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Coou.edu.ngAntimicrobial Activities of A. sativum, Z. officinale and O. gratissimum Extracts on Plant and Fish Pathogens
Department of Microbiology, Department of Applied Department of Microbiology, Microbiology and Brewing, Ojukwu University, Anambra Nnamdi Azikiwe University, Awka Ojukwu University, Anambra State, State, Nigeria. Anambra State, Nigeria The spoilage of Tomato (Lycopersicon esculentum) and Orange (Citrus sinensis) from Eke Umuoma market in Uli, Anambra State, South-Eastern Nigeria were investigated. Healthy and Spoilt samples of orange and tomato were brought to the laboratory and analyzed for the presence of pathogens using the pathogenicity test. The isolation of fungi from the plant samples was carried out on Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) respectively. The most common fungi associated with fruit decay were R. stolonifer (found in orange and tomato), Fusarium solani, Aspergillus niger and P. digitatum . Characterization of various isolates from fish and fish products based on biochemical tests like oxidase, citrate, coagulase and Triple-Sugar-Iron test used on Thiosulphate-Citrate- Bile-Salts, Salmonella-Shigella Agar revealed S. aureus, V. vulnificus, Salmonella sp. and Vibrio sp. The method employed for the bioactive component determination and antimicrobial activity are phytochemical analysis and food poisoning technique. The result revealed the presence of flavonoids, phenolics, saponins, tannins, glycosides, phlobatannins, resins, steroids, alkaloids and quinones. Also, the results revealed that the combination of ethanolic extracts of A. sativum and Z. officinale, O. gratissimum, O. gratissimum, O. gratissimum, and combination of A. sativum and Z. officinale had the highest growth inhibition percentages of 37.84, 38.61 ,40.42, 34.10 and 56.43 at concentrations of 5%,15%,15%,15% and 15% on the fungal pathogens P. digitatum, R. stolonifer, F. solani, A. niger and R. stolonifer respectively. Moreso, spectrophotometric results of the percentage inhibition of fish pathogens by the plant extracts showed that ethanolic extracts of Z. officinale, A. sativum, O. gratissimum, combination of Z. officinale and A. sativum, and combination of Z. officinale and O. gratissimum at 55.34, 57.71, 58.46, 71.26 and 76.61 had the highest percentage inhibition on S. aureus, Vibrio, S. aureus, S. aureus and S. aureus respectively.
Thus, there is evidence of fungistatic and bacteriostatic attributes of O. gratissimum, A. sativum and Z. officinale and could be further developed pharmacologically for controlling post harvest rot of tomatoes, oranges and fishery products by several pathogenic fungi and bacteria.
Key words: Pathogenicity, Lycopersicon esculentum, Citrus sinensis, Inhibition, Z. officinale, A. sativum, O.
The ubiquity of pathogenic organisms leaves us open to developing food-borne illness, chronic conditions or deadly diseases. When it comes to the food supply, pathogenic organisms can be introduced in a number of different ways.
Common vectors include water, soil, waste or fecal matter, humans and animals. For these reasons, the study and control of pathogenic organisms comprises a large part of our food safety systems.
Human infections caused by pathogens transmitted from fish or the aquatic environment are quite common and depend on the season, patients' contact with fish and related environment, dietary habits and the immune system status of the exposed individual. They are often bacterial species facultatively pathogenic for both fish and human beings and may be isolated from fish without apparent symptoms of the disease (Acha and Szyfres, 2003).
Pathogenic and potentially pathogenic bacteria associated with fish and shell fish include mycobacteria, Vibrio vulnificus, Vibrio spp; Salmonella spp. and other (Chattopadhyay, 2000). Plant pathogens of fungal origin isolated from orange and tomato respectively associated with plant spoilage were studied on Potato Dextrose Agar (PDA) and Malt Extract Agar (MEA) respectively. Plants are the richest source of organic chemicals on the earth and produce a wide variety of secondary metabolites, which can be used as defensive weapons. Fungi are the most important of the various groups of pathogen that attack plants. Our agricultural fields, forests, grasslands and urban landscapes are diminished in economic value and beauty by this important group of plant pathogens. Some fungal diseases, such as chest-nut blight, make it impossible for hosts such as the America chestnut to grow where the disease is present African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
Number of plants has been found to possess antimicrobial properties. These are Garlic (Allium sativum), Ginger (Zingiber officinale) and Scent leaf (Ocimum gratissimum).
