Mathuresh Singh

Possible Escape of a Recombinant Isolate of Potato virus Y by Serological Indexing and Methods of its Detection

Surveys of commercial and seed potato fields for virus diseases (1998 to 2002) in Manitoba established that Potato virus Y (PVY) is of concern in seed potato production. To determine the prevalence of PVY strains, PVY-infected tubers identified by reverse transcription-polymerase chain reaction (RT-PCR) from surveys (2000 to 2001) were grown for symptom expression and strain characterization by strain-specific RT-PCR, bioassays, and serological assays. Of the samples collected (2000 to 2001) and tested by RT-PCR, 4.0% contained PVY. Further analysis of the PVY-positive samples by a duplex RT-PCR facilitating the simultaneous detection of common (PVYO) and tobacco veinal necrosis strains (PVYN/NTN) indicated that 37.5% contained PVYO and 63.5% contained PVYN-type isolates. Analysis of the PVYN-type samples using three monoclonal antibodies (MAbs) showed that all reacted with only the PVYO MAbs and not with the PVYN-specific MAb. Partial nucleotide sequences of both ends of PVY-RNA showed that the PVYN-type isolates resembled those reported in 1996 from Manitoba. These isolates are designated as PVYN:O. In view of the increased incidence of PVYN:O in one production area, seed tubers imported from other provinces of Canada and the neighboring United States were analyzed for PVYN:O. The PVYN:O was detected in imported seeds from Minnesota, Montana, and North Dakota.

Sodium sulphite inhibition of potato and cherry polyphenolics in nucleic acid extraction for virus detection by RT-PCR.

Phenolic compounds from plant tissues inhibit reverse transcription-polymerase chain reaction (RT-PCR). Multiple-step protocols using several additives to inhibit polyphenolic compounds during nucleic acid extraction are common, but time consuming and laborious. The current research highlights that the inclusion of 0.65 to 0.70% of sodium sulphite in the extraction buffer minimizes the pigmentation of nucleic acid extracts and improves the RT-PCR detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) in potato (Solanum tuberosum) tubers and Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV) in leaves and bark in the sweet cherry (Prunus avium) tree. Substituting sodium sulphite in the nucleic acid extraction buffer eliminated the use of proteinase K during extraction. Reagents phosphate buffered saline (PBS)-Tween 20 and polyvinylpyrrolidone (PVP) were also no longer required during RT or PCR phase. The resultant nucleic acid extracts were suitable for both duplex and multiplex RT-PCR. This simple and less expensive nucleic acid extraction protocol has proved very effective for potato cv. Russet Norkotah, which contains a high amount of polyphenolics. Comparing commercially available RNA extraction kits (Catrimox and RNeasy), the sodium sulphite based extraction protocol yielded two to three times higher amounts of RNA, while maintaining comparable virus detection by RT-PCR. The sodium sulphite based extraction protocol was equally effective in potato tubers, and in leaves and bark from the cherry tree.

Factors affecting detection of PVY in dormant tubers by reverse transcription polymerase chain reaction and nucleic acid spot hybridization

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY(O), but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY(O)-specific probe. As little as 1 pg of purified PVY(O)-RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY(O) amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY(O) concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and did not result in reduced detection of PVY(O) by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.

Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids.

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95-99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT-PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.

Duplex RT-PCR: reagent concentrations at reverse transcription stage affect the PCR performance

Test conditions for the simultaneous detection of potato leafroll virus (PLRV) and potato virus Y (PVY) in dormant tubers and leaves by reverse transcription-polymerase chain reaction (RT-PCR) were optimized. Various factors optimized at the reverse transcription (RT) stage rather than at the amplification (PCR) stage affected the outcome. In the simplex RT-PCR a onefold dNTPs concentration (0.5 mM) was sufficient in yielding a PLRV or PVY band. In contrast, the duplex RT-PCR required a minimum twofold dNTPs concentration (1.0 mM) during RT to produce distinct bands in PCR. Similarly, various proportions of antisense primers of PLRV and PVY used during RT affected subsequent duplex RT-PCR. Optimal amplification of both viruses were obtained at a ratio of 0.90:0.49 microM of PLRV:PVY antisense primers. An interaction of dNTPs and RNA template concentration was observed. A higher concentration of RNA was required at onefold dNTPs concentration than at twofold dNTPs. Dilutions down to 1:300 of RNA template yielded distinct bands of both viruses at twofold dNTPs concentration. At optimized conditions of the duplex RT-PCR both viruses were reliably detected in composite samples at a ratio of one part infected sap mixed with 399 parts of sap from healthy tubers. Application of optimized conditions to singly- and doubly-infected tubers detected both viruses from naturally infected field-grown tubers. A nearly perfect correlation (r(2)=0.99) was observed between visible plant symptoms and the virus detection from leaves and tubers by the duplex RT-PCR.

