Impact of Potassium-Solubilizing Microorganisms with Potassium Sources on the Growth, Physiology, and Productivity of Wheat Crop under Salt-Affected Soil Conditions

Abstract

Salinity adversely affects plant growth and productivity worldwide. To overcome salinity and other abiotic stresses, safe, ecofriendly biofertilizers that stimulate productivity have been experiencing rising demand, alongside decreasing use of mineral fertilizers. The purpose of this study was to examine changes in the growth, yield, physiological and biochemical parameters of wheat crop as a response to two potassium-solubilizing microorganisms (KSMs), Trichoderma asperellum and Bacillus circulans, with 50% or 75% of the recommended amount of K fertilizer (potassium sulphate), combined with no or 25% feldspar as well as 1.5% potassium sulphate (K-leaf) as foliar application, and all treatments were compared with a control treatment under salt-affected soil conditions, during two winter seasons in 2019–2020 and 2020–2021. The randomized complete block design (RCBD) was used to set up the experiment. Results showed that the vegetative growth, yield, physiological and biochemical parameters were affected under salt-affected soil conditions. Nevertheless, inoculation with T. asperellum and B. circulans with potassium application mitigated the deleterious effect of salt-affected soil conditions by improving growth parameters, photosynthetic pigment content, antioxidant enzymes (superoxide dismutase, ascorbate peroxidase, catalase and peroxidase) content, total soluble sugars, relative water content, potassium content in leaves, grains yield, and some biochemical constituents in the grains and straw. Meanwhile, these treatments decreased proline content, Na⁺ content in leaves, and the Na⁺/K⁺ ratio as compared to the control treatment. The most pronounced treatment, inoculation by B. circulans with 1.5% K-leaf as foliar application, significantly increased grain yield by 16.41% relative to the control treatment. It could be concluded that inoculating wheat with KSMs T. asperellum and B. circulans with 1.5% K-leaf as foliar application will increase wheat salinity tolerance and wheat productivity and decrease the detrimental effect of salinity on wheat growth and grain yield quantity and quality under salt-affected soils conditions.

1. Introduction

Salinity is a major abiotic stressor that restricts plant development and productivity in the world’s arid and semiarid regions and is caused by irrigation with poor-quality water and soil salinization [1]. Depending on the intensity and duration of the stress, salinity stress has a negative effect on several physiological and metabolic plant processes and eventually reduces agricultural production [2]. Salinity adversely affects plant growth and yield through lower osmotic water availability, ion toxicity, nutritional imbalance, enzyme reduction, lower photosynthetic efficiency, and other physiological problems. Under salt stress, membrane components and cell organelles are damaged by the increase in reactive oxygen species (ROS) production as a result of osmotic stresses, which eventually lead to plant death [3].

Potassium (K) is one of the most vital nutrients for plants. It is crucial for protein and starch synthesis, water and nutrient transport under salinity stress, and the activation of enzymes, osmoregulation, and stomatal closure and opening [4]. Potassium is one of the nutrients that is most significantly reduced during salt stress due to ion toxicity and the osmotic effect, which inhibits plant root growth [5]. Potassium elements represent 2.6% of the weight of the Earth’s crust. Only around 2% of K is in its soluble form, while the remaining 98% is found in insoluble forms such as minerals (biotite, feldspar, mica, muscovite, and vermiculite), which are unavailable for plant uptake. Soil K can be divided into four categories according to its accessibility to crops [6,7]: (a) The main source of K absorbed by plant roots is soil solution K; its concentration depends on soil weathering, previous cropping, and K fertilization techniques. (b) Exchangeable K is held on soil clay and organic matter by their negative charges exchange sites and ranges from 40 to 600 mg kg⁻¹. (c) Non-exchangeable K is retained as fixed ions in clay mineral lattices and that which is incorporated into mineral structures. (d) Mineral K is present in soils containing K-bearing minerals and is mostly dependent on the parent material’s source.

Soil microorganisms affect soil fertility by influencing several soil processes via many mechanisms such as decomposition, immobilization, and mineralization. A crucial role in the natural K cycle is played by soil microorganisms, especially potassium-solubilizing microorganisms (KSMs). KSMs can solubilize K-bearing minerals, micas, feldspar, muscovite, illite, orthoclase, and biotite and convert the K into a soluble form [8]. Utilizing KSMs as a biofertilizer can boost crop productivity and the availability of plant nutrients. KSMs include Aspergillus spp., Flavobacterium spp., B. pumilus, B. mucilaginosus, Rhizobium spp., B. edaphicus, Agrobacterium tumefaciens, B. circulans, and B. subtilis [9], as well as plant-growth-promoting fungi (Trichoderma), which are environmentally friendly, induce plant resistance to abiotic stresses, and support agricultural development. Trichoderma plays an important role in salinity alleviation [10].

Wheat (Triticum aestivum L.) is considered an important cereal crop with regard to daily human food intake, containing very important minerals, vitamins, amino acids, and proteins [11,12,13,14]. Under salt-affected soil conditions, wheat plants have shown many physiological and biochemical changes in their metabolism, which had an impact on both the qualitative and quantitative yield parameters [15].

The aim of this study was to improve wheat salt tolerance, growth, and productivity and to reduce the consumption of mineral fertilizers by using eco-friendly biofertilizers. This was accomplished by investigating the effect of applying Trichoderma asperellum OR234761 and B. circulans strains as KSMs in combination with different sources and doses of K on wheat under salt-affected soil conditions and evaluating the various physiological and biochemical parameters of wheat.

2. Materials and Methods

Both microorganisms, namely T. asperellum OR234761 and B. circulans, were provided by Biofertilizer Production Unit, Agric. Microbiol. Department, Soil, Water and Environmental Research Institute, Agriculture Research Center, Giza, Egypt. Individually, T. asperellum was cultured on potato dextrose agar and B. circulans on nutrient broth [16]. The cultures were incubated at 28 °C on a rotary shaker for 5 days.

