In the present study, randomly selected a total of five rhizosphere soil samples were collected from the fungicide applied sugarcane field near the Sugarcane Research Institute, Guangxi Academy of Agricultural Sciences, Nanning, China. From collected rhizospheric soil samples, stone, soil debris, and plant parts were removed and subjected to shade drying for 24–48 h. The composite sieved rhizospheric soil sample was enriched in mineral salt medium (MSM) amended with 500 μg/ml of CBZM (50% w/v of the active ingredient) for 96 h for the isolation of fungicide tolerant bacteria. After incubation of the fungicide enriched soil sample, the bacteria isolation was carried out by the serial dilution method on mineral salt agar plates supplemented with 500 μg/ml CBZM and incubated for 72–96 h at 28°C. After the incubation, a total of 18 dominant bacterial colonies (ANCB-1 to ANCB-18) were screened and conserved in 50% glycerol at −80°C for further use.

The intrinsic fungicide tolerance ability of the obtained 18 rhizobacterial isolates was determined through minimal salt agar (MSA) medium (g/L: KH₂PO₄ 1.0 g, K₂HPO₄ 1.0 g, NH₄NO₃ 1.0 g, MgSO₄.7H₂O 0.2 g, FeSO₄.7H₂O 0.01 g, CaCl₂.2H₂O 0.02 g, agar powder 18.0 g, and pH 6.5). Briefly, actively growing fresh rhizobacterial cultures (10⁸ cells/ml) were spot inoculated on MSA plates containing varying concentration of fungicide CBZM, viz., 500, 1,000, 1,500, 2,000, 2,500, and 3,000 μg/ml. After the inoculation, all the inoculated medium plates were incubated at 28 ± 2°C for 48–72 h. Followed by highest fungicide, tolerant rhizobacteria was selected for further study.

The selected bacteria isolate ANCB-12 showed a higher level of CBZM tolerance was morphologically and biochemically characterized by following Bergey’s Manual of Determinative Bacteriology (Tindall et al., 2010). Furthermore, isolate ANCB-12 was metabolically characterized by carbon source utilization pattern using BIOLOG GNIII MicroPlate™ (Biolog, Inc., Hayward, CA, USA) following the manufacturer’s instruction.

By adopting the standard protocol of Pospiech and Neumann (1995), genomic DNA was extracted from the actively grown rhizobacterial culture. To amplify the 16S rRNA gene, the universal 16S rRNA gene primers PA (5′-AGA GTT TGA TCC TGG CTC AG-3′) and PH (5′-AAG GAG GTG ATC CAG CCG CA-3′) were used (Sharma et al., 2015). The 50 μl PCR reaction mixture contains 50 ng of DNA template, 10× reaction buffer, 2.5 mM dNTPs, 10 pM of each primer, and 3 U of Taq polymerase. PCR condition in the thermal cycler was as follows: at 94°C initial denaturations for 4 min, followed by denaturation at 94°C for 45 s of 35 cycles, annealing at 51°C for 1.5 min, and at 72°C elongations for 90 s, and 72°C final extensions for 7 min. The Amplified 16S rRNA gene PCR product was purified using the BioFlux PCR purification kit (China) by following the manufacturer’s protocol. The purified PCR product was sequenced and analyzed for percent (%) similarities with the available sequences in the GenBank database at the NCBI website. The nucleotide sequence of the bacteria ANCB-12 was deposited in the GenBank sequence database (ON878101). Based on evolutionary distances and Jukes–Cantor coefficient calculation, a neighbor-joining (NJ) phylogenetic tree was constructed through MEGA-X software (Sharma et al., 2019).

Among the all tested bacterial isolates, isolate ANCB-12 showed the highest tolerance of carbendazim and was selected for growth kinetic study. The selected isolate was inoculated in liquid MSM containing 0, 500, 1,000, 2,000, and 3,000 μg/ml concentration of CBZM and incubated at 150 rpm for 60 h at 28 ± 2°C. To make the growth kinetic curve, the optical density at 600 nm was taken every 12 h.

Cell viability in terms of CFU count was carried out by spreading the 100 μl aliquots of 48 h grown culture of each CBZM stress level. CFU was counted after 48 h of incubation at 28 ± 2°C. The CFU was calculated as:

The fungicide-induced bacterial membrane integrity and mortality were determined by confocal laser scanning microscopy (Xia et al., 2022). In brief, bacteria culture was inoculated in MSM liquid medium supplemented with 3,000 μg/ml CBZM fungicide and incubated in a rotatory shaker for 48 h at 28°C. After the incubation of the treated and un-treated cultures, the pellet was collected by centrifugation and 2–3 times washed with PBS (phosphate buffer saline). In 100 μl of fungicide treated and un-treated bacterial suspension, 5 μl of propidium iodide (PI) and 5 μl of acridine orange (AO) were added and incubated for 10 min at 28 ± 2°C. Then the samples were centrifuged at 5,000 rpm for 10 min to remove the unbound dyes, and the pellets were resuspended in PBS buffer. Samples were transferred to glass slides and observed under confocal laser scanning microscope (CLSM) (Leica Confocal Microscope, Germany).

All the PGP activities of CBZM fungicide treated and un-treated rhizobacteria were assayed by using a standard method as given in the below section.

The total protein content of fungicide treated and un-treated rhizobacterial cells was estimated by following the standard method of Bradford (1976). The amount of protein in the rhizobacterial cells was determined spectrophotometrically at 595 nm, and protein content was calculated by using the standard curve of bovine serum albumin (BSA).

The lipid peroxidation (LPO) was analyzed by measuring the end product malondialdehyde (MDA), according to the method of Heath and Packer (1968). Finally, the optical density of the obtained supernatant was measured at 532 nm (all lipids) and 600 nm (all lipids except for MDA), and the amount of MDA was expressed in nmol/ml.

Freshly grown fungicide-treated and un-treated rhizobacterial cultures were centrifuged at 10,000 rpm for 10 min, and cell pellets were harvested. In the collected cell pellet, Tris–HCl was added to obtain a homogenous bacterial suspension. Furthermore, rhizobacterial cell suspensions were sonicated, and then sonicated cell suspensions were centrifuged (at 12,000 rpm for 10 min) to eliminate the cell debris. The supernatant containing cellular extract was used for the assay of antioxidant enzyme activity (Srivastava et al., 2017). Antioxidant enzymes like superoxide dismutase (SOD) (ADS-W-KY011), catalase (CAT) (ADS-W-KY002), glutathione peroxidase (GPX) (ADS-W-G003), ascorbate peroxidase (APX) (ADS-W-VC005), and glutathione reductase (GR) (ADS-W-FM029), activities were measured using an enzyme-linked immune sorbent assay (ELISA) kit (Jiangsu, China) by following the manufacturer’s protocol.

Data obtained from this study are the mean of three individual replications ± standard error. SPSS (ver. 16.0) statistical software (IBM, Armonk, NY, USA) was used to analyze the data through ANOVA (analysis of variance) and DMRT (Duncan’s multiple range test) at p ≤ 0.05.

In the present study, a total of 18 dominant rhizobacterial colonies, i.e., from ANCB-1 to ANCB-18, were obtained from the collected rhizosphere soil sample of sugarcane on MSA medium plate supplemented with 500 μg/ml carbendazim. On the basis of the initial fungicide stress and plant growth promoting traits assay, a multiple PGPR bacterial isolate ANCB-12, which showed its growth at a higher carbendazim concentration, was selected for further study. The selected isolate ANCB-12 was subjected to phenotypic, biochemical, and metabolic characterization. The obtained rhizobacterial strain ANCB-12 was a Gram-positive, motile, rod-shaped, endospore-forming rhizobacteria based on phenotypic characteristics. The biochemical characteristics demonstrated that isolate ANCB-12 showed positive results for citrate utilization, catalase, methyl red, oxidase, indole, lysine utilization, nitrate reduction, and ornithine utilization but was found negative for the Ortho-Nitrophenyl-β-galactoside (ONGC), Voges–Proskauer test, arginine, phenylalanine deaminase, and H₂S production (Table 1). The great metabolic flexibility of the bacterial isolate allows them to inhabit variable environments such as those reported in the present study. Similarly, by referring to Bergey’s Manual of Systematic Bacteriology Al-Enazi et al. (2022) characterized pesticide-tolerant Pseudomonas sp. from the rhizosphere soil of Vigna radiata (L.).

