Potato Virus Y Isolates Research Articles

Full-genome analyses of a Potato Virus Y (PVY) isolate infecting pepper (Capsicum annuum L.) in the Republic of South Africa.

Potato Virus Y (PVY) is a pathogen of economic importance in pepper and other major crop species in the family Solanaceae. Three major PVY strain groups: O, C, and N, have been distinguished on the basis of genome sequencing. In this study, the first full-genome sequence of a PVY isolate (JVW-186) infecting pepper from the province of KwaZulu-Natal, Republic of South Africa is reported. The complete genome sequence of JVW-186 was assembled from overlapping RT-PCR clones using MEGA 5 software. Two ORFs were identified at position 186 and 2915 of the sequence encoding the viral polyprotein and the frameshift translated protein P3N-PIPO, respectively. RDP4 software confirmed three recombination breakpoints at position 343, 1365, and 9308 of the sequence. At each recombination event, a 1,021-bp fragment at the 5' end in the region of the P1/HC-Pro protein and a 392-bp fragment in the region of the coat protein shared a high sequence similarity of 91.8 and 98.89 % to the potato borne PVY(C) isolate PRI-509 and the PVY(O) isolate SASA-110, respectively. The non-recombinant fragment 1 (342-bp) clustered within the C clade of PVY isolates; however, the large 7,942-bp fragment 3 did not cluster within any of the clades. This suggests the possibility of a PVY isolate that has evolved due to the dynamics of selection pressure or the likelihood of an ancestral PVY strain.

Assessment of Genes R1 and R3 Conferring Resistance to Late Blight and of Gene R Ysto Conferring Resistance to Potato Virus Y in Two Wild Species Accessions and their Hybrid Progenies/ Izturîbas Pret Lakstu Puvi Gçnu R1 Un R3 Un Izturîbas Pret Kartupeïu Vîrusu Y Gçna Rysto Novçrtçjums Divu Savvaïas Sugu Lînijâs Un To Hibrîdos

Abstract The oomycete Phytophthora infestans Mont. (de Bary), which causes potato late blight, and Potato Virus Y (PVY) are economically important potato pathogens. More virulent P. infestans strains have evolved and are able to overcome resistance genes introgressed earlier to cultivated potato from wild Solanum species, especially from S. demissim Lindl. Potato cultivars resistant to the previously common type of PVYN may be susceptible to the more virulent isolates of PVY. In previous research, S. guerreroense Corr. (grr) and S. neoantipoviczii Buk. (nan) accessions were rated as resistant to P. infestans and to two (grr) and three (nan) strains of PVY. These parental accessions and their respective hybrid offspring were screened using polymerase chain reaction (PCR) tests to detect alleles conferring resistance to P. infestans (R1 and R3) and to PVY (Rysto). Screenings were made in detached leaflet tests (P. infestans), and with the aid of ELISA tests (PVY). The resistance of two hybrids derived from grr was rated as high, mostly due to a hypersensitivity reaction. The allele R3 was detected in only one grr plant among 20 plant populations for each grr and nan accession. R1 and R3 alleles were more frequently detected in one hybrid of grr (grr × B.d.R5.). The resistance allele Rysto was found in nan and provided host plant resistance to three strains of PVY, including PVYNTN. Hybrids derived from this accession were characterised by a high frequency of plants bearing the resistant allele Rysto. In the parental clone tbr × phu, as well as in grr and its hybrids, only the susceptible allele at Rysto locus was detected.

Screening Potato Cultivars for new Sources of Resistance to Potato virus Y

Potato virus Y (PVY) strains have been defined based on genetic reactions in potato indicators expressing hypersensitive reaction (HR) response due to the presence of three different N genes, and also based on genomic information. Nine strains are known currently, with five PVY strains defined biologically, PVYO, PVYC, PVYZ, PVYN, and PVYE. The genetic background of the majority of North American potato cultivars has so far been poorly characterized for the presence of N genes inducing HR towards different PVY strains. Here, the HR response was studied in eight potato cultivars, elicited by five strains of PVY circulating in North America. These PVY isolates included representative isolates of PVYN-Wi, PVYNA-N, PVYO, PVYZ, and PVYN strains. Potato cultivars tested included Russet Burbank, Russet Norkotah, Shepody, Ranger Russet, Western Russet, Alturas, Rio Grande Russet, and Yukon Gem, grown in the U.S., and standard indicators Desiree and Maris Bard with the known genetic background. Three additional strains, PVYN:O, PVY-NE11, and PVYE, were tested on Yukon Gem. Virus-free potato plants were mechanically inoculated with PVY inoculum, and local and systemic foliar symptoms were observed for 8 weeks post-inoculation under different climate-controlled conditions. Virus status of the inoculated plants was tested starting at 3 weeks post-inoculation, by serotype-specific ELISA and RT-PCR, in order to monitor successful infections and confirm the identity of the inoculated PVY isolate. This systematic approach allowed us to identify Nytbr and Nztbr genes present in several North American cultivars. Two more new, putative N genes were postulated to be expressed in the cultivar Yukon Gem, and one additional putative N gene was postulated to be expressed in two cultivars, Yukon Gem and Rio Grande Russet. These N genes may represent valuable sources of resistance against multiple strains of PVY.

First Report of Recombinant Potato virus Y Strains Infecting Potato in Jordan.

Potato (Solanum tuberosum L.) is an important vegetable crop in Jordan, occupying second position after olives. In 2012, potatoes were planted on about 6,000 ha with a production of about 141,000 t (2). Potato virus Y (PVY) is a serious problem for potato production worldwide. Recombinant strains of the virus were reported to cause tuber necrotic ringspot disease (PTNRD) in many potato-growing regions of the world. In the last few years, a new recombinant PVYNTN-NW that belongs to PVYZ (3) has been reported in the neighboring Syria. It included three recombination patterns, SYR-I, SYR-II, and SYR-III, and caused severe PTNRD (1). Since PVY is easily transmitted from one region to another by aphid vectors and infected potato seeds, this study was initiated to investigate the possible occurrence of PVY strains in Jordan. In October 2013, 33 leaf samples were collected from symptomatic potato plants cv. Spunta from Wadi Rum, Jordan (GPS coordinates 29°31'37.76″ N, 35°42'48.75″ E), the largest potato-producing area in Jordan. Sampled plants displayed leaf mottling and yellowing, symptoms similar to those caused by PVY. All samples were tested for PVY by DAS-ELISA using the ELISA kit (monoclonal cocktail) developed by BIOREBA (Reinach, Switzerland) to detect all PVY isolates. Twenty-nine samples were found positive for PVY by ELISA. To confirm virus infection, total RNA was extracted from all ELISA-positive samples and used as template in uniplex RT-PCR using strain-specific primers (1). The band pattern of PCR amplicons showed that 12 samples were infected with PVYNTN-NW genotype SYR-III and produced bands of 1,085, 441, and 278 bp. One sample was infected with PVYNTN (A) and produced bands of 1,307, 633, and 441 bp, and one other sample was infected with PVYNTN-NW genotype SYR-II and produced bands of 1,085 and 441 bp. Mixed infection with PVYNTN-NW genotype SYR-III and PVYNTN (B) was also detected in one sample producing bands of 278, 441, 1,085, and 1,307 bp. To confirm infection with the recombinant strains, PCR fragments of 278 bp amplified from three samples and 1,085 bp obtained from another three samples were directly sequenced and sequences were deposited in GenBank under accession numbers KJ159968, KJ159969, and KJ159970 for the 278-bp fragment and KJ159974, KJ159975, and KJ159976 for the 1,085-bp fragment. Sequence comparison with other PVY strains available in the NCBI database showed that the 278-bp fragment had the highest nucleotide sequence identity (100%) with PVY isolates SYR-III-A26 (AB461467) and SYR-III-2-4 (AB461457) from Syria. BLAST searches also showed that the 1,085-bp fragment shared 99% nucleotide identities with PVY isolates SYR-II-L3 (AB461482) and SYR-II-Be4 (AB461474) from Aleppo, Syria. To our knowledge, this is the first report of PVY recombinants in Jordan, and the first report of PVYNTN-NW recombinants infecting potato crop outside Syria. Since Europe is the main supplier of potato seeds for farmers in Jordan and Syria, the introduction of PVYNTN-NW to the region could have happened through infected potato seeds. Results of this study create new challenges for potato growers in Jordan as well as other countries in the region.

Evidence of a Monogenic Nature of the Nz Gene Conferring Resistance Against Potato virus Y Strain Z (PVYZ) in Potato

Hypersensitive resistance (HR) to Potato virus Y (PVY) in potato (Solanum tuberosum) is conferred by strain-specific N genes. Two such genes have been identified in potato so far, Ny tbr conferring HR to PVYO, and Nc tbr conferring HR to PVYC. A third, putative gene Nz tbr was proposed to confer HR against a distinct strain PVYZ. However, due to the scarcity of the PVYZ isolates of PVY, no formal proof of the monogenic nature of this new gene, Nz tbr , was available until now. Here, we report on a genetic study of the Nz tbr inheritance in three crosses between cultivars Maris Bard (Ny:Nz) and King Edward (ny:nz), and Maris Bard (Ny:Nz) and Russet Norkotah (ny:nz). A fully-sequenced PVYZ isolate, L26, was used to screen the parents and progeny for a virus-induced HR phenotype in foliage. Based on the phenotypic analysis of 203 progeny, segregation of HR phenotype in the PVYZ-infected plants was found to be 1:1, indicating a monogenic, dominant nature of the Nz tbr gene. Since the PVYZ strain includes PVYNTN isolates associated with tuber necrotic ringspot disease (PTNRD) in susceptible potato cultivars, the Nz tbr gene represents a valuable source of HR against PTNRD-inducing PVY isolates. This is the first demonstration that Nz tbr is a single, dominant N gene in potato conferring resistance to the PVYZ-NTN strain.

Characterization of a Novel Resistance-Breaking Isolate of Potato Virus Y in Nicotiana Tabacum

Potato virus Y (PVY) was identified from a PVY-resistance flue-cured tobacco variety KF120 showing vein necrosis at Buron province, Korea. Biological properties of the isolate named PVY-ToBR1 was characterized using various host plants with another isolate PVY-ToJC37. The isolated PVY-ToBR1 induced systemic vein necrosis symptoms on a PVY-resistant tobacco cultivar (VAM) harboring potyvirus resistant va gene, though tissue printing showed the systemic movement of virus was slightly delayed. By contrast, the isolate PVY-ToJC37 failed to infect VAM plants and the virus was not detected on inoculated leaf and systemic leaves in VAM plants. Similarly, the isolated PVY-ToBR1 induced distinctly systemic vein necrosis symptoms on PVY-resistant tobacco cultivars (V.SCR, PBD6, TN86, TN90, Virgin A Mutant, NC744, and Wislica) that have the recessive potyvirus resistance gene va, but PVY-ToJC37 did not infect systemically infect these tobacco cultivars, suggesting that PVY-ToBR1 is a novel resistance-breaking isolate in tobacco. The coat protein (CP) genes of PVY-ToBR1 and PVY-ToJC37 were amplified using RT-PCR assays with specific primers for PVY isolates and nucleotide sequences of the CP genes were determined. The isolate PVY-ToBR1 showed 88.4% - 99.4% and 86.6% - 99.4% CP identities to the 46 different PVY isolates at the nucleotide and amino acid, respectively. Phylogenetic relationship from CP comparisons showed that PVY-ToBR1 isolate clustered with PVYNTN isolates and PVY-ToBR1 isolate more closely related to the isolates from European than from North American PVY NTN.

Surface plasmon resonance for monitoring the interaction of Potato virus Y with monoclonal antibodies

Surface plasmon resonance (SPR)-based biosensors have been widely utilized for measuring interactions of a variety of molecules. Fewer examples include higher biological entities such as bacteria and viruses, and even fewer deal with plant viruses. Here, we describe the optimization of an SPR sensor chip for evaluation of the interaction of the economically relevant filamentous Potato virus Y (PVY) with monoclonal antibodies. Different virus isolates were efficiently and stably bound to a previously immobilized polyclonal antibody surface, which remained stable over subsequent injection regeneration steps. The ability of the biosensor to detect and quantify PVY particles was compared with ELISA and RT-qPCR. Stably captured virus surfaces were successfully used to explore kinetic parameters of the interaction of a panel of monoclonal antibodies with two PVY isolates representing the main viral serotypes N and O. In addition, the optimized biosensor proved to be suitable for evaluating whether two given monoclonal antibodies compete for the same epitope within the viral particle surface. The strategy proposed in this work can help to improve existing serologic diagnostic tools that target PVY and will allow investigation of the inherent serological variability of the virus and exploration for new interactions of PVY particles with other proteins.

