Towards Sustainable Agriculture—Agronomic and Economic E ﬀ ects of Biostimulant Use in Common Bean Cultivation

: Today, one of the greatest challenges faced by the agriculture industry is the development of sustainable and environmentally-friendly systems to meet nutritional demands of the continuously growing global population. A number of research studies have recently been undertaken with the aim to indicate types of parameters used in plant production that would be able to improve plant growth as well as the e ﬀ ectiveness and quality of yield, and to help plants cope with environmental stress. The aim of this study was to verify a hypothesis that the implementation of a sustainable agricultural technology, based on the use of synthetic biostimulants, will allow not only increasing crop yield and quality but also improving the cost-e ﬀ ectiveness of common bean cultivation. The ﬁeld experiment was conducted in three growing seasons (2016–2018). In the growing season, the plants were treated with Atonik and Tytanit biostimulants in the form of single or double spraying. We determinated biometric traits, seed yield, seed number, and 1000-seed weight. Further analyses included contents of nutraceutical potential. The economic e ﬀ ect of using biostimulants was also calculated. The results of our experiment allowed verifying a hypothesis that the implementation of a sustainable agricultural technology based on the use of synthetic preparations was an e ﬀ ective method to increase plant productivity and, consequently, economic proﬁts to farmers.


Introduction
Today, one of the greatest challenges faced by the agriculture industry is the development of sustainable and environmentally-friendly systems to meet nutritional demands of the continuously growing global population. Considering the diminishing area of arable lands and the depleting genetic potential of crops, one of the solutions that would enable increasing crop yields and protecting produce would be the implementation of novel agricultural technologies and the development of the existing effective plant protection against biotic and abiotic factors as they contribute to the improvement of biochemical, morphological, and physiological processes in plants [25][26][27][28][29][30]. The composition of synthetic PBSs includes, most of all, plant growth regulators, polyphenolic compounds or plant stimulants such as inorganic salts or essential elements. Synthetic PBSs include, among others, preparations with commercial names Atonik and Tytanit. Atonik is also referred to as Asahi SL or Chapperone. Its composition includes three phenolic compounds: sodium para-nitrophenolate (0.3%), sodium ortho-nitrophenolate (0.2%), and sodium 5-nitroguaiacolate (0.1%), dissolved in water. However, the results obtained by many researchers showed that nitrophenolates have significant potential as protective agents, growth regulators and biostimulants, and further study is warranted. Presently, there is no evidence suggesting that nitrophenolates are toxic to pollinators, mammals, or humans and plants [31,32] and soil and water are residue-free shortly after application [33,34]. In turn, Tytanit contains 0.85% of a titanium complex, 5% of MgO, and 10% of SO 3 . Its formula was developed and implemented into the agricultural practice in countries of the Central and Eastern Europe as an agronomic tool for the improvement of crop productivity through stimulating the activity of certain enzymes (peroxidase, catalase), increasing chlorophyll content, stimulating photosynthesis, promoting nutrient uptake, increasing tolerance to stress, and improving crop yield quality [35].
In terms of cultivation area, common bean is ranked second in the world among the leguminous plants. India accounts for over 1/3 of its global acreage, whereas Brazil and Guatemala for ca. 20%. In Europe, it is cultivated mainly in Belarus, Romania, Portugal [36]. As a legume species, common bean provides significant amounts of nitrogen to the soil. The nitrogen is one of the key nutrients that determines the development and health of crops, whereas cost-effective practices of its management are indispensable for efficient production. For this reason, common bean is often cultivated as an inter-crop or included into crop rotation systems in biofarming [37]. Even though the yield potential of common bean is high, its seed yield is the lowest among all legumes [38]. Its lower seed yield is mainly due to its sensitivity to increasingly often occurring stress factors (drought, frosts, salinity, environment pollution with heavy metals, activity of pests and pathogens). In such cases, it seems justified to follow sustainable agrotechnical technology based on various types of biostimulants.
However, the supervision over economic outcomes of implemented technologies is necessary in every production process. The ultimate goal is to achieve some short-term or long-term economic effects not only in the production activity per se but also in scientific research. The implementation and dissemination of the cutting-edge technologies in the agricultural production must also be based on estimations of their economic effectiveness. These calculations aim to offer every farmer the possibility of choosing optimal technological variants, ensuring high profits per area unit, high yield quality, and low expenditures [39]. In common bean cultivation, a comparative analysis of the effectiveness of using various biostimulants and methods of their application should underlie the choice of an appropriate cropping system for this plant. Available literature provides sparse works into the effects of biostimulant administration methods on the profitability and effectiveness of cultivation.
This study was undertaken in order to verify a hypothesis that the implementation of a sustainable agricultural technology, based on the use of synthetic biostimulants, will allow not only increasing crop yield and its quality but also improving the cost-effectiveness of common bean cultivation. Hence, this study aimed to determine the biostimulating activity of two preparations, under real field conditions, and then to evaluate crop quality. Analyses were carried out to establish the morphological and chemical response as well as selected metabolic parameters of Mexican Black common bean plants.
In this manuscript, we also evaluate economic effects resulting from biostimulant application in bean cultivation because the appropriate effectiveness and profitability coupled with reduced pesticide use will support both environmental and economic sustainability.

