Open field trials were performed during two growing seasons; the spring season (SS) and the autumn season (AS) of 2020 in the horticultural experimental station of Fayoum University, Fayoum, Egypt (29°17’38”N 30°54’55”E). The climatic conditions in the local area are arid based on the aridity index classification (Ponce et al., 2000). The soil analysis in Table 1 indicates that the soil is strongly saline (9.6 dS m^-1), and slightly alkaline (pH 7.76). The soil has a coarse (sandy loam) texture and is moderately deep, and is classified as Typic Torripsamments, siliceous, hyperthermic (Soil Survey Staff USDA, 2014).

Seeds of hot pepper (cv. Omega F1) were sown on 25 January and 25 July 2020, in 209-cell polystyrene trays filled with a mixed substrate of peat moss and vermiculite (1:1) and the trays were then placed in the greenhouse. Transplantation took place on 10 March and 5 September 2020 (when seedlings form four true leaves) in beds of 10 m in length and one-meter width in an open field. The seedlings were transplanted 0.50 m apart, with one row of plants per bed. Each plot area included three beds. The incorporation of nutrients during the entire growth period of the hot pepper crop were applied at the recommended rates in the area; including 80 kg P ha^-1, and 170 kg K ha^-1. Proper agronomic management and pest control for hot pepper crop was applied following the bulletin of the Egyptian agricultural research center.

Experiments were carried out in a split-plot in a randomized complete block design (RCBD) with two factors; EMs were allocated the main plots, and different N levels were allocated in the subplots. Three N levels (N₁₂₀ with 120 kg N unit ha^-1, N₁₅₀ with 150 kg N unit ha^-1, and N₁₈₀ with 180 kg N unit ha^-1) were applied as ammonium nitrate (NH₄NO₃) form by fertigation from the third week after transplantation. Thereafter, each N level was divided into two weekly doses during the hot pepper growing season as follows; 15, 25, 50, and 10% of the N quantity were applied during the vegetative growth, early flowering and bearing, harvesting, and late harvest stages, respectively (Condés, 2017). The total quantity of N applied as N₁₂₀, N₁₅₀, and N₁₈₀ were 360 kg ha^-1, 455 kg ha^-1, and 545 kg ha^-1 of ammonium nitrate. According to the bulletin of the Egyptian Research Center, N₁₅₀ is the recommended level for hot pepper production.

The EMs were not applied (EMs^-) or applied (EMs⁺) at three different times after 15, 35, and 55 days of transplantation as a soil drench (simultaneously with irrigation). The EMs were used in a formulation that included five types of beneficial microorganisms, including photosynthetic bacteria (Rhodobacter sphaeroides and Rhodopseudomonas palustris), lactic acid bacteria (Lactobacillus casei, Lactobacillus plantarum, and Streptococcus lactis), yeast (Candida utilis and Saccharomyces cerevisiae), fermenting fungi (Penicillium sp., Aspergillus oryzae, and Mucor hiemalis), and actinobacteria (Streptomyces griseus and Streptomyces albus). The photosynthetic bacteria were cultivated on Luria-Bertania broth medim (LB; Lab M Limited, Lancashire, UK)), pH 7.0 at 30°C for 72 h in a shaking incubator at 200 rpm (Kang et al., 2015). The lactic acid bacteria were cultivated on MRS broth medium (Lab M Limited), pH 6.2 at 37°C for 48 h in anaerobic incubator (De Man et al., 1960). The yeasts were cultivated on YPD growth medium (Lab M Limited), pH 5.5-5.8 at 30°C for 72 h in a shaking incubator at 150 rpm (Sherman et al., 1986).

The fungi used in this study were cultivated on Czapek broth medium (Lab M Limited), pH 7.3 ± 0.2 at 28°C for 7 days in a shaking incubator at 150 rpm (Khan et al., 2015). The actinobacteria were cultivated on tryptic soy broth medium (TSB) (Lab M Limited), pH 7.3 ± 0.2 at 30°C for 7 days in a shaking incubator at 200 rpm (Nonthakaew et al., 2022). The mixture was prepared in equal proportions as an EM stock solution at the Ministry of Agriculture and Land Reclamation, Giza, Egypt, and was diluted to 1:1000 (EM: water, v/v) with a final concentration of microorganisms of 1.0×10⁸ – 2.0×10⁸ colony forming units (CFU) ml⁻¹ at the application time. Each plant beside the root was then inoculated with 100 ml of suspension of each application.

