Biocontrol Potential of Bacillus amyloliquefaciens against Botrytis pelargonii and Alternaria alternata on Capsicum annuum

The aim of this study was to assess the ability of Bacillus amyloliquefaciens, to augment plant growth and suppress gray mold and leaf spot in pepper plants. Morphological modifications in fungal pathogen hyphae that expanded toward the PGPR colonies were detected via scanning electron microscope. Furthermore, preliminary screening showed that PGPR could produce various hydrolytic enzymes in its media. Treatments with B. amyloliquefaciens suppressed Botrytis gray mold and Alternaria leaf spot diseases on pepper caused by Botrytis pelargonii and Alternaria alternata, respectively. The PGPR strain modulated plant physio-biochemical processes. The inoculation of pepper with PGPR decreased protein, amino acid, antioxidant, hydrogen peroxide, lipid peroxidation, and abscisic acid levels but increased salicylic acid and sugar levels compared to those of uninoculated plants, indicating a mitigation of the adverse effects of biotic stress. Moreover, gene expression studies confirmed physio-biochemical findings. PGPR inoculation led to increased expression of the CaXTH genes and decreased expression of CaAMP1, CaPR1, CaDEF1, CaWRKY2, CaBI-1, CaASRF1, CaSBP11, and CaBiP genes. Considering its beneficial effects, the inoculation of B. amyloliquefaciens can be proposed as an eco-friendly alternative to synthetic chemical fungicides.


Introduction
Pepper (Capsicum annuum L., family Solanaceae) is one of the most economically important vegetable crops and consumed spices around the world. Pepper fruits can be consumed in fresh, dry, and processed forms and numerous health benefits are associated with their consumption. They are a rich source of vitamins, minerals, and antioxidants and help to hinder inflammation, cancer, and cell damage and improve the immune system. Their compounds are used in commercial medicinal products to treat muscle pains, arthritis, stomach ulcers, etc. [1]. Pepper consumption has surged in the last 20 years [2] and further increases are expected due to higher demand by consumers. However, this crop is highly susceptible to a broad range of pests and diseases; thus, plant yield and productivity are affected by such stresses, leading to economic losses [3].
Botrytis and Alternaria spp. attack a wide range of crops, including more than 1000 species of vascular plants. Geographically, they occur wherever their host plants grow, ranging from extreme cold areas to hot desert regions. They trigger diseases on all parts of the plant, including the seed, flower, fruit, leaf, and shoot, and impose serious medium at 25 • C for further analysis. The PGPR (B. amyloliquefaciens) was obtained from our previous study (accession no. MW599955).

Molecular Characterization of Fungal Isolates
Genomic DNA was obtained from fresh fungal cultures (seven days old) as per the methods of Al-Sadi et al. [36]. Amplification reactions were performed using the BioFACT™ 2X Multi-Star PCR Master Mix (BIOFACT, Daejeon, Korea) and a combination of primers (ITS1/ITS4, RPB2-5F2/fRPB2-7cR, Alt-for/Alt-rev, and gpd 1/gpd 2) according to the defined conditions [37][38][39] (Table S1). PCR products were purified and sequenced at SolGent Co., Ltd. (Daejeon, Korea). Sequence data for the ITS, GAPDH, and Alt a 1 were obtained for the isolate of Alternaria (ALT), which resulted in the GenBank accession numbers, MW793507, MW803061, and MW803062, respectively. The RPB2 gene sequences obtained for Botrytis isolate (BOT) were logged in the GenBank database under accession number MW803063. Two different datasets were used to estimate two phylogenies: a species tree in Alternaria section Alternaria based on combined ITS, GAPDH, and Alt a 1 gene and a Botrytis species tree based on RPB2 gene region. The phylogenetic analyses were performed using the raxml GUI v1.3 [40] and the dendrogram was created with MEGA v7.0.26 software.

Pathogenicity of Fungal Isolates in Pepper Seedlings
The pathogenicity test of isolated fungi was carried out on the pepper cultivar Geumsugangsan (Takii Korea Ltd., Seoul, Korea). Inoculation with fungal pathogens (ALT and BOT) was conducted by pipetting individual droplets of fungal suspension on the surface of healthy leaves. Control plants were treated with sterile distilled water (SDW) only. Inoculated plants were maintained in a humid chamber (250 µmol photons m −2 s −1 PAR, 70% relative humidity, 25 ± 2 • C, 16:8 h light:dark cycle). After symptoms appeared on the seedlings, the fungus was re-isolated from symptomatic tissues and its identity was confirmed by morphological and molecular studies.

In Vitro Evaluation of the Antifungal Activity of the Bacterial Strain Against Fungal Pathogens
The ability of the bacterial strain to antagonize ALT and BOT isolates was examined via a dual culture method [41]. The inoculated plates were kept at 28 ± 2 • C until the leading edge of the fungus in the control plate reached the edge of the plate. The antagonistic effect of the bacterial strain against ALT and BOT was confirmed by the formation of an inhibition zone. Moreover, the effect of the chosen bacterial strain on the hyphal morphology of ALT and BOT was visualized using a scanning electron microscope (SEM; Hitachi SU8220, Tokyo, Japan). The SEM sample preparation was carried out as defined by Heckman et al. [42].

Determination of Hydrolytic Enzyme Activity of the Bacterial Strain
The ability of the bacterial strain to produce amylase, protease, pectinase, and cellulase was determined [43]. The catalase activity test was conducted by adding three-to-four drops of hydrogen peroxide (H 2 O 2 ) to the bacterial culture, which was grown on trypticase soy agar medium. The effervescence confirmed the catalase activity of the bacterial strain [44]. To determine phytase activity, bacterial strain was inoculated onto medium containing sodium phytate [45]. Qualitative lipase activity was evaluated on Tween agar medium. The appearance of a white precipitate indicated positive lipolytic activity [46,47]. The activity of laccase was detected in the medium supplemented with gallic acid. The formation of a dark brown color around the colony was the result of laccase activity [48]. The method of Balasubramanian et al. [49] was used for the visual detection of glucanase activity of the bacterial strain.

In Vivo Evaluation of the Antifungal Activity of the PGPR against ALT and BOT Preparation of the Fungal and Bacterial Inocula
The fungal inoculum was established as described previously [50]. Conidia were suspended in a solution containing glucose and potassium phosphate (10 mM, pH 6) to stimulate the infection on pepper leaves.
The chosen bacterial strain was used to prepare the bacterial inoculum suspension at the optical density (600 nm). The bacterial strain was cultured in lysogeny broth (120 rpm, 24 h, 28 ± 2 • C) and bacterial cells were collected via centrifugation (5000 rpm, 10 min, 4 • C). The obtained pellet was washed three times with SDW. Afterward, the obtained cell pellets were suspended in 0.03 M MgSO 4 (10 8 -10 9 CFU/mL), vortexed, and used for plant treatment (50 mL/pot).

Plant Material and Growing Conditions
Seeds of C. annuum cv. Geumsugangsan were disinfected by washing with 70% ethanol (1 min) and 1.5% sodium hypochlorite (5 min) followed by rinsing three times with SDW. Thereafter, the disinfected seeds were evaluated for efficiency of the sterilization process [51] and viability [52]. Pre-germinated seeds were placed in sterilized plastic pot trays (28 × 54 cm) containing autoclaved sterilized soil (Shinsung Mineral Co., Ltd., Chungcheongbuk-do, Korea). One seed was sown in each pot. Seeds were grown for three weeks in a humid chamber as described above and watered daily.

Experimental Design
Various treatments were applied to three-week-old seedlings two days after transplanting. The seedlings were split into the two following groups: the normal control group irrigated with SDW (50 mL/pot) and the PGPR group irrigated with bacterial inoculum suspension (50 mL/pot). Each group was treated for seven days, after which the seedlings with or without PGPR treatment were further split into two groups with an equal number of seedlings. This formed six experimental groups, which are described in Table 1. The pepper seedlings were exposed to selected biotic stresses, and sampling was performed after eight days [53,54]. The harvested samples were either immediately used or rapidly deactivated in liquid nitrogen and stored at −80 • C. The moisture level (70%), pH value (~7), and EC (≤1.2) of the bulk soil samples were recorded before the pot experiment. The soil moisture level of each pot was monitored daily using a humidity tester (Model DM-5, Takemura Electric Works, LTD., Tokyo, Japan). Furthermore, at harvesting, a soil sample from certain pots per treatment was used to determine pH, EC, and moisture using the humidity tester and conductivity meter (YSI Model 32, Yellow Spring, OH, USA) (Table S2).

Physio-Biochemical Attributes of the Pepper Plant Plant Growth Characteristics and Photosynthetic Pigments
To assess the effect of each treatment on the seedlings, multiple plant growth parameters were evaluated. These parameters comprised plant height, stem diameter, leaf area (length/width), total plant fresh weight, and number of leaves, which were recorded after eight days. A digital Vernier caliper and a ruler were used to measure the stem diameter, leaf area (length and width), and plant height.
Leaf chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll, and carotenoid contents were determined by the spectrophotometric analysis of chemically extracted pigments [55]. Briefly, the freeze-dried ground leaves were extracted in 80% ethanol at room temperature after centrifugation. Pigment absorption was measured spectrophotometrically at 663, 645, and 480 nm (Thermo Fisher Scientific, Waltham, MA, USA).
2.11. Phytohormone Analysis; Abscisic acid (ABA) and Salicylic Acid (SA) The pepper ABA content was extracted and analyzed according to the previously described method [56]. Nitrogen gas (N 2 ) was added to dry out the resultant extract, and its methylation was achieved using diazomethane (CH 2 N 2 ). ABA content was quantified by GC-MS (Agilent 6890N Gas Chromatograph, Santa Clara, CA, USA). ThermoQuest software (Manchester, UK) was used to observe the responses to ions (m/e of 162 and 190 for Me-ABA and 166 and 194 for Me-[ 2 H 6 ]-ABA).
The level of SA in the pepper plants was estimated as outlined previously [57,58]. Briefly, the freeze-dried sample (0.1 g) was extracted with methanol (90% and 100%) by centrifugation (12,000 rpm for 15 min at 4 • C). The combined methanol extracts were then vacuum-dried. The dried residue was dissolved in 5% trichloroacetic acid and centrifuged at 10,000 rpm for 10 min. The supernatant was partitioned with ethyl acetate/cyclopentane/isopropanol (49.5:49.5:1, v/v). The top layer of the aqueous solution was dried and used for SA quantification using high-performance liquid chromatography.

Amino Acid Content of the Leaves
The amino acid content was determined by hydrolyzing freeze-dried leaves (50 mg) in 1 mL of hydrochloric acid (6N HCl) for 24 h at 110 • C [59]. Then, the extraction was condensed and dried with a vacuum at 80 • C for 24 h. Afterward, the residue was diluted with deionized water (2 mL) and evaporated twice. Finally, the concentrated residue was dissolved with hydrochloric acid (0.02 N HCl, 1 mL) and the mixture was passed through a 0.45 µM filter membrane. The solution was analyzed using a Hitachi L-8900 Amino Acid Analyzer (Hitachi High-Technologies Corporation, Tokyo, Japan).

Estimation of the Leaf Protein and Sugar Content
The soluble protein content subjected to different treatments was quantified following the methods of Ashraf and Iram [60], and bovine serum albumin was used as a standard. The leaf samples (0.1 g) were crushed and later blended with 1 mL of phosphate buffer (50 mM, pH 7.0). The mixture was then centrifuged at 10,000 rpm for 10 min at 4 • C. Afterward, the appropriate reagent was added to the obtained supernatant and the absorbance of each sample was recorded at 595 nm.
The total sugar content was determined as described by Khan et al. [61]. Specifically, freeze-dried leaves were ground and extracted with 80% ethanol followed by vacuum drying. The dried remnant was re-dissolved in 1 mL of deionized water and passed through 0.45 µM Nylon-66 syringe filters. Furthermore, the filtered samples were injected into a high-performance liquid chromatograph (Millipore Co., Waters Chromatography, Milford, MA, USA).

Enzymatic and Nonenzymatic Antioxidant Activity
The antioxidant enzyme assays for peroxidase (POD) and polyphenol oxidase (PPO) were performed using the method of Putter [62]. Superoxide dismutase (SOD) content was analyzed by the method proposed by Sirhindi et al. [63]. To determine flavonoid content, DPPH radical scavenging activities, and total polyphenol samples, were processed following previously described procedures [64][65][66][67]. The mixture activity and absorbance were measured at selected wavelengths using the Multiskan™ GO UV/Vis microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Hydrogen Peroxide and Lipid Peroxidation (Malondialdehyde (MDA)) Contents
The H 2 O 2 level of plants subjected to various treatments was determined as per the methods described previously [68,69]. The frozen samples were freeze-dried then ground finely. The powdered sample (0.3 g) was homogenized with 3 mL of ice-cold phosphate buffer (50 mM, 1 mM EDTA, 1% PVP, pH 7.0) and centrifuged at 13,000 rpm for 20 min. The supernatant (2 mL) was blended with 1 mL of 20% (v/v) H 2 SO 4 containing 0.1% titanium chloride and the mixture was then centrifuged at 13,000 rpm for 20 min. The supernatant intensity was measured at 410 nm with a T60 UV-Vis Spectrophotometer (PG instruments Ltd., Wibtoft, UK).
The method of López-Serrano et al. [70] was employed to estimate the amount of lipid peroxidation. The MDA content was calculated using its extinction coefficient. The lipid peroxidation content was expressed as the level of MDA created per gram of tissue.

Quantification of the Nutrient Content in Pepper Plants
To examine the nutrient contents of the pepper plants, samples were freeze-dried and processed into a powder. Eventually, the prepared samples were subjected to the quantification of nutrient (potassium, K; phosphorus, P; calcium, Ca) uptake in pepper plants using ICP-MS (Optima 7900DV, Perkin-Elmer, Akron, OH, USA). Treatments without bacterial inoculation were performed to determine the initial concentration of the nutrients.

cDNA Synthesis and Real-Time PCR Analysis
Total RNA was extracted from the pepper leaves harvested at the end of the experiment. The obtained RNA was employed for cDNA synthesis and quantitative PCR following the previously described procedure [71]. Specifically, 1 µg of RNA was consumed to synthesize cDNA using the BioFACT TM RT-Kit (BIOFACT, Daejeon, Korea) following the manufacturer's standard protocol. The synthesized cDNA was used as a pattern in a two-step qRT-PCR reaction carried out to determine the transcript quantity with an Illumina Eco TM system (Illumina, San Diego, CA, USA) (Table S3).

