Impact of altitudinal variations on plant growth dynamics, nutritional composition, and free living rhizospheric N₂ fixing bacterial community of Eruca sativa

Abstract

High-altitude environments present unique abiotic stresses, yet their impact on the growth, nutritional quality, and rhizospheric interactions of E. sativa remains underexplored. Here, we investigate the altitudinal variations in growth dynamics, nutritional composition, and rhizospheric free-living N₂-fixing bacteria (NFBs) of E. sativa (Arugula) grown at higher (3,524 m, Leh-Ladakh) and lower (321 m, Chandigarh) altitudes. Results revealed significant physiological adaptations to high-altitude conditions, with increased concentrations of magnesium (748.84 ± 4.06 mg/100 g), iron (189.83 ± 2.16 mg/100 g), and manganese (8.48 ± 0.27 mg/100 g), while potassium (3,400.83 ± 3.82 mg/100 g), sodium (175.83 ± 1.44 mg/100 g), and copper (1.69 ± 0.01 mg/100 g) were higher at lower-altitude. Zinc content remained unchanged. Notably, dietary nitrate was higher (155.67 ± 22.12 mg/100 g) at high-altitudes. Rhizospheric NFBs were isolated and functionally characterized for N₂-fixation efficacy along with various plant growth-promoting (PGP) attributes; viz., production of ammonia, siderophores, HCN, IAA and phosphate solubilization. Field inoculation with selected strains significantly enhanced nitrogen content and plant growth. Soil chemical analysis further revealed significant differences between the altitudes. A total of twenty-seven NFBs belonging to Actinobacteria (77%), Proteobacteria (11%), Firmicutes(8%), and Bacteroidetes(4%) were isolated, with Streptomyces being the predominant genus, exhibiting distinct species at different altitudes. Remarkably, high-altitude strains showed significantly higher N₂-fixing efficiencies (88.15 ± 17.41 µgN mL^-1) than lower-altitude (65.7 ± 14.36 µgN mL^-1) along with superior PGP traits. Overall, these findings suggest that E. sativa, enriched in key nutrients at high-altitudes, could be a valuable functional food crop, addressing the dietary needs of high-altitude populations. Furthermore, the rhizospheric NFBs identified in this study may be potentially beneficial for the development of novel bio-fertilizers, promoting eco-friendly agricultural practices through improved N₂-fixation. Further field trials are recommended to validate their potential for sustainable crop production.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-98242-2.

Introduction

High-altitude (HA) environments are characterized by low barometric pressure (Pb), increased UV radiation, low humidity, strong wind velocity, extreme temperature fluctuations, and reduced oxygen availability. These conditions not only impact human health but also significantly influence plant ecosystems^1,2. As altitude increases, environmental conditions can become more challenging, posing unique challenges to plant morphology, growth patterns, and nutrient uptake, and requiring plants to adjust their physiology and morphology to survive³. To enhance their resource utilization strategy under varying environmental situations, plants may exhibit phenotypic and anatomical plasticity⁴. Plasticity in morphology and anatomical structures helps plants to appropriately adjust to long-term climatic change⁵. Common tactics for dealing with low temperatures include minimizing heat loss by reducing leaf-area and improving leaf-thickness, as well as modifications to epidermal and mesophyll tissues, to improve boundary-layer resistance in higher elevations^3,4. However, some research reports contradictory findings, such as decreased leaf thickness in C. polyantha and increased leaf area in Kobresia capillifolia at higher elevations^6,7. These findings indicate that plants have a diverse set of adaptation strategies. Therefore, understanding how plants respond to different altitudes might give useful insights into the complex-interactions that exist between environmental conditions and the biology of plants.

One of the many complicated phenomena that are related to altitude is dietary nitrate, which stands out as a potential moderator. Interestingly, nitrate derived from plants is vital for increasing systemic nitric oxide (NO) levels, which helps maintain cardiovascular and cognitive function, especially in high-altitude environments⁸. However, it is crucial to note, that the dietary nitrate content of a plant is in proportion to the quantity of biologically assimilable forms of nitrogen available in the growth medium, which in turn depends on the free-living and plant-associated N₂-fixing microbes called diazotrophs. Thus plant’s dietary nitrate enrichment is related to its ecological association with unique diazotrophs under specific soil and environmental conditions. In this regard, E. sativa has been extensively used worldwide as a nitrate-rich veggie food, medicinal plant, and herb⁹. And as a dietary nitrate crop, has the potential to provide cardiovascular advantages, and other potent phyto-compounds it contains may be significantly advantageous for addressing specific dietary needs at high elevations¹⁰.

Since no study is available on the comparative cultivation, nutritional composition, and rhizospheric N₂-fixing bacterial community of E. sativa (Arugula) grown plants at higher and lower altitudes, this study was undertaken. We investigated how altitude influences E. sativa’s growth, nutritional profile, and rhizospheric NFBs. Specifically, we examined how altitude influences dietary nitrate levels, shaped by soil chemistry, microbial associations, and micro-environmental factors. To understand these dynamics, we isolated NFBs from the rhizosphere, assessed their nitrogen-fixing potential and plant growth-promoting (PGP) traits, and developed bacterial consortia to evaluate their impact on plant growth. Additionally, soil chemical properties were analyzed to understand NFBs interactions. Overall, the current study included (i)Comparative analysis of growth patterns and morphological parameters (i.e. leaf width, plant height, leaf length and root length) of E. sativa grown plants at high-altitude [Leh-Ladakh, India; 3,524 m above mean sea level (MSL)] vs. lower-altitude (Chandigarh, India; altitude of 321 m above MSL) (ii)analysis of dietary nitrate and nutritional value of E. sativa (iii)the isolation and molecular characterization of rhizospheric free-living NFBs associated with E. sativa (iv) Further, NFBs strains isolated from both altitudes were assessed for N₂-fixation efficiency along with various PGP traits, which included the production of ammonia, siderophores, IAA, HCN and phosphate solubilization, (v) Based on the N₂-fixation efficacy and most favourable PGP traits, selected isolates were used to prepare bacterial consortia; subsequently, examine their effects on the E. sativa plant development and total nitrogen content, (vi) The soil’s chemical properties were also examined in order to understand the interaction that exists between the soil and the NFBs at both high and lower altitudes. Thus, this study enhances our understanding of Eruca sativa’s resilience and adaptation mechanisms across different environmental conditions. Based on our current knowledge, this is the first study that explores the rhizospheric NFBs isolates of Eruca sativa. The outcome will give new insight to understand the NFBs population at high and lower altitudes. In addition, may also be advantageous for the formulation of novel bio-fertilizers and also useful for the fortification of nitrate-rich foods.

Materials and methods

Plant material, cultivation practices and collection of plant

Seeds of E. sativa were procured from The Seed Store (India), (https://theseedstore.in/; Socorro, Bardez, North Goa, Goa, INDIA – 403501). E. sativa seeds were cultivated in the open-experimental field area of DIHAR-DRDO at two different stations i.e. firstly at Leh (3,524 m above MSL; 34°08.313ʹ N, 077°34.377ʹ E) in the months of June and July and secondly at Chandigarh (321 m above MSL; 30°41ʹ31ʹʹ N and 76°47ʹ10ʹʹ E) in the months of November and December, since it is a winter crop. The investigation utilized a fully randomized approach with three different replications conducted at both locations, i.e., high and lower altitudes. At each site, twelve plots of about 2.5 m (length) X 1.5 m (width) were made, with irrigation canals separating the plots to keep a 25 cm spacing between them. The seeds were sown in rows with a 5-centimeter gap from plant to plant and 15 centimeters from row to row. The watering was accomplished by flooding the area at three-day intervals. Mechanical and manual methods were used to clear weeds, no pesticides or chemical fertilizers were utilized at either site. E. sativa samples of plants were randomly collected after 15 days, 25 days, and 35 days from each site. They were dried, properly mixed, and ground into powder. Finally, samples were kept in sealed plastic zip bags at 4ºC until analysis.

Morphological and growth parameters

The plant morphological characteristics (i.e. leaf width, plant height, leaf length and root length) were measured after 15 days, 25 days and 35 days intervals, following the guidelines outlined by the “National Bureau of Plant Genetic Resources”, with slight modification¹¹. The height of the plant (in centimeters) was recorded using a ruler scale, extending from the surface to the apex of the tallest leaves. The ruler scale was used to measure leaf width, leaf length and root length. The leaves number per plant was measured by the manual counting method. The chlorophyll amount present in the leaves was determined using a chlorophyll meter (Opti-Sciences, CCM-200 plus Inc., USA). An oven-drying method was used to estimate the moisture content¹². The following equation determined moisture content(%):

Where F.W. stands for the sample’s fresh weight; D.W. represents the sample’s dry weight.

