Molecular diagnostic assays based on cpn60 UT sequences reveal the geographic distribution of subgroup 16SrXIII-(A/I)I phytoplasma in Mexico

Edel Pérez-López,Douglas Rodríguez-Martínez,Tim J Dumonceaux,Mauricio Luna-Rodríguez,Chrystel Y Olivier

doi:10.1038/s41598-017-00895-1

Abstract

Geographically diverse samples from strawberry exhibiting symptoms of Strawberry Green Petal (SbGP), periwinkle plants with virescence, and blackberry, blueberry, and raspberry plants displaying yellowing and inedible fruits, were assayed for the presence of phytoplasma DNA. PCR targeting the 16S rRNA-encoding gene and chaperonin-60 (cpn60) showed that the plants were infected with phytoplasma subgroup16SrXIII-(A/I)I (SbGP/MPV). To examine the geographic distribution of this pathogen in Mexico, we designed an array of cpn60-targeted molecular diagnostic assays for SbGP/MPV phytoplasma. A fluorescent microsphere hybridization assay was designed that was capable of detecting SbGP/MPV phytoplasma in infected plant tissues, successfully differentiating it from other known phytoplasma cpn60 UT sequences, while identifying a double infection with SbGP/MPV and aster yellows (16SrI) phytoplasma. Two quantitative assays, quantitative real-time PCR (qRT-PCR) and droplet digital PCR (ddPCR), gave similar results in infected samples. Finally, a loop-mediated isothermal amplification (LAMP) assay provided rapid detection of SbGP/MPV phytoplasma DNA. Application of these assays revealed that SbGP/MPV phytoplasma is widely distributed in Central Mexico, with positive samples identified from eleven localities within three states separated by hundreds of kilometres. These results also provide tools for determining the presence and geographic distribution of this pathogen in plant and insect samples in other localities.

Highlights

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are wall-less bacteria that were first described as mycoplasma-like organisms[1] with a small, A-T rich and distinctive genome, that are taxonomically classified as Mollicutes[2, 3]
We previously determined that Strawberry green petal (SbGP) disease affecting strawberry plants and Mexican periwinkle virescence disease (MPV) affecting periwinkle plants (Catharanthus roseus) in Mexico is associated with the heterogeneous 16SrXIII-(A/I)I phytoplasma[30]
The cpn[60] UT sequences determined from 11 strawberry (KY061173 to KY061183), 1 blueberry (KY061168), 4 raspberry (KY061169 to KY061172), and 2 blackberry (KY061184, KY061185) samples were identical to the SbGP/MPV phytoplasma cpn[60] UT reported previously[30]

Summary

Introduction

Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are wall-less bacteria that were first described as mycoplasma-like organisms[1] with a small, A-T rich and distinctive genome, that are taxonomically classified as Mollicutes[2, 3]. The group 16SrXIII or Mexican periwinkle virescence was first identified in Catharanthus roseus from Mexico[27] and represents a new ‘Ca. Phytoplasma’ species, ‘Ca. Phytoplasma hispanicum’[28]. Other plants, such as potato, have been identified as hosts, but members of this phytoplasma group have not been described outside of the Americas[29]. We previously determined that Strawberry green petal (SbGP) disease affecting strawberry plants and Mexican periwinkle virescence disease (MPV) affecting periwinkle plants (Catharanthus roseus) in Mexico is associated with the heterogeneous 16SrXIII-(A/I)I phytoplasma[30]. The results reveal the minimum extent of the geographic distribution of this pathogen in Mexico and provide a set of tools for determining the prevalence and distribution of the pathogen in other geographic locations

Methods

Results

Conclusion