Arbuscular Mycorrhizae Confers Salinity Tolerance to Medicago sativa L.

Abstract

Alfalfa, a crop cultivated worldwide for use as livestock feed, exhibits great adaptability to various environmental conditions. This study evaluates the biomass production, lipid peroxidation, photosynthetic pigments and osmo-compatible response in Medicago sativa var CW660 mycorrhizae (AM) and non-mycorrhizae (NM) plants with Rhizophagus intraradices and when subjected to salinity (100 mM and 200 mM of NaCl). They were evaluated using the following morphological parameters: foliage fresh weight (FFW), foliage dry weight (FDW), root fresh weight (RFW), root dry weight (RDW), foliage length (FL) and root length (RL), chlorophylls a and b, proline and malondialdehyde (MDA) in AM and NM plants treated with different concentration of NaCl. The LFW and the LDW were higher in M plants. The RFW and the RDW increased in control AM plants, and under different saline treatments there were no differences between AM and NM in either parameter. FL increased in the control and at 100 mM of NaCl in M plants. Chlorophyll a decreased 27–30% and b decreased 1–50% in AM and NM plants at 200 mM NaCl. The proline level increased four times and promoted a defense in AM plants at 200 mM of NaCl. Lipid peroxidation decreased in AM plants by 10% at maximum salinity. M. sativa CW660 is sensitive to salinity stress, and inoculation with arbuscular mycorrhizal fungi (AMF) regulates its physiology and performance under such conditions, with osmotic protection and membrane protection.

1. Introduction

Soil degradation due to salinity has a strong impact on agricultural areas, affecting the physical–chemical properties of the soil and decreasing its fertility and productivity, mainly in semi-arid and arid areas [1]. The excessive application of fertilizers and herbicides and poor drainage systems and inadequate irrigation practices increase the levels of salinization in the soil, which reduces the infiltration rate and decreases the availability of water for crops [2,3]. Salt stress induces morphological, physiological and biochemical responses in plants. The effects are a consequence of osmotic and ionic factors. Salinity affects photosynthesis and respiration and produces oxidative damage and osmotic stress. The ions absorbed by the plant caused imbalances in mineral nutrition and have toxic effects on metabolism, germination and growth [4]. This impacts the productivity of extensive arable areas; therefore, it is necessary to find solutions to mitigate the effects of salt stress in agriculture. Alfalfa is widely used as livestock feed due to its low production cost and high-quality forage, meeting the nutritional needs of animals with high requirements [5], and it is cultivated on more than 30 million hectares around the world. Alfalfa exhibits great adaptability to various environmental conditions, growing in different soils and climates throughout the year [6].

Plants form symbiotic associations with arbuscular mycorrhizal fungi (AMF). These improve mineral nutrition and water uptake thanks to hyphae, while plants provide nutrients and a microhabitat for mycorrhizae [7]. Numerous studies demonstrate that the association with AMF allows the adverse effects caused by salinity to be mitigated, improving nutrient uptake, increasing osmotic adjustment and minimizing oxidative damage through antioxidant responses in the host plant. These effects result in improved growth, establishment and survival of the host plant in stressful environments. Additionally, mycorrhizal fungi may be potential tools to combat soil salinization in ecological conservation and restoration efforts [8,9,10]. The mycorrhizae–plant (AM) relationship is beneficial for both, as they contribute to the absorption of nitrogen and phosphorus, in addition to some micronutrients that are found in trace amounts, such as some metals, whose limitations in plants causes poor plant growth [11].

Studies have shown that inoculation with AMF, Rhizophagus intraradices in M. sativa L. var. Dekalb (DK166), influenced physiological parameters and antioxidant defense in plants under salt, drought and cold stress. Stomatal conductance, photosynthetic efficiency and aerial and root dry weight in mycorrhizal plants increased under drought and salinity, showing greater tolerance. Antioxidant defense increased in mycorrhizal plants and abiotic stress was mitigated [12]. Hormones are involved in mycorrhizal control, and jasmonic acid (JA) showed a significant increase in the AM plant of M. sativa L. (DK 166) under drought and salinity stresses [13]. The proteomic study reported that the accumulation pattern of PIP1 aquaporin protein was greater in NM plants than in AM alfalfa plants, thus ensuring the provision of water in symbiotic plants and a better water balance under stress [13]. Micronutrients such as Fe, Mn and macronutrients such as Ca and Mg increased in AM plants; in contrast, the concentration of heavy metals such as Cu, Pb, Ti, Hg, Bi, Cr, As and Be was lower under salt and drought stress [13]. In previous studies on the genotypes CW 197, Trinidad 87, CW 660 and Salina PV and levels of saline (NaCl) and cadmium (CdCl₂) stress, factorial ANOVA was performed to identify the influence of stress conditions on the measured variables, and multiple comparisons analysis of means were performed when a significant effect was found. Under salinity, the CW660 genotype was tolerant to salinity at germination, but sensitive during growth [14].

We hypothesize that the alfalfa variety CW 660, sensitive to salt during growth in symbiosis with AM, can improve its hydraulic condition and tolerance to salinity. The objective was to study the effect of the symbiosis of Medicago sativa L. (CW660)-Rhizophagus intraradices against salt stress. The tolerance of M. sativa in symbiosis under salt stress was evaluated through the analysis of morphological parameters, photosynthetic pigments, the osmo-compatible response and oxidative damage.

2. Results

2.1. Percentage of Colonization

The application of salinity stress did not affect root colonization. The mycorrhization percentages of R. intraradices to M. sativa L. were 56.8% in non-stress plants, 46.3% in the 100 mM NaCl treatment and 69.8% in the 200 mM NaCl treatment; there were no significant differences between them (

Table 1. Mycorrhization percentage of Medicago sativa L. roots in control, 100 and 200 mM NaCl.

Treatments	Percentage of Mycorrhization
Control	56.8% a
100 mM NaCl	46.3% a
200 mM NaCl	69.8% a

).

2.2. Germination

According to International Rules for Seed Testing (ISTA) [15], GE is an indicator of vigor and measures how quickly seeds germinate, providing information about the lot’s performance under various environmental conditions and its storage potential. The GP is expressed as the percentage of seeds that germinate and develop normal seedlings under optimal conditions of temperature, light and moisture. In the study of the germination of AM and NM seeds of M. sativa L. subjected to saline stress, those under 200 mM NaCl decreased significantly with respect to the control and seeds under 100 mM NaCl, both in the parameters of GE and those of GP. If the germination percentage of AM seeds is compared with that of NM seeds, there are no differences at low salt concentrations and in the control, but at 200 mM NaCl the AM seeds had a higher germination percentage than the NM seeds, both in the GE and GP parameters (Figure 1).

2.3. Morphological Parameters

Comparing the morphological values between AM and NM plants, it was observed that foliage fresh weight (FFW) and foliage dry weight (FDW) increased in all treatments in AM plants. Root fresh weight (RFW) and root dry weight (RDW) increased in the control AM plants. Foliage length (FL) was increased in the control and the saline treatment with 100 mM NaCl, and root length (RL) in the no treatment group (

Table 2. Morphological parameters in Medicago sativa L. AM and NM plants in control, 100 and 200 mM NaCl groups. Different letters mean significant differences (p ≤ 0.05) as determined by the ANOVA test (n = 3).

