1. Introduction

Salinity is considered the scourge of intensive agriculture [1]. The costs, associated with soil salinity are potentially enormous, and the effects of salinity may impact heavily on agriculture, biodiversity and the environment [2]. Salinity is one of the major factors that reduce crop productivity worldwide. High amounts of salts in soil reduced the seed germination [3, 4] and plant growth [5].

Resulting from high concentrations of sodium ions in the soil, Seed germination is decreased under saline conditions either by creating osmotic potential or by toxic effects [6]. These concentrations of soluble salts through their high osmotic pressures affect plant growth by restricting the uptake of water and balance absorption of essential nutritional ions by the roots thus, reduce the rate of germination and may retard plant development [7, 8].

Nearly 40% of world’s surface, suffer from salinity problems [9]. In Egypt, two to three m3 of saline drainage wastewater are annually used for irrigating about 405.000 hectares of land [10] and the majority of salt-affected soils are located in the Northern-Center part of the Nile Delta. Soil in Delta is classified according to alluvial that varies in texture from light (30% clay content) to heavy (80% clay content) [11]. Kafr El-Sheikh governorate was chosen as the study area because of its terrain and climatic conditions.

Increase in salinity tolerance for the world’s two major crops, wheat, and rice is an important goal as the world’s population is increasing more rapidly than the area of agricultural land [12]. Wheat is considered a major staple food crop for more than one-third of the world population and it is the main staple food of Asia [13], originated in South Western Asia and has been a major agricultural commodity since prehistoric times. Total production area in Pakistan is 8.2 mha and the average yield is 2170 kg/hectare [14]. The average production of wheat (Tritium aestival) in Egypt 2.745 tons/feddan, Pakistan 2660 Kg ha^-1, China 4710 Kg ha^-1 and India (2910 Kg ha^-1) [15].

There are biotechnological approaches, used to improve crop productivity in saline soil [16, 17]. Salt-tolerant plant growth promotes Rhizobacteria (ST-PGPR) as Azotobacteria helps to improve soil fertility through decomposition of organic matter and nutrient cycling, by fixation of atmospheric nitrogen or production of growth hormones [18-21]. Growth of wheat plants performs better under saline environment as inoculated with different rhizobia strains due to the production of ethylene under stressed conditions; reduction in sodium uptake by the utilization of different rhizobia strains under saline environment is a positive sign to mitigate salt stress biologically [22]. Azotobacter is a useful organism in many ways as it can fix atmospheric nitrogen, producing plant growth hormones like gibberellins, auxins and cytokinins and can solubilize phosphate which reduces the use of fertilizers [23, 24]. Azotobacter can increase seed’s germinating ability by 20–30% because of the production of the plant growth-promoting compounds [25]. Inoculation by A.chroococcum substantially reduced the effect of salt stress on plant growth parameters such as root length, plant height, fresh shoot, root weight, dry shoot and root weight [26]. The most appropriate solution in salinization problem is to use salt-tolerant useful bacteria in developing strategies to facilitate plant growth in saline soils. Therefore, this study aimed at improving wheat yield and solving the problem of low growth, low productivity for crop plants, caused by soil salinity by using local salt tolerant bacterial inoculants with Osmoadaptive strategies to increase tolerance to high salt, capable of producing plant growth promoting substances to facilitate wheat growth in saline soils.

2. Material and Methods

2.1. Soil Sampling and Preparation

Soil samples were collected from different cultivated areas in Kafr El-Sheikh governorate, including the following towns, mentioned below:

Region (I): Desouk city, West Kafr El-Sheikh

Region (II): Sidi Salem town, North Kafr El-Sheikh

Region (III): University, South of Kafr El-Sheikh

Region (IV): Baltim and Hamul, North East of Kafr El-Sheikh

Soil samples were obtained after removal of 5 cm of surfaces and collected in clean plastic bags then, samples were air dried, ground in a mortar and sieved through 2 mm sieve. Water suspensions of soil were prepared and spread on the surface of solid nitrogen free media in Petri dishes [27].

2.2. Measuring Electrical Conductivity (EC) dS/m of Soils

Electrical conductivity (E.C dSm^-1) of soil extract for the collected samples was measured according to Richards [27].

2.3. Isolation and Purification of Azotobacter sp.

Soil suspensions were prepared to spread on the surface of solid nitrogen-free media Vancura and Macura [28] (Sucrose 30gm, K₂HPO₄ 0.16gm, NaCl 0.2gm, MgSO₄.7H₂O 0.2gm, CaCO₃ 2gm, Fe₂(SO₄)₃ 0.005gm, NaMoO₄.2H₂O 0.005gm, NaBO₃ 0.005gm, Dist.H₂O 1000 ml) in Petri dishes and incubated at 30°C with inverted position for 4-6 days, the colonies which appeared on the surface of plates were transferred to new plates containing fresh media for more purification. Several subcultures were carried out in order to purify the isolated bacteria [29].

2.4. Characterization and Identification of Azotobacter Isolates

Isolates were characterized morphologically, examined for Gram stain to identify Azotobacter sp. according to Bergey’s Manuals Systematic of bacteriology [30].

2.5. 16s Ribosomal RNA (rRNA) Identification for the Two Isolated Strain

Genomic DNA was extracted as described by Carozzi [31] and the 16S rRNA gene of the bacterial isolates was amplified by PCR using 16S rRNA primers. PCR was performed for 5 min by initial denaturation at 95°C, followed by 35 cycles for each denaturation process at 95°C for 30 sec, annealing at 65°C for 1 min, extending at 72°C for 1.30 min, and a final extension at 72°C for 10 min. The PCR product of 0.5 kb was extracted from the gel using Ferments gel extraction kit (Thermo), and the amplified products were electrophoresed on 1% agarose gel. Sequencing was carried out by the Genetic Analysis System model Gene JET™ PCR Coulter Inc., Fullerton, CA, USA. The 16S rRNA gene sequences were compared with others found by BLAST searching (Basic Local Alignment Search Tool) in the database of National Centre for Biotechnology Information. Then, phylogenetic analysis was performed using neighbourhood joining method [32] to identify strains selected under this study.

2.6. Osmoadaptive Strategies to Increase Salt Tolerance of Isolates

Fixed amount of selected Azotobacter isolates were inoculated to free nitrogen media Vancura and Macura supplemented with different concentration of NaCl range from (0.009, 5 and 20 g/100ml) and incubated at 30°C with inverted position for 4-6 days then, growth was measured in each culture by measuring Optical Density (OD) to raise salt tolerance, reaching the maximum available tolerance against salt concentration of the media.

2.7. Evaluation of Production of Indole-3Acetic Acid (IAA) by the Isolated Bacteria

One ml of inoculums were transferred to a flask containing 50 ml of (NFB) medium, supplemented with 0.3 g/L tryptophan and incubated at 28°C with agitation at 100 rpm in the dark. Clusters were centrifuged at 4000 rpm for 15 min and two millilitres of supernatants were mixed with one millilitre Pilet-Chollet reagent consisting of 12g FeCl₃/1 of 7.9 H₂SO₄ and the mixtures were lifted in the dark for 30 minutes at room temperature. The appearance of pink colour indicates that IAA was produced. The absorption spectra of mixtures were determined at 530 nm [33] for isolates previously exposed to different salt concentrations compared with non-salted one and IAA production was determined from the standard curve.

2.8. Studying the Effect of the Most Tolerated Bacteria on the Cultivation of the Wheat Plants

This study was based on Split Plot Design [34, 35] with two different factors in both field and pots experiments to investigate the changes in plant growth criteria under the effect of osmoadaptive bacteria and different concentration of N-fertilizer. The most tolerated bacteria were inoculated to wheat plant through soil during seeding stage to study the effect of bacteria on crop production of wheat compared with uninoculated one grown in salinized soil in field and pots experiments. The experimental design was 3 lines of articulating squares each of which 1m², one line represented inoculated plant with A.chroococcum, the second one represented control (uninoculated plant) and the third represented inoculated plant with A.vinelandii with different replicates and different fertilizer concentrations. The field experiment shown in photos 4, 5, 6, 7, and 8. The same experimental design used for pots experiment shown in photos 13, 14, 15, 16, 17, and 18.

