Figure 1.
Breadth of detection of the cpn60-targeted PCR assays for ‘Ca.Phytoplasma’ spp.
A. Samples amplified using primer set H279p/H280p. B. Samples amplified using primer set D0317/D0318. C. 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 (‘Ca.P. mali’); lane 2, Peach yellow leaf roll (‘Ca.P. pyri’); lane 3, European stone fruit yellows (‘Ca.P. prunorum’); lane 4, Bois noir – isolate Pyrenées Orientalis (‘Ca.P. solani’); lane 5, AY strain OY-M (‘Ca.P. asteris’); lane 6, AY strain COL (‘Ca.P. asteris’); lane 7, AY strain CVB (‘Ca.P. asteris’); lane 8, AY strain AY-WB (‘Ca.P. asteris’); lane 9, Brazilian huanglongbing phytoplasma (‘Ca.P. phoenicium’); lane 10, Flavescence dorée (‘Ca.P. ulmi’); lane 11, Bois noir – isolate VL-06-1-20, Lebanon (‘Ca.P. solani’); lane 12, Rubus stunt (‘Ca.P. ulmi’); lane 13, Ash yellows (‘Ca.P. fraxini’); lane 14, no template control.
Figure 2.
Molecular phylogeny (maximum likelihood) of ‘Ca.Phytoplasma’ spp. based on the sequences of the cpn60 UT.
Reference strains are indicated by public database accession numbers (cpnDB id before strain name and GenBank accession numbers in parentheses). Numbers next to the nodes indicate bootstrap support based on 1000 replicates. The cpn60 UT sequence obtained from the genome of Acholeplasma laidlawii strain PG-8A (GenBank accession no NC_010163.1) is included as the outgroup. See S1 Table for a list of strain abbreviations used on this tree. Groupings correspond to those suggested by Chung et al. [32].
Table 1.
Sensitivity and specificity of cpn60-targeted PCR assay compared to 16S–23S PCR.
Table 2.
Limit of detection (LOD) of PCR assays.
Figure 3.
cpn60-targeted fluorescent microsphere hybridization assay to detect ‘Ca.Phytoplasma’ spp.
Results are shown for a 4-plex assay (format used for analysis of 192 B. napus 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 S1 Table (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.
Figure 4.
Expanded 11-plex fluorescent microsphere hybridization assay.
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 (S1 Table).
Table 3.
Summary of diagnostic assays conducted on 192 field-collected samples of B. napus from the 2012 growing season.
Table 4.
Determination of Phytoplasma subtypes from host plants grown in 2012 at a single location in Saskatoon, SK, Canada (52.13° N, 106.68° W).