*3.9. E*ff*ect on Number of Fruit Plant*<sup>−</sup>*1, Fruit Fresh Weight Plant*<sup>−</sup>*<sup>1</sup> and Total Fruit Yield (Ton Hectare*<sup>−</sup>*1).*

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−<sup>1</sup> (7.7 and 4.8 fruit), fresh weight of fruit plant−<sup>1</sup> (524.5 and 356.4 g) and total fruit yield hectare−<sup>1</sup> (7.05 and 5 ton) as the mean of the both seasons when compared to control plants (15.7 fruit plant<sup>−</sup>1, 974 g plant<sup>−</sup><sup>1</sup> and 14.9 ton hectare<sup>−</sup>1). However, *B. thuringiensis* and chitosan significantly increased the number of fruit plant<sup>−</sup>1, fruit fresh weight (g plant<sup>−</sup>1) and total fruit yield (ton hectare<sup>−</sup>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−<sup>1</sup> (14.9 and 12.7), fruit fresh weight plant−<sup>1</sup> (911 and 527 g plant<sup>−</sup>1), and total fruit yield (14 and 10.8 ton hectare<sup>−</sup>1) as the mean of the both seasons.

**Figure 5.** Effect of *B. thuringiensis* and chitosan on number of fruit plant<sup>−</sup>1(**A**), fruit fresh weight plant−<sup>1</sup> (**B**) and total fruit yield (ton hectare<sup>−</sup>1) (**C**) under two salinity concentrations in sweet pepper during two seasons. Data is the mean (±SE) of four replicates. Different letters above the data columns indicate significant differences between the samples determined by ANOVA, Duncan's multiple range test at 0.05 level.

#### *3.10. 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), H2O2 (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 H2O2 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).

**Figure 6.** Circle of correlation between variables and factors for sweet pepper.

### **4. 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 [82–84]. 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, O2 <sup>−</sup>, and H2O2. 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, O2 <sup>−</sup> and H2O2 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 O2 <sup>−</sup> and H2O2 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 O2 <sup>−</sup> 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<sup>−</sup>1, fruit fresh weight plant<sup>−</sup>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−<sup>1</sup> 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].

#### **5. Conclusions**

The present research concluded that seeds treated with *B. thuringiensis* and foliar application of chitosan 30 mg dm−<sup>3</sup> 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 (O2 <sup>−</sup> and H2O2) 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.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/8/1180/s1, Table S1: Correlation matrix among different treatments and quantitative traits of sweet pepper.

**Author Contributions:** Conceptualization, K.A.A.A., Y.M.H., K.A.A., and M.D.F.A.; methodology, K.A.A.A., Y.M.H., K.A.A., M.D.F.A., A.M.E., and M.A.M.A.; software, K.A.A.A., Y.M.H., K.A.A., M.D.F.A., and M.A.M.A.; formal analysis, K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A., and N.K.; investigation, K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A.M.A., and N.K.; resources, K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A.M.A., and N.K.; data curation, K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A.M.A., and N.K.; writing-original draft preparation, K.A.A.A., Y.M.H., K.A.A., A.M.E., M.A.M.A., M.D.F.A., and N.K.; writing-review and editing, K.A.A.A., Y.M.H., A.M.E., M.A., N.K., and M.D.F.A.; visualization, K.A.A.A., Y.M.H., and K.A.A.; funding acquisition, M.D.F.A., K.A.A.A., M.A., A.M.E., and K.A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University through the Fast-track Research Funding Program.

**Acknowledgments:** The authors extend their appreciation to all members of PPBL and EPCRS excellence center, Faculty of Agriculture, Kafrelsheikh University, and the Deanship of Scientific Research at Princess Nourah bint Abdulrahman University Fast-track Research Funding Program.

**Conflicts of Interest:** The authors declare no conflict of interest
