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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 O

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.

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.

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.

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.

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.

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.

In-field management of

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus

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).

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.

Potato virus Y (PVY) is an economically important disease agent in potatoes with worldwide distribution. It is mainly transmitted vegetatively by tubers and between plants by an aphid vector. PVY management is primarily through reducing viral inoculum in seed tubers and other sources and controlling transmission by aphids. Seed certification programs are designed to comprehensively test and restrict levels of PVY within a region, enforcing low levels of PVY in marketed seed lots and allowing growers informed choice to plant low-PVY crops. Sanitation techniques on the farm can also reduce volunteer, weed, and neighboring field sources of PVY. Major techniques to reduce aphid-mediated PVY spread include mineral oil foliar sprays, especially combined with insecticides. Spray timing is important, starting early after first plants emerge, continuing weekly through season to vine-kill, with additional sprays during rapid growth and periods of aphid abundance. Other practices for PVY reduction include avoiding mechanical transmission by field equipment, planting crop borders or intercropping as a barrier or sink for PVY, breeding PVY-resistant potato varieties, and roguing symptomatic plants from fields. Major remaining challenges in combatting PVY are management complacency, proliferation of PVY strains circumventing resistance and roguing, and informal trade of untested potatoes.

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

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.

Potato virus Y (PVY) transmission was studied in six commercial potato fields in 2015 and 2016 in New Brunswick, Canada. Plants emerged from PVY-positive tubers, or PVY-free plants that were artificially inoculated with PVY shortly after emergence, were used as PVY inoculum plants in the study. In all trials, equal numbers of PVY inoculum plants from each of three strains common in the region, PVYO, PVYN:O and PVYNTN, were used. PVY inoculated into tractor-traffic rows showed 2 to 7 times as much PVY spread to previously virus-free plants by the end of the season (up to 48.5% in one tractor row, compared to a maximum of 16.3% in a distant control row unaffected by tractor traffic). Evidence supporting a hypothesis that tractor traffic enhances PVY transmission through aphid disturbance was observed by PVY spread in both directions along the rows, not biased in the direction of tractor travel, and that the ratio of spread of the three strains was nearly indentical in control and tractor rows. However, the lack of spread to immediately adjacent rows, and statistically significant spatial pattern matching the circumference of tractor wheels specific to each field support the hypothesis that direct mechanical transmission of wounded plant sap could also be a factor in the enhanced PVY transmission.

Potato virus Y (PVY) is a major cause of yield and quality loss in potato crops worldwide. Recently, populations of PVY strains have shifted dramatically toward recombinant strains such as PVYNTN and PVYN:O. A 2010 to 2016 survey of PVY strains in commercial fields of New Brunswick (NB), Canada, and five field trials tracking PVY spread in NB and Manitoba, were conducted to study the current status of PVY strains and their relative rates of spread. In NB, PVYO dropped from 82% of infections in 2010 to 14% in 2016, replaced mostly by PVYNTN (64%) and PVYN:O (22%).In field trials with Russet Burbank and Gold rush varieties, PVYNTN spread most effectively compared to PVYN:O and PVYO. Strain-specific PVY spread varied with the potato variety, possibly due to selective PVYO resistance in Goldrush, mostly expressed at the plant-to-plant transmission level with little difference in transduction to tubers in infected plants. Relevance of in-field differences in spread of strains to changes in regional PVY populations, and potential mechanisms responsible, are discussed.

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.

