Antibacterial activity of medicinal plant extracts against Ralstonia solanacearum ( Smith ) that causes bacterial wilt in hot pepper ( Capsicum annuum L . )

This study was conducted to evaluate the in vitro antibacterial activity of some medicinal plants against Ralstonia solanacearum. Bioactive chemicals were extracted from Burcea antidysenterica, Eucalyptus citriodora, Justicia schimperiana, Lantana camara, Melia azedarach and Ricinus communis leaves using maceration method. The bioassay was evaluated by disc diffusion method. The pathogen was isolated from infected Capsicum annuum plants using Casamino acid-Peptone-Glucose agar (CPG) medium. The isolate was identified using cultural, biochemical characteristics, pathogenicity test and found to be R. solanacearum. The methanol extracts had different composition, percentage extract yield, antibacterial activity and relative percentage inhibition. Unlike others, extracts of E. citriodora and R. communis consisted of all the tested secondary metabolites. All species showed antibacterial activity except M. azedarach. Significant differences were recorded in antibacterial activity among species and test concentrations. The highest antibacterial activity and the lowest bacteriostatic and bactericidal concentrations were found from E. citriodora and R. communis extracts. The higher potency of E. citriodora and R. communis extracts suggested the potential of the two species as a biocide to control bacterial wilt. However, further in vivo studies on these species extracts are compulsory.


Introduction
Hot pepper (Capsicum annuum L.) is one of the cultivated species of the genus Capsicum in the family Solanaceae.The genus originated in Central and South America (Grubben & Denton, 2004).The combination of four lines of evidence indicated that domestication of C. annuum could have occurred in Mexico (Kraft et al., 2014).The Spanish and Portuguese have brought capsicum pepper to Europe, from where especially hot pepper was widely spread to all tropical and sub-tropical areas of the world (Grubben & Denton, 2004).C. annuum has been grown as a popular vegetable and spice ubiquitous in the tropics and many very distinct types and landraces had been developed at the end of the 17 th C (Grubben & Denton, 2004).
Hot pepper is cultivated so widely in Africa that African people consider it as a traditional African vegetable or spice (Grubben & Denton, 2004).Similarly, the history of hot pepper in Ethiopia is perhaps the most ancient than the history of any other vegetable product and established as a national spice in the country.It has been grown extensively under wide range of environmental conditions, from lowlands to 3000 meters above sea levels (Grubben & Denton, 2004;Yadeta, Belew, Gebreselasie, & Marame, 2011).The crop is a warm season crop requiring an optimum day and night temperatures of 20-30 and 15-20°C, respectively.Hot pepper grows better on loam or sandy loam soil with a pH of 6-7.It is grown as a rainfed crop in areas receiving 850 -1200 mm of annual rainfall (Zeleke & Derso, 2015).Evidences showed that the world's average yield of hot pepper is 1.7 tons ha -1 for dry pod and 16.3 tons/ha for green pod (Food and Agriculture Organization of the United Nations Statistic [FAOSTAT], 2012).In the same cropping season, the average yield of hot pepper dry and green pod was found to be 2.3 and 8.1 tons/ha, respectively (Central Statistical Agency of Ethiopia [CSA], 2007).The average green pepper yield decreased from 6.5 to 6.2 Acta Scientiarum.Biological Sciences, v. 41, e45402, 2019 tons/ha from 2016 to 2017.Similarly, the average red pepper yield has been reduced from 1.8 tons/ha in 2016 to 1.7 tons/ha in 2017 (Central Statistical Agency of Ethiopia [CSA], 2010).
In Ethiopia, hot pepper has nutritional, medicinal and economic values.Such importance of the vegetable is entrenched in the chemical composition of the fruits (capsaicin and oleoresin).Nutritionally, hot pepper is produced as spice flavoring and color to food while providing essential vitamins and minerals (Shumeta, 2012;Zeleke & Derso, 2015).It is one component of the daily diet of Ethiopian people.The average daily consumption of hot pepper by an Ethiopian adult is estimated to be 15 g, which is higher than tomatoes and most other vegetables (Marcelis, Heuvelink, Hofman-Eijer, Bakker, & Xue, 2004).It is used in different ways and forms.The fine powdered pungent product is a necessary flavoring and coloring ingredient in the common traditional sauce 'Wot' while mature green pods ('karia') are eaten as salads (Shumeta, 2012).Moreover, hot pepper is rich source of soluble and insoluble fibers, vitamins (C, niacin, A, E, K and folic acid) and minerals (calcium, sodium, magnesium, phosphorus and copper) (Grubben & Denton, 2004;Emmanuel-Ikpeme, Henry, & Okiri, 2014).Ethiopians believe that a person who frequently consumes hot pepper has resistance to various diseases (Shumeta, 2012).This is attributed to the chemical composition of hot pepper fruit in that it contains higher amount of tannins, flavonoid, saponin, terpenoid and carotenoids as compared to other species of capsicum (Emmanuel-Ikpeme et al., 2014).Besides, capsaicin has shown anti-oxidant, antimutagenic, anticarcinogenic and immunosuppressive activities.It also inhibits bacterial growth and platelet aggregation (Grubben & Denton, 2004).It is an important cash crop for small holder farmers that generate income both in domestic and export markets (Lin et al., 2013).It also creates employment to urban and rural workers (Shumeta, 2012).
Despite its importance, the production of hot pepper in Ethiopia has shown a progressive decrease through years.This is caused by several abiotic and biotic factors (Welderufael, 2016).One of the major constraints that contribute to the low productivity of hot pepper is bacterial wilt disease, caused by a soil borne bacterium R. solanacearum (Smith) (Yabuuchi, Kosako, Yano, Hotta, & Nishiuchi, 1995).The pathogen is the most destructive disease of the crop.It is xylem inhabiting that clogs the tissue and blocks water and mineral transport in the plant.This results in rapid wilting especially young and succulent plants.
The first visible symptom appears on the youngest leaves followed by stunting, downward curling of leaflets and petioles (Singh, Singh, Kumar, & Birah, 2014).Currently, the common management strategies include mechanical removal of infected plant materials and the application of bactericidal chemicals.However, the continuous use of chemicals results in indiscriminate damage and accumulation of harmful residues in the soil, water and grains (Grubben & Denton, 2004).This shows the need to investigate alternative, ecofriendly, biodegradable and easily accessible disease controlling strategies.Thus, this study was aimed to evaluate the in vitro antibacterial activity of methanol leaf extracts from Burcea antidysenterica, Eucalyptus citriodora, Justicia schimperiana, Lantana camara, Melia azedarach and Ricinus communis and characterize the chemical composition of the extracts.

