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Determination of Synergy/Antagonism Impact of Mineral Nutrients on Salinity Resilience During Germination and Root Establishment: Use of Hierarchical Clustering and Heat Map Analysis

A
Assia Chaker-Haddadj1,*
H
Hamida Sadji-Ait Kaci1
F
Fahima Nabi2
F
Farida Taguett1
M
Massakib Bekkaye1
1Laboratory of Biology and Physiology of Organisms, Faculty of Biological Sciences, Team of Soils Biology, University of Sciences and Technology Houari Boumediene, Algiers, Algeria.
2Laboratory of Biotechnology and Valorization of Biological Resources, Faculty of Sciences, Department of Nature and Life Sciences, Yahia Farès Medea University, Médéa 26100, Algiers, Algeria.

ABSTRACT

Background: Salinity is one of the most significant environmental restrictions threatening global food security. This study aimed to evaluate the effects of mineral nutrients, on germination traits, seedling parameters, root system development and tolerance to salinity of Vicia faba L.

Methods: Seeds were immersed in solutions containing KNO3 (K), CaO-MgO (D) and P2O5 (P), applied individually or in combination. The impact (synergistic or antagonistic) on germination homogenization and root growth under salinity conditions (0 mM, 100 mM, 150 mM and 200 mM of NaCl) was studied.

Result: Various salinity levels have affected all germination parameters and seedlings growth compared to control set. Root establishment was gradually decreased with increasing salinity levels. Remarkably, the combination between mineral elements enhanced the emergence traits of Vicia faba L. seeds more effectively. Inversely, the individual mineral elements had beneficial effects on root length compared to mineral combination in stressed seedlings. Synergistic effects were observed with PK, DP, DK and DPK nutrients which improved the germination percentage and volume of the root system of seedlings at 150 mM and 200 mM of salinity levels. Principal component analysis (PCA), heat map and cluster analysis showed the resilience of faba bean to salt stress during the early stages of seedling establishment. This study found under salinity; the combined mineral nutrients induced a synergistic impact on germination attributes and an antagonistic effect on root system formation.

INTRODUCTION

Faba bean is an important legume for human and animal food and sustainable agriculture due to the ability of root nodules to improve soil fertility by fixing atmospheric nitrogen (Rubiales, 2010). Faba bean seeds are rich in proteins, carbohydrates, polyphenols, carotenoids and L-dopa (levadopa), a dopamine precursor used in pharmacology for the treatment of Parkinson’s disease (Fuentes-Herrera  et al., 2025). In addition, its roles in plant anchorage, raw sap absorption, nutrient storage and yield stability in stressful environments, the performance of agricultural crops depends on the roots, which are considered the first barrier exposed to stress. Mass and Hoffman (1977) classified faba bean as sensitive to abiotic stresses such as salinity. Salinity has negative effects on the developmental stages of the plant. Germination of seeds and seedling establishment are the most vulnerable and lead to reduced crop yield (Batool et al., 2022; Nabi et al., 2020).
       
In arid and subarid areas where rainfall is scarce and erratic, increasing agricultural yields necessitates the utilization of resistant species to abiotic stresses, as well as the use of well-balanced fertilization based on the selection of suitable mineral nutrients (e.g. NO3, PO-24, K+, Ca+2, Mg+2) found in soil which significantly influence the mineral nutrition and plant development (Radulov and Berbecea, 2024). In fact, the nitrogen is a crucial for photosynthesis, respiration and protein synthesis while phosphorus is implied in energy transfer and root development. Physiologically, the potassium balances membrane potential, activates enzymes and regulates osmotic pressure (Cao et al., 2025). Whereas, the calcium is crucial for maintaining cell wall integrity, membrane functions and root development (Sahin et al., 2025) and Mg2+ acts as a cofactor for various enzymes and being a core component of chlorophyll in green tissues (Tang and Luan, 2017).
       
Mineral nutrition is one of the most effective a way to mitigate the negative impact of abiotic stresses in crop and improve plant resilience (Ahmad et al., 2016; Liu et al., 2025; Bouchenafa et al., 2026). Understanding how mineral elements interact with biotic and abiotic stress responses is crucial for designing plant responses under stressful conditions.
       
Thus, we hypothesized that using different mineral elements separately or combined could result in synergistic and/or antagonistic interactions that control nutritional homeostasis and salt tolerance. As few or no works have investigated this strategy as result the aims of this study were to determine the effectiveness of KNO3, Snezhinka CaO-MgO and P2O5 separately or combined on seed germination attributes and root system establishment to enhance seed emergence of the faba bean in salt-affected soils. The contribution of each nutrient in the improvement of the plant resilience to salinity and rational fertilization were also explored.

