At harvest, the shoots were cut off at the soil surface. All the roots were carefully collected. Roots were shaken to remove loose soil and then submerged in water to remove the attached soil. The alfalfa height, branching, stem diameter, and nodules were recorded. Root and shoot samples were dried in an oven at 70°C for 72 h and weighed.

Shoot δ¹⁵N and N concentration were measured using an isotope facility (Iso- prime100, Elementar, Germany). Root N concentration was measured using an elemental analyzer (Elementar vario MACRO cube, Germany). The %Ndfa of alfalfa was calculated following the equation: %Ndfa = (δ¹⁵Nreference plant − δ¹⁵Nalfalfa/δ¹⁵Nreference plant − β) × 100, where β is the δ¹⁵N of alfalfa when wholly reliant on N fixation for its N nutrition (Fan et al., 2006). The amount of N fixed by the alfalfa was calculated using the following equation: N fixed = %Ndfa (%) × shoot dry weight × shoot N concentration (%). The N fixation efficiency of nodules was calculated following the following equation: N fixation efficiency = amount of N fixed/nodule weight (Döbereiner, 1966).

The CP content was determined by a laboratory N concentration analysis, from which the CP content can be calculated by multiplying the N concentration by 100/16, or 6.25 (Mulder, 1839). The NDF and ADF contents were determined by the Van Soest method (Van Soest et al., 1991).

All parameters were analyzed using analysis of variance by SAS (v8, SAS Institute Inc., Cary, NC, USA). When effects were statistically significant, the least significant difference (LSD) at p = 0.05 is presented. Figures were produced in SigmaPolt software (v10.0, Systat Software, San Jose, CA, USA).

In experiment 1, alfalfa growth decreased significantly with increasing salt levels ( Figures 1A–C ). The decrease in alfalfa height ranged from 20.1% to 77.1% in salt treatments compared with the control ( Figure 1A ). Stem diameters of alfalfa in 100, 150, and 200 mmol Na₂SO₄ L^–1 levels were significantly lower than those in the control by 35.1%, 49.9%, and 66.0%, respectively ( Figure 1B ). However, a salt level of 50 mmol L^–1 did not change stem diameter compared with the control. Branching number decreased significantly when the salt level was above 100 mmol L^–1. The fewest branches were observed in the treatment of 200 mmol Na₂SO₄ L^–1, which produced 2.38 branches per plant ( Figure 1C ).

In experiment 2, although plant height, stem diameter, and branching were significantly inhibited by salt, N supply partially countered these effects ( Figures 1D–F ). In contrast, N supply did not improve the plant height, stem diameter, or branching of alfalfa when compared with alfalfa that was not treated with N supply and did not suffer salt stress. Plant height and stem diameter were higher (13%–18%) in alfalfa with N supply treatment than in alfalfa with no N supply that suffered salt stress ( Figures 1D, E ). There was no difference in plant height and stem diameter between the alfalfa treated with 100 and 200 mg N kg_-1 supply. However, an increase in branching was observed only in the alfalfa treated with 100 mg N kg^–1 supply, which was higher by 13% than in the alfalfa treated with no N supply ( Figure 1F ).

In experiment 1, there was no difference in shoot and root dry weight between the control and the treatment of 50 mmol Na₂SO₄ L^–1( Figures 2A, B ). However, salt addition at levels of 100, 150, and 200 mmol Na₂SO₄ L^–1significantly decreased shoot dry weight compared with the control by 63%, 76%, and 86%, respectively ( Figure 2A ). Like shoot dry weight, root dry weight decreased by 43%, 53%, and 79% under the same treatments, respectively. Alfalfa root dry weight did not significantly further decrease when salt exceeded 100 mmol Na₂SO₄ L^–1 ( Figure 2B ).

In experiment 2, N supply did not change shoot and root dry weight in alfalfa treated without salt stress ( Figures 2C, D ), whereas salt stress significantly decreased shoot and root dry weight by 49%–67% when compared with treatments without salt stress. In alfalfa treated with N supply, shoot and root dry weights were greater than in alfalfa without N supply when the plants suffered salt stress ( Figures 2C, D ). Shoot dry weight significantly increased by 40% and 45% in alfalfa treated with 100 and 200 mg N kg^–1, respectively, compared with alfalfa treated without N supply ( Figure 2C ). Root dry weight significantly increased by 23% and 29% in alfalfa treated with 100 and 200 mg N kg^–1, respectively, compared with alfalfa treated without N supply ( Figure 2D ).

In experiment 1, salt addition did not change shoot and root N concentration, which was 3.98% on average until the salt level was above 100 mmol Na₂SO₄ L^–1 ( Figures 3A, B ). Shoot N concentration decreased by 31.2% in alfalfa treated with 150 mmol Na₂SO₄ L^–1 and by 37.2% in alfalfa treated with 200 mmol Na₂SO₄ L^–1, compared with that in the control ( Figure 3A ). The difference observed in shoot N concentration between these two treatments was not statistically significant. Like shoot N concentration, root N concentration decreased by 9.7% in alfalfa treated with 150 mmol Na₂SO₄ L^–1 and 10.8% in alfalfa treated with 200 mmol Na₂SO₄ L^–1 compared with that in the control ( Figure 3B ).

In experiment 2, salt level significantly increased shoot N concentration by 11.7%–15.2% compared with treatments without salt but did not change root N concentration ( Figures 3C, D ). There was no difference in shoot N concentration among different N supply treatments, regardless of salt stress. The same result was found in root N concentration.

In experiment 1, there was no difference in shoot and root N content between the control and alfalfa treated with 50 mmol Na₂SO₄ L^–1 ( Figures 4A, B ). However, salt addition significantly decreased shoot N content compared with that in the control by 64%, 83%, and 91% at salt levels of 100, 150, and 200 mmol Na₂SO₄ L^–1, respectively ( Figure 4A ). In addition, root N contents of alfalfa treated with 100, 150 and 200 mmol L^–1 Na₂SO₄ levels were 58%, 79%, and 66% significantly lower than that in the control, respectively ( Figure 4B ).

In experiment 2, N supply partially recovered the loss of shoot N content caused by salt addition ( Figures 1D–F ). Shoot N content was higher (10%–28%) in alfalfa treated with N supply than in alfalfa treated without N supply when they suffered the same salt stress ( Figure 4C ). In contrast, N supply did not change root N content ( Figure 4D ).

In experiment 1, nodule formation was inhibited when the salt level was above 100 mmol Na₂SO₄ L^–1 ( Figures 5A, B ). There were 38 and 76 nodules per pot in the control and the alfalfa treated with 50 mmol Na₂SO₄ L^–1, respectively, and nodule weight ranged from 0.13 to 0.21 g pot^–1. In addition, the corresponding %Ndfa ranged from 35% to 42%, and amount of N fixed ranged from 18.08 to 23.72 mg pot^–1 when the salt level was belove 50 mmol Na₂SO₄ L_–1 ( Figure 5C ; Table 1 ). The N fixation efficiency showed the same result. There was no difference in number of root nodules, weight, %Ndfa, or amount of N fixed between the control and alfalfa treated with 50 mmol Na₂SO₄ L^–1 ( Figure 5 ; Table 1 ).

In experiment 2, the number of nodules decreased owing to salt stress only in the treatment with no N supply. Although salt stress did not change the nodule number, it did significantly decrease %Ndfa by 18%–33% compared with the alfalfa treated without salt stress. Salt addition significantly decreased the amount of N fixed by 41%–55%. A decrease of N fixation efficiency caused by salt stress was observed in alfalfa treated with a supply of 100 and 200 mg N kg^–1 ( Table 2 ). The number and weight of nodules significantly increased by 80% and 78%, respectively, in alfalfa treated with 200 mg N kg^–1 compared with alfalfa treated without N supply and subjected to salt stress ( Figures 5D, E ). The %Ndfa was significantly higher by 53% and 42% in alfalfa treated with 100 and 200 mg N kg^–1 than in alfalfa with no N supply ( Figure 5F ). The N supply improved the amount of N fixed by approximately 60% but had no effect on the N fixation efficiency of nodules ( Table 2 ).

