Skip to main content
PMC home page
Plants logoLink to Plants
. 2021 Jan 7;10(1):110. doi: 10.3390/plants10010110

Strategy of Salt Tolerance and Interactive Impact of Azotobacter chroococcum and/or Alcaligenes faecalis Inoculation on Canola (Brassica napus L.) Plants Grown in Saline Soil

Arafat Abdel Hamed Abdel Latef 1,*, Amal M Omer 2, Ali A Badawy 3,*, Mahmoud S Osman 3,*, Marwa M Ragaey 4
PMCID: PMC7825586  PMID: 33430173

Abstract

A pot experiment was designed and performed in a completely randomized block design (CRBD) to determine the main effect of two plant growth-promoting rhizobacteria (PGPR) and their co-inoculation on growth criteria and physio-biochemical attributes of canola plants (Brassica napus L.) plant grown in saline soil. The results showed that inoculation with two PGPR (Azotobacter chroococcum and/or Alcaligenes faecalis) energized the growth parameters and photosynthetic pigments of stressed plants. Moreover, soluble sugars’ and proteins’ contents were boosted due to the treatments mentioned above. Proline, malondialdehyde (MDA), and hydrogen peroxide (H2O2) contents were markedly declined. At the same time, antioxidant enzymes, viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase (POD), were augmented due to the inoculation with Azotobacter chroococcum and/or Alcaligenes faecalis. Regarding minerals’ uptake, there was a decline in sodium (Na) and an increase in nitrogen (N), potassium (K), calcium (Ca), and magnesium (Mg) uptake due to the application of either individual or co-inoculation with the mentioned bacterial isolates. This study showed that co-inoculation with Azotobacter chroococcum and Alcaligenes faecalis was the most effective treatment and could be considered a premium tool used in facing environmental problems, especially saline soils.

Keywords: plant growth-promoting rhizobacteria, salinity, canola, osmolytes, antioxidant activity

1. Introduction

Soil salinity directly affects crops. It is one of the most destructive abiotic stresses due to its disastrous effect on agricultural areas and reducing crops’ quality and productivity [1,2,3,4]. Moreover, there is also an abnormal increase in saline soils. This increase is due to several reasons, including some unfavorable agricultural practices, irrigation with saline water, and high surface evaporation rate [5].

Salinity of the soil limits crop plants’ productivity depending on the crop plants’ sensitivity to salts concentrations. Salt-stressed soils reduce plant growth [6,7]. They can also interfere with nitrogen (N) nutrition in the plant in direct or indirect ways, usually at the inorganic nitrogen compounds’ assimilation pathway. Besides, where high concentrations of salts are present in soils, the capacity for NO3 leaching in soil may boost because the plants’ efficiency in absorbing or utilizing the applied N from the soil is reduced under salinity stress [8]. One of the most severe problems that depend on salinity is the accumulation of reactive oxygen species (ROS) that leads to oxidative stress causing oxidative damage of proteins, pigments, and DNA of salt-stressed plants [7,9,10,11]. The higher concentration of salt in soil affects the plant’s potency and efficiency to absorb water and essential nutrients by roots. High sodium concentration inside the plant cell leads to many disturbances that lead to a decrease in plant growth [12,13]. Excess salt concentration decreased photosynthetic pigments in plant leaves, leaf area, and photosynthetic efficiency [3,14]. Furthermore, salinity stress caused oxidative stress due to the accumulation of hydrogen peroxide (H2O2), which induces cell shrinkage, DNA fragmentation, and induce malondialdehyde (MDA) accumulation, which is represented as an indicator for lipid peroxidation [15].

Plant growth-promoting bacteria (PGPB) play a direct or indirect useful role in enhancing plant growth, yield, and nutrient uptake through various action mechanisms [4,16]. These bacterial strains directly regulate plant physiology by promoting the nutrient uptake through phytohormone production (e.g., auxin, gibberellins, and cytokinin), increasing nitrogen and mineral availability in the soil and/or producing siderophores [17]. The PGPB containing 1-aminocyclopropane 1-carboxylate (ACC) deaminase are located in various soils and offer a promising approach for improving plant growth, particularly under stressed environmental conditions. Plants inoculated with ACC-deaminase containing PGBR showed a decrease in stimulated ethylene due to the diminishing impact of salt stress on ethylene. Plants with a lower level of ethylene showed more excellent resistance to abiotic stress [18]. Therefore, it could be mentioned that plants treated with ACC-deaminase containing PGPB help different plants to face different types of abiotic stresses [19,20].

Azotobacter genus is characterized as a free-living, aerobic, nitrogen-fixer, heterotrophic, Gram-negative bacteria in the class γ-proteobacteria. The first described species in Azotobacter genus was A. chroococcum [21]. Inoculation with A. chroococcum improves crop resistance to salinity through increasing plant content of soluble sugars, soluble proteins, and proline in shoots and roots. Moreover, it stimulates plant growth by increasing the dry weights of root and shoot [22,23,24]. Alcaligenes faecalis was isolated first in 1896, an Alcaligenaceae family member. This species is Gram-negative rods that are aerobically motile, flagellated, slightly curved non-spore-forming, slowly growing, and capsule-forming bacteria [25]. Alcaligenes faecalis is considered PGPB due to its ability to produce indole acetic acid (IAA), ACC-deaminase, and phosphate solubilization and fix atmospheric nitrogen [26]. Also, [27] showed that Alcaligenes sp. could be used as a biofertilizer to enhance the growth and yield of different plants under typical and different stress types. Inoculation with Alcaligenes faecalis containing ACC-deaminase ameliorates the salinity stress effect on growth, biochemistry, and yield of plants [28]. The most prevalent reason for the impact of Alcaligenes faecalis on plants is based on the production of phytohormones that alter plant morphology and metabolism, leading to improved water and mineral absorption [26].

Canola (Brassica napus L.), also known as oilseed rape, is one of the most important oilseed crops globally and was ranked globally as third in the term of oilseed crop production following soybean and palm oil [29,30]. At the same time, it ranks first among field oil crops that tolerate stressed conditions [31]. Canola seeds contain 40–42% oil, 60% oleic acid, 8.8% linoleic acid, and 25% protein [32,33,34]. Cultivation of canola in Egypt can introduce an opportunity to beat several deficiencies in edible oil production. Additionally, canola could be successfully cultivated in newly reclaimed land out of the old Nile Valley areas to avoid competition with other crops inhabiting the old cultivated lands [35,36].

Because of damage caused by salinity to crops and due to increases in saline land area, overcoming this problem in Egypt became one of the most critical challenges; therefore, it was necessary to use one of the appropriate approaches to meet this challenge. So, the use of plant growth-promoting rhizobacteria (PGPR) was considered as an alternative tool to alleviate salinity stress of essential oil crops like canola. In this line, we examined the possible role of PGPR strains Azotobacter chroococcum and Alcaligenes faecalis (individually or in co-inoculation) in enhancing salinity tolerance in canola plants by evaluating their impact on growth attributes, the contents of photosynthetic pigments, osmolytes, oxidative stress, and minerals as well as the antioxidants’ enzyme activities.