MATERIALS AND METHODS

Experiment setup
 
The experiments were conducted at soils biology laboratory, University of Sciences and Technology Houari Boumediene in Algiers, Algeria, during 2023 and 2024. The faba bean cv Sidi Aich seeds were obtained from the Technical Institute of Field Crops of Elkhroub in Algeria. Uniform and healthy seeds were selected and sterilized using 1% sodium hypochlorite for 4-5 min and then rinsed five times with sterilized water to eliminate chemical and biological contaminations.
 
Mineral seed preparation
 
The selected seeds were immersed for 3h in eight different mineral solutions such as: distilled water (C’); K (0.5% KNO3); D (0.3% CaO-MgO); P (0.9% P2O5); DK; DP; PK and DPK (Mariyapillai and Kulanthaivel, 2024). The control treatments C (distilled water) and non-immersed seeds were used as to evaluate the effects of these solutions on seed germination and root growth.
 
Salinity treatment
 
The mineral immersed and control seeds were subjected to salt stress with different concentrations of NaCl (0 mM as control, 100 mM, 150 mM and 200 mM of NaCl). An average of 12 seeds from each treatment were placed in Petri dishes containing layers of filter paper moistened with 15 mL of saline solution and incubated in darkness at 25°C during 7 days in complete randomized block design (CRBD) with three replications. Germination was considered to have occurred when the radicals were 2 mm long (Tanveer et al., 2010).
 
Germination parameters calculation
 
Final germination percentage (FGP) was calculated using the following equation (Bajji et al., 2002).
 
 
  
With
Ng = Number of the germinated seeds on the last day of counting.
Nt = Total seeds in each treatment.
       
Mean daily germination (MDG) was calculated using Osborne and Mercer (1993) method:
 
  
        
Germination speed (SG) was calculated by using the equation below as described by Shahba et al., (2008).
   
  
Where,
gi = Total germination percentage on an incubation day.
i = Total germination percentage on the previous day g.
(i-1) = Divided by the incubation day i.
 
Chemical determination of soak media
 
The electric conductivity (EC) and pH of the soak solution was estimated after seed germination and seedling stages using conductometer and pH meter.
 
Seedling growth parameters
 
After germination, three seedlings from each treatment were transferred into hydroponic system using vacuums containing 20 mL of the same mineral solutions used in germination test: distilled water, K (0.5% KNO3); D (0.3% CaO-MgO); P (0.9% P2O5) separately and DK; DP; PK and DPK as combined elements (de la Rosa-Rodríguez  et al., 2025). Salinity was applied using the NaCl at different concentrations (0 mM, 100 mM, 150 mM and 200 mM). 
 
Root parameters determination
 
In order to determine the effect of salinity and nutrients mineral on root’s growth, the primary root length (RL), primary root diameter (IRD), lateral roots number (SNR) and root fresh weight (FRW) were measured for each treatment.
 
Mineral nutrients interactions determination
 
Under salinity, interactions among mineral nutrients could influence the germination and seedling establishment process. This approach was assessed by investigating the relationship between concentrations of mineral elements supply under stressful conditions using the following formula (Robson and Pitman, 1983; Wallace, 1990):
Eab/EC = (Ea/EC) x (Eb/EC) as zero-interactions between elements
Eab/EC < (Ea/EC) x (Eb/EC) as antagonism between elements
Eab/EC > (Ea/EC) x (Eb/EC) as synergy between elements
 
Where,
Eab = Indicated effect of combined mineral nutrient.
Ea = Effect of nutrient a and Eb was effect of nutrient b.
EC = Corresponded to effect of control condition.
 
Statistical analysis
 
Statistical study was analyzed by ANOVA test, principal component analysis (PCA), Person’s correlation and cluster analysis. All data were performed by the least significant difference (LSD) at p≤0.05 using SPSS 25.0 software.

