**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**

**Muneera D. F. ALKahtani 1, Kotb A. Attia 2, Yaser M. Hafez 3, Naeem Khan <sup>4</sup> , Ahmed M. Eid 5, Mohamed A. M. Ali <sup>6</sup> and Khaled A. A. Abdelaal 7,\***


Received: 15 July 2020; Accepted: 1 August 2020; Published: 12 August 2020

**Abstract:** 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<sup>−</sup>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 <sup>−</sup>) 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 <sup>−</sup>, and H*2*O2 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<sup>−</sup>1, fruit fresh weight plant<sup>−</sup>1, and total fruit yield of sweet pepper grown under saline conditions.

**Keywords:** sweet pepper; salinity; *Bacillus*; chitosan; chlorophyll fluorescence; fruit yield

#### **1. 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<sup>+</sup> was associated with the ROS accumulation such as H2O2 and O2<sup>−</sup>. 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 [14–16].

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 [20–23], 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 [26–29], 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 [48–50]. 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.

#### **2. Materials and Methods**

### *2.1. 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 106–8 CFU/cm3) and foliar spray with chitosan 30 mg·dm−<sup>3</sup> 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 106–8 CFU/cm3 [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 cm3 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<sup>−</sup>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−<sup>3</sup> twice after 20 and 40 days from transplanting. The compound fertilizer containing nitrogen, phosphorus, and potassium (NPK) (135:40:35 kg·ha<sup>−</sup>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.


**Table 1.** Chemical and physical characters of the experimental soil before conducting the experiments in 2019 and 2020 seasons.

#### \* EC = Electrical conductivity.
