The Impact of Organic, Inorganic Fertilizers, and Biochar on Phytochemicals Content of Three Brassicaceae Vegetables

: The need for soil fumigants of natural origin such as glucosinolates (GSLs) has increased due to the general prevention of manmade soil fumigants. GSLs and other phytochemicals (vitamin C and phenols) present in Brassica vegetables such as turnips, arugula, and mustard have antioxidant properties, and hence have important health attributes. The study examined how different soil amendments (chicken manure CM, vermicompost Vermi, horse manure HM, sewage sludge SS, elemental inorganic fertilizer Inorg, organic fertilizer Org, and biochar) impact the concentrations of glucosinolates (GSLs), vitamin C, phenols, and reducing sugars in three varieties of turnips (Purple Top White Globe PTWG, Scarlet Queen Red SQR, and Tokyo Cross TC), arugula, and mustard greens grown under ﬁeld conditions. The results showed that mustard greens contained higher concentrations of GSLs (974 µ g g − 1 fresh shoots) than arugula (651 µ g g − 1 fresh shoots), and the TC variety of turnip had the highest concentrations of GSLs, vitamin C, and sugars. Additionally, amending the soil with SS, CM, and HM signiﬁcantly increased the vitamin C content in mustard shoots by 82%, 90%, and 31%, respectively, and the total phenols by 77%, 70%, and 36%, respectively, compared to the control treatment. The increased inorganic fertilizers cost, and availability of large amounts of animal manure made animal manure application to cropland an attractive disposal option.


Introduction
Fertilization is the most important and controllable factor affecting the phytochemicals and nutritional value of vegetables.Biofumigation, an approach aimed at controlling pests and pathogens in soil, involves incorporating residues of Brassica plants into the soil.As these residues decompose, they release toxic degradation products known as isothiocyanate (ITC), which can effectively combat various pests and soil-borne pathogens.GSLs are secondary metabolites present in seeds, roots, stems, and leaves of Brassicaceae vegetables including arugula, mustard, turnips, radish, broccoli, cabbage, cauliflower, and horseradish.GSLs are non-nutritional organic anions present in 16 dicotyledonous plant families in about 120 different chemical structures [1].The concentration of GSLs has become an important parameter in developing plant varieties for human and animal welfare.They have a range of properties, such as antioxidant, herbicidal, and anticarcinogenicity [2].GSLs-thioglucosidase (myrosinase) chemical defense system is the bioactive mechanism in the Brassicaceae family.GSLs and thioglucosidase (the enzyme that breaks down GSLs) are located in two different compartments in Brassica cells that in combination release plant chemical defensive products [3,4].GSLs are water-soluble compounds deposited in the Brassica cell vacuole, whereas thioglucosidase (myrosinase) is located in the phloem parenchyma cells and the mesophyll or cortex [5].As a result, Brassica tissue interruption due to infestation or attack by arthropods causes the discharge of a complex mixture of GSLs that interrupt hydrolysis products of which ITCs (Figure 1) are powerful antimicrobial agents.Myrosinase in Brassica breaks the β-thioglucoside bond of the GSL molecule and releases one mole of ITC.Accordingly, one molecule of GSL yields one molecule of ITC and one molecule of glucose upon hydrolysis.ITCs might be useful as alternative agents for pest control as a replacement for the toxic metam sodium, a common fungicide [6], and/or methyl bromide and ethylene dibromide synthetic fumigants.Intact GSLs molecules have no toxic effect on fungi, whereas the hydrolysis products of GSLs inhibit the development of Sclerotinia sclerotiorum and Rhizoctonia solani causal of plant stem rot [7].Several investigators found that degradation products of the cut leaf or stem part of Brassica plants prevented the development of a diversity of soil diseases such as Rhizoctonia solan, Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, and Fusarium sambucinam [8] and pathogenic bacteria [9].
Appl.Sci.2023, 13, x FOR PEER REVIEW 3 of 19 impact of animal manure (SS, CM, and HM) as organic fertilizers on the concentrations of phytochemicals (vitamin C, total phenols, and soluble sugars in three varieties of fieldgrown turnips, Brassica rapa; arugula, Eruca sative; and mustard, Brassica juncea greens.

