Construction of an interactive online phytoplasma classification tool, iPhyClassifier, and its application in analysis of the peach X-disease phytoplasma group (16SrIII).

Yan Zhao,Xiaobing Suo,Wei Wei,R E Davis,Jonathan Shao,I.-M Lee

doi:10.1099/ijs.0.010249-0

Abstract

Phytoplasmas, the causal agents of numerous plant diseases, are insect-vector-transmitted, cell-wall-less bacteria descended from ancestral low-G+C-content Gram-positive bacteria in the Bacillus–Clostridium group. Despite their monophyletic origin, widely divergent phytoplasma lineages have evolved in adaptation to specific ecological niches. Classification and taxonomic assignment of phytoplasmas have been based primarily on molecular analysis of 16S rRNA gene sequences because of the inaccessibility of measurable phenotypic characters suitable for conventional microbial characterization. In the present study, an interactive online tool, iPhyClassifier, was developed to expand the efficacy and capacity of the current 16S rRNA gene sequence-based phytoplasma classification system. iPhyClassifier performs sequence similarity analysis, simulates laboratory restriction enzyme digestions and subsequent gel electrophoresis and generates virtual restriction fragment length polymorphism (RFLP) profiles. Based on calculated RFLP pattern similarity coefficients and overall sequence similarity scores, iPhyClassifier makes instant suggestions on tentative phytoplasma 16Sr group/subgroup classification status and ‘Candidatus Phytoplasma’ species assignment. Using iPhyClassifier, we revised and updated the classification of strains affiliated with the peach X-disease phytoplasma group. The online tool can be accessed at http://www.ba.ars.usda.gov/data/mppl/iPhyClassifier.html.

Highlights

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Montana potato purple top phytoplasma PPT-MT117-1, pecan bunch phytoplasma PB1, goldenrod yellows phytoplasma GR1 and Alaska potato purple top phytoplasma PPT-AK6 determined in this study are FJ226074, FJ376626, FJ376627 and FJ376629
After evolutionary divergence from an Acholeplasma-like last common ancestor, phytoplasmas emerged as a discrete clade and a large number of widely divergent phytoplasma lineages have evolved in adaptation to a broad range of bio- and geo-ecological niches (Lee et al, 1992a, b, 1993a, 2000; Marcone et al, 1999; Davis et al, 2005; Martini et al, 2007; Wei et al, 2007, 2008a, b; Cai et al, 2008)
Sequence information held in 16S rRNA genes has served as a baseline for ‘Candidatus Phytoplasma’ candidate species delineation as recommended by the Phytoplasma Taxonomy Group of the International Research Program on Comparative Mycoplasmology (IRPCM Phytoplasma/Spiroplasma Working Team – Phytoplasma taxonomy group, 2004)

Summary

Program components

The current version of iPhyClassifier contains the following three program modules: a sequence similarity search and pairwise sequence similarity score calculation module (PM1), an intelligent sequence trimming and virtual RFLP analysis module (PM2) and a virtual electrophoresis gel image plotting module (PM3). While VGelME generates virtual electrophoresis gel images resulting from in silico digestions of a single input sequence (an F2nR2 fragment from a single phytoplasma strain) by 17 individual enzymes, VGelMS produces gel images resulting from in silico digestions of multiple input sequences (F2nR2 fragments from multiple phytoplasma strains) by a single restriction enzyme. The latter helps to identify key restriction enzymes that distinguish different group and subgroup patterns.

Operational process

Critical issues