The soil samples were collected from the experimental farm of ICAR–Central Soil Salinity Research Institute situated in the western Indo—Gangetic Alluvial Plains, Panipat, Haryana, India (29°19'7.09'' to 29°19'10.0'' N latitude and 76°47'30.0'' to 76°48' 0.0'' E longitude, elevation of 230 to 231 m above mean sea level). The site has complex saline and saline—sodic soil with sandy loam soil texture and ustic soil moisture regime. The soil of the experimental farm is classified as Hasplustepts. The EC_e of the sites ranged from <4 to >30 dS m⁻¹ and pH_1:2 <8.2 to 9.0. A total of 15 rhizospheric soils of four crops (wheat cv. KRL 210 (Triticum aestivum L.), Pearl millet cv. HHB 197 (Pennisetum glaucum L.), sorghum cv. Hybrid Mayur (Sorghum bicolour L), and mustard cv. CS 56 (Brassica juncea L)) were collected at the flower initiation stage. The three adjacent plants of each crop were uprooted carefully to collect rhizosphere soil [21]. The soils adhering on the roots were collected aseptically and stored at <4°C for microbial isolations and other biochemical analysis. For physiochemical properties, bulk soil samples were collected adjacent to the plants collected for rhizosphere soils. The bulk soil samples were air–dried and sieved through a two mm sieve before analysis.

The pH and electrical conductivity (EC) were estimated using a digital pH meter (Systronics ^μpH system 362), and EC meter (Systronics conductivity meter 306^μc), respectively in 1:2 (w/v) aqueous soil suspension. Electrical conductivity of saturation extract (EC_e) was determined by removal of aqueous extract of soil paste under suction at 0.88 kg cm⁻² [3]. Walkley and Black oxidizable organic C, KMnO₄ oxidizable–N, Olsen’s P and NH₄OAc extractable K were estimated following standard procedures [35]. The soil texture and saturation extract parameters were determined for the composite soil sample taken from all 15 sites (Table S1). The Shannon–Weiner diversity index (SDI) is an index of measuring the diversity of species in a habitat. The bacteria, actinobacteria and fungi population [20] were estimated in rhizosphere soil samples for computing the rhizosphere SDI [20] as described in Eq. (1).

where pi is proportion of the species i in population, and S is the total number of species.

Pikovskaya agar medium supplemented with 5% NaCl was used for enumerating phosphate solubilizing fungi (PSF) and bacteria. Streptomycin (30 μg L⁻¹) and amphotericin B (50 μg L⁻¹) were supplemented to the medium to control bacteria and fungi growth for isolation of PSF and PSB, respectively. The isolates on Pikovskaya agar medium demonstrating halo zones around their colonies after 5 days were counted (Fig. S1a, b). Fungi with the highest zone of solubilization in each rhizospheric sample were picked on potato dextrose agar (PD) slants for further study after purification. A total of 15 distinct salt–tolerant phosphate solubilizing fungal isolates were selected for further study.

The culture condition variation alters the expression of the biosynthestic gene expression responsible for structural diversity and production of the metabolites [36]. Therefore, the isolated fungi were incubated at 25°C for seven days on the agar medium such as Czapek–Dox (CZ), Potato–Dextrose (PD), Malt Extract (ME), and Yeast Extract Sucrose (YES), to identify the fungal–media combinations having the ability to metabolically utilize various substrates such as carbohydrates, and compounds as sole sources of carbon. Fungi have greater variability in the growth rate, mechanisms of sporulation, and nutrient requirements on different growth media, hence colony growth, colony obverse and reverse colors were recorded following standard procedure [37, 38]. The microscopic study was carried out on slides prepared from the fragment of a fungal colony (approximately 1–2 mm from the periphery) grown on MEA medium stained with lactophenol cotton blue (Himedia®) [39]. Microscopic characteristics focusing on conidiophore branching patterns were examined to identify the genus. The physiological characterization was carried out by monitoring fungal growth on Czapek Dox agar medium at pH 5 and 8. Fungal growth was also observed at different temperatures (25–40°C) [40–42].

