Effect of organic waste material and rhizobacteria on growth and physiology of Brassica juncea (L.) Czern. under salinity stress

doi:10.21203/rs.3.rs-5306513/v1

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Effect of organic waste material and rhizobacteria on growth and physiology of Brassica juncea (L.) Czern. under salinity stress

https://doi.org/10.21203/rs.3.rs-5306513/v1

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Salinity is one of the major environmental problems in arid and semi-arid region which affect the plant physiological and yield attributes. Organic waste materials such as banana peels and eggshells contain essential nutrients that are necessary for plant growth and development. The inoculation of rhizobacteria allows to decrease the hazardous effects of salinity stress. The present study was conducted to evaluate the potential of organic waste material and rhizobacteria on growth and physiology of Brassica juncea (L.) Czern. under salinity stress. The experiment consisted of various treatments including eggshells (20g), banana peel (20g), rhizobacteria (30ml Bacillus megaterium) and NPK(0.033kgh^− 1). In the current study, salt stress 0 mM and 150 mM NaCl concentration was applied. From outcome of current study, it is shown that salt stress caused changes in morphological, physiological, biochemical and yield parameters. Peel powder treatment and Bacillus megaterium strain play major role as growth regulators for plant development under salt stress. Among the treatments, eggshells and banana peels significantly enhanced the antioxidant activity (CAT, POD, SOD and total soluble protein), total phenolic, leaf proline, reduced the oxidative stress markers (MDA, H₂O₂) and promoted membrane stability and yield attributes. However, carotenoids content increased under NPK treatment.

Salinity

antioxidant

yields

Bacillus megaterium

lipid peroxidation

Among biotic and abiotic stresses in the environment salt stress is the most restrictive for plant distribution. Various concentrations of salt can exert harmful effects on plant growth [1]. Salinity stress adversely impacts soil fertility, biodiversity and stability. Salt stress affecting various crop growth and productivity, ultimately overall food production reduced [2]. Soil salinization eventuates when excess amount of soluble salts accumulates in land. This may be naturally due to weathering of saline rocks, scarcity of rainfall, or the flow of saline water from seas and oceans, anthropogenic activities such as inadequate irrigation practices, overuse of fertilizers [3]. Soil salinity intensification provokes multitude of negative responses in plants by the production of higher levels of reactive oxygen species (ROS) that lead to oxidative damage to cellular biomolecules [4]. In a major enzymatic reaction, the accumulation of toxic sodium (Na⁺) ion displace potassium (K⁺) ions in the cytoplasm and causes the overproduction of reactive oxygen species, resulting to changes in nutrients, protein synthesis, photosynthesis and redox homeostasis in plants [5].

Brassica juncea (L.) Czern. known as Indian mustard, an amphiploid species within the Brassicaceae family. It is primarily cultivated as an oilseed and food crop, but also for medicinal purposes. Brassica juncea is the richest source of iron, vitamins A and C, and also contains thiamine riboflavin, potassium, calcium and β-carotene [6]. Indian mustard is the third-largest crop which source of vegetable oil globally, after soybeans crops and palm oil. Indian mustard stands out for its rapid seed germination rate, high yield potential and resistance to abiotic stress [7]. It is utilized in soaps, paints, lubricants, hair oils, and condiment in pickles as a commercially. Traditionally, it has been used to skin eruptions, treat rheumatism, and ulcers. Indian mustard residual parts can be used as feed for cattle and poultry [8].

Plant responses against salt stress rely by PSII activity, along with biochemical adjustments adjacent PSII, comprising activation of xanthophyll cycle which helps to release excess heat energy [9]. PSII stability maintained by accumulation of osmo-protectants such as proline, glycine and betaine [10]. PSII, a multi-subunit protein-pigment complex that extremely susceptible to salinity stress [11]. In PSII all the protein complexes, D1 protein is a primary target for salt-induced ROS leading to photo-inhibition of PSII. Moreover, salt-induced ROS hampers the repair cycle of the D1 protein [12].

The utilization of Plant Growth-Promoting Rhizobacteria (PGPR) is an effective strategy to elevate plant salt tolerance [13]. PGPR can survive within the plant’s parts or in the rhizosphere by forming mutualistic relationships that lead to boost plant growth [14] PGPR also increased field crop productivity under specific environmental conditions, but some species commercially available for direct soil inoculation. Bacillus megaterium, particular is well-known for its abundance in soils and its capability to promote plant growth [15]. The strain of Bacillus megaterium can effectively colonize the soil early after inoculation and remarkably increasing the richness of soil bacterial and fungal communities. It also famous to solubilize k and p in soil [16]. B. megaterium, along with other Bacillus species, reveals an exceptional capability to colonize the rhizosphere of grasses. It can solubilize phosphate via the release of inorganic and organic acids and mineralize phosphate, proton extrusion, by synthesizing phosphatases, and produce phytohormones and siderophores [17, 18].

Calcium (Ca) and potassium (K) are vital nutrients that impact various biochemical and physiological processes, thereby supporting plant growth and metabolism. These elements play a crucial role in the persistence of plants under different environmental stresses [19]. In many state of world, nutrient deficiencies in various crops is a prevalent issue due to infertile soil. Essential nutrients such as Nitrogen, Calcium, Phosphorus, Potassium, Calcium, Sulphur, and Iron are crucial for plant existence. While these nutrients are already present in soil, they are not in such a form that plants can easily absorb [20]. Farmers are interested to foster their land and are motivated by the enrichment of soil fertility soil, instigation them to find alternate ways to elevate and improve it for better crop yields [21].

Undeniably, chemical fertilizers comprising Nitrogen, Phosphorus, and Potassium have significantly benefited in recent cropping systems. Though, synthetic fertilizer arises with many drawbacks [22]. Overutilization of these synthetic fertilizers can lead to environmental degradation, such as air, water, and soil pollution resulting to eutrophication [23]. Synthetic fertilizer harms agriculture soil quickly by disturbing beneficial plant growth promoting bacteria present within soil as a result it is restricted and lowered plant yield. Despite the well-known health risks of chemicals, farmers are regularly employed synthetic fertilizer to boost up soil fertility and shield plants from pests [21].

Researchers are mostly concentrating and focusing on emerging and using fertilizers to address environmental and health concerns accompanying to the extensive use of synthetic fertilizers [24]. As the increasing demand for healthy and safe food, in a sustainable environment, agronomists are frequently exploring alternative like eco-friendly techniques to restore soil nutrients and restrain these nutrients [25]. Fertilizer using them lessens the reliance on expensive and harmful agrochemicals, conserves soil microflora, boosts overall soil fertility, and improves the nutrient levels in the soil [26]. Some mechanisms of rhizobacteria are elevating stress confrontation, boosting nutrient availability in soil and producing siderophores, so plant growth-promoting bacteria become effective fertilizers [27].

PGPR has been employed in numerous crops to boost growth of plant, seeds germination rate, and ultimately crop yield [28]. Eggshell waste is used as a soil modification and natural fertilizer. It can combined with banana peels as a substitute to create organic fertilizer [29]. Therefore, the objective of this study were, to explore the potential role of rhizobacteria in the enhancement of growth and yield of Brassica juncea (L.) Czern. To assess the performance of Brassica juncea (L.) Czern under salt stress. To evaluate different biochemical and physiological characters of Brassica juncea (L.) Czern under the application of organic waste material and rhizobacteria.

2.1 Experimental layout and design

Super Raya mustard seeds were acquired from Ayub Agricultural Research Institute in Faisalabad, Pakistan. Soil samples were collected from the Main Nursery at the University of Education, Lahore, Pakistan. These samples were ground, homogenized, sieved through a 2 mm sieve, and thoroughly mixed. Pot was filled with 5kg soil. The soil was loamy and coarse in texture, with a pH of 7.1 and an electrical conductivity (EC) of 820 dS/m. Experiment were performed in research area of botanical garden University of Education, Township campus Lahore. Eggshells and banana peels were collected, cleaned, air-dried, and ground into fine powder. The PGPR strain (Bacillus megaterium STB1) was obtained from the institute of molecular biology and biotechnology (IMBB) University of Lahore's microbiology lab. The bacterial inoculum optical density (OD) was 0.7 (measured at 600 nm wavelength). The experimental work aimed to evaluate the effect of rhizobacteria, eggshell waste, and banana peel on Brassica juncea L. under salt stress. Treatments included individual and combined applications of these materials, with salinity stress induced by 150 mM NaCl. The treatments consisted of fine eggshell powder (20 g), banana peel powder (20 g), NPK (0.033 kg ^h−1) and Bacillus megaterium solution (50 ml) as given in Table 1. For seeds priming mustard seeds were soaked in Bacillus megaterium solutions for three hours. And remaining mustard seeds were placed in distilled water for same duration. Subsequently seeds were kept in blotting paper to absorb excess moisture.

Table 1

Treatment used in this experiment.
Plant sets	Treatment	Levels of treatment
S1O1B1F1	Control	0 g
S1O2B1F1	Peels powder	40 g
S1OB2F1	PGPR	30 ml
S1O2B2F1	Peels + B. megaterium	40 g + 30 ml
S1OB1F2	NPK Fertilizer	0.033 kg ^h−1
S2O1B1F1	NaCl	150 mM
S2O2B1F1	NaCl + Peels powder	150 mM + 40 g
S2O1B2F1	NaCl + B. megaterium	150 mM + 30 ml
S2O2B2F1	NaCl + Peels + B. megaterium	150 mM + 40 g + 30 ml
S2O1B1F2	NaCl + NPK Fertilizer	150 mM

2.2 Assessment of growth parameters

One plant from each of the 30 pots was uprooted after two weeks of salt stress. The plants were carefully plucked and the roots were washed to remove soil. The remaining plants were retained for assessing yield attributes, including seeds yield per plant and 100 seeds weight. Tissue paper was used to dry the samples and roots were detached from shoots with scissors. The lengths of shoots and roots were measured immediately, then placed into labeled bags to avoid mixing. Fresh weights of shoots and roots were recorded using a digital balance and these samples were dried in an oven (Pol-Eko SLN 32 Smart SN32200069) at 70°C for five days to assess dry biomass. The total number of leaves, leaf area and number of pods were also recorded.

