Next Article in Journal
Unravelling Phytochemical and Bioactive Potential of Three Hypericum Species from Romanian Spontaneous Flora: H. alpigenum, H. perforatum and H. rochelii
Next Article in Special Issue
Meta-Analysis of the Effect of Nitric Oxide Application on Heavy Metal Stress Tolerance in Plants
Previous Article in Journal
Drought-Induced Responses in Maize under Different Vapor Pressure Deficit Conditions
Previous Article in Special Issue
Effect of Phytoplasma Associated with Sesame Phyllody on Ultrastructural Modification, Physio-Biochemical Traits, Productivity and Oil Quality
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 11
Issue 20
10.3390/plants11202769
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress

by
Muhammad Ayaz
1,†,
Qurban Ali
1,†,
Qifan Jiang
1,
Ruoyi Wang
1,
Zhengqi Wang
1,
Guangyuan Mu
2,
Sabaz Ali Khan
3,
Abdur Rashid Khan
1,
Hakim Manghwar
4,
Huijun Wu
1,
Xuewen Gao
1 and
Qin Gu
1,*
1
Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Department of Plant Pathology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China
2
Shenzhen Batian Ecological Engineering Co., Ltd., Shenzhen 518057, China
3
Biotechnology Department, College of Environmental Sciences, COMSATS, Abbottabad 22060, Pakistan
4
Lushan Botanical Garden, Chinese Academy of Sciences, Jiujiang 332000, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2022, 11(20), 2769; https://doi.org/10.3390/plants11202769
Received: 21 September 2022 / Revised: 9 October 2022 / Accepted: 11 October 2022 / Published: 19 October 2022
(This article belongs to the Special Issue Physiological Aspects of Plant Response to Pathogens and Abiotic Stress)
Browse Figures
Review Reports Versions Notes

Abstract

:
Soil salinity is a major constraint adversely affecting agricultural crops including wheat worldwide. The use of plant growth promoting rhizobacteria (PGPR) to alleviate salt stress in crops has attracted the focus of many researchers due to its safe and eco-friendly nature. The current study aimed to study the genetic potential of high halophilic Bacillus strains, isolated from the rhizosphere in the extreme environment of the Qinghai–Tibetan plateau region of China, to reduce salt stress in wheat plants. The genetic analysis of high halophilic strains, NMCN1, LLCG23, and moderate halophilic stain, FZB42, revealed their key genetic features that play an important role in salt stress, osmotic regulation, signal transduction and membrane transport. Consequently, the expression of predicted salt stress-related genes were upregulated in the halophilic strains upon NaCl treatments 10, 16 and 18%, as compared with control. The halophilic strains also induced a stress response in wheat plants through the regulation of lipid peroxidation, abscisic acid and proline in a very efficient manner. Furthermore, NMCN1 and LLCG23 significantly enhanced wheat growth parameters in terms of physiological traits, i.e., fresh weight 31.2% and 29.7%, dry weight 28.6% and 27.3%, shoot length 34.2% and 31.3% and root length 32.4% and 30.2%, respectively, as compared to control plants under high NaCl concentration (200 mmol). The Bacillus strains NMCN1 and LLCG23 efficiently modulated phytohormones, leading to the substantial enhancement of plant tolerance towards salt stress. Therefore, we concluded that NMCN1 and LLCG23 contain a plethora of genetic features enabling them to combat with salt stress, which could be widely used in different bio-formulations to obtain high crop production in saline conditions.

1. Introduction

Wheat (Triticum aestivum L.) is grown on 17% of cultivated land worldwide [1]. It is considered to be an important cereal crop that provides 82% of protein and 85% of calories to the world population. It is an essential staple food for approximately 41 countries, but it can be cultivated worldwide due to its unique adaptability nature [2]. Staple food demand is increasing day by day, so it is necessary to produce wheat in abundance to fulfil the needs of the current increasing population throughout the globe [3]. Wheat is reported to be adversely affected by abiotic stresses worldwide [4]. Among abiotic stresses, soil salinity is known to be a major constraint in wheat production that results in terrible yield loss [5]. Salinity has a detrimental effect on wheat growth by inducing physiological and metabolic disorders that lead to oxidative stress, osmotic stress, nutritional abnormalities, membrane dysfunction, reduced photosynthetic activity and improper hormone function [6,7]. Plants under salt stress often overproduce reactive oxygen species (ROS), i.e., superoxide (O2−) and hydrogen peroxide (H2O2) leads to protein, cell wall and nucleic acid damage [8,9]. Salinity has become more notorious than ever before due to global warming, along with possibly undesirable effects on the economy, such as in agriculture and food security, mainly in developing nations [10]. Around 300 million hectares of cultivated irrigated land is affected by salinity, and half of it lies in Pakistan, India, China and USA [11].
The application of Plant Growth Promoting Rhizobacteria (PGPR) has been documented to reduce abiotic stresses in plants that will enhance sustainable agriculture [12,13]. The use of PGPR to enhance plant growth under salt stress is an emerging technology [14]. Many researchers are working on the potential of PGPR for salt stress alleviation in agricultural crops [15]. The cross-talk between microbes and plants provides multifaceted mechanisms that induce salt stress resistance in plants [16]. Previous studies revealed that PGPRs such as Pesudomonas spp. and Bacillus spp. isolated from saline soil can promote plant growth under saline conditions [6,17]. This idea has attracted the attention of researchers to discover efficient biotechnological approaches involving the application of PGPR as a bio-resource to reduce salinity effects and enhance plant growth under salt stress [17,18]. Recently, many significant results have been achieved through the application of PGPR for salt stress reduction in wheat plants, leading to enhanced growth and productivity in the saline area [16]. PGPR or certain compounds can help to withstand salt stress by modulating hormonal, photosynthetic and ROS scavenging pathways in plants [8,10]. Among PGPR, Bacillus strains have attracted significant focus because of their important characteristics such as gram-positivity, ability to make spores, colonise roots and promote plant growth [19,20]. Bacillus is an important and well-known group of microbes that can produce multiple products with the potential to show resistance to unfavourable environmental conditions, due to their natural rigidness and stability [21,22]. Bacillus strains have been used to alleviate abiotic stresses in plants, however, the genetic features enabling them to show resistance were rarely studied under extreme conditions [20,23]. Therefore, it is important to develop high salinity-tolerant Bacillus strains with salt-resistant genetic features, which simulate the production of bioactive osmotic compatible solutes that cope with hostile salt stress conditions [24].
The expression analysis of key genetic features in the genome of Bacillus enable them to show tolerance under stress conditions [25]. The genes that belong to different families, i.e., membrane transport, osmotic regulation, signal transduction and oxidative stress and antioxidant enzymes are mainly attributed to the potential of Bacillus strains to endure salt stress [25,26,27]. The genes expressed under salt stress result in certain transcriptional changes to improve membrane function, antioxidant enzyme activity, and water maintenance inside the cell through regulating various signalling pathways [28]. In addition to these essential genetic features, the formation of biofilm by Bacillus is an important property to colonise and produce secondary metabolites under adverse conditions [29].
The stress signals in plants, such as proline accumulation, lipid peroxidation and abscisic acid, can regulate stress in plants, as these indigenous signal molecules can stimulate various plants biological processes both distantly and locally [30]. Few PGPRs, including Bacillus, have the potential to regulate these stress signalling molecules and lead to stress alleviation in plants [15]. Under salt stress conditions, abscisic acid (ABA), which promotes root growth and regulates water balance in plants [31], is also reported to be downregulated by the application of PGPRs [32]. Malondialdehyde (MDA) is a well-known marker for oxidative stress injury and membrane lipid peroxidation under stressful conditions in plants [33]. Interestingly, PRGR was also able to decrease the level of MDA in plants [9]. Similarly, the use of PGPR could positively modulate the activity of Proline, which is an osmoprotectant which plays a vital role in plant defence against salt stress [34,35]. The Bacillus strains can also regulate the plant stress response by regulating phytohormones in plants [36]. The presence of the ACC deaminase gene in Bacillus can lower ethylene concentration under abiotic stresses that results in plant growth promotion [37]. The upregulation of phytohormones such as cytokinin, gibberellin, and auxin by Bacillus under stress conditions in plants results in wheat plant growth promotion [38]. The main plant cell wall regulator expansin is reported to be stimulated by PGPR under stress conditions that results in plant growth promotion [16].
Therefore, the aim of the present study was to evaluate the potential of Bacillus strains isolated from the Qinghai–Tibet region of China to enhance plant growth and reduce the adverse effect of saline stress on wheat. The genetic screening and expression analysis revealed Bacillus spp. key genetic features that make them capable of resistance under salt stress conditions by regulating various metabolic processes. The halophilic Bacillus strains (salt-loving) were found to have positive effects on wheat growth parameters by minimizing the adverse effect of salinity.

2. Results

2.1. Bacterial Growth and Biofilm Potential under Various Salt Concentrations

The selected strains and FZB42 were able to grow on up to 10% NaCl LB medium. However, two strains, i.e., NMCN1 and LLCG23, were further able to grow on up to 18% NaCl and 14% NaCl, respectively, and showed high resistance under salt stress conditions. NMCN1 and LLCG23 were then grown in LB liquid medium with different NaCl concentrations (1–18%) and the growth pattern was observed for each strain. Significant growth was observed for NMCN1 at up to 16% NaCl, followed by LLCG23 up to 14% NaCl medium. Furthermore, less growth of NMCN1 on 18% NaCl was observed than on 16% NaCl medium, as shown in Figure 1A,B.
Biofilm formation potential results of Bacillus strains (NMCN1, LLCG23 and FZB42) under different NaCl concentration (1%, 4%, 8%, 10%, 16%, and 18%) at 37 °C for 5 days showed that all the strains were able to form the finest biofilm up to at 8% however the potential of FZB42 to form biofilm at 10% was reduced and stopped at high NaCl concentrations of 14–18%. The NMCN1 was observed as the best strain to form biofilm continuously up to on 18% NaCl followed by LLCG23. The weak biofilm was noticed at 18% NaCl for NMCN1 while LLCG23 was unable to grow at the same conditions as shown in Figure 1C.

