Current status and future perspective of molecular marker-based identification, classification, and species delineation of phytoplasmas

Authors: Zhao, Yan; Davis, Robert; Wei, Wei

Submitted to: International Organization for Mycoplasmology
Publication Type: Abstract Only
Publication Acceptance Date: 4/16/2018
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Phytoplasmas are mycoplasma-like pathogenic agents that infect plants. Parasitizing phloem sieve tubes and being transmitted by phloem-feeding insects, phytoplasmas are responsible for numerous plant diseases worldwide. Despite decades of efforts and scattered reports of success, cultivation of phytoplasmas in axenic medium remains a persistent challenge. Consequent inaccessibility of measurable phenotypic characters has prevented phytoplasmas from being characterized using polyphasic taxonomic approach. Rather, phytoplasma identification has been achieved through selective amplification of phytoplasma DNA and subsequent analysis of nucleotide sequence markers present in the amplicons. Such molecular marker-based genotyping approaches, including 16S rDNA RFLP profiling and sequence typing of multiple housekeeping genes with varying degrees of conservation, have unveiled the breadth of genetic diversity in phytoplasmas. Easy access of DNA sequence data and evolving data-mining technologies have unleashed a surge in discoveries of novel phytoplasmas. Rapid expansion of the 16S rDNA RFLP analysis-based classification scheme emphasizes the need to establish a platform for group and subgroup registration. While current genotyping approaches have served well in providing valuable markers for classification of phytoplasmas into groups/subgroups and for fine differentiation of closely related lineages, key molecular markers for phytoplasma species delineation remain to be identified. Per existing guidelines for reporting new ‘Candidatus Phytoplasma’ species, a novel taxon can be recognized only if the phytoplasma’s 16S rRNA gene sequence shares less than 97.5% similarity to that of any previously described ‘Ca. Phytoplasma’ species unless the phytoplasma “clearly represents an ecologically separated population”. We hypothesize that reliable markers capable of distinguishing ecologically separated populations must be present in the genomes of respective phytoplasmas. We further reason that such distinguishing makers are likely embedded in genes encoding surface-localized membrane proteins (SLMPs) since they are at the interface of phytoplasma-plant host-insect vector three-way interactions, the outcome of which determines divergent selection and ecological separation of populations (speciation). Our preliminary results from a search of genome sequences of 24 phytoplasma strains have revealed a comprehensive set of SLMP genes that may serve as markers for future delineation of new phytoplasma species. Based on these SLMP markers, at least three new ‘Ca. phytoplasma’ species can be recognized. Each of the new taxa has a lower than 95% whole genome average nucleotide identity (ANI) score with the previously established closest ‘Ca. phytoplasma’ species, validating the delineation. The applicability of this SLMP marker-based species delineation approach is supported by the observation that these SLMP genes have conserved genomic contexts, and therefore are readily accessible via PCR amplifications with universal primers.