Combined Biological and Chemical Control of Sclerotinia sclerotiorum on Oilseed Rape in the Era of Climate Change

Abstract

This study investigates the biocontrol potential of Trichoderma asperellum and Coniothyrium minitans against the pathogen Sclerotinia sclerotiorum, which causes yield losses in many plants, including oilseed rape (Brassica napus) cultivation. This research emphasizes the promising alternative of hybrid control, specifically using T. asperellum and C. minitans in strategy with synthetic fungicides. In vitro experiments demonstrated that T. asperellum effectively inhibited S. sclerotiorum mycelial growth, especially when combined with synthetic fungicides such as azoxystrobin. Field trials conducted over two years revealed that pre-sowing applications of T. asperellum and C. minitans, followed by fungicide treatments during the flowering stage, significantly reduced plant infection rates and improved both yield and seed quality across different oilseed rape cultivars. The results indicated an efficacy range of 81% to 100% in controlling the pathogen and highlighted the synergistic effects of combining biological and chemical controls. Overall, the research findings support the integration of T. asperellum and C. minitans into sustainable agricultural practices for oilseed rape, offering a viable strategy to enhance disease management while reducing reliance on chemical fungicides. This research underscores the importance of adopting innovative biocontrol approaches to improve crop health and productivity.

1. Introduction

Sclerotinia sclerotiorum is a soilborne pathogen that causes significant disease in a wide range of crops, including oilseed rape (Brassica napus). This fungal pathogen is responsible for the disease known as Sclerotinia stem rot or white mold, which can lead to yield losses in oilseed rape cultivation. Climate change, characterized by rising global temperatures and altered precipitation patterns, is expected to profoundly affect the biology and control of this pathogen, with implications for disease incidence and management strategies worldwide. Climate change affects S. sclerotiorum both directly and indirectly. The direct effects are related to the immediate responses of the pathogen to altered temperature and moisture conditions, influencing processes such as sclerotial germination and apothecia formation [1,2]. Indirect effects involve changes in the host plant phenology, notably the acceleration of flowering periods in crops like oilseed rape due to warming, which in turn alters the window of susceptibility to infection [2].

Weather conditions such as rainfall and relative humidity are also crucial in disease development. High moisture levels promote ascospore germination and infection, while dry conditions limit disease progress [3]. Forecasting models integrating temperature, moisture, and other meteorological data have been developed to predict apothecial risk and optimize fungicide application timing, improving disease management efficiency [1,4]. However, climate variability and extremes challenge these models, necessitating continuous adaptation of management strategies

The pathogen produces sclerotia, which are fungal structures that can survive in the soil for even 10 years, making its control challenging. In oilseed rape cultivation, S. sclerotiorum can infect various parts of the plants, including stems, leaves, and flowers, leading to decay and wilting. The pathogen can develop in conditions of high humidity and moderate temperatures, often leading to outbreaks during flowering stage when the plants are most susceptible [5]. Studies made by Brand and Zamani-Noor [6] demonstrates that temperatures above 15 °C combined with soil moisture exceeding 75% provide optimal conditions for sclerotia germination, whereas temperatures between 5 °C and 15 °C promote apothecial formation. Thus, the optimal temperature for the development of apthecia is 5–15 °C [7] The presence of sclerotia in the soil serves as a long-term source of inoculum, contributing to the recurring nature of the disease in fields where oilseed rape is grown. Cultural practices such as crop rotation, proper field drainage, and the use of resistant varieties are commonly implemented to manage S. sclerotiorum in oilseed rape production. However, these methods often provide limited efficacy due to the pathogen’s broad host range and persistent nature [8]. Moreover, the reliance on fungicides for control has raised concerns regarding the development of resistance and the environmental impact of chemical treatments [9]. In the era of withdrawal of active substances of fungicides, there is a need to deliver new solutions in S. sclerotiorum control. One alternative strategy is the use of biocontrol agents such as Trichoderma, which can effectively inhibit the pathogen’s growth and reduce disease severity through mechanisms such as mycoparasitism and competition [10]. For instance, Trichoderma harzianum has demonstrated significant potential in biocontrol against S. sclerotiorum, providing an environmentally friendly approach in managing white mold in oilseed rape [11]. Hybrid plant protection, which involves combining the use of biological products with synthetic active ingredients, offers many advantages. One of them is the reduced use of chemical products, which contributes to environmental protection. The use of bioproducts, also through combined application with fungicides, fits into integrated pest management (IPM) strategies. Thanks to pest-targeted products, it is possible to tailor actions to a specific location and timing. Moreover, well-designed hybrid protection is more effective, offering better control efficacy at lower long-term costs.

In the last few years, new Trichoderma strains were developed. One of the most common and registered for agricultural production is T. asperellum., which is a filamentous fungi of the genus Trichoderma that is widely known as a biocontrol agent against various plant pathogens. The fungus is predominantly obtained from soil and decaying plant material and has been linked to its ability to promote plant growth and enhance soil health through multiple mechanisms [12,13]. T. asperellum is fast-growing and has the capability of producing conidia, which are spores for its distribution and are produced asexually. Fungi are so flexible in nature that they can easily grow in almost all kinds of different environmental conditions, and because of this, they are a good option for the agricultural industry. As a biocontrol agent, T. asperellum employs a number of mechanisms to suppress plant pathogens. These include competition for nutrients and space, the production of antifungal metabolites, and mycoparasitism, where T. asperellum infects and degrades the mycelium of other fungi [14]. T. asperellum has been shown to induce systemic resistance in plants, which enhances their resistance to a range of biotic stresses [15,16]. Due to its efficiency and environmentally friendly nature, T. asperellum is a subject of study for sustainable agriculture that offers an alternative to chemical fungicides and healthier crop production [17]. T. asperellum can be also used with other microbial products, like Bacillus spp., with potential synergistic activity in disease control [18]. One of the most common methods involves the incorporation of T. asperellum into the soil. This can be done by mixing the fungal spores with organic fertilizers or soil amendments before sowing the plants. This method enhances root colonization and promotes plant growth by improving nutrient uptake and disease resistance [19]. T. asperellum can be also applied as a seed treatment. Seeds are coated with a solution of spores or a talc-based formulation. This method helps in the early establishment of the fungus in the rhizosphere, providing protection against soil-borne pathogens [20]. T. asperellum can be used for controlling foliar pathogens and enhancing plant health by inducing systemic resistance [21]. The introduction of novel microencapsulation for T. asperellum in various matrices reduces the impact of environmental stressors on its shelf life and effectiveness [22].

Coniothyrium minitans is a fungal mycoparasite that specifically targets the sclerotia of S. sclerotiorum. The pathogen survives in soil primarily through sclerotia, which germinate to infect plants, leading to significant yield losses. C. minitans parasitizes the sclerotia of S. sclerotiorum by penetrating their melanized rind through enzymatic degradation and physical pressure, eventually destroying the sclerotia and reducing the inoculum potential in the soil. The fungus produces enzymes such as β-1,3-glucanase and chitinases that degrade the fungal cell walls of the pathogen, facilitating effective colonization and mycoparasitism [23]. This mode of action makes C. minitans highly specific and effective against S. sclerotiorum sclerotia. However, environmental factors such as soil moisture and temperature influence its efficacy and persistence [24].

The aim of this study was to evaluate the influence of T. asperellum on S. sclerotiorum mycelium growth inhibition in vitro and T. asperellum and C. minitans on S. sclerotiorum control in strategy with fungicides under field conditions in oilseed rape cultivation including its impact on yield and quality of the seeds.

2. Materials and Methods

2.1. In Vitro Experiment

In an in vitro experiment, the growth of mycelium after the use of Trichoderma asperellum containing product was assessed. This research used a preparation based on prothioconazole (Promino 300 SC; Helm Polska Sp. z o.o., Warsaw, Poland); tebuconazole (Tebu 250 EW; Helm Polska Sp. z o.o., Warsaw, Poland); and azoxystrobin (Amistar 250 SC; Syngenta Polska Sp. z o.o., Warsaw, Poland); and a preparation based on T.asperellum T-34 strain (Xilon, Kwizda Agro GMBH; Wien, Austria) (

Table 1. Treatment list of tested products in vitro.

No.	Combination	Dose per 200 L of Water
1	Untreated	-
2	Prothioconazole (300 g/L)	0.5 L
3	Tebuconazole (250 g/L)	1.0 L
4	Azoxystrobin (250 g/L)	0.8 L
5	T. asperellum T34 strain	10 kg
6	Tebuconazole (250 g/L) + T. asperellum T34 strain	1.0 L + 10 kg
7	Prothioconazole (300 g/L) + T. asperellum T34 strain	0.5 L + 10 kg
8	Azoxystrobin (250 g/L) + T. asperellum T34 strain	0.8 L + 10 kg

).

Pieces of stems with disease symptoms were disinfected and then placed on PDA medium. Fungal colonies growing from fragments of plant material were transplanted and subjected to further incubation. Fungi were identified by assessing the morphological characteristics of colonies and sclerotia, based on available mycological keys. Isolates od S. sclerotiorum are stored in the Laboratory of Mycology Department of the Institute of Plant Protection—NRI. The tested substances were added to sterile agar–glucose–potato medium (PDA) cooled to 45 °C in such quantities as to obtain the appropriate concentration of the active substance corresponding to the field dose. The obtained medium mixture and the agents were poured into Petri dishes. The experiment was performed in five repetitions. The control combination was pure PDA medium (without the addition of tested substances). Discs of individual culture of 20-day-old isolate of S. sclerotiorum with formed sclerotias with a diameter of 4 mm were placed on the solidified medium in Petri dishes, in their central part. The plates were incubated in the controlled conditions of a Binder chamber (Sanyo Electric Japan—Incubator MIR-254, PHC Europe BV, Etten-Leur, Netherlands). Incubation conditions were 20 °C for 14 h during the day/14 °C for 10 h at night. The diameter of the cultures in each combination was measured after the mycelium had overgrown the surface of the medium in a given control object (after 7–10 days). The average growth of mycelium in millimeters was calculated. All experiments were performed in two independent series. The results from individual series were averaged and summarized in tables.

2.2. Field Experiment

This study was conducted on two oilseed rape varieties (Graf and Harry) and four fungicides containing active substances from triazoles and SDHI group: metconazole (Caramba 60 SL; Basf Polska Sp. z o.o.; Warsaw; Poland); difenoconazole + tebuconazole (Magnello 350 EC; Syngenta Polska Sp. z o.o., Warsaw, Poland); fluopyram + prothioconazole (Propulse 250 SE; Bayer Polska Sp. z o.o.; Warsaw, Poland) and preparation based on T. asperellum T34 strain (Xilon; Kwizda Agro GMBH; Wien, Austria), Coniothyrium minitans (Lalstop Contans, Danstar Ferment AG, Zurich, Switzerland) (

Table 2. Characteristic of products applied in the field trials.

No.	Combination	Content of Active Ingredient per l/kg of Product	Application Timing	Dose per Hectare (L/kg)
1	Untreated	-	-	-
2	Metconazole	60 g·L−1	BBCH 16	0.7
	Difenoconazole + tebuconazole	100 g·L−1 + 250 g·L−1	BBCH 39	0.8
	Fluopyram + prothioconazole	125 g·L−1 + 125 g·L−1	BBCH 65	1.0
3	Trichoderma asperellum	10 g·kg−1	BBCH 00 (during sowing)	10.0
	Metconazole	60 g·L−1	BBCH 16	0.7
	Difenoconazole + tebuconazole	100 g·L−1 + 250 g·L−1	BBCH 39	0.8
	Fluopyram + prothioconazole	125 g·L−1 + 125 g·L−1	BBCH 65	1.0
4	Trichoderma asperellum	1 × 107 CFU/kg	BBCH 00 (during sowing)	10.0
	Metconazole	60 g·L−1	BBCH 16	0.7
	Difenoconazole + tebuconazole	100 g·L−1 + 250 g·L−1	BBCH 39	0.8
5	Coniothyrium minitans	1 × 107 CFU/kg	before sowing	2.0
	Metconazole	60 g·L−1	BBCH 16	0.7
	Difenoconazole + tebuconazole	100 g·L−1 + 250 g·L−1	BBCH 39	0.8
	Fluopyram + prothioconazole	125 g·L−1 + 125 g·L−1	BBCH 65	1.0
6	Coniothyrium minitans	1 × 107 CFU/kg	before sowing	2.0
	Metconazole	60 g·L−1	BBCH 16	0.7
	Difenoconazole + tebuconazole	100 g·L−1 + 250 g·L−1	BBCH 39	0.8

BBCH—Biologische Bundesanstalt, Bundessortenamt and Chemical industry scale of growth.

).

The selection of treatments for field experiments resulted from the need to adapt plant protection strategies to changing climatic factors, the increasing pressure of S. sclerotiorum, and the lack of up-to-date scientific data regarding hybrid protection approaches. The applied treatments aimed to demonstrate differences in the reduction of S. sclerotiorum depending on the timing of application (biological control agent applied pre-sowing and/or foliar spraying at the BBCH 65 growth stage). The remaining treatment combinations (BBCH 16 and BBCH 39) were intended to exclude the influence of other pathogenic factors on the health status of winter oilseed rape plants. When selecting the treatments, both the potential effectiveness of the overall strategies under field conditions and the safety of the cultivated plants were taken into account.

The field research was conducted in 2022–2023 based on the experiment established at the Field Experimental Station (PSD) Winna Góra, Institute of Plant Protection—National Research Institute in Poznań (Poland). The meteorological conditions during the tests are given in Appendix A. The size of the experimental plots was 20 m². A one-factor experiment was established in four replications, in a randomized block design. Trials were carried out using natural infection without artificial inoculation. Biological products were applied during sowing of oilseed rape plants according to producer’s methodology. The varieties were sown in the last decade of august in each of the experimental years. Application of the chemical products was performed using a Wachowiak backpack sprayer equipped with a compressed air cylinder and a spray boom with 4 nozzles (DG TeeJet 110–02 V). The spray liquid output was set at 200 L·ha⁻¹. The operating pressure during the treatment was 2.5 bar. The distance between the spray boom and the plant surface was 50 cm. Plants were harvested in full maturity stage BBCH 99 (15.07.2022 and 22.07.2023).

Trials were conducted according to EPPO methodology nr: PP 1/181 (4), 1/78 (4), 1/80 (4) [25,26]. The degree of infestation of the stems by the S. sclerotiorum was determined and then the infection index [%] was calculated. One assessment was carried out: 25.06.2022, 30.06.2023 for each year of the experiment. The disease severity index (DI) for S. sclerotiorum was calculated using the following formula:

$$\mathrm{D}\mathrm{I}={\displaystyle \frac{\mathsf{\Sigma }\left(a \times b\right)}{N\times c}} \times 100\%$$

(1)

where a is the number of plants in a given severity class, b is the numerical value of the severity class, N is the total number of evaluated plants, and c is the maximum value of the adopted rating scale. The index was expressed as a percentage (%).

Used classes: 1 0–50% infection, 2 > 50% infection, but strength of stem unaffected, 3 > 50% infection, stem weakened, 4 <50% −100%.

Efficacy was calculated according to the Abbott formula [%]. Assessment of the phytotoxic effect of the agents on winter oilseed rape plants was made by visually comparing the condition of the plants on the plots treated with fungicide with the plants on the control plots (without fungicides). The oilseed rape was harvested with the Wintersteiger Classic harvester. The weight of seeds collected from each plot was determined and the humidity was measured. The yield was converted into tons per hectare, assuming a standard seed moisture of 11%. Randomly collected 0.5-liter seed samples. Three batches of 200 seeds were counted from each sample and their weight was determined with an accuracy of 0.01 g using a Sartorius scale. The result is shown as the average weight of 1000 seeds in grams. Oil content was measured using an Agroscan analyzer. The obtained results were analyzed statistically.

2.3. Statistical Analysis

The normality of the distributions of the values of mycelium growth of S. sclerotiorum was tested using Shapiro–Wilk’s normality test [27]. One-way analysis of variance (ANOVA) was carried out to determine the effects doses on the variability of mycelium growth of S. sclerotiorum. The arithmetic means, minimal and maximal values, as well as standard deviations of observed trait were calculated. Moreover, Fisher’s least significant difference (LSD) was estimated at a significance level of α = 0.05. Homogeneous groups for the analyzed trait were determined based on LSD. All the analyses were conducted using the GenStat 23.1 statistical software package.

3. Results

3.1. In Vitro Experiment

In order to determine the suitability of a biofungicide containing T. asprellum and synthetic active substances contained in fungicides and their mixtures for limiting the development of the S. sclerotiorum agent, in vitro studies were conducted, followed by field experiments. Laboratory studies measured the growth of S. sclerotiorum mycelium after adding T. asperellum, prothioconazole, metconazole, azoxystrobin, and their mixtures to the medium (Figure 1). The addition of a.i. tebuconazole and T. asperellum to the PDA medium showed the highest efficacy against S. sclerotiorum (

Table 3. The effect of protection using biopreparations and fungicides on the occurrence of Sclerotinia sclerotiorum on the Graf cultivar in the years of the study.

