Soil characteristics Soil samples were taken from the upper (0–30 cm) horizon of the soil profile before being air-dried. They were sieved (via a 2-mm sieve) and blended in appropriate ways. The soil characteristics including texture (Beretta et al., 2014), pH (Bargrizan et al., 2017), electrical conductivity (EC) (Aboukila and Norton, 2017), organic matter (OM) (Miyazawa et al., 2000), cation exchange capacity (CEC) (Meimaroglou and Mouzakis, 2019), the available amount of phosphorus (Shen et al., 2019), total nitrogen quantity (Guebel et al., 1991), potassium content (Kumar and Gill, 2018), and micronutrients’ concentrations such as Zn, Fe, Cu, and Mn (Lindsay and Norvell, 1978) were determined via referenced methods. The physicochemical characteristics of the soil are presented in Table S1.
This experiment was carried out in the College of Agriculture, Shiraz University. The seeds of G. glabra were collected from Eghlid (Aspas village, 52° 23′ 58″ E and 30° 38′ 31″ N), a region in the north of Fars province, Iran. The seeds were scarified by soaking in concentrated H2SO4 (97%- Merck) for 10 min, washed with running water several times, and immediately sown in transplant trays (Peat moss and perlite mix, 2:1). In each cell of the trays, two seeds were sown. They were allowed to grow in the greenhouse after sowing (Day: 27±1°C, night: 23±1°C, humidity: 70±3%, light: 40000 Lux). A month after germination, the plantlets were transferred into small plastic pots containing 250 mL of field soil and sand mixture (2:1) (Lang et al., 2019). Bigger pots (50 cm height × 29 cm diameter) were used for transplanting 6-month-old seedlings in a sandy medium (soil and sand mixture (2:1)) (Lang et al., 2019). Each pot had two seedlings.
Preparation of microbial inoculation
C. etunicatum was supplied by the soil biology lab at Shiraz University, department of soil science, and was previously separated by Dr. Mehdi Zarei. This fungus was propagated in a sterilized mixture of soil and sand (1:1) for five months using Sorghum bicolor as the host plant. The inoculum potential for infectivity as root colonization was estimated at 85%. A number of 10 spores existed per gram of substrate. The inoculum (250 g) was added to the root zone before doing the final transplantation. Each mycorrhizal pot was filled with soil containing fungal spores, mycorrhizal roots, and mycelia of C. etunicatum. Meanwhile, non-mycorrhizal pots were supplied with an equal amount of washed sand (Zarei et al., 2020).
Experimental design and growth conditions
A pot experiment was performed in a factorial experiment that was arranged in a completely randomized design with three factors, including five levels of drought stress (i.e. 100 (control), 80, 60, 40 and 20% of field capacity, represented by W (i.e. W100 (control), W80, W60, W40, and W20), two levels of AMF, denoted by F (i.e. F0: without inoculation, F1: inoculated), and two levels of Si solute in irrigation water (i.e. Si0: without Si application, Si1: 300 mg/ L of SiO2). Four replications were considered. After two weeks of adaptation and plant establishment in pots, irrigation treatments were carried out for two months. Irrigation was performed differently to make the soil reach the five different levels of FC. The weighting approach was used for adjusting the stress treatments (Abbaszadeh et al., 2020). As the plants consumed water and as evaporation occurred, the weight of pots decreased gradually through the course of observations. For each treatment group, the five drought levels were applied. In the case of Si nutrition, SiO2 was dissolved in the irrigation water. Every other day, water was added to the pots after weighing the pots so that the amount of soil moisture could be measured indirectly and the specifications of each treatment could be upheld. After eight weeks, and under the respective conditions of growth, the plants were harvested for analysis.
The plants were entirely removed from the pots at the end of the experiment. After cleaning the roots, various parameters were examined and measured. Root length was measured by a scientific ruler and root diameter was measured by a caliper (0.01 mm accuracy) (Shan et al., 2018). The root fresh weight (FW) was determined promptly after harvesting the plants. Then, the roots were oven-dried at 80 °C for 24 h before measuring their dry biomass. Dry and fresh weights of the roots were measured with a four-digit scale (Lamacque et al., 2020). Root volume was calculated according to the volume displacement method (Harrington et al., 1994). Root area was measured by MATLAB software (Jadhav, 2021).
