1 Introduction

Onion (Allium cepa L.) is one of the most popular vegetables that is consumed worldwide throughout the year, it is rich in minerals, carbohydrates, protein, and fibers (El-Sherbeny et al. 2022). It represents a rich source of vitamins (A, B1, B2, B3, B6, C, and E), and various antioxidant compounds especially flavonoids (Sami, et al. 2021). These compounds are accountable for the quality of onions in terms of their distinctive taste, aroma, and the majority of their biological traits. As well as strengthen immunity and contribute to the treatment and avoidance of several serious diseases such as reducing the risks of obesity, cholesterol, cardiovascular disease, and cancer (Younes et al. 2021). These days, the world faces a great challenge in providing crops, especially in the light of the successive population increases which are expected to reach 9.7 billion in 2050 (Gaigbe et al. 2022), rapid climate changes, and the dropping in soil quality, especially the calcareous one. The amount of calcareous soil in Egypt is around 0.65 million acres (0.26 million hectares) which makes up 25–30% of the entire area (Taalab et al. 2019). This soil type has several chemical, and physical problems including high pH, calcium carbonate, infiltration rate, soil crusting, and hardening of the subsurface layer. It also has low availability of macro and micronutrients (especially Mg, K, and Ca), organic matter content, water holding capacity, poor structure, and low microbial activity (Zahran et al. 2020; Hefzy et al. 2020; Mahmoud et al. 2023). All the previous issues reflect on the crop's physiological and biochemical processes resulting in decreasing the crop's yield and quality and increasing the infestation by pests and pathogens (Rai et al. 2021). Several efforts are required to develop strategies for the conversion of these soils into healthful productive soils by using cost-efficient and easy-to-use techniques, particularly when planting onions which are considered an important strategic crop in sandy calcareous soils.

Plant hormones play a critical role during all the plant stages of growth and development. Gibberellic acid (GA3) has wide consideration all over the world for its valuable and promising application in the agricultural field (Pandya et al. 2023). Gibberellic acid is a natural phytohormone produced by plants and microorganisms with a tetracyclic dihydroxy-γ-lactone acid and chemical formula C19H22O6 (Camara et al. 2018). Microorganisms represent a promising industrial source for GA3 production, especially fungi and bacteria with endophytic origins (Camara et al. 2018). The highest producers of GA3 are filamentous fungi related to the genus Fusarium includes F. chlamydosporum, F. oxysporum, F. sacchari, F. moniliforme, F. solani, F. konzum, F. verticilloides, and F. glutinans (Mohmed and Mahmoud 2018; Gupta et al. 2021). Gibberellic acid stimulates plant root formation, increases water and mineral absorption (especially K, P, and N), and enhances the germination rates of the seeds even in stressful conditions (Parveen et al. 2023). Also, it regulates cell division, and elongation, improves the development of chloroplasts, prevents the breakdown of chlorophyll, and reduces the levels of reactive oxygen species that lead to cell death (Alharby et al. 2021; Ritonga et al. 2023). Foliar applications of various plant growth regulators like GA3 are a good remedy to regulate the nutrient uptake of plants and increase the crop's yield, and quality (Leilah and Khan 2021). External applications of GA3 with low amounts of phytohormones have a significant impact on plant growth regulation, whereas excessive concentrations have the opposite effect (Islam et al. 2023). Administering GA3 externally at the proper dose was found to have enhancement effects on crop development, nutrient absorption, and yield quality (Miceli et al. 2019).

Vitamins are one of the most significant organic compounds for promoting plant development, cofactors for numerous critical enzymes, and major players in plants' metabolic processes (Li et al. 2021). Water soluble vitamins, especially vitamins B1, B2, and C, play influential roles in changing the redox chemistry of plants by acting as antioxidants which enhances the plant defense system (Kausar et al. 2023). These vitamins are crucial for lowering oxidative stressors brought by abiotic and biotic stress and are used for reactive oxygen species (ROS) detoxification as ROS scavengers (Sadak 2023), which improve agricultural production and withstand biotic and abiotic stresses. The chemical synthesis of plant growth enhancers has many economic, environmental and safety concerns including high energy consumption, high cost, utilization of non-renewable chemicals materials, generation of toxic residues, and hazardous waste (Wang et al. 2021). Microbial production of these products is the safe, low-cost, green and sustainable way for both the environmental, and the economic standpoints. Yeasts especially Saccharomyces cerevisiae and Zygosaccharomyces bailii are common ascorbic acid (vitamin C) producers using sorbose, galactose, and glycerol media, while Gluconobacter oxydans, Ketogulonigenium vulgare, and Bacillus endophyticus bacteria produce it on sorbose medium (Sugisawa et al. 2005; Ma et al. 2019). Ashbya gossypii, and Bacillus subtilis are the common microbial producers of riboflavin (Chu et al. 2022; Chen et al. 2022). However, bacterial production now utilizes metabolic-engineered strains of Bacillus subtilis in large-scale riboflavin fermentations (Liu et al. 2023). Microbial thiamine is not commonly produced by microorganisms and is produced by a genetic transformation process using E. coli (Cardinale et al. 2017). External applications of ascorbic acid enhance the plant's tolerance to biotic and abiotic stress, plant development, absorption of nutrients, yield, and quality of garlic (Ali 2017), barley (Yaseen et al. 2021), potato (Selem et al. 2022), and wheat (El-Hawary et al. 2023). Spraying thiamine (vitamin B1) on plants enhances the plant tolerance to stress, nutrients uptake, quality and yield of potato (Gouda et al. 2015), faba bean (El-Metwally and Sadak 2019), cauliflower (Jabeen et al. 2022), and pea (Kausar et al. 2023). Riboflavin (vitamin B2) as a spray in the appropriate quantity on the plant's exterior led to the same positive trend in some crops, such as roselle (Azooz 2009), and maize (Xin Chi et al. 2021).