Data Analysis
Statistical analysis was performed using R software (v4.0.3) and Microsoft Excel 2017. Treatments were compared via analysis of variance using the least significant difference test at a 5% probability level (p < 0.05). The graphs were prepared using GraphPad Prism software (v6.01, San Diego, CA, USA). A completely randomized design was applied for all experiments, with three replications and three repetitions.

Identification of Fungal Isolates
Two pathogenic fungi were isolated from diseased pepper plants. The phylogenetic analysis based on combined sequences of ITS, GAPDH, and Alt a 1 strongly supported our Alternaria isolates to be the Alternaria alternata ( Figure 1). Further, results of the RPB2 tree for the genus Botrytis demonstrated that the isolate BOT formed a strongly supported clade with the Botrytis pelargonii ( Figure 2).

Bacillus amyloliquefaciens Antifungal Hydrolytic Enzyme Activity and Effect on the Morphology of B. pelargonii and A. alternata
The antagonism test showed substantial suppression of B. pelargonii and A. alternata in the PDA plate under the influence of B. amyloliquefaciens. This suppression was demonstrated by the production of an inhibition zone ( Table 2). Scanning electron microscopy examination showed that B. amyloliquefaciens induced notable changes in the general appearance of B. pelargonii and A. alternata hyphae. The morphology of both fungal hyphae was modified after being exposed to B. amyloliquefaciens. As shown in Figure 3A,B, the hyphae of these fungi were irregular, ruptured, wrinkled, and deformed compared to those of the control. In terms of hydrolytic enzyme activity, B. amyloliquefaciens was positive for all examined hydrolytic enzymes ( Figure 4, Table 2).

Pepper Seedling Response to B. amyloliquefaciens Inoculant under Biotic Stress Soil Properties
Soils from different treatments were examined to evaluate the impact of PGPR and pathogen on the soil moisture level, pH value, and EC content. The pH value of all soil samples was found to be alkaline (pH = 6.9-7.9), while the EC content ranged from 0.3 to 2.9 mS (Table S2). The EC content was slightly higher in PGPR-inoculated plants subjected to non-stress and stress conditions. The level of moisture was significantly higher in PGPR treated plants with or without biotic stresses.

Impact of PGPR on Plant Growth Attributes
The impact of the PGPR strain on the growth promotion of the pepper seedlings under no stress as well as biotic stress conditions was evaluated through pot trials ( Figure S2A,B). The unfavorable consequences of pathogen invasion led to the reduction in growth parameters; namely, plant height, stem diameter, leaf area (length/width), total plant fresh weight, and the number of leaves of the pepper plants, in comparison with non-diseased, un-inoculated pepper plants (Table 3). In contrast, the application of PGPR increased plant height, stem diameter, leaf area (length/width), and total fresh weight in the inoculated plants exposed to pathogen stress. The plant height improved by 32.40% in the Botrytis treatment group and by 18.13% in the Alternaria treatment group compared to those of the corresponding un-inoculated stressed plants (p < 0.05). Similarly, in PGPR-treated plants, the total plant fresh weight increased by 34.83% and 26.22% in Botrytis and Alternaria treatment groups, respectively, compared to those of the control group of stressed plants (Table 4).

Chlorophyll and Carotenoid Contents
The chlorophyll and carotenoid contents were determined for pepper plants under both normal and stressed conditions. Biotic stresses adversely influenced the photosynthetic pigments of pepper plants. The analysis of plant pigments showed that the Chla/Chlb and carotenoid levels increased in infected plants inoculated with the bacterial strain compared to those that were infected but not inoculated. Likewise, all the PGPRtreated infected plants showed higher total chlorophyll levels than those of the control infected plants ( Table 3). Decreases of 9.96% and 40.34% in total chlorophyll content were observed in Botrytis and Alternaria-stressed plants compared to those of control plants. PGPR inoculation was effective (p < 0.05) and caused approximately 31.07% and 57.88% in-creases under Botrytis and Alternaria stress conditions compared to those of control infected plants, respectively (Table 3).

Phytohormones; ABA and SA Accumulation
Plant hormone analysis showed the differential accumulation of ABA and SA in PGPRinoculated pepper plants under control and biotic stress conditions over eight days. Biotic stresses caused increases in ABA in the pepper seedlings. The PGPR treatment decreased the ABA levels in pepper plants compared to those of control plants in the absence of biotic stress. Upon exposure to Botrytis and Alternaria stresses, PGPR-inoculated plants exhibited significantly reduced ABA content (73.82% and 67.74%, respectively) compared with those of non-inoculated stressed plants ( Figure 5A). In contrast to the stressed plants without treatment, non-inoculated plants displayed decreases in SA concentrations of 75.05% (Botrytis) and 76.52% (Alternaria), respectively, compared to those of the control plants. As shown in Figure 5B, seedlings inoculated with PGPR for eight days showed remarkable 74.68% and 68.30% increases in SA contents in pepper plants under Botrytis and Alternaria stresses compared with those of non-inoculated stressed plants. The results suggested that PGPR inoculation enhanced the SA content in pepper seedlings with or without stress.

