Interactive effects of phosphorus fertilization and salinity on plant growth, phosphorus and sodium status, and tartrate exudation by roots of two alfalfa cultivars

Abstract

Background and Aims

Soil phosphorus (P) deficiency and salinity are constraints to crop productivity in arid and semiarid regions. Salinity may weaken the effect of P fertilization on plant growth. We investigated the interactive effects of soil P availability and salinity on plant growth, P nutrition and salt tolerance of two alfalfa (Medicago sativa) cultivars.

Methods

A pot experiment was carried out to grow two cultivars of alfalfa in a loess soil under a combination of different rates of added P (0, 40, 80 and 160 mg P kg⁻¹ soil as monopotassium phosphate) and sodium chloride (0, 0.4, 0.8 and 1.6 g NaCl kg⁻¹ soil). Plant biomass, concentrations of P ([P]), sodium ([Na]) and potassium ([K]) were determined, and rhizosheath carboxylates were analysed.

Key Results

There were significant interactions between soil P availability and salinity on some, but not all, of the parameters investigated, and interactions depended on cultivar. Plant growth and P uptake were enhanced by P fertilization, but inhibited by increased levels of salinity. Increasing the salinity resulted in decreased plant P-uptake efficiency and [K]/[Na]. Only soil P availability had a significant effect on the amount of tartrate in the rhizosheath of both cultivars.

Conclusions

Increased salinity aggravated P deficiency. Appropriate application of P fertilizers improved the salt tolerance of alfalfa and increased its productivity in saline soils.

INTRODUCTION

Phosphorus (P) is an essential macronutrient for plant growth and agricultural production (Ma et al., 2016). Although total P concentrations in soil are not always low, much of this P is poorly available, and P deficiency is widespread and frequently limits plant growth (Hinsinger, 2001). Crop yields on ~30–40 % of the world’s arable land are limited by P availability (Ros et al., 2020). Soil P occurs both as inorganic P (Pi) and organic P (Po) (Shen et al., 2011). Organic P may account for ~80 % of the total soil P, but it cannot be taken up by plants (Wang et al., 2007; Zribi et al., 2014). The only form of P that can be absorbed by plants, Pi, is often present in the soil solution at a very low concentration, due to its sorption to various soil minerals. Therefore, the P-uptake and -utilization efficiencies of plants are usually low (Hinsinger, 2001; Zribi et al., 2017).

Large amounts of P fertilizers are applied to soil to improve agricultural productivity, but only 15–30 % of applied fertilizer P is taken up by crops in the year of its application (Veneklaas et al., 2012). Long-term fertilization causes accumulation of a large amount of P in soils, and may lead to eutrophication of waterways (Johnston et al., 2014). Furthermore, phosphate rock, from which P fertilizers applied in modern agriculture are produced, is a non-renewable resource (Cordell et al., 2009). Therefore, it is of great importance to improve the P-uptake and -utilization efficiency of crops, in order to reduce the reliance on a natural resource and minimize the adverse impact of over-application of P fertilizers on the environment (Johnston et al., 2014; Cong et al., 2020).

Phosphorus deficiency is a major factor limiting crop production in a number of low-input agricultural systems worldwide, including many regions where calcareous and alkaline soils prevail (Zribi et al., 2011). Plants have various adaptations to obtain adequate P under P deficiency, including changes in root morphology and architecture, and secretion of protons, low-molecular-weight organic acids, and phosphatases into the rhizosphere (Vance et al., 2003). The rhizosphere is a key zone in which plants interact with each other, and with soil and microorganisms. Organic compounds such as mucilage and organic acids (e.g. citric acid, malic acid, oxalic acid, succinic acid and malonic acid), which are secreted by the root system, can greatly change the rhizosphere environment, thus affecting P availability and its uptake by plants (Shen et al., 2011).

More than 6 % of the world’s land and 20 % of the irrigated areas worldwide are facing salinity threats. In addition to drought and limited availability of nutrients (notably P), soil salinity is among the most damaging abiotic constraints to crop productivity (Munns and Tester, 2008; Bertrand et al., 2015). Potential adverse effects of salt stress on plants include osmotic stress caused by low external water potential, toxicity of sodium (Na) and/or chloride (Cl) ions, and nutritional imbalance caused by interference with the absorption and transportation of nutrients (Farooq et al., 2015; Long et al., 2019). Increasing salinity levels can significantly decrease plant uptake of nutrients; poor soil physical conditions and high Na and Cl concentrations have a negative effect on the availability of nutrients (especially P) (Zahedi et al., 2021). Salinity decreases P-uptake efficiency of crops such as chickpea (Cicer arietinum) (Kaci et al., 2018), although it may enhance the release of carboxylates by roots of some plants, e.g. some Acacia species (Abbas et al., 2014). High concentrations of Na in plants can compete with other nutrients, especially potassium (K), thus resulting in nutritional imbalances and often a decrease of the K/Na ratio (Hauser and Horie, 2010; Sofy et al., 2020). Salinity can inhibit important metabolic processes in which K is involved, and consequently affect plant growth and development (Farooq et al., 2015; Ashrafi et al., 2018). Maintaining a high K/Na ratio is directly related to greater salt tolerance and better growth of plants under salt stress (Flowers and Colmer, 2015).

Application of sufficient P to saline soil can alleviate the negative effects of salinity on plant growth and development, and improve the salt tolerance of plants (Bargaz et al., 2016; Zribi et al., 2018). Different crop cultivars or genotypes may have different strategies for P acquisition and utilization (Cong et al., 2020). The growth and physiological responses of different cultivars or genotypes to salinity can vary greatly; the salt tolerance and tolerance mechanisms of different cultivars or genotypes also vary (Dugasa et al., 2021; Feng et al., 2021). Furthermore, soil P availability and salinity may affect plant P acquisition and salt tolerance in an interactive way (Abbas et al., 2018). Investigating the interactive effects of P fertilization and salinity on plant growth and physiology of different cultivars or genotypes and identifying underlying mechanisms of the effects are important for improving the utilization efficiency of P fertilizers and increasing the productivity of crops on saline soils.

Alfalfa (Medicago sativa) is a widely distributed and long-cultivated forage legume in China. It has great economic value and good ecological effects, and plays a pivotal role in the establishment of cultivated grasslands in China (Fan et al., 2015). The objective of this study was to investigate the effects of P availability, salinity level and their interaction on plant growth, P nutrition and salt tolerance of alfalfa. We carried out a pot experiment to grow alfalfa in a loess soil under a combination of different rates of added P and sodium chloride (NaCl), and tested the following hypotheses: (1) adding P would have a positive effect on plant growth and P concentration ([P]), while adding NaCl would have a negative effect on plant growth and [P]; (2) P-uptake efficiency would decline with increasing P-addition rate, and also with increasing NaCl-addition rate; (3) plant sodium concentration ([Na]) would increase with increasing NaCl-addition rate, but decrease with increasing P-addition rate, and the ratio of [K] to [Na] ([K]/[Na]) in plants would decrease with increasing NaCl-addition rate, but increase with increasing P-addition rate; (4) the amounts of rhizosheath carboxylates would be greater at lower P-addition rate, but greater at higher NaCl-addition rate; and (5) increasing NaCl-addition rate would weaken the effect of P addition on the above-mentioned parameters, and vice versa.

MATERIALS AND METHODS

Soil preparation and plant cultivation

A loess soil was collected from a farmland that had been abandoned for a few years and fertilized before abandonment, in Yangling, Shaanxi Province, China, and used for the pot experiment. The physicochemical properties of the soil were determined (Table 1). The soil was air-dried and sieved through a 2-mm mesh before filling the pots. About 2 kg air-dried soil was added to each plastic pot lined with a plastic bag inside. To the soil in each pot, external P was added as an aqueous KH₂PO₄ solution at four rates, i.e. 0, 40, 80 and 160 mg P kg⁻¹ dry soil (hereafter referred to as 0, 40, 80 and 160P, respectively); NaCl was added at four rates, i.e. 0, 0.4, 0.8 and 1.6 g kg⁻¹ dry soil (hereafter referred to as 0, 0.4, 0.8 and 1.6NaCl, respectively). The experiment was set up as a two-factor completely random design, and each treatment was replicated four times. Other nutrients, including K and nitrogen (N), were added to the soil in each pot. External K was added at 100 mg K kg⁻¹ dry soil as an aqueous K₂SO₄ and KCl solution, in which the molar ratio of K₂SO₄ to KCl was 1:2. External N was added at 100 mg N kg⁻¹ dry soil as an aqueous NH₄NO₃ solution. After addition of the above-mentioned nutrients, soil water content was maintained at 60 % of the field capacity of the soil in each pot for 2 weeks by weighing the pots and replenishing with deionized water every 2 or 3 d. Then the soil in each pot was air-dried, sieved through a 2-mm mesh separately and thoroughly mixed again, and filled back to the pot lined with a plastic bag inside to ensure homogeneous distribution of added chemicals.

Two cultivars of alfalfa. i.e. Medicago sativa L. ‘Golden Empress’ and Medicago sativa L. ‘Salt-tolerant Star’, were used in this study. For each cultivar, seeds were sterilized in 10 % (v/v) hydrogen peroxide (H₂O₂) for 20 min, rinsed five times with deionized water and then soaked overnight before sowing. On the next day, 30 seeds were sown in each pot, and seedlings were thinned to 20 plants per pot after 2 weeks. Plants were grown from April to July in 2019 in a greenhouse at Northwest A&F University (34°16′19″N, 108°04′20″E), Yangling, Shaanxi, China. Soil water content was maintained at 60 % of the water-holding capacity of the soil in each pot by weighing the pot and replenishing deionized water every 2 or 3 d, and no drainage was allowed from the pots. During the experiment, the average temperature in the greenhouse was 26/18 °C (day/night), the minimum night-time temperature was 15 °C and the maximum daytime temperature was 30 °C. Plants were grown under natural light with a photoperiod of 9−11 h, and the relative humidity in the greenhouse was 40−60 %.

Plant harvest, collection, and analyses of rhizosheath carboxylates

Plants were harvested 106 d after sowing. Shoots were severed at the soil surface, and leaves and stems were collected separately; leaves from the same pot were pooled as one sample for further analyses, and stems were treated in the same way. The plastic bag lining inside each pot was lifted out of the pot to separate the roots from the soil. For the plants in each pot, ~1 g fine roots and rhizosheath soil were collected and transferred into a glass beaker containing 20 mL 0.2 mm CaCl₂. The roots and rhizosheath soil were soaked in the solution for ~5 min and gently stirred to remove the rhizosheath soil as much as possible and to ensure cell integrity. About 1 mL of a subsample of the rhizosheath extract was filtered by a 0.22-μm syringe filter attached to a 10-mL syringe and injected into a 1-mL high-performance liquid chromatography (HPLC) vial, into which a drop of concentrated phosphoric acid was added to acidify the extract and prevent microbial degradation of the carboxylates. The pH of the remaining extract was measured in the beaker. The vials containing the rhizosheath extracts were stored at –20 °C until analysis of carboxylates using HPLC (He et al., 2020a). The soaked roots were collected and washed with tap water, and oven-dried at 60 °C to constant weight. The dry masses of leaves, stems and roots that were not soaked were determined separately after the samples were oven-dried at 60 °C for at least 48 h until constant weight. The dry mass of leaves and that of stems were summed to obtain the shoot dry mass (SDM); the dry mass of soaked roots and that of roots not soaked were summed to obtain the root dry mass (RDM).

Analysis of rhizosheath carboxylates was performed using a Waters E2695 HPLC equipped with a Waters 2998 detector and Waters Symmetry C18 reverse phase column (Waters, Milford, MA, USA). The working standards included acetic acid, malonic acid, tartaric acid, malic acid, succinic acid and citric acid (all analytical pure, Sinopharm Chemical Reagent Co. Ltd, Shanghai, China) to identify carboxylates at 210 nm. The mobile phase included 20 mm KH₂PO₄ (adjusted to pH 2.5 with concentrated H₃PO₄) and 100 % methanol, and the flow rate of the KH₂PO₄ solution and methanol were 0.6 and 0.01 mL min⁻¹, respectively. The injection volume of each sample was 10 μL, and the run time for each sample was 18 min (He et al., 2020a). Acetate, malonate, malate, succinate and citrate were only detected in a few samples, and their results are not presented. Tartrate was detected in all samples, and its amount was expressed in mmol per unit root dry mass.