Soil microorganisms require some basic nutrients (such as carbon and nitrogen) for their growth and survival and to maintain their metabolic functions. Thus, microorganisms’ ferment or metabolize a wide range of simple sugars, complex carbohydrates, and amino acids for their growth and energy. The utilization patterns of carbon and amino acid sources may reveal information about the microbial biochemical pathways for various biological activities as well as for comparing the various microorganisms. With this account, in the present study, the metabolic profile through the C-utilization pattern of the isolate ANCB-12 was analyzed through “BIOLOG phenotype micro-array™ GNIII-carbon plate.” The results of the BIOLOG assay revealed that isolate ANCB-12 was positive for 19 sugars, 2 reducing sugars, chemically sensitive for 13 substrates, 6 amino acids, 7 hexose-PO₄, 7 hexose acids, 10 carboxylic acids, esters, and fatty acids (Supplementary Table 1). The results of carbon utilization through BIOLOG assay showed that the tested rhizobacterial isolate was metabolically active. The results are in line with the study of Li et al. (2017) and Singh et al. (2020) who used BIOLOG based C-utilization assay to the exploration of metabolically active rhizobacteria of sugarcane.

Molecular identification of the rhizobacteria was carried out through structural 16S rRNA gene sequencing analysis. The comparison of the obtained nearly ∼1.5 kb 16S rRNA gene sequences through BLAST search with the available bacterial sequences in the NCBI Genbank public databases indicates that the isolate ANCB-12 is closely related to Priestia megaterium with 100% sequence similarity. The obtained 16S rRNA gene nucleotide sequence of the rhizobacterial isolate ANCB-12 was submitted to GenBank with accession number ON878101. Figure 1 shows a phylogenetic tree made from the isolate ANCB-12 and similar bacterial 16S RNA gene sequences of NCBI database. The tree was made using the neighbor-joining method and 1,000 bootstrap samples.

16S rRNA gene sequencing analysis is one of the most widely used methods for bacterial identification and phylogeny/taxonomy analysis (Jamali et al., 2020). In accordance with our results, Sharma et al. (2019, 2021) have also isolated rhizospheric bacteria from rhizospheres of chickpea and sugarcane from different agro-climatic regions, and molecularly identified them through 16S RNA partial gene sequencing analysis.

