Soil is a mixture of different components, organic matter (5%), minerals (45%), gases (25%), and liquids (25%). Besides this, several organisms which support and maintain life on Earth are also found in the soil. Soil provides several ecosystem services by maintaining biogeochemical cycles and protection against erosion (Tahat et al. 2020). Healthy soil acts as a dynamic living system that delivers multiple ecosystem services, such as plant productivity, sustaining water quality and controlling soil nutrient recycling decomposition. Soil health is closely associated with sustainable agriculture, because soil microorganism diversity and activity are the main components of the soil health (Tahat et al. 2020). Anthropogenic and industrial activities, facilities and products such as mining of natural resources, electroplating, smelting, fertilizers, pesticides, tanneries add a large number of contaminants to our natural environment. These contaminants affect the physiological and biological properties of soil (Arivalagan et al. 2014). Among different contaminants, heavy metals such arsenic (As), cadmium (Cd), lead (Pb), copper (Cu), chromium (Cr), nickel (Ni), zinc (Zn), aluminum (Al) and manganese (Mn) are the most common (Ullah et al. 2015). Heavy metals have shown negative impacts on soil fertility and soil microbial community (Chu 2018). They are not soluble in water and are not readily degradable; therefore, they remain insoluble in the soil. Later on, they are assimilated by plant roots. In this way, heavy metals are transferred to different trophic levels of the ecosystem and contaminate the food chain (Tchounwou et al. 2012).
Zinc is an essential micronutrient which promotes plant growth and development (Hefferon 2019); however, higher concentration of Zn causes root blunt, cell wall thickening, reduction in cell division, cell elongation, and disruption in cell organelles and an increase in the number of the nucleoli (Glińska et al. 2016, Taghizadeh et al. 2018). Plants have different defense mechanisms or strategies for tolerance or detoxification, whenever they are exposed to heavy metal stress condition. In addition, microorganisms from soil assist plants to degrade a huge variety of contaminants. In contaminated soil, microorganisms can modify and adopt various mechanisms that convert toxic substances into a less toxic form (Jaiswal et al. 2017). These mechanisms include biosorption, bioaccumulation, biotransformation, and bio-mineralization. Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus and Mycobacterium have the potential to detoxify contaminants in nature (Vergani et al. 2017). Some bacterial species such as Bacillus and Pseudomonas are generally used for the remediation of heavy metals from contaminated water and soil, as these bacterial strains have more metal binding affinities (Arivalagan et al. 2014, Ullah et al. 2015). Plant growth-promoting bacteria increase plant growth and development by increasing accessibility of nutrients (Bulgarelli et al. 2013), which contributes to plant health and increases their biomass and absorbing capacity to overcome stress (Weyens et al. 2013). Rhizobia have the potential to remove organic and metal contamination by degrading organic contaminants (Teng et al. 2015). Besides, bacteria have the ability to produce phytohormones such as auxins, cytokinins and gibberellins, which control all aspects of plant growth and development (Glick 2010). Plant growth-promoting bacteria enhance plant growth in stress conditions through various mechanisms including nitrogen (N) and phosphorus (P) solubilization, siderophore production and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production (Fatima et al. 2017).
Increased concentration of heavy metals inhibits normal plant functioning by disturbing protein structure; this alteration hampers metabolic processes (Hall 2002). The change in in protein structure can cause mutation in various physiological processes in plants such as photosynthesis, respiration, and enzymatic activities (Hossain et al. 2012). Plants have different defense mechanisms or strategies for tolerance or detoxification whenever they are exposed to elevated concentration of heavy metals. The first step in metal tolerance is inhibition or restriction of heavy metal entry into plant root (Viehweger 2014). Failure of the first step activates the mechanism of phytosequestration which permits heavy metals to enter in intracellular compartments (Patra et al. 2004), which produce different substances like phytochelatins (PCs), organic acid, polysaccharides and metallothionein (MTs), which are the two best-characterized metal-binding ligands in plant cells that help a plant to detoxify heavy metals (Dalvi and Bhalerao 2013). If all these strategies fail, plants activate their antioxidant defense mechanism which can help the plant by inducing reactive oxygen species (ROS), which inhibit most cellular processes at various levels of metabolism. ROS, being highly unstable, could play dual role (1) damaging cellular components and (2) act as an important secondary messenger for inducing plant defense system. Cells are equipped with enzymatic and non-enzymatic defense mechanisms to counteract this. (Sytar et al. 2013, Manara 2012).
