Phytoplasma classification and phylogeny based on in silico and in vitro RFLP analysis of cpn60 universal target sequences

Abstract

Phytoplasmas are unculturable, phytopathogenic bacteria that cause economic losses worldwide. As unculturable micro-organisms, phytoplasma taxonomy has been based on the use of the 16S rRNA-encoding gene to establish 16Sr groups and subgroups based on the restriction fragment length polymorphism (RFLP) pattern resulting from the digestion of amplicon (in vitro) or sequence (in silico) with seventeen restriction enzymes. Problems such as heterogeneity of the ribosomal operon and the inability to differentiate closely related phytoplasma strains has motivated the search for additional markers capable of providing finer differentiation of phytoplasma strains. In this study we developed and validated a scheme to classify phytoplasmas based on the use of cpn60 universal target (cpn60 UT) sequences. Ninety-six cpn60 UT sequences from strains belonging to 19 16Sr subgroups were subjected to in silico RFLP using pDRAW32 software, resulting in 25 distinctive RFLP profiles. Based on these results we delineated cpn60 UT groups and subgroups, and established a threshold similarity coefficient for groups and subgroups classifying all the strains analysed in this study. The nucleotide identity among the reference strains, the correspondence between in vitro and in silico RFLP, and the phylogenetic relationships of phytoplasma strains based on cpn60 UT sequences are also discussed.

Identification of grass white leaf disease associated with a ‘Candidatus Phytoplasma asteris’-related phytoplasma strain (16SrI-B and cpn60 I-IIIB) in Mexico

“…2b). RFLP analysis of the cpn60 UT sequence (Pérez-López et al 2016b) showed that phytoplasma strain GWL-Ver was a member of the previously described (Pérez-López et al 2016b) cpn60 UT I-IIIB subgroup (not shown).…”

A PCR-Based Method for Distinguishing between Two Common Beehive Bacteria, Paenibacillus larvae and Brevibacillus laterosporus

“…This created misleading genetic similarities among unrelated taxa at the 16S rRNA gene. In general, these single-locus similarities often cannot fully reflect whole-genome relationships due to the horizontal transfer of genes, especially between bacteria that share the same ecological niche (for a sampling of recent discussions on the issues surrounding the use of 16S rRNA sequencing and the necessity for analysis of multiple loci, see references [29][30][31][32][33][34][35][36][37][38].…”

“…Although 16S rRNA sequencing is a powerful tool for assigning taxonomy of many bacteria, there are several pitfalls when using single-gene analyses to distinguish between bacterial strains (29)(30)(31)(32)(33)(34)(35)(36)(37)(38). The presence of both B. laterosporus and P. larvae in beehives, along with their taxonomic relatedness in the Paenibacillaceae family, may facilitate horizontal gene transfer, complicating the identification of bacterial isolates from beehives.…”

Genome Sequence of a Plant-Pathogenic Bacterium, “Candidatus Phytoplasma asteris” Strain TW1

“…The restriction digestion pattern of one of these genes corresponded to 16SrI-B ( F = 1.00) using the i PhyClassifier ( 10 ), while the other copy corresponded to 16SrI-A ( F = 0.97). The single-copy phytoplasma taxonomic marker cpn60 ( 11 ) typed as I-IB ( F = 1.00) using random fragment length polymorphism (RFLP) analysis ( 12 ). Other taxonomic markers, including secY , secA , nusA, and rp , were also found in a single copy in the genome sequence.…”