2. Results

2.1. Microbiological Characteristics in the Canola Rhizosphere

Results in Table 1 show the positive effect of bacterial inoculation on both the microbial community’s abundance and activity in the canola rhizosphere. Under salinity-stress regimes, total microbial count in canola rhizosphere was enhanced in response to inoculation with Azotobacter chroococcum and Alcaligenes faecalis and their mixture by 65.5%, 110.3%, and 113.7%, respectively. Moreover, nitrogen fixer count was increased in the treatments, as mentioned earlier, by 3.6%, 3.9%, and 3.9%, respectively. Dehydrogenase activity in the rhizosphere of Azotobacter chroococcum- and Alcaligenes faecalis-inoculated plants was increased by 63.3% and 116.6%, while co-inoculation recorded an increase in dehydrogenase activity by 112%.

Table 1.

Effect of bacterial inoculation on the microbial characteristics of the rhizosphere. TBC: total bacterial count, NFC: nitrogen fixer count.

Treatment TBC * × 105 CFU/gm Dry Soil TBC Increasing % NFC × 103 CFU/gm Dry Soil NFC Increasing % Dehydrogenase (μg TPF/g Dry Soil/24 h) Dehydrogenase Increasing %
Saline soil control 78 34.4 2.3 109 112 26.9
A. chroococcum 96 65.5 3.6 227 144 63.3
A. faecalis 122 110.3 3.9 254 191 116.6
A. chroococcum + A. faecalis 124 113.7 3.9 254 187 112
Open in a new tab

2.2. PGPR Enhance Canola Plant Growth under Salinity Stress

The results in Figure 1 show that the rhizobacterial inoculation with A. chroococcum, A. faecalis, and their co-inoculation enhanced the different canola growth parameters such as lengths of shoot and root, fresh and dry weights of shoot, fresh and dry weights of root, and number of leaves. Significant increases recorded by the individual inoculation with A. chroococcum were observed in shoot length by 25%, root length by 54%, shoot fresh weight by 165.7%, shoot dry weight by 182.8%, and root fresh weight by 66.70% as compared to un-inoculated canola plants grown in saline soil control. Moreover, the inoculation with A. faecalis recorded significant increases in shoot length, root length, shoot fresh weight, shoot dry weight, and root fresh weight by 65.8%, 75.5%, 157.5%, 93.1%, and 183.3%, respectively. The co-inoculation between the two mentioned strains showed significant increases reached to 64.6% in shoot length, 69.8% in root length, 248.4% in shoot fresh weight, 282.8% in shoot dry weight, 233.3% in root fresh weight, and 200% in root dry weight of salinity-stressed canola plants in comparison with saline soil control. Results in Figure 1 show that bacterial inoculation with A. chroococcum, A. faecalis, and their mixture insignificantly enhanced the number of leaves in canola plants under salinity-stress conditions.

Figure 1.

Figure 1

Open in a new tab

(A) shoot length; (B) root length; (C) shoot fresh weight; (D) shoot dry weight; (E) root fresh weight; (F) root dry weight; and (G) number of leaves of canola plant inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05.

2.3. PGPR Protect Photosynthetic Pigments in Leaves of Canola Plant under Salinity Stress

Both bacterial strains, A. chroococcum and A. faecalis, markedly accumulated fresh leaves’ contents of chlorophyll a (by 16% and 39%), chlorophyll b (by 14.1% and 44.6%), total chlorophyll (by 15.1% and 41.6%), and carotenoids (by 19.2% and 90.4%), respectively, when compared to saline soil control (Figure 2). The results exhibited that the highest significant increases in chlorophyll a (by 47.4%), chlorophyll b (by 52.7%), total chlorophyll (by 50%), and carotenoids (by 109.6%) were recorded in response to the co-inoculation with the two strains when compared to saline soil control.

Figure 2.

Figure 2

Open in a new tab

(A) Chlorophyll a; (B) chlorophyll b; (C) chlorophyll a + b; and (D) carotenoids’ content in canola plant fresh leaves inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05. FW: fresh weight.

2.4. PGPR Regulate Osmolytes’ Contents in Salinity-Stressed Canola Plants

Soluble sugars’ content of stressed canola plants was insignificantly enhanced in response to the individual inoculation with A. chroococcum or A. faecalis isolate, while soluble sugars’ content was significantly augmented, by 94% in the case of the two isolates’ interaction when compared with saline soil control (Table 2). The inoculation with PGPR changed the soluble proteins’ content of canola plants that were cultivated in saline soil. The inoculation of stressed canola plants with A. chroococcum, A. faecalis, or their interaction respectively recorded significant increases by 45.5%, 50.9%, and 55% of soluble proteins content (Table 2). Proline content was insignificantly decreased due to the treatment with Azotobacter chroococcum strain (Table 2). The decreases of proline content in salinity-stressed canola plants were recorded by 7.8% and 10.9% when the plants were inoculated with Alcaligenes faecalis and the interaction (Table 2).

Table 2.

Organic solutes contents (mg g−1 DW) in canola plant inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05. DW: dry weight.

Treatments Soluble Sugars Soluble Proteins Proline
Saline soil control 116.7 ± 3.85 b 16.9 ± 0.26 b 0.64 ± 0.04 a
A. chroococcum 135.3 ± 11.85 b 24.6 ± 0.48 a 0.62 ± 0.009 ab
A. faecalis 157.1 ± 9.76 b 25.5 ± 0.70 a 0.59 ± 0.006 bc
A. chroococcum + A. faecalis 226.5 ± 6.69 a 26.2 ± 0.38 a 0.57 ± 0.023 c
Open in a new tab

2.5. PGPR Lessen MDA and H2O2 Contents in Leaves of Salinity-Stressed Canola Plants

Comparing with the non-inoculated plants, MDA and H2O2 contents of salinized canola plants were inhibited due to the application of A. chroococcum about 12% and 7%, A. faecalis by 13.5% and 4.7%, and their interaction by 19.6% and 2.3%, respectively (Figure 3).

Figure 3.

Figure 3

Open in a new tab

(A) Malondialdehyde (MDA) content and (B) hydrogen peroxide (H2O2) content in canola plant fresh leaves inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05. FW: fresh weight.

2.6. PGPR Stimulate Antioxidant Enzymes under Salinity-Stress Conditions

The inoculation with Azotobacter chroococcum insignificantly stimulated the levels of SOD and APX of salinity-stressed canola plants, while it significantly stimulated POD by 121.7% (Figure 4). Regarding the inoculation with Alcaligenes faecalis, the activities of SOD, APX, and POD were insignificantly enhanced. In the interaction treatment with the two bacterial strains, there was a significant enhancement in SOD by 228.6%, APX by 29.3%, and POD by 130.4% of canola plants grown in saline soil in comparison with un-inoculated plants (Figure 4).

Figure 4.

Figure 4

Open in a new tab

(A) Superoxide dismutase (SOD), (B) ascorbate-peroxidase (APX), and (C) peroxidase (POD) activity in canola plant fresh leaves inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05. FW: fresh weight.

2.7. PGPR Regulate Mineral Uptake in Salinity-Stressed Canola Plants

Under salinity-stress conditions, canola plants inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation significantly decreased sodium (Na) content by 50.31%, 37.62%, and 57%, respectively (Table 3). However, the content of potassium (K) was increased dramatically due to the treatments as mentioned above by 51.98%, 77.98%, and 41.47%, respectively (Table 3), as compared to saline soil control. The contents of nitrogen (N) and calcium (Ca) were significantly increased in response to inoculation with Azotobacter chroococcum by 57.46% and 11.76%, respectively, while the content of magnesium (Mg) was decreased by 21.19% (Table 3). Moreover, inoculation with microbial strain Alcaligenes faecalis significantly increased contents of N, Ca, and Mg by 46.84%, 33.82%, and 80.50%, respectively (Table 3). Co-inoculation with Azotobacter chroococcum and Alcaligenes faecalis enhanced the contents of N, Ca, and Mg by 79.28%, 175%, and 55.08% versus saline soil control (Table 3).

Table 3.

Minerals’ contents (mg g−1 DW) in canola plant inoculated with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation under salinity-stress conditions. Bars show means of three independent replications (n = 3) ± standard error. Means with the same letter are not significantly different at p < 0.05. DW: dry weight.

Treatments Na K N Ca Mg
Saline soil control 14.81 ± 0.96 a 2.34 ± 0.10 d 1.34 ± 0.06 d 0.22 ± 0.05 b 0.39 ± 0.04 b
A. chroococcum 7.36 ± 0.35 c 3.56 ± 0.06 c 2.12 ± 0.09 b 0.25 ± 0.04 b 0.31 ± 0.03 b
A. faecalis 9.24 ± 0.70 b 4.17 ± 0.08 b 1.97 ± 0.07 c 0.30 ± 0.04 b 0.71 ± 0.05 a
A. chroococcum + A. faecalis 6.37 ± 0.11 d 5.66 ± 0.09 a 2.41 ± 0.05 a 0.62 ± 0.08 a 0.61 ± 0.02 a
Open in a new tab

3. Discussion

Salinization of water and soil plays a crucial role in limiting crops’ growth and productivity [3,4,37]. Soil salinity is a vast problem that spreads in most areas over the world, so it is imperative to found solutions for plants to have the ability to grow in these salinized areas [38]. In recent years, light has been shed on the use of natural sources and microorganisms (bacteria, fungi, algae, plant extracts, etc.) to cope with salt stress and mitigate its harmful effects on plant life [4,7,39,40,41,42]. PGPB have the colossal ability to lessen salt stress and improve plant development, playing a critical role in food security by boosting the productivity of crops. Use of PGPB under salinity stress enhances plant growth in several ways, including ACC deaminase activity, synthesis of plant hormones as IAA, gibberellic acid (GA), abscisic acid (ABA), cytokinin, and exopolysaccharides [43]. PGPR stimulate plant growth directly by enhancing the uptake of nutrients through phytohormone production (e.g., auxin, gibberellins, and cytokinin) or by lowering plant ethylene levels enzymatically [17]. It has been suggested that the production of auxins by root-associated microbes is one of the most important mechanisms through which microbes regulate plant growth. Also, specific beneficial endophytes can produce auxin and/or display ACC deaminase activity that can aid host plant growth in dangerous areas [44].

Soil salinity as a vital stress factor harms the microbial process, diminishing bacterial diversity and controlling microbial wealth, composition, and functions [28]. Plants’ inoculation can mitigate this negative impact of salinity with our tested PGPR Azotobacter chroococcum, Alcaligenes faecalis, and co-inoculation. Bacterial treatment of canola plants with plant growth-promoting rhizobacteria had a remarkable stimulation effect on the rhizosphere’s microbial population [45]. Dehydrogenase activity by indigenous microorganisms in soil can serve as a valuable marker of microbial activity, which indicates the relative effectiveness of microbes with plant rhizosphere in soils [46]. Our results are in harmony with results [47] that stated that the combined inoculation with Azospirillum sp. and Bacillus sp. increased the dehydrogenase at all growth plant stages. Plant growth-promoting rhizobacteria can enhance the tolerance of plants to various abiotic stresses, including salinity.

Our study demonstrated enhancements in salinity-stressed canola plants’ growth parameters in response to inoculation with Azotobacter chroococcum and Alcaligenes faecalis. Similar improvements in plant growth due to inoculation with halotolerant plant growth-promoting bacterium Alcaligenes faecalis were evidenced in the study of [48], which stated that vegetative growth characteristics of salinity-stressed rice and wheat plants, respectively, were increased. These results documented that PGPR inoculation appeared to reinforce canola’s growth by relieving the suppression caused by salinity stress [49]. The utilization of PGPR was recommended to boost the growth of different salinity-stressed crops [50,51,52,53]. Ref. [54,55] linked the augmentation of plant growth with the ability of PGPR to produce some plant growth regulators, solubilize phosphate, and fix nitrogen. These features are found in the selected isolates and generally increase a plant’s ability to absorb nutrients from the soil and improve its growth, especially under salinity-stress conditions.

Photosynthetic pigments are a fundamental physiological trait directly associated with photosynthesis ability under abiotic stresses. Our results observed increases in chlorophylls and carotenoids in canola plants cultivated in saline soil. These increases were due to the soil supplementation with the tested PGPR. Similar results recorded enhancements in the photosynthetic pigments in PGPR-inoculated plants under different saline conditions [56,57,58]. The augmentation in photosynthetic pigments in PGPR-inoculated plants suggests the potency of bacterial inoculation to nullify the harmful impacts of salinity stress by improving the activities of electron transporters associated with photosynthesis [59] as well as the biosynthesis of proteins and enzymes that related to pigment stabilization [60].

In the present study, the inoculation with plant growth-promoting rhizobacterial strains Azotobacter chroococcum and Alcaligenes faecalis, especially the co-inoculation between them, led to enhancements in soluble sugars’ content in canola plants cultivated in saline soil. These enhancements were evidenced in several studies [61,62,63,64,65]. Recently, the inoculation with A. chroococcum exhibited increases in sugar contents in maize plants cultivated in salt-affected soil [4]. They documented that sugar content rising is considered as a vital osmolyte that maintains the plant against salinity stress. The current study clarified that soluble proteins’ content in salinity-stressed canola plants was increased due to the inoculation with PGPR. Various studies on crop plants have well documented the positive impacts of rhizobacterial inoculation on increasing the soluble protein content [4,63,64,65]. A possible strategy behind this increase could be that bacterial inoculation might inhibit the activity of protein-hydrolyzing enzymes in addition to the ability of bacteria in promoting the efficiency of proline in protecting soluble proteins and, thus, increasing their amounts under the salt-stress conditions [63,65,66].

The plant faces environmental stressors by accumulating some osmolytes like proline, which acts as a solute for osmoregulation [67]. However, the accumulation of proline in plants has been documented as an environmental stress indicator [68]. The proline level in the present study was inhibited in salinity-stressed canola plants that were inoculated with the tested PGPR containing ACC-deaminase. This result is in harmony with the findings of [69,70]. The current results may imply that PGPR alleviated the severity of salinity stress on the plant and, thus, the proline content (a marker of stress) in canola shoots also lessened.

Our study showed a reduction in the contents of MDA and H2O2 in salinized canola plants that were inoculated with PGP rhizobacterial isolates. Our findings on the efficacy of PGPR in decreasing the contents of MDA and H2O2 in plants cultivated in conditions of salinity stress are in harmony with the studies of [52,70,71]. Thus, PGPR could prevent canola plants from oxidative destruction caused by salinity stress.

To mitigate the oxidative stress induced by salinity stress, the plants developed a group of physiological and biochemical strategies made of various enzymes that can scavenge the ROS species. Antioxidant enzymes act in a network to achieve the detoxification of ROS species [10,72,73]. In our study, we noticed different increases in SOD, APX, and POD activities in the inoculated canola plants with the mentioned PGPR under saline conditions. Our findings comply with the reports of [74] on mung bean.