Turnips Field and Experimental Design
The study involved 126 field plots arranged in a randomized complete block design with three replicates.Three different turnip varieties and 14 treatments were utilized.Each treatment had dimensions of 3 feet (0.91 m) in width and 4 feet (1.22 m) in length.The field was exploited to cultivate three varieties of turnips: Brassica rapa var.Purple Top White Globe PTWG, var.Scarlet Queen Red SQR, and var.Tokyo Cross TC.The 14 soil treatments were employed in the experiment and included sewage sludge SS, horse manure HM, chicken manure CM, vermicompost Vermi (worm castings), commercial inorganic fertilizer (NPK 19:19:19), commercial organic fertilizer (Nature Safe NPK 10:2:8), as well as no-manure native soil).Additionally, biochar was added to the SS, CM, HM, Vermi, Org, Inorg, and UA treatments.The native soil in the experimental plots was Bluegrass-Maury Silty Loam with a pH of 6.1 and an organic matter content of 2.2%.The soil had 56% silt, 38% clay, and 6% sand, along with other properties listed in Table 1.The chemical structure of GSLs can be categorized into three main groups: aliphatic GSLs, which primarily originated from methionine; aromatic GSLs, derived mainly from phenylalanine or tyrosine; and indoles GSLs, derived from tryptophan.All GSLs produce isothiocyanate (ITC) and their production of ITCs is greatest at pH 7, whereas at acidic pH nitriles are mainly produced (Figure 1).Thioglucosidase hydrolyzes GSLs in plants, insects, and fungi and is frequently found in soil [10].Metam sodium, a synthetic commercial soil fumigant in the United States [6,11], also degrades to methyl ITC, which is utilized for the control of soil-borne fungi and nematodes.However, metam sodium is an extremely toxic compound that causes severe damage to the immune system [6].
Numerous research studies have explored the potential use of Brassica plants as environmentally friendly fumigants [12][13][14][15][16][17].Antonious [16] found that the levels of GSLs in collard plants (Brassica oleracea cv.Top Bunch) grown in soil treated with sewage waste sludge (SS) and chicken manure (CM) were significantly higher compared to those grown in native control soil.Additionally, the organic matter content in the soil increased from 2% in native soil to 4 and 6.5% in soil supplemented with SS and CM, respectively.The presence of SS or CM amendments in the soil resulted in higher GSL levels in collard plants compared to those grown under controlled conditions.This finding suggests that collard leaves cultivated in soil amended with SS or CM could be used as an alternative method for managing soil-borne diseases in agricultural systems.Furthermore, Antonius et al. [18] found that different ratios of far-red and red light reflected by colored plastic mulches used for water conservation irrigation systems affected turnip plants (Brassica rapa L. cv.Purple Top).Turnip roots grown in blue plastic mulch exhibited the highest total GSL concentrations among the various colored mulches.Although turnip roots grown with white plastic mulch were larger in size than those grown with blue or green plastic mulch, they showed lower GSL concentrations on a fresh weight basis.Another study [14] highlighted two approaches to increase ITC concentrations: growing Brassica plant varieties with high GSL content or disrupting the cells of Brassica plants when incorporating their tissues into the soil.This disruption causes the GSLs to break down, resulting in a straightforward increase in ITC hydrolysis.In this current study, the aim is to investigate the impact of various organic and inorganic fertilizers on the internal composition of Brassica plants, including GSLs, vitamin C, total phenols, and soluble sugars.Additionally, the pyrolysis process, which involves burning material without oxygen, produces biochar.The potential benefits of biochar in improving agricultural soil quality have been investigated.It has been observed that biochar enhances the soil's capacity to retain nutrients and water, potentially leading to accelerated plant growth [19].Furthermore, biochar can serve as a habitat for soil microorganisms, aiding in the breakdown of organic matter in the soil [20].
The present investigation aimed to: (1) Assess the composition of animal manures used for growing turnips, arugula, and mustard.(2) Assess the variation in total GSLs concentration among varieties of turnip, Brassica rapa (Tokyo Cross, Scarlet Queen Red, and Purple Top White Globe.(3) Assess the impact of fourteen ( 14) soil treatments on the concentration of GSL, one of the unique phytochemicals in Brassica family.(4) Test the impact of animal manure (SS, CM, and HM) as organic fertilizers on the concentrations of phytochemicals (vitamin C, total phenols, and soluble sugars in three varieties of fieldgrown turnips, Brassica rapa; arugula, Eruca sative; and mustard, Brassica juncea greens.

Turnips Field and Experimental Design
The study involved 126 field plots arranged in a randomized complete block design with three replicates.Three different turnip varieties and 14 treatments were utilized.Each treatment had dimensions of 3 feet (0.91 m) in width and 4 feet (1.22 m) in length.The field was exploited to cultivate three varieties of turnips: Brassica rapa var.Purple Top White Globe PTWG, var.Scarlet Queen Red SQR, and var.Tokyo Cross TC.The 14 soil treatments were employed in the experiment and included sewage sludge SS, horse manure HM, chicken manure CM, vermicompost Vermi (worm castings), commercial inorganic fertilizer (NPK 19:19:19), commercial organic fertilizer (Nature Safe NPK 10:2:8), as well as no-manure native soil).Additionally, biochar was added to the SS, CM, HM, Vermi, Org, Inorg, and UA treatments.The native soil in the experimental plots was Bluegrass-Maury Silty Loam with a pH of 6.1 and an organic matter content of 2.2%.The soil had 56% silt, 38% clay, and 6% sand, along with other properties listed in Table 1.
To standardize the nitrogen (N) content across different soil treatments, each soil amendment was mixed with the native soil in the experimental plots at a rate of 5% nitrogen on a dry weight basis at the amounts shown in Table 2.The SS was obtained from the Metropolitan Sewer District in Louisville, KY, USA, while the CM (composted manure) was sourced from the Department of Animal and Food Sciences at the University of Kentucky in Lexington, KY, USA.HM was collected from the Kentucky Horse Park in Lexington, KY, USA, and Vermicompost (Vermi) was obtained from Worm Power in Montpelier, Vermont, USA.Organic and inorganic fertilizers were purchased from Southern States Stores in Lexington, KY, USA.Prior to planting in the experimental plots, each soil amendment was added to the native soil and mixed thoroughly to a depth of 15 cm of the topsoil using a root tiller.Turnip seeds (Brassica rapa) were then manually planted in the freshly tilled soil with an 18-inch spacing between rows.Drip irrigation was employed as necessary.In the remaining seven treatments, the soil was mixed with 10% (w/w) biochar obtained from Wakefield Agricultural Carbon in Columbia, MO, USA.The biochar used in this study had the following characteristics: total inorganic carbon content of 0.34%, particle size less than 0.5 mm, pH of 7.4, biochar surface area of 366 m 2 /g (dry weight basis), 88%, total organic carbon content, bulk density of 480.6 kg/m 3 , and moisture content of 54%.Throughout the growing season, routine agricultural tasks were performed as needed.
During the growing season, two insecticides, namely esfenvalerate (sold as Asana XL) and Baythroide XL (containing β-cyfluthrin), were applied three times using the recommended application rate [21].At the time of harvest, three different turnip varieties (PTWG, SQR, and TC) were taken out of the soil.Their shoots and roots were cleaned with tap water and then separated utilizing a sharp knife.The concentrations of GSLs (glucosinolates), vitamin C, total phenols, and soluble sugars in the shoots and roots were determined as explained below.