In the 2014 and 2015 crop seasons, the efficacies of different types, rates and combinations of mineral oil and insecticide foliar sprays for reducing Potato virus Y (PVY) spread were tested in controlled field trials in New Brunswick (NB), Canada. Experimental plots were planted with certified PVY-free Goldrush, supplemented with known virus-infected seed to raise PVY inoculum to 2.3% and 3% at the beginning of the 2014 and 2015 seasons, respectively. Treatments consisted of mineral oil-only sprays at different application rates, insecticide-only sprays of differing numbers, and several combined mineral oil and insecticide spray regimes, all compared to a no-spray control treatment. PVY spread to 18% (2014) and 22% (2015) of initially virus-free plants in no-spray control plots, with significant reductions observed in PVY spread in several treatments. Greatest PVY reductions, as low as 4% (2014) and 12% (2015), were in combined mineral oil and insecticide spray treatments, followed by oil-only sprays; while insecticide-only sprays did not significantly reduce PVY spread. As well as measuring PVY spread to marked test plants and randomly collected post-harvest tuber sample from the plots, exhibited similar treatment pattern for PVY incidence. Multiple logistic regression modeling confirmed the relative efficacy of combined oil and insecticide sprays for reducing PVY spread, while accounting for variable inoculum and aphid factors. Modeling also highlighted the importance of planting low-PVY seed initially, and of early application of foliar sprays. Local best management practice recommendations for reduction of in-field PVY spread were discussed.

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.

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

A field isolate of Potato virus Y (PVY) was collected in New Brunswick, Canada in 2007 due to unusual symptoms observed on different potato cultivars. To unveil the PVY strain identity, tobacco and potato bioassays, PVY

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.

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.

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.

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.

Molecular markers reported to be linked to extreme resistance (ER) against Potato virus Y (PVY) were evaluated in potato germplasm. YES3-3A and YES3-3B, markers linked to Rysto, were detected in ‘Barbara’ and its three descendants that exhibit ER to PVY; RYSC3, a marker linked to Ryadg, was detected in breeding clones NY121 and NY123. Assessment of RYSC3 as a marker for selection for Ryadg-mediated ER validated its efficacy in identification of selections with ER to PVY.

In this study, on-farm within-season spread of Potato virus Y (PVY) was measured in 56 seed, processing and tablestock potato fields in New Brunswick, Canada between 2010 and 2014. These represented 13 potato varieties, managed by 16 growers employing a wide range of PVY control techniques. Many aspects of management techniques were quantified, as well local aphid abundances and climatological data. PVY spread, measured in leaves and tubers (by ELISA and RT-PCR), was tracked through the season in individually marked plants. Over the five seasons under study, on-farm PVY spread overall has declined substantially. Across all 56 fields, however, it has varied widely, from 10 fields not showing any spread during the crop season, up to as high as 76 % spread in one 2012 field. Factors correlated with increased PVY spread included seed-borne PVY inoculum planted in the field, aphid abundance early in the season, and to a lesser degree temperatures in July and over the preceding winter. Factors correlated with decreased spread included numbers of foliar mineral oil and insecticide sprays (especially of the lambda-cyhalothrin and flonicamid types), later crop planting and earlier first spraying dates. A mid-season leaf test for PVY was shown to be strongly indicative of ultimate PVY spread at harvest, and may prove a useful test in advising growers. A set of locally-relevant best management practices based on these results is discussed.

The complete genome comprising three genomic RNAs of three Canadian and two Chinese isolates of Potato mop-top virus were sequenced and analysed. Two open reading frames (ORFs) were found in RNA1 of 6.1 kb, encoding a readthrough RNA-dependent RNA polymerase (RdRp). A coat protein (CP)-readthrough protein was encoded by RNA2 of 3.1 kb. Four ORFs that encoded the triple gene block proteins (TGBps) and a cysteine-rich protein were found in RNA3 (2.9 kb) of the Chinese isolate ‘Yunnan’; whereas in the remaining isolates (three Canadian isolates and the Chinese isolate ‘Guangdong’), only three ORFs encoding TGBps were observed in RNA3. A single nucleotide mutation of A2462 to G2462 abolished the start codon ‘AUG’ for the fourth putative ORF in RNA3 of these isolates. Based on phylogenetic and sequence similarity analysis of these isolates as well as those reported by others at the complete RNA sequence level, each of RNA1, RNA2 and RNA3 could be divided into at least two groups. In Canadian isolates ‘Ch9’, ‘Ch10’ and ‘Ch20’ and Chinese isolate ‘Guangdong’, all genomic RNAs belonged to group A; and in Chinese isolate ‘Yunnan’, all of its RNA belonged to group B. Interestingly, in Swedish isolate ‘Sw’, RNA1 and RNA2 belonged to group A while RNA3 belonged to group B. A duplex RT-PCR for differentiating groups A and B of RNA3 was developed and evaluated. All PMTV samples collected in Guangdong, China, and New Brunswick, Canada, possessed a RNA3 belonging to group A; whereas the samples collected in Yunnan, China, possessed a RNA3 belonging to group B.

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.

The complete genome sequences of four new isolates of strawberry mild yellow edge virus (SMYEV) were determined and analysed. The isolates, designated as AB41-01, AB41-02, NB1165 and NS26, were found from strawberry fields showing strawberry decline symptoms in eastern Canada. AB41-01 and AB41-02 were from a single-plant sample originating from Prince Edward Island, while NB1165 came from New Brunswick and NS26 from Nova Scotia. Nucleotide sequence identities are 95.8% between AB41-01 and NB1165, 99.6% between AB41-02 and NS26, and 84% between AB41-01/NB1165 and AB41-02/NS26. The four isolates share nucleotide sequence identities of 83.5–89.9% to two previously identified SMYEV isolates, namely MY18 and D7. Phylogenetic analysis indicates that the four Canadian isolates represented two new SMYEV strain types and the strain divergences were not likely from recombination events among all presently known SMYEV isolates.

Potato virus Y (PVY) undergoes continuous nucleotide mutation and genome recombination, leading to novel strains with varying pathogenicity on host plants. Little is known about how tomato plants respond to infections with different PVY strains, especially the emerging strains such as PVYNTN and PVYN:O, and in combination with other viral species such as Potato virus X (PVX). In this study, the response of tomato (cultivar Rutgers) plants to single and mixed infections with PVY and PVX was investigated. Plants infected singly with PVYO, PVYN:O, PVYN or PVYNTN developed mosaic symptoms and leaf deformation; whereas plants infected with PVX developed mild local lesion and varying degrees of mosaic and leaf deformation symptoms. Mixed PVX+PVY infections induced more severe symptoms, which include severe local and systemic leaf necrosis, leaf drop and severe leaf deformation, compared to single infections with PVX or PVY, indicating PVX-PVY synergism. The level of PVX-PVY synergism was affected significantly by PVY strains, with PVYNTN being the greatest effector. Quantitative analysis of viral titre indicated that mixed PVX+PVY infections elevated PVX level and reduced PVY level. A comparative transcriptional analysis of 46 key genes involved in signal molecule synthesis and signal transduction using NanoString technology revealed diverse gene expression profiles in both single and mixed infections. Two hierarchical expression clusters, one up-regulated and the other down-regulated, were observed during the infections. In general, mixed infections led to more genes being differentially regulated than single infections. The expression alteration of genes involved in oxidative stress and salicylic acid (SA)-pathways is particularly noteworthy, as the mixed infections, especially PVX+PVYNTN infections, led to a further elevation of reactive oxygen species (ROS) and SA producing abilities and a reduction of ROS scavenging ability over single infections.

A preliminary screening for garlic viruses in garlic plants in Hunan, China, using existing monoplex (simplex) reverse transcription-polymerase chain reaction (RT-PCR) procedures detected four viruses/virus groups. These viruses/virus groups were Onion yellow dwarf virus (OYDV), Leek yellow stripe virus (LYSV), Shallot latent virus (SLV) and allexiviruses (e.g., garlic viruses A, B, C, D, E, X). Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D (GarV-D), which shared 92–97% sequence identities with various isolates from the world. A multiplex RT-PCR (mRT-PCR) was therefore developed to simultaneously detect and differentiate the four viruses/virus groups. To achieve this, four primer pairs targeting allexiviruses, OYDV, LYSV and SLV were designed. The anticipated amplicon sizes are 183 bp (allexiviruses), 265 bp (OYDV), 404 bp (LYSV) and 592 bp (SLV), respectively. All primer pairs produced virus-specific fragments in both simplex and multiplex formats, thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses. The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan. Allexiviruses, OYDV, LYSV and SLV were detected in 50.9, 40.3, 28.3 and 58.5% of leaf samples, respectively; and mixed infections with two or more viruses accounted for 54% of the garlic samples. The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays. In conclusion, this newly developed mRT-PCR provides a rapid, sensitive and reliable method for the detection and identification of major garlic viruses.

