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Article

Isolation and Identification of Multi-Traits PGPR for Sustainable Crop Productivity Under Salinity Stress

by
Md. Injamum-Ul-Hoque
1,†,
Muhammad Imran
2,†,
Nazree Zainurin
1,
Shifa Shaffique
1,
Sang-Mo Kang
1,3,
S. M. Ahsan
4,
Peter Odongkara
1 and
In-Jung Lee
1,*
1
Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2
Biosafety Division, National Institute of Agricultural Sciences, 370, Jeonju 54874, Republic of Korea
3
Institute of Agricultural Science and Technology, Kyungpook National University, Daegu 41566, Republic of Korea
4
Department of Plant Medicals, Andong National University, Andong 36729, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Sustainability 2024, 16(21), 9263; https://doi.org/10.3390/su16219263
Submission received: 24 September 2024 / Revised: 17 October 2024 / Accepted: 22 October 2024 / Published: 25 October 2024
(This article belongs to the Special Issue Sustainable Agriculture and Restoration of Agroecosystem Affected by Climate Change)
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Abstract

:
High salinity poses a significant threat to arable land globally and contributes to desertification. Growth-promoting rhizobacteria assist plants in mitigating abiotic stresses and enhancing crop productivity through the production of siderophores, exopolysaccharides (EPS), solubilisation of phosphate, indole-3-acetic acid (IAA), and other secondary metabolites. This study aimed to isolate, identify, and characterise bacteria that exhibit robust growth-promoting properties. A total of 64 bacterial isolates from the rhizosphere of Miscanthus sinensis were evaluated for plant growth-promoting (PGP) traits, including IAA, EPS, siderophores, and solubilisation of phosphate. Among them, five isolates were selected as plant growth-promoting rhizobacteria (PGPR) based on their PGP features and identified via 16S rRNA sequencing: Enterococcus mundtii strain INJ1 (OR122486), Lysinibacillus fusiformis strain INJ2 (OR122488), Lysinibacillus sphaericus strain MIIA20 (OR122490), Pseudomonas qingdaonensis strain BD1 (OR122487), and Pseudomonas qingdaonensis strain MIA20 (OR122489), all documented in NCBI GenBank. BD1 demonstrated a higher production of superoxide dismutase (SOD) (17.93 U/mg mL), catalase (CAT) (91.17 U/mg mL), and glutathione (GSH) (0.18 U/mg mL), along with higher concentrations of IAA (31.69 µg/mL) and salicylic acid (SA) (14.08 ng/mL). These isolates also produced significant quantities of amino and organic acids. BD1 exhibited superior PGP traits compared to other isolates. Furthermore, the NaCl tolerance of these bacterial isolates was assessed by measuring their growth at concentrations ranging from 0 to 200 mM at 8-h intervals. Optical density (OD) measurements indicated that BD1 and INJ2 displayed significant tolerance to salt stress. The utilisation of these isolates, which enhances plant growth and PGP traits under salt stress, may improve plant development under saline conditions.