2.3 Assessment of leaf water relations

A fully third young expanded leaf from top of each plants was cut at 10: am to 12: am and plant leaf water potential were conducted using a pressure chamber (PMS Model 670). Same leaf of plants utilized for measuring leaf water potential, was frozen for five days at − 18°C in deep freezer. After that, the frozen leaf samples were thawed and a glass rod was used to press the leaf material to release the sap. The sap was utilized directly in Osmometer to determine the osmotic potential (Löser Messtechnik Osmometer). Leaf turgor potential was determined by subtracting the osmotic potential from the water potential values.

2.4 Measurement of gas exchange parameters

Gas exchange attributes were measured by using an ADC LC pro-SD System infra- red Gas analyzer (IRGA). For each replica choose healthy and youngest plant. Place the third intact leaf in the chamber of IRGA. Measurements were conducted between 12:30 to 2.00 hours with the following environmental adjustments; leaf surface area of 6.25 cm², leaf chamber temperature varying from 16 to 18°C, leaf chamber molar gas flow rate (U) at 200.5 µmol s⁻¹, ambient CO₂ concentration (C_ref) at 447 µmol mol⁻¹, ambient pressure (P) at 998 kPa, and PAR (Q_leaf) at the leaf surface reaching a maximum of 1305 µmol m⁻².

2.5 Determination of chlorophyll fluorescence

To assess Photosystem II (PSII) efficiency, the third healthy, young, and mature leaves from top of each plant were selected. Chlorophyll fluorescence measurements were conducted on these dark-adapted leaves using an OS + 30P (Opti-sciences, Inc. | Hudson, NH 03051, USA) chlorophyll fluorometer. Dark adaptation was achieved by attaching clips to the leaves for 15 minutes prior to fluorescence measurements. Dark adapted clips avert the light from reaching the leaf. Chlorophyll fluorescence quenching analysis has provided substantial advancements in detecting photoinhibition of PSII. The important parameter was F_v/F_m ratio to detect photoinhibition of PSII caused by a stress factor. To determine this ratio a weak modulated beam was applied to evaluate minimal fluorescence (F₀) in a dark-adapted leaf, tracked by a saturating flash to measure maximal fluorescence (Fm).

2.6 Measurement of SPAD value

To determine total chlorophyll, first calibrate the SPAD meter (atleaf). Then, select healthy, mature plant leaves. Turn on the SPAD meter and position the leaf (from top, mid, or base) between the measurement heads, ensuring it is flat and fully covered. Initiate the measurement process by pressing the measurement button to obtain a reading. Record the SPAD value, which reflects the chlorophyll content in SPAD units.

2.7 Determination of chlorophyll and carotenoids content.

Chlorophyll a and b contents were determined following [30] method. Collect youngest fresh plant leaves from every replicate and weigh 0.5 grams of leaf tissue. Homogenize these leaf tissue with 80% acetone (10 ml) by using a mortar and pestle. Then homogenize mixture were filtered with help of filter paper and kept these samples in freezer at 4°C for 24 h. After that allocation the homogenate to a centrifuge tube, and subsequently Centrifuge at 10,000 rpm for 10 minutes to obtain the chlorophyll-containing supernatant. Measuring the absorbance of the supernatant at 663 nm, 645 nm, and 652 nm by using a spectrophotometer (Hitachi-U2001, Tokyo, Japan).

Use the following formulas to calculate the chlorophyll content

Chl. a (mg g-1 FW) = [12.7 (OD663) – 2.69 (OD645)] x V/1000 x W

Chl. b (mg g-1 FW) = [22.9 (OD645) – 4.68 (OD663)] x V/1000 x W

Where, V represents the volume of extract (mL), W is the weight of fresh leaf, and OD stands for optical density.

Carotenoid contents were measured using the formula described by [31].

Carotenoids = (1000 x OD480) – (1.9 x Chl a – 63.14 x Chl b)/214

2.8 Determination of relative water content

From every replica leaf, all of equal size, were gathered, weighed, and swiftly set afloat on distilled water. Following this, leaves were submerged in distilled water for three hours at a temperature of 25–25 degrees Celsius. Three hours later, the leaves swelled, and their turgid weight was recorded. Subsequently, the leaves were dried in an oven at 80 degrees Celsius for 24 hours, and their weight when dry was measured. Relative water content (RWC) of each replica was determined using the formula provided by [32].

RWC (%) = [(FW – DW) / (TW – DW)] x 100

In this context;

FW stands for Fresh Weight

DW stands for Dry Weight

TW stands for Turgid Weight

2.9 Determination of relative membrane permeability

Mature and youngest leaves of consistent size were gathered from every replica. The leaves were cut up and placed into test tubes containing 20 ml of deionized water. The blend was briefly mixed using a vortex for 5 seconds, and the electrical conductivity (EC₀) was determined using Milwaukee MW805 MAX pH/EC/TDS/Temperature Portable Meter. Subsequently, the tubes were kept in a refrigerator at 4 degrees Celsius for 24 hours, and EC₁ was noted. The stored samples were then subjected to an autoclave at 120 degrees Celsius for 20 minutes to obtain readings for EC₂. The RMP percentage was calculated using the formula provided by [33].

RMP (%) = [(EC₁ – EC₀)/(EC₂-EC₀)] x 100

2.10 Assessment of malondialdehyde content

The protocol outlined [34] was followed with some modifications. Each replicate involved harvesting fresh leaves (0.5 grams weighed using a balance), which were then homogenized in 3 milliliters of 1.0% TCA acid at 4 degrees Celsius. Following the transfer of the homogenate to conical tubes, it underwent centrifugation at 20,000 rpm for 15 minutes. After centrifuging, test tubes were filled with 0.5 milliliters of the filtrate, which was then mixed with solution of 0.5 percent thiobarbituric acid prepared in 20% TCA acid solution (3 milliliters). Subsequently, all the samples underwent heating at 95 degrees Celsius for 50 minutes using a shaking water bath (Model: HSW-1/06). The reaction ceased by promptly cooling using an ice water bath. Subsequently, all samples underwent an additional centrifugation at 10,000 rpm for 10 minutes, and the UV/VIS spectrophotometer measured absorbance at 532 and 600 nm. The levels of MDA (in nanomoles) for each sample were determined using formula as mentioned.

MDA level (nmol) = [(A_532nm – A_600nm)/1.56 x 10⁵] x V/W x 1000000

2.11 Hydrogen peroxide determination

H₂O₂ was determined following protocol as suggested by [35]. Fresh foliage was collected from each sample, weighing 0.5 g, then thoroughly blended with trichloroacetic acid (0.1%, 5 ml) using a mortar and pestle (pre-chilled). The resultant solutions were shifted to conical tubes. Centrifuging the extracts occurred at 12,000 rpm for 15 minutes at 4 degrees Celsius using HERMLE Z 326 K Universal Centrifuge. Test tubes were filled with half a milliliter of potassium phosphate buffer at a pH of 7, one milliliter of potassium iodide, and half a milliliter of the obtained supernatant. After vortexing the mixture, the UV/VIS spectrophotometer measured the absorbance at 390 nanometers.

2.12 Determination of total soluble protein

Protein levels in samples were assessed following the specified protocol [36]. Fresh leaf was gathered from every replica, weighed (0.5 g), and mixed in 10 ml of pre-cooled phosphate buffer (50 mM, pH 7.8). After centrifugation at 6,000 g for 20 minutes at 4 degrees Celsius, supernatant was separated from residue and stored in the freezer. Solution of Bradford was prepared by dissolving 100 mg of coomassie brilliant blue in 50 milliliters of 95% ethanol, followed by the addition of this solution to 100 milliliters of 85% phosphoric acid. Distilled water was then added to reach a total volume of one liter. Subsequently, 5 milliliters of the Bradford solution were combined with 0.1 milliliters of previously prepared leaf extracts (which had been frozen in a deep freezer). UV/VIS spectrophotometer was used to determine optical density 595 nm wavelength. Bovine albumen serum was utilized to make standard curve and estimating protein level.

2.13 Determination of catalase (CAT) and peroxidase (POD) activity

The concentration of catalase and peroxidase was determined by analyzing the reaction solution of 50 mM of phosphate buffer having pH 7.0 = 1 ml, 5.9 mM of H₂O₂ = 1.9 ml Enzyme extract 0.1 ml, Total = 3 ml. CAT absorbance was assessed at a wavelength of 240 nm, with measurements taken every 30 seconds over a 120-second period using a spectrophotometer [37].

For POD, absorbance readings were conducted at a wavelength of 470 nm, with measurements taken every 30 seconds over a 150-second period using the same spectrophotometer.

2.14 Determination of ascorbate peroxidase activity (APX)

Each replicate involved preparing a reaction mixture for APX, totaling 3 milliliters. This mixture comprised 2.70 ml of phosphate buffer (50 mM,) along with 0.10 ml each of ascorbic acid (7.50 mM), hydrogen peroxide (H₂O₂) (300 mM,) and enzyme extract. Subsequently, the APX reaction mixture was analyzed using a UV/VIS spectrophotometer, with absorbance readings taken at a wavelength of 290 nm every 30 seconds over a 60-second period (Nakano & Asada, 1981).

2.15 Determination of superoxide dismutase (SOD) activity

For SOD, reaction mixture composed of 0.3 ml of methionine (130 MM), nitro blue tetrazolium (50 µM), 0.3 ml of EDTA-Na2 (100 µM), 0.3 ml of riboflavin (20 µM). To this, 0.05 ml of enzyme solution was added, followed by exposure to 4000 lux light continuously for thirty minutes. Subsequently, the samples were analyzed for absorbance at 560 nanometers using spectrophotometer [38].

2.16 Determination of total phenolics

Total phenolic content were assessed by following the method [39]. In which 0.50 grams of dried leaf samples were weighted and transferred into centrifuge tubes. Following this, the samples were homogenized in aqueous acetone (10 ml of 80%) for one minute, after which the mixture underwent centrifugation at 4000 rpm for 15 minutes at 4 degrees Celsius. Supernatant was then decanted, and the resulting dried solid residue was utilized for subsequent steps. This dried solid residue was subsequently combined with 10 ml of methanol. The total phenolic contents in the prepared sample was determined following the Folin-Ciocalteu protocol. Two milliliters of prepared samples from each replica were placed in tubes, and then 1 ml of Folin-Ciocalteu reagent and 0.8 ml of Na₂CO₃ solution (7.5%) were vigorously mixed and stand for about 30 minutes. Using UV/VIS spectrophotometer absorbance at 765 nm were measured. The total phenolic contents were quantified in terms of gallic acid equivalents per gram of dry material.

