1. Introduction
Crop plants, such as rice, maize, wheat, soybean, and sorghum, are widely cultivated for food and feed in large quantities. The excessive use of agricultural land affects crop yield, and a wide variety of biotic and abiotic stresses can decrease the growth of these plants. The most important challenge in agriculture is to increase the nutrient use efficiency (NUE) and crop productivity through better crop management [
1]. To achieve a high yield of crop plants in agricultural practice, a large variety of chemical fertilizers are used, with serious consequences, such as deterioration of the soil and environmental quality, affecting the health of living organisms. The use of biofertilizers based on plant-growth-promoting rhizobacteria (PGPR) is an alternative method for sustainable agriculture [
2]. These bacterial strains can stimulate the growth and productivity of plants by improving the absorption and assimilation efficiency of nutrients and abiotic stress tolerance. Generally, biostimulants affect primary metabolism by increasing photosynthetic activity and generating compounds and can also induce secondary metabolism by activating specific biosynthetic pathways [
3].
After molecular communication, PGPR colonizes the roots and rhizosphere of plants and can increase plant growth, development, and nutrient use efficiency. However, chemical fertilizers decrease their number and persistence [
1,
4]. The symbiotic and free-living beneficial microorganisms belong to the genera
Rhizobiaceae,
Pseudomonas,
Bacillus,
Azotobacter,
Arthrobacter,
Serratia,
Variovorax, and
Azospirillum [
1,
5].
PGPR help adjust the growth, development, and defense of plants by promoting plant growth through a variety of mechanisms, including biological nitrogen fixation, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, phosphate solubilization, siderophore production, and hormonal compounds (indole-3-acetic acid, ethylene, gibberellin, and cytokinin) [
6,
7].
Plants need several nutrients for their normal growth and development, such as macro- (Ca, Mg, O, C, H, S, N, P, K) and micronutrients (B, Cu, Mn, Fe, Mo, Zn, Ni, Co, Cl) [
7]. In addition, other metals are naturally present in the soil as compounds or simple ions. Trace metals are elements derived from parent rock and are found in the Earth’s crust; they have metallic characteristics such as conductivity, stability, and ligand specificity [
8]. Anthropogenic activities, such as manufacturing and mining metals, pesticide application, and irrigation with untreated sewage water and sludge can cause environmental problems in the soil [
9,
10]. The most commonly used trace metals are As, Cr, Zn, Cu, Cd, Hg, Al, Be, and Pb [
10].
Cd is considered the most phytotoxic trace element because it is highly soluble in water; therefore, it is easily taken up by plants. Thus, Cd enters the food chain and causes serious human health hazards. Cadmium generally occurs in the lithosphere (0.2 mg/kg), sedimentary rocks (0.3 mg/kg), and the soil (0.53 mg/kg). The use of phosphate fertilizers is one of the major anthropogenic Cd sources in agricultural soils, and the concentration of Cd in phosphate fertilizers can reach the range of 0.1–120 mg Cd (kg P
2O
5)
−1. The highest cadmium concentrations in soil were reported in France (16.7 mg/kg), Belgium (7.61 mg/kg), and China (7.43 mg/kg) [
11]. Sewage irrigation has contributed to Cd pollution in China over the past six decades, with more than 125.893 tons of Cd released into the environment [
12].
The toxic effects of Cd on most plants range between 5 and 10 μg Cd/g dry matter [
11]. Even at low concentrations, cadmium can severely alter several enzyme activities involved in the Calvin cycle, carbohydrate and phosphorus metabolism, and CO
2 fixation. These effects result in stunted growth, chlorosis, leaf epinasty, alterations in chloroplast ultrastructure, inhibition of photosynthesis, pollen germination and tube growth, the induction of lipid peroxidation, the disruption of antioxidant machinery, and alterations in nitrogen and sulfur metabolism [
13].
In contrast to Cd, the essential nutrient Zn plays an important role in the regulation of several physiological and biochemical processes in plants [
14]. Cadmium and zinc have similar chemical and physical properties; therefore, these two metals compete for cell membrane transporters involved in uptake, translocation, sequestration, and remobilization [
15]. The zinc concentration in soil varies between 10 and 300 mg/kg, and the average zinc content is approximately 50–55 mg/kg. Most plants contain high concentrations (30–100 mg/kg); however, concentrations higher than 300 mg/kg are generally toxic to plants [
16]. On the other hand the majority of soils are zinc deficient; for example, in Indian soils, 0.01–52.9 mg/kg zinc is available, which is less than 1% of total required zinc content for plants [
17]. Therefore, high concentrations of toxic trace metals, especially cadmium in soil, or a deficit of essential micronutrients such as zinc, can be an abiotic stress factor for plants.