2.2. Relative Expression Profiling of Predicted Genes through qPCR

The signal transduction genes, i.e., DegS and DegU, through which bacteria can perceive and respond to abiotic stress were detected in the selected strains. The gene superoxide dismutase SodA plays an important role to minimise the effect of free radicals produced in bacteria under stress conditions detected in selected strains. The OhrR gene was also predicted in the selected strains that plays an important role in the regulation of hydrogen peroxide in the bacterial cell under salt stress. The OstB gene, which regulated osmotic potential in the bacteria and plays an important role in salt stress, was detected in the selected strains. The glycine betaine genes (OPuD and OpuAC) are also responsible for osmotic regulation, and were detected in selected strains. The expression profile of selected genes involved in salt resistance was studied in NMCN1 and LLCG23 in comparison to FZB42 grown in different NaCl concentrations (1%, 10%, 14% and 16%) for four days. The results showed a linear upsurge in the expression of salt-resistant genes in NMCN1 and LLCG23, as compared with control. The expression of salt-resistant genes in FZB42 was also stimulated up to 10% NaCl, however, with increase in saline conditions, i.e., 14% NaCl and 16% NaCl, FZB42 was unable to stimulate the expression of salt-resistant genes. The high expression of the selected salt resistant genes was noticed in NMNC1 in linear order with increased saline conditions, followed by LLCG23. The gene involved in signal transduction, i.e., DegU showed high expression in saline conditions (16% NaCl) as compared with LLCG23, which might impart efficient sensing potential to the NMCN1 and makes it able to induce high resistance to salt stress. The SodA gene was noticed to be highly expressed in bacteria at 16% NaCl saline condition followed by LLCG23 to regulate ROS inside the strains. The OpuAC gene responsible for glycine betaine involved in osmotic protection and regulation also showed increased expression in NMCN1, followed by LLCG23 under 16% NaCl concentration. The OstB and OhrR genes, which are reported to play an important role in osmotic regulation under high saline conditions, were also observed to be highly overexpressed in NMCN1. The ComA genes involved in quorum sensing regulation were also noticed to be highly stimulated in NMCN1 and LLCG23. The overall results showed that all the selected salt-resistant genes showed an upsurge in the expression level with increasing saline conditions in the selected strains as shown in Figure 2.

2.3. ROS Regulation Potential of Bacillus Strains under Salt Conditions

Bacillus strains cultured in 16% NaCl LB media for four days were studied for ROS regulation through the staining method. The bacterial cells stained with dye for ROS detection were observed under microscope. The results showed that the salt-resistant strains NMCN1 and LLCG23 maintained lower levels of ROS grown for four days in 16% NaCl LB media at 37 °C, whereas the FZB42 strain was observed with a significant increase in ROS level grown in the same conditions, as shown in Figure 3.

2.4. Seedling Growth and Root Morphological Parameters

Wheat seedlings’ growth along with different root morphological parameters were noticed to be enhanced by Bacillus strains under different NaCl concentrations (0 mmol–200 mmol). The FZB42 was observed to have high growth-promoting potential under normal conditions (0 mmol) as compared to NMCN1 and LLCG23, however, a decrease in growth-promoting potential for FZB42 was observed at a high saline condition of 200 mmol. The halophilic strain NMCN1 showed maximum growth potential under 200 mmol, followed by LLCG23 and FZB42. The vigour index (VI) calculated from the data of total seedlings’ length and percent germination was noticed to be maximum under high NaCl concentration (200 mmol) in wheat seedlings inoculated with NMCN1 (VI = 1289), found to be significantly higher than control seedlings and the seedlings inoculated with LLCG23 and FZB42. The second highest VI was observed for LLCG23 grown under 200 mmol NaCl stress. Moreover, under normal conditions (0 mmol) the best result for FZB42 strain was noticed in growth promotion and vigour index of wheat seedlings having V1 value (1700) higher than un-inoculated control seedlings VI as shown in Figure 4.
Moreover, the data related to morphological root parameters such as root diameter, root volume, root length, root tips and root surface area were observed to be increased for NMCN1-inoculated wheat seedlings, and several tips were found to be increased in the case of seedlings inoculated with NMCN1, followed by LLCG23 and FZB42 under high stress conditions (200 mmol NaCl) in comparison with non-treated seedlings grown in the same conditions. The overall data demonstrated that wheat seedlings treated with selected Bacillus strains have increased root morphological parameters as compared with control under normal conditions (200 mmol), whereas NMCN1 and LLCG23 were found to be more efficient to alleviate the risk of high salt stress conditions (200 mmol) as compared with FZB42 as shown in Figure 5.

2.5. Plant Stress Response Parameters Quantification and Expression Analysis

Plant stress response parameters were observed to be regulated by Bacillus strains. The UPLC results for ABA level showed significant reduction for NMCN1-inoculated wheat plants followed by LLCG23 under high saline conditions (200 mmol NaCl) in comparison with non-inoculated plants as control. The ABA level in wheat plants inoculated with NMCN1 was 560 ng/g FW and 640 ng/g FW for LLCG23 in comparison with 1200 ng/g FW ABA level in control plants. Under normal conditions, FZB42 was observed to reduce ABA level significantly, i.e., 432 ng/g FW compared with 750 ng/g FW of ABA level in control. The wheat plant inoculated with NMCN1 and LLCG23 also showed reduced ABA level under normal conditions as shown in Figure 6.
The Malondialdehyde (MDA) level was measured through the spectrophotometric method in inoculated and un-treated Bacillus strain wheat plants under saline conditions (200 mmol NaCl). In inoculated wheat plants the MDA level was noticed to be significantly reduced, i.e., 0.493 µmole MDA/g FW for NMCN1 and 0.544 µmole MDA/g FW for LLCG23 in comparison with control MDA content of 0.897 µmole MDA/g FW under salt stress, as shown in Figure 6. The MDA content of wheat plants treated with FZB42 under salt stress conditions (200 mmol) was 0.797 µmole MDA/g FW that is slightly high in comparison with halophilic strains (NMCN1 and LLCG23). Under normal conditions, measured MDA content for all treatments showed no significant increase when compared with control.
In the present study proline content in wheat plants was noticed to be significantly increased in wheat seedlings inoculated with NMCN1 and LLCG23 under salt stress conditions (200 mmol NaCl). The inoculated wheat plants showed a maximum proline level, i.e., 1.272 µg/g FW and 1.145 µg/g FW, respectively, in comparison with control 0.293 µg/g FW under saline condition. The measured proline content for all treatments under normal conditions (0 mmol) showed no significant increase as compared to their control. The candidate genes involved in the different stress response parameters were observed to be highly regulated by halophilic Bacillus strains. The keys genes involved in the MDA and ABA pathways were observed to be significantly downregulated in NMCN1 and LLCG23 treated wheat plants under high saline conditions. Moreover, the proline encoding P5CS was observed to be upregulated in the plants treated with the selected strains. The maximum increase in the expression of P5CS in plants under saline condition was noticed for NMCN1, followed by LLCG23. The results showed that stress-responsive genes are significantly regulated in wheat plants by salt resistance strains (MNCN1 and LLCG23) as shown in Figure 6.

2.6. Bacillus Strains Enhance Plant Growth under Saline Conditions

In the current study plant growth and root morphological parameters were noticed to be significantly increased in wheat plant inoculated with bacteria under salt stress as shown in Figure 7 and Figure 8. The high halophilic strain NMCN1 followed by LLCG23 were observed for significant improvement in wheat plant growth, one-fold increase in shoot/root length and fresh/dry weight under salt stress (200 mmol NaCl) whereas the strain FZB42 slightly enhanced plant growth under the same condition. Furthermore, all the Bacillus strains were noticed for nearly equal enhancement in wheat pants under normal conditions. An increase in salt stress conditions, i.e., 100 mmol–200 mmol NaCl concentrations negatively affected the growth of un-inoculated wheat plants however the effect was reduced when the plants were inoculated with halophilic strains. The NMCN1 was observed to highly alleviate the negative effect of increased saline condition. Overall salt stress was observed to be alleviated in the presence of halophilic strains, i.e., NMCN1 and LLLCG23 in wheat plants.

2.7. ROS Reduction in Wheat Roots by Bacillus Strains under Salt Conditions

The halophilic strain NMCN1 was observed to highly alleviate ROS production in wheat roots grown under the high salt condition as compared to FZB42. The strains NMCN1 played an efficient role in ROS reduction in wheat roots and thus might reduce the severe effect of salt stress on wheat roots, as shown in Figure 9.

2.8. Regulation of Photosynthesis in Wheat by BACILLUS Strains

Wheat plants inoculated with Bacillus strains under salt stress conditions were studied for photosynthesis and stomatal conductance regulation. The current findings showed that all the strains were able to regulate the photosynthesis and stomatal conductance in inoculated and un-inoculated wheat plants under saline or normal conditions. The NMCN1 strain was noticed to significantly improve the photosynthesis rate and stomatal conductance in wheat plants under high saline conditions (200 mmol NaCl). The second important results under salt stress for wheat photosynthetic and stomatal conductance enhancement were noticed for LLCG23 strain. Bacillus strain FZB42 was also observed for the most significant improvement in the photosynthetic activity, as well as stomatal conductance compared with control plants under normal conditions, as shown in Figure 10.

2.9. Expression Analysis of Growth and Salt Related Genes in Wheat

The expression of genes related to growth promotion and salt resistance in wheat plants under saline conditions were analysed in the presence of Bacillus strains (NMCN1, LLCG23 and FZB42). The current results showed that all the strains have the potential to efficiently regulate the growth promoting and salt resistance genes in wheat plants under saline conditions (200 mmol NaCl). The most significant results were obtained for NMCN1 which possessed high potential to upregulate the genes involved in growth promotion and salt tolerance in wheat plants. The plants inoculated with NMCN1 under salt stress (200 mmol NaCl) significantly overexpressed the genes related to expansin (expA1), cytokinin (CKX2) and auxin (ARF), followed LLCG23 and FZB42. The expression of ethylene encoding gene (ERF) was noticed to be highly downregulated in wheat plants treated with NMCN1 strain grown under the same stress condition. Under normal conditions the plants inoculated with FZB42 were noticed for high upregulation of growth-related genes. Overall high upregulation in the expression of growth promoting genes (CKX2, expA1 and ARF) was observed, while significant down-regulation was observed for ethylene encoding gene (ERF) in wheat plants treated with high halophilic strains, NMCN1 and LLCG23, under the saline conditions (200 mmol NaCl), as shown in Figure 11. The expression of salt stress-related genes in wheat plants inoculated with Bacillus strains was observed to be significantly stimulated under saline conditions, i.e., (200 mmol NaCl). The wheat plants treated with highly halophilic bacteria, NMCN1, were noticed to highly over-express the salt-resistant genes (MYB, DREB2, HKT1 and WRKY17), followed by LLCG23 and FZB42. The NMCN1 was found to highly upregulate the expression of selected genes with 4–5-fold increase in plants grown under high salt stress as compared with control. The FZB42 was also observed to increase the expression of the selected genes but less than NMCN1 and LLCG23 as shown in Figure 11.

3. Discussion

The plant faces many challenges due to its sessile nature in the form of abiotic and biotic stresses [39,40]. Soil salinity is considered as a major constraint among abiotic stresses that reduces crop productivity worldwide [40,41]. Through molecular breeding and genetic engineering plants’ defence mechanisms have been improved for the purpose of alleviating the adverse effects of abiotic stresses on important agricultural crops [6,24], but these methods are mostly laborious and time-consuming [10,20]. Therefore, the use of PGPR is considered as a fast and eco-friendly approach among the researchers to reduce salt stress in plants [18,42]. Plants’ abiotic stress tolerance mechanism can be improved by inoculating them with PGPR [2,43]. In the present study the Bacillus spp. strains, i.e., NMCN1 and LLCG23 isolated from the extreme environment of Qinghai–Tibetan region of China showed high salinity stress tolerance and were able to grow at up to 16% and 18% NaCl concentrations. The reference Bacillus velezensis FZB42 strain was also observed to grow on LB medium under 10% NaCl concentration.
In the present study, the halophilic strains were explored for their genetic features that could take part in alleviating salt stress condition. The results showed that key genes responsible for membrane transport, osmotic regulation, regulation of reactive oxygen species, cell signalling and fatty acid and lipid metabolism that enable the bacteria to combat with salt stress were mostly detected in NMCN1 and LLCG23, as compared with FZB42. All these genetic features (SodA, OpuAC, OPuD, OstB, ComA and DegU) previously reported in the literature were known to play an important role in abiotic stress (Cold and Salt) resilience [17,20]. The whole genome genetic analysis of the halophilic strains (NMCN1 and LLCG23) also showed that the selected strains possess a plethora of genes to combat high salinity stress. The gene SodA linked with ROS regulation was noticed to be present in the selected strains. The presence of the ROS regulation genes help Bacillus strains to reduce the adverse effects of oxidative stress induced after salt stress. Moreover, the most important signal transduction genes DegS and DegU were also detected in halophilic strains. The predicted genes in halophilic strains linked with signal transduction suggested that our bacteria have the ability to sense different stresses that enable the strains to show response to such environmental shocks at early stage. The genes related to osmotic stress regulation, i.e., OpuAC, OPuD and OstB were also predicted in salt-resistant Bacillus strains that play an important role in glycine betaine synthesis which is considered as an important osmoprotectant [44]. The ComA gene was also found in the halophilic strains which is an important regulator of quorum-sensing previously reported in the literature.
The upregulation in the expression of salt-resistant genes found in the halophilic strains (NMCN1 and LLCG23) reveals their potential to survive under high saline conditions. The FZB42 was unable to survive under high salt stress conditions due to lower expression of selected genes involved in salt stress tolerance. Moreover, the high expression of quorum-sensing regulator gene (ComA) was for the first time noticed in the halophilic strains that could help them to survive under saline conditions, however, it was previously reported as an important transcriptional activator of other physiological responses in bacteria [45]. The genes involved in the signal transduction pathway, i.e., DesS and DegU, were also observed to be highly expressed in NMCN1 and LLCG23, showing their potential to sense and respond rapidly to the changing environmental stresses and thus support the previous findings that documented the main role of these genes in Bacillus Pumilus 7P strain under salt stress. The high upsurge in the expression of the signal transduction genes of NMCN1 and LLCG23 reveal that the strains were able to perceive salt stress signals in an effective manner as compared to FZB42, which was unable to grow under high saline conditions due to lower expression of the mentioned genes. The genes, i.e., OhrR and OpuAC, play an important role in osmotic regulation and were also noticed to be over-expressed in NMCN1 and LLCG23 under high salt stress conditions, and thus support the previous findings related to the activation of these transcription factors upon abiotic stress [46]. The genetic screening and expression analysis of the predicted genes involved in abiotic stress resistance suggested that the halophilic Bacillus strains under study contain several genetic features supporting them to survive under saline stress condition.
The Reactive Oxygen Species (ROS) produced in bacteria under salt–stress conditions disrupt the cellular function that leads to cell death [33,47]. In the present study the halophilic strains (NMCN1 and LLCG23) were found to have less ROS as compared with FZB42, indicating that these strains have a high potential to regulate oxidative stress for the purpose to well survive under salt stress condition. Furthermore, biofilm formation is another important characteristic of Bacillus strains that help them endure environmental stresses. The growth of bacteria in the self-secreted matrix for biofilm formation help them survive under adverse conditions [48]. Previous study showed the distortion in bacterial biofilm structures under high salt stress conditions [49], but little evidence is reported about the development of biofilm under high saline conditions. The current research first time showed thick and fine biofilm formation by halophilic strains (NMCN1 and LLCG23) for the purpose to survive under high saline conditions. Salinity affected plant growth, inducing reductions in the roots system [50]. We assume that the selected halophilic Bacillus stains possess the unique potential of biofilm formation and producing important metabolites under salt stress. The biofilm formation of halophilic PGRR can be linked with their ability to colonize plant roots in order to alleviate the adverse effects of salt stress on plants.
The halophilic strains (NMCN1 and LLCG23) were further noticed for their important role in reducing the adverse effects of salinity on wheat plants grown under saline conditions. The strains have the potential to regulate stress responsive parameters, such as proline accumulation, lipid per-oxidation and abscisic acid (ABA) in salt stress challenged wheat plants. The halophilic strains were observed for efficiently, regulating the mentioned parameters as compared with FZB42 strain. The ABA level was noticed to be high in plants grown under saline conditions but wheat plants inoculated with halophilic strains were observed to have decreased ABA levels for the purpose of mitigating salinity stress that supports the previous findings of ABA level reduction in inoculated Bacillus strains plants facing salt stress [51]. Furthermore, the accumulation of proline and reduction in malondialdehyde (MDA) levels are important indicators in plants to combat various environmental stresses, as proline is an important osmoprotectant [52], whereas the decreased level of MDA indicated less membrane damage of plants inoculated with halophilic Bacillus strains under saline conditions [53]. The halophilic strains in the current study were also found to regulate proline and MDA levels in a very efficient manner in wheat plants grown under salt stress conditions. The results showed an increased accumulation of proline and decreased MDA level in inoculated halophilic bacteria wheat plants grown under saline conditions. Moreover, expression of the key genes responsible for these stress responsive parameters were also noticed to have the same pattern that further supports our claim of positive regulation for alleviating salt stress in bacteria inoculated plants. Besides regulation of stress responses, the current study also reported the important role of halophilic Bacillus strains (NMCN1 and LLCG23) in the positive regulation of the photosynthesis rate and stomatal conductance of wheat plants under salt stress. There have been some studies suggesting the improvement in these parameters by inoculated bacteria in normal conditions [54] but no previous study has indicated such results under salt stress in wheat plants.
The use of PGPR to promote plant growth has been previously well documented in the literature [55]. There are many studies related to plant growth promotion by PGPR under drought stress conditions [56]. Similarly, the application of Pseudomonas spp. enhances the growth of pea plant under drought stress [57]. We hereby report the halophilic strains (NMCN1 and LLCG23) for their major role in enhancing wheat growth through seed inoculation under saline conditions. Furthermore, all the selected Bacillus strains (NMCN1, LLCG23 and FZB42) were observed to have a high potential of wheat plant growth promotion under normal conditions, however, under salt stress conditions (200 mmol) high reduction in plant growth promoting potential of FZB42 strain was observed for the first time as compared to other halophilic strains NMCN1 and LLCG23. Previously, Bacillus and Pseudomonas strains were studied for their positive effects on seed germination, vigour index and root morphology of young growing plants [58,59], but in the current study significant improvements in vigour index and root morphological parameters of wheat seedlings were first time reported through inoculation of highly salt-tolerant Bacillus spp. strains NMCN1and LLCG23. The growth parameters such as fresh/dry biomass, root and shoot length of wheat inoculated with the selected bacteria were noticed to be significantly increased as compared to control plants.
The genes related to phytohormones, i.e., cytokinin (CKX2), auxin (ARF) and expansin (exPA1) were observed to be upregulated while ethylene gene (ERF) was noticed to be downregulated in wheat plants inoculated with selected Bacillus strains under salt stress conditions, as there are many studies elaborating the importance of phytohormones in plant growth promotion [24,60]. Previous studies also showed upregulation of phytohormones, i.e., cytokinin, auxin and expansin related genes and downregulation of the ethylene gene in abiotic stress plants inoculated with PGPR [20,61]. The regulation of genes related to phytohormones in the same pattern by halophilic Bacillus strains NMCN1 and LLCG23 significantly showed their main role in wheat growth promotion under salt stress conditions. Thus, we noticed that wheat plant growth promotion through inoculated bacteria under saline conditions is an accumulative effect of their ability to regulate phytohormones and salt resistance genes. Moreover, few detailed studies related to salt stress reduction in wheat plants by halophilic Bacillus strains through regulation of salt-resistant genes (MYB, DREB2, HKT1 and WRKY17) were reported in the literature [20,40]. The present study evaluated the potential of Bacillus strains to enhance plant growth and reduced the adverse effect of saline stress on wheat through regulation of salt resistant genes. Furthermore, these halophilic Bacillus strains (salt-loving) were found to have positive effects on wheat growth parameters and minimize the adverse effects of saline conditions.

4. Materials and Methods

4.1. Growth and Biofilm Formation of Bacillus Strains

Bacillus strains were obtained from Laboratory of Biocontrol and Bacterial Molecular Biology, Nanjing Agricultural University. These strains were isolated from Qinghai–Tibet plateau region of China. These strains were screened for their growth and biofilm formation on Luria-Bertani (LB) agar plates under different salt (NaCl) concentrations (1–18%) at 37 °C for 4 days along with Bacillus velezensis FZB42, a model plant growth promoting and bio-control strain as a control. Two strains (NMCN1 and LLCG23) that showed high salt resistance were then grown in liquid culture to study their growth pattern at optical density OD600 for different time intervals (maximum up to 96 h) at 37 °C through a spectrophotometer. The growth pattern of each strain was determined by making a growth curve for the different time intervals. Furthermore, to know the effects of different salt concentrations on the biofilm formation, the selected strains were grown on LB liquid culture. The selected salt-resistant strains were grown in a 20 mL flask in LB at 37 °C till 1.0 OD600 and then 4 μL of each bacterial culture was mixed with LB with different salt concentrations in 12 wells sterile costar® culture plates. After sealing the plates were kept at 37 °C for 5 days. The experiment was repeated thrice with three replicates for each treatment.

4.2. Genetic Analysis of Bacillus Strains and Expression of Predicted Salt Related Genes

Each strain was screened for predicted salt tolerance genes in its genome using the bioinformatics tool RAST. The predicted genes were amplified through PCR with primers designed through the Primer Quest tool and detected on gel electrophoresis, as previously described [20]. Total RNA was isolated from each PGPR, i.e., NMCN1, LLCG23 and FZB42, grown under different NaCl concentrations (1%, 10% 14% and 16%) in liquid LB culture at 37 °C for 4 days in a shaker incubator. The samples were harvested after 4 days post-inoculation for the expression pattern of the selected salt resistance genes. The RNA from each sample was extracted through (Bio-tek-OMEGA, Inc., Norcross, GA, USA) kit. The purity and concentration of extracted RNA was calculated using Nanodrop (Nanodrop 1000, Thermo Scientific, Wilmington, DE, USA) at an absorbance of 260/280 nm. The RNA sample of each strain was then subjected to cDNA synthesis using a 5× All-In-One RT Master Mix (with AccuRT Genomic DNA Removal Kit) by Applied Biological Materials Inc. (abm®, Beijing, China), according to the given protocol.
The expression profile of each salt resistant gene in the selected strain was observed by RT-qPCR performed in a Step One Real-Time PCR System (Applied BioSystems, Foster City, CA, USA) machine based on the modulation in fluorescence relative to the cyclic increase in PCR products. The Syber Green (Takara Bio, Beijing, China) was used as a detector that emits fluorescence while binding to cDNA. The fluorescence was recorded with threshold cycle ©. The PrimerQuest tool of IDT was used to design primers for the selected salt resistant genes listed in Table S1. The rpsJ genes, previously mentioned as a housekeeping gene in Bacillus strains, were used in this current study [20,62]. The 20 µL volume of PCR mixture contained 10 µL 2X SYBR premix Ex Taq (Takara Bio, Beijing, China) (TilRNaseH Plus) with reference dye ROX (0.4 µL), 0.4 µL forward and reverse primers (20 mmol), 2 µL cDNA (100 ng) and 6.8 µL ddH2O. The PCR was programmed as: 95 °C for 30 secs as initial denaturation temperature, 40 cycles of 95 °C for 5 secs and 34 secs at 60 °C. The T melt curve was observed to find out the accuracy of the reactions at the end of the PCR process. At the end comparative C method of 2−ΔΔCT reported by [63] was used to analyse the data.

4.3. ROS Detection in the Selected Strains Grown under Saline Conditions

The severity of cell damage or dysfunction is closely related to the production of ROS under unfavourable conditions [64]. As an indicator of cell damage or cell disturbance, ROS was detected in each strain grown under different salt concentrations. For ROS detection NMCN1, LLCG23 and FZB42 strains were grown on LB medium with different salt concentrations and incubated at 37 °C for 4 days. The cells of each strain were harvested after 4 days of inoculation in 1.5 mL Eppendorf tube through centrifugation at 1000 rpm. The harvested cells were incubated at 25 °C for 30 min in a dichloro-dihydro-fluorescein diacetate (DCFH-DA) (JianCheng Bioengineering, Nanjing, China [20,65] and 10 mM sodium phosphate buffer with 7.4 pH. The damage Bacillus cells containing ROS was stained with chemical dye and the fluorescence was detected with microscope Olympus1 × 71 and Image-Pro express software v.6.2 (Olympus, Tokyo, Japan).

4.4. Determination of Salt Stress Effects on Vigour Index of Wheat Seedlings

The effect of salt stress on wheat germination and seedlings growth was observed through vigour index (VI) measurement. Surface sterilisation of wheat seeds was performed with 5% sodium hypochlorite and then washed with 70% ethanol and autoclaved water. The sterilised seeds were inoculated with high salt-resistant strains i.e., NMCN1 and LLC23 and well-known high salt-sensitive plant growth-promoting bacteria FZB42. The seeds were transferred to sterilised 9 cm Petri plates with 0. 3% water agar of different NaCl concentrations (0 mmol, 100 mmol, 150 mmol, and 200 mmol) sealed with parafilm and incubated at 25 °C in dark. The seeds simply dipped in water were used as control and were placed on different salt concentrations of 0.3% water agar. The germination was observed when the radicals were half of the shoot length. Five germinated seedlings were then transferred from each treatment to Petri plates containing water agar (0.3%) of different salt concentrations and allow them to grow for 7 days at 25 °C to find the effects of salt stress on seedlings stage in the presence of the selected strains. The percent germination and VI were calculated by the formula previously reported [58].

4.5. Measuring Effects of Salt Stress on Seedling Roots

The ability of the PGPR inoculated wheat seedlings was determined to grow under different salt concentrations of 0.3% agar water for 7 days at 25 °C in sealed petri plates (9 cm). Briefly, the seeds were surface sterilised and inoculated with different PGPRs i.e., MNCN1, LLG23, and FZB42. The seeds without inoculation were used as control. The seeds were then allowed to germinate and transferred to petri plates (9 cm) having different salt concentrations of 0.3% water agar and allowed to grow for 7 days at 25 °C. To study the root morphology three seedlings were then taken from each treatment. The root length, surface area, root diameter, root volume, and the number of each root seedling tips were calculated through Rhizoscanner (EPSON Perfection V700 Photo, Epson America, Long Beach, CA, USA), equipped with WinRHIZO software offered by Regent Instruments Co., (Sainte-Foy, Quebec, QC, Canada) [58].

4.6. Salt Stress Alleviation and Plant Growth Promotion by Bacillus Strains

Seven days’ wheat seedlings were transplanted into pots containing sterilised soil of equal size placed in a greenhouse. The NMCN1, LLCG23 and FZB42 strains were grown in the LB medium for 24 h at 37 °C. The cells were recovered by centrifugation and resuspended in the sterilised water until the final OD600 of 1.0 (1 × 107 CFU/mL). After one day of inoculation, 20 mL NaCl solutions of different concentrations (0 mmol, 100 mmol, 150 mmol and 200 mmol) were added to the inoculated and non-inoculated wheat plants. The non-inoculated plants grown with simple water or in the presence of different salt concentrations were used as control. Each treatment was performed in triplicate with three wheat seedlings in each pot. After 9 days’ post-inoculation, the effects of salt stress were recorded. The growth parameters, i.e., biomass were measured and the data were used for indication of growth promotion.

4.7. Rhizoscanning of Wheat Plant Roots for Morphological Traits

The root morphological study is mostly carried out in PGPRs-inoculated plants under stress conditions to find their role in stress alleviation. To study the effects of PGPRs (MNCN1, LLCG23 and FZB42) on wheat root morphology in the presence of salt stress an automated Rhizoscanner, equipped with WinRHIZO software offered by Regent Instruments Co. was used to calculate different morphological root parameters, i.e., root length, surface area, volume, diameter and number of root tips for each wheat plant. Three wheat plants were randomly selected from each treatment in triplicate after 9 days’ post-inoculation for Rhizoscanning and the average values were calculated for the above parameters to see the effects of each PGPR on wheat root morphology under salt stress.

4.8. Determination of Photosynthetic Potential of Plants under Salt Conditions

Wheat plants grown at 25 °C under different NaCl concentrations were used for analysing photosynthesis rate and stomatal conductance performance by taking readings from five leaves of each plant using an open IRGA LI-COR 6400 XT portable photosynthesis system (LI6400, Li-Cor Inc., Lincoln, NE, USA). Net photosynthetic rate (Pn) and stomatal conductance (gs) were taken under saturated light conditions at a photosynthetic photon flux density of 1000 µmoL photons m–2 s–1 and 380 mol–1 CO2 concentrations.

4.9. Quantification of Plant Stress Response Parameters and Their Expression Profiling

The plant induces a response to salt stress in the presence of different stress responsive parameters. The wheat plants were studied for these stress responsive parameters under salt stress conditions. The treated and control plants were grown in the greenhouse in a controlled environment and analysed for proline accumulation, lipid peroxidation and abscisic acid (ABA). Seven days’ wheat seedlings were transplanted to pots inoculated with selected PGPRs grown under different salt stress conditions. The non-inoculated wheat seedlings were used as control. The leaves were randomly harvested in triplicates from each salt treatment after 7 days.

4.9.1. Abscisic Acid (ABA) Determination

To quantify ABA in salt stress treated wheat plants, 0.1 g of each sample was ground into a fine powder with pestle and mortar in the presence of liquid nitrogen followed by transferring to 2 mL autoclaved tubes and stored at −80 °C to proceed later. The ground samples were then homogenised in 80% methanol and kept at 4 °C overnight in a stirring machine at 300 rpm. The samples were then centrifuged at 14,000 rpm at 4 °C followed by the collection of supernatants of each sample in new 2 mL tubes. The supernatants were dried through vacuum evaporation for 4 h at room temperature. The extract of each sample was then dissolved in 80% and subjected to UPLC for ABA detection and Quantification [66].

4.9.2. Quantification of Proline in Wheat Plants under Salt Stress

Proline overproduction in plants is an important stress response indicator for study in different environmental stresses [67]. Fresh wheat leaves samples (0.1 g) grown under salt conditions and untreated as control were taken at 7 days and homogenised completely in 70% ethanol [68]. Briefly, in a 2:1 ratio, the ethanolic extract and a solution containing 60% acetic acid and 20% ethanol were mixed to form a homogenised leaf mixture of each sample. The volume of 100 µL reaction mixture of each sample was taken in a 1.5 mL test tube, heated up to 95 °C for 20 min followed by cooling at room temperature and the absorbance was measured at 520 nm in a microplate reader (Spectrum max plus; Molecular Devices, Sunnyvale, CA, USA).

4.9.3. Estimation of MDA Level (Lipid Peroxidation) in Salt-Treated Wheat Plants

Lipid peroxidation is estimated in terms of malondialdehyde (MDA) levels studied in plants under stress conditions. Briefly, fresh leaves (0.1 g) of PGPRs-inoculated wheat plants were grown under salt conditions and untreated as a control was taken and homogenised in 0.1% (w/v) TCA 500 µL solution. The mixture was then centrifuged at 4 °C and 13,000 rpm. The supernatant of each treatment was then mixed with 1.5 mL of 0.5% TBA solution and heated to 95 °C in a water bath for 25 min. To stop further reactions, the mixture was placed on ice for 5 min. The absorbance of the reaction was recorded at 532 nm and 600 nm by using a microplate reader (Spectrum max plus; Molecular Devices, Sunnyvale, CA, USA).

4.9.4. Expression Analysis of Growth and Salt Stress Related Genes in Wheat Plant

To find the expression pattern of the above stress responsive genes, i.e., P5CS for proline, 4-HNE for MDA and ABARE for ABA and phytohormones genes (ARF, ERF, expA1, CKX2) and genes responsible for salt resistance (DREB2, MYB, HKT1, and WRKY17) was carried out through RT q-PCR in wheat plants grown under salt stress conditions (Table S1). The selected gene sequences were taken from NCB1, followed by designing primers through the PrimerQuest tool. The RNA was extracted from PGPRs inoculated wheat plants grown under salt stress conditions at 7 days using the TRizol method [69]. The RNA of each sample was used for cDNA synthesis according to the protocol given with the cDNA kit. The cDNA of each sample was used in RT-qPCR to find out each gene expression profile. RT-qPCR was performed to find the expression profile of these genes in PGPRs inoculated or uninoculated wheat plant through Quant Studio Real-Time Thermocycler (Thermo Fisher Scientific, San Jose, CA, USA).

4.10. Statistical Analysis

All the in vitro and in planta experiments in this study were conducted completely randomised and repeated thrice. The data were expressed in ±SD (standard deviations) of three replicates (n = 3). One-way analysis of variance (ANOVA) was used to statistically evaluate the experimental data by using Statistix software ver. 8.1. The least significant difference test (Tukey HSD) was used to observe the significance among the treatment means at the probability level of p ≤ 0.05 [70].

5. Conclusions

The significance of the current research is to explore the genetic features of potential halophilic bacterial strains, NMCN1 and LLCG23, that make them able to perform efficient metabolic and physiological functions under high saline conditions. The ability of these bacteria to alleviate salt stress in wheat plants and promote plant growth through modulation of phytohormones and salt resistance genes. These beneficial plant microbe interactions achieved more attention for further research to use them efficiently in making promising bio-fertilisers or biopesticides for the purpose of obtaining better agricultural crops’ production in an extreme saline environment.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants11202769/s1, Table S1: Primers used in this study.

Author Contributions

X.G., M.A. and Q.G. planned and designed the research; M.A. performed research and contributed to the methodology, writing, and editing; Q.A., A.R.K., Z.W. and S.A.K. assisted in analysis and compiled the data and results; Q.J., G.M. and R.W. assisted in experiments and improved the manuscript; X.G., H.M., H.W. and Q.G. critically revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Key Project of NSFC regional innovation and development joint fund (U20A2039), the Natural Science Foundation of Jiangsu Province, China (grant BK20181322), and, the Ningxia Hui Autonomous Region Introduction Team Project (2021RXTDLX10), the Key Research and Development project of the Ningxia Hui Autonomous Region (2021BBF02023).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Sharma, D.; Singh, R.; Tiwari, R.; Kumar, R.; Gupta, V.K. Wheat Responses and Tolerance to Terminal Heat Stress: A Review; Springer: Berlin/Heidelberg, Germany, 2019; ISBN 9789811368820. [Google Scholar]
  2. Rahaie, M.; Xue, G.-P.; Schenk, P.M. The Role of Transcription Factors in Wheat under Different Abiotic Stresses. Abiotic Stress. Responses Appl. Agric. 2013, 367–385. [Google Scholar] [CrossRef][Green Version]
  3. Mahalingam, R. Consideration of Combined Stress: A Crucial Paradigm for Improving Multiple Stress Tolerance in Plants. In Combined Stresses in Plants; Springer: Berlin/Heidelberg, Germany, 2015; pp. 1–25. [Google Scholar]
  4. Genc, Y.; Taylor, J.; Lyons, G.H.; Li, Y.; Cheong, J.; Appelbee, M.; Oldach, K.; Sutton, T. Bread Wheat with High Salinity and Sodicity Tolerance. Front. Plant Sci. 2019, 10, 1280. [Google Scholar] [CrossRef][Green Version]
  5. Martinez-Beltran, J.; Manzur, C.L. Overview of salinity problems in the world and FAO strategies to address the problem. In Proceedings of the 3rd International Salinity Forum, Riverside, CA, USA, 25–27 April 2005; pp. 311–313. [Google Scholar]
  6. Yasin, N.A.; Akram, W.; Khan, W.U.; Ahmad, S.R.; Ahmad, A.; Ali, A. Halotolerant Plant-Growth Promoting Rhizobacteria Modulate Gene Expression and Osmolyte Production to Improve Salinity Tolerance and Growth in Capsicum annum L. Environ. Sci. Pollut. Res. 2018, 25, 23236–23250. [Google Scholar] [CrossRef]
  7. Jacoby, R.P.; Che-Othman, M.H.; Millar, A.H.; Taylor, N.L. Analysis of the Sodium Chloride-Dependent Respiratory Kinetics of Wheat Mitochondria Reveals Differential Effects on Phosphorylating and Non-Phosphorylating Electron Transport Pathways. Plant Cell Environ. 2016, 39, 823–833. [Google Scholar] [CrossRef][Green Version]
  8. Quintela, V.; Walter, S.; Serra, F.; Ynglety, M.; Pereira, C.; Ricardo, B.; Maria, E.; Guedes, S.; Mohammed, L.; Alyemeni, N.; et al. Pretreatment with 24-Epibrassinolide Synergistically Protects Root Structures and Chloroplastic Pigments and Upregulates Antioxidant Enzymes and Biomass in Na+-Stressed Tomato Plants. J. Plant Growth Regul. 2022, 41, 2869–2885. [Google Scholar] [CrossRef]
  9. Srivastava, S.S.; Bist, V.; Srivastava, S.S.; Singh, P.C.; Trivedi, P.K.; Asif, M.H.; Chauhan, P.S.; Nautiyal, C.S. Unraveling Aspects of Bacillus amyloliquefaciens Mediated Enhanced Production of Rice under Biotic Stress of Rhizoctonia Solani. Front. Plant Sci. 2016, 7, 587. [Google Scholar] [CrossRef][Green Version]
  10. Bharti, N.; Barnawal, D.; Awasthi, A.; Yadav, A.; Kalra, A. Plant Growth Promoting Rhizobacteria Alleviate Salinity Induced Negative Effects on Growth, Oil Content and Physiological Status in Mentha Arvensis. Acta Physiol. Plant. 2014, 36, 45–60. [Google Scholar] [CrossRef]
  11. Dresselhaus, T.; Hückelhoven, R. Biotic and Abiotic Stress Responses in Crop Plants. Agronomy 2018, 8, 267. [Google Scholar] [CrossRef][Green Version]
  12. Ayaz, M.; Ali, Q.; Farzand, A.; Khan, A.R.; Ling, H.; Gao, X. Nematicidal Volatiles from Bacillus atrophaeus GBSC56 Promote Growth and Stimulate Induced Systemic Resistance in Tomato against Meloidogyne Incognita. Int. J. Mol. Sci. 2021, 22, 5049. [Google Scholar] [CrossRef]
  13. Hadi, F.; Ayaz, M.; Ali, S.; Shafiq, M.; Ullah, R. Comparative Effect of Polyethylene Glycol and Mannitol Induced Drought on Growth (in Vitro) of Canola (Brassica napus), Cauliflower (Brassica oleracea) and Tomato (Lycopersicon esculentum) Seedlings. Int. J. Biosci. 2014, 6655, 34–41. [Google Scholar] [CrossRef]
  14. Grover, M.; Ali, S.Z.; Sandhya, V.; Rasul, A.; Venkateswarlu, B. Role of Microorganisms in Adaptation of Agriculture Crops to Abiotic Stresses. World J. Microbiol. Biotechnol. 2011, 27, 1231–1240. [Google Scholar] [CrossRef]
  15. Ansari, F.A.; Ahmad, I.; Pichtel, J. Growth Stimulation and Alleviation of Salinity Stress to Wheat by the Biofilm Forming Bacillus pumilus Strain FAB10. Appl. Soil Ecol. 2019, 143, 45–54. [Google Scholar] [CrossRef]
  16. Smith, D.L.; Gravel, V.; Yergeau, E. Editorial: Signaling in the Phytomicrobiome. Front. Plant Sci. 2017, 8, 8–10. [Google Scholar] [CrossRef][Green Version]
  17. Ali, Q.; Ayaz, M.; Mu, G.; Hussain, A.; Yuanyuan, Q.; Yu, C.; Xu, Y.; Manghwar, H.; Gu, Q.; Wu, H.; et al. Revealing Plant Growth-Promoting Mechanisms of Bacillus strains in Elevating Rice Growth and Its Interaction with Salt Stress. Front. Plant Sci. 2022, 13, 1–17. [Google Scholar] [CrossRef]
  18. Khan, M.A.; Asaf, S.; Khan, A.L.; Adhikari, A.; Jan, R.; Ali, S.; Imran, M.; Kim, K.M.; Lee, I.J. Halotolerant Rhizobacterial Strains Mitigate the Adverse Effects of NaCl Stress in Soybean Seedlings. Biomed Res. Int. 2019, 2019, 9530963. [Google Scholar] [CrossRef][Green Version]
  19. Wu, L.; Wu, H.-J.; Qiao, J.; Gao, X.; Borriss, R. Novel Routes for Improving Biocontrol Activity of Bacillus Based Bioinoculants. Front. Microbiol. 2015, 6, 1395. [Google Scholar] [CrossRef][Green Version]
  20. Zubair, M.; Hanif, A.; Farzand, A.; Sheikh, T.M.M.; Khan, A.R.; Suleman, M.; Ayaz, M.; Gao, X. Genetic Screening and Expression Analysis of Psychrophilic Bacillus Spp. Reveal Their Potential to Alleviate Cold Stress and Modulate Phytohormones in Wheat. Microorganisms 2019, 7, 373. [Google Scholar] [CrossRef][Green Version]
  21. Borriss, R.; Chen, X.-H.H.; Rueckert, C.; Blom, J.; Becker, A.; Baumgarth, B.; Fan, B.; Pukall, R.; Schumann, P.; Spröer, C.; et al. Relationship of Bacillus amyloliquefaciens Clades Associated with Strains DSM 7T and FZB42T: A Proposal for Bacillus amyloliquefaciens Subsp. Amyloliquefaciens Subsp. Nov. and Bacillus amyloliquefaciens Subsp. Plantarum Subsp. Nov. Based on Complete Genom. Int. J. Syst. Evol. Microbiol. 2011, 61, 1786–1801. [Google Scholar] [CrossRef][Green Version]
  22. Nicholson, W.L. Roles of Bacillus Endospores in the Environment. Cell. Mol. Life Sci. C. 2002, 59, 410–416. [Google Scholar] [CrossRef]
  23. Ali, Q.; Ayaz, M.; Yu, C.; Wang, Y.; Gu, Q.; Wu, H.; Gao, X. Cadmium Tolerant Microbial Strains Possess Different Mechanisms for Cadmium Biosorption and Immobilization in Rice Seedlings Chemosphere Cadmium Tolerant Microbial Strains Possess Different Mechanisms for Cadmium Biosorption and Immobilization in Rice Seedlings. Chemosphere 2022, 303, 135206. [Google Scholar] [CrossRef]
  24. Das, P.; Behera, B.K.; Meena, D.K.; Azmi, S.A.; Chatterjee, S.; Meena, K.; Sharma, A.P. Priyanka Das Salt Stress Tolerant Genes in Halophilic and Halotolerant Bacteria: Salt Stress Adaptation and Osmoprotection. Int. J. Curr. Microbiol. Appl. Sci. 2015, 4, 642–658. [Google Scholar]
  25. Ranea, J.A.G.; Buchan, D.W.A.; Thornton, J.M.; Orengo, C.A. Evolution of Protein Superfamilies and Bacterial Genome Size. J. Mol. Biol. 2004, 336, 871–887. [Google Scholar] [CrossRef] [PubMed]
  26. Hoffmann, T.; Boiangiu, C.; Moses, S.; Bremer, E. Responses of Bacillus subtilis to Hypotonic Challenges: Physiological Contributions of Mechanosensitive Channels to Cellular Survival. Appl. Environ. Microbiol. 2008, 74, 2454–2460. [Google Scholar] [CrossRef][Green Version]
  27. Wood, J.M. Bacterial Osmoregulation: A Paradigm for the Study of Cellular Homeostasis. Annu. Rev. Microbiol. 2011, 65, 215–238. [Google Scholar] [CrossRef] [PubMed][Green Version]
  28. Cui, P.; Liu, H.; Islam, F.; Li, L.; Farooq, M.A.; Ruan, S.; Zhou, W. OsPEX11, a Peroxisomal Biogenesis Factor 11, Contributes to Salt Stress Tolerance in Oryza Sativa. Front. Plant Sci. 2016, 7, 1357. [Google Scholar] [CrossRef][Green Version]
  29. Yan, F.; Yu, Y.; Gozzi, K.; Chen, Y.; Guo, J.; Chai, Y. Genome-Wide Investigation of Biofilm Formation in Bacillus cereus. Appl. Environ. Microbiol. 2017, 83, e00561-17. [Google Scholar] [CrossRef][Green Version]
  30. Kumar, P.P. Regulation of Biotic and Abiotic Stress Responses by Plant Hormones. Plant Cell Rep. 2013, 32, 943. [Google Scholar] [CrossRef][Green Version]
  31. Cutler, S.R.; Rodriguez, P.L.; Finkelstein, R.R.; Abrams, S.R. Abscisic Acid: Emergence of a Core Signaling Network. Annu. Rev. Plant Biol. 2010, 61, 651–679. [Google Scholar] [CrossRef][Green Version]
  32. Bharti, N.; Pandey, S.S.; Barnawal, D.; Patel, V.K.; Kalra, A. Plant Growth Promoting Rhizobacteria Dietzia natronolimnaea Modulates the Expression of Stress Responsive Genes Providing Protection of Wheat from Salinity Stress. Sci. Rep. 2016, 6, 34768. [Google Scholar] [CrossRef][Green Version]
  33. Gill, S.S.; Tuteja, N. Reactive Oxygen Species and Antioxidant Machinery in Abiotic Stress Tolerance in Crop Plants. Plant Physiol. Biochem. 2010, 48, 909–930. [Google Scholar] [CrossRef]
  34. Tavakoli, M.; Poustini, K.; Alizadeh, H. Proline Accumulation and Related Genes in Wheat Leaves under Salinity Stress. J. Agric. Sci. Technol. 2016, 18, 707–716. [Google Scholar]
  35. Ayaz, M.; Ahmad, R.; Shahzad, M.; Khan, N.; Shah, M.M.; Khan, S.A. Drought Stress Stunt Tomato Plant Growth and Up-Regulate Expression of SlAREB, SlNCED3, and SlERF024 Genes. Sci. Hortic. 2015, 195, 48–55. [Google Scholar] [CrossRef]
  36. Shahzad, R.; Waqas, M.; Khan, A.L.; Asaf, S.; Khan, M.A.; Kang, S.M.; Yun, B.W.; Lee, I.J. Seed-Borne Endophytic Bacillus Amyloliquefaciens RWL-1 Produces Gibberellins and Regulates Endogenous Phytohormones of Oryza Sativa. Plant Physiol. Biochem. 2016, 106, 236–243. [Google Scholar] [CrossRef] [PubMed]
  37. Glick, B.R.; Cheng, Z.; Czarny, J.; Duan, J. Promotion of Plant Growth by ACC Deaminase-Producing Soil Bacteria. Eur. J. Plant Pathol. 2007, 119, 329–339. [Google Scholar] [CrossRef]
  38. Naz, I.; Bano, A. Tamoor-Ul-Hassan Isolation of Phytohormones Producing Plant Growth Promoting Rhizobacteria from Weeds Growing in Khewra Salt Range, Pakistan and Their Implication in Providing Salt Tolerance to Glycine Max L. African J. Biotechnol. 2009, 8, 5762–5768. [Google Scholar] [CrossRef]
  39. Peck, S.; Mittler, R. Plant Signaling in Biotic and Abiotic Stress. J. Exp. Bot. 2020, 71, 1649–1651. [Google Scholar] [CrossRef][Green Version]
  40. Mahajan, S.; Tuteja, N. Cold, Salinity and Drought Stresses: An Overview. Arch. Biochem. Biophys. 2005, 444, 139–158. [Google Scholar] [CrossRef]
  41. Palaniyandi, S.A.; Damodharan, K.; Yang, S.H.; Suh, J.W. Streptomyces Sp. Strain PGPA39 Alleviates Salt Stress and Promotes Growth of “Micro Tom” Tomato Plants. J. Appl. Microbiol. 2014, 117, 766–773. [Google Scholar] [CrossRef]
  42. Hontzeas, N.; Richardson, A.O.; Belimov, A.; Safronova, V.; Abu-Omar, M.M.; Click, B.R. Evidence for Horizontal Transfer of 1-Aminocyclopropane-1-Carboxylate Deaminase Genes. Appl. Environ. Microbiol. 2005, 71, 7556–7558. [Google Scholar] [CrossRef][Green Version]
  43. Gagne-Bourque, F.; Mayer, B.F.; Charron, J.-B.; Vali, H.; Bertrand, A.; Jabaji, S. Accelerated Growth Rate and Increased Drought Stress Resilience of the Model Grass Brachypodium Distachyon Colonized by Bacillus subtilis B26. PLoS ONE 2015, 10, e0130456. [Google Scholar] [CrossRef]
  44. Kempf, B.; Bremer, E. OpuA, an Osmotically Regulated Binding Protein-Dependent Transport System for the Osmoprotectant Glycine Betaine in Bacillus subtilis. J. Biol. Chem. 1995, 270, 16701–16713. [Google Scholar] [CrossRef] [PubMed][Green Version]
  45. Dogsa, I.; Choudhary, K.S.; Marsetic, Z.; Hudaiberdiev, S.; Vera, R.; Pongor, S.; Mandic-Mulec, I. ComQXPA Quorum Sensing Systems May Not Be Unique to Bacillus subtilis: A Census in Prokaryotic Genomes. PLoS ONE 2014, 9, e96122. [Google Scholar] [CrossRef] [PubMed]
  46. Helmann, J.D.; Wu, M.F.W.; Gaballa, A.; Kobel, P.A.; Morshedi, M.M.; Fawcett, P.; Paddon, C. The Global Transcriptional Response of Bacillus subtilis to Peroxide Stress Is Coordinated by Three Transcription Factors. J. Bacteriol. 2003, 185, 243–253. [Google Scholar] [CrossRef] [PubMed][Green Version]
  47. Ramu, V.S.; Paramanantham, A.; Ramegowda, V.; Mohan-Raju, B.; Udayakumar, M.; Senthil-Kumar, M. Transcriptome Analysis of Sunflower Genotypes with Contrasting Oxidative Stress Tolerance Reveals Individual-and Combined-Biotic and Abiotic Stress Tolerance Mechanisms. PLoS ONE 2016, 11, e0157522. [Google Scholar] [CrossRef][Green Version]
  48. Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol. 2010, 8, 623–633. [Google Scholar] [CrossRef]
  49. Feldman, M.; Ginsburg, I.; Al-Quntar, A.; Steinberg, D. Thiazolidinedione-8 Alters Symbiotic Relationship in C. Albicans-S. Mutans Dual Species Biofilm. Front. Microbiol. 2016, 7, 140. [Google Scholar] [CrossRef][Green Version]
  50. Silva, B.R.S.; Batista, B.L.; Lobato, A.K.S. Anatomical Changes in Stem and Root of Soybean Plants Submitted to Salt Stress. Plant Biol. 2021, 23, 57–65. [Google Scholar] [CrossRef]
  51. Jain, D.; Chattopadhyay, D. Analysis of Gene Expression in Response to Water Deficit of Chickpea (Cicer arietinum L.) Varieties Differing in Drought Tolerance. BMC Plant Biol. 2010, 10, 24. [Google Scholar] [CrossRef][Green Version]
  52. Song, S.-Y.; Chen, Y.; Chen, J.; Dai, X.-Y.; Zhang, W.-H. Physiological Mechanisms Underlying OsNAC5-Dependent Tolerance of Rice Plants to Abiotic Stress. Planta 2011, 234, 331–345. [Google Scholar] [CrossRef]
  53. Weber, H.; Chételat, A.; Reymond, P.; Farmer, E.E. Selective and Powerful Stress Gene Expression in Arabidopsis in Response to Malondialdehyde. Plant J. 2004, 37, 877–888. [Google Scholar] [CrossRef]
  54. Tahir, H.A.S.S.; Gu, Q.; Wu, H.; Raza, W.; Hanif, A.; Wu, L.; Colman, M.V.; Gao, X. Plant Growth Promotion by Volatile Organic Compounds Produced by Bacillus subtilis SYST2. Front. Microbiol. 2017, 8, 171. [Google Scholar] [CrossRef] [PubMed][Green Version]
  55. Adesemoye, A.O.; Torbert, H.A.; Kloepper, J.W. Enhanced Plant Nutrient Use Efficiency with PGPR and AMF in an Integrated Nutrient Management System. Can. J. Microbiol. 2008, 54, 876–886. [Google Scholar] [CrossRef] [PubMed]
  56. Etesami, H.; Maheshwari, D.K. Use of Plant Growth Promoting Rhizobacteria (PGPRs) with Multiple Plant Growth Promoting Traits in Stress Agriculture: Action Mechanisms and Future Prospects. Ecotoxicol. Environ. Saf. 2018, 156, 225–246. [Google Scholar] [CrossRef] [PubMed]
  57. Arshad, M.; Shaharoona, B.; Mahmood, T. Inoculation with Pseudomonas Spp. Containing ACC-Deaminase Partially Eliminates the Effects of Drought Stress on Growth, Yield, and Ripening of Pea (Pisum sativum L.). Pedosphere 2008, 18, 611–620. [Google Scholar] [CrossRef]
  58. Rasul, M.; Yasmin, S.; Zubair, M.; Mahreen, N.; Yousaf, S.; Arif, M.; Sajid, Z.I.; Mirza, M.S. Phosphate Solubilizers as Antagonists for Bacterial Leaf Blight with Improved Rice Growth in Phosphorus Deficit Soil. Biol. Control 2019, 136, 103997. [Google Scholar] [CrossRef]
  59. Suleman, M.; Yasmin, S.; Rasul, M.; Yahya, M.; Atta, B.M.; Mirza, M.S. Phosphate Solubilizing Bacteria with Glucose Dehydrogenase Gene for Phosphorus Uptake and Beneficial Effects on Wheat. PLoS ONE 2018, 13, e0204408. [Google Scholar] [CrossRef] [PubMed][Green Version]
  60. Linić, I.; Šamec, D.; Grúz, J.; Vujčić Bok, V.; Strnad, M.; Salopek-Sondi, B. Involvement of Phenolic Acids in Short-Term Adaptation to Salinity Stress Is Species-Specific among Brassicaceae. Plants 2019, 8, 155. [Google Scholar] [CrossRef][Green Version]
  61. Hayat, R.; Ali, S.; Amara, U.; Khalid, R.; Ahmed, I. Soil Beneficial Bacteria and Their Role in Plant Growth Promotion: A Review. Ann. Microbiol. 2010, 60, 579–598. [Google Scholar] [CrossRef]
  62. Velho, R.V.; Caldas, D.G.G.; Medina, L.F.C.; Tsai, S.M.; Brandelli, A. Real-Time PCR Investigation on the Expression of SboA and ItuD Genes in Bacillus spp. Lett. Appl. Microbiol. 2011, 52, 660–666. [Google Scholar] [CrossRef]
  63. Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  64. Kim, T.Y.; Jang, J.Y.; Jeon, S.J.; Lee, H.W.; Bae, C.H.; Yeo, J.H.; Lee, H.B.; Kim, I.S.; Park, H.W.; Kim, J.C. Nematicidal Activity of Kojic Acid Produced by Aspergillus oryzae against Meloidogyne incognita. J. Microbiol. Biotechnol. 2016, 26, 1383–1391. [Google Scholar] [CrossRef] [PubMed]
  65. Massawe, V.C.; Hanif, A.; Farzand, A.; Mburu, D.K.; Ochola, S.O.; Wu, L.; Tahir, H.A.S.; Gu, Q.; Wu, H.; Gao, X. Volatile Compounds of Endophytic Bacillus Spp. Have Biocontrol Activity against Sclerotinia Sclerotiorum. Phytopathology 2018, 108, 1373–1385. [Google Scholar] [CrossRef] [PubMed]
  66. Pavlović, I.; Petřík, I.; Tarkowská, D.; Lepeduš, H.; Vujčić Bok, V.; Radić Brkanac, S.; Novák, O.; Salopek-Sondi, B. Correlations between Phytohormones and Drought Tolerance in Selected Brassica Crops: Chinese Cabbage, White Cabbage and Kale. Int. J. Mol. Sci. 2018, 19, 2866. [Google Scholar] [CrossRef][Green Version]
  67. Sithtisarn, S.; Harinasut, P.; Pornbunlualap, S.; Cha-Um, S.; Carillo, P.; Gibon, Y. PROTOCOL: Extraction and Determination of Glycine Betaine Initiating Author Name. Kasetsart J. Nat. Sci. 2009, 43, 146–152. [Google Scholar]
  68. Mustafiz, A.; Sahoo, K.K.; Singla-Pareek, S.L.; Sopory, S.K. Metabolic Engineering of Glyoxalase Pathway for Enhancing Stress Tolerance in Plants. In Plant Stress Tolerence; Springer: Berlin/Heidelberg, Germany, 2010; Volume 639, ISBN 9781607617013. [Google Scholar]
  69. Rio, D.C.; Ares, M.; Hannon, G.J.; Nilsen, T.W. Purification of RNA Using TRIzol (TRI Reagent). Cold Spring Harb. Protoc. 2010, 5, pdb.prot5439. [Google Scholar] [CrossRef] [PubMed]
  70. Saddique, M.A.B.; Ali, Z.; Sher, M.A.; Farid, B.; Ikram, R.M.; Ahmad, M.S. Proline, Total Antioxidant Capacity, and OsP5CS Gene Activity in Radical and Plumule of Rice Are Efficient Drought Tolerance Indicator Traits. Int. J. Agron. 2020, 2020, 8862792. [Google Scholar] [CrossRef]
Plants 11 02769 g001 550
Figure 1. Bacillus spp. strains’ growth and biofilm formation under different saline conditions. (A) The visual representation of strains’ growth on solid LB media (a–h) with different NaCl concentrations (1–18%). Each number indicates strain: 1. FZB42, 2. B168, 3. EZ-1509, 4. CJCL2, 5. LLCG23, 6. NMCN1, 7. SYST2, 8. LNXM65, 9. TS1, 10. LSSC3, 11. GBSW11, 12. LNXM37, 13. LLTC93, 14. GBAC46, 15. NMCC4, 16. LNXM10, 17. LLTC96, 18. GBWC18. (B) Shows the graphical presentation of optical density at different time intervals calculated at (OD600) of high halotolerant strains (MNCN1 and LLCG23) as compared to FZB42 grown in Liquid LB media with different salt concentrations. (C) The biofilm formation potential of selected strains grown under different NaCl concentrations (1–18%) for 5 days at 37 °C. The experiment was repeated thrice with similar results.
Figure 1. Bacillus spp. strains’ growth and biofilm formation under different saline conditions. (A) The visual representation of strains’ growth on solid LB media (a–h) with different NaCl concentrations (1–18%). Each number indicates strain: 1. FZB42, 2. B168, 3. EZ-1509, 4. CJCL2, 5. LLCG23, 6. NMCN1, 7. SYST2, 8. LNXM65, 9. TS1, 10. LSSC3, 11. GBSW11, 12. LNXM37, 13. LLTC93, 14. GBAC46, 15. NMCC4, 16. LNXM10, 17. LLTC96, 18. GBWC18. (B) Shows the graphical presentation of optical density at different time intervals calculated at (OD600) of high halotolerant strains (MNCN1 and LLCG23) as compared to FZB42 grown in Liquid LB media with different salt concentrations. (C) The biofilm formation potential of selected strains grown under different NaCl concentrations (1–18%) for 5 days at 37 °C. The experiment was repeated thrice with similar results.
Plants 11 02769 g001
Plants 11 02769 g002 550
Figure 2. Expression analysis of predicted genes in Bacillus strains FZB42, NMCN1 and LLCG23 grown in different NaCl concentrations (1%, 10%, 14% and 16%). The genes analysed were: DegU (a), OstB (b), OhrR (c), ComA (d), SodA (e) and OpuAC (f). The error bars on the columns indicate the standard deviation of the mean (n = 3). Letters above the columns represent significant differences between treatments at p ≤ 0.05. The expression studies were repeated thrice with three replicates for each treatment.
Figure 2. Expression analysis of predicted genes in Bacillus strains FZB42, NMCN1 and LLCG23 grown in different NaCl concentrations (1%, 10%, 14% and 16%). The genes analysed were: DegU (a), OstB (b), OhrR (c), ComA (d), SodA (e) and OpuAC (f). The error bars on the columns indicate the standard deviation of the mean (n = 3). Letters above the columns represent significant differences between treatments at p ≤ 0.05. The expression studies were repeated thrice with three replicates for each treatment.
Plants 11 02769 g002
Plants 11 02769 g003 550
Figure 3. The intensity of green fluorescence indicates the level of Reactive Oxygen Species (ROS) in selected Bacillus strains grown at 37 °C in saline condition for 4 days. The less intense fluorescence observed in NMCN1 and LLCG23 strains describes lower ROS production in their cells under salt stress while the high intensive green fluorescence in FZB42 strain displays more ROS production under saline condition. The experiment was repeated thrice similar results.
Figure 3. The intensity of green fluorescence indicates the level of Reactive Oxygen Species (ROS) in selected Bacillus strains grown at 37 °C in saline condition for 4 days. The less intense fluorescence observed in NMCN1 and LLCG23 strains describes lower ROS production in their cells under salt stress while the high intensive green fluorescence in FZB42 strain displays more ROS production under saline condition. The experiment was repeated thrice similar results.
Plants 11 02769 g003
Plants 11 02769 g004 550
Figure 4. Wheat seedlings inoculated with Bacillus strains FZB42, NMCN1 and LLCG23 were grown for 7 days at 25 °C under different NaCl concentrations (0 mmol–200 mmol). (A) The visual representation showing the effect of selected strains on wheat growth promotion under different saline conditions. (B) The calculation of seedlings’ length and percent germination is represented in graph as vigour index for wheat seeds treated with different Bacillus strains as compared with control (seeds without bacteria). The bars on the column represent mean standard deviation (n = 3). The small letters on the bars indicate significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05 and the experiment was repeated thrice with three replicates for each treatment.
Figure 4. Wheat seedlings inoculated with Bacillus strains FZB42, NMCN1 and LLCG23 were grown for 7 days at 25 °C under different NaCl concentrations (0 mmol–200 mmol). (A) The visual representation showing the effect of selected strains on wheat growth promotion under different saline conditions. (B) The calculation of seedlings’ length and percent germination is represented in graph as vigour index for wheat seeds treated with different Bacillus strains as compared with control (seeds without bacteria). The bars on the column represent mean standard deviation (n = 3). The small letters on the bars indicate significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05 and the experiment was repeated thrice with three replicates for each treatment.
Plants 11 02769 g004
Plants 11 02769 g005 550
Figure 5. Rhizoscanning of wheat seedlings grown under different saline conditions (0 mmol–200 mmol NaCl) at 25 °C for 7 days insulated with Bacillus spp. strains. (A) Rhizoscanning in image form of wheat seedlings grown under different saline conditions at 25 °C. (B) Morphological root parameters such as (a) Root Tips, (b) Root Length (mm), (c) Root Diameter (cm), (d) Root Area (mm2), (e) Root Volume (cm3). The small letters on the bars indicate significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05 and the experiment was repeated thrice with three replicates for each treatment.
Figure 5. Rhizoscanning of wheat seedlings grown under different saline conditions (0 mmol–200 mmol NaCl) at 25 °C for 7 days insulated with Bacillus spp. strains. (A) Rhizoscanning in image form of wheat seedlings grown under different saline conditions at 25 °C. (B) Morphological root parameters such as (a) Root Tips, (b) Root Length (mm), (c) Root Diameter (cm), (d) Root Area (mm2), (e) Root Volume (cm3). The small letters on the bars indicate significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05 and the experiment was repeated thrice with three replicates for each treatment.
Plants 11 02769 g005
Plants 11 02769 g006 550
Figure 6. Relative expression and quantification of Abscisic acid, Proline and Lipid peroxidisation. (a) Expression profiling of abscisic acid (ABA) encoding gene ABARE, (b) ABA quantification through UPLC analysis in wheat plants under salt stress (200 mmol NaCl), (c) expression analysis of MDA encoding gene (4-HNE), (d) quantification of MDA content through spectrophotometric method in wheat plants grown under saline condition, (e) expression analysis of proline encoding gene P5CS, (f) spectrophotometric quantification of proline. The error bars on the columns represent the standard deviation of the mean (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Figure 6. Relative expression and quantification of Abscisic acid, Proline and Lipid peroxidisation. (a) Expression profiling of abscisic acid (ABA) encoding gene ABARE, (b) ABA quantification through UPLC analysis in wheat plants under salt stress (200 mmol NaCl), (c) expression analysis of MDA encoding gene (4-HNE), (d) quantification of MDA content through spectrophotometric method in wheat plants grown under saline condition, (e) expression analysis of proline encoding gene P5CS, (f) spectrophotometric quantification of proline. The error bars on the columns represent the standard deviation of the mean (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Plants 11 02769 g006
Plants 11 02769 g007 550
Figure 7. Effect of Bacillus spp. strains (FZB42, NMCN1 and LLCG23) on wheat plants grown under different salt conditions (0 mmol–200 mmol). (A) Visual representation showing the effect of the selected strains on wheat plants under saline conditions; (B) graphical detail of different plant parameters, i.e., (a) fresh weight of wheat plants inoculated with Bacillus strains grown in different salt stress conditions; (b) plants dry weight; (c) plant total shoot length. Standard deviations of mean (n = 3) were used to indicate error bars on the columns. The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Figure 7. Effect of Bacillus spp. strains (FZB42, NMCN1 and LLCG23) on wheat plants grown under different salt conditions (0 mmol–200 mmol). (A) Visual representation showing the effect of the selected strains on wheat plants under saline conditions; (B) graphical detail of different plant parameters, i.e., (a) fresh weight of wheat plants inoculated with Bacillus strains grown in different salt stress conditions; (b) plants dry weight; (c) plant total shoot length. Standard deviations of mean (n = 3) were used to indicate error bars on the columns. The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Plants 11 02769 g007
Plants 11 02769 g008 550
Figure 8. Wheat roots rhizoscanning image under different saline conditions (0 mmol–200 mmol) for 10 days treated with Bacillus strains (NMCN1, FZB42 and LLCG23) in a greenhouse experiment. (A) Visually gives the information regarding the effect of Bacillus strains on root morphology. The effect of selected strains on root morphological parameters (B) i.e., (a) wheat root total root length (mm) post inoculation of Bacillus strains grown under different salt stress conditions, (b) roots diameter (mm), (c) root total surface area (cm2), (d) number of root tips for each treatment. The error bars shown on the graphs represent the mean standard deviation of each treatment (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Figure 8. Wheat roots rhizoscanning image under different saline conditions (0 mmol–200 mmol) for 10 days treated with Bacillus strains (NMCN1, FZB42 and LLCG23) in a greenhouse experiment. (A) Visually gives the information regarding the effect of Bacillus strains on root morphology. The effect of selected strains on root morphological parameters (B) i.e., (a) wheat root total root length (mm) post inoculation of Bacillus strains grown under different salt stress conditions, (b) roots diameter (mm), (c) root total surface area (cm2), (d) number of root tips for each treatment. The error bars shown on the graphs represent the mean standard deviation of each treatment (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was repeated thrice with three replicates for each treatment.
Plants 11 02769 g008
Plants 11 02769 g009 550
Figure 9. ROS reduction in wheat roots inoculated with Bacillus strains (NMCN1 and FZB42). The halophilic strains NMCN1 were found to highly reduced ROS in wheat roots and thus alleviate the risk of cell damage in roots. The experiment was repeated thrice with three replicates.
Figure 9. ROS reduction in wheat roots inoculated with Bacillus strains (NMCN1 and FZB42). The halophilic strains NMCN1 were found to highly reduced ROS in wheat roots and thus alleviate the risk of cell damage in roots. The experiment was repeated thrice with three replicates.
Plants 11 02769 g009
Plants 11 02769 g010 550
Figure 10. Effect of bacterial strains on photosynthesis rate and stomatal conductance regulation under salt stress. (a) Photosynthesis regulation of wheat plants inoculated with Bacillus strains under saline conditions; (b) Bacillus strains effects on stomatal conductance of wheat plants under different saline environment. The error bars represent the mean standard deviation of each treatment (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was performed in triplicate with similar results.
Figure 10. Effect of bacterial strains on photosynthesis rate and stomatal conductance regulation under salt stress. (a) Photosynthesis regulation of wheat plants inoculated with Bacillus strains under saline conditions; (b) Bacillus strains effects on stomatal conductance of wheat plants under different saline environment. The error bars represent the mean standard deviation of each treatment (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment was performed in triplicate with similar results.
Plants 11 02769 g010
Plants 11 02769 g011 550
Figure 11. Effect of Bacillus strains on the expression of growth-promoting and salt-related genes in wheat plants grown under salt stress conditions (200 mmol). (A) Shows the expression profile of growth promoting genes (a–d) in wheat plants. (B) Indicates the expression analysis of salt-related genes (e–h) in wheat plants inoculated with Bacillus strains under saline condition (200 mmol). The error bars on the graphs indicate the standard deviation of the mean (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment has repeated three times with three replicates for each treatment.
Figure 11. Effect of Bacillus strains on the expression of growth-promoting and salt-related genes in wheat plants grown under salt stress conditions (200 mmol). (A) Shows the expression profile of growth promoting genes (a–d) in wheat plants. (B) Indicates the expression analysis of salt-related genes (e–h) in wheat plants inoculated with Bacillus strains under saline condition (200 mmol). The error bars on the graphs indicate the standard deviation of the mean (n = 3). The small letters were used to show significant difference among the treatments calculated through Tukey’s HSD test at p ≤ 0.05. The experiment has repeated three times with three replicates for each treatment.
Plants 11 02769 g011
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Ayaz, M.; Ali, Q.; Jiang, Q.; Wang, R.; Wang, Z.; Mu, G.; Khan, S.A.; Khan, A.R.; Manghwar, H.; Wu, H.; et al. Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress. Plants 2022, 11, 2769. https://doi.org/10.3390/plants11202769

AMA Style

Ayaz M, Ali Q, Jiang Q, Wang R, Wang Z, Mu G, Khan SA, Khan AR, Manghwar H, Wu H, et al. Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress. Plants. 2022; 11(20):2769. https://doi.org/10.3390/plants11202769

Chicago/Turabian Style

Ayaz, Muhammad, Qurban Ali, Qifan Jiang, Ruoyi Wang, Zhengqi Wang, Guangyuan Mu, Sabaz Ali Khan, Abdur Rashid Khan, Hakim Manghwar, Huijun Wu, and et al. 2022. "Salt Tolerant Bacillus Strains Improve Plant Growth Traits and Regulation of Phytohormones in Wheat under Salinity Stress" Plants 11, no. 20: 2769. https://doi.org/10.3390/plants11202769

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop