Application of Azolla spp. as a Growing Medium Component for Melon Grown in a Soilless Culture System

Abstract

Azolla spp. is a prominent macrophyte and one of the world’s fastest growing plants, making it a highly important resource that has been widely exploited as a biofertilizer, food and feed, and biofuel production. As a result, the uses, promotion, and application of Azolla spp. in sustainable agriculture would be of significant interest. The objectives of this study were (i) to assess the feasibility of using Azolla as a growing media of melons with a reduction in fertigation concentration in a soilless culture system, and (ii) to compare the agronomic and fruit quality traits of melons grown in different Azolla treatments. The experiment was carried out using a completely randomized design with 4 treatments including: (1) control treatment (1:1 combination of chopped coconut mesocarp (CCM) and coconut coir (CC) with recommended fertigation concentration (RFC)), (2) dried Azolla (1:1:2 combination of CCM, CC, and dried Azolla with 75% of RFC), (3) fermented Azolla (1:1:2 combination of CCM, CC, and fermented Azolla with 75% of RFC), and (4) fresh Azolla (1:1:2 combination of CCM, CC, and fresh Azolla with 75% of RFC). Seven melons per treatment were recorded on the agronomic traits (stem length, stem diameter, leaf width, leaf length, and leaf number) and fruit quality (fruit weight, fruit height, fruit perimeter, rind thickness, pulp thickness, fruit cavity, pulp firmness, pulp color (L*, a*, b*, Hue, and c*), total soluble solid, juice electrical conductivity, juice pH, and juice titratable acidity). Statistical analyses revealed the differences among melon agronomic and yield traits under different Azolla treatments. Nutrient analysis results showed that fermented and fresh Azolla had a high nutritional value as compared to dried Azolla. Most fruit traits from fermented and fresh Azolla treatments with a fertilizer reduction were not significantly different as compared to control. Both treatments were recommended for melon production in a soilless culture system, since these treatments can reduce fertigation use by 25% while maintaining growth and fruit quality when compared to conventional practice.

1. Introduction

Melon, Cucumis melo L., is a popular annual fruit crop of the Cucurbitaceae family. Melon fruit is a rich source of nutrients and phytochemicals, i.e., dietary fibers, minerals, vitamin C, folic acid, provitamin A, phenolic compounds, and cucurbitacin, which may assist with preventing human diseases [1]. In general, the high-quality melon fruit has a high marketable price and is required by customers of all ages. It is used worldwide as a fresh cut fruit or as an ingredient in desserts like cake, ice cream, juice, melon bingsu (Korean shaved ice with melon), and many more products. Melon fruit quality is classified primarily by sweetness, acidity, color, aroma, and shelf life [1,2].

In melon production, a soilless culture system in a greenhouse is widely used to produce high-quality fruit. A plant-growing technique known as a “soilless culture system” does not use soil as the growing medium. Melon absorbs nutrients from irrigation water, which is called “nutrient solution”. This system has advantages in the reduction of soil-borne infections, the capacity to regulate the concentration of plant nutrients and water used, and the reduction in nutrient unavailability form [3]. This technique can be utilized in the production of several horticultural crops and vegetables, such as lettuce, strawberry, tomato, and melon [4]. Coconut fiber is generally used as a growing medium. However, it contains low nutrients which do not meet the plant needs and requires them to be mixed with other components such as organic fertilizers, cow manure, green manure, etc. [5]. Because animal manure can occasionally be a cause of plant disease, foul odor, greenhouse dirt, and some human pathogenic microorganisms that pose potential hazards to human health [6], it may not be advised to use it as a component of the melon growing medium. Green manure might be suitable for the component. Fertilizer is a high proportion cost (38–50% of variable cost) for our previous cultivation, and one of the key successes for melon production. Thus, the alternate source of fertilizer is crucial for reducing production costs by minimizing the use of fertilizer in melon production.

Azolla spp. is a water fern that is used for several purposes in agriculture, such as biofertilizer, animal feed, growing media, etc. It is the host of N-fixing cyanobacteria (Anabaena spp.) and occurs in fresh water of tropical to warm-temperate regions [7]. The symbiosis of Azolla–Anabaena is capable of fixing atmospheric nitrogen at 1.1 kg N ha⁻¹ day⁻¹. Azolla spp. has a low C/N ratio which releases minerals faster than other organic fertilizers. It provides 60–80% of nitrogen in a couple of weeks when incorporated in soil with logged water [8]. Azolla spp. contains high organic matter, crude protein, vitamins, and multiple plant nutrients, especially trace elements [9]. Azolla spp. has generally been used in rice cultivation as a green fertilizer. In addition, it was also used as a biofertilizer in various crops such as tomatoes, taro, bananas, wheat, corn, etc. Azolla spp. has a positive effect on soils in both physical and chemical features by lowering the ammonia volatility that occurs after the use of chemical nitrogen fertilizers [10]. Currently, the utilization of Azolla spp. as a biofertilizer has been used in several crop productions because it has several advantages as compared to chemical fertilizers. The advantages are (1) it is inexpensive, making use of freely available solar energy, atmospheric nitrogen, and water; (2) it utilizes renewable resources, as opposed to chemical fertilizer production, which is dependent on petroleum; and (3) it provides for plant nutrients and other substrates that are advantageous to plants or some animals, such as growth promoter intermediaries, vitamins (vitamin A, beta-carotene, and vitamin B12), amino acids, etc. [11]. From the positive aspects of Azolla spp., it might be beneficial for melon growers if Azolla spp. can be used as a growing medium with a reduction in fertilizer used. Hence, the objectives of this study were (i) to assess the feasibility of using Azolla spp. as a growing media of melons with a reduction in fertigation concentration in a soilless culture system and (ii) to compare the agronomic and fruit quality traits of melons grown in different Azolla treatments. The findings of this study can be applied further by researchers and farmers, potentially impacting food security and sustainability.

2. Materials and Methods

2.1. Experimental Design

Azolla filiculoides and Azolla pinnata, known as Azolla collectively, were harvested from our cultivation (mixing) and used in the experiment. Dried Azolla was prepared by dehydrating with sunlight for 2 days. The fermented Azolla was prepared by fermenting it spontaneously for 7 days under aerobic conditions. The samples from the Azolla treatment were analyzed for nutrient status, including organic matter, nitrogen, phosphorus, potassium, calcium, and magnesium. The procedure for nutritional analysis followed Motsara and Roy [12].

The experiment was set up using a completely randomized design (CRD) with 4 treatments of planting media and fertigation. The treatments were control (a one-to-one mixture of chopped coconut mesocarp (CCM) and coconut coir (CC) with recommended fertigation concentration), dried Azolla (1:1:2 combination of CCM, CC, and dried Azolla with 75% of recommended fertigation concentration), fermented Azolla (1:1:2 combination of CCM, CC, and fermented Azolla with 75% of recommended fertigation concentration), and fresh Azolla (1:1:2 combination of CCM, CC, and fresh Azolla with 75% of recommended fertigation concentration). The coconut mesocarp was obtained from 12-year-old coconut trees in Tha Sala District, Nakhon Si Thammarat Province, Thailand.

The recommended fertigation concentration was followed by previous report of Khomphet et al. [13]. The fertigation prepared by the fertilizer was made from calcium nitrate (89.5 g Ca(NO₃)₂ 100 L⁻¹), potassium nitrate (74 g KNO₃ 100 L⁻¹), magnesium sulfate (40 g MgSO₄ 100 L⁻¹), monoammonium phosphate (19.2 g NH₄H₂PO₄ 100 L⁻¹), Fe-EDTA (1.63 g 100 L⁻¹), manganese sulfate (0.19 g MnSO₄ 100 L⁻¹), zinc sulfate (0.14 g ZnSO₄ 100 L⁻¹), copper sulfate (0.08 g CuSO₄ 100 L⁻¹), sodium borate (0.06 g Na₂[B₄O₅(OH)₄] 100 L⁻¹), and sodium molybdate (0.013 g Na₂MoO₄ 100 L⁻¹). The melons received fertilizer four times a day at 7 a.m., 10 a.m., 1 p.m., and 4 p.m. The experiment was carried out in the greenhouse at the School of Agriculture Technology and Food Industry, Walailak University, Nakhon Si Thammarat, Thailand.

