Plant growth promoting bacteria and citric acid promote growth and cadmium phytoremediation in ryegrass

Abstract Based on the growth-promoting effect of plant growth promoting bacteria on plants and the mobilization of Cd by citric acid, an experiment was designed in which the combined treatment of Bacillus megaterium and citric acid promoted ryegrass to repair Cd-contaminated soil. This study aimed to evaluate the effects of different treatments on the antioxidant enzyme activity, photosynthesis intensity, Cd accumulation, and rhizosphere cadmium migration under cadmium contamination conditions. And the soil morphology and structure changes were studied by infrared spectroscopy FourierTransformInfrared(FT-IR) and scanning electron microscope Energy Dispersive Spectrometer(SEM-EDS) before and after different treatments. The results show that the combined treatment of Bacillus megaterium and citric acid significantly improved the oxidative stress defense and plant photosynthesis and increased of rye biomass. rye biomass 1.28 times higher than CK treatment. Joint treatment significantly increased the amount of shoot accumulation of Cd, 2.31 times higher than CK treatment, increased the migration and accumulation of cadmium. FTIR and SEM-EDS also showed that the organic constituents such as O-H, C-O and C-N in soils as a major mechanism for mobilization of the heavy metal Cd. Thus, the combined treatment of Bacillus megaterium and citric acid can promote plant growth, improve the damage to ryegrass caused by single organic acid addition, and improve the plant extraction efficiency, which is a feasible way to repair Cd-contaminated soil through activated extraction system. NOVELTY STATEMENT The novelty of this study is the combined application of bacteria and chelating agents to ryegrass to improve phytoremediation efficiency. Bacillus giganosus has a good role in promoting the growth of ryegrass. As citrate, a small molecule chelate, can activate heavy metal cadmium and detoxify heavy metals, so it was selected. This study revealed in detail the response of ryegrass to the heavy metal Cd after exogenous addition of Bacillus gigansus and citrate, which is important for the application of cadmium removal by phytoremediation. HIGHLIGHT Exogenous Bacillus megaterium has a pro-growth effect on ryegrass. The combined treatment of Bacillus megaterium and citric acid significantly improved oxidative defense mechanisms in ryegrass to alleviate Cd stress in plants. FTIR and SEM-EDS showed that the joint treatment significantly improved the soil nutrient content. Combined application of citric acid and Bacillus megaterium showed the highest removal rate of cadmium.


Introduction
Pollution from heavy metals in soil is an alarming environmental issue (Lu et al. 2015;Van Thinh et al. 2021;Song et al. 2022).Due to its high toxicity and non-degradability, soil contamination with the heavy metal Cd can have negative impacts on soil ecosystems and sustained productivity (Peng et al. 2018), and Cd in soil can threaten human health through soil-crop systems and food chain pathways (Aksoy et al. 2014;Xu et al. 2014).Therefore, mitigation of Cd contamination in agricultural fields has become an urgent need to save animals and humans from harmful pollution.
Remediation of heavy metal-contaminated soils through phytoremediation has become popular and it is considered cost-effective and eco-friendly (Yang et al. 2022).In addition, phytoremediation is free from secondary contamination and the harvested heavy metal-contaminated biomass can be treated by various methods such as composting, pyrolysis, plant excavation, hydrothermal extraction, and liquid extraction.However, the phytotoxic effects and low bioefficacy of heavy metals as well as slow plant growth and low biomass remain major limiting factors for remediation efficiency in the field application of phytoextraction (Arjun et al. 2022).These limitations are addressed by a range of ancillary techniques, such as (1) chelation-enhanced plant extraction (Wang et al. 2019), (2) genetic engineering (Gao et al. 2021) (3) microbial-assisted phytoremediation (Liu et al. 2022).Among these means, we focused on the effectiveness of plant growth-promoting bacteria (PGPB) and chelators to enhance phytoextraction.
Citric acid (CA) is extremely biodegradable and cheap compared to expensive synthetic and persistent chelators, greatly reducing concerns about the risk of heavy metal leaching and possible ecotoxicity.Many studies have shown that CA has great potential to be used as a chelator for many heavy metals (Farid et al., 2017) and to improve plant antioxidant enzyme activity (Zaheer et al. 2015).
PGPB are widely used in the phytoremediation of heavy metal-contaminated soils (Chen et al. 2014;Bhanse et al. 2022;Liu et al. 2022) and play a key role in converting unavailable nutrients into plant-available nutrients through oxidation, nitrification, ammonification, nitrogen fixation, and other nutrient cycling processes.Moreover, several studies have shown that plant growth-promoting bacteria can enhance plant photosynthesis (Guo and Chi 2014;Ma et al. 2016;Ke et al. 2021;Tanwir et al. 2021).The main plant endophytes with good heavy metal resistance that have been reported at home and abroad are Bacillus spp, Actinomyces spp, Pseudomonas spp, and Microbacterium spp (Alves et al. 2022).Bacillus spp.are resistant to external harmful factors, widely distributed, and have been widely used in agricultural soil research due to their rapid reproduction, high viability, which are frequently seen in various studies.Bacillus spp., can promote plant growth through colonization, phosphorus solubilization, siderophore, and indoleacetic acid production (Ashraf et al. 2017;Ahmad et al. 2018).On the other hand, Bacillus spp.can also detoxify heavy metal Cd and improve plant extraction efficiency by increasing antioxidant enzyme activity and oxidative stress capacity in plants (Manoj et al. 2020;Ke et al. 2021).
A large number of articles have been published on the promotion of heavy metal uptake by plants by PGPB and a large number of studies on the enhancement of phytoextraction by chelating agents, but there are fewer studies on the promotion of phytoremediation by the combination of chelating agents and bacteria, so this paper investigates the effectiveness of PGPB combined with CA in promoting plant uptake of the heavy metal Cd.The main objective was to evaluate the effectiveness of the combined application of Bacillus megaterium þ CA in enhancing phytoextraction of Cd from contaminated soil.
Moreover, ryegrass is a turfgrass with a well-developed root system, rapid growth, high biomass, and high tolerance for Cd and Pb remediation.It was found that turfgrass, as a widely used green plant, has the characteristics of fast growth, adaptability, high biomass, resistance and soil fixation.Some studies have shown that the concentration of heavy metal ions in the soil can be gradually reduced by repeated mowing and harvesting of ryegrass, and more heavy metals are absorbed and treated more efficiently (Li et al. 2020;Ke et al. 2021).
Therefore, the objectives of this study were (1) to investigate the effect of the combination of Bacillus megaterium and CA in promoting Cd uptake by ryegrass (2) to investigate the physiological changes of Bacillus megaterium and CA on growth and chlorophyll fluorescence of ryegrass (3) to investigate the effect of Bacillus megaterium and CA on antioxidant enzyme activity of ryegrass.

Preparation of cadmium-doped soil
Surface soils (0-20 cm) were collected from Qingdao Agricultural University.The basic characteristics were as follows: organic matter 10.2 g/kg, alkali-hydrolyzable nitrogen 3.5 mg/kg, available phosphorus 21.3 mg/kg, available potassium 55.6 mg/kg, total cadmium 0.18 mg/kg, and pH 8.2 (1:2.5 w/v water).Weigh 30 kg of air-dried soil with a 2 mm sieve and add CdCl 2 at 10 mg/kg Cd 2þ dosage for poisoning.The pots of 18 Â 11cm were selected and each pot was filled with 1 kg of soil.

Preparation of Bacillus megaterium suspension
The Bacillus megaterium species were purchased from the microbial storage center with CICC number 20665.The strains were inoculated in NB medium and shaken at 37 C for 24 h in a shaker at 150 rpm, and the strains reached the late logarithmic growth phase.The bacterial solution was transferred to sterile centrifuge bottles and the organisms were collected.The bacteria were resuspended in sterile water to make a suspension of (i.e., 1OD) 10 9 cfu/mL and set aside.

Experimental design
The pot experiments were conducted in the research intelligent greenhouse of Qingdao Agricultural University.Four treatments: blank treatment 1 ryegrass, Bacillus megaterium suspension 1 ryegrass, citric acid 1 ryegrass, citric acid 1 Bacillus megaterium 1 ryegrass.Citric acid was set at 4 concentrations: 2.5 mmol/kg (CA1), 5 mmol/kg (CA2), 7.5 mmol/kg (CA3), 10 mmol/kg (CA4).Three parallel experiments were set up in each group.Seven days after germination, 50 mL of Bacillus megaterium suspension was applied to the rhizosphere soil area in each pot.And after 24 days of germination, CA was added to the rhizosphere soil area of each pot.The pre-experimental results showed that the CA concentration of 7.5 mmol/kg showed the highest shoot cadmium accumulation in ryegrass and the best plant extraction effect (Figure A.1).Therefore, 7.5 mmol/kg CA was used to further study the effect of different treatments on the extraction effect of heavy metal Cd and the physiological indicators and growth of ryegrass.The treatment groups for the pot experiments were: control (CK), Bacillus megaterium (T1), 7.5 mmol/kg CA (T2), 7.5 mmol/ kg CA 1 Bacillus megaterium (T3).
The ryegrass seeds were sterilized and sown at 100 seeds per pot.All treatments were repeated three times with uniform water and temperature management, the light was natural.After ryegrass germination, 60 healthy and uniform ryegrass shoots were retained.40 days were harvested.
Determination of cadmium concentration in ryegrass 0.5 g of dried plant samples were digested with nitric acid and perchloric acid (v:v ¼ 9:1), and 0.1 g of dried inter-root soil samples were digested with total hydrochloric acid-nitric acid-hydrofluoric acid-perchloric acid.Then the volume was fixed and the heavy metal concentration in the ablation solution was determined by inductively coupled plasma mass spectrometry (NexlON 1000, USA).The translocation factor (TF) of each metal was determined using Equation (1) with the following equation:

Measurement of ryegrass growth and photosynthesis indicators
Biomass: Rinse well with deionized water.Fresh samples were killed at 105 C for 30 min and then dried at 80 C for 24 h to constant weight, and the dry weight was measured.

Determination of malondialdehyde and antioxidant enzyme activities in ryegrass
Malondialdehyde (MDA) concentration (Dipierro and Leonardis 1997): A 0.5 g of chopped ryegrass was weighed, adding 5 ml of 10% TCA (trichloroacetic acid) solution and a small quartz sand, ground until homogenized, centrifuged at 4000 r/min for 10 min, and the supernatant was an enzyme extract. 1 mL of enzyme extract (control plus 1 mL of water) was taken, 2 mL of 0.67% TBA (thiobarbituric acid) was added, sealed and subjected to boiling water bath for 15 min, rapidly cooled, centrifuged at 4000 r/min for 20 min, and the supernatant was taken at 600, 532 and 450 nm for colorimetric analysis.
Determination of antioxidant enzyme activity: weigh about 0.5 g of fresh leaves, add 2 mL of phosphate buffer (pH ¼ 7.8) and grind in an ice bath.After grinding was completed, the leaves were poured into a centrifuge tube, and the bowl was washed with 2 mL of phosphate buffer and poured into a centrifuge tube, equilibrated and centrifuged at 4 C for 20 min at low temperature (10,500 r/min).Peroxidase (POD) activity was determined by the guaiacol method (Polle et al. 1994); superoxide dismutase (SOD) activity by the nitrogen blue tetrazolium (NST) method; catalase (CAT) activity by the ammonium molybdate method (Aebi 1984).The enzyme activity was expressed as grams of H 2 O 2 decomposed per minute per gram of fresh sample.

Soil characterization
The sample before and after treatments were glued directly onto the conductive substrate with adhesive and sprayed with gold for 45s using an Oxford Quorum SC7620 (Czech Republic) sputter coater at 10 mA.The sample was then photographed using a TESCAN MIRA LMS scanning electron microscope for morphology and energy spectrum mapping tests.An acceleration voltage of 3 kV and 15 kV was used for SEM analysis energy spectrum mapping, respectively, using an SE2 secondary electron detector.
The samples before and after treatments were placed on a Nicolet iS 10 FT-IR spectrometer (Shanghai, China) and scanned over the wavenumber range of 4000-400 cm À1 at a resolution of 4 cm using 32 scans.

Quality control
The determination of Cd concentration of the samples was carried out under strict quality control, and the containers used were immersed in 10% HNO 3 solution 24h before the experiments, and the experimental reagents were of high purity.Plant standard samples (DB32/T 5852009) were used for quality control.The recoveries of heavy metals in plants studied in this paper ranged from 89% to 115%, and the standard deviations (RSDs) were all controlled within 5% when parallel samples were set up during the experiments.

Statistical analysis
All treatments were performed with three replications, and the chi-square test and significance of differences test were performed using Origin 2018 and SPSS 26.0 with a significant difference level of p ¼ 0.05.

Biomass and Cd accumulation in ryegrass
In the T2 treatment, plant height was significantly reduced (Figure 1A).As shown in Figure 1B, there were significant differences in the biomass of ryegrass under different treatments, and the T1 and T3 treatments significantly promoted the growth of plants, which increased 1.11 and 1.28 times than that of the CK treatment, respectively.In addition, the shoot Cd concentration and root Cd concentration and aboveground Cd accumulation of ryegrass increased significantly in different treatment groups (Figure 1D).Among them, T3 cadmium accumulation increased the most, which had an obvious synergistic effect.The transfer factor (TF) is the ratio of the metal concentration in the shoots of the plant to that in the roots.It is used to assess the ability of ryegrass to transfer Cd from roots to shoots.In this study, the TF of ryegrass was significantly increased by either the addition of a chelating agent or microbial treatment (Figure 1F).
The shoot Cd accumulation in T3 treatment increased significantly.On the one hand, Bacillus megaterium secreted various substances to promote the growth of ryegrass; on the other hand, CA formed a Cd-CA complex with Cd to promote Cd activation in soil and detoxify Cd contamination in plants.Functional groups of organic acids (e.g.,carboxyl and hydroxyl groups) are important binding sites for metals (Ghnaya et al. 2013), and CA has three carboxyl groups and one hydroxyl group, so CA and Cd can form Cd-CA complexes.Some studies have shown that the bioaccumulation of Cd in plants was significantly enhanced by the addition of CA (Ma et al. 2019;Yang et al. 2022).This is consistent with the results of the present study.The biomass of ryegrass was not reduced after exogenous addition of CA, probably because Cd-CA is much less toxic than Cd 2þ (Aravind and Prasad, 2005).Moreover, it has also been shown that certain concentrations of CA can stimulate plant growth (Farid et al., 2017).CA formed metal chelates with Cd, which not only increased the mobility of Cd in plants and promoted the accumulation of Cd in plants, but also was able to alleviate the toxic effects of Cd ions on the plant body.It is consistent with the results of previous studies (Ma et al. 2020).
Moreover, biomass is also an important factor affecting the efficiency of phytoremediation.This study demonstrated a good promoting effect of Bacillus megaterium on ryegrass.Many studies have shown that Bacillus megaterium, one of the most common plant growth-promoting bacteria, is able to regulate plant physiological characteristics and promote plant growth (Funes et al., 2017).Bacillus can promote effective phosphate absorption of ryegrass by dissolving inorganic phosphate, and siderophore to make iron available to plants in metal-contaminated soil environments.It can also significantly reduce the plant growth inhibition caused by metal Cd stress by secreted ACC deaminase and indoacetate IAA, which greatly promote the growth of plants in metal-contaminated soil by stimulating root growth and their ability to absorb water and nutrients (Etesami and Maheshwari, 2018;Del Carmen Orozco-Mosqueda et al. 2020;Nanjani et al. 2022).Moreover, it has been shown that Bacillus is the dominant group of IAA-producing bacteria (Bishnu Maya et al. 2021).Erturk, Y. et al. showed that Bacillus simplex RC19 and Bacillus polymyxa RC05 produced high levels of IAA, 33.6 and 32.8 ug/ml, respectively (Erturk et al. 2010).The study by He et al. showed that two strains of Bacillus sp.QX8 and QX13 had strong Cd and Pb resistance and various probiotic properties including IAA, siderophore and ACC deaminase production and phosphate solubilization.(He et al. 2020).In addition to the probiotic effect of Bacillus megaterium on ryegrass, it may also be due to the secretion of organic acids and surface active substances by the bacteria, among others (Ma et al. 2013;Wang et al. 2018), which allows a portion of Cd to enter the soil liquid phase from the soil solid phase and promotes the activation of heavy metal Cd in soil.Bacillus megaterium directly or indirectly increased the effective heavy metal Cd concentration in the soil and promoted the uptake of heavy metal Cd in the soil by ryegrass.
In conclusion, T3 treatment improved both the effectiveness of heavy metals and increased the biomass of ryegrass.It can simultaneously promote the growth of ryegrass and cadmium absorption and transport, thus improving the phytoremediation efficiency of cadmium.

