PVY NTN Research Articles

Management of Potato virus Y (PVY–NTN) causing potato tuber necrotic ringspot disease (PTNRD) in potato by prior treatment with a mild PVY strain

Management of Potato virus Y (PVY–NTN) causing potato tuber necrotic ringspot disease (PTNRD) in potato by prior treatment with a mild PVY strain

Identificación molecular de Potyvirus infectando cultivos de papa en el oriente de Antioquia (Colombia)

Los potyvirus son uno de los grupos de virus más limitantes en los cultivos de papa (Solanum tuberosum y S. phureja) en el mundo, siendo PVY, PVV y PVA las especies más prevalentes. En este trabajo se evaluó la presencia de estos potyvirus en cuatro lotes de S. tuberosum cv. Diacol-Capiro y cuatro lotes de S. phureja cv. Criolla-Colombia ubicados en el oriente de Antioquia, analizando la cápside viral mediante RT-PCR/secuenciación Sanger y secuenciación de nueva generación (NGS) para S. tuberosum. Los resultados indicaron la ocurrencia de los potyvirus PVY y PVV en las muestras de S. tuberosum y S. phureja, respectivamente; siendo detectadas mediante cebadores específicos la presencia de tres diferentes cepas de PVY (PVYN, PVYNTN y PVYO) en la región de estudio. Este hallazgo fue confirmado por NGS, obteniendo las secuencias completas de los genomas de estas tres cepas, lo que representa el primer reporte de PVYO en Colombia. Por su parte, los análisis de secuencias de la región CP de PVV indicaron niveles de identidad superiores a 99% con respecto a aislamientos del linaje PVVPhu reportado previamente en Antioquia. Estos hallazgos evidencian la necesidad de ajustar los sistemas de detección de virus en los programas de certificación de tubérculo-semilla de papa adelantados en el país.

Upcoming Plant Pathological Techniques in the Disease Diagnosis

Serodiagnostics for the detection of plant pathogens particularly the plant viruses started in the fifties and had a quantum jump in application after the introduction of enzyme conjugated antibodies in enzyme linked immunosorbent assays EIA in the s Improvements and modifications in the original EIA made its application routine in the large scale indexing of vegetatively cultivated crops It is still the methods of choice for many purposes However its limited sensitivity as compared to nucleic acid based diagnostics particularly PCR restricts its use in critical decision making related to pathogens of quarantine significance like ring rot of potato caused by Corynebacterium sepedonicum tuber necrosis strains of PVY PVYNTN etc Serodiagnostics due to limited sensitivity and specificity in the detection of fungal and bacterial pathogens has not become popular for the detection of many of these pathogens Recent developments in the field of genomics of microorganisms and PCR based assays of microbes have revolutionized diagnostics A combination of immunocapture of pathogen and PCR has made the detection simpler and more sensitive than PCR alone Combination of immunocapture with multiplex PCR further reduces cost of detection Combination of nucleic acid probes and ELISA has the potential of its adoption for large scale indexing of propagating stocks WTO agreements related to trade in agricultural commodities need internationally acceptable diagnostic tools for the detection of pathogens of quarantine significance An internationally recognized monitoring organization is the urgent need The organization will be responsible for the evaluation of reagents and protocols for diagnostics and their regular update The issue was mooted in a recent symposium titled Moving Plant Disease Detection from the Ivory Towers to the Real World where a ring test process was described to validate and standardize new diagnostic procedures As more and more techniques are validated by the scientific community and their reliability and cost effectiveness demonstrated molecular based techniques will play a greater and more important role in the detection of pathogens

Resistance of potato clones to necrotic recombinant strains of potato virus y (pvy)

The Ry adg allele is widely used by breeders to confer extreme resistance to all strains of PVY. However, the necrotic strain has increased recombination resulting in recent considerable losses in productivity. Thus far, not all necrotic recombinant strains of PVY have been tested for their reaction to the Ry gene. The objective of this study was to identify potato clones carrying the resistant allele and to assess their reaction to the following recombinant strains: NTN (PVY NTN), Wilga (PVY N-Wi), and "curly top" (PVY E). Advanced clones from the potato breeding program at Universidade Federal de Lavras were evaluated through a specific molecular marker for the Ry adg allele. The clones carrying the resistance allele were grafted on tobacco plants infected with necrotic recombinant strains of PVY. The clones carrying the allele for resistance to PVY were not infected with any of the recombinants during the grafting test. These results confirm that resistance to necrotic recombinant strains has not yet been overcome and that the Ry adg allele also confers resistance to the three recombinant strains tested.

