Organic acids induce by metal-tolerant Pantoea sp. WP-5 and biogas residues enhanced phytostabilization of cadmium in soil

This study investigated the phytoremediation potential of maize (Zea mays L.) in Cd contaminated soil through co-inoculation of metal tolerant plant benecial rhzobacteria (MtPBR: Pantoea sp. strain WP-5) with organic manures (PM: poultry manure and BGR: biogas residues). The objectives of this study were to i) examine comparative eciency of MtPBR, PM and BGR alone or in combined form to improve maize biomass and physiology, and ii) understand the role of organic acid production in root exudates of maize for Cd accumulation and translocation. Pantoea sp. WP-5 showed tolerance to high Cd concentration (1000 mg L − 1 ), thereby inoculated to maize seeds sown in soil spiked with 75 mg Cd kg − 1 soil and 500 g each of the organic manures per pot. The co-inoculation of MtPBR + BGR signicantly (P < 0.05) increased chlorophyll contents, root/shoot dry weight, photosynthetic rate, stomatal conductance and relative water contents, whereas decreased electrolyte leakage, malondialdehyde contents, ascorbate peroxidase and catalase activity in maize over the control treatment. The co-inoculation of MtPBR + BGR produced signicantly (P < 0.05) higher concentrations of acetic and citric acid (52.7 ± 0.5 and 22.8 ± 0.08 µg g − 1 root fwt, respectively) in root exudates of maize, which immobilized Cd within plant roots inferred by the positive relation (root Cd vs. organic acids; R 2 = 0.80–0.92) and reduced Cd translocation to shoots inferred by the negative relation (shoot Cd vs. organic acids; R 2 = 0.81–0.90). It is concluded that the application of MtPBR + BGR enhanced organic acid induced phyto-stabilization and accumulation of Cd in roots and restricted its translocation to shoots.


Introduction
Soil, the only growth medium for arable crops, is now becoming useless due to deposition of heavy metals. Heavy metals (HMs) like cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), arsenic (As), zinc Maize (Zea mayz L.) due to its better Cd accumulation capacity and high biomass production potential has been widely used as probable candidate for management of Cd-contaminated soils (Rizwan et al., 2016). Maize is resistant to moderate level of Cd stress in soil. However, seed germination, mineral nutrition, photosynthesis, and growth/yields of maize may be reduced due to higher level of Cd  Chen et al. (2020) found higher accumulation of Cd in sorghum grass in the presence of citric acid and maltose. However, studies on the inoculation of organic acid producing bacteria to maize and their effect on root exudates under Cd stress is remain elusive. Production of organic acids like acetic acid, oxalic as well as citric acid by the plant bene cial rhizobacteria and the role of these acids in P solubilization has been reported in our previous studies (Tahir et al., 2013(Tahir et al., , 2015. We hypothesized that use of the organic acid producing MtPBR along with organic treatments such as BGR and PM enhance the phytoextraction and bioaccumulation of Cd in Zea mays L. under Cd contaminated soil. The objectives of this study were to i) examine comparative e ciency of PBR, PM and BGR alone or in combined form to improve maize biomass and physiology and ii) understand the role of organic acid production in the root exudates of maize for Cd accumulation and translocation.

Source of PBR, PM and BGR
Plant bene cial bacterial strain Pantoea sp. WP-5 (accession no. HE661627; Tahir et al., 2015) obtained from NIBGE Biotech Resource Centre. Recently this strain was characterized positive for IAA, produced organic acids and solubilized P in culture medium . The organic manures such as PM and BGR collected from local industry were characterized for nutritional status using the standard procedures. The PM contains 2.13% N, 1.91% P and 1.52% K, while the BGR contains 1.71% N, 0.96% P, 1.26% K.

Cd tolerance measurement of PBR
The plant bene cial bacterial strain Pantoea sp. WP-5 was tested for Cd tolerance through measuring the minimum inhibitory concentration (MIC) of Cd. The strain was grown in nutrient-agar medium amended with 0-2000 mg L -1 Cd using CdCl 2 as salt. MIC for the Cd was calculated by observing the growth of the strain after 7 days of incubation at 28 ± 2 ºC. The minimum concentration on which the growth of the strain Panotea sp. WP-5 was inhibited, considered as MIC.