PGPRs have mostly been used to assist plants in the uptake of large amounts of nutrients from the soil environment, as well as in the bioremediation of organic metal pollutants. Bacteria play a role in the immobilization of trace elements and limit their translocation to plants through precipitation, complex formation, and adsorption.
The aims of this study were multiple: (i) the isolation of indigenous trace-metal-tolerant bacterial strains; (ii) the assessment of the PGP activity of isolated bacterial strains under stress conditions; (iii) the inoculation effect of trace-metal-tolerant PGP bacteria on seed germination, plant growth parameters (length, weight, and dry matter content), physiology (photosynthetic activity), stress management (GPOX activity), and the accumulation and translocation of Cd and Zn in trace-metal-treated maize; (iv) the evaluation of trace metal uptake in the microbially inoculated maize.
2. Materials and Methods
2.1. Isolation of Trace Metal Resistant Bacterial Strains
Microbial isolates were recovered from a trace-metal-polluted soil from a spoil tip (Bălan area, Romania) using the serial dilution procedure. The isolation of soil bacteria was accomplished using agar media containing trace metals (0.5 mM Cd2+ + 1 mM Zn2+). One mL of soil suspension from dilutions 10−1–10−10 was added to sterile Petri plates and incubated at 28 °C for 72–120 h. Following the incubation, distinct colonies were picked up and transferred to Nutrient agar media and stored at 4 °C in a refrigerator for later use. A total of 13 isolates were obtained.
2.2. Assessment of PGP Traits under Trace Metal Stress
The mobilization ability of organic and inorganic phosphorus and the production of siderophores, indole acetic-3-acid (IAA), and exopolysaccharide (EPS) of trace-metal-tolerant bacterial strains were determined. These properties are the basis for the selection of bacterial strains for plant experiments.
2.2.1. Determination of Siderophore Production
The ability of the bacterial strains to produce siderophores was examined on solid culture media containing chrome azurol S (CAS), as described by Oldal et al. (2002), amended with trace metals [
18]. Bacterial suspensions (OD
590 = 0.3) were obtained in physiological solution from cultures grown overnight. The media were inoculated with 10 µL of the bacterial suspension using spot inoculation. Samples were prepared in triplicate and incubated at 28 °C for 72 h. The plates were examined for the development of a yellow zone (halo) around the colonies, indicating the release of siderophores. The diameter of the halo zone was measured using digital calipers.
2.2.2. Mobilization of Organic and Inorganic Phosphorus
Organic phosphorus mobilization ability was tested on solid culture media containing Na-phytate (glucose 15 g/L, (NH4)2SO4 5 g/L, KCl 0.5 g/L, MgSO4·7H2O 0.1 g/L, NaCl 0.1 g/L, CaCl2·2H2O 0.1 g/L, FeSO4·7H2O 0.01 g/L, MnSO4 0.01 g/L, Na-phytate 5 g/L, agar 20 g/L) amended with trace metals. The medium was inoculated with 10 µL of the bacterial suspension (OD590 = 0.3) using spot inoculation and incubated at 28 °C for 72 h. The plates were examined for the development of a clear zone (halo) around the colonies, indicating the production of phytase. The diameter of the halo zone was measured using digital calipers. The samples were prepared in triplicate.
Inorganic phosphorus mobilization was determined using spot inoculation on Pikowskaya solid medium (yeast extract 0.5 g/L, glucose 10 g/L, calcium phosphate 5 g/L, ammonium sulfate 0.5 g/L, KCl 0.2 g/L, MgSO
4 0.1 g/L, MnSO
4 0.0001 g/L, FeSO
4 0.0001 g/L, agar 20 g/L) amended with trace metals, as mentioned before [
19].
2.2.3. Evaluation of Indole-3-Acetic Acid Production
The indole-3-acetic acid (IAA) secretion by the isolated bacterial strains was measured using the methodology described by Becze et al. (2021) [
20]. Bacterial strains were grown in 5 mL TSB (tryptone–soy broth) amended with trace metals and incubated at 28 °C and 150 rpm for 72 h in a shaking incubator. After incubation, 1.5 mL of the bacterial cultures was centrifuged (5.000 rpm, 15 min) and 1 mL of the supernatant was mixed with 2 mL of Salkowsky reagent (300 mL of 98% H
2SO
4, 15 mL of 0.5 M FeCl
3, and 500 mL of distilled water). For color development, the prepared samples were incubated at room temperature in the dark for 30 min. The absorbance of the samples was measured at 570 nm by measuring the pink color that emerged during the reaction. The indole-3-acetic acid concentration was determined using IAA as a reference (calibration curve).
2.2.4. Swimming and Swarming Motility
The swimming and swarming motility of the isolated bacterial strains was determined using Luria Bertani (LB) agar media (NaCl 10 g/L, peptone 10 g/L, yeast extract 5 g/L, pH = 7). For the swarming test, the medium was supplemented with 0.5% agar, and for the swimming test, it was supplemented with 0.3% agar [
21]. The bacterial strains were grown overnight on Nutrient agar media, and the Petri plates were inoculated in triplicates. After five days of incubation at 28 °C, the diameters of swimming and swarming zones were measured, and the results were compared.
2.3. Seed Germination Experiments
The selected bacterial strains were used in the maize germination experiments. To sterilize maize seeds, they were first treated with absolute ethanol for one minute, followed by three washes with distilled water. The seeds were then treated with a hypochlorite solution for 20 min and washed three times with distilled water. The surface-sterilized seeds were soaked for 30 min in bacterial suspension (OD
590 = 1) for bacterial treatment and in nutrient broth for the control. Germination was carried out in Petri dishes (10 seeds each, 8 replicates) in the dark at 28 °C for eight days, and the seeds were sprinkled twice with distilled water (2 mL) on days four and six. The number of germinated seeds was counted every two days during the experiment. At the end of the germination experiment, the following germination indices were calculated: germination rate (G), mean germination time (MGT), coefficient of velocity of germination (CVG), mean germination rate (MGR), germination rate index (GRI), germination index (GI), and Timson Germination Index (TGI) [
22].
G = (total seeds germinated at the end of trial/total number of initial seeds) × 100.
where F is the number of seeds germinated on day x.
where N is the number of seeds germinated every day and T is the number of days from seeding corresponding to N.
where T is the mean germination time and CVG is the coefficient of velocity.
where G1 is the germination percentage on day 1, G2 is the germination percentage on day 2; and so on.
where N1, N2, …, N8 are the number of germinated seeds on the first, second and subsequent days until 8th day, and the multipliers (for example 8 and 6) are the weights assigned to the days of germination.
where G is the percentage of seeds germinated per day and T is the germination period.
2.4. Identification of Bacterial Isolates
Two bacterial strains were selected from the 13 trace-metal-tolerant bacterial strains, which were marked as BB2.A.1 and BB2.1.1. Pure bacterial cultures were obtained on Nutrient agar plates by the streak plate method. Bacterial cells were stained with methylene blue (1%) and observed under an optical microscope (Olympus BX53, Olympus Corporation, Tokyo, Japan). Pictures were obtained using labSens 1.1. software. These bacterial strains were identified by 16S rDNA sequence analysis. The sequences were edited and aligned using Chromas 2.6.6. software. The sequences were compared with those found in the NCBI database and deposited in the EMBL database. The phylogenetic tree was constructed using MEGA 11.0 software.
2.5. Effect of PGP Bacterial Strains in Plant Growth Experiments
The effects of PGP bacterial strains were tested on maize (Zea mays MV3122 certified variety) plants under stress conditions. Maize seeds were surface sterilized with absolute ethanol for 1 min and then washed with distilled water three times. The seeds were then soaked in a hypochlorite solution for 20 min and rinsed several times with distilled water. Germination was performed in the dark on wet filter paper in Petri dishes at 28 °C for five days, and water was replenished once with 2 mL of distilled water. In the next phase, the sterilized soil (121 °C, 40 min) was placed in sterile polypropylene boxes. Twenty equally developed plant seeds were planted in each pot, and the bacterial suspension (0.5 mL/seed, OD590 = 1.5) was pipetted into the seed environment. After that, the soil was treated with different concentrations of trace metal (0.5 mM Cd2+, 1 mM Zn2+, 0.1 mM Cd2+ + 0.5 mM Zn2+). Finally, the seedlings were placed in a plant growth chamber (Sanyo MLR-351 Versatile Environmental Test Chamber, SANYO Electric Co., Ltd., Osaka, Japan) for 21 days under controlled conditions: 22 °C, 70% relative humidity, and 12 h/day 2500 lx light.
2.6. Plant Growth Parameters
Plant growth parameters were measured at the end of the experiment. The weights of the shoots and roots were determined using a digital scale (KERN 440-33, KERN & Sohn GmbH, Balingen, Germany), and the length was measured using a digital caliper. The numbers of leaves and root hairs were also counted.
2.7. Chlorophyll Content of the Maize Plants
The extraction of total chlorophyll (chlorophyll-α and chlorophyll-β) was carried out using an ammonia–acetone solution (1 L of 80% acetone, 1.5 mL of 25% ammonia solution). First, the plant leaf samples (0.15 g) were destroyed in a mortar and pestle with an ammonia acetone solution. The plant extracts were centrifuged at 5000 rpm for 5 min in centrifuge tubes, and the supernatants were transferred into new tubes and filled up to 5 mL with the ammonia–acetone solution. The absorbance of the prepared samples was measured with a spectrophotometer (HACH DR 6000, Hach Company, Loveland, Colorado, USA) at 800, 730, 664, and 647 nm. Measurements at 800 nm and 730 nm are required as correction factors for acetone absorption; 664 nm is chlorophyll-α, and 647 nm is the chlorophyll-β absorption maximum. The amounts of chlorophyll-α and chlorophyll-β were calculated from the absorbance values based on the method described by Porra et al. [
23].
2.8. Determination of GPOX Enzyme Activity
During sample preparation, 0.15 g of plant shoots and roots were weighed, which was first disrupted in a mortar with a pestle, and then treated with 1 mL of QB buffer (pH 7.8, 100 mM KPO4, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, DTT added only before use) and centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatant was stored at −20 °C.
The total protein concentration was determined using the Bradford method [
20]. Guaiacol peroxidase (GPOX) enzyme activity was measured using a spectrophotometric method. The reaction mix contained the following solutions: 885 µL 1 M Na-phosphate buffer, 50 µL 50 mM guaiacol solution, 50 µL H
2O
2, and 15 µL extracted plant sample. The absorbance was measured (Agilent Technologies Cary 60 UV-VIS, Agilent, Santa Clara, CA, USA) at 436 nm after 5 s for 15 min [
24].
2.9. Determination of Trace Metal Accumulation in Plants
To determine the accumulation of trace metals, samples were dried to a constant weight at 105 °C in a heat sterilizer. The pre-dried samples were placed in a porcelain jar and incinerated in a Gefran 1001 incinerator at 450 °C for 4 h, with a continuous increase in temperature from 250 °C to 100 °C per hour. The resulting ash was dissolved in 5 mL of 25% HNO
3, filtered through a filter paper, and filled with distilled water to a volume of 10 mL [
20]. The trace metal content of the solution was determined using a Varian Spectra AA 110 atomic absorption spectrophotometer. Concentrations were determined using a calibration curve obtained using standard solutions [
20]. To compare the root-to-shoot translocation of Zn and Cd between the different treatments, the translocation factor (TF) was determined as the ratio of shoot/root Zn and Cd concentrations.
2.10. Evaluation of Inoculum Persistence
The number of bacterial cells was determined by re-isolation. The importance of re-isolation was to detect the presence of bacteria after plant experiments and to compare the changes in the number of bacterial cells. The number of colonies was determined using the spread plate method for colony counting.
2.11. Statistical Analysis
Data presentation and statistical analysis were carried out using Microsoft Excel 2019 and the Past.exe 4.03 statistical program. One-way ANOVA was used to compare the data series between treatments and controls, followed by Tukey’s test and Pearson correlation analysis.
3. Results
3.1. Bacterial Strains
In this study, 13 bacterial strains from trace-metal-polluted soil from a spoil tip (Bălan area, Transylvania, Romania) were isolated, and 11 of the isolates were assessed for their beneficial traits. Five strains proved to be strong siderophore producers, seven strains were able to mobilize inorganic phosphorus, and four strains produced higher amounts of IAA. All strains produced EPS and showed both types of motilities (
Table 1). Each beneficial trait was converted to a value from 0 to 5, the values were added together and compared. From the 11 isolated bacterial strains, two strains (BB2A1 and BB211) with the highest values based on all beneficial traits were selected for further analysis.
