Aflatoxin contamination of food and feed crops poses a major global threat to health and food safety. Surveillance studies report incidence rates ranging from 37% to 92%, with mean concentrations between 1.15 and 107.9 µg kg^-1 across countries (Kabak et al., 2006). In Europe, 80% of samples contain detectable mycotoxins and about 20% exceed legal limits (EFSA, 2020). In India, contamination is equally concerning: maize from Bihar showed 47% infection during kharif and 43% in storage, often surpassing the 20 µg kg^-1 safety threshold (Reddy and Raghavender, 2007). National surveys report that 50-74% of maize exceeds permissible limits (FSSAI, 2020). Acute outbreaks, such as the 1974 Gujarat incident that caused over 200 deaths (Krishnamachari et al., 1975), highlight the public health risks. With projected temperature rises of 2-5^oC, aflatoxin prevalence is expected to intensify in tropical and subtropical regions (Sarmah et al., 2023), emphasizing the need for integrated field-level mitigation.
       
Maize (Zea mays L.) is one of the most important cereals, contributing 42% of global grain production (>1.2 billion tonnes annually) (FAOSTAT, 2022). In India, maize covers ~10 million ha and yields over 31 million tonnes, ranking third after rice and wheat (DAC and FW, 2023). However, its quality is threatened by Aspergillus flavus and A. parasiticus, which produce aflatoxins under warm, humid conditions (Mishra et al., 2019). Aflatoxin B (AFB), classified  as a Group 1 carcinogen (IARC, 2012), contributes to food safety, trade and health risks worldwide. More than 25% of food crops are affected by mycotoxins annually, leading to losses exceeding USD 1 billion (WHO, 2018). In India, poor post-harvest infrastructure exacerbates the problem, with surveys reporting up to 74% of market maize samples exceeding 20 µg kg^-1 (FSSAI, 2020). Traditional approaches such as drying, storage improvement, fungicides and resistant varieties have limited success under climate variability (Paterson and Lima, 2010).
       
Attention has shifted towards activating plant innate immunity. Jasmonic acid (JA) and salicylic acid (SA) are central defence regulators: JA mediates induced systemic resistance (ISR) against necrotrophic pathogens, while SA governs systemic acquired resistance (SAR), effective against biotrophic fungi (Kuchlan et al., 2023). Exogenous JA and SA prime defence pathways, enhancing PR proteins (β-1,3-glucanase, POD, SOD, CAT) that degrade fungal cell walls and detoxify ROS (Wasternack and Hause, 2013).
       
At the same time, intercropping with legumes such as soybean improves soil nitrogen, microbial diversity and microclimate, thereby reducing fungal infestation (Vandermeer, 1989; Singh et al., 2019). Maize-soybean systems lower canopy temperature, vapor pressure deficit and fungal colonization risk (Zhou et al., 2020). Soybean root isoflavonoids may also trigger maize defence responses via root-root or microbe-mediated signalling (Moraes et al., 2021). Despite evidence for individual success, little is known about the synergistic effects of combining intercropping with JA and SA priming on aflatoxin suppression.
       
Therefore, this study was undertaken to assess the efficacy of an integrated approach under field conditions. The objectives were to: (i) evaluate the impact of soybean intercropping and hormonal priming on aflatoxin B1 and B2  levels in maize; (ii) quantify endogenous JA and SA accumulation and antioxidant enzyme activities (POD, SOD, CAT, β-1,3-glucanase); (iii) examine the expression of defence genes (PR1, PR5) and (iv) analyse rhizospheric microbial diversity and soil parameters. This research provides insights into host-pathogen interactions and offers a biologically intensive strategy for aflatoxin management within climate-resilient maize production systems in South Asia and beyond.

A field experiment was conducted during the kharif seasons of 2022 and 2023 at the Agronomy Research Farm of Lovely Professional University, Punjab, India (31.25^oN latitude, 75.58^oE longitude, 234 m above mean sea level). The site falls under the subtropical humid agroclimatic zone, characterized by hot summers and moderately cool winters. The study aimed to evaluate the effects of soybean intercropping and foliar application of defence-inducing phytohormones on aflatoxin contamination and defence responses in maize.
       
The experiment was laid out in a randomized complete block design (RCBD) with three replications. For field feasibility, four representative treatment combinations were evaluated: T₁ - Sole maize (control), T₂ - Sole maize + jasmonic acid (JA, 100 µM), T₃ - Maize + soybean intercropping (2:2 row arrangement) and T₄ - Maize + soybean intercropping + JA (100 µM) + salicylic acid (SA, 1 mM). Before sowing, land preparation involved one deep ploughing followed by two harrowing and levelling to obtain a uniform seedbed. The maize hybrid variety NMH 589 and the soybean variety JS-9560 were used as test crops. In sole cropping, maize was sown at 40 cm x 25 cm spacing, while soybean was spaced at 30 cm x 5 cm. In the intercropping system, maize and soybean were planted in a 2:2 paired row arrangement, maintaining the respective intra-row spacings. Each net plot measured 4 m x 5 m (20 m²).
       
The recommended dose of fertilizer (RDF) was applied to maize (80:60:20 NPK kg ha^-1) and soybean (25:60:20 NPK kg ha^-1). Nitrogen for maize was applied in three splits: 25% as basal, 50% at 30 days after sowing (DAS) and the remaining 25% at 60 DAS. Phytohormones were applied as foliar sprays at two crop growth stages: 45 DAS (vegetative) and 60 DAS (early reproductive). Jasmonic acid (JA, 100 µM) and salicylic acid (SA, 1 mM) were freshly prepared in distilled water with 0.1% Tween-20 as surfactant. Treatments T₂ and T₄ received JA sprays, whereas only T₄  received both JA and SA. Foliar applications were carried out using a hand-held knapsack sprayer in the early morning to ensure maximum absorption and to minimize photooxidative degradation.
       
At harvest maturity, maize cobs were manually harvested, sun-dried and shelled. Grain samples were stored under standardized low-moisture conditions before analysis. Aflatoxin B₁ and B₂ concentrations were determined using competitive ELISA kits (Romer Labs, Austria; Catalogue No. COKAQ1). Grain extracts were prepared by homogenizing 20 g of maize grain in 100 mL of 80% methanol, shaking for 30 min at room temperature and centrifuging at 4000 g for 10 min. The supernatant was diluted according to the manufacturer’s protocol and absorbance was measured at 450 nm using a microplate reader. The detection limit of the assay was 1.0 µg kg^-1 and results were expressed as µg kg^-1.
       
For hormonal profiling, leaf tissues sampled at 45 and 60 DAS were frozen in liquid nitrogen and stored at -80^oC until analysis. JA and SA were quantified using high-performance liquid chromatography (HPLC, Shimadzu, Japan) equipped with a C18 column (250 x 4.6 mm, 5 µm). The mobile phase consisted of methanol: water (80:20 v/v) with 0.1% formic acid at a flow rate of 1.0 mL min^-1 and detection was performed at 230 nm. Standard curves were prepared using authentic JA and SA standards (Sigma-Aldrich, USA) and concentrations were expressed as ng g^-1 fresh weight.
       
Biochemical assays of antioxidant enzymes were conducted using spectrophotometric methods. POD activity was assayed using 20 mM guaiacol and 10 mM H₂O₂ as substrates, recording absorbance at 470 nm (extinction coefficient = 26.6 mM^-1 cm^-1). CAT activity was determined by monitoring the decomposition of 10 mM H₂O₂  at 240 nm (extinction coefficient = 39.4 mM^-1 cm^-1). SOD activity was measured by inhibition of NBT (50 µM) reduction at 560 nm, using riboflavin–methionine–NBT assay. β-1,3-glucanase activity was estimated from the release of glucose equivalents from 4 mg mL^-1 laminarin substrate incubated at 37^oC, with absorbance measured at 540 nm. Each assay was performed with three biological replicates, analysed in triplicate.
       
