Effect of Vermicompost on Growth, Plant Nutrient Uptake and Bioactivity of Ex Vitro Pineapple (Ananas comosus var. MD2)

Abstract

Vermicompost is a nutrient-rich organic waste produced from earthworms that is beneficial in enhancing the soil condition and has been reported to aid in improving the crop yield and quality. In the present study, a field trial was conducted using a randomized complete block design with four replicates to elucidate the effects of vermicompost application (compared to supplementation with chemical fertilizer and no fertilizer) on the productivity of ex vitro MD2 pineapple plants. Vermicompost was applied on the sandy loam soils at transplanting followed by a second application at 7 months after planting (MAP) at the rate of 10 t·ha⁻¹, while chemical fertilizer was applied based on the recommended cultivation practice. Data analysis revealed that there was no significant difference between the plants treated with vermicompost and chemical fertilizer in terms of the plant height, number of leaves, length and width of D-leaves, stomatal density and stomatal size. However, the fruits produced with vermicompost amendment were smaller in size but contained higher total soluble solids, titratable acidity, total solids, ascorbic acid and total chlorophyll content compared to the fruits produced from plants supplied with chemical fertilizer. Based on the DPPH, ABTS and FRAP assays, the methanolic fruit extracts from the control plants showed the highest antioxidant potential, followed by those of plants treated with vermicompost and chemical fertilizer. On the other hand, the application of vermicompost reduced soil acidity and produced macro- and micronutrient contents (N, P, K, Mg, Ca, S, Fe, Zn, B and Al) in the soil and plants that were comparable to or higher than those produced by the chemical fertilizer treatment. However, some of the nutrient contents observed in all treatments were lower than the recommended range for pineapple plant growth, suggesting that vermicompost or chemical fertilizer should not be used alone as a source of nutrients for ex vitro MD2 pineapple plants under these soil and field conditions. However, vermicompost can be used as a supplement to increase the fruit chemical quality and maintain the soil quality for agricultural sustainability.

1. Introduction

Pineapple (Ananas comosus (L.) Merr) is the leading edible member of the Bromeliaceae family. In terms of its importance in global production, it ranks second among four major fresh tropical fruits, where it comes after mango and is followed by papaya and avocado [1]. The world production of pineapple was increased by 30% between 2007 (20 million tons) to 2017 (25.9 million tons), with Costa Rica, Brazil and the Philippines as the top three pineapple producers in the world [1]. In Malaysia, the production of pineapple is dominated by N36, Josapine, MD2 and Moris varieties [2]. Pineapple (Ananas comosus) has been listed as one of seven high-value nonseasonal tropical fruits (alongside jackfruit, starfruit, banana, rock melon and papaya) prioritized for production for premium market under Malaysia’s National Key Economic Areas (NKEA) for agriculture and the Economic Transformation Program (ETP) [3]. This is due to its robust international demand and significant domestic consumption, as well as its various quality characteristics. It is reported to have sweeter taste, blemish-free flesh, high fiber content, cylindrical shape with solid golden-yellow pulp, a very pleasant aroma when ripe, low acidity (0.4–0.45%) and longer shelf life compared to other varieties [4,5]. It is also a good source of health-promoting antioxidants such as ascorbic acid, β-carotenes and phenolic compounds [6,7]. Furthermore, it has the highest price rate per kg fruit when compared to other varieties (e.g., N36, Josapine, Moris), which makes cultivating the MD2 pineapple attractive to farmers [3].

However, the production of suckers of MD2 pineapple is expensive and unable to meet the demand for planting materials, as 43,000 propagules are required per hectare (for planting density of 30 × 60 × 90 cm). A single mother plant of MD2 pineapple only produces 2–3 propagules per cycle (18–20 months). In order to produce the plantlets on a large scale, researchers have turned to utilizing tissue culture techniques to aid the cultivation of MD2 pineapple [5,8,9,10,11]. According to Hamid, Bukhori and Jalil [10], an average of five plantlets per explant was produced in just one month of culture in the initiation media, which then further multiplied in the shoot-multiplication media to produce three shoots per plantlet. In addition, the usage of in vitro plantlets can minimize the problematic early natural flowering occurrence and reduce the amount of pesticide used on the plantlets prior to planting [12]. Thus, in vitro micropropagation approaches may be a way to overcome the shortage of MD2 pineapple plantlets for commercial production. However, very few research studies and published reports can be found on ex vitro acclimatization of MD2 pineapple plants under field conditions and its fruit quality.

Moreover, to improve the quality of fruits produced as well as to protect environmental and ecological resources, fertilization plays an important role in crop management. Pineapple plants have a large nutrient uptake demand, especially for potassium (K), followed by nitrogen (N), sulfur (S), calcium (Ca), magnesium (Mg) and phosphorus (P) [13]. Macro- and micronutrients can be obtained by supplementation of inorganic or organic fertilizers such as vermicompost. Vermicompost is a slow-release fertilizer and is rich with essential plant nutrients produced by the joint action of certain species of earthworms (especially Eisenia fetida or Eudriculus eugeniae) and microorganisms in the decomposition of organic waste such as agro-wastes [14], sewage sludge [15] and food wastes [16]. Several studies have shown that vermicompost amendment can directly increase plant production through increasing available plant nutrients and indirectly promote soil quality by improving soil structure and stimulating microbial activities, relative to conventional chemical fertilization [17,18]. For example, Baldotto et al. [19] reported that in vitro grown ‘Victoria’ pineapple plantlets treated with 15 mmol·L⁻¹ vermicompost-derived humic acids showed better growth characteristics and nutrient uptake during ex vitro acclimatization in the greenhouse. In their study, a significant accumulation of N, P, K, Ca and Mg was observed in the roots and shoots of ex vitro ‘Victoria’ pineapple plants compared to the control [19]. In the experiment, the pineapple plantlets were placed in baby food glass pots containing 15 mmol·L⁻¹ humic acids [19]. Vermicompost has also been reported to improve the yield parameters of wheat [20], maize [21] tomatoes [22] and peppermint [23]. Besides that, the peppermint plants grown on vermicompost also produced higher amounts of chlorophyll a, chlorophyll b and carotenoids compared to the plants supplemented with inorganic fertilizers and unfertilized plants [23]. Moreover, vermicompost can retain nutrients for a long time and has high soil porosity (24% higher than unfertilized soil) and high water-holding capacity compared to conventional compost due to its humus content, thus reducing the irrigation requirement by 30–40% [14]. Other than that, the usage of vermicompost has also been reported to result in the production of healthier plants with better resistance towards pests and diseases [24,25]. The vast benefits of vermicompost have garnered the attention of farmers as a greener and more sustainable replacement for chemical fertilizers.

Nowadays, the pollution of soil and water sources due to the excessive use of chemical fertilizers is of great concern; therefore, shifting towards the application of organic fertilizers as an alternative to chemical fertilizers is the way forward to support sustainable agriculture. In this study, the effect of vermicompost supplementation compared to conventional practices using chemical fertilizers on the growth productivity of ex vitro MD2 pineapple plants was evaluated. The accumulation of nutrients in the D-leaves (most recently matured leaf with maximum physiological activity) and the bioactive compound contents and antioxidant potential of the resulting fruits were also analyzed. This paper provides essential information that improves the knowledge and understanding on the effects of vermicompost on A. comosus var. MD2 plants and thus allows the usage of tissue-culture-produced pineapple plants to be recommended for commercial production.

2. Materials and Methods

2.1. Study Area and Biological Materials

The field trial was conducted on sandy loam soil located at the Glami Lemi Biotechnology Research Centre, Jelebu, Negeri Sembilan, Malaysia (3°3′ N, 102°3′ E) from January 2015 until December 2016. The experiment area is well known as the warmest area in Malaysia, with a mean annual precipitation of 130 mm and monthly average temperatures ranging from 23 to 33 °C. The highest peaks of monthly precipitation as recorded at the nearest meteorological station were in April, October and November, while the lowest was in January [26]. The vermicompost and chemical fertilizer were bought from a local company (Earthworm, Synergy Resources, Malaysia). The soil chemical properties of the planting sites prior to the experiment are shown in

Table 1. Chemical properties of the soil at the planting sites prior to the experiment, along with the composition of vermicompost and chemical fertilizer used in the study.

Sample	Chemical Properties
Soil (prior to experiment)	pH 5.65, 57.90% electrical conductivity, 0.06% total nitrogen, 0.51% total carbon, 92.28 mg·kg−1 available P, 0.21 cmol (+) kg−1 exchangeable K, 0.85 cmol (+) kg−1 exchangeable Ca and 0.18 cmol (+) kg−1 exchangeable Mg.
Vermicompost	Total nitrogen (N) 1.54%, total phosphorus (P) 0.64%, total potassium (K) 6.31%, total magnesium (Mg) 0.58%, total calcium (Ca) 1.39%, total sulfur (S) 0.34%, total zinc (Zn) 0.01%, total boron (B) 0%, total iron (Fe) 0.76% and total aluminum (Al) 1.04%.
Chemical fertilizer	(1) Fertilizer granules at 1, 3, 7 and 14 MAP: NPK (15:15:15). (2) Foliar fertilizer mix at 1.5 MAP: 640 g hydrated lime, 42 g copper sulfate, 42 g zinc sulfate and 21 g ferrous sulfate in 18 L water. (3) Foliar fertilizer mix at 4.5 MAP: 640 g hydrated lime, 42 g copper sulfate, 42 g zinc sulfate, 21 g ferrous sulfate and 640 g urea in 18 L water.

.

2.2. Experimental Design

The experimental design was arranged in randomized complete block design (RCBD) with three treatments and four replicates consisting of ex vitro pineapple plants supplemented with vermicompost (EPV), ex vitro pineapple plants supplemented with chemical fertilizer (EPF) and control ex vitro pineapple plants not supplied with any chemical fertilizers or vermicompost products (EPC). Vermicompost was applied twice (10 t·ha⁻¹) during transplanting and 8 months after planting (MAP) by mixing with the topsoil. The nutrient availability in the vermicompost is described in Table 1. The rate of chemical fertilizer application was based on the recommended dose by the Malaysian Pineapple Industry Board (MPIB) [27]. The NPK fertilizer granules (Table 1) were applied at the rate of 20 g/plant at 1, 3, 7 and 14 MAP. A foliar fertilizer mix was sprayed twice (50–100 mL/plant) at 1.5 MAP and 4.5 MAP (Table 1).

The cultivation techniques and field management were based on the pineapple cultivation guidelines provided by the Malaysian Pineapple Industry Board [27]. Prior to planting, the site was ploughed and furrowed to acquire a good tilth. A drain flow (1.5 × 1.2 × 1.2 m) was built around the field to prevent standing water in the plot from affecting the plant growth. Individual plot size was 3.0 m × 2.0 m. The beds and blocks were separated with a spacing of 1.0 m to ensure uninterrupted flow of irrigation for each individual plot. An average of fifteen plants were planted in double rows for each plot with plant-to-plant spacing of 60 cm × 30 cm. Prior to planting, shades were constructed in each plot and were used until 12 MAP (Figure 1). The plants were watered when necessary using a sprinkler water system, while the weeds were controlled intermittently. Flowering was induced after 16 MAP based on the crop development stage by spraying with 50 mL of Ethrel (2-chloroethyl phosphonic acid) solution (15 mL Ethrel and 90 g urea in 9 L of water) at the center of the pineapple plants. After 30–45 days, the plants were checked for the success of flowering induction. When the fruits were fully developed, the fruits were covered to avoid sun scorch. The fruits were harvested when they were one-third ripe (about 120 days after flowering was successfully induced).

2.3. Morphophysiology of Ex Vitro MD2 Pineapple Plants

Three plants from each plot were randomly tagged for data collection. For vegetative data collection, the number of leaves, plant height and length and width of D-leaves (cm) were measured every month, starting from 2 MAP and continuing until the flowering stage. The chlorophyll content was measured with a Minolta chlorophyll meter (SPAD-502, Konica Minolta, Japan) every two months by averaging the soil plant analysis development (SPAD) values from the base, middle and tip of the D-leaves [28]. The SPAD values were taken before 9:00 a.m. or after 5:30 p.m. under the shade, as the values are more accurate if recorded when the irradiance is low.

The D-leaf surface structure, stomatal density, stomatal size, stomatal length and width, pore length and stomatal aperture were determined at 9 MAP using a field emission scanning electron microscope (FE-SEM; Quanta FEG 450, FEI, Australia). For preparation of the sample, fresh D-leaves were collected from the plants. Then, they were washed with distilled water and blot-dried with tissues. The leaf surface (both abaxial and adaxial) was scrubbed lightly by using a sharp scalpel to remove the trichomes attached on the leaf surface to observe the stomata. The leaf was cut into 0.3 cm × 0.3 cm (taken 30 cm from the bottom of the leaf and away from the edge). Then, the samples were attached with black double-sided tape on the specimen stubs to prevent the samples from moving. The prepared stubs with the samples were immediately put in the FE-SEM. The images of the cross-section and transverse section of the D-leaf were taken to show the general structure of the MD2 pineapple leaf. For morphological stomatal features, the measurements were made on four leaves (one leaf per plant) on both adaxial (upper) and abaxial (lower) leaf surfaces for each treatment. The number of stomata was counted on a field of view of 0.295 mm² (area of leaf) at a magnification of 500 times, and the stomatal density (d) was calculated as d = number of stomata/leaf area. The stomatal size was calculated as the product of stomatal length and stomatal width (µm²). The stomatal length, stomatal width, pore length and pore aperture were measured as described by Savvides et al. [29]. All parameters were measured using the xT Microscope Control imaging software. The results were compared with those of in vivo MD2 pineapple plants grown under similar conditions [30].

2.4. Physical Characteristics of Fruits

The yield and physical properties of the fruits such as the fruit weight, fruit weight without crown, fruit diameter and crown weight and length were determined. The pineapple fruit was cut into two parts. At the bottom, middle and top of the fruits, the pulp firmness was measured using a fruit hardness tester (Nippon Optical Works, Japan) and expressed as kilogram force (kg f) required to penetrate the tissue. The core diameter was measured using a ruler at three different places: the bottom, middle and top of fruits.