Six representative plants from each plot area were sampled at 80 days after transplantation, and the growth parameters were determined. Plant height and number of leaves and branches plant^-1 were recorded. Plant leaf area was assessed by washing the leaf surface after that ten discs were taken and dried at 75°C in the air-forced oven for 24 h and the disc’s dry weight was determined (DDW; g). The leaf area was determined as follows:

Where, LDW denotes the leaf dry weight; DDW denotes the disc’s dry weight; and DA denotes the disc’s area. The plant leaves and branches were dried to constant weight in the oven at 75°C, after which the dry weight for each part and the total dry weight (called shoots) were determined. Ten plants from each experimental plot were harvested to determine the number of fruits plant^-1, average fruit weight, and total fruit yield.

Relative water content (RWC, %) and membrane stability index (MSI, %) of leaf were assessed as described by Abdelkhalik et al. (2019). Leaf MSI was determined with 0.2 g of fully expanded leaves as follows;

The leaves samples were inserted into a tube with 10 ml of distilled water and incubated in the water bath for 30 min at 40°C, and the solution’s electrical conductivity (EC) was measured (EC1). After heating the samples for 10 min at 100°C, the solution EC was measured (EC2).

Leaf RWC was assessed with 10 discs taken from fully expanded leaves in each plot area and calculated after obtaining the disc’s fresh (FW), saturated (SW), and dry (DW) weight as follows;

The photosynthetic efficiency of the PSII was determined as maximal quantum yield (F_v/F_m ) and calculated with the formula: (F_m -F₀ )/F_m (Maxwell and Johnson, 2000), and the index of photosynthetic performance (PI_abs) as described by Clark et al. (2000). The readings were taken on a sunny day between 9:00-11:00 AM from fully expanded one leaf plant^-1 by a fluorometer (Hansatech Instruments Ltd., Kings Lynn, UK). Before conducting the measurements, the leaves were kept for 20 min for dark adaptation with light-blocking clips (Hansatech Instruments Ltd.). The relative chlorophyll content (SPAD value) was measured in fully developed leaves using a SPAD-502 chlorophyll meter (Minolta, Osaka, Japan).

Hot pepper total protein was determined in the leaves using Folin phenol as a reagent following the methodology of Lowry et al. (1951). Free proline assessment was obtained according to the Bates et al. (1973) method. Hot pepper plant tissues were homogenized in 10 ml of C₇H₆O₆S (3%) and filtered. Two ml of the filtrate was mixed with 2 ml of CH3COOH + 2 ml of C₉H₆O₄ reagent. Simultaneously, the samples were heated for 60 min at 100°C with the caps on. The resulting solution was harvested and strenuously mixed in a test tube with 4 ml toluene for 15-20 sec. Toluene-containing chromophore was withdrawn from the aqueous phase and left to ambient temperature. At 520 nm, absorbance was measured using toluene as a blank. Using the calibration curve, the proline content of the samples (mg g^-1 fresh weight) was quantified.

TSS were quantified using ethanol (80%) extract from fresh hot pepper leaves (Irigoyen et al., 1992). The alcoholic extract (1 ml) was mixed with 3 ml of anthrone reagent [freshly prepared as 150 mg C₁₄H₁₀O plus 100 ml of 72% (v/v) H₂SO₄] and then placed in a boiling water bath 10 min, followed by cooling. The reading was read with a spectrophotometer at 625 nm. Total capsaicin was extracted and determined from the dry fruits following the methodology reported by Trejo-González and Tamirano (1973).

