Isolation and identification of a new Bacillus glycinifermentans strain from date palm rhizosphere and its effect on barley seeds under heavy metal stress

Abstract

Soil contamination by heavy metals is one of the major problems that adversely decrease plant growth and biomass production. Inoculation with the plant growth-promoting rhizobacteria (PGPR) can attenuate the toxicity of heavy metals and enhancing the plant growth. In this study, we evaluated the potential of a novel extremotolerant strain (IS-2 T) isolated from date palm rhizosphere to improve barley seedling growth under heavy metal stress. The species-level identification was carried out using morphological and biochemical methods combined with whole genome sequencing. The bacterial strain was then used in vitro for inoculating Hordeum vulgare L. exposed to three different Cr, Zn, and Ni concentrations (0.5, 1, and 2 mM) in petri dishes and different morphological parameters were assessed. The strain was identified as Bacillus glycinifermentans species. This strain showed high tolerance to pH (6–11), salt stress (0.2–2 M), and heavy metals. Indeed, the minimum inhibitory concentrations at which bacterium was unable to grow were 4 mM for nickel, 3 mM for zinc, more than 8 mM for copper, and 40 mM for chromium, respectively. It was observed that inoculation of Hordeum vulgare L. under metal stress conditions with Bacillus glycinifermentans IS-2 T stain improved considerably the growth parameters. The capacity of the IS-2 T strain to withstand a range of abiotic stresses and improve barley seedling development under lab conditions makes it a promising candidate for use as a PGPR in zinc, nickel, copper, and chromium bioremediation.

Introduction

Over exploitation for mineral resources has overloaded the endurance capability of the environment; the pollution caused by the release of heavy metals during mining is getting more and more serious and has emerged as one of the most concerns in achieving sustainable agricultural production worldwide [1, 2]. The heavy-metal-polluted soils limit plant growth and productivity, hamper agricultural economy, and lead to serious land degradation in many countries [3]. In fact, high levels of heavy metal in the soil can cause damage to plant’s root system, photosynthetic process, and membrane permeability, limiting plant growth and lowering crop yield and quality [4].

Furthermore, heavy metals, unlike organic pollutants, are not biodegradable and accumulated in soil, then absorbed and accumulated in plants, and transferred into human body through food chains [5]. Which is seriously harmful for human health, some cancers and diseases are hence induced [1]. The majority of heavy metals are hazardous; however, their toxicity varies widely depending on the metal [6]. Many heavy metals, such as nickel (Ni), cobalt (Co), cadmium (Cd), zinc (Zn), lead (Pb), mercury (Hg), chromium (Cr), and copper (Cu), are classified by the World Health Organization as priority metals that require immediate attention because of their effect on human health [7]. Since the 1980s, several technologies have been used to remediate contaminated soils using both chemical and physical methods. These technologies can remediate the polluted soil, to a certain extent, but the procedures are complex and secondary pollution might be caused [1]. As a result, it is critical to develop trustworthy, low cost, eco-friendly, little harmfulness for environment, and high efficiency methods for reducing heavy metal pollution in agricultural fields. Compared with physical and chemical methods, bio-restoration technology is gotten attention considerably. The technology with the combination application of plant-microorganism is a valuable strategy that allows promotion of plant productivity and yield under heavy metals conditions [8].

Large quantity of microorganisms which can resist heavy metals are isolated from soil and most of them are bacteria. Indeed, many telluric bacteria from the genera Azotobacter, Enterobacter, Bacillus, Aeromonas, Pseudomonas, and Klebsiella are known to improve plant development even in difficult environmental conditions [9].

PGPRs (plant growth promoting rhizobacteria), which can endure, resist, and absorb heavy metals, provide a viable alternative for reducing the harmful impact of heavy metal pollution on plants [10]. Recently, some heavy metals tolerant-PGPRs have been found to ameliorate plant growth in metal-contaminated soils while also lowering hazardous metal bioavailability and uptake by plants, leading in safe food production [11]. Stimulation of plant growth by PGPR would be provided through a variety of mechanisms, including producing of plant hormones [12], enzymes (phosphatase/phytase,1-aminocyclopropane-1-carboxylate deaminase…), hydrocyanic acid (HCN), extracellular polysaccharide (EPS), siderophores, and solubilizing phosphates; secreting phytostimulants (indole acetic acid); and reducing reactive oxygen species (ROS) [13].

