Abstract: Raw and processed products of Ocimum gratissimum reportedly contain some antibacterial effects. In this study, raw plant extracts and essential oils derived from leaves of Ocimum gratissimum were screened for their antibacterial properties on Ralstonia solanacearum, the causal agent of bacterial wilt in Irish potato at varying concentrations. The raw extracts were obtained through sequential cold extraction using methanol solvent while the essential oils were extracted through steam distillation. The test products were used at concentrations of 0.4, 0.2, 0.1, 0.05 and 0.025 mg mL-1. Antibacterial tests were done using paper disc diffusion inhibition method and antibacterial activity was determined by measuring the size of inhibition zones. The results showed that the five different concentrations of essential oils and plant extracts exhibited highly significant (p<0.0001) differences on their effects against growth of R. solanacearum. The study confirmed the antibacterial effects of O. gratissimum and further demonstrated that the active compound (s) against Ralstonia solanacearum is (are) concentrated in the essential oils.
INTRODUCTION
Irish potato or cultivated potato (Solanum tuberosum L.) is an important crop worldwide providing food, income and employment to over 800 million people globally (Hoffler and Ochieng, 2008). Kenya is the fifth biggest potato producer in Sub-Saharan Africa, with an output of 790,000 tonnes in 2006 (Muthoni and Nyamongo, 2009). The crop ranks as the second most important food crop after maize in Kenya (Felix et al., 2010). It grows in cool and high altitude parts of the country where rainfall is well distributed including slopes of Mt. Kenya and both sides of the Aberdare ranges. The crop is also grown on the highlands on Mau Escarpment, Tinderet, Nandi Escarpment and Cherangani hills (Muthoni and Nyamongo, 2009). The cultivated potato provides high energy content and is easy to produce making it an important part of agriculture (Hoffler and Ochieng, 2008). Potato production is much higher in developed than in developing countries and diseases have been cited as one of the major limiting factors (Muthoni and Nyamongo, 2009; Lemaga et al., 2001; Mureithi, 2000; Otipa et al., 2003).
One of the most destructive diseases known to attack potato plants is bacterial wilt caused by Ralstonia solanacearum race 3, biovar 2 (Momol et al., 2000). The disease, also known as brown rot, southern wilt, sore eye or Jammy eye, is a major limiting factor to potato crop production in most countries. Other hosts of R. solanacearum include economically important crops such as tobacco, pepper, banana, beans, tomato and eggplant (Hayward, 1991; Norman et al., 2009). The pathogen invades the roots of the host plant and aggressively colonizes the xylem vessels causing a lethal wilting (Tahat et al., 2008). In Kenya, bacterial wilt was first reported in 1940 s and since then it has spread to most potato growing regions. It has been reported that the diseases causes losses ranging from 30 to 70% at altitudes ranging from 1800 to 2800 m (Otipa et al., 2003). The disease is considered more serious than even late blight as there are no chemical control procedures and many farmers do not know how to control it (Muthoni and Nyamongo, 2009). In addition, some of the recommended control measures such as crop rotation, use of clean seeds, planting in non-infested soils and growing tolerant varieties (Tahat and Sijam, 2010) have individual practical, technological and economic limitations (Lemaga et al., 2001; Muthoni and Nyamongo, 2009; Jinnah et al., 2002; Khalequzzaman et al., 2002).
The increasing antibiotic resistance of some pathogens that are associated with diseases has increased the interest in the development of new types effective and nontoxic antimicrobial compounds (Sobhy and El-Feky, 2007). Subsequently, the use of agrochemicals is becoming less favourable (Bonjar et al., 2006) while the use of plant extracts and phyto-products is gaining attention due to their proven nature specificity, biodegradability, low toxicity and minimum residual toxicity in the ecosystem (Ogbo and Oyibo, 2008; Sobhy and El-Feky, 2007). Much curiosity has been devoted to the Lamiaceae family, especially Ocimum gratissimum L. considered as one of the main sources of potential active metabolites (Louis et al., 2011). Previous studies have shown that O. gratissimum) contains antimicrobial principles (Wagura et al., 2011; Lemos et al., 2005; Iwalokun et al., 2003; Adamu et al., 2009). Ocimum oil has been reported to be active against plant pathogens such as Phytophthora palmivora, Alternaria brassicicola, Aspergillus flavus, Bipolaris oryzae, Fusarium moniliforme, Fusarium proliferatum, Pyricularia grisea and Rhizoctonia solani (Piyo et al., 2009). Ocimum oil has also been shown to contain active principles against several species of animal and human bacteria (Sunita and Mahendra, 2008; Akinyemi et al., 2004; Lemos et al., 2005; Lopez et al., 2005).
Guided by the information on potato yield losses caused by bacterial wilt disease, difficulties in the control of the pathogen and the reported antimicrobial activities of O. gratissimum, this study was set up to compare the antibacterial effects of raw extracts and essential oils of O. gratissimum.
MATERIALS AND METHODS
The study was conducted at Maseno University between September, 2006 and November, 2007. Approximately 4 kg fresh leaves of O. gratissimum were collected from Maseno region in Western Kenya and divided into two heaps; one for extraction of plant extracts using methanol solvent and the other for extraction of essential oils. The plucked leaves were washed thoroughly 2 to 3 times with running tap water and once with sterile distilled water.
Extraction of plant extracts: One heap of leaves was shredded and dried as described by Okigbo and Ogbonnaya (2006). They were then ground into fine powder at Kenya Sugar Research Foundation in Kisumu, Kenya in readiness for solvent extraction. Cold extraction of the powdered plant materials using methanol solvent was done sequentially following the method of Eaton (1989). Known quantity of dry ground leaf material was soaked in the solvent in Erlenmeyer flask and left for four days with occasional shaking. The liquid portion was then filtered using Whatman No.1 filter paper. The filtrate was then concentrated in vacuo in a round-bottomed flask using rotary evaporator at 60°C (Junaid et al., 2006). The extracts obtained were kept in vials in readiness for bioassay tests (Eaton, 1989; Llorach et al., 2003).
Extraction of essential oils: The second heap of leaves was air dried under shade, chopped into small pieces and subjected to steam distillation for three h. The distillate was then extracted with petroleum ether which was removed carefully and 34 g of essential oil obtained. The essential oils were diluted with dimethyl sulphoxide to make test concentrations of 0.4, 0.2, 0.1, 0.05 and 0.025 mg mL-1 and then stored at-20°C until required (Mbata and Saikia, 2005).
Isolation of wilt bacteria from infected potato tubers: Infected tubers were obtained from a test plot at the National Agricultural Research Laboratories (NARL) fields, Nairobi. These were cleaned under running water to remove adhering soil, air-dried, then cleaned using 97% ethanol to remove any microorganism on its surface. The skin at the end of the stolon was removed using a disinfected scalpel to make vascular tissues visible. A bacterial suspension was prepared using the method described by Priou et al. (1999). Approximately 0.5 mL of the bacterial suspension was spread on nutrient agar in Petri dishes. The plates were incubated for 48 h at 28°C and bacterial colonies that were fluidal, flat, pearly white and irregular identified.
Pathogenicity test: The method of Koch’s postulates was performed with Solanum tuberosum var. Tigoni 381381 as the host. After a 24 h period without water, one side of some potato roots were injured one centimetre from the stem and approximately 20 mL of an aqueous suspension of R. solanacearum of 1x107 cfu mL-1 was poured around the base of the stem. Five days after inoculation (after the wilting symptoms were exhibited), vascular flow test was run by cutting a piece of potato stem (5 cm long) and suspending it in clear water in a glass container. The cut stem was held with a clip to keep it in a vertical position until smoke like threads streamed downwards from the cut stem (Priou et al., 1999).
Antibacterial assay: The antibacterial effects of the raw extracts and essential oils against R. solanacearum were evaluated using the method described by Barry et al. (1979) and Souza et al. (2005). Inoculation was done by rubbing a sterile cotton swab containing the pathogen on the surface of solidified agar as described by Linnette et al. (1974).
Experimental design, data recording and analysis: The experiments were laid down in Randomized Complete Block Design (RCBD) with four replications. The antibacterial activity was recorded as the width (in mm) of clear zones of inhibition surrounding the diffusion discs after 48 h (Reiner, 1982; Baker et al., 1983; Deans and Ritchie, 1987). The data were subjected to ANOVA using SAS version 9.1 and effects declared significant at 5% level. Separation of means was done only for those parameters where the ANOVA was significant, using Least Significant Difference at 5% level of significance [LSD5%] (Steel and Torrie, 1980).
RESULTS
Results of the inhibitory effects of essential oils and plant extracts of O. gratissimum against R. solanacearum are shown in Fig. 1. The presence of inhibition zones (Fig. 2a-c) depicted antibacterial activity of O. gratissimum extracts and essential oil on Ralstonia solanacearum.
| Fig. 1: | Antibacterial effects of O. gratissimum extracts and essential oils |
| Fig. 2 (a-c): | Inhibitory effects of O. gratissimum essential oils; (a) 0.05 mg mL-1, (b) 0.2 mg mL-1 and (c) 0.4 mg mL-1 |
Analysis of variance showed that the five different concentrations (0.4, 0.2, 0.1, 0.05 and 0.025 mg mL-1) of essential oils and plant extracts exhibited highly significant (p<0.0001) differences on their effects against growth of R. solanacearum (Fig. 1). In both cases, antibacterial activity of the test materials increased as their concentration increased but essential oils were more effective than the plant extracts (Fig. 1).
