All 28 halophilic archaeal isolates were assessed qualitatively and quantitatively for solubilization of phosphorus (P), potassium (K), and zinc (Zn). The HRA medium was modified by adding 5.0 g L⁻¹ tricalcium phosphate to estimate P solubilization (HPS medium), and KCl was replaced with 5.0 g L⁻¹ potassium aluminum silicate to estimate K solubilization (HKS medium). Similarly, Zn solubilization was quantified using two sets of HRA medium supplemented with zinc oxide and zinc carbonate, respectively (added at 1.0 gL⁻¹) (HZS medium). For qualitative estimation of P, K, and Zn solubilization, 10 μl of log phase cultures were spot-inoculated on respective modified HRA agar and incubated at 37 °C. The formation of the halo zone around colonies was recorded periodically until the 10th day of incubation and the D/D ratio was calculated, i.e., the diameter of the zone of solubilization/colony diameter. The cultures which solubilized the P, K, and Zn with a D/D ratio higher than one were selected for quantifying the extent of their solubilization. For this, 1 ml inoculum (10⁸ CFU ml⁻¹) of the selected cultures was inoculated in 50 ml (for P solubilization) and 100 ml (for K and Zn solubilization) of respective HRA broth and incubated at 37 °C at 130 rpm. After 10 d of incubation, the pH of the culture broth was recorded and subjected to centrifugation at 10, 000 g for 5 min. The supernatant was used for quantitative estimation of soluble P, K, and Zn by the ascorbic acid method (Yadav et al., 2015), flame photometer (Banerjee and Prasad, 2020), and atomic absorption spectrophotometer (ZEEnit 700 P, Analytik Jena, Gmbh) (Saravanan et al., 2004), respectively. The experiment was performed in triplicates, and un-inoculated control was maintained.

The production of indole-3-acetic acid (IAA) by 28 isolates of halophilic archaea was quantified by culturing them in HRA broth supplemented with L-tryptophane (at 500μg ml⁻¹). The 50 ml broth was inoculated with 1 ml of actively growing inoculum (10⁸ CFU ml⁻¹) and incubated at 37 °C for 10 d at 130 rpm. Following incubation, the culture was centrifuged at 5,500 g for 10 min, and 2 ml supernatant was used for spectrophotometric estimation of IAA production by Salkowski's reagent method (Goswami et al., 2014). The concentration of IAA in the samples was inferred by comparison with the standard curve. The un-inoculated medium was used as blank, and the experiment was conducted in triplicates.

Plant growth-promoting rhizobacteria (PGPR) play a big role in the mitigation of abiotic stresses by improving the expansion of soil colonization by plant roots. This can be reflected in higher affordability to retain the needed nutrients for plant growth and development (Širić et al., 2022). To determine whether the halophilic archaea can grow in soils having slight (4.0 to 8.0 dsm⁻¹) to moderate (8.0 to 30.0 dsm⁻¹) salinity, a modified halophilic broth was prepared using soil extract, where the water component was replaced with soil extract. The soil extract was prepared from the soil sample collected from a wheat field (15 cm depth) located at the village Chhapra Khera (29°40′42.3^′′N and 77°02′50.7^′′E), Karnal, Haryana, India. The soil was sandy loam in texture with an EC of 6.2 dsm⁻¹, pH 8.1, total carbon: 0.48 %, total nitrogen: 0.051%, exchangeable K: 173.61 mg Kg⁻¹, and available P: 4.21 mg Kg⁻¹. For the preparation of soil extract, 400 g of fine soil was mixed with 1600 ml of tap water and mixed thoroughly to make a uniform solution. The mixture was heated at 121°C for 15 min. After cooling to room temperature, the mixture was filtered through a sterile muslin cloth followed by filter paper (Whatman No. 1). The filtrate was used as a water component in the preparation of halophilic broth. The HRA broth was modified to develop halophilic soil extract (HSE) broth having EC and nutrient mineral composition (gL⁻¹) as shown in Table 1. The composition of major mineral ingredients (NaCl, MgCl₂.6H₂O, MgSO₄.7H₂O, KCl, NaHCO₃, NaNO₃, and CaCl₂.2H₂O) was subjected to change to achieve desired EC levels, and the composition of remaining minor mineral nutrients (KH₂PO₄, NH₄Cl, FeSO4.7H2O, and MnSO4.7H2O) was kept constant. The medium was denoted as HSE-1 to HSE-9 (EC: 7.13–30.84 dsm⁻¹). HRA medium (EC: 119.8 dsm⁻¹) was used as a reference control for comparing the growth and PGP properties of selected halophilic archaea. The HSE broth (HSE-1 to HSE-9) was used to quantify the growth and PGP attributes of H. pelagica CDK2.

Based on the results of the experiment as described in Section 2.2, the best performing halophilic archaeon was selected for evaluating its growth, nutrient solubilization (P, K, and Zn) potential, and IAA production at different strengths of EC in HSE broth. The growth was quantified by estimating protein by Bradford's protein assay (Marion, 1976) as described above in Section 2.1. For quantifying IAA production and the solubilization of P, K, and Zn, the HSE broth was amended with tryptophan, tricalcium phosphate, potassium aluminum silicate, ZnO, and ZnCO₃ similarly as described above in Section 2.1 for the modification of HRA medium. The quantification of IAA production and P, K, and Zn solubilization was carried out as per the method described in Section 2.1.

The inoculum for in planta studies was raised by culturing H. pelagica CDK2 in 250 ml of HRA1 broth for 15 days. The exponentially growing culture was centrifuged at 7,500 g for 15 min, and the supernatant was discarded. The pellet was washed twice with phosphate buffer saline (PBS) whose electrical conductivity was maintained at 8 dSm⁻¹. The washed pellet was suspended in PBS (prepared using soil extract instead of water; EC: 8dsm⁻¹) to maintain the cell density of ~10⁹ cells ml⁻¹. The PBS with archaeal cells was mixed with charcoal (sterilized at 121°C for 20 min for 3 consecutive days and buffered with calcium carbonate) and homogenized. This mixture was air-dried overnight at room temperature under sterile conditions and was coated over wheat seeds immediately before sowing in the pots. For the soft mineral salt agar experiment, the seeds were soaked directly in the PBS with archaeal cell suspension for 30 min and were sown in the soft agar tubes.

Five wheat cultivars, namely two tolerant (K65 and KRL210) and three susceptible (HD2380, HD3086, and HD2687), were procured from ICAR-Indian Institute of Wheat and Barley Research, Karnal, India. A 21-day wheat-halophilic archaea interaction experiment was carried out in soft agar test tubes under controlled conditions to assess the effect of inoculating H. pelagica CDK2 on seed germination. Seeds of all the wheat cultivars were treated with cells of H. pelagica CDK2 suspended in PBS (halophilic archaeal soil extract phosphate buffer saline suspension; EC: 8 dsm⁻¹) for 30 min. The mineral salt soil extract soft agar (MSSE) was prepared by dissolving various macro- and micronutrients, vitamins, and amino acids in previously prepared soil extract as per composition. The composition of MSSE was as follows: a) macronutrients (mg L⁻¹): 1650.0 NH4NO3, 332.0 CaCl₂, 180.690 MgSO₄, 1900.0 KNO3, and 170.0 KH₂PO₄, b) micronutrients (mg L⁻¹): 6.20 H₃BO₃, 0.025 CoCl₂.6H₂O, 0.025 CuSO₄·5H₂O, 37.30 disodium EDTA dihydrate, 27.800 FeSO₄.7H₂O, 16.90 MnSO₄·H₂O, 0.213 Na₂MoO₄·2H₂O, 0.830 KI, 8.60, and ZnSO₄.7H₂O, c) vitamins (mg L⁻¹): 100.0 myoinositol, 0.50 nicotinic acid, 0.50 pyridoxine HCl, and 0.10 thiamine HCl, and d) amino acid (mg L⁻¹): 2.00 glycine and agar 8.0 gL⁻¹ (pH 6.8 and EC: 8 dSm⁻¹). The treated seeds were placed in a 50 ml test tube having 35 ml of MSSE soft agar medium (Filek et al., 2010). The tubes were incubated in the dark until seed germination and were later transferred to a glasshouse of Phytotron facility, IARI, New Delhi, under controlled conditions (glasshouse conditions were as follows; temperature: 20-24° C, humidity: 50%, and white LED lights, 15000 lux with 10 h of daily illumination). The un-inoculated control was maintained, and the experiment was carried out in three replications. Observations on vegetative growth were recorded after 21 days of incubation.

The wheat–H. pelagica CDK2 interaction was further evaluated in a pot experiment to verify the effect of inoculating H. pelagica CDKA on wheat growth and yield parameters in an un-sterile rooting medium. The study was conducted in the net house of the Division of Microbiology, ICAR-Indian Agricultural Research Institute, New Delhi. The treatment included two salt-tolerant (K65 and KRL210) and three susceptible (HD2380, HD3086, and HD2687) wheat cultivars in triplicates. The seeds were coated with the halophilic archaeal-charcoal-based inoculum (as described in section 2.4) and were sown in 14-inch pots each containing 10 kg of un-sterile soil. The electrical conductivity of the soil was maintained at 8 dSm⁻¹ using a mineral salt solution. An un-inoculated control was maintained for all the wheat cultivars in triplicates. The pots were fertilized with the recommended dose of NPK (120:60:60). The full dose of P, K, and 50% of N was applied as a basal dose before sowing. The remaining half dose of nitrogen was applied in 2 split doses at 21 d and 45 d. Observations on vegetative growth, osmolyte accumulation, and biochemical and yield parameters were recorded after 45 days of sowing and at the time of harvesting.