Zróżnicowanie polskich izolatów wirusa Y ziemniaka (Potato virus Y, PVY) z pomidora

In 2012 and 2013 the presence of Potato virus Y on tomato in greenhouses and fields was analyzed. A total number of 176 samples displaying different type of symptoms: deformation, discoloration, necrosis of leaves and growth retardation were collected. The presence of viral infection using enzyme-linked immunosorbent assay (ELISA) and transmission electron microscopy (TEM) was confirmed in 147 samples. Biological and genetic diversity of the virus isolates was also analyzed. The analyses revealed that the particular strains cause different symptoms on the tested plants such as Solanum lycopersicum cv. Moneymaker, S. melongena and Datura inoxia. In contrast to previous years, in which only isolates belonging to PVYNWi-P strains were observed, four different strains were identified: PVYNWi-P, PVYNN242, PVYNTN and PVYO. The isolates belonging to PVYNWi-P strain remained dominant in the population. W latach 2012 i 2013 przeprowadzono monitoring występowania wirusa Y ziemniaka na pomidorze. Pozyskano 176 prób z pomidora szklarniowego i gruntowego, charakteryzujących się różnymi objawami chorobowymi w postaci deformacji blaszek, przebarwień, nekroz liści oraz karłowacenia pędów. W 147 próbach potwierdzono obecność wirusa za pomocą testu immunoenzymatycznego oraz transmisyjnej mikroskopii elektronowej. Analizowano zróżnicowanie biologiczne i genetyczne pozyskanych izolatów. Wykazano, iż poszczególne szczepy mogą powodować odmienne objawy chorobowe na roślinach testowych: Solanum lycopersicum cv. Moneymaker, S. melongena oraz Datura inoxia. W przeciwieństwie do lat ubiegłych, w których obserwowano jedynie izolaty należące do szczepu PVYNWi-P, w badanym materiale zidentyfikowano cztery różne szczepy PVY: PVYNWi-P, PVYNN242, PVYNTN i PVYO. Izolaty należące do szczepu PVYNWi-P pozostały dominujące w populacji.

Virus resistance of transgenic potato plants (Solanum tuberosum L.) bearing PVY NTN -CP gene of potato virus Y coat protein

The PVY NTN -CP coat protein gene from a necrotic strain of potato virus Y (PVY NTN ) has been transferred into two potato Solanum tuberosum L. cultivars, Mindenes and Somogyi kifli, via agrobacterial transformation. Expression of the PVY NTN -CP gene was revealed in 33 (89%) of 37 and 3 (75%) of 4 kanamycin-resistant regenerates of these cultivars, respectively. The level of virus resistance against two virus strains (PVY O , PVY NTN ) of independent lines of transgenic potatoes varied from extreme resistance to full susceptibility. The three independent lines of transgenic potatoes proved to be extreme resistant to both PVY strains.

The simultaneous differentiation of Potato virus Y strains including the newly described strain PVY NTN-NW by multiplex PCR assay

New recombinant strain and genotype of PVY, designated as PVY NTN-NW and SYR-III, respectively, shared properties with PVY NTN and PVY NW has been reported recently. PVY NTN-NW predominated in potato fields in Syria and was able to induce potato tuber necrotic ringspot disease (PTNRD). Due to the rapid spread of the recombinant strains of PVY which might be the case of PVY NTN-NW, a specific and reliable detection method is an essential step to control this strain and minimize its spread. The shared properties of PVY NTN-NW and SYR-III with PVY NTN and PVY NW, however, complicate their identification involving multiple detection methods. Therefore, a multiplex polymerase chain reaction (PCR), that relies on a combination of previously published and newly designed primers was developed for the detection and identification of PVY NTN-NW and SYR-III in single or mixed infections with the main PVY strains, PVY O, PVY N, PVY NTN and PVY NW. In addition, the present PCR assay was able to detect the recombination points in the P1 region enabling the differentiation of the variable genotypes of the recombinant strains PVY NTN-NW, PVY NTN and PVY NW. The reliability of this PCR assay was confirmed using a significant number of well characterized PVY isolates collected from Syria and Japan including those of PVY NTN-NW, SYR-III, PVY O, NA-PVY N, PVY NW and PVY NTN.

PVYNTN-CP coat protein gene mediated virus resistance of transgenic potato plants

PVYsup style="line-height:1.6em"NTN/sup-CP span style="line-height:1.6em"coat protein gene from a necrotic strain of potato virus /spanY (pvysup style="line-height:1.6em"ntn/sup) span style="line-height:1.6em"has been transferred into two potato /spanSolanum tuberosum L. span style="line-height:1.6em"cultivars /spanMindenes span style="line-height:1.6em"and /spanSomogyi kifli via Agrobacterium tumefaciens span style="line-height:1.6em"transformation. Expression of integrated PVY/spansup style="line-height:1.6em"NTN/supspan style="line-height:1.6em"-CP gene were confirmed for 33 (89 %) of 37 and 3 (75 %) of 4 kanamycin-resistant regenerants of potato cultivars Mindenes and Somogyi kifli respectively. The level of virus resistance against two virus strains /span(PVY°, PVYsup style="line-height:1.6em"NTN/sup) span style="line-height:1.6em"of independent lines of transgenic potatoes varied between extreme resistance to susceptibility. The three independent lines of transgenic potatoes proved to be extreme resistant against both PVY strains./span

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

The complex Potato virus Y classification, including groups (PVY N and PVY O) and variants (PVY NTN and PVY N-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/C 9259, A/C 2271, G/C 8573 and A/G 2213 PVY polymorphic nucleotides. The “TAGA”, “CCCG”, “CACA” and “CAGA” four-digit codes associated with tested samples allowed identification of PVY N, PVY O, PVY N-W and PVY NTN 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/PVY O dual characterization for mixed samples containing PVY N/PVY O 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.

Potato virus Y induced changes in the gene expression of potato ( Solanum tuberosum L.)

The tuber necrotic strain of Potato virus Y (PVY NTN) causes potato tuber necrotic ringspot disease in sensitive potato cultivars. Gene expression in the disease response of the susceptible potato ( Solanum tuberosum L.) cultivar Igor was investigated at different times after infection, using subtractive hybridization, cDNA microarrays and real-time PCR. The most pronounced change in the expression pattern of functionally diverse groups of genes was detected in systemically infected leaves 14 days after inoculation, and in leaves of plants grown from infected tubers. The expression of several stress-related genes during the infection process, including those for heat shock proteins, catalase 1, β-1,3-glucanase, wound inducing gene, and genes involved in photosynthesis, suggests their role in the susceptible potato–PVY NTN interaction.

Oligonucleotide-based microarray: A new improvement in microarray detection of plant viruses

Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY NTN and PVY O strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.

Scanning electron microscopic investigation of different types of necroses in potato tubers

SEM was used to follow some changes in potato tissue resulting from pathogen infection and differences between different types of necroses in cultivars: Irga, Kuba, Rosalind and Vineta. Irga showed superficial necrotic symptoms caused by Fusarium spp., Alternaria solani, Streptomyces scabies, Rhizoctonia spp., and Phytophthora infestans. Fungal necrosis caused strong disintegration of cell walls with cell lysis. Bacteria affected deep lesion inside phloem and starch damages. Inoculation of Kuba with PVY NTN by grafting resulted in internal point necroses with damaged of phloem tissue and storage material The Rosalind and Vineta showed external necrotic lesions after mechanical inoculation with PVY NTN.

Spread of potato virus Y NTN in potato cultivars ( Solanum tuberosum L.) with different levels of sensitivity

The aim of this work was to correlate the appearance of the symptoms, multiplication and spread of virus after mechanical inoculation of potato ( Solanum tuberosum L.) cultivars showing different levels of susceptibility and sensitivity to Potato virus Y NTN (PVY NTN). The potato cultivars used were the resistant cultivar Sante and susceptible cultivars Igor, Pentland squire and Désirée. The spread of the virus PVY NTN in infected plants was monitored using different methods: DAS-ELISA, tissue printing, immuno-serological electron microscopy and real-time PCR. In all three susceptible cultivars, the virus was detected in the inoculated leaves 4–5 days after inoculation. From there virus spread rapidly, first into the stem, then more or less simultaneously to the upper leaves and roots. Real-time PCR was shown to be very sensitive and enabled viral RNA to be detected in non-inoculated leaves of susceptible cultivar Igor earlier than other methods. Therefore, for exact studies of plant–virus interaction, a combination of methods which detect viruses on the basis of their different properties (coat protein, morphology or RNA) should be used to monitor the spread of viruses.

Specific differentiation of recombinant PVY N:O and PVY NTN isolates by multiplex RT-PCR

The recombinant isolates of tobacco veinal necrotic strain of Potato virus Y (PVY N) and potato tuber necrotic group (PVY NTN) contain segments of the PVY O and the PVY N genome. Three major recombinant junctions (RJ) are present in the genome of the recombinant PVY NTN at sites HC/Pro-P3, 6K2–NIa, and the C-terminal region of CP gene and one RJ at HC/Pro-P3 site in some recombinant PVY N isolates (termed PVY N:O). Protocols for specific differentiation of the recombinant PVY NTN and PVY N:O from the non-recombinant PVY N are described. Specific primer pairs were designed to target the three RJs so that sense and antisense primers completely matched the nucleotide sequences at either side of the RJ. In a uniplex reverse transcription-polymerase chain reaction (RT-PCR), the first primer pair amplified a fragment of 641 bp from the recombinant PVY NTN and PVY N:O. The second and third primer pairs exclusively amplified fragments of 448 and 290 bp, respectively from the recombinant PVY NTN. In a multiplex (triplex) RT-PCR, when all three primer pairs were used simultaneously, the three fragments (641, 448 and 290 bp) were amplified exclusively from the recombinant PVY NTN, while only one fragment (641 bp) was amplified from the PVY N:O isolates, clearly differentiating the two recombinant isolates. No amplification was observed from the non-recombinant PVY, including PVY O and North American (NA)-PVY N/NTN. For further improvement of the multiplex RT-PCR, effects of cDNA preparation using specific antisense primers, random primers or oligo(dT) plus random primers were investigated. The cDNA prepared by random primer plus oligo(dT) increased the overall band intensity.

Tagging of viral RNA transcripts with strain-specific oligonucleotides: characterization and application

Binding(‘tagging’) of a virus-specific oligonucleotide ‘sticker’ to RNA transcripts copied from PCR products caused retardation of transcript mobility in gel. This enables detection of specific sequences within the RNA transcripts, and a virus strain (PVY NTN) could thus be positively identified. We have demonstrated further that oligonucleotides that contained virus sequences originated from different genomic locations varied in their inhibitory effect on the rate of transcript migration in gel; thus, the most effective oligonucleotide could be chosen. Combinations of different strain-specific oligonucleotides had additive retarding effects on transcript migration. The conditions for annealing oligonucleotides to the RNA transcripts were studied, including concentrations of oligonucleotides and salt. A higher electrophoresis temperature (up to 45 °C) reduced the gel retardation phenomena, which indicated a conformation mechanism. The applicability of ‘tagging’ of RNA transcripts with a strain-specific oligonucleotide for virus strain differentiation is discussed.

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.

Differentiating PVY NTN from PVY N by annealing to reference RNA transcripts

A method for the differentiation of virus strains based on the shift in electrophoretic mobility of partially annealed RNA transcripts is described. Oppositely oriented RNA transcripts of the NTN- and N-strains of PVY, complementary at their 3′-end variable (strain-specific) region, were annealed to form a partial duplex which moved more slowly in gel than heterologous (NTN+N) unpaired transcripts. Thus, the two virus strains could be identified by annealing to a known reference transcript. The rate of duplex migration was correlated with transcript lengths and could be tightly controlled thereby. Thus, a higher degree of resolution was obtained than with transcript conformation polymorphism, which is empirical and unpredictable in nature.

Reverse-transcription polymerase chain reaction for the detection of viruses from plants and aphids

A reverse transcription polymerase chain reaction (RT-PCR) protocol used for the detection of potato viruses in dormant tubers and leaves and in an aphid vector is described. Problems in plant sample preparation from different hosts, uneven distribution or low concentration of viruses and the presence of PCR inhibitors in plant extracts are discussed and various ways to eliminate their effect are described. Using Potyviridae viruses, it has been shown that RT-PCR in various modified forms can be used to differentiate viruses at the level of family, genus, species, strains and their subtypes or serotypes. The specificity of primer pairs and PCR modifications has been used to separate closely related potato viruses A and PVY strains (PVY°, PVY N and PVY NTN) from a known mixture.

Strain specific recombinant antibodies to potato virus Y potyvirus

Single chain Fv antibody fragments have been selected from a synthetic phage-antibody library following three and four rounds of affinity selection with purified potato virus Y, common strain (PVY O). The selected fragments were highly specific for PVY and detected seven out of nine isolates of PVY O whilst failing to detect three isolates of PVY N and 12 isolates of PVY NTN. Nucleotide sequence of the scFv genes showed the variable heavy fragments belonged to the human VH4 family, whilst the variable light fragments belonged to the Vλ1 family. The fragments were used in ELISA to detect virus at concentrations of 50 ng/ml in plant sap and in comparisons with commercially available PVY monoclonal antibodies were shown to have similar limits of detection. This is the first report of the selection of a scFv specific for a member of the potyviridae, and its use in detecting and differentiating strains of PVY in infected plant sap. The results highlight the potential of the technology for the selection of strain specific antibodies with an avidity equivalent to traditional monoclonal antibodies raised against viral pathogens and their use for viral diagnosis.