Effects of Utilizing Plasma-Activated Water as a Nitrate Source on Growth and Flowering of Vanda Orchids

Abstract

The application of cold plasma technology in agriculture includes its use as a nitrate fertilizer, offering an alternative to traditional chemical fertilizers. This study investigated the effects of using plasma-activated water (PAW) as a nitrate source on the growth and flowering of Vanda orchids through two integrated experiments. Plants were treated with different nitrate concentrations (0, 100, 200, 300, and 400 mg/L) and fertilizing frequencies (weekly vs. biweekly), in combination with varying plant ages (1-, 2-, and 3-year-old plants). The analysis focused on several variables, including plant height, the number of leaves, fresh and dry biomass, and flowering traits, such as time to bloom, inflorescence length, floret number, floret diameter, and vase life. The leaf nitrate, total nitrogen, and gas exchange parameters were also recorded. The results demonstrate that the plants receiving 100 mg/L PAW-NO₃⁻ exhibited significantly greater plant height, fresh weight, and dry weight than the control (0 mg/L), with a trend toward a higher leaf number. Flowering occurred earlier in the 100 mg/L treatment group, with the first, second, and third inflorescences emerging at 208, 284, and 304 days after treatment, respectively. Additionally, this concentration produced the highest floret number per inflorescence and the longest vase life (12.63 days). Weekly fertilization resulted in more pronounced vegetative growth than biweekly application, particularly in 3-year-old plants—the only group to flower. Fertilizing frequency, however, had no effect on flower quality regarding the inflorescence length, floret number, or floret size. These findings suggest that 100 mg/L nitrate from plasma-activated water, applied weekly, optimally enhances growth and flowering performance in Vanda orchids.

1. Introduction

In recent years, cultivating plants in greenhouses or extensive gardens has become increasingly crucial for enhancing yield and quality, particularly in economically significant species, such as Vanda orchids. These orchids hold substantial economic value in numerous countries, making it essential to develop new techniques to improve their growth and flowering.

Orchids are distinctive ornamental plants due to their esthetic appeal, vibrant colors, and unique characteristics [1]. Vanda is a genus of monopodial orchids which is extensively grown in Thailand for both local utilization and exports as cut flowers. They have incredible economic and export value, generating more than THB 2 billion annually in revenue. Thailand’s major orchid trading partners include the United States, Japan, the Netherlands, China, and Italy. The orchid species with the most significant export volume is Dendrobium, followed by Oncidium, Mokara, Vanda, Aranda, and others. In addition, Thailand also exports orchids in various forms, such as rooted and rootless orchid cuttings, seedlings, and orchid plants.

The production of large quantities of high-quality orchids for export requires careful practices, especially in terms of fertilizing or nutrient management. However, most commercial fertilizers are not specifically developed for individual orchid species. As a result, the output quality is not as good as it should be. Meanwhile, the cost of production is rising. Therefore, assessing plant nutrient requirements is essential to increase production efficiency and yield quality [2]. Water-soluble fertilizing is beneficial for orchids, as they can control nutrient concentration and fertilizing frequency according to the needs of each orchid species [3,4]. Orchids generally require large amounts of nitrogen and potassium [5]. However, chemical preparation for nutrient solutions is integral to increasing production costs. Although orchid cultivation technology has dramatically advanced in terms of new hybrid species and greenhouse management, nutrient or fertilizer management is still required.

One of the innovative approaches gaining attention in modern agriculture is the application of plasma-activated water (PAW). PAW is generated by discharging cold plasma into water, forming reactive nitrogen species—primarily nitrate (NO₃⁻)—in forms that are more readily available for plant uptake. This feature makes PAW a promising alternative to conventional nitrogen fertilizers, offering enhanced nutrient efficiency with potentially reduced environmental impacts. In addition, PAW exhibits antimicrobial properties that suppress soil-borne pathogens, lowering plant disease incidence [6,7].

PAW has been shown to improve a wide range of physiological and developmental traits in plants. Studies have reported enhancements in plant height, fresh and dry biomass, photosynthetic pigment concentration, and photosynthetic activity following PAW application [8,9]. Furthermore, PAW has been effective in promoting early-stage development, including improved seed germination, stem and root elongation, leaf expansion, and chlorophyll content in several species [10,11]. These physiological benefits indicate that PAW serves as a nutrient source and contributes to overall plant vigor.

Multiple experimental findings have supported the agricultural value of PAW. For instance, Ruamrungsri et al. [7] demonstrated that PAW significantly improved the growth of green oak lettuce (Lactuca sativa L.), suggesting its potential as a sustainable substitute for synthetic nitrate fertilizers. Similarly, Chuea-uan et al. [12] found that PAW generated via air-gliding arc discharge enhanced the germination of rice (Oryza sativa L.) seeds and increased nitrate and nitrite accumulation. More recent studies have extended the application of PAW across various cultivation systems. In lettuce, PAW treatments significantly increased foliar biomass and leaf area, particularly under elevated nitrate concentrations and with greater substrate volumes [13]. In nursery-grown horticultural crops, PAW enhanced plant height, biomass accumulation, and nutrient uptake efficiency [14]. Additionally, PAW has been validated as an effective nitrogen source in hydroponic systems, stimulating both root and shoot growth in radish plants [15]. Although plasma-activated water (PAW) is not a new agricultural concept, its application to specific horticultural crops remains highly interesting. As a sustainable alternative to conventional fertilizers, PAW can improve nutrient use efficiency while reducing chemical inputs and minimizing environmental impact—particularly in high-value ornamental crops such as Vanda orchids.

Despite the recognized economic importance of Vanda orchids, research on the use of PAW in their cultivation remains limited. This study investigates the effects of PAW, applied as a nitrate source, on the growth and flowering of Vanda orchids. The findings are expected to support the development of more efficient and sustainable cultivation practices for this commercially significant species.

2. Materials and Methods

Both experiments were conducted at the King’s Initiative Centre for Flower and Fruit Propagation, Chiang Mai, Thailand (18.7142° N, 98.9205° E), between February 2023 and March 2024. The climate of the area is classified as tropical savanna (Aw) according to the Köppen–Geiger system, with an average annual temperature of 25.9 °C.

