Characterization and Application of Endophytic Bacteria for Enhancing Nitrogen Uptake in Vanda Orchids

Abstract

Vanda orchids are a commercially significant genus in the global floriculture industry, yet their cultivation often depends on substantial chemical fertilizer inputs, which raise both economic and environmental concerns. Endophytic bacteria offer a promising, sustainable alternative by promoting plant growth and enhancing nutrient acquisition. This study aimed to characterize native endophytic bacteria and assess their potential to improve nitrogen uptake and growth in Vanda orchids. Three potent nitrogen-fixing bacterial isolates (2R13, 3S19, and 3R14) were selected for this research. Through 16S rRNA sequencing, they were identified as Curtobacteriumcitreum, Stenotrophomonas panacihumi, and Bacillus subtilis, respectively. The efficacy of these isolates was evaluated in both controlled in vitro and practical greenhouse conditions using various dilution ratios. Scanning electron microscopy confirmed the successful colonization of isolate 3S19 within the root tissue of inoculated Vanda plantlets. The results revealed a significant interaction between the bacterial treatments and the growing environment. In vitro, isolate 3S19 applied at a 1:25 ratio yielded the highest total nitrogen content (12.46 mg g⁻¹ DW). Conversely, in the greenhouse experiment, isolates 2R13 and 3S19 were most effective at a 1:50 ratio, achieving nitrogen contents of 11.18 and 10.83 mg g⁻¹ DW. Furthermore, bacterial inoculation in the greenhouse generally led to significant improvements in plant growth parameters, including height, leaf count, and root development, compared to non-inoculated controls. These findings highlight the potential of these endophytic bacteria as effective biofertilizers for Vanda orchid cultivation. The contrasting outcomes between the two experimental settings underscore the critical importance of optimizing application rates based on specific environmental conditions to maximize benefits in commercial production.

1. Introduction

The Vanda genus is considered one of the most popular and commercially important orchids standing alongside other well-known genera including Phalaenopsis, Cattleya, and Dendrobium [1]. Vanda is a genus of large, evergreen, tropical epiphytic orchids with uniquely colored flowers indigenous to the tropical and subtropical regions of Asia and the Pacific [2,3]. Unlike many orchids, Vandas do not have pseudobulbs to store food and their aerial roots are exposed, preventing them from accessing nutrients slowly released from a potting medium. Consequently, they depend on frequent fertilization to remain healthy [3]. Chemical inputs are expensive, increasing the overall cost of production for growers. They can also have notable environmental impacts, contributing to soil and water pollution.

To address these sustainability challenges, research has pivoted towards the orchid microbiome. Traditionally, Orchid Mycorrhizal Fungi (OMF) such as Tulasnella and Serendipita have been recognized as vital partners that enhance nutrient uptake and stress resistance throughout the orchid’s life cycle [4]. Recent evidence suggests a more complex tripartite interaction, where nitrogen-fixing cyanobacteria colonizing the velamen may indirectly supply nitrogen to OMF species that cannot metabolize mineral nitrogen themselves [5].

Beyond fungal associations, endophytic Plant Growth-Promoting Bacteria (PGPB) have emerged as a sustainable alternative to chemical inputs. These bacteria reside within host tissues and facilitate growth through various mechanisms, most notably the conversion of atmospheric nitrogen (N₂) into plant-available forms via nitrogenase activity—a process critical for epiphytes with limited access to terrestrial nitrogen sources. Specifically, nitrogen-fixing bacteria (NFB) such as Bacillus and Pseudomonas have been identified within the roots of various orchid species, with their prevalence influenced by seasonal shifts and mycorrhizal presence [6]. Studies have shown that specific strains, such as Stenotrophomonas maltophilia and Bacillus subtilis, not only improve nitrogen levels but also act as biotic elicitors that boost overall biomass and root development in genera like Cymbidium and Vanda [7,8]. Consistent with the study of Cymbidium sp., inoculation with Herbaspirillum frisingense isolates was shown to improve nutrient uptake; specifically, nitrogen (N) content was elevated by 68% in plantlets during nursery acclimatization [9].

Despite the documented potential of PGPB in enhancing orchid growth, there remains a significant knowledge gap regarding the specific characterization and application of these bacteria in Vanda orchids, particularly within commercially popular hybrid varieties. Understanding how these endophytes can be leveraged to optimize nitrogen uptake is essential for developing eco-friendly cultivation protocols. Therefore, this study aims to isolate, characterize, and evaluate the application of endophytic bacteria to enhance nitrogen uptake and growth performance in Vanda orchids, providing a biotechnological framework for sustainable orchid production.

2. Materials and Methods

The isolation of endophytic bacteria residing within the tissues of Vanda orchids was conducted by Inkaewpuangkham in 2021 [10]. The Vanda ‘Manuvadee’ was dissected into leaves (L), stems (S), and roots (R) for separation. The top three isolates exhibiting the highest nitrogen fixation rates, measured by the Acetylene Reduction Assay (ARA), were 3S19, 2R13, and 3R14, with rates of 24.32, 22.63, and 16.22 µmol C₂H₄/tube/24 h, respectively.

2.1. Molecular Identification by 16S rRNA Sequencing and Phylogenetic Analysis

The three pure isolates (2R13, 3S19, and 3R14) of the most effective nitrogen fixation endophytic bacteria were sent to Macrogen’s Humanizing Genomics company in South Korea for molecular identification by 16S rRNA sequencing. The 16S rRNA gene was amplified using PCR with primer 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′ and 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′. Next, the 16S rDNA gene sequences were sequenced with primer 785F 5′ (GGA TTA GAT ACC CTG GTA) 3′ and 907R 5′ (CCG TCA ATT CMT TTR AGT TT) 3′. To analyze sequence similarity, the 16S rDNA gene sequences obtained were aligned using the BLAST program (version 2.6.0+) (http://blast.ncbi.nlm.nih.gov, accessed on 23 February 2021). The phylogenetic tree was created in MEGA 11 using the neighbor-joining method, and sequence divergences between strains were quantified using the maximum composite likelihood.

2.2. In Vitro Analysis of Endophytic Bacterial Colonization and Nitrogen Content in Vanda

Isolate 2R13, 3S19, and 3R14 endophytic bacteria were cultured in nutrient broth media (NB) for 5 days. The initial bacterial density in the form of the suspension was 1 × 10⁸ CFU mL⁻¹. Liquid bacteria were diluted with sterilized water to 4 ratios: 1:1, 1:10, 1:25, and 1:50. A Factorial Completely Randomized Design was conducted. There were 2 factors, the first factor being the endophytic bacteria isolates (2R13, 3S19, and 3R14). The second factor was the ratios of bacterial solution (liquid bacteria:sterilized water as 1:1, 1:10, 1:25, and 1:50) compared to no bacteria treatment, where we applied only sterilized deionized water as a control treatment, with four replications per treatment (3 plants per replication). Eight-month-old Vanda plants obtained from the shoot meristem culture of Vanda ‘Pachara Delight’ were immersed in each bacterial solution for 30 min and subsequently transferred to Vacin and Went medium [11] for 4 months under the average temperature of 25 °C (throughout the experiment), 16 h of daylight and 8 h of darkness with light intensity of 45 µmol·m⁻²·s⁻¹. After 6 months of inoculation, the total nitrogen content in Vanda plantlets was determined by the modified indophenol method using Kjeldahl digestion solution [12]. The best isolate was selected for colonization. The assessment of endophytic bacteria colonization was performed in Vanda tissue. The tissue of Vanda plantlets that demonstrated the most effective growth was fixed in 2.5% (v/v) glutaraldehyde for 4 h and postfixed in 1% (w/v) osmium tetroxide (OsO4) for 1 h. After dehydration with an increasing-concentration ethanol series (30, 50, 70 and 100%), the intermediate fluid was removed from the samples by Critical Point Drying. Before investigation, specimens were coated with coal and gold evaporation. Observation for endophytic bacteria colonization was made using an LV scanning electron microscope (JSM 5910 LV; JEOL Ltd., Tokyo, Japan).