Sequence variation and protein structure of pipo gene in Potato virus Y

The objectives of this study were to understand the sequence variation and the putative protein structure of pipo gene in the Potato virus Y (PVY) collected from Solanum tuberosum. The pipo gene in PVY was cloned using a pair of degenerate primers designed from its conserved region and its sequences were used to re-construct phylogenetic tree in Potyvirus genera by a Bayesian inference method. An expected fragment of 235 bp was amplified in all 20 samples by RT-PCR and the pipo genes in the 20 samples assayed shared more than 92% nucleotide sequence similarity with the published sequences of PVY strains. Among the 20 pipo gene sequences, 13 polymorphic sites were detected, including 4 parsimony informative sites and 9 singleton variable sites. These results indicate that PVY pipo gene is highly conserved but some sequence variations exist. Further analyses suggest that the pipo gene encodes a hydrophilic protein without signal peptide and transmembrane region. The protein has theoretical isoelectric points (pI) ranging from 11.26 to 11.62 and contains three highly conserved regions, especially between aa 10 and 59. The protein is likely located in the mitochondria and has a-helix secondary structure. Bayesian inference of phylogenetic trees reveals that PVY isolates are clustered in the same branch with high posterior probability, while Sunflower chlorotic mottle virus (SoCMoV) and Pepper severe mosaic virus (PepSMV) are closely related, consisting with the classification of Potyvirus genera using other approaches. Our analyses suggest that the pipo gene can be a new marker for phylogenetic analysis of the genera. The results reported in this paper provide useful insights in the genetic variation and the evolution of PVY and can stimulate further research on structure and function of the PIPO protein.

An Improved Multiplex IC-RT-PCR Assay Distinguishes Nine Strains of Potato virus Y.

A multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay was previously developed to identify a group of Potato virus Y (PVY) isolates with unusual recombinant structures (e.g., PVYNTN-NW and SYR-III) and to differentiate them from other PVY strains. In the present study, the efficiency of this multiplex RT-PCR assay was validated and extended considerably to include five additional strains and strain groups not tested before. To make the multiplex RT-PCR assay more applicable and suitable for routine virus testing and typing, it was modified by replacing the conventional RNA extraction step with the immunocapture (IC) procedure. The results obtained using well-characterized reference isolates revealed, for the first time, that this multiplex RT-PCR assay is an accurate and robust method to identify and differentiate the nine PVY strains reported to date, including PVYO (both PVYO and PVYO-O5), PVYN, PVYNA-N, PVYNTN, PVYZ, PVYE, PVY-NE11, PVYN-Wi, and PVYN:O, which is not possible by any of the previously reported RT-PCR procedures. This would make the IC-RT-PCR procedure presented here a method of choice to identify PVY strains and assess the strain composition of PVY in a given area. The IC-RT-PCR protocol was successfully applied to typing PVY isolates in potato leaf tissue collected in the field.

The effects of co‐infection by different Potato virus Y (PVY) isolates on virus concentration in solanaceous hosts and efficiency of transmission

The influence of co‐infection on concentration and accumulation of genetically different isolates of Potato virus Y (PVY) in potato and tobacco plants and the efficiency of transmission by Myzus persicae of PVY isolates from doubly versus singly infected plants were evaluated. The vector ability to simultaneously transmit two virus isolates was examined. Eight PVY isolates represented three strain groups: PVYO (pathotype and serotype O), PVYNW (pathotype N and serotype O), and PVYNTN (pathotype and serotype N). Different diagnostic methods, including DAS‐ELISA, multiplex RT‐PCR, aphid transmission tests and bioassays, were applied to detect the presence of PVY isolates in source and assay plants. Significant reductions in concentrations of certain PVY isolates during co‐infection with other isolates were found both in potato and tobacco plants. The observed effects were both isolate‐ and host‐dependent in form. The highest rates of virus transmission by single aphids were recorded with PVYNTN isolates, and the lowest ones with PVYO isolates. Individual aphids of M. persicae were able to simultaneously transmit two PVY isolates. The frequency of transmission was generally low, but it reached as high as 20% for one of the isolate combinations. The findings presented in the work provide proof for antagonistic within‐plant interactions between isolates of PVY, with some implications of these interactions for virus transmission by aphid vectors. Consequently, this research contributes to a better understanding of the epidemiology of the disease caused by PVY.

First Report of Recombinant Potato virus Y Strains in Potato in Jalisco, Mexico.

