Specific detection of the PVY(N)-W variant of Potato virus Y

PVY(N)-W is one of the variant populations of Potato virus Y (PVY). This variant is of concern in seed potato production and requires a specific diagnosis since it induces more or less symptomless infections and is not detectable easily in field inspections. Moreover, this variant is serologically indistinguishable from the common strain PVY(O). This study describes a simple and specific molecular detection test for the PVY(N)-W variant using a PCR protocol based on the recombinant point within the HC-Pro/P3 region of PVY(N) variants (PVY(NTN), PVY(N)-W). To avoid both detection of recombinant PVY(NTN) and PVY(N)-W isolates, a forward PVY(N)-like primer located in the HC-Pro region coupled to a reverse PVY(O)-like primer located in the NIa region was designed to amplify a specific PCR product of 4114 nt from PVY(N)-W isolates. This technique was assessed on 41 PVY reference and field isolates. Only isolates referenced as PVY(N)-W were amplified and gave the expected PCR product of 4114 nt, whereas no band was obtained from PVY(N), PVY(NTN) or PVY(O) isolates. In conclusion, this PVY(N)-W diagnosis tool is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato certification.     

Probable geographical grouping of PVY(N) and PVY(NTN) based on sequence variation in P1 and 5'-UTR of PVY genome and methods for differentiating North American PVY(NTN)

An increasing number of countries in recent years have reported the occurrence of potato tuber necrotic ringspot disease (PTNRD), caused by tobacco veinal necrosis strain of Potato virus Y (PVY(N)), belonging to the sub-group tuber necrosis (PVY(NTN)). Methods for the differentiation of PVY(NTN), based on primer sequences often detect isolates of European (EU) type but not the North American (NA) type. To resolve this problem, the nucleotide sequence of 5'-untranslated region (5'-UTR) and the P1 gene of 11 isolates of PVY(N) and PVY(NTN) from the European Union and North America was determined. Sequence comparison and phylogenetic analysis of 5'-UTR and P1 region indicated that PVY(N) isolates from the European Union and North America formed their own separate groups. Intra-group sequence identity for all except one was over 98%, as opposed to the inter-group identity of 90%. Additionally, the PVY(NTN) isolates from the European Union and North America clustered with their respective PVY(N) isolates. This indicates a possible evolution of PVY(NTN) isolates from the PVY(N) isolates of a geographical region. With this information of regional relationships of PVY(NTN) and PVY(N) isolates, two approaches were developed based on a competitive RT-PCR and a restriction pattern, for the differentiation of NA-PVY(NTN) from the local PVY(N) and from EU-PVY(NTN). Thus sequencing of the P1 gene and use of competitive RT-PCR approach could be applicable for determining the possible origin of new occurrences of PVY(NTN) from other geographical regions.     

The detection of tuber necrotic isolates of Potato virus Y, and the accurate discrimination of PVY(O), PVY(N) and PVY(C) strains using RT-PCR

Potato tuber necrotic ringspot disease (PTNRD) is a damaging disease of potatoes, causing unsightly necrotic rings on the surface of tubers. The causal agent is thought to be tuber necrotic isolates of Potato virus Y, known as PVY(NTN). The disease spoils tubers for processing and table use, and the lack of a diagnostic method makes control especially difficult. The development of an RT-PCR assay for the reliable detection of PVY(NTN) and discrimination of all the main strains of PVY (PVY(O), PVY(N) and PVY(C)) is described. An assay was developed, exploiting a recombination site in the coat protein of PVY(NTN), allowing more reliable diagnosis of these isolates. Although the conserved nucleotide differences observed between the strains was very small, competitive PCR and mutagenically separated PCR were both employed in the development of a robust assay. The assay was found to be more reliable than the most commonly used RT-PCR method, and should prove to be an important tool in the confirmation of symptoms and for the detection of PVY(NTN) in symptomless tissue, in disease surveys and seed health schemes.     

A multiple single nucleotide polymorphisms interrogation assay for reliable Potato virus Y group and variant characterization

The complex Potato virus Y classification, including groups (PVYN and PVYO) and variants (PVYNTN and PVYN-W), is based mainly on biological properties of isolates. Published PVY detection tools targeting markers not associated with biological properties could fail to assign correctly isolates in the current classification. To improve PVY detection tools, a single nucleotide polymorphism (SNaPshot) detection assay was developed. The technique was adapted to target the T/C9259, A/C2271, G/C8573 and A/G2213 PVY polymorphic nucleotides. The "TAGA", "CCCG", "CACA" and "CAGA" four-digit codes associated with tested samples allowed identification of PVYN, PVYO, PVYN-W and PVYNTN isolates, respectively. The PVY SNaPshot procedure is efficient and reliable for PVY detection and characterization in samples containing as few as 10(2) viral RNA copies. Moreover, PVY group assignment is possible for fractions containing only 10 copies of a PVY RNA genome. Finally, the SNaPshot assay allows PVY(N)/PVYO dual characterization for mixed samples containing PVYN/PVYO quantity ratios in the range of 0.1-10. This innovative SNaPshot tool improved clearly PVY diagnostic assays described previously by targeting simultaneously major functional markers and sequence unlinked to biological properties used separately in PVY detection tools available currently.     

Rapid identification of potato virus Y strains by one-step triplex RT-PCR

A one-step triplex RT-PCR method was characterised that allows rapid, strain-specific detection of potato virus Y (PVY) occurring on potato: PVY^N, PVY^O, PVY^NTN (recombinant isolates), PVY^NWi and PVY^C. Three specific primer pairs were designed on aligned PVY sequences available from genomic data banks. The specificity of the selected primers was first examined by simplex RT-PCR with a large number of PVY reference isolates. Two fragments of 0.44 and 1.11 kb were amplified for PVY^N and non-recombinant PVY^NTN isolates, two fragments of 0.53 and 0.66 kb for PVY^O isolates, a single fragment of 0.44 kb for recombinant PVY^NTN isolates, a 0.66 kb fragment for PVY^C isolates and a 0.53 kb fragment for PVY^NWi isolate. The primers were then combined in a one-step triplex RT-PCR reaction, optimised stepwise and validated with the reference isolates. The great similarity between the genomes of PVY^N and non-recombinant PVY^NTN prevented their differentiation using this method. No fragments were amplified with samples infected by non-related potato viruses, as well as with samples from healthy tobacco and potato plants. The one-step triplex RT-PCR described here fastens specific detection of PVY strains that are otherwise only distinguishable by combined serological and biological assays.     

Single-step RT real-time PCR for sensitive detection and discrimination of Potato virus Y isolates

Potato virus Y (PVY) has a worldwide distribution and infects several economically important crops from the Solanaceae family. The emergence and spread of the PVYNTN strain, which is the causative agent of potato tuber necrotic ringspot disease (PTNRD), has lead to large economic losses and highlighted the need for accurate discrimination of the different PVY strains. Detection and differentiation of PVY isolates is mainly based on a combination of ELISA, RT-PCR and bioassays; however, PVYNTN isolates are particularly difficult to differentiate from standard PVYN without the use of time-consuming bioassays. A strong correlation has been identified previously between the ability to induce PTNRD and the presence of a recombination point in the virus coat protein. An RT real-time PCR assay has been developed to enable detection of isolates with the recombination point, therefore, enabling rapid differentiation between potentially tuber necrotic PVYNTN isolates and standard PVYN isolates. The assay is also able to detect the presence of PVYO isolates. To aid with routine testing, immuno-capture and post-ELISA virus release were introduced; when coupled with RT real-time PCR the sensitivity of the assays were up to seven orders of magnitude higher than ELISA. The assay was shown to be a suitable method for rapid large-scale diagnostic testing of PVY in different types of plant material including tubers, and specific screening for potentially tuber necrotic recombinant isolates.     

Within plant distribution of Potato Virus Y in hairy nightshade (Solanum sarrachoides): an inoculum source affecting PVY aphid transmission