2.3. Effects of Endophytic Bacteria on the Growth and Nitrogen Uptake of Vanda Orchids Under Greenhouse Conditions

Six-month-old Vanda plants raised via tissue culture were immersed in individual bacterial solutions (as with the previous experiment) for 30 min while those of the control were soaked in sterilized deionized water for the same period. Inoculated plantlets, including those of the control, were then transferred to cultivated baskets without growing media to be placed under greenhouse conditions for 6 months, with 15 replications per treatment (1 plant per replication). The average greenhouse temperature, humidity and light intensity were 28 °C, 70% RH and 45 µmol·m⁻²·s⁻¹, respectively. The plants were watered daily with no fertilizer application throughout the experiment.

Records were made on total nitrogen content in Vanda plants 6 months after inoculation. Plant height (cm), number of leaves, root length (cm) and number of roots were also recorded monthly for the first 6 months following inoculation.

Total nitrogen content and plant growth data were then calculated via analysis of variance (ANOVA) using the Swx program (Analytical Software version 8.0). Indication of significant difference was measured according to least significant difference (LSD) analysis at 0.05 probability level.

3. Results

3.1. Phylogeny of Endophytic Bacteria Isolated from Vanda ‘Pachara Delight’

Phylogenetic analysis based on 16S rRNA gene sequencing was conducted to identify the three selected endophytic isolates (Figure 1). The results showed 100% sequence similarity for all three isolates to known bacterial species. The research found that isolate 2R13 showed 100% similarity to Curtobacteriumcitreum, 3S19 showed 100% similarity to Stenotrophomonas panacihumi and 3R14 showed 100% similarity to Bacillus subtilis. The identification of these three isolates as C. citreum, S. panacihumi, and B. subtilis strongly supports their observed potential as plant growth-promoting (PGP) agents. All three genera are well-documented in literature for their endophytic nature and beneficial associations with various host plants, including orchids.

3.2. Colonization and the Potential of Endophytic Bacteria on Nitrogen Content in Vanda: A Comprehensive In Vitro Analysis

3.2.1. Total Nitrogen Concentration (mg g⁻¹ DW)

Analysis of total nitrogen (N) concentration revealed no statistically significant effect from the bacterial isolate when considered as an independent factor. The N concentrations in plantlets treated with various isolates were not significantly different from the uninoculated control (

Table 1. Effects of endophytic bacteria and ratios of bacterial suspension on nitrogen content (mg g⁻¹ dry weight) of Vanda ‘Pachara Delight’ at 6 months after inoculation.

Factors		Nitrogen Content (mg g−1 Dry Weight)
Isolates	Ratios	In Vitro
2R13	1:1	9.98 ± 0.13 b
	1:10	7.94 ± 0.28 cd
	1:25	9.32 ± 0.62 bc
	1:50	8.18 ± 0.52 cd
3S19	1:1	7.92 ± 0.12 cd
	1:10	7.80 ± 0.32 de
	1:25	12.46 ± 0.91 a
	1:50	6.38 ± 0.12 e
3R14	1:1	8.23 ± 0.63 cd
	1:10	8.15 ± 0.71 cd
	1:25	10.12 ± 0.50 b
	1:50	8.23 ± 0.73 cd
Deionized water (Control)		7.62 ± 0.15 de
isolates		ns
ratios		*
isolates × ratios		*
LSD0.05		1.47
%CV		11.89

* Means in the same column followed by different letters are significantly different (p < 0.05) by LSD. ^ns, non-significant differences.

). In contrast, the inoculation ratio was a significant factor. Treatments using the 1:25 ratio yielded the highest average N concentration, recorded at 10.63 mg g⁻¹ DW. Furthermore, a significant interaction effect between the isolate and its ratio was observed. The single treatment of isolate 3S19 at a 1:25 ratio was the top performer, producing a maximum N concentration of 12.46 mg g⁻¹ DW, which was significantly higher than all other treatment combinations (Table 1).

