Neil C. Gudmestad

A Multiplex PCR Assay to Characterize Potato virus Y Isolates and Identify Strain Mixtures

Potato virus Y (PVY) has become a serious problem for the seed potato industry, with increased incidence and rejection of seed lots submitted for certification. New PVY strains and strain variants have emerged in recent decades in Europe and North America, including the PVYN strain that causes veinal necrosis in tobacco, and strain variants that represent one or three recombination events between the common strain (PVYO) and PVYN. Several reverse transcription-polymerase chain reaction (RT-PCR) assays have been described that characterize PVY isolates as to strain type, but they are limited in their ability to detect some combinations of mixed strain infections. We report here the development of a single multiplex RT-PCR assay that can assign PVY strain type and detect mixed infections with respect to the major strain types. Validation of this assay was achieved using 119 archived PVY isolates, which had been previously characterized by serology and bioassay and/or previously published RT-PCR assays. Results for single-strain isolates were comparable to previous results in most cases. Interestingly, 16 mixed infections were distinguished that had previously gone undetected. The new multiplex RT-PCR assay will be useful for researchers and seed production specialists interested in determining PVY infection type using a single assay.

Baseline Sensitivity of Ascochyta rabiei to Azoxystrobin, Pyraclostrobin, and Boscalid

Ascochyta rabiei, causal agent of Ascochyta blight on chickpea (Cicer arietinum), can cause severe yield loss in the United States. Growers rely on applications of fungicides with site-specific modes of action such as the quinone outside inhibiting (QoI) fungicides azoxystrobin and pyraclostrobin, and the carboximide fungicide boscalid, to manage disease. In all, 51 isolates collected prior to QoI fungicide registration and 71 isolates collected prior to boscalid registration in the United States were tested in an in vitro assay to determine the effective fungicide concentration at which 50% of conidial germination was inhibited (EC50) for each isolate-fungicide combination. The effect of salicylhydroxamic acid (SHAM) on conidia of A. rabiei in the presence and absence of azoxystrobin also was assessed to determine whether the fungus is capable of using alternative respiration. Five of nine A. rabiei isolates tested had significantly higher (P ≤ 0.05) EC50 values when SHAM was not included in media amended with azoxystrobin, indicating that A. rabiei has the potential to use alternative respiration to overcome fungicide toxicity in vitro. EC50 values of azoxystrobin and pyraclostrobin ranged from 0.0182 to 0.0338 μg/ml and from 0.0012 to 0.0033 μg/ml, with mean values of 0.0272 and 0.0023 μg/ml, respectively. EC50 values of boscalid ranged from 0.0177 to 0.4960 μg/ml, with a mean of 0.1903 μg/ml. Establishment of these baselines is the first step in developing a monitoring program to determine whether shifts in sensitivity to these fungicides are occurring in the A. rabiei pathogen population.

Variability in Virulence Among Asexual Progenies of Phytophthora infestans

ABSTRACT One hundred two single zoospore isolates of Phytophthora infestans, derived asexually from four parental isolates of US-8 genotype and one isolate of US-1 genotype, were characterized for their virulence phenotypes to determine changes in virulence during asexual reproduction. Potato differentials, each containing a major gene for resistance to P. infestans (R1 to R11), were used to characterize the virulence patterns. Isolates were also characterized for mating type, glucose-6-phosphate isomerase (Gpi) banding pattern, and DNA fingerprints using probe RG57 to determine any genotypic changes in the single zoospore isolates. A subset of these single zoospore isolates was tested for response to mefenoxam to determine any shifts in sensitivity. Results showed that single zoospore isolates derived from parent PI-1 (US-8, 11 isolates) were identical to their parental virulence. Isolates derived from parent PI-191 (US-8, 29 isolates) showed some differences in virulence, mainly toward R8 and R9. Isolates derived from parent PI-126 (US-8, 14 isolates) demonstrated a higher level of virulence diversity. Isolates derived from parents PI-52 (US-1, 28 isolates) and PI-105 (US-8, 20 isolates) showed the highest level of virulence variability among the single zoospore isolates. Mating type, Gpi banding pattern, and DNA fingerprints for the single zoospore isolates were, in most cases, identical to the parental isolates. Single zoospore isolates showed different levels of sensitivity to mefenoxam. Virulence and other genetic changes during asexual reproduction are likely to play a major role in changing the race structure of P. infestans populations. This continuous change in the race structure is a serious problem and now poses a new challenge for utilization of race-specific resistance to manage late blight of potato.

