Agricultural Certification Services

Fredericton, Canada

Agricultural Certification Services

Fredericton, Canada
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Nie X.,Agriculture and Agri Food Canada | Sutherland D.,Agriculture and Agri Food Canada | Dickison V.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services | And 3 more authors.
Phytopathology | Year: 2016

Sequence analysis of the chromosome region harboring the sequencetagged 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 x 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. © Her Majesty the Queen in Right of Canada, as represented by the Minister of Agriculture and Agri-Food Canada, 2016.


Singh R.P.,Agriculture and Agri Food Canada | Dilworth A.D.,Agriculture and Agri Food Canada | Ao X.,Hunan Agricultural University | Singh M.,Agricultural Certification Services | Misra S.,University of Victoria
European Journal of Plant Pathology | Year: 2010

Tomato chlorotic dwarf viroid (TCDVd) manually inoculated to transgenic (cv.'Desiree') potato plants containing antimicrobial cationic peptides failed to develop symptoms in above ground plant parts, but infected tubers were symptomatic. Plants from the infected tubers (second generation plants) emerged as either severely stunted (bushy stunt isolate, BSI) or tall and symptomless. Molecular characterization of BSI isolates showed TCDVd sequence variants 95 to 98% identical to TCDVd sequences from the database, while a viroid variant identical to TCDVd type isolate (acc # AF162131) was cloned from symptomless plants. The TCDVd BSI variants had novel U165C, GU177-178AA, and UCAC181-184CUUU nucleotide substitutions in the terminal right (TR) domain of the viroid molecule. The cloned viroid cDNAs of the BSI were infectious to experimental (cv. 'Sheyenne') tomato plants causing stunted plants with profuse auxiliary shoots. Visual evaluation of the susceptibility of the BSI to 18 potato and 21 tomato cultivars revealed severe symptoms in most cultivars of both species. The progeny variants accumulating in each potato and tomato cultivar exhibited the same novel TR domain in most cultivars, with only a slight variation in a few. The severity of the stunting symptoms induced by TCDVd from BSI isolates in both potato and tomato cultivars has not been noted previously with other TCDVd isolates and, as such, it is proposed that this new isolate be recognized as a distinct genotype. Emergence of this type of sequence variant in commercial fields or commercial tomato greenhouses could potentially cause relevant losses in both crops. © 2009 KNPV.


Li X.-Q.,Agriculture and Agri Food Canada | Sveshnikov D.,BioAtlantech | Zebarth B.J.,Agriculture and Agri Food Canada | Tai H.,Agriculture and Agri Food Canada | And 4 more authors.
American Journal of Potato Research | Year: 2010

In-season chemical or optical measures of crop N status can be effective tools in optimizing potato fertilizer N management. The feasibility of using a gene expression as an alternative approach for early detection of potato nitrate deficiency was examined using three potato cultivars (Shepody, Russet Norkotah, and Red Pontiac) with abundant (7.5 mM NO 3), limited (0.75 mM NO 3) or deficient (0 mM NO 3) nitrate supply in nutrient culture over a 7 d period. RNA was extracted from the last fully expanded leaf and quantified using realtime RT-qPCR. Reduced nitrate supply had no measurable effect on shoot dry weight or leaf chlorophyll concentration, but decreased petiole nitrate concentration. Under deficit nitrate supply, down-regulation of nitrate reductase and nitrite reductase was measured within 3 days for all cultivars, and down-regulation of asparagine synthetase was measured in two cultivars. Nitrate supply had no effect on expression of ammonium transporter. In this experimental system, plant gene expression markers detected a reduction of nitrate supply prior to measureable reductions in plant growth or in N status measured using common chemical or optical methods. © Potato Association of America 2009.


Fageria M.S.,Agricultural Certification Services | Singh M.,Agricultural Certification Services | Nanayakkara U.,Agriculture and Agri Food Canada | Pelletier Y.,Agriculture and Agri Food Canada | And 2 more authors.
Plant Disease | Year: 2013

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. © 2013 The American Phytopathological Society.


Nie B.,Huazhong Agricultural University | Nie B.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services | Murphy A.,Agriculture and Agri Food Canada | And 3 more authors.
Plant Disease | Year: 2012

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. © 2012 Department of Agriculture and Agri-Food, Government of Canada.