Chlorophyll fluorescence parameters
Maximal quantum yield of PSII (Fv/Fm), (Fv/F0), absorption light energy-based performance index (PI abs), light energy absorbed per unit reaction center (ABS/RC), and the efficiency of the energy absorbed by the antenna transferred below QB (ETo/ABS) vary significantly between treatments.Among them, the fluorescence parameters of T3 treatment increased the most, being 1.03, 1.12, 1.28, 1.15 and 1.08 times that observed for CK treatment, respectively (Figure 2).
Moreover, PGPB inoculation can promote plant photosynthesis (Wu et al. 2020).The present study also demonstrated that T3 treatment can successfully promote host growth by stimulating photosynthesis in ryegrass.Although T3 treatment caused significantly higher accumulated cadmium in plants than other treatments, its fluorescence index still increased significantly, which just explains the protection and biopromoting effect of Bacillus gigasum on ryegrass, which can reduce the heavy metal Cd stress on plants.The feasibility of T3 treatment to promote this behavior of cadmium phytoremediation was demonstrated.T3 treatment can promote photosynthesis in ryegrass by a higher maximum quantum showed that plants inoculated with Bacillus SDA-4 under Cd stress showed a significant increase in plant biomass accumulation, along with a decrease in proline and MDA concentration (Shahid et al. 2020).This is consistent with the results of this experimental study.
In conclusion, T3 treatment can significantly increase the chlorophyll fluorescence parameters in ryegrass, promote photosynthesis, and promote soil Cd absorption in ryegrass, either directly or indirectly.

Antioxidant enzyme activity
Superoxide dismutase (SOD) is an important antioxidant enzyme that scavenges excess activated oxygen species (ROS) in plants under pollutant stress.Catalase (CAT) is ubiquitous in plant tissues and acts to remove H 2 O 2 produced in metabolism to avoid oxidative damage from H 2 O 2 accumulation.Peroxidase (POD) is a common and highly active enzyme in plants.It is closely related to respiration, photosynthesis and the oxidation of auxin.Compared with CK, SOD, CAT, and POD enzymes activity was significantly increased with T3 treatment, which was 1.22, 2, 1.49-fold higher than with CK treatment, respectively (Figure 3A,B,C).Malondialdehyde (MDA) is the main product of membrane lipid peroxidation, and it is often used to indicate the degree of cell membrane damage induced by oxidative stress.And the T3 treatment had the lowest malondialdehyde content.
Moreover, plant exposure to adverse environments, such as heavy metal stress, can lead to dysregulation of oxygen metabolism in plants, resulting in the accumulation of reactive oxygen species in plants (Zulfiqar and Muhammad., 2022).Excessive production of reactive oxygen species leads to the production of more MDA and the oxidation of important cellular structures, including lipids, proteins, and nucleic acids (Hasanuzzaman et al. 2012), which can cause a range of plant damage, such as inhibition of plant growth.Previous studies have shown that PGPB can help plants detoxify Cd by altering antioxidant enzyme activity and  phytohormone secretion in superenriched plants (Ashraf et al. 2017).The results of this study showed that T3 treatment greatly increased SOD, POD and CAT enzyme activities in ryegrass, while MDA was significantly decreased, indicating that T3 treatment can increase plant growth by reducing cell damage and increasing host plant tolerance.SOD, POD and CAT constitute the protective enzyme system for reactive oxygen species scavenging in ryegrass.The main role of SOD is to scavenge superoxide anion radicals O 2 --in the ryegrass and protect cells from oxidative damage, generating O 2 and H 2 O 2 , the first-line defense mechanism.The reaction product H 2 O 2 is further broken down by CAT to produce H 2 O and O 2 (Gill et al. 2015).The three coordinate with each other to form a protective system that maintains reactive oxygen species at low levels in ryegrass.Similar to the results of this experimental study, two plant growth-promoting bacteria, strains NT1 and NCT-2, could affect antioxidant enzyme activity and alleviate oxidative stress, thereby detoxifying Cd and promoting the growth of Lobelia.(Chi et al. 2022).It has been shown that the endophyte Bacillus amyloid AW3 can confer resistance by inducing the accumulation of defense-related enzymes/genes (Zhang et al. 2022).The study of Sebastian showed that exogenous addition of citric acid significantly increased the accumulation of antioxidants such as anthocyanins and glutathione (GSH) under Cd stress (Sebastian and Prasad 2018).The study of Mahmud found that CA as an exogenous protectant significantly reduced Cd toxicity by upregulating antioxidant defense system and glyoxal system (Mahmud et al. 2018).Excess heavy metals lead to the accumulation of H 2 O 2 in plants through oxidative stress (Ivanova et al. 2021).The addition of exogenous Bacillus megaterium or CA activated the ryegrass antioxidase enzyme system to control superoxide and H 2 O 2 levels.The combined treatment of organic acids and bacteria maintained the highest antioxidant enzyme activities despite the significant increase of shoot and root Cd in ryegrass, which fully demonstrated the dominance of T3 treatment.As for its synergistic mechanism, further studies are needed.MDA is the final decomposition product of membrane lipid peroxidation, and its accumulation can cause serious damage to the cell membrane and cells, which to some extent reflects the environmental stress of plant cells (Khan et al. 2015).The results showed that the T3 treatment resulted in the highest shoot Cd accumulation in ryegrass, but the lowest in vivo malondialdehyde concentration.It showedthat T3 treatment significantly reduced cell membrane lipid peroxidation and detoxified the heavy metal Cd in ryegrass.
In conclusion, T3 treatment can significantly slow down the Cd stress damage caused by ryegrass, promote healthy plant growth, and improve the Cd plant repair efficiency of ryegrass.