Fungal biomass under saline conditions was estimated in Pikoskaya’s broth containing 1 and 5% NaCl upto 15^th day. The salt–tolerance index (STI) was determined on PDA supplemented with 5% NaCl [43]. The STI was calculated by following formulae. (2):

The PSP was confirmed by inoculating isolated fungal strains on Pikovskaya’s agar medium supplemented with 1 and 5% NaCl. Fungal culture disc was inoculated on Pikovskaya’s agar medium under aseptic condition and incubated at 28±2°C for 5 days. The halo zones developed around the colonies showed PSP. The results were represented as the diameter of zone in mm as phosphorus solubilization potential. The quantitative phosphorus solubilization by fungal isolates were studied under stationary conditions in Pikovskaya’s medium [44]. About twenty ml of growth medium supplemented with 1 and 5% NaCl (w/v) and tricalcium phosphate (TCP) (0.1% w/v) were inoculated with the 7 days old grown two disc (diameter 6 mm) of mycelia of fungi and incubated for 15 days at 28±2°C. The amount of the P released in media was monitored up to 15 days by the ascorbic acid blue color method [45]. The phosphorus solubilization ratio (PSR) was calculated by the following formulae. (3):

where PS_5N is the phosphorus solubilized at 5% NaCl salinity level, and PS_1N is the phosphorus solubilized at 1% NaCl salinity level.

The organic acid (OA) production was determined by titrating culture broth with 0.05 N NaOH and was expressed as equivalent g tartaric acid L⁻¹ [46]. Change in pH was estimated by a digital pH meter.

Amylase, cellulose [47], and protease [48] activities of fungal isolates were screened on medium containing carboxy methyl cellulose, soluble starch, and skimmed milk power. Enzymatic index (EI) was calculated as described [49] earlier using following eq. (4):

where, R = diameter halo zone, and r = diameter of fungal colony.

For the estimation of acid and alkaline phosphatase enzymes [50], 100 ml Erlenmeyer flask containing sterilized 25 ml Czapek Dox medium was inoculated with 2 mm disc of 5 day–old grown culture for 5 days at 25°C. The fungal mat and culture broth were centrifuged for 10 min at 12000 g. The cell–free culture filtrate was used to determine the extracellular acid and alkaline phosphatase. The biomass obtained was homogenized in 25 ml phosphate–buffered saline (PBS) aseptically and filtered with Whatmann 42 filter. The filtrate obtained was used to estimate the intracellular acid and alkaline phosphatases.

Indole acetic acid (IAA) [51] and hydrogen cyanide (HCN) [52] estimation was carried out in tryptophan and glycine–supplemented medium, respectively. Chrome azurol S dye was used to detect siderophores production [53]. The ammonia production was determined by incubating the isolates in peptone water [54]. Pikovskaya agar medium supplemented with 0.1% zinc oxide has been used to determine zinc solubilization [55].

The bioinoculant potential of fungi was evaluated on four different crops (wheat cv. KRL 210 (Triticum aestivum L.), pearl millet cv. HHB 197 (Pennisetum glaucum L.), sorghum cv. Hybrid Mayur (Sorghum bicolour L), and mustard cv. CS 56 (Brassica juncea L)) through in vitro germination assay. After surface sterilization, seeds of crops were treated with culture filtrate of each fungus. The culture filtrate of fungi was harvested at the log stage obtained after the fifth day of incubation in potato dextrose broth. The 5 ml of 1% NaCl in sterilized distilled water (w/v) was applied to seeds to maintain salinity. Germination was considered when the radicles were half of the seed length. The germination percentage, and root and shoot length were measured [6]. The germination rate and vigour index were calculated with the formulae [5]:

The SPSS software was used for all the statistical analysis. Shapiro Wilk’s and Bartlett’s Tests were applied for evaluating the normality and heterogeneity of the variance, respectively. The Kruskal-Wallis test was performed for analysis of variance (ANOVA) to characterize the effects of the crop rhizosphere, and fungal isolates. Similarly, the Mann Whitney U test was carried out to test the effect of fungus genera. Post–hoc analyses pertaining to treatments means were performed using the p values adjusted for Benferroni correction for multiple tests. The cluster analysis was carried out using R. The relationship between different soil variables with microbial population and diversity index in the study was established using Redundancy analysis (RDA). The Monte Carlo permutation test was carried out to verify the significance of RDA models. Contributions of the fungal properties, including plant growth potential and salt tolerance to the total variation in germination and vigor of four crops (wheat (Triticum aestivum L.), pearl millet (Pennisetum glaucum L.), sorghum (Sorghum bicolor L), mustard (Brassica juncea L) were determined by partial redundancy analysis (pRDA) using the Vegan package in R [56].