In this study, PGPR’s positive role appeared in removing the harmful effect of salinity stress by limiting the uptake of Na and increasing the uptake of essential minerals such as N, K, Ca, and Mg. This positive role may be attributed to the finding that PGPR facilitate the entity of essential elements in the soil to be easily absorbed by the plant [13,75] or due to roots’ exudates initiated by PGPR, increasing the availability of some micronutrients [57,76]. Moreover, increasing nitrogen content in canola shoots is attributed to the ability of PGPR in increasing nitrogen and mineral availability in the soil [17].

4. Materials and Methods

4.1. Isolation, Identification, and Description of PGPR (Salt-Tolerant Bacteria)

Two halophilic bacterial strains were used for alleviating the salt stress in canola plants. The first halophilic strain is rhizospheric bacteria isolated, purified on Ashby’s media as selective media [77] from the rhizosphere of wheat plant cultivated in saline soil at Sahl El-Tina, Sinai, Egypt (Electrical conductivity (EC 6000–7000 ppm), and identified to its molecular level as Azotobacter chroococcum strain NBRC using partial 16S rRNA gene sequence technique according to [78] in Sigma Scientific Services Co. (Giza, Egypt). The second strain is rhizospheric bacteria isolated, purified on King’s media as selective media [79] from the barley plant’s rhizosphere cultivated in saline soil at Ras Sudr, Sinai, Egypt (EC 5000–6000 ppm), and identified to molecular level as Alcaligenes faecalis strain NBRC 13111. Two strains were assigned in Gene Bank NCBI with accession number as NR 113606.1 and NR114167, respectively.

4.2. Pot Experiment

A pot experiment was conducted in the greenhouse of the microbiological unit of Desert Research Center, Cairo, Egypt. Canola (Brassica napus L. cv. Pactol) was provided by Agricultural Research Center (ARC), Giza, Egypt. Microbial inoculants of Azotobacter chroococcum, Alcaligenes faecalis, and a mixture of them were used for treating canola plants. The experimental design was performed in a complete randomized block design (CRBD) with three replications. Climate conditions were: average day/night temperature cycle of 23/12, light 10/14 h, and air humidity between 39% and 58%. Ten Seeds of canola were planted into 10-kg pots containing saline soil collected from Sahl El-Tina, Sinai, Egypt. The physical and chemical characteristics of the soil were soil depth 0–15 cm−1, total sand 30%, silt 10.2%, clay 59.8%, texture clay, EC 11.5 mmhos cm−1, pH 7.6, HCO3- 18.5 mg g−1, Cl- 51.6 mg g−1, SO4− 9.8 mg g−1, Ca2+ 21.1 mg g−1, Mg2+ 15.1 mg g−1, Na+ 56.2 mg g−1, and K+ 0.64 mg g−1. For bacterial treatments, seeds were coated with bacterial inoculum using carboxymethyl cellulose (CMC) solution (1%) in the ratio of 1 kg seeds/250 mL of inoculum (106 CFU/mL) mixed with 50 g−1 of CMC before application to get a thin, uniform coating of bacterial inoculum on seeds. Inoculated seeds were dried in shade before sowing [80]. Untreated control seeds were maintained. After seed germination, plants were thinned1to five plants per pot then each pot was inoculated with 10 mL of microbial inoculum (106 CFU/mL) of an individual strain and mixture of them. Pots were arranged as follows: (1) saline soil control, (2) saline soil control + Azotobacter chroococcum, (3) saline soil control + Alcaligenes faecalis, and (4) saline soil control + co-inoculation with Azotobacter chroococcum and Alcaligenes faecalis. Pots were irrigated two times weekly. After 66 days of planting, the plants were harvested to determine lengths of shoots and roots, fresh and dry weights of shoots and roots, and biochemical parameters.

4.3. Microbiological Analysis of Canola Rhizosphere

Total microbial count and populations of Azotobacter chroococcum and Alcaligenes faecalis in the rhizosphere samples were estimated using yeast extract agar medium [81], Ashby’s [77], and King’s media [79]. Soil dehydrogenase activity (μg TPF/g dry soil/24 h) was analyzed by the reduction of 2,3,5-triphenyl tetrazolium chloride (TTC) to triphenyl formazan (TPF) as described by [82].

4.4. Determination of Photosynthetic Pigments

Chlorophyll content in fresh leaves of canola plants was estimated according to methods described by [83]. In this method, 100 mL of acetone (80%) were used for pigments’ extraction from fresh leaves (1 g). Then, the extract was filtered and the green color was measured at 470, 649, and 665 nm using spectrophotometer. Photosynthetic pigments calculated according to the following equations: Chl a (mg g−1 FW) = 11.63(A665) − 2.39(A649), Chl b (mg g−1 FW) = 20.11(A649) − 5.18(A665); Chl a + b (mg g−1 FW) = 6.45 (A665) +1 7.72(A649); carotenoids’ contents (mg g−1 FW) = {(1000 ×A470) − (1.82 × Chl a) − (85.02 × Chl b)}/198, according to [84].

4.5. Determination of Osmolyte Contents

Soluble sugars’ content of dried canola plants’ shoot was estimated according to [85]. One g from the dried sample was placed with 5 mL of 2% phenol and 10 mL of 30% trichloroacetic acid for extraction. Two mL of the filtered extract were mixed with 4 mL of anthrone reagent (2 g anthrone/L of 95% sulfuric acid). At 620 nm we measured the developed blue-green color.

Soluble proteins’ content was estimated according to methods of [86] in the dried shoot of canola plants. Sample (0.1 g) was extracted in 5 mL of 2% phenol and 10 mL of distilled water. One mL of extract was added to 5 mL of alkaline reagent (50 mL from 2% Na2CO3 prepared in 0.1 N NaOH and 1 mL from 0.5% CuSO4.5H2O prepared in 1% sodium potassium tartrate) and mixed thoroughly. Then, 0.5 mL of folin reagent (diluted 1:3 v/v) was added. The developed color after 30 min was measured at 750 nm.

The described method of [87] was used for determination of proline contents. In such method, a half gram of the dried shoot of canola plants was homogenized in 10 mL (3%) sulfosalicylic acid. The homogenate was filtered and 2 mL of it were reacted with 2 mL of acid ninhydrin (warm 1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6M phosphoric acid) and 2 mL of glacial acetic acid for one hour in a boiling water bath. Then, the reaction was placed in an ice bath. Four mL of toluene was added to the mixture 4. Then, we read the absorbance at 520 nm.

4.6. Estimation of Malondialdehyde Content

MDA content in canola fresh leaves was estimated according to the described method of [88]. In this method, fresh leaf samples (0.5 g) were extracted with 5% trichloroacetic acid and centrifugated at 4000× g for 10 min. Then, 2 mL of the extract were mixed with 2 mL of 0.6% Thiobarbituric acid (TBA) solution. Then, the mixture was placed in a water bath for 10 min. After cooling, the absorbance of the devolved color was at 532, 600, and 450 nm subsequently. MDA content was calculated according to the following equation: 6.45 × (A532 − A600) − 0.56 × A450.

4.7. Determination of Hydrogen Peroxide (H2O2) Content

Estimation of hydrogen peroxide content in the leaves of canola plants was according to methods described by [89], in which fresh samples (0.05 g) were extracted with 4 mL cold acetone. An aliquot (3 mL) of the extracted solution was mixed with 1 mL of 0.1% titanium dioxide in 20% (v:v) H2SO4 and the mixture was then centrifuged at 6000 rpm for 15 min. The intensity of the yellow color of the supernatant was measured at 415 nm.

4.8. Extraction and Assay of Antioxidant Enzymes

For the extraction of antioxidant enzymes, terminal buds with first true leaves of canola plants were used, according to methods described by [89], for the extraction of POD and SOD. A method described in [4] was used to extract ascorbate peroxidase (APX).