Plant Material and Growth Conditions
The experimental material originated from a field experiment conducted in the years 2016-2018 in Perespa village (50º66'N; 23º63'E, Poland) with common bean (Phaseolus vulgaris L.) of Mexican Black cultivar. The experiment was designed and performed in a random block system in 4 replications, on experimental plots with areas of 10 m 2 . Bean was cultivated on the soil belonging to the Gleyic Phaeozems, which was characterized by an alkaline pH (pH in 1M KCl: 7.4-7.5). Contents of available nutrients in the soil were at medium levels: P (12.6-14.2 mg P 2 O 5 in 100 g of soil), K (15.3-17.1 mg K 2 O in 100 g of soil), Mg (6.2-6.8 mg Mg in 100 g of soil), and N (8.1-9.3 mg N-NO 3 + N-NH 4 in 100 g of soil). In each experimental year, winter wheat was used as the previous crop. Bean seeds were sown on the 2 May, 2016, 2017, and 2018, with 3.5 cm gaps in rows with 45 cm spacing. Weeds were eradicated mechanically and manually. In the growing season, the plants were treated with Atonik and Tytanit biostimulants (composition provided in Table 1) according to the scheme presented in Table 2. Plants sprayed with water (being a solvent for biostimulants) served as the control sample. Biostimulants were used when the foliar application of microelements is recommended. Their doses were established based on recommendations for other crops due to a lack of producer's information regarding the recommended terms of application and concentrations of working solutions in common bean cultivation. The terms of PBS application were established also based on the authors' experience from previous investigations [39].
Average temperatures and rainfall in three growing seasons of bean are presented in Table 3. Determinations were conducted for: pod number per m 2 , 1000-seed weight, seed number per m 2 , and seed weight.

Protein Content
Protein content was determined in bean seed extracts using a Bradford reagent with the method described by Redmile-Gordon et al. [40] with own modifications. A volume of 150 µL of the Bradford reagent was transferred onto a microplate, then 50 µL of either the sample or standard protein (bovine serum albumin (BSA)) was added. Afterwards, the samples were shaken at room temperature for 15 min, and the absorbance was read at a wavelength of 595 nm using an Epoch Microplate Spectrophotometer (BioTek, USA). Protein content was expressed in mg per g of fresh matter (FM).

Extract Preparation
Ground bean seeds were subjected to the extraction process following the method presented byŚwieca et al. [41]. Extraction was performed with a mixture of acetone, water, and hydrochloric acid (70:29:1, v/v/v). The prepared samples were centrifuged for 10 min (6800× g). The process of extraction was conducted in three replications for each analyzed combination. After centrifugation, the supernatant was collected and used for further laboratory analyses.