For molecular analysis, leaf samples collected at 60 DAS were used for RNA extraction with TRIzol reagent (Invitrogen, USA). Following cDNA synthesis, quantitative real-time PCR (qRT-PCR) was conducted using SYBR Green chemistry on a Bio-Rad CFX96 system. Primer sequences for PR1, PR5 and the internal reference gene ZmAct1 are provided (Table 1). The PCR cycling program was 95^oC for 3 min; 40 cycles of 95^oC for 15 s and 60oC for 30 s. Relative fold change was calculated using the 2^-ΔΔCt method (Livak and Schmittgen, 2001). Each treatment was represented by three biological replicates, with three technical replicates per sample. Rhizospheric soil samples were collected from all plots at 60 DAS. Microbial populations were quantified by serial dilution up to 10^-6, with 0.1 mL aliquots plated on nutrient agar (for bacteria) and potato dextrose agar (for fungi). Plates were incubated at 28±2^oC for 48-72 h and counts were expressed as log CFU g^-1 soil.


 
Statistical analysis
 
Data were analysed using OPSTAT software (CCS HAU, Hisar). One-way analysis of variance (ANOVA) was performed on treatment means and comparisons were made using the least significant difference (LSD) test at P£0.05. Results are presented as mean ± standard error (SE), n = 3 and different letters indicate significant differences among treatments. Graphs were generated using OriginPro 2024 software. ANOVA indicated significant treatment effects: aflatoxin B₁ + B₂  content differed significantly among treatments (F_3,8  = 12.45, p < 0.01), endogenous JA (F_3,8  = 15.32, p < 0.01) and SA (F_3,8  = 14.87, p<0.01) levels also varied strongly and antioxidant enzyme activities (POD, SOD, CAT), β-1,3-glucanase and defence gene expression (PR1, PR5) all showed significant differences (p<0.05).

Aflatoxin contamination in maize grains
 
Aflatoxin contamination, particularly by aflatoxin B₁ (AFB) and B₂, remains a critical food safety concern in maize-producing regions. In the present study, maize grains from different treatment combinations exhibited significant variation (P<0.05) in aflatoxin content at harvest. The untreated control (T₁: sole maize) recorded the highest mean aflatoxin concentration at 38.6 µg kg^-1, far exceeding the safety threshold of 20 µg kg^-1 prescribed by the Food Safety and Standards Authority of India (FSSAI, 2020). This observation aligns with earlier reports indicating that maize monocultures, particularly under warm and humid conditions, are highly vulnerable to Aspergillus flavus colonization and subsequent mycotoxin accumulation (Kabak et al., 2006; Reddy and Raghavender, 2007). The treatment involving sole maize with jasmonic acid foliar spray (T₂) showed a moderate reduction in aflatoxin levels (28.4 µg kg^-1), indicating partial induction of systemic resistance. A more pronounced decrease was recorded in T₃ (maize + soybean intercropping), where aflatoxin concentration declined to 21.7 µg kg^-1, possibly due to microclimate modification, increased canopy shading and allelopathic influences from soybean root exudates. The greatest reduction was achieved under the integrated treatment (T₄: maize + soybean intercropping + JA + SA), where aflatoxin levels dropped to 16.9 µg kg^-1 (Fig 1). This reduction of more than 56% compared with the control highlights the effectiveness of combining intercropping with phytohormonal priming and underscores the role of synergistic JA-SA defence activation in suppressing aflatoxin biosynthesis, as also suggested by Paterson and Lima (2010).
 