Any plant used for phytoremediation should not be metal-tolerant, and must be fast growing with the potential to produce high biomass. Maize is mainly used in phytoextraction due to the fact that it is a common and rapidly growing crop, which possesses an extensive fibrous root system and can tolerate stress conditions (Farooq et al. 2015). Therefore, maize plants are suitable for the extraction of heavy metals from contaminated soil (Lu et al. 2015). There is insufficient information about the fate of bacterial inoculation in maize edible and non-edible parts to enhance maize tolerance in heavy metal soils (Shahzad et al. 2016). Henceforth, present investigation is aimed to evaluate the role of Bacillus cereus strain in enhancing the phytoextraction ability of maize plant under Zn contaminated soil conditions.
Materials and methods
Bacillus cereus (Accession no. KR232400) was selected on the basis of its plant growth promoting potential (Shahzad et al. 2020). The strain was obtained from phyto-hormone laboratory at Quaid-i-Azam University, Pakistan. Bacterial strain was previously identified by 16S rRNA gene sequencing (Shahzad et al. 2016).
Preparation of heavy metal solution
Three different concentrations of Zn solution (20, 40 and 60 mg kg-1) for maize plant treatments were prepared in pure distilled water by dissolving zinc sulfate (ZnSO4). We selected these three concentrations on the basis of previous scientific data (Long et al. 2003, Yang et al. 2005). Pure distilled water was used as the control for the experiment. 200 mL of each solution was added in 1 kg of potted soil.
Preparation of inoculum
For the preparation of inoculum, five ml of nutrient broth medium (Oxoid, UK) was autoclaved in test tubes for cultivation of bacterial strain. Bacteria were incubated in a shaker incubator (EXCELLA E24 Germany) at 37 ºC and 150 rpm for 48-72 h. Thereafter, the culture was centrifuged at 3000 rpm for 10 min. The pellet was re-suspended in autoclaved distilled water and the optical density (OD) was adjusted to 0.100 at 660 nm with UV-VIS spectrophotometer (UV-VIS Double Beam Spectrophotometer, Model NO: AE-S90-2D A & E Lab (UK) Co., Ltd.). Bacterial density was 106 cells mL-1 at optical density of 0.10 at 660 nm. The optical density was adjusted by adding about 200 ml sterile water to pure bacterial pellets.
Preparation of treatment applications and seed inoculation
Kashmir Gold variety maize (Zea mays L) seeds were collected from the National Agricultural Research Centre, Islamabad, Pakistan. The seeds were surface sterilized by washing with 95% ethanol, following by soaking in 10% Clorox for 2-3 minutes. The seeds were washed successively 2-3 times in autoclaved distilled water.
For experiment preparation, seeds were soaked in the B. cereus inoculum for 2-3 hrs, after that about 10 seeds were sown in each pot. Different concentrations of Zn solutions (100, 200 and 300 mg L-1) and distilled water was added to each pot. Eight different treatments with three replicates were made. Detailed explanation of treatments is given in Table 1
The percentage germination was noted after seven day of sowing. Maize seedlings were uprooted after 21 days of sowing, after which root length (cm), plant height (cm), leaf length (cm), number of leaves and fresh plant weight parameters were recorded. Each treatment contained three replicates and 5 plants from each replicate were measured.
