Effects of the Arbuscular Mycorrhizal Fungus Gigaspora albida (Gigasporaceae) on the Physiology, Growth, and Na/K Balance of Creole Corn (Poaceae) Under Different Salinity Levels

Abstract

Arbuscular mycorrhizal fungi (AMFs) can alleviate salt stress in plants by promoting growth. The mitigating effect of the AMF Gigaspora albida on the physiology, growth, and Na⁺/K⁺ balance in heirloom maize under different dilutions of saline wastewater was evaluated. The study was conducted in a greenhouse under a completely randomized design (CRD) in a 3 × 4 factorial scheme, with six replicates. The treatments consisted of three mycorrhizal conditions (M1—control plants without the AMF; M2—plants inoculated with G. albida; and M3—plants inoculated with G. albida plus the soil microbiota) and four levels of electrical conductivity (ECw): 0.5, 1.8, 3.1, and 4.4 dS m⁻¹. The results indicate that saline wastewater affects the physiology of heirloom maize. The symbiosis in M2 and M3 mitigated the stress in PSII by dissipating heat. The M3 treatment alleviated ionic stress in maize, reduced the Na⁺/K⁺ ratio in the aerial part, and increased the MSPA, MSRA, AP, and DC at EC_a levels of 1.8 and 3.1 dS m⁻¹. The M1 plants adapted by investing in root growth to tolerate the high salinity. In M2, the plant–AMF interaction did not mitigate the effects of high salinity, showing the worst growth performance. The saline wastewater reduced the percentage of G. albida colonization. An EC_a of 2.9 dS m⁻¹ favored a high spore density.

1. Introduction

Water scarcity is a paramount global concern, which is particularly pronounced in regions like Brazil, where irrigation often becomes the sole recourse to ensure agricultural productivity, especially in hot, arid climates such as the semi-arid Northeast region [1]. The arid and semi-arid areas’ scarcity underscores the imperative for sustainable water management technologies, particularly in agriculture. Among these, water reuse emerges as a prevalent practice, notably through irrigation with saline water sourced from agricultural drainage wells and brackish water treatment plant effluents. When effectively implemented, this practice fulfills crop water requirements while reducing the demand for freshwater resources [2]. However, without proper management techniques, it poses a significant challenge for agriculture. High salt concentrations in reused water can severely limit agricultural outputs, reducing crop yields to economically unviable levels [3,4].

Restrictions on plant development stem from the osmotic and ionic stress induced by excessive salt levels, hindering water absorption, nutrient assimilation, and transport [5,6]. Osmotic stress, characterized by Na⁺ and Cl⁻ accumulation in tissues, triggers nutritional stress and tissue cytotoxicity [7,8], disrupting metabolic, physiological, and biochemical pathways [9] and leading to a redox imbalance and biomolecule damage via lipid peroxidation [10].

Crop responses to salinity, in terms of sensitivity and tolerance, exhibit variability [11,12]. Adaptation mechanisms include alterations in photosynthetic pathways, the synthesis of compatible osmolytes, the activation of enzymatic and non-enzymatic antioxidant systems, and selective ion absorption, enhancing plant survival in saline environments [13,14].

Selective ion absorption is a crucial strategy in salinity tolerance, allowing plants to preferentially absorb essential nutrients even in the presence of higher concentrations of non-essential ions [15]. Simultaneously, it diminishes sodium uptake and accumulation in tissues [16], thus maintaining a favorable K⁺/Na⁺ ratio that is essential for sustained plant growth [17].

Among the strategies to utilize saline waste for irrigation, employing arbuscular mycorrhizal fungi (AMFs) has emerged as an effective approach to mitigate saline stress in plants [18,19,20]. Symbiosis with AMFs promotes the expression of osmoregulatory substances such as proline, glycine betaine, and polyamines, aiding in the regulation and maintenance of cellular water potential. This leads to improved water use efficiency, the maintenance of cell turgor, gas exchange, and subsequently, enhanced photosynthetic rates [21,22]. Nutritionally, this symbiosis promotes nutrient absorption and, under saline conditions, can reduce the uptake of toxic ions such as Na⁺ and Cl⁻, which compromise the ionic balance in photosynthetic tissues [23,24,25].

In this context, Gigaspora albida, an AMF belonging to the Gigasporaceae family [26], has been reported in the literature to enhance the growth of various crops. In a more recent study, G. albida improved the quality of Dipteryx alata seedlings by promoting increases in height, diameter, and dry biomass accumulation [27]. Under high salinity conditions (10, 15, and 20 dS m⁻¹), G. albida increased the tolerance of eucalyptus seedlings by maintaining the relative water content (RWC), enhancing nutrient uptake, particularly nitrogen, phosphorus, and potassium, and improving the K/Na ratio by reducing sodium accumulation [28]. For maize, although several studies in the literature highlighted the benefits of symbiotic relationships with AMFs, there is still a gap regarding the interaction of Gigaspora albida, associated with the original soil microbiota, with Creole maize varieties under saline stress.

The benefits of AMFs result from strengthening the plant’s resistance to salinity, increasing the absorption area for nutrients and water, promoting the selective uptake of elements, enhancing the efficiency of the photosynthetic apparatus, and reinforcing antioxidant defense mechanisms [29,30]. However, the interaction between non-native AMFs and soil microbiota remains poorly understood. According to [31], native soil microorganisms have a greater potential to increase plant yield. This potential can be attributed to the beneficial growth-promoting properties of these microorganisms, the harmonious symbiosis within the community, and their strong colonization ability compared to non-native ones.

AMFs and soil microbiota interact in complex ways that can enhance plant growth. Ref. [32] found that the interaction between AMFs and Bacillus spp. promotes greater phosphate solubilization and the absorption of phosphorus, zinc, and copper. Additionally, this interaction leads to an increased production of phytohormones that protect against biotic and abiotic stresses compared to isolated strains.

There is evidence that plant growth-promoting rhizobacteria stimulate the growth of AMFs. Among them, those in the genus Pseudomonas are more frequently found in the rhizosphere, while Arthrobacter and Bacillus are more common in the AMF hyphosphere. Some species of Rhizobium and Pseudomonas attach to fungal spores and hyphae; however, the colonization capacity varies considerably among different bacteria. Although AMFs can contribute to an increase in the nutritional status of the mycorrhizosphere by decomposing organic nitrogen (N₂) compounds, in the presence of nitrogen-fixing bacteria, there is a considerable increase in N₂ fixation, which is one of the main benefits of this interaction [33].

Under high salinity conditions, inoculation with endophytic Bacillus subtilis, both alone and in combination with AMFs, increased the levels of N, P, K, Mg, and Ca, as well as phosphatase activity in plant tissues. This confirmed that the enhanced nutrient uptake resulting from this interaction supports plant species development under salt stress. Additionally, reductions in Na⁺ and Cl⁻ levels were observed, demonstrating mitigation of the deleterious effects of salts [34]. Thus, native soil microorganisms associated with AMFs can be used as an alternative to optimize production in agroecosystems affected by high salinity.

The improvement of soil physical properties through the addition of AMFs is primarily attributed to the production of organic acids and glomalin, which protect against soil erosion, chelate heavy metals, enhance carbon sequestration, and stabilize soil macroaggregation. By recruiting bacteria that produce alkaline phosphatase, an enzyme involved in soil mineralization and associated with organic phosphorus availability, AMFs also enhance the soil’s chemical activity. Additionally, dead mycelia contribute to organic matter accumulation. AMFs influence the composition, diversity, and activity of soil microbial communities through antagonism or cooperation [29].

In the long term, the addition of AMFs can increase the organic carbon accumulation in agricultural soils; however, the successful establishment of AMFs depends on the soil properties, host plant, inoculum type, and experimental conditions [35]. The increased resistance of plants inoculated with AMFs to high salinity conditions can also be attributed to various biochemical and physiological mechanisms, which, according to [30], can be categorized into three groups: (1) enhanced nutrient uptake, maintenance of ionic homeostasis, improved water absorption, and osmotic balance; (2) increased photosynthetic efficiency and protection of the photosynthetic apparatus; and (3) modulation of the plant’s hormonal profile and induction of the antioxidant system to prevent ROS-induced damage.

In this context, we hypothesized that combining the AMF Gigaspora albida with soil microbiota can ameliorate the effects of saline stress on the growth, physiology, and Na⁺ and K⁺ balance of Creole corn (Ibra variety) when irrigated with saline waste from reverse osmosis. Hence, this study aimed to assess the impact of the arbuscular mycorrhizal fungus (AMF) Gigaspora albida on the growth, physiology, and Na⁺ and K⁺ balance of Creole corn (Ibra variety) under various levels of electrical conductivity from saline waste.

2. Materials and Methods

2.1. Location and Characterization of the Area

The study was conducted at the Universidad Federal Rural do Semi-Árido (UFERSA) in Mossoró, Rio Grande do Norte/RN (5°12′2.03″ N and 37°19′36.32″ W). The experiment was carried out in a greenhouse environment, using pots, from January to March 2022. The greenhouse covered an area of 126 m², with a ceiling height of 4.0 m, and was constructed with a metallic frame and a transparent plastic cover, while the walls were shaded at 50%. The average maximum temperature recorded was 37.4 °C, with daytime fluctuations, and the average minimum temperature hovered around 31 °C. The average relative humidity (RH) was 97.7% with a standard deviation of 86%.

2.2. Experimental Design

The experimental design followed a completely randomized model, in a 3 × 4 factorial scheme, with six replications; the plots contained polyethylene pots with a capacity of 30 L, totaling 72 experimental units. The treatments consisted of three mycorrhizal conditions (M1—control plants without the fungal inoculum; M2—plants with the G. albida fungal inoculum; and M3—plants with the G. albida fungal inoculum plus soil microbiota) and saline waste with four electrical conductivity levels (EC_a): 0.5, 1.8, 3.1 and 4.4 dS m⁻¹ (conductivity of 0.5 dS m⁻¹ is from the supply water, which was used as the control). The saline concentrations of the irrigation water were established by diluting reverse osmosis saline waste with supply water from the Rio Grande do Norte Water and Sewage Company (CAERN), Mossoró, Brazil.

2.3. Soil, Saline Waste, Plant, and Mycorrhizal Materials

Soil material with a sandy texture was gathered from the upper soil layer, approximately 0–30 cm deep, at the Rafael Fernandes Experimental Farm of UFERSA, situated in the rural vicinity of Mossoró/RN. For physical–chemical characterization, disturbed samples were collected and subsequently analyzed using the Embrapa methodology [36], as detailed in

Table 1. Physicochemical characterization of the eutrophic red Oxisol.

pH	CEes	OM	P	K+	Na+	Ca2+	Mg2+	Al3+	H + Al	SB	t	CEC
(water)	dS m−1	g/kg	________ mg/dm3 ______			_____________________ cmolc/dm3 _________________________
7.4	0.80	31.9	112.2	791.7	160.1	6.5	4.8	0.0	0.0	14.0	14.56	14.0
V	m	ESP	Sand			Silt			Clay
_______ % ______			_____________________ (g kg−1) _______________________
100	0.0	5.5	89.25			2.79			7.79

.

The pH in water was determined using a soil–water ratio of 1:2.5. CEes is the electrical conductivity of the soil–water extract at a ratio of 1:2.5. The elements P, Na⁺, and K⁺ were extracted using a Mehlich-1 extractor at a soil–extractor ratio of 1:10. The elements Ca²⁺, Mg²⁺, and Al³⁺ were extracted with 1 mol/L KCl using a soil–extractant ratio of 1:10. H + Al is the potential acidity extracted with 0.5 mol/L calcium acetate using a soil–extractor ratio of 1:15. SB is the sum of bases. t is the effective CEC. CEC is the soil CEC or CEC at pH 7.0. V is the base saturation. m is the aluminum saturation. ESP is the percentage of exchangeable sodium.

The saline waste was obtained from the water treatment facility of the Jurema Rural Settlement, situated along the RN-012 highway, which links Mossoró to the municipality of Tibau, RN. The physicochemical attributes of both the saline waste and the supply water are outlined in

Table 2. Physicochemical characteristics of the supply water (ABT) and the concentrated saline waste (RSC) used in the experiment.

	pH	CEes	K+	Na+	Ca2+	Mg2+	Cl	CO32−	HCO3−	RAS
	(H2O)	(dS m−1)	-------------------------------mmolc/L------------------------------
ABT	7.5	0.54	0.31	3.78	0.84	1.20	2.40	0.61	3.21	3.76
RSC	7.11	9.50	0.83	54.13	36.80	24.2	116	0	3.39	9.71

CEes—electrical conductivity of the soil saturation extract; RAS—sodium adsorption ratio.

.

Creole corn seeds, specifically of the Ibra variety, were procured from the 2021 harvest in the municipality of Umarizal. The Gigaspora albida arbuscular mycorrhizal fungi (AMF) inoculants were sourced from the Laboratory of Plant Physiology and Biochemistry (LFBP) at the State University of Rio Grande do Norte (UERN).

2.4. Experimental Stage

Soil Preparation, AMF Propagation, and Experimental Setup

To propagate the fungus, a substrate comprising soil and sand in a 3:1 (v/v) ratio was utilized; it was sterilized in an autoclave at 121 °C and 1 ATM for two hours, with a 24 h interval between successive autoclaving sessions. In a greenhouse, the sterilized substrate was transferred to polyethylene pots with an 8 L capacity, along with 8 g of inoculum soil containing fragments of colonized roots and propagules of the G. albida species. Subsequently, Panicum milaceum (millet) seeds were sown as trap plants to facilitate the proliferation of the AMF. After 60 days, soil portions weighing 1.5 kg containing the inoculum were collected and preserved in plastic bags at 4 °C for future use.

The substrate employed in the experiment was a blend of soil and organic compounds (0.7% (w/w) N, 0.5% (w/w) P2O5, 0.5% (w/w) K2O, maximum humidity of 50% (w/w), 15% (w/w) total organic carbon, CTC of 250, C/N (maximum) of 18, pH 8.0) in a 2:1 (v/v) ratio. This substrate underwent sterilization following the aforementioned protocol, except for the M3 treatment. Post-sterilization, the substrate was allocated into polyethylene pots with a 30 L capacity. Inoculation with 100 g of soil containing the AMF inoculum (675 spores/50 dm³ of soil) was conducted prior to sowing, which was positioned approximately 3 cm below the seeds. To maintain uniform microbial populations across all treatments, each plot of non-mycorrhizal plants received 100 mL of crude soil filtrate devoid of AMF spores. This filtrate was obtained from a suspension of 50 dm³ of soil, which was filtered through a sieve with pores measuring 0.053 mm in diameter, and subsequently filtered twice through qualitative filter paper to eliminate AMF propagules, as outlined in [37].

2.5. Irrigation and Fertilization

Irrigation was conducted on a daily basis to maintain a soil moisture close to its maximum retention capacity. Saline water irrigation commenced 20 days after thinning, in accordance with the electrical conductivity (EC_a) of each treatment. To induce salt stress, the diluted saline waste was progressively applied until the desired concentration was reached. The EC_a of the saline waste in each treatment was monitored using a portable conductivity meter (model Instrutherm CD-860) every two days. The irrigation was manually administered.

