Synergistic effects of magnesium nano-oxide and mycorrhizal fungi on metabolite profiles in sesame (Sesamum indicum) under varying water regimes

Abstract

This study aimed to evaluate the effects of nano magnesium oxide (MgO NPs, OM2) and mycorrhizal fungi species, Funneliformis mosseae (MI2) and Rhizophagus irregularis (MI3), on sesame plants under different environmental conditions. This study was conducted using a factorial field experiment with three replicates over two years (2023–2024) across four environments conditions with varying irrigation levels (optimal and deficit) and rainfall patterns The results showed that OM2 significantly enhanced antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT), by approximately 29% and 32%, respectively, as well as increased primary metabolites such as soluble sugars (CBD) and proline (PRN). Secondary metabolites like phenolic compounds (PNL) and anthocyanins (ANT) were also elevated, contributing to improved stress tolerance.The mycorrhizal fungi (MI2 and MI3) facilitated nutrient uptake and enhanced root development, particularly under water-limited conditions. In environments with optimal irrigation and high rainfall, the OM2MI2 treatment yielded the highest grain production (1117.52 kg ha^-1, a 29% increase over the control), while under water-limited conditions, OM2MI2 achieved 795.76 kg ha^-1 (28% higher than the control). The results indicated that OM2 significantly boosted antioxidant enzyme activities (SOD and CAT activities increased by approximately 29% and 32%, respectively, as well as increased primary metabolites such as soluble sugars (CBD) and proline (PRN). Secondary metabolites like phenolic compounds (PNL) and anthocyanins (ANT) were also elevated, contributing to improved stress tolerance The biplot results showed that OM2 and mycorrhizal fungi (MI2) had high yield effects in different environments studied, and had the same position in the two-dimensional principal component diagram. Also, the cluster analysis dendrograms showed that the treatments were grouped together (the group that had the highest yield). Cluster and biplot analyses confirmed that OM2 and mycorrhizal fungi (MI2) effectively enhanced both primary and secondary metabolism, supporting increased stress resistance in sesame plants. This study emphasizes the potential benefits of combining nano magnesium oxide with mycorrhizal fungi to enhance sesame productivity and quality under diverse environmental challenges.

Introduction

Sesame (Sesamum indicum L.) is cultivated on approximately 7.9 million hectares worldwide, producing around 4 million tons annually¹. Iran contributes about 28,000 tons from 40,000 hectares¹. Sesame seeds are highly valued for their rich oil content, which constitutes 50–60% of the seed and contains high levels of oleic acid (47%), linoleic acid (39%), and omega-6 fatty acids. This makes sesame an essential resource for edible, cosmetic, and health industries². Given its economic and nutritional importance, optimizing sesame cultivation practices, especially in regions facing environmental stressors, is critical. Sesame is predominantly grown in arid and semi-arid regions, where limited water resources, irregular rainfall, and high temperatures pose significant challenges³. Water scarcity, which is increasingly recognized as a major issue for agricultural productivity, particularly in Iran and other similar regions⁴. Water restriction is one of the most severe abiotic stressors affecting crop yields, reducing growth, photosynthesis, and overall yield^5,6. The impact of water stress on sesame includes reduced seed yields and altered physiological processes, with plants often exhibiting increased antioxidant activity as a stress response, which can influence seed and oil quality⁵.

Abiotic stresses such as drought, temperature extremes, and nutrient imbalance disrupt vital physiological processes in plants, including photosynthesis, respiration, and cell division, ultimately reducing growth and yield^7,8,9. These stressors can also impair water and nutrient uptake, causing osmotic stress and cellular damage¹⁰. At the biochemical level, abiotic stress leads to the overproduction of reactive oxygen species (ROS), resulting in oxidative stress and lipid peroxidation. Markers like hydrogen peroxide and malondialdehyde (MDA) significantly increase under severe conditions, indicating cellular damage^11,12. To cope with stress, plants accumulate osmolytes such as proline and activate antioxidant systems, including enzymes like SOD, CAT, and APX^7,13,14. These responses help maintain osmotic balance and protect cells from oxidative injury¹⁵.

To mitigate water restriction, various strategies have been explored, including physiological, molecular, and genetic approaches. Nonetheless, these strategies often involve high costs and long-term implementation¹⁶. Deficit irrigation, a water-saving strategy involving reduced water supply compared to the plant’s full requirement, can be effective if properly implemented. Improper use, however, may worsen water stress¹⁷. The physiological link between deficit irrigation and magnesium deficiency is well-established. Beyond water scarcity, drought impairs root function and ion transport, creating a hidden nutritional bottleneck especially for magnesium, a vital element in chlorophyll synthesis and stress-response enzymes. Water restriction significantly affects magnesium uptake from the soil, leading to deficiencies that exacerbate the negative effects of water limitation. Magnesium-deficient plants under water stress show reduced CO₂ assimilation and energy consumption, resulting in excess energy within the photosystem^18,19. Magnesium enhances plant tolerance to water stress by promoting root growth and nutrient/water uptake^20,21, sustaining photosynthesis and reducing oxidative damage. As the second most abundant cellular cation, it activates key enzymes like Rubisco, PEP carboxylase, and ATPases^22,23. Deficiency severely limits growth and yield^4,24,25. Magnesium also regulates metabolic pathways for defense-related secondary metabolites, including phenolic compounds^10,26,27. by increasing endogenous organic acid levels in root tissues, supporting osmotic balance and nutrient availability^26,27.

Magnesium oxide nanoparticles (MgO NPs) have shown promise in agricultural applications, particularly in improving plant defense mechanisms and stress responses²⁸. MgO NPs enhance enzyme activities related to defense, such as phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and superoxide dismutase (SOD), while increasing salicylic acid content, which is crucial for plant defense responses²⁹. Nano-fertilizers, including magnesium nanoparticles, have shown potential to improve plant tolerance to abiotic stress via physiological and metabolic modulation^30,31,32,33. Studies indicate that magnesium nanofertilizers can improve sesame growth under water restriction⁴. Due to their higher surface-to-volume ratio, MgO nanoparticles have greater solubility, reactivity, and mobility across plant cell membranes than bulk forms, potentially improving the transport of magnesium, an essential element for chlorophyll synthesis, enzyme activation, and photosynthetic efficiency. In addition, nano-MgO has unique effects at low concentrations, such as growth stimulation or stress reduction, whereas conventional MgO often requires larger amounts for application and has slower dissolution kinetics, which can lead to nutrient leaching or fixation in the soil.

Arbuscular mycorrhizal fungi (AMF) play a vital role in enhancing plant nutrient uptake, especially phosphorus, under stress conditions. Inoculation with AMF, such as Funneliformis mosseae, can increase sesame seed yield by up to 115%, oil yield by 55%, and protein yield under severe water stress³⁴. Mycorrhizal symbiosis reprograms primary metabolic pathways, impacting carbon metabolism by increasing photosynthetic activity and sugar transport to the roots³⁵. It also affects amino acid composition and the tricarboxylic acid cycle, leading to increased organic acid production, which supports enhanced nutrient uptake and stress tolerance^36,37. Similar to AMF and nanoparticles, biochar has also been found to enhance crop stress tolerance via soil-plant interaction improvement³⁸. Moreover, mycorrhizal colonization induces changes in plant secondary metabolism, activating multiple biosynthetic pathways and altering the production of secondary metabolites. These changes depend on the specific fungal species involved, with some increasing phenolic acid levels and others reducing them^39,40. In medicinal plants, AMF colonization can significantly influence the production of bioactive compounds at different growth stages⁴¹. Microbial fertilizers, including arbuscular mycorrhizal fungi (AMF), have also been shown to modulate primary and secondary metabolism under abiotic stress⁴². Recent findings also showed ploidy-related differences in drought response, reflecting genotype-specific metabolic adaptation⁴³.Despite extensive research on mycorrhizal fungi and nano-fertilizers, there is a lack of comprehensive studies on the combined effects of magnesium nanooxide and mycorrhizal fungi in mitigating deficit irrigation stress in sesame.

The selection of Magnesium Nano-Oxide (MgO NPs) and Arbuscular Mycorrhizal Fungi (AMF) as drought-mitigating treatments is scientifically essential due to their complementary, synergistic roles in enhancing plant resilience under water deficit. MgO NPs, owing to their nano-scale bioavailability, ensure sustained chlorophyll synthesis, photosynthetic efficiency, osmotic adjustment, and antioxidant defense by delivering magnesiumdirectly to cellular sites even under limited water uptake, while AMF extend the root system via hyphal networks to improve water and nutrient (especially phosphorus) acquisition from drought-affected soils, modulate phytohormonal balance, enhance soil structure through glomalin secretion, and prime antioxidant systems for oxidative stress mitigation; together, they form a holistic strategy that simultaneously targets internal physiological optimization (via MgO NPs) and external resource mobilization (via AMF), making their combined application not merely advantageous but necessary to comprehensively address the multifaceted nature of drought stressthereby offering a sustainable, nano-bio integrated solution for improving crop performance under increasingly prevalent arid conditions. Haghaninia et al., (2025) examined the combined effects of AMF and nanoparticles on reducing drought stress in oilseed crops that support this integrated approach.

However, no study has simultaneously investigated the effects of MgO nanoparticles, conventional MgO, and two AMF species on primary and secondary metabolites in sesame under different irrigation regimes.

This study aims to evaluate the combined effects of nano magnesium oxide (MgO NPs), conventional magnesium oxide, and two mycorrhizal fungi species (Funneliformis mosseae and Rhizophagus irregularis) on sesame grain yield, primary metabolites (soluble sugars, proline), and secondary metabolites (phenolic compounds, anthocyanins) under optimal and deficit irrigation conditions. By investigating these interactions across four environmental conditions, this research seeks to identify sustainable strategies for enhancing sesame productivity and stress tolerance in water-limited environments.

Materials and methods

Permission to collect plant material

The sesame cultivar Oltan was chosen for this study, which investigated three experimental factors. The Oltan cultivar was chosen due to its widespread use in Iran, adaptability to semi-arid conditions, and high oil content, making it representative for studying stress responses (supported by Ghasemi et al., 2023)³. The identity of the sesame cultivar was verified by a specialist botanist affiliated with Shahrood University. All local and national regulations were strictly followed.

Plant culture

A field experiment was conducted as a factorial design with three replicates over two consecutive agricultural years (2023–2024) at the research facility of Gonbad Kavous University’s Faculty of Agriculture and Natural Resources (55°21′E, 37°26′N, 45 m elevation). Before planting, soil samples were collected and analyzed for physical and chemical properties, including magnesium content (Table 1).

Table 1 Soil characteristics of Gonbad Kavous university farm in the range of test.

• 1. Irrigation regime: Two levels- optimal irrigation (8-day interval) and deficit irrigation (16-day interval). The optimal (8-day interval) and deficit (16-day interval) irrigation regimes were selected based on local agricultural practices in Iran and previous studies on sesame water requirements, which indicate that 16-day intervals induce moderate drought stress without complete crop failure (Liu et al. (2018)⁴⁴.

• 2. Magnesium oxide application: Three levels- control (no spray), nano magnesium oxide (2%), and conventional magnesium oxide (2%). Mg concentrations (2%): The 2% concentration for both nano and conventional MgO was chosen based on prior studies (e.g., Khordadi Varamin et al. (2018)⁴ demonstrating that this level effectively enhances sesame growth and stress tolerance without causing toxicity. MgO treatments (nano and conventional) were applied as foliar sprays at 2% concentration once at the flowering stage using a handheld sprayer, applying approximately 400 L ha⁻¹ in the late evening to avoid leaf scorching.

• 3. Mycorrhizal fungi: Three levels- control, Funneliformis mosseae (formerly Glomus mosseae), and Rhizophagus irregularis (formerly Glomus intraradices). These AMF species were selected due to their proven efficacy in enhancing nutrient uptake and drought tolerance in sesame and other crops, as reported by Gholinezhad and Darvishzadeh (2019)⁴⁵ and Wu et al. (2021)⁴⁰.

These factors resulted in a total of 54 experimental plots. Fertilizer application rates were determined based on soil analysis results. Field operations, including plowing, discing, leveling, and plot demarcation, were completed in May, followed by planting in late May. Each plot consisted of five 3-m rows spaced 40 cm apart, with a planting depth of 2 cm and a target density of 50 plants per square meter. A 1-meter buffer zone was maintained between plots. Irrigation was uniformly applied via drip systems until the plants reached the 6-leaf stage, after which deficit irrigation treatments were introduced. Water volumes were recorded using temporary meters, and soil moisture was monitored weekly. Weed, pest, and disease management practices were consistently applied across all plots.

Treatment implementation

Treatment application

Commercial mycorrhizal fungi were applied pre-planting by placing spores into 7 cm-deep furrows, covering them with 3 cm of soil, and then placing the seeds on top. Each treatment received 7 g of inoculated soil containing ~ 70 spores/g, placed in 7 cm-deep furrows before sowing seeds. Initial colonization success was confirmed microscopically 21 days post-sowing in randomly sampled roots. The mycorrhiza was sourced from Turan Shahrood Biotechnology Company, Shahrood, Iran. Both nano and conventional MgO were sprayed at the onset of flowering (during sunset to prevent leaf burn). Nano-MgO was purchased from Iranian Nanomaterials Pioneers Company. The mean particle size (~ 40 nm) and morphology were confirmed via SEM imaging. Purity (> 99%) and crystalline structure were verified by supplier data and independent SEM and XRD analyses.

Irrigation scheduling treatments involved fixed intervals of 8 days (optimal) and 16 days (deficit) irrigation cycles. Soil moisture content before and after irrigation was measured using time-domain reflectometry (TDR) 24–72 h post-irrigation. These measurements were used to calculate irrigation water requirements and soil water storage in the root zone. Water volumes applied were monitored using flume-type flowmeters installed at field inlets, ensuring precise quantification of irrigation inputs and soil water dynamics⁴⁶.

Harvesting and sampling

To assess photosynthetic pigments, physiological traits, and antioxidant enzyme activities in the leaves, samples were collected from young leaves at the seed-filling stage, 71 days after planting. For grain yield measurement, all plants within a 1-square-meter area from the center of each plot were harvested at maturity. Grain yield was then calculated in kg ha^{− 1}. To investigate mycorrhizal symbiosis and determine the percentage of sesame root colonization, root staining was performed using the Phillips and Hayman (1970)⁴⁷ method. Root colonization was determined microscopically after staining using the Phillips and Hayman (1970) method. The percentage of root colonization was calculated using the gridline intersect method.

Measurement of antioxidant enzymes

Anthocyanin measurement (ANT)

Fresh tissue (0.1 g) was homogenized in acidic methanol (99:1 methanol: HCl ), centrifuged (4,000 rpm, 10 min), and absorbance at 550 nm was recorded⁴⁸. Anthocyanin concentration was calculated using ε = 33,000 cm⁻¹ mol⁻¹.

Proline content (PRN)

In this study, proline content was determined following the method established by Bates et al. (1973)⁴⁹. Initially, 0.1 g of terminal leaf tissue was homogenized in 2 mL of 3% sulfosalicylic acid, followed by centrifugation at 10,000 g for 15 min. Subsequently, 2 mL of the resultant supernatant was added to 2 mL of ninhydrin reagent (prepared by dissolving 1.25 g of ninhydrin in a mixture of 30 mL of glacial acetic acid and 20 mL of 6 M phosphoric acid) and 2 mL of glacial acetic acid. The mixture was heated in a water bath at 100 °C for 1 h, then promptly cooled on ice. To each sample, 4 mL of toluene was added, followed by vortexing for 30 s. After phase separation, the absorbance of the upper toluene phase was measured at 520 nm using a spectrophotometer. Proline content was determined from a standard curve prepared with proline standard solutions ranging from 0 to 60 mg L⁻¹.

Soluble sugars (CBD)

Ethanol-extracted sugars were quantified using the phenol-sulfuric acid method⁵⁰. Absorbance at 485 nm was converted to mg glucose g⁻¹ dry weight via standard curve.

Phenolic compounds (PNL)

Ethanol extracts were reacted with Folin-Ciocalteu reagent⁵¹, and absorbance at 650 nm was expressed as mg gallic acid equivalent per 100 g fresh weight.

Superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), Guaiacol peroxidase (GPX) and polyphenol oxidase (POX)

Radical scavenging capacity was calculated using Equation⁵².

I (%) = 100 × (Ao- As) / A0 equation. Here Ao is the absorbance of the control (containing all reactive components without the sample) and As is the absorbance of the sample. The activity of SOD was measured via inhibition of NBT reduction under light⁵³. Absorbance at 560 nm inversely correlated with enzyme activity. The activity of the CAT was measured at a temperature of 25 °C using a spectrophotometer. For this purpose, H₂O₂ decomposition was monitored at 240 nm⁵⁴. APX activity was assayed at 290 nm⁵⁵. The reaction mixture contained 500 µl of 0.1 mM EDTA, 2100 µl of 50 mM phosphate buffer with pH 7, 350 µl of 0.5 mM ascorbic acid, 4 µl of 30% H2O2, and 100 µl of enzyme extract. The activity of the GPX in leaf tissue was measured using the method of Dionisio-Sese and Tobita (1984)⁵⁶. The activity of the GPX was calculated based on the amount of absorption of the orange compound tetraguaiacol per milligram of protein concentration. The activity of the POX in leaf tissue was performed according to the method of Pizzocaro et al. (1993)⁵⁷. The activity of this enzyme was measured at 25 °C by injecting 0.5 ml of the enzyme extract into 2.5 ml of catechol (50 mM). The curve of changes in absorbance at a wavelength of 420 nm was read for two minutes. Total protein was quantified via Bradford method (1976)⁵⁸.

Root colonization measurement (CPR)

To investigate mycorrhizal symbiosis and determine the percentage of sesame root colonization, root staining was performed according to the Phillips and Hyman method (1970)⁴⁷.