3.2.2. Colonization of Isolate 3S19 in Inoculated Vanda ‘Pachara Delight’ Tissue

Among all treatments, inoculation with isolate 3S19 at a 1:25 ratio produced the most prominent and superior results. This treatment combination significantly outperformed all other isolates and ratios across overall total nitrogen content in the Vanda plantlets. To determine if this superior performance was linked to the bacteria’s ability to live within the plant, root sections from the 3S19 (1:25) treatment were analyzed. Using Scanning Electron Microscopy (SEM; JEOL Ltd., Tokyo, Japan), we visually confirmed evidence of bacterial colonization. As shown in Figure 2, distinct colonies of isolate 3S19 were observed within the root tissues of the inoculated Vanda plantlets, confirming a successful endophytic relationship had been established. In contrast, the colonies of bacteria were not found in uninoculated plants (Figure 3).

3.3. Influence of Endophytic Bacteria on the Vegetative Growth and Nitrogen Concentration in Vanda ‘Pachara Delight’ Under Greenhouse Conditions

3.3.1. Plant Growth

Analysis of vegetative growth parameters (

Table 2. Effects of endophytic bacteria and ratios of bacterial suspension on growth of Vanda ‘Pachara Delight’ at 6 months after inoculation.

Factors		Number of Leaves	Plant Height (cm)	Number of Roots	Root Length (cm)
Isolates	Ratios
2R13	1:1	5.4 ± 0.23 cd	9.5 ± 0.23 a	5.2 ± 0.31 cd	2.6 ± 0.34 fg
	1:10	5.8 ± 0.17 bc	9.0 ± 0.24 abc	4.9 ± 0.21 de	4.0 ± 0.16 bc
	1:25	6.4 ±0.12 a	9.2 ± 0.12 abc	6.1 ±0.07 a	4.5 ± 0.11 ab
	1:50	5.0 ± 0.14 d	9.4 ± 0.17 ab	5.0 ± 0.19 cde	2.8 ± 0.29 efg
3S19	1:1	5.8 ± 0.14 c	8.9 ± 0.20 bc	5.4 ±0.13 bcd	4.7 ± 0.26 a
	1:10	5.4± 0.27 cd	9.2 ± 0.27 abc	5.9 ± 0.24 ab	2.9 ± 0.30 ef
	1:25	6.3 ± 0.12ab	8.6 ± 0.15 cd	5.6 ± 0.13 abc	4.0 ± 0.14 bc
	1:50	4.3 ± 0.19 e	8.2 ± 0.16 d	3.9 ± 0.27 f	1.6 ± 0.18 h
3R14	1:1	5.6 ± 0.13 c	9.5 ± 0.23 a	5.1 ± 0.15 cd	3.3 ± 0.16 de
	1:10	5.8 ± 0.26 bc	9.4± 0.19 ab	4.5 ± 0.42ef	2.2 ± 0.21 g
	1:25	6.6 ± 0.13 a	8.7 ± 0.20 cd	5.9 ± 0.20 ab	3.2 ± 0.13 def
	1:50	6.3 ± 0.16 ab	9.1± 0.25 abc	4.9 ± 0.28cde	2.6 ± 0.18 fg
Deionized water (Control)		5.7± 0.17 c	8.7± 0.12 cd	5.4 ± 0.15bcd	3.7 ± 0.15 cd
Isolates		*	*	ns	*
Ratios		*	*	*	*
isolates × ratios		*	*	*	*
LSD0.05		0.50	0.56	0.64	0.59
%CV		12.15	8.59	17.04	25.44

* Means in the same column followed by different letters are significantly different (p < 0.05) by LSD. ^ns Not significant.

) revealed that the bacterial isolate, the inoculation ratio, and the interaction between these two factors all had a statistically significant effect on the development of Vanda plantlets.

A strong promotive effect on leaf numbers was observed in specific treatments. Plantlets inoculated with isolate 2R13 (1:25 ratio), isolate 3S19 (1:25 ratio), and isolate 3R14 (1:25 ratio) produced the highest number of leaves, with ranges of 4.83–6.40, 4.70–6.33, and 4.80–6.57, respectively, over the 6-month period. In contrast, isolate 3S19 at a 1:50 ratio yielded the minimum number of leaves per plant from the third month of growth onward.