RESULTS AND DISCUSSION

Statistical analysis indicated a highly significant effect of the salinity (P<0.01, F=179.21) and the mineral nutrients (P<0.01, F=3.896) on seeds germination percentage. Without salinity and on the second day, the germination rate was on average over 47% using mineral nutrients versus 16% in control seeds (Table 1), but after three days, these germination rates exceeded 86% under all treatments. Under control conditions and low salinity (100 mM), seeds exhibited an increase in germination percentage, whereas excessive salinity inhibited seed emergence (-15%) (Table 1). Salinity induced disruption of cellular mechanisms caused by low water availability in cells following ionic and osmotic stress which decreased the capacity to absorb water and essential nutrients necessary for embryo survival, triggering of the germination process, growth and development of plants (Khadraji et al., 2020; Nabi et al., 2025). Application of mineral nutrients under salinity on germination seeds under salinity revealed significant interaction affecting germination seeds (P<0.05, F = 1.72). From 3rd day, the 100 mM of NaCl induced the maximum of germination using PK combination (89%), DP combination (89%) and DPK combination (86%) compared with P (52%) and D (58%) alone (Table 1). This improvement of germination rates could be attributed to synergistic relationship between P, K and D (Table 1). Analysis of these results might suggest that P combined with others elements would have positive effect on germination process compared to P applied alone.  P would have contributed to the earlier and enhanced commencement of metabolic activities (Zhang et al., 2020). Based on Wallace (1990), the P, K and D combinations would induce a synergistic interaction between them referring to the response that is greater than individual responses (Table 1).

Table 1: Effects of different mineral elements on the germination percentage (GP) of faba bean seeds under saline conditions over 6 days.


       
Under low salinity, application of K and DP mineral nutrients significantly (P<0.001, F=16.54) improved MDG by about 27% and 22% respectively compared to none salinity. However, increasing the NaCl concentrations (200 mM) decreased the MDG of seeds (Table 2). Remarkably, the use of the P individually induced the lowest rate of MDG (9.72%) could be due to the storage of phosphorus in the form of phytate in seeds which are considered as antagonists limiting the absorption of other bio-available micronutrients such as Fe and Zn (Raboy, 2020). Nevertheless, the MDG parameter was enhanced when P was combined to DP, DK and DPK recording a higher rate, 13.43%, 12.96% and 12.5% respectively supporting the synergistic interactions between the three nutrients at germination stage. In fact, the calcium and magnesium have beneficial influence on starch synthesis and accumulation, as well as the accumulation of phytohormones (indole-3- acetic acid) and gibberellic acid) on maize growth improving the activity of catalytic enzymes on starch for embryo development (He et al., 2023).

Table 2: Effect of different mineral elements on the mean daily germination (MDG%) and speed of germination (SG) of faba bean seeds under salinity.


       
Under salinity (200 mM), the synergistic effect between the PK, DP and DPK combinations was confirmed by high germination speed of seeds (+ 42%) particularly using PK treatment. However, the interaction of salinity x mineral nutrients (P=0.173, F=1.37) was not significant at 5% (Table 2). The positive effect of PK combination could be explained by the stimulatory effects of K on establishment of hormonal balance, reduction of growth inhibitors like IAA and GA (Manono, 2026) and an increase of activities of total soluble sugars and phenolic compounds (Ali et al., 2020). It could activate the biological defense mechanisms, including antioxidant enzymes (Catalase, Ascorbate Peroxidase, Superoxide Dismutase, Glutathione Peroxidase and Polyphenol Oxidase and antioxidant metabolites (DPPH antioxidant activity, phenolic, flavonoid, ascorbic acids), thus mitigating the effects of oxidative stress (Mamedi et al., 2022). Moreover, the KNO3 and CaCl2 were helpful in improving seed germination and seedling growth by protecting the membrane integrity through detoxification of over-accumulated ROS in the stressful conditions (Salles et al., 2019; Liu et al., 2025).
       
Salinity induced significant effects on seedling root traits (p≤0.05).  In fact, ANOVA analysis revealed a highly significant effect of the salinity (P<0.001, F=385.915), mineral nutrients (P<0.001, F=5.28) and the interaction of salinity x mineral nutrients (P<0.001, F=3.406) on primary root length. The K and D seedlings exhibited better root length (9.6 mm and 5.9 mm respectively) under stressful conditions compared to those obtained with DK (4.8 mm) and DPK (4.6 mm) (Fig 1).  Remarkably, the moderate and severe salinity combined to DPK % decreased root length by about 50% but increased the root diameter suggesting an antagonistic interaction between mineral nutrients applied (Fig 1). These results were in alignment with those obtained with Acikbas et al., (2021) on the forage pea and by Mohammad et al., (2024) on the rice. They reported that the reduction in length primary root is caused by salt ion accumulation, which inhibits root elongation and decreases the root meristem size affecting the formation of root hairs and overall root development (Robin et al., 2016).

Fig 1: Effects of different mineral elements combined or no on primary root length (cm), primary root diameter (cm), pH and electrical conductivity (EC) of the soaking solution of studied faba bean seedlings under salinity.


       
Chemical analysis of hydroponic solution revealed an increase of pH from acidity (pH≤3) to neutrality (pH ~ 6-7) indicating an increase of final pH under salinity and using the mineral nutrients separately and in combinations (Fig 1). Whereas, the electric conductivity recorded a high level in all treatments compared to solution control. Correlation matrix revealed a negative relationship between conductivity (EC) of the medium and germination parameters, seedling traits and pH of the medium. Nevertheless, seed emergence was strongly and positively correlated with plant primary root traits (r≥0.7) but both parameters were slightly influenced by the pH of seed soaking solution (Table 3). These could suggest that the increase of pH medium was in favor of improvement of germination seeds response and consequently the rooting establishment of seedlings under salinity as previously demonstrated in chickpea under salinity (Nabi et al., 2025; Sadji-Ait Kaci  et al., 2022). Ebone et al., (2019) reported that the chemical and osmotic effects due to exposure to minerals modified the metabolic and physiological behavior at the seedling stage probably for better resilience to stress. The PCA analysis showed that germination and root growth traits were scattered across the four quarters of the biplot, indicating diversity in the response of each plant developmental stage to mineral solutions under salt stress. Three groups were defined: the first component (CP1) corresponding to SG, MDG and GP positively impacted by individual and combined mineral solutions, especially PK0 under 100 mM and to a lesser extent to 150 mM of NaCl concentration. The second cluster (CP2) revealed by root attributes RL, SNR, IRD, FRW and pH’s medium positively stimulated by mineral solutions into non-salinity. The third component (CP3) clustered initial and final conductivity (Fin EC, In EC) of the soaking medium (Fig 2). Furthermore, the first axis revealed that minerals used separately or in combination under 150 mM salinity were close to the barycenter and opposite to the germination and root growth response, indicating their low impact on these parameters. Conversely, 200 mM combined to mineral solutions induced a negative impact on germination and root growth parameters, as shown by their distant and opposite position to axis 1. Axis 2 showed that the root growth parameters located in the fourth quarter were more sensitive to salinity than germination traits, but positively influenced by mineral under control conditions (Fig 2). Result was confirmed by high-speed germination, germination kinetics and germination percentage as shown in Vicia faba and chickpea (Chaker-Haddadj  et al., 2014; Sadji-Ait Kaci  et al., 2018).

Table 3: Pearson’s correlation coefficients between germination traits and root attributes under salinity combined with seed mineral solutions.



Fig 2: Principal components Analysis (PCA) of germination traits and roots establishment of faba bean under salinity.


       
Heat map analysis includes eight categories from thirty-six treatments and reveled three groups: under salinity, DP15 and DP1 mineral solutions significantly influenced the fresh root weight, P15 and DK1 had an impact on the primary root diameter while C0 and PK0 treatments acted on all root traits (Fig 3). Exogenous applied Ca2+ could repair the salinity inhibited uptake of K+ to sufficient level protecting cell membranes from Na+ toxicity, avoid cytosolic potassium leakage and could regulate the microRNAs playing a crucial role in plant hormones metabolism for enhancing germination performance (Ahmad et al., 2016).

​

Fig 3: Cluster heat map of 36 mineral pretreatments combinations on germination and root traits of faba bean growing under 4 levels of salinity. Hierarchical clustering analysis expresses the representation of indifferent groups based on similarity as well as the correlation with different traits.

CONCLUSION

To conclude, seed exposed to different mineral nutrients, either alone or in combination, revealed the diversity of responses during the germination phase and root development under salt. Under salinity, our previous hypothesis was confirmed by results showing a synergistic impact of combined mineral nutrients used on germination parameters of seeds while antagonistic interactions were induced by combined mineral elements on rooting establishment of seedlings. These results showed the importance of mineral nutrition as a way to estimate plant needs at various stages of growth in order to improve salinity resilience.

ACKNOWLEDGEMENT

The authors received no external funding for this work.
 