Plant materials
Medicinal plants were selected on the basis of their traditional use in treating human ailments and antimicrobial activity reports in the literature.Accordingly, fresh leaf samples of B. antidysenterica (JF.Mill), E. citriodora (Hook), J. schimperiana (Hochst.ex Nees) T. Anderson, L. camara L., M. azedarach L. and R. communis L. were collected from Bahir Dar city, Ethiopia and the surrounding areas and brought to the laboratory.The leaf samples were washed with running tap water to remove dust and other debris.The sample was dried to constant weight under shade at room temperature.The dried leaf samples were milled to fine powder using an electrical grinder.The powder was sieved through 0.5 mm mesh and stored in polythene (plastic) bags.The bags were tightly closed and preserved at 4°C until needed for extraction.

Extraction and phytochemical of screening of methanol extracts
The bioactive ingredients of medicinal plant leaf samples were extracted using maceration method as described in Rai, Pai, Kedilaya, and Hegde (2013).Methanol was used for extraction.The leaf powder of each species and solvent were added into separate conical flasks in the ratio of 1:10 (for every gram of leaf powder, 10 mL of solvent).The flasks were tightly closed and placed on an orbital shaker at a speed of 170 rpm.The content of each flask was shaken for 72 hours under room temperature.The extract of each species was decanted to new flasks in the presence of four layers of cheese cloth and cotton.The residue was squeezed to the flasks and discarded.The crude extract was further passed through Whatman no. 1 filter paper.The solvent was evaporated using rotary evaporator.Dried extracts of each species were stored at 4ºC until used for antibacterial testing.Extract yield (%) was determined gravimetrically using the dry weight of the extract and the initial weight of the leaf powder as follows: Extract yield (%) = * 100 Qualitative analysis was performed to confirm the presence or absence of major secondary metabolites in the methanol leaf extracts.Phytochemical screening was carried out for phenolic compounds, terpenoids, tannins, flavonoids, alkaloids and saponins using standard chemical methods as described in Mendonça-Filho (2006) and Ayoola et al. (2008).

Isolation and identification of R. solanacearum
Infected plant samples were collected from one of the major hot pepper growing areas, in Mankusa, Amhara Regional State.Sampling sites were selected based on number of hot pepper plants showing wilting symptoms.Accordingly, a site with wider prevalence and higher severity was used for sample collection.Infected plants were uprooted from these sites and separately put in polythene bags and thoroughly washed with water to remove soil particles and other debris.The collected samples were preserved in an ice box and transported to Bahir Dar University for further processing.
The collected infected hot pepper plants were surface sterilized by soaking in 5% sodium hypochlorite solution.These materials were rinsed three times in sterilized distilled water.Infected plant parts were cut into small pieces and macerated with mortar and pestle in the presence of 0.85% NaCl sterile saline solution.Serial dilutions were prepared from the bacterial suspension and inoculated to separate Petri plates poured with CPG growth medium.The Petri plates were incubated at 28 ± 1°C for 72 hours.After this period, suspension was prepared from isolated white creamy colonies and stored in screw caped vials at 4ºC.This suspension was inoculated to separate Petri plates filled with CPG growth medium.Sub culturing was made until pure culture of R. solanacearum is found.The isolate was regularly renewed by plating on CPG medium.The pathogen was identified based on colony characteristics, biochemical and pathogenicity tests.
Different biochemical tests were performed to characterize R. solanacearum.The Gram staining test was performed as described by Schaad (1980) and confirmed that the bacterium isolated was Gram negative.R. solanacearum colonies were aseptically removed from Petri plates with an inoculating wire loop and put on glass slide in a drop of 3% KOH solution.This was stirred for 10 seconds and observed for the formation of slime threads (Suslow, Schroth, & Isaka, 1982).
Catalase test was carried out by mixing a loop full of a fresh bacterial culture with two drops of solution of 3% H 2 O 2 on a microscope slide according to the method described by He, Sequeria, and Kelman (1983).Bubble formation indicated a positive test for catalase.
Nutrient agar plates containing 0.2% soluble starch (w v -1 ) were streaked with R. solanacearum suspension and incubated at 28 ± 1ºC until heavy growth occurred.Then the plates were flooded with KI solution.A clear zone around a colony was record as positive reaction for starch hydrolysis (Sands, 1990).

Pathogenicity test
The pathogenicity test was carried out according to Mimura, Yoshikawa, and Hirai (2009).Seeds of hot pepper were sown and grown in plastic buckets for three months and half under glasshouse conditions.The bucket soil was infested by pouring 10 mL of R. solanacearum suspension containing 1.5 x 10 8 CFU mL -1 around the root.Plants in the control were irrigated with equal volume of sterilized water.The development of disease symptoms was monitored for 6 weeks for every other day in the greenhouse.Plants with at least one wilted leaf were classified as wilted (Figure 1).The pathogen was re-isolated from infected plants using CPG agar medium.The colonies were compared with the initial colonies and found to be similar.

Antibacterial activity assay
The antibacterial activity of methanol leaf extract of selected medicinal plants against R. solanacearum was tested in vitro using disc diffusion method.Serial test concentrations (100, 50, 25, 12.5, and 6.25 mg mL -1 ) of each species extract were prepared first by dissolving the dry extract in 2 mL of methanol and then adding one mL sterilized distilled water to reach to the required test concentration.Discs of 5 mm in diameter were prepared from Whatman no. 1 filter paper using paper borer.These discs were impregnated to each test concentration for 24 hours.Then after, the discs were taken out from the extract and dried under aseptic condition.
Ralstonia solanacearum suspension was prepared from 72 hours old pure culture of R. solanacearum.Sterilized distilled water was used to prepare the suspension.The optical density of the suspension was adjusted to 0.132 at 600 nm using a spectrophotometer (Jenway, Models 7310 & 7315; United Kingdom).This is equivalent to 0.5 McFarland turbidity standards.The number of bacterial population at this turbidity standard is 1.5 x 10 8 CFU mL -1 .Approximately 0.5 mL of the bacterial suspension was aseptically inoculated to CPG medium and uniformly distributed using cotton swab.The dried discs were placed on the inoculated Petri plates within 15 minutes.Sensitive discs of tetracycline at one concentration (30 μg mL -1 ) were used as standard controls.Discs soaked in methanol were used as negative controls.All the Petri plates were incubated at 28 ± 1ºC.Each test concentration was replicated thrice, and the test was performed three times.The antibacterial activities of extracts were evaluated in terms of diameter of zone of inhibition.The inhibition zone was measured using a transparent ruler including the disc.
The minimum inhibitory concentration (MIC) of leaf extracts was determined using agar dilution method as described in European Committee for Antimicrobial Susceptibility Testing EUCAST (2000).One mL of each test concentration of each methanol extract was thoroughly mixed with 19 mL of CPG molten growth medium and poured to Petri plates.The medium was allowed to solidify at room temperature and inoculated with 0.1 mL of R. solanacearum suspension adjusted to 0.5 McFarland turbidity standards and evenly spread.The Petri plates were incubated at 28 ± 1 °C for 72 hours.Petri plates without extracts and the results were compared against these controls.
Minimum bactericidal concentration (MBC) of leaf extracts was determined as described in Njinga et al. (2014).Serial test concentrations of each species extract were prepared.CPG growth medium was prepared and autoclaved at 121°C for 15 minutes.One milliliter of each test concentration was mixed with 19 mL of molten CPG medium and poured to sterile Petri plates.This was allowed to cool and solidify.R. solanacearum suspension was prepared from the MIC Petri plates that did not show growth or showed growth less than 80% of the controls and sub-cultured onto the medium.The Petri plates with their contents were incubated at 28 ± 1 °C for 72 hours.Test concentrations that didn't show bacterial growth were considered as minimum bactericidal concentrations.
The relative percentage inhibition of the leaf extracts with respect to the positive control was calculated as described by Kumar & Arya (2006).

Relative percentage inhibition of the test extract = ( ) ( )
where: X: total area of inhibition of the test extract Y: total area of inhibition of the solvent Z: total area of inhibition of the standard antibiotic.The total area of inhibitions was calculated by using = ; where, r = radius of zone of inhibition.

Statistical analysis
All data were subjected to analysis of variance (ANOVA).This was done after carrying out test of homogeneity and normal distribution of each measured parameter.Mean values were compared with Tukey's Honestly Significant Difference (HSD) test at p < 0.05.All the statistical analyses were computed with SPSS Software for Windows version 20, IBM, NY, USA.

Identification of R. solanacearum
The bacterium isolated from infected C. annum plants was identified using cultural, biochemical characters and pathogenicity test.White creamy colored, irregularly round and non-transparent colonies were formed on CPG medium which matches with the reports of Alemu, Lemessa, Wakjira, and Berecha (2013), Popoola et al. (2015) and Din et al. (2016).According to the Gram staining test, the isolate was found to be Gram negative.This was corroborated by the positive KOH solubility test result, whereby drops of KOH mixed with the isolate suspension produced viscous solution that causes a thin strand of slime when lifted with a wire loop.Moreover, the bacterium showed catalase activity and hydrolyzed starch.The result of the present study agrees with Murthy and Srinivas (2012), Alemu et al. (2013), Popoola et al. (2015) and Kataky, Tamuli, Teron, and Sarma (2017), who reported Gram-negativity, KOH solubility, catalase activity and starch hydrolysis of R. solanacearum.

Extract yield
Plants produce plethora of secondary metabolites with biological activities and thus can be used as source for the development of pesticides (Jeyaseelan, Pathmanathan, & Jeyadevan, 2010).The search for new biocides from plant materials is dependent on the productivity of extraction and the type of solvent.There were variations in percentage extract yield between medicinal plant species (Figure 2).It ranged from 23.75-34%.The highest was obtained from the leaves of E. citriodora followed by R. communis, while the lowest percentage extract yield was found from J. schimperiana.The remaining species had an intermediate percentage extract yield (Figure 2).Maximum extract yield has been reported when methanol is used as a solvent since methanol is the most potent and all-purpose solvent that dissolves most secondary metabolites in plants and enhances the release of these chemicals from cellular matrix (Moteriya, Padalia, Rathod, Menpara, & Chanda, 2014;Gaitonde & Ramesh, 2016).Moreover, the differences in extract yield are also due to the genes that dictate the types of phytochemicals present, while environment such as biotic and abiotic factors influence the expression and relative abundance of secondary metabolites (Moore, Andrew, Kulheim, & Foley, 2014).
The phytochemical analysis showed the presence of different secondary metabolites in the methanol leaf extracts of the studied species.Extracts of E. citriodora and R. communis found to have alkaloids, flavonoids, phenols, saponins, tannins and terpenoids (Table 1).Javed, Shoaib, Mohmood, Mushtaq, and Iftikhar (2012) and Suurbaar, Mosobil and Donkor (2017) have found out similar constituents of E. citriodora (except tannins) and R. communis leaf extracts, respectively.Unlike the present study, alkaloids, tannins and terpenoids are absent from the methanol leaf extract of E. citriodora (Pathmanathan, Uthayarasa, Jeyadevan, & Jeyaseelan, 2010).Alkaloids are also absent in the extract of R. communis (Minakshi et al., 2016).Moreover, extracts of J. schimperiana and L. camara had alkaloids, flavonoids, phenols, tannins and terpenoids (Table 1).Similar compositions of these Acta Scientiarum.Biological Sciences, v. 41, e45402, 2019 species extracts have been reported by Abebe, Kibret, and Haile (2014) and Sousa, Rodrigues, Campos, and Costa (2015).Contrary to this study, Feyera, Deressa, Deyou, and Abdisa (2018) have found alkaloids, flavonoids, phenols, saponins and terpenoids in the methanol leaf extract of M. azedarach.The deviations in the composition of these species extracts might be ascribed to the difference in site of sample collection, analysis methodology and reagent used.

Antibacterial activity assay
The in vitro bioassay studies demonstrated a significant growth inhibition of R. solanacearum.Statistically significant (p < 0.05) variations were observed among test concentrations of each species and between species extract (Table 2).Antibacterial activity was not detected by the negative control.Most species extracts showed a progressive increase in growth inhibition zone in a dose dependent manner.Significantly greater growth inhibition zone was produced by the leaf extracts of E. citriodora at the higher two test concentrations than the remaining concentrations.In R. communis extract, the highest test concentration had remarkably greater growth inhibition than the lower concentrations (Table 2).Similar antibacterial activity of E. citriodora (Pathmanathan et al., 2010;Ghaffar et al., 2015) and R. communis (Suurbaar et al., 2017) leaf extracts has been reported against both Gram negative and positive bacterial pathogens of humans such as Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Aspergillus niger and Rhizopus solani.Moreover, the two upper test concentrations (100 and 50 mg mL -1 ) of L. camara and J. schimperiana revealed significantly higher growth inhibition within a species than the remaining lower test concentrations (Table 2).Alemu et al. (2013) and Revathi, Narayanaswamy, Patil, Naik, and Shindhe (2017) have described moderate growth inhibition of L. camara leaf extract against R. solanacearum.
Furthermore, significant differences were also documented between species at the different test concentrations (Table 2).Accordingly, wider growth inhibition zones were formed by the extracts of E. citriodora and R. communis at all test concentrations as compared to other species (Table 2).The differences among the remaining species extracts were nonsignificant.The methanol leaf extract of M. azedarach did not show growth inhibition against R. solanacearum at all test concentrations, which agrees with the result of Din et al. (2016) against the same pathogen.The same lower-case letters in a row and upper case letters in a column indicate the absence of statistical differences between means at p < 0.05.
The potency of the extracts was also evaluated through bacteriostatic and bactericidal concentrations and found to give different results.Comparatively lower bacteriostatic (MIC) and bactericidal (MBC) concentrations were recorded by the extracts of E. citriodora and R. communis (Figure 3).Opposite to the present study, Lekganyane, Matsebatlela, Howard, Shai and Masoko (2012) and Sousa et al. (2015) have discovered low MIC values of R. communis and L. camara extracts, respectively, against human pathogens.Similarly, MIC values ranging from 3.125-6.25mg mL -1 and MBC from 25-50 mg mL -1 have been reported from methanol leaf extracts of L. camara against different human pathogens (Dubey & Padhy, 2013).The stronger antibacterial activity of E. citriodora and R. communis extracts might be explained by the presence of diverse and abundant number of secondary metabolites.Crude extracts are complex blends of phytochemicals where some have specific role on single target and most interfere with several targets on the pathogen in a pleiotropic manner (Wink, 2008).The constituents of crude extracts have both additive and synergistic bioactivity against bacterial pathogens and employ their antibacterial activity into two different ways; by killing the bacteria and by impairing the key events in the pathogenic process (Wink, 2008).It is well-known that multiple constituents in crude extract act at different sites thus contributing to the overall activity of the extract.Accordingly, alkaloids intercalate with DNA causing impaired cell division and consequently death (Savoia, 2012).Flavonoids are diverse and have ability to stick to and produce complex with bacterial cell walls and soluble proteins; inactivating enzymes of the pathogen (Ghosh et al., 2011).Tannins on the other hand involved in deactivation of cell envelope transport proteins and microbial adhesins (Sher, 2004).Moreover, terpenoids are ascribed to disruption of membranes in microbes (Termentzi, Fokialakis, & Skaltsounis, 2011).In general, the diverse phenolic compounds adversely affect microbes by reducing pH, increasing membrane permeability and changing efflux pumping (Emeka, Badger-Emeka, & Fateru, 2012).
The relative percentage inhibition (RPI) indicates the growth inhibition capacity of extracts with respect to the standard antibiotic, tetracycline.According to Gaitonde & Ramesh (2016) RPI of 50% or more is considered as active product for further study.In this regard, methanol extracts of all the studied species showed above 50% RPI indicating high potency of the extracts (Table 3).The highest RPI was produced by the extract of E. citriodora followed by extracts of R. communis and B. abyssinica as compared to other species (Table 3).The same lower-case letters in a row and upper case letters in a column indicate the absence of statistical differences between means at p < 0.05.

Conclusion
The methanol leaf extracts of the studied medicinal plant species showed different antibacterial activity attributed to the concentration and diversity of secondary metabolites.Although not quantified, extracts with abundant amount and large number of secondary metabolites had higher antibacterial activity.Accordingly, extracts of E. citriodora and R. communis had higher antibacterial activity and the potential as biocide formulations in controlling hot pepper bacterial wilt.However, the antibacterial activity of the extracts should be evaluated under greenhouse and field conditions to draw a solid conclusion.

Figure 2 .
Figure 2. Percentage extract yield of the medicinal plant leaf samples tested.

Figure 3 .
Figure 3. Minimum inhibitory and bactericidal concentrations of methanol leaf extracts of medicinal plants against Ralstonia solanacearum.

Table 1 .
Phytochemical screening of methanol leaf extracts of medicinal plants used in the bioassay.

Table 2 .
Antibacterial activity of methanol leaf extracts of medicinal plants against Ralstonia salanacearum evaluated in terms of bacterial growth inhibition zone (mm).

Table 3 .
Relative percentage inhibitions of medicinal plant methanol leaf extracts against Ralstonia solanacearum as compared to the standard antibiotic, tetracycline.