Endogenous hormonal induction: JA and SA accumulation
 
Quantification of endogenous jasmonic acid (JA) and salicylic acid (SA) in leaf tissues revealed substantial variation among the treatment combinations. In the control (T₁: sole maize), basal levels of 120.5 ng g^-1 (JA) and 95.3 ng g^-1 (SA) were recorded. The treatment involving sole maize with JA foliar spray (T₂ ) increased JA content to 310.2 ng g^-1, while SA remained nearly unchanged, reflecting the specificity of exogenous JA application. The intercropping treatment (T₃ : maize + soybean) enhanced both JA (265.4 ng g^-1) and SA (220.7 ng g^-1), which may be attributed to soybean-maize root interactions and microbiome-mediated cross-activation. The integrated treatment (T₄: maize + soybean + JA + SA) recorded the highest hormonal levels-320.6 ng g^-1 (JA) and 310.9 ng g^-1 (SA)-indicating robust hormonal crosstalk and comprehensive defence activation (Fig 1). This hormonal profile supports the model of parallel JA and SA signalling in defence regulation, particularly in modulating host responses against necrotrophic pathogens such as A. flavus (Glazebrook, 2005; Zhang et al., 2015).


 
Antioxidant defence enzymes
 
The oxidative burst, a hallmark of plant immunity, triggers rapid accumulation of reactive oxygen species (ROS), which are detoxified by antioxidant enzymes including peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT). In the present study, the control treatment (T₁ : sole maize) exhibited the lowest activity levels: POD (0.85 U mg^-1 protein), SOD (1.05 U mg^-1 protein) and CAT (0.92 U mg^-1 protein). The treatment with sole maize + JA foliar spray (T₂) increased enzyme activities moderately, whereas the intercropping treatment (T₃: maize + soybean) resulted in comparatively higher activities, likely due to rhizosphere-mediated biotic interactions. The integrated treatment (T₄: maize + soybean + JA + SA) recorded the maximum activities-POD (2.85 U mg^-1 protein), SOD (3.02 U mg^-1 protein) and CAT (2.77 U mg^-1 protein)-demonstrating that the combination of intercropping and hormonal priming most effectively stimulated systemic antioxidant responses. These observations consistent with earlier findings (Aebi, 1984) and highlight that enhanced ROS detoxification capacity is central to restricting aflatoxin biosynthesis and fungal spread.
 
PR protein response: β-1,3-glucanase activity
 
β-1,3-glucanase is a hallmark pathogenesis-related (PR) protein involved in fungal cell wall degradation. In the present study, its activity showed a progressive increase across the treatment combinations, with the lowest value recorded in the control (T₁: 1.21 µg glucose eq. mg^-1 protein) and the highest in the integrated treatment (T₂: 3.54 µg glucose eq. mg^-1 protein). Intermediate activities were observed in T₃ (sole maize + JA: 2.43 µg glucose eq. mg^-1 protein) and T₄ (maize + soybean intercropping: 3.01 µg glucose eq. mg^-1 protein) (Fig 2). These results indicate that while T₂  and T₃  each enhanced glucanase activity to a certain extent, the combined strategy in T₄ exerted the strongest induction, reflecting a synergistic effect of intercropping with hormonal priming. The role of β-1,3-glucanase in resistance to A. flavus has been well documented in oilseeds and cereals (Velazhahan and Vidhyasekaran, 2000), which supports the present findings.


 
Gene expression of PR1 and PR5
 
Defence gene expression analysis using qRT-PCR revealed significant upregulation of PR1 and PR5 genes in the treated maize plants. Expression was normalized using the actin gene (ZmAcT1) as an internal reference and relative fold change was calculated using the 2-ΔΔCt method (Livak and Schmittgen, 2001). In the control (T₁: sole maize), baseline expression was maintained at 1.0 for both genes. The treatment with sole maize + JA foliar spray (T₂) increased expression to 2.3-fold for PR1 and 1.8-fold for PR5. The intercropping treatment (T₃: maize + soybean) further enhanced expression to 2.8-fold (PR1) and 2.7-fold (PR5). The maximum induction was observed under the integrated treatment (T₄ : maize + soybean + JA + SA), where PR1 and PR5 reached 3.6- and 3.3-fold, respectively (Fig 3). These results demonstrate that T₄  elicited the most robust transcriptional activation of defence-related genes. Since PR1 and PR5 are well-established markers of systemic acquired resistance (SAR) pathways (Glazebrook, 2005), these findings confirm the successful activation of systemic immunity under the integrated treatment. The mechanistic basis of this coordinated defence activation, driven by jasmonic acid-salicylic acid signalling crosstalk and its role in suppressing aflatoxin biosynthesis, is schematically illustrated (Fig 3).