2.17 Estimation of proline content

The method followed by [40] was used to assess the free leaf proline content, utilizing toluene as the solvent. Fully developed plant leaves were harvested for sampling. An acid ninhydrin solution was prepared by heating 2.5 g of ninhydrin with a mixture of 60 mL of glacial acetic acid and 40 mL of 6 M ortho-phosphoric acid until complete dissolution, with continuous agitation. About 0.2 g of fresh plant material was homogenized in 5 mL of 3% aqueous sulfosalicylic acid and subsequently filtered through filter paper. Following this, two milliliters of the filtrate were combined with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid in a test tube, and the reaction was allowed to proceed for 1 hour at 100°C incubated oven (POL-EKO-APARATURA SP.J. Type: SLN 32 IG SMART). Termination of the reaction was achieved by placing the tube in ice. The resulting mixture was then subjected to extraction with 4 mL of toluene, vigorously stirred with help of test tube stirrer for 15–20 seconds. Toluene colored layer was then separated from aqueous phase allowed to reach room temperature, and its absorbance was measured at 520 nm, with toluene used as the blank.

2.18 Estimation of inorganic mineral ions content

Root and shoot dry samples were finely ground using pre-chilled mortar and pestle. To digest 0.1 grams of shoot and root samples, H₂SO₄ was utilized. Subsequently, these samples were placed in test tubes, to which 2 ml of H₂SO₄ was added. 24-hours later, the samples were heated on a hot plate (Model: 85–2) and H₂O₂ was added gradually until the mixture became colorless. Following this step, distilled water was incorporated, and the final volume was adjusted to 50 ml. The resulting mixture was filtered, and readings for Ca^2+, K⁺, and Na⁺ and ions were obtained using a flame photometer (Sherwood model 360).

2.19 Determination of yield parameters

After four months of germination, yield from the mature plants was attained and the seeds were stored. The measured yield parameters included the number of pods per plant and the weight of 100 seeds (in grams).

2.20 Statistical Analysis

During the research, ANOVA and least significant difference (LSD) mean compare tests for all studied parameters was conducted using the statistics program R Studio Software (R 4.3.3). Graphs were generated using MS Excel 365.

3.1 Effect of organic waste material and rhizobacteria on growth parameters of Brassica juncea under salinity stress

3.1.1 Shoot fresh weight

The shoot fresh weight of Brassica juncea showed highly significantly results in both fertilizer and salt stress treatment, but their interaction was not significant (Table 2). Peels (eggshell + banana peel) treatment notably increased shoot fresh weight by 9%, whereas the combination of peels and Bacillus megaterium resulted in only a 1% increase under non-stress condition. Compared to the control, the combined treatment of B. megaterium with peels and NPK was statistically significant. Under 150 mM salt stress, shoot fresh weight decreased by 43% relative to the control (Fig. 1A). However, all treatments showed a positive response to salt stress. The peels treatment revealed the highest increase in shoot fresh weight by 37%, while the NPK treatment had the lowest increase by 4%. Overall, all treatments significantly enhanced shoot fresh weight under salt stress condition.

Table 2

Effect of organic waste material and rhizobacteria on Shoot fresh weight, Shoot dry weight, Root fresh weight, Root dry weight, Shoot length, Root length, Leaf area and Leaf number under salinity stress.
Source	Df	Shoot fresh weight		Root fresh weight	Root dry weight	Shoot length	Root length	Number of leaves	Leaf area
Fertilizer	4	57.064167 ***	0.1265***	0.317955***	0.0046967***	267.9***	17.234917***	18625***	18625***
NaCl	1	1133.4453 ***	6.88323***	2.7240533***	0.0048133***	1604.5453***	114.66075 ***	290083.33***	290083.33***
Fertilizer*NaCl	4	13.936167*	0.0177133 ns	0.024995*	2.1333e-4 ns	22.968667*	0.6149167 ns	3208.3333 ns	3208.3333 ns
Error	20	3.521<-	0.0066067<-	0.0065467<-	1.9667e-4<-	7.347<-	0.4799167<-	1250<-	1250<-

3.1.2 Shoot dry weight

In case of shoot dry weight of Brassica juncea revealed highly significant effects under salt stress and fertilizer treatment, while the interaction between fertilizer and NaCl was non-significant (Table 2). Compared to the control, all treatments positively enhanced shoot dry weight under non-stress conditions, with the maximum increase of 7% observed in the peel powder treatment and the minimum increase of 2% in the NPK treatment. Salt stress significantly reduced shoot dry weight by 32%. However, peels, B. Megaterium, their combination and NPK treatment mitigated the harmful effects of salt stress and improved shoot dry weight compared to the control. Under salt stress, the peels treatment resulted in the maximum increase of 24% in shoot dry weight, while B. megaterium treatment showed a minimum increase of 6% compared to control (Fig. 1B).

3.1.3 Root fresh weight

The analysis of variance for the root fresh weight of Brassica juncea showed that NaCl treatment and fertilizer had a highly significant effect, including their interaction (Table 2). Compared to the control, peels treatment showed highest increase by 22% in root fresh weight, while smallest increase by 3% from NPK fertilizer. under non-stress conditions. All treatments (peels, B. megaterium, peel + B. megaterium, and NPK fertilizer) increased root fresh weight under no stress. Under salt stress, root fresh weight decreased by 28% compared to the control. However, all treatments improved the root fresh weight by 43%, 12%, 12%, and 9%, respectively, under salinity stress (Fig. 1C). Among these, the peel treatment showed a significant effect under salt stress, while B. megaterium combined with peel and NPK treatments were not statistically significant.

3.1.4 Root dry weight

Root dry weight of Brassica juncea showed highly significant effects under salinity stress and fertilizer application, but their interaction was non-significant (Table 2). Compared to the control, all treatments enhanced root dry weight under non-stress conditions, with the maximum increase of 50% observed in peels treatment and the minimum increase of 21% in the NPK treatment. Peels treatment reveals statistically highly significant effect, while B. megaterium and NPK treatment showed non-significant effect under non-stress conditions. Root dry weight was reduced by 19% under NaCl stress compared to the control (Fig. 1D). Under salt stress, all treatments positively affected root dry weight, with the peel’s treatment showing the highest increase of 54% and NPK showed the lowest increase of 41% compared to the control.

3.1.5 Shoot length

Highly significant effects on shoot length in Brassica juncea were observed under salinity stress and fertilizer treatment. The interaction between fertilizer and NaCl was significant (Table 2). All treatments increased shoot length compared to the control without salt stress, with highest increase (18%) was observed from the combine treatment. Peels treatment alone increased shoot length by 17%, B. megaterium by 5%, and NPK by 2%. NaCl decreased shoot length by 28%. Under salt stress, all treatments improved shoot length, with peels treatment having the most significant effect, increasing shoot length by 44%. B. megaterium increased shoot length by 18%, the peel powder and B. megaterium combination by 28%, and NPK by 5% under salt stress (Fig. 2A).

3.1.6 Root length

The root length of Brassica juncea exhibited highly significant results under both fertilizer and salt stress, with a non-significant interaction between fertilizer and NaCl (Table 2). The combined treatment of peels and B. megaterium resulted in the maximum increase of 29% in root length compared to the control under non-stress conditions. Peels, B. megaterium and NPK treatments increased root length by 23%, 21%, and 6%, respectively, compared to the control without stress (Fig. 2B). B. megaterium, peels, and their combination combine treatment showed statistically non-significant with each other. Root length decreased by 36% under salt stress compared to the control. All treatments positively affected root length under salt stress, with the combined treatments showing the highest increase of 55%, and NPK showing the smallest increase of 1%. Peels and B. megaterium treatments showed non-significant effect, while their combination was highly significant effect under salt stress.

3.1.7 Number of leaves plant^− 1

The analysis of variance data showed that both salt stress and fertilizer treatments had highly significant effects on the number of leaves in Brassica juncea. The interaction between salinity and fertilizer was non-significant (Table 2). All treatments positively affected leaf area compared to the control without salt stress. The maximum increase in leaf number (22%) was observed with peel treatment, while the minimum increase (3%) was with NPK fertilizer. B. megaterium and its combination with peel treatment increased leaf number by 3% and 13%, respectively, compared to the control without stress. Salt stress reduced the number of leaves by 22%. Under salinity stress, peels treatment increased the number of leaves by 28%, B. megaterium by 8%, NPK increased 12% compared to control (Fig. 2C).

3.1.8 Total leaf area plant^− 1

In case of leaf area of Brassica juncea showed highly significant effects under salinity stress and fertilizer treatment, with a non-significant interaction between salinity and fertilizer (Table 2). All treatments increased leaf area compared to the control without salt stress. The maximum increase in leaf area (27%) was observed with peels treatment, while the minimum increase (14%) was with the combination of peels and B. megaterium treatment (Fig. 2D). B. megaterium and NPK treatments increased leaf area by 20% and 16%, respectively, compared to the control without stress. Salt stress significantly reduced leaf area by 16%. Under salt stress, peel powder treatment increased leaf area by 6%, B. megaterium by 8%, and their combination by 3%. NPK and the combination treatment were statistically non-significant under salt stress.

3.2 Effect of organic waste material and rhizobacteria on Physiological parameters of Brassica juncea under salinity stress.

3.2.1 Net CO₂ assimilation rate

Brassica juncea plant showed highly significant effect under fertilizer and NaCl treatment in net CO₂ assimilation rate (A). Interaction between fertilizer and NaCl also highly significant (Table 3). All treatments elevated the assimilation rate when compared with control no addition of stress. Maximum increase 67% in CO₂ assimilation rate was observed with peels treatment. In relation to control, B. megaterium treatment increase 53%, its combine form increased 44% and NPK treatment increase 51% in CO₂ assimilation rate with no addition of stress. NaCl showed negative response and decrease 29% in CO₂ assimilation rate in relation to control. But all treatments were improved the CO₂ assimilation rate when compared with control under salt stress. B. megaterium treatment showed highly significant effect and it resulted maximum increase 86% in CO₂ assimilation rate when compared with control under salt stress. Peels treatment increase 26%, combine treatment increase 76% and NPK increase 49% in CO₂ assimilation rate when compared with control under salt stress. (Fig. 3A).

Table 3

Effect of organic waste material and rhizobacteria on SPAD value, Intercellular CO₂ concentration (ci), Stomatal conductance (gs), Rate of photosynthesis (A), Rate of transpiration (E), Variable fluorescence (Fv), Maximal fluorescence (Fm), Maximum quantum yield of photosystem II (Fv/Fm), Minimal fluorescence (Fo) and Fv/Fo ratio, Water potential, Osmotic potential and turgor potential under salinity stress.
Source	Df	SPAD value		gs	A	E	Fo	Fm	Fv	Fv/Fo	Fv/Fm	Water potential	Osmotic potential	Turgor potential
Fertilizer	4	33.5753***	2813.1333 ***	0.010521***	99.642872***	0.038845***	262.86667***	9056.2833***	12008.58***	2.0050083***	0.014227***	830***	13429.917***	8589.9167***
NaCl	1	168.033***	3763.2***	0.0644033***	361.08821***	0.28812***	720.3 ***	82477.633***	104430***	5.0939681***	0.14686***	6750***	65894.533***	30464.533***
Fertilizer*NaCl	4	7.69333**	369.7 **	0.00346**	30.186988***	0.039761***	70.8 ns	5834.7167***	6628.083***	0.1443325ns	0.0014378*	33.333333ns	1110.2833*	1061.95*
Error	20	1.6633333<-	82.566667<-	7.1e-4<-	2.4293033<-	0.0038267<-	36.3<-	511.86667<-	572.3<-	0.0763984<-	4.564e-4<-	56.666667<-	323.56667<-	319.23333<-

3.2.2 Stomatal conductance (gs)

The stomatal conductance of Brassica juncea plants was affected and showed highly significant results in both fertilizer and NaCl treatment. Interaction between fertilizer and NaCl was significant (Table 3). In comparison to control, all treatments showed positive response and enhanced the stomatal conductance with no addition of stress. Maximum increase (65%) of stomatal conductance was observed in peel treatment while minimum increase 40% was found in combine treatment with no addition of stress (Fig. 3B). Compared to control B. megaterium treatment increase 42% while NPK treatment increase 43% with no addition of stress. NaCl treatment showed highly significant results and effectively reduced 42% of stomatal conductance. Maximum increase 39% of stomatal conductance was observed in combine treatment and minimum increase 20% was observed in both peel and NPK treatment under salt stress respective to their control.

3.2.3 Intercellular CO₂ concentration (Ci)

In case of intercellular CO₂ (ci) concentration Brassica juncea plant showed highly significant result in both NaCl and fertilizer treatment. Interaction between NaCl and fertilizer was significant (Table 3) All treatments showed positive response and increased the ci concentration compared to control without stress plant. Maximum increase (25%) in ci was observed with treatment of peels and minimum increase 20% was found in NPK treatment with no addition of stress. B. megaterium treatment enhanced 24% ci concentration and its combine form enhanced 22% compared with control no addition of stress. Ci concentration was remarkably affected due to salt stress. Ci concentration reduced 4% in parallel to control (Fig. 3C). Under salt stress all treatments improved the ci concentration. Ci concentration of Brassica juncea increased by 12%, with Peels treatment. B. megaterium treatment increased by 21%, its combine form increased 24% and NPK treatment increased by 9% under salt stress.

3.2.4 Transpiration rate

Salt stress significantly affected the transpiration rate of Brassica juncea and reduced particularly 14% in parallel to control. Interaction between fertilizer and NaCl was highly significant (Table 3). As compare to control, peels treatment improved the transpiration rate. Maximum increase (38%) of transpiration rate was observed in peels treatment compared to control with no addition of stress. Moreover, B. megaterium treatment increase 27%, its combine form increased 5% and NPK treatment increase 21% of transpiration rate in contrast to control with no addition of stress (Fig. 3D). Under salt stress all treatments showed highly significant effect and elevated the rate of transpiration. Maximum increase (22%) of transpiration rate found with combine treatment under salt stress. However, B. megaterium treatment increase 14%, peels treatment increased 6% and NPK treatment increase 19% rate of transpiration under salt stress.

3.2.5 Total chlorophyll content (SPAD value)

In Brassica juncea plant the SPAD value revealed highly significant effect under salt stress and fertilizer treatment. Interaction between salinity and fertilizer was significant (Table 3). All treatments showed remarkable response and increase the SPAD value relative to control with no addition of salt stress. Maximum increase 15% in SPAD value was observed with NPK treatment. When compare to control plant, peels treatment increased 14%, B. megaterium treatment increase 6% and combine treatment increase 6% in SPAD value with no addition of salt stress (Fig. 4A). In comparison NPK and B. megaterium showed statistically non-significant results while peels treatment and its combine form showed significant effect in contrast to control to with no addition of salt stress. NaCl showed negative response and reduced 10% in SPAD value opposed to control. But all treatments were improved the SPAD value respective to their control in salt stress. Peel treatment showed highly significant effect and it resulted maximum increase 14% in SPAD value under salt stress. B. megaterium increased treatment increase 7%, combine treatment increase 12% and NPK increase 7% in SPAD value under salt stress.

3.2.6 Water Potential

The result showed that Brassica juncea exhibited highly significant changes in water potential under both NaCl and fertilizer treatments. The significant interaction between them (Table 3). Compared to control, peels treatment showed the greatest increase in water potential (66%), while the lowest increase (22%) was observed with NPK in the absence of stress. B. megaterium treatment enhanced water potential by 33%, and the combined treatment (B. megaterium + peels) increased it by 44% compared to control without stress. Under salt stress, water potential decreased by 39% relative to the control, but all treatments still showed positive effects. Peels treatment resulted in the highest increase in water potential (73%), while B. megaterium and NPK treatment caused the lowest increase (27%) under salt stress (Fig. 4B). Statistically significant differences were observed among B. megaterium, peels, their combination, and NPK treatments under salt stress conditions.

3.2.7 Osmotic potential

In case of osmotic potential of Brassica juncea showed highly significant effects under both NaCl and fertilizer treatments, with a significant interaction between NaCl and fertilizer (Table 3). Compared to control, peels and its combine treatment resulted highest increase in osmotic potential (16%), whereas NPK showed the lowest increase (6%) without stress. B. megaterium treatment improved osmotic potential by 11%, compared to control without stress. Under salt stress, osmotic potential decreased by 13% relative to the control, but all treatments still showed positive effects. B. megaterium treatment led to greatest increase in osmotic potential (18%), peels treatment increase (17%), while NPK resulted in the lowest increase (10%) under salt stress (Fig. 4C).

3.2.8 Turgor pressure

Turgor potential of Brassica juncea plants showed highly significant result under fertilizer and NaCl treatments. The interaction was significant between NaCl and fertilizer (Table 3). All treatment showed positive results compared to control. Combine treatment had highest increase in turgor potential (14%), while NPK treatment had lowest increase (5%) without stress plant. B. megaterium treatment improved turgor potential by 9%, and the peel treatment increased it by 12% relative to the control without stress. Under salt stress, turgor potential decreased by 10% compared to the control, but all treatments still revealed positive effects relative to this stress condition. The B. megaterium treatment achieved the maximum increase in turgor potential (17%), whereas the minimum increase was observed with peels and NPK treatment (9%) and combine treatment increase (16%) under salt stress (Fig. 4D).

3.2.9 Chlorophyll fluorescence

Chlorophyll fluorescence parameters responded variably to different treatments, including Bacillus megaterium, peels, their combined form, and NPK, as well as under salt stress (Table 3). The minimal fluorescence (F₀) showed weak modulation across all treatments, had a negligible effect on the basal level of PSII activity and F₀ increase by (17%) under salt stress (Fig. 5A). However, maximum fluorescence (Fm) elevated with treatment of Bacillus megaterium, peels, their combination and NPK, showed an improvement in the efficiency of the photosynthetic apparatus. Fm greatest increase was observed by (34%) in peels treatment without stress plant. Salt stress affected the Fₘ and it decrease 10% due to detrimental impact of salinity on PSII efficiency (Fig. 5B). But all treatments respond positively under salt stress. Combined treatment increased by 23% of Fm under salt stress. In case of Variable fluorescence Fv all treatments behaved positively and enhanced. Maximum Fv found in peels treatment by 52% compared to control without stress plant. Salinity stress reduced the Fv by 20% but all treatment still responds positively under salinity stress (Fig. 5C). Maximum Fv was observed in combined treatment by 42% under salinity stress. Furthermore, the Fv/F₀ ratio denotes variable fluorescence to minimal fluorescence, reduced by 32% under salt stress may be reduction in the photochemical efficiency of PSII. This decline emphasizes the adverse effect of salinity on the photosynthetic process. while the treatments elevated system performance and showed maximum Fv/F₀ ratio by 42% compared to control without stress and 66% in peels treatment under salinity stress (Fig. 6A). The F_V/Fm ratio revealed maximum increase by 19% in NPK treatment compared to control, while Bacillus megaterium and peels treatment showed increases by 13% and 17% respectively under no salt stress. Under NaCl stress all treatments improved F_V/Fm ratio, with peels treatment showing the highest increase by 17%. Bacillus megaterium, combined and NPK treatment resulted in 17%, 14%, and 4% increases, respectively, under salt stress. Salt stress reduced F_V/Fm ratio by 17% (Fig. 6B).

3.2.10 Chlorophyll a

Chlorophyll a content in Brassica juncea exhibited highly significant effects under salt stress and fertilizer treatments. The interaction between salinity and fertilizer also being highly significant (Table 4). All treatments positively affected chlorophyll a content compared to the control without salt stress. The maximum increase of 18% in chlorophyll a was observed with the combined treatment, while the minimum increase of 2% was observed with NPK fertilizer. Peels treatment increased chlorophyll a by 17%, B. megaterium by 5%, and NPK by 2% compared to the control without stress. NaCl treatment significantly reduced chlorophyll a by 28% compared to the control (Fig. 7A). Under salt stress, all treatments improved chlorophyll a compared to the control. Peels treatment showed the highest increase (44%), B. megaterium (18%), the peel + B. megaterium combination (28%), and NPK (5%). B. megaterium and their combination treatment were statistically significant under salt stress, whereas NPK was non-significant.