The daily irrigation depth (LD) was determined via drainage lysimetry, calculated every two days using lysimeters corresponding to each salinity level (average of two vessels). The calculation considered the volume applied (Va) via irrigation per vessel, which was obtained by subtracting the previous applied lamina (La) from the average drained volume (Vd), divided by the number of vessels (n), as per Equation (1).

Table 3. Volumes of saline waste applied to Creole corn (Zea mays L.) plants under the influence of an AMF as a function of EC_a.

	V. Irrigation (L Plant−1)
ECa (dS m−1)	M1/M2/M3
0.5	36,350
1.8	39,520
3.1	32,700
4.4	31,500

M1—control plants without fungal inoculum; M2—plants with fungal inoculum of G. albida; and M3—plants with fungal inoculum of G. albida plus soil microbiota.

presents the volumes of saline waste applied during cultivation.

$$Va=\left(La-Vd\right)/n$$

(1)

To ensure adequate nutrition, two applications of Hoagland and Arnon’s [38] solution, which is devoid of phosphorus, were administered at full concentration at 15-day intervals post-germination. At 34 days, fertigation as recommended in [39] was applied, which consisted of 72.73 kg of N ha⁻¹ and 39.28 kg of KCl ha⁻¹, split into two fractions. The fertilizer sources utilized were urea (45% N) and potassium chloride (60% K₂O).

The saline treatment irrigation commenced on the 20th day after thinning. Growth analyses were conducted following the methodology outlined in [40] after 30 days. The plant height (AP) was measured, using a measuring tape, as the distance between the ground and the youngest leaf; the stem diameter (DC) was recorded with a digital caliper (Digital Caliper 150 mm model) using the first internode of the plant as the measurement point; the number of leaves (NF) was determined by counting the leaves with fully expanded limbs.

2.6. Plant Analyses

2.6.1. Gas Exchange and Chlorophyll Fluorescence

Physiological analyses were conducted at the onset of the bolting phase (50 days after sowing). The readings were obtained in the morning, between 7:00 am and 9:00 am, from the fully expanded third leaf of the apical meristem, using a portable infrared gas analyzer (IRGA), specifically the LCPro Portable Photosynthesis System (ADC BioScientific Limited, Hertfordshire, UK). The IRGA maintained a controlled temperature of 25 °C, irradiation of 1200 photomotonic m⁻² s⁻¹, and an air flow of 200 mL min⁻¹. The quantified variables included transpiration (E) (mmol (H₂O) m⁻² s⁻¹), stomatal conductance (gs) (mol (H₂O) m⁻² s⁻¹), net assimilation rate (A), and leaf temperature (TI) (°C).

Subsequently, chlorophyll fluorescence readings were taken using a pulse-modulated fluorometer, specifically the OptiScience OS5p model (Marconi Manufacturer, Piracicaba, SP, Brazil. The Fv/Fm protocol was employed for assessments under dark conditions, following a 30 min dark adaptation of the leaves using accessory clips from the device to ensure that all reaction centers were open [41]. From these readings, the initial fluorescence (Fo), maximum fluorescence (FM), variable fluorescence (Fv = Fm − Fo), maximum quantum efficiency of photosystem II (PSII) (Fv/Fm), basal quantum yield of photochemical processes in PSII (Fo/Fm), and photochemical efficiency in PSII (Fv/Fo) were estimated.

The evaluations were conducted under light conditions utilizing the Yield protocol. The readings were taken by applying an actinic light source with a saturating multi-flash pulse that was connected to a clip for determining the active photosynthesis radiation (PAR-Clip). From these measurements, the following parameters were estimated: the initial fluorescence before the saturation pulse (F’), maximum fluorescence after adaptation to saturated light (Fm’), electron transport rate (ETR) (μmol (photons) m⁻² s⁻¹), and quantum efficiency of photosystem II Y(II). Additionally, the minimum fluorescence of illuminated plant tissue (Fo’) [42], photochemical extinction coefficient by the lake model (qL), regulated photochemical extinction yield (YNOP), and unregulated photochemical extinction yield (YNO) [43] were calculated from these data.

2.6.2. Growth Analysis

The leaf area (FA) was estimated by extracting leaf discs (area of 1.78 cm²) from the basal portion of the leaf, leaving the central vein intact. Subsequently, both the leaf discs and the remaining leaf tissues were dried in a forced air circulation oven at 70 °C until a constant weight was reached. The leaf area was calculated using the formula AF = PF × AD/PD, where AF represents the estimated leaf area, PF is the dry mass of the leaf, AD is the area of the removed leaf disc (1.78 cm²), and PD is the dry mass of the leaf discs, as per [44,45].

The dry matter of the shoot (MSPA) and root (MSR) was determined by dehydrating the fresh biomass in a forced air circulation oven at 70 °C until a constant weight was achieved. Prior to drying, a 1 g sample of fresh root was collected from each plot, washed, and preserved in an FAA solution (5% formaldehyde, 90% ethyl alcohol, and 5% acetic acid) for the subsequent analysis. Using the obtained dry mass data, the root-to-shoot ratio (R/PA) was calculated by dividing the dry mass of the root by the mass of the shoot, following the method described in [46].

2.6.3. Accumulation of Na and K in the Plant

The extraction of Na⁺ and K⁺ ions from the leaf, stem, and root tissues involved digesting 0.5 g of dry biomass in a muffle furnace at 500 °C. The biomass was combined with a nitric acid solution (containing nitric acid and 1 mol HCl) to produce an extract. The extract was then subjected to flame spectrophotometry to determine the concentrations of Na and K ions. Subsequently, the Na/K ratio was calculated based on the obtained Na and K concentrations, following the methodology described in [36].

2.6.4. Mycorrhizal Colonization Rate

The root colonization rate (TC) was assessed by examining roots that were diaphanized in 10% KOH, acidified with 1% HCl, and stained with 0.05% Trypan blue, following the protocol outlined in [47]. The percentage of root colonization was determined using the gridline intersection method on a checkered plate, using a binocular magnifying glass with a 40× magnification, as described in [48]. Roots were considered colonized by arbuscular mycorrhizal fungi (AMFs) if they exhibited at least one mycorrhizal structure (e.g., vesicles, arbuscules, or hyphae).

2.7. Variables Evaluated in the Soil

Soil Spore Density

The spore density in the soil was determined using the wet sieving method described in [49] for spore extraction. Following extraction, the spores were centrifuged in a 50% sucrose solution at 1106 g for three minutes, as outlined in [50]. The spore density per gram of soil was estimated from a diluted aliquot of the sample, which was then observed under a stereoscopic microscope (40×), Leica Microsystems manufacturer, Wetzlar, Germany, following the procedure described in [51].

2.8. Statistical Analysis

The results were subjected to analysis of variance using the F test. For the mycorrhizal condition factor, Tukey’s test was applied at a 5% level of significance, and for the EC_a factor of saline waste, regression analysis was applied. Data that did not present a normal distribution were transformed into square roots (SQRTs). The analyses were carried out using the statistical software SISVAR, version: 5.6 [52].

