Effects of arbuscular mycorrhizal fungi on carbon assimilation and ecological stoichiometry of maize under combined abiotic stresses

Abstract

Arbuscular mycorrhizal fungi (AMF) enhance plant tolerance to abiotic stresses like salinity and improve crop yield. However, their effects are variable, and the underlying cause of such variation remains largely unknown. This study aimed to assess how drought modified the effect of AMF on plant resistance to high calcium-saline stress. A pot experiment was performed to examine how AMF inoculation affects the growth, photosynthetic activity, nutrient uptake and carbon (C), nitrogen (N) and phosphorus (P) stoichiometric ratio (C:N:P) of maize under high calcium stress and contrasting water conditions. The results showed that high calcium stress significantly reduced mycorrhizal colonization, biomass accumulation, C assimilation rate and C:N stoichiometric ratio in plant tissues. Besides, the adverse effects of calcium stress on photosynthesis were exacerbated under drought. AMF inoculation profoundly alleviated such reductions under drought and saline stress. However, it barely affected maize performance when subjected to calcium stress under well-watered conditions. Moreover, watering changed AMF impact on nutrient allocation in plant tissues. Under well-watered conditions, AMF stimulated P accumulation in roots and plant growth, but did not induce leaf P accumulation proportional to C and N, resulting in increased leaf C:P and N:P ratios under high calcium stress. In contrast, AMF decreased N content and the N:P ratio in leaves under drought. Overall, AMF inoculation improved maize resistance to calcium-salt stress through enhanced photosynthesis and modulation of nutrient stoichiometry, particularly under water deficit conditions. These results highlighted the regulatory role of AMF in carbon assimilation and nutrient homeostasis under compound stresses, and provide significant guidance on the improvement of crop yield in saline and arid regions.

摘要

复合非生物胁迫下丛枝菌根真菌对玉米碳同化和生态化学计量的影响

丛枝菌根真菌(arbuscular mycorrhizal fungi, AMF)能够提高植物对非生物胁迫(如盐碱)的耐受性，并改善作物产量。然而，这种影响是不稳定的，导致这种变异的原因仍不清楚。本研究旨在评估干旱如何改变AMF对植物抵抗高钙盐胁迫的影响，采用盆栽实验探究AMF接种如何影响玉米在高钙胁迫和两种水分条件下的生长、光合、养分吸收以及C:N:P化学计量比。结果显示，高钙胁迫显著降低了菌根侵染率、生物量积累、C同化速率以及植物组织中的C:N化学计量比。此外，干旱进一步加剧了钙胁迫对光合作用的抑制。在干旱和钙盐胁迫下，AMF接种在很大程度上缓解了这些负面效应。然而，在充足灌溉条件下，当受到高钙胁迫时，AMF几乎不影响玉米的生长。此外，水分影响了AMF对植物组织中养分分配的调控。在充足水分条件下，AMF刺激了根部的P积累和植物生长，但未造成叶片P与C/N比的增长，导致在高钙胁迫下叶片C:P和N:P增加。相反，在干旱条件下，AMF降低了叶片N含量和N:P比。总体而言，AMF通过增强光合作用和调节养分化学计量，提高了玉米对钙盐的抵抗力，这种效应在水分亏缺条件下更为显著。该研究结果强调AMF在复合胁迫下碳同化和养分稳态调控中的调节作用，并为在盐碱和干旱地区作物产量提升提供了科学依据。

INTRODUCTION

Salt-affected soils cover more than 7% of the topsoil of the earth and are gradually increasing at an estimated growth rate of 0.3–1.5 million hectares of farmland annually, which profoundly limits global crop production by more than 20%, particularly in some arid and semiarid regions (Evelin et al. 2009; Manuel Ruiz-Lozano et al. 2012). Increasing salinity hampers plant growth by causing osmotic and ionic stresses that affect their physiological and biochemical homeostasis, such as protein synthesis, photosynthesis, enzyme activity and mineral nutrition (Iqbal et al. 2015). A growing body of studies has reported that the principal global threats to forthcoming food security is environmental stresses (Battisti and Naylor 2009), and the world population is estimated to increase from the current 7.2 to 9.6 billion in 2050 (Gerland et al. 2014). The increasing human population and deteriorated soils for crop cultivation pose a severe threat to agricultural sustainability (Kumar and Verma 2018; Shahbaz and Ashraf 2013).

To achieve sustainable agriculture, it is necessary to increase plant productivity and develop environmentally friendly biofertilizers to prevent plants from abiotic and biotic stresses (Noble and Ruaysoongnern 2010; Yang et al. 2009). In recent years, increasing attention has been paid to the mutual relationships between plants and mycorrhizal symbiosis related to stress tolerance and yield improvement (Mei and Flinn 2010). Notably, beneficial microbes associated with plant roots play a prominent role in improving plant resilience against abiotic stresses including drought and salinity (Etesami et al. 2017; Meena et al. 2017). Such beneficial microorganisms have the potential to mitigate stress responses in various crops, offering a promising approach to sustainable agricultural production (Etesami et al. 2017; Shrivastava and Kumar 2015). Arbuscular mycorrhizal fungi (AMF) are a vital component of the beneficial microbial community in soil, present in over 80% of terrestrial plant roots (Akiyama et al. 2005; Evelin et al. 2009; Rosendahl 2008). AMF can produce an extensive hyphae network in the rhizosphere soil during the symbiosis with host plants, and fungal hyphae can extend the root–soil interface to promote water and nutrients uptake. Under stressed environments, AMF could facilitate host plants’ establishment and growth efficiently through a complex series of communications between the fungi and the host (Porcel and Ruiz-Lozano 2004), such as facilitation of underground resource acquisition (Tang et al. 2022), regulation of plant physiological activities (Mbodj et al. 2018) and improvement of soil structure (Rillig and Mummey 2006). For instance, studies have demonstrated that AMF can promote plant tolerance to salinity through the formation of mutualistic relationships with plants when exposed to high salt concentrations (Ye et al. 2019). It has also been hypothesized that the effects of AMF on salinity tolerance are attributed to improved P nutrition, which can maintain photochemical capacity and enhance antioxidant enzyme activities (Shrivastava and Kumar 2015). Due to the significant agricultural and ecological importance of symbiotic interactions between plants and mycorrhizal fungi, AMF has been recognized as a novel agent for promoting plant stress tolerance and resistance in harsh environments.

The role of AMF is variable due to the interactions among fungi, plants and the environment in a complex way. A multitude of studies have documented that AMF inoculation could positively mitigate plant physiological responses to salt stress, but the magnitude of the effects greatly differs (Munns 2002; Porcel et al. 2012). The direction of plant–fungal symbiosis is generally dependent on abiotic and biotic factors, and even the same fungal taxa confer different benefits or costs under varying conditions (Worchel et al. 2013). Under stressful conditions, plant root growth is inhibited and certain edaphic processes such as soil aggregation will be drastically altered, which in turn affects the uptake capacity of belowground resources and AMF colonization in the rhizosphere. These factors can directly impact the success of various mycorrhizal types (Begum et al. 2019; Kilpelainen et al. 2017). Drought is a prevalent abiotic factor that profoundly hampers the growth and productivity of cultivated crops, particularly in semiarid and arid regions (Bodner et al. 2015; Naveed et al. 2014). In addition to the fungi responses, drought also modifies mycorrhizal formation through the host responses, including reduced photosynthesis, stomatal closure and altered growth allocation and assimilation (Lehto and Zwiazek 2011). Drought may cause changes in the composition, activity or abundance of plant-associated microbial communities (Compant et al. 2010). The microbial community may be an important buffer against disturbance-mediated environmental changes; however, such effects are highly variable, and whether drought stress is responsible for such variation in a saline environment remains largely unknown. Therefore, the influence of drought on the microbial community and its subsequent effects on plant systems should be further explored.

Soil salinity is primarily attributed to Na⁺; however, substantial accumulations of Ca²⁺, Mg²⁺ and K⁺, commonly referred to as salt cations, are also observed (Gamalero et al. 2020). This occurs in various desert regions and agricultural areas irrigated with hard water (Borer et al. 2012). In Ca-deficient areas, AMF could interact with Ca supplements, enhancing mycorrhizal proliferation and fostering host plant growth by releasing AMF-induced exudates and reshaping the rhizosphere environment (Doubková et al. 2012; Fu et al. 2023). Nonetheless, an excess of Ca can be cytotoxic, causing osmotic stress and reducing rhizospheric bioavailable Ca through precipitation with inorganic phosphate. Excessive Ca uptake by plants can also disturb ion homeostasis balance, leading to changes in cytosol pH and decreases in the solubility of ions such as iron (Hanikenne et al. 2021). While current research has explored AMF mechanisms in enhancing plant tolerance to salinity, the majority concentrates on NaCl-induced salt stress, neglecting calcium-salt stress (Chandrasekaran et al. 2019; Porcel et al. 2012). Additionally, numerous studies have investigated how AMF influences plant sense to environmental changes and root nodule symbiosis, treating Ca as a signal transduction factor and second messenger rather than a stress factor (Navazio et al. 2007; Parniske 2008). These studies primarily adopt physio-biochemical approaches and often overlook how AMF alleviates plant photosynthesis and elemental stoichiometry in environments with excessive external Ca (Evelin et al. 2009; Fu et al. 2023). Therefore, this study aims to investigate how AMF mitigates the effects of combined drought and calcium-salt stress on maize photosynthesis and ecological stoichiometry. We hypothesized that an excess of external calcium may stimulate AMF performance, thereby enhancing maize photosynthesis and internal homeostasis under combined stress. This research will contribute to a better understanding of crop–microbe interactions in response to adverse stress and the improvement of crop yield.

MATERIALS AND METHODS

Soil preparation

The calcareous soil for the experiment was collected from the surface layer (0–20 cm) at Chongqing Jigong Mountain, China (29°39ʹ51″ N, 107°10ʹ26″ E). The soil had a pH value of 6.81, an organic matter content of 26.8 g kg⁻¹, alkali-hydrolyzed nitrogen of 68.35 mg kg⁻¹, available potassium of 108.41 mg kg⁻¹, exchangeable calcium of 2.33 g kg⁻¹, total potassium of 14.62 g kg⁻¹, total phosphorus of 0.46 g kg⁻¹ and total nitrogen of 1.34 g kg⁻¹. The soil was air-dried, sieved to 2 mm and then mixed with sand at a 1:4 (w/w) ratio as the growth medium. Therefore, the background value of exchangeable Ca in the substrate was 466 mg kg⁻¹. The sand–soil mixture was autoclave-sterilized for 2 h at 121°C to remove indigenous mycorrhizal propagules and other microbes. 200 mg kg⁻¹ of basal nutrient (N:P:K = 13:6:5) was carefully mixed into the growth medium to ensure an adequate supply of nutrients during plant growth.

AMF and plant material

Glomus mosseae strain (BGC XJ02, GM) was used as the AMF in this study. The isolated strain was provided by the Institute of Plant Nutrition and Resources, Beijing Academy of Agriculture and Forestry Sciences, China. In brief, the AMF was propagated onto a soil–sand mixture (1:1, w/w) with white clover (Trifolium repens L.) for 3 months before the experiment (Hao et al. 2021). The subsequent inoculum encompassed spores, extra radical mycelium and colonized fragments of roots present in the cultivation substrate, which contained approximately 52 spores per 1 g of soil.

Maize (Zea mays) seeds were collected from a typical karst region, Youyang County, China. After surface sterilization with 10% (v/v) hydrogen peroxide (H₂O₂) for 10 min, washed with deionized water and pregerminated at 25°C for 48 h with moist filter paper until the appearance of radicles.

Pot experiment

A pot experiment was conducted in a greenhouse at Southwest University, Chongqing, under natural light conditions. The experiment employed a 2 × 2 × 2 factorial design, containing three treatment factors: salt levels (0 and 1 g Ca kg⁻¹ dry soil), water conditions (well-watered and drought) and mycorrhizal inoculation treatments (with or without GM). To achieve the high calcium exposure condition (S), the soil was homogeneously mixed with 2.775 g of CaCl₂ solution to obtain a 0.1% Ca level (excluding the background value). Each treatment group was tested in triplicate, resulting in a total of 24 pots that were randomly arranged. In mycorrhizal treatments, 15 g of fresh inoculum was injected into the plants. The non-mycorrhizal plants were treated with 10 ml of sterilized inoculum, which was obtained by filtering unsterilized inoculum through a 10 mm pore-size filter. This was done to ensure a similar microflora. Each plastic pot (top diameter 19 cm, bottom 13 cm and height 16 cm) was filled with 2 kg of sterilized substrates. Four disinfected seeds were sown in each pot, and the surface layer was covered with 500 g of sterilized soil. After 7 days, each pot was thinned to two seedlings. To ensure AMF colonization and plant growth, deionized water was supplied to maintain the gravimetric substrate water content at 20% before initiating the drought treatment. Drought treatment began after 4 weeks, and the gravimetric substrate water content of drought pots was kept at 10% by daily weighing (Machado and Paulsen 2001). During the experiment, the temperature was 35°C/25°C during daytime/night and the relative humidity was 30%–65%.