Table 4

Effect of organic waste material and rhizobacteria on Relative water content (RWC), Relative membrane permeability (RMP), Malondialdehyde (MDA), Hydrogen peroxide (H₂O₂), Chlorophyll A (ch a), Chlorophyll B (ch b) and carotenoids under salinity stress.
Source	Df	RWC		MDA	H₂O₂	Ch a	Ch b	Carotenoids
Fertilizer	4	269.38333 ***	204.13333***	8.425013***	0.1322144***	4.3987e-4***	1.5469e-4***	1.4591704 ***
NaCl	1	1128.5333***	496.13333***	138.5824***	0.5837238***	0.0027906***	0.001509***	30.02731***
Fertilizer*NaCl	4	19.95*	41.966667***	4.1386825***	0.0588049***	1.5745e-4 ***	1.1287e-5ns	0.2159214 ns
Error	20	4.8<-	4.8333333<-	0.3862144<-	5.4988e-4<-	1.1086e-5<-	4.4393e-6<-	0.0940647<-

3.2.11 Chlorophyll b

In case of chlorophyll b of Brassica juncea exhibited highly significant results in both fertilizer and NaCl treatments. The interaction between salinity and fertilizer being notably significant (Table 4). Compared to the control, all treatments enhanced chlorophyll b levels. The peels treatment revealed maximum increase of 38%, while the B. megaterium treatment showed a minimum increase of 26% without stress plant. Under NaCl treatment, chlorophyll b levels significantly decreased by 34%. However, treatments with peels, B. megaterium, their combined and NPK mitigated the negative effects of NaCl stress and improved chlorophyll b content relative to their control. Among these, the peels treatment resulted in increase of 34%, whereas the B. megaterium treatment showed a minimum increase of 27%, under salt stress (Fig. 7B).

3.2.12 Carotenoids

The carotenoid content in Brassica juncea was affected by salt stress. Analysis of variance data showed highly significant results for both fertilizer and NaCl treatments, while the interaction between NaCl and fertilizer was non-significant (Table 4). All treatments showed to an increase in carotenoids compared to the control without salt stress. The NPK treatment showed the maximum increase of 29% in carotenoids, Peels increased by 25%, B. megaterium increased by 25%, their combined form increased by 13 with no stress plant. NaCl treatment alone had a negative impact, reducing carotenoid content by 25% compared to the control. However, under NaCl stress, all treatments improved carotenoid content compared to the control (Fig. 7C). Among these, Peels treatment showed a highly significant effect, resulting in an 18% increase in carotenoids. B. megaterium increased by 10%, their combined form enhanced by 1% and NPK enhanced by 17% under NaCl stress.

3.2.13 Relative water content

In case of relative water content of Brassica juncea showed highly significant results for fertilizer and salt stress. The interaction between them was non-significant (Table 4). Peels treatment increased relative water content by 27%, while NPK showed a 5% increase without stress. B. megaterium treatment resulted in a 13% increase, but the combination of B. megaterium and peels had no effect compared to the control (Fig. 7D). Under salt stress, relative water content decreased by 17% compared to the control. However, all treatments showed a positive response under salt stress, with peels treatment revealed by 30% increase and NPK a 22% increase compared to the control under salt stress

3.3 Effect of organic waste material and rhizobacteria on Membrane stability of Brassica juncea under salinity stress.

3.3.1 Malondialdehyde

The interaction between fertilizer and salinity was highly significant (Table 4). Malondialdehyde (MDA) content, a negative indicator of plant stress, increased with stress. Without stress, all treatments reduced leaf MDA compared to the control. The maximum decrease of 31% in leaf MDA was observed with peels treatment, while the minimum decrease of 4% was found with the combined treatment of B. megaterium and peels (Fig. 8A). B. megaterium alone reduced leaf MDA by 2%, and NPK reduced it by 19% compared to the control without stress. NaCl significantly increased leaf MDA content, with a maximum increase of 103% under salt stress. However, all treatments improved leaf MDA content under salt stress, with peels treatment reducing it by 33% and B. megaterium treatment by 20% under salinity stress.

3.3.2 Hydrogen peroxide (H₂O₂) content

In case of Hydrogen peroxide activity Brassica juncea plants revealed highly significant results for fertilizer and salinity treatments. The interaction between salinity and fertilizer was also highly significant (Table 4). Compared to the control, all treatments reduced H₂O₂ activity without added stress. The maximum decrease of 42% in H₂O₂ activity was observed with B. megaterium treatment, while the minimum decrease of 13% was found with the combined treatment of B. megaterium and peels. Peels treatment reduced H₂O₂ activity by 18%, and NPK reduced it by 24% compared to the control without stress. All treatments were statistically significant compared to each other with no stress. NaCl significantly increased H₂O₂ activity, with a maximum increase of 67% under salt stress (Fig. 8B). However, all treatments improved H₂O₂ activity under salt stress, with the combined treatment reducing it by 43% and B. megaterium the treatment by 30% under salt stress.

3.3.3 Relative membrane permeability

The relative membrane permeability of Brassica juncea showed highly significant effect under salt stress and fertilizer treatment, with a highly significant interaction between salinity and fertilizer (Table 4). Relative membrane permeability, a negative characteristic indicating cell membrane leakage, increased with stress. Without stress, all treatments reduced relative membrane permeability compared to the control. The maximum decrease of 31% was observed with the combined treatment of peels and B. megaterium, while the minimum decrease of 13% was found with B. megaterium treatment alone (Fig. 8C). Salinity stress significantly increased relative membrane permeability by 31% compared to the control. Under salt stress, all treatments improved relative membrane permeability, B. megaterium showed the maximum decrease of 35% and NPK showed the minimum decrease of 20% under salt stress.

3.4 Effect of organic waste material and rhizobacteria on biomolecules parameters of Brassica juncea under salinity stress.

3.4.1 Activity of catalase

The catalase activity of Brassica juncea plants showed highly significant effects both in fertilizer and NaCl treatment. Interaction between salt stress and fertilizer was significant (Table 5). In comparison to control, all treatments showed positive response and elevated catalase activity without addition of stress. Maximum increase by 47% of catalase activity was observed in B. megaterium treatment while minimum increase 27% was found in NPK when compared to control with no addition of stress. Peels treatment increased by 29%, while its combine form (B. megaterium + peels) increase by 36% of catalase activity in contrast to control with no addition of stress. Highly significant effect was obtained with salt stress and it resulted maximum increase by 46% of catalase activity in parallel to control. Under salinity stress all treatments showed positive response and enhanced catalase activity. Peels treatment increase by 28%, B. megaterium increase by 13% and its combine form increase 21% while, NPK increased by 7% of catalase activity of Brassica juncea plants under salt stress. (Fig. 9A).

Table 5

Effect of organic waste material and rhizobacteria on Total soluble protein (TSP), Catalase (CAT), Peroxidase (POD), Super oxide dismutase (SOD), Ascorbate peroxidase (APX), Total phenolic content (TPC), Leaf proline (LP) under Salinity stress.
Source	Df	TSP		APX	POD	SOD	TPC	LP
Fertilizer	4	9.202e-7***	4.6464e-5 ***	0.0096967***	4.6161e-5***	1754459.4***	188346.08***	0.1168826***
NaCl	1	2.0973e-5***	3.7725e-4 ***	0.1860422***	0.002677***	13310050***	3547092.4 ***	2.4328381***
Fertilizer*NaCl	4	2.3767e-7**	1.192e-5*	9.1728e-4*	2.0612e-6ns	1367629.7***	22291.913*	0.03590547***
Error	20	4.8727e-8<-	3.856e-6<-	3.0756e-4<-	3.0118e-6<-	33318.304<-	6472.2885<-	0.0014372<-

3.4.2 Peroxidase activity

In case of peroxidase activity of Brassica juncea plants revealed highly significant effects under salt stress and fertilizer treatments. The interaction between salt stress and fertilizer was non-significant (Table 5). Compared to the control, all treatments boosted peroxidase activity without stress plants. The maximum increase by 32% was observed with combined treatment of B. megaterium and peels, while the minimum increase by 1% was found with NPK fertilizer. Peels treatment alone increased peroxidase activity by 28%, and B. megaterium by 10%. Under salt stress, there was a highly significant effect, resulting in a maximum increase by 96% in peroxidase activity compared to the control. All treatments elevated peroxidase activity under salinity stress. Peels by 21%, B. megaterium by 9%, the combined form by 19%, and NPK by 9% (Fig. 9B).

3.4.3 Superoxide dismutase activity

In case of superoxide dismutase activity of Brassica juncea plants showed highly significant results with both salt stress and fertilizer treatments. The interaction between NaCl and fertilizer was also highly significant (Table 5). Compared to the control, all treatments elevated superoxide dismutase activity without stress plants. The maximum increase by 78% was observed with NPK fertilizer, while the minimum increase of 14% was found with peels treatment. B. megaterium elevated activity by 49%, and the combined treatment with peels increased it by 75% without stress plants. Under salinity stress, superoxide dismutase activity enhanced significantly, with a maximum increase by 84% compared to the control (Fig. 9C). Plants treated with peels, the combined treatment of B. megaterium and peels, and NPK all enhanced superoxide dismutase activity under salt stress.

3.4.4 Ascorbate peroxidase activity

The ascorbate peroxidase activity of Brassica juncea plants shown statistically highly significant results with both fertilizer and NaCl stress treatments. Compared to the control, all treatments enhanced ascorbate peroxidase activity without stress. The maximum increase by 58% was observed with B. megaterium treatment, while the minimum increase by 43% was found with peels treatment without stress condition. The interaction between fertilizer and NaCl was significant (Table 5). NaCl stress had a highly significant results, it was maximum increase by 93% in ascorbate peroxidase activity compared to the control. Under salt stress, all treatments boosted ascorbate activity. Peels treatment enhanced it by 6%, B. megaterium by 22%, the combined form by 16%, and NPK by 15% (Fig. 9D).

3.4.5 Total phenolic

The addition of NaCl treatment increased the accumulation of phenolics content in Brassica juncea plants. Under salinity stress, the application of peels and B. megaterium shown highly significant effect on total phenolic content. The interaction between fertilizer and salt stress was significant (Table 5). All treatments elevated total phenolic content positively. The maximum increase by 59% was observed with the combined treatment of peels and B. megaterium compared to the control without stress plant. Peels treatment alone enhanced total phenolic content by 39%, B. megaterium by 23%, and NPK by 38% compared to the control without stress. NaCl treatment alone increased total phenolic content by 79% compared to the control. Under salt stress, the combined treatment of peels and B. megaterium resulted in the maximum increase of 19% in total phenolics compared to the control. Peel powder treatment increased total phenolics by 13%, B. megaterium by 11%, and NPK by 5% under NaCl stress (Fig. 10A).