Factors influencing PVY development and disease expression in three potato cultivars

Studies were performed to investigate factors affecting symptom expression of potato virus Y infection in three potato cultivars, Russet Norkotah, Shepody, and Red LaSoda. Quantitative enzyme-linked immunosorbent assay (ELISA) results revealed few differences in the relative virus titer among cultivars tested. Potato virus Y (PVY) titers developed as rapidly in Russet Norkotah as in Shepody and Red LaSoda. Additional studies were performed to determine the effect of light intensity and infections of PVY and potato virus X (PVX), alone and in combination, on the expression of mosaic symptoms in these three cultivars. Low light intensity (270-330 uE/m2/sec) significantly increased plant heights and severity of mosaic disease among the cultivars compared to high light intensity (100-200 uE/m2/sec). PVX and PVY, as well as the combination of PVX and PVY in the same plant, decreased plant height compared to the uninoculated (healthy) controls. Low light intensity and dual infections of PVX and PVY significantly increased mosaic disease severity in Shepody and Red LaSoda, but not in Russet Norkotah. Results of these studies refute the suggestion that Russet Norkotah is resistant to PVY infection since virus titers in this cultivar are similar to the known susceptible cultivars Shepody and Red LaSoda. These results further suggest that while Russet Norkotah is fully susceptible to infection by PVY, it resists symptom expression.ResumenEste estudio se realizó con el fin de investigar los factores que afectan la expresión de los síntomas del virus Y de la papa en tres cultivares, Russet Norkotah, Shepody y Red LaSoda. Los resultados del ensayo inmunoabsorbente ligado a la enzima cuantitativa (ELISA) revelaron algunas diferencias en el título relacionado del virus entre los cultivares examinados. El título del virus Y de la papa (PVY) se desarrolló rápidamente tanto en el cv. Russet Norkotah como en los cvs. Shepody y Red LaSoda. Se realizaron estudios adicionales para determinar los efectos de la intensidad de la luz y de las infecciones de PVY y del virus X (PVX), solos y en combinación, sobre la expresión de los síntomas del mosaico en esos tres cultivares. La baja intensidad de la luz (270–330 uE/m2/sec) incrementó significativamente la altura de la planta y la severidad de la enfermedad del mosaico entre los cultivares en comparación con la alta intensidad de la luz (100–200 uE/m2/sec). La presencia de PVX y PVY así como la combinación de ambos en la misma planta, redujo la altura de la planta en comparación con los controles no inoculados (sanos). La baja intensidad de la luz y la infección dual de PVX y PVY incrementaron significativamente la severidad de la enfermedad del mosaico en los cvs. Shepody y Red LaSoda, pero no en el cv. Russet Norkotah. Los resultados del estudio refutan la sugerencia de que Russet Norkotah es resistente a la infección del PVY, ya que el título del virus de este cultivar es similar a los cultivares de Shepody y Red Lasoda de susceptibilidad conocida. Estos resultados sugieren ampliamente que mientras el cv. Russet Norkotah es altamente susceptible a la infección por PVY, es resistente a la expresión de los síntomas.

Monitoring Fungicide Sensitivity of Cercospora beticola of Sugar Beet for Disease Management Decisions

Cercospora leaf spot, caused by the fungus Cercospora beticola Sacc., is the most serious and important foliar disease of sugar beet (Beta vulgaris L.) wherever it is grown worldwide. Cercospora leaf spot first caused economic damage in North Dakota and Minnesota in 1980, and the disease is now endemic. This is the largest production area for sugar beet in the United States, producing 5.5 to 6.0 million metric tons on approximately 300,000 ha, which is 56% of the sugar beet production in the United States. This Plant Disease feature article details a cooperative effort among the participants in the sugar beet industry in this growing area and represents a successful collaboration and team effort to confront and change a fungicide resistance crisis to a fungicide success program. As a case study of success for managing fungicide resistance, it will serve as an example to other pathogen-fungicide systems and provide inspiration and ideas for long-term disease management by fungicides.

Russet Norkotah: A new russet-skinned potato cultivar with wide adaptation

Russet Norkotah is a long, smooth, shallow-eyed, russet-skinned potato cultivar with wide adaptation. It has a smooth golden russet-skin and produces a high percent of medium sized U.S. No. 1 tubers that are good count-carton size.CompendioRusset Norkotah es un cultivar de papa de amplia adaptación, con tubérculos de forma alargada, ojos superficiales y cubierta lisa de color rojizo. Tiene la cáscara de un tinte rojizo dorado y produce un alto porcentaje de tubérculos US No. 1 de tamaño mediano, apropiados para embalaje reducido.

Sporulation of Helminthosporium solani and infection of potato tubers in seed and commercial storages

Silver scurf has become a major reason for rejection of fresh and processing potatoes in recent years. Control of the disease by chemical or cultural practices or resistant cultivars has been difficult. Observations have shown spread and increase of disease of potatoes in storage, but this has not been extensively studied. The objective of this study was to document Helminthosporium solani conidia production, dispersal, and tuber infection in potato storages. Spore samplers placed in seed, processing, and table stock storages collected conidia ranging from 0 to 12,000 conidia per day in seed and table stock storages (4°C), and from 0 to 24,000 conidia per day in processing storages (10°C). Conidia were detected soon after tubers entered storage and increased progressively during the storage period, with the maximum conidia numbers found during the time of tuber handling. Greenhouse-produced minitubers placed in storages for 1, 2, 3, or 4 weeks were infected by H. solani spores. Infection was significantly higher in those exposed for 4 weeks than in those exposed for 1 week. Results document the buildup of H. solani spores throughout the storage period, and that this inoculum is important in disease epidemiology. Control of this inoculum could lead to disease reduction.

