Detection and Quantification of Spongospora subterranea Sporosori in Soil by Quantitative Real-Time PCR.

Powdery scab on potato tubers is caused by the obligate soilborne biotroph Spongospora subterranea and is known to cause substantial losses in potato production. The pathogen also infects roots of susceptible hosts, forming galls which can negatively affect root function. S. subterranea is also the vector of Potato mop-top virus, which causes a tuber necrosis disease that can, depending on temperature and cultivar, render potato tubers unmarketable. In this study, we adapted a published protocol to develop a sensitive and robust quantitative real-time PCR (qPCR) assay using specific primers and probes for detecting and quantifying S. subterranea sporosori in soil types that differ in physical properties, including organic matter content and soil pH. For the first time, an external control was utilized and applied directly to the soil prior to DNA extraction, which facilitated normalization of S. subterranea sporosori soil levels from sample to sample. The duplex qPCR protocol was demonstrated to be highly sensitive, capable of detecting and quantifying as few as 1 sporosorus/g of soil, with consistently high qPCR efficiency and the coefficient of determination (R2) values ranging from 94 to 99% and 0.98 to 0.99, respectively. The protocol was successfully implemented in enumerating S. subterranea sporosori in naturally infested field soil collected from several states and in artificial potting mixes with high organic matter content ranging from 64 to 71%. The qPCR method developed can be useful for potato growers to avoid agricultural soils highly infested with S. subterranea and in the development of risk assessment models in the future that incorporate cultivar susceptibility to powdery scab and soil infestation levels.

Quantifying Control Efficacy of Fungicides Commonly Applied for Potato Early Blight Management.

Early blight is an economically important foliar disease of potato in the United States. Because of the lack of resistant potato cultivars, fungicides are applied extensively to obtain adequate control. To manage early blight, standard protectant fungicides and single-site mode-of-action "specialty" fungicides are applied either alone or incorporated into a fungicide rotation program. Control efficacy at two crop growth stages (tuber initiation/early bulking and late bulking/tuber maturation) and the overall tuber yield response to standard and specialty fungicides were assessed using network metaanalytic models. Control efficacy of fungicides ranged from moderate to very high (>30 to 75%) compared with the nontreated control. For both potato growth stages, specialty fungicides performed better than standard protectant fungicides. Furthermore, control efficacy of both fungicides was higher (3 to 9%) at late bulking and tuber maturation when compared with early bulking crop growth stage. Specialty fungicide programs increased overall tuber yields by 4 and 9% over standard fungicides and nontreated control, respectively. Based on the results, more precise fungicide use recommendations and fungicide programs can be developed for early blight management.

Assessing Potato Cultivar Sensitivity to Tuber Necrosis Caused by Potato mop-top virus.

Potato mop-top virus (PMTV) causes mop top disease in potato. This disease can result in a decline in tuber quality causing economic losses to growers due to the production of necrotic lesions and discolored tissue in infected tubers. Due to the soilborne nature of PMTV, identifying and developing host resistance against the virus is considered the best disease management option. Very little is known about the sensitivity of U.S. potato cultivars to PMTV-induced tuber necrosis. The current study is aimed at investigating the sensitivity of a large number of potato cultivars to PMTV-induced tuber necrosis. Sixty-three cultivars representing all market-types were evaluated in North Dakota over a 2-year period for virus-induced tuber necrosis incidence and severity. PMTV-induced tuber necrosis (P < 0.0001) and severity (P < 0.0001) were significantly different among cultivars. Cultivars were categorized into sensitive, insensitive, and moderately sensitive/insensitive groups based on the virus-tuber induced necrosis data from both years. Based on data from ND trials, six cultivars (Red Endeavor, Viking, Dakota Jewel, Dark Red Norland, Nicolet, and Modoc) were rated as sensitive and 43 were rated as insensitive to PMTV-induced tuber necrosis. Four cultivars, including Bannock Russet, Gemstar Russet, Lelah, and Waneta showed zero PMTV incidence over 2 years. These results will help growers in making individual or coordinated decisions for the management of PMTV-induced tuber necrosis under field and storage conditions.

Potential Impact of Fluopyram on the Frequency of the D123E Mutation in Alternaria solani.

Succinate dehydrogenase-inhibiting (SDHI) fungicides have been widely applied in commercial potato (Solanum tuberosum L.) fields for the control of early blight, caused by Alternaria solani Sorauer. Five-point mutations on three AsSdh genes in A. solani have been identified as conferring resistance to SDHI fungicides. Recent work in our laboratory determined that A. solani isolates possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were collected at successively higher frequencies throughout a 3-year survey. In total, 118 A. solani isolates previously characterized as possessing the D123E mutation were evaluated in vitro for boscalid and fluopyram sensitivity. Over 80% of A. solani isolates with the D123E mutation evaluated were determined to be highly resistant to boscalid in vitro. However, effective concentration at which the fungal growth is inhibited by 50% values of isolates with the D123E mutation to fluopyram, ranging from 0.2 to 3 µg/ml, were sensitive and only slightly higher than those of baseline isolates to fluopyram, which ranged from 0.1 to 0.6 µg/ml. Five A. solani isolates with the D123E mutation were further evaluated in vivo for percent disease control obtained from boscalid and fluopyram compared with two wild-type isolates, three isolates possessing the F129L mutation, two isolates possessing the H134R mutation, two isolates possessing the H133R mutation, and one isolate with the H278R mutation. Relative area under the dose response curve values for boscalid and fluopyram were significantly lower for all five D123E-mutant isolates, demonstrating reduced disease control in vivo. In field trials, the frequency of A. solani isolates with the D123E mutation recovered from treatments receiving an in-furrow application of fluopyram ranged from 5 to 37%, which was significantly higher compared with treatments receiving foliar applications of standard protectants, in which the frequency of the D123E mutation in isolates ranged from 0 to 2.5%. Results suggest that A. solani isolates possessing the D123E mutation have a selective advantage under the application of fluopyram compared with SDHI-sensitive isolates, as well as isolates possessing other mutations conferring SDHI resistance. These data illustrate the importance of implementing fungicide resistance management strategies and cautions the use of fluopyram for in-furrow applications that target other pathogens of potato.