For the extraction of glutathione reductase (GR), ascorbate peroxidase (APX), catalase (CAT), and superoxide dismutase (SOD), fresh samples of 500 mg were homogenized with a pre-chilled pestle and mortar in ice-cold 0.1 M phosphate buffer (pH=7.5) containing 0.5 mM EDTA. Each resulting mixture was transmitted to centrifuge tubes and centrifuged at 15000 x g, at 4°C for 15 min in a Beckman refrigerated centrifuge. The supernatant was used to quantify the enzyme’s activity (Bradford, 1976). The SOD activity was calculated by measuring the enzyme’s decrease in the absorbance of the superoxide-nitro blue tetrazolium complex (Kono, 1978). The absorbance was measured at 560 nm. The activity of CAT was assessed as described by Aebi (1984). The amount of H₂O₂ decomposed was used to calculate the enzyme activity. The reaction began with the addition of H₂O₂, and the reduction in the absorbance was measured at 240 nm for 1 min. The APX activity was determined following the criteria illustrated by Yoshimura et al. (2000). The GR activity was measured by measuring the increase in the absorbance when the oxidized glutathione (GSSG) and dithionitrobenzoic acid (DTNB) were present (Sairam et al., 2002).

Glutathione (GSH) was estimated using Ellman’s reagent as described by Anderson (1985). Under cold conditions, GSH was extracted with 500 mg of hot pepper tissues in 1% picric acid (w/v). At 412 nm, the absorbance was measured with a spectrophotometer. The content of ascorbate (AsA) was estimated using the Folin phenol reagent as outlined by Jagota and Dani (1982). About 500 mg of plant tissues were ground in oxalic acid and centrifuged for 5 min at 15000 x g, at 4°C for 15 min. After diluting 0.2-0.5 ml supernatant to 2 ml with distilled water, 0.2 ml of diluted Folin reagent was added to the extract. The tubes were shaken vigorously and the absorbance was mesured after 10 min at 760 nm.

The determination of nutrients concentration in the hot pepper tissues were performed in leaves, which were dried and milled before chemical analysis. The leaf samples were digested using a mixture of HNO₃ and HClO₄ (at 3:1, v/v), and the digest solution was used for the quantification of the nutrients. The N content in hot pepper leaves was assessed according to the procedures of AOAC (2000) with a micro-Kjeldahl device (Ningbo Medical Instruments Co., Ningbo, China). The blue color method (Jackson, 1973) was used for the P assessment. Using flame photometer apparatus (Perkin-Elmer Model 52-A, Glenbrook, Stamford, CT, USA) the K⁺, Ca²⁺ and Na⁺ contents were quantified as outlined in the methods of Page et al. (1982).

The stem and leaf samples (the sixth top internode from the stem with its leaf) were taken at 80 days after transplantation for anatomical observations. The samples of leaves and stems were fixed in a formalin-acetic acid-alcohol (FAA) solution for 72 h to kill and fix the tissue sample according to Mohamed et al. (2020). The following steps of clearance, dehydration, embedding in paraffin wax, and cutting of the cross-sections by a rotary microtome (Zhejiang Jinhua Kedi Co., Ltd. China) were performed (Mohamed et al., 2020). Thereafter, the sections were stained with a crystal violet erythrosine combination and decolored sequentially by placing into 50%, 70%, and 90% gradient alcohols for 5–10 sec, then carbol xylene, and inserted in Canada balsam (Sass, 1958). The sections of stems and leaves were observed using an optical microscope instrument (AxioPlan, Zeiss, Jena, Germany) connected with a digital camera (Nikon DS-U3, Tokyo, Japan). Furthermore, image analysis was performed using the CaseViewer 2.3 program (3DHISTECH Ltd., Budapest, Hungary).

The analysis of variance (ANOVA) as well as the homogeneity of error variance for all features were performed. The statistical analysis for all data was carried out based on the split-plot in CRBD with Genstat software. The collected data from the two planting seasons were combined since both growing seasons behaved similarly. Differences between the single effects of growing seasons, EMs and N levels, and the interaction effect of EMs × N levels were compared using Fisher’s least-significant difference (LSD) test at P ≤ 0.05.

First, we determined the growth characteristics of hot pepper, including plant height, number of leaves and branches, shoot dry weight, and leaf area in response to the growing seasons, EMs, N levels, and EMs × N interaction ( Table 2 ). Hot pepper plants transplanted in spring recorded greater growth trait values than those transplanted in autumn, despite having the same number of branches plant^-1.

The EMs supplemented plants grown in saline soil (EMs⁺) enhanced plant height, number of leaves, number of branches, leaf area, and shoot dry weight by an increase of 9.5, 33.3, 11.0, 24.7, and 22.3%, respectively, in comparison with non-EMs-treated plants grown under the same conditions (EMs^-). In addition, the fertilization with N₁₅₀ and N₁₈₀ increased growth parameters relative to plants that received a lower N dose (N₁₂₀). The co-application of EMs⁺ and N showed a stimulatory effect on hot pepper growth under saline soil conditions. The combined EMs⁺ × N₁₅₀ or EMs⁺ × N₁₈₀ increased plant height (by 10.6 or 12.9%, respectively), leaf number (by 41.9 or 30.8%, respectively), number of branches (by 2.5 or 12.5%, respectively), leaf area (by 18.1 or 21.2%, respectively), and shoot dry weight (by 24.2 or 20.6%, respectively), compared to those received the recommended N dose and non-amended with EMs (EMs^- × N₁₅₀) ( Table 2 ).

Hot pepper plants cultivated in spring recorded higher fruit yield than those cultivated in autumn; while both seasons recorded similar fruit number and average fruit weight ( Table 2 ). Under high salt stress conditions, plants not treated with EMs (EMs^-) induced damages to yield-related parameters; however, soil application of EMs ameliorated salt stress-mediated damages to the yield. This was evidenced from the increased in number of fruits, average weight and cumulative yield of fruits by 14.6, 16.7, and 21.6%, respectively ( Table 2 ). In comparison to hot pepper plants grown in salt-affected soil and supplemented with a low N dose (N₁₂₀), the application of N at the rate of 150 or 180 kg ha^-1 improved fruit number by 11.8 or 8.8%, fruit weight by 10.4 or 12.5%, and fruit yield by 12.6 or 15.5%, respectively ( Table 2 ). However, the integration of EMs and N (particularly at higher levels) showed significant increases in the yield-related attributes. In this regards, the number, average weight and yield of fruits increased by 14.4 or 17.0%, 20.8 or 20.8%, and 28.4 or 27.5% in hot pepper plants treated with EMs⁺ × N₁₅₀ or N₁₈₀, respectively, compared to the EMs^- × N₁₅₀ treatment ( Table 2 ).

Growing season, EMs, N rates, and EMs × N level were induced modulation in hot pepper membrane integrity (MSI), water status (RWC), and photosynthetic capacity; SPAD chlorophyll, Fv/Fm, and PI ( Table 3 ). The MSI, RWC, and SPAD chlorophyll were significantly influenced by the two growing seasons. However, there were no variations in the Fv/Fm or PI between the two growing seasons ( Table 3 ).

For EMs application, hot pepper plants exposed to salinity stress and not treated with EMs markedly triggered a reduction in the aforementioned traits. Nevertheless, the application of EMs alleviated the salinity damage and elevated these parameters by 6.4, 5.6, 8.3, 8.0, and 10.0%, respectively ( Table 3 ). For N fertilization, the results showed increasing N levels to 150 or 180 kg ha^-1 in comparison to 120 kg ha^-1, and significantly boosted the MSI by 4.4 or 4.7%, RWC by 5.6 or 10.5%, SPAD chlorophyll by 6.8 or 5.7%, Fv/Fm by 8.2 or 11.0%%, and PI by 19.1 or 19.1%%, orderly. Intriguingly, there were no appreciable differences between the N levels of 150 or 180 kg ha^-1 ( Table 3 ).

Regarding to the EMs × N interaction, the co-application of EMs and high levels of N fertilization improved the physio-biochemical attributes of salt-stressed hot pepper ( Table 3 ). Compared to the EMs^– × N₁₅₀ treatment, applying EMs along with N at 150 or 180 kg ha^-1 increased the MSI (by 4.3 or 5.7%), RWC (by 10.5 or 11.5%), SPAD (by 4.9 or 5.4%), Fv/Fm (by 9.2 or 9.2%), and PI (by 11.3 or 11.3%), respectively ( Table 3 ). Notably, in hot pepper plants treated with EMs⁺, there was no statistical differences between the two N levels of 150 or 180 kg ha^-1 for the aforementioned physio-biochemical parameters ( Table 3 ).

The proline, TSS, and total protein contents were higher in hot pepper plants grown during the spring season, while the total capsaicin content was similar in both growing seasons ( Table 4 ). Salt-stressed hot pepper plants supplemented with EMs⁺ significantly increased the accumulation of total capsaicin by 11.6%, total protein by 12.5%, TSS by 7.4%, and proline by 20.0% more than the non-EMs-treated plants ( Table 4 ). In saline soil, N supplied at a rate of 150 kg ha^-1 significantly increased total capsaicin, total protein, TSS, and proline contents by 32.2, 8.3, 3.8, and 15.0%, respectively. While increasing N fertilization at 180 kg ha^-1 further increased all previously mentioned attributes (except for proline) by 39.0, 12.5, 7.7, and 15.0%, respectively, as compared to those that received 120 kg ha^-1 ( Table 4 ).

The integrative effect of EMs and different N fertilization on the contents of total capsaicin, total protein, TSS, and proline were significant ( Table 4 ). The integrative EMs⁺ × N₁₈₀ resulted in higher values for the aforementioned attributes by 20.8, 16.7, 7.4, and 14.3%, respectively than the integrative EMs^- × N₁₅₀. Whereas, the combined EMs⁺ × N₁₅₀ treatment yielded the highest proline content followed by the combined EMs⁺ × N₁₈₀ ( Table 4 ).

The contents of GSH and AsA in hot pepper plants were similar in the spring and autumn seasons ( Table 5 ). EMs⁺ supplementation of salt-stressed hot pepper plants increased GSH by 29.4%, and AsA by 25.0% compared with the non-EMs-treated plants ( Table 5 ). In comparison to low N fertilization (N₁₂₀), higher N levels of N₁₅₀ or N₁₈₀ notably elevated the GSH by 33.3 or 53.3%, and AsA by 21.1 or 36.8% ( Table 5 ).

Salt-stressed hot pepper plants supplemented with EMs⁺ and a high N rate of 180 kg ha^-1 displayed higher GSH (by 44.4%), and AsA (by 38.1%) compared to non-supplemented plants with EMs and received recommended N dose (N₁₅₀) ( Table 5 ). The activities of APX, SOD, and GR were greater in hot pepper plants grown during the spring season, but the CAT activity was lower when compared to the enzymes activity in plants grown during the autumn season ( Table 5 ). EMs-supplemented hot pepper plants showed greater activities of CAT, SOD, and GR than the salt-damaged hot pepper plants non- supplemented with EMs ( Table 5 ). Regarding N fertilization, as N rates increased, the activities of all the analyzed antioxidant enzymes substantially increased ( Table 5 ). Otherwise, the activities of CAT, SOD, and GR in the leaves of salt-stressed hot pepper were notably up-regulated by the ameliorating action of the integration EMs and N at N₁₅₀ or N₁₈₀, showing higher activities of these enzymes by 23.8 or 28.6%, 28.6 or 28.6%, and 66.7 or 77.8%, respectively, as compared to EMs^- × N₁₅₀ treatment ( Table 5 ).

Data of the nutrients acquisition in hot pepper plants in response to the growing season, EMs, N rate, and the EMs × N interaction are shown in Table 6 . Hot pepper plants growing during the spring season showed higher P, K⁺, Ca²⁺, and Na⁺ contents, whereas both growing seasons showed similar N values. Application of EMs promoted the nutrients acquisition; N, P, K⁺, Ca²⁺, and Na⁺ in hot pepper plants than in non-applied EMs plants grown in saline soil. In contrast to hot pepper plants that received N₁₂₀, fertilization at N₁₅₀ or N₁₈₀ significantly mediated increases in N by 10.6 or 18.0% and P by 11.1 or 13.9%, K⁺ by 6.7 or 6.6%, and Ca²⁺ by 9.2 or 13.1%, while decreased Na⁺ by 9.4 or 16.1%, respectively ( Table 6 ).

The nutrients acquisition was significantly decreased in salt-stressed hot pepper untreated with EMs and fertilized with low N dosage (EMs^- × N₁₂₀). However, co-application of EMs and higher N rates corrected these impairments in nutrient contents in hot pepper. The integrative EMs⁺ × N₁₅₀ and EMs⁺ × N₁₈₀ increased N (by 30.2 and 41.2%), P (by 13.2 and 5.3%), K⁺ (by 20.9 and 17.3%), Ca²⁺ (by 10.4 and 12.8%) associated with reduced Na⁺ (by 10.1 and 22.0%) contents, respectively, compared with the integrative EMs^- × N₁₅₀ treatment ( Table 6 ).

In gereal, the data indicated that saline soil caused a negative influence on the anatomical features of the stems and leaves of hot pepper plants. However, the application of EMs and/or higher N dosages (N₁₅₀ and N₁₈₀) reversed the previous pattern and mediated improvements in the anatomical features in hot pepper ( Figure 1 ).

In comparison to the combined EMs^- with recommended N dose (N₁₅₀), the combined EMs⁺ × N₁₅₀ or EMs⁺ × N₁₈₀ showed substantial improvements in the stem dimensions, including length by 14.0 or 19.7% ( Figure 2A ) and width by 4.5 or 7.9% ( Figure 2B ), respectively. In addition, there was an increase by 8.7 or 15.0% in the number of cortical layers ( Figure 2C ), and 24.2 or 29.1% in the thickness of the vascular cylinder ( Figure 2D ) when hot pepper plants were treated with EMs⁺ × N₁₅₀ or EMs⁺ × N₁₈₀, respectively, compared with that in EMs^- × N₁₅₀. The same treatments of EMs⁺ with either high levels of N (N₁₅₀ or N₁₈₀) also increased xylem vessel diameter, number of pith layers, pith width and length up to 18.4 or 29.6% ( Figure 2E ), 16.3 or 18.6% ( Figure 2F ), 16.7 or 21.3% ( Figure 2G ), and 19.5 or 23.4% ( Figure 2H ), respectively, in comparison of EMs^- combined with N₁₅₀.

Similarly, the anatomy of leaf features was altered in response to EMs⁺ and N₁₅₀ or N₁₈₀ fertilization ( Figure 3 ). In this regard, application of EMs⁺ × N₁₅₀ or EMs⁺ × N₁₈₀ increased the leaf blade thickness and mid vein thickness by 33.6 or 35.5% ( Figure 4A ) and 14.9 or 19.9% ( Figure 4B ), respectively, when compared to EMs^- × N₁₅₀ treatment. In addition, the width of vascular bundles increased by 13.8 or 24.2% ( Figure 4C ), the length of the leaf vascular bundles increased by 35.3 or 58.8% ( Figure 4D ) in plants treated with the combination of EMs⁺ and N₁₅₀ or EMs⁺ and N₁₈₀, respectively, compared to EMs^- × N₁₅₀ treatment. In comparison with EMs^- × N₁₅₀, the number and diameter of leaf xylem vessels also improved by 12.7 or 21.9% ( Figure 4E ), and 29.5 or 38.3% ( Figure 4F ), in N₁₅₀ or N₁₈₀ combined with EMs⁺, respectively. We also observed an increase in the thickness of palisade and spongy tissues in EMs⁺ × N₁₅₀ or EMs⁺ × N₁₈₀ by 31.0 or 31.7% ( Figure 4G ), and 35.26 or 39.0% ( Figure 4H ), respectively when compared to EMs^- × N₁₅₀ treatment.