Determination of <i>Phytoplasma</i> subtypes from host plants grown in 2012 at a single location in Saskatoon, SK, Canada (52.13° N, 106.68° W).

<p>Determination of <i>Phytoplasma</i> subtypes from host plants grown in 2012 at a single location in Saskatoon, SK, Canada (52.13° N, 106.68° W).</p

Error trade-offs in OTU assembly optimization.

<p>A. Total error (left ordinate) for <i>de novo</i> assemblies of <i>cpn</i>60 UT sequence reads from a synthetic community of 20 cloned targets, using a minimum identity value of 92% and a range of minimum overlap lengths (50–400 nucleotides). Raw total error (blue line), as well as error remaining after post-assembly primer trimming and clustering (red line), and after chimera removal (green line). Light blue bars indicate the percent of sequence reads identified as singletons in each assembly (right ordinate). B. Number of OTU assembled at each minimum overlap length. Each coloured segment of the stacked bar indicates a different member of the panel of 20 community members. The total number of OTU assembled is indicated on the top of each stack.</p

Breadth of detection of the <i>cpn</i>60-targeted PCR assays for ‘<i>Ca.</i>Phytoplasma’ spp.

<p><b>A.</b> Samples amplified using primer set H279p/H280p. <b>B.</b> Samples amplified using primer set D0317/D0318. <b>C.</b> Samples amplified using an optimized cocktail consisting of a 1∶7 molar ratio of primer sets H279p/H280p:D0317/D0318. For all panels: Lane 1, Apple proliferation (‘<i>Ca.</i>P. <i>mali</i>’); lane 2, Peach yellow leaf roll (‘<i>Ca.</i>P. <i>pyri</i>’); lane 3, European stone fruit yellows (‘<i>Ca.P. prunorum</i>’); lane 4, Bois noir – isolate Pyrenées Orientalis (‘<i>Ca.</i>P. <i>solani</i>’); lane 5, AY strain OY-M (‘<i>Ca.</i>P. <i>asteris</i>’); lane 6, AY strain COL (‘<i>Ca.</i>P. <i>asteris</i>’); lane 7, AY strain CVB (‘<i>Ca.</i>P. <i>asteris</i>’); lane 8, AY strain AY-WB (‘<i>Ca.</i>P. <i>asteris</i>’); lane 9, Brazilian huanglongbing phytoplasma (‘<i>Ca.</i>P. <i>phoenicium</i>’); lane 10, Flavescence dorée (‘<i>Ca.</i>P. <i>ulmi</i>’); lane 11, Bois noir – isolate VL-06-1-20, Lebanon (‘<i>Ca.</i>P. <i>solani</i>’); lane 12, Rubus stunt (‘<i>Ca.</i>P. <i>ulmi</i>’); lane 13, Ash yellows (‘<i>Ca.</i>P. <i>fraxini</i>’); lane 14, no template control.</p

Sensitivity and specificity of <i>cpn</i>60-targeted PCR assay compared to 16S–23S PCR.

<p>Positives and negatives were defined by the results of the P1/Tint assay and the corresponding numbers of positive and negative samples identified by the <i>cpn60</i>-targeted assay are indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116039#pone.0116039-Banoo1" target="_blank">[34]</a>. The sensitivity of the <i>cpn60</i> PCR using P1/Tint as a gold standard was 0.927 (114 positives of 123), with a 95% confidence interval of 0.046. The specificity of the <i>cpn60</i> PCR using P1/Tint as a gold standard was 0.435 (30 negatives of 69), with a 95% confidence interval of 0.117.</p>a<p>assayed using primer set H279p/H280p.</p>b<p>CI, confidence interval.</p><p>Sensitivity and specificity of <i>cpn</i>60-targeted PCR assay compared to 16S–23S PCR.</p

Expanded 11-plex fluorescent microsphere hybridization assay.

<p>All templates were plasmid DNA controls (10⁸ copies/2 µl). Probe identities are shown in the legend. Beads with a positive hybridization signal in each sample are identified (*). Abbreviations: PD, Pear decline; AY, Aster yellows; AP, Apple proliferation; ESFY, European stone fruit yellows; BN, Bois noir, FD, Flavescence dorée; AshY, Ash yellows. CVB and COL are strains of Aster Yellows (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116039#pone.0116039.s005" target="_blank">S1 Table</a>).</p

<i>cpn</i>60-targeted fluorescent microsphere hybridization assay to detect ‘<i>Ca.</i>Phytoplasma’ spp.

<p>Results are shown for a 4-plex assay (format used for analysis of 192 <i>B. napus</i> DNA extracts) on plasmid DNA controls (10⁷ copies/PCR) and on genomic DNA extracted from various infected plant tissues. Beads with a positive hybridization signal in each sample are identified (*). Samples from infected plant tissues are those described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116039#pone.0116039.s005" target="_blank">S1 Table</a> (onion, item #25; flax, item #41; vinca1, item #33; vinca2, item #35; carrot, item #15). Abbreviations: MFI, median fluorescence intensity; BN, Bois Noir; ESFY, European Stone Fruit Yellows; AY, Aster Yellows.</p

Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns

<div><p>Phytoplasmas (‘<i>Candidatus</i> Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (<i>cpn</i>60) gene sequence from a diverse array of ‘<i>Ca.</i>Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of <i>cpn</i>60 from ‘<i>Ca.</i>Phytoplasma’ spp. and used to amplify <i>cpn</i>60 gene fragments from various reference samples and infected plant tissues. Forty three <i>cpn</i>60 sequences were thereby determined. The <i>cpn</i>60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using <i>cpn</i>60 sequences was congruent with that reported for 16S rRNA-encoding genes. The <i>cpn</i>60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, <i>Brassica napus</i> and <i>Camelina sativa</i>, displayed different phytoplasma infection patterns.</p></div

Sequence diversity across the 16S rRNA gene and <i>cpn</i>60 UT.

<p>Median percent identity of each of sequence to its nearest neighbour among the 16S rRNA <i>cpn</i>60 UT sequences from 1,394 bacterial genomes. Median percent identity was calculated for each 120 bp window along the length of the targets and identity values are plotted for the midpoint of each window. Due to target length variation, particularly among 16S rRNA genes, data is shown for windows for which at least 95% of the genomes could be included.</p

Summary of diagnostic assays conducted on 192 field-collected samples of <i>B. napus</i> from the 2012 growing season.

<p>Complete results for each sample are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116039#pone.0116039.s007" target="_blank">S3 Table</a>.</p>a<p>assayed using H279p/H280p.</p>b<p>only PCR products from samples that gave discordant results between P1/Tint (neg) and H279p/H280p (pos) were directly sequenced. One reaction failed so no typing information is available. For the fluorescent microsphere assays, all positive samples returned a typing result.</p>c<p>ND, not determined.</p><p>Summary of diagnostic assays conducted on 192 field-collected samples of <i>B. napus</i> from the 2012 growing season.</p

Barcode gap analysis for 16S rRNA and <i>cpn</i>60 targets.

1<p>Median length of the target region, between amplification primer annealing sites.</p>2<p>Barcode gap is the difference between the median inter-specific distance and median intra-specific distance.</p>3<p>Distance is expressed in terms of substitutions/site.</p