Growth substrate
Growth substrate consisted of sand and vermiculite (1:1, V/V). Sand was allowed to pass through a 2-mm sieve to remove impurities and further rinsed under running tap water until the water was clear. Further, the sand was rinsed once with deionized water and sterilized at 121°C for 2 h to eliminate all possible mycorrhizal propagules and other micro-organisms. The sieved and sterilized sand as a growth substrate has the following properties: pH 8.69, organic matter content 1.18 g·kg− 1, total nitrogen content 0.073 g·kg− 1, hydrolytic nitrogen content 12 mg·kg− 1, total phosphorus content 0.505 g·kg− 1, effective phosphorus content 2.44 mg·kg− 1, total potassium content 22.9 g·kg− 1, effective potassium content 82.3 mg·kg− 1, and conductivity 334 µs·cm− 1. Vermiculite was autoclaved (121°C, 2 h), mixed well with the prepared sand (1:1, V/V), and used as the growth substrate in this study.
Plant And Fungal Inoculum
The seeds of A. sparsifolia were collected in August 2020 at the natural habitat restoration demonstration area of the lower Tarim River at the Insu section (40°25.918' N, 87°56.458' E) and were stored at 4°C. Before sowing, the seeds were polished with sandpaper to release dormancy, disinfected with 75% alcohol for 10 min, washed with sterile water, and dried using a filter paper.
Claroideoglomus etunicatum and Funneliformis mosseaedominant, the dominant AMF species in the roots of A. sparsifolia, were obtained from Beijing Academy of Agricultural and Forestry Sciences. They were mixed in a ratio of 1:1 (w/w) to prepare the inoculum. The inoculum contained spores (14–20 spores per g inoculum), mycelia, root fragments, and sand.
Experimental Design
The experiment was conducted in the greenhouse of Xinjiang Normal University, China, with an average temperature of 27°C and a relative air humidity of 30.5%. The experiment consisted of six group of treatments: 1) control/CK-NM (without salt or drought stress and no AMF inoculation), 2) D-NM (drought stress and no AMF inoculation), 3) S-NM (salt stress and no AMF inoculation), 4) CK-AM (AMF inoculation and no salt or drought stress), 5) D-AM (drought stress and AMF inoculation), and 6) S-AM (salt stress and AMF inoculation). Each treatment had six replicates, with a total of 36 plastic pots.
Uniformly sized, full-grained A. sparsifolia seeds were selected and sown in plastic pots (24-cm length, 16-cm width, and 18-cm depth). When the seedlings grew to a height of approximately 5 cm, they were transferred to plastic pots sterilized with 75% ethanol. Each plastic pot contained 3 plants and 4 kg of fixed growth substrate. According to the AMF inoculation method, 20 g of inoculum was evenly spread flat at 10 cm from the soil surface. In the control group, equal weight of growth substrate was added instead. During the first 30 days, seedlings were grown without drought or salt stress to obtain plants with functional mycorrhizas and to avoid stress effects on the establishment of symbiosis with AMF. Drought stress was set at 30% ± 5% of the field water holding capacity, and salt stress was set at 0.6% of the weight of growth substrate to keep the A. sparsifolia seedlings under stress but allowing not to die. Soil water content was measured using a WET-2 portable rapid moisture meter. The relative soil water content was adjusted to fall within the range of the drought stress treatment by weighing and rehydrating with an electronic scale at 8:00 pm each day. To avoid osmotic shock, sodium chloride solution was gradually introduced by successively adding 100 mL of prescribed solution of salts in distilled water every 3 days, starting at day 30 after sowing until the sodium chloride content was 0.6% of the weight of the growth substrate. To the groups without salt or drought stress, equal volume of distilled water was added, ensuring that no excess leaching occurred from the pots. A saucer was placed under each pot to retain salt and other nutrients. A total volume of 300 mL of the corresponding salt solution or distilled water was added to each pot. The indicators were measured after 60 days of drought or salt stress. Throughout the experiment, the moisture content of the growth substrate in the groups without drought stress was maintained at 70% ± 5% of the field holding capacity.