Spatial and Temporal Distribution of Mutations Conferring QoI and SDHI Resistance in Alternaria solani Across the United States.

The application of succinate dehydrogenase inhibiting (SDHI) and quinone outside inhibiting (QoI) fungicide chemistries is a primary tactic in the management of early blight of potato, caused by Alternaria solani. Resistance to QoIs in A. solani has been attributed to the F129L mutation, while resistance to SDHIs is conferred by five different known point mutations on three AsSdh genes. In total, 1,323 isolates were collected from 2013 through 2015 across 11 states to determine spatial and temporal frequency distribution of these mutations. A real-time polymerase chain reaction (PCR) was used to detect the presence of the F129L mutation. Molecular detection of SDHI-resistant isolates was performed using SDH multiplex PCR specific for point mutations in AsSdhB, AsSdhC, or AsSdhD genes and mismatch amplification analysis PCR detecting the point mutations in AsSdhB. Previous work in our research group determined that substitutions of histidine for tyrosine (H278Y) or arginine (H278R) at codon 278 on the AsSdhB gene were the most prevalent mutations, detected in 46 and 21% of A. solani isolates, respectively, collected in 2011 to 2012, and uniformly distributed among six sampled states. In contrast, the substitution of histidine for arginine (H134R) at codon 134 in the AsSdhC gene was the most prevalent mutation in 2013 through 2015, identified in 36% of isolates, compared with 7.5% of isolates recovered in 2011 to 2012. Substitutions of histidine for arginine (H133R) at codon 133 and aspartic acid for glutamic acid (D123E) at codon 123 in the AsSdhD gene were detected in 16 and 12%, respectively, in the A. solani population by 2015 and were recovered across a wide range of states, compared with 15 and 1.5% of isolates collected in 2011 to 2012, respectively. Overall, SDHI- and QoI-resistant isolates were detected at high frequencies across all years, with evidence of significant spatial variability. Future research will investigate whether these results are due to differences in parasitic fitness.

Assessing Potato Cultivar Sensitivity to Tuber Necrosis Caused by Tobacco rattle virus.

Tobacco rattle virus (TRV) causes the economically important corky ring spot disease in potato. Chemical control is difficult due to the soilborne nature of the TRV-transmitting nematode vector, and identifying natural host resistance against TRV is considered to be the optimal control measure. The present study investigated the sensitivity of 63 cultivars representing all market types (evaluated at North Dakota and Washington over 2 years) for the incidence of TRV-induced tuber necrosis and severity. This article also investigates the cultivar-location interaction (using a mixed-effects model) for TRV-induced necrosis. TRV-induced tuber necrosis (P < 0.0001) and severity (P < 0.0001) were significantly different among cultivars evaluated separately in North Dakota and Washington trials. Mixed-effects model results of pooled data (North Dakota and Washington) demonstrated that the interaction of cultivar and location had a significant effect (P = 0.03) on TRV-induced necrosis. Based on the virus-induced tuber necrosis data from both years and locations, cultivars were categorized into sensitive, moderately sensitive, insensitive, and moderately insensitive groups. Based on data from North Dakota, 10 cultivars, including Bintje, Centennial Russet, Ciklamen, Gala, Lelah, Oneida Gold, POR06V12-3, Rio Colorado, Russian Banana, and Superior, were rated as insensitive to TRV-induced tuber necrosis. Similar trials assessing TRV sensitivity among cultivars conducted in Washington resulted in a number of differences in sensitivity rankings compared with North Dakota trials. A substantial shift in sensitivity of some potato cultivars to TRV-induced tuber necrosis was observed between the two locations. Four cultivars (Centennial Russet, Oneida Gold, Russian Banana, and Superior) ranked as insensitive for North Dakota trials were ranked as sensitive for Washington trials. These results can assist the potato industry in making cultivar choices to reduce the economic impact of TRV-induced tuber necrosis.

Individual Participant Data Meta-Analysis of Foliar Fungicides Applied for Potato Early Blight Management.

Foliar fungicides continue to be the primary means of early blight management on potato in the United States. Both premium-priced, single-site mode-of-action "specialty" fungicides and standard protectant multisite fungicides are applied, either alone or incorporated into fungicide rotation programs to combat early blight. Individual participant data meta-analysis was conducted to compare overall fungicide efficacy against early blight on potato, quantify tuber yields, and identify the most efficacious timing for fungicide applications. In this study, the specialty fungicide-based applications were compared against the standard fungicides chlorothalonil and mancozeb applied alone. Type 3 fixed effects indicated that there was a significant difference (P < 0.0001) in overall efficacy and yield among the treatments applied to manage early blight in potato. There was a significant difference (P < 0.0001) among treatments in early blight development during the growing season. Applications incorporating specialty fungicides, when compared with standard fungicides, significantly affected disease severity from vegetative growth initiation (P = 0.0139) to tuber maturation (P = 0.0009). Results demonstrate that the higher cost, specialty-fungicide-based applications were most effective for early blight management in North Dakota and Minnesota.