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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.

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.

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.

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.

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.

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.

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.

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 ...

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.

International audience; The management of Potato virus Y (PVY) in potato crops poses a con-tinual challenge due to the non-persistent mode of transmission of the virus and the propagation of seed potato tubers over several generations in the field. While PVY- resistant cultivars remain the most efficient way to protect potato crops against PVY, a vast majority of cultivars grown do not display significant resistance to PVY. Due to the short time period for PVY transmission by non-colonising aphid vectors, efficient control of PVY relies on preventing aphids landing on a crop and on adopting precautionary measures by ensuring that crops are grown in areas of low aphid and low virus pressure and limiting field generation. Prophylactic mea-sures such as roguing and early haulm destruction limit PVY spread but are not efficient alone. Among all existing control methods, spraying potato crops with mineral oils can offer significant protection against PVY spread, but their efficacy do vary in field conditions. The combination of several control methods such as mineral oil treatments, crop borders, intercropping, straw mulching or insecticide treatments can increase protection. These emphasise the importance of controlling virus through appropriate monitoring methods and crop management enforced by seed certification schemes through the use of ‘clean’ input seed and, when possible, the segregation of seed and ware crops to minimise the risk of virus transmission. This chapter presents and discusses the most widely used techniques of control and management of PVY, their effectiveness and their mode of action. This chapter also presents the history, objectives and principles of seed potato certification schemes and their role in minimising the spread of viruses within potato crops worldwide.

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.

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.

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 NO3), limited (0.75 mM NO3) or deficient (0 mM NO3) 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.

Pink rot of potatoes caused by Phytophthora erythroseptica is known to infect all underground potato (Solanum tuberosum) tissues such as roots, stolons, tubers and basal stems. Potato leaves and stems do not normally exhibit symptoms except when extensive infection of underground plant parts results in plant wilt, chlorosis and necrosis. This is the first study to investigate the spread of P. erythroseptica in above-ground potato tissues using both traditional isolation and PCR methods originally developed to detect the pathogen in tubers. P. erythroseptica was detected in 66% of leaf and stem tissue samples originated from artificially-inoculated and naturally-infected tubers of ‘Yukon Gold’ and ‘Shepody’ by PCR methods. However, it was only recovered in pure cultures from 38% of stem and leaf tissue samples. The pathogen was also detected in leaf and stem tissues and aerial tubers of plantlets grown in potting mixtures infested with P. erythroseptica. Pure cultures of P. erythroseptica obtained from stem and leaf tissues, together with the 95% of the detection with the real-time PCR assay and 45% with the conventional PCR assay confirmed the viability of the pathogen in above-ground potato tissues. Furthermore, the pathogen was detected in progeny tubers and stolons produced by the infected potato plants. P. erythroseptica was also detected in a few samples of debris taken from naturally senesced above-ground potato tissue after harvest.

Correct identification of potato cultivars and selections is essential to a large and diverse user group. This group includes curators of germplasm repositories, breeders and other researchers, certification program officials, commercial growers, processing industry managers and for some cultivars, the public. Agencies involved in cultivar registration and plant breeders' rights (or patenting) also have a vested interest in correct identification. DNA fingerprinting is an important tool that can be used to describe new or existing cultivars, verify cultivar identity, and resolve cultivar mixtures. Gel-based fingerprints are usually preferred because they are visual and within the technical capacity of most molecular laboratories. In this study, a multiplex PCR protocol "Multiplex SUP" and an improved version "Multiplex SUPN" were developed using four primer pairs (STEM0014 and genes of starch synthase, patatin, and UDP-glucose pyrophosphorylase). The agarose-gel-based Multiplex SUP was successfully used in identifying cultivars from blind samples in a collaborating laboratory, and in pilot tests to verify the identity of introduced cultivars for seed potato production. The Multiplex SUPN, using native polyacrylamide gel electrophoresis (PAGE) with GelRed or ethidium-bromide staining, generated more than 38 polymorphic markers among the potato cultivars tested. The method distinguished 116 cultivars that included many of the public potato cultivars registered in Canada and several protected cultivars that were fingerprinted with permission. The Multiplex SUPN-PAGE method is user friendly and effective, and is recommended for routine potato cultivar verification and identification. Key words: cultivar identification, database development, DNA fingerprinting, GelRed staining, genotyping, polymorphism, Solanum tuberosum L.

