*3.7. E*ff*ect of PGPR Inoculation on the Biomass of Wheat (Triticum aestivum L.) Plants Grown under Salinity Stress*

The overall decrease of 30% in the plant biomass of wheat plants was observed due to salt stress. However, the bacterial isolates exerted a significant positive influence on wheat growth and resulted in an increase in the biomass of plants in the control and stressed conditions, respectively. The relative increase in the fresh and dry biomass due to bacterial isolates ranged between 39% and 67% as compared to the uninoculated plants under saline conditions.

The best results were obtained when plants were inoculated with a consortium of all four isolated strains, which caused an increase of 93% in stress and 60% in controlled conditions. Moreover, pronounced results were also encountered for dry biomass, when plants were inoculated with a consortium, which resulted in an increase of 65.4% in salt stress and 78.7% in control conditions (Table 3).


**Table 3.** Effect of inoculation of halotolerant PGPR on the fresh and dry biomass and leaf area of wheat plants grown under salinity stress.

This data displays the means and standard deviation (*n* = 3). Different letters show significant differences (*p* < 0.05). (SR1: Inocualted with *Bacillus* sp; SR2: Inocualted with *Azospirillum brasilense*; SR3: Inocualted with *Azospirillum lipoferum*; SR4: Inocualted with *Pseudomonas stutzeri*; Consortium is a combination of all four strains *Bacillus* sp, *Azospirillum brasilense*, *Azospirillum lipoferum*, *Pseudomonas stutzeri*).

#### *3.8. E*ff*ect on the Membrane Stability Index and Water Content*

Results of the percent electrolytic leakage showed that the inoculation remains significant under stress as well as normal conditions However, co-inoculation with bacterial consortium successfully decreased (34%) the ionic discharge at the 150 mM NaCl level compared to the control (Table 4). Furthermore, the percent of water content showed a significant reduction of 33% in wheat plants under salt stress as compared to the uninoculated control plants. More pronounced results were obtained with SR2 and SR3, causing an increase of 10.5% and 17.54% in the stress condition. The consortiuminoculated plants recorded the maximum amount of water of 21% and 17.64% in the stress and control conditions. A similar trend was observed by SR1 and SR4 (Table 4).

**Table 4.** Effect of inoculation of halotolerant PGPR strains on the leaf water content and electrolyte leakage of wheat plants grown under salinity stress.


This data displays the means and standard deviation (*n* = 3). Different letters show significant differences (*p* < 0.05). Treatment details are the same as in Table 3.

#### *3.9. Chlorophyll Contents*

Salinity stress negatively affected the photosynthetic pigments of wheat plants. A considerable decrease of 30.4%, 22%, and 25% was observed in chlorophyll a, b, and total chlorophyll. The response to the consortium was effective (*p* ≤ 0.05) and resulted in a 13.23%, 12.49%, 12.9%, and 11.76% increase as compared to the control under salt-stress conditions (Table 5).

**Table 5.** Effect of halotolerant PGPR on the chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents of wheat plants grown under salinity stress.


This data displays the means and standard deviation (*n* = 3). Different letters show significant differences (*p* < 0.05). Treatment details are the same as in Table 3.

#### *3.10. Proline Contents*

Salinity stress increased proline accumulation in wheat plants. A considerable increase of 50% in the proline content of wheat plants was recorded in saline stress conditions as compared to their respective control. Inoculation with halotolerant PGPR increased the levels of proline in the leaves. All four inoculants increased the proline contents in the range of 18–36%, respectively. The accumulation of proline was maximum in consortium-treated plants, with an increase of 46.67% under stress conditions (Table 6).


**Table 6.** Effects of halotolerant PGPR on the total soluble sugar, amino acid, protein, and proline contents of wheat plants grown under salinity stress.

This data displays the means and standard deviation (*n* = 3). Different letters show significant differences (*p* < 0.05). Treatment details are the same as in Table 3.

#### *3.11. Amino Acid Content*

The amino acid content was highest in the consortium of halotolerant PGPR strains, with an increase of 19.29% and 15.54% under salt stress and control conditions. Moreover, plants inoculated with SR2 and SR3 contained 10% and 14.1% greater concentrations of amino acids as compared to the uninoculated stressed plants (Table 6).

#### *3.12. Total Soluble Sugar*

Salinity stress produced a significant increase of 12.5% for the soluble sugar contents of wheat plants as compared to the control. The best outcomes were obtained when plants were inoculated with SR2 and SR3, which resulted in an increase of 9.52% and 15.87%, respectively, under stress conditions. However, a more prominent effect was revealed with the inoculation of a consortium of strains, with an increase of 28.57% and 23.2%, respectively, under the stress and control condition (Table 6).

#### *3.13. Antioxidants Enzyme Assay*

The antioxidant enzymes of the wheat plants showed a significant increase under salinity stress. Inoculation with all four halotolerant PGPR improved the production of antioxidant enzymes in plants. However, the best results were shown by the consortium of all strains. The consortium increased the superoxide dismutase activity by 21.4% as compared to stressed plants. Similarly, a significant increase of 16% in the catalase activity was recorded by the inoculation with the consortium. A significant increase of 34.4% in the peroxidase content of plants was recorded as compared to the control (Table 7).


**Table 7.** Effects of halotolerant PGPR on the antioxidant enzymes activity of wheat plants grown under salinity stress.

This data displays the means and standard deviation (*n* = 3). Different letters show significant differences (*p* < 0.05). Treatment details are the same as in Table 3.

#### *3.14. Heatmap Responses of Pearson's Correlation Coe*ffi*cient (r)*

From the heat map analysis, the data of the osmolyte production, electrolyte leakage, chlorophyll contents, antioxidant enzymes, and halotolerant PGPR showed positive correlations (Figure 4). A comparative analysis of the parameters related to salinity tolerance (presented by green boxes)

showed that salinity tolerance had a positive correlation with amino acid, osmotic potential, soluble sugars, proline, SOD, POD, and CAT activities (Figure 5).

**Figure 4.** Heatmap of the correlation coefficient (r) for the antioxidant enzymes, stress determinants, and relative water content of wheat leaves treated with bacterial isolates and their consortium. Whereas, EL = Electrolyte leakage, Pro = Proline, POD = Peroxidase, SOD = Superoxide dismutase, CHL = Total chlorophyll, TAA = Total amino acids, TSS = Total soluble sugars, RWC = relative water content.

**Figure 5.** Principle component analysis (PCA) of phytohormones, proline, total soluble sugars, and reducing sugars of halotolerant bacterial isolates and their consortium grown under salt stress in culture conditions. Whereas, IAA = Indole acetic acid, GA = gibberellic acid, CK = Trans zeatin riboside, RS = Reducing sugars, TSS = Total soluble sugars.

#### **4. Discussion**

Soil bacteria associated with rhizosphere have been known as growth promotors as well as biotic and abiotic stress alleviators [8]. Bacteria associated with the roots of halophytes and saline soil, capable of tolerating higher levels of salts, are termed as halotolerant [39]. In the current study, bacterial isolates SR1, SR2, SR3, and SR4 showed the best salt tolerance abilities among all 50 bacterial isolates from the roots–soil interface of plants growing in the saline area. Phenotypic and molecular genotyping (16S RNA sequencing) of four potent isolates proved that SR2 and SR3 strains belong to

the *Azospirillum* genus (*Azospirillum brasilense* and *Azospirillum lipoferum*) and the other two (SRI and SR4) belong to the genus *Bacillus* (*Bacillus sturtezi)* and *Pseudomonas* (*Paeudomonas stutzeri*) (Table 2). These beneficial PGPR belonged to different genera, which indicate that plant growth promotion has been distributed across different taxons Halotolerant strains from the genera of *Pseudomonas*, *Bacillus*, *Azospirillum*, *Klebsiella*, and *Ochromobacter* have shown remarkable performance in the amelioration of salt stress in a wide range of crops [40].

Halotolerant PGPR has been reported to promote plant growth as well as mitigate salinity stress [41]. In the current study, we attempted to identify the key mechanisms used by halotolerant strains to alleviate the salinity stress in wheat plants by regulating plant defense mechanisms. The ability of halotolerant PGPR to produce phytohormones is associated with improved growth of plants under saline conditions [42]. The halotolerant PGPR produced IAA, GA, CK, and ABA. The results showed that *Azospirillum* strains produced higher amounts of GA, IAA, and CK than those of *Bacillus* and *Pseudomonas* strains in liquid media (Figure 1). The production of hormones by halotolerant PGPR is thoroughly supported by previous literature and many halotolerant strains of *Azotobacter*, *Bacillus*, *Arthrobacter*, *Azospirillum*, and *Pseudomonas* have been shown to produce IAA, GA, CK, and ABA [43]. These phytohormones regulate the stress defense responses in plants. They influence all aspects of plant growth, like cell wall elongation (IAA), cell division (CK), germination (gibberellin), and stress tolerance (ABA) [44–46]. Various reports suggest that these phytohormones produced under salinity stress help plants to survive and impart tolerance in them under abiotic stresses [46].

Here, the results proved that rhizobacteria secrete more compatible solutes (soluble sugars and proline) in culture media supplemented with a higher NaCl (10%) content. Various studies documented that bacterial cells can accumulate a considerable amount of compatible solutes inside their cells, acting as osmolytes and helping them to survive under severe osmotic stress [47].

Salinity is one of the common factors that can limit agricultural productivity due to its effects on seed germination, plant growth, and crop yield. Wheat is an important staple crop, but as it is a moderately salt-tolerant crop, high salt stress strictly limits its growth and development. Salt stress ultimately reduces the crop yield and nutritive value of wheat. The regulation of physiological, enzymatic, and biochemical changes in plants after inoculation with PGPR helps to alleviate salt or drought stress [40,48].

We demonstrated that salinity reduced the growth and development and relative water content of wheat plants. It also caused curling and wilting of leaves, early leaf senescence, and ultimately a reduction in the growth of plants. This is consistent with what was found in a previous study that salinity restricts cell differentiation and the cell cycle due to osmotic and ionic stress, deficiency of nutrients, oxidative damage, and limited water uptake, which affects plant germination, growth development, and physiological processes, ultimately leading to growth inhibition [49].

In this study, a consortium of four strains produced a prominent result for the dry biomass and leaf area than the control and individual inoculants. These results are in line with Walker et al. [50], who reported that inoculation with a consortium of *Azospirillum-Pseudomonas-Glomus* improved the root architecture in maize under salinity. A better adaptability of PGPR to stress conditions is correlated with efficient root colonization, phosphate solubilization, and nitrogen fixation abilities [51]. From the results, it is clear that salinized plants inoculated with halotolerant strains and their consortium exhibited a higher relative water content of leaves. Rakshapal et al. [52] also observed that PGPR-treated plants not only cope with stress but also that these microbes help to maintain higher water levels in comparison to control plants.

Salinity decreases the photosynthetic efficiency of plants and results in the production of reactive oxygen species (ROS), which cause damage to DNA, proteins, and membranes [53]. We described the results of photosynthetic pigments of wheat plants, which showed that treatment with a consortium showed a pronounced effect of reducing the damage caused by salinity on the photosynthetic apparatus. A similar pattern of results was reported by El-Esawi et al. [54], who observed an increase in the photosynthetic efficiency of plants by PGPR inoculation under salinity.

Salt stress can develop more discharge of electrolytes through the misplacement of Ca associated with membranes. As a result, the permeability of the membrane is destroyed and accumulates a higher efflux of electrolytes inside plant cells/tissue [55]. In the current study, the successive increase in the electrolyte leakage of wheat plants was observed at 150 mM salt stress than the control. These results are inconsistent with the Bojórquez-Quintal et al. [56], who found salt stress enhances electrolyte leakage and the generation of reactive oxygen species (ROS), having a detrimental effect on plant growth. Our results showed that inoculation with halotolerant PGPR tends to decrease the injurious effect of saline stress and decrease the potential electrolytic leakage of ions in stress-treated plants. This is consistent with what was found in previous studies [57,58].

In the present study, the concentration of compatible solutes was also increased in inoculated wheat plants under salt stress (Table 7). The accumulation of compatible solutes, particularly proline, free amino acid, and soluble sugar, is correlated with the adaptability of the plant to stress conditions. We reported that halotolerant PGPR produces compatible osmolytes, which help the plants to maintain their ionic balance. PGPR also induce osmolyte accumulation [59] and phytohormone signaling [40], which facilitates plants in overcoming the initial osmotic shock after salinization. In a previous study, it was found that rice inoculation with salt-tolerant *Bacillus amyloliquefaciens* under salinity increased the plant's salt tolerance and affected the expression of genes involved in osmotic and ionic stress response mechanisms [60].

Proline is the most important osmolyte, which is produced in plants by the hydrolysis of proteins under osmotic stress [61]. From the results, it is clear that a consortium of halotolerant PGPR plants improved proline levels under salt stress. These results are in line with Wang et al. [62]. The production of osmolytes helps the plant to maintain a high turgor potential, prevent oxidative damage by scavenging reactive oxygen species, and protect the membrane structure [63].

We also reported a pronounced increase in the production of soluble sugars with a consortium of halotolerant strains in wheat under salinity stress. PGPR can stimulate carbohydrate metabolism and transport, which results in changes in the source–sink relations, photosynthesis, and growth rate. In previous reports, seeds inoculated with *B. aquimaris* strains showed an increased production of total soluble sugars in wheat under salinity conditions, which resulted in higher biomass and plant growth [64].

An increase in the antioxidant enzyme activity of wheat plants grown under salinity stress was observed by a consortium of halotolerant PGPR strains. This indicates that these bacteria can help the plant to combat the deleterious effects of ROS generated during salinity stress. These results tie well with the previous studies, where an increase in antioxidant enzyme activity under salinity stress was proven to be associated with salt tolerance [65]. Moreover, Wang et al. [66] reported that the application of PGPR strains alleviates the oxidative damage induced by abiotic stresses, including salinity, by augmenting the activity of antioxidant enzymes.

#### **5. Conclusions**

In summary, crop inoculations with halotolerant PGPR consortium can serve as a potential tool for alleviating salinity stress. Halotolerant PGPR strains have developed several mechanisms to cope with salinity, particularly the potential to produce phytohormones and compatible solutes. Halotolerant PGPR strains can induce salinity tolerance in plants by activating key defense mechanisms like the production of osmoregulators as well as activating ROS scavenging enzymes. Natural microflora adapted to saline conditions can be used for the development of microbial consortia for crop inoculation, ultimately leading to the formulation of biofertilizer for salt-stressed areas. However, further investigation is needed to observe their performance in field conditions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/7/989/s1, Table S1: Morphology of isolates from rhizosphere of plants from saline soil, Table S2: Preliminary screening data of isolated strains (+ indicates groeth, − indicates no growth), Table S3: Growth characters of isolated strains, Table S4: Effect of Isolates on germination attributes of wheat, Table S2: Carbon/Nitrogen source utilization pattern

determined by QTS -24 kits, Figure S1: Phylogenetic analysis of strain SR1, Figure S2: Phylogenetic analysis of strain SR2, Figure S3: Phylogenetic analysis of strain SR3, Figure S4: Phylogenetic analysis of strain SR4.

**Author Contributions:** Conceptualization, N.I. Writing—Original Draft, N.I. and R.M. Formal Analysis, H.Y. and W.K. Investigation, R.M. Proofreading, H.E.E. and D.J.D. Editing, H.Y., W.K. and S.I. Formatting, D.J.D. Writing—Review, H.Y., S.I. and W.K. Supervision, N.I. Facilitation, H.E.E. Review, H.E.E. and D.J.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by Allcosmos Industries Sdn. Bhd. through research project No. R.J130000.7344.4B200. The APC was supported by UTM-TNCPI research fund.

**Conflicts of Interest:** Authors have no conflict of interest.

**Ethical Statement:** Not applicable.

#### **References**


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