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Article

Enhancing Sustainable Cultivation of Organic Bell Pepper through Fulvic Acid (FA) Application: Impact on Phytochemicals and Antioxidant Capacity under Open-Field Conditions

by
Pinkky Kanabar
,
Ying Wu
and
Dilip Nandwani
*
Department of Agriculture and Environmental Sciences, College of Agriculture, Tennessee State University, Nashville, TN 37209, USA
*
Author to whom correspondence should be addressed.
Sustainability 2024, 16(16), 6745; https://doi.org/10.3390/su16166745
Submission received: 10 July 2024 / Revised: 31 July 2024 / Accepted: 6 August 2024 / Published: 7 August 2024
(This article belongs to the Special Issue Advances in Sustainable Agricultural Crop Production)
Browse Figures
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Abstract

:
Fulvic acid (FA) is an eco-friendly solution for reducing the reliance on agrochemicals and enhancing crop quality. The study aimed to investigate the impact of soil and foliar application of FA on the phytochemical content and antioxidant properties of organically grown bell peppers at both the green and red stages. Bell pepper cv. Revolution was grown under open-field conditions at the certified organic farm at Tennessee State University, Nashville, with nine treatments and three replications. FA was applied as a soil drench and foliar spray at four different rates (0, 2.3, 5.5, 7.8, 10.9 mL L−1). The fruits were harvested 55 days (green stage) and 86 days after transplantation (red stage). The study showed significant differences (p ≤ 0.05) between the treatment and maturation stage. The foliar treatment of 7.8 mL L−1 resulted in the highest phenolic content in green bell peppers. The highest total flavonoid content in red bell peppers was achieved with a soil treatment of 7.8 mL L−1. Additionally, the highest level of vitamin C in red bell peppers occurred with a soil concentration of 5.5 mL L−1. In conclusion, applying FA as a biostimulant can enhance the quality of organically grown bell peppers, offering promising opportunities for sustainable agricultural practices.

Graphical Abstract

1. Introduction

Bell peppers, scientifically known as Capsicum annum L., are widely recognized as a valuable source of vital bioactive compounds and antioxidants. These include polyphenolic compounds like flavonoids, vitamin C, vitamin A (β-carotene), and carotenoid pigments, such as lycopene and zeaxanthin [1]. According to the study by Deepa et al. [2], phenolics exhibit antioxidant capacity because of their capability to quench singlet oxygen, donate hydrogen, and serve as a reducing agent. Antioxidants are crucial in removing harmful free radicals that trigger oxidative stress and hinder cell damage. Additionally, well-known antioxidants are essential for safeguarding against various degenerative diseases, including cancer, cardiovascular disease [3], cataracts, diabetes, neurodegenerative diseases [4], and inflammatory disorders [5]. According to various studies, extracts from bell peppers are known for their antibacterial, antifungal, and chemotherapeutic properties, as reported by [6]. Fresh sweet peppers are an abundant source of vitamin C, with high levels of approximately 76 to 243 mg per 100 g of FW. As per Zhuang et al. [7] and El-Ghorab et al. [8], the consumption of just 100 g of these peppers can provide a total ascorbic acid Recommended Dietary Allowance (RDA) of 60 mg/day of daily intake. The mature green color is primarily attributed to carotenoids and chlorophyll [9]. Meanwhile, the red color is mainly due to carotenoid pigments, such as β-carotene with provitamin A and oxygenated carotenoids, like capsanthin, capsorubin, and capsanthin 5,6-epoxides. These pigments are predominantly found in the Capsicum genus [10]. Red pepper also has lycopene and is believed to reduce the risk of certain cancers [11]. During the development of peppers, a range of biochemical and physiological changes occur. These changes result from alterations in the synthesis, transportation, and degradation of various metabolites [12]. Bell peppers are known for their rich bioactive compounds like carotenoids and phenolic content, but several factors can affect them. These factors include genotypes, production practices (organic or conventional), fruit maturity, and postharvest handling conditions [4]. The plant response to stress depends on genetics, affecting the phytochemical content and antioxidant capacity [2]. Numerous studies have revealed that peppers contain more bioactive compounds when grown organically [13]. However, conflicting reports have also been published on this matter [14]. The harvesting time and the maturation stage can also affect the quality and antioxidant capacity of peppers. These are essential factors to consider when choosing different pepper varieties for a healthy diet [15]. Bell peppers have gained popularity globally due to their economic and nutritional significance [16].
Fulvic acid (FA) is a significant component of humic substances that plays a vital role in promoting the absorption of mineral nutrients [17,18,19]. FA, due to its low molecular weight, can effectively penetrate the pores of plant cell membranes. This unique characteristic allows them to serve as carriers for essential nutrients, essentially acting as natural chelating agents [20,21]. Fulvic acid is a soluble component of soil organic matter (SOM) that remains in the soil solution even under high salt concentrations and across a wide pH range [22]. As the world’s population continues to grow, the challenge of providing food to people without harming the environment has become a paramount concern for agricultural sciences. The intensification of agriculture has now reached a critical stage, highlighting the need for new technologies to facilitate the sustainable intensification of agricultural production [18,23]. Farmers can reduce their reliance on agrochemicals and fertilizers by utilizing plant biostimulants containing FA while improving crop yield and enhancing fruit quality. This eco-friendly strategy is a novel approach to plant nutrition that helps increase nutrient uptake and overall crop health [24]. Recent studies have demonstrated the effectiveness of FA on plant growth and production. For e.g., when treated with FA in field and greenhouse experiments, yarrow showed improvements in its growth factors, photosynthetic pigments, and antioxidant capacity [25]. Likewise, in a study, it was found that applying FA as drenches to peppers grown in field conditions enhanced fruit quality and antioxidant activity [26]. In addition, wheat plants exposed to salinity stress demonstrated increased antioxidant capacity and improved growth and production when treated with fulvic acid [27]. Moreover, fulvic acid has been found to enhance produce quality by increasing secondary metabolites, antioxidants, vitamins, and mineral content, supporting the findings of previous studies [28].
Phytochemicals, found in vegetables, fruits, unrefined grains, and other plant-based foods, are natural compounds produced by plants. Due to their antioxidant properties, they are believed to have health-promoting effects that may help to reduce the risk of chronic diseases [16]. As we strive to better understand the nutritional value of foods and how they contribute to our daily requirements, it is essential to investigate how the nutritional composition of fruits and vegetables changes as they ripen and mature. Regrettably, there is insufficient comprehensive data in the literature concerning the impact of fulvic acid on the phytochemical composition and antioxidant potential of sweet bell pepper cultivated under field conditions at various maturation stages. Therefore, we aim to explore the impact of soil and foliar application of fulvic acid at varying concentrations on the phytochemical content and antioxidant activity of organic bell pepper cv. Revolution at both the green and red maturation stages (Figure 1).