3.4.6 Leaf Proline

Leaf proline content of Brassica juncea plants revealed highly significant effect under salt stress and fertilizer treatments. All treatments elevated leaf proline levels. Compared to the control, the maximum increase by 149% was observed with the combined treatment of peels and B. megaterium, while the minimum increase by 56% was found with B. megaterium treatment without stress plants (Fig. 10B). NPK increased leaf proline by 99%, and B. megaterium treatment with peels increased it by 66% compared to the control without stress. The interaction between NaCl and fertilizer was highly significant (Table 5). Salt stress had a highly significant effect, enhancing leaf proline levels by 201% compared to the control. Under salt stress, all treatments increased leaf proline levels. The maximum increase by 22% was observed with the combined treatment of peels and B. megaterium, while the minimum increase by 2% was found with NPK fertilizer. Peels treatment increased leaf proline by 22%, and B. megaterium treatment increased it by 11% under salt stress.

3.4.7 Total soluble protein

In the case of total soluble protein in Brassica juncea plants had highly significant results with both fertilizer and NaCl treatments. The interaction between NaCl and fertilizer was also significant. Compared to the control, all treatments positively enhanced protein content without stress plant (Table 5). The maximum increase by 31% in protein content was observed with the combined treatment of peels and B. megaterium, while the minimum increase by 2% was found with NPK and B. megaterium compared to the control without stress. The peels treatment alone increased protein levels by 14% compared to the control without stress. Under salt stress, there was a highly significant effect noted resulting in a maximum increase of 69% in protein content compared to the control. All treatments showed a positive response and enhanced protein content under salt stress. Specifically, peels treatment increased protein content by 35%, B. megaterium by 1%, their combined form by 25%, and NPK by 4% under salt stress (Fig. 10C).

3.5 Effect of organic waste material and rhizobacteria on inorganic ion of Brassica juncea under salinity stress.

3.5.1 K⁺ ion in shoots

Under salt stress, the concentration of K⁺ ions in the shoots of Brassica juncea decreased as the NaCl concentration increased. A highly significant effect was observed for both fertilizer application and salt stress, and their interaction was also significant (Table 6). All treatments resulted in a positive increase in potassium ion levels in the shoots compared to the control with no stress addition. The maximum increase in K⁺ ions by 38%, was observed with peels treatment, while the minimum increase by 18% was found with NPK fertilizer, compared to the control with no stress addition. Combined treatment with B. megaterium and peels increased K⁺ ions by 36%, and B. megaterium alone increased by 23%, compared to the control with no stress addition. K⁺ ion uptake in shoots decreased by 29% compared to the control under salinity stress. However, all treatments under salt stress showed increases in K⁺ ion levels by 46% with peel powder, 26% with NPK, 23% with B. megaterium and 29% with combined B. megaterium and peels treatment (Fig. 11A).

Table 6

**Effect of organic waste material and rhizobacteria on Shoot Na**⁺, **Shoot k** ^+, **Root k** ⁺, **Shoot Ca**⁺⁺, **Root Ca**⁺⁺ **ion, Number of pods and seeds weight under salinity.**
Source	Df	Shoot Na⁺		Shoot Ca⁺⁺	Root Ca⁺⁺	Shoot K	Root K	No. of pods	Seeds weight
Fertilizer	4	195.23983***	260.30772***	173.93943***	173.93943***	142.5129***	212.30772***	454.36667***	0.11217***
NaCl	1	1241.6618***	974.55337***	295.72068***	295.72068***	1208.1644***	489.55337***	4737.6333***	o.797073***
Fertilizer*NaCl	4	16.680103*	31.4774907***	71.824578***	71.824578***	12.026437*	19.4774907*	125.46667***	0.0.01862***
Error	20	5.2766614<-	4.0508181<-	4.6657263<-	4.6657263<-	4.1068444<-	5.05317664<-	5.6<-	4.5333e-4-<-

3.5.2 K⁺ ion in roots

K⁺ ion in roots of Brassica juncea was affected under salt stress. K⁺ ion in roots was decreased when NaCl concentration increased. Analysis of variance data showed highly significant effect under salt stress and fertilizer. Interaction between NaCl and fertilizer was significant (Table 6). All treatment showed positive result and elevated potassium ion in roots compared to control with no addition of stress. Maximum increase by 39% of K⁺ ion in roots was obtained in peels treatment while its minimum increase by12% in B. megaterium treatment in contrast to control with no addition of stress. Furthermore, combine treatment (B. megaterium + peels) increase 26% and NPK increase by 16% compared to control with no addition of stress Fig. 11B). Salinity stress considerably reduced the K⁺ ion in root and it resulted decrease by 26% as opposed to control. But in salinity stress all treatments showed increase 48%, 46%, 40% and 74% respectively K⁺ ion in roots.

Ca ²⁺ ion in shoots

Ca²⁺ ion in shoot of Brassica juncea showed highly significant effects under salt stress and fertilizer treatment. The Interaction between NaCl and fertilizer was highly significant (Table 6). All treatment showed positive result and increase calcium ion in shoots when compared to control with no addition of stress. Maximum increase 19% of calcium ion in shoot was obtained in peels treatment while minimum increase 1% was found in NPK when compared to control with no addition of stress. B. megaterium increased 1% and its combine form increased 13% in comparison to control with no addition of stress (Fig. 11C). Salt stress lowered the calcium ion in shoot and it resulted decrease by 19% in comparison to control. Maximum increase 92% of calcium ion was observed in combine treatment (Peels + B. megaterium) when compared to control under salt stress. B. megaterium increased by 58%, peels increased by 88% and NPK increase by 58% of Ca²⁺ ion in shoots in comparison to control under salt stress.

3.5.3 Ca²⁺ ion in roots

In case of ca²⁺ ion in roots of Brassica juncea showed highly significant results under salinity and fertilizer treatment. Interaction between salt stress and fertilizer was significant (Table 6). All treatments showed positive response and enhanced calcium ion uptake in roots when compared to control with no addition of stress. Maximum increase by 14% of calcium ion in root was obtained in peels treatment while minimum increase by 3% was found in NPK compared with control without stress plant. B. megaterium increase by 8% and its combine form increased 9% compared to control with no addition of stress. There was remarkable reduction of calcium ion in root was observed under salt stress and it resulted decrease 20% in corresponding to control. Maximum increase 64% of calcium ion was observed in combine treatment (Peel + B. megaterium) compared with control under salt stress. B. megaterium increase by 42%, peels increase by 62% and NPK increase by 39% of Ca²⁺ ion in roots compared with control under salt stress (Fig. 11D).

3.5.4 Na⁺ ion in shoot

Na⁺ ion in shoot of Brassica juncea plant showed highly significant effects in both fertilizer and salt stress treatment (Table 6). Interaction between salinity and fertilizer was significant. In comparison to control, plants treated with Peel, B. megaterium, its combination, NPK reduced the Na⁺ ion uptake with the no addition of stress. Maximum decrease by 31% of sodium ion in shoot of Brassica juncea was noted in combined treatment (Peels + B. megaterium) while minimum decrease by2% was observed in NPK when compared to control with no addition of stress. Moreover, B. megaterium treatment decrease by 12% and peels treatment decrease 24% of Na⁺ ion in shoots in parallel to control with no addition of stress. Na⁺ ion in shoot of Brassica juncea was increase 52% when compared with control under salt stress. But all treatments lowered the Na⁺ ion by 23%, 20%, 23% and 19% compared to control under salt stress (Fig. 12A).

3.5.5 Na⁺ ion roots

Na⁺ ion in roots of Brassica juncea showed highly significant results in both fertilizer and NaCl treatment. Interaction between salinity and fertilizer was highly significant (Table 6). In comparison to control, plants treated with peels, B. megaterium, its combination, NPK reduced the Na⁺ ion uptake with no addition of stress. Maximum decrease by 29% of Na⁺ ion in Brassica juncea roots was observed in peels treatment while minimum decrease 11% was observed in NPK when compared to control with no addition of stress. Moreover, B. megaterium treatment decrease by 18% and combined treatment decrease 19% of Na⁺ ion in roots when compared to control with no addition of stress (Fig. 12B). Na⁺ ion in shoot of Brassica juncea was increased 35% when compared with control under salt stress. But all treatments lowered the Na⁺ ion by 28%, 26%, 28% and 22% in comparison to control under salt stress.

3.6 Effect of organic waste material and rhizobacteria on yield parameters of Brassica juncea under salinity stress.

3.6.1 Number of pods plant^− 1

Statistical analysis data revealed that both NaCl and fertilizer treatments significantly affected the number of pods in Brassica juncea plant. The highly significant interaction between salinity and fertilizer (Table 6). All treatments showed a positive response, increasing the number of pods compared to control with no salt stress. The maximum increase in the number of pods was 66% with peels treatment, while minimum increase by 27% with NPK in the absence of stress (Fig. 13A). B. megaterium treatment enhanced the number of pods by 37%, and the combined treatment of B. megaterium and peels increased by 49%, compared to the control with no stress. Under salt stress, the number of pods decreased by 22% compared to the control. However, peels treatment increased the number of pods by 23%, B. megaterium treatment by 13%, the combined treatment by 26%, and NPK by 4% under salt stress.

3.6.2 Weight of 100 Seeds

In seeds weight of Brassica juncea plant showed highly significant effect in fertilizer and salt stress. Interaction between fertilizer and salt stress was highly significant (Table 6). All treatments demonstrated positive results and enhanced the seeds weight of Brassica juncea. Peels treatment showed remarkable results and enhanced the seeds by 98%, B. megaterium enhance 72%, combine treatment (Peel + B. megaterium) enhance 92% and NPK enhance by 40% seed weight of Brassica juncea plant in relation to control with no addition of stress. Salt stress negatively impeded the seeds weight and its reduced 36% in contrasted to control (Fig. 13B). All treatments revealed positive results and increased the seeds weight under salt stress. Maximum seeds weight was observed in peels treatment under salt stress. B. megaterium enhanced seeds weight by 40%, combine treatment enhance 66% and NPK enhance by 36% as compare to their respective control under salt stress.