2.1. Investigating Trichoderma asperellum and Bacillus circulans as Plant-Growth-Promoting Microorganisms (PGPM)

2.1.1. Phosphate Solubilization by the Two Microbial Strains

The solubilization of phosphate by the two microbial strains was quantitatively estimated on 50 mL of modified Pikovskaya PVK broth (2.5 g insoluble tricalcium phosphate, 13 g glucose, 0.2 g NaCl, 0.1 g MgSO₄.7H₂O, 0.5 g (NH₄)SO₄, 0.2 g KCl, 0.5 g yeast extract, FeSO₄.7H₂O trace, MnSO₄ trace, 20 g agar per one liter of distilled water, adjusted to pH 7.2), which was inoculated with 500 μL of the microbial cultures. At 28 °C and 180 rpm, the flasks were incubated in an incubator shaker. The sterilized media was used as a control. After 6 days, 10 mL of each broth culture was centrifuged at 8000× g rpm for 15 min, and P content was determined according to Watanabe and Olsen [17]. The P values were calculated as μg mL⁻¹ over control. Solubilization was quantitatively measured according to Pikovskaya [18].

2.1.2. Potassium Solubilization by the Two Microbial Strains

The K solubilization conducted by two microbial strains was quantitatively estimated using modified Aleksandrov medium (5.0 g glucose; 2.0 g Ca₃(PO₄)₂; 0.5 g MgSO₄.7H₂O; 0.006 g FeCl₃; 0.1 g CaCO₃; 3.0 g potassium aluminum silicate; and 20.0 g agar in 1 L of deionized sterile water). To adjust the pH to 7.2, 1 N NaOH was used, and the mixture was then incubated at 28 ± 2 °C for 3–4 days [16]. The supernatant from centrifuging the culture broth was utilized to quantify the K solubilization using flame photometry. The standard curve for K quantification was made by preparing several KCl solution concentrations [19]. Mica is typically utilized in this assay as a supply of the insoluble form of K.

2.1.3. Indole Acetic Acid Production by the Two Microbial Strains

Salkowski reagent was used to quantitatively estimate the production of Indole-3-acetic acid (IAA) in nutrient broth media supplemented with DL-tryptophan (0.1%) [20]. Broth medium was inoculated with 100 μL of 24 h old microbial cultures and then incubated for 48 h at 28 °C in the dark. After two days for the bacteria and five days for the fungi, the cultures were centrifugated at 10,000× g rpm for 20 min, and 1 mL of culture supernatant was mixed with 4 mL of Salkowski reagent and left for 10 min in the dark before the intensity of the developed pink color was measured at 535 nm spectrophotometrically (Jenway67-series spectrophotometer). The IAA values were calculated as μg mL⁻¹ based on the standard curve.

2.1.4. Siderophore Production by the Two Microbial Strains

The siderophore production for the two microbial strains was estimated quantitatively on Chrome Azurol sulphonate (CAS) broth [21]. The CAS broth media were inoculated with the microbial cultures and incubated at 28 °C for five days. Optical density was measured at 630 nm, and siderophore content was calculated by the method of Sayyed et al. [22].

2.1.5. Ammonia Production by the Two Microbial Strains

The ammonia (NH₃) production of the two microbial strains was qualitatively detected by adding Nessler’s reagent to microbial strains, which were grown on peptone broth media for 24 h. Ammonia production was indicated by the color changing from yellow to orange or dark brown [23].

2.1.6. Hydrogen Cyanide (HCN) Production by the Two Microbial Strains

The microbial strains were spotted on nutrient agar media supplemented with 4% glycine. Filter paper discs were immersed in picric acid solution (0.5% picric acid in 2% sodium carbonate) and placed under the petri plate covers. The plates were incubated for three days for the bacteria and five days for the fungi at 28 °C. The color of the filter paper discs changing from orange to brown indicated the production of HCN [24].

2.2. Field Experiment

In South El Husainia Plain, El Sharkia Gov., Egypt (31°00′13″ N 32°08′14″ E), two field experiments were carried out during the 2019–2020 and 2020–2021 winter seasons to examine the physiological and yield parameters of wheat plants cultivated under salt-affected soil and its response to biofertilizer inoculations with both different K doses and sources. Therefore, growth parameters, soluble sugar content, photosynthetic pigment content, antioxidant enzymes activities, yield, yield components, Na⁺/K⁺ ratio, and proline (Pro) and protein content in wheat plant grains were investigated.

A randomized complete block design (RCBD) with four replicates for each treatment was used for the experiment. Each plot size was 12 m². The experiment was conducted in the same location during both seasons and watered using a flood irrigation approach. The physical and chemical characteristics of soil at the experimental location in the two seasons in 2019–2020 and 2020–2021 are displayed in

Table 1. The soil physical and chemical characteristics of the experimental location in both seasons (2019–2020 and 2020–2021) of study.

Season	EC (dS m−1) in (1:5) Extract	pH (1:2.5)	Texture	O.M %	Cations (mL Equivalent L−1)				Anions (mL Equivalent L−1)			CEC (cmol kg−1) *
					Ca2+	Mg2+	Na+	K+	HCO3−	Cl	Ca2+
2019–2020	6.8	8.20	Clay	1.15	10.0	17.9	57.98	1.54	1.68	63.95	21.95	38.9
2020–2021	6.5	8.20	Clay	1.17	10.0	17.7	54.81	1.55	1.68	60.74	21.76	38.6

* CEC: Cation exchange capacity of soil layer 0–20 cm.

.

Wheat grains cv. Masr 1 were planted on 15th and 18th November in the 2019–2020 and 2020–2021 seasons, respectively. Air temperature in the farm varied from 9.9 to 24.8 °C in the 1st season and from 10.5 to 24.6 °C in the 2nd season. The perennial average precipitation was 73 and 55 mm in the two winter seasons, respectively. For soil preparation, 35 kg P₂O₅ ha⁻¹ in the form of calcium super phosphate (15.5% P₂O₅) was applied. Prior to irrigation, 3 recommended dosages of 215 kg nitrogen (N) ha⁻¹ of urea (46% N) were applied.