The shoot and root length (cm) and shoot and root biomass (g) were measured manually using standard laboratory protocols. The plants at 21 d from MSSE and at 45 d from pot experiments were sampled destructively. The roots were washed thoroughly with water to remove adhering medium and soil, respectively. The above-ground part (shoots) was separated from the roots and allowed to air dry for 1 h. The fresh weight (biomass) and the length of the shoot and roots were measured manually with a balance and sliding caliper, respectively.

Crude protein was extracted from 100 mg wheat leaves homogenized with the 5 ml salt-alkaline phosphate extraction buffer (0.1 M NaOH in 3.5% NaCl; pH 7.4). The homogenate was incubated at 60 °C for 90 min followed by centrifugation at 4,000 g for 20 min at room temperature. A total of 1 ml of supernatant was used for the quantification of crude protein following Bradford's quantitative protein extraction method (Marion, 1976). The protein content was expressed as μg protein mg⁻¹ of wheat tissue. The quantity of protein in the sample was extrapolated from a standard curve prepared using different concentrations of bovine serum albumin (BSA).

Chlorophyll from the leaf sample was estimated using the DMSO method (Blanke, 1992). A 100 mg fresh plant leaf sample was placed in a 100 ml volumetric flask containing 10 ml DMSO (Dimethyl sulfoxide). The flask was allowed to stand overnight before measuring the spectroscopic absorbance at 663 and 645 nm. DMSO without leaf sample was used as blank, and un-inoculated wheat leaf was used as control. The total chlorophyll content of the wheat leaf was calculated using the formula given below.

Total sugars in wheat leaf samples were quantified using the phenol-sulfuric acid method (Dubois et al., 1951). D-glucose was used as a standard for the preparation of the standard curve, and a run without a sample was used as a blank. The amount of proline synthesized in wheat seedlings in response to salinity stress was estimated using an acid ninhydrin-sulfosalicylic acid method as described by Ábrahám and László Erdei (2010). A blank was run without the sample, and a standard graph was prepared using L-proline.

The antioxidant enzymes, such as ascorbate peroxidase (APox), catalase (CAT), and superoxide dismutase (SOD) accumulation, were quantified in wheat leaves. The enzyme extract was prepared by freezing 1 g of wheat leaf sample in liquid nitrogen to arrest the proteolytic activity followed by grinding with 10 ml of extraction buffer (0.1 M phosphate buffer, pH 7.5, containing 0.5 mM EDTA with 1 mM ascorbic acid). The mixture was passed through four-layered muslin cloth followed by centrifugation at 15,000 g for 20 min. The resultant filtrate was used as an enzyme in the enzyme assay. The APox activity was determined by measuring the decrease in ascorbic acid content as described by Nakano and Asada (1981). Superoxide dismutase activity was quantified by measuring the decrease in the absorbance of formazan generated by superoxide radicles and nitroblue tetrazolium (NBT) dye as per the method described by Dhindsa et al. (1981). A unit of SOD activity was expressed as the amount of enzyme, which reduced the absorbance to 50% as compared to tubes lacking enzyme. Catalase antioxidant enzyme activity was analyzed by calculating the amount of H₂O₂ decomposed by comparing it to a standard curve of known concentrations of hydrogen peroxide (Aebi, 1984).

Analysis of changes in root physiological parameters at the harvesting was performed using a root scanner LA2400 Instruments REGENT, equipped with WinRhizo software. Freshly harvested roots were washed under running tap water to remove all the soil particles attached to the roots. After washing, the roots were kept immersed in water in a 500 ml beaker and taken for root imaging. Roots were placed horizontally on the scanner block filled with a minimal amount of distilled water to keep the roots submerged. The entangling of roots was removed using the forceps.

Based on the halo zone formation observed around spot-inoculated colonies in specific solubilization media (HPS, HKS, and HZS medium), we qualitatively assessed the ability of 28 halophilic archaea to solubilize phosphorus (P), potassium (K), and zinc (Zn) (Supplementary Table 2). Among the isolates, 17 showed significant zones of P solubilization. The highest D/D ratio (cm) for P solubilization was observed in H. pelagica CDK2 (1.8), followed by Halobacterium sp. IARI-SNS3 (1.75), Natronoarchaeum mannanilyticum IARI-SSAB3 (1.6), H. borinquense IARI-WRAK9 (1.6), Halolamina pelagica IARI-CSK1 (1.6), and Natronomonas pharaonis IARI-MAAB4 (1.6). The halophilic archaea N. mannanilyticum IARI-SSAB3, H. borinquense IARI-WRAK9, H. pelagica IARI-CSK1, and N. pharaonis IARI-MAAB4 exhibited similar D/D ratios for P solubilization. Conversely, the lowest D/D ratio was observed in Halorubrum sp. IARI-WRAB4 (1.16), followed by Halostagnicola kamekurae IARI-TWAK7 (1.2) and Halosarcina sp. IARI-WRAB3 (1.25), respectively. Furthermore, 21 isolates displayed significant K solubilization zones, with D/D ratios ranging from 1.25 to 1.68. The highest D/D ratios for K solubilization were recorded in Haloterrigena sp. IARI-SOAB2, N. pharaonis IARI-MAAB4, and H. pelagica CDK2 (1.68, 1.66, and 1.65, respectively). In contrast, only a few isolates demonstrated the ability to solubilize Zn, with six isolates solubilizing ZnO and three isolates solubilizing ZnCO3 in the respective HZS medium. The D/D ratio for all the Zn solubilizing isolates was just above 1, indicating the formation of very small clearing zones around the spot-inoculated colonies.

We conducted quantification of P solubilization and measured the reduction in pH of the medium for the selected 17 isolates, grown in HPS broth. On the 10th day of growth, a significant decrease in pH was observed, ranging from 3.6 to 4.8, compared to the initial pH of 7.2. The greatest reduction in pH was observed in Haloarcula tradensis IARI-WRAK3 and H. pelagica CDK2 (pH: 3.6), while the least reduction occurred in Halorubrum sp. IARI-WRAB4 and Haloarcula argentinensis IARI-SOAB1 (pH: 4.8) (Figure 1A). Regarding P solubilization in HPS broth, the values ranged from 15.98 ± 1.46 to 61.10 ± 2.56 mg L⁻¹ among the 17 isolates. H. pelagica IARI-CDK2 demonstrated the highest P solubilization, significantly measuring 61.10 ± 2.56 mg L⁻¹. Close behind were N. mannanilyticum IARI-SSAB3 with 55.17 ± 1.36 mg L⁻¹, Halogeometricum rufum IARI-WRAK7 with 50.04 ± 1.56 mg L⁻¹, and N. pharaonis IARI-MAAB4 with 49.69 ± 1.45 mg L⁻¹ (as depicted in Figure 1A). Conversely, the lowest P solubilization was observed in Halorubrum sp. IARI-WRAB4 (15.98 ± 1.46 mg L⁻¹), H. kamekurae IARI-TWAK7 (16.20 ± 1.21 mg L⁻¹), Halopenitus persicus IARI-MAAB3 (16.30 ± 1.06 mg L⁻¹), and H. argentinensis IARI-SOAB1 (19.68 ± 1.35 mg L⁻¹) (Figure 1A).

Similarly, a significant reduction in the pH of HKS broth was observed on the 10th day of incubation, compared to the initial pH of 7.4, during the solubilization of P. The highest pH reduction was observed in Haloferax sp. IARI-MAAK4, H. rufum IARI-WRAK7, and H. pelagica CDK2 (pH: 3.6), followed by Haloferax alexandrinus IARI-MAAB1 (pH: 3.68) and Natrinema sp. IARI-WRS9 (pH: 3.7) (as depicted in Figure 1B). Conversely, the least decrease in pH was observed in Halopenitus persicus IARI-MAAB3 and Haloterrigena hispanica IARI-SGAB3 (pH: 4.8), followed by Halobacterium sp. IARI-SNS3 and N. pharaonis IARI-MAAB4 (pH: 4.7) (refer to Figure 1B). For potassium solubilization in HKS broth, the halophilic archaea exhibited a range of solubilization from 27.89 ± 0.30 to 74.36 ± 2.74 mg L⁻¹. H. pelagica CDK2 (74.36 ± 2.74 mg L⁻¹), N. pharaonis IARI-MAAB4 (73.85 ± 1.26 mg L⁻¹), and H. persicus IARI-MAAB3 (73.16 ± 1.84 mg L⁻¹) solubilized a significantly high amount of K. On the other hand, H. argentinensis IARI-SOAB1 (27.89 ± 0.30 mg L⁻¹), Haloarcula sp. IARI-DWAK3 (33.64 ± 0.39 mg L⁻¹), and H. kamekurae IARI-TWAK7 (38.19 ± 0.06 mg L⁻¹) solubilized a significantly low amount of K (Figure 1B).

The solubilization of Zn by six isolates in the HZS medium using ZnO as the source ranged from 4.96 ± 0.03 mg L⁻¹ to 7.21 ± 0.29 mg L⁻¹. Additionally, in the HZS medium with ZnCO3 as the source, three positive isolates exhibited Zn solubilization ranging from 4.91 ± 0.15 to 5.84 ± 0.25 mg L⁻¹. Notably, H. borinquense IARI-WRAK9, H. pelagica CDK2, and Natrinema pallidum IARI-WRAK8 were capable of solubilizing both insoluble sources of Zn (ZnO and ZnCO3), whereas H. alexandrinus IARI-MAAB1, N. pharaonis IARI-MAAB4, and Halorubrum sp. IARI-WRAB4 solubilized only ZnO. Among the ZnO-containing HZS media, H. pelagica CDK2 exhibited the maximum quantity of Zn solubilization (7.21 ± 0.29 mg L⁻¹), which was statistically comparable to Halorubrum sp. IARI-WRAB4 (7.14 ± 0.21 mg L⁻¹) and significantly higher than IARI-MAAB4 (5.72 ± 0.09 mg L⁻¹), IARI-MAAB1 (5.28 ± 0.98 mg L⁻¹), IARI-WRAK8 (5.4 ± 0.20 mg L⁻¹), and IARI-WRAK9 (4.96 ± 0.23 mg L⁻¹). In contrast, N. pallidum IARI-WRAK8 exhibited the highest solubilization of Zn in the HZS media containing ZnCO3 (5.84 ± 0.25 mg L⁻¹), which was significantly higher than the solubilization by H. pelagica IARI-CDK2 (5.27 ± 0.12 mg L⁻¹) and IARI-WRAK9 (4.91 ± 0.15 mg L⁻¹). A significant reduction in pH was observed in the HRA medium containing ZnO and ZnCO3 compared to the initial pH of 7.4, ranging from 3.6 ± 0.15 to 4.2 ± 0.13.

The ability of all halophilic archaea isolates to produce the phytohormone IAA (indole-3-acetic acid) was assessed by culturing them in HRA media supplemented with tryptophan. Out of the isolates, 17 exhibited significant production of IAA, ranging from 17.33 ± 0.43 μg ml⁻¹ to 49.31 ± 1.79 μg ml⁻¹ (Figure 1C). Among them, the highest amount of IAA was produced by H. rufum IARI-WRAK7 (49.31 ± 1.79 μg ml⁻¹), which was significantly greater than the IAA production of all other isolates. Following closely were H. kamekurae IARI-TWAK7 (29.97 ± 0.79 μg ml⁻¹), H. hispanica IARI-SGAB3 (29.06 ± 0.82 μg ml⁻¹), and IARI-SNS3 (28.49 μg ml⁻¹). The isolate Halolamina pelagica CDK2, which exhibited significant solubilization of P, K, and Zn, also produced IAA (23.86 ± 0.52 μg ml⁻¹). On the other hand, Halococcus sp. IARI-BGAK2 produced the lowest amount of IAA, which was significantly measured at 17.33 ± 0.43 μg ml⁻¹.

Based on the obtained results, H. pelagica CDK2 was chosen for further investigation. The study aimed to assess its ability to thrive in lower electrical conductivity (EC) conditions, compared to its natural habitat with EC ranging from 75.25 to 106.7 dSm⁻¹. Additionally, the study aimed to evaluate its potential in enhancing wheat growth by managing nutrient availability and mitigating salinity stress. A modified growth medium suitable for halophilic organisms, supplemented with soil extract (HSE), was utilized. The HSE medium was prepared with different levels of EC, as shown in Table 1. The growth and plant growth-promoting (PGP) attributes of H. pelagica CDK2 were quantified at 8-, 15-, and 21-day intervals, as outlined in Table 2. Observations revealed that H. pelagica CDK2 exhibited growth even at the lowest EC of 7.13 dSm⁻¹. Protein production by the haloarchaeon increased significantly as the electrical conductivity (EC) of the HSE broth increased (Table 2). Notably, at the highest EC level of 119.8 dSm⁻¹, H. pelagica CDK2 produced the highest amount of protein throughout the incubation period. Specifically, protein production reached 220.80 ± 2.35 μg ml⁻¹, 243.25 ± 10.40 μg ml⁻¹, and 263.45 ± 10.10 μg ml⁻¹ at 8, 15, and 21 days, respectively (Table 2 and Figure 2A).

H. pelagica CDK2 exhibited the ability to solubilize insoluble tricalcium phosphate across various salinity levels, ranging from EC: 7.13 to 30.84 dSm⁻¹ (HSE-1 to HSE-9). The solubilization of phosphorus (P) in different EC levels of HSE broth ranged from 8.12 ± 0.19 mg L⁻¹ (EC: 7.13 dSm⁻¹) to 31.44 ± 1.17 mg L⁻¹ (EC: 30.84 dSm⁻¹) after 10 days. The lowest values of P solubilization in HSE-1 broth (EC: 7.13 dSm⁻¹) were statistically similar to those observed in HSE-2, with a P solubilization of 8.12 ± 0.20 mg L⁻¹ at an EC of 9.73 dSm⁻¹. However, a significant increase in P solubilization was observed as the EC level increased from 12.20 to 30.84 dSm⁻¹. The highest solubilization, reaching 31.44 ± 1.17 mg L⁻¹, was observed in HSE-9 with an EC of 30.84 dSm⁻¹. It is worth noting that the P solubilization in HSE-9 was significantly lower compared to the solubilization observed in the control HRA media (61.10 ± 2.56 mg L⁻¹) with an EC of 119.8 dSm⁻¹ (Table 2). A similar pattern was observed for potassium (K) solubilization, with the exception that H. pelagica CDK2 did not exhibit K solubilization in the HSE-1 medium with the lowest EC of 7.13 dSm⁻¹. However, K solubilization began at a level of 3.67 ± 0.036 mg L⁻¹ in HSE-2 with an EC of 9.73 dSm⁻¹, and this was statistically similar to the K solubilization (4.92 ± 0.12 mg L⁻¹) observed in HSE-3 with an EC of 12.20 dSm⁻¹. A significant increase in K solubilization was observed as the EC increased from 16.59 to 30.84 dSm⁻¹, reaching a maximum yield of 42.09 ± 1.44 mg L⁻¹ in HSE-9 medium with an EC of 30.84 dSm⁻¹. However, this value was significantly lower than the K solubilization observed in the control HRA medium (74.36 ± 2.74 mg L⁻¹) with an EC of 119.8 dSm⁻¹ (Table 2).

In contrast to P and K solubilization, the solubilization of zinc (Zn) by H. pelagica CDK2 in HSE media using ZnO as the source of Zn was not observed at lower EC levels of 7.13, 9.73, and 12.20 dSm⁻¹ (HSE broth 1, 2, and 3, respectively). Solubilization of ZnO was statistically similar (ranging from 1.25 ± 0.02 to 2.96 ± 0.05 mg L⁻¹) in media with ECs ranging from 16.59 to 27.63 dSm⁻¹ (HSE-4 to HSE-8). The maximum solubilization of zinc oxide (ZnO) was observed in the medium with an EC of 30.84 dSm⁻¹, specifically in the HSE-9 medium, with a solubilization level of 3.54 ± 0.10 mg L⁻¹. However, this value was significantly lower than the solubilization observed in the control HRA medium with an EC of 119.8 dSm⁻¹. Furthermore, H. pelagica CDK2 did not exhibit solubilization of zinc carbonate (ZnCO3) at any of the lower EC levels. It only showed solubilization in the medium with an EC of 30.84 dSm⁻¹, with a solubilization level of 2.47 ± 0.015 mg L⁻¹. Again, this value was significantly lower than the solubilization observed in the control HRA medium with an EC of 119.8 dSm⁻¹ (Table 2).

The production of indole acetic acid (IAA) was observed at all EC levels in different HSE broths. There was a significant increase in indole-3-acetic acid (IAA) production with each subsequent increase in the electrical conductivity (EC) level, as indicated in Table 2. The IAA production in HSE broth supplemented with tryptophan ranged from 2.64 ± 0.027 μg ml⁻¹ in HSE-1 medium with an EC of 7.13 dSm⁻¹ to 16.98 ± 0.20 μg ml⁻¹ in HSE-9 medium with an EC of 30.84 dSm⁻¹ on the 10th day of incubation. Notably, the IAA production in the control HRA medium (23.85 ± 0.52 μg ml⁻¹) was significantly higher compared to the values observed in the HSE-9 medium (16.98 ± 0.20 μg ml⁻¹) (Table 2).

Inoculation of the halophilic archaea H. pelagica CDK2 had a significant positive impact on the germination and initial vegetative growth of all wheat cultivars grown in soft agar (EC 8 dSm⁻¹) compared to the un-inoculated control (Figure 2A). The germination time of the inoculated wheat cultivars was significantly reduced by 2 to 3 days compared to the germination observed on the 8th day in the un-inoculated control treatment. Among the inoculated treatments, wheat cultivar K65 exhibited the fastest germination on the 5th day, followed by germination on the 6th day for all other cultivars. On the 21st day of germination, shoot and root growth parameters were measured (Supplementary Table 3). Inoculation resulted in a significant increase in the total shoot weight and length of all wheat cultivars compared to their respective un-inoculated treatments. The maximum percent increase in shoot weight due to inoculation was observed in wheat cultivar HD2687 (21.23%), followed by HD2380 (18.48%), K65 (16.96%), HD3086 (15.93%), and KRL210 (13.78%) compared to their respective un-inoculated wheat cultivars. Similarly, the maximum percent increase in shoot length due to inoculation was observed in wheat cultivar K65 (25.19%), followed by HD3086 (21.45%), HD2687 (18.80%), HD2380 (17.82%), and KRL210 (15.24%) compared to their respective un-inoculated wheat cultivars.

Likewise, inoculation of H. pelagica CDK2 in different wheat cultivars significantly improved the root growth parameters compared to their respective un-inoculated wheat cultivars. Inoculation significantly increased the percent root weight of wheat cultivar K65 by 67.18%, followed by HD3086 (47.06%), HD2687 (46.56%), KRL210 (42.30%), and HD2380 (42.22%) compared to their respective un-inoculated wheat cultivars. The number of lateral roots also increased compared to the un-inoculated control (Figure 2A). A similar effect was observed on total root length, and inoculation of H. pelagica CDK2 significantly increased the percent root length of wheat cultivar HD2380 by 39.93%, followed by HD2687 (37.81%), HD3086 (33.53%), K65 (16.45%), and KRL210 (14.62%) compared to their respective un-inoculated wheat cultivars.

Inoculation with H. pelagica CDK2 significantly increased the fresh shoot biomass and shoot length of all wheat cultivars compared to their respective un-inoculated treatments on the 45th day after seed germination (Table 3). The wheat cultivar HD3086 showed the maximum percent increase in shoot biomass due to inoculation (47.13%), followed by HD2380 (32.44%), KRL210 (32.37%), HD2687 (30.65%), and K65 (28.81%) compared to their respective un-inoculated cultivars. Similarly, the maximum percent increase in shoot length due to inoculation was observed in wheat cultivar KRL210 (10.32%), followed by HD3086 (9.30%), K65 (6.32%), HD2380 (4.94%), and HD2687 (4.25%) over their respective un-inoculated treatments (Table 3). The differences in shoot biomass and shoot height in response to halophilic archaeal inoculation were statistically significant among the wheat cultivars at p ≤ 0.05. A representative image of the pot experimental study is shown in Figure 2B.

In a similar manner to the shoot parameters, inoculation of H. pelagica CDK2 significantly improved root growth in different wheat cultivars compared to their respective un-inoculated treatments (Table 3). The maximum significant increase in root biomass (%) due to inoculation was observed in wheat cultivar HD3086 (52.08%), followed by HD2687 (46.41%), KRL210 (38.96%), K65 (33.66%), and HD2380 (29.10%) compared to their respective un-inoculated treatments (Table 3). The percent increase in root biomass in inoculated HD3086 was significantly higher than the percent increase observed in other wheat cultivars. Similarly, inoculation significantly increased the root length of all wheat cultivars compared to the un-inoculated treatments. The maximum root length due to inoculation was observed in wheat cultivar HD3086 (27.48 cm). Among the inoculated cultivars, KRL210, K65, and HD2380 showed statistically similar root lengths (25.16 cm, 25.06 cm, and 25.06 cm, respectively). The least increase in root length was observed in cultivar HD2687 (19.36 cm). The percent increase in root length due to inoculation was maximum in HD3086 (41.21%), followed by HD2687 (28.81%), K65 (14.63%), KRL210 (13.18%), and HD2380 (11.13%) compared to their respective un-inoculated wheat cultivars (Table 3).

The root scanning revealed significant increases in the number of lateral roots, total root surface area, and root volume due to the inoculation of H. pelagica CDK2 compared to the un-inoculated control at harvest time (Figure 3). The root surface area in the inoculated wheat treatments at harvest time ranged from 148.34 ± 6.41 cm² to 241.25 ± 3.91 cm², which was significantly higher than their respective un-inoculated control treatments (ranging from 91.67 ± 1.15 cm² to 140 ± 2.88 cm²). The overall percent increase in root surface area ranged from 51.34% to 73.75%. The wheat cultivar HD3086 showed the maximum percent increase in root surface area (73.75%), followed by K65 (71.96%), KRL210 (61.82%), HD2687 (56.42%), and HD2380 (51.34%) compared to their respective un-inoculated control (Figure 3A).