Plant height, measured at 6 months after inoculation, ranged from 8.2 to 9.5 cm. Inoculation with all three endophytic bacteria generally increased plantlet height compared to the control plants. The greatest average heights (9.5 cm) were observed in plantlets treated with isolate 2R13 (1:1 ratio) (Figure 4A) and isolate 3R14 (1:1 ratio) (Figure 4I). However, the height recorded for isolate 2R13 at a 1:1 ratio was not significantly different from the same isolate at 1:25 or 1:50 ratios. The isolate 3S19 (1:50 ratio) (Figure 4H) treatment produced the lowest plant height, which was comparable to the control plantlets (Figure 4M).

Root growth was significantly influenced by the treatments. An increased number of roots was recorded in plantlets treated with 2R13 (1:25 and 1:50 ratios) and 3R14 (1:25 ratio), with ranges of 3.57–6.10, 3.31–5.00, and 3.53–5.87, respectively. Regarding root length, the maximum lengths were achieved by isolate 2R13 (1:25 ratio) at 4.55 cm and isolate 3S19 (1:1 ratio) at 4.73 cm. Consistent with all other metrics, isolate 3S19 at a 1:50 ratio yielded the minimum average root number and root length throughout the experiment (Table 2).

3.3.2. Total Nitrogen Concentration (mg g⁻¹ DW)

Analysis of total nitrogen (N) concentration in Vanda plantlets revealed that the bacterial isolate as a standalone factor did not cause significant variation. However, the inoculation ratio and, critically, the interaction between the isolate and its ratio, had a statistically significant effect on total N concentration (

Table 3. Effects of endophytic bacteria and ratios of bacterial suspension on nitrogen content (mg g⁻¹ dry weight) of Vanda ‘Pachara Delight’ at 6 months after inoculation under greenhouse conditions.

Factors		Nitrogen Content (mg g−1 Dry Weight)
Isolates	Ratios	Greenhouse Conditions
2R13	1:1	8.62 ± 0.23 bcd
	1:10	8.48 ± 0.11 cde
	1:25	7.49 ± 0.18 ef
	1:50	11.18 ± 0.42 a
3S19	1:1	8.22 ± 0.17 cdef
	1:10	9.62 ± 0.66 b
	1:25	7.32 ± 0.26 f
	1:50	10.83 ± 0.66 a
3R14	1:1	9.18 ± 0.43 bc
	1:10	7.78 ± 0.38 def
	1:25	8.27 ± 0.35 cdef
	1:50	8.61 ± 0.28 bcd
Deionized water (Control)		7.31 ± 0.21 f
isolates		ns
ratios		*
isolates × ratios		*
LSD0.05		1.07
%CV		8.65

* Means with the same letter within the same column are not significantly different by LSD (p ˂ 0.05). ^ns Not significant.

). The highest total N concentrations were recorded in plantlets inoculated with isolate 2R13 at a 1:50 ratio (11.18 mg g⁻¹ DW), followed by isolate 3S19 at a 1:50 ratio (10.83 mg g⁻¹ DW). Conversely, isolate 3S19 at a 1:25 ratio yielded the minimum average total N concentration (7.32 mg g⁻¹ DW), a level that was not significantly different from the uninoculated control (Table 3).

4. Discussions

(a)

Phylogenetic Identification and Functional Significance of Endophytic Isolates from Vanda ‘Pachara Delight’

The phylogenetic analysis, based on 16S rRNA gene sequencing, provided high taxonomic confidence for the three selected isolates. All three isolates exhibited 100% sequence similarity to their respective reference strains in the database, indicating that the isolates are genetically identical or extremely closely related to the established Type Strains, ensuring the identification process’s accuracy. The identification of these specific taxa is highly significant, as all three genera are well-recognized in botanical literature for their roles as Plant Growth-Promoting (PGP) agents. Their presence as endophytes suggests a specialized symbiotic relationship where these bacteria may contribute to the host orchid’s nutrient uptake, pathogen defense, or environmental resilience.