2.2. Plant Materials

Melon cultivar “Melon Cat 697” from Chia Tai Co., Ltd. (Bangkok, Thailand), was purchased and used for the experiment. Melon seeds were soaked with warm water at 40 °C for an hour, covered with wet paper for 48 h, and transferred to plastic tray using peatmoss as the germination medium. Water was applied to the seeds twice daily using a foggy system. The 15-day seedlings were used for the experiment. The seedlings were transplanted into plastic pots (25.4 cm in diameter and 15.1 in height) with different Azolla treatments as a growing medium. The plastic pots were spaced at 60 cm between plants and 50 cm between rows.

2.3. Data Collection, Experimental Places, and Weather Conditions

For data collection, seven melons per treatment were recorded on the agronomic traits at 1–5 weeks after planting and harvestable fruits were assessed the fruit quality at 6 weeks after pollination. The agronomic traits were stem length, stem diameter (measured at the middle of stem), leaf width, leaf length (measured at the newest mature leaf), and leaf number (counted only mature leaves). The fruit traits were fruit weight, fruit height, fruit perimeter, rind thickness, pulp thickness, fruit cavity, pulp firmness, pulp color, total soluble solid (TSS), juice electrical conductivity (EC), juice pH, and juice titratable acidity (TA).

The pulp’s color was measured using a portable Hunterlab ColorFlex^®EZ device (Hunter Assoc. Laboratory, Reston, VA, USA) in tristimulus mode, where L* stands for lightness and is measured on a scale of 0 to 100, a* for redness and greenness, and b* for yellowness and blueness. Hue is referred to as a color’s appearance parameters, while chroma (c*) corresponds to the saturation level of a color, with clear, bright, and brilliant being possible chroma values.

The TSS was assessed by a brix refractometer (Sinotech (Thailand) Co., Ltd. (Bangkok, Thailand), model RHB-18ATC) and the results were expressed as °Brix.

Melon juice’s EC and pH were assessed using EC and pH meters (Hanna Instruments, Inc. (Smithfield, RI, USA), edge^® model HI2030).

Juice extract was titrated to pH 8.1 with 0.1 M NaOH to determine the TA, and the percentage of citric acid in the samples was then measured [14].

The fruit quality of melon was analyzed at The Center for Scientific and Technological Equipment, Walailak University. The experiment was conducted during April to June 2023. During the experiment, rainfall varied from 0.6 to 143.8 mm, and relative humidity varied from 77.6 to 87.6%, with June observing the highest levels of both rainfall and humidity and May having the highest relative humidity. The temperature ranged between 23.7 and 37.7 °C, with April as the hottest month. The weather data was collected from the greenhouse’s weather station.

2.4. Data Analysis

The observed traits were assessed via an analysis of variance. The mean of traits was compared according to Duncan’s new multiple range test and the differences were reported at p < 0.05 or 0.01. All statistical analyses were performed by R software (version 4.1.2) with Agricolae package [15] and the boxplot was performed by Microsoft Excel.

3. Results and Discussion

3.1. Nutrient Status of Azolla

The nutrient analysis of different Azolla treatments is presented in

Table 1. Nutrient analysis of different Azolla treatments.

Nutrients	Dried Azolla	Fermented Azolla	Fresh Azolla
Organic matter	73.58%	84.66%	78.65%
Nitrogen	4.81%	6.32%	5.15%
Phosphorus	0.33%	0.84%	0.41%
Potassium	1.17%	1.21%	1.35%
Calcium	1.88%	2.15%	1.93%
Magnesium	0.59%	0.45%	0.58%

