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In Vitro Antibacterial Activity of Traditionally Used Medicinal Plants Against Xanthomonas Campestris Pv. Musacearum in Ethiopia

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In Vitro Antibacterial Activity of Traditionally Used Medicinal Plants Against Xanthomonas Campestris Pv. Musacearum in Ethiopia BIODIVERSITAS ISSN: 1412-033X Volume 20, Number 2, February 2019 E-ISSN: 2085-4722 Pages: 555-561 DOI: 10.13057/biodiv/d200235 In vitro antibacterial activity of traditionally used medicinal plants against Xanthomonas campestris pv. musacearum in Ethiopia GETAHUN YEMATA1,♥, BRUKTAWIT DESTA2, MASRESHA FETENE3 1Department of Biology, College of Science, Bahir Dar University. P.O. Box. 79, , Bahir Dar, Ethiopia. email: [email protected] 2Department of Biology, College of Natural Science, Ambo University. Ambo, Ethiopia 3Department of Plant Biology and Biodiversity Management, College of Natural and Computational Sciences, Addis Ababa University. P. O. Box. 1176, Addis Ababa, Ethiopia. Manuscript received: 17 October 2018. Revision accepted: 30 January 2019. Abstract. Yemata G, Desta B, Fetene M. 2019. In vitro antibacterial activity of traditionally used medicinal plants against Xanthomonas campestris pv. musacearum in Ethiopia. Biodiversitas 20: 555-561. In Ethiopia, traditional medicinal plants have long been used to treat human and livestock ailments. Nevertheless, studies about the use of these plant extracts to control crop diseases are scarce. Therefore, the aim of the present study was to evaluate the antibacterial activity of traditionally used medicinal plants against Xanthomonas campestris pv. musacearum (Xcm). The bioactive chemicals from leaf samples were drawn using methanol by maceration method. Total phenolic content of the extracts was determined by Folin Ciocalteau reagent. The antibacterial activity of leaf extracts was evaluated by disc diffusion method. The phytochemical analysis revealed the presence of alkaloids, flavonoids, phenolic compounds, terpenoids, tannins and saponins. Extracts with higher total phenolic content had greater antibacterial activity. The extract of each species showed antibacterial activity against Xcm on a dose dependent manner. Significant differences were recorded between species and test concentrations. The extract of Bersama abyssinica exhibited the strongest antibacterial activity at 200 mg/mL followed by Ricinus communis, Eucalyptus citriodora and Acokanthera schimperi. At lower test concentrations, extracts of E. citriodora and R. communis revealed higher antibacterial activity. Moreover, these species had lower bacteriostatic and bactericidal concentrations. The results showed the potential potency of E. citriodora and R. communis leaf extracts in controlling enset bacterial wilt. However, further studies on the identification of chemical compounds and in vivo evaluation of the extracts are recommended. Keywords: Antibacterial, enset, Ensete ventricosum, leaf extract, Xanthomonas campestris pv. musacearum INTRODUCTION disease, caused by Xcm. Recent survey studies showed that EBW is increasing both in severity and distribution in most Plant diseases caused by phytopathogenic bacteria of the enset growing agroecologies (Handoro 2017). Xcm represent an emerging threat to global food security. is soil borne and infested fields should remain fallow for at Xanthomonas is a large genus of Gram-negative bacteria least 6 months to avoid re-infestation (Tripathi et al. 2009). that cause disease in several host plants leading to EBW causes up to 100 % yield loss (Handoro, 2017), considerable yield losses (Bajpai et al. 2011). Pathogenic which severely affects food security and livelihoods in species and pathovars within species show high degree of enset based farming households (Blomme et al. 2017). host plant specificity. Several of these exhibit tissue The pathogen is transmitted mechanically through specificity, invading either the vascular system or the contaminated farming tools, infected plant materials, water mesophyll tissue of the host (Ryan et al. 2011). flash and insects (Tripathi et al. 2009; Addis et al. 2010). Xanthomonas campestris pv. musacearum is a member of The management of Xcm has relied on cultural practices the genus that causes a destructive disease in enset (Ensete such as burying infected plants and sterilizing farming tools ventricosum) and banana (Blomme et al. 2017; Nakato et that keep the pathogen population at tolerable levels (Addis al. 2017). et al. 2010; Nakato et al. 2017). However, these methods Ensete ventricosum (Welw.) Cheesman) is a multi- are not effective as farmers are inconsistent and reluctant to purpose root crop that has been produced for thousands of employ labor-intensive disease controlling measures years only in Ethiopia. It serves as a staple/co-staple food (Tripathi et al. 2009). Furthermore, no bactericide has been supporting the livelihood of approximately 20 million suggested against Xcm (Bajpai et al. 2011). In Ethiopia, people in the south and southwestern part of the country traditional medicinal plants have long been used to treat (Magule et al. 2014). As a food crop, enset has several human and livestock ailments. However, studies about the merits. It produces high yield per unit area as compared to use of these medicinal plants extracts to control plant other crops; tolerant to prolonged drought and can be diseases are absent. Thus, the aim of this study was to harvested at any developmental stage if people face food evaluate the antibacterial activity of traditional medicinal shortage. Thus, the crop is considered as a field bank for plants in controlling EBW. food (Tsegaye and Struik 2002). However, enset production is threatened by enset bacterial wilt (EBW) 556 BIODIVERSITAS 20 (2): 555-561, February 2019 MATERIALS AND METHODS room temperature to constant weight. The dried samples were ground to fine powder with a mechanical grinder. Plant material collection and extraction Powder of each species was passed through 0.6 mm mesh Leaf samples and specimens of traditionally used and put in a tightly closed bag. Powdered leaf sample of medicinal plants namely; Acokanthera schimperi (A. DC.) each species was macerated with 99.8% methanol in the Schwein, Albizia schimperiana Oliv., Bersama abyssinica ratio of 1:10 (w/v) and shaken for 72 h on an orbital shaker Fresen, Brucea antidysenterica JF. Mill, Eucalyptus at a speed of 200 rpm. The extract was filtered first with citriodora (Hook), Fagaropsis angolensis (Engl.) Dale, four layers of cheese cloth and cotton followed by Indigofera arrecta Hochst. ex A. Rich, Laggera crispata Whatman’s filter paper. All the activities were performed (Vahl) Hepper & Wood, Olinia rochetiana A. Juss., under room temperature. Methanol was evaporated using Peponium vogelii (Hook.f) Engl., Ricinus communis L., rotary evaporator at 42°C and the dry extract was stored at Rumex abyssinicus Jacq and Solamu incanum L. were 4°C. Percent extract yield was calculated gravimetrically collected from Gedeo, Sidama and Wolaita zones (Figure by dividing the dry weight of the extract to the weight of 1). Plant species most frequently used by the people were the leaf powder multiplied by 100. selected and used in this study. The pressed plant specimens were placed in a drier box with an in-built 60W Phytochemical analysis of extracts light bulb. The drier has adequate ventilation. The dried Qualitative analysis was performed to affirm the specimens were trimmed to fit the mounting sheet, presence or absence of major secondary metabolites in the carefully put and attached with a few dots of glue on the methanol crude leaf extract of each species. The presence under sides of plant parts. The specimens were identified at of alkaloids, flavonoids, phenolic compounds, saponins, a species level by the taxonomist expert and compared tannins and terpenoids was checked using Wagner test, visually with the authenticated plant specimens in the Alkaline reagent, Foam test, Ferric Chloride test and National Herbarium (ETH). The voucher specimens were Salkowski test, respectively as described by (Tiwari et al. deposited at the National Herbarium (ETH), Addis Ababa 2011; Rai et al. 2013). University. The leaf samples were washed with tap water to remove dust and other debris, and air dried under shade at Study zones Figure 1. Map of the region where the study zones are Wolaita (Welayita), Sidama and Gedeo, Ethiopia YEMATA et al. – Traditionally used medicinal plants against Xanthomonas campestris pv musacearum 557 Estimation of total phenolic content of extracts and agar (1.5%) medium (YPSA). The upper surfaces of Extract of each species (0.5 g) and 10 ml of distilled several isolated (pure) colonies were swabbed with cotton water were added in separate test tubes, shaken and swab and suspended in distilled water in a test tube. The centrifuged. An aliquot of (0.1 ml) of the supernatant was content of the test tube was thoroughly shaken until a taken and diluted to 3 ml with distilled water. homogenous suspension was formed. The absorbance of Consecutively, 0.25 ml of Folin Ciocalteau reagent was the bacterial suspension was measured with a added. After 3 minutes, 1 ml of 20 % (w/v) sodium spectrophotometer (NV202, Sunny) at 600 nm and adjusted carbonate was added and thoroughly mixed. The tubes to 0.132 equivalent to the density of 0.5 McFarland were placed in boiling water for 1 min and cooled. The standards (Sutton 2011). The bacterial population at 0.5 absorbance of the resulting solution was measured at 650 McFarland standards is approximately equal to 1.5 × 108 nm against a reagent blank using a spectrophotometer CFU/mL. (NV202 Spectrophotometer, Sunny). The blank was composed of 3 ml of distilled water, 0.25 ml of Folin Antibacterial activity of leaf extracts Ciocalteau and 1 ml of 20 % sodium carbonate. The Extracts
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