Free Amino Acid Content
Six amino acids were detected with different concentrations in pepper seedlings ( Figure 6). Biotic stresses increased the amino acid content of the pepper seedlings compared to those under normal conditions over eight days. Proline (Pro) levels increased by 69.44% and 75.92% in Botrytisand Alternaria-stressed plants, respectively. However, plants with PGPR treatment showed a decrease in Pro levels. Amino acid levels decreased eight days after the application of PGPR to stressed plants. For instance, Pro levels decreased by 65.83% and 69.36% in PGPR-treated plants under Botrytis and Alternaria stress, respectively, compared to those of stressed plants without treatment ( Figure 6). Values show the means ± standard error (n = 3) and significant differences are indicated at p < 0.05 in accordance with the least significant difference test. Bars with different letters are significantly different from each other.

Soluble Protein and Sugar Contents
The protein levels increased in diseased seedlings after eight days. In uninfected conditions, protein levels increased by 26.35% upon PGPR inoculation compared with plants non-inoculated with the bacterium. The protein production rates improved by 50.60% and 67.00% under Botrytis and Alternaria stresses, respectively, compared to those of non-stressed plants without treatment (p < 0.05) ( Figure 7A). Conversely, a decrease in protein levels was detected in PGPR-treated plants when subjected to Botrytis (54.01%) and Alternaria (65.49%) stresses compared with those of plants subjected to biotic stresses. A decrease in sugar levels occurred after exposure to pathogen attack ( Figure 7B). As shown in Figure 7B, the sugar contents were alleviated in response to Botrytis (27.20%) and Alternaria (24.80%) stresses compared to those of plants under normal conditions. The optimum outcomes were obtained once plants were treated with PGPR, which contributed to an increase in sugar contents of 36.25% and 29.32% under Botrytis and Alternaria stress conditions, respectively, compared with those of untreated stressed plants ( Figure 7B).

H 2 O 2 and MDA Content
The H 2 O 2 levels were assessed to determine whether PGPR application attenuated the effects of stress on pepper seedlings. Biotic stresses caused substantial modification in H 2 O 2 contents in pepper plants ( Figure 7C) As illustrated in Figure 7D, stress conditions led to the increase in MDA production in untreated pepper plants. The MDA levels increased by 81.05% and 75.71% under Botrytis and Alternaria stresses, respectively. Compared with the untreated plants, the decreases in MDA levels in the PGPR-treated plants were approximately 66.89% under Botrytis and 73.04% under Alternaria stress conditions (p < 0.05).

Antioxidant Content
SOD activity augmentation occurred under stress conditions. SOD activity decreased by 61.96% and 64%, respectively, in PGPR-treated plants exposed to Botrytis and Alternaria stresses compared to those of untreated stressed plants ( Figure 8A). POD, PPO, and flavonoid activities followed similar patterns; their activities increased under stress conditions. PGPR treatment reduced POD, PPO, and flavonoid levels under stress conditions compared to those of untreated stressed plants. For instance, their activities decreased (POD, 59.77%; PPO, 46.21%; flavonoid, 63.67%) in PGPR-inoculated seedlings subjected to Botrytis stress (p < 0.05) ( Figure 8B-D).
DPPH levels in pepper seedlings increased under stress, while they decreased in PGPR-treated seedlings exposed to biotic stress. PGPR inoculation assisted in lowering DPPH contents by 57.12% and 58.29% under Botrytis and Alternaria stresses, respectively, compared with those of untreated stressed plants ( Figure 8E).
Total polyphenol levels increased slightly under stress conditions. On the other hand, these levels decreased upon PGPR inoculation under stress conditions ( Figure 8F).

Nutrient Content in Plants
To determine the effects of the PGPR inoculant on the nutrient levels of pepper plants, three elements; Ca, K, and P, were examined (Table 4). In uninfected plants, increases were observed in the concentrations of K and P of plants inoculated with PGPR compared to those of control plants. Additionally, Ca levels increased in inoculated plants compared to those of uninfected control plants. Compared with the stressed plants and PGPR-inoculated plants, the inoculated infected plants showed increases in the concentrations of Ca, K, and P.

Effect of B. amyloliquefaciens Treatment on the Regulation of Biotic Stress Responsive Genes
The expression of biotic stress responsive genes was investigated in pepper seedlings. Overall, 12 genes (Table S3) were assessed for their change in expression under biotic stresses and PGPR application in pepper plant seedlings.

Antimicrobial and Defense-Related Protein (CaAMP1, CaPR1, and CaDEF1)
The transcription pattern of CaAMP1, CaPR1, and CaDEF1 was evaluated in pepper plants treated with either biotic stresses or PGPR. As illustrated in Figure 9A-C   Values show the means ± standard error (n = 3) and significant differences are indicated at p < 0.05 in accordance with the least significant difference test. Bars with different letters are significantly different from each other.

