Transcriptome analysis of the synergistic mechanisms between two strains of potato virus Y in Solanum tuberosum L.

Many viruses employ a process known as superinfection exclusion (SIE) to block subsequent entry or replication of the same or closely related viruses in the cells they occupy. SIE is also referred to as Cross-protection refers to the situation where a host plant infected by a mild strain of a virus or viroid gains immunity against a more severe strain closely related to the initial infectant. The mechanisms underlying cross-protection are not fully understood. In this study, we performed a comparative transcriptomic analysis of potato (Solanum tuberosum L.) leaves. The strains PVYN-Wi-HLJ-BDH-2 and PVYNTN-NW-INM-W-369-12 are henceforth designated as BDH and 369, respectively. In total, 806 differentially expressed genes (DEGs) were detected between the Control and JZ (preinfected with BDH and challenge with 369) treatment. Gene Ontology (GO) analysis showed that the response to external biological stimulation, signal transduction, kinase, immunity, redox pathways were significantly enriched. Among these pathways, we identified numerous differentially expressed metabolites related to virus infection. Moreover, our data also identified a small set of genes that likely play important roles in the establishment of cross-protection. Specifically, we observed significant differential expression of the A1-II gamma-like gene, elongation factor 1-alpha-like gene, and subtilisin-like protease StSBT1.7 gene, with StSBT1.7 being the most significant in our transcriptome data. These genes can stimulate the expression of defense plant genes, induce plant chemical defense, and participate in the induction of trauma and pathogenic bacteria. Our findings provided insights into the mechanisms underlying the ability of mild viruses to protect host plants against subsequent closely related virus infection in Solanum tuberosum L.

Susceptibility factor StEXA1 interacts with StnCBP to facilitate potato virus Y accumulation through the stress granule-dependent RNA regulatory pathway in potato.

Plant viruses recruit multiple host factors for translation, replication, and movement in the infection process. The loss-of-function mutation of the susceptibility genes will lead to the loss of susceptibility to viruses, which is referred to as 'recessive resistance'. Essential for potexvirus Accumulation 1 (EXA1) has been identified as a susceptibility gene required for potexvirus, lolavirus, and bacterial and oomycete pathogens. In this study, EXA1 knockdown in potato (StEXA1) was found to confer novel resistance to potato virus Y (PVY, potyvirus) in a strain-specific manner. It significantly compromised PVYO accumulation but not PVYN:O and PVYNTN. Further analysis revealed that StEXA1 is associated with the HC-Pro of PVY through a member of eIF4Es (StnCBP). HC-ProO and HC-ProN, two HC-Pro proteins from PVYO and PVYN, exhibited strong and weak interactions with StnCBP, respectively, due to their different spatial conformation. Moreover, the accumulation of PVYO was mainly dependent on the stress granules (SGs) induced by StEXA1 and StnCBP, whereas PVYN:O and PVYNTN could induce SGs by HC-ProN independently through an unknown mechanism. These results could explain why StEXA1 or StnCBP knockdown conferred resistance to PVYO but not to PVYN:O and PVYNTN. In summary, our results for the first time demonstrate that EXA1 can act as a susceptibility gene for PVY infection. Finally, a hypothetical model was proposed for understanding the mechanism by which StEXA1 interacts with StnCBP to facilitate PVY accumulation in potato through the SG-dependent RNA regulatory pathway.

Anaerobiosis modulation of two phytoglobins in barley (Hordeum vulgare L.), and their regulation by gibberellin and abscisic acid in aleurone cells.

