Ameliorative effect of indole-3-acetic acid- and siderophore-producing Leclercia adecarboxylata MO1 on cucumber plants under zinc stress

This study investigated the plant growth-promoting effects of Leclercia adecarboxylata MO1 for Zn stress mitigation and plant growth improvement in Zn-contaminated soil. Results demonstrated that L. adecarboxylata MO1 produced siderophores that could solubilize Zn and silicate, had a tolerance to elevated levels of Zn supplementation (2 and 5 mM) in growth mediums, and produced significant amounts of indole-3-acetic acid (IAA). It was also found to promote plant growth under both control conditions and Zn toxicity (2 and 5 mM). Furthermore, L. adecarboxylata MO1 positively regulated physiochemical attributes by decreasing hydrogen peroxide (H2O2) and Zn uptake in both roots and shoots, improving antioxidant systems (e.g. catalase (CAT), peroxidase (POD), polyphenol peroxidase (PPO), superoxide anion (SOA), lipid peroxidation (MDA), and glutathione (GSH)), and reducing stress-responsive endogenous abscisic acid (ABA) and salicylic acid (SA) in plants grown under Zn toxicity of 2 and 5 mM, compared with non-inoculated plants. ARTICLE HISTORY Received 25 August 2020 Accepted 9 December 2020


Introduction
Rapid industrialization has led to remarkable increases in metal pollutants in the environment that have even been so high to contaminate water and render the soil unfit for agricultural use (Rai et al. 2019;Francová et al. 2020;Hou et al. 2020). These increases in heavy metal toxicity in agricultural land pose a serious threat to both human and plant health (Okereafor et al. 2020), as metal toxicity directly and indirectly affects plant growth and production (Tomáš et al. 2019). Among the various heavy metals, zinc (Zn) is excessively used in different industries without recovery, which has resulted in rapid increases in Zn toxicity that has become a serious threat (Nagajyoti et al. 2010;Mishra et al. 2019).
Zn is an important trace element for all living organisms on earth (Frassinetti et al. 2006;Hanif et al. 2017); however, elevated Zn levels are extremely toxic to living organisms (Mossa et al. 2020;Okereafor et al. 2020). Its high accumulation in agricultural soil and its high absorption by plants disturb the metabolism, causes stunted growth, and reduces productivity Bilal et al. 2018). Zn is not a redox element, and therefore, the generation of reactive oxygen species (ROS) when Zn levels are elevated causes oxidative stress to organisms, which is one of the primary reasons why elevated Zn levels eventually evoke plant defense systems (Pandey et al. 2009;Lin and Aarts 2012). Nutritional imbalance, production of stress response hormones, chlorosis, and poor growth are also important features of Zn toxicity (Rout and Das 2009;Nagajyoti et al. 2010).
It is well recognized that soil microbes, particularly plant growth-promoting rhizospheric bacteria (PGPR), can influence plant growth and be used for toxic metal remediation (Kong and Glick 2017;Jan et al. 2019). PGPR mobilize heavy metals by solubilizing and acidifying rhizospheres and expanding root surfaces (Hayat et al. 2010;Pii et al. 2015). PGPR can both increase and decrease the bioavailability of metallic nutrients (Khan and Bano 2018;Uzoh and Babalola 2020). Recently, microbes have been reported to promote plant growth and decrease heavy metal bioavailability and absorption by plants, which has resulted in the safer production of crops under heavy metal toxicity conditions Bilal et al. 2018;Shahzad et al. 2019a). Among vegetable crops, cucumber is one of the top 10 vegetable crops with significant economic importance and nutritional value; it is used for food and medicinal purpose and is sensitive to abiotic stress (Yan et al. 2016;Kim et al. 2019). In South Korea, cucumber is an important part of daily meal and used in several traditional recipes such as soups, kimchee, and salad (Kang et al. 2014). However, cucumber yield and quality are frequently affected by different types of abiotic stresses that cause a decline in cucumber output (Wang et al. 2012;Kang et al. 2014;Nadeem et al. 2016;Al-Harbi et al. 2018;Chen et al. 2020;Kartik et al. 2020). Accordingly, several authors have reported about the use of plant growth-promoting microbes for enhancing the growth and mitigating the abiotic stress in cucumber (Wang et al. 2012;Kang et al. 2014;Nadeem et al. 2016;Kartik et al. 2020) Zinc-solubilizing bacteria are a type of PGPR that produce organic acids, which sequester zinc cations and decrease soil pH (Kumar et al. 2019). It has also been reported that anions may contribute to zinc chelation (Kour et al. 2019;Kumar et al. 2019). Zinc solubilization requires siderophore production and proton oxidoreductive chelating ligands (Chung et al. 2005;Kumar et al. 2019). Leclercia adecarboxylata MO1 is a PGPR used in the present study that has been isolated from tomato rhizospheres and has also been reported to potentially promote plant growth and mediate salinity stress . In this study, we evaluated L. adecarboxylata MO1 for Zn resistance, indole-3-acetic acid (IAA) production, siderophore production, plant growth influence, metal accumulation, antioxidant enzyme activities, and stress response-induced abscisic acid (ABA) regulation under Zn toxicity conditions.

Bacterial growth conditions
The plant growth-promoting L. adecarboxylata MO1 used in the present study was previously isolated and reported for plant growth promotion and salinity stress reliance ). In the present study, L. adecarboxylata MO1 was grown in Luria-Bertani (LB) broth at 28°C in a shaking incubator at a speed of 120 rpm.
Siderophore production, silica and zinc solubilization L. adecarboxylata MO1 was incubated on a chrome azurol S agar (CAS) plate as previously described by Kim et al. (2017) to evaluate its siderophore-producing potential. Briefly, an actively growing single colony of L. adecarboxylata MO1 was streaked onto a CAS plate and incubated at 28°C for 7 days. The formation of yellow zones around the streaked bacteria was considered as a positive sign for siderophore production.

Stress resistance
The Zn+ stress tolerance capabilities of L. adecarboxylata MO1 were evaluated according to a method reported by Shahzad et al. (2019a). Briefly, L. adecarboxylata MO1 was grown in 100 mL LB (Luria-Bertani) broth supplemented with 2 and 5 mM Zn+ and incubated at 28°C in a shaking incubator at a speed of 120 rpm for 5 days. After 5 days, the cell growth was determined by measuring the cell density at OD 600 using a spectrophotometer.
IAA production under Zn toxicity IAA production in the culture medium of L. adecarboxylata MO1 was evaluated under normal conditions, and Zn toxicity (2 and 5 mM) was quantified after 3 days according to a previously described protocol . Briefly, the L. adecarboxylata MO1 culture was centrifuged after 3 days to separate cells into a free culture, which was further acidified to a pH of 2.8 and supplemented with 40 µL [D5]-IAA as an IAA internal standard. The acidified and standard supplemented free culture cells were extracted, methylated, and injected into a GC-MS SIM column for identification and quantification of IAA.

Determination of Zn content in plants
The zinc contents in the shoots and roots of L. adecarboxylata MO1-inoculated and -non-inoculated plants grown under 2 and 5 mM Zn+ stress were extracted and quantified according to the method described by Shahzad et al. (2019a) using inductively coupled plasma mass spectrometry (ICP-MS; Optima 7900DV Perkin-Elmer, USA).

Catalase
Catalase activity (CAT) (EC 1.11.1.6) in L. adecarboxylata MO1-inoculated and -non-inoculated plants was evaluated according to the method described by Bilal et al. (2018). Briefly, 200 mg of plant leaves were ground in 50 mM Tris HCl (pH 7.0), 3 mM MgCl 2 , 1 mM EDTA, and 1.0% PVP and then centrifuged to obtain the supernatant. To the supernatant, 0.5 mL of 0.2 M H 2 O 2 in 10 mM phosphate buffer (pH 7.0) was added, and the resulting absorbance was measured at 240 nm wavelength. The CAT was calculated using a standard curve.

