In vitro evaluation of the antibacterial activity of some medicinal plant extracts against Xanthomonas campestris pv . musacearum

Enset bacterial wilt caused by Xanthomonas campestris pv. musacearum (Xcm) is a destructive disease of Ensete ventricosum (Welw.) Cheesman) in Ethiopia. The antibacterial activities of methanol leaf extracts of Achyranthes aspera, Agarista salicifolia, Datura stramonium, Melia azedarach, Pycnostachys abyssinica and Vernonia amygdalina were evaluated in vitro against Xcm. Fresh leaves were collected, dried under shade and ground to fine powder. Extraction was carried out using maceration method. The antibacterial activity of extracts was evaluated by disc diffusion method. Total phenolics content was estimated using Folin Ciocalteau method. The result revealed that higher percent extract yield was obtained from A. salicifolia followed by P. abyssinica. Extracts of all species showed antibacterial activity except M. azedarach. Significant differences in inhibition zone diameter were recorded between species and among test concentrations. The widest inhibition zone was recorded by A. salicifolia followed by P. abyssinica. Unlike others, extract of A. salicifolia had abundant amount of alkaloids, flavonoids, phenols, terpenoids, saponnins, tannins and cardiac glycosides. The remaining species lacked one or more of these metabolites and the existing ones occurred either in small or moderate amount. Moreover, the extract of A. salicifolia was found to have the highest total phenolics content and this was positively correlated with inhibition zone diameter at all test concentrations suggesting its potential antibacterial activity. This shows that the extract of A. salicifolia has the potential for further bioformulation and commercialization as biocide with broad spectrum activity. However, further research should be conducted to identify the active compounds responsible for such antibacterial activity.


INTRODUCTION
Enset (Ensete ventricosum (Welw.)Cheesman) is a perennial staple food crop widely cultivated in the South and Southwestern Ethiopia.It supports the lives of approximately 20 million people (Temesgen Addis, 2005).It is a multipurpose crop providing a range of services such as food, forage, medicine, and environment protection (Dereje Fekadu, 2009).As a food crop enset has several advantages.First, the plant can be harvested at from about 1200 to 3100 meters above sea level (Temesgen Addis, 2005).This allows farmers to grow the crop in all parts of the country including areas not suitable for cereal cultivation.Fifth, it is considered tolerant to environmental conditions (Dereje Fekadu, 2009).
The disease is a major constraint of enset based agriculture, affecting the lives of millions of people in Ethiopia (Afza et al., 1996;Gizachew Welde-Michael et al., 2008).In addition to enset, the disease also affects other Musa species like banana which is the main staple food source in the whole of east Africa.The disease poses serious food insecurity in the region (Mwangi et al., 2007).
Unlike other diseases, enset bacterial wilt is both extreme and rapid causing gradual increasing losses over years.The economic impact of bacterial wilt is due to death of the mother plant that would otherwise contribute to the continuation of enset production cycles.Fields infested with Xcm cannot be replanted for at least 6 months due to carryover of soil borne inoculum (Tripathi et al., 2009).
The major transmission means of the disease across or within fields are insects, contaminated tools and infected plant materials (Mwangi et al., 2007).The spread of the disease is prevented by cultural disease management practices such as burying infected plants, restricting movement of infected plant materials and sterilizing farming tools (Biruma et al., 2007;Mwangi et al., 2007;Gizachew Wolde-Michael et al., 2008;Tripathi et al., 2009;Temesgen Addis et al., 2010).However, these methods are not effective as farmers are inconsistent and reluctant to employ labor-intensive disease control measures (Tripathi et al., 2009).
Moreover, the disease is systemic and hence surface application of chemicals has little or no use to control the disease (Smith et al., 2008).Thus, investigations for alternative disease controlling strategies that are effective, eco-friendly, longlasting, low cost, and easy to prepare have prime importance.
In recent years, much interest has been developed in the antimicrobial effects of medicinal plants for plant disease control.Plant extracts are important sources of new agrochemicals for the control of plant diseases.Furthermore, biocides of plant origin are nonphytotoxic, systemic and easily biodegradable (Kagale et al., 2004;Guleria and Tiku, 2009).It is now known that various plant extracts can reduce populations of foliar pathogens and control disease development.Consequently, they have a potential as environmentally safe alternatives and components in integrated disease management programs (Bowers and Locke, 2004).
Therefore, the aim of the present study was to evaluate the antibacterial activity of medicinal plant extracts against Xcm and see the potential to control enset bacterial wilt disease.(Asteraceae) were collected from different areas.