Pot experiment
A pot experiment was carried out at Agriculture research farm of Bahauddin Zakariya University Multan (30°11′52″N 71°28′11″E), Pakistan. For this purpose, soil was collected from an experimental eld of the Agriculture research farm of Bahauddin Zakariya University Multan from the depth of 0-15 cm using auger. The soil was dried up in air, minced and sieved through a 2 mm sieve. A composite soil sample before pot experimentation was examined for physico-chemical characteristics (saturation percentage, total nitrogen, organic matter, soil texture, available phosphorus, extractable potassium, total Cd, electrical conductivity and pH) by following standard analytical methods as mentioned in ICARDA manual. The proportion of sand, silt and clay in the soil was 25.6%, 53.1% and 18.4% respectively. The pH, saturation percentage (SP) and electrical conductivity (EC) values were estimated as 8.2, 30%, and 3.41 dS m -1 , respectively. The organic matter (OM), total N, available P, extractable K and Cd contents values were measured as 0.6%, 0.04 mg kg -1 , 6.2 mg kg -1 , 198 mg kg -1 and 0.2 mg kg -1 , respectively.
The homogenized soil (10 kg) was lled in earthen pots of area 0.035 m 2 . The soil was spiked with 75 mg Cd kg -1 soil using CdCl 2 as salt ve days prior to start of the experiment. After ve days of spiking, all the treatments (PBR, PM, BGR, PBR+PM, PBR+BGR, PM+BGR, PBR+PM+BGR) along with control without PBR, BGR and PM) were applied in Cd contaminated (75 mg Cd kg -1 soil) and normal soil. The manures PM and BGR were applied @ of 0.5 kg/pot. To inoculate the PBR strain (Pantoea sp. WP-5), the seeds of maize hybrid ICI-9091 (obtained from Ayyub Agriculture Research institute, Faisalabad) were mixed with mixture of over-night grown bacterial culture of Pantoea sp. WP-5 with strength of 10 9 colony forming units (cfu) per mL and the sugar cane lter mud (at the rate of 0.5 g per 100 g seed). The seeds (10 seeds per pot) were sown in the earthen pots on March 20, 2017. Crop was fertilized with NPK at 200, 150 and 150 kg ha -1 , respectively using urea (NH 2 ) 2 CO, diammonium phosphate (NH 4 ) 2 HPO 4 and SOP (K 2 SO 4 ) as source of N, P 2 O 5 and K 2 O, respectively. Completely randomized design (CRD) was used in laying out the experiment. Five replications (n=5) were used for each treatment. Thinning was done at 20 days after sowing and three seedlings was maintained in each pot. A standard criterion for irrigation was followed (Ayer's and Westcot, 1985). Maize crop was harvested manually on May 20, 2017.

Data collection 2.3.1 Germination count (%) at 8 days after sowing
After the eight days of sowing, germinating seedlings were counted, and germination percentage was noted by using the following formula:

Physiological parameters and enzymatic analysis
Fresh leaf samples of maize were obtained at 40 days after sowing to measure the chlorophyll a & b contents, carotenoids contents, antioxidant enzyme activity, photosynthetic rate, stomatal conductance, electrolyte leakage and relative water contents in leaves.

Chlorophyll content determination
Chlorophyll contents were measured by grinding the 250 mg of fresh leaf samples in liquid nitrogen. A 10 mL volume of acetone/water (80/20: v/v) were added in the sample and kept at 4ºC for 24 h. After the 24 h, the samples were centrifuged at 10000 g for 10 min and the supernatant was collected in falcon tubes. Absorbance of the samples was recorded at 470, 646 and 663.2 nm. Chlorophyll contents were determined by using the procedure adopted by Lictenthaler (1987).