Estimation of peroxidase (POD) and polyphenol peroxidase (PPO) levels
Peroxidase (POD) (EC-Number 1.11.1.7) and polyphenol peroxidase (PPO) (EC-Number 1.10.3.1) levels in L. adecarboxylata MO1-inoculated and -non-inoculated plants under normal and Zn stress (2 and 5 mM) conditions were examined and calculated according to the protocol reported by Bilal et al. (2018). Briefly, 500 mg of plant samples were homogenized in a 0.1 M potassium phosphate buffer (pH 6.8) and then centrifuged at 5000 rpm for 15 min at 4°C to obtain the supernatant. Next, 100 μL of the supernatant was mixed with a reaction mixture (0.1 M potassium phosphate buffer (pH 6.8), 50 μl pyrogallol (50 μM), and 50 μl H 2 O 2 (50 μM)) and incubated for 5 min at room temperature (25°C) to initiate reaction. The initiated reaction was stopped with the addition of 5% H 2 SO 4 solution. The formation of purpurogallin in result of reaction was determined by measuring the absorbance at 420 nm. Similarly, polyphenol peroxidase (PPO) was estimated using the same reaction mixture of POD, excluding H 2 O 2 , and the resulting reaction was measured at 420 nm wavelength (Khan et al. 2019c).

Superoxide anion (SOA)
Superoxide anion (SOA) levels in L. adecarboxylata MO1inoculated and -non-inoculated plants under normal and Zn stress conditions were measured according to the method described by  and Khan et al. (2019b). Briefly, 200 mg of samples was homogenized in an extraction solution (0.05% (w/v) NBT and 10 mM sodium azide (NaN 3 ) in a 0.01 M phosphate buffer (pH 7.0)) and then incubated at room temperature for 60 min. After incubation, the extraction solution was heated at 85°C in a preheated water bath for 15 min and then cooled and vacuum-filtrated. The absorbance of the filtered solution was used to estimate the reduction of exogenously supplied nitroblue tetrazolium (NBT) to determine the amount of superoxide anions at 580 nm, which was calculated using the following formula:

Lipid peroxidation
Lipid peroxidation was measured as malondialdehyde (MDA) (EC) production in L. adecarboxylata MO1-inoculated and -non-inoculated plants under normal and Zn stress (2 and 5 mM) conditions according to the method described by Bilal et al. (2020). Briefly, 500 mg of plant samples was extracted in 10 mM phosphate buffer (pH 7) and then centrifuged to separate the supernatant. To the resulting supernatant, a reaction mixture (0.2 mL of 8.1% sodium dodecyl sulfate, 1.5 mL of 20% acetic acid (pH 3.5), and 1.5 mL of 0.81% thiobarbituric acid aqueous solution) was added and then incubated in preheated boiling water for 60 min. The heated samples were then cooled down to room temperature. Next, 5 mL of butanol:pyridine (15:1 v/v) was added to the reaction mixture, and the resulting upper layer (MDA) was removed and measured at 532 nm wavelength.

Glutathione (GSH)
Glutathione (GSH) (EC 1.8.4.4) level was measured according to the method described previously by Shahzad et al. (2019a). Briefly, 0.2 g of samples was ground and homogenized in 3 mL of 5% trichloroacetic acid (TAC) and then centrifuged to collect the supernatant. Next, 0.1 mL of the supernatant was mixed with 3 mL of 150 mM monosodium phosphate buffer (pH 7.4) and 0.5 mL of Ellman's reagent. The solution mixture was incubated at 30°C for 5 min, and the absorbance was measured at 412 nm.

Abscisic acid
The endogenous ABA content in L. adecarboxylata MO1inoculated and -non-inoculated plants grown under 2 and 5 mM Zn+ toxicity was extracted and quantified according to the method described by Shahzad et al. (2016). Briefly, 0.5 g of freeze-dried plant samples was extracted with 95% isopropanol and 5% glacial acetic acid and supplemented with 20 ng of [(±)−3,5,5,7,7,7-d6]-ABA as an internal ABA standard. The final extracted samples were methylated with diazomethane for gas chromatography-mass spectrometry (GC-MS) analysis using a selected ion monitoring (SIM) 6890N network GC system and the 5973 network mass-selective detector (Agilent Technologies, Palo Alto, CA, USA). The monitored responses to ions at m/z of 190 and 162 for Me-ABA and 194 and 166 for Me-[ 2 H 6 ]-ABA were obtained using the Lab-Base (ThermoQuest, Manchester, UK) data system software.