Biofertilizers and K treatments were as follows: inoculation by T. asperellum OR234761 and B. circulans, combined with different doses and sources of K (50% recommended K (potassium sulphate, K₂SO₄); 50% K plus 25% recommended feldspar (F); 75% K; 75% K plus 25% F and 1.5% K-leaf (commercial potassium contains 50% K₂O) foliar spray) for each biofertilizer and the control, 100% recommended potassium (115 Kg K ha⁻¹) as K₂SO₄. The wheat grains were coated with T. asperellum and B. circulans (10⁹ cells ml⁻¹) before sowing; after that, it was added to soil at rate of 12 L ha⁻¹ at 15, 25, 40, and 55 days after sowing (DAS) according to the different treatments, while K as K₂SO₄ and feldspar (potash feldspar constituent in

Table 2. Percentage of potash feldspar constituent.

Feldspar Constitutes	Total SiO2	Al2O3	Fe2O3	Na2O	K2O	MgO	TiO2	CaO
%	67.00	18.00	0.68	2.10	11.00	Nil	Nill	0.15

) were added to the soil. Foliar spray of 1.5% K-leaf was applied two times at 35 and 50 DAS, as these were suitable times for foliar spray.

According to the Egyptian Field Crops Research Institute’s recommendations for cultivating wheat in the area, all cultural practices were followed.

2.2.1. Growth Parameter Measurements

Growth parameters were assessed as follows; six plants were randomly sampled from each plot at 65, 80, and 95 DAS (the highest vegetative growth period), and at the same time, additional plant samples were collected and oven dried at 70 °C to a consistent weight. Leaf area index (LAI), crop growth rate (CGR), and net assimilation rate (NAR) were estimated in accordance with Hunt formulas [25]:

LAI = leaf area of plant/land area occupied by plant.

CGR (g m⁻² day⁻¹) = (W₂ − W₁)/(t₂ − t₁).

NAR (g m⁻² day⁻¹) = (W₂ − W₁)(log_e A₂ − log_e A₁)/(A₂ − A₁)(t₂ − t₁).

where:

A₂ − A₁ = the difference in leaf area between two taken samples;

W₂ − W₁ = the difference between two samples in accumulated dry weight (g) of whole plants;

t₂ − t₁ = the number of days between two consecutive samples (day);

Log_e = natural logarithm.

2.2.2. Photosynthetic Pigment Content

The content of photosynthetic pigments (carotenoids, chlorophyll a, and chlorophyll b) were measured at 80 DAS in accordance with Lichtenthaler and Buschmann [26].

2.2.3. Assessment of Antioxidant Enzymatic Activities

To prepare the enzyme extract, 0.2 g of fresh leaf samples were ground and homogenized with 5 mL of 100 mM potassium–phosphate (K–P) buffer (pH 7.0), which contained 0.1 mM disodium-EDTA and 0.1 g polyvinylpyrrolidone, in a chilled mortar using liquid nitrogen. The samples were filtered and centrifuged for 10 min at 14,500× g and 4 °C. The resulting supernatants were used for measuring antioxidant enzyme activity.

Superoxide dismutase (SOD) activity was measured in accordance with Scebba et al. [27]. For this method, three ml of a solution containing 50 mM K–P buffer (pH 7.8), 13 mM L-methionine, 75 µM nitro blue tetrazolium, 0.1 mM EDTA, and 2 µM riboflavin was combined with 2 ml of leaf extract. The mixture was exposed to cool white, fluorescent light for 15 min and the reaction occurred. The blue color that resulted was measured spectrophotometrically at 560 nm.

Catalase (CAT) activity was measured in accordance with Aebi [28]. For assaying CAT activity, 3 mL of the reaction mixture was used. The mixture consists of leaf extract, 50 mM phosphate-P buffer (pH 7.0), and 30% H₂O₂ (w/v). The changes in the H₂O₂ absorbance were evaluated at 240 nm to assess the CAT activity.

The enzymatic activity of peroxidase (POD) was measured by utilizing guaiacol in accordance with Maehly and Chance [29]. A measure of 0.5 mL of leaf extract was added to 3 mL of the reaction solution that contained 10 mM H₂O₂, 10 mM K–P buffer (pH 7.0), and 20 mM guaiacol. Tetraguaiacol was created as a result of the reaction, causing an increase in absorbance, which was measured at 470 nm.

Ascorbate peroxidase (APX) activity was assessed following Chen and Asada’s method. Measures of 3 ml of the reaction mixtures containing leaf extract, 50 mM K–P buffer (pH 7.0), 0.5 mM ascorbic acid, and 0.5 mM H₂O₂ were used [30]. The APX activity was evaluated by measuring the decrease in absorbance at 290 nm that resulted from ascorbate oxidation.

2.2.4. Soluble Sugar Evaluation

The leaves’ soluble sugars content was assessed in accordance with Yemm and Willis’s modified method [31]. To extract soluble sugars, dry plant leaf samples were dipped in 10 mL of 80% ethanol (v/v) at 25 °C for the entire night while being periodically shaken. Next, 0.1 mL of the extract was combined with 3.0 mL of anthrone reagent (150 mg anthrone + 100 mL 72% H₂SO₄ (v/v)), which had been just made as a reacting reagent, and heated in a water bath for 10 min. The soluble sugar contents of the leaf extracts were evaluated as a glucose equivalent.

2.2.5. Evaluation of Relative Water Content

Under both control and salt stress conditions, the relative water content (RWC) of each Masr 1 wheat cv. was assessed. At noon, the uppermost leaves were collected and used. The leaves were cut at the base of their blade, wrapped immediately in plastic bags, and stored in an icebox before being swiftly moved to the laboratory. Immediately upon removing the leaves from the plastic bags, the fresh weight (FW) of each treatment was measured. To measure the leaves’ turgid weight (TW), the leaves were soaked in distilled water for 24 h at room temperature (about 20 °C) and under dim light conditions in the lab, then rapidly and gently dried using paper tissues. The leaf samples were oven dried for 72 h at 70 °C to determine their dry weight (DW). RWC was evaluated according to Schonfeld et al. [32].