Nutritional analysis

Determination of macro-micro elements and dietary nitrate content

To study the chemical properties of samples, dry plant powdered sample (200 mg) was digested with a micro digester (Analytik Jena AG, Germany) using nitric acid (HNO₃) and hydrochloric acid (HCL) at a 3:1 ratio. After the digestion process was complete, the samples were diluted with distilled water to make the required volume (50 mL), and then they were filtered using Whatman paper (grade 1). This filtrate was then used for the analysis. All the samples were digested in triplicates. Potassium (K) and sodium (Na) contents were analyzed by flame photometer (Jenway PFP7, UK), while the zinc (Zn), iron (Fe), copper (Cu), magnesium (Mg), and manganese (Mn) were evaluated by an atomic-absorption spectrophotometer (ZEEnit 700 plus, analytic-Jena, Germany)¹³. In addition, dietary nitrate concentration was determined by using ion-exchange (Metrohm IC 930 system) chromatography¹⁴. Three replicates were prepared for each sample.

Quantitative Estimation of total nitrogen content

The Kjeldahl method, as described by the Bureau of Indian Standards, was used to determine the sample’s total nitrogen content, with some modifications¹⁵. For this, 200 mg of each sample was taken in a digestion flask to which two Kjeldahl catalyst tablets (2.5gm, Fluka, Sigma-Aldrich, US) and 10 mL of concentrated H₂SO₄ were added. All the contents were thoroughly mixed by a gentle rotation followed by complete digestion in the digestion unit (Electric Speed Digester K-425 Buchi, Germany). Further, steam distillation of the digested sample was performed using the Kjeldahl distillation unit (k-355 Buchi, Germany). After that, samples were made alkaline using 60 mL of 32% sodium hydroxide solution after being diluted with 50 mL of distilled water. Finally, the sample mixture was distilled into 60 mL of 4% boric acid solution (pH 4.65) containing bromocresol green and methyl red mixture as an indicator. Distillate boric acid solution thus obtained was titrated with 0.25 M sulphuric acid. Finally, content was measured using the following formula:

whereas W_(N) : Nitrogen weight fraction, Vol_blank : mean titrant volume for the blank (mL), Vol_samp : volume of titrant for sample (mL), f: titrant factor, c: titrant concentration (molL^− 1), z: molar valence factor (2 for H₂SO₄), Mn: nitrogen molecular weight (14.007gm mol^− 1), m_Sample: sample volume (gm), 1000: conversion factor (mLL^− 1)

Comprehensive nutrient composition

Comprehensive nutrient composition, which includes total energy, carbohydrates, fat, crude protein, dietary fiber, ash, fatty acids, and vitamin content, was estimated in E. sativa from both the field stations i.e., higher and lower altitudes. The quantitative profiling of test samples was outsourced to the “Punjab Biotechnology Incubator Agri and Food Testing Laboratory (PBTI), S.A.S. Nagar (Mohali), Punjab”.

Rhizospheric N₂-fixing bacteria of E. sativa: high altitude vs. lower altitude

Rhizospheric soil samples collection

In our study, as per stranded protocol, we have collected twelve numbers of rhizospheric soil samples (6 from each location) from the E. sativa plant after fifteen days of seed sowing at two different altitudes, i.e., high altitude and lower altitude, respectively. For each plant, approximately 10 g of rhizospheric soil was collected from the root zone, ensuring a representative sample by gently shaking the roots and collecting the soil adhering to them. Following collection, the samples were thoroughly mixed to obtain uniformity, labelled, and transported to the testing lab in cold conditions (4 °C) and processed immediately upon arrival. All the rhizospheric soils were divided into two parts, one part of the soil sample used for microbial culture study and another for chemical analysis, as discussed below.

Estimation of rhizospheric soil chemical properties

To study the chemical properties of rhizospheric soils, we have considered the following parameters, i.e., pH, total Kjeldahl nitrogen (TKN), electrical conductivity (EC), organic carbon (OC), available sulphur (S), potassium (K), phosphorous (P) and micronutrients, viz., boron(B), iron(Fe), and manganese (Mn). The nitrogen content of the soil was determined by employing the Kjeldahl method, with a slight modification¹⁵. Soil pH, EC, OC, and available P, S, K, Fe, B and Mn were determined using the Soil Test and Fertilizer Recommendation (STFR) meter as per the manufacturer’s instructions (IARI Pusa, New Delhi).

Isolation of NFBs from rhizospheric soils

To isolate the NFBs from the rhizospheric soils, we employed the serial dilution method. Briefly, 0.1 mL of aliquots were collected from each (10^− 2 to 10^− 6) tube and uniformly spread on the Petri plates comprising solid nitrogen-free Jensen’s medium (Himedia, India). Subsequently, the plates were then kept for 21 days at 28 °C. Bacteria were isolated at different time intervals ranging from 5 to 21 days. These bacterial isolates were catalogued based on variations in colony appearance. Finally, the pure cultures of selected isolates were prepared and stored at -80 °C in glycerol stocks (20%) for further studies.

Molecular identification of the bacteria isolated from rhizospheric soils

At the onset, the DNA extraction was carried out from the freshly grown pure bacterial cultures using a commercial DNA isolation kit (Zymo Research Kit, USA). Universal primers “27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’)” were used to successfully amplify the 16 S rRNA gene in an automated PCR thermocycler (Eppendorf, USA)⁶. The PCR was as follows: “initial denaturation at 95 °C for 4 min, 30 cycles at 94 °C for 1min, 55 °C for 30 s and 72 °C for 1.5 min and elongation at 72 °C for 12 min”. To purify the PCR-products, gel purification kit (Qiagen, Germany) was employed, followed by sequencing PCR. The PCR (sequencing) was as follows: “initial denaturation at 96 °C for 1 min, 24 cycles at 96 °C for 10s, 50 °C for 5s, and elongation at 60 °C for 15 min”. After conducting sequencing PCR amplification, the resulting PCR product underwent analysis with the ABI 3130 × 1 sequencer (Thermo-Fisher Scientific, USA). The FinchTV (Version 1.4.0, Geospiza, Inc.; Seattle, WA; http://www.geospiza.com) software was employed to analyze the nucleotide sequences.

Phylogenetic analysis and accession numbers

The EzTaxon(“https://www.ezbiocloud.net/”) web-server has been used to analyze the gene (16 S rRNA) sequences¹⁷. Gene sequences exhibiting high similarity in EzTaxon were obtained, and the embedded Muscle algorithm was used to align them. Finally, the aligned files were used as input to build a neighbor-joining phylogenetic tree (> 500 bases) using Molecular evolutionary genetics analysis (MegaX, Version 10.1.5; https://www.megasoftware.net/) software¹⁸. The Maximum Composite Likelihood method was used to calculate the evolutionary distances¹⁹. A total of one thousand boot strap replicates were performed, which was expressed as a percentage. Furthermore, all sequences have been deposited in the “NCBI GenBank database”, and their respective accession numbers can be found in the supplemental material (Supplementary Table S1 and S2).

Quantitative estimation of N₂-fixation by isolates

The N₂-fixing potential of bacterial isolates was determined using the Kjeldahl method with some modifications¹⁵. Firstly, a loop-full activated culture of each selected isolate was inoculated separately into an Erlenmeyer flask containing 50 mL of sterilized nitrogen-free media in triplicates and incubated at 28 °C with a rotational speed of 120 rpm. Then, the flasks were incubated for seven days, subsequently followed by the measurement of the total nitrogen content of each sample using the Kjeldahl method. Finally, content was calculated using the subsequent equation:

Whereas W_(N): Nitrogen weight fraction, Vol_blank: mean titrant volume for the blank (mL), Vol_samp: volume of titrant used for the sample (mL), Mn: nitrogen molecular weight (14.007gm mol^− 1), f: titrant factor, c: titrant concentration (mol L^− 1), z: molar valence factor (2 for H₂SO₄), m_Sample : sample volume (mL), 1000: conversion factor (mLL^− 1)

Study the PGP activities of the bacterial isolates

IAA production

The quantification of IAA production by bacterial isolates was conducted using the method described by Mohite (2013) with minor modifications. Briefly, this experiment was conducted by inoculating a loop of activated culture into YM broth (25 mL) with L-tryptophan (0.2%) and incubating for seven days at 28 °C with 120 rpm. The supernatant was subsequently collected by centrifuging the sample at 8000 g for a duration of 15 min. Further, the collected supernatant of each bacterial sample was mixed with the Salkowski reagent (1:2), followed by a 25-minute incubation at RT. Then, the absorbance of the developed pink color (showing the presence of IAA) was determined spectrophotometrically at 530 nm²⁰. The amount of IAA (µg mL^− 1) produced in the sample was determined using indole-3- acetic acid as the reference standard (y = 0.0202x − 0.0185, R² = 0.9998).