The cultural and morphological characterization of the fungi was carried out by observing colony features under the two or more growth conditions. Different colony size of fungal isolates was observed on four growth media (Table 1). The morphological features of the fungal growth on PD, YE and CZ media and microscopic features on the ME media indicated that the fungal isolates belonged to the genus Penicillium and Aspergillus (Table 1; Fig. 1). Out of 15 fungal isolates, eleven were from Penicillium while the other four were related to Aspergillus. The rhizospheric soil samples collected for determining the microbial assemblage had different EC, pH, SOC, and available N, P, and K content (Table 2). The soil under the pearl millet had greater EC, SOC, and NH₄OAc–K, while KMnO₄–N was greater under the wheat crop. The EC was lowest under the wheat, while soil pH was similar in rhizosphere of all the crops. The highest rhizosphere microbial population was observed for pearl millet and lowest for wheat. However, the relative abundance of the P solubilizing bacteria (PSB) and fungi (PSF) were lowest for pearl millet compared to sorghum, mustard, and wheat. The rhizospheric PSF assemblage was greater than PSB in all the crops. The Shannon–Weiner diversity index ranged from 0.20–0.21 in all rhizospheric soils (Table 2). The association of Penicillium spp. with crops was greater in soils of higher salinity than Aspergillus. Penicillium was also a preferred partner in different crops under nutrient deprivation compared to Aspergillus. Sorghum inhabiting Penicillium as dominant P solubilizers had a greater population of the actinobacteria, bacteria, fungi, and P solubilizers but the relative proportion of the PSF and PSB were greater in sorghum with rhizospheric dominance of Aspergillus. Different soil parameters like EC_e, EC, pH, N, P, K, and SOC explained about 51.6% variability (Monte Carlo permutation test, p≤ 0.001) in the rhizospheric assemblage of the bacteria, fungi, actinomycetes, Shannon–Weiner diversity index and percentage of phosphorus solubilizing microbes (Fig. 2). RDA1 and RDA2 explained about 48.7 and 2.4% variability and EC, pH and KMnO₄–N had a significant marginal effect (p≤ 0.05; Table S2).

Variations in the growth–pattern and size of fungal isolates were observed under different growth conditions. All the fungal isolates demonstrated temperature tolerance upto 40°C and pH tolerance upto 8.0 (Fig. S2, 3). Aspergillus and Penicillium demonstrated comparable temperature and pH–tolerance. Both the fungi showed an increase in the colony diameter with an increase in temperature (Fig. S2). At each temperature, fungal strains from sorghum rhizosphere had greater colony diameter while from pearl millet had smaller colony diameter. The SA1 and SP4 had maximum colony diameter. Rhizosphere of sorghum and mustard has highly tolerant fungal isolates than pearl millet and wheat. The colony diameter of the fungi was lower in acidic pH compared to alkaline pH (Fig. S3). At both the pH, the colonies of the Aspergillus and Penicillium fungal strains from pearl millet were smaller compared to the sorghum, wheat and mustard.

All the fungi isolated from saline soils demonstrated a high–salt tolerance index (STI) of 0.86–0.95. The strain’s STIs were almost identical across the crops except for P2 (mustard). The salt tolerance index of P2 (mustard) was 0.86 compared to P1 (0.94) with an increase in the salinity at 5% NaCl in the growth media (Fig. 3a). Similarly, Aspergillus and Penicillium demonstrated indistinguishable STI. These salt–tolerant fungi exhibited variable zone of PS on Pikovskaya’s medium (Fig. S1c, d). The P solubilization zone varied among the fungal isolates and ranged from 2.20–5.07 mm, and isolate PP3 demonstrated the greater solubilization zone (Fig. 3b). Aspergillus showed better solubilization potential than Penicillium. The P solubilization zones of pearl millet, sorghum, and wheat rhizospheric fungi were similar but greater than the mustard.

All the fungal isolates demonstrated salt–tolerance, and the ratio of biomass produced in media supplemented with 5% NaCl compared to 1% NaCl was similar except for a few isolates from pearl millet and sorghum rhizosphere (Fig. 3c). Although the fungal biomass produced in 5% NaCl media were greater for SA1, PP3, PP2 and SP2 (12.3–13.5 dry wt g⁻¹) (Table S3) but WP2 exhibited the least decline in P solubilization at high salt–concentration (Fig. 3c). The salinity of the growth media affected the P–solubilization potential of different isolates. The ratio of the P solubilization in 5% NaCl enriched media compared to the 1% NaCl media was < 1 for all the fungal isolates. All the crops had PSF with differential response to salinity. The WP2, SP2, PA1 and MP2 showed greater P solubilization ratio (p≤ 0.05) (Fig. 4a). But the P solubilization in 5% NaCl media was greater for PP3, SA1, WP1 and SP2 (27.1–28.9 mg ml⁻¹) (Table S3). The organic acid (OA) secretion in the growth media slightly increased with an increase in the salinity. The ratio of the OA released at 5% NaCl concentration compared to 1% NaCl media was >1 for mustard, pearl millet and wheat (Fig. 4b). Aspergillus and Penicillium showed similar response to salinity for OA production in growth media (Table S3). These fungal isolates declined the pH of the growth media with the increase in the incubation period (Table S4). The decline in pH was less at 5% NaCl concentration compared to 1%. The pH decline was greater for the pearl millet isolates than for other crops. The pH reduction was greater for the isolates PP2, PP3, SP2, SA1 and WP1 at both salinity levels.

The fungal isolates varied in production of phosphatase enzymes. Except for extracellular acid phosphatase, Aspergillus demonstrated higher phosphatase activity than Penicillium (Table 3). The PSF from the mustard rhizosphere showed lower activities of phosphatases compared to the pearl millet, sorghum and wheat. Isolates from Aspergillus genus also showed higher phosphatase compared to Penicillium. The fungal isolates PP3 and WA1 recorded greater acidic and alkaline phosphatase activity, respectively. Isolates from the mustard and wheat rhizosphere showed greater enzyme index (EI) for cellulase, amylase, and protease than sorghum and pearl millet (Table 3). The EI was almost similar for both the genera. Among the isolates, WA1 showed maximum EI for cellulase, amylase, and protease. Although the PSF from the Penicillium genus showed greater Zn solubilization, siderophore (Fig. S1e, f), ammonia, and IAA production, these isolates did not vary from one crop to another. Among the isolates, SP1 showed greater Zn solubilization, siderophore, IAA, and ammonia production ability than other PSF. In contrast to other growth–promoting attributes, the HCN secretion was greater in Aspergillus compared to Penicillium. The PSF from sorghum rhizosphere produced the maximum HCN, while pearl millet produced the lowest HCN.

In vitro, germination and vigor including shoot and root length of different crops were substantially influenced by the application of the salt–tolerant fungi under saline conditions (Table S5). The PSF from the Penicillium genus had better plant growth promotion in wheat, whereas the sorghum and pearl millet’s root length and vigor index were higher with PSF from Aspergillus. The SA1 and SP1 were the most effective PSF for sorghum and wheat, respectively. Similarly, PP2 was the most effective PSF for promoting plant growth parameters of mustard crops. The redundancy analysis showed that siderophore, IAA, phosphatase, HCN and biomass production affected the variability 33% variability (Monte Carlo permutation test, p≤ 0.001) in the germination and vigor of sorghum, pearl millet, mustard, and wheat (Fig. 5; Table S6). Siderophore, HCN, and biomass production ability of the culture in saline condition were the most important parameters affecting the plant performance and showed a strong positive correlation with different growth parameters of the culture. These parameters caused 85% variation in the germination and vigor (Monte Carlo permutation test, p≤ 0.001; Fig. 5) (Table S6).

The rhizosphere, the junction where plant, soil, and microbial interactions converge in soil ecology, is home to an extensive array of microorganisms [57]. Plant exerts some selectivity in shaping the microbial composition of the rhizosphere by altering the rhizosphere soil environment through the secretion of low molecular weight organic acids and energy–rich exudates [58, 59]. Crop, specific variation in the microbial population and relative abundance of the PSB and PSF in different crops were mainly because of the selectivity of the host plants enabled by the exudation of metabolites to host the microbes capable of alleviating nutrient deprivation, and water and salt stresses [60, 61]. The numerous stressors present in the SAS threaten the emergence of crops. Plants acclimatize by forming a mutualistic interaction with particular beneficial microorganisms in such stressed soil settings to develop tolerance to the prevalent stresses [62, 63]. The greater assemblage of the PSF compared to the PSB in the saline soils highlights the plasticity and ability of the fungi to adapt in unfavorable environment [64]. Aspergillus and Penicillium were the most common genera among the salt–tolerant fungi that could survive in the severe salinity of the soils used in the current study. They are frequently found in soil and are crucial to the disintegration of organic carbon and the cycling of nutrients [24, 26, 31]. The relatively greater population of the Penicillium at higher soil salinity also corroborates the earlier studies highlighting the contribution of this genera in P solubilization and plant growth promotion in extreme saline soils [32–34]. High salinity, nutrient deprivation, and water stress favor the selective assemblage of the PSF with greater adaptability. The present study also established the importance of soil parameters like EC_e, EC, pH, N, P, K, and SOC explained about 52% variability in the rhizosphere assemblage of different crops in the saline soils.