RWC (%) = ((FW − DW)/(TW − DW)) × 100

2.2.6. Determination of Proline

Leaf Pro content was measured in accordance with Bates et al. [33]. After homogenizing 0.25 g of fresh leaves in 2.5 mL of 3% sulfosalicylic acid, the mixture was centrifuged at 10,000× g for 10 min. For each treatment, 2.5 mL of sulfosalicylic acid and 1 mL each of acid ninhydrin solution and glacial acetic acid were added to 2.5 mL supernatant in the test tube and kept for 1 h in the incubator at 100 °C. The reaction was quickly stopped by rapidly adding 2 mL of toluene to the mixture and stirring it constantly for 15 to 20 s. The toluene-containing chromophore layer was separated, and the absorbance was evaluated at 528 nm.

2.2.7. Assessment of Na⁺ and K⁺ Ion Contents

In accordance with Allen et al. [34], the contents of Na⁺ and K⁺ ions were measured as mmole kg⁻¹ DW.

2.2.8. Assessment of Yield and Yield Attributes

The following parameters were measured at harvest time: plant height (cm), 1000-grain weight (g), spike weight (g), harvest index, spike length (cm), and grain and straw yields (t ha⁻¹).

2.2.9. Nitrogen, Phosphorus, Potassium, and Protein Percentages in Wheat

The percentages of N, P, and K in grains and straw were measured. As described by Motsara and Roy, the Micro-Kjeldahl method was employed to measure the total N content, while P was calorimetrically measured [35]. Following the Mac Lean method, K was measured using a flame photometer [36], and K content was multiplied by yield to estimate K uptake. The total N readings were multiplied by 6.25 to estimate the protein content.

2.2.10. Potassium Content in Soil

Soil K analysis was determined by flame photometry as follows: water-soluble K was determined in water extract (1:2), while exchangeable K was assessed using (NH₄)OAC extract (1:2.5) [37] and the total K was estimated using the method of Jackson [38].

2.3. Statistical Analysis

As per Steel and Torrie, statistical analysis of variance was conducted on the data obtained from the two seasons [39]. Fisher’s least significant difference (LSD) test was used to compare the treatment averages at the 0.05 level of significance.

3. Results

3.1. Trichoderma asperellum and Bacillus circulans as PGPMs

According to data obtained from IAA production, the highest amount of IAA was produced by T. asperellum, followed by B. circulans (

Table 3. Screening for T. asperellum and B. circulans as PGPMs.

Microbial Strains	Phosphate Solubilization (μg mL−1)	Potassium Solubilization (μg mL−1)	IAA Production (μg mL−1)	Ammonia Production *	Siderophore Production %	HCN Production
T. asperellum OR234761	107.13	3.58	109.11	+	45.72	++
B. circulanse	123.15	5.11	79.03	++	11.42	+++

* + moderate producer, ++ high producer, and +++ very high producer.

). The produced amounts were 109.11 and 79.03 µg ml⁻¹, respectively. The two microorganism strains that were tested demonstrated the ability to solubilize tricalcium phosphate, which was supplemented to Pikovskaya broth medium, and solubilize insoluble K (mica) to soluble form on Aleksandrov medium. Obtained results revealed that B. circulans showed strong phosphate and K solubilization, with the amounts of available P measured at 123.15 and 5.11μg ml⁻¹, followed by T. asperellum at 107.13 and 3.85 μg ml⁻¹. T. asperellum exhibited superiority in siderophore production in comparison with B. circulans, recording 45.72 and 11.42%, respectively. B. circulans exhibited the highest activity in hydrogen cyanid (HCN) and NH₃ production in comparison with T. asperellum.

3.2. Field Experiment Results

3.2.1. Growth Parameters

The plant height, crop growth rate (CGR), and net assimilation rate (NAR) of wheat grown under salt-affected soil conditions were decreased in both seasons (

Table 4. Growth parameters (plant height, CGR, and NAR) of wheat affected by T. asperellum and B. circulans inoculation with various rates of potassium application during the 2019–2020 and 2020–2021 seasons.

Treatments	Plant Height (cm)	CGR (g m−2 Day−1)		NAR (g m−2 Day−1)
		65–80 Day	80–95 Day	65–80 Day	80–95 Day
2019–2020
Control	91.50 de *	14.61 f	17.77 cd	10.91 d	11.59 e
T+50%K	90.75 e	14.16 g	17.05 e	10.27 f	11.33 f
T+50%K+25%F	91.50 de	14.80 f	17.51 de	10.81 d	11.58 e
T+75%K	92.25 cde	15.61 cd	17.64 cd	11.02 d	11.89 cd
T+75%K+25%F	92.50 cd	15.89 bc	18.20 bc	11.32 c	12.03 bc
T+75%K-leaf	93.00 cd	16.51 a	18.58 ab	11.58 ab	12.50 a
B+50%K	91.50 de	15.17 e	17.39 de	10.59 e	11.55 e
B+50%K+25%F	92.75 cd	15.38 de	17.83 cd	10.98 d	11.79 de
B+75%K	93.50 bc	16.04 bc	18.22 bc	11.21 c	12.04 bc
B+75%K+25%F	94.50 b	16.12 b	18.42 ab	11.40 bc	12.20 b
B+75%K-leaf	95.75 a	16.77 a	18.90 a	11.71 a	12.66 a
LSD 0.05	1.09	0.362	0.425	0.190	0.188
2020–2021
Control	92.50 e	15.29 e	18.09 cd	11.10 de	11.77 e
T+50%K	92.00 e	14.54 f	17.43 e	10.48 g	11.52 e
T+50%K+25%F	93.00 de	15.41 e	17.75 de	10.91 ef	11.73 e
T+75%K	94.25 cd	16.05 cd	18.13 cd	11.10 de	12.07 cd
T+75%K+25%F	95.00 c	16.30 cd	18.63 bc	11.50 bc	12.32 b
T+75%K-leaf	94.75 c	16.97 ab	19.00 ab	11.79 a	12.72 a
B+50%K	94.00 cd	15.81 de	17.78 de	10.75 f	11.71 e
B+50%K+25%F	94.50 c	15.99 cd	18.34 c	11.19 d	11.98 d
B+75%K	95.50 c	16.38 cd	18.65 bc	11.33 cd	12.23 bc
B+75%K+25%F	96.75 b	16.53 bc	19.04 ab	11.58 b	12.38 b
B+75%K-leaf	98.25 a	17.25 a	19.41 a	11.86 a	12.89 a
LSD 0.05	1.06	0.482	0.411	0.196	0.197

* Letters indicate significant differences between treatments at the p < 0.05 level.