Mycorrhizal Colonization
After harvesting A. sparsifolia, fresh roots were selected and rinsed with tap water. The roots with diameter < 2 mm were further selected and cut into 1-cm-long fragments for fixation, dissociation, acidification, and staining. Before observation and photography, the roots were decolorized with lactic acid and glycerol solution (1:1, V/V), and the stained root samples were squashed with a cover glass onto a glass slide. Mycorrhizal colonization rate and intensity were determined using the Asma method (2020). In total, 20 g soil sample was randomly selected from the root system of A. sparsifolia seedlings, and the number of AMF spores was counted by decantation using a wet sieve.
Plant Growth
Plant height and basal stem length were measured from the A. sparsifolia seedlings at days 0 and 60 of salt or drought stress. Statistical analyses of root length, root surface area, and root tip number were conducted using WinRHZIO root image analysis software(Zealquest Scientific Technology Co., Ltd, Shanghai, China). The relative growth rates and other parameters were calculated as follows:
Growth rate of plant height = (plant height at day 60 − plant height at day 0)/60 × 100%
Growth rate of basal stem = (basal stem length at day 60 − basal stem length at day 0)/60 × 100%
Specific root length (m/g) = root length/root dry weight
Root to shoot ratio = aboveground biomass/belowground biomass
Biomass And Nutrient Accumulation
During the harvest, the plants were cut from the basal stem, and the above- and belowground parts were divided. Their fresh weight was separately measured. Further, they were placed in a constant-temperature drying oven at 95°C for 15 min to destroy the enzymatic activity in the fresh leaves, followed by drying at 80°C till constant weight was obtained, which was measured using an electronic balance. The nitrogen (N) and phosphorus (P) contents were evaluated at the Institute of Botany, Chinese Academy of Sciences, after drying and grinding the above- and belowground parts. P content (%) was measured using the molybdenum phosphate method, and N content (%) was determined using Kjeldahl method.
P content (mg) = %P × weight of biomass
N content (mg) = %N × weight of biomass
Physiological Measurements
The initial fluorescence (Fo), maximum fluorescence (Fm), maximum photochemical efficiency (Fv/Fm), and potential photochemical efficiency (Fv/Fo) of leaves of A. sparsifolia seedlings were measured every 15 days during the treatment period using a MINI-Pam portable modulated chlorophyll fluorometer(Zealquest Scientific Technology Co., Ltd, Shanghai, China). The data were collected from the same seedlings at the same sites on different leaves from the bottom up. The parameters were evaluated using the following formulae.
Maximum photochemical efficiency (Fv/Fm) = (Fm − Fo)/Fm
Potential photochemical efficiency (Fv/Fo) = (Fm − Fo)/Fo
At the harvest, fresh leaf samples (0.1 g) were cleaned using deionized water to remove any surface contamination. Chlorophyll content was determined using the acetone method. The activities of superoxide dismutase (SOD), peroxisome (POD), and catalase (CAT) were determined using the nitrogen blue tetrazolium, guaiacol, and trace methods, respectively. Malondialdehyde (MDA), proline (Pro), and soluble sugar (SS) contents were determined using the thiobarbituric acid, sulfosalicylic acid, and anthrone colorimetric methods, respectively. Each treatment was repeated three times for each physiological index. The kits for determining SOD, POD, and CAT activities and chlorophyll, MDA, Pro, and SS contents were purchased from Beijing Solaibao Technology Co.(Beijing, China) and used as per the manufacturer’s instructions. The levels of hormones [strigolactones (SLs), indole acetic acid (IAA), gibberellic acid (GA), and abscisic acid (ABA)] were measured using enzyme-linked immunosorbent assay (ELISA; EnzymeLink Biologics, Shanghai, China) as per the manufacturer’s instructions. The content of each hormone indicator was evaluated using 3 replicates using a microplate reader.
Statistical analysis
Data analysis was performed using the Statistical Package for the Social Sciences (SPSS 21.0). Potential differences among various treatments were analyzed using Duncan’s multiple-comparison tests (P < 0.05). All data in the figures and tables are the original data presented as mean ± standard error (mean ± SE). P < 0.05 was considered significant. The results were plotted using origin 2019 and TB tools.