#Isolates In 2016, and 2017, we conducted a survey of S. sclerotiorum from soybean in NE, IA, WI, and MI, resulting in a collection of 1,369 isolates, which included farmer fields and fungicide field trials. In 2003 to 2012, 366 isolates were collected and characterized previously from white mold screening nurseries or producer fields throughout dry bean and snap bean production areas across the US, with others collected in prior years. From these, we randomly selected 207 isolates: 93 from dry beans (18 ND, 16 MI, 16 WA, 15 CO, 11 NE, 2 DE, 2 MN, 2 WI, 1 OR, 6 from Brazil and, 4 from Mexico) and 114 from soybeans (48 NE, 25 MI, 24 IA, and 17 WI). Isolates were from farmer fields (FF), fungicide field trials (FFT), and fields without fungicide applications, the latter were called baseline. Fungicide sensitivity was determined for four technical products representing different modes of action: thiophanate methyl (TM) in group 1, tetraconazole in group 3, boscalid in group 7, and picoxystrobin in group 11. First, we determined fungicide sensitivity of 21 baseline isolates plus 21 additional isolates from FF and FFT. We used serial dilution with 6 concentrations for TM (0.75, 1.00, 1.50, 2.00, 2.50, 10.00 ppm), for tetraconazole (0.5, 1.0, 2.0, 3.0, 5.0 ppm), for boscalid (0.025, 0.050, 0.100, 0.200, 0.800 ppm), and picoxystrobin (0.01, 0.02, 0.04, 0.06, 0.10 ppm), with 4 repetitions per isolate and 2 experimental replications. Plates were incubated in darkness at 23 ± 2 °C for 42 h and diameter measured with digital calipers. A dose-response curve was fit to estimate the EC50 for each fungicide. Second, we identified the concentration with the best prediction of EC50 for each fungicide, known as discriminatory concentration (DC), by linear regression of % mycelial growth vs. log(EC50). The concentration yielding the highest coefficient of determination (r2) was selected as the DC for each fungicide. For the remaining isolates among the 207, growth on the DC was used to estimate EC50, which is termed EC50(D).
We sought to determine if there is a difference in fungicide sensitivity between isolates from dry bean and soybean and those exposed to intensive, moderate, and no fungicide, in fungicide field trials (FFT), farmer fields (FF), and white mold nurseries (WMN), respectively. We also sought to determine if reduced sensitivity is linked to use of fungicides with a single active ingredient. Initially, fungicide sensitivity was assessed to thiophanate methyl (MBC), tetraconazole (DMI), boscalid (SDHI), and picoxystrobin (QoI) using serial dilution for 42 isolates and a dose-response curve was fit to estimate the EC50. Linear regression of % mycelial growth at each concentration vs. logEC50 was used to identify the best predictor of EC50, used as the discriminatory concentration (DC). Thiophanate methyl had a qualitative DC of 10 ppm that classified as sensitive or resistant. Others were 2.0 ppm for tetraconazole, 0.2 ppm for boscalid, and 0.01 ppm for picoxystrobin. These concentrations will be used to assess sensitivity to all four fungicides for an additional 165 isolates (1-3 per field) to compare isolates grouped by source (FF, FFT, WMN) and also by host (dry bean and soybean). An additional 188 isolates (5-10 per field) from fields with applications of single-site fungicides will be used to assess if there is reduced sensitivity to fungicide used in each field.
In 2015, 2016, and 2017, we conducted a survey of S. sclerotiorum from soybean in NE, IA, WI, and MI, resulting in a collection of 1,362 isolates, which included farmer fields and fungicide research trials. At least 2 sclerotia from mostly 10 different plants/field or per plot was collected when dried at harvest season. GPS, area, incidence of the disease, crop history and fungicide history past 5 years (current season and previous season) were collected from those as information. Also isolates from two sources: white mold screening nurseries using no chemical or cultural treatments against S. sclerotiorum and employing standardized protocols for screening new cultivars for resistance to white mold or producer fields in dry bean production areas across the United States over the years (Steadman Lab).
We randomly selected 154 isolates: 86 from dry beans (from 9 states and 2 states from Brazil, with 1-17 isolates) and 66 from soybeans (6 NE, 18 IA, 13 WI & 18 MI and 4 from Mexico) were selected from the collection (baseline, farmer fields and fungicide field trials) plus 5 same isolates used in the baseline approach. A fungicide sensitivity was done using the DC for each fungicide under the conditions below mentioned. The outliers for each isolate within an experimental replication were removed. The outliers of each grouping by fungicide were preselected as field to be tested. Since at least 10 isolates per field are needed to compensate the heterogeneity among isolates within a field, if fields from the preselection have > 9 isolates/field, they were selected widely distributed by state and year for future studies.
Four technical products of fungicides representing each one different mode-action group: thiophanate methyl (TM) as group 1, tetraconazole (T) as group 3, boscalid (B) as group 7, and picoxystrobin (P) as group 11.
The first goal was to determine the baseline through the evaluation and comparison fungicide sensitivity from those isolates with and without fungicide field exposure from the collection, called baseline isolates. From 42 isolates: 15 soybean fields isolates, 6 soybean fungicide trials isolates, and 21 baseline isolates (from white mold nurseries of dry beans) were used to determine the EC50. Fungicide sensitivity was determined using the serial dilution method with 6 concentrations for TM (0.75, 1.00, 1.50, 2.00, 2.50, 10.00 ppm & control), for T (0.5, 1.0, 2.0, 3.0, 5.0 ppm & control), for B (0.025, 0.050, 0.100, 0.200, 0.800 ppm & control), and (0.01, 0.02, 0.04, 0.06, 0.10 ppm & control) was done by 4 repetitions for each isolate and 2 experimental replications. Plates were incubated in darkness at 23 ± 2 °C for 42 h and measure diameter with digital calipers. A dose-response curve was fit to estimate the EC50 for each fungicide. The second goal was to identify the concentration with the best prediction of EC50 for each fungicide known as discriminatory concentration (DC) by linear regression of % mycelial growth vs. log(EC50) at each concentration. The concentration yielding the highest coefficient of determination (r2) was selected as DC for each fungicide. Then, as a validation, a linear regression of EC50 from plate dilution and EC50(D) from discriminatory concentration was carried out.