In experiment 1, salt addition did not change alfalfa CP, which was 23% on average until the salt level was above 100 mmol Na₂SO₄ L^–1. Alfalfa CP significantly decreased by 31% and 37% in alfalfa treated with 150 and 200 mmol Na₂SO₄ L^–1, respectively, compared with the control ( Figure 6A ). Alfalfa ADF and NDF decreased significantly with increasing salt levels. Compared with the control, ADF did not change in alfalfa treated with 50 mmol Na₂SO₄ L^–1 but showed a significant decrease of 18% in alfalfa treated with 100 mmol Na₂SO₄ L^–1 ( Figure 6B ). Salt addition significantly decreased the NDF of alfalfa by 20% and 34% in treatments of 50 and 100 mmol Na₂SO₄ L^–1, respectively, compared with the control ( Figure 6C ).

In experiment 2, salt stress significantly increased CP by 11%–15% and decreased ADF and NDF by 15%–24% compared with alfalfa treated with the same N levels but without salt stress. The N supply did not change alfalfa CP content, which was 19.1% and 21.7% on average in the alfalfa treated without and with salt stress, respectively ( Figure 6D ). The same results were found for alfalfa ADF and NDF. The ADF was 37.2% and 29.5%, and NDF was 43.1% and 35.1% on average in the alfalfa treated without and with salt stress, respectively ( Figures 6E, F ).

Salt stress had a negative effect on alfalfa growth and biomass in experiment 1. Additionally, experiment 2 also confirmed the results and showed N supply alleviated the negative effects of salt stress. Like chickpeas, alfalfa can bear a light salt stress that was less than 100 mmol Na₂SO₄ L^–1 (Qurashi and Sabri, 2013). One reason for this could be the accumulation of soluble sugars and proline in plants experiencing light salt stress, which facilitates the maintenance of the cytoplasmic osmotic pool for growth stabilization and cellular metabolism in plants (Farooq et al., 2017). However, once salt concentration exceeds the critical level, plant growth is inhibited by salt toxicity (Samineni et al., 2011). The results show that the height, stem diameter, and branching number of alfalfa significantly decreased when the salt level was more than 100 mmol Na₂SO₄ L^–1 ( Figures 1A–C ). These responses were attributed to the decline in the water potential of tissue, which results in the closure of stomata and sequentially decreases photosynthesis, resulting in reduced growth (Garg and Manchanda, 2009; Garg and Bhandari, 2016). Root growth is more vulnerable to salt stress than shoot growth for safflower (Kaya, 2003). Our results did not support this difference between root and shoots for alfalfa, as they showed a similar response to salt stress ( Figures 2C, D ).

Alfalfa growth inhibited by salt stress was partially overcome by treatments with N supply ( Figures 1D–F ). This finding was consistent with previous studies in which soybeans suffering salt stress could still achieve high plant biomass when supplied with 120 mg N kg^–1 soil in a pot experiment (Abdel Wahab and Abd Alla, 1995). Sikder et al. (2020) found that N supply increases plant water status, photosynthetic pigment synthesis, and gas exchange attributes, and further improves plant growth in salt-stressed conditions. In addition, N supply also increases the activity of defense enzymes and decreases salt ion concentrations to alleviate the negative effect on plant growth of legumes suffering salt stress (Kirova, 2020; Hashemi et al., 2022). In this study, however, N supply did not have any stimulation for alfalfa growth except where salt stress was induced ( Figures 2C, D ). This is consistent with the results of He et al. (2018), who found no significant difference in the shoot growth of alfalfa among treatments comprising different N application rates when plants were not suffering salt stress in the field.

Salt accumulation in the rhizosphere causes a nutritional imbalance in plants (Miransari and Smith, 2007), including the inhibition of N adsorption (Rabie and Almadini, 2005). Our results showed that alfalfa shoot and root N concentrations significantly decreased when the salt level was above 150 mmol Na₂SO₄ L^–1 ( Figures 3A, B ), but the shoot and root N contents were more sensitive to salt addition than concentrations ( Figures 4A, B ). Previous studies show decreased biomass accumulation leads to a higher nutrient concentration in plants because of the concentration effect (Jarrell and Beverly, 1981; Li et al., 2010). We suspect that this is why shoot N concentrations became higher when the alfalfa suffered salt stress in experiment 2 ( Figures 3C, D ; Figures 4C, D ). The N supply significantly increased shoot and root N contents through accelerating alfalfa growth in salt-affected soil ( Figures 4C, D ). This indicates that salt stress induced N deficiency in the alfalfa in this study. The N supply improved plant N nutrition when alfalfa suffered salt stress, in line with previous studies (Wortmann et al., 2000; Abbasi et al., 2011).

Salt stress significantly suppresses nodulation formation in legumes (Li et al., 2021). For instance, salt stress has been found to substantially decrease the activity and density of nodules in the pigeon pea by two to three times (Garg and Manchanda, 2008). The nodule number and weight in alfalfa were also significantly reduced by increasing salt levels in this study ( Figures 5A–C ). The reasons for this due to salt stress could include poor root growth, fewer available photosynthates, fewer hairs, and lower respiration rate (Manchanda and Garg, 2008; Cornacchione and Suarez, 2015; Díaz et al., 2018). These factors are necessary for nodule formation, and the effects of salt stress on them further lead to the negative impacts on the colonization of rhizobia in the root (Del Pilar Cordovilla et al., 1999; Arora, 2015). Furthermore, salt stress decreased nodule number but not nodule weight in alfalfa treated without N supply ( Figures 5D, E ). This indicates that salt stress inhibited nodule emergence rather than nodule growth. In addition, N fixation still requires N from the soil for the early stage of nodule formation (Bordeleau and Prevost, 1994; Lindström and Mousavi, 2020). Thus, N supply did not increase alfalfa nodule formation and the amount of fixed N owing to there being sufficient soil N in the no-salt-stress condition ( Figures 5D–F ; Table 2 ). However, as discussed above, the N fixation processes of alfalfa were disturbed by salt stress. Thus, it was not unexpected that N supply increased the nodule number and weight of alfalfa in salt-affected soil in this study because N supply provides the starting N for nodules and enhances the N fixation efficiency of rhizobia through increasing the activity of nitrogenase and nitrate reductase when legumes suffer salt stress (Abdel Wahab and Abd Alla, 1995; Liu et al., 2022). This result aligns with previous studies (Undersander, 2011; Elgharably and Benes, 2021). Salt stress also reduced the N fixation efficiency of nodules, indicating that N fixation processes were disturbed by the salt.

Salt stress decreased the CP, ADF, and NDF of alfalfa in this study ( Figures 6A–C ), which is consistent with the results of Yan et al. (2005). Similar results have also been found in marvel grass (Kumar et al., 2018). As per the discussion above, salt addition decreases CP concentration in alfalfa because of the inhibition of N uptake and N fixation (El-Sharkawy et al., 2017). The low cell wall and lignin concentrations caused by salt addition are responsible for the decreased ADF and NDF (Oliveira et al., 2020). A meta-analysis showed that the CP concentration of alfalfa increases with N supply, which is due to the higher activity of key enzymes for N metabolism (Geisseler et al., 2010; Wan et al., 2022). However, N supply did not change the CP concentration of alfalfa in this study ( Figure 6D ). This may be explained by the alfalfa growing so fast that it caused the dilution effect (Jarrell and Beverly, 1981). Our results showing that alfalfa ADF and NDF were not responsive to N supply ( Figures 6E, F ), are also consistent with the results of Cherney et al. (1994).