Used of three halophilic plant growth promoting bacteria (PGPB) strains to mitigate salt stress and increase the performance of Foeniculum vulgare  under salt stress condition

ABSTRACT
This study evaluated the effects of biological fertilizers on growth, yield (fed) and quantity of fennel an experiment in two winter seasons 2023-2024 and 2024-2025 ,at the El-Sabahia Agricultural Research Station (Latitude 31o 13' 4.76" N and longitude 29o 58"26.94" E), in Alexandria Governorate, Egypt. The experimental was a completely randomized design with three replications. The eight experimental treatments included Control, Nitratireductor shengliensis Li1 PQ058684, Roseibium album Li8 PQ059401 and Halomonas elongate Sar13 PQ059853, (Li1+ Li8), (Li1 +Sar13), (Li8 + Sar13) and (Li1 +Li8 + Sar13) (Mixing treatment) theses strains obtained from previous study the bacterial strains were added in two methods: Soil drenches (S) and foliar application (F). The results indicated that the significant differences between treatments in terms of vegetative growth traits, yield attributes, applications exhibited the highest significant effects on most vegetative growth traits and yield traits with increment percentage (plant height), (Umbels number), (number of branches), order comparing with the general control.  All treatments application exhibited the highest significant effect on some chemical constituents, oil content and carbohydrates contents with increment percentage comparing with the general control. Nutrient contents in the fresh, dry tissues and seeds were also increased positively change in N, P, K content. 
Keywords: Soil salinization, osmoregulation, Membrane instability, nutritional imbalance
Introduction
          Soil salinization is a global problem that has affected 833 million ha of agricultural land in over 100 countries (Zaman et al., 2018). About 833 million hectares of soil worldwide are impacted by salt (FAO, 2021). Salt affects about 20% of the world's farmed land and 33% of irrigated land. (Machado and Serralheiro, 2017). Soil salinization is spreading at the rate of 1–2 million ha year-1 globally, affecting a significant portion of crop production and making land unsuitable for cultivation (Abbas et al., 2013; Hopmans et al., 2021). Globally, soil Salinity is the main abiotic problem facing agricultural productivity and food security across the world. So, the accumulation of different salts especially sodium and chloride ions, causes the salinization of soil and decreases the fertility of agricultural lands. Moreover, the plant goes to wilt, dry and die, at the end led to low yield and productivity (Krishnamoorthy et al., 2022). Salinity is one of the major factors reducing plant growth in most parts of the world. Because of osmotic stress and partial stomata closure, salinity stress also reduces photosynthetic potential.  Membrane instability and general nutritional imbalance can affect plants. In order for osmoregulation to take place and safeguard enzyme activity, salt-stressed plants store a variety of compounds found in organic matter, such as proline, glucose, glycin, betaine, etc., in the cell membrane. High salt concentrations in the soil reduce the water potential, making it difficult for plants to absorb water. This leads to osmotic stress, which inhibits cell expansion and division, ultimately stunting plant growth. Recent studies have shown that osmotic stress triggers the production of reactive oxygen species (ROS), causing oxidative damage to cellular components (Zhou et al., 2023). Soil is the major environmental factor that affected any grown plants. So every metabolite happened in the growing soil reflects on plant productivity and its bioactive compounds accumulate in its tissue. So, healthy soil equals healthy plant led to great productivity (Elyemni et al., 2022). Bio-fertilizers are environmentally friendly and be an effective and cost-effective substitute for chemical fertilizers with lower capital and energy input Bhardwaj et al., (2014). Furthermore, microalgae can produce metabolites such as phytohormones, polysaccharides, and so on, which can benefit agricultural production. By stimulating soil microbial activity, this fascinating group of organisms can help to improve nutrient availability, maintain soil organic carbon and fertility, and enhance plant growth and crop yield Mazhar et al., (2013).
        The Apiaceae family (Umbelliferaceae) is an important aromatic family which cultivated worldwide on a large scale due to their medical properties and various uses. Foeniculum vulgare Mill.is a member of this family (Umbelliferaceae). The essential oil found in mature fennel fruits (seeds) is used to flavor foods, including cheese, bread, pickles, liqueurs, and pastries.  Additionally, they are a component of pharmaceutical and cosmetic items. Fennel is one of the major essential oil plants cultivated and the essential oil concentration in the plants is between 3-6% in the mature fruit (Telci et al., 2009). Fennel herbs rich in active compounds and minerals (vitamin C, antioxidants, phenols, flavonoids, phosphorus, calcium and potassium).  Also, its essential oil had great medical benefits in many cultures around the world (Barzegar et al., 2020) (1).
        Nowadays, the application of halotolerant and halophilic rhizobacteria (HT- PGPRB) is used as an eco-friendly solution to face the soil salinity due to their active metabolites to alleviate and decrease the salt stress (Elkamshoush and Enas, 2024). Those halotolerant and halophilic rhizobacteria (HT- PGPRB) modulate salt tolerance in plants through different strategies; production of antioxidant compounds, phytohormones, expolysaccharides and  sugars as well as, ion homeostasis, improve soil structure and mineral uptake (Riseh et al., 2021). 

Material and methods
1- Experiment location and soil analysis 
A calcareous salt-affected soil located at the El-Sabahia Agricultural Research Station (Latitude 31o 13' 4.76" N and longitude 29o 58"26.94" E), in Alexandria Governorate, Egypt was chosen to be the soil of study due its calcareous nature and salinity. 