Graft inoculation of potato (Solanum tuberosum) ‘Shepody’ plants with Potato virus A (PVA) results in hypersensitive resistance responses (HR) in tubers with characteristic necrosis. Increasing the number of PVA-containing scions or increasing the duration of scions on the ‘Shepody’ rootstock plants resulted in an increased number of tubers showing necrosis. Transgenic ‘Shepody’ plants with a sense or an antisense copy of PVA coat protein (CP) gene under the direction of the CaMV 35S promoter were developed. The plants were subjected to graft inoculation with PVA in the greenhouse and were monitored by detecting PVA using enzyme-linked immunosorbent assays and reverse transcriptase polymerase chain reaction during the growing season. No PVA was detected in the wild type or the antisense-CP transgenic lines. However, PVA was detected from one of the two sense-CP transgenic lines. Potato tubers from graft-inoculated plants were harvested 3 months after inoculation and were examined for necrosis. In...

A rapid and simple protocol for preparing viral RNA from aphids ( Myzus persicae ) and potato ( Solanum tuberosum L.) tissue (leaves, sprouts, and tubers) for reverse transcription–polymerase chain reaction (RT–PCR) was developed. The four-step method involves: (1) preparing plant crude sap or aphid macerates in a buffered detergent (Triton XL-80N) solution; (2) immobilizing clarified sap on a nitrocellulose membrane; (3) performing reverse transcription using eluted water extract from cut-out spot from membrane; and (4) amplifying cDNA through PCR. The entire procedure from tissue grinding to RT–PCR can be completed within 6 h. The protocol was applied successfully for the detection of individual potato viruses: carlavirus Potato virus S (PVS), potexvirus Potato virus X (PVX), potyvirus Potato virus Y (PVY), and polerovirus Potato leafroll virus (PLRV). PLRV was also detected from individual viruliferous aphids or composites of viruliferous and healthy aphids. PVY and PLRV were detected from extracts immobilized on nitrocellulose membranes, stored for more than 65–273 days at room temperature (25 °C). The protocol was companed with the ‘long protocols’ involving enzyme and phenol extraction for aphids or sodium sulfite and phenol extraction for tubers. The simplified protocol was found comparable in sensitivity to these long procedures, and is especially suitable for those regions where specialized PCR laboratories are only available in centralized locations. Viral RNA immobilized membranes can be mailed out for detection by RT–PCR to these centralized laboratories from remote areas of the country.

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.

The rising ambient temperature by plant cells is crucial for the timely activation of various molecular defences before the appearance of heat damage. The heat-threshold level varies considerably at different developmental stages. With a view to survive under heat stress, mechanisms of regulation at the molecular level enable plants to prosper. Traditional breeding contributed for improving heat tolerance meagrely. The genetic transformation approach needs to be accelerated that can mitigate harmful effects by developing improved thermotolerance of crop plants. In this background, a thorough understanding of physiological responses of plants to high temperature, mechanisms of heat tolerance and possible strategies is vital. Temperature changes are sensed through cellular responses due to signal transduction into the cell. A key ingredient for plants temperature sensing ability is a specialized histone protein, dubbed H2A.Z that wraps DNA into a more tightly packed structure. Further, metabolic reactions and internal conductance to CO 2 decrease Rubisco activation, inhibition of Rubisco activase and subsequently slowing down net photosynthesis (Pn). Importance of internal conductance to CO 2 , heat inducible transcription factor for heat shock response (HSR) involve different HSR genes. The mechanisms, which are regulated at the molecular level, facilitate plants to thrive under heat stress. Exogenous applications of protectants such as osmoprotectants, phytohormones, signaling molecules, trace elements etc. have shown beneficial effects on plants growing under high temperature, due to the growth promoting and antioxidant activities of these compounds. There is acute necessity of crop biotechnology research for evolving genetically modified (GM) crops for a high CO 2 world at this juncture. So, attaining thermotolerance is a lively process to escape damages caused by heat stress.

Detection of the common strain of PVY (PVYO) in field-grown dormant potato tubers, by nucleic acid spot hybridization (NASH) and reverse-transcription polymerase chain reaction (RT-PCR) was compared with diagnosis by visual symptoms and enzyme linked immunosorbent assay (ELISA) on leaves of eight potato cultivars, differing in their symptom expression and virus content. NASH and RT-PCR readily detected PVYO in dormant tubers. The percent agreement between NASH and RT-PCR ranged from 77.6 to 100 for cvs. included in this study (AC Novachip, Atlantic, Norchip, Red Pontiac, Russet Burbank, Russet Norkotah, Shepody and Superior). ELISA on leaves was almost as good as NASH and RT-PCR on dormant tubers, but required dormancy break. Only two cultivars, namely Red Pontiac and Russet Burbank, could accurately be assessed, based on the visual symptoms. A very high degree of discrepancy was observed for PVYO diagnosis between visual symptoms and other detection procedures for varieties like Russet Norkotah and Shepody, which are known to be latent carriers of PVYO. The use of NASH and RT-PCR in large-scale post-harvest testing is feasible.

