Effect of mono- or bi-species Arbuscular mycorrhizal symbioses in improving the salt stress tolerance in Sorghum bicolor (L.) Moench.

Effect of mono- or bi-species Arbuscular mycorrhizal symbioses in improving the salt stress tolerance in Sorghum bicolor (L.) 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Moench. Ashutosh Kundu, Prashanta Kumar Mitra, Vivekananda Mandal This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8810660/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background and Aims: Salt stress is one of the most significant environmental constraints to agriculture, resulting in decreased crop productivity. The study aims to investigate the salt-stress alleviation effects of two potential Arbuscular Mycorrhizal Fungi (AMF), viz . Funneliformis mosseae and Rhizophagus intraradices , on the growth and metabolic changes of Sorghum plants. Methods A pot experiment was conducted under greenhouse conditions for 120 days in two different salt stress conditions (NaCl at 150 mM, EC 7.32 dS/m, and 300 mM, EC 12.03 dS/m) with/without AMF species, either alone or in combination. The AMF colonization and biochemical parameters were estimated at 30-day intervals. Results The total biomass, chlorophyll, carbohydrate, phosphate, nitrate, and proline contents increased in combined AMF-colonized plants exposed to 150 mM NaCl stress compared to the AMF-untreated control. In contrast, 300 mM salt stress significantly reduced AMF colonization and plant growth parameters. In 150 mM NaCl-stressed plants, the combined AMF-treated plants exhibited higher SOD, CAT, and APX activity compared to the AMF-untreated control plants, and lowered MDA and H 2 O 2 contents. The essential mineral contents were increased, while the uptake of Na+ ions was decreased. Conclusion The study concludes that dual-species AMF-treated plants (Fm + Ri) ameliorated salt stress more effectively by modulating ion uptake, enhancing stress defense enzymes, and minimizing redox factors, compared with salt-stressed AMF-untreated control plants and single-species AMF inoculations, resulting in a collective improvement in plant growth parameters. Thus, a consortium of AMF species ( R. intraradices and F. mosseae ) could be an efficient ameliorator of salt stress in sorghum cultivation. Salt stress tolerance Sorghum bicolor consortium Funneliformis mosseae Rhizophagus intraradices Antioxidant enzymes Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Plants are often exposed to various environmental stresses, which result in significant alterations in their growth and metabolism, ultimately reducing plant yield (Ahanger et al., 2018 ; Czarnocka and Karpiński, 2018 ). Salinity stress is one of the primary stresses that can impede plant growth and development (Grover et al., 2011 ; Banerjee and Roychoudhury, 2017 ; Hashem et al., 2018 ). It is estimated that increased salinization of arable land adversely affects germination, growth, and plant reproduction, consequently diminishing crop yield (Chinnusamy et al., 2005 ). Worldwide, saline soil can cause annual agricultural losses of up to $ 27 billion (Wang et al., 2021 ). It is expected to have devastating global effects on food productivity, resulting in a 30% loss of land within the next 25 years (Saxena et al., 2014, 2017 ). Soil salinity is a widespread problem, affecting over one billion hectares of land and spreading by more than 2 million Hectares annually (Tian et al., 2020 ; Hopmans et al., 2021 ; Singh, 2022 ). Soil salinity, measured in decisiemens per meter (dS/m), relates to the presence of high content of soluble salts and low exchangeable sodium ions [EC e > 4 dS/m; ESP < 15%; pH < 8.5; SAR < 13] (Eswar et al., 2021 ). Different salts, such as NaCl, Na2CO3, MgCl2, CaSO4, MgSO4, and Na2SO4, contribute to soil salinity. Among these, sodium chloride is most common in arid and semiarid soils (Flowers et al., 1977 ). Most salinized areas are found in India, China, the USA, Sudan, Pakistan, and Turkey (Singh, 2022 ). Globally, over one-fifth of the total irrigated land is salt-affected (Adejumobi et al., 2016 ). In India, approximately 6.46 million hectares of soil are expected to have high salt content among the 23 million hectares of arable land affected by salinity/alkalinity/acidification factors (Kumar and Sharma, 2020 ; Mandal et al., 2023 ). In the highly productive Indo-Gangetic Plain, sodic soils with high sodium (Na) content and a pH exceeding 8, in some cases even 10, occupy 1.37 million hectares (Mha) (Sharma et al., 2012 ; Bhardwaj et al., 2016). Therefore, a significant portion of India's landmass is unsuitable for agriculture due to salinity. Soil salinity has been grouped according to five classes, i.e., 0–4 dS/m (normal), 4–8 dS/m (marginally affected), 8–12 dS/m (moderately affected), 12–16 dS/m (severely affected), and more than 16 dS/m (extremely affected) (Datta and Jong 2002). Saline soils having an electrical conductivity (EC e ) of more than 4 dS/m and an osmotic pressure of -0.2 MPa are equal to about 40 mM NaCl (Santander et al., 2019 ). Plants exhibit different tolerance strategies across these salinity levels/classes; however, no crops were grown on land with an electrical conductivity (ECe) exceeding 16 dS/m. Soil salinity has an adverse effect on seed germination and agricultural productivity. It retards plant development by imposing osmotic stress and specific-ion toxicity, thereby restricting root development (Singh, 2022 ). These may lead to oxidative stress by generating reactive oxygen species (ROS), including the hydroxyl radical (. OH), H 2 O 2 , O2.-, and 1 O 2 , as well as nutritional deficiencies (Arzani et al., 2023 ). The toxic effects of specific ions, Na + and Cl − , damage cell organelles, cause membrane dysfunction, disrupt general metabolic activities, disrupt the structure of enzymes and other macromolecules, inhibit protein synthesis, and induce ion deficiency, leading to a decline in plant growth, rendering the plants weak and unproductive (Evelin et al., 2012 ; Saxena et al., 2017 , 2022 ). This hinders plant growth and metabolism. Plants can eliminate these deleterious forms of ROS through a range of defense mechanisms, commonly called the antioxidant system, which is controlled by several enzymes such as catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) and thus diminution of oxidative damage (Costa et al., 2005 ; Munns and Tester 2008 ; Porcel et al., 2012 ). In addition, some produce the indices of oxidative stress parameters (polyphenol oxidase, hydrogen peroxide, and malondialdehyde). Therefore, monitoring these oxidative stress parameters, ROS-scavenging enzymes, and growth parameters could help understand the ecophysiological status of the targeted crops under salinity stress. Sorghum, an ancient grain belonging to the family Poaceae, is a crucial climate-resilient crop that has been a staple food worldwide for thousands of years and is the fifth most widely cultivated cereal crop, after rice, wheat, maize, and barley, predominantly in tropical countries (Hossain et al., 2022 ; Ostmeyer et al., 2022 ). It is grown worldwide in over 86 countries, covering approximately 38 million hectares, with an annual production of 58 million tonnes (FAO, 2018). It has been the dietary foundation of over 500 million people in 30 countries (Majzoobi et al., 2023 ). Sorghum is also a significant grain crop consumed in India after wheat ( Triticum aestivum ) and rice ( Oryza sativa ) ( https://www.millets.res.in/ ). India uses it as food and fodder due to its high grain and green biomass yields and its good nutritional composition. Sorghum is a feed ingredient in several pet food brands and the aquaculture industry. In addition to grain, sorghum stover serves as a crucial feed for dairy and draft animals in India's livestock industry, particularly during the dry seasons (Rao 2019 ). It is also used in the high-quality distilling industry and serves as a key raw material in biofuel ethanol production (Kelley and Rao, 1994; Ibrahim, 2004 ; Mansour et al., 2021 ). Therefore, understanding the impact of salt stress on Sorghum cultivars, their responses, and tolerance strategies would help guide the selection of suitable cultivars for saline soils and identify biomarkers for crop improvement. Moreover, further investigations are required to determine the role of beneficial microbes in mitigating salt stress in the rhizosphere. Among the plant growth-promoting microbes (PGPM), arbuscular mycorrhizal fungi (AMF) are crucial because they can establish symbiosis with 80% of plants (Basu et al., 2018 ). AMF survives in most environments and provides various ecological services, significantly enhancing the rhizosphere's chemical and physical properties, thereby strengthening ecosystem function and increasing host plant growth and performance (Frosi et al., 2017 ). It has been reported that the AMF spore density in sodic soils of rice-wheat cropping systems is greater, with the dominant AMF species being Funneliformis mosseae and F. geosporum (Chandra et al., 2022 ). Another interesting comparative study examining a wide range of C3 and C4 plant species and their AMF symbiotic relationships found that C3 and C4 plants responded positively to AMF inoculation under saline conditions (Chandrasekaran et al., 2016 ). Among the AMF, Rhizophagus irregularis had a positive effect on C3 plants, whereas F. mosseae had a positive impact on C4 plants than other species (Chandrasekaran et al., 2016 Therefore, it is very crucial to envisage the effect of these two AMF species, R. irregularis , the model species for AMF, with the F. mosseae that have different host group preferences and modulation of the host physiology on the targeted crop Sorghum bicolor under saline stress conditions (Guo et al., 2021 ; Tang et al., 2023 ; Kokkoris et al., 2024 ). The present study aimed to investigate the effects of AMF inoculation with R. intraradices and F. mosseae , either alone or in consortia, on development, physiological, and biochemical characteristics, as well as the antioxidant enzymes and lipid peroxidation in forage sorghum cultivar ("CSV 53F") plants under salinity stress that simulates the salinity status of arable soil. Materials and Methods Arbuscular mycorrhizal inoculation in Sorghum seedlings Several varieties of Sorghum grow in India, with planting between February and July depending on the farm's location. A single-cut forage sorghum variety, Sorghum bicolor (L.) Moench "CSV 53F" (SPV 2705), developed by the Forage Section, Department of Genetics & Plant Breeding, CCS Haryana Agricultural University, Hisar, India, and cultivated during the Kharif season, has been selected as the experimental plant. It is a salt-sensitive, drought-tolerant cultivar, tolerant to shoot fly and stem borer (Kumari et al., 2023 ). The seeds were surface-sterilised by dipping for 15 minutes in a 0.2% solution of Mercuric Chloride (HgCl 2 , w/v), followed by three washes with sterile distilled water. They were then immersed in 70% ethanol for 30 seconds and further rinsed three times with sterile distilled water. The surface-sterilised seeds were placed in a petri dish with moist blotting paper at the base and were placed in a plant growth chamber maintained at 25 ± 2°C temperature, with an 18-hour photoperiod and 70–75% relative humidity for 3–5 days for germination. The seedlings were placed in a Hosco (small hole container for seedling growth, PP 100, of 30 cc with a 3.0 cm diameter and 6.4 cm depth, Horticultural Supplies Co., Mumbai, India) containing a sterilised (121°C for 2 hrs in autoclaved conditions) sand-soil mix (1:3, v/v). The monosporal AMF used in the current experiment was propagated using the trap culture methodology outlined by Stutz and Morton ( 1996 ). A single spore of the monosporal AMF isolates R. intraradices (syn. Glomus intraradices ) and F. mosseae (syn. Glomus mosseae ) was added separately to each place of Hosco (PP 100, Horticultural Supplies Co., Mumbai, India) and maintained in the plant growth chamber in the same light, humidity, and temperature control for 30 days. The AMF used in this study are the agriculture field isolates from Malda district (25°32'08"N to 24°40'20"N Latitude and 88°28'10"E to 87°45'05"E Longitude), West Bengal, India, with soil properties as no salt-affected (pH < 8.5, EC e (dS/m) < 4.0, Sodium Adsorption Ratio (SAR) < 13.0, Exchangeable Sodium Percentage (ESP) < 15.0) (categorized as per Gharaibeh et al., 2021 ). The seedlings were then transferred to larger plastic pots with a 25 cm diameter and 20 cm depth (~ 3 kg soil capacity), filled with autoclaved soil and sand (1:2, v/v). The plantlets were maintained for 120 days in a greenhouse and watered with half-strength Hoagland's solution (Hoagland and Arnon, 1950 ) at 7-day intervals, and with water every 3rd day. AMF colonization was checked at every 30-day interval. Application of salt stress on AMF-colonized sorghum plantlets A complete randomised design (CRD) was used to experiment with five biological replicates. Two distinct NaCl concentrations (150 mM, EC 7.32 dS/m, and 300 mM, EC 12.03 dS/m) were used as salt stress. Different salt concentrations were first applied at 15 days of plantlet growth (50 mL per pot). After that, the same volume and concentration of salt solution was applied after every 30 days, meaning at 45 days, 75 days, and 105 days. The soil's electrical conductivity and TDS were measured before and after the salt treatment, i.e., ± 5 days apart. The control pot had sorghum seedlings that were not inoculated with spores. The experimental pots were divided into four groups: (C0) control plant (no AMF) + 0 mM NaCl; (C150) control plant (no AMF) + 150 mM NaCl; (C300) control plant (no AMF) + 300 mM NaCl; (Fm0) plants inoculated with F. mosseae (Fm) + 0 mM NaCl; (Fm150) plant inoculated with Fm + 150 mM NaCl, (Fm300) plants inoculated with Fm + 300 mM NaCl; (Ri0) plants inoculated with R. intraradices (Ri) + 0 mM NaCl; (Ri150) plants inoculated with Ri + 150 mM NaCl; (Ri300) plants inoculated with Ri + 300 mM NaCl and (Fm+Ri0) plants inoculated with both Fm + Ri+0 mMNaCl treatment, (Fm+Ri150) plants inoculated with Fm + Ri+150 mM NaCl, and (Fm+Ri300) plants inoculated with Fm + Ri+300 mM NaCl stress. The roots and leaves were collected and processed for physio-biochemical investigation following the standard protocols. Measurement of soil salinity and electrical conductivity The soil salinity, TDS, and EC of the AMF-treated and AMF-untreated pots' soil were measured using a PCSTestr 35 (Eutech PCSTEST35-01X441506, Eutech Instruments Pte Ltd., USA, and Oakton 35425-10, Oakton PCSTestr 35 Waterproof pH/Conductivity/TDS/Salinity Tester, Cole-Parmer India, Mumbai, India) machine. The equipment was calibrated according to the system's respective standards. The experiment was repeated three times, and the average values were recorded. Estimation of AMF colonization The root samples were collected from the AMF-treated and untreated plants, washed in running tap water to remove soil particles, and then preserved in FAA solution (a mixture of 50 ml of 95% ethyl alcohol, 2.5 ml of glacial acetic acid, 5.5 ml of formaldehyde, and 42 ml of double-distilled water). For colonization measurement, the roots were cleared in 10% KOH solution (w/v), placed in a water bath (70°C) for 20–30 min, and cooled at room temperature. These were washed with water, acidified with 1% HCl (v/v), and stained with 0.05% Trypan blue (w/v) for 30 mins. The excess stain was removed by washing with water, followed by a 5% acetic acid solution (v/v), and further destained with a lactophenol solution (comprising phenol crystals, 12.5 g; glycerol, 50 ml; lactic acid, 25 ml; and 70% ethanol, 25 ml) (Sun and Tang 2012 ). Randomly cut 50 root fragments from the tip portion for each sample (1 cm long) were mounted on slides in a Polyvinyl Lacto Glycerol (PVLG, comprising 8.4 g of Polyvinyl alcohol (PVA), dissolved in 50 ml of lactic acid, 5 ml of glycerol, and 45 ml of distilled water, in a dark bottle) and examined for AM colonization under a Leica DM 750 phase contrast microscope (Germany) fitted with LAS EZ camera software. The percentage of root colonization was calculated using the formula provided by Ho-Plágaro et al. ( 2020 ). The abundance of arbuscules, vesicles, and total colonization was observed, and photomicrographs were taken. Ten microscopic fields were observed per set, and the average values were recorded. Assay for physio-biochemical parameters Measurement of stomatal movement upon salt stress Fresh leaves (3rd ) from all treatment set plants were collected to count stomatal opening and closing. The cut leaves were immediately floated, abaxial side down, in a Petri dish containing MES/KOH buffer (5 mM KCl, 10 mM MES (2-N-morpholinoethanesulfonic acid), 50 µM CaCl 2 , pH 6.15). The Petri dishes were maintained under constant light conditions. The lower leaf surface was peeled and observed for colonization under a Leica DM 750 phase-contrast microscope (Germany) equipped with LAS EZ camera software (Wu and Zhao, 2017 ). Ten stomata were observed randomly in triplicate to check the stomatal opening and closing. Estimation of chlorophyll content The total chlorophyll content of every 3rd leaf (top) was estimated by a chlorophyll meter SPAD-502Plus (Konica Minolta, USA). The SPAD-502Plus measures the absorbance of the leaf in the red and near-infrared regions and represents a numerical SPAD value proportional to the amount of chlorophyll present in the leaf. Ten SPAD data values were recorded from each leaf sample, and the average values were calculated. Estimation of total carbohydrate The total carbohydrate content of the plant samples was estimated using the Anthrone method (Yemm and Willis, 1954 ). 0.5 g of leaf (fresh weight) samples were crushed with 5 ml of 80% ethanol (v/v) and then heated in a boiling water bath for 20 min. The extract was centrifuged at 4,032 x g for 10 min at room temperature. The supernatant was collected, and the pellet was re-crushed and then re-extracted with the same solvent. The final volume of the supernatant was adjusted to 5 ml using double-distilled water. To 1 ml of suitably diluted supernatant under ice-cold conditions, 2 ml of Anthrone reagent (200 mg/100 ml ice-chilled conc. H 2 SO 4 ) was added, and the mixture was gently mixed. Then, it was placed in a boiling water bath for 10 minutes. The solution's blue-green absorbance was measured at 620 nm in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The experiment was repeated three times. The total carbohydrate content was estimated from the standard curve of D-Glucose (100 µg/ml) using the following formula. Total carbohydrate content/g of tissue = Total carbohydrate obtained from the standard curve × volume makeup (ml) × dilution factor/weight of sample (g). Estimation of total Phosphate and Nitrate The total nitrogen content of the plant sample was measured using the Salicylic Acid method (Cataldo et al., 1975 ), and the phosphorus content was quantified according to the method of Ames et al. ( 1960 ). Briefly, 50 mg of fresh leaves were crushed with 500 µl of distilled water for total nitrogen estimation, and then centrifuged at 5,478 × g for 10 min. After collection, the supernatant (0.2 ml) was mixed with 0.8 ml of 5% salicylic acid solution (dissolved in conc. H2SO4) and incubated at room temperature. After that, 1.9 ml of 2N NaOH was added to the mixture, and the absorbance was read at 410 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). For the estimation of total inorganic phosphate, 10 mg of fresh tissue was mixed with 1 part of 10% ascorbic acid and 6 parts of 0.42% ammonium molybdate, then heated at 45°C for 20 min. This was followed by incubation for 1 hour at 37°C. The extract was collected by centrifugation (Remi 12C Plus, Mumbai, India) at 5,478 × g for 10 min, and the absorbance was measured at 820 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The mineral content was measured using the following formula: Nitrate/phosphate content (µM/mg tissue) = Total nitrate/phosphate obtained from standard curve × volume of sample × dilution factor/sample weight (mg). Assay of total proteins and antioxidant enzymes 0.5 g of fresh leaves was crushed using a mortar and pestle, with a gradual addition of 5 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 g of polyvinyl polypyrrolidone (PVPP), and was kept at 4°C for 30 minutes. The extract was then centrifuged at 8,928 x g for 10 min at 4°C, and the supernatant was collected. The protein content of the extract was determined using the Lowry et al. method (1951) with BSA as a standard at 750 nm on a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The superoxide dismutase (SOD, EC 1.15.1.11) activity was determined following the Beyer and Fridovich method (1987) to assess the enzyme's capacity to impede the photochemical reduction of nitro blue tetrazolium (NBT). The enzyme units (EU) for SOD activity were measured as the amount of protein required to achieve a 50% reduction in NBT in the presence of SOD. The catalase (CAT, EC 1.11.1.6) activity was estimated by measuring H 2 O 2 decomposition over three minutes, as indicated by the absorbance at 240 nm in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) (Aebi, 1984 ). The reaction mixture consisted of 0.10 mM EDTA, 0.10 M potassium phosphate buffer (pH 7.0), 20 mM H 2 O 2 , and 100 µL of enzyme extract, for a total volume of 2 mL. Enzyme activity was calculated in EU/mg of protein. The ascorbate peroxidase activity (APX, EC 1.11.1.11) was measured using the method described by Nakano and Asada ( 1981 ). Briefly, 0.1 ml of enzyme extract, 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H 2 O 2 , and 1 ml of potassium phosphate buffer (0.1 M, pH 7.0) were all included in the test mixture. The absorbance of ascorbate reduction was measured at 290 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The activity was expressed as EU/mg protein. Estimation of MDA content Malondialdehyde (MDA) was quantified using the method of Heath and Packer ( 1968 ) to measure lipid peroxidation. 0.5 g of fresh leaves were homogenized in a mortar and pestle with 10 ml of 0.1% aqueous trichloroacetic acid (TCA, w/v), then centrifuged (Remi 12C Plus, Mumbai, India) at 11,200 x g for 10 min at 4°C. The supernatant was collected, and the volume was adjusted to 10 ml with 0.1% aqueous TCA. After adding 4.0 ml of 0.5% (w/v) Thio barbituric acid (TBA) to the supernatant (1.0 ml), the mixture was heated to 95°C for 30 min, cooled in an ice bath, and centrifuged (Remi 12C Plus, Mumbai, India) at 10000 x g for 5 min at 4°C. The supernatant's absorbance was measured at 532 and 600 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The following formula was used to determine the MDA content: MDA (µmol/g FW) = [(A 532 - A 600 )/156] ×10 3 × dilution factor, Where A 532 and A 600 stands for absorbance at 532 and 600 nm. Estimation of H 2 O 2 content 0.5 g of fresh leaves was homogenized in a mortar and pestle with 5 ml of 1% aqueous trichloroacetic acid (TCA, w/v) solution, then centrifuged (Remi 12C Plus, Mumbai, India) at 11,200 × g at 4°C for 10 min. The supernatant was collected, and the volume was adjusted to 5 ml with 1% aqueous TCA. An equal volume (1 ml) of 0.1 M potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide were mixed with 0.5 ml of the supernatant. The samples were vortexed gently, and the absorbance was measured at 390 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) (Velikova et al., 2000 ). The H 2 O 2 content was determined from the standard curve of H 2 O 2 conc. grades from 50 to 700 µl of 163 µM of H 2 O 2 stock solutions. Each tube was filled with 1 ml of 1 M potassium iodide. After that, the final volumes were increased to 2 ml with 0.1 M phosphate buffer, and the contents were expressed as µM/g FW. All these preparations were taken in amber containers or under light-controlled conditions. Estimation of Proline content The proline content was determined following the method of Bates et al. ( 1973 ). The fresh tissue (0.5 g) was homogenized in a mortar and pestle with 5 mL of 3% sulfosalicylic acid solution (w/v). The homogenate was then centrifuged in a refrigerated centrifuge (RM-12C Plus, Remi, Mumbai, India) at 10,000 × g at 4°C for 10 min. The supernatant was collected the volume was adjusted to 5 ml with 3% sulfosalicylic acid solution. After that, 1 ml of supernatant was combined with 1 ml of glacial acetic acid, and 1 ml of acid ninhydrin reagent (1.25 g ninhydrin in 30 ml glacial acetic acid, w/v) was incubated in a boiling water bath for 1 hr. The tube was cooled in an ice bath for 15 min to stop the reaction, then 2 mL of toluene was added and vortexed. The mixture was allowed to stand for a short time to let the two phases separate. The absorbance of the top chromophore toluene layer was determined in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) at 520 nm, and the proline concentration was determined from the standard curve of proline using toluene as a blank. Estimation of mineral content The macro-elements, such as sodium (Na), potassium (K), and calcium (Ca), were estimated using the procedure of Johnson and Ulrich ( 1959 ) with some modifications. Here, 500 µl of H 2 O 2 was added to the acid-digested sample to decompose organic matter, making the reaction more efficient and faster. At first, 0.5g dry leaf samples were ashed in a muffle furnace at 600°C for 4 hrs, after which the ash was suspended in a 50 ml volumetric flask. Acid digestion was performed using a diacid mixture (nitric acid and perchloric acid, 4:1 v/v) in a boiling water bath until the ash particles were completely dissolved. The samples were filtered through Whatman No. 42 filter paper and diluted to 20 mL with deionized water. Calcium, sodium, and potassium were determined using a µ-Controller-based Flame photometer with a compressor (Flame Photometer 128, Systronics, Kolkata, India) (Kravić et al., 2012). The microelements, including iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn), in the acid-digested samples were estimated using an atomic absorption spectrophotometer (AA-6880, Shimadzu, Japan) with some modifications. Here, 500 µl of H 2 O 2 was added to the acid-digested sample to decompose organic matter, making the reaction more efficient and faster (Keeney and Nelson, 1982 ). The sample preparation was identical to that used in the macro-element analysis method. The content was expressed as µg/g of sample. The mineral content was measured using the following formula: Mineral content (µg) / gm of tissue = mg/l flame photometer or AAS result × volume of sample × dilution factor/weight of sample (gm) Statistical analysis ANOVA was performed to analyze the significant differences. For analysing correlations, linear regression was performed, a correlation matrix was created, and Principal Component Analysis (PCA) was conducted. A distance matrix (Euclidean-normalised) was created for clustering similar treatment groups, and hierarchical clustering (Ward) was done. All analyses were performed in R and Orange3. Results Effect of salt stress on AMF colonization, growth, and biomass of Sorghum plant The microscopic observations for the mycorrhizal colonization of the sorghum plant at 30-day intervals revealed the presence of all the expected mycorrhizal fungi structures (hyphae, vesicles, and arbuscules) in AMF-inoculated plant roots under salt-non-stressed and salt-stressed (150 mM and 300 mM) samples (Table 1 ; Fig. 1 ). It was observed that soil salinity at 300 mM NaCl significantly reduced AMF colonization in sorghum roots. The number of vesicles, arbuscule, and total colonization formation was reduced to 46.66%, 63.63%, and 61.29%, respectively, in the ' Fm + Ri' treatment at 300 mM NaCl concentration after 120 days (Table 1 ; Fig. 1 ). A similar result was also observed at 30 days, 60 days, and 90 days. Conversely, total root colonization increased by about 4% in 150 mM salinity stress compared to non-treated (0 mM) plants in the respective AMF species. Total colonization was also the highest in combined AMF (Fm + Ri) treatment in 150 mM salt stress conditions after 60 days and 90 days (Table 1 ; Fig. 1 ). The formation of a more prominent arbuscule was observed at 30 days in F. mosseae than in R. intraradices with the 150 mM salt stress (panel Fm150 and Ri150) than in the salt-untreated set-up (panel Fm0 and Ri0). The vesicle formation was also higher at 60 days in F. mosseae than in R. intraradices . However, 300 mM salt stress reduced the arbuscular and vesicle formation in R. intraradices compared to F. mosseae (panels Fm300 and Ri300). Moreover, the size of the vesicles also increased with the increase in salt stress (panel Fm150, Fm300, Ri150, and Ri300), and the number of vesicular formations was higher in 150- and 300-mM salt treatment in R. intraradices (panel R150 and R300) than in F. mosseae (panel Fm150 and Fm300). This indicates that F. mosseae is a faster (30 and 60 days) colonizer with more arbuscule formation than R. intraradices at moderate salt stress, but R. intraradices had more robust vesicle formation at prolonged incubation time (90 and 120 days). In contrast, the combination of both isolates (Fm + Ri) in 150 mM and 300 mM salt treatment had more significant colonization, arbuscule, and vesicle formation in all the observation times (panel Fm + Ri 150 and Fm + Ri 300) as compared to salt-unstressed conditions (panel Fm+Ri0). Thus, the study revealed that these AMF isolates exhibit synergistic properties, enabling them to colonize the host plant and tolerate moderate to high salt stress conditions by forming more arbuscule or vesicles (Table 1 ; Fig. 1 ). Salt stress also negatively affected various morphological characteristics in sorghum plants. Compared with the AMF non-colonized plants (control) at varying salt concentrations, all employed AMF significantly improved overall sorghum plant development (Figs. 2 and 3 ). The application of salt stress (150 mM and 300 mM NaCl) reduced the plant height compared to the salt-unstressed control plant; however, in the AMF-colonized plants, plant shoot length and root length grew in the 150 mM NaCl salt-stressed condition but decreased in the 300 mM salt stress condition (Figs. 2 and 3 ). Plant biomass increased by 21.42%, 16.37%, and 36.44% in 120 days in the Fm, Ri, and Fm + Ri colonized plants in the 0 mM NaCl sets compared to the AMF-non-inoculated salt-untreated control plants. However, under 300 mM NaCl stress, both the control and AMF-colonized plants experienced a decline in plant biomass. Consequently, in both the presence and absence of salt stress, applying various AMF inocula increased the stomatal opening (Fig. 2 ). The highest stomatal opening was found in 0mM salt-stressed conditions in control as well as AMF colonized plants 0 mM NaCl (Figs. 2 and 3 ). However, AMF inoculation under salt stress improved stomatal opening in plants subjected to 150 mM and 300 mM NaCl stress compared to non-AMF plants under the same salt stress (Figs. 2 and 3 ). The ANOVA analysis shows that there is an overall significant difference found between four AMF species in the parameters arbuscule (p = 9.7e-10), carbohydrate (p = 1.3e-06), nitrate (p = 2e-04), phosphate (p = 2e-10), total colonization (p = 7.8e-13), and vesicles (p = 6.8e-13). However, no significant difference was found in the parameters biomass (p = 0.5), chlorophyll (p = 0.41), conductivity (p = 0.96), proline (p = 0.97), root length (p = 0.23), salinity (0.99), shoot length (p = 0.083), stomata opening (p = 0.079) and TDS (p = 0.92) (Fig. 2 ). There is an overall significant difference found between the two AMF species in the parameters arbuscule (p = 9.7e-10), carbohydrate (p = 1.3e-06), nitrate (p = 2e-04), phosphate (p = 2e-10), total colonization (p = 7.8e-13), and vesicles (p = 6.8e-13). However, no significant difference was found in the parameters of biomass (p = 0.5), chlorophyll (p = 0.41), conductivity (p = 0.96), proline (p = 0.97), root length (p = 0.23), salinity (p = 0.99), shoot length (p = 0.083), stomata opening (p = 0.079), and TDS (p = 0.92) (Fig. 2 ). Effect of salt stress on biochemical parameters of the sorghum plant The chlorophyll content in 'Fm + Ri' colonized plants under 150 mM NaCl stress after 90 days of culture was 38.46% higher than that of the AMF-non-colonized plants (Fig. 2 ). However, the chlorophyll content of both AMF-colonized and non-colonized plants sharply dropped from 60 to 90 days under 300 mM NaCl stress conditions (Figs. 2 and 3 ). A similar observation was also found in 90 to 120 days (Figs. 2 and 3 ). In the Fm-treated plant, nitrate content increased from 0.46, 0.21, 1.4, 1.09 µM/10 mg to 0.92, 1.41, 1.65, and 1.94 µM/10 mg, and in the Ri-inoculated plant, it increased from 0.46, 0.21, 1.4, 1.09 µM/10 mg to 1.27, 1.07, 1.72, and 1.22 µM/10 mg in 30 days, 60 days, 90 days, and 120 days, respectively in 0 mM salt-stressed conditions than the AMF untreated control plant. In the combined 'Fm + Ri’-treated plant, the nitrate content increased to 63.2%, 79%, 53.48%, and 50.9% in 30 days, 60 days, 90 days, and 120 days, respectively, in 0 mM salt-stressed conditions than AMF untreated control plant (Fig. 2 ). In 150 mM salt-stressed condition, the Fm-treated plant, the nitrate content increased to 73.98%, 77.14%, 74.73%, and 75.8%, and in the Ri-inoculated plant, nitrate content increased to 73.92%, 60.17%, 57.77%, and 63.19%, in 30 days, 60 days, 90 days and 120 days respectively than AMF-untreated control plant. In the combined 'Fm + Ri' treated plant, the nitrate content increased to 64%, 59.61%, 57.64%, and 75% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to AMF untreated control plant (Fig. 3 ). In 300 mM salt-stressed conditions, Fm-treated plant nitrate content increased to 18.16%, 21.24%, 26.63%, and 17.18%. In Ri-inoculated plants, nitrated content increased to 11.31%, 13.86%, 16.68%, and 9.63% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to AMF-untreated control plants (Fig. 3 ). In the combined 'Fm + Ri' treated plant, plant nitrate content increased to 21.23%, 23.82%, 28.24%, and 21.02% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plant (Figs. 2 and 3 ). Similarly, under 0 mM salt-stressed conditions, the plant phosphate content of Fm-treated plants increased to 18.13%, 19.27%, 45.53%, and 47.86%. Ri-treated plant phosphate content increased to 5.68%, 21.17%, 20.45%, and 19.73% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF-untreated control plant (Figs. 2 and 3 ). In the combined AMF-treated plant, the phosphate content increased to 10.24%, 52.81%, 57.18%, and 56.27% after 30 days, 60 days, 90 days, and 120 days, respectively, under 0 mM salt-stressed conditions, compared to the AMF-untreated control plant. In 150 mM salt-stressed conditions, the phosphate content increased to 31.62%, 46.41%, 66.19%, and 69.13% and 40.24%, 46.41%, 60.1%, and 64.6% in Fm and Ri-inoculated plants in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plants (Fig. 3 ). In the combined AMF treatment, the plant phosphate content increased to 35.24%, 67.05%, 72.1%, and 71.36%, respectively, compared to AMF-untreated control plants (Figs. 2 and 3 ). In 300 mM salt stressed condition, the Fm-treated plant, phosphate content increased from 1.28, 1.07, 0.38 and 0.32 µM/10 mg to 1.47, 2.2, 2.69 and 1.53 µM/10 mg and Ri-inoculated plant phosphate content increased from 1.28, 1.07, 0.38 and 0.32 µM/10 mg to 1.81, 1.84, 1.92 and 1.6 µM/10 mg and the combined AMF treated plants, plant phosphate content increased to 14.87%, 26.62%, 29.28% and 19.85% in 30 days, 60 days, 90 days and 120 days, respectively than untreated control plant (Figs. 2 and 3 ). In 0 mM salt-stressed conditions, the total carbohydrate content in Fm-treated plants increased from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 6.12, 10.1, 14.3 and 13.2 mg/100g and in the Ri-inoculated plant, total carbohydrate content increased from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 5.58, 11.4, 14.1 and 13.2 mg/100gm in 30 days, 60 days, 90 days and 120 days, respectively, than AMF untreated control plants (Figs. 2 and 3 ). In the combined Fm + Ri-treated plant, the total carbohydrate content increased to 42.48%, 50.54%, 51.63%, and 55.86% after 30 days, 60 days, 90 days, and 120 days, respectively, under 0 mM salt-stressed conditions, compared to the AMF-untreated control plant (from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 7.32, 14.8, 18.4 and 17.9 mg/100gm). While the 150 mM salt-stressed condition, the Fm-treated plant increased total carbohydrate content to 48.3%, 56.77%, 53.03%, and 56.06%, and the Ri-inoculated plant increased to 38.26%, 54.05%, 49.59%and 50%, and the Fm + Ri-inoculated plant the total carbohydrate content increased to 62.67%, 67.72%, 62.87%, and 64.19%in 30 days, 60 days, 90 days and 120 days, respectively than in the AMF untreated control plant. Whereas, in 300 mM salt-stressed conditions, the total carbohydrate content in Fm-inoculated plants increased to 23.21%, 24.11%, 17.62%, and 11.62%, and in Ri-inoculated plants, it increased to 17.68%, 22.68%, 17.68%, and 10.68%. In the Fm + Ri-inoculated plants, the total carbohydrate content increased to 23.72%, 25.36%, 24.79%, and 15.68% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plant (Fig. 2 , 3 ). In 30 days, proline content increased to 5.88%, 4.37%, and 5% in the 'Fm',' Ri', and 'Fm + Ri' inoculated plants compared with the AMF untreated control in 0mM salt-stressed conditions. Meanwhile, proline content increased to 25%, 16%, and 38.23% in 'Fm',' Ri', and 'Fm + Ri' inoculated plants, respectively, in 150 mM salt-stressed conditions compared with the AMF untreated control in 30 days at and increased to 53.73%, 35.89% and 46.8% in 'Fm',' Ri', 'Fm + Ri' inoculated plant respectively in compared with the untreated control in 30 days at 300 mM salt stressed condition (Fig. 2 , 3 ). The ANOVA analysis and linear regression analysis of the plant parameter change with changing salt concentration and incubation days shows that the arbuscle percentage in treated groups decreased with increasing salt concentration (Fig. 3 a) but increased with days, except Ri, which showed a slight decrease with days (b); there is no change in the control group (Fig. 3 a, b). For all the groups, biomass decreased with increasing salt concentration (Fig. 3 a) but increased with days (Fig. 3 b). For all the groups, carbohydrates decreased with increasing salt concentration (Fig. 3 a) but increased with days (Fig. 3 b); the noticeable thing is that the amplitude of Fm and Ri is similar (Fig. 3 a, b). For all the groups, chlorophyll decreased with increasing salt concentration and days (Fig. 3 a, b). For all groups, conductivity increased with increasing salt concentration (Fig. 3 a). However, over time, conductivity increased for the control, decreased for the AMF-treated groups, and the reduction was most pronounced for Fm + Ri (Fig. 3 b). Nitrate levels decrease with increasing salt concentration for all parameters (Fig. 3 a) and increase with time (Fig. 3 b). phosphate in the control group decreases with