The United States is the second-largest producer of soybean (Glycine max) globally, with Iowa serving as a key contributor in the Midwest (USDA-NASS, 2022). Iowa ranks as the second-highest soybean-producing state, with a yield of 15.59 million metric ton in

3.97 million ha (USDA-NASS, 2022). Sudden death syndrome (SDS) caused by Fusarium virguliforme, and white mold (WM), caused by Sclerotinia sclerotiorum, are the second and third most impactful diseases contributing to soybean yield losses of 0.81 and 0.71 million metric ton, respectively, in the northern USA (CPN, 2025b).

Sclerotia are fungal structures produced by members of the Sclerotiniaceae family. Disease incidence can be significantly reduced if sclerotia formation is prevented or if they are destroyed in the soil or plant residues (Xia et al., 2019). Previous research has examined white mold sclerotia production in the field following fungicide applications (Miorini et al., 2017; Vieira et al., 2003). Similarly, other studies have investigated the impact of benzovindiflupyr and flusilazole fungicides on in vitro sclerotia production of WM (Huang et al., 2019b; Lu et al., 2015).

Pydiflumetofen is a second-generation fungicide from the succinate dehydrogenase inhibitor (SDHI)-(FRAC) group 7 (FRAC, 2024), known for its broad-spectrum activity against a wide range of plant pathogens (Duan et al., 2019; Huang et al., 2019a). It has been labeled for use on soybean in the USA since 2019, both as a seed treatment (Kandel et al., 2019) and in commercial premix formulations for foliar application to manage diseases such as SDS and WM. While limited research has been conducted on fungicide sensitivity in the USA isolates of Fv and Ss, pydiflumetofen has demonstrated efficacy against Fusarium graminearum (from wheat, corn, dry bean, and soybean), Cercospora sojina and Corynespora cassiicola (from soybean) in the USA, Sclerotinia sclerotiorum from oil seed rape, cucumber, tomato, eggplant, kidney bean, pepper and pumpkin in China (Breunig and Chilvers, 2021; Duan et al., 2019; Huang et al., 2019a; Neves and Bradley, 2021; Xu et al., 2020), Botrytis cinerea from tomato in China (Li et al., 2022) and Bipolaris maydis from corn in China (Sun et al., 2024).

The package “drc” (Ritz et al., 2015) was explicitly written to estimate dose-response relationships across many disciplines, included plant pathology. It is a robust package describing symmetric and asymmetric dose-response relationships, including those describing hormetic effects (Knezevic et al., 2007). Symmetric dose-response curve describes the relationships that can be modeled, showing symmetry around an inflection point, whereas asymmetric dose-response curves do not have this property (Noel et al., 2018). To estimate the EC50, dose-response models generally use the following parameters: the upper asymptote, lower asymptote, and slope (Noel et al., 2018). There are then two definitions that describe the EC50: relative and absolute. The relative EC50 is the inflection point in a dose-response curve, whereas the absolute EC50 is the concentration at which 50% maximal growth occurs (more consistent with the FRAC-defined EC50; Noel et al., 2018).

The objective was to estimate the absolute EC50 for mycelial growth of Fusarium virguliforme from Iowa. Additionally, to determine the absolute EC50 values for both mycelial growth and sclerotial production of Sclerotinia sclerotiorum from Iowa.

Media preparation. One-third strength potato dextrose agar (PDA) was prepared by dissolving 1.3 g of PDA (Difco Laboratories, Detroit, MI) and 1.0 g of agar (Bacto Laboratories, NSW, Australia) in 100 mL of distilled water. The medium was autoclaved for 1 hour. A stock solution of Saltro^? (pydiflumetofen 41.7%, Syngenta; flowable concentrate formulated for seed treatment) at 417,000 ppm was diluted with sterile distilled water and incorporated into the PDA medium at 50 °C to achieve final concentrations of 0.0008, 0.0042, 0.0083, 0.0417, 0.0834, 0.8340, and 834 ppm through standard serial dilution. Medium without fungicide served as the control. Amended media were stirred thoroughly and poured into 10 cm diameter petri plates. For consistency, 20 mL of medium was dispensed into each plate using a sterile Falcon tube (Fisher Scientific, Waltham, MA).

