Chlorophyll Fluorescence Parameters and Antioxidant Defense System Can Display Salt Tolerance of Salt Acclimated Sweet Pepper Plants Treated with Chitosan and Plant Growth Promoting Rhizobacteria

Salinity stress deleteriously affects the growth and yield of many plants. Plant growth promoting rhizobacteria (PGPR) and chitosan both play an important role in combating salinity stress and improving plant growth under adverse environmental conditions. The present study aimed to evaluate the impacts of PGPR and chitosan on the growth of sweet pepper plant grown under different salinity regimes. For this purpose, two pot experiments were conducted in 2019 and 2020 to evaluate the role of PGPR (Bacillus thuringiensis MH161336 106–8 CFU/cm3) applied as seed treatment and foliar application of chitosan (30 mg dm−3) on sweet pepper plants (cv. Yolo Wonder) under two salinity concentrations (34 and 68 mM). Our findings revealed that, the chlorophyll fluorescence parameter (Fv/Fm ratio), chlorophyll a and b concentrations, relative water content (RWC), and fruit yield characters were negatively affected and significantly reduced under salinity conditions. The higher concentration was more harmful. Nevertheless, electrolyte leakage, lipid peroxidation, hydrogen peroxide (H2O2), and superoxide (O2−) significantly increased in stressed plants. However, the application of B. thuringiensis and chitosan led to improved plant growth and resulted in a significant increase in RWC, chlorophyll content, chlorophyll fluorescence parameter (Fv/Fm ratio), and fruit yield. Conversely, lipid peroxidation, electrolyte leakage, O2−, and H2O2 were significantly reduced in stressed plants. Also, B. thuringiensis and chitosan application regulated the proline accumulation and enzyme activity, as well as increased the number of fruit plant−1, fruit fresh weight plant−1, and total fruit yield of sweet pepper grown under saline conditions.