The present investigation aimed to recover bacterial isolated from date palm rhizospheric soil of the Tunisian oasis ecosystem (IS-2 T). Then, the selected bacterium was evaluated for different stress, including temperature, pH, high NaCl concentration, and heavy metals. Finally, the effect of the B. glycinifermentans IS-2 T strain inoculation on barley seedlings grown under Cr, Zn, and Ni stress in vitro conditions was tested.

Materials and methods

Isolation and characterization of thermotolerant rhizobacteria

The rhizospheric soil for bacterial strain isolation was obtained from date palm trees localized in three different regions of Tunisia: Gabes, Kebili, and Desert of Kebili. The bacterial isolates were obtained by adopting serial dilution technique. Briefly, 10 g of each soil sample was shaken with 90 mL of sterile distilled water at 160 rpm and 45 °C for 30 min [14]. A serially diluted aliquot (0.1 mL) was spread on Luria–Bertani agar medium (peptone 10.00 g/L, NaCl 5.00 g/L, yeast extract 5.00 g/L, and agar 20 g/L) and incubated at a range of temperatures between 45 and 60 °C for 48 h. A high incubation temperature was chosen in consideration of Tunisia’s desert climate. After incubation, various morphologically distinct colonies were selected, purified, and preserved in glycerol stocks. One thermo-tolerant stain, namely B. glycinifermentans IS-2 T, isolated from the desert of Kebili, was selected for further study. A voucher specimen [MB02] of the B. glycinifermentans IS-2 T strain was deposited in the microbial culture collections of the faculty of sciences of Sfax. This strain was assessed biochemically with API 50CHB galleries (the kit was used according to the instructions of the manufacturer), amylase, protease, and mannanase production and physiologically with pH range determination and NaCl tolerance. Resistance to antibiotics and heavy metals was also tested.

For salt resistance, an overnight grown culture of the selected bacterium was inoculated on Luria–Bertani agar plates supplemented with various concentrations of sodium chloride (NaCl 0.2, 0.4, 0.6, 0.8, 1, 1.5, and 2 M) and further incubated for 24 h at 60 °C for 3 days [15, 16].

pH tolerance was also determined by regulating the pH of the LB agar plates at various levels (7, 8, 9, 10, and 11). The isolate ability to withstand pH and salt was demonstrated by observing its development on LB agar media. Positive strain growth was confirmed by observing colonies on LB agar media.

Protease and amylase activities were evaluated using LB medium containing 4% of milk protein and 1% starch agar, respectively. The selected strain was spot inoculated and then incubated at 60 °C for 48 h [17, 18]. Mannanase activity of the strain was assessed on the LBG medium according to Guenoun et al. [19]. The plates were stained with Congo-Red after 48 h of incubation. Protease, amylase, and mannanase production is indicated by the appearance of a clear zone around the colony.

Sensitivity to antibiotics

Antibiotic sensitivity of the bacterial strain was assessed using the disk diffusion method [16, 20–22]. A fresh grown culture was swabbed onto LB agar plates, and then the disks with varied antibiotic concentrations, cefotaxime (CTX 30 µg/mL), levofloxacin (LEV 5 µg/mL), ofloxacin (OFX 5 µg/mL), amikacin (AK 30 µg/mL) and streptomycin (S 10 µg/mL), were put on the plates. The appearance of inhibition zones surrounding the antibiotics disks was measured in millimeters (mm) [23]. Based on inhibitory zone diameter, the bacterium was classified as susceptible or resistant by using by the clinical laboratory standards institute CLSI guidelines [24, 25].

Determination of minimum inhibitory concentration

The resistance to heavy metals of the selected bacterium was evaluated using LB agar plates supplemented with a low concentration (0.5 mM) of CuSO₄, K₂CrO₄, ZnSO₄, and NiCl₂. The minimal inhibitory concentration (MIC) is the lowest metal concentration at which a microorganism cannot proliferate [26]. The minimal inhibitory concentration of the chosen bacteria was tested using LB agar medium containing increasing concentrations of the said heavy metals ranging from 1 to 40 mM. LB agar plates devoid of metal ions were used as the control. All plates were then incubated for 7 days at 60 °C [27, 28]. The results were visually examined after incubation, and the MIC was considered as the lowest of heavy metal concentrations that prevents the bacterial growth [29].

Genome sequencing and annotation

The total genomic DNA of the strain B. glycinifermentans IS-2 T cultivated in LB medium was isolated using a DNA isolation kit (Promega) following the manufacturer’s instructions. The quantity and quality of the gDNA were estimated spectrophotometrically (Nanodrop). The gDNA library preparation was performed using TruSeq DNA PCR-Free Kit (Illumina) and was sequenced with paired-end strategy on the Hiseq Illumina platform. FastQC (v 0.11.9) was used to check the overall quality of the reads [30]. Raw reads were further trimmed by cutadapt v 2.2 [31]. De novo assembly of short-read fragments was carried out using Unicycler (v 0.4.8) [32]. Open reading frames (ORFs) and annotation were predicted using RAST (Rapid Annotation using Subsystem Technology) tool kit (RASTtk) [33] with default parameters against the reference genome B. glycinifermentans strain SRCM103574 (Genbank Accessions: CP035232.1, Assembly Accession (GCA_004103615.1).

Phylogenetic analyses

Fifty single-copy gene sequences from 17 requested genomes were aligned with MAFFT [34] in PATRIC [35], and the nucleotides for each of those sequences were mapped to the protein alignment. Concatenated amino acid and nucleotide data matrices were analyzed under the maximum likelihood criterion using a generalized time-reversible model and a gamma distribution (GTRGAMMA) in RAxML version 8.2.11 [36] and with fast bootstrapping [37] to generate the support values in the tree. Visualization of the phylogenetic tree was produced with FigTree version 1.4.4 (https://github.com/rambaut/figtree/releases).

Additional overall genome relatedness index (OGRI) comparisons based on variations of average nucleotide identity (ANI) were performed using GTDB-Tk—v 1.7.0 online tool available through KBase [38] and digital DNA-DNA hybridization (dDDH) with GGDC 3.0 (http://ggdc.dsmz.de/). Fast genome and metagenome distance estimation was performed using Mash/MinHash [39] available through PATRIC [35].

In vitro germination of barley seedlings and determination of morphological parameters

For surface sterilization, barley seeds were imbibed in 50, 20, and 10% sodium hypochlorite for 3, 10, and 30 min, respectively, and then rinsed three times with sterile water. Bacteria were grown in LB medium at 60 °C for 48 h. After incubation, the cells were centrifuged and then resuspended in sterilized distilled water. The optical density was adjusted to 10⁸ CFU/mL (OD 0.3 at 595 nm) utilizing a UV–Vis spectrophotometer [26, 40]. Briefly, half of the sterilized barley seeds were coated with the washed bacterial suspension for 2 h. The remaining half of the control seeds (without bacteria) was treated with distillated water [41, 42]. Inoculated and control seeds were placed on petri plates covered with filter paper, and 10 mL of different concentration of Zn, Cr, and Ni (0.5, 1, and 2 mM) were applied on each plate. Distilled water was applied only to control plates. Morphological parameters were determined after 10 days of growth including shoot and root length (cm), germination rate, seedling vigor index (SVI), and root tolerance index (RLSI). The formula was as follows [43, 44]:

$$\mathrm{Germination}\mathrm{percentage}=\frac{\mathrm{number}\mathrm{of}\mathrm{seeds}\mathrm{germinated}}{\mathrm{total}\mathrm{number}\mathrm{of}\mathrm{seeds}}\times 100$$

$$\mathrm{Vigor}\mathrm{index}=\left[\right)\mathrm{mean}\mathrm{of}\mathrm{root}\mathrm{length}(\text{cm})+\mathrm{mean}\mathrm{of}\mathrm{shoot}\mathrm{length}(\text{cm})]\times \mathrm{percentage}\mathrm{of}\mathrm{seed}\mathrm{germination}$$

$$\mathrm{RLSI}(\%)=\frac{\mathrm{root}\mathrm{length}\mathrm{of}\mathrm{stressed}\mathrm{plants}}{\mathrm{root}\mathrm{length}\mathrm{of}\mathrm{control}\mathrm{plants}}\times 100$$

Statistical analysis

The SPSS program (IBM SPSS Statistics, version 20.0) was utilized for statistical analysis. Tukey’s multiple comparisons test was used to determine the significance of difference between inoculated and non-inoculated seedling shoot and root length. The non-parametric test was used to compare between tolerance index and vigor index of inoculated and non-inoculated seeds. Analyses were performed at the P < 0.05.

