Influence of Biofungicides Containing Microorganisms Such as Pythium oligandrum and Bacillus subtilis on Yield, Morphological Parameters, and Pathogen Suppression in Six Winter Pea Cultivars

Abstract

Field peas (Pisum sativum L.) are a valuable source of protein and help to support crop biodiversity in a sustainable agriculture system. To maintain varied crop rotation in sustainable production, it is advisable to include the winter form of pea, which is an excellent alternative to the spring form. However, the prolonged development of winter peas when weather patterns are unfavorable can adversely affect the morphological features and the health of the plants. The literature lacks studies on this issue. The objective of this study was to evaluate the morphological characteristics, yield, and canopy health of selected cultivars of winter peas. The study was conducted at the Prusy Experimental Station of the University of Agriculture in Krakow, located near Krakow, Poland (50°07′28″ N, 20°05′34″ E), during two growing seasons. The study evaluated six cultivars of winter peas and two means of protecting the canopy, with biological products containing Pythium oligandrum (Polyversum WP) or Bacillus subtilis (Serenade ASO). The yield, yield structure, efficiency of N uptake, and health of the plants were assessed. Crop protection treatments using Polyversum WP and Serenade ASO were shown to effectively protect winter peas against Fusarium wilt, which occurred only in the Specter and Arkta cultivars sprayed with Serenade. Polyversum WP increased the productivity of winter peas on average by 0.5 t ha⁻¹ and increased the efficiency of nitrogen uptake on average by 10 kg ha⁻¹ in comparison to the control. The Aviron and Arkta cultivars are recommended for cultivation in the conditions of Central Europe due to their high yield potential, high efficiency of nitrogen uptake, and good canopy health.

1. Introduction

The pea plays a very important role around the world as a versatile species with applications for food, fodder, and industry [1,2]. According to FAO (Food and Agriculture Organization of the United Nations) data [3], the area of cultivation of this species in 2022 was about 10 million ha (7.04 million ha of dry peas and 2.590 million ha of green peas), and pea production is estimated at 33 million tons (12.403 million tons of dry peas and 20.529 million tons of green peas). The FAO reports an upward trend in dry pea seed production primarily in macroregions with high economic potential (Canada 3.184 Mt; Russia 1.677 Mt; China 1.256 Mt). In the EU-27, the total pea production (dry and green) amounts to 850,000 tons, which is 38.8% of the global market production of dry peas and 7.4% that of green peas.

In temperate climates, field pea is an invaluable legume species for sustainable or organic agriculture due to its high production potential and versatility [4,5,6].

In the intensive pea breeding conducted in the second half of the 20th century, the species underwent significant genetic improvement. Expanding the sources of variation made it possible to breed varieties with improved botanical and agricultural traits, i.e., a shorter stem, shorter growing period, and afila leaves instead of traditional leaves. This is aimed at improving the productivity of the plants and the qualitative characteristics of the crop, such as the protein content in the seeds. Currently, the leading direction of breeding is to improve the winter hardiness of pea cultivars. The first winter pea cultivars were imperfect in this regard, but biological progress is ongoing [7,8]. According to Święcicki [8], the biochemical and genetic mechanisms of resistance of plants to low temperatures are not well known, and therefore scientific research aimed at improving winter legume technology continues, including cultivation of winter peas in mixed crops with cereals or selection of varieties for adaptation to cultivation in given habitat conditions.

Great hope for expanding the area of pea cultivation in northern Europe is placed in the introduction of winter pea cultivars with significantly improved yield characteristics. Autumn sowing of peas prolongs the period of vegetative growth (germination to flowering), naturally resulting in an increased biomass and more seeds [9]. However, peas sown in autumn develop at suboptimal temperatures, and in order to survive the winter they require adaptive mechanisms for winter hardiness, e.g., acclimation to cold, response to vernalization, and sensitivity to light [10].

Winter pea cultivars are imperfect, because due to the long development cycle and weather conditions (low temperatures and periodic rainfall) they face two threats: freezing and fungal diseases such as Ascochyta blight [11]. Breeders have created new breeding lines with an increased resistance to freezing, but the problem of resistance to pathogens remains.

Ascochyta blight is one of the most important diseases affecting field peas. It occurs in nearly all pea-growing regions of the world and can cause significant crop losses when conditions are favorable for an epidemic [12,13]. Even a low level of infection can cause significant losses in both production and quality [14]. Ascochyta blight causes spots or lesions on the leaves, stems, and pods, as well as root rot [15,16]. Ascochyta blight has been shown to affect the seed weight, reducing the final yield by as much as 30% [11,17].

Fusarium wilt is an economically important disease, causing losses of about 30–40%. The first symptoms are usually a yellowing of the lower leaves and stunting of plant growth, ultimately causing the plants to wilt. F. oxysporum penetrates pea roots and infects the vascular system at any growth stage [18]. Pressure from pathogens and their negative impact on the health and yield of pea necessitate the use of plant protection treatments during vegetative growth. In the modern era, pesticides are an indispensable element of agricultural practices. Extensive use has increased crop yields and significantly reduced harvest losses, thereby increasing food availability. On the other hand, indiscriminate use of these chemicals has had environmental implications and adverse effects on human health [19,20,21]. The use of biopesticides could help to increase crop production without compromising human health [22]. Several factors indicate that biopesticides are excellent alternatives to synthetic pesticides. Specifically, they are highly effective, are target specific, and have fewer environmental risks. Current research into biopesticides focuses on improving their action spectra, including mechanisms to replace the use of chemical pesticides in Integrated Pest Management (IPM) plans [23]. Biopesticides are considered a natural, safe, and environmentally friendly alternative in organic agricultural production [24].

Biopesticides use such microorganisms as Pythium oligandrum and Bacillus subtilis [24].

P. oligandrum protects plants from biotic stressors and promotes plant growth. It colonizes the rhizosphere of many crop species and helps to control diseases caused by a number of soil-borne fungal pathogens. It exhibits mycoparasitic activity through direct contact with fungal plant pathogens. P. oligandrum parasitizes Fusarium oxysporum by producing enzymes (cellulases or chitinases) that degrade the pathogen’s cell walls. It also promotes plant growth by producing tryptamine, an auxin-like compound, and induces systemic resistance in many plants [25].

Rhizobacteria, including B. subtilis, influence plant growth and nutrition, promoting early bloom; increasing the health, vigor, and height of plants; increasing the shoot and root biomass and chlorophyll content; and enhancing nodulation in legumes. The beneficial influence of rhizobacteria on plant development is multi-faceted. Fixed nitrogen in legumes increases the supply of other nutrients, e.g., iron, copper, sulfur, and phosphorus, induces the production of plant hormones, increases the activity of beneficial microbes, and acts as antagonist of pathogenic fungi and bacteria [26].

No studies have been carried out to test the effect of biological fungicide applications on yield, morphological parameters, and the health of selected winter pea cultivars. The aim of this study was to verify the effectiveness of biological products in protecting plants against Ascochyta blight and Fusarium wilt and to assess crop yield and nitrogen uptake in selected winter pea cultivars.

2. Material and Methods

2.1. Weather Conditions

To characterize the temperature and moisture conditions during plant growth, the hydrothermal coefficient (K) proposed by Selyaninov [27] was used (Table 1) according to the following formula:

$$\mathrm{K}=\frac{\mathrm{P}}{\sum \mathrm{T}\mathrm{p}}\times 0.1$$

where:

P—precipitation total for a given 10-day period (mm);

∑Tp—sum of average daily air temperatures for a given 10-day period (°C) > 0 °C.

Then, the temperature and precipitation conditions during pea growth were characterized according to the ranges of values proposed by Skowera and Puła [28]: K ≤ 0.4—extremely dry (ed); 0.4 < K ≤ 0.7—very dry (vd); 0.7 < K ≤ 1.0—dry (d); 1.0 < K ≤ 1.3—quite dry (qd); 1.3 < K ≤ 1.6—optimal (o); 1.6 < K ≤ 2.0—quite wet (qw); 2.0 < K ≤ 2.5—wet (w); 2.5 < K ≤ 3.0—very wet (vw); and K > 3.0—extremely wet (ew). Selyaninov’s coefficient was used to define the optimal moisture and periods of drought [29]. The coefficient was used to characterize the years in terms of soil drought.

Table 1. Selyaninov’s hydrothermal coefficients during the growing season (April to August) of peas in 10 day periods.

Year/Month	April			May			June			July			August
	I	II	III	I	II	III	I	II	III	I	II	III	I	II	III
2018	0.23	0.13	0.17	0.60	3.52	0.00	1.67	0.51	2.67	0.21	4.36	1.43	0.40	1.06	1.21
2019	0.93	0.81	4.62	0.55	6.99	7.01	0.00	0.19	0.81	0.06	1.04	1.43	2.43	1.92	0.03

Table 1. Selyaninov’s hydrothermal coefficients during the growing season (April to August) of peas in 10 day periods.

Year/Month	April			May			June			July			August
	I	II	III	I	II	III	I	II	III	I	II	III	I	II	III
2018	0.23	0.13	0.17	0.60	3.52	0.00	1.67	0.51	2.67	0.21	4.36	1.43	0.40	1.06	1.21
2019	0.93	0.81	4.62	0.55	6.99	7.01	0.00	0.19	0.81	0.06	1.04	1.43	2.43	1.92	0.03

2.2. Experimental Design

A two-year (2017/2018 and 2018/2019) field experiment was conducted at the Experimental station of the University of Agriculture in Krakow, located in Prusy near Krakow (47°24′ N, 7°19′ E, 300 m above sea level). The experiment was established on Haplic Phaeozem. The soil was heavy (36% < 0.02 mm fraction), with an acid reaction (pH_KCl 6.2), a low content of sulfur, and medium contents of available phosphorus (68 mg kg⁻¹ DW) and potassium (125 mg kg⁻¹ DW). A two-factor field experiment was established in a randomized block design in triplicate. The first factor was the application of a biological agent as a fungicide (Serenade ASO—Bacillus subtilis; Polyversum WP—Pythium oligandrum; control) and the second factor was the cultivars. Six different cultivars, i.e., Baltrap, Myster, Specter, Dexter, Arkta, and Aviron, were used in the study. The environmental conditions specific to Central Europe are favorable for growing winter peas. To test the productivity and plant health of winter peas, we selected all cultivars available on the market for sale to farmers.

The plot size was 10 m². The sowing density for each cultivar was 100 seeds m⁻². The crop preceding pea was winter rape. Harvesting of the crop was followed by disking, ploughing, harrowing, and cultivation of the soil for sowing with a tillage machine consisting of a cultivator and a string roller. Prior to the experiment, phosphorus and potassium fertilizers were applied at 70 kg P₂O₅ and 100 kg K₂O ha⁻¹ in the form of triple superphosphate and potassium salt. Sowing was carried out in the first 10 days of September. Fungicides (Serenade ASO at 5 L ha⁻¹ or Polyversum WP at 0.15 kg ha⁻¹) were applied twice (at 7 day intervals): at the seedling stage and in the first 10 days of October. The biological agent Serenade ASO contains the active substance Bacillus subtilis strain QST 713–13.96 g/L (1.34%) (minimum concentration 1.042 × 10¹² CFU/L). The holder of the authorization for the product is Bayer AG (Leverkusen, Germany). The entity responsible for the final packaging and labeling of the plant protection product is AGROPAK Company (Jaworzno, Poland). Biofungicide Polyversum WP contains the active substance Pythium oligandrum, with 10⁶ oospores of the fungus in 1 g of the product. The holder of the authorization for the product is BIOPREPARATY Company Ltd. (Praha, Czech Republic). The entity responsible for the final packaging and labeling of the plant protection product is Target Company (Katowice, Poland).

Harvesting was carried out in the last 10 days of June each year.

The plants were observed during the growing period. Their density was estimated in autumn and after winter. At full maturity, prior to harvest, 30 plants were collected from each treatment for morphological parameter assessment, which included plant height, the height of the first pod setting, pod number, seed number, pod weight, seed weight, and 1000 seed weight (TSW).

2.3. Assessment of the Incidence of Fusarium wilt and Ascochyta Blight in Pea Plants

Foliar diseases were identified based on visual symptoms as described in the literature and by microscopic observations of causal pathogens. The symptoms of Ascochyta (Ascochyta pisi) blight on the leaves are slightly sunken, circular, tan-colored lesions with dark brown margins [30,31]. The lesions caused by F. oxysporum (Fusarium wilt) appear on plants as chlorotic leaflets which curl downward and become flaccid, after which the plant wilts and turns yellowish-brown [30,31]. The health status of the pea plants was evaluated at the end of the flowering stage (BBCH stage 69). The disease severity was recorded in 20 sample plants in the middle rows on each plot. Plants were evaluated visually for disease severity and rated on a scale of 0 to 4: 0 = no symptoms or lesions; 1 = some minor lesions present on 1% to 10% of the leaf; 2 = lesions present on 11% to 25% of the leaf; 3 = 26% to 50% of the leaf; 4 = 51% or more of the leaf covered with lesions and withered and dead leaves. The disease index (DI) was calculated for the diseases as a percentage, where

DI (%) = [sum (class frequency × score of rating class)]/[(total number of plants) × (maximum scale value)] × 100.