Measurement of bulk soil pH

Bulk soil samples were collected from each pot when plants were harvested, after all soil in each pot was passed through a 2-mm sieve and mixed. The pH of air-dried bulk soil was determined in a 1:5 (w:v) soil:water suspension using an FG2 pH-meter (Mettler Toledo, Shanghai, China) (Zhang et al., 2021).

Analyses of P, Na and K concentrations in plants

Oven-dried roots that were not soaked to extract carboxylates, stems and leaves were finely ground with a mortar and pestle. For each sample, ~1 g of subsample was weighed and digested in a 4:1 (v:v) HNO₃:HClO₄ mixed acid solution. The [P] in the solution was assayed using the vanado-molybdate yellow method (Gupta et al., 1993). The [Na] and [K] in the solution were determined by flame emission photometry (Zribi et al., 2011).

Calculation of P-uptake efficiency

Plant P content per pot was calculated as the sum of leaf P content (leaf [P] × leaf dry mass), stem P content (stem [P] × stem dry mass) and root P content (root [P] × RDM). For each treatment supplied with external P, P-uptake efficiency was calculated as the difference in plant P content per pot under a certain P rate and that with no added P and NaCl, i.e. 0P + 0NaCl treatment, divided by the difference in the amount of P applied between this treatment and the 0P treatment (He et al., 2017).

Statistical analyses

All data were statistically analysed using R software version 4.1.0. The effects of P-addition rate, NaCl-addition rate and their interaction on the above-mentioned parameters were analysed by performing a two-way (P × NaCl) analysis of variance (ANOVA) for the two cultivars separately; the effects were determined significant at P < 0.05. After the two-way ANOVA, if the effect of P-addition rate on a parameter was significant, Fisher’s protected least significant difference (LSD) test for post hoc means comparison was made among different P treatments by performing one-way (P treatment) ANOVA, and significance was determined at P < 0.05. If the results of the two-way ANOVA showed a significant effect of NaCl treatment or a significant interaction between P and NaCl on a parameter, an LSD test for post hoc means comparison was made among NaCl treatments within the same P treatments after performing one-way (NaCl treatment) ANOVA, and significance was determined at P < 0.05.

RESULTS

Plant growth

In general, adding P enhanced plant growth, while adding NaCl inhibited plant growth (Figs 1 and 2). For neither cultivar was there a significant interaction between soil [P] and [NaCl] on SDM (Fig. 2A, D, Table 2). For both cultivars, adding P significantly increased SDM (both P < 0.001), with the 160P treatment increasing SDM the most. The effects of NaCl on SDM were significant for both cultivars (both P ≤ 0.002). Adding NaCl reduced SDM of ‘Golden Empress’ in most cases, and always reduced SDM of ‘Salt-tolerant Star’, with the 1.6NaCl treatment reducing SDM the most. The interaction between soil P and NaCl on RDM was significant for ‘Salt-tolerant Star’ (P = 0.003) (Fig. 2E), but it was not significant for ‘Golden Empress’ (Fig. 2B). The RDM of ‘Golden Empress’ generally increased with increasing P-addition rate (P < 0.001), while it decreased considerably with increasing NaCl-addition rate (P < 0.001). For ‘Salt-tolerant Star’, RDM increased with increasing P-addition rate at all NaCl-addition rates, except 1.6NaCl; RDM decreased with increasing NaCl-addition rate at 0P and 160P, but increased at 0.4NaCl, and then decreased with increasing NaCl-addition rate at 40P and 80P. For neither cultivar was there a significant interaction between soil [P] and [NaCl] on RMR (Fig. 2C, F). The RMR always increased when more P was added (P < 0.001), while it declined in almost all cases when NaCl was added (P < 0.001, Fig. 2C, F).

Plant P concentration

The interaction between soil [P] and [NaCl] on leaf [P] was not significant for either cultivar (Fig. 3A, C, Table 2). For both cultivars, the effect of soil [P] on leaf [P] was significant (P = 0.035 for ‘Golden Empress’ and P = 0.002 for ‘Salt-tolerant Star’). Leaf [P] of ‘Golden Empress’ decreased, rather than increased, in almost all cases when P was added, while leaf [P] of ‘Salt-tolerant Star’ increased in almost all cases when P was added. Leaf [P] of ‘Golden Empress’ was not considerably affected by adding NaCl, but leaf [P] of ‘Salt-tolerant Star’ decreased with increasing NaCl addition. The interaction between soil [P] and [NaCl] was significant for stem [P] of ‘Golden Empress’ (P = 0.026), but not for that of ‘Salt-tolerant Star’ (Fig. 3B, D). For ‘Golden Empress’, stem [P] increased when more P was added in almost all cases, except at 40P + 0.8NaCl, while it decreased when more NaCl was added in almost all cases, except at 160P + 0.4NaCl. Stem [P] of ‘Salt-tolerant Star’ was not significantly affected by P-addition rate, but markedly reduced by adding NaCl, and decreased the most at 1.6NaCl.