Arugula and Mustard Field Experimental Design
Arugula, Eruca sative Mill variety Astro and mustard, Brassica juncea L. variety Kranti (Family: Brassicaceae) seedlings were grown in freshly tilled soil.The plants were developed in 30 ft. × 144 ft.(9.14 m × 43.89 m) beds.Each bed (12 ft.× 30 ft.) (3.66 m × 9.14 m) contained three replicates established in a randomized complete block design (RCBD) with four soil treatments.The study area contained 24 experimental plots (2 crops × 3 replicates × 4 treatments).The four treatments were (1) SS amended with native soil, (2) CM amended with native soil, (3) HM amended with native soil, and (4) no-manure (NM) native control soil.Animal manures were applied to native soil at 15 tons acre −1 on a dry weight basis and mixed with the top 15 cm soil and the plants were grown following Kentucky agricultural guidelines [21] and no other fertilizers were applied during the growing season.

Glucosinolates (GSLs) Analysis
The GSLs present in the plant's crude extracts were separated by adsorption on a diethyl amino-ethyl ether ion exchange resin.The quantification of GSLs was performed colorimetrically by measuring the enzymatically released glucose.The separation and quantification of total GSLs were carried out using an inexpensive and accurate method developed by Antonious [16] in which total GSLs in turnip root, shoot, arugula, and mustard greens were extracted with boiled methanol to prevent endogenous thioglucosidase (the enzyme that hydrolyzes GSLs) from inhibiting GSLs.The extracts underwent vacuum filtration to eliminate the methanol solvent, and the resulting water extract was further filtered using celite powder through disposable pipette tips to obtain a purified and homogeneous aqueous extract.The quantification of GSLs concentrations was determined by measuring the enzymatically released glucose after hydrolyzing sinigrin (2-propenyl-glucosinolate) since each molecule of GSLs liberates one mole of ITC (isothiocyanate) and one mole of glucose (Figure 1).Sinigrin (a GSL molecule) standard calibration curve in the range of 100-500 µg sinigrin mL −1 of aqueous solution was used for the determination of glucose liberated after hydrolysis of sinigrin by the enzyme thioglucosidase.

Vitamin C, Total Phenols, and Free Sugars Analysis
Before bolting (flowering), turnip root, shoot, arugula, and mustard greens were harvested.To extract phenols, representative samples of plant tissue (20 g) were mixed with 150 mL of ethanol.The resulting homogenates were then filtered through Whatman No. 1 filter paper.For the determination of total phenols, 1 mL aliquots of the filtrate were utilized and determined by the Folin-Ciocalteu method [22].The method used a standard calibration curve ranging from 10 to 80 µg mL −1 of chlorogenic acid (obtained from Fisher Scientific Company, Pittsburgh, PA, USA).A total of 20 g of each plant tissue was blended with 100 mL of a 0.4% (w/v) oxalic acid solution [23] for the extraction of vitamin C (ascorbic acid).The quantification of vitamin C was carried out colorimetrically using the potassium ferricyanide method [24].A standard curve within the range of 90-300 µg mL −1 of ascorbic acid (with a purity of 99%) was employed.To extract soluble glucose from plant tissue samples, crude extracts were treated with 80% ethanol.The quantification of soluble glucose was performed using a calibration curve ranging from 100 to 800 µg mL −1 , based on standard-grade glucose [25].Concentrations of total GSLs, vitamin C, phenols, and glucose contents in each plant tissue were compared using a one-way analysis of variance (ANOVA) and Duncan multiple range test for mean comparisons using SAS (SAS Institute, 2016) [26].The concentrations of each parameter in each plant tissue were compared using ANOVA.Initially, the response variables were plotted to visualize the distribution of the data.The normality of the data was tested using the Shapiro-Wilk test in SAS.The homogeneity of variance across the groups was checked by Levine's test.

Soil Enzymes Analysis
Urease activity (the enzyme that hydrolyzes urea fertilizers into NH 3 and CO 2 ) in soil and its energetic part in ruling N to plants following the addition of soil amendments was determined as described by [27].Invertase activity was determined in soil by Balasubramanian et al. [28].Invertase is important for releasing simple carbon for the growth and multiplication of soil microorganisms.Phosphatases, the enzyme that converts organic phosphate esters in the soil to orthophosphate ions for plant uptake was determined using Tabatabai and Bremner [29] procedure.

Results and Discussion
Regardless of turnip variety, CM-amended soil significantly (p ≤ 0.05) increased the shoot, root, and plant weight (490, 303, and 793 g, respectively) compared to the yield obtained from inorganic fertilizers (422, 267, and 689 g, respectively) (Figure 2).CM is a natural, affordable, and locally available organic fertilizer that vegetable growers can obtain.The increased size along with the required periodic cleanup of several poultry farm operations on a timely basis would ensure sufficient supplies for crop needs.Poultry litter is made of raw poultry manure and bedding materials, such as sawdust wood shavings, grass cuttings, leaves, or rice hulls.This blend provides an excellent source of N, P, K, and S for growing plants.On the contrary, inorganic fertilizers are relatively costly and can potentially harm the environmental quality due to their uncontrolled release of N and P nutrients in surface water (eutrophication) following natural rainfall events.The properties of organic and inorganic fertilizers used in agricultural production systems differ considerably.Inorganic fertilizers are prepared to be rapidly released from the soil to grow plants, whereas nutrients in organic fertilizers, such as animal manures, are usually discharged in a slow-released process during which soil microorganisms break down complex forms of nutrients through mineralization.Producers seeking cost-effective N, P, and K nutrients source have found animal dung use as organic fertilizer a cost-effective way to meet their needs [30].The poultry industry is growing, and poultry manure is available in increasing quantities.Compared to other livestock manures, poultry has relatively high dietary requirements for sulfur amino acids, methionine, and cysteine [31].Antonious et al. [32] reported that due to the addition of sulfur (S) into hens' diets during industrial breeding operations, CM could be exploited in growing plants, such as onions, with health-promoting properties.Concentrations of dipropyl disulfide and dipropyl trisulfide were significantly higher (p ≤ 0.05) in onion bulbs (Allium cepa var.Super Star F1) of plants grown in soil amended with CM compared to SS and yard waste compost treatments.S absorbed from soil mixed with CM by plants enters the plant actively as sulfate ions (SO 2 −4 ).SO 2 −4 ions are transported to the leaves, where it can be stored in the cell vacuole or be reduced to sulfide and assimilated into cysteine in the chloroplast [33].Cysteine is therefore either incorporated into plant proteins or used in the synthesis of methionine and glutathione [34] having health-promoting activity such as the anticarcinogenic properties of Allium vegetables.Accordingly, CM can not only decrease dependence on synthetic inorganic fertilizers but also supports the production of healthy food.These findings support [35] and [36] who reported a positive correlation between GSLs (S-containing phytochemicals) and S fertilizer supply used in growing mustard, turnip, kale, and broccoli.
On the other hand, one disadvantage of using CM in agricultural production systems is that large-scale chicken farms in the U.S. and worldwide are concentrated animal feeding operations (CAFO) that might have a million animals raised up in one capacity, which requires heavy use of antibiotics that can cause severe local air and water pollution.In these facilities, arsenic (As) in the form of roxarsone (4-hydroxy-3-nitrobenzenearsonic acid) and arsenic acid (4-aminophenylarsonic acid) are added to the feed to eliminate parasites (coccidia) and increase hens' weight improvement.Arsenic is typically mixed with a hen's diet at 22.7-45.4g per ton, or 0.0025-0.005%[37].Roxarsone that passes through the bird's digestive system ends in its feces unchanged.Each broiler releases around 150 mg of roxasone during a period of 42-day growth [38,39].As is an environmental pollutant with severe carcinogenic impacts on human beings [40].In addition, more than 70% of the As in saved piles of poultry litter was dissolved by rainfall events and actually leached into lakes and streams, making this application practice unsustainable and not in line with the national organic standards [41].Accordingly, As may reach agricultural soils when CM is applied as a fertilizer.The maximum acceptable As levels in the U.S. soils fluctuate extensively from one state to another state.The As concentration is 2.4 in Arkansas's soil and 500 ppm in Montana's agricultural soil, whereas this level in residential soils ranges from 0.4 ppm in Illinois to 40 ppm in Colorado.In South Carolina, which has a great hen industry, has set the maximum concentration of As in hens' litter at 41 ppm (dry weight basis) with no more than a total of 37 pounds of As to be applied ever [42].In Italy, the maximum permissible concentration of As in compost is 10 ppm, and it is 13 ppm in Canada, 15 ppm in the Netherlands, and 25 ppm in Denmark [43], whereas the detected levels of As in Kentucky soil ranged from 0.1-10 ppm based on analyzing samples from across the state [44].
Results revealed that CM mixed with native soil used in the present investigation contained < 9 ppm arsenic (As) (Table 1).Accordingly, As does not represent any hazardous issues for growing plants in the study area.However, Bellows [41] reported a phytotoxic effect on plants growing in soil that contains As at levels of 10 ppm.Table 1 also revealed a significant increase in invertase activity in all the amended soil compared to the control treatment.In terms of urease activity, Vermi displayed a significantly greater increase compared to the SS-, CM-, and HM-amended soils, as well as the native control soil.Both Vermi and SS displayed a significant increase in acid phosphatase activity compared to the CM-and HM-amended soils.CM was found to be the most effective at increasing alkaline phosphatase activity among the treatments.Conversely, Vermi and SS displayed the lowest alkaline phosphatase activity, which was even below that of the native soil.These results could be due to the variability of the nutritional composition of each of the soil amendments used.
Results in Table 3 listed the total GSLs concentrations in roots and shoots of each of the three turnip varieties investigated in this study.About 120 different GSLs are reported in the literature review in [1].Based on the release of glucose molecules used in the quantification of GSLs, total GSLs in turnip variety PTWG grown in elemental inorganic fertilizer (Inorg) and SS-amended soil were significantly (p ≤ 0.05) increased compared to plants grown in the control treatment (NM native soil).SS elevated the total concentration of GSLs in variety PTWG from 919 in the control to 2024 µg g −1 fresh tissue, which is about a 120% increase.Similarly, Inorg fertilizer elevated the total concentration of GSLs in variety PTWG from 919 in the control to 1736 µg −1 fresh tissue, which is about an 89% increase.This means there is a greater release of ITC and greater mycelia suppression in agricultural fields infested with soil-borne diseases when extract of PTWG is used for soil pest control.
Studies revealed a strong inverse relationship between dietary ingesting of cruciferous vegetables and occurrences of cancer due to the chemopreventive and chemotherapeutic role of ITCs against numerous tumor types [45] including pancreatic, breast, prostate, and ovarian [46][47][48][49][50].
Table 3 also revealed that none of the amendments tested increased the GSLs concentrations in the shoots of the three turnip varieties (PTWG, SQR, and TC) tested, whereas all amendments increased GSLs in the roots of variety PTWG and only SS and Inorg amendments increased total GSLs in the roots of variety TC compared to the control treatments.Regardless of the soil amendments used in this investigation, variety TC significantly increased GSLs and consequently ITCs concentration upon the hydrolysis of GSLs in the roots, shoots, and total GSLs compared to the SQR and PTWG varieties (Figure 3).Accordingly, TC extracts have great potential for use as an alternative pest control agent to metam sodium, a common fungicide, due to its high GSL content.Hence, turnip varieties can be organized based on GSLs composition in the order of TC > SQR > PTWG.Results in Table 3 listed the total GSLs concentrations in roots and shoots of each of the three turnip varieties investigated in this study.About 120 different GSLs are reported  Studies revealed a strong inverse relationship between dietary ingesting of cruciferous vegetables and occurrences of cancer due to the chemopreventive and chemotherapeutic role of ITCs against numerous tumor types [45] including pancreatic, breast, prostate, and ovarian [46][47][48][49][50].
Table 3 also revealed that none of the amendments tested increased the GSLs concentrations in the shoots of the three turnip varieties (PTWG, SQR, and TC) tested, whereas all amendments increased GSLs in the roots of variety PTWG and only SS and Inorg amendments increased total GSLs in the roots of variety TC compared to the control treatments.Regardless of the soil amendments used in this investigation, variety TC significantly increased GSLs and consequently ITCs concentration upon the hydrolysis of GSLs in the roots, shoots, and total GSLs compared to the SQR and PTWG varieties (Figure 3).Accordingly, TC extracts have great potential for use as an alternative pest control agent to metam sodium, a common fungicide, due to its high GSL content.Hence, turnip varieties can be organized based on GSLs composition in the order of TC > SQR > PTWG.Regardless of soil amendment type, results revealed that biochar treatments lowered GSLs concentration in variety TC from 5857 to 3894 µg g-1 turnip fresh tissue, indicating a 34% reduction related to plants grown in treatment not amended with biochar.On the contrary, biochar increased GSLs concentration in variety SQR by 150% compared to SQR Regardless of soil amendment type, results revealed that biochar treatments lowered GSLs concentration in variety TC from 5857 to 3894 µg g −1 turnip fresh tissue, indicating a 34% reduction related to plants grown in treatment not amended with biochar.On the contrary, biochar increased GSLs concentration in variety SQR by 150% compared to SQR grown in soil not amended with biochar, whereas no significant differences were shown in GSLs concentration in a variety of PTWG whether grown in treatment mixed or not mixed with biochar (Figure 4).Regardless of turnip variety, SS and Inorg significantly (p ≤ 0.05) increased the concentration of GSLs compared to the control treatment (NM native soil) (Figure 5). Figure 5 also showed that the concentration of GSLs was significantly greater in HM amended with biochar in relation to HM not amended with biochar.