2.5. Chemical Properties of Fruits

The pH of the pineapple juice, total soluble solids and total titratable acidity were measured as described by Dadzie and Orchard [31]. Briefly, the pH and the total soluble solids of the samples were measured at room temperature (25 ± 2 °C) using an electronic pH meter (Mettler Toledo, Switzerland) and a digital refractometer (PR-1, Atago, Japan), respectively. The refractometer was standardized with distilled water, and the results were expressed in standard ⁰Brix unit. The titratable acidity was determined by titration method. The titer volume of 0.1 N NaOH added was recorded and multiplied by the citric acid factor (0.07) to obtain the total titratable acidity. The results were expressed as g citric acid per kg of pineapple (g·kg⁻¹). The sugar:acid ratio was derived as the ratio of total soluble solids to total titratable acidity. The percentage of total solids (%) was analyzed based on the AOAC [32] method. Ascorbic acid content was estimated by 2,6–dichlorophenolindophenol visual titration method [32]. The blank used in the measurement consisted of distilled water, while the standard solution was pure ascorbic acid. The ascorbic acid content of the samples was expressed in terms of µg ascorbic acid/g of sample fresh weight. All measurements were performed in triplicate.

2.6. Soil and Plant Nutrient Analysis

Before sampling, three core soil samples were randomly collected from 0–15 cm topsoil by using a Dutch Auger and bulk to form a composite. The samples were air-dried, crushed using a mortar and pestle and allowed to pass through a 2.0 mm sieve for soil pH analysis and then 0.25 mm sieves for total elements analysis. The nutrients in the plants were determined using the finely ground dried D-leaves as the samples, where an average of three whole D-leaves per plot were used. The D-leaves were identified by gathering all the leaves by hand to form a vertical “bundle” in the center of the plant, of which the D-leaves are the longest ones. The sampling of both the soil and D-leaves was conducted on the same day at six months after planting (S1; 6 MAP) and during the red bud stage (S2; 17 MAP).

The soil pH was measured at a soil to distilled water ratio of 1:2.5 using a pH meter (PB-10, Sartorius, Germany). The total nitrogen (N) of the D-leaf samples was determined via dry combustion using a Nitrogen Determinator (FP-528, LECO, United Kingdom) [33], while the total nitrogen (N) in the soil was determined by the Kjeldahl method [34]. The contents of other total elements, namely phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), zinc (Zn), iron (Fe), boron (B) and aluminum (Al), were determined by aqua regia digestion method [35]. On the other hand, the elements in the D-leaves were determined by the method of dry ashing and digestion with nitric acid [36,37]. The filtrated solution of both extracts was analyzed using an inductively coupled plasma optical emission spectrometer (725-ES ICP-OES, Varian, Australia). A blank digest was carried out in the same way and used as the control.

2.7. Determination of Bioactive Compounds and Antioxidant Potential in Fruit Extracts

2.7.1. Sample Extraction

Five-gram portions of the freeze-dried samples were subjected to solvent extraction with 150 mL of 99.8% methanol for 48 h at room temperature under dark conditions using an orbital shaker (722-2T, Protech, Malaysia) at 100 rpm. The extracts were filtered using Whatman No. 2 filter papers, and the collected filtrate was stored at −20 °C. The residue was re-extracted and filtered. The extracts were pooled, centrifuged at 9000 rpm and 4 °C for 5 min and the supernatant was collected before being concentrated to dryness using a Rotavapor (R-3, Büchi Labortechnik AG, Switzerland) at 45 °C. Then, 99.8% methanol was used to adjust the concentration of the solvent-free extract to 20 mg/mL prior to storage in an air-tight container at −20 °C until further analysis. As far as possible, all extraction procedures were performed under daylight protection.

2.7.2. Determination of Chlorophyll and Total Carotenoid Contents

The methanolic solutions of fruit extracts were analyzed using a UV-Vis spectrophotometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) at 470, 652.4 and 665.2 nm. Chlorophylls (a and b) and total carotenoid concentrations were calculated based on the formula by Lichtenthaler and Buschmann [38]:

C_a (mg/L) = 16.72 A_665.2 − 9.16 A_652.4

(1)

C_b (mg/L) = 34.09 A_652.4 − 15.28 A_665.2

(2)

C_(x+c) (mg/L) = [(1000 A₄₇₀ − (1.63 C_a − 104.96 C_b)]/221

(3)

2.7.3. Measurement of Total Phenolic Content

The total phenolic content (TPC) of the fruit methanolic extracts was determined using the Folin–Ciocalteu (FC) method as described by Singleton et al. [39]. The absorbance of the samples was read using a UV-Vis spectrophotometer (Lambda 25, Perkin Elmer, Waltham, MA, USA) at 765 nm. A standard solution of gallic acid was used to prepare the calibration curve (r² = 0.99). The TPC of the samples was expressed in terms of mg gallic acid equivalent/g of dried extract.

2.7.4. DPPH (2, 2-Diphenyl-1-picrylhydrazyl) Free Radical Scavenging Activity Assay

The DPPH free radical scavenging activity was determined as described by Yusof et al. [40], with slight modifications. Briefly, 150 µL of 3 mM solution of DPPH radical solution in methanol was added into 50 µL of methanolic fruit extract (2 to 12 mg/mL), standard solution (0.01 to 1.0 mg/mL) and control (99.8% methanol) in different wells for triplicates. Then, the solution was left to stand for 30 min in the dark at 27 °C. The changes in the absorbance of the samples were measured at 515 nm using a microplate spectrophotometer (Multiskan GO, Thermo Scientific, MA, USA). The percentage of DPPH radical scavenging activities was calculated as follows:

DPPH radical scavenging activity (%) = [(A₀ − A₁)/A₀] × 100

(4)

where A₀ is the absorbance of the control and A₁ is the absorbance of samples.

A linear regression line was plotted between the percentage of inhibition and the concentration (r² = 0.99). The results were reported as the concentration of sample required to reduce 50% of DPPH (IC₅₀) in mg/mL. A more potent antioxidant was denoted by a lower IC₅₀ value. The positive control used was ascorbic acid.

2.7.5. ABTS (2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) Radical Scavenging Activity Assay

For the ABTS assay, the procedure described by Miller et al. [41] was followed, with few modifications. The stock solutions (7.4 mM ABTS and 2.6 mM potassium persulfate solutions) were separately prepared prior to the analysis and were used in preparing the working solution. For the analysis, 1:1 ratio of the stock solutions was mixed and incubated for 12–16 h at room temperature in the dark before use. The solution was then diluted with deionized water (18.2 MΩ·cm⁻¹) until an absorbance of 0.70 ± 0.02 units at 734 nm was obtained using a spectrophotometer. A fresh ABTS solution was prepared for each assay. Fruit extracts (20 µL) at six different concentrations (2.0 to 12.0 mg/mL) were allowed to react with 200 µL of ABTS solution in the dark for 10 min. Then, the absorbance was read at 734 nm using a microplate spectrophotometer (Multiskan Go, Thermo Scientific, MA, USA), and the assay was performed in triplicate. A linear regression line was plotted between the percentage of inhibition and concentration (r² = 0.99). The results were reported in terms of 50% inhibition concentration (IC₅₀) values in mg/mL.

2.7.6. Ferric Reducing Antioxidant Power (FRAP) Assay

The FRAP assay was performed based on the method described by Benzie and Strain [42], with some modifications. Stock solutions consisting of 300 mM acetate buffer (3.1 g C₂H₃NaOO and 16 mL C₂H₄O) at pH 3.6, 10 mM TPTZ (2, 4, 6 tripyridyl-s-triazine) solution in 40 mM HCl and 20 mM FeCl₃.6H₂O solution were separately prepared before the analysis. Prior to each analysis, a fresh working solution consisting of 10:1:1:1.2 of acetate buffer, TPTZ solution, FeCl₃.6HO solution and distilled water was prepared and warmed at 37 °C before use. The fruit extracts (10 µL) were allowed to react with 300 µL of FRAP solution in the dark for 30 min. The absorbance readings of the colored product (ferrous tripyridyltriazine complex) were read at 593 nm using a microplate spectrophotometer (Multiskan Go, Thermo Scientific, USA). The standard curve was plotted using a linear regression between 0.01 and 0.10 mg/mL of ferrous sulfate FeSO₄.7H₂O (r² = 0.99). The FRAP values were expressed in milligrams of ferrous equivalent Fe (II) per gram of dried extract.