3.7 Pearson’s correlation and principal component analysis

Pearson’s correlation results shown in Fig. 14 revealed that Na⁺ ions in the shoot and root of B. juncea is negatively correlated with most of growth and physiological attributes. This depicts that elevated level of Na in response to salt stress resulted in reduced growth and physiological activities in B. juncea plants. Furthermore, Na contents in the plants are in positive relation with stress markers like MDA, H₂O₂ and RMP. This indicates that salt stress caused oxidative stress in plants in the form of increased lipid peroxidation and membrane leakage. The positive relation among Na, MDA, H₂O₂, RMP, CAT, POD, APX, SOD, proline and phenolics showed that oxidative stress caused due to salt stress also triggered antioxidant defense mechanism in plants resulting in increased activities of enzymatic and non-enzymatic antioxidants activities as first line of defense to scavenge oxidative stress. On the other side, it is depicted that soil amendment with organic waste and NPK fertilizers reduced Na⁺ uptake through roots and its subsequent translocation in shoot (Fig. 12). This lowered the effect of salt stress on growth and physiological activities of plants. PCA biplot results (Fig. 15) showed that all studied parameters can be divided into two groups, the aligned with PC1 and the other with PC2. PC2 contains mainly stress indicators and antioxidants which are negatively correlated with parameters of PC1 including growth, physiology and yield related attributes. PCA results showed that all the parameters were successively explained in the first two components (PC1 & PC2) with contribution of 86.82% in total. Dots with numerics represent various treatments employed in this study. Where 1 represents control plants and 6 represents salt stressed plants. As shown in Fig. 15, salt stress (6) is separated well from all others treatments that means it had a clearly different effect on all studied parameters compared to remaining treatments.

Salinity is a significant environmental factor impacting plant growth and yield. Therefore, increasing demand for oil seeds the utilization of salt-resistant crops can be a strategic approach to deal with this problem [41]. This aim of current study was to assess the impacts of organic waste material and rhizobacteria on growth and physiological events of Brassica juncea under salinity stress. Abiotic stresses upset the plant natural endogenous hormone level. Rhizobacteria have been observed to synthesize the phytohormones such as indole acetic acid and gibberellins that can have boosted the plants existing hormone levels and promote root and shoot development [42]. B. megaterium, along with other Bacillus species, reveals an exceptional capability to colonize the rhizosphere of grasses. It can solubilize phosphate via the release of inorganic and organic acids and mineralize phosphate, proton extrusion, by synthesizing phosphatases, and produce phytohormones and siderophores [17, 18].

From results of recent study, it was shown that Brassica juncea plants were adversely affected due to salt stress imposition. Salt stress impedes the growth parameters such as root fresh weight, dry weight of root, shoot length, root length, shoot fresh weight, dry weight of shoot and number of leaves in Brassica juncea. This may be due to nutritional imbalance, low osmotic potential and particular ionic action all contribute to the detrimental consequences of salinity [2]. Conversely, harmful ions accumulate at higher concentration in shoots compared to roots, plants leaves are highly susceptible to salt stress than roots [43]. Similar results were described about the detrimental effects of salinity stress on both root and shoot fresh and dry weights of barley plants [44]. Salinity stress triggered a remarkable reduction in Pisum sativum cultivars [45]. The inhibition of plant growth by salt-induced stress may have disturbed a range of physiological and biochemical processes at cellular or tissue or whole plant level. Harmful impact of salinity stress on plant growth attributes was observed in previous studies [46, 47]. Leaf area played a major role in physiological attributes, which in turn increased crop growth and the amount of photo assimilates that were accumulated from source to sink, thus increasing grain output [48]. Leaf area of maize plants significantly decreased with salt stress. From results of present study, it was shown that salt stress affected leaf area of Brassica juncea plants and reduced the leaf area. Leaf area of Brassica juncea plants decrease with salt stress might have been due to high osmotic pressure, reducing water availability, stomatal closure, reducing transpiration and photosynthesis. In this study, highest leaf area of Brassica juncea plant was observed with eggshells and banana peels treatment. The maize plant treated with combination treatment (Eggshells + fruit peels) enhanced the leaf area because they contain K and Ca ion and nutrients from both treatments [49].

In recent study, eggshells and banana peels treatment enhanced the growth attributes of Brassica juncea. The application of eggshells to plants resulted impressive growth of Arachis hypogea L.[50]. The okra plant when cultivated in soil fertilized with a mixture of banana peel and eggshells powder then better growth observed. There were notable differences in plant size and amount of okra fruit compare to without treatment [51].

In the present study, the total phenolic contents of Brassica juncea plant increased under salt stress. High phenolic contents may be due to for having strong antioxidant qualities, which are crucial in removing singlet oxygen and which improve tolerance towards salinity. Under salinity stress, the phenylpropanoid biosynthetic pathway is initiated, leading to improved production of diverse phenolic compounds. The levels of polyphenols in various plant tissues escalates with higher salinity levels. The total phenolic content in red pepper increases moderately with elevated salinity levels [52]. Phenolic compounds stabilize cellular membranes by chelating and scavenging, thereby reducing the impact of salt stress on cells [53]. Consequently, these compounds react to stressful conditions by retaining diverse adaptive mechanisms. Phenolic compounds synthesized via phenylpropanoid or shikimic pathway play a vital role in mitigating numerous types of stress owing to their antioxidant properties [54, 55].

Our recent study revealed that malondialdehyde content of Brassica juncea plant increased with salt stress. High MDA content of Brassica juncea plant might be due to high salt concentration promote reactive oxygen species causing oxidative stress, salt stress triggers the lipid peroxidation in plant cell membranes. High MDA content reflects the extent of lipid peroxidation. A similar were found in Brassica juncea plant at different NaCl levels caused elevation in the MDA content [56]. In a salt-stressed cell, malondialdehyde accumulation due to membrane lipid peroxidation is an indication of membrane degradation [57]. A similar result found that under salt stress MDA level were raised in both Pisum sativum accessions [58]. The MDA level of Daucus carota accessions was increased under salinity stress [59]. From results of current investigation, it is shown that B. megaterium reduced the MDA content under salt stress in Brassica juncea plant. Inoculation with B. megaterium markedly inhibits the elevation in MDA content, generally stimulated by salinity stress.

Elevated salt stress disturbs the electron transport chain causing oxidative damage to plants. Extreme energy synthesized during electrochemical reactions that can be release via Mehler reaction, causing reactive oxygen species overproduced etc. H₂O₂ [43]. In this study, oxidative stress markers such as cell membrane and H₂O₂ content were high under salt stress when compared with control plants. This could be that H₂0₂ and electrolyte leakage with the chain of free radicals generating, which can harm macromolecules, cellular structures, reduce membrane fluidity, disrupt the cellular redox balance and result cell membrane raptured and swift desiccation. Electrolyte leakage and H₂O₂ were increased under high salinity [43]. The concentration of hydrogen peroxide (H₂O₂) was rise under salt stress e.g. in P. cathayana [60]. H₂O₂ act as signaling molecules when stress tolerance process happened, but its higher concentration inactivates the numerous enzymes involved in the Calvin cycle and antioxidant systems and directly linked to pigment and membrane damage, which in turn lowers the amount of photosynthetic machinery that produces more radicals [61].

From outcomes of current study, activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (Apx) were increased when Brassica juncea plant underwent the salt stress. Superoxide dismutase works as the primary defense mechanism, converting the harmful superoxide radical into H₂O₂ within the chloroplasts. Subsequently, with the help of other peroxidase, H₂0₂ is further detoxified into water. In chloroplast Apx typically uses ascorbate to transform H₂O₂ into water whereas CAT and GPX typically works in the cytoplasm [62]. The activity of antioxidant enzymes (CAT, POD and SOD) markedly enhanced under salt stress in Brassica juncea plant [63]. Plants generate different types of antioxidants like superoxide dismutase, ascorbate and peroxidase to lessen reactive oxygen species [64].

Recent study results showed that B. megaterium treatment can increase the superoxide dismutase activity, catalase activity, peroxidase activity and ascorbate peroxidase activity under salt stress, thereby mitigating the toxic effect of reactive oxygen species. Bio-priming with rizhobacteria can boost the activity of antioxidant enzymes like superoxide dismutase, ascorbate and peroxidase in chickpea [65]. Similar results were found in canola plants by inoculation of HSNJ4 strain increased the CAT, POD and SOD activity under salinity stress [66]. SOD in potato plants increase due to inoculated with PGPR under abiotic stress [67]. In plants, ascorbate peroxidase act as the enzyme responsible for scavenging hydrogen peroxide [68]. The antioxidant capacity of banana peel was assessed through hydroxyl radical scavenging activity [69].

Based on the findings of the current investigation, it is revealed that the eggshells and B. megaterium treatment enhance the chlorophyll a, chlorophyll b and carotenoid content. Different PGPR and consortium primed seeds of canola can enhance the photosynthetic pigment under saline stress. The increase in chlorophyll content and other pigments in seeds inoculated with microbial bio-agents highlights the role of rhizobacteria in enhancing the functions of electron transporters linked to photosynthesis [70]. and carotenoids content increased due to rhizobacterial inoculation. Potassium and phosphorus increase photosynthesis rate by elevating adenosine triphosphate synthesis in plants, thereby enhancing their growth rate [71].

Based on recent investigation, it is shown that Na⁺ ions increased while Ca²⁺ and K⁺ ion decreased under salinity stress. The root/shoot distribution of potassium (K⁺) and sodium (Na⁺) ions disrupted by rising rhizospheric salinity. In shoot of plant, the proportion of Na⁺ significantly increased, but it was lowered in root, in contrast condition of K⁺ was antipodal under salinity [43]. In this study, high Na⁺ accumulation in plants decreased K⁺ uptake may cause ion competition on the K⁺ transporter over the nonselective cation channel. This condition may create plasma membrane disintegration and depolarization, which displace necessary mineral ions Mg^2+, K⁺, Ca²⁺ [72]. Similar results were found that Na⁺ ions accumulation increased while K⁺ ion uptake reduced in Capsicum annum plants under NaCl stress [52] and Pisum sativum [58].

From results of recent study, it is revealed that rhizobacteria treatment reduced the Na⁺ ion accumulation both in shoot and root while enhanced the K⁺ and Ca²⁺ ion uptake. Similar results were found that inoculation of rhizobacterial strains inhibited Na⁺ ion accumulation in leaves of wheat plants [72]. Furthermore, elevated levels of Ca²⁺ and K⁺, along with reduced Na⁺ levels in wheat plants, resulted in enhanced responses to stress signals and synthesis of amino acids and metabolites, ultimately elevating salt tolerance [73]. In Brassica species Ca²⁺ signaling may be crucial in controlling the initiation of the antioxidant defense system along with ions transport and homeostasis (Na⁺, K⁺) to adapt salt tolerance [74].

