INFLUENCE OF THE METABOLITES OF THREE PAECILOMYCES SPECIES ON THE GERMINATION AND SEEDLING DEVELOPMENT OF TWO GHANAIAN MAIZE VARIETIES (ABELEEHI AND OBAATANPA) A THESIS PRESENTED BY ANDREW AMEGBEDZI \1L\AMOR BSc (HONS) IN FULFILMENT OF THE REQUIREMENT FOR THE MASTER OF PHILOSOPHY DEGREE OF THE UNIVERSITY OF GHANA OCTOBER, 1995 FROM: THE DEPARTMENT OF BOTANY UNIVERSITY OF GHANA LEGON University of Ghana http://ugspace.ug.edu.gh TABLE OF CONTENTS PAGE DECLARATION DEDICATION 11 ABSTRACT m I. INTRODUCTION AND LITERATURE REVIEW 1 II. MATERIALS AND METHODS 5 (i) MATERIALS 5 (ii) GENERAL METHODS 5 A Maize samples kept under humidity chamber 5 B Direct - Plating Method 5 C. Identification of Fungi 6 D. Serial-Dilution Method 6 E. Maintenance of Stock Cultures 6 F. Preparation of Media 6 G Method of Inoculation 8 H. Assessment of Growth 8 I Seed viability Test 8 J IN vitro studies on the effect of Fungal M etabolites on Germination and Radicle Development 9 K. Influence of Fungal Metabolites on Vegetative Growth aud Dry M atter Accumulation by Maize Seedlings 10 L Chlorophyll content n M Anatomical Studies 11 University of Ghana http://ugspace.ug.edu.gh N Effect of Culture Filtrates 011 Germination and Radicle Development of Maize Grains 11 O Preparation of Polythene Bags and Field for Growing of Seeds 12 P Isolation of Rhizosphere and Non-Rhizosphere Fungi 13 Q Methods of Sterilization 13 R Experiment Precautions 14 III. EXPERIMENTAL PROCEDURE 15 A. Mycoflora of Maize Grains Abeleehi and Obaatanpa Varieties Incubated at 55-95% ERH for 36 days at 28-31°C 15 B. Radial Growth of Three Paecilomyces species Isolated from Abeleehi and Obaatanpa Varieties on Five Different M edia. 15 C. Radial Growth of Three Aspergillus spp Isolated From Abeleehi and Obaatanpa Varieties on Five Different Media 16 D. Radial Growth of Penicillium digitatum and Fusarium moniliforme Isolated from Abeleehi and Obaatanpa Varieties on Five Different Media. 16 E. In Vitro Studies on the Effect of Metabolites of Three Paecilomyces species on Germination and Radicle Development of Abeleehi and Obaatanpa Varieties 17 F. In Vitro Studies on the Effect of Metabolites of Aspergillus alutaceus (A. ochraceus) on Germination and Radicle Development of Maize (Abeleehi and Obaatanpa Varieties). 17 G. In Vitro Studies on the Effect of Metabolites of Penicillium digitatum and Fusarium moniliforme on Germination and Radicle Development Maize of (Abeleehi and Obaatanpa Varieