Evaluation of Combination Fungicides for Charcoal Rot and Collar Rot Management in Soybean

Abstract

Soil-borne diseases, including charcoal rot (Macrophomina phaseolina) and collar rot (Sclerotium rolfsii), threaten global soybean production. Four fungicide combinations were tested as seed treatments at three concentrations (1, 1.5, and 2 g or ml per kg of seed) under controlled conditions to address the challenges posed by these diseases. Under controlled conditions, the combination of thiophanate methyl + pyraclostrobin at a rate of 2 mL/kg of seed significantly alleviated disease symptoms caused by both pathogens. Additionally, it enhanced shoot and root weights by over 50% in plants affected by S. rolfsii. Field trials were conducted for two years at two distinct locations to assess the efficacy of three selected combination seed treatment fungicides against M. phaseolina and S. rolfsii. Both inoculated and uninoculated controls were included for the comparison. Among the fungicides, thiophanate-methyl + pyraclostrobin and trifloxystrobin + penflufen proved the most effective for suppressing both diseases under epiphytotic field conditions across the years and locations. This study also highlighted the benefits of these chemical combinations in enhancing agronomic traits, maintaining yield, and ensuring the economic viability of soybeans.

1. Introduction

The first and foremost principle of disease management is growing healthy and clean seeds [1]. Achieving healthy and clean seed production is difficult because some fungi are seed-borne and asymptomatic [2,3]. Using effective fungicides in seed treatment is an efficient and economical approach to managing plant diseases effectively. The soybean crop is renowned for its rich nutritional content and significant industrial value and has the unique ability to achieve the highest protein production per hectare. Furthermore, it plays a pivotal role by contributing to half of the global edible oil consumption [4].

Soybean diseases pose a persistent threat to annual crop yields and seed quality. Multiple factors contribute to their development, including the timeline of disease occurrences within fields, environmental conditions, agricultural practices, and cultivar selection. Understanding and managing these elements is critical for extenuating disease impact and ensuring sustainable soybean production [5]. In recent years, soybean cultivation has faced many challenges due to biotic stresses. Charcoal rot, collar rot, and anthracnose are the most dangerous and destructive diseases confronting soybean cultivators. These diseases pose significant coercion to crop health and yield, necessitating attentive management strategies to defend soybean production [6,7,8].

Macrophomina phaseolina is a necrotrophic fungus responsible for causing charcoal rot in soybeans. It survives in soil, plant debris, and seeds by producing microsclerotia, which is the primary inoculum for disease initiation [9,10]. The phytopathogen initially establishes a parasitic relationship with the soybean during the seedling stage. However, favorable epidemiological conditions, including elevated temperatures and drought-like situations at the flowering stage, lead to the development of characteristic symptoms such as leaf yellowing, wilting, and leaf drop. During the reproductive stage, epidermal and subepidermal tissues of the taproot and lower portion of stems exhibit silvery discoloration, with abundant microsclerotia [10].

Charcoal rot disease has led to a decline of 0.25 million tons of soybean yield globally. This loss accounts for roughly 4.2% of worldwide yield reduction attributed to various phytopathogens [11]. Under favorable epidemiological conditions, high temperatures, and drought-like situations, soybean charcoal rot has the potential to result in complete crop failure [4]. Even under irrigated conditions, it can lead to significant yield losses ranging from 6% to 33% in susceptible cultivars [12]. The epidemic of soybean charcoal rot in India caused an 80% yield loss in soybeans, particularly in Central India [13].

Sclerotium rolfsii, a pathogenic fungus, incites collar rot disease in soybeans. It is a necrotrophic seed and soil-borne phytopathogen that produces sclerotial bodies as primary inoculums. Particularly in regions with high temperatures and heavy rainfall, sclerotial bodies germinate and produce white mycelium. The white cottony growth establishes pathogenesis through direct penetration and induces damping-off during soybeans’ pre- and post-emergence stages [14]. It shows light brown encircling the collar region with white mycelium and enormous mustard-sized sclerotial bodies [15].

Collar rot in soybeans accounts for roughly 0.36% of worldwide yield reduction. This disease is considered to be of minor importance in major soybean-growing countries [11]. However, collar rot in India causes significant yield losses in soybeans annually, ranging from 30% to 40% [16]. Additionally, susceptible cultivars of soybeans were at risk of experiencing even higher yield losses, with potential losses ranging from 1.02% to 46.17% [17].

Soybean commercial cultivars resistant to soil-borne disease are unavailable in India [15,18]. Seed treatment with fungicides and crop rotation are the two most common methods soybean growers use to manage these diseases. The broad host range of M. phaseolina and S. rolfsii restricted the effectiveness of the crop rotations method [9]. Seed treatment with fungicides protects soybeans against soil-borne diseases and mitigates yield losses caused by these pathogens [19,20,21,22].

Various chemicals are available for seed treatment to control these soil-borne fungal pathogens. The application of the combination product thiophanate methyl + pyraclostrobin [23], azoxystrobin + metalaxyl [19], thiophanate methyl + pyraclostrobin + metalaxyl [21], fipronil + pyraclostrobin + thiophanate-methyl [22], trifloxystrobin + tebuconazole [24], thiram + carboxin and metalaxyl + mancozeb [25] significantly decreased the severity of soil-borne diseases and improved yield in soybean.

Only four registered and recommended seed-dressing fungicides are available to soybean farmers in India following the government’s ban on fungicides. These include thiophanate methyl + pyraclostrobin, penflufen + trifloxystrobin, carboxin + thiram, and carbendazim + mancozeb. Against this backdrop, the current study was carried out to evaluate the efficacy of new authorized and recommended seed treatment combination fungicides for managing the primary soil-borne disease of soybeans at two locations (Indore and Jorhat, India) during 2020 and 2021.