Determination of yield

At harvest, seed yield (GRY), biological yield, and yield components (branches/plant, capsules/plant, seeds/capsule, 1000-seed weight) were measured from 1 m² subplots.

Statistical analysis

Bartlett’s test is a statistical procedure designed to assess the homogeneity of variances across different populations. It operates under the null hypothesis that these variances are equal, making it particularly relevant in contexts where such an assumption is critical, such as in ANOVA and regression analyses. The test is derived from the likelihood ratio test and is most effective when the data follows a normal distribution. Bartlett’s test is essential for validating the assumption of equal variances before conducting ANOVA, ensuring the reliability of the results, Data were tested for violation of assumptions underlying the ANOVA^59,60. Data for the two years was tested for homogeneity using Bartlett’s (1937)⁶¹ test of homogeneity and it was found to be hetrogeneous so the data were separate for analysis⁶¹.

MANOVA was performed to assess multivariate treatment effects. MANOVA is a statistical technique used to test whether there are significant differences between two or more groups on multiple dependent variables in cluster analysis, in this method, ratio of variance between groups and within groups calculated. This ratio helps decide how many clusters ideally, the number that maximizes separation between groups while minimizing variation within each group. A high between-group variance relative to within-group variance suggests that the clusters are well-separated and distinct. Cluster analysis doesn’t automatically tell you the “best” number of clusters. So, researchers try different numbers of clusters and use statistical tools like MANOVA and variance ratios to evaluate which number produces the most meaningful and distinct groups. After MANOVA shows that there are significant differences among groups, the LSD post-hoc test is used to compare all possible pairs of group means to see exactly which groups differ from each other. All analyses were performed with XLSTAT software.

Graphical illustrations

Prior to conducting multivariate statistical analyses, all measured variables were standardized to ensure comparability across different scales and units of measurement. Standardization was performed by transforming each variable to have a mean of zero and a unit standard deviation z = - $\:\frac{\text{X}-\stackrel{-}{\text{X}}}{\text{S}}$, thereby eliminating the influence of heterogeneous variances and measurement units on subsequent analyses. Principal Component Analysis (PCA) was then applied to the standardized dataset using the variance–covariance matrix as the basis for eigen-decomposition. Although the use of the correlation matrix is common in PCA when variables are on disparate scales, the explicit standardization of the data rendered the variance–covariance matrix mathematically equivalent to the correlation matrix, ensuring that the principal components reflected the underlying correlation structure among variables rather than artifacts of scale. Pairwise Pearson product-moment correlation coefficients were computed to quantify the strength and direction of linear relationships among all measured variables. These correlations provided complementary insight into variable interdependencies and aided in the interpretation of PCA loadings and clustering patterns. To visualize multivariate patterns across experimental treatments, three complementary graphical outputs were generated: (i) heatmaps depicting the magnitude and direction of standardized variable values across all samples, with hierarchical clustering applied to both rows (variables) and columns (samples) using Euclidean distance and Ward’s linkage criterion; (ii) dendrograms derived from agglomerative hierarchical cluster analysis based on the same distance and linkage metrics, illustrating the degree of similarity among treatment replicates; and (iii) PCA biplots, which simultaneously display the positions of sample points (scores) and variable vectors (loadings) in the reduced-dimensional space defined by the first two principal components, thereby facilitating the identification of variables that contribute most to treatment differentiation.

All multivariate analyses including data standardization, PCA, correlation computation, hierarchical clustering, and graphical visualization were performed using XLSTAT (version 2023.4, Addinsoft, Paris, France), with each treatment represented by three independent biological replicates to account for within-treatment variability and enhance the robustness of statistical inference⁶².

Result and discussion

A combined analysis was initially conducted for all the studied traits. Bartlett’s test revealed that the variances of experimental errors were not uniform. Additionally, the interaction between year and treatment was significant for the studied traits. As a result, the analysis was carried out separately for each year. Further analysis of each year showed that the interaction between different irrigation managements and the treatments was significant in each case. Upon examining the meteorological conditions (Table 2), it was determined that the non-uniformity of error variances was due to the combination of irrigation type and rainfall amount. Consequently, the experimental environment was divided into four sections based on the cultivation conditions, as outlined in Table 3.⁶³

Table 2 Temperature and precipitation statistics in the test years.

Table 3 General experimental environment based on cultivation conditions.

Analysis of variance

Magnesium oxide (MgO) treatments significantly impacted grain yield across all environments (p < 0.01), highlighting the essential role of magnesium in plant physiology. Beyond its well-documented function in photosynthesis, magnesium acts as a cofactor for over 300 enzymes involved in various metabolic pathways, enhancing plant performance under diverse environmental conditions²⁴. PRN accumulation, a key indicator of osmotic adjustment under stress, was significantly affected by MgO treatments in environments E2 and E3 (p < 0.05 or p < 0.01). Higher proline levels were observed under OM2 and OM3 treatments, suggesting their efficacy in mitigating stress through osmotic regulation. Similarly, CBD content increased significantly under OM2 and OM3 treatments (p < 0.01) in most environments, further supporting their role in stress adaptation (Table 4). These physiological enhancements align with the findings of Ali et al. 45 who demonstrated that MgO-NPs not only contribute to chlorophyll synthesis and stomatal regulation but also activate metabolic pathways associated with drought and salinity tolerance in various crops⁴⁴.

MgO treatments also boosted the activity of antioxidant enzymes, including POX, SOD, CAT, APX, and GPX, across all environments (p < 0.01). One of the key mechanisms by which magnesium oxide nanoparticles (MgO-NPs) protect plants is by upregulating antioxidant enzyme activities (Table 4). Research has demonstrated that MgO-NPs can significantly increase the activities of key antioxidant enzymes, such as SOD and CAT, with reported increases of 29% and 32%, respectively, under stress conditions^45,64. These findings highlight the potential of MgO-NPs in enhancing plant resilience to abiotic stress. Such upregulation plays a central role in reactive oxygen species (ROS) detoxification, particularly under water-deficit scenarios, as noted by Sheteiwy et al.⁶⁵ who observed similar trends in chickpea and ryegrass under metal-induced oxidative stress.

In addition to MgO treatments, mycorrhizal fungi (MF) played a crucial role in enhancing plant physiology and stress tolerance. Specifically, Funneliformis mosseae (MI2) and Rhizophagus irregularis (MI3) significantly affected root colonization, phenolic compound content (p < 0.01), anthocyanin production (p < 0.01), and antioxidant enzyme activities (Table 4). Mycorrhizal fungi demonstrated a consistent and significant impact on root colonization across all conditions, aligning with findings by Gholinejad and Darvishzadeh (2021)⁶⁶ in sesame under varied treatments. By enhancing the antioxidant defense system, mycorrhizal fungi help plants manage oxidative stress induced by drought⁶⁷. This system effectively eliminates harmful reactive oxygen species (ROS) and maintains cellular integrity during drought stress⁶⁸. This can be explained by improved photosynthesis as well as increased osmoregulation of the protectors, namely the sucrose and proline biosynthesis pathways⁶⁵. Moreover, the ability of AMF to modulate phytohormone levels such as abscisic acid and jasmonic acid has been linked to improved drought sensing and signal transduction, enhancing overall stress adaptation capacity, as reported in recent meta-analyses.

The interaction between MgO and MF treatments was significant for several key traits, including GRY, PRN, CBD, ANT, PNL, and antioxidant enzyme activities (p < 0.01 or p < 0.05) (Table 4). Factorial analysis of variance revealed significant interactions between magnesium oxide treatments, mycorrhizal fungi types, and planting environments, highlighting their combined influence on sesame growth and physiology. Nano magnesium oxide (OM2) and mycorrhizal fungi (MI2 and MI3) emerged as particularly effective tools for enhancing crop performance under both optimal and water-stressed conditions. These findings provide valuable insights into the synergistic effects of combining MgO and MF treatments, offering a promising approach to optimizing sesame cultivation practices and improving food security in the context of climate change. These results corroborate earlier studies indicating that integrated nutrient and microbial management can enhance physiological robustness in arid and semi-arid agriculture. Notably, Sheteiwy et al.⁴² observed improved seed quality and biochemical stability in Guar when microbial fertilizers were combined with macro-nutrient treatments.

Studies have consistently shown that combining multiple stress-mitigation strategies, such as MgO supplementation and mycorrhizal fungi inoculation, is more effective in minimizing the adverse impacts of stress on crop production and quality compared to single interventions⁶⁹. The integration of arbuscular mycorrhizal fungi (AMF) and magnesium supplements represents a sustainable and effective strategy for improving plant performance under drought conditions⁷⁰. This integrative approach aligns with global strategies promoting nature-based solutions for sustainable intensification, where the combined use of nanotechnology and beneficial microbes is recognized as a frontier in resilient crop production systems. By leveraging these synergistic interactions, farmers can enhance crop resilience and productivity, contributing to global efforts to address food security challenges in an era of changing climatic conditions.