3. Results

Salinity affected the photosynthesis and the symbiotic relationship between the plants and AMF. The association with the AMF reduced the damage to the photosystems. The interaction of the EC of the saline waste and AMF produced a significant difference in the electron transport rate (p < 0.05) and regulated photochemical quenching quantum yield (p < 0.05). The EC_a of the saline waste significantly affected the stomatal conductance (p < 0.01), transpiration (p < 0.05), leaf temperature (p < 0.05), CO₂ assimilation rate (p < 0.05), intrinsic water use efficiency (p < 0.001), minimum fluorescence of illuminated plant tissue (p < 0.001), PSII quantum efficiency (p < 0.001), and maximum PSII quantum efficiency (p < 0.05). The FMA condition was significant for sweating (p < 0.05). The unregulated photochemical quenching quantum yield was not significant for either factor (

Table 4. Summary of analysis of variance for stomatal conductance (gs), transpiration (E), leaf temperature (Tl), CO₂ assimilation rate (A_N), intrinsic water use efficiency (A/gs), instantaneous water use efficiency (A/E), minimum fluorescence of illuminated plant tissue (Fo’), PSII quantum efficiency (Y), maximum PSII quantum efficiency (Fv/Fm), electron transport rate (ETR), photochemical quenching quantum yield regulated (YNPQ) and unregulated photochemical quenching quantum yield (YNO) of Creole corn plants as a function of EC_a and the AMF.

		Test F
		Gas Exchange
FV	GL	gs †	E	Tl	AN †	A/gs †	A/E
AMF	2	1709 ns	5180 *	0.626 ns	1483 ns	0.653 ns	0.667 ns
ECa (d Sm−1)	3	8569 **	3559 *	3141 *	3327 *	8929 ***	1131 ns
AMF × ECa	6	0.809 ns	1372 ns	0.952 ns	2112 ns	1375 ns	1929 ns
		Chlorophyll fluorescence
		Fo’ †	Y	Fv/FM	ETR †	Y(NPQ)	YNO
AMF	2	0.180 ns	0.666 ns	2657 ns	1106 ns	0.701 ns	0.576 ns
ECa (d Sm−1)	3	22.053 ***	22.569 ***	3339 *	6674 **	23.300 ***	2103 ns
AMF × ECa	6	2103 ns	2643 ns	1258 ns	2427 *	2517 *	1503 ns

^† Data transformed into square roots (SQRTs); FV—source of variation; GL—degrees of freedom; AMF—arbuscular mycorrhizal fungus; (EC_a)—electrical conductivity of saline wastewater; *** p < 0.001; ** p < 0.01; * p < 0.05; and ns—not significant.

).

The stomatal conductance (gs) of the irrigated corn exhibited a linear decrease in response to increasing electrical conductivity levels from the saline waste (EC_a). The highest and lowest values of corn gs were recorded as 0.631 and 0.414 mol (H₂O) m⁻² s⁻¹ at EC_a levels of 0.5 and 4.4 dS m⁻¹, respectively, representing a 34.38% decrease in gs (Figure 1A). When comparing the optimal gs results for corn, the plants irrigated with the saline waste demonstrated a reduction in gs of at least 24.4% when compared to those irrigated with freshwater.

The transpiration rate (E) of the corn plants showed variability with changes in the electrical conductivity of the saline waste. At an EC_a level of 1.91 dS m⁻¹, the transpiration rate peaked at 8.52 mmol (H₂O) m⁻² s⁻¹, representing a 3.4% increase compared to the 0.5 dS m⁻¹ level (Figure 1B). Comparing the plants irrigated with an EC of 0.5 dS m⁻¹ to those irrigated with the saline waste, the increase was 2.75%. When examining the average transpiration rates for the treatments with the AMF, the highest average was observed in the M2 plants, reaching 8.53 mmol (H₂O) m⁻² s⁻¹, which was statistically different from the M3 treatment with a value of 7.86 mmol (H₂O) m⁻² s⁻¹, representing a difference of 8.5% (Figure 1C). The transpiration rate of the M1 plants did not exhibit statistical differences compared to that of the M2 and M3 plants, with an average transpiration rate of 8.33 mmol (H₂O) m⁻² s⁻¹.

The CO₂ assimilation rate (AN) of the corn plants decreased with increasing EC_a of the saline waste. The corn plants irrigated with an EC_a of 0.5 dS m⁻¹ demonstrated a higher CO₂ assimilation rate compared to those irrigated with saline waste with a higher EC_a (Figure 1D). The difference was 5.938 μmol (CO₂) m⁻² s⁻¹ between the 0.5 dS m⁻¹ level and the 4.4 dS m⁻¹ level.

The leaf temperature (T_l) of the corn plants exhibited a linear increase with rising electrical conductivity of the saline waste. The difference in average Tl values between the corn plants at EC levels of 0.5 and 4.4 dS m⁻¹ was 0.94 °C (Figure 1E).

The intrinsic water use efficiency (A/gs) increased linearly with the rise in EC_a of the saline waste, with a unit increase of 10.062 (μmol CO₂ m⁻² s⁻¹)/(mmol H₂O m⁻² s⁻¹) (Figure 1F).

The minimum fluorescence of illuminated plant tissue (Fo’) increased with higher EC_a levels of the saline waste, showing a unit increase of 0.6996 µmol (photons) m⁻² s⁻¹ (Figure 2A. Fo’ increased by 63.93% at the 4.4 dS m⁻¹ level compared to the EC_a level of 0.5 dS m⁻¹ (Figure 2A).

The quantum efficiency of PSII (y) exhibited a linear reduction with the increase in electrical conductivity of the saline waste. At the 0.5 dSm⁻¹ level, the average value was 0.504; when compared to the plants irrigated with saline waste with an EC of 4.4 dSm⁻¹, the reduction was 19.84% (Figure 2B). The maximum efficiency of photosystem II (Fv/FM) as a function of the increase in the electrical conductivity of the saline waste was fitted to a quadratic regression model, with its maximum point at an EC of 1.7 dS m⁻¹. Beyond this point, there was a reduction of 3.85% at the highest EC_a of the saline waste at 4.4 dS m⁻¹ (Figure 2B).

The electron transport rate (ETR) of the corn plants in response to increasing EC_a levels in mycorrhizal treatments M1 and M2 was fitted to a quadratic regression model, (Figure 3C). The ETR results in the M1 and M2 plants were comparable, with their maximum points occurring around EC levels of 2.2 and 2.3 dS m⁻¹, reaching values of 86.03 and 94.76 µmol (photons) m⁻² s⁻¹, respectively. In contrast, the ETR of the M3 plants at an EC level of 4.4 dSm⁻¹ decreased by 26.96% compared to the control at 0.5 dSm⁻¹ (Figure 2C). Among the mycorrhizal treatments, at an EC_a level of 0.5 dSm⁻¹, the M3 plants exhibited the highest ETR at 91.683 µmol (photons) m⁻² s⁻¹, differing significantly from M2 by an average of 61.1 µmol (photons) m⁻² s⁻¹, representing an increase of 33.35% (Figure 2C).

The quantum yield of regulated photochemical quenching (YNPQ) increased in all mycorrhizal treatments as the EC_a levels increased (Figure 2D). In treatment M1, the linear regression equation was not significant, yielding an average of 0.45 across all saline levels. The plants from the M2 and M3 groups at the highest EC_a level of 4.4 dSm⁻¹ exhibited unit increases in YNPQ of 0.0672 and 0.0425, respectively. Among the highest averages, the M2 plants at an EC_a of 4.4 dS m⁻¹ recorded an average of 0.608, which was statistically different from that of the M1 mycorrhizal plants (0.483). However, the YNPQ of the M3 plants did not differ significantly from that of the M1 and M2 plants, with an average value of 0.531.

Salt stress significantly impacted the plant growth parameters. The interaction of salinity and the presence of the AMF resulted in significant differences (p < 0.001) in the growth parameters such as shoot dry matter (MSPA), root dry matter (MSR), and the root/shoot ratio (R/PA). Additionally, there was an of both factors on the plant height (AP) and number of leaves (NF), and an effect of mycorrhizal association (AMF) on the stem diameter (DC) (p < 0.5) (

Table 5. Analysis of variance for the growth parameters shoot dry mass (MSPA), root dry mass (MSRA), root/shoot ratio (R/PA), total dry mass (MST), leaf area (AF), height of the plant (AP), stem diameter (DC), and number of leaves (NF) of Creole corn (v. Ibra) of Creole corn plants as a function of EC_a and the AMF.

		Teste F
FV	GL	MSPA	MSRA		R/PA
AMF	2	185.42 ***	73.223 ***		32.513 ***
ECa (d Sm−1)	3	38.69 ***	29.270 ***		22.963 ***
AMF × ECa	6	13.16 ***	47.989 ***		32.555 ***
		AF	AP	DC	NF
AMF	2	1.385	3.414 *	4.514 *	4.333 *
ECa (d Sm−1)	3	28.332 ***	5.393 **	2.394 ns	5.127 *
AMF × ECa	6	6.469 **	1.371 ns	1.427 ns	0.841 ns

FV—source of variation; GL—degrees of freedom; AMF—arbuscular mycorrhizal fungus; EC_a—electrical conductivity of irrigation water; *** p < 0.001; ** p < 0.01; * p < 0.05; and ns—not significant.

).

The dry mass of the aerial part (MSPA) of the corn plants exhibited variation across the different mycorrhizal conditions and electrical conductivity levels of the saline waste. In the M3 condition, the MSPA increased with the rise in EC_a, reaching its highest value at an EC_a of 2.5 dS m⁻¹, with an average of 120.92 g per plant (Figure 3A). Treatments M1 and M2 showed similar trends, following a quadratic regression model with their maximum points at EC_a levels of 1.4 and 1.9 dS m⁻¹, corresponding to 87.83 and 68.09 g per plant, respectively.

When considering mycorrhizal conditions under the influence of the saline waste, significant differences were observed among treatments. Both the M1 and M3 treatments showed notable disparities from M2 (Figure 3A). At the highest EC_a level of 4.4 dS m⁻¹, M3 exhibited the highest MSPA at 88.57 g per plant, which was statistically different from that of M1 and M2, which had average values of 64.67 and 48.62 g per plant, respectively. This represents decreases of 26.98% and 45.11%, respectively, in comparison to M3 (Figure 3A).

The root dry mass (MSR) of the M3 plants exhibited a quadratic regression pattern, with its peak at an EC_a of 2.0 dS m⁻¹, averaging 114.2 g per plant (Figure 3B). In contrast, the plants from the M1 and M2 groups showed their highest MSR values at EC_a levels of 4.4 and 0.5 dS m⁻¹, respectively, with values of 113.96 and 83.10 g per plant (Figure 3B). At the highest EC_a level (4.4 dS m⁻¹), the corn plants under the different mycorrhizal conditions differed significantly: M1 had the highest value at 115.87 g per plant, followed by M2 with 61.05 g per plant, and M3 with 50.17 g per plant (Figure 3B).

Regarding the root/shoot ratio (R/PA ratio), it was higher in the plants from the M1 condition at an EC_a level of 4.4 dS m⁻¹, with an average of 1.78 (Figure 3C). However, in the M3 plants, the R/PA ratio decreased linearly with increasing EC_a of the saline waste, with a unit reduction of 3.16 g per plant. The difference between the highest and lowest EC_a levels (4.4 and 0.5 dS m⁻¹) was 41.09% (Figure 3C). For the corn plants irrigated with the water supply, the M2 plants had a higher R/PA ratio, averaging 1.39, statistically differing from the M1 and M3 plants. At the highest saline waste level (4.4 dS m⁻¹) among the mycorrhizal treatments, the M2 and M3 plants differed significantly, with a reduction in the R/PA ratio of 0.58 compared to M1 (Figure 3C).

The height of the corn plants (AP) decreased with increasing electrical conductivity levels of the saline waste, peaking at an EC_a of 1.8 dS m⁻¹, with an average of 191.14 cm (Figure 4A). Beyond this EC_a, there was a significant reduction in AP with increasing salinity, showing a difference of 10.91% (Figure 4A).

In terms of mycorrhizal treatments, the M3 plants exhibited greater height, averaging 189.23 cm, which was statistically different from that of M2 only, with a difference of 13.23 cm. The M1 plants had an average height of 184.03 cm, showing no statistical difference from that of the M2 plants (Figure 4B).

The response of the number of leaves (NF) of the corn plants to the increase in electrical conductivity fit a quadratic regression model, reaching an average of 14.41 leaves at its peak, which occurred at an EC_a of 2.3 dS m⁻¹ (Figure 4C). In terms of mycorrhizal treatments, the M3 plants exhibited a higher NF, averaging 14.33 leaves, with no significant differences from that of the M1 plants (average of 14.25 leaves), but differing from M2 plants, where there was a reduction of 5.79%, equivalent to 0.83 leaves (Figure 4D).

The leaf area (FA) of the corn plants did not significantly differ between the mycorrhizal treatments in response to the increasing electrical conductivity of the saline waste, except at an EC_a of 3.1 dS m⁻¹, where M1 had the highest average FA of 1640.46 cm². Compared to the M2 and M3 treatments, this represented an increase of 29.58% and 23.97%, respectively (Figure 4E). The maximum leaf area of the corn plants in M2 at different EC_a levels of the saline waste was 1155.15 cm² at an EC_a of 2.1 dS m⁻¹, while in M3, it peaked at an EC_a of 2.4 dS m⁻¹, with an average leaf area value of 1247.19 cm².

The corn stalk diameter (DC) exhibited a similar trend to that of the AP and NF, with the M3 plants having higher values compared to the M2 plants but not differing from the M1 plants (Figure 4F). The M3 plants had an average DC of 2.4 mm, approximately 0.27 mm higher, representing an increase of 11.25% compared to the M2 plants (Figure 4F).

The interaction between the electrical conductivity of the irrigation water (EC_a dSm⁻¹) and the mycorrhizal condition significantly affected the sodium content in the leaf (p < 0.05), stem, and root (p < 0.001), as well as the potassium content in the stem and root (p < 0.001), and the sodium-to-potassium ratio in the leaf, stem, and root (p < 0.01). Additionally, there was an effect of the EC_a levels on the sodium-to-potassium ratio in the root (p < 0.01) (

Table 6. Analysis of variance for the sodium (Na⁺) and potassium (K⁺) content in the leaf, stem, and root. Sodium and potassium ratio (Na/K) in the leaves, stems, and roots of Creole corn plants as a function of the EC_a of the saline waste and the AMF.

					Teste F
FV	GL	Na (folha)	Na (colmo)		Na (raiz)	Na/K (folha) †
AMF	2	29.910 ***	86.367 ***		51.937 ***	1.617 ns
ECa (d Sm−1)	3	65.373 ***	274.226 ***		229.684 ***	0.345 ns
AMF × ECa	6	2.862 *	25.310 ***		21.147 ***	21.485 ***
		K (folha)	K (colmo)	K (raiz)	Na/K (colmo)	Na/K (raiz) †
AMF	2	7.683 *	17.578 ***	0.438 ns	68.526 ***	4.441 *
ECa (d Sm−1)	3	7.094 **	23.352 ***	6.479 **	11.455 **	2.773 ns
AMF × ECa	6	2.132 ns	40.669 ***	10.811 ***	51.162 ***	8.430 ***

^† Data transformed into square roots (SQRTs). FV—source of variation; GL—degrees of freedom; AMF—arbuscular mycorrhizal fungus; EC_a—electrical conductivity of irrigation water; *** significant at 0.1% probability level (p < 0.001); ** significant at the 1% probability level (p < 0.01); * significant at the 5% probability level (p < 0.05) and ns—not significant.

