In Vitro Antibacterial Activity and Mode of Action of Piper betle Extracts against Soft Rot Disease-Causing Bacteria

Soft rot disease affects a range of crops in the field and also during transit and storage, resulting in significant yield losses and negative economic impacts. This study evaluated the in vitro antibacterial activities and mode of action of Piper betle extracts against the soft rot disease-causing bacteria, Erwinia caratovora subsp. caratovora (ECC). Dried leaves of P. betle were extracted with water, ethanol, and hexane solvents and evaluated for their antibacterial activity. The results showed the highest antibacterial activity against ECC in the ethanol extract, followed by hexane and water extracts with minimum inhibitory concentration (MIC) 1.562, 6.25, and more than 12.50 mg/mL, respectively. The time-kill assay indicated a bactericidal mode of action. ECC growth was destroyed within 6 and 8 hours after treatment with the ethanol extract at 4-fold MIC and 2-fold MIC, respectively. The ethanol extract of P. betle showed promising activity against ECC, with the potential for further development as a novel alternative treatment to control phytobacteria.


Introduction
Bacterial soft rot is one of the most serious global plant diseases that afects crops both in the feld and during transit and storage.Tis disease devastates a wide variety of crops including potato [1,2], dragon fruit [3], cabbages [4], cucumbers [4], and tomato [5], with an estimated 15-30% reduction in agricultural production per annum [4].
Bacteria associated with soft rot disease have been extensively studied due to their huge negative economic impact.Erwinia caratovora subsp.caratovora (ECC), a Gramnegative pectolytic bacteria, belongs to the Pectobacteriaceae family and causes softening, wetting, and rotting of internal plant tissues.Tis pathogen spreads through drain water, sprinkler irrigation, and also manually.It can survive on feld weeds and plant debris for a long time, resulting in difculties in prevention and control [1,4].Te pathogen also spreads during crop transportation and storage.ECC accesses plant tissues through wounds and natural openings such as stomata and, subsequently, produces accumulative amounts of plant cell wall-degrading enzymes such as cellulase, protease, pectate lyase, polygalacturonase, and pectinmethylesterase. Tese enzymes then degrade plant cell walls, leading to extensive plant tissue maceration and the development of infection.Symptoms have similar appearances on each host, beginning with water-soaked lesions that rapidly enlarge to other areas, often accompanied by an ofensive smell [6,7].
Several strategies are employed to prevent soft rot disease such as plant rotation, improvement of farm management, and selection of healthy crops for culture or storage.However, none of these remedies are entirely successful and are both time-and labor-consuming [2].Chemicals and antibiotics are also used to control disease distribution in agricultural felds, but this method is expensive and negatively impacts the environment, while also leading to bacterial resistance and health concerns.Chemicals and antibiotics are also unsuitable and not allowed for organic agriculture [8].Terefore, alternative sustainable methods to control ECC are required.Medicinal plants have long been used to manage microbial diseases with promising results.Tese plants are inexpensive, have reduced side efects, and cause less environmental pollution.Many studies have shown that medicinal plants contain a broad spectrum of constituents to inhibit bacteria, fungi, and yeast which cause human, animal, and plant diseases [9][10][11].
Piper betle, commonly known as betel vine, belongs to the Piperaceae family.In Tailand, it is called Phu and is widely distributed as a popular ethnomedicine in many Asian countries.P. betle leaves are used in traditional medicine to cure skin conditions, treat oral and dental problems, headaches, arthritis, and joint discomfort as well as a mouthwash to curb bad breath.Traditional uses of P. betle leaves relate to their antibacterial ability [12][13][14].Several investigations have revealed additional drug-related properties of P. betle including antioxidant, anti-infammatory, antimalarial, antidiabetic, and gastro-and hepatoprotective activities [12,[14][15][16].Although few studies have assessed the efect of P. betle extract on the control of phytopathogenic bacteria and fungi.
Tus, this study examined the in vitro antibacterial activity of P. betle extracts and the mode of action against ECC.Te knowledge gained can be used to promote a safe alternative treatment for ECC management.

Materials and Methods
2.1.Plant Material and Extraction.Leaves of P. betle as 500 g samples were randomly collected from three areas in Srisaket Province, Tailand.A single composite sample was prepared by combining 500 g of samples for each representative area and then homogenizing to obtain a uniform single composite sample.Te sample was dried in a hot air oven at 60 °C for 3 days before milling.Ground samples were extracted with water, ethanol, and hexane at a ratio of 1 : 10 (sample: solvent) on an orbital shaker at 150 rpm for 24 hours [17].Te extracts were fltrated, evaporated at 60 °C by a rotary evaporator, and then kept at 4 °C for anti-ECC activity testing.