The experiment was done, during the two successive winter seasons of 2023/2024- 2024/2025 The seeds (fruits) of fennel were obtained from the Medicinal and Aromatic Plants Section, Horticultural Research Institute, Agricultural Research Center, Giza, Egypt. Sown in 15th November and 18th for the two seasons (2023 and 2024) in sandy soil at distances of 30 cm between hills (2-3 seeds/hill), 75 cm between rows. After germination, seedlings were thinned leaving one plant per hill after two weeks of germination. The recommended rate of phosphorus fertilizer in the form of monocalcium super phosphate (15.5% P2O5) was added rate 200 (kg/fed) during soil preparation.
The experimental design was a completely randomized design with three replicates. Prior to plant cultivation, a representative surface sample (0-30 cm depth) was collected from the soil under study, air dried, crushed using a wooden roller, sieved through a 2 mm sieve then used for determining soil physical and chemical properties following the standard methods shown thereafter. Particle size distribution was carried out by the international pipette method as outlined by Klute (1986) in which sodium hexametaphosphate is used as a dispersing agent. Soil pH was determined in 1:2.5 soil: water suspension by the pH meter (ICM 71150).Soil EC was estimated in the saturated soil paste extract using EC meter (Orion Expandable ion analyzer EA920). According to Sparks et al. (1996), the modified Walkley and Black method was used to calculate the organic carbon content. CaCO3 content was determined volumetrically using Collins calciminer, (Jackson 1973). Soluble cations (Ca2+, Mg2+, Na+ and K+) and anions (CO2-, HCO-, Cl-) were determined in the saturated soil paste extract following the methods outlined by Sparks et al. (1996).i.e. Carbonate (CO2-) and bicarbonate (HCO-) by titration with HCl; calcium and magnesium by the versenate technique; sodium and potassium by flame photometer (JENWAY PFP7 flame). Sulfates were calculated by subtracting the total determined soluble anions from the total determined soluble cations. The results are displayed in Table 1.
Table 1:Physical and chemical properties of the soil under investigation
Particle size distribution	Chemical analysis	
Coarse sand%	12.58	pH:(1:2.5(soil:water suspension)	7.8	
Fine sand%	41.90	EC dSm-1 in the soil water paste extract	6.47	
Silt%	23.01	O.M %	1.91	
Clay%	22.51	CaCO3 mg kg-1	126.8	
pH was determined in soil:water (1:2.5) suspension, while EC was determined in soil paste extract
Chemical analysis	
Available N g kg-1	28.198	
Available P mg kg-1	2 .189	
Available K mg kg-1	187.897	

2- Bio- fertilizers inoculants and treatments
Three halophilic bacterial strains obtained from previous study according to Elkamshoush and Enas (2024), identified by 16S rRNA gene recorded at international gene bank with accession number (Nitratireductor shengliensis Li1 PQ058684, Roseibium album Li8 PQ059401 and Halomonas elongate Sar13 PQ059853), were used as green and ecofriendly application to alleviate the salt stress on fennel plants grown under salt stress conditions. The fermented culture of strains used in this study was prepared in unit of bio-fertilisers, faculty of agriculture, Ain Shams University. Strains cultures were added in two ways: Soil drenches (S) and foliar application (F). The first dose of treatments was applied after two weeks of seed sowing and every 30 days until the end of the experiment. The input application of strains culture was 1 liter fermented culture from every strain (108 CFU/ ml) / 10 meter2 soil. The mixing treatments consist of three strains in equal volume to give 1 liter volume in final application.
Treatments including 
1-	Control	2-	Li1	
3-	Li8	4-	Sar13	
5-	Li1+ Li8	6-	Li1 +Sar13	
7-	Li8 + Sar13	8-	Li1 +Li8 + Sar13 (Mixing)	
Studied plant characters:
a- Vegetative characters:
1- Plant height (cm).                                     2- No. of branches plant-1
3- No. of umbels plant-1.
b- Yield characters:
1-	Seed weight plant-1 (g) 
2-	1000-seed weight (g).                             
3- Chemical analysis
A-	Soil analysis
A representative surface sample (0-30 cm depth) was collected from the soil under study. This sample was air dried, crushed using a wooden roller, sieved through a 2 mm sieve then analysed for soil physical and chemical properties as shown thereafter. Particle size distribution was analyzed using the international pipette method described by Klute (1986), with sodium hexametaphosphate employed as a dispersing agent. Soil pH was measured in a 1:2.5 soil-to-water suspension using a pH meter (ICM 71150). Electrical conductivity (EC) was measured in the saturated soil paste extract using an EC meter (Orion Expandable Ion Analyzer EA920). Organic matter content was determined by the modified Walkley and Black method, as detailed by Sparks et al., (2020). Calcium carbonate (CaCO₃) content was determined volumetrically using a Collins calcimeter (Jackson, 1973). Soluble cations (Ca²⁺, Mg²⁺, Na⁺, and K⁺) and anions (CO₃²⁻, HCO₃⁻, and Cl⁻) were determined in the saturated soil paste extract following the methods outlined by Sparks et al., (2020). Calcium and magnesium were determined using the versenate method. Sulfate (SO₄²⁻) content was calculated by subtracting the total determined soluble anions from the total determined soluble cations. Available nitrogen was determined after being extracted by 1% K2SO4, by Kjeldahl method (Page, 1982). Phosphorus and potassium were determined in sodium bicarbonate and NH4OAC solutions, according to Jackson (1973).Fe, Mn, and Zn were extracted by DTPA according to Soltanpour and Schwab (1977) and were determined by using an Atomic Absorption Spectro-photometer (novaAA 350 analytikjena). The results of these analyses are summarised in Table 1.