Antioxidant enzyme analysis
For the analysis of antioxidants enzymes, fresh leaves of maize were minced in liquid nitrogen. The grinded sample was homogenized in phosphate buffer (0.05 M and pH=7.1). The homogenized sample was ltered and centrifuged at 16000g for 10 min at 4 °C. By adopting the procedure designated by Aebi (1984), activity of catalase enzyme was measured. In this procedure, the enzyme extract was mixed with 300 mM H 2 O 2 , phosphate buffer (50 mM) and CA (2.0 mM, pH 7.0). Due to H 2 O 2 disappearance (ε ¼ 39.4 mM −1 cm −1 ), diminution in absorbance at 240 nm was measured.
Activity of ascorbate peroxidase (APX) enzyme was measured using the method described by Nakano and Asada (1981). For this method, 100 μL enzyme extract was mixed with equal volume of 7.5 mM ascorbate and 300 mM H 2 O 2 , 2.7 mL of 25mM potassium phosphate buffer and 2.0 mM ethylenediaminetetraacetic acid (C 10 H 16 N 2 O 8 ) (neutral pH). To measure the ascorbate oxidation activity, wavelength at 290 nm was noted.
Peroxidation of lipids in leaf tissue is represented by malondialdehyde (MDA) contents and this was measured by using thiobarbituric acid-reactive-substances assay (TBARS; Heath and Packer, 1968). For this, 0.25 g of fresh leaves were mixed with trichloro acetic acid (TCA) and centrifugation was done at 12000g for 15 min. The supernatant was separated, and 1.0 mL of the supernatant was mixed with 4.0 mL of 20% TCA comprising 0.5% thiobarbituric acid (TBA). The mixture was placed at 95 °C for 30 min.
After the heating, it was rapidly cooled in an ice bath and centrifuged at 10,000 g for 10 min. The supernatant was analyzed on spectrophotometer at 532 nm and value for nonspeci c absorbance at 600 nm was deducted. Using extinction coe cient of 155 mM −1 cm −1 , the calculation of MDA contents was made.

Photosynthetic rate and stomatal conductance
Stomatal conductance and rate of photosynthesis were measured on portable Infra-red Gas Analyzer leaves of maize were taken and weighed to get leaf fresh biomass. The leaves were then placed in water for overnight. After that, weight of the leaves was noted (i.e. fully turgid weight). To measure the oven dried biomass, the fully turgid leaves were dried in an oven at 70±2 °C for 48 h.
The method described by Ahmad et al. (2014) was used to measure the electrolyte leakage (ELL). In this method, completely extended ag leaf was cut into pieces of about 5 mm length. These pieces were placed in test tubes lled with 10 mL doble distilled water. The tubes were kept on shaking at 150g and 30 °C for 4 h. After that, the electrical conductivity (EC1) of the medium was noted. Then all the test tubes were autoclaved by placing them in an autoclave and cooled down to 25 °C. Afterwards, electrical conductivity (EC2) of the cooled samples was measured. ELL was calculated by:

Measurement of growth data
Maize plants were harvested on May 20, 2017 i.e. at 60 days after sowing. At the time of harvesting, maize plants were uprooted, and roots were separated from each plant. The separated roots were washed in tap water and placed separately on a transparent polyethylene sheet. The sheet along with the roots were placed on desktop scanner. The roots were scanned, root image was created on computer and the root length was measured using root image analysis program. Shoot length of each plant was measured with measuring tape. At the time of harvesting, fresh weight of both root and shoot were measured separately with the help of laboratory weighing balance. After measuring the fresh weight, root and shoot were kept in an oven at 70 °C for 48 h and weighed separately on digital balance to measure dry weight.

Organic acids detection in root exudates of maize
Root samples were collected from each treatment at ag leaf stage. The roots were placed in 30 mL sterilized distilled water and kept on shaking at 150g for 5 days. Thereafter, supernatant of samples was collected through centrifuge at 6000g for 8 min and concentrated to 1.5 mL in a concentrator (Concentrator 5301, Eppendorf, Germany) and ltered over a 0.2 μm lter (Orange Scienti c GyroDisc CA-PC, Belgium). To determine the organic acids (acetic acid and citric acid), the ltrates were analyzed by HPLC, using a Perkin Elmer series 200 with 20 μL auto-sampler PE NELSON 900 series interface, PE NELSON 600 series link and Perkin Elmer NCI 900 Network Chromatography interface using Diode-array detector at 210 nm and their UV spectra (190-400 nm), Microgaurd Cation-H Precolumn and an Aminex HPx-87H analytical column for separation. Sulfuric acid (0.001N) was used as mobile phase with ow rate of 0.6 mL min -1 . The run was isocratic, and the run time of each sample was 20 min. Solutions (100 μg L -1 ) of acetic and citric acid (Daejung, Korea) were used as standards.

Cadmium concentration in plant tissue (root and shoot)
The root and shoot samples were kept in oven at 70 °C for 24 h to get oven dried. The dried samples were weighed on digital balance. After weighing, the samples were mashed, and digested with nitric acid (HNO 3 ) and perchloric (HClO 4 ) acid (3:1 ratio). The digested samples were placed on a hot plate for heating at 350 °C until the dense white fumes produced. The samples were then cooled, passed through a Whatman 40, and stored. The Cd in the ltered samples was determined on atomic absorption spectrophotometer wit detection limit of 0.002 µg L -1 (PerkinElmer, 100 Analyst, Waltham, USA). Translocation factor (TF) was measured according to Ahmad et al. (2014).

Statistical analysis
Normal distribution (P>0.05) test on all the data collected was performed according to Shapiro-Wilk test. Levene test was performed to verify the homogeneity of variance (P>0.05) of sample data. Statistix v8.1 was used to analyze the data statistically using one-way analysis of variance (ANOVA). The data presented here is the average of ve replicates (n=5) ± standard error. Linear regression analysis was applied to determine relation of Cd concentration in maize plant to organic acids concentration in root exudates of maize. When the overall, main effect was signi cant, treatments mean was further compared using LSD test at P<0.05 probability level (Steel et al., 1997).

Minimum inhibitory concentration of cadmium
The PBR strain Pantoea sp. WP-5 showed tolerance up to 1000 mg L − 1 of Cd, further increase in Cd concentration inhibited the growth of this strain.

Pot experiment
3.2.1. Germination count (%) at 8 days after sowing Analysis of data revealed that BGR treatment gave signi cantly higher germination percentage (60%) under Cd stress conditions at 8 days after sowing (DAS; Fig. 1a). The treatments MtPBR + BGR, MtPBR + PM and MtPBR + BGR + PM resulted in 57% germination count in Cd contaminated soil. Minimum values of germination percentage i.e. 43% was recorded in control treatment in Cd contaminated soil. Under no Cd stress, the MtPBR + BGR resulted in higher germination count (60%) at 8 DAS (Fig. 1a).

Plant height (cm) and dry weight (g per plant)
Under normal soil conditions, MtPBR + BGR gave higher plant height (104 cm) and plant dry weight (25.9 g per pot) as compared to all other treatments ( Table 1) (Table 1). However, the treatment MtPBR + BGR + PM increased the photosynthetic rate, stomatal conductance and RWC by 20%, 38% and 15% over control treatment in Cd contaminated soil. Application of MtPBR as sole treatment in normal soil also performed well and increased the photosynthetic rate by 10%, stomatal conductance by 4% and RWC by 11% over the control treatment.
While in Cd contaminated soil the MtPBR as sole application increased the photosynthetic rate by 13%, stomatal conductance by 26% and RWC by 7% over the control treatment. Minimum values of these parameters were recorded in the control treatment under normal as well as Cd stress conditions (Table 1).
Under normal soil conditions, the application of PM alone and MtPBR + BGR + PM gave signi cantly (P < 0.05) higher chlorophyll a content i.e. 2.07 and 2.02 mg g − 1 leaf fresh weight respectively, as compared to all other treatments (Fig. 1b). In the Cd contaminated soil, the treatment MtPBR + BGR gave higher chlorophyll a content i.e. 1.87 mg g − 1 leaf fresh weight as compared to other treatments in Cd contaminated soil (Fig. 1b). The control treatment resulted in minimum chlorophyll a content under normal as well as under Cd stress conditions.
Application of MtPBR as sole treatment gave signi cantly (P < 0.05) higher chlorophyll b content (1.5 mg g − 1 leaf fresh weight) as compared to other treatments under normal soil conditions (Fig. 1c). After the MtPBR treatment, the treatments BGR, BGR + PM and MtPBR + BGR + PM produced chlorophyll b content 0.8, 0.76 and 0.78 mg g − 1 leaf fresh weight respectively, under the normal soil conditions. In Cd contaminated soil, the MtPBR as sole treatment gave signi cantly (P < 0.05) higher chlorophyll b content (0.95 mg g − 1 leaf fresh weight) as compared to the other treatments. After the MtPBR treatments, the treatments BGR, MtPBR + BGR, BGR + PM and MtPBR + BGR + PM also increased the chlorophyll b contents by 93%, 73%, 118% and 103% respectively, over the control treatment in Cd contaminated soil (Fig. 1c).
In normal soil, the application of MtPBR as sole treatment resulted in signi cantly (P < 0.05) higher carotenoid contents i.e. 2.01 mg g − 1 leaf fresh weight. After the MtPBR treatment, the treatment MtPBR + BGR produced higher carotenoids content (1.13 mg g − 1 leaf fresh weight) in normal soil (Fig. 1d). In Cd contaminated soil, the treatment MtPBR + BGR gave signi cantly (P < 0.05) higher carotenoid contents (1.06 mg g − 1 leaf fresh weight) as compared to the other treatments. Minimum carotenoids were measured in the control treatments under normal as well as under Cd stress conditions.
Membrane damage was determined by measuring the ELL. It was noted that ELL was signi cantly (P < 0.05) higher in plants grown in Cd contaminated soil as compared to that of the plants in normal soil (Fig. 2a). Among the treatments, the values of ELL were signi cantly (P < 0.05) higher in control treatment i.e. 61.6% in Cd contaminated soil and 40.5% in normal soil. Application of organic amendments either alone or in combined form tended to decrease the ELL value. In normal soil, the treatment MtPBR + BGR reduced the ELL value up to 28.2% while in Cd contaminated soil the same treatment reduced the ELL value up to 45% (Fig. 2a).

Antioxidant enzyme analysis
Effect of Cd doses and organic amendments was observed signi cant (P < 0.05) on APX, catalase and MDA contents in leaves of maize. Analysis of data indicated APX production was higher in plants grown in Cd contaminated soil as compared to that in normal soil. Among the treatments, the control treatment resulted in signi cantly higher APX concentration (162.0 g − 1 fwt and 57.8 g − 1 fwt respectively in Cd contaminated and normal soil) in leaves of maize as compared to all other treatments (Fig. 2b) Cd contaminated soil, the values of APX were 128.2 g − 1 fwt and 129.5 g − 1 fwt, respectively due to application of MtPBR + BGR and MtPBR + BGR + PM (Fig. 2b). The application of MtPBR as sole treatment gave APX value 132.5 g − 1 fwt in Cd contaminated soil and 33.5 g − 1 fwt in normal soil.
Catalase activity was recorded higher (80.9 g − 1 fwt and 70.8 g − 1 fwt, respectively under Cd stress and normal soil conditions) in control treatment as compared to all other treatments (Fig. 2c) The control treatment produced signi cantly higher contents of MDA i.e., 11.6 µM g − 1 fwt and 6.0 µM g − 1 fwt, respectively in leaves of plants grown under Cd stress and normal soil conditions (Fig. 2d)  3.2.6. Organic acid concentration in root exudates of maize (µg g -1 root fwt) Acetic and citric acid concentrations were recorded higher in root exudates of maize plants grown in Cdcontaminated soil as compared to that of normal soil (Fig. 2e & 2f). Among the treatments, the application of MtPBR strain Pantoea sp. WP-5 either alone or in combination with BGR and PM resulted in higher secretion of acetic and citric acid concentrations in root exudates of maize (Fig. 2e & 2f).
Application of MtPBR + BGR produced signi cantly (P < 0.05) higher amount of acetic acid i.e. 52.7 and 40.5 µg g − 1 root fresh weight, respectively in Cd contaminated and normal soil. The treatment MtPBR + BGR + PM produced acetic acid 51.7 µg g − 1 in Cd contaminated while 39.8 µg g − 1 root fresh weight in normal soil. The sole application of MtPBR produced acetic acid by 39.7 and 30.5 µg g − 1 root fresh weight in Cd and normal soils, respectively, which was higher than control treatment under both the soil conditions.
The treatment MtPBR + BGR gave signi cantly higher amount of citric acid i.e. 22.8 µg g − 1 root fresh weight in Cd contaminated soil and 17.5 µg g − 1 root fresh weight in normal soil as compared to all other treatments (Fig. 2e & 2f). The values of citric acid produced in the treatment MtPBR + BGR + PM were at par with that of the treatment MtPBR + BGR under both the soil conditions. The sole application of MtPBR produced 16.5 and 12.7 µg g − 1 root fresh weight citric acid in Cd contaminated and normal soil, respectively which were higher than that of the control treatment under both the soil conditions.