The activity of SOD was estimated according to methods described by [90]. The solution (10 mL) consisted of 3.6 mL of distilled water, 0.1 mL of enzyme, 5.5 mL of 50 mM phosphate buffer (pH 7.8), and 0.8 mL of 3 mM pyrogallol (dissolved in 10 mM HCl). The rate of pyrogallol reduction was measured at 325 nm with UV-spectrophotometer.

The described method of [91] was followed to estimate APX activity, in which 0.5 mM AsA, 0.8 mL of potassium phosphate buffer (50 mM, pH 7), 0.1 mM H2O2, and 0.2 mL enzyme extract were mixed. The changes in absorbance were read at 290 nm.

The activity of POD was estimated according to methods described by [92]: 5.8 mL of 50 mM phosphate buffer (pH 7.0), 0.2 mL of the enzyme extract, and 2 mL of 20 mM H2O2 after addition of 2 mL of 20 mM pyrogallol. The rate of increase in absorbance as pyrogallol was determined spectro-photometrically by UV-visible spectro-photometer within 60 s at 470 nm.

4.9. Determination of Mineral Contents

Dry shoot samples (0.1 g) were acid digested with 80% perchloric acid (HCLO4) and sulfuric acid (H2SO4) 1:5 solution for 12 h. A method described by [93] was used to determine Na, K, Ca, and Mg in the digested sample. Nitrogen content was determined in digested sample according to a modified micro-Kjeldahl method [94].

4.10. Statistical Analysis

Data were statistically analyzed by analysis of variance (ANOVA), to determine a significant difference between different treatments using CoStat (CoHort software, Monterey, CA, USA). Least significant difference (LSD) at p ≤ 0.05 was used to indicate a significant difference among treatments. Results were shown as mean ± standard error (SE) of three independent replications for each treatment (n = 3).

5. Conclusions

From the outcome of the obtained results, it seems likely to conclude that using of Azotobacter chroococcum and Alcaligenes faecalis brought about enhancements in different growth indices of canola plants grown in saline soil. The co-inoculation with both bacterial isolates brought about significant improvements in most morphology parameters, photosynthetic pigments and carotenoids, soluble sugars, and soluble protein contents. Also, proline, malondialdehyde, and hydrogen peroxide contents were inhibited, indicating less salt-stress toxicity. Additionally, ascorbate peroxidase, peroxidase, and superoxide dismutase activities were promoted as a reason for the single inoculation and co-inoculation with the mentioned isolates, thus boosting the tolerance of plants to cope with salinity stress. Moreover, mineral contents (except Na+) were enhanced in salinity-stressed canola plants in response to inoculation with Azotobacter chroococcum, Alcaligenes faecalis, and their co-inoculation. We suggest using the co-inoculation with Azotobacter chroococcum and Alcaligenes faecalis producing IAA, solubilizing phosphate, and containing ACC-deaminase as an effective and important approach for ameliorating salinity stress.

Author Contributions

Conceptualization, A.A.B., M.S.O., and A.M.O.; methodology, A.A.B., M.S.O., and A.M.O.; software, A.A.B. and M.S.O.; validation, A.A.B. and M.S.O.; formal analysis, A.A.B. and M.S.O.; investigation, A.A.B. and M.S.O.; resources, A.A.B., M.S.O., A.M.O. and M.M.R.; data curation, A.A.B., M.S.O., and M.M.R.; writing—original draft preparation, A.A.B., M.S.O., and A.M.O.; writing—review and editing, A.A.H.A.L.; visualization, M.S.O.; supervision, A.A.H.A.L.; funding acquisition, A.A.H.A.L. and M.M.R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Taif University Researchers Supporting Project number (TURSP-2020/72), Taif University, Taif, Saudi Arabia.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare that there are no conflicts of interest related to this article.

