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

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.

Molecular Detection of Blueberry Stunt Phytoplasma in Eastern Canada: a Multi-year Study

“…The time to positive found by Castro et al 33 investigating a rapid diagnosis of Zika virus was found to be in the same 13-15 minute range as our work described here. An inverse relationship between time to positive and input copy number that was linear over several orders of magnitude was also demonstrated for a Strawberry Green Petal phytoplasma (16SrXIII) LAMP assay 15 .…”

“…Results were analyzed using a 2x2 table and sensitivity and speci city along with 95% con dence intervals calculated as described 25 . Samples that were discordant (LAMP positive/nested PCR negative) were re-analyzed using BBSP-speci c real-time qPCR as described here (Table S1) as well as using nested PCR targeting the ribosomal protein (rp) operon 15 . For rp operon ampli cation, primers rpF1/rpR1 37 were used in the rst round, then the PCR product was diluted 1:30 and 2 µl used in a second round with primers rp(I)FIA/rp(I)RIA 38 .…”

“…Phytoplasmas are further classi ed into groups and subgroups according to RFLP analysis of their 16S rRNA-encoding loci, with more than 35 16Sr groups described to date 13 . Of those, members of the groups 16SrI, 16SrXIX, and 16SrXIII have been reported to affect blueberries in North America 14,15 . Although the 16S rRNA-encoding locus is regarded as the 'gold standard' for phylogenetic characterization of phytoplasmas, there are shortcomings to the use of this target.…”

Compilación de oligonucleótidos para la detección y clasificación de fitoplasmas

“…Inicialmente la identificación de fitoplasmas se realizó mediante técnicas como Western Blot, hidrólisis de ADN, separación mediante centrifugación, Cromatografía Líquida de Alta Resolución (HPLC, por las siglas en inglés de High Performance Liquid Chromatography), hibridación, entre otras (Kollar y Seemüller, 1989). Sin embargo, la inclusión de la técnica de PCR y de sus variantes han permitido complementar su diagnóstico, además de corroborar, cuantificar e incluso identificar nuevos fitoplasmas (Torres et al, 2005;Obura et al, 2011;Pérez-López et al, 2017).…”

Commentary: Phyllostomid bat microbiome composition is associated to host phylogeny and feeding strategies

“…Mollicutes (Phylum Firmicutes ) are a class of microorganisms that include phytoplasmas (‘ Candidatus Phytoplasma’) that are being increasingly recognized for their role in plant diseases such as sapodilla little leaf, yellow leaf roll disease of peach, strawberry green petal and sugarcane white leaf syndrome. These diseases affect a diverse array of economically and ecologically important plant hosts around the world (Vesterinen et al, 2013 ; Pérez-López et al, 2016 , 2017 ). Mollicutes are highly diverse and are made up of five orders, Acholeplasmatales, Anaeroplasmatales, Entoplasmatales, Haloplasmatales, and Mycoplasmatales.…”