Disclaimers

The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
This study involved plant materials only. No experiments involving humans or animals were conducted; there, ethical approval and informed consent were not required.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Determination of Synergy/Antagonism Impact of Mineral Nutrients on Salinity Resilience During Germination and Root Establishment: Use of Hierarchical Clustering and Heat Map Analysis

    A
    Assia Chaker-Haddadj1,*
    H
    Hamida Sadji-Ait Kaci1
    F
    Fahima Nabi2
    F
    Farida Taguett1
    M
    Massakib Bekkaye1
    1Laboratory of Biology and Physiology of Organisms, Faculty of Biological Sciences, Team of Soils Biology, University of Sciences and Technology Houari Boumediene, Algiers, Algeria.
    2Laboratory of Biotechnology and Valorization of Biological Resources, Faculty of Sciences, Department of Nature and Life Sciences, Yahia Farès Medea University, Médéa 26100, Algiers, Algeria.

    ABSTRACT

    Background: Salinity is one of the most significant environmental restrictions threatening global food security. This study aimed to evaluate the effects of mineral nutrients, on germination traits, seedling parameters, root system development and tolerance to salinity of Vicia faba L.

    Methods: Seeds were immersed in solutions containing KNO3 (K), CaO-MgO (D) and P2O5 (P), applied individually or in combination. The impact (synergistic or antagonistic) on germination homogenization and root growth under salinity conditions (0 mM, 100 mM, 150 mM and 200 mM of NaCl) was studied.

    Result: Various salinity levels have affected all germination parameters and seedlings growth compared to control set. Root establishment was gradually decreased with increasing salinity levels. Remarkably, the combination between mineral elements enhanced the emergence traits of Vicia faba L. seeds more effectively. Inversely, the individual mineral elements had beneficial effects on root length compared to mineral combination in stressed seedlings. Synergistic effects were observed with PK, DP, DK and DPK nutrients which improved the germination percentage and volume of the root system of seedlings at 150 mM and 200 mM of salinity levels. Principal component analysis (PCA), heat map and cluster analysis showed the resilience of faba bean to salt stress during the early stages of seedling establishment. This study found under salinity; the combined mineral nutrients induced a synergistic impact on germination attributes and an antagonistic effect on root system formation.

    INTRODUCTION

    Faba bean is an important legume for human and animal food and sustainable agriculture due to the ability of root nodules to improve soil fertility by fixing atmospheric nitrogen (Rubiales, 2010). Faba bean seeds are rich in proteins, carbohydrates, polyphenols, carotenoids and L-dopa (levadopa), a dopamine precursor used in pharmacology for the treatment of Parkinson’s disease (Fuentes-Herrera  et al., 2025). In addition, its roles in plant anchorage, raw sap absorption, nutrient storage and yield stability in stressful environments, the performance of agricultural crops depends on the roots, which are considered the first barrier exposed to stress. Mass and Hoffman (1977) classified faba bean as sensitive to abiotic stresses such as salinity. Salinity has negative effects on the developmental stages of the plant. Germination of seeds and seedling establishment are the most vulnerable and lead to reduced crop yield (Batool et al., 2022; Nabi et al., 2020).
           
    In arid and subarid areas where rainfall is scarce and erratic, increasing agricultural yields necessitates the utilization of resistant species to abiotic stresses, as well as the use of well-balanced fertilization based on the selection of suitable mineral nutrients (e.g. NO3, PO-24, K+, Ca+2, Mg+2) found in soil which significantly influence the mineral nutrition and plant development (Radulov and Berbecea, 2024). In fact, the nitrogen is a crucial for photosynthesis, respiration and protein synthesis while phosphorus is implied in energy transfer and root development. Physiologically, the potassium balances membrane potential, activates enzymes and regulates osmotic pressure (Cao et al., 2025). Whereas, the calcium is crucial for maintaining cell wall integrity, membrane functions and root development (Sahin et al., 2025) and Mg2+ acts as a cofactor for various enzymes and being a core component of chlorophyll in green tissues (Tang and Luan, 2017).
           
    Mineral nutrition is one of the most effective a way to mitigate the negative impact of abiotic stresses in crop and improve plant resilience (Ahmad et al., 2016; Liu et al., 2025; Bouchenafa et al., 2026). Understanding how mineral elements interact with biotic and abiotic stress responses is crucial for designing plant responses under stressful conditions.
           
    Thus, we hypothesized that using different mineral elements separately or combined could result in synergistic and/or antagonistic interactions that control nutritional homeostasis and salt tolerance. As few or no works have investigated this strategy as result the aims of this study were to determine the effectiveness of KNO3, Snezhinka CaO-MgO and P2O5 separately or combined on seed germination attributes and root system establishment to enhance seed emergence of the faba bean in salt-affected soils. The contribution of each nutrient in the improvement of the plant resilience to salinity and rational fertilization were also explored.

    MATERIALS AND METHODS

    Experiment setup
     
    The experiments were conducted at soils biology laboratory, University of Sciences and Technology Houari Boumediene in Algiers, Algeria, during 2023 and 2024. The faba bean cv Sidi Aich seeds were obtained from the Technical Institute of Field Crops of Elkhroub in Algeria. Uniform and healthy seeds were selected and sterilized using 1% sodium hypochlorite for 4-5 min and then rinsed five times with sterilized water to eliminate chemical and biological contaminations.
     
    Mineral seed preparation
     
    The selected seeds were immersed for 3h in eight different mineral solutions such as: distilled water (C’); K (0.5% KNO3); D (0.3% CaO-MgO); P (0.9% P2O5); DK; DP; PK and DPK (Mariyapillai and Kulanthaivel, 2024). The control treatments C (distilled water) and non-immersed seeds were used as to evaluate the effects of these solutions on seed germination and root growth.
     
    Salinity treatment
     
    The mineral immersed and control seeds were subjected to salt stress with different concentrations of NaCl (0 mM as control, 100 mM, 150 mM and 200 mM of NaCl). An average of 12 seeds from each treatment were placed in Petri dishes containing layers of filter paper moistened with 15 mL of saline solution and incubated in darkness at 25°C during 7 days in complete randomized block design (CRBD) with three replications. Germination was considered to have occurred when the radicals were 2 mm long (Tanveer et al., 2010).
     
    Germination parameters calculation
     
    Final germination percentage (FGP) was calculated using the following equation (Bajji et al., 2002).
     
     
      
    With
    Ng = Number of the germinated seeds on the last day of counting.
    Nt = Total seeds in each treatment.
           
    Mean daily germination (MDG) was calculated using Osborne and Mercer (1993) method:
     
      
            
    Germination speed (SG) was calculated by using the equation below as described by Shahba et al., (2008).
       
      
    Where,
    gi = Total germination percentage on an incubation day.
    i = Total germination percentage on the previous day g.
    (i-1) = Divided by the incubation day i.
     
    Chemical determination of soak media
     
    The electric conductivity (EC) and pH of the soak solution was estimated after seed germination and seedling stages using conductometer and pH meter.
     
    Seedling growth parameters
     
    After germination, three seedlings from each treatment were transferred into hydroponic system using vacuums containing 20 mL of the same mineral solutions used in germination test: distilled water, K (0.5% KNO3); D (0.3% CaO-MgO); P (0.9% P2O5) separately and DK; DP; PK and DPK as combined elements (de la Rosa-Rodríguez  et al., 2025). Salinity was applied using the NaCl at different concentrations (0 mM, 100 mM, 150 mM and 200 mM). 
     
    Root parameters determination
     
    In order to determine the effect of salinity and nutrients mineral on root’s growth, the primary root length (RL), primary root diameter (IRD), lateral roots number (SNR) and root fresh weight (FRW) were measured for each treatment.
     
    Mineral nutrients interactions determination
     
    Under salinity, interactions among mineral nutrients could influence the germination and seedling establishment process. This approach was assessed by investigating the relationship between concentrations of mineral elements supply under stressful conditions using the following formula (Robson and Pitman, 1983; Wallace, 1990):
    Eab/EC = (Ea/EC) x (Eb/EC) as zero-interactions between elements
    Eab/EC < (Ea/EC) x (Eb/EC) as antagonism between elements
    Eab/EC > (Ea/EC) x (Eb/EC) as synergy between elements
     
    Where,
    Eab = Indicated effect of combined mineral nutrient.
    Ea = Effect of nutrient a and Eb was effect of nutrient b.
    EC = Corresponded to effect of control condition.
     
    Statistical analysis
     
    Statistical study was analyzed by ANOVA test, principal component analysis (PCA), Person’s correlation and cluster analysis. All data were performed by the least significant difference (LSD) at p≤0.05 using SPSS 25.0 software.