2.8. Statistical Analysis

All data related to plant growth parameters (plant height; number of leaves; and length, width and SPAD value of the D-leaves) obtained in this study were analyzed using repeated measures ANOVA (rANOVA), and the sphericity assumption was tested using Mauchly’s test. Meanwhile, other data were analyzed using analysis of variance (ANOVA). The normality was also assessed prior to conducting ANOVA analysis by plotting a histogram. The differences between treatment means were separated using Duncan’s multiple range test (DMRT) at 5% significance level. The correlations among data were calculated using Pearson’s correlation coefficient in bivariate correlations. All statistical analysis was done by using SPSS software version 24.

3. Results and Discussion

3.1. Effect of Vermicompost on Morphophysiology of Plants

The height of the ex vitro MD2 pineapple plants were measured from 2 months after planting (MAP) until 18 MAP. The plant height ranged from 11 to 95 cm (Figure 2A). Based on repeated measures ANOVA, it was shown that the height of ex vitro plants supplemented with vermicompost (EPV) was comparable to that of plants supplemented with chemical fertilizer (EPF) (

Table 2. The morphophysiology of field-grown ex vitro MD2 pineapple plants supplemented with different types of fertilizers.

Parameters	Samples
	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
Plant height (cm)	47.1 ± 2.4 b	57.3 ± 2.4 a	57.0 ± 2.4 a
Number of leaves	43 ± 1 b	51 ± 1 a	44 ± 1 b
Length of D-leaves (cm)	38.0 ± 1.9 b	44.5 ± 1.9 a	46.0 ± 1.9 a
Width of D-leaves (cm)	3.8 ± 0.1 b	4.3 ± 0.1 a	4.2 ± 0.1 a
SPAD value	69.2 ± 1.0 a	71.2 ± 1.0 a	64.8 ± 1.0 b

Means ± standard error followed by different letters in a row are significantly different based on repeated measures ANOVA and Duncan’s multiple range test at p ≤ 0.05, n = 12.

). The plants treated with each fertilizer type were also significantly taller than the control plants (EPC). However, the supplementation with chemical fertilizer produced the highest number of leaves (51 leaves per plant) when compared to vermicompost (44 leaves per plant) and control (43 leaves per plant) (Figure 2B). The length of the D-leaves of EPF plants was also found to be not significantly different from that of EPV plants, but both were significantly longer than the control (EPC). Nevertheless, data analysis revealed that the length of the D-leaf of EPV plants showed a significantly marked increase compared to EPF plants after 13 MAP, which was possibly due to the second supplementation of vermicompost to the soil (Figure 2C). Similar results were obtained in previous research on pineapple plants of the Queen variety, where the influence of vermicompost (20 tonnes·ha⁻¹·year⁻¹) was clearly greater in the second year of growth [14]. At 18 MAP, the length of the D-leaves was observed to decrease for all treatments as the plants started to produce flowers.

Moreover, the D-leaf width of EPV was comparable to that of EPF, and both treatments produced wider leaves than the control (Table 2). The effect of vermicompost application on the width of the D-leaves can be seen after its second application (after 8 MAP), where the width of the D-leaves gradually increased until 12 MAP (Figure 2D). This may also be due to the weather conditions, as the width of the D-leaves had drastically increased during the rainy season in the months of April (3 MAP, 15 MAP), October (9 MAP) and November (10 MAP). The widths of the D-leaves were observed to decrease during 13–15 MAP (February to April 2016), possibly due to the super El Nino event of 2015/2016 that had caused drought to occur nationwide. The super El Nino event 2015/2016 was reported to be among the strongest since 1997/1998 [43]. The removal of the shades from the plots at 12 MAP could have also contributed to the decrease of the D-leaf width. The plants grown in warmer and drier climates tend to have smaller leaves to reduce water loss through transpiration, while larger leaves are more prevalent in wetter environment with low light intensity [44,45]. During these extreme conditions (13–15 MAP), EPV plants showed the highest D-leaf width when compared to EPF and control (EPC) plants. This indicates that the high water-holding capacity of vermicompost further helped with the water economy of the pineapple plants. Similarly, the water-holding capacity of soils in which black gram was grown was reported to increase when vermicompost was added, compared to the control (untreated) [46].

A nondestructive and rapid method to estimate the chlorophyll and nitrogen status in the leaves is the use of a chlorophyll meter (SPAD-502). The SPAD meter values show the relative greenness of the crop canopy, and values lower than 40 indicate an impairment in the photosynthesis process [28]. The SPAD values of ex vitro MD2 pineapple plants grown in the field with different types of fertilizers are depicted in Figure 2E. The SPAD values of all treatments ranged between 45.9 to 88.7, and the highest reading was obtained during the red bud stage. In this study, data analysis revealed that the plants supplemented with vermicompost (EPV) exhibited the lowest chlorophyll content when compared to chemical fertilizer (EPF) and control (EPC) (Table 2). These results are in agreement with those obtained by El-Hassan et al. [47], where the chlorophyll content of green bean plants supplied with vermicompost was lower than that of plants supplemented with fertilizers. Alaboz et al. [48] also reported that pepper (Capsicum annuum) plants treated with 0.75 w/w vermicompost contained lower chlorophyll content (60.7 SPAD) compared to the unfertilized plants (64.9 SPAD) under field capacity with 80% soil moisture level. Moreover, the chlorophyll content (based on the SPAD values) of the ex vitro MD2 pineapple plants was observed to drastically reduce at 12 MAP, when the precipitation level was the lowest of the year. The chlorophyll content will decrease in the event of water shortage [49] due to lesser leaf water content, which in turn reduces the rate of chlorophyll synthesis in the leaves [50].

A cross-section of the MD2 pineapple leaves (Figure 3A,B) shows that the leaf structure consists of an upper epidermis covered with a thick and smooth cuticle, i.e., the water-storage tissue, which encompasses nearly half of the leaf thickness (depending on the water status of the plant) and the lower hypodermis with the stomata covered with dense, flat and shield-shaped trichomes that give the leaf its silvery appearance and protect the plant from excessive transpiration and intense sunlight [51,52]. As shown in Figure 3C, no stomata were observed on the adaxial (upper) epidermis, while rows of stomata were observed on the abaxial (lower) surface, where they were located in furrows that were parallel to the longitudinal axis of the leaf (Figure 3D).

The stomatal density and stomatal characteristics (e.g., stomatal size and stomatal length) are indicators of acclimation and adaptation to environmental changes, such as changes in light intensity [53], temperature [54], water status [55], leaf nutrients and soil nutrient contents [54]. In general, the stomatal density of the pineapple leaves was low, with about 80 stomata per mm² [52]. Data analysis showed that the stomatal density of EPV leaves (76.49 stomata per mm²) was significantly higher than that of the EPC (control) plants (56.78 stomata per mm²) (

Table 3. The parameters of stomata on the lower epidermis of the D-leaves from 9-month-old field-grown ex vitro MD2 pineapple plants supplemented with different types of fertilizers.