Ajayi and Olufolaji, (2008) reported that extracts of Zingiber officinale and Gmelina aborea significantly inhibited the mycelial growth and sporulation of Colletotrichum capcici (in vitro), the causal organism of brown clotch of cowpea. Also, Wokocha and Okereke, (2005) reported that extracts of neem seeds, bitter kola, pawpaw roots and rhizomes of ginger effectively inhibited the sclerotial germination of Sclerotium rolfsii, in vitro, the causal organism of the basal rot disease of tomato. The objective of this study is to determine the inhibitory effects of A. sativum, O. gratissimum and Z. officinale extracts on isolated pathogens of plants and fishery products.
Materials and Methods
The fresh leaves of scent leaf were collected from Akwa-Ibom, Uyo State. The fish, shellfish, periwinkle were bought from Onne, Port-Harcourt, Rivers State. The rhizome of ginger and bulb of garlic, orange and tomato fruits were bought from Eke Umuoma market, Uli, Ihiala, Local Government Area, Anambra State (Akintobi et al.,2011).
Isolation of Test Fungi from Orange and Tomato
A total of 12 randomly selected fungal infected fruits and 12 unblemished, healthy and clean looking fruits were purchased. Fruits were surface sterilized by exposing them in 1min 90% ethanol and then 3 min to 1% sodium hypochlorite and then rinsed three times in sterile distilled water. Segments (3-5cm) of tissues from the margins of the rotted areas were cut out with a sterile scalpel ad serial dilution is carried out with the spoilt parts dissolved in the water inside the test tube. The middle tube (10-2) of three tubes containing bad oranges and bad (spoilt) tomatoes respectively is taken and 1 ml is dropped onto previously prepared potato dextrose agar (three Petri-dishes respectively i.e spoilt tomato and orange in MEA and PDA respectively) and incubated at 28± 10C for 5 days under 12 h photoperiod (9.8 g of PDA in 250 ml of distilled water and 0.01 of chloramphenicol for orange) and 6.3g of MEA, 2.8 g of glucose, 0.7g of peptone, 2g of Agar-agar and 0.01g of chloramphenicol for tomato). After 2-3 days, colony forming unit (CFU) was counted (Akintobi et al., 2011) Each of the fresh fruits were washed and sterilized with 75% ethanol. Then a little portion of each of the fruits was inoculated into 90 ml of sterile distilled H20 in a test tube (serial dilution). I ml is then taken from the second tube and dropped on sterile PDA and MEA plates (three each). All plates were incubated at 300C for 72 hours (Akintobi et Identification of Fungi Isolates
The pure isolated fungi were identified using cultural and morphological features according to the most documented keys in fungal identification (Klich, 2002).
This was carried out as described by Baiyewu et al., (2007). Each of the fungal isolates was tested on healthy fruits for its ability to induce spoilage. Six healthy fruits (orange and tomato) were washed with tap water and rinsed with distilled water after which they were surface sterilized with 75% ethanol. A sterile 4mm cork borer was used to make holes in each of the fruits. A colony of fungi isolate (from each pure culture) was used to inoculate the fruits and the core of the fruits were replaced. The point of inoculation was sealed with petroleum jelly to prevent contamination.
Controls consisted of six fruits each of orange and tomato, wounded with the sterilized cork borer but not inoculated.
The inoculated fruits and the control were placed in clean poly ethylene bag (one fruit per bag) each moistened with wet balls of absorbent cotton wool to create a humid environment and incubated at 30±10C for 5 days (Akintobi et al., 2011). After 72 h, the inoculated fruits were observed for symptom development. The causal agents were re- isolated from the infected orange and tomato fruits and compared with the original isolates.
Isolation, Identification and Characterization of Pathogenic Bacteria from Shellfish and Fish Products
Sample preparation were prepared . The part of the fresh fish body were scraped and swab stick was used to swab the shell fish etc. body and inserted into first test tube containing 9 ml of sterile distilled water as a stock, and five other test tubes also containing 9 ml of distilled water were arranged serially in the test tube rack. I ml of the stock was collected using a pipette to the first test tube and from the first test tube to the second test tube up to the fifth test tube i.e. 10-1, 10-2, 10-3, 10-4, and 10-5 respectively. 10-4 and 10-5 were used as the dilution factor and 1 ml was taken from African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
each factor into sterilized Petri-dish in duplicate. All plates were incubated at a temperature of 370C for 24 hours, before colony counting and isolation procedures. Identification and characterization of various isolates were based on gram-staining technique and biochemical tests. The biochemical tests include oxidase test, citrate test (Dark green), coagulase test and TSI (red) (Triple sugar Iron test, oxidase test makes use of Thiosulfate-Citrate-Bile-Salt (TCBS), Citrate test make use of Salmonella-Shigella Agar, Coagulase test make use of Mannitol Salt Agar while Thiosulfate- Citrate-Bile-Salts makes use of Salmonella-Shigella Agar. The isolates were also identified by comparing their characteristics with those of known taxa, as described by Holt et al (1994).