    RESULTS AND DISCUSSION

    Statistical analysis indicated a highly significant effect of the salinity (P<0.01, F=179.21) and the mineral nutrients (P<0.01, F=3.896) on seeds germination percentage. Without salinity and on the second day, the germination rate was on average over 47% using mineral nutrients versus 16% in control seeds (Table 1), but after three days, these germination rates exceeded 86% under all treatments. Under control conditions and low salinity (100 mM), seeds exhibited an increase in germination percentage, whereas excessive salinity inhibited seed emergence (-15%) (Table 1). Salinity induced disruption of cellular mechanisms caused by low water availability in cells following ionic and osmotic stress which decreased the capacity to absorb water and essential nutrients necessary for embryo survival, triggering of the germination process, growth and development of plants (Khadraji et al., 2020; Nabi et al., 2025). Application of mineral nutrients under salinity on germination seeds under salinity revealed significant interaction affecting germination seeds (P<0.05, F = 1.72). From 3rd day, the 100 mM of NaCl induced the maximum of germination using PK combination (89%), DP combination (89%) and DPK combination (86%) compared with P (52%) and D (58%) alone (Table 1). This improvement of germination rates could be attributed to synergistic relationship between P, K and D (Table 1). Analysis of these results might suggest that P combined with others elements would have positive effect on germination process compared to P applied alone.  P would have contributed to the earlier and enhanced commencement of metabolic activities (Zhang et al., 2020). Based on Wallace (1990), the P, K and D combinations would induce a synergistic interaction between them referring to the response that is greater than individual responses (Table 1).

    Table 1: Effects of different mineral elements on the germination percentage (GP) of faba bean seeds under saline conditions over 6 days.


           
    Under low salinity, application of K and DP mineral nutrients significantly (P<0.001, F=16.54) improved MDG by about 27% and 22% respectively compared to none salinity. However, increasing the NaCl concentrations (200 mM) decreased the MDG of seeds (Table 2). Remarkably, the use of the P individually induced the lowest rate of MDG (9.72%) could be due to the storage of phosphorus in the form of phytate in seeds which are considered as antagonists limiting the absorption of other bio-available micronutrients such as Fe and Zn (Raboy, 2020). Nevertheless, the MDG parameter was enhanced when P was combined to DP, DK and DPK recording a higher rate, 13.43%, 12.96% and 12.5% respectively supporting the synergistic interactions between the three nutrients at germination stage. In fact, the calcium and magnesium have beneficial influence on starch synthesis and accumulation, as well as the accumulation of phytohormones (indole-3- acetic acid) and gibberellic acid) on maize growth improving the activity of catalytic enzymes on starch for embryo development (He et al., 2023).

    Table 2: Effect of different mineral elements on the mean daily germination (MDG%) and speed of germination (SG) of faba bean seeds under salinity.


           
    Under salinity (200 mM), the synergistic effect between the PK, DP and DPK combinations was confirmed by high germination speed of seeds (+ 42%) particularly using PK treatment. However, the interaction of salinity x mineral nutrients (P=0.173, F=1.37) was not significant at 5% (Table 2). The positive effect of PK combination could be explained by the stimulatory effects of K on establishment of hormonal balance, reduction of growth inhibitors like IAA and GA (Manono, 2026) and an increase of activities of total soluble sugars and phenolic compounds (Ali et al., 2020). It could activate the biological defense mechanisms, including antioxidant enzymes (Catalase, Ascorbate Peroxidase, Superoxide Dismutase, Glutathione Peroxidase and Polyphenol Oxidase and antioxidant metabolites (DPPH antioxidant activity, phenolic, flavonoid, ascorbic acids), thus mitigating the effects of oxidative stress (Mamedi et al., 2022). Moreover, the KNO3 and CaCl2 were helpful in improving seed germination and seedling growth by protecting the membrane integrity through detoxification of over-accumulated ROS in the stressful conditions (Salles et al., 2019; Liu et al., 2025).
           
    Salinity induced significant effects on seedling root traits (p≤0.05).  In fact, ANOVA analysis revealed a highly significant effect of the salinity (P<0.001, F=385.915), mineral nutrients (P<0.001, F=5.28) and the interaction of salinity x mineral nutrients (P<0.001, F=3.406) on primary root length. The K and D seedlings exhibited better root length (9.6 mm and 5.9 mm respectively) under stressful conditions compared to those obtained with DK (4.8 mm) and DPK (4.6 mm) (Fig 1).  Remarkably, the moderate and severe salinity combined to DPK % decreased root length by about 50% but increased the root diameter suggesting an antagonistic interaction between mineral nutrients applied (Fig 1). These results were in alignment with those obtained with Acikbas et al., (2021) on the forage pea and by Mohammad et al., (2024) on the rice. They reported that the reduction in length primary root is caused by salt ion accumulation, which inhibits root elongation and decreases the root meristem size affecting the formation of root hairs and overall root development (Robin et al., 2016).