Salicylic acid
The salicylic acid (SA) content in the shoots and roots of L. adecarboxylata MO1-inoculated and -non-inoculated plants grown under 2 and 5 mM Zn+ toxicity was extracted and quantified according to the method described by Shahzad et al. (2017a). Briefly, 0.2 g of freeze-dried plant samples were extracted and subjected to high-performance liquid chromatography (HPLC), which was performed using a Shimadzu device outfitted with a fluorescence indicator (Shinadzu RF-10AxL) with excitation and emission wavelengths of 305 and 365 nm, respectively, and filled with a C18 reverse-phase HPLC column (HP Hypersil ODS, particle size 5 μm, pore size 120 Å, Waters). The flow rate was maintained at 1.0 mL/min.

Statistical analysis
Data were collected in triplicate and subjected to Duncan's multiple range test using the SAS software (version 9.2, Cary, NC, United States) and also to the t-test using the GraphPad Prism software (version 6.01, San Diego, CA, United States) where appropriate, followed by Two-Way Anova (Supplementary Table 1). Graphical presentation of the data was performed using the GraphPad Prism software (version 6.01, San Diego, CA, United States).

Siderophore production, silica and zinc solubilization
The siderophore production, as well as the silica-and zincsolubilizing potential, of L. adecarboxylata MO1 in vitro showed positive results (Figure 1). After 7 days of inoculation on CAS, the Zn-and silica-supplemented agar medium exhibited siderophore production, as indicated by a color change from blue to orange, and Zn and silica solubilization, as indicated by a clear zone formation around the inoculated bacterial colony.

Stress resistance
Treatment with 2 mM Zn showed no significant difference in the growth of L. adecarboxylata MO1; however, 5 mM Zn toxicity did significantly influence L. adecarboxylata MO1 by decreasing its growth by 27% compared with control ( Figure 2).

Estimation of Zn content
Estimation of the Zn content in L. adecarboxylata MO1inoculated and -non-inoculated plants showed significant differences when grown under 2 and 5 mM Zn toxicity (Figure 4). Under normal conditions, nonsignificant differences in Zn accumulation were recorded in the roots and shoots of L. adecarboxylata MO1-inoculated and -non-inoculated plants. Under 2 mM Zn toxicity, L. adecarboxylata MO1 inoculation significantly hindered Zn accumulation in the roots and shoots by a decrease of 69.45% and 54.89%, respectively, compared to that in noninoculated plants. A similar trend of reduced Zn accumulation by 69.22% and 67.22% was recorded in the roots and shoots of L. adecarboxylata MO1-inoculated plants, respectively, under 5 mM Zn toxicity compared to that in noninoculated plants (Figure 4).

Hydrogen peroxide (H 2 O 2 )
The ameliorative effect of L. adecarboxylata MO1 inoculation on hydrogen peroxide (H 2 O 2 ) accumulation in cucumber leaves under the three conditions was also examined ( Figure 5). The results demonstrated excessive hydrogen peroxide (H 2 O 2 ) accumulation under 2 and 5 mM Zn toxicity; however, H 2 O 2 accumulation was significantly decreased by L. adecarboxylata MO1 inoculation. Under control conditions, no hydrogen peroxide (H 2 O 2 ) accumulation was recorded in both inoculated and non-inoculated leaves ( Figure 5).

Catalase
Catalase regulates stress responses and maintains intracellular redox states by reacting with ROS. Under normal growth conditions, the concentrations of catalase were significantly enhanced upon L. adecarboxylata MO1 inoculation by 41.67% compared to those in non-inoculated plants. There was a negative trend of decreased catalase concentrations in L. adecarboxylata MO1-inoculated plants under 2 and 5 mM Zn toxicity, with the decreases being 16.32% and 36.09%, respectively ( Figure 6).

Peroxidase (POD) and polyphenol oxidase (PPO)
The stress ameliorative potential of L. adecarboxylata MO1 was also investigated, and the results showed that under normal growth conditions, L. adecarboxylata MO1-inoculated and -non-inoculated plants exhibited nonsignificant differences in PPO levels. Under 2 and 5 mM Zn stress, L. adecarboxylata MO1 inoculation significantly reduced the PPO levels by 15.35% and 19.55%, respectively, compared to those in non-inoculated plants ( Figure 6).
Under normal conditions, L. adecarboxylata MO1-inoculated and -non-inoculated plants exhibited nonsignificant differences in PPO levels. Under 2 and 5 mM Zn toxicity, L. adecarboxylata MO1 inoculation significantly reduced the PPO levels by 45.10% and 35.53%, respectively, compared to those in non-inoculated plants ( Figure 6).

Superoxide anion (SOA)
The evaluation of SOA levels in L. adecarboxylata MO1inoculated and -non-inoculated plants grown under normal conditions and Zn (2 and 5 mM) toxicity revealed significant differences ( Figure 6). Under normal growth conditions, SOA levels were significantly increased by 36.05% with L. adecarboxylata MO1 inoculation. A negative trend of reduced SOA levels was observed under Zn toxicity. Under 2 and 5 mM Zn stress, the SOA levels were reduced by 44.57% and 18.93%, respectively, in L. adecarboxylata MO1-inoculated plants compared to those in non-inoculated plants ( Figure 6).

Lipid peroxidation
The extent of lipid peroxidation due to metal stress was estimated by measuring the levels of malondialdehyde (MDA). Under normal conditions, nonsignificant differences in LPO levels were observed in L. adecarboxylata MO1-inoculated plants compared to those in non-inoculated plants. However, under 2 mM Zn stress, L. adecarboxylata MO1 inoculation reduced the LPO levels by 40% and 15.57%, respectively, compared to those in non-inoculated plants ( Figure 6).

Glutathione (GSH)
Glutathione (GSH) is considered as one of the primary mechanisms of antioxidant defense against ROS. Under normal conditions, L. adecarboxylata MO1-inoculated and -non-inoculated plants showed nonsignificant differences in GSH levels. However, under 2 and 5 mM Zn stress, the GSH levels were significantly decreased by 18.68% and 28.01% in L. adecarboxylata MO1-inoculated plants, respectively, compared to those in non-inoculated plants (Figure 6).   5 mM) conditions. The leaves were incubated in 1% solution of DAB (Sigma Aldrich) and fully covered until the light and dark brown spots showed up. After incubation, leaves were bleached by boiling a decolorizing solution (acetic acid: glycerol: 96% ethanol = 1:1:3) to clearly visualize blue and brown spots, respectively, and after which they were photographed. All solutions were prepared in potassium phosphate buffer at pH 7.4.

Endogenous abscisic acid
Stress-responsive, endogenous ABA levels were significantly regulated in L. adecarboxylata MO1-inoculated plants under Zn toxicity (Figure 7). Under control conditions, L. adecarboxylata MO1 inoculation did not cause a significant difference compared with non-inoculated plants. Under Zn toxicity (2 and 5 mM), the plants accumulated a significant amount of ABA; however, L. adecarboxylata MO1 inoculation significantly decreased the ABA accumulation compared to that in non-inoculated plants ( Figure  7). Under 2 mM Zn stress, L. adecarboxylata MO1 inoculation significantly reduced the ABA accumulation by 38.97% compared to that in non-inoculated plants. Similarly, under 5 mM Zn stress, the ABA levels were significantly lower (22.63% decrease) in L. adecarboxylata MO1-inoculated plants than in non-inoculated plants (Figure 7).