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Machado R.M.A., Serralheiro R.P. Soil salinity: Effect on vegetable crop growth. Management practices to prevent and mitigate soil salinization. Horticulturae. 2017;3:30. doi: 10.3390/horticulturae3020030. [DOI] [Google Scholar]
  • 2.Abdel Latef A.A.H., Srivastava A.K., El-sadek M.S.A., Kordrostami M., Tran L.P. Titanium dioxide nanoparticles improve growth and enhance tolerance of broad bean plants under saline soil conditions. Land Degrad. Dev. 2018;29:1065–1073. doi: 10.1002/ldr.2780. [DOI] [Google Scholar]
  • 3.Abdel Latef A.A.H., Mostofa M.G., Rahman M.M., Abdel-Farid I.B., Tran L.-S.P. Extracts from yeast and carrot roots enhance maize performance under seawater-induced salt stress by altering physio-biochemical characteristics of stressed plants. J. Plant Growth Regul. 2019;38:966–979. doi: 10.1007/s00344-018-9906-8. [DOI] [Google Scholar]
  • 4.Abdel Latef A.A.H., Alhmad M.F.A., Kordrostami M., Abo–Baker A.-B.A.-E., Zakir A. Inoculation with Azospirillum lipoferum or Azotobacter chroococcum reinforces maize growth by improving physiological activities under saline conditions. J. Plant Growth Regul. 2020;39:1293–1306. doi: 10.1007/s00344-020-10065-9. [DOI] [Google Scholar]
  • 5.Jadhav G.G., Salunkhe D.S., Nerkar D.P., Bhadekar R.K. Isolation and characterization of salt-tolerant nitrogen-fixing microorganisms from food. EurAsian J. Biosci. 2010;4:33–40. doi: 10.5053/ejobios.2010.4.0.5. [DOI] [Google Scholar]
  • 6.Paul D. Osmotic stress adaptations in rhizobacteria. J. Basic Microbiol. 2013;53:101–110. doi: 10.1002/jobm.201100288. [DOI] [PubMed] [Google Scholar]
  • 7.Osman M.S., Badawy A.A., Osman A.I., Latef A.A.H.A. Ameliorative impact of an extract of the halophyte Arthrocnemum macrostachyum on growth and biochemical parameters of soybean under salinity stress. J. Plant Growth Regul. 2020 doi: 10.1007/s00344-020-10185-2. [DOI] [Google Scholar]
  • 8.Bowman D.C., Devitt D.A., Miller W.W. The effect of moderate salinity on nitrate leaching from bermudagrass turf: A lysimeter study. Water Air Soil Pollut. 2006;175:49–60. doi: 10.1007/s11270-006-9110-5. [DOI] [Google Scholar]
  • 9.Abdel Latef A.A.H. Changes of antioxidative enzymes in salinity tolerance among different wheat cultivars. Cereal Res. Commun. 2010;38:43–55. doi: 10.1556/CRC.38.2010.1.5. [DOI] [Google Scholar]
  • 10.Ahammed G.J., Li Y., Li X., Han W.-Y., Chen S. Epigallocatechin-3-gallate alleviates salinity-retarded seed germination and oxidative stress in tomato. J. Plant Growth Regul. 2018;37:1349–1356. doi: 10.1007/s00344-018-9849-0. [DOI] [Google Scholar]
  • 11.Shokri-Gharelo R., Noparvar P.M. Molecular response of canola to salt stress: Insights on tolerance mechanisms. PeerJ. 2018;6:e4822. doi: 10.7717/peerj.4822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Singh R.P., Jha P., Jha P.N. The plant-growth-promoting bacterium Klebsiella sp. SBP-8 confers induced systemic tolerance in wheat (Triticum aestivum) under salt stress. J. Plant Physiol. 2015;184:57–67. doi: 10.1016/j.jplph.2015.07.002. [DOI] [PubMed] [Google Scholar]
  • 13.Kumar A., Singh S., Gaurav A.K., Srivastava S., Verma J.P. Plant Growth-Promoting Bacteria: Biological Tools for the Mitigation of Salinity Stress in Plants. Front. Microbiol. 2020;11 doi: 10.3389/fmicb.2020.01216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ashraf M., Harris P.J.C. Photosynthesis under stressful environments: An overview. Photosynthetica. 2013;51:163–190. doi: 10.1007/s11099-013-0021-6. [DOI] [Google Scholar]
  • 15.Zhu J., Fan Y., Shabala S., Li C., Lv C., Guo B., Xu R., Zhou M. Understanding mechanisms of salinity tolerance in barley by proteomic and biochemical analysis of near-isogenic lines. Int. J. Mol. Sci. 2020;21:1516. doi: 10.3390/ijms21041516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Vejan P., Abdullah R., Khadiran T., Ismail S., Nasrulhaq Boyce A. Role of plant growth promoting rhizobacteria in agricultural sustainability—A review. Molecules. 2016;21:573. doi: 10.3390/molecules21050573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Kohler J., Caravaca F., Carrasco L., Roldan A. Contribution of Pseudomonas mendocina and Glomus intraradices to aggregate stabilization and promotion of biological fertility in rhizosphere soil of lettuce plants under field conditions. Soil Use Manag. 2006;22:298–304. doi: 10.1111/j.1475-2743.2006.00041.x. [DOI] [Google Scholar]
  • 18.Glick B.R. Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase. FEMS Microbiol. Lett. 2005;251:1–7. doi: 10.1016/j.femsle.2005.07.030. [DOI] [PubMed] [Google Scholar]
  • 19.Kausar R., Shahzad S.M. Effect of ACC-deaminase containing rhizobacteria on growth promotion of maize under salinity stress. J. Agric. Soc. Sci. 2006;2:216–218. [Google Scholar]
  • 20.Nadeem M.A., Nawaz M.A., Shahid M.Q., Doğan Y., Comertpay G., Yıldız M., Hatipoğlu R., Ahmad F., Alsaleh A., Labhane N. DNA molecular markers in plant breeding: Current status and recent advancements in genomic selection and genome editing. Biotechnol. Biotechnol. Equip. 2018;32:261–285. doi: 10.1080/13102818.2017.1400401. [DOI] [Google Scholar]
  • 21.Robson R.L., Jones R., Robson R.M., Schwartz A., Richardson T.H. Azotobacter genomes: The genome of Azotobacter chroococcum NCIMB 8003 (ATCC 4412) PLoS ONE. 2015;10:e0127997. doi: 10.1371/journal.pone.0127997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Aly M.M., El-Sabbagh S.M., El-Shouny W.A., Ebrahim M.K.H. Physiological response of Zea mays to NaCl stress with respect to Azotobacter chroococcum and Streptomyces niveus. Pak. J. Biol. Sci. 2003;6:2073–2080. doi: 10.3923/pjbs.2003.2073.2080. [DOI] [Google Scholar]
  • 23.Ravikumar S., Kathiresan K., Alikhan S.L., Williams G.P., Gracelin N.A.A. Growth of Avicennia marina and Ceriops decandra seedlings inoculated with halophilic azotobacters. J. Environ. Biol. 2007;28:601. [PubMed] [Google Scholar]
  • 24.Chaudhary D., Narula N., Sindhu S.S., Behl R.K. Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains. Physiol. Mol. Biol. Plants. 2013;19:515–519. doi: 10.1007/s12298-013-0178-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Austin B. In: The Family Alcaligenaceae. Rosenberg E., DeLong E.F., Lory S., Stackebrandt E., editors. Springer; New York, NY, USA: 2014. pp. 729–757. [Google Scholar]
  • 26.Neethu S., Vishnupriya S., Mathew J. Isolation and functional characterisation of endophytic bacterial isolates from curcuma longa. Int. J. Pharm. Biol. Sci. 2016;7:455–464. [Google Scholar]
  • 27.Akhtar S., Ali B. Evaluation of rhizobacteria as non-rhizobial inoculants for mung beans. Aust. J. Crop Sci. 2011;5:1723. [Google Scholar]
  • 28.Omer A.M. Inducing plant resistance against salinity using some rhizobacteria. Egypt. J. Desert Res. 2017;67:187–208. doi: 10.21608/ejdr.2017.6498. [DOI] [Google Scholar]
  • 29.Ashraf M., McNeilly T. Salinity tolerance in Brassica oilseeds. CRC Crit. Rev. Plant Sci. 2004;23:157–174. doi: 10.1080/07352680490433286. [DOI] [Google Scholar]
  • 30.Abdel Latef A.A.H. Ameliorative effect of calcium chloride on growth, antioxidant enzymes, protein patterns and some metabolic activities of canola (Brassica napus L.) under seawater stress. J. Plant Nutr. 2011;34:1303–1320. doi: 10.1080/01904167.2011.580817. [DOI] [Google Scholar]
