1. Introduction
Globally, the production rate of agriculture is far less than the estimated food requirement of the ever-increasing population and the gap will be widened over time [
1] (GAP Report, 2018). Agro-ecosystems are influenced by environmental and climatic conditions, farming techniques, and management practices. It is estimated that internationally, salinity affects 22% of the total cultivated and 33% of the total irrigated agricultural area, which is increasing at an alarming rate of 10% annually. Pakistan is also facing severe salinity issues and a total area of 6.30 million hectares is salt affected, out of which 1.89 million hectares is marked as saline [
2].
Due to a higher concentration of sodium chloride (NaCl), plants growing in salt-affected soils suffer from both hyperosmotic and hyperionic effects. These stresses result in reduced water uptake; altered ion and mineral absorption rates; increased production of reactive oxygen species, causing disorganization of the cell membrane; and reduction of metabolic activities [
3]. Halophytes adapt themselves to saline conditions by adjusting their physiological activities, maintaining their water balance by osmotic adjustments, producing compatible solutes, and modifying the antioxidant system [
4]. Some plants overcome salinity stress through the production of osmolytes, particularly glycine betaine, proline, soluble sugars, and proteins [
5].
Improvement in the crop yield of saline soils requires a multidimensional approach consisting of salt-tolerant varieties or amelioration by chemical neutralizers, but there is a dire need for eco-friendly sustainable approaches. Rhizobacteria, showing potential to improve plant growth, are termed as plant growth-promoting rhizobacteria (PGPR) [
6]. PGPR have the potential to improve plant growth through various mechanisms, including better plant growth, the production of phytohormones, and amelioration of stresses [
7]. Due to the natural coping mechanisms of PGPR, their inoculation can help the amelioration of various abiotic stresses in plants. PGPR inoculation can help to improve the growth and yield of crops, particularly in regions prone to drought and salt stress [
8,
9]. Natural halotolerant PGPR strains have better potential for the amelioration of salt stress in regional crops for sustainable yields. These native PGPR strains are well acclimated to indigenous conditions and the plant–microbe interactions can help the plants to tolerate stress [
10].
In this study, native halotolerant PGPR strains were isolated from local saline soils, and their ability to promote plant growth when inoculated under salt stress was investigated. The objective of the present research was to focus on the evaluation of isolated bacterial strains to stimulate salinity tolerance and the promotion of wheat growth, as well as the identification and characterization of the candidate strain both bio-physiochemically and genetically. This study provides a basis to identify and characterize PGPR from natural saline conditions and testing their potential for improving salinity tolerance in wheat, the major staple crop across the world.
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 SR
1, SR
2, SR
3, and SR
4 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 SR
2 and SR
3 strains belong to the
Azospirillum genus (
Azospirillum brasilense and
Azospirillum lipoferum) and the other two (SR
I and SR
4) 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,
45,
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.