2.9. Determination of Plant Growth Criteria

Determination of plant growth criteria (stem high, 1m² weight, grains weight, straw weight and 100 grains weight) in the inoculated and uninoculated plants were done by collecting crops and measuring length and weight by sensitive balance and meter.

2.10. Statistical Analysis

Numerical variables are expressed by descriptive statistics as mean, standard deviation. Two ways" ANOVA and post hock test (tukey)" were used to compare quantitative data. Correlation analyses showed a relation between parameters. P-value <0.05(*) was considered significant difference and P-value <0.001(**) was considered highly significant difference. Statistical analyses were performed using Statistical Package for Social Sciences (SPSS version 23).

3. Results and Discussion

3.1. Soil Sampling

Eight Soil samples were collected from four regions in Kafr El-Sheikh governorate where Region (I) 3 samples, Region (II) 2 samples, Region (III) 1 sample, and Region (IV) 2 samples.

3.2. Electrical Conductivity (EC) dS/m of Soil Samples

Table (1). Electrical Conductivity of the collected soil samples

According to the standard classification of soil depending on salinity from AGRIS.FAO organization [36], soil is slightly consider as a saline from (2–4) dS/m and considered as a very strongly saline when EC reach more than (16 dS/m). Result showed that sample number 2 in region (I) and sample number 8 from region (IV) showed high electrical conductivity, so they are considered as a strong saline.

3.3. Isolation and Purification of Azotobacter sp.

Twenty isolates were collected from soil suspension of different cultivated regions of Kafr El-Sheikh governorate:

Table (2). Number of isolates

3.4. Characterization of Azotobacter Isolates

Table (3). Characterization of Azotobacter isolates

According to the results of electrical conductivity which determined salinity degree of soil samples and the morphological characterizations of the bacterial isolates, two different isolates from two high saline regions were selected to be under this experiment:

Isolate (no. 17) from region (I) and soil sample (no. 2) was labelled Isolate (A)

Isolate (no. 9) from region (IV) and soil sample (no. 8) was labelled Isolate (B)

3.5. 16s rRNA Identification

16s ribosomal RNA (rRNA) sequence for the two isolated strains in the present study were used for identification of strain level. Phylogenetic analysis using neighbourhood joining method shown in figure 1 and figure 2.

Table (4). 16s rRNA Sequence of isolates

Figure 1. Phylogenetic tree constructed from the 16S rRNA gene sequence with 99% similaritie percentage to Azotobacter chroococcum

Figure 2. Phylogenetic tree constructed from the 16S rRNA gene sequence with 99% similaritie percentage to Azotobacter vinelandii

Results showed that the two species selected under this study were:

Isolate (no. 17) from region (I) and soil sample (no.2), Isolate (A) was Azotobacter chroococcum.

Isolate (no. 9) from region (IV) and soil sample (no.8), Isolate (B) was Azotobacter vinelandii.

3.6. Osmoadaptive Strategies to Increase Salt Tolerance of Isolates

Increasing the ability of the selected species to tolerate salt was achieved by exposing isolates to gradually increase in salt concentrations in the media and the result shown in Table (5). R is correlation coefficient in the statistical results for Table (5), when r is (-) it is mean there is negative relation between two groups (one increase while the other decrease), when r is (+) it is mean there is positive relation between two groups (the 2 groups increase together or decrease together). If r <0.5 there is a weak relation, r>0.5 there is a strong relation.

Table (5). Effect of different NaCl concentrations on Azotobacter growth

Results as illustrated in Table (5) showed that there was a negative strong relation between salt concentrations of media & A.chroococcum or A.vinelandii growth: when salt concentration increase, the growth of A.chroococcum and A.vinelandii decrease. Increase of NaCl concentraion in the media reduce the bacterial growth, where there was a gradual decrease in the bacterial growth, accompanied with as an increase of NaCl concentration until the maximum concentration (5g/100ml) of salt gets peak, after which the growth began to decrease sharply, indicating that osmoadaptive bacteria reached the maximum available tolerance against salt concentration of the media.