Siderophore production

To study siderophore production qualitatively, we have employed the method described by Alexander and Zuberer (1991) with minor modifications. According to this method, bacterial culture with the ability to grow in the presence of 8-hydroxyquinoline (50 mg L^− 1) containing TSA (10%) agar plates indicated the production of siderophore, and it was depicted as + ve (presence) and –ve (absence)²¹.

Phosphate solubilization

The solubilization of phosphate in selected bacterial strains was examined qualitatively. This was accomplished by inoculating activated bacterial cultures on Pikovskaya’s agar (Himedia, India) and incubating for 7–10 days at a temperature of 28 °C. Halo zones formed around the growth showed the potential for phosphate solubilization²².

HCN production

Plates with 10% Tryptone Soy Agar (Himedia, India) and 4.4 gm L^− 1 of glycine were inoculated with bacterial isolates.

And a piece of filter paper saturated with Na₂CO₃ (2%) and picric acid (0.5%), was carefully positioned on the lid of the Petri plate. Subsequently, the plates underwent incubation at 28 °C for a duration of five days alongside filter paper.

Finally, the shift in the filter paper’s color from yellow to a shade of orange-brown indicated the presence of HCN²³.

Ammonia production

The ammonia production was evaluated qualitatively using peptone-water. Briefly, the bacteria were grown in peptone water broth (10 mL) and incubated at 28 °C for a duration of three to five days. Afterward, 0.5 mL of reagent (Nessler) was added and observed brownish color to the pale yellow or deep yellow formation, indicating ammonia production²⁴.

In-vivo studies

The experiment was conducted to evaluate the in-vivo PGP activities of selected bacterial isolates. Isolates demonstrating superior N₂-fixation efficiency and favorable PGP traits were selected for the development of bacterial consortia. These consortia were subsequently applied to evaluate their effects on plant growth and total nitrogen content. The soil at the experimental site had a loamy sand texture with the following characteristics: OC (0.44 ± 0.02%), EC (0.28 ± 0.01 ms cm^− 1), TKN (0.15 ± 0.01%), available P (75.53 ± 3.57 kg ha^− 1), available S (16.11 ± 0.21 mg kg^− 1), available K (460.73 ± 9.95 kg ha^− 1), available Fe (4.75 ± 0.45 mg kg^− 1), and pH (8.44 ± 0.06).

At the onset, inocula of selected bacterial strains were prepared by transferring a single pure colony into respective media broths (i.e., YM and NB). The bacterial culture was grown in an incubator shaker for 2–7 days at 28 ± 2 °C (120 rpm). Then, cells were diluted to 1 × 10⁷ CFU ml^− 1 using 0.85% saline; after that, 200 gm of soil and 50 mL of inocula were mixed thoroughly, and this mixture was spread homogenously in the plots. Likewise, ten different inoculations of soil were carried out with following nomenclature i.e. Control [T1], L1/CT3 [T2], L1/CT9 [T3], L1/CT11 [T4], CHD_R_CT6 [T5], CHD_R_CT11 [T6], CHD_CT10 [T7], L1 (CT3 + CT9 + CT11) [T8], Chd (R_CT6 + R_CT11 + CT10) [T9], {L1 (CT3 + CT9 + CT11) + Chd (R_CT6 + R_CT11 + CT10)} [T10]. Normal saline (0.85%) was used as a control.

Initially, the seeds of E. sativa were surface-sterilized by immersing them in ethanol (70% solution). Subsequently, they were rinsed four times with sterile distilled water and then dried by blotting on clean filter paper. The experiment followed an entirely-randomized design. Plots with dimensions of 0.8 m (width) by 1 m (length), were prepared, and irrigation channels were used to separate the plots, ensuring a spacing of 25 cm between neighboring plots. The seeds of E. sativa were sown in rows, maintaining a gap of 15 cm between rows and 5 cm between individual plants. Subsequently, flood irrigation was carried out after seed sowing. The plant morphological characteristics (i.e., plant height, leaf area, root length, etc.) were measured after 15 days in accordance with the instructions provided by “National Bureau of Plant Genetic Resources”, with minor modification¹¹. The chlorophyll amount present in the leaves was determined using a chlorophyll meter (Opti-Sciences, CCM-200 plus Inc., USA). At the end of the experiment, the Kjeldahl method, as described by the Bureau of Indian Standards, was used to determine the sample’s total nitrogen content¹⁵. The moisture content was determined using the methodology provided in ‘Morphological and growth parameters’ Section.

Statistical analysis

The experiment findings have been depicted in the form of mean ± Standard Deviation (SD). To identify the significant difference, we have employed the one-way Analysis of variance (ANOVA) with Duncan’s multiple range tests and independent t-test by SPSS 17.0 (SPSS Corporation, Chicago, IL). Bacterial diversity was evaluated using the Shannon Diversity Index. Also, the findings were analyzed with the aid of computer programs, viz., MS Excel and Graph Pad Prisma 8.0.2.

Results and discussion

Morphological and growth parameters of E. sativa at high vs. lower altitude

Eruca sativa samples grown at two different altitudes, i.e., high and lower altitudes were evaluated for growth and morphological characteristics at different time intervals, i.e., 15, 25, and 35 days. The findings, summarized in Table 1, reveal significant variation in plant growth between the two altitudinal locations. In both high and lower altitude sites, the plant grew gradually with the maturation stage. However, it was also observed that during the first stage of plant development, the plant height was considerably lesser at high altitude than at lower altitude. For instance, after 35 days, the height of the plant at the lower altitude (15.13 ± 2.57 cm) was noticeably higher than at the high altitude (10.33 ± 1.05 cm) (p ≤ 0.05). Leaf morphology also varied considerably between the two altitudes. Plants from lower altitudes exhibited larger leaves, as evidenced by greater leaf width, length, area, and number compared to their high-altitude counterparts. For instance, leaf width, leaf length, leaf area and leaf number of E. sativa samples after 35 days from lower altitude (3.86 ± 0.35 cm, 9.8 ± 0.65 cm, 38.17 ± 5.95 cm, and 9.33 ± 0.57) were found to substantially greater than E. sativa samples from the higher altitude (2.86 ± 0.20 cm, 6.5 ± 1.74 cm, 18.86 ± 6.49 cm, and 6.33 ± 0.57), (p ≤ 0.05), respectively. The findings align with earlier studies where similar plant-size responses have also been shown in other investigations²⁵. This might be owing to the impacts of abiotic stresses at high altitude, including cold, salt, high wind velocity, drought, low O₂ levels, and high UV-radiations, etc²⁶. Furthermore, the size or dimensions of the leaf could reduce the quantity of solar energy absorbed and the rate of transpiration, thereby decreasing the harm imposed by elevated UV irradiation and intense winds. In short, small leaf size is a crucial adaptation enabling plants to survive the cold temperatures, high amounts of wind exposure, and intense solar radiation characteristic of high altitudes.

Table 1.

Comparative analysis of various morphological and growth parameters of E. sativa plants from leh (high altitude) and Chandigarh (lower altitude).

Location	TMI	MC	PH	LW	LL	LA	NOL	RL	CC
Leh	15 Days	86.32 ± 0.91a**	2.8 ± 0.36a	1.23 ± 0.05a	1.1 ± 0.1a	1.35 ± 0.08a	4.33 ± 0.57a	3.9 ± 0.52a	6.26 ± 1.38a**
	25 Days	86.39 ± 0.49a***	3.8 ± 0.36a*	1.7 ± 0.2b*	2.6 ± 0.36a**	4.47 ± 1.14a***	5.66 ± 0.57b	6.366 ± 0.47b	22.36 ± 2.27b**
	35 Days	85.20 ± 0.46a***	10.33 ± 1.05b*	2.86 ± 0.20c*	6.5 ± 1.74b*	18.86 ± 6.49b*	6.33 ± 0.57b**	8.43 ± 1.006c	23.36 ± 1.006b***
Chandigarh	15 Days	90.01 ± 0.24b	2.7 ± 0.20a	1.4 ± 0.1a	1.2 ± 0.11a	1.78 ± 0.28a	4.3 ± 0.57a	3.1 ± 0.51a	12.06 ± 0.90a
	25 Days	89.34 ± 0.29ab	8.06 ± 1.80b	2.36 ± 0.25b	5.8 ± 0.79b	13.60 ± 0.44b	6.33 ± 0.57b	3.1 ± 1.00a	12.06 ± 0.80a
	35 Days	88.97 ± 0.67a	15.13 ± 2.57c	3.86 ± 0.35c	9.8 ± 0.65c	38.17 ± 5.95c	9.33 ± 0.57c	7.5 ± 1.10b	14.56 ± 0.32b

Mean values in each column with the different superscript (within group) is showed significantly different by Duncan multiple comparison tests (P < 0.05).

Mean values in each column with different p value (***p ≤ 0.001, **p ≤ 0.01,*p ≤ 0.05; between group) is showed significantly different by independent t-test.

TMI: Time intervals; MC: Moisture content (%); PH: Plant height (cm); LL: Leaf length (cm); LA: Leaf area (cm²); LW: Leaf widths (cm); NOL: No. of leaves; RL: Root length (cm); CC: Chlorophyll content (CCI).

Interestingly, the total chlorophyll contents exhibited a notable increase at high altitude (23.36 ± 1.006 CCI) than the samples from lower altitude (14.56 ± 0.32) (p ≤ 0.001), respectively. The high mountainous region of Leh Ladakh experiences elevated light intensity and significant ultraviolet radiation, potentially increasing chlorophyll concentration in plants²⁷. In addition, the intensity and duration significantly influence the chlorophyll concentration of the plant. The findings align with the observations made by Begum et al. in 2021, who noted that UV radiation has the potential to enhance the chlorophyll content in E. sativa plants²⁸. In contrast, the moisture content of samples at high altitudes ranged from 85.20 ± 0.46% to 86.39 ± 0.49%, whereas the moisture content of samples at lower altitudes ranged from 88.97 ± 0.67% to 90.01 ± 0.24%. This is consistent with the average moisture content of vegetables reported in earlier studies^29,30. In summary, our results highlight significant altitudinal variations in the growth and physiological responses of E. sativa, which are likely adaptations to the challenging environmental conditions present at higher elevations.

Dietary nitrate quantification of E. sativa at high vs. lower altitude

In this study, E. sativa samples grown at two different altitudes, i.e. high and lower altitudes, were quantified for dietary nitrate content across three growth stages: 15, 25, and 35 days. The results revealed significant variations, as depicted in Fig. 1. It was observed that plants are rich in dietary nitrate at both altitudes in the early stage of the plant. However, concentration was significantly higher at high altitude compared to lower altitude. The maximum nitrate content found at high-altitude plants was 155.67 ± 22.12 mg/100 g. Our findings are consistent with prior research indicating that E. sativa leaves are an excellent source of dietary nitrate, a compound known to contribute to cardiovascular health by promoting nitric oxide production^9,31. Increased NO production enhances blood circulation, facilitates oxygen delivery to tissues, and improves hypoxic tolerance, thereby supporting physiological adaptation and overall well-being³¹. The elevated nitrate levels at higher altitudes could offer significant health benefits, particularly for populations residing in these regions, where dietary nitrate plays a vital role in adapting to hypoxic conditions and supports cardiovascular health⁹. It is important to note, however, natural nitrate enrichment in food crops is influenced by complex interactions between soil chemistry, ecological association with soil microbes, and micro-environmental factors³². To better understand these dynamics, we isolated free living nitrogen-fixing bacteria from the rhizosphere of E. sativa and assessed their nitrogen-fixing potential, along with their PGP traits including, phosphate solubilization, production of ammonia, siderophores, IAA and HCN, to evaluate their contribution to nitrate accumulation in the plants (Section ‘Rhizospheric N₂-fixing bacteria of Eruca sativa: high altitude vs. lower altitude’).

Fig. 1.

Nitrate analysis of E. sativa samples (*p ≤ 0.05).

Nutritional analysis

The nutritional value of food lies in its ability to provide essential nutrients and energy that are vital for the proper functioning of the human body. Insufficient intake or deficiencies of these essential minerals has been associated with various health conditions, including compromised immune function and cardiovascular and neurological diseases. Therefore, they must be obtained through the diet, primarily from food and water³³. This study compares the nutritional composition of E. sativa cultivated at two distinct altitudes.

Estimation of macro and micro elements at high vs. lower altitude

Samples of E. sativa from both high and low altitudes exhibited notable differences, as illustrated in Table 2, indicating the impact of altitude on plant physiology and nutrient absorption. Across all growth stages, particularly at 15 days, the plant exhibited enriched nutrient content, with several key differences between the two altitudes. For instance, the potassium content of E. sativa at both altitudes was within 2232.50 ± 5.00 to 3400.83 ± 3.82 mg/100 g range which is remarkably higher than range of 165 mg/100 g to 459 mg/100 g reported for various leafy vegetables³⁴, but lower than 3460 mg/100 g in Amaranthus viridis³⁵. Similarly, sodium content at both altitudes was within 97.50 ± 2.50 to 175.83 ± 1.44 mg/100 g range, comparable to the reported Brassica oleracae vegetable (176.00 ± 1.16 mg/100g)³⁶. These sodium and potassium are crucial cations found both inside and outside cells, playing significant roles in the regulation of muscle contraction, acid-base balance, and plasma volume. Further, the ratio of sodium ions to potassium ions also less than one (Na⁺/K⁺ < 1), which has been recommended for the prevention of high blood pressure³⁷. Thus, the consumption of E. sativa may help reduce the risk of cardiovascular diseases, particularly in high-altitude populations.

Table 2.

Comparative analysis of various element content of E. sativa plants grown at leh (high altitude) and Chandigarh (lower altitude).

Location	TMI	Macronutrients			Micronutrients
		N	K	Mg	Na	Zn	Cu	Fe	Mn
Leh	15 Days	52.4 ± 1.13c***	2415.00 ± 13.23c***	748.84 ± 4.06c***	150.83 ± 1.44b***	3.97 ± 0.38b	1.52 ± 0.08b*	189.83 ± 2.16c***	8.48 ± 0.27b***
	25 Days	41.90 ± 0.53b***	2390.00 ± 10.90b***	686.42 ± 3.98b***	97.50 ± 2.50a***	3.02 ± 0.34a	1.38 ± 0.09ab*	115.55 ± 1.74b***	4.62 ± 1.18a
	35 Days	34.32 ± 1.60a**	2232.50 ± 5.00a***	636.10 ± 8.73a***	99.17 ± 1.44a	2.93 ± 0.10a	1.34 ± 0.08a	89.91 ± 1.48a**	4.22 ± 0.65a
Chandigarh	15 Days	44.12 ± 0.35c	3400.83 ± 3.82c	680.77 ± 6.87c	175.83 ± 1.44c	3.93 ± 0.38a	1.69. ± 0.01b	163.42 ± 1.75b	5.88 ± 0.40b
	25 Days	32.45 ± 1.01b	2880.83 ± 5.20b	662.47 ± 3.06b	149.17 ± 1.44b	3.22 ± 0.63a	1.58 ± 0.05ab	82.83 ± 1.55a	4.29 ± 0.50a
	35 Days	27.43 ± 1.73a	2733.83 ± 12.79a	502.91 ± 4.49a	98.75 ± 1.25a	3.02 ± 0.93a	1.34 ± 0.24a	83.07 ± 0.89a	5.23 ± 1.03ab

• Mean values in each column with the different superscript (within the group) are showed significantly different by Duncan multiple comparison tests (P < 0.05).

• Mean values in each column with different p value (***p ≤ 0.001, **p ≤ 0.01,*p ≤ 0.05; between groups) showed significantly different by independent t-test.

• The results were expressed on average dry weight bases as: Nitrogen (N) (mg/g); Potassium (K) (mg/100 g): Sodium (Na) (mg/100 g); Iron (Fe) (mg/100 g); Manganese (Mn) (mg/100 g); Zinc (Zn) (mg/100 g); Copper (Cu) (mg/100 g); and Magnesium (Mg) (mg/100 g). TMI: Time intervals.

Magnesium (Mg) content from high-altitude samples was found to be in the range of 636.10 ± 8.73 to 748.84 ± 4.06 mg/100 g, which was higher than the range of 502.91 ± 4.49 to 680.77 ± 6.87 mg/100 g, samples collected from low altitude. These values are far greater than those reported for other leafy vegetables³⁸, indicating that E. sativa is a particularly rich source of magnesium, a mineral essential for muscle and nerve function, as well as energy production. Among the studied minerals, nitrogen was the most abundant in E. sativa samples in both samples. However, samples from high altitude have greater TKN, viz., 34.32 ± 1.60 to 52.4 ± 1.13 mg/g, as compared to samples collected from low altitude, viz., 27.43 ± 1.73 to 44.12 ± 0.35 mg/g, respectively. The increased N concentration indicates a higher potential for nitrogen fixation in high-altitude soils, likely influenced by microbial interactions, such as nitrogen-fixing bacteria. In addition to that, Fe content from high altitude samples was found to be in the range of 89.91 ± 1.48 to 189.83 ± 2.16 mg/100 g, which was higher than the range of 83.07 ± 0.89 to 163.42 ± 1.75 mg/100 g, samples collected from low altitude. These values are significantly higher than those in various leafy vegetables³⁴, indicating that E. sativa can serve as an excellent dietary source of iron, that supports red blood cell production and helps prevent hypoxia. Further, Mn content from high altitude samples was found to be in the range of 4.22 ± 0.65 to 8.48 ± 0.27 mg/100 g, which was comparable to range of 4.29 ± 0.50 to 5.88 ± 0.40 mg/100 g, samples collected from low altitude, respectively. The levels observed in this study are comparable to those reported in other Brassica species³⁹, indicating that E. sativa is a reliable source of manganese, irrespective of the growing altitude. Given its essential role in antioxidant defense and metabolic regulation, the manganese content in E. sativa further enhances its nutritional significance. Cu, on the other hand, followed an opposite pattern, with concentrations much greater at lower altitudes than at higher ones. At lower altitudes, the highest Cu concentration was 1.69. ± 0.01 mg/100gm, which is greater than the 0.05 ± 0.003 mg/100 g in Brassica oleracae var. capitata L³⁶. Further, no significant difference was found in Zn content at high altitude (2.93 ± 0.10 to 3.97 ± 0.38 mg/100 g) and lower altitude 3.02 ± 0.93 to 3.93 ± 0.38 mg/100 g). The result is greater than 0.64 mg/100 g in cauliflower and 0.95 mg/100 g in broccoli³⁹, but comparable to 2.11 ± 0.04 mg/100 g in Brassica oleracae var. capitata L³⁶. Zinc plays a crucial role in enhancing immune function and mitigating oxidative stress, further underscoring the nutritional value of E. sativa. The uniform concentrations of Zn in E. sativa at varying elevations suggest that altitude does not significantly affect the plant’s ability to uptake, and accumulate zinc from the soil. This could be an outcome of the possible exclusion of zinc from the Eruca leaf cells or its unavailability in the free form via amino acid complexation or phosphate precipitates as previously explained by Terzano et al. (2008)⁴⁰.

Variation in concentration at both altitudes (i.e. higher and lower altitudes) may be attributable to the difference in anatomical structures of the plants, environmental conditions, soil composition, microbial activity in the rhizosphere, and genetic variation that modifies the nutritional composition of plants. In addition, the variance may be attributable to the ability of plants to acquire resources from their surroundings, either to meet physiological requirements or as a precautionary strategy. Hence, some plants may function as biomonitors to assess pollution levels in the surrounding environment⁴¹. Furthermore, the concentration of some minerals, such as Mg, Cu, and Ca is influenced by both the season and the plant tissue. Overall, it can be stated that E. sativa grown at high altitude was, in general, enriched in micronutrients in comparison with plants grown at lower altitude. Thus, locally grown E. sativa could prove to be a diet of choice for those who live in high-altitude environments.

Comprehensive nutrient composition

The E. sativa samples from both altitudes have almost the same fat content (2.16% at high, 2.15% at lower). This is consistent with the widespread finding that leafy greens have a low-fat content and play an important part in reducing the risk of obesity³⁵. However, these values are lower than the 3.82% of fat in E. sativa Mill. leaves reported by Nurzyńska-Wierdak, (2015)⁴². Further, the fiber content of samples from high altitude (37.61%) was higher than that of samples from lower altitude (30.89%), and these values are relatively higher than the amounts (13.38–16.99%) reported in the same plant by Nurzyńska-Wierdak, (2015)⁴². The high fiber content of E. sativa makes it an important source of dietary fiber, which can help in preventing diseases like diabetes and cardiovascular disorders.

Ash content, indicating mineral composition, was higher at high altitudes (20.92%) than at lower altitudes (18.95%). The earlier findings showed that E. sativa Mill. leaves have ash content in the ranged of 15.91–21.83%, which is very close to this study⁴². Further at high altitude (32.75 g/100gm), the crude protein concentration was substantially greater than at lower altitude (27.57 g/100gm), reinforcing the role of altitude in enhancing protein levels. This makes E. sativa an excellent source of plant protein, particularly for populations in high-altitude regions where protein intake is critical due to environmental stress. In contrast, the carbohydrate content of low-altitude (52.59%) samples was greater than that of high-altitude (44.18%) ones, contributing to the energy value of E. sativa, which falls within the range of other leafy greens⁴³. According to reports, green vegetables are an excellent source of energy, the determined energy value of E. sativa at high altitude (276.14 kcal/100 g) and lower altitude (308.35 kcal/100 g), respectively, aligning with those reported for other leafy vegetables⁴³.

Further, significant altitude-dependent variation in vitamin profile of E. sativa was also observed. At high altitudes, the concentrations of riboflavin (B2-vitamin), niacin (B3-vitamin), pyridoxine (B6-vitamin), folic acid (B9-vitamin), and cobalamin (B12-vitamin) were 1.01 mg/100 g, 15.21 mg/100 g, 0.14 mg/100 g, 93.4 µg/100 g, and 0.04 µg/100 g, respectively. In contrast, lower-altitude plants exhibited lower values: riboflavin (0.56 mg/100 g), niacin (7.33 mg/100 g), pyridoxine (0.13 mg/100 g), folic acid (91.3 µg/100 g), and cobalamin (0.03 µg/100 g). Similar trends were observed for omega-6 and omega-3 fatty acids, with high-altitude plants containing 0.63% and 0.22%, compared to 0.51% and 0.21% at lower altitudes. Conversely, vitamin A (543.6 µg/100 g), ascorbic acid (217.07 mg/100 g), tocopherol (30.69 mg/100 g), and beta-carotene (8154.2 µg/100 g) were more concentrated in plants grown at lower altitudes in comparison to higher altitudes, 483.4 µg/100 g, 138.11 mg/100 g, 26.19 mg/100 g, and 7250.3 µg/100 g, respectively. The elevated concentrations of B-vitamins such as B2, B3, B6, B9, and B12, and essential fatty acids in high-altitude plants suggest their enhanced role in supporting energy metabolism and cardiovascular health, moreover, the presence of vitamins A, C, E, and beta-carotene in E. sativa plants indicate greater antioxidant capacity, crucial for immune support and protection against oxidative stress, which is particularly beneficial for populations in high-altitude regions where energy demands are increased. Thus our results are consistent with the previous reports where rocket leaves are considered to be a valuable source of mineral nutrients, L-ascorbic acid, carbohydrates, and proteins and have a high nutritional value^44,45. Again, variations in outcomes may be attributable to plant type, soil composition, and environmental conditions and genetic variation that modifies the chemical composition of most edible plants. Overall, it can be stated that E. sativa is characterized by a high nutritional composition and should be considered a potential diet component for those who live in high altitude environments.

Rhizospheric N₂-fixing bacteria of Eruca sativa: high altitude vs. lower altitude

Chemical profiling of rhizospheric soils

Chemical profiling of rhizospheric soil samples from different altitudes revealed significant variations in several key parameters, as depicted in Table 3. Determining the pH of the soil is a critical factor since it has a significant influence on plant development and growth because it impacts both nutrient availability and diversity, including soil microorganisms associated with nutrient transformations⁴⁶. Soils from higher altitudes exhibited a near-neutral pH (7.65 ± 0.11), whereas those from lower altitudes were mild alkaline (8.55 ± 0.04). Moreover, minerals are readily available to plants if the soil pH is close to neutral (6.5 to 7.5) and facilitate the abundance of the microbial species in the soils as compared to both acidic and alkaline soils⁴⁶. This difference in pH is likely a major factor contributing to the observed variations in nutrient content and microbial activity between the soils from the two altitudes. These differences in pH are closely related to variations in EC and TOC levels between the altitudes. Soil samples from high altitude have greater EC and TOC, viz., 0.31 ± 0.01 ms cm^− 1 and 0.95 ± 0.04%, as compared to lower altitude samples, viz., 0.27 ± 0.01 ms cm^− 1 and 0.36 ± 0.01%, respectively. The higher TOC content in high-altitude soils suggests a positive correlation between altitude and organic carbon accumulation, which is likely due to the lower temperatures at higher altitudes slowing down the decomposition of organic matter. According to previous reports, higher nitrogen and carbon concentrations in soil correlate with altitudes in mountainous regions⁴⁷; which corroborates with the results of the present study, in which a high TOC co-existed with higher nitrogen content in high altitude (0.31 ± 0.02%) samples compared to lower altitude (0.15 ± 0.01%). The increased nitrogen content in high-altitude soils suggests that the nitrogen fixing bacteria, associated with E. sativa are likely more efficient at nitrogen fixation than the lower altitude nitrogen fixers and support the plant growth, despite the variations in other nutrient levels.

