An in-field heat treatment to reduce Cercospora beticola survival in plant residue and improve Cercospora leaf spot management in sugarbeet.

Introduction: Sugarbeets account for 55 to 60% of U.S. sugar production. Cercospora leaf spot (CLS), primarily caused by the fungal pathogen Cercospora beticola, is a major foliar disease of sugarbeet. Since leaf tissue is a primary site of pathogen survival between growing seasons, this study evaluated management strategies to reduce this source of inoculum. Methods: Fall- and spring-applied treatments were evaluated over three years at two study sites. Treatments included standard plowing or tilling immediately post-harvest, as well as the following alternatives to tillage: a propane-fueled heat treatment either in the fall immediately pre-harvest or in the spring prior to planting, and a desiccant (saflufenacil) application seven days pre-harvest. After fall treatments, leaf samples were evaluated to determine C. beticola viability. The following season, inoculum pressure was measured by monitoring CLS severity in a susceptible beet variety planted into the same plots and by counting lesions on highly susceptible sentinel beets placed into the field at weekly intervals (fall treatments only). Results: No significant reductions in C. beticola survival or CLS were observed following fall-applied desiccant. The fall heat treatment, however, significantly reduced lesion sporulation (2019-20 and 2020-21, P < 0.0001; 2021-22, P < 0.05) and C. beticola isolation (2019-20, P < 0.05) in at-harvest samples. Fall heat treatments also significantly reduced detectable sporulation for up to 70- (2021-22, P < 0.01) or 90-days post-harvest (2020-21, P < 0.05). Reduced numbers of CLS lesions were observed on sentinel beets in heat-treated plots from May 26-June 2 (P < 0.05) and June 2-9 (P < 0.01) in 2019, as well as June 15-22 (P < 0.01) in 2020. Both fall- and spring-applied heat treatments also reduced the area under the disease progress curve for CLS assessed the season after treatments were applied (Michigan 2020 and 2021, P < 0.05; Minnesota 2019, P < 0.05; 2021, P < 0.0001). Discussion: Overall, heat treatments resulted in CLS reductions at levels comparable to standard tillage, with more consistent reductions across year and location. Based on these results, heat treatment of fresh or overwintered leaf tissue could be used as an integrated tillage-alternative practice to aid in CLS management.

Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

First report of Geotrichum candidum causing rubbery rot of potato (Solanum tuberosum) in Michigan.

In April 2018, rotted 'Lamoka' tubers were received from a commercial storage facility (<1% incidence) in St. Joseph County, Michigan by the MSU Potato & Sugar Beet Pathology and Plant & Pest Diagnostics programs. Dense circular colonies of white fungal-like growth were observed on the surface of the tubers, and internal tissues were watery and spongy with gray to brown discoloration (Supplemental Figure 1). Tubers had a strong, sweet alcoholic odor. External and internal tuber tissues were surface disinfested in 0.825% sodium hypochlorite for 1 min, rinsed twice in sterile distilled water, blotted on sterile filter paper, and placed onto 1.5% (w/v) water agar (WA). After 3 days at 21-24°C and ambient light conditions, septate, branched mycelia and hyaline, cylindrical, single-celled conidia 5.2-8.9 µm x 3.6-5.2 µm (n=20 arthrospores) were observed singly or in chains (Supplemental Figure 2A&B). On potato dextrose agar (PDA), colonies were white, circular, and dense (Supplemental Figure 2C). These observations matched morphological descriptions of Geotrichum candidum (Carmichael 1957). No Pythium or Phytophthora spp. were detected. A mono-conidial isolate of the fungus was obtained and maintained on PDA. DNA was extracted from mycelia using a DNeasy plant mini kit (QIAGEN). Fragments of the internal transcribed spacer (ITS) and 18S ribosomal RNA gene regions were amplified using primers ITS1F/4 primers and NS3/8, respectively (White et al. 1990). Purified PCR products (QIAquick PCR purification kit, QIAGEN) were submitted for Sanger sequencing at the Genomics Research Technology Support Facility (East Lansing, MI). The ITS1F/4 and NS3/8 consensus sequences (OP142324 and OP153873) aligned with GenBank accessions of G. candidum KY103456.1 (100% identity) and JF262193.1 (99.75% identity), respectively. Healthy 'Lamoka' tubers were rinsed with tap water, surface disinfested in 0.825% sodium hypochlorite for 15 min, rinsed twice in sterile distilled water, and blotted dry on sterile paper towel. Ten tubers were inoculated by placing 10-mm diameter fully colonized agar plugs, excised from the margin of a 9-day-old PDA culture, onto the surface of each tuber (Duellman et al. 2021). Ten tubers were mock-inoculated using sterile PDA. Tubers were placed in a moist chamber and incubated in the dark at room temperature. After nine days, inoculated tubers exhibited white colony growth on tuber surfaces and an alcoholic scent was present. After 27 days, internal tissues were rubbery, but no discoloration was observed. No rubbery rot symptoms were observed on the control tubers. Samples were excised 1 cm laterally from and vertically beneath the inoculation site. Tissues were surface disinfested as described above and plated on 1.5% WA. After 9 days, a Geotrichum sp. identical to the original isolate was confirmed in 50% of samples from inoculated tubers. No Geotrichum sp. were detected from mock-inoculated tubers. Since 2018, G. candidum has been confirmed in commercial storages in three counties in the Lower Peninsula (incidences up to 1-2%). Geotrichum candidum was recently reported causing rubbery rot of potato in Idaho (Duellman et al. 2021); however, to our knowledge this is the first report of rubbery rot in Michigan. Despite increasing detection frequencies, incidences remain low and spread in storage appears limited. Seed decay leading to stand loss (incidence 1-3%) was observed after planting infected lots, which should be avoided or minimized.