3.7. Evaluation of Production Indole-3Acetic Acid (IAA) by Isolates

Referring to IAA standard curve (Figure 3), result in table (6) showed that osmoadaptive species of bacteria previously exposed to different salt concentration produced more Indole-3Acetic Acid (IAA) than controlled one which were not exposed to salt previously in both species A.chroococcum and A.vinelandii which indicate that osmoadaptive isolates produce more auxins and can be used as salt-tolerant plant growth promoting rhizobacteria (ST-PGPR).

Many authors reported that IAA has a positive effect on plants and play a major role in the growth. The IAA producing bacteria significantly raaised seedling root growth up to 25% in non-salinated conditions and up to 52% at 100 mM NaCl, compared with control plants. It is concluded that growth regulators considerably alleviated salinity-induced dormancy of wheat seeds. Root colonizing bacteria produce phytohormone to alleviate salt stress of wheat grown under conditions of soil salinity [37].

Table (6). Effect of osmoadaptation (exposure to salt) of bacteria on Indole-3Acetic Acid (IAA) producing ability

Figure 3. IAA Standard Curve

It also reported that pre-sowing wheat seeds with plant growth regulators like IAA, gibberellins alleviated the growth inhibiting effect of salt stress [38-41]. Worth mentioning, it was observed that the plant growth stimulating substances can alleviate the effect of salinity on germination [42-45]. It is also possible that under high salt concentrations, natural present hormones may be suppressed [46, 41]. Salinity results in a progressive decline in the level of IAA in the root system of plants [47]. In this condition, seed soaking with plant growth regulators and an application of additional natural phytohormones supplied sufficient hormones for normal plant development and a growth in saline conditions [46, 41]. Inoculation of the soil with osmoadaptive isolates selected under this study during seedling stage of wheat plant raised the production of IAA and reduced the salt stress.

3.8. Effect of Using Tolerated Bacteria on the Wheat Plant Productivity

It was found that Azotobacter could increase seed’s germinating ability and reduced the effect of salt stress on plant growth parameters such as root length, plant height, fresh shoot and root weight and dry shoot and root weight [25, 26]. In this study, both species Azotobacter chroococcum or Azotobacter vinelandii were inoculated to wheat plant, growth criteria were determined as showen in Figures (9, 10, 11, and 12) for field experiments and Tables (7, 8, 9, and 10) for pots experiments as:

Figure 4-8

Figure 4, 5, 6, 7, and 8 represent stages of wheat planting in field experiments.

There were 3 lines of articulating squares represented A.chroococcum, control, and A.vinelandii with different replicates for different fertilizer concentrations and were based on Split Plot Design.

3.9. Results of Field Experiment

Figure 9. Effect of A.chroococcum and A.vinelandii on Stem length

Figure 10. Effect of A.chroococcum and A.vinelandii on Grains weight

Figure 11. Effect of A.chroococcum and A.vinelandii on Straw weight

Figure 12. Effect of A.chroococcum and A.vinelandii on 100 grains weight

The result as illustrated in Figure (9) showed that there was a significant increase in stem length of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated (control) especially with using A.chroococcum. On other hand, the increase of N-fertilizer in combination with osmoadaptive species led to increase of stem length especially with A.vinelandii.

In recent study, it was reported that inoculation of crop plants with certain strains of PGPR at an early stage of development improves biomass production through direct effects on root and shoot growth [48]. Soil inoculation with PGPB had a positive impact on plant growth in combination with the organic fertilizer that was added [49]. Osmoadaptive strains selected in this study produced phytohormones as IAA which influenced the growth and length of the shoot of wheat to tolerate the effect of salt stress so, inoculated wheat with Azotobacter species give better results in shoot length than inoculated one.

Inoculation of crop plants with certain strains of PGPR at an early stage of development improves biomass production [48]. There was a significant increase in grains weight of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated one (control) especially with using A.vinelandii. On other hand, the increase of N-fertilizer with using these osmoadaptive species led to increase in weight of wheat. Similar enhancement reported by inoculation of PGPB during wheat cultivation [56].

Results as illustrated in Figure (11) showed that there was a significant increase in straw weight and biomass of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated (control) on both 2 species where results were better with species A.vinelandii, on the other hand, the increase of N-fertilizer with using these osmoadaptive species led to increase of straw weight of wheat.