zymes as bioindicators of soil health had been investigated [57,58].Antonious et al. [51] reported no increase in soil alkaline phosphatase activity with biochar addition to CM. Biochar added to native soil reduced the activity of soil alkaline phosphatase by 41% more than the control treatment.Trace metal contamination removes some microorganisms and multiplies others, resulting in changes in soil quality and functionality.For example, alkaline phosphatase activity is significantly inhibited with Cd [52].Similarly, the breakdown of GSLs into ITC and glucose is dependent on the activity of thioglucosidase.
As described earlier, using Brassica crops as green manures reduces soilborne pests and pathogens such as Rhizoctonia solani, and Sclerotinia sclerotiorum, the two most damaging soilborne pathogens [59].Brassica plants are well adapted in Kentucky and other states and hence can be used for pest control either by spreading dried Brassica plant powder or using green manure by incorporating plants into the soil to release ITCs upon hydrolysis of their CSLs content.The process of releasing ITCs from GSL molecules could be improved by ( 1) selecting high-ITC-content Brassica varieties (i.e., turnip variety TC), (2) releasing more GSLs by increasing plant tissue cellular disruption, and (3) maintaining good soil moisture to facilitate the breakdown of GSLs and release ITCs breakdown products.Concentrations of soluble sugars (3894 µg g −1 fresh turnip tissue) and total phenols (610 µg g −1 fresh turnip tissue) were greater in variety TC compared to varieties SQR and PTWG, whereas no significant differences in vitamin C content were found among the three varieties (Figure 6).Soil quality and composition of plants depend on soil biology, where microorganisms via organic matter decomposition, nutrient cycling, and enzymatic activity play an important role in soil fertility and plant composition.The impact of biochar on the activity of soil enzymes was reported by Antonious et al. (2020) [51].During the preparation of biochar, the thermal preparation pyrolysis process increases heavy metals concentration in biochar.Heavy metals impact soil microorganisms and their secreting enzymes.For example, Zn inhibits soil urease activity [52] and lowers the concentrations of GSLs observed in plants with higher foliar Cd concentrations, whereas Ni in plants induced GSLs at elevated levels [53].In contrast, several studies did not support biochar's impact on soil productivity and nutritional crop composition.Investigators reported controversy in their findings, with biochar having positive, negative, or insignificant effects on soil microbial communities depending on the biochar source and soil types [54][55][56].
Table 1 listed the activity of urease, invertase, acid, and alkaline phosphatase in each of the animal manures used in growing turnips, arugula, and mustard plants.Soil enzymes as bioindicators of soil health had been investigated [57,58].Antonious et al. [51] reported no increase in soil alkaline phosphatase activity with biochar addition to CM. Biochar added to native soil reduced the activity of soil alkaline phosphatase by 41% more than the control treatment.Trace metal contamination removes some microorganisms and multiplies others, resulting in changes in soil quality and functionality.For example, alkaline phosphatase activity is significantly inhibited with Cd [52].Similarly, the breakdown of GSLs into ITC and glucose is dependent on the activity of thioglucosidase.
As described earlier, using Brassica crops as green manures reduces soilborne pests and pathogens such as Rhizoctonia solani, and Sclerotinia sclerotiorum, the two most damaging soilborne pathogens [59].Brassica plants are well adapted in Kentucky and other states and hence can be used for pest control either by spreading dried Brassica plant powder or using green manure by incorporating plants into the soil to release ITCs upon hydrolysis of their CSLs content.The process of releasing ITCs from GSL molecules could be improved by (1) selecting high-ITC-content Brassica varieties (i.e., turnip variety TC), (2) releasing more GSLs by increasing plant tissue cellular disruption, and (3) maintaining good soil moisture to facilitate the breakdown of GSLs and release ITCs breakdown products.
Concentrations of soluble sugars (3894 µg g −1 fresh turnip tissue) and total phenols (610 µg g −1 fresh turnip tissue) were greater in variety TC compared to varieties SQR and PTWG, whereas no significant differences in vitamin C content were found among the three varieties (Figure 6).Concentrations of soluble sugars (3894 µg g −1 fresh turnip tissue) and total phenols (610 µg g −1 fresh turnip tissue) were greater in variety TC compared to varieties SQR and PTWG, whereas no significant differences in vitamin C content were found among the three varieties (Figure 6).Phytochemicals in plants (GSLs, vitamin C, phenols, etc.) have antioxidant properties as important food quality features.Results revealed significant variability among phytochemicals in plant species and among plants grown under different animal manures.Ascorbic acid (vitamin C) and phenols concentrations in arugula were superior in plants grown in SS, CM, and HM amended soils in comparison to plants grown in no-manure (NM) control soil.The concentration of soluble sugars in arugula shoots (greens) revealed no significant differences among all the three animal manures tested, but greater than plants grown in NM control soil.Whereas SS and CM-amended soil increased total phenols in arugula compared to HM-amended soil (Figure 7A).Mustard grown in SS-, CM-, and HM-mixed soil contained the uppermost concentrations of ascorbic acid, total phenols, and soluble sugars (Figure 7B) compared to the NM control treatment.plants grown in NM control soil.Whereas SS and CM-amended soil increased total phenols in arugula compared to HM-amended soil (Figure 7A).Mustard grown in SS-, CM-, and HM-mixed soil contained the uppermost concentrations of ascorbic acid, total phenols, and soluble sugars (Figure 7B) compared to the NM control treatment.GSLs concentrations in mustard shoots were larger (1424 µg g −1 fresh shoot) compared to arugula shoots (1250 µg g −1 fresh shoot).This might be due to the greater proportion of the above-ground biomass of mustard related to arugula biomass.SS mixed with native soil elevated the concentration of GSLs in arugula and mustard shoots from 1006 and 927 µg −1 fresh tissue in NM native soil to 1250 and 1424 µg −1 fresh tissue, respectively (Figure 8), whereas GSLs in arugula and mustard shoots of plants grown in CM or HM amended soils were significantly lower compared to SS amended soil.Kleinwachter and Selmar [60] described a remarkable activity of the enzyme myrosinase (glucosidase) by ascorbic acid and in some situations, myrosinase is entirely inactive in the absence of ascorbic acid.GSLs concentrations in mustard shoots were larger (1424 µg g −1 fresh shoot) compared to arugula shoots (1250 µg g −1 fresh shoot).This might be due to the greater proportion of the above-ground biomass of mustard related to arugula biomass.SS mixed with native soil elevated the concentration of GSLs in arugula and mustard shoots from 1006 and 927 µg −1 fresh tissue in NM native soil to 1250 and 1424 µg −1 fresh tissue, respectively (Figure 8), whereas GSLs in arugula and mustard shoots of plants grown in CM or HM amended soils were significantly lower compared to SS amended soil.Kleinwachter and Selmar [60] described a remarkable activity of the enzyme myrosinase (glucosidase) by ascorbic acid and in some situations, myrosinase is entirely inactive in the absence of ascorbic acid.The greater content of ascorbic acid (vitamin C), phenols, and soluble sugars in arugula and mustard plants grown in animal manure-amended soil could be due to the higher synthesis of these water-soluble phytochemicals by Brassica leaves.It might also be due to improved absorption from the soil rhizosphere by the plant roots.This increase might also be due to increased soil organic matter and microbial activity after the addition of animal manures.SS, CM, and HM contain several enzyme substrates that activate either urease, invertase, and phosphatase, respectively, and other hydrolyzing enzymes.Regardless of crop type, Figure 9 also revealed a significant increase in ascorbic acid (vitamin c), total phenols, and sugars in plants grown in SS, CM, and HM compared to NM control soil.Accordingly, the pronounced increase in these three phytochemicals could be due to increased microbial activity and their enzyme excretions after mixing native soil with animal manures.Many reasons were reported in the literature review for this increase, but none were extensively investigated.Studies carried out by Antonious et al. [61] revealed that animal manure increased soil enzyme activity.Soil enzymes secreted by soil microorganisms also promote many soil processes such as the synthesis of humus substances and the breakdown of organic matter in soil and the release of nutrients to growing plants.The greater content of ascorbic acid (vitamin C), phenols, and soluble sugars in arugula and mustard plants grown in animal manure-amended soil could be due to the higher synthesis of these water-soluble phytochemicals by Brassica leaves.It might also be due to improved absorption from the soil rhizosphere by the plant roots.This increase might also be due to increased soil organic matter and microbial activity after the addition of animal manures.SS, CM, and HM contain several enzyme substrates that activate either urease, invertase, and phosphatase, respectively, and other hydrolyzing enzymes.Regardless of crop type, Figure 9 also revealed a significant increase in ascorbic acid (vitamin c), total phenols, and sugars in plants grown in SS, CM, and HM compared to NM control soil.Accordingly, the pronounced increase in these three phytochemicals could be due to increased microbial activity and their enzyme excretions after mixing native soil with animal manures.Many reasons were reported in the literature review for this increase, but none were extensively investigated.Studies carried out by Antonious et al. [61] revealed that animal manure increased soil enzyme activity.Soil enzymes secreted by soil microorganisms also promote many soil processes such as the synthesis of humus substances and the breakdown of organic matter in soil and the release of nutrients to growing plants.
Among the phytochemicals that provide health benefits in human nutrition are polyphenols, flavonoids, isoflavonoids, anthocyanins, phytoestrogens, terpenoids, carotenoids, vitamin C, limonoids, phytosterols, glucosinolates, isothiocyanates (ITCs), and fibers.However, some animal manures, such as SS, are sometimes associated with inorganic and organic toxic compounds, such as heavy metals and pesticides [62,63] that when incorporated into soil establish a pollution problem and consequently cause toxic effects to soil microorganisms, which reduces the nutrients available to plants.Accordingly, the monitoring of soil enzymes secreted by the variety of soil microorganisms and the antioxidant contents of plants grown in animal manure-amended soil is suggested when using animal dung for growing vegetables and other edible plants.