Treatments	NM Plants	AM Plants
	FFW (g)
Control	0.43 + 0.10 c	0.82 + 0.15 a
100 mM NaCl	0.50 + 0.06 b	0.63 + 0.05 a
200 mM NaCl	0.49 + 0.06 b	0.65 + 0.06 a
	FDW (g)
Control	0.08 + 0.06 b	0.15 + 0.032 a
100 mM NaCl	0.10 + 0.01 b	0.14 + 0.02 a
200 mM NaCl	0.10 + 0.06 b	0.14 + 0.01 a
	RFW (g)
Control	0.34 + 0.04 b	0.97 + 0.19 a
100 mM NaCl	0.71 + 0.11 a	0.73 + 0.07 a
200 mM NaCl	0.61 + 0.05 a	0.72 + 0.04 a
	RDW (g)
Control	0.03 + 0.03 c	0.096 + 0.01 b
100 mM NaCl	0.09 + 0.00 b	0.083 + 0.08 b
200 mM NaCl	0.13 + 0.03 a	0.107 + 0.01 a
	FL (cm)
Control	15.16 + 0.40 b	20.00 + 1.00 a
100 mM NaCl	14.93 + 1.06 b	19.33 + 2.08 a
200 mM Na Cl	18.07 + 1.50 a	17.67 + 0.57 a
	RL (cm)
Control	13.67 + 1.52 a	15.33 + 0.57 a
100 mM NaCl	13.00 + 1.00 a	13.33 + 2.30 a
200 mM NaCl	13.67 + 0.57 a	15.67 + 0.57 a

).

2.4. Pigments

Chlorophyll a decreased 27–30% (Figure 2A) and chlorophyll b decreased around 1–50% (Figure 2B) significantly at 200 mM NaCl, in both AM and NM plants. Chlorophyll photosynthetic pigments help to maintain photosynthesis even under salt stress. In this study, mycorrhizae did not show an increase in chlorophylls (a and b); they were affected in the same way in AM plants as in NM plants, decreasing their content.

2.5. Proline and Malondialdehyde (MDA)

Proline is an important osmotic protective substance that plays an indispensable role in maintaining the normal function of cells and in protecting the structure of cell membranes and the stability of biological macromolecule structures [16]. In NM plants, it did not act as a protector, with similar values without significant differences in all treatments. In AM at 200 mM NaCl, it was significantly increased (Figure 3A).

MDA is a natural antioxidant that plays an important role in protecting plants against oxidative stress [17]. MDA has been shown to regulate plant growth and development by modulating hormone activity [18]. MDA can protect plants against dehydration by regulating stomatal closure and reducing water loss [19].

The MDA content decreases significantly in shoots of AM plants when subjected to 200 mM NaCl compared to the NM group. This difference denotes the protection of mycorrhizae against the salt stress of 200 mM NaCl since MDA is an indicator of membrane lipid peroxidation (Figure 3B).

3. Discussion

Salinity causes three types of stress, osmotic, ionic and oxidative, which alter agricultural productivity [12]. It produces a decrease in water potential, reduces plant biomass and disturbs mineral nutrition, and high concentrations of Na⁺ and Cl⁻ cause ionic toxicity [20,21,22]. Germination is influenced by many genetic, endogenous and environmental factors [23,24]. The dry seeds’ imbibition (water absorption) triggers the metabolic mechanisms that initiate germination [25].

Salt stress decreases in water uptake during imbibition and may cause excessive uptake ions, delaying seed germination and seedling establishment [26,27,28]. AMF are able to make the plants tolerate the stress and alter their morphology and physiology in such a way that the plant can resist the stress [29]. Under stress, the growth and activity of fungi and plants could be affected. The homeostasis of phytohormones such as abscisic acid and gibberellic acid that participate in the regulation of germination [16] can suffer imbalances due to the toxic effect exerted by ions, altering the functionality of the membrane and the cell wall of the embryo [30]. In our studies, saline treatment significantly decreased germination in NM plants and the germination percentage significantly increased in AM plants. Various studies demonstrate that colonization by AMF has a protective effect on host plants, significantly increasing biomass and improving nutrient and water exchange [31,32]. However, salt stress can generate negative impacts on root colonization by AM fungi due to the accumulation of toxic substances in the rhizosphere, which can limit spore germination and hyphal development [33,34]. Our results showed that the degree of mycorrhization was not affected by the increase in salinity. The results obtained showed that when Medicago sativa L. var. CW 660 was mycorrhized, it increased the FW and DW of leaves both in control conditions and in the two levels of salt stress; however, the FWR only increased in the control, and the DW did so in the control and 100 mM NaCl groups. FL was greater in AM plants, but in the roots there were no differences between AM and NM plants. This is in agreement with the findings reported in citrus by [35] and in Viburum tinus [36] under salinity conditions, which demonstrate the protective effect that AMF have against saline stress [8]. Salt stress reduces the contents of photosynthetic pigments; this is attributed to the degradation of pigments affecting photosynthesis and growth [8,37]. At 200 mM NaCl, the contents of chlorophyll a and b were significantly reduced in both NM and AM. For tests carried out on M. sativa var. CW197 and CW660, a similar decrease was observed in NM plants [38].