Table 3.

Comparative soil chemical properties of high altitude and lower altitude soil.

Sr no.	Test name	High altitude soil sample (average ± sd)	Lower altitude soil sample (average ± sd)
1	pH	7.65 ± 0.11**	8.55 ± 0.04
2	EC (ms cm− 1)	0.31 ± 0.01 ***	0.27 ± 0.01
3	Organic Carbon (%)	0.95 ± 0.04***	0.36 ± 0.01
4	Nitrogen (%)	0.31 ± 0.02***	0.15 ± 0.01
5	Boron (mg kg− 1)	2.66 ± 0.03***	0.21 ± 0.01
6	Phosphorus (kg ha− 1)	243.80 ± 10.45***	68.47 ± 2.67
7	Potassium (kg ha− 1)	311.47 ± 8.71***	471.40 ± 10.63
8	Sulphur (mg kg− 1)	9.02 ± 0.29***	15.35 ± 0.40
9	Manganese (mg kg− 1)	9.82 ± 0.86*	8.47 ± 0.56
10	Iron (mg kg− 1)	0.10 ± 0.00***	4.34 ± 0.51

(***p ≤ 0.001, **p ≤ 0.01,*p ≤ 0.05)

These variations are further illustrated by the differences in the availability of macronutrients such as potassium, phosphorus, and sulfur. K and S were more abundant in lower altitude soils (471.40 ± 10.63 kg ha⁻¹ and 15.35 ± 0.40 mg kg⁻¹), compared to higher altitude soils (311.47 ± 8.71 kg ha⁻¹ and 9.02 ± 0.29 mg kg⁻¹), respectively. In contrast, P was more available in high-altitude soils (243.80 ± 10.45 kg ha⁻¹) compared to low-altitude soils (68.47 ± 2.67 kg ha⁻¹). These variations in macronutrient availability are likely influenced by the differences in soil pH and organic matter content between the altitudes, as well as the temperature-dependent dynamics of nutrient cycling. The interplay between soil pH, organic matter, and altitude is also evident in the differences in micronutrient content, particularly boron, manganese, and iron. High-altitude soils contained higher concentrations of boron (2.66 ± 0.03 mg kg⁻¹) and manganese (9.82 ± 0.86 mg kg⁻¹) compared to low-altitude soils (0.21 ± 0.01 mg kg⁻¹ and 8.47 ± 0.56 mg kg⁻¹, respectively). However, iron content was significantly higher in low-altitude soils (4.34 ± 0.51 mg kg⁻¹) than in high-altitude soils (0.10 ± 0.00 mg kg⁻¹). These differences highlight the complex interactions between soil chemistry and altitude, where the mildly alkaline pH of low-altitude soils may reduce the availability of certain micronutrients like boron and manganese while favoring the retention of iron. Conversely, the near-neutral pH at higher altitudes supports the retention of boron and manganese but may limit the availability of iron, reflecting the intricate balance of soil nutrients influenced by altitude and environmental conditions.

Isolation of culturable NFBs from rhizospheric soils

The total number of culturable diazotrophs recovered in the soil samples collected from high-altitude was higher (1.66 × 10⁷ CFU ml^− 1) than in the soil samples collected from lower-altitude (1.21 × 10⁷ CFU ml^− 1). In our study, a total of 27 free-living NFBs (14 isolates from high altitude and 13 isolates from lower altitude, respectively) were isolated from rhizospheric soils of E. sativa on a selected medium and were differentiated based on their unique morphology. Besides, a close analysis of identified bacterial isolates revealed that all the bacteria isolated from both altitudes were found to be distinct. Supplementary Fig. S1 and Fig. S2 showed the purified bacterial colonies isolated from rhizospheric soil of high and lower altitude.

The majority of the free-living NFBs isolates from soil samples were related to Actinobacteria, followed by Proteobacteria, Firmicutes and Bacteroidetes. The members of Actinobacteria and Proteobacteria were found to be prominently distributed in both altitudes; however, Bacteroidetes were found exclusively in the soil samples collected from high altitude. Similarly, members of Firmicutes were present only in soil samples collated from lower altitude (Fig. 2). These findings suggest an altitude-driven shift in the microbial community structure. At the genus level, the high-altitude soil samples exhibited genera of Streptomyces, Microbacterium, Promicromonospora, Pseudarthrobacter, Ensifer, Lysobacter and Sphingobacterium. In contrast, rhizospheric soil from lower altitude has shown the genera Streptomyces, Bacillus, Arthrobacter, and Sphingomonas. The presence of several isolates representing the genus of Streptomyces across both altitudes highlights its ecological significance in the rhizosphere of E. sativa, although the species diversity was distinct between the two altitudes. Furthermore, bacterial diversity was evaluated using the Shannon Diversity Index⁴⁸, Evenness, and Richness. The diversity index was 1.02 at high altitude and 1.04 at low altitude, indicating comparable microbial diversity. Evenness values were similarly high (0.74 at high altitude and 0.75 at low altitude), suggesting a uniform distribution of bacterial populations. Additionally, richness remained constant at 4 across both altitudes, reflecting a similar number of distinct bacterial populations at both sites.

So far, a very limited study reported on the genera of Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetesas, major culturable soil microbes from the high altitudes region of Leh-Ladakh. We also obtained similar findings identified by Yadav et al. 2015, Kumar et al. 2019^49,50; although these previous reports do not provide any information on rhizospheric or plant-associated NFBs found in the cold desert of Leh-Ladakh. Nevertheless, our findings provide novel insights into the culturable N₂-fixing bacteria in the rhizospheric soil of E. sativa in this unique ecological niche. This study advances our understanding of the rhizospheric microbiota associated with nitrogen fixation in high-altitude agroecosystems. Further exploration of the functional roles of these bacteria, particularly under environmental stress conditions, could provide valuable insights into their contributions to sustainable agriculture in challenging environments.

Phylogenetic analysis

To understand the phylogenetic relationship of NFBs found at high altitude and lower altitude, we reconstructed the phylogenetic trees using the neighbor-joining method (Supplementary Fig. S3 and Fig. S4). The results demonstrated that the species were systematically arranged according to their respective genera, in conjunction with members of other genera that were noted to be grouped together. Further, based on the evolutionary distance scores and topological structure, the high altitude phylogenetic tree can be categorized into two primary clusters, 1 and 2. Cluster 1 (Microbacterium thalassium, Streptomyces sporoverrucosus, Lysobacter panacisoli, Pseudarthrobacter polychromogenes, Streptomyces glomeroaurantiacus, Ensifer morelensis, Streptomyces griseoflavus and Streptomyces scabiei ) is sister to cluster 2, that has the rest of the species. The isolates of cluster 1 underwent further subdivision into clades 1 and 2, determined by their branching topology. Within cluster 1, sub-clade I (M. thalassium, S. sporoverrucosus, L. panacisoli, P. polychromogenes, S. glomeroaurantiacus) is sister to sub-clade 2 (Ensifer morelensis, Streptomyces griseoflavus and Streptomyces scabiei). Likewise, the phylogenetic tree of lower altitude can be categorized into two primary clusters, 1 and 2, according to the scores of evolutionary distance and topological structure. Cluster 1 (S. viridochromogenes, S. nigra, S. muensis, Bacillus subtilis subsp. Stercoris, S. misionensis, A. nitrophenolicus) is sister to cluster 2, which contains the remaining species. The microbes in cluster 1 were subsequently categorized into clades 1 and 2 according to their branching topology. Within cluster 1, sub-clade I (S. viridochromogenes, S. nigra, S. muensis) is sister to sub-clade 2 (Bacillus subtilis subsp. Stercoris, Streptomyces misionensis, Arthrobacter nitrophenolicus). Furthermore, all sequences obtained in this study have been deposited in the NCBI database^51,52, with accession numbers provided in Supplementary Table S1 and S2.

