Microbial Biopreparations and Their Impact on Organic Strawberry (Fragaria x ananassa Duch.) Yields and Fungal Infestation

Abstract

Growing consumer awareness of the importance of food quality on their health is the main driving force for increasing the market for sustainable agricultural products. This makes sustainable, environmentally friendly production methods into non-chemical plant protection products against pathogens, including microbial biopreparations, increasingly important among farmers. Strawberry fruits (Fragaria x ananassa Duch.) are often negatively affected by fungal pathogens. The aim of this study was to evaluate the impact of five combinations of newly developed microbial biopreparations (from K2 to K6) on fungal pathogens and the yield of three strawberry cultivars. The research was conducted on a certified organic strawberry plantation in Poland in 2020–2021. In the first year of the study, no statistically significant positive impact of tested treatments on strawberry yields have been found. At the same time, yields of ‘Honeoye’ treated with K4 combination showed a tendency (with no statistical significance) to have higher yields than the control object by about 33%. In the second year of the study, yields of ‘Honeoye’ and ‘Rumba’ treated with K4 combination (containing Bacillus sp. Sp116AC*, Bacillus sp. Sp115AD, Bacillus sp. AF75BC and Bacillus subtilis AF75AB2, humic acids, yeast culture effluent, micronized dolomite, and mustard and rapeseed oil) significantly increased by 79% and 49%, respectively. Fruit infestation by fungal pathogens was reduced under some microbial treatments; however, the effect varied between years, cultivars, and tested biopreparations. The K2 combination showed a tendency (with no statistical significance) to limit B. cinerea infestation rate by 23% in 2020 and 21% in 2021, C. acutatum by 16% in 2021, and P. cactorum infestation rate by 30% in 2021. Tested microbial biopreparations showed a positive impact on the yield of strawberries and (to some extent) on disease infestation, with an impact on pathogen infestation strongly dependent on the year, variety, and biopreparation tested.

1. Introduction

The strawberry (Fragaria x ananassa Duch.) is a species that originated about 250 years ago after crossing the Fragaria chiloensis L. with Fragaria virginana Duch. [1]. It is cultivated mostly in temperate and cool, sometimes in subtropical, climates. It grows best and has the highest yield on fertile soils with regulated water relations, in well-sunned sites [1]. The quality of strawberry fruits is reflected by the presence of health-promoting active substances [2,3]. Flavonols, flavonoids (responsible for fruits’ aroma), phenols, ellagic acid, and anthocyanins [4,5,6,7,8] can contribute to the anti-inflammatory [5,9,10], anti-neurodegenerative [5], and anticancer [6,8,10,11,12,13] properties of strawberry fruits. The global production of strawberries reached 8.8 million tons in 2020 [14]. Currently, Poland is the second largest producer of strawberries in the EU and is among the top ten producers worldwide [1,14,15]. Globally, nearly 10% of all strawberry plantations are located in Poland [14]. The high volume of production as well as the crop value makes it one of the most important berry crops in the country [16,17,18]. In 2021, there were 389.7 thousand hectares of strawberry plantations worldwide, while in Poland, they were grown on 33.9 thousand hectares. Moreover, the importance of strawberries in Poland seems to be increasing, as in 2021, the cultivated area increased year by year by 900 hectares [19]. In 2022, the harvest of strawberries was 185.1 thousand tons. In the same year, the largest cultivation area of strawberries was found in the Mazowieckie voivodeship (15.8 thousand ha), which is the region were the present study has been carried out. The second largest strawberry producer was Lubelskie region (3.8 thousand ha) [20,21]. Lubelskie is one of the regions with the highest yields of strawberries, reaching an average of 8.21 t ha⁻¹ in 2020 [22]. According to PIORIN (

Table 1. A list of acronym names used in the text.

Acronym Names	Explanation
EM	Effective microorganisms
GA3	Gibbelleric acid
K1	Control
K2	Combination with P3 preparation only
K3	Combination of preparations P3 and P1
K4	Combination of preparations P3 and P2
K5	Combination of preparations P3 and P1 and P2
K6	Combination of preparations P1 and P2
n.s.	Non-significant
P1	Preparation containing: Bacillus subtilis AF75AB2 and Bacillus sp. Sp115AD, on a carrier consists of plant extracts (nettle, horsetail, calendula), humic acids in liquid formulation in 2020 and in micronized dolomite (Inco S.A.) in 2021,
P2	Preparation containing: Bacillus sp. Sp116AC*, Bacillus sp. Sp115AD, humic acids, yeast culture effluent in liquid formulation in 2020 and in micronized dolomite (Inco S.A.) in 2021,
P3	Preparation containing: In 2020: Bacillus sp. AF75BC and Bacillus subtilis AF75AB2, on a carrier consists of wheat bran, dry humic acids, mustard, rapeseed oil, clove oil in a pellet formulation and on micronized dolomite (Inco S.A.) instead of wheat bran in 2021.
PIORIN	Plant Health and Seed Inspection Service
SD	Standard deviation

) data [23], the most popular certified strawberry cultivars in 2021 included ‘Camarosa’, ‘Nabila’, ‘Flair’, ‘Honeoye’, and ‘Grandarosa’. Moreover, Poland was the largest producer of certified organic strawberries in the European Union in 2020 in terms of both the cultivated area (1048 ha) and production volume of organic strawberries (8013 tons) [24].

The growing ecological awareness of the society, as well as the increasing demands of consumers regarding food quality, are important reasons for the development of sustainable agricultural management strategies, like organic farming system [25,26]. Organic farming is a production system based on few environmentally friendly principles, including the use of diversified crop rotation, the abandonment of the use of synthetic mineral fertilizers, and chemical plant protection products [27]. Biodiversity conservation, protection of the environment, and the prevention of water, soil, and air pollution are also important goals of the organic agriculture [28]. Under this system, the use of natural, technologically unprocessed inputs that create a positive impact on soil fertility and ensure yields of high biological quality is preferred [29]. Preventive measures to reduce the occurrence of diseases, pests, and weeds are also recommended. Certified plant protection products, which are based on the natural substances, are allowed to be used if other means are unsuccessful [30,31]. Organic agriculture is gaining importance as an alternative, sustainable food source due to its ability to improve ecosystem service delivery, reduce environmental pollution, lower carbon footprints, mitigate greenhouse gas emissions, and produce safe food of high quality [32,33]. Studies have shown that organic agriculture is in line with sustainable development principles by providing not only private goods, such as organic food, but also environmental public goods, such as landscape preservation and biodiversity conservation [34]. In 2022, the total number of organic farms in Poland (both in conversion process and certified) was 21,193, and the total area of organic farmlands reached 554,632 hectares [20].

Organic food is gaining importance because of its high nutritional value, which can be beneficial for human health due to higher content of bioactive substances (polyphenols, antioxidants) compared to fruits cultivated on conventional plantations [35,36,37]. Organic farming principles excludes the use of chemical plant protection products [31], so farmers have to look for new management strategies, including biological preparations (biopreparations), to maintain plantation quality and control pathogen infestations. Biopreparations are products that contain natural microorganisms and/or naturally occurring compounds to support agricultural production. Grzyb et al. [38] defines biopreparations as ‘consortia of microorganisms with species composition and activity adapted to carry out a specific process’. According to Pylak et al. [39], biopreparations may contain plant extracts, polysaccharides, humic substances, and beneficial bacteria and fungi.