Phosphorus-uptake efficiency

No significant interaction between soil [P] and [NaCl] on P-uptake efficiency was found for either cultivar (Fig. 4A, B, Table 2). Both adding P and adding NaCl caused a significant decline in P-uptake efficiency for both cultivars (all P < 0.001), and P-uptake efficiency of ‘Salt-tolerant Star’ showed a more obvious declining trend with increasing P-addition rate, as well as NaCl-addition rate, than that of ‘Golden Empress’.

Plant Na concentration

The interaction between soil [P] and [NaCl] on both leaf [Na] and stem [Na] was not significant for either cultivar (Fig. 5A–D, Table 2). The effect of soil [P] on leaf [Na] was only significant for ‘Golden Empress’ (P < 0.001), of which leaf [Na] decreased when more P was added. Adding NaCl had a significant effect on leaf [Na] of both cultivars. Leaf [Na] of ‘Golden Empress’ increased in almost all cases when NaCl was added, and it increased the most at 1.6NaCl. For ‘Salt-tolerant Star’, leaf [Na] increased with increasing NaCl addition. The effect of soil [P] on stem [Na] was only significant for ‘Salt-tolerant Star’ (P = 0.003), of which stem [Na] decreased with increasing P addition in most cases. The effect of [NaCl] on stem [Na] was significant for both cultivars (P < 0.001 for both cultivars), with stem [Na] increasing with increasing NaCl addition in most cases.

Plant [K]/[Na]

For neither cultivar was the interaction between soil [P] and [NaCl] on leaf [K]/[Na] significant (Fig. 6A, C, Table 2). Adding more P resulted in a significant increase in leaf [K]/[Na] of ‘Golden Empress’ (P = 0.001), but did not cause considerable variation in leaf [K]/[Na] of ‘Salt-tolerant Star’. Adding NaCl caused a significant decline in leaf [K]/[Na] of both cultivars (both P < 0.001), with leaf [K]/[Na] of ‘Golden Empress’ in most cases and that of ‘Salt-tolerant Star’ always declining with increasing NaCl addition. The interaction between soil [P] and [NaCl] on stem [K]/[Na] was significant for both cultivars (P < 0.001 for ‘Golden Empress’ and P = 0.029 for ‘Salt-tolerant Star’) (Fig. 6B and D). For ‘Golden Empress’, stem [K]/[Na] at 40P and 80P declined with increasing NaCl addition, but that at 0P was similar at 0.4NaCl and 0.8NaCl, and that at 160P was also similar at 0.4NaCl and 0.8NaCl. For ‘Salt-tolerant Star’, stem [K]/[Na] declined with increasing NaCl addition at all P-addition rates, except 80P, at which stem [K]/[Na] was similar at 0.4NaCl and 0.8NaCl.

Rhizosheath tartrate

The amount of tartrate in the rhizosheath of both cultivars was only markedly affected by soil [P] (both P < 0.001) (Fig. 7A, B, Table 2). For ‘Golden Empress’, the amount of tartrate was 10.7–88.5 mmol g⁻¹ RDM, and it decreased by 15, 42 and 74 % at 40P, 80P and 160P, respectively, when compared with that at 0P. For ‘Salt-tolerant Star’, the amount of tartrate was 15.0–55.4 mmol g⁻¹ RDM, and it increased by 20 % at 40P, but decreased by 23 and 51 % at 80P and 160P, respectively, when compared with that at 0P.

The pH of the bulk soil and rhizosheath extract

For both cultivars, the pH of the bulk soil after plants were harvested was significantly affected by the interaction between soil [P] and [NaCl] (both P = 0.001, Tables 2 and 3). However, soil [P] and [NaCl] and their interaction did not considerably affect the pH of the rhizosheath. The pH of bulk soil ranged from 7.05 to 8.37, while the pH of the rhizosheath ranged from 7.23 to 7.84. The pH of the rhizosheath was lower than the pH of the bulk soil in some, but not all treatments.