To control the plants’ diseases and to enhance agriculture production, an overdose of pesticides and fungicides imposes a negative effect on non-targeted beneficial microorganisms. Thus, in the present study, the effects of carbendazim fungicide on the growth of 18 isolated rhizobacterial (ANCB-1 to ANCB-18) were evaluated. At the initial screening level, all 18 isolates were spot inoculated on the MSA medium plates stressed by adding different concentrations of fungicide ranging from 0, 0.05% (500 μg/ml), 0.1% (1,000 μg/ml, recommended dose), 0.2% (2,000 μg/ml), and 0.3% (3,000 μg/ml). After the incubation time, results showed that all the tested rhizobacteria did not show growth. Only one isolate, ANCB-12, was found to be significantly tolerant to higher fungicide stress, so isolate ANCB-12 was chosen for a growth kinetic study in a liquid medium with varying concentrations of carbendazim (Figure 2A). Generally, pesticides can change the growth of tolerant or degrading bacteria (Peters et al., 2014). In the present study, the growth of the isolate ANCB-12 revealed a distinct pattern of growth when exposed to increasing concentrations of fungicide. Results in the form of a growth curve showed that in an un-treated medium, the isolate revealed luxurious growth (Figure 2A), while as the concentration of fungicide increased, the growth was sluggish as an adaptation period and a long lag phase was observed before reaching exponential growth. Isolate tolerates up to 0.25% fungicide concentration, whereas compared to the lower fungicide concentration, the higher concentration had an adverse toxic effect on bacterial life. Results showed the maximum concentration (0.3%) of fungicide has a killing effect on bacterial cell CFU. Lower growth and CFU with increasing concentration of fungicide may be due to the intake of fungicide, which influences the damage of the cell membrane and metabolic process of the bacteria (Figure 2B; Reshma et al., 2018). Tolerance to fungicides is assumed to be a distinctive characteristic among soil-living beneficial microorganisms, which are led by unique physiological, metabolic, and genetic features (Khan et al., 2020). In 2012, from the mustard rhizosphere, Ahemad and Khan isolated Pseudomonas putida PS9 which exhibited a different level of tolerance to various fungicides ranging from 1,400 to 3,200 μg ml^–1 concentrations. Similarly, a Bacillus subtilis BC8 strain isolated from the cabbage rhizosphere soil showed a different growth pattern under different concentrations of pesticides and fungicides ranging from 0 to 3,200 μg ml^–1 (Shahid et al., 2019). The results of the current study are also in accordance with the observation of Nagaraju et al. (2017), who reported the lethal effect of fungicides, herbicides, and zinc solubilizing bacteria.

In the present study, an advanced confocal laser scanning microscopy (CLSM) technique was used to analyze the toxic effects of the fungicide CBZM on rhizobacterial membrane permeability. Results of CLSM analysis showed that as the fungicide treatment level increased, a huge number of red fluorescence releasing rhizobacterial cells were observed (Figure 3). This indicated the highly toxic effect on the viability of rhizobacterial cells, which led to cell damage over controlled un-treated cells (Figure 3). However, no red-colored cells were detected in CLSM images of control CBZM un-treated cells (Figure 3A). This is because toxic fungicides increase cell permeability, allowing dyes to enter the cell and bind with intracellular components and nucleic acids. The results of the present study are in line with the study of Kudoyarova et al. (2017), who differentiated between dead and live cells of B. subtilis strain BC8 in CLSM analysis. Similar to our results, similar observations were obtained in the studies of Shahid et al. (2019; 2021a,b), who reported an increasing number of dead cells of Bradyrhizobium japonicum, Pseudomonas sp., and Enterobacter cloacae after treatment with increasing concentrations of fungicides.

Bacterial membrane integrity in terms of LPO due to stress conditions is a major stress marker (Heath and Packer, 1968). Therefore, considering this fact, in the present study, the membrane integrity of the isolate ANCB-12 under fungicide CBZM stress was further analyzed by MDA content. Results showed that MDA levels of the isolate ANCB-12 was 0.54 nmol/ml under control conditions (T-1) without any fungicide stress (Figure 5A). However, with increasing fungicide concentration, the cellular toxicity increased, which was evident by increasing MDA levels (Figure 5A). CBZM stress showed severe cellular toxicity and MDA content was increased to 1.48, 2.36, and 4.56 nmol/ml at 1,000 (T-2), 2,000 (T-3), and 3,000 μg/ml (T-4) CBZM concentration treatments, respectively. Results demonstrated that the levels nearly doubled with increasing fungicide treatments (p < 0.05; Figure 5A). The generation of MDA is evidence of oxidative stress within the cells. The findings are consistent with those of Shahid and Khan (2018), who found an increase in MDA levels in B. subtilis under CBZM and kitazin stress. In a recent study, Rovida et al. (2021) also found that herbicide-treated Pseudomonas sp. had a higher level of MDA.