The transcript levels of the phytoglobin (Pgb) genes Pgb1 and Pgb3, and the protein content of Pgb1 were responsive to anaerobiosis in several tissues of barley (Hordeum vulgare L.). Oxygen deficiency induced the level of both Pgb transcripts and protein in aleurone layers and coleoptiles, as well as up-regulated both Pgb1 and Pgb3 in leaves, apexes and more strongly in roots of barley seedlings. In O2-depleted aleurone cells the induction of the Pgb transcript-protein pair was reversed by re-supplying O2. Based on this observation, it is suggested that Pgb1 and Pgb3 are inducible in all tissues. In aleurone cells, gibberellic acid (GA) induced Pgb1 and Pgb3 together with α-amylase, whereas abscisic acid (ABA) eliminated the GA stimulating effects on both α-amylase and Pgb1 and Pgb3 expression. While GA had no effects on alcohol dehydrogenase (Adh1, Adh2 and Adh3) transcripts, ABA induced all three Adh genes. It is concluded that Pgb and α-amylase in seeds are regulated reciprocally with the ethanolic fermentation pathway, and that Pgb induction is mediated by GA. Nitric oxide turnover and scavenging mediated by Pgb represents an important alternative to fermentation under anoxia.

Global Screening and Functional Identification of Major HSPs Involved in PVY Infection in Potato.

HSP40 (also known as DnaJ), HSP70, and HSP90 are major heat shock protein (HSP) families that play critical roles in plant growth and development and stress adaption. Recently, several members of the three HSP families were reported to be widely involved in the plant host-virus interactions. However, their global expression profiles and core members recruited by viruses are largely unknown. In this study, a total of 89 StDnaJs were identified from a genome-wide survey, and their classification, phylogenetic relationships, chromosomal locations, and gene duplication events were further analyzed. Together with 20 StHSP70s and 7 StHSP90s previously identified in the potato genome, the global expression patterns of the members in 3 HSP families were investigated in 2 potato cultivars during Potato virus Y (PVY) infection using RNA-seq data. Of them, 16 genes (including 8 StDnaJs, 6 StHSP70s, and 2 StHSP90s) were significantly up- or downregulated. Further analysis using qRT-PCR demonstrated that 7 of the 16 genes (StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5) were remarkably upregulated in the potato cultivar 'Eshu 3' after PVY infection, implying their potential roles in the potato-PVY compatible interaction. Subsequent virus-induced gene silencing (VIGS) assays showed that silencing of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in Nicotiana. benthamiana plants dramatically reduced the accumulation of PVY, which indicated the four genes may function as susceptibility factors in PVY infection. This study provides candidate genes for exploring the mechanism of potato-PVY compatible interaction and benefits breeding work aiming to produce new cultivars with the ability to grow healthily under PVY infection.

Potato Virus Y Strain N-Wi Offers Cross-Protection in Potato Against Strain NTN-NW by Superior Competition.

Potato virus Y (PVY) is one of the most economically important pathogens of potato. PVY exhibits different phenotypes in dissimilar potato cultivars. Previously, we observed that two recombinant isolates, PVYN-Wi-HLJ-BDH-2 (BDH) and PVYNTN-NW(SYR-II)-INM-W-369-12 (369), exhibited different virulence levels in potato cultivar Kexin 13 despite high genome sequence identity. Indeed, 369 induced severe necrosis and plant death in severe cases in Kexin 13 and severe mosaic in cultivar Yanshu 8, whereas BDH caused mainly mosaic symptoms on the plants of both cultivars. We hypothesized that preinfection of plants with BDH could cross-protect them from 369 infection, and not vice versa. Challenge inoculation, either by mechanical wounding or through grafting, with 369 on plants that were preinfected with BDH did not augment the symptom expression in both cultivars. Reverse transcription quantitative PCR analysis showed that, after challenge inoculation with 369, the titer of the isolate on BDH-preinfected plants remained at a low level (about 3 × 104 copy/µl) during the tested time course (0 h to 30 days). In contrast, in plants that were preinoculated with buffer (mock) and challenge inoculated with 369, the titer of 369 increased continuously until reaching its highest level of about 2 × 107 (Yanshu 8) and about 4 × 108 (Kexin 13) during the time course. Surprisingly, in plants that were preinfected with 369 and challenge inoculated with BDH, the accumulation of BDH reached nearly the same level as that in plants that were preinoculated with buffer and challenge inoculated with BDH. Taken together, these results suggest that PVYN-Wi mediated cross-protection against PVYNTN-NW(SYR-II) by superior competition and better fitness.