Conclusions
Our literature review revealed a lack of information on testing the impact of organic amendments on plants' phytochemical composition and their nutritional and antioxidant properties.Investigators have focused on the plant yield and soil's physical and chemical characteristics following the incorporation of organic and inorganic fertilizers with very little information on the plant's nutritional and antioxidant contents.The use of animal manure as fertilizer has properties that cannot be acquired from the use of synthetic inorganic fertilizers.Health benefits of Brassicaceae plants are attributed to glucosinolates (GSLs) vitamin C, and phenol compounds due to their antioxidant activity.The presence of microorganisms in animal manures facilitates the slow release of the three main plant nutrients, N, P, and K, from soil organic matter to growing plants.Animal manure contains algae, protozoa, fungi, and bacteria.These microorganisms' secrete a variety of hydrolyzing enzymes such as invertase, dehydrogenase, cellulase, amylase, urease, and acid and alkaline phosphatase capable of degrading hazardous chemicals and of the mineralization of organic compounds in soil systems resulting in the release of nutrients for plant uptake.On average and regardless of the turnip variety tested, turnip shoot, root, and plant weight gained from chicken manure (CM)-amended soil was significantly (P ≤ 0.05) greater (490, 303, and 793 g, respectively) compared to the yield obtained from inorganic fertilizers (422, 267, and 689 g, respectively).
Results revealed that phytochemical concentrations varied significantly among plant species and among plants of the same species cultivated under different animal manures.Ascorbic acid (vitamin C) and phenol concentrations in arugula were greater in plants grown in SS-, CM-, and HM-amended soils compared to plants grown in the control treatment.Animal manures used as organic amendments are exceptional fertilizers.However, there is an emerging concern regarding the impact of endocrine-disrupting compounds (EDCs) in municipal sewage sludge (SS), chicken manure (CM), and horse manure (HM).Most livestock raised worldwide are raised in large-scale concentrated animal-feeding operations, which require the use of antibiotics, and inorganic and organic compounds (heavy metals, hormones, antibiotics, and pesticides) that when incorporated into soil might constitute a pollution problem and therefore might impact the activity of soil microorganisms and their enzymatic secretions.The increasing consumer awareness of the value of vegetables in the human diet requires continuous investigation and monitoring of plants' phytochemicals composition and the growth factors that impact the antioxidant content of the different varieties of edible plants grown under different agricultural soil management practices such as animal manures used in land farming as an alternative to inorganic fertilizers.