DISCUSSION

We carried out a pot experiment with two cultivars of alfalfa grown in a loess soil with different rates of added P and NaCl, to investigate the interactive effects of P availability and salinity on plant growth and P and Na status, and tested several hypotheses. The results of the present study support some of our hypotheses.

Our first hypothesis – that adding P would have a positive effect on plant growth and [P], while adding NaCl would have a negative effect on plant growth and [P] – was partly supported. We observed significant increases in SDM, RDM and RMR when P was added, but decreases in these parameters when NaCl was added, indicating that increasing P availability enhanced plant growth, while increasing salinity inhibited plant growth. Therefore, the hypothesis regarding the effects of P and salinity on plant growth was fully supported. Phosphorus fertilization can improve plant growth and production for most plants, including alfalfa (He et al., 2017, 2020a, b; Lu et al., 2020), and salinity reduces biomass production of many plants (Al-Farsi et al., 2020). According to Bertrand et al. (2015), the negative effect of salinity on alfalfa growth is more pronounced on SDM than on RDM, and our study confirmed this. The hypothesis regarding the effects of P and salinity on plant [P] was not fully substantiated, as adding P reduced leaf [P] of ‘Golden Empress’, and did not markedly affect root [P] of ‘Golden Empress’ (Supplementary Data Table S1, Fig. S1A) and stem [P] of ‘Salt-tolerant Star’, while salinity did not considerably affect root [P] of ‘Golden Empress’ (Supplementary Data Table S1, Fig. S1A). Increased plant [P] with increasing soil P addition (He et al., 2020a, b) and decreased plant [P] with increasing salinity are often reported (Zribi et al., 2011). The results of the present study suggest that the effects of soil [P] and salinity on plant [P] depend on cultivar as well as plant organ.

The second hypothesis – that P-uptake efficiency would decline with increasing P-addition rate, and also with increasing NaCl addition – was fully supported. With increasing salinity, the availability of P in soil would be reduced due to the ionic strength effect, which reduces phosphate activity under salinity (Martinez et al., 1996). Physical and chemical changes at the membrane level and inhibition of P uptake and accumulation in shoots have been speculated to be the result of competition between chlorine (Cl) and P, as well as interactions other than competition (Papadopoulos and Rendig, 1983). Inorganic P uptake depends on Pi transporters; salinity may decrease P acquisition at sufficient P supply, possibly due to its negative effect on Pi transporters (Zribi et al., 2015). Kochian (2000) suggested that the activity of Cl reduces the activity of Pi transporters. Furthermore, increasing salinity inhibited plant growth, and the P demand by plants therefore decreased, leading to reduced P-uptake efficiency. The significant reduction in root growth suggests that plants had less surface area for P uptake, which might also contribute to the decline in P-uptake efficiency.

The third hypothesis – that plant [Na] would increase with increasing NaCl addition, but decrease with increasing P addition, and [K]/[Na] in plants would decrease with increasing NaCl addition, but increase with increasing P addition – was not fully supported. Our results show that plant [Na] generally increased with increasing salinity, as reported in other studies (Farooq et al., 2015; Tavakoli et al., 2019). Plant [Na] may decrease in response to P fertilization under salt stress (Bargaz et al., 2016; Yarahmadi et al., 2018). However, in the present study, the hypothesis that plant [Na] would decrease with increasing P addition was only supported for root [Na] of both cultivars (Supplementary Data Table S1, Fig. S1B, F), leaf [Na] of ‘Golden Empress’, and stem [Na] of ‘Salt-tolerant Star’. Potassium is involved in a range of metabolic processes in plants, and salt stress often reduces plant [K], resulting in lower [K]/[Na]. In the present study, increasing the degree of salinity considerably reduced root [K] of both cultivars (Supplementary Data Table S1, Fig. S1C, G), but only significantly reduced leaf [K] and stem [K] of ‘Salt-tolerant Star’ (Supplementary Data Table S1, Fig. S2C, D). However, [K]/[Na] in leaves, stems and roots of both cultivars (Fig. 6, Supplementary Data Table S1, Fig. S1D, H) was markedly reduced when salinity was increased. Maintaining a high [K]/[Na] in plants, especially in leaves, is important for plant salt tolerance (Boughanmi et al., 2005; Tavakoli et al., 2019). In most cases, adding P significantly increased leaf and stem [K]/[Na] of ‘Golden Empress’, and also stem and root [K]/[Na] of ‘Salt-tolerant Star’ (Supplementary Data Table S1, Fig. S1H), indicating that P fertilization enhanced the salt tolerance of both cultivars of alfalfa. The [K]/[Na] in two cultivars of Phaseolus vulgaris were significantly higher as P supply was increased, while salinity significantly increased [Na] and reduced the [K]/[Na] considerably (Bargaz et al., 2016).