). The inoculation of wheat with T. asperellum and B. circulans and application of K significantly increased growth parameters. The increase in growth parameters with B. circulans application was higher than with T. asperellum application in both seasons. The highest significant increase in plant height (6%), CGR (12% at 65–80 days 12% and 7% at 80–90 days), and NAR (7% at 65–80 days and 10% at 80–90 days) was observed with B. circulans plus K-leaf application as compared to the control treatment.

The leaf area index (LAI) of wheat plants at 65, 80, and 95 DAS were affected under salt-affected soils conditions in both seasons (Figure 1). The inoculation of wheat with plant-growth-promoting microorganisms significantly enhanced the plant growth and increased the LAI. The increase in LAI was achieved with 75% of recommended K with 25% feldspar. However, the increase in LAI was high with B. circulans. The highest LAI values were obtained after inoculation and foliar application of 75% K-leaf in both seasons.

3.2.2. Plant Photosynthetic Pigments

In uninoculated plants under salt-affected soil conditions, photosynthetic pigments were affected, and there was a noticeable decline in chlorophyll a (Chl a) and b (Chl b) and carotenoid content in both seasons. The T. asperellum and B. circulans strains significantly improved the photosynthetic pigments, especially with increasing K dose (Figure 2). However, the application of 75% K with or without 25% feldspar on inoculated plants showed a significant increase in photosynthetic pigments. The highest significant increase in photosynthetic pigments was achieved with B. circulans inoculation plus 75% K-leaf application (Chl a 21%; Chl b 43%; carotenoids 55%) as compared with the control treatment.

3.2.3. Antioxidant Enzyme Activities

Antioxidant enzyme activities in uninoculated wheat plants decreased under salt-affected soil conditions (Figure 3). Inoculating wheat plants with KSMs significantly increased the antioxidant enzyme activity. The increase with B. circulans was higher than with T. asperellum, and increasing the K dose increased the activities of antioxidant enzymes in both seasons. The highest increase in antioxidant enzyme activities was achieved when plants were treated with B. circulans and 75% K-leaf (SOD 22%; CAT 8%; APX 4% and POD 15%) compared to the control.

3.2.4. Soluble Sugar Content

The overall soluble sugar content in plant leaves was affected by salt-affected soil conditions (control). The content increased as a result of inoculation with T. asperellum and B. circulans (Figure 4). There was a significant positive correlation found between the increase in the soluble sugar content and the increase in K dose applied with microorganism inoculation in both seasons. The highest significant increase in soluble sugar content was achieved by application of B. circulans with 1.5% K₂SO₄ (K-leaf) foliar spray, and the increase was 5% compared to the control treatment. On the contrary, the lowest value for soluble sugar content was recorded when wheat plants were treated with T. asperellum and 50% K compared with the control in both seasons

3.2.5. Relative Water Content (%)

Wheat leaves’ relative water content was affected by salt-affected soil conditions (Figure 5). The inoculation of wheat plants with T. asperellum and B. circulans increased RWC in the leaves in both seasons. B. circulans inoculation resulted in a higher RWC than T. asperellum inoculation in the two seasons. The lowest significant RWC was obtained as a result of T. asperellum and B. circulans inoculation with 50% K (K₂SO₄). However, RWC was corelated positively with K dose. The highest significant RWC was achieved as a result of T. asperellum and B. cerculance inoculation with K-leaf in both seasons. T. asperellum and B. cerculance inoculation with K-leaf foliar application resulted in increasing RWC by 6 and 8%, respectively.

3.2.6. Proline Content

Proline content in wheat plants leaves was affected by the salt-affected soil conditions (Figure 6). Inoculation with T. asperellum and B. circulans decreased Pro content in wheat leaves in both seasons, and the higher decrease was achieved with B. circulans treatments. The highest significant Pro content was achieved by T. asperellum and B. circulans inoculation with 50% K in both seasons. The lowest significant Pro content resulted from T. asperellum and B. circulans inoculation with K-leaf treatment in both seasons. B. circulans inoculation with K-leaf application decreased Pro by 23% as compared to the control.

3.2.7. Ion Homeostasis

Salt-affected soil conditions had an impact on the concentration of Na⁺ and K⁺ along with the Na⁺/K⁺ ratio in wheat plants (Figure 7). However, Na⁺ content and Na⁺/K⁺ ratio decreased with the inoculation of wheat with T. asperellum and B. circulans. With increasing K dose, the reduction rate rises. The inoculation of wheat with T. asperellum or B. circulans alongside K-leaf application resulted in the lowest values among all treatments. However, B. circulans inoculation with K-leaf application considerably reduced the Na⁺ content by 13% and Na⁺/K⁺ ratio by 21% in comparison with the control. Wheat leaves’ K⁺ concentration rose simultaneously with T. asperellum and B. circulans inoculation and K application. Treatments with increased K concentrations considerably accelerate the enhancement rate. Of all the treatments, B. circulans inoculation with K-leaf application produced the highest K⁺ content values. It markedly raised the K⁺ content level by 10% in comparison to the control.

3.2.8. Yield and Yield Components

During the two growing seasons, salinity stress had a considerable impact on wheat yield and yield traits under salt-affected soil conditions (

Table 5. Yield and yield features of wheat which were affected by T. asperellum and B. circulans inoculation with application of different doses of potassium in both 2019–2020 and 2020–2021 seasons.