Due to the population rates increasing and needs, the agricultural areas dropping, and the crop productivity decreasing, we desperately need to reclaim all soil types especially the new ones with low fertility with basic crops like onions to increase our agricultural sources. Also, we need to enhance the crop's productivity and quality by using natural plant growth enhancers with eco-friendly, and safe properties to preserve soil sustainability and environmental safety. To our knowledge, prior research has not taken into account the idea of applying GA3 and different vitamins generated by microbial processes in onion plants. Therefore, the current study was set up to investigate the synergetic effects of two plant growth enhancers on onion growth and the yield quality in newly reclaimed soil. We evaluated the impacts of spraying the microbial phytohormone (GA3) and microbial vitamins (C, and B2) during two successive seasons on the growth traits (plant height, number of leaves, plant fresh weight, bulb diameter, and neck diameter), yield, chemical (N, P, and K) and biochemical composition (total sugars, total proteins, total antioxidants, vitamin C, total phenols, and total flavonoids) of the onion plants in sandy calcareous soil.

2 Materials and Methods

2.1 Microbial treatments preparations

2.1.1 Microbial Gibberellic Acid (GA3)

Endophytic Fusarium incarnatum ASU19 was recovered from onion roots on potato dextrose agar medium, identified genetically using ITS1 and ITS4 primers, and deposited in the National Centre of Biotechnology Information (NCBI) with accession number MK387876. For GA3 production, F. incarnatum MK387876 was grown in PDA medium for five days at 28 ± 1 °C, then the conidia were collected in sterilized triton X as desperation agent (0.1%), and then diluted to 106 conidia/ml as a final concentration (Mahmoud et al. 2021). Mineral broth medium containing yeast extract, 0.5%; glucose, 3%; KH2PO4, 0.1%; NaNO3, 0.3%; KCl, 0.05%, and MgSO4.7H2O 0.05%, was prepared in sterilized dist. Water. The medium was supplemented with 2% triton X-conidia solution, and incubated for one week at 28 ± 1 °C (Mohmed and Mahmoud 2018). After incubation, the fungal broth was collected, centrifuged, and extracted by ethyl acetate with 2:1 (v:v) after diminishing the pH to 2.5 using 1 M HCL. The absorbance was measured at 254 nm and the concentration was measured from the GA3 standard curve according to Berríos et al. (2004).

2.1.2 Microbial Vitamins

Bacillus subtilis KU559875 was recovered from Egyptian clover seeds on nutrient agar medium, identified genetically using 16S rRNA gene sequencing, and deposited in the National Centre of Biotechnology Information (NCBI) with accession number KU559875. For riboflavin production, B. subtilis KU559875 was grown in nutrient broth medium for 24 h at 30 ± 1 °C, centrifuge for 15 min at 6,000 xg, the cells cleaned twice, and then re-dispersed in saline solution with 105 CFU/ml final concentration. Mineral broth medium contains glucose, 5%; NaNO3, 0.5%; KH2PO4, 0.15%; K2HPO4,0.05%; MgSO4.7H2O 0.05%, and ZnSO4 0.001%, was prepared in sterilized dist. water (Shi et al. 2009). The medium was supplemented with 2% inoculum, and incubated for 72 h. at 30 ± 1 °C. After incubation the broth was collected, and centrifuged then mixed with 1 M NaOH and centrifuged again to remove the bacterial cells, then neutralized with buffer solution (acetic acid sodium-acetate, 50 mM pH 5) (Mahmoud and Bashandy 2021). The absorbance was measured at 444 nm and the concentration was calculated from the riboflavin standard curve according to Tajima et al. (2009) and Nafady et al. (2015). Saccharomyces cerevisiae ASU211 was grown in yeast malt extract medium for 24 h at 30 ± 1 °C, centrifuge for 15 min at 6,000 xg, cells cleaned twice, and then re-dispersed in saline with 105 CFU/ml final concentration. For ascorbic acid production the yeast was grown on a modified yeast-peptone medium containing yeast extract, 0.5%; peptone, 0.5%; glucose, 2%; galactose, 0.3%; MgSO4.7H2O 0.05%, was prepared in sterilized dist. water with an initial pH 5 (Banjo et al. 2020). The medium was supplemented with 2% inoculum, and incubated for 72 h. at 30 ± 1 °C. After incubation, the fungal broth was collected, and centrifuged for 15 min at 6,000 xg at 4 °C (Nowosad et al. 2022). For ascorbic acid determination, collected supernatant was analyzed using 2,6-dichloroindophenol and the absorbance was measured at 515 nm. The concentration was calculated from the ascorbic acid standard curve according to Guimarães et al. (2009).