2. Materials and Methods

2.1. Experimental Design

This study was carried out at a certified organic farm at Tennessee State University, Nashville, TN (36°10′34.19″ N and 86°49′27.23″ W), during the summer of 2021 and 2022. The field was sectioned into 6 rows using black plastic mulch laid out by a machine. In the month of June, seedlings were transplanted by hand for both seasons. The plants were spaced 72 inches (1.83 m) apart in rows and 18 inches (0.46 m) apart within the rows. The experimental plot spanned an area of 90 × 37 ft2 (309.37 m2). Sweet bell pepper cv. Revolution seeds (Harris Seeds, Rochester, NY, USA) were used for the study. All the guidelines and practices related to culture and management were followed, based on the National Organic Program (NOP) standards. Based on organic pest management standards, the following products were used to manage pest and disease pressure during the growing seasons: M-Pede (Gowan®, Yuma, AZ, USA, OMRI listed), Monterey B.T. (Fresno, CA, USA, OMRI listed), and PyGanic (OMRI listed). The experiment followed a randomized complete block design (RCBD), comprising nine treatments and three replications. These treatments included a control group with no fulvic acid application, and four concentrations (0, 2.3, 5.5, 7.8, 10.9 mL L−1) of FA were applied through a soil drench and a foliar spray. FA application commenced 14 days after transplanting and continued thrice a week until the fruiting stage.

Chemicals

Fulvic acid (FUL-POWER, sourced from Faust Bio-Agricultural Services Inc., BIOAG, Independence, OR, USA, OMRI, and OIM listed), derived from leonardite, is a humic acid isolate using a commercial biological fermentation process. Methanol (70%), Folin–Ciocalteu reagent, sodium bicarbonate, gallic acid, sodium hydroxide, aluminum chloride, metaphosphoric acid, bromine water, thiourea, 2,4-Dinitrophenylhydrazine (DNPH), ascorbic acid, and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) were purchased from VWR International, LLC, (Radnor, PA, USA). Catechin, ascorbic acid, sodium nitrite, acetic acid, sulfuric acid, sodium phosphate, iron(III) (ferric) chloride, and trichloroacetic acid were purchased from Sigma Aldrich Chemical Co. (St. Louis, MO, USA). All chemicals and reagents used in our study were of analytical grade.

2.2. Sample Preparation

Samples of bell pepper cv. Revolution were harvested at two distinct stages of maturity: green (BBCH 79) and red (BBCH 89). The green-stage fruits were picked 55 days after transplantation, while the red-stage fruits were harvested 86 days after transplantation. Three replications of five fruits of each color were randomly obtained from each treatment and then subjected to phytochemical analysis. The harvested fruit samples were washed with deionized water, precisely cut into uniform small pieces, and then stored in a chilling environment at 4 °C. The next phase involved lyophilizing frozen samples using a state-of-the-art freeze dryer (Labconco, Bulk Tray Dryer with 6-Port Manifold 115V, Kansas City, MO, USA). After this, the freeze-dried samples underwent grinding using a kitchen blender (Ninja BL660 Professional Compact Smoothie and Food Processing Blender, SharkNinja Operating LLC, Needham, MA, USA). The samples were finely ground and stored in plastic tubes, then preserved at −18 °C until they were ready for analysis.

2.3. Preparation of Methanolic Extracts

The extraction process involved using 0.5 g of ground green and red bell pepper samples mixed with 20 mL of 70% methanol as a solvent. The mixture underwent agitation in an orbital shaker (Thermo Scientific MaxQ 4000, Waltham, MA, USA) for 1 h at 20 °C (250 rpm), followed by centrifugation (4696× g, 4 °C) for 20 min (Thermo Scientific Sorvall™ Legend™ X1 Centrifuge Series, USA). The supernatants were collected, and further extraction steps were repeated, followed by centrifugation. The resulting supernatants were combined and increased to a volume of 50 mL. These extracts were then refrigerated at 4 °C for further analysis, focusing on the total phenolics, total flavonoids, and antioxidant activities, such as the reducing power and DPPH radical scavenging activity.

2.4. Total Phenolic Content (TPC)

The total phenolic content (TPC) was measured using the Folin–Ciocalteu method as outlined by [7], with some modifications. A 100 μL of methanolic extract was combined with 200 μL of 10% Folin–Ciocalteu reagent (v/v), followed by adding 800 μL of 7.5% sodium bicarbonate solution. The mixtures were then incubated at room temperature for 2 h, and the absorbance was measured at 765nm (Thermo Scientific GENESYS™ 150 UV–Visible Light Spectrophotometer). The results were recorded in milligrams of gallic acid equivalents per gram of dry extract weight (mg GAE g−1 d.w.), with gallic acid as the standard (Figure A1).

2.5. Total Flavonoid Content (TFC)

The analysis of the total flavonoid content (TFC) utilized the aluminum chloride method with catechin as a reference, as described by [29], with certain modifications. To elaborate, 1 mL of methanolic extract was combined with 100 μL of 5% sodium nitrite. After 6 min, 10% aluminum chloride was introduced, and 1 mL of 5% sodium hydroxide was added after another 5 min. The resulting solution was vortexed, and the absorbance was measured spectrophotometrically at 510 nm (Thermo Scientific GENESYS™ 150 UV–Visible Light Spectrophotometer, USA). The findings were reported as the mg catechin equivalent per gram of dry weight of the extract (mg CE g−1 d.w.) (Figure A2).

2.6. Vitamin C

The method for determining the concentration of vitamin C in bell peppers, based on the procedure described by [30,31], involved mixing 1 g of freeze-dried samples with 2.5 mL of 5% metaphosphoric acid and 2.5 mL of 10% acetic acid solution, followed by centrifugation at 5000× g for 20 min (Thermo Scientific Sorvall™ Legend™ X1 Centrifuge Series USA). The supernatant was filtered using Whatman® filter paper (WHA5230090, Sigma-Aldrich Corp.).
Next, a few drops of bromine water and 10% thiourea were added to the filtrate solution to clarify it, followed by adding 0.1 mL of 2,4 DNPH solution. The sample was then incubated at 37 °C for 3 h (New Brunswick™ I 24 Benchtop Incubator Shaker Series, VWR International, LLC, Radnor, PA, USA). After incubation, the sample solution was cooled in an ice bath and treated with 0.25 mL of 85% sulfuric acid, with continuous stirring.
The red-colored solutions were then used to measure the absorbance at 521 nm (Thermo Scientific GENESYS™ 150 UV–Visible Light Spectrophotometer USA). The concentration of vitamin C was determined by creating a calibration curve with ascorbic acid as a standard (Figure A3), and the results were expressed as the mg of vitamin C per 100 g−1 of the dry weight (mg Vitamin C/100 g d.w.).

2.7. Antioxidant Quantities

2.7.1. DPPH (1,1-Diphenyl-2-picrylhydrazyl Radical) Assay

The radical scavenging capacity of the methanolic extracts was assessed following the method outlined by [32], with some minor adjustments. In this procedure, 400 μL of extract samples with concentrations ranging from 100 μL to 600 μL were mixed with 1 mL of methanol solution of DPPH (0.004%). The deep violet color was changed to a light-yellow solution after allowing the mixture to stand at room temperature for 30 min. The absorbance was then measured at 517 nm (Thermo Scientific GENESYS™ 150 UV VWR International, LLC –Visible Light Spectrophotometer, USA). Similarly, a control solution was prepared using methanol instead of the extract. Ascorbic acid was included as a positive control due to its well-known antioxidant activity. The DPPH radical scavenging percentage was calculated using the following equation:
DPPH Scavenging effect (%) = 100 × (1 − Absample/Abcontrol)
Absample is the absorbance of the sample extract and DPPH reagent, while Abcontrol is the absorbance at 520 nm of methanol and DPPH reagent.

2.7.2. Reducing Power Assay

The methanolic extract’s reducing power was evaluated using the method outlined by Guilherme et al. [33]. Moreover, 500 μL of the extract (at concentrations ranging from 100 μL to 600 μL) was combined with 0.5 mL of 0.1 M sodium phosphate buffer (pH 7.0) and 0.5 mL of 1% (w/v) potassium ferricyanide. The mixtures were then placed in a thermostatic water bath (VWR® Digital General-Purpose Water Bath, VWR International, LLC, Radnor, PA, USA), and incubated for 20 min at 50 °C. Following incubation, 1 mL of 10% (w/v) trichloroacetic acid was added. The resulting sample solution was vortexed, and then 1 mL of deionized water and 0.1 mL of 0.1% ferric chloride were added. In this assay, the presence of antioxidants in the pepper extracts cause the ferric chloride and ferricyanide complex to transform into a blue-colored ferrous complex solution. Deionized water was utilized as a blank, and the absorbance was measured at 700 nm using a Thermo Scientific GENESYS™ 150 UV–Visible Light Spectrophotometer, USA, to assess the reducing capacity. Ascorbic acid was selected as the positive control due to its outstanding antioxidant activity. A higher absorbance of the mixture indicates a greater reducing power [34].

2.8. Statistical Analysis

The statistical analysis was performed using R statistical software (R 4.0.2 for Windows). All phytochemical determinations were carried out in triplicate, and the results were presented as mean ± standard deviation (SD) of three replicates. A two-way analysis of variance (ANOVA) was performed to assess significant differences among the treatments. If there were notable variations (p < 0.05), a post hoc comparison using the least significant difference (LSD) test (α < 0.05) was performed. Additionally, the impact of the two maturation stages (green and red) was considered, and a two-way ANOVA was employed to identify significant differences between green and red bell peppers. The contents of the bell pepper extracts were reported on a dry matter basis, as the study was conducted on freeze-dried samples.

3. Results and Discussion

3.1. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The total phenolic content (TPC) in green and red bell peppers was significantly affected by soil and foliar application of FA (Table 1). In green bell pepper, foliar 7.8 mL L−1 showed the highest TPC (3.58 mg GAE g−1 d.w.) among all the treatments, followed by a significantly higher TPC at soil 7.8 mL L−1 (3.39 mg GAE g−1 d.w.) compared to the control. The TPC of green bell pepper increased with an increasing concentration of FA (2.3 to 7.8 mL L−1) and then decreased in response to the highest concentration (10.9 mL L−1) in both the foliar and soil treatments. In red bell pepper, soil 2.3 and 5.5 mL L−1 showed significantly higher TPC (3.53 mg GAE g−1 d.w.) than the control and other treatment groups. No significant differences were observed between the foliar treatments and the control (Figure 2).
The color of the sweet bell pepper influenced the TPC content. Overall, green bell pepper had significantly higher TPC than red bell pepper. The TPC values from our results are in line with the findings by Thuphairo et al. [35], who reported that green bell peppers extracted with 70% (v/v) aqueous methanol showed significantly higher TPC (339 mg GAE 100 g−1 d.w.) compared to red bell peppers (321 mg GAE 100 g−1 d.w.) cultivated under a controlled environment in greenhouse conditions. Comparable results were observed by Blanco-Ríos et al. [4], who found that green bell pepper had the highest TPC with no significant variation among red, yellow, and orange cultivars grown under greenhouse conditions. The decreasing trend in the TPC from the green to the red stage can be attributed to amino acid synthesis in the initial stage of fruit maturity, which enters the metabolic pathway, a precursor in the formation of phenolic acids. This increases the phenolic content in green fruits [27]. Our results showed an opposite trend when compared with the study by Chávez-Mendoza et al. [16], who observed a higher concentration of TPC (111.26 mg 100 g−1 d.w.) in red bell pepper (Fascinato/Robusto) compared to green bell pepper (Sweet/Robusto), with lower TPC content (70.39 mg 100 g−1 of d.w) produced undercover, using net shading.
The total flavonoid content (TFC) in green and red bell peppers ranged from 10.39–13.94 mg CE g−1 d. w. and 11.73–15.62 mg CE g−1 d. w., respectively, (Table 1). In green bell pepper, the TFC at soil 10.9 mL L−1 (13.94 mg CE g−1 d. w.) was significantly higher (p < 0.05) than the control. No significant differences were observed for the foliar treatments (2.3, 7.8, and 10.9 mL L−1) and soil treatments (5.5 and 7.8 mL L−1) (Figure 3). In red bell pepper, the soil (2.3 and 7.8 mL L−1) and foliar treatments (2.3, 5.5, and 10.9 mL L−1) showed significantly higher amounts of TFC than the controls. However, the soil (5.5 and 7.8 mL L−1) and foliar treatments (2.3, 5.5, and 10.9 mL L−1) were not statistically different. Treatments with foliar 7.8, soil 5.5, and 10.9 mL L−1 were not statistically different from the control. In the present study, the TFC for red bell pepper was significantly higher than green bell pepper. The higher flavonoid content in red bell peppers than in green bell peppers can be ascribed to the maturation process. Studies have shown that as fruits ripen, a series of intricate biochemical processes unfold, leading to the synthesis and accumulation of secondary metabolites, such as flavonoids. These compounds are well-regarded for their antioxidant properties. Notably, the phenolic composition of the fruit undergoes significant changes during the maturation process, with the red stage typically exhibiting a richer profile of these beneficial compounds [33,36].
Like the results in our study, Aminifard et al. [26] recorded a TFC range from 132 to 199 mg QE 100 g−1 d. w. in red bell peppers grown using organic production methods under open-field conditions. Abdalla et al. [34] reported a higher TFC for green bell peppers (41.69 mg QE g−1 d. w.) than red (39.19 mg QE g−1 d. w). Moreover, these values were higher than the amount of TFC in green and red bell peppers recorded in the present study. Finally, it is well-known that several factors, such as the production system; pepper variety; analytical conditions, such as extraction methods; agro-climatic conditions; ripening stage; and postharvest handling, may explain the variation in the total phenolic and flavonoid content values from those reported in the literature [33].

3.2. DPPH and Reducing Power

The antioxidant efficiency of green and red bell peppers is presented (Table 2). The antioxidant potential of these extracts was determined by their ability to decolorize DPPH (% inhibition). The antioxidant efficacy comes from its ability to donate its own electron to the free radical, which enables it to break the chain reaction of oxidation [37]. In the case of green bell peppers, the DPPH radical scavenging activity ranged from 88 to 90%, while in red bell peppers, the inhibition ranged from 89 to 92%. The study showed no significant difference between the treatments (soil and foliar at different concentrations) and the control in terms of the DPPH radical scavenging activity in green and red bell peppers.
However, it was found that red bell pepper had a significantly higher DPPH than green bell pepper, which is in line with the findings by Cisternas-Jamet et al. [38], who observed an increase in DPPH activity with an increase in the maturity of fruit. The results from the present study were higher than those reported by Howard et al. [39], who found antioxidant activity in the 20–71.7% range for the pepper variety Parker and Flamingo grown under greenhouse conditions. In addition, Chávez-Mendoza et al. [16] reported the antioxidant activity of red bell pepper Fascinato/Robusto to be 79.65%, cultivated using net shading. These variations may be due to differences in pepper cultivars, maturation stages, and environmental conditions [8]. To assess the antioxidant activity of Capsicum, researchers have used different assay systems [40,41]. It does not allow us to make suitable comparisons due to inconsistencies in the assay systems [2]. Regarding the reducing power, foliar 7.8 and soil 2.3 mL L−1 exhibited an absorbance value of 0.50, significantly higher than the control and other foliar treatments. In contrast, soil 2.3 mL L−1 showed significantly higher absorbance than other soil treatments in green bell pepper. In red bell pepper, treatment at soil 10.9 mL L−1 showed a maximum absorbance of 0.51, significantly higher than that of the control and other soil and foliar treatments. In contrast, foliar 5.5 mL L−1 showed an absorbance of 0.49, significantly higher than other foliar treatments. However, it is not significantly different from the control treatment. Our study revealed that green bell pepper extracts’ reducing power capacity was significantly higher than red bell pepper extracts. The absorbance values were in line with the study by Abdalla et al. [34], who recorded the highest reducing power capacity that ranged from 0.1 to 0.6 for red, yellow, and green pepper extracts. Our findings were also in agreement with the variation in the reducing power capacity of colored peppers in a recent study by Mohammad Salamatullah et al. [42], who reported the highest reducing content in green pepper (2.305), followed by yellow (1.905) and red pepper (1.857). However, the absorbance values were much higher than the ones recorded in the present study. The antioxidant activity in bell peppers is attributed to phenolic compounds [43].