 
Rhizospheric microbial diversity, soil moisture and temperature effects
 
Soil microbiological and physicochemical attributes play a critical role in crop resilience and disease suppression under intercropping systems. In the present study, rhizospheric microbial diversity, soil moisture content and soil temperature were monitored across the four treatment combinations (T₁ -T₄) (Fig 4). A progressive enhancement in microbial diversity was observed, with the control (T₁: sole maize) recording the lowest value (6.1 log CFU g^-1) and T₂ (sole maize + JA) showing a slight increase to 6.5 log CFU g^-1. Higher microbial proliferation was noted in the intercropping treatment (T₃: maize + soybean, 7.4 log CFU g^-1), while the integrated treatment (T„ : maize + soybean + JA + SA) achieved the maximum value (8.2 log CFU g^-1). The enrichment of microbial diversity under T₄ may be attributed to increased rhizospheric root exudates and hormone-induced systemic resistance, which together promote the colonization of beneficial microbes (Mendes et al., 2013; Bhattacharyya and Jha, 2023). Soybean root exudates are known to support populations such as Trichoderma spp. and Pseudomonas fluorescens, potentially contributing to the suppression of aflatoxigenic fungi (Moraes et al., 2021).


       
Soil moisture also increased progressively among the treatments. T₁ maintained the lowest moisture level (14.8%), while T₂ and T₃ recorded 16.3% and 18.2%, respectively. The integrated treatment (T₄ ) displayed the highest soil moisture (20.4%), likely due to canopy shading from soybean, improved soil aggregation by microbial exudates and reduced evapotranspiration (Reddy, 2020). These findings align with reports that diversified cropping systems enhance water retention by increasing organic matter and improving root structure (Sharma et al., 2021).

Conversely, soil temperature showed a declining trend across treatments. T₁ exhibited the highest mean temperature (32.4^oC), followed by T₂ (31.9^oC) and T3 (30.7 ^oC), while the lowest value was recorded in T₄ (29.8^oC). This reduction is likely attributable to greater canopy cover under intercropping, reduced soil albedo and improved moisture conservation, which buffer against temperature fluctuations and support microbial activity (Singh et al., 2022). Collectively,  these results demonstrate that the integrated treatment (T₄) not only maximizes microbial community richness but also modifies the soil microenvironment in ways that strengthen plant defence and minimize aflatoxin accumulation.

The integrated treatment (T4: maize + soybean intercropping + JA + SA) significantly reduced aflatoxin contamination to 16.9 µg kg^-1, below the FSSAI limit. It also achieved the highest JA (320.6 ng g^-1) and SA (310.9 ng g^-1) accumulation. Antioxidant enzyme activities (POD, SOD, CAT) and PR gene expression (PR1, PR5) were maximized in T4. Soil microbial diversity, moisture content and microclimate conditions were most favourable under this treatment. These results highlight the potential of integrating intercropping with phytohormonal priming for aflatoxin suppression. Future research should focus on multi-location trials and full factorial designs to confirm scalability of this integrated approach under diverse agroclimatic conditions.

The author greatly acknowledges Lovely Professional University (LPU), Jalandhar, Punjab, for providing facilities to conduct research during Ph.D. programme at Division of Agronomy and School of Agriculture (SOA). 
 
Disclaimers
 
The opinions and conclusion outlined in this article reflect those of the author and should not be considered as representing the perspectives of their affiliated institutions. While the authors have ensured the accuracy and completeness of the information presented, they disclaim any responsibility for direct or indirect damages that may result from the application of this content.
 
Ethical statement
 
This study did not involve human participants or vertebrate animals. Field experiments were conducted in accordance with institutional and national agricultural research guidelines.

The authors declare no conflict of interest.