The total root volume of the different wheat cultivars significantly increased compared to their respective un-inoculated cultivars. The root volume in the treated wheat cultivars at harvest time ranged from 1.85 ± 0.061 cm³ to 2.91 ± 0.12 cm³, which was significantly higher than their respective control treatments (ranging from 1.20 ± 0.018 cm³ to 2.19 ± 0.080 cm³) (Figure 3A). The overall percent increase in root volume ranged from 21.97% to 131.83%. The maximum percent increase in root volume due to inoculation was observed in wheat cultivar HD2687 (131.83%), followed by HD3086 (71.96%). Cultivar HD2380, KRL210, and K65 showed significantly less percent increase due to inoculation (30.14%, 25.11%, and 21.97%, respectively) compared to HD2687 and HD3086. The average root diameter in each of the H. pelagica CDK2-treated wheat cultivars (ranging from 0.68 ± 0.026 mm to 1.76 ± 0.025 mm) increased significantly compared to their respective non-treated wheat cultivars (ranging from 0.53 ± 0.02 mm to 1.02 ± 0.021 mm) (Figure 3B). The average root diameter also varied significantly among inoculated wheat treatments. The maximum root diameter due to inoculation was observed in HD2687 (173.37%), followed by HD3086 (126.87%) and HD2380 (50.06%). In comparison, the salinity-tolerant wheat cultivars, K65 and KRL210, showed significantly less improvement in root diameter upon halophilic archaeal inoculation (20.78% and 12.89%, respectively). The overall effect of halophilic archaeal inoculation seems to be higher in salinity-susceptible wheat varieties compared to tolerant varieties.

Inoculation with halophilic archaea resulted in a significant increase in both grain yield per spike and grain yield per plant across all wheat cultivars compared to their respective untreated controls (Table 3). The number of grains per spike ranged from 51.67 ± 1.15 to 58.00 ± 2.01 in the inoculated wheat cultivars, while it ranged from 44.00 ± 1.00 to 52.00 ± 1.01 in the untreated controls. The wheat cultivars HD2687 (20.00%) and HD3086 (19.69%) showed the maximum percentage increase in grain per spike due to inoculation, which was statistically similar to each other. These values were significantly higher than the percentage increase in grain yield per spike observed in HD2380 (13.98%), K65 (11.40%), and KRL210 (7.69%) compared to their respective untreated controls (Table 3).

Similarly, the grain yield per plant also exhibited a significant increase as a result of inoculation with H. pelagica CDK2 in various wheat cultivars compared to their respective untreated controls. The grain yield per plant in the inoculated cultivars ranged from 3.68 g to 5.21 g, while it ranged from 2.70 g to 3.47 g in the untreated treatments (Table 3). The increase in grain yield showed significant variation among different wheat cultivars. The wheat cultivar K65 demonstrated the highest percentage increase in grain yield (50.28%), followed by KRL210 (47.91%), HD2687 (41.85%), HD3086 (38.48%), and HD2380 (30.03%). It is worth noting that the salinity-tolerant varieties of wheat displayed a stronger response to inoculation with halophilic archaea in terms of grain yield per plant (Table 3).

Seed treatment with halophilic archaea resulted in a significant reduction in antioxidant enzyme activity in all wheat cultivars. The inoculated wheat cultivars showed a decrease in ascorbate peroxidase activity ranging from 20.0% to 57.43%, while the reduction in superoxide dismutase activity ranged from 59.22% to 75.99%. The decrease in ascorbate peroxidase activity was statistically similar among the susceptible cultivars HD2687 (57.43%), HD2380 (56.08%), and HD3086 (55.17%) but significantly higher than the reduction observed in the salinity-tolerant wheat cultivars, KRL210 (23.33%) and K65 (20.17%). The highest percentage reduction in SOD activity was observed in wheat cultivars HD3086 (75.99%), followed by HD2687 (75.82%), HD2380 (68.55%), KRL210 (60.16%), and K65 (59.22%) (Table 4). Catalase activity also exhibited a significant reduction in susceptible wheat cultivars (HD2380: 61.14%, HD2687: 52.86%, and HD3086: 49.89%) compared to tolerant ones (K65: 32.50% and KRL210: 29.88%) when compared to their respective un-inoculated treatments (Table 3). The overall impact of halophilic archaeal inoculation was more pronounced in susceptible wheat cultivars than in tolerant ones. This suggests that the inoculation of halophilic archaea H. pelagica CDK2 has assisted wheat cultivars in mitigating salinity stress.