Results

Screening of thermo-tolerant rhizobacteria

In this study, ten bacterial strains isolated from the rhizosphere of date palm were screened for their ability to grow at a temperature of 45 °C. The thermo-resistance of the selected isolates was evaluated by incubating them at different temperature ranges (45–60 °C) for 48 h. Results indicated that only B. glycinifermentans IS-2 T showed a good growth at high temperatures, with a maximum level of 60 °C. The bacterial strain was further tested for its tolerance toward other abiotic stresses. The growth potential of the B. glycinifermentans IS-2 T strain under pH (7–12) and salt (0–2 M) stress in LB agar plates was examined. Moreover, B. glycinifermentans IS-2 T strain was highly tolerant to pH and salt stress with maximum tolerance levels of 10 and 2 M, respectively (Fig. 1).

Fig. 1.

Effect of A pH and B NaCl concentration on bacterial growth

Characterization of bacterial strain

The morphological and biochemical characterization through gram staining and API 50 CHB test kit showed that the bacterial strain was gram positive, aerobic, and capable of fermenting a large range of carbon sources. Moreover, the bacterial strain ability to produce extracellular hydrolytic enzymes on LB agar plates supplemented with their substrates showed the presence of amylolytic, proteolytic, and mannanase enzymes. The presence of these enzymes was determined through the formation of clear zones around the colonies (Fig. 2).

Fig. 2.

In vitro extracellular enzyme A amylase, B protease, and C mannanase activities of B. glycinifermentans IS-2 T strain

Determination of minimum inhibitory concentration of heavy metals

To investigate the ability of B. glycinifermentans IS-2 T strain to grow under various metals concentrations, the bacterial plate’s culture was supplemented with heavy metal concentrations ranging from 0 to 40 mM. B. glycinifermentans IS-2 T showed a highly tolerance capability to heavy metals such as nickel, zinc, copper, and chromium. The whole trend of toxicity toward these showed as Zn²⁺ > Ni²⁺  > Cu²⁺  > Cr²⁺. The lowest heavy metal concentrations (minimum inhibitory concentrations) that inhibited the bacterial isolate growth were then determined. In fact, the minimum inhibitory concentrations (MICs) to Ni²⁺ were 4 mM, to Zn²⁺ were 3 mM, to Cu²⁺ were more than 8 mM, and to Cr²⁺ were up to 40 mM. The results indicate that the B. glycinifermentans IS-2 T strain was a multi-heavy metal-tolerant bacteria and had stronger tolerance capability to the chromium than the others heavy metals.

Antibiotic resistance

The antibiotic sensitivity of the selected bacterium was determined against five antibiotics using disk diffusion method. The results showed that the B. glycinifermentans IS-2T stain was sensitive to four antibiotics with zones of inhibition of 27, 24, 19, and 16 mm for levofloxacin (LEV 5 µg/mL), ofloxacin (OFX 5 µg/mL), amikacin (AK 30 µg/mL), and streptomycin (S 10 µg/mL), respectively (Fig. 3). It was resistant only to one antibiotic: cefotaxime (CTX 30 µg/mL).

Fig. 3.

Antibiotic susceptibility test of B. glycinifermentans IS-2 T strain against cefotaxime (CTX 30 µg/mL), levofloxacin (LEV 5 µg/mL), ofloxacin (OFX 5 µg/mL), amikacin (AK 30 µg/mL), and streptomycin (S 10 µg/mL)

Genomic characterization

Whole genome sequencing of strain B. glycinifermentans IS-2 T was performed with paired-end strategy on the HiSeq Illumina platform. De novo assembly and annotation were produced using Unicycler and RARTtk tools, respectively. Genome assembly of strain B. glycinifermentans IS-2 T yielded 119 contigs indicative of one 4,465,826 bp long chromosomes with GC content of 46.15%, an L50 value of 16, and an N50 of 91,059 bp. This whole genome shotgun project has been deposited at GenBank under the accession JASHHQ000000000. In total, strain B. glycinifermentans IS-2 T genome contains 5044 protein coding sequences (CDS), 73 genes of transfer RNA (tRNA), and three genes of ribosomal RNA (rRNA). The annotation included 1289 (25.55%) hypothetical proteins and 3755 (74.44%) proteins with functional assignments.