Fungal isolates are ecological generalists about salinity, as shown by their wide range of tolerances [65]. Specialists are limited in scope; generalists are frequently omnipresent and highly adaptable in harsh circumstances. The PSF isolated from different rhizosphere also showed a high salt, temperature, and alkalinity tolerance. This suggested the greater plasticity in adaptation and the presence of mechanisms enabling resistance to sodium toxicity and water loss [66]. Several researchers had previously established the ability of Penicillium spp. to survive in severe circumstances [67]. Although these PSF could survive in high salt environments, results from the present study pointed out that the fungal biomass declined with increased salinity stress. The decline in fungal biomass and hyphal density with increased salinity was also observed earlier [68]. The reduction in growth with increasing NaCl concentrations might be because of altered cytoplasmic water activities and loss of intracellular water. Reports also highlighted the reduced successful colonization of PSF in saline soils because of the disruption in cell walls, reduced metabolic activities, increased ionic concentration in cell sap and disorganization of nucleic acid structure under osmotic stress [69, 70]. In the stressed environment, microbes also spend some energy producing the osmolytes such as ectoine, betaine, and proline, to balance the osmotic pressure in and outside the cell mass [71]. Likewise, for mycorrhizal fungi, the increased colonization of the AMF in salt–affected soil was associated with increased secretion of the glomalin [8]. Variation in fungal growth on growth media used in the study indicated the differential ability of these isolates to utilize variable nutrient source. Different media supported luxuriant growth of fungi which indicates the diverse medium can be utilized for the development of fungi as bio–inoculants for their bulk production.

Cellulases, amylases, and proteases are three important ex tracellular non–pectinolytic hydrolases produced by fungi to maintain the proper balance of carbon and nutrients in the soil [72]. These enzymes also impact rhizosphere colonization by regulating physiological processes directly affecting the signaling, protein synthesis, and gene regulation [73]. Soil fungi can be categorized as ecosystem regulators involved in compound transformations and organic matter decomposition based on their functional characteristics [74–77]. In the present study, the P solubilization and pH decline was lower in–spite of increased organic acid secretion by different isolates at increased salinity level. The observed conundrum in P solubilization and organic acid secretion at high salinity might be because of the salinity–induced changes in the composition of organic acid secreted by the fungi as an adaptive response. These organic acids vary in their pKa value had varied effects on the pH of the media. The hydrolysis of organic acids with lower pKa produced pH lower than the acids with higher pKa [78, 79]. But all PSF isolates in high saline media exhibited about 75% potential of P solubilization in non–saline conditions. The intrinsic ability to solubilize phosphates under substantially high saline conditions appeared to exist in the strains as they were isolated from SAS. PSF was able to convert insoluble Pi into soluble orthophosphate forms (PO₄³⁻_, HPO₄²⁻, and H₂PO₄⁻) as they possess the potential to emanate various organic or inorganic acids that release H⁺ and reduce the medium's pH. The organic acids (OA) have carboxyl groups that bind to P by substituting for cations or by competing with them for P adsorption sites, increasing Pi solubilization and improving soil absorption of PO₄³⁻ [80]. The PSF isolates display notable levels of OA synthesis and Pi solubilization efficiency coupled with the pH decline of the growth media (r= 0.4–0.6; p≤ 0.05).

The study also noted a variation in the pH decline and PSI, highlighting the variability of the PSF associated with rhizosphere of different crops. Fungi that solubilize phosphate, can create extracellular phosphatase, an enzyme that can convert organic phosphate to inorganic phosphate. Siderophores are another organic low molecular weight molecule secreted by the PSF to facilitate the chelation of the Ca and other metal cations [81]. The fungal isolates examined in the current study could release IAA, a hormone found in plants. The observed variation in the rhizospheric assemblage and P solubilization index of the PSF from different crop rhizosphere was probably driven by the combined adaptive response of the plant–PSF interaction to a given salinity. The salinity–induced biochemical response of different PSF provided two distinct clusters (Fig. S4). The cluster analysis also indicates the nearest Euclidean distance of OA secretion, and PSP, for cluster I, while STI and fungal enzymatic profiles for cluster II. Thus, any of these two groups of variables can be used for segmenting different isolates for selecting the PSF with suitable characteristics for P solubilization and plant growth promotion. The SP1, SP2, PP2, SA1 and WA1 isolates were almost similar with respect to P solubilization and salinity–tolerance while different from the other isolates clustered in second group. Any of these isolates can be utilized for P solubilization at a targeted salinity condition. The PSF’s extracellular secretion of the siderophore and phytohormones enhances plant growth and development of roots. This was the reason for identifying the siderophore as the most important parameter, controlling about 14% variability in the plant growth promotion. The maximum vigor index of crops was shown by fungi producing large concentrations of siderophore and IAA.