Effect of Organic and Bio-Fertilization on Fruit Yield, Bioactive Constituents, and Estragole Content in Fennel Fruits

Abstract

Fennel fruits (Foeniculum vulgare Mill.) represent one of the plant-based natural spices. This study aims to improve the fruit yield and essential oil (EO) quality by reducing the undesirable component, estragole, under different fertilizer treatments. The fertilizers included chemical nitrogen, phosphorus, and potassium (NPK), and also the organic additive, rabbit manure (RM). For bio-fertilization, plants were inoculated with a mixture of N-fixing bacteria, and P- and K-solubilizing bacteria with/without vesicular-arbuscular mycorrhizal fungi. The results showed that fruit and EO yield parameters, total phenolic content (TPC), total flavonoid content (TFC), and DPPH scavenging activity of fruit extracts were enhanced by fertilizer treatments in both growing seasons. NPK at 150% of the recommended dose (NPK150) and RM at 60 m³/fed (RM60) recorded the highest values of plant height, umbel number/plant, 100-fruit weight, fruit yield, chlorophyll, carbohydrates, N and P content, EO content, and yield. TPC and TFC were enhanced by using biological fertilizers. DPPH scavenging activity was higher in organically and biologically fertilized fennel. The GC-MS analyses of EO revealed higher contents of the desirable trans-anethole in the organically and chemically fertilized fennel. However, the highest proportion of estragole, the undesirable compound, was recorded for NPK150 and unfertilized plants. On the contrary, increases in the EO content and yield of fennel fruits were achieved by RM along with a reduction in estragole, enhancement in trans-anethole, and increments in other favorable compounds such as fenchone and limonene. In addition, the inhibition of estragole formation was recorded with bio-fertilizers, which also increased the trans-anethole content. Furthermore, the trans-anethole/estragole ratio was significantly higher with the application of organic and bio-fertilization. Hence, organic and bio-fertilizer resources can produce high-quality fennel fruit and EO. The reduction in the use of chemical fertilizers can help to reduce environmental pollution.

1. Introduction

Fennel (Foeniculum vulgare Mill.) is a medicinal, aromatic herbaceous plant of the family Apiaceae native to the Mediterranean region. The fruits, foliage, as well as bulbs of fennel, are used medicinally and as vegetables worldwide. Fennel fruits (also seeds) are known for their essential oil (EO). Biopharmacological studies of the phytochemicals present in fennel fruit proved their efficacy as an antioxidant, anticancer, antibacterial, antifungal, antithrombotic, anti-inflammatory, chemopreventive, hepatoprotective, memory-enhancing, anti-aging, antidiabetic, and insecticide [1,2].

Fruits and seeds are the essential oil-producing organs in Apiaceae plants. The content of EO in fennel fruits ranges from 1 to 4% depending on the cultivar, fruit maturity, climate conditions, and agricultural practices (e.g., irrigation, fertilization) [3,4,5]. Trans-anethole (25–85%), estragole (2–50%), fenchone (0.60–28%), and limonene (0.90–18%) are the main constituents of fennel EO that determine the quality of the oil [6,7,8,9,10,11]. In addition, trans-anethole has been reported to have anti-inflammatory, anti-obesity, anti-cancer, anti-diabetic, and hepatoprotective activities [12,13,14].

Estragole is a phenylpropanoid constituent of the EOs of several aromatic plants such as F. vulgare, Artemisia dracunculus, Ocimum basilicum, Ravensara anisate, Agastache rugosa, and Pimpinella anisum. Estragole may act as a genotoxic hepatocarcinogen in rodent livers by producing DNA adducts responsible for the genotoxicity of estragole [15,16]. Hence, the quality of the EO decreases as the proportion of estragole increases, causing a decrease in Egyptian exports of fennel oil [8,17]. Reducing the proportion of estragole in the EO is important as fennel cannot be free of it due to the biosynthetic association with anethole formation [6]. Recent reports indicated that the level of estragole in the volatile oil might change in response to treatment with nutrients of different origins [3,9,11,18,19]. However, minimal studies compared the components of the EO produced from organically, biologically, or chemically fertilized fennel. On the other side, the benefit of the increase in the contents of phenolic and flavonoid compounds in fennel fruits becomes significant when the levels of the carcinogenic estragole and other harmful substances in food increase. Phenols and flavonoids act as natural antioxidants and protect human cells from harmful molecules such as estragole [20]. The content of these bioactive antioxidants can change dramatically between fennel ecotypes [21] as well as nutrient treatments [19,22].

Previous investigations showed that adding the macronutrients nitrogen (N), phosphorus (P), and potassium (K) to cultivated plants increased crop productivity and the content of various bioactive constituents such as EO, vitamin C, phenols, and flavonoids in the apiaceous plants [23,24,25]. Chemical fertilizers are a primary and quick-release source of macro and micronutrients for various crops. However, chemical fertilization has been proven to have negative environmental effects, increasing the cost of environmental protection [26,27], and assessment of nutrient uptake and recovery is crucial for crop yield, sustainability, and environmental aspects [28]. Therefore, the rational use of chemical fertilizers is crucial for sustainable agriculture to reduce the adverse impacts of agriculture on the environment [29].

In an organic farming system, the use of synthetic chemical fertilizers and pesticides is largely avoided or excluded, and organic or bio-fertilizers are used as an alternative in providing nutrients to plants and sustaining agricultural ecosystems [30,31]. In a bio-fertilization system, preparations containing specialized microorganisms are added to the soil, whereby its fertility improves, the availability and uptake of nutrients in it increases, and accordingly, the productivity of crops is improved. Microorganisms in this bio-system fix N, dissolve soil-P and soil-K, produce molecules that promote plant growth, and protect it from pathogens and biotic and abiotic stresses [30,32,33]. Natural organic additives such as manure and processed bio-fertilizers represent natural sources of essential plant nutrients in organic farming. There are many benefits of organic fertilization, including improving soil health, maintaining soil fertility because it helps to compensate for the loss of organic matter, reducing environmental damage without reducing productivity, and achieving sustainable agricultural production [31], in addition to alleviating excessive fertilization and the risks of groundwater pollution [34].

The research on adding organic manure and microbes to the soil is an important area of applied agricultural research in sustainable development, especially if this is associated with reducing the content of some undesirable plant substances such as estragole [19]. There is a need to achieve economic productivity of the fruit and EO crop of fennel accompanied by high quality, such as low estragole content and an increase in anethole, pharmaceutical active ingredients, and antioxidant activity. Very few studies have evaluated the EO constituents in organically, biologically, or chemically fertilized fennel fruits. Therefore, the current study aims at comparing the productivity levels and quality parameters of fennel fruits and their EOs under bio-, organic, and chemical fertilization and reducing the estragole proportion in fennel fruit EO.

2. Materials and Methods

2.1. Experiment Site and Design

Two field studies were conducted during the two successive seasons, 2019/2020 and 2020/2021, at a private farm located in El-Santa, Gharbia, Egypt (30°44′23.3″ N 31°09′02.1″ E). The experiment took place in a site that had been without cultivation for two years, and grassy crops such as wheat and corn had been cultivated prior to that. Before planting, the experimental soil was analyzed physically and chemically. The soil used was 58.50% clay, 13.84% coarse sand, 16.45% silt, 9.10% fine sand, pH 6.10, E.C. 6.25 mM, and contained 3.36% N, 4.42% P, 5.40% K, 1.50% Na, 2.20% Mg, 4.30% Ca, 3.20% SO₄, 4.55% Cl, 3.30% HCO₃, and 20.00% SP.

Fruits of sweet fennel (F. vulgare cv “Florence”) were sourced from the Agricultural Research Center, Giza, Egypt. During the first week of November in the 2019/2020 and 2020/2021 seasons, the fruits were sown in nursery beds. After 45 days, seedlings were transplanted to the open field in plots (1.5 × 2 m) of 3 rows of 50 × 50 cm. A complete randomized block design was employed for the experiment design. Each treatment consisted of 3 replicates (plots), and each replicate contained 12 plants. In the second season, the location of the experiment was changed to very close plots within the same field to avoid the effect of fertilizer residues from the first season.

2.2. Treatments

For bio-fertilization, N-fixing bacteria (Azospirillum brasilense) (Az), P-solubilizing bacteria (Bacillus megaterium var. phosphaticum) (Bm), and K-solubilizing bacteria (B. circulans) (Bc) were obtained from Soil Microbiology Laboratory, National Research Center (NRC), Egypt. The cultures of bacterial strains were grown following Abd-el-Malek and Ishac [35] and Dobereiner et al. [36]. A total of three fungal strains (Glomus etunicatum, G. intraradices, and G. fasciculatum), known as vesicular arbuscular mycorrhizal (VAM) fungi, were inoculated in the soil after transplanting (200 VAM spores/plant). Plants were inoculated separately after transplanting with a 3 mL mixture of bacterial suspension alone (Az + Bm + Bc) or with fungal strains (Az + Bm + Bc + VAM).

Rabbit manure (RM) was applied as organic fertilizer. The pH value of RM was 5.45, E.C. 6.15 mM, and contained 91 g/kg N, 22.1 g/kg P, 11.9 g/kg K, 130 mg/kg Fe, 151 mg/kg Mn, 85 mg/kg Cu, 95 mg/kg Zn, and the C/N ratio was 8.3/1. Two doses of 30 and 60 m³/fed (RM30 and RM60, respectively) of organic manure were applied before planting.

The sources of NPK fertilizers were (NH₄)₂SO₄ (20.6% N) 100 kg/fed (Feddan is an area unit common in Egypt that is equivalent to 0.42 Hectare), calcium superphosphate (15.5% P₂O₅) 50 kg/fed, and K₂SO₄ (48% K₂O) 50 kg/fed, respectively, as the recommended dose (RD). NPK chemical fertilization was added as two levels of 100 and 150% of the RD (NPK100 and NPK150, respectively). N and K fertilizers were amended to the soil on days 15, 30, and 45 after transplanting. During soil preparation, calcium superphosphate was applied once before planting. The unfertilized plot was considered as a control.

A level of 50–75% of field capacity was maintained for soil moisture, and weeds, pests, and diseases were controlled following the practices following the recommendations of the Ministry of Agriculture and Land Reclamation of Egypt.

2.3. Measurements

Fennel fruits were harvested at the end of the growing seasons, at 130–140 days after transplanting, when the fruits were yellowish in color. The recorded parameters were plant height (cm), umbel number/plant, the weight of 100 fruits (g), fruit yield g/plant, and fruit yield t/fed. Chlorophyll content (mg/g FW) was determined spectrophotometrically in the acetone leaf extract as described by Dere et al. [37]. The content of total carbohydrates in the mixture of dried stems and leaves was estimated according to Dubois et al. [38] by following the phenol-sulfuric acid method. Nitrogen, phosphorus, and potassium (%) were also determined in the mixture of dried stems and leaves. N was estimated by following the protocol presented in AOAC [39], while P and K were assayed according to Cottenie et al. [40].

2.3.1. Determination of Total Phenolics

Dried fruit powder (100 mg) was immersed in ethanol (5 mL; 95%) and shaken for 4 h, then placed under 0 °C for 48 h. Then, the tubes containing ethanol and fruit powder were homogenized and centrifuged for 10 min. Total phenolic content (TPC) was assayed in the supernatants by following the Folin-Ciocalteu method according to Singleton and Rossi [41] and Elateeq et al. [42]. One ml of the supernatant was then mixed with 1 mL of 95% ethyl alcohol, distilled water (5 mL), and 0.5 mL of 50% Folin-Ciocalteu reagent. Next, after 5 min, 1 mL of 5% Na₂CO₃ was added and stirred well. The resultant solution was incubated at 25 °C for an hour. At the wavelength of 725 nm, the absorbance was measured against a blank using a Jenway 6800 UV/Vis spectrophotometer (Bibby Scientific Ltd., Staffordshire, UK). Dilutions of gallic acid were read to draw the standard curve. TPC was expressed as mg gallic acid equivalents (GAE)/g dry weight (DW) of fennel fruits.

2.3.2. Determination of Total Flavonoids

Fennel fruit powder (100 mg DW) was mixed with ethanol 95% (5 mL), shaken for 4 h, and then placed for 24 h at room temperature. Total flavonoid content (TFC) was determined after filtration using the aluminum chloride (AlCl₃) colorimetric method as described by Chang et al. [43] and Elateeq et al. [42]. Ethanol extract (0.5 mL) was mixed with 1.5 mL of ethanol (95%), 0.1 mL of AlCl₃ (10%), 0.1 mL of potassium acetate (1 M), and 2.8 mL of distilled water. The mixture was incubated at 25 °C for 30 min. The absorbance was measured at 415 nm against a blank using a JENWAY 6800 UV/Vis spectrophotometer. The calibration curve was established using quercetin dilutions. Then, TFC was expressed as mg QE (quercetin equivalents) per g DW of fennel fruits.

2.3.3. Determination of Free Radical Scavenging Activity

The antioxidant activity of fennel fruit extracts was estimated using the DPPH (2,2-diphenyl-1-picrylhydrazyl) test as reported by Elateeq et al. [44]. The ethanol extract (0.7 mL) of the fruit samples was mixed with 3 mL of 200 µM DPPH ethyl alcohol solution. The mixture was shaken and incubated in the dark at 25 °C for 30 min. The absorbance was recorded at 517 nm using a Jenway 6800 UV/Vis spectrophotometer. Ascorbic acid was used as a positive control. The DPPH radical scavenging activity (%) was calculated by the following formula:

$$\mathrm{DPPH} \mathrm{activity}\left(\mathrm{\%}\right)=\frac{\mathrm{A} \mathrm{control}-\mathrm{A}\mathrm{sample}}{\mathrm{A} \mathrm{control}}\times 100$$

where A sample is the absorbance of fennel fruit extract or ascorbic acid solution mixed with DPPH solution, and A control is the absorbance of the DPPH solution with a 0.7 mL ethanol (95%) free sample.

2.3.4. Extraction of Essential Oil

The EO was extracted from fennel fruits by the hydro-distillation method using Clevenger-type apparatus. The dried fruits (100 g) were placed in 1 L of distilled water in a 2-L round bottom flask and boiled for 3 h so that all EO was extracted. The collected EO was dried chemically using anhydrous sodium sulfate. The extracted EO was kept at 4–5 °C for further analysis. The EO is expressed as a relative percentage (v/w).

2.3.5. GC-MS Analysis of Essential Oil

The GC-MS (gas chromatography-mass spectrometry) analysis of the EO samples was carried out at the Department of Medicinal and Aromatic Plants Research, National Research Center, Egypt. TRACE GC Ultra Gas Chromatographs (Thermo Scientific Corp., San Jose, CA, USA), coupled with a Thermo mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer) were used in this study. A TG-WAX MS column (30 m × 0.25 mm i.d., 0.25 m film thickness) was installed in the GC-MS system. The following temperature program was used for the analyses: 60 °C for 1 min; 3.0 °C/min increase to 240 °C; maintained for 1 min. Helium gas was used as the carrier gas at a flow rate of 1.0 mL/min and a split ratio of 1:10. At 240 °C, the injector and detector were maintained. Always, 1 L of the diluted samples (1:50 hexane, v/v) of the mixes were injected. By utilizing a spectral range of m/z 40–450 and electron ionization (EI) at 70 eV, mass spectra were produced. The analytical technique of mass spectra was used to identify the majority of substances (authentic chemicals, Wiley spectral library collection, and the NSIT library).

2.4. The Statistical Analysis

A complete randomized block design was used in all essays. The statistical analysis was conducted using the ANOVA method (Analysis of Variance). This is followed by DMRT (Duncan’s Multiple Range Test) [45] at p ≤ 0.05 using version 6.4 of CoStat software (CoHort software, Monterey, CA, USA), according to Snedecor and Cochran [46].

3. Results

3.1. Effect of Different Fertilizers on Plant Height and Umbel Number of Fennel Plant

Results obtained from the growing seasons (2019/2020 and 2020/2021) showed that fertilizer types and levels significantly affected (p ≤ 0.05) the height of fennel plants and umbel number/plant (

Table 1. Effect of bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM), organic (RM30 and RM60), and chemical fertilizers (NPK100 and NPK150) on plant height (cm) and umbel number/plant of fennel plants during seasons 2019/2020 (1st season) and 2020/2021 (2nd season).

Treatments	Plant Height (cm)		Umbel Number/Plant
	1st Season	2nd Season	1st Season	2nd Season
Control	117.78 e	126.82 d	19.40 g	22.93 g
Az + Bm + Bc	138.10 d	132.57 d	26.40 f	28.73 f
Az + Bm + Bc + VAM	139.20 d	144.02 c	32.83 e	35.27 e
RM30	146.17 c	152.48 c	39.33 d	41.47 d
RM60	160.31 b	166.92 ab	47.03 b	48.30 b
NPK100	151.77 c	161.51 b	42.73 c	44.77 c
NPK150	167.12 a	173.48 a	50.47 a	51.30 a

Az: N-fixing bacteria (Azospirillum brasilense), Bm: P-solubilizing bacteria (Bacillus megaterium var. phosphaticum), Bc: K-solubilizing bacteria (B. circulans), VAM: vesicular arbuscular mycorrhiza (Glomus etunicatum, G. intraradices, and G. fasciculatum). RM30 and RM60 are rabbit manure at 30 and 60 m³/fed, respectively. NPK100 and NPK150 are NPK at 100% and 150% of recommended dose, respectively. Unfertilized plants were considered as control. Mean values with different letters in the column are statistically different according to DMRT (p ≤ 0.05).

). Fennel plants that received organic or chemical fertilizers were the tallest and produced more umbels compared with bio-fertilized and unfertilized (control) plants. The highest significant values of plant height (167.12 and 173.48 cm) and umbel number/plant (50.47 and 51.30 umbels) were observed in fennel plants fertilized with NPK150, followed by RM60; 160.31 and 166.92 cm for plant height, and 47.03 and 48.30 umbel number/plant, in the first and second seasons, respectively. Both bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM) increased plant height and umbel number/plant compared with the control.

3.2. Effect of Different Fertilizers on Total Chlorophyll and Total Carbohydrate Contents of Fennel Plants

Applying chemical, organic, and bio-fertilization exhibited a significant accumulation (p ≤ 0.05) of chlorophyll in the experimental seasons (Figure 1A) compared to unfertilized plants. Higher rates of organic manure (RM60) and NPK (NPK150) recorded the highest significant chlorophyll content in both seasons. The percentage of total carbohydrates in fennel leaves and stems also increased significantly in response to fertilizers (Figure 1B). The topmost carbohydrate content was observed for RM60 (61.59 and 69.01%) and NPK150 (67.18 and 71.75%) in the first and second seasons, respectively, with non-significant differences between each other in the second season. Inoculation of fennel plants with Az + Bm + Bc + VAM microbes produced a higher carbohydrate content than Az + Bm + Bc.

3.3. Effect of Different Fertilizers on N, P, and K Contents of Fennel Plants

Applying NPK fertilizer at 150% of RD (NPK150) led to significantly (p ≤ 0.05) higher N content; 4.14% and 4.37% in the first and second seasons, respectively, followed by RM60 and NPK100 (Figure 2A). On the contrary, organic fertilizer (RM30 and RM60) was superior to NPK in increasing the P content (Figure 2B). Individual inoculation of Az + Bm + Bc did not affect the content of N, while the mixture of Az + Bm + Bc + VAM increased the content of N, P, and K compared to control and Az + Bm + Bc (Figure 2A–C). The potassium content of fennel fertilized organically and biologically was better than those that received chemical fertilizers and the control (Figure 2C). The maximum significant content of K was recorded for the treatment RM60 (4.32 and 4.66% in the two seasons, respectively).

3.4. Effect of Different Fertilizers on Weight and Yield of Fennel Fruits

The data displayed in

Table 2. Effect of bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM), organic (RM30 and RM60), and chemical fertilizers (NPK100 and NPK150) on weight of 100 fruits (g) and fruit yield (g/plant and t/fed) of fennel plants during seasons 2019/2020 (1st season) and 2020/2021 (2nd season).

Treatments	Weight of 100 Fruits (g)		Fruit Yield (g/Plant)		Fruit Yield (T/Fed)
	1st Season	2nd Season	1st Season	2nd Season	1st Season	2nd Season
Control	1.11 e	1.23 e	56.98 f	54.43 f	0.93 f	0.89 f
Az + Bm + Bc	1.31 e	1.80 d	64.45 e	68.30 e	1.05 e	1.11 e
Az + Bm + Bc + VAM	1.53 d	1.89 d	68.06 d	72.26 d	1.11 d	1.18 d
RM30	2.18 c	2.23 c	74.11 c	78.96 c	1.20 c	1.29 c
RM60	2.85 a	3.01 a	86.15 a	84.65 b	1.41 a	1.38 b
NPK100	2.31 c	2.36 c	77.85 b	82.21 b	1.27 b	1.34 b
NPK150	2.58 b	2.78 b	88.09 a	92.30 a	1.44 a	1.50 a

Az: N-fixing bacteria (Azospirillum brasilense), Bm: P-solubilizing bacteria (Bacillus megaterium var. phosphaticum), Bc: K-solubilizing bacteria (B. circulans), VAM: vesicular arbuscular mycorrhiza (Glomus etunicatum, G. intraradices, and G. fasciculatum). RM30 and RM60 are rabbit manure at 30 and 60 m³/fed, respectively. NPK100 and NPK150 are NPK at 100% and 150% of recommended dose, respectively. Unfertilized plants were considered as control. Mean values with different letters in the column are statistically different according to DMRT (p ≤ 0.05).

show that the weight of 100 fruits and fruit yield responded significantly (p ≤ 0.05) to fertilization, especially the forms of organic RM and NPK. In both seasons, RM60 achieved the highest significant 100-fruit weight, which reached 2.85 and 3.01 g/100 fruits in the 2019/2020 and 2020/2021 seasons, respectively, representing 2.57 and 2.45-fold higher than unfertilized plants. NPK150 came in the order of significance after RM60. Az + Bm + Bc + VAM significantly increased the 100-fruit weight, while Az + Bm + Bc enhanced the 100-fruit weight, as compared to the control in the second season.

In general, the fruit yield was higher in fertilized fennel than in the unfertilized control by about 1.13–1.70-fold during the growing seasons (Table 2). The yield of fruits in fennel that received organic manure and NPK was significantly higher than that inoculated with the plant growth-promoting microbes. In this context, the highest significant amount of fennel fruits was harvested from plants fertilized with NPK150 (88.09 and 92.30 g/plant; 1.44 and 1.50 t/fed), in the first and second seasons, respectively, and RM60 (86.15 g/plant; 1.41 t/fed) in the first season, representing a 1.55, 1.70, and 1.51-fold increase than in unfertilized fennel. Both bio-fertilizers significantly increased the fruit yield in both seasons. Fennel inoculated with Az + Bm + Bc achieved 64.45 g and 68.30 g fruit/plant (1.05 and 1.11 t/fed) compared to the control (56.98 and 54.43 g/plant; 0.93 and 0.89 t/fed), in the first and second seasons, respectively. This fruit yield was significantly increased to 68.06 and 72.26 g/plant (1.11 and 1.18 t/fed) in the growing seasons, respectively, when the bacterial mixture was combined with VAM (Az + Bm + Bc + VAM).

3.5. Effect of Different Fertilizers on TPC and TFC of Fennel Fruit

Fennel plants inoculated with Az + Bm + Bc achieved the topmost significant value (p ≤ 0.05) of TPC (16.87 and 15.95 mg GAE/g DW) and TFC (16.64 and 18.68 mg QE/g DW) in the fruit extract, which was higher than the control by 132.52% and 120.29% for TPC, and by 169.28% and 194.58% for TFC in the first and second seasons, respectively (

Table 3. Effect of bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM), organic (RM30 and RM60), and chemical fertilizers (NPK100 and NPK150) on total phenolic content (TPC) (mg GAE/g DW), and total flavonoid content (TFC) (mg QE/g DW) in fruit extract, fruit essential oil (EO) (%), and EO yield (mL/plant and L/fed) of fennel plants during seasons 2019/2020 (1st season) and 2020/2021 (2nd season).

Treatments	TPC (mg GAE/g DW)		TFC (mg QE/g DW)		Fruit EO (%)		EO Yield (mL/Plant)		EO Yield (L/Fed)
	1st Season	2nd Season	1st Season	2nd Season	1st Season	2nd Season	1st Season	2nd season	1st Season	2nd Season
Control	12.73 e	13.26 c	9.83 e	9.60 d	2.60 e	2.72 e	1.48 g	1.50 g	24.10 g	24.11 g
Az + Bm + Bc	16.87 a	15.95 a	16.64 a	18.68 a	3.02 d	3.19 d	1.95 f	2.18 f	31.71 f	35.53 f
Az + Bm + Bc + VAM	15.77 b	14.85 b	14.89 b	15.86 b	3.21 c	3.31 c	2.18 e	2.39 e	35.56 e	38.97 e
RM30	15.35 b	14.77 b	13.77 bc	14.56 b	3.42 b	3.51 b	2.54 d	2.77 d	41.33 d	45.11 d
RM60	14.95 bc	14.53 b	12.94 c	11.99 c	3.63 a	3.70 a	3.13 b	3.13 b	50.96 b	51.00 b
NPK100	14.43 cd	14.74 b	11.56 d	11.93 c	3.49 b	3.55 b	2.72 c	2.92 c	44.31 c	47.61 c
NPK150	13.89 d	14.14 b	9.87 e	10.64 cd	3.70 a	3.76 a	3.26 a	3.47 a	53.06 a	56.60 a

Az: N-fixing bacteria (Azospirillum brasilense), Bm: P-solubilizing bacteria (Bacillus megaterium var. phosphaticum), Bc: K-solubilizing bacteria (B. circulans), VAM: vesicular arbuscular mycorrhiza (Glomus etunicatum, G. intraradices, and G. fasciculatum). RM30 and RM60 are rabbit manure at 30 and 60 m³/fed, respectively. NPK100 and NPK150 are NPK at 100% and 150% of recommended dose, respectively. Unfertilized plants were considered as control. Mean values with different letters in the column are statistically different according to DMRT (p ≤ 0.05).

). Bio-fertilizer treatment in the form of Az + Bm + Bc + VAM and organic additives (RM30 and RM60) gave the same statistical values of TPC in both growing seasons. Moreover, the combination of VAM with Az + Bm + Bc decreased the TFC without significant differences with RM30. Compared with bio- and organic fertilization, TPC decreased in NPK150-fertilized fennel. However, this reduction was not statistically significant in the second season. Heavy addition of RM (RM60), as well as both levels of NPK, significantly decreased the TFC in the two growing seasons.

3.6. Effect of Different Fertilizers on EO Content and EO Yield of Fennel Fruit

The EO content in fennel fruits significantly responded to different fertilizers ranging from 3.02% to 3.76% compared to nonfertilized plants (2.60% to 2.72%) (Table 3). Fruits of plants that received NPK150 and RM60 contained the maximum significant EO; 3.70% and 3.76% for NPK150, and 3.63% and 3.70% for RM60, in the first and second seasons, respectively. These values represent 1.42, 1.38, 1.40, and 1.36 times of that of the control, respectively. The EO content was also significantly elevated with bio-fertilizer application and Az + Bm + Bc + VAM was better than Az + Bm + Bc.

The yield of EO (expressed as mL/plant or L/fed) was improved in all fertilized fennels, representing 1.32–2.31-fold higher than control. Data presented in Table 3 indicate that the highest significant (p ≤ 0.05) yield of EO (3.26 and 3.47 mL/plant; 53.06 and 56.60 L/fed) was gained from fennel plants that received NPK150 compared to the control (1.48 and 1.50 mL/plant; 24.10 and 24.11 L/fed) in the two growing seasons, respectively, followed by RM60. The combination of VAM and Az + Bm + Bc was more effective when compared to Az + Bm + Bc.

3.7. Effect of Different Fertilizers on EO Composition of Fennel Fruit

The GC-MS analysis of EO identified 19 compounds (accounting for 94.37–97.34% of the total compositions), among which trans-anethole represented the main component (22.25–26.75%) (

Table 4. Variations of essential oil (EO) components (%) of fennel fruits and trans-anethole/estragole ratio in response to bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM), organic (RM30 and RM60), and chemical fertilizers (NPK100 and NPK150).

No	EO Components	RT (min)	RI	Control	Az + Bm + Bc	Az + Bm + Bc + VAM	RM30	RM60	NPK100	NPK150
1	α-pinene	5.6	937	12.24 a	8.72 d	9.69 bc	9.85 b	9.93 b	7.57 e	8.79 cd
2	Camphene	7.9	955	0.11 d	0.41 c	0.55 bc	0.67 b	0.92 a	0.68 b	0.97 a
3	β-pinene	8.7	981	0.12 d	0.35 c	0.56 b	0.49 b	0.71 a	0.54 b	0.68 a
4	Phellandrene	10	1004	5.15 cd	5.70 bc	6.79 a	5.94 b	5.01 d	5.82 b	5.11 cd
5	Limonene	11.5	1030	10.22 bc	11.84 a	10.12 bc	9.54 c	9.94 bc	11.57 a	10.54 b
6	γ-terpinene	12.7	1060	0.94 c	0.59 cd	0.56 d	0.71 cd	0.79 cd	1.98 a	1.51 b
7	Sabinene	13.8	980	0.02 b	0.03 b	0.06 b	1.01 a	1.03 a	0.08 b	0.09 b
8	α-terpinene	14.3	1039	5.68 b	6.38 a	6.18 a	5.09 c	6.31 a	5.21 c	4.67 d
9	Fenchone	14.4	1075	2.09 d	2.68 b	2.31 c	2.41 c	2.84 a	2.91 a	2.78 ab
10	Myrcene	14.5	950	0.08 c	0.14 ab	0.13 ab	0.12 b	0.15 ab	0.13 ab	0.16 a
11	Linalool	17.99	1114	5.45 e	7.98 bc	8.25 b	8.13 b	7.64 cd	7.32 d	8.70 a
12	Camphor	19.38	1185	6.47 ef	7.68 b	7.34 c	8.12 a	7.07 d	6.69 e	6.24 f
13	trans-anethole	19.64	1380	22.25 c	23.31 bc	23.87 b	26.54 a	26.35 a	25.64 a	26.75 a
14	Fenchyl acetate	19.8	1235	5.98 b	6.98 a	6.38 b	5.28 c	4.31 d	2.94 e	3.21 e
15	Terpinen-4-ol	21.8	1175	0.51 a	0.26 c	0.39 b	0.28 c	0.36 b	0.39 b	0.48 a
16	Cymene	22.25	1030	2.58 c	3.57 b	4.06 a	3.54 b	4.02 a	3.95 a	3.85 ab
17	Estragole	28.1	1194	13.54 a	7.91 c	6.45 d	7.21 cd	7.95 c	10.93 b	11.87 b
18	cis-carveol	28.9	1226	1.25 a	0.31 c	0.38 c	0.32 c	0.41 c	0.59 b	0.52 b
19	Germacrene D	47.4	1490	0.29 cd	0.26 d	0.30 cd	0.35 bc	0.37 ab	0.39 ab	0.42 a
Total identified (%)				94.97	95.10	94.37	95.60	96.11	95.33	97.34
Monoterpene hydrocarbons (1–8, 10, 16)				37.14	37.73	38.70	36.96	38.81	37.53	36.37
Oxygenated monoterpene (9, 11, 12, 14, 15, 18)				21.75	25.89	25.05	24.54	22.63	20.84	21.93
Sesquiterpene hydrocarbons (19)				0.29	0.26	0.30	0.35	0.37	0.39	0.42
Phenylpropene derivatives (13, 17)				35.79	31.22	30.32	33.75	34.30	36.57	38.62
Trans-anethole/estragole ratio				1.65 e	2.95 c	3.71 a	3.69 a	3.31 b	2.35 d	2.25 d

RT: Retention time, RI: Retention index. Az: N-fixing bacteria (Azospirillum brasilense), Bm: P-solubilizing bacteria (Bacillus megaterium var. phosphaticum), Bc: K-solubilizing bacteria (B. circulans), VAM: vesicular arbuscular mycorrhiza (Glomus etunicatum, G. intraradices, and G. fasciculatum). RM30 and RM60 are rabbit manure at 30 and 60 m³/fed, respectively. NPK100 and NPK150 are NPK at 100% and 150% of recommended dose, respectively. Unfertilized plants were considered as control. Mean values with different letters in the row are statistically different according to DMRT (p ≤ 0.05).

). In addition to trans-anethole, other predominant components that were identified include estragole (6.45–13.54%), α-pinene (7.57–12.24%), and limonene (9.54–11.84%). The main component groups in the EO were monoterpene hydrocarbons (α-pinene, camphene, β-pinene, phellandrene, limonene, α-terpinene, γ-terpinene, sabinene, myrcene, and cymene) and phenylpropene derivatives (trans-anethole and estragole), which account for 36.37–38.81% and 30.32–38.62% of the total EO composition, respectively.

All treatments of fertilization increased the trans-anethole percentage and reduced the estragole compound compared to unfertilized plants. The highest significant proportion (p ≤ 0.05) of trans-anethole (26.54, 26.35, 25.64, and 26.75%) was recorded by applying RM30, RM60, NPK100, and NPK150, respectively, compared with the control (22.25%) and bio-fertilizer application. However, the undesirable estragole compound was increased significantly in NPK-fertilized fennel and control treatment compared to organic and bio-fertilized plants. The lowest significant percentage of estragole (7.91, 6.45, 7.21, and 7.95%) was detected with Az + Bm + Bc, Az + Bm + Bc + VAM, RM30, and RM60, respectively, while the higher contents of estragole (13.54, 11.87, and 10.93%) were found for control, NPK150, and NPK100 treatments, respectively. On the other side, the ratio of trans-anethole/estragole ranged 1.65–3.71 (Table 4). The highest significant ratio of trans-anethole/estragole (3.71 and 3.69) was found for Az + Bm + Bc + VAM and RM30, respectively, followed by RM60 (3.31) and Az + Bm + Bc (2.95). Unfertilized fennel and both NPK levels recorded the lowest significant ratio of trans-anethole/estragole.

3.8. DPPH Radical Scavenging Activity of Fennel Fruits

Overall, fruit extracts exhibited 66.79–80.27% scavenging activity of DPPH compared to ascorbic acid, which exhibited 93.60–95.80% (Figure 3). In the first season, the treatment of Az + Bm + Bc achieved the highest significant value of antioxidant activity (80.27%) when compared to other treatments and the control (68.50%), followed by Az + Bm + Bc + VAM and organic manure. In the second growing year, the fruit of biologically and organically fertilized fennel exhibited higher scavenging activity than the control and NPK. Furthermore, the heavy addition of synthetic NPK (NPK150) decreased the antioxidant capacity of fruit extract in both seasons.

3.9. Pearson’s Correlations Analysis of Important Parameters

The Pearson correlations analysis indicated significant correlations (positive or negative) between important traits of fennel (Figure 4). Average data of the two growing seasons (2019/2020 and 2020/2021) were used. A positive correlation can be seen with red circles, while a negative correlation with blue circles was shown in the same row. Positive correlations (p < 0.05 and 0.01) were observed between growth and yield parameters. The umbel number, weight of 100 fruits, fruit yield, EO content, and EO yield were positively correlated with the nutritional content parameters (N, P, K, and carbohydrates %) of fennel plants. The bioactive constituents (TPC and TFC) were positively correlated with each other as well as with the DPPH scavenging capacity of fennel fruit extracts. However, negative correlations were recorded between estragole content and TPC, TFC, and DPPH scavenging activity. Moreover, the K content of fennel was negatively correlated with the estragole content in EO.

4. Discussion

The current study aims to find out the most suitable fertilizer to enhance growth and yield parameters, phenolics, and flavonoid accumulation, and reduce estragole content in the EO of fennel (F. vulgare Mill.) fruits. A decrease in the height of fennel plants was noticed with the application of bio-fertilizers compared to organic and chemical fertilizers. In a previous study on caraway plants belonging to the Apiaceae family [47], a decrease in the height of caraway plants was also observed when applying a single application of bio-fertilizer (A. chroococcum, B. megaterium var. phosphaticum, and S. cerevisiae). Previous studies showed that P-solubilizing bacteria (B. megaterium) has a slight promoting effect on the growth, development, and yield of fennel compared to mineral phosphorus [48]. However, in the current study, bio-fertilization improved the plant height compared to the unfertilized plants (control). Additionally, El-Serafy and El-Sheshtawy [9] reported longer stems in F. vulgare spp. vulgare upon inoculation with N-fixing bacteria (B. polymyxa, A. chroococcum, and A. lipoferum). Likewise, mycorrhizal treatment of fennel plants produced taller plants than non-inoculated fennel [49]. Previous reports on medicinal and aromatic crops confirmed the stimulating effects of arbuscular mycorrhizal fungi on plant growth and productivity [50,51,52,53,54]. The highest significant value of plant height was measured for plants fertilized with NPK150. Waskela et al. [55] observed that plant height, biomass weight, and the number of branches were improved by increasing NPK rates in sweet fennel plants. A positive correlation was observed between plant height and content of N, P, K, and carbohydrates in fennel plants under different treatments (Figure 4).

The highest significant number of umbels was counted for plants fertilized with NPK150 followed by the high organic additive RM60. Likewise, Ehsanipour et al. [56] observed that the umbel number/plant in fennel was increased by raising N rates. The bio-fertilizers (Az + Bm + Bc and Az + Bm + Bc + VAM) increased umbel number/plant compared to the control. In line with this, Ghaderimokri et al. [11] reported that bio-fertilization increased the umbel number in fennel by 16.5% compared with the control, and humic acid recorded the largest number of umbels (51.5 umbels/plant), which is also similar to our findings with RM60 and NPK150 (47.03–51.30 umbels/plant). Similar observations were made by El-Sayed et al. [57] in Anethum graveolens, and Youssef et al. [47] in Carum carvi. An increase in the number of umbels per plant is positively associated with an increase in the productivity of fruits and EOs (Figure 4).

The chlorophyll content is an indicator of the plant’s nutrient availability and the plant’s health status [58]. Previous studies also showed that N, P, and K levels were positively correlated with chlorophyll content in fennel [24,55]. The improvement in vegetative parameters as a result of the application of NPK may result in more extensive canopy growth and increased chlorophyll content as nutrients actively contribute to its formation [55], and thus increased carbohydrate content. Potassium plays a prominent role in increasing the chlorophyll content in plant tissues and processes closely related to photosynthesis, thus improving the efficiency of photosynthesis and increasing the accumulation of carbohydrates [59]. Inoculation of fennel plants with Az + Bm + Bc + VAM microbes was superior to Az + Bm + Bc. These findings were in close conformity with those of Nada [60] on Calendula officinalis, as a combination of VAM with Az + Bm + Bc was more effective than Az + Bm + Bc and Az + VAM in enhancing carbohydrate accumulation.

Compared with the unfertilized plants, the content of the macronutrients N, P, and K increased in fennel plants that were fertilized regardless of the fertilizer source (Figure 2). Merlin et al. [53] mentioned that P and N content in coarse mint increased with the inoculation of arbuscular mycorrhizal fungi. Faridvand et al. [25] reported that organic fertilizers improved the growth of fennel due to their effect on the availability of most of the micro and macronutrients compared to chemical fertilizers. Eisa [61] found that organic additives (farmyard and chicken manure) and mineral NPK (50 and 100% of RD) resulted in the highest N, P, and K content in fennel plants. Previous studies proved that the accumulation of N, P, and K in plants in response to chemical, organic, and bio-fertilizations as reported in caraway [47], chamomile [62], collard [63], moringa [32], kale [64], and coriander [65]. Increasing the content of carbohydrates, N, P, and K in the above-ground parts of the fennel had a similar enhancing effect on fruit and EO yields. Therefore, a positive correlation was observed between the nutrition content variables (N, P, K, and carbohydrates %) and the yield of fruits and EO (Figure 4).

A higher level of organic manure (RM60) achieved the highest significant fruit weight. Al-Enzy et al. [66] also noticed a positive effect of organic fertilizers (sheep and ostrich manures) on the weight of 1000 fruits of fennel compared to the synthetic fertilizer (9.19, 9.90, and 8.87 g/1000 fruits, respectively). A decrease in the 1000-seed weight of the caraway plant was also reported due to individual application of bio-fertilization when compared with NPK and its combination with bio-fertilizer [47]. In contrast, Ghaderimokri et al. [11] recorded an increase in the 1000-seed weight of fennel subjected to a mixture of N-fixing bacteria, P-, and K-solubilizing bacteria compared to the control. These results indicate that applying bio-, organic, and chemical fertilization to fennel plants affected physiological processes like photosynthesis, which led to the accumulation of nutrients and phytochemicals in the fruits and the full filling of the fruits [67].

Crop yield is directly related to a plant’s health status and growing conditions. Fennel fruit yield (g/plant and t/fed) was highly improved due to the application of bio-fertilizers, organic, and chemical fertilizers (Table 2). Fennel inoculated with the bacterial mixture and VAM (Az + Bm + Bc + VAM) achieved high productivity of fruits compared to Az + Bm + Bc. VAM has symbiotic associations with plant roots and promotes nutrient capture [68], resulting in improved growth and yield. Ghaderimokri et al. [11] also noticed a significant improvement in the fruit yield of bio-fertilized fennel compared to controls. Since the fruit yield is the final expression of vegetative and flowering growth, the increase in the fruit yield is mainly due to the higher plant height, phytonutrient content, umbels per plant, and 1000-fruit weight with positive correlations between these parameters according to Pearson correlations analysis (Figure 4). Similar results were reported by Ayub et al. [67] in fennel and El-Sayed et al. [57] in dill.

Previous studies on fennel have reported that applying N, P, and K is important to enhance plant growth and fruit yield [55,56,67]. Potassium plays an important role in photosynthesis, carbohydrate formation, stomata conduction, increased transport of photoassimilates to various plant organs, nitrogen metabolism, and other nutrient absorptions, besides enzyme activation in plant cells [69,70]. However, the excessive application of chemical fertilizers of chemical origin increases the severity of environmental pollution and leads to high nitrate concentrations in the consumed parts of vegetable and medicinal plants [71,72]. Organic fertilizers, which are considered one of the cheapest sources of organic additives, improve the soil structure, increase the soil’s ability to hold water, and improve aeration and drainage, which helps the sound growth of roots and better absorption of nutrients [31]. All these are reflected positively on improving plant growth and crop productivity in general.

Among the many factors that cause fluctuations in crop yield are plant cultivar, surrounding environmental conditions, plant health, and soil fertility. The yield values of fennel fruit obtained in this study (0.89–1.50 t/fed) (2.20–3.71 t/ha) are close to that achieved in previous studies by Mahfouz and Sharaf-Eldin [73] (2.27–3.29 t/ha), Zardak et al. [49] (1177–3733 kg/ha), and higher than that reported by Ehsanipour et al. [56] (447.69–2120.73 kg/ha), Waskela et al. [55] (0.76–1.25 t/ha), Abdelkader et al. [74] (525.37–800.33 kg/fed), El-Serafy and El-Sheshtawy [9] (1600–2600 kg/ha), and Ghaderimokri et al. [11] (1960–2233 kg/ha).

Phenolics and flavonoids are valuable bioactive constituents in F. vulgare fruits that act as antioxidants and have various protective and therapeutic effects [22,75,76]. Data from Table 3 show that application of different fertilizers positively enhanced the TPC and TFC compared to unfertilized fennel in both seasons. Indeed, these fertilization treatments increased the rates of photosynthesis and significantly improved the accumulation of carbohydrates, which stimulated the biosynthesis of carbon-based secondary metabolites, such as phenolic and flavonoid compounds [24].

Inoculating medicinal and aromatic plants with VAM can enhance the content of various secondary metabolites including phenols, flavonoids, and EOs [51,77]. Compared with bio- and organic fertilization, the TPC decreased significantly in the NPK-fertilized fennel during the first growing season. However, this reduction was not statistically significant in the second season. The study of Barzegar et al. [24] on sweet fennel bulbs revealed the increase of total phenol and flavonoid content by NK fertilizer compared to the control. The TPC decreased by increasing the dose of NPK (NPK150). The observation was also reported by Biesiada et al. [78] that heavy N fertilization reduced the content of phenolics in Lavandula angustifolia flowers while increasing it in leaves.

Heavy addition of rabbit manure and both levels of NPK significantly decreased the TFC in both seasons. Similar results were found by Liu et al. [79] that increasing N fertilization in Chrysanthemum morifolium decreased flavonoid accumulation. The values of TPC and TFC reported in this study for fennel fruits (12.73–16.87 mg GAE/g DW and 9.60–18.68 mg QE/g DW, respectively) are close to that found in fruit extracts in the study of Ahmad et al. [75] (15.74–32.61 mg GAE/g extract and 5.22–18.59 mg rutin/g extract, respectively) and Khammassi et al. [21] (6.00–29.86 mg GAE/g dry extract and 5.17–16.42 mg QE/g dry extract, respectively). Obtaining fennel fruits with a high content of phenols and flavonoids limits the negative effects caused by harmful substances that may be present in food. In addition, these high-value health compounds balance the damage of harmful compounds that may be present with them, such as estragole.

According to the second edition of the European Pharmacopoeia monograph, the EO of sweet fennel accounts for at least 2.0% v/m [20]. A combination of VAM with bacterial inoculation (Az + Bm + Bc + VAM) was better than Az + Bm + Bc alone in enhancing the EO content. VAM has symbiotic associations with the plant roots that may cause changes in the biosynthesis of plant secondary metabolites, including EO [52]. Consistent with our results, Akbari and Gholami [80], Arango et al. [81], Rydlová et al. [50], Amiri et al. [51], José de Almeida et al. [52], and Merlin et al. [53] reported that arbuscular mycorrhizal fungi improved plant growth and increased the EO content of fennel, peppermint, dill, geranium, chamomile, and coarse mint plants, respectively. However, the percentage of fruit EO of fennel plants fertilized with NPK and organic fertilizer was higher compared to bio-fertilization. Similarly, the EO percentage of Carum carvi seeds decreased with the application of bio-fertilizer alone [47]. Other studies did not find a positive effect of biological fertilizers on the EO percentage in fennel [82,83].

The positive effects of organic additives on growth, flowering, and crop yield are related to several factors including improving the nutritional status of plants. The significant increase in the percentage of EO that was recorded for the RM60 and NPK150 treatments is mainly related to the increase in carbohydrates, N, and P content for these treatments. Overall, the EO content and yield were positively correlated with the nutritional content parameters (N, P, K, and carbohydrates %) of fennel plants (Figure 4). This nutritional state of the plant prior to umbel and fruit formation may have aided the improved synthesis of the EO. Herein, the biosynthesis of EOs depends on the P content in the plant [81], where P participates in forming the EOs’ precursors isopentenyl diphosphate and its isomer dimethylallyl diphosphate [84].

The contents of the isolated EO in the present experiment (2.60 to 3.76%) are close to the EO values reported in previous studies on fennel fertilization such as Ayub et al. [67] (2.59–2.75%), Moradi et al. [82] (2.20–2.90%), Ehsanipour et al. [56] (1.20–2.78%), Delfieh et al. [83] (2.35–3.43%), Zardak et al. [49] (2.85–3.51%), Abdelkader et al. [74] (3.22–3.60%), Machiani et al. [85] (2.76–3.78%), El-Serafy and El-Sheshtawy [9] (1.00–2.30%), and Ghaderimokri et al. [11] (2.38–4.22%).

Although the higher dose of NPK (NPK150) boosted EO yield compared to organic and bio-fertilizers, excessive use of chemical /inorganic fertilizers results in human health problems and affects the surrounding environment [71]. The preference for organic and chemical fertilizers for EO productivity is due to their stimulating effect on the EO content of the fruit as well as the fruit yield compared to microbial inoculation. Delfieh et al. [83] also showed a decrease in the EO yield from bio-fertilized fennel fruits compared to organic and chemical N fertilizer.

The yield of fennel fruit EO in our study (24.1–56.6 L/fed) (59.55–139.86 L/ha) is higher than that achieved in some previous reports such as Mahfouz and Sharaf-Eldin [73] (29.60–52.50 L/ha), Moradi et al. [82] (20.00–29.90 L/ha), Abdelkader et al. [74] (16.92–28.83 L/fed), and El-Serafy and El-Sheshtawy [9] (17.00–59.00 L/ha) in response to different nutrients.