Assessment of AMF colonization
Fresh root segments (2 cm long) were dyed as described by Rahimi et al., 2021 with minor modifications so that root colonization by AMF could become measurable. Stained root segments were selected randomly for evaluation under the microscope and according to a relevant approach in the literature (Trouvelot et al., 1986). A Nikon Eclipse E200 microscope was used for examining the segments (at 40× magnification). The mycorrhizal inoculation parameters and confirmation were calculated utilizing Mycocalc (http://www2.dijon.inra.fr/mychintec/Mycocalc-prg/MYCOCALC.EXE) (Etemadi et al., 2014).
Glycyrrhiza glabra root extraction and quantitative analysis by HPLC
The well-ground powder was prepared from the dried roots of each sample (500 mg) and its extract was taken by ethanol: water (70:30). After adding the solvent to the powder and after sonicating them for half an hour in an ultrasonic bath, the extracts were centrifuged and injected into High-Performance Liquid Chromatography (HPLC). Extraction and injection were done in three replicates (Esmaeili et al., 2019).
The glycyrrhizic acid content in the licorice roots was evaluated by Knauer liquid chromatography device consisting of a 2695 Separations Module (Germany), equipped with a 100 μl loop and Photodiode Array Detectors (PDA) using a C18 column (Knauer, 25 cm × 4.6mm Eurospher 100-5), with water that contained 0.3% H3PO4 (solvent A) and acetonitrile (solvent B) as the mobile phase. The flow rate was 1 ml/min. The samples were monitored at a wavelength of 250 nm in the case of glycyrrhizic acid (Esmaeili et al., 2019).
Antioxidant activity and 50% inhibition (IC-50) determination
Total antioxidant activity was calculated according to the protocol of Taban et al., 2021. The absorbance was read at 517 nm (Taban et al., 2021). According to the DPPH free-radical scavenging method, the sample concentration was assessed in relation to IC-50 (Half maximal Inhibitory Concentration). The inhibition (%) was plotted against sample concentrations and each test was performed three times (Taban et al., 2018).
Determination of total phenols and flavonoids
The total phenolic quantity of the ethanolic extracts was assessed by the method described by Haghighi and Saharkhiz, 2021. The absorbance read by a spectrophotometer at 750 nm (Epoch Biotek, Winooski, VT, USA) . The absorbance of ethanolic extracts to estimate the concentration of total flavonoids done by the method described by Taban et al., 2020. The absorbance was measured at 510 nm by a microplate reader in the spectrophotometer (Taban et al., 2020).
Extraction and profile assay of polyphenolic compounds by HPLC
The extraction was carried out using a solvent (85% methanol/15% acetic acid) with a concentration with the method described by Gholami et al., 2018 with little modifications. The polyphenols were collected using a syringe that had a filter on its head (0.45 µm). Then, they were transferred to glass containers and remained frozen until HPLC analysis (Gholami et al., 2018).
Polyphenolic components in G. glabra extracts were separated, identified, and quantified by HPLC analysis on an Agilent 1200 series (USA), equipped with a reverse-phase Zorbax Eclipse (XDB)-C18 column (10 cm × 5 mm i.d.; 150 mm film thickness) and with the assistance of a photodiode array detector (PAD). The method completely described by (Mousavi et al., 2021).
Mineralogical analysis by X-ray Diffraction
The root powder ash (1g) of each sample was prepared at 500°C and then became subjected to X-ray powder diffraction (XRD) analysis. The quantity of N, K, P, and Si in the samples was measured by X-ray diffraction (XRD) (Bruker's X-ray Diffraction Model: D8 Advance; Germany) while Cu Kα radiation had a wavelength of 1.54 Å in the three replicates. The relative abundance of minerals was measured using peak height intensity as an indication. Scanning was carried out from 15 to 75° 2θ, with a step interval of 0.05° and with a counting time of 1 s per step. Quantitative analysis of mineral abundance was carried out using the X'Pert HighScore software (Oliveira et al., 2012).
This research mostly involved a pot experiment in a factorial arrangement with four replications in a completely randomized design. It had three factors, i.e. drought, AMF, and Si. A general linear model was employed for statistical analysis and for dealing with the entire experimental data using Minitab software (version 17). Statistical significance was defined at p<0.05 (Tukey’s test). In the case of significant interactions, the slice method was applied to obtain mean comparisons. Principal component analysis was also used according to a correlation matrix by Minitab software. Corrplot was drawn by R software version 4.1.1 (corrplot package) and path analysis was performed by IBM SPSS (version 27).