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

¹ Dipartimento di Biologia Applicata alla Difesa delle Piante, University of Udine, 33100 Udine, Italy
² Molecular Plant Pathology Laboratory, USDA, ARS, Beltsville, MD 20705, USA
³ Dipartimento di Scienze e Tecnologie Agroalimentari, University of Bologna, 40127 Bologna, Italy
⁴ FLREC, University of Florida, Fort Lauderdale, FL, USA
⁵ Dipartimento di Scienze Farmaceutiche, University of Salerno, I-84084 Fisciano, Italy
⁶ Department of Crop Science, College of Agricultural and Marine Sciences, Sultan Qaboos University, Al-Khod 123, Oman

Correspondence
I.-M. Lee
leeim{at}ba.ars.usda.gov

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.

The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are given in Fig. 1.

Figures presenting the position of the degenerate primers and the analysis of putative restriction sites in ribosomal protein operon sequences are available as supplementary material with the online version of this paper.

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