Plant sampling and analysis

AMF colonization and element uptake assay

Plants were harvested 6 weeks after sowing. The harvested plants were meticulously uprooted, cleansed with deionized water and segregated into leaves, stems and roots. All samples were subjected to an oven-drying procedure for 30 min at 105°C, followed by further drying at 80°C until a constant weight was attained. The weight of the dry matter content was then measured.

The roots were cut into 1 cm segments and mixed thoroughly. Root fragments were immersed in a 10% KOH bath for 90 min at 90°C and then stained with trypan blue (Millar and Bennett 2016). Thirty stained root sections were mounted onto the slides in polyvinylalcohol lactic acid glycerol, and the arbuscule, vesicule and hyphae in the roots were examined using a light microscope. Finally, the infected root segments ratio to the total was evaluated as root colonization percent (Rai et al. 2001).

Determination of gas exchange parameters

Before the harvest, net photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and intercellular CO₂ concentration (Ci) were determined on the top third mature of intact plants using a photosynthesis system Li-6400 (Li-COR, USA). The photosynthetic photon flux density of the Li-6400 source was 1200 μmol m⁻² s⁻¹ during the measurements. The relative humidity in the sample chamber was kept at 65% and the CO₂ concentration was relative at 470 ± 10 μmol mol⁻¹. Measurements were subsequently performed in the morning (09:00–12:00 a.m.) in a random order. Three replicates of each treatment were randomly selected while both two plants per pot were determined. The calculation of water use efficiency (WUE) was subsequently performed using the formula:

Determination of C, N and P content and eco-stoichiometry

The determination of carbon (C) and nitrogen (N) content in plant tissues was conducted using an elemental analyzer (Vario MAX, Germany). To assess the phosphorus (P) content in the roots and shoots, 0.5 g of dried crushed root or shoot sample was digested with HNO₃ (guaranteed reagent) for 12 h, and then microwave accelerated reaction was performed using a microwave accelerated reaction system for 35 min (Mars, CEM Corp., USA). Later, P content was measured by an ICP-OES (Prodigy, Teledyne Leeman, USA). The ecological stoichiometric ratio of C, P and N in the plant tissues was calculated.

Statistical analysis

The SPSS (v16.0) was used for data analysis. Three-way analysis of variance (ANOVA) was conducted to determine the effects of mycorrhizal (M), salinity (S) and water intensity (W) on maize growth parameters, photosynthetic activities and stoichiometric ratios, followed by Duncan’s multiple range test at P < 0.05. Tukey honestly significant difference (HSD) test was applied to compare differences in C, N, and P concentrations and their ratios between plant organs in a given treatment group. Before the analysis, data normality and homogeneity of variance were verified and log-transformed when necessary. The means were reported with their standard errors.

RESULTS

AMF colonization and plant biomass

As shown in Fig. 1, no AMF colonization was found in the uninoculated plant roots, and salinity markedly decreased AMF colonization from 67.3% to 36.7% under both drought stress and well-watered conditions (P < 0.05), whereas drought had little impact on the colonization rate (Fig. 1a; Table 1). Plant biomass was also drastically influenced by the treatments, and salinity exerted a greater effect than drought and AMF inoculation on both shoot and root biomass (Table 1). Specifically, when compared with CK, the shoot and total biomass of maize increased by 40.1% and 32% by AMF inoculation in the well-watered treatment, respectively (P < 0.05), while AMF and drought hardly affected the shoot and root biomass (Fig. 1b and c). The root-to-shoot ratio was particularly inhibited by salinity under water deficit combined with no AMF (Fig. 1d).

Photosynthetic parameters and WUE

Calcium, drought stress, AMF inoculation and their interactions had significant effects on plant Pn, Tr, Gs and Ci (P < 0.01, Table 1). Moreover, Ci was also notably affected by the interactions of drought stress with calcium and with inoculation (P < 0.05, Table 1). The leaf chlorophyll content was significantly restrained by drought, and AMF greatly mitigated this inhibition under both drought and well-watered conditions (Fig. 2a). Furthermore, the least Pn, Gs and Tr were observed in non-AMF inoculated plants subject to calcium stress combined with drought, and AMF inoculation significantly increased the Pn, Gs and Tr under both water regimes (P < 0.01). Precisely, AMF increased the Pn, Gs and Tr by 2.3-, 1.6- and 1.46-fold when compared with the CK under the drought, respectively (Fig. 2b, c and e). In contrast, the opposite trend was observed for the Ci, in which AMF significantly decreased the Ci under each paired treatment group (Fig. 2d). Plant WUE was significantly affected by calcium, inoculation, drought stress, drought stress–inoculation interactions and both three interaction factors (P < 0.01, Table 1). The least WUE was observed in non-AMF inoculated plants in the presence of calcium combined with drought (Fig. 2f). Under non-CaCl₂ treatment, inoculation with AMF significantly improved the WUE under both water regimes, as compared with the uninoculated plants (P < 0.01, Table 1). Additionally, under CaCl₂ treatment, AMF inoculation markedly increased the WUE of maize by 4.8-fold under drought conditions compared with the uninoculated plants, but the same trend was not observed under well-watered conditions (Fig. 2f).

C, N and P concentrations in plant tissues

As shown in Fig. 3, C content in different tissues was comparable, while the content of N and P exhibited significant differences among tissues. High calcium stress greatly impacted C and N content and distribution in all plant tissues (Fig. 3; Table 2). Under both water regimes, AMF had little impact on the C content in maize tissues, regardless of the salinity stress. In contrast, high calcium exposure significantly decreased plant C, especially in stems and leaves (P < 0.05, Fig. 3a). Moreover, N in stems was also greatly impacted by the water regime and their interactions of treatments (P < 0.05, Table 2). For N and P, the content in plant tissues was generally in the descending order of stem > leaf > root. The N concentration in plant tissues was barely affected by AMF or soil moisture, while high calcium stress significantly increased plant N content in each paired treatment group (P < 0.05, Fig. 3b). Although AMF inoculation did not affect N content in plant organs under well-watered conditions, it decreased leaf N by 21% under drought (Fig. 3b).

In all treatments, P content in stems was markedly higher than that in leaves, followed by that in roots. Moreover, root P content was markedly affected by inoculation, drought stress and the interaction of calcium with inoculation, while stem P content was only impacted by salinity (P < 0.01, Table 2). AMF inoculation significantly increased the content of P in roots in both water regimes, regardless of salinity exposure (P < 0.05, Fig. 3c). A water deficit also increased P accumulation in maize roots in all paired treatment groups. In contrast, leaf P content was decreased by AMF by 21% under the well-watered condition. High calcium alone increased the P content by 32% and 29% in stems under both water conditions, respectively.

C:N:P stoichiometry

The ratio of C:N in the stem was evidently affected by the three main factors and their interactions, while C:N in the leaf was only impacted by water condition, calcium and their interaction (P < 0.05, Table 2). In comparison, the root C:N ratio was affected by only calcium. It can be seen that Ca stress significantly reduced the C:N ratio, regardless of plant organs; however, AMF inoculation had little influence on the C:N ratio when plants were exposed to high Ca (Fig. 4a).

The ratio of C:P in the roots was dramatically affected by calcium, inoculation, drought stress and their interactions, while those in the stems and leaves of maize were dramatically affected by calcium and the interaction of calcium with inoculation (P < 0.01, Table 2). Under CaCl₂ treatment, inoculation with AMF decreased the C:P ratios in the roots by 62%, while those in the leaves of maize increased by 24.8% under well-watered conditions; however, variations in the C:P did not drastically fluctuate in plant organs under drought stress (Fig. 4b).

The ratio of N:P in roots was evidently affected by calcium, drought stress, inoculation and their interactions, while those in stems were markedly affected by calcium and the interaction of calcium with inoculation. In contrast, the ratio of N:P in the leaves of maize was remarkably affected by calcium and the inoculation–drought stress interactions (P < 0.01, Table 2). As shown in Fig. 4c, AMF significantly decreased the N:P ratio in the roots, regardless of the water condition or whether it was salt-affected. Specifically, under CaCl₂ treatment, inoculation with AMF decreased the N:P ratios in the roots by 62% and increased those in the leaves by 31% under well-watered conditions; however, drought stress did not greatly affect those in the leaves or roots. The ratio of N:P in the leaves of salt-stressed maize was significantly increased by AMF inoculation under well-watered conditions, whereas the mycorrhizal effect exhibited an opposite trend under drought stress (Fig. 4c).

DISCUSSION

Root colonization with microbiomes has a differential effect on plant resistance in response to abiotic stresses including water deficit or salt, and the ecological effects of plant microbiota can be either positive or negative (Fadiji et al. 2023). As expected, this study showed that water deficit was one crucial factor modulating the outcome of the interactions among maize growth, AMF colonization and salt stress. There are variable results when studying ecological interactions, especially when not all factors affecting these interactions are understood (Heil 2014). Considering that drought is identified as a driver for variability in our system, more research is warranted to clarify other biological and abiotic factors that drive this variation, so as to predict the ultimate outcome of interactions between microbial, plant and salt stress.

AMF colonization ameliorated salt-induced abiotic stresses in maize

The colonization of AMF has laid the foundation for improving the growth and physiologic status of plants under salt stress (Porcel et al. 2012). With regard to AMF traits, salinity has strongly affected plant root colonization, which is consistent with numerous previous observations (Estrada et al. 2013; Evelin et al. 2011). It was evident from Fig. 1a that root colonization rates declined under saline conditions, which can be attributed to the inhibition of hyphal growth and spore germination, or a reduction in the spread of mycorrhizal colonization (Hajiboland et al. 2010; Yarahmadi et al. 2018). Moreover, drought stress had little impact on mycorrhizal infection, regardless of the Ca exposure (Fig. 1a). In response to drought or other abiotic stresses, the rapid hyphal growth and resilient spores of AMF could be formed, in cooperation with plant roots, to optimize belowground resource absorption from the soil (Auge 2001; Bergmann et al. 2020).

Biomass is an important parameter for the salt tolerance of plants. We found that maize biomass production was negatively affected by high calcium stress, which could be attributed to excessive intracellular free calcium. Similar to the generation of $PO43−$ precipitate, which interferes with the related process of phosphorus metabolism and blocks the transmission of calcium signals. Munns (2002) reported that ionic stress disturbed intracellular ion homeostasis and led to disorders in hormonal status, nutrient translocation, photosynthesis and other metabolic processes. Under non-stress conditions, plant shoot biomass is usually increased by mycorrhizal inoculation, which is partially attributed to enhanced P nutrition in host plants mediated by mycorrhiza (Alqarawi et al. 2014). Generally, AMF can increase plant biomass under abiotic stresses such as salinity or drought. In our study, AMF did increase plant shoot and root biomass by 33.7% and 82.8% under the combined drought and salt stresses, respectively, while there were no statistically significant differences between S and MS under drought (Fig. 1b and c). This may be correlated with the carbon drain effect or metabolism dysfunction in maize (Liu et al. 2004). In addition, it is reported that AMF barely affects biomass accumulation and sometimes even decreases the biomass of arbuscular mycorrhizal (AM) plants, which depends on the specific fungi species and phenological stage of the plants (Klironomos 2003; Smith and Smith 2011).

Photosynthetic activity was improved by AMF under compound stresses

It has been demonstrated that AM symbiosis can enhance salinity tolerance by improving nutrient absorption, increasing photosynthetic activity and WUE (Santander et al. 2017). Photosynthesis is a direct indicator of the physiological sensitivity of plants to abiotic stresses (Chaves et al. 2009). The present study found that salt stress significantly decreased the leaf Gs, Pn and Tr of maize seedlings, and drought plus high calcium stress additively enhanced the negative effects on Pn and Gs. These results suggest that salt stress alone or compound stresses can suppress the growth of maize via the reduction of leaf gas exchange in the early seedling stage. However, mycorrhizal plants had higher chlorophyll content and higher Gs, Pn and Tr, but lower Ci, indicating that AMF colonization sustains as exchange capacity relatively high by reducing stomatal resistance while enhancing CO₂ assimilation and transpiration fluxes (Zhu et al. 2011). The increase in Ci obliquely suggests that the photosynthetic apparatus was destroyed, since the salt-induced decrease in Gs and the passivation of enzymes can trigger CO₂ accumulation in the intercellular areas (Munns 2002; Sheng et al. 2008). However, the promotive role of AMF in plant photosynthesis is quite dependent on soil moisture, as this study showed that AMF had a greater facilitation of photosynthetic parameters under drought than under well-watered conditions (Fig. 2). This can be attributed to the variations in nutrient composition in the substrate, the suitability of plants to the substrate, and the interaction of AMF matrix moisture with other environmental factors (Sheng et al. 2008). The role of plant growth-promoting bacteria (PGPB) in alleviating abiotic stress depends on several factors, such as the type of stress, microbial species, plant genus and the type of relationship between microorganisms and plants. Combined exposure to salinity and drought results in plant secondary responses such as oxidative and osmotic stress. PGPB can induce plant tolerance to drought and salinity stresses by modulating physiological and biochemical processes, thereby inducing systemic resistance at the individual scale (Kumar and Verma 2018).

A higher WUE is beneficial for transporting water from roots to evaporating surfaces and maintaining leaf hydronic balance. Inoculation with AMF not only increases the gas exchange performances but also ameliorates plant water status under salt stress (Bahadur et al. 2019). Similarly, under the common effect of drought and salt stress, this study showed that the WUE was higher in mycorrhizal plants than those of non-mycorrhizal plants. Notably, mycorrhizal plants had better water status could be explained by their improved nutrient status, which facilitates their abilities to effectively absorb soil water. Due to the considerable relevance of WUE and gas exchange, our results showed that their response of WUE to AMF inoculation under drought was also more obvious than that under well-watered conditions. This is consistent with the results of photosynthesis that AMF would exert a greater facilitative role in harsher environments such as compound stresses.

The modulatory role of AMF in eco-stoichiometric characteristics under combined stresses

Nutrient availability, competitive absorption, transport or partitioning in plants can be affected by salt stress (Munns and Tester 2008). C, N and P are essential components of cells in plants, and their dynamics will substantially impact on cell structure and functions. Our results indicated that salt stress specifically decreased C contents in plant organs (Fig. 3). Salt stress-induced reduction in plant photosynthetic C assimilation may be the cause of the inhibitory effect on C accumulation (Boldaji et al. 2012). N and P in plants can be easily affected by the external environment, which is why they are considered crucial indicators for plant adaptation (Li et al. 2014; Rong et al. 2015). Mycorrhizal symbiosis may improve plant nutrition, and AMF can enhance the uptake of mineral nutrients by the host through their extensive extraradical mycelium network, especially for N and P (Ortiz et al. 2015; Zhao et al. 2015). Therefore, they can increase Gs, Pn, leaf chlorophyll content and photosynthetic efficiency, which has been generally regarded as an important mechanism of salt tolerance (Auge et al. 2007; Wu and Xia 2006). AMF facilitates biological nitrogen fixation through symbiosis with diazotrophs and boosts the bioavailability of micronutrients, including phosphorus (Hodge and Storer 2015; Yu et al. 2021). This study showed that AMF only reduced leaf N in maize under drought but had little impact on root or stem N. Currently, the effects of AMF colonization on N distribution are inconsistent, exhibiting a promotive or no effect (Govindarajulu et al. 2005; Johnson 2010; Wang et al. 2011). It is also reported that AMF symbiosis may have indirect effects on N metabolism (Lee et al. 2012). P is necessary for rRNA biosynthesis, while mycorrhizal colonization enables P allocation in cells and improves plant P status (Chen et al. 2010; Elser et al. 2003). The results displayed that under two water regimes, fungal hyphae can access smaller water-filled pores than roots, which significantly increased P content in the roots of AMF seedlings. This may be due to the formation of the extensive network of extraradical hyphal, which allows mycorrhizal plants to explore more soil space and acquire available P. Another explanation is that inoculating mycorrhiza can induce an enhancement of soil acid phosphatase activity, which is considered a crucial mechanism that contributes to the mycorrhizal effect on plant nutrition (Hu et al. 2013). The enhancement of P mobilization in the inoculated plants may provide more RNAs for protein synthesis and plant growth (Marulanda-Aguirre et al. 2008; Matzek and Vitousek 2009). AMF significantly reduced leaf P under well-watered conditions, but had no obvious effect under drought in this study. These results demonstrate that P and N in roots and leaves respond differently to water and microorganisms, indicating that there are contrasting differences in the accumulation patterns of N and P in plant tissues via chemical or biochemical pathways. Roots and leaves are vital organs acquiring above- and belowground resources, respectively, and function contrastingly as well as their growing environments exhibiting significant differences in the physicochemical properties (Walter et al. 2009).

Studies of ecological chemometrics focused on understanding the interactions and equilibrium of various elements between organisms and their environments (Austin and Vitousek 2012). The proportions of essential elements play a crucial role in defining the key characteristics of organisms. One important indicator of these characteristics is the ratio of carbon, nitrogen and phosphorus (C:N:P), which is closely linked to their biochemical processes and functions (Khan et al. 2014). In this study, under drought and well-watered conditions, the C:N ratios in maize organs were significantly reduced by high calcium stress. This may be due to the increased synthesis of nitrogenous metabolites as an adaptation strategy to the calcareous environment. Ecological stoichiometry varies among mycorrhizal plants and is dependent because the availability of one element in the soil under different moisture conditions affects the interactions between plants and fungi in acquiring the other elements (Milleret et al. 2009). In the current study, compared with non-AM inoculated plants, microbial inoculation did not affect the C:N ratios in plant organs under high calcium treatments, regardless of the soil moisture. In contrast, the N:P ratios in the leaves of plants subjected to salt stress showed the opposite trend. This may be ascribed to the changes caused by AMF-dependent modifications in the relation between the plant and soil nutrients (Milleret et al. 2009). However, it is necessary to further investigate the biochemical mechanism behind the N:P responses mediated by AMF. The stoichiometry of C:N:P in the tissues of symbionts is a result of the evolutionary interactions between plants and microorganisms. In conclusion, the AMF-induced adjustment of plant C:N:P stoichiometry improves resistance to salt stress and plant growth. This study also showed that the colonization of AMF changed the C:N:P stoichiometry in maize, demonstrating that there was a possible pathway between biogeochemical and symbiosis cycling. These effects may be strongly correlated with the physiological and ecological characteristics of microorganisms utilized under adverse conditions. The potential mechanisms underlying these phenomena include the significant influence of AMF formation on carbon allocation in plant roots, particularly when underground resources are more limited compared to other resources.

CONCLUSIONS

This study showed that soil and water conditions greatly changed the effect of mycorrhizal fungi on photosynthetic performance as well as the nutrient accumulation and distribution of maize subjected to the combined abiotic stresses. This indicates that the mediating role of AMF in promoting plant salt defenses is greater when encountering soil water deficit than under normal water supply. The shoot biomass accumulation preserved a higher priority under salt exposure, and AMF mitigated salt-induced inhibition of photosynthesis via chlorophyll synthesis, efficient carbon assimilation and WUE, particularly under drought. Although AMF had little impact on the concentrations of C and P in maize tissues, it significantly modified the ecological stoichiometric ratio of maize under combined stresses. It is most notable that P was predominantly transported from aerial parts to the roots and more N would be accumulated into stems and leaves due to the modulating of AMF. Nevertheless, further study focusing on addressing additional aspects such as the timing of the interaction or the intensity of drought will shed more light on the mechanism of action of the modulators for microbe–plant–salt interactions, which would be helpful and instructive for crop yield improvement under a compound stressful environment.

Funding

This work was financially supported by China Postdoctoral Science Foundation (2021M703137), Chongqing Postdoctoral Science Foundation (cstc2021jcyj-bshX0195), Postdoctoral Foundation of Jiangsu Province of China (1501014B), Education Department of Sichuan Province (17ZB0211), the Ecological Security and Protection Key Laboratory of Sichuan Province (07144812) and the Scientific Research Foundation of Chongqing University of Technology (2021ZDZ022).

Acknowledgements

We are grateful to the anonymous reviewers for their valuable comments and suggestions.

Conflict of interest statement. The authors declare that they have no conflict of interest.

REFERENCES