Fungal isolates, inoculation and incubation. Monosporic isolates were isolated from roots of soybean plants exhibiting typical SDS symptoms using a modified Nash and Snyder medium by the procedure of Cho et al. (2001). The F. virguliforme isolates were morphologically and molecularly identified by the procedure of Chilvers and Brown- Rytlewski (2010). Isolations of S. sclerotiorum were carried out from a single sclerotium from the soybean stem with WM symptoms during the harvest season, following the procedure of Otto-Hanson et al. (2011). The S. sclerotiorum isolates grew fast with cottony- white aerial mycelia and formed irregular sclerotia on the border of the PDA petri plate.

Two isolates of F. virguliforme and three isolates of S. sclerotiorum were used in total (Table 1). Historical isolates are considered isolates collected before pydiflumetofen introduction in the market (2019) (Fv1^his), whereas contemporary isolates are considered isolates collected currently and after pydiflumetofen introduction in the market (2019). We were able to obtain historical isolates for F. virguliforme, but not for S. sclerotiorum. Actively growing cultures of F. virguliforme (seven days after inoculation, DAI) and S. sclerotiorum (48 hours after inoculation, HAI) on 1/3 strength PDA were used. Agar plugs (0.6 cm diameter) were taken from the colony edge and placed the center of each amended medium-plate. Plates were sealed with film parafilm^? (Amcor corporate, Zürich, Switzerland) and placed in an incubator at 25 °C dark. After the incubation period (7 DAI for F. virguliforme and 48 HAI for S. sclerotiorum), plates were scanned to quantify the mycelial growth at 400 dpi with a blue background and a small ruler as a scale-without removing the parafilm^?. Colony diameters (equatorial and polar) were measured using ImageJ (National Institutes of Health, Bethesda, MD), and the S. sclerotiorum plates were further kept at 25 °C dark.

Sclerotia Production. These aforementioned S. sclerotiorum plates were incubated at 25

°C dark for 21 days to allow sclerotia formation. All sclerotia were per plate were collected, dried in an oven at 50 °C for 48 hours, and counted and weighed, following the procedure of Lu et al. (2015).

Data Analysis. EC50 values for mycelial growth of both fungus (F. virguliforme and S. sclerotiorum) and for sclerotial production of S. sclerotiorum were estimated by fitting three models: the three-parameter log-logistic (LL.3), four-parameter log-logistic (LL.4), and four-parameter Weibull type I (W1.4) and type II (W2.4) dose-response models using the “drc” package in R (Ritz et al., 2015). The best-fitting model was selected based on Akaike’s Information Criterion (AIC), F-tests for lack-of-fit, and examination of residual

variance, following Noel et al. (2018), using the “mselect” function in the same package. All fungicide concentrations, including controls, had three replications of a media plate (experimental unit). The petri plates were incubated in a completely random design, and the experiment was performed twice. ANOVA and Fisher’s Least Significant Difference (LSD) post hoc tests were conducted using the “agricolae” package (de Mendiburu, 2023). All data analyses were performed in R studio version (R Development Core Team, 2024). The best-fit models for mycelial growth were the Weibull type II (W2.4) for Fv1 and the four-parameter log-logistic (LL.4) for Fv3. For S. sclerotiorum, the best models were W2.4 for Ss1, and Weibull type I (W1.4) for both Ss2 and Ss4 (Table 1). The absolute EC50 values of pydiflumetofen for mycelial growth of F. virguliforme isolates were 0.045 ppm (Fv1) and 0.048 ppm (Fv3) (Table 1). For S. sclerotiorum, the absolute EC50 values for

mycelial growth ranged from 0.011 to 0.046 ppm (Table 1).