The inhibitory effects of essential oils increased almost exponentially with every double increase in concentration with each concentration producing significantly different (p<0.05) effect from all the rest. The inhibition zones obtained were also distinctively clear and easy to measure (Fig. 2). The highest concentration of 0.4 mg mL-1 demonstrated the highest antibacterial activity with inhibition zone of 10.12 mm while the lowest concentration of 0.025 mg mL-1 resulted in the smallest inhibition zone of 2.75 mm (Fig. 1). There was no inhibition zone around the discs treated with pure dimethyl sulphoxide (not shown).
The plant extracts were not as effective as the essential oils though they also demonstrated some inhibitory/antibacterial effects against R. solanacearum. In fact, the highest concentration of the plant extracts performed statistically the same as the lowest concentration of essential oils. However, like essential oils, the effects of plant extracts were also found to increase with increase in concentration though the increase was not as pronounced as in the case of essential oils. The two lowest concentrations of 0.025 and 0.05 mg mL-1 exhibited statistically similar activity where inhibition zone measured 1.84 mm and 1.88 mm respectively. Their efficiency increased marginally to 2.3 mm and then to 2.6 mm when the concentration was raised to 0.1 and 0.2 mg mL-1, respectively. The best results of 3.13 mm were obtained at 0.4 mg mL-1 (Fig. 1).
DISCUSSION
The study confirmed the antibacterial effects of O. gratissimum as previously reported by other researchers. The presence of antibacterial substances in the plant extracts which caused the inhibition of radial growth in vitro agree with reports of other studies (Olayinka, 2009; Mbata and Saikia, 2005). Antibacterial effects of O. gratissimum extracts have also been reported by Ntezurubanza et al. (1984), Nakamura et al. (1999), Iwalokun et al. (2003) and Lemos et al. (2005). In a study to determine the effects of plant extracts of O. gratissimum and other plants on post harvest pathogen of Persea americana, Ogbo and Oyibo (2008) observed 100% inhibition with ethanolic extract and over 60% inhibition when unsterilized water was used for extraction. Orafidiya et al. (2000) demonstrated that the oil extract of O. gratissimum was active against enteroaggregative E. coli. The study demonstrated that antibacterial activity of O. gratissimum increased with increase in concentration of the active compound. Similar trend have also been reported by Mbata and Saikia (2005).
The inhibitory effects of essential oils were more pronounced than those of plant extracts. Similar observation was made by Adebolu and Salau (2005). The significantly higher activity of essential oils compared to raw extracts was an indication that the oil components of O. gratissimum could be the active compounds with antibacterial principles. Akinyemi et al. (2004), Lemos et al. (2005) and Lopez et al. (2005) reported that Ocimum oil is active against several species of bacteria (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Shigella, Salmonella and Proteus) and fungi (Trichophyton rubrum T. mentagrophytes, Cryptococcus neoformans, Penicillium islandicum and Candida albicans). The difference observed in antibacterial activity of the essential oils and raw plant extracts can therefore be attributed to the high volatibility of the oil, leading to the escape or evaporation of the oil during leaf drying as well as insufficient release of the oil during extraction. It has been reported that the active principles present in plants are influenced by many factors which include the age of plant, extracting solvent, method of extraction and time of harvesting plant materials (Ajalie and Okigbo, 2005; Okigbo et al ., 2005; Okigbo and Ogbonna, 2006).
Previous studies have shown that various species of the genus Ocimum produce oils of diverse characteristics and most with antimicrobial properties (Louis et al., 2011; Ntezurubanza et al., 1984; Nakamura et al., 1999; Lemos et al., 2005; Reuveni et al., 1984). Matasyoh et al. (2008) reported that essential oils derived from leaves of O. gratissimum collected from Meru region contained eugenol, methyl eugenol, cis-ocimene and trans-ocimene. A study carried out to analyze essential oils of O. gratissimum by Gas Chromatography (Masada, 1976) showed that they contain compounds such as thymol, eugenol and d-limoneme among others. The oils have been reported to be active against species of bacteria and fungi (Matasyoh et al., 2008; Mbata and Saikia, 2005; Iwalokun et al., 2003; Malik and Singh, 2010). Though the study did not ascertain the chemical compound present in the oil that has the antibacterial properties, eugenol was suspected as the most likely candidate. This component has been demonstrated to have both antibacterial (Nakamura et al., 1999) and antihelmintic activities (Pessoa et al., 2002).
CONCLUSION
The study confirmed the antibacterial effects of O. gratissimum and further demonstrated that the active compound (s) against Ralstonia solanacearum is contained in the essential oils. Biologically active essential oils represent a rich potential source of an alternative and perhaps environmentally more acceptable disease management compounds. Further research is therefore recommended to identify the specific chemical compound(s) that is active against the pathogen.
ACKNOWLEDGMENTS
The authors wish to acknowledge Maseno University for providing laboratory space, equipments and technical assistance. Thanks are due to Prof. George Odhiambo of Department of Botany and Horticulture, Maseno University, for his valuable assistance in data analysis.