The results revealed a significant increase in 100 grains weight of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated (control) on both 2 species espicially with species A.vinelandii.

It was reported that the use of PGPB could be a frontier goal to achieve a positive effect on plants and reduce the negative impact of chemical and fertilizers on the environment [50]. Plant had a higher grain yield with the application of liquid phosphor-bacteria followed by Azotobacter [51]. Azotobacter plays different beneficial roles by producing different types of secondary metabolites in the soil such as vitamins, amino acids, plant growth hormones, antifungal substances, hydrogen cyanide and siderophores. These secondary metabolites have direct influence on growth of shoot, root and seed germination of many agriculture crops [52]. The production of auxins as IAA by selected isolates of Azotobacter stimulate plant growth and improve its productivity under saline soil.

3.10. Results of Pots Experiment

Figure 13-18

Figures 13, 14, 15, 16, 17, and 18 represent stages of wheat planting in pots experiments.

There were 3 lines of articulating squares represented A.chroococcum, control, and A.vinelandii with different replicates for different fertilizer concentrations and were based on Split Plot Design.

Table (7). Effect of Azotobacter species on Stem length

Table (8). Effect of Azotobacter species on Grains weight

Table (9). Effect of Azotobacter species on Straw weight

Table (10). Effect of Azotobacter species on 100 grains weight

Results in Table (7) showed that there was a significant increase in stem length of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated one (control) especially with A.vinelandii. On other hand, increase of N-fertilizer with using these osmoadaptive species led to increase of stem length which indicated that, inoculation with Azotobacter spp. give better results on wheat cultivation under saline soil.

Results as illustrated in Table (8, 9, and 10) indicated that, there was a significant increase in grains weight, straw weight, and 100 grains weight of wheat grown in salinized soil inoculated with Azotobacter spp. than uninoculated one (control) on both A.chroococcum and A. vinelandii. On other hand, increase of N-fertilizer with using these osmoadaptive species led to increase of grains weight, straw weight, and 100 grains weight of wheat which indicated that inoculation with Azotobacter spp. give better results on wheat cultivation under saline soil.

Plant growth promoting bacteria PGPB are the most studied phytohormone producers [53] because the use of PGPR is an efficient and cheaper method that induces salt stress tolerance in plants [54]. The application of indole acetic acid synthesizing plant growth-promoting bacteria may represent an important alternative approach to decrease the impact of salt stress on crops [55]. PGPB have a favourable effect on plant growth, tolerance against stresses. Moreover, they are considered as a promising alternative to inorganic fertilizer for promoting plant growth, yield and quality. PGPR colonize at the plant root, raised germination rates, promote root growth, yield, leaf area, chlorophyll content, nitrogen content, protein content, tolerance to drought, shoot and root weight, and delayed leaf senescence [56].

Osmoadaptive bacteria selected under this study were exposed to gradually increasing salt concentrations in order to increase its availability of salt tolerance, when inoculated with wheat plant during seedling stage, it showed the ability to produce more growth regulating hormones as IAA than non-adaptive bacteria, led to enhance the wheat productivity in biomass and shoot length. Results of field and pots experiments showed that using of osmoadaptive salt tolerant bacteria (A.chroococcum or A.vinelandii) gave better results in growth criteria in inoculated wheat plant than uninoculated plant, some criteria were better with using A.chroococcum and others with using A.vinelandii. On other hand, increase of N-fertilizer gave better results with both species, such biofertilizers can be used as a proper tool for increasing wheat yield under salinity condition [57].

4. Conclusions

The obtained results demonstrated that, crop productivity of wheat grown in salinized soil was improved by using osmoadaptive salt tolerant Azotobacter spp. There was a significant increase in plant growth criteria (stem high, grains weight, straw weight and 100 grains weight) in the inoculated plants especially with osmoadaptive bacteria (A.chroococcum or A.vinelandii) during seeding stage, the improvement ability was correlated to production of plant growth regulators as auxins (IAA) by bacteria which indicated that applying of salt-tolerant Azotobacter spp. was beneficial and effective in improving the productivity of wheat under salinity stress conditions and suggested the ability of using of osmoadaptive bacterial species during cultivation of wheat in salinized soils.