Preparation of the Plant Extracts
Fresh leaves of Ocimum gratissimum, the bulbs of garlic and rhizomes of ginger were thoroughly washed with distilled water (sterile and air-dried at 28°C for 2 hours. Washed garlic bulbs and ginger rhizomes were cut into small pieces using a sharp sterilized knife. They were respectively ground into power using pestle and mortar. Exactly 10g of each of the pulverized garlic, ginger and Ocimum gratissimum were respectively introduced into 100 ml of cold distilled water and 100 ml of ethanol for 48 hours at room temperature to allow for maximum extraction of the components.
This was followed by evaporation of the filtrate to eliminate the solvent. The residue was used as crude extract for each of the test plants (Wabale and Kharde, 2010).
Qualitative Determination of the Presence of Phytochemicals
The plant extract was dissolved in dilute Hcl and filtered. The filtrate was used to test for the presence of alkaloid using Dragendroff's reagent which indicated red precipitate (Kagbo and Ejebe, 2010).
1g of the extract was dissolved in 20ml of distilled water and filtered. 2 or 3 drops of 10% FeCl3 was added to 2ml of the filtrate. Blackish-blue or blackish-green showed positive result (Kagbo and Ejebe, 2010) Small amount of the extract was shaken with little quantity of water. Formation of 1cm layer of foam which persisted for 10minutes indicated the presence of saponins (Kagbo and Ejebe, 2010).
A few pieces of magnesium metal were added to 5ml of the extract and concentrated HCl. A reddish colouration indicated a. positive test for flavonoids (Kagbo and Ejebe, 2010).
50mg of the plant extract was dissolved in 5ml of distilled water. A few drops of neutral ferric chloride solution were cooled. A dark green colour indicated the presence of phenolics (Kagbo and Ejebe, 2010).
0.2g of the powder sample was extracted with 15ml of 96% ethanol. The extract was then poured into20ml of distilled water in a beaker. A precipitate occurred indicated the presence of resins (Kagbo and Ejebe, 2010).
0.5g of the extract was dissolved in 3ml of chloroform and filtered. Few drops of concentrated H2SO4was added to the filtrate to form a lower layer. A reddish brown colour was taken as positive for steroid ring (Kagbo and Ejebe, Four milliliters (4.0ml) of aqueous leaf extracts was mixed with 2ml 1% aqueous HCl and boiled for 5 minutes. The formation of precipitate was an indication of phlobatannin (Kagbo and Ejebe, 2010).
Effect of Plant Extract on Fungal growth
Different concentrations of the extracts were prepared (5% means 1 ml of extract + 19ml PDA; 10% means 2 ml of extract + 18ml of PDA and 15% means 3ml of extract + 17ml of PDA). The control contained 20ml of PDA without addition of extracts, but contained the fungal pathogens. Ethanolic extracts of ginger, garlic and scent leaf were used respectively and also in combination on all the Petri-dishes (Sharma and Borah, 2003). 58.5 g of PDA was dissolved in 1500 ml of distilled water and poured into 80 Petri dishes. The agar-extract mixture was allowed to solidify and then inoculated centrally with a 5mm diameter mycelia disc obtained from the colony of 10 day old cultures of each of the test fungi using a sterile inoculating needle. PDA plates inoculated with the test fungus but without the extract served as the control. All the plates were incubated at 270C, after which the zones of inhibition was measured for 5 days. Percentage inhibition was determined according to Vincent (1947).
Percentage growth inhibition = C-T/C X 100 Where, C = Growth of pathogen in control after incubation T = Growth of pathogen in treatment after incubation.