Plants associated with AM accumulate more proline and sugars compared to NM plants [38,39]. These provide better resistance through osmoregulation, playing a role in stress tolerance in plants [40]. At 200 mM NaCl, a significant increase was observed in AM plants compared to NM plants subjected to the same salt concentration. This increase may be mediated by AMF to reduce the damage to cells under salt stress [37]. This behavior was reported in Vigna radiate [41], Glycine max [42] and Medicago sativa var. Dekalb [12].

Symbiosis protects plants by reducing pathways for ROS production, maintaining membrane integrity and stabilizing proteins and enzymes [43]. Malondialdehyde is a product of lipid peroxidation; its accumulation is related to the damage caused by reactive oxygen species under stressful conditions [17]. In our studies, MDA content remained unchanged in the control in both NM and AM. At 200 mM NaCl in AM plants, the MDA decreased significantly in relation to those NM plants; this means that the damage in AM plants is lower thanks to the fungus protection. These results coincide with those reported for Viola prionantha [44] and Medicago sativa L. var. Dekalb [12] associated with AMF against salt stress.

4. Materials and Methods

4.1. Germination

Medicago sativa var. CW660 seeds were divided into two groups: inoculated with Rhizophagus intraradices and non-inoculated. They were sown in Petri dishes, each containing 20 seeds arranged in 3 replicates per treatment. The dishes were lined with double-moistened filter paper and kept in darkness at 25 °C. They were irrigated with water as the control and saline solutions containing 100 mM and 200 mM of NaCl. Germination energy (GE) and germination power (GP) were measured on the 3rd and 7th day, respectively, using the following calculation: germination percentage (%) = number of germinated seeds/number of total seeds × 100.

4.2. Inoculation with Rhizophagus Intraradices

Inoculation was carried out using 1 cm³ of granular arbuscular mycorrhizae per pot in recipients of 500 mL with a sterilized mixture of soil/perlite substrate (1:1).

4.3. Treatments, Experimental Design and Growing Conditions

Three treatments were compared under a randomized complete block design with five replications. Treatments came from the combination of two factors: (1) inoculated with Rhizophagus intraradices and non-inoculated (control), and (2) growing conditions of non-stressed (control) or with one of two stresses (100 mM and 200 mM NaCl). The salinity levels used corresponded to 10 and 20 dS/m, which are common values in salinized soils.

The experimental units consisted of 50 pots of 500 mL each, and one seed per pot was sown in the sterilized mixture of soil/perlite (Figure 4). Plants were grown in a culture chamber at 25 °C with a photoperiod of 16 h light and 8 h darkness. When the plants reached a height of 10 cm, in the fourth week, stress treatments were started with different solution irrigation conditions, twice a week for one month.

4.4. Mycorrhizal Development

Rhizophagus intraradices colonization was estimated by a visual inspection of fungal structures after treatments with different stresses, with the clearing of roots in 10% KOH and staining with 0.05% (w/v) trypan blue in lactic acid [45]. The percentage of R. intraradices colonization was calculated according to the grid line intersections method [46]. Three boxes per treatment were analyzed, counting the mycorrhizal roots in the vertical and horizontal lines.

