Julie S. Pasche

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.

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.

Biological Significance of Mefenoxam Resistance in Phytophthora erythroseptica and Its Implications for the Management of Pink Rot of Potato

Tubers from plants treated with in-furrow and foliar applications of mefenoxam were inoculated with eight isolates of Phytophthora erythroseptica having varying levels of sensitivity to the fungicide. Two isolates with effective concentration causing 50% reduction of mycelial growth (EC50) values of 0.02 and 0.04 μg ml-1 were categorized as being mefenoxam sensitive. Isolates with EC50 values >1.0 μg ml-1 were designated as insensitive to mefenoxam and were grouped two each into low intermediate (EC50 = 1.1 and 5.3 μg ml-1), high intermediate (EC50 = 26 and 74 μg ml-1), and resistant (EC50 ≥ 100 μg ml-1). The biological significance of these isolates was examined by quantifying disease control. P. erythroseptica isolates classified in the resistant group infected a significantly greater proportion of untreated tubers than isolates in any other group. Mefenoxam reduced infection frequency of sensitive isolates by as much as 37%. Mefenoxam did not provide disease control of any isolate possessing insensitivity to the fungicide, with the greatest decrease in control observed with the low intermediate group. Aggressiveness indices, representing tuber infection frequency and depth of penetration, were calculated for untreated and mefenoxam-treated tubers. According to these indices, both isolates classified in the resistant group and high intermediate isolate 252-4 were more aggressive than sensitive isolates in the absence of mefenoxam pressure, and significantly so in the presence of mefenoxam. These results suggest that pink rot may become more severe in fields known to contain P. erythroseptica populations with mefenoxam EC50 values >1.0 μg ml-1 if the fungicide is applied. These factors should be considered when developing strategies to manage pink rot and mefenoxam-resistant populations of P. erythroseptica.

Management of soilborne diseases of potato

A large number of soilborne diseases that affect potato are important in the United States. In the Midwestern USA, early dying, involving Verticillium dahliae and Colletotrichum coccodes and the storage rots, pink rot and leak tuber rot, caused by Phytophthora erythroseptica and Pythium ultimum are among the most serious. Various aspects of the biology and aetiology of these pathogens have been investigated to improve our understanding of the factors involved in disease development. These studies have addressed knowledge gaps and have assisted in the development of management strategies and tactics for each disease. Disease management integrates cultural practices, use of agrochemicals and to some degree, host resistance. Unfortunately, cultural management practices alone are currently inadequate to control these diseases, causing the potato industry to become over-reliant on the use of agrochemicals for effective management. Current research efforts are directed at the identification and incorporation of genetic resistance into cultivars with acceptable horticultural characteristics to provide more effective disease management.

Resistance to QoI Fungicides in Ascochyta rabiei from Chickpea in the Northern Great Plains

Ascochyta blight, caused by Ascochyta rabiei (teleomorph: Didymella rabiei), is an important fungal disease of chickpea (Cicer arietinum). A monitoring program was established in 2005 to determine the sensitivity of A. rabiei isolates to the QoI (strobilurin) fungicides azoxystrobin and pyraclostrobin. A total of 403 isolates of A. rabiei from the Northern Great Plains and the Pacific Northwest were tested. Ninety-eight isolates collected between 2005 and 2007 were tested using an in vitro spore germination assay to determine the effective fungicide concentration at which 50% of conidial germination was inhibited (EC50) for each isolate-fungicide combination. A discriminatory dose of 1 μg/ml azoxystrobin was established and used to test 305 isolates from 2006 and 2007 for in vitro QoI fungicide sensitivity. Sixty-five percent of isolates collected from North Dakota in 2005, 2006, and 2007 and from Montana in 2007 were found to exhibit a mean 100-fold decrease in sensitivity to both azoxystrobin and pyraclostrobin when compared to sensitive isolates, and were considered to be resistant to azoxystrobin and pyraclostrobin. Under greenhouse conditions, QoI-resistant isolates of A. rabiei caused significantly higher amounts of disease than sensitive isolates on azoxystrobin- or pyraclostrobin-amended plants. These results suggest that disease control may be inadequate at locations where resistant isolates are present.