Both selected bacterial strains were identified using 16S rDNA sequence analysis performed by BIOMI Ltd. (Godollo, Hungary). GenBank accession numbers are given in parentheses after the species names. The two bacterial strains identified were
Pseudomonas koreensis BB2.A.1 (OP748250) and
Serrratia liquefaciens BB2.1.1 (OP748249). The cell and colony morphologies of both the strains are presented in
Figure 1. The phylogenetic trees of the two bacterial strains are presented in
Figure 2 and
Figure 3.
3.2. Plant Growth Promoting Potential of the Selected Strains under Trace Metal Stress
The siderophore production ability of both strains was modified in the presence of trace metals. The presence of Zn and lower Cd concentrations with Zn resulted in significantly higher values compared to the control (
Table 2). The presence of 0.5 mM Cd lowered siderophore production in both strains. The phosphorus mobilization ability of the selected strains was also studied at different trace metal concentrations. Organic phosphorous mobilization ability was observed only in plates containing trace metals. Inorganic phosphorus mobilization in
P. koreensis BB2.A.1 was lowered by the presence of 1 mM Zn and 0.5 mM Cd, whereas in the presence of both trace metals, the halo diameter was similar to that observed in the control samples. The inorganic phosphorus mobilization ability of the
Serratia liquefaciens BB2.1.1. strain was not significantly affected by the presence of trace metals. Indole acetic acid production was slightly affected by the presence of both metals in both the bacterial strains. Exopolysaccharide production was completely inhibited by Cd in
P. koreensis BB2.A.1, whereas the higher Cd concentration inhibited EPS production in
Serratia liquefaciens BB2.1.1. too. Both the stimulatory and inhibitory effects of trace metal treatments on plant-growth-promoting characteristics were observed; however, total limitation was observed only in EPS production. Most of the PGP characteristics were retained despite the presence of Zn and/or Cd. Regarding bacterial trace metal stress reduction, the two important traits that may prevent trace metals from entering the plant cell, siderophore production and EPS production, were affected by metal concentration.
3.3. Germination of Bacteria-Treated Maize Seeds
Seed germination and seedling emergence are two of the most critical and delicate stages in the life cycle of a crop, as they lay the foundation for the plant’s future growth and development. Therefore, the selected bacterial strains were used to treat maize seeds.
Table 3 shows the mean variations for the seven germination indices as affected by seed inoculation. Seeds treated with the
Pseudomonas koreensis BB2A.1. strain increased by 2.47%, whereas inoculation with
Serratia liquefaciens BB2.1.1 increased the seed germination rate by 12.47%. The average coefficient of velocity of germination (CVG) was 21.76 for control seeds and 24.43 (112.27%) and 29.99 (137.72%) for
P. koreensis BB2A.1.- and
S. liquefaciens BB2.1.1-treated seeds, respectively. The Timson Germination Index (TGI) was higher in inoculated seeds than in the control. The highest seed germination indices were achieved when inoculated with
S. liquefaciens BB2.1.1, followed by that of
P. koreensis BB2A.1. inoculation. However, the differences in the values were not statistically significant.
3.4. Growth and Development of the Maize Plants with PGPR Inoculation
3.4.1. The Length of the Shoot and the Root of Maize Plants
Figure 4 and
Table 4 shows the shoot and root lengths of maize plants. The
Pseudomonas koreensis BB2.A.1 bacterial strain reduced the shoot and root length growth compared to the treated controls (1 mM Zn, 0.5 mM Cd, 0.5 mM Zn + 0.1 mM Cd). Samples treated and inoculated with the
Pseudomonas koreensis BB2.A.1 bacterial strain were slightly longer than the inoculated control in the case of the shoot, but the root length decreased in the presence of used treatments.
The Serratia liquefaciens BB2.1.1 bacterial strain increased (by 6.66%) the shoot length of maize plants when 1 mM zinc concentration was applied, compared with the uninoculated and treated control. Nonetheless, in the presence of cadmium and combined treatment, the S. liquefaciens BB2.1.1 bacterial strain decreased shoot length (by 11.51%, 14.63%). However, these differences were not statistically significant. A decreasing tendency can be observed in the case of root length, because the S. liquefaciens BB2.1.1 bacterial strain and the concentrations used reduced the examined parameter (by 10.37%–45.12%) compared to the inoculated and uninoculated controls.
3.4.2. The Weight of the Shoot and the Root of the Maize Plants
The shoot and root biomass results are shown (
Figure 5 and
Table 5). The
Pseudomonas koreensis BB2.A.1 bacterial strain had a similar effect on the shoot and root biomass, and the bacterial inoculation decreased the biomass in the presence of 1 mM Zn treatment and increased it in the case of the 0.5 mM Cd and combined treatment (0.5 mM Zn + 0.1 mM Cd). These differences were observed compared to the uninoculated and treated samples and relative to the inoculated control.
The Serratia liquefaciens BB2.1.1 bacterial strain decreased the shoot biomass in all treatments compared to the uninoculated and treated samples, whereas the root biomass decreased only in the presence of 0.5 mM Cd treatment. The combined treatment increased the root biomass, whereas the zinc treatment had no effect on this parameter. The treatments had a different effect on the root biomass compared to the inoculated control, because the zinc concentration increased (22.41%), while the Cd (11.11%) and combined treatments (4.44%) slightly decreased the biomass values.
3.4.3. The Dry Matter Content (DMC) of the Shoot and the Root of the Maize Plants
The DMC results are shown in
Figure 6. Zinc treatment (1 mM Zn) resulted in a remarkably high (50%) dry matter content (DMC) in the shoot of maize plants inoculated with
Pseudomonas koreensis BB2.A.1 bacterial strain, compared to the uninoculated and treated control, as well as to the uninoculated and untreated controls. The differences were statistically significant. Inoculation with Cd (41.66%) or combined (30%) treatment resulted in a decreasing trend of DMC in the shoot, compared to the uninoculated and treated samples, as well as to the uninoculated and untreated control (12.5%). In the case of root DMC, the same results were observed: inoculation increased the DMC in the presence of zinc treatment (8.33%), whereas cadmium (25%, 45.45%) and combined treatment (36.36%) lowered DMC, compared to the uninoculated and treated samples, as well as to the uninoculated and untreated control.
When inoculated with Serratia liquefaciens BB2.1.1, the bacterial strain increased DMC in the shoot in the presence of zinc treatment (27.27%), whereas the cadmium (8.33%) and combined treatment (20%) resulted in lower values compared to the uninoculated and treated samples. In the presence of bacteria, the Zn and Cd treatments increased (27.27%), whereas the combined treatment did not influence the DMC values in the shoot compared to the uninoculated and untreated controls. In the case of the root DMC values, we observed that inoculation increased the parameter in the presence of zinc and combined treatment (18.18%), while the cadmium concentration decreased (27.27%) the DMC values compared to the uninoculated and treated samples. Regarding the uninoculated and untreated controls, the DMC in the roots varied as follows: inoculation decreased the DMC in the presence of zinc (18.18%) or combined treatment (9.09%).
3.5. Determination of Trace Metal Accumulation in Plants
In the control and samples treated with both bacterial strains (lower Cd concentrations), the presence of Cd in plant tissues was not detectable. Although the measured values of Cd accumulation in the shoots and roots of maize plants in different treatments showed changes, these were not statistically significant (
Figure 7). The presence of the
Pseudomonas koreensis BB2.A.1 bacterial strain increased the accumulation of toxic metals in the shoot (47.61%) and the root (75.93%), in the case of the 0.5 mM Cd treatment, compared to the uninoculated and treated samples. The
Serratia liquefaciens BB2.1.1 bacterial strain decreased (36.36%) the accumulation of Cd in the shoot in the presence of the 0.5 mM Cd treatment, compared to the uninoculated and treated samples. The presence of
S. liquefaciens BB2.1.1 slightly increased (11.11%) the amount of accumulated cadmium in the roots in the case of the Cd treatment, whereas in the presence of the combined treatment, it decreased (40.74%) the accumulation of cadmium.
The results of zinc accumulation measurements are shown in
Figure 8. The
Pseudomonas koreensis BB2.A.1 bacterial strain slightly increased the accumulation of zinc in the shoot and roots in the presence of Zn treatment, while in the case of the combined treatment, a slightly decreasing trend was observed compared to the uninoculated and treated samples. A statistically significant difference was observed only in Zn-treated samples (1 mM Zn), where the
Serratia liquefaciens BB2.1.1 bacterial strain stimulated the accumulation of Zn in the shoot compared to the inoculated control (131.49%) samples. Inoculation with the
Serratia liquefaciens BB2.1.1 bacterial strain and the Zn treatment significantly increased zinc accumulation in the roots compared to that in the inoculated control (101.56%).
A plant is efficient in metal translocation from the root to the shoot when the translocation factor (TF) is higher than one, owing to its efficient metal transport system. If the TF value is less than one, metal transfer is inhibited.
The translocation factor values for Cd were not obtained in the case of the control samples and combined treatments. The translocation values for control and inoculated samples treated with 0.5 mM Cd were low (0.08–0.16) because of the inhibited translocation from the root into the shoot.
The translocation of zinc as an essential micronutrient was determined, and the transport of accumulated zinc from root into the shoot was realized to a large extent (1.65 for the control, 1.14 for 1 mM Zn, and 0.64 for the combined treatment). The P. koreensis BB2.A.1 bacterial strain increased the TF of zinc in the case of 1 mM Zn (14.28%) and combined treatments (34.69%). The S. liquefaciens BB2.1.1 bacterial strain decreased the TF in the presence of 1 mM Zn treatment (46.49%), whereas the combined treatment increased the TF (47.10%). Both bacterial strains increased the TF of Zn in the combined treatment, stimulating the translocation of essential micronutrients to the shoot.
3.6. Chlorophyll Content of the Maize Plants
No statistically significant differences were found in the total chlorophyll content of the maize plants (
Figure 9). The
Pseudomonas koreensis BB2.A.1 bacterial strain decreased (5.27–25%) the chlorophyll content of the maize plants in all the treatments, compared to the uninoculated and treated samples and also compared to the inoculated control. The
S. liquefaciens BB2.1.1 bacterial strain slightly increased the chlorophyll content in the case of the 1 mM Zn (3.16%) and 0.5 mM Cd treatment (10.38%), whereas in the presence of the combined treatment, a decrease (20.02%) was observed. Inoculation with
S. liquefaciens BB2.1.1 in all applied treatments, compared with the inoculated control, increased the chlorophyll content (26.23–30.61%).
The Chla/b ratio showed a reverse trend compared to the total chlorophyll. Both bacterial strains increased the Chla/b ratio of the control samples, whereas when the trace metal treatments were applied, the inoculation with P. koreensis BB2.A.1 bacterial strain increased the ratio in all treatments.
3.7. Determination of GPOX Enzyme Activity
GPOX enzyme activity in the roots of maize plants was higher than that in the shoots. The differences in GPOX enzyme activity in the shoots of the maize plants were not statistically significant (
Figure 10). The most significant effect on GPOX values in the shoots was caused by
Pseudomonas koreensis BB2.A.1 bacterial strain, which increased the enzyme activity in the presence of 1 mM Zn (44.44%) and 0.5 mM Cd (12.50%) compared to the inoculated control. Inoculation with the above-mentioned bacterial strain in the case of the combined treatment had no significant effect on GPOX activity in the shoot compared to the inoculated control. The
Pseudomonas koreensis BB2.A.1 inoculation lowered the root GPOX enzyme activity in the presence of the 1 mM Zn treatment and the combined treatment compared to uninoculated, treated samples (68.62% and 29.72%, respectively) in the inoculated control (60.77% and 36.58%, respectively). Inoculation and Cd treatment increased GPOX activity in the roots compared to that in the uninoculated and treated samples (29.41%).
The Serratia liquefaciens BB2.1.1 bacterial strain increased the GPOX activity in the shoot in the case of Zn (16.66%) and combined treatment (16.66%), whereas the Cd treatment (42.85%) decreased the activity compared to the uninoculated and treated samples. Bacterial inoculation decreased GPOX activity in the shoot in the presence of Cd (33.33%) compared to the inoculated control. The Serratia liquefaciens BB2.1.1 bacterial strain decreased GPOX activity in the roots in the presence of Zn (39.21%) and combined treatment (56.75%) compared to the uninoculated and treated samples. Regarding the inoculated control, the zinc treatment increased (16.12%), while the combined treatment (38.46%) decreased GPOX enzyme activity in the root.