Antioxidant enzymes such as SOD, CAT, GPX, and GR have been found in almost all cellular compartments and play a significant role in the neutralization or detoxification of toxic ROS molecules. SOD is an enzyme that is activated by ROS generation and the breakdown of singlet oxygen radical (¹O₂^–) or superoxide radical (*O₂) into H₂O₂ and O₂ (Hasanuzzaman et al., 2020). However, this SOD induced end product H₂O₂ retains its potential to induce cell damage by conversion of H₂O₂ into hydroxyl radical (*OH). Thus subsequently, CAT, APX, and GPX detoxifies the H₂O₂ into H₂O and O₂, as well as inhibited LPO and protect cell damage (Sharma et al., 2012; Ighodaro and Akinloye, 2018). On the other hand, GR kept the intracellular glutathione pool (GSH) in a reduced state, which acts as a direct and indirect antioxidant for scavenging ROS. In the present study, we assessed the effects of different doses of fungicide CBZM on the activities of antioxidant enzymes, which are directly related to the detoxification of ROS molecules (Figure 5). Results showed that the addition of CBZM causes oxidative stress in the ANCB-12 rhizobacterial strain. SOD activity under non-stress conditions (0 μg/ml: T-1) was maximal (48.4 U/ml). CBZM stress gradually reduces SOD activity by 26.9, 62.5, and 82.3% at the recommended doses of T-2 (1,000 μg/ml), T-3 (double dose: 2,000 μg/ml), and T-4 (triple dose: 3,000 μg/ml) levels of CBZM (Figure 5B). Similarly, CAT activity was highest at normal conditions, i.e., 182.6 μmol/ml, which was significantly decreased by 27.4, 42.9, and 61.4% when exposing them to T-2, T-3, and T-4 CBZM stress concentrations, respectively, over non-CBZM stressed control (Figure 5C). Similar to SOD and CAT activities, GPX activity also decreased with an increasing dose of fungicide. Results showed that GPX activity maximally declined by 76.1% when exclusively exposed to the highest stress of CBZM (T-4: 3,000 μg/ml), followed by 54.5 and 32.6% at T-3 and T-4 CBZM stress levels, respectively, over control (T-1; 0 μg/ml CBZM) (Figure 5D). Furthermore, similar trends were also observed in APX activity, where activity maximally decreased by 81.6% at T-4 treatment of CBZM concentration, followed by 49.3 and 30.3% decreases under T-3 and T-2 CBZM stress conditions, respectively, over the non-stress control (T-1) condition (Figure 5E). In addition, results showed that the GR activity of bacterial cells decreased by 84.8, 68.0, and 41.3% at T-4, T-3, and T-2 CBZM stress conditions, respectively, as compared to the T-1 non-stress level of CBZM (Figure 5F). Overall, the results of the present study showed that with increasing fungicide stress, all the ROS scavenging antioxidants SOD, CAT, GPX, APX, and GR enzyme activity were gradually downregulated. The decrease in the activities of these enzymes is related to the increase in the free movement of H₂O₂, leading to increased cell permeability, LPO (MDA), and disruption of nuclear materials, which causes direct damage to the cell. These results are directly related to our growth kinetics, CLMS analysis, and increased level of LPO (MDA) under a gradient dose of fungicide. Similar damaging effects on antioxidant enzymes were observed with an increasing dose of pesticide stress by Shahid and Khan (2018). Our observations are also supported by similar results in Pseudomonas spp., Enterobacter asburiae, and Mesorhizobium ciceri under different pesticide stress conditions (Martins et al., 2011; Rovida et al., 2021; Shahid et al., 2021a).

Heatmap analysis revealed that the plant growth promoting attributes, LPO, and antioxidant enzyme variables displayed differential responses in the integrated heatmap against the various dosage of carbendazim fungicide treatments (Figure 6). The results of heatmap multivariance analysis clearly demonstrate that CBZM stress imposed adverse effects on all the tested functional and metabolic attributes of the beneficial rhizobacteria.