Phylogenetic evidence

A maximum likelihood tree was obtained from concatenated amino acid and nucleotide data matrices analyzed by fast bootstrapping and with GTRGAMMA model using RAxML. Strain IS-2 T is located within the B. glycinifermentans clade of RAxML genomic phylogenetic trees, indicating its close relationship to that species, and not with other clades, with strong bootstrap support (Fig. 4). OGRI comparisons using ANI confirm the close relatedness of strain IS-2 T to B. glycinifermentans species, particularly to B. glycinifermentans strain GO-13 T (GCA_001042475.2) with an ANI of 96.06% within the defining limits of 95–96%. In silico dDDH analysis comparing strains IS-2 T and GO-13 T yielded a value of 82.60% (78.7–85.9%) greater than 70%, indicating that both strains belonged to the same species. Using the MinHash algorithm, the closest genome to strain IS-2 T was B. glycinifermentans strain BGLY (GCA_900093775.1) with K-mer match count of 960/1000.

Fig. 4.

A RAxML phylogenetic tree constructed using 50 aligned single copy amino acid and nucleotide sequences. Accession numbers for genome assemblies in GenBank are shown in parentheses. The strain IS-2 T is unambiguously located in the B. glycinifermentans clade. The scale bar equals 0.07 substitution

Effect of B. glycinifermentans IS-2T strain on barley seeds growth

The effects of the bacterial strain inoculation on barley seed germination under heavy metal stress are shown in Fig. 5. The experiments were done in vitro (in Petri dishes) without or with three nickel, zinc, copper, and chromium concentrations (0.5, 1, and 2 mM). Our results showed that barley seeds resisted to different concentrations of heavy metals. In fact, at the highest dose of Cr, Ni, and Zn, germination rate was 84, 84, and 97%, respectively (Fig. 6).

Fig. 5.

Effect of B. glycinifermentans IS-2 T strain inoculation on shoot and root elongation under ZnSO₄ (A), K₂CrO₄ (B), and NiCl₂ (C) stress after 5 days of germination

Fig. 6.

Effect of the B. glycinifermentans IS-2 T strain on germination under A ZnSO₄, B K₂CrO₄, and C NiCl₂ stress

On the other hand, analysis of shoots and roots length of barley seeds showed a significant (P < 0.05) decrease of these growth parameters under heavy metal stress. While the inoculation with the B. glycinifermentans IS-2T bacterium increased shoot and root elongation compared with uninoculated seeds, under both normal and stressful conditions (Fig. 7).

Fig. 7.

The inoculation effect of B. glycinifermentans IS-2 T strain on A shoot and B root length on barley seedlings after 5 days of germination under heavy metal stress. Different letters indicate significant differences among the treatments at P < 0.05

The application of the B. glycinifermentans IS-2T isolate enhanced the shoot elongation by 1.65, 1.36, and 1.22 folds and roots by 1.61, 1.42, and 1.35 times compared to uninoculated seeds under 0.5 mM of Cr, Zn, and Ni, respectively. Furthermore, the most pronounced adverse effect on seed growth was seen under Ni treatment, while barley seeds were able to germinate at the highest concentration. However, the growth of the shoots and roots was completely blocked. The inoculation with B. glycinifermentans IS-2T strain appears to have no effect on the seed’s growth under this concentration.

Other parameter like heavy metal tolerance index was also enhanced in presence of B. glycinifermentans IS-2T under heavy metal stress (Fig. 8A). It was noted that heavy metal tolerance index was decreased from 100% in control seedlings to 11.2, 39.91, and 19.61% at the highest concentration of Cr, Zn, and Ni. An augmentation in the tolerance index was observed when stressed seedlings were amended with bacteria.

Fig. 8.

Effect of the B. glycinifermentans IS-2 T strain on tolerance index (A) and vigor index (B). Different letters indicate significant differences among the treatments at P < 0.05

The vigor indices of germinated seeds (Fig. 8B) defined as the level of activity and the durability of seeds were significantly (P < 0.05) enhanced after inoculation by bacteria under heavy metal stress. In this study, it was observed that the vigor index decreased as metal concentrations increased. The lowest index was found in uninoculated seeds grown at the highest concentrations of metals. The vigor index of barley seeds was enhanced by bacterium inoculation. The highest values were found in uninoculated seeds without heavy metal amendment. Then, the better seedling health was obtained by B. glycinifermentans inoculation.

Discussion

Heavy metals negatively affected seeds by reducing germination, shoot and root elongation, altering protein, and sugar metabolism, causing membrane alteration and oxidative damage and nutrient loss [45]. Telluric bacteria were designed as PGPR which positively affect plants by enhancing health and growth, improving root elongation, and increasing their resistance to environmental stresses [46].

In this study, a novel strain B. glycinifermentans (IS-2T) exhibiting high temperature tolerance was isolated from the date palm rhizosphere. The physiological characterization showed that B. glycinifermentans IS-2T strain was able to endure other abiotic stress such as high pH and salt level. Similar results were reported by Bokhari et al. [47], who isolated several Bacillus sp. from a desertic environment that could grow under salt (1.5 M) and heat (50 °C) stress conditions. Mishra et al. [48] also stated that Pseudomonas guariconensis IIPRMKCP-9 was able to grow at 42 °C. According to the biochemical characterization using API 50 CH testes, the B. glycinifermentans IS-2T strain was able to grow in the presence of a variety of substrates, including glucose, L-arabinose, sucrose, maltose, trehalose, salicin, lactose, D-melibiose, and D-cellobiose, which are common substrates in root exudates and may be used as indicators for PGPR screening. According to Shi et al. [49], metabolizing common root substrates by rhizobacterial isolates aids in their colonization of the rhizosphere and may be serving as PGPR traits for screening. The assimilation of melibiose by bacteria was linked with the trophic benefit in bacteria-plant interaction [50]. Rameshkumar et al. [51] revealed that sucrose, glucose, and maltose are the main important nutrients utilized by rhizobacteria. Additionally, Meyer et al. [50] results showed that these sugars enhance the strain’s potential to solubilize phosphorus. In addition to serving as microbial carbon sources, several of these substrates, including salicin and trehalose, may also help plants respond to pathogen [49]. Trehalose is found in a wide range of species, including bacteria, fungi, yeast, invertebrates, plants, and insects, and linked with several of physiological processes [52]. Lebeis et al. [53] reported that salicin is utilized as immune signal molecules and is crucial for controlling the root-colonizing microbiome. Added to this, Shi et al. [49] suggested that certain PGPR isolates’ capacity to degrade salicin to control plant immune responses was essential for their effect on plants.

Our findings revealed that B. glycinifermentans IS-2T strain secreted protease, mannanase, and amylase and displayed several metal resistance and antibiotic sensitivity. Similar to our results, Ahmed et al. [54] isolated from the Red Sea two halophilic bacteria Enterobacter cloacae W1 and B. glycinifermentans S3, capable of producing amylase and protease. In fact, PGPR produce metabolites and enzymes to enhance plants’ grow and defense [55]. Wu et al. [56] showed that amylase and protease are two PGP substances secreted by rhizobacteria. These two enzymes, secreted during germination, may play a significant role in germinability [57]. Previous research showed that rhizobacteria’s ability to produce enzymes such as mannanase and protease may be related to their antagonistic activities against pathogenic fungi [58, 59]. In this study, we found that B. glycinifermentans IS-2T strain exhibited high tolerance to metals, particularly to chromium. The minimum inhibitory concentrations of zinc, nickel, copper, and chromium was 3 mM, 4 mM, more than 8 mM, and 40 mM, respectively, indicating that the B. glycinifermentans IS-2T strain was a multi-heavy metal-tolerant bacteria. These results are consistent with the previous reports which showed that Paenibacillus sp. ISTP10 strain has tolerance against the same metals with MICs of 3.3, 1.8, 0.36, and 2.5 mM, respectively [60]. Moreover, Kocuria sp. CRB15 strain isolated from the rhizosphere of Saccharum spontaneum was resistant to Zn²⁺, Ni²⁺, Pb²⁺, Cd ²⁺, and Cu²⁺ [61]. Previous study revealed that two B. glycinifermentans strains A4 and A10 were found to exhibit resistance to iron and zinc, respectively [62]. Our results are similar to those reported by Shahid et al. [63], who found that the plant growth promoting bacteria Priestia aryabhattai BPR-9 was able to produce a variety of enzymes and was resilient to a variety of abiotic stresses, such as heavy metals, salt, and antibiotics.