A visual assessment of disease was confirmed in the laboratory. Infected stem tissue samples from wilted plants and leaf samples from plants showing typical disease symptoms were taken to the laboratory. Pathogens were identified under a microscope using aqueous mounting media. The species of fungi were identified on the basis of mycological keys and monographs [32,33,34]. Identification of each isolate was conducted twice.

2.4. Statistical Analysis

A three-factorial analysis of variance was performed with cultivar, biological product, and year as the factors, with subsequent multiple comparisons of means, using Statistica version 13.1. Means were separated by least significant differences (LSD) when the F-test indicated factorial effects at p < 0.05. Results are presented for the cultivars in both years. The main effects are noted within the figure. Significant factor interactions are shown by p-value.

2.5. Chemical Analysis

The nitrogen content in the seeds was analyzed by the Kjeldahl method. The results were used to calculate nitrogen uptake by the seeds (in kg ha⁻¹) according to Witt et al. [35].

3. Results

3.1. Weather Conditions

In Prusy, there was high variation in Selyaninov’s coefficient during the growing period between years, months, and even 10 day periods (Table 1).

During the field experiment in Prusy, the starts of the growing season (April) in 2018 and June of 2019 were extremely dry (Figure 1 and Table 1). Drought was noted in April, May, and August of 2018 and in June and July of 2019. The optimal soil moisture was recorded in June, July, and September of 2018 and August of 2019. Temperature and precipitation conditions throughout the growing period of pea plants, i.e., from April to September, were fairly dry in 2018 according to the classification in [28], whereas the corresponding period in 2019 was characterized as wet (Table 1).

3.2. Plant Density

Plant density was estimated twice—after emergence in autumn (mid-November) and after winter in early spring (March) (

Table 2. Pea plant density during the growing seasons dependent on biological agent application.

Cultivar	2017/2018				2018/2019
	Plant Density (pc m−2) in Autumn	Plant Density (pc m−2) in Early Spring			Plant Density (pc m−2) in Autumn	Plant Density (pc m−2) in Early Spring
		Control	Serenade ASO	Polyversum WP		Control	Serenade ASO	Polyversum WP
Aviron	95 ± 3.2	54 ± 5.4	59 ± 6.7	61 ± 7.2	94 ± 2.4	74 ± 6.5	78 ± 8.4	79 ± 9.1
Baltrap	96 ± 2.3	42 ± 4.6	43 ± 5.4	47 ± 6.4	95 ± 3.3	74 ± 7.8	75 ± 9.2	77 ± 7.3
Myster	95 ± 3.1	42 ± 4.3	43 ± 5.1	45 ± 5.2	95 ± 3.3	79 ± 6.3	78 ± 7.3	79 ± 7.8
Dexter	98 ± 1.2	55 ± 5.8	53 ± 6.2	56 ± 6.5	97 ± 2.1	75 ± 7.3	76 ± 7.3	77 ± 7.5
Specter	97 ± 2.2	41 ± 4.3	53 ± 6.8	57 ± 6.2	96 ± 2.5	75 ± 6.9	75 ± 7.1	76 ± 6.3
Arkta	98 ± 1.3	48 ± 5.1	59 ± 5.7	65 ± 7.4	98 ± 2.3	74 ± 7.4	75 ± 7.3	77 ± 6.9
Mean	96 ± 2.2	47 ± 4.9	52 ± 6.1	55 ± 6.5	96 ± 2.7	75 ± 7.1	76 ± 7.8	77 ± 7.5

). A high plant density was clearly shown in autumn, and losses were shown after winter.

The greatest losses were observed in the control, in which no biological agents were applied.

The weather in winter (December, January, and February) significantly affected the plant density. Conditions were less favorable in the first year of the study, i.e., 2017/2018, with temperatures below 0 °C and light snowfall.

3.3. Seed Yield, Yield Structure, and N Uptake

The morphological characteristics of the pea plants are presented in

Table 3. Morphological characteristics of peas depending on the choice of cultivar and biological agent application.

Treatment	Plant Height (cm)	Height to First Node (cm)	No. of Pods per Plant	No. of Seeds per Plant
Year (Y)
2018	64.2 ± 36.4	40.6 ± 27.7	11.9 ± 3.84	54.0 ± 17.9
2019	115.5 ± 43.1	65.7 ± 30.8	14.4 ± 4.13	57.5 ± 14.3
p value	0.001	0.001	0.001	0.014
Cultivar (C)
Aviron	83.6 ± 35.7	47.9 ± 18.2	13.4 ± 4.36	58.7 ± 20.5
Baltrap	69.3 ± 27.6	38.3 ± 16.6	13.1 ± 4.30	57.8 ± 18.5
Myster	72.0 ± 28.5	37.9 ± 20.1	13.1 ± 4.32	53.8 ± 16.3
Dexter	69.6 ± 26.2	38.2 ± 17.9	10.9 ± 3.12	53.7 ± 12.0
Specter	84.1 ± 19.1	53.3 ± 13.5	13.1 ± 4.27	52.9 ± 14.9
Arkta	160.5 ± 58.9	103.3 ± 37.1	15.7 ± 3.39	57.6 ± 13.7
p value	0.001	0.001	0.001	ns
Protection (P)
Control	97.2 ± 55.1	60.1 ± 36.4	12.4 ± 3.95	54.3 ± 18.9
Serenade ASO	92.7 ± 53.5	53.9 ± 36.1	13.3 ± 4.54	54.2 ± 14.5
Polyversum WP	79.6 ± 26.8	45.5 ± 17.7	14.1 ± 3.91	58.8 ± 14.7
p value	0.001	0.001	0.001	0.012
Y × C	0.001	0.001	ns	0.001
Y × P	0.001	0.001	ns	ns
C × P	0.001	0.001	0.001	0.001

ns—not significant.

. Among the cultivars compared in the study, Arkta stood out in terms of stem length, forming the longest stems (160.5 cm), while the Baltrap cultivar formed the shortest stems (69.3 cm). The variation in plant size corresponded to the height of the first pod setting above the soil surface. In terms of the number of pods formed, the most productive cultivar was Arkta (15.5 pods) and the least productive was Dexter (10.9).

The high pod productivity of the Arkta cultivar translated to seed weight (8.43 g) and thus to seed yield (5.71 t ha⁻¹) (Table 3). The Arkta cultivar formed seeds of lower weight than the Myster cultivar, with a TSW of 147 g, compared to 152 g for the Myster cultivar. The highest yielding cultivar was Aviron (5.73 t ha⁻¹), producing on average nearly 1 ton more than the less productive Myster cultivar (4.77 t ha⁻¹).

The biological products were shown to significantly affect the biometric features of the plants (Table 3). Polyversum WP significantly limited stem length, with a difference of 17.6 cm compared to the control, while the difference for the height of the first pod setting was 14.6 cm. Application of Serenade ASO had little effect on these parameters, as no significant differences were shown between treatments. The use of Polyversum WP positively influenced the pod number per plant (on average by more than two), the seed number per plant (on average by more than four) (Table 3), and the pod weight per plant (on average by more than 3 g) (

Table 4. Morphological characteristics, yield and N uptake of peas depending on the choice of cultivar and biological agent application.

Treatment	Weight of Pods (g)	Weight of Seeds Mass (g)	1000 Seed Weight (g)	Seed Yield (t ha−1)	N Uptake (kg ha−1)
Year (Y)
2018	14.9 ± 8.62	8.09 ± 2.72	148.4 ± 14.5	4.17 ± 1.52	132.6 ± 50.9
2019	14.9 ± 6.93	8.43 ± 2.16	151.2 ± 17.9	6.32 ± 1.62	213.8 ± 66.2
p value	ns	ns	ns	0.001	0.001
Cultivar (C)
Aviron	16.8 ± 8.59	8.65 ± 2.77	149.3 ± 17.4	5.73 ± 1.87	189.1 ± 71.1
Baltrap	15.6 ± 8.06	8.56 ± 2.84	149.8 ± 20.1	5.06 ± 2.01	180.7 ± 79.4
Myster	16.4 ± 9.24	8.13 ± 3.05	152.4 ± 28.5	4.77 ± 1.96	147.3 ± 76.1
Dexter	16.9 ± 8.14	7.92 ± 1.67	149.1 ± 18.1	5.14 ± 1.42	157.9 ± 50.3
Specter	14.2 ± 5.61	7.86 ± 2.12	151.1 ± 19.2	5.06 ± 1.99	171.6 ± 72.8
Arkta	9.93 ± 3.64	8.43 ± 1.95	147.1 ± 14.2	5.71 ± 1.92	192.4 ± 67.3
p value	0.001	ns	ns	0.019	0.001
Protection (P)
Control	14.0 ± 7.25	8.25 ± 3.15	152.4 ± 18.3	5.03 ± 2.11	182.8 ± 82.3
Serenade ASO	13.9 ± 7.36	8.01 ± 2.14	150.1 ± 24.0	5.14 ± 1.91	164.4 ± 71.1
Polyversum WP	17.0 ± 8.43	8.51 ± 1.89	147.1 ± 18.1	5.56 ± 1.62	172.4 ± 59.1
p value	0.006	ns	ns	0.001	0.001
Y × C	0.001	0.001	ns	0.001	0.001
Y × P	ns	ns	ns	0.001	0.001
C × P	0.001	0.001	ns	0.001	0.001

ns—not significant.

) relative to the control. In addition, application of Polyversum WP significantly increased plant yield, on average by 0.5 t ha⁻¹, compared to the control. Serenade ASO had a much smaller influence on pea yield than Polyversum WP.

Polyversum WP and Serenade ASO negatively affected nitrogen uptake by the seeds. However, the use of Polyversum WP increased nitrogen uptake on average by 10 kg ha⁻¹ relative to Serenade ASO.

In addition, the interaction of the factors (cultivars and biological agents) and the weather were shown to influence the biometric features (Table 3 and Table 4). Significantly more seeds per plant were formed by the Aviron and Arkta cultivars following application of Polyversum WP (Figure 2a). On the other hand, significantly more seeds were produced by the Specter cultivar following application of Serenade ASO (Figure 2a). Significantly more pods were formed by the Arkta and Specter cultivars following application of Polyversum WP (Figure 2b), while the Aviron and Myster cultivars formed significantly more pods following the use of Serenade ASO (Figure 2b).

The interaction between the choice of cultivar and biological agent on seed yield significantly influenced the seed yield (Figure 2c). The use of Polyversum WP significantly increased the yield of the Aviron and Arkta cultivars, while Serenade significantly increased the yield of the Specter cultivar (Figure 2c). The biological agents were not shown to significantly influence the yield of the Myster cultivar (Figure 2c).

The seed weight per plant was significantly determined by the interaction of the choice of cultivar and biological agent (Figure 2d). The interaction of the factors significantly increased the seed weight of the Aviron and Arkta cultivars in the case of application of Polyversum WP and in the Specter cultivar following application of Serenade ASO relative to the control. The Myster, Dexter, and Baltrap cultivars responded negatively to the biological agents, attaining a much lower seed weight than the control.

The choice of cultivar and biological agents significantly affected the efficiency of nitrogen uptake by the seeds (Table 4). Significantly more nitrogen was taken up by pea plants in the second year of the study, when the rainfall distribution was optimal for their development; the difference was 81.2 kg ha. The most efficient cultivar in terms of nitrogen uptake was Arkta (192.4 kg) and the least efficient was Myster (147.3 kg); the difference between cultivars was 45.1 kg (Table 4). The biological agents strongly reduced the efficiency of some of the pea cultivars (Myster and Dexter) in their uptake of nitrogen (Figure 2f). A significantly lower efficiency of nitrogen uptake was noted for the Myster cultivar following application of Polyversum WP and for the Myster and Dexter cultivars following the use of Serenade ASO. A significant positive effect of the use of Polyversum WP was noted only for the Aviron cultivar, while Serenade ASO positively influenced only the Arkta cultivar.

3.4. Pathogen Suppression

Fusarium wilt was found to pose a greater threat in the 2017/2018 season (Figure 3a and Figure 4). In both years, the Arkta cultivar was significantly more severely infected with F. oxysporum (Figure 3b). On the other hand, in unprotected control objects, the disease significantly took the strongest hold on the Specter variety (Figure 4 and Figure 5). In the 2017/2018 season, the Fusarium disease index ranged from 5% (in the protected treatments) to 87.5% (control) (Figure 4). The use of plant protection significantly decreased infection in all pea cultivars (Figure 3c,d). Protection using the biological agents was most effective for the Specter cultivar and least effective for Arkta. In the 2017/2018 season, Polyversum WP was more effective at reducing Fusarium wilt (Figure 3c and Figure 4).

In the 2018/2019 season, significantly, the highest disease index was 37.5% for the Specter cultivar in the treatment without protection (Figure 5), while the disease was not observed in the Aviron cultivar. Application of Polyversum WP and Serenade ASO effectively protected the plants against Fusarium wilt. The disease was observed only in the Specter and Arkta cultivars sprayed with Serenade ASO. The plants in the other treatments were healthy (Figure 5).

Ascochyta blight affected the pea cultivars more severely in 2018 (Figure 6a). In that season, the A. pisi disease index ranged from 5% (protected treatments) to 77.5% (control) (Figure 7). In 2019, the disease index ranged from 0% (protected treatments) to 50.0% (control) (Figure 8). The Baltrap cultivar was significantly the most severely diseased by A. pisi in both years of the experiment, followed by Specter and Arkta (Figure 6b). In both seasons, the use of protection significantly reduced infection of all pea cultivars with A. pisi (Figure 6c,d), but the biological products provided better protection in 2018 (Figure 7 and Figure 8).