African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
Turbidity Test for Bacterial Pathogens
Nutrient broth medium was prepared (7.15 g in 550ml of distilled water) and dispensed in 10 ml aliquots into labeled test tube was covered with cotton wool and sterilized in an autoclave. The tubes were inoculated with the test organisms while the control was also inoculated. Two drops of both the water and ethanolic extracts were added into the test tubes except the control. All tubes were incubated at 370C for 24h and their absorbance measured at 570 nm wavelength. The difference in absorbance between the test culture and control was determined and express as a percentage. The percentage inhibition was calculated as shown below: % inhibition = AS – AU/AS X 100% Where AS = Absorbance of control; AU = Absorbance of test culture RESULTS AND DISCUSSION
Table 1: Component of plant extract and their qualitative analysis
Component analysed = Low presence; ++ = Moderate presence; +++ = High presence; - = Not present Table 2: Colonial, morphological and cellular characteristics of fungi associated with the spoilage of fruits
Colonial characteristics Morphology and cellular A bundle of sporangiophores Colonies light grey, growing Rhizopus stolonifer stretching around from rhizoids. extreme rapidly and filling the Many spores present. Sporangia petri dish with dense cottony globose or sub-globose with mycelia producing mass of some flattened base Growth moderately rapid, Oval micro conidia produced. Fusarium solani covering agar plate within 4 Micro conidia produced on days with sparse, floccose richly branched conidiophores grayish-white mycelium. A cylindrical to facilitate discolouration developed in the Colonies velvety yellow, green Penicillia Penicillium digitatum irregularly and asymmetrically sparse metulae with phialides on them, smooth conidia Colonies with loose white to Vesicles light yellow brown Aspergillus niger rapidly darkbrown changing to dark brown to black phialides. Primary phialides and upon conidia development secondary vesicles are both Colonies light grey-sporangia Colonies light grey as in the Rhizopus stolonifer globose and subglobose with tomato isolate some flattened base etc African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
Table 3: Biochemical properties of test organisms (Fish pathogens)
Keys: SSA = Salmonella-shigella agar; TCBS = Thiosulfate – Citrate – Bile-Salts Agar; MSA = Mannitol salt agar; SSA = Salmonella Shigella Agar; TSI = Triple sugar Iron testS1 = Shell fish = Marine fish 1 and 2; P1, 2 = Periwinkles 1 and 2; CF1,2 = Crayfish 1 Table 4: Effect of various plant extracts on the growth of Penicillium digitatum at various concentrations
5% Concentrations 10% Concentration.
Average colony Growth Average colony Growth Average colony Growth diameter(mm) Inhibition % diameter(MM) Inhibition % Allium sativum (Ethanolic extract) (Ethanolic extract) officinale 22.30 (ethanolic extract) Combination of 1 & 3 (ethanolic extract) Combination of 2&3 (ethanolic extract) Key: E.E. = Ethanolic extra African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
Table 5: Effect of various plants extracts on the growth of Rhizopus stolonifer at various concentrations
5% Concentrations 10% Concentration 15% Concentration Average colony Growth Average colony Growth Average colony Growth diameter(mm) Inhibition % diameter(mm) Inhibition % diameter(mm) inhibition % A. sativum (E.E) O. gratissimum E.E Z. Officinale E.E Combination of 1&3 35.30 Combination of 2 and 3 45.70 Key: E.E. = Ethanolic extract Table 6: Effect of various plant extracts on the growth of Fusarium solani at various concentrations
5% Concentrations 10% Concentration 15% Concentration Average colony Growth Average colony Growth Average colony Growth diameter(mm) Inhibition % diameter(mm) Inhibition % diameter (mm) inhibition % A. Sativum E.E O. gratissimum E.E Z. officinale E.E Combination of 1&3 Combination of 2 and 3 63.30 Key: E.E. = Ethanolic extract Table 7: Effect various plant extract on the growth of Aspergillus niger at various concentrations
5% Concentrations 10% Concentration Average colony Growth Average colony Growth Average colony Growth diameter(mm) Inhibition % diameter(mm) Inhibition % diameter (mm) Inhibition % A. sativum O. gratissimum E.E Z. officinale E.E Combination of 1 and 3 20.30 Combination of 2 and 3 20.00 Key: E.E. = Ethanolic extract African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
Table 8: Effect of various plants extracts on the growth of Rhizopus stolonifer at various concentrations
5% Concentrations 10% Concentration 15% Concentration Average colony Growth Average colony Growth Average colony Growth diameter(mm) Inhibition % diameter(mm) Inhibition% diameter (mm) Inhibition % O. gratissimum E.E Z. officinale E.E Combination of 1 and 3 41.70 Combination of 2 and 3 40.00 Key: E.E. = Ethanolic extract Table 9: Percentage Inhibition of test organisms by the plants extract determined spectrophotometrically at
Staph.aureus (P1) Vibrio (P2) aureus (S1) vulnificus Keys: GgEE- Ginger ethanolic extract, Ggwe- Ginger water Extract; GlEE- Garlic ethanolic extract, GLWE- Garlic Water extract; SLEE- Scent leaf ethanolic extract, SLWE -Scent leaf Water extract; GgEE+GLEE- Ginger Ethanolic extract plus Garlic ethanolic extract; GgEE+SLEE,- Ginger Ethanolic extract plus Scent leaf ethanolic extract P1 = Periwinkles 1& 2, MF1 = Marine fish 1; S1 = Shell fish, MF2 = Marine fish 2.
This study reveals the phytochemical and antimicrobial effect of Ocimum gratissimum, Zingiber officinale and Allium sativum on both plant (fungal) and fish (bacterial) pathogens. The importance of phytochemical agents in plant had been a subject of discussion amongst the intellectual, traditional medicine practitioners and herbal trade fare across the globe. The pharmaceutical quality and the vast spectrum of activity of some plant active principles have been reported by Umar et al. (2000) and Adenisa et al., (2000) in their studies reported on the medicinal values of some of these plants which corroborate the result of the present study. The higher susceptibility of the test isolates to ethanol extracts is not surprising as previous studies have reported methanol and ethanol to be a better solvent than water (Obi and Onuoha, 2000).
The results of the phytochemical analysis revealed the presence of tannins, phlobatannins, steroids, saponins, flavonoids and alkaloids in Ocimum gratissimum, Allium sativum and Zingiber officinale extracts. The presence of these phenolic compounds in these extracts indicates that these plants can serve as antimicrobial agents. This is because phenol and phenolic compounds have been extensively used in disinfection and remain the standard with which other fungicides are compared (Doherty et al., 2010). Phenolic compounds act as electron donors and are readily oxidized to phenolate ion or quinine, an electron acceptor (Doherty et al., 2010). The antimicrobial activity of the oils is believed to be associated with the phytochemical components of these plants (Matasyoh et al., 2007) which diffuse into and damage cell membrane structures.
The results of the effect of various plant extracts on the growth of plant pathogen clearly revealed the pronounced activities of the extracts against the tested organism. The ethanol extract was used as the active phytochemical constituents of the leaf extract had more ability to dissolve in ethanol (organic solvent) than in water (inorganic solvent) as stated above (Table 1). The effect of the different plant extracts varied on different fungal pathogens i.e combination of Allium sativum and Zingiber officinale had a pronounced effect on Rhizopus stolonifer (orange) while Allium sativum had the highest inhibitory effect on A. niger at 5% concentration but lowest in the 10 and 15% concentration respectively (Table 4 – 8). This shows that effect of plant extracts on pathogens both fungal African Journal of Education, Science and Technology, April, 2016 Vol 3, No. 2
and bacterial, varies with concentration, the active components in the plant extract and the susceptibility of the pathogen to the extract. Wabale, A.S.and Kharde, M.N. (2010) reported that extracts of Boswellia serrata leaves were found significantly superior in inhibiting the mycelial growth 24.51 %, 30.42 % and 47.89 % of F. moniliforme at 5 per cent, 10 per cent and 15 per cent, respectively. Extract of Woodfordia fruticosa and Ocimum americanum were found second best after Boswellia serrata, while extracts of Gnidia glauca and Mundulea sericea were least effective in growth inhibition as compared to other plant extracts at all the three concentrations The results of the percentage inhibition of fish pathogen by the plant extracts determined spectrophotometrically (Table 9) shows that these plant extracts are able to inhibit the growth of the test organisms irrespective of the method or means of its exposure to microbes, studies should be directed toward ascertaining the identity of the active principle. Such findings if harnessed will help in the preservation of our fruits, canned and fresh fishes, and studies should be directed in that direction. The antimicrobial mechanism of the test plant extracts is not fully understood, but like other antimicrobial agents, their effectiveness may be associated with the damage of cell wall or membrane, inhibition of protein synthesis, inhibition of nucleic acids replication of the pathogen amongst The traditional spices studied in this work were found to have antimicrobial properties against known pathogens often implicated in fruits and fishes e.g. shellfish, periwinkles, etc. Since these spices are used in traditional food preparations in Nigeria especially south-eastern Nigeria. Therefore, the use of these spices as seasoning in food preparation is highly recommended as it helps combat against both fungal and bacterial pathogens in our foods and our bodies. The phytochemical properties of these spices revealed that they do not contain harmful phytochemicals like cyanide as HCN, but rather contain phenolic compounds which are being used extensively in disinfection and serves as a benchmark for fungicides and bactericides.
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LAB #: U CLIENT #: PERCENTILE per g creatinine 2.5th 16th 50th 84th 97.5th 3,4-Dihydroxyphenylacetic acid (DOPAC) 3-Methoxytyramine (3-MT) Norepinephrine, free Epinephrine, free 5-Hydroxyindolacetic acid (5-HIAA) Phenethylamine (PEA) <dl: less than detection limit