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Nanayakkara U.N.,Agricultural Certification Services Inc. | Singh M.,Agricultural Certification Services Inc. | Peters R.D.,Agriculture and Agri Food Canada
American Journal of Potato Research | Year: 2010

Pink rot caused by Phytophthora erythroseptica is found in most major potato-growing regions of the world. The pathogen can survive for many years in soil by means of oospores which are disseminated from diseased potato tissues. The ability to detect the pathogen in soil could be a valuable management tool that enables growers to plan control strategies depending on the presence of pathogen propagules in a particular field. However, soils are one of the most challenging environmental matrices to obtain microbial DNA that will support PCR. A method was developed that combined traditional baiting technique with PCR methods to detect P. erythroseptica in infested soil samples. Hairy nightshade (Solanum sarrachoides Sendt.) and bitter nightshade (Solanum dulcamara L.) two leaf stage (TLS) seedlings and cotyledon leaves successfully baited P. erythroseptica from zoospore suspensions, artificially inoculated soils and naturally infested soils. The pathogen was detected in the bait tissue with PCR methods. PCR increased the precision of the bait test. However, time was still required for the pathogen to infect and develop on the bait tissues. Although P. erythroseptica was detected from some bait plants only after 2 days of incubation, 10 days of incubation produced consistent results across the replicates with hairy and bitter nightshade cotyledon leaves and TLS seedlings. © Potato Association of America 2009.


Hu X.,Hunan Agricultural University | Dickison V.,Agriculture and Agri Food Canada | Lei Y.,Hunan Agricultural University | He C.,Hunan Agricultural University | And 4 more authors.
Canadian Journal of Plant Pathology | Year: 2016

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. The contribution of Virginia Dickison and Xianzhou Nie is © 2016 Her Majesty the Queen in Right of Canada, as represented by Agriculture and Agri-Food Canada (AAFC). Xinxi Hu, Yan Lei, Changzheng He, Mathuresh Singh, Yanli Yang and Xingyao Xiong hereby waive their right to any copyright in the Article but not their right to be named as co-Authors of the Article.


Singh M.,Agricultural Certification Services Inc. | Singh R.P.,Agriculture and Agri Food Canada | Fageria M.S.,Agricultural Certification Services Inc. | Nie X.,Agriculture and Agri Food Canada | And 2 more authors.
American Journal of Potato Research | Year: 2013

A TaqMan real-time reverse transcription-PCR (real-time RT-PCR) procedure was developed, optimized, and compared with other routine methods to detect Potato virus Y (PVY) in dormant potato tubers. Three pairs of primers and probes were designed and evaluated for their suitability to facilitate the real-time RT-PCR detection of PVY for all strain groups including PVYO, PVYN, PVYN:O (= PVYN-Wi) and PVYNTN. Among the primer and probe combinations tested, the combination PVY-1 produced the lowest threshold cycle (Ct) value of 25. 75. The procedure was further optimized by adjusting various parameters including primer/probe concentration, reaction volume, amplification cycles, and master mixes from different sources. The real-time RT-PCR was then employed to detect PVY from dormant tubers of different cultivars and potato fields, and the results were compared with those obtained from conventional RT-PCR, enzyme-linked immunosorbent assay (ELISA) on sprouts and grow-out testing. Out of 1,069 single-virus infected (PVY only) tubers tested, both formats of RT-PCR detected PVY in 52 samples, ELISA on sprouts in 45, ELISA on leaves in 54 and visual observations in 53. However, in 61 multiple-virus infected tubers tested, both formats of RT-PCR, and ELISA on both sprouts and leaves detected a similar number of positives, thus, making all the methods equally sensitive. Considering that ELISA requires sprouting of dormant potato tubers for PVY testing, grow-out testing takes approximately 6-8 weeks to obtain results, and conventional RT-PCR needs post-PCR processing, real-time RT-PCR offers a speedy alternative for large scale detection of PVY from dormant tubers. The method is therefore recommended for testing of PVY in potato tubers on a commercial scale in a diagnostic laboratory. © 2012 Potato Association of America.


Fageria M.,Agricultural Certification Services Inc | Nie X.,Agriculture and Agri Food Canada | Gallagher A.,Agricultural Certification Services Inc | Singh M.,Agricultural Certification Services Inc
American Journal of Potato Research | Year: 2015

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. © 2014, The Potato Association of America.


MacKenzie T.D.B.,Agricultural Certification Services Inc. | Fageria M.S.,Agriculture and Agri Food Canada | Nie X.,Agriculture and Agri Food Canada | Singh M.,Agricultural Certification Services Inc.
Plant Disease | Year: 2014

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 enzymelinked 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. © 2014 The American Phytopathological Society.

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