The transmission of PVY strains, PVYO, PVYN:O, and PVYNTN via tuber cutting and plant wounding was investigated in PVY susceptible cultivars Shepody and Russet Norkotah. For the tuber cutting experiment, after one infected tuber was cut with a knife, four uninfected tubers were cut sequentially with the same knife without disinfecting it between the cuts. In the plant wounding experiments, wounds were induced in the healthy and infected plants of Shepody by bouncing, brushing, hammering, squeezing, and Carborundum rubbing treatments. These treatments allowed exchange of sap between the healthy and infected plants. Results demonstrated that seed cutting did not transmit PVY, whereas plant wounding treatments caused varying levels of PVY transmission, depending on the wounding treatment. Plant bouncing was the least effective whereas hammering was the most effective.

Viroids are highly structured small circular RNAs that do not encode proteins but are able to replicate and cause diseases in plants. Characteristic symptoms caused by a viroid in a specific indicator host are the foundation of biological assay (bioassay) for detection and identification of the viroid. For bioassay of Solanaceae -infecting pospiviroids, a panel of indicator hosts are needed as similar types of symptoms may be induced by different viroids. For bioassay of citrus viroids, both citrus and herbaceous species are used. Many factors including environmental conditions, viroid strains, host species and genotype, and whether or not another viroid/virus is present, affect the bioassay. Combining bioassays with molecular assays for viroid detection and identification are also discussed.

International audience; As obligate parasites, plant viruses, require in order to survive, to be transmitted to another plant. Experimentally, viruses such as Potato virus Y (PVY) can be transmitted by mechanical means such as wounding and grafting. In its natural environment, PVY transmission is mediated by sap-feeding aphid vector, or vegetatively through propagated organs such as potato tubers. A vast number of aphid species have been reported to transmit PVY in a non-persistent manner with variable efficiency to a large number of solanaceous and non-solanaceous plant species including weeds and ornamentals. Several sensory stimuli will influence host selection and feeding behaviour of the aphid and will strongly influence virus epidemiology. The interactions between the virus, its vector, and the environment are complex and are the focus of many studies aiming to understand the molecular basis of these interactions and their impact on disease development. This chapter will present the current knowledge of PVY transmission, epidemiology, and its management in different countries.

Potato cultivar Exploits and breeding clone F87084 have been considered resistant to Potato virus Y (PVY). To further explore the degree of resistance and whether the resistance is strain specific, these materials, together with cultivar Rochdale Gold-Doree and breeding line F02010, were investigated for their response to different PVY strains including PVYO, PVYN:O, PVYNTN and PVYN. Both F02010 and Rochdale Gold-Doree were readily infected with all tested PVY strains after either mechanical or graft inoculation, indicating susceptibility of the materials to PVY. F87084 was unable to be infected by any of the tested PVY strains as no ELISA-detectable level of PVY were found in plants after either mechanical or graft inoculation, demonstrating extreme resistance in F87084 to all strains of PVY tested. Exploits was infected with PVYN:O/PVYNTN/PVYN after either mechanical or graft inoculation, indicating susceptibility of the cultivar to these PVY strains. The cultivar was also infected readily with PVYO after graft inoculation. However, despite induction of local lesions on the inoculated leaves, mechanical inoculation with PVYO may or may not lead to systemic symptoms and ELISA-detectable level of PVY, depending on temperature. At low temperature (e.g., 22 °C), no visible systemic symptoms or ELISA-detectable level of PVY was found in the plants; whereas at high temperature (e.g., 30 °C), systemic symptoms and high level of PVY were detected in the plants. These results demonstrate that Exploits possesses temperature-dependent hypersensitive resistance to PVYO. Analysis of a segregating population of F87084 × F02010 revealed that the ER in F87084 is controlled by a dominant resistance gene.

