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

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. Phytoplasmas ('Candidatus Phytoplasma' spp.) are wall-less bacteria that were first described as mycoplasma-like organisms 1 with a small, AT rich and distinctive genome, that are taxonomically classified as Mollicutes 2, 3. Phytoplasmas are insect-vectored plant pathogens that infect a very wide variety of plants, including most crop species, causing developmental alterations leading to leaf-like floral structures and aborted seed production 4-6. Detection of phytoplasma infection in plant and insect tissues, along with proper classification and identification, is therefore critical for disease surveillance and control 2. However, phytoplasmas are unculturable microorganisms and the detection and classification criteria are based on the use of molecular approaches 7. Phytoplasma detection has relied on PCR amplification of targets within and surrounding the 16 S rRNA-encoding gene 8. Identification and classification has typically used restriction fragment length polymorphism (RFLP) analysis of this locus 9 , resulting in the identification of over thirty 16Sr groups described as16SrI-16SrXXXIII 10. However, other genes have been used as additional markers, including the groEL or cpn60 gene 11, 12. The cpn60 universal target (cpn60 UT), a sequence of approximately 550 bp, is located within the Cpn60-encoding gene 13. This