4.5. Plant Growth and Determination of Photosynthetic Pigments

At the end of the treatments, the plants were harvested and the FL, RL, FFW and FDW, RFW and RDW were measured in quintuplicate. To calculate the DW, the samples were placed in an oven at a temperature of 30 °C for 7 days. The determination of chlorophylls a and b was carried out according to [47]. Briefly, 100 mg of fresh aerial material (leaves) was collected, crushed in a mortar with 10 mL of 80% (v/v) acetone and filtered. The extract was kept at 4 °C until the spectrophotometer reading. The absorbance of photosynthetic pigments was measured at a wavelength of 646.6 nm (chlorophyll a) or 663.6 nm (chlorophyll b).

4.6. Proline Content

The Bates method (1973) was used for proline determination [48]. We took 0.5 g of fresh aerial material (leaves) and crushed it with a mortar and pestle with 10 mL of 3% aqueous sulfosalicylic acid solution. The homogenate was filtered and mixed with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid. The mixture was boiled in a water bath at 100 ºC for 1 h at constant temperature and the reaction was stopped by immersing the tube in cold water. Then, 0.5 mL of the samples was taken and the absorbances were measured at 520 nm in a spectrophotometer, and the proline concentration in each was determined. The calibration curve was produced with the standard L-PROLINE p.a. C₅H₉NO₂, with which the ug/mL of proline in the sample was calculated.

4.7. Oxidative Damage to Lipids

To determine the oxidative damage to lipids, 500 mg of FW of leaves was ground in a mortar on ice with 6 mL of 100 mM of potassium phosphate buffer (pH 7) and the homogenate was filtered with a Miracloth layer and centrifuged at 15,000 rpm for 20 min. The chromogen was formed by mixing 200 μL of supernatants with 1 mL of a reaction mixture containing 15% (w/v) trichloroacetic acid (TCA), 0.375% (w/v) 2-thiobarbituric acid (TBA), 0.1% (w/v) butylhydroxytoluene and 0.2 N HCl, and the mixture was incubated at 100 °C for 30 min [49]. After cooling at room temperature, tubes were centrifuged at 800× g for 5 min and the supernatant was measured at 532 nm in the spectrometer. The standard used was 1,1,3,3-Tetramethohoxypropane Malonaldehyde bis (dimethyl acetal) (C₇H1₄O₄). Lipid peroxidation was estimated as the content of 2-thiobarbituric acid-reactive substances (TBARS) and expressed as equivalents of malondialdehyde (MDA) according to Halliwell and Gutterige (1989) [49].

4.8. Statistical Analysis

Statistical analyses were performed with Graph Pad Prism Version 8.0.2 (263). The morphological parameters were analyzed with Student’s T parametric test and the percentage of colonization, germination, pigments, proline and MDA results were analyzed using multifactorial ANOVA. Significant differences among treatments were identified using Tukey’s B test (p < 0.05).

5. Conclusions

Medicago sativa var. CW660 is affected by salinity, and its association with arbuscular mycorrhizae improves growth under these conditions. This research allows us to understand the responses of plants to abiotic stress and how the use of microorganisms is a sustainable alternative for the development of alfalfa crops in saline soils. Under salt stress, AM plants showed better performance than the NM in almost all measured parameters. The AM plants had greater growth than the NM plants in the parameters of leaf and root length, in the fresh and dry weight in the aerial and the root part. At concentrations of 200 mM NaCl, all morphological parameters and biochemicals decreased in NM plants. Chlorophyll a and b contents decreased at 200 mM in both AM and NM plants. The proline content associated with osmo-compatible protection increased at 200 mMNaCl in AM plants. MDA content increased in NM plants at 100 mM and 200 mM of NaCl, showing lipid peroxidation of the membranes. AM plants maintained similar MDA levels in all treatments, evidencing the protection of symbiosis with arbuscular mycorrhizae.

If the symbiosis of forage plants with mycorrhizae favors tolerance to salt stress, it would be a good biotechnological tool and would promote better practices that are more sustainable for the environment.