Abstract: Bio-control methods have become a common practice in horticulture and crop husbandry due to the attendant negative impact of fungicides and other chemical agents on soil, vegetation and environment. Most fungicides accumulate in plant tissues and subsequently trigger carcinogenic effects in animals and humans. The leaf extracts of Ficus thonningii, F. saussureana, F. exasperata and F. sur were screened for antimicrobial properties on eight (8) fungal species which included Aspergillus flavus, A. niger, Botryodiploidia theobromae, Fusarium oxysporum, F. solani, Penicillium chrysogenum, P. oxalicum and Rhizopus stolonifer and two (2) bacterial species viz; Pseudomonas spp. and Klebsiella spp. which were isolated from the rot portions of tubers of Dioscorea rotundata. The extracts from the Ficus species had low antimicrobial effect at 25 and 50 mg mL-1 concentrations while a significant arrest of mycelia growth was observed at 75 and 100 mg mL-1 concentrations. The presence of alkaloids, flavonoids and cardiac glycosides in the leaves of these species may have conferred the antimicrobial properties on these species. Application of the fungal pathogens isolated on healthy tubers and the subsequent development of rots confirmed these organisms as the natural pathogens of this crop. The extracts from all the four Ficus species exerted significant antimicrobial effect on all the test organisms at 75 and 100 mg mL-1 concentrations and its application at these concentrations would help to minimise infection and spoilage during and after storage and improve farmers’ revenue.
INTRODUCTION
The cultivated yams belong to the genus Dioscorea with over 200 varieties (Norman et al., 1995; FAO, 2005) of which six (6) species are the most common foods in tropical countries across the West Africa, Caribbean and North Central of the South east Asia (Ajalie and Okigbo, 2005). The most important and widely cultivated member of this group is Dioscorea rotundata (White yam) (Asadu and Akamigbo, 1996). The 21, 814 million tons of yam produced in Nigeria annually (FAO, 1998; Ajalie and Okigbo, 2005) may be eaten boiled, fried or roasted or as yam pastes by the Yorubas and as ingredients for few confectionaries (Okaka et al., 1991).Yams are valued food crops, a major source of carbohydrates, vitamins and dietary fibres (Ekefan et al., 1999; Ogaraku and Usman, 2008) and equally known to contain medicinal properties for the treatment of diabetes mellitus and hyperchosterolemia (Okigbo and Ogbonna, 2006). Postharvest losses account for about 40% of valuable yam stock (Awuah and Akrasi, 2007) and rots from microbial infestation of healthy tubers reduce their table quality and renders them unappealing to consumers (Amusa, 1999). Microbial infestation and the spread of rots are severe in the tropics where temperature and humidity are high. The fungal and bacterial pathogens implicated in the development and spread of rots are Aspergillus flavus ex Fr., A. niger, A. tamari, Botryodiploidia theobromae, Cladosporium herbarium, C. sphaerospermum and Cylindrocarpon radiocola while Corynebacterium spp. Serratia spp. and Erwinia spp are the representative bacteria involved in yam rot diseases (Okigbo, 2004).
The incidence of rot varies with the species of fungi and bacteria, the varieties of yam and the physical or health state of tubers. Infection is accentuated by a breakdown of tissues of the peridermal layer through wounds inflicted by rodents, nematodes and farm implements or improper handling of tubers (Ogaraku and Usman, 2008). The management and control of rot disease has been a major challenge to yam farmers the world over. The use of chemicals such as Sodium o-phenylphenate, Borax, Captan, Thiobendazole, Naphthalene acetic acid and Sodium hypochlorite have been quite effective. However, their uses have been associated with a number of deleterious and physiological effects on plant tissues (Amusa and Ayinla, 1997).
A number of plant extracts have been proven useful as biological control agents without side effects on both humans and environment (Omoseyindemi, 2003; Okwute, 1992). The extracts from few leaves including Carica papaya, Cassia alata, Cassia fistula and stems of Citrus limon inhibited the growth of Collectotrichum gloeosporiodes (Banos et al., 2002) and Microsporum gypseum, Trichophyton rubrum and Penicillum marneffei (Suleiman et al., 2008). Srivastava et al. (2011) observed a significant reduction in the growth of Fusarium oxysporium with 15% concentration of extracts from leaves of both Neem and Lantana plants. The application of wood ash and palm oil to cut surfaces of yam tubers prevented rot diseases caused by Aspergillus niger, Penicillum chrysogenum and Rhizopus stolonifer (Awuah and Akrasi, 2007). Ashraf and Khan (2007) reported effectiveness of the extracts from Gliocladium virens on the growth and multiplication of Meloidogyne javanica (nematode) infecting some eggplants. Equally, the extracts from leaves of Zingiber officinale, Vernonia amygdalina, Senna alata, Cymbopogon citrates and Ricinus communis are well documented antifungal agents against yam rot (Amusa, 2001; Nahunnaro, 2008).
The Nigerian forest is replete with over 45 different species (Sonibare et al., 2005) of the 800 members of the genus Ficus already characterised (Zerega et al., 2005). Members of this group can be found in savanna and rainforest ecosystems and beside rivers and streams. They are known for their medicinal properties and pharmacological values (Kubmarawa et al., 2007). Some phenolic compounds with pharmacological properties including funarocoumarins (psoralen and bergapten), flavonoids (rutin, phenolic acids,) and also phytosterol (Teixeira et al., 2006) were found to be abundant in this species. Ficus carica is rich in phenols, essential oils and flavonoids and other bioactive compounds such as arabinose, β-amyrins, β-carotenes, glycosides, β-setosterols and xanthotoxol (Aref et al., 2010).
The efficacy of Ficus extracts has not been documented on known pathogens of tuber crops, particularly among the members of Dioscorea. Consequently, liquid extracts from leaves of four (4) of the forty five (45) Ficus species in Nigeria were tested for their antimicrobial properties on isolated fungal and bacterial pathogens of Dioscorea rotundata with the view of providing a cheap and ready source of biological control to improve the market value of this very important tuber crop in Nigeria.
MATERIALS AND METHODS
Collection of yam tubers and plant materials: Yam tubers with symptoms of rots were collected along with fresh healthy ones from markets and distribution points in four towns in each of Ekiti and Lagos States, Nigeria in 2009 and 2010. A total of 160 yam tubers samples were collected for the study. These samples were sealed and transported in sterile polythene bags and later stored at room temperature in the laboratory.The whole shoot and foliages of Ficus thonningii (Blume), F. saussureana (De Candolle), F. exasperata (Vahl) and F. sur (Forssk) were collected from the Biological garden of the Department of Botany and Microbiology, University of Ibadan, Nigeria and fields across the ancient city of Ibadan, Oyo State, Southwest Nigeria. The plants were air dried at room temperature and stored at 24°C until ready for use.
Preparation of culture media: The Potato Dextrose (PDA) and Mueller Hilton agar for the respective culturing of fungi and bacteria were prepared according to the manufacturer’s instruction and following the techniques described by Arora and Arora (2008) for the purpose of obtaining pure cultures.
Isolation of fungal pathogens: The yam tubers with symptoms of rot were first rinsed with deionised water, surface sterilized for a minute with Sodium hypochlorite and air dried. The technique of isolating the fungal pathogens was as described by Willey et al. (2008).
Isolation of bacterial pathogens: The yam tubers with symptoms of rot were first rinsed with deionised water, cut open and pieces with a dimension of 3x3x2 mM of the infested portions were lifted. The pure culture of the test organisms was obtained using the techniques described by Arora and Arora (2008) on Mueller Hilton agar. The test was set up in triplicate.
Biochemical test analysis: The different biochemical tests analysis included Gram staining procedure as described by Cheesbrough (2002), citrate utilization test (MacWilliams, 2009), Hydrogen sulphide production test (Lehman, 2009) and Sugar fermentation and oxidase tests (Cowan and Steel, 2002). The isolates were later identified using Bergey’s manual as described by Brenner et al. (2005).
Preparation and application of plant extracts: Cold extraction of the powdered plant materials was done with ethanol as organic solvent. Following the techniques of Junaid et al. (2006), specific quantity of the powdered plant materials was soaked in the extraction solvent, filtered using Whatman No. 1 filter paper and filtrate was concentrated using rotary evaporator at 60°C.
Antimicrobial activity assay: The technique of Okigbo and Ogbonna (2006) was adopted for the screening of the plant extracts on the isolates with the 20 uL of the different concentrations (100, 75, 50 and 25 mg mL-1) poured in the agar wells. Dimethylsulfoxide solvent and 10 μg mL-1 cefoxitin were respectively applied as negative and positive controls for both treatments. The diameters of zones of inhibition were measured (mM) using a ruler. The experiment was set up in triplicate.
Minimal Inhibitory Concentration (MIC): The determination of the minimum inhibitory concentration of the extracts was as described by Adeniyi and Ayepola (2008) using 12.5-50 mg mL-1 concentrations of the extracts. The extract concentration(s) around which there was least visible growth was regarded as the minimal inhibitory concentration.
Qualitative phytochemical analysis: Aqueous extracts of the four Ficus species were screened for the presence of bioactive chemicals including alkaloids using Mayer’s reagents, tannins and saponins using ferric salt and frothing tests respectively in line with standard procedures described by Harborne (1998) and Parekh and Chanda (2007). Cardiac glycosides were determined by Keller-Keliani’s tests while sodium hydroxide test was employed for the detection of flavonoids as described by Trease and Evans (2002). Two hundred milligrams of the extracts was dissolved in appropriate solvents: methanol for alkaloid determination and deionised water for both tannins and saponins.
Pathogenicity test: The yam tubers were prepared for Pathogenicity tests following the techniques described by Priou et al. (1999). The inoculated yam tubers were covered with the yam tissue 6 cm in diameter, sealed with a wax and left for six weeks.
Statistical analysis: Percentages and statistical mean were employed to analyse the data generated. The different effects of Ficus extracts on the test organisms was analysed using ANOVA.
RESULTS AND DISCUSSION
The fungal pathogens isolated from rot portions of the different yam tubers included Aspergillus flavus, A. niger, Botryodiploidia theobromae, Fusarium oxysporium, Penicillium chrysogenum, P. oxalicum and Rhizopus stolonifer (Table 1). A similar profile of fungal species was obtained from some yam tubers (Okigbo, 2004; Okigbo and Nmeka, 2005) in Southeast, Nigeria. However, Mucor circinelliodes and Sclerotium rolfsii were consisted in related group of fungal pathogens isolated from some yam tubers also from Southeast, Nigeria (Nahunnaro, 2008). Fusarium solani was the rare inclusion in the similar fungal profile isolated from yam tubers in Owerri, Imo State, Southeast, Nigeria. A similar fungal isolates from Yola town in Adamawa State, Northeast, Nigeria (Yusuf and Okusanya, 2008) and Keffi in Nassarawa State, North central, Nigeria (Ogaraku and Usman, 2008) consisted of Sclerotium rolfsii and Rhizoctonia spp. and F. solani as rare inclusions, respectively. These rare inclusions may be linked to the differences in soil and climatic conditions and as well as the prevailing and dominant fungi spores at harvest.The percentage occurrence of Fusarium oxysporium and Penicillium oxalicum was 100% and they were found in all the plates examined (Table 1) while the percentage occurrence for Fusarium solani was 92 and 89% for both Aspergillus niger and Penicillum chrysogenum. Ezeibekwe et al. (2009) reported similar spread and observed that Fusarium oxysporium and Botryodiplodia theobromae occurred 33.3% more than other organisms.
| Table 1: | The percentage occurrence of the isolated bacterial and fungal pathogens in the sampled yam | |
| Table 2: | Induced infection on yam tubers using the bacterial and fungal pathogens | |
| Table 3: | Organisms and associated diseases | |
The Pseudomonas spp. and Klebsiella spp. are the two bacteria pathogens isolated from this study (Table 1). This result differs from the ones reported by Okigbo (2004) for the sampled yam tubers from the Southeast Nigeria and his results also tallied with the observations of Amusa (1999) who reported similar bacterial profile for some yam tubers from North central Nigeria. This is the first time a different bacteria profile is being implicated in the rot disease of yam tubers in the Southwest Nigeria. However, the Pathogenicity test could not confirm these bacteria as being majorly responsible for the rot disease (Table 2). They are likely opportunistic organisms found in association with the isolated fungi species.
The isolated fungal species were confirmed as the natural pathogens responsible for the rot disease in the sampled yam tubers (Table 2). The intrinsic ability of some isolated fungal pathogens at causing significant rot of exposed yam tubers has equally been reported (Okigbo and Ogbonna, 2006). The average spread of the rotted area (48 mM-64 mM) was observed for all the fungal species. Penicillium chrysogenum exhibited a wider rotted area (62 mM) followed by Fusarium solani with 60 mm spread (Table 2). A significantly different result was reported by Ezeibekwe et al. (2009) in which they observed that Rhizopus oryzae exhibited a 25.37 cm spread and a 17.20 cm and 12.65 cm spread by Botryodiplodia theobromae and Fusarium solani, respectively. These implicated organisms posed a significant threat to the revenue of farmers and the health of consumers at large.
The consequence of this contamination may be severe, as these fungi and bacteria have been implicated in a number of gastro-intestinal, respiratory and related health complications in humans (Table 3). The soil may have been contaminated by sewage and other domestic and household wastes. The sanitary state of most storage facilities is deplorable and farm produce are often exposed to contamination while in storage. Regular fumigation and cleaning of these facilities will minimise the incidence of spoilage and ensure good market value for these products.
| Table 4: | The antimicrobial effects of Ficus extracts on bacterial and fungal pathogens a, b, c, d = diameter of inhibition in millimeter | |
| 50-100 mg mL-1 concentrations inhibited fungal growth (p = 0.05) significantly while growth of bacterial pathogens was successfully inhibited at 75-100 mg mL-1 when compared with positive controls | ||
There is paucity of data on bacteria induced yam rot, particularly in Nigeria and the volume of reports from local studies has been on fungal pathogens which are otherwise considered major pathogens that can affect the table and market values of tuber crops in the tropics. The combined analysis of variance (ANOVA) showed that the extracts from the four Ficus species exhibited a significant (p = 0.05) difference against the growth of all the Fungi species studied at 75-100% concentrations (Table 4). The minimum inhibitory concentration (2-12 mM) at 25 mg mL-1 was equally effective at interfering with the growth of all the fungal pathogens identified. The strong antimicrobial activity of these extracts confirms their efficacy as bio-control agents against the rot disease and the causative agents. There was no visible inhibition of fungal growth around the two wells designated as positive and negative controls.
Lopez et al. (2005) reported that Ocimum oil was active against several species of bacteria (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Shigella and some Salmonella spp. and Proteus spp.) and fungi (Trichophyton rubrum, T. mentagrophytes, Cryptococcus neoformans, Penicillium islandicum and Candida albicans). Alhussaen et al. (2011) reported a significant reduction in the growth of Pythium ultimum on the foliages of tomato seedlings exposed to undiluted garlic extract. Extracts from foliages of some plants (Sharma et al., 2003; Nahunnaro, 2008; Kubmarawa et al., 2009) confirmed them as effective bio-control against few fungal pathogens of yam tubers. The popular Azadirachta indica proved efficacious on some fungal pathogens of yam tubers (Okigbo and Ogbonna, 2006). Boughalleb et al. (2010) reported resistance to Fusarium isolates by Cucurbita species grafted with Citrullus lanatus (watermelon). Plant extracts are relatively low in toxic substances and exert no deleterious and mutagenic effects on plants tissues (Kubmarawa et al., 2009) and hence, their use is gaining more importance among the local farmers.
The presence of alkaloids, flavonoids and cardiac glycosides in the four Ficus species screened may have conferred the antimicrobial properties on these groups of plants and which helped transform them as effective bio-control agents against the isolated bacterial pathogens. Flavonoids and phenolic compounds including psoralen and bergapten (Teixeira et al., 2006) and essential oils and other bioactive compounds such as arabinose, β-amyrins, β-carotenes and glycosides (Aref et al., 2010) have been found to be in abundant in some Ficus species. The essential oil of pepper fruit and activity of the phenolic acids have been shown (Ejechi et al., 1999) to be effective against tomato rot fungi. They suggested that the presence of b-phenylnitroethane may be responsible for the pungent properties of this essential oil.
Ejechi and Akpomedaye (2005) equally reported the efficacy of some essential oils against some food borne microorganisms including Staphylococcus aureus, Salmonella spp. Pseudomonas aeruginosa, Proteus spp. Escherichia coli, Enterococcus faecalis, Serratia spp. Bacillus spp. Clostridium spp. Penicillium spp. and Aspergillus flavus isolated from food products. The leaf extracts from the species of Ficus demonstrated a broad-spectrum of activity against Pseudomonas and Klebsiella spp and the fungal pathogens and which confirms them as antimicrobial agents for the control of rot diseases of yam tubers, particularly in the tropics and similar environment.
CONCLUSION
The profile of fungi and bacteria pathogens implicated in the yam rots may be dependent on the prevailing fungal spores at harvest and the severity of rot was accentuated by soil and prevailing climatic conditions. The contamination of soil through domestic and household wastes and poor sanitary condition of the storage facilities were suggested as sources of fungi infection. The fungal pathogens isolated included Aspergillus A. niger, Botryodiploidia theobromae, Fusarium oxysporium, Penicillium chrysogenum, P. oxalicum and Rhizopus stolonifer and were confirmed as causative agents of yam rots in southwest, Nigeria. The bacteria isolated viz: Pseudomonas spp. and Klebsiella spp. were regarded as opportunistic organisms as they could not be confirmed as rot agents. The leaf extracts at 50 mg mL-1-100 mg mL-1 concentrations had profound antimicrobial properties on the fungal and bacterial organisms. These extracts contained alkaloids, flavonoids and cardiac glycosides and may have conferred the antimicrobial properties on this group of plants.ACKNOWLEDGMENT
The financial assistance and facilities for the successful completion of this study was provided by the Redeemer’s University, Nigeria. Miss O. O. Fatoki typed the manuscript.