Isolate 2R13 (C. citreum) from Vanda is consistent with previous findings. The previous study reveals that isolate 2R13 from Vanda ‘Manuvadee’ yielded the highest amount of IAA (152.63 mg/L) when cultured with L-tryptophan [13]. A Curtobacterium species was recently isolated from a surface-sterilized orchid (Chiloschistasegawae), where it was noted for its potential in salinity stress alleviation [14]. Furthermore, C. citreum has demonstrated direct growth-promoting capabilities; a strain isolated from tea leaves was shown to significantly enhance the growth and vigor of sunflower and tomato seedlings [15].

Isolate 3S19 (S. panacihumi) is particularly relevant to plant growth. This genus is known for producing high quantities of auxin, a key hormone for stimulating root development, as observed in poplar cuttings [16]. Gontijo et al. [9] reported that Stenotrophomonas maltophilia isolated from Cymbidium orchid roots increased the plant’s total dry matter by 29% compared to uninoculated controls. This efficacy is often linked to the genus’s ability to produce a wide array of phyto-stimulants, including IAA, ammonia, siderophores, and lytic enzymes [17].

B. subtilis (isolate 3R14) is one of the most widely recognized and utilized PGP endophytes. Its association with orchids is well-established, with strains being isolated from various terrestrial Mediterranean orchid species [18]. A recent study by Chand et al. [8] not only isolated B. subtilis from the Vanda cristata orchid but also confirmed its ability to produce beneficial compounds, including the growth hormone IAA, ammonia, and soluble phosphate. The application of this Vanda-derived B. subtilis to Cymbidium aloifolium to plantlets resulted in enhanced root and shoot development, as with the PGP effects observed in our experiment. The identification of these isolates confirms they belong to genera known to colonize internal plant tissues and promote host growth through mechanisms such as N₂ fixation and IAA production.

(b)

Optimizing Inoculum Density for Effective Endophytic Colonization in Vanda ‘Pachara Delight’ In Vitro

Our results find that the concentration of the initial inoculum plays a greater role on nitrogen enhancement in tissue-cultured Vanda plantlets than the choice of bacteria isolate alone. The superior performance of the 1:25 ratio suggests an optimal concentration range is required for the bacteria to effectively colonize the host and perform their metabolic functions. This finding is supported by literature on bioformulation. Khan et al. [19] suggested that an optimum bioformulation must contain a minimum microbial threshold (at least 1 × 10⁷ CFU/g) to be effective. This aligns with our results, where lower-density ratios (e.g., 1:50) may have failed to establish a sufficient population.

Conversely, there is also a risk of excessive concentration. Oliveira et al. [20] reported a clear dose-dependent effect in sugarcane. They found that low inoculum levels (1 × 10²–1 × 10⁴ CFU mL⁻¹) had only a minor positive impact, while high levels (1 × 10⁸ CFU mL⁻¹) significantly reduced the root surface area. The authors hypothesized that such heavy colonization could block the plant’s xylem vessels at an early stage, impairing water and nutrient transport and stunting plant growth. Our findings support this concept of an optimal concentration of inoculation. The 1:25 ratio for isolate 3S19 likely achieves the ideal balance, providing a sufficient bacterial population to colonize the plant and enhance N content without reaching a harmful concentration that could damage the plantlet’s sensitive vascular tissues.

The confirmation of in vitro colonization by isolate 3S19 strongly indicates that the enhanced growth and improved nitrogen status of plantlets in this treatment were a direct consequence of the bacteria’s ability to colonize the host tissue. The physical presence of the endophyte within the root cells allows for the efficient transfer of fixed nitrogen and other growth-promoting compounds to the plant. This observation aligns with findings from similar research. Shah et al. [21] also used scanning electron microscopy to verify endophytic colonization in their work. They reported that their isolate, PVL1, was successfully observed inside the plant cells of Vanda leaves after inoculation.

Therefore, our results, supported by the microscopic evidence, demonstrate that the efficacy of isolate 3S19 as a plant growth promoter is directly linked to its capacity to colonize the Vanda root system.

(c)

Optimal Inoculum Density: Balancing Vegetative Development and Efficient Nitrogen Uptake in Vanda Under Greenhouse Conditions

The application of growth-promoting bacteria has demonstrated significant benefits across various orchid genera. The results showed that inoculation with three specific isolates enhanced the vegetative growth of Vanda plantlets in terms of leaf number, plant height, root number, and root length. Similarly, a study of endophytic bacteria isolated from the meristem of Cymbidium eburneum revealed that the genus Paenibacillus can effectively promote the growth of Cattleya loddigesii during the critical acclimatization phase [22]. Regardless of the inoculation ratio, it clearly demonstrates that all three tested endophytic bacterial isolates possess plant growth-promoting (PGP) capabilities, significantly enhancing the vegetative growth of Vanda plantlets compared to the uninoculated controls. A critical finding is the significant interaction effect, which indicates that the inoculation density (ratio) is a decisive factor in the success of the inoculation, with this effect varying between isolates. The 1:25 ratio consistently emerged as highly effective for promoting the generation of new tissues, specifically increasing the number of leaves (for all three isolates) and the number of roots (for 2R13 and 3R14). In contrast, higher-density ratios like 1:1 appeared to favor elongation growth, as seen in the maximum plant height (2R13, 3R14) and root length (3S19). Azarbad and Junker [23] suggested that inoculum density is one of the most important experimental factors that can change the outcome of an inoculation. Vuolo et al. [24] note that the effects of inoculation can vary widely based on levels of the introduced inoculant. Too low a density may fail to establish a large enough colony to produce a growth-promoting effect. Conversely, an inoculum that is too dense can sometimes lead to competition for nutrients or even trigger plant defense responses.

The performance of isolate 3S19 demonstrates this complex interaction. It was a superior performer for leaf production at a 1:25 ratio and for root length at a 1:1 ratio. However, at the 1:50 (lowest density) ratio, it was the worst performer across all measured parameters. This suggests that while 3S19 is a potent growth promoter, it may have a narrow optimal concentration range and becomes ineffective or fails to establish at lower inoculation densities.

These findings underscore the necessity of optimizing inoculation density for each specific bacterial isolate to maximize its PGP benefits for in vitro and greenhouse orchid production.

A clear difference emerged when comparing the in vitro N concentration data with the greenhouse plant growth performance. The 3S19 isolate at a 1:25 ratio, which produced the lowest tissue N concentration in vitro, subsequently promoted the most significant plant growth and biomass accumulation under greenhouse conditions. This contradiction, where the treatment resulting in the best growth also shows a lower N concentration, is an example of the growth dilution effect [25]. This phenomenon occurs when a bacterial inoculation treatment successfully stimulates a rapid increase in plant biomass. This new growth dilutes the concentration of a given mineral element within the plant’s tissue, even as the total uptake of that element by the plant has increased. This finding is consistent with previous studies, such as Riedell [26], who observed in maize that a significant increase in shoot dry weight corresponded with a decrease in the concentrations of all mineral elements in the shoot. The low N concentration recorded for the 3S19 (1:25) treatment may not be interpreted as nitrogen fixation failing, but instead as an indicator of its success in promoting rapid biomass, which was faster than the rate of N accumulation per gram of dry weight.

Regardless of inoculation ratio, all three isolates resulted in higher N content when compared to control treatment. This indicates that these three endophyte bacteria improve N uptake in Vanda. Although orchids naturally rely on mycorrhizal fungi for growth promotion, nutrient acquisition, and stress tolerance, the integration of nitrogen-fixing endophytic bacteria may further enhance the efficiency of host nutrient uptake mechanisms.

5. Conclusions

This research identifies Curtobacteriumcitreum (2R13), Stenotrophomonas panacihumi (3S19), and Bacillus subtilis (3R14) as effective endophytic biofertilizers for Vanda orchids. These bacteria successfully colonized host root tissues, leading to significant improvements in plant height, leaf count, and root development under greenhouse conditions. A key finding was the environmental dependence of the optimal bacterial concentration; a 1:25 ratio was superior for nitrogen uptake in vitro, whereas a 1:50 ratio proved more effective in the greenhouse. This demonstrates that while these isolates are a viable, sustainable alternative to chemical fertilizers, their application rates must be optimized for specific cultivation environments to achieve the best results. Further validation through large-scale field trials is recommended to facilitate commercial implementation and promote more sustainable orchid cultivation practices.