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.

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.

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.

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.

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.

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.

Increased sensitivity of RT-PCR for Potato virus Y detection using RNA isolated by a procedure with differential centrifugation.

The sensitivity of reverse transcription-polymerase chain reaction (RT-PCR) for virus detection is influenced by many factors such as specificity of primers and quality of templates. These factors become extremely important for successful detection when virus concentration is low. Total RNA isolated from Potato virus Y (PVY)-infected potato plants using the sodium sulfite RNA isolation method or RNeasy plant mini kit contains a high proportion of host RNA and may also contain trace amount of phenolic and polysaccharide residues, which may inhibit RT-PCR. The goal of this study was to enhance the sensitivity of PVY detection by reducing host RNA in the extract by differential centrifugation followed by extraction using an RNeasy mini kit (DCR method). One-step RT-PCR had relatively low amplification efficiency for PVY RNA when a high proportion of plant RNA was present. SYBR Green-based real time RT-PCR showed that the RNA isolated by the DCR method had a higher cycle threshold value (Ct) for the elongation factor 1-α mRNA (Ef1α) of potato than the Ct value of the RNA extracted using the RNeasy plant mini kit, indicating that the DCR method significantly reduced the proportion of potato RNA in the extract. The detectable amount of RNA extracted using the DCR method was <0.001ng when plant sap from 10 PVY-infected and PVY-free potato leaflets in a 1.5:100 fresh weight ratio was extracted, compared with 0.01 and 0.02ng of RNA using the RNeasy plant mini kit and sodium sulfite RNA isolation methods, respectively.

Host recovery and reduced virus level in the upper leaves after Potato virus Y infection occur in tobacco and tomato but not in potato plants.

In this study, the recovery phenomenon following infection with Potato virus Y (PVY) was investigated in tobacco (Nicotiana tobaccum), tomato (Solanum lycopersicum) and potato (Solanum tuberosum) plants. In tobacco plants, infection of severe strains of PVY (PVYN or PVYN:O) induced conspicuous vein clearing and leaf deformation in the first three leaves above the inoculated leaves, but much milder symptoms in the upper leaves. The recovery phenotype was not obvious in tobacco plants infected with PVY strain that induce mild symptoms (PVYO). However, regardless of the virus strains, reduction in PVY RNA levels was similarly observed in the upper leaves of these plants. Removal of the first three leaves above the inoculated leaves interfered with the occurrence of recovery, suggesting that the signal(s) mediating the recovery is likely generated in these leaves. In PVYN or PVYN:O but not in PVYO-infected tobacco plants, the expression of PR-1a transcripts were correlated with the accumulation level of PVY RNA. Reduced level of PVY RNA in the upper leaves was also observed in infected tomato plants, whereas such phenomenon was not observed in potato plants. PVY-derived small RNAs were detected in both tobacco and potato plants and their accumulation levels were correlated with PVY RNA levels. Our results demonstrate that the recovery phenotype following PVY infection is host-specific and not necessarily associated with the expression of PR-1a and generation of PVY small RNAs.

RT-PCR and real-time RT-PCR methods for the detection of potato virus Y in potato leaves and tubers.

Potato virus Y (PVY) is a major threat to potato crops around the world. It is an RNA virus of the family Potyviridae, exhibiting many different strains that cause a range of symptoms in potato. ELISA detection of viral proteins has traditionally been used to quantify virus incidence in a crop or seed lot. ELISA, however, cannot reliably detect the virus directly in dormant tubers, requiring several weeks of sprouting tubers to produce detectable levels of virus. Nor can ELISA fully discriminate between the wide range of strains of the virus. Several techniques for directly detecting the viral RNA have been developed which allow rapid detection of PVY in leaf or tuber tissue, and that can be used to easily distinguish between different strains of the virus. Described in this chapter are several protocols for the extraction of RNA from leaf and tuber tissues, and three detection methods based upon reverse-transcription-PCR (RT-PCR). First described is a traditional two-step protocol with separate reverse transcription of viral RNA into cDNA, then PCR to amplify the viral cDNA fragment. Second described is a one-step RT-PCR protocol combining the cDNA production and PCR in one tube and one step, which greatly reduces material and labor costs for PVY detection. The third protocol is a real-time RT-PCR procedure which not only saves on labor but also allows for more precise quantification of PVY titre. The three protocols are described in detail, and accompanied with a discussion of their relative advantages, costs, and possibilities for cost-saving modifications. While these techniques have primarily been developed for large-scale screening of many samples for determining viral incidence in commercial fields or seed lots, they are also amenable to use in smaller-scale research applications.

Molecular characterization of a Chinese isolate of potato virus A (PVA) and evidence of a genome recombination event between PVA variants at the 3'-proximal end of the genome.

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.

Effects of Crop Management Practices on Current-Season Spread of Potato virus Y.

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.

Analysis of sequence polymorphism and population structure of tomato chlorotic dwarf viroid and potato spindle tuber viroid in viroid-infected tomato plants.

The sequence polymorphism and population structure of Tomato chlorotic dwarf viroid (TCDVd) (isolate Trust) and Potato tuber spindle viroid (PSTVd) (isolate FN) in tomato plants were investigated. Of the 9 and 35 TCDVd clones sequenced from 2 different TCDVd-infected plants, 2 and 4 sequence variants were identified, respectively, leading to a total of 4 sequence variants of 360 nucleotides in length. Variant I was identical to AF162131, the first TCDVd sequence to be reported, and the rest exhibited 1 to 3 nucleotide differences, all in the T(R) domain, from AF162131/variant I. Of the 33 and 29 PSTVd clones sequenced from 2 different PSTVd-infected plants, 8 and 9 sequence variants were found, respectively, leading to a total of 15 variants ranging in length from 356 to 359 nucleotides. The variant I was identical to EF044303, a PSTVd reported in Russia. The rest exhibited 1 to 11 nucleotide differences scattering in all five domains from EF044303/variant I. The results demonstrated for the first time that TCDVd, like many other viroids including PSTVd, exists in host plants as a collective group comprised of various sequence variants. However, in comparison to PSTVd, TCDVd is less polymorphic in tomato plants as fewer variants and lower haplotype/nucleotide diversities were observed.

Molecular characterization and detection of recombinant isolates of potato virus Y from China.

Although potato virus Y (PVY) is one of the most economically important pathogens of potatoes in China, few studies have been carried out to characterize the virus in that country. Using reverse transcription-polymerase chain reaction (RT-PCR)-based genotyping developed previously, two types of recombinant PVY were identified in China for the first time. One resembled the European (Eu) type of potato tuber necrosis strain (Eu-PVY(NTN)), possessing three widely recognized recombinant joints (RJs 1-3) of the common strain (PVY(O)) and the Eu- type tobacco veinal necrosis strain (Eu-PVY(N)). The other, on the other hand, appeared to have only RJ1 and RJ2. The complete genome of a representative isolate, PVY-HN2, from the second type was subsequently sequenced. Comparison of the sequence of 'HN2' with those of PVY(O) and Eu-PVY(N) not only confirmed the recombinant nature of 'HN2' but also revealed the existence of three recombinant events in the isolate, similar to that in PVY(NTN)-Hun. However, the two isolates differed significantly at RJ1 (PVY(NTN)-Hun vs. HN2, nt 2419 vs. nt 2521) and RJ3 (PVY(NTN)-Hun vs. HN2, nt 9183 vs. nt 8572) and slightly at RJ2 (PVY(NTN)-Hun vs. HN2, nt 5844 vs. nt 5867). A primer pair was developed to facilitate the detection of the alternative RJ3. Using the newly and previously designed RJ primers, all targeted RJs were detected. Interestingly, tests of the available PVY samples indicated that two were doubly infected with both types of recombinant PVY, further confirming the effectiveness of the detection. Further analysis of these samples using enzyme-linked immunosorbent assay and bioassay revealed that 'HN2' possesses a PVY(O) serotype, a PVY(N) pathotype in tobacco and a PVY(NTN) pathotype in potato.

Development of universal primers for detection of potato carlaviruses by RT-PCR.

To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two sense primers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained.