Detection of potato viruses A,M, S,X, Yand leafroll and potato spindle tuber viroid from tissue culture plantlets using single leaf discs

Using enzyme-linked immunosorbent assay (ELISA) and dotimmunobinding assay (DIBA) for potato viruses A (PVA), M (PVM), S (PVS), X (PVX), Y^N (PVY^N), Y^O (PVY^O) and leafroll (PLRV) and nucleic acid spot hybridization (NASH) for potato spindle tuber viroid (PSTVd), virus and viroid were detected reliably from single leaf discs (6 mm) of tissue-culture plantlets. Leaf discs taken from leaf positions (1 to 8) (bottom to top) can be used for detection of all viruses except PLRV where the lower leaves had higher concentrations of virus than the leaves from the upper part of the plantlet. Virus cultures were maintained for 1 to 4 years in several potato cultivars. The levels of virus remained reproducible except for PVM concentration, which was found to be very low in cv. Green Mountain. Using densitometry software, the DIBA spots were quantified and results were comparable to A₄₀₅ values obtained by ELISA. PSTVd concentration as measured by densitometry from spots of NASH indicated no loss of viroid over 1–4 years in tissue culture in two potato cultivars.     

Purple top roll disease of potato

A graft transmissible disease of potato termed purple top roll is prevalent in several potato varieties grown in the hilly tracts of India. The common symptoms are rolling of the basal parts of younger leaves, sometimes accompanied by pink or purple pigmentation. The plants are stunted with profuse growth of axillary branches and aerial tubers. The losses in yield varied from 40 to 70%. Five to 20% of the tubers from diseased plants developed hairy sprouts. Tuber transmission of the pathogen varied from 6 to 33%, depending on the variety.Datura, tomato and white clover are among susceptible plants.N. rustica was tolerant or a symptomless carrier. Sunnhemp, Petunia andSesamum were immune to the pathogen, which was transmitted by the leaf hopper,O. albicinctus Distant but not byCuscuta spp. or by sap inoculation. Application of Chloramphenicol, Tetracycline and Antiamoebin resulted in remission of symptoms and increased tuber yields, indicating possible association of a mycoplasma-like organism.     

Association of viral,bacterial and fungal pathogens with vascular discoloration of Russet Burbank potato tubers

Russet Burbank potatoes from the 1979 and 1980 crop years, collected from Chicago, IL repack warehouses and retail markets, were sampled for vascular discoloration. The amount of discoloration varied among sampling months and states of origin and decreased from 7.7% in 1979 to 1.6% in 1980. Highest levels of vascular discoloration were detected in December and January samples. Vascular discolored and non-discolored (control) tubers were assayed for the presence of potato leafroll, potato virus X, and beet western yellows viruses by the enzyme-linked immunosorbent assay (ELISA). Tubers were also assayed for 2 subspecies ofErwinia carotovora and forVerticillium albo-atrum andV. dahliae. Potato leafroll virus was detected in 31 of 831 vascular discolored tubers.V. albo-atrum was detected in 1 of 180 discolored tubers. Beet western yellows virus was not detected in discolored or non-discolored tubers. Two subspecies ofE. carotovora and potato virus X were equally common in discolored and non-discolored tubers.     

Enzyme-linked immunosorbent assay with single or combined antisera for viruses S and X in potato tubers and plants

Enzyme-linked immunosorbent assay (ELISA), with single or combined antisera was effective for diagnosing potato virus S (PVS), potato virus X (PVX) or both viruses in plants grown in the greenhouse or field. In dormant tubers, stolon, middle and apical end composite sampling with or without eyes and sprouted tubers produced reliable positive assays for PVX. Only tuber pieces with sprouts resulted in consistently reliable assays for PVS. Composite sampling of potato foliage was effective for detecting one PVX infected plant in a total of 100 Kennebec, Norland, or Russet Burbank plants. There were some false negative results and greater variability in composite PVS assays, but on average, one PVS infected plant can be detected in composites of 10 Kennebec, Norland, or Russet Burbank plants. Sodium diethyldithiocarbamate (0.01M NaDIECA) in phosphate buffered saline + 0.5% Tween (PBS-T) added to plant extracts enhanced specific reactions for either virus. Onceor twiceused enzyme conjugate was effective in ELISA of either virus from potato foliage.     

Effect of time of inoculation with potato leafroll virus on development of net necrosis and stem-end browning in potato tubers

The Green Mountain cultivar was used in field tests to determine the effects of inoculating potato plants at various times with the potato leafroll virus (PLRV) on development of internal necrosis of tuber tissue. Viruliferous apterae of the green peach aphid,Myzus persicae (Sulz.), were placed on each stem in all hills to be inoculated in each 3.0 m single-row plot. Planting and inoculation dates were varied in all field experiments and, in one, several vine-killing dates were also included. All harvested tubers were stored for approximately four months at 10°C to enhance development of internal necrosis prior to examination. Similar but smaller greenhouse studies involving both apterous and alate green peach aphids were also conducted using Green Mountain, Irish Cobbler, and Russet Burbank cultivars. All results showed that as inoculation was delayed relative to plant development, more net necrosis (NN) occurred. Conversely, when plants were inoculated early, stem-end browning (SEB) rather than NN predominated. A high percentage of naturally occurring SEB tubers (cv. Russet Burbank) were found by ELISA to contain PLRV. Plants produced by these tubers only rarely developed leafroll symptoms. These findings suggest a previously unsuspected causal relationship between SEB and PLRV. Implications of this apparent relationship on the epidemiology of potato leafroll in Maine are discussed.     

Potato leafroll virus purification and antiserum preparation for enzyme-linked immunosorbent assays

Potato leafroll virus (PLRV) was purified from potato foliage and stems with an average yield of 0.14 mg of PLRV/kg of potato. Modifications of an existing purification procedure are reported. Five low dosage (38-118 μg of PLRV) intravenous injections were used to produce a PLRV antiserum for use in enzyme-linked immunosorbent assays (ELISA) from tubers. PLRV was readily detected in ELISA testing of potato tubers and leaves and inPhysalis floridana Rybd. Non-specific reactions were low with all tissues. In parallel tests, a Canadian antiserum produced higher nonspecific reactions with tuber and leaf tissue. The results indicated that the use of low dosage-intravenous injections might be necessary methodology for producing PLRV antiserum for use in ELISA diagnostic tests with tuber tissue where high non-specific reactions have been reported.     

A method for large scale laboratory inoculation of potato tubers with late blight fungus

As part of a project to investigate the possibility that teratogenic factors are formed in potato tubers infected with the late blight fungus,Phytophthora infestans, a method was devised which allowed quick, efficient inoculation of large numbers of potato tubers. Tubers were placed in specially constructed trays, wounded by puncturing the periderm with flower “pin frogs”, and sprayed with a zoospore suspensionof P. infestans prepared from cultures grown on rye agar at 20C (68F) for 10 days. Approximately 95% of Katahdin and 93% of Russet Sebago tubers inoculated by this method exhibited typical late blight lesions after two weeks incubation at 20C, 80–90% RH. Eight tons of potatoes were processed in this manner.     

Relationship of virus concentration with the field resistance to potato virus Y in potatoes

The concentration of potato virus Y (PVY) was determined, using ELISA values (A405 nm), in twenty-six potato cultivars belonging to five resistance groups, grown in the field and in the greenhouse. On the basis of virus concentration, potato cultivars of group A, B, and C did not differ significantly and constitute the most susceptible group; those of group D and E differ significantly with each other and with group A, B, C, and constitute moderate and highly resistant groups, respectively. In the second year of infection, virus concentration was higher in each group, irrespective of resistance level. Thus, the infected plants of resistant groups, in a second year of growth, could be as rich sources of virus to aphids as plants from susceptible groups.     

Prestile: A new round white potato variety

Prestile is a late maturing potato variety with attractive, round to oblong, white-skinned, white-fleshed tubers with shallow eyes. Its major use is expected to be for tablestock in areas where Katahdin and Ontario are now grown. Prestile tubers are slow to sprout in storage and do not show the net necrosis caused by potato leafroll virus, but are quite susceptible to heat necrosis. This variety is immune to potato virus X, and is moderately resistant to common scab (Streptomyces scabies), verticillium wilt (Verticillium albo-atrum), and early blight (Alternaria solani). Prestile is susceptible to skinning and shatter bruise, and is difficult to kill, particularly when heavily fertilized.     

Purification of potato virus A and its detection in potato by enzyme-linked immunosorbent assay (ELISA)

Potato virus A (PVA) was purified fromNicandra physaloides by a simple method that omitted organic solvent clarification and consisted of differential centrifugation followed by equilibrium centrifugation in CsCl. An antiserum was produced that specifically detected PVA in potato leaf sap using either the SDS-agar test or enzyme-linked immunosorbent assay (ELISA). No heterlogous reaction of the antiserum with potato virus Y was detected. Purified PVA was highly infectious; it had an A 258/280 nm absorbance ratio of 1.28. The particles had a normal intact appearance in the electron microscope. Detection of PVA in potato sprouts and foliage by ELISA was compared with the local lesion assay onPhysalis angulata L. plants. ELISA was superior over an indicator plant test when sprout tissue was used. PVA antiserum reacted similarly with mild and crinkle isolates.     

Use of enzyme-linked immunosorbent assay to detect potato leafroll virus in field grown potato,cv. Russet Burbank

Use of enzyme-linked immunosorbent assay (ELISA) in detecting potato leafroll infections in field grown potato, cv. Russet Burbank, was studied from 1986 to 1988 at Rosemount, Minnesota. The objective was to determine relative reliability of current season foliage ELISA, tuber tissue ELISA, and tuber progeny foliage ELISA. Serological tests were most accurate when foliage of tuber progenies was tested. ELISA underestimated total leafroll infection when current season foliage from the inoculated plant was used, in those plants inoculated during late tuber bulking stage. Current season foliage ELISA tests using newly expanded terminal leaflets were more reliable than were tests using older leaflets. Leafroll infection was detected in the current season foliage and tuber progenies (tuber tissue as well as tuber progeny foliage) of some plants seven days after inoculation. Most current season foliage infections were detected by day 14–28 depending on year. Differences among years were most likely caused by variation in quality of virus source plants and numbers of vectors used in inoculation. ELISA tests on tuber tissue were almost as effective as ELISA tests on tuber progeny foliage in detecting potato leafroll 20 days after inoculation, but ELISA on tuber tissue substantially underestimated infection if plants were sampled earlier. Maximum percent tuber infection occurred 20 days or more after inoculation. Movement of the virus from the inoculated stem to other stems decreased with increased plant age at inoculation. Percent infected tubers declined with increased plant age at inoculation. Action thresholds developed for aphids in managing potato leafroll virus should take into account the temporal change in percent infected tubers.     

Anatomical evidence for the existence of roots on potato tubers and stolons

Roots on potato tubers and stolons displayed the normal root anatomy which consisted of a central vascular cylinder surrounded by endodermis with Casparian strips, the cortex and epidermis. Tuber roots appear to initiate from the parenchyma cells adjacent to the vascular tissue. Shoot tips were similar to normal apical meristems. These observations support our research demonstrating the growth of functional roots from potato tubers and stolons.     

Correlation of phytotoxin production with pathogenicity ofStreptomyces scabies isolates from scab infected potato tubers

A total of 23 isolates obtained from scab infected potato tubers representative of six sampling areas in Eastern and Central Canada and five ATCCStreptomyces strains were screened for pathogenicity on the basis of their ability to initiate scab development on aseptically cultured minitubers and plant generated tubers. The results were then correlated with any associated generation of the scab inducing phytotoxin, Thaxtomin A. In all cases a positive correlation was demonstrated between the pathogenicity of variousStreptomyces scabies isolates and their ability to produce the phytotoxin.     

A rapid method for determining blackspot susceptibility of potato clones

Two methods of determining susceptibility of potato clones to blackspot were compared: (1) bruising by weight dropping and (2) bruising by abrasive peeling. A highly significant positive correlation was obtained between the intensity of enzymatic discoloration following abrasive peeling and the amount of blackspot that developed by weight dropping (r=0.93). Abrasive peeling was more rapid than the weight-dropping method. Tuber samples were abraded 30 sec and the amount of enzymatic discoloration evaluated after 24 hr. The need for individually bruising and hand peeling of tubers was eliminated with this method. Because of the rapidity of the abrasive peeling method, it can be used effectively in potato breeding programs to screen large numbers of clones for blackspot susceptibility. Results indicate that tuber maturity affects enzymatic discoloration and blackspot susceptibility. Immature tubers, dug while the vines are still green, are more resistant to blackspot than mature tubers. Tuber maturity therefore must be considered when screening clones for susceptibility to blackspot.     

Purification of potato leaf roll virus and an evaluation of methods for its diagnosis

To evaluate four diagnostic methods for potato leaf roll virus (PLRV), an antiserum was prepared against a virus preparation purified from infectedDatura stramonium L. by an exudation method. The antiserum had a titer of 1:64 in microprecipitin tests. In a procedure developed subsequently PLRV was purified by a method that involved grinding liquid nitrogen frozen stems, petioles, and veins from different solanaceous hosts. A chloroform and n-butanol clarification was done followed by two cycles of differential centrifugation. On sucrose gradients, these preparations formed one band containing spherical particles 25 nm in diameter. In Ouchterlony double-diffusion plates, the antiserum reacted positively with virus preparations from frozenPhysalis, Datura or potato tissue partially purified by two cycles of differential centrifugation. No visible reaction was obtained between the antiserum and the virus preparation from density gradient centrifugation. Antiserum neutralized infectivity of 40–50% of the virions in a partially purified preparation. No virus peak was detected in the scanning patterns obtained after a mixture of antiserum and virus was centrifuged on a sucrose density gradient. A few virus particles were seen attached to serologically specific grids. Methods to diagnose PLRV in infected potato tubers were then compared. In the first, aphids were used to acquire the virus from tuber sprouts. They transmitted the virus to 100% of thePhysalis test plants. Results of the second method, visual inspection of symptoms on potato plants, were similar to those of the first method. Sarkar’s electron microscopic method and immuno-specific grids were less efficient than aphid transmission to diagnose PLRV in tuber sprouts. The low virus concentration in infected tissue made these last two methods unreliable.     

A method for estimating the decrease in marketable tubers caused by potato late blight

A regression analysis was used to develop a method to estimate the decrease in marketable tubers caused by late blight of potato (Phytophthora infestans), based on estimates of percentage loss in total tuber yield computed from a published method. The economic loss due to late blight in table stocks is the relative percentage marketable yield loss, calculated from the expression: {fx19-01} when total yield losses (in percentage) did not exceed 40%. A graphical method is presented as an alternative. Yield results from seven field experiments conducted in the Maritime Provinces of Canada during the period 1953 to 1971 were used in the study, where marketable tubers were defined as 2 1/4 inch or more in diameter.     

Influence of calcium on internal heat necrosis of Atlantic potato

While Atlantic is the cultivar of choice of potato chip processors for the mid-Atlantic region, susceptibility to internal heat necrosis (IHN) greatly increases grower risk of economic loss from unsalable tubers. Yield, tuber calcium levels, and percentage of tubers with IHN of several cultivars from a common seed source were examined at two locations. The percentage of tubers with IHN were significantly higher for Atlantic while the tuber Ca concentration was lower than those of Superior. Both the percentage of tubers with IHN and tuber Ca concentration was higher in New Jersey than Virginia. Lower Ca levels were found in tubers with necrotic medullary tissue than in those without. Studies were completed in Virginia and New Jersey to evaluate the influence of Ca source, Ca rate and location within the hill as well as nitrogen rate on the onset and progression of IHN of Atlantic potato. Tuber yield was not significantly influenced by either Ca source (calcium carbonate (CaCO₃) or calcium sulfate (CaSO₄)) or Ca rate (0-1800 kg Ca/ha). Calcium rate had no influence on size distribution. As expected, a significantly greater percentage of large tubers were noted with delayed harvest. In a short growing season (<110 days) yield was not influenced by nitrogen rates ranging from 84 to 252 kg/ha. However, with delayed harvest, yield was increased and IHN slightly reduced with 168 or 252 kg N/ha when compared to 84 kg/ha. The influence of applied Ca on IHN varied between years, but the addition of Ca or N did not reduce IHN sufficiently to avoid out-of-grade because of internal defects. Location of applied Ca within the hill had no effect on yield, specific gravity, IHN, or tissue Ca concentrations. Slightly higher leaf Ca was found when using CaCO₃ than CaSo₄. The influence of Ca rate in 1987 and 1988 on leaf, periderm, or medullary Ca concentrations was not consistent. Nitrogen had no significant influence on tissue Ca levels.