SEM-EDS
Figure 4 shows the SEM-EDS images of the cross-sectional area of the soil samples under the different treatments.It is clear that after T1, T2, and T3 treatment, the soil has a reduced aperture, less folds, and a relatively flat surface.Among them, T3 treatment changes were the most obvious, which reduced the specific surface area and active site of the soil, weakened the adsorption of heavy metal ions, and favored the mobilization of cadmium in the soil.The possible reason for this is the possible interaction with the biosurfactant in the T3 treatment group during Cd mobilization.The binding of heavy metals to the biosurfactant was stronger than the binding between soil and particles, thus facilitating elution from the soil.Moreover, from the EDS results, T3 treatment significantly increased N, P, O content.Therefore, the soil surface topography and elemental analysis results showed that T3 treatment significantly improved soil nutrient content and promoted soil cadmium mobilization.

FTIR
Figure 5 shows the FTIR images of the cross-sectional area of the soil samples under the different treatments.After treatment with T1, T2, and T3, the O-H at 3628 cm À1 were stretched vibrate to 3621 cm À1 , 3625 cm À1 , 3630 cm À1 , respectively.The sudden peak at 3425 cm À1 , 3422 cm À1 , 3387 cm À1 is also due to the stretching vibration of the O-H (Bartos et al. 2020).The new peak occurring at 1643 cm À1 , 1635 cm À1 , 1651 cm À1 , is due to the stretching vibration of the C ¼ O and C-N bonds of the protein.The peak at 1026 cm À1 was shifted to 1030 cm À1 ,1033cm À1 ,1031cm À1 , respectively, and was considered as the tensile vibration of C-O-C and C-OH of polysaccharides (Sheng et al. 2013).Some frequency bands in the fingerprint region (<1000 cm À1 ) may indicate the presence of a phosphate group, a functional group of nucleic acids (Hou et al. 2013).
In conclusion, organic constituents such as O-H, C-O and C-N in soils as a major mechanism for mobilization of the heavy metal Cd.the changes of the soil functional groups before and after the treatment may affect the effectiveness of cadmium in the soil or the soil properties, and then affect the phytoremediation effect of cadmium.

Conclusion
In this study, we improved the phytoremediation efficiency of ryegrass by combining CA with Bacillus megaterium (T3).The results show that T3 treatment significantly improved the oxidative stress defense and plant photosynthesis and increased of rye biomass.rye biomass 1.28 times higher than CK treatment.Joint treatment significantly increased the amount of shoot accumulation of Cd, 2.31 times higher than CK treatment, increased the migration and accumulation of cadmium.FTIR and SEM-EDS also showed that the organic constituents such as O-H, C-O and C-N in soils as a major mechanism for mobilization of the heavy metal Cd.Thus, the combined treatment of Bacillus megaterium and citric acid can promote plant growth, improve the damage to ryegrass caused by single organic acid addition, and improve the plant extraction efficiency, which is a feasible way to repair Cd contaminated soil through an activated extraction system.

Figure 1 .
Figure 1.Different treatments treatment on growth and cadmium accumulation of ryegrass; (A) shoot height; (B) shoot dry weight; (C) shoot Cd accumulation; (D) shoot Cd concentration; (E) root Cd concentration; (F) IF.

Figure 2 .
Figure 2. Effects of Bacillus megaterium and citric acid treatment on fluorescence parameters and chlorophyll concentration of ryegrass.

Figure 3 .
Figure 3. Effects of Bacillus megaterium and citric acid treatment on antioxidant enzyme activity and MDA concentration of ryegrass; (A) SOD; (B) CAT; (C) POD; (D) MDA.