Antibacterial activity of tannins isolated from Sapium baccatum extract and use for control of tomato bacterial wilt

In the search for new antibacterial agents from natural sources, we revealed that a crude methanol extract of Sapium baccatum was highly active against Ralstonia solanacearum, a causal agent of a serious disease called bacterial wilt of tomato. The bioassay-guided fractionation of this extract resulted in the isolation of seven known active compounds, including gallic acid, methyl gallate, corilagin, tercatain, chebulagic acid, chebulinic acid, and quercetin 3-O-α-L-arabinopyranoside. Their chemical structures were determined by electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy. An in vitro antibacterial bioassay using a broth microdilution method revealed that, except for quercetin 3-O-α-L-arabinopyranoside (MIC = 250 μg/mL), the isolated compounds exhibited strong antibacterial activity against R. solanacearum (MIC = 26–52 μg/mL). Among the seven compounds, methyl gallate exhibited the strongest broad-spectrum activity against most of the plant pathogenic bacteria tested (MIC = 26–250 μg/mL). In the in vivo experiments, the crude extract of S. baccatum at 2000 and 1000 μg/mL reduced the development of tomato bacterial wilt by 83 and 63%, respectively, under greenhouse conditions after 14 days of infection. The results suggested that the extracts of S. baccatum or isolated tannins could be used as natural bactericides for the control of bacterial wilt of tomato.


Introduction
Phytopathogenic bacteria cause serious economic losses by reducing the yield of marketable quality crops [1]. Ralstonia solanacearum, which causes vascular wilt disease, is one of the most destructive pathogens [2]. This soil-born pathogen is found worldwide and has a large range of hosts, comprising more than 200 plant species, including tomato, potato, pepper, peanut, tobacco, and banana [2][3][4]. The direct yield losses caused by R. solanacearum differ widely a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 according to the host, cultivar, climate, soil type, cropping pattern, and strain; the losses were up to 90% for tomato, potato, and banana [2].
The control of bacterial wilt has been considered difficult owing to the complex nature of the pathogen: endophytic growth patterns, survival in soil, transport in water, and a wide host range and biological diversity [2,5]. The investigation into various methods for the control of bacterial wilt disease has spanned several decades. Yuliar [2] reported that studies of these methods conducted between 1984 and 2014 predominantly described the biological methods (54%), followed by cultural practices (21%), chemical methods (8%), and physical methods (6%). Plant disease control has been largely dependent on the use of chemical pesticides. Unfortunately, the use of chemical pesticides has been strongly limited owing to their association with environmental pollution, poisonous effects, and antibiotic resistance [2,6,7]. As the demand for environmentally acceptable pesticides is increasing, the development of safe and effective antimicrobial agents is essential for the treatment of plant disease.
Plants contain chemically diverse compounds that can be used directly as pesticides with reduced harmful effects [8]. Globally, the use of botanicals as alternatives to synthetic pesticides has increased annually. In particular, the growing demand for botanical pesticides has been much higher in developed or industrialized countries because of the increase in organic food production [9]. Botanicals have many advantages over synthetic chemicals, such as less or no residues on food because of rapid degradation, little or no harmful effect in humans or on the environment, and cost effectiveness [9]. However, botanical pesticides have some limitations, such as slow and lower efficacy compared with chemical pesticides and less efficacy when applied to fields; thus, the development of novel formulations with enhanced efficacy and longer shelf life is required [10][11][12]. Nowadays, nanotechnology has been applied in formulating, thus plant parts such as fruit, leaf, bark, seed, and stem extracts have been used for synthesis of nanoparticles, as effective formulations for the phytopathogens control [12]. There are many botanical pesticides such as plant extracts and their compounds possess antibacterial activities against phytopathogenic bacteria including R. solanacearum [12]. Most current studies that have focused on botanicals for the control of R. solanacearum utilized in vitro assays or potted plants [13][14][15][16]. To the best of our knowledge, there are no commercialized botanical products currently available for the control of bacterial wilt of tomato. Therefore, the search and development of highly active botanical pesticides for the control of tomato bacterial wilt are quite necessary.
In the search for antibacterial agents from Vietnamese plants, we found that the methanol extract of aerial parts of Sapium baccatum was highly active against R. solanacearum. S. baccatum is widespread across South Asia and has been used as a traditional medicine in Malaysia [17,18]. Although several compounds, including bukittingine, lupeol, betulin, β-taraxerol, taraxerone, aleuritolic acid, 3-acetoxy-aleuritolic acid, 1-hexacosanol, β-sitosterol, stigmasterol, docosyl trans-isoferulate, and docosanoic acid 2',3'-dihydroxypropyl ester have been extracted from S. baccatum [17][18][19], limited information is available about the antimicrobial activities of these components or whether S. baccatum has additional active compounds. The aims of this study were: 1) to isolate and identify antibacterial compounds from S. baccatum; 2) to examine the antibacterial spectra of the isolated compounds against plant pathogenic bacteria; and 3) to evaluate the disease control efficacies of the methanol extract of S. baccatum on bacterial wilt of tomato under greenhouse conditions.