B-Plant analysis:
Samples of the collected plant materials were oven dried at 70 oC for 72 h, ground in a Willy Mill then plant portions (equivalent to 0.2g of the dried plant leaves and seeds) were digested with H2SO4 - H2O2 according to Parkinson and Allen (1975). Total contents of macro nutrients were assessed in plant digests as follows: N by micro-Kjeldahel apparatus, P spectro-photometrically via Spectrophotometer (JENWAY 6405 UV/Vis) and total K by flame photometer (JENWAY PFP7 flame). One milliliter of ethanol extracts of fennel seeds after homogenies and grinded (1 gram soaked in twenty five milliliter of ethanol alcohol 99% kept at room temperature for three days) was used to determined total phenols as described by Manzoor et al., (2019), while, the antioxidants activity using 2, 2’diphenylpicrylhydrazyl (DPPH) reagent and quercetin as a positive control used to determine the % antioxidants in fennel seeds according to (Burits and Bucar, 2000). Proline determined spectrophotometry by Bates et al. (1973). Dried seeds of fennel plants of each treatment were used to determine total carbohydrates as described by Smith et al. (1956). Oil content in seeds was determined according to the standard method described by AOAC. (1990).
4- Statistical analysis:
The analysis of variance method outlined by Snedecor and Cochran (1982) was used to all of the collected data. The least significant difference (LSD) at 5% level of significance to compare differences among means
Results
Characteristics of vegetative growth and yield: 
The present study demonstrates that drenching soil and foliar applications methods significantly affected the quality, growth and yield of fennel plants. The response of fennel (Foeniculum vulgare Mill.) plants growth performance of the plants was indicated by recording in plant height (cm), No. of branches/plant, No. of umbels/plant seed weight per plant, weight (g), 1000-seed weight (g).  In saline calcareous soils, results obtained that all growth and production characteristics improved in all treatments that received bacteria in both single and mixed applications, but mixed applications improved more than single applications Tables (2 and 3). 
The response of fennel plant cultivars to foliar spray of during vegetative development appears to be positive. The yields of cultivar single Sar13 were significantly (P < 0.05) influenced by soil fertilization and foliar applications in 2023, 2024 and for cultivar Li1 +Li8 + Sar13, the greatest yield occurred in Li1 +Li8 + Sar13 applications with 64.72 and 60.22 g. seed plant-1 in successive years, respectively.
Data in Tables (2 and 3) showed that the application of Sar13 resulted in the highest values in the plant height (cm), No. of branches/plant, No. of umbels/plant seed weight per plant, weight (g), 1000-seed weight (g). as compared with a single application. While applied treatment of Li8 + Sar13 increased plant height (cm), No. of branches/plant, No. of umbels/plant seed weight per plant, weight (g), 1000-seed weight (g), and more than the other doubles treatments. Generally, the mixing treatments of Li1 +Li8 + Sar13 were a superior treatment showed the highest values in plant height (cm), No. of branches/plant, No. of umbels/plant seed weight per plant, weight (g), 1000-seed weight (g). as compared with all treatments. Also Data in Tables 2 and 3) showed that the application of soil drenches (S) recorded high values compared to foliar application within all treatments the two winter seasons under saline calcareous soil conditions. These results, similarly with Zaki et al,. (2019) elucidated that the application of Bacillus megaterium gave a positive effect on the fresh and dry weight of bulb of fennel plants. Also, Kearl et al., (2019) said that inoculating alfalfa seedlings with Halomonas sp. and Bacillus sp. then growing those seedlings in soil watered with % NaCl, the Bacillus sp. treatments gave higher growth in shoots and roots compared with other treatments. Cappellari and Banchio (2020) found that inoculation of Mentha piperita L. with Bacillus amyloliquefaciens reduces the negative effect of salt stress on treated plants and increases the growth.