3.8. Evaluation of Inoculum Persistence
Inoculum persistence was evaluated to monitor the change in the number of inoculated bacteria during the plant experiment. At the beginning of the experiment, the samples were inoculated with 816 × 106 CFU/mL Pseudomonas koreensis BB2.A.1 bacterial strain and 231 × 106 CFU/mL of Serratia liquefaciens BB2.1.1 bacterial strain.
Both the bacterial strains (Pseudomonas koreensis BB2.A.1 and Serratia liquefaciens BB2.1.1) were successfully isolated at the end of the plant experiments from control samples to the following extent: 968 × 106 CFU/mL and 166.4 × 106 CFU/mL. In the presence of 1 mM Zn, 0.5 mM Cd, and 0.5 mM Zn + 0.1 mM Cd treatments, the Pseudomonas koreensis BB2.A.1 bacterial strain was isolated at the following colony densities: 928 × 106 CFU/mL, 536 × 106 CFU/mL, and 1056 × 106 CFU/mL. The Serratia liquefaciens BB2.1.1 bacterial strain was isolated with the following concentrations of trace metals: 1 mM Zn (312 × 106 CFU/mL), 0.5 mM Cd (424 × 106 CFU/mL), and 0.5 mM Zn + 0.1 mM Cd (1248 × 106 CFU/mL). Two substantial differences in microbe number were observed: Cd treatment (0.5 mM Cd) decreased the quantity of the Pseudomonas koreensis BB2.A.1 bacterial strain at 34.31%, whereas the combined treatment increased the number of colony forming units of Serratia liquefaciens BB2.1.1.
3.9. Pearson Correlation Analysis
Figure 11 shows the Pearson correlation results for the shoot (a) and root (b) parameters in the context of
Pseudomonas koreensis BB2.A.1 bacterial strain inoculation. The growth parameters (length, biomass, and total biomass) showed a positive correlation between them, because biomass changed in the same way with the length and, respectively, with the total biomass. The shoot biomass decreased compared to the DMC values; that is, the DMC and shoot biomass had a strong negative correlation. Similar results were observed for total biomass and DMC, which can be explained by the higher water content of the shoot.
Considering the growth and physiological parameters, a positive correlation was observed between biomass, DMC, and the chlorophyll content of the shoot. GPOX activity was strongly negatively correlated with the shoot growth parameters (length and biomass). This can be explained by the fact that if there is less stress in the shoot, growth is greater.
The growth parameters and metal accumulation in the majority were positively correlated. Zn accumulation was positively correlated with DMC values, whereas Cd accumulation showed a strong positive correlation with the total biomass of the plants. The two metal accumulation values were strongly negatively correlated with the chlorophyll content of the plants. In the presence of high metal concentrations, chlorophyll faded into the background.
In the case of the root, we observed a similar tendency between growth parameters (length, biomass, total biomass, and DMC) as in the case of the shoot, because DMC remained strongly negatively correlated with biomass. These results indicate that the difference in weight originates from the higher water content of the samples. The root relationships are the opposite of those experienced in the shoot.
Physiological and growth parameters were positively correlated. The GPOX activity values varied just as the growth parameters (length, biomass, and total biomass) did, indicating that the presence of the antioxidant system had a stimulating effect on plant growth parameters.
Considering the growth and accumulation parameter results, we observed the following correlations. A negative correlation was observed between Zn and Cd accumulation and the growth parameters (length and DMC). This phenomenon occurred because the accumulation of these metals in the shoot was lower than that in the roots. The relationship between root biomass and Cd accumulation in roots showed the same positive correlation as in the case of shoots. This correlation can be seen in the fact that Cd mostly accumulated in the roots, increasing the biomass. Zn accumulation was negatively correlated with GPOX activity. Zinc is an essential micronutrient that does not cause high stress in plants.
In
Figure 12, Pearson correlation results can be observed for the shoot (a) and root (b) parameters based on
Serratia liquefaciens BB2.1.1 bacterial strain inoculation. Biomass was negatively correlated with DMC values because of the high water content of shoots. Growth (length, biomass, total biomass, and DMC) and physiological parameters (GPOX, chlorophyll) were not strongly correlated.
Growth parameters (length, biomass, total biomass, and DMC) and accumulation were strongly negatively correlated. In the case of high metal accumulation, the growth parameter values were lower, whereas the low accumulation values produced higher growth.
The GPOX activity of the root was positively correlated with root biomass, and both parameter values decreased. The root growth parameters and metal accumulation were strongly negatively correlated, as in the case of the shoot.