Bacterial strains and culture conditions
The following strains of plant pathogenic bacteria were used for antibacterial activity assays: Acidovorax avenae subsp. cattleyae SL4351, the causal agent of bacterial brown spot in Phalaenopsis (Phalaenopsis sp.); Agrobacterium tumefaciens SL2434, the causal agent of crown gall in apple (Malus domestica); Burkholderia glumae SL4269, the causal agent of bacterial panicle blight in rice (Oryza sativa); Clavibacter michiganensis subsp. michiganensis SL4135, the causal agent of bacterial wilt and canker in tomato (Solanum lycopersicum); Pectobacterium carotovorum subsp. carotovorum SL290, the causal agent of bacterial soft rot in potato (Solanum tuberosum); Pectobacterium chrysanthemi SL3218, the causal agent of bacterial leaf rot in aloe (Aloe vera); Pseudomonas syringae pv. actinidiae CJW7, the causal agent of bacterial canker in kiwifruit (Actinidia deliciosa); Pseudomonas syringae pv. lachrymans SL308, the causal agent of cucumber angular leaf spot (Cucumis sativus); R. solanacearum SL1944, the causal agent of bacterial wilt in tomato (S. lycopersicum); and Xanthomonas arboricola pv. pruni SL4370, the causal agent of bacterial spot of peach (Prunus persica). All of these bacteria were isolated from the infected tissues by Dr SD Lee of the National Academy of Agricultural Sciences [4], except for P. syringae pv. actinidiae CJW7, which was isolated by Prof. YJ Koh of Sunchon National University [20] and R. solanacearum, which was isolated by Prof. SW Lee of Dong-A University [4,21]. All strains were grown on tryptic soy agar (TSA; Becton, Dickinson and Co., Sparks, MD, USA) or tryptic soy broth (TSB). P. syringae pv. actinidiae and X. arboricola pv. pruni were grown at 25˚C for 18-36 h and all other strains were cultured at 30˚C for 18-36 h.

Plant material
The aerial parts of S. baccatum were collected by the Department of Phytochemistry, Vietnam Institute of Industrial Chemistry (Hanoi, Vietnam). Plant species were identified by Dr The Bach Tran from the Institute of Ecology and Biological Resources (Hanoi, Vietnam) and voucher specimens were deposited in the laboratory.

Extraction and isolation of antibacterial compounds
The dry powdered material of S. baccatum (200 g) was extracted twice with 90% methanol (2 × 3 L) for 48 h at room temperature. The extracts were filtered through Whatman No. 1 filter paper and the filtrates were concentrated by using a rotary evaporator under vacuum to yield a crude extract (24.5 g). A portion of the methanol extract (15 g) was suspended in 500 mL distilled water and then successively partitioned twice with n-hexane, ethyl acetate (EtOAc), and n-butanol (BuOH). Of the four layers, the EtOAc and BuOH layers showed strong antibacterial activity against R. solanacearum as determined by the broth microdilution method [4]. Therefore, further isolation of active compounds was conducted from these two layers.

Structure determination of antibacterial compounds
The chemical structures of the active components were determined by using mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and comparison with values reported in the literature. The electrospray ionization mass spectra (ESI-MS) of the isolated compounds were recorded on an MSD1100 single-quadruple mass spectrometer equipped with an electrospray ionizer (Hewlett-Packard Co., Palo Alto, CA, USA). 1 H and 13 C NMR spectra were measured using methanol-d 4 , dimethyl sulfoxide (DMSO)-d 6 or acetone-d 6 (E. Merck) with a Bruker AMX-500 spectrometer (Bruker, Analytische Messtechnik Gmbh, Rheinstetten, Germany). Chemical shifts were calculated using tetramethylsilane as the internal standard.

In vitro antibacterial activity
The antibacterial activities of the isolated compounds (compounds 1-7) were measured with the broth microdilution method against various plant pathogenic bacteria, including R. solanacearum. Briefly, suspensions of plant pathogenic bacteria in TSB media (100 μL) with inocula of 10 6 CFU/mL were added to the wells of sterile 96-well plates. The stock solutions of the isolated compounds (25 mg/mL) were diluted 100-fold in the first wells and then subjected to two-fold serial dilutions in the growth media. The final concentrations of the isolated compounds were in the range 7.8-250 μg/mL. DMSO (1%), which corresponded to the highest concentration and did not affect the bacterial growth, was used as the negative control. Streptomycin sulfate was used as the positive control.
The inoculated plates were incubated at 30˚C (except for P. syringae pv. actinidiae and X. arboricola pv. pruni, which were incubated at 25˚C) for 18-36 h after shaking at 300 rpm for 10 min on a microplate shaker. The minimum inhibitory concentration (MIC) was defined as the lowest concentration that completely inhibited the growth of the bacteria. The assay was performed three times with three replicates for each extract at all concentrations tested.

Disease control efficacy of the methanol extract of S. baccatum against bacterial wilt of tomato
To evaluate the efficacy of the methanol extract of S. baccatum in controlling tomato bacterial wilt caused by R. solanacearum SL1944 (race 1, biovar 4) [4,21], we used 3-week-old 'Seokwang' tomato plants at the four-to five-true-leaf stage. The plants were grown in vinyl pots with a volume of 90 mL in a greenhouse and were then transplanted into vinyl pots with a volume of 180 mL (one plant per pot). Two different amounts of starting material (100 and 200 mg) of the methanol plant extract were dissolved in 2 mL MeOH and then diluted in 98 mL distilled water containing 250 μg/mL Tween-20 to obtain test concentrations of 1000 and 2000 μg/mL. R. solanacearum was grown in TSA Petri dishes at 30˚C for 48 h, harvested with distilled water, and adjusted to an optical density at 600 nm of 0.1 (approximately 1.5 × 10 8 CFU/mL). The two methanol extract solutions were applied to the soil of each pot (20 mL per pot). After 3 h of treatment, a cell suspension (20 mL) of R. solanacearum was inoculated into the soil of each pot. Streptomycin sulfate (200 μg/mL) was used as the positive control and distilled water solutions containing Tween-20 (250 μg/mL) and MeOH (2%) were used as negative controls. The plants were maintained in a controlled climate at 30 ± 2˚C and a relative humidity of 70-80%. The pots were arranged as a randomized complete block with five replicates per treatment. The experiment was repeated three times [4,22].
The disease severity was ranked daily for 14 days and recorded on a scale of 0-4 as described by He et al. (1983): 0, no symptoms; 1, one leaf wilted; 2, two or three leaves wilted; 3, four or more leaves wilted; and 4, plant dead [4,23]. The control value was calculated by using following formula [22]: Control value ð%Þ ¼ 100 Â ðdisease severity of control À disease severity of treatmentÞ=disease severity of control

Statistical analysis
The data were assessed by one-way analysis of variance (ANOVA) and the significance of the treatments was determined by Tukey's honest significant difference (HSD) for multiple comparisons (p = 0.05). Statistical analyses were performed by using SAS software (version 12.0, SAS Institute, Cary, NC). Differences were considered statistically significant for p values less than 0.05.

In vitro antibacterial activity
The antibacterial activity of the compounds isolated from S. baccatum is presented in Table 1. Among the seven compounds, methyl gallate (compound 2) exhibited the strongest broadspectrum activity against most of the plant pathogenic bacteria tested, with MIC values between 26.0 and 250 μg/mL (except against A. avenae subsp. cattleyae). Of the glucoside gallates (compounds 3-6), corilagin (compound 3) and chebulagic acid (compound 5) showed a slightly stronger activity than that of tercatain (compound 4) and chebulinic acid (compound 6), respectively. Gallic acid was less active than other hydrolysable tannins (compounds 2-6) and quercetin 3-O-α-L-arabinopyranoside (compound 7) was the least active compound (Table 1).
Methyl gallate (compound 2) was much more active than gallic acid (compound 1). The measurements of antibacterial activity in our study agreed with those of previous reports [4,37,38]. Compounds with similar structures, such as 3 and 4, and 5 and 6, had similar antibacterial activity and spectra (Table 1). Corilagin (compound 3) has been reported against Acinetobacter baumannii [39], methicillin-resistant Staphylococcus aureus [40,41], and Escherichia coli [41]. Chebulagic acid (compound 5) and chebulinic acid (compound 6) showed moderate antibacterial activity against A. baumannii [39]. The antibacterial activity of quercetin-3-O-α-L-arabinopyranoside or guaijaverin (compound 7) against Streptococcus mutans has been reported [36]. To the best of our knowledge, information on the antibacterial activities of tercatain (compound 4) is unavailable. In addition, this is the first report of the antibacterial activities of the isolated compounds (with the exception of compounds 1 and 2) against plant pathogenic bacteria [4,42].

Effect of the methanol extract on tomato bacterial wilt
In the in vivo experiment, wilt symptoms were observed 5 days after inoculation. The extracts efficiently suppressed the development of tomato bacterial wilt in a dose-dependent manner. At concentrations of 1000 and 2000 μg/mL, the methanol extract showed control efficacies of 100 and 100% after 7 days of inoculation, and 63 and 83% after 14 days of inoculation, respectively. The disease control efficacies of the extract at 1000 μg/mL were higher than those of 200 μg/mL streptomycin sulfate after 7 and 14 days of inoculation (Fig 2 and S6 Table). No phytotoxic symptoms appeared on the treated plants.
Recent studies revealed that several plant-derived products also exhibited potential antibacterial activity against R. solanacearum in in vivo tests. Allium fistulosum extract at concentrations of 50 and 100% significantly reduced the incidence of bacterial wilt of tomato: only 6 and 14% of the plants were affected, respectively, whereas the disease affected 61% of the plants in the untreated control [43]. The leaf extract of Eichhorina crassipes reduced the severity index of the bacterial wilt by more than 91% [44]. Several essential oils, such as cinnamon and clove oils [13], lemongrass and palmarosa oils and their components such as thymol [45,46], were found to effectively reduce the R. solanacearum populations and incidence of bacterial wilt of tomato grown in infested soil. Methyl gallate at a concentration of 500 μg/mL showed a control efficacy of 65.2% in greenhouse conditions [42]. It is difficult to compare the results of our study with those of previous studies owing to a number of factors, such as different experimental design conditions, plants species, and bacterial species. In this study, the control efficacy of S. baccatum at a concentration of 2000 μg/mL was similar to that of the wettable powder formulation of the ethyl acetate layer of S. takesimense at a 200-fold dilution, as described in our previous study [4].

Conclusions
In this study, seven antibacterial compounds were isolated from the methanol extracts of S. baccatum. The compounds showed potent in vitro antibacterial activities against R. solanacearum, except for quercetin 3-O-α-L-arabinopyranoside. In addition, most compounds exhibited strong antibacterial activities against X. arboricola pv. pruni and B. glumae. Methyl gallate and corilagin showed the strongest activities. These results suggested that the extracts from S. baccatum or their isolated compounds were promising antibacterial agents for the control of bacterial wilt of tomato.
Botanical pesticides are a promising alternative to reduce the harmful effects caused by use of synthetic pesticides. They have become more attractive after the increase in demand for organic food. Further research into production, formulation, and delivery may greatly assist and promote the development of botanical pesticides. Therefore, further studies are necessary to examine the toxicity of the S. baccatum extract, evaluate the disease control efficacy of the extract in various fields, and develop optimum formulations of the crude extracts for the control of tomato bacterial wilt. Supporting information S1 Table. NMR data of gallic acid and methyl gallate isolated from Sapium baccatum in methanol-d 4 .