Increasing the favorable components of fennel fruit EO and reducing harmful compounds are of great importance to both the pharmaceutical and food industries. The unique odor of fennel fruit, leaf, and bulb is due to anethole, the most common constituent in fennel EO [1]. Moreover, the quality of the EO decreases as the estragole content increases, causing a decrease in Egyptian exports of fennel EO [8,17]. GC-MS analysis of fennel fruit EO identified 19 compounds, among which, trans-anethole represented the main component (Table 4). In addition to trans-anethole, other predominant components identified include estragole, α-pinene, and limonene. Likewise, previous studies demonstrated that anethole and estragole are the main compounds in fennel EO [3,85]. However, the proportion of such components fluctuated according to cultivar and growth conditions.

It is recommended that the proportion of estragole in fennel EO does not exceed 10% [20]. Estragole content was increased significantly in NPK-fertilized fennel. Our previous study on fennel bulbs also showed an increase in estragole content in bulb EO of fennel plants fertilized with NPK compared to organic and bio-fertilized bulbs [19]. In a similar study, a higher level of N increased the undesirable estragole proportion and reduced linalool content in sweet basil EO [86]. This might be attributed to the N effect on the synthesis of phenylalanine, nucleic acids, and O-methyltransferase, which are involved in the biosynthesis of volatile phenylpropanoids, mainly estragole [6,87]. Furthermore, foliar spraying with L-phenylalanine increased the estragole content in bitter fennel EO [18]. These reports may support our results that an increase in the rate of chemical fertilizer (NPK150) resulted in an increase in the N content of fennel, followed by an increase in the estragole percentage. The difference in the level of estragole (=methyl chavicol) due to different treatments may be associated with the level of O-methyltransferase activities that catalyzes the estragole biosynthesis pathway [87]. The decrease in the proportion of estragole as a result of using organic manure was also observed in the study of Atta-Aly [88] on fennel bulbs. However, further additives of synthetic N fertilizers increased the estragole percentage. Rioba et al. [89] also reported a significant effect of N and P on the EO composition of sage (Salvia officinalis L.).

In the current study, the productivity of fennel fruits and EO was decreased by bio-fertilizers compared with organic and chemical fertilizers, however, the lowest proportion of estragole was detected in fennel inoculated with Az + Bm + Bc + VAM, which was supported by the findings of El-Serafy and El-Sheshtawy [9]. They found that fennel inoculated with N-fixed bacteria represented the most effective bio-stimulants in decreasing the estragole percentage and increasing anethole proportion in fruit EO. Moreover, our findings revealed a significant increase in the ratio of trans-anethole/estragole in biologically fertilized fennel. The slow release of N and other essential elements in bio-fertilization treatments may explain the reduced growth, fruit, and EO yield. Interestingly, however, this slow release of N leads to a decrease in the rate of estragole biosynthesis [3]. In contrast to organic and bio-fertilizers, synthetic NPK quickly supplies plants with N and hence elevates the biosynthesis capacity of estragole [3]. The same findings were displayed in Sakr et al.’s [90] paper on sweet basil, in which methyl chavicol content reached the highest value with the full dose of NPK and the lowest value with the bio-fertilizer Microbein^®.

The increase in the EO content and yield of fennel fruits achieved by organic manure was associated with a reduction in the unfavorable oil component, estragole, and an enhancement in the favorable compounds trans-anethole, α-pinene, linalool, camphor, and cymene. In addition, the inhibition of estragole formation recorded with the treatment of plant growth-promoting microbes was accompanied by an increase in trans-anethole, phellandrene, α-pinene, limonene, α-terpinene, linalool, camphor, fenchyl acetate, and cymene. Similarly, Ghazal and Shahhat [91] indicated that bio-organic fertilizers could contribute to higher anethole and lower estragole contents in F. vulgare var. vulgare. Additionally, the ratio of trans-anethole/estragole was higher with the application of organic and bio-fertilizers compared to chemical fertilizers. This indicates that organic and bio-fertilizer systems for fennel can produce high-quality fruit EO that provides the desired health benefits, thus avoiding chemical fertilizers and helping to reduce environmental pollution.

Determining the biological and antioxidant activities of natural products is a major concern worldwide for their use in the manufacture of safe functional foods. EOs and the plant organs they contain are good candidates in this regard [92]. The higher scavenging activity of fruit extracts of fertilized fennel may be due to the increased content of phenols and flavonoid phytomolecules. Possibly, the increased accumulation of phenols in the fruits of plants treated with Az + Bm + Bc may be the reason for their superiority. According to the Pearson correlations analysis, bioactive constituents-associated parameters (TPC and TFC) were positively correlated with DPPH scavenging capacity (Figure 4), inferring that phenols and flavonoids have antioxidant activity and that increasing their accumulation in fennel fruits will add value to the direct consumption of them or their total extract. Amiri et al. [51] recorded an increase in the antioxidant activity, TPC, and TFC in Pelargonium graveolens because of arbuscular mycorrhizal fungi inoculation. However, the higher dosage of NPK (NPK150) reduced the fruit antioxidant capacity. Other reports also demonstrated the inhibition of antioxidant activity by increasing N levels [78,79]. Based on the data shown here, fennel fruits exhibit potential as natural antioxidants, which may help protect the human body from free radical damage. Confirming our results, fruit extracts of different fennel populations showed strong antioxidant activity in the study of Khammassi et al. [21]. In this study, methanol extracts of fennel fruits exhibited effective reducing power, DPPH-radical scavenging, ABTS scavenging, and total antioxidant capacity.

5. Conclusions

In this study, fennel growth, fruit, and EO yield, accumulation of bioactive constituents (TPC and TFC), and antioxidant capacity (DPPH) positively responded to bio-fertilizer, organic, and chemical fertilizers. However, the percentage of the EO components differed markedly according to the source of the fertilizer. Heavy addition of NPK and RM maximized growth and yield parameters of fruits and EO, i.e., plant height, umbel number/plant, 100-fruit weight, fruit yield, chlorophyll, carbohydrates, N and P content, EO content, and yield. The most favorable compound in fennel EO, trans-anethole, had a high proportion in the fruits harvested from organically and chemically fertilized fennel. However, the unfavorable component, estragole, was elevated with the application of synthetic NPK and the ratio of trans-anethole/estragole was also decreased compared to the organic and bio-fertilization. The increase in the EO content and yield of fennel fruits achieved by RM was accompanied by a reduction in estragole proportion and an increase in trans-anethole, fenchone, limonene, and trans-anethole/estragole ratio. In addition, the application of plant growth-promoting microbes can produce high-quality EO from fennel fruits. The inhibition of estragole formation recorded with bio-fertilizers was accompanied by an increase in trans-anethole, trans-anethole/estragole ratio, and accumulation of phenols and flavonoids. It can be concluded that organic agriculture and bio-fertilization for sweet fennel and avoiding synthetic fertilizers ensures high productivity and quality of fruits and EO with improved health benefits and less environmental pollution.