No.	Treatment *	Infestation DI [%]	Efficacy [%]	Infestation DI [%]		Efficacy [%]
		2022		2023
1	Untreated	34.2 a	-	36.9 a
2	Metconazole	16.0 b	53	14.7 b	60
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
3	Trichoderma asperellum	5.3 b	85	7.1 b	81
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
4	Trichoderma asperellum	18.7 b	46	22.2 c	40
	Metconazole
	Difenoconazole + tebuconazole
5	Coniothyrium minitans	11.1 b	68	11.6 b	69
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
6	Coniothyrium minitans	20.9 b	39	20.1 c	43
	Metconazole
	Difenoconazole + tebuconazole

* Fungicide application dates are given in Table 2. Different letters a–c indicate statistically different mean values (α = 0.05).

). The use of a.i. prothioconazole caused a small growth of mycelium—an average of 1.83 mm. The values obtained in individual series and repetitions ranged from 0 to 3 mm. These values did not differ statistically from those obtained after the use of a.i. tebuconazole and T. asperellum. The use of mixtures of a.i. tebuconazole and prothioconazole with T. asperellum also allowed the effective limitation of mycelium development, which for the combination T. asperellum + tebuconazole was 0.67 mm, and for the mixture of T. asperellum with a.i. prothioconazole was on average 2.17 mm. The obtained mycelium growth values were slightly higher in comparison to those obtained after the use of these a.i. solo. Weaker inhibition, in comparison with other objects, but statistically significantly different in comparison to the control, was observed after the use of a.i. azoxystrobin and its mixture with T. asperellum. In the case of this a.i., the opposite trend was observed, when only a.i. azoxystrobin was applied, the average mycelium growth was 44.33 mm, and with the addition of T. asperellum, the mycelium diameter was significantly smaller and averaged 10.33 mm. The studies with most of the a.i. used in laboratory tests were continued under field conditions.

3.2. Field Experiment

The weather conditions during the experimental years varied, which was reflected in the level of disease observed in the untreated combinations. A higher severity of infection in both tested cultivars was recorded in 2023. A detailed analysis of meteorological data revealed a 10% higher humidity in April 2023 compared to 2022. In May the difference amounted to 5%. The product containing T. asperellum was used in selected combinations before sowing rape. For comparison, a biofungicide registered in Poland for the control of S. sclerotiorum containing the fungus Coniothyrium minitans was used at the same time in paralell combination. In the experiment, fungicides were used at standard BBCH stages during the growing season—in autumn (BBCH 16) and in spring after the start of vegetation (BBCH 39) and in selected combinations in the full flowering stage (BBCH 65). The fungicide treatment at this stage is in practice dedicated to limiting the occurrence of S. sclerotiorum. In both years of the study, a higher index of plant infection by S. sclerotiorum was recorded on the Graf variety, compared to the Harry variety (Table 3 and

Table 4. The effect of protection using biopreparations and fungicides on the occurrence of Sclerotinia sclerotiorum on the Harry cultivar in the years of the study.

No.	Treatment *	Infestation DI [%]	Efficacy [%]	Infestation DI [%]		Efficacy [%]
		2022		2023
1	Untreated	24.0 a	-	33.8 a
2	Metconazole	5.8 b	76	4.4 b	87
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
3	Trichoderma asperellum	0.0 b	100	0.4 b	99
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
4	Trichoderma asperellum	4.9 b	82	4.0 b	88
	Metconazole
	Difenoconazole + tebuconazole
5	Coniothyrium minitans	3.6 b	85	4.4 b	87
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
6	Coniothyrium minitans	4.9 b	82	8.9 b	74
	Metconazole
	Difenoconazole + tebuconazole

* Fungicide application dates are given in Table 2. Different letters a–b indicate statistically different mean values (α = 0.05).

). The use of all the tested preparations in both oilseed rape varieties allowed for a statistically significant reduction in the infection index. For both rape varieties, the highest effectiveness in reducing the occurrence of S. sclerotiorum was noted in object 3, after the application of T. asperellum before sowing, and in the oilseed rape flowering phase of the fungicide containing the active substances prothioconazole and fluopyram. The effectiveness for both varieties in the years of the study ranged from 81 to 100%. In combinations where C. minitans was used before sowing, and then the fungicide was used in the flowering phase (object no. 5), the effectiveness was ranged from 68 to 87%. The effect of not applying fungicide in the flowering phase on plots where biofungicides were applied before sowing varied depending on the variety. In the case of the variety less susceptible to infection of S. sclerotiorum (Harry), the infection index ranged from 4.0 to 8.9%, and the effectiveness from 74 to 88%. On the other hand, for the Graf variety, the infection index ranged from 18.7 to 22.2%, and the effectiveness of pathogen reduction, in this case, was insufficient and ranged from 39 to 46%.