2. Materials and Methods

2.1. Isolation and Identification of Plant Pathogens

The plant pathogens M. phaseolina and S. rolfsii were isolated from soybean plants showing typical symptoms of charcoal rot and collar rot diseases at the Indian Council of Agricultural Research (ICAR)-Indian Institute of Soybean Research (IISR), Indore, India. Small bits (5 to 6 mm) of the infected stem were surface sterilized with 0.1% sodium hypochlorite solution for 45 s, followed by rinsing with sterile distilled water and transferring to potato dextrose agar (PDA) containing 100 ppm of streptomycin. After four days of incubation at 26 ± 1 °C, the Petri plates exhibited the growth characteristic of M. phaseolina (Supplementary Figure S1). These included light gray colony, cottony mycelial growth, brown mycelia, and abundant black microsclerotial bodies [9,26]. Simultaneously, the isolate of S. rolfsii was obtained from our previous studies [15,27], having white cottony mycelia, white colony and 0.32 mm size brown color sclerotia [9]. The sclerotium of S. rolfsii was collected from Petri plates through visual observation and transferred to PDA plates acidified with 0.2% lactic acid to establish a pure culture. A similar protocol was followed to purify M. phaseolina, where a single sclerotium was collected from Petri plates using a compound microscope.

2.2. Evaluation of Seed Treatment Fungicides Against M. phaseolina and S. rolfsii in Controlled Environment

Four seed treatment fungicides registered in India, namely, thiophanate methyl + pyraclostrobin (Xelora, BASF India Ltd., Mumbai 400 079, India), penflufen + trifloxystrobin (EverGOL Xtend, Bayer India, Thane (West) 400 607, India), carboxin + thiram (Vitavax Power, Dhanuka Agritech Ltd., Gurugram 122002, India) and carbendazim + mancozeb (Sprint, Indofil Industries Ltd., Mumbai 400059, India) were used to identify most effective seed treatment fungicide through controlled evaluation at ICAR-IISR, Indore against M. phaseolina and S. rolfsii.

We treated the susceptible cultivar Shivalik with four seed treatment fungicides individually at three different concentrations: 1 g or mL/kg of seed, 1.5 g or mL/kg of seed, and 2 g or mL/kg of seed. Slurries of seed treatment fungicides were prepared separately at three different doses. The slurries were applied to the seeds using a seed dresser to ensure a uniform fungicide coating on the seed surface. Natural gum (Camlin, Kokuyo Camlin Ltd., Mumbai 400 093, India) was used as a sticker to enhance adhesion.

The treated seeds were spread out on clean paper and dried in a cool, shaded area. Seeds treated with four different seed treatment fungicides at three concentrations were used as the treatments, while untreated seeds served as a control. Seeds from each treatment group and the control were sown separately in pots filled with a sterilized mixture of sand, soil, and vermicompost in equal proportions. The experiment was performed in a completely randomized design with three replications for each treatment. Each replication consists of three pots. The cut-stem inoculation method was followed to assess the efficacy of fungicides [28].

We used a sterilized laser-sharp blade to make a precise cut with a blunt end 25 mm above the unifoliate node in 15-day-old plants to test the efficacy of seed treatment fungicides against M. phaseolina. A four-day-old culture of M. phaseolina was placed on the apex of the blunted end of the soybean stem with the help of an autoclaved 20 µL pipette tip (Thermo Fisher Scientific India pvt. Ltd., Ahmadabad, India). The pathogen culture was appropriately inserted to minimize the risk of disease escape. The disease was measured in terms of lesion length (cm) at 2, 4, and 8 days after inoculation (DAI). Infections caused by M. phaseolina at the stem apex often result in rotting and eventual detachment of the stem.

To determine the total length of necrosis, lesion length was measured starting from the unifoliolate node and added to a baseline of 25 mm. If necrosis did not reach the unifoliolate node, the distance from the node to the necrosis was measured and subtracted from the initial 25 mm. The area under disease progress curve (AUDPC) was calculated through a modified trapezoidal method through lesion length according to the formula,

$$\mathrm{A}\mathrm{U}\mathrm{D}\mathrm{P}\mathrm{C}={\sum }_{\mathrm{t}=1}^{\mathrm{n}-1}\left({\displaystyle \frac{{\mathrm{L}}_{\mathrm{i}}+{\mathrm{L}}_{\mathrm{i}+1}}{2}}\right)\left({\mathrm{t}}_{\mathrm{i}+1}-{\mathrm{t}}_{\mathrm{i}}\right)$$

where ${\mathrm{L}}_{\mathrm{i}}$ lesion length (cm) at the ith observation was a time (days) in ith observation, and n is the total number of observations [29]. The entire experiment was carried out inside of a controlled condition, in the most suitable environment (30 ± 2 °C with 14 h of photoperiod), for symptom expression of charcoal rot.

A similar experiment was conducted with the same experiment treatments to determine the most effective seed treatment fungicides against S. rolfsii. The nine-day-old culture was used to inoculate 15-day-old plants as the method prescribed by Ramteke [15]. A mycelial disc of 8 mm was kept near the collar region of the plant in two directions to avoid disease escape in a 15-day-old plant. The collar rot disease was measured in terms of incidence percentage at 7 and 15 DAI with the following formula:

Incidence percentage = ((Total number of plant − no of plant infected)/Total number of plant)) × 100)

Fresh shoots and root weights of every plant were also measured in each treatment after 15 DAI. The experiment was controlled under the most suitable environment (27 ± 2 °C with 14 h of photoperiod) for symptom expression of collar rot.

2.3. Evaluation of Seed Treatment Fungicides Against M. phaseolina and S. rolfsii in Epiphytotic Field Conditions

The field evaluation experiment was performed at the Plant Pathology Field of ICAR-IISR, Indore, for M. phaseolina and at the Department of Plant Pathology of AAU, Jorhat, for S. rolfsii. The seeds of the moderately susceptible genotypes JS 20–29 (for M. phaseolina) and JS 335 (for S. rolfsii) were sown on 27 June 2020, and 1 July 2021, at Indore and on 11 September 2020, and 19 August 2021, at Jorhat. A randomized block design with four replications was utilized. Each replication comprised six lines, each 3 m in length. The rows were spaced 45 cm apart, with 10 cm spacing between plants within each row. Management of insect pests and nutrients was followed as per ICAR-IISR Indore’s recommendation [30,31].

The study evaluated three new combination fungicide seed treatments at two locations, with the treatments defined as follows: trifloxystrobin + penflufen applied at 1 mL/kg of seed, thiophanate-methyl + pyraclostrobin at 2 mL/kg of seed, and carboxin + thiram at 2 g/kg of seed. Additionally, two controls were included: uninoculated and inoculated controls. Fungicide was not applied to seed treatments in either control.

M. phaseolina was incorporated into the soil in all treatments except the uninoculated control treatment at Indore. In contrast, S. rolfsii was incorporated into the soil in all treatments except the uninoculated control treatment at Jorhat. Field inoculation was performed using the method prescribed by Reznikov [23]. For inoculation in field trials at Indore, sterilized sorghum seeds were inoculated with M. phaseolina and incubated for 15 days at 26 ± 1 °C in 14 h of darkness to promote the production of microsclerotia. A similar inoculation was followed for S. rolfsii in field trials at Assam. The fine powder of inoculated sorghum was added at 5 g per meter of linear line length and was placed near the seed at planting depth by hand at sowing time.

The severity of charcoal rot disease was observed at the R₇ stage of soybean in 20 pre-tagged randomly selected plants using 1 to 5 scales and converted to percent disease index (PDI) [32]. The disease incidence was measured as the percentage of death of plants 30 days after sowing (DAS) [15]. The percent disease control (PDC) for both diseases over the inoculated control was calculated [33]. Soybean yield (kg/ha) was measured at harvest. The percentage increase in yield (IY) was calculated over the inoculated control treatment with the formula

IY% = ((Soybean yield (kg/ha) in individual treatment − Soybean yield (kg/ha) in inoculated control) × 100/Soybean yield (kg/ha) in inoculated control))

The seed index, defined as the weight of 100 seeds (g), was measured at harvest. The soybean price was considered based on the minimum support price for the 2021–2022 seasons. The 1 United States dollar (USD) exchange rate was 74.87 Indian rupees. The net return (USD/ha) and BC ratio were calculated to identify the most economical seed treatment fungicide [34].

2.4. Data Analysis

Analysis of variance (ANOVA) and least significant difference (LSD) test were carried out using the R package “agricolae” [35]. Principle component analysis (PCA) was performed for treatment and pathological and agronomical parameters as variables in R Studio version 4.3.0 using the “factoextra” package [36].

3. Results

3.1. Evaluation of New Combination Seed Treatment Fungicides Against M. phaseolina in a Controlled Environment

In the three sampling dates, a highly significant difference (p < 0.001) was observed for lesion length in all the evaluated seed treatment fungicides compared to the control (

Table 1. Lesion length (cm) at 2 DAI, 4 DAI, 8 DAI, and AUDPC in M. phaseolina-inoculated soybean plant treated with new combination seed treatment fungicides in a controlled environment.

Treatment	Doses (g or mL/kg of Seed)	Lesion Length (cm) at 2 DAI	Lesion Length (cm) at 4 DAI	Lesion Length (cm) at 8 DAI	AUDPC
Penflufen + trifloxystrobin	1	1.20 c–f	2.57 e–g	6.58 ij	23.44 d–j
Penflufen + trifloxystrobin	1.5	1.23 cd	2.62 c–f	7.04 c–e	24.56 b–g
Penflufen + trifloxystrobin	2	1.45 b	2.93 b	6.76 g–i	25.24 b–d
Thiophanate methyl + pyraclostrobin	1	0.98 h–j	2.23 k	5.10 k	18.80 k
Thiophanate methyl + pyraclostrobin	1.5	0.98 h–j	2.20 kl	4.94 k	18.52 k
Thiophanate methyl + pyraclostrobin	2	0.85 j	1.96 m	4.86 k	18.04 k
Carboxin + thiram	1	1.25 c	2.79 c	7.18 cd	25.52 bc
Carboxin + thiram	1.5	1.22 c–e	2.69 c–e	7.24 c	25.24 b–e
Carboxin + thiram	2	1.25 c	2.78 cd	7.01 c–f	25.14 b–f
Carbendazim + mancozeb	1	1.18 c–g	2.54 e–i	6.89 e–h	23.94 b–h
Carbendazim + mancozeb	1.5	1.09 d–i	2.47 fj	6.90 e–g	23.68 b–i
Carbendazim + mancozeb	2	1.12 c–h	2.56 e–h	7.68 b	25.60 b
Control	-	2.57 a	3.33 a	8.38 a	30.08 a

The data represent the mean value of three replicates per treatment. Different superscript letters are statistically different at p < 0.05 based on ANOVA and Fisher’s protected Least Significant Difference test.

). Each concentration of every fungicide applied as seed treatment significantly reduced disease symptoms. The seed treatment with thiophanate methyl + pyraclostrobin at 2 mL/kg of seed resulted in the lowest mean lesion length of 0.85 cm at 2 days after inoculation (DAI). It was significantly lower than the control (2.57 cm).

At 4 DAI, seed treatment with thiophanate methyl and pyraclostrobin at 2 mL/kg of seed showed superior efficacy in managing charcoal rot. It resulted in the lowest lesion length of 1.96 cm compared to 3.33 cm in the control. Interestingly, at 8 DAI, the seed treatment with thiophanate methyl + pyraclostrobin at 2 mL/kg of seed was the most effective. It resulted in a minimum lesion length of 4.86 cm, which was significantly lower than the control (8.38 cm). This treatment was statistically comparable to those with thiophanate methyl + pyraclostrobin at 1.5 mL/kg (4.94 cm) and 1 mL/kg (5.10 cm) of seed.

Overall, the lowest AUDPC of 18.04 was recorded with the seed treatment of thiophanate methyl + pyraclostrobin at 2 mL/kg of seed. It achieved a 40.02% reduction in disease compared to the control. This treatment was statistically on par with seed treatment of thiophanate methyl + pyraclostrobin at 1.5 mL/kg (AUDPC of 18.52) of seed and at 1 mL/kg (AUDPC of 18.80) of seed.

3.2. Evaluation of New Combination Seed Treatment Fungicides Against M. phaseolina at Epiphytotic Field Condition

During 2020, the analysis of charcoal rot disease severity did not reveal a significant difference among the treatments included in the present study (p > 0.05) (

Table 2. Mean charcoal rot disease severity (PDI) (%), PDC (%), mean crop yield (Kg/ha), IY (%), net return (USD/ha), BC ratio, mean seed index (g) in epiphytotic field condition after seed treatment of fungicides in Indore during 2020 and 2021 season.