2.2 Experimental Location

Through the experimental farm of Arab Al-Awammer Research Station, Agricultural Research Center (ARC), Assiut, Egypt, two consecutive winter seasons, 2021/2022 and 2022/2023, were planted with tested treatments. The experimental farm is 71 m above sea level and situated at the intersection of 27°, 03° N latitude and 31°, 01° E longitude. The average monthly meteorological data of the Assiut weather station during the two growth seasons are shown in Table 1. The soil type was calcareous sandy and classified as Typic Torripsamments according to Soil Taxonomy (Soil Survey Staff 2022). A composite soil sample was collected from 0–0.25 m from the experimental site, air-dried, crushed, and passed through a 2 mm sieve. Some physical and chemical properties of the experimental soil are shown in Table 2, which was determined in line with standard methods by Jackson (1973), Page et al. (1982), and Burt (2004). A constant sprinkler irrigation system with a squared spacing pattern (12 m × 12 m) was used to irrigation in the test location. The revolving sprinklers had a flow rate of 1.2–1.4 m3 per hour at 2–3 bars, and they were 1.0 m over the ground.

Table 1 Average monthly meteorological data of Assiut weather station during the two growth seasons of 2021/2022 and 2022/2023
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Table 2 Some physical and chemical properties of the experimental soil at depth 0–25 cm
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2.3 Experimental Layout

The present investigation was set up in spilt-plot design, with three replicates per treatment. The main plot was assigned to microbial gibberellic acid (with or without GA3) at the rate of 150 mg l−1 and the sub-plots were assigned to vitamins treatments (control (Ck), chemical thiamine (CT), microbial ascorbic acid (MA), chemical ascorbic acid (CA), microbial riboflavin (MR), and chemical riboflavin (CR) at the rate of 100 mg l−1 for each treatment. The chemical thiamine, riboflavin, and ascorbic acid were purchased from Alfa Chemical, China. Hence, the total plots of the experiment were 36 plots, each plot contained seven ridges, and the plot area was 10.5 m2. The application method for applied treatments was the foliar spray method. The treatment solution was sprayed at a rate of 500 ml on each plot each time. The plots were sprayed twice; once after 45 days from transplanting, and the second treatment one month later (after 75 days from transplanting). All of the treatments received the wetting agent Triton B during the spraying and a knapsack hand sprayer was used to spray onion plants.

2.4 Irrigation Water Applied

The amounts of actual irrigation water were determined according to James (1988) using the following equation:

$$\text{I}.\text{Ra}=(\text{ETc}+\text{Lf})/\text{Er}$$

I. Ra = total actual irrigation water applied mm/ interval, ETc = Crop evapotranspiration using Penman–Monteith equation. The CROPWAT model was used to calculate the Penman–Monteith equation (Smith 1991), Lf = leaching factor 10%, and Er = irrigation system efficiency. The monthly irrigation water applied during the growth season are shown in Fig. 1, irrigation water applied varied according to the monthly growth stage, which was lowest in December and reached the maximum value in March then decreased in April on the late-season stage in both seasons. The total amount of irrigation water applied during the first season (7157.24 m3/ha) was higher than the second season (6739.08 m3/ha) due to the effect of wind speed in the first season (13.64 km/h as an average of the season) was higher than second season (11.12 km/h).

Fig. 1
figure 1

The amount of irrigation water (m.3/ha) in each month of onion growth during the two seasons 2021/2022 and 2022/2023

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2.5 Plant Growth Conditions

Healthy and uniform onion seedlings were planted on both sides of each ridge at 7 cm in between (approximately 300 seedlings per plot) during the first week of December in both seasons. Phosphorus was given in one dose at the rate of 107.1 kg P2O5/ha in the form of granular superphosphate (15% P2O5) during soil preparation. Nitrogen was added at the rate of 285.7 kg N/ha, which was divided into seven equal doses every 12 days, started after 20 days from transplanting), and nitrogen fertilizer was supplied in the form of ammonium nitrate (33.5% N). K2SO4 fertilizer (50% K2O) was used as a source of K at the rate of 119 kg K2O/ha separated into four equal doses (45, 65, 75, and 85 days after transplanting). Cheated Zn, Mn and Fe were applied as a foliar spray at a rate of 476 L/ha, twice, in a liquid solution comprising 150 ppm of each of them and triton B as a wetting agent. The other agricultural practices were carried out under the onion extension guide published by the Egyptian Ministry of Agriculture.

2.6 Phenotypic Criteria, and Yield Measurement

To test the effect of the treatments on onion phenotypic criteria, and yield, two samples were taken from onion plants, the first sample after 125 days from transplanting and the second sample was taken at the end of the season. The first sample consisted of 5 plants taken randomly from each plot, and were utilized in recording the number of plant leaves, plant height (cm), and fresh weight of the whole plant (g). At the end of the season the bulb diameter (mm), and neck diameter (mm) were estimated in the onion bulbs. Also, bulbs from each plot were weighted after removing their shoots and the data were converted into total bulb yield in ton/ha.