Comparison of Mineral Oil, Insecticidal, and Biopesticide Spraying Regimes for Reducing Spread of Three Potato virus Y Strains in Potato Crops.

In-field management of Potato virus Y (PVY) faces challenges caused by the changing availability and environmental acceptability of chemical agents to control aphid vectors of the virus and by proliferation of PVY strains with different symptoms and rates of spread. From 2018 to 2020, foliar spray treatments were compared in field experiments in New Brunswick, Canada, to measure effectiveness at reducing spread of PVYO, PVYN:O, and PVYNTN strains. Mineral oil, insecticide, combined oil and insecticide spray, and a biopesticide (i.e., LifeGard WG) were compared. Insecticide-only and mineral oil-only treatments were not effective, but several combined oil and insecticide treatments and biopesticide treatments significantly reduced PVY spread. The biopesticide was proportionately more effective with recombinant PVYN:O and PVYNTN strains, possibly by exciting the plant's hypersensitive resistance response, caused naturally only in cultivar 'Goldrush' by PVYO. Pesticide residue analysis showed that mineral oil increased the retention of pyrethroid insecticide in the potato foliage longer than with insecticide applied alone, which may explain the beneficial synergistic effect of combined sprays for reducing PVY spread. Tuber yields were generally unchanged in chemical insecticide treatments but were slightly lower in biopesticide treatment. The cost per PVY treatment was competitive across all effective treatments, including biopesticide; however, there was some revenue loss from lower yield with the biopesticide. This biopesticide is certified organic, however, and thus a small premium on the price for organic production could offset this yield deficit.

Coleus blumei viroid 7: a novel viroid resulting from genome recombination between Coleus blumei viroids 1 and 5.

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

Extreme Resistance to Potato Virus A in Potato Cultivar Barbara is Independently Mediated by Ra and Rysto.

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus Potyvirus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained. Phenotyping of the F1 and BC1 populations by graft inoculation with PVA showed segregation ratios of 3:1 and 1:1 (resistant:susceptible), respectively. These results suggest that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

Development of High-Resolution DNA Melting Analysis for Simultaneous Detection of Potato Mop-Top Virus and Its Vector, Spongospora subterranea, in Soil.

In this study, a set of duplex reverse transcription PCR (RT-PCR)-mediated high-resolution DNA melting (HRM) analyses for simultaneous detection of potato mop-virus (PMTV) and its protist vector, Spongospora subterranea f. sp. subterranea (Sss), was developed. The infestation of soil by PMTV was detected with a tobacco-based baiting system. Total RNA extracted from the soil led to successful RT-PCR gel electrophoresis detection of both PMTV and Sss. To facilitate more efficient detection, newly designed primer pairs for PMTV RNA species (i.e., RNA-Rep, RNA-CP, and RNA-TGB) were analyzed together with the existing Sss primers via real-time RT-PCR. The resulting amplicons exhibited melting profiles that could be readily differentiated. Under duplex RT-PCR format, all PMTV and Sss primer combinations led to successful detection of respective PMTV RNA species and Sss in the samples by HRM analyses. When the duplex HRM assay was applied to soil samples collected from six fields at four different sites in New Brunswick, Canada, positive detection of PMTV or Sss was found in 63 to 100% samples collected from fields in which PMTV-infected tubers had been observed. In contrast, the samples from fields where neither PMTV- nor Sss-infected tubers had been observed resulted in negative detection by the assay. Bait tobacco bioassay for PMTV and Sss produced similar results. Of the soil samples collected from PMTV-infested fields, 63 to 83% and 100% led to PMTV and Sss infections in the bait tobacco plants, respectively, whereas no PMTV- or Sss-infected plants were obtained from soil samples collected from PMTV- and Sss-free fields.

Genetic diversity of strawberry mild yellow edge virus from eastern Canada.

Strawberry mild yellow edge virus (SMYEV) is a member of the genus Potexvirus, family Alphaflexiviridae. It is one of the most common pathogenic viruses infecting cultivated strawberries worldwide. In this study, we investigated the genetic diversity of SMYEV in strawberry fields that were severely affected by strawberry decline disease in the eastern Canadian provinces of New Brunswick, Nova Scotia, Prince Edward Island and Quebec. A total of 134 SMYEV coat protein (CP) gene sequences, representing 85 nucleic acid haplotypes, were identified in 56 field samples. A highly divergent SMYEV population was found in all four provinces, but there was little genetic differentiation among the populations, and moreover, the Canadian SMYEV isolates formed a unique dissimilar, genetically divergent population group when compared to those reported in other countries. Phylogenetic analysis revealed three new SMYEV subclades that consisted mainly of Canadian variants and were composed of 76 sequence haplotypes (76/85, 88%). Mixed infections by different SMYEV variants were observed in 38 samples (38/56, 68%). Evolutionary analysis suggested that the SMYEV strains in eastern Canada possibly originated outside of Canada but adapted to conditions in the region through genetic mutations.

Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato.

In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6-98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

Potato Tuber Necrosis Induced by Alfalfa Mosaic Virus Depends on Potato Cultivar Rather Than on Virus Strain.

Alfalfa mosaic virus (AMV) was identified as the causal agent of internal tuber necrosis in the potato cultivar Innovator in New Brunswick, Canada. Further pathological characterization of the isolate (designated as isolate CaM) was performed on six potato cultivars and one breeding clone. Upon mechanical inoculation, four cultivars (Innovator, Yukon Gold, Rochdale Gold-Dorée, and Shepody) showed needle-sized necrotic spots and increasing calico symptoms on new leaves, whereas the remaining cultivars only developed calico symptoms on new leaves. All tubers of CaM-infected Innovator and Shepody plants developed sporadic internal necrotic spots, as did ca. 23 and 8% tubers of CaM-infected Yukon Gold and Rochdale Gold-Dorée, respectively. Sequence analysis of the CP gene of CaM with AMV isolates from potato, all presumed belonging to the "non-necrotic" strain and retrieved from GenBank, indicated that CaM shared >97.1% sequence identity with all but four Egyptian isolates. At the complete genome level, phylogenetic analysis of all available sequences demonstrated that RNA 1 and RNA 3 can be grouped into three major clades each, whereas RNA 2 can be clustered into two clades. CaM and Ca175-1, an AMV isolate that was deemed non-necrotic in a previous study, had different phylogenetic clade patterns, indicating different RNA 1-RNA 2-RNA 3 haplotypes: IA-I-IB (CaM) versus Ca175-1 (IB-II-IA). Despite the difference in haplotype composition, CaM and Ca175-1 induced similar levels of internal necrosis in tubers of Innovator and its parent Shepody. The results suggest that the internal necrosis in AMV-infected tubers depends on potato cultivar rather than on AMV strain/haplotype, and CaM is just a "regular" isolate of AMV.

Molecular Evolutionary Analysis of Potato Virus Y Infecting Potato Based on the VPg Gene.

Potato virus Y (PVY) is an important plant pathogen infecting solanaceous crops, causing significant losses to global potato and tobacco production. Some aspects of the plant pathology and molecular biology of PVY have been studied intensively, but the evolutionary dynamics of this virus are poorly understood. Here, we performed a comprehensive set of rigorous evolutionary analyses using 177 nucleotide sequences of the viral genome linked protein (VPg) gene, which interacts with the plant eukaryotic translation initiation factor 4E (eIF4E). Our Bayesian analysis reveals that the VPg gene of PVY has been evolving at a rate of 5.60 × 10-4 subs/site/year (95% credibility interval 3.35 × 10-4-8.17 × 10-4), which is equivalent to those of other plant-infecting RNA viruses. We identified different evolutionary constraints on the two clades of PVY, clade N and clade O, whose diverge time were estimated at the year 1861 CE (95% credibility interval 1750-1948 CE). We also found that genetic variations were correlated with geographic regions, suggesting that the evolution of this pathogen is strongly affected by geographical associated factors. Taken together, the results of our study have potential implications for the control strategies of PVY.

Proliferation of Recombinant PVY Strains in Two Potato-Producing Regions of Canada, and Symptom Expression in 30 Important Potato Varieties with Different PVY Strains.

Potato virus Y (PVY) exists as several strains with distinct symptomology and tuber yield effects in different potato varieties. Recently, new recombinant strains have proliferated and dominated local populations around the world. In this study, PVYO, PVYN:O, PVYN-Wi, and PVYNTN strains were tracked across Canada from 2014 to 2017, showing rapid evolution of populations away from the traditionally dominant PVYO to recombinants PVYN-Wi (western Canada) and PVYNTN (eastern Canada). Simultaneously, 30 potato varieties were inoculated with PVYO, PVYN:O, and PVYNTN in controlled greenhouse experiments. Foliar symptoms of primary (mechanical inoculation mimicking aphid infection) and secondary (tuber seedborne) infection were cataloged, and tuber yield measured. On average, and generally similar in primary and secondary infection, symptom expression and yield reduction were most severe with PVYO, followed by PVYN:O and PVYNTN. Strong mosaic symptoms were most commonly expressed with PVYO infection, and only seen with PVYN:O or PVYNTN in 15 and 3 varieties, respectively. Across variety-strain combinations, yield reduction was correlated with symptom severity, most strongly in PVYO-infected plants (e.g., AC Chaleur, Beljade, Envol, Norland, and Pacific Russet), and four varieties exhibited tuber necrotic ringspot disease with PVYNTN (AC Chaleur, Envol, Pacific Russet, and Yukon Gold).

Genetic Diversity of Potato virus Y in Potato Production Areas in Northeast China.

In 2011-2014, ELISA or nucleic acid spot hybridization (NASH) testing for common potato viruses or Potato spindle tuber viroid (PSTVd) was performed on 500 leaf samples collected in potato fields in the northeast provinces Heilongjiang and Inner Mongolia, China. The results revealed that 38.4% (Heilongjiang) and 27.7% (Inner Mongolia) were positive for Potato virus Y (PVY). To unveil the strain composition and population structure of PVY in the region, the multiplex RT-PCR described by Chikh-Ali et al. was performed on all of the ELISA-PVY-positive samples. Of the 158 samples whose PVY strain scenarios could be determined, PVYNTN-NW-SYR-II and PVYN-Wi were the most abundant strains, occurring in 58.9 and 47.5% samples, followed by PVYNTN-NW-SYR-I (31.0%), PVYN:O (19.6%), Eu-PVYNTN (7.6%), NA-PVYN (1.3%), and PVYO (0.6%). In the 84 single-strain-infected samples, PVYN-Wi accounted for 41.7%, PVYNTN-NW-SYR-II for 40.5%, PVYNTN-NW-SYR-I for 14.3%, and PVYN:O and Eu-PVYNTN for 3.6% each. Seven isolates representing PVYNTN-NW-SYR-I (HLJ-6-1 and HLJ-9-4), PVYNTN-NW-SYR-II (INM-W-369-12 and SC-1-1-2), PVYN:O (HLJ-30-2), and PVYN-Wi (HLJ-BDH-2 and HLJ-C-429) were sequenced and analyzed molecularly. Whereas the sequence identities for isolates belonging to the same strain group were >98.5%, they fell for isolates belonging to different strain groups to 92.7-98.1% at the genome level and 96.1-98.4% at the polyprotein level. Interestingly, the exact location of the recombination events varied among isolates within a strain group. Phylogenetic analysis of all 42 full length PVY sequences from China indicated that most clustered to various recombinant groups, despite the fact that the PVY isolates were isolated from at least five host species. Pathological analysis of four isolates representing PVYN:O, PVYN-Wi, PVYNTN-NW-SYR-I, and PVYNTN-NW-SYR-II revealed that the PVYNTN-NW-SYR-II isolate incited the most severe symptoms on potato cultivar Kexin 13, followed by PVYNTN-NW-SYR-I, PVYN:O and PVYN-Wi. The PVYNTN-NW-SYR-I and PVYNTN-NW-SYR-II isolates also caused necrotic ringspots on the tubers of Kexin 13, indicating their ability to induce the potato tuber necrotic ringspot disease in potato.