Polyphenolics are a widely distributed class of plant constituents implicated in the inhibition of the polymerase chain reaction (PCR). Since chlorogenic acid forms the bulk of phenolics in the potato tuber, a range of chlorogenic acid concentrations were tested for inhibition of reverse transcription (RT) and PCR steps. The RT step was inhibited at chlorogenic acid concentrations of 6.0 to 7.5 microg, while the PCR step was inhibited at much lower concentrations of 1.2 to 1.8 microg. Incorporation of 1.2% citric acid at the nucleic acid extraction step prevented darkening of the extract and neutralized the inhibitory effect of chlorogenic acid.

Three Canadian isolates of potato virus X (PVX) that produced distinctly different symptoms in tobacco (Nicotiana tabacum cv. Samsun) were analyzed for differences in their coat proteins using SDS-PAGE, denaturing isoelectric focusing, peptide analysis and nucleotide sequencing. The amino acid sequence of the coat protein in a mild isolate differed from that of isolates that produced severe or intermediate symptoms at two locations. However, the amino acid sequences of the Canadian isolates that produced severe or intermediate symptoms were identical and were the same as the coat proteins of PVX isolates from Argentina, China, The Netherlands, and United Kingdom. These results suggest that regions of the PVX genome in addition to the coat protein gene may be involved in viral-host interactions.

(1996). Nucleotide sequence and genome organization of a Canadian isolate of the common strain of potato virus Y (PVY°) Canadian Journal of Plant Pathology: Vol. 18, No. 3, pp. 209-224.

A reverse transcription polymerase chain reaction (RT-PCR) protocol was developed using two 20-mer primers located in nuclear inclusion genes NIa and NIb of potato virus Y (PVY). A 1017 bp PCR-product was detected in dormant potato tubers, infected with PVY O , but not in tubers from healthy plants. The PCR product was specific to PVY, as determined by Southern blot detection by hybridization with a PVY O -specific probe. As little as 1 pg of purified PVY O -RNA can be detected after RT-PCR amplification. The presence of phenolics or polysaccharides in tuber nucleic acids inhibited PVY O amplification, which was eliminated by diluting nucleic acid preparations prior to cDNA synthesis, modifying the nucleic acid extraction procedure by isopropanol precipitation and using phosphate-buffered saline-Tween in the cDNA mix. Potato cultivars differed in PVY O concentration in tubers as much as 128-fold. Tuber parts used for nucleic acid extractions were important in potato cultivars with low virus titres and combining of saps from bud and stem ends improved the PVY O detection. Storage of tubers up to 90 days at 4 °C did not result in reduced detection of PVY O by both nucleic acid spot hybridization and RT-PCR, but RT-PCR band intensity was lower at longer storage periods. The primer pair developed in this study exhibited broad specificities with field isolates from Peru, Scotland and North America.

An isolate (Y139) of the potato virus Y strain PVYO, common in the potato-growing regions of eastern Canada, was cloned and segments of about 1800 bases from the 3'-end of the PVY-genome were sequenced. These clones were used to prepare molecular detection probes by labelling with digoxigenin. Five methods reported to yield nucleic acid preparations from tuber tissue were compared. Two of the methods failed to yield nucleic acids from tubers suitable for use in dot-blot hybridization. The other three methods yielded satisfactory nucleic acids which could be successfully detected with the PVYO probes. A method of nucleic acid extraction from tuber tissues was selected which is simple, reproducible and does not use phenol. Using this standardized procedure, PVY was detected in freshly harvested, greenhouse-grown tubers of 8 potato cultivars; the probe was specific to PVY; and the sensitivity of detection using purified viral RNA was down to 10 pg. The sensitivity of detection in tuber extracts was down to a dilution of 1:64, while that from potato leaves was to a dilution of 1:1024. Nucleic acids prepared by the standardized procedure and denatured prior to storage at 4 degrees C, -20 degrees C or at -70 degrees C remained suitable for hybridization for over 2 weeks.

Solanum microdontum (PI 558098) develops diagnostic symptoms when inoculated with plant sap containing potato virus A (PVA). The symptoms consist of local lesions (2–4 mm), followed by systemic necrosis of leaf veins, bronzing of leaf surface, and leaf drop. Symptoms develop in a temperature range of 20–29 C with 4 to 8 Klux light intensity of 14 h daylength. Local lesions are visible within 1 wk of inoculation and can be caused by PVA containing sap dilutions of up to 1:100. Plants multiplied by shoot cuttings as well as those produced from true potato seed are equally sensitive. True potato seed production occurs under normal greenhouse conditions in cross-pollinated plants.