. The highest organic matter (OM) was obtained from fermented Azolla (80.66%), while dried Azolla shows the lowest OM (76.58%). Fermented Azolla also provides the highest percentage of nitrogen (6.32%), phosphorus (0.84), and calcium (2.15%); however, the fresh and dried Azolla shows higher values of magnesium (0.58 and 0.59%), and fresh Azolla provides the highest potassium (0.35%). The nutrients of Azolla treatments are similar or higher than some previous report on Azolla nutrient components. Cherryl et al. [16] assessed the nutritive value of Azolla pinnata, and reported that the Azolla contained organic matter at 75.73%, phosphorus at 0.26%, and calcium at 2.58%. Alalade and Iyayi [17] determined the chemical composition of Azolla pinnata used as feeding of egg-type chicks. They reported that the Azolla provided calcium at 1.16%, phosphorus at 1.29%, potassium at 0.35%, and some trace elements (copper, zinc, manganese, and iron) at 16.74–775.73 ppm. The nutritional value of Azolla pinnata as a feed for animals was evaluated by Anitha et al. [9]. They found that Azolla contained organic matter at 82.66%, calcium at 1.64%, potassium at 2.71%, phosphorus at 0.34% and other trace elements (nickel, cobalt, copper, boron, zinc, iron, and manganese) at 5.3–2418 ppm. The different nutritive values in Azolla might depend on several factors such as genotype, culture method, fertilizer supply, and water quality [18].

3.2. Agronomic Trait of Melons

The analysis of variance of agronomic traits of melons is presented in

Table 2. Analysis of variance of agronomic traits of melons grown under different Azolla treatments.

Traits	Weeks after Planting	MSTr	MSE	CV (%)
Stem length (cm)	1st	214.269 **	11.16	26.02
	2nd	786.26 *	184.59	35.84
	3rd	420.91 ns	723.76	28.17
	4th	849.38 ns	892.07	16.31
	5th	2231.16 *	696.45	10.29
	6th	580.75 ns	637.87	8.60
Stem diameter (cm)	1st	0.08 **	0.004	10.62
	2nd	0.06 **	0.004	7.31
	3rd	0.001 ns	0.004	7.67
	4th	0.01 ns	0.01	9.44
	5th	0.02 ns	0.01	9.66
	6th	0.02 *	0.01	10.85
Leaf width (cm)	1st	8.95 *	2.18	13.19
	2nd	12.26 **	2.65	12.13
	3rd	5.35 *	1.34	7.61
	4th	4.62 *	1.24	6.89
	5th	7.83 **	1.48	8.71
	6th	19.19 **	2.02	14.71
Leaf length (cm)	1st	8.95 *	2.18	13.19
	2nd	10.71 *	2.95	12.37
	3rd	15.88 **	1.81	8.74
	4th	8.57 **	1.33	6.77
	5th	9.76 **	1.77	8.87
	6th	20.25 **	2.11	13.41
Leaf number (no.)	1st	5.86 **	0.46	14.78
	2nd	18.43 **	2.99	18.62
	3rd	7.98 ns	3.76	11.18
	4th	1.66 ns	5.60	9.29
	5th	2.96 ns	4.99	6.94
	6th	9.80 ns	6.19	6.37

^ns, *, ** are not significantly different, significantly different at p < 0.05 and 0.01 (Duncan’s test), respectively.

. All attributes were significantly different among the Azolla treatments in most observed times. The boxplot of agronomic traits of melons grown under different Azolla treatments is illustrated in Figure 1.

Six weeks after planting, stem lengths were between 284.60 ± 16.12–302.69 ± 17.25 cm. The longest stem was obtained from samples with dried Azolla and the shortest stem was obtained from the samples with fermented Azolla.

Stem diameters were 0.68 ± 0.08–0.77 ± 0.08 cm. The biggest stem was obtained from samples in control treatment and the smallest stem was obtained from samples with fresh Azolla.

Leaf widths were 8.21 ± 2.20–11.25 ± 1.52 cm. The widest leaf was obtained from sample with dried Azolla and the narrowest leaf was obtained from sample with fresh Azolla.

Leaf lengths were 9.15 ± 2.38–12.05 ± 0.98 cm. The longest leaf was obtained from samples with fermented Azolla and the shortest leaf was obtained from samples with fresh Azolla.

Leaf numbers were 37.70 ± 3.27–39.70 ± 3.27 leaves, The most leaf numbers was obtained from samples with fermented Azolla and the least leaf numbers was obtained from samples with fresh Azolla.

3.3. Melon Fruit Traits

Table 3. Analysis of variance of fruit traits of melons grown under different Azolla treatments.

Fruit Traits	MSTr	MSE	CV (%)
Fruit weight (kg)	0.54 **	0.04	13.34
Fruit height (cm)	4.23 **	0.79	5.87
Fruit perimeter (cm)	7.45 **	1.06	6.96
Rind thickness (cm)	0.24 **	0.03	20.42
Pulp thickness (cm)	1.19 *	0.26	17.32
Fruit cavity (cm)	1.80 **	0.20	7.92
Pulp firmness (N)	0.02 ns	0.01	31.86
Lightness (L*)	34.06 *	11.44	4.69
Redness (a*)	10.53 *	4.98	29.11
Yellowness (b*)	47.01 *	36.47	15.16
Color appearance (Hue)	2.21 *	1.72	3.58
Color saturation (c*)	54.00 *	39.60	15.50
Total soluble solid (°Brix)	10.59 **	2.14	14.03
Juice electrical conductivity (µS/cm)	763,548 ns	318,938	9.78
Juice pH	0.03 ns	0.02	2.10
Juice titratable acidity (%)	0.01 **	0.00	16.95

^ns, *, ** are not significantly different, significantly different at p < 0.05 and 0.01 (Duncan’s test), respectively.

showed the analysis of variance of fruit traits of melons grown under different Azolla treatments. Fruit weight (0.54 **), fruit height (4.23 **), fruit perimeter (7.45 **), rind thickness (0.24 **), pulp thickness (1.19 *), fruit cavity (1.80 **), lightness (34.06 *), redness (10.53 *), yellowness (47.01 *), color appearance (2.21 *), color saturation (54.00 *), total soluble solid (10.59 **), were significantly different among the Azolla treatments. However, there is a nonsignificant difference found in the pulp firmness (0.02 ^ns), juice electrical conductivity (763,548 ^ns), and juice pH (0.03 ^ns).

Table 4. Mean comparison of fruit traits of melons grown under different Azolla treatments.

Treatments	FP (cm)	FH (cm)	FW (kg)	RT (cm)	PT (cm)	FC (cm)	PF (N)	TSS (°Brix)	EC (µS/cm)	pH	TA (%)
Control	15.83 ± 0.75 a	15.50 ± 0.84 a	1.69 ± 0.12 a	0.67 ± 0.16 b	3.41 ± 0.38 a	5.76 ± 0.38 a	0.33 ± 0.14	12.02 ± 1.47 a	6173.33 ± 341.33	6.34 ± 0.14	0.16 ± 0.02 a
Dried Azolla	13.20 ± 1.64 b	13.80 ± 1.10 b	1.00 ± 0.34 b	0.82 ± 0.20 b	2.29 ± 0.98 b	4.80 ± 0.75 b	0.39 ± 0.14	9.62 ± 1.89 b	5752.00 ± 996.93	6.16 ± 0.11	0.09 ± 0.01 b
Fermented Azolla	15.36 ± 0.85 a	15.79 ± 0.81 a	1.64 ± 0.18 a	1.13 ± 0.22 a	2.89 ± 0.16 ab	6.14 ± 0.21 a	0.36 ± 0.08	11.26 ± 0.94 ab	5348.57 ± 457.73	6.28 ± 0.13	0.10 ± 0.02 b
Fresh Azolla	14.43 ± 0.84 a	15.21 ± 0.86 a	1.44 ± 0.12 a	0.87 ± 0.13 ab	3.08 ± 0.34 a	5.70 ± 0.41 a	0.44 ± 0.12	11.53 ± 1.55 ab	5870.00 ± 383.54	6.28 ± 0.14	0.09 ± 0.02 b
F-test	**	**	**	**	*	**	ns	**	ns	ns	**

ns, *, ** are not significantly different, significantly different at p < 0.05 and 0.01 (Duncan’s test), respectively. Means followed by the same letter do not differ significantly. RT: rind thickness, PT: pulp thickness, FC: fruit cavity, PF: pulp firmness, TSS: total soluble solid, EC: juice electrical conductivity, pH: juice pH, TA: juice titratable acidity, FP: fruit perimeter, FH: fruit height, FW: fruit weight.