3.3. Vitamin C

Vitamin C is an important antioxidant in our diet. Pepper fruits, like most vegetables, are low in calories but have lots of vitamins, especially vitamin C [34]. The effect of soil and foliar application of fulvic acid showed significant differences in the vitamin C content in both green and red bell pepper stages (Table 3). In green bell pepper, the soil and foliar treatments showed significantly different levels of vitamin C, with foliar 7.8 mL L−1 being the highest (209 mg 100 g−1 d. w.) among all the treatments, followed by soil 5.5 mL L−1 (190.05 mg 100 g−1 d. w.). No significant differences were recorded between foliar 2.3, 5.5 mL L−1 and the control (Figure 4). In the case of red bell peppers, soil 5.5 mL L−1 showed the highest vitamin C content (255.71 mg 100 g−1 d. w.) than the control and the other treatments. The vitamin C content decreased at higher soil concentrations, 7.8 and 10.9 mL L−1, with values varying from 240.43 and 209 mg 100 g−1 d. w., respectively. On the other hand, in the case of foliar treatment, there was an observed increase in the vitamin C content with higher concentrations of fulvic acid, notably at 10.9 mL L−1 (240.2 mg 100 g−1 d.w.), in comparison to the control and the other foliar treatments (Figure 4).
It was observed that the ascorbic acid content increased with ripening and maturation. The red bell pepper extract showed significantly higher vitamin C content than the green. The results were consistent with Hallmann and Rembiałkowska. [13], who reported an increase in vitamin C content for organically grown cv. Roberta (18.9 g kg−1 DW) than conventionally grown cv. Spartacus (19.3 g kg−1 d.w.) and cv. Berceo (18.1 g kg−1 DW), cultivated undercover in polytunnels. Another study by Abdalla et al. [34] on the variation in vitamin C content in colored peppers showed the superiority of red bell pepper (180.56 mg/g f.w.) over green bell pepper (83.30 mg/g f.w.). The results from the current study were slightly lower than those reported by Hamed et al. [44]. They reported a wide variation in the ascorbic acid content of different colored field-grown pepper cultivars that ranged from 222.55 to 945.36 mg/100 g d.w. at the green stage, while a range from 314.87 to 752.54 mg/100 g DW was recorded at the red stage. The higher level of ascorbic acid at the red stage is associated with carbohydrate metabolism [44]. This mechanism plays a crucial role in providing the necessary energy and materials to produce ascorbic acid. Studies have shown that as the fruit matures, there is increased synthesis of ascorbic acid, which is influenced by genetic and environmental factors. This heightened production of ascorbic acid is important for the plant’s defense mechanisms and the overall quality of the fruit [45,46]. Differences in the concentration of ascorbic acid have been reported in many pepper cultivars. However, there is no agreement on the extent of the changes, which appear to depend on the pepper variety, maturity stage, genetics, growth, and climatic conditions [40].
Our findings align with previous research that showed the beneficial impact of humic substances on tomatoes [47], lettuce [41], and yarrow [25]. A field trial by [28] on organic pepper was conducted under open-field conditions to investigate the effect of FA on fruit quality and antioxidant activity. The study found that fulvic acid positively affected various fruit quality parameters, including total phenolics, antioxidant activity, total soluble solids, carotenoids, and capsaicin. Moreover, it has been reported that the amount of humic compounds (humic and fulvic acids) present in the soil can influence the antioxidant activity; the greater the content of humic compounds in the soil, the stronger the antioxidant activity [48]. One hypothesis explained plant responses to insects, pathogens, and weeds through increased organic acids and polyphenols in the organic production system. In organic production systems, such as in our experiment, the limited use of synthetic insecticides, pesticides, and fungicides can lead to plant stress. There are limits to the prohibition of synthetic insecticides, pesticides and fungicides in organic production systems (like our experiment), which may induce plant stress. This may require plants to allocate resources to synthesize their enzymatic and non-enzymatic defense mechanisms. Therefore, higher amounts of antioxidants, such as polyphenols, may be attributed to their production during plant defense [49].

