Seven PVM isolates (Ca5, Ca99, Ca102, Ca128, Ca414, Ca508 and Ca513) from potatoes (Solanum tuberosum cv. Shepody) grown on an experimental farm in the province Prince Edward Island, Canada and four PVM isolates (CL1, 2, 3, 4) from CFIA-CL virus collection, were identified and characterized, on the basis of bioassay and serological reactions (Table 1). All isolates were maintained in infected potato plants that were grown under greenhouse conditions and progeny tubers were harvested and stored at 4°C. Dormancy breaking was done by maintaining tubers at room temperature. Tubers were sampled according to the methods described previously . Leaf and sprout samples were directly used for extracting tissue sap. All samples were macerated in extraction bags (Bioreba, Reinach, Switzerland) by pounding the tissue with a hammer and the tissue sap was subjected to different tests as described below. Plant sap was kept at -20°C if it was not used within 12 hours. A second aliquot of the sap from RT-PCR positive samples was re-tested to confirm the initial results. All PCR amplicons were subjected to restriction digestion and the RFLP pattern was used to determine whether they matched PVM patterns. Positive samples were then subjected to bioassay (see below).
Tissue sap from PVM positive samples based on initial ELISA and RT-PCR tests were inoculated onto healthy potato (Russet Burbank) and indicator (C. quinoa) plants. Tissue sap was diluted (1:2) in phosphate buffer (0.1 M, pH7.2) and inoculated onto leaves of potato and indicator plants by rubbing carborundum dusted leaves. Inoculated plants were grown in a greenhouse at 18°C - 22°C and observed every other day for the development of symptoms. All inoculated plants (regardless of whether or not symptoms developed) were tested for PVM by ELISA and RT-PCR. All progeny tubers were tested by RT-PCR followed by RFLP analysis for confirmation.
Potato tubers and leaves as well as indicator plants were screened for the presence of PVM by a standard double antibody sandwich ELISA using commercial coating antibody, conjugate, positive control and all necessary reagents from Neogen Europe Ltd. (Auchincruive, Scotland, UK), following the procedures recommended by the supplier. Tuber or leaf tissues were homogenized in 0.1 M phosphate buffer containing 0.02% NaN3, 0.1% Tween 20 and 0.1% skim milk powder (pH 7.4) at a sample to buffer ratio of 1:5 (w:v) and 100 μl of extracted sap was loaded in duplicate on microtitre plates. A panel of positive, negative, and buffer controls, in addition to the controls supplied with the ELISA kit, were included on each plate. Absorbance values (A405 nm) of 4 times of the healthy control reading was used as the positive threshold, but if absorbance of the healthy control was < 0.030, a positive threshold of 0.100 was used.
From each macerated sample, 100 μl of sap was extracted with the Tri-Reagent (Molecular Research Center, Inc, Cincinnati. OH) as described by the manufacturer. Subsequently the RNA was extracted with chloroform, precipitated with isopropanol, washed with ethanol and suspended in 25 μl (for tuber samples) or 50 μl (for sprouts, leaves and tissue culture plantlets) of RNase-free and DNase-free water according to the procedures described previously . RNA extracts from leaves and tubers of healthy potato plants were used as negative controls. RNA extracts from foliage and/or tubers of potato plants infected with other viruses including AMV, PMTV, TRV, PVS, PVX, Potato aucuba mosaic virus (PAMV, Potexvirus), PVYO, the tobacco vein necrotic strain of PVY (PVYN), the potato tuber necrotic strain of PVY (PVYNTN), Potato virus A (PVA, Potyvirus), Potato latent virus (PotLV, Carlavirus), Potato leafroll virus (PLRV, Polerovirus) and PSTVd were also included in RT-PCR tests to determine primer specificity.
Two sets of primers were designed based on sequence analysis of known PVM isolates obtained from the NCBI website including the isolates Hangzhou (China, AJ437481), M57 (Poland, AY692075), Uran (Poland, AY311394), German isolate (X57440), Italy tomato strain (X85114), Idaho strain (USA, AF023877) and two Russian PVM isolates (D14449, Table 2). Primers PVM1 (Reverse: CTTCATTTGTTATTCGACTT) and PVM2 (Forward: ATGGGAGATTCAACRAAGAA) were used for amplifying the entire CP gene and the nucleotide sequences of the amplicons (917 bp) were then determined in both directions using the same primer set. PVM3 (Reverse: TGAGCTCGGGACCATTCATAC) and PVM4 (Forward: ACATCTGAGGACATGATGCGC) were used in RT-PCR and real-time RT-PCR to yield an amplicon of 520 bp.
First strand cDNA synthesis was carried out using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen Canada, Burlington, ON, Canada) using the antisense primer (PVM1 or PVM3). The procedure for the two step RT-PCR were essentially the same as described previously . A reaction containing no cDNA template was included in all PCR tests as a blank control. The temperature regime for amplification reactions was as follows: initial denaturation for 5 min at 95°C, followed by 35 cycles of 94°C for 45 seconds, 58°C for 45 seconds, and 72°C for 45 seconds. The final extension was at 72°C for 7 min. A GeneAmp 9700 thermocycler (Applied Biosystems, Foster City, CA) was used for RT-PCR amplifications. Optimal annealing temperature of primers was determined using a temperature gradient thermocycler (Watman Biometra, Goettingen, Germany). PCR products were separated on a 1.2% agarose gel, stained with ethidium bromide, and visualized under UV light.
Several restriction endonucleases MscI, NdeI, PstI, PvuII and TaqI (New England Biolabs, Pickering, ON, Canada) were evaluated for a direct digestion of PCR amplicons generated with primers PVM3/PVM4 and MscI was selected for all RFLP tests in this study. Five microliters of PCR amplicons were digested with 5 units of the restriction enzyme at 37°C for 1 hour in a reaction volume of 20 μl using the buffer recommended by the enzyme supplier. RFLP patterns were analysed by agarose gel electrophoresis using 1.5% agarose-1000 (Invitrogen Canada, Burlington, ON, Canada) stained with ethidium bromide and visualized under UV light.
The CP gene amplicons generated in RT-PCR from test samples using primer PVM1/2 and PVM3/4 were purified using QIAquick PCR purification kit (Qiagne Inc., Mississauga, ON, Canada) following the procedures recommended by the provider and the dsDNAs amplified in a typical RT-PCR reaction (50 μl) were eluted with 30 μl of water (DNase-free and RNase-free). Purified PCR amplicons from the CP gene of PVM isolates CL1, CL3, CL4, Ca5, Ca128, Ca508 and Ca513 were sequenced in both orientations by automated cycle sequencing (York University, Toronto, ON, Canada) using primers PVM1/PVM2 and/or PVM3/PVM4 depending on the templates. Sequences of Canadian PVM isolates were compared with PVM sequences in the NCBI database with the program BLAST. Nucleotide and amino acid sequences were aligned using Clustal G (V1.1) and GeneDoc Multiple Sequence Alignment Editor and Shading Utility (V2.5.000). The phylogenetic relationship of the Canadian potato isolates of PVM and 8 other known PVM strains/isolates (Table 2) were deduced using the Bootstrap Neighbour-Joining (N-J) methods (random number generator seed: 111, number of bootstrap trails: 1000) in the Phylip formatted Clustal W (V1.82). The trees were visualized and the dendrograms were displayed using the program TreeView (V1.5).