As a microbial treatment, Azospirillum brasilense (Az) inoculum formulation was applied to work as a plant growth-promoting rhizobacterium. Az was grown in LB medium (10g L^-1triptone, 5g L^-1NaCl, 10g L^-1 yeast extract) for 4 days under continuous shaking at 30°C [22]. The bacteria were then centrifuged for 15 minutes at 180 rpm and washed four times with a sterile solution (NaCl 0.85% w/v). After that, the Azospirillum bacteria were ready for injection into the seeds and soil. The inoculant was prepared by diluting 200 ml of bacterial suspension in 800 ml of distilled water at pH 7.0 to produce a final concentration of 20 mg C L⁻¹ and a final bacterial concentration of 5 × 10⁸ cells ml⁻¹.

Vermicompost was obtained from a red wiggler /ENC mix worm farm in Alexandria, Egypt. It was produced as a by-product from the beer industry residues (spent grain) by brandling worm red wiggler (1500 worms m²). The chemical properties of the spent grain and vermicompost are shown in Table 2.

Soluble-humic substances were extracted from vermicompost, produced as described in 2.2.2 with 0.1 M NaOH in a 1:20 solid-liquid (w:v). The extraction samples were shaken at 2000 rpm for 4 hours and left overnight. The supernatant was centrifuged at 5000 rpm and filtered through a Whatman no. 42 filter paper to show the Az production's humate. The HS characteristics were characterized as indicated in Table 2.

The micronutrients were prepared with 1.64% (Fe-EDTA) solution (4.0 mL), and KOH (4.5 g) was mixed with one liter of the micronutrient solution containing CuSO₄ (0.4 g), ZnSO₄·7H₂O (0.12 g), H₃BO₃ (1.4 g), Na₂MoO₄·2H₂O (1.0 g), and MnSO₄·H₂O (1.5 g). The pH of the solution was adjusted to 5.8.

The field experiment included six treatments comprising CK (control without amendment); mineral fertilizers (NPK), (38 g/m² of urea (46%); 10 g/m² of calcium phosphate (45%); 12 g/m² of potassium sulfate (50%)); Azospirillum sp. (Az), a cultivated suspension of plant growth-promoting rhizobacteria seeds, soaked in the suspension for 4 hours before sowing and introduced three times into the soil with irrigation water at 0.7 ml/m²; vermicompost (VC) was applied to the soil at a rate of 2.5 kg/m²; humic substances + micronutrients (HS-M), neutral HS with micronutrients solution 1.0 ml / m² soil inoculation; and humic substances (HS) 0.5 ml /m² for foliar spray application on plants and soil. All treated plots were sown in November 2020 with wheat (TriticumaestivumL.). The wheat plants were grown under experimental field conditions for 6 months. The treatment descriptions and application rates are shown in Table 3.

The total trial area had a size of 500 m² comprising 18 plots, each of the six treatment variants was tested in three replicate plots; the plot size was (5.5m x 5m) distributed in a randomized complete block design. The mass seeds (weight) were 10.7 kg/ 500 m², about 22 g (500 seeds) per m². The cultivation method included scattering seeds in the soil before irrigation water was supplied. To maintain adequate seed germination moisture, all plots were watered from the Nile River to the soil saturation capacity. The water source was the limited source, and on the research farm, we stored water every 2-3 months for research experiments. Therefore, there were restrictions on saving water constantly. The vermicompost and HS characteristics are characterized as indicated in Table 2.

In each fertilizer treatment, the leaves of 10 plants located in the central area of each plot were selected. Leaf samples were collected three months after seed sowing on January 20^th, 2020, to determine N, P, and K concentrations. Moreover, the grain yield was collected at harvest six months after sowing, which occurred on April 16^th, 2020, by selecting ears.

The measurements of plant parameters included the weight of 1000 seeds (g), grain yield (ton/ha), biological yield (ton/ha), and N, P, and K (%) contents [37]. All the wheat in each experimental plot was collected; each plot's grain yield of wheat ton ha ^-1 was calculated.

