Plant, insect and bacterial material
Phytoplasma strains for specificity testing were cultivated in Catharanthus roseus and were kindly provided by Dr R. Osler from the University of Udine, Italy. The strains included: elm yellows phytoplasma strain EY1 (EY1, 16SrV-A), aster yellows phytoplasma (AY, 16SrI-B), a stolbur phytoplasma isolate SE (SE, 16SrXII-A, original host: celery), Western X-disease transferred to C. roseus (WX, 16SrIII-A), apple proliferation phytoplasma strain AP15 (AP, 16SrX-A), pear decline phytoplasma (PD, 16SrX-C) and the European stonefruit yellows phytoplasma transferred to C. roseus from plum (ESFY, 16SrX-B). MA phytoplasma (16SrIII-B), originating from Chrysanthemum leucanthemum (Marguerite Daisy) cultivated on Tanacetum cinerariifolium, was also included in the study.
Several strains of plant pathogenic bacteria were obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB), York, UK for specificity testing. These were: Xylophilus ampelinus (NCPPB 2217, type strain of the species), Pseudomonas syringae pv. syringae (NCPPB 281), Erwinia amylovora (NCPPB 683, type strain of the species) and Clavibacter michiganensis subsp. sepedonicus (NCPPB 4053). Agrobacterium vitis (KIS Av 13-2) was obtained from the collection of the Agricultural Institute of Slovenia, Ljubljana, Slovenia. Some of these strains are present in the grapevine microflora. Additionally, 29 bacterial isolates from plant extracts of various grapevine cultivars grown on nutrient agar (NA) (Bacto Nutrient Agar, Difco) or YPGA (yeast extract 7·0 g, proteose peptone 5·0 g, glucose 10·0 g, agar 15·0 g, distilled water 1 L, pH 7·0) were used. An additional five strains of grapevine pathogenic bacteria employed in this study were obtained from the collection of the Instituto Valenciano de Investigaciones Agrarias (Valencia, Spain) and were characterized as Agrobacterium vitis, A. tumefaciens, A. rhizogenes, Xylella fastidiosa Fetz and X. fastidiosa Staps.
Grapevine leaf samples were collected in the field from 148 grapevine plants (17 cultivars: Chardonnay, Beli Pinot, Sivi Pinot, Modri Pinot, Merlot, Refošk, Modra Frankinja, Sauvignon, Rebula, Šipon, Renski Rizling, Rumeni Muškat, Žametovka, Malvazija, Kerner, Zweigeld and Syrah). All the main coastal and continental winegrowing regions of Slovenia (Primorje, Posavje and Podravje) were represented. Three field-collected samples of field bindweed (Convolvulus arvensis) were also tested. Healthy field-collected grapevine plants were used as negative controls in real-time PCR reactions (cvs Barbera, Chardonnay, Sivi Pinot, Merlot and Refošk). The following sweep-captured insects were also tested: Hyalesthes obsoletus (four samples of at least 20 adults), Scaphoideus titanus (10 samples of at least 20 adults and one sample of at least 20 nymphs), Euscelis incisus (one sample of seven adults) and Reptalus panzeri (three samples of at least 20 adults). Sampling of grapevine plants was carried out from July to October 2005. An additional 12 samples of field-collected apple trees (Malus domestica, leaf veins) that contained apple-proliferation-group phytoplasmas (16SrX) were tested. An additional 159 grapevine leaf samples (the same range of cultivars and geographical locations as in 2005) were collected from July to October 2006 and were analysed with real-time PCR only.
DNA samples from FD-infected (four samples) and BN-infected grapevine plants (one sample) were provided by Dr P. Ermacora of the University of Udine, Italy.