Figure 1 .
Figure 1.Chemical structure of glucosinolate (GSL) molecule as potassium salt.Note that every GSL molecule has a central carbon (C) atom bonded to a sulfur atom in the glucose group and via a N atom to an O-sulfated thiohydroximate.The side group R differs among diverse GSLs' structures and biological activities.

Figure 1 .
Figure 1.Chemical structure of glucosinolate (GSL) molecule as potassium salt.Note that every GSL molecule has a central carbon (C) atom bonded to a sulfur atom in the glucose group and via a N atom to an O-sulfated thiohydroximate.The side group R differs among diverse GSLs' structures and biological activities.

Figure 2 .
Figure 2. Overall average weight (n = 3) ± standard deviation of turnip root (A), shoot (B), and weight of turnip plants (C) collected at harvest under seven soil treatments, regardless of turnip variety.Statistical differences were accompanied among seven soil treatments for each plant part or total weight.Bars with dissimilar letter(s) indicate significant variances at p ≤ 0.05 using Duncan's multiple range test in SAS [26].

Figure 2 .
Figure 2. Overall average weight (n = 3) ± standard deviation of turnip root (A), shoot (B), and weight of turnip plants (C) collected at harvest under seven soil treatments, regardless of turnip variety.Statistical differences were accompanied among seven soil treatments for each plant part or total weight.Bars with dissimilar letter(s) indicate significant variances at p ≤ 0.05 using Duncan's multiple range test in SAS [26].

