Combined use of Enterobacter sp. MN17 and zeolite reverts the adverse effects of cadmium on growth, physiology and antioxidant activity of Brassica napus

The objective of the study was to evaluate role of zeolite and Enterobacter sp. MN17 on Cd uptake, growth, physiological and biochemical responses of Brassica napus on Cd-contaminated soil. A sandy clay loam soil in plastic pots was spiked with Cd (0 and 80 mg kg-1) and amended with zeolite (0 and 10 g kg-1). Seeds of B. napus were inoculated with Enterobacter sp. MN17. Both inoculated and non-inoculated seeds of B. napus were sown and plants were harvested after 60 days of growth and data were collected. Although sole application of zeolite and seed inoculation reverted adverse effects of Cd in B. napus plants, the combined use resulted in even higher growth and physiological responses compared to control plants. The combined use under Cd stress increased plant height, root length, dry biomass of shoot and root up to 32%, 57%, 42% and 64%, respectively compared to control. The different physiological attributes (photosynthetic rate, chlorophyll content, transpiration rate, stomatal conductance) of B. napus were improved from 6% to 137%. Moreover, combined use of zeolite and seed inoculation on Cd-contaminated soil reduced the stress to plants as antioxidant activities decreased up to 25–64%, however enzyme activities were still higher than plants grown on normal soil. Root and shoot analysis of B. napus for Cd content depicted that zeolite and bacterium decreased Cd uptake from soil. It is concluded that combined use of zeolite and strain MN17 reduces Cd uptake from soil and improves physiological and biochemical responses of B. napus which is helpful to alleviate Cd toxicity to plants.

Introduction Heavy metal pollution of water and soil resources is an emergent issue of the modern industrial world. Among heavy metals, cadmium (Cd) is considered as one of the most toxic, nonessential element. It enters into soils through anthropogenic activities, atmospheric deposition, volcanic eruptions and weathering of parent materials [1,2,3]. The accumulation of Cd in soil deteriorates its quality, affects soil microbes and interferes uptake of plant essential elements [4,5,6], thus seriously limiting crop productivity. Cadmium can cause several morpho-physiological and biochemical disorders in plants [7]. Moreover, food chain contamination with Cd threatens human health [8,9,10].
This situation demands the development of suitable strategy to remediate Cd-contaminated soils. Various approaches such as physical, chemical and/or biological have been employed to remediate such soils [11,12,13]. Physical methods involve replacement of contaminated soils with healthy soils. This technology is large in working volume, costly and is suitable for soil with small area and polluted severely [14]. Among the chemical approaches, different extractants and leachants have been used to remediate Cd-contaminated soils [15]. The leaching and washing of soil for Cd removal has low scope as it causes loss of soil nutrients, ground and surface water pollution. Moreover, success highly depends on soil properties. Hence, it is necessary to develop heavy metal remediation technologies having low cost and environment friendly. Under the scenario, biological remediation approach involving bioremediation, phytoremediation or combination is getting attention worldwide [16]. The main advantages over physico-chemical approaches include cost effectiveness, environment friendly nature, in situ remediation and produces little or no secondary residues [17,18]. The use of metal tolerant microbes and plant species in combination to remediate Cd-contaminated soils is getting popular worldwide. The bacteria associate with plant roots and enhance plant growth through multifarious mechanisms. Such microbes ameliorate heavy metal stress by minimizing their uptake through reduced bioavailability in rhizosphere [19,20]. Low bioavailability of metals in rhizospher favors better root proliferation and ultimately the plant growth. These metal resistant microbes may immobilize/ modify the availability of metals to plants by synthesis of exopolysaccharides (EPS), siderophores, acidification and/or metal phosphates [21]. The second main factor which may immobilize/ adsorb Cd in soil is the use of organic and inorganic amendments. One of the adsorbents is zeolite, a class of alumino-silicate minerals with a negative charge embedded in its porous structure and can adsorb multiple exchangeable cations [22]. The efficiency to adsorb cations depends on exchange capacity and porosity of a particular zeolite [22]. Due to this property such materials are known as molecular sieves and have been studied for Cd removal from aqueous solution/ wastewaters [23,24]. However, there is very limited work on investigating role of zeolite amendment in reducing plant Cd uptake and retrieval of adverse effects on plant growth [24,25]. Moreover, there are contrasting observations regarding Cd uptake from zeolite amended soils. For instance, Eshghi et al. [25] reported that zeolite reduced Cd accumulation in soybean plant, whereas Mollaei et al. [26] found non-significant increase in Cd uptake in spinach. So, there is need to do further investigations in understanding the role of zeolite on Cd uptake from Cd-contaminated soils. In addition, no information is available regarding physiological and biochemical responses of crops to zeolite application on Cd-contaminated soils. There is also need to investigate the role of using metal tolerant endophytic bacteria and zeolite together on Cd uptake, growth, physiological and biochemical responses of crops grown in Cd-contaminated soil.
It is hypothesized that combined use of metal-tolerant endophytic bacterium, Enterobacter sp. MN17, and zeolite may reduce plant Cd uptake from Cd-spiked soil and retrieve Cdinduced growth retardation and physiological disturbances of B. napus, a commonly used plant species in phytoremediation studies.

Endophytic bacterium and experimental soil
The previously isolated endophytic bacterium, Enterobacter sp. MN17 (accession number KT375575), was obtained from the Environmental Sciences Laboratory of Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Pakistan. The soil used in study was collected from research field of Institute of Soil and Environmental Sciences, University of Agriculture Faisalabad, Pakistan (31.439052˚N, 73.069335˚E). A subsample of sieved soil was analyzed for various physico-chemical properties (Table 1). Particle size analysis was done using hydrometer method of Gee and Bauder [27]. The obtained values of soil particles (% clay, % silt, and % sand) were plotted against a textural triangle to get the soil textural class. Saturation percentage (SP) of the soil was determined by following the method of Richard [28]; SP ¼ Wt:of soil paste ðgÞ À Wt:of dry soil paste ðgÞ=Wt:of oven dry soil paste ðgÞ The pH of the soil was measured in a saturated soil paste using pH meter (Kent Eil 7015, UK) [28]. Electrical conductivity of the saturated soil paste extract measured by conductivity meter (Jenway 4070, UK) [28]. Organic matter was estimated by following the method described by Moodie et al. [29]. Total nitrogen was analyzed by Ginning and Hibbard's method and Kjeldahl's apparatus was used for digestion [29]. Soil plant available phosphorus content was determined by sodium bicarbonate (NaHCO 3 ) extraction method [30]. Soil plant available potassium was estimated through analysis on flame photometer (Jenway PFP7, UK) after extraction by ammonium acetate (NH 4 C 2 H 3 O 2 ) [31]. Available Cd concentration in soil was extracted with diethylene triamine penta acetic acid (DTPA), and Cd in the extract was directly measured using atomic absorption spectrometer (PerkinElmer, AAnalyst 100, Waltham, USA).

Heavy metal tolerance
Cadmium resistance of the bacterium MN17 was determined following the method of Ahmad et al. [32]. Cadmium nitrate salt was used to develop various levels of Cd (0, 40, 80, 120, 160 and 200 mg mL -1 ) in tryptic soy agar (TSA) plates. The bacterial growth was checked on TSA plates in triplicate. For this purpose, 100 μl inoculum having optical density of 0.50 at 600 nm was poured on to TSA agar plates and the inoculated plates were incubated at 30˚C in dark. For strain MN17, the lowest concentration (120 mg mL -1 ) that inhibited visible growth at incubated temperature within 5 days was determined.

Seed inoculation
The pure culture of strain MN17 was inoculated into 100 mL of sterilized tryptic soy broth (TSB) medium taken in a 250 mL Erlenmeyer flask and placed on an orbital shaker (Firstek Scientific, Tokyo, Japan) at 120 rpm for 24 hours at 28˚C. The culture was harvested by centrifugation at 6000 rpm at 21˚C for 20 minutes. The population of bacteria was established by turbid metric method and optical density (O.D) was maintained to about 10 8 cells mL -1 broth (OD~660 = 0.08) [33]. Seeds of B. napus were surface sterilized with 5% sodium hypochlorite solution for 5 minutes and washed thrice with distilled water to remove residues of sterilizing agent [34]. Seeds were inoculated with the broth mixed with 10% sugar solution, peat and clay mixture. Peat to clay ratio was used as 1:1 w/w [35]. The seeds were shaken well till fine coating appeared on seeds. Control was treated with sterilized peat plus clay mixed with sterilized broth and sugar solution. Inoculated seeds were placed over night for drying under laboratory conditions.