Treatment	PDI%			PDC (%)	Yield (kg/ha)			IY (%)	Net Return ($/ha)	BC Ratio	Seed Index (g)
	2020	2021	Pooled		2020	2021	Pooled				2020	2021	Pooled
Penflufen + trifloxystrobin	5.09 a	51.25 c	28.17 c	29.98	1301.75 a	1820.04 a	1560.89 a	32.02	494.09	3.13	12.70 a	13.14 ab	12.92 a
Thiophanate methyl + pyraclostrobin	4.28 a	50.00 C	27.14 c	32.54	1329.21 a	1827.40 a	1578.31 a	33.50	499.34	3.12	12.81 a	13.24 ab	13.03 a
Carboxin + thiram	5.16 a	64.00 b	34.58 b	14.04	1311.93 a	1545.19 c	1428.56 b	20.83	432.05	2.86	12.58 a	12.54 b	12.56 b
Uninoculated control	4.75 a	32.25 d	17.44 d	56.65	1367.88 a	1830.11 a	1586.50 a	34.19	508.64	3.21	12.87 a	13.77 a	13.32 a
Inoculated Control	5.71 a	74.75 a	40.23 a	-	1211.11 a	1153.47 d	1182.29 d	-	343.82	2.50	12.34 a	10.96 c	11.65 c

The data represent the mean value of four replicates per treatment. Different superscript letters are statistically different at p < 0.05 based on ANOVA and Fisher’s protected Least Significant Difference test. The same superscript letters are statistically nonsignificant at p < 0.05 based on ANOVA. Details of treatments were provided in Materials and Methods.

). Interestingly, among seed treatment fungicides, thiophanate methyl + pyraclostrobin achieved 25.04% more disease control (PDC) than the inoculated control. It recorded the lowest PDI of 4.28%. No significant differences in crop yield or seed index were observed among the treatments in this study (p > 0.05) (Table 2). Interestingly, among seed treatment fungicides thiophanate methyl + pyraclostrobin produced 118.10 kg/ha more soybean grain yield than the inoculated control (1211.11 kg/ha). It also provided a maximum seed index of 12.81 g.

During 2021, the analysis of charcoal rot disease PDI, soybean yield and seed index exhibited a significant difference among the treatments (p < 0.0001) (Table 2). This indicated that the PDI of charcoal rot disease, soybean yield and seed index changed significantly due to seed treatment with different fungicides. The inoculated control showed the maximum charcoal rot disease PDI of 74.75%. It showed a significant difference compared to the uninoculated control (32.25%), thiophanate methyl + pyraclostrobin (50.00%), penflufen + trifloxystrobin (51.25%) and carboxin + thiram (64.00%). Concerning crop yield, treatments with the uninoculated control (1830.11 kg/ha), thiophanate methyl + pyraclostrobin (1827.40 kg/ha), and penflufen + trifloxystrobin (1820.04 kg/ha) differed statistically compared to treatments with carboxin + thiram (1545.19 kg/ha) and the inoculated control (1153.47 kg/ha). Seed treatment with thiophanate methyl + pyraclostrobin produced 673.93 kg/ha more soybean crop yield than inoculated control. In comparison to inoculated control, penflufen + trifloxystrobin and carboxin + thiram showed an increase of 666.57 kg/ha and 391.72 kg/ha, respectively. As for seed index, the uninoculated control (13.77 g) differed statistically from carboxin + thiram (12.54 g) and the inoculated control (10.96 g). It did not show a significant difference with thiophanate methyl + pyraclostrobin (13.24 g) and penflufen + trifloxystrobin (13.14 g) (Table 2).

The pooled analysis of charcoal rot disease PDI exhibited a significant difference among the treatment (p < 0.0001), year (p < 0.0001), and treatment × year (p < 0.0001) included in the present study (

Table 3. Analysis of variance to identify effective seed treatment fungicide against charcoal rot and collar rot diseases of soybean.

Source of Variation	Charcoal Rot				Collar Rot
	PDI%				Incidence%
	Degree of Freedom	Mean Sum Square	F Value	p > F	Degree of Freedom	Mean Sum Square	F Value	p > F
Replication	3	5.7	0.49	NS *	3	12.49	1.64	NS
Treatment	4	587.5	51.74	0.0001	4	272.35	35.80	0.0001
Time	1	24,874.7	2190.96	0.0001	1	37.78	4.96	NS
Treatment × Time	4	444.4	39.14	0.0001	4	0.32	0.04	NS
Yield (Kg/ha)
Replication	3	19,374	5.94	NS	3	2427.00	0.22	NS
Treatment	4	262,633	80.65	0.0001	4	600,699.00	55.63	0.0001
Year	1	1,266,391	388.92	0.0001	1	109,685.00	10.21	NS
Treatment × Time	4	217,574	66.81	0.0001	4	10,157.00	0.94	NS
Seed index (g)
Replication	3	0.32	1.32	NS	3	1.82	2.19	NS
Treatment	4	3.31	13.68	0.0001	4	3.37	4.06	NS
Time	1	0.05	0.21	NS	1	0.59	0.71	NS
Treatment × Time	4	1.53	6.32	0.001	4	0.24	0.29	NS

* NS is non significance.

). This revealed that charcoal rot disease PDI changed significantly across all treatments, evaluated by year, and the interaction between treatments and year. Charcoal rot disease PDI was significantly higher during the year 2021 (54.45%) compared to the year 2020 (4.57%). The uninoculated control showed minimum charcoal rot disease PDI 17.44% with 56.65% of PDC (Supplementary Figure S2). Among seed treatment fungicides thiophanate methyl + pyraclostrobin (27.14%) and penflufen + trifloxystrobin (28.17%) showed significantly lower charcoal rot disease PDI than the inoculated control (40.23%) and carboxin + thiram (34.58%) (Table 2).

The pooled analysis of soybean yield exhibited a significant difference among the treatment (p < 0.0001), year (p < 0.0001), and treatment × year (p < 0.0001) included in the present study (Table 3). This showed that soybean yield changed significantly across all treatments, evaluated year, and the interaction between treatments and year. Soybean yield was significantly higher in 2021 (1635.24 kg/ha) than in 2020 (1299.38 kg/ha). The seed treatment fungicide thiophanate methyl + pyraclostrobin (1578.31 kg/ha) and penflufen + trifloxystrobin (1560.89 kg/ha) did not differ statistically compared to the uninoculated control (1586.50 kg/ha). It showed statistically significant higher soybean yield than the inoculated control (1182.29 kg/ha) and carboxin + thiram (1428.56 kg/ha) (Supplementary Figure S3).