Soil nutrient analyses
Determination of macro- and micro-nutrients (Na, Ca, Mg, K, P, N, Fe, Cu, Cr, Co, Zn, and Mn) was carried out by following the ammonium bicarbonate and diethylenetriamine pentaacetic acid (DTPA) method as described by Soltanpour and Schwab (1977). The 0.005 M ammonium bicarbonate and (DTPA) solution was prepared by adding 1.97 g of DTPA to 800 mL of distilled water. About 2 mL of ammonium hydroxide (NH4OH) was added to facilitate dilution and to prevent effervescence due to ammonium bicarbonate addition. When DTPA was dissolved completely, 79.06 g of ammonium bicarbonate (NH4HCO3) was added. The pH of the solution was adjusted to 7.6 by adding 2 mL of ammonium hydroxide. Final volume was adjusted to 1 L with the addition of distilled water. For the analysis of different nutrient, 10 g of soil sample was mixed with 15 mL of distilled water. The suspension was stirred for 30 minutes on a magnetic stirrer. After stirring step, suspension was filtered. Two mL of filtrate was removed and 18 mL of distilled water was added for nutrient analysis.
Plant nutrient analysis
The perchloric-acid digestion scheme was used to determine the content of various nutrients in maize plants (Allen et al. 1974). A solution of nitric acid, sulfuric acid, and perchloric acid in the ratio 5:1:0.1 (6.5 mL) was added to 0.25 g of plant material in a 50 mL flask and the mixture was heated on the hot plate in the fume hood to complete the digestion process. The formation of white fumes in the mixture confirmed the completion of digestion of the plant extract. Thereafter, distilled water (few drops) was poured into the flask and the mixture was allowed to cool. The digested plant extract samples were transferred to the volumetric flasks (50 mL) and the volume was raised up to 50 mL by the addition of distilled water. The samples were filtered with Whatman no.42 filter paper and were stored for further element analysis. The concentration of different elements/nutrients present in the plant samples were measured by atomic absorption spectrophotometer (Shimadzu AA-670 Germany).
The experiment was conducted in a completely randomized design (CRD) by using Statistic 8.1.1. (https://statistix.informer.com/8.1/). The results are the compare means and standard error of means of three replicates of a treatment. Five plants from each replicate were measured.
Effects on seed germination
Percentage germination of maize seeds has shown variations among inoculated and uninoculated seeds (Fig. 1a). Maximum seed germination (%) was observed in B. cereus-inoculated seeds in combination with 20 mg kg-1 ZnSO4 (treatment T3), which was significantly higher than control (39%) and the majority of the treatments. High germination percentage was also recorded in B. cereus-inoculated seeds in combination with 40 mg kg-1 ZnSO4 (treatment T4), which was significantly higher than the treatment of uninoculated seeds with 60 mg kg-1 ZnSO4 (treatment T8), where the lowest values were obtained (germination reduction of 9% as compared to control).
Effects on root length, leaf length and number of leaves
The root length was only mildly affected by investigated treatments (Fig. 1b). The maximum increase in the root length (15%) was observed in B. cereus inoculated seeds in combination with 20 mg kg-1 ZnSO4 (treatment T3), although it was not significant compared to control. Similar increase in root length (14%) was also recorded for B. cereus-inoculated seed (treatment T2 treatments). On the contrary, the 19% reduction in root length was observed in the treatment of uninoculated seeds exposed to60 mg kg-1 ZnSO4 (treatment T8), which was not significant compared to control, but was significantly reduced in comparison to treatments T2 and T3. Other treatments exhibited values similar to those of control
Maximum increase in leaf length (17%) was observed in B. cereus-inoculated seeds in combination with 20 mg kg-1 ZnSO4 (treatment T3), although it was not significant as compared to control and other treatments of inoculated seeds; however, it was significantly higher in comparison to result obtained for uninoculated seeds (Fig. 1c). Namely, all treatments of uninoculated seeds resulted in reduction in leaf length, which was particularly after exposure to 20 mg kg-1 ZnSO4 (treatment T6).
Maximum number of leaves (1% increase compared to control) was observed in B. cereus-inoculated seeds in combination with 40 mg kg-1 ZnSO4 (treatment T4), although this value was not significantly different as compared to the majority of the treatments; the only exception was the treatment of uninoculated seeds exposed to 20 mg kg-1 ZnSO4 (treatment T6), which exhibited the lowest value (7% decrease compared to control) (Fig. 1d).
Effects on plant height, fresh weight and zinc uptake
The inoculation of maize seeds with B. cereus significantly increased the fresh weight and height of maize plants (Fig. 2). A significantly higher increase in fresh weight was observed upon exposure of inoculated seeds to 20 mg kg-1 ZnSO4 (treatment T3, 80% increase) and 40 mg kg-1 ZnSO4 (treatment T4, 62% increased) in comparison to control and the treatments of uninoculated seeds. On the contrary, a significant reduction in plant fresh weight as compared to control was observed in all treatments of uninoculated seeds, with the maximum reduction of 27% recorded upon exposure to 60 mg kg-1 ZnSO4 (treatment T8) (Fig. 2b).
Exposure of inoculated seeds to ZnSO4 slightly improved the plant height (treatments T3, T4 and T5) as compared to control; the maximum value (12% increase as compared to control) was obtained upon exposure to 20 mg kg-1 Zn (treatment T3). On the other hand, exposure of uninoculated seeds to the same Zn concertation (20 mg kg-1) resulted in a reduction in plant height up to 19% (Fig. 2a).
Zinc uptake was reduced in the majority of the treatments with inoculated seeds as compared to control; significantly for treatments T2 and T3 (exposure to 100 mg L-1 ZnSO4), and notably for treatment T4 (exposure to 49 mg kg-1); reduced Zn uptake was 49%, 44% and 22% in T2, T3 and T4 respectively (Fig. 2c). However, the exposure of inoculated seeds to the highest tested Zn concentration (treatment T5) resulted in a significantly higher Zn uptake as compared to control. Among treatments with uninoculated seeds, exposure to 20 mg kg-1 (treatment T6) resulted in a value similar to that of control, while upon treatment with 40 and 60 mg kg-1 Zn (treatments T7 and T8, respectively) Zn uptake was significantly higher; the maximum increase (75% as compared to control) was observed in treatment T8 (Fig. 2c).
Nutrients status of soil and accumulation of nutrients by maize plant
Different nutrients including Ca, Mg, K, Na, Fe, Cd, Cu, Cr, Zn, Co and Ni were detected in soil samples (Tab. 2). The plant nutrient status (Tab. 3) showed that the Cu content decreased in majority of the treatments as compared to control; the only exceptions were treatments T3 and T8. The Mn content was significantly elevated in the treatment of plants from inoculated seeds exposed to 20 mg kg-1 ZnSO4 (treatment T3), while the exposure of plants from uninoculated seeds to 40 mg kg-1 ZnSO4 (treatment T7) resulted in significantly lower Mn content as compared to control. Reduced Ni and Co contents were recorded in the majority of treatments as compared to control. Seed inoculation with B. cereus in general decreased the Na content as compared to values obtained in uninoculated plants, since the higher values were obtained in treatments of plants from uninoculated seeds (treatments T6, T7 and T8) as compared to treatments of plants from inoculated seeds to the corresponding Zn concentrations. Seed inoculation with B. cereus increased Cr content in plants as compared to control as well as to the treatments of plants from uninoculated seeds upon exposure to 20 and 40 mg kg-1 ZnSO4 (treatments T3 and T4) as compared to treatments T6 and T7, respectively). Fe content was significantly elevated in plants from inoculated seeds upon exposure to 40 and 60 mg kg-1 ZnSO4 compared to control as well as to the exposure of plants from uninoculated seeds to the same Zn concentration. Seed inoculation with B. cereus in general increased the Ca and Mg uptake in all investigated treatments as compared to control, with higher values obtained in plants from inoculated seeds compared to the uninoculated ones. K content was significantly higher in plants from inoculated seeds as compared to the control as well as to plants from uninoculated seeds exposed to the corresponding Zn concentrations.