both increasing salt concentration and days (Fig. 3 a, b), while in treated groups with increasing salt concentration, slightly increases in Ri, not unchanged in Fm, and decreases in Fm + Ri; and with increasing days, increases in Fm, Fm + Ri, and decreases in Ri (Fig. 3 a, b). Proline increased with increasing salt concentration and days (Fig. 3 a, b). Root length decreased with increased salt concentration (Fig. 3 a) and increased with days (Fig. 3 b). salinity increased with increasing salt concentration and days for all the groups (Fig. 3 a, b). Shoot length decreased with increasing salt concentration and increased with days (Fig. 3 a, b). Stomatal opening decreased with increasing salt concentration (Fig. 3 a), decreased with increasing days in the treatment groups, and remained constant in the control group (Fig. 3 b). TDS increased with both increasing salt concentration and days (Fig. 3 a, b). Total colonization decreased with increasing salt concentration (Fig. 3 a) and increased with increasing days (Fig. 3 b); the pattern is similar in Fm and Fm + Ri (Fig. 3 a, b). Vesicles decreased with increasing salt concentration (Fig. 3 a) and increased with increasing days (Fig. 3 b); the notable thing is that the increasing and decreasing pattern is similar in Fm and Fm + Ri (Fig. 3 a, b). The correlation matrix analysis between parameters at significance values of p < 0.05, p < 0.01, and p < 0.001, shows that many of the parameters are significantly positively correlated, and some are significantly negatively correlated, as shown in Fig. 4 . The PCA analysis shows that proline, shoot length, and carbohydrate contributed the highest in data variation and TDS; the stomatal opening was found to be the least contributing in data variation (Fig. 5 a). The control group was quite distinct from AMF-treated groups; the effects of FM and FM + RI on plant parameters were similar, and FM + RI was found to affect biomass, shoot length, and colonization the highest (Fig. 5 b). Plant parameters were also found quite similar in 90 and 120 days, while 30 and 60 days are quite different, probably an indication of adaptation with longer incubation time (Fig. 5 c). Different concentrations of salt-affected plant parameters differently, while the control group was found with higher values of stomatal opening chlorophyll; the group with 150 mM salt concentration was found to affect biomass, vesicles majorly; the group with 300 mM salt concentration proline, salinity, and TDS (Fig. 5 d). Effect of salt stress on proteins and antioxidant enzyme parameters of the sorghum plant The expression of antioxidants in sorghum plants during the salt and AMF treatments is shown in Fig. 6 (a-f). When compared to AMF untreated control plants in 300 mM salt stress conditions, the combined AMF-treated plants showed higher SOD, CAT, and APX activity (32.64%, 49.61%, and 33.69%, respectively) in 60 days (Fig. 6 ). Salt stress significantly impacted total protein content (TPC). TPC of AMF-treated plants increased (from 8.44 mg/g to 15.47, 15.87, and 17.84 mg/ g in Fm, Ri, and Fm + Ri treated plants, respectively) at 150 mM salt-stressed conditions compared to the AMF-untreated control sets at 60 days. However, salt stress (150 mM and 300 mM) increased, and TPC decreased across all treatment conditions; however, in AMF-treated plants, TPC increased when comparing treated and AMF-untreated plants. Among AMF-treated plants, the highest TPC was observed in 'Fm + Ri' treated plants in different salt-stressed conditions (Fig. 6 f). The higher malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) content in plant tissues indicates oxidative stress associated with reactive oxygen species production. The results show that H 2 O 2 and MDA concentrations did not differ between non-AMF and AMF-inoculated plants under the control condition (Fig. 6 c, e). The combined AMF-treated plants showed lower MDA and H2O2 levels in response to 300 mM salt stress than the untreated salt-stressed control plants (51.10% and 31.14%, respectively) after 60 days. In contrast, single AMF-treated plant 'Fm' showed higher activity (42.23% and 22.73%, respectively) in identical conditions. This indicates that AMF-treated plants alleviate salt stress conditions by reducing MDA and H 2 O 2 activity, unlike the AMF-untreated control plants (Fig. 6 ). Hierarchical clustering based on salt stress-affected enzymes (Fig. 7 a) and other plant parameters (Fig. 7 b), which indicated that the control group was quite different from the treatment groups (Fig. 7 a,b). However, treatment groups were not quite separable, and a cluster of mixed treatment groups (FM, RI, FM + RI) was found in both the enzymatic (Fig. 7 a) and other plant parameters (Fig. 7 b). The overall analysis shows that different salt concentrations induce different effects (in terms of parameters) and control plant parameters and enzymes differently. Regarding the impact of AMF on counteracting salt stress, it was observed that different salt concentrations elicit distinct effects. Consequently, the AMF groups also attempted to mitigate these effects accordingly. Over time, the plant adapts to salt stress, thereby reducing its harmful effects. FM + RI was more effective than FM or RI in enhancing salt tolerance across several aspects, while FM and RI were more effective in others. RI complements FM well in reducing salt stress (Fig. 7 ). Effect of salt stress on the mineral content of the sorghum plant Salt stress raised the Na + salt concentration in AMF-untreated control sorghum leaf tissues. It significantly decreased the absorption of vital elements such as potassium (K + ), calcium (Ca 2+ ), iron (Fe 3+ ), zinc (Zn 2+ ), manganese (Mn 2+ ), and copper (Cu 2+ ) (Fig. 8 ). Maximum reduction of nutritional minerals was observed in the AMF-untreated control at 300 mM NaCl stress. However, AMF inoculation increased the content of vital minerals in both salt-stressed conditions. The content of minerals like 'Cu, Zn, Mn, Fe, K, and Ca in 'Fm + Ri' inoculated plants increased by 40.70%, 33.78%, %, 53.27%, %, 14.76%, and 15.58%, respectively, 'Fm’-inoculated plants increased by 36.89%, 25.99%, 30.25%, 12.15%, 12.79% and 21.02%, and 'Ri’-inoculated plants by 10.23%, 10.93%, 13.72%, 7.79%, 9.14% and 8.35% than the AMF-untreated control in 0 mM NaCl stressed at 60 days. In individual Fm-inoculated and Ri-inoculated plants, mineral uptake such as Cu, Zn, Mn, Fe, K, and Ca was increased by 36.89%, 25.99%, 30.25%, 12.15%, 12.79% and 21.02%, and 10.23%, 10.93%, 13.72%, 7.79%, 9.14%, and 8.35%, respectively which were lower than 'Fm + Ri' treated plant. The Na + content decreased up to 18.26% in 'Fm + Ri’-inoculated plant, while in 'Fm' inoculated plant, it decreased up to 16.03% while in 'Ri’-inoculated plant, it decreased up to 10.81% compared to the AMF-untreated control at 150 mM salt-stressed conditions in 60 days (Fig. 8 ). This indicates that AMF colonization significantly regulates the Na + uptake. Colonization also substantially minimizes the Cu/Na, Zn/Na, Mn/Na, Fe/Na, K/Na, and Ca/Na ratios, thus minimizing the Na + -associated mineral uptake and transport processes (Fig. 8 ). Moreover, it has been observed that the vesicle and total colonization development were the highest in Fm+Ri150. At the same time, it affected the arbuscule development (Fig. 8 ). The PCA analysis shows that 150 mM salt stress greatly influences the arbuscule, vesicle, and total colonization and mineral uptake by the AMF species (Fig. 8 ). In contrast, the colonization minimizes the salt stress associated with mineral transport in the plant system. The hierarchical cluster analysis reveals that 300 mM salt stress significantly reduces plant AMF colonization and mineral uptake, thereby forming a distinct clade. In contrast, Fm+Ri150 and Fm150 formed a separate clade and the C0, Fm0, and Ri0 formed a separate clade (Fig. 8 ). No significant difference was found between AMF treatments for all the minerals measured (Fig. 8 a). However, a significant difference was found between AMF treatments for all the minerals measured (Fig. 8 b). There is a decreasing trend with salt concentration for all the AMF treatment groups for Ca, Cu, Fe, K, Mn, and Zn, but an increasing trend for Na (c) (Fig. 8 ). PCA significance analysis shows that Cu, Mn, and Zn were the most significantly contributing parameters in data variation. PCA-biplot also showing FM treatment contributes to Fe, K, Ca accumulation, RI treatment contributes to Na, Ca, K accumulation, FM + RI treatment contributes in Cu, Mn, Zn accumulation (Fig. 8 b) Na found with significantly negatively correlated with all the parameters, rest of the parameters (Ca, Mn, Zn, Fe, K, and Ca) are significantly positively correlated among themselves (Fig. 8 c). Discussion Salinity stress adversely affects agricultural productivity by disrupting water uptake. It has a profoundly detrimental effect on plant growth, biomass, and overall biochemical parameters by altering the soil solution's water potential, making it more negative and leading to physiologically dry soil (Razzaq et al., 2020 ). It also affects the uptake of nutrients, like N, P, Mg, and Fe. This leads to unfavorable Na+/Ca2 + and Na+/K+ ratios, as well as reduced relative water content (RWC) and membrane stability index (MSI) in plants (Romero-Munar et al., 2019; Mansour and Hassan, 2022 ; Zhang et al., 2023 ). In a study on salt-tolerant sorghum varieties, Bavei et al. ( 2011 ) observed a decrease in fresh weight, higher ion accumulations, proline content, and peroxidase activity. In another study with two Kenyan grain sorghum varieties [ S. bicolor (L.) Moench], Serena and Seredo, Netondo et al. ( 2004a , b ) found that the 250 mM NaCl treatment significantly reduced the relative shoot growth rates by 75 and 73%, respectively, and stem dry weight by 75 and 53%. It also strongly inhibited the accumulation of K + and Ca 2+ in the roots, stems, and leaves. They noted that Chlorophyll a and b, net CO 2 assimilation, stomatal conductance, and transpiration rate decreased significantly with the increase in salinity. It also affects AMF infectivity, colonisation, spore density, spore germination, and hyphal development (Evelin et al., 2009 ; Porcel et al., 2012 ; Barajas González et al., 2023 ). However, AMF ameliorated the deleterious effects of salt stress and also enhanced biomass in maize (Romero-Munar et al., 2019; Huang, 2024) and millet (Umashankar et al., 2023 ) compared to non-mycorrhizal plants. Similar to the above observations, the present study also revealed that salt stress, specifically 150 mM NaCl (EC 7.32 dS/m), increased sorghum plant AMF colonization and increased vesicle and arbuscle formation (Fig. 1 ; Table S1 ). However, under 300 mM NaCl stress (EC 12.03 dS/m), hyphal growth, arbuscular formation, and total colonization decreased (Fig. 1 ; Table S1 ). The extent of stomatal opening is significant for CO 2 uptake and photosynthesis per unit leaf area (Qin et al., 2024 ). The lower chlorophyll content and stomatal opening under stress conditions indicate the detrimental effects of stress factors on the photosynthetic efficiency of the plants. It has been reported that lipid peroxidation and reactive oxygen species (ROS) play a crucial role in damaging various photosynthetic membranes where photosynthetic pigments are bound (Kang et al., 2012; Xing et al., 2013) and in causing thylakoid swelling (Kafi, 2009; Yamane et al., 2008). Moreover, under this salinity-stress condition, chlorophyll degradation and reduced chlorophyll synthesis result in reduced chlorophyll content. While AMF colonization (Fm + Ri) reduces lipid peroxidation and ROS generation, it thereby achieves greater membrane stability. In addition, AMF colonization reduces Na+ uptake and increases the levels of beneficial minerals essential for chlorophyll synthesis and stability (Wang et al., 2009), thereby enhancing chlorophyll content under salt stress. Salt stress depletes turgor pressure in leaves, leading to stomatal closure and thereby inhibiting plant CO2 uptake and reducing photosynthesis (Qin et al., 2024 ). Moreover, it has been shown that, because of their similar physicochemical properties, Na+ ions compete with other minerals at their transport sites (Evelin et al., 2012 ; Hedrich and Shabala, 2018 ). However, it has been observed that AMF-colonised plants maintain a favourable K + /Na + ratio by minimizing Na + uptake. Thus, it protects photosynthetic tissues from inhibiting Na + uptake, thereby enhancing overall development and productivity under saline conditions (Colmer et al., 2006 ; Munns and Tester, 2008 ; Cuin et al., 2011 ). A similar observation was noted in the present experiment (Fig. 2 ). In an experiment, sorghum plants colonized by Glomus intraradices (Gi), Gigaspora margarita (Gm), or a mixture of AM species during a sustained drought following exposure to salinity treatments (NaCl stress) Cho et al. ( 2006 ) observed that combined NaCl and drought stress exposure, stomata of Gi plants remained open 17–22% longer than in non-AM plants. They hypothesized that AMF-plants modulated cellular metabolism to produce various osmotic adjustment substances, such as proline (Pro) and total soluble solids (TSS), within cells, thereby maintaining normal cell expansion, growth, and water absorption, and ensuring osmoregulation (Liu et al., 2021 ). The higher content of TSSs in salt-stressed AMF-plants might be due to the accumulation of osmoprotectant or to the higher synthesis of TSS from starch and sucrose (Schrader and Sauter, 2002 ; Garg and Bharti, 2018 ; Evelin et al., 2019 ; Mostafaie et al., 2024 ). Salinity also decreases the solubility and mobility of other micronutrients (Zn, Cu, and Fe), creating a depletion zone around the roots and thereby affecting the plant's acquisition of these micronutrients (Grattan and Grieve, 1992 ). However, AMF plants showed higher concentrations of these micronutrients than non-AMF plants (Evelin et al., 2012 ). The present study shows that the contents of minerals like 'Ca, Zn, Mn, Fe, K, and Cu in 'Fm + Ri' inoculated plants were increased compared to the AMF-untreated control in 0 mM NaCl stressed (Fig. 9 ). At the same time, the Na + concentration decreased up to 18.26% in the 'Fm + Ri’-inoculated plant, up to 16.03% in the 'Fm'-inoculated plant, and decreased up to 10.81% in the 'Ri’-inoculated plant compared to the AMF-untreated control at 150 mM salt stressed condition. A greater relative N, P, Ca, Mg, Mn, and Fe absorption rate was also observed in AMF-inoculated wheat plants under salt stress than in non-AMF seedlings (Huang et al., 2023 ). This may be attributed to the development of extraradical hyphae to reach distant areas of the rhizospheric zones, and to the upregulation of transporter gene expression for these nutrients. (Burleigh et al., 2003 ). The qRT-PCR analysis of rice seedlings under NaCl stress, treated with AMF, showed overexpression of genes related to ion homeostasis compared with the non-inoculation treatment group (Zhang et al., 2023 ). The mechanism of such selective enhancement of minerals in the AMF plants could be due to compartmentalizing Na + into the vacuole via up-regulation of OsNHX3 (sodium/hydrogen exchanger) and efflux of Na + from the cytosol to apoplastic spaces via higher expression of OsSOS1 (salt overly sensitive) and OsHKT2;1 (high-affinity potassium transporter) and also induce more NHXs (vacuolar Na + /H + antiporters present in roots and leaves) that help sequester Na + in the vacuole (Davenport et al., 2007 ; Porcel et al., 2016 ) and SOS1 (plasma membrane Na + /H + antiporters) responsible for secretion of Na + from the cytosol beyond plasma membrane (Olías et al., 2009; Evelin et al., 2013 ; Liu et al., 2023 ). Soil salinisation also causes precipitation of inorganic phosphorus (P) with other cations, such as Ca2+, Mg2+, and Zn2+, depending on soil pH, thereby limiting the element's availability to plants (Wu, 2018 ; Iqbal et al., 2020 ). However, AMF colonization enhances P acquisition in AMF plants either by secretion of acid and alkaline phosphatases, by forming polyphosphates inside the hyphae, or by the expression of high-affinity phosphate transporter genes (GvPT, GiPT, and GmosPT) (Evelin et al., 2012 , 2013 ). This is also attributed to maintaining the integrity of the cell membrane, reducing ion leakage, and preventing the compartmentalization of toxic ions in vacuoles, thereby reducing the adverse effects of salinity (Evelin et al., 2012 , 2013 ). Therefore, it is assumed that the enhanced phosphorus content in the 150 mM and 300 mM salt-stressed sorghum plants by the AMF F. mossae and R. irregularis may be due to one or more of these mechanisms that need to be explored. Plants absorb N as nitrate (NO 3 2− ) and ammonium (NH 4 + ) ions (Frechilla et al., 2001). However, salinity conditions interfere with their uptake by immobilizing them (Hodge and Fitter, 2010 ; Miransari, 2010 ). While NO 3 2− uptake is challenged by Cl − , NH 4 + absorption faces competition from Na + at the membrane (Evelin et al., 2019 ). A low flux of NO 3 2− from soil to roots leads to reduced activity of nitrogen reductase (NR) (Hoff et al., 1992 ). A higher expression of nitrate (NRT1.1, NAR2.2) and ammonium transporters (AMT1.1 and AMT1.2) was observed in Triticum aestivum plants colonized with R. irregularis and F. mosseae (Evelin et al., 2019 ). The present study also shows that N-content was higher in Fm-treated, Ri-treated, and 'Fm + Ri-treated plants than AMF-untreated control plants in 150 mM and 300 mM salt-stressed conditions (Fig. 2 ; Table S3). A higher N content in the AMF plants might be due to the AMF-facilitated maintenance of membrane stability and increased NR activity (Talaat and Shawky, 2014 ). The AMF exerts its beneficial stress-ameliorating effect on crops by enhancing the acquisition of several essential mineral elements (P, N, K, Ca, Mg, Zn, Fe, Mn, and Cu) and significantly reducing deleterious Na + ion uptake. Thus, AMF colonization helps maintain the Na+/Ca2 + and Na+/K+ ratios, as well as physiological and biochemical changes, and the selective expression of genes (PIP, Na + /H + antiporters, Lsnced, Lslea, and LsP5CS) (Evelin et al., 2009 ). Salt stress leads to the production of large amounts of reactive oxygen species (ROS) in plants, which can impact plant growth and development, and even result in plant death (Czarnocka and Karpiński, 2018 ; Zhang et al., 2023 ). The higher malondialdehyde (MDA) and hydrogen peroxide (H 2 O 2 ) levels in plant tissues under salt stress indicate oxidative stress associated with ROS production. Antioxidant enzyme (SOD, APX, and CAT) activity is usually greater in AMF plants than in non-AMF plants (Elhindi et al., 2017 ). A similar observation was also noted in the AM-infested plants under salt stress in this study (Fig. 6 a, b, and d). This suggests that salt stress induces oxidative stress, which is mitigated by the increased activity of these ROS enzymes. The combined AMF (Fm + Ri) treatment in 150 mM and 300 mM salt stress conditions exhibited the highest antioxidant enzyme activity compared to the single AMF-colonized plant (Fig. 6 ; Table S4). The higher CAT, APX, and SOD activity associated with higher salinity stress indicates a more effective detoxification of ROS (Benavídes et al., 2000). These redox enzymes stabilize subcellular components of cell membranes, such as lipids and proteins, quenching free radicals, and buffer cellular redox potential under salinity stress (Yang et al., 2009 ; Evelin et al., 2012 ; Frosi et al., 2017 ). Several studies