Seed treatments with fungicides thiophanate-methyl + pyraclostrobin, penflufen + trifloxystrobin, and carboxin + thiram resulted in increased yields (IY) of 33.50%, 32.02%, and 20.83%, respectively, compared to the inoculated control. The highest IY of 34.19% was observed in the uninoculated control over inoculated control. The maximum net return of USD 508.64/ha and BC ratio of 3.21 were observed with uninoculated control. Seed treatments with fungicides thiophanate-methyl + pyraclostrobin, penflufen + trifloxystrobin, and carboxin + thiram, increased net returns by USD 155.52/ha, USD 150.27/ha, and USD 88.23/ha, respectively, compared to the inoculated control. The seed treatment fungicides thiophanate-methyl + pyraclostrobin and penflufen + trifloxystrobin resulted in BC ratios that were 0.08 and 0.09 less than the uninoculated control, respectively. However, they have provided 0.63 and 0.62 higher BC ratios compared to the inoculated control, respectively. The pooled analysis of the seed index exhibited a significant difference between the treatment (p < 0.0001) and treatment × time (p < 0.01). It did not show a significant difference with time (p > 0.01), which was included in the present study (Table 3). This revealed that the seed index changed significantly across all treatments and interactions between treatments and years, but it did not change with the year of evaluation. The seed treatment fungicide thiophanate methyl + pyraclostrobin (13.03 g) and penflufen + trifloxystrobin (12.92 g) did not differ statistically to the uninoculated control (13.32 g). It showed a statistically significant higher seed index than the inoculated control (11.65 g) and carboxin + thiram (12.56 g) (Table 2).

3.3. Evaluation of New Combination Seed Treatment Fungicides Against S. rolfsii in Controlled Environment

The statistical analysis of the incidence of disease over two time periods, shoot and root weight, showed significant differences (p < 0.001) among the treatments included in the present study (

Table 4. Incidence (%) at 7 DAI, 15 DAI, shoot weight (g), and root weight (g) in S. rolfsii-inoculated soybean plant treated with new combination seed treatment fungicides in a controlled environment.

Treatment	Doses (g or mL/kg of Seed)	Incidence% at 7 DAI	Incidence% at 15 DAI	Shoot Weight (g)	Root Weight (g)
Penflufen + trifloxystrobin	1	26.19 f–h	31.75 g–j	1.35 hi	0.91 g–i
Penflufen + trifloxystrobin	1.5	27.30 f–g	33.97 fg	1.22 ij	0.83 ij
Penflufen + trifloxystrobin	2	28.97 e	37.30 e	1.17 i–k	0.72 j
Thiophanate methyl + pyraclostrobin	1	24.52 g–j	32.86 g–h	1.47 f–h	1.26 a–d
Thiophanate methyl + pyraclostrobin	1.5	20.95 kl	25.71 l	1.81 ab	1.26 a–d
Thiophanate methyl + pyraclostrobin	2	16.19 m	20.95 m	1.98 a	1.38 a
Carboxin + thiram	1	27.78 f	36.11 ef	1.61 b–g	1.01 fg
Carboxin + thiram	1.5	25.56 f–i	32.22 g–i	1.65 b–f	1.13 d–f
Carboxin + thiram	2	21.75 k	27.30 k	1.69 b–e	1.19 b–e
Carbendazim + mancozeb	1	50.00 bc	55.56 bc	1.75 b–d	1.28 a–c
Carbendazim + mancozeb	1.5	44.29 d	49.05 d	1.75 b–d	1.31 ab
Carbendazim + mancozeb	2	51.11 b	57.78 b	1.76 bc	0.98 gh
Control	-	68.25 a	73.81 a	0.96 k	0.69 j

The data represent the mean value of four replicates per treatment. Different superscript letters are statistically different at p < 0.05 based on ANOVA and Fisher’s protected Least Significant Difference test.

). All the fungicides applied as seed treatment significantly reduced collar rot disease incidence. The seed treatment with thiophanate methyl + pyraclostrobin at a rate of 2 mL/kg of seed achieved the lowest collar rot disease incidence, with 16.19% at 7 DAI and 20.95% at 14 DAI. It was significantly outperforming all other treatments. This treatment reduced disease symptoms by 68.25% at 7 DAI and 73.81% at 14 DAI compared to control. All seed treatment fungicides included in the present study enhanced inoculated shoot and root weight. The seed treatment with thiophanate methyl + pyraclostrobin at 2 mL/kg of seed achieved a maximum shoot weight of 1.98 g and a root weight of 1.38 g. This treatment was significantly superior to all others for both shoot and root weight. Exceptions treatment thiophanate methyl + pyraclostrobin at 1.5 mL/kg for both shoot (1.81 g) and root weight (1.26 g) or 1 mL/kg for root weight (1.26 g, which did not show signifcnace difference to thiophanate methyl + pyraclostrobin at 2 mL/kg of seed).

3.4. Evaluation of Combination Seed Treatment Fungicides Against S. rolfsii at Epiphytotic Field Condition

During both Kharif 2020 and 2021, the analysis of collar rot disease incidence and soybean yield revealed a significant difference (p < 0.0001) among the treatments included in the present study (Table 3). This indicated that collar rot disease incidence and soybean yield differed significantly due to the different seed treatment fungicides used in both years.

During Kharif 2020, the inoculated control exhibited maximum collar rot disease incidence 19.42%. It showed a significant difference with the uninoculated control (4.10%), thiophanate methyl + pyraclostrobin (7.32%), penflufen + trifloxystrobin (8.11%) and carboxin + thiram (10.16%). Concerning crop yield, uninoculated control (2410.69 kg/ha), thiophanate methyl + pyraclostrobin (2353.47 kg/ha), penflufen + trifloxystrobin (2331.94 kg/ha), and carboxin + thiram (2284.72 kg/ha) did not differ statistically from each other. They showed a significant higher yield than the inoculated control (1887.50 kg/ha). Seed treatment with thiophanate methyl + pyraclostrobin increased yield by 465.97 kg/ha. Penflufen + trifloxystrobin increased yield by 444.44 kg/ha, while carboxin + thiram increased it by 397.22 kg/ha compared to the inoculated control. All three seed treatment fungicides did not differ statistically from each other regarding collar rot disease incidence and crop yield.