Nie X.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services
Canadian Journal of Plant Pathology | Year: 2013

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 (PVY NTN) 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 co-infections. Virus-free plantlets of potato 'Kennebec' were mechanically inoculated with PVX, PVY°, and PVYNTN alone or with PVX+PVY° or PVX+PVY NTN combinations in the greenhouse. Single infections with PVY° or mixed-infections with PVX+PVY° or PVX+PVYNTN incited severe mosaic symptoms and systemic necrosis soon after inoculation; whereas single infections with PVX and PVYNTN induced mild to intermediate mosaic symptoms only. The most severe symptoms occurred in the mixed-inoculation with PVX+PVYNTN, demonstrating dramatic synergism between these strains. Similarly, profound PVX and PVYNTN synergism was also found in tobacco and Physalis floridana plants, suggesting that the strain of PVY plays an important role in the level of synergistic reactions between PVX and PVY on host plants. © 2013 The contribution of Xianzhou Nie.


Nie X.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services | Pelletier Y.,Agriculture and Agri Food Canada | McLaren D.,Agriculture and Agri Food Canada
American Journal of Potato Research | Year: 2013

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. © 2012 Potato Association of America.


Nanayakkara U.N.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services | Pelletier Y.,Agriculture and Agri Food Canada | Nie X.,Agriculture and Agri Food Canada
American Journal of Potato Research | Year: 2012

The strain diversity and variant population of Potato virus Y (PVY) was investigated in potato lots with high PVY incidence in 2009 in New Brunswick, Canada, using reverse transcription (RT)-PCR, enzyme-linked immunosorbent assay (ELISA) and biological assays. Twenty lots with PVY infection rates < 3% were chosen following post-harvest testing for further investigation. Assays using multiplex RT-PCR revealed the existence of the common (PVY O), the recombinant N:O (PVY N:O) and the recombinant (European) potato tuber necrosis (Eu-PVY NTN) strains. PVY O was the predominant strain while PVY N:O and Eu-PVY NTN were widespread in these potato lots. PVY N:O and Eu-PVY NTN, either present alone or mixed with PVY O, were found in 19 and 13 of the 20 lots, respectively. Neither the tobacco veinal necrosis strain (PVY N) nor the non-recombinant (North American) PVY NTN was detected in the potatoes. Among the PVY O strain group, PVY O-Oz/-FL variant type was the predominant followed by the uncharacterized PVY O and the PVY O-139/-RB types. Biological assays of representative PVY positive-samples using tobacco and Yukon Gold were consistent with the findings of RT-PCR and serological assays. © 2012 Potato Association of America.


Nie B.,Agriculture and Agri Food Canada | Nie B.,Huazhong Agricultural University | Singh M.,Agricultural Certification Services | Sullivan A.,Plant Propagation Center | And 3 more authors.
Plant Disease | Year: 2011

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, PVYO and PVYN-specific antibody-based enzyme-linked immunosorbent assays, and reverse-transcription polymerase chain reaction (PCR)-based genotyping were carried out. All the assays demonstrated that the isolate, designated as PVYO-FL in this study, belonged to the PVYO strain group. Greenhouse tests with the potato cvs. FL 1533 and Jemseg confirmed the severe nature of infection by PVYO-FL. The complete genome sequences of PVYO-FL and PVYO- RB, the latter a mild PVYO isolate, were determined. BLAST analysis revealed that the two isolates shared 97 and 98% sequence identities at the nucleotide and polyprotein levels, respectively. Further BLAST analysis unveiled that PVYO-FL shared 99.7% nucleotide sequence identity with PVYO-Oz, an isolate reported in New York, United States, whereas the PVYO-RB isolate shared 99.2% sequence identity with PVYO-139, a PVYO isolate reported in New Brunswick, Canada. A phylogenetic tree of available, full-length sequences of PVY isolates demonstrated two subgroups within the PVYO branch, one clustered with PVYO-RB and the other with PVY O-FL. Group-specific sense primers for differentiation of the two subgroups were developed and evaluated. A limited survey of potato tubers collected from a field plot at the Potato Research Centre, Agriculture and Agri-Food Canada, using the newly developed PCR primers, indicated that 65.3 and 2.4% of the PVYO-positive tubers were infected with PVYO isolates belonging to the PVYO-FL and PVYO-RB subgroups, respectively. Assessment of the pathogenicity of three representative isolates from each subgroup on the potato cv. Jemseg demonstrated that severe and mild symptoms were induced by the PVYO-FL-like and PVYO-RB-like isolates, respectively.


PubMed | Agricultural Certification Services
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2014

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.

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