Bacillus pumilus induced tolerance of Maize (Zea mays L.) against Cadmium (Cd) stress

Heavy metals contaminate the soil that alters the properties of soil and negatively affect plants growth. Using microorganism and plant can remove these pollutants from soil. The present investigation was designed to evaluate the induced effect of Bacillus pumilus on maize plant in Cadmium (Cd) contaminated soil. Three different concentrations of Cd (i.e. 0.25, 0.50 and 0.75 mg kg−1) were applied in soil under which maize plants were grown. The germination percentage, shoot length, leaf length, number of leaves, root length, fresh weight and nutrient uptake by maize plant were determined. The experiment was conducted by using complete randomized design (CRD) with three replicates. The result indicated that germination percentage, Shoot length, leaf length, root length, number of leaves, and plant fresh weight were reduced by 37, 39, 39, 32 and 59% respectively at 0.75 mg kg−1 of CdSO4 concentration but when maize seeds inoculated with Bacillus pumilus significantly increased the germination percentage, shoot length, leaf length, number of leaves, plant fresh weight at different concentrations of CdSO4. Moreover, the plant protein were significantly increased by 60% in T6 (0.25 mg kg−1 of CdSO4 + inoculated seed) and Peroxidase dismutase (POD) was also significantly higher by 346% in T6 (0.25 mg kg−1 of CdSO4 + inoculated seed), however, the Superoxide dismutase (SOD) was significantly higher in T5 (0.75 mg kg−1 of CdSO4 + uninoculated seed) and was 769% higher as compared to control. The Cd contents in Bacillus pumilus inoculated maize roots and shoots were decreased. The present investigations indicated that the inoculation of maize plant with Bacillus pumilus can help maize plants to withstand Cd stress but higher concentration of Cd can harm the plant. The Bacillus pumilus has good potential to remediate Cd from soil, and also have potential to reduce the phyto availability and toxicity of Cd.

www.nature.com/scientificreports/ short term disease like nervous, kidney, cardiovascular, and bone diseases 6 . Soil adulteration with heavy metals is a common problem for world which is alarming threat for human health 7 . Cd is an unnecessary and greatly noxious heavy metal, which present in environment due to anthropogenic activities. Cd inhibit the plant to absorb important nutrients, in result plant growth is reduced which indicates Cd phytotoxicity 8 . Cd is non-amphoteric in nature and not properly dissolves in base solution 9 . The development of plant organs bears harmful effect of heavy metals like lead (Pb) and Cd which reduce biomass of various plant species 10 .
The plant species grown in contaminated soil having high concentration of pollutant reduce plant organ formation 11 . The crop which are produced in contaminated soil, absorb contaminants in their tissues and are very toxic for living organisms when are used as food 12 . Different plant species accumulate different types of heavy metals in their tissues from contaminate site 13 . Industrial pollutants contaminate water and play harmful impact on organisms. Uptake of toxic metals in plants effects variations in plant species, plants growth stage and translocation of metals 14 . These heavy metals damage molecular structure of plant and animals 15 . To eliminate contamination of non-degraded partials, phytoextraction is used which increase biomass and bio-concentration of plants 16 . There are different types of technologies used in present time to eliminate contaminants from polluted areas to reestablish natural condition. Phytoremediation is one of the best technology in which plant absorbs toxic substances from soil and water. Only selected plants are utilized for this purpose 16 . Phytoremediation is an ecofriendly technology to remove toxic metals 17 .
Numerous bacterial species are known that play vital role to tolerate plants under stress condition which can detoxify, transfer and collect heavy metals. Microorganisms and plants combine together against toxic effect of heavy metals by using rhizoremediation and phytoremediation mechanism. Microbes enhance the growth of plant in heavy metals stress 18 . Plant absorbs heavy metals in soil and transport from root to shoot via xylem tissue after physiological process accumulates into grains. Plants having different genotype and capacity to detoxify heavy metals stress 19 . Plant microbe's interaction decomposes various pollutants and increase plant development and growth 20 . A bulk of enzymes from bacteria, have been reported to be concerned in the biodegradation of toxic organic pollutants and remove the soil contamination 21 .
Previous reports demonstrated that several species of Bacillus can beneficially promote growth and enzyme system which may help the plants to overcome the biotic stresses 22 . The application of several Bacillus strains in soil contaminated with heavy metals soil can help to reduce the harmful effects of heavy metals and enhances the plant growth. The Bacillus spp also have ability to accelerate the plant growth by increasing water uptake and reducing electrolyte leakage to mitigate Cd stress 23 . B. licheniformis enhances Cu, Zn, Cd, Cr and Pb accumulation and distribution in plants grown in heavy metal-contaminated soil, which leads to reduced levels of toxic metals in soil 24 . Similarly, higher concentration of Cd in soil reduce nutrient (P, Fe, Zn, and Mn) uptake in plants. B. pumilus is a promising plant growth promoting bacteria and previous reports 25 demonstrated that B. pumilus affected metal toxicity in tomato and rapeseed (Brassica napus L.) The application of Bacillus spp. alleviate stress effect by reducing lipid peroxidation and SOD activity and increasing amylase and protease to promote plant growth in heavy metal-polluted soil 26 . Similarly, Bacillus spp. support plant tolerance against Zn and Cu stress by enhancing the activities of ROS scavenging enzymes, such as POD, SOD, CAT, APX, and DHAR 27 . The regulation of antioxidants in cells inhibits oxidative stress damage and triggers plant growth-promoting substances to enable plants to adapt to metal stress. Bacillus-mediated plant tolerance against Ni and Cr stresses is achieved through the enhancement of photosynthetic pigments and leghemoglobin, which leads to increased crop yield 28 . However, the effect of B. pumilus on Cd uptake by plants has received lesser attention. It is not clear whether plant physiological processes work independently or together with other mechanism like antioxidant system of plant under cd stress. In this context, the present study was therefore performed to investigate the potential of Bacillus pumilus to induce growth and antioxidant enzymes of maize plants under Cd stress.

Materials and method
Preparation of heavy metal solution. Three different concentration of cadmium sulphate (CdSO 4 ) solution (0.25, 0.50 and 0.75 mg mL −1 ) were prepared for different treatment in pure distilled water by dissolving the cadmium sulfate (CdSO 4 ). The different concentrations of CdSO 4 were selected on the basis of the previous scientific data 29 . Pure distilled water was used as control for the experiment. 100 mL of each solution was added in 1 kg of potted soil.
Preparation of bacterial inoculum. The Bacillus Pumilus (Acc KF859972) used in this study was taken from phytohormone Lab Quaid-i-Azam University,Islamabad, Pakistan, on the basis of its plant growth indorsing latent 30 . For the preparation of inoculum, the nutrient broth was purchased from OXOID-UK. The nutrient broth was sterilized at 121 °C for 20 min. The isolated strain was inoculated in nutrient broth and incubated in shaker incubator (EXCELLA E24 Germany) at 150 rpm for 48-72 h. After that, the culture was centrifuged for 10 min at 3000 rpm. The pellet was again suspended in double distilled water and optical density (O.D) was adjusted to 0.100 at 660 nm with UV-VIS spectrophotometer. The inoculum was prepared by culture of bacterial strain having O.D 0.100 at 660 nm and bacterial density (10 6 cells/ml).

Seed inoculation.
Maize (Zea mays L.) seeds (KASHMIR GOLD) was obtained from NARC (National Agricultural Research Centre) Islamabad, Pakistan. The seeds were washed with ethanol (95%) for surface sterilization, following by soaking in 10% Chlorox for 2-3 min and subsequently the seeds were washed successively 2-3 times with autoclaved distilled water 31 . Moreover all the methods were performed in accordance with the relevant guidelines given by the national agriculture research center for the cultivation of maize plants. Parameter measured. The germination percentage was observed after four day of sowing whereas, maize were harvested after 28 day of sowing. In order to remove non-aggregated soil, seedlings were slightly shaken.
The following parameters were studied 32 . Shoot and root lengths were measured from the root initiation up to the tip of the longest shoot and root. It was measured in centimeters 33 . Leaf size was measured in cm, from node to tip of the leaf 34 . Root length was measured from the junction of root and stem towards the tip of the longest root. It was measured in centimeters 35 . After harvesting plants from the pots, they were shaken to remove extra soil other than aggregates, the weight measured in grams 36 . Each plant of each replicate was measured and their mean value was used to compare the treatments.
Leaf proline. Proline content of maize plant leaves was determined by the method of 37 about 0.5 g of fresh maize leaves were used to determine the proline content.
where the K value is 19.6.
Peroxidase dismutase assay. The POD activity of maize leaves was measured by the method of 38 about 1 g of fresh maize leaves were used to determine the POD enzyme.
Superoxide dismutase assay. The SOD activity of maize leaves was measured by the method of 39 . The activity of SOD was expressed as units/100 g fresh weight. About 0.2 g of fresh maize leaves were used to determine the SOD enzyme.
Plant nutrient analysis. The per chloric-acid digestion method was used to determined presence of the nutrients in the plant organs like root leaves and shoot 40 . Maize leaves (0.25 g) were used for nutrient analysis.

Results
The experiment was carried out in pots with complete randomize design (CRD) and plants were harvested after 28th day of seed sowing and results were analyzed. Different parameter were observed i.e. fresh biomass, root length, shoot length, leaf size and number of leaves, Cd contents in roots, shoots and seeds germination .
Effect of cadmium (Cd) on maize seed germination percentage. The germination percentage was significantly increased with the inoculation of Bacillus pumillus (T2), however the inhibition in germination was observed at all concentration of Cd as compared to the control. About 39% reduction in seed germination percentage was observed in T5 (0.75 mg kg −1 CdSO 4 + uninoculated seed) as compared to control. While inoculation of Bacillus pumillus in the presence of Cd increased the germination percentage however this increase was non-significant (Fig. 1a). The germination percentage was increased in Bacillus pumillus inoculated seeds Proline =K × Dilution factor × optical density/weight of sample www.nature.com/scientificreports/ as compared to control and uninoculated seeds. The maximum seed germination was observed in T2 (Bacillus pumillus inoculated seed) which was 40% higher than control.
Effect of cadmium (Cd) on maize shoot length (cm). The Bacillus pumilus inoculation (T2) significantly induced shoot length of maize plant as compared to control (T1) but cadmium (Cd) inhibited the shoot length and maximum reduction (37%) in shoot length was observed in (T5) however Bacillus pumilus inoculation significantly increased the shoot length (Fig. 1b). The maximum shoot length was observed in treatment T2 (Bacillus pumilus inoculated Seeds.) which was 39% higher than control while 37% reduction in shoot length was observed in T5 (0.75 mg CdSO 4 kg −1 + uninoculated seed) as compared to control. Cd also affected leaf length of maize plants. However, the inoculation of maize seeds with B. pumillus significantly enhanced the leaf length of maize plant at different concentration of Cd as compared to control and uninoculated maize plants.
Moreover, 40% increase in leaf length was observed in T2 (Bacillus pumillus inoculated seed) as compared to control while, seeds showed 39% reduction in leaf length in T5 (0.75 mg CdSO 4 kg −1 + uninoculated seed) as  www.nature.com/scientificreports/ compared to control (Fig. 1c). The Fig. 1d shows the root length of maize plant affected by Cd ; however, the root length of inoculated maize seeds with Bacillus pumilus significantly increased when grown at different concentrations of Cd . The maximum (40%) root length was observed in T2 (Bacillus pumilus + seed) which were 40% higher than control and uninoculated plants, as compared to control. The reduction in root length was observed at different concentration of Cd and about 39% reduction in root length was observed in T5 (0.75 mg CdSO 4 kg −1 + uninoculated seed) as compared to control, however inoculation of Bacillus pumilus significantly enhanced the rood length at different concentrations of Cd. The Cd affected the number of leaves in maize plants (Fig. 1e), while inoculation of maize seeds with Bacillus pumilus enhanced the number of leaves in maize plants at different concentrations of Cd. The number of leaves were increased by 42% in T2 (Bacillus pumilus inoculated Seed) as compared to control and uninoculated seeds, while uninoculated seeds T5 (0.75 mg CdSO 4 kg −1 + uninoculated seed) showed 32% reduction in number of leaves as compared to control. The result presented in Fig. 1 shows the Cd affected fresh weight of maize plants. However, inoculation of maize seeds with Bacillus pumilus notably enhanced the fresh biomass of maize plant at different concentration of Cd. The maximum (34%) plant fresh weight was observed in T2 (Bacillus pumilus inoculated seed) as compared to control, while 59% reduction in plant fresh biomass was observed in T5 (0.75 mg CdSO 4 kg −1 + uninoculated seed) as compared to control.
Effect of cadmium (Cd) on plant protein. Though  On the other hand, a reduction of 13% in POD activity was observed in treatment T2 (Bacillus pumilus). (Fig. 2b).

Accumulation of cadmium (Cd) in maize roots (mg/g). There was variation in the accumulation of
Cd contents in maize roots which was observed in all the treatment as shown in Fig. 3a. The maximum 45% Cd uptake was found in T5 (0.75 mg CdSO0 4 kg −1 + uninoculated Seed) as compared to control. However, the Cd accumulation was reduced in all treatments when inoculated with Bacillus pumilus as compared to uninoculated seeds. The minimum 21% Cd contents in maize plant were observed in T6 (0.25 mg CdSO0 4 kg −1 + inoculated Seed). Fig. 3b showed variation in Cd contents in maize plant leaves in all the treatment, however the maximum 90% Cd uptake was found in T5 (0.75 mg CdSO0 4 kg −1 + uninoculated Seed) as compared to control. While Cd concentration was reduced in all treatments when inoculated with Bacillus pumilus as compared to control and uninoculated seeds. (The minimum 45% Cd contents in maize plants observed in T6 (0.25 mg CdSO0 4 kg −1 + inoculated Seed). Table 2 showed Cu content increased in all the treatments when inoculated with Bacillus pumilus as compared to uninoculated plants.

Discussion
The Cd contaminant adversely affects plants and animals directly and indirectly however, trace amount of Cd in soil did not harm plants 12 . Cd enters into soil in different anthropogenic activities as well as by natural process. Heavy metals present in soil and air remain untreated and enters plant body through dust and moisture contents, which first impacts seeds, and roots of plants, afterwards damages shoots and leaves respectively 41 . It is obvious that germination or growth of plants is increased in inoculated treatments and the growth of maize plant affected by high concentration of Cd, however the effect is minimized by inoculating with Bacillus pumilus. During the present study, the germination was improved with the inoculation of Bacillus pumilus when grown over Cd and these findings are in agreement with 42 , who reported that the inoculation of plant seeds with microorganism species like Pseudomonas, Pasteurella, Salmonella, Bacillus and Burkholderia have the ability to resist. The result finding are also supported by 43 , who reported that Cd toxicity has decreased seed germination percentage. The removal of heavy metals contaminants from contaminated site, the combined application of plant and microbe is a successful method as compared to the use of plant or bacteria separately 44 . The higher concentration of lead (Pb) reduces the flower production 45 . In the present study, Cd affected the maize plant in the same way. The higher concentration of Cd can cause plant toxicity and reduction in growth through interference with mineral and Cd absorption, and movement of necessary elements 8 T1  T2  T3  T4  T5  T6  T7  T8

SOD acƟvity units/min/g FW of leaves
Treatments (c) www.nature.com/scientificreports/ with these results. The Cd concentration reduced the plant growth and prompted phytochelatin (PC), Cd destructively lowers plant growth because it is non-essential element 46 . Inoculation of seeds with Bacillus pumilus also enhanced plant growth, this increase in plant length might be due to the production of phytohormones 47 . It is also reported that the growth in bacterial inoculated seeds with different Cd concentrations showed significant leaf growth, which showed that bacterial inoculation can promote the tolerant capacity of plants which are in  www.nature.com/scientificreports/ agreement with our findings in which the seed inoculated with Bacillus pumilus showed better leaf growth under Cd stress 48 . Cd transported from soil to all parts of plants tissue, damages the tissues in various ways, so size of contaminated leaf stunted. Likewise 49 , reported visual symptoms of chlorosis and necrosis in tomato plant when applied up to 25 and 50 μM of CdCl2. We also got same result when 75 mg dose of Cd on maize plant caused wilting in uninoculated treatment but inoculated treatment did not showed these symptoms because Bacillus pumilus inhibit toxic symptoms by providing tolerance ability. Root is the first organ of plant which is affected by Cd and Cd adversely affects the root length. The study of 50 , showed similar findings which showed decreased root length in the presence of Cd without any inoculation, because Cd destroyed the protein structure however root length showed better growth when inoculated with Bacillus pumilus and accumulation of Cd in roots of Barlay plant was 25% more than stem which inhibited the normal growth of plant root 51,52 . The effects of heavy metals depend on type of environment and toxic substances uptake by plants. Greater the toxic substance in soil will cause reduction in plants growth 53 also confirmed our finding that in high level of Cd the maize plant showed reduced growth.
The Cd stress in maize plant produce free radicals which damage membrane and cause leakage of electrolyte 50 , therefore number of leaves decreased in Cd stress 54 reported soybean plant change its physiology as well as morphology like number, shape and size of leaf against Cd is agreement of our present finding in which the inoculation Bacillus pumilus significantly change the structure of bacterial community which enhance growth as compare to control after 15 days of experiment 55 confirmed our findings that Bacillus pulmilus promote the tolerance capacity of plants.
In this study Bacillus pumilus also enhanced plant fresh weight by producing phytohormones like IAA and GA 56 . These hormones increase the plant root and shoot length, and leaf volume which promote fresh weight of maize plant. The Bacillus species also responsible for bioavailability of macro and micro nutrients from soil 57 have beneficial effect on plant fresh weight.
Root secretions have vital function in altering metal bioavailability, these secretions have various compounds that combine with metals and restrict their movement in soil. These rhizo secretions also provide essential elements to microbial communities that enhance their growth and survival ability. Root secretions have different enzymes and protons that make the soil acidic and increase the heavy metal bioavailability 58 .
Maize plant accumulate Cd in shoots and inhibit the growth of shoot by damaging cell membrane which remove ions from damage site Cd 59 . Result presented in this experiment shows that Cd uptake by maize plant decrease in all treatments that were inoculated with Bacilus pumillus as compared to control and stressed plants. The reduction in Cd uptake was observed in plants that were inoculated with Bacillus pumilus and highest Cd uptake was observed in uninoculated plants. Bacillus pumilus converts Cd in to unavailable form in soil, and also reduces its toxicity. Previous studies also supported these results that inoculation with Bacilus species reduces Cd bioavailability [60][61][62] .
The plant possessess a well-organized antioxidant defense system. The accumulation of Cd toxicity was observed in maize cultivar with various treatments with B. pumillus and without B.pumillus inoculation in order to discern their ability to tolerate different concentration levels of Cd. The present study revealed that antioxidant activities (POD and SOD) stimulated at the higher concentration of Cd. The higher Cd concentrations in maize cause an increase in enzymatic activities because of the activation of enzymes that are already present in plants [63][64][65][66] . Comparable changes in the enzymatic activities under different concentrations of heavy metals specifically Cd toxicity have been reported earlier 67,68 . However, some of the studies are in deviation with our results reporting a decrease in SOD activity under the higher concentration of Cd level [69][70][71] . The deviation in results could ensue due to the difference in the time duration of Cd stress applied, the intensity of Cd, and specifically plant stage and cultivar. Moreover, no significant increase was observed in maize plants treated with Bacillus pumilus 72 .
Present study depicted an increased SOD and POD activity at higher concentrations suggesting that both of these enzymes act simultaneously to avert the formation of OH ions and remove H 2 O 2 71,73 . Therefore, the increased enzymatic (particularly SOD) activity at a higher concentration of Cd is considered a good indication for defensive mechanism stimulation 74 . In addition to this, it was observed in a study that the SOD activity was higher at the lower concentration of Cd in soil (20-25 mg/kg), normal when the concentration ranges between 50 and 75 mg/kg Cd in the soil and start to decrease when the soil Cd toxicity levels reached to 100 mg/kg 71 . The decrease in the enzymatic activity perhaps might be attributed to inhibition caused by accelerating H 2 O 2 75,76 . Thus, heavy metal stress causes an induction of SOD and POD enzymes which in return provides protection and membrane integrity.
It is a known phenomenon that Cd stress leads to the denaturation of proteins. The present study validated the phenomenon that with the gradual increase in the Cd toxicity level the protein content started to decrease. The results are in agreement with the preceding studies demonstrating the reduction of protein content in maize due to Cd stress [77][78][79] .
Heavy metals like aluminum, nickel, lead, and Cd accumulate in root of plants and effect metabolisms of plant by reducing cell elongation and new cell formation 80 , so plant cannot promote their growth. Similarly in our present study plants treated with Cd showed stunted growth and accumulates maximum Cd in their roots 81 also reported that most plant species like cucumber, rice, maize and etc. hold chief Cd concentration in their roots which reduced the plant growth by disturbing their metabolic activity. Cd. Soil polluted with Cd impacts roots of plants directly which disturb roots to uptake essential nutrients for metabolic activities of plants. However different plant species have tolerance capacity against specific heavy metals 82 . www.nature.com/scientificreports/