The applied products influenced a statistically significant increase in the yield of winter rape, but it was significant only in the first year of experiments (2022) on both evaluated varieties (

Table 5. Effect of biopreparations and fungicides on the parameters of seed yield—Graf variety.

No.	Treatment *	Yield (t/ha)		TGW (g)		Oil Content (%)
		2022	2023	2022	2023	2022	2023
1	Untreated	4.9 b	5.7 a	4.33 a	4.75 a	47.8 b	48.5 a
2	Metconazole	5.0 a	6.1 a	4.32 a	5.12 a	48.5 ab	48.7 a
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
3	Trichoderma asperellum	5.2 a	6.1 a	4.40 a	5.16 a	48.9 a	48.9 a
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
4	Trichoderma asperellum	5.1 a	6.0 a	4.40 a	4.81 a	48.6 ab	48.5 a
	Metconazole
	Difenoconazole + tebuconazole
5	Coniothyrium minitans	5.1 a	6.0 a	4.56 a	4.76 a	48.8 a	49.0 a
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
6	Coniothyrium minitans	5.0 a	6.0 a	4.35 a	4.92 a	48.5 ab	48.7 a
	Metconazole
	Difenoconazole + tebuconazole

* Fungicide application dates are given in Table 2. Different letters a–b indicate statistically different mean values (α = 0.05). TGW—thousand grain weight.

and

Table 6. Effect of biopreparations and fungicides on the parameters of seed yield—Harry variety.

No.	Treatment *	Yield (t/ha)		TGW (g)		Oil content (%)
		2022	2023	2022	2023	2022	2023
1	Untreated	4.4 b	5.0 a	4.95 a	4.71 b	47.5 b	51.4 a
2	Metconazole	4.8 a	5.4 a	5.09 a	5.90 a	48.2 ab	51.7 a
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
3	Trichoderma asperellum	4.8 a	5.4 a	5.25 a	5.02 a	48.5 a	51.6 a
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
4	Trichoderma asperellum	4.7 a	5.3 a	5.08 a	5.01 a	47.9 ab	51.6 a
	Metconazole
	Difenoconazole + tebuconazole
5	Coniothyrium minitans	4.8 a	5.3 a	4.95 a	4.97 a	48.1 ab	51.5 a
	Metconazole
	Difenoconazole + tebuconazole
	Fluopyram + prothioconazole
6	Coniothyrium minitans	4.7 a	5.2 a	5.04 a	4.72 a	48.0 ab	51.5 a
	Metconazole
	Difenoconazole + tebuconazole

* Fungicide application dates are given in Table 2. Different letters a–b indicate statistically different mean values (α = 0.05). TGW—thousand grain weight.

). Application of T. asperellum before sowing and in the oilseed rape flowering stage of a fungicide containing a.s. prothioconazole and fluopyram influenced the highest yield increase compared to the untreated plots for the Graf variety in both years of the study and in 2023 in the combination in which a fungicide containing a.i. prothioconazole and fluopyram was used in the flowering stage. In the experiments conducted on the Harry variety, the highest yield increase compared to the control in both years of the study was obtained in combinations in which a fungicide containing a.i. prothioconazole and fluopyram (object no. 3) and only fungicides (object no. 2) were used. In 2022, at the same level, the yield was harvested from the combination in which C. minitans was used, and then a fungicide containing a.i. prothioconazole and fluopyram in the flowering phase was applied. The application of both biofungicides before sowing without fungicide treatment in the flowering stage resulted in a significant increase in yield in 2022 for both evaluated varieties from 0.1–0.3 t/ha in comparison to the control. In the second year of investigation, differences were observed (0.2–0.3 t/ha)s, but they were not significant.