Potato virus Y (PVY) is a serious problem for potato production worldwide. The virus reduces both tuber yield and quality, and recent spread of recombinant strains of PVY in potato production areas is largely credited with the spread of potato tuber necrotic ringspot disease (PTNRD) (1). In Mexico, recombinant strains of PVY were reported in at least two states, Chihuahua (4) and the State of Mexico (3); however, no surveys have been conducted in other potato-producing areas, and the spectrum of PVY isolates circulating in the country has remained uncharacterized. In October 2011, a small-scale survey of seed potato was conducted in the state of Jalisco, Mexico, to identify PVY isolates present in fields. Twelve seed potato fields were inspected visually. These represented various generations of seed potato, from nuclear to G2. Leaf samples were collected from plants displaying mosaic, crinkling, and yellowing symptoms, and were tested for PVY. Fifty samples were collected from cultivars Fabula, Mondial, Fianna, Gigant, Caesar, and Adora. Of the 50 leaf samples collected, seven were PVY-positive using the Immuno-strip Kit (Agdia, Elkhart, IN), and six of these were determined to have a N-serotype according to the typing by the Pocket Diagnostics lateral flow kit (Forsite Diagnostics, Ltd., York, UK). PVY-positive samples came from cultivars Fabula (2 with N serotype), Mondial (4 with N serotype), and Fianna (1 with O serotype). Extracts of the seven PVY-positive leaf samples were applied to Whatman FTA cards (Sigma, St. Louis, MO), dried, and transported to the Plant Virology Laboratory at the University of Idaho for further characterization. All samples immobilized on FTA cards were subjected to RNA extraction and standard reverse transcriptase (RT)-PCR typing using a set of PVY-specific primers (2) to determine the strain type. All PVY isolates were recombinant. The six N-serotype samples were found to contain recombinant PVYNTN isolates and produced characteristic bands of 181 and 452 bp in RT-PCR, which indicated the presence of two recombination junctions in the HC-Pro/P3 and VPg regions typical of European PVYNTN isolates. The one O-serotype sample was identified as a recombinant PVYN-Wi/N:O isolate, and produced 181 and 689 bp bands in RT-PCR, which indicated the presence of one recombination junction in the HC-Pro/P3 region. Sequence analysis of RT-PCR products amplified from five samples with N serotype identified them as PVYNTN isolates, and from the one with O serotype identified it as PVYN-Wi/N:O isolate. Sequence comparisons confirmed that N serotype samples contained PVY isolates most closely related to typical PVYNTN sequences (Accession No. EF026075), while the O serotype sample contained the PVY isolate most closely related to PVYN-Wi from Europe (HE608963). The data obtained suggest the presence of two different types of PVY recombinants, PVYNTN and PVYN-Wi, in seed potato in Jalisco. Additional surveillance for these recombinant isolates may be needed, as well as a survey of their effects on tuber quality in production areas. This is the first report of recombinant isolates of PVY often associated with PTNRD circulating in seed potato in Jalisco, Mexico.

Sources and Effectiveness of Potato PVY Resistance in IHAR’s Breeding Research

Resistance to Potato virus Y (PVY) is one of the most important traits in seed and ware potato production. Currently the tobacco-necrosis inducing PVYN strain including PVYN-Wi and PVYNTN dominates in the PVY population in Poland. The PVYO strain which dominated in early 1980s is rarely detected. Since early 1950s, breeding for PVY resistance in IHAR was a part of the program developing parental lines, which resulted in selecting lines extremely resistant to PVY. The selection of these lines was improved by the application of developed molecular markers. Moreover, the group of genes conferring comprehensive hypersensitive resistance (HR) to all strains of PVY was identified and mapped. The cultivars obtained from parental lines have enhanced level of resistance to PVY. For registered cultivars, the level of resistance to PVY is monitored in field exposure tests, while the reaction to four different isolates of PVY is examined by artificial inoculations. The 36 cultivars with field resistance scores 7.5 and 9 expressed resistance to all tested isolates and 23 of them originated from IHAR parental lines. Several moderately resistant cultivars varied in sensitivity to the PVY isolates and in majority they were more sensitive to isolates Wi (collected in 1984) and 12/94 (collected in 1994), than to older isolates LW and Ny collected in 1970-ties.

Differentiation of PVY isolates depending on criterion of evaluation

In 2000-2001 35 isolates of Potato virus Y(PVY) were characterized by serological and biological methods. These isolates represented 5 distinct strains: PVY<sup>C<sup>, PVY<sup>O<sup>, PVY<sup>N<sup>, PVY<sup>N<sup> W, PVY<sup>NTN<sup>. Among isolates belonging to PVY<sup>N<sup> W and PVY<sup>C<sup>NYN isolates which are able to overcome the resistance in three tobacco cultivars (VAM, Wiślica, PBD6) were found. They were obtained from tobacco plants grown in Poland or Hungary. Isolates obtained from potato plants were able to cause only mild symptoms on cvs. Wiślica and PBD6.

Molecular and Serological Typing of Potato virus Y Isolates from Brazil Reveals a Diverse Set of Recombinant Strains.

In Brazil, Potato virus Y (PVY) currently presents a significant problem for potato production, reducing tuber yield and quality. Recombinant tuber necrotic isolates of PVY had been reported to occur in the country but no systematic study of the PVY isolate diversity was conducted thus far. Here, a panel of 36 PVY isolates, randomly collected in Brazil from potato between 1985 and 2009, was subjected to a systematic molecular and serological typing using reverse-transcription polymerase chain reaction and a series of PVYO- and PVYN-specific monoclonal antibodies. The data collected were combined with biological characterization of the same isolates in tobacco. Of the 36 isolates tested, 3 were typed as PVYO, 10 as PVYN:O/N-Wi, 21 as PVYNTN, and 2 as "unusual" or inconclusive. Of the 10 isolates from the recombinant PVYN:O/N-Wi strain group, 1 isolate, MAF-VOY, was found to have an unusual serological profile identical to the nonrecombinant PVYO-O5 strain group. The 21 tested PVYNTN isolates included 1 isolate that did not induce vein necrosis in tobacco and 2 isolates with an unusual serological profile (i.e., displaying negative reactivity to one commercial PVYN-specific monoclonal antibody). Whole genome sequences were determined for four PVY isolates from Brazil, representing PVYO, PVYNTN, and PVYN-Wi strains. The genome of the MAF-VOY isolate was found to be recombinant, having characteristic N-Wi structure with two recombinant junctions and carrying a single mutation in the capsid protein at position 98, which led to an unusual O5 serological reactivity. Taken together, the data obtained suggest that the two recombinant strains, PVYNTN and PVYN:O/N-Wi, now are apparently dominant in the Brazilian potato crop. The data also suggest that recombinant isolates in Brazil often have unusual serological reactivity which may hamper their correct identification by conventional typing based on enzyme-linked immunosorbent assay.

Response of Potato Cultivars to Five Isolates Belonging to Four Strains of Potato virus Y.

The responses of 14 potato cultivars to five Potato virus Y (PVY) isolates belonging to four strains (ordinary [PVYO], tobacco veinal necrosis [PVYN], N:O group [PVYN:O], and nonrecombinant potato tuber necrotic [PVYNTN]) were studied in primary and secondary infections. For the primary infection experiments, foliage symptoms were monitored daily after mechanical inoculation with a PVY isolate until harvest; and, for the secondary infection experiments, foliage symptoms were monitored regularly from plant emergence until harvest. Tuber symptoms (namely, tuber necrotic ringspots) were checked at harvest and monthly postharvest for up to 4 months. In both infections, symptoms varied significantly depending on potato cultivar and virus strain or isolate. In primary infections, local lesions occurred on inoculated leaves of 'AC Chaleur', 'Eramosa', 'Goldrush', 'Jemseg', 'Katahdin', 'Ranger Russet', and 'Yukon Gold' after inoculation with PVYO isolates, followed by systemic necrosis on latterly emerged uninoculated leaves. In contrast, plants of 'CalWhite', 'La Rouge', 'Red LaSoda', 'Russet Burbank', 'Russet Norkotah', and 'Superior' did not exhibit any visible symptoms on inoculated leaves but developed mild to severe mosaic on latterly emerged leaves after infection with PVYO isolates. In all cultivars, near-symptomless to mild mosaic was induced by PVYN and mild to severe mosaic by PVYN:O. PVYNTN induced mild to severe mosaic in plants of all cultivars except AC Chaleur, 'Cherokee', and Yukon Gold, which developed visible systemic necrosis. Necrotic ringspots were observed in tubers of PVYNTN-infected plants of AC Chaleur, Cherokee, and Yukon Gold. The tuber symptoms were also incited by PVYN-Jg on Cherokee. In secondary infections, the symptoms were generally more severe than primary infections even though the symptom types did not alter. As in the greenhouse, a clear symptom severity pattern (PVYO-FL > PVYO-RB > PVYNTN-Sl > PVYN:O-Mb58 > PVYN-Jg) was observed in AC Chaleur, Cherokee, Eramosa, Goldrush, Jemseg, Katahdin, Ranger Russet, and Yukon Gold in the field.