2.7 Physiological and Biochemical Analysis

Fresh samples of onion bulbs were collected randomly from each plot and transferred to the laboratory to analyze the quality of onion bulbs. Bulb extraction was performed following Jang et al. (2013). Ten grams were taken from each replicate and mixed with 100 ml of 80% methanol solution, and left overnight at 4 °C. Samples were then vortexed for 5 min, filtrated, centrifuged at 4 °C for 15 min., and stored at—80 °C for further analysis. Sugar contents were measured using dinitro-salicylic acid (DNS) as stated by Saqib and Whitney (2011). One ml of prepared extract mixed with one ml of DNS reagent, and boiled in a 100 °C water bath for 5 min. After cooling the generated red color was measured at 540 nm using a spectrophotometer and the values were calculated from the glucose standard curve. For vitamin C determination, ten grams of bulbs were extracted using 20 ml of 1% metaphosphoric acid, homogenized by a blender. One ml filtrate was mixed with 9 ml 2,6-dichloroindophenol and the absorbance was measured at 515 nm. The concentration was calculated from the ascorbic acid standard curve following Guimarães et al. (2009). Total antioxidants were measured by phosphor-molybdenum method following Prieto et al. (1999) using ammonium molybdate reagent, the color was measured at 695 nm by spectrophotometer and the values were calculated from ascorbic acid standard curve as an antioxidant agent. Total phenolic contents were measured using Folin-Ciocalteu as described by Singleton et al. (1999) and Sidhu et al. (2019). The blue color was measured at 765 nm by spectrophotometer and the values were calculated from the gallic acid standard curve. The total flavonoids were measured using an aluminum chloride reagent following the Da Silva et al. (2015) method. The yellow color was measured at 415 nm by spectrophotometer and the values were calculated from the quercetin standard curve. To determine the total N, K, and P in onion bulbs, the bulbs were chopped and dried in a hot air oven at 70 °C until constant weight. The digestion mixture was prepared from selenium (0.42 g), LiSO4.H2O (14 g), hydrogen peroxide (350 ml), and concentrated sulfuric acid (420 ml). Each 0.5 g of dried onion utilized 15 ml of the digestion mixture, and the samples were digested to the end (Parkinson and Allen 1975). Total nitrogen was measured using the micro-Kjeldahl method (Jackson 1973) and total proteins were calculated by multiplying nitrogen content by 6.25 (AOAC 2000). Phosphorus was determined colorimetric using the stannous chloride reagent following Jackson (1973) method. Potassium was determined by the flame photometric procedure (Page et al. 1982), and NPK collected data were calculated in g kg−1 of onion dry matter.

2.8 Data Analysis

The treatments were set out in a split-plot design with three replicates. To check the significance of differences among the treatments, two-way analysis of variance (ANOVA) was utilized and then Duncan’s multiple range tests were used to compare the means at 1% and 5% level of probability. Data are tabled as the means ± standard deviations (SD). Data statistical analyses, Duncan’s multiple range test, and standard deviations (SD) were performed using IBM SPSS Statistics version 8.1 (Analytical Software 2005).

3 Results

3.1 Phenotypic Criteria of Onion

The effects of microbial gibberellic acid (GA3), vitamins, and their interaction on onion (Allium cepa L.) average plant height (cm), number of leaves, and whole plant fresh weight (g) during the two growth seasons of 2021/2022 and 2022/2023 are shown in Table 3. The analysis of variance showed that microbial GA3 treatment has achieved a significant increase (p < 0.05) in plant height compared with microbial gibberellic acid-free treatments for the plant length, number of leaves, and plant fresh weight in both seasons. The combination of vitamins and microbial GA3 had achieved a highly significant increase (P < 0.01) in the plant height, number of leaves, and plant fresh weight in the two seasons. For the plant height, the results indicated that treated plants with microbial GA3 and vitamins achieved longer plant heights with significant effect (p < 0.01) compared with vitamin treatments (without GA3) and the control samples. The highest plant height was observed in the interaction between microbial GA3 and microbial ascorbic acid giving 56.56 cm (1st season) and 60.59 (2nd season), followed by the interaction of microbial GA3 with microbial riboflavin giving 56.1 cm (1st season) and 59.99 cm (2nd season), while the control treatment recorded 48.9 cm (1st season), and 52.5 cm (2nd season). Moreover, the chemical riboflavin and chemical thiamin give close results of 54.3 and 54 cm during 1st season and 59.2 and 58.7 cm during 2nd season, respectively as cleared in Table 3.

Table 3 Effect of microbial gibberellic acid (GA3), vitamins, and their interaction on onion (Allium cepa L.) average plant height (cm), number of leaves, and whole plant fresh weight (g) during two growth seasons of 2021/2022 and 2022/2023
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The highest number of plant leaves was observed also in the combination of the microbial GA3 and microbial ascorbic acid giving 9.16 (1st season) and 9.71 (2nd season), followed by the interaction of microbial GA3 with microbial riboflavin 8.97 (1st season) and 9.69 (2nd season), while the control treatment recorded 8.13 (1st season) and 8.55 (2nd season). However, chemical thiamine, ascorbic acid, and riboflavin recorded 8.83, 8.7, and 8.5 in 1st season and 9.58, 9.4, and 9.24 in 2nd season, respectively as cleared in Table 3. Fresh weight of onions showed a significant (P < 0.05) increase by microbial GA3 treatment and a highly significant (P < 0.01) in the interaction with vitamin treatments as compared with the control treatment. The highest plant fresh weight was observed in the combination between the microbial GA3 and microbial ascorbic acid giving 169.87 g (1st season) and 184.5 g (2nd season), followed by the interaction with microbial riboflavin 163.11 g (1st season) and 177.65 g (2nd season), while the control treatment recorded 133.1 g (1st season) and 142.42 g (2nd season). However, chemical ascorbic acid, thiamine, and riboflavin recorded 158.64, 157.78, and 153.55 g in 1st season and 172.77, 171.23, and 168.29 in 2nd season, respectively as cleared in Table 3.

3.2 Yield Quality of Onion

The effects of microbial GA3, vitamins, and their interactions on onion (Allium cepa L.) bulb diameter (mm), and neck diameter (mm) during the two growth seasons of 2021/2022 and 2022/2023 as cleared in Table 4. Results showed that the bulb diameter, and neck diameter significantly (p < 0.05) improved by microbial GA3 treatment in both seasons. In the same manner, it was found that the vitamins and the combination treatments of microbial GA3 and vitamins have high significant (p < 0.01) improvement in the bulb and neck diameter. Data revealed that the combination of microbial GA3 and vitamins gives higher bulb diameter (mm) and neck diameter (mm) of onion compared with vitamins treatment (without GA3) and the control samples. It was observed that the interaction of microbial GA3 with microbial ascorbic acid and microbial riboflavin gives close enhancing effects on onion bulb with 70.94, 70.36 mm (1st season) and 76.84, 75.79 mm (2nd season), respectively. However, chemical ascorbic acid, riboflavin, and thiamine recorded 68.96, 68.53, and 67.72 mm in 1st season and 74.09, 74.03, and 73.29 mm in 2nd season, respectively, while the control treatment recorded 62.12 mm (1st season) and 66.8 mm (2nd season) as cleared in Table 4. The data of onion neck diameter (mm) revealed similar directions of the bulb diameter (mm), where the interaction of microbial GA3 with microbial ascorbic acid and microbial riboflavin gives very close enhancing effects on onion bulb with 18.36, 19.77 mm (1st season) and 18.31, 19.96 mm (2nd season), respectively. However, ascorbic acid, chemical thiamine, and riboflavin recorded 17.92, 17.67, and 16.67 mm in 1st season and 19.47, 18.99, and 17.74 mm in 2nd season, respectively, while the control treatment recorded 15.31 mm (1st season) and 16.67 mm (2nd season) as cleared in Table 4.

Table 4 Effect of microbial gibberellic acid (GA3), vitamins, and their interaction on onion (Allium cepa L.) bulb diameter (mm), and neck diameter (mm) during two growth seasons of 2021/2022 and 2022/2023
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The effects of microbial GA3, vitamins, and their interactions on onion (Allium cepa L.) onion yield (ton ha−1) during two growth seasons of 2021/2022 and 2022/2023 as cleared in Fig. 2. Onion yield showed a significant (P < 0.05) increase by microbial GA3 treatment and highly significant (P < 0.01) in the interaction with vitamin treatments. The highest onion yield was observed in the interaction treatment of the microbial GA3 and microbial ascorbic acid giving 33.16 (1st season) and 36.96 ton ha−1 (2nd season), followed by the interaction of microbial GA3 with microbial riboflavin 32.12 ton ha−1 (1st season) and 35.48 (2nd season), while the control treatment recorded 26.31 (1st season) and 29.31 ton ha−1 (2nd season). However, chemical thiamine, ascorbic acid, and riboflavin recorded 31.29, 29.39, and 28.55 ton ha−1 in 1st season and 35.01, 32.91, and 31.19 in 2nd season, respectively.

Fig. 2
figure 2

Effect of microbial gibberellic acid (GA3), vitamins, and their interactions on onion yield (ton ha−1) during the first season (a) and second season (b). Ck: control, CT; chemical thiamine (vit. B1), MA; microbial ascorbic acid (vit. C), CA; chemical ascorbic acid (vit. C), MR: microbial riboflavin (vit. B2), and CR: chemical riboflavin (vit. B2). Data are presented as means, line bars indicate ± standard errors, and the different upper letters demonstrate significant differences at P < 0.05 level

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3.3 Biochemical Quality of Onion Bulbs

The effect of gibberellic acid (GA3), vitamins, and their interaction on onion (Allium cepa L.) total nitrogen, phosphorus, and potassium contents (g kg onion dry matter−1) during two growth seasons of 2021/2022 and 2022/2023 as cleared in Table 5. In both seasons of study, statistical analysis of NPK content revealed that the major effect of microbial GA3 and vitamin factors recorded significant differences (p < 0.05). The interaction of microbial GA3 and vitamin treatments in the first and second seasons achieved highly significant differences (p < 0.01) in phosphorus content whilst NK content had non-significant results. The majority of interaction treatments have significant differences between them and control (Ck). Regarding nitrogen content, the microbial riboflavin treatment, which measured 27.3 and 28.4 (g kg−1), achieved the highest N concentration among all vitamin treatments and increased N content by 8.9 and 6.9%, respectively, in comparison to the control (Ck) during the two seasons. On the other hand, the largest P content was recorded by microbial ascorbic acid treatment, which was 3.6 and 3.8 (g P kg−1) in both seasons, with significant differences between it and the control treatment which recorded 3.3 and 3.4 (g P kg−1) in both seasons. Concerning potassium content, microbial riboflavin increased K content by 7.6 and 6.5% through the two seasons, respectively, in comparison to the control treatment, which recorded 22.2 and 23.1(g kg−1) in both seasons.