During Kharif 2021, the inoculated control exhibited the highest collar rot disease incidence at 21.25%, which was significantly higher compared to the uninoculated control (5.41%), thiophanate methyl + pyraclostrobin (9.31%), penflufen + trifloxystrobin (10.44%), and carboxin + thiram (12.41%). Concerning crop yield, the uninoculated control (2392.94 kg/ha), thiophanate methyl + pyraclostrobin (2347.37 kg/ha), and penflufen + trifloxystrobin (2275.66 kg/ha) differed statistically with treatments of carboxin + thiram (2158.79 kg/ha) and the inoculated control (1684.91 kg/ha). Seed treatment with fungicide thiophanate methyl + pyraclostrobin produced 662.47 kg/ha more soybean grain yield than the inoculated control. In comparison, penflufen + trifloxystrobin and carboxin + thiram showed an increase of 590.75 kg/ha and 473.88 kg/ha, respectively. Among seed treatment fungicides, thiophanate methyl + pyraclostrobin and penflufen + trifloxystrobin showed significantly lower collar rot disease incidence and higher crop yield than carboxin + thiram. During both Kharif 2020 and 2021, the analysis of the seed index did not reveal a significant difference among the treatments included in the present study (p > 0.05). Interestingly, the uninoculated control treatment recorded the highest seed index in both years, with 12.46 g in 2020 and 12.29 g in 2021.

The pooled analysis of collar rot disease incidence (p < 0.0001) and soybean yield (p < 0.0001) exhibited a significant difference among the treatments. They did not change significantly over the evaluated year, and the interaction between treatments and year (p > 0.05) (Table 3). This revealed that collar rot disease incidence and soybean yield changed significantly across all treatments. The uninoculated control showed a minimum collar rot disease incidence of 4.75% with a maximum PDC of 76.64% (Supplementary Figure S2). Among the seed treatment fungicides thiophanate methyl + pyraclostrobin (8.32%) exhibited significantly lower collar rot disease incidence compared to the inoculated control (20.33%) and carboxin + thiram (11.29%). However, it did not differ statistically from penflufen + trifloxystrobin (9.27%) (

Table 5. Mean collar rot incidence (%), PDC (%), mean crop yield (Kg/ha), IY (%), BC ratio, mean seed index (g) in epiphytotic field condition after seed treatment of fungicides in Jorhat during 2020 and 2021 season.

Treatment	Incidence (%)			PDC (%)	Yield (Kg/ha)			IY (%)	Net Return (USD/ha)	BC Ratio	Seed Index (g)
	2020	2021	Pooled		2020	2021	Pooled				2020	2021	Pooled
Penflufen + trifloxystrobin	8.11 bc	10.44 bc	9.27 bc	54.40	2331.94 a	2275.66 ab	2303.80 ab	28.98	840.40	4.62	11.89 a	11.65 a	11.77 a
Thiophanate methyl + pyraclostrobin	7.32 bc	9.31 c	8.32 c	59.08	2353.47 a	2347.37 a	2350.42 a	31.59	858.98	4.65	12.26 a	12.19 a	12.23 a
Carboxin + thiram	10.16 b	12.41 b	11.29 b	44.47	2284.72 a	2158.79 b	2221.75 b	24.38	801.63	4.44	11.80 a	11.27 a	11.54 a
Uninoculated control	4.10 c	5.41 d	4.75 d	76.64	2410.69 a	2392.94 a	2401.82 a	34.47	888.49	4.87	12.46 a	12.29 a	12.38 a
Inoculated control	19.42 a	21.25 a	20.33 a	-	1887.50 b	1684.91 c	1786.20 c	-	601.86	3.62	11.54 a	11.31 a	11.43 a

The data represent the mean value of three replicates per treatment. Different superscript letters are statistically different at p < 0.05 based on ANOVA and Fisher’s protected Least Significant Difference test. The same superscript letters are statistically nonsignificant at p < 0.05 based on ANOVA. Details of treatments were provided in materials and methods.

).

The seed treatment fungicide thiophanate methyl + pyraclostrobin (2350.42 kg/ha) did not differ statistically to the uninoculated control (2401.82 kg/ha) and penflufen + trifloxystrobin (2303.80 kg/ha). It showed statistically significant higher soybean yield than the inoculated control (1786.20 kg/ha) and carboxin + thiram (2221.75 kg/ha) (Supplementary Figure S3). Seed treatments with the fungicides thiophanate-methyl + pyraclostrobin, penflufen + trifloxystrobin and carboxin + thiram resulted in increased yields (IY) of 31.59%, 28.98% and 24.38%, respectively, compared to the inoculated control.

The highest IY of 34.47% was observed in the uninoculated control over inoculated control. The maximum net return of USD 888.49/ha and BC ratio of 4.87 were observed with uninoculated control. Compared to the inoculated control seed treatments with fungicides thiophanate-methyl + pyraclostrobin, penflufen + trifloxystrobin and carboxin + thiram increased net returns by USD 257.12/ha, USD 238.54/ha and USD 199.77/ha, respectively. The seed treatment fungicides thiophanate-methyl + pyraclostrobin and penflufen + trifloxystrobin resulted in BC ratios of 0.22 and 0.25 lower than the uninoculated control. Still, they were 1.03 and 1.00 higher, respectively, than the inoculated control.

The pooled analysis of the seed index did not exhibit a significant difference between the treatment, evaluated year, and the interaction between treatments and year (p > 0.05). This revealed that the seed index did not change significantly across all treatments, evaluated year, and the interaction between treatments and year. The maximum seed index of 12.38 g was recorded in the inoculated control. It was 0.95 g higher than that of the uninoculated control (Table 5).