Cadmium concentration in shoots and roots (mg kg -1 dwt)
Analysis of data revealed that control treatment showed higher concentration of Cd i.e. 179.7 ± 1.5 mg kg − 1 dwt in Cd contaminated while 6.63 ± 0.5 mg kg − 1 dwt in normal soil in shoots of maize plants (Table 2). Poultry manure application either alone or with the BGR were the treatment after the control treatment that showed maximum concentration of Cd i.e. 135.3 mg kg − 1 dwt in Cd contaminated while 5.23 mg kg − 1 dwt in shoots of plants grown in normal soil. Minimum concentration of Cd i.e. 70.3 ± 1.2 and 0.50 ± 0.1 mg kg − 1 dwt of shoots in Cd contaminated and normal soils respectively, was noted due to application of MtPBR + BGR treatment. The treatment MtPBR + BGR + PM gave Cd concentration 103.7 ± 1 mg kg − 1 dwt of shoot in Cd contaminated soil while 1.33 ± 0.2 mg kg − 1 dwt in normal soil ( Table 2).
The sole application of MtPBR gave Cd concentration 108.3 ± 0.5 mg kg − 1 dwt in Cd contaminated while 2.33 ± 0.5 mg kg − 1 dwt of shoot in normal soils.
The MtPBR + BGR treatment showed higher concentration of Cd in roots i.e. 385 ± 2.6 mg kg − 1 dwt in Cd contaminated while16.07 ± 1 mg kg − 1 dwt in normal soil ( Table 2) . Correlation analysis indicated that the signi cant negative correlation (R 2 = -0.9 and − 0.8 respectively, in Cd contaminated and normal soil; P < 0.05) exist between the organic acid concentration in root exudates and Cd concentration in shoots of maize. Progressive reduction in TF factor of Cd in maize when exposed to MtPBR + BGR treatment was observed in our study and has been reported  Application of PM resulted in decreased plant growth and dry matter yield in our study while Shumba et al. (2014) reported an increase in dry matter yield of maize due to PM application in sandy soil. This might be due to higher translocation of Cd to shoot by the PM, which inhibits photosynthetic activity and thus reduced the dry weight of maize plant. We have tried to nd the role of bacterial produced organic acids in Cd concentration in roots and its translocation to shoot of maize. Positive correlation between organic acid contents in root exudates and Cd uptake in roots while the negative in case of shoots strengthen our hypothesis that organic acid production in the root exudates of maize remediate Cd in soil.
Positive relationship of organic acids in root exudates and Cd uptake by roots has already been reported (Najeeb et al., 2011).

Conclusions
It is concluded that the application of organic acid producing MtPBR either alone or in combination with BGR ameliorate Cd contamination in soil through stabilization in maize roots and restrict translocation to shoot, which improve maize biomass and physiology. Therefore, it is conferred that this technique may be used to grow maize in Cd contaminated soil without compromising its biomass yield and quality.
Further, the application of PM alone or in combination with BGR increased the Cd translocation to shoot. It is obvious from results that organic acid production in maize root exudates is responsible for stabilization of Cd in roots and translocation to shoot. The high production of organic acids in response to MtPBR + BGR increased whereas low production in response to PM decreased Cd concentration in root.
Based on our ndings, we can recommend combined application of MtPBR + BGR for improving maize biomass and phyto-management of Cd contaminated soil.   Effect of Pantoea sp. strain WP-5 and organic amendments on translocation factor from root to shoot in maize. Values are the means (±SE) of ve replicates (n=5).