Phenolics Content and Antioxidant Capacity Determination
The content of total phenolic compounds (TPC) was determined according to the method of Singleton and Rossi [42] using Folin-Ciocalteau reagent. In brief, 0.5 mL of water and then 2 mL of the Folin-Ciocalteau reagent (1:5 H 2 O) were added to 0.5 mL of the extract. After 3 min, 10 mL of 10% sodium carbonate was added to the mixture. After 30 min, the absorbance of the samples was measured at a wavelength of 724 nm using a UV-VIS spectrophotometer. Total phenolic compounds content was expressed in mg of gallic acid equivalents (GAE) per g of dry matter (DM).
The total flavonoid content (TFC) was determined acc. to the method proposed by Lamaison and Carnet [43]. Seed extract was mixed with a 2% methanolic solution of AlCl 3 x 6H 2 O (1:1, v/v).
After 10 min of incubation at room temperature, the absorbance of the solutions was measured at a wavelength of 430 nm using a UV-VIS spectrophotometer. The content of flavonoids was expressed in mg of quercetin equivalents (QE) per g of dry matter (DM).
The content of anthocyanins (TAC) in bean seeds was determined acc. to the method described by Fuleki and Francis [44]. Determinations were carried out using solutions of potassium chloride and sodium acetate at two pH values, i.e., 1.0 and 4.5. The solutions were mixed with bean seed extracted in a ratio of 20:1 (v/v). After 15 min, the absorbance of the samples was measured at wavelengths of 520 nm and 700 nm. After absorbance value correction at various pH values, the content of anthocyanins was computed and expressed in mg of cyanidin 3-glucoside equivalents (Cy3-GE) per g of dry matter (DM).
The reducing power was determined using the method of Pulido et al. [45]. Bean seed extracts were mixed with a phosphate buffer (200 mM, pH 6.6) and a 1% aqueous solution of K 3 [Fe (CN 6 )], in a ratio of 1:1:1 (v/v/v). After sample incubation at a temperature of 50 • C for 20 min, 0.5 mL of trichloroacetic acid (TCA) was added. Afterwards, the samples were centrifuged (6800× g, 10 min), and the resultant supernatant was mixed with distilled water and a 1% aqueous solution of iron (III) chloride (III) (2.5:2.5:0,5, v/v/v). The absorbance of the samples was measured at a wavelength of 700 nm using a UV-VIS spectrophotometer. The reducing power was expressed in mg of Trolox equivalents per g of dry matter (DM).
Ferric ion reducing ability (FRAP) was determined following the method provided by Jimenez-Alvarez et al. [46] with some modifications. The FRAP mixture was obtained by mixing an acetate buffer (3.6 pH), 2,4,6-tripyridyl-s-triazine (TPTZ-10 mM dissolved in 40 mM hydrochloric acid), and FeCl 3 × 3H 2 O (10:1:1, v/v/v). A volume of 250 µL of the FRAP reagent and 25 µL of the sample were transferred to a 96-well microplate, and the mixture was incubated at room temperature for 8 min. Sample absorbance was measured at a wavelength of 593 nm. The calibration curve was plotted based on a Trolox solution with concentrations from 90 to 540 µM/mL, prepared in the extraction mixture.
The antioxidant activity was analyzed with the TEAC (Trolox equivalent antioxidant capacity) method following the procedure described by Sancho et al. [47] with own modifications. An earlier prepared ABTS + solution (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) was adjusted to that concentration, ensuring the absorbance of 0.7 ± 0.02 at 734 nm. Then, 20 µL of the sample, acetone mixture (blank sample) or Trolox (calibration solutions) was transferred to a 96-well microplate and mixed with 280 µL of ABTS + . The absorbance was measured after 6 min, at a wavelength of 734 nm, at room temperature.
Proline content was determined acc. to the method of Carillo and Gibon [48]. Bean seed extract was mixed with a reaction mixture (1% solution of ninhydrin in 60% acetic acid and 20% ethyl alcohol) and incubated at 95 • C for 20 min. Absorbance was measured at a wavelength of 520, and total proline content was expressed in µM per mL.
Ground bean seeds were determined for content of neutral-detergent fiber (NDF), acid-detergent fiber (ADF), and lignin (ADL), in three replications acc. to the method described by Van-Soest et al. [49]. Filtration bags and Ankom apparatus (Ankom 220, USA) were used. NDF fraction content was assayed using the solution of a neutral detergent (sodium-lauryl sulfate, ethylenediaminetetraactic salt, sodium borate, disodium phosphate, triethylene glycol), alpha-amylase (17,400 liquid units/mL, FAA Ankom Technology), and sodium sulfite (FSS Ankom Technology). ADF fraction content was determined using the solution of an acid detergent (trimethylammonium bromide, normalized sulfuric (VI) acid). Lignin content in bean seed samples was determined using the solution of sulfuric (VI) acid (VI) (Ankom Technology, FSA 72). The content of hemicellulose was computed from the difference between contents of the NDF and ADF fractions. In turn, cellulose content in been seed samples was computed from the difference between contents of ADF fraction and lignin (ADL) [49].

Economic Analysis
The economic effect of biostimulant application was computed based on the value of yield increase resulting from the use of biostimulants and costs of their application [50].
Income growth resulting from the use of biostimulants (O sb ) was calculated from the following formula: where: The value of yield increase (W pp ) was computed acc. to the following formula: where: P nb -seed yield from the combination with biostimulant application, (t·ha −1 ), P nk -seed yield from the control combination, (t·ha −1 ), C n -average price of seeds in a given study year, (EUR·t −1 ).
Costs of the use of biostimulants (K sb ) were computed acc. to the following formula: where: k b -cost of biostimulant purchase, (EUR·ha −1 ), k w -cost of water used for the treatment, (EUR·ha −1 ), k z -cost of performing the treatment, (EUR·ha −1 ).
The average purchase price was established based on information from wholesale markets ( Table 4). The cost of purchasing biostimulants was calculated as a mean price from 3 wholesale companies supplying farms. Water use cost was calculated based on the price of 1 m 3 of tap water in a village community in the Lubelskie Province. The cost of treatment performance was calculated as a mean price of the spraying service with a 1000-L tractor-mounted sprayer.

Statistical Analysis
The materials for analyses originated from three growing seasons (2016-2018). All analyses were performed in three replications. The Shapiro-Wilk test was used to evaluate the normal distribution of data. The results were analyzed using the one-way analysis of variance. The significance of differences between mean values was estimated based on Tukey confidence intervals (significance level of p < 0.05). The statistical analysis was performed using Statistica 13.3 software (StatSoft, Inc.).

Effect of Biostimulants on Biometric Traits
The use of Atonik and Tytanit biostimulants in the cultivation of the common bean of Mexican Black cultivar modified the biometric traits and yield of seed in particular years of the study. In 2016, the double application of Atonik preparation in the lower concentration increased 1000-seed weight compared to the control and to other variants of application. In 2017 and 2018, the single application of Atonik in the lower concentration decreased 1000-seed weight (Table 5). In the other combinations, no significant changes were noted in the value of this trait compared to the control sample. In turn, the application of Tytanit preparation in particular study years caused significant differences in 1000-seed weight by either increasing or decreasing it (compared to the control) in particular variants of biostimulant application. The application of both Atonik and Tytanit contributed to a significant increase in bean seed yield compared to the control. In the case of Atonik, the most beneficial effect was achieved upon double treatment of plants with this biostimulant in its higher concentration. Whereas in the case of Tytanit, the most desirable effect was achieved after its single application in the higher concentration. The increase in bean seed yield was due to the positive response of plants to treatments with both biostimulants that was manifested by an increased pod number and seed number compared to the control combination.  421a  428a  400a  350a  421a  428a  400a  LSS  598b  605c  588d  597b  471b  606c  598d  558bc  HSS  613bc  530b  538c  560b  527c  497b  512b  512ab  LDS  627cd  473a  486b  529b  535c  584c  575c  565bc  HDS  644d  627c  623e  631b  670d  617c  618d  635c Number of seeds 1492b  2719c  2850d  2354a  1775d  2522c  2480b  2259a  HSS  1720c  2325b  2400b  2148a  1617c  2648c  2670c  2312a  LDS  1849d  2305b  2421b  2192a  1548b  2711c  2753d  2337a  HDS  2012e  2644c  2685c  2447a  1605c  2299b  2478b  2127a Abbreviations: C, control; LSS, lower concentration single spraying; LDS, lower concentration double spraying; HSS higher concentration single spraying; HDS, higher concentration double spraying. Means in the columns, concerning the selected traits, followed by different small letters are significantly different at p < 0.05.

Effect of Biostimulant Treatment on Nutraceutical Quality
The use of biostimulants in bean cultivation caused changes also in the protein content of bean seeds. Tytanit preparation increased protein content compared to the control variant, irrespective of the number of treatments and biostimulant concentration ( Table 6). Plant treatment with this biostimulant in its higher concentration resulted in a higher protein content compared to its lower concentration. An increase in protein content of the seeds was also observed in plants after foliar application of Atonik preparation. The greatest increase in protein content, compared to the control, was determined after double application of this preparation in its higher concentration. Also, the growing season had a significant effect on protein content of bean seeds, which was the lowest in 2017. The evaluation of the effects of the number of doses and concentrations of the biostimulants tested on the antioxidant activity of bean seeds was based on determinations of their capability to scavenge an ABTS + cation-radical and their capability to reduce a TPTZ-Fe (III) complex to a TPTZ-Fe (II) complex.
When considering the antioxidant activity of bean seeds in particular combinations in terms of their ABTS + radical scavenging capability, the use of Tytanit and Atonik preparations in the growing season contributed to the enhancement of this capability, irrespective of the number of treatments and solution concentration (Table 6). A statistically significant increase in the radical scavenging capability was determined in the combination in which plants were double sprayed with the higher concentration of Tytanit.
A significant increase in the antioxidant activity of bean seeds was also obtained after plant treatment with Atonik biostimulant in the case of its double application in the lower concentration. Considering growing seasons, the highest antioxidant activity of bean seeds was determined in 2017, compared to the other study years.
The ferric ion reducing capability of bean seeds was found to depend on the type of biostimulant, and on the number of treatments and solution concentration. Statistically significant differences were observed in the case of the higher and the lower concentration of Tytanit preparation after its double application. Double plant spraying with its solution in the concentration of 0.07% decreased the FRAP value by ca. 14.64% compared to the control. In turn, double plant treatment with Tytanit in its higher concentration contributed to FRAP value increase by 14.31% on average, compared to the control.
The use of Atonik biostimulant in higher concentrations contributed to FRAP values decrease compared to the control variant. The greatest decrease in FRAP value was noted after double plant spraying with this biostimulant. However, the higher concentrations of Atonik solution increased FRAP values compared to the control.

Effect of Biostimulant Treatment on the Antioxidant Potential
A complex evaluation of the effects the tested biostimulants had on the antioxidant activity of bean seeds assayed with ABTS and FRAP tests shows that higher values of the activity were achieved after plant treatment with Atonik than with Tytanit.
No statistically significant differences were determined in proline content, irrespective of the number of doses and concentrations of the analyzed biostimulants. A significant difference was, however, noted in particular study years, which suggests that proline content of bean seeds is determined by weather conditions in a given growing season.
Despite a lack of significant differences in proline content of bean seeds, its decrease was noted compared to the control in all combinations studied. The greatest decrease in the content of this amino acid occurred after single plant spraying with Tytanit in its lower concentration (0.07%).
The content of anthocyanins in bean seeds after plant treatment with Atonik biostimulant was the same as in the control samples, except for the combination with double plant spraying with the lower concentration of its solution, in which anthocyanins content decreased compared to the control. In the case of Tytanit, decrease was observed in anthocyanins content in bean seeds after double plant treatment with its solution in the higher concentration (Table 7). In the other combinations, the content of these compounds increased compared to the control samples, but the differences were statistically insignificant. Atmospheric conditions occurring in the growing season of 2017 resulted in a lower content of anthocyanins in bean seeds compared to the other study years. The complex analysis of biostimulant use in common bean cultivation in terms of anthocyanins content shows that Tytanit preparation had a more beneficial effect on their content in bean seeds.
The content of flavonoids in bean seeds depended on both the number of treatments and concentration of the biostimulants. The double application of Tytanit in its lower concentration resulted in the lowest flavonoid content in bean seas. However, single plant spraying with this biostimulant in the same concentration caused the highest content of these compounds in bean seeds. The use of Atonik biostimulant in the form of single plant spraying with its lower concentration caused an insignificant decrease in flavonoids content of the seeds (Table 7). In contrast, a significant increase in their content compared to the control was determined after double plant spraying with Atonik in the higher concentration (0.2%). Flavonoids content of bean seeds was also significantly determined by the growing season of the field experiment.
The content of polyphenolic compounds in bean seeds differed in particular study years. Plant treatment with Tytanit increased polyphenol content compared to the control, regardless of the number of treatments and solution concentration. In the case of Atonik biostimulant, only double plant treatment with its solution in the lower concentration caused an insignificant decrease in the content of these compounds compared to the control material. However, the same number of treatments but with the solution in the higher concentration allowed achieving the highest content of polyphenols in bean seeds compared to the control, and the differences observed were statistically significant.
The complex analysis of biostimulants effect on polyphenol content demonstrated Tytanit preparation to be more effective in increasing polyphenol content in bean seeds.
The antioxidant potential of bean seeds was also analyzed based on the reducing power (RP). Its values were determined not only by the type, number of treatments, and concentration of the biostimulant, but also by meteorological conditions in a given growing season.
The use of the biostimulant, containing mainly phenolic compounds, in its higher concentration resulted in RP value increase compared to the control samples. However, single plant spraying with Atonik in the concentration of 0.1% caused a 2-fold decrease in RP value. The double application of this biostimulant in its lower concentration had a negligible effect on reducing power of the seeds, and its mean values achieved were similar to those determined in the control samples.
The foliar application of Tytanit also caused differences in the reducing power of bean seeds. An increase in RP value was observed only after single plant treatment with this biostimulant in its lower concentration. In the other combinations, RP values decreased slightly compared to the control and differences noted were statistically insignificant.

Effect of Biostimulant Treatment on Fiber Content
The use of Tytanit biostimulant in common bean cultivation caused a decrease in the content of neutral-detergent fiber (NDF) in seeds compared to the control samples ( Table 8). None of the combinations tested caused NDF content to approach that determined in the untreated plants. The greatest, over 2-fold, decrease in NDF content occurred after single plant spraying with this biostimulant in its lower concentration.
Plant treatment with Atonik preparation also resulted in a decreased content of the NDF fraction in seeds compared to the control samples, except for the single spraying with the higher concentration after which NDF content increased in seeds by ca. 17.57%.
Contents of acid-detergent fiber (ADF) varied depending on the type of biostimulant, number of treatments, and solution concentration. Meteorological conditions occurring in that growing season also determined ADF content in bean seeds.
Plant treatment with Tytanit preparation increased ADF content only in one of the analyzed combinations, i.e., after double plant spraying with this biostimulant in its higher concentration. ADF contents determined in the other combinations were similar to these found in the control samples.
The greatest, nearly 1.5-fold, decrease in the content of acid-detergent fiber fraction was determined after double plant spraying with Atonik in its higher concentration. In turn, the plants responded with the greatest increase (by 21.98% on average compared to the control) of the ADF fraction content after single application of this biostimulant in its lowest concentration.
Lignin content analysis showed that double plant spraying with Atonik in the lower concentration (0.1%) caused a nearly 2.5-fold increase in its level in bean seeds compared to the control sample (Table 8). An opposite observation was made after double plant spraying with this biostimulant in its higher concentration, which resulted in almost 2-fold decrease in ADL fraction content. The single foliar application of Tytanit in the lower concentration caused an increase in lignin content compared to the untreated samples. This increase was also noted after double plant treatment with this preparation in its higher concentration. Analyses conducted in the other combinations showed a decrease in ADL fraction content. The differences observed in lignin content of bean seeds after plant treatment with Tytanit biostimulant were statistically insignificant.
The Atonik biostimulant was more effective in increasing the lignin content of bean seeds compared to the control treatment. Lignin content was additionally determined by weather conditions occurring in a given growing season.
The analysis of hemicelluloses (HCEL) in bean seeds after plant treatment with Atonik biostimulant demonstrated their content to increase by 40.13% on average only in the combination in which plants were sprayed with Atonik solution in the higher concentration. An opposite observation was made after the use of Tytanit, where the content of hemicelluloses did not increase compared to the control samples in any of the combinations tested. In all combinations, HCEL content was observed to decrease. The greatest, over 7.5-fold, decrease was determined after double plant treatment with this preparation in its higher concentration.
The foliar application Atonik biostimulant during common bean growing season caused changes in cellulose (CEL) content in its seeds. An increase in cellulose content by 15.19% on average was determined after single application of Atonik in its lower concentration. In the other combinations, CEL content was lower than in the control samples.
The greatest increase in cellulose content of bean seeds was due to the double plant treatment with Tytanit in its higher concentration and reached 18.46% on average compared to the control material. A decrease in the CEL content of bean seeds represented plant response to the single application of this preparation in the lower concentration, i.e., 0.07%.
In 2017, the mean cellulose content determined in bean seeds was higher than in the other study years.

Economic Analysis
A comparative analysis of the economic effects of biostimulant use and modes of application in common bean cultivation should underlie decision about their use. The conducted analyses confirmed the seed yield increase caused by the applied biostimulants to be the factor improving the profitability of bean production for dry seeds. In all combinations tested, the application of the studied biostimulants had a positive effect on bean seed yield and increased the cost-effectiveness of the cultivation. In 2016, the profitability of biostimulant use ranged from 49.51 to 1059.96 EUR·ha −1 (Figures 1 and 2). The highest profitability was demonstrated in the case of the double foliar application of Atonik preparation in both concentrations tested. An increase in incomes after the use of Tytanit biostimulant was relatively high and the highest after plant treatment with working solutions in the lower concentrations.
After the second year of bean production, the analysis of the effects of biostimulant application on the economic effectiveness of the production process (Figures 1 and 2) demonstrated increased profitability of the use of both preparations. In 2017, the total profitability from using Atonik biostimulant ranged from 721.61 (double plant spraying with the lower dose) to 1010.51 EUR·ha −1 (twice as high dose). Different economic effects were observed after the application of Tytanit, as the average higher profitability was due to the single application of this preparation in both concentrations tested.
In the third year of cultivation, the highest profitability of biostimulant use was demonstrated after single plant spraying with Tytanit in both concentrations. For Atonik, the greatest economic profits were noted after double plant treatment with its higher concentration.

Discussion
The results of our study indicate a positive effect of biostimulants tested not only on bean seed yield but also on the yield quality traits. Both protein content and the antioxidant potential of seeds, produced with the use of biostimulants, were significantly increased compared to the control samples. The more beneficial effect on average was due to the application of the biostimulant

Discussion
The results of our study indicate a positive effect of biostimulants tested not only on bean seed yield but also on the yield quality traits. Both protein content and the antioxidant potential of seeds, produced with the use of biostimulants, were significantly increased compared to the control samples. The more beneficial effect on average was due to the application of the biostimulant containing titanium compounds. The use of the Atonik preparation resulted in increased antioxidant capability (FRAP) and reducing power (RP) compared to the control samples and to the second plant growth regulator tested in the study. This could stem from the effect of this biostimulant on the increased inhibition of IAA oxidase which enhances natural synthesis of endogenous auxins in plants [33,[51][52][53]. This is due to Atonik composition, because the phosphorylated form of para-nitrophenolate, being a substrate for phosphatases, determines IAA activity enhancement [54] because it exhibits effects analogous to ATP [55]. According to Przybysz et al. [56], plant treatment with Atonik influences nitrogen metabolism in these plants, which may stimulate the activity of nitrate reductase.
The concentration of antioxidants in plants is extremely important to their protection against oxidative damage induced by reactive oxygen species (ROS). The analysis of the antioxidant capability of bean seeds demonstrated the positive effect of biostimulants tested, which may indicate that they supported the development of the plant antioxidative system, composed of both antioxidant enzymes and antioxidant organic molecules [57,58]. Noteworthy is the fact that seeds of bean treated with biostimulants had a higher content of flavonoids. A high amount of these compounds is accumulated in vacuoles. Flavonoids are commonly believed to be strong antioxidants which effectively scavenge hydrogen peroxide and other ROS [59,60]. It needs to be emphasized, however, that the antioxidant system of plants is a vast and complicated network of metabolites and biomolecules which are capable of both ROS production and elimination. In addition, this dualism of nature of antioxidant biomolecules facilitated the selection and evolution of ROS as signaling molecules. For this reason, the evolution of the antioxidant system allowed ROS to play a double role. Today, owing to the antioxidant system, which had initially been perceived as detrimental, ROS have become important signaling molecules. This, in turn, suggests that they play a meaningful role both in plant growth and development as well as in the fight with environmental stress. The latest conclusions from scientific research indicate, however, that the demonstrated signaling role of ROS will require redefining the notion of 'oxidative stress' considering 'oxidative signaling' [61].
Plants respond to environmental stress at many levels, including at biochemical, cellular, and molecular levels. This response may be triggered by the activation of stress hormones, production of osmolytes, elimination of reactive oxygen species (ROS) or accumulation of proteins protecting against stress [62,63]. According to Ashraf and Foolad [64], proline is one example of plant osmolytes; its synthesis is stimulated by several stress conditions. Its roles include, most of all, osmotic regulation, stabilization of subcellular structures, scavenging free radicals, buffering the redox potential, and gene induction. Proline content in plants is determined by multiple factors, including plant species and cultivar, plant organs and phenological stage, as well as by the type and intensity of abiotic stress [64][65][66][67]. Plants exposed to stress exhibited increased accumulation of proline [68]. Investigations conducted by Hong et al., [69] demonstrated even that the elimination of enzyme feedback inhibition from proline metabolism in the Arabidopsis plants led to a significant accumulation of this osmolyte, which in turn ensured increased plant tolerance to the extreme osmotic stress. It needs to be emphasized, however, that-according to many authors-proline accumulation does not always occurs in plants under conditions of abiotic stress. However, a strong correlation is often observed in higher plants between tolerance to stress factors and accumulation of this osmolyte [64,67,69]. Therefore, considering the study results and speculations, a hypothesis has been advanced that plants that superproduce proline will be more resistant to drought stress, which may result in their higher productivity compared to the control plants or in the maintenance of the assumed productivity even under the exposure to the stress factor. However, based on results of his study, Carvalho [70] concluded that proline metabolism is regulated through the elicitor mechanism. According to Djanaguiraman et al., [53,71], Atonik also affects the production of proline and polyols in plants, i.e., two extremely important and compatible metabolites involved in anti-stress mechanisms. The results of our study demonstrated a similar effect of Tytanit biostimulant, because proline content decreased in bean seeds upon its use compared to the untreated samples. Nevertheless, proline content cannot be directly perceived as a specific marker of tolerance to one specific stress condition because its accumulation reflects only the response to general abiotic stress [72]. Szabados and Savouré, [73] demonstrated that the high content of proline allows plants to maintain a low water potential through the additional uptake of water from the environment. However, a study conducted by Patanè et al. [74] proved that the genotypes of tomatoes characterized by a higher sensitivity to water shortages in the soil responded to the drought-induced stress with suppressed synthesis of proline in leaves. Therefore, according to Goñi et al. [75], the high accumulation of proline cannot be treated as a general response of crops exposed to stress or to the use of biostimulants. In addition, the study conducted by this author demonstrated that the magnitude of this osmolyte accumulation may depend on biostimulant type, the method of its application or crop class. Nevertheless, scientists do agree that proline plays a key role in plants regeneration after stress [75].
Investigations addressing the effects of the use of biostimulants on contents of dietary fiber fractions are relatively new. Therefore, sparse information may be found in the available literature concerning results of such analyses. The concept of the division and analysis of the fractions of cell walls, neutral-detergent fiber (NDF) and acid-detergent fiber (ADF), has been proposed by Van Soest. He advanced a hypothesis that feedstuffs contain cell wall constituents (CWC) and cell contents (CC); and that the CWS, i.e., NDF and ADF, are factors which reduce their intake, digestibility, and energy value [76]. An unexpected outcome of our study was the finding that the NDF content decreased in almost all analyzed combinations of plant treatment with Tytanit and Atonik biostimulants, compared to the control samples. In the case of the ADF fraction, a similar tendency was observed after plant treatment with Atonik preparation. In turn, the use of the titanium-containing biostimulant resulted in the increased ADF content, especially after the double treatment. However, the differences observed were statistically insignificant. Some scarce study results show that the use of biostimulants can determine the content and technological characteristics of dietary fiber [77]. But according to Wang et al. [78], the process of dietary fiber biosynthesis is extremely complicated and determined not only by the nutritional status of plants but also by the effects of multiple abiotic factors. The stage of its biosynthesis itself entails the enhanced synthesis of gibberellins which directly determine the micronaire, length or strength of the fiber [78]. In the opinion of Silva et al. [77], the foliar application of biostimulants may lead to the increased accumulation of gibberellins in plants, which in turn triggers changes in the production process of both dietary fiber and its fractions. Apart from ADF and NDF, the main cell wall components include cellulose, hemicelluloses, and lignin [79][80][81].
Our study demonstrated that their contents in bean seeds originating from the combinations with biostimulants were lower than in the control samples. Only in the case of Tytanit was the cellulose content increased, but differences observed were statistically insignificant. It needs to be emphasized, however, that cellulose is not digested by monogastric animals, but may be degraded, to some extent, in the gastrointestinal tract upon fermentation by intestinal microflora [81,82]. Worthy of notice is also the reduced content of hemicelluloses in bean seeds, because this group of compounds includes polysaccharides exhibiting anti-nutritional effects and are non-homogenous in terms of their chemical structure and physicochemical properties [83]. Some of the compounds exist in an insoluble form in the environment of the gastrointestinal tract of animals, but the majority of these compounds are soluble which is determined, to a large extent, by the presence of L-arabinose which-next to D-glucose, D-galactose and D-glucuronic acid-represents a branch of the main chain of this polysaccharide [81,84,85].
Even though biostimulants offer novel possibilities and although their use in agriculture is in many cases deemed beneficial for improved crop yielding, the exact mechanism of their action still remains unknown, which is indicated by the presented results concerning the contents of dietary fiber in bean seeds [86,87], because the effect of biostimulants on plants is not only a consequence of their direct role in metabolism regulation, but also of their multi-faceted action. The most important, however, is the fact that-unlike hormones-biostimulants improve metabolic processes in plants without modifying their natural pathways [87,88].
The analysis of the average income from cultivation during the three-year experiment demonstrated that in the case of Atonik preparation used in higher concentrations, its double application at appropriate developmental stages of plants contributed to the achievement of the greatest seed yield increase, and thus to the stability of incomes gained by farmers. For this reason, this method of biostimulant application seems to be the most beneficial from an economic perspective. The size and value of crop yields as well as costs incurred on their cultivation determine the profitability of common bean cultivation, but most of all affect incomes [89]. According to Santoso et al. [90], the application of biostimulants will allow farmers to achieve additional incomes from higher crop yields achieved owing to the use of these preparations. These additional incomes will be significantly higher than the costs of the preparations and labor and may exceed even 20 times the expenditures incurred. In the available literature, there are few papers on the impact of the biostimulant application on profitability and crop efficiency. However, the results of economic analysis are reflected in the research of Zarzecka et al. [89] and Mystkowska [91]. These authors stated that the factor determining profitability of potato production was the value of harvested crops, which depended on their quality. Zarzecka et al. [89] showed that the highest economic effect was obtained after the application of the Atonik. Gugała et al. [92] also concluded that the use of such preparations not only favorably stimulates the yield increase, but also determines the quality of seeds, which is connected with a higher selling price. Anderson et al. [93] proved that the use of biostimulants in sweetcorn cultivation led to yield increase. However, financial analysis showed that applying biostimulants to seeds is a very significant cost. Therefore, for greater profitability, these researchers recommended the use of biostimulants using appropriate methods.

Conclusions
The application of the tested biostimulants induced the beneficial responses of common bean plants that were manifested in terms of size and the quality of seed yield. The results of the present study point to the need for more exhaustive explanations of the basic mechanisms responsible for the positive effects of these preparations on the analyzed crops, which will represent a highly important issue in future research. However, the use of the tested preparations in order to improve crop productivity seems to be a justified agronomical approach in the context of food safety, sustainable development, and the effective use of expenditures. The increased protection of plants against environmental factors after the use of biostimulants, which results in increased productivity and profitability of crop production, represents a potential form of sustainability support in agricultural farms. The results of our experiment allowed verifying a hypothesis that the implementation of a sustainable agricultural technology based on the use of synthetic preparations was an effective method to increase plant productivity and, consequently, economic profits to farmers. However, it is not only crop yields and economic aspects that should determine the use of specific biostimulants. Nowadays, ecological considerations come to the fore. Due to the fact that synthetic biostimulants, including Atonik, are mixtures of potentially toxic compounds, scientific research should indicate a specific range of concentrations for working solutions that can be used by farmers in practice. This will enable sustainable crop management with minimal impact on the natural environment.
Author Contributions: A.S. and S.K. conceived and designed the research. A.S., S.K. and E.C. performed the experiments. A.S., S.K., E.C., M.K., Z.K. and D.K. prepared the materials. A.S., M.K., S.K. and Z.K. analyzed the data. A.S. and S.K. wrote the paper. M.K., Z.K. and D.K. revised the manuscript. All authors read and approved the final manuscript.