Integration of Row Spacing, Seeding Rates, and Fungicide Applications for Control of Sclerotinia Stem Rot in Glycine max.

Soybean (Glycine max) farmers in the Upper Midwest region of the United States often experience severe yield losses due to Sclerotinia stem rot (SSR). Previous studies have revealed benefits of individual management practices for SSR. This study examined the integration of multiple control practices on the development of SSR, yield, and the economic implications of these practices. Combinations of row spacings, seeding rates, and fungicide applications were examined in multisite field trials across the Upper Midwest from 2017 to 2019. These trials revealed that wide row spacing and low seeding rates individually reduced SSR levels but also reduced yields. Yields were similar across the three highest seeding rates examined. However, site-years where SSR developed showed the highest partial profits at the intermediate seeding rates. This finding indicates that partial profits in diseased fields were reduced by high seeding rates, but this trend was not observed when SSR did not develop. Fungicides strongly reduced the development of SSR while also increasing yields. However, there was a reduction in partial profits due to their use at a low soybean sale price, but at higher sale prices fungicide use was similar to not treating. Additionally, the production of new inoculum was predicted from disease incidence, serving as an indicator of increased risk for SSR development in future years. Overall, this study suggests using wide rows and low seeding rates in fields with a history of SSR while reserving narrow rows and higher seeding rates for fields without a history of SSR.

Meta-Analytic and Economic Approaches for Evaluation of Pesticide Impact on Sclerotinia Stem Rot Control and Soybean Yield in the North Central United States.

As complete host resistance in soybean has not been achieved, Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum continues to be of major economic concern for farmers. Thus, chemical control remains a prevalent disease management strategy. Pesticide evaluations were conducted in Illinois, Iowa, Michigan, Minnesota, New Jersey, and Wisconsin from 2009 to 2016, for a total of 25 site-years (n = 2,057 plot-level data points). These studies were used in network meta-analyses to evaluate the impact of 10 popular pesticide active ingredients, and seven common application timings on SSR control and yield benefit, compared with not treating with a pesticide. Boscalid and picoxystrobin frequently offered the best reductions in disease severity and best yield benefit (P < 0.0001). Pesticide applications (one- or two-spray programs) made during the bloom period provided significant reductions in disease severity index (DIX) (P < 0.0001) and led to significant yield benefits (P = 0.0009). Data from these studies were also used in nonlinear regression analyses to determine the effect of DIX on soybean yield. A three-parameter logistic model was found to best describe soybean yield loss (pseudo-R2 = 0.309). In modern soybean cultivars, yield loss due to SSR does not occur until 20 to 25% DIX, and considerable yield loss (-697 kg ha-1 or -10 bu acre-1) is observed at 68% DIX. Further analyses identified several pesticides and programs that resulted in greater than 60% probability for return on investment under high disease levels.

