Combined Effects of Inoculating Serendipita Indica on Soybean Growth and Soil Health Under Cd Stress

Cadmium (Cd) pollution in the soil is a global environmental problem. Plants-microbial technology has been regarded as a potential technique for the remediation of Cd polluted soils. Here, we aimed to explore the combined effects of inoculating (Serendipita indica) S. indica on soybean growth and soil health under Cd stress. Therefore, a pot experiment was conducted to investigate the S. indica on soybean growth and the soil enzyme activities, pH and chemical forms of Cd in the soil under Cd 0, 10, 20, 30 mg/kg soil teatments. Results reected that compared to non-inoculated ones, the application of S. indica can still enhance the dry weight (66.57%), shoot height (90.35%) and promote the net photosynthesis rate (72.18%), transpiration ratio (80.73%), and stomatal conductance (119.05%) photosynthesis of soybean under Cd 30 mg/kg soil. Furthermore, The pH, phosphatase (116.39%) and catalase (4.17%) activities in the S. indica treatments were increased under Cd 10 mg/kg soil. Meanwhile, inoculated S. indica treatments signicantly shifted Cd from exchangeable fraction to other more stable fractions, primarily decreased Cd concentrations (23.66%) under Cd 20 mg/kg soil. The Cd pollution assessment in soil indicated that S. indica could effectively reduce Cd pollution in the Cd 10 mg/kg soil treatments. This work suggests that S. indica may be a potential method for not only promoting plant growth, but also relieving the phytotoxicity of Cd and remediating Cd contaminated soil.


Introduction
The root endophytic fungus, S. indica, can colonize in the roots of plenty of plant species and promote the absorption of nutrients. Besides, the antioxidant defense system of the plant can be enhanced by the fungus, which has a vital role in resistance against biotic and abiotic stresses ( The symbiotic relationship between fungi and host plants can produce a synergistic effect on Cd phytoremediation. There were few reports on the physiological characteristics and soil health of soybean under Cd stress. Therefore, in this research, we hypothesized that S. indica could promote soybean growth and transform Cd into a form, which is not easily utilized by plants under high Cd stress, thus reducing the risk of Cd owing into the food chain. We conducted pot experiments to determine the effects of Cd stress and S. indica on soybean growth, soybean physiological characteristics, the enzyme activities and the accumulation of Cd in the soil. In addition, the ecological risk of soybean soil inoculated with S. indica was analyzed to reveal the application prospect of S. indica in the remediation of Cd contaminated farmland.

Plant and fungal materials
Heinong 48 soybean seeds purchased from seed station, Harbin, China. Seeds were surface sterilized by soaking in 70% ethanol for 2 min and 4% sodium hypochlorite for 10 min, and then washed with double distilled water for 4 times. S. indica, is a strain preserved and propagated in the laboratory, was cultured in Petri dishes on a Hill & Käfer (2001) medium at 30 ± 1°C in the dark for 14 days. The mycelium plugs of S. indica (10 mm) were taken from the edge of the fungus culture plates (7.3×10 4 spore / plug). The fungal plugs of non-S. indica treatments were the same as S. indica treatments but it was autoclaved.

Soil preparation
The soil of pot experiment was collected from farmland soil around Nangang District of Harbin City, Heilongjiang Province, PR China, is a typical black soil, and then physicochemically characterized: The soil pH was 7.8, organic matter content was 24.3 g/kg, total nitrogen was 1.8 g/kg, available nitrogen was 50.3 mg/kg, available potassium was 213.1 mg/kg, available phosphorus was 9.2 mg/kg, and background Cd concentration is 0.128 mg/kg soil. Air-dried soil was sieved with a 2 mm sieve and autoclaved sterilized 3 times at 100°C for 1 h to eliminate AM fungal spores and other microorganisms activities. After that, four concentrations of Cd (0, 10, 20 and 30 mg/kg soil) were added to the soil in the form of CdCl 2 ·5H 2 O aqueous solution. Then the soil samples were incubated at 20°C for 30 days to make the Cd evenly distributed and stabilized in the soil solid phase.

Experimental design
The greenhouse experiment was conducted in a 2 × 4 factorial design with a completely random design. Two S. indica treatments were -S (non-inoculation / control) and + S (S. indica); Four soil Cd concentrations were Cd 0 mg/kg soil (CK), Cd 10 mg/kg soil (LH), Cd 20 mg/kg soil (MH), and Cd 30 mg/kg soil (HH). Each treatment was repeated 10 times.

Sampling
Each experimental plastic pot (30×15×15 cm 3 ) was lled with 5 kg four added Cd concentrations soil and ve soybean seeds with one fungal plug below. Every pot was watered once every four days. The soybean plants were harvested after about 120 days of planting (June 4 th -October 4 th, 2018). The shoots and roots were rinsed with distilled water, wiped with tissue paper and weighted. Then, shoot height was determined, and nally dried at 75°C for 48 h to determine the dry weights and Cd concentrations. Furthermore, root subsamples were stored in 50% ethanol for root colonization assessment. The rhizosphere soil samples were collected from each basin, thoroughly mixed, passed through 10, 60 and 100 mesh nylon screens, and divided into two parts for storage: one part was air-dried for the determination of soil pH, Cd content and chemical form, and one part was preserved at 4°C for the determination of soil enzyme activity.

Root colonization
The tryphan blue was used to estimated root colonization according to Phillip's (1970) method. After 60 days of co culture, 3 samples were randomly selected from each treatment. The roots were washed with distilled water, cut into 1cm root pieces, and put in 10% KOH solution overnight, then washed with distilled water for 3-5 times. After soaking in 1% HCl for 3-5 min, 0.05% trypan blue staining was performed for 1 min, After that, the root pieces were washed in distilled water for 8-10 times. Slides were prepared and observed under the light microscope, and took photographs.

Soil enzyme activities
The soil urease activity was assayed by phenol sodium colorimetric method, and the results were expressed as the number of milligrams of NH3-N released in 1 g of soil after incubation at 37 ℃ for 24 h (mg / g). The soil sucrase activity was measured by 3, 5 -dinitrosalicylic acid colorimetry (DNS method), and the results were expressed as the number of milligrams of glucose hydrolyzed in 1 g of soil after incubation at 37 ℃ for 24 h (mg / g). The soil phosphatase activity was assayed by sodium diphenyl phosphate colorimetry method, and the results were expressed as the number of milligrams of phenol released in 1 g of soil after incubation at 37 ℃ for 24 h (mg / g). The soil catalase activity was determined by UV spectrophotometry (240 nm), and the results were expressed as the number of milligrams of hydrogen peroxide consumed in 1 g of soil after incubation at 20 ℃ for 0.5 h (mg / g) (Yang et al. 2007;Ge et al. 2017;Trasar. 1999).

Cd determination
Cd content was analyzed according to Vieira et al. (2005). The dried soil samples (0.25 g) were digested with HNO 3 and HClO 4 (5:1) in a microwave oven. The Cd concentrations were estimated by an inductively

Assessment methods of heavy metal pollution
The single pollution index method (Hakanson 1980) was used to evaluate the risk of heavy metal pollution in soybean soil. The formula of the single pollution index method was as follows: In the formula: I j represents the single factor index of the pollution; C j is the measured concentration of the pollution (mg/kg); C 0 is the assessment standard of the pollution (mg/kg).
The contribution of S. indica in reducing Cd pollution was expressed by the decrease rate of Cd. Result Establishment of the symbiotic relationship between S. indica and soybean S. indica can infect soybean roots, especially establish symbiotic relationships with mature root soybean ( Fig. 1). The spore in soybean roots is a typical pear type. The results showed that with the CK, LH, MH, and HH treatments, the colonization rate of S. indica was 83.81%, 67.46%, 47.62% and 34.92% after inoculation ( Table 1). The root colonization rate decreased signi cantly with the increase of Cd content in soil (P < 0.01). The infection rate of S. indica was the lowest under HH treatment (Table 1). Though the colonization rate decreased, the S. indica was still functional. Still, the promotion effect of S. indica on soybean growth, soil enzyme activity and so on was limited at high Cd concentration ( Table 1).