Crop border and mineral oil sprays used in combination as physical control methods of the aphid‐transmitted potato virus Y in potato

BACKGROUND The objectives of this work were to determine if the control of potato virus Y (PVY, genus Potyvirus, family Potyviridae) in seed potato could be improved by combining border crops and mineral oil sprays, and if the border crop acts as a barrier or a virus sink. RESULTS Field tests over 3 years confirmed that mineral oils alone are an effective barrier to PVY, and showed that borders alone act as a PVY sink. Combining the familiar mineral oil and the more recent crop border methods was almost twice as effective in reducing PVY incidence as either one used alone. The combination provided consistently high PVY control compared with the variable and often lower level of control by either method alone. The contribution of the oil to PVY reduction was similar whether it was applied to the border, the center seed plot, or both. Oil application to the border alone should not affect efficacy and would help keep control costs down. CONCLUSION Combining border and oil provided the best reduction in PVY incidence 3 years out of 3, providing producers with a tool to reduce year-to-year variation in the effectiveness of crop borders or oil sprays used separately.

Use of citric acid for neutralizing polymerase chain reaction inhibition by chlorogenic acid in potato extracts.

Polyphenolics are a widely distributed class of plant constituents implicated in the inhibition of the polymerase chain reaction (PCR). Since chlorogenic acid forms the bulk of phenolics in the potato tuber, a range of chlorogenic acid concentrations were tested for inhibition of reverse transcription (RT) and PCR steps. The RT step was inhibited at chlorogenic acid concentrations of 6.0 to 7.5 microg, while the PCR step was inhibited at much lower concentrations of 1.2 to 1.8 microg. Incorporation of 1.2% citric acid at the nucleic acid extraction step prevented darkening of the extract and neutralized the inhibitory effect of chlorogenic acid.

Specific Detection of Potato Virus A in Dormant Tubers by Reverse-Transcription Polymerase Chain Reaction

A reverse-transcription polymerase chain reaction (RT-PCR) protocol was developed for the detection of potato virus A (PVA) in dormant tubers. A 255-bp amplified product was produced using a primer pair from the P1 gene of the PVA genome. The 255-bp product was detected in nucleic acids from leaves, tubers, and purified virions and was specific to PVA as determined by Southern blot tests and detection by a PVA-specific probe. When presented with seven potato virus/strain nucleic acids and a viroid, singly and in mixed infections, the primer pair did not amplify any products. Its specificity to PVA was further demonstrated by RT-PCR detection of PVA from the known mixtures of PVA and potato virus Y samples. PVA was detected in foliage nucleic acids at a dilution of 1:1024-1:4096 and tuber nucleic acids at 1:256-1:1024. It was uniformly present in various parts of the potato tuber. PVA was detected in composite tuber samples containing a ratio of infected to healthy sap of 1:29 and was readily detected in tubers of several cultivars or breeding lines, in dormant as well as in sprouting tubers stored at 20-25°C for 4 months.

Recognition and Molecular Discrimination of Severe and Mild PVYO Variants of Potato virus Y in Potato in New Brunswick, Canada

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVYO and PVYN-specific antibody-based enzyme-linked immunosorbent assays, and reverse-transcription polymerase chain reaction (PCR)-based genotyping were carried out. All the assays demonstrated that the isolate, designated as PVYO-FL in this study, belonged to the PVYO strain group. Greenhouse tests with the potato cvs. FL 1533 and Jemseg confirmed the severe nature of infection by PVYO-FL. The complete genome sequences of PVYO-FL and PVYO-RB, the latter a mild PVYO isolate, were determined. BLAST analysis revealed that the two isolates shared 97 and 98% sequence identities at the nucleotide and polyprotein levels, respectively. Further BLAST analysis unveiled that PVYO-FL shared 99.7% nucleotide sequence identity with PVYO-Oz, an isolate reported in New York, United States, whereas the PVYO-RB isolate shared 99.2% sequence identity with PVYO-139, a PVYO isolate reported in New Brunswick, Canada. A phylogenetic tree of available, full-length sequences of PVY isolates demonstrated two subgroups within the PVYO branch, one clustered with PVYO-RB and the other with PVYO-FL. Group-specific sense primers for differentiation of the two subgroups were developed and evaluated. A limited survey of potato tubers collected from a field plot at the Potato Research Centre, Agriculture and Agri-Food Canada, using the newly developed PCR primers, indicated that 65.3 and 2.4% of the PVYO-positive tubers were infected with PVYO isolates belonging to the PVYO-FL and PVYO-RB subgroups, respectively. Assessment of the pathogenicity of three representative isolates from each subgroup on the potato cv. Jemseg demonstrated that severe and mild symptoms were induced by the PVYO-FL-like and PVYO-RB-like isolates, respectively.

SCREENING BY A 3-PRIMER PCR OF NORTH AMERICAN PVYN ISOLATES FOR EUROPEAN-TYPE MEMBERS OF THE TUBER NECROSIS-INDUCING PVYNTN SUBGROUP

Oligonucleotide primers for a 3-primer polymerase chain reaction (PCR) were optimized by adjusting their Tm values to identify European-type members of the potato tuber necrotic ringspot disease (PTNRD) inducing PVYNTN sub-group of PVYN. Fiftyseven North American isolates of PVYN were screened using this assay and one, originating from garden potato tubers in Ontario, was found to be positive. This isolate was confirmed to induce the diagnostic PTNRD, using a panel of 20 potato cultivars. AC Chaleur, AC Novachip, Century Russet, Cherokee, and Frontier Russet reacted severely and developed PTNRD either at the time of harvest or during storage. About 800 tubers collected from New Brunswick and Prince Edward Island fields, from plants exhibiting mosaic and leafroll symptoms were also screened for various strains of PVY using PCR primers. Results indicate that primer pairs developed are specific for PVY spp., PVYN, and PVYNTN.