1. Introduction

The increase in the global population and the concurrent decrease in accessible land for crop production pose significant challenges for sustainable agriculture [1]. In addition, several abiotic factors, including salinity, water stress, heat stress, and chilling, pose serious challenges to sustainable agriculture across the world [2,3,4]. Salinity significantly limits crop production and has widespread effects on agriculture worldwide [5]. The FAO estimates that 424 million hectares of topsoil (0–30 cm) and 833 million hectares of subsoil (30–100 cm) are affected by salt stress globally [6]. Saline soils are often categorised based on their electrical conductivity (ECe), which is measured at 4 dS/m or above. This level of electrical conductivity is equivalent to a concentration of 40 mM NaCl and leads to an osmotic pressure of around 0.2 MPa [7]. The accumulation of excess salt can disrupt the ionic equilibrium and increases the Na+/K+ ratio, leading to osmotic stress in plants, which adversely affects plant morphology, physiology, and biochemistry [8,9,10]. Salt stress, on the other hand, raises ROS levels, which, in turn, causes oxidative damage, which, in turn, damages plant cells and metabolism [11,12].
Researchers are working on ways to better control resources and create salt-tolerant plant types, such as transgenic plants [13,14]. These methods take too much effort and cost and need more efficient, scalable, and affordable options. Plant growth-promoting rhizobacteria (PGPR) are beneficial soil microorganisms that colonise the plant rhizosphere (a dynamic ecological niche that harbours numerous microbial species) and are influenced by root exudates [15]. These bacteria are recognised as a potential strategy to enhance various crops’ ability to withstand salt stress [16,17,18] through diverse mechanisms [3,19,20]. Microbial biosurfactants (low and high molecular biosurfactants), produced by microorganisms, are surface-active compounds with emulsifying properties that solubilise microbial biofilms [21]. These compounds enhance the interface between PGPR and plant roots, resulting in improved colonisation and increased yield [22]. Biosurfactants also facilitate bacterial cells in forming complexes with essential metal ions and micronutrients in soil by enhancing nutrient chelation [23]. Furthermore, their penetrating, gelling, wetting, and amphiphilic properties render them effective dispersing agents [24], thus benefitting plant root colonisation and increasing the availability of phytohormones and siderophores to plants. Various bacterial strains have been found, including Bacillus spp., Burkholderia, Acinetobacter, Enterobacter, Frankia spp., Halomonas, Microbacterium, Pseudomonas spp., Planococcus, Rhizobia spp., and Serratia, for their capacity to facilitate plants in acclimating to diverse environmental stressors, particularly salinity [5,17,25,26,27,28] by creating biofilms [29,30] and indole-3-acetic acids (IAA) [31]. In addition, exopolysaccharides (EPS) produced by PGPR under salt stress are essential for plant protection through the chelation of soil Na+, limitation of Na+ uptake, promotion of biofilm formation, and enhancement of soil stability [32]. PGPR with aminocyclopropane-1-carboxylate (ACC) deaminase activity degrades the plant ethylene precursor ACC, reducing the stress-induced ethylene levels in plants [33,34], siderophore (enhances iron uptake availability) [17,35,36], and through P solubilisation (availability of phosphorus uptake) [37]. Additionally, these bacteria possess a sodium ion export system that expels excess sodium ions that are detrimental to microbial cells at high concentrations [38]. Certain halotolerant cells accumulate potassium/chloride ions to counterattack salt stress [39]. Specific salt-tolerant strains produce anionic phospholipases to protect against high osmotic pressure and maintain the water balance in the plasma membrane. Furthermore, numerous halotolerant cells produce compatible solutes (amino and organic acids) to maintain the electrolytes concentration and water content under high osmotic pressure [40]. Consequently, the presence of salt-tolerant microbes with PGPR attributes in the rhizosphere contributes significantly to the mitigation of salt stress during plant growth [41]. Recent literature suggests that PGPR can act like endophytes, conferring various benefits to plants by providing growth-promoting traits, enhancing osmotic, antioxidants, and phytohormonal signalling pathways and improving nutrient absorption efficiency [42]. Previous research has demonstrated that Pseudomonas sp. inoculation can enhance the resilience of canola to salt stress, thereby improving its growth [43]. A study by Kumawat et al. [44] revealed that the co-inoculation of halotolerant Pseudomonas sp. and Enterococcus sp. could improve salt tolerance and yield in Vigna radiata. Similarly, Damodaran et al. [45] showed that the bacterial consortia of Lysinibacillus sp. promoted tomato plant growth under salt stress conditions. Furthermore, an additional study found that Lysinibacillus fusiformis mitigated salt stress and improved various growth parameters in rice seedlings [46]. Despite the extensive research on PGPR, there is a significant gap in understanding the full spectrum of PGPR traits, particularly regarding the identification and characterisation of halotolerant bacterial strains that can produce multiple plant growth-promoting (PGP) compounds under saline conditions. While several studies have reported individual strains or consortia of bacteria improving plant tolerance to salt stress, the diversity and multifunctionality of PGPR in saline environments remain underexplored. Furthermore, research on the synthesis of key compounds such as phytohormones, antioxidants, amino acids, and organic acids by halotolerant PGPR is still limited. Increasing salinity resulting from global warming has led to significant crop failures; consequently, PGPR capable of alleviating salt stress are essential for mitigating the adverse effects of salinity. However, there is a paucity of research on microbial strains that can enhance plant growth and salt tolerance. In light of this perspective, the current study sought to isolate and identify halotolerant bacterial strains capable of producing qualitatively measuring PGP traits (in vitro) using various media assays. Furthermore, the study evaluates the identified bacterial strains’ capacity to synthesise phytohormones, antioxidants, amino acids, and organic acids. The identification of bacteria possessing diverse beneficial traits could provide a sustainable approach to enhance crop productivity under challenging soil conditions, thereby addressing food security concerns in salt-affected regions.

2. Materials and Methods

2.1. Collection of Rhizospheric Soil Samples

A total of 9 soil samples were collected from the root zone of the Miscanthus sinensis plant at the Bullocheon River (35.902140′ N, 128.626900′ E) in Daegu, the Republic of Korea. The samples were taken to the Crop Physiology Laboratory at Kyungpook National University in Daegu, the Republic of Korea, being kept at a temperature between 0 °C and 6 °C with ice. Each 1 g of soil sample was placed in a 0.85% NaCl solution in the lab and serially diluted until it reached a concentration of 10−8. Each dilution (100 μL) was spread onto Luria–Bertani (LB) agar medium (Becton, Dickinson and Company, Le pont de claix, France) and incubated at a temperature ranging from 28 to 30 °C until microbial colonies were visible. After culturing, individual colonies were carefully selected after culture and replated on Luria–Bertani (LB) agar medium. Out of 64 randomly selected bacterial isolates, 5 isolates were chosen based on their ability to promote plant growth-promoting (PGP) traits such as the production of EPS (exopolysaccharides), siderophores, phosphate solubilisation, and secretion of indole acetic acid (IAA).

2.2. Qualitative Measurement of Exopolysaccharides, Siderophore, Indole-3-Acetic Acid (IAA), and Phosphate Solubilisation Produced in Isolates

The Congo red agar plate test was used to detect bacteria that produce EPS. The test medium consisted of LB broth (18 g/L), agar (2%), sucrose (5%), and Congo red (0.8 g/L). After mixing, the material was autoclaved and poured into plates. Microorganisms were then grown on the plates at 28–30 °C for 5 days. The colonies appeared black against a red background, which may be due to iron interaction, confirming EPS production [47,48]. For the production of siderophore of the isolate, an agar diffusion technique was used. For this reason, 2 μL of pure bacterial culture was carefully placed on a petri dish with Chromeazurol S-supplemented agar medium. After growing isolates at 28–30 °C for 5 days, orange halos around the bacterial colonies were observed, indicating siderophores production [49].
To assess the presence of IAA, Salkowski’s solution was prepared by blending 50 mL of 35% HClO4 (perchloric acid) with 1 mL of 0.5 M FeCl3 (ferric chloride). This mixture was combined in a 1:1 ratio with pure bacterial isolates. The reaction occurred in darkness for 30 min, and a change in colour from white to pink indicated the presence of IAA [50].
The phosphate solubilising capacity of the bacterial isolates was tested following the guidelines of [51] with slight modifications. The phosphate solubilising media was prepared by mixing glucose (10 g/L), ammonium sulphate [(NH4)2SO4] (0.5 g/L), sodium chloride [NaCl] (0.3 g/L), potassium chloride [KCl] (0.3 g/L), ferrous sulphate [FeSO4] (0.03 g/L), magnesium sulphate [MgSO4] (0.03 g/L), manganese (II) sulphate [MnSO4] (0.03 g/L), calcium phosphate [Ca3(PO4)] (5 g/L), and agar (20 g/L), then autoclaved for 1 h. One millilitre of pure bacteria was cultivated on a trypticase soy agar media using tricalcium phosphate [Ca3(PO4)2] and incubated for seven days at a temperature range of 25 °C to 30 °C. The solubilisation of phosphate by the isolates was evaluated by frequent monitoring for translucent halos around bacterial populations, indicating phosphorus solubility in the isolates.

2.3. Screening of Salt-Tolerant and Identification Using 16S rRNA Gene Sequence

The isolates were assessed for their tolerance to NaCl by exposing them to different concentrations of NaCl (0, 50, 100, 150, and 200 mM) in 10-mL test tubes. This was followed by 24 h of incubation at 25 °C to 30 °C. The optical density at 600 nm (OD600) was measured using UV spectrophotometers (PG Instruments Ltd., Lutterworth, UK) every eight hours (8, 16, 24, and 32). The 16S rRNA gene underwent amplification and sequencing, and its identity was confirmed by comparing it to the NCBI (National Center for Biotechnology Information) sequence using BLAST. The universal primer pair 27F (5′-AGAGTTTGATCACTGGCTCAG-3′) and 1492R (5′-CGGCTTACCTTGTTACGACTT-3′) was used for the amplification of the 16S rRNA. The neighbour-joining method was used to construct a phylogenetic tree, and very similar sequences were aligned using MEGA 11.0 software (CLUSTAL-W program). One thousand bootstrap replications were used to determine statistical support for the nodes in the analysis. The sequencing data was deposited in GenBank accessions, and the procedure was followed as described in [52].

2.4. In Vitro Quantification of Antioxidant Enzyme Activities in Bacterial Isolates

Superoxide dismutase activity was measured by centrifuging a 4-day-old bacterial culture at 10,000× g. A 50-μL supernatant was mixed with 150 µL of extraction buffer, and then, 50 µL of pyrogallol (7.2 mM) was added. Following a 10-min incubation period at 25 °C, the reaction was terminated using 50 μL of 1 N hydrochloric acid, and a modified technique ascertained the spectrophotometric absorbance at 420 nm [17]. Superoxide dismutase (SOD) activity was assessed using the following equation:
SOD activity (%) = [1 − (A − B)/C × 100]
where A, B, and C are pyrogallol-containing, free, and buffer solution controls, respectively.
The catalase activity in the chosen isolates was measured using a modified approach described by [53]. A bacterial culture (4 days old) was centrifuged at 10,000× g, and 50 μL of the supernatant was combined with 50 μL of H2O2 (Hydrogen peroxide) and 100 μL of phosphate buffer. The spectrophotometric absorbance was recorded at 240 nm. The Ellman technique, as described by [54], was used to assess the activity of glutathione.

2.5. In Vitro Quantification of IAA and Salicylic Acid (SA) Produced by Isolates

The selected bacteria were cultured in LB medium for three days. After successful growth, the medium was centrifuged at 5000× g for 15 min to remove cells from the culture broth following the technique described by [55]. The amount of IAA in the broth was determined by using GC-MS in the ion monitoring mode, comparing the peak regions of IAA to established standards as recommended by [56,57]. To determine the concentration of salicylic acid, 5-day-old cultures were mixed with 90% methanol and centrifuged at 13,000× g for 7 min. Rotating evaporation was done on the supernatant left over in a round-bottomed flask. After that, 2.5% TCA, ethyl acetate, cyclopentane, and 1 mL of isopropanol were added in equal amounts. The solution was then dried using nitrogen. The salicylic acid levels were quantified using HPLC [58].

2.6. In Vitro Quantification of Organic and Amino Acids Composition

The organic acid content of the selected isolates was analysed by carefully filtering the culture broth through a precise 0.45-μm Millipore filter (DISMIC-25CS, ADVANTE, Tokyo, Japan). Following this, a little amount of 10 μL from each sample was gently injected into a HPLC column (Waters 600 E; column: RSpakKC-811 (8.0 × 300 mm2); eluent: 0.1% phosphoric acid/water; flow rate: 1.0 mL/min; temperature: 40 °C). To ensure accuracy, we used standards provided by Sigma-Aldrich, St. Louis, MO, USA, and compared retention periods and peak regions within the chromatograms [59].

2.7. Statistical Analysis

The investigation used a fully randomised design for all experiments and analysed the data using GraphPad Prism, version 6.0 (Dotmatics, San Diego, CA, USA). The means were subjected to statistical analysis using Duncan’s Multiple Range test, with a significance level of p < 0.05. The mean value of the results was presented, along with the corresponding standard error, derived from three replications (mean ± standard error).

3. Results

3.1. Preliminary Recognition of PGPR by Means of PGP Characteristics

In this study, a total of 64 bacterial strains were successfully identified. Among these isolates, a total of 32 bacterial isolates showed various growth-promoting activities, including the siderophore (RSS1, RSS3, RSS19, RSS20, RSS22, CR4, CR24, CR28, CR36, CR39, CR41, CRR9, CRR25, CRR37, CRR38, and CRR47); EPS synthesis (RSS3, RSS14, RSS16, RSS17, RSS19 CR4, CR6, CR8, CR24, CR25, CR27, CR28, CR36, CR39, CR41, CRR5, CRR9, CRR25, CRR27, CRR34, CRR35, CRR36, CRR37, and CRR38); and phosphate solubilisation (RSS1, RSS3, RSS14, RSS16, RSS17, RSS19, RSS20, RSS22, CR4, CR5, CR8, CR24, CR25, CR27, CR28, CR35, CR36, CR39, CR41, CR43, CRR5, CRR6, CRR25, CRR27, CRR34, CRR35, CRR36, CRR37, CRR38, and CRR47) (Figure S1). Additionally, five bacterial isolates, namely INJ1 (CR4), BD1 (CR24), MIA20 (CR41), INJ2 (CRR37), and MIIA20 (RSS19), were selected due to their remarkable ability to enhance plant growth-promoting attributes (Figure 1).

3.2. Molecular Identification of Bacterial Isolates

The chosen isolates were finally identified using the 16s rRNA sequencing technique and subsequently submitted to NCBI. The gene sequence submitted to the NCBI GenBank under isolates with accession numbers were obtained for five bacterial isolates and were identified as Enterococcus mundtii strain INJ1 (OR122486), Lysinibacillus fusiformis strain INJ2 (OR122488), Lysinibacillus sphaericus strain MIIA20 (OR122490), Pseudomonas qingdaonensis strain BD1 (OR122487), and Pseudomonas qingdaonensis strain MIA20 (OR122489). Through neighbour-joining, a phylogenic tree was constructed using MEGA 11 software, accompanied by 1000 bootstrap replications. The evolutionary tree in Figure 2 illustrates the taxonomic classification and ecological development of these five distinct strains and shows a similarity of 100% with Enterococcus mundtii, 99% with Lysinibacillus fusiformis, 100% with Lysinibacillus sphaericus, 87% with Pseudomonas qingdaonensis, and 100% similarity with Pseudomonas qingdaonensis, respectively (Figure 2).

3.3. Measurement of Antioxidant Activity by Bacterial Isolates

The antioxidant potential such as SOD, CAT, and GSH of these five different isolates, namely INJ1, BD1, MIA20, MIIA20, and INJ2, were also assessed, as shown in Figure 3. The results showed that BD1 isolate produced a significant amount of superoxide dismutase (SOD) activity, measuring at 17.93 U/mg mL. This was followed by INJ2, INJI, MIIA20, and MIA20 at 16.14, 13.76, 13.46, and 12.48 U/mg mL, respectively. On the other hand, similar trends were obtained in terms of catalase (CAT) activity, as seen in Figure 3B. The isolate BD1 exhibited a significant amount of catalase activity at levels of 91.17 U/mg mL. Moreover, INJ2, INJI, MIIA20, and MIA20 exhibited catalase activity levels of 80.28, 62.51, 61.20, and 50.09 U/mg mL, respectively. In addition, isolates INJ1, BD1, MIA20, MIIA20, and INJ2 exhibited glutathione (GSH) activity levels of 0.16, 0.17, 0.14, 0.15, and 0.17 U/mg mL, respectively. The isolate BD1 produced the highest amount of GSH activity compared to the other isolates. These findings suggest that all five isolates have the ability to mitigate negative impacts on plants through the induction of antioxidant activities.

3.4. Determination of Phytohormone Produced by Bacterial Isolates

The bacterial isolates INJ1, BD1, MIA20, MIIA20, and INJ2 showed a wide range of indole-3-acetic acid (IAA) synthesis. Among all the isolates, BD1 had a significantly higher indole-3-acetic acid (IAA) production, with a concentration of 31.69 µg/mL. The isolates INJ2, INJ1, MIA20, and MIIA20 produced indole-3-acetic acid (IAA) at concentrations of 26.51, 21.52, 19.57, and 16.12 µg/mL, respectively. These findings suggest that BD1 is capable of producing a larger quantity of indole-3-acetic acid (IAA), an important plant growth hormone, particularly under salt stress conditions (Figure 4A). On the other hand, five isolates were discovered to produce salicylic acid (SA), a phytohormone known for its role in plant defence. Among these isolates, BD1 showed a significant increase in SA production compared to the others. The SA was produced by the isolates INJ1, BD1, MIA20, MIIA20, and INJ2 at concentrations of 9.09, 14.08, 6.13, 6.08, and 12.12 µg/mL, respectively (Figure 4B). The above findings suggest that bacterial isolates capable of producing phytohormones could be important for mitigating salt stress.

3.5. Quantification of Organic Acid Produced by Isolates

The bacterial isolates were further examined to determine the levels of organic acids present. The bacterial isolates produced a wide range of organic acids, such as citric acid, malic acid, succinic acid, lactic acid, acetic acid, and propionic acid (Figure 5A). Citric acid production was highest in the BD1 isolate (447.36 ppm), followed by INJ2 (411.64 ppm), MIA20 (361.26 ppm), and INJ1 (337.33 ppm). Similarly, BD1 produced a higher amount of malic acid at 72.53 ppm, followed by INJ2 (60.98 ppm), MIIA20 (38.52 ppm), and INJ1 (33.26 ppm) (Figure 5B). Succinic acid was produced significantly in the BD1 isolate (297.26 ppm), followed by INJ2 (225.67 ppm), MIA20 (135.33 ppm), and INJ1 (113.67 ppm) (Figure 5C). BD1 produced significantly higher amounts of lactic acid (301.67 ppm), acetic acid (1974.33 ppm), and propanoic acid (515.67 ppm) compared to the others. Lactic acid was produced by the isolates INJ1 (116.67 ppm), INJ2 (231.67 ppm), MIA20 (81 ppm), and MIIA20 (163.67 ppm), respectively (Figure 5D). Acetic acid was produced by INJ1 (1039.67 ppm), INJ2 (1694.67 ppm), MIA20 (1043.67 ppm), and MIIA20 (1258.67 ppm) (Figure 5E). Propanoic acid was produced by INJ1 (225.67 ppm), INJ2 (195.825 ppm), MIA20 (357.01 ppm), and MIIA20 (405.67 ppm), respectively (Figure 5F).

3.6. Determination of Amino Acid Produced by Bacterial Isolates

Five bacterial isolates were analysed for their amino acid production profiles (Figure 6). The levels of aspartic acid synthesised by INJ1, BD1, MIA20, MIIA20, and INJ2 were 4.72, 7.67, 3.53, 5.71, and 6.98 mg/g, respectively. Threonine production in the same isolates measured 2.2, 4.42, 1.37, 2.10, and 4.01 mg/g, respectively. Serine was generated at 1.26, 2.79, 1.62, 1.84, and 2.74 mg/g, respectively. Glutamic acid production was 13.30, 23.56, 16.51, 16.97, and 21.19 mg/g, respectively. The glycine levels were 1.93, 3.16, 1.26, 1.84, and 2.76 mg/g, respectively. The alanine production measured 1.99, 4.75, 1.13, 2.31, and 4.24 mg/g, respectively. Cystine was produced at 3.78, 2.67, 1.36, 4.76, and 2.34 mg/g, respectively. Valine production was 4.13, 6.89, 2.42, 4.64, and 6.06 mg/g, respectively. The methionine levels were 1.94, 2.41, 0.84, 1.68, and 2.68 mg/g, respectively. The isoleucine levels in the selected isolates were 2.61, 5.90, 1.52, 2.97, and 5.03 mg/g, respectively. Leucine production was 2.12, 9.63, 1.43, 2.42, and 6.38 mg/g, respectively. Tyrosine was produced at 1.94, 4.14, 1.10, 1.42, and 4.05 mg/g, respectively. Phenylalanine production was 1.63, 4.30, 0.88, 1.43, and 3.38 mg/g, respectively, while lysine was produced at 9.85, 8.04, 7.62, 6.99, and 6.90 mg/g, respectively. Histidine production was 2.13, 1.91, 2.79, 3.43, and 2.87 mg/g, respectively. Arginine was produced at 1.82, 2.78, 1.03, 1.43, and 2.28 mg/g, respectively, while the proline levels were 15.83, 19.62, 12.14, 12.05, and 17.03 mg/g, respectively. This study shows that the isolates have diverse amino acid biosynthetic capabilities, providing valuable insights into their metabolic diversity.

3.7. Screening of Salt Tolerance Assay

Five bacterial isolates (INJ1, BD1, MIA20, INJ2, and MIIA20) were screened for their ability to tolerate high levels of NaCl. This characteristic may help them adapt to and withstand salt-induced stress. In the study, we monitored the optimum bacterial growth and its degree of tolerance at different concentrations (0, 50, 100, 150, and 200 mM) of NaCl at regular 8-h intervals (Figure 7). The obtained OD data indicate that the BD1 and INJ2 isolates performed exceptionally well under varying degrees of salt stress.

4. Discussion

Salt stress is a widely recognised factor that significantly diminishes global agricultural productivity. However, the conventional approaches we have relied on, such as chemical-based fertilisers and pesticides, come with their own limitations and drawbacks [60], including adverse environmental impacts, high cost, and implications for human health. Breeding and genetic alterations are also expensive and limited by societal norms. However, the interaction between plants and microorganisms may help plants tolerate the negative consequences of salt exposure [61]. PGPRs have garnered interest due to their capacity to enhance nutrient levels and facilitate crop growth, resulting in increased agricultural yields [62]. To investigate the plant growth-boosting properties, 64 bacterial isolates were initially selected. Out of these, a total of thirty-four bacterial isolates showed some PGP characteristics. Five of these isolates had many plant growth-promoting (PGP) characteristics, such as the ability to produce siderophores, solubilise phosphate, produce exopolysaccharides (EPS), and synthesise indole-3-acetic acid (IAA). Siderophores are released by the majority of bacteria in the rhizosphere under low iron stress, which helps in chelating ferric iron [63]. Biofilm formation is always accompanied by EPS synthesis, which is a crucial trait for bacteria to tolerate salt. Exopolysaccharides can help mitigate osmotic stress, increasing the plant water content, dry weight, and fresh weight [64]. Rhizobacteria that secrete EPS and promote plant development can also chelate carious cations, including Na+. Bacteria exposed to salt stress can bind Na+ ions through EPS production, reducing soil toxicity [65,66]. Rhizobacteria can enhance phosphorus (P) absorption by stimulating the activity of plasma membrane ion channels in the roots, thereby increasing phosphate availability [67]. Therefore, the PGPR approach, through mechanisms like siderophore production, EPS secretion, phosphate solubilisation, and IAA synthesis, represents a promising natural solution to enhance plant growth under salt stress. This holistic interaction between plants and beneficial microbes can mitigate the limitations of chemical fertilisers and pesticides while contributing to sustainable agricultural practices.
Salt stress impairs plant active oxygen metabolism, producing ROS and peroxidising membrane lipids. This, in turn, disrupts biochemical processes and reduces plant growth [68]. The antioxidant enzymes responsible for scavenging ROS in plants include SOD, POD, GSH, APX, and CAT. However, most plants are unable to produce enough antioxidants to mitigate oxidative damage under stress, and while these enzymes play a vital role in protecting plants from oxidative damage, many plants under severe salt stress are unable to produce enough antioxidants to fully counteract the excess ROS, leading to oxidative damage and reduced growth. Therefore, five bacterial isolates in this investigation produced a diverse range of SOD, CAT, and GSH, consistent with previous research, showing that bacterial isolates can increase the antioxidant activity under salt stress [69].The identification of five PGPR isolates was achieved through the analysis of the 16S rRNA gene sequence. The NCBI database indicated their closest similarity as Enterococcus mundtii, Lysinibacillus fusiformis, Lysinibacillus sphaericus, and Pseudomonas qingdaonensis. These well-known species have been isolated and tested as PGPR on diverse crops to mitigate salt stress [45,70,71,72,73]. In conclusion, the identification of these PGPR isolates suggests that they have potential as biostimulants for improving plant tolerance to salt stress by enhancing the antioxidant enzyme activity. By supporting the plant’s defence mechanisms against oxidative stress, PGPR can significantly improve plant resilience, growth, and productivity under saline conditions. This contributes to the development of environmentally friendly agricultural practices aimed at improving crop yields in challenging environments.
Plant hormones govern cellular growth and development, division, and the absorption of nutrients, while also playing a pivotal role in aiding plants to resist both biotic and abiotic challenges [74]. IAA is one of the hormones that controls plant growth and root development in response to abiotic stress [50]. Evidence suggests that the synthesis of indole-3-acetic acid (IAA) might promote relationships among plants and microorganisms [75]. The present investigation identified five bacterial isolates that exhibited varying concentrations of IAA, which aligns with previous research showing that bacterial isolates produce significant amounts of IAA in plants under salt stress [76]. The production of indole-3-acetic acid (IAA) by PGPR is an essential process that increases root development and the surface area, which, in turn, helps plants absorb water and nutrients from the soil [77,78]. Another important plant hormone, salicylic acid (SA), helps plants tolerate and adapt to environmental stress. This study found that five distinct bacterial isolates exhibited varying levels of SA production, consistent with the previous research showing significant SA production by bacterial isolates under salt stress in different crops [79]. In summary, the production of IAA and SA by PGPR is a significant factor in plant growth promotion and stress tolerance. IAA increases root development and nutrient uptake, while SA boosts the plant’s ability to withstand stress. The study’s identification of PGPR isolates that produce both IAA and SA under salt stress conditions highlights their potential as bioinoculants to improve crop resilience in saline soils. These findings support the growing body of research advocating for the use of PGPR to enhance plant growth and productivity in environmentally stressful conditions.
Plants rely on organic acids for a variety of essential functions, including carbon and energy production and the modulation of their tolerance to abiotic stress [80]. A variety of organic acids can be synthesised by the bacterial strains used in this investigation. These findings are consistent with the results reported by [51], who also found that bacterial isolates capable of producing organic acids had a positive impact on plant development under salt-induced stress. An essential role that amino acids play is as intermediates or precursors to metabolites that help reduce the effects of biotic and abiotic stresses [81,82]. The present investigation identified five bacterial isolates that exhibited distinct amino acids. This finding confirms that bacteria living within the soil release amino acids to promote the development of plants, with the subsequent absorption of these amino acids by plants through essential root pathways [83]. This research emphasises the potential impact of a particular PGPR strain in enhancing agricultural productivity by synthesising amino acids, organic acids, phytohormones, and antioxidants under salt stress conditions. These bacterial metabolites play complementary roles in promoting plant growth, enhancing nutrient absorption, and improving stress tolerance, making PGPR a promising tool for sustainable agriculture in salt-affected soils.

5. Conclusions and Future Prospects

In conclusion, the microbial strains investigated in this study exhibited a significant capacity to synthesise essential plant hormones, including salicylic acid (SA) and indole-3-acetic acid (IAA), which are integral to plant growth promotion and stress resilience. Furthermore, these strains demonstrated the ability to enhance plant antioxidant defence mechanisms by upregulating the activity of key enzymes, such as SOD, CAT, and GSH. Collectively, these effects contribute to the mitigation of oxidative stress in plants. These isolates also produced substantial quantities of organic and amino acids. The selected isolates were evaluated for their salinity tolerance at various concentrations. BD1 and INJ2 exhibited superior resistance to salt stress. Future research should encompass large-scale field trials to validate these findings across diverse crop species and environmental conditions. Additionally, investigating the potential synergistic interactions between these microbes and other beneficial soil organisms, as well as optimising application methodologies, may further enhance their efficacy. The integration of these microbial strains into comprehensive crop management strategies holds promise for improving agricultural sustainability, enhancing plant health, and reducing the dependence on chemical inputs.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/su16219263/s1: Figure S1: Initial screening of bacterial isolates for their plant growth-promoting (PGP) features, including (A) siderophore production, (B) EPS generation, (C) phosphate solubilization, (D) qualitative evaluation of IAA production.