Endogenous salicylic acid
Stress responsive, endogenous SA levels were significantly regulated in L. adecarboxylata MO1-inoculated plants under Zn toxicity (Figure 7). Under control conditions, L. adecarboxylata MO1 inoculation resulted in nonsignificant differences compared with non-inoculated plants. Under Zn toxicity (2 and 5 mM), the plants exhibited significant accumulation of endogenous SA; however, L. adecarboxylata MO1 inoculation significantly decreased the SA accumulation compared to that in non-inoculated plants (Figure 7). Under 2 mM Zn stress, L. adecarboxylata MO1 inoculation significantly reduced the SA accumulation by 53.22% compared to that in non-inoculated plants. Similarly, under 5 mM Zn stress, the SA levels were significantly lower (72.25% decrease) in L. adecarboxylata MO1-inoculated plants than in non-inoculated plants (Figure 7).

Discussion
Heavy metal contamination due to rapidly increasing anthropogenic activities is a serious concern for the environment and human health, considering the transport of metals through food chain (Mishra et al. 2019;Hembrom et al. 2020). The ingestion of excessive metals may cause various human and animal health issues (e.g. poor immunological responses and high prevalence of upper gastrointestinal diseases) (Järup 2003;Duruibe et al. 2007;Tchounwou et al. 2012). Reducing metal accumulations in the environment and avoiding agricultural contamination with metals can protect human lives Rai et al. 2019). Several researchers have been attempting to develop sustainable, environmentally friendly, and manageable approaches to reduce heavy metal accumulations in agricultural soil (Wong et al. 2018;Bilal et al. 2019). One of the best alternatives for heavy metal removal is the use of plant growth-promoting microorganisms (Etesami 2018), which have been well reported to be heavy, resistant microbes that improve plant growth and mediate stress (Ayangbenro and Babalola 2017;Shahzad et al. 2019a). Microorganisms have developed various bioremediation strategies, such as phosphate solubilization and siderophore and phytohormone production (Kong and Glick 2017;Verma and Kuila 2019;Yin et al. 2019).
In the present study, we hypothesized that the previously isolated plant growth-promoting L. adecarboxylata MO1 ) could be used in soils with an overabundance of Zn to facilitate plant growth. We selected L. adecarboxylata MO1 because of its potential to produce siderophores and auxins, as well as its properties of Zn solubility and Zn resistance. These properties are the consequence of the potential of microbes to solubilize various metals and secrete phytohormones and siderophores not only to facilitate plant growth but also to mediate metal stresses by various physiochemical modulations Kong and Glick 2017;Verma and Kuila 2019). The present study has revealed the potential of L. adecarboxylata MO1 to produce IAA, solubilize Zn, and resist elevated levels of Zn toxicity. L. adecarboxylata is metabolically diverse and can produce phytohormones, synthesize extracellular enzymes, degrade hydrocarbons, and solubilize minerals, which have been widely reported to promote plant growth and mediate various stresses (Sarma et al. 2004;Shahzad et al. 2017c;Kang et al. 2019b).
The IAA-and siderophore-producing potential of L. adecarboxylata MO1 (Figures 1 and 2) is consistent with the reports of Shahzad et al. (2017c), Kang et al. (2019b), and Kumawat et al. (2019). In addition, L. adecarboxylata MO1 was found to produce significant amounts of IAA under Zn toxicity (Figure 2). Moreover, the growth-promoting capability of L. adecarboxylata has been widely reported (Sarma et al. 2004;Shahzad et al. 2017c), and L. adecarboxylata has the potential to mitigate different abiotic stresses such as salinity stress ) and drought stress (Danish and Zafar-ul-Hye 2019;Danish et al. 2020). Nevertheless, the role of L. adecarboxylata in metal stress mitigation, and more specifically Zn stress, has not explored in detail, which allows for an appropriate investigation of its potential role in stress mitigation and improving stress tolerance. The plant growth-promoting effect and Zn tolerance capability of L. adecarboxylata are consistent with the reports of Kang et al. (2019b), Sarma et al. (2004), Shahzad et al. (2019a), andHan et al. (2019). They linked the plant growth-promoting potential and stress-mitigating capability of L. adecarboxylata to its distinguishing abilities of nitrogen fixation, mineral solubilization, phytopathogen inhibition, siderophore production, and indole-3-acetic acid production; this association was also observed in the present study.