High Resolution DNA Melting Assays for Detection of Rx1 and Rx2 for High-Throughput Marker-Assisted Selection for Extreme Resistance to Potato virus X in Tetraploid Potato.

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.

Strawberry vein banding virus isolates in eastern Canada are molecularly divergent from other isolates.

The complete sequence of a strawberry vein banding virus (SVBV) isolate collected in Nova Scotia, Canada, and designated NS8, was determined. The 7,856-nucleotide circular double-stranded DNA genome contains seven open-reading frames (ORFs), which is consistent with other SVBV isolates and other members of the genus Caulimovirus. Comparison of NS8 with other whole-genome sequences retrieved from databases revealed that NS8 shares the highest sequence similarity (96.5% identity) with isolate China (accession number HE681085) and the lowest (88.3% identity) with clone pSVBV-E3 (accession number X97304). Despite the overall high sequence similarity between NS8 and China, the coat protein encoding ORF IV of NS8 shares only 90.9% sequence identity with the China isolate. Phylogenetic analysis at the complete-genome level placed NS8 and all Chinese isolates in one clade and clone pSVBV-E3 in a separate clade. Interestingly, phylogenetic analysis of all available ORF IV sequences, including those retrieved from databases and newly sequenced samples in this study from Canada, revealed three distinct clades. All Canadian isolates grouped together as one clade, pSVBV-E3 and several others from Europe, Egypt and the USA grouped as a second clade, and isolates from China formed a third clade. These results demonstrate that SVBV is more divergent than previously reported.

Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto.

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato 'Barbara' (Rrrr) and 'AC Chaleur' (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate.

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

RT-PCR Differentiation, Molecular and Pathological Characterization of Andean and Ordinary Strains of Potato virus S in Potatoes in China.

A survey of potatoes in a field in Hubei, China, for common potato viruses revealed that Potato virus S (PVS) was the most abundant virus. To unveil the strain identity of the virus, primers specific to the ordinary and/or Andean strains of PVS (i.e., PVSO and PVSA) were designed. RT-PCR using these primers successfully detected PVSO and PVSA in the samples. Sequence analysis of the amplicons confirmed the correctness of the RT-PCR assay. Two isolates, PVS HB24 and PVS HB7, representing PVSO and PVSA, respectively, were chosen for molecular and biological characterization. Both isolates contained a genome of 8,453 nt in length with six open reading frames. They shared a sequence identity of 79.5% at the complete genome sequence level. Phylogenetic analysis placed PVS HB24 and PVS HB7 to PVSO and PVSA clades, respectively. PVS HB24 induced chlorotic local lesions on the inoculated leaves but no visible symptom on the upper uninoculated leaves of Chenopodium quinoa after mechanical inoculation, whereas PVS HB7 induced both local and systemic symptoms on C. quinoa. ELISA and RT-PCR confirmed that PVS HB7 infected C. quinoa systemically whereas PVS HB24 failed to do so. Both isolates infected potato cv. Shepody and Solanum chacoense asymptomatically, but did not infect Nicotiana occidentalis and N. tobaccum cv. Samsun.