Microbial biopreparations can be utilized as a measure to lower the risk of pathogenic bacteria or fungi infection; however, they are not always registered as plant protection products (PPPs). Such products can still have a beneficial impact on plant growth and development and hence lower the risk of the pathogen infestation of plantations [40]. Recently, the use of this type of product in integrated and conventional agriculture has also been increasing due to the restrictions introduced by the EU to the list of authorized active substances of PPPs and the significant increase in the price of mineral fertilizers and chemical plant protection products. This resulted in a greater demand for alternative products [41,42].

According to Pylak et al. [39], in many cases, biopreparations can be at least as effective as commercially available chemical PPPs; however, their effectiveness varies and is highly dependent on environmental factors such as precipitation, soil moisture content, and air humidity. Kosicka et al. [43] states numerous advantages of using biopreparations, such as improvements in the humus-forming capacity, a reduction in decay processes, improvements in the growth and quality of agricultural crops, and an increase in the availability of hard-to-access nutrients for plants and the elimination of pathogens. All this makes microbial preparations highly effective in improving the overall quality of fruits. In the last two decades, numerous scientific articles have been published on strawberry cultivation and pest control, including those concerning the effect of microbial fertilizers on the vegetative growth of strawberry [44,45]; applications of nanofertilizers [46] and nanotechnology in strawberry cultivation [47]; the effects of the composition of the growing medium enriched with biofertilizer on the growth and quality of strawberry plants [48]; the effect of growing media on growth, flowering, and fruit weight of strawberry [49]; the effect of zinc preparations on strawberry biomass [50,51]; and silicon preparations on growth and yield [52]. Moreover, some authors studied the effects of biostimulants [53], biopreparations and biocontrol agents [54,55,56,57,58], protective and stimulating effects of selected microorganisms [59], the nutrient and health-promoting content of strawberry fruits [1,60], and leaf diseases [54,55] in strawberry cultivation. At the same time, relatively few papers deal with organic strawberry cultivation [61,62,63]. According to Feledyn-Szewczyk et al. [64], one of the most important factors influencing both the quantity and quality of the crop yield in an organic system is the selection of cultivars (crop genotype). This is also the reason why the selection of the best possible genotype has an impact on farmers’ agricultural income and food security. The development and testing of new formulations of biopreparations can help farmers to increase the yields and provide consumers with healthy and safe food. The aim of the research was to determine the effect of newly developed biopreparations containing beneficial microorganisms and plant extracts on the yield and health of selected strawberry cultivars grown under the organic farming system conditions. In addition, the authors wanted to investigate whether there is an interaction between biopreparations and cultivars.

While previous studies have evaluated multiple aspects of strawberry cultivation and the use of microbial preparations, there is still a gap in our understanding of how the newly developed microbial biopreparations affect organic strawberry production, particularly in terms of the yield and pathogen infestation of different cultivars. It was hypothesized that the newly developed microbial biopreparations will significantly improve the strawberry yield and reduce pathogen infestation under organic farming conditions, with effects varying among cultivars. This study is unique in that it focuses on organic farming practices, the specific combination of beneficial microorganisms and plant extracts in our biopreparations, and the study of cultivar-specific responses.

2. Materials and Methods

2.1. Characteristics of the Experiment

The field study was conducted at the Institute of Soil Science and Plant Cultivation-State Research Institute in Puławy at the Agricultural Experimental Station in Grabów (51°21′17.1″ N 21°39′14.0″ E) (Poland, Mazowieckie region). The experiment with strawberry cultivation was established in 2019 on a certified organic field with area of 1150 m². The experimental factors were (1) combinations of biopreparations with beneficial microorganisms and plant extracts and (2) three strawberry cultivars with different susceptibility to fungal pathogens: ‘Honeoye’, ‘Vibrant’, and ‘Rumba’. The experiment was set up in a split-block design in 4 replication (3 cultivars × 6 combination of biopreparations × 4 replications, n = 72 plots). The experiment was set up on Luvisols on a gray-brown podzolic soil made of strong loamy sands on light loam. The forecrop for strawberries was red clover (Trifolium pratense L.). The experiment was set up in the third decade of May 2019. The experimental area was covered with fleece to reduce weed growth and evaporation, and no irrigation was used in this part of the experiment. Plants were planted at 30 cm × 100 cm spacing at a density of 30,000 plants·ha⁻¹. The area of each single plot of strawberries was 16 m² (4 × 4 m) and 48 strawberry plants were planted on it. Certified frigo strawberry seedlings from the professional Agronom Plants horticultural and nursery farm were grown. In addition to the tested biopreparations, fertilizers approved for organic farming were applied before (2018–2019) and during the years of research (2020–2021) (

Table 2. Fertilizers and other means of production used on strawberry plantation in organic system.

Trade Name	Manufacturer	Chemical Composition	Application Date	Dose Applied
	2018
Cattle manure	-	-	19,11,2018	30 t·ha−1
Patentkali®	K+S Polska Sp. z o.o., Poland	Potassium sulphate with magnesium: SO3 42.5%, K2O 30% water-soluble potassium	16,11,2018	250 kg·ha−1
Potassium salt	K+S Minerals and Agriculture GmbH, Germany	60% K2O	16,11,2018	100 kg·ha−1
	2019
Bioilsa N 12.5	NaturalCrop Poland Sp. z o.o., Poland	12.5% organic nitrogen, 40% organic carbon, SO3 4.4%, B 100 mg·kg−1, Zn 61 mg·kg−1, Cu 20 mg·kg−1, Mn 18 mg·kg−1, other mineral compounds (P, K, Ca, Mg, Fe)	11,04,2019	150 kg·ha−1
	2020
Redarom Activstart	Biodevas Laboratoires ZA de l’ L’Épine, France,	Aromatic plant extract. Mixture of micronutrients, guaranteed zinc content 0.7%.	29,04,2020	1.5 L·ha−1
Olibio	Biodevas Laboratoires ZA de l’ L’Épine, France	Silicon extracts from field horsetail (Equisetum arvense) and nettle (Urtica dioica): B 0.02%, Cu 0.5%, Fe 0.5%, Mn 0.5%, Mo 0.1%	29,04,2020	2.0 L·ha−1
Patentkali®	K+S Polska Sp. z o.o., Poland	Potassium sulphate with magnesium: SO3 42.5%, K2O 30% water-soluble potassium	05,08,2020	150 kg·ha−1
Aminosol	AZELIS POLAND Sp. z o. o., Poland	N 9.4%, K2O 1.1 %, S 0.25%, Na 1.28%, 66.3% organic matter (56–58% aminoacids and peptides)	21,07,2020	2.0 L·ha−1
	2021
Redarom Activstart	Biodevas Laboratoires ZA de l’ L’Épine, France,	Aromatic plant extract. Mixture of micronutrients, guaranteed zinc content 0.7%.	30,04,2021	1.5 L·ha−1
Olibio	Biodevas Laboratoires ZA de l’ L’Épine, France	Silicon extracts from field horsetail (Equisetum arvense) and nettle (Urtica dioica): B 0.02%, Cu 0.5%, Fe 0.5%, Mn 0.5%, Mo 0.1%	30,04,2021	2.0 L·ha−1
Aminosol	AZELIS POLAND Sp. z o. o., Poland	N 9.4%, K2O 1.1%, S 0.25%, Na 1.28%, 66.3% organic matter (56–58% aminoacids and peptides)	07,05,2021	3.0 L·ha−1

Table prepared on the basis of product/fertilizer labels and IUNG-PIB fertilizer search engine.

).

2.2. Biopreparations

The strawberries were treated with five organic biopreparations containing beneficial microorganisms and plant extracts, established as a result of ECOFRUITS project (BIOSTRATEG3/344433/16/NCBR/2018) (

Table 3. Composition of biopreparations. Source: Drobek et al., modified [65].

Components	Biopreparation Combinations Used in the Experiment
	K1	K2	K3	K4	K5	K6
	Control (sprayed with water)
(P1) Bacillus subtilis AF75AB2 and Bacillus sp. Sp115AD, on a carrier consists of plant extracts (nettle, horsetail, calendula), humic acids in liquid formulation in 2020 and in micronized dolomite in 2021.			x		x	x
(P2) Bacillus sp. Sp116AC*. Bacillus sp. Sp115AD, humic acids, yeast culture effluent in liquid formulation in 2020 and in micronized dolomite in 2021.				x	x	x
(P3) In 2020: Bacillus sp. AF75BC and Bacillus subtilis AF75AB2, on a carrier consists of wheat bran, dry humic acids, mustard, rapeseed oil, clove oil in a pellet formulation and on micronized dolomite instead of wheat bran in 2021.		x	x	x	x

The “x” sign indicates the presence of the preparation component.

).

The P3 preparation was applied to the treatments: K2, K3, K4, and K5. Biopreparation P1 was applied in treatments K3, K5, and K6, while biopreparation P2 was used in treatments K4, K5, and K6 by spraying the plants. On the same dates, plants in control objects were sprayed with water (K1) at a rate equivalent to the water used to dissolve the biopreparations. In 2021, the biopreparations were improved compared to 2020 (the carrier was changed from organic (bran) to mineral (dolomite)). In 2020, growing season biopreparations were used twice during the growing season, whereas in 2021, they were used three times to increase the effect. In 2020, the preparations were applied at a rate of 35 kg·ha⁻¹ of preparation diluted in 700 L·ha⁻¹ of water, whereas in 2021, the preparations were applied at a rate of 50 kg·ha⁻¹ of preparation dissolved in 700 L·ha⁻¹ of water to improve the effect on plants. On the next development stage of the products, market product will be 10 times more concentrated for ease of use by farmers. The tested products were applied using a tractor sprayer with a Fragaria type boom (spraying both from above and from sides).

2.3. Strawberry Fruit Analysis

Strawberry fruits were harvested during the second (2020) and third (2021) seasons of strawberry growth. Fruits were picked up successively every 3–4 days from the marked 8 plants in each plot. The fruits from each plot were counted and weighed. Total yield of strawberries was calculated per plant and per 1 ha.

At each harvest, an assessment of fruit infection by fungal pathogens, such as strawberry fruit leathery rot (Phytophthora cactorum), strawberry fruit gray mold (Botrytis cinerea), and strawberry anthracnose (Colletotrichum acutatum), was carried out. The percentage share of infested fruits in each combination was calculated. The percentage of fruit infected by individual pathogens was determined in each sample. Four train people were assigned to carry out fruit infestation assessment. The training took place directly before 1st season of harvest and 2nd season of harvest (2020 and 2021). No other inter-rater reliability measures have been undertaken.

2.4. Analysis of Biometric Parameters of Strawberry Plants

The assessment of the impact of tested biopreparations on the selected morphological features and biometric parameters of strawberry plants were carried out. This included the determination of the number of crowns developed by strawberry plants and dry weight of aboveground parts of plants. The analyses were conducted postharvest, in the first decade, in July of each year of the study. The measurement was performed on 5 randomly selected plants collected from each of 72 plots tested.

2.5. Meteorological Conditions

Average monthly temperatures in spring (April and May) in both years of the study were close to the multi-year average, while average temperatures for June and July were above the multi-year average (

Table 4. Meteorological conditions at experiment location in 2020–2021 growing seasons.

Month	Average Temperature [°C]			Precipitation [mm]
	2020	2021	Multi-Annual Average	2020	2021	Multi-Annual Average
April	8.6	6.4	7.5	15.6	51.2	42.0
May	11.3	12.5	12.4	76.5	49.9	53.0
June	18.3	19.5	16.7	157.8	70.1	110.0
July	18.6	21.8	17.8	38.3	61.7	105.0

). Year 2020 was characterized by an uneven distribution of the precipitation. Rainfall in May and June was higher compared to the multi-year average and 2021 season, while April and July 2020 were dry with a precipitation of about 37% and 36.5% of average multiannual precipitation. July 2021 was especially warm, with a mean temperature 4 °C higher than the multi-year average. With the exception of April, the 2021 season was warmer than the 2020.

2.6. Statistical Analysis

The data were analyzed using STATISTICA software (Statistica v.10, Statsoft Inc., Tulsa, OK, USA). In order to compare the influence of biopreparations (n = 6) and cultivars (n = 3) and their interaction effects on strawberry yield and health status, a one-way analysis of variance ANOVA was made. The significance of the mean differences of the study factors was evaluated using a post hoc Fisher’s test at a significance level of α = 0.05.

In order to check the normality of the distributions, the Shapiro–Wilk test was used. Since the data on pathogen infestation did not meet the requirements for parametric tests, a logarithmic transformation log (x + 0.1) of the data was carried out before the analysis of variance.

For the purpose of this study, no method was used to fill in missing data (there were no missing data for all included parameters).

3. Results and Discussion

3.1. The Influence of Biopreparations on Strawberry Yielding

Among three tested strawberry cultivars in 2020, only ‘Honeoye’ responded positively by increasing the fruit yield under the K4 combination treatment; the yield was 119.4% higher when compared to the control (

Table 5. The influence of the tested biopreparations on yields (g·plant⁻¹).

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	240.2 ab (±21.2)	227.30 a (±65.2)	440.2 ab (±79.0)	527.2 b (±184.0)	373.6 ab (±157.6)	419.1 ab (±260.4)	383.2 A (±176.1)
‘Rumba’	621.0 b (±0.70)	370.6 a (±108.2)	374.3 a (±58.6)	512.4 ab (±132.4)	599.0 b (±129.1)	606.6 b (±111.1)	504.2 B (±143.0)
‘Vibrant’	389.1 ab (±255.2)	216.6 a (±25.2)	341.7 ab (±72.2)	393.0 b (±54.3)	358.5 ab (±134.5)	260.7 ab (±62.4)	320.9 A (±109.7)
average	416.8 ab (±206.3)	271.5 a (±99.5)	385.4 ab (±76.7)	477.5 b (±137.0)	443.7 b (±171.6)	428.8 b (±211.4)	402.8 (±162.5)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	368.9 a (±32.8)	519.3 ab (±56.6)	588.8 ab (±136.1)	660.6 b (±33.8)	496.8 ab (±188.3)	590.6 ab (±148.2)	552.8 A (±135.2)
‘Rumba’	443.9 a (±25.8)	578.8 ab (±89.2)	574.9 ab (±77.4)	661.1 b (±119.4)	560.1 ab (±77.7)	602.3 ab (±84.5)	581.7 A (±95.6)
‘Vibrant’	663.2 ab (±137.6)	576.2 ab (±83.2)	688.5 b (±49.0)	649.9 ab (±122.7)	622.2 ab (±104.0)	503.5 a (±121.7)	613.1 A (±110.4)
average	492.0 a (±151.1)	558.1 ab (±75.9)	617.4 bc (±100.7)	657.2 c (±91.3)	559.7 ab (±130.9)	565.5 ab (±118.7)	582.5 (±115.8)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

). On average, ‘Rumba’ yielded significantly higher (504.2 g·plant⁻¹; i.e., 15.2 t·ha⁻¹) than ‘Honeoye’ (383.2 g·plant⁻¹; i.e., 11.5 t·ha⁻¹) and ‘Vibrant’ (320.9 g·plant⁻¹; 9.6 t·ha⁻¹). A low level of the ‘Vibrant’ yield was linked with flower damage by frost in May 2020. ‘Vibrant’ flowers raised high above the plant resulted in a high risk of direct contact with frost on the surface of the white agro-textile that covered the field to protect it from local spring frost damage. Interestingly, K2 and K3 treatment in the ‘Rumba’ cultivar resulted in a significant yield loss compared to the control object.

In 2021, the ‘Honeoye’ and ‘Rumba’ cultivars yielded significantly higher when treated with the K4 biopreparations (by 79% and 49% higher than the control) (Table 5). Although there was no significant impact, a tendency was visible for the ‘Honeoye’ cultivar to have higher yields when treated with the K3 and K6 biopreparations. In 2021, the impact of the tested combinations of biopreparations on the strawberry yield of the tested cultivars was positive or, at the worst, neutral (there was no negative impact on the yield for any of the tested treatments). In both years of the study, ‘Honeoye’ was the cultivar that was under the strongest positive influence of biopreparation, with significant yield increases for the K4 combination in 2021 and a visible tendency for a yield increase (however, not proven statistically) for the other tested treatments.

The results correspond with Kuś et al.’s [66] research, who found that using effective, beneficial microorganisms (EM biopreparations) resulted in an increase in the winter wheat yield in a conventional monoculture, whereas in an organic system, the effect of EM preparations was insignificant. It suggests that biopreparations with beneficial microorganisms work best in difficult, problematic habitats. An organic farming system may have higher initial biodiversity than other farming systems, and thus, the impact of introduced biopreparations might be lower compared to the conventional system. Soltaniband et al. [3], comparing the performance of six biostimulants under conventional cultivation in a high tunnel and greenhouse and in organic greenhouse conditions found that biostimulant use resulted in an increased productivity in the conventional high tunnel, while the highest yields were obtained in the conventional greenhouse after the application of citric acid. The authors hypothesized that the different impacts of biopreparations in 2020 and 2021 may be due to habitat stabilization, as the positive impact of microbial-based biopreparations can build up over time, stabilizing the microbial community. Moreover, the doses of biopreparations and the frequency of usage were increased in 2021 compared with 2020. Similarly, Mikiciuk et al. [67] reported a positive effect of the tested mycorrhizal biopreparations on the strawberry yield and fruit weight.

According to Mohamed et al. [68], the yield of strawberries and its quality depends on nutrient provision, soil fertility, and water availability during the growing season. Hindersah et al. [48] cite growth media compositions among the factors affecting yield, which while influencing seedling quality, also affect the yield. Ahmed et al. [69], in a 3-year study conducted in Bangladesh, obtained yields averaging from 6.88 to 9.59 t·ha⁻¹ depending on the fertilization rate when grown in a conventional farming system. Those yields were lower than the results obtained in the present study in organic conditions (average yields of 12.1 t·ha⁻¹ in 2020 and 17.3 t·ha⁻¹ in 2021), which suggest that the level of crop yields in an organic system can reach or even increase the yield of conventional crops [70]. Mohamed et al. [68] indicated significantly higher yields (65.5 t·ha⁻¹) after the application of 50% mineral N + 100% organic N and the foliar spray of Ca+B in comparison to the control (44 t·ha⁻¹). You et al. [71] obtained a 40% increase in yield as well as a prolongation of fruiting time and an increase in the share of high quality fruits after the application of a fermented biofertilizer containing straw, cattle manure, antagonistic actinomycetes, and solar disinfection.

Yields per plant in 2020 ranged from about 271.5 g per plant (K2) to 477.5 g (K4), while in 2021, they ranged from 492.0 g (control) to 657.2 g (K4) (Table 5). In both years of the study, the highest yields were obtained in the combination with the K4 formulation (477.5 g·plant⁻¹ and 657.2 g·plant⁻¹ on average for all cultivars). Interestingly, the yield of the control treatment in 2021 was greater than the yield of the best treatments of 2021. This shows the importance of local weather conditions for strawberry productivity. Wójcik-Seliga et al. [18] assessed the production potential of 23 strawberry cultivars and average yields for individual cultivars ranging from 45 g·plant⁻¹ for the ‘Plahuelfre’ cultivar to 580 g·plant⁻¹ for the ‘Alfa Centauri’ cultivar. ‘Honeoye’ yields were on average 320 g·plant⁻¹. In the present study, ‘Honeoye’ yielded, on average for all combinations, 383.2 g·plant⁻¹ in 2020 and 552.8 g·plant⁻¹ in 2021. ‘Honeoye’ yielded the highest when treated with K4, 660.6 g·plant⁻¹ in 2021.

Similar levels of yields were obtained by Sener and Turemis [72], who studied the effect of different types of mulch on the yield of several strawberry cultivars grown under organic farming conditions. The authors observed the highest yields from plants mulched with agro-textile (486.11 g·plant⁻¹), while among the cultivars, ‘Monterey’ yielded significantly the highest (696.90 g·plant⁻¹).

Kalnina et al. [73] in a field experiment conducted in Latvia obtained ‘Rumba’ cultivar yields of 245.9 g·plant⁻¹ (2010), 710.7 g·plant⁻¹ (2011), and 433.0 g·plant⁻¹ (2012). In the present experiment with the same cultivar, the average yield for all tested bioformulations was at 504.2 g·plant⁻¹ in 2020 and 581.7 g·plant⁻¹ in 2021. In contrast, in the experiment described in the same paper [73] conducted in 2015 in Germany in a tunnel where strawberry plants were mulched with black plastic mulch, yields of 249.3 g·plant⁻¹ for ‘Honeoye’ and 208.1 g·plant⁻¹ for ‘Rumba’ were obtained. Those yields were lower than obtained in the present study for both ‘Honeoye’ and ‘Rumba’ under organic conditions in both the years of the study (Table 5).

Kilic et al. [74] tested the effect of different fertilizers on strawberries grown under an organic system and reported lower levels of strawberry yields than in the present experiment. The highest yield of 190.61 g·plant⁻¹ was obtained after vermicompost application and slightly lower after humic–fulvic acid application (182.92 g·plant⁻¹), while a significantly lower yield was obtained after manure application (95.30 g·plant⁻¹). The authors concluded that the fertilization with vermicompost and humic–fulvic acids in organic strawberry cultivation had a positive effect on yields. Develi et al. [75] recorded that after the application of vermicompost at a rate of 45 g per plant, strawberries yielded 972.8 g, which was 153.2% higher than the control (384.2 g). Filipczak et al. [52] obtained the highest increase in strawberry yielding after the application of silicic acid on plants of the ‘Elkat’ cultivar (1300 g of fruit per plant—15% more than the control), while the ‘Elsanta’ cultivar yielded 850 g per plant after the application of calcium silicate (70% more than the control).

In comparison to organic systems, Khunte et al. [12] recorded the highest yields of strawberries in the conventional system—330.18 g·plant⁻¹ after the application of poultry manure at 5.50 t·ha⁻¹ and triacontanol (150 ppm). Singh et al. [50] found that three applications of 0.2% FeSO₄ + 0.3% ZnSO₄ at monthly intervals on the strawberry cultivar ‘Chandler’ resulted in the highest total yield of 236.57 g·plant⁻¹.

Some authors studied the effect of gibberellic acid application on the yield and strawberry fruit quality. Sharma and Singh [76] reported a decrease in the number of deformed fruits and increase in the total yield and marketable yield in relation to the control. Treatment with GA₃ resulted in yields of 529.2 g·plant⁻¹, which was 256% more than yields on the control object. Similarly, Saima et al. [77], who tested the effect of plant bioregulators on strawberry yield traits, noted that the ‘Chandler’ cultivar yielded the most after the application of GA₃ in an amount of 75mg L⁻¹ (356.56 g·plant⁻¹). According to Kosicka et al. [43], biopreparations are less effective and take longer to act on pathogens than pesticides, but unlike pesticides, they do not cause biotic imbalances and improve the humus-forming properties of the soil, resulting in higher crop yields.

3.2. The Influence of Biopreparations on Fungus Infestation of Strawberry Fruits

Strawberry yields, as any crop, are significantly influenced by various pathogens. The impact of a pathogen infestation can cause a major yield loss in strawberry fruits. The primary pathogens affecting strawberry plants include fungi such as Botritis cinerea, Colletotrichum acutatum, Phytophtora cactorum, Fusarium oxysporum, and Verticillium dahliae, which are known to cause diseases that can reduce yields by as much as 20–80% depending on the pathogen and environmental conditions [78,79,80,81]. Most fungal pathogens can accumulate in the soil on plantations where strawberries are grown repeatedly in the same location for a long time, leading to increased disease pressure, reduced plant health, and, in turn, yield and economic losses [82,83]. Plant diseases caused by some fungus species are recognized as one of the most important drivers of yield losses worldwide, including strawberry plantations. Depending on the region, host plant or fungus species, losses range from 8 to as much as 41% [78]. Ziedan [84] reports that crop losses of even up to 50–75% are due to soil-borne pathogenic fungi, such as Rhizoctonia spp., Fusarium spp., Verticillum spp., Sclerotinia spp., Phytium spp., and Phytophtora spp. The most common strawberry pathogens are fungi of the genera: Botrytis, Verticillium, Phytophthora, Colletotrichum, Penicillium, Alternaria, Cladosporium, Rhizopus, Aureobasidium, and Cryptococcus [85]. Infections of fruit leading to rotting can occur between flowering and maturity or even at the harvest stage. The use of microorganisms for plant protection requires the disruption of one of the stages of the disease or the life cycle of the pathogen. This mostly means the prevention of infection by the reduction of cell colonization or sporulation and survival of the pathogen. In order to effectively prevent infection by phytopathogenic fungi, the antagonist should be on the surface before the infection occurs [86].

A review of the literature shows that some biopreparations, especially those containing microorganisms, have a potential to improve the health of strawberry plants and fruits [52,87,88,89,90]. In the present study, preparations containing Bacillus spp., Paenibacillus spp. bacterial strains, plant extracts, humic acids, ground mustard, xanthan gum, bran, and vegetable oils were tested to improve the health of strawberry fruits in an organic system.

In the present study, on average for all tested cultivars, there was no positive effect on decreasing significantly the infestation rate of Botritis cinerea, Colletotrichum acutatum, or Phytophthora cactorum in relation to the control treatment. The effectiveness of the tested combinations in relation to each other depended significantly on both used combinations, the tested cultivar, and the year of the experiment. Although the results of the statistical analysis did not show a statistically significant effect on the reduction of disease infestation, some trends were visible on their impact on the reduction of the infestation rate. Some of the tested treatments showed trends (statistically non-significant) of the reduction of the infestation rate often exceeding 30% compared to the control object.

3.2.1. Botrytis cinerea (Gray Mold)

Botrytis cinerea, commonly known as gray mold, is one of the most common fungal pathogens of many crops. This pathogen is notorious for causing extensive damage to strawberries, both the plant and the flowers and fruits. It also can damage fruits during postharvest storage, leading to substantial economic losses for producers. Nutrient-rich strawberry fruits, with high moisture content, make strawberries an ideal host for the pathogen [91,92,93,94]. The pathogen’s high reproduction capabilities are reflected by its ability to produce a large number of conidia. In turn, its genetic variability contributes to its general resilience and capacity to develop resistance to fungicides—which makes effective management an issue [95,96].

In 2020, there was a tendency to reduce the incidence of Botrytis cinerea on strawberry fruits by the tested microbiological preparations, especially in combination of K6 (by 29.5% in comparison with the control), but the differences were not significant (

Table 6. The influence of biopreparations on the Botrytis cinerea (gray mold) infestation rate (% of fruit infected).

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	1.49 a (±0.98)	2.07 a (±1.34)	4.47 a (±3.75)	2.79 a (±0.60)	3.19 a (±3.11)	1.93 a (±2.21)	2.76 A (±2.33)
‘Rumba’	4.85 a (±3.22)	2.80 a (±2.54)	4.49 a (±2.28)	3.38 a (±1.00)	4.03 a (±1.84)	3.58 a (±2.97)	3.77 A (±2.12)
‘Vibrant’	4.43 ab (±1.13)	3.48 ab (±1.41)	5.10 b (±3.20)	2.69 ab (±0.82)	3.33 ab (±1.53)	2.08 a (±1.63)	3.44 A (±1.93)
average	3.59 ab (±2.28)	2.78 a (±1.78)	4.69 b (±2.85)	2.96 a (±0.81)	3.52 ab (±2.09)	2.53 a (±2.25)	3.32 (±2.14)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	1.10 ab (±1.55)	0.13 a (±0.15)	0.29 ab (±0.28)	1.06 b (±1.00)	0.17 ab (±0.14)	0.26 ab (±0.32)	0.45 A (±0.67)
‘Rumba’	0.12 a (±0.17)	0.50 a (±0.56)	0.20 a (±0.07)	0.15 a (±0.19)	0.30 a (±0.32)	0.31 a (±0.22)	0.28 A (±0.30)
‘Vibrant’	0.20 a (±0.28)	0.49 a (±0.56)	0.36 a (±0.59)	0.74 a (±0.69)	0.57 a (±0.93)	0.69 a (±0.86)	0.54 A (±0.65)
average	0.47 a (±0.86)	0.37 a (±0.46)	0.28 a (±0.35)	0.65 a (±0.75)	0.34 a (±0.55)	0.42 a (±0.53)	0.42 (±0.57)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

). In 2020, Botrytis cinerea remained at a low level, with an average of 3.32% for the experiment. A positive trend in reducing the infestation of fruit by gray mold for the ‘Rumba’ cultivar after using all combinations of preparations was found. In 2021, there was no clear effect of biopreparations in reducing fruit infestation by Botrytis cinerea, with a generally very low pressure of this pathogen at an average of 0.42% for the whole experimental site (Table 6). A positive trend of all tested biopreparations on protection against this pathogen was observed on the ‘Honeoye’ plantation, but the differences were not significant.