Table 5 Effect of microbial gibberellic acid (GA3), vitamins, and their interaction on onion (Allium cepa L.) total nitrogen, phosphorus, and potassium contents (g kg onion dry matter−1) during two growth seasons of 2021/2022 and 2022/2023
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3.4 Physiological Quality of Onion Bulbs

Total sugars, proteins, antioxidants, phenols, flavonoids, and vitamin C were measured in onion bulbs for quality assessments as cleared in Figs. 3, 4 and 5. In both seasons of study, the statistical analysis of total sugar and total protein contents (Fig. 3) revealed that the effect of microbial GA3 and vitamins factor achieved significant differences (p < 0.05) in both seasons, however, the interaction among microbial GA3 and vitamins treatments in the first and second seasons were non-significant for both sugars and proteins. Regarding the major effect, the microbial GA3 treatment had the highest sugar content in the presence of microbial ascorbic acid followed by microbial riboflavin with values of 76.05 and 71.5 mg/100 g d.wt. total sugars in the first season and 95.9 and 91.9 mg/100 g d.wt. total sugars in the second season, respectively. For total protein contents also the foliar spraying with microbial GA3 treatment had the highest protein content, especially in the presence of microbial riboflavin and microbial ascorbic acid with values of 176.1 and 174.6 g/ kg total proteins in the first season, respectively. However, in the second season the highest effects were in microbial riboflavin followed by chemical thiamine with 183.2 and 181.2 g/ kg total proteins, respectively.

Fig. 3
figure 3

Effect of microbial gibberellic acid (GA3), vitamins, and their interactions on onion total sugars (mg/100 g d.wt.) during the first season (a) and second season (b) and total proteins (mg/g d.wt.) during the first season (c) and second season (d). Ck: control, CT; chemical thiamine (vit. B1), MA; microbial ascorbic acid (vit. C), CA; chemical ascorbic acid (vit. C), MR: microbial riboflavin (vit. B2), and CR: chemical riboflavin (vit. B2). Data are presented as means, line bars indicate ± standard errors, and the different upper letters demonstrate significant differences at P < 0.05 level

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Fig. 4
figure 4

Effect of microbial gibberellic acid (GA3), vitamins, and their interactions on onion total antioxidants (mg/100 g d.wt.) during the first season (a) and second season (b) and vitamin C (mg/100 g f.wt.) during the first season (c) and second season (d). Ck: control, CT; chemical thiamine (vit. B1), MA; microbial ascorbic acid (vit. C), CA; chemical ascorbic acid (vit. C), MR: microbial riboflavin (vit. B2), and CR: chemical riboflavin (vit. B2). Data are presented as means, line bars indicate ± standard errors, and the different upper letters demonstrate significant differences at P < 0.05 level

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Fig. 5
figure 5

Effect of microbial gibberellic acid (GA3), vitamins, and their interactions on onion total flavonoids (mg/g d.wt.) during the first season (a) and second season (b) ad total phenols (mg/g d.wt.) during the first season (c) and second season (d). Ck: control, CT; chemical thiamine (vit. B1), MA; microbial ascorbic acid (vit. C), CA; chemical ascorbic acid (vit. C), MR: microbial riboflavin (vit. B2), and CR: chemical riboflavin (vit. B2). Data are presented as means, line bars indicate ± standard errors, and the different upper letters demonstrate significant differences at P < 0.05 level

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For total antioxidants and vitamin C contents, the statistical analysis revealed that the effect of microbial GA3 and vitamins factor achieved significant differences (p < 0.05) in total antioxidants and vitamin C in both seasons, however, the interaction among microbial GA3 and vitamins treatments in the first and second seasons were non-significant for total antioxidants and vitamin C contents (Fig. 4). Foliar spraying with microbial GA3 reveals high total antioxidants and vitamin C contents, especially in the presence of microbial ascorbic acid and microbial riboflavin with values of 89.3 and 80.6 mg/100 g d.wt. total antioxidants in the first season and 82.4 and 72.7 mg/100 g d.wt. total antioxidants in the second season, respectively. Vitamin C contents were also higher in the microbial GA3 treatments, especially in the presence of microbial ascorbic acid and microbial riboflavin with values of 118 and 102 mg/100 g f.wt. (1st season), and 124 and 116 mg/100 g f.wt. (2nd season), respectively.

For total flavonoids and total phenols contents, the statistical analysis revealed that the effect of microbial GA3 and vitamins factor achieved significant differences (p < 0.05) in total flavonoids and phenols in both seasons, also the interaction among GA3 and vitamins treatments in the first and second seasons were significant (p < 0.05) for both (Fig. 5). The highest significant interactions of total flavonoids were demonstrated with the presence of microbial ascorbic acid and microbial riboflavin with 155.8 and 151.4 mg/g d.wt. in the first season and 122 and 107.3 mg/g d.wt. total flavonoids in the second season, respectively. However, total phenol contents were also higher in the GA3 foliar spraying treatments, especially in the presence of microbial ascorbic acid and microbial riboflavin with 32.7 and 31.1 mg/g d.wt. (1st season), and 28.6 and 26.6 mg/g d.wt. (2nd season), respectively.

4 Discussion

With increasing urbanization and industrialization every day, environmental suffering has increased, causing shrinks of the agricultural land, declines in plant growth, and losses in crop yields which represent a great threat to global food security. Egypt is one of the world's driest countries with 86% hyper-arid geographical area (Hussein 2011), and about 25–30% of their soil were calcareous soil characterized by high pH, and low availability of macro and micronutrients (Taalab et al. 2019; Mahmoud et al. 2023). Although enormous progress has been conducted in utilizing various soil types in agriculture and improving crop productivity, the role of microbial technologies has been acknowledged for agriculture sustainability, and environmental health (Egamberdieva et al. 2017).