Introduction
Sweet pepper belongs to Solanacease family. It is an annual plant in the cultivated lands in many countries, however it is grown as a perennial plant in tropical areas. It is one of the most widespread and popular vegetables, and has a greatest economic importance worldwide [1]. It is the richest source of different antioxidants and vitamins and has several health benefits [2]. However, salinity is a very significant factor that threatens the production of economic plants such as sweet pepper [1], strawberry plants [3], and cucumber plants [4]. Salinity damages plant growth and proliferation by creating water stress and cytotoxicity due to the excess in uptake of ions, such as sodium and chloride. Furthermore, salinity is usually accompanied by oxidative stress due to the generation of reactive oxygen species [5,6]. Salinity stress adversely affects morpho-physiological characters of sweet pepper such as plant height and leaf area which are significantly reduced [7]. Likewise, chlorophyll a and b as well as RWC were reduced under salinity in cucumber [4]. Photosynthesis is harmfully affected by salinity through the reduction in stomatal conductance. Also, salinity led to increased ion toxicity and negatively affected nutrients uptake, especially potassium uptake, so the salt stressed plants showed low membrane stability [8]. The chlorophyll fluorescence parameters were adversely affected with salinity and the content of chlorophyll pigments significantly decreased in cucumber [9]. Also, the study of Misra et al. [10] pointed out that salt stress causes photoinhibition in PSII and decreases its activity. Salt stress led to decreased chlorophyll concentrations, leaf area and mungbean yield [11] and led to an increase in the accumulation of Na + , decreasing the uptake of mineral nutrients such as nitrogen and potassium [12]. The high level of Na + was associated with the ROS accumulation such as H 2 O 2 and O 2− . The excessive formation of ROS causes protein oxidation and lipid peroxidation under several stresses mainly under salinity stress [1,13]. Previous studies have shown that the adverse effects of salinity stress on leaf number, plant length, fresh and dry weights of shoots, and plant yield also increases with the increase in NaCl concentration [14][15][16].
According to salinity concentrations, the plants are classified to euhalophytes or glycophytes. Euhalophytes have the salinity thresholds of 250 mM NaCl, i.e., euhalophytes are able to complete their life cycle upon salinities exceeding 250 mM NaCl. Glycophytes cannot grow under high salinity concentrations and their response to salinity differs in terms of osmotic regulation, photosynthetic electron transport, chlorophyll content, and reactive oxygen species (ROS) formation as well as antioxidant defense system [1,7]. The excessive accumulation of ROS under stress, such as salinity [1,17], drought [18,19], and biotic stress factors [20][21][22][23], results in the activation of the enzymatic and non-enzymatic antioxidant system to enhance stress tolerance in plants to cope with increased accumulation of ROS [24]. The antioxidative system also consists of some of the non-enzymatic systems, such as salicylic acid and carotenoids. Nonetheless, the enzymatic defense system contains ascorbate peroxidases (APX), glutathione reductases (GR), superoxide dismutases (SOD), catalases (CAT), and peroxidases (POD), which protect the plant tissues against stress factors [25]. Also, the plants have adaptive mechanisms to salinity stress through morphological, anatomical, and biochemical changes. Euhalophytes can cope with salinity stress through different mechanisms, such as salt exclusion, salt elimination, salt succulence and salt redistribution [7]. Furthermore, EL%, lipid peroxidation, and ROS were increased significantly under salinity, as these parameters are signals to various stresses, such as salinity, drought, and heat [26][27][28][29], that enable plants to respond to a particular stress. Some plants protect themselves from salinity stress by maintaining ion homeostasis and transportation of the excess salt to the vacuole or sequestering in the older tissues which ultimately are sacrificed, thereby defending itself from salinity stress [30]. Meanwhile, other plants keep the ion concentration in the cytoplasm at a low level. Membranes along with their linked components play an essential role in retaining ion concentration within the cytosol during the period of stress by regulating ion uptake and transport [31,32]. Chlorophyll fluorescence is a fast method for photosynthetic processes measurements [33] and provides a lot of information about the plant status under abiotic and biotic stresses to understand the mechanisms of photosynthesis and how plants respond to various stresses [34]. Chlorophyll fluorescence parameters are important indicators used to measure the quantum yield of photosystem II (PSII), display the plant response to stress and the harmful effects, particularly on photosynthesis and chlorophyll concentrations [35].
Chitosan or chitin is a natural polysaccharide consisting of two molecules of D-glucosamine and naturally present in the cell walls of many organisms such as crabs, shrimp, fungi, and the exoskeleton of insects [36]. In the agricultural field, it improves the morpho-physiological parameters and alleviates the injurious effect of abiotic stresses through stress transduction pathway [37]. Application of chitosan led to increased plant tolerance to many stresses in various plants [38,39], enhance growth characters and improve germination rate of many plants [38,40]. The fruit yield of tomato plants was improved with chitosan treatments [41]. Under drought, barley plants treated with chitosan showed a significant increase in chlorophyll, RWC, total soluble sugar, and grain yield [42]. Plant growth-promoting rhizobacteria (PGPR) can prompt plant tolerance to stress through some chemical and physical changes which are identified as induced systemic tolerance [43]. The application of PGPR led to improved growth and yield production [44]. Under stress conditions, PGPR can improve the injurious impacts and enhance the yield production under salt conditions [45], as a bio-fertilizer in sugar beet and sweet sorghum plants [20,46,47] and as a bio-control agent [48][49][50]. There are many PGPR strains, such as Bacillus, Azotobacter, Azospirillum, Pseudomonas, Rhizobium, and Serratia, which can be used in improving plant growth even under various stress factors [51,52] by the production of antioxidants, phytohormones and vitamins [53]. There is a lot of information about the effect of PGPR, nevertheless studies about chitosan and its effects on plants under salinity stress are still scarce and have not yet been fully understood. Hence, in this research, we focus on the effect of chitosan and Bacillus thuringiensis MH161336 in alleviating the harmful effect of salinity to improve chlorophyll fluorescence parameters, chlorophyll concentration, enzymes activity, and fruit yield of sweet pepper.

Experiments Preparation and Plant Materials
Two pot experiments were conducted at Kafrelsheikh University, Agricultural Botany Department during two summer seasons 2019 and 2020, to evaluate the effect of seed treatment with plant growth promoting rhizobacteria (B. thuringiensis MH161336 10 6-8 CFU/cm 3 ) and foliar spray with chitosan 30 mg·dm −3 on sweet pepper plants under salinity (sodium chloride at 34 and 68 mM). The physio-biochemical characters were done at Plant Pathology & Biotechnology Lab., and EPECRS Excellence Center, Kafrelsheikh University. The seeds of sweet pepper (Capsicum annuum L.) cv. Yolo Wonder (obtained from a private agricultural company) were divided into three groups (the first group was treated with B. thuringiensis and the others without treatments). Seed treatment was done with B. thuringiensis. Thereby, the seeds underwent surface sterilization by sodium hypochlorite 2.5% for 5 min, 70% ethanol for 1 min, and were then washed 5 times by sterile distilled water. B. thuringiensis MH161336 which was isolated from the halophytic plant Spergularia marina (obtained from Dr. Ahmed Eid), B. thuringiensis pure cultures were grown in nutrient broth at 35 ± 2 • C on a shaker at 180× g. Bacterial cultures were diluted in sterilized distilled water to reach a final concentration of 10 6-8 CFU/cm 3 [54]. Sterilized seeds were incubated with bacterial suspensions at room temperature for 6 h and sown in the nursery in foam trays on 7th and 3rd January in the two seasons, respectively. After forty-five days from the sowing, the transplantation was done in pots 50 cm 3 in diameter, each one containing two seedlings and the pots were divided into three groups (control, B. thuringiensis treatment and chitosan treatment 30 mg·dm −3 ). The plants irrigated with two concentrations (34 and 68 mM) of saline water (was prepared from NaCl) and the group of chitosan treatment was treated with chitosan 30 mg·dm −3 twice after 20 and 40 days from transplanting. The compound fertilizer containing nitrogen, phosphorus, and potassium (NPK) (135:40:35 kg·ha −1 ) was used as recommended in two doses, the first dose after 12 days from transplanting and the second at the flowering stage initiation. The experiments were in a completely randomized design with 4 replicates, the physiological and biochemical studies were done at 80 days from transplanting. The chemical and physical characters of experimental soil were determined [55] and are presented in Table 1.

Biochemical and Physiological Characters
The physiological and biochemical characters were recorded at 80 days from transplanting.

Relative Water Content (RWC%)
According to Sanchez et al. [56], twenty leaf discs were used to determine RWC, the fresh weight (FW) for the discs was determined, the same discs were saved in petri dishes containing distilled water for 1 h to determine the turgid weight (TW), after that the discs were dried for 24 h at 80 • C to determine the dry weight (DW). Relative water content (RWC%) was calculated as follows: RWC = (FW − DW)/(TW − DW) × 100.

Determination of Chlorophyll a and b Concentrations
The extraction was done using N-N Dimethyl formamide, whereby 5 cm 3 N-N Dimethyl formamide was added to 1 g fresh leaves and kept in the refrigerator overnight, and the absorbance was measured at 647 and 664 nm according to Moran [57].

Electrolyte Leakage Assay (EL%)
Ten discs (1 cm 2 ) of sweet pepper leaves were placed into flasks containing deionized water (25 cm 3 ). Flasks were shaken for 20 h, initial electrical conductivity was recorded for each vial and then flasks were immersed in a water bath at 80 • C for 1 h. The vials were shaken for 20 h at 21 • C. Final conductivity was measured for each flask. Electrolyte leakage % was calculated according to Szalai et al. [58] with the following formula: initial conductivity/final conductivity × 100.

Chlorophyll Fluorescence Parameter
Chlorophyll a fluorescence parameter (Fv/Fm ratio) was measured at 80 days from the sowing using a chlorophyll fluorometer (PEA, Hansatech Instrument Ltd., version 1.21, Norfolk, UK).
According to Schreiber [59], middle-aged sweet pepper leaves were placed in the dark for 30 min to stimulate the reaction of photosystem II. The minimum chlorophyll fluorescence (Fo) was measured using a measuring beam of <0.1 µmol m −2 ·s −1 . The maximum fluorescence (Fm) was determined after a 1 s saturating pulse (>3500 µmol·m −2 ·s −1 ). Variable fluorescence (Fv) was determined by the difference between the maximum fluorescence and the minimum fluorescence (Fm − Fo). The maximum efficiency of PSII was determined as the ratio of (Fv) to (Fm) as follows: Fv/Fm = (Fm − Fo)/Fm.

Proline Determination
According to Bates et al. [60], proline was assayed in sweet pepper plants, 0.5 g fresh leaf in 3% sulphosalicylic acid and centrifuged for 20 min at 3000× g. Then, 2 cm 3 of glacial acetic acid and 2 cm 3 ninhydrin reagent was boiled with 2 cm 3 supernatant for 1 h, the reaction was completed in an ice bath, and proline was separated using toluene. Proline was determined as µg·g −1 FW using a spectrophotometer at 520 nm.

Determination of Lipid Peroxidation
According to Davenport et al. [61], lipid peroxidation was measured as malondialdehyde (MDA) using 100 mg fresh leaves in 1% trichloro acetic acid and centrifuged at 10,000× g for 5 min. 0.5% thiobarbituric acid was then added, and mixture was boiled at 95 • C for half an hour. The samples were placed on an ice bath and centrifuged for 5 min at 5000× g, the measurements were done using spectrophotometer at 532 and 600 nm.

Determination of Superoxide (O 2 − ) and Hydrogen Peroxide (H 2 O 2 )
Sweet pepper leaves were vacuum infiltrated with 10 mM potassium salicylate buffer containing 0.1 w/v% nitro blue tetrazolium (NBT) or 0.1 w/v% 3,3-diaminobenzidine (DAB). The leaves were incubated in the light for 140 min and two hours, respectively. The samples were cleared with trichloroacetic acid in ethanol: chloroform 4:1 v/v for 1 day, the samples were washed and placed in 50% glycerol. O 2 − and H 2 O 2 were determined as nmol·g −1 FW according to Huckelhoven et al. [62] using a ChemiImager 4000 digital imaging system (Alpha Innotech Corp., San Leandro, CA, USA).

Assay of Enzymes Activity
For the determination of enzymes, 0.5 g fresh leaves were homogenized in 3 cm 3 of 50 mM Tris buffer at 0-4 • C, containing 1 mM EDTA-Na 2 and 7.5% polyvinyl pyrrolidone. The samples were centrifuged 12,000× g for 20 min at 4 • C and the total soluble enzyme activities were measured using spectrophotometer in the supernatant [63]. Catalase activity (CAT) was determined through the decomposition of H 2 O 2 by catalase results in the decrease of the ultraviolet absorption of H 2 O 2 at 240 nm, catalase activity can be calculated from this decrease. The reaction mixture contained 2.15 cm 3 , 2 cm 3 0.1 M Na-phosphate buffer, 100 µL H 2 O 2 , and 50 µL leaves extract. The solution is mixed, and the absorptions were recorded at 240 nm according to Aebi [64]. Peroxidase activity (POX) was calculated according to Hammerschmidt et al. [65]. The reaction mixture consisted of 2.9 cm 3 of a 100 mM sodium phosphate buffer containing 0.25% (v/v) guaiacol and 100 mM H 2 O 2 . The reaction was done with adding 100 µL of crude enzyme extract, the changes in absorbance were recorded every 30 s intervals for 3 min at 470 nm, the activity was determined for min −1 ·g −1 fresh weight. Activity of superoxide dismutase (SOD) was measured according to Mishra et al. [66]. Then, we add 290 µL of a mixture containing 100 mM potassium phosphate buffer, 0.1 mM EDTA, 11 mm 3 xanthine, cytochrome-c, and 0.002 units of xanthine oxidase to 20 µg of protein extracts was prepared. Xanthine oxidase regulation produced an increase in the absorbance due to the reduction of cytochrome-c (0.025 ± 0.005 min −1 ). Activity of SOD was stated by McCord and Fridovich [67]. According to Goldberg and Spooner [68], GR activity was measured, approximately 0.05 cm 3 enzyme extract was mixed with 1 cm 3 phosphate buffer combined with EDTA, 0.1 cm 3 glutathione, and 0.1 cm 3 NADPH, the absorbance was determined at 340 nm.

Fruit Yield
The harvest date starts at 120 days from transplanting to determine number of fruit plant −1 , fruit fresh weight plant −1 (g), and total fruit yield (ton hectare −1 ).