Phylogenetic studies of selected isolates of Potato virus Y (PVY) infecting vegetable crops in KwaZulu-Natal, Republic of South Africa

Phylogenetic relationships of Potato virus Y (PVY) isolates infecting vegetable crops in KwaZulu-Natal, Republic of South Africa, were investigated. A 1 067 bp amplicon covering part of the coat protein gene and the 3′ non-translated region (NTR) of three PVYO isolates infecting tomato (Solanum lycopersicum L.), one PVYO isolate infecting pepper (Capsicum annuum L.) and one PVYN Wilga isolate infecting potato (Solanum tuberosum L.) were amplified, cloned and sequenced. The 5′ NTR, P1, HC-Pro and part of the P3 regions (2 559 bp) of a PVYN isolate infecting potato were amplified, cloned and sequenced. Sequence data were compared with sequences of PVY isolates from different geographical locations and subjected to phylogenetic analyses. The PVYN isolate clustered with the European sublineage N and has five unique amino acid residues. The PVYN Wilga isolate branched with the American PVYO isolate in the O lineage. All PVYO isolates infecting tomato and pepper were grouped in a new sublineage within the O lineage.

Cloning and sequencing of the genes encoding coat protein (CP) in Potato virus Y from Thai Nguyen province

Potato (Solanum tuberosum L.) is the most widely distributed crop in tropical and subtropical zones of the world. Among diseases potato viruses play major role in reducing the yield. PVX (Potato virus X), PVY (Potato virus Y), PLRV (Potato leaf roll virus), PVA (Potato virus A), PVM (Potato virus M), PVS (Potato virus S) are major viral diseases occurring in Vietnam. PVY (Potato virus Y) is the type member of the genus Potyvirus of the family Potyviridae. The virus has a long filamentous particle containing one single-stranded, positive sense RNA genome of approximately 9.7 kb. PVY is an important pathogen infecting potato, tobacco, tomato and other solanaceous plant species. Different potato PVY isolates occur that can be categorized in 5 groups: PVYO, PVYN, PVYNTN, PVYNWi and PVYC. In this paper we report cloning and sequencing of coat protein (CP) gene of PVY from Thai Nguyen potato. The CP gene of this isolate was found to be 801 nucleotides long, coding for 267 amino acids. Comparison amino acid of CP protein in potato from Thai Nguyen with ten sequences on NCBI showed that amino acid was 92.1-98.9% homology.

In silico prediction of virus-derived small interfering RNAs and their putative host messenger targets in Solanum lycopersicum infected by different potato virus Y isolates

Motivations. RNA silencing, or post-transcriptional gene silencing (PTGS), is a conserved mechanism in a broad range of eukaryotes. In plants, PTGS acts as an antiviral system and a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with RNA and DNA viruses and provide specificity to this RNA-mediated immune system. High-throughput sequencing has contributed to expanding our previously knowledge of siRNA populations better describing their abundance, complexity and diversity in infected tissues. It is now known that siRNAs from virus-infected plants are extraordinarily abundant and diverse, and are widespread in near saturation at any region of either positive and negative genomic RNAs. However, certain regions of viral genomes ("hot spots") are usually more represented than others in sequenced siRNA populations. A gene involved in chlorophyll biosynthesis has been shown targeted by a siRNA derived from viral satellite RNA, revealing PTGS mechanism at basis of the symptom. Potato virus Y (PVY) is the type species of Potyvirus, a genus of agricultural importance belonging to the largest plant virus family, Potyviridae. The potyviral genome is a single-stranded, positive-sense RNA of ca. 10 kilobases (kb). PVYc-to and PVY-SON41 are two isolates of PVY that infect solanaceous hosts. While PVYc-to induces severe leaf distortion in different cultivars of tomato (Solanum lycopersicum), PVY-SON41 produces in the same host only a mild mosaic, followed by recovery. In order to elucidate the molecular mechanism underlying the different symptoms induced by PVYc-to and PVY-SON41 infections on tomato, we set up an in silico approach, mining genomic regions of PVY isolates and looking at possible RNA-based mechanisms where siRNA putatively generated from the PVY genome could target and suppress accumulation of host messenger RNA (mRNA), leading to dysfunctional biological processes that could explain different disease phenotypes. Methods. A Perl script was used to extract the complete datasets of 21-mers from isolates PVYc-to and PVY-SON41 complete genomes, leading the scanning of the complete sequence, shifted by one base at the time. MySQL was used for identification of the 21-mers shared and unique between the two viral genomes, highlighting sequence differences that could be at the base of diverse induced symptoms. The data obtained in the previous step were used to build the genomic map of identical regions, by fancyGene. A BLAST analysis was conducted with the identified 21-mer dataset, considering only the dissimilar sequences between the two isolates (blastn-short identity > 94%, max 2 mismatch or gap, alignment length 21 bp). The 21-mer were used as query and the tomato mRNA database (Solgenomics, release ITAG2.3) was used as target. The results were further used as input in a gene ontology analysis, through Blast2GO. Results. Despite the high identity (> 91%) shared by the two viral genomes, the 21-mer dataset produced a high degree of variation, as the analyses showed 1750 identical and 9671 and 9680 dissimilar sets of 21-mer, for PVYc-to and PVY-SON41, respectively. Data showed that 315 and 381 tomato mRNA were complementary to, and thus possible targets of, the in silico-generated siRNAs deduced from of PVYc-to and PVY-SON41 sequences, respectively. The GO analysis showed different putative gene targets, involved in several metabolic pathways indicating specific targets for each isolate. Of particular interest, for instance, are putative target mRNAs of transcription factors with a known role concerning leaf development and symmetry. These genes, if differentially modulated during the infections of the two different PVY isolates, could cause the leaf malformations observed as major differential symptoms between the two PVY isolates. To validate the bioinformatic approach, target genes with highly significant complementarity have been selected from the in silico data for experimental validation in tomato plants mechanically infected by virus isolates.