Plant experiment
A pot experiment was conducted in wirehouse to investigate the efficacy of seed inoculation with Enterobacter sp. MN17 and zeolite on plant growth, physiology and reduce uptake of Cd in B. napus in Cd contaminated soil. The soil was air dried, ground and, after passing through a 2 mm sieve, it was thoroughly mixed. Each pot was filled with 8 kg sieved soil. Soil in plastic pots was spiked with Cd (0 and 80 mg kg -1 ) two weeks before seeds sowing, and amended with zeolite (0 and 10 g kg -1 ). To develop desired Cd level in soil, a stock solution of 5000 mg Cd L -1 was prepared in distilled water using cadmium nitrate. The volume of distilled water to fully saturate the soil taken in pots was calculated and 640 mg Cd was added into it from stock solution. The seeds of B. napus were very kindly provided by Ayub Agriculture Research Institute (AARI) Faisalabad, Pakistan. Five seeds (inoculated or non-inoculated) were sown in each pot and the seedlings were thinned to 2 plants per pot on germination. The treatments were applied according to completely randomized design (CRD)-factorial arrangements with three replicates. Recommended doses of nitrogen, phosphorus (P 2 O 5 ) and potassium (K 2 O) were applied at the rate of 90, 60 and 75 kg ha -1 , respectively as urea, single super phosphate (SSP) and sulfate of potash (SOP) during sowing time [36]. The quantity of N added through cadmium nitrate in Cd-spiked soils was subtracted from total N per pot and remaining quantity was added through urea. Soil in pots was maintained at about field capacity during the experiment by daily observations. All the standard agronomic as well as insect and pest management strategies were adopted for this pot experiment. Plants were harvested after 60 days and plant growth parameters such as plant height, root length, root and shoot dry biomass were recorded. Plant height was taken from the soil surface to the tip of plant by meter rod and average was taken. After harvesting root and shoot samples were placed in an oven for drying at 65˚C for 72 hours to determine their dry weights.

Measurement of physiological and biochemical attributes
Physiological attributes. Plant physiological parameters such as photosynthetic rate (A), transpiration rate (E) and stomatal conductance (gs) of the leaves of B. napus were measured using infra-red gas analyzer (model LCA-4, Germany). Whereas, chlorophyll contents (SPAD) were measured by using chlorophyll meter (SPAD 502, Minolta, Japan).
Relative water content and electrolyte leakage. Relative water content (RWC) of plant leaves (one cm 2 piece without midrib) were determined according to method described by Mayak et al. [37].
Electrolyte leakage (EL) was determined following the method of Lutts et al. [38]. Plant leaf (one cm 2 piece without midrib) was placed in test tubes containing 10 mL of distilled water and recorded the electrical conductivity, EC 1 with EC meter. The tubes were placed on shaker for 2 hours and recorded the EC 2 . Then the tubes were autoclaved at 120˚C and after cooling EC 3 were recorded.

Measurement of Cd concentration in soil and plant tissues
For determining DTPA extractable Cd content in soils receiving different treatments, 10 g air dried soil sample was suspended in 20 mL DTPA extractant solution, suspension was shaken for 15 min at 200 rpm on reciprocal shaker [45]. Samples were filtered through Whatman No. 42 filter paper. Cadmium in extract was directly measured using atomic absorption spectrometer (PerkinElmer, AAnalyst 100, Waltham, USA). The shoot and root samples were dried in an oven at 65˚C to a constant weight, ground in Wiley mill and 0.5 g of ground material was digested using HNO 3 : HCLO 4 (3:1) mixture. After digestion, the contents in flasks were cooled, filtered properly and stored in washed plastic bottles for subsequent analysis of Cd on atomic absorption spectrometer following the method described by Yong et al. [46].

Statistical analysis
The collected data were analyzed statistically by employing the Fisher's analysis of variance technique [47], and treatment's mean were compared by using LSD test at 5% probability level.

Heavy metal tolerance of Enterobacter sp. MN17
The tolerance of endophytic bacterium, Enterobacter sp. MN17, to Cd was estimated in TSA medium ( Table 2). The Enterobacter sp. strain MN17 was able to survive and grow well on TSA medium up to 120 mg Cd mL -1 , however, no bacterial growth was observed at 200 mg Cd mL -1 .

Plant growth parameters
The individual and combined effect of zeolite and Enterobacter sp. MN17 on growth attributes of B. napus in normal and Cd contaminated soil is shown in Table 3. The plant growth (plant height, root length, shoot dry biomass and root dry biomass) significantly (p<0.05) decreased in Cd-contaminated soil than normal soil. Application of zeolite and Enterobacter sp. MN17 individually as well as in combination retrieved Cd-induced growth suppression of B. napus. However, better growth retrieval response of plants was observed on their combined application, even better growth was observed than control (no Cd, no zeolite and no inoculation).
Zeolite and strain MN17 also significantly (p<0.05) improved growth on normal soil. The combined application of zeolite and Enterobacter sp. MN17 increased plant height up to 29% and 32%, root length up to 54% and 57%, dry biomass of shoot up to 34% and 42%, and dry biomass of root up to 51% and 64% in normal and Cd contaminated soil, respectively. In general, zeolite and bacterium improved the growth more on Cd contaminated soil than normal soil as higher percent increases in different growth attributes were observed.

Physiological parameters
Like growth attributes, photosynthetic rate (A), transpiration rate (E), stomatal conductance (g s ) and SPAD chlorophyll content of leaves of B. napus were modified by Cd stress (

Water relations
The RWC of plant leaves and EL due to membrane injury significantly (p<0.05) varied between B. napus plants grown on normal and Cd contaminated soils. In control plants (no amendment), a decrease in RWC up to 52% and 43% (Fig 1) and increase in EL up to 7.5 and 8.7% (Fig 2) was observed in normal and Cd contaminated soil, respectively. Compared to control treatment, the application of zeolite and Enterobacter sp. MN17 improved RWC of 59-74%, and decreased EL of 6.9-7.8%, respectively on normal and Cd-contaminated soils. The application of zeolite and Enterobacter sp. MN17 improved the RWC to 83.0 and 71.1% and EL to 5.1 and 6.4% in normal and Cd contaminated soil, respectively. The use of zeolite and Enterobacter sp. MN17 together produced higher response in terms of RWC and lower in terms of EL in Cd contaminated than normal soil.

Antioxidant activities
In Cd stress environment, the activity of antioxidants including CAT, APX, SOD, GR, GPX and GST was modified, and it increased from 22

Discussion
This study evaluates the potential of amending soil with zeolite, an inorganic adsorbent, and seed inoculation with endophytic metal tolerant bacterium, Enterobacter sp. MN17, to mitigate the Cd induced changes in growth, physiology and antioxidant activity of B. napus. Moreover, potential of zeolite and strain MN17 to immobilize Cd in soil with reduced plant uptake has been investigated.
The strain MN17 was found as Cd tolerant bacterium as it can grow in the presence of Cd up to 120 mg mL -1 on TSA medium. This endophytic bacterium is more tolerant to Cd than other Cd-tolerant bacteria, Pseudomonas Serratia sp. LRE07, Pseudomonas aeruginosa ZM-130, Micrococcus sp. MU1, Klebsiella sp. BAM1 and Bacillus sp. L14 [48][49][50][51]. High tolerance to Cd makes this bacterium a suitable candidate to be used on soils contaminated with Cd to induce metal tolerance in crop plants. Results of the greenhouse study depicted that growth of B. napus was significantly (p<0.05) reduced on Cd-spiked soil than normal soil (Table 3). These results are in accordance with previous reports on B. napus grown on Cd-contaminated soils [52][53][54]. Cadmium reduces growth of plants by interfering with nutrient absorption through roots by occupying exchange sites, reduce microbial growth and activities, production of reactive oxygen species (ROS) and physiological disturbances [55][56][57]54]. Along with significant reduction in growth of B. napus, Cd stress reduced photosynthetic rate, chlorophyll content and gas exchange characteristics in leaves of 60-day old B. napus plants, whereas electrolyte leakage was increased (Table 4; Fig 2). Cadmium affects rate of photosynthesis by disturbing the function of enzymes involved in the photosynthesis and chlorophyll synthesis [58,59]. Electrolyte leakage accompanies plant response to stresses and is related to potassium ion efflux from plant cells [60]. The other modification in normal response of B. napus was increased antioxidant activities (CAT, APX, SOD, GR, GPX and GST) ( Table 5). The signals of stress trigger the production of different antioxidants to scavenge ROS [54]. The modification of biochemical response of B. napus in the presence of Cd has already been observed by others [61,62]. Moreover, it was found that plants grown on Cd-spiked soils had high Cd content in root and shoot of B. napus ( Table 6). The high uptake of Cd in root and shoot was associated with more availability of Cd in spiked soil.
Application of zeolite improved plant growth, physiological and biochemical responses of B. napus under Cd stress. There are scientific reports on mitigating Cd-induced growth retardation of soybean and spinach by the application of zeolite [25,26], however no information is available regarding usefulness of zeolite application on ameliorating Cd-induced growth inhibition of B. napus. This growth retrieval response of B. napus to zeolite application might have occurred due to decrease in Cd uptake from the plant rhizosphere. Zeolite is a strong adsorbent of metals and has more binding capacity due to its negatively charged ions [63]. Moreover, absorption and retention capacity for major nutrients released from fertilizer and maintenance of adequate water supply might be the reason behind this increase [25]. Zeolite might also improve B. napus growth by excluding Cd interferences on absorption of other micronutrients. In contrast to our findings Mollaei et al. [26] observed slight increase in Cd uptake in spinach by zeolite application. We also found that zeolite application improved photosynthetic rate, chlorophyll content, stomatal conductance and transpiration rate in both normal and Cd-contaminated soils (Table 4). Recently, De Smedt et al. [64] also observed beneficial effect of foliar application of zeolites on physiological responses of apple trees. Furthermore, it was found that zeolite application decreased antioxidant activities in B. napus leaves under Cd stress (Table 5). This shows that zeolite amendment has ameliorated the Cdstress to plant; probably this occurred due to phenomena of immobilization of Cd in rhizosphere. Nozari et al. [65] observed that zeolite application alleviates water stress-induced antioxidant activities in soybean. However, modifications in physiological responses and antioxidant activities of B. napus by zeolite application in Cd-contaminated soils have not been studied so far.
Although, toxic effects of Cd on soil micro-biota are well-recognized as Cd negatively affects soil biota and cease their activity [66,67]. But our findings clearly demonstrated the improvement in growth of B. napus in pot conditions by seed inoculation with plant growth promoting Cd-tolerant endophytic bacterium (Enterobacter sp. MN17) both on normal and Cd-contaminated soils. Plant growth promoting endophytic bacteria have also been tested for improving growth of different crops in Cd-contaminated soils [68,69]. However, there is no report of inducing Cd-resistance to B. napus through endophytic bacteria. The bacterium, strain MN17, used in this study carried a number of plant growth promoting traits such as production of auxins, cytokinins, gibberellins, siderophores, ACC-deaminase activity, inorganic P solubilization, N fixation and exopolysaccharide (EPS) production [34,70]. These traits might have helped the plants to perform better in normal and Cd-contaminated soils. Like zeolite, seed inoculation with Enterobacter sp. MN17 reduced the uptake of Cd in B. napus tissues. The EPS producing biofilm forming bacteria are popular to adsorb metals and reduce their absorption through roots of crop plants [71]. The reduced uptake of Cd might have resulted in better physiological and biochemical response of B. napus, which ultimately reduced Cdinduced growth retardation. The improvement in physiological attributes might be due to the fact that microbes helped to improve the nutrients uptake which further upsurge the chlorophyll from photo-oxidative destruction [72]. Increased chlorophyll contents in leaves may have a positive effect on plant photosynthesis and growth under Cd stress condition.
The combined use of zeolite and bacterial strain MN17 was found superior in improving growth, physiological and biochemical responses of B. napus grown on normal and Cd contaminated soils. The response was the additive effect of zeolite and seed inoculation with strain MN17. Zeolite might have immobilized the Cd thus rendering them unavailable for the plant uptake, whereas in addition to immobilizing Cd, bacterium might have increased the availability of mineral nutrients to growing plants and improved the root proliferation. Previously, there is no information available in the literature regarding efficacy of combined application of zeolite and Cd-tolerant endophytic bacterium in inducing Cd-resistance to B. napus,

Conclusions
In conclusion, application of zeolite and Enterobacter sp. MN17 significantly enhanced the plant growth, improved physiological and biochemical responses of B. napus in normal and Cd contaminated soils. Moreover, both zeolite amendment and seed inoculation with strain MN17 decreased Cd uptake in root and shoot of B. napus. However, combined application of zeolite and Enterobacter sp. MN17 was found more effective in improving Brassica napus plant height, root length, dry biomass of root and shoot, chlorophyll content, photosynthetic rate, transpiration rate and stomatal conductance in Cd contaminated soil. The antioxidant activity of CAT, APX, SOD, GR, GPX and GST were decreased. Likewise, zeolite and strain MN17 together reduced Cd uptake in root and shoot of B. napus on Cd-contaminated soil.