Quantification of nitrogen-fixation by bacterial isolates

The nitrogen-fixing efficacy of bacterial isolates was evaluated over a seven-day incubation period under controlled laboratory conditions. In this study, bacteria found in rhizospheric soil samples from high and lower altitudes were found to fix nitrogen in the range of 57.57-121.39 µgN mL^− 1 and 38.9-85.59 µgN mL^− 1, respectively. The results of the assays presented in Fig. 3(a), clearly indicate a significantly higher nitrogen fixation capacity among high-altitude bacterial isolates compared to those from lower altitudes. This enhanced nitrogen fixation ability may be attributed to the adaptive mechanisms these microorganisms have developed to cope with the harsh conditions of high-altitude environments, which often include lower temperatures, low oxygen availability, and nutrient limitations. On the other hand, less N₂-fixing efficiency of lower-altitude isolates, may be because the environmental conditions there are not as harsh as compared to high altitudes. These conditions may not apply the same selection pressures as those that are seen in high-altitude settings.

Fig. 2.

Graph showing the percentage relative abundance of genera at both high and lower altitudes.

A notable finding was the predominance of the Streptomyces genus among N₂-fixing bacteria at both the altitudes. The most effective nitrogen fixers were Streptomyces sporoverrucosus (L1/CT10), Streptomyces griseoflavus (L1/CT11), Streptomyces fulvissimus (L1/CT1), Streptomyces badius (L2/CT2) and Pseudarthrobacter polychromogenes (L1/CT9). The findings of this study are consistent with those of prior studies that show that Streptomyces also makes a substantial contribution to nitrogen fixation in diverse environments^53,54. Collectively, our results indicate that the diazotrophic isolates, especially those of the Streptomyces species, highlights their potential as bioinoculants for sustainable agriculture, particularly in regions with low soil fertility or environmental stress^53,55. These isolates provide a potentially useful alternative for enhancing the health of the soil and the yield of crops, therefore contributing to agricultural methods that are more environmentally friendly.

Evaluate the PGP potential of diazotrophic isolates

Efficient PGP bacteria and diazotrophic diversity are the foundation of the agro-ecosystem since they play vital roles extending from nitrogen fixation, nutrient bioavailability, soil formation, and plant growth promotion. These microorganisms exert their effect through indirect or direct mechanisms, which encompass the modulation of phyto-hormones, nutrients solubilization, chelation of metallic ions, and biocontrol of phytopathogens⁵⁶. Over the years, various PGP rhizobacteria have been identified and used in the advancement of agriculture sector output. Our study extends this knowledge by evaluating diazotrophic isolates for their in-vitro nitrogen-fixing potential along with various PGP characteristics such as the production of IAA, siderophores (iron-binding ligands), HCN, ammonia, and phosphate solubilization. The results of the aforementioned assays are described in Tables 4 and 5 and Supplementary Fig S5.

Table 4.

Plant growth promoting activities of bacterial isolates from high altitude soil sample.

Sr no	Strain ID	Bacterial isolates	SPR	PS	HCN	AP	IAA (µg ml− 1)	NFE (µgN ml− 1)
1	L1/CT1	Streptomyces fulvissimus	+	-	+	+	17.34 ± 1.68 j	107.38 ± 4.67l
2	L1/CT3	Microbacterium thalassium	+++	-	+	+	91.55 ± 1.90 n	99.60 ± 2.70 l
3	L1/CT4	Ensifer morelensis	-	-	+	+	24.67 ± 1.31l	70.03 ± 4.67 cdef
4	L1/CT5	Streptomyces indoligenes	-	-	+	+	9.52 ± 0.69 cdef	84.04 ± 4.67 ijk
5	L1/CT6	Sphingobacterium anhuiense	-	-	+	+	6.67 ± 1.34 abc	90.27 ± 2.70k
6	L1/CT7	Promicromonospora alba	++	-	+	+	10.94 ± 1.30 ef	77.81 ± 5.39 fghi
7	L1/CT9	Pseudarthrobacter polychromogenes	+++	+	++	+	8.12 ± 1.16 abcde	121.39 ± 4.67 m
8	L1/CT10	Streptomyces sporoverrucosus	-	+	+	-	20.82 ± 1.34 k	105.82 ± 5.39 l
9	L1/CT11	Streptomyces griseoflavus	+	-	++	+	35.20 ± 1.69 m	102.71 ± 4.67 l
10	L2/CT1	Streptomyces anulatus	++	-	+	+	19.74 ± 1.20 jk	80.93 ± 7.13 hij
11	L2/CT2	Streptomyces badius	+++	-	+	+	9.44 ± 1.55 cdef	87.15 ± 2.70 jk
12	L2/CT6	Streptomyces scabiei	++	-	++	+	7.56 ± 1.99 abcd	76.26 ± 5.39 efghi
13	L2/CT9	Streptomyces glomeroaurantiacus	-	-	+	-	6.84 ± 1.77 abc	73.14 ± 2.70 defgh
14	L2/CT10	Lysobacter panacisoli	+	+	+	+	5.43 ± 1.66 ab	57.58 ± 2.70 b

Single positive sign (+): normal activity showed; double positive sign (++): moderately high activity showed; triple positive sign (+++): strong activity showed of particular properties like SPR, PS, HCN, and AP by bacterial strains; negative sign (-): no activity. Mean values in each column with the same superscript (s) do not differ significantly but different superscript is showed significantly different between each treatments by Duncan multiple comparison tests (p < 0.05). SPR: Siderophore production; PS: Phosphate solubilization; HCN: Hydrogen cyanide production; AP: Ammonia production; IAA: Indole acetic acid production; NFE: Nitrogen fixation efficacy.

Table 5.

Plant growth promoting activities of bacterial isolates from lower altitude soil sample.

Sr no	Strain ID	Bacterial isolates	SPR	PS	HCN	AP	IAA (µg ml− 1)	NFE (µgN ml− 1)
1	CHD/R1	Bacillus megaterium	++	-	+	+	6.77 ± 1.54 ijk	84.04 ± 4.67 abc
2	CHD/R2	Streptomyces viridochromogenes	+	-	+	+	8.45 ± 1.78 cde	68.45 ± 5.41bcde
3	CHD/R4	Streptomyces pseudogriseolus	++	-	+	+	17.09 ± 1.97 b	57.58 ± 2.70ij
4	CHD/R5	Streptomyces misionensis	++	-	+	+	10.46 ± 1.70 bc	62.25 ± 7.13 def
5	CHD/R6	Streptomyces nigra	+	-	+	+	16.68 ± 1.46 defg	71.59 ± 2.70 hij
6	CHD/R8	Sphingomonas leidyi	-	-	++	-	4.98 ± 1.68 cde	68.47 ± 2.70 a
7	CHD/R11	Bacillus subtilis subsp. stercoris	+++	+	+	+	12.12 ± 1.96 defg	71.59 ± 5.39fg
8	CHD/CT6	Streptomyces carpinensis	-	-	+	-	17.95 ± 1.48 ghij	79.15 ± 5.00jk
9	CHD/CT7	Streptomyces nogalater	+	+	+	+	14.23 ± 1.64 b	54.46 ± 2.70 ghi
10	CHD/CT10	Arthrobacter nitrophenolicus	+++	-	+	-	89.00 ± 3.72 cd	66.92 ± 2.70 n
11	CHD/CT11	Streptomyces iakyrus	-	+	+	+	17.47 ± 1.57 a	38.91 ± 2.70 j
12	CHD/CT12	Streptomyces muensis	+	+	+	-	14.01 ± 1.57 jk	87.15 ± 5.39 gh
13	CHD/CT14	Streptomyces pseudovenezuelae	+	+	+	+	9.27 ± 2.09 a	43.58 ± 2.70 cdef

Single positive sign (+): normal activity showed; double positive sign (++): moderately high activity showed; triple positive sign (+++): strong activity showed of particular properties like SPR, PS, HCN and AP by bacterial strains; negative sign (-): no activity. Mean values in each column with the same superscript (s) do not differ significantly but different superscript is showed significantly different between each treatments by Duncan multiple comparison tests (p < 0.05). SPR: Siderophore production; PS: Phosphate solubilization; HCN: Hydrogen cyanide production; AP: Ammonia production; IAA: Indole acetic acid production; NFE: Nitrogen fixation efficacy.