Compare means

In the first environment (E1), characterized by optimal irrigation and high rainfall, nano magnesium oxide (OM2) and conventional magnesium oxide (OM3) significantly outperformed the no-spray treatment (OM1) in terms of grain yield. Specifically, OM2 yielded 1077.64 kg/ha, and OM3 yielded 1082.55 kg/ha, compared to OM1’s 866.69 kg/ha (Table 5). This improvement can be attributed to the enhanced nutrient absorption and strengthened defense systems facilitated by magnesium oxide foliar spraying⁷¹. Enzymatic activities, including APX, CAT, SOD and POX, were notably higher in OM2 and OM3 compared to OM1. Additionally, secondary metabolites such as PNL and ANT, as well as CBD, demonstrated superior performance under OM2 and OM3 treatments.

Mycorrhizal fungi (MI2 and MI3) also played a significant role in improving CPR and GRY compared to the no-fungi treatment (MI1). MI2, in particular, achieved the highest root colonization rate (32.55%) and grain yield (1041.16 kg/ha) (Table 5). The combination of nano magnesium oxide (OM2) and conventional magnesium oxide (OM3) with mycorrhizal fungi enhanced enzyme activities and secondary metabolism, contributing to higher grain yields even under optimal conditions. This suggests that these treatments support both primary and secondary metabolic processes, even in the absence of significant water stress. Mycorrhizal fungi enhance nutrient acquisition and water use efficiency through mechanisms such as increased water uptake via enhanced root systems and aquaporins, regulation of transpiration through abscisic acid-mediated stomatal control, and improved stress resistance through biochemical pathways⁷². However, under optimal irrigation and high rainfall, the effects of mycorrhizal fungi were less pronounced, as plants were able to allocate resources more efficiently toward growth and metabolism. While both nano magnesium oxide and mycorrhizal fungi enhance productivity under these conditions, their benefits appear complementary rather than synergistic. This is consistent with findings by Sheteiwy et al.⁶⁵, who reported that under non-stress conditions, AMF improved physiological traits but did not strongly amplify yield unless combined with a limiting factor such as drought or salinity.

In the second environment (E2), characterized by low irrigation and high rainfall, grain yields dropped significantly compared to E1. Despite this decline, OM2 remained the most effective treatment, yielding 767.39 kg/ha, followed by OM3 (750.68 kg/ha) and OM1 (619.38 kg/ha) (Table 5). This reduction in yield aligns with findings by Kouighat et al. (2023)⁷³, who reported decreased sesame GRY under drought stress conditions. Enzymatic activities, including GPX, APX, CAT, SOD, and POX, were markedly higher in OM2 and OM3, reflecting their critical role in stress tolerance⁷¹.

Mycorrhizal fungi (MI2 and MI3) maintained relatively high grain yields under low irrigation, with MI2 yielding 743.75 kg/ha and MI3 yielding 727.64 kg/ha (Table 5). Root colonization (CPR) was significantly higher in MI2 and MI3 compared to MI1, indicating enhanced nutrient uptake under water-limited conditions. Arbuscular mycorrhizal fungi (AMF) employ several mechanisms to improve plant drought tolerance, including enhanced hydraulic conductivity and water transport. Fungal hyphae can access water in small soil pores beyond the reach of root hairs, increasing root hydraulic conductivity by two to three times compared to non-mycorrhizal plants⁷⁴. Remarkably, AMF can access water below the permanent wilting point, further supporting plant survival under drought conditions⁷⁵. Similar conclusions were drawn by Ali et al.⁴⁴ who emphasized that MgO-NPs activate ROS-scavenging enzymes and modulate hormonal signaling, enhancing drought resilience in crops. Moreover, the partial overlap between the AMF and MgO mediated pathways suggests a potential for additive or synergistic effects, especially under moderate water stress, as supported by previous studies in legumes and cereals.

Nano magnesium oxide (OM2) continued to excel in promoting enzymatic activities and grain yield under low irrigation, highlighting its effectiveness in mitigating oxidative stress caused by water scarcity. Mycorrhizal fungi (MI2 and MI3) played a crucial role in maintaining productivity by improving root colonization and nutrient acquisition, thereby compensating for the lack of water. Low irrigation introduces mild water stress, which triggers antioxidant defenses and underscores the importance of mycorrhizal associations. The combination of AMF and magnesium supplements represents a sustainable and effective strategy for improving plant performance under drought conditions⁷⁰. Together, these treatments work synergistically to maintain plant health and productivity, offering valuable insights for optimizing crop cultivation in water-limited environments.

In the third environment (E3), characterized by optimal irrigation and very low rainfall, grain yield was highest in the conventional magnesium oxide treatment (OM3) at 1078.06 kg/ha, followed by nano magnesium oxide (OM2) at 1038.41 kg/ha and the no-spray treatment (OM1) at 841.61 kg/ha (Table 5). Elevated enzymatic activities, including APX, CAT, SOD, and POX, were observed in OM2 and OM3, underscoring their role in mitigating drought stress. Mycorrhizal fungi (MI2 and MI3) demonstrated high CPR (MI2: 33.91%, MI3: 37.03%) and grain yields comparable to the magnesium oxide treatments (MI2: 1035.63 kg/ha, MI3: 979.47 kg/ha) (Table 5). Mycorrhizal colonization induces significant changes in plant secondary metabolism, leading to both quantitative and qualitative alterations in various metabolite classes⁷⁶. Conventional magnesium oxide (OM3) outperformed nano magnesium oxide (OM2) in this environment., likely due to its sustained release characteristics under prolonged drought conditions changed to Conventional magnesium oxide (OM3) outperformed nano magnesium oxide (OM2) in this environment. Mycorrhizal fungi (MI2 and MI3) exhibited strong resilience, enhancing nutrient acquisition and root development despite very low rainfall. This supports the findings by Sheteiwy et al.⁴² where AMF was shown to significantly influence biochemical pathways, particularly under early-stage drought scenarios. Early-stage water stress, caused by very low rainfall, necessitates reliance on enzymatic defenses and mycorrhizal associations to sustain productivity, even when optimal irrigation is provided later in the growth cycle.

In the fourth environment (E4), characterized by low irrigation and very low rainfall, grain yields dropped significantly compared to other environments. OM2 performed best, yielding 770.68 kg/ha, followed by OM3 (736.06 kg/ha) and OM1 (593.52 kg/ha) (Table 5). This decline aligns with findings by Kouighat et al. (2023)⁷³, who reported reduced sesame seed yields under drought stress conditions. Enzymatic activities, including GPX, APX, CAT, SOD, and POX, were highest in OM2 and OM3, reflecting their critical role in stress management. Mycorrhizal fungi (MI2 and MI3) maintained moderate grain yields (MI2: 730.99 kg/ha, MI3: 713.59 kg/ha) despite extreme water scarcity. CPR was highest in MI3 (24.95%), highlighting its importance in resource acquisition under severe stress (Table 5). Nano magnesium oxide (OM2) remained effective in promoting enzymatic activities and grain yield, demonstrating its versatility under extreme conditions. Mycorrhizal fungi (MI2 and MI3) played a vital role in sustaining productivity by enhancing root colonization and nutrient uptake, even under severe water stress.

The combination of low irrigation and very low rainfall imposes significant water stress, forcing plants to prioritize enzymatic defenses and mycorrhizal associations for survival, often at the expense of grain yield. Nano magnesium oxide (OM2) consistently enhanced enzymatic activities (APX, CAT, SOD, POX) and GRY across all environments, demonstrating exceptional performance under both optimal and stressful conditions. Conventional magnesium oxide (OM3) showed superior performance under prolonged drought (E3), likely due to its sustained release properties. Foliar application of magnesium has been shown to increase photosynthetic efficiency and reduce the accumulation of harmful reactive oxygen species (ROS) under drought stress conditions⁷³.

The differences in sesame yield observed across the four environments can be primarily attributed to variations in soil moisture, irrigation regime, and rainfall distribution. Environment 1, characterized by optimal irrigation (8-day interval) combined with relatively high early-season rainfall (38.6 mm) and moderate rainfall later (16.9 mm), provided the most favorable moisture conditions for sesame growth, resulting in the highest yield. Conversely, Environment 4, which experienced deficit irrigation (16-day interval) along with very low early-season rainfall (0.7 mm) and relatively higher rainfall later (52.4 mm), had the lowest yield, likely due to prolonged water stress during critical early developmental stages. The intermediate yields observed in Environments 2 and 3 further confirm that both irrigation frequency and timing of rainfall events significantly influence soil water availability, ultimately affecting plant performance and productivity. These results highlight the importance of adequate and timely water supply, through both irrigation and natural precipitation, in optimizing sesame yield under varying climatic conditions.Mycorrhizal fungi (MI2 and MI3) significantly improved root colonization and nutrient acquisition under water-limited conditions (E2, E3, E4), playing a crucial role in maintaining productivity under severe water stress. Early-stage rainfall profoundly impacts germination, emergence, and seedling establishment, while later-stage rainfall influences ripening and GRY. Mycorrhizal fungi support long-term resilience by producing glomalin, a glycoprotein that improves soil structure and water-holding capacity⁷⁷. These insights align with a growing body of literature emphasizing integrated soil–plant–microbe management approaches as climate-resilient strategies in sustainable agriculture Collectively, these results underscore the value of combining nano-based foliar nutrition with microbial bio-inoculants to buffer plants against environmental extremes. Such integrative strategies not only optimize yield and plant health under variable water availability but also contribute to long-term soil health and resilience in arid and semi-arid agroecosystems.

Arbuscular mycorrhizal fungi (AMF) in synergy with beneficial nanoparticles (NPs) enhance plant drought tolerance through multiple interconnected physiological and biochemical mechanisms. AMF extend the root absorptive surface via extraradical hyphae, significantly improving water and nutrient (particularly phosphorus and zinc) uptake under limited soil moisture. Concurrently, certain nanoparticles act as nano-elicitors that upregulate antioxidant defense systems (e.g., superoxide dismutase, catalase, and ascorbate peroxidase), thereby mitigating oxidative damage caused by drought-induced reactive oxygen species (ROS). NPs such as SiO₂ also reinforce cell wall integrity and modulate stomatal conductance, reducing transpirational water loss, while AMF colonization enhances osmotic adjustment through the accumulation of compatible solutes like proline and soluble sugars. Furthermore, the AMF–NP consortium often amplifies phytohormonal signaling (e.g., abscisic acid and strigolactones), improving root architecture and stress-responsive gene expression. Together, these interactions lead to improved photosynthetic efficiency, membrane stability, and biomass retention under water-deficit conditions Haghaninia et al. (2025)⁷.

Interaction effects

In the first environment (E1), characterized by optimal irrigation and high rainfall, nano magnesium oxide (OM2) significantly enhanced enzymatic activities and secondary metabolism, contributing to higher grain yields under favorable conditions. The application of OM2 improved the plant’s ability to manage oxidative stress and optimize metabolic processes, even in the absence of significant water stress. Funneliformis mosseae (MI2) further boosted productivity by improving root colonization and nutrient acquisition, demonstrating the complementary benefits of combining magnesium oxide treatments with mycorrhizal fungi (Table 6). Under optimal irrigation and high rainfall, plants can allocate resources more efficiently toward growth and metabolism, reducing the need for stress mitigation mechanisms. The combination of nano magnesium oxide (OM2) and MI2 maximizes productivity under these conditions, highlighting their synergistic potential in enhancing crop performance. Gholinezhad and Darvishzadeh (2019)³⁴, reported up to 115% yield increases with AMF under severe stress, noting that our moderate stress conditions may explain the lower percentage. This synergy between nanonutrition and microbial symbiosis suggests a multitargeted enhancement of plant physiological efficiency, not merely additive but interactive in modulating secondary metabolism and root architecture, even under well-watered conditions.

In the second environment (E2), characterized by low irrigation and high rainfall, nano magnesium oxide (OM2) continued to excel in promoting enzymatic activities and stress tolerance under mild water stress. The treatment enhanced the activity of key antioxidant enzymes, such as APX, CAT, SOD, and POX, which are critical for mitigating oxidative damage caused by reduced water availability. Samia Faiz et al. (2021)⁷⁸ but highlighted differences due to crop-specific responses (sesame vs. carrot). Mycorrhizal fungi (MI2) maintained high root colonization and nutrient uptake efficiency, compensating for the lack of irrigation (Table 6). Low irrigation introduces mild water stress, which triggers antioxidant defenses and increases the importance of mycorrhizal associations. The combination of nano magnesium oxide (OM2) and MI2 effectively mitigates stress and sustains productivity, demonstrating their adaptability to varying environmental conditions. These findings align with Sheteiwy et al.⁴² who observed increased stress resilience in Guar when microbial fertilizers were combined with nutrient treatments. The consistency across crops highlights a broader relevance of integrated strategies.

In the third environment (E3), characterized by optimal irrigation and very low rainfall, conventional magnesium oxide (OM3) demonstrated superior performance under prolonged drought conditions (Table 6). This is likely due to its sustained release properties, which provide a steady supply of magnesium over time, supporting plant health and metabolic functions. Mycorrhizal fungi (MI2) improved root colonization and nutrient acquisition, further enhancing plant resilience under very low rainfall. Early-stage water stress, caused by very low rainfall, significantly impacts germination, emergence, and seedling establishment, even when optimal irrigation is provided later in the growth cycle. Plants rely heavily on enzymatic defenses and mycorrhizal associations to sustain productivity under these conditions, underscoring the importance of integrating stress-mitigation strategies. Such findings reinforce the importance of early root and microbial establishment, as AMF-induced modulation of aquaporins and hydraulic conductivity (Junqin Li et al., 2019)⁷⁰ enhances drought tolerance at critical developmental stages.

In the fourth environment (E4), characterized by low irrigation and very low rainfall, nano magnesium oxide (OM2) remained effective in promoting enzymatic activities and stress tolerance under severe water stress (Table 6). The treatment consistently enhanced the activity of antioxidant enzymes, such as APX, CAT, SOD, and POX, which are critical for managing oxidative stress under extreme conditions. Mycorrhizal fungi (MI2) played a vital role in maintaining productivity by enhancing CPR and nutrient uptake, even under severe water scarcity. Low irrigation combined with very low rainfall imposes significant water stress, forcing plants to prioritize enzymatic defenses and mycorrhizal associations for survival, often at the expense of grain yield. The production of glomalin by AMF, a glycoprotein that improves soil aggregation and water retention, may also contribute to the sustained performance under these extreme conditions⁷⁹.

Across all environments, nano magnesium oxide (OM2) consistently enhanced enzymatic activities and GRY, demonstrating exceptional performance under both optimal and stressful conditions. This highlights its robustness and versatility as a tool for improving crop resilience. Conventional magnesium oxide (OM3) showed superior performance under prolonged drought (E3), likely due to its sustained release properties, which provide a steady supply of magnesium over time (Table 6). Mycorrhizal fungi (MI2 and MI3) improved root colonization and nutrient acquisition under water-limited conditions (E2, E3, E4), with MI2 consistently outperforming MI3 and MI1 in terms of GRY and CPR (Table 6). Together, MgO nanoparticles and AMF act through distinct but complementary mechanisms: MgO enhances enzymatic antioxidant systems (e.g., SOD, CAT) via magnesium-mediated enzyme activation²⁹, while AMF improves nutrient uptake, modulates secondary metabolites (e.g., phenolic compounds), and induces metabolic reprogramming. This dual action supports sustainable sesame production under increasing climatic variability⁶⁹.

The application of magnesium oxide and mycorrhizal fungi has been shown to increase phenolic compounds, which play a critical role in enhancing antioxidant mechanisms and plant defense responses to environmental stresses⁸⁰. Early-stage rainfall profoundly impacts germination, emergence, and seedling establishment, while later-stage rainfall influences ripening and grain yield. Mycorrhizal fungi support long-term resilience by improving nutrient acquisition and water use efficiency, even under challenging environmental conditions. These findings underscore the importance of integrating magnesium oxide treatments and mycorrhizal fungi to enhance crop resilience and productivity, offering valuable insights for sustainable agricultural practices in water-scarce regions (Table 7).

Table 4 Analysis of variance of studied traits.

Table 5 Compare means of OM and MI treatments for studied traits.

Table 6 Compare means of combined treatments for studied traits.

Table 7 Percentage increase and decrease in grain yield and other studied traits from the best environment compared to the worst environment.

MgO NPs enhanced antioxidant enzyme activities (e.g., SOD, CAT) by improving magnesium availability, which acted as a cofactor for enzymatic reactions and stabilized cellular membranes under stress (supported by Samia Faiz et al., 2021)⁴⁵. We also discussed their role in upregulating salicylic acid and defense enzymes like PAL and PPO²⁹.

AMF improved nutrient (e.g., phosphorus) and water uptake via extensive hyphal networks, increasing root hydraulic conductivity and aquaporin expression⁷⁰. Our result showed role of modulating secondary metabolism (e.g., phenolic compounds) through metabolic reprogramming⁷⁹. MgO NPs enhanced enzymatic defenses, and AMF improved resource acquisition, leading to higher grain yields and stress tolerance⁶⁹ .

The comparison between environments E1 (first year, 8-day irrigation interval) and E4 (second year, 16-day irrigation interval) revealed significant variations in physiological and biochemical traits of sesame plants. Environment E4 exhibited the highest levels of proline, soluble sugars, anthocyanins, phenols, and five different antioxidant activities, indicating a stronger stress response under longer irrigation intervals and differing climatic conditions. Conversely, overall treatment performance, particularly in terms of growth and yield, was superior in E1. Notably, root colonization percentage was higher in E4 when no fungal inoculation was applied, suggesting enhanced natural colonization under stress. However, with fungal inoculation combined with magnesium oxide and nano magnesium oxide foliar sprays, E1 showed increased root colonization, highlighting the beneficial interaction between treatments and irrigation conditions in promoting symbiotic relationships. These findings suggest that while stress-related metabolites increase under harsher conditions (E4), optimal irrigation and treatment combinations in E1 favor better overall plant performance and symbiotic colonization.

Correlations

In the first environment, characterized by optimal irrigation and high rainfall, strong correlations with GRY suggest that these conditions enhance enzymatic activities involved in oxidative stress management (Fig. 1.a). Antioxidant enzymes, including POX, SOD, CAT, APX, and GPX, play a critical role in protecting cells from damage during active growth, indirectly supporting higher grain production. CBD and PRN are crucial for osmotic adjustment and energy storage, with moderate correlations indicating their supportive role in maintaining plant health. However, under these favorable conditions, they may not be as critical as antioxidant enzymes. Anthocyanins and phenolic compounds (secondary metabolites, ANT and PNL) showed moderate correlations, suggesting that secondary metabolism is less prioritized when plants are not under significant stress. The establishment of mycorrhizal symbiosis triggers substantial reprogramming of plant primary metabolic pathways⁸¹, and mycorrhizal fungi can further enhance primary metabolism, particularly improving sugar, starch, and galactose metabolism⁸². The weak correlation of CPR by mycorrhizal fungi implies that it is less important under optimal irrigation and high rainfall, likely because nutrient acquisition is already sufficient. Sufficient nutrient availability under optimal irrigation reducing AMF dependency, contrasting with Wu et al. (2021)⁴⁰. This environment minimizes water stress, allowing plants to allocate resources toward rapid growth and primary metabolism. Antioxidant enzymes dominate the correlation pattern, reflecting their importance in maintaining cellular integrity during vigorous growth (Fig. 1.a). These findings align with previous reports Ali et al.⁴⁴ demonstrating that nano magnesium oxide nanoparticles enhance nutrient uptake and antioxidant capacity, thereby improving plant growth under optimal moisture conditions. The synergistic effects observed between MgO-NPs and mycorrhizal fungi emphasize the importance of integrating nanomaterials and microbial symbionts for sustainable crop management.

In the second environment, characterized by low irrigation and high rainfall, PNL exhibited the strongest correlation with GRY, indicating that secondary metabolism becomes more prominent under mild water stress (Figure 1b). Phenolic compounds contribute to stress tolerance and improve overall plant performance, playing an important role in combating oxidative environmental stresses and enhancing plant defense responses, particularly under drought and irrigation stress⁸⁰. Similar to the first environment, antioxidant enzymes (POX, SOD, CAT, APX, GPX) were strongly correlated with GRY. However, their importance increases under low irrigation, as plants must cope with fluctuating water availability. CBD and ANT showed moderate correlations, suggesting that carbohydrate metabolism and pigmentation play supportive roles in stress adaptation (Fig. 1.b). Research by Chen et al. (2022)⁸³ corroborates these findings regarding the role of ANT. PRN and CPR had weak correlations, possibly because early-stage rainfall mitigated some effects of low irrigation, reducing the need for osmoprotectants and enhanced nutrient uptake. Low irrigation introduces mild water stress, which triggers secondary metabolism and strengthens antioxidant defenses. Under these conditions, mycorrhizal fungi appear less critical due to the buffering effect of early-stage rainfall. The effectiveness of nano magnesium oxide in combination with mycorrhizal fungi under suboptimal irrigation supports the hypothesis that these treatments mitigate oxidative damage and improve phosphorus uptake⁴². This interaction likely facilitates improved water-use efficiency and metabolic resilience, crucial for maintaining yield under intermittent water stress.

In the third environment, characterized by optimal irrigation and very low rainfall, the strongest correlation with GRY highlights the importance of CBD in energy storage and osmotic adjustment under drought-like conditions. Plants prioritize carbohydrate metabolism to survive water scarcity. Antioxidant enzymes (POX, SOD, CAT, APX, GPX) remain crucial for managing oxidative stress caused by water scarcity, with their strong correlations underscoring their role in maintaining cellular function (Figure.1.c). Rahman et al. (2024)⁸⁴ emphasized the importance of plant antioxidants in combating reactive oxygen species under various abiotic stresses, highlighting their relevance in crop management and genetic modification. Secondary metabolites (ANT, PNL) show moderate correlations, indicating their contribution to stress tolerance without being as dominant as under mild water stress. PRN is moderately correlated with GRY, reflecting its role in osmoregulation and cellular protection. Plants employ amino acids including proline to maintain cellular osmotic and antioxidant balance under environmental stress. Proline is also known as a stress defense molecule, it is a compatible solute and can play a role in ROS scavenging. The glutamate and ornithine pathways can independently feed proline biosynthesis. In the glutamine pathway, glutamine is converted to glutamate by glutamine synthetase (GS), reduced to glutamate-5-semialdehyde by pyrroline-5-carboxylate synthetase (P5CS), which spontaneously converts to pyrroline-5-carboxylate (P5C). In ornithine pathway, ornithine is generated from arginine by arginase (ARG), and transaminated to P5C by ornithine-d-aminotransferase (OAT)⁶⁵. The weak correlation of CPR suggests that optimal irrigation offsets the need for enhanced nutrient acquisition through mycorrhizal associations (Fig. 1.c). Very low rainfall during early growth stages creates significant water stress, even with optimal irrigation later, forcing plants to rely heavily on CBD, antioxidant enzymes, and secondary metabolites to sustain growth and productivity. The distinct roles of nano magnesium oxide and mycorrhizal fungi observed here suggest a complementary mechanism where MgO-NPs directly stimulate enzymatic antioxidant defenses, while mycorrhizal fungi enhance nutrient scavenging and secondary metabolism under drought. Such differentiation highlights the potential to tailor treatment combinations based on environmental water availability⁸⁵.

In the fourth environment, characterized by low irrigation and very low rainfall, PNL again exhibit the strongest correlation with GRY, emphasizing their importance under extreme water stress conditions (Fig. 1.d). Phenolic compounds play a critical role in combating oxidative environmental stresses⁸⁰. Biofertilizers inoculation improved secondary metabolites accumulation – phenolics⁶⁵. Secondary metabolism plays a central role in protecting plants from oxidative damage and environmental challenges. Antioxidant enzymes (POX, SOD, CAT, APX) are indispensable for combating oxidative stress under severe drought, with their very strong correlations reflecting their critical role in maintaining cellular integrity. CBD and ANT show moderate correlations, suggesting that carbohydrate metabolism and pigmentation support stress tolerance but are secondary to antioxidant defense mechanisms. PRN, GPX, and CPR had weak correlations, possibly because plants prioritize other mechanisms, such as secondary metabolism and enzymatic defenses, under extreme conditions (Fig. 1.d). Low irrigation combined with very low rainfall imposes severe water stress, forcing plants to focus on secondary metabolism and antioxidant defense to survive, while mycorrhizal associations and osmoprotectants become less critical. Under extreme drought stress, the complementary actions of nano magnesium oxide and mycorrhizal fungi appear vital for plant survival and physiological stability. This supports the emerging view that integrating nanotechnology with beneficial microbes provides a promising strategy for enhancing crop resilience in the face of climate variability⁴⁴.

Across all environments, antioxidant enzymes (POX, SOD, CAT, APX, GPX) consistently showed strong correlations with grain yield, highlighting their universal importance in stress management and plant health. Secondary metabolites (PNL) became increasingly prominent under water stress, particularly in the third and fourth environments, reflecting their protective role in adverse conditions. CBD played a key role in energy storage and osmotic adjustment, especially under very low rainfall. PRN showed moderate correlations in most environments, indicating its importance as an osmoprotectant but not as a primary determinant of GRY. CPR had weak correlations in all environments, suggesting that mycorrhizal fungi are less critical when water stress is mitigated by irrigation or early-stage rainfall (Fig. 1.a.b.c.d).

The relationship between plants, primary and secondary metabolites, and yield is multifaceted, involving the roles these metabolites play in plant growth, defense, and interaction with the environment. Primary metabolites, such as carbohydrates, amino acids, and nucleic acids, are essential for basic life functions, while secondary metabolites contribute to plant survival and can enhance agricultural productivity through various mechanisms. Secondary metabolites, also known as phytoconstituents, are derived from primary metabolites through metabolic pathways influenced by environmental factors such as light, temperature, and metals. The strong correlations between antioxidant enzymes and yield across environments underscore the central role of reactive oxygen species (ROS) mitigation in stress tolerance. The observed shifts in secondary metabolite accumulation further suggest adaptive metabolic reprogramming, aligning with findings from Wu et al.⁴⁰ on stress-responsive biochemical pathways in crops.

Fig. 1

Heatmaps of correlation between GRY and other studied traits in (a) E1, (b) E2, (c) E3, (d) E4. Positive correlations of traits with grain yield are shown in green and negative correlations of grain yield with other traits are shown in red. The intensity of the colors indicates the strength of the correlation. The environments E1, E2, E3, E4 are defined in Table 3, Grain yield: GRY, Leaf proline content: PRN, Soluble leaf sugars: CBD, Anthocyanin: ANT, Phenolic compound content: PNL, Root colonization percentage: CPR, Polyphenol oxidase: POX, Superoxide dismutase: SOD, Catalase: CAT, Ascorbate peroxidase: APX, Guaiacol peroxidase: GPX., OM: magnesium oxide treatments, MI: Mycorrhizal fungi treatments.

Clustering of treatment

Based on the cluster analysis results, the performance of treatments combinations of magnesium oxide types and mycorrhizal fungi can be compared and discussed under four distinct environmental conditions. The clustering grouped treatments into three classes (Class 1, Class 2, and Class 3), which exhibited differences in physiological responses and GRY (Table 8). Treatments assigned to Class 1 (e.g., OM1MI1, OM1MI2, OM1MI3) generally showed higher values for traits such as GRY, PRN, CBD, ANT, PNL, and antioxidant enzyme activities (POX, SOD, CAT, APX, GPX) (Fig. 2). These traits are closely associated with plant productivity, stress tolerance, and antioxidant defense mechanisms. The application of magnesium oxide nanoparticles (MgO-NPs) significantly enhances the activity of plant enzymes and defense-related compounds. For instance, treatment with MgO-NPs increases the activity of defense-related enzymes such as phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and POX²⁹ .

Treatments in Class 2 (e.g., OM2MI1, OM3MI1) exhibited intermediate values for most traits but showed the highest CPR, indicating better root colonization by mycorrhizal fungi (Table 8). This enhanced root colonization improves nutrient uptake and drought tolerance, even when other physiological traits are not maximized. Sheteiwy et al.⁴² achieved similar results as us. Treatments in Class 3 (e.g., OM2MI2, OM3MI2, OM2MI3, OM3MI3) had lower values for most traits, except for CPR, which remained relatively high compared to Class 1(Fig. 2). This suggests that while these treatments may not excel in all physiological metrics, they still benefit from effective mycorrhizal associations. The distinct clusters identified reinforce the notion that physiological and biochemical traits are integrally linked to treatment efficacy under varying environmental stresses. This stratification can guide the development of precision agriculture approaches that optimize treatment combinations for specific field conditions.

In the first environment, characterized by optimal irrigation and high rainfall, treatments in Class 1 performed well despite the absence of magnesium oxide sprays (OM1). High values for GRY, PRN, CBD, ANT, PNL, and antioxidant enzyme activities (POX, SOD, CAT, APX, GPX) suggest that favorable growing conditions compensated for the lack of magnesium supplementation. Treatments such as OM2MI1 and OM3MI1 in Class 2 showed high CPR, indicating strong root colonization by mycorrhizal fungi (Table 8). This colonization triggers widespread metabolic changes in the host plant, affecting both primary metabolism processes, such as photosynthesis and water absorption, and secondary metabolism pathways⁸¹. These changes enhance nutrient uptake and water-use efficiency, even though other physiological traits remained intermediate. Treatments in Class 3, such as OM2MI2, OM3MI2, OM2MI3, and OM3MI3, had lower values for most traits but still performed well due to the combined effects of nano magnesium oxide (OM2) and effective mycorrhizal associations (MI2, MI3) (Fig. 2).

In the second environment, characterized by low irrigation and high rainfall, treatments in Class 1 again performed well due to high early-season rainfall, despite reduced irrigation. However, the absence of magnesium oxide (OM1) limited their stress tolerance during later growth stages. In Class 2, treatments such as OM2MI1 and OM3MI1 showed high CPR, which improved nutrient uptake and water-use efficiency under reduced irrigation (Table 8). Treatments in Class 3, including OM2MI2, OM3MI2, OM2MI3, and OM3MI3, had lower values for most traits but maintained good performance due to the synergistic effects of nano magnesium oxide (OM2) and mycorrhizal fungi (MI2, MI3) (Fig. 2). The combination of arbuscular mycorrhizal fungi (AMF) and magnesium supplementation represents a promising integrated approach for enhancing plant drought tolerance. Studies have shown that combining multiple strategies is more effective in minimizing stress impacts on crop production and quality compared to single interventions⁴².

In the third environment, characterized by optimal irrigation and very low rainfall, treatments in Class 1 relied heavily on optimal irrigation to compensate for very low early-season rainfall. High values for GRY, PRN, CBD, ANT, PNL, and antioxidant enzyme activities (POX, SOD, CAT, APX, GPX) indicate good overall performance (Table 8), consistent with findings by Rajput et al. (2021)⁸². In Class 2, treatments such as OM2MI1 and OM3MI1 showed high CPR, which improved nutrient uptake and drought tolerance under water-limited conditions. In Class 3, treatments such as OM2MI2, OM3MI2, OM2MI3, and OM3MI3 had lower values for most traits but still performed well due to the combined effects of nano magnesium oxide (OM2) and mycorrhizal fungi (MI2, MI3) (Fig. 2).

In the fourth environment, characterized by low irrigation and very low rainfall, treatments in Class 1, such as OM1MI1, OM1MI2, and OM1MI3, struggled under extreme water scarcity (Table 8). The absence of magnesium oxide (OM1) limited their ability to cope with drought stress. Treatments in Class 2, such as OM2MI1 and OM3MI1, showed high CPR, which improved nutrient uptake and water-use efficiency, enabling plants to survive under low irrigation and very low rainfall (Fig. 2). Treatments in Class 3, including OM2MI2, OM3MI2, OM2MI3, and OM3MI3, had lower values for most traits but maintained the best overall performance due to the synergistic effects of nano magnesium oxide (OM2) and effective mycorrhizal associations (MI2, MI3)⁴². Treatment performance in third and fourth of environments highlight the crucial interaction between nano magnesium oxide and mycorrhizal fungi in enhancing drought tolerance, consistent with Sheteiwy et al.⁶⁵ who demonstrated similar synergistic effects on plant physiological responses under metal stress. Such findings emphasize the broader applicability of these treatments beyond water stress.

Early growth stages, including germination, emergence, and seedling establishment, were critical for developing strong root systems and efficient nutrient uptake. Treatments with high CPR (Class 2) likely benefited from better root colonization during these stages. Later growth stages, including juvenile development, first bud formation, and ripening, required robust antioxidant defense mechanisms (SOD, CAT, APX, GPX) to mitigate oxidative stress caused by water scarcity.

Among the mycorrhizal fungi treatments, Funneliformis mosseae (MI2) consistently demonstrated high performance across environments, particularly in terms of CPR, GRY, and stress tolerance. These findings align with previous research by Gholinejad et al., (2021)⁶⁶ in sesame. The symbiotic relationship between MI2 and plant roots improved nutrient uptake and water-use efficiency. Rhizophagus irregularis (MI3) also performed well but was slightly less effective than MI2, especially under water-limited conditions. Treatments without mycorrhizal fungi (MI1) resulted in poor performance, underscoring the importance of mycorrhizal associations for sesame cultivation. Among the magnesium oxide treatments, nano magnesium oxide (OM2) enhanced enzyme activities (GPX, APX, CAT, SOD) and stress tolerance, making it the most effective treatment across all environments. MgO-NPs enhance the activity of defense-related enzymes, such as PAL, PPO, POD, and SOD, while increasing salicylic acid content, which is crucial for plant defense responses²⁹. OM3 performed well but was slightly less effective than OM2, likely due to its lower bioavailability. Treatments without magnesium oxide supplementation (OM1) limited plant performance, particularly under stressful conditions, highlighting the importance of magnesium supplementation for optimizing crop resilience and productivity. The superior performance of Funneliformis mosseae (MI2) corroborates previous studies showing its effectiveness in enhancing nutrient uptake and stress resilience⁶⁵. The enhanced bioavailability of nano magnesium oxide further supports its utility in improving enzymatic defenses, highlighting the combined potential of these biostimulants in sustainable agriculture.

Fig. 2

Dendrograms of nine treatments based of ward algorithm and all studied traits. Cutting point determined based F test. A high between-group variance relative to within-group variance suggests that the clusters are well-separated and distinct. The environments E1, E2, E3, E4 are defined in Table 3., Grain yield: GRY, Leaf proline content: PRN, Soluble leaf sugars: CBD, Anthocyanin: ANT, Phenolic compound content: PNL, Root colonization percentage: CPR, Polyphenol oxidase: POX, Superoxide dismutase: SOD, Catalase: CAT, Ascorbate peroxidase: APX, Guaiacol peroxidase: GPX., OM: magnesium oxide treatments, MI: Mycorrhizal fungi treatments.

Table 8 Means of studied traits for classes of cluster analysis.

BiPlot analysis

The biplot analysis reveals how nine treatments influence primary and secondary metabolites, as well as grain yield, across four distinct environments characterized by irrigation levels and rainfall patterns (Fig. 3).

This analysis points to two basic points: 1-BiPlot analysis and metabolite associations. 2-environmental impact on treatment efficacy. In the first environment, characterized by optimal irrigation and high rainfall, nano magnesium oxide (OM2) demonstrated its high bioavailability, enhancing nutrient uptake and metabolic activity. Specifically, the treatment OM2MI1 (nano magnesium oxide without mycorrhiza) showed strong correlations with antioxidant enzymes (POX, GPX, SOD) and CBD, indicating improved stress tolerance and carbohydrate metabolism. The application of magnesium oxide nanoparticles (MgO-NPs) significantly influences plant enzyme activities and defense-related compounds (Fig. 3). Treatment with MgO-NPs increases the activities of defense-related enzymes such as phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (POD), and POX, while also elevating the levels of antioxidant enzymes like SOD and CAT. Additionally, MgO-NPs treatment can substantially increase salicylic acid content in plants, further enhancing stress resilience²⁹. PRN accumulation suggests enhanced osmotic adjustment, which is beneficial even under optimal conditions, as it prepares plants for potential future stress. GRY was also positively influenced, highlighting the efficacy of nano magnesium oxide in promoting biomass production.

Treatments combining conventional magnesium oxide (OM3) with mycorrhizal fungi (MI2, MI3), such as OM3MI2, OM3MI3, OM2MI2, and OM2MI3, were associated with secondary metabolites like ANT, PNL, and CPR (Fig. 3). Mycorrhizal fungi improve nutrient acquisition, particularly phosphorus, which can enhance secondary metabolism. The fungal colonization triggers widespread metabolic changes in the host plant, affecting both primary metabolism processes, such as photosynthesis and water absorption, and secondary metabolism pathways⁸⁵. Additionally, the presence of conventional magnesium oxide (OM3) likely supported enzymatic activities related to detoxification (CAT, APX), contributing to overall plant health. Optimal irrigation and high rainfall reduce water stress, allowing plants to allocate resources toward growth and secondary metabolism. These observations suggest that nano magnesium oxide (OM2) excels in promoting primary metabolites and yield, while mycorrhizal fungi (MI2, MI3) enhance secondary metabolism and root colonization (Fig. 3).

In the second environment, characterized by low irrigation but high rainfall, nano magnesium oxide treatments (OM2MI3, OM2MI1) maintained their effectiveness in enhancing antioxidant defense mechanisms (APX, CAT, POX) and osmoprotectants (PRN, CBD) (Fig. 3). This indicates that nano magnesium oxide helps mitigate oxidative stress caused by fluctuating water availability. Treatments such as OM3MI2, OM3MI3, and OM2MI2 were linked to secondary metabolites (PNL, ANT) and GRY. Mycorrhizal fungi play a crucial role in improving water-use efficiency and nutrient uptake under suboptimal irrigation conditions. As noted by Stallmann et al. (2021)⁸⁶, the symbiosis between plants and arbuscular mycorrhizal fungi (AMF) is so effective that AMF create an additional nutrient uptake pathway, significantly contributing to plant phosphorus acquisition even when no obvious growth benefits are observed⁸⁷. Conventional magnesium oxide (OM3) likely complements this effect by supporting enzyme activities involved in stress alleviation (GPX, SOD). Low irrigation increases water stress, but high rainfall during critical growth stages mitigates some adverse effects. The results show that both nano magnesium oxide and mycorrhizal fungi are effective in maintaining plant performance under such conditions, albeit through different pathways.

This analysis highlights the different but complementary contributions of magnesium oxide nanoparticles and mycorrhizal fungi in modulating primary and secondary metabolism. These findings support a model in which nanomaterials and microbial symbionts collaboratively optimize plant metabolic pathways under different environmental stresses. In the third environment, characterized by optimal irrigation and very low rainfall, OM2MI1 again stood out for its ability to maintain primary metabolites (CBD, GRY) and antioxidant defenses (SOD, GPX, POX)) (Fig. 3). The combination of nano magnesium oxide and no mycorrhiza appears particularly advantageous when rainfall is scarce, possibly due to the direct enhancement of enzymatic activities and osmoregulation by nano magnesium oxide²⁹.Secondary metabolites (ANT, PNL) and enzymatic activities (APX, CAT) were dominant in treatments such as OM3MI2, OM3MI3, OM2MI2, and OM2MI3) (Fig. 3). Mycorrhizal fungi likely played a significant role in scavenging limited nutrients from the soil, enabling plants to invest more resources into protective compounds under drought-like conditions⁸¹. Very low rainfall necessitates efficient water use and stress tolerance. Nano magnesium oxide (OM2) supports primary metabolism and yield, while mycorrhizal fungi focus on secondary metabolism and nutrient acquisition.

In the fourth environment, characterized by low irrigation and very low rainfall, nano magnesium oxide treatments (OM2MI3, OM2MI1) maintained antioxidant defenses (GPX, APX) and osmoprotectant synthesis (PRN, CBD) despite extreme water scarcity) (Fig. 3). This highlights the robustness of nano magnesium oxide in combating oxidative stress and ensuring basic physiological functions²⁹. Treatments such as OM3MI2, OM3MI3, and OM2MI2 focused on secondary metabolites (ANT, PNL), CPR, and general stress responses (SOD, POX, CAT) ) (Fig. 3). Mycorrhizal fungi likely facilitated nutrient uptake and root development, enabling plants to survive under severe water stress. GRY was still observed, albeit at lower levels compared to other environments. This environment represents the most challenging condition for sesame growth. The results indicate that nano magnesium oxide prioritizes survival mechanisms, while mycorrhizal fungi support long-term resilience and resource acquisition.

Across all environments, nano magnesium oxide (OM2) consistently enhanced primary metabolites (CBD, PRN, GRY) and antioxidant enzymes (POX, GPX, SOD), demonstrating exceptional performance under both optimal and stressful conditions. This underscores its versatility as a tool for improving crop resilience) (Fig. 3). Conventional magnesium oxide (OM3) primarily supported secondary metabolites (ANT, PNL) and enzymatic activities (APX, CAT), showing greater effectiveness when combined with mycorrhizal fungi, suggesting synergistic effects. Mycorrhizal fungi (MI2, MI3) improved CPR and secondary metabolism (ANT, PNL) under water-limited conditions, enhancing nutrient uptake and stress tolerance while complementing the effects of magnesium oxide treatments) (Fig. 3). Early-stage rainfall, particularly during the first month, had a profound impact on germination, emergence, and seedling establishment. Later-stage rainfall influenced ripening and grain yield, with mycorrhizal fungi playing a critical role in maintaining productivity under prolonged drought⁸⁵. The critical influence of rainfall timing on treatment outcomes emphasizes the need to consider environmental variability when designing crop management strategies. Integrating nano magnesium oxide and mycorrhizal fungi offers a resilient approach to sustain productivity under changing climatic patterns, aligning with current trends in agroecological research.

Fig. 3

Biplot of nine treatments and all studied traits. Principal Component Analysis (PCA) was then applied to the standardized dataset using the variance–covariance matrix as the basis for eigen-decomposition. PCA biplots, simultaneously displayed the positions of sample points (scores) and variable vectors (loadings) in the reduced-dimensional space defined by the first two principal components, thereby facilitating the identification of traits that contribute most to nine treatments differentiation. The environments E1, E2, E3, E4 are defined in Table 3, Grain yield: GRY, Leaf proline content: PRN, Soluble leaf sugars: CBD, Anthocyanin: ANT, Phenolic compound content: PNL, Root colonization percentage: CPR, Polyphenol oxidase: POX, Superoxide dismutase: SOD, Catalase: CAT, Ascorbate peroxidase: APX, Guaiacol peroxidase: GPX, OM: magnesium oxide treatments, MI: Mycorrhizal fungi treatments.

Conclusion

This study presents a physiology-informed strategy for enhancing sesame resilience and productivity under water-limited conditions through the combined application of nano-magnesium oxide (MgO NPs; specifically the OM2 formulation) and the arbuscular mycorrhizal fungus Funneliformis mosseae (AMF; isolate MI2). While prior research has examined nano-fertilizers or microbial symbionts in isolation, our work demonstrates, for the first time in sesame, how their synergistic integration can concurrently optimize stress mitigation and metabolic efficiency. The OM2 + MI2 treatment consistently outperformed other combinations across four controlled water regimes (E1–E4), with the greatest yield benefits observed under moderate drought (E2 and E3). This synergy is mechanistically underpinned by AMF-enhanced nutrient and water uptake via extended hyphal networks, coupled with MgO NP–mediated upregulation of key antioxidant enzymes (SOD, CAT, APX) and osmoprotectants (proline, soluble sugars), which collectively stabilized photosynthetic performance, reduced oxidative damage, and maintained membrane integrity—ultimately translating into higher seed yield and biomass. Notably, the efficacy of the combined treatment varied with stress intensity; under extreme drought (E4), yield gains were attenuated, highlighting a threshold beyond which physiological buffering capacity is overwhelmed. These findings support the potential of tailored nano-biofertilizer formulations as a low-input, agroecologically aligned approach for semi-arid regions where sesame serves as both a vital food and cash crop. Nevertheless, the current evidence is confined to greenhouse and semi-controlled environments, and real-world applicability requires validation under field conditions with greater climatic and edaphic variability. Future research should prioritize (i) long-term field trials assessing the persistence of AMF colonization and soil health impacts of repeated MgO NP applications, (ii) economic feasibility and farmer-led validation of dosage tuning across drought gradients, and (iii) mechanistic dissection of MgO NP–AMF crosstalk—particularly the role of Mg²⁺ signaling, ROS-scavenging pathways, and strigolactone-mediated symbiosis—in coordinating stress resilience and growth. This work thus establishes a foundational framework for developing next-generation, physiology-guided nano-biofertilizers for oilseed crops in a changing climate.