Treatments	Spike Length (cm)	Spike Weight (g)	1000-Grain Weight (g)	Grain Yield (t ha–1)	Straw Yield (t ha–1)	Harvest Index
	2019–2020
Control	11.75 bcd *	4.33 d	52.55 d	7.65 cd	11.30 de	40.37 cd
T+50%K	9.88 e	3.76 e	48.41 g	6.99 e	11.04 e	38.76 f
T+50%K+25%F	11.38 d	4.39 d	51.50 e	7.54 d	11.42 cd	39.78 e
T+75%K	12.00 abcd	4.53 c	53.25 c	7.75 cd	11.49 bcd	40.28 d
T+75%K+25%F	12.25 ab	4.63 bc	53.63 c	7.95 c	11.73 abc	40.38 cd
T+75%K-leaf	12.50 ab	4.75 ab	54.24 b	8.86 a	11.86 ab	42.77 a
B+50%K	10.25 e	3.86 e	49.41 f	7.21 e	10.98 e	39.62 e
B+50%K+25%F	11.50 cd	4.40 d	52.22 d	7.70 cd	11.39 cd	40.32 cd
B+75%K	12.13 abc	4.61 bc	53.55 c	7.88 cd	11.49 bcd	40.66 c
B+75%K+25%F	12.38 ab	4.74 ab	54.12 b	8.21 b	11.85 ab	40.95 b
B+75%K-leaf	12.75 a	4.87 a	54.83 b	8.96 a	11.97 a	42.80 a
LSD 0.05	0.516	0.116	0.367	0.239	0.262	0.259
	2020–2021
Control	12.00 bc	4.49 c	53.14 e	7.79 e	11.39 cd	40.61 e
T+50%K	10.00 d	3.81 e	49.16 h	7.13 g	11.23 d	38.83 i
T+50%K+25%F	11.50 c	4.46 c	52.42 f	7.67 e	11.46 cd	40.07 g
T+75%K	12.25 ab	4.58 c	53.70 d	7.85 e	11.61 c	40.33 f
T+75%K+25%F	12.50 ab	4.74 b	54.20 c	8.30 c	11.93 ab	41.04 c
T+75%K-leaf	12.88 a	4.86 ab	54.80 b	8.96 a	11.98 ab	42.79 a
B+50%K	10.50 d	3.96 d	50.23 g	7.34 f	11.40 cd	39.17 h
B+50%K+25%F	11.75 bc	4.53 c	52.82 e	7.83 e	11.55 cd	40.40 f
B+75%K	12.38 ab	4.74 b	54.14 c	8.07 d	11.70 bc	40.83 d
B+75%K+25%F	12.50 ab	4.81 b	54.83 b	8.54 b	12.10 a	41.36 b
B+75%K-leaf	13.00 a	4.95 a	55.42 a	9.07 a	12.10 a	42.83 a
LSD 0.05	0.553	0.106	0.349	0.189	0.252	0.140

* Letters indicate significant differences between treatments at the p < 0.05 level.

). However, T. asperellum and B. circulans inoculation with K application boosted the spike length, grain yield, spike weight, harvest index, straw yield, and 1000-grain weight for wheat in both seasons. Increasing K application with microorganism inoculation enhanced the increase in yield and yield attribute parameters. The highest significant values for yield and yield components were achieved from T. asperellum or B. circulans inoculation with 1.5% K-leaf foliar application in both seasons.

3.2.9. NPK and Protein Content in Wheat

Nitrogen, P, K, and protein contents in wheat grains and straw were significantly affected by salt-affected soil conditions (

Table 6. NPK and protein percentages in wheat plants at harvest as affected by T. asperellum and B. circulans inoculation with various rates of potassium application during the 2019–2020 and 2020–2021 seasons.

Treatments	N %	P %	K % in	Protein %	N %	P %	K %	Protein %
	Grains				Straw
	2019–2020
Control	1.87 c *	0.40 e	0.46 d	11.67 c	0.49 d	0.105 de	0.85 de	3.08 d
T+50%K	1.74 d	0.35 f	0.41 f	10.84 d	0.37 g	0.085 f	0.66 g	2.32 g
T+50%K+25%F	1.84 c	0.38 e	0.45 d	11.47 c	0.46 e	0.101 e	0.82 ef	2.85 e
T+75%K	1.88 bc	0.42 d	0.47 d	11.72 bc	0.49 d	0.110 cd	0.88 de	3.05 d
T+75%K+25%F	1.89 bc	0.44 bcd	0.50 c	11.78 bc	0.52 c	0.116 bc	1.04 c	3.22 c
T+75%K-leaf	1.97 ab	0.46 ab	0.53 b	12.28 ab	0.55 b	0.121 ab	1.17 b	3.41 b
B+50%K	1.76 d	0.36 f	0.43 e	11.02 d	0.40 f	0.087 f	0.75 f	2.52 f
B+50%K+25%F	1.87 bc	0.39 e	0.46 d	11.70 bc	0.47 e	0.103 de	0.92 d	2.93 e
B+75%K	1.90 bc	0.43 cd	0.49 c	11.84 bc	0.50 cd	0.110 cd	1.03 c	3.14 cd
B+75%K+25%F	1.92 bc	0.45 abc	0.52 b	11.97 bc	0.54 b	0.119 ab	1.14 b	3.38 b
B+75%K-leaf	2.00 a	0.47 a	0.55 a	12.52 a	0.58 a	0.125 a	1.30 a	3.60 a
LSD 0.05	0.063	0.019	0.018	0.292	0.017	0.006	0.082	0.104
	2020–2021
Control	1.89 c	0.41 e	0.48 d	11.81 c	0.50 de	0.107 ef	0.88 ef	3.14 de
T+50%K	1.76 d	0.37 f	0.43 f	10.98 d	0.37 h	0.089 g	0.72 h	2.28 h
T+50%K+25%F	1.88 c	0.40 e	0.47 d	11.73 c	0.48 f	0.0103 f	0.86 fg	2.97 f
T+75%K	1.90 bc	0.44 d	0.49 d	11.89 bc	0.52 d	0.114 cd	0.96 de	3.22 d
T+75%K+25%F	1.92 bc	0.46 bcd	0.52 c	12.02 bc	0.55 c	0.120 bc	1.11 c	3.41 c
T+75%K-leaf	2.00 ab	0.48 ab	0.55 b	12.47 ab	0.59 ab	0.125 ab	1.21 b	3.66 ab
B+50%K	1.78 d	0.38 f	0.45 e	11.14 d	0.40 g	0.090 g	0.78 gh	2.47 g
B+50%K+25%F	1.90 bc	0.41 e	0.48 d	11.88 bc	0.50 e	0.107 def	0.98 d	3.09 e
B+75%K	1.93 bc	0.45 cd	0.51 c	12.05 bc	0.54 c	0.113 cde	1.09 c	3.34 c
B+75%K+25%F	1.96 bc	0.47 abc	0.54 b	12.25 bc	0.58 b	0.124 ab	1.20 b	3.59 b
B+75%K-leaf	2.05 a	0.49 a	0.58 a	12.78 a	0.60 a	0.129 a	1.38 a	3.72 a
LSD 0.05	0.065	0.018	0.019	0.408	0.019	0.007	0.082	0.118