2.1. Experiment 1: Effect of Nitrate Levels Produced by Plasma-Activated Water on the Growth of Vanda Orchids

2.1.1. Experimental Design and Plant Growing Conditions

This experiment followed a completely randomized design (CRD) with five treatments and five replications, each consisting of four plants. Plasma-activated water was generated using a pinhole plasma jet at varying discharge durations to produce five nitrate concentrations: 0, 100, 200, 300, and 400 mg/L.

Hybrid Vanda orchids (var. Virat Gordon), approximately three years old and previously flowered, with uniform height and leaf number, were selected for this study. These plants were acquired from a local orchid grower who propagated them using tissue culture techniques. All experimental plants were derived from the same tissue culture batch and maintained under uniform cultivation conditions for three years before the experiment. The plants were cultivated under a suspension system in a 50% shaded greenhouse. A volume of 100 mL of plasma-activated water was sprayed weekly onto the shoot system, including the leaves and aerial roots.

All experimental units received the same nutrient formulation, excluding nitrate (NO₃⁻), as specified in

Table 1. Concentration of nutrients other than NO₃⁻ used in the experiments’ nutrients recipe stock solution (dilution ratio = 1:100).

Macronutrients	Concentration (mg/L)	Micronutrients	Concentration (mg/L)
NH4+	58	B	0.24
P	100	Mn	0.226
K	200	Zn	0.11
Ca	100	Mo	0.005
Mg	100	Cu	0.01
S	212	FeEDTA	0.30

. The nutrient solution was maintained at a pH of 5.8–6.0 throughout the study, using HCl and NaOH to adjust and stabilize the pH as needed.

2.1.2. Installing Pinhole Plasma Jet

The plasma machines used in this project can produce plasma-activated water inside the device. This process increases the rate of production of plasma water and hydroxyl free radicals. The machine’s design considers user convenience, and its operation is not complicated. The plasma dispenser consists of a glass tube on the inside. The inner glass tube is responsible for creating plasma. The positive electrode (+HV) is connected to a 125-watt neon transformer. One transformer is used for one plasma dispenser. The glass tube also serves as the ingress for gases used for plasma production. The outer glass tube is responsible for distributing free radicals into plasma water. The negative electrode (-HV) induces the plasma generated by the inner glass tube to disperse in the water contained in the outer glass tube through a 1 mm diameter pinhole. The water in the glass tube is circulated from the plasma dispenser and passed to the water container. The generated plasma water is sent back and forth between the plasma dispensers and water storage containers until the desired concentration of free radicals is achieved.

2.1.3. Plasma-Activated Water Generation

Plasma-activated water (PAW) was prepared by discharging plasma into 3 L of tap water using air as the working gas. Discharge durations of 30, 60, 90, and 120 min were applied to achieve nitrate concentrations of 100, 200, 300, and 400 mg/L, respectively. To verify the nitrate levels, a 10 mL sample of PAW was collected and analyzed using the HI3874-0 nitrate reagent (

Table 2. Properties of plasma-activated water at discharge times of 0–120 min.

Time (min)	Temperature (°C)	pH	Conductivity	ORP (mV)	NO3− Content (mg/L)
0	24.0 ± 0.00	6.98 ± 0.25	191.4 ± 1.44	247.0 ± 1.25	0.00 ± 0.00
30	29.2 ± 1.39	7.84 ± 0.14	197.4 ± 4.89	216.0 ± 3.86	100.75 ± 15.52
60	32.0 ± 1.08	7.05 ± 0.26	203.5 ± 4.49	220.0 ± 8.06	200.28 ± 22.56
90	33.0 ± 0.47	7.85 ± 0.01	209.4 ± 3.19	218.0 ± 6.70	300.59 ± 8.39
120	33.5 ± 0.71	7.68 ± 0.19	222.4 ± 6.28	229.0 ± 8.60	400.38 ± 45.67

). The concentration of plasma-generated nitrate was determined via UV-Vis spectrophotometry (Genesys 10S, Thermo Scientific, Waltham, MA, USA at a wavelength of 372 nm.

2.1.4. Data Collection

Data on plant growth were collected monthly in terms of plant height and the number of leaves. The flowering information recorded included the number of days it took to flower (1st, 2nd, and 3rd cycles) and flower quality (the number of inflorescences per plant, florets per inflorescence, flower stem length (cm), floret diameter (cm), and flower vase life in days). Plant photosynthesis attributes were recorded after 12 months of experimental treatment. The leaf gas exchange parameters of the Vanda plants were measured using a portable photosynthesis system (LI-6800^®, LI-COR Inc., Lincoln, NE, USA). Because they are CAM plants, the net assimilation rate (Pn), transpiration (E), stomatal conductance (Gs), and intercellular CO₂ concentration (Ci) measurements of the Vanda plants were conducted during the early morning hours (3–5 am). Plant biomass was assessed in terms of the total plant fresh and dry weights (g) after 12 consecutive monthly PAW-NO₃⁻ treatments.

The total nitrogen (%) and nitrate (mg/kg DM) contents in the Vanda orchid leaves were also analyzed. Sampled plants were thoroughly washed with tap water and later rinsed with DI water, allowing some time for the water to vaporize under shade. The samples were weighed, and separated leaves were dried in an oven for 168 h at 70 °C. The dried leaves were weighed, ground, and sifted to a fine mesh. The content of total nitrogen in the leaf was evaluated using the modified Kjeldahl method, as described by Ohyama [16]. The nitrate concentration in the Vanda leaves was determined using the salicylic acid method described by Cataldo et al. [17]. A 100 mg sample of ground leaf was mixed with 10 mL of distilled water and incubated at 45 °C for an hour. The mixture was then centrifuged at 5000× g for 15 min. Next, 0.2 mL of the supernatant was combined with 0.8 mL of 5% (w/v) salicylic acid in concentrated H₂SO₄ and left at room temperature for 15 min. After that, 19 mL of 2N NaOH was added. Once the mixture cooled to room temperature, the absorbance was measured at 410 nm using a spectrophotometer. The results were expressed as mg/kg FW.

2.2. Experiment 2: Study on the Effect of Plant Age and Application Frequency of Nitrate Produced by Plasma-Activated Water (PAW-NO₃⁻) on Growth and Flowering of Vanda Orchids

A 3 × 2 factorial experiment laid out in CRD was performed to investigate the effect of plasma nitrate on the growth and flowering characteristics of Virat × Gordan Vanda orchid hybrids. The two factors studied were Vanda plant age (1-, 2- and 3-year-old plants) and the application frequency of 100 mL of plasma nitrate (weekly and biweekly). A schematic diagram of the plasma system and the experimental treatments applied in both trials is provided in Figure 1, illustrating the PAW generation process and application methods.

The plant selection, growth conditions, and data collection were the same as in Experiment 1, except that the growth parameters, i.e., the number of leaves and plant height, were calculated as the monthly change rate from the initial readings. This is because of the variation in the ages of the plants. The change could be positive or negative for the number of leaves per plant (+/−) due to periodic leaf shedding. For these parameters, the rate of change was the observed average increase/decrease throughout observation. For illustration, the change in the number of leaves was calculated as follows:

$$\Delta in number of leaves={\displaystyle \frac{NLx-NL1}{\mathrm{n}}}$$

where

NL1 = the initial number of leaves;

NLx = the present number of leaves;

n = the number of months.

The same procedure was used to calculate the change in plant height in the three-, two-, and one-year-old Vanda orchid plants.

2.3. Data Analysis

The data from both experiments were subjected to statistical analysis using Statistix software, version 9.0 [18]. Analysis of variance (ANOVA) was performed to determine the significance of the treatment effects. Where significant differences were detected, the treatment means were separated using the least significant difference (LSD) test at p < 0.05.

3. Results and Discussion

3.1. Experiment 1

3.1.1. Plant Height

The results of plant height in response to the treatments are presented in

Table 3. Plant height and number of leaves of Vanda orchids in response to plasma-activated water.

PAW-NO3− (mg/L)	Plant Height (cm)				Number of Leaves per Plant
	Months After Treatments (MAT)
	3	6	9	12	3	6	9	12
0	18.25 ± 2.08	18.70 ± 1.94 b	20.30 ± 2.33 c	20.82 ± 1.83 b	13.67 ± 2.23 b	14.27 ± 1.80 b	16.00 ± 1.92 b	14.33 ± 2.13
100	20.41 ± 2.20	20.92 ± 1.98 a	23.23 ± 2.41 ab	24.03 ± 2.56 a	15.40 ± 1.64 a	16.27 ± 2.13 a	18.20 ± 2.26 a	16.53 ± 2.13
200	20.53 ± 2.14	21.50 ± 1.96 a	23.64 ± 2.10 ab	22.90 ± 3.01 a	14.67 ± 1.53 ab	16.40 ± 1.73 a	18.13 ± 1.92 a	16.47 ± 2.53
300	20.23 ± 2.22	19.95 ± 1.91 ab	21.82 ± 2.35 bc	23.75 ± 3.00 a	13.93 ± 1.37 b	15.07 ± 2.45 ab	16.67 ± 2.18 ab	16.86 ± 3.83
400	19.71 ± 2.19	19.85 ± 1.61 ab	21.40 ± 2.54 c	24.25 ± 2.16 a	13.87 ± 1.31 b	14.80 ± 1.83 b	16.40 ± 1.98 ab	16.27 ± 1.83
LSD0.05	ns	*	*	*	*	*	*	ns

* Denotes that the result is statistically significant (p < 0.05); a, b, c = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05); ns = not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as the mean ± standard error (SE).

and Figure 2. Vanda orchid plants showed a response to the PAW-NO₃⁻ treatments. At 3 months after treatment (MAT), the differences in plant height were not statistically significant (p < 0.05). Similar results were observed among all treatments at 6 and 9 MAT, indicating no significant differences (p < 0.05). However, at 12 MAT, plants treated with 100, 200, 300, and 400 mg/L PAW-NO₃⁻ were significantly higher (24.03, 22.90, 23.75, and 24.25 cm, respectively) compared to the control group (20.82 cm) (p < 0.05). This suggests that Vanda plants’ height responds gradually to PAW-NO₃⁻, with 100 mg/L being the most effective and economical concentration. Increasing the concentration beyond this level did not result in further height increases. These findings are consistent with previous studies reporting improved height in response to PAW-NO₃⁻ application [19]. Similar results were also reported by Wang and Konow [4].

3.1.2. Number of Leaves

Our results show a significant difference (p < 0.05) between the treatments and the control (0 mg/L NO₃⁻) in terms of the mean number of leaves per plant (Table 3, Figure 2). The control group exhibited the smallest number of leaves at 12 MAT. In contrast, plants treated with 100 mg/L PAW-NO₃⁻ consistently maintained the highest number of leaves throughout the experiment, although the difference was not significant at 3, 6, and 9 MAT compared to the other treatments. At 12 MAT, the number of leaves in the four PAW-NO₃⁻ treatments did not significantly differ from the control, likely due to seasonal leaf fall. These results suggest that PAW-NO₃⁻ application can enhance vegetative growth in Vanda plants in both the short and long term. The increased leaf number may result from the remobilization of nitrates to actively growing tissues, leading to the development of new leaves in Vanda orchid plants. Similar findings were reported by Wang et al. [20], who observed an increase in the number of leaves in lettuce following PAW-NO₃⁻ application.

3.1.3. Fresh and Dry Weights, Leaf Nitrate, and Total Nitrogen Contents

Our results demonstrate a statistically significant effect of the PAW-NO₃⁻ treatments on both the fresh and dry weights of Vanda orchids, as presented in

Table 4. Total fresh and dry weights and total leaf nitrate and nitrogen contents of Vanda orchids at 12 months after being supplied with different levels of nitrate produced by plasma-activated water (PAW-NO₃⁻).