    Fig 1: Effects of different mineral elements combined or no on primary root length (cm), primary root diameter (cm), pH and electrical conductivity (EC) of the soaking solution of studied faba bean seedlings under salinity.


           
    Chemical analysis of hydroponic solution revealed an increase of pH from acidity (pH≤3) to neutrality (pH ~ 6-7) indicating an increase of final pH under salinity and using the mineral nutrients separately and in combinations (Fig 1). Whereas, the electric conductivity recorded a high level in all treatments compared to solution control. Correlation matrix revealed a negative relationship between conductivity (EC) of the medium and germination parameters, seedling traits and pH of the medium. Nevertheless, seed emergence was strongly and positively correlated with plant primary root traits (r≥0.7) but both parameters were slightly influenced by the pH of seed soaking solution (Table 3). These could suggest that the increase of pH medium was in favor of improvement of germination seeds response and consequently the rooting establishment of seedlings under salinity as previously demonstrated in chickpea under salinity (Nabi et al., 2025; Sadji-Ait Kaci  et al., 2022). Ebone et al., (2019) reported that the chemical and osmotic effects due to exposure to minerals modified the metabolic and physiological behavior at the seedling stage probably for better resilience to stress. The PCA analysis showed that germination and root growth traits were scattered across the four quarters of the biplot, indicating diversity in the response of each plant developmental stage to mineral solutions under salt stress. Three groups were defined: the first component (CP1) corresponding to SG, MDG and GP positively impacted by individual and combined mineral solutions, especially PK0 under 100 mM and to a lesser extent to 150 mM of NaCl concentration. The second cluster (CP2) revealed by root attributes RL, SNR, IRD, FRW and pH’s medium positively stimulated by mineral solutions into non-salinity. The third component (CP3) clustered initial and final conductivity (Fin EC, In EC) of the soaking medium (Fig 2). Furthermore, the first axis revealed that minerals used separately or in combination under 150 mM salinity were close to the barycenter and opposite to the germination and root growth response, indicating their low impact on these parameters. Conversely, 200 mM combined to mineral solutions induced a negative impact on germination and root growth parameters, as shown by their distant and opposite position to axis 1. Axis 2 showed that the root growth parameters located in the fourth quarter were more sensitive to salinity than germination traits, but positively influenced by mineral under control conditions (Fig 2). Result was confirmed by high-speed germination, germination kinetics and germination percentage as shown in Vicia faba and chickpea (Chaker-Haddadj  et al., 2014; Sadji-Ait Kaci  et al., 2018).

    Table 3: Pearson’s correlation coefficients between germination traits and root attributes under salinity combined with seed mineral solutions.



    Fig 2: Principal components Analysis (PCA) of germination traits and roots establishment of faba bean under salinity.


           
    Heat map analysis includes eight categories from thirty-six treatments and reveled three groups: under salinity, DP15 and DP1 mineral solutions significantly influenced the fresh root weight, P15 and DK1 had an impact on the primary root diameter while C0 and PK0 treatments acted on all root traits (Fig 3). Exogenous applied Ca2+ could repair the salinity inhibited uptake of K+ to sufficient level protecting cell membranes from Na+ toxicity, avoid cytosolic potassium leakage and could regulate the microRNAs playing a crucial role in plant hormones metabolism for enhancing germination performance (Ahmad et al., 2016).

    ​

    Fig 3: Cluster heat map of 36 mineral pretreatments combinations on germination and root traits of faba bean growing under 4 levels of salinity. Hierarchical clustering analysis expresses the representation of indifferent groups based on similarity as well as the correlation with different traits.

    CONCLUSION

    To conclude, seed exposed to different mineral nutrients, either alone or in combination, revealed the diversity of responses during the germination phase and root development under salt. Under salinity, our previous hypothesis was confirmed by results showing a synergistic impact of combined mineral nutrients used on germination parameters of seeds while antagonistic interactions were induced by combined mineral elements on rooting establishment of seedlings. These results showed the importance of mineral nutrition as a way to estimate plant needs at various stages of growth in order to improve salinity resilience.

    ACKNOWLEDGEMENT

    The authors received no external funding for this work.
     
    Disclaimers

    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    This study involved plant materials only. No experiments involving humans or animals were conducted; there, ethical approval and informed consent were not required.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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