Author Contributions

Conceptualisation: I.-J.L. and M.I.; Methodology: M.I.; Formal analysis and investigation: M.I.-U.-H., N.Z. and P.O.; Writing—original draft preparation: M.I.-U.-H.; Writing—review and editing: M.I., S.S., S.M.A. and S.-M.K.; Funding acquisition: I.-J.L.; Resources: I.-J.L.; Supervision: I.-J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No. 2022R1A2C1008993).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Qualitative evaluation of five bacterial isolates to assess their strong performance in generating PGP characteristics.
Figure 1. Qualitative evaluation of five bacterial isolates to assess their strong performance in generating PGP characteristics.
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Figure 2. The isolates of the bacteria were analysed to create a phylogenetic tree using the neighbour-joining technique, based on their 16S rRNA sequences of genes. The selected isolates have been taxonomically classified into three distinct genera, specifically Enterococcus, Lysinibacillus, and Pseudomonas, and these groups were visually represented by pink, green, and violet, respectively.
Figure 2. The isolates of the bacteria were analysed to create a phylogenetic tree using the neighbour-joining technique, based on their 16S rRNA sequences of genes. The selected isolates have been taxonomically classified into three distinct genera, specifically Enterococcus, Lysinibacillus, and Pseudomonas, and these groups were visually represented by pink, green, and violet, respectively.
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Figure 3. Antioxidative enzymatic activities of isolates, including the activities of (A) SOD, (B) CAT, and (C) GSH. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
Figure 3. Antioxidative enzymatic activities of isolates, including the activities of (A) SOD, (B) CAT, and (C) GSH. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
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Figure 4. Quantitative measurement of phytohormones, namely (A) IAA and (B) SA, produced by bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
Figure 4. Quantitative measurement of phytohormones, namely (A) IAA and (B) SA, produced by bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
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Figure 5. Quantification of organic acids, namely (A) Citric acid, (B) Malic acid, (C) Succinic acid, (D) Lactic acid, (E) Acetic acid, and (F) Propanoic acid produced by bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
Figure 5. Quantification of organic acids, namely (A) Citric acid, (B) Malic acid, (C) Succinic acid, (D) Lactic acid, (E) Acetic acid, and (F) Propanoic acid produced by bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
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Figure 6. Determination of amino acids, (A) Aspartic acid, (B) Threonine, (C) Serine, (D) Glutamic acid, (E) Glycine, (F) Alanine, (G) Isoleucine, (H) Cystine, (I) Valine, (J) Tyrosine, (K) Methionine, (L) Lysine, (M) Histidine, (N) Leucine, (O) Phenylalanine, (P) Arginine, and (Q) Proline produced by the selected bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
Figure 6. Determination of amino acids, (A) Aspartic acid, (B) Threonine, (C) Serine, (D) Glutamic acid, (E) Glycine, (F) Alanine, (G) Isoleucine, (H) Cystine, (I) Valine, (J) Tyrosine, (K) Methionine, (L) Lysine, (M) Histidine, (N) Leucine, (O) Phenylalanine, (P) Arginine, and (Q) Proline produced by the selected bacterial isolates. ND denotes “not detected”. The data are the means of three replicates ± SEM. Identical letters reflect no statistically significant differences (p < 0.05), whereas distinct letters imply substantial differences.
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Figure 7. Growth curve of five bacterial isolates at every 8 h interval (A) 8 h, (B) 16 h, (C) 24 h, and (D) 32 h, at different salinity concentrations. The data are the means of three replicates ± SEM.
Figure 7. Growth curve of five bacterial isolates at every 8 h interval (A) 8 h, (B) 16 h, (C) 24 h, and (D) 32 h, at different salinity concentrations. The data are the means of three replicates ± SEM.
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MDPI and ACS Style

Injamum-Ul-Hoque, M.; Imran, M.; Zainurin, N.; Shaffique, S.; Kang, S.-M.; Ahsan, S.M.; Odongkara, P.; Lee, I.-J. Isolation and Identification of Multi-Traits PGPR for Sustainable Crop Productivity Under Salinity Stress. Sustainability 2024, 16, 9263. https://doi.org/10.3390/su16219263

AMA Style

Injamum-Ul-Hoque M, Imran M, Zainurin N, Shaffique S, Kang S-M, Ahsan SM, Odongkara P, Lee I-J. Isolation and Identification of Multi-Traits PGPR for Sustainable Crop Productivity Under Salinity Stress. Sustainability. 2024; 16(21):9263. https://doi.org/10.3390/su16219263

Chicago/Turabian Style

Injamum-Ul-Hoque, Md., Muhammad Imran, Nazree Zainurin, Shifa Shaffique, Sang-Mo Kang, S. M. Ahsan, Peter Odongkara, and In-Jung Lee. 2024. "Isolation and Identification of Multi-Traits PGPR for Sustainable Crop Productivity Under Salinity Stress" Sustainability 16, no. 21: 9263. https://doi.org/10.3390/su16219263

APA Style

Injamum-Ul-Hoque, M., Imran, M., Zainurin, N., Shaffique, S., Kang, S.-M., Ahsan, S. M., Odongkara, P., & Lee, I.-J. (2024). Isolation and Identification of Multi-Traits PGPR for Sustainable Crop Productivity Under Salinity Stress. Sustainability, 16(21), 9263. https://doi.org/10.3390/su16219263

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