Isolate Ss2 did not produce sclerotia in the control and was therefore excluded from subsequent sclerotia analyses. The best-fit models for sclerotia number and dried-weight were LL.3 for Ss1 and Ss4. Absolute EC50 values for sclerotia number ranged from 0.004 to 0.019 ppm, and for dried-weight sclerotia ranged from 0.101 to 0.137 ppm (Table 1).

The historical isolate F. virguliforme Fv1 isolate (NE305) showed a lower EC50 value than the contemporary Fv3 isolate (FV-RD-0024), indicating greater sensitivity to pydiflumetofen, as expected. Notably, the highest tested concentrations (0.834 and 834 ppm) did not completely inhibit F. virguliforme mycelial growth (Figure 1).

A significant effect of pydiflumetofen concentration on sclerotia number was observed (p < 0.001). Concentrations of 0.0042, 0.0083, 0.0417, 0.0834, 0.834, and 834 ppm significantly reduced the number of sclerotia compared to the control (14.6 sclerotia). The highest concentrations, 0.834 and 834 ppm, resulted in the lowest sclerotia counts recorded

(0) in the study (Table 2; Figure 2). Due to noticeable variation in both the number and size of sclerotia within the same treatment, sclerotia dried-weight was also assessed as a more consistent metric. Pydiflumetofen concentration had a significant effect on sclerotia dried- weight as well (p < 0.001), with concentrations of 0.834 and 834 ppm significantly reducing sclerotia dried-weight (0 g) compared to the control (0.066 g) (Table 2; Figure 2).

Figure 1

Table 1

Fungus	ID	Isolate	EC50 M(ppm)	SEM	EC50 N(ppm)	SEN	EC50 D(ppm)	SED	Model	Collection location	Collection year
Fusarium virguliforme	Fv1his	NE305 (Gongora-Canul et al., 2012) Mueller’s lab	0.045	0.004	-	-	-	-	W2.4Md	Nevada	2006
F. virguliforme	Fv3con	FV-RD- 0024	0.048	0.006	-	-	-	-	LL.4Mb	Roland	2024
S. sclerotiorum	Ss1	Leandro’s lab WM- 2024	0.011	0.001	0.004	0.005	0.101	0.014	W2.4Md; LL.3Na; LL.3Da	Unknown in IA	Unknown
S. sclerotiorum	Ss2	#630 Mueller’s lab	0.046	0.004	-	-	-	-	W1.4Mc	Unknown in IA	2020
S. sclerotiorum	Ss4	WM-CO-0024	0.041	0.005	0.019	0.012	0.137	0.019	LL.3Na; LL.3Da	Colo	2024

hisHistorical isolate = Collected before pydiflumetofen introduction in the market; ^conContemporary isolate = Collected currently and after pydiflumetofen introduction in the market; ^MMycelial growth; ^NNumber sclerotia; ^DDried-weight sclerotia.

Table 2

Concentration (ppm)	Number sclerotia	Groupsz	Dried-weight sclerotia (g)	Groupsz
0	14.600	a	0.066	a
0.0008	10.00	ab	0.071	a
0.0042	7.167	b	0.068	a
0.0083	7.333	b	0.064	a
0.0417	7.333	b	0.057	a
0.0834	8.667	b	0.051	a
0.834	0	c	0	c
834	0	c	0	c

zConcentrations with the same letter are not significantly different by Fisher’s Least Significant Difference (LSD) at p<0.05

Figure 2

Pydiflumetofen reduced the mycelial growth of Fusarium virguliforme. The EC50 mycelial growth values for F. virguliforme in this study (0.045-0.048 ppm) fall within the previously reported range of 0.011-0.4602 ppm from soybean isolates in the USA (Hamilton et al., 2024; Olaya et al., 2021). The best-fitting models for mycelial growth were four-parameter types: The Weibull type II (asymmetric) and the log-logistic (symmetric) distributions. The use of the Weibull type II model is consistent with Hamilton et al. (2024), who studied the same fungus and active ingredient. However, their maximum

tested concentration was 100 ppm, whereas this study used concentrations up to 834 ppm. This difference may be attributed to our use of a commercial formulation, while Hamilton et al. (2024) used the pure active ingredient. Nevertheless, the highest concentrations tested (0.834 and 834 ppm) in this study cannot be considered the minimum inhibitory concentration (MIC).