Potato virus Y (PVY) is vectored by several potato-colonizing and non-colonizing aphid species in a non-persistent manner and has a wide host range. It occurs naturally in several plant families. Myzus persicae and Macrosiphum euphorbiae are the most efficient potato-colonizing aphid vectors of PVY. Rhopalosiphum padi, a cereal aphid that migrates in large numbers through potato fields during the middle of the growing season, does not colonize potato plants but can transmit PVY. Hairy nightshade, Solanum sarrachoides, a prevalent annual solanaceous weed in the Pacific Northwest (PNW) of the United States, is an alternative host for PVY and a preferred host for M. persicae and M. euphorbiae. Hence, hairy nightshade plants might play an important role as an inoculum source in the epidemiology of PVY. We looked at titre accumulation and distribution of PVY(O), PVY(N:O) and PVY(NTN) in S. sarrachoides and potato after aphid inoculation with M. persicae and studied the transmission of PVY(O) and PVY(NTN), by M. persicae, M. euphorbiae and R. padi from hairy nightshade to potato plants. Virus titre at different positions on the plant was similar in S. sarrachoides and potato plants with strains PVY(O) and PVY(N:O). Titres of PVY(NTN) were similar in S. sarrachoides and potato but differences in titre were observed at different positions within the plant depending on the plant phenology. Percentage transmission of PVY(NTN) by M. persicae and M. euphorbiae was twice as high (46 and 34%, respectively) from hairy nightshade to potato than from potato to potato (20 and 14%). Percentage transmission of PVY(O) by M. persicae and M. euphorbiae was not affected by the inoculum source. No effect of the inoculum source was observed in the transmission of either PVY strain by R. padi. These results show that hairy nightshade may be an equal or better virus reservoir than potato and thus, important in the epidemiology of PVY.     

A single nucleotide polymorphism-based technique for specific characterization of YO and YN isolates of Potato virus Y (PVY)

One of the most important properties used to classify Potato virus Y (PVY) isolates is their ability to induce (PVY(N)) or not (PVY(O)) veinal necrosis symptoms on the indicator host plant Nicotiana tabacum cv. Xanthi. As an alternative to biological assays, several serological and molecular detection tools have been developed for PVY detection and characterization and these have evolved as our knowledge of PVY has improved. However, the assays that have been previously published are all based on the use of neutral markers (antigenic determinants, sequence data, recombination sites or restriction enzyme cleavage sites), which are unlinked to the biological property being characterized (e.g. veinal necrosis). Using the recently identified molecular determinants of the tobacco leaf necrosis symptom induced by PVY(N) isolates, a one-step fluorescent [TaqMan] RT-PCR assay, based on a single nucleotide polymorphism (SNP) linked to the necrosis property of PVY isolates, has been designed. This assay reliably detects and distinguishes PVY(N) and PVY(O) isolates. The method is simple (leaf soak extraction process, gel-free, no post-PCR manipulations), rapid (96 tests in less than 3h from plants sampling to diagnostic results), sensitive (threshold in a range of 10(4)-10(5) PVY copies), reliable (correctly assigns 42 PVY isolates in their respective group) and allows co-detection of mixed samples containing close to equivalent PVY(N) and PVY(O) quantities. All these characteristics suggest that the newly developed SNP assay could be used to reliably classify PVY isolates, as a substitute for biological assays performed on N. tabacum cv. Xanthi.     

Detection of different strains of Potato virus Y and their mixed infections using competitive fluorescent RT-PCR

A competitive fluorescent RT-PCR assay (CF RT-PCR) was developed for the rapid and reliable detection and discrimination of the two most common strains of Potato virus Y (PVY) found in potato (necrotic and ordinary). The assay incorporates two strain specific primers labelled with fluorescent labels, used in conjunction with a universal PVY primer. The strain specific primers compete for the same annealing site which further increases specificity. Discrimination is conferred by the fluorescent labels; green PCR products for PVY(O) and red for PVY(N), whilst mixed infections are detected as orange PCR products without the need for staining agarose gels. The assay can be scaled up for the processing of 96 samples simultaneously, with the detection of PCR products directly using a fluorescent microtitre plate reader. The assay successfully discriminated between 20 isolates of PVY tested, and could be used for the direct detection of PVY in potato tubers.     

Virus strain discrimination using recombinant antibodies

Most routine testing for plant viruses is currently carried out using monoclonal and polyclonal antibodies. Traditional methods of antibody production however can be time consuming and require the use of expensive cell culture facilities. Recombinant antibody technology however is starting to make an impact in this area, enabling the selection of antibody fragments in a few weeks compared with the many months associated with traditional methods and requires only basic microbiological facilities. Single chain Fv antibody fragments (scFv) have been selected from a synthetic phage-antibody library by affinity selection with purified Potato virus Y, ordinary strain (PVYO). The scFv selected was specific for PVY and detected 7 out of 9 isolates of PVYO whilst it did not detect 15 isolates from the closely related necrotic strains PVYN and PVYNTN. In ELISA the scFv could be used to detect virus at concentrations of 50 ng/ml in plant sap and was shown to have similar limits of detection as commercially available PVY monoclonal antibodies. These results highlight the potential of the technology for the selection of strain specific antibodies with an affinity and assay sensitivity similar to traditional monoclonal antibodies and their use in viral diagnostics.