Genetic Variability in the Potato Pathogen Colletotrichum coccodes as Determined by Amplified Fragment Length Polymorphism and Vegetative Compatibility Group Analyses

ABSTRACT Amplified fragment length polymorphism (AFLP) using three primer sets was used to characterize 211 Colletotrichum coccodes isolates from North America, 112 of which were assigned to six vegetative compatibility groups (VCGs) using nitrate nonutilizing (nit) mutants. These isolates clustered into five corresponding groups by unweighted pairgroup method with arithmetic means-based cluster analysis of AFLP banding patterns. Isolates of C. coccodes belonging to NA-VCG1 and NA-VCG3 were closely related, as were isolates belonging to NA-VCG2 and NA-VCG5. Based on bootstrap analysis of AFLP data, the two isolates originally assigned to NA-VCG4 clustered with isolates belonging to NA-VCG2 and NA-VCG5. C. coccodes isolates that clustered with two isolates belonging to NA-VCG6 were the most diverged from other groups, including seven isolates collected from hosts other than potato. As opposed to the bootstrap analysis, a quadratic discriminant analysis (QDA) of AFLP data correctly categorized the two isolates of NA-VCG4. Furthermore, in isolates where VCG determinations had been made, this model correctly classified isolates of all VCGs. QDA classifications were identical to those made by the bootstrap analysis, with the exception of VCG4. Overall, classifications made by the QDA model were strongly correlated (r = 0.970, P < 0.001) to the VCGs assigned by traditional methods. All 99 C. coccodes isolates evaluated only by AFLP also were subjected to QDA, leading to the assignment of a presumptive VCG for each isolate. No isolates of VCG4 or VCG6 were identified by QDA within this population. Symptoms of black dot developed in plants inoculated with isolates collected from both potato and non-potato hosts. However, total yield was not significantly reduced by infection with non-potato isolates. The lack of any additional groups identified by AFLP analysis may be an indicator of a limited level of genetic variation among North American C. coccodes isolates. AFLP is a much more efficient technique for subspecific characterization in C. coccodes than VCG analysis utilizing nit mutants and will provide an effective means by which the population biology of this pathogen can be further investigated worldwide.

Development and Validation of a Real-Time PCR Assay for the Quantification of Verticillium dahliae in Potato

An increase in the stringency for higher quality potato tubers and restrictions on the use of soil fumigants, among other factors, has garnered renewed interest in Verticillium wilt, particularly in russet-skinned cultivars grown for processing. In response to the needs of producers, breeders have increased efforts in the development of potato cultivars with resistance to Verticillium dahliae Kleb., the primary cause of Verticillium wilt. These efforts have resulted in the release of numerous russet-skinned cultivars with purported resistance to the pathogen. However, because efficient and effective methods to screen germplasm for true resistance do not exist, breeders typically have reported resistance based on the development of wilt symptoms alone. The studies reported here demonstrate the efficiency and practicality of a QPCR method for quantification of V. dahliae in potato stem tissue. This method, developed to detect the target trypsin protease gene of the pathogen, was compared with traditional methods for V. dahliae quantification which involve plating stem tissue or sap onto semi-selective media, as well as to a recently developed QPCR assay which amplifies a region of the β-tubulin gene of V. dahliae. The QPCR assay developed in the studies reported here was demonstrated to be sensitive to 0.25 pg of DNA. Use of the duplex real-time PCR assay, utilizing the potato actin gene to normalize quantification, resulted in clearer differentiation of levels of resistance among eight russet-skinned potato cultivars inoculated in greenhouse trials when compared with traditional plating assays. However, relative levels of resistance among cultivars were similar between traditional plating and QPCR methods, resulting in correlation coefficients greater than 0.93. The assay described here also detected the pathogen in inoculated stem tissue at higher frequencies than both traditional plating assays and a previously developed QPCR assay. The QPCR assay developed here demonstrates rapid, efficient, and accurate quantification of V. dahliae, providing a tool amenable for use by breeding programs on large numbers of clones and selections, and will aid researchers evaluating other control strategies for Verticillium wilt.

Resistance to phytophthora erythroseptica and pythium ultimum in a potato clone derived from s. berthaultii and s. etuberosum

Tubers of several potato clones and cultivars were screened for susceptibility to infection by zoospores of Phytophthora erythroseptica (causal agent of pink rot) and mycelia of Pythium ultimum (causal agent of leak) over a three-year period, from 2003-2005. Incidence of infected tubers (%) and penetration of rot (mm) were the parameters used to determine the susceptibility of each potato clone. Responses of each potato clone were compared to cultivars with known resistance or susceptibility to these pathogens. Tubers of cultivars Atlantic and Snowden have moderate resistance to infection and colonization by P. erythroseptica and P. ultimum, respectively, and were used as the resistant checks. Cultivars Russet Norkotah and Red Norland are susceptible to infection by both pathogens. A number of potato clones demonstrated resistance to pink rot equal to or greater than the control cultivar Atlantic, including Etb 6-5-2, ND5822C-7, ND6956b-13, ND7443Ab-44, ND7443Ab-181, ND7818-1Y and J101K6A22. In addition to demonstrating the highest resistance to pink rot, Etb 6-5-2 was the only clone that demonstrated resistance to leak greater than or equivalent to the resistant cultivar Snowden. Etb 6-5-2 is a backcross derivative from a somatic hybrid of Solanum etuberosum and Solanum berthaultii and will be investigated further as a potential source of resistance to these two storage rot diseases.ResumenLos tubérculos de varios clones y cultivares de papa fueron tamizados para susceptibilidad con zoosporas de Phytopthora erythroseptica (agente causal de pudricion rosada) y con micelio Pythium ultimum (agente causal de pudricion acuosa o gotera) en un periodo de tres aÑos, del 2003 al 2005. La incidencia de tubérculos infectados (%) y la profundidad de la pudriciön (mm) fueron los parámetros usados para determinar la susceptibilidad en cada clon de papa. Las respuestas de cada clon fueron comparadas con las de cultivares de conocida resistencia o susceptibilidad a estos patögenos. Tubérculos de los cultivares Atlantic y Snowden que tienen resistencia moderada a la infecciön y colonizacion por P. erythroseptica y P. ultimum respectivamente, fueron usados como testigo resistente. Los cultivares de Russet Norkotah y Red Norland son susceptibles a la infeccion de ambos patögenos. Varios clones de papa mostraron resistencia a la pudriciön rosada, igual o mayor que el testigo Atlantic, incluyendo, Etb 6-5-2, ND5822C-7, ND6956b-13, ND7443Ab-44, ND7443Ab-181, ND7818-1Y y J101K6A22. Además de demostrar muy alta resistencia, Etb 6-5-2 fue el único clon que demoströ resistencia a la gotera en mayor proporciön o equivalente al cultivar resistente Snowden. Etb 6-5-2 es producto de una retrocruza derivada de un híbrido somático de Solanum etuberosum y S. berthaultii y será investigada adicionalmente como fuente potencial de resistencia a estas dos pudriciones de tubérculos almacenados.

Influence of Tillage and Method of Metam Sodium Application on Distribution and Survival of Verticillium dahliae in the Soil and the Development of Verticillium Wilt of Potato