Studies have reported that elevated Zn concentrations inhibit the survival of plant PGPR in Zn-contaminated soil (Vivas et al. 2006;Kang et al. 2017). Similarly, Marques et al. (2013) demonstrated the inhibition of PGPR in agricultural soil supplemented with elevated Zn levels. Therefore, L. adecarboxylata was evaluated for Zn tolerance, because the survival of microbes under stressed conditions is important to help us understand plant survival in stressed environments (Shahzad et al. 2017b). In the present study, L. adecarboxylata MO1 was found to tolerate Zn toxicity (Figure 2), which could be the reason for the survival of MO1 in Zn-supplemented soil and the promotion of plant growth under Zn toxicity ( Figure 3 and Table 1). Zinc stress causes biochemical and physiological disturbances within plants by inhibiting their growth, photosynthesis, nutrient uptake, enzyme activation or deactivation, and phytohormone production (Rout and Das 2009). In the present study, cucumber plants exposed to zinc stress showed inhibited growth, root/shoot length, and biomass production (fresh/dry weight) (Figure 3 and Table 1). However, the zinc-tolerant isolate MO1 mitigated zinc toxicity in cucumber plants by enhancing the cucumber growth attributes such as root/shoot length and biomass (fresh/dry weight) ( Figure 3 and Table 1). Previously, several researchers have reported about different bacteria such as Serratia, Sphingomonas, and Rhizobium sp. in maize, sedum, and lentil, which increased the plant growth, biomass, and tolerance to zinc toxicity (Ahmad Wani et al. 2008;Chen et al. 2014;Jain et al. 2020). Similarly, chlorophyll has a vital role in photosynthesis, and its synthesis was found to be inhibited under zinc stress. However, cucumber plants inoculated with the isolate MO1 showed a markedly increased chlorophyll content compared to that in zinc-treated non-inoculated control plants ( Figure 2). These results are in agreement with previous findings (Joshi et al. 2013;Islam et al. 2014;Jain et al. 2020), wherein the authors reported similar observations in maize and wheat seedlings under zinc stress.
It is important to determine the distribution and accumulation of metal uptake in the roots and shoots of plants when evaluating plant survival under metal stress Kang et al. 2017). In the present study, L. adecarboxylata MO1 inoculation significantly protected the plants from the inhibitory effects of higher concentrations of Zn by reducing the Zn content . The reduction of Zn content at higher Zn toxicity levels by this bacterium might be due to Zn removal through adsorption and desorption mechanisms, as well as solubilization and leaching mechanisms of the bacterium (Pirhadi et al. 2016;Jafarian and Ghaffari 2017). Enhanced Zn uptake triggered Fe deficiency (Lešková et al. 2017) that reduced root and shoot growth and chlorophyll content as well as chlorosis in young leaves (Marschner 2012;Lešková et al. 2017). The results of plant growth and chlorophyll content in our study under Zn stress might be due to Fe deficiency. A similar observation was made by Fukao et al. (2011) who reported that excessive Zn levels reduce chlorophyll content and cause iron deficiency (Lešková et al. 2017).
It has been reported that zinc toxicity in plants induces oxidative damage and causes cellular damage, which in turn alter the plant antioxidative systems (Rout and Das 2009;Radić et al. 2010). In general, Zn toxicity can induce several alterations in plant cells, such as binding of cell membrane proteins and enzymes to sulfhydryl groups, increases in lipid peroxidation, and disturbances in essential elements (Castillo-González et al. 2018). In the present study, the induction of oxidative stress was found in plants grown in Zn-contaminated soil, which was evident by the increase in the antioxidant activities of plants ( Figure 6). However, L. adecarboxylata MO1 inoculation reduced the antioxidant activities of plants, suggesting that improved plant tolerance under heavy metal stress can occur after rhizobacterial inoculation (Mesa et al. 2015;Ahemad 2019). Rhizobacterial bioaugmentation in the present study also diminished the activities of CAT, POD, PPO, SOA, MDA, and GSH in the plants compared to those in non-inoculated plants, which also supports the conclusion that Zn accumulation in plant roots and shoots was reduced ( Figure 6). With higher metal accumulation and toxicity, an increase in ROS generation and, consequently, antioxidant enzyme activity could be expected. Although the upregulation of antioxidants in plants under stress conditions has been reported by several researchers (Kang et al. 2019a;AbdElgawad et al. 2020), our data support that L. adecarboxylata MO1 inoculation may contribute to the mediation of Zn stress not by increasing the antioxidants but by reducing the metal toxicity ( Figure 6). For instance, L. adecarboxylata MO1 produced siderophores and indole-3-acetic acid. Studies have reported that siderophores released by the rhizobacterial consortium are chelators that may bind metals, thus alleviating their toxicity (Dimkpa et al. 2009a(Dimkpa et al. , 2009b. In the same manner, selected rhizobacteria produce indole-3-acetic acid, which has a bioprotective effect (Mesa et al. 2015). Collectively, the findings of this study suggest that L. adecarboxylata MO1 inoculation causes a reduction in the formation of cell-damaging free radicals, thus lessening the need of plant enzymatic defences (Dimkpa et al. 2009a(Dimkpa et al. , 2009b. The regulation of stress-responsive plant endogenous hormones, specifically ABA, is well known to be a stress coping mechanism (Shahzad et al. 2017b;Khan et al. 2019a). However, higher accumulation of ABA under conditions of stress causes stomata closure, which further results in leaf senescence and photosynthesis reduction (Verma et al. 2016). Interestingly, in the present study, L. adecarboxylata MO1 inoculation resulted in a significant reduction of endogenous ABA levels in plants grown under Zn stress compared to those in non-inoculated plants (Figure 7). Moreover, the reduced ABA levels in L. adecarboxylata MO1-inoculated plants were associated with a decrease in antioxidant contents, because enhanced ROS generation led to the higher accumulation of endogenous ABA for stress mitigation (Kar 2011;Leitão and Enguita 2016). The reduction of endogenous ABA levels is accredited to the potential of L. adecarboxylata MO1 to enhance stress tolerance and reduce metal toxicity in plants.
Endogenous SA is regulated in plants upon exposure to various biotic and abiotic stresses (Shahzad et al. 2017a(Shahzad et al. , 2017bKhan et al. 2020). It has been reported that SA protects plants from heavy metal-induced toxicity due to ROS production by antioxidant regulation Kang et al. 2017). In the present study, endogenous SA was significantly reduced in MO1-inoculated plants under 2 and 5 mM levels of Zn compared to that in non-inoculated plants (Figure 7). In general, endogenous SA is regulated and synthesized from chorismate-derived l-phenylalanine through various reactions of enzymes initially catalyzed by phenylalanine ammonia lyase (PAL) (Vlot et al. 2009). Furthermore, Zn toxicity has been reported to enhance PAL activity, which resulted in the enhancement of endogenous SA levels in plants exposed to Zn toxicity (Luo et al. 2010;Kang et al. 2017). The inoculation of MO1 in plants grown under Zn toxicity may regulate PAL activity and subsequently decrease endogenous SA synthesis to exert its protective role in plants.

Conclusion
The present study has demonstrated interesting results and identified L. adecarboxylata MO1 as a promising alternative for plant growth promotion and also showed that MO1 application may be an effective method to mitigate metal toxicity in plants. Our results demonstrated that L. adecarboxylata MO1 inoculation enhanced Zn stress tolerance and reduced metal toxicity in plants by the mechanisms of Zn stress tolerance, as well as by siderophore and IAA production, which positively regulated Zn distribution in the plant tissues, regulated the antioxidant levels (H 2 O 2 , CAT, POD, PPO, SOA, MDA, and GSH), and modulated the production of stress-responsive phytohormones (ABA and SA). These findings illustrate that L. adecarboxylata MO1 application is a viable strategy for improving plant growth and reducing metal toxicity. These results also encourage the utilization of secondary metabolite-producing PGPR as an alternative to enhance plant growth, improve stress tolerance, and reduce metal toxicity in plants grown in metal-contaminated fields. Further experiments are required to assess the role of the isolate MO1 under Zn stress on iron accumulation and its effect on the growth attributes of Zn-Fe-MO1 in cucumber plants at the molecular level.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01065443).