Identification of new Potato virus Y (PVY) molecular determinants for the induction of vein necrosis in tobacco.

Two tobacco vein necrosis (TVN) determinants, the residues K(400) and E(419) , have been identified previously in the helper component-protease (HC-Pro) protein sequence of Potato virus Y (PVY). However, since their description, non-necrotic PVY isolates with both K(400) and E(419) necrotic determinants have been reported in the literature. This suggests the presence in the viral genome of other, as yet uncharacterized, TVN determinant(s). The identification of PVY(N) pathogenicity determinants was approached through the replacement of genomic regions of the necrotic PVY(N) -605 infectious clone by corresponding sequences from the non-necrotic PVY(O) -139 isolate. Series of PVY(N/O) chimeras and site-directed PVY mutants were constructed to test the involvement of different parts of the PVY genome (from nucleotide 421 to nucleotide 9629) in the induction of TVN symptoms. The analysis of both the genomic characteristics and biological properties of these mutants made it possible to highlight the involvement, in addition to residues K(400) and E(419), of the residue N(339) of the HC-Pro protein and two regions in the cytoplasmic inclusion (CI) protein to nuclear inclusion protein a-protease (NIa-Pro) sequence (nucleotides 5496-5932 and 6233-6444) in the induction of vein necrosis in tobacco infected by PVY isolates.

Epitope Mapping for Monoclonal Antibodies Recognizing Tuber Necrotic Isolates of Potato Virus Y

Potato virus Y (PVY) is an important viral pathogen of potato responsible for reducing tuber yield and quality across the globe. The PVYN and PVYNTN strains, the latter of which induces potato tuber necrotic ringspot disease (PTNRD), are regulated for international potato trade, and have been routinely detected using monoclonal antibodies (MAbs) that discriminate between PVYN and PVYO serotypes. Here, we identify the distinct binding sites in the capsid protein of PVY for three of the four main PVYN-specific MAbs, Bioreba-N, SASA-N, and Neogen-N, available commercially. These binding domains were mapped through a combination of TAS-ELISA testing of MAbs on multiple reference isolates of PVY, sequence analysis, heterologous expression of capsid protein fragments, and synthetic peptide binding experiments. All three MAbs were found to bind linear epitopes located within the first 31 N-terminal amino acids of the capsid protein. Bioreba-N MAb epitope spanned aa 1-17 and included three positions, aa 1, aa 11, and aa 17, which differ between PVYN and PVYO serotypes. Both SASA-N and Neogen-N epitopes spanned aa 22-30, and included two positions, aa 24 and aa 29, which differ between PVYN and PVYO serotypes. Epitopes for SASA-N and Neogen-N MAbs are likely to be identical or overlapping. Examination of available sequences for tuber necrotic isolates of PVY that do not react with PVYN-specific MAbs SASA-N and Neogen-N indicated possible selection for substitutions in corresponding epitopes leading to the loss of reactivity towards these antibodies. The data obtained suggested that testing with more than one PVYN serotype-specific MAb could assure a reliable serological identification of a PVYN or PVYNTN isolate.