UNIVERSITY OF GHANA LIBRARY QK865. Ap 2 blthr C.l G325747 The Balme Library llilll 3 0692 1078 6809 1 University of Ghana http://ugspace.ug.edu.gh EVALUATION OF POSSIBLE FUNGITOXIC AND ANTIBACTERIAL ACTIVITY OF SOME LOCAL PLANTS A thesis presented by ALFRED KOFI APETORGBOR, B.Sc.(HONS) in part fulfilment of the requirements for the M .Phil. Degree of the University o f Ghana. December, 1991 From: The Department of Botany University of Ghana Legon. University of Ghana http://ugspace.ug.edu.gh DECLARATION I, the undersigned, ALFRED KOFI APETORGBOR, do hereby declare that except references to other peoples’ work which have been duly cited, this work is the result o f my own original research and that the thesis has never been presented, either in part or whole for another degree elsewhere. (Dr. G.T. Odamtten) Supervisor (Alfred K. Apetorgbor, B.Sc.(Hons) University of Ghana Legon. University of Ghana http://ugspace.ug.edu.gh ABSTRACT 12 families on some aspects o f the physiology of five fungi (Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii, Nigrospora sp .), and five bacterial species (Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella (Group C,), Escherichia coli (EPEC 0 :43), E. coli (ETEC OK5)) were investigated in vitro in either liquid or agar medium amended with varying dilutions (1:1 - 1:5 v/v) of the extracts. Scopulariopsis brevicaulis grew best at a temperature of 30°C and is being recorded for the first time in soil from Ghana. Comparatively higher fungistatic effects were found in both the water and the methanol extracts of leaves of Cassia rolundifolia, Pergularia daemia, Ahernanthera pungens, Voacanga africana, Launaea taraxacifolia, Tridax procumbens, Zanthoxylum xanthoxyloides, Oxalis corniculata, Azadirachta indica, Desmodium triflorum, Euphorbia heterophylla and Crotalaria retusa (in decreasing order) when the five test fungi were cultured on solid agar medium. Vegetative growth of mycelial discs of test fungi (Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii, Nigrospora sp.) immersed in 1:1 v/v dilution of plant extracts for varying periods (1/4, 1/2, 1, 3, 12, 24, 48h) were variably depressed by the extract after transfer to extract-free (Potato Dextrose Broth) medium. The longer the period of immersion, the greater the depression of vegetative growth. Oxalis corniculata showed the highest antibacterial activity among the eight plants (Oxalis corniculata, Pergularia daemia, Desmodium triflorum, Ahernanthera pungens, Voacanga africana, Cassia rolundifolia, Zanthoxylum xanthoxyloides, Azadirachta indica) tested; the inhibitory effect of 0 . corniculata was akin to what was obtained when standard antibiotics (35 /xg/ml Streptomycin, 5 /xg/Chloramphenicol, 5 /xg/ml Oxytetracycline) were used. The effects of water and methanol extracts of 19 Ghanaian plants belonging to University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS Page I. IN TRODUCT ION ......................................................................................................................... 1 n . LITERATURE REVIEW ...................................................................................................... 14 m . MATERIALS AND GENERAL M E TH O D S ................................................................... 23 (i) Materials: ................................................................................................. 23 (ii) General M e th o d s ................................................................................................................. 24 (a) Preparation of Plant e x tra c ts ................................................................................ 24 (b) Composition of culture media: ........................................................................... 24 (c) Method of sterilization .................................................................................... 25 (d) Assessment of vegetative growth by oven-dry m ethod................................. 25 (e) Assessment of vegetative growth of test fungi on Agar ........................ 25 (f) Antibacterial activity of plant e x t r a c t s ............................................................. 26 (g) Statistical A na ly s is .................................................................................................. 27 IV. EXPERIMENTAL PROCEDURE ...................................................................................... 28 A. Effect of temperature on growth of Scopulariopsis brevicaulis ................ 28 B. Vegetative growth of Scopulariopsis brevicaulis in basal medium Amended with water extract of six plants ......................................................................... 28 C. Vegetative growth of five test fungi on solid medium amended with water and methanol extracts of plants in the family C om po s ita e ...................... 29 D. Vegetative growth of five test fungi on solid medium amended with water and methanol extracts of plants in the family Leguminosae (tribe Papilionoidae and Caesalpinoidae)........................................................................... 29 E. Vegetative growth of five test fungi on solid medium amended with water and methanol extracts of plants in the family Rutaceae and Meliaceae............................................................................................................................ 30 F. Studies on the effect of water and methanol extracts o f plants in the family Malvaceae, Amaranthaceae, Nyctaginaceae and Oxalidaceae on vegetative growth of five fungi............................................................................... 30 G. Studies on the vegetative growth of five fungi on solid medium amended with water and methanol extracts of plants from the family Apocynaceae, Asclepiadaceae and Euphorbiaceae.......................................................................... 31 H. Comparative fungistatic activity of extracts of nineteen plants on vegetative growth of five selected fungi................................................................ 31 I. Growth o f five fungi stored in water and methanol extracts o f nine Plants............................................................................................................................................. 32 University of Ghana http://ugspace.ug.edu.gh J. Antibacterial activity of selected plants 37 V. RESULTS ................................................................................................................................ 39 A. Effect of temperature on growth of Scopulariopsis brevicaulis . . . 39 B. Vegetative growth of Scopulariopsis brevicaulis in basal liquid medium amended with water extract o f six plants.................... 41 C. Vegetative growth of five test fungi on agar (solid medium) amended with water and methanol extract of plants in the family Compositae..................................................................................................................... 43 D. Vegetative growth of five test fungi on agar (solid medium) Amended' with water and methanol extracts of plants in the family Leguminosae (tribe Papilionoidae and Caesalpinoidae) ...............67 E. Vegetative growth of five test fungi on agar (solid medium) amended with water and methanol extracts of plants in the family Rutaceae and Meliaceae............................................................................................89 F. Studies on the effect of water and methanol extracts of plants in the family Malvaceae, Amaranthaceae, Nyctaginaceae and Oxalidaceae on vegetative growth of five fu n g i ..............................................99 G. Studies on the vegetative growth of five fungi in medium amended with water and methanol extracts of plants from the family Apocynaceae, Asclepiadaceae and Euphorbiaceae ..................... 120 H. Comparative fungistatic activity o f extracts of nineteen plants on vegetative growth of five selected fungi ............................................... 137 I. Growth of five fungi stored in water and methanol extracts of nine plants.............................................................................................................142 J. Antibacterial activity of eight selected p la n ts .............................................. 148 VI. GENERAL DISCUSSION ........................................................................................ 151 VII. SUMMARY...........................................................................................................159 ACKNOWLEDGEMENTS................................................................................................ 164 VIII. LITERATURE CITED ........................................................................ 165 APPENDICES 180 University of Ghana http://ugspace.ug.edu.gh There are several examples of plant diseases caused by microorganisms. These include storage rots (Fusarium spp.), seedling diseases, root rots, gall diseases (Plasmodiophora brassicae), vascular wilts (Fusarium oxysporum f. cubense), leaf blights (Alternaria solani), rusts (Puccinia spp.), smuts (Ustilago spp.), powdery and sooty mildews (Leveillula taurica, Meliola cojfeae, etc.) and viral diseases (Tobacco mosaic virus, Cocoa swollen shoot virus, etc) (Clerk, 1974; Tarr, 1972; Butler and Jones, 1949; Wood, 1967). The potential of disease affecting the economy of a country i s vast. For example, approximately 10 per cent of the world’s agricultural production is lost through the attack of crops in the field or their products in transit or storage by plant diseases (Clerk, 1974). The major pests and pathogens taking a heavy toll on agricultural crops in the field and in storage are insects and fungi. The idea of controlling them by the use of chemical eradicants and protectants is not new. The first naturally occurring insecticide, nicotine - from extracts of tobacco leaves - v/as used to control the plum curculio (Conotrachelus nenuphar) and the lace bug (Steplianitis sp.) (Cremlyn, 1978). About 141 years ago two important natural insecticideswere introduced; rotenone from the roots of the derris plant (Derris sp.) and pyrethrum from the flower heads of a species of Chrysanthemum. The 1930s really represent the beginmcjof modem era of the synthetic organic pesticides and fungicides. Important examples include an organic fungicide salicylanide (Shirlan), in 1931; dithiocarbamate, valuable as foliar sprays for the control of a range of pathogenic fungi including scabs (Elsinoe fawcetti) and rots of fruit (Sclerotium rolfsii, Penicillium digitatum) and potato blight (Phytophthora infestans) I. INTRODUCTION 1 University of Ghana http://ugspace.ug.edu.gh in 1934; 2,4 - dinitro 6 - ( l ’-methyl-n-heptyl) phenyl crotonate or dinocap in 1946 and, chloranil tetrachloro - 1,4-Benzoquinone in 1938. These were called protectant fungicides, the former was especially valuable against powdery mildew (Cremlyn, 1973). By 1966 the major classes o f systemic fungicides developed were the oxathins, benzimidazoles, thiophanates, and pyrimidines. Other effective systemic fungicides currently in use include antibiotics, morpholines, and organophosphorus compounds (Cremlyn, 1978). Many of the original chemical fungicides developed were generally injurious to the environment with non-specific activity. Fungicides such as sulphur, Bordeaux mixture and organomercurials tended to be comparatively non-specific in their toxicity towards fungi. Later work led to the discovery of less poisonous and more selective organic chemical fungicides like the methylthio fungicides (Cremlyn, 1978). There is now greater awareness of the dangers of environmental pollution arising from the widespread application of chemical pesticides and fungicides (Carson, 1963) and candidate chemicals have to pass increasingly stringent tests on their toxicity, and residue formation before they can be marked as pesticides and fungicides in many countries. This has provided impetus in research on new agrochemicafand fungicides with the view of applying naturally derived compounds which are safer and more selective in their action and do not affect non-target organisms. Naturally occuring substances in plants have been used in medical preparations and herbal therapy. It is known that in the early nineteenth century, 80 per cent of the drugs were originally prepared from plants (Moss.\ et u i , 1983). 2 University of Ghana http://ugspace.ug.edu.gh Most plants contain secondary metabolites having peculiar individual properties, and often having no known relation to the metabolism or functions of the plant as a whole. Such plant constituents may vary from one species to another. When such a substance exerts an influence on the structure or function of another organism it is known as an "active principle" (Githens, 1949). It is the presence of such principles which provide the therapeutic value of plants. Penso (1982) estimated that about 20,000 plants are used medically for treating various ailments. The need to evaluate the biological activities of plant extracts and their phytochemical constituents is not only important for the development of new therapeutic agents but the new chemicals isolated from the plants with some biological activity become a springboard for chemists exploring to manufacture possible synthetic analogues from these naturally occuring compounds. The use of angiosperm plant extracts to control microorganisms has been reported (Almagboul er al. 1985 a, b; Saksena and Tripathi, 1986; Tomes et a l., 1986; Chiappeta et al., 1988). For example, Saksena and Tripathi (1986) studied 15 aromatic plants for their fungistatic activity against some common fungi present in the air, namely Aspergillus fumigatus, A. niger, Rhizopus arrhizus, Mucor mucedo and Alternaria alternate!. They showed that the spore concentration of these fungi in the air could be controlled by the volatile compounds from the various plant species. The leaf, flowers, stem and root extracts of two varieties of Cc tharanthus rose us were antifungal in their effect against Hebninthosporium nodulosum, Sderotium rolfsii, Fusarium oxysporutn, and Aspergillus niger which parasitise green gram, brinjal, tobacco, and groundnut respectively. The extracts inhibited spore germination, sporulation and mycelial growth 3 University of Ghana http://ugspace.ug.edu.gh of the test fungi. The extracts markedly differed in their fungistatic activity. Leaf extractswere generally more repressive than ex trac t from flower, stem and roots. As part of an on-going research in this laboratory, the phytochemical effect of the extract of plant parts of the flora of West Africa have been tried on many fungi including Sclerotium rolfsii, Aspergillus niger, A. flavus, Phytophthora palm ivora, etc. (Ahiabu, 1985; Okyere, 1986; Boateng, 1986; Myles, 1986; Otoo, 1987). Encouraging results spurred on further studies using a w ider spectrum o f plants and test microorganisms. The following plant species were screened for their antifungal and antibacterial activity against selected microorganisms. Plant Materials The plants were selected from the following families: Compositae, Leguminosae (tribes Papilionoideae and Caesalpinioideae), Rutaceae, Meliaceae, Malvaceae, Amaranthaceae, Nyctaginaceae, Oxalidaceae, Euphorbiaceae, Asclepiadaceae and Apocynaceae. Information on their medicinal and phytochemical composition are readily available in the pertinent literature (Oliver, 1960; Kokwaro, 1976; Odebeyi & Sofowora, 1979; Rizk, 1982, Sofowara, 1982; Ampofo, 1983; Ayitey-Smith, 1989; etc.) 1. Pergularia daemia (Forsk) Chiov. : P. daemia is used as a cure for various ailments. The whole plant is used for rheumatism and for any eye complaint; as an emenagogue for regulating menstruation; antiseptic, anaesthetic or analgesic, for fever remedies and sores (Sofowora, 1982). It is used for infantile and general diarrhoeae (Oliver, 1959); as an anthelmintic for 4 University of Ghana http://ugspace.ug.edu.gh gu inea w o rm , as exp ec to ran t and em e tic (So fow ora , 1982); fo r h ead ach es , sm a ll pox in fec tion , as com ponen t o f soap fo r g en e ra l d eb ility and to rem ove excessive o ils from a b aby ’s sk in , as an ana lg esic o r to re lea se w eakness o r slugg ishness in p re g n an t w om en , and as an enem a fo r in fan tile te tanus (E lew ude, 1979). P ou ltic e o f leaves o f P. daem ia is app lied to bo ils and abscesses (O liver, 1959). T he ju ic e o r la tex is used as as thm a rem edy and fo r so re eyes; the cha rred stem is used fo r cough , w h ile the b a rk is fried in oil w hich is then rubbed on the sk in fo r treating craw -craw in L ib e ria by the M ano tribe , and fo r superfic ia l d ry skin le sions (Sofowora, 1982). In E ast A frica , the ro o t is chewed o r pounded and soaked in cold w ater, o r bo iled , o r used in the fo rm o f an in fusion fo r coughs, and an in fu sion o f the roo t is taken for stom ach pains and as an abortifac ien t (Kokwaro, 1976). A decoction o f the leaves o f P. daem ia is given to ch ild ren fo r asthm a while the ju ice o f the leaves is used to treat in fan tile d iarrhoea, catarrhal in fections, am enorrhoea and dysm enorrhoea (Chopra et al, 1958). Investigations showed the presence o f many cardenolides like calactin , calo tropagenin , uzarin and coroglaucigenin , methyl sterols like a -am yrin , p -am yrin and lupeol and p - sitosterol in various parts o f the p lant including leaf, stem , roo t, etc. (M ishuhashi and Sasaki, 1963; Rhakit et al, 1959; Pattabiram an and Barua, 1958). 2. A ltem anthera pungens (L inn.) L ink .: Dokosi (1969) reported decoction o f the whole plant is taken as gonorrhoea remedy; it is used as an enema for abdom inal pains in pregnant women; together with certain ingredients, it is abortifacient. The poultice is used externally in treating snake bite. The poultice o f the leaves and seeds o f Aframomum melegueta to which the ju ice of lime has been added is taken for sore-throat. Poultice inserted in the anus of 5 University of Ghana http://ugspace.ug.edu.gh children stops protrusion of the anus. According to Dalziel (1936) it is used for constipation with griping and is applied as enema for diarrhoeic conditions. The plant is also used to treat dysentery and rheumatism (Ampofo, 1983) and in recipe for abortion, decoction for headache; is lactogenic, curing neuralgia, and as enema (Abbiw, 1990). 3. Euphorbia heterophylla (Linn.): The juice of Euphorbia contains acrid resins and perhaps other principles which cause acute gastroenteritis if swallowed, and intense local inflammation and necrosis if injected. It is used by Native Africans for poisoning arrows, for destroying vermin, as ordeal and homicidal poisons (Githens, 1949). Associated with a resin and perhaps combined with it is euphorbon, found in the latex of many species of Euphorbia. The latex or parts containing it, is used as purgative, expectorant, emetic, and vermifuge, and as an application to ringworm and other skin lesions (Githens, 1949). The flower of E. heterophylla has been found to have antibacterial activity on Mycobacterium tuberculosis (Ayitey-Smith, 1989). Rizk (1982) found that the whole plant of E. heterophylla above ground contained alkaloids, coumarins, flavonoids sterols and/or terpenes. 4. Zanthoxylum (Fagara) xanthoxyloides (Lam.) Waterman: Investigations carried out (El-Said et a l . , 1970) on the antimicrobial activity of Z. xanthoxyloides on its use as chewing stick show that it possesses antibacterial activity against oral microbial flora. The antimicrobial activity of Z. xanthoxyloides has been shown to be due to benzoic acid derivatives (Odebeyi & Sofowora, 1979). Z. xanthoxyloides also contain some active principles (alkaloids) called fagaridine, 6 University of Ghana http://ugspace.ug.edu.gh chelerythrine, skimmianine, dihydrochelerythrine and arterine (9-ethoxychelerythrine) (Torto et al., 1969). The root and bark of this plant is used for treating whooping cough (Ampofo, 1983), and in Ghana the root bark is commonly used for dental dressing. It is also used for the treatment of ganglia, swellings and rheumatic pains where ointment made from extracts of its roots and those of Clausena anisata and Piper guineensis is applied topically (Ayitey-Smith, 1989). The root extracts are used as fish and rat poison. 5. Tridax procumbens Linn.: The essential oils isolated from T. procumbens showed inhibitory activities against houseflies (Musca domestica), mosquito (Culex fatiguns) larvae, Dysdercus similis and cockroaches (Supella spp.). The oil also contained strong insect repellent activity when tested against three varieties of an ts : Cataulacus taprobunae Sm ., Componotus compressus Fabr. and Solenopsis geminata Sm. (Pathak and Dexit, 1988). During their field studies Pathak and Dexit (1988) observed that the plant was neither affected by insects nor grazed by cattle. T. procumbens is often regarded as a weed. 6. Desmodium triflorum (Linn.) DC.: A paste of the bruised leaves of D. triflorum is applied to itches and indolent sores. Fresh leaves are used as galactagogue and for diarrhoea, abscesses, and wounds (Datta and Benerjee, 1979). It is known to confiin tannin (Githens, 1949). The plant is used as lawn, green manure and sometimes regarded as a weed. 7 University of Ghana http://ugspace.ug.edu.gh 7. Sida acuta Burm. f.: S. acuta is well known for its variety of medicinal uses in the Ayurvedic system of medicine (Dastur, 1956). The leaf juice is used as an anthelmentic, abortifacient and to heal abscesses. The root is bitter tonic, astringent, antipyretic and used in urinary and nervous diseases (Rao et al, 1984). In Ghana S. acuta is used to treat venereal diseases (Blatter et al, 1975). The herb is mashed in water and the liquid used as an enema for paralysed children to help them walk. The leaves are freqently used to cause abortion. The plant is used in Cuba and Jamaica to dispel colic. In Haiti, it is used as an enema and also as a sedative, whereas in Venezuela, it is employed in the treatment of conjunctivitis (Morton, 1981). It is also considered as astringent, cooling, and tonic; the roots are used in urinary and nervous diseases as well as disorders of bile and blood (Datta and Banerjee, 1979). The leaves serve as abortifacient in Africa and for making a poultice for sores in the Philippines. The active constituents in S. acuta are cryptolepine and vasicine (Gunatilaka et al., 1980) 8. Aspilia africana (Pers.) C.D. Adams: Leaves or flowers of A. africana are used for cleaning cut wounds, ulcers, bums (Ampofo, 1983; Ayitey-Smith, 1989) and for cataract of the eye (Ampofo, 1983). 9. Azadirachta indica A. Juss.: A. indica is claimed to possess various therapeutic values in Ayurveda in India University of Ghana http://ugspace.ug.edu.gh (Singh et al; 1987). The bark is used against cough and cold, fever, gastric and inflammatory disorders; flowers are used as anthelminthic (Chopra et a l., 1956; Kirkitar & Basu, 1965). The in vitro inhibitory action of leaves has also been reported against Plasmodium falciparum (Sudaratana et a l., 1985), The plant is used in most West A f rican countries for malaria fever (Sofowora, 1982; Ayitey-Smith, 1989). Decoction of the stem bark or leaves is drunk for this purpose and has been reported to possess antimalarial and antipyretic properties. The aqueous extract is also used in the treatment o f zone inflammatory skin diseases and malaria (Obaseki et al., 1985). It is also used as a chewing stick with some antibacterial activity. A. indica is known to contain nimbolin, tannin and various glucosides (Ayitey- Smith, 1989), nimbin and nimbidon (Oliver-Bever, 1986). 10. Emilia sonchifolia (Linn.) DC.: E. sonchifolia is used to treat convulsion in children and diarrhoea of infancy (Ampofo, 1983). In vitro antibacterial activities suggest that the plant has low antibacterial activity against some bacteria (Wong-Leung, 1988). It is used in China as folk medicinal herb for treating respiratory tract infections, pneumonia, enteritis, bacillary dysentery and urinary tract infections (Cheong & Li, 1983; Anonymous, 1979). 11. Chromolaena odorata (Eupatorium odoratum) (L.) King and Robinson: Leaves of C. odorata are known to arrest bleeding, function as antiseptic, and help to accelerate the healing of wounds. According to Bruce (1988) preparations of C. odorata could cure typhoid fever, cataract of the eye, rheumatic joints, jaundice, 9 University of Ghana http://ugspace.ug.edu.gh vaginal infections and also used as a diuretic. It is also used to cure piles (Mensah, 1988). Sarpong (1988) identified some of the active constituents o f C. odorata as terpenoids, alkaloids, flavonoids, thymol, tannins, odoratin, anisic acid, salvegenin, essential oils, and isosakuretin. 12. Boerhavia diffusa Linn: Bedi (1979) reported that root decoction of B. diffusa is used to cure fever, and the leaves are used as vegetable in India. In most parts of West Africa, the plant is used in traditional folk-medicine for the treatment of many ailments (Dalziel, 1955). In N igeria the Yoruba - speaking people usually employ infusions of B. diffusa as mild laxatives and as a febrifuge for children (Ojewole & Adesina, 1985). The roots are used for prevention, management and/or teatment of convulsive disorders in babies (Ojewole & Adesina, 1985). They reported further that concoctions of the roots are used as expectorants and for treating asthma. The thick roots, softened by boiling, are applied as a poultice to draw abscesses and to encourage the extraction of guinea worms (Dalziel, 1937). Phytochemical studies have led to the isolation and characterization of hentriacontane, sistesterol, ursolic acid (Mizra & Tiwari, 1971) and hypoxanthine-9-L- arabinofuranoside (Ahmad & Afzal,1968) from the leaves. Also an alkaloid, punamavine has been extracted as well as boerhavic acid, reducing sugars, potassium nitrate and tannins including phlobaphenes (Oliver, 1960). 10 University of Ghana http://ugspace.ug.edu.gh 13. Voacanga africana Stapf. In Senegal a root-decoction of V. africana is given to women orally to ward off the ontoward consequences of premature or precipitant parturition. The same prescription is used for painful hernias. The extract of the bark is used for washing sores (Kerharo & Adam, 1963, 1964). The latex is also applied to wounds in Senegal (Kerharo & Adam, 1964, 1974) and into a carious tooth in Nigeria (Dalziel, 1937). A root-decoction is taken in Tanzania for dysmenorrhoea and bark-decoction or root-decoction for heart-troubles (Hardi et al., 1974). In Cote d ’Ivoire a leaf-decotion is taken by enema for diarrhoea for general oedema, by friction and draughts for leprosy, and in a lotion for convulsion in infants; sap of the leaves is given as nose-drops in insanity (Bouquet & Debray, 1974). For the treatment of fatigue due to shortness of breath, a decoction of the leaves is taken orally (Kerharo & Adam, 1964). V. africana is rich in alkaloids. Alkaloids isolated include voacamine (major), voacangine, voacangrine, voacorine and vebtusine which are all hypotensive. There are also tannins and flavonoids present (Kerharo & Adam, 1974; Thomas & Bieman, 1968). 14. Griffonia simplicifolia (Vahl ex DC.) Baill.: This plant is of reputed medical value. The leaves are fed to sheep and goats in West Africa to stimulate reproduction (Odamtten et al., 1988). All parts of the plant especially the seeds, are very rich in 5-Hydroxy-L-tryptophan and 5-Hydroxytryptamine or serotonin (Odamtten et al., 1988). 11 University of Ghana http://ugspace.ug.edu.gh Test microorganisms selected for screening the plant species mentioned above for antifungal and antimicrobial activity are as follows: Test Fungi Fungal species used are known to cause plant and/or human diseases. Scopulariopsis brevicaulis (Sacc.) Bainier: This fungus is found growing on all kinds of decomposing organic matter and, unlike many moulds, flourishes on substances containing a high percentage of protein, such as meat and ripening cheese (Smith, 1960). It is found as a human parasite, causing a serious infection of the nails. Most species of Scopulariopsis (and Paecilomycis) can liberate arsenic in the form of very poisonous gaseous compounds from any substrate containing even a trace of this element. In the past there have been one or two serious cases of arsenic poisoning due to the growth of S. brevicaulis in wallpapers coloured with paris green, and it has been proposed to use this species for detecting minute traces o f arsenic in suspected materials, instead of employing the usual chemical methods (Smith, 1960). S. brevicaulis was isolated from tomato fruit harvested from and experimental farm near the Botany Department. It is the first record of this fungus in the country (Piening, 1962) I. Sclerotium rolfsii Sacc.: S. rolfsii is the most frequently encountered fruit rot pathogen of tomato (Lycopersicon esculentum Mill) in Ghana (Leather, 1959). It has been estimated that losses due to fruit rot in tomato by this fungus is about 30 per cent or more (Addison and Chona, 1971). S . 12 ( t University of Ghana http://ugspace.ug.edu.gh fruit rot in tomato (Lycopersicon esculentum Mill), bulb rot in onion (Allium cepa L.) * and wilt in groundnut (Aracliis hypogea L.) and potato (Solatium tuberosum L. ) (Leather, 1959). 3. Aspergillus niger Van Tiegham: Piening (1962) listed crops in Ghana which are attacked by A. niger as Allium ascalonicum, Aracliis hypogea, Butyrospermum parkii, Cocos nucifera, Elaeis guineense and Theobroma cocao. The crown and collar rot disease widespread in groundnut (Arachis hypogea) is caused by A. niger. The brown germ disease of oil palm (Elaeis guineense) caused by A. niger can kill embryos before they emerge. A. niger is also a seed-borne pathogen of stored grains and cereals and is a well known component o f the soil microflora in many parts o f the world. 4. Aspergillus flavus Link: A. Jlavus produces secondary metabolites called aflatoxins in grains, grain products and seeds. It also produces other mycotoxins like aflatram and cyclopiazonic acid in rice and wheat cultures. Aflatoxins are carcinogenic and contaminate many staple foods; they are the best known, most studied and most widespread o f all mycotoxins. Afedzi (1985) recorded the deleterious effect of A. Jlavus on Amaranthus hybridus. A. Jlavus metabolites were repressive in their effect on the length of leaves, seed germination and dry weight o f stem and roots. The metabolites of A. Jlavus also had repressive effect on seed germination of tomato (Lycopersicon esculentum seedlings (Nutsugah, 1985). Similar observations have been made by Tagoe (1987) for Cajanus cajan seedlings. 13 University of Ghana http://ugspace.ug.edu.gh 5. Nigrospora sp. Zimm.: This fungus was suspected to be a dermatophyte. Dermatophytes infect the skin, hair or nails. This fungus was scraped from the palm of an infected man. Dermatophyte infections may be highly infectious and irritating (Clegg and Clegg, 1973). Normally species of Nigrospora are also known to be parasitic or saprophytic on plants (Barnett & Hunter, 1972). Test microorganisms (Bacteria) Bacterial species used in the screening of plants for antimicrobial activity are all by and large medically important. 1. Pseudomonas aeruginosa (Gram negative): This is a common saprophyte but is frequently found as a secondary or primary invader of wounds or ulcers that have not healed properly in man and animals (Frobisher, 1968). P. aeruginosa also causes outbreaks of diarrhoea in adults and especially in newborn children; it is responsible for a leaf-rot disease in tobacco and lettuce and a fatal disease in poultry (Frobisher, 1968). 2. Staphylococcus aureus (Gram positive): S. aureus causes mastitis of women, boils, carbuncles, infantile impetigo, inter abscesses and food poisoning (Frobisher, 1968). 14 University of Ghana http://ugspace.ug.edu.gh 3. Salmonella (Group C,) (Gram negative): Salmonella spp. cause infections (commonly called salmonellosis) in man and many species of domestic animals. It may include typhoid fever, food poisoning and paratyphoid (Frobisher, 1968). 4. Escherichia coli (Gram negative): Two strains of E. coli were used - EPEC 0 :43 and ETEC 0K5. E. coli occurs commonly in the intestinal tract o f man and animals. Certain strains of E. coli cause mild to severe diarrhoea especially in infants. E. coli is found in sewage and material polluted by faeces (Frobisher, 1968). The effect of water and alcohol (methanol) extracts of 19 plants belonging to 12 families on some aspects of the physiology of the listed microorganisms were investigated and are reported in this thesis. 15 University of Ghana http://ugspace.ug.edu.gh II LITERATURE REVIEW Naturally occurring antifungal and antibacterial compounds in plants have received much attention in recent years because of the increasing anxiety o f the effect of agrochemicals on the ecosystem. Biological and phytochemical evaluation have also been carried out on many higher plants. There are several reports in the pertinent literature on the antifungal and antibacterial effects of many essential oils from plants (Maruzella & Liguori, 1958; Maruzella & Sieurella, 1960; Vanhaelen, 1973; Sharma & Singh, 1979). Essential oils of 28 plants were tried for antifungal activity against some test fungi (Microsporum gypseum, Trichophyton equinum and T. rub rum). The oils of Anethum graveolus, Artemisia officinalis, A. nilagarica, Cymbopogon Jlexuosus, C. winterianus, Cyperus scariosus, Melissa officinalis, Santalum album, Trachyspermum ammi and Vetiveria zizaniodes were toxic against one, two or all the test organisms (Dikshit & Husain, 1984). Out of these active oils, five (C. Jlexuosus, C. winterianus, M. officinalis, T. ammi, and V. zizaniodes) were found to be fungicidal. Batra and Mehta (1985) extracted oil from the seed of Argyreia speciosa and found the main component to be oleic acid. The oil had moderate antiseptic activity against several Gram positive and Gram negative bacteria and phytopathogenic fungi. Moleyar and Narasimham (1986) evaluated 15 essential oil components for antifungal activity towards five spoilage-causing fungi. In liquid shake cultures, unsaturated aldehydes (citral, cinnamic aldehyde and citronellal) followed by geraniol, an unsaturated alcohol,were most inhibitory to Aspergillus niger, Fusarium oxysporum and PeniciUium digitatunv, their minimal inhibitory concentrations (MIC) was 100 ^g/m l. 16 University of Ghana http://ugspace.ug.edu.gh Methol, a terpene alcohol was strongly inbitory to Khizopus stolonifer, and Mucor sp. with a MIC of 20 /ig/ml. Hydrocarbons like camphene, limonene and a-terpinene were least inhibitory. When incorporated in agar medium different patterns of activity were found. Thus citral, cinnamic aldehyde, citronellal, geraniol and methol not only failed to completely inhibit A. niger, F. oxysporum and P. digitatum but were more active against R. stolonifer and Mucor sp. than in liquid medium. The differences were due to vapour of the volatile test compounds which accumulated over the agar medium. Salih and Nadir (1984) investigated some Iraqi plants for possible antifungal activities against Candida albicans. About 47 total extracts, representing 38 species, were studied for their anticandidal activity. Only eight o f these extracts (from Calligonum comosum, Hypericum triquetrifolium, Myrtus communis, Propsis farcta, Pteropyrum qucheria and Quercus infectoria) were found active against the four Candida species (C. tropicalis, C. utilis, C. albicans, C. pseudotropicalis). Recently Gundidza (1986) tested 60 extracts from twelve plants in vitro for antifugal properties. Candida albicans was used as the test microorganism. Among the investigated plants, Monotes engleiri, Vernonia glabra, Musa sapienrum, Carica papaya, "ZAziphus mucronata and Cyphostemma junceum showed greatest antifungal properties. Kumar and Nene (1968) showed that leaf extracts of Cleome isocandra completely . . r inhibited the growth of several fungi, namely Helminthosporium maydis, Altejiaria solani, Glomerella cingulata, H. turcicum and Sclerotium rolfsii. Growth of Aspergillus niger and Rhizopus nigricans, however, were not inhibited. Root, stem, seed and flower extracts of the plants also prevented vegetative growth of the listed fungi. The leaf 17 University of Ghana http://ugspace.ug.edu.gh extracts were inhibitory even at a dilution of 1:200. The active ingredient appears to be quite stable as it could withstand the effect o f heat, pH and ageing. The benzene extracts of the dried seeds of C. isocandra yield a fixed oil which on standing deposits palmitic,, myristic and Wscosic acids(Gupta and Dutt, 1950). From the alcoholic extracts Gupta and Dutt (1950) obtained a flavone, viscosin, which is a monomethoxy-trihydroxy flavone. The yellow pigments of flavone type were suggested by Newton and Anderson (1929) to be the main phenolic compounds partly responsible for the resistance which some varieties of wheat show against the stem rust fungus. The activities of the crude extracts, three purified groups of compounds and the volatile oil of Aframomum melegueta were tested against several bacterial and fungal strains (Oloke et al., 1988). The crude extract showed considerable fungicidal activities against Candida albicans, Trichophyton mentagrophytes, Aspergillus niger, Botryodiplodia theobromae and species of Cladosporium cladicsporioides at very low concentrations. Similarly, the three purified groups of compounds, namely gingerols, paradols and shagaols and the volatile oil showed appreciable activities against these microorganisms. The paradols showed the greatest activity followed by the shagaols and the gingerols. The activity of the volatile oil was comparable to that o f the paradols and both compared favourably with the activity of trimethoprim - sulphamethoxazole and ticonazole. The alcoholic extracts of 32 Egyptian plants were investigated bv Ross et al. (1980 a) for their antimicrobial properties against four bacteria and five fungi. About 62 per cent of these plants showed antibacterial activity while 15 per cent exhibited a marked antifungal property. The alcoholic extracts were proved to be the most active. 18 University of Ghana http://ugspace.ug.edu.gh Ross et al. (1980 b) also found that the ethanolic extract of the seeds of Peganum hannala demonstrated a marked antimicrobial activity. They found that P. harmala contained the alkaloids harmaline and harmalol with antimicrobial activity. Almagboul et al. (1988 b) investigated 102 extracts of 18 plants, belonging to 12 families, for their antifungal activity towards Aspergillus niger and Candida albicans. Out of the extracts 32 (31 per cent) exhibited inhibitory effect against the two mentioned fungi. In Ghana very little work has been done on antifungal properties of plant extracts. Okyere (1986) found some Ghanaian weeds (Commelina vogeli, Euphorbia heterophylla, Synedrella nodiflora and Tridax procumbens) suppressing growth of sclerotia of Sclerotium rolfsii. Okyere (1986) also reported the killing of sclerotia of S. rolfsii in soil and in vitro by leaves of Tapinanthus bangwensis. Earlier, Ahiabu (1985) found that the leaf extract of T. bangwensis could prevent the growth and sporulation of Aspergillus flavus. The pertinent literature is replete with examples of the antibacterial activity of plant extracts. Kumar et al. (1988) investigated essential oils of 24 different species of k o ik a s ft-s ruXi-pcktkva t i n c Eucalyptus for their antimicrobial activities. The oils were effective againsybScteria. In general Gram positive organisms were found to be more sensitive to the oils than Gram negative; of all the essential oils tested that of Eucalyptus teriticarnis, E. camaldulensis and E. grandis were found to be effective against 13 of the 15 organisms. Bacillus subtilis and Micrococcus glutamicus were found sensitive to almost all the oils. Eugenia uniflora is a volatile oil-containing plant, native of Northeast Brazil. In Uruguay, tea made from the young leaf and an alcoholic liqueur flavoured with the leaf 19 University of Ghana http://ugspace.ug.edu.gh and fruit of E. uniflora are used for all disorders of the digestive tract (Coppetti & Gonzales, 1922). These activities were suggested to be due to the volatile oil content. In Mauritius the hot water extract of the dried leaves is given orally to adult female as an emmenagogue (Sussman, 1980) and in Nigeria as a febrifuge and antimalarial (Adebajo, 1988). Antimicrobial activities have been reported for volatile oils and expressed juice of the Eugenia genus (Garg, 1974; Rao& Nigam, 1970; Bushnell et al., 1950). Adebajo et al. (1989) assessed the antimicrobial efficacy of the essential oils of E. uniflora and their transformed microbial products collected at different times. The results obtained especially against Proteus vulgaris, Pseudomonas aeruginosa, Klebsiella pneumoniae, Candida albicans and Trichophyton mentagrophytes may be a biological confirmation of the variation of the oils. Most of the oils were inactive against Staphylococcus aureus, Serratia marcescens and Yersinia enterocolitica. P. aeruginosa was the most sensitive bacterium while T. mentagrophytes was the most susceptible fungus. The results may provide a scientific explanation for the folkloric uses of E. uniflora against digestive tract disorders. The steam distillates of leaves and flowers of Tanacetum macrophyllum were 'tasted for antimicrobial activity against five widely occurring and medically important bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus spp. and Salmonella spp (Thomas, 1989). The oils exhibited antibacterial property. Rao et al. (1989) studied the antifungal and antibacterial activities of ten essential oils of Limnophila gratissima in vitro on some Gram positive, Gram negative bacteria and fungi. Its activity was compared with those of standard antibiotics. The antibacterial activity was found to be comparable with that of streptomycin and chloramphenicol and 20 University of Ghana http://ugspace.ug.edu.gh the antifungal activity was higher than that of griseofulvin. L. gratissima possesses the odour of turpentine and yields 0.13 per cent of an essential oil containing d-limonene and d-perillaldehyde as the principal constituents. It is regarded as antiseptic, galactogogue and aperient. Plants belonging to Lamiaceae are reputed for their medicinal uses. Due to their content of essential oils, several species of this family show antimicrobial activity (Allegrini et al., 1974; El-Keltawi et a l., 1980; Dikshit and Husain, 1984; Melegari et al., 1985). However, investigations on plants utilized in folk medicine demonstrate that the antimicrobial activity is not only attributable to essential oils, but also to non-volatile substances, ie. tannins (Duquenois & Greib, 1955) and flavonoids (Kubo et a l., 1981; Miski et al., 1983). Diaz et al. (1988) examined 43 species of Spanish Lamiaceae for phytochemical content and antibacterial activity. The plants contained alkaloids, volatile oils, tannins, saponins, flavonoids, leucoanthocians, cyanogenetic heterosides and anthraquinones. The petrol and methanol-water extract of Jatropha .podagrica possesses both antimicrobial and antifungal activities. In the petrol extract the presence of citral, thymol and carvocrol was observed and a flavonoid compound, 5-hydroxy - 7, 4 ’ - dimethoxyflavone, was isolated from the methanol extract (Odebeyi, 1985). The effects showed by these compounds were however not significant when compared with o grise/ulvin, nystatin, streptomycin sulphate and chloramphenicol. From the stems o f / . podagrica the presence of tannins and alkaloid tetramethylpyrazine was observed (Odebeyi & Sofowora, 1978; Odebeyi, 1980). The antimicrobial, neuromuscular and cardiovascular actions of the plant extract were attributed to the presence of this alkaloid 21 University of Ghana http://ugspace.ug.edu.gh (Ojewole & Odebeyi, 1980, 1981). Farouk et al. (1983) screened certain Sudanese plants used in folkloric medicine for different purposes for antibacterial activity. 76 extracts of 31 plants belonging to 21 families were investigated for their antibacterial activity. Out of these extracts 64 (84 per cent) exhibited inhibitory effects against at least one organism. The results showed that when the activity is present it is not restricted to any morphological part o f the plant. The tested microganisms were Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Almagboul et al. (1985 a) carried out investigation on 198 extracts of 40 plants belonging to 18 families for antibacterial activity against four different bacterial species (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa). Out of the 198 extracts 134 (68 per cent) exhibited inhibitory effects against one or more of the microorganisms. In the same year they carried out two more investigations against the same microorganisms. Almagboul et al. (1985 b) investigated 135 extracts of 31 plants belonging to 15 families. Out of the 135 extracts, 101 (75 per cent) exhibited inhibitory effects against one or more of the microorganisms. Further studies by the same workers (1985 c) on 126 extracts o f 24 plants, belonging to 21 families showed that out of the 126 extracts, 97 (77 per cent) exhibited inhibitory effects against one or more of the microorganisms. The plants which exhibited marked antibacterial activity were shown to be rich in flavonoids, tannins and alkaloids. Later Almagboul et al. (1988 a) made 102 extracts of 15 plant species, belonging to six families. Out of these extracts 87 (85 per cent) exhibited inhibitory effects against one or more of the microorganisms. Preliminary phytochemical screening of the most active seven plants showed that five 22 University of Ghana http://ugspace.ug.edu.gh plants are rich in tannins and/or flavonoids. Alkaloids were detected in four plants but only one plant was rich in alkaloid. An active antimicrobial fraction isolated from Pleropyrum aucheri had been tested against Micrococcus luteus, Staphylococcus aureus, Escherichia coli and Bacillus subtilis (Mubarak et al. 1988). The Minimum Inhibitory Concentration (MIC) has been determined and found to be 0.40 mg/ml against E. coli and 1.2 mg/ml against S. aureus. P. aucheri is used by the people o f Al-Jezira (Iraq) as a paste for the treatment of injuries. The antimicrobial activity of several extracts from leaves and flowers of three Hypericum species was evaluated against eight different microorganisms (Sakar et al., 1988). The extracts of H. salsugineum and II. origanifolium exerted antimicrobial activity against Gram positive bacteria, the fungus (Candida utilis) and Mycobacterium smegmatis only. Generally, Hypericum species contain hypericin, pseudohypericin (Brockman, 1957), flavonoids (Kitanov, 1985; Berghofer and Hbzl, 1986), volatile oils (Mathias &. Qurisson, 1964; Mctthela & Mathela 1984), xanthones (Cardona et al., 1985; Ctien & Chen, 1983) pyrone (Kikuchi er j l . , 1985a), hyperenor. B (Kikuchi et al., 1985 b), n-alkanes (Brondz et al., 1983) n-l-alkanols (Brondz et al., 1983) and tannins (Michahok et al., 1956). Antimicrobial activity of other Hypericum species are also known (Nadir & Salih, 1985; Tanker et al . i980). The antimicrobial activity of H. perforatium and H. hircinum, all of Sicilian origin, was investigated by Barbagallo and Chisari (1987). H. perforatium was found to be most active and showed activity against Gram positive and Gram negative bacteria. Aqueous, petrol, chloroform and dichloromethane extracts of both the bark and 23 University of Ghana http://ugspace.ug.edu.gh leaves of Helimis integrifolius were tested for activity against S. aureus and C. albicans (Gundidza, 1987). The aqueous extracts showed significant activity against S. aureus and C. albicans. Wong-Leung (1988) tested 40 Hong Kong plants against selected Gram positive and Gram negative bacteria. These plants have been used traditionally in folk medicine as antibacterial agents. Four of these plants Glochidion eriocarpum , Cratoxylum ligustrinum, Psychotria rubra and Desmodium triquetrum) showed high level in vitro antibacterial activities against two or more strains of bacteria. Six of the tested herbs including Emilia sonchifolia showed no appreciable in vitro antibacterial actions although there were clinical records in China reporting that two of these herbs (Plantago major and Eclipra prostrata) were effective in treating bacillary dysentery. The extracts of different parts of Solanum viarum showed antibacterial and antifungal activities (Chiappeta et a l., 1988). Fruits and seeds showed activity against Gram positive, Gram negative, and acid-fast bacteria and yeast. The leaf and root extracts appeared to be ineffective against the microorganisms. The ethanolic and methanolic stem extracts inhibited all the test microorganisms except the acid-fast bacteria, Mycobacterium smegmatis. The roots o f Aegle marmelos are known in ethnomedicine, having properties of anti-diarrhoeitic and antidote to snake venom (Kirtikar & Basu,-1935). The presence of alkaloids, coumarins and sterols had been described in this drug (Karawya et al., 1982). Pitre and Srivastava (1987) found that ethanoate extract o f the root of A. marmelos showed moderate to appreciable activity against Vibrio cholerae, Salmonella typhimurium, Klebsiella pneumoniae, Candida albicans, Aspergillus fwnigatus and 24 University of Ghana http://ugspace.ug.edu.gh Trichophyton mentagrophytes. From this extract a coumarin named marmin was isolated. The alcohol extract of Peltophorum pterocarpum had been tested by Sethuraman et al., (1984) for its anti-inflammatory and antibacterial effects. At high doses (200 and 400 mg/kg) the extract exhibited significant anti-inflammatory activity. The extract was also effective against Streptococcus pyosenes (Gram positive). The bark of P. pterocarpum is used to cure dysentery, gargle for sore-throat and to make tooth powders. A lotion of the bark is employed as external application in muscular pains (C .S .I.R ., 1966; Hooker, 1954). Tomes et al. (1986) screened a number of Argentine higher plants for antimicrobial activity. Petrol extract of Qomplirena boliviana, G. meyeniana, Poa heucu, Wedelia glauca and Erythroxylon argentinum were active against Mycobacterium phlei. Petrol extracts of Vigna luteola was active against Micrococcus luteus. Chloroformic percolates of Senecio pampeanus and S. bonariensis were active against Micrococcus luteus and Mycobacterium phlei. The petrol extract o f Wedelia glauca was also active against Streptococcus faecalis, Micrococcus luteus and Bacillus subtilis. Jaffer et al. (1988) extracted Withania somnifera plant parts (fruits, leaves, stem and roots) separately using petrol, chloroform and methanol. The resulted twelve extracts were tested against representative Gram positive, Gram negative and two Candida species. None of these extracts showed any antimicrobial actions against Gram negative bacteria. However, leaf chloroformic, leaf methanolic and stem chloroformic extracts were the most significant anti-Gram positive bacterial agent. Leaf methanolic extract gave the highest yield in comparison with the plant’jo th e r parts. Some phytochemical studies on this plant resulted in isolation of many constituents such as 25 University of Ghana http://ugspace.ug.edu.gh tannins, sugars, phytosterols, alkaloids and steroidal lactones (Kirson et cil., 1970, 1971, Schwarting, 1963; Schroter et a l., 1966; Atal & Schwarting, 1960). Recently, Sabir et al. (1987) carried out in vitro antibacterial studies with hexane, chloroform and methanol extracts of the leaves, stems and seeds of Ardisia solanacea. The ethanol extracts showed a good activity against most of the Gram positive and Gram negative microorganisms. Qualitative phytochemical tests indicated that the plant might contain alkaloids, steroids, resins, tannins as well as reducing sugars. Ikram and Inam (1984) studied the antimicrobial activity of extracts of 42 plants of Pakistan origin. Only nine of the 42 plants including Peganum harmala showed significant activity. It is interesting to note that only the alkaloid part of P. harmala is active while the non-alkaloidal part has very little activity. The African savannah grass {Hyparrherm sp.) produces a thermolabile, partly water-soluble toxin in soil in Ghana and Zimbabwe that inhibit growth of Nitrobacter and Nitrosomonas (Meiklejohn, 1962; Clark & Paul, 1970). Nineteen Ghanaian plants belonging to twelve families were tested for antifungal and antibacterial activity in vitro using both water and methanol extracts. There is hardly any information in the pertinent literature on their potential use for controlling diseases of microbial origin in Ghana. 26 University of Ghana http://ugspace.ug.edu.gh III. MATERIALS AND GENERAL METHODS (i) Materials: The leaves of the following plants were used: PLANT Pergularia daemia (Forsk.) Chiov. Oxalis corniculata Linn. Cassia rotundifolia Altemanthera pungens (Linn.) Link. Euphorbia heterophylla Linn. Launaea (Lactuca) taraxacifolia (Willd.) Schum. ex Hornemann L13 FAMILY Asclepiadaceae Oxalidaceae urruaosae— C ae s« i|) i nic i cfeQ€» Amaranthaceae Euphorbiaceae Compositae Zanthoxylum (Fagara) xanthoxyloides (Lam.) Waterman Rutaceae Tridax procumbens Linn. Crotalaria retusa Linn. Desmodium triflorum (Linn.) DC. Sida acuta Burn.f. Aspilia africana (Pers.) C.D. Adams Azadirachta indica A. Juss. Boerhavia diffusa Linn. Emilia sonchifolia (Linn) DC. Synedrella nodiflora Gaertn. Chromolaena odorata [Eupatorium odoratum) (L.)King and Robinson. Voacanga africana Stapf. Compositae L e ^ u m i a o s a - e , — f k p JiQMuf&ci-e. — Pi Malvaceae Compositoe Meliaceae Nyctaginiaceae Compositae Compositae Compositae Apocynaceae Griffonia simplicifolia (Vahl ex DC.) Baill. L ec ju rru ryasA 'e— CMe^cJ^cvuc 27 University of Ghana http://ugspace.ug.edu.gh The test fungi used were Scopulariopsis brevicaulis, Sclerotium rolfsii, Aspergillus niger, A. flavus and Nigrospora sp. The following test microorganisms (bacteria) were also used: Pseudomonas aeruginosa (Gram negative), Staphylococcus aureus (Gram positive), Salmonella (Group C, ), and two strains of Escherichia coli: Enteropathogenic E. coli (EPEC 0 :43) and Enterotoxigenic E. coli (ETEC 0K5) (Gram positive). (ii) General Methods: (a) Preparation of Plant extracts 1. WATER EXTRACT then blended with distilled water. The supernatant liquid was strained with cheese cloth and then with filter paper using a vacuum pump (Compton Vacuum Pump, Type D/351 VM). The resulting clear liquid was made up to 1,000 ml. Dilutions o f the filtrate were made as required. 2. ALCOHOL (METHANOL) EXTRACT The leaves of the plants were sun-dried and then coarsely powdered. Alcohol extract of 50g of the powdered material was obtained using Soxhlet apparatus (Plate 1) with methanol as the solvent. The solvent was evaporated to dryness on a Rotary evaporator (Eyela Vacuum Evaporator NE) and the gummy residue suspended in 1,000 ml sterile distilled water. 28 University of Ghana http://ugspace.ug.edu.gh P la t e 1 Photograph showing apparatus used for Sohxlet extraction of methanol extract of plants. (x 0.08) University of Ghana http://ugspace.ug.edu.gh (b) Composition of culture media: 1. POTATO DEXTROSE AGAR (PDA) 200g of peeled Irish potato was boiled in 500 ml of distilled water, strained and made up to 1,000 ml. 20g dextrose and 20g agar were added. 2. POTATO DEXTROSE BROTH (PDB) 200g of peeled Irish potato was boiled in 500 ml of distilled water, strained and made up to 1,000 ml. 20g dextrose was added. 3. BASAL MEDIUM Composition of basal medium was as follows: Glucose; lOg Yeast extract; l.Og KH2P 0 4; 0.5g FeCl3; O.OOlg Distilled water; 1000 ml. (c) Method of sterilization All glassware were sterilized by heating in an electrically heated oven (Gallenkamp Oven 300, Plus series) at 165°C for 8 hours. All culture media were autoclaved at 1.05 kg/cm2 steam pressure (121°C for 15 minutes). Cotton wool plugs were covered with oluminium foil or cellophane paper to prevent penetration of any condensed water during autoclaving. 29 University of Ghana http://ugspace.ug.edu.gh (d) Assessment of vegetative growth bv oven-dry method. Growth in the liquid cultures was assessed by estimating the dry weight o f the harvested mycelium at the end of the required period of incubation indicated at the appropriate places in the text. The mycelium was collected on a previously dried and weighed filter paper and then dried for 24 hours in an electrically heated oven o f temperature 75°C. The filter paper with the dried mycelium was weighed, after being allowed-to cool in a dessicator. (e) Assessment of vegetative growth of test fungi on Agar The stock plant extract was autoclaved at 1.05 kg/cm2 steam pressure (121 °C for 10 minutes) before use. The extract was then amended with sterile basal medium containing 20g agar to obtain 1:1, 1:2 and 1:5 v/v dilutions o f the extract. 20 ml of the mixture was made to mix uniformly in the Petri plate by swirling. The plates were inoculated with 3 -mm discs of the mycelium from the growing edges of 7-day old cultures of the appropriate fungus growing on PDA. The Petri plates were inoculated at the centre along two diameters drawn at the bottom of the plates. There were two replicates for each dilution level. Unamended agar medium containing only basal medium served as control. The plates were incubated at 30°C for those inoculated with Scopulariopsis brevicaulis, Aspergillus Jlavus, Sclerotium rolfsii and Nigrospora sp. and at 35°C for Aspergillus niger. Diameters of fungal colonies were measured daily for up to 8 days. 30 University of Ghana http://ugspace.ug.edu.gh (0 Antibacterial activity of plant extracts This was done using the paper diffusion disc method (Fairbrother and Rao, 1957; Egorov, 1985). The filter paper discs (6mm in diameter) were obtained by punch perforation of Whatman No. 1 filter paper and sterilized at 160°C for 1 hour. The sterile filter paper discs were immersed in the stock plant extracts. The pH o f the extracts were adjusted to neutral by 2% ammonia solution. The test microorganisms were first incubated in 0.1% bacteriological peptone solution for 24 hours at 37°C before use. Sterile Nutrient agar (about 20 ml ) was poured into each Petri plate. The test microorganisms were evenly spread over the cooled agar surface by streaking with a sterile inoculating loop. Three sterile filter paper discs were immersed in each plant extract and placed on the plates containing the microorganisms with sterile forceps. There were four replicates for each plant extract used. Filter paper discs immersed in 5 /xg/ml Chloramphenicol, 35 pig/ml Streptomycin and 5 pig/ml Oxytetracycline served as reference standards. The Petri plates were incubated at 37°C for 24 hours. Zones of inhibition between the discs and the growing culture of bacteria were measured in millimetres. (g) Statistical Analysis Tke data^where appropriate3were analysed statistically and the results quoted at the 5% and 1% levels of significance. 31 University of Ghana http://ugspace.ug.edu.gh rv. EXPERIMENTAL PROCEDURE A. EFFECT OF TEMPERATURE ON GROWTH OF SCOPULARIOPSIS BREVICAULIS . The optimum temperature for growth o f fungi differs from one geographical region to another. The optimum recorded in connection with any specific factor has not been the same for each species and it appears desirable to examine separately each isolate used in any studies. Scopulariopsis brevicaulis isolated from soil in Ghana for the first time in the investigations reported in this thesis, is of pathological importance. The optimum temperature for the isolate of S. brevicaulis from Ghana may have a different optimum temperature for growth. This was investigated in this experiment to provide the temperature to be adopted in subsequent investigations. About 30 ml of the basal liquid medium (lOg glucose, l.Og yeast extract, 0.5g KH2P 0 4, O.OOlg FeCl3, 1000 ml distilled water) was used in each conical flask. The flasks were inoculated with 3-mm agar discs of the test fungus and then incubated at 15°, 20°, 25°, 30°, 35° and 40°C respectively. At each predetermined incubation period of 2, 4, 8, 12 andl5 days, four flasks were harvested from each temperature treatment and vegetative growth was determined using the oven-dry weight method (see materials and general methods, page 30). Results are presented in Fig. 1 cmd in B. VEGETATIVE GROWTH OF SCOPULARIOPSIS BREV ICAULIS IN BASAL MEDIUM AMENDED WITH WATER EXTRACT OF SIX PLANTS In this preliminary experiment water extract of six plants, Altemanthera pungens (Amaranthaceae), Euphorbia heterophylla (Euphorbiaceae), Oxalis corniculata (Oxalidaceae), Sida acuta (Malvaceae), Crotalaria retusa. (Papilionaceae) and Tridax 32 University of Ghana http://ugspace.ug.edu.gh procumbens (Compositae) was used in amending the basal medium used in Procedure A to obtain dilution of 1:1, 1:2 and 1:5V/V of the plant extracts. Approximately 30 ml of the amended medium was poured in E rlenmeyer flasks. Vegetative growth of S. brevicaulis was assessed after 2, 4, 8, 12 and 15 days. Results are presented in Fig. 2 and in A p p en d ic e s £>—G . C. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON SOLID MEDIUM AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY COMPOSITAE Plants contain many compounds that have been used in herbal therapy and in inhibiting growth of microorganisms. Test fungi (Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii and Nigrospora sp.) used in this Procedure are of economic importance to man. It was anticipated that if extracts of the plants belonging to the family Compositae (Launaea teraxacifolia, Tridax procumbens, Aspilia africana, Emilia sonchifolia, Synedrella nodiflora, and Giromolaena odorata) are able to depress vegetative growth and sporulation of the fungal species, the plants could provide active ingredients to be used in biocontrol of pathogenic fungi. Agar media amended with either water or methanol extracts o f the listed plants were inoculated with 3-mm discs of mycelium of test fungi and the vegetative growth was measured along two diameters. Results are presented in Figs. 3-8 and Tables 1-4 33 University of Ghana http://ugspace.ug.edu.gh fl-enrc. D. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON/(SOLID MEDIUM) AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY LEGUMINOSAE (TRIBE PAPILIONOIDEftfi AND CAESALPINDID B hE ) The experiments in Procedure C were repeated, this time using plants in the family Leguminosae (Tribe Papilionoideae and Caesalpinbideae). The plants are Crotalaria retusa, Desmodium triflorum (Papilionoideae), Cassia rotundifolia, Griffonia simplicifolia (Caesalpinbideae). The same test fungi, namely Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii and Nigrospora sp. were used. Results obtained are presented in Figs. 9-12 and Tables 5-9 E. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON AGAR (SOLID MEDIUM) AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY RUTACEAE AND MELIACSVE. Two plants Zanthoxylum xanthoxyloides (Rutaceae) and Azadirachta indica (Meliaceae) were used. Their inhibitory effect on growth of test fungi (Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii and Nigrospora sp.) were ascertained in the same way as in Procedures C and D. Results obtained are presented in Figs. 13-14 and in Tables 10-11 34 University of Ghana http://ugspace.ug.edu.gh F. STUDIES ON THE EFFECT OF WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY MALVACEAE, AMARANTHACEAE, NYCTAGINACEAE AND OXALIDACEAE ON VEGETATIVE GROWTH OF FIVE FUNGI. The screening of plants for antifungal principles was extended to cover selected plants from the families Malvaceae, Amaranthaceae, Nyctaginaceae and Oxalidaceae. The plants are Sida acuta (Malvaceae), Altemanthera pungens (Amaranthaceae), Boerhavia diffusa (Nyctaginaceae) and Oxalis corniculata (Oxalidaceae). The same procedure followed in C and D was followed. Vegetative growth on solid agar amended with water and methanol extracts (1:1 - 1:5 v/v dilution) were recorded and are presented in Figs. 15 - 18 and in Tables 12-16 G. STUDIES ON THE VEGETATIVE GROWTH OF FIVE FUNGI ON SOLID MEDIUM AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS FROM THE FAMILY APOCYNACEAE, ASCLEPIADACEAE AND EUPHORBIACEAE. It was observed in previous experiments (C-F) that the inhibitory effect of the plant extracts was variable and marginal in some instances. The wider the number of plants examined from different families the better the chance of encountering higher plants with considerable antifungal principles. The experiments were extended to include plants of the family Apocynaceae, Asclepiadeceae and Euphorbiaceae The same procedure was adopted and results are presented on Figs. 19-21 and in Tables 17-20. 35 University of Ghana http://ugspace.ug.edu.gh H. COMPARATIVE FUNGISTATIC ACTIVITY OF EXTRACTS OF NINETEEN PLANTS ON VEGETATIVE GROWTH OF FIVE SELECTED FUNGI. The ability of plant extracts to exert inhibitory effect on fungal growth is also measured by the mathematical relationship: Diameter of colony in control medium E/C = ____________________________________________________ Diameter of colony in medium amended with extract at same temperature and growth period. The experiments in Procedures C-G were repeated and the E /C ratio were determined for each fungal species tested in either the water o r the methanol extract o f the listed nineteen angiosperm plants. High E /C ratio, > 1 .0 shows some measurable inhibition of growth of the fungus. Results are presented in Tables 21-24 I. GROWTH OF FIVE FUNGI STORED IN WATER AND METHANOL EXTRACTS OF NINE PLANTS. It is possible that the metabolism of the selected fungi subjected to possible toxic effect of the water and methanol extracts from the nine selected plants may be permanently impaired or modified. The shorter the period to achieve impairment of metabolism, the better the antifungal principle exuding from the extract. An investigation was designed to find out whether mycelium of the five fungi would recover from the depressing effect of the plant extracts. The plant extracts were from Zanthoxylumxanthoxyloides (Rutaceae) Azadirachta indica (Meliaceae), Desmodium 36 University of Ghana http://ugspace.ug.edu.gh triflorum (Leguminosae-Papilionoideae), Altemanthera pungens (Amaranthaceae), Voacanga africana (Apocynaceae), Cassia rotundifolia } Griffonia simplicifolia (Leguminosae-Caesalpinoideae), Oxalis corniculata (Oxalidaceae) and Pergularia daemia (Asclepiadaceae). The nine plants were those with the highest fungistatic activity among the nineteen plants used. The investigation was carried out by studying the growth rate of 3-mm discs of the mycelium obtained from the growing edge of the appropriate test fungus after immersion in 30 ml of 1:1 7 V dilution of either the water or methanol extract of the plants for varying periods from 15 minutes to 48 hours. After the period of immersion in the extract, the mycelium in each case was washed in three changes of sterile distilled water to remove traces of the extract from the surface of the hyphae. The mycelium discs were then used to inoculate 250 ml Erlenmeyer flasks containing 30 ml of Potato Dextrose Broth (PDB). Control cultures received no plant extract treatment. The flasks were incubated at 30 °C for those incubated with Scopulariopsis brevicaulis, Aspergillus flavus, Sclerotium rolfsii and Nigrospora sp. and at 35 °C for Aspergillus niger. Vegetative growth was assessed by the dry weight method after 10 days growth. Results obtained are presented in Figs. 22-26. J. ANTIBACTERIAL ACTIVITY OF SELECTED PLANTS There are several reports of antibacterial activities of plant extracts(Dikshit and Hussain, 1984; Batra and Mehta, 1985; Kumar et a l., 1988; Adebajo et a l., 1989; etc) but the antibacterial activity of the Ghanaian plants used in this thesis has not been tried. 37 University of Ghana http://ugspace.ug.edu.gh The experiments on the antibacterial activity of both water and methanol extracts of Oxalis corniculata (Oxalidaceae), Pergularia daemia (Asclepiadaceae), Desmodium triflorum (Leguminosae-Papilionoideae), Alternanthera pungens (Amaranthaceae), Voacanga africana (Apocynaceae), Cassia rotundifolia (Leguminosae-Caesalpinoideae), Zanthoxylum xanthoxyloides (Rutaceae) and Azadirachta indica (Meliaceae) were carried out with the following test bacteria: Pseudomonas aeruginosa (Gram negative), Staphylococcus aureus (Gram positive), Salmonella (Group C, ) (Gram negative), Escherichia coli (EPEC 0:43), (Gram negative), E. coli (ETEC OK5) (Gram negative), these. The eight plants were^with the highest fungistatic activity among the nineteen plants used. Solutions of standard antibiotics, Streptomycin (35 ^ig/ml), Chloramphenicol (5 jtg/ml) and Oxytetracycline (5 jug/ml) served as reference. Discs of the solutions on sterile filter paper (6 mm in diameter) were placed on culture of the listed bacterial species. Diameter of inhibition zones were measured after 24 hours. Results are presented in Table 25 38 University of Ghana http://ugspace.ug.edu.gh V. RESULTS A. EFFECT OF TEMPERATURE ON GROWTH OF SCOPULARIOPSIS BREVICAULIS Results are presented in Fig. 1 and Appendix A. The best vegetative growth of the fungus was between 25° - 30°C (Fig. 1). Optimum growth (60 mg) in liquid culture was obtained in 12 days at 30°C and thereafter declined. Vegetative growth at 35° and 40°C was clearly inferior as the lowest vegetative growth (25.0 mg and 10-Offvg respectively) were recorded at these temperatures. Vegetative growth of the fungus at 15°C can be described as relatively good (32.5 mg; about half of what was obtained at 30°C) and was intermediate between growth at 30°C and J£>°C (F ig .l). The pH of the medium drifted from 5.2 to 6.1 in 15 days at 30°C and to pH 5.8 and pH 4.7 at 35°C and 40°C respectively (Appendix A). 39 University of Ghana http://ugspace.ug.edu.gh DR Y W E IG H T OF M Y C E LI U M (m g ) 70 2 4 6 8 10 12 14 16 PER IO D OF I N C U BA T IO N (DAYS) Fig-1• Vegetative grcwth of Scopulariopsis brevicaulis in basal liquid medium at indicated temperatures. 40 University of Ghana http://ugspace.ug.edu.gh B. VEGETATIVE GROWTH OF SCOPULARIOPSIS BREVICAULIS IN BASAL LIQUID MEDIUM ^MENDED WITH WATER EXTRACT OF SIX PLANTS. Results obtained are presented in F ig .2 and Appendices B-G. Water extract of Oxalis corniculata depressed growth of S. brevicaulis. 1:1 v/v dilution of the extract depressed growth of the fungus by 41.7 per cent in 15 days and prevented sporulation. This inhibitory effect was gradually removed with increasing dilution of the extract. The pH in this medium increased from an initial 3.0 to 7.7. The extracts o f the other plants namely Alternanthera pungens, Sida acuta, Euphorbia heterophylla and Crotalaria retusa promoted growth of S. brevicaulis. The highest dry weight of 87.5 mg was recorded in the 1:1 v/v dilution of Tridax procumbens after 15 days growth. This stimulation or increased vegetative growth was removed with increasing dilution of the extract. The pH of the medium in all instances drifted from acid to basic side. 41 University of Ghana http://ugspace.ug.edu.gh M Y C E L IU M (m g ) 1:1 1 :2 1 :5 C on t ro l 80 - 70 - 60 - 50 - 40 - 30 - 20 - 10 0 - S l d Q QCU t Q 8 10 12 14 16 80 70 60 50 40 30 20 10 0 J L P E R I O D OF I N CUB A T I O N ( DAYS) 8 10 12 14 16 Fig.2. Vegetative growth of Scopulariopsis brevicaulis in different concentrations of water exlpacts of indicated plants at 30°C 42 University of Ghana http://ugspace.ug.edu.gh C. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON AGAR (SOLID MEDIUM) ^MENDED WITH WATER AND METHANOL EXTRACT OF PLANTS IN THE FAMILY COMPOSITAE. Results obtained are summarised below: 1. Launaea taraxacifolia (a) Water extract There was no statistical difference (p_< 0.05) between vegetative growth of all test fungi on agar medium in the control and in the media ammended with water extract of this plant (Fig.3). Growth of the test fungi Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii and Nigrospora sp. in the various dilutions of the extract (1:1-1:5 v/v) was close to the control. The water extract of L. taraxacifolia therefore does not seem to exert any inhibitory effect on the test fungi. (b) Methanol extract The effect of the methanol extract on the test fungi on agar medium was variable (Fig.3 and Tables la - le). The methanol extract (1:1 v/v dilution) of L. taraxacifolia depressed vegetative growth of S. brevicaulis by about 67.0 per cent, A flavus by about 34.0 per cent and Nigrospora sp. by about 51.5 per cent. Its effect on S. rolfsii was neglegible. Statistical analysis of the results are presented in Tables la - le. 2. Tridax procumbens (a) Water extract Except for S. brevicaulis there was no statistical difference (p.<. 0.05) between ottier vegetative growth of the^test fungi in the control and in the media amended with the 43 University of Ghana http://ugspace.ug.edu.gh (a) Methanol extract The methanol extract of this plant had significant inhibitory effect on the vegetative growth of S. brevicaulis, A. niger and S. rolfsii (p_< 0.05) in all dilutions (Fig. 4; Tables 3a - 3e). Extracts of 1:1 v/v dilution depressed growth of S. brevicaulis, A. niger and S. rolfsii by 25.0, 28.6 and 38.0 per cent respectively. Vegetative growth of A. flavus in medium ammended with methanol extract did not differ significantly from what obtained in the unammended control. The methanol extract of T. procumbens enhanced the vegetative growth of Nigrospora sp. 3. Aspilia africana Both the water and the methanol extracts of this plant had no significant inhibitory effect on the vegetative growth of the five test fungi (Fig. 5; Tables 4a - 4e ). The water extract on the other hand improved vegetative growth of S. brevicaulis (Plate 3), A. niger and A. flavus\ the methanol extract enhanced growth of S. brevicaulis (Fig. 5). 4. Emilia sonchifolia Both the water and the methanol extracts suppressed vegetative growth of S. brevicaulis. Water extract of 1:1 v/v dilution depressed growth of the fungus by about 23.2 per cent while the methanol extract depressed its growth by 11.9 per cent. water extract of T. procumbens (Fig. 4; Tables 2a-2e). The water extract (1.1 v/v dilution) depressed vegetative growth of S. brevicaulis by about 36.5 per cent (Plate 2). 44 University of Ghana http://ugspace.ug.edu.gh nutrient medium anmended with indicated concentrations o of water extract of Tridax procumbens at 30 C. (Note the depression of vegetative growth in 1:1 and 1:2 v/v concentrations after 8 days.) P la t e 2 . V e g e ta t iv e g row th o f S c o p u la r io p s is b r e v i c a u l i s in University of Ghana http://ugspace.ug.edu.gh Plate 3 Vegetative growth of Scopulariopsis brevicaulis in nutrient medium ammended with indicated concentrations of water extract of Aspilia africana at 30 C for 8 days. University of Ghana http://ugspace.ug.edu.gh Growth of the other fungi (A. niger, A. jlavus, S. rolfsii and Nigrospora sp.) was not very much affected by any of the concentrations of the two extracts. 5. Synedrella nodiflora (a) Water extract The extract had no significant inhibitory effect on the vegetative growth of the test fungi. (b) Methanol extract The effect of the extract was significant on the vegetative growth of S. rolfsii. Extract of 1:1 v/v dilution depresed growth of the fungus by 31.2 per cent. The effect on the other fungi can be described as marginal. 6. Chromolaena odorata (a) Water extract The water extract depressed vegetative growth to different extent depending on the fungus. Effect on S. rolfsii was marginal, but it suppressed growth of Nigrospora sp. by 14.1 per cent. Vegetative growth of A. niger and A. flavus after 7 days was depressed by 10.3 and 13.5 per cent respectively. * 45 University of Ghana http://ugspace.ug.edu.gh The effect of the extract on vegetative growth of the test fungi also varied. Depression of vegetative growth was highest on A. Jlavus (19.6 per cent). This was followed by that of S. rolfsii (12.5 per cent), and by Nigrospora sp. (12.1 per cent) and 5. brevicaulis (11.8 per cent) (Fig. 8). (b) Methanol extract 46 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) WATER E X T R A C T -----------K 2 1 : s ---------------- contro l M ETH A N O L E X T R A C T J - PERIOD OF I N C U B A T I O N ( D A Y S ) F ig .3 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Launaea ( L a c tu c a ) t a r a x a c i f o l i a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i. 47 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) 1:1 1 : 2 i : 5 -----------------C o n t r o l P ER I OD O F ' I N C U B A T I O N ( DAYS ) F ig .3 . (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f Launaea ( L a c tu c a ) t a r a x a c i f o l i a on v e g e ta t iv e g row th o f i n d ic a t e d fu n g i . 48 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R o f C O LO N Y (m m ) ---------------- C o n t r o l WATER E X TRAC T M E T H A N O L E X T R A C T ) PER IOD OF I N C U B A T I O N ( DAYS ) F ig .4 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f L ridax procumbens on v e g e t a t i v e g row th o f i n d ic a t e d f u n g i . 49 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) K 2 .'5 PER IOD OF I N C U 3 A T I 0 N ( D A Y S ) F ig .4 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f T rid ax procumbens on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 50 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y (m m ) WATER EX TRA CT i : 2 - i :s C o n t r o l M E T H A N O L E X T RA C T PERIOD OF I NCU B A T I O N (DAYS) F ig .5 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f A s p i l i a a f r i c a n a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 51 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) i : 2 i ;s i : i WATER EXTRACT — C o n t r o l M E T HANO L EXTRACT PER I OD O F ' I N T U B A T I O N ( DAYS ) F ig .5 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f A s p i l i a a f r i c a n a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 52 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y 1m m ) . i ----------------- Control PER IOD OF I N C U B A T I O N ( DAYS ) F ig .6 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Elm l i a s o n c h i f o l i a on v e g e t a t i v e g row th o f in d ic a te d fu n g i. 53 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) : 1 : 2 : 5 P E R I O D OF I N C U B A T I O N ( D A Y S ) Fig.6 (Cont'd). Effect of varying dilutions of water and methanol extracts of Bnilia sonchifolia on vegetative growth of indicated fungi. 54 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R of C O LO N Y (m m ) S. b r e v i c a u l i sS bre vi e a uI i t A . t l a v u s A. f l a v u s .................. !: s —. — — — C on t ro l WATER E X TRACT f / . g T W A N O L E X T R A C T — i ; i PER I OD O F I N C U B A T I O N ( D A Y S ) l ' i g .7 . Et f e e t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f S y n e d re l la n c x i if lo ra on v e g e t a t i v e grow th o f in d ic a te d f i in q i . 55 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y (m m ) .............. 1; 5 PER I OD OF I N C U B A T I O N ( DAYS) F ig .7 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f S y n e d re l la n o d i f lo r a on v e g e t a t i v e grow th o f in d ic a t e d fu n g i . 56 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF c o l o n y (m m ) ............... 1:5 __________ C o n t r o l WATER EXTRACT M E T H A N O L E X T R A C T --------------------i : i PER I OD OF I N C U B A T I O N ( DAYS ) F i g . 8 . E f f e c t o f v a r y i n g d i l u t i o n s o f w a t e r a n d m e t h a n o l e x t r a c t s o f C h r o m o l a e n a o d o r a t a on v e g e t a t i v e g r o w t h o f i n d i c a t e d f u n g i . 57 University of Ghana http://ugspace.ug.edu.gh fiuuM iM m m -in r.5 Y^y\TEF ^ EXTRACT — — — C o n t r o l M ETHANO L EXTRACT PER IOD OF I N C U B A T I O N ( DAYS ) Fig.8 (Cont'd). Effect of varying dilutions of water and methanol extracts of Chromolaena odorata on vegetative growth of indicated fungi. 58 University of Ghana http://ugspace.ug.edu.gh TABLE la lhanol extract o f Launaca tanixacifolia on vegetative growth of Scopulariopsis brevicaulis at 30°C Degrees of freedom Sum of squares Mean square F-value 43 9839.55 10 6616.30 661.630 22.61 ract 3 2345.32 781.773 26.71 30 877.93 29.264 ty 1 586.28 586.277 58.30 29 291.65 10.057 Table F-value at p = 0.05 is 2.92 p = 0.01 is 4.51 TABLE lb thanol extract o f Launaea uiraxacifolia on vegetative growth o f Aspergillus niger at 35°C. Degrees of Sum of Mean F-value freedom squares square 39 9380.27 9 8567.65 951.961 88.54 ract 3 522 32 174.108 16.19 27 290.30 10.752 ity 1 280.24 280.244 724.57 26 10.06 0.387 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 TABLE lc ;thunol extract of Launaea uiraxacifolia on vegetative growth o f Aspergillus flavus at 30°C. Degrees of Sum of Mean F-value freedom squares square 39 14959.19 9 13790.26 1532.251 84.25 tract 3 677.87 225.956 12.42 27 491.07 18.188 ity 1 469.08 469.081 28.62 26 21.99 0.846 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 59 University of Ghana http://ugspace.ug.edu.gh TABLE Id thanol extract o f Launaea taraxacifolia on vegetative growth of Sclerotium rolfsii at 30 C. Degrees of Sum of Mean F-value freedom squares square 23 13709.83 5 13605.71 2721.142 589.06 :ract 3 34.83 11.611 2.51 15 69.29 4.619 ity 1 44.31 44.311 24.83 14 24.98 1.784 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 ithanol extract of Launaea taraxacifolia TABLE le on vegetative growth o f Nigrospora sp. at 30°C. Degrees of Sum of Mean F-value freedom squares square 35 19451.19 8 12395.88 1549.484 11.23 ;tract 3 3744.58 1248.192 9.05 24 3310.74 137.947 /ity 1 978.75 978.753 9.65 23 2331.98 101.391 Table F-value at p = 0.05 is 3.01 p = 0.01 is 4.72 60 University of Ghana http://ugspace.ug.edu.gh TABLE 2a of water extract of Tridax procumbens on vegetative growth of Scopulariopsis brevicaulis at 30°C of Degrees ol Sum o f Mean F-value jn freedom squares square 31 15148.47 ates 7 11680.59 1668.656 49.70 of extract 3 2762.78 920.927 27.43 21 705.09 33.576 Jditivity 1 498 06 498.061 48.11 lal 20 207.03 10.352 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 of water extract of Tridax procumbens on TABLE 2b vegetative growth o f Aspergillus niger at 35°C. : of on Degrees of freedom Sum of squares Mean square F-value »> 27 3686.68 :ates 6 3668.43 611.405 ‘ 1388.05 of extract 3 10.32 3.440 7.81 18 7.93 0.440 ■dditivity 1 0.69 0.685 1.61 ual 17 7.24 0.426 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 [ of water extract o f Tridax procumbens TABLE 2c on vegetative growth o f Aspergillus flavus at 30°C. e of ion Degrees of freedom Sum of squares Mean square F-value 27 10335.93 cates 6 10199.30 1699.884 828.97 of extract 3 99.71 33.238 16.21 18 36.91 2.051 idditivity 1 23.16 23.165 28.62 ual 17 13.76 0.809 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 61 University of Ghana http://ugspace.ug.edu.gh TABLE 2d ect of water extract o f Tridax procumbens on vegetative growth of Sclerotium rolfsii at 30 C. irce of Degrees of Sum of Mean F-value iation freedom squares square Lai 23 12937.33 Dlicates 5 12760.21 2552.042 487.39 nc. of extract 3 98.58 32.861 6.28 or 18 78.54 5.236 n-additivity 2 62.27 65.266 68.83 iidual 17 19.28 0.948 Table F-value at p = 0.05 is 5.42 p = 0.01 is 3.29 ect of methanol extract of Tridax procumbens TABLE 2e on vegetative growth o f Nigrospora sp. at 30°C. irce of iation Degrees of freedom Sum of squares Mean square F-value tal 35 17216.50 plicates 8 17164.50 2165.663 30.94 nc. of extract 3 35.39 11.795 17.04 :or 24 19.61 0.692 'n-additivity 1 1.28 1.284 1.93 sidual 23 15.33 0.866 Table F-value at p = 0,05 is 3.01 p = 0.01 is 4.72 62 University of Ghana http://ugspace.ug.edu.gh TABLE 3a Ifcct of methanol extract o f Tridca procumbens on vegetative growth of Scopulariopsis brevicaulis at 30°C mrce of .nation Degrees of freedom Sum of squares Mean square F-value )tal jplicates Dnc. of extract Tor 43 10 3 30 26249.70 24960.76 459.34 829.10 2496.076 153.278 27.637 90.32 5.55 on-additivity 1 591.14 591.141 72.04 jsidual 29 236.96 8.206 Table F-value at p = 0.05 is 2.92 p = 0.01 is 4.51 Ffect of methanol extract of Tridcix procumbens TABLE 3b on vegetative growth o f Aspergillus niger at 35°C. )urce of Degrees of Sum of Mean F-value iriation freedom squares square 3tal jplicates 29 9 9073.90 8106.27 900.697 69.71 one. of extract 3 618.75 206.250 15.96 rror 27 348.88 12.921 on-additivity 1 314.68 314.684 239.29 esidual 26 34.19 1.315 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 ffect of methanol extract of Tridax procumbens TABLE 3c on vegetative growth o f Aspergillus flavus at 30°C. Kurce of Degrees of Sum of Mean F-value ination freedom squares square otal eplicates 39 9 17707.60 17531.73 1947.969 494.20 one. of extract 3 69.45 23.150 5.87 rror 27 106.42 3.942 on-additivity 1 75.58 75.583 63.72 esidual 26 30.84 1.186 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 63 University of Ghana http://ugspace.ug.edu.gh TABLE 3d feet of methanol extract o f Tridax procumbens on vegetative growth of Sclerolium rolfsii at 30 C. urce of riation Degrees of freedom Sum of squares Mean - - square F-value tal plicates me. of extract 23 5 9929.99 8969.55 1793.910 66.10 13 553.36 184.455 6.80 ror 15 407.07 27.138 >n-additivity 1 386.96 386.960 269.34 sidual 14 20.11 1.437 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 feet of methanol extract of Tridax procumbens TABLE 3e on vegetative growth o f Nigrospora sp. at 30°C. urce of Degrees of Sum of Mean F-value riation freedom squares square tal plicates 31 7 14749.72 13948.22 1992.603 309.03 me. of extract 3 666.09 222.031 34.48 ror 21 135.41 6.448 )n-additivity 1 100.41 100.414 57.39 sidual 20 34.99 1.750 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 64 University of Ghana http://ugspace.ug.edu.gh TABLE 4a ect o f w a fe r extract o f Aspilia africana on vegetative growth o f Scopulariopsis brevicaulis at 30 C. irce o f iation Degrees o f freedom Sum o f squares Mean square F-valu lal jlicates 31 7 15558.43 13684.74 1954.963 104.94 nc. o f extract 9 1482.46 494.154 26.52 or 21 391.23 18.630 n-additivity 1 303.00 302.996 68.68 iidual 20 88.23 4.412 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 ect o f w a ifc i* extract o f A sp ilia africana TABLE 4b on vegetative growth o f Asperg illu s n ig er at 35°C. irce o f Degrees o f Sum o f Mean F-valu iation freedom squares square tal plicates 27 6 9567.74 9534.80 1589.134 1975.15 nc. o f extract 3 18.46 6.152 7.65 or 18 14.48 0.805 n-additivity 1 6.30 6.300 13.09 sidual 17 8.18 0.481 Table F-value at p = 0.05 is 3 .29 p = 0.01 is 5 .42 feet o f Wflfer- extract o f A sp ilia a fr icana on TABLE 4c vegetative growth o f A sp e rg illu s f la v u s at 30°C . urce o f Degrees o f Sum o f Mean F-vali nation freedom squares square tal plicates 27 6 9827 .24 9773 .18 1628.863 2047 .25 ■nc. o f extract 3 39 .74 13.247 16.65 ror 18 14.32 0 .796 m-aJditivity 1 6 .08 6 .080 12.54 sidual 17 8 .24 0 .485 Table F-vaiue al p = 0.05 is 3.16 p = 0.01 is 5 .09 65 University of Ghana http://ugspace.ug.edu.gh TABLE 4d ;t of v'fatl r extract o f Aspilia africana on vegetative growth of Scleriotiuin rolfsii at 30 C. ce of Degrees of Sum of Mean F-value ition freedom squares square 1 23 14106.00 icates 5 14095.38 2819.075 10048.19 of extract 3 6.42 2.139 7.62 r 15 4.21 0.281 -additivity 1 1.71 1.705 9.54 dual 14 3.50 0.179 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 4e et of extract of Aspilia africana on vegetative growth o f Nigrospora sp. at 30°C. rce of Degrees of Sum of Mean F-value ation freedom squares square al 31 18319.47 'licates 7 18212.47 2601.781 1480.44 ic. of extract 3 70.09 23.365 13.29 )r 21 36.91 1.757 i-additivity 1 0.05 0.050 0.03 idual 20 36.86 1.843 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 66 University of Ghana http://ugspace.ug.edu.gh D. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON AGAR (SOLID MEDIUM) AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY LEGUMINOSAE (TRIBE PAPELIONOIDEAE AND CAESALPINOIDEAE) Results obtained are summarised below: 1. Crotalaria retusa (Papilionoideae) (a) Water extract Effect of the water extract from C. retusa on the test fungi was variable (Fig. 9). The depressing effect on vegetative growth of Scopulariopsis brevicaulis, Aspergillus flavus and Nigrospora sp. were significant at all the concentrations of the extract (1:1-1:5 v /v dilutions) as compared with the control. Water extract dilution (1:1 v/v) o f the plant suppressed vegetative growth of S. brevicaulis by 20.0 per cent and that o f A. flavus by 22 .6 per cent; growth of Nigrospora sp. was depressed by 10.1 per cent. Vegetative growth of A. niger and Sclerotium rolfsii in water extract at all the concectrations (1:1-1:5 v/v dilutions) were close to that in the control ( U twnended medium). (b) Methanol extract Vegetative growth of S. brevicaulis and A. niger were significantly depressed by 1:1 v/v dilution of the extract. Growth in the 1:2 and 1:5 v/v dilutions were close to that in the control. Radial diameter of mycelium of A. flavus, S. rolfsii and Nigrospora sp. in all the dilutions of the extract were also close to what obtained in the control. 67 University of Ghana http://ugspace.ug.edu.gh 2. Desmodium triflorum (Papilionoldeae) Both water and methanol extract were quite repressive on growth of the fungi (Fig. 10; Tables 5a-5e, 6a-6e). (a) Water extract Vegetative growth of A. niger, A. flavus, S. rolfsii and Nigrospora sp. was significantly inhibited by this extract (Fig. 10; Tables 5a - 5e). The highest effect was on A. flavus where vegetative growth was suppressed in the 1:1 7 V dilution of the extract by 41.4 per cent. Radial diameter of A-niger, S. rolfsii and Nigrospora sp. was depressed by 24.2, 17.3 and 12.9 per cent respectively. (b) Methanol extract This extract significantly (p_<. 0.05) suppressed vegetative growth of A. niger, A. flavus, S. rolfsii and Nigrospora sp. (Fig. 10 ; Tables 6a - 6e). The highest inhibition was 27.9 per cent on growth of A. flavus followed by nearly the same depression of growth of S. rolfsii (27.1 per cent). Vegetative growth of A. niger and Nigrospora sp. was depressed by 26.9 and 16.5 per cent respectively. 3. Cassia rotundifolia (CaesalpinoideaeJ (a) Water extract The effect of this extract on the fungi was variable (Fig. 11). Vegetative growth of S. brevicaulis was the most affected. In the 1:1 7 V dilution of the extract radial diameter of the 68 University of Ghana http://ugspace.ug.edu.gh fungus was suppressed by 86.9 per cent; 1:2 v/v and 1:5 v/v dilutions of the extract depressed vegetative growth of S. brevicaulis by 32.1 and 16.6 per cent respectively. Radial diameter of A. niger, Nigrospora sp ., A. flavus and S. rolfsii was depressed by 20.0, 19.0. 15.6 and 10.6 per cent respectively by 1:1 v/v dilution of the water extract o f this plant. (b) Methano1 extract This extract also affected the test fungi to varying extent (Fig. 11; Tables 7a-7e). The effects on S. brevicaulis and A. flavus were significant. The highest repression o f growth by 41.1 per cent was obtained on Nigrospora sp., followed by 32.8 per cent on A. niger. In all instances, the inhibitory effect was gradually removed with increasing dilution. 4. Griffonia simplicifolia (Caesalpinoideoe.) (a) Water extract Water extract (1:1 - 1:5 v/v dilution) of G. simplicifolia stimulated vegetative growth of A. niger. Radial diameter o f the fungi in the 1:1 v/v dilution was increased by 34.3 per cent (Fig. 12; Tables 8a - 8e). Growth of the other test fungi in all dilutions of the extract was only marginally depressed as compared to what obtained in the control. 69 University of Ghana http://ugspace.ug.edu.gh The extract suppressed growth of the fungi to varying extent (Fig. 12; Tables 9a - 9e). Inhibition of growth of A. niger, S. rolfsii and A. flavus was quite negligible. The highest inhibition of 28.6 per cent was on Nigrospora sp.\ vegetative growth of S. brevicaulis was depressed by 20.8 per cent. (b) Methanol extract 70 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y (m m ) .......... i.:s ------------------- C o n t r o l 1 :1 P E R I O D O F I N C U B A T I O N ( DAYS ) F ig .9 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f C r o t a l a r i a r e tu s a on v e g e t a t i v e grow th o f in d ic a te d fu n g i . 71 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y (m m ) — — i: 2 ............. 1 .5 -------------C o n t r o l PER IOD OF^ IN C U B A T IO N ( DAYS ) F ig .9 ( Co n t ' d ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f C r o t a l a r i a r e t u s a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 72 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R - OF c o l o n y (m m ) WATER E X T R A C T I ' . i i: 2 j: s C o n t r o l M E T H A N O L E X T R A C T PERIOD OF I NCUBAT I ON (DAYS) l g . 0 . E f f e c t o f v a ry in g d i l o t i o n s o f w a te r and m e th ano l e x t r a c t s o f Desmodium t r i f o l i u m on v e g e t a t i v e grow th o f in d ic a t e d fu n g i . 73 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O L O N Y (m m ) -------------- I i I --------------- l i 2 i:s _________ C o n t r o l P ER IO D OF I N C U B A T I O N ( D A Y S ) Fig.10 (Cont'd). Effect of varying dilutions of water and methanol extracts of Desmodium trifolium on vegetative growth of indicated fungi. 74 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O LO N Y (m m ) ........... I : s ------------- C o n t r o l WATER EXTRACT M E T H A N O L E X T R A C T ---------------------i : i PE R I O D OF I N C U B A T I O N (DAYS j F i q . l l . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f C a s s ia r o t u n d i f o l i a on v e g e t a t i v e grow th o f in d ic a te d fu n g i . 75 University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) i ; 2 i :s PER I OD O F I N C UB A T I O N ( DAYS ) F ig .11 ( c o n t ' d ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f C a s s ia r o t u n d i f o l i a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 76 University of Ghana http://ugspace.ug.edu.gh ................. i : s ------------------------- C o n t r o l W ATER E X T R A C T M E T H A N O L E X T R A C T PERIOD OF I N C U B A T I O N (DAYS, ) 2. E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e thano l e x t r a c t s c f G r i f f o n ia on v e g e ta t iv e g row th o f in d ic a te d l u ng i . University of Ghana http://ugspace.ug.edu.gh D IA M E TE R OF C O LO N Y (m m ) I : 5 PER I OD OF I N C U B A T I O N ( D A Y S ) F ig .12 ( Co n t ' d ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f G r i f f o n ia s im p l i c i f o l i a on v e g e t a t i v e grow th o f i n d ic a t e d fu n g i . 78 University of Ghana http://ugspace.ug.edu.gh TABLE 5a ter extract of Desmodium triflorum on vegetative growth of Scopulariopsis brevicaulis at 30 C. Degrees of Sum of Mean F-value freedom squares square 23 11483.96 5 11377.21 2275.442 1536.88 ract 3 84.54 28.181 19.03 15 22.21 1.481 ty 1 11.06 11.056 13.88 14 11.15 0.797 le F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 5b iter extract o f Desmodium triflorum on vegetative growth of Aspergillus niger at 35°C. Degrees of Sum of Mean F-value freedom squares square 31 7045.99 7 6434.68 919.240 109.23 tract 3 434.59 144.862 17.21 21 176.73 8.416 ity 1 140.23 140.228 76.34 20 35.50 1.825 5le F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE 5c ater extract o f Desmodium triflorum on vegetative growth o f Aspergillus flavus at 30°C. Degrees of Sum of Mean F-value freedom squares square 31 8807.12 7 7627.43 1089.633 41.76 (tract 3 631.77 210.59 18.07 21 547.91 26.091 /ity 1 478.39 478.386 137.61 20 69.53 3.476 ble F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 79 University of Ghana http://ugspace.ug.edu.gh TABLE 5cl ter extract o f Desmodium triflorum on vegetative growth o f Sclerotium rolfsii at 30 C. Degrees of freedom Sum of squares Mean square F-value 19 7815.20 4 7469.20 1867.300 922.12 ract 3 321.70 107.233 52.95 12 24.30 2.025 ty 1 22.26 22.256 119.80 11 2.04 0.186 le F-value at p = 0.05 is 3.49 p = 0.01 is 5.95 TABLE 5e iter extract of Desmodium triflorum on vegetative growth of Nigrospora sp. at 30°C. Degrees of freedom Sum of squares Mean square F-value 27 13260.17 6 12962.23 2160.372 915.37 tract 3 255.46 85.152 36.08 18 42.48 2.360 ity 1 23.85 23.846 21.75 17 18.64 1.096 le F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 80 University of Ghana http://ugspace.ug.edu.gh TABLE 6a t of m ethanol extract of Desmodium triflorum on vegetative growth o f Scopulariopsis brevicaulis at 30°C. e o f ion Degrees o f freedom Sum o f squares Mean square F-value 27 13194.50 ;ates 6 12982.00 2163.667 1110.48 o f extract 3 117.43 59.143 30.35 18 35.07 1.948 dditivity 1 10.22 10.224 6.99 jal 17 24.85 1.462 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 6b o f methanol extract o f Desmodium triflorum on vegetative growth o f Aspergillus niger at 35°C. ; of Degrees o f Sum o f Mean F-value on freedom squares square 27 6659.96 ates 6 6037.46 1006.244 124.03 of extract 3 476.46 158.821 19.58 18 146.04 8.113 ld itivity 1 124.29 124.289 97.16 ' al 17 21.75 1.279 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.01 TABLE 6c j f methanol extract o f Desmodium triflorum on vegetative growth o f Aspergillus it 30°C. of Degrees o f Sum o f Mean F-value n freedom squares square 27 7533.75 6 6795.63 1132.604 96.78 f extract 3 527.46 175.821 15.02 18 210.66 11.703 litiv ity 1 194.94 194.942 210.83 1 17 15.72 0.925 Table F-value at p = 0 .05 is 3.16 p = 0.01 is 5.01 81 University of Ghana http://ugspace.ug.edu.gh TABLE 6d hanol extract o f Desmodium triflorum on vegetative growth of Sclerotium rolfsii at 30 C. Degrees of Sum of Mean F-value freedom squares square 23 11074.74 5 10174.30 2034.860 146.52 act 3 692.11 230.705 16.61 15 208.32 13.888 ;y 1 177.72 177.719 81.80 14 30.60 2.186 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.01 lhanol extract of Desmodium triflorum TABLE 6e on vegetative growth o f Nigrospora sp. at 30°C. Degrees of Sum of Mean F-value freedom squares square 27 13827.18 6 13157.80 2192.884 538.73 ract 3 596.61 198.869 48.86 18 73.27 4.070 iy 1 52.70 52.698 43.55 17 20.57 1.210 . . le F-value at p = 0.05 is 3.16 p = 0.01 is 5.01 82 University of Ghana http://ugspace.ug.edu.gh TABLE 7a anol extract o f Cassia rolundifolia on vegetative growth o f Scopulciriopsis brevicaulis at 30 C. Degrees of Sum of Mean F-value freedom squares square 31 8831.38 7 8493.63 1213.375 599.15 ict 3 294.50 98.167 47.66 21 43.25 2.060 1 8.20 8.203 4.68 20 35.05 1.752 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE 7b - - tanol extract o f Cassia rolundifolia on vegetative growth o f Aspergillus niger at 35°C. Degrees of Sum of Mean F-value freedom squares square 27 5120.74 6 4337.93 722.988 52.75 act 3 536.10 178.699 39.04 18 246.71 18.706 y 1 222.82 222.821 158.54 17 23.89 1.405 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 7c hanol extract o f Cassia rolundifolia on vegetative growth of Aspergillus flavus at 30°C. Degrees of Sum of Mean F-value freedom squares square 27 9477.81 6 8110.88 1351.813 197.17 •act 3 243.53 81.176 11.94 18 123.41 6.856 ty 1 38.33 38.329 7.66 17 85.08 5.005 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 83 University of Ghana http://ugspace.ug.edu.gh TABLE 7d lethanol extract o f Cassia rolundifolia on vegetative growth of Sclerotium rolfsii at 30 C. Degrees o f freedom Sum of squares Mean square F-value 23 10024.96 5 9184.96 1836.992 306.59 ctract 3 750.13 250.042 41.73 15 89.88 5.992 /ity 1 19.60 19.600 3.90 14 70.27 5.020 Table F-value at lethanol extract o f Cassia roluiulifolia on p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 7e vegetative growth o f Nigrospora sp. at 30°C. Degrees of freedom Sum of squares Mean square F-value 31 11277.47 7 9402.84 1343.263 77.37 xtract 3 1510.00 503.344 28.99 21 364.59 17.362 vity 1 224.39 224.391 32.01 20 140.20 7.010 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 84 University of Ghana http://ugspace.ug.edu.gh TABLE 8a 'ater extract o f Griffonia simplicifolia on vegetative growth of Scopulariopsis brevicaulis at 30 C. Degrees of Sum of Mean F-value freedom squares square 23 5555.33 5 5464.33 1092.867 336.27 xtract 3 42.25 14.083 4.33 15 48.75 3.250 vity 1 21.05 21.048 10.64 14 27.70 1.979 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 8b vater extract o f Griffonia simplicifolia on vegetative growth o f Aspergillus niger at 35°C. Degrees of Sum of Mean F-value freedom squares square 23 6133.63 5 5201.25 1040.250 55.23 :xtract 3 649.88 216.625 11.50 15 282.50 18.833 ivity 1 267.78 267.776 254.61 14 14.72 1.052 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 water extract of Griffonia simplicifolia on TABLE 8c vegetative growth of Aspergillus flavus at 30°C. Degrees of freedom Sum of squares Mean square F-value , 23 7572.49 - - 5 7531.30 1506.260 1169.28 extract 3 21.86 7.288 5.66 15 19.32 1.288 tivity 1 8.32 8.319 10.58 14 11.00 0.786 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 85 University of Ghana http://ugspace.ug.edu.gh TABLE 8d r extract o f Griffonia simplicifolia on vegetative growth of Sclerotium rolfsii at 30 C. Degrees of freedom Sum of squares Mean square F-value 23 14686.83 5 14333.58 2866.717 957.35 ict 3 308.33 102.778 34.32 15 44.92 2.994 1 21.77 21.770 13.17 14 23.15 1.653 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 8e :r extract o f Griffonia simplicifolia on vegetative growth o f Nigrospora sp. at 30°C. Degrees of Sum of Mean F-value freedom squares square 27 11945.71 6 11741.59 1956.932 747.48 act 3 157.00 52.333 19.99 18 47.13 2.618 1 1 34.02 34.023 44.15 17 13.10 0.771 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 86 University of Ghana http://ugspace.ug.edu.gh TABLE 9a lanol extract o f Griffonia simplicifolia on vegetative growth of Scopulariopsis brevicaulis at 30°C. Degrees of Sum of Mean F-value freedom squares square 27 3918.36 6 3499.73 583.289 77.74 ict 3 283.57 94.524 12.60 18 135.05 7.503 1 53.58 53.582 11.18 17 81.47 4.792 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 9b lanol extract o f Griffonia simplicifolia on vegetative growth o f Aspergillus niger at 35 °C. Degrees of Sum of Mean F-value freedom squares square 31 5157.49 7 5116.93 730.990 1663.76 act 3 31.34 10.445 23.77 21 9.23 0.439 y i 0.70 0.696 1.63 20 8.53 0.427 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE 16c hanol extract o f Griffonia simplicifolia on vegetative growth o f Aspergillus flavus at 30°C. Degrees of Sum of Mean F-value freedom squares square 31 17280.72 7 17001.09 2428.728 582.27 act 3 192.03 64.010 15.35 21 87.59 4.171 :y 1 30.31 30.309 10.58 20 S I .29 2.864 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 87 University of Ghana http://ugspace.ug.edu.gh TABLE 9d thanol extract o f Griffonia simplicifolia on vegetative growth of Sclerotium rolfsii at 30 C. Degrees of freedom Sum o f squares Mean square F-value 23 14516.83 5 14306.08 2861.217 1009.84 :ract 3 168.25 56.083 19.79 15 42.50 2.233 ity 1 33.86 33.860 54.87 14 8.64 0.617 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 . _ TABLE 16e ;thanol extract of Griffonia simplicifolia on vegetative growth o f Nigrospora sp. at 30°C. Degrees o f freedom Sum of squares Mean square F-value 35 17828.47 8 16172.10 2021.512 360.08 tract 3 1521.64 507.213 90.35 24 134.74 5.614 ity 1 128.01 128.010 437.76 23 6.73 0.292 Table F-value at p = 0.05 is 3.01 p = 0.01 is 4.72 University of Ghana http://ugspace.ug.edu.gh E. VEGETATIVE GROWTH OF FIVE TEST FUNGI ON AGAR (SOLID MEDIUM) 'AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY RUTACEAE AND MELIACEAE. A summary of results obtained are as follows: 1. Zanthoxylum xanthoxyloides (Rutaceae) (a). Water extract The various dilutions of the water extract decreased vegetative growth of test fungi (Scopulariopsis brevicaulis, Aspergillus niger, A. flavus, Sclerotium rolfsii and Nigrospora sp.) (Fig. 13). The inhibitory effect was, however, removed with increasing dilution of the extract (Plate 4). (b). Methanol extract Growth of the test fungi were variably affected by the methanol extract o f this plant. The highest inhibition was on the growth of S. rolfsii. Dry matter accumulation was depressed by 28.4 per cent (Fig. 13). The other test fungi also showed decline in vigour of vegetative growth. Thus S. brevicaulis was depressed by 26.2 per cent, Nigrospora sp by 22.4 per cent and A. flavus by 14.9 per cent. Again the severity of the inhibition was removed with increasing dilution of the extract. 2. Azadirachta indica (Meliaceae) (a). Water extract Vegetative growth of Nigrospora sp. in the 1:1 v/v dilution o f the extract was depressed by 24.4 per cent after 4 days. Growth of the other fungi in all the extract dilution was only marginally inferior to the control. Conversely, the water extract at 1:1 v/v dilution improved growth of S. brevicaulis (Fig. 14, Tables 10a - lOe). University of Ghana http://ugspace.ug.edu.gh Plate 4. Vegetative growth of Scopulariopsis brevicaulis in nutrient medium ammended with water extract of o Zanthoxylum xanthoxyloides at 30 C. (Note the depression of radial growth by all (1:1 - 1:5 v/v) dilutions after 7 days as well as the change in cultural characteristics of the fungus). University of Ghana http://ugspace.ug.edu.gh (b). Methanol extract Statistical analyses showed that the depression in vegetatw tgrowth o f S. rolfsii and Nigroapora sp. in 1:1 v/v dilution were significant (P_<_ 0.05) (Tables 11a - l ie ) and that further dilution of the extract gradually removed the inhibitory effect. The highest depression in vegetative growth of 41.8 per cent on Nigrospora sp. was obtained in 1:1 v/v methanol extract of A. indica. The depression in vegetative growth was less severe (22.4 per cent) on S. rolfsii. - ~ 90 University of Ghana http://ugspace.ug.edu.gh WATER EXTRACT l ! 5 M E T H A N O L E X T R A C T C o n t r o l 1:1 1:2 P E R I O D OF I N C U B A T I O N ( DAYS ) F ig .13. E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Zanthoxylum x an th o x y lo id e s on v e g e t a t i v e g row th o f i n d ic a t e d fu n g i . 91 University of Ghana http://ugspace.ug.edu.gh OF C O L O N Y (m m ) .................. 1!5 ------------------ C o n t r o l WATER E X T R A C T M E T H A N O L E X T R A C T — 1:1 F ig .13 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Zanthoxylum x a n th o x y lo id e s on v e g e t a t i v e grow th o f i n d ic a t e d f u n g i . 92 University of Ghana http://ugspace.ug.edu.gh --------------- -- i : t ------------- 1:2 ............. 1:5 _____________ C o n t r o l WATER E X T R A C T M E T H A N O L E X T R A C T P E R I O D O F I N C U B A T I O N ( D A Y S ) |. 14. E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f A z a d ira c h ta in d ic a on v e g e t a t i v e g row th o f i n d ic a t e d fu n g i . 93 University of Ghana http://ugspace.ug.edu.gh O F C O L O N Y (m m ) WATER E X T R A C T 1:1 1:2 1:5 C o n t r o l M E T H A N O L E X T R A C T P E R I O D O F I N C U B A T I O N ( D A Y S ) Fig. 14 (Cont'd). Effect of varying dilutions of water and methanol extracts of Azadirachta indica on vegetative growth of indicated fungi. 94 University of Ghana http://ugspace.ug.edu.gh TABLE 10a ixtract of Azadirachta indica on vegetative growth o f Scopulariopsis brevicaulis at 30°C. Degrees Sum of Mean F-value o f freedom squares square 39 20004.48 9 19496.35 2166.261 343.65 3 337.93 112.642 17.87 27 170.20 6.301 1 145.78 145.78 155.21 26 24.42 0.939 le at p = 0.05 is 2.96 p = 0.01 is 4.60 TABLE 10b extract o f Azadirachta indica on vegetative growth o f Aspergillus niger at 35°C. Degrees Sum of Mean F-value o f freedom squares square 27 4668.43 6 4507.05 751.176 568.03 t 3 137.09 45.857 34.68 18 23.80 1.322 1 1.96 1.963 1.53 17 21.84 1.285 Table F value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 10c extract of Azadirachta itulica on vegetative growth o f Aspergillus flavus at 30°C. Degrees Sum of Mean F-value o f freedom squares square 27 10917.21 6 10706.46 1784.411 865.59 t 3 173.64 57.881 28.08 18 37.11 2.062 1 23.34 23.338 28.82 17 13.77 0.810 Table F value at p = 0.05 is 3.16 p = 0.01 is 5.09 95 University of Ghana http://ugspace.ug.edu.gh TABLE lOd - - extract o f Azadirachta indica on vegetative growth of Sclerotium rolfsii at 30 C. Degrees o f freedom Sum of squares Mean square F-value 27 20335.18 6 20170.05 3361.676 1071.99 :t 3 108.68 36.226B 18 56.45 3.136 1 37.97 37.970 34.94 17 18.48 1.087 Table F value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE lOe extract o f Azadirachta indica on vegetative growth o f Nigrospora sp. at 30°C. Degrees Sum of Mean F-value o f freedom squares square 35 20482.74 8 18982.43 2372.804 200.08 :t 3 1215.69 405.229 34.17 24 284.62 11.859 1 172.59 172.592 35.43 23 112.03 4.871 Table F value at p = 0.05 is 2.78 p = 0.01 is 4.22 96 University of Ghana http://ugspace.ug.edu.gh TABLE 11a al extract of Azadirachta iiulica on vegetative growth of Scopulariopsis brevicaulis at 30 C. Degrees of freedom Sum of squares Mean square F-value 31 7 6135.99 5954.80 850.686 ' 192.53 3 88.40 29.466 6.67 21 92.79 4.419 1 32.60 32.596 10.83 20 60.19 3.010 Table F value at ol extract o f Azadirachta indica p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE l ib on vegetative growth o f Aspergillus niger at 35°C. Degrees Sum o f Mean F-value o f freedom squares square 31 7 6216.87 6084.55 869.222 1001.49 3 114.09 38.029 43.82 21 18.23 0.868 1 6.69 6.688 11.59 20 11.54 0.577 Table F value at p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE 11c lol extract o f Azadirachta indica on vegetative growth o f Aspergillins flavus at 30°C. Degrees Sum of Mean F-value o f freedom squares square 27 6 11155.71 10996.21 1832.702 1137.54 ; 3 130.50 43.500 27.00 18 29.00 1.611 1 19.34 19.341 37.04 17 9.66 0.568 Table F value at p = 0.05 is 3.16 p = 0.01 is 5.09 97 University of Ghana http://ugspace.ug.edu.gh TABLE l id 10I extract o f Azadirachta indica on vegetative growth of Sclerotium rolfsii at 30 C. Degrees o f freedom Sum of squares Mean square F-value 23 12421.74 5 11617.80 2323.560 232.11 ; 3 653.78 217.927 21.77 15 150.16 10.010 1 103.03 103.029 30.61 14 47.13 3.365 Table F value at p = 0.05 is 3.29 p = 0.01 is 5.42 - TABLE l ie 'extract o f Azadirachta indica on vegetative growth o f Nigrospora sp. at 30°C. Degrees Sum of Mean F-value o f freedom squares square 35 17150.64 8 14442.01 1805.252 80.38 t 3 2169.64 723.213 32.20 24 538.99 22.458 1 479.39 479.387 185.00 23 59.60 2.591 Table F value at p = 0.05 is 3.01 p = 0.01 is 4.72 98 University of Ghana http://ugspace.ug.edu.gh F. STUDIES ON THE EFFECT OF WATER AND METHANOL EXTRACTS OF PLANTS IN THE FAMILY MALVACEAE, AMARANTHACEAE, NYCTAGINACEAE AND OXALIDACEAE ON VEGETATIVE GROWTH OF FIVE FUNGI A summary of the results is as follows: 1. Sida acuta (Malvaceae) (a) Water Extract. Water extract o f this plant significantly depressed vegetative growth of Sclerotium rolfsii (Fig. 15). Growth of the other fungi in all dilutions (1:1-1:5 v/v) of the extract was very close to those in the control media. (b) Methanol Extract The extract did not suppress growth of Aspergillus niger and A. flavus (Fig. 15; Tables 12a-12e). It rather promoted the growth of these fungi. The highest inhibition of 24.4 per cent of the extract (1:1 v/v dilution) was on vegetative growth of S. rolfsii. Its effect on S. brevicaulis and Nigrospora sp. was not significant. Dilutions 1:5 and 1:2 v/v of the extract stimulated growth. Extract o f 1:1 v/v dilution, however, suppressed growth of S. brevicaulis by 13.3 per cent and that of Nigrospora sp. by 7.6 per cent. 2. Alternanthera pungens (Amaranthaceae) a) Water Extract Effect of this extract on test fungi was significant but varied from species to species (Fig. 16; Tables 13a-13e). The highest inhibitory effect was obtained on S. 99 University of Ghana http://ugspace.ug.edu.gh brevicaulis', vegetative growth was depressed by 52.9 per cent. This was followed by 45.2 per cent on A. niger and 36.5 per cent on S rolfsii. Vegetative growth o f A. flavus in 1:1 v/v dilution was depressed by 23.9 per cent. (b) Methanol Extract. Inhibition of vegetative growth of the test fungi by this extract was also variable but significant (Fig. 16; Tables 14a-14e). Vegetative growth of A. niger was depressed by 47.0 per cent by 1:1 v/v dilution of the extract. Vegetative growth of S. rolfsii was depressed by 36.7 per cent, S. brevicaulis by 30.0 per cent and A. flavus by 23.9 per cent. 3. Boerhavia diffusa (Nyctaginaceae) (a) Water Extract All dilutions (1:1-1:5v/v) of the water extract of B. diffusa did not have any significant effect on any of the test fungi (Fig. 17). Growth in all dilutions o f the extract were close to those in the controls. (b) Methanol Extract . _ The methanol extract too did not suppress vegetative growth of any of the test fungi to any significant extent (Fig. 17). The extract improved vegetative growth of S. brevicaulis and Nigrospora sp. (Fig. 17). 100 University of Ghana http://ugspace.ug.edu.gh 4. Oxalis corniculata (Oxalidaceae) (a) Water extract The extract had very significant effect on growth of S. brevicaulis and A. niger. Vegetative growth of A. Jlavus, S. rolfsii and Nigrospora sp. was also significantly suppressed (Fig. 18, Tables 15a-15e). Vegetative growth o f A. niger was suppressed by 43.1 per cent while vegetative growth of S. brevicaulis in 1:1 v/v dilution was depressed by 41.7 per cent (Plate 5). (b) Methanol Extract \ Vegetative growth of S. brevicaulis, A. niger and A. Jlavus was only marginally inhibited by the methanol extract o f O. corniculata. On the other hand dry matter accumulation o f Nigrospora sp. and S. rolfsii in the presence of 1:1 v/v dilution o f the methanol extract was depressed by 12.9 and 32.9 per cent respectively^Ta f l t s lloa— I b e J - 101 University of Ghana http://ugspace.ug.edu.gh Plate 5. Vegetative growth of Scopulariopsis brevicaulis in nutrient medium anmended with water extract of o Oxalis corniculata at 30 C. (Note the depression of radial growth by all (1:1 - 1:5 v/v) dilutions after 8 days.) University of Ghana http://ugspace.ug.edu.gh O F C O L O N Y 1:1 1 1 2 ............. i:5 ------------------Co n t r o l WAT ER E X T R A C T M E T H A N O L E X T R A C T P ER I O D OF I N C U B A T I O N ( D A Y S ) F ig .15. E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f S id a a c u ta on v e g e ta t iv e g row th o f i n d ic a t e d f u n g i . 102 University of Ghana http://ugspace.ug.edu.gh WATER EXTRACT ----------- i : i 1:2 1:5 --------------------- C o n t r o l M E T H A N O L E X T R AC T P E R I O D OF I N C U B A T I O N ( D A Y S ) 15 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l ex tra c ts , o f S id a a c u ta on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 103 University of Ghana http://ugspace.ug.edu.gh l : i t : 2 1:5 C o n t r o l WATER E X T R A C T M E T H A N O L E X T R A C T P E R I O D O F IN C U B A T I O N ( D A Y S ) F ig . 16. ^ d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s funi — ° n ^ g e t a t i v e g row th o f i n d ic a t e d 104 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O L O N Y (m m ) ........... i :5 --------------- Control WATER E X T R A C T M E T H A N O L E X T R A C T . 111 P E R I O D OF I N C U B A T I O N ( D A Y S ) Fig.16 (Cont'd). Effect of varying dilutions of water and methanol extracts of Altemathera pungens on vegetative growth of indicated fungi. ' ' 105 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R O F C O L O N Y (m m ---------— i: i 1:2 1:5 --------------Cont rol WATER E X T R A C T M E T H A N O L E X T R A C T Fl9' 17' o fS rS v in ilu S 1";1;” f "ater “» -W l-O l ve9e t a t i v e g row th o f i n d ic a t e d f u n g i . P ER I O D OF I N C U B A T I O N ( D A Y S ) 106 University of Ghana http://ugspace.ug.edu.gh OF C O L O N Y (m m ) ----------- i : i 1:2 1:5 --------------------- C o n t r o l WATER E X T R A C T M E T H A N O L E X T R A C T F ig .17 (C on t d ) . E f f e c t o f v a r y in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Boerh a v ia d i f f u s a on v e g e t a t i v e q row th o f i n d i c a t e d f u n g i . 107 University of Ghana http://ugspace.ug.edu.gh C O L O N Y (m m S. b r ev i c au l i sS. b r e v i c au l i s n i ge rni ger A. f l a v u sA. f l a v u s P E R I O D OF I N C U B A T I O N ( D A Y S ) i : i 1:2 1:5 ' C o n t r o l WA TE R E X T R A C T M E T H A N O L E X T R A C T 9 0 100 Fl9-18- « « * . ---------------------------- 2 v e g e t a t i v e g row th of irelicated fungi. 108 University of Ghana http://ugspace.ug.edu.gh CO LO N Y (m m ) WAT ER E X T R A C T — 1:1 1:2 >>•••• 1 : 5 C o n t r o l M E T H A N O L E X T R A C T P E R I O D OF I N C U B A T I O N ( D A Y S ) F ig .18 (C o n t 'd ) . E f f e c t o f v a r y in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f O x a l is c o r n i c u l a t a on v e g e t a t i v e g row th o f in d ic a t e d f u n g i . 109 University of Ghana http://ugspace.ug.edu.gh TABLE 12a anol extract o f Sida acuta on vegetative growth of Scopulariopsis brevicaulis at 3CPC Degrees Sum of Mean F-value o f freedom squares square 39 21153.69 9 17355.76 1928.417 36.62 et 3 2376.27 792.090 15.04 27 1421.67 52.654 1 860.55 860.555 39.87 26 561.11 21.581 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 TABLE 12b lanol extract o f Sida acuta on vegetative growth o f Aspergillus niger at 35°C. Degrees Sum of Mean F-value o f freedom squares square 39 7365.98 9 7207.35 800.817 269.60 ict 3 78.43 26.142 8.80 27 80.20 2.970 1 1 65.90 65.904 119.86 26 14.30 0.550 Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 TABLE 12c lanol extract of Sida acuta on vegetative growth o f Aspergillus flavus at 30°C Degrees Sum of Mean F-value of freedom squares square 39 18320.47 9 18127.72 2015.192 476.11 act 3 78.52 26.175 6.19 27 114.23 4.231 y 1 33-47,0. IO-7S 26 SO 'T S £>‘ ioip Table F-value at p = 0.05 is 2.96 p = 0.01 is 4.60 110 University of Ghana http://ugspace.ug.edu.gh TABLE 12d thanol extract of Sida acuta on vegetative growth of Sclerotium rolfsii at 30°C Degrees of freedom Sum o f squares Mean square F-value 23 11318.24 5 10802.68 2160.535 142.96 ract 3 288.86 96.288 6.37 15 226.70 15.113 ty 1 217.09 217.092 316.40 14 9.61 0.686 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 12e thanol extract of Sida acuta on vegetative growth o f Nigrospora sp. at 30°C Degrees Sum o f Mean F-value o f freedom squares square 31 16669.43 7 14533.49 2076.213 127.26 tract 3 1793.34 597.779 36.64 21 342.60 16.314 ity 1 278.54 278.544 86.97 20 64.06 3.203 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 f 111 University of Ghana http://ugspace.ug.edu.gh TABLE 13a ter extract o f AUernanthera pungens on vegetative growth of Scopulariopsis brevicaulis at 3CPC Degrees o f freedom Sum of squares Mean square F-value 27 10314.24 6 5245.43 874.238 14.10 tract 3 3952.53 1317.509 21.24 18 1116.29 62.016 ity 1 1046.68 1046.678 255.63 17 69.61 4.095 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 13b iter extract o f AUernanthera pungens on vegetative growth o f Aspergillus niger at 3ffC Degrees Sum of Mean F-value of freedom squares square 27 4535.03 6 3767.21 627.869 30.78 tract 3 400.60 133.533 6.55 18 367.21 20.401 fity 1 300.06 300.064 75.97 17 67.15 3.950 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 13c ater extract of Ahernanthera pungens on vegetative growth o f Aspergillus fTavus at 30°C Degrees Sum of Mean F-value o f freedom squares square 27 6319.17 6 5964.23 994.039 154.65 xtract 3 239.24 79.747 12.41 18 115.70 6.428 vity 1 94.72 94.718 76.75 17 20.98 1.234 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 112 University of Ghana http://ugspace.ug.edu.gh TABLE 13d er extract o f Altemanthera pungens on vegetative growth of Sclerotium rolfsii at 3CPC Degrees o f freedom Sum o f squares Mean square F-value 23 9850.49 5 8741.68 1748.335 63.76 ract 3 697.53 232.510 8.48 15 411.28 27.419 ty 1 365.67 365.673 112.25 14 45.61 3.258 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 13e ter extract o f Altemanthera pungens on vegetative growth o f Nigrospora sp. at 30°C Degrees o f freedom Sum o f squares Mean square F-value 23 8299.74 5 8045.68 1609.135 326.04 ;ract 3 180.03 60.010 12.16 15 74.03 4.935 ity 1 55.89 55.891 43.13 14 18.14 1.296 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 113 University of Ghana http://ugspace.ug.edu.gh ta b l e 14a thanol extract o f Alternanthera pungens on vegetative growth of Scopulariopsis brevicaulis at 30^0 Degrees o f freedom Sum of squares Mean square F-value 27 12770.03 6 10844.21 1807.369 90.98 ;ract 3 1568.24 522.747 26.31 IS 357.57 19.865 ity 1 238.70 238.699 34.14 17 118.87 6.992 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 14b :thanol extract of Alternanthera pungens on vegetative growth o f Aspergillus niger at 35°C Degrees Sum o f Mean F-value o f freedom squares square 27 5104.96 6 4177.46 696.244 33.54 tract 3 553.89 184.631 8.90 18 373.61 20.756 ity 1 316.74 316.742 94.69 17 56.87 3.345 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 14c ethanol extract o f Alternanthera puneens on vegetative growth o f Aspergillus flavus at 30°C Degrees Sum of Mean F-value o f freedom squares square 27 6401.11 6 5953.23 992.205 157.48 ttract 3 334.46 111.488 17.69 18 113.41 6.301 /ity 1 96.90 96.901 99.78 17 16.51 0.971 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 114 University of Ghana http://ugspace.ug.edu.gh TABLE 14d ;thanol extract o f Altemanthera pungens on vegetative growth of Scopulariopsis brevicaulis at 3CfC Degrees o f freedom Sum of squares Mean square F-value 23 9889.50 5 8464.63 1692.925 56.67 tract 3 976.75 325.583 10.90 15 448.13 29.875 ity 1 403.64 403.640 127.03 14 44.48 3.177 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 14e ethanol extract o f Altemanthera pungens on vegetative growth o f Nigrospora sp. at 30°C Degrees Sum of Mean F-value o f freedom squares square 23 8592.96 5 8218.21 1643.642 415.82 ; tract 3 315.46 105.153 26.60 15 59.29 3.953 dty 1 46.44 46.440 50.59 14 12.85 0.918 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 115 University of Ghana http://ugspace.ug.edu.gh TABLE 15a r extract o f Oxalis corniculata on vegetative growth of Scopulariopsis brevicaulis at 3CPC Degrees Sum of Mean F-value of freedom squares square 23 10025.33 5 7728.46 1545.692 45.57 act 3 1788.08 596.028 17.57 15 508.79 33.919' ' y 1 443.70 443.699 95.43 14 65.09 4.649 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 15b er extract o f Oxalis corniculata on vegetative growth o f Aspergillus niger at 35°C Degrees Sum of Mean F-value of freedom squares square 27 4940.03 6 3841.84 640.307 30.41 ract 3 719.24 239.747 11.39 18 378.95 21.053 ty 1 307.43 307.430 73.08 17 71.52 4.207 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 15c ter extract o f Oxalis corniculata on vegetative growth o f Aspergillus flavus at 3 0 ^ Degrees Sum o f Mean F-value of freedom squares square 27 10491.93 6 10236.80 1706.134 479.98 ;ract 3 191.14 63.714 17.92 18 63.98 3.555 ity 1 41.78 41.779 31.99 17 22.20 1.306 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 116 University of Ghana http://ugspace.ug.edu.gh TABLE lSd rater extract o f Oxalis corniculata on vegetative growth of Sclerotium rolfsii at 3CFC Degrees Sum of Mean F-value o f freedom squares square 19 9423.24 4 9398.68 2349.669 5197.42 :xtract 3 19.14 6.379 14.11 12 5.43 0.452 ivity 1 0.01 0.009 0.02 11 5.42 0.492 Table F-value at p = 0.05 is 3.49 p = 0.01 is 5.95 TABLE 15e ,vater extract o f Oxalis corniculata on vegetative growth o f Nigrospora sp. at 30PC Degrees Sum o f Mean F-value o f freedom squares square 31 18411.80 7 17934.87 2562.124 948.49 sxtract 3 420.21 140.070 51.85 21 56.73 2.701 ivity 1 5.26 5.262 2.09 20 51.47 2.573 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 117 University of Ghana http://ugspace.ug.edu.gh TABLE 16a nethanol extract o f Oxalis corniculata on vegetative growth o f Scopulariopsis brevicaulis at 3 CPC Degrees Sum o f Mean F-value o f freedom squares square 27 14937.81 6 14756.25 2459.375 789.00 extract 3 125.46 41.818 13.42 18 56.11 3.117 ivity 1 14.77 14.773 6.08 17 41.33 2.431 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 16b methanol extract o f Oxalis corniculata on vegetative growth o f Aspergillus niger at 35°C Degrees Sum o f Mean F-value o f freedom squares square 27 6189.21 6 6099.46 1016.577 1006.59 extract 3 71.57 23.857 23.62 18 18.18 1.010 livity 1 11.21 11.213 27.37 17 6.97 0.410 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 16c methanol extract o f Oxalis corniculata on vegetative growth o f Aspergillus Jlavus at 30°C Degrees Sum o f Mean F-value o f freedom squares square 27 14734.86 5 6 14581.73 2430.289 1377.03 extract 3 121.36 40.452 22.92 18 31.77 1.765 tivity 1 2.55 2.553 1.49 17 29.22 1.719 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 118 University of Ghana http://ugspace.ug.edu.gh TABLE 16d methanol extract o f Oxalis corniculata on vegetative growth of Sclerotium rolfsii at 30°C Degrees o f freedom Sum of squares Mean square F-value 19 9006.45 4 8371.70 2092.925 94.52 extract 3 369.05 123.017 5.56 12 265.70 22.142 ;ivity 1 227.41 227.407 65.32 11 38.29 3.481 Table F-value at p = 0.05 is 3.49 p = 0.01 is 5.95 TABLE 16e methanol extract o f Oxalis corniculata on vegetative growth o f Nigrospora sp. at 30°C Degrees Sum of Mean F-value o f freedom squares square 31 18200.30 s 7 17828.99 2546.999 480.61 extract 3 260.02 86.674 16.36 21 111.29 5.299 tivity 1 57.71 57.706 21.54 20 53.58 2.679 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 119 University of Ghana http://ugspace.ug.edu.gh G. STUDIES ON THE VEGETATIVE GROWTH OF FIVE FUNGI ON SOLID MEDIUM AMENDED WITH WATER AND METHANOL EXTRACTS OF PLANTS FROM THE FAMILY APOCYNACEAE, ASCLEPIADACEAE AND EUPHORBIACEAE A summary of the results is as follows: 1. Euphorbia heterophylla (Euphorbiaceae) (a) Water Extract. The only inhibitory effect of significance of the extract on the test fungi was that on Scopulariopsis brevicaulis (Fig. 19). Vegetative growth of S. brevicaulis was inhibited by 37.5 per cent when cultured in the l : lv /v dilution of the extract. Growth in all the dilutions (1:1- l:5v/v) were close to those in the controls in the remaining test fungi namely Aspergillus niger, A. flavus, Scelerotiwn rolfsii and Nigrospora sp. (b) Methanol Extract The extract had no significant inhibitory effect on A. niger, A . flavus and Nigrospora sp. (Fig. 19). On the other hand, r a d i a l d'lo.-m&'u-r of S. brevicaulis and S. rolfsii in the presence of 1:1 v/v dilution of the extract was depressed by 35.2 and 21.2 per cent respectively. 2. Pergularia daemia (Asclepiadaceae) (a) Water Extract Vegetative growth of all test fungi was significantly depressed by this water extract (Fig. 20; Tables 17a-17e). R .adia.1 diamettr- jyp.S'. brevicaulis was depressed by 47.6 per cent in 1: lv /v dilution whilst the same extract depressed dry matter accumulation of A. niger by 40.2 120 University of Ghana http://ugspace.ug.edu.gh per cent. Vegetative growth of Nigrospora sp. was inhibited by 15.5 per cent while that of A. flavus was by 13.8 per cent. (b) Methanol Extract. This extract also significantly (p< 0.05) suppressed vegetative growth of all the test fungi (Fig. 20; Tables 18a-18e). Growth of S. brevicaulis was depressed by 37.7 per cent and that of A. niger by 34.3 per cent, in the l : lv /v dilution of extract. Vegetative growth of S. rolfsii and A. flavus was depressed by 16.1 per cent and 10.0 per cent respectively whilst that of Nigrospora sp. was depressed by 19.5 per cent. 3. Voacanga africana (Apocynaceae) (a) Water Extract Growth of S. rolfsii and Nigrospora sp. were very significantly depressed in the water extract of V. africana (Fig. 21; Tables 19a-19e). The depression of growth of A. niger and A. flavus by this extract can be described as marginal in all dilutions ( l : l - l :5 v /v ) . Growth of S. rolfsii was depressed by 37.2 per cent followed by 28.4 per cent inhibition of growth by Nigrospora sp. in the 1:1 v/v dilution. Vegetative growth of S. brevicaulis was depressed by 22.8 per cent. 121 University of Ghana http://ugspace.ug.edu.gh (b) Methanol Extract The methanol extract was more potent against S. brevicaulis, S. rolfsii and Nigrospora sp. (Fig. 21; Tables 20a-20e). The effect of this methanol extract on A . niger and A. flavus was only marginally sigrificant. Interestingly, vegetative growth of Nigrospora sp., S. rolfsii and S. brevicaulis was inhibited by 44.0, 33.3 and 32.7 per cent respectively by l: lv /v dilution of this extract. 122 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O L O N Y (m m ) — 1:1 1!2 ................. 1:5 ------------- Cont rol W A T E R E X T R A C T M E T H A N O L E X T R A C T P E R I O D O F I N C U B A T I O N ( D A YS ) F ig .19. E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f E upho rb ia h e te r o p h y l la on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 123 University of Ghana http://ugspace.ug.edu.gh O F C O L O N Y (m m ) ----------- t : 1 1:2 ............. i :5 '• ------------------ C o n t r o l WAT E R E X T R A C T ' M E T H A N O L E X T R A C T P E R I O D OF I N C U B A T I O N ( D AYS ) Fig.19 (Cont'd). Effect of varying dilutions of water and methanol extracts of Euphorbia heterophylla on veqetative arowth of indicated fungi. 124 University of Ghana http://ugspace.ug.edu.gh (m m ) >- P E R I O D OF I N C U B A T I O N ( D A Y S ) Fig.20. Effect of varying dilutions of water and methanol extracts of Pergulana daemia on vegetative growth of indicated fungi. 125 i: i i : 2 1:5 C o n t r o l m e t h a n o l e x t r a c t S. b r ev i c ou l i s WATER EXTRACT S. b r ev i cau l i s University of Ghana http://ugspace.ug.edu.gh C O L O N Y ( mm ) WATER EXTRACT ............... i ; 5 --------------------- C o n t r o l M E T H A N O L E X T R A C T 1:1 1 2 3 I N C U B A T I O N ( D A Y S ) h- TO UJ 5 6 0 < I 5 0 P E R I O D OF F ig .20 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f P e r g u la r i a daem ia on v e g e t a t i v e g row th o f in d ic a t e d f u n g i . 126 University of Ghana http://ugspace.ug.edu.gh WATER EXTRACT i: i 1:2 t : 5 Con t r o l M E T H A N O L E X T R A C T P E R I O D O F I N C U B A T I O N ( D A Y S ) F ig .21 . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th an o l e x t r a c t s o f Voacanga a f r i c a n a on v e g e t a t i v e g row th o f in d ic a t e d fu n g i . 127 University of Ghana http://ugspace.ug.edu.gh ----------- i : i 1:2 1 :5 ---------------------C o n t r o l WA T E R E X T R A C T M E T H A N O L E X T R A C T 3 P E R I O D I N C U B A T I O N ( D A Y S ) >- z o -I o o li. o 100 r Q £ 80 - UJ h LkJ 5 < F ig .21 (C o n t 'd ) . E f f e c t o f v a ry in g d i l u t i o n s o f w a te r and m e th ano l e x t r a c t s o f Voacanga a f r i c a n a on v e g e t a t i v e g row th o f in d ic a te d fu n g i . 128 University of Ghana http://ugspace.ug.edu.gh TABLE 17a Effect of water extract o f Pergularia daemia on vegetative growth of S. brevicaulis at 3CPC Source of variation Degrees o f freedom Sum of squares Mean square F-value Total Replicates 27 6 12107.24 8919.68 1486.613 21 A 3 Cone, of extract 3 2212.10 737.366 13.61 Error 18 975.46 54.192 Non-additivity 1 897.13 897.135 194.71 Residual 17 78.33 4.608 Table F-value at Effect of water extract o f Pergularia daemia on p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 17b vegetative growth o f Aspergillus niger at 35°C. Source of Degrees Sum of F -v a tu e . variation o f freedom squares Total Replicates 27 6 5105.96 4387.96 7 £ l '3 £ 7 Cone, of extract 3 403.54 1 7 « 7 0 Error 18 314.46 1 7 * ^ 7 0 Non-additivity 1 243.03 S 7 v 9 f Residual 17 71.43 Table F-value at Effect of water extract o f Pergularia daemia on p = 0.05 is 3.16 p = 0.01 is 5.OS TABLE 17c vegetative growth o f Aspergillus flavus at 30°C Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total Replicates 27 6 9763.037 9550.59 1591.765 624.08 Cone, of extract 3 166.53 55.509 21.76 Error 18 45.91 2.551 Non-additivity 1 33.08 33.083 43.84 Residual 17 12.83 0.755 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 129 University of Ghana http://ugspace.ug.edu.gh TABLE m Effect of water extract o f Pergularia daemia on vegetative growth of Sclerotium rol/sii at 3CTC Source of variation Degrees o f freedom Sum o f squares Mean square F-value Total 15 8562.77 . - Replicates 4 8505.00 2126.251 5260.83 Cone, of extract 3 54.53 18.178 44.98 Error 8 3.23 O -'fry t Non-additivity 1 1.05 1 - 0 ^ 7 3 - t o Residual 7 'W' 2.18 0 3 1 1 Table F-value at Effect of water extract o f Pergularia daemia on p = 0.05 is 4.07 p = 0.01 is 7.59 TABLE 17e vegetative growth o f Nigrospora sp. at 30oC Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total Replicates 23 5 9670.244 9344.93 1868.985 823.80 Cone, of extract 3 291.28 97.094 42.80 Error 15 34.03 2.269 Non-additivity 1 13.02 13.019 8.67 Residual 14 21.01 1.501 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 130 University of Ghana http://ugspace.ug.edu.gh TABLE 18a Effect of methanol extract o f Pergularia daemia on vegetative growth of Scopulariopsis brevicaulis at 3CPC Source of variation Degrees o f freedom Sum of squares Mean square F-value Total Replicates 27 6 10678.68 8612.18 1435.363 41.39 Cone, of extract 3 1442.32 480.774 13.86 Error 18 624.18 34.677 Non-additivity 1 211.92 211.921 8.74 Residual 17 412.26 24.250 Table F-value at Effect of methanol extract o f Pergularia daemia p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 18b on vegetative growth o f Aspergillus niger at 35°C Source of Degrees Sum of Mean F-value variation o f freedom squares square Total Replicates 27 6 6284.50 5496.25 916.042 85.48 Cone, of extract 3 595.36 198.452 18.52 Error 18 192.89 10.716 Non-additivity 1 183.76 183.765 342.24 Residual 17 9.13 0.537 Table F-value at Effect of methanol extract o f Pergularia daemia p = 0.05 is 3.16 p = 0.01 is 5.09 TABLE 18c on vegetative growth o f Aspergillus fla vus at 30°C Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total Replicates 27 6 12202.50 11873.00 1978.885 387.03 Cone, of extract 3 239.79 70.929 16.04 Error 18 89.71 4.984 Non-additivity 1 47.67 47.670 Residual 17 42.05 2.473 Table F-value at p = 0.05 is 3.16 p = 0.01 is 5.09 131 University of Ghana http://ugspace.ug.edu.gh TABLE 18d Effect of methanol extract of Pergularia daemia on vegetative growth of Sclerotium rolfsii at 3 (PC Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total 23 12868.49 Replicates 5 12485.05 2497.010 371.49 Cone, of extract 3 282.61 94.205 14.02 Error 15 100.82 6.722 Non-additivity 1 83.76 83.757 68.71 Residual 14 17.07 1.219 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 18e Effect of methanol extract o f Pergularia daemia on vegetative growth o f Nigrospora sp. at 30PC Source of Degrees Sum of Mean F-value variation o f freedom squares square Total 23 9400.74 Replicates 5 8982.55 1796.510 449.83 Cone, of extract 3 358.28 119.427 29.90 Error 15 59.91 3.994 Non-additivity 1 22.99 22.987 8.72 Residual 14 36.92 2.637 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 132 University of Ghana http://ugspace.ug.edu.gh TABLE 19a Effect of water extract o f Voacanga africana on vegetative growth o f Scopulariopsis brevicaulis at 3CPC Source of variation Degrees o f freedom Sum o f squares Mean square F-value Total Replicates 23 5 5635.83 5414.33 1082.867 179.48 Cone, of extract 3 131.00 43.667 7.24 Error 15 90.50 6.033 Non-additivity 1 23.39 23.387 4.88 Residual 14 67.11 4.794 Table F-value at Effect of water extract o f Voacanga africana on p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 19b vegetative growth o f Aspergillus niger at 35°C Source of Degrees Sum of Mean F-value variation o f freedom squares square Total Replicates 23 5 2246.99 2135.80 427.160 175.20 Cone, of extract 3 74.61 24.872 10.20 Error 15 36.57 2.438 Non-additivity 1 30.99 30.991 77.74 Residual 14 5.58 0 -39& Table F-value at Effect of water extract o f Voacanga africana on p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 19c vegetative growth o f Aspergillus tis ^>0°C Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total 23 7305.50 ' " Replicates 5 7153.00 1430.600 486.78 Cone, of extract 3 108.42 36.139 12.30 Error 15 44.08 2.939 Non-additivity 1 41.23 41.230 202.28 Residual 14 2.85 0.204 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 133 University of Ghana http://ugspace.ug.edu.gh TABLE 19(1 Effect of water extract o f Voacanga africana on vegetative growth of Sclerotium rol/sii at 3 (PC Source of Degrees Sum of Mean F-value variation o f freedom squares square Total 23 10861.46 Replicates 5 9311.83 1862.367 44.80 Cone, of extract 3 926.04 308.681 7.43 Error 15 623.58 41.572 Non-additivity 1 599.85 599.852 353.87 Residual 14 23.73 1.695 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 - TABLE 19e Effect of water extract o f Voacanga africana on vegetative growth o f Nigrospora sp. at 30PC Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total 38 18125.93 Replicates 9 16725.66 1858.406 210.02 Cone, of extract 3 1170.21 390.071 44.08 Error 26 230.06 8.849 Non-additivity 1 151.01 151.013 m b Residual « 25 79.05 Table F-value at p = 0.05 is 2.98 p = 0.01 is 4.64 134 University of Ghana http://ugspace.ug.edu.gh TABLE 20a iffect of methanol extract o f Voacanga africana on vegetative growth of Scopulariopsis brevicaulis at 3CPC lource of wation Degrees o f freedom Sum of squares Mean square F-value Total Replicates 31 7 7500.00 6483.38 926.196 69.82 2onc. of extract 3 738.06 246.012 18.55 Error 21 278.56 13.265 Non-additivity 1 194.33 194.333 46.14 Residual 20 84.23 4.211 Table F-value at Effect of methanol extract o f Voacanga africana p = 0.05 is 3.07 p = 0.01 is 4.87 TABLE 20b on vegetative growth o f Aspergillus niger at 35°C Source of Degrees Sum of Mean F-value vination o f freedom squares square Total Replicates 31 7 5370.38 5319.76 759.964 515.85 Cone, of extract 3 19.69 6.563 4.45 Error 21 30.94 1.473 Non-additivity 1 2.71 2.709 1.92 Residual 20 28.23 1.411 Table F-value at Effect of methanol extract o f Voacanga africana p = 0.05 is 3.07 p = 0.01 is 4.87 . . TABLE 20c on vegetative growth o f Aspergillus flavus at 30°C Source of Degrees Sum of Mean F-value variation o f freedom squares square Total Replicates 31 7 16735.47 16575.47 2367.924 859.67 Cone, of extract 3 102.16 34.052 12.36 Error 21 57.84 2.754 Non-additivity 1 17.87 17.870 8.94 Residual 20 39.97 1.999 Table F-value at p = 0.05 is 3.07 p = 0.01 is 4.87 135 University of Ghana http://ugspace.ug.edu.gh TABLE 2Qd Effect of methanol extract o f Voacanga africana on vegetative growth of Sclerotium rolfsii at 3ff’C Source of Degrees Sum of Mean F-value variation o f freedom squares square Total 23 11494.66 Replicates 5 10367.59 2073.519 64.47 Cone, of extract 3 644.61 214.872 6.68 Error 15 482.45 32.163 Non-additivity 1 454.30 454.296 225.92 Residual 14 28.15 2.011 Table F-value at p = 0.05 is 3.29 p = 0.01 is 5.42 TABLE 20e Effect of methanol extract o f Voacanga africana on vegetative growth o f Nigrospora sp. at 30PC Source of Degrees Sum o f Mean F-value variation o f freedom squares square Total 35 14917.64 Replicates 8 11397.39 1424.674 61.99 Cone, of extract 3 2968.69 989.565 43.06 Error 24 551.56 22.981 Non-additivity 1 509.18 509.179 276.36 Residual 23 42.38 1.842 Table F-value at p = 0.05 is 3.01 p = 0.01 is 4.72 136 University of Ghana http://ugspace.ug.edu.gh H. COMPARATIVE FUNGISTATIC ACTIVITY OF EXTRACTS OF NINETEEN PLANTS ON VEGETATIVE GROWTH OF FIVE SELECTED FUNGI Results obtained are presented in Tables 21-24. Fungistatic activity of the plant extracts on each fungus varied from plant to plant. There was no relationship between the effect of extracts from plants from the same family on any of the test fungi. There were differences in the activity of the water and methanol extracts from each plant. None of the plant extracts exhibited a broad spectrum activity over all the five selected test fungi. The highest fungistatic activity of 7.7 was obtained with the water extract of Cassia rotundifolia on Scopulariopsis brevicaulis (Table 22). This was followed by 2.6 and 2.2 by Alternaruhera pungens and Tridax procumbens respectively on S. brevicaulis (Tables 23 and 22). Pergularia daemia and Oxalis corniculata each exerted fungistatic activity of 2.0 on S. brevicaulis. The water extracts of Launaea taraxacifolia, Aspilia africana, Emilia sonchifolia, Synedrella nodiflora, Zanthoxylum xanthoxyloides and Boerhavia diffusa all exerted fungistatic activities that were below 1.5 on any of the test fungi. The most susceptible fungus to the water extracts was S. brevicaulis whilst the most resistant was Aspergillus flavus. 137 University of Ghana http://ugspace.ug.edu.gh ! § .a C/5 Do z On VO ^ vn O ^ fS vn o o o o O rn V) o O vi ri o ri cm —• (N r4 o o o o o q © *n © vnO ^ rl ^ rH n fS ri ri « •^ ■3 ^ g §^ • 1 5 3 S s & & | | ^ Cl, Ci, -2ia I t s t s i o o inin in o o vn o vn^ >o h o n—• *-< r- o —• © © vn vn —n »n vn © © in ^ vn vd r-i Oo o o o.c_ri r* —• o\ £\0 _ _ 0»n o o »n SS 2 id 8 90 O - H >00 © © © *S5 S s 2 o© *n o o 3aa ‘| -° fc i 3.3 -SP a r<| s E? sCl *>ST Cx, Cj, . a % ^ I 1 o' I t 3.5, <15 & s I s , ^ 2 I I Ci, 4fSr o X «o £ & 1 as n »n o ^ ~ -« *+ >n o vn vn vn O' (N •—i O >n o o o o •n «n vn o oc*^ Tt- rn n n cs cs cs i t | := ■a & I #S. c ^ £■s 3 3 § 3 5 1 ■3 f® P® S §■ S. S . i E & Sc op ul ar io ps is br ev ic au lis 19 .5 22 .0 0.9 25 .0 21 .0 As pe rg ill us ni ge r 37 .0 34 .0 1.2 54 .5 46 .0 Ch ro m ol ae na od or at a As pe rg ill us J la vu s 21 .5 19 .0 1.1 22 .0 18 .0 Sc ler ot iu m ro lfs ii 43 .0 41 .0 1.1 23 .0 18 .0 Ni gr os po ra sp . 23 .0 15 .5 1.5 35 .0 26 .0 University of Ghana http://ugspace.ug.edu.gh E£ U 3 u3 • a * § :> c/i DU2e si 00 (N CN (N m o o in o o OOOO i/l O O O Y"> o in »n $ 2 I ■S b I s .a -2f a ^8 » « , £ f i l l*3 g? § 8- S. S.-S _ •2 .3 H3cx n m 't >o in »n in o o o o ri K \o n n N in o in iocn >0 't ri 't— *n t}- rj- (N in o in o o ^ os o >o cs»-. r*i -3- in o o vn o ■a -Sp o -S' S. K ^ £ ! I I ! cl ^ h S' g. o. -Sin O m »n o m O O 0 1 * 0 * I ! 1 2 5^i ? S' .9 »- *1 £° £°D V ^^ o ' a , a , ^ Si) Ia s ■9 I .5a. oo cs (S n ^^ ^ ^ ^ CS O m m m o o in in o in h 6 n Kcs (N >n (N ^ o o in (N O in in »n o in (N in >o ttn m -h cs m © o o o a 43 3 •§ .O5 s )sis bn ni ge r fla vu s ro lfs ii sp . ■a t.a -2? s c I l l i 1 1 J•f ? ? 3 | 1 &o, 1) ^ wS' a, a, *, §■ §., 0 J2 ‘O < j U jr r to < *3 T £ ^ I I « 5 •2 a, o•p1 cn 6 Si in o o o m in o o n o n N N n N o m o o o o o o o in 1 1 3^ s I I £? | S.-S •a r-c aKc 5 6 3DQC N N (S m a o m m o o O O m m m o ^ rs M f ' o o m m m O m in m m 0 \cn S. c f ! ! -2 Sc Sc V K> t-a. a. -Si*3 oX X n © m in o O in O m o in O <3 .a -2? S c | 3 I ^ I is, O J? S I I on - ex. Ci, -Sf ^ T T ^ On CS CN r> n o in -* >C >n n SVO s5 — CN « o * I s ■8 - r ^ t . I I I E £ University of Ghana http://ugspace.ug.edu.gh o ■a u 5 uj ■a » § :> U- a S - S H aj U < E C a 'J .2 H cjs 3ra X Q T3 w OV- > O oc uO ‘ i UJ <5 H EM | < S c £ <3 Do z £ 5i >r (S - i in rf o o o o o in vn o o */-> 't 00 U\ (N 0\ m in © o m * fc I :5•S -2? q S is « «, s ■ I j 3 g 3 3 !"3 ?« !*> c? O' Q, Q, 4l Si £B- .§ §■U4 B o o o o o o VO Q cs »o m in in q o o 3 'd o oo o in m o o in .1 3 3 1»w 5: 2s 3 I & 2? I s 8- §. S.-5 I I- a•Si ' i I< University of Ghana http://ugspace.ug.edu.gh I. GROWTH OF FIVE FUNGI STORED IN WATER AND METHANOL EXTRACTS OF NINE PLANTS. Results obtained are presented in Figs. 22-26. In all cases mycelia of the fungi resumed growth when placed in Potato Dextrose Broth (PDB) medium after immersion in the water or methanol extract of the plants for the varying periods (15 minutes to 48 hours). The longer the period of immersion in the extract the severer the decline in dry matter accumulation by the fungus. The only exception was with the water extract of Griffonia simplicifolia (Fig. 26) where the extract appeared to have stimulated growth of Aspergillus niger and A. Jlavus because of the increase in their dry matter accumlation. 142 University of Ghana http://ugspace.ug.edu.gh DRY WEIGHT OF MYCELIUM (mg) WATER EXTRACT I I <00 METHANOL EXTRACT WATER [" I 200 EXTRAC.T|J 100 METHANOL 100 ■ e x t r a c t Fi9‘22' anfr^hVe f OVT °f fUn9i in 1:1 v/v dilution of water transferrS toX^ t CtS S Pl3ntS f°r indicated Periods and then transferred to Potato Dextrose Broth for 10 days at 30°C. 143 University of Ghana http://ugspace.ug.edu.gh WATER extract! WATER EXTRACT DRY WEIGHT OF MYCELIUM (mo) i0° ■ METHANOL '00 I EXTRACT METHANOL '00 ■ EXTRACT transferred to * £ 2 3 0 ^ 144 University of Ghana http://ugspace.ug.edu.gh DRY WEIGHT OF M YCEL IUM (mg) C ASS IA ROTUND I FO L IA WATER T e x t r a c t L methanol EXTRACT WATER ["I EXTRACTU Fig. 24. Vegetative growth of fungi stored in 1:1 v/v dilution of water and methanol extracts of plants for indicated periods and then transferred to Potato Dextrose Broth for 10 days at 30°C. 145 University of Ghana http://ugspace.ug.edu.gh WATER e x t r a c t WATER EXTRACT DRY WEIGHT OF M.YCELIUM (mg) METH ANO L E X T R A C T METHANO L 1 0 0 ■ E X TR AC T Fig. 25. Vegetative growth of fungi .stcr-ad in 1:1 v/v dilution of water and methanol extracts of plants for indicated periods and then transferred to Potato Dextrose Droth for 10 days at 30 °C. 146 University of Ghana http://ugspace.ug.edu.gh Fig.26. Vegetative growth of fungi .stored m 1:1 v/v dilution of f c r ^ d i c a S 10 d^s at 3?c? transferred to Potato Dextrose Broth for 147 University of Ghana http://ugspace.ug.edu.gh J. ANTIBACTERIAL ACTIVITY OF EIGHT SELECTED PLANTS The results are presented in Table 25. Most o f the plant extracts tested did not have any significant antimicrobial activity against the test microorganisms. Oxalis corniculata had the highest antimicrobial activity amongst the eight plants tested. Its water extract in most instances produced wider diameter of zones of inhibition than those of the standard antibiotics. The methanol extract of this plant however, failed to exert any significant inhibitory effect as compared to the water extract (Table 25). Inhibition of growth of test microorganisms was related to the type o f antibiotic used. Oxytetracycline was not effective against Staphylococcus aureus, Salmonella (Group C,) and Escherichia coli (ETEC OKS). The water extract of O. corniculata was more inhibitory to Salmonella (Group C,) than was Streptomycin but the inhibition of E. coli by O. corniculata was about the same as obtained for Streptomycin. The inhibitory effect of Chloramphenicol on Pseudomonas aeruginosa (1.0mm diameter) was significantly inferior to what existed in O. corniculata water extract (3.8mm diameter). However, 0 . corniculata did not have any inhibitory effect on S. aureus. The same water extract o f O. corniculata could adversely affect growth of E. coli (ETEC OK5). 148 University of Ghana http://ugspace.ug.edu.gh o o o o o o o o X ft. 5 o o o o o o o o o o o o o o o o o o o o o o o a | 1 z M £ z o cc m 5 X0 zO "a U-.ij o £ W 8 £ zo N u. o u. w H W i 3 'So a. 5 o £ , 60 t i I !n ©U J3 Q >N I I ft- “ £ .3- Son OOSO 0BC u u. o w z o N u, O06 S w o o vo o o o o o tj- (N O o o d o o o o o o o o o o o o o o o