Parameters	Samples
	Standard (In Vivo Plants)	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
Stomatal density (mm2)	85.60 ± 2.13 a	56.78 ± 1.62 c	73.45 ± 3.20 b	76.49 ± 0.72 b
Stomatal size (µm2)	606.32 ± 19.77 b	678.91 ± 19.45 a	658.54 ± 27.08 a,b	614.81 ± 6.02 b
Stomatal length (µm)	26.41 ± 0.14 a	27.79 ± 0.25 a	27.48 ± 0.73 a	26.43 ± 0.62 a
Stomatal width (µm)	22.96 ± 0.69 a	22.42 ± 0.48 a	23.94 ± 0.43 a	23.29 ± 0.46 a
Stomatal pore length (µm)	9.10 ± 0.26 a	9.68 ± 0.01 a	9.58 ± 0.45 a	9.90 ± 0.21 a
Stomatal pore aperture (µm)	4.60 ± 0.18 a	5.22 ± 0.11 a	5.03 ± 0.42 a	4.91 ± 0.36 a

Means ± standard error followed by different letters in a row are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 4.

). The higher stomatal density showed by the EPV plants could be attributed to their higher water-holding capacity, which had in turn reduced the water-stress level. This had also resulted in the EPV plants having wider stomatal pore length than other treatments.

3.2. Effect of Vermicompost on Physicochemical Properties of Fruits

Figure 4 illustrates the different sizes of field-grown ex vitro MD2 pineapple fruits harvested from the plants supplemented with different types of fertilizers. The results of the physical analysis conducted on the fruits harvested from ex vitro grown MD2 pineapple plants are shown in

Table 4. Morphological characteristics of fruits of ex vitro grown MD2 pineapple plants supplemented with different types of fertilizers.

Parameters	Samples
	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
Estimated yield (t·ha−1)	64.74 ± 3.58 b	90.46 ± 4.62 a	85.55 ± 4.26 a
Fruit weight (g)	1248 ± 51 c	1734 ± 63 a	1540 ± 77 b
Fruit weight without crown (g)	865 ± 62 c	1436 ± 68 a	1195 ± 78 b
Crown weight (g)	398 ± 23 a	288 ± 22 b	337 ± 5 a,b
Diameter of fruit (cm)	10.5 ± 0.3 b	11.9 ± 0.3 a	12.0 ± 0.3 a
Length of fruit (cm)	12.3 ± 0.6 b	15.0 ± 0.6 a	14.5 ± 0.7 a
Length of crown (cm)	27.6 ± 1.0 a	22.7 ± 1.2 b	27.8 ± 0.8 a
Core size (cm)	1.8 ± 0.1 a	1.7 ± 0.1 a	1.8 ± 0.1 a
Pulp firmness (kg f)	0.72 ± 0.02 a	0.69 ± 0.03 a	0.68 ± 0.02 a

Means ± standard error followed by different letters in a row are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 12.

. The fruit yield was not significantly different between plants supplied with vermicompost (EPV) and the plants treated with chemical fertilizer (EPF), but both produced significantly higher fruit yield than the control plants (EPC). The fruit weight ranged from 1248 to 1734 g and was heavier than that of the commercialized MD2 pineapple fruits reported by a previous study (1132.8 g) [56]. The EPF plants produced the largest fruits, followed by EPV and control plants, but the EPF plants produced the lowest crown weight. Based on the weight of the resulting fruits, the fruits can be classified into A grade A (>1.7 kg), grade B (1.3 to 1.6 kg) or grade C (<1.3 kg) [57]. The EPF plants produced grade A fruits (1734 g), the EPV plants produced grade B fruits (1540 g) and the control (EPC) plants produced grade C fruits (1248 g). However, based on the diameter and length of the fruits, there was no significant difference between the fruits produced by plants supplied with chemical fertilizer and plants supplied with vermicompost. Overall, the ex vitro MD2 pineapple plants treated with chemical fertilizer were observed to produce the highest fruit yield and the largest fruit with smaller crown and core size, followed by the plants supplied with vermicompost and the control plants.

The physicochemical characteristics of the fruits are shown in

Table 5. The chemical analysis of fruits of ex vitro grown MD2 pineapple plants supplemented with different types of fertilizers.

Parameters	Samples
	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
pH	4.86 ± 0.15 a	4.48 ± 0.07 b	4.42 ± 0.04 b
Total soluble solid (⁰Brix)	12.6 ± 0.3 a	12.1 ± 0.2 a	12.6 ± 0.4 a
Titratable acidity (g·kg−1)	0.30 ± 0.03 b	0.32 ± 0.03 a,b	0.39 ± 0.03 a
Sugar:acid ratio	42.00	37.81	32.31
Total solid (%) w/w	18.044 ± 0.530 b	17.804 ± 1.012 b	20.841 ± 1.023 a
Ascorbic acid (µg AA/g FW fruit)	37.477 ± 1.452 a	7.896 ± 1.404 b	44.577 ± 7.467 a

Means ± standard error followed by different letters in a row are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 12. AA, ascorbic acid; FW, fresh weight.

. Data analysis showed that the pH of the juice was decreased when the titratable acidity increased. There was a significant strong negative correlation between the titratable acidity and the pH of the juice, with value of r² = −0.673 at p ≤ 0.01. Based on Table 5, the pH of the fruit juice from the EPV and EPF plants was found to be more acidic than that of fruits produced from unfertilized plants (control; EPC). The results of this study showed that the fruit acidity was significantly influenced by fertilization. The lowest acidity of fruit recorded was produced by EPC (0.300 g·kg⁻¹), and highest acidity of fruit recorded was produced EPV (0.39 g·kg⁻¹); the acidity of fruit recorded for EPF fell between these values. These results are lower than those reported by Lu et al. [56]. A similar trend was observed for the ratios of soluble solids to acid. There was a significant negative correlation between the sugar-to-acid ratio and the titratable acidity (r² = −0.758, p ≤ 0.01). According to Soler [58] and Lu et al. [56], the sugar-to-acid ratio recorded in this study fell within the recommended range for obtaining high-quality pineapple fruits (20 to 40). The percentage of the total solids was higher in the fruits harvested from EPV plants (20.841% w/w) compared to fruits harvested from the EPF and EPC plants, with total solids of 17.804% w/w and 18.044% w/w, respectively.

The nutritional value of the fruits is characterized by their contents of antioxidants such as ascorbic acid (vitamin C). The ascorbic acid content was highest in the fruits harvested from the EPV plants (44.577 µg AA/g FW), followed by fruits from the EPC plants (37.477 µg AA/g FW fruit) and the EPF plants (7.896 µg AA/g FW). These results are in agreement with reports in previous studies [59]. In terms of the chemical characteristics, the fruits from the EPV plants were found to produce competitive results when compared to those of the EPF plants but have significantly higher contents of total solids and ascorbic acid.

3.3. Effect of Vermicompost on Soil pH and Nutrient Contents

Soil pH has a direct impact on the availability of soil nutrients for plant growth. Based on data analysis, the soil pH was found to vary when different types of fertilizers were applied (

Table 6. The pH and concentrations of nutrients in the soils in which ex vitro MD2 pineapple plants were grown, measured at 6 months after planting (S1) and during red bud stages (S2) (n = 4).

pH/Total Elements	Samples
	Control (EPC)		Chemical Fertilizer (EPF)		Vermicompost (EPV)
	S1	S2	S1	S2	S1	S2
pH	5.21 ± 0.10 a,b	3.66 ± 0.08 b	4.95 ± 0.04 b	3.68 ± 0.05 b	5.80 ± 0.30 a	4.90 ± 0.29 a
N (%)	0.06 ± 0.02 c	0.10 ± 0.02 b,c	0.06 ± 0.01 c	0.13 ± 0.02 b	0.06 ± 0.01 c	0.18 ± 0.02 a
P (%)	0.02 ± 0.00 b	0.02 ± 0.00 b	0.02 ± 0.00 b	0.03 ± 0.01 a,b	0.03 ± 0.01 a,b	0.04 ± 0.00 a
K (%)	0.06 ± 0.00 b,c	0.04 ± 0.00 c	0.07 ± 0.00 a	0.06 ± 0.01 b,c	0.07 ± 0.01 a,b	0.05 ± 0.01 c
Mg (%)	0.04 ± 0.00 c	0.03 ± 0.00 c	0.04 ± 0.00 b,c	0.04 ± 0.01 c	0.05 ± 0.01 a,b	0.06 ± 0.00 a
S (%)	0.01 ± 0.00 b	0.01 ± 0.00 b	0.01 ± 0.00 b	0.01 ± 0.00 b	0.02 ± 0.00 b	0.02 ± 0.00 a
Ca (%)	0.05 ± 0.01 b	0.03 ± 0.01 b	0.05 ± 0.00 b	0.04 ± 0.01 b	0.07 ± 0.02 a,b	0.09 ± 0.01 a
Fe (%)	0.61 ± 0.04 a	0.59 ± 0.03 a	0.65 ± 0.07 a	0.66 ± 0.08 a	0.67 ± 0.07 a	0.69 ± 0.01 a
Zn (mg·kg−1)	34.32 ± 2.98 a	28.38 ± 3.13 a	33.49 ± 2.14 a	27.62 ± 2.25 a	47.31 ± 17.33 a	41.40 ± 8.08 a
B (mg·kg−1)	4.39 ± 1.16 a	0.74 ± 0.10 c	2.90 ± 0.73 a,b	1.74 ± 0.58 b,c	2.52 ± 0.20 a,b,c	1.90 ± 0.19 b,c
Al (%)	2.41 ± 0.35 a	2.44 ± 0.47 a	2.80 ± 0.47 a	3.29 ± 0.60 a	2.94 ± 0.71 a	2.81 ± 0.70 a

Means ± standard error followed by different letters in a row are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 4.

). The supplementation with vermicompost leads to a significant increase in the pH of soil when compared to the soil treated with chemical fertilizer and unfertilized (control) soils (for both samplings). Based on previous studies, the acidity of the soil was found to decrease with increased levels of vermicompost application [14,60], parallel to the findings reported in this paper. However, the soil pH was found to be more acidic during the red bud stages (S2) when compared to after 6 months of planting (S1) for all treatments. Nevertheless, the pH of soils supplemented with vermicompost was found to be within the recommended pH range for pineapple plants, i.e., pH 4.5–5.5 [61]. The soil pH of unfertilized plants drastically decreased from pH 5.21 to 3.66, where it was the lowest among all treatments. The decrease in the soil pH over time with continuous application of chemical fertilizer was in agreement with the findings from previous studies, which showed that the supplementation of NPK fertilizer decreased the soil pH [62]. This might be due to the composition of chemical fertilizer used, with 9% ammonium (NH₄⁺) and 6% nitrate (NO₃⁻) as the source of nitrogen. The leaching of NO₃⁻ and increasing H⁺ accumulation in the soils (released from NH₄⁺) can accelerate soil acidification [63].

The nutrient contents in the soils in which ex vitro MD2 pineapple plants were grown are also presented in Table 6. At S1, the Mg content in the soil supplemented with vermicompost was significantly higher than that of unfertilized (control) soil. Moreover, the K content in the soil showed a significant difference between the soil supplied with chemical fertilizer (0.07%) and unfertilized soil (0.06%) at p ≤ 0.05. No difference was observed among the treatments for other nutrients. However, the nutrient contents during S2 were found to increase when compared to the nutrient contents during S1, especially in the soil supplied with vermicompost, where the total N content had increased by two-fold. Furthermore, after the second application of vermicompost, all macronutrient contents were found to be significantly higher than those of unfertilized soils. Similar results were reported by Zaman et al. [60], where the total N; available P; exchangeable K, Ca and Mg; and available S, Zn and B were observed to be significantly increased with vermicompost application (10 t·ha⁻¹) when compared to the unfertilized soils.

The difference in contents of macronutrients (N, Mg, S and Ca) was also found to be statistically significant at p ≤ 0.05 between the soils amended with vermicompost and the soils supplied with chemical fertilizer at S2. The K, Zn and B contents were decreased in all treatments compared to S1. Surprisingly, only the soils supplied with vermicompost showed an increase in Ca content and a decrease in Al content. This could be due to its higher soil acidity compared to the soils supplied with chemical fertilizer and unfertilized soil (Table 6). These results are in agreement with a previous study reported by Angelova et al. [64], where an increment in the soil pH was observed when the soil was amended with 10 g·kg⁻¹ vermicompost, and this was highly correlated with its exchangeable Ca (r² = 0.90). Poorly buffered soils would exhibit reduced soil pH, and this condition could worsen if a high rate of N is applied to sandy soils. On the other hand, the decrease in the soil acidity could also reduce the potential of root tips being injured by aluminum (Al) and manganese (Mn) toxicity [65].

In this study, the accumulation of macronutrients by the ex vitro plants showed a similar pattern for all treatments at 6 MAP (S1), with the following decreasing order of uptake: K > N > Ca > Mg > P > S. Based on

Table 7. Concentration of macronutrients (%) in the D-leaves of ex vitro MD2 pineapple plants at 6 months after planting (S1) and during red bud stages (S2).

Samples		Macronutrients
		N	P	K	Ca	Mg	S
S1	EPC	0.68 ± 0.05 a	0.21 ± 0.04 b,c	2.33 ± 0.23 a	0.29 ± 0.03 a,b	0.22 ± 0.01 b	0.07 ± 0.01 a
	EPF	0.75 ± 0.10 a	0.13 ± 0.01 c	1.80 ± 0.10 b,c	0.26 ± 0.02 a,b	0.15 ± 0.01 c,d	0.05 ± 0.00 b
	EPV	0.77 ± 0.05 a	0.15 ± 0.01 b,c	2.17 ± 0.11 a,b	0.29 ± 0.03 a,b	0.20 ± 0.03 b,c	0.06 ± 0.00 a
S2	EPC	0.55 ± 0.07 a,b	0.17 ± 0.01 b,c	1.53 ± 0.19 c	0.30 ± 0.03 a	0.24 ± 0.01 b	0.06 ± 0.01 a
	EPF	0.66 ± 0.08 a	0.23 ± 0.04 a,b	1.75 ± 0.04 b,c	0.21 ± 0.02 b	0.13 ± 0.02 d	0.07 ± 0.00 a
	EPV	0.42 ± 0.08 b	0.30 ± 0.03 a	1.55 ± 0.14 c	0.26 ± 0.02 a,b	0.37 ± 0.04 a	0.07 ± 0.00 a
Malavolta (1)		1.5–1.7	0.23–0.25	3.9–5.7	5.0–7.0	0.18–0.20	-
Dalldorf and Langenegger (2)		1.5–1.7	±0.10	2.2–3.0	0.8–1.2	±0.3	-
Ramos et al. (3)		1.48/0.66	0.14/0.07	2.3/1.16	0.44/0.13	0.23/0.09	0.15/0.06

Means ± standard error followed by different letters in a column are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 4. ⁽¹⁾ Ideal concentrations at 4 months (whole leaf) [66]. ⁽²⁾ Ideal concentrations at inflorescence emergence (whole leaf). ⁽³⁾ Ideal concentrations/deficiency concentrations at floral induction [68]. EPC, control; EPF, chemical fertilizer; EPV, vermicompost.

, the N content in the D-leaves of ex vitro MD2 pineapple plants at S1 ranged between 0.68 and 0.77%, and these values were lower than the ideal concentration of N during vegetative stages (4 months after planting) reported by Malavolta [66] (1.5 to 1.7%). Similar trends were observed for the P, K and Ca concentrations. However, there was no significant difference observed between the N, P, K and Ca contents in the D-leaves of the EPV plants and those of the EPF and EPC plants. According to Malavolta [66], the Mg content in the EPV plants (0.20%) showed an ideal concentration (0.18 to 0.20%) for pineapple growth. Moreover, the D-leaves of the EPV plants contained significantly higher S amounts than those of the EPF plants. Although vermicompost was supplied 6 months before sampling, the nutrient uptake of the plants grown on vermicompost-amended soils was found to be comparable to that of the plants supplied with chemical fertilizer. This was probably due to vermicompost being a ‘slow-release fertilizer’ with properties that allowed the plants to absorb these nutrients over time [18]. The nutrient contents of different plant components such as roots, shoots and fruits also were also found to improve when vermicompost was supplied to the soils [67].

The N content in the D-leaves of the ex vitro plants (for all treatments) decreased during S2 and was considered as inadequate for pineapple plants at floral induction based on values recommended by Ramos et al. [68]. However, the low N uptake by plants during flowering resulted in better fruit chemical properties (Table 5). According to Ramos and Pinho [69], the deficiency of N led to the increase of total soluble solids (TSS) of Jupi pineapple by 11.2%, titratable acidity by 85% and vitamin C content by three-fold compared to the fruits supplied with complete nutrient solution. Similar results were obtained by Omotoso and Akinrinde [70], where 40.1% reduction of fruit juice acidity (relative to control) was observed with high N fertilization rates. The highest TSS content was also recorded in the crops that received the lowest N fertilizer; 50 kg·N·ha⁻¹ [70]. In this study, the Pearson’s correlation coefficient analysis revealed that the N content showed a significant negative correlation with TSS (r² = −0.795, p ≤ 0.01), titratable acidity (r² = −0.750, p ≤ 0.01) and ascorbic acid content (r² = −0.669, p ≤ 0.05). Similar trends were observed for the K, Ca and S concentrations, but all these values were still considered as adequate to support plant growth. Other than that, the levels of P and Mg contents in the D-leaves were observed to be higher in the EPV plants than in other treatments. Soil pH had a significant positive correlation with P content (r² = 0.588, p ≤ 0.05) and Mg content (r² = 0.778, p ≤ 0.01) in the D-leaves at the red bud stage (S2). The soil acidity for the EPF and EPC plants were found to be too acidic for pineapple plant growth and hence reduced the availability of nutrients in the soil, which further decreased the plant’s nutrient uptake.

In terms of the micronutrient contents, at both S1 and S2, the Fe contents (for all treatments) in the ex vitro plants were observed to be lower than the ideal concentration required for pineapple plants (

Table 8. Concentration of micronutrients and Al content (mg·kg⁻¹) in the D-leaves of ex vitro MD2 pineapple plants at 6 months after planting (S1) and during red bud stages (S2).

Samples		Micronutrients/Al Content
		Fe	Zn	B	Al
S1	EPC	127.37 ± 38.55 a	43.41 ± 5.65 a,b	9.48 ± 0.72 a	67.14 ± 28.59 a
	EPF	45.50 ± 9.46 b	32.16 ± 2.50 b	4.96 ± 0.23 b	32.70 ± 11.38 a
	EPV	69.97 ± 22.86 a,b	46.99 ± 6.47 a	8.00 ± 0.89 a	37.52 ± 30.71 a
S2	EPC	40.65 ± 5.14 b	32.18 ± 1.87 b	8.43 ± 0.45 a	53.28 ± 12.55 a
	EPF	45.93 ± 9.58 b	13.95 ± 1.39 c	7.24 ± 1.00 a	39.90 ± 22.33 a
	EPV	41.77 ± 9.73 b	14.14 ± 2.41 c	9.49 ± 0.69 a	22.61 ± 3.83 a
Malavolta (1)		600–1000	17–39	-	-
Dalldorf and Langenegger (2)		100–200	±10	30	-
Ramos et al. (3)		-	-	20/5.6	-

Means ± standard error followed by different letters in a column are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 4. ⁽¹⁾ Ideal concentrations at 4 months (whole leaf) [66]. ⁽²⁾ Ideal concentrations at inflorescence emergence (whole leaf) [71,72]. ⁽³⁾ Ideal concentrations/deficiency concentrations at floral induction [68]. EPC, control; EPF, chemical fertilizer; EPV, vermicompost.

). Nevertheless, the EPV plants at S1 showed a higher Zn concentration (46.99 mg·kg⁻¹) than the required range recommended by Malavolta [66] (17 to 39 mg·kg⁻¹). Although the EPC plants contained more Zn than the plants supplied with chemical fertilizer and vermicompost during S2, their range of Zn concentration was still considered to be adequate for plants during flowering. In contrast, the B concentration in the ex vitro plants (for all treatments) was lower than the ideal concentration during flowering and was near the B-deficiency level for the plants. Overall, the EPV plants showed higher content of micronutrients during S1. During S2, the micronutrient contents recorded in the EPV plants were found to be comparable to those of the EPF plants.

As the soil pH drops below 5, Al is solubilized into toxic forms [73]. The excess Al³⁺ in the soil enters roots and then inhibits the root growth, which limits water and interferes with the uptake, transport and utilization of most mineral elements. Under Al stress, the deficiency symptoms of some essential nutrients, including phosphorus (P), calcium (Ca²⁺), magnesium (Mg²⁺), potassium (K⁺) and iron (Fe), can be easily detected [73]. According to Mota et al. [74], the increment in Al concentration reduced the accumulation of K and Mg in the roots; K in the stems; and N, P and K in the fruits of the ‘Vitoria’ pineapple plants. In this study, the D-leaves of ex vitro plants of all treatments showed lower N, P, K, Ca and Fe contents than the recommended range. In contrast, the P and Mg contents were higher than the recommended range. The EPV plants showed the lowest accumulation of Al, which is possibly related to the mechanism of defense to Al toxicity. For example, P can help retard the entry of Al in the apoplast through the formation of insoluble compounds such as Al₄(PO₄)₃ [74].

3.4. Determination of Bioactive Compounds and Antioxidant Capacity

The chlorophyll and carotenoid contents in the methanolic fruit extracts produced from ex vitro MD2 pineapple plants grown in the field with different types of fertilizers were also investigated. Based on

Table 9. Chlorophyll, total carotenoid and phenolic contents (µg/g) of the methanolic fruit extracts of ex vitro MD2 pineapple fruits grown in the field with different types of fertilizers.

Parameters	Samples
	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
Ca (µg/g)	0.525 ± 0.014 b	0.627 ± 0.026 b	0.977 ± 0.086 a
Cb (µg/g)	2.349 ± 0.077 b	2.128 ± 0.098 b	3.094 ± 0.136 a
Ca + Cb (µg/g)	2.874 ± 0.074 b	2.754 ± 0.072 b	4.071 ± 0.70 a
C(x+c) (µg/g)	2.834 ± 0.030 b	3.080 ± 0.060 a	2.890 ± 0.054 b
Ca/Cb ratio	0.224 ± 0.011 a	0.297 ± 0.026 a	0.319 ± 0.043 a
Ca + Cb/C(x+c) ratio	1.015 ± 0.032 b	0.896 ± 0.041 b	1.410 ± 0.049 a
Total pigments Ca + Cb + C(x+c) (µg/g)	5.708 ± 0.065 b	5.834 ± 0.012 b	6.961 ± 0.031 a
Total phenolic content (mg GAE/g dE)	8.212 ± 0.567 a	6.083 ± 0.273 b	6.055± 0.141 b

Means ± standard error followed by different letters in a column are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 3. C_a, chlorophyll a; C_b, chlorophyll b; C_a + C_b, total chlorophyll a and b; C_(x+c) total carotenoids (xanthophyll and carotene); GAE, gallic acid equivalent; dE, dried extract.

, the fruit extracts from the EPV plants contained significantly higher chlorophyll a (0.977 µg/g), chlorophyll b (3.094 µg/g) and total chlorophyll contents (4.071 µg/g) than those from other treatments. On the other hand, the EPF plants produced fruits with significantly higher amounts of total carotenoids (3.080 µg/g). The carotenoid contents are influenced by several pre- and postharvesting factors such as the ripening time, production practice and growing locations, as well as the climatic conditions such as light and temperature [75]. In a previous study conducted on tomato plants, it was found that K fertilization can affect carotenoid biosynthesis [76]. The ratio of chlorophyll to carotenoid content changes during ripening, where the chlorophyll content will decrease with increasing carotenoid content as the fruit ripens [77]. This is in line with the findings obtained in this study, where the pineapple fruits produced with vermicompost contained significantly higher total chlorophyll content, thus yielding a lower carotenoid content.

The phenolics are formed to protect the plants from reactive oxygen species (ROS), photosynthetic stress and herbivory [78]. They could also be produced to provide protection against abiotic stresses such as UV-B irradiation, heat stress, low water potential or mineral deficiency [78]. In this study, the methanolic fruit extract obtained from the EPV plants contained the lowest total phenolic content (6.055 mg GAE/g dried extract), followed by EPF (6.083 mg GAE/g dried extract) and EPC plants (8.212 mg GAE/g dried extract) (Table 9). Similar results were reported in a previous study, in which the total phenolic content of C. nutans leaves was found to be significantly higher in the control plants (unfertilized) than in plants supplied with chemical fertilizer and plants supplied with vermicompost [40]. On the other hand, this could also be due to Al stress, as the control (EPC) plants were found to contain the highest Al accumulation (Table 8). According to Meriño-Gergichevich et al. [79], Al toxicity triggers an increase in ROS, which may then increase or inhibit antioxidant ROS-scavenging activities.

The fruit pulp extract was also examined for its radical scavenging and antioxidant activities. For a more complete picture of the antioxidant capacity of the pineapple fruit extracts, more than one method was used. The antioxidant capacity was measured by DPPH, ABTS and FRAP assays. These methods measure the ability of the antioxidants to scavenge for specific radicals, to inhibit lipid peroxidation or to chelate metal ions [80]. The IC₅₀ values for DPPH and ABTS assays and the FRAP values of the methanolic extracts of fruits produced from ex vitro MD2 pineapple plants are shown in

Table 10. Antioxidant capacities of the methanolic extracts of fruits produced from ex vitro MD2 pineapple plants grown with different types of fertilizers.

Antioxidant Capacities	Standard/Samples
	Ascorbic Acid (Standard)	Control (EPC)	Chemical Fertilizer (EPF)	Vermicompost (EPV)
DPPH, IC50 (mg/mL)	0.050 ± 0.001 d	6.022 ± 0.036 c	8.660 ± 0.102 a	8.250 ± 0.035 b
ABTS, IC50 (mg/mL)	0.065 ± 0.002 c	7.361 ± 1.775 b	10.502 ± 1.791 a,b	12.559 ± 0.126 a
FRAP (mg FE/g dE)	29.074 ± 4.800 a	0.301 ± 0.030 b	0.181 ± 0.014 b	0.220 ± 0.021 b

Means ± standard error followed by different letters in a column are significantly different based on Duncan’s multiple range test (DMRT) at p ≤ 0.05, n = 3. FE, ferric equivalent; dE, dried extract.

. The highest antioxidant potential (denoted by the lowest IC₅₀) against DPPH radicals was recorded in fruit extracts produced from the control (EPC) plants (IC₅₀ of 6.022 mg/mL), followed by those of EPV (IC₅₀ of 8.250 mg/mL) and EPF (IC₅₀ of 8.660 mg/mL). The EPV fruit extracts also showed the lowest scavenging activity against ABTS radicals. In addition, the FRAP reducing power exhibited by the EPV fruits was observed to be comparable to that of EPF fruits, but both showed lower reducing power than the control (EPC) fruits. Overall, the antioxidant capacities of the methanolic fruit extracts, arranged in decreasing order, were EPC > EPV > EPF. Similarly, the application of vermicompost has been reported to enhance the antioxidant activities of field-grown cassava when compared to the application of inorganic fertilizer (NPK) [81].

A correlation analysis was also done between the antioxidant capacities of the extracts and the total phenolic contents of the fruits. Based on the Pearson’s correlation coefficient, a strong significant correlation was found between the total phenolic content and antioxidant capacities determined by DPPH (r² = −0.876, p ≤ 0.01), ABTS (r² = −0.819, p ≤ 0.01) and FRAP (r² = 0.897, p ≤ 0.01) assays. These results are in agreement with several previous studies that showed that the TPC of pineapple fruit extract correlates with its DPPH radical scavenging potential [56,82,83].

4. Conclusions

The preceding results showed that the utilization of vermicompost at the rate 10 t ha⁻¹, applied twice throughout the planting period, produced competitive results (in terms of the growth of the ex vitro MD2 pineapple plants) when compared with those obtained using conventional cultivation practice through regular supplementation with chemical fertilizer. The EPV plants had higher stomatal density and smaller stomata sizes, but they had lower SPAD values. Moreover, the application of vermicompost produced fruits that were smaller in size but contained higher TSS, titratable acidity, total solids, ascorbic acid content, chlorophyll content and antioxidant capacities than fruits produced with chemical fertilizer. The results of the soil analysis showed that the application of vermicompost significantly increased the soil pH and was able to retain the nutrient contents in the soils. Although the uptakes of some of the nutrients by the plants were lower than the ideal concentrations required for pineapple growth, these were similar to when chemical fertilizer was used. Thus, it could be deduced that both types of fertilizers (chemical fertilizer and vermicompost) could not supply the ideal concentration of nutrients required by pineapple plants when they were used as the sole nutrient provider for the sandy loam soil. Therefore, further research needs to be carried out to identify the best ratio of combination between vermicompost and chemical fertilizer to support plant growth and development, ensure agricultural sustainability and further reduce environmental pollution.