Xyloglucan Endotransglucosylase/Hydrolase (XTH)
The expressions of CaXTH1 and CaXTH2 genes in pepper seedlings under biotic stresses and PGPR inoculation are shown in Figure 9E

BCL2-Associated x Protein (BAX) Inhibitor 1 (BI-1)
The expression of the BI-1 gene (CaBI-1 gene) under biotic stress was assessed in pepper seedlings that had been inoculated with PGPR ( Figure 9J). We identified minor differences in expression between the control and PGPR-inoculated uninfected plants; higher expression was identified in stressed plants. PGPR-inoculated plants showed decreased BI-1 expression (40.89% under Botrytis and 34.76% under Alternaria stress conditions) compared to that of untreated stressed plants.

RING-Type E3 Ligases (ASRF1)
Enhanced CaASRF1 expression was observed in pepper seedlings subjected to biotic stress. The CaASRF1 expression level raised by 25.25% (Botrytis) and 51.98% (Alternaria) compared to the unstressed control plants ( Figure 9K). However, PGPR-inoculated pepper plants showed reduced CaASRF1 expression under stress condition. Botrytisand Alternariainfected plants exhibited 78.26% and 79.01% lower CaASRF1 expression compared to the untreated stressed plants.

Squamosa Promoter Binding Protein (SBP)
The CaSBP11 expression level in pepper seedlings under biotic stresses and PGPR application is depicted in Figure 9L. Decrease in CaSBP11 expression level was detected in Botrytisand Alternaria-stressed plants by 51.20% and 61.37%, respectively, as compared to the unstressed plants. On the other hand, application of PGPR increased the CaSBP11 expression content in Botrytis (77.36%) and Alternaria (74.79%) diseased pepper plants in comparison with the corresponding untreated stressed plants.