Our fourth hypothesis – that the amounts of rhizosheath carboxylates would be greater at lower P addition rate, but greater at higher NaCl addition rate – was not fully supported. The results show that tartrate was the major carboxylate in all treatments, and its amount was markedly affected by soil P in both cultivars, but not by the level of salinity for either cultivar. The amount of tartrate was greater at lower P-addition rate, similar to the results in our previous study (He et al., 2020a). Release of carboxylates is a P-mining strategy to enhance desorption or solubilization of organic and inorganic P in soil, although release of more carboxylates is not invariably accompanied with a more acidic rhizosphere (Lambers et al., 2006). The release of organic acids is considered a plant P-stress tolerance mechanism (Larsen et al., 1998). Abbas et al. (2014) observed increased concentrations of a few organic acids, including tartaric acid, with increasing degree of salinity in different Acacia species. However, the results of our study do not support that release of tartrate is a salt-tolerance mechanism.

Based on the results and the discussions above, the fifth hypothesis – that increasing NaCl addition would weaken the effect of P addition on the above-mentioned parameters, and vice versa – was only partly supported. Increased soil salinity may cause imbalances of mineral nutrients (including P) in plants, due to competitive absorption of elements via roots, and translocation or redistribution of elements within plants. In this study, increased salinity likely aggravated P deficiency and the plant’s demand for P (Ashrafi et al., 2018). Appropriate application of P fertilizers may be a promising strategy to improve the salt tolerance of crops and increase productivity (Bargaz et al., 2016).

In conclusion, interactions between soil P and salinity on plant growth, P nutrition and salt tolerance are complex, and the interactions depend on crop cultivar. However, increased salinity negatively affected plant P uptake and aggravated P deficiency, thus further limiting plant growth, which is often limited by P. Soil P supply affects Na uptake by plants and Na translocation within plants; appropriate application of P fertilizers can improve the salt tolerance of crops and increase their productivity in saline soils. At present, there is no consensus on the interactive effects of soil P availability and salinity, and the mechanisms underlying the interactions between P availability and salinity warrant further investigation.

SUPPLEMENTARY DATA

Supplementary data are available online at https://dbpia.nl.go.kr/aob and consist of the following. Table S1: statistical levels of significance of the two-way ANOVA (NaCl × P) for root [P], [Na], [K] and [K]/[Na], and leaf [K] and stem [K]. Figure S1: root [P], [Na], [K] and [K]/[Na] of alfalfa plants grown in a loess soil with different rates of added P and NaCl.

Figure S2: leaf [K] and stem [K] of alfalfa plants grown in a loess soil with different rates of added P and NaCl.

ACKNOWLEDGEMENTS

Rhizosheath carboxylates were analysed using The Biology Teaching and Research Core Facility at College of Life Sciences, Northwest A&F University. We thank Xiyan Chen for helping the analysis of rhizosheath carboxylates using HPLC, and Fuping Tian for providing seeds for the study. H.H. and R.S. designed the experiment. R.S. and Z.Z. set up the experiment and cultivated the plants. R.S., Z.Z., C.C., Q.P. and X.C. harvested the plants, analysed the samples, and collected the data. R.S and Z.Z analysed the data and wrote the manuscript. J.P., H.H. and H.L. interpreted the data and revised the manuscript. There is no conflict of interest to declare.

FUNDING

This work was supported by The Natural Science Basic Research Program of Shaanxi Province (2019JM-411), and The National Key Research and Development Plan of China (2017YFC0504504).

LITERATURE CITED

Author notes