Potato plants that exhibited mosaic symptoms were collected in Xiangxi, Hunan province, China. Multiplex RT-PCR screening for common viruses revealed the presence of potato virus A (PVA) in these samples. ELISA with virus-specific antibodies confirmed infection by PVA in the plants. Rod-shaped virions of ~750 nm in length and ~13 nm in width were observed by transmission electron microscopy. One virus isolate (designated PVA-Hunan) was subjected to molecular characterization. The viral genome consisted of 9,567 nucleotides, excluding the poly(A) tail, and encoded a polyprotein of 3,059 amino acids. A second characteristic potyvirus open reading frame (ORF), pretty interesting Potyviridae ORF (pipo), was located at nucleotides 2,834-3,139. The isolate shared 84 % to 98 % and 93 % to 99 % sequence identity with other PVA isolates at the nucleotide and amino acid level, respectively. Phylogenetic analysis demonstrated that, within the PVA group, PVA-Hunan clustered most closely with the Finnish isolate Her, then with isolates 143, U, Ali, M and B11. The isolate TamMV stood alone at a separate branch. However, scanning of complete genome sequences using SimPlot revealed 99 %-sequence identity between PVA-Hunan and TamMV in the 3′-proximal end of the genome (~nt 9,160 to the 3′end) and a 50 %-94 % (average ~83 %) identity upstream of nt 9,160. In contrast, 98 % identity between PVA-Hunan and isolates M and B11 was detected for nucleotides 1 to ~9,160, but only ~94 % for the 3′-proximal region, suggesting a genome recombination event (RE) at nt 9,133. The recombination breakpoint also was identified by the Recombination Detection Program (RDP). The RE was further confirmed by analysis of the CP gene, where the apparent RE was located.

The current-season spread of Potato virus Y (PVY) was monitored in 19 fields under various management practices in New Brunswick, Canada, through the 2011 and 2012 growing seasons. The focus of this study was to evaluate the role of seedborne PVY inoculum, aphid vector abundance, and the numbers, timing, and types of insecticide and mineral oil sprays, and to confirm the reliability and forecasting capacity of midseason PVY testing. In each field, 100 to 110 virus-free plants were identified shortly after emergence and were assessed four times from early July to early September (after top-kill) with enzyme-linked immunosorbent assay (ELISA) and reverse-transcription polymerase chain reaction (RT-PCR) to track PVY spread. In addition, tubers harvested during development in August and after top-kill were grown-out in the greenhouse for ELISA testing. PVY spread to selected virus-free plants varied widely, ranging from 0 to 76.2% across all studied fields. Of the 19 fields over two seasons, 10 fields were planted with no detectable seedborne PVY, and they showed 0 to 8.7% (mean 2.9%) PVY spread by harvest. The remaining nine study fields with 0.9 to 5.8% seedborne PVY showed 1 to 76.2% (mean 15.2%) PVY spread by harvest. PVY spread was detected in most fields during midseason testing with ELISA and RT-PCR; all tests correlated well with final PVY rates after top-kill, though RT-PCR detection in developing tubers was most sensitive and correlated. Logistic regression modeling was used to identify major factors in PVY spread, including seedborne PVY, early-season aphid abundance, and the numbers of insecticide and mineral oil sprays. The best-fitting model, constructed using these factors as well as a measurement of July PVY incidence (ELISAJuly), strongly explained PVY spread by harvest, with the most significant management factor being the number of mineral oil sprays supplemented with insecticide used during the growing season. A similar model fitted without the ELISAJuly did not adequately predict ultimate PVY spread. The analysis suggests that mineral oil alone was effective at lowering PVY spread, and more effective when combined with insecticide, particularly when used early in the season. No evidence was found for differences in PVY spread across the eight cultivars used or across the range of mineral oil application rates, whereas some evidence was found for differences in the effectiveness of different insecticide types.