The weight of one thousand seeds collected from the experiments was higher after the use of all combinations in Graf variety cultivation without combination no. 5, and all combinations of Harry variety without combinations no. 6. The largest seeds of the Harry variety in both years of the study were collected in research object no. 3, in which T. asperellum was used before sowing, and then fungicide in the flowering stage were applied, and in 2023 in combination no. 4, where T. asperellum was applied before sowing, and no fungicides were used in the flowering stage. For the Graf variety, the largest seeds in 2022 were collected from the combination in which C. minitans was used before sowing, and a fungicide containing a.s. prothioconazole and fluopyram was used in the flowering stage. In 2023, however, it was also used in case of combined disease control. Analysis of the oil content showed its increase after the use of all products. It is worth mentioning the increase in oil content in the case of the Graf variety in both years of the study after the application of C. minitans before sowing, fungicide containing a.s. prothioconazole and fluopyram in the flowering stage, and in 2022 from combination no. 3. In the studies conducted on the Harry variety, the highest oil content in both years of the study was shown in object no. 3, in which T. asperellum was used before sowing, and then the synthetic fungicides were used in the flowering stage.

4. Discussion

T. asperellum used without synthetic fungicides completely inhibited the growth of S. sclerotiorum mycelium in in vitro conditions. The combined use of a biofungicide containing T. asperellum with an active substance from the triazole chemical group significantly limited mycelium growth in relation to the control object. The addition of T. asperellum to an active substance from the strobilurin chemical group significantly increased the effectiveness of limiting the growth of S. sclerotiorum mycelium in comparison to azoxystrobin used alone. Zamani-Noor [28] also tested the influence of the azoxystrobine and selected triazoles on S. sclerotiorum mycellium growth and noted comperable level of S. sclerotiorum control by strobilurins.

In soybean crops, strains of T. asperelloides (closely related to T. asperellum) have also been assessed for their antagonistic effects on various strains of S. sclerotiorum. Results obtained by authors correspond to these obtained by Sumida et al. [29]. The researchers reported that T. asperelloides strains T25 and T42 effectively reduced disease incidence and severity in field-grown soybeans, outperforming commercial products based on T. harzianum. The study found that T25 strain and T42 strain decreased disease incidence by 39.9% and 29.9%, respectively, demonstrating the potential of T. asperelloides as a viable biocontrol agent for managing white mold disease in soybeans. Moreover, Elias et al. [30] highlighted the effectiveness of T. asperellum in controlling S. minor and S. sclerotiorum in lettuce seedlings. Their findings revealed that T. asperellum isolates significantly reduced incidence rates of both pathogens, showcasing the versatility of this species in various crop systems.

Abo-Elyousr et al. [31] emphasize the antagonistic potential of T. asperellum against plant pathogens, demonstrating its ability to inhibit the growth of Alternaria porri, thereby showcasing its role as an effective biocontrol agent. This aligns with findings by Benítez et al. [14], who elaborate on the biocontrol mechanisms of Trichoderma strains, asserting that T. asperellum can significantly reduce pathogen populations through direct competition for nutrients and space.

The occurrence of S. sclerotiorum in natural conditions depends on the cultivated variety, agroclimatic factors and chemical protection. The choice of cultivar plays a crucial role in managing pathogens, particularly as breeding programs focus on developing resistant varieties. However, the polygenic nature of resistance to S. sclerotiorum poses challenges, as many commercial cultivars currently lack effective resistance [32]. The meteorological data provided in Appendix A include average temperatures, air humidity percentages, and total rainfall amounts for the experimental period spanning from August 2021 through July 2023. This data is critical for understanding the environmental context in which the experiments on controlling S. sclerotiorum in oilseed rape were conducted. Average temperatures in spring months (April–May) were mostly within or near the optimal range for S. sclerotiorum development. April 2022 had temperatures averaging 7.94 °C with air humidity around 68.84%, and May 2022 averaged 14.84 °C with 63.90% humidity. In 2023, April and May showed similar or slightly fluctuating conditions, with some months exhibiting higher humidity (up to ~79% in April 2023). These findings are consistent with the results of Jajor et al. [33], which demonstrated a highly significant correlation between average air humidity and the average percentage of oilseed rape plants infected by the causal agent of white mold. These conditions align also with other researchers [34,35,36] observations that higher humidity and moderate temperatures promote ascospore germination and infection, which is consistent with the higher infection rates observed in 2023 compared to 2022. The total rainfall during critical months varied. May 2022 recorded 51.20 mm and May 2023 had 28.40 mm, indicating sufficient moisture availability for pathogen development. The meteorological data support this, as years or months with higher humidity corresponded with more severe disease outbreaks in the field experiments (higher infection indices in 2023).

The integration of the application of bio-control agents with conventional fungicides can lead to synergistic effects, enhancing disease control while minimizing fungicide usage. Field trials carried out by Authors indicated that pre-sowing applications of T. asperellum, and C. minitans followed by comprehensive fungicide treatments during flowering, resulted in a significant reduction in plant infection rates by S. sclerotiorum, achieving effectiveness rates between 81% and 100% for T. asperellum and 39–87% for C. minitans. Research has demonstrated that combining Trichoderma with fungicides such as thiophanate-methyl can improve disease suppression in crops like soybean, while also reducing the likelihood of pathogen resistance development [37]. In the study conducted by Hu et al. [38], the combined use of a preparation based on Trichoderma sp. Tri-1 with a fungicide used at a reduced dose (75% of the recommended dose) allowed the protection of oil seed rape against S. sclerotiorum at a level similar to the efficacy of the full dose of fungicide. The use of Trichoderma sp. together with a chemical fungicide therefore allows the dose of the synthetic protection agent to be reduced, which is beneficial for the natural environment.

Excessive fungicide application can lead to the development of resistant pathogen populations, as seen in S. sclerotiorum, which has shown resistance to multiple fungicide classes [39,40]. By incorporating biological control strategies, farmers can potentially reduce the frequency and amount of chemical fungicides applied, thus mitigating the risk of resistance and preserving the efficacy of available fungicides [41]. Additionally, the environmental benefits of adopting biological controls cannot be overstated. The use of BCAs helps reduce chemical residues in agricultural products and the surrounding ecosystem, aligning with consumer demand for more sustainable and organic farming practices [42]. This shift is not only beneficial for the environment but can also enhance marketability and consumer confidence in agricultural products.

Research has consistently demonstrated that the application of T. asperellum can lead to significant increases in the yield of oilseed rape. For instance, Kowalska and Remlein-Starosta [43] reported that treatments with T. asperellum resulted in statistically significant yield increases compared to untreated control plants, with yields reaching up to 3.2 t·ha⁻¹ in treated plots versus 2.6 t·ha⁻¹ in untreated plots This yield enhancement is attributed to several mechanisms, including improved root growth, enhanced nutrient uptake, and increased resilience to biotic stresses. In the authors’ research, pre-sowing use of T. asperellum and full fungicide protection including treatments in the flowering period allowed for obtaining the highest yield values of winter rape and thousand seed weight of both varieties.

Application of C. minitans to soil or crop residues has been shown to significantly reduce sclerotial survival and subsequent apothecial production, thus lowering disease incidence [44]. C. minitans can survive in soil for extended periods (up to two years), maintaining viability and infecting newly incorporated sclerotia, which is crucial for long-term disease suppression. Application methods include colonized solid substrate inocula or spore suspensions, with spore suspensions often providing better distribution and infection rates [24].

Climate change is expected to affect the epidemiology of S. sclerotiorum by altering temperature and humidity conditions that are conducive to its development. Research indicates that rising temperatures may enhance the infection window for S. sclerotiorum, particularly during the flowering stage of oilseed rape when the plant is most susceptible to infection [45]. For instance, a study by Wójtowicz and Wójtowicz [2] found that under climate change scenarios (RCP4.5 and RCP8.5), the occurrence and severity of Sclerotinia stem rot were projected to change across various regions in Poland. This suggests that while some regions may benefit from reduced disease pressure, others could experience heightened risks, necessitating localized management strategies. That is why appropriate control methods of S. sclerotiorum in oilseed rape cultivation are necessary.

5. Conclusions

In in vitro studies, 100% inhibition of S. sclerotiorum mycelial growth was observed after adding a bioproduct containing T. asperellum to the PDA medium. The addition of active substances from the triazole group to the biofungicide significantly limited mycelial growth compared to the untreated combination. Interestingly, the combination of T. asperellum with an active substance from the strobilurin chemical group significantly increased the efficacy of inhibiting S. sclerotiorum mycelial growth compared to azoxystrobin used separately. Field studies with two winter oilseed rape cultivars demonstrated that combining methods—applying T. asperellum before sowing and then implementing comprehensive fungicide protection treatments during the flowering stage—effectively reduced plant infection by S. sclerotiorum. This was reflected in the yield quantity and its quality. Research conducted under in vitro and field conditions confirmed the effectiveness of T. asperellum and Coniothyrium minitans in reducing S. sclerotiorum and highlighted the usefulness of biofungicides as an element of Integrated Pest Management. Integrating T. asperellum into agricultural practices could contribute to reducing use of fungicides and promoting sustainable approaches. These actions are particularly important in the context of a changing climate, which may affect the biology of pathogens and their harmfulness to plants.