status of phytoplasma and classified them as the class Mollicutes (Lee et al 2000).…”