Phytohormones are categorized among the high important plant growth stimulators; through their positive effects on plant metabolism, and the plant defense system. Exogenous supplementation of phytohormone has improved effects on plant growth, physiological traits, and its metabolism in various stress conditions (Islam et al. 2023). From phytohormones, GA3 has been utilized commercially to improve the morpho-physiological and yield features of several vegetable and crops (Miceli, et al. 2019). The common way of exogenous applications of GA3 was foliar application (as we used in our research), GA3 naturally synthesized with low concentrations in young leaves, which makes it more suitable in foliar application (Dayan, et al. 2012). In our study, under calcareous sandy soil, onion yield and the development parameters such as plant height, number of leaves, fresh weight, onion bulb and nek diameter were improved significantly with the application of microbial GA3. Gibberellic acid has a vital role in stimulating the plant growth, seed dormancy, and lateral shoot growth under various stress conditions (Olszewski et al. 2002; Ahmad 2010). GA3 demonstrated to control the cell division and elongation, cell proliferation, hyperplasia, and increased flexibility of cell walls (Ritonga, et al. 2023). In agreement with our findings, Sarkar et al. (2018) found that the external use of GA3 (60 ppm) by root soaking and foliar spray at 30 and 60 days after transplanting increased shoot biomass, plant height, bulb biomass, and dry matter accumulation in onions compared to control. Dwivedi et al. (2019) clarified that the maximum plant height, leaf area, leaf width, leaf length, number of leaves, fresh and dry weight of the plant, and polar and equatorial diameter of the onion bulb were recorded under 100 ppm GA3 foliar spray at 80 days after transplanting. Additionally, researchers have verified considerable improvements in plant growth, yield components and yield as a result of the foliar application of a proper dose of GA3 in numerous crops other than onions, including mungbean (Islam, et al. 2023), and jojoba (Atteya, et al. 2018).

Onion quality traits including sugar content, protein, antioxidants, vitamin C, phenols, and flavonoid were improved significantly with the application of microbial GA3. Remarkably, GA3 has a part in promoting plant metabolic activities, promoting the development and expansion of chloroplasts, encouraging the photosynthesis of photosynthetic pigments and raising the efficiency of representation photosynthesis (Chandel, et al. 2023). It has a promoting effect on DNA, RNA, protein synthesis, polyribosomes, and ribose multiplication, which makes it capable of altering the plant growth pattern depending on its concentration (Miceli, et al. 2019). It was found that foliar application of GA3 increased the protein content in shoot and roots of wheat (Alharby et al. 2021), and maize (Tuna et al. 2008). Manjili et al. (2012) found that gibberellic acid has direct role in enhancing the plant antioxidant system and decreasing the levels ROS. Moreover, Taș et al. (2021) stated that foliar applications of 100 ppm GA3 raised the total phenols, and vitamin C in the strawberry fruits. In our study, d GA3 treatments significantly (P < 0.05) increased the NPK contents in onion bulbs compared to the control. This finding agreed with the opinion of several researchers who confirmed increased in nutrients and protein content of treated plants by a proper dose of GA3 as a foliar application. Gibberellic acid has critical role in enhancing the nutrient uptake and ion partitioning of the plant system, and maintaining the plant metabolism in normal and stress conditions (Iqbal and Ashraf 2013). Khan et al. (2004) found that exogenous application gibberellic acid has directly enhancing effects on the plant growth, yield, and NPK uptake in tomato. Tuna et al. (2008) revealed that N, P, K, Mg, Ca, Fe, Mn and Zn contents in shoot and root of maize were increased after foliar applications of GA3. Atteya et al. (2018) recorded the NPK content high contents in jojoba leaves after GA3 applications. Alharby et al. (2021) found that foliar application of GA3 increased P, K, Mn and Fe content in roots, shoots and grains of wheat.

Vitamins are essential compounds not only for the human physiological process but also for the plant’s metabolism, due to their redox, antioxidant properties, and cofactor potentials. Vitamins have essential roles in the plant defense system against biotic and a biotic stress and in increasing the plant yield (Boubakri et al. 2016), which reflects on the enhancement of agricultural productivity and global food security under the current climatic changes. The addition of vitamins to plants found to enhance the plants growth, development, and their physiological activities (Hanson et al. 2016). Exogenous vitamin application plays a significant role in reducing ROS and retarding ROS-responsive gene expression (Li et al. 2021). Moreover, it reduces cell damage and enhances the membrane stability of plant cells, increasing the resistance of plants to chilling stress, and drought (Ahmad et al. 2015; Xin Chi et al. 2021). In the current study, the application of foliar vitamins significantly increased the onion yield, and their phenotypic and quality properties including plant height, number of leaves, fresh weight, bulb diameter, and neck diameter. However, the microbial ascorbic acid and microbial riboflavin recorded higher results and best treatments, especially in the combination of microbial GA3. Ascorbic acid addition to plants found to have enhancing effects on plant development and productivity (Mehrshahi et al. 2010), seedling growth (Foyer et al. 2020), and reduces the reactive oxygen species produced in plant leaves (Li et al. 2009). Increasing vitamin C in plants by external fortification could increase its concentration in plant human food sources, the postharvest shelf life of crops, and the plant resistance to diseases (Paciolla et al. 2019). In agreement with our findings, El-Hifny and El-Sayed (2011) stated that sweet pepper grown under sandy loam soil conditions has maximum growth characteristics such as the number of branches, plant height, fresh and dry weight of plant, and number of fruits per plant as a result of foliar spray of 400 ppm ascorbic acid in combination of microbial Saccharomyces cerevisiae and Azotobacter chroococcum. Ali (2017) found that foliar spraying of garlic with ascorbic acid (200 ppm) cultivated in newly reclaimed soil has significant enhancement in plant growth traits, such as leaf area per plant, plant height and plant fresh and dry weight. Selem, et al. (2022) proved that exogenously application of ascorbic acid on potatoes cultivated in loam soil significantly enhanced the plant height, shoot fresh and dry weight, and leaf area. Plants treated with ascorbic acid showed an increase in their root length, fresh and dry weights in maize (Loutfy et al. 2020), barley (Yaseen et al. 2021), and hybrid chillies (Zahid, et al. 2023).

The addition of thiamine and riboflavin enhances the division of meristem cells, and the initiation of cells in new organs especially the non- photosynthetic ones (Martinis et al. 2016). Rice treatment with thiamine enhanced its resistance to sheath blight disease (Bahuguna et al. 2012). Riboflavin stimulated the rooting of Eucalyptus globulus (Trindade and Pais 1997), rooting, and cell division in Common Bean (Phaseolus vulgaris L.) (Boland et al. 1989). The addition of riboflavin to tomato, Arabidopsis, and tobacco enhances their defense properties against plant pathogens, Pseudomonas syringae, Peronospora parasitica, and Alternaria alternate, respectively (Zhang et al. 2009; Abdel‐Monaim 2011). In agreement with our findings, Saeidi-Sar et al. (2007) revealed that under both normal and Nickel stress conditions, the combination of ascorbic acid and GA3 increased the dry weights of the shoot and root in soybeans. Saeidi-Sar et al. (2013) reported that supplication of GA3 with ascorbic acid improved the fresh weights of shoot and root and dry weights of shoot and root in common beans under both normal and salt stress conditions. Gouda et al. (2015) stated that foliar application of individual vitamin C, B1, and B2 at 100 ppm increased the number of leaves, plant height, leaf area and yield of potato plants. Exogenous application of riboflavin to maize (Zea mays L.) seedlings enhanced the seedling growth, germination rate, sugars, and antioxidants (Xin Chi et al. 2021). Kausar et al. (2023) found that the foliar application of thiamine with 250 and 500 ppm enhances shoot fresh and dry weight, root fresh and dry weight, leaf area, and number of pods of pea plants grown under sandy loam soil.

The combination of microbial GA3 and microbial ascorbic acid and microbial riboflavin significantly enhanced the biochemical and physiological traits of onion quality including NPK content, sugar content, protein, antioxidants, vitamin C, phenols, and flavonoid compared with the control samples. It was found that vitamin C as a non-enzymatic antioxidant has essential roles in the plants progress, growth, physiological process, and cell differentiation (Akram et al. 2017). Athar et al. (2008) found that exogenous application of ascorbic acid enhanced the antioxidant contents of wheat and alleviated the salt stress. El-Hifny and El-Sayed (2011) stated that the application of GA3 on sweet pepper plant increased NPK contents, while Ali (2017) demonstrated an increased in NPK contents in garlic plants in response to the GA3 application. El-Hawary, et al. (2023) recorded an increased in the protein content of wheat in response to the GA3 application. Mohamad et al. (2023) declared that spraying maize with ascorbic acids improved its physiological traits and productivity. Plant supplementation with thiamine, and riboflavin significantly decreased the early‐seedling‐lethal phenotypes (Hanson and Gregory 2011), reduced the cell membrane permeability which increased the accumulation of the photosynthetic pigments, antioxidant, and enhanced the maize growth (Kaya et al. 2015). Riboflavin has been stated as an initiator for flavocoenzymes that has a critical role in the metabolism of proteins, lipids, ketone bodies, and carbohydrates, which provided the majority of the energy for living things (Kausar et al. 2023). Application of riboflavin to plants improved the nutrient content of plants and enhanced the physiological properties of potato, and Hibiscus sabdariffa (Azooz 2009; Gouda et al. 2015). Vitamin B complex (B1 + B2 + B6 + B12) was found to have a positive role in increasing the concentration of NPK in Grapevines (Abdelaziz et al. 2021). Xin Chi et al. (2021) stated that exogenous application of riboflavin to maize seedlings enhanced the sugar content and antioxidants.

5 Conclusion

These days sustainable agriculture faces several key challenges and issues that have direct impacts on agriculture especially the excessive use of chemical plant enhancers which decrease soil fertility, biodiversity, and accumulate in food causing long-term health problems. Integrated microbial products into agriculture is an effective, economical, and environmentally natural solution. Phytohormones and vitamins are produced from microorganisms using low-cost methods and available microbes like fungi and bacteria. It was found that the effectiveness of these products is no less than those manufactured chemically, but on the contrary, they may be more effective as natural products have other bioactive groups that enhance their effectiveness. Foliar spraying of microbial gibberellic acid increased the onion productivity and quality. The interaction between microbial gibberellic acid and vitamins especially with ascorbic acid gives the highest productivity and enhances the phenotypic and physiological quality of onion. The combined application of gibberellic acid and foliar spraying of microbial ascorbic acid, with proper management of irrigation and fertilization, is an appropriate strategy to increase the growth and quality of onion plants under sandy calcareous soil conditions. More studies are needed under different environmental conditions and different soil types to explore the effects of these valuable natural products on different crops.