Synergistic Effect of Bacillus thuringiensis IAGS 199 and Putrescine on Alleviating Cadmium-Induced Phytotoxicity in Capsicum annum

Plant growth-promoting bacteria (PGPB) and putrescine (Put) have shown a promising role in the mitigation of abiotic stresses in plants. The present study was anticipated to elucidate the potential of Bacillus thuringiensis IAGS 199 and Put in mitigation of cadmium (Cd)-induced toxicity in Capsicum annum. Cadmium toxicity decreased growth, photosynthetic rate, gas exchange attributes and activity of antioxidant enzymes in C. annum seedlings. Moreover, higher levels of protein and non-protein bound thiols besides increased Cd contents were also observed in Cd-stressed plants. B. thuringiensis IAGS 199 and Put, alone or in combination, reduced electrolyte leakage (EL), hydrogen peroxide (H2O2) and malondialdehyde (MDA) level in treated plants. Synergistic effect of B. thuringiensis IAGS 199 and Put significantly enhanced the activity of stress-responsive enzymes including peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD). Furthermore, Put and microbial interaction enhanced the amount of proline, soluble sugars, and total soluble proteins in C. annum plants grown in Cd-contaminated soil. Data obtained during the current study advocates that application of B. thuringiensis IAGS 199 and Put establish a synergistic role in the mitigation of Cd-induced stress through modulating physiochemical features of C. annum plants.

reported that PGPB reduce metal uptake, phytotoxicity and enhance growth and biomass production in inoculated plants. Furthermore, the PGPB capable of inducing metal precipitation, bioaccumulation, chelation and complexation diminish phytotoxic effects in supplemented plants [36].
Keeping this in view, it was hypothesized that seed priming and subsequent application of B. thuringiensis IAGS 199 may improve the growth of C. annum plants exposed to Cd stress. The research was designed to evaluate the potential of B. thuringiensis IAGS 199, alone or in combination with Put, in the alleviation of Cd stress in C. annum seedlings. Furthermore, the effect of Put and B. thuringiensis IAGS 199 on the synthesis of metal-chelating biochemicals such as protein and non-protein bound thiols and total thiols were also evaluated. The use of biostimulants reduces the need for fertilizers [37][38][39]. Foliar application of fertilizer and micronutrient could be a better alternative to fast action [40,41].

Cadmium Immobilization and Putrescine Concentration in Solution
As shown in Figure 1, water-soluble Cd concentration did not differ significantly in the absentia of B. thuringiensis IAGS 199 strain. However, water-soluble Cd concentration reduced significantly in the presence of a microbe. The pH ranged from 6.24 to 6.85 in the presence as well as absentia of the strain. Figure 2 explains that Put concentration elevated in the presence of a microbe. B. thuringiensis IAGS 199 strain produced Put. After 7 days, the peak value of Put was 59 mg L −1 . Moreover, Cd content on the cell surface of B. thuringiensis IAGS 199 strain increased with time, ranging from 0.03 mg g −1 to 0.09 mg g −1 between 1 and 7 days of incubation. Intracellular Cd content also varied from 0.10-0.17 mg g −1 .

Analysis of Growth Parameters
The results showed that Cd toxicity significantly reduced germination percentage, root fresh weight, shoot fresh weight, root dry weight and shoot dry weight by 43.85%, 86.56%, 58.73%, 40.8% and 55.47%, respectively, as compared to control treatment. Application of B. thuringiensis IAGS 199 and Put enhanced growth attributes in normal and Cd-contaminated soil. In case of C. annum seedlings grown in Cd-contaminated soil, the combined application of B. thuringiensis IAGS 199 and Put significantly enhanced growth attributes of C. annum seedlings as compared to individual treatment of B. thuringiensis IAGS 199 and Put (Table 1).

Quantification of Chlorophyll Contents
Cadmium stress reduced Chl content in C. annum seedlings as compared to control. Individual treatments of B. thuringiensis IAGS 199 and Put enhanced Chl a, Chl b and total chlorophyll content in C. annum seedlings. Nevertheless, co-application of B. thuringiensis IAGS 199 and Put significantly enhanced Chl contents in C. annum seedlings under Cd contaminated conditions ( Table 2).

Analysis of Growth Parameters
The results showed that Cd toxicity significantly reduced germination percentage, root fresh weight, shoot fresh weight, root dry weight and shoot dry weight by 43.85%, 86.56%, 58.73%, 40.8% and 55.47%, respectively, as compared to control treatment. Application of B. thuringiensis IAGS 199 and Put enhanced growth attributes in normal and Cd-contaminated soil. In case of C. annum seedlings grown in Cd-contaminated soil, the combined application of B. thuringiensis IAGS 199 and Put significantly enhanced growth attributes of C. annum seedlings as compared to individual treatment of B. thuringiensis IAGS 199 and Put (Table 1).

Quantification of Chlorophyll Contents
Cadmium stress reduced Chl content in C. annum seedlings as compared to control. Individual treatments of B. thuringiensis IAGS 199 and Put enhanced Chl a, Chl b and total chlorophyll content in C. annum seedlings. Nevertheless, co-application of B. thuringiensis IAGS 199 and Put significantly enhanced Chl contents in C. annum seedlings under Cd contaminated conditions (Table 2).

Determination of Photosynthetic Rate and Gas-Exchange Parameters
Cadmium stress decreased photosynthetic rate in C. annum plants by 28%, as compared to control. Application of B. thuringiensis IAGS 199 and Put enhanced photosynthetic rate in normal and Cd-polluted soil, as compared with control and Cd-treatment, respectively. However, the synergistic application of B. thuringiensis IAGS 199 and Put significantly enhanced the photosynthetic rate of C. annum plants, as compared to alone treatments of B. thuringiensis IAGS 199 and Put ( Figure 3).

Determination of Photosynthetic Rate and Gas-exchange Parameters
Cadmium stress decreased photosynthetic rate in C. annum plants by 28%, as compared to control. Application of B. thuringiensis IAGS 199 and Put enhanced photosynthetic rate in normal and Cd-polluted soil, as compared with control and Cd-treatment, respectively. However, the synergistic application of B. thuringiensis IAGS 199 and Put significantly enhanced the photosynthetic rate of C. annum plants, as compared to alone treatments of B. thuringiensis IAGS 199 and Put ( Figure 3).

Analysis of Lipid Peroxidation and Hydrogen Peroxide Content
Cadmium stress significantly enhanced MDA content (24.44%) in C. annum plants, in comparison with control treatment. Alone treatment of B. thuringiensis IAGS 199 and Put reduced MDA content in C. annum plants grown in normal and Cd-contaminated soil. Nevertheless, synergistic treatment of B. thuringiensis IAGS 199 and Put reduced MDA content in the case of C. annum seedlings grown in Cd-contaminated soil ( Figure 5).
Cadmium toxicity augmented H2O2 contents (18.75%) in C. annum plants, as compared to control treatment. In the case of C. annum seedlings grown in non-contaminated conditions, alone treatments of Put and B. thuringiensis IAGS 199 reduced H2O2 contents significantly as compared to Cdtreatment. In Cd-contaminated soil, synergistic application of Put and B. thuringiensis IAGS 199 significantly reduced H2O2 contents in comparison with alone treatments of Put and B. thuringiensis IAGS 199 ( Figure 5).

Determination of Total Soluble Protein and Soluble Sugars
Cadmium stress decreased total soluble proteins by 55.55%, as compared to control treatment. Individual treatments of Put and B. thuringiensis IAGS 199 elevated total soluble proteins content in C. annum seedlings grown in normal and Cd-polluted soil. Nevertheless, the co-application of Put and B. thuringiensis IAGS 199 significantly enhanced total soluble proteins under Cd-stress. Conversely, Cd stress enhanced the soluble sugar level in C. annum seedlings. Individual treatments of Put and B. thuringiensis IAGS 199 enhanced soluble sugar content in normal and Cd-polluted soil. The highest soluble sugar content was found when treated with B. thuringiensis IAGS 199 in normal soil ( Table 2).

Determination of Electrolyte Leakage (EL)
Cadmium stress significantly enhanced EL (38.70%) as compared to control treatment. B. thuringiensis IAGS 199 and Put reduced EL in C. annum plants grown in normal and Cd-toxic soil. Nevertheless, synergistic application of B. thuringiensis IAGS 199 and Put significantly reduced EL in C. annum plants grown in Cd-toxic soil ( Figure 6). Cadmium toxicity augmented H 2 O 2 contents (18.75%) in C. annum plants, as compared to control treatment. In the case of C. annum seedlings grown in non-contaminated conditions, alone treatments of Put and B. thuringiensis IAGS 199 reduced H 2 O 2 contents significantly as compared to Cd-treatment. In Cd-contaminated soil, synergistic application of Put and B. thuringiensis IAGS 199 significantly reduced H 2 O 2 contents in comparison with alone treatments of Put and B. thuringiensis IAGS 199 ( Figure 5).

Determination of Total Soluble Protein and Soluble Sugars
Cadmium stress decreased total soluble proteins by 55.55%, as compared to control treatment. Individual treatments of Put and B. thuringiensis IAGS 199 elevated total soluble proteins content in C. annum seedlings grown in normal and Cd-polluted soil. Nevertheless, the co-application of Put and B. thuringiensis IAGS 199 significantly enhanced total soluble proteins under Cd-stress. Conversely, Cd stress enhanced the soluble sugar level in C. annum seedlings. Individual treatments of Put and B. thuringiensis IAGS 199 enhanced soluble sugar content in normal and Cd-polluted soil. The highest soluble sugar content was found when treated with B. thuringiensis IAGS 199 in normal soil (Table 2).

Determination of Electrolyte Leakage (EL)
Cadmium stress significantly enhanced EL (38.70%) as compared to control treatment. B. thuringiensis IAGS 199 and Put reduced EL in C. annum plants grown in normal and Cd-toxic soil. Nevertheless, synergistic application of B. thuringiensis IAGS 199 and Put significantly reduced EL in C. annum plants grown in Cd-toxic soil ( Figure 6).

Determination Proline Contents
Cadmium stress enhanced the activity of proline content (59.09%) in C. annum plants as compared to control treatment. In the case of C. annum plants grown in Cd-toxic conditions, synergistic application of B. thuringiensis IAGS 199 and Put significantly enhanced proline levels in C. annum plants as compared to Cd-treatment ( Figure 9).

Determination Proline Contents
Cadmium stress enhanced the activity of proline content (59.09%) in C. annum plants as compared to control treatment. In the case of C. annum plants grown in Cd-toxic conditions, synergistic application of B. thuringiensis IAGS 199 and Put significantly enhanced proline levels in C. annum plants as compared to Cd-treatment ( Figure 9).

Determination Proline Contents
Cadmium stress enhanced the activity of proline content (59.09%) in C. annum plants as compared to control treatment. In the case of C. annum plants grown in Cd-toxic conditions, synergistic application of B. thuringiensis IAGS 199 and Put significantly enhanced proline levels in C. annum plants as compared to Cd-treatment ( Figure 9).  Table 3 shows that Cd content decreased in shoots of C. annum seedlings, as compared to Cd content in the root. Co-treatment of B. thuringiensis IAGS 199 and Put significantly decreased Cd contents in the shoot of C. annum seedlings, as compared to alone treatments of B. thuringiensis IAGS 199 and Put in Cd-contaminated conditions. In the case of C. annum seedlings grown in normal and Cd-contaminated conditions, alone treatment of B. thuringiensis IAGS 199 in normal conditions and combined application of B. thuringiensis IAGS 199 and Put in Cd-stressed conditions showed significantly higher value of MTI, respectively.

Determination of Metal Chelating Compounds
Cadmium stress enhanced total thiol content (54.16%) in C. annum seedlings as compared to control treatment. Alone application of Put and B. thuringiensis IAGS 199 significantly enhanced total thiol content in Cd-toxic soil, as compared to C. annum seedlings grown in non-contaminated soil. In the case of C. annum seedlings grown in Cd-contaminated soil, synergistic application of Put and B. thuringiensis IAGS 199 significantly enhanced total thiol content as compared to alone treatments of Put and B. thuringiensis IAGS 199. Moreover, non-protein thiol and protein thiol content was also significantly enhanced by the co-application of Put and B. thuringiensis IAGS 199 in Cd-contaminated soil, as compared to other treatments (Table 4).

Discussion
Put is an essential biogenic cationic polyamine in microbes, plants, and animals [42]. Beatriz et al. [43] reported that Lactococcus lactis is able to synthesize Put to induce alkalinization of the culture solution. During current research, B. thuringiensis IAGS 199 enhanced Put and increased pH yet reduced Cd concentration in the culture solution. It may be assumed that B. thuringiensis IAGS 199 immobilized Cd by enhancing Put synthesis and pH in the culture solution ( Figure 1). The Cd was adsorbed on the cell surface, intracellular and extracellular regions of B. thuringiensis IAGS 199 ( Figure 2). B. thuringiensis IAGS 199 enhanced Put synthesis causing immobilization of Cd in the solution (Figure 1). Bacteria tolerate metal toxicity through (1) sequestrating metal on their cell walls or by employing metallothioneins and phytochelatins which are intracellular metal-binding proteins and peptides (2) amending uptake paths thus hindering metal uptake; (3) converting toxic form of metal to a less toxic form by enzymatic activities; and (4) reducing the intracellular metal concentration through engaging a particular efflux scheme or through compartmentalization [44]. The PGPB-plant association affects the availability and uptake of Cd in plants through chelation, precipitation, bioaccumulation and complexation [36]. The bacterial strains capable to synthesize siderophores, auxin and ACCD improve growth in metal-stressed C. annum plants [45]. Jebara et al. [46] also revealed that PGPB synthesizing phytohormones such as auxin and capable to solubilize P and fix atmospheric N assist companion plants to improve growth and alleviate metal stress. The ACCD activity of PGPB maintains the synthesis of stress ethylene in plants under metal stress [47]. The arginine decarboxylase synthesizing PGPR induce numerous physiochemical changes in metal-stressed plants to alleviate respective stress [48]. B. thuringiensis IAGS 199 inoculated C. annum plants exhibited improved growth in comparison with the control in the non-contaminated and Cd spiked soils ( Figure 2). B. thuringiensis IAGS 199 revealed different plant growth-promoting attributes responsible for Cd stress alleviation and growth promotion of treated C. annum plants (Figure 4). Bacterial inoculation may induce enhanced Put synthesis, Cd immobilization and reduced Cd contents in crop plants [49,50]. Plants inoculated with B. thuringiensis IAGS 199 decreased the amount of soil available Cd ( Figure 5), reduced Cd uptake in plants as compared to the control ( Figure 4). Moreover, reduced translocation factor of B. thuringiensis IAGS 199 applied plants may be ascribed to the declined Cd accumulation in inoculated plants compared to the control (Table 1). B. thuringiensis IAGS 199 enhanced the concentration of Put and ADPB in soils, causing increased pH. The high pH results from the insolubilization of Cd by making hydroxides, organic complexes and carbonates precipitates [51].
Madhaiyan et al. [52] also reported reduced Ni and Cd bioavailability and uptake in tomato plants treated with Magnaporthe oryzae CBMB20 and Burkholderia sp. CBMB40, respectively. Similarly, Lin et al. [53] observed that Cd resistant B. megaterium H3 decreased Cd bioavailability, uptake and accumulation in rice plants. The metal-resistant PGPR reduce the bioavailability of metal through binding metal on their cell surfaces or accumulating in their cells (Ledin et al. 1999). The Cd precipitation, intracellular and extracellular adsorption accumulation by B. thuringiensis IAGS 199 (Figures 2 and 3), caused a decrease in bioavailable Cd in the soil. Metal-resistant PGPR may chelate metal ions through their siderophores and reduce the bioavailability of these metals [54,55]. B. thuringiensis IAGS 199 may have synthesized siderophores causing chelation and binding of Cd resulting decreased Cd bioavailability in soil and reduced Cd uptake and bioaccumulation in inoculated plants (Table 1, Figure 5). Furthermore, B. thuringiensis IAGS 199 enhanced root growth which ultimately increased synthesis of root exudates and concentration of organic matter in the rhizospheric soil. The higher concentration of organic matter content enhanced ADPB in B. thuringiensis IAGS 199 supplemented soils compared to the controls. The increased synthesis of polyamines assists plants to mitigate heavy metal stress [56,57]. The higher concentration of organic matter besides increased ADPB and Put in B. thuringiensis IAGS 199 inoculated rhizospheric soils may be responsible for improving the growth of C. annum plants and the immobilization of Cd in the Cd amended soil.
Polyamines (PAs) assist plants to alleviate stresses through modulation of physiochemical activities and reduction of toxic metals uptake [58]. Our current study reveals the role of Put and B. thuringiensis IAGS 199 in the alleviation of Cd-induced phytotoxicity in C. annum. The uptake and accretion of Cd impede cellular growth besides the activity of proton pumps causing variations in the growth configurations and physiological activities [59]. Nevertheless, Put-treated C. annum seedlings were capable to ameliorate Cd-induced stress and exhibited improved growth. Put application triggered an improvement in seed germination, root length, shoot length and biomass production in alfalfa [60]. Zhao et al. [61] demonstrated that γ-Aminobutyric acid (GABA) regulates lipid production and Cd uptake in plants. Since GABA induces synthesis of PAs. Therefore, it is assumed that increased biosynthesis of Put in response to GABA alleviated Cd-induced toxicity in C. annum plants through the reduction of lipid peroxidation. Some other researchers have also supported the metal stress mitigation capability of PAs in different plants [62].
Li et al. [6] reported that B. thuringiensis HC-2 reduced Cd content in radish roots. The results of our experiments also showed that B. thuringiensis IAGS 199, alone or in combination with Put, reduced Cd uptake in shoots of C. annum seedlings.
Equally, the reduced levels of protein contents were observed in Cd supplemented plants (Table 2), while an improved amount of amino acids and proline also appears to be a tactic of seed plants to alleviate Cd toxicity through scavenging a higher amount of ROS by employing osmoregulators. Put residues are a source of spermidine and spermine biosynthesis [63]. Spermine mitigates abiotic plant stress through modulation of glyoxalase system and antioxidative machinery [64]. Likewise, spermidine alleviates Cd phytotoxicity through intonation of nitrogen metabolism and antioxidative machinery [14]. It was detected that Put treatment enhanced photosynthetic pigments, growth parameters, soluble protein contents and proline contents in C. annum seedlings under Cd regimes.
The reduced level of electrolyte leakage and Cd content was perceived in Put applied C. annum seedlings. The improved level of growth attributes in Put-treated Cd-stressed seedlings may be a result of reduced uptake and translocation of Cd in plant tissues (Table 3). Potassium acts as an osmolyte and helps in the maintenance of membranous stability. Put maintains the dimensions of K + channel and porosity in guard cells and regulates transpiration in plants [65]. Catabolites of Put regularize uptake and translocation of Ca 2+ and K + , and therefore maintain bioenergetics of chloroplast and mitochondria under stress [66]. Rahdari et al. [67] also found that Put regularizes the biosynthesis of photosynthetic pigments in plants subjected to abiotic stresses. Higher K + ions help in the maintenance of membrane stability and reduce the level of electrolyte leakage in stressed seedlings [68]. By the same token, higher uptake of macronutrient cations like N, P and K perhaps restricted Cd uptake [69]. Higher K + ions altered the biosynthesis level of endogenous hormones and in return enhanced chlorophyll fluorescence [70] in Put-treated seedlings. Polyamines maintained water integrity, reduced Cd content in Put-treated seedlings confer beneficial role of this PA for LRWC. Improved membranous integrity and stress alleviation in PAs applied plants was reported by Groppa et al. [57]. Cadmium phytotoxicity adversely affected water relations and biomass production of subjected C. annum plants (Table 1). Metal toxicity reduces water uptake, transpiration, growth and biomass production in the stressed plant was reported by Rady et al. [71].
B. thuriengiensis is involved in the enhancement of Ca ++ , Mg ++ , Zn ++ , Mn ++ , and Cu ++ , and enhancement of nutritional content assisted in drought stress management in Lavandula angustifolia and Salvia divinorum [72]. Calcium is involved in membrane stabilization and Mg ++ accumulation in chloroplasts [73]. Current research also depicts the involvement of B. thuringiensis IAGS 199, alone or in combination with Put, in the enhancement of photosynthetic rate and pigment content in C. annum seedlings.
Li et al. [74] found that Put application assisted in membranous stability and standardization of the active oxygen biosynthesis; by this means, Put application defended plants from acid toxicity and improved their stress tolerance. A higher amount of EL was observed in Cd-stressed plants (Figure 4), confirming the involvement of Cd-induced phytotoxicity in the stability and permeability of the cellular membrane. Hassan et al. [75] demonstrated that exogenously applied Put alleviate plant stress through the protection of chloroplast and membranous structure. Our study also showed that Pu application, alone or in combination with B. thuringiensis IAGS 199, protected C. annum seedlings from Cd toxicity by stabilization of photosynthetic apparatus.
A higher level of osmoregulators including proline and soluble sugars and reduced level of protein content was observed in Cd-stressed C. annum seedlings. Put treatment further enhanced proline contents in Cd-stressed plants. Higher proline contents in Put applied plants confer linkage between PAs metabolism to proline biosynthesis [76,77]. Our results are analogous to Sharma and Dietz [78] who reported protein degradation resulting in an upgraded level of amino acids in stressed plants. Higher biosynthesis of proline helps in stress mitigation through chelation and metal detoxification, ROS scavenging, osmoregulation, enzymatic defense, and modulation of cytosolic acidity. Sun et al. [79] revealed that exogenous Put application declined MDA content while the enhanced amount of photosynthetic pigments, proline content and activity of antioxidant enzymes in Anthurium andraeanum under chilling stress. The increased synthesis of osmoregulators including soluble sugars, proline and free amino acids helps to alleviate drought stress and improve the growth of Put-treated plants [80]. In the same way, the increased amount of free amino acids and proline in Put applied seedlings assisted in the reduction of Cd-induced toxicity. Further enhancement of total free amino acids in Put-treated seedlings enabled plants to manage Cd stress in a better way.
B. subtilis improved Cd tolerance in Triticum aestivum through enhancing the activity of POD, CAT besides reducing the level of MDA [81]. In another study, Jan et al. [82] reported that B. cereus enhanced growth of Oryza sativa seedlings under Cd stress, due to enhanced activity of antioxidant enzymes and reduced EL. Current research also showed that B. thuringiensis IAGS 199, alone or in combination with Put, alleviated Cd toxicity through enhanced activity of antioxidative enzymes and reduction of EL.
The results of the current study demonstrated that Cd stress enhanced the amount of corresponding chelating biochemicals including protein and non-protein bound thiols as well as total thiols. The sulfhydryl groups of these chelating biochemicals successfully make compounds with metals causing a reduction in bioavailability and uptake of metals by plants [83]. Heavy metal transporting ATPases including Zip and Nramp enable the transport of phytochelatins bounded metal complexes in plant vacuoles [84]. Transformation, immobilization and mobilization of metal ions decrease metal uptake and toxicity in PGPB assisted plants by bio-accumulating phenomenon comprising sequestration, biosorption and bioexclusion, complexation and exclusion, with metal-binding proteins [85]. The amino acids and organic acids present in root exudates engage ligands including MTs and phytochelatins in the process of chelation and precipitation to influence bioavailability, solubilization and mobilization of metal ions in growing media [86,87]. The results of current research are in agreement with the findings of Aly and Mohamed [88] in Zea mays plants growing under copper regimes. Nagalakshmi and Prasad [89] also observed that higher Cu contents induced modulation in glutathione synthesis resulting in binding and sequester of Cu in stressed Scenedesmus bijugatus. Awasthi et al. [90] reported that thiol metabolism comprising phytochelatins, glutathione, protein and non-protein thiols (NBTs) make metal compounds, reduce metal uptake and sequester it in plant vacuoles resulting in the mitigation of metal toxicity in plants. Additionally, NBT persuades antioxidative activities in plants. Cysteine synthesized during sulphur assimilation of thiol metabolism improves biosynthesis of glutathione and phytochelatins. Awasthi et al. [91] reported metal complexation in tissues resulted in a higher level of phytochelatins, glutathione, and NBTs in P. putida inoculated rice plants under As stress. Hassan and Bano [92] observed that exogenous Put enhanced uptake and bioaccumulation of Mg, K and Ca in plants subjected to salinity stress. The improved uptake of essential nutrients may have enhanced the synthesis of thiols and successively increased biosynthesis of metal chelating biochemical in microbe-assisted plants.

Procurement of Capsicum annum L. Germplasm and Growth Conditions
The experiment was conducted in the Department of Botany, University of Narowal, Narowal with temperature 23 ± 4 • C, humidity: 70-76% and light: 500-550 mmol m −2 s −1 light. Seeds of the commonly cultivated Capsicum annuum var. fasciculatum were obtained from Punjab Seed Corporation Pakistan and were used as per requirement of the study. The obtained C. annum seeds with initial moisture contents of 9.8% were sterilized through submerging in sodium hypochlorite solution (0.5%) for 3 min followed by washing thoroughly with distilled water [93]. These seeds were primed with 1 mM Put at 25 • C for 15 h under dark conditions. Seeds were dried by placing over blotting paper at 25 • C for 24 h before sowing in pots.
The soil used for the present study was obtained from Botanical Garden, University of the Narowal. Soil was autoclaved at 121 • C and 1.5 bars. Soil contents were measured before sterilization. Soil was unified by mixing it thoroughly and for pot experiment contained 0.35 g Potassium, 0.06 mg cadmium, 0.35 mg chromium, 2.7 g organic matter and 1.76 g nitrogen. Subsequently, soil was contaminated with Cd (50 mg kg −1 ) and 2 kg soil was filled in polystyrene pots. For this purpose, cadmium chloride (CdCl 2 ) was thoroughly mixed in the soil. The Cd amended and non-spiked soil samples were placed in the shade for 15 days. Pots were placed under greenhouse conditions. Five seeds were sown in each pot. Soil samples were filled in plastic pots (6 × 5 ). Pots were provided 50 mL full strength Hogland's solution alone or contaminated with Cd during each alternative day [94]. Pots that received full-strength Hoagland's solution were regarded as control. Plants were watered on alternate days. Distilled sterilizer water was used for watering plants. After one month of sowing, seedlings were harvested carefully and were submerged in 20 mM EDTA for 15 min to eradicate adsorbed metal ions. Plant samples were frozen by applying liquid-N and stored at −80 • C for the upcoming physiochemical examination.

Procurement and Characteristics of Bacterial Agent
B. thuringiensis IAGS 199 was obtained from the bacterial conservatory, University of the Punjab, Lahore. Indole acetic acid synthesis capability of microbe was evaluated with the help of the method described by Glickmann and Dessaux [95]. For this purpose, B. thuringiensis IAGS 199 was grown in LB medium having tryptophan (500 mg) for 1 d. Afterwards, culture was centrifuged and 1 mL supernatant was mixed with 50 mL orthophosphoric acid (10 mM) and 2 mL of Salkowski's reagent for 0.5 h. The absorbance value was determined at 530 nm and was calibrated with a known standard value of IAA. As far as IAA production capability of microbe is concerned, B. thuringiensis IAGS 199 was capable to synthesize IAA (13 µg mL −1 ).
Cadmium tolerance was examined by inoculating 10 mL of B. thuringiensis IAGS 199 inoculum on LB agar plates supplemented with 0, 50, 75, 100 mg kg −1 Cd at 25 • C for 4 d. The plates without Cd showed 5 mm bacterial colony. Bacterial growth was not observed above 75 mg kg −1 Cd.
A pilot experiment was also conducted to evaluate the effectiveness of seed priming with different concentrations of Put (0.25, 0.5, 1, 1.5 mM Put) in the alleviation of 50 mg kg −1 Cd concentration in C. annum seedlings. The survival percentage of C. annum seedlings increased significantly in C. annum seedlings treated with 1 mM Put. The survival percentage of C. annum seedlings treated with 0.25, 0.5, 1, 1.5 mM Put-treated seedlings were 65%, 58%, 84%, 72%, respectively, under 50 mg kg −1 Cd concentration.

Effect of B. thuringiensis IAGS 199 on Concentration of Water Soluble Cd in Solution
Method of Chen [96] was adopted to analyze the immobility characterization of water-soluble Cd in B. thuringiensis IAGS 199 solution besides the concentration of water-soluble Cd and Put concentration in the solution [96]. B. thuringiensis IAGS 199 was inoculated in culture flasks containing 1.0 mg L −1 Cd 2+ supplemented LB media. The un-inoculated 1.0 mg L −1 Cd 2+ supplemented LB media was regarded as control. Treated flasks were placed at 28 • C for 0, 1, 3, 5 and 7 d. Afterwards, B. thuringiensis IAGS 199 growth from culture solution was measured. The pH of the B. thuringiensis IAGS 199 culture solution was also observed by using pH meter. The B. thuringiensis IAGS 199 culture solution was subjected to centrifugation for 5 min at 12,000 rpm. The amount of Cd present in the supernatant was measured by using inductively coupled plasma-optical emission spectrometer.

Assessment of Put in the Culture Solution
The method adopted by Ebeed et al. [97] with a slight amendment was employed for Put estimation. A 4-mL sample from all aforementioned flasks was subjected to centrifugation for 10 min at 10,000 rpm at 4 • C. B. thuringiensis IAGS 199 cells were removed and the supernatant was passed through a 0.22 µm filter paper. The 2 mL filtrate was mixed with 2 mL of cold perchloric acid (10% v/v) before keeping at 4 • C for 60 min. The aliquot of the supernatant was vortexed with 15 mL of benzoyl chloride along with 2 mL of 2 mol L −1 NaOH and placed at 38 • C for 0.5 h. To stop the reaction, 4 mL saturated NaCl solution was added. Subsequently, the benzoyl Put was removed by using 3 mL cold diethyl ether. Then, 1.5 mL of the ether phase was evaporated to dryness after which 1 mL methanol was added. The mixture was vortexed and passed through a 0.45 µm porous membrane filter (Millipore). Put concentration from 2 µL filtrate was determined through HPLC (ACQUITY UPLC H-Class Core System; Waters: USA) supplemented with the column (ACQUITY UPLC HSS T3 (2.1 by 100 mm) 1.8 µm-pore-size). The UV-2487 detector, adjusted at 230 nm was used as a detection device. The acetonitrile/water (44:56, v/v) solution passed through a 0.22 µm pore size membrane filter was employed as a mobile phase which was supplied at 0.45 mL min −1 flow rate. The peak of Put was recognized and measured by comparing with known HPLC values of Put standard. The Put detection limit was 0.1 µg mL −1 .

Experimental Treatments
The treatments used for the present study are as follows: 1.
Put: Seeds primed with Put and grown in un-inoculated soil without Cd contamination 4.
Cd + Put: Seeds primed with Put and grown in un-inoculated soil having 50 mg kg −1 Cd contamination 6.
Cd + Put + PGPB: Seeds primed with Put and grown in inoculated soil with 50 mg kg −1 Cd contamination.
Put (1 mM) primed seedlings of C. annum plants were grown in B. thuringiensis IAGS 199 inoculated soil contaminated with Cd (50 mg kg −1 ) for 30 d.

Analysis of Plant Samples
Harvested plant samples were separated into leaves, shoot and roots for different biochemical analyses. The growth parameters of treated plants for instance root fresh weight, shoot fresh weight; root dry weight and shoot dry weight were analyzed according to Anwaar et al. [98]. Fresh biomass of plant samples was measured followed by drying these samples till constant weight in an oven at 70 • C for 48 h to analyze dry biomass.

Estimation of Chlorophyll Contents
For assessment of total chlorophyll contents (Chl a and Chl b), 100 mg fresh weight of foliage sample was grounding along with 8 mL of acetone 80% (v/v) in a pre-chilled mortar. The mixture of extract was filtered and the volume adjusted to 10 mL by adding cold acetone. The colorimetric value of the supernatant was observed at 663.2, 646.8 nm regarded as the amount of Chl a, Chl b correspondingly [99,100].

Determination of Photosynthetic Rate and Gas-Exchange Parameters
The net photosynthesis rate (A), transpiration rate (E) and intercellular CO 2 concentration was determined early in the morning (at 9 am) in the leaves with the help of portable gas-exchange system (Li-COR Inc., Biosciences, Lincoln, NE, USA) according to the methodology of Holá et al. (2010).

Determination of Total Soluble Protein
The magnitude of total soluble protein was assessed at 595 nm by using bovine serum albumin according to Bradford [101].

Assessment of Soluble Sugars
Phenol sulphuric acid method was employed for the evaluation of soluble sugars as described by Dubois et al. [102]. Homogenized 0.5 g plant sample was added in test tubes containing 80% ethanol (10 mL). This solution was heated over water bath at 80 • C for 60 min and mixture (0.5 mL) was transferred to other tubes. An equal volume of deionized water along with 1 mL of 18% phenol was mixed. Tubes were permitted to cool at 25 • C for 30 min after which absorbance was noted at 490 nm. The number of total sugars present in the sample was estimated according to the following equation:

Sugar =
Sample absorbance × dilution factor × K value weight of fresh plant tissue

Determination of Electrolyte Leakage (EL)
The totally extended topmost leaves were randomly selected and cut into 0.5 cm sections. The leaf section submerged into glass tubes containing 7 mL sterilized water. Tubes containing these leaves section were retained over rotary shaker for 1 day at 25 • C. The readings for primary conductivity (ECi) of the leaf sections were calculated by autoclaving the section holding tubes for 30 min at 120 • C. Values for maximum conductivity (ECmax) from a solution containing leaves section were estimated at 25 • C for measuring of EL in line with the following formula described by Li et al. [103]:

Evaluation of Antioxidant Enzymatic Activities
The antioxidative enzymes including SOD, POD and CAT were estimated with the help of MagNA Lyser and 1 mM ASC (Roche, Vilvoorde, Belgium). For evaluation of SOD activity, fresh leaves sample (100 mg) was homogenized along with solutions including 0.25% (v/v) Triton X-100, 10% (w/v) polyvinylpyrrolidone (PVP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mL of 50 mM potassium phosphate buffer at pH 7.0. Decrease in NBT (nitroblue tetrazolium) was measured at 560 nm to estimate SOD activity [104].
For the estimation of POD activity, leaf samples (1 g) were vortexed with 3 mL of KH 2 PO 4 buffer 100 mM (pH = 7). The homogenate was subjected to centrifugation at 12,000 rpm for 20 min at 4 • C. The reaction mixture was arranged by overtaxing 100 mL supernatant with 50 mL of guaiacol solution having 3 mL of KH 2 PO 4 buffer and 30 mL of H 2 O 2 . The absorbance of the mixture was estimated by using spectrophotometer at 436 nm according to Putter [105].
For evaluation of CAT activity, 1.0 g leaf sample was homogenized with 3 mL of 100 mM KH 2 PO 4 buffer at pH = 7 followed by 20 min centrifugation at 12,000 rpm at 4 • C. The supernatant (70 mL) was homogenized with 1500 mL 50 mM KH 2 PO 4 buffer and 930 mL of 15 mM. H 2 O 2 . The amount of H 2 O 2 decomposed at 240 nm was estimated to assess CAT activity [106].
Before centrifugation, filtration of the solution was carried out with the help of nylon mesh. The solution was centrifuged at 5 • C for 20 min. The supernatant (0.2 mL) obtained was mixed with ascorbic acid (0.25 mM), EDTA (0.1 mM), phosphate buffer (25 mM) at neutral pH. Then H 2 O 2 (1 mM) was added and level of oxidation carried out by ascorbate was observed at 290 nm. Oxidation was again recorded after 1 min. The difference obtained in both the spectrometrically calculated values was then divided to molar co-efficient of ascorbate [107].

Determination Proline Contents
For estimation of proline, 1.0 g of leaf tissues was homogenized in 10 mL (3%) sulfosalicylic acid and kept at 100 • C for 15 min. Afterwards, 4 mL ninhydrin and 4 mL glacial acetic acid were dissolved and reserved for 60 min at 90 • C. After cooling, 8 mL toluene was mixed and absorbance was observed at 520 nm [108].

Analysis of Cadmium Content
Plant samples were uprooted and washed by using distilled H 2 O. Plant samples were oven-dried for 48 h (hrs). Digestion of oven-dried plant samples was carried out in HNO 3 : HClO 4 . Cadmium content in digested plant samples was quantified with the help of atomic absorption spectrophotometer. The quantity of Cd content in digested plant samples was carried out by multiplication of dry weight with Cd content in plant tissues.
For assessment of translocation factor (TF), Cd content in the shoot was divided into the root Cd content according to Mattina et al. [109]. Metal tolerance index (MTI) was measured by the following equation: where DWPS = dry weight of PGPB-treated plant, DWNP = dry weight of control seedlings.

Determination of Total Thiols
Sedlak and Lindsay [110] method was employed for the assessment of total thiols. For this, plant sample (0.5 g) was homogenized in ascorbate buffer (20 mM) prepared with the help of EDTA (20 mm). Afterwards, centrifugation was accomplished at 12,000× g for 20 min at 4 • C. The supernatant (0.5 mL) attained was mixed with Tris HCl (200 Mm) and 10 mM of 5,5-dithio-bis-[2-nitrobenzoic acid]. The mixture was permitted to stand for 20 min and absorbance was calculated at 412 nm.

Determination of Non-Protein Thiols
Plant sample (0.5 g) was macerated in 5% sulphosalicylic acid (3 mL). Afterwards, the samples were centrifuged at 12,000× g for 20 min at 4 • C. The extract (100 µL) obtained was mixed with 0.1 M potassium phosphate buffer and 0.5 mL of 1 Mm 5,5-dithio-bis-[2-nitrobenzoic acid]. The mixture was then allowed to stand for 20 min and absorbance value was calculated at 412 nm [111].

Determination of Protein Bound Thiols
Protein-bound thiols were calculated by subtraction of non-protein thiol from total thiols.

Statistical Analysis
One-way analysis of variance (ANOVA) was conducted and seven treatment means were compared using Duncan's multiple range test (DMRT) [112]. The differences were considered significant when p-value was at least ≤ 0.05. DSASTAT statistical package software was employed for statistical analysis.

Conclusions
The results of the current study demonstrate that the presence of Cd in the rhizospheric area has detrimental effects on germination, seedling growth and biomass production of C. annum. Nevertheless, an affirmative effect of seed priming with Put and application of B. thuringiensis IAGS 199 significantly reinstates the physiological and morphological growth parameters of plants. Higher Cd tolerance in Put and B. thuringiensis IAGS 199-treated seedlings was attributed to the increased biosynthesis of osmoregulators including proline in consort with improved total soluble proteins and soluble sugars. Additionally, the reduced level of EL and MDA in plant tissues also abridged the Cd-induced toxicity. Furthermore, Put and B. thuringiensis IAGS 199 reduced bioavailability and uptake of Cd through binding it within root tissues by synthesizing chelating compounds including protein and non-protein thiols. Consequently, the present study advocates the application of Put and B. thuringiensis IAGS 199 as phytostabilizers for the successful cultivation of C. annum under Cd contaminated conditions. At the moment, it would be of pronounced interest to evaluate the transcriptome based strategy(s) by which Put and B. thuringiensis IAGS 199 maintain the physiochemical activities of plants under Cd stress.