).

The leaf sodium accumulation exhibited a linear increase with increasing EC_a, with the highest accumulations observed in the control plants (M1) at all studied levels. The highest concentration was 7.18 g kg⁻¹ at an EC_a of 4.4 dS m⁻¹. In contrast, the plants from the M2 and M3 groups showed lower sodium accumulations at all levels compared to the M1 plants. The highest concentrations obtained were 5.19 g kg⁻¹ and 4.180 g kg⁻¹ for M3 and M2, respectively, at an EC_a of 4.4 dS m⁻¹. The corn plants under mycorrhizal influence (M2 and M3) reduced their leaf sodium accumulation by 41% and 28%, respectively, compared to M1 at the same EC_a of 4.4 dS m⁻¹ (Figure 5A).

The sodium concentrations in the stalk increased with the EC_a of the saline waste. The plants from the M2 and M1 groups exhibited the highest accumulations at an EC_a of 4.4 dS m⁻¹, with averages of 24.13 g kg⁻¹ and 23.12 g kg⁻¹, respectively (Figure 5B). The M1 plants differed from the M3 plants only at EC_a levels of 0.5, 3.1, and 4.4 dS m⁻¹. The M3 plants, at an EC_a of 3.1 dS m⁻¹, had a reduction in the sodium content in the stem of 62% and 52% compared to the M2 and M1 plants, respectively. At an EC_a of 4.4 dS m⁻¹, there was a reduction of 66% and 37% compared to M1 and M2, respectively, under the same EC_a condition (Figure 5B).

The sodium concentrations in the root increased linearly with the electrical conductivity levels. In the M1 and M3 plants, the highest sodium concentration of 21.0 g kg⁻¹ and 21.8 g kg⁻¹, respectively, corresponded to the highest EC_a level of 4.4 dS m⁻¹ (Figure 5C).

Among all treatments, the M2 plants accumulated less sodium in their roots. When comparing the plants from the M3 and M1 groups at the same level (EC 4.4 dS m⁻¹), there was a reduction of 18.4% and 15.2%, respectively, in the sodium content in their root. For the M3 plants, the lowest root sodium concentration (10.56 g kg⁻¹) occurred at the minimum point of the curve (1.9 dSm⁻¹), which was only statistically different compared to that of the M1 plants at the same saline level (Figure 5C).

The interaction of factors resulted in a higher potassium (K⁺) content in the stem of the control plants (M1), which differed from that of the M2 and M3 plants only at the level of 1.8 dS m⁻¹ (Figure 5D). The highest concentration in M1 was 44.43 g kg⁻¹ at an EC_a of 2.7 dS m⁻¹. In the M2 plants, the potassium content increased with the EC_a levels, reaching the highest concentration of 43.53 g kg⁻¹ at an EC_a of 4.4 dS m⁻¹, differing only from that of M1. In M3, the highest concentration occurred at the highest level (4.4 dS m⁻¹), reaching 40.93 g kg⁻¹ of K⁺ in the stem (Figure 5D). At the highest EC_a condition (4.4 dS m⁻¹), the M2 and M3 plants were more efficient in concentrating potassium in the stalk, with an increase of 60% and 50.6%, respectively, compared to the control.

The potassium accumulation in the roots of non-inoculated plants (M1) decreased with increasing EC_a levels. Among the mycorrhizal treatments, the highest concentration (14.56 g kg⁻¹) occurred at an EC_a of 0.5 dS m⁻¹, which was statistically different from that of M2 under the same condition (Figure 5E). The M2 plants increased their root potassium content with increasing EC_a levels, with the largest accumulation (17.94 g kg⁻¹) occurring at an EC_a of 4.4 dS m⁻¹ (Figure 5E). For the potassium accumulation in M3, no regression models were fitted; the plants had an average of 11.90 g kg⁻¹ of potassium at all EC_a levels. The M2 plants showed the best results for K⁺ accumulation, increasing their root potassium content by 90% and 56%, respectively, compared to the M1 and M3 plants (Figure 5E).

The leaf potassium (K⁺) concentration decreased quadratically with increasing EC_a (Figure 6A).

The lowest accumulation occurred at an EC_a of 2.9 dS m⁻¹, corresponding to 36 g kg⁻¹ of potassium in the leaf (Figure 6A). For the AMF treatments, the highest concentration of K⁺ occurred in the M1 and M2 plants, with an average of 42.0 g kg⁻¹, compared to the M3 plants, which obtained an average of 35.0 g kg⁻¹ (Figure 6B). The increase in M1 and M2 was 20% compared to M3 (Figure 6B).

The sodium to potassium ratio in the M1 corn leaves (Na⁺/K_leaf⁺) increased quadratically as a function of the EC_a level. The highest (0.16) and lowest (0.03) ratios occurred at EC_a levels of 2.3 and 4.4 dS m⁻¹, respectively, representing a decrease of 76.2% in the Na⁺/K⁺_leaf ratio at the highest level (Figure 7A). For the M2 plants, the Na⁺/K⁺_leaf ratio did not differ between the EC_a levels, with an average of 0.08 (Figure 7A). The Na⁺/K⁺_leaf ratio in the M3 plants was fitted to a quadratic regression model. The highest and lowest ratios (0.16 and 0.04) occurred at EC_a levels of 4.4 and 2.4 dS m⁻¹, respectively, representing an increase of 284.6% at the highest level and a reduction of 74% at an EC_a level of 2.5 dS m⁻¹. Among plants, the highest Na⁺/K⁺_leaf ratio occurred at the 4.4 dS m⁻¹ level in the M3 plants, corresponding to an increase of 231.4% and 97% compared to the M1 and M2 plants, respectively (Figure 7A).

The relationship of sodium and potassium in the M1 corn stalks (Na/K_culm) was fitted to a quadratic regression model, with the highest and lowest ratios being 0.73 and 0.26 at EC_a levels of 2.3 and 4.4 dS m⁻¹, respectively, representing a reduction of 64.3% (Figure 7B). The Na/K_culm ratio in the M2 plants could not be fitted to any of the tested regression models; it showed an average of 0.31 for all EC_a levels (Figure 7B). The Na/K_culm ratio in M3 showed a quadratic behavior but it was not significant based on the regression analysis, it showed an average of 0.27 for all levels. The highest ratio (0.71) was observed in the M1 plants at the 1.8 dS m⁻¹ level, corresponding to increases of 359% and 134% compared to the M2 and M3 plants, respectively (Figure 7B).

The relationship of sodium and potassium in the root (Na/K_root) in M1 did not fit any of the regression models tested; it showed an average of 1.36 for all EC_a levels. The highest and lowest Na/K⁺_root ratios in M1 were 2.22 and 1.26 at EC_a levels of 1.8 and 4.4, respectively (Figure 7C). The Na/K⁺_root ratio in M2 fit a quadratic model but it was not significant based on the regression analysis; it showed an average of 1.04 for all EC_a levels. In M3, the highest Na/K⁺_root ratios were observed at EC_a levels of 0.5 and 4.4 dS m⁻¹, corresponding to 1.88 and 1.95, respectively. The largest reduction was 0.84 at the 2.5 dS m⁻¹ level. The best results for the Na/K⁺_root was observed in the M1 plants, with increases in the Na/K⁺_root ratio of 139% and 137% compared to the M2 and M3 plants, respectively.

The interaction of the EC_a and AMF was significant for the root colonization rate (p < 0.001), number of spores in the soil (p < 0.001), and easily extractable glomalin content (p < 0.001) (

Table 7. Analysis of variance for root colonization rate (TCR) (A) and soil spore density (B) of landrace corn plants as a function of EC_a (dS m⁻¹) and the AMF.