have shown that salt stress often leads to proline accumulation (Evelin et al., 2012 ; Yooyongwech et al., 2013 ). Proline is a crucial osmoprotectant that maintains tissue water potential, protein integrity, and function, thereby reducing oxidative damage to cells (Parvaiz and Satyawati, 2008 ). The present work also confirmed that AMF inoculation increased proline content under salt stress (Fig. 2 ; Table S3). It has been shown that in AMP-colonized plants, Proline synthesis is enhanced due to upregulation of the delta1-pyrroline-5-carboxylate synthetase gene, which encodes the rate-limiting enzyme in proline biosynthesis, MeP5CS, under stressful conditions, with a considerable drop in its catabolism (Huang et al., 2013 ; Mansour and Ali, 2017 ). Thus, the elevated proline can scavenge ROS, stabilize DNA, proteins, and membranes, and reduce NaCl-induced enzyme denaturation (Evelin et al., 2019 ). Therefore, it is assumed that the enhanced proline level in salt-stressed AMF-colonizing plants in this study may result from upregulated proline synthesis, providing a better tolerance mechanism against abiotic stress (Miransari, 2010 ; Wu et al., 2016 ; Caruso et al., 2018 ). A tripartite interaction study among AMF, R. intraradices , and associated bacteria (Massilia sp.) in maize demonstrated enhanced salt stress tolerance in seedlings compared with biotrophs alone (Krishnamoorthy et al., 2016 ). Therefore, it is observed that the application of a single AMF species, alone or in combination with other AMF species or functional microorganisms, significantly affects the physiobiochemical parameters of the targeted crops and exerts a stress-ameliorating effect compared to a single species (Parvin et al., 2020 ). Conclusion The current study concludes that the selected AMF species, F. mosseae and R. intraradices , significantly influence the physiobiochemical parameters of the Sorghum variety "CSV 53F" more than the non-AMF sets. Moreover, it alleviated salt stress (150 mM and 300 mM) in sorghum plants by enhancing mineral uptake, increasing antioxidant enzyme activity, and improving physiological and biochemical parameters, thereby improving overall growth and biomass. The current study concludes that the use of a combination of AMF ( F. mosseae and R. intraradices ) is an effective biofertilizer for enhancing sorghum growth in saline agricultural areas. Furthermore, large-scale field trials and more in-depth molecular studies may explore the vivid mechanisms by which these AMF species mitigate salinity stress in Sorghum plants, thereby helping to enhance crop productivity in current adverse environmental /soil conditions. Declarations Competing Interests: The authors declare that they have no conflict of interest. Funding: This work was supported by the West Bengal Biodiversity Board, Dept. of Environment, Govt. of West Bengal (Memo No. 437/3K(Bio)-6/2019; dated 30.06.2022). Author's Contribution: Conceptualization: Vivekananda Mandal; Methodology: Ashutosh Kundu; Formal analysis and investigation: Ashutosh Kundu and Prashanta Kumar Mitra; Writing - original draft preparation: Ashutosh Kundu; Writing - review and editing: Kiran Sunar, Vivekananda Mandal; Funding acquisition: Vivekananda Mandal; Resources: Kiran Sunar, Vivekananda Mandal; Supervision: Kiran Sunar, Vivekananda Mandal Acknowledgments: The authors are grateful to the West Bengal Biodiversity Board, Dept of Environment, Govt of West Bengal (Memo No. 437/3K(Bio)-6/2019; dated 30.06.2022) for the financial support to carry out the study. The authors are also grateful to the WB DST-BT-supported BOOST program 2017–2018 ( vide Ref. No. 1089/BT(Estt)/1P-07/2018; dated 24.01.2019) for the equipment grant to the department. We are also grateful to Dr Malay Das, Department of Biological Sciences, Division of Botany, Presidency University, 86/1 College Street, Kolkata-700073, West Bengal, India, for providing infrastructural support to measure the soil salinity, TDS and electrical conductivity (EC) by PCSTestr 35 (Eutech PCSTEST35-01X441506/Oakton 35425-10) and chlorophyll meter SPAD-502Plus machines. We are also grateful to Sri Arup Mandal, Research Scholar, and Dr Dipak Nayak, Sr. Scientist & I/c RRS (Hort.) Fruit Science, ICAR-CISH Regional Research Station, Malda, W.B., for providing the facilities to analyze the minerals using Flame Photometer and AAS. Data Availability Statement: The data supporting this study's findings are available from the corresponding author upon reasonable request. References Adejumobi MA, Awe GO, Abegunrin TP, Oyetunji OM, & Kareem TS (2016). 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Wang F, Yang S, Wei Y, Shi Q, Ding J (2021) Characterizing soil salinity at multiple depth using electromagnetic induction and remote sensing data with random forests: A case study in Tarim River Basin of southern Xinjiang, China. Science of the Total Environment. 754:142030.https://doi.org/10.1016/j.scitotenv.2020.142030 Wu (2018) Plant salt tolerance and Na+ sensing and transport. The Crop Journal. 6(3):215-25. https://doi.org/10.1016/j.cj.2018.01.003 Wu N, Li Z, Wu F, Tang M. (2016) Comparative photochemistry activity and antioxidant responses in male and female Populus cathayana cuttings inoculated with arbuscular mycorrhizal fungi under salt. Sci Rep 6:37663. https://doi.org/10.1038/srep37663 Wu S, Zhao B (2017) Using clear nail polish to make Arabidopsis epidermal impressions for measuring the change of stomatal aperture size in immune response. Plant pattern recognition receptors: methods and protocols. 243-8. https://doi.org/10.1007/978-1-4939-6859-6_20 Yang SL, Lan SS, Gong M (2009) Hydrogen peroxide-induced proline and metabolic pathway of its accumulation in maize seedlings. J Plant Physiol 166(15):1694-9. https://doi.org/10.1016/j.jplph.2009.04.006 Yemm EW, Willis A (1954) The Estimation of carbohydrates in plant extracts by Anthrone. Biochem J 57(3):508. https://doi.org/10.1042%2Fbj0570508 Yooyongwech S, Phaukinsang N, Cha-Um S, Supaibulwatana K (2013) Arbuscular mycorrhiza improved growth performance in Macadamia tetraphylla L. Grown under water deficit stress involves soluble sugar and proline accumulation. Plant Growth Regul. 69: 285–293. https://doi.org/ 10.1007/s10725-012-9771-6 s Zhang B, Shi F, Zheng X, Pan H, Wen Y, Song F (2023) Effects of AMF compound inoculants on growth, ion homeostasis, and salt tolerance-related gene expression in Oryza sativa L. under salt treatments. Rice. 16(1):18.https://doi.org/10.1186/s12284-023-00635-2 Tables Table 1 is available in the Supplementary Files section. Supplementary Files Fig.GraphicalabstractMargedpic4.tif MSSISaltstress07.02.2026.docx Table113.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8810660","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":592599728,"identity":"cbbb7ac8-0860-4009-af3c-13d15ddb2aad","order_by":0,"name":"Ashutosh Kundu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ashutosh","middleName":"","lastName":"Kundu","suffix":""},{"id":592599729,"identity":"7ea516c7-6f39-4641-85fb-23850283bc21","order_by":1,"name":"Prashanta Kumar Mitra","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Prashanta","middleName":"Kumar","lastName":"Mitra","suffix":""},{"id":592599730,"identity":"877125b8-8fd0-4e98-ae2a-6cc1aef5eccc","order_by":2,"name":"Vivekananda Mandal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYJACCYYCBh4GBjbGB0AODx9xWgzAWpgNQFrYiNUCBGxsEmCKkHLd9rMHb/wwOCxjcLwtrfJrjp0MGwPzw0c38GgxO5OXbNljcJjH4MyxY7dltyUDHcZmbJyDT8uBHDMJHoM0HskZ6W23JbcxA7XwsEnj1XL+jZnkH5CW+c/biiW31ROh5UaOmTSPgQ0PvwTbMcaP2w4To+WNsbUMSAtPWrI047bjPGzMhPxyPsfw5psKCXs29mOGH39uq7bnZ29++BifFhTAzAMmiVUOAow/SFE9CkbBKBgFIwYAAIaYP4aXpCg6AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-6523-8069","institution":"University of Gour Banga","correspondingAuthor":true,"prefix":"","firstName":"Vivekananda","middleName":"","lastName":"Mandal","suffix":""}],"badges":[],"createdAt":"2026-02-06 20:29:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8810660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8810660/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102997164,"identity":"0eb58eec-ddee-4401-9779-02a751756528","added_by":"auto","created_at":"2026-02-19 12:25:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1946908,"visible":true,"origin":"","legend":"\u003cp\u003eAMF colonization of Sorghum roots in different NaCl concentrations and day variations. Here, '\u003cstrong\u003eC\u003c/strong\u003e' indicates Control plant, '\u003cstrong\u003eFm\u003c/strong\u003e' indicates \u003cem\u003eF. mosseae\u003c/em\u003e(Fm)\u003cem\u003e, \u003c/em\u003e'Ri' means\u003cem\u003e R. intraradices \u003c/em\u003e(Ri), and\u003cem\u003e '\u003c/em\u003e\u003cstrong\u003eFm\u003c/strong\u003e+\u003cstrong\u003eRi\u003c/strong\u003e\u003cem\u003e' \u003c/em\u003eindicates\u003cem\u003e F. mosseae\u003c/em\u003e (Fm)\u003cem\u003e+ R. intraradices \u003c/em\u003e(Ri). Here, the labels' v','a','ih','is','es', and 'hc' indicate vesicle, arbuscle, intraradical hyphae, intraradical spore, extraradical spore, and hyphal coil, respectively.\u003c/p\u003e","description":"","filename":"Figure1Colonization.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/3d8e6174086a66c1b52616e3.png"},{"id":102997174,"identity":"91c71346-6d01-4e38-878a-d4151051d85a","added_by":"auto","created_at":"2026-02-19 12:25:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1944137,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot shows a significant difference in parameters. Here, '\u003cstrong\u003eC\u003c/strong\u003e' indicates Control plant, '\u003cstrong\u003eFm\u003c/strong\u003e' indicates \u003cem\u003eF. mosseae\u003c/em\u003e(Fm)\u003cem\u003e, \u003c/em\u003e'Ri' means\u003cem\u003e R. intraradices \u003c/em\u003e(Ri), and\u003cem\u003e '\u003c/em\u003e\u003cstrong\u003eFm\u003c/strong\u003e+\u003cstrong\u003eRi\u003c/strong\u003e\u003cem\u003e' \u003c/em\u003eindicates\u003cem\u003e F. mosseae\u003c/em\u003e (Fm)\u003cem\u003e+ R. intraradices \u003c/em\u003e(Ri). Here, the values are the average of triplicate trials ± SE.\u003c/p\u003e","description":"","filename":"Figure2StatisticalAnalysisSBox1.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/5bb23e9b4519018c0590add4.png"},{"id":102997221,"identity":"f319d77d-35b6-407f-b026-30766f48bda3","added_by":"auto","created_at":"2026-02-19 12:25:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5518601,"visible":true,"origin":"","legend":"\u003cp\u003eLinear regression showing the trend in plant parameter change with changing salt concentration and incubation days. (a) the variations in salt concentration, and (b) the result variables in terms of days of culture. Here, 'Fm' indicates \u003cem\u003eF. mosseae\u003c/em\u003e (Fm)\u003cem\u003e, \u003c/em\u003eRi means\u003cem\u003e R. intraradices \u003c/em\u003e(Ri), and\u003cem\u003e '\u003c/em\u003eFm+Ri\u003cem\u003e' \u003c/em\u003eindicates\u003cem\u003eF. mosseae\u003c/em\u003e (Fm)\u003cem\u003e+ R. intraradices \u003c/em\u003e(Ri). Here, the values are the average of triplicate trials ± SE.\u003c/p\u003e","description":"","filename":"Figure3SSRegression.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/c1b8534b8055ef11315f1a4d.png"},{"id":102997170,"identity":"46cd37ad-a93f-4154-865b-dedea79ef15e","added_by":"auto","created_at":"2026-02-19 12:25:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3784446,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation matrix showing correlation between parameters (*: p \u0026lt; 0.05, **: p \u0026lt; 0.01, ***: p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4SSCorrelationMatrix.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/fc795ac329301203b0846d34.png"},{"id":102997242,"identity":"f399ce24-a994-4d48-bc1e-d6a6d8e08c0e","added_by":"auto","created_at":"2026-02-19 12:25:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7120209,"visible":true,"origin":"","legend":"\u003cp\u003ePCA contribution plot showing the contribution of parameters in data variation (a), PCA biplot showing a correlation between plant parameters and how they are related to AMF (b), day (c), and salt concentration (d).\u003c/p\u003e","description":"","filename":"Figure5SSPCA.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/5ef46b3c2f872f98240dd5da.png"},{"id":102997212,"identity":"5733a4fc-3a24-44f1-baf4-201b605ae397","added_by":"auto","created_at":"2026-02-19 12:25:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3342955,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of salinity on the activity of antioxidative enzymes in sorghum plants at 60 days, represented as a box plot.\u003cstrong\u003e \u003c/strong\u003e(a)\u0026nbsp; CAT activity; (b) APX activity; (c) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content; (d) SOD activity; (e) MDA content; and (f) Total protein content.\u003c/p\u003e","description":"","filename":"Figure6StatisticalAnalysisAntioxidantsEnzymesBox.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/ab783ba7dcadcfec64f71e8d.png"},{"id":102997216,"identity":"77ce8ad7-1e77-4b3c-9e03-4365c334a797","added_by":"auto","created_at":"2026-02-19 12:25:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2080674,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering showing similar groups of different AMF treatments based on salt stress-affected enzymes (a) and other plant parameters (b).\u003c/p\u003e","description":"","filename":"Figure7SESSHcluster.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/e67ac5040727fb3adac90f9f.png"},{"id":102997239,"identity":"2ddb4511-a0b7-42d3-a3b2-c8d4a1196c6a","added_by":"auto","created_at":"2026-02-19 12:25:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3274775,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplots of mineral contents showing differences between treatment (a) and salt concentration (b), along with the trend of different AMF treatments in increasing salt concentration (c).\u003c/p\u003e","description":"","filename":"Figure8StatisticalAnalysisMineralsDL.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/f037a4b4bd3c0011355f2412.png"},{"id":102997187,"identity":"b0b3b8f8-5c70-4d53-a266-d2ae400d3068","added_by":"auto","created_at":"2026-02-19 12:25:36","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":3331442,"visible":true,"origin":"","legend":"\u003cp\u003ePCA-significance plot showing the contributing parameters in data variation (a), PCA-biplot showing correlations and effect of AMF treatment (b), Correlation matrix showing significant correlations (c), hierarchical clustering showing clusters of similar treatment groups (d), and concentration effects(e).\u003c/p\u003e","description":"","filename":"Figure9SDLCorrelations2425.png","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/7d81d462dcd18a5c3ed7919f.png"},{"id":105905461,"identity":"4a9cc652-c033-4864-bfe2-11f553778934","added_by":"auto","created_at":"2026-04-01 10:12:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32554858,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/b6e711a7-7345-4d5e-96d7-b87d38f649a0.pdf"},{"id":102997173,"identity":"fd210782-478b-41e4-9bc3-b68a64666bd3","added_by":"auto","created_at":"2026-02-19 12:25:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":45142440,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.GraphicalabstractMargedpic4.tif","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/cb6c6f3779e3877f2ea862a0.tif"},{"id":102997168,"identity":"901ff5ac-b49f-494d-8a9a-aab570f2faa9","added_by":"auto","created_at":"2026-02-19 12:25:29","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38493,"visible":true,"origin":"","legend":"","description":"","filename":"MSSISaltstress07.02.2026.docx","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/cc81d3a24cd3482becea3721.docx"},{"id":103028797,"identity":"57aef45e-f281-422e-96c0-a13a024f497d","added_by":"auto","created_at":"2026-02-19 21:27:55","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":19459,"visible":true,"origin":"","legend":"","description":"","filename":"Table113.docx","url":"https://assets-eu.researchsquare.com/files/rs-8810660/v1/1ef5fe43a18d5b5b4b3333f0.docx"}],"financialInterests":"","formattedTitle":"Effect of mono- or bi-species Arbuscular mycorrhizal symbioses in improving the salt stress tolerance in Sorghum bicolor (L.) Moench.","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants are often exposed to various environmental stresses, which result in significant alterations in their growth and metabolism, ultimately reducing plant yield (Ahanger et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Czarnocka and Karpiński, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Salinity stress is one of the primary stresses that can impede plant growth and development (Grover et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Banerjee and Roychoudhury, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Hashem et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It is estimated that increased salinization of arable land adversely affects germination, growth, and plant reproduction, consequently diminishing crop yield (Chinnusamy et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Worldwide, saline soil can cause annual agricultural losses of up to \u003cspan\u003e$\u003c/span\u003e27\u0026nbsp;billion (Wang et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It is expected to have devastating global effects on food productivity, resulting in a 30% loss of land within the next 25 years (Saxena et al., 2014, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil salinity is a widespread problem, affecting over one billion hectares of land and spreading by more than 2\u0026nbsp;million Hectares annually (Tian et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Hopmans et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Singh, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Soil salinity, measured in decisiemens per meter (dS/m), relates to the presence of high content of soluble salts and low exchangeable sodium ions [EC\u003csub\u003ee\u003c/sub\u003e \u0026gt; 4 dS/m; ESP\u0026thinsp;\u0026lt;\u0026thinsp;15%; pH\u0026thinsp;\u0026lt;\u0026thinsp;8.5; SAR\u0026thinsp;\u0026lt;\u0026thinsp;13] (Eswar et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Different salts, such as NaCl, Na2CO3, MgCl2, CaSO4, MgSO4, and Na2SO4, contribute to soil salinity. Among these, sodium chloride is most common in arid and semiarid soils (Flowers et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1977\u003c/span\u003e). Most salinized areas are found in India, China, the USA, Sudan, Pakistan, and Turkey (Singh, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Globally, over one-fifth of the total irrigated land is salt-affected (Adejumobi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In India, approximately 6.46\u0026nbsp;million hectares of soil are expected to have high salt content among the 23\u0026nbsp;million hectares of arable land affected by salinity/alkalinity/acidification factors (Kumar and Sharma, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Mandal et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In the highly productive Indo-Gangetic Plain, sodic soils with high sodium (Na) content and a pH exceeding 8, in some cases even 10, occupy 1.37\u0026nbsp;million hectares (Mha) (Sharma et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Bhardwaj et al., 2016). Therefore, a significant portion of India's landmass is unsuitable for agriculture due to salinity.\u003c/p\u003e \u003cp\u003eSoil salinity has been grouped according to five classes, i.e., 0\u0026ndash;4 dS/m (normal), 4\u0026ndash;8 dS/m (marginally affected), 8\u0026ndash;12 dS/m (moderately affected), 12\u0026ndash;16 dS/m (severely affected), and more than 16 dS/m (extremely affected) (Datta and Jong 2002). Saline soils having an electrical conductivity (EC\u003csub\u003ee\u003c/sub\u003e) of more than 4 dS/m and an osmotic pressure of -0.2 MPa are equal to about 40 mM NaCl (Santander et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Plants exhibit different tolerance strategies across these salinity levels/classes; however, no crops were grown on land with an electrical conductivity (ECe) exceeding 16 dS/m. Soil salinity has an adverse effect on seed germination and agricultural productivity. It retards plant development by imposing osmotic stress and specific-ion toxicity, thereby restricting root development (Singh, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These may lead to oxidative stress by generating reactive oxygen species (ROS), including the hydroxyl radical (. OH), H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, O2.-, and \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e, as well as nutritional deficiencies (Arzani et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The toxic effects of specific ions, Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, damage cell organelles, cause membrane dysfunction, disrupt general metabolic activities, disrupt the structure of enzymes and other macromolecules, inhibit protein synthesis, and induce ion deficiency, leading to a decline in plant growth, rendering the plants weak and unproductive (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Saxena et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This hinders plant growth and metabolism. Plants can eliminate these deleterious forms of ROS through a range of defense mechanisms, commonly called the antioxidant system, which is controlled by several enzymes such as catalase (CAT), peroxidase (POX), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) and thus diminution of oxidative damage (Costa et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Munns and Tester \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Porcel et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In addition, some produce the indices of oxidative stress parameters (polyphenol oxidase, hydrogen peroxide, and malondialdehyde). Therefore, monitoring these oxidative stress parameters, ROS-scavenging enzymes, and growth parameters could help understand the ecophysiological status of the targeted crops under salinity stress.\u003c/p\u003e \u003cp\u003eSorghum, an ancient grain belonging to the family Poaceae, is a crucial climate-resilient crop that has been a staple food worldwide for thousands of years and is the fifth most widely cultivated cereal crop, after rice, wheat, maize, and barley, predominantly in tropical countries (Hossain et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ostmeyer et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). It is grown worldwide in over 86 countries, covering approximately 38\u0026nbsp;million hectares, with an annual production of 58\u0026nbsp;million tonnes (FAO, 2018). It has been the dietary foundation of over 500\u0026nbsp;million people in 30 countries (Majzoobi et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Sorghum is also a significant grain crop consumed in India after wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) and rice (\u003cem\u003eOryza sativa\u003c/em\u003e) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.millets.res.in/\u003c/span\u003e\u003cspan address=\"https://www.millets.res.in/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). India uses it as food and fodder due to its high grain and green biomass yields and its good nutritional composition. Sorghum is a feed ingredient in several pet food brands and the aquaculture industry. In addition to grain, sorghum stover serves as a crucial feed for dairy and draft animals in India's livestock industry, particularly during the dry seasons (Rao \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is also used in the high-quality distilling industry and serves as a key raw material in biofuel ethanol production (Kelley and Rao, 1994; Ibrahim, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Mansour et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, understanding the impact of salt stress on Sorghum cultivars, their responses, and tolerance strategies would help guide the selection of suitable cultivars for saline soils and identify biomarkers for crop improvement. Moreover, further investigations are required to determine the role of beneficial microbes in mitigating salt stress in the rhizosphere.\u003c/p\u003e \u003cp\u003eAmong the plant growth-promoting microbes (PGPM), arbuscular mycorrhizal fungi (AMF) are crucial because they can establish symbiosis with 80% of plants (Basu et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). AMF survives in most environments and provides various ecological services, significantly enhancing the rhizosphere's chemical and physical properties, thereby strengthening ecosystem function and increasing host plant growth and performance (Frosi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It has been reported that the AMF spore density in sodic soils of rice-wheat cropping systems is greater, with the dominant AMF species being \u003cem\u003eFunneliformis mosseae\u003c/em\u003e and \u003cem\u003eF. geosporum\u003c/em\u003e (Chandra et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Another interesting comparative study examining a wide range of C3 and C4 plant species and their AMF symbiotic relationships found that C3 and C4 plants responded positively to AMF inoculation under saline conditions (Chandrasekaran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Among the AMF, \u003cem\u003eRhizophagus irregularis\u003c/em\u003e had a positive effect on C3 plants, whereas \u003cem\u003eF. mosseae\u003c/em\u003e had a positive impact on C4 plants than other species (Chandrasekaran et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003eTherefore, it is very crucial to envisage the effect of these two AMF species, \u003cem\u003eR. irregularis\u003c/em\u003e, the model species for AMF, with the \u003cem\u003eF. mosseae\u003c/em\u003e that have different host group preferences and modulation of the host physiology on the targeted crop \u003cem\u003eSorghum bicolor\u003c/em\u003e under saline stress conditions (Guo et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Tang et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kokkoris et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The present study aimed to investigate the effects of AMF inoculation with \u003cem\u003eR. intraradices\u003c/em\u003e and \u003cem\u003eF. mosseae\u003c/em\u003e, either alone or in consortia, on development, physiological, and biochemical characteristics, as well as the antioxidant enzymes and lipid peroxidation in forage sorghum cultivar (\"CSV 53F\") plants under salinity stress that simulates the salinity status of arable soil.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eArbuscular mycorrhizal inoculation in\u003c/b\u003e \u003cb\u003eSorghum\u003c/b\u003e \u003cb\u003eseedlings\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eSeveral varieties of Sorghum grow in India, with planting between February and July depending on the farm's location. A single-cut forage sorghum variety, \u003cem\u003eSorghum bicolor\u003c/em\u003e (L.) Moench \"CSV 53F\" (SPV 2705), developed by the Forage Section, Department of Genetics \u0026amp; Plant Breeding, CCS Haryana Agricultural University, Hisar, India, and cultivated during the Kharif season, has been selected as the experimental plant. It is a salt-sensitive, drought-tolerant cultivar, tolerant to shoot fly and stem borer (Kumari et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe seeds were surface-sterilised by dipping for 15 minutes in a 0.2% solution of Mercuric Chloride (HgCl\u003csub\u003e2\u003c/sub\u003e, w/v), followed by three washes with sterile distilled water. They were then immersed in 70% ethanol for 30 seconds and further rinsed three times with sterile distilled water. The surface-sterilised seeds were placed in a petri dish with moist blotting paper at the base and were placed in a plant growth chamber maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C temperature, with an 18-hour photoperiod and 70\u0026ndash;75% relative humidity for 3\u0026ndash;5 days for germination. The seedlings were placed in a Hosco (small hole container for seedling growth, PP 100, of 30 cc with a 3.0 cm diameter and 6.4 cm depth, Horticultural Supplies Co., Mumbai, India) containing a sterilised (121\u0026deg;C for 2 hrs in autoclaved conditions) sand-soil mix (1:3, v/v). The monosporal AMF used in the current experiment was propagated using the trap culture methodology outlined by Stutz and Morton (\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). A single spore of the monosporal AMF isolates \u003cem\u003eR. intraradices\u003c/em\u003e (syn. \u003cem\u003eGlomus intraradices\u003c/em\u003e) and \u003cem\u003eF. mosseae\u003c/em\u003e (syn. \u003cem\u003eGlomus mosseae\u003c/em\u003e) was added separately to each place of Hosco (PP 100, Horticultural Supplies Co., Mumbai, India) and maintained in the plant growth chamber in the same light, humidity, and temperature control for 30 days. The AMF used in this study are the agriculture field isolates from Malda district (25\u0026deg;32'08\"N to 24\u0026deg;40'20\"N Latitude and 88\u0026deg;28'10\"E to 87\u0026deg;45'05\"E Longitude), West Bengal, India, with soil properties as no salt-affected (pH\u0026thinsp;\u0026lt;\u0026thinsp;8.5, EC\u003csub\u003ee\u003c/sub\u003e (dS/m)\u0026thinsp;\u0026lt;\u0026thinsp;4.0, Sodium Adsorption Ratio (SAR)\u0026thinsp;\u0026lt;\u0026thinsp;13.0, Exchangeable Sodium Percentage (ESP)\u0026thinsp;\u0026lt;\u0026thinsp;15.0) (categorized as per Gharaibeh et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe seedlings were then transferred to larger plastic pots with a 25 cm diameter and 20 cm depth (~\u0026thinsp;3 kg soil capacity), filled with autoclaved soil and sand (1:2, v/v). The plantlets were maintained for 120 days in a greenhouse and watered with half-strength Hoagland's solution (Hoagland and Arnon, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1950\u003c/span\u003e) at 7-day intervals, and with water every 3rd day. AMF colonization was checked at every 30-day interval.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eApplication of salt stress on AMF-colonized sorghum plantlets\u003c/h2\u003e \u003cp\u003eA complete randomised design (CRD) was used to experiment with five biological replicates. Two distinct NaCl concentrations (150 mM, EC 7.32 dS/m, and 300 mM, EC 12.03 dS/m) were used as salt stress. Different salt concentrations were first applied at 15 days of plantlet growth (50 mL per pot). After that, the same volume and concentration of salt solution was applied after every 30 days, meaning at 45 days, 75 days, and 105 days. The soil's electrical conductivity and TDS were measured before and after the salt treatment, i.e., \u0026plusmn;\u0026thinsp;5 days apart. The control pot had sorghum seedlings that were not inoculated with spores. The experimental pots were divided into four groups: (C0) control plant (no AMF)\u0026thinsp;+\u0026thinsp;0 mM NaCl; (C150) control plant (no AMF)\u0026thinsp;+\u0026thinsp;150 mM NaCl; (C300) control plant (no AMF)\u0026thinsp;+\u0026thinsp;300 mM NaCl; (Fm0) plants inoculated with \u003cem\u003eF. mosseae\u003c/em\u003e (Fm)\u0026thinsp;+\u0026thinsp;0 mM NaCl; (Fm150) plant inoculated with Fm\u0026thinsp;+\u0026thinsp;150 mM NaCl, (Fm300) plants inoculated with Fm\u0026thinsp;+\u0026thinsp;300 mM NaCl; (Ri0) plants inoculated with \u003cem\u003eR. intraradices\u003c/em\u003e (Ri)\u0026thinsp;+\u0026thinsp;0 mM NaCl; (Ri150) plants inoculated with Ri\u0026thinsp;+\u0026thinsp;150 mM NaCl; (Ri300) plants inoculated with Ri\u0026thinsp;+\u0026thinsp;300 mM NaCl and (Fm+Ri0) plants inoculated with both Fm\u0026thinsp;+\u0026thinsp;Ri+0 mMNaCl treatment, (Fm+Ri150) plants inoculated with Fm\u0026thinsp;+\u0026thinsp;Ri+150 mM NaCl, and (Fm+Ri300) plants inoculated with Fm\u0026thinsp;+\u0026thinsp;Ri+300 mM NaCl stress. The roots and leaves were collected and processed for physio-biochemical investigation following the standard protocols.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMeasurement of soil salinity and electrical conductivity\u003c/h3\u003e\n\u003cp\u003eThe soil salinity, TDS, and EC of the AMF-treated and AMF-untreated pots' soil were measured using a PCSTestr 35 (Eutech PCSTEST35-01X441506, Eutech Instruments Pte Ltd., USA, and Oakton 35425-10, Oakton PCSTestr 35 Waterproof pH/Conductivity/TDS/Salinity Tester, Cole-Parmer India, Mumbai, India) machine. The equipment was calibrated according to the system's respective standards. The experiment was repeated three times, and the average values were recorded.\u003c/p\u003e\n\u003ch3\u003eEstimation of AMF colonization\u003c/h3\u003e\n\u003cp\u003eThe root samples were collected from the AMF-treated and untreated plants, washed in running tap water to remove soil particles, and then preserved in FAA solution (a mixture of 50 ml of 95% ethyl alcohol, 2.5 ml of glacial acetic acid, 5.5 ml of formaldehyde, and 42 ml of double-distilled water). For colonization measurement, the roots were cleared in 10% KOH solution (w/v), placed in a water bath (70\u0026deg;C) for 20\u0026ndash;30 min, and cooled at room temperature. These were washed with water, acidified with 1% HCl (v/v), and stained with 0.05% Trypan blue (w/v) for 30 mins. The excess stain was removed by washing with water, followed by a 5% acetic acid solution (v/v), and further destained with a lactophenol solution (comprising phenol crystals, 12.5 g; glycerol, 50 ml; lactic acid, 25 ml; and 70% ethanol, 25 ml) (Sun and Tang \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Randomly cut 50 root fragments from the tip portion for each sample (1 cm long) were mounted on slides in a Polyvinyl Lacto Glycerol (PVLG, comprising 8.4 g of Polyvinyl alcohol (PVA), dissolved in 50 ml of lactic acid, 5 ml of glycerol, and 45 ml of distilled water, in a dark bottle) and examined for AM colonization under a Leica DM 750 phase contrast microscope (Germany) fitted with LAS EZ camera software. The percentage of root colonization was calculated using the formula provided by Ho-Pl\u0026aacute;garo et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The abundance of arbuscules, vesicles, and total colonization was observed, and photomicrographs were taken. Ten microscopic fields were observed per set, and the average values were recorded.\u003c/p\u003e \u003cp\u003e\u003cimg 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\" width=\"609\" height=\"88\"\u003e\u003c/p\u003e\n\u003ch3\u003eAssay for physio-biochemical parameters\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of stomatal movement upon salt stress\u003c/h2\u003e \u003cp\u003eFresh leaves (3rd ) from all treatment set plants were collected to count stomatal opening and closing. The cut leaves were immediately floated, abaxial side down, in a Petri dish containing MES/KOH buffer (5 mM KCl, 10 mM MES (2-N-morpholinoethanesulfonic acid), 50 \u0026micro;M CaCl\u003csub\u003e2\u003c/sub\u003e, pH 6.15). The Petri dishes were maintained under constant light conditions. The lower leaf surface was peeled and observed for colonization under a Leica DM 750 phase-contrast microscope (Germany) equipped with LAS EZ camera software (Wu and Zhao, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Ten stomata were observed randomly in triplicate to check the stomatal opening and closing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of chlorophyll content\u003c/h2\u003e \u003cp\u003eThe total chlorophyll content of every 3rd leaf (top) was estimated by a chlorophyll meter SPAD-502Plus (Konica Minolta, USA). The SPAD-502Plus measures the absorbance of the leaf in the red and near-infrared regions and represents a numerical SPAD value proportional to the amount of chlorophyll present in the leaf. Ten SPAD data values were recorded from each leaf sample, and the average values were calculated.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEstimation of total carbohydrate\u003c/h3\u003e\n\u003cp\u003eThe total carbohydrate content of the plant samples was estimated using the Anthrone method (Yemm and Willis, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e1954\u003c/span\u003e). 0.5 g of leaf (fresh weight) samples were crushed with 5 ml of 80% ethanol (v/v) and then heated in a boiling water bath for 20 min. The extract was centrifuged at 4,032 x g for 10 min at room temperature. The supernatant was collected, and the pellet was re-crushed and then re-extracted with the same solvent. The final volume of the supernatant was adjusted to 5 ml using double-distilled water. To 1 ml of suitably diluted supernatant under ice-cold conditions, 2 ml of Anthrone reagent (200 mg/100 ml ice-chilled conc. H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e) was added, and the mixture was gently mixed. Then, it was placed in a boiling water bath for 10 minutes. The solution's blue-green absorbance was measured at 620 nm in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The experiment was repeated three times. The total carbohydrate content was estimated from the standard curve of D-Glucose (100 \u0026micro;g/ml) using the following formula.\u003c/p\u003e \u003cp\u003eTotal carbohydrate content/g of tissue\u0026thinsp;=\u0026thinsp;Total carbohydrate obtained from the standard curve \u0026times; volume makeup (ml) \u0026times; dilution factor/weight of sample (g).\u003c/p\u003e\n\u003ch3\u003eEstimation of total Phosphate and Nitrate\u003c/h3\u003e\n\u003cp\u003eThe total nitrogen content of the plant sample was measured using the Salicylic Acid method (Cataldo et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1975\u003c/span\u003e), and the phosphorus content was quantified according to the method of Ames et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1960\u003c/span\u003e). Briefly, 50 mg of fresh leaves were crushed with 500 \u0026micro;l of distilled water for total nitrogen estimation, and then centrifuged at 5,478 \u0026times; g for 10 min. After collection, the supernatant (0.2 ml) was mixed with 0.8 ml of 5% salicylic acid solution (dissolved in conc. H2SO4) and incubated at room temperature. After that, 1.9 ml of 2N NaOH was added to the mixture, and the absorbance was read at 410 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). For the estimation of total inorganic phosphate, 10 mg of fresh tissue was mixed with 1 part of 10% ascorbic acid and 6 parts of 0.42% ammonium molybdate, then heated at 45\u0026deg;C for 20 min. This was followed by incubation for 1 hour at 37\u0026deg;C. The extract was collected by centrifugation (Remi 12C Plus, Mumbai, India) at 5,478 \u0026times; g for 10 min, and the absorbance was measured at 820 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The mineral content was measured using the following formula:\u003c/p\u003e \u003cp\u003eNitrate/phosphate content (\u0026micro;M/mg tissue) = Total nitrate/phosphate obtained from standard curve \u0026times; volume of sample \u0026times; dilution factor/sample weight (mg).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAssay of total proteins and antioxidant enzymes\u003c/h2\u003e \u003cp\u003e0.5 g of fresh leaves was crushed using a mortar and pestle, with a gradual addition of 5 ml of 0.1 M potassium phosphate buffer (pH 7.0) containing 0.1 g of polyvinyl polypyrrolidone (PVPP), and was kept at 4\u0026deg;C for 30 minutes. The extract was then centrifuged at 8,928 x g for 10 min at 4\u0026deg;C, and the supernatant was collected. The protein content of the extract was determined using the Lowry et al. method (1951) with BSA as a standard at 750 nm on a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The superoxide dismutase (SOD, EC 1.15.1.11) activity was determined following the Beyer and Fridovich method (1987) to assess the enzyme's capacity to impede the photochemical reduction of nitro blue tetrazolium (NBT). The enzyme units (EU) for SOD activity were measured as the amount of protein required to achieve a 50% reduction in NBT in the presence of SOD. The catalase (CAT, EC 1.11.1.6) activity was estimated by measuring H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e decomposition over three minutes, as indicated by the absorbance at 240 nm in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) (Aebi, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). The reaction mixture consisted of 0.10 mM EDTA, 0.10 M potassium phosphate buffer (pH 7.0), 20 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 100 \u0026micro;L of enzyme extract, for a total volume of 2 mL. Enzyme activity was calculated in EU/mg of protein. The ascorbate peroxidase activity (APX, EC 1.11.1.11) was measured using the method described by Nakano and Asada (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). Briefly, 0.1 ml of enzyme extract, 0.1 mM EDTA, 0.5 mM ascorbate, 0.1 mM H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and 1 ml of potassium phosphate buffer (0.1 M, pH 7.0) were all included in the test mixture. The absorbance of ascorbate reduction was measured at 290 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The activity was expressed as EU/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of MDA content\u003c/h2\u003e \u003cp\u003eMalondialdehyde (MDA) was quantified using the method of Heath and Packer (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) to measure lipid peroxidation. 0.5 g of fresh leaves were homogenized in a mortar and pestle with 10 ml of 0.1% aqueous trichloroacetic acid (TCA, w/v), then centrifuged (Remi 12C Plus, Mumbai, India) at 11,200 x g for 10 min at 4\u0026deg;C. The supernatant was collected, and the volume was adjusted to 10 ml with 0.1% aqueous TCA. After adding 4.0 ml of 0.5% (w/v) Thio barbituric acid (TBA) to the supernatant (1.0 ml), the mixture was heated to 95\u0026deg;C for 30 min, cooled in an ice bath, and centrifuged (Remi 12C Plus, Mumbai, India) at 10000 x \u003cem\u003eg\u003c/em\u003e for 5 min at 4\u0026deg;C. The supernatant's absorbance was measured at 532 and 600 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA). The following formula was used to determine the MDA content:\u003c/p\u003e \u003cp\u003eMDA (\u0026micro;mol/g FW) = [(A\u003csub\u003e532\u003c/sub\u003e- A\u003csub\u003e600\u003c/sub\u003e)/156] \u0026times;10\u003csup\u003e3\u003c/sup\u003e \u0026times; dilution factor, Where A\u003csub\u003e532\u003c/sub\u003e and A\u003csub\u003e600\u003c/sub\u003e stands for absorbance at 532 and 600 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content\u003c/h2\u003e \u003cp\u003e0.5 g of fresh leaves was homogenized in a mortar and pestle with 5 ml of 1% aqueous trichloroacetic acid (TCA, w/v) solution, then centrifuged (Remi 12C Plus, Mumbai, India) at 11,200 \u0026times; g at 4\u0026deg;C for 10 min. The supernatant was collected, and the volume was adjusted to 5 ml with 1% aqueous TCA. An equal volume (1 ml) of 0.1 M potassium phosphate buffer (pH 7.0) and 1 ml of 1 M potassium iodide were mixed with 0.5 ml of the supernatant. The samples were vortexed gently, and the absorbance was measured at 390 nm using a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) (Velikova et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content was determined from the standard curve of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e conc. grades from 50 to 700 \u0026micro;l of 163 \u0026micro;M of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e stock solutions. Each tube was filled with 1 ml of 1 M potassium iodide. After that, the final volumes were increased to 2 ml with 0.1 M phosphate buffer, and the contents were expressed as \u0026micro;M/g FW. All these preparations were taken in amber containers or under light-controlled conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of Proline content\u003c/h2\u003e \u003cp\u003eThe proline content was determined following the method of Bates et al. (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1973\u003c/span\u003e). The fresh tissue (0.5 g) was homogenized in a mortar and pestle with 5 mL of 3% sulfosalicylic acid solution (w/v). The homogenate was then centrifuged in a refrigerated centrifuge (RM-12C Plus, Remi, Mumbai, India) at 10,000 \u0026times; g at 4\u0026deg;C for 10 min. The supernatant was collected the volume was adjusted to 5 ml with 3% sulfosalicylic acid solution. After that, 1 ml of supernatant was combined with 1 ml of glacial acetic acid, and 1 ml of acid ninhydrin reagent (1.25 g ninhydrin in 30 ml glacial acetic acid, w/v) was incubated in a boiling water bath for 1 hr. The tube was cooled in an ice bath for 15 min to stop the reaction, then 2 mL of toluene was added and vortexed. The mixture was allowed to stand for a short time to let the two phases separate. The absorbance of the top chromophore toluene layer was determined in a UV-Vis spectrophotometer (Cary 60, Agilent Technologies, USA) at 520 nm, and the proline concentration was determined from the standard curve of proline using toluene as a blank.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEstimation of mineral content\u003c/h2\u003e \u003cp\u003eThe macro-elements, such as sodium (Na), potassium (K), and calcium (Ca), were estimated using the procedure of Johnson and Ulrich (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1959\u003c/span\u003e) with some modifications. Here, 500 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added to the acid-digested sample to decompose organic matter, making the reaction more efficient and faster. At first, 0.5g dry leaf samples were ashed in a muffle furnace at 600\u0026deg;C for 4 hrs, after which the ash was suspended in a 50 ml volumetric flask. Acid digestion was performed using a diacid mixture (nitric acid and perchloric acid, 4:1 v/v) in a boiling water bath until the ash particles were completely dissolved. The samples were filtered through Whatman No. 42 filter paper and diluted to 20 mL with deionized water. Calcium, sodium, and potassium were determined using a \u0026micro;-Controller-based Flame photometer with a compressor (Flame Photometer 128, Systronics, Kolkata, India) (Kravić et al., 2012). The microelements, including iron (Fe), manganese (Mn), copper (Cu), and zinc (Zn), in the acid-digested samples were estimated using an atomic absorption spectrophotometer (AA-6880, Shimadzu, Japan) with some modifications. Here, 500 \u0026micro;l of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was added to the acid-digested sample to decompose organic matter, making the reaction more efficient and faster (Keeney and Nelson, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1982\u003c/span\u003e). The sample preparation was identical to that used in the macro-element analysis method. The content was expressed as \u0026micro;g/g of sample. The mineral content was measured using the following formula:\u003c/p\u003e \u003cp\u003eMineral content (\u0026micro;g) / gm of tissue\u0026thinsp;=\u0026thinsp;mg/l flame photometer or AAS result \u0026times; volume of sample \u0026times; dilution factor/weight of sample (gm)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eANOVA was performed to analyze the significant differences. For analysing correlations, linear regression was performed, a correlation matrix was created, and Principal Component Analysis (PCA) was conducted. A distance matrix (Euclidean-normalised) was created for clustering similar treatment groups, and hierarchical clustering (Ward) was done. All analyses were performed in R and Orange3.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of salt stress on AMF colonization, growth, and biomass of Sorghum plant\u003c/h2\u003e\n \u003cp\u003eThe microscopic observations for the mycorrhizal colonization of the sorghum plant at 30-day intervals revealed the presence of all the expected mycorrhizal fungi structures (hyphae, vesicles, and arbuscules) in AMF-inoculated plant roots under salt-non-stressed and salt-stressed (150 mM and 300 mM) samples (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). It was observed that soil salinity at 300 mM NaCl significantly reduced AMF colonization in sorghum roots. The number of vesicles, arbuscule, and total colonization formation was reduced to 46.66%, 63.63%, and 61.29%, respectively, in the \u0026apos; Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; treatment at 300 mM NaCl concentration after 120 days (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A similar result was also observed at 30 days, 60 days, and 90 days. Conversely, total root colonization increased by about 4% in 150 mM salinity stress compared to non-treated (0 mM) plants in the respective AMF species. Total colonization was also the highest in combined AMF (Fm\u0026thinsp;+\u0026thinsp;Ri) treatment in 150 mM salt stress conditions after 60 days and 90 days (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The formation of a more prominent arbuscule was observed at 30 days in \u003cem\u003eF. mosseae\u003c/em\u003e than in \u003cem\u003eR. intraradices\u003c/em\u003e with the 150 mM salt stress (panel Fm150 and Ri150) than in the salt-untreated set-up (panel Fm0 and Ri0). The vesicle formation was also higher at 60 days in \u003cem\u003eF. mosseae\u003c/em\u003e than in \u003cem\u003eR. intraradices\u003c/em\u003e. However, 300 mM salt stress reduced the arbuscular and vesicle formation in \u003cem\u003eR. intraradices\u003c/em\u003e compared to \u003cem\u003eF. mosseae\u003c/em\u003e (panels Fm300 and Ri300). Moreover, the size of the vesicles also increased with the increase in salt stress (panel Fm150, Fm300, Ri150, and Ri300), and the number of vesicular formations was higher in 150- and 300-mM salt treatment in \u003cem\u003eR. intraradices\u003c/em\u003e (panel R150 and R300) than in \u003cem\u003eF. mosseae\u003c/em\u003e (panel Fm150 and Fm300). This indicates that \u003cem\u003eF. mosseae\u003c/em\u003e is a faster (30 and 60 days) colonizer with more arbuscule formation than \u003cem\u003eR. intraradices\u003c/em\u003e at moderate salt stress, but \u003cem\u003eR. intraradices\u003c/em\u003e had more robust vesicle formation at prolonged incubation time (90 and 120 days). In contrast, the combination of both isolates (Fm\u0026thinsp;+\u0026thinsp;Ri) in 150 mM and 300 mM salt treatment had more significant colonization, arbuscule, and vesicle formation in all the observation times (panel Fm\u0026thinsp;+\u0026thinsp;Ri 150 and Fm\u0026thinsp;+\u0026thinsp;Ri 300) as compared to salt-unstressed conditions (panel Fm+Ri0). Thus, the study revealed that these AMF isolates exhibit synergistic properties, enabling them to colonize the host plant and tolerate moderate to high salt stress conditions by forming more arbuscule or vesicles (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSalt stress also negatively affected various morphological characteristics in sorghum plants. Compared with the AMF non-colonized plants (control) at varying salt concentrations, all employed AMF significantly improved overall sorghum plant development (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). The application of salt stress (150 mM and 300 mM NaCl) reduced the plant height compared to the salt-unstressed control plant; however, in the AMF-colonized plants, plant shoot length and root length grew in the 150 mM NaCl salt-stressed condition but decreased in the 300 mM salt stress condition (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Plant biomass increased by 21.42%, 16.37%, and 36.44% in 120 days in the Fm, Ri, and Fm\u0026thinsp;+\u0026thinsp;Ri colonized plants in the 0 mM NaCl sets compared to the AMF-non-inoculated salt-untreated control plants. However, under 300 mM NaCl stress, both the control and AMF-colonized plants experienced a decline in plant biomass. Consequently, in both the presence and absence of salt stress, applying various AMF inocula increased the stomatal opening (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest stomatal opening was found in 0mM salt-stressed conditions in control as well as AMF colonized plants 0 mM NaCl (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). However, AMF inoculation under salt stress improved stomatal opening in plants subjected to 150 mM and 300 mM NaCl stress compared to non-AMF plants under the same salt stress (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe ANOVA analysis shows that there is an overall significant difference found between four AMF species in the parameters arbuscule (p\u0026thinsp;=\u0026thinsp;9.7e-10), carbohydrate (p\u0026thinsp;=\u0026thinsp;1.3e-06), nitrate (p\u0026thinsp;=\u0026thinsp;2e-04), phosphate (p\u0026thinsp;=\u0026thinsp;2e-10), total colonization (p\u0026thinsp;=\u0026thinsp;7.8e-13), and vesicles (p\u0026thinsp;=\u0026thinsp;6.8e-13). However, no significant difference was found in the parameters biomass (p\u0026thinsp;=\u0026thinsp;0.5), chlorophyll (p\u0026thinsp;=\u0026thinsp;0.41), conductivity (p\u0026thinsp;=\u0026thinsp;0.96), proline (p\u0026thinsp;=\u0026thinsp;0.97), root length (p\u0026thinsp;=\u0026thinsp;0.23), salinity (0.99), shoot length (p\u0026thinsp;=\u0026thinsp;0.083), stomata opening (p\u0026thinsp;=\u0026thinsp;0.079) and TDS (p\u0026thinsp;=\u0026thinsp;0.92) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). There is an overall significant difference found between the two AMF species in the parameters arbuscule (p\u0026thinsp;=\u0026thinsp;9.7e-10), carbohydrate (p\u0026thinsp;=\u0026thinsp;1.3e-06), nitrate (p\u0026thinsp;=\u0026thinsp;2e-04), phosphate (p\u0026thinsp;=\u0026thinsp;2e-10), total colonization (p\u0026thinsp;=\u0026thinsp;7.8e-13), and vesicles (p\u0026thinsp;=\u0026thinsp;6.8e-13). However, no significant difference was found in the parameters of biomass (p\u0026thinsp;=\u0026thinsp;0.5), chlorophyll (p\u0026thinsp;=\u0026thinsp;0.41), conductivity (p\u0026thinsp;=\u0026thinsp;0.96), proline (p\u0026thinsp;=\u0026thinsp;0.97), root length (p\u0026thinsp;=\u0026thinsp;0.23), salinity (p\u0026thinsp;=\u0026thinsp;0.99), shoot length (p\u0026thinsp;=\u0026thinsp;0.083), stomata opening (p\u0026thinsp;=\u0026thinsp;0.079), and TDS (p\u0026thinsp;=\u0026thinsp;0.92) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of salt stress on biochemical parameters of the sorghum plant\u003c/h2\u003e\n \u003cp\u003eThe chlorophyll content in \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; colonized plants under 150 mM NaCl stress after 90 days of culture was 38.46% higher than that of the AMF-non-colonized plants (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). However, the chlorophyll content of both AMF-colonized and non-colonized plants sharply dropped from 60 to 90 days under 300 mM NaCl stress conditions (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). A similar observation was also found in 90 to 120 days (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the Fm-treated plant, nitrate content increased from 0.46, 0.21, 1.4, 1.09 \u0026micro;M/10 mg to 0.92, 1.41, 1.65, and 1.94 \u0026micro;M/10 mg, and in the Ri-inoculated plant, it increased from 0.46, 0.21, 1.4, 1.09 \u0026micro;M/10 mg to 1.27, 1.07, 1.72, and 1.22 \u0026micro;M/10 mg in 30 days, 60 days, 90 days, and 120 days, respectively in 0 mM salt-stressed conditions than the AMF untreated control plant. In the combined \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026rsquo;-treated plant, the nitrate content increased to 63.2%, 79%, 53.48%, and 50.9% in 30 days, 60 days, 90 days, and 120 days, respectively, in 0 mM salt-stressed conditions than AMF untreated control plant (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). In 150 mM salt-stressed condition, the Fm-treated plant, the nitrate content increased to 73.98%, 77.14%, 74.73%, and 75.8%, and in the Ri-inoculated plant, nitrate content increased to 73.92%, 60.17%, 57.77%, and 63.19%, in 30 days, 60 days, 90 days and 120 days respectively than AMF-untreated control plant. In the combined \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; treated plant, the nitrate content increased to 64%, 59.61%, 57.64%, and 75% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to AMF untreated control plant (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In 300 mM salt-stressed conditions, Fm-treated plant nitrate content increased to 18.16%, 21.24%, 26.63%, and 17.18%. In Ri-inoculated plants, nitrated content increased to 11.31%, 13.86%, 16.68%, and 9.63% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to AMF-untreated control plants (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the combined \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; treated plant, plant nitrate content increased to 21.23%, 23.82%, 28.24%, and 21.02% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plant (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSimilarly, under 0 mM salt-stressed conditions, the plant phosphate content of Fm-treated plants increased to 18.13%, 19.27%, 45.53%, and 47.86%. Ri-treated plant phosphate content increased to 5.68%, 21.17%, 20.45%, and 19.73% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF-untreated control plant (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the combined AMF-treated plant, the phosphate content increased to 10.24%, 52.81%, 57.18%, and 56.27% after 30 days, 60 days, 90 days, and 120 days, respectively, under 0 mM salt-stressed conditions, compared to the AMF-untreated control plant. In 150 mM salt-stressed conditions, the phosphate content increased to 31.62%, 46.41%, 66.19%, and 69.13% and 40.24%, 46.41%, 60.1%, and 64.6% in Fm and Ri-inoculated plants in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plants (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the combined AMF treatment, the plant phosphate content increased to 35.24%, 67.05%, 72.1%, and 71.36%, respectively, compared to AMF-untreated control plants (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In 300 mM salt stressed condition, the Fm-treated plant, phosphate content increased from 1.28, 1.07, 0.38 and 0.32 \u0026micro;M/10 mg to 1.47, 2.2, 2.69 and 1.53 \u0026micro;M/10 mg and Ri-inoculated plant phosphate content increased from 1.28, 1.07, 0.38 and 0.32 \u0026micro;M/10 mg to 1.81, 1.84, 1.92 and 1.6 \u0026micro;M/10 mg and the combined AMF treated plants, plant phosphate content increased to 14.87%, 26.62%, 29.28% and 19.85% in 30 days, 60 days, 90 days and 120 days, respectively than untreated control plant (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn 0 mM salt-stressed conditions, the total carbohydrate content in Fm-treated plants increased from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 6.12, 10.1, 14.3 and 13.2 mg/100g and in the Ri-inoculated plant, total carbohydrate content increased from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 5.58, 11.4, 14.1 and 13.2 mg/100gm in 30 days, 60 days, 90 days and 120 days, respectively, than AMF untreated control plants (Figs. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In the combined Fm\u0026thinsp;+\u0026thinsp;Ri-treated plant, the total carbohydrate content increased to 42.48%, 50.54%, 51.63%, and 55.86% after 30 days, 60 days, 90 days, and 120 days, respectively, under 0 mM salt-stressed conditions, compared to the AMF-untreated control plant (from 4.21, 7.32, 8.9 and 7.9 mg/100 gm to 7.32, 14.8, 18.4 and 17.9 mg/100gm). While the 150 mM salt-stressed condition, the Fm-treated plant increased total carbohydrate content to 48.3%, 56.77%, 53.03%, and 56.06%, and the Ri-inoculated plant increased to 38.26%, 54.05%, 49.59%and 50%, and the Fm\u0026thinsp;+\u0026thinsp;Ri-inoculated plant the total carbohydrate content increased to 62.67%, 67.72%, 62.87%, and 64.19%in 30 days, 60 days, 90 days and 120 days, respectively than in the AMF untreated control plant. Whereas, in 300 mM salt-stressed conditions, the total carbohydrate content in Fm-inoculated plants increased to 23.21%, 24.11%, 17.62%, and 11.62%, and in Ri-inoculated plants, it increased to 17.68%, 22.68%, 17.68%, and 10.68%. In the Fm\u0026thinsp;+\u0026thinsp;Ri-inoculated plants, the total carbohydrate content increased to 23.72%, 25.36%, 24.79%, and 15.68% in 30 days, 60 days, 90 days, and 120 days, respectively, compared to the AMF untreated control plant (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In 30 days, proline content increased to 5.88%, 4.37%, and 5% in the \u0026apos;Fm\u0026apos;,\u0026apos; Ri\u0026apos;, and \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; inoculated plants compared with the AMF untreated control in 0mM salt-stressed conditions. Meanwhile, proline content increased to 25%, 16%, and 38.23% in \u0026apos;Fm\u0026apos;,\u0026apos; Ri\u0026apos;, and \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; inoculated plants, respectively, in 150 mM salt-stressed conditions compared with the AMF untreated control in 30 days at and increased to 53.73%, 35.89% and 46.8% in \u0026apos;Fm\u0026apos;,\u0026apos; Ri\u0026apos;, \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; inoculated plant respectively in compared with the untreated control in 30 days at 300 mM salt stressed condition (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe ANOVA analysis and linear regression analysis of the plant parameter change with changing salt concentration and incubation days shows that the arbuscle percentage in treated groups decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) but increased with days, except Ri, which showed a slight decrease with days (b); there is no change in the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). For all the groups, biomass decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) but increased with days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). For all the groups, carbohydrates decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) but increased with days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb); the noticeable thing is that the amplitude of Fm and Ri is similar (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). For all the groups, chlorophyll decreased with increasing salt concentration and days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). For all groups, conductivity increased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea). However, over time, conductivity increased for the control, decreased for the AMF-treated groups, and the reduction was most pronounced for Fm\u0026thinsp;+\u0026thinsp;Ri (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Nitrate levels decrease with increasing salt concentration for all parameters (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) and increase with time (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). phosphate in the control group decreases with both increasing salt concentration and days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b), while in treated groups with increasing salt concentration, slightly increases in Ri, not unchanged in Fm, and decreases in Fm\u0026thinsp;+\u0026thinsp;Ri; and with increasing days, increases in Fm, Fm\u0026thinsp;+\u0026thinsp;Ri, and decreases in Ri (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Proline increased with increasing salt concentration and days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Root length decreased with increased salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) and increased with days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). salinity increased with increasing salt concentration and days for all the groups (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Shoot length decreased with increasing salt concentration and increased with days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Stomatal opening decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea), decreased with increasing days in the treatment groups, and remained constant in the control group (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). TDS increased with both increasing salt concentration and days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Total colonization decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) and increased with increasing days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb); the pattern is similar in Fm and Fm\u0026thinsp;+\u0026thinsp;Ri (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b). Vesicles decreased with increasing salt concentration (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) and increased with increasing days (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb); the notable thing is that the increasing and decreasing pattern is similar in Fm and Fm\u0026thinsp;+\u0026thinsp;Ri (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e\n \u003cp\u003eThe correlation matrix analysis between parameters at significance values of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, shows that many of the parameters are significantly positively correlated, and some are significantly negatively correlated, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. The PCA analysis shows that proline, shoot length, and carbohydrate contributed the highest in data variation and TDS; the stomatal opening was found to be the least contributing in data variation (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). The control group was quite distinct from AMF-treated groups; the effects of FM and FM\u0026thinsp;+\u0026thinsp;RI on plant parameters were similar, and FM\u0026thinsp;+\u0026thinsp;RI was found to affect biomass, shoot length, and colonization the highest (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb). Plant parameters were also found quite similar in 90 and 120 days, while 30 and 60 days are quite different, probably an indication of adaptation with longer incubation time (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec). Different concentrations of salt-affected plant parameters differently, while the control group was found with higher values of stomatal opening chlorophyll; the group with 150 mM salt concentration was found to affect biomass, vesicles majorly; the group with 300 mM salt concentration proline, salinity, and TDS (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of salt stress on proteins and antioxidant enzyme parameters of the sorghum plant\u003c/h2\u003e\n \u003cp\u003eThe expression of antioxidants in sorghum plants during the salt and AMF treatments is shown in Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e(a-f). When compared to AMF untreated control plants in 300 mM salt stress conditions, the combined AMF-treated plants showed higher SOD, CAT, and APX activity (32.64%, 49.61%, and 33.69%, respectively) in 60 days (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Salt stress significantly impacted total protein content (TPC). TPC of AMF-treated plants increased (from 8.44 mg/g to 15.47, 15.87, and 17.84 mg/ g in Fm, Ri, and Fm\u0026thinsp;+\u0026thinsp;Ri treated plants, respectively) at 150 mM salt-stressed conditions compared to the AMF-untreated control sets at 60 days. However, salt stress (150 mM and 300 mM) increased, and TPC decreased across all treatment conditions; however, in AMF-treated plants, TPC increased when comparing treated and AMF-untreated plants. Among AMF-treated plants, the highest TPC was observed in \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; treated plants in different salt-stressed conditions (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ef). The higher malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) content in plant tissues indicates oxidative stress associated with reactive oxygen species production. The results show that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and MDA concentrations did not differ between non-AMF and AMF-inoculated plants under the control condition (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003ec, e). The combined AMF-treated plants showed lower MDA and H2O2 levels in response to 300 mM salt stress than the untreated salt-stressed control plants (51.10% and 31.14%, respectively) after 60 days. In contrast, single AMF-treated plant \u0026apos;Fm\u0026apos; showed higher activity (42.23% and 22.73%, respectively) in identical conditions. This indicates that AMF-treated plants alleviate salt stress conditions by reducing MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e activity, unlike the AMF-untreated control plants (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eHierarchical clustering based on salt stress-affected enzymes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea) and other plant parameters (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb), which indicated that the control group was quite different from the treatment groups (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea,b). However, treatment groups were not quite separable, and a cluster of mixed treatment groups (FM, RI, FM\u0026thinsp;+\u0026thinsp;RI) was found in both the enzymatic (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003ea) and other plant parameters (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eb). The overall analysis shows that different salt concentrations induce different effects (in terms of parameters) and control plant parameters and enzymes differently. Regarding the impact of AMF on counteracting salt stress, it was observed that different salt concentrations elicit distinct effects. Consequently, the AMF groups also attempted to mitigate these effects accordingly. Over time, the plant adapts to salt stress, thereby reducing its harmful effects. FM\u0026thinsp;+\u0026thinsp;RI was more effective than FM or RI in enhancing salt tolerance across several aspects, while FM and RI were more effective in others. RI complements FM well in reducing salt stress (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of salt stress on the mineral content of the sorghum plant\u003c/h2\u003e\n \u003cp\u003eSalt stress raised the Na\u003csup\u003e+\u003c/sup\u003e salt concentration in AMF-untreated control sorghum leaf tissues. It significantly decreased the absorption of vital elements such as potassium (K\u003csup\u003e+\u003c/sup\u003e), calcium (Ca\u003csup\u003e2+\u003c/sup\u003e), iron (Fe\u003csup\u003e3+\u003c/sup\u003e), zinc (Zn\u003csup\u003e2+\u003c/sup\u003e), manganese (Mn\u003csup\u003e2+\u003c/sup\u003e), and copper (Cu\u003csup\u003e2+\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). Maximum reduction of nutritional minerals was observed in the AMF-untreated control at 300 mM NaCl stress. However, AMF inoculation increased the content of vital minerals in both salt-stressed conditions. The content of minerals like \u0026apos;Cu, Zn, Mn, Fe, K, and Ca in \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; inoculated plants increased by 40.70%, 33.78%, %, 53.27%, %, 14.76%, and 15.58%, respectively, \u0026apos;Fm\u0026rsquo;-inoculated plants increased by 36.89%, 25.99%, 30.25%, 12.15%, 12.79% and 21.02%, and \u0026apos;Ri\u0026rsquo;-inoculated plants by 10.23%, 10.93%, 13.72%, 7.79%, 9.14% and 8.35% than the AMF-untreated control in 0 mM NaCl stressed at 60 days. In individual Fm-inoculated and Ri-inoculated plants, mineral uptake such as Cu, Zn, Mn, Fe, K, and Ca was increased by 36.89%, 25.99%, 30.25%, 12.15%, 12.79% and 21.02%, and 10.23%, 10.93%, 13.72%, 7.79%, 9.14%, and 8.35%, respectively which were lower than \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026apos; treated plant.\u003c/p\u003e\n \u003cp\u003eThe Na\u003csup\u003e+\u003c/sup\u003e content decreased up to 18.26% in \u0026apos;Fm\u0026thinsp;+\u0026thinsp;Ri\u0026rsquo;-inoculated plant, while in \u0026apos;Fm\u0026apos; inoculated plant, it decreased up to 16.03% while in \u0026apos;Ri\u0026rsquo;-inoculated plant, it decreased up to 10.81% compared to the AMF-untreated control at 150 mM salt-stressed conditions in 60 days (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). This indicates that AMF colonization significantly regulates the Na\u003csup\u003e+\u003c/sup\u003e uptake. Colonization also substantially minimizes the Cu/Na, Zn/Na, Mn/Na, Fe/Na, K/Na, and Ca/Na ratios, thus minimizing the Na\u003csup\u003e+\u003c/sup\u003e-associated mineral uptake and transport processes (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). Moreover, it has been observed that the vesicle and total colonization development were the highest in Fm+Ri150. At the same time, it affected the arbuscule development (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The PCA analysis shows that 150 mM salt stress greatly influences the arbuscule, vesicle, and total colonization and mineral uptake by the AMF species (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). In contrast, the colonization minimizes the salt stress associated with mineral transport in the plant system. The hierarchical cluster analysis reveals that 300 mM salt stress significantly reduces plant AMF colonization and mineral uptake, thereby forming a distinct clade. In contrast, Fm+Ri150 and Fm150 formed a separate clade and the C0, Fm0, and Ri0 formed a separate clade (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). No significant difference was found between AMF treatments for all the minerals measured (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ea). However, a significant difference was found between AMF treatments for all the minerals measured (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb). There is a decreasing trend with salt concentration for all the AMF treatment groups for Ca, Cu, Fe, K, Mn, and Zn, but an increasing trend for Na (c) (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). PCA significance analysis shows that Cu, Mn, and Zn were the most significantly contributing parameters in data variation. PCA-biplot also showing FM treatment contributes to Fe, K, Ca accumulation, RI treatment contributes to Na, Ca, K accumulation, FM\u0026thinsp;+\u0026thinsp;RI treatment contributes in Cu, Mn, Zn accumulation (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003eb) Na found with significantly negatively correlated with all the parameters, rest of the parameters (Ca, Mn, Zn, Fe, K, and Ca) are significantly positively correlated among themselves (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSalinity stress adversely affects agricultural productivity by disrupting water uptake. It has a profoundly detrimental effect on plant growth, biomass, and overall biochemical parameters by altering the soil solution's water potential, making it more negative and leading to physiologically dry soil (Razzaq et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It also affects the uptake of nutrients, like N, P, Mg, and Fe. This leads to unfavorable Na+/Ca2\u0026thinsp;+\u0026thinsp;and Na+/K+ ratios, as well as reduced relative water content (RWC) and membrane stability index (MSI) in plants (Romero-Munar et al., 2019; Mansour and Hassan, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In a study on salt-tolerant sorghum varieties, Bavei et al. (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) observed a decrease in fresh weight, higher ion accumulations, proline content, and peroxidase activity. In another study with two Kenyan grain sorghum varieties [\u003cem\u003eS. bicolor\u003c/em\u003e (L.) Moench], Serena and Seredo, Netondo et al. (\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2004a\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003eb\u003c/span\u003e) found that the 250 mM NaCl treatment significantly reduced the relative shoot growth rates by 75 and 73%, respectively, and stem dry weight by 75 and 53%. It also strongly inhibited the accumulation of K\u003csup\u003e+\u003c/sup\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e in the roots, stems, and leaves. They noted that Chlorophyll a and b, net CO\u003csub\u003e2\u003c/sub\u003e assimilation, stomatal conductance, and transpiration rate decreased significantly with the increase in salinity. It also affects AMF infectivity, colonisation, spore density, spore germination, and hyphal development (Evelin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Porcel et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Barajas Gonz\u0026aacute;lez et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, AMF ameliorated the deleterious effects of salt stress and also enhanced biomass in maize (Romero-Munar et al., 2019; Huang, 2024) and millet (Umashankar et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) compared to non-mycorrhizal plants. Similar to the above observations, the present study also revealed that salt stress, specifically 150 mM NaCl (EC 7.32 dS/m), increased sorghum plant AMF colonization and increased vesicle and arbuscle formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). However, under 300 mM NaCl stress (EC 12.03 dS/m), hyphal growth, arbuscular formation, and total colonization decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe extent of stomatal opening is significant for CO\u003csub\u003e2\u003c/sub\u003e uptake and photosynthesis per unit leaf area (Qin et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The lower chlorophyll content and stomatal opening under stress conditions indicate the detrimental effects of stress factors on the photosynthetic efficiency of the plants. It has been reported that lipid peroxidation and reactive oxygen species (ROS) play a crucial role in damaging various photosynthetic membranes where photosynthetic pigments are bound (Kang et al., 2012; Xing et al., 2013) and in causing thylakoid swelling (Kafi, 2009; Yamane et al., 2008). Moreover, under this salinity-stress condition, chlorophyll degradation and reduced chlorophyll synthesis result in reduced chlorophyll content. While AMF colonization (Fm\u0026thinsp;+\u0026thinsp;Ri) reduces lipid peroxidation and ROS generation, it thereby achieves greater membrane stability. In addition, AMF colonization reduces Na+ uptake and increases the levels of beneficial minerals essential for chlorophyll synthesis and stability (Wang et al., 2009), thereby enhancing chlorophyll content under salt stress.