4. Conclusions

In conclusion, this study demonstrates that applying fulvic acid (FA) through soil and foliar methods at specific concentrations can significantly enhance the antioxidant activity and phytochemical content in organically grown green and red bell peppers. The findings indicate that FA treatment influenced the total phenolic content (TPC), total flavonoid (TFC), and vitamin C content, with varying effects on green and red bell peppers. The research indicates that using FA at optimal concentrations has the potential to enhance the nutritional value of organic bell peppers in an environmentally sustainable way. FA can serve as a safe and efficient substitute for chemical fertilizers in bell pepper nutrition, facilitating the production of safe food, preserving soil fertility, and minimizing environmental contamination. Additional research and field experiments could further validate the practical application of FA for enhancing the nutritional value of bell peppers.

Author Contributions

P.K. conceptualization, methodology, investigation, writing—original draft, data analysis; Y.W. conceptualization, methodology, investigation; D.N. conceptualization, writing—review, supervision, project administration, funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

The Tennessee State University (TSU) Cooperative Extension Program funded this work for Dr. Dilip Nandwani (#223199).

Institutional Review Board Statement

Not Applicable.

Informed Consent Statement

Not Applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors thank Shahid Chowdhury, ZaDarrheyal Wiggins, and Eddie Williams for their field assistance and Myles for lab assistance. The authors would also like to acknowledge Faust Bio-Agricultural Services, Inc. (Aka BioAg Inc., www.bioag.com) for providing the fulvic acid for the trials.

Conflicts of Interest

The authors declare no conflict of interest.