	Test F
FV	GL	% TCR	Number of Spores
AMF	2	447.963 ***	298.393 ***
ECa (d Sm−1)	3	4.582 *	85.332 ***
AMF × ECa	6	13.412 ***	42.446 ***

FV—source of variation; GL—degrees of freedom; AMF—arbuscular mycorrhizal fungus; (EC_a)—electrical conductivity of irrigation water; *** significant at a probability level of 0.001 (p < 0.001); * significant at a probability level of 0.05 (p < 0.5).

).

The colonization rate (%TCR) in the M3 plants followed a quadratic regression model (Figure 8A). Colonization decreased with increasing EC_a levels in the waste. The lowest percentage occurred at the 3.2 dS m⁻¹ level, corresponding to 63.5%. In M2, the percentage increased linearly as a function of the EC_a level. The highest value, 46%, was observed at the 4.4 dS m⁻¹ level (Figure 8A). The best results were observed in the M3 plants, with increases in the colonization rate of 83% and 53% at levels of 1.8 and 3.1 dS m⁻¹, respectively, compared to the M2 plants under the same EC_a condition (Figure 8A).

The spore density was higher in the M3 plants compared to the M2 and M1 plants at all EC_a levels (Figure 8B). The highest spore density in M3 occurred at the 2.9 dS m⁻¹ level, corresponding to 650 spores per 50 dm³ of soil. At the 4.4 dS m⁻¹ level, there was a 29% reduction in the number of spores compared to the 3.1 dS m⁻¹ level. Treatments M1 and M2 did not differ from each other (Figure 8B).

4. Discussion

4.1. Physiological Response of Maize Irrigated with Saline Waste and Inoculated with the AMF G. albida

Irrigation with saline waste reduced the gas exchange and photochemical efficiency in all the maize plants, regardless of the mycorrhizal treatment, limiting the gs, AN, and E, while increasing the A/gs and leaf temperature.

Irrigation with saline water affected the photosynthetic tissues, reduced photosynthesis, and consequently, plant productivity [19]. This reduction occurred due to variations in the osmotic potential caused by excess salts, leading to a decrease in the water content within plant cells, directly affecting physiological processes [53].

As indicated by the results, the reduction in gs, AN, and E in maize is due to stomatal closure, one of the plant’s first responses to osmotic stress induced by an increase in soil EC_a [54]. This response is a physiological strategy that plants use to reduce transpiration and limit water loss. However, stomatal closure also reduces CO₂ influx, since assimilation depends on substomatal air spaces for carboxylation sites [55].

The increase in leaf temperature in all the maize plants was due to stomatal closure and reduced E, which is also a pathway that aids in the dissipation of leaf heat. These results correspond to the findings of [56], who observed reductions in E, gs, and AN, as well as an increase in Tl in maize plants as a result of increasing irrigation water EC_a.

Damage to the photosystems is reinforced by the decrease in ETR, which showed a significant interaction with the EC_a levels and mycorrhizal conditions in the soil. The M1 and M2 plants’ ETR increase with the EC_a level up to 2.3 dS m⁻¹, demonstrating a high amount of energy, whereas the M3 condition showed a linear decrease as a function of saline waste EC_a, indicating a greater capacity for ETR adaptation to salinity levels. Ref. [57] states that the effects of salinity can reduce the electron transport rate (ETR), probably due to a higher consumption rate of ATP and NADPH, leading to reduced CO₂ assimilation [58].

Damage to the photosystems was also confirmed by the reduction in quantum efficiency (Y) and the maximum quantum efficiency of PSII (Fv/Fm) at higher EC_a levels. The reductions in these parameters indicate a restriction in the photochemical capacity of maize plants [59,60]. Similarly, the Fv/Fm ratio indicated disturbances to the photosynthetic system. Its decrease indicated a decline in PSII photochemical efficiency [61].

The increase in Fo’ and YNPQ in the maize plants also supported the hypothesis of damage to the photosynthetic apparatus. The accumulation of energy in chlorophyll, indicated by Fo’, explains the increase in YNPQ, suggesting that energy dissipation occurred in the form of heat through the regulatory photoprotective mechanism, the xanthophyll cycle [62]. This result suggests a high photoprotective capacity in maize plants, especially at higher salinity levels, with a more intense response in plants exposed to G. albida alone (M2).

Indeed, our findings indicate that irrigation with saline waste affects the physiology of Creole maize plants. However, the level of damage to photosynthesis was considered low compared to other reports in the literature. In one study [63], at a similar EC_a level (4.5 dS m⁻¹), the gs, E, and A/gs values were lower than those found in this study, with respective increases of 150%, 25%, and 400% in Creole maize. These results are likely due to the characteristics of the Creole variety itself, which, by its nature, has greater adaptive capacity to conditions in its cultivation areas [64]. Additionally, it was noted that interaction with G. albida produced a better performance in terms of energy dissipation as heat, a characteristic already present in Creole maize and intensified by G. albida.

4.2. Na/K Balance in the Growth and Development of Creole Corn Irrigated with Saline Waste and Inoculated with the AMF Gigaspora albida

Saline waste contains high concentrations of Na⁺ and Cl⁻ ions in its composition. Irrigation with this residue affects plant development [65] and disrupts ionic homeostasis in photosynthetic tissues, causing damage to plant physiological processes [66]. Our findings indicate that the interaction between G. albida and soil microbiota helped mitigate the effects of high salinity in different parts of the plants, alleviating salt stress through a reduction in ionic stress.

Corn plants irrigated with saline waste exhibited an increased sodium content due to the higher electrical conductivity (EC_a). However, the association with the AMF G. albida (M2) and G. albida combined with soil microbiota (M3) reduced the Na⁺ content while maintaining K⁺ influx in leaf tissues, promoting ionic balance and reducing the Na⁺/K⁺ ratio in the leaves, particularly at intermediate EC_a levels. This suggests a mitigating effect that reduced ionic stress in photosynthetic tissues.

In the control plants (M1), the sodium accumulation in the leaf tissues and other plant parts was the highest compared to the other treatments. This clearly indicates that plants without association with G. albida were less selective in sodium uptake. Even though they maintained a high potassium influx, the excessive sodium accumulation increased the foliar Na⁺/K⁺ ratio, negatively impacting plant growth.

A study [28] demonstrated that G. albida improved the tolerance of eucalyptus seedlings by enhancing the K⁺/Na⁺ ratio and reducing sodium accumulation. These results reinforce our findings, suggesting that G. albida may contribute to reducing sodium levels in corn leaves under high EC_a conditions.

The sodium accumulation in the stalk increased linearly with the EC_a level in all the corn plants. However, the Na⁺ concentration was modulated by the mycorrhizal treatments. Plants in the M3 group accumulated less sodium than those in the M1 and M2 groups, indicating a higher degree of selectivity for this ion in the stalk due to the interaction between G. albida and soil microbiota. On the other hand, the K⁺ concentration varied among the mycorrhizal treatments. In M1, the K⁺ levels significantly increased up to 2.7 dS m⁻¹ but did not remain high at elevated EC_a levels (4.4 dS m⁻¹), suggesting possible competition with Na⁺ ions, which may have limited K⁺ uptake [66].

For the M2 plants, the K⁺ accumulation was linear, whereas in M3, it occurred at the highest salinity level (4.4 dS m⁻¹). This supports the idea that G. albida may have induced an adaptive response in corn plants by enhancing K⁺ absorption and maintaining a lower Na⁺/K⁺ ratio, particularly at higher salinity levels. This suggests an effort to maintain cellular osmotic potential and sustain stomatal turgor pressure, thereby improving the photosynthetic processes [67,68].

