Effect of Dark Septate Endophytes on Plant Performance of Artemisia Ordosica and Associated Soil Microbial Community Under Salt Stress

Background: Fungal endophytes can improve plant tolerance to abiotic stress, however, the role of these plant–fungal interactions in desert species ecology and their management implications remain unclear. This study aimed to assess whether dark septate endophytes (DSE) can shift the performance of Artemisia ordosica and associated soil microbial community under salt stress. Methods: We investigated the effects of three DSE (Alternaria chlamydosporigena [AC], Paraphoma chrysanthemicola [PC] and Bipolaris sorokiniana [BS]) isolated from desert habitats on plant morphology, physiology and rhizosphere soil microhabitat of Artemisia ordosica seedlings under different NaCl concentrations (0 %, 0.1 %, 0.2 %, and 0.3 %) in a growth chamber. Results: Three DSE strains could colonize the roots of A. ordosica, and the symbiotic response with host plants depended on DSE species and NaCl concentration. The greatest benets associated with DSE occurred under 0.1 % NaCl. Specically, AC improved root morphology, and increased total biomass and superoxide dismutase (SOD) activity; PC increased root morphology, root biomass, and glutathione (GSH) and indoleacetic acid (IAA) contents; and BS promoted SOD activity and GSH and IAA contents. DSE reduced the root Na + content. Interestingly, BS promoted gram-positive (G +) and gram-negative (G −) bacteria under 0.1 % NaCl and the abundance of AM fungi under 0.2 % and 0.3 % NaCl. PC positively affected fungi, AM fungi, G − bacteria and actinomycetes under 0.2 % and 0.3 % NaCl, while AC increased the abundance of all examined microbes under 0.3 % NaCl. A structural equation modeling (SEM) demonstrated that DSE not only positively affects A. ordosica performance but also directly or indirectly impacts soil microbes by regulating the soil organic carbon (SOC), available phosphorus (AP), and alkaline nitrogen (AN) content. Conclusions: DSE isolated from A. ordosica enhanced the root development of host

not only provide important habitats for their associated microbiome but also provide photosynthates for microbial growth [11,12]. Moreover, the soil-associated microbiome has an important function in ecological functions and nutrient availability, further affecting plant growth and productivity [13,14].
Previous studies have shown that plant tolerance to stress, such as drought, salinity and high temperature, is closely related to the colonization of endophytic fungi [15,16]. Hence, the resource excavation of these associated endophytic fungi containing dark septate endophytes (DSE) is vital for improving the health and productivity of plants, especially for plant growth and vegetation restoration in degraded soils [17,18].
DSE represent a diverse group of endophytic fungi mainly involving ascomycetes, which propagate via conidia or asexual reproduction. These fungi inhabit the root tissues of healthy plants, and the dark septal hyphae and microsclerotia grown intracellularly and intercellularly are their main characteristics [19][20][21]. DSE have been shown to harbor an extensive range of hosts and have a broad ecological distribution; they are typically found in plants growing in stressful habitats, including those experiencing drought, salt, or heavy metal stress [22][23][24][25][26][27][28]. Previous studies have shown that host plant responses to DSE are variable, including positive, neutral and negative responses [29]. Indeed, DSE can alter plant growth, alter the accumulation of auxin and other chemical components, and prevent the development of abiotic resistance in host plants [30][31][32]. However, the effects of DSE on host plant performance and tness remain unknown, especially under salt-stress conditions. DSE isolated from wetlands or coastal areas have been found to alleviate the adverse effects of salt stress on plant growth [33][34][35][36]. Pan et al. [33] reported that Curvularia spp. isolated from Suaeda salsa could alleviate the adverse effects of salt stress on Populus tomentosa by increasing both antioxidant enzyme activity and the content of chlorophyll and proline in the leaves. Farias et al. [34] reported that inoculation with DSE improved the absorption of nitrogen and phosphorus in Vigna unguiculata under salt stress, which was bene cial for increasing plant growth and net photosynthesis. In addition, rhizosphere-associated microbes have important functions in the maintenance of soil nutrient availability, which have positive impacts on the tolerance of host plants to salt stress [37,38]. Similar results have also been reported in other stressful environments. He et al. [15] reported that DSE improved the root development and antioxidant enzyme activity of Glycyrrhiza uralensis and altered the soil organic nutrition content and microbiota composition, which may also promote plant growth and survival under water de cit conditions. To date, the effects of DSE on the rhizospheric microbial community of host plants under salt stress have not been studied.
Artemisia ordosica (Asteraceae) is one of the dominant sand-xing shrub species that is widely distributed in the arid and semiarid regions of northwestern China and is an important medicinal and feed plant species. This species is tolerant to drought, soil infertility, wind erosion and sand burial and has been widely used for phytoremediation as a wind break and to sand x, maintaining biodiversity and ecosystem stability in northwestern China [39,40]. Excessive amounts of Na + in the soil have been found to be a major cause of plant damage and restrict vegetation restoration in arid and semiarid regions [1,41]. Investigating the response of host plants and soil microbial communities to DSE under salt-stress conditions is highly important to improve the bioremediation of saline soils in arid and semiarid regions.
In this study, we conjecture that DSE fungi could provide more bene cial effects on promoting the growth of A. ordosica and altering the soil microbial community and physicochemical properties in the rhizosphere of A. ordosica under conditions of salt stress. Based on our conjecture, we researched the response of (1) the growth of plants, (2) the antioxidant system, (3) Na + and K + ions, (4) soil physical and chemical properties, and (5) the composition of soil microbes to DSE inoculation under conditions of salt stress. We expect that the results will reveal the impact mechanism of DSE inoculation resistance to salinization conditions on the growth and production of plants and further explore the potential of these fungi to enhance plant stress tolerance and symbiotic function during vegetation restoration in saline desert regions.

DSE colonization analysis
After harvest, typical DSE structures including dark septate hyphae and microsclerotia in inoculated plants were observed (Fig. 1). The total colonization rate was 10%-60% (Fig. 2). The colonization rate of Paraphoma chrysanthemicola (PC) peaked under 0.1% NaCl. The maximum colonization rate of Bipolaris sorokiniana (BS) occurred under 0.3% NaCl, and no signi cant differences were found among the different NaCl treatments. However, the total colonization by Alternaria chlamydosporigena (AC) in the A. ordosica roots signi cantly decreased with increasing NaCl concentration.

Plant biomass production
The shoot, root and total biomass and root:shoot ratio of host plants were signi cantly in uenced by the interactions between DSE species and NaCl stress treatment (Table 1). Inoculation with AC resulted in an increase in the shoot biomass and total biomass under 0.1% NaCl compared to the control plants. No signi cant differences in shoot and total biomass were found in the other treatments (Fig. 3a, c). DSE increased the root biomass under 0% NaCl compared with the control plants. With increasing NaCl concentration, compared with that of the control plants, only inoculation with PC increased the root biomass under 0.1% and 0.3% NaCl, whereas BS inoculation decreased the root biomass under 0.2% and 0.3% NaCl (Fig. 3b).
The root:shoot ratio was signi cantly enhanced by DSE under 0% NaCl compared to control plants. With increasing NaCl concentration, only inoculation with PC increased the root:shoot ratio under 0.3% NaCl. However, inoculation with AC and BS decreased the root:shoot ratio under 0.1% NaCl (Fig. 3d).
Plant morphological traits DSE species and NaCl stress treatment signi cantly in uenced plant height. Furthermore, the interaction between DSE species and NaCl stress treatment signi cantly affected shoot branching (Table 1).
Inoculation with AC positively affected plant height and was signi cant under 0.2% and 0.3% NaCl (Fig. 4a). A signi cant increase in shoot branching occurred only under 0% NaCl. However, with increasing NaCl concentration, inoculation with AC reduced shoot branching compared with that of the control plants under 0.1% and 0.3% NaCl (Fig. 4b).
Interactions between DSE species and NaCl stress treatment signi cantly in uenced the root length, root volume and root surface area of A. ordosica (Table 1). Inoculation with DSE promoted the root growth of A. ordosica under 0% NaCl. In addition, under 0.1% NaCl, AC and PC inoculation enhanced the root length, root volume and root surface area compared to control plants. As the NaCl concentration increased, only inoculation with PC signi cantly enhanced the root length, root volume and root surface area under 0.3% NaCl. Furthermore, no signi cant differences in root diameter were found under any treatment (Fig. 4c, d, e, f).

Antioxidant enzyme activities in the leaves
Interactions between DSE species and NaCl stress treatment on the superoxide dismutase (SOD) activity and glutathione (GSH) content in host plants were signi cant ( Table 2). DSE inoculation resulted in increased SOD activity and GSH content of the leaves under NaCl stress. AC and BS inoculation increased SOD activity under NaCl stress compared with that of the control plants, while PC inoculation improved SOD activity only under 0.2% NaCl (Fig. 5a). BS inoculation increased the GSH content compared to the control plants under NaCl stress, PC inoculation increased the GSH content only under 0.1% NaCl, and AC inoculation increased the GSH content under 0.2% and 0.3% NaCl (Fig. 5b). Indoleacetic acid (IAA) content in the roots DSE species signi cantly in uenced IAA production in A. ordosica roots regardless of the NaCl stress treatment ( Table 2). Inoculation with PC and BS increased the IAA content compared to the control plants under all NaCl treatments. Moreover, AC inoculation had a positive effect on IAA content under 0.2% and 0.3% NaCl, but there were no signi cant differences (Fig. 5c). Na + and K + content DSE species and NaCl stress treatment had signi cant interaction effects on the K + content in the shoots of A. ordosica. Moreover, the Na + content in the shoots was remarkably in uenced by DSE species and NaCl stress treatment ( Table 2). BS inoculation increased the Na + and K + content under NaCl stress compared with that in the control plants. No signi cant difference in the Na + :K + ratio was found under NaCl stress treatment (Fig. 6a, c, e). DSE species and NaCl stress treatment had signi cant interaction effects on the Na + and K + content and the Na + :K + ratio in the roots of A. ordosica (Table 2). Compared to the controls, the Na + content in roots of DSE-inoculated plants decreased under NaCl stress treatments. Inoculation with AC increased the K + content in the roots under 0.2% and 0.3% NaCl compared to control plants. Furthermore, inoculation with AC and PC decreased the Na + :K + ratio under 0.2% and 0.3% NaCl (Fig. 6b, d, f).

Soil physicochemical properties
Interactions between DSE species and NaCl stress treatment remarkably in uenced the soil organic carbon (SOC) content, while the soil available phosphorus (AP) content was signi cantly affected by DSE species and NaCl stress treatment. Moreover, the soil alkaline nitrogen (AN) content was signi cantly affected only by DSE species (Table 3). PC inoculation reduced the SOC content under all NaCl treatments and reduced the AP content under 0.1% NaCl compared with that in the control plants. AC inoculation reduced the SOC content only under 0.3% NaCl. However, BS inoculation reduced the content of SOC and AP under both 0.1% and 0.3% NaCl and increased the AN content under both 0% and 0.3% NaCl (Fig. 7). The partial eta squared (η p 2 ) presented the effect size of different factors. Signi cant P-values (P < 0.05) are in bold. SOC indicates soil organic carbon; AN indicates alkaline nitrogen; AP indicates available phosphorus; G − bacteria indicates gram-negative bacteria; G + bacteria indicates grampositive bacteria.

Soil microbial community composition
Interactions between DSE species and NaCl stress treatment signi cantly affected the microbial community composition in the rhizospheric soil of A. ordosica (Table 3). Under 0.1% NaCl, BS inoculation enhanced gram-negative (G −) and gram-positive (G +) bacterial abundance compared with those of the control plants. Under 0.2% NaCl, PC inoculation increased the abundance of G -bacteria, actinomycetes, fungi and arbuscular mycorrhizal (AM) fungi compared to controls, whereas BS inoculation only enhanced the abundance of AM fungi. When the NaCl concentration reached 0.3%, compared with the control, the plants inoculated with AC presented an increased abundance of soil microbes; the plants inoculated with PC presented an increased abundance of G − bacteria, actinomycetes, fungi and AM fungi, whereas BS inoculated plants presented an enhanced abundance of fungi and AM fungi (Fig. 8).

Correlation analysis
Spearman's rank correlation test indicated that signi cant relationships existed among DSE species, NaCl stress, soil factors, soil microbial composition, and the growth parameters of A. ordosica (Table S1). On the basis of the correlation coe cients (R-value), structural equation modeling (SEM) further revealed the relative effects of DSE species, NaCl stress and soil factors on the growth of A. ordosica and on the rhizospheric microbial composition (χ 2 = 78.527, df = 60, P = 0.055, CFI = 0.969, TLI = 0.953, and RMESA = 0.081; Fig. 9). The SEM indicated that DSE directly and signi cantly positively in uenced the plant hormone (IAA) content and antioxidant enzyme (SOD) activity and directly negatively affected the root Na + content. Moreover, DSE signi cantly indirectly affected plant hormone (IAA) content, antioxidant enzyme (SOD) activity, and root growth (root length and biomass) by affecting soil factors (SOC, AP, and AN). In addition, DSE directly and signi cantly positively in uenced the abundance of G + bacteria and fungi, whereas DSE had signi cant indirect effects on the abundance of G − bacteria by affecting soil factors (SOC). In addition, G -bacteria and root biomass directly negatively affected the Na + content in the roots.

Discussion
The current study reported for the rst time the effects of DSE isolated from an arid desert environment on the growth of A. ordosica under NaCl stress. In all inoculated plants, typical DSE structures (dark septate hyphae and microsclerotia) in roots were observed, revealing that these DSE can be regarded as effective colonizers of the roots of A. ordosica under all NaCl treatments. DSE hyphae are important media for water transport and nutrient exchange between plants and DSE, while microsclerotia are also considered to be propagules or hypopus, which constitute important ecological strategies to plants for tolerating environmental stress [23,42]. However, different DSE strains showed variable colonization patterns with increasing NaCl concentrations, and similar results of studies of DSE improving the heavy metal tolerance of plants have been reported [43][44][45]. The primary reason may be that the stress affected the growth and physiological metabolism of the DSE hyphae [46,47], thus regulating the infection ability of the DSE on plant roots. In addition, the growth response of A. ordosica to DSE colonization was also strain dependent, which is consistent with previous ndings [29,46]. Speci cally, AC resulted in a signi cant increase in A. ordosica biomass accumulation under 0.1% NaCl, while PC exerted bene cial effects under both 0.1% and 0.3% NaCl. However, BS inoculation decreased the root biomass under 0.2% and 0.3% NaCl. Therefore, the variable effects of different DSE on plants may be related to the different colonization rates and DSE species, which need further investigation.
The root system is the primary plant part that senses salt stress, and roots can respond rapidly through changes in elongation [48] and function [49]. In the present study, DSE promoted the growth of the root system under NaCl stress, although the effects depended on the DSE species. Under the low-stress treatment (0.1% NaCl), AC and PC inoculation enhanced the root length, root volume and root surface area compared to control plants. As the stress increased, only PC inoculation signi cantly promoted root growth under 0.3% NaCl. Pan et al. [33] also reported that DSE had positive in uences on root morphological characteristics under salt-stress conditions. Moreover, studies have shown that DSE can promote plant root growth under water de ciency and ion stress [50,51]. Extensive and deep roots are conducive to increasing the water and nutrient intake of plants, and eventually in uence biomass [31].
Thus, the relatively large root length, root volume and root surface area of A. ordosica in this study may be bene cial to improve the adaptability of plants to NaCl stress.
Previous studies have shown that fungal endophytes can produce phytohormones, the process of which is bene cial to host plants to counteract the adverse effects of abiotic stress [52]. IAA, an important plant hormone, plays an important function in plant responses to salt stress [53]. In our study, the content of IAA in PC-and BS-inoculated plant roots was signi cantly greater than that in the control plants regardless of the NaCl treatment. The SEM also showed that DSE species was the dominant impact factor and had a signi cant direct effect on the IAA content in the roots of A. ordosica. In previous studies, several DSE species were reported to produce IAA, thereby promoting plant growth and increasing plant stress tolerance [54,55]. Qiang et al. [56] reported that inoculation with Alternaria alternata (LQ1230) increased the IAA content, increased the growth of wheat and increased drought tolerance. These results indicated that DSE may also promote the growth of A. ordosica by regulating the plant root IAA content, thereby improving the salt stress tolerance of host plants.
An appropriate Na + :K + ratio in plant tissues represents an important basis for the normal physiological metabolism of plants [8]. Under salt stress, the substitution of Na + for K + in plants often results in an increased Na + content, which in turn damages cells [57]. In the present study, with increasing NaCl stress, the Na + content and Na + :K + ratio in the shoots of A. ordosica tended to increase. However, no signi cant changes were observed between inoculated A. ordosica (AC, PC) and control plants. These results indicate that DSE did not seem to limit the translocation of Na + to the aboveground plant parts, which agrees with the results of Farias et al. [34]. In addition, the DSE signi cantly directly affected the Na + content in the roots of A. ordosica. Some DSE promoted the absorption of K + in the roots of A. ordosica and decreased the root Na + content regardless of NaCl stress. The signi cantly decreased Na + content in the roots inoculated with DSE may be related to DSE promoting the upregulation of RpSOS1 in the roots and increased Na + export to the soil [57,58]. Moreover, the SEM analysis showed that root biomass had direct negative effects on the Na + content in the roots. DSE promoted the growth of plant roots and caused a dilution effect, which may be an important reason for the decrease in Na + in the roots [59]. In addition, G − bacteria had direct negative effects on the Na + content in roots. These ndings are consistent with those of Mohamed et al. [33], in which G − bacteria decreased the Na + in the roots of plants under salt stress. Mendpara et al. [60] also reported that most salt-tolerant bacteria are G − bacteria. Studies have shown that bacteria can secrete exopolysaccharides (EPSs) to bind excess Na + and restrict the in ux of Na + into the roots. Moreover, these bacteria can also increase the absorption of K + and the export of Na + by the roots, thus improving plant salt tolerance [61].
Generally, salt stress causes oxidative damage to plant cells, thereby negatively affecting plant growth [62]. In this study, DSE enhanced the antioxidant enzyme activity in the leaves to remove reactive oxygen species (ROS) under salt-stress conditions. SOD, an important antioxidant enzyme, can eliminate ROS generated from oxidative damage [63]. Compared with the control plants, the plants inoculated with AC and BS presented increased SOD activity under NaCl stress, and the plants inoculated with PC presented increased SOD activity only under 0.2% NaCl. Similar results have been reported by Pan et al. [33], in which DSE increased SOD activity in the leaves of plants under salt stress. As an antioxidant and a ligand peptide, GSH plays an important role in mitigating salt-induced damage [64]. In our study, BS inoculation increased the GSH content of plants under NaCl stress, and PC inoculation increased the GSH content in plants under 0.1% NaCl; however, an increase in GSH content in the AC-inoculated plants was detected only under 0.2% and 0.3% NaCl. Similar results in which DSE could activate GSH metabolism in Zea mays under heavy metal stress have been reported [65]. These results indicated that, by regulating the accumulation of antioxidants and the Na + :K + balance, DSE can alleviate the adverse effects of NaCl stress on plants.
Plant symbiotic fungi normally play important roles in the regulation of the soil microenvironment [12,66]. Here, DSE had direct negative in uences on the soil AP and SOC content but signi cant direct in uences on the soil AN content. BS and PC inoculation reduced the SOC and AP content under 0.1% and 0.3% NaCl, and AC inoculation decreased the SOC content under 0.3% NaCl. These ndings indicated that DSE may regulate the balance of soil nutrients under NaCl stress. As a bridge between plants and the soil, DSE have been determined to convert soil organic carbon and nitrogen and insoluble phosphorus mineralization into effective forms, which can greatly expand plant organic nutrient pools [67][68][69][70]. Moreover, DSE may increase the contact area between plants and the soil through the extension of hyphae, increasing the absorption of nutrients by host plants [22,23]. In addition, PC inoculation promoted the abundance of G -bacteria, actinomycetes, fungi and AM fungi in the soil under 0.2% and 0.3% NaCl, and AC inoculation increased the abundance of soil microbes under 0.3% NaCl, while BS increased the abundance of soil bacteria and AM fungi under 0.1% and 0.2% NaCl, respectively. These ndings indicated that DSE increased the abundance of soil microorganisms under salt stress. Similar results have been reported by He et al. [15], in which DSE increased the abundance of fungi, bacteria and actinomycetes under drought stress. Previous research has also shown that fungi and G − bacteria have more adaptive capacity to stress environments, and G + bacteria may positively in uence AM fungi propagation [60,71,72]. Actinomycetes are also an important part of the rhizosphere microbial community, which can promote plant growth and soil nutrient cycling [73]. In our study, DSE not only signi cantly directly affected the abundance of fungi and G + bacteria but also indirectly affected the abundance of G − bacteria and fungi by in uencing the SOC content. These results indicated that DSE may release increased amounts of organic nutrients into the soil, thereby promoting the growth of salttolerant microorganisms. In addition, the in uence of DSE on the composition and quantity of host plant root exudates may be another factor in uencing the microbial community composition. Further studies associated with the effects of DSE on plant growth and soil micro ora under salt-stress conditions will help us better understand the ecological functions of DSE in stressful ecosystems.

Conclusion
In the present study, three DSE isolated from A. ordosica could effectively colonize the roots of host plants under different NaCl concentrations. Although derived from the identical environment, these DSE strains exerted considerably different effects on the growth of plants. The effect of DSE inoculation on plant function ranges from bene cial to neutral with increasing NaCl concentrations and depends on the DSE species and NaCl treatment. Under NaCl stress, inoculation with DSE reduced the Na + content and Na + :K + ratio in plant roots and increased the SOD activity, GSH content and IAA content, as well as the root growth of A. ordosica, which alleviated the negative effects of NaCl stress on the host. DSE also affected the rhizospheric soil microbial community by The seeds of A. ordosica were sterilized in ethanol (70%) for 3 min followed by sodium hypochlorite (2.5%) for 10 min, after which they were rinsed three times in sterilized water. The sterilized seeds were germinated on 10 g/L agar-water media at 27 °C. After that, three seedlings of uniform size were selected and transplanted into a sterile pot (11.5 cm height, 13.6 cm diameter at the top and 9.5 cm diameter at the base) containing 1,000 g of growth media, which consisted of a 1:1 (v/v) mixture of river sand and soil (< 2 mm). The soil was collected from farmland in North China. The growth media included 9.94 mg/g soil organic carbon (SOC), 16.55 mg/kg alkaline nitrogen (AN), and 11.26 mg/kg available phosphorus (AP). With respect to the DSE inoculation treatments, two 9 mm diameter discs obtained from the margins of an active colony were inoculated within 1 cm from the roots of A. ordosica seedlings. With respect to the noninoculated treatments, two 9 mm discs were removed from PDA medium without any fungi. All plants were grown in a greenhouse at 27 °C/22 °C day/night temperature, 14 h/10 h photoperiod and 60% mean relative humidity.
Thirty days after inoculation, NaCl stress treatments were imposed by the application of NaCl solutions to the pots. To avoid osmotic shock, NaCl stress was applied in increments [75]; that is, each pot received 50 mL of 0.34 mol/L NaCl solutions at 5-day intervals successively. Fifty milliliters of sterile water without NaCl was applied to the controls each time to ensure consistent soil moisture. NaCl solutions were applied such that the desired 0%, 0.1%, 0.2% and 0.3% NaCl levels were reached [76]. Finally, the plants were harvested after 60 days of treatment.

Plant biomass and morphological parameters
Prior to harvest, the plant height and shoot branching in each replicate (three plants/pot) were recorded.
The roots and shoots in each replicate were subsequently harvested separately. The cleaned roots were scanned utilizing an Epson Perfection V800 Photo scanner. The root morphological parameters, including root length, root volume, root diameter and root surface area, were measured using the WinRHIZO image analysis system (Regent Instruments, Quebec, Canada). Fresh roots were subsequently collected and used to estimate the DSE colonization levels and indoleacetic acid (IAA) concentration. Similarly, fresh leaves were used to measure physiological parameters, such as superoxide dismutase (SOD) activity and glutathione (GSH) content. The remaining fresh shoots and roots were dried to a constant weight at 70 °C to con rm the plant biomass and the Na + and K + contents. Soil samples from each pot were sieved (2mm mesh size) and then split into two parts: one subsample was stored at -80 ℃ to analyze the community composition of soil microbes, and the other subsample was dried at room temperature to analyze soil physicochemical properties.

DSE colonization analyses
Fresh roots of A. ordosica were randomly sampled from each pot to determine DSE colonization levels. First, the roots were cleaned under running water. The root segments (0.5 cm) were clari ed in potassium hydroxide (10%, w/v) and then dyed in acid fuchsin (0.5%, w/v) according to the methods of Phillips and Hayman [77]. Thirty root segments of each sample were randomly selected and pressed onto slides, and then observed under a light microscope (Olympus BX51) with 20 × and 40 × eyepieces [78]. The DSE total colonization rate (%) was expressed as the percentage of infected root segments per root sample.

Determination of SOD activity and GSH content
The activity of SOD in the leaves was assayed by measuring the diminution in the opticaldelnsity of the nitro blue tetrazolium (NBT) complex according to Elavarthi and Martin [79]. Brie y, fresh leaves (0.2 g) were ground together with phosphate buffered saline (PBS) (50 mM, 5 mL; pH 7.8) in an ice bath and then centrifuged for 10 min at 4,000 × g and 4 °C. The reaction solution (5 mL) contained 0.1 mL of enzyme extract, 30 mL of ribo avin (33 µmol/L), 0.3 mL of NBT (1.25 mmol/L), 0.3 mL of methionine (220 mmol/L) and 4 mL of PBS (50 mM). One unit of SOD was de ned as enzyme activity that inhibited NBT reduction by 50% at 560 nm.
The crude extract was subsequently centrifuged for 10 min at 10,000 × g. The reaction mixture consisted of PBS (100 mM, 0.6 mL), DTNB (40 µL) and the supernatant (0.5 mL). The opticaldelnsity of the supernatant was determined at 421 nm. The standard curve was used to calculate GSH content.

Measurement of IAA content
The

Soil physicochemical properties analysis
The SOC content was measured according to the potassium dichromate oxidation method [81,82]. A dried soil sample (approximately 1 g) was placed in a tube containing K 2 Cr 2 O 7 solution (0.8 mol/L, 5 mL) and H 2 SO 4 (98%, 5 mL) and then boiled for 5 min at 170 ℃. After cooling, the solution was transferred to a conical ask, and the volume was adjusted to 60 mL by dilution with distilled deionized water. The excess dichromate was subsequently titrated with 0.2 mol/L FeSO 4 with diphenylamine used as an indicator.
The AN content was determined according to the alkaline hydrolysis diffusion method [82]. Approximately 2 g of dried soil sample was uniformly distributed in the outer chamber of the diffusion dish. H 3 BO 3 indicator (20 g/L, 2 mL) and NaOH (1 mol/L, 10 mL) were added to the inner and outer chambers, respectively. The diffusion dishes were then sealed and incubated at 40 °C for 24 h. Titration was subsequently performed via 0.01 mol/L H 2 SO 4 solution.
The AP content was measured by the chlorostannus-reduced molybdophosphoric blue color method according to the methods of Olsen et al. [83]. Brie y, dried soil samples (approximately 1 g) were placed into a conical ask containing both NaHCO 3 solution (0.5 mol/L, 50 mL) and phosphor-free activated carbon (0.1 g). After the mixture had been shaken for 30 min, the ltrate (10 mL) and molybdenum vanadate solution (5 mL) were mixed together, followed by dilution with distilled deionized water to 50 mL. The mixed reagent was then nurtured for 30 min, after which the absorption values were determined at 700 nm via a spectrometer.

Soil microbial community composition
The composition of the rhizosphere soil microbial community was determined by phospholipid fatty acid w9c. The fatty acid 16:1 w5c was determined to be a biomarker for AM fungi [15].

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