Appendix A

Sustainability 16 06745 g0a1
Figure A1. Gallic acid standard curve.
Figure A1. Gallic acid standard curve.
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Figure A2. Catechin standard curve.
Figure A2. Catechin standard curve.
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Figure A3. Ascorbic acid standard curve.
Figure A3. Ascorbic acid standard curve.
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Figure 1. Phytochemical analysis of organically grown green and red bell pepper.
Figure 1. Phytochemical analysis of organically grown green and red bell pepper.
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Figure 2. Effect of FA on TPC content of bell pepper at the green and red stage. a–d Different letters indicate statistical difference (p ≤ 0.05).
Figure 2. Effect of FA on TPC content of bell pepper at the green and red stage. a–d Different letters indicate statistical difference (p ≤ 0.05).
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Figure 3. Effect of FA on TFC content of bell pepper at the green and red stage. a–d Different letters indicate statistical difference (p ≤ 0.05).
Figure 3. Effect of FA on TFC content of bell pepper at the green and red stage. a–d Different letters indicate statistical difference (p ≤ 0.05).
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Figure 4. Effect of FA on vitamin C of bell pepper at the green and red stage. a–g Different letters indicate statistical difference (p ≤ 0.05).
Figure 4. Effect of FA on vitamin C of bell pepper at the green and red stage. a–g Different letters indicate statistical difference (p ≤ 0.05).
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Table 1. Effect of FA on total phenolic content (TPC) and total flavonoid content (TFC) of green and red bell pepper.
Table 1. Effect of FA on total phenolic content (TPC) and total flavonoid content (TFC) of green and red bell pepper.
GreenRedGreenRed
Treatment (mL L−1)TPC (mg GAE g−1 d. w.)TFC (mg CE g−1 d. w.)
Control2.82 ± 0.29 cd2.58 ± 0.01 d11.77 ± 0.88 bc11.85 ± 2.70 c
Foliar 2.33.09 ± 0.22 bc2.67 ± 0.28 d12.40 ± 0.11 b14.32 ± 0.73 ab
Foliar 5.53.13 ± 0.07 bc2.69 ± 0.25 d10.39 ± 0.32 d14.69 ± 0.68 a
Foliar 7.83.58 ± 0.11 a2.72 ± 0.06 d11.35 ± 0.11 c12.42 ± 0.17 c
Foliar 10.92.90 ± 0.30 cd2.76 ± 0.25 d11.58 ± 0.26 c15.57 ± 0.00 a
Soil 2.32.83 ± 0.16 cd3.34 ± 0.26 ab10.51 ± 0.44 d15.34 ± 0.24 a
Soil 5.53.21 ± 0.12 abc3.53 ± 0.13 a11.58 ± 0.45 c11.73 ± 1.25 c
Soil 7.83.39 ± 0.29 ab3.03 ± 0.11 c11.87 ± 0.16 bc15.62 ± 0.16 a
Soil 10.92.58 ± 0.28 d3.13 ± 0.05 bc13.94 ± 0.30 a12.83 ± 0.08 bc
Values are presented as mean ± standard deviation. Different letters for each treatment indicate significant differences (p ≤ 0.05).
Table 2. Effect of FA on DPPH (% radical scavenging activity) and reducing power capacity of green and red bell pepper.
Table 2. Effect of FA on DPPH (% radical scavenging activity) and reducing power capacity of green and red bell pepper.
GreenRedGreenRed
Treatment (mL L−1)DPPH (%)Reducing Power
Control88.82 ± 1.27 bc90.04 ± 2.53 ab0.48 ± 0.02 de0.46 ± 0.01 bcd
Foliar 2.386.59 ± 2.19 c90.65 ± 2.14 ab0.47 ± 0.01 e0.43 ± 0.01 de
Foliar 5.588.28 ± 3.36 bc89.29 ± 3.64 b0.48 ± 0.01 e0.49 ± 0.02 ab
Foliar 7.890.44 ± 1.41 ab92.88 ± 0.88 a0.50 ± 0.01 bc0.43 ± 0.02 de
Foliar 10.989.02 ± 1.61 bc91.73 ± 1.05 ab0.43 ± 0.01 f0.44 ± 0.02 cde
Soil 2.390.17 ± 2.28 ab92.55 ± 1.03 ab0.50 ± 0.02 ab0.41 ± 0.01 e
Soil 5.590.24 ± 1.59 ab91.05 ± 2.81 ab0.48 ± 0.01 cde0.47 ± 0.03 bc
Soil 7.889.02 ± 2.19 bc91.33 ± 0.82 ab0.48 ± 0.01 cde0.46 ± 0.03 cd
Soil 10.988.48 ± 3.77 bc91.53 ± 1.12 ab0.50 ± 0.02 bcd0.51 ± 0.01 a
Values are presented as mean ± standard deviation. Different letters for each treatment indicate significant differences (p ≤ 0.05).
Table 3. Effect of FA on vitamin C in green and red bell pepper.
Table 3. Effect of FA on vitamin C in green and red bell pepper.
GreenRed
Treatment (mL L−1)Vitamin C (mg 100 g−1 d. w.)
Control142.74 ± 3.61 f154.69 ± 0.97 f
Foliar 2.3143.76 ± 0.70 f148.11 ± 0.73 g
Foliar 5.5144.59 ± 1.13 f148.85 ± 1.37 g
Foliar 7.8209.13 ± 1.31 a233.21 ± 0.85 c
Foliar 10.9162.65 ± 1.97 c240.24 ± 0.42 b
Soil 2.3156.91 ± 0.85 d187.65 ± 2.56 e
Soil 5.5190.05 ± 1.73 b255.71 ± 2.24 a
Soil 7.8156.63 ± 1.58 d240.43 ± 1.85 b
Soil 10.9153.30 ± 1.25 e209.22 ± 1.21 d
Values are presented as mean ± standard deviation. Different letters for each treatment indicate significant differences (p ≤ 0.05).
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Kanabar, P.; Wu, Y.; Nandwani, D. Enhancing Sustainable Cultivation of Organic Bell Pepper through Fulvic Acid (FA) Application: Impact on Phytochemicals and Antioxidant Capacity under Open-Field Conditions. Sustainability 2024, 16, 6745. https://doi.org/10.3390/su16166745

AMA Style

Kanabar P, Wu Y, Nandwani D. Enhancing Sustainable Cultivation of Organic Bell Pepper through Fulvic Acid (FA) Application: Impact on Phytochemicals and Antioxidant Capacity under Open-Field Conditions. Sustainability. 2024; 16(16):6745. https://doi.org/10.3390/su16166745

Chicago/Turabian Style

Kanabar, Pinkky, Ying Wu, and Dilip Nandwani. 2024. "Enhancing Sustainable Cultivation of Organic Bell Pepper through Fulvic Acid (FA) Application: Impact on Phytochemicals and Antioxidant Capacity under Open-Field Conditions" Sustainability 16, no. 16: 6745. https://doi.org/10.3390/su16166745

APA Style

Kanabar, P., Wu, Y., & Nandwani, D. (2024). Enhancing Sustainable Cultivation of Organic Bell Pepper through Fulvic Acid (FA) Application: Impact on Phytochemicals and Antioxidant Capacity under Open-Field Conditions. Sustainability, 16(16), 6745. https://doi.org/10.3390/su16166745

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