Post-harvest screening of potato ‘Kennebec’ revealed a Potato virus Y (PVY) incidence of 15.8%, a rate that is unusually high for a cultivar possessing a high level of field resistance to the virus. Randomly selected tubers were planted in a field plot and the resulting plants were monitored. Approximately 16% of plants developed symptoms ranging from mild mosaic symptoms to severe necrosis/rugosity/stunting. ELISA and RT–PCR analysis revealed that infections with Potato virus S (PVS), Potato virus X (PVX) and PVY, mostly in mixed-infections, occurred commonly in 14 sampled plants. Two strains, namely the common strain (PVY°) and the recombinant tuber necrotic strain (PVYNTN) were identified in the PVY-positive plants. In general, mild mosaic was associated with infections with PVX and PVS; intermediated mosaic was associated with PVS and PVYNTN infections; whereas severe leaf deformation/necrosis/drop symptoms were associated with PVYNTN and PVX co-infections, or with PVY° and either PVS or PVX ...

Potato tubers exhibiting necrotic rings/arcs were found in a winter potato crop ‘Favorita’ in the subtropical area Huidong county, Guangdong province, China, in 2012. When the symptomatic tubers were cut crosswise, light to dark brown necrotic arcs were observed in the tuber flesh. A mini-survey of the crop found 8% symptomatic tubers in an area of 10 meters2. To reveal the causal agent of the disease, reverse transcription–polymerase chain reactions (RT-PCR) targeting 9 common potato viruses including those viruses (e.g, Potato mop-top virus (PMTV), Tobacco rattle virus, Tomato spotted wilt virus, and Potato virus Y tuber necrosis strain) known to be capable of induction of tuber necrosis, were carried out. Except for PMTV, no other viruses were detected. Sequencing of the 460 bp amplicon revealed that the fragment exhibited a 99-100% sequence identify with most PMTV coat protein sequences deposited in GenBank, which was confirmed by a further phylogenetic analysis. Enzyme linked immunosorbent a...

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

Significant progress has been made in recent years in understanding pathological, serological and molecular properties of various strains of PVY and the aphid-mediated transmission. PVYO and PVYN appear to be the basic strain groups. Through genome recombination between these two groups, progeny groups whose genome possess one (e.g., PVYN:O or PVYN-Wi) to three (e.g., recombinant PVYNTN or European-PVYNTN) recombinant joints of PVYO and PVYN emerged. PVYN:O causes PVYN-like veinal necrosis in tobacco, but reacts to PVYO-specific antibody. PVYNTN causes potato tuber necrotic ringspot diseases in sensitive potato cultivars, and PVYN-like necrosis in tobacco plants, and reacts to PVYN-specific antibody. Through single nucleotide mutation(s), non-recombinant PVYNTN (or North American PVYNTN) also emerged from PVYN. It is also noteworthy that PVYN isolates originated from North America and Europe may have evolved separately; and to date most recombinant strains appear to be progenies of Eu-PVYN and PVYO. Several RT-PCR-based methodologies have been developed to characterize and detect various strains of PVY. A field survey revealed that PVYN:O has become a predominant strain in Manitoba and neighbouring states in USA. Moreover, three distinct variant groups inciting severe, intermediate and mild veinal/petiole/stem necrosis, respectively, on tobacco plants were observed within the PVYN:O isolates collected in Manitoba. Pathological and molecular diversity within PVYO strain group were revealed in New Brunswick, represented by PVYO-FL as a severe variant and by PVYO-RB as a mild variant. Studies on the transmission of PVY by various species of aphids revealed that aphid behavior plays an important role in the vector-mediated transmission. Application of mineral oil on the growing crop, especially in combination with use of crop borders, reduces aphid-mediated PVY transmission. Based on recent PVY research studies, Bartlett Superior 70 Oil was approved in 2011 for application to potato crops in Canada.