Medicinal plant material collection
The study species were selected on the basis of follow-up of antimicrobial activity reports and ethnomedical or traditional uses against diseases (Fabricant and Farnsworth, 2001).Specimens of each species were freshly pressed, mounted and identified by experts and authenticated specimens in the National Herbarium, Department of Plant Biology and Biodiversity, Addis Ababa University.
Voucher specimens of each species have been deposited in the National Herbarium.Collection number was designated using the first letters from the full name of the collector and order of collection time.Accordingly, voucher specimens of A. aspera, D. stramonium, M. azedarach, A. salicifolia, P. abyssinica, and V. amygdalina were given GY-01, GY-02, GY-03, GY-04, GY-05 and GY-06 collection numbers, respectively.
The leaves were washed with running tap water to remove dust and other debris, and air dried under shade at room temperature to constant weight.
The dried leaves were ground to fine powder using mechanical grinder.The powder was sieved through 0.6 mm wide mesh, homogenized and used for extraction.

Preparation of crude leaf extract
Extraction was performed using maceration method (Rai et al., 2013).Leaf powder and the solvent (methanol) were added into conical flasks at the ratio of 1:10 (w/v).The flasks were tightly closed and the mixture was shaken for 72 h using orbital shaker at a speed of 250 rpm under room temperature.After 72 h, the extract was filtered first by four layer of cheese cloth and cotton followed by Whatman No.1 filter paper.The extract was dried and concentrated by evaporating methanol using rotary evaporator.
The concentrated extract was stored at 4°C 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 (%) dry weight of extract initial powder weight = x 100

Infected enset material collection
Newly infected enset pseudostem samples were collected from a homegarden at Wondo Genet College of Forestry and Natural Resources.
The samples were cut into small pieces of approximately 2-5 mm 2 in size using sterilized knife.Each piece was placed in separate plastic bags and kept as cool as possible in an ice box to prevent drying, microbial degradation and avoid tissue decomposition.The specimens were then transported to the laboratory for Xcm isolation.

Isolation of the pathogen
Infected pseudostem specimens were further cut into smaller pieces using sterilized scalpel.The pieces were surface disinfected by dipping in 5 % sodium hypochlorite solution for one minute and immediately immersed in distilled water three times to remove the disinfectant.Then after, the cut pieces were immersed into a test tube containing 5 mL of sterilized water and allowed to stand for 5 minutes until the bacterial population diffuses out of the cut tissue into the sterilized water and form a suspension.A loopful of bacterial suspension was streaked to sterilized semi-selective growth medium composed of yeast extract (10 g L -1 ), peptone (10 g L -1 ), sucrose (10 g L -1 ), agar (15 g L -1 ), cephalexin (50 mg L -1 ) and amphotericin (150 mg L -1 ) as developed by Tripathi et al. (2007).Cephalexin and amphotericin were used to inhibit the growth of most saprophytes and fungal contaminants, respectively.The streaked Petri dishes were incubated in an inverted position at 28°C for 72 h.Isolated colonies were selected and streaked on a newly prepared yeast extract, peptone, sucrose and agar (YPSA) growth medium.
The sub-culturing of the bacterium was carried out using streak plate method.In this method, loopful of bacteria was directly taken using wire loop from growth plates that contain uniform colonies of Xcm.Sub-culturing was done several times until pure colonies were produced.The pure culture grown on YPSA was stored at 4°C, and every time activated at 28°C before use (Schaad and Stall, 1988).

Pathogenicity test
Plastic buckets filled with soil, sand and manure in the ratio of 2:1:1 were prepared in the glasshouse and suckers of a susceptible enset clone were transplanted.After establishment, individual enset plants were inoculated with 10 ml of Xcm suspension adjusted to 1.5x10 8 CFU/mL (0.5 McFarland standard) at the base of midrib in three replications.The negative control was inoculated with the same amount of distilled water using syringes with metal needle.A week after inoculation, symptom development was monitored for every other day.Yellowing of the inoculated leaf was seen after three weeks.Pseudostem of the infected plants was taken and the bacterium was re-isolated using the standard isolation procedure (Kidist Bobosha, 2003).

Antibacterial susceptibility testing of leaf extracts
The antibacterial activity of crude leaf extracts against Xcm was evaluated by disc diffusion

Inoculum preparation and inoculation
The inoculum was prepared from 72 h old bacteria grown on YPSA medium.The upper surfaces of several pure culture colonies were swabbed with cotton swab and mixed with distilled water in a test tube.The content of the test tube was thoroughly shaken until a homogenous suspension was formed.
The absorbance of the inoculum was measured with a spectrophotometer (NV202, Sunny) at 600 nm and adjusted to 0.132.This value is equivalent to turbidity of a 0.5 McFarland standard (Sutton, 2011).The bacterial population equivalent to a 0.5 McFarland standard turbidity is approximately 1.5 x 10 8 CFU/mL.
Cotton swab was used for inoculation.The Zone of complete inhibition was measured using transparent ruler at the longest possible diameter including the disc (Ortez, 2005).

Minimum inhibitory concentration (MIC)
The minimum inhibitory concentration (MIC) of leaf extracts was determined using agar dilution method as described in EUCAST, (2000).One mL of each test concentration of each extract was thoroughly mixed with 19 mL of YPSA molten growth medium and poured to Petri dishes.The medium was then allowed to solidify at room temperature.The inoculum adjusted to turbidity of a 0.5 McFarland standard (0.3μl) was inoculated at four points on each Petri dish.The inoculated Petri dishes were incubated at 28°C for 72 h.Parallel to this, Petri dishes without extract were used as controls and the results were compared against these controls.

Minimum bactericidal concentration (MBC)
Minimum bactericidal concentration (MBC) of leaf extracts was determined as described in Njinga et al. (2014).YPSA growth medium was prepared and autoclaved at 121°C for 15 minutes.
The medium was poured into sterile Petri-dishes and allowed to cool and solidify.The contents of the MIC Petri-dishes that did not show growth or showed growth less than 80 % of the control were sub-cultured onto the prepared Petri-dishes.The Petri-dishes were then incubated at 28°C for 72 h.Then after, the Petri-dishes were observed for growth.The Petri-dishes without growth represent the minimum bactericidal concentration (MBC).
After 72 h, the results were recorded and taken as MBC.

Phytochemical screening of crude leaf extracts
Qualitative analysis was performed to confirm the presence or absence of major secondary metabolites in the crude leaf extracts.The screening was carried out following standard chemical methods (Harborne, 1998;Tiwari et al., 2011;Jones and Kinghorn, 2006;Wadood et al., 2013;Kumar et al., 2013;Rai et al., 2013).

Test for alkaloids: Wagner's test
Each extract (20 mg) was dissolved in 2% HCl and filtered with glass funnel.The filtrate was used for the detection of alkaloids.Wagner's reagent was prepared by dissolving 1.27 g of iodine and two gram of potassium iodide (PI) in 100 mL of distilled water.One mL of Wagner's reagent was added to two mL of the filtrate.The formation of redish or brown solution was an indication for the presence of alkaloids.

Test for flavonoids
Sample from each plant extract (0.5 g) was taken in separate test tubes and 10 mL of distilled water was added, shaken and filtered.Five mL of diluted ammonia solution (10%) was mixed with 3 mL of the aqueous filtrate followed by the addition of 1 mL of concentrated sulphuric acid.Yellow color was formed showing a positive test for flavonoids.

Test for phenolic compounds: Ferric Chloride (FeCl 3 )
To two mL of the extract filtrate, 3-4 drops of 5% Ferric Chloride solution were added.Bluish black color production indicated the presence of phenols.

Test for terpenoids
Half gram of each species leaf powder was added in separate test tubes and poured with 10 mL of methanol.The content was shaken well and centrifuged.Five mL of the supernatant was mixed with two mL of chloroform followed by the addition of three mL of sulphuric acid to the solution.Reddish brown layer production between chloroform and sulphuric acid was an indicator for the presence of terpenoids.

Test for saponins: Froth test
One gram of each extract was boiled in five mL of distilled water and filtered with glass funnel.Three mL of distilled water was added to one mL of the filtrate and vigorously shaken for about 5 minutes.
Frothing that persisted for 15 minutes was taken as an evidence for the presence of saponins.

Test for tannins
Each extract (0.2 g) was dissolved in 5 mL of distilled water in separate test tubes, warmed in a water bath and filtered.Two mL of 5 % ferric chloride solution prepared in distilled water was added to one mL of the filtrate.The appearance of deep blue color affirmed the presence of tannins.

Test for cardiac glycosides
Approximately 20 mg of each extract was dissolved in 3 mL of 2% HCl and filtered.One mL of the filtrate was treated with five drops of glacial acetic acid and two drops of 5% ferric chloride solution and thoroughly mixed.Subsequently, two mL of concentrated sulfuric acid was added and two layers were formed; the lower white layer and upper acetic acid layer which turned bluish green indicating a positive test for glycosides.

Estimation of total phenolics content
Half gram of each extract and 10 mL of distilled water were added to a test tube, shaken and centrifuged.An aliquot of the supernatant (0.1 mL) was taken and diluted to three mL with distilled water.Consecutively, 0.25 mL of Folin Ciocalteau reagent was added.After three minutes, one mL of 20% (w/v) sodium carbonate was added and thoroughly mixed.The tube was warmed in boiling water for one min and cooled.The absorbance of the resulting solution was measured at 650 nm against a reagent blank using a spectrophotometer (NV202 Spectrophotometer, Sunny).The blank was composed of three mL of distilled water, 0.25 mL of Folin Ciocalteau reagent and one mL of 20% sodium carbonate.The absorbance of the blank was subtracted from each reading.Catechol was used to prepare the standard calibration curve from which the amount of total phenols in the sample was calculated.The amount of total phenols was expressed in mg catechol equivalent of phenol/g of sample (Zieslin and Ben-Zaken, 1993).

Statistical analysis
All data were subjected to analysis of variance  thus, MIC and MBC were not determined (Figure 3).

Phytochemical screening
The preliminary phytochemical screening results revealed that the chemical constituents of extracts vary between species (Table 1).Accordingly, extracts of A. salicifolia and P. abyssinica had abundant amount of the tested secondary metabolites.However, cardiac glycosides were absent in the extract of P. abyssinica.Extract of V. amygdalina on the other hand had abundant amount of phenols, terpenoids and tannins, and moderate amount of alkaloids, flavonoids and saponnins (Table 1).

Total phenolics content
The result of the quantitative chemical analysis of extracts showed that total phenolics content was significantly different between the studied plant species (Figure 4).The highest total phenolics content was recorded by the leaf extract of A.
stramonium, M. azedarach, P. abyssinica, and V. amygdalina, respectively.Extract of V. amygdalina ranked second and extract of P. abyssinica ranked third in terms of total phenolics content.The total phenolics content of the remaining extracts was negligible (lower than the minimum concentration used to construct the standard calibration curve).

DISCUSSION
The exploitation of higher plant products as novel chemical treatments in plant protection has gained emphasis recently and some plant products are being used globally as ecofriendly biocontrol agents (Gurjar et al., 2012).The present study was conducted to test the in vitro antibacterial activity of some medicinal plants and antibiotics against enset pathogen, Xcm.The tested plants are used in traditional medicine for the treatment of many diseases either in unprocessed or extracted forms.
Efficiency of extraction is an important step in the discovery of bioactive components from plant materials.In the present study, A. salicifolia produced the highest extract yield followed by P.
abyssinica.Extract yield of V. amygdalina was moderate.Similarly, Odey et al. (2012) found out 13.6 and 12 % extract yields of root and stem of V.
amygdalina, respectively, which is twofold lower than the leaf extract yield in the present study.
The differences in the extract yields might be ascribed to the different availability of extractable components, resulting from the varied chemical composition of plants (Sultana et al., 2009).
Moreover, Zongo et al. (2011) and Moteriya et al. (2014) reported maximum percent extract yield when methanol is used as a solvent.Methanol is an all purpose solvent that dissolves most secondary metabolites in plants and enhances the release of these chemicals from cellular matrix (Visht and Chaturvedi, 2012;Moteriya et al., 2014).different mechanisms (Kotzekidou et al., 2008).Accordingly, alkaloids intercalate into cell wall and hinder its formation (Gurjar et al., 2012).
Flavonoids are antimicrobial substances against a wide array of microorganisms in vitro and have the ability to bind and complex with bacterial cell walls and soluble proteins.This inactivates enzymes (Marjorie, 1999;Ghosh et al., 2011;Gurjar et al., 2012).Terpenoids on the other hand are responsible for disbanding of the cell wall of microorganisms by deteriorating the membranous tissue (Marjorie, 1999;Ghosh et al., 2011;Jasmine et al., 2011;Gurjar et al., 2012).
Similarly, saponins have the ability to cause leakage of proteins and certain enzymes from the cell (Marjorie, 1999).Moreover,  (Mamtha et al., 2004;Igbinosa et al., 2009;Ghosh et al., 2011;Gurjar et al., 2012).Overall, extracts that possess diversity of secondary metabolites in relatively large amount revealed high antibacterial activity against Xcm.
In the present study, extracts of most plant species inhibited the growth of the test bacterium in vitro, indicating the presence of antibacterial compounds in the extracts.These compounds are secondary metabolites that belong to various chemical categories in plant materials and work either individually or in combination to produce an antibacterial activity (Ghosh et al., 2011).The quantitative analysis revealed that the leaf extract of A. salicifolia contain the highest amount of total phenolics content as compared to others and thus the maximum potency against the test bacterium.This is evident from the strong positive correlation between total phenolics content and bacterial growth inhibition zone.Similar correlation was observed in extracts of P. abyssinica at all test concentrations.Therefore, the antibacterial activity of the two species may be explained by the total phenolics content.Many research reports reveal that phenolic compounds contribute to the antimicrobial properties of plant extracts whereby the extent of inhibition depends on the concentration of these compounds (Taguri et al., 2006;Rhouma et al., 2009;Rodriguez et al., 2009;Tian et al., 2009;Zongo et al., 2011;Moteriya et al., 2014).
The biological activities of phenolic compounds are associated to the presence of hydroxyl groups and phenolic ring in their molecular structure.
Membrane proteins of bacteria interact with hydroxyl groups of phenolics by hydrogen bonding.
This interaction changes the membrane permeability that in turn causes loss of cellular constitutes and cell destruction (Marcucci et al., 2001;Kang et al., 2011).They also bind to adhesins, complex with cell wall and inactivate proteolytic macerating enzymes used by plant pathogens (Mohanta et al., 2007;Gurjar et al., 2012).From this it can be presumed that phenolic compounds act at two different points (cell membrane and cell wall), affecting the growth and metabolism of bacteria (Taguri et al., 2006;Stefanovic et al., 2012).Similar results have also been reported in other studies (Rajeshwar et al., 2005;Kuete et al., 2007).Extract of V. amygdalina contain high amount of total phenolics next to A.
salicifolia.However, the antibacterial activity of the extract is low.This may be due to the moderate  and Batra, 2012).

CONCLUSION
This study demonstrated that extracts of A.
sterilized swab was dipped into the bacterial suspension and the excess fluid was removed by turning the swab against the inside of the test tube.This avoids over inoculation of the Petri dish.The inoculum was spread evenly over the entire surface of the Petri dish by swabbing in three directions.The dried discs were applied to the inoculated Petri dish within 15 minutes of inoculation.During application, the discs were pressed downward and the Petri dish was kept in normal position until the discs got wet.Discs rinsed in methanol were used as negative controls.Streptomycin sulphate was used as a positive control.All the Petri dishes were inverted and incubated at 28°C for 72 h.Inhibition zone was measured after 72 h of incubation.

Figure 2 :
Figure 2: Growth inhibition zone of methanol crude leaf extracts at various test concentrations.Bars at each test concentration followed by different letters are significantly different at p < 0.01 (n = 6).

Figure 3 :
Figure 3: Minimum inhibitory and bactericidal concentrations of crude leaf extracts of medicinal plants of different secondary metabolites in the leaf extracts of the studied species.The variation is both in type and amount of chemical categories.The high antibacterial activity of methanolic extract of A. salicifolia and P. abyssinica may be due to the abundance of alkaloids, flavonoids, phenols, tannins, saponins and terpenoids.In addition, A. salicifolia extract contains glycosides.Each bioactive constituent exert antibacterial activity through

Figure 4 :
Figure 4: Total phenolics content (mg of catechol equivalent of phenol/g of fresh weight) of medicinal plant leaf extracts.Bars followed by different letters are significantly different at P < 0.01.Values represent means of six replicates amount of flavonoids and saponins in the leaf extract of V. amygdalina and weak synergistic effect of all kinds of phenolic compounds on Xcm.The minimum inhibitory concentration (MIC) is the lowest concentration value that inhibits the growth of microorganisms.It is the measure of assaying the effectiveness of antimicrobial plant extracts and predictive of the inhibitive outcomes.Extracts with low antibacterial activity against Xcm give high MIC and MBC, Viz. A. aspera, D. stramonium, M. azedarach and V. amygdalina.To the contrary, extracts with high antibacterial activity yield low MIC and MBC, Viz. A. salicifolia and P. abyssinica.Similarly, various MIC and MBC concentrations of methanol extracts of medicinal plants against both Gram positive and negative bacteria have been reported by Kang et al. (2011).Unlike the present study, M. azedarch showed MIC against several human pathogens (Sen salicifolia and P. abyssinica with high antibacterial activity in vitro against X. campestris pv.musacearum could be used for biological control of Enset bacterial wilt.The maximum percent extract yield plus the highest anti-Xanthomonas activity of the two species could be designated as distinctive features of these medicinal plants for further development, formulation and commercialization as biocides with broad spectrum activity.Such developments in the use of natural products from plants in disease control will minimize or avoid environmental and health hazards caused by synthetic chemical pesticides.However, further researches into these extracts should be conducted to identify the active compounds responsible for their antibacterial activity and to evaluate the performance under field conditions.

Table 1 .
Classes of secondary compounds found in leaf extracts of medicinal plants in a preliminary Taguri et al.
(2006), Rodriguez et al. (2009) and Boulekbache-Makhlouf et al. (2013) reported that hydrolysable tannins have potent antibacterial effects on various bacteria.They bind and form irreversible complexes with proline rich protein and cause inhibition of cell wall synthesis