The phytoplasma-enrichment procedure described by Ahrens & Seemüller (1992) was used, with some modifications; 1·5 g of leaf veins (whole leaves in the case of C. roseus and C. arvensis) were used as starting material, homogenized in 20 mL ice-cold grinding buffer and centrifuged for 5 min at 2500 g (4°C) followed by centrifugation of the supernatant for 25 min at 18 000 g (4°C). Lysis was performed in modified CTAB buffer (4% CTAB, 1% PVP MW 10 000, without 2-mercaptoethanol) at 65°C. After chloroform/isoamyl alcohol (24:1) extraction the DNA was precipitated with cold (–20°C) isopropanol, incubated at room temperature for 20 min and centrifuged for 15 min at 15 000 g. The pellet was then washed with cold 80% ethanol (–20°C), dried at room temperature and re-suspended in 50 µL TE buffer (10 mm Tris-HCl, 1 mm EDTA, pH 8·0) overnight at 4°C. Quantity and quality of DNA were analysed using gel electrophoresis (8 µL). The DNA concentrations of all phytoplasma and bacterial strains used in specificity testing and of several diagnostic samples (to estimate the range of concentrations) were measured spectrophotometrically with NanoDrop (NanoDrop Technologies). Tenfold dilutions of the samples were shown to be sufficient to keep the DNA load below 100 ng in real-time PCR reactions.
Real-time PCR primers and assay design and setup
Publicly available phytoplasma sequences from the GenBank® database were aligned in Vector NTI software (Invitrogen) to find suitable regions for amplicon design. Primers and probes for the universal detection of phytoplasmas and for specific detection of BN and FD were then designed using primer express© (Applied Biosystems). TaqMan® MGB probes were labelled with 6–carboxyfluorescein (FAM) at the 5′ end and a non-fluorescent quencher (NFQ) with minor groove binder (MGB) at the 3′ end (Table 2). The specificities of the designed amplicons were tested in silico with a Basic Local Alignment Search Tool (blast) search of public databases.
Table 2. Primers and MGB probes for Flavescence dorée (FD)-specific (FDgen), Bois noir (BN)-specific (BNgen) and universal phytoplasma (UniRNA) amplicons
|Name||Orientation||Sequence (5′–3′)||Amplicon length||Location|
|UniRNA||Forward||AAA TAT AGT GGA GGT TAT CAG GGA TAC AG||73 bp||16S rRNA|
|Reverse||AAC CTA ACA TCT CAC GAC ACG AAC T|
|Probe||5′FAM-ACG ACA ACC ATG CAC CA-3′NFQa|
|FDgen||Forward||TTA TGC CTT ATG TTA CTG CTT CTA TTG TTA||85 bp||sec Y gene|
|Reverse||TCT CCT TGT TCT TGC CAT TCT TT|
|Probe||5′FAM-ACC TTT TGA CTC AAT TGA-3′NFQa|
|BNgen||Forward||AAG CAG GTT TAG CGA TGG TTG T||71 bp||Stol11 genomic fragment|
|Reverse||TGG TAC CGT TGC TTC ATC ATT T|
|Probe||5′FAM-TTA ATA CCA CCT TCA GGA AA-3′NFQa|
All real-time PCR reactions were performed on an ABI PRISM® 7900 HT Sequence Detection System (Applied Biosystems) in optical 384-well plates with optical adhesive covers (both Applied Biosystems) using universal cycling conditions (2 min at 50°C, 10 min at 95°C, followed by 45 cycles of 15 s at 95°C and 1 min at 60°C, with 9600 Emulation mode) which allowed running of all reactions on the same plate. Real-time PCR was performed in a final reaction volume of 10 µL containing 2 µL of sample DNA, 900 nm primers, 250 nm probe and 1× TaqMan® Universal PCR Master Mix (Applied Biosystems), which includes ROXTM as a passive reference dye. Each sample DNA was tested with all three amplicons for phytoplasmas and an amplicon for an endogenous control (in separate real-time PCR reactions): cytochrome oxidase (COX) for plants (Weller et al., 2000) or 18S rRNA for insects (eukaryotic 18S rRNA TaqMan endogenous control, Applied Biosystems). Endogenous controls amplified plant or insect DNA co-extracted with phytoplasmic DNA. All reactions were performed in two replicate wells in two dilutions (10- and 100-fold) on the same 384-well plate. An automated liquid handling system (Multiprobe® II PLUS EX, PerkinElmer) was used for pipetting a large number of DNA samples and master mixes onto the 384-well plates.
The software sds 2·2 (Applied Biosystems) was used for fluorescence acquisition and calculation of threshold cycles (Ct). For this calculation, the baseline was automatically set and the fluorescent threshold was set manually to intersect with the linear part of amplification curves of all amplicons, resulting in the final Ct value for each well.
Efficiency, limit of detection (LOD) and sensitivity of amplicons
Elm yellows phytoplasma strain EY1 is an American isolate of the elm yellows phytoplasma (‘Ca. Phytoplasma ulmi’). It was used to develop the real-time PCR tests because it has the same target sequence as FD (Figs 1 and 3) and because no FD isolate was available.
Figure 1. Schematic representation of the FDgen amplicon within aligned nucleotide sequences of the rpl15/secY gene (AY197685, FD9 fragment) obtained from GenBank. Accession numbers marked with suffix FD are Flavescence dorée phytoplasma sequences; the rest are other strains from the elm yellows group (16SrV). AY197690 is isolate EY1. The position of primers for detection of FD with conventional PCR FD9f/r (Daire et al., 1997) and FD9f3b/r2 (Clair et al., 2003) are also shown in AY197685.
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Figure 3. Schematic representation of the UniRNA amplicon within the aligned nucleotide sequences of the 16S rRNA gene (AY197643) obtained from GenBank. Sequences belonging to the elm yellows group (16SrV) are marked with the prefix EY and those marked by the suffix FD are Flavescence dorée phytoplasmas. AY197655 is isolate EY1. Sequences belonging to the stolbur group (16SrXII) are marked with the prefix Stol and those marked with the suffix BN belong to Bois noir phytoplasmas isolated from grapevine. Positions of universal PCR primers for detection of phytoplasmas, P1 (Schneider et al., 1995), 6F (modified P1 primer, Dr P. Ermacora, University of Udine, personal communication) and U5 (Lorenz et al., 1995) are shown in AY197643 (the corresponding reverse primers are situated outside the sequence shown). Thirty-five additional sequences of the 16 rDNA gene were included in the alignment, but are not shown in the figure because they were identical to at least one of the sequences already present.
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Limit of detection (LOD) for all three real-time PCR amplicons was performed on 10-fold serial dilutions of EY1 DNA (340 ng µL−1) and SE DNA (830 ng µL−1) in water ranging from undiluted DNA to a 109-fold dilution factor. Five replicate wells per dilution were used. The slope (k) of the linear regression line between logarithmic values of relative DNA concentrations (y-axis) and Ct values (x-axis) was used to calculate the amplification efficiency, E = (10[–1/k])–1, where a value of one corresponded to 100% amplification efficiency (Pfaffl, 2001). The squared regression coefficient after the linear regression (R2) was also determined. Dynamic range, i.e. the range of concentrations for which Ct values were in linear relationship with logarithms of concentrations and range of detection, were also determined.
Sensitivities of PCR and real-time PCR were compared on EY1 and SE dilution series. Each dilution series was prepared by mixing the diluted phytoplasmic DNA with undiluted DNA isolated from leaf veins of several healthy grapevine plants. This healthy grapevine material had already been tested with real-time PCR and confirmed to be BN- and FD-negative. Thus, PCR sensitivity was evaluated for potential effects of host-material inhibition. Two separately prepared sets of dilution series were used, employing the same EY1 and SE DNA, but adding these to a different pool of DNA from healthy grapevine leaf veins. The concentration of DNA in both pools was 300–400 ng µL−1.
Data mining approach
Decision trees were generated using the weka machine learning workbench (Frank et al., 2004) and its algorithm J48, which is weka's implementation of the C4·5 algorithm. As described in Larose (2005), the algorithm visits each possible decision node, in this case all conducted assays, selecting the optimal split, until no further splits are possible. Decision trees were drawn for the PCR-based detection system and the newly-developed real-time PCR assays, and evaluated using 10-fold cross validation. The percentage of correctly classified instances was calculated for both methods (PCR and real-time PCR).
Diagnostic sensitivity and specificity were also calculated for evaluation of both detection systems according to Dinnes et al. (2005). Diagnostic sensitivity (proportion of diseased samples that gave positive results) was calculated as true positives/total diseased and diagnostic specificity (proportion of non-diseased samples that gave negative test results) as true negatives/total non-diseased. Results from all three amplicons were used for calculations.