3.5. Principle Component Analysis (PCA)

PCA was performed to recognize the key independent variables and enumerate the total variance explained by select components. The PCA was performed with pathological and agronomical parameters obtained through field experiments over consecutive years for both diseases. The analysis unveiled principal components, each accompanied by their respective proportions of explained variation, as determined by eigenvectors. The first dimension of principal components (PCI and PC II) explained 97.30% of the total variation given by variables, which captured the maximum variation present in pathological and agronomical parameters. Four clusters were created through PCA.

The PCA analysis categorized the treatments into four clusters (Figure 1). Cluster I comprised the seed treatment fungicide carboxin + thiram, which showed a positive association with charcoal rot disease parameters. Cluster II included the inoculated control, which was positively associated with collar rot disease parameters but negatively associated with agronomic traits. Cluster III included the seed treatment fungicide thiophanate methyl + pyraclostrobin and the uninoculated control, both positively associated with the seed index at Jorhat and Indore in 2020, percentage disease control of charcoal rot and negatively associated with charcoal rot disease parameters. Cluster IV consisted of the seed treatment fungicide penflufen + trifloxystrobin, which was positively associated with the yield at both locations and years, the percentage disease control of collar rot, as well as with the seed index at Indore, economic parameters at both locations, but negatively associated with collar rot disease parameters.

4. Discussions

The effective management of soil-borne diseases in soybeans through resistant cultivars has been challenging due to the non-availability of commercial cultivars with robust and consistent resistance. Simultaneously, the shift from traditional soybean–wheat crop rotations to soybean–chickpea crop rotations in India has enhanced soil-borne diseases [15]. Subsequently, the use of combination seed treatment fungicides, which protect the seeds and seedlings from early infections caused by M. phaseolina and S. rolfsii, has become a more common management practice in soybean cultivation against soil-borne diseases [19,20,21,22,23,37,38,39].

Therefore, the current study was focused on evaluating new combination seed treatment fungicides against M. phaseolina and S. rolfsii that could be supportive in minimizing the incidence of soil-borne pathogens and enhancing agronomical parameters in soybean crops. The present study revealed that all the tested concentrations of three combination seed-treatment fungicides, carboxin + thiram, trifloxystrobin + penflufen, and thiophanate methyl + pyraclostrobin, significantly reduced disease symptoms under controlled and field conditions.

The inhibition of phytopathogens is attributed to trifloxystrobin and pyraclostrobin binding to the quinol oxidation site of cytochrome b in mitochondria. This action disrupts respiration by blocking electron transfer from cytochrome b to cytochrome c1, halting energy synthesis and reducing phytopathogen growth [40]. They are highly effective before infection or during the initial stages of disease development. This is due to their strong antagonistic effects on spore germination. This stage is a high-energy-intensive stage of fungal growth and development [40].

On the other hand, penflufen and carboxin inhibit the enzymatic activity of succinate dehydrogenase, which leads to energy production by complete inhibition of oxidative phosphorylation and tricarboxylic acid cycle in fungi [41]. They were also found effective in seed dressing by inhibiting or delaying spore germination in phytopathogenic fungi. Succinate dehydrogenase inhibitor compounds can also induce apoptosis in cells of phytopathogens by disrupting the cell wall and cell membrane [41].

The fungicide thiophanate methyl is initially converted into carbendazim. Carbendazim binds to β-tubulin, a protein component of microtubules. This binding inhibits the polymerization of microtubules, which are necessary for chromosome separation during mitosis. Ultimately, it leads to abnormal growth and distortion of germ tubes in fungal spores, thereby inhibiting the growth and reproduction of the fungi [42]. The fungicide thiram exhibits multisite activity by inhibiting metal and sulfhydryl enzymes critical for fungal growth and reproduction. It interferes with the respiratory processes in the mitochondria of fungal cells and disrupts their lipid metabolism, compromising the integrity of their cellular membranes [42].

Under controlled conditions, the combination of thiophanate methyl + pyraclostrobin at a rate of 2 mL/kg of seed significantly reduced the AUDPC of charcoal rot disease by 40.02% and decreased the incidence of collar rot by 71.61%. Moreover, it also promoted significant growth in S. rolfsii-infected plants, increasing shoot weight by 51.51% and root weight by 50.00%. Thiophanate methyl + pyraclostrobin applied at concentrations of 1 mL/kg and 1.5 mL/kg of seed exhibited comparable efficacy in significantly reducing the AUDPC of charcoal rot disease. Supporting our study, a combination of seed treatment fungicides thiophanate-methyl and pyraclostrobin significantly reduced M. phaseolina lesion length in soybeans compared to the control under controlled environmental conditions [43].

Similarly, in addition to reducing root rot incidence in coleus caused by M. phaseolina, thiophanate methyl has also been reported to enhance tuber length and weight in infected plants [44]. In another study, seed treatments with thiophanate methyl significantly reduced S. rolfsii infection in chickpeas under controlled conditions and improved crop agronomical parameters [45]. Several combination seed treatment fungicides, including various fungicide groups such as strobilurin + benzimidazole, strobilurin + imidazole, and dithiocarbamate + benzimidazole effectively protected groundnut plants against S. rolfsii under controlled conditions, and resulting in complete inhibition of sclerotia production [46].

Under epiphytotic field conditions, seed treatment with the combination fungicides thiophanate methyl + pyraclostrobin or penflufen + trifloxystrobin was equally effective in reducing the severity of charcoal rot disease and the incidence of collar rot disease. Both treatments significantly enhanced soybean yield and seed index compared to the inoculated control. PCA analysis revealed that the combination fungicide thiophanate methyl + pyraclostrobin was closely associated with charcoal rot management. At the same time, penflufen + trifloxystrobin was associated with improving agronomic and economic parameters and effectively managing collar rot disease. Seed treatment with thiophanate methyl + pyraclostrobin reduced the effective and essential population of M. phaseolina to establish a parasitic relationship with the soybean host and enhanced soybean yield and emergence percentage [23]. In soybean crops, other combination seed treatment fungicides carboxin + thiram, thiophanate methyl, and carbendazim + mancozeb were also found effective in the management of charcoal rot disease severity in soybean and enhancing soybean yield [47,48].

The application of pyraclostrobin reduced the number of the sclerotial body in S. rolfsii through the reduction in oxalate secretion and protein synthesis [49]. Seed treatments with thiophanate methyl + T. asperellum and pyraclostrobin + Trichoderma asperellum provided 85.83% and 73.25% control of fruit rot disease caused by S. rolfsii in pumpkins, respectively, compared to the untreated control. Both treatments also significantly enhanced fruit yield [50]. Combination seed treatment fungicides also significantly reduced the lesions developed by seedling soil-borne pathogens in soybeans [19,20,21,22,37,38,39].

The seed treatment fungicides used in this study improved soybean plants’ yield and seed index, which were challenged by soil-borne phytopathogens. Seed treatment with systemic fungicides may improve plant health, reducing lesion length and incidence of phytopathogens compared to untreated inoculated plants. Previous research has shown systemic fungicides effectively inhibit fungal growth and development by being absorbed into germinating soybean seeds and translocating to various parts of the seedling, including the roots, stems, and leaves [51]. Furthermore, systemic fungicides, particularly those from the strobilurin/QoI group, improved photosynthesis, leading to increased yields [52].

Under epiphytotic field conditions at Indore, the severity of charcoal rot disease was significantly lower in 2020 compared to 2021. The disease did not reach its full potential in 2020, making seed treatment fungicides less effective. Consequently, seed treatment with fungicides showed no significant impact on crop yield or seed index in 2020 compared to 2021. This outcome was influenced by weather conditions, as the cropping season in 2020 experienced significantly higher rainfall, with 901.19 mm received between June and August. In contrast, a drought spell occurred during the same period in 2021, with only 396.24 mm of rainfall. Under drought or drought-like conditions at the maturity stage, the phytopathogen M. phaseolina becomes more virulent, resulting in the typical symptoms of charcoal rot infection [10,12,13].

Drought or drought-like conditions, combined with high-temperature stress during the R6 stage, created an ideal environment for charcoal rot infection [13]. The seed treatment fungicide combination thiophanate methyl + pyraclostrobin demonstrated greater efficacy against charcoal rot under drought or drought-like conditions [23]. In contrast, high rainfall and waterlogged, low-lying soil conditions favored the predominance of collar rot [15]. The efficacy of the seed treatment combination fungicides fludioxonil + mefenoxam and azoxystrobin + metalaxyl against soil-borne pathogens was significantly higher under high-moisture conditions. These pathogens thrived under low-moisture conditions [19].

Seed treatment with a combination fungicide penflufen + trifloxystrobin was moderately effective against both diseases in a controlled environment but highly effective under epiphytotic field conditions. Similarly, findings have been demonstrated previously that the combination fungicide penflufen + trifloxystrobin + metalaxyl was found to be less effective in controlling soil-borne seedling diseases of soybeans under controlled conditions compared to field conditions [21]. This is because, in controlled conditions, environmental factors such as temperature, relative humidity, and soil moisture are managed carefully, which improves the efficacy of fungicides against pathogens. In contrast, in field conditions, variable environmental factors, including rainfall, fluctuating relative humidity, wind, and changing soil and air temperatures, can reduce the effectiveness of fungicides in controlling pathogens [53,54,55]. In controlled conditions, the inoculum levels of phytopathogens are fixed and maintained within regulated environmental parameters. In contrast, in field conditions, the level of phytopathogens is influenced by the soil microbiome and various edaphic and non-edaphic factors affecting the soil [3].

Across locations, the average net return from using combination fungicides as seed treatments was USD 654.41/ha, showing an increase of USD 181.57/ha compared to the net return from the inoculated control with untreated seeds. The higher net returns from fungicide seed treatments in soybeans align with findings from previous studies. Seed treatments using combination fungicides, such as Fludioxonil + Mefenoxam and Azoxystrobin + Metalaxyl, generated an additional USD 22/ha in net return compared to untreated seeds in the disease field [19]. In another study, seed treatments with fungicides in soybean crops increased growers’ profits by USD 43.71/ha [56].

Among the seed treatment fungicides, thiophanate methyl + pyraclostrobin provided the highest average net return and BC ratio across locations. However, these values were USD 19.41/ha and 0.16 lower, respectively, compared to the uninoculated control with untreated seeds. This is because the disease pressure did not increase up to the economic threshold level. Therefore, the cost of fungicide was not compensated by enhanced yield. In this regard, it was observed that yield enhancement in susceptible cultivars could not compensate for the increased cost of fungicide due to additional fungicide requirements [57]. Previous research has also shown that when disease pressure is low, the application of fungicides results in only a slight increase in soybean yield, making it less profitable for managing soybean diseases [58,59]. The lower BC ratio was attributed to India’s higher cost of plant protection chemicals. A previous study found that using expensive fungicides on susceptible soybean cultivars in India significantly increased the cost of cultivation for managing soybean anthracnose [34].

This study provided evidence that seed treatments with combination fungicides, thiophanate-methyl + pyraclostrobin or penflufen + trifloxystrobin, were highly effective. These fungicides showed high efficacy under climatic conditions that favored the proliferation of soil-borne pathogens affecting the soybean. These findings will be valuable for Indian farmers as they will help them develop more effective management strategies to combat charcoal rot and collar rot in soybean cultivation. Further studies can be conducted across various soybean-growing regions of India to evaluate the efficacy of these chemical treatments against other soil-borne pathogens and to understand their interactions under diverse environmental conditions. It was essential to acknowledge that these trials assessed combination fungicides as seed treatments on a single soybean cultivar, and the response to seed treatments varied across different cultivars. Therefore, further investigations can be focused on understanding the interaction between climate, chemical treatments, pathogens, and soybean varieties.

5. Conclusions

In the current study, seed treatment with a combination of fungicides thiophanate methyl + pyraclostrobin or penflufen + trifloxystrobin was most effective in managing charcoal rot and collar rot diseases. Apart from disease management, seed treatment with combination fungicides also enhanced the agronomical parameters of soybeans, such as root weight, shoot weight, and yield. Results obtained from this study on soybeans will be helpful for farmers in developing more efficient management strategies for charcoal rot and collar rot in India. The efficacy of these combination seed treatment fungicides depended upon climatic conditions favorable for the proliferation of soil-borne pathogens. There is a need to shift the focus from seed treatment fungicides to integrated disease management strategies. This includes developing and using moderately resistant cultivars and incorporating beneficial microbes that enhance plant and soil health to reduce disease incidence.