Effects of S. indica on soybean growth and development
If there is excessive Cd accumulated in the soil, the growth and development of plants will be incredibly restricted. However, these studies have suggested that inoculated S. indica can signi cantly alleviate this inhibition (Table 1). Compared with the control, CK, LH, MH and HH treatments with S. indica increased the plant height of soybean by 11.00%, 13.52%, 31.52% and 90.35%. After treatment with S. indica, the dry weights of soybean under Cd concentration gradient were increased by 137.35% 116.60% 84.07% and 66.57%, respectively. The maximum dry weight of soybean was 43.91 g, obtained after treatment with CK and S. indica. The effect of the S. indica on the dry weight and plant height of soybean had different trends. Cd treatment and fungal treatment had signi cant main effects and interaction effects (P < 0.01) on root colonization and growth parameters of soybean (Table 1).

Effects of S. indica on pH and the decreasing rate of Cd in soil
The contents of Cd and pH in the soil after harvest have shown in Fig. 3. The increase of Cd content decreased the soil pH, and the soil pH of S. indica inoculated treatments were signi cantly higher than those of the control. The results were indicative that the Cd content in all S. indica treatments was lower than that of control, especially in the MH treatment. There was a signi cant difference between the inoculated S. indica treatment and the control (P < 0.01), inoculated S. indica decreased the Cd content in soil by 23.66%. Figure 3 showed the distribution of Cd in the soil tested by the Tessier sequential extraction. The result was re ected that soil Cd mainly combined with the exchangeable form, accounting for 40.78% ~ 54.86% of the total Cd in the rhizosphere soils. However, the exchangeable Cd in the rhizosphere soil was transformed into carbonate-bound and reducible iron and manganese forms in the S. indica treatments. Especially in the soil treatment of MH, the decline rate of the control group was the largest (Fig. 4):

Redundancy analysis of soybean growth index and environmental factors
The relationship of soybean physiological indexes, soil enzyme activities and Cd chemical forms were analyzed through redundancy analysis (Fig. 5). The exchangeable form Cd accounted for 67.4%, which can be considered as the main driver, the cumulative rate was 80.8%, which could explain all variables. The growth indexes (dry weight, shoot height) of soybean and photosynthesis indexes (Gs, Pn, Tr) were in the second quadrants; The soil enzyme activities indexes (urease, phosphatase, sucrase and catalase) and pH were in the third quadrants. The chemical forms of Cd in soil were negatively correlated with growth, photosynthesis and soil enzymes and had a greater impact on pH and soil enzyme activities.

Assessment of heavy metal pollution in soil
The assessment of the contents of Cd in soybean soil revealed that the contents of Cd in the soil of inoculated and uninoculated S. indica treatments were higher compared to the limitation standards. Besides, the contents of Cd in inoculated S. indica treatments were signi cantly lower than those in control (Table 3)   Our result expounded that compared with the control, treatments with exogenous Cd signi cantly decreased Pn, Tr, Gs, and Ci (Table 2). This change may be caused by the decrease of stomatal conductance and the obstruction of CO 2 entering leaves when soybeans were stressed by Cd, which led the decrease of Pn, which was the stomatal limitation of photosynthesis ). The Ci value of the treatment with inoculation of S. indica decreased may be because CO 2 was the raw material of photosynthesis, the less CO 2 between cells, the more carbon dioxide consumed in photosynthesis, the  (Table 2). S. indica can effectively improve the stability of Cd and reduce the inhibition of Cd on photosynthesis.

Effects of S. indica on the activities of soil enzymes under Cd stress
The soil enzyme is an important biocatalyst in soil, which reveals ecosystem perturbations and plays an irreplaceable role in the detoxi cation process of pollutants. It can also facilitate the biogeochemical cycle of nutrients, maintain soil structure, and produce the necessary compounds for microorganisms and plants (Gelsomino et al. 2006;Topac et al. 2009;Hu et al. 2014). Many studies demonstrated that Cd had adverse effects on soil enzyme activities (Ali et al. 2020). On the one hand, pollutants inhibit enzyme activity by silencing catalytic active groups that led to protein conformational denaturation. On the other hand, pollutants may compete with the enzyme's substrate, thus hindering the enzyme from functioning (Kizilkaya and Bayrakli 2005). Among the different soil enzymes, soil urease, sucrase and phosphatase are often used to evaluate the nutrient absorption of plants and organic matter transformation, and catalase was often used to evaluate the detoxi cation ability of soil ecosystem.
In this study, we also observed that Cd contamination had adverse effects on soil enzyme activities. The activities of urease, sucrase and catalase in the LH, MH and HH treatments were signi cantly lower than those of control. The decrease of soil enzyme activities was due to the increase of heavy metal content and decreased pH value (Fig. 5). A result showed that the soil enzyme activities decreased with the increase of heavy metal concentration, and Cd inhibited the activities of urease and catalase (Wang et al. 2020). On the one hand, the reason for S. indica to played a role might be that S. indica promoted the growth of soybean (Table 2), stimulated the secretion of plant root metabolites, and directly enhanced soil enzyme activity. Similar to endophytic plant growth promoting bacteria and arbuscular mycorrhizal fungi, S. indica might improve the activity of urease, sucrase and phosphatase in soil to absorb su cient C / N / P from soil, and signi cantly increased soybean biomass (Table 2), which can make the root system absorb and accumulate Cd; on the other hand, S. indica stimulated soil microorganisms, which increased the biomass and activity of microorganisms, and indirectly increased the activity of soil enzymes. The effect of microorganism on soil enzyme activity is more complex. We observed that the soil enzyme activity decreased with the increase of Cd concentration, and plants inoculation with S. indica in the root, which increased soil enzyme activities, in turn. The phosphatase and catalase activities in soybean soil, which inoculated S. indica were signi cantly increased under MH. It is similar to the conclusion of Xiao et al. (2021), which in highly Cd-polluted soils, Trifolium repens with mycorrhizal inoculation and straw treatment, phosphatase activity and catalase activity were promoted, and reduced Cd toxicity via a dilution effect.
Heavy metal ecotoxicity and soil enzyme activity are closely related to soil physical and chemical properties (Heidari et al. 2020), especially can be signi cantly affected by soil pH value. RDA results demonstrated a positive correlation between soil enzyme activity and soil pH value. That is, the decrease of soil pH value could represent the adverse effect of Cd on soil enzyme activity. The change of soil pH value in S. indica treatment was the result of multiple factors. The increase of Cd content led to more organic acids secreted by plant roots, decreased soil pH and increased metal availability, making plants absorb more heavy metals (Zeng et al. 2020). Notably, in HH treatments, the soil pH was enhanced from 7.60 to 7.68 with S. indica, indicating that S. indica may reduce the content of soil organic acid to effectively inhibit of soil acidi cation process. (Fig. 5d). The research of Yang et al. (2020) found that the citric and malic acids in rhizosphere soil of inoculating AMF were signi cantly higher than the control under Cd stress. Besides, the decrease of exchangeable Cd form may be due to the signi cant reduction of organic acid release from soybean roots by S. indica (Fig. 4).

Effects of S. indica on soil Cd fraction
The toxicity of heavy metals was mainly related to the exchangeable form, which was highly mobile and easy to enter into plants. On the contrary, carbonate form and reducible iron and manganese form were relatively stable components, which were not easy to enter the plants . These results were confrmed by RDA analysis. The chemical forms of Cd in soil were negatively correlated with the content of DW, SH and photosynthetic parameters of soybean, indicating that the accumulation of Cd in soil greatly inhibited the physiological indexes of soybean. Indeed, the photosynthetic parameters of soybean was positively correlated with urease, sucrase, phosphatase and catalase in soil. In this study, S. indica caused a positive effect on the growth and development of soybean to attenuate the toxic effects of Cd on soybean, and ultimately enhanced soil enzyme activities of soybean soil to reduce the accumulation of Cd.

Conclusions
Pot experiments were conducted to examine S. indica on soil enzyme activities, physiological characteristics of soybean and the potential risks of heavy metal pollution to soybean. According to the investigation results, the following conclusions can be drawn: (1) Cd contamination in the soil caused physiological dysfunctions of plants. Cd reduced root colonization and growth and also affected the photosynthesis of soybean plants. Inoculation of S. indica mitigated the negative impact of Cd by enhancing growth and improvement in Pn, Tr, Gs.
(2)The increase of Cd concentration lowered soil pH, which not only inhibited the activities of soil enzymes, but also increased the risks of heavy metals pollution. Besides, S. indica can reduce Cd toxicity by enhancing soil pH, promote soil enzyme activities, and reduce exchangeable Cd in soil.

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