Validating Sclerotinia sclerotiorum Apothecial Models to Predict Sclerotinia Stem Rot in Soybean (Glycine max) Fields.

In soybean, Sclerotinia sclerotiorum apothecia are the sources of primary inoculum (ascospores) critical for Sclerotinia stem rot (SSR) development. We recently developed logistic regression models to predict the presence of apothecia in irrigated and nonirrigated soybean fields. In 2017, small-plot trials were established to validate two weather-based models (one for irrigated fields and one for nonirrigated fields) to predict SSR development. Additionally, apothecial scouting and disease monitoring were conducted in 60 commercial fields in three states between 2016 and 2017 to evaluate model accuracy across the growing region. Site-specific air temperature, relative humidity, and wind speed data were obtained through the Integrated Pest Information Platform for Extension and Education (iPiPE) and Dark Sky weather networks. Across all locations, iPiPE-driven model predictions during the soybean flowering period (R1 to R4 growth stages) explained end-of-season disease observations with an accuracy of 81.8% using a probability action threshold of 35%. Dark Sky data, incorporating bias corrections for weather variables, explained end-of-season disease observations with 87.9% accuracy (in 2017 commercial locations in Wisconsin) using a 40% probability threshold. Overall, these validations indicate that these two weather-based apothecial models, using either weather data source, provide disease risk predictions that both reduce unnecessary chemical application and accurately advise applications at critical times.

Spatiotemporal Distribution Pattern of Sclerotinia sclerotiorum Apothecia is Modulated by Canopy Closure and Soil Temperature in an Irrigated Soybean Field.

Identifying the optimal timing for fungicide application is crucial in order to maximize the control of Sclerotinia stem rot (SSR), which is caused by Sclerotinia sclerotiorum. In this study, the impact of canopy closure and soil temperature on apothecia production was investigated to optimize fungicide application timing. Replicated soybean plots with a row spacing of 0.36 and 0.38 or 0.76 m were established in 2015 and 2016 in an irrigated soybean field at Michigan State University's Montcalm Research Center. The number of apothecia and ascospores and the incidence of SSR were monitored two times per week for 10 to 12 weeks. In both row-spacing trials, apothecia were observed earlier in 2016 (before the R1 growth stage) than in 2015 (between R1 and R2). The maximum number of apothecia was 50 times higher with the 0.36-m row spacing than with the 0.76-m row spacing in 2015 but was 2.5 times higher with the 0.76-m row spacing than with the 0.38-m row spacing in 2016, though the overall numbers were much lower in 2016. The apothecia distribution pattern was synchronized with the canopy closure pattern and the soil temperature profile. The peak number of apothecia was observed when canopy closure reached at least 50% and when average soil temperature in the row was between 21.5 and 23.5°C. In 91% of the cases, the presence of apothecia was observed when the percentage of light blocked was 70%, and no apothecia germinated in the absence of light or under full light exposure. During the first 50 days after plant emergence, the rate of canopy closure was higher in 2016 than in 2015, and the first diseased plant was observed earlier in 2016 (R2) than in 2015 (R5). Canopy closure and the distance of the sampling point from the soybean row explained much of the variability in the number of apothecia. These results can partially explain the inconsistent efficacy of fungicide applications based on the soybean growth stage and will be helpful for informing disease models and fine-tuning fungicide application strategies.

Weather-Based Models for Assessing the Risk of Sclerotinia sclerotiorum Apothecial Presence in Soybean (Glycine max) Fields.

Sclerotinia stem rot (SSR) epidemics in soybean, caused by Sclerotinia sclerotiorum, are currently responsible for annual yield reductions in the United States of up to 1 million metric tons. In-season disease management is largely dependent on chemical control but its efficiency and cost-effectiveness depends on both the chemistry used and the risk of apothecia formation, germination, and further dispersal of ascospores during susceptible soybean growth stages. Hence, accurate prediction of the S. sclerotiorum apothecial risk during the soybean flowering period could enable farmers to improve in-season SSR management. From 2014 to 2016, apothecial presence or absence was monitored in three irrigated (n = 1,505 plot-level observations) and six nonirrigated (n = 2,361 plot-level observations) field trials located in Iowa (n = 156), Michigan (n = 1,400), and Wisconsin (n = 2,310), for a total of 3,866 plot-level observations. Hourly air temperature, relative humidity, dew point, wind speed, leaf wetness, and rainfall were also monitored continuously, throughout the season, at each location using high-resolution gridded weather data. Logistic regression models were developed for irrigated and nonirrigated conditions using apothecial presence as a binary response variable. Agronomic variables (row width) and weather-related variables (defined as 30-day moving averages, prior to apothecial presence) were tested for their predictive ability. In irrigated soybean fields, apothecial presence was best explained by row width (r = -0.41, P < 0.0001), 30-day moving averages of daily maximum air temperature (r = 0.27, P < 0.0001), and daily maximum relative humidity (r = 0.16, P < 0.05). In nonirrigated fields, apothecial presence was best explained by using moving averages of daily maximum air temperature (r = -0.30, P < 0.0001) and wind speed (r = -0.27, P < 0.0001). These models correctly predicted (overall accuracy of 67 to 70%) apothecial presence during the soybean flowering period for four independent datasets (n = 1,102 plot-level observations or 30 daily mean observations).

Case Study of an Epidemiological Approach Dissecting Historical Soybean Sclerotinia Stem Rot Observations and Identifying Environmental Predictors of Epidemics and Yield Loss.

Sclerotinia sclerotiorum is a significant threat to soybean production worldwide. In this study, an epidemiological approach was used to examine 11 years of historical data from a soybean management performance trial in order to advance our understanding of Sclerotinia stem rot (SSR) development and to identify environmental predictors of SSR epidemics and associated yield losses. Recursive partitioning analysis suggested that average air temperature and total precipitation in July were the most significant variables associated with disease severity. High levels of SSR disease severity index were observed when the average temperature in July was below 19.5°C and total precipitation in July was moderate, between 20 and 108.5 mm. A biphasic sigmoidal curve accurately described the relationship between SSR disease severity index (DSI) and yield, with a DSI threshold of 22, below which minimal yield loss was observed. A 10% increase in the DSI, from 22.0 to 24.2, led to an 11% decrease in yield, from 3,308.14 to 2,951.29 kg/ha. Also, a yield threshold (3,353 kg/ha) that was higher than the annual U.S. average soybean yield (3,039.7 kg/ha) was suggested as an expected yield under low or no SSR pressure in the U.S. Midwest. These thresholds can allow soybean stakeholders to assess the value of disease control and establish an SSR baseline for cost-effective management to protect yields. Because S. sclerotiorum has more than 400 plant host species, and because having quantitative information concerning crop losses is crucial for decision making, this study shows the usefulness of historical data on SSR and, hence, can serve as a model in other SSR pathosystems (canola, dry bean, potato, pea, and so on).

Expression, purification, and characterization of a carbohydrate-active enzyme: A research-inspired methods optimization experiment for the biochemistry laboratory.

The development and implementation of research-inspired, discovery-based experiences into science laboratory curricula is a proven strategy for increasing student engagement and ownership of experiments. In the novel laboratory module described herein, students learn to express, purify, and characterize a carbohydrate-active enzyme using modern techniques and instrumentation commonly found in a research laboratory. Unlike in a traditional cookbook-style experiment, students generate their own hypotheses regarding expression conditions and quantify the amount of protein isolated using their selected variables. Over the course of three 3-hour laboratory periods, students learn to use sterile technique to express a protein using recombinant DNA in E. coli, purify the resulting enzyme via affinity chromatography and dialysis, analyze the success of their purification scheme via SDS-PAGE, assess the activity of the enzyme via an HPLC-based assay, and quantify the amount of protein isolated via a Bradford assay. Following the completion of this experiment, students were asked to evaluate their experience via an optional survey. All students strongly agreed that this laboratory module was more interesting to them than traditional experiments because of its lack of a pre-determined outcome and desired additional opportunities to participate in the experimental design process. This experiment serves as an example of how research-inspired, discovery-based experiences can benefit both the students and instructor; students learned important skills necessary for real-world biochemistry research and a more concrete understanding of the research process, while generating new knowledge to enhance the scholarly endeavors of the instructor.