Table 2: Effect of applicants in first season winter 2023 -2024 on some vegetative parameters of fennel plants   
treatments	Plant height at 70 DAS	Plant height at harvest	No. of branches/plant	No. of umbels/plant	Seed per plant (g)	Weight of 1000 seeds (g)	
	S	F	S	F	S	F	S	F	S	F	s	F	
control	41.3a	65.2a	3.60a	41.2a	39.11a	2.82a	
Li1 	48.0b	40.0b	119.8b	99.2b	6.7b	4.70b	67.15b	54.5b	48.50b	42.6b	3.83b	3.65b	
Li8	53.3c	44.3c	121.7b	104.1b	7.8b	5.92c	70.28c	58.10c	49.72c	44.7c	3.90c 	3.70b	
Sar13	60.3d	49.0d	130.8c	112.3c	9.1c	7.10d	73.15d	61.22d	51.21d	48.5d	3.92c	3.74b	
Li1+ Li8	75.5e	56.2e	139.8d	119.6c	12.7e	9.22e	77.89e	69.27e	55.60e	51.10f		4.13d 	4.02c	
Li1 +Sar13	72.0f	60.0f	144.8e	127.2d	13.6f	10.0e	82.35f	74.34f	59.91f	54.3f	4.37e	4.13c	
Li8 + Sar13	79.0g	67.4g	152.6f	135.7f	14.1f	12.10f	84.00g	79.5g	61.34g	57.30g	4.41f	4.22d	
Li1 +Li8 + Sar13	82.5h	74.5h	158.8g	143.2g	17.8g	15.11g	89.24h	83.21h	64.72h	60.22h	4.60g	4.39e	
L.S.D 	2.12	1.2	3.26	5.34	1.22	0.82	0.77	0.82	0.32	0.81	0.12	0.1	
Soil drenches (S) and foliar application (F)
Table 3: Effect of applicants in second season winter 2024 -2025 on some vegetative parameters of fennel plants   
treatments	Plant height at 70 DAS	Plant height at harvest	No. of branches/plant	No. of umbels/plant	Seed per plant (g)	Weight of 1000 seeds (g)	
	S	F	S	F	S	F	S	F	S	F	s	F	
control	39.8a	62.1a	4.30a	29.25a	35.10a	2.906a	
Li1 	54.3b	44.1b	111.6b	75.2b	7.9b	5.00b	63.71b	40.52b	45.54b	40.5b	3.52b	3.11b	
Li8	63.1c	49.2c	115.2c	80.1c	8.8c	6.30c	67.89c	43.46c	46.11c	43.0c	3.76c	3.27b	
Sar13	75.3d	54.2d	123.3d	87.7d	9.0c	7.49d	70.51d	47.25e	46.52d	44.2d	4.23d	3.92c	
Li1+ Li8	83.2e	67.2e	131.6e	95.9e	10.9d	8.72e	73.22e	52.00d	51.10e	48.10e	4.43e	4.12d	
Li1 + Sar13	89.5f	69.0f	139.4f	101.2f	12.1e	9.90f	78.85f	59.46f	56.73f	51.3f	4.63f	4,42e	
Li8 + Sar13	109.0g	73.4g	145.9g	115.7g	13.3f	10,00f	80.73g	66.42g	58.94g	53.10g	4.70g	4.53f	
Li1 +Li8 + Sar13	139.4h	78.5h	151.2h	130.5h	16.2g	12.26g	86.42h	71.66h	63.55h	56.55h	4.82h	4.63g	
L.S.D 	2.16	1.22	3.20	5.40	1.23	0.91	0.89	0.88	0.30	0.79	0.11	0.1	
Soil drenches (S) and foliar application (F)
The studied treatments significantly affected the 1000-seed weight (Table 2 and 3). Means of the showed that the highest 1000-seed weight (4.6, 4.39 g in the first season and 4.82, 4.63 g in the second season, respectively) were associated with the control treatment, that is, non-application of the bio-fertiliser.
Leaf mineral contents:
        Soil fertilisation and elevated foliar applications increased the content of almost all nutrients in leaves of both fennel plant cultivars in both experiment years. Nutrient contents in the fresh and dry tissues were also measured to assess nutrient content in fennel plants. Saline-stressed fennel plants (control) exhibited low levels of N, P, K (Tables 4 and 5). On the other hand, all different treatment methods increased the content of N, P and K, respectively, compared to control plants grown in saline calcareous soil in both soil drenches (S) and foliar application. 
Table 4: Effect of applicants in first season winter 2023 -2024 on Macronutrients content of fennel plants
Treatments	N %	P%	K mg.kg-1	N %	P%	K mg.kg-1	
	Fresh leaves	Dry leaves	
	S	F	S	F	S	F	S	F	S	F	S	F	
Control	2.76a	1.30a	238.32a	0.16a	0.176a	83a	
Li1	3.03b	3.68b	2.86b	2.76b	314.99 b	246.38b	0.321b	0.20b	0.278b	0.199b	160b	88b	
Li8	3.19c	3.76c	2.87b	2.82b	317.38 b	259.21c	0.357c	0.22c	0.281c	0.210c	168c	91b	
Sar13	3.27c	3.81c	3.14c	3.00c	317.90 b	276.95d	0.382d	0.25d	0.283c	0.213c	174d	98c	
Li1+ Li8	3.36d	4.72d	3.18d	3.19d	338.50 c	273.67d	0.423e	0.28e	0.287d	0.230e	187e	101c	
Li1 + Sar13	3.47e	4.80e	3.23e	3.30e	350.02 d	281.43d	0.432f	0.29e	0.288d	0.238e	191e	106e	
Li8 + Sar13	3.52e	4.84e	3.24e	3.41f	355.33d	289.54d	0.440g	0.30e	0.294e	0.244f	200f	115f	
Li1 +Li8 + Sar13	3.64f	5.21f	3.29f	3.46f	422.50e	303.63e	0.465g	0.33f	0.320f	0.251g	210g	122g	
L.S.D 	0.16	0.07	0.03	0.07	8.71	8.33	0.02	0.01	0.02	0.01	5.2	4.6	
Table 5: Effect of applicants in second season winter 2024 -2025 on Macronutrients content of fennel plants
Treatments	N %	P%	K mg.kg-1	N %	P%	K mg.kg-1	
	S	F	S	F	S	F	S	F	S	F	S	F	
	Fresh	Dry	
Control	1.75a	1.66a	170a	0.195a	0.127a	72a	
Li1 	2.46b 	1.92b	2.41 	2.00	447b	189b	0.357b	0.300b	0.272b	0.259b	159.0b	76b	
Li8	2.52b	2.02b	2.54 	2.17	449b	205c	0.360c	0.321c	0.279b	0.262c	173.8c	94.5c	
Sar13	2.90c	2.20c	2.60 	2.26	453b	215d	0,363d	0.322c	0.285b	0.266c	179.2d	99.4d	
Li1+ Li8	3.22c	2.55d	2.76 	2.31	464c	222e	0.398e	0.343d	0.298c	0.276d	184.7e	107e	
Li1 + Sar13	3.46d	2.63d	3.00 	2.37	473d	235f	0.424e	0.356e	0.321d	0.278d	198.3f	112f	