Statistical Analysis
Statistical analysis was done using analysis of variance (ANOVA) procedures according to the method of Gomez and Gomez [69] using the MSTAT-C statistical software package. The means between treatments were compared by Duncan [70] when the difference was significant (P ≤ 0.05). The correlation analysis was done using XLSTAT 2014.5.03 statistical software.

Effect on Relative Water Content (RWC%)
The presented results in Figure 1A showed a significant decrease in RWC in sweet pepper under two salinity concentrations (57.6% at the low concentration (34 mM) (S1) and 52% at the high concentration (68 mM) (S2) comparing with control plants (74.6%) as the mean of the two seasons. Likewise, the results in Figure 1 revealed that seed treatment with B. thuringiensis showed a significant increase in RWC in stressed plants (65.7% compared with 57.6% at the low concentration and 60.8% compared with 52% at the high concentration). Furthermore, chitosan application at 30 mg dm −3 caused a significant increase in RWC (71.5% compared with 57.6% at the low concentration of salinity) and (67.1% compared with 52% at the high concentration) as a mean of both seasons in the stressed plants. The best treatment under salinity conditions was chitosan at 30 mg·dm −3 which achieved 71.5% when compared with control plants 74.6% without any significant difference.

Effect on Chlorophyll a and b Concentrations
It is obvious from the achieved results in Figure 1B-C that chlorophyll was significantly reduced in stressed plants; chlorophyll a significantly decreased at low concentration of salinity (2 mg·g −1 FW −1 ) compared with control (2.85 mg·g −1 FW −1 ) as the mean of both seasons. Furthermore, the high salinity concentration caused a significant reduction in chlorophyll a (1.25 mg·g −1 FW −1 ) in stressed plants compared to control (2.85 mg·g −1 FW −1 ). Similarly, salinity stress led to a significant decrease in chlorophyll b concentration, the two concentrations caused significant decreases (0.84 and 0.55 mg·g −1 FW −1 respectively) compared with control (2.85 mg·g −1 FW −1 ). Nonetheless, seed treatment with B. thuringiensis and chitosan application led to significant increases in chlorophyll a and b. The greatest result was obtained with chitosan (S1 + Chitosan) treatment (2.85 mg·g −1 FW −1 ) in the stressed plants with the low salinity concentration compared to the stressed plants (S1) without treatments (2 mg·g −1 FW −1 ).

Effect on Electrolyte Leakage (EL%)
The presented data in Figure 1D exhibited that EL% significantly increased in the stressed plants, the low salinity concentration caused significant increase (42.3%) comparing with control (13.8%) as the mean of two seasons. Furthermore, the high salinity concentration was more harmfully effective and caused a significant increase in EL% (52.6%) compared with control (13.8%). Nevertheless, chitosan application 30 mg dm −3 and seed treatment with B. thuringiensis led to significant decrease in EL% in the stressed plants under the two concentrations. Seed treatment with B. thuringiensis caused a positive effect and significant decrease in EL% (30.2% and 37.6%) in the stressed plants at the two concentrations compared with untreated plants (42.3% and 52.6%), respectively. Furthermore, EL% was reduced significantly in the stressed treated plants with chitosan 30 mg dm −3 (21.7% and 27.2%) that compared with the stressed untreated plants (42.3% and 52.6%).

Effect on Proline Concentration
It could be noted from Figure 2A that the exposed plants to salinity at (S1) and (S2) caused a significant increase in proline concentration, the high concentration of salinity (S2) achieved the high concentration of proline (24 µg·g −1 FW) comparing to control (

Effect on Proline Concentration
It could be noted from Figure 2A that the exposed plants to salinity at (S1) and (S2) caused a significant increase in proline concentration, the high concentration of salinity (S2) achieved the high concentration of proline (24 μg·g −1 FW) comparing to control (

Effect on Chlorophyll Fluorescence Parameter (Fv/Fm)
Our results in Figure 2B

Effect on Chlorophyll Fluorescence Parameter (Fv/Fm)
Our results in Figure 2B

Effect on Lipid Peroxidation as Malondialdehyde
According to the findings in Figure  Agronomy 2020, 10, x FOR PEER REVIEW 9 of 20 two concentrations, chitosan caused a significant increase in Fv/Fm ratio. The best treatment was chitosan at the low salinity concentration (0.815) compared with control (0.822).

Effect on Lipid Peroxidation as Malondialdehyde
According to the findings in Figure

Effect on Number of Fruit Plant −1 , Fruit Fresh Weight Plant −1 and Total Fruit Yield (Ton Hectare −1 ).
In the present study, the results in Figure 5  In the present study, the results in Figure 5 point out that salinity at the both concentrations caused a significant decrease in number of fruit plant −1 (7.7 and 4.8 fruit), fresh weight of fruit plant −1 (524.5 and 356.4 g) and total fruit yield hectare −1 (7.05 and 5 ton) as the mean of the both seasons when compared to control plants (15.7 fruit plant −1 , 974 g plant −1 and 14.9 ton hectare −1 ). However, B. thuringiensis and chitosan significantly increased the number of fruit plant −1 , fruit fresh weight (g plant −1 ) and total fruit yield (ton hectare −1 ) in the stressed plants compared with untreated plants. Interestingly enough, under the both salinity concentrations, chitosan application gave the best results and significantly increased the number of fruit plant −1 (14.9 and 12.7), fruit fresh weight plant −1 (911 and 527 g plant −1 ), and total fruit yield (14 and 10.8 ton hectare −1 ) as the mean of the both seasons. thuringiensis and chitosan significantly increased the number of fruit plant −1 , fruit fresh weight (g plant −1 ) and total fruit yield (ton hectare −1 ) in the stressed plants compared with untreated plants. Interestingly enough, under the both salinity concentrations, chitosan application gave the best results and significantly increased the number of fruit plant −1 (14.9 and 12.7), fruit fresh weight plant −1 (911 and 527 g plant −1 ), and total fruit yield (14 and 10.8 ton hectare −1 ) as the mean of the both seasons.

Correlation Studies
In the present study chlorophyll a was positively and significantly correlated with chlorophyll b (r = 0.99), number of fruits (r = 0.98), RWC (r = 0.97), GR (r = 0.80) and MDA (r = 0.75). Among the treatment it has a negative correlation with salinity stress @ 34 mM (r = −0.05), salinity stress @ 68 mM (r = −0.02), however, a positive correlation was noted among the chlorophyll a and treatments of Bacillus sp. and chitosan ( Figure 6 and Supplementary Table S1

Correlation Studies
In the present study chlorophyll a was positively and significantly correlated with chlorophyll b (r = 0.99), number of fruits (r = 0.98), RWC (r = 0.97), GR (r = 0.80) and MDA (r = 0.75). Among the treatment it has a negative correlation with salinity stress @ 34 mM (r = −0.05), salinity stress @ 68 mM (r = −0.02), however, a positive correlation was noted among the chlorophyll a and treatments of Bacillus sp. and chitosan ( Figure 6 and Supplementary Table S1). A similar trend of relationship was shown by chlorophyll b. Proline showed highly positive correlation with MDA (r = 0.98), H 2 O 2 (r = 0.97), SOD (r = 0.96) and GR (r = 0.96) but was negatively correlated with the treatments Bacillus sp. (r = −0.04) and chitosan (r = −0.05). A very similar correlation was observed among all the studies. Antioxidant enzymes and H 2 O 2 concentration that were highly correlated with each other also showed a negative correlation with the treatments of Bacillus sp. and chitosan. The number of fruits showed a highly significant correlation with chlorophyll a and b (r = 0.98) and with RWC (r = 0.95). However, this trait was inversely related to the treatments of salinity @ 34 mM (r = − 0.15) and @ 68 mM (r = − 0.25).
1 Figure 6. Circle of correlation between variables and factors for sweet pepper.