* Letters indicate significant differences between treatments at the p < 0.05 level.

). The inoculation of wheat with T. asperellum and B. circulans in combination with K sources significantly increased N, P, K, and protein contents of grains and straw in both seasons. Increasing K application with microorganism inoculation enhanced the increase in N, P, K, and protein contents in wheat. The greatest significant values for N, P, K, and protein contents were achieved from T. asperellum or B. circulans inoculation with K-leaf (75% of recommended dose) foliar application in both seasons. However, B. circulans with K-leaf significantly increased the percentage N by 9% and 20%, the percentage P by 20% and 21%, the percentage K by 21% and 50%, and the percentage protein by 8% and 20% in wheat grains and straw, respectively.

Potassium uptake in wheat plants was affected by salt-affected soil conditions (Figure 8). However, the inoculation of wheat by T. asperellum and B. circulans with different K sources significantly increased K uptake as compared with the control in both seasons. At the same time, increasing K dose increases K uptake in wheat plants. The highest significant values for K uptake were achieved by T. asperellum or B. circulans inoculation with K-leaf foliar application in both seasons.

3.2.10. Potassium Content in Soil

Under salt-affected soil conditions, K soil pools clearly showed changes in water-soluble K (Figure 9). The variations in water-soluble K concentration were noted when various K fertilizer sources and biofertilizer were used, in comparison with the control. The highest significant values for water-soluble K concentration were achieved with B. circulans and T. asperellum inoculation and 75% of the recommended K–leaf foliar application and 25% feldspar after the harvest stage. The exchangeable K at a depth of 0–30 cm ranged between 308.6 mg L⁻¹ and 401.8 mg L⁻¹. The highest significant increase in exchangeable K was achieved with B. circulans inoculation combined with 75% K₂SO₄ application and 25% feldspar, followed by T. asperellum with the same potassium application (75% K₂SO₄ and 25% feldspar) compared to the control. The application of 75% K₂SO₄ with 25% feldspar on inoculated plants showed a significant increase in exchangeable K. The highest significant increase in total K content was achieved by B. circulans inoculation plus 75% K₂SO₄ application and 25% feldspar (0.1415% at depth of 0–30 cm), followed by T. asperellum combined with the same K application (75% K₂SO₄ and 25% feldspar) compared to the control (0.07% at depth of 0–30 cm).

4. Discussion

Salinity, in the Mediterranean region, imposes a harmful impact on soil properties and plant growth, leading to a reduction in plant productivity ranging from 25 to 30% [40]. However, salt-affected soil is considered to be a significant abiotic stressor that lowers crop productivity. Wheat plant is sensitive to salinity stress. The development of the plant growth rate is affected by osmotic stress [41]. Under salt-affected soil conditions, the decrease in growth rate is observed in plant height, leaf area, and dry weight.

The use of different K sources in combination with T. asperellum and B. circulans inoculation mitigates the salinity stress and promotes the growth parameters of the wheat plant. This outcome may be explained by the roles played by K, T. asperellum, and B. circulans in nutrients and water cell transport systems [42]. The capacity of microorganisms as plant growth promoters correlates directly to their phosphate production and K-solubilizing enzymes; plant growth promoting enzymes, such as 1-aminocyclopropane-1-carboxylate deaminase (ACC-deaminase); siderophores, which are used by plants as a source of iron [43]; phytohormones, especially IAA and cytokinins (CKs) [44,45,46]; and HCN [47]. Phytohormones regulate plant responses and maintain normal plant physiology under salinity stress [48]. Siderophores have a considerable affinity for Fe and function as chelating agents for ferric compounds. By creating a Fe-siderophore complex, siderophores increase the amount of iron available to plants. Under soil salinity conditions, salinity limits the plant’s iron uptake [49]. Volatile substances such as ammonia, which is produced intermediately during root exudate amino acid hydrolysis, can help the host plants and supply them with nitrogen [50]. Under salt stress conditions, Trichoderma species have been shown to demonstrate a symbiotic relationship with the plant and are used to alleviate stressors and improve plant metabolism [51]. It is well known that Trichoderma spp. generates various types of organic acids that enhance plant vigor and growth and can also boost the plant’s tolerance by alleviating the toxicity levels of salts in the plant under salinity conditions [52,53].

Potassium, Trichoderma, and Bacillus are capable of promoting the growth and yield of wheat crop [54,55] and increasing the phytosubstance content in derived products that have nutraceutical value [56].

Under salt-affected soil conditions, the photosynthetic pigment content in wheat plant leaves decreased, and this may be due to the degradation of the thylakoid membranes, with more chlorophyll breakdown occurring than its synthesis [57,58]. Photosynthesis inhibition by Na⁺ and Cl^- ion accumulation in the chloroplast may also be a reason [59]. Therefore, the inoculation of wheat plants with K-solubilizing microorganisms combined with K application improves photosynthetic system by enhancing the quantity and size of leaves, decreasing osmotic stress, and stimulating the production of the enzyme ATPase in leaves [60]. However, the improvement in photosynthetic pigment content in inoculated plants with different K sources is related to the increased Mg, N, and Fe-uptake [61] and the decrease in ethylene production [62].

Increasing antioxidant enzymes is one of antioxidant techniques used by plants to decrease the harmful effects of salinity. However, the best and strongest antioxidant enzymes reduce lipid peroxidation to a minimum and safeguard against oxidative damage to combat the negative effects of salt-affected soil conditions. Also, plants need to have antioxidant enzymatic activities to overcome the ROS-damaging effects [63,64]. In this study, the inoculation of wheat plants by KSMs with K application under salt-affected soil conditions increased the activities of the antioxidant enzymes SOD, CAT, APX, and POD [65]. However, under stress conditions, K application can lower ROS production and eventually improve the plant’s state. Moreover, K participates in plant signaling systems that activate antioxidant defense systems to assist plants in withstanding certain stressors [66].

According to our study, Pro accumulation increases under salt-affected soil conditions, and this increase plays a crucial role in mitigating the negative effects of salt stress and promoting wheat growth [67]. The osmoprotectant Pro is a useful water-soluble amino acid for preserving cell turgor [68]. However, inoculation by KSMs with the application of different K doses and sources decreased Pro accumulation under salt-affected soil conditions. These results are supported by other studies where biofertilizers and K decreased the Pro content in plants under salinity stress [55,69,70,71].

Increasing the absorption of Na⁺ for plants grown in salt-affected soils limits plant growth and development. However, the relative increase in Na⁺ uptake enhances the Na⁺/K⁺ ratio in the plant, limiting growth under salt-affected soil conditions [72]. In this study, wheat plants inoculated with KSMs combined with K applications in different forms and doses had increased K⁺ uptake, decreased Na⁺ uptake, and decreased Na⁺/K⁺ ratio in plant leaves. But decreasing the Na⁺/K⁺ ratio with KSMs in plants cultivated in salt-affected soil alleviates the damaging impacts of salt stress [73]. This increase in K⁺ uptake may be due to the expression of a high-affinity K⁺ transporter in AtHKT1 being altered in plants [74,75]. However, K-solubilizing microorganisms mediate the selective transport capacity for K⁺ over Na⁺ and consequently decrease the Na⁺/K⁺ ratio under salt-affected soil conditions [76]. KSM polysaccharides bond to available Na⁺ in the soil, preventing its absorption by the plants, and subsequently reduce the salinity stress effect [77]. KSMs lower the soil pH by releasing protons and organic acids. Moreover, some isolates dissolve K in soil by producing malic and syringic acids that increase K⁺ uptake by the plant [78]. Also, KSMs are considered potent natural decomposing agents that might help in improving nutrient availability in soils [79]. At the same time, the foliar application of K increases the uptake of K⁺ and decreases Na⁺, subsequently decreasing the Na⁺/K⁺ ratio in plant leaves under salt- affected soils conditions [80,81].

The reduction in wheat crop yield under salt-affected soil conditions could be due to the antagonistic impact of salinity stress on physiological functions and vegetative growth parameters, including photosynthesis, water absorption, cell division, and grain filling [82,83]. Furthermore, under salinity stress, the decrease in yield and its attributes may be an outcome of a decrease in photoassimilation assembly and motivation that lowers the harvest index values [84]. Nevertheless, the inoculation of wheat with KSMs with K application improved plant tolerance to salt stress and promoted yield and its components, especially with B. circulans inoculation with foliar application of K; however, Elbagory [85] confirmed that the combined application of B. circulans and compost tea on wheat under salt-affected soils improved growth, yield, and the uptake of nutrients.

The K-uptake by wheat, NPK content, and protein content in wheat grains and straw were affected by salt-affected soil conditions due to the inhibitory impact of salinity stress on growth and its chemical contents. However, the changes related to salinity were showed in wheat productivity and its attributes, nutrient uptake, and nutrient content, as well as in the protein percentage content. Inoculation with KSMs alongside the application of different K sources, especially B. circulans with foliar K treatment, significantly increase the nutrient uptake and content as well as protein percentage [85,86].

Microorganisms release various organic substances during their metabolic processes [87]. These substances include metabolic byproducts, extracellular enzymes, and chelates, as well as both simple and complex organic acids. These improve the dissolution of the feldspar by lowering the surrounding pH. One possible explanation for the mobilization of soil K reserves is the formation of biofilms on mineral surfaces in the rhizosphere by certain bacterial strains [88]. Biofilm offers protection to the microbial community from external factors as well as antibiotics. They also control heavy metal transportation as well as potential other ions and nutrients to microbes. Furthermore, they act as a barrier between mineral weathering and nutrient uptake by bacteria, effectively isolating these processes from the rest of the soil.

Balogh-Brunstad et al. [88] found that ectomycorrizal hyphal networks and the root hairs of non-ectomycorrhizal trees were contained within biofilms for transferring nutrients to their host. These findings indicate that biofilms play a role in accelerating the weathering of minerals such as biotite and anorthite, thereby increasing the uptake of nutrients by plants.

5. Conclusions

The inoculation of wheat by KSMs alongside the application of different K sources demonstrated their important role in mitigating the adverse effects of salinity stress on physiological characteristics, growth, and yield. However, under salt-affected soil conditions, KSM inoculation alongside the application of different K doses remarkably enhanced the growth and yield of wheat by boosting the stress tolerance, enzyme production, and solubilizing K; increasing availability and uptake of nutrients; and increasing the plant enzyme activity. Furthermore, the results obtained suggest that, in salt-affected soil conditions, inoculation with T. asperellum and B. circulans with 1.5% K-leaf (75% of recommended dose) foliar application could be recommended for wheat growers to mitigate salinity stress and to improve its growth and productivity, and thus decrease the usage of mineral nutrients. But, to inoculate the different wheat genotypes with KSMs and different K sources under salt-affected soil conditions, further research should be conducted to optimize the application techniques. The potential to select salinity-tolerant wheat genotypes at early growth stages of the wheat crop might be made possible by examining the application of different K sources with KSM inoculation of wheat in different vegetative growth stages.