Li8 + Sar13	3.65e	2.85e	3.35 	2.41	480e	244g	0.429e	0.379f	0.332e	0.279d	200.3f	120g	
Li1 +Li8 + Sar13	4.13f	3.19f	3.87 	2.46	562f	262h	0.475f	0.393g	0.355f	0.324e	220.0g	129h	
L.S.D 	0.11	0.11	0.10	0.02	4.2	3.2	0.02	0.01	0.02	0.01	2.1	2.0	
Soil drenches (S) and foliar application (F)

        Data in Tables (4 and 5) showed that the nitrogen, phosphorus and potassium contents in plant leaves. In respect of the presence of bacteria, single and mixed treatments in the presence had higher contents than other treatments and the control. Results in the above-mentioned Tables (4 and 5). Showed that there were increased in the nitrogen (N) content, Phosphorus (P) content and potassium (K) values. The highest level of N, P, K The largest nitrogen, phosphorus, and potassium ratios were 4.13 %, 3.87 % and 562 mg.kg-1 in fresh leaves, respectively as a soil drenches, while dry leaves recorded 0.475, 0.355 and 220.0 mg.kg-1 to the nitrogen (N) content, Phosphorus (P) content and Potassium content (K), respectively. The same trend showed in foliar application treatments. A single Sar13 treatment showed high content of N, P, K in compared with other single treatments Li1 or Li8.
The superior treatment of bio-fertiliser application in macronutrients N, P and K was the mixing culture of Li1 +Li8 + Sar13. The lowest value was obtained by the control treatment in two seasons. These results are consistent with the availability of nutrients in the soil resulting from the activity of the bacteria used in the treatments. The vegetative parameters were enhanced in this study due to the application of biofertilizers of halophilc bacteria. Which improved the growth of fennels plants by different mechanisms chelating the elements by production of organic acid to facilitate the nutrient in soluble form for plants to be adsorbed led to maximize the photosynthesis phytohormones production specially the indole acetic acid play a certical role in plant development, ion exchange, stomatal conductance and improve plant water capacity in soil by producing exopolysaccharides that keep the water available around the roots and deleted the bad effect of Na+ ion available for plants uptake Gupta et al., (2019) and (Khairnar et al., 2022). All these physiological processes led to high quantity of strong vegetative growth with a satisfactory yield at the final.  

Chemical constituents: 
Soil salinity affects plant chemical constituents, which help plants to endure difficult circumstances. The edible components of fennel contain compounds that have been found to have hepatic, antioxidant, and antibacterial qualities. Fennel plants with applied Li8+ Sar13 recorded the highest level of Antioxidants, while plants untreated showed the highest level of Total Phenols and Proline. Also, the application of Li1+Li8+Sar13 (Mixing) recorded the highest level of Carbohydrates and Oil % all treatments in comparison to plants in (Table 6 and 7). On the other hand data showed that the foliar application recoded high values in the seeds, Chemical constituents of fennel plants compared with drench soil application in two seasons under this experimental condition.    

Table 6: Effect of applicants in first season winter 2023 -2024 on some chemical composition of seeds fennel plants
Treatment 	Antioxidants (%)	 Total Phenols (mg/100 g.d.w)	Carbohydrates (mg/100 g.d.w)	Proline 
(mg/g.d.w)	Oil %	
	S	F	S	F	S	F	S	F	S	F	
Control 	49.8a	50.21a	27.7f 	29.4f 	1.6a	1.8a	150.8h	152.6h	1.99c	1.82c	
Li1	63.6c	64.6c	16.2e	18.4e	7.5b 	8.3b 	96.6g	98.0g	2.70c	2.42c	
Li8	62.6c	63.3c	16.6e	18.9e	9.0bc	9.8bc	59.5f	61.2f	2.85c	2.55c	
Sar13	65.9d	69.4d	13.7d	16.2d	9.9cd	11.2cd	35.1e	37.2e	2.99b	2.74b	
Li1+Li8	54.6b	55.9b	9.4c	11.0c	9.5c	11.1c	20.2d 	21.6d 	3.12a	3.00a	
Li1+Sar13	50a 	53a 	4.4b	5.7b	9.8c	12.0c	15.9c	17.0c	3.21a	3.17a	
Li8+Sar13	74.1e	75.5e	1.2a	1.5a	11.6d	12.8d	9.56b	10.6b	3.28a	3.24a	
Li1+Li8+Sar13 (Mixing)	65.9d	67.2d	0.7a	0.86a	11.6d	13.0d	4.25a	5.11a	3.89	3.55	
L.S.D 	1.6	1.43	1.8	1.67	1.8	1.63	2.1	1.88	1.32	1.17	
Soil drenches (S) and foliar application (F

Table 7: Effect of applicants in second season winter 2024 -2025 on some chemical composition of seeds fennel plants
Treatment 
	Antioxidants (%)	Total Phenols (mg/100 g.d.w)	Carbohydrates (mg/100 g.d.w)	Proline 
(mg/g.d.w)	Oil %	
	S	F	S	F	S	F	S	F	S	F	
Control 	50.8a	51.1a	29.2g	31.5g	1.4a	1.6a	156.4h	157.2h	2.12c	1.92c	
Li1	64.4c	66.3c	15.1e	16.7e	6.3b	7.1b	98g	100.1g	2.90c	2.50c	
Li8	63.5c	67.6c	17.8f	19.1f	8.4c	8.9c	56.5f	58.1f	2.96c	2.69c	
Sar13	66.7d	69.1d	12.4d	13.5d	8.9cd	9.5cd	39.7e	40.5e	3.04b	2.74b	
Li1+Li8	55.3b	59.3b	7.6c	8.4c	10.5de	11.7de	19.3d	21.1d	3.43a	3.10a	
Li1+Sar13	55b 	55a 	5.7b	6.4b	10.5de	11.9de	16.9c 	17.4c 	3.55a	3.35a	
Li8+Sar13	75.2e	76.5e	1.87a	2.00a	12ef	13.2ef	9.36b	10.6b	3.63a	3.43a	
Li1+Li8+Sar13
(Mixing)	66.6d	69.4d	0.89a	1.09a	12.4f	13.7f	4.9a	5.6a	4.01	3.81	
L.S.D 	1.8	1.41	1.6	1,34	1.8	1.61	2.1	1.66	1.34	1.19	
Soil drenches (S) and foliar application (F
Antioxidant activity:
Data in Tables (6 and 7) represented the high percentages of antioxidants activity in all treatments compared to control treatment (49.8- 50.8%) and (50.21- 50.1%) in 2023-2024 and 2024-2025 seasons in soil and foliar applications, respectively, the mixing treatments (Li8+Sar13) in two seasons with soil and foliar application was the best (74.1- 75.2%) and (75.5- 76.5%), respectively then the mixing treatment of (Li1+Li8+ Sar13) gave (65.9- 66.6 %) and (67.2- 76.5%), respectively. Salama et al. (2015) observed that organic and bio-organic fertilisation improved the vitamin C ranged 32.88- 34.00 mg/100gram fresh weight, antioxidant activity, and total phenolics and total flavonoids in fennel bulbs. Also, the stressed plants by biotic and abiotic stresses led to the generation and accumulation of reactive oxygen species (ROS). These ROS had a strongly oxidising form on the cell compounds. Antioxidant compounds play an effective role in detoxifying the ROS effect (Khan et al., 2021) and (Saberi Riseh et al., 2021).
Total phenols.
Tables (6 and 7) showed the significant reaction of all treatments compared to control, the reduction of total phenols in inoculated fennel plants ranged from (0.7- 16.2 mg gallic acid/ 100 g.d.w) and (0.89- 15.1 mg gallic acid/ 100 g.d.w) compared to control plants (27.7 mg/gallic acid / 100g.d.w) and (29.2 mg/gallic acid / 100g.d.w). The best value was in mixing treatments of three strains (0.7 mg gallic acid/ 100 g.d.w) and (0.89 mg gallic acid/ 100 g.d.w) with drench soil application in first season 2023-2024 and the second season 2024-2025, respectively. On the other hand, the total phenols content was the best in foliar application compared to drench soil application. Our results in agree with the study done by Beyazen et al., (2017), who confirmed that the water extract of fennel seeds had a low total phenolic compound value as compared to methanol. Another study by Rajić et al., (2018) found that the ethanol extract of fennel seed contains a higher phenolic compound compared to control. Always, the first barriers induced by different stresses were total phenols and proline, especially in salt stress. Proline an amino acid which accumulates during the growth of plant in salt condition plays a significant role to protect the cell proteins from damage. Also, proline increases the photosynthetic activity and plant growth (Liu et al., 2017) and (Saberi Riseh et al., 2021). 

Total carbohydrates.
Data of total carbohydrates in tables (6 and 7) recorded significant differences between all treatments in drench soil application ranged from (7.5-11.6 mg/100g.d.w) and (6.3-12.4 mg/100 g.d.w). While in foliar application (8.3-13.0 mg/100g.d.w) and (7.1- 13.7 mg/100g.d.w), compared to control treatment (1.6- 1.4 mg/100g.d.w) and (1.8- 1.6 mg/100g.d.w), in both seasons respectively. While the highest total carbohydrates recorded in the mixing treatment (Li1+Li8+ Sar13). These results are similar to those reported by some researchers Nada (2014) confirmed this, as a combination of Vescular Aarbscular Myccohyraa  (VAM) with Azos+Bm+Bc was superior to Azotobacter sp.+ Bacillus megaterium + Bacillus circulans and Azotobacter sp. + Vescular Aarbscular Myccohyraa in accumulating total carbohydrates in Calendula officinalis. The vegetative parameters enhanced in this study due to the application of biofertilizers of halophilc bacteria. Which improved the growth of fennels plants by different mechanisms chelating the elements by production of organic acid to facilitate the nutrient in soluble form for plants to be adsorbed led to maximize the photosynthesis phytohormones production specially the indole acetic acid play a certical role in plant development, ion exchange, stomatal conductance and improve plant water capacity in soil by producing exopolysaccharides that keep the water available around the roots and deleted the bad effect of Na+ ion available for plants uptake Gupta et al. (2019) and (Khairnar et al., 2022). All these physiological processes led to high quantity of carbohydrates and strong vegetative growth with a satisfactory yield at the final.  Increase happened in yield and total carbohydrates of Dill plants was observed by Darzi et al. (2012).
Proline.
In both seasons, generally, the treatments gave positive significant results compared with control treatments, tables (6 and 7) the control plant grown under salt stress conditions recorded maximum proline (150.8-156.4 mg/g.d.w) and (152.6- 157.2 mg/g.d.w)in soil and foliar experiments compared with the values of proline in all bioinoculated treatments. In contrast, the lowest proline in two seasons determined with mixing treatments (Li1+Li8+Sar13) were (4.25-4.9 mg/ g.d.w) and (5.11- 5.6 mg/g.d.w), in soil and foliar application treatments, respectively. Mostafa (2015) found that improved the growth of fennel plants under salt stress condition by using biostimulants, which decreased the proline compared with control. Haroon et al. (2021) found that the inoculation with plant growth promoting bacteria for example Burkholderia sp., Azospirillum sp., Arthrobacter sp., Rhizobium sp., Bacillus sp. and Pseudomonas sp. help plants to generate osmolytes such as proline and betaine helped plants to resist salt stress conditions. Similar study was conducted by (Egamberdieva et al., 2019). 
Oil percentage
The oil percentage illustrated in Tables 6 and 7 proved that the applications, foliar or soil applied, increased oil percentage. The level oil percentage reached maximum of 3.89 and 3.55 when treated with Li1+Li8+Sar13 (Mixing) in first season and 4.01 and 3.81 in second season, respectively for soil application and foliar application, compared to their controls and other treatments. That increase in oil seeds percentage was a result related to the quality plants growth and its had resistant for soil salinity factor by affected of bacteria to plants as foliar or on soil, which increases nutrient availability in the soil.
Table 8: Effect of applicants in first season winter 2023 – 2024 and 2024 -2025 on content of NPK of fennel seeds.
	N	P	K	N	P	K	
	Frist 	Second	
Control	0.220a	0.215a	0.341a	0.235a	0.23a	0.31a	
Li1 	0.338b	0.321b	0.263b	0.248b	0.407b	0.385b	0.372b	0.348b	0.26b	0.25b	0.37b	0.33b	
Li8	0.350c	0.347b	0.271c	0.257b	0.424c	0.416c	0.386c	0.356c	0.28b	0.26b	0.44c	0.34b	
Sar13	0.355c	0.352b	0.280d	0.263b	0.431d	0.418c	0.389c	0.375d	0.31c	0.27b	0.48d	0.40c	
Li1+ Li8	0.382d	0.373c	0.294e	0.277c	0.459e	0.440d	0.412d	0.386e	0.33c	0.29c	0.50d	0.45d	
Li1 + Sar13	0.390d	0.381c	0.317f	0.288d	0.465f	0.445d	0.419d	0.399f	0.36d	0.30c	0.53e	0.47e	
Li8 + Sar13	0.398d	0.387c	0.329g	0.294d	0.480g	0.449d	0.427f	0.418g	0.37d	0.33d	0.55e	0.49e	
Li1 +Li8 + Sar13	0.421e	0.400d	0.327h	0.310f	0.524h	0.495f	0.440g	0.429h	0.41e	0.39e	0.58f	0.54f	
L.S.D 	0.011	0.011	0.01	0.01	0.012	0.011	0.012	0.010	0.02	0.01	0.022	0.01	

Macronutrient contents N P K:
Seed nutrient contents N, P, K percentages were decreased by soil salinity. Data presented in Table (8) showed that the biofertilizer attained the maximum content of N for plants grown in salt soil and with Li1 +Li8 + Sar13 treatment. also, P and K in two seasons. In general, data showed that the soil applications increased the seed nutrient contents N, P, K percentages more than that of foliar applications. 
Data in table (8) showed the ratio of nitrogen, phosphorus and potassium contents in seeds. As affected by bacteria, single and mixed treatments in the presence had higher contents than other treatments and the control. Results in the above-mentioned Table (8). Showed that, the all treatments were increased in the nitrogen (N) content, Phosphorus (P) content and potassium (K) values. The highest levels of N, P, K were shown as affected of the superiority treatment of bio-fertilizer application in N% P, and character was bio Li1 +Li8 + Sar13. The lowest value was obtained by the control treatment in two seasons.
The largest nitrogen, phosphorus, and potassium ratios were 0.421 %, 0.33 % and 0.524% in seeds, respectively as a soil drench, while dry leaves recorded 0.4% 0.31 and 0.495% to the nitrogen (N) content, Phosphorus (P) content and potassium (K), respectively as foliar application. The same trend showed in foliar application treatments. A single Sar13 treatment showed high content of N, P, K in compared with other single treatments Li1 or Li8. These results were consistent with the plants' leaves content of nutrients which were affected by the availability of nutrients in the soil resulting from the activity of the bacteria used in the treatments. 

Table (9) Effect of applicants in two season’s winter 2023 – 2024 and 2024 -2025 on salt calcareous soil affected (mean of two seasons). 
Treatments	EC	pH	O.M%	N	P	K	Fe	Mn	Zn	
				mg.kg-1 soil	
	Soil application	
Control	6.42a		1.16a	36.74a	2.21a	166 a	0.18a	0.12a	0.08a	
Li1	6.31b	7.55	1.21b	40.84b	2.78b	177.22b	0.23b	0.16b	0.11b	
Li8	6.29c	7.51	1.22b	57.34c	3.12c	192.44c	0.31c	0.19c	0.14c	
Sar13	6.29c	7.48	1.24c	60.26d	3.32d	200.04c	0.37c	0.23d	0.17d	
Li1+ Li8	6.10d	7.43	1.29d	64.34e	3.50e	220.00d	0.42e	0.28e	0.22e	
Li1 + Sar13	6.09d	7.46	1.24d	64.67e	3.54e	195.50c	0.40e	0.24d	0.23e	
Li8 + Sar13	6.08d	7.39	1.27e	69.49f	3.59e	221.48d	0.44e	0.28e	0.29f	
Li1 +Li8 + Sar13	5.87e	7.35	1.31f	72.66g	3.65e	236.28e	0.47f	0.32f	0.31g	
L.S.D	0.12		0.01	2.16.	0.22	8.28	0.02	0.01	0.01	
	Foliar application	
Control	6.42a	7.69	0.20a	30.36a	2.78a	177.05a	0.20a	0.16a	0.10a	
Li1	6.40a	7.64	0.20a	32.67b	2.79a	177.34a	0.22a	0.16a	0.11a	
Li8	6.40a	7.64	0.22b	34.14c	2.89b	180.74b	0.23a	0.17a	0.12a	
Sar13	6.40a	7.63	0.23b	36.28d	2.97b	182.26b	0.25b	0.19b	0.13b	
Li1+ Li8	6.38b	7.63	0.20a	34.33c	2.81a	178.00a	0.22a	0.17a	0.11a	
Li1 + Sar13	6.37b	7.64	0.20a	37.62e	3.02c	182.10b	0.22a	0.17a	0.12b	
Li8 + Sar13	6.37b	7.63	0.21a	35.33d	2.77a	180.00b	0.20a	0.17a	0.12b	
Li1 +Li8 + Sar13	3.32c	7.62	0.23b	56.84f	3.22d	191.44c	0.30c	0.17a	0.11a	
L.S.D	0.11		0.01	1.21.	0.14	2.2	0.02	0/01	0.01	

Results present in Table (9) investigated that the applied of applications and treatments were affected in soil chemical properties and availability of macro and micronutrients. while the soil application showed high values compared with foliar application, which showed slight effects for organic matter, macro and micronutrients soil content values. Also, in the soil pH and soil E.C.    
Data in Table (9) showed that the soil application of a single treatment of sar1 recorded high increase in organic matter, macro and micronutrients soil content reaching 0.24 mg.kg-1 soil for soil organic matter and 60.26 mg.kg-1, 3.32 mg.kg-1 and 200.04 mg.kg-1 for N, P and K, respectively. Also, micro nutrients Fe, Mn and Zn reached to 0.37, 0.23 and 0.17 mg.kg-1. respectively. and showed a decrease in soil pH and E.C. compared with Li1 or Li8. Also, treatment of Li8 and Sar1 showed an increase in available macro and micronutrient soil content for compared with other double treatments. On the other hand, the mix treatment of (Li1 + Li8 + Sar1) showed the highest organic matter, macro and micronutrients of soil content reaching 0.31 mg.kg-1 soil for soil organic matter and 72.66 mg.kg-1, 3.45 mg.kg-1 and 236.28 mg.kg-1 for N, P and K, respectively. Also, micro nutrients Fe, Mn and Zn reached to 0.47, 0.32 and 0.31 mg.kg-1. and high decreased for soil pH and E.C. values as soil application under the experimental condition. As the treatments showed a beneficial effect in soil pH and soil E.C. as affected by Bactria used.     
The changes in soil pH, E.C., organic matter percentage and some nutrient contents such as nitrogen, potassium, phosphorus, and micronutrients Fe, Mn and Zn as increasing in organic matter, macro and micronutrients of soil content, may be due to the activity of bacteria used. Also, maybe it could fixed air oxygen. 
This alternative is provided by biological nitrogen fixation (BNF), 
a microbiological procedure that transforms atmospheric nitrogen into a form that plants can utilize. Nitrogen-fixing systems provide a financially appealing and environmentally responsible way to reduce external inputs and enhance internal resources (Rashid et al., 2016).  Barney, B. M. (2020). And also, it compounds products of its activities release potassium from soil and converting phosphorus to an available form in soil. On the other side that the compounds products of Bactria used decreased soil pH and soil E.C., the biological activity and their released activity substances that enhancing nutrients solibilization from both native and added sources, besides the favorable biological conditions that are keeping them in available forms and their mobility for uptake by plant roots Li et al., (2015a), whose studies the mechanisms for solubilization of various insoluble phosphates and activation of immobilized phosphates in different soils by an efficient and salinity-tolerant. Jain et al., (2022) studied the effect of potassium-solubilising microorganisms as soil health engineers. Also, the effect of bacteria in decreased soil pH help on increased solubilization of phosphorus, potassium Iron, Zinc and Manganese in the soil solution.         
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