4. Discussion

4.1. Wintering, Yield, and N Uptake

The experiment confirmed the value of conducting research in Central Europe (southern Poland) on the adaptation of winter legume plants to cultivation. The weather conditions during the research period were favorable to the wintering of winter peas. This resulted in a satisfactory density in spring and before harvest, ranging from 55% to 75%. In the conditions of Western Europe, Neugschwandtner et al. [36] showed a high percentage of plants surviving the winter in Austria, from 80% to 100% depending on the cultivar and weather conditions. The authors reported that the Aviron cultivar showed much higher frost resistance than other French winter pea cultivars (Isard, James, Enduro, and Curling).

In our study, the pea yield was determined by the weather conditions during the growing season. Higher yields were noted in the year with more rainfall, which confirms results reported by Klimek-Kopyra et al. [37] on the dependency of plant yield on weather conditions in the growing season. Among the winter pea cultivars compared in the study, Arkta and Aviron were the most productive in the conditions of southern Poland, with higher pod weight and seed weight (Aviron) or higher pod numbers and seed numbers (Arkta and Aviron). The least productive were the Myster and Dexter cultivars. Neugschwandter et al. [36] showed variation in winter pea yield between years. Winter pea cultivars with access to a lot of water in winter and less water in spring produced higher yields. In these conditions, the yields of the Cherokee and James cultivars were highest, while the Aviron cultivar produced the lowest yield.

Our study showed that weather conditions and the choice of cultivar strongly influenced the efficiency of nitrogen uptake by the plants. The efficiency of plants was much higher in the year with more favorable weather conditions. The choice of cultivar mattered as well, as the Arkta and Aviron cultivars took up more nitrogen than the Myster cultivar. Neugschwandtner et al. [38] showed that the efficiency of nitrogen uptake is determined by the yield of the cultivars rather than by the amount of nitrogen accumulated. In a study comparing the yield of winter and spring forms of faba beans and peas, the authors showed that the grain nitrogen yields of winter peas and winter faba beans were 1.83-fold and 1.35-fold higher compared to their spring forms, respectively, with a higher value for winter peas.

Our study confirmed the value of the biological agents used to maintain the productivity of pea plants. The biological fungicides had a pronounced effect on the morphological characteristics of the plants. After treatment with application of Serenade ASO, the plants were slightly larger than those after treatments with Polyversum WP. The use of these agents slightly increased the number of pods and seeds relative to the control, while Polyversum WP had a significant positive effect on pod weight, seed weight, 1000 seed weight, and seed yield. We showed a significant increase in pea yield, on average by 0.5 t ha⁻¹, in the treatments with Polyversum WP. Our results are confirmed by experiments conducted by Georgieva et al. [39], who showed that the application of the organic products Biofa, Polyversum, NeemAzal, and Pyrethrum positively influenced the productivity of forage peas. The highest yield was obtained following the application of Polyversum in combination with the bioinsecticide Pyrethrum at the budding and flowering stages. According to the producer of Polyversum (Biopreparáty spol. s r.o., Praha, Czech Republic), the fungus Pythium oligandrum directly inhibits the growth of phytopathogenetic microorganisms in the plant and promotes the production of growth-stimulating substances. Our study confirmed the positive effect of the biological agents on plant productivity, especially on the pod number and seed number. However, we noted a positive and/or negative interaction of the product with the choice of pea cultivar. Not all pea cultivars responded in the same way to the presence of Pythium oligandrum in the soil. Polyversum was most effective in the Aviron cultivar, which produced the highest yields and showed a relatively low level of infection with pathogenic fungi.

4.2. Biological Agents and Plant Health

In our study, the diseases Ascochyta blight and Fusarium wilt were observed in the pea cultivars in both years of the experiment. Both diseases were more severe in the 2017/2018 season. The disease severity of Ascochyta blight can vary depending on the temperature and the duration of leaf wetness. The development of the disease is favored by temperatures of 20–21 °C and a high relative humidity. It normally will not develop at temperatures below 4 °C or above 35 °C or when periods of leaf wetness are shorter than 6 h [40]. A soil temperature of 23–27 °C is most favorable to Fusarium wilt development [41]. In earlier research, infection of peas with various pathogens in a field experiment was strongly influenced by the weather conditions during the growing season [42]. In the present study, the weather conditions in May of 2019 deviated from the long-term average. The last three weeks of the month were colder and extremely wet compared to the long-term average and the previous year. These conditions were not favorable for the development of Fusarium wilt, which develops well when the temperature is high during the flowering stage and pod-filling stage [31]. On the other hand, June of 2019 was drier and warmer compared to the previous year and the long-term average, which was unfavorable for the development of A. pisi. In a study by Olle and Sooväli [43], an experimental year with colder temperatures than average combined with a lot of rainfall in the same period provided good conditions for pod spot (A. pisi) on field pea plants. Marcinkowska et al. [44] tested the seeds of 10 pea varieties for Ascochyta blight fungi and found that all fungal species occurred less frequently in years when temperatures were higher than average, especially in June.

In the present study, the cultivar most severely infected with F. oxysporum in the treatments without protection was Specter, while A. pisi most severely infected the Baltrap cultivar, followed by Specter.

An important means of controlling pathogenic fungi is to use resistant cultivars. The choice of cultivar influences the incidence of disease in field peas. Despite extensive research, only moderate resistance is available in pea cultivars, and this has proven inadequate in controlling the disease [45]. Olle and Sooväli [43] found that different genotypes influenced the incidence of pod spot in field peas. The cultivars most damaged by Ascochyta blight were Vitra (Latvia) and Onward (Greece), while the least damaged were Bruno (Latvia) and Clara (Sweden). Winter peas are more sensitive to Ascochyta blight than spring peas, and thus their development is more limited by it [46]. Due to differences in plant architecture, Ascochyta blight may spread at different speeds in different genotypes. The plant architecture may affect spore dispersal on the plant by modifying the distances between the source and target organs. Leaf height and orientation can modify spore deposition, reducing disease development. Moreover, the rosette-type growth habit may affect the canopy architecture, leading to the development of a microclimate unfavorable for disease development. The rosette-type growth habit and the production of small leaflets may restrict the dispersal of spores in water droplets [47].

In the present study, protective treatments involving the application of Polyversum WP and Serenade ASO were very effective at protecting the plants against Fusarium wilt and Ascochyta blight. These microbial biocontrol products contain the bacterium Bacillus subtilis (Serenade ASO) and the fungus Pythium oligandrum (Polyversum WP), which are counted among biocontrol agents [48]. The greater the plant density and the shorter the internode length, the more susceptible the plant is to infection by fungal pathogens [15].

Research by Patkowska [49] showed that Polyversum applied as seed dressing and sprayed onto pea plants markedly improved their emergence, health, and yield. It also decreased the amount of plant pathogenic fungi, such as F. oxysporum, A. alternata, and B. cinerea, isolated from the plants. In earlier research, Patkowska [50] suggests that P. oligandrum can effectively protect germinating bean and pea seeds and seedlings from soil-borne fungi, which present a serious threat, especially species such as A. alternata, B. cinerea, F. culmorum, F. oxysporum, F. solani, P. irregulare, P. exigua, and R. solani.

Bacillus spp., such as B. subtilis, induce systemic resistance in plants, thereby significantly reducing disease. Bacillus pumilis has been found to induce the accumulation of phenolic compounds when pea roots are attacked by F. oxysporum [51]. The phenolic compounds enhance the mechanical strength of the plant cell wall and inhibit fungal growth, as they are toxic to fungi. El-Shennawy and Rania [52] showed that B. subtilis used as a bioagent was effective against the causal pathogens of faba bean wilt and root-rot diseases, i.e., Rhizoctonia solani, Fusarium oxysporum, and F. solani, by reducing the growth of the fungus. Moreover, application of B. subtilis in greenhouse conditions significantly reduced pre- and post-emergence damping-off infection and increased the number of healthy surviving faba bean plants compared with the control treatment. The bioagent improved some growth characteristics of faba bean plants infected under greenhouse conditions, increasing the plant height, fresh weight, and dry weight.

5. Conclusions

The yield of winter peas is influenced by a combination of the weather conditions during the growing seasons and the choice of cultivar. Pea cultivars with higher winter survival rates produce significantly higher yields. Biological agents containing Pythium oligandrum (Polyversum WP) or Bacillus subtilis (Serenade ASO) improve the health of winter peas, which translates to a higher productivity. Protective treatments involving the use of Polyversum WP or Serenade ASO effectively protect the plants against Fusarium wilt and Ascochyta blight.

The Aviron and Arkta cultivars are recommended for cultivation in the conditions of Central Europe due to their high yield, nitrogen uptake efficiency, and canopy health. The study indicates a clear need to continue field studies dedicated to the monitoring and the assessment of winter pea health and productivity using biological agents containing Pythium oligandrum or Bacillus subtilis microorganisms, compared to standard chemical agents.