Genome Sequence of a Plant-Pathogenic Bacterium, "Candidatus Phytoplasma asteris" Strain TW1.

A draft genome sequence is presented for a strain of "Candidatus Phytoplasma asteris" affecting canola plants in Saskatoon, Canada. This phytopathogenic bacterium was determined to be a 16SrI strain and features 16S rRNA-encoding gene sequence heterogeneity.

Aster leafhopper survival and reproduction, and Aster yellows transmission under static and fluctuating temperatures, using ddPCR for phytoplasma quantification.

Aster yellows (AY) is an important disease of Brassica crops and is caused by Candidatus Phytoplasma asteris and transmitted by the insect vector, Aster leafhopper (Macrosteles quadrilineatus). Phytoplasma-infected Aster leafhoppers were incubated at various constant and fluctuating temperatures ranging from 0 to 35 °C with the reproductive host plant barley (Hordium vulgare). At 0 °C, leafhopper adults survived for 18 days, but failed to reproduce, whereas at 35 °C insects died within 18 days, but successfully reproduced before dying. Temperature fluctuation increased thermal tolerance in leafhoppers at 25 °C and increased fecundity of leafhoppers at 5 and 20 °C. Leafhopper adults successfully infected and produced AY-symptoms in canola plants after incubating for 18 days at 0-20 °C on barley, indicating that AY-phytoplasma maintains its virulence in this temperature range. The presence and number of AY-phytoplasma in insects and plants were confirmed by droplet digital PCR (ddPCR) quantification. The number of phytoplasma in leafhoppers increased over time, but did not differ among temperatures. The temperatures associated with a typical crop growing season on the Canadian Prairies will not limit the spread of AY disease by their predominant insect vector. Also, ddPCR quantification is a useful tool for early detection and accurate quantification of phytoplasma in plants and insects.

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

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.

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

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.

The underestimated diversity of phytoplasmas in Latin America.

Phytoplasmas ('Candidatus Phytoplasma') are insect-transmitted, cell-wall-less, plant-pathogenic bacteria that cause economically important crop diseases. Because phytoplasmas are difficult or impossible to culture in vitro, they are classified taxonomically according to the convention used for unculturable micro-organisms. The first coherent scheme of classification of phytoplasmas, based on the RFLP pattern of the 16S rRNA-encoding gene generated with 17 restriction endonucleases, was updated several times until the development of the iPhyClassifier. iPhyClassifier is an interactive online tool capable of determining the species, group and subgroup of 'Candidatus Phytoplasma' of unknown samples using the 16S F2nR2 sequence. Latin America, an important geographical area in relation to food production, has a high incidence of plant diseases caused by phytoplasmas. However, many phytoplasmas associated with these diseases have not been properly classified. An extensive literature review and the use of iPhyClassifier allowed us to identify two new tentative groups (16SrXXXIII-A and 16SrXXXIV-A) and the following tentative new subgroups among Latin American strains that were either previously unclassified or misclassified: six in 16SrI, six in 16SrII, one in 16SrIII, one in 16SrVII, one in 16SrIX, one in 16SrXII and two in 16SrXIII.