PAW-NO3− (mg/L)	Fresh Weight (g)	Dry Weight (g)	Nitrate (mg/Kg DW)	Total Nitrogen (%)
0	181.58 ± 7.42 d	37.75 ± 1.54 b	231.25 ± 10.59 d	0.66 ± 0.01 c
100	256.08 ± 7.70 a	45.25 ± 5.28 a	330.69 ± 5.18 b	0.77 ± 0.05 a
200	227.82 ± 5.76 b	41.30 ± 2.23 ab	309.22 ± 13.45 bc	0.71 ± 0.01 b
300	212.10 ± 7.22 c	45.38 ± 3.90 a	321.08 ± 16.17 c	0.75 ± 0.00 ab
400	218.74 ± 9.70 bc	43.38 ± 2.30 ab	352.72 ± 6.85 a	0.71 ± 0.00 b
LSD0.05	*	*	*	*

* Denotes that the result is statistically significant (p < 0.05); a, b, c, d = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05). Values are presented as mean ± standard error (SE).

. Plants treated with 100 mg/L PAW-NO₃⁻ exhibited the highest fresh weight (256.08 g), followed by 200, 300, and 400 mg/L (227.82 g, 212.10 g, and 218.74 g, respectively), while the control group (0 mg/L) showed the lowest value (181.58 g). Similarly, the treatment levels significantly affected the dry weight, with the highest values observed at 100 and 300 mg/L PAW-NO₃⁻ (45.25 g and 45.38 g) and the lowest in the control group (37.75 g). As nitrate is a key component of proteins, amino acids, and nucleic acids, sufficient NO₃⁻ supply can enhance cell division and elongation, thereby increasing biomass production [19,21,22,23]. However, Han et al. [24] reported reduced biomass in spinach with excessive nitrate application. The absence of a significant difference between the 400 mg/L treatment and the other PAW-NO₃⁻ levels may be attributed to nitrogen oversupply or an imbalanced nitrogen-to-carbon ratio, which can suppress plant growth [25].

Table 4 also shows that PAW-NO₃⁻ significantly influenced the nitrate and total nitrogen content in Vanda leaves after 12 months of treatment. The lowest nitrate concentration was found in the control (231.25 mg/kg DW), and increased PAW-NO₃⁻ levels resulted in higher nitrate accumulation, peaking at 400 mg/L (352.72 mg/kg DW). The 100 mg/L PAW-NO₃⁻ treatment resulted in the highest total nitrogen percentage (0.77%), which was statistically similar to the 300 mg/L treatment (0.75%), but significantly higher than those at 200 and 400 mg/L (both 0.71%), as shown in Table 4. These findings suggest that increasing the rate of PAW-NO₃⁻ does not necessarily translate to proportional nitrogen assimilation, potentially due to limitations in uptake or imbalanced NO₃⁻/NH₄⁺ ratios.

Furthermore, a positive correlation between the nitrate and total nitrogen content was observed up to a specific concentration. The total nitrogen content increased with rising nitrate levels and peaked at 100 mg/L PAW-NO₃⁻ (0.77%). Beyond this point, the values plateaued or declined slightly. At 200 and 400 mg/L, the total nitrogen content remained relatively stable at 0.71%, despite higher nitrate concentrations (309.22 and 352.72 mg/kg DW, respectively). A moderate increase was observed at 300 mg/L (0.75%). These findings indicate that PAW-NO₃⁻ enhances nitrogen accumulation in Vanda leaves only up to an optimal level, after which further nitrate application fails to increase nitrogen content, likely due to limited assimilation efficiency [26].

3.1.4. Photosynthesis

Table 5. The transpiration rate, stomata conductance, and photosynthesis rates of Vanda orchids at 12 months after being supplied with different levels of nitrate produced by plasma-activated water (PAW-NO₃⁻).

PAW-NO3− (mg/L)	Transpiration Rate (mol/m2/s1)	Stomatal Conductance (mol/m2/s1)	Photosynthetic Rate (µmol/m2/s1)
0	0.532 ± 0.065	0.0480 ± 0.0064	1.1340 ± 0.63
100	0.538 ± 0.061	0.0460 ± 0.0062	2.4200 ± 0.61
200	0.640 ± 0.062	0.0540 ± 0.0059	2.2880 ± 0.58
300	0.682 ± 0.062	0.0620 ± 0.0053	1.6820 ± 0.63
400	0.610 ± 0.060	0.0560 ± 0.0057	1.0560 ± 0.65
LSD0.05	ns	ns	ns

ns: Not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as mean ± standard error (SE).

presents the gas exchange properties of Vanda orchids in response to PAW-NO₃⁻ treatments after 12 months of weekly applications. Data analysis revealed no statistically significant (p < 0.05) differences among the treatments for the three parameters recorded: the leaf transpiration rate (E), stomatal conductance (Gs), and the net single leaf CO₂ assimilation rate (Pn). This implies that PAW-NO₃⁻ might not be effective in varying these specific physiological parameters in Vanda orchids.

3.1.5. Flowering and Flower Quality

The data in

Table 6. Flowering and flower quality of Vanda orchids at the flowering stage after being supplied with different levels of nitrate produced by plasma-activated water (PAW-NO₃⁻).

PAW-NO3− (mg/L)	Days to Bloom (Cycle)			Flower Stalk Length (cm)	Florets per Inflorescence	Floret Diameter (cm)	Vase Life (Days)
	1st	2nd	3rd
0	233.67 ± 6.53 a	297.88 ± 5.34 a	326.50 ± 6.66 a	29.66 ± 5.23	4.13 ± 0.35 c	9.94 ± 1.52	7.13 ± 1.36 c
100	208.22 ± 10.93 d	284.00 ± 8.35 b	304.75 ± 5.56 d	30.59 ± 5.80	5.38 ± 0.52 a	10.54 ± 1.29	12.63 ± 1.77 a
200	226.11 ± 3.67 bc	290.88 ± 6.00 ab	312.88 ± 2.12 bc	29.78 ± 1.86	5.25 ± 0.46 ab	9.93 ± 0.84	11.00 ± 2.45 ab
300	222.11 ± 5.00 c	285.88 ± 6.03 b	307.88 ± 1.41 cd	27.64 ± 5.58	4.75 ± 0.46 b	10.00 ± 1.20	11.88 ± 1.36 ab
400	232.00 ± 4.36 ab	293.88 ± 3.95 a	315.88 ± 1.73 b	30.20 ± 2.43	3.88 ± 0.83 c	9.74 ± 1.38	10.50 ± 1.51 b
LSD0.05	*	*	*	ns	*	ns	*

* Denotes that the result is statistically significant (p < 0.05); a, b, c, d = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05); ns = not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as mean ± standard error (SE).

indicate statistically significant variations in the flowering and flower quality attributes of Vanda orchids treated with PAW-NO₃⁻. The time to bloom demonstrated statistically significant (p < 0.05) differences across the treatments. The shortest blooming period was observed at 100 mg/L (208.22 days in the first cycle), while the longest was at 0 mg/L (233.67 days in the first cycle). Higher PAW-NO₃⁻ concentrations (300 mg/L and 400 mg/L) generally resulted in longer blooming periods, though less extensive than the control. The blooming trends in Vanda orchids in the first, second, and third cycles are displayed in Table 6. No statistically significant (p < 0.05) differences among the treatments were observed in the flower stalk length. Floret count per inflorescence showed a significant (p < 0.05) response to the varying PAW-NO₃⁻ concentrations. The highest number of florets per inflorescence was observed at 100 mg/L (5.38), while the control (0 mg/L) and 400 mg/L treatments had the lowest counts (4.13 and 3.88, respectively). In addition, the intermediate concentrations (200 mg/L and 300 mg/L) showed better floret counts per inflorescence than the control and the highest PAW-NO₃⁻ concentration (Figure 3). Conversely, the diameter of florets (mm) did not vary significantly (p < 0.05) across the PAW-NO₃⁻ treatments. Optimal flowering responses were observed at 100–200 mg/L, while higher concentrations delayed blooming and reduced floret numbers, suggesting that high NO₃⁻ application is detrimental to flowering. Previously, research reported that applying nitrogen at specific levels can affect the duration of flowering, spike length, number of spikes per plant, and vase life of the snapdragon [27]. Nitrogen status was reported as a dominant factor affecting flowering time, with a complex network integrating N status and photoperiod conditions into the internal regulation of flowering time and flowering traits in plants [28].

The vase life days differed significantly (p < 0.05) among the PAW-NO₃⁻ treatments, as shown in Table 6. Flowers from plants treated with 100 mg/L PAW-NO₃⁻ had the most extended vase life (12.63 days), comparable to the 200, 300, and 400 mg/L PAW-NO₃⁻ treatments, while the shortest vase life was observed in the control treatment (7.13 days). The extension of flower vase life observed under the 100 mg/L PAW-NO₃⁻ treatment can be attributed to the enhanced physiological stability conferred by nitrate-derived nitrogen. Nitrogen plays a vital role in maintaining postharvest quality by supporting the synthesis of amino acids, nucleic acids, proteins, and coenzymes essential for cell maintenance and stress tolerance [29,30]. Vishwakarma and Kumar [30] demonstrated that nitrogen application significantly extended the vase life in tuberose by reducing floret drooping and maintaining spike turgidity, with the maximum vase life observed at 200–300 kg N/ha. Similarly, Malik et al. [29] reported that increasing the nitrogen levels up to 150 kg/ha in chrysanthemum cultivars resulted in enhanced vegetative vigor, which may contribute to their postharvest longevity through better nutrient reserves and delayed senescence. Furthermore, Verma et al. [27] found that nitrogen at optimal levels improved floret appearance and extended vase life in the snapdragon. These findings are consistent with our results and suggest that nitrate supplied through PAW supports physiological processes that prolong flower life by reducing senescence and sustaining tissue hydration.

3.2. Experiment 2: Study on the Effect of Plant Age and Application Frequency of Nitrate Produced by Plasma-Activated Water (PAW-NO₃⁻) on Growth and Flowering of Vanda Orchids

3.2.1. Plant Height and Number of Leaves per Plant

Table 7. The rate of increase/decrease in the number of Vanda leaves, as affected by plant age and frequency of plasma nitrate application.

Factors	Plant Height (cm)				Number of Leaves
	Months After Treatment (MAT)				Months After Treatment (MAT)
	3	6	9	12	3	6	9	12
Plant age(years)
1	0.15 ± 0.10 b	0.12 ± 0.09 b	0.33 ± 0.15	0.78 ± 0.67	0.44 ± 0.63	0.94 ± 0.70	−1.1 ± 0.80 b	0.86 ± 0.65
2	0.57 ± 0.12 a	0.17 ± 0.11 b	0.34 ± 0.10	0.79 ± 0.33	0.83 ± 0.65	0.83 ± 0.68	0.58 ± 0.45 a	0.79 ± 0.60
3	0.37 ± 0.09 ab	0.40 ± 0.14 a	0.34 ± 0.13	0.92 ± 0.66	0.99 ± 0.36	0.62 ± 0.50	0.59 ± 0.46 a	0.86 ± 0.62
LSD0.05	*	*	ns	ns	ns	ns	*	ns
Fertilizing frequency
Weekly	0.28 ± 0.11	0.25 ± 0.10	1.83 ± 0.73	1.01 ± 1.20	0.63 ± 0.58	0.74 ± 0.59	0.58 ± 0.46 a	1.19 ± 0.95 a
Biweekly	0.44 ± 0.18	0.21 ± 0.08	1.64 ± 0.66	0.65 ± 0.43	0.88 ± 0.56	0.86 ± 0.56	0.20 ± 0.13 b	0.48 ± 0.31 b
LSD0.05	ns	ns	ns	ns	ns	ns	*	*
Plant age × frequency of fertilizing
1 year × weekly	0.14 ± 0.09	0.18 ± 0.11	0.47 ± 0.19	0.96 ± 0.98	0.25 ± 0.52	1.00 ± 0.70	0.13 ± 0.15	1.29 ± 0.90
1 year × biweekly	0.16 ± 0.10	0.06 ± 0.03	0.47 ± 0.19	0.66 ± 0.30	0.63 ± 0.74	0.88 ± 0.62	−0.13 ± 0.13	1.14 ± 0.80
2 years × weekly	0.32 ± 0.13	0.22 ± 0.09	0.47 ± 0.19	1.40 ± 1.51	0.50 ± 0.53	0.83 ± 0.58	0.88 ± 0.62	1.14 ± 0.80
2 years × biweekly	0.82 ± 0.33	0.12 ± 0.06	0.50 ± 0.20	0.60 ± 0.62	1.17 ± 0.79	0.83 ± 0.58	0.30 ± 0.21	0.43 ± 0.30
3 years × weekly	0.40 ± 0.16	0.36 ± 0.14	0.47 ± 0.19	0.91 ± 1.33	1.13 ± 0.00	0.38 ± 0.27	0.80 ± 0.56	0.43 ± 0.31
3 years × biweekly	0.34 ± 0.14	0.44 ± 0.18	0.50 ± 0.20	0.44 ± 0.28	0.86 ± 0.49	0.86 ± 0.60	0.43 ± 0.30	0.57 ± 0.40
LSD0.05	ns	ns	ns	ns	ns	ns	ns	ns

* Denotes that the result is statistically significant (p < 0.05); a, b = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05); ns = not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as mean ± standard error (SE).

provides an overview of the data analysis on the effect of plant age and PAW-NO₃⁻ fertilizing frequency on the increase in Vanda orchid plant height and the number of leaves. At 3 months after treatment (MAT), the plant height increase rate was significantly (p < 0.05) higher for the two-year-old plants (0.57 cm) compared to the one-year-old plants (0.15 cm). At 6 MAT, the three-year-old plants (0.40 cm) were significantly (p < 0.05) taller than the one-year-old (0.12 cm) and two-year-old (0.17 cm) plants. No significant (p < 0.05) differences in the plant height increase rate were observed between ages 9 and 12 MAT. Additionally, no statistically significant (p < 0.05) differences were observed between the weekly and biweekly fertilizing frequencies at 3, 6, 9, and 12 MAT. There were also no significant (p < 0.05) interaction effects between the plant age and the fertilizing frequency on plant height throughout the experiment.

The trend in plant height gain during the 12 months of 100 mg/L PAW-NO₃⁻ weekly application can be divided into three distinct phases: the initial phase (3–6 MAT), the middle phase (6–9 MAT), and the final phase (9–12 MAT).

Initial Phase (3–6 MAT): The height of the 1-year-old plants slightly decreased before stabilizing. The 2-year-old plants also experienced a decline, although less pronounced than the 1-year-old plants. In contrast, the 3-year-old plants maintained a relatively constant height with only minor fluctuations.

Middle Phase (6–9 MAT): All age groups showed an upward trend in height gain. The 1-year-old plants began to increase in height, although less steeply. The 2-year-old plants displayed a moderate rise, while the 3-year-old plants experienced a significant increase.

Final Phase (9–12 MAT): There was a substantial increase in height across all age groups. The 1-year-old plants saw a dramatic rise, the 2-year-old plants continued to grow steadily, and the 3-year-old plants exhibited the most substantial and steepest increase in height.

This progressive growth suggests that while the younger plants initially showed slower growth rates, they underwent significant growth in later stages. Despite a slower start, the older plants ultimately displayed the most pronounced growth response to the nitrate treatment over an extended period.

These results can be attributed to higher accumulated deposits of nitrogen, which could be mobilized to new growing regions for growth and differentiation. Additionally, older plants, through their aged leaves, regulate photosynthetic capacity over the leaf’s lifespan, affecting the distribution of nitrogen (N) among leaves and the overall plant carbon gain [31]. These enhanced capabilities could improve plant height, the number of leaves, biomass, and overall plant growth.

Table 7 also presents an analysis of the rate of change in the number of leaves of Vanda orchid plants in response to the plant age and PAW-NO₃⁻ fertilizing frequency. At 9 months after treatment (MAT), plant age significantly (p < 0.05) affected the rate of change in the number of leaves. The one-year-old plants had a decreased number of leaves (−1.1), while the two-year-old and three-year-old plants showed favorable leaf yield rates (0.58 and 0.59, respectively). However, the effect of plant age was not statistically significant (p < 0.05) at 3, 6, and 12 MAT.

The practical implications of this research are significant. For instance, weekly fertilizing with 100 mg/L PAW-NO₃⁻ led to a significantly (p < 0.05) higher rate of leaf increase (0.58) compared to biweekly application (0.20) at 9 MAT. At 12 MAT, weekly fertilizing also resulted in a significantly (p < 0.05) higher rate of leaf increase (1.19) compared to biweekly applications (0.48). This suggests that weekly application of plasma nitrate ensures an adequate supply of the essential element for metabolism, leading to higher leaf gain, as shown in Figure 4.

3.2.2. Fresh Weight, Dry Weight, Leaf Nitrate, and Total Leaf Nitrogen Contents

The data presented in

Table 8. Fresh weight, dry weight, leaf nitrate content, and total nitrogen contents as affected by plant age and frequency of plasma nitrate application at 12 months after treatment.

Factor	Fresh Weight (g)	Dry Weight (g)	NO3− Content (mg/Kg DW)	Total N Content (%)
Plant age  (years)
1	70.98 ± 16.03 c	15.38 ± 3.15 c	251.59 ± 13.35	0.52 ± 0.03 c
2	247.59 ± 31.94 b	47.41 ± 4.57 b	246.51 ± 8.23	0.54 ± 0.02 b
3	393.39 ± 53.23 a	76.73 ± 9.61 a	257.81 ± 18.80	0.62 ± 0.02 a
LSD0.05	*	*	ns	*
Fertilizing frequency
Weekly	228.59 ± 129.04	44.55 ± 24.23	256.68 ± 12.05	0.57 ± 0.05 a
Biweekly	246.05 ± 151.87	48.46 ± 28.79	247.26 ± 15.09	0.55 ± 0.05 b
LSD0.05	ns	ns	ns	*
Plant age × Frequency of fertilizing
1 year × weekly	77.50 ± 14.38 d	15.89 ± 2.55 d	260.07 ± 9.44	0.54 ± 0.006 cd
1 year × biweekly	64.46 ± 16.28 d	14.86 ± 3.90 d	243.12 ± 11.87	0.50 ± 0.021 c
2 years × weekly	242.12 ± 45.95 c	45.68 ± 4.55 c	245.94 ± 11.54	0.52 ± 0.021 d
2 years × biweekly	253.06 ± 10.50 c	49.14 ± 4.33 c	247.07 ± 5.95	0.55 ± 0.010 c
3 years × weekly	420.62 ± 31.62 a	81.39 ± 10.02 a	264.02 ± 9.33	0.63 ± 0.006 a
3 years × biweekly	366.1659.51 ± b	72.08 ± 7.29 b	251.59 ± 26.10	0.60 ± 0.006 b
LSD0.05	*	*	ns	*

* Denotes that the result is statistically significant (p < 0.05); a, b, c, d = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05); ns = not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as mean ± standard error (SE).

summarize the fresh weight, dry weight, leaf nitrate content, and total leaf nitrogen content of Vanda orchids treated with 100 mg/L PAW-NO₃⁻ over 12 months, as influenced by the plant age and PAW-NO₃⁻ fertilizing frequency (weekly or biweekly). The results indicate no statistically significant (p < 0.05) effects of either factor on the Vanda orchid plants’ total leaf nitrate content.

Plant age significantly (p < 0.05) affected the total plant fresh and dry weights and the total leaf nitrogen contents. The one-year-old plants had the lowest fresh and dry weights (70.98 g and 15.38 g, respectively), followed by the two-year-old plants, with significantly higher fresh and dry weights (247.59 g and 47.41 g, respectively). The three-year-old plants had the highest fresh and dry weights of 393.39 g and 76.73 g, respectively. Similarly, the one-year-old Vanda plants demonstrated the lowest total leaf nitrogen content (0.52%), followed by the two-year-old plants (0.54%), with the highest content in the three-year-old plants (0.62%). The older plants’ higher biomass accumulation is possibly due to age-related enhancements in physiological processes and the remobilization of accrued nitrogen for growth, including protein and acid synthesis, cell division and elongation, and chlorophyll production for photosynthesis [31].

The impact of weekly PAW-NO₃⁻ application on the total leaf nitrogen content was significant. Vanda plants that received weekly applications of 100 mg/L PAW-NO₃⁻ had a significantly (p < 0.05) higher total nitrogen content (0.57%) compared to the lower value (0.55%) observed in plants treated biweekly. Frequently applying 100 mg/L of plasma nitrate contributed to better accumulation of total leaf nitrogen in the Vanda plants, enhancing their metabolic processes. This finding sheds more light on the effective fertilizing frequency for enhancing nitrogen accumulation in Vanda plants.

The role of plant age in determining nitrogen-use efficiency is a key finding of the experiment. The weekly application of 100 mg/L PAW-NO₃⁻ to three-year-old plants resulted in the highest biomass, with fresh and dry weights of 420.62 g and 81.639 g, respectively. Conversely, the one-year-old plants had the lowest fresh and dry weights regardless of the PAW-NO₃⁻ fertilizing frequency (Table 8). Similarly, weekly application of PAW-NO₃⁻ to three-year-old plants led to a significantly (p < 0.05) higher total leaf nitrogen accumulation (0.63%) compared to all other treatment combinations. Plant age is a determinant of N-use efficiency [32]. Therefore, the older plants that received plasma nitrate every week had the highest biomass accumulation from the contribution of the treatment to the total N concentration, which in turn promoted anabolic processes in the plants.

3.2.3. Photosynthesis

Table 9. The transpiration rate, stomatal conductance, and photosynthesis rate of Vanda orchids as affected by plant age and frequency of plasma nitrate application at 12 months after treatment.

Factors	Transpiration Rate (mol/m2/s1)	Stomatal Conductance (mol/m2/s1)	Photosynthetic Rate (µmol/m2/s1)
Plant age (years)
1	0.6663 ± 0.070	0.0575 ± 0.006	2.1387 ± 0.32
2	0.6238 ± 0.062	0.0600 ± 0.006	2.5087 ± 0.38
3	0.7275 ± 0.073	0.0800 ± 0.007	1.9913 ± 0.30
LSD0.05	ns	ns	ns
Fertilizing frequency
Weekly	0.6450 ± 0.07	0.06090 ± 0.006	2.7750 ± 0.42
Biweekly	0.7000 ± 0.068	0.07080 ± 0.005	2.1483 ± 0.33
LSD0.05	ns	ns	ns
Plant age x Frequency of fertilizing
1 year x weekly	0.6375 ± 0.06	0.0525 ± 0.0053	1.5125 ± 0.23
1 year x biweekly	0.6200 ± 0.05	0.0600 ± 0.0050	3.0550 ± 0.46
2 years x weekly	0.6775 ± 0.07	0.0700 ± 0.0071	2.2650 ± 0.34
2 years x biweekly	0.6950 ± 0.07	0.0625 ± 0.0067	2.7650 ± 0.41
3 years x weekly	0.6275 ± 0.06	0.0600 ± 0.0058	1.9625 ± 0.29
3 years x biweekly	0.7775 ± 0.08	0.0900 ± 0.0092	1.7175 ± 0.26
LSD0.05	ns	ns	ns

ns: Not statistically significant, according to the least significant difference (LSD) test (p < 0.05). Values are presented as mean ± standard error (SE).

presents the transpiration rate (E), stomatal conductance (Gs), and photosynthetic rate (Pn) of Vanda orchids after 12 months of treatment. There were no statistically significant (p < 0.05) effects of plant age or PAW-NO₃⁻ fertilizing frequency on any observed gas exchange parameters. While the transpiration rate had an increasing trend in the three-year-old plants (0.7275 mol/m²/s¹), the plants fertilized every two weeks had a slightly higher transpiration rate (0.7000 mol/m²/s¹) compared to those fertilized weekly (0.6450 mol/m²/s¹). These results suggest that the variations in the transpiration rates among the different plant age and fertilizing frequency combinations might be due to natural variability rather than a direct effect of the treatments.

Regarding stomatal conductance, the three-year-old plants exhibited a trend toward increased conductance (0.0800 mol/m²/s¹). The plants fertilized biweekly also showed higher conductance (0.0708 mol/m²/s¹) than those fertilized weekly (0.0609 mol/m²/s¹).

Regarding the photosynthetic rate, the highest rate was recorded in the two-year-old plants (2.5087 µmol/m²/s¹). The plants fertilized weekly had a higher photosynthetic rate than those fertilized every two weeks, which may indicate increased photosynthetic efficiency with more frequent fertilization. However, there was no statistically significant difference (Table 9).

The lack of statistically significant differences in gas exchange parameters, such as E, Gs, and Pn, may reflect the physiological adaptation of Vanda orchids to stable environmental conditions and their inherent capacity for resource conservation. As epiphytic plants with velamen-covered aerial roots, Vanda orchids possess specialized water retention and gas exchange adaptations that may buffer them against treatment variations [33].

Moreover, stomatal conductance is known to play a crucial role in regulating photosynthetic activity and water use efficiency. However, its responsiveness varies among species and growth conditions. Although some trends were observed in our study, they were not significant, possibly due to the orchid’s intrinsic stomatal behavior under controlled conditions [34].

Previous research has shown that photosynthesis peaks in the early morning in Vanda species and declines during midday due to stomatal regulation and temperature stress [35]. This behavior could explain the overall limited variation in the measured photosynthetic rates. Orchid species also exhibit photosynthetic activity in their roots, contributing to the complexity of gas exchange interpretation [35].

3.2.4. Flowering and Flower Quality

Table 10. Flowering and flower quality characteristics of Vanda orchids as affected by plant age and frequency of plasma nitrate application at flowering stage.

Treatments	Days to Bloom (Cycle)			Flower Stalk Length (cm)	Florets per Inflorescence	Floret Diameter (cm)	Vase Life (Days)
	1st	2nd	3rd
Plant age × Frequency of fertilizing
1 year × weekly	NF	NF	NF	NF	NF	NF	NF
1 year × biweekly	NF	NF	NF	NF	NF	NF	NF
2 years × weekly	NF	NF	NF	NF	NF	NF	NF
2 years × biweekly	NF	NF	NF	NF	NF	NF	NF
3 years × weekly	185.16 ± 2.3 b	276.00 ± 9.20	296.00 ± 1.00 b	24.80 ± 0.23	5.71 ± 1.10	12.00 ± 0.01	13.57 ± 0.79 a
3 years × biweekly	193.25 ± 4.3 a	279.29 ± 9.34	306.56 ± 9.34 a	27.79 ± 1.39	5.85 ± 0.24	10.87 ± 1.70	9.86 ± 4.10 b
t-test 0.05	*	ns	*	ns	ns	ns	*

* Denotes that the result is statistically significant (p < 0.05); a, b = means followed by the same superscript letters in the same column are not statistically significantly different from each other (p < 0.05); ns = not statistically significant, according to the least significant difference (LSD) test (p < 0.05); NF = no flowering. Values are presented as mean ± standard error (SE).

presents the effects of plant age and plasma nitrate fertilization frequency on the flowering and flower quality characteristics of Vanda orchids. Notably, only the three-year-old plants fertilized with 100 mg/L PAW-NO₃⁻, either weekly or biweekly, exhibited flowering (Figure 5). In contrast, the one- and two-year-old plants did not flower, likely due to physiological immaturity and insufficient accumulation of carbohydrates and nitrogen reserves necessary for initiating reproductive development.

The absence of flowering in younger plants aligns with findings that adequate carbohydrate and nitrogen reserves are critical for floral initiation in orchids. Specifically, studies have shown that the accumulation of total non-structural carbohydrates (TNCs) and an appropriate carbon-to-nitrogen (C/N) ratio are essential for the transition from vegetative to reproductive stages in Vanda orchids [36].

A significant difference was observed in the days of the first flower bloom. Three-year-old plants fertilized weekly bloomed earlier (185.16 days) than those fertilized biweekly (193.25 days). However, no statistically significant difference was noted between the treatments regarding the days to bloom during the second flowering cycle. In the third cycle, the weekly fertilized three-year-old plants bloomed at 296 days, significantly earlier than the biweekly fertilized counterparts, which bloomed at 306.56 days.

These findings suggest that more frequent fertilization may accelerate the flowering process due to enhanced nutrient availability, facilitating faster developmental transitions. This is consistent with the understanding that Vanda orchids, lacking pseudobulbs, rely heavily on external nutrient supply, and frequent fertilization supports continuous growth and timely flowering [37].

Significant differences were observed in flower longevity among the treatments. Flowers from the three-year-old plants fertilized weekly had a longer vase life (13.57 days) than those fertilized biweekly (9.86 days). Enhanced vase life with more frequent fertilization may be attributed to improved plant vigor and nutrient status, improving flower quality and longevity.

These results underscore the importance of plant maturity and fertilization frequency in optimizing flowering and flower quality in Vanda orchids. Implementing appropriate fertilization regimes can enhance the timing of flowering and the esthetic and commercial value of the blooms.

4. Conclusions

This study demonstrated that the weekly application of 100 mg/L nitrate derived from plasma-activated water (PAW-NO₃⁻) effectively enhanced the vegetative growth and flowering performance of Vanda orchids. The most notable improvements were observed in three-year-old plants, which exhibited increased biomass, earlier floral initiation, and extended vase life. These results support the potential of PAW-NO₃⁻ as a sustainable and efficient nitrate source for improving yield and quality in the context of commercial orchid production.