Experimental design
The pot experiment was conducted in a glasshouse of Xiashu Forest Farm, Nanjing Forestry University, China, from March to October 2018. The experiment consisted of a completely randomized block design with two inoculation treatments: non-mycorrhizal control plants and plants inoculated with the model AMF, Funneliformis mosseae (C. Walker & A. Schüßler). Each inoculation treatment comprised 48 replicates, totaling to 96 pots (1 plant per pot). The 48 pots in each treatment were randomly divided into four groups (12 pots per group), and each group was subjected to one of the four NaCl concentrations (0, 50, 100, and 150 mM NaCl).
Plant material and soil
The seeds of G. sinensis were provided by Jiangsu forestry station. All the seeds were soaked in concentrated H2SO4 for 10 min until the color of the seeds turned crimson, and then washed with sterile distilled water until the pH of residual water on the surface of the seeds turned to about 7.0. After that, the seeds were soaked in warm water for 2 days. The inflated seeds were embedded in wet yellow sand (which was previously sterilized in an autoclave for 2 h at 0.14 MPa and 121 °C) and incubated in plant incubator under dark condition at 25 °C.
Loamy soil was collected from Xiashu Forest Farm of Nanjing Forestry University, China, sieved (2 mm), mixed with yellow sand (< 2 mm) and vermiculite (1:1:1, topsoil/sand/vermiculite, v/v/v), autoclaved at 0.14 MPa and 121 °C for 2 h, and used as nursery substrates. The soil mixture was tested for its physicochemical properties: total C, 1.55%; total N, 0.03%; total P, 570.48 mg·kg− 1; total K, 15.18 g·kg− 1; available P, 10.00 mg·kg− 1; available K, 101.39 mg·kg− 1; electrical conductivity, 0.23 mS·cm− 1 (soil:water ratio, 1:5); and pH, 7.15 (soil:water ratio, 1:5).
Inoculation treatment
F. mosseae (isolate number: BGC G201C) was obtained from the Beijing Academy of Agriculture and Forestry Science, China. The inoculum was bulked in an open-pot sterilized yellow sand culture together with maize and clover as trap plants. After 3 months, the aboveground was cleared, and the roots were chopped into small pieces and mixed with the sand of the culture pot. This sand-based inoculum, consisting of yellow sand, infected root fragments, and mycorrhizal spores (> 7 g− 1), were collected and used in this study. The uniform seedlings (5 cm in length) were transported to the pots (1 seedling per pot). Before transportation, the pots were soaked in 0.3% KMnO4 solution for 3 h and washed with tap water. About 2.5 kg of the autoclaved nursery substrates were dispensed into each pot and 80 g of sand-based inoculum were added 5 cm below the surface of the nursery substrates. The non-inoculated control pots contained the fungal inoculums filtration and the same dosage of sterilized inoculum to provide the same microbial community (except for AMF) with inoculated treatment.
Growth conditions
The seedlings were grown in the glasshouse under the following conditions: 18 °C night/30 °C day temperature, 50–80% relative humidity, and 14 h/10 h diurnal light/dark cycles with a photosynthetic photon flux density of about 700–1,000 µmol m− 2·s− 1. Water was supplied adequately during the entire period of the experiment to avoid any drought effects, and modified Hoagland’s nutrient solution containing only 25% P concentration (300 mL per pot every time) was irrigated every month. The seedlings were cultivated for about 4 months prior to salinization to allow adequate plant growth and symbiotic establishment. Subsequently, the four groups of non-mycorrhizal control and mycorrhizal treatments were respectively gradually supplemented with aqueous NaCl solution (300 mL per pot) at the concentrations of 0, 50, 100, and 150 mM NaCl every week for 2 months. In order to avoid salt shock, all three salt treatments (50, 100, and 150 mM NaCl treatments) were treated with 50 mM NaCl for the first week; salt treatment (50 mM NaCl treatment) were treated with 50 mM NaCl, and salt treatments (100 and 150 mM NaCl treatments) were treated with 100 mM NaCl for the second week; salt treatments (50, 100, and 150 mM NaCl treatments) were treated with 50, 100, and 150 mM NaCl, respectively, from week 3 onwards, the seedlings were harvested and analyzed for growth and biochemical parameters.
Plant harvest and chemical analyses
Before and after salt stress, the seedling height was measured using a steel ruler, and basal diameter was measured using calipers. After harvesting, the plants were rinsed with tap water, and separated into leaf, stem, and root. The leaf area and root system characteristics (root length, root surface area, and root tip number) were determined using a LA2400 Scanner (Expression 12000XL, EPSON, Long Beach, CA, USA). The dry weights of plant tissues (leaf, stem, and root) were recorded after drying the plant tissues in an oven at 70 °C to a constant weight. The mycorrhizal dependency was calculated using the formula (Wang et al. 2018): mycorrhizal dependency (%) = (dry weight biomass of inoculated seedlings – mean of dry weight biomass of non-inoculated seedlings) / dry weight biomass of inoculated seedlings × 100%.
The dried plant tissues were ground separately, sieved through a 0.5-mm sieve. 50 mg of each sample was weighed to determine the concentrations of N using an elemental analyzer (Vario MACRO cube, Elementar Trading Shanghai, Shanghai, China). 0.2 g of each sample was digested in 10 mL of acid mixture (HClO4:HNO3, 1:5), and diluted with double-distilled water. The concentrations of P were ascertained spectrophotometrically using ammonium molybdate blue method, the concentrations of K+, Ca2+, Mg2+, and Na+ were ascertained with an atomic absorption spectrophotometer (AA900T, Perkin Elmer, Norwalk, CA, USA) (Allen 1989), and the K+/Na+, Ca2+/Na+, and Mg2+/Na+ ratios in the tissues were calculated using K+, Ca2+, Mg2+, and Na+ data.
Estimation of root mycorrhizal colonization
For the quantification of mycorrhizal colonization, the washed fine roots were cut into 1-cm-long segments. The root segments were clarified with 10% (w/v) KOH at 90 °C for 1 h, stained with basic H2O2 (containing 30 mL of 10% (v/v) H2O2, 3 mL of concentrated NH4OH, and 60 mL of water) for 25 min, soaked in 1% (w/v) HCl for 3 min, and stained with 0.05% (w/v) Trypan Blue solution as described by Philips and Hayman (1970). Subsequently, the root segments were soaked in lactic acid-glycerol (1:1) to eliminate excess Trypan Blue solution, and microscopically examined for AMF colonization based on the presence of arbuscules, vesicles, hyphae, and spores (Giovannetti and Mosse 1980).
Determination of chlorophyll contents and photosynthetic parameters
The chlorophyll contents (Chl) in leaves were determined according to Lichtenthaler (1987) with minor modification. Fresh mature leaves (0.1 g) of each plant were cut into small pieces and completely submerged in acetone solution (0.5 mL of pure acetone and 15 mL of 80% acetone). The samples were incubated at 35 °C under dark condition. After the leaf turned white in color, the samples were diluted with 80% acetone to 25 mL. The absorbance of the extracts was determined using an ultraviolet spectrophotometer (UV 2700, Shimadzu) at 663, 645, and 470 nm, respectively.
Leaf gas exchange (Gs) was evaluated on the mature expanded leaf using an infrared gas analyzer (LI-6400, LI-COR, Lincoln, NE, USA) during the day between 09:30 and 11:30 am under the following condition: photosynthetically active radiation, 1000 µmol m− 2 s− 1; CO2 concentration, 390 µmol mol− 1; leaf temperature, 25 °C; leaf humidity, 35–50%; and air flow rate, 0.5 dm3 min− 1. Leaf net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), room CO2 concentration (CO2R) and transpiration rate (Tr) were simultaneously recorded, and leaf limiting value of stomata (Ls) was calculated using the formula: Ls = 1 - Ci/ CO2R.
Measurement of relative water content and membrane stability
The leaf relative water content (RWC) was measured according to the previous method described by Wang et al. (2019) using the following formula: RWC = (FW - DW) / (TW - DW) x 100%, where FW is fresh weight, DW is dry weight, and TW is turgid weight obtained after the leaf was soaked for 24 h in deionized water. The membrane stability index (MSI) was estimated according to the method described by Talaat and Shawky (2014) using the formula: MSI = (1 - C1/C2) x 100%, where C1 is the electrical conductivity bridge after the leaves were heated at 40 °C for 30 min in a water bath and C2 is the electrical conductivity bridge after the leaves were boiled at 100 °C in a boiling water bath for 10 min.
Determination of lipid peroxidation and proline content
Lipid peroxidation in leaves and roots was estimated by measuring the concentration of malondialdehyde (MDA) as described by Hodges et al. (1999) with minor modification. The leaves and roots samples were homogenized and dissolved with quartz powders in 5% (w/v) trichloroacetic (TCA) solution under cold condition. The homogenate was centrifuged at 12,000 rpm for 10 min at 4 °C. The reaction mixture containing 2.0 mL of supernatant and 2.0 mL of 0.6% (w/v) thiobarbituric acid (TBA) was heated in a water bath at 95 °C for 30 min. Then, the boiled reaction mixture was immediately cooled in an ice bath and centrifuged at 3,000 rpm for 10 min. The absorbance of the supernatant was measured at 532, 600, and 450 nm, respectively. The concentration of MDA was calculated by using the formula given by Hodges et al. (1999). The concentration of proline (Pro) generated was ascertained via ninhydrin reaction as described by Bates et al. (1973). The leaves and roots were cut into small pieces, completely submerged in 3% (w/v) sulfosalicylic acid solution, and heated in a water bath at 100 °C for 15 min. Then, 2 mL of the extract were added to 2 mL of glacial acetic acid and 2 mL of 2.5% ninhydrin solution, and heated in a water bath at 100 °C for 15 min. Subsequently, the reaction mixture was cooled down and 5 mL of methylbenzene were added to it and placed under dark condition. After the mixture completely separated into different layers, the absorbance of methylbenzene layer was measured at 520 nm.
Soluble proteins and antioxidant enzymes assay
Crude enzymes were extracted from the leaf and root samples homogenized in an ice bath with 50 mmol·L− 1 sodium phosphate buffer (pH 7.0) containing 1% (w/v) PVP-40 (polyvinylpyrrolidone). The mixture was centrifuged at 12,000 rpm for 20 min at 4 °C, and the supernatant was collected for soluble proteins (SP) measurement and antioxidant enzymes analyses. The SP contents of leaves and roots were determined using the method of Coomassie Brilliant Blue G250 (Blakesley and Boezi 1977), and a commercial Bradford reagent (Sigma) and BSA (Merck) were employed as standard. Superoxide dismutase (SOD) activity was assayed using nitro blue tetrazolium (NBT) reduction test by measuring the ability of SOD to inhibit photochemical reduction of NBT (Giannopolitis and Ries 1977). A 50% inhibition of NBT reduction was considered as one unit of SOD activity at 560 nm. Peroxidase (POD) activity was assayed using guaiacol test and spectrophotometrically determined at 470 nm (Chance and Maehly 1955). Catalase (CAT) activity was ascertained by monitoring the decrease in the absorbance of H2O2 at 240 nm (Chance and Maehly 1955), and ascorbate peroxidase (APX) activity was determined by examining the decrease in the absorbance of ascorbate at 290 nm (Nakano and Asada 1981).
Statistical analysis
The data obtained were analyzed using SPSS19.0 (SPSS Inc., Chicago, IL, USA). Two-way ANOVA was used to determine the effects of NaCl levels, AMF inoculation, and their interactions. Multiple comparisons of means were performed by Tukey’s test (P ≤ 0.05). All the figures were derived using Origin 8.5 (Origin Lab, Northampton, USA), and all data are presented as mean ± standard deviation of at least three plants.