\u003c/p\u003e \u003cp\u003eSalt stress depletes turgor pressure in leaves, leading to stomatal closure and thereby inhibiting plant CO2 uptake and reducing photosynthesis (Qin et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Moreover, it has been shown that, because of their similar physicochemical properties, Na+ ions compete with other minerals at their transport sites (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Hedrich and Shabala, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, it has been observed that AMF-colonised plants maintain a favourable K\u003csup\u003e+\u003c/sup\u003e/Na\u003csup\u003e+\u003c/sup\u003e ratio by minimizing Na\u003csup\u003e+\u003c/sup\u003e uptake. Thus, it protects photosynthetic tissues from inhibiting Na\u003csup\u003e+\u003c/sup\u003e uptake, thereby enhancing overall development and productivity under saline conditions (Colmer et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Munns and Tester, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Cuin et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). A similar observation was noted in the present experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In an experiment, sorghum plants colonized by \u003cem\u003eGlomus intraradices\u003c/em\u003e (Gi), \u003cem\u003eGigaspora margarita\u003c/em\u003e (Gm), or a mixture of AM species during a sustained drought following exposure to salinity treatments (NaCl stress) Cho et al. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) observed that combined NaCl and drought stress exposure, stomata of Gi plants remained open 17\u0026ndash;22% longer than in non-AM plants. They hypothesized that AMF-plants modulated cellular metabolism to produce various osmotic adjustment substances, such as proline (Pro) and total soluble solids (TSS), within cells, thereby maintaining normal cell expansion, growth, and water absorption, and ensuring osmoregulation (Liu et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The higher content of TSSs in salt-stressed AMF-plants might be due to the accumulation of osmoprotectant or to the higher synthesis of TSS from starch and sucrose (Schrader and Sauter, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Garg and Bharti, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Evelin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Mostafaie et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalinity also decreases the solubility and mobility of other micronutrients (Zn, Cu, and Fe), creating a depletion zone around the roots and thereby affecting the plant's acquisition of these micronutrients (Grattan and Grieve, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). However, AMF plants showed higher concentrations of these micronutrients than non-AMF plants (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The present study shows that the contents of minerals like 'Ca, Zn, Mn, Fe, K, and Cu in 'Fm\u0026thinsp;+\u0026thinsp;Ri' inoculated plants were increased compared to the AMF-untreated control in 0 mM NaCl stressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). At the same time, the Na\u003csup\u003e+\u003c/sup\u003e concentration decreased up to 18.26% in the 'Fm\u0026thinsp;+\u0026thinsp;Ri\u0026rsquo;-inoculated plant, up to 16.03% in the 'Fm'-inoculated plant, and decreased up to 10.81% in the 'Ri\u0026rsquo;-inoculated plant compared to the AMF-untreated control at 150 mM salt stressed condition. A greater relative N, P, Ca, Mg, Mn, and Fe absorption rate was also observed in AMF-inoculated wheat plants under salt stress than in non-AMF seedlings (Huang et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This may be attributed to the development of extraradical hyphae to reach distant areas of the rhizospheric zones, and to the upregulation of transporter gene expression for these nutrients. (Burleigh et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The qRT-PCR analysis of rice seedlings under NaCl stress, treated with AMF, showed overexpression of genes related to ion homeostasis compared with the non-inoculation treatment group (Zhang et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The mechanism of such selective enhancement of minerals in the AMF plants could be due to compartmentalizing Na\u003csup\u003e+\u003c/sup\u003e into the vacuole via up-regulation of OsNHX3 (sodium/hydrogen exchanger) and efflux of Na\u003csup\u003e+\u003c/sup\u003e from the cytosol to apoplastic spaces via higher expression of OsSOS1 (salt overly sensitive) and OsHKT2;1 (high-affinity potassium transporter) and also induce more NHXs (vacuolar Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e antiporters present in roots and leaves) that help sequester Na\u003csup\u003e+\u003c/sup\u003e in the vacuole (Davenport et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Porcel et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and SOS1 (plasma membrane Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e antiporters) responsible for secretion of Na\u003csup\u003e+\u003c/sup\u003e from the cytosol beyond plasma membrane (Ol\u0026iacute;as et al., 2009; Evelin et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSoil salinisation also causes precipitation of inorganic phosphorus (P) with other cations, such as Ca2+, Mg2+, and Zn2+, depending on soil pH, thereby limiting the element's availability to plants (Wu, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Iqbal et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, AMF colonization enhances P acquisition in AMF plants either by secretion of acid and alkaline phosphatases, by forming polyphosphates inside the hyphae, or by the expression of high-affinity phosphate transporter genes (GvPT, GiPT, and GmosPT) (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This is also attributed to maintaining the integrity of the cell membrane, reducing ion leakage, and preventing the compartmentalization of toxic ions in vacuoles, thereby reducing the adverse effects of salinity (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Therefore, it is assumed that the enhanced phosphorus content in the 150 mM and 300 mM salt-stressed sorghum plants by the AMF \u003cem\u003eF. mossae\u003c/em\u003e and \u003cem\u003eR. irregularis\u003c/em\u003e may be due to one or more of these mechanisms that need to be explored.\u003c/p\u003e \u003cp\u003ePlants absorb N as nitrate (NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e) and ammonium (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) ions (Frechilla et al., 2001). However, salinity conditions interfere with their uptake by immobilizing them (Hodge and Fitter, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Miransari, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). While NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e uptake is challenged by Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e absorption faces competition from Na\u003csup\u003e+\u003c/sup\u003e at the membrane (Evelin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). A low flux of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e from soil to roots leads to reduced activity of nitrogen reductase (NR) (Hoff et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). A higher expression of nitrate (NRT1.1, NAR2.2) and ammonium transporters (AMT1.1 and AMT1.2) was observed in \u003cem\u003eTriticum aestivum\u003c/em\u003e plants colonized with \u003cem\u003eR. irregularis\u003c/em\u003e and \u003cem\u003eF. mosseae\u003c/em\u003e (Evelin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The present study also shows that N-content was higher in Fm-treated, Ri-treated, and 'Fm\u0026thinsp;+\u0026thinsp;Ri-treated plants than AMF-untreated control plants in 150 mM and 300 mM salt-stressed conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table S3). A higher N content in the AMF plants might be due to the AMF-facilitated maintenance of membrane stability and increased NR activity (Talaat and Shawky, \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The AMF exerts its beneficial stress-ameliorating effect on crops by enhancing the acquisition of several essential mineral elements (P, N, K, Ca, Mg, Zn, Fe, Mn, and Cu) and significantly reducing deleterious Na\u0026thinsp;+\u0026thinsp;ion uptake. Thus, AMF colonization helps maintain the Na+/Ca2\u0026thinsp;+\u0026thinsp;and Na+/K+ ratios, as well as physiological and biochemical changes, and the selective expression of genes (PIP, Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e antiporters, Lsnced, Lslea, and LsP5CS) (Evelin et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalt stress leads to the production of large amounts of reactive oxygen species (ROS) in plants, which can impact plant growth and development, and even result in plant death (Czarnocka and Karpiński, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The higher malondialdehyde (MDA) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) levels in plant tissues under salt stress indicate oxidative stress associated with ROS production. Antioxidant enzyme (SOD, APX, and CAT) activity is usually greater in AMF plants than in non-AMF plants (Elhindi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). A similar observation was also noted in the AM-infested plants under salt stress in this study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b, and d). This suggests that salt stress induces oxidative stress, which is mitigated by the increased activity of these ROS enzymes. The combined AMF (Fm\u0026thinsp;+\u0026thinsp;Ri) treatment in 150 mM and 300 mM salt stress conditions exhibited the highest antioxidant enzyme activity compared to the single AMF-colonized plant (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S4). The higher CAT, APX, and SOD activity associated with higher salinity stress indicates a more effective detoxification of ROS (Benav\u0026iacute;des et al., 2000). These redox enzymes stabilize subcellular components of cell membranes, such as lipids and proteins, quenching free radicals, and buffer cellular redox potential under salinity stress (Yang et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Frosi et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral studies have shown that salt stress often leads to proline accumulation (Evelin et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Yooyongwech et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Proline is a crucial osmoprotectant that maintains tissue water potential, protein integrity, and function, thereby reducing oxidative damage to cells (Parvaiz and Satyawati, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The present work also confirmed that AMF inoculation increased proline content under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e; Table S3). It has been shown that in AMP-colonized plants, Proline synthesis is enhanced due to upregulation of the delta1-pyrroline-5-carboxylate synthetase gene, which encodes the rate-limiting enzyme in proline biosynthesis, MeP5CS, under stressful conditions, with a considerable drop in its catabolism (Huang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Mansour and Ali, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, the elevated proline can scavenge ROS, stabilize DNA, proteins, and membranes, and reduce NaCl-induced enzyme denaturation (Evelin et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, it is assumed that the enhanced proline level in salt-stressed AMF-colonizing plants in this study may result from upregulated proline synthesis, providing a better tolerance mechanism against abiotic stress (Miransari, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Caruso et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). A tripartite interaction study among AMF, \u003cem\u003eR. intraradices\u003c/em\u003e, and associated bacteria (Massilia sp.) in maize demonstrated enhanced salt stress tolerance in seedlings compared with biotrophs alone (Krishnamoorthy et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Therefore, it is observed that the application of a single AMF species, alone or in combination with other AMF species or functional microorganisms, significantly affects the physiobiochemical parameters of the targeted crops and exerts a stress-ameliorating effect compared to a single species (Parvin et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current study concludes that the selected AMF species, \u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eR. intraradices\u003c/em\u003e, significantly influence the physiobiochemical parameters of the Sorghum variety \"CSV 53F\" more than the non-AMF sets. Moreover, it alleviated salt stress (150 mM and 300 mM) in sorghum plants by enhancing mineral uptake, increasing antioxidant enzyme activity, and improving physiological and biochemical parameters, thereby improving overall growth and biomass. The current study concludes that the use of a combination of AMF (\u003cem\u003eF. mosseae\u003c/em\u003e and \u003cem\u003eR. intraradices\u003c/em\u003e) is an effective biofertilizer for enhancing sorghum growth in saline agricultural areas. Furthermore, large-scale field trials and more in-depth molecular studies may explore the vivid mechanisms by which these AMF species mitigate salinity stress in Sorghum plants, thereby helping to enhance crop productivity in current adverse environmental /soil conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the West Bengal Biodiversity Board, Dept. of Environment, Govt. of West Bengal (Memo No. 437/3K(Bio)-6/2019; dated 30.06.2022).\u003c/p\u003e\n\u003ch2\u003eAuthor\u0026apos;s Contribution:\u003c/h2\u003e\n\u003cp\u003eConceptualization: Vivekananda Mandal; Methodology: Ashutosh Kundu; Formal analysis and investigation: Ashutosh Kundu and Prashanta Kumar Mitra; Writing - original draft preparation: Ashutosh Kundu; Writing - review and editing: Kiran Sunar, Vivekananda Mandal; Funding acquisition: Vivekananda Mandal; Resources: Kiran Sunar, Vivekananda Mandal; Supervision: Kiran Sunar, Vivekananda Mandal\u003c/p\u003e\n\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\n\u003cp\u003eThe authors are grateful to the West Bengal Biodiversity Board, Dept of Environment, Govt of West Bengal (Memo No. 437/3K(Bio)-6/2019; dated 30.06.2022) for the financial support to carry out the study. The authors are also grateful to the WB DST-BT-supported BOOST program 2017\u0026ndash;2018 (\u003cem\u003evide\u003c/em\u003e Ref. No. 1089/BT(Estt)/1P-07/2018; dated 24.01.2019) for the equipment grant to the department. We are also grateful to Dr Malay Das, Department of Biological Sciences, Division of Botany, Presidency University, 86/1 College Street, Kolkata-700073, West Bengal, India, for providing infrastructural support to measure the soil salinity, TDS and electrical conductivity (EC) by PCSTestr 35 (Eutech PCSTEST35-01X441506/Oakton 35425-10) and chlorophyll meter SPAD-502Plus machines. We are also grateful to Sri Arup Mandal, Research Scholar, and Dr Dipak Nayak, Sr. Scientist \u0026amp; I/c RRS (Hort.) Fruit Science, ICAR-CISH Regional Research Station, Malda, W.B., for providing the facilities to analyze the minerals using Flame Photometer and AAS.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdejumobi MA, Awe GO, Abegunrin TP, Oyetunji OM, \u0026amp; Kareem TS (2016). Effect of irrigation on soil health: a case study of the Ikere irrigation project in Oyo State, southwest Nigeria. Environmental monitoring and assessment, 188(12), 696. https://doi.org/10.1007/s10661-016-5628-1\u003c/li\u003e\n \u003cli\u003eAebi, H (1984). Oxygen Radicals in Biological Systems. 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Biochem J 57(3):508. https://doi.org/10.1042%2Fbj0570508\u003c/li\u003e\n \u003cli\u003eYooyongwech S, Phaukinsang N, Cha-Um S, Supaibulwatana K (2013) Arbuscular mycorrhiza improved growth performance in \u003cem\u003eMacadamia tetraphylla\u003c/em\u003e L. Grown under water deficit stress involves soluble sugar and proline accumulation. Plant Growth Regul. 69: 285\u0026ndash;293. https://doi.org/ 10.1007/s10725-012-9771-6 s\u003c/li\u003e\n \u003cli\u003eZhang B, Shi F, Zheng X, Pan H, Wen Y, Song F (2023) Effects of AMF compound inoculants on growth, ion homeostasis, and salt tolerance-related gene expression in \u003cem\u003eOryza sativa\u003c/em\u003e L. under salt treatments. Rice. 16(1):18.https://doi.org/10.1186/s12284-023-00635-2\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Salt stress tolerance, Sorghum bicolor, consortium, Funneliformis mosseae, Rhizophagus intraradices, Antioxidant enzymes","lastPublishedDoi":"10.21203/rs.3.rs-8810660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8810660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Aims:\u003c/h2\u003e \u003cp\u003eSalt stress is one of the most significant environmental constraints to agriculture, resulting in decreased crop productivity. The study aims to investigate the salt-stress alleviation effects of two potential Arbuscular Mycorrhizal Fungi (AMF), \u003cem\u003eviz\u003c/em\u003e. \u003cem\u003eFunneliformis mosseae\u003c/em\u003e and \u003cem\u003eRhizophagus intraradices\u003c/em\u003e, on the growth and metabolic changes of Sorghum plants.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA pot experiment was conducted under greenhouse conditions for 120 days in two different salt stress conditions (NaCl at 150 mM, EC 7.32 dS/m, and 300 mM, EC 12.03 dS/m) with/without AMF species, either alone or in combination. The AMF colonization and biochemical parameters were estimated at 30-day intervals.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe total biomass, chlorophyll, carbohydrate, phosphate, nitrate, and proline contents increased in combined AMF-colonized plants exposed to 150 mM NaCl stress compared to the AMF-untreated control. In contrast, 300 mM salt stress significantly reduced AMF colonization and plant growth parameters. In 150 mM NaCl-stressed plants, the combined AMF-treated plants exhibited higher SOD, CAT, and APX activity compared to the AMF-untreated control plants, and lowered MDA and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e contents. The essential mineral contents were increased, while the uptake of Na+ ions was decreased.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe study concludes that dual-species AMF-treated plants (Fm\u0026thinsp;+\u0026thinsp;Ri) ameliorated salt stress more effectively by modulating ion uptake, enhancing stress defense enzymes, and minimizing redox factors, compared with salt-stressed AMF-untreated control plants and single-species AMF inoculations, resulting in a collective improvement in plant growth parameters. Thus, a consortium of AMF species (\u003cem\u003eR. intraradices\u003c/em\u003e and \u003cem\u003eF. mosseae\u003c/em\u003e) could be an efficient ameliorator of salt stress in sorghum cultivation.\u003c/p\u003e","manuscriptTitle":"Effect of mono- or bi-species Arbuscular mycorrhizal symbioses in improving the salt stress tolerance in Sorghum bicolor (L.) Moench.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-19 12:24:43","doi":"10.21203/rs.3.rs-8810660/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b4aa877b-8289-43ee-a234-baef635aab1e","owner":[],"postedDate":"February 19th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-31T21:12:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-19 12:24:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8810660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8810660","identity":"rs-8810660","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}