The results of the current study revealed that all bacterial isolates were capable of producing IAA, with concentrations ranging from 4.98 to 91.54 µg mL^− 1. Interestingly, 28% of isolates from high altitude and only 7% of isolates from lower altitude were capable of producing more than 20 µg mL^− 1 of IAA (Fig. 3b). This suggests that high-altitude bacteria may possess enhanced abilities to synthesize IAA, possibly as a response to the harsher environmental conditions. Among the isolates, Actinobacteria were observed as dominant IAA producing species, with Microbacterium thalassium (L1/CT3) from high altitudes and Arthrobacter nitrophenolicus (CHD_CT10) from lower altitude soil samples being the highest IAA producers. This observation is consistent with previous findings indicating that rhizospheric soil bacteria can produce IAA to support plant development^57,58. In terms of siderophore production, 21% of bacterial isolates from high altitude showed strong siderophore activity, and 42% of bacterial isolates have shown normal to moderately high activity. Whereas isolates from lower altitude, only 15% of bacteria showed strong activity, and 61% showed normal to moderately high activity. Siderophores are crucial for plant iron acquisition, particularly in iron-deficient soils, and their production is an important trait for PGP bacteria⁵⁵.

Fig. 3.

(a) Scatter plots showing nitrogen fixation potential of bacterial isolates (high altitude vs. lower altitude). (b) Scatter plots showing indole-acetic acid (IAA) production of bacterial isolates (high altitude vs. lower altitude).

Phosphate solubilization is another critical trait observed in PGP bacteria. In this study, eight bacterial isolates were identified as efficient phosphate solubilizers. Phosphate solubilization improves the availability of phosphorus, a key nutrient for plant growth, especially in soils where phosphorus is present in insoluble forms. The ability of these bacteria to convert insoluble phosphates into bioavailable forms supports plant nutrition, which has been documented in various studies⁵⁹. HCN production was ubiquitous among all isolates, and ammonia production was observed in 85% of high-altitude isolates and 69% of those from lower altitudes. Ammonia generation provides an additional nitrogen source for plants, further enhancing their growth, particularly in nutrient-deficient soils. The widespread HCN production also suggests potential biocontrol capabilities, as HCN is known to suppress the growth of plant pathogens⁶⁰.

Overall, it can be concluded that diazotrophic bacteria isolated from the rhizospheric soil of high altitude were efficient nitrogen fixers with superior PGP activities than isolates from lower altitude. This enhanced ability of these isolates may be an adaptive response to the extreme conditions of high-altitude environments, whereas the relatively milder conditions at lower altitudes impose weaker selection pressures. Collectively, these results suggested that these diazotrophic bacterial isolates coupled with multiple PGP traits, highlight their potential as valuable bioinoculants for promoting plant growth, particularly in marginal or challenging agroecosystems and underscore their importance in sustainable agriculture.

In-vivo studies: a field experiment

This experiment was conducted as a preliminary study to assess the in-vivo plant growth-promoting activities of chosen bacterial isolates. Bacterial consortia, prepared from various isolates based on their diazotrophic and PGP traits (mentioned in Section ‘In-Vivo studies’), were tested for their impact on E. sativa growth and total nitrogen content. The plants grown on fields inoculated with different isolates (alone or in combination) responded with varying degrees to plant growth characteristics viz., plant height, leaf area, leaf length, no. of leaves, root length and chlorophyll content etc. The results of the aforementioned assays are presented in Fig. 4 and Supplementary Fig. S6. This growth enhancement could be attributed to increased production of IAA and ammonia by the isolates, as well as the improved availability of macronutrients such as nitrogen and phosphorus under different treatments.

Fig. 4.

Preliminary bio-fertilizer formulation findings using soil microbes (a) Moisture content; (b) Plant height; (c) Leaf area; (d) Root length; (e) Chlorophyll content; (f) total kjeldahl nitrogen, Control [T1];L1/CT3 [T2]; L1/CT9 [T3]; L1/CT11 [T4]; CHD_R_CT6 [T5]; CHD_R_CT11 [T6]; CHD_CT10 [T7]; L1 (CT3+CT9+CT11) [T8]; Chd (R_CT6+ R_CT11+ CT10) [T9]; {L1 (CT3+CT9+CT11)+ Chd (R_CT6+ R_CT11+ CT10)} [T10]; Values with the same superscript (s) do not differ significantly but different superscript is showed significantly different between each treatments by Duncan multiple comparison tests (p < 0.05).

The TKN content in plants served as a crucial measure of the effectiveness of bacterial inoculation. It was observed that plants growing in soil inoculated with diazotrophic isolates exhibited significantly higher TKN levels (0.0491 ± 0.0004 gmN gm^− 1) compared to control plants, particularly in the T10 treatment group. The plants inoculated with T10 treatment exhibited significantly better plant height (6.17 ± 0.25 cm), root length (5.56 ± 0.55 cm), and the number of leaves (6.33 ± 0.58) as compared with control and other treatment groups. These findings align with previous research by Singh et al. (2021), which reported that N₂-fixing bacteria significantly enhance plant physiological and growth parameters⁶¹. The findings indicate that bacterial inoculation had a significant impact on plant development, which is associated with the diazotrophic and PGP characteristics of the isolates. The current study further supports the role of diazotrophic isolates in boosting total nitrogen content in E. sativa plants. These tested isolates, which exhibit single, binary, or multiple PGP traits, may promote plant growth both directly and indirectly while also contributing to bio-fortification by increasing dietary nitrate content in crops. While the results are promising, further experiments are warranted to fully understand their mechanisms and optimize their application for sustainable agriculture.

Conclusions

Considering the present findings, this is the first report on the comparative investigation of altitudinal variations in growth dynamics, nutritional composition, and rhizospheric NFBs of E. sativa grown at high vs. lower altitudes. In addition, the study highlights the potential of NFBs, along with showcasing their utility for bio-fortification and plant growth promotion using green technologies. Results revealed significant physiological adaptations of the E. sativa plants to high-altitude conditions, with increased concentration of dietary nitrate at high-altitude. Notably, the study’s results indicated that the genus Streptomyces was dominant among NFBs in rhizospheric soil samples of E. sativa from both sampling locations, despite the species being unique. Furthermore, these isolated NFBs exhibit multiple PGP characteristics simultaneously, such as phosphate solubilization, production of ammonia, siderophores, IAA, and HCN, suggesting that these strains may enhance plant growth via multiple mechanisms. Therefore, the bacterial strains with the most advantageous PGP characteristics were examined in a field inoculation experiment and shown to have the capacity to increase TKN content and plant growth parameters significantly. Moreover, these isolated NFBs may be potentially beneficial, but, they should be tested more in field conditions with E. sativa to confirm their potential for application as bio-fertilizers. Overall, these findings suggest that E. sativa exhibits significant altitudinal variations in growth and physiology, likely as adaptations to high-altitude stress, which also influence nutrient absorption. Its high nutritional value underscores its potential as a functional food for high-altitude populations. Furthermore, the present investigation on potential NFBs is a stepping stone in the development of novel bio-fertilizers to improve nitrogen fertilization of the high mountain cultivation fields and endorse the use of microbiological resources in eco-friendly agriculture.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

AMS

Acute mountain sickness

B

Boron

CFU

Colony forming unit

Cu

Copper

D.W

Dry weight

EC

Electrical conductivity

F.W

Fresh weight

Fe

Iron

HA

High-altitude

HACE

High-altitude cerebral edema

HAPE

High-altitude pulmonary edema

HCN

Hydrogen cyanide

IAA

Indole−3-acetic acid

K

Potassium

Mg

Magnesium

Mn

Manganese

MSL

Mean sea level

Na

Sodium

NA

Nutrient agar,

NB

Nutrient broth

NFBs

N₂-fixing bacteria

NO

Nitric oxide

NO₂⁻

Nitrite

NO₃^-

Nitrate

OC

Organic carbon

P

Phosphorous

PGP

Plant growth-promoting

rRNA

Ribosomal ribonucleic acid

S

Sulphur

TKN

Total Kjeldahl nitrogen

TSA

Tryptic soy agar

Zn

Zinc

Author contributions

Nitish Kumar contributed to the design of the research, collected samples, conducted lab work, analyzed the data and wrote the manuscript with support from Deepika Sharma. Madhu Khatri, Suresh Korpole, and Shweta Saxena conceived the idea of this research work and as a supervisor contributed to the design and implementation of the research. Somen Acharya contributed to data interpretation and supervised the study. Bhupinder Kaur, Shardulya Shukla, Pushpender Bhardwaj and Manoj Kumar Patel helped conduct the experiments. All authors have read and approved the final version of the manuscript.

Funding

This work was supported by “Defence Institute of High Altitude Research (DIHAR), Defence Research and Development Organization (DRDO), Ministry of Defence, Govt. of India, C/O 56 APO, Leh-Ladakh, Pin-194101, India”.

Data availability

The sequences obtained in this study have been submitted to the NCBI database, and their corresponding accession numbers are provided in the supplemental material (Supplementary Table S1 and S2).

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study does not include any research or experiments involving human participants or animals.