Differences in Etiology Affect Mefenoxam Efficacy and the Control of Pink Rot and Leak Tuber Diseases of Potato

Data supplementing a previously published survey of North American isolates of Phytophthora erythroseptica and Pythium ultimum demonstrated that the proportion of the populations sensitive to mefenoxam remains high, 79.6 and 96.9% with EC50 sensitivities ranging from <0.01 to 0.9 µg ml-1 and <0.01 to 0.8 µg ml-1, respectively. Mefenoxam should provide control of these pathogens in most potato production areas. Factors affecting the development of pink rot and leak in potato tubers and the efficacy of mefenoxam to control these diseases with different etiologies were examined. Results confirmed that P. erythroseptica is capable of directly infecting potato tubers causing pink rot, whereas Pythium ultimum requires a wound to infect and cause leak. Mefenoxam was applied to replicated field plots as a single in-furrow application at planting, as an in-furrow application at planting followed by an additional sidedress application 3 weeks after planting, as a single foliar application when tubers were 7 to 8 mm in diameter, and as two foliar applications when the tubers were 7 to 8 mm in diameter and 14 days later. The recommended label rate plus two additional lower application rates were used with each method. For tubers challenge-inoculated after harvest, mefenoxam was found to be more effective in controlling pink rot relative to leak over all application methods. The greatest level of pink rot control (89%) was attained with the in-furrow at planting and sidedress application. All rates tested provided similar levels of control with this application method, but this method provided only a modest level of leak control (35%), and leak was not controlled by foliar applications of mefenoxam at any rate tested. In contrast, the foliar applications of mefenoxam resulted in 10 to 50% control of pink rot. Since the isolates of both pathogens were highly sensitive to me-fenoxam, disease-specific control was attributed to differences in disease etiology. Therefore, the use of mefenoxam to control pink rot in the field and storage appears to be well founded.

Assessment of Resistance of Tubers of Potato Cultivars to Phytophthora erythroseptica and Pythium ultimum

Tubers of 34 potato cultivars were examined for their susceptibility to infection by zoospores of Phytophthora erythroseptica and mycelia of Pythium ultimum. Incidence of infected tubers (%) and penetration of rot (mm) were the parameters used to determine the susceptibility of each cultivar. Tubers of cv. Atlantic appeared to have some resistance to infection and colonization by Phytophthora erythroseptica. Cvs. Russet Norkotah and Snowden were the most susceptible to infection by P. erythroseptica. Snowden was found to be highly susceptible to P. erythroseptica, but it was the most resistant to Pythium ultimum. Cvs. FL-1625 and FL-1867 also were less susceptible to P. ultimum than the other cultivars. Cvs. Superior, Itasca, and Dark Red Norland were the most susceptible to P. ultimum. Cultivar susceptibility should be considered when making disease management decisions, particularly in fields where these soilborne diseases are a recurring problem.

Comparison of PCR, ELISA, and DNA hybridization for the detection of Clavibacter michiganensis subsp. sepedonicus in field-grown potatoes

Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot, was specifically detected in field-grown potatoes by means of the polymerase chain reaction (PCR). A 20-bp synthetic oligomer, derived from an inverted repeat region of the repeated sequence of the bacterial plasmid pCS1, was used as the PCR primer. In assays of pure bacterial cultures, this system gave positive reactions with all strains of C. michiganensis subsp. sepedonicus tested and had a sensitivity of <10 CFU per PCR reaction. With the exception of 4 out of 12 strains of C. michiganensis subsp. insidiosus, no other species of bacteria tested produced a specific PCR product. The ability of PCR to detect C. michiganensis subsp. sepedonicus in field material was evaluated by testing sections of potato stems that were grown in New York and North Dakota from seed pieces of potato cultivars BelRus and Russet Burbank inoculated with 0, 10 2 , or 10 9 CFU C. michiganensis subsp. sepedonicus and destructively sampled at 35 and 90 days after planting (DAP). Parallel tests of these samples by enzyme-linked immunosorbent assay (ELISA) and DNA hybridization assay (DHA) were conducted for comparative purposes. Overall, 36.2, 35.8, and 29.1% of inoculated samples tested positive by PCR, ELISA, and DHA, respectively. Each assay was affected significantly by inoculum dose, cultivar, and sampling date (P = 0.0001), with detection approaching 100% for the combination of the following parameters: 90 DAP, susceptible cv. Russet Burbank, and 10 9 CFU inoculum level. None of the buffer-inoculated plants tested positive with either PCR or DHA, whereas ELISA results were highly dependent upon the positive-negative threshold used.