Agar-Disc Difusion Method.
Te ECC strain used in this study was obtained from the Department of Plant Production Technology, Faculty of Agricultural Technology, Kalasin University, Tailand.Dried extracts were dissolved in dimethyl sulfoxide (Loba, India) at a concentration of 50 mg/mL.Dimethyl sulfoxide was used as a cosolvent to dissolve dried extract because of its destring property.It enhances compound solubility as it is miscible with water and organic solvents, and it is not toxic to the test organism [18].Te agar-disc difusion method was performed for anti-ECC activity screening according to the method described by Charirak and Ratananikom (2022) with some modifcations [17].Briefy, sterile blank discs 6 mm in diameter were placed on nutrient agar plates covered with 100 μL of ECC.Ten microliters of each extract were allowed to penetrate through the sterile discs into the ECC.Te plates were then incubated at 30 °C for 24 hours.Te determination of antibacterial activity was performed as fve replicates.Te inhibition zone diameter of each extract against ECC was measured in millimeters.Dimethyl sulfoxide was used as a negative control and kanamycin (30 μg/disc) (Termo Scientifc, England) was used as a positive control [19].

Minimum Inhibitory Concentration.
Te minimum inhibitory concentration (MIC) of each extract was evaluated by the resazurin microtiter assay [20] because the resazurin microtiter assay is more sensitive than colorimetric assay such as the MTTassay and XTTassay in measuring cell viability [21].One hundred microliters of nutrient broth were pipetted into each well of a 96-well plate.A two-fold dilution was performed to prepare the extracts in various concentrations.One hundred microliters of ECC culture were added to the mixture of the extracts.Te 96-well plates were incubated at 37 °C for 24 hours, and then 30 μL of 0.02% resazurin was added.Te plates were further incubated at 37 °C for 16-18 hours to obtain the MIC, defned as the lowest concentration of extract with no color change of resazurin, indicating the lowest concentration of P. betle extract that inhibited microbial growth [20].

Time-Kill Assay.
Te ethanol extracts were subjected to time-kill curve analysis following Su et al. (2015) with some modifcations [22].An inoculation of 10 6 CFU/mL of ECC culture, harvested from a colony grown overnight, was mixed with ethanol extracts at fnal concentrations of 2 and 4-fold MIC.One hundred microliters of the mixture were taken at 0, 2, 4, 6, 8, 10, and 12 hours, serially diluted in nutrient broth, and then plated on nutrient agar.After 24 hours of incubation, the number of colonies was counted to determine the total number of visible bacteria.All procedures were performed in triplicate, and a graph of log 10 (CFU/mL) was plotted against time.Growing cells of ECC in nutrient broth without the addition of ethanol extract was used as the control.

Statistical Analysis.
Te data was expressed as the mean ± standard deviation (S.D.).Statistix version 8 was used for statistical analysis.Statistical analysis was carried out using one-way analysis of variance (ANOVA), with diferences between means calculated using the least signifcant diference (LSD).In all cases, p < 0.05 was considered signifcant. 1 show the results of anti-ECC screening by agar-disc difusion.Anti-ECC activities showed diferent degrees of efciency in all extracts.Kanamycin as the positive control gave the highest anti-ECC activity with an inhibition zone of 17.72 ± 0.15 mm, while dimethyl sulfoxide as the negative control showed no 2 Scientifca inhibitory efect against ECC.Te ethanol extract gave the second most efcient anti-ECC activity (8.48 ± 0.69 mm), followed by the hexane and water extracts at 7.89 ± 0.74 and 7.07 ± 0.74 mm, respectively.

MIC Determination.
Table 2 demonstrates the MICs of P. betle extracts against ECC.Te MIC results showed different values and agreed with those from antibacterial screening by agar-disc difusion.Te ethanol extract exhibited the highest activity against ECC with the lowest MIC at 1.562 mg/mL, followed by the hexane extract with a MIC 6.250 mg/mL.Conversely, the highest concentration of water extract showed no inhibitory efect toward the ECC culture.

Mode of Action of P. betle Extracts against ECC.
Ethanol extracts of P. betle at concentrations of 2-fold MIC and 4-fold MIC were used to study its mode of action.Te growth curve of ECC increased continuously over time, while a remarkable decline was found with incubation of ECC with the ethanol extracts.ECC viability was completely destroyed within 6 and 8 hours after incubation with the ethanol extracts at concentrations of 4-fold MIC and 2-fold MIC, respectively (Figure 2).

Discussion
Herbal extracts are a great alternative to harmful chemicals and a safe, eco-friendly way to prevent plant diseases [14,16].Te identifcation of an efcient plant extract against ECC was conducted.Relatively few botanical extracts have been reported to control soft rot pathogens, while numerous studies have shown the antibacterial action of herbal extracts against many other bacterial pathogens [23,24].Results demonstrated that the ethanol extract of P. betle signifcantly inhibited the growth of soft rot bacteria in vitro.Te MIC of the ethanol extract was 1.562 mg/mL, and it behaved in a bactericidal manner.Interestingly, the time-kill kinetics of the ethanol extract were dose-dependent.ECC were completely destroyed within 6 and 8 hours when utilizing ethanol extracts at concentrations of 4-fold MIC and 2fold MIC, respectively.Tis result concurred with previous studies indicating that plant crude extracts had bactericidal or bacteriostatic properties.Te mode of action of the antibacterial agents was identifed using the MBC/MIC ratio or time-kill kinetic [17,25,26].If the ratio of MBC/MIC is ≤4, it is defned as a bactericidal agent, with a bacteriostatic mode of action considered when the ratio is ≥4.In this study, the bactericidal efect of the ethanol extract of P. betle was proved by the time-kill assay as a bactericidal agent.Tis fnding was corroborated by earlier studies revealing that essential oil from P. betle showed only a bactericidal efect while P. betle extracts were both bacteriostatic and bactericidal [14,[27][28][29].Diferences in mode of action among P. betle extracts were attributed to several factors including type of solvent, extraction method, type of indicator strain, dose of the extracts, and phytochemical constituents in the extracts [15].Signifcant anti-ECC activity of P. betle occurred a result of the synergistic efect of the antibacterial components found in the ethanol extract.One beneft of employing crude plant extracts to suppress pathogenic bacteria is that numerous active ingredients provide a variety of antibacterial actions.A combination of active ingredients is difcult for bacteria to resist; however, synthetic antibacterial agents typically cause bacteria to evolve resistant mechanisms [17].Our results indicated that the active constituents against ECC growth found in P. betle showed moderate hydrophilic properties.Te most suitable extraction solvent for obtaining the highest anti-ECC property was ethanol, with a polarity less than that of water but higher than that of hexane.Tis result indicated that the extraction solvent also plays an important role in the isolation of active components from plants and was supported by earlier studies that determined organic solvents to be typically more efective at extracting antimicrobial compounds than aqueous-based techniques [14,17].Ethanol extracts from P. betle also demonstrated excellent antibacterial properties over Gram-positive and Gram-negative bacteria, including those classifed as multidrug resistant such as metallo-β-lactamase-producing P. aeruginosa and A. baumannii, MRSA, and VRE [14].Some phytogenic bacteria and fungi are also inhibited by the extract of P betle such as Xanthomonas axonopodis p.v. malvacearum [24], X. axonopodis pv.vericatoria [24], Xanthomonas oryzae [24], Xanthomonas campestris p.v. campestris [24], Fusarium oxysporum [30], Plasmopara viticola [23], and Candida albicans [31].Te ethanol extract of the P. betle showed a broad spectrum toward pathogenic microbes.Te exact chemical constituents in the ethanol extract were not evaluated in this study, although signifcant chemical components have previously been identifed in P. betle extracts.Aoki et al. (2019) [23] examined the inhibitory efect of methanol extract from P. betle leaves on grape downy mildew.Teir results showed that the methanol extract suppressed grape downy mildew pathogens, and they identifed four components in the methanol extract including 4allylpyrocatechol, eugenol, α-pinene, and β-pinene.Singtongratana et al. (2013) [32] recorded major constituents in P. betle extract as hydroxychavicol or allypyrocatechol.Tese chemical components have been reported for antimicrobial activity against several strains, causing plasma membrane damage, coagulation of nucleoid, plasma membrane permeability alteration, and proton pumping inhibition [14,15,31,33,34].Terefore, these components may be found in the ethanol extract due to the relatively similar polarity of the extraction solvent used in this study, leading to ECC death.
P. betle is ubiquitously found in Tailand and the leaves are easily accessible and cost-free.Te decomposition of P. betle leaves also contributes to increased soil fertility.Terefore, employing P. betle leaf extract has no negative phytotoxin impacts on plants.Te success in eliminating ECC provides fresh information regarding the most efective way to use P. betle extract to manage soft rot disease.

Conclusion
P. betle showed the potential for antimicrobial application in vitro.Its ethanol extract demonstrated a bactericidal mode of action to completely inhibit the ECC, soft rot diseasecausing bacteria.It could be said that this extract can be used as an alternative to chemical bactericides in organic agriculture.

Table 1 :
Inhibition zone diameters of P. betle extracts against ECC.