On the other hand, the effect of B. glycinifermentans IS-2T strain on morphological growth parameters of barley seedlings grown under heavy metal stress was assessed in vitro. Although the toxic effects of metals on plant growth are well known, plant responses and the degree of phytotoxicity vary widely depending on heavy metal types, plant genotypes, and concentrations, all of which are crucial for the development, growth, and physiological functions of the target plants [64]. Our results showed that barley seeds exhibited resistance against different metals as seen by their 84, 97, and 84% germination rate at the highest dose of Cr, Zn, and Ni, respectively. Similar results were reported by El Rasafi et al. [65] that showed arsenic, nickel, and copper have no effect on the germination of barley seeds. Similarly, a significant reduction in shoot and root length of barley seeds was observed with different metal concentrations. Previous studies revealed that other species’ root and shoot length decreased in response to Zn, Cr, and Ni stress [66–69]. Our results are also supported by Rolón-Cárdenas et al. [70], who found that root elongation of A. thaliana seedlings was affected by different cadmium concentrations. The biological function of the elements Cd, Ni, and Cr in plants is uncertain. They have been linked to plant death due to chlorosis, decreased CO₂ fixation, root growth, electron transport, and chlorophyll production, and among other effects [71].

Furthermore, the bacterial inoculation improved root and shoot length than uninoculated seeds. De O. Nunes et al. [72] revealed that B. subtilis and B. licheniformis strains application increased the root length of tomato seeds under normal conditions. Similarly, Liaquat et al. [73] found that Cd-tolerant Stenotrophomonas maltophilia inoculation improved shoot and root elongation of seeds of Cd-treated Capsicum annuum L. seeds. Indeed, the prominent effect of B. glycinifermentans IS-2T strain was obtained at 0.5 mM of different metals. Shreya et al. [74] and Islam et al. [75] also reported that the utilization of Cr-resistant strains B. thuringiensis, B. subtilis, and S. maltophilia and Zn-resistant bacterium P. aeruginosa enhanced the shoot and root length of various spices. Similar results were reported by applying Ni-resistant bacterium Bacillus megaterium under Ni stress [76]. Moreover, Ghosh et al. [12] investigated the effects of an arsenic-resistant PGPR Bacillus aryabhattai MCC3374 on the stimulation of rice seedling growth under arsenic stress. In the current experiment, the growth of the root and shoot was inhibited under 2 mM of Ni. The B. glycinifermentans IS-2T strain inoculation had no impact on the seeds’ development at this concentration. Our results agree with those of Madline et al. [4] who suggested that heavy metal treatment completely inhibited the elongation of lettuce seed roots.

Other parameter like heavy metal tolerance index was also affected by different concentration of Cr, Zn, and Ni. An improvement in this index was obtained on the bacterized seeds as compared to those uninoculated. This result agrees with other studies [77, 78]. The vigor index reflects the seedlings’ health, including their capacity to endure a range of diverse stress factors. It was also significantly enhanced after inoculation by bacteria under heavy metal stress. Two previous studies revealed that the vigor index of rice seedlings was also increased with the addition of Klebsiella pneumoniae K5 and Pantoea dispersa strain under cadmium and arsenic stress, respectively [78–80].

Conclusion

The B. glycinifermentans IS-2T strain isolated from rhizospheric soil of date palm trees significantly improved in vitro barley seed growth in terms of germination, root and shoot elongation, and vigor index under heavy metal stress (Zn, Ni, and Cr). In conclusion, this isolated strain has impressive potential for application as a PGPR under stressful conditions.

Author contribution

MB and BK: conceptualization, validation, investigation, writing—original draft, writing—review and editing. AF: conceptualization, validation, resources, data curation, writing—original draft preparation, writing—reviewing and editing. HEA: formal analysis, data curation, visualization. ZC: supervision, resources, methodology. FB: investigation and writing the genomic characterization section. AE: resources, review and editing, funding acquisition. IF: supervision, writing—reviewing and editing.

Funding

This research was supported by PRIMA program (BENEFIT-Med project).

Declarations

Ethics approval

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.