Figure 3 .
Figure 3. Concentrations of glucosinolates in roots, shoots, and whole plant of three varieties of turnips, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG) grown under field conditions, regardless of soil amendment type.Statistical analysis was conducted among three varieties using Duncan's multiple range test in SAS [26].Values with different letters reveal statistical differences at p ≤ 0.05.

Figure 3 .
Figure 3. Concentrations of glucosinolates in roots, shoots, and whole plant of three varieties of turnips, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG) grown under field conditions, regardless of soil amendment type.Statistical analysis was conducted among three varieties using Duncan's multiple range test in SAS [26].Values with different letters reveal statistical differences at p ≤ 0.05.

Figure 4 .
Figure 4.The concentration of glucosinolates among three commercial varieties of turnip, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG) grown in biochar-and no-biochar-amended soil, regardless of soil amendments.Statistical analysis was conducted among three varieties using Duncan's multiple range test in SAS [26].Values with different letters indicate statistical differences at p ≤ 0.05.

Figure 4 . 19 Figure 5 .
Figure 4.The concentration of glucosinolates among three commercial varieties of turnip, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG) grown in biocharand no-biochar-amended soil, regardless of soil amendments.Statistical analysis was conducted among three varieties using Duncan's multiple range test in SAS [26].Values with different letters indicate statistical differences at p ≤ 0.05.Appl.Sci.2023, 13, x FOR PEER REVIEW 12 of 19

Figure 5 .
Figure 5.The overall concentration of glucosinolates in turnip, Brassica rapa grown under soil treatments amended and not amended with biochar, regardless of crop varieties.Statistical analysis was performed among crop varieties via Duncan's multiple range test in SAS [26].Values with the same letter(s) indicate no significant differences (p ≥ 0.05).

19 Figure 5 .
Figure 5.The overall concentration of glucosinolates in turnip, Brassica rapa grown under soil treatments amended and not amended with biochar, regardless of crop varieties.Statistical analysis was performed among crop varieties via Duncan's multiple range test in SAS [26].Values with the same letter(s) indicate no significant differences (p ≥ 0.05).

Figure 6 .
Figure 6.Concentrations of soluble sugars, AA (vitamin C), and total phenols in three commercial varieties of turnip, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG), irrespective of treatments.Statistical analysis was performed among turnip varieties via Duncan's multiple range test in SAS [26].Values with the same letter(s) indicate significant differences (p ≥ 0.05).Phytochemicals in plants (GSLs, vitamin C, phenols, etc.) have antioxidant properties as important food quality features.Results revealed significant variability among phytochemicals in plant species and among plants grown under different animal manures.Ascorbic acid (vitamin C) and phenols concentrations in arugula were superior in plants

Figure 6 .
Figure 6.Concentrations of soluble sugars, AA (vitamin C), and total phenols in three commercial varieties of turnip, Brassica rapa Tokyo Cross (TC), Scarlet Queen Red SQR), and Purple Top White Globe (PTWG), irrespective of treatments.Statistical analysis was performed among turnip varieties via Duncan's multiple range test in SAS [26].Values with the same letter(s) indicate significant differences (p ≥ 0.05).

Figure 7 .
Figure 7. Concentrations of soluble sugars, ascorbic acid (AA), total phenols ± std.error in arugula (A) and mustard (B) grown under different soil management practices.Statistical analysis comparisons were carried out among four soil treatments of each parameter.Statistical analyses were done via Duncan's multiple range test in SAS [26].Values with the same letter are not significantly different (p ≥ 0.05).

Figure 7 .
Figure 7. Concentrations of soluble sugars, ascorbic acid (AA), total phenols ± std.error in arugula (A) and mustard (B) grown under different soil management practices.Statistical analysis comparisons were carried out among four soil treatments of each parameter.Statistical analyses were done via Duncan's multiple range test in SAS [26].Values with the same letter are not significantly different (p ≥ 0.05).

Figure 8 .
Figure 8. Overall concentrations of glucosinolates ± std.error in arugula and mustard cultivated under four soil management practices (sewage sludge, SS; chicken manure, CM; horse manure, HM; and no manure, NM).Statistical comparisons were performed among four soil treatments.Bars associated with the same letter are not significantly different (p ≥ 0.05) using Duncan's multiple range test.

Figure 8 .
Figure 8. Overall concentrations of glucosinolates ± std.error in arugula and mustard cultivated under four soil management practices (sewage sludge, SS; chicken manure, CM; horse manure, HM; and no manure, NM).Statistical comparisons were performed among four soil treatments.Bars associated with the same letter are not significantly different (p ≥ 0.05) using Duncan's multiple range test.

Figure 9 .
Figure 9. Overall concentrations of soluble sugars, ascorbic acid, and phenols ± std.error under four soil management practices (sewage sludge, SS; chicken manure, CM; horse manure, HM; and no manure, NM), regardless of crop type.Statistical analysis was performed among four soil management practices.Values with the same letter are not significantly different (p ≥ 0.05).

Table 1 .
Properties and composition of soil amended with animal manures used for growing turnips, arugula, and mustard under field conditions (Lexington, Kentucky, USA).

Table 1 .
Properties and composition of soil amended with animal manures used for growing turnips, arugula, and mustard under field conditions (Lexington, Kentucky, USA).
The value represents averages of three replicates ± standard error.Statistical analyses were conducted among the glucosinolate concentrations in the shoots, roots, and total amount in each of the three turnips varieties.Values accompanied by a different letter(s) in each plant part of total plant weight are statistically different at p ≤ 0.05.Appl.Sci.2023, 13, x FOR PEER REVIEW 10 of 19