|Nutrient||Soil (mg kg-1)|
|Ca||19.6 ± 1.11|
|Mg||2.46 ± 0.23|
|K||1.67 ± 0.21|
|Na||22.39 ± 0.66|
|Fe||3.18 ± 0.12|
|Cd||4.39 ± 0.19|
|Cu||11.20 ± 0.44|
|Cr||10.5 ± 0.12|
|Zn||8.9 ± 0.11|
|Co||0.19 ± 0.02|
|Ni||2.23 ± 0.15|
|Mn||6.22 ± 0.27|
Zinc is a trace element that performs a vital role in plant growth and development. However, at higher concentrations, it reduces seed germination, length of seedlings and stem as well as contents of chlorophyll, carotenoid, sugar, and amino acids (Todeschini et al. 2011, Glińska et al. 2016). [REMOVED HYPERLINK FIELD]In this study, the effects of zinc toxicity on plant height, leaf length, number of leaves, root length, fresh weight, and nutrient content was observed. Our results indicated that high Zn concentrations inhibited seed germination as compared to the control. Previous studies found a negative impact of Zn on plant growth parameters. Current findings are in agreement with previous studies (Islam et al. 2014, Glińska et al. 2016, Boi et al. 2020), in these studies, they reported that increased heavy metal concentration causes decrease in plant biomass and growth. Nonetheless, maize inoculated with B. cereus showed increases in seed germination, plant height and fresh weight as well as nutrient uptake as compared to control and uninoculated plants. Enhanced root growth results due to inoculation of B. cereus under higher concentrations of zinc and thus a positive impact of B. cereus on root growth was observed, which may be due to the stress tolerance capacity of B. cereus (Hassan 2018). Improvement in plant root development under zinc concentration resulted in an increase in nutrient uptake, which increased seed germination, plant height, leaf length, number of leaves, root length and plant fresh weight. Inoculated seeds showed an increase in leaf growth and number of leaves as compared to un-inoculated plants grown in the soil supplemented with zinc.
We argue that gibberellins play a vital role in plant growth development. Scientific data suggests that B. cereus helps the plant by producing different plant promoting hormones i.e gibberellic acid (GA). Therefore, the increase in some plant growth parameters due to inoculation of B. cereus might be because of production of certain plant promoting enzymes, which can stimulate seed germination by enhancing growth ability (Finch-Savage and Leubner-Metzger 2006, Tuan et al. 2018). Gibberellins increase leaf area by suppressing peroxidase secretion (de Souza and MacAdam 2001), and induce cell mitotic division (Takatsuka and Umeda 2014, Fonouni-Farde et al. 2019).[REMOVED HYPERLINK FIELD] Moreover, some researchers suggested that B. cereus induces production of indole acetic acid (IAA), which may help to increase root length, plant height, and leaf area (Shafi et al. 2017). Henceforth, phytohormones produced by B. cereus might have played vital role in maize growth. Zn uptake decreased in B. cereus-inoculated maize plants. Common logic suggests that microbes including Bacillus species have ability to convert Zn into smaller, inaccessible and immoveable form in the soil (Saravanan et al. 2004, Li et al. 2016). Another explanation for reduced Zn uptake is production of metal binding proteins in the rhizosphere. Bacillus cereus produces metal binding proteins to counter heavy metal toxicity and these proteins have ability to restrict phytoavailability of heavy metals (Islam et al. 2014). We also demonstrated the accumulation of macro and micronutrients in maize plant owing to B. cereus. Bacillus cereus showed increase uptake of Cu, Mn, Ni, Na, Cr, Fe, Ca, and Mg in maize plant. Tiwari et al. (2013) also demonstrated that Bacillus species can enhance the phytoavailbility of nutrients Fe, Cd, Pb, Cr, Ni, Cu.
It is concluded from the present investigation that maize seeds inoculated with B. cereus, in the presence of 20 mg kg-1 of zinc showed significant increases in seed germination, plant height, leaf length, number of leaves; however, higher concentrations of zinc in uninoculated seeds indicated reduction in plant growth. Furthermore, the uptake of zinc in maize is decreased in the presence of B. cereus in the soil, which reduces the mobility of zinc leading to lower zinc accumulation in the maize plant. Our study demonstrated the positive impact of B. cereus on maize growth at different levels of Zinc toxicity. The present investigation revealed that B. cereus inoculation can be used as bio-fertilizer in Zinc-stressed soil.