Discussion
During crop cultivation, biotic stress resulting from plant pathogens is a serious challenge that causes enormous economic losses for growers. Different agrochemicals are currently employed to control plant diseases. However, their usage is problematic due to public concern regarding dangerous residues, the selection of resistant strains of the pathogens, and increased expenses for plant protection. The development of microbe-based control methods could produce effective substitutes for managing crop disease. At present, free-living, nonpathogenic, root-colonizing bacteria are implemented in a broad range of agricultural production systems as bioinoculants in a variety of economically important plants [72,73].
Our study showed antagonistic activity of B. amyloliquefaciens, a plant growth promoting bacterium, against Botrytis pelargonii and Alternaria alternata under in vitro conditions. The activity was mediated by hydrolytic enzyme activity. The influence of this interaction was apparent under in vivo conditions for diminished fungal diseases. Inoculation with B. amyloliquefaciens resulted in increased growth and improved health of the pepper plants with or without Botrytis pelargonii and Alternaria alternata infection. Augmentation in the chlorophyll and carotenoid contents was observed in the leaves of PGPR-inoculated plants exposed to stress conditions. In addition, we found that the treatment with B. amyloliquefaciens increased Ca, K, and P levels compared to non-inoclulated plants. The increases in Ca levels are known to increase resistance to fungal infections in many crops [74,75]. Inoculation with plant growth promoting rhizobacteria (PGPRs) inoculation enhances the photosynthetic pigments in plants during stress conditions [76,77]. This corresponds to improved nutrient uptake from the rhizosphere, which sustains plant growth under stressful conditions [78,79]. It should be noted that inoculation with B. amyloliquefaciens improved the soil moisture level in diseased and healthy pepper plants. In agreement with our results, previous studies indicated that PGPRs promote soil moisture content, thus enhancing plant growth and survival [80,81]. This could be due to the formation of exoploysaccharides and biofilm by PGPRs [80].
Xyloglucan endotransglucosylase/hydrolase (XTHs) are responsible for the regulation of various physiological processes, including the elongation of plant cells [82]. Furthermore, they modulate plant response to environmental stimuli, including salinity, water deficit, and heat [83][84][85]. Our results revealed reduced XTH expressions in diseased plants. This could be due to fungal attack mechanisms, which causes decreased cell wall extensibility and growth reduction of seedlings. Muñoz-Bertomeu and Lorences [86] showed that the expressions of various XTHs decrease as pathogen infection progresses. PGPR-inoculated plants showed higher XTH expressions under pathogen attack, which induced improved plant height and leaf area. Overexpression of XTHs enhances plant tolerance toward abiotic stresses [84,87]. Our results confirm that the high expression of XTHs (CaXTH1 and CaXTH2) is associated with plant maintenance and tolerance in diseased pepper seedlings without unfavorable effects.
Phytohormones synergistically or antagonistically function in a complicated network to modulate multiple facets of plant growth, reproduction, and immunity [88]. Salicylic acid (SA) contributes to the photosynthetic and growth parameters as well as antagonized oxidative damage in plants in response to natural attackers [89,90]. Melotto et al. [91] showed antagonistic interactions between SA and abscisic acid (ABA) signaling in response to pathogens. Our results showed that pathogen stress increased ABA levels but decreased SA levels, which is in agreement with the results of previous studies [92,93]. These results indicate that PGPR application relieves pathogen stress in pepper seedlings by decreasing their ABA levels and increasing their SA levels.
Protein post-translational modification events, such as ubiquitination, have been detected during plant stress responses, growth, and development [94]. It has been proven that various ABA signaling convertors are exposed to the modulation via ubiquitination. Several kinds of E3 ligases have been recognized that modulate ubiquitination of ABA receptors [94]. Joo et al. [95] indicated that CaASRF1 gene (Capsicum annuum ABA Sen-sitive RING Finger E3 ligase 1) assuredly regulates ABA signaling pathway and plant development. They found that CaASRF1 gene alters drought stress endurance via ABA signaling. Our findings showed enhanced CaASRF1 expression in diseased plants, which was along with boosted ABA level. This confirmed that CaASRF1 gene certainly regulate ABA signaling and biotic stress response in diseased pepper plants.
SBP-box genes (SBP) have a crucial role in plant development, signal conduction, and reaction to abiotic and biotic stresses [96,97]. Previous study reported that SBP5 improved resistance to Erysiphe necator through SA disease resistance signaling mechanisms [98]. In our study, reduction in CaSBP11 level was detected in infected plants. In contrast, PGPRinoculated plants showed enhanced CaSBP11 expression under Botrytis and Alternaria attack. Zhang et al. [99] confirmed that CaSBP11 increased pepper plant defense response to Phythophthora capsici by regulating SA and jasmonic acid signaling mechanisms. Based on the obtained results, it is speculated that CaSBP11 positively modulates SA signaling pathways to enhance disease resistance in Botrytisand Alternaria-infected plants.
WRKY, a major transcription factor family, was found to be involved in numerous developmental and physiological functions comprising abiotic and biotic stress signaling pathways [100][101][102]. WRKY expression increased upon pathogen stress conditions, which was consistent with the results of a previous study [103]. Additionally, a number of WRKY transcription factors act in ABA and SA signaling pathways. Xie et al. [104] found that ABA either negatively or positively modulates the transcripts of some WRKYs. We found that in the presence of ABA, WRKY2 expression increased in diseased pepper plants. These results indicated that ABA positively mediates the expression of WRKY2. Upon applying PGPR, the SA level increased and the ABA and WRKY2 levels decreased under stress condition. Dong et al. [105] found that upon stimulating the SA-dependent defense, WRKYs revealed various modulations comprised of suppression or augmentation. These results indicated that PGPR application allows the stressed plants to face various biotic stresses.
Adverse environmental cues have a negative influence on plant growth and development and induce protein denaturation or misfolding [106,107]. Endoplasmic reticulum stress is triggered by misfolded proteins that accumulate in the endoplasmic reticulum under harmful environmental situation and lead to programmed cell death [108]. Misfolded or unfolded protein augmentation in endoplasmic reticulum has impact on cellular protein function and localization [109]. BiPs play a crucial role in protein quality monitoring by distinguishing and refolding misfolded proteins. Furthermore, by alleviating the amount of unfolded protein, BiPs balance immune receptors to ease plant defense [110]. In this study, biochemical and molecular approaches identified an increase in the protein value of diseased plants ( Figures 3A and 5A-C). Protein augmentation caused BiP genes (CaBiP1, CaBiP2, and CaBiP3) induction in Botrytis and Alternaria-infected plants. It has been shown that the aggregation of unfolded proteins promotes BiP induction to ameliorate plant endurance to abiotic and biotic stresses [109]. In contrast, BiPs were restrained in PGPR-inoculated plants exposed to pathogen stress. Together, these results suggest that B. amyloliquefaciens mitigates the soluble protein content in diseased plants. This might be due to the stress-soothing effect of this PGPR, which results in protein catabolism.
Plant exposure to biotic and abiotic stresses leads to the generation of reactive oxygen species (ROS) to trigger the stress response and defense pathways. ROS overaccumulation causes oxidative damage, impaired membrane lipid functions, enzyme inactivation, impeded metabolic activities, and, ultimately, plant death [111]. We found that H 2 O 2 and MDA levels increased in infected plants, which was in accordance with the results of previous studies [111]. PGPR-inoculated plants showed lower H 2 O 2 and MDA levels when under pathogen attack. This suggested that PGPR application might restrain the production of ROS, successfully inhibiting cell damage under oxidative stress conditions [112][113][114].
ROS are key players in programmed cell death (PCD); a cell suicide process that inhibits the pathogen spread in plants and eliminates damaged cells. BAX is a pivotal modulator of PCD and is stabilized by the function of the anti-PCD factor BI-1 [115]. BI-1 demonstrates inducement as opposed to numerous types of biotic and abiotic environmen-tal stresses and provides endurance in plants against these stressors [116][117][118]. Increased expression of CaBI-1 was observed in diseased pepper plants. CaBI-1 expression was decreased in PGPR-inoculated plants exposed to pathogen invasion. This validated the potency and attenuating influence of PGPR in pepper plants affected by Alternaria or Botrytis stress.
Plants have defensive mechanisms comprised of antioxidants with enzymatic or nonenzymatic activity to survive oxidative damage and neutralize immoderate oxidation [119]. PGPR increases the activity of enzymatic/non-enzymatic antioxidants. Consistently, our results have validated the amelioration of antioxidant activity in healthy plants. In the current study, the activity of antioxidants increased in the diseased plants, whereas their activity decreased in PGPR-inoculated plants subjected to Botrytis or Alternaria stress. This decreased activity implies progress in scavenging over accumulated ROS and minimizing oxidative damage, which maintains optimal protection of the plant [119].
Environmental stress lowers leaf sugar content, leading to physiological and biochemical modifications as sugar sustains macromolecules and membrane structure during stress [120]. Accumulated soluble sugars can induce pathogen resistance in plants as the soluble sugars or sugar byproducts can play a role as osmoprotectants under stress conditions [121]. In the present study, an increase in leaf sugar content was identified in PGPR-inoculated plants under normal as well as stress conditions. Soluble sugars function as metabolic resources and structural constituents of cells and modulate many processes connected with plant development under stress conditions [122]. Sugar accumulation in leaves also triggers the expression of genes connected to photosynthetic activities [123]. In this study, PGPR triggered major sugar accumulation, which potentially acted as an osmoprotectant in the photosynthetic organs and assisted with the retention of photosynthetic performance, leading to improved growth and defense mechanisms under Botrytis and Alternaria attack.
Amino acids act as precursors for metabolite synthesis and modulate plant responses to environmental stress [124]. Our results showed an increase in amino acid contents in infected plants. This augmentation indicates that they play a role in plant defense in addition to their roles in metabolism [125,126]. Previous studies have indicated that amino acid levels increase under pathogen stress [126,127]. In the present study, the PGPR treatment decreased the amino acid level in Botrytisand Alternaria-stressed plants. The accumulation of Pro, which acts as an osmolyte and ROS scavenger, could be a strategy for withstanding pathogen invasion [127,128]. The reduced Pro content in PGPR-inoculated plants might be caused by osmotic adjustment, which leads to improved plant survival under pathogen attack.
Plants trigger a series of responses toward microbial attacks, which induce a range of antimicrobial defenses both locally and systematically [129]. These defense responses include the strengthening of mechanical barriers, oxidative burst, and the production of antimicrobial and defensive compounds [130,131]. The antimicrobial protein gene CaAMP1 was strongly induced in infected pepper plants to enhance tolerance to fungal disease, which is in agreement with the results of previous studies [132][133][134][135]. AMPs play a crucial role in constitutive or triggered tolerance to various pathogens by deteriorating fungal cell walls, stimulating membrane channels, and preventing DNA synthesis [134,136]. Moreover, infection of Botrytis and Alternaria spp. highly activated the expression of defense-related genes; namely, CaDEF1 and CaPR1, in the pepper plants. Previous studies have demonstrated the expression of defense-related genes in pepper and tomato plants upon infection with pathogens, such as Phytophthora capsici, Xanthomonas campestris, and Clavibacter capsici [137,138]. In contrast, CaAMP1, CaDEF1, and CaPR1 expressions were strongly decreased in PGPR-inoculated plants subjected to biotic stress. Together, these results confirm the involvement of CaAMP1, CaDEF1, and CaPR1 in resistance to fungal pathogens and demonstrated the stress-relieving effect of PGPR.

Conclusions
Along with enhancing pepper growth, the bacterium B. amyloliquefaciens stimulates resistance to Botrytis pelargonii and Alternaria alternata infections. This bacterial strain can secrete hydrolytic enzymes and solubilize nutrients, thus promoting host growth and alleviating the disease in the pepper plant. Additionally, PGPR treatment influences the host biochemistry increasing resistance to the infections by fungal pathogens. PGPR application triggered the expression of stress-related genes, namely, CaAMP1, CaDEF1, CaPR1, CaXTH, CaWRKY2, CaBI-1, CaASRF1, CaSBP11, and CaBiP. This study is the first report, to our knowledge, of the suppressive effects of B. amyloliquefaciens on Alternaria leaf spot and Botrytis gray mold of C. annum. The results of this study indicate that B. amyloliquefaciens is beneficial for C. annum under biotic stress and may be suitable as a candidate for the management of crop diseases.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7060472/s1, Figure S1: Symptomatic pepper plant collected from the farm, Figure S2: Effect of plant growth-promoting rhizobacteria (PGPR) inoculation on the pepper plants grown under normal and biotic stress conditions after eight days, Table S1: Primers used in this study for PCR amplification of the 18S rDNA fungal isolates, Table S2: Physiochemical properties of the soil samples over eight days of treatment, Table S3: Primers used for relative gene expression analysis.