Molecular diagnostic tools for detection and differentiation of phytoplasmas based on chaperonin-60 reveal differences in host plant infection patterns

Abstract

Phytoplasmas ('Candidatus 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 (cpn60) gene sequence from a diverse array of 'Ca.Phytoplasma' spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from 'Ca.Phytoplasma' spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 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, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.

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. .

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).