The historical isolate Fv1 (NE205), collected in 2006-prior to the introduction of pydiflumetofen in the USA-and the contemporary isolate Fv3 (FV-RD-0024) had EC50 values of 0.045 and 0.048 ppm, respectively. Both values fall within the baseline range of 0.04-0.24 ppm for unexposed isolates in the USA (Hamilton et al., 2024).

Pydiflumetofen also had an effect on the mycelial growth of Sclerotinia sclerotiorum. The EC50 values obtained in this study were within the baseline ranges previously reported: 0.0058-0.09 ppm (soybean isolates; Duan et al., 2019) and 0.0015-0.0350 ppm (non- soybean isolates from six different crops; Huang et al., 2019a), both from China. However, it is worth noting that Huang et al. (2019a) used broth media for in vitro testing, while our study used solid media. The best fit models for S. sclerotiorum mycelial growth were the W1.4 and W2.4 distributions (asymmetric), but these model types were not reported in previous studies using this fungus and fungicide, limiting direct comparisons.

In addition to inhibiting mycelial growth, pydiflumetofen significantly reduced sclerotia production in S. sclerotiorum. The EC50 values for sclerotia number and dried-weight (0.004-0.019 ppm and 0.101-0.137 ppm, respectively) overlap with the ranges reported by Gao et al. (2020) in China for non-soybean baseline isolates (mean EC50 values of 0.023 ppm for number and 0.003 ppm for dried-weight). In this study, sclerotia number was significantly reduced starting at 0.0042 ppm, while sclerotia dried-weight was significantly reduced starting at 0.834 ppm. These results are consistent with previous findings that concentrations between 0.2 and 0.8 ppm significantly reduced both the number and dried- weight of sclerotia when using benzovindiflupyr, another second-generation SDHI fungicide (Huang et al., 2019b). Based on our results, the sclerotial inhibitory concentration in vitro appears to be 0.834 ppm.

The EC50 values for mycelial growth of Fusarium virguliforme isolates from Iowa fall within the previously reported range for the USA soybean isolates (Hamilton et al., 2024; Olaya et al., 2021). Similarly, EC50 values for Sclerotinia sclerotiorum isolates from Iowa are consistent with those reported for isolates from outside the USA (Duan et al., 2019; Huang et al., 2019a). These findings suggest that pydiflumetofen has potential to reduce sclerotia production, an important survival structure in the disease cycle of S. sclerotiorum. Therefore, additional studies under field conditions (in planta) are warranted to evaluate its effectiveness in disrupting disease progression by targeting sclerotia formation. Commercial fungicide formulations containing this active ingredient (7%) in mixture with more active ingredients, are currently labeled for foliar application on soybean to manage

S. sclerotiorum in the USA (CPN, 2025a).

The EC50 mycelial growth of F. virguliforme Iowa isolates fall in the range of previous reported for the USA. The EC50 mycelial growth of S. sclerotiorum Iowa isolates fall in the range previous reported outside the USA. Pydiflumetofen has the potential to diminish the sclerotial production but more studies are needed. This study did not assess the environmental or non-target effects of pydiflumetofen, which should be addressed in future research. To our knowledge, this is the first report estimating the EC50 of pydiflumetofen

on both mycelial growth and sclerotia production of S. sclerotiorum soybean isolates from the USA.