The effect of plowing and deep-rip tillage, in combination with chemigation or shank injection of metam sodium, onVerticillium dahliae populations and disease development was assessed in two fields with differing soil types and potato rotations. Soil samples were collected on a geo-referenced basis at depths of 0 to 10 cm and 10 to 20 cm before tillage, after tillage, after chemical application, and before planting and assayed for the presence of the pathogen. Propagules ofV. dahliae were detected at 140 of 141 sites sampled prior to tillage. Most (74.4% in heavy, sandy loam; 63.1% in light, loamy sand) were concentrated in upper 10 cm of the soil profile. Plowing redistributed inoculum vertically while deep-rip tillage did not. In the non-chemical treated areas of both fields, theVerticillium population reached a maximum between 25 July and 8 August before declining to near pre-tillage levels. Overall, the population generally was lower in the field with heavy soil, higher organic matter content, and a 3-year crop rotation. Metam sodium appeared to be most effective when shank injected, as the levels of inoculum in both fields declined by 60% to 80% following this application method. Chemigation was ineffective in the lighter soil, but the inoculum density in the deep-rip tillage area of the field with the heavier soil declined by nearly 20% in the upper and 60% in the lower strata following this treatment. The number ofV. dahliae propagules at the 10- to 20-cm depth in the plowed area of the same field was reduced by 25% following chemigation, but remained unchanged in the upper strata. Wilt was reduced in both fields by as much as 50% with shank injection of metam sodium with concomitant increases in total yield, marketable yield and gross income. Increases in total yield were significant (P<0.05) for the main effect of chemical, in the plowed area of the field with the heavier soil type following shank injection. These data suggest that growers might benefit from altering their tillage and chemical application practices as part of an integrated approach to managing Verticillium wilt.ResumenSe probó en dos campos con diferentes tipos de suelo y rotaciones de papa, el efecto de labranza y de aradura profunda, en combinación con la aplicación de productos químicos por el agua de riego e inyección (shank injection) de metasodio en el suelo, sobre poblaciones deVerticillium dahliae y desarrollo de la enfermedad. Las muestras de suelo se colectaron por zonas geográficas, a profundidades de 0 a 10cm y 10 a 20 cm antes del barbecho, después del barbecho, después de la aplicación de productos químicos y antes de la siembra y se hizo la prueba de presencia del patógeno. Los propágulos deV. dahliae se detectaron en 140 de los 141 lugares de muestreo antes del barbecho. La mayoría (74.4% en suelos areno-arcillosos pesados; 63.1% en suelos areno-arcillosos ligeros) estaban concentrados en los 10cm superiores del suelo. La labranza redistribuyó el inóculo verticalmente, mientras que la aradura profunda no lo hizo. En las áreas de ambos campos que no fueron tratadas químicamente, la población deVerticillium alcanzó el máximo entre el 25 de julio y el 8 de agosto antes de disminuir a niveles de pre-barbecho. Sobre todo, la población fue generalmente más baja en campos con suelo pesado, alto contenido de materia orgánica y una rotación de tres años. El metasodio se mostró más efectivo cuando se inyectó, ya que los niveles de inóculo en ambos campos disminuyeron en un 60–80% con este método de aplicación. La aplicación de productos químicos por medio del riego no fue efectiva en suelo más ligero pero la densidad de inóculo en áreas de suelo con aradura profunda y suelo más pesado disminuyó cerca de 20% en la parte superior y 60% en el estrato inferior después de este tratamiento. El número de propágulos deV. dahliae a 10 a 20 cm de profundidad en el área arada del mismo campo se redujo después de la aplicación de productos químicos por riego en un 25% pero permaneció igual en el estrato superior. La marchitez se redujo en ambos campos en un 50% con el metasodio inyectado al suelo con el concomitante incremento del rendimiento total, rendimiento comerciable e ingresos totales. Los incrementos en el rendimiento total fueron significativos (P<0.05), principalmente por el efecto del producto químico, en áreas del campo con suelo pesado después de inyección de producto químico. Estos datos sugieren que los productores pueden obtener beneficios alterando la labranza y las prácticas de aplicación de productos químicos como parte de un manejo integrado para marchitez por Verticillium.

Effect of Application Method and Rate on Residual Efficacy of Mefenoxam and Phosphorous Acid Fungicides in the Control of Pink Rot of Potato

Experiments were conducted to examine the effectiveness of rate and method of phosphorous acid application for controlling pink rot of potato (Solanum tuberosum) caused by Phytophthora erythroseptica. Replicated small-plot and replicated split commercial field trials were established in commercial production fields in Minnesota from 2006 to 2009. Fungicides were applied in-furrow at planting, or as one, two, or three foliar applications via ground sprayer, irrigation system (chemigation), or fixed-wing aircraft. Phosphorous acid efficacy was compared to mefenoxam, the fungicide commonly utilized to manage pink rot, either by determining natural infections in the field or by inoculating eyes of harvested tubers using a mefenoxam-sensitive and -resistant isolate of P. erythroseptica via postharvest challenge inoculation. In replicated small plot trials, both in-furrow and two foliar applications of mefenoxam controlled tuber rot in the field, and significantly controlled tuber rot in storage. Phosphorous acid also reduced tuber rot in the field when applied two or three times to the foliage. Although phosphorous acid was ineffective when applied in-furrow, one, two, and three foliar applications and a postharvest application of phosphorous acid controlled mefenoxam-sensitive and -resistant isolates of P. erythroseptica during storage for 187 days, while mefenoxam failed to control the resistant isolate. In replicated split commercial field trials, two aerial applications of phosphorous acid were as effective as three applications in reducing pink rot incidence in tubers inoculated postharvest. Three aerial applications were as effective as three chemigation applications in replicated split commercial field trials in 2008, but provided significantly greater protection than chemigation in 2009.