Bacillus subtilis CBR05 for Tomato (Solanum lycopersicum) Fruits in South Korea as a Novel Plant Probiotic Bacterium (PPB): Implications from Total Phenolics, Flavonoids, and Carotenoids Content for Fruit Quality

Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil biota which benefit plants by improving plant productivity and immunity. The aim of the present work was to evaluate the effect of the inoculation of PGPR strain, Bacillus subtilis CBR05 on the quality of tomato fruits produced under greenhouse conditions. Results were compared with mock-inoculated control and market sample. We found a significant increase in total phenol and flavonoid contents of tomato fruits in PGPR strain B. subtilis CBR05 inoculated plants compared to those of mock-inoculated control and market sample. Moreover, B. subtilis CBR05 inoculation stimulated antioxidant activities and levels of carotenoid (β carotene and lycopene) content in plants. In addition, the inoculation of the strain B. subtilis CBR05 produced the highest content of lycopene (21.08 μg/g FW) in tomato fruits as compared to mock-inoculated plants. Our results show that the PGPR strain B. subtilis CBR05 is a versatile soil bacterium that enhances tomato production by elevating antioxidant activities and carotenoid (β carotene and lycopene) levels in fruit.


Introduction
Tomato (Solanum lycopersicum) is regarded as the second most vegetable crop next to potato in the agricultural implications of human consumption. According to agricultural statistics, tomatoes along with sweet corn and snap beans constitute 93% of crop production and processing strategies (Agricultural Statistics, United States Department of Agriculture (USDA), 2016). The positive benefits of tomato consumption have been rigorously proved against a variety of diseases like chronic degenerative diseases, owing to the escalated content of significant phytochemicals with potent health benefits, The inoculum mixture of the strain was prepared by culturing in nutrient broth and incubating at 28 • C with constant shaking at 130 rpm. The bacterial cells were centrifuged at 10,000 g for 10 min at 4 • C. The cell precipitate was resuspended in 10 mM MgCl 2 and the cell concentration of bacterial suspensions of B. subtilis CBR05 was adjusted to 10 8 colony-forming unit (CFU)/mL (OD 600 = 1.0) for further studies.

Plant and Growth Conditions
Tomato seeds (Korean cultivar, Kwangbok) utilized for this study were obtained from a Korean seed resource center, Seoul, South Korea. They were surface sterilized in sodium hypochlorite, rinsed several times with distilled water, and planted onto pots containing sterilized growth media (Peat moss with perlite in a ratio of 3:1). Two sets of plants (three plants per set) were maintained, one without PGPR (mock-inoculated control) inoculation and the other with PGPR strain, B. subtilis CBR05 inoculum. All treatments were placed randomly in the greenhouse and replicated 3 times. Plant growth-promoting B. subtilis CBR05 were applied under sterile conditions to the base of the plants close to the roots to ensure better colonization. Plants were maintained under greenhouse conditions at a temperature of 25 • C with watering carried out every alternate day, to make a better availability for nutrition and plant growth promotion.

Antioxidant Assays
2.4.1. DPPH Assay 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was performed to assess the fractions exhibiting scavenging property of free radicals in vitro [27]. Then, 0.2 mM solution of DPPH in ethanol was added to the fraction of aliquot at concentrations (100 µg/mL). The mixture was allowed to stand for 30 min and the absorbance was measured at 517 nm using a UV-Visible spectrophotometer. The percent scavenging activity was determined and Trolox was used as the standard.

Determination of Total Phenolic Contents
The Folin-Ciocalteu method was used to estimate various concentrations concerned with the total phenolic content. The extracts were dissolved in absolute methanol and later 200 µL of the extract was mixed with 800 µL of 1 N Folin-Ciocalteu reagent (1: 10). After 5 min at room temperature, 3 mL of sodium carbonate (15%) was added to the extracts. Following incubation for 30 min at room temperature, the absorbance was read at 765 nm using a UV-spectrophotometer. A standard curve for gallic acid equivalents (GAE) (milligrams per gram of extract (mg GAE/ge)) was utilized to evaluate the concentration of total phenolic compounds. Analyses were performed in triplicate per each extract.

Determination of Total Flavonoid Contents
Screening the content of flavonoids was done by a modified protocol as reported previously [29]. The extracts were dissolved in absolute methanol. In a 15 mL conical tube, 1 mL of a sample was mixed with 0.3 mL of 5% sodium nitrite, followed by incubation for 5 min. After incubation, 0.3 mL of aluminum chloride (10%) and 2 mL of sodium hydroxide (1 mol/L) were added to the reaction mixture, and the absorbance was read at 496 nm with a UV-spectrophotometer, using catechin as the standard. Quercetin equivalents (QE) present per g of extract (mg QE/ge) was used to quantify the expression levels.

Extraction and Quantification of Carotenoids
Carotenoids were extracted in triplicates and quantified according to previously established protocol with minor modifications [30,31]. All the preparations were performed in low light conditions to avoid the degradation of carotenoids. Three independent biological samples were extracted separately. Briefly, one whole tomato fruit was finely chopped and mixed thoroughly. Five grams of chopped fruits (exact to 0.001 g) from each treatment were separately transferred into test tubes containing 20 mL of acetone and 0.1% butylated hydroxytoluene (BHT: w/v). The samples were homogenized with a mechanical homogenizer and centrifuged at 5000× g (5 min at 4 • C temperature). The supernatant was recovered and pelleted samples were repeatedly extracted until the pallets became colorless. Supernatants from all extractions were pooled and vacuum-dried in a rotary evaporator (Temperature < 35 • C) (Büchi RE 111, Switzerland).

HPLC Analysis
The extract was recovered with 10 mL of methylene chloride (CH 2 Cl 2 ) containing 0.1% BHT and transferred to an amber color HPLC vial for HPLC analysis. The chromatographic separation was achieved using an Agilent Model 1100 HPLC instrument (Agilent Technologies Canada Inc., Mississauga, ON, Canada) equipped with a degasser, autosampler, dual pump, and diode array detector (DAD). Samples were scanned (200-800 nm) with 0.05 min (1 s) response time, 8.0 mm slit width, and a detection wavelength of 450 (for most of the carotenoids) and 470 nm (for lycopene). The bandwidth was ±16 nm for all detection wavelengths. Similarly, 600 nm was used as a reference wavelength with ±50 nm bandwidth in all detections. The column used was a YMC, C30 carotenoid column, 250 × 4.6 mm, 5 µm (YMC, Wilmington, NC, USA), and the chromatographic data were recorded with ChemStation LC 3D software. The column thermostat was maintained at 25 • C temperature. Then, 20 µL of standards and samples were injected with an autosampler. The solvent system consisted of Methanol: methyl tertiary butyl ether (MTBE): water (81:15:4) (Mobile phase A) and MTBE: Methanol (91:9) (Mobile phase B). The gradient elution was 0%-100% B in 90 min, and 5-min post-run at a flow rate of 1 mL/min.

Statistics
All of the experiments were conducted in triplicate and results were tabulated as the Mean ± standard deviation (SD). Statistical significance of the data was determined using one-way analysis of variance (one-way ANOVA) followed by Fisher LSD (Least Significant Difference) test. Data analyses were performed using Sigmastat v8.02 (Systat Software Inc., San Jose, CA, USA). A p value of ≤ 0.05 was considered significant.

Results
In the present investigation, the effects of the PGPR strain, B. subtilis CBR05 inoculation on the maintenance of carotenoids, total phenolics, flavonoid contents, and antioxidant properties were evaluated. The results revealed that the PGPR strain, B. subtilis CBR05 has the capacity to improve the plant growth and change some of the tomato fruit quality characteristics under greenhouse conditions. Tomato plants treated with PGPR strain, B. subtilis CBR05 showed significantly higher biomass compared with mock-inoculated controls. Significant increases in root length and dry weight, over mock-inoculated controls, were achieved in green-house conditions (data not shown). In addition, antioxidant activities of three tomato fruits (mock-inoculated, B. subtilis CBR05 inoculated, and market fruit) were determined using both DPPH and ABTS radical scavenging method. The extract of fruits from plants inoculated with B. subtilis CBR05 strain was the most active against DPPH and ABTS radical and that of fruits from the market, the differences being significant when compared to mock-inoculated control ( Figure 1). The total phenolic content of the fruit extracts is shown in Figure 2. Among the fruits, the PGPR strain, B. subtilis CBR05 inoculated plants had the highest phenolic content followed by market fruits. Moreover, the flavonoid contents were also elevated in PGPR treated tomato fruits as compared to those of both mock-inoculated control and market fruits ( Figure 2). We did not find any significant differences between mock-inoculated control and market fruit. The results of this study show that B. subtilis CBR05 is an effective probiotic agent for the promotion of tomato fruit quality. The carotenoids, such as lycopene and β-carotene, were extracted and quantified ( Table 1). The contents and composition of carotenoids were found to differ among control, B. subtilis CBR05 inoculated, and market fruits (Figure 3). Carotenoid levels (lycopene) in tomato fruits in plants treated with PGPR strain, B. subtilis CBR05 were higher than both the control and market fruits (Table 1; Figure 3). The total phenolic content of the fruit extracts is shown in Figure 2. Among the fruits, the PGPR strain, B. subtilis CBR05 inoculated plants had the highest phenolic content followed by market fruits. Moreover, the flavonoid contents were also elevated in PGPR treated tomato fruits as compared to those of both mock-inoculated control and market fruits ( Figure 2). We did not find any significant differences between mock-inoculated control and market fruit. The results of this study show that B. subtilis CBR05 is an effective probiotic agent for the promotion of tomato fruit quality. The total phenolic content of the fruit extracts is shown in Figure 2. Among the fruits, the PGPR strain, B. subtilis CBR05 inoculated plants had the highest phenolic content followed by market fruits. Moreover, the flavonoid contents were also elevated in PGPR treated tomato fruits as compared to those of both mock-inoculated control and market fruits ( Figure 2). We did not find any significant differences between mock-inoculated control and market fruit. The results of this study show that B. subtilis CBR05 is an effective probiotic agent for the promotion of tomato fruit quality. The carotenoids, such as lycopene and β-carotene, were extracted and quantified ( Table 1). The contents and composition of carotenoids were found to differ among control, B. subtilis CBR05 inoculated, and market fruits (Figure 3). Carotenoid levels (lycopene) in tomato fruits in plants treated with PGPR strain, B. subtilis CBR05 were higher than both the control and market fruits (Table 1; Figure 3). The carotenoids, such as lycopene and β-carotene, were extracted and quantified ( Table 1). The contents and composition of carotenoids were found to differ among control, B. subtilis CBR05 inoculated, and market fruits (Figure 3). Carotenoid levels (lycopene) in tomato fruits in plants treated with PGPR strain, B. subtilis CBR05 were higher than both the control and market fruits (Table 1; Figure 3). All-E-β-Carotene 3.53 ± 0.12 a All-E-Lycopene 21.08 ± 0.32 b In each experiment, means followed by different superscript letters are significantly different among the treatment groups (p ≤ 0.05). Values are mean ± standard deviation from three replicates.

Treatment Carotenoids Content (μg/g FW) Mock inoculated control
All-E-β-Carotene 5.63 ± 0.28 a All-E-Lycopene 7.48 ± 1.96 a Market tomato All-E-β-Carotene 4.65 ± 0.96 a All-E-Lycopene 10.51 ± 3.34 a Bacillus subtilis CBR05 All-E-β-Carotene 3.53 ± 0.12 a All-E-Lycopene 21.08 ± 0.32 b In each experiment, means followed by different superscript letters are significantly different among the treatment groups (p ≤ 0.05). Values are mean ± standard deviation from three replicates. The present investigation extends their results by comparing the contents of carotenoids from the fruits using HPLC-DAD ( Figure 4). Using this methodology, all-E-β -carotene and all-E-lycopene were identified as the major carotenoids in tomato fruits, based on retention time with standards and by comparing the peak spectra recorded with a DAD during the analysis. The chromatograms (470 nm) and the peak spectra of major identified peaks were shown in Figure 4. The other minor carotenoids were not quantified due to the unavailability of standard compounds. We did not find any significant differences in β-carotene. However, in the present study, we have recorded a significantly higher amount of lycopene (All-E-lycopene) in fruits during ripening in PGPR strain, B. subtilis CBR05 inoculated plants than those of both mock-inoculated control and market fruits. These results validate the productive roles of the B. subtilis CBR05 in enhancing the nutritional potential of tomato fruits. The present investigation extends their results by comparing the contents of carotenoids from the fruits using HPLC-DAD ( Figure 4). Using this methodology, all-E-β -carotene and all-E-lycopene were identified as the major carotenoids in tomato fruits, based on retention time with standards and by comparing the peak spectra recorded with a DAD during the analysis. The chromatograms (470 nm) and the peak spectra of major identified peaks were shown in Figure 4. The other minor carotenoids were not quantified due to the unavailability of standard compounds. We did not find any significant differences in β-carotene. However, in the present study, we have recorded a significantly higher amount of lycopene (All-E-lycopene) in fruits during ripening in PGPR strain, B. subtilis CBR05 inoculated plants than those of both mock-inoculated control and market fruits. These results validate the productive roles of the B. subtilis CBR05 in enhancing the nutritional potential of tomato fruits.

Discussion
The use of PGPR is increasing in agriculture and may offer an attractive alternative to synthetic chemicals and fertilizers. Plant growth-promoting microorganisms are efficient microbial competitors that can promote plant growth by producing phytohormones and/or by increasing available nutrients through production of secondary metabolites or act as biocontrol agents to protect plants from infection by phytopathogens [19,[21][22][23][24]. There have been many reports on PGPR and their effective roles [19][20][21][22][23][24]. Insufficient experimental work has been reported to speculate on the mechanisms of PGPR effects on fruit quality. In the present study, PGPR strain, B. subtilis CBR05 isolated from rice were used as inoculants for tomato plants grown under greenhouse conditions. Our results showed that PGPR inoculations significantly increased the total biomass and root length compared to those in the control. B. subtilis CBR05 appears to impart plant growth promotion effects that are distinct from other commercial biocontrol agents. Tomato fruits are a good source of antioxidant compounds that can reduce harmful oxidation reactions in the human body, thus preventing various diseases associated with free radical oxidation, such as cardiovascular and neurological disorders and cancer [1][2][3][4].
Antioxidant activity has been widely used to test the ability of plant extracts to act as free radical scavengers [32]. In the present investigation, B. subtilis CBR05 had a net positive effect on the antioxidant activity measured by the DPPH and ABTS scavenging capacity, which seems to indicate that the bacteria acted as a regulator of the synthesis of antioxidant compounds in the plant ( Figure  1). Strong scavenging of ion radical was exhibited by the inoculated tomato fruits, thus showing that B. subtilis CBR05 inoculation increased the radical scavenging capacity of tomato fruits. In a previous study, B. licheniformis inoculated plants had increased antioxidant profiles in tomato plants under greenhouse conditions [32]. Similar results concerned with enhanced fruit quality and marketable grade have been reported for other crops under the influence of PGPR [33] but this report is the first of its kind to specifically address PGPR strain, B. subtilis CBR05 in improvement of fruit quality. Moreover, PGPR enhances fruit characteristics based on the mediation of increased availability of nutrients to plants like phosphorous and iron, enhancing the nutritional status of the plants in the rhizosphere [33][34][35].
Phenolic contents perform an essential role in plant resistance and defense against phytopathogens, which are closely linked with reactive oxygen species (ROS). This study reveals that among the selected tomato fruits, PGPR strain, B. subtilis CBR05 inoculated tomato fruits had the highest amount of phenolics ( Figure 2). Some phenolic compounds may prevent oxidative damage in vivo and thus protect against the development of the disease such as cardiac disease and cancer [1,2]. This might be considered as useful for health purposes. In addition, inoculation of B. subtilis

Discussion
The use of PGPR is increasing in agriculture and may offer an attractive alternative to synthetic chemicals and fertilizers. Plant growth-promoting microorganisms are efficient microbial competitors that can promote plant growth by producing phytohormones and/or by increasing available nutrients through production of secondary metabolites or act as biocontrol agents to protect plants from infection by phytopathogens [19,[21][22][23][24]. There have been many reports on PGPR and their effective roles [19][20][21][22][23][24]. Insufficient experimental work has been reported to speculate on the mechanisms of PGPR effects on fruit quality. In the present study, PGPR strain, B. subtilis CBR05 isolated from rice were used as inoculants for tomato plants grown under greenhouse conditions. Our results showed that PGPR inoculations significantly increased the total biomass and root length compared to those in the control. B. subtilis CBR05 appears to impart plant growth promotion effects that are distinct from other commercial biocontrol agents. Tomato fruits are a good source of antioxidant compounds that can reduce harmful oxidation reactions in the human body, thus preventing various diseases associated with free radical oxidation, such as cardiovascular and neurological disorders and cancer [1][2][3][4].
Antioxidant activity has been widely used to test the ability of plant extracts to act as free radical scavengers [32]. In the present investigation, B. subtilis CBR05 had a net positive effect on the antioxidant activity measured by the DPPH and ABTS scavenging capacity, which seems to indicate that the bacteria acted as a regulator of the synthesis of antioxidant compounds in the plant (Figure 1). Strong scavenging of ion radical was exhibited by the inoculated tomato fruits, thus showing that B. subtilis CBR05 inoculation increased the radical scavenging capacity of tomato fruits. In a previous study, B. licheniformis inoculated plants had increased antioxidant profiles in tomato plants under greenhouse conditions [32]. Similar results concerned with enhanced fruit quality and marketable grade have been reported for other crops under the influence of PGPR [33] but this report is the first of its kind to specifically address PGPR strain, B. subtilis CBR05 in improvement of fruit quality. Moreover, PGPR enhances fruit characteristics based on the mediation of increased availability of nutrients to plants like phosphorous and iron, enhancing the nutritional status of the plants in the rhizosphere [33][34][35].
Phenolic contents perform an essential role in plant resistance and defense against phytopathogens, which are closely linked with reactive oxygen species (ROS). This study reveals that among the selected tomato fruits, PGPR strain, B. subtilis CBR05 inoculated tomato fruits had the highest amount of phenolics ( Figure 2). Some phenolic compounds may prevent oxidative damage in vivo and thus protect against the development of the disease such as cardiac disease and cancer [1,2]. This might be considered as useful for health purposes. In addition, inoculation of B. subtilis significantly increased flavonoid content compared to those of both control and market fruits. Similarly, increases in total flavonoids content by B. licheniformis have been reported for tomato fruits [32]. In our previous studies, we also found that B. subtilis CBR05 inoculation enhanced the accumulation of peroxidase and polyphenol oxidase enzymes, which are involved in the metabolism of phenols and flavonoids [22,23]. Hence, this shows that B. subtilis colonization induces resistance against biotic and abiotic stress agents. The results of antioxidant assays also revealed that tomatoes are a rich source of antioxidants, thus their habitual consumption can potentially help to combat the oxidative stress.
Regulation of carotenoid biosynthesis and high-accumulation lycopene during tomato fruit development is widely studied [36][37][38]. In the present study, we found a significantly higher amount of lycopene (All-E-lycopene) in B. subtilis CBR05 inoculated tomato fruits (Figures 3 and 4). Lycopene possesses the highest antioxidant potential among the carotenoids and several other antioxidants found in fruits and vegetables [39]. Thus, the addition of PGPR enhances lycopene content in tomato fruits and can potentially contribute to antioxidant levels of diets. This potent antioxidant activity of lycopene protects from a variety of ROS and reactive nitrogen species (RNS), thus helping in preventing chronic diseases in humans [31,36]. Similar to the lycopene contents, the DPPH and ABTS antioxidant activity of B. subtilis CBR05 inoculated tomato fruits was much higher than both the control and market fruits. Earlier, we also reported that defense-related enzymes in tomato after treatment with B. subtilis CBR05 efficiently combated X. campestris pv, vesicotoria [40] and induction of defense-related enzymes like superoxide dismutase, catalase, peroxidase, and polyphenol oxidase assessment revealed the up-regulation of glucanase and phenyl ammonia lyase indicating induced systemic resistance (ISR) in tomato. The earlier results established that the antioxidant capabilities of tomatoes are naturally present. Further, it was specifically proved that B. subtilis CBR05 mechanism of disease resistance against X. campestris pv, vesicotoria was confirmed for the involvement of the de novo pathway involved in Vitamin B6 biosynthesis [41]. In addition, known bacterial elicitors of ISR are microbial associated molecular patterns triggering immunity. When this fails, microbial effector-triggered immunity is induced and leads to programmed cell death. It increases the plant's systemic resistance to subsequent pathogen challenge by PGPR. Moreover, plant probiotic bacterium (PPB) could be used to reduce the use of chemicals (fertilizers, pesticides) in agriculture. This could lead to improved quality at reduced costs and could provide the basis for more sustainable agriculture [42]. Organic agriculture has been widely promoted and adopted to establish better sustainability in food production and crop protection. Micromonospora has been regarded as a PPB due to rhizobia helper bacteria properties in Medicago sativa L. [43]. Similarly, Phyllobacterium and B. licheniformis also show promising benefits for increasing vitamin C content across various functional foods and have been considered as PPB, devoid of economic loss [32,44].
In the present investigation, we extracted and quantified phenols, flavonoids, and carotenoids, in the mock-inoculated control, B. subtilis CBR05 inoculated plants and market fruits. Among them, PGPR strain, B. subtilis CBR05 inoculated tomato fruits were found to have the richest source of lycopene, total phenolics, and flavonoids contents. Additionally, the PGPR strain, B. subtilis CBR05 inoculated tomato fruits showed potent antioxidant activities. The significantly higher lycopene content and radical-quenching activity of PGPR strain, B. subtilis CBR05 inoculation confer tomato fruits with more nutritional value, and their consumption can minimize oxidative stress-mediated chronic diseases. Thus, biofertilizers based on PGPR may be a viable alternative to improve the nutraceutical quality of greenhouse-produced tomato fruits. Funding: This research received no external funding.

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