  • 31.Lohani N., Jain D., Singh M.B., Bhalla P.L. Engineering Multiple Abiotic Stress Tolerance in Canola, Brassica napus. Front. Plant Sci. 2020;11 doi: 10.3389/fpls.2020.00003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Hu X., Sullivan-Gilbert M., Gupta M., Thompson S.A. Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.) Theor. Appl. Genet. 2006;113:497–507. doi: 10.1007/s00122-006-0315-1. [DOI] [PubMed] [Google Scholar]
  • 33.Milazzo M.F., Spina F., Vinci A., Espro C., Bart J.C.J. Brassica biodiesels: Past, present and future. Renew. Sustain. Energy Rev. 2013;18:350–389. doi: 10.1016/j.rser.2012.09.033. [DOI] [Google Scholar]
  • 34.Carré P., Pouzet A. Rapeseed market, worldwide and in Europe. Ocl. 2014;21:D102. doi: 10.1051/ocl/2013054. [DOI] [Google Scholar]
  • 35.Ghallab K.H., Sharaan A.N. Selection in canola (Brassica napus L.) germplasm under conditions of newly reclaimed land. II. Salt tolerant selections. Egypt. J. Plant Breed. 2002;6:15–30. [Google Scholar]
  • 36.El Sabagh A., Omar A.E., Saneoka H., Barutçular C. Evaluation agronomic traits of canola (Brassica napus L.) under organic, bio-and chemical fertilizers. Dicle Univ. J. Inst. Nat. Appl. Sci. 2015;4:59–67. [Google Scholar]
  • 37.Abdel Latef A.A.H., Kordrostami M., Zakir A., Zaki H., Saleh O.M. Eustress with H2O2 facilitates plant growth by improving tolerance to salt stress in two wheat cultivars. Plants. 2019;8:303. doi: 10.3390/plants8090303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Deinlein U., Stephan A.B., Horie T., Luo W., Xu G., Schroeder J.I. Plant salt-tolerance mechanisms. Trends Plant Sci. 2014;19:371–379. doi: 10.1016/j.tplants.2014.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Abdel Latef A.A.H., Chaoxing H. Effect of arbuscular mycorrhizal fungi on growth, mineral nutrition, antioxidant enzymes activity and fruit yield of tomato grown under salinity stress. Sci. Hortic. (Amsterdam) 2011;127:228–233. doi: 10.1016/j.scienta.2010.09.020. [DOI] [Google Scholar]
  • 40.Abdel Latef A.A.H., Alhmad M.F.A., Abdelfattah K.E. The possible roles of priming with ZnO nanoparticles in mitigation of salinity stress in lupine (Lupinus termis) plants. J. Plant Growth Regul. 2017;36:60–70. doi: 10.1007/s00344-016-9618-x. [DOI] [Google Scholar]
  • 41.Abdel Latef A.A.H., Abu Alhmad M., Ahmad S. Foliar application of fresh moringa leaf extract overcomes salt stress in fenugreek (Trigonella foenum-graecum) plants. Egypt. J. Bot. 2017;57:157–179. [Google Scholar]
  • 42.Abdel Latef A.A.H.A., Srivastava A.K., Saber H., Alwaleed E.A., Tran L.-S.P. Sargassum muticum and Jania rubens regulate amino acid metabolism to improve growth and alleviate salinity in chickpea. Sci. Rep. 2017;7:1–12. doi: 10.1038/s41598-017-07692-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Bhise K.K., Dandge P.B. Mitigation of salinity stress in plants using plant growth promoting bacteria. Symbiosis. 2019;79:191–204. doi: 10.1007/s13199-019-00638-y. [DOI] [Google Scholar]
  • 44.Foo E., Plett J.M., Lopez-Raez J.A., Reid D. The Role of plant hormones in plant-microbe symbioses. Front. Plant Sci. 2019;10 doi: 10.3389/fpls.2019.01391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Ashrafuzzaman M., Hossen F.A., Ismail M.R., Hoque A., Islam M.Z., Shahidullah S.M., Meon S. Efficiency of plant growth-promoting rhizobacteria (PGPR) for the enhancement of rice growth. Afr. J. Biotechnol. 2009;8 [Google Scholar]
  • 46.Mathew M., Obbard J.P. Optimisation of the dehydrogenase assay for measurement of indigenous microbial activity in beach sediments contaminated with petroleum. Biotechnol. Lett. 2001;23:227–230. doi: 10.1023/A:1005613406948. [DOI] [Google Scholar]
  • 47.Abou-Aly H.E. Stimulatory effect of some yeast applications on response of tomato plants to inoculation with biofertilizers. Ann. Agric. Sci. Moshtohor. 2005;43:595–609. [Google Scholar]
  • 48.Fatima T., Mishra I., Verma R., Kumar N. Mechanisms of halotolerant plant growth promoting Alcaligenes sp. involved in salt tolerance and enhancement of the growth of rice under salinity stress. 3 Biotech. 2020 doi: 10.1007/s13205-020-02348-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Li H., Lei P., Pang X., Li S., Xu H., Xu Z., Feng X. Enhanced tolerance to salt stress in canola (Brassica napus L.) seedlings inoculated with the halotolerant Enterobacter cloacae HSNJ4. Appl. Soil Ecol. 2017;119:26–34. doi: 10.1016/j.apsoil.2017.05.033. [DOI] [Google Scholar]
  • 50.Shukla P.S., Agarwal P.K., Jha B. Improved salinity tolerance of Arachishypogaea (L.) by the interaction of halotolerant plant-growth-promoting rhizobacteria. J. Plant Growth Regul. 2012;31:195–206. doi: 10.1007/s00344-011-9231-y. [DOI] [Google Scholar]
  • 51.Ullah S., Bano A. Isolation of plant-growth-promoting rhizobacteria from rhizospheric soil of halophytes and their impact on maize (Zea mays L.) under induced soil salinity. Can. J. Microbiol. 2015;61:307–313. doi: 10.1139/cjm-2014-0668. [DOI] [PubMed] [Google Scholar]
  • 52.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. 2016:1–16. doi: 10.1038/srep34768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Orhan F. Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum) Braz. J. Microbiol. 2016;47:621–627. doi: 10.1016/j.bjm.2016.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Majeed A., Abbasi M.K., Hameed S., Imran A., Rahim N. Isolation and characterization of plant growth-promoting rhizobacteria from wheat rhizosphere and their effect on plant growth promotion. Front. Microbiol. 2015;6:198. doi: 10.3389/fmicb.2015.00198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Etesami H., Glick B.R. Halotolerant plant growth–promoting bacteria: Prospects for alleviating salinity stress in plants. Environ. Exp. Bot. 2020:104124. doi: 10.1016/j.envexpbot.2020.104124. [DOI] [Google Scholar]
  • 56.Yildirim E., Turan M., Ekinci M., Dursun A., Cakmakci R. Plant growth promoting rhizobacteria ameliorate deleterious effect of salt stress on lettuce. Sci. Res. Essays. 2011;6:4389–4396. [Google Scholar]
  • 57.Kang S.-M., Khan A.L., Waqas M., You Y.-H., Kim J.-H., Kim J.-G., Hamayun M., Lee I.-J. Plant growth-promoting rhizobacteria reduce adverse effects of salinity and osmotic stress by regulating phytohormones and antioxidants in Cucumis sativus. J. Plant Interact. 2014;9:673–682. doi: 10.1080/17429145.2014.894587. [DOI] [Google Scholar]
  • 58.Aslam F., Ali B. Halotolerant bacterial diversity associated with Suaeda fruticosa (L.) forssk. improved growth of maize under salinity stress. Agronomy. 2018;8:131. doi: 10.3390/agronomy8080131. [DOI] [Google Scholar]
  • 59.Pinnola A., Staleva-musto H., Capaldi S., Ballottari M., Bassi R. Biochimica et Biophysica Acta Electron transfer between carotenoid and chlorophyll contributes to quenching in the LHCSR1 protein from Physcomitrella patens. Biochimica et Biophysica Acta (BBA)—Bioenergetics. 2016:1870–1878. doi: 10.1016/j.bbabio.2016.09.001. [DOI] [PubMed] [Google Scholar]
  • 60.Enebe M.C., Babalola O.O. The influence of plant growth-promoting rhizobacteria in plant tolerance to abiotic stress: A survival strategy. Appl. Microbiol. Biotechnol. 2018;102:7821–7835. doi: 10.1007/s00253-018-9214-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 61.Chen L., Liu Y., Wu G., Veronican K., Shen Q., Zhang N. Induced maize salt tolerance by rhizosphere inoculation of Bacillus amyloliquefaciens SQR9. Physiol. Plant. 2016:34–44. doi: 10.1111/ppl.12441. [DOI] [PubMed] [Google Scholar]
  • 62.Hmaeid N., Wali M., Mahmoud O.M.-B., Pueyo J.J., Ghnaya T., Abdelly C. Efficient rhizobacteria promote growth and alleviate NaCl-induced stress in the plant species Sulla carnosa. Appl. Soil Ecol. 2019;133:104–113. doi: 10.1016/j.apsoil.2018.09.011. [DOI] [Google Scholar]
  • 63.Egamberdieva D., Lugtenberg B. Use of Microbes for the Alleviation of Soil Stresses. Volume 1. Springer; New York, NY, USA: 2014. Use of plant growth-promoting rhizobacteria to alleviate salinity stress in plants; pp. 73–96. [Google Scholar]
  • 64.Pan J., Peng F., Xue X., You Q., Zhang W., Wang T., Huang C. The growth promotion of two salt-tolerant plant groups with PGPR inoculation: A meta-analysis. Sustainability. 2019;11:378. doi: 10.3390/su11020378. [DOI] [Google Scholar]
  • 65.Hamdia M.A.E.-S., Shaddad M.A.K., Doaa M.M. Mechanisms of salt tolerance and interactive effects of Azospirillum brasilense inoculation on maize cultivars grown under salt stress conditions. Plant Growth Regul. 2004;44:165–174. doi: 10.1023/B:GROW.0000049414.03099.9b. [DOI] [Google Scholar]
  • 66.Beneduzi A., Ambrosini A., Passaglia L.M.P. Plant growth-promoting rhizobacteria (PGPR): Their potential as antagonists and biocontrol agents. Genet. Mol. Biol. 2012;35:1044–1051. doi: 10.1590/S1415-47572012000600020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Silveira J.A.G., de Almeida Viégas R., da Rocha I.M.A., Moreira A.C.D.O.M., de Azevedo Moreira R., Oliveira J.T.A. Proline accumulation and glutamine synthetase activity are increased by salt-induced proteolysis in cashew leaves. J. Plant Physiol. 2003;160:115–123. doi: 10.1078/0176-1617-00890. [DOI] [PubMed] [Google Scholar]
  • 68.Rai S., Luthra R., Kumar S. Salt-tolerant mutants in glycophytic salinity response (GSR) genes in Catharanthus roseus. Theor. Appl. Genet. 2003;106:221–230. doi: 10.1007/s00122-002-1026-x. [DOI] [PubMed] [Google Scholar]
  • 69.Han H.S., Lee K.D. Physiological responses of soybean-inoculation of Bradyrhizobium japonicum with PGPR in saline soil conditions. Res. J. Agric. Biol. Sci. 2005;1:216–221. [Google Scholar]
  • 70.Khan M.H., Panda S.K. Alterations in root lipid peroxidation and antioxidative responses in two rice cultivars under NaCl-salinity stress. Acta Physiol. Plant. 2008;30:81. doi: 10.1007/s11738-007-0093-7. [DOI] [Google Scholar]
  • 71.Samaddar S., Chatterjee P., Choudhury A.R., Ahmed S., Sa T. Interactions between Pseudomonas spp. and their role in improving the red pepper plant growth under salinity stress. Microbiol. Res. 2019;219:66–73. doi: 10.1016/j.micres.2018.11.005. [DOI] [PubMed] [Google Scholar]
  • 72.Abbas T., Pervez M.A., Ayyub C.M., Ahmad R. Assessment of morphological, antioxidant, biochemical and ionic responses of salttolerant and salt-sensitive okra (Abelmoschus esculentus) under saline regime. Pakistan J. Life Soc. Sci. 2013;11:147–153. [Google Scholar]
  • 73.Abdelgawad H., Zinta G., Hegab M.M., Pandey R. High Salinity Induces Different Oxidative Stress and Antioxidant Responses in Maize Seedlings Organs. Front. Plant Sci. 2016;7:276. doi: 10.3389/fpls.2016.00276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Islam F., Yasmeen T., Arif M.S., Ali S., Ali B., Hameed S., Zhou W. Plant growth promoting bacteria confer salt tolerance in Vigna radiata by up-regulating antioxidant defense and biological soil fertility. Plant Growth Regul. 2016;80:23–36. doi: 10.1007/s10725-015-0142-y. [DOI] [Google Scholar]
  • 75.Etesami H., Mirsyed Hosseini H., Alikhani H.A. In planta selection of plant growth promoting endophytic bacteria for rice (Oryza sativa L.) J. Soil Sci. Plant Nutr. 2014;14:491–503. doi: 10.4067/S0718-95162014005000039. [DOI] [Google Scholar]
  • 76.Jaiswal D.K., Verma J.P., Prakash S., Meena V.S., Meena R.S. Potassium Solubilizing Microorganisms for Sustainable Agriculture. Springer; New York, NY, USA: 2016. Potassium as an important plant nutrient in sustainable agriculture: A state of the art; pp. 21–29. [Google Scholar]
  • 77.Abd-el-Malek Y., Ishac Y.Z. Evaluation of methods used in counting azotobacters. J. Appl. Bacteriol. 1968;31:267–275. doi: 10.1111/j.1365-2672.1968.tb00367.x. [DOI] [PubMed] [Google Scholar]
  • 78.Berg G., Roskot N., Steidle A., Eberl L., Zock A., Smalla K. Plant-dependent genotypic and phenotypic diversity of antagonistic rhizobacteria isolated from different Verticillium host plants. Appl. Environ. Microbiol. 2002;68:3328–3338. doi: 10.1128/AEM.68.7.3328-3338.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 79.King E.O., Ward M.K., Raney D.E. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 1954;44:301–307. [PubMed] [Google Scholar]
  • 80.Samasegaran P., Hoben H., Halliday J. The NIFTAL (Nitrogen Fixation in Tropical Agricultural Legumes) Manual for Methods in Legume Rhizobium Technology. US Agency for International Development; Washington, DC, USA: 1982. [Google Scholar]
  • 81.Allen O.N. Experiments in Soil Bacteriology/Soil Bacteriology. Burgess; Minneapolis, MN, USA: 1959. [Google Scholar]
  • 82.Pepper I.L., Gerba C.P., Brendecke J.W. Environmental Microbiology: A Laboratory Manual. Academic Press; Cambridge, MA, USA: 1995. [Google Scholar]
  • 83.Vernon L.P., Seely G.R. The Chlorophylls. Academic Press; New York, NY, USA: 1966. [Google Scholar]
  • 84.Lichtenthaler H.K. [34] Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes. Methods Enzymol. 1987;148:350–382. [Google Scholar]
  • 85.Umbreit W.W., Burris R.H., Stauffer J.F. Manometric Techniques: A Manual Describing Methods Applicable to the Study of Tissue Metabolism. Burgess Publishing Co.; Minneapolis, MN, USA: 1957. [Google Scholar]
  • 86.Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275. [PubMed] [Google Scholar]
  • 87.Bates L.S., Waldren R.P., Teare I.D. Rapid determination of free proline for water-stress studies. Plant Soil. 1973;39:205–207. doi: 10.1007/BF00018060. [DOI] [Google Scholar]
  • 88.Zhang Z.L., Qu W.J. The guidance of plant physiology experiments. Chin. Agric. Sci. Technol. Press. Beijing. 2004:120–135. [Google Scholar]
  • 89.Mukherjee S.P., Choudhuri M.A. Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol. Plant. 1983;58:166–170. doi: 10.1111/j.1399-3054.1983.tb04162.x. [DOI] [Google Scholar]
  • 90.Marklund S., Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem. 1974;47:469–474. doi: 10.1111/j.1432-1033.1974.tb03714.x. [DOI] [PubMed] [Google Scholar]
  • 91.Chen G.-X., Asada K. Inactivation of ascorbate peroxidase by thiols requires hydrogen peroxide. Plant Cell Physiol. 1992;33:117–123. [Google Scholar]
  • 92.Bergmeyer H.U. Methods of Enzymatic Analysis. Verlag Chemie; Hoboken, NJ, USA: 1974. [Google Scholar]
  • 93.Williams V., Twine S. Flame photometric method for sodium, potassium and calcium. Mod. Methods Plant Anal. 1960;5:3–5. [Google Scholar]
  • 94.Pregl F. Quantitative Organic Microanalysis. JA Churchill Ltd.; London, UK: 1945. [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No new data were created or analyzed in this study. Data sharing is not applicable to this article.

Articles from Plants are provided here courtesy of Multidisciplinary Digital Publishing Institute (MDPI)

RESOURCES