showed the mean comparison of fruit traits of melons grown under different Azolla treatments. As compared to control treatments, the treatments of fermented and fresh Azolla were not significantly different in the traits of fruit perimeter (15.36 ± 0.85 and 14.43 ± 0.84 cm), fruit height (15.79 ± 0.81 and 15.21 ± 0.86 cm), fruit weight (1.64 ± 0.18 and 1.44 ± 0.12 kg), pulp thickness (2.89 ± 0.16 and 3.08 ± 0.34 cm), fruit cavity (6.14 ± 0.21 and 5.70 ± 0.41 cm), and total soluble solid (11.26 ± 0.94 and 11.53 ± 1.55 °Brix). Whereas the fruits from the treatments of dried Azolla were the lowest of those traits.

The radar chart of melon fruit pulp is illustrated in Figure 2. The melon fruit pulp from control treatment shows the highest value of redness (a*; 9.26), yellowness (b*; 43.45), and color saturation (c*; 44.46), whereas it shows the lowest values of lightness (L*; 68.73) and color appearance (Hue; 36.24). The melon fruit pulp from dried Azolla treatment shows the highest value of lightness (L*; 73.88) and color appearance (Hue; 37.58), however, it was the lowest value of redness (a*: 5.90), yellowness (b*; 36.34), and color saturation (c*; 36.84).

Several studies have reported that the nutrients supplied impacted fruit quality and pulp color. Piñero et al. [14] investigated the fish water with and without synthetic fertilizers for melon production. According to their findings, treatments using fish water and foliar supplements increased fruit size and weight but decreased pulp lightness and had lower levels of total amino acids, proteins, nitrate, and potassium ion. Ferrante et al. [19] also reported that the increase in lightness and yellowness in melon pulp was influenced by a higher supply of nitrogen fertilizer.

The quality of melon fruits depends on multiple factors such as variety, pollination, soil property, irrigation, fertilizer management, and growing season [20]. Herein, the utilization of Azolla as growing medium component is successful for melon production, especially the treatment of fermented and fresh Azolla. Both Azolla treatments provide a higher percentage of organic matter (78.65 and 84.66%), nitrogen (5.15 and 6.32%), and calcium (2.15 and 1.93%), than the treatment of dried Azolla. An organic matter release from Azolla promotes the growth and fruit quality of melons due to enhancing of pH and nutrient level in growing media [21]. Organic matter accounts for 50–90% of cation absorbing power, which improves the availability of plant nutrients especially potassium, calcium, magnesium, and other cation forms. It is a major indigenous source of available nitrogen, phosphorus, and some among of sulfur. Organic matter also functions as pH buffering capacity, which controls the fluctuation of pH [22]. Nitrogen and calcium play an important role in melon cultivation since both affect yield and fruit size, as well as texture and sugar content [14]. Moreover, Azolla has the ability to provide some micronutrients to plant. Several micronutrients (B, Cu, Fe, Mn, Mo, and Zn) are associated with fruit quality traits essential rind cracking incident. The lack of these nutrients is resulting in a reduction in fruit quality [23].

There are a few studies related to the use of Azolla as growing media in plants. According to Petruccelli et al. [10], they used the quantity of Azolla at 0, 25, 50, and 100% as a growing medium component in the nursery production of olive trees. They reported that a quantity of 50% Azolla was suitable for growing the olive tree because it displayed the best total biomass production and linear growth. This study is the first report on the use of Azolla as a growing medium component of melon production. The results from this study might be used as basic information. The use of Azolla in combination with various fertilizer levels or other crops is needed for further research.

4. Conclusions

The use of Azolla as a plant growing medium for melons revealed no significant variations in several agronomic and fruit attributes when compared to the control treatment. Fermented and fresh Azolla showed a high nutritional value as compared to dried Azolla and both Azolla treatments with a fertilizer reduction were not significantly different in most fruit traits (fruit perimeter, fruit height, fruit weight, pulp thickness, fruit cavity, and total soluble solid) as compared to control. When compared to typical practice, the treatment of fermented and fresh Azolla can be proposed for melon production in a soilless culture system, since it can reduce fertigation demand by 25% while maintaining growth and fruit quality.