Discussion
Salinity stress adversely affects plant growth, inhibiting plant development and reducing fruit yield of sweet pepper. The present data revealed the deleterious effects of salinity at the two different concentrations (34 and 68 mM) on RWC. This might be due to the injurious influence of salinity on the cell wall structure [71], thereby increasing ethylene concentration, which reduces the growth of roots [44]. This effect causes changes in cell wall properties, the reduction in osmotic potential, and the decrease in water balance [72], consequently reducing RWC in sweet pepper [1]. These deleterious impacts of salinity were overcome by seed treatment with B. thuringiensis and treating stressed sweet pepper with chitosan. The pivotal role of B. thuringiensis under salinity stress could be due to the formation of Indole-acetic acid which causes enhancement of root growth and increased water uptake [73]. Likewise, PGPR can produce exopolysaccharides (EPSs) which aggregate with soil particles and improve soil structure as well as water uptake [74]. Further, the application of PGPR causes a decay in the soil bulk density and enhances the availability of soil water. Chitosan application positively affects RWC in stressed plants, this progressive effect of chitosan could be due to the positive role of chitosan on water availability in stressed plants. These valuable effects were documented in barley under drought [19].
Chlorophyll a and b are very important pigments in the process of photosynthesis, in this process, two reactions take place. One such reaction is the light reaction, in which NADPH and ATP are produced, and the second is the dark reaction, in which carbon dioxide is fixed [75]. Demonstrated data revealed a significant decrease in chlorophyll content under the two salinity concentrations, this decrease in chlorophyll was more considerable at the high concentration (68 mM) than at lower concentration (34.mM) and this might be due to the damaging effect of salinity on the chloroplast structure [3,76], that decrease energy transport from PSII to PSI [77] and, consequently, reduce the chlorophyll formation in stressed sweet pepper plants. The harmful effect of salinity on the content of chlorophyll was also due to reduction in stomatal conductance and destruction of biochemical processes [78]. These findings are in accordance with those reported by Abdelaal et al. [1] in sweet pepper under salinity stress. Also, Asrar et al. [79] indicated that a high salinity concentration caused harmful effects on PSII and decreased chloroplast proteins as well as chlorophyll concentrations. This decrease in chlorophyll concentrations is related to the reduction in RWC under high salt concentration.
Conversely, inoculation of seeds with B. thuringiensis mitigates the adverse effects of salinity on the content of chlorophyll that improve the overall growth and proliferation of plants under stressful environments [80]. Beside this, the application of chitosan had also synergistic effects on the contents of chlorophyll a and b. This increase in the content of chlorophyll with the application of chitosan may be attributed to the fact that chitosan is a rich source for amino acids which increase the chloroplast number and chlorophyll formation. These results are in harmony with the findings of Possingham [81]. During the present study, a significant increase was found in EL% under two different salt concentrations mainly. The higher salt concentration was more effective and significantly increased the EL%. This negative influence of salinity on EL% may be due to its damaging impacts on the cytoplasmic membrane and permeability process. Previously, a similar result was reported by Abdelaal et al. [1] in sweet pepper. Contrariwise, EL% significantly reduced in stressed plants as a result of seed treatment with B. thuringiensis and chitosan, these valuable effects of B. thuringiensis treatment and chitosan application is attributed to the positive roles of B. thuringiensis and chitosan on membrane stability and an improvement in the selective permeability of cell plasma membrane.
In the present study, the chlorophyll fluorescence parameter was adversely affected under two salinity concentrations. Salinity stress causes a significant decrease to maximum efficiency of PSII (Fv/Fm). This adverse effect of salinity on (Fv/Fm) might be due to its role in the inhibition of electron transport and the reaction centers at the PSII sites as well as destroys the oxygen-evolving complex [82][83][84]. Also, salinity stress has a negative effect on enzymes activity and decreases the activity of water splitting enzyme complexes and electron transport chains resulting in decrease Fv/Fm [85]. However, seed treatment with B. thuringiensis and the application of chitosan caused a significant increase Fv/Fm ratio in the stressed plants. These results are credited to the helpful role of B. thuringiensis and chitosan in increasing the production of protective metabolites, increasing N and K content as well as the number of chloroplasts under stress [81,86], and consequently, improving the chlorophyll fluorescence parameter. The obtained results indicated that proline significantly increased in the stressed plants under both the salinity concentrations (34 and 68 mM). This impact of salinity may be due to its role in reducing the proline oxidation to glutamate, consequently increasing the proline content [87]. Proline is one of the most important osmoprotectants, plays a key role in osmotic regulation, and protects the plants under stress [1,8]. Chitosan application and seed treatment with B. thuringiensis regulated proline content under salinity conditions. Seed inoculation with B. thuringiensis positively regulated proline content under stress because this species regulates the osmotic balance under saline conditions. Similar results for proline production under saline conditions were also reported by Egamberdieva et al. [88].