Due to the high variability of the results, there were few significant statistical differences in terms of the ability of the tested combinations of biopreparations to reduce the Botritis cinerea infestation. At the same time, some of the tested combinations may have the potential to reduce this infestation—which is indicated by the percentage of reduced infestation rate compared to the control object: the Botritis cinerea infestation rate was reduced by 29% by the K6 treatment in 2020 (11% in 2021), while in 2021, the use of the K3 combination resulted in a 40% decrease in the infestation rate of Botritis cinereal (interestingly, the K3 treatment in 2020 resulted in an increase in the infestation rate of 31%; however, in this year, the infestation of Botritis cineral on the whole field was very low, with an average of only 0.42%). The use of the K2 combination resulted in a decreased infestation rate of Botritis cinerea of 23% in 2020 and 21% in 2021. This shows how important the weather conditions of a given year are for the development of pathogens and the effectiveness of their control by microbiological agents.

According to Stachowiak and Ratajczyk [78], gray mold can cause up to a 60% loss in the strawberry yield. The authors noticed that the growth of phytopathogens in the presence of cultures and Bacillus circulans supernatants added to the nutrient solution was reduced by 53% to almost 90%. This leads to the conclusion that B. circulans may prove to be a promising alternative to chemical fungicides [78]. In a study by Kowalska [87], a preparation containing a strain of Trichoderma asperellum was tested for the protection of strawberries grown in an organic system and the symptoms of gray mold on the harvested and stored fruits as well as the plants itself were found sporadically. In the second year of the experiment, significantly higher yields were recorded on the objects where the preparation of T. asperellum was applied compared to the control object. According to Kowalska [87], biopreparations with Trichoderma spp. may be useful to organic processors and producers as an alternative to synthetic protection agents, the use of which is prohibited in certified organic farms.

Laboratory studies on the effect of Bacillus subtilis BS-2 and peppermint oil as a biocontrol agent against the pathogen Botrytis cinerea showed that supernatants obtained from Bacillus subtilis BS-2 inhibited the mycelial growth of B. cinerea by up to 57%, while the application of peppermint oil at the highest concentration (4%) reduced mycelial growth by 32% [57]. Biological methods of protection against Botrytis sp., such as the use of microorganisms or metabolites as well as plant extracts or essential oils, can be an effective alternative or supplement to conventional disease control methods [57]. In a study by Meszka and Bielenin [88] laminarin, a compound extracted from the brown alga Laminaria digitata used to stimulate plant resistance reduced the severity of strawberry gray mold by up to 50 to 80%, depending on the severity of the disease. In a study by Filipczak et al. [52], it was shown that the soil application of silicic acid also inhibited Botritis cinerea. The authors concluded that fertilization with silicic acid preparations is economically viable, effective, and safe in strawberry cultivation. The study of Wachowska et al. [89] showed that all of the preparations, Signum 33 WG, Vaxiplant SL, and a solution of cells of the fungus Aureobasidium pullulans, reduced the infestation of flowers by Botrytis cinerea. The application of Vaxiplant SL also reduced leaf infestation. In contrast, none of the tested preparations significantly reduced the infestation of strawberry fruits and had no effect on the increase in plant yields. On the other hand, Oliveira Filho et al. [90] found that an inhibition of Botrytis cinerea on strawberries of up to 100% and 83% after the application of M. spicata and C. martini essential oils, respectively. Aquilar-Gonzàlez et al. [97], when they tested the antifungal activity of mustard (Brassica nigra) and clove (Syzygium aromaticum) essential oils against gray mold on strawberry fruits, found that the application of vapors of these oils together or individually inhibited the growth of B. cinerea both in vitro and in vivo. At the same time, they found that the combined use of clove and mustard oil produced a synergistic antifungal effect. The combined use of these two oils may be an alternative to synthetic antimicrobials.

3.2.2. Colletotrichum acutatum (Anthracnose of Strawberry, Black Spot of Strawberry)

Colletotrichum acutatum can cause anthracnose disease across a cultivar of crops, including strawberries. This pathogen is particularly notorious for its ability to induce severe fruit rot, root necrosis, and crown rot [98,99,100]. The disease manifests as black spots on leaves and brown, collapsed spots on the fruits, which can severely affect both the yield amount and marketability of fruits [99,101]. Effective management strategies for C. acutatum include the use of plant protection products and management practices aimed at reducing humidity and improving air circulation around the plants [102,103].

Anthracnose of strawberries is caused by three fungi of the species Colletotrichum: C. acutatum J.H. Simmonds, C. fragariae A.N. Brooks, and C. Gloeosporoides (Penz.) Penz. and Sacc [101]. It manifests itself by the appearance of brown dry or rot spots on the fruit and by the browning and drying of the flowers and shoots [104,105]. According to Miller-Butler et al. [101], the cultivation of cultivars resistant to anthracnose is a promising way to reduce fungicides on strawberry plantations, especially in organic farming.

Colletotrichum acutatum was, in general, poorly controlled by tested combinations in both years of the study. However, in 2021, the K5 treatment shows a tendency to limit by a 27% infestation rate of C. acutatum. However, in 2020, the same combination showed a 20% increase in the infestation rate (average for all tested cultivars). The use of the K4 combination resulted in moderately good results with a 12% increase in the infestation rate (2nd best score of the year) in 2020 and a 16% decrease in the infestation rate of C. acutatum in 2021.

In 2020, there were no significant differences in infestation between different combinations (

Table 7. The influence of biopreparations on the Colletotrichum acutatum (anthracnose) infestation rate (% of fruit infested).

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	3.0 a (±4.24)	7.7 a (±11.26)	8.0 a (±3.90)	9.4 a (±8.45)	4.8 a (±2.93)	5.4 a (±7.44)	6.7 A (±6.68)
‘Rumba’	17.0 ab (±0.01)	26.5 b (±10.83)	18.7 ab (±7.16)	14.2 a (±9.15)	18.9 ab (±3.61)	11.8 a (±3.80)	17.9 B (±8.00)
‘Vibrant’	16.2 a (±0.58)	21.4 a (±8.37)	16.0 a (±3.75)	17.2 a (±2.96)	20.0 a (±7.80)	18.6 a (±5.91)	18.4 B (±5.57)
average	12.1 a (±7.29)	18.5 a (±12.42)	14.2 a (±6.67)	13.6 a (±7.48)	14.6 a (±8.64)	11.9 a (±7.74)	14.3 (±8.65)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	7.4 a (±7.12)	9.4 a (±4.17)	5.5 a (±3.66)	4.7 a (±1.83)	4.0 a (±2.10)	9.0 a (±5.50)	6.6 A (±4.13)
‘Rumba’	8.0 a (±0.24)	11.7 a (±5.19)	13.0 a (±4.77)	9.1 a (±1.99)	10.0 a (±2.34)	9.8 a (±2.18)	10.5 B (±3.41)
‘Vibrant’	10.1 ab (±2.29)	9.7 b (±2.29)	9.7 b (±3.92)	7.4 ab (±2.21)	4.5 a (±1.34)	9.9 b (±4.59)	8.4 AB (±3.38)
average	8.5 abc (±3.57)	10.2 b (±3.83)	9.4 bc (±4.94)	7.1 ac (±2.61)	6.2 a (±3.33)	9.6 bc (±3.93)	8.5 (±3.94)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

). In 2020, the least infected cultivar was ‘Honeoye’ (6.7% of infected fruit), whereas the infestation rate of ‘Vibrant’ and ‘Rumba’ was about three times higher than ‘Honeoye’. In 2021, ‘Vibrant’ was the only cultivar to show a trend of reduction in the infestation for all tested combinations in relation to the control. The lowest infection rate was recorded after the K5 treatment; on the ‘Vibrant’ plantation, a 55.5% lower infection was noted after the K5 application compared to the control. Similarly, on average for all cultivars, formulation K5 was the most effective in reducing anthracnose infection (27.1% lower than in the control).

According to Marian et al. [106], anthracnose is considered one of the most important and, unfortunately, also most common diseases of strawberries, causing more than 50% of yield losses in production plantations and even up to 80% of strawberry plant deaths in nurseries. The authors concluded that the Streptomyces sp. MBFA-172 isolate has a great potential for use as an alternative or complement to conventional chemical fungicides in the protection of strawberries against anthracnose. The application of the Streptomyces sp. MBFA—172 isolates resulted in a significant reduction in the frequency and severity of strawberry anthracnose, and the results obtained were statistically comparable to those obtained after the application of the chemical fungicide Propineb [106]. Alijani et al. [107] reported a positive effect of Bacillus spp. bacterial isolates in reducing postharvest fruit anthracnose, with the highest inhibition found for the isolate MarD40 (86% of reduction) and MarD35 (82% of reduction).

3.2.3. Phytophthora cactorum (Leather Rot)

Phytophthora cactorum, a fungal-like oomycete pathogen, can infect various parts of the strawberry plant, including roots, crowns, and fruits [108,109]. The disease often manifests rapidly as wilting, stunting, and ultimately plant collapse, particularly in susceptible cultivars [110,111]. The pathogen’s adaptability and ability to overcome host defenses complicate management strategies, making an integrated approach—including the utilization of resistant cultivars, effective plant protection, and soil and water management—a key in controlling this pathogen in strawberry cultivation [112,113].

Phytophtora cactorum (Lebert and Cohn) J. Schröt. is one of the most important pathogens in strawberry cultivation [114]. The appearance of this pathogen in the plantation can result in a significant yield and economic losses. Meszka and Michalecka [115] concluded that the P. cactorum is the most common accidental infection agent of strawberries in Poland. At temperatures above 20 °C and high humidity, it causes whole-plant rot and significant losses in strawberry fruit yields.

Phytophthora cactorum infestation showed a tendency to be well managed by K3 treatment in both years of the study. The infestation rate was reduced by this treatment by 46% in 2020 and 27% in 2021. Moreover, the K2 combination showed a tendency to limit the P. cactorum infestation rate by 30% in 2021 (and to increase the infestation rate by 17% in 2020). In 2020, the most effective combination in reducing P. cactorum on strawberry fruits was K3 (

Table 8. The influence of biopreparations on the Phytophthora cactorum (leather rot) infestation rate (% of fruit infested).

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	1.84 a (±1.03)	2.33 a (±2.64)	0.92 a (±1.21)	2.01 a (±2.07)	1.80 a (±1.84)	1.37 a (±1.38)	1.70 A (±1.69)
‘Rumba’	3.70 a (±2.02)	5.38 a (±1.64)	3.07 a (±1.92)	3.46 a (±0.40)	3.09 a (±2.10)	4.11 a (±2.36)	3.81 B (±1.81)
‘Vibrant’	4.99 a (±4.07)	4.58 a (±3.41)	1.68 a (±1.38)	2.99 a (±1.58)	2.45 a (±2.96)	4.53 a (±4.85)	3.40 B (±3.03)
average	3.51 ab (±2.52)	4.10 b (±2.76)	1.89 a (±1.67)	2.82 ab (±1.51)	2.45 ab (±2.20)	3.34 ab (±3.25)	2.97 (±2.41)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	3.29 ab (±2.29)	2.05 ab (±0.70)	1.34 a (±1.24)	1.95 ab (±1.18)	3.96 b (±0.90)	2.35 ab (±2.12)	2.42 B (±1.52)
‘Rumba’	2.00 a (±0.97)	1.02 a (±0.69)	1.16 a (±1.05)	1.89 a (±2.21)	1.95 a (±1.36)	1.12 a (±1.01)	1.48 A (±1.25)
‘Vibrant’	2.83 ab (±0.38)	2.60 a (±0.36)	3.40 ab (±0.55)	4.67 b (±2.32)	2.78 a (±0.49)	2.57 a (±1.15)	3.17 B (±1.29)
average	2.70 a (±1.27)	1.89 a (±0.87)	1.97 a (±1.39)	2.84 a (±2.24)	2.90 a (±1.24)	2.01 a (±1.52)	2.36 (±1.51)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

). In 2021, the combination K2, K3, and K6 were found to be highly effective in reducing the infestation of strawberry fruits by P. cactorum (by 26–30% on average compared to the control). Although in both years of the study there were no significant differences between combinations of tested biopreparations and control object, generally, the infestation level was low (below 5.5% of infested fruits). The least infested cultivar was ‘Honeoye’ in 2020 and ‘Rumba’ in 2021.

The combinations of microbial biopreparations tested in this study included Bacillus spp., particularly Bacillus subtilis; humic acids; and some other organic compounds (Table 3). All of those were present in the K4 combination, which proved to be one of the most universal combinations among the tested ones. Bacillus species are recognized for their plant growth-promoting properties, among them, enhancing the nutrient availability, stimulating root development, and releasing phytohormones (indole-3-acetic acid and gibberellins) [116,117]. These mechanisms can positively impact not only plant flowering but also fruit size and, in turn, the overall yield of strawberries [118,119]. Bacillus spp. have been proven to have the potential to control Botrytis cinerea and Phytophthora cactorum, which are the major threats to strawberry crops. The antimicrobial properties of Bacillus, attributed to the production of secondary metabolites and enzymes, help suppress these pathogens, thereby reducing disease incidence and enhancing plant health [120,121]. Humic acids can positively impact strawberries by improving the soil structure, enhancing the nutrient uptake, and stimulating the microbial activity in the rhizosphere [122]. The synergistic effect of Bacillus spp. and humic acids can lead to improved resilience against environmental stresses and pathogens to promote the yield quantity and better fruit quality [117].

Other authors have investigated the efficacy of preparations containing Bacillus strains on strawberry plant diseases. Pastrana et al.’s [123] study showed that two commercial microbial-based preparations: the Prodygy® (AMC Chemical, Sevilla, Spain) formulation with Trichoderma asperellum T18 and the Fusbact® (AMC Chemical, Sevilla, Spain) formulation with two strains of the Bacillus (B. megaterium and B. laterosporus) were found to be effective in controlling strawberry sclerotial rot (charcoal rot). Dipping strawberry roots in the formulation with T. asperellum reduced the incidence of crown and root rot caused by Fusarium solani by 81% under field conditions and even up to 100% in the greenhouse.

Filipczak et al. [53] concluded that biostimulators can be used as an effective, safe, and economically viable alternative to mineral nitrogen fertilization and plant protection measures against pathogenic microorganisms such as Salmonella sp., Escherichia coli, and Listeria monocytogenes, which pose a direct threat to human health.

3.3. The Influence of Biopreparations on Biometric Parameters of Strawberry Plants

All treatment combinations showed a positive trend to increase the number of crowns per plant compared to the control in both years of the study (with the exception of the K2 treatment in 2021), but due to high variability between replications, this effect was not statistically significant when compared to the control (

Table 9. The effect of biopreparations on the number of crowns developed by strawberry plants.

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	4.5 a (±0.71)	4.5 a (±1.00)	4.8 a (±1.50)	7.5 a (±3.00)	5.8 a (±2.75)	5.3 a (±2.06)	5.5 A (±2.15)
‘Rumba’	7.5 a (±3.54)	8.5 a (±4.36)	9.5 a (±6.24)	6.3 a (±2.50)	6.3 a (±1.50)	5.8 a (±2.22)	7.3 B (±3.59)
‘Vibrant’	5.0 a (±1.41)	7.8 a (±0.96)	6.5 a (±2.08)	5.3 a (±1.89)	5.8 a (±2.22)	6.5 a (±1.29)	6.2 AB (±1.77)
average	5.7 a (±2.25)	6.9 a (±3.00)	6.9 a (±4.08)	6.3 a (±2.46)	5.9 a (±2.02)	5.8 a (±1.80)	6.3 (±2.69)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	6.5 ab (±2.12)	4.5 a (±2.08)	7.8 ab (±1.71)	9.8 b (±2.63)	6.0 a (±1.83)	6.8 ab (±3.50)	6.9 B (±2.72)
‘Rumba’	3.5 a (±0.71)	5.3 a (±2.63)	5.8 a (±2.22)	7.5 a (±3.11)	6.3 a (±1.89)	5.5 a (±1.91)	5.8 AB (±2.30)
‘Vibrant’	6.5 a (±2.12)	5.3 a (±1.89)	5.0 a (±3.46)	4.5 a (±0.58)	5.5 a (±1.91)	5.5 a (±2.52)	5.3 A (±2.05)
average	5.5 ab (±2.07)	5.0 a (±2.04)	6.2 ab (±2.62)	7.3 b (±3.11)	5.9 ab (±1.73)	5.9 ab (±2.54)	6.0 (±2.44)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

).

In the first year of the experiment, the use of all the tested biopreparations resulted in a trend towards the increased dry weight of the aboveground parts of strawberries in relation to the control, but due to high variability between replications, this effect was not statistically significant (

Table 10. Effect of biopreparations on dry weight of aboveground parts of plants (g·plant⁻¹).

Years and Combinations
2020
	K1	K2	K3	K4	K5	K6	Average
‘Honeoye’	61.5 a (±13.08)	59.5 a (±14.95)	81.5 a (±43.86)	108.1 a (±59.31)	116.9 a (±86.11)	106.9 a (±49.69)	91.6 A (±52.43)
‘Rumba’	113.4 a (±45.32)	115.0 a (±64.96)	118.8 a (±58.65)	105.0 a (±34.16)	133.6 a (±58.14)	146.0 a (±61.24)	122.7 B (±50.85)
‘Vibrant’	100.4 a (±22.84)	112.0 a (±40.19)	126.7 a (±51.37)	118.3 a (±30.03)	102.1 a (±42.98)	96.8 a (±27.15)	110.2 AB (±35.38)
average	91.7 a (±33.66)	95.5 a (±48.58)	109.0 a (±51.05)	110.5 a (±39.49)	117.5 a (±60.24)	116.6 a (±48.86)	108.2 (±47.90)
2021
	K1	K2	K3	K4	K5	K6	average
‘Honeoye’	35.5 a (±14.59)	31.6 a (±12.03)	44.9 a (±15.77)	48.1 a (±12.16)	31.5 a (±6.05)	35.5 a (±10.16)	38.1 B (±12.40)
‘Rumba’	34.1 ab (±11.42)	51.1 b (±23.52)	32.4 ab (±10.57)	47.8 b (±2.09)	33.6 ab (±12.95)	26.5 a (±10.09)	37.9 B (±15.10)
‘Vibrant’	38.5 a (±23.20)	22.0 a (±8.44)	26.1 a (±11.64)	23.6 a (±3.62)	27.5 a (±7.62)	22.3 a (±3.84)	25.6 A (±9.46)
average	36.0 ab (±13.43)	34.9 ab (±19.23)	34.5 ab (±14.22)	39.8 b (±13.74)	30.9 ab (±8.85)	28.1 a (±9.64)	33.8 (±13.67)

Biopreparation × cultivar interaction—n.s. Values marked with different lowercase letters indicate significant differences between combinations. Values marked with uppercase letters indicate significant differences between cultivars. Values in brackets indicated standard deviation (SD).

). In the second year, this effect was highly dependent on the cultivar and biopreparation used. This can be important information, as the observed increase in strawberry fruit yields may have been supported by a higher crown number and weight of the aboveground parts of the strawberry.

The present study corresponds with the results of Kowalska [87], which noted that an increased biomass of the plants was recorded after the application of a preparation containing a strain of Trichoderma asperellum. Jamiołkowska et al. [124] reported an increase of 11–15% in the number of tomato leaves per plant after root inoculation with mycorrhizal fungi (Rhizophagus intraradices, Claroideoglomus etunicatum). The authors found that the treatments applied did not have a significant effect on marketable yields but, by improving plant health, reduced the share (ratio) of infested fruits [124]. In recent years, there has been a growing interest among both scientists and farmers in natural, pro-ecological methods of cultivation and plant protection, with an emphasis on increasing plant resistance to pathogens, pests, and stress conditions caused by unfavorable weather conditions, drought, or frosts. This is due to the growing awareness and demands of consumers to use the best quality products [125,126,127] with positive impacts on human health [125,126]. It is believed that quality food is supposed to provide consumers with their energy needs, but it is also supposed to provide the necessary macro- and micronutrients and keep consumers healthy without the need for the additional supplementation of essential vitamins and minerals in the form of tablets. Food should also be safe and therefore not contain residues of chemical pesticides or fertilizers used during plant cultivation, which could then negatively affect the health and longevity of consumers. In addition, the increased interest in seeking more natural, often innovative solutions for agriculture and horticulture is linked to the adoption of the European Green Deal strategy by the European Commission, and the consequent introduction of measures in member states aimed at increasing care for the environment and climate as well as increasing the share of organic farming [128]. The list of active substances permitted for use in agriculture has also been reduced, which creates the need to look for new solutions, substances, preparations, and fertilizers that will enable farmers to produce food that is healthy and safe for consumers while at the same time being economically viable [129,130,131].

4. Conclusions

The cultivars of strawberries reacted differently to the tested biopreparations in terms of the yield and infestation rate. The largest significant increase in the strawberry fruit yield was obtained in 2021 with the combination of K4 containing Bacillus sp. Sp116AC*, Bacillus sp. Sp115AD, Bacillus sp. AF75BC and Bacillus subtilis AF75AB2, liquid and dry humic acids, yeast culture effluent, micronized dolomite, mustard, and rapeseed oil (33% higher yield compared to the control). Yields of the ‘Honeoye’ cultivar were showing the increase when treated with microbial biopreparations. ‘Honeoye’ was also the most resistant to Colletotrichum acutatum (anthracnose) in both years of the study. With the exception of the K2 and K3 treatments in ‘Rumba’ in 2020, none of the tested bioformulations caused a significant decrease in strawberry yields. In 2020, all biopreparation combinations reduced infestation by the fruit leathery rot pathogen of the ‘Vibrant’ cultivar in relation to the control, with the K3 combination being the most effective. All treatment combinations showed a positive trend to increase the number of crowns per plant compared to the control in both years of the study, with the exception of the K2 treatment in 2021. The research on microbial organic biopreparations needs to be continued in subsequent years in order to select the combination of preparations with the most beneficial effect on plant health and yields to support sustainable food production. Future studies should be carried out with more years included and with verification of how microbial preparations affect the diversity of microorganisms in the soil, especially in the root zone of plants.