The electrical conductivity (EC) was measured in soil extract in water (1:5) using an EC meter; the pH was measured in a soil: water suspension (1:2.5 w/v) [28]. The total nitrogen (N) was determined using the Micro-Kjeldahl method; extractable phosphorus (P) was extracted with 0.5N NaHCO₃ and measured using a spectrophotometer at 670 nm wavelength. Available potassium (K) was extracted with 1 N ammonium acetate solution and measured by flame photometry. N, P, and K were measured as explained previously [23], [24], and [25]. Micronutrients (Fe, Zn, Mn, Cu, and B) were extracted from DTPA solution as explained in a previous study [30], then measured with inductively coupled plasma (ICP) atomic emission spectroscopy

The soil dehydrogenase activity was determined by tri-phenyl-tetra-zolium chloride (TTC) following a method described previously [26]. Soil urease (S-UE) was determined using previously published methods [27], involving: 5 g of soil was placed in 100-ml Erlenmeyer flasks and wetted with 2.5 ml of a 0.08 M aqueous urea solution. The flasks were stoppered and placed in an incubator at 37 °C. After 1 hr., the stoppers were removed, 50 ml 1N of KC1 to 0.01N of HC1 was added and the mixtures were shaken on a shaker for 30 min. The resulting suspensions were filtered, and ammonia in the filtrates was determined calorimetrically. 1 ml of filtrate was diluted to 10 ml with distilled water, and 5 ml of sodium salicylate and 2 ml of 0.1% sodium dichloroisocyanurate were successively added. The sodium salicylate solution was prepared for use by mixing 100 ml of 0.12% sodium nitroprusside, 100 ml of 17% sodium salicylate, and 100 ml of distilled water. Optical density was determined at 690 nm after a 30 min incubation at room temperature.

Data (Fig. 1) show the effect of soil treatment and foliar fertilizer on the wheat yield, which increased significantly during the season 2019/2020. When studying the impact of each factor, it was found that the soil treatments with HS-M, HS, and VC before sowing resulted in the highest yield records of wheat of 9.58, 9.22, and 8.84 t ha^-1, respectively, after six months of seed sowing compared to the CK and NPK treatments. The water use efficiency (kg grain / m³) values increased by 279, 268, 262, 258, and 139% for HS-M, HS, VC, Az, and NPK treatments, respectively, compared to the control treatment. The water use efficiency calculated was more significant in the higher grain yield than in the HS-M, HS, and VC treatments. The wheat productivity increased when using a preparation supplemented with micronutrients (Fig. 1). On the other hand, there were differences between the effects of fertilizers. Compared to the control treatment, the plant productivity increased by 260% and 138% with Azospirillum and NPK treatments. These results were in agreement with the previous findings [30].

Fig. (1) shows soil treatment and HS foliar fertilizers on the water use efficiency for wheat plants during the research season. The inoculation of Azospirillum with seeds or HS, HS-M, and CV treatments and application of VC increased the plant growth and grain yield during the growing season. The VC as an organic treatment increased soil water retention, increasing water use efficiency during the research season. The positive effect of organic fertilizers on the water use efficiency was noticed in plant growth during the research season [31]. The treatment of wheat plants with NPK, VC, Az, HS, and HS-M increased the water use efficiency by 139%, 262%, 258%, 269%, and 279%, respectively, compared to the control, respectively. On the other hand, the foliar fertilizing effect of HS-M and HS increased by 1.34 and 1.29 kg m^-3, respectively, compared to the control. While the VC and Az treatments increased 1.26 and 1.24 kg m^-3 compared to the control and NPK treatments, these results were in agreement with the findings of other studies [32, 34].

Fig. (2) shows the effect of soil treatment and HS foliar fertilizer on the 1000 grain weight of wheat. Data showed a positive impact on the weight of 1000 grains for the wheat plant when using the HS, HS-M, VC, and az treatments compared to control and NPK treatments. The maximum average 1000 grain weight increased by 149.53, 135.39, 152.30, 120.65, 99.61% for HS-M, HS, VC, Az, and NPK treatments, respectively, compared to CK treatments. The VC treatment significantly (p ≤ 0.05) increased the weight of 1000 grains of the wheat plant. The direct relationship between the weight of 1000 grains and grain yield (tons/ ha) is an indicator of HS-M and VC treatment applications that can increase wheat plant productivity in arid regions. Thousand-grain weight is regarded as one of the most important factors concerning wheat quality (Table 3). It has been generally accepted that the plant seeds should have as large a 1000-grain weight as possible because such grain has larger amounts of storage substances and more developed embryos. This enables plants to develop and grow faster, which is important in arid and semi-arid regions. The weight of 1000 grains as a final component of grain yield depends on many parts developed during the previous phases of ontogenesis. As there is a high production of all plant organs in each stage of wheat plant growth, it is possible to influence the weight of 1000 grains [35, 36].

Soil pH is a critical factor that affects the solubility and availability of plant nutrients. Increasing the availability of certain plant nutrients, such as P, can be achieved by decreasing soil pH in calcareous soils. The soil analysis showed that soil pH was significantly reduced relative to the control (Fig. 4). The calcareous soil pH values treated with organic and bioorganic ameliorants decreased by 2.84, 3.19, 5.91, 7.90, and 12.77% for HS, HS-M, NPK, Az, and VC treatments, respectively, compared to CK treatment. The VC and Az treatments resulted in the lowest pH levels in calcareous soil. No significant (p ≤ 0.05) difference was observed in CK, HS, and HS-M treatments compared to VC and Az applications. In the VC treatment, decreased soil pH was enhanced by organic matter content in the initial vermicompost source. The type of amendment influenced the reduction of pH values. The increasing application of Az and VC enhanced pH reduction. However, soil pH increase may induce the Ca²⁺ to become more alkaline and, therefore, more sodic as calcium solubility is suppressed; this was also proposed in a study conducted previously [41]. In arid and semi-arid climates, the solubility of Ca²⁺ and Mg²⁺ becomes low; Na⁺ and K⁺ ions accumulate in soil solution when CO₃^-2 and HCO₃^- increase [42]. It has been found that the applications of organic amendments, such as spent grain and Azospirillum, might reduce pH in calcareous soil [38]. A long-term soil incubation experiment was conducted to examine the effect of different organic amendments on the chemical properties of soil, which positively impacted soil pH in calcareous soil [43]. Found applications of organic amendments to soil decreased the soil pH and enhanced phosphorus availability in sandy soil.

Fig. (3). Influence of soil and foliar spray treatment on the plant macronutrients concentrations of the wheat plant after three months of seed sown. Columns and error bars represent mean values ± standard error of the mean (n = 3).

Fig. (4). Influence of soil and plant foliar spray treatment on soil pH in the calcareous soil under wheat plant growth after 6 months of seed sown. Columns and error bars represent mean values ± standard error of the mean (n = 3).

Fig. (5). Influence of soil and foliar spray treatment on micronutrient concentration (mg kg-1) of calcareous soil under wheat plant growth after 6 months of seed sown. Columns and error bars represent mean values ± standard error of the mean (n = 3).

The soil additives were found to have a significant effect on the extractability of micronutrients. Fig. (5) shows Fe, Zn, Mn, Cu, and B concentrations six months after sowing. The Fe, Zn, Mn, Cu, and B concentrations in the soil after wheat harvest followed the order HS-M > Az > VC > HS > CK > NPK, respectively. The soil's higher concentrations of micronutrients were observed in variants with HS-M and Az treatments, while the lowest increases were observed with NPK and CK treatments after six months of seed sowing. The Az and HS-M application rates were superior to soil fertility than HS and NPK treatments for micronutrient status (Fig. 5). The superiority of HS-M and Az was due to their effect on lowering soil pH and solubility and microelement chelating the soil treatments.

Accordingly, Az application to enrich the soil with micronutrients is highly recommended in calcareous soils [43]. The Az mineralization of organic matter leads to a decrease in soil pH, which increases micronutrient availability in calcareous soil. The presented results agree with the findings presented in a study [41] that reported that the available micronutrients improve using plant growth-promoting bacteria and organic source fertilization. The organic amendments, especially HS-M, increased the concentrations of most of the essential elements. One-way ANOVA showed a significant (p ≤ 0.05) effect of microbial amendments and humic substances on micronutrient status in calcareous soil. The Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, and B⁺ significantly (p ≤ 0.05) increased the wheat plant growth in Az, HS-M, and HS amended to soil and plant. The applications of NPK fertilizer had lower Fe²⁺, Zn²⁺, Mn²⁺, Cu²⁺, and B⁺ concentrations than most microbial and organic treatments [41].