Salinity could hamper plant growth and increase lipid peroxidation, O 2 − , and H 2 O 2 . A significant increase was noted in the mentioned parameters during the present study. These reactive compounds can damage lipids and proteins, essential for the process of photosynthesis and electron transport chain. Islam et al. [18] noted similar results in two wheat cultivars grown under saline conditions. However, in the present study, a significant decrease was noted in the lipid peroxidation upon treatment with chitosan. This may be due to the involvement of chitosan in cell protection from oxidative stress under salinity conditions. Similarly, O 2 − and H 2 O 2 were significantly reduced with chitosan due to the presence of hydroxyl and amino groups which react with ROS, thus chitosan can scavenge superoxide radicals [89]. Chitosan derived from the pathogen is recognized by a specific cellular receptor resulting in enhancing the defense response to abiotic and biotic stresses [90]. The positive effect of chitosan in the plant cell protection was also noted in plants under drought stress [20]. Interestingly, seed treatment with B. thuringiensis led to improved cell membrane stability and decreased the formation of MDA in the stressed sweet pepper, this effect of B. thuringiensis is due to its improved phenol content and defense enzyme system [91]. Also, B. thuringiensis causes decreases in O 2 − and H 2 O 2 by increasing reactive oxygen scavenging enzyme activity [92]. Enzymes up-regulation (CAT, POX, SOD, and GR) is involved in the mitigation of salinity stress in sweet peppers compared with control plants. The significant increase in these enzymes is a natural defense system, which helps to cope with salinity stress and reduces the osmotic and toxic effects by scavenging ROS. Our results are in agreement with those reported by Abdelaal et al. [17] and Foyer et al. [93]. Nevertheless, it was clear from our results that the application of seed treatment with B. thuringiensis led to improved and regulated up-regulation of CAT, POX, SOD, and GR in the stressed sweet pepper. The induction of these enzymes is involved in the mitigation of salt stress in sweet pepper treated with Bacillus. A similar trend of enzyme activity was recorded in the findings of Kohler et al. [94]. Likewise, chitosan application causes an increase in enzymes activity to protect the plant from oxidative damage and reduce lipid peroxidation as well as scavenge O 2 − due to its structure and protective role in sweet pepper plants subjected to salinity stress. These results are in agreement with those reported by Hafez et al. [19]. The presented study showed that two salinity concentrations caused a significant reduction in the number of fruit plant −1 , fruit fresh weight plant −1 , and total fruit yield. This harmful impact of salinity may be due to the decrease in reproductive organs, such as pollen grains in stressed plants [95], and also due to the decrease in water absorption, nutrients uptake, and chlorophyll content [1,4], resulting in a significant decrease in fruit yield [96]. The vital role of B. thuringiensis might be due to the formation of growth regulators such as gibberellins, auxin, and cytokinins, as well as an increase in proline content [87], up-regulation of essential enzymes and solubilization of nutrients [89], and an increase in the number of fruits and fruit yield hectar −1 in sweet pepper. These findings are in agreement with the previous results reported by Hafez et al. [19], Katiyar et al. [36], and Hidangmayum et al. [37].

Conclusions
The present research concluded that seeds treated with B. thuringiensis and foliar application of chitosan 30 mg dm −3 on sweet pepper plants under two salinity concentrations (34 and 68 mM) led to an improvement of the adverse effects of salinity and enhanced the growth and yield of sweet pepper. RWC, chlorophyll a and b concentrations, chlorophyll fluorescence parameters, and fruit yield characters significantly increased with B. thuringiensis and chitosan treatments in sweet pepper under two salinity concentrations. Conversely, lipid peroxidation, electrolyte leakage, and reactive oxygen species (O 2 − and H 2 O 2 ) were decreased significantly as a result of B. thuringiensis and chitosan treatments. Overall, seed treatment with B. thuringiensis and chitosan foliar application was an effective and cheaper approach to cope with the deleterious effects of salinity on sweet pepper by improving the chlorophyll fluorescence parameters, proline accumulation, and up-regulation of enzymes activity as well as the enhancement of fruit yield characters.