The Na⁺ concentration in the roots of the M1 and M2 plants increased linearly with EC_a. However, in M3, the highest concentration was only observed at the highest EC_a level (4.4 dS m⁻¹), which increased the root Na⁺/K⁺ ratio. The allocation of Na⁺ to root cells may act as a defense mechanism to reduce ionic toxicity in photosynthetic tissues while increasing potassium influx, lowering root osmotic potential, and improving water uptake [6]. This enhances the plant’s tolerance to high salinity effects [16].

The results in the M3 plants may reflect the benefits conferred by the AMF–soil microbiota interactions. Salinity conditions can induce higher expression of genes encoding membrane transporter proteins, which directly contribute to Na⁺ extrusion into the soil solution and K⁺ influx into the xylem, thereby maintaining a favorable Na⁺/K⁺ ratio [19,69].

For corn, the literature indicates that the expression of genes such as ZmAKT2, ZmSOS1, and ZmSKOR in the roots contributes to K⁺ and Na⁺ homeostasis [70]. Additionally, under high salt stress conditions, another significant contribution of AMFs is the expression of aquaporins, proteins that regulate water flow across membranes in both leaves and roots, thereby improving plant water availability [71].

The lower Na⁺/K⁺ ratio in the aerial parts and higher ratio in the roots of the M3 plants may have provided favorable water and nutrient conditions, enhancing growth [19].

Our findings suggest that G. albida in combination with soil microbiota improved plant growth and development in the M3 plants. When comparing biomass production in M1 with that of M3, the results indicate that the increased dry shoot mass (MSPA), plant height (AP), number of leaves (NF), and stem diameter (DC) observed in this treatment resulted from improved ionic homeostasis induced by the symbiotic relationship between the corn plants, G. albida, and soil microbiota interactions. However, despite the high Na⁺/K⁺ ratio in the leaves and stems of M1 plants, these plants showed the second-best performance in terms of growth and biomass accumulation, demonstrating the strong adaptive capacity of Creole corn.

The benefits of G. albida have been widely reported in the literature. To improve the quality of Dipteryx alata seedlings, Ref. [26] investigated the benefits of G. albida in seedling development and found that its symbiotic interaction enhanced the relative water content (TRA) in plants. It was reported that, under severe salinity conditions (10, 15, and 20 dS m⁻¹), G. albida increased eucalyptus seedling tolerance by maintaining the RWC above 60% at the highest level (20 dS m⁻¹), with even higher percentages at lower salinity levels [28].

The increased MSPA, AP, NF, and DC in the M3 corn plants likely resulted from improved water and nutrient conditions induced by the symbiotic relationship between G. albida and the soil microbiota, particularly under intermediate salinity conditions (2.5 dS m⁻¹) where the most significant gains were observed. In M2, under the effect of EC_a, the MSPA, root dry mass (MSRA), AP, NF, and DC were the least significant among treatments, even though the foliar Na⁺/K⁺ ratio was reduced. This suggests that inoculation with G. albida alone (without the complete soil microbiota) was insufficient to mitigate the negative effects of high salinity.

Under high EC_a levels, the root growth in the corn plants was modulated by the mycorrhizal treatments. The difference in the MSRA between M3 and M1 (control) at 2.0 dS m⁻¹ was associated with the beneficial effects of G. albida and native soil microbiota interactions. According to the literature, mycorrhizal plants under intermediate salinity conditions may develop a more extensive root system to improve soil exploration, water uptake, and nutrient absorption [72].

The increased root development in M1 at 4.4 dS m⁻¹ may indicate an adjustment mechanism in the allocation of photoassimilates, clearly represented in the root-to-shoot ratio (R/PA). This suggests that the M1 plants directed more energy toward root development, explaining the reduction in shoot dry mass (MSRA). The lower root investment in M3 may result from a more linear adjustment and greater adaptation to Na⁺ accumulation in root cells. Sodium accumulation may have induced better osmotic adjustment and an improved root water status, explaining the increased SDM even at the highest EC_a level (4.4 dS m⁻¹).

4.3. Gigaspora albida’s Response to the Reverse Osmosis Brine

Soil salinity affects not only plants but can also interfere with hyphal growth and development, the colonization capacity, and the spore germination of AMFs [73]. In this study, the colonization percentage of G. albida in the M2 maize plants increased as a function of the EC_a of the saline brine. On the other hand, the plants in the M3 group showed a gradual adjustment, with a tendency toward higher colonization at higher saline levels. In saline environments, several factors can influence colonization, affecting spore germination, reducing fungal growth, and impairing the propagation of hyphae and arbuscules [74,75].

A recent study [76] showed that the interaction between AMFs and soil bacteria modulated maize growth and increased AMF colonization by 80% under high salinity conditions. Ref. [77] using strains of the AMF Claroideoglomus claroideum (Cc), observed an increase in the colonization of lettuce (Lactuca sativa) plants, resulting in higher yields and improved nutritional conditions for the crop.

In this study, plant colonization in the M2 group was lower compared to that of M3. This reduction was due to the presence of a single AMF, G. albida. However, high salinity was not a limiting factor for colonization. In M3, colonization decreased with increasing EC_a but showed a tendency to rise again. Nonetheless, compared to the M2 plants, colonization in the M3 plants was higher, which may be attributed to the interaction of G. albida with native AMF species in the soil and other organisms that possibly enhanced the mycorrhizal effect in maize plants, helping to alleviate the salt stress.

The tolerance of G. albida to high salinity levels (10, 15, and 20 dS m⁻¹) in eucalyptus clones was also confirmed in [28], where greater root colonization and spore density with G. albida were observed.

Spore density was affected by the salinity of the brine at the highest level. However, at an intermediate level (2.9 dS m⁻¹), there was a greater AMF response in terms of spore production. This behavior was similar across all three mycorrhizal conditions, indicating that this salinity level stimulated the highest sporulation potential of AMF in this study. Comparing the mycorrhizal treatments, the significant increase in spore density in M3 compared to M2 was due to the combination of spores of G. albida with the spores of native AMFs in the soil. Although there was no statistical difference between M1 and M2 at 2.9 dS m⁻¹, the number of spores in M2 increased by 53%, indicating that G. albida was also sporulating. These results suggest that intermediate salinity levels may induce higher spore production in G. albida, which may also positively affect native AMFs in the soil.

The results of Ref. [78] support our findings; they observed an increase in AMF spore numbers in a rotational maize and bean crop irrigated with saline water. Ref. [79] found that the increase in spore numbers under high salinity conditions was due to the adaptive capacity of AMF communities. There are few literature reports on the effects of high salinity on arbuscular mycorrhizal fungal spore induction. In this study, the evaluation period was short (50 days), and the results are still preliminary, requiring further long-term field studies to better understand the effects of high salinity on spore production and to determine the salinity threshold beyond which germination may be adversely affected.

5. Conclusions

Irrigation with saline wastewater produced using reverse osmosis affects the physiology of criollo maize. The symbiotic relationship in both mycorrhizal treatments, M2 and M3, mitigated the effects of excess energy in PSII by promoting heat dissipation.

The interaction between G. albida and the soil microbiota mitigated the ionic stress in the aerial part of criollo maize by favoring a lower Na⁺/K⁺ ratio and higher MSPA, MSRA, AP, and DC between the levels of 1.8 and 3.1 dS m⁻¹.

Criollo maize irrigated with saline wastewater adapted by investing in root growth to tolerate the high salinity.

The interaction of G. albida with criollo maize was not sufficient to mitigate the negative effects of high salinity, resulting in the lowest growth performance.

Irrigation with wastewater reduced the colonization percentage of G. albida in the roots. An EC of 2.9 dS m⁻¹ increased the spore density in the soil.