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

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.

Fig. 1.

Diagrammatic representation of the operational process of iPhyClassifier. Rectangles represent input and output files, squares represent databases, diamonds represent computational operations and ovals represent recommendations on tentative 16Sr group/subgroup classification status and ‘Ca. Phytoplasma’ species assignment.

Fig. 2.

Screenshot of the iPhyClassifier web page.

Fig. 3.

Virtual RFLP patterns derived from in silico digestions of 16S rRNA gene F2nR2 fragments from representative strains of novel and previously delineated 16SrIII subgroups. Recognition sites for the following 17 restriction enzymes were used in the simulated digestions: AluI, BamHI, BfaI, BstUI (ThaI), DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, Sau3AI (MboI), MseI, RsaI, SspI and TaqI. Lanes MW, φX174 DNA digested with HaeIII.

Fig. 4.

Phylogenetic tree inferred from analysis of 16S rRNA gene sequences. Minimum-evolution analysis was conducted using the close neighbour interchange (CNI) algorithm implemented in mega4 (Tamura et al., 2007). The initial tree for the CNI search was obtained by the neighbour-joining method. The reliability of the analysis was subjected to a bootstrap test with 1000 replicates. The taxa used in the phylogenetic tree reconstruction included reference strains of each phytoplasma 16Sr group, reference strains of subgroups belonging to the peach X-disease phytoplasma group and reference strains of each ‘Ca. Phytoplasma’ species (‘Ca. Phytoplasma allocasuarinae’ and ‘Ca. Phytoplasma lycopersici’ were not included because the available 16S rRNA gene sequences did not encompass the entire F2nR2 region. Reference strains of subgroups 16SrIII-E, 16SrIII-H and 16SrIII-I were not included because complete sequence information is not available). The sequence of Acholeplasma palmae J233^T served as an outgroup during phylogenetic tree reconstruction. Bar, 0.01 nucleotide substitutions per site. †In the report by Jung et al. (2002), ‘Ca. Phytoplasma castaneae’ was assigned to group VI according to DNA sequence similarity, rather than results from RFLP analysis. In accordance with the more widely accepted RFLP-based classification system, this phytoplasma was reassigned to group 16SrXIX by Wei et al. (2007). ‡The original reference (Al-Saady et al., 2008) reported ‘Ca. Phytoplasma omanense’ as the reference member of a novel group designated group 16SrXIX. However, the group number 16SrXIX had been published previously (Wei et al., 2007) to accommodate a different phytoplasma, ‘Ca. P. castaneae’. Therefore, we assign ‘Ca. P. omanense’ to a new group, 16SrXXIX, subgroup 16SrXXIX-A.

Fig. 5.

Key restriction enzymes that distinguish novel 16SrIII subgroup pattern types. Lanes MW, φX174 DNA digested with HaeIII.