In recent study, proline content increased under salt stress. Exposure to salinity treatment led to a notable rise in proline levels, serving as a mechanism for osmo-protection. Under salinity stress, the non-enzymatic antioxidant proline increases, enhancing the plant's antioxidant system and facilitating the restoration of energy compensation in plants. Proline mitigates damage leading by reactive oxygen species and promote plant resistance by reducing the need for detoxifying ROS generated under salinity stress. Proline accumulation suggested to regulates the membranes as a result maintains protein confirmation under salt environment. Proline play important role to prevents photo-damage in thylakoids membrane by scavenging the superoxide radical [75]. In salt tolerant cultivars, mostly high level of proline accumulated in Cynodon dactylon [76], sugar cane [77], pea [45], and proso millet [78] and Brassica juncea [75]. From the results of current study, it is revealed that the treatment of peels and rhizobacteria enhance the proline content. There was substantial increase in proline content due to fertilizer application [79]

Our study results shown that gaseous exchange parameters such as rate of photosynthesis, transpiration and CO₂ assimilation significantly decrease under salt stress. Generally, plant face abiotic stresses express reduction in growth that is mostly linked with rate of photosynthesis. However, decrease in photosynthetic rate may be attributed due to metabolic factor or stomatal factor. In all Panicum populations salinity stress had deleterious effect on gaseous exchange attributes and greatly reduced conductance of stomata, net carbon dioxide assimilation rate, transpiration rate and intercellular concentration [80]. Salinity stress drastically reduced conductance of stomata, transpiration rate and CO₂ assimilation rate in mustard cultivars. It is commonly known that closure of stomata may be salt induced abscisic acid accumulation an important factor contributing to retardation of photosynthetic process [81, 82].

Recent study outcomes revealed that the application of peels, NPK, rhizobacteria and combined treatment enhanced the leaf gas exchange parameters such as conductance of stomata, carbon dioxide assimilation rate and transpiration rate. Similar results were found that the application of PGPRs enhanced the photosynthetic effectiveness and gaseous exchange parameters under salt stress and in Arabidopsis inoculation of Pseudomonas knackmussii increased the leaf gas exchange attributes under saline stress [83]. The correlation between salinity tolerance in B. napus and stomatal aperture is strong, as it helps maintain tissue water potential at high levels and enhances the rates of photosynthesis, CO₂ assimilation, and transpiration [84].

Water status of plant considered an important mechanism for controlling cellular metabolism and it can be determined by relative water content and three inter-related factors, that is osmotic potential, water potential and turgor potential [85]. From results of current study, it is shown that leaf water potential, relative water content and osmotic potential decrease under salt stress. This might be NaCl has deleterious impact on osmotic pressure, disrupts metabolic processes, reduces energy needs, and interferes with cell division [86, 87]. Salinity stress enhanced the chlorophyllase activity and reduced the leaf water potential and photosynthetic efficiency [73]. The osmotic potential of leaves decreased in six Panicum populations when exposure to salinity stress [80]. Relative water content decreased in mustard cultivars under salt stress [88]. similar were found in Olive [89], Brassica rapa [90], pea [81]. Salt stress reduced osmotic and water potential under the consequences of rising osmotic stress [91].

In salty environments, protein accumulation in plants may work as nitrogen storage for post-stress utilization and aid in osmotic adjustments. This elevation in total soluble protein during stress is linked with higher production and accumulation of numerous stress-response enzymes [92, 93]. Our result is contradictory to previous findings that salt stress tends to reduce the soluble protein level in black mung [94]. Total soluble proteins were increased under NaCl stress [52]. The PGPR treatment under salt stress activates the accumulation of proteins which is strongly involved in imparting salt stress. Biochemical parameters like, amino acid and protein increased with rising concentration of eggshells [95].

The effect of salt stress on photosynthesis in plants can be elucidated by chlorophyll a fluorescence parameter, which signpost damage to the donor or acceptor side of PSII, inhibition of electron transport, and modifications in the oxidation and reduction of primary acceptor quinone (QA), secondary acceptor quinone (QB) and photochemical quenching (PQ). Based on recent investigation, a significant change was detected in Fv/Fm under salinity stress. An increase in Fo, as shown by current study, suggests a reduced energy trapping capacity of PSII, while the decline in Fm values may be due to inactive reaction centers affected by salinity stress. High salinity situations remarkable reduction was observed in Fv/Fm in tested mustard cultivars [79]. Salt stress significantly increased Fo, indicating instability in the light-harvesting complex and the physiological state of PSII. In contrast, Fm, Fv, Fv/Fo, and Fv/Fm significantly decreased reflecting a deterioration in PSII efficiency and photochemical yield [96]. A photochemical process reduced along with remarkable elevation in photochemical quenching in tomato plant [97]. Decreased in Fv/fm may be indicated the presence of photo- inhibitory damage [98]. Salinity stress could potentially disrupt pigments within the reaction center and decrease the trapping efficiency of PSII [80]. Fv/Fo sensitive indicator of photosynthetic ETC (electron transport chain). Reduction in Fv/Fo suggests a declined efficiency of electron donation from OEC to donor side of PSII under salinity stress Likewise, the decline in Fv/Fm reflects damage to PSII reaction centers, rendering them photochemically inactive due to the stress. This could also be attributed to a diminished capacity of PSII to transport electrons under saline conditions. These findings align with previous studies conducted on mustard [99] canola [100, 101].

In recent era tremendous raise of salinity level due to natural and anthropogenic activities that extremely effect the crops. To address these challenges, the application of peels, B. megaterium alone as well as combined form and NPK used in our study, can effectively enhance B. juncea growth. In recent study results shown that salt stress caused remarkable reduction in morphological, biochemical, physiological and yield attributes. This might be due to ion toxicity, low k⁺ and ca⁺ ion uptake. Our study revealed that peels treatment that rhizobacteria play major role as a growth regulator for plant development under salt stress. Peels and B. megaterium treatment enhanced the antioxidants activity (CAT, POD, SOD and total soluble protein), total phenolics, leaf proline, reduced the oxidative stress markers (MDA, H₂O₂) and promoted membrane stability and yield attributes. However, NPK increased the carotenoids content and SPAD value. In conclusion peels and B. megaterium alone as well as combined form are effective under salinity stress.

Availability of data and materials

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval and consent to participate

We declare that the manuscript reporting studies do not involve any human participants, human data or human tissues. So, it is not applicable.

Our experiment follows the relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable

Clinical trial number: Not applicable

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Funding

Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

Author Contributions

NK; Experimentation and Methodology, MR & ZN; Conceptualization, Supervision and Validation, SU; Statistical analysis, AAS; Resource acquisition and Investigation, FK, AI & AR; writing-original draft preparation, and MKG & SS; Data curation and Formal analysis. All authors read and approved the final manuscript.

Acknowledgments: Authors are thankful to Researchers Supporting Project number (RSP2024R393), King Saud University, Riyadh, Saudi Arabia.

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Competing interest reported. One of the authors have conflict of interest with Pervaiz Ahmed, the editor BMC plant biology. Rest of the authors declare no conflict of interest.

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Effect of organic waste material and rhizobacteria on growth and physiology of Brassica juncea (L.) Czern. under salinity stress

Status:

Version 1

Abstract

Figures

1 INTRODUCTION

2 METHODOLOGY

2.1 Experimental layout and design

2.2 Assessment of growth parameters

2.3 Assessment of leaf water relations

2.4 Measurement of gas exchange parameters

2.5 Determination of chlorophyll fluorescence

2.6 Measurement of SPAD value

2.7 Determination of chlorophyll and carotenoids content.

2.8 Determination of relative water content

2.9 Determination of relative membrane permeability

2.10 Assessment of malondialdehyde content

2.11 Hydrogen peroxide determination

2.12 Determination of total soluble protein

2.13 Determination of catalase (CAT) and peroxidase (POD) activity

2.14 Determination of ascorbate peroxidase activity (APX)

2.15 Determination of superoxide dismutase (SOD) activity

2.16 Determination of total phenolics

2.17 Estimation of proline content

2.18 Estimation of inorganic mineral ions content

2.19 Determination of yield parameters

2.20 Statistical Analysis

3 RESULTS

3.1.1 Shoot fresh weight

3.1.2 Shoot dry weight

3.1.3 Root fresh weight

3.1.4 Root dry weight

3.1.5 Shoot length

3.1.6 Root length

3.1.7 Number of leaves plant− 1

3.1.8 Total leaf area plant− 1

3.2.1 Net CO2 assimilation rate

3.2.2 Stomatal conductance (gs)

3.2.3 Intercellular CO2 concentration (Ci)

3.2.4 Transpiration rate

3.2.5 Total chlorophyll content (SPAD value)

3.2.6 Water Potential

3.2.7 Osmotic potential

3.2.8 Turgor pressure

3.2.9 Chlorophyll fluorescence

3.2.10 Chlorophyll a

3.2.11 Chlorophyll b

3.2.12 Carotenoids

3.2.13 Relative water content

3.3.1 Malondialdehyde

3.3.2 Hydrogen peroxide (H2O2) content

3.3.3 Relative membrane permeability

3.4.1 Activity of catalase

3.4.2 Peroxidase activity

3.4.3 Superoxide dismutase activity

3.4.4 Ascorbate peroxidase activity

3.4.5 Total phenolic

3.4.6 Leaf Proline

3.4.7 Total soluble protein

3.5.1 K+ ion in shoots

3.5.2 K+ ion in roots

3.5.3 Ca2+ ion in roots

3.5.4 Na+ ion in shoot

3.5.5 Na+ ion roots

3.6.1 Number of pods plant− 1

3.6.2 Weight of 100 Seeds

3.7 Pearson’s correlation and principal component analysis

4 DISCUSSION

5 Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

3.1.7 Number of leaves plant^− 1

3.1.8 Total leaf area plant^− 1

3.2.1 Net CO₂ assimilation rate

3.2.3 Intercellular CO₂ concentration (Ci)

3.3.2 Hydrogen peroxide (H₂O₂) content

3.5.1 K⁺ ion in shoots

3.5.2 K⁺ ion in roots

3.5.3 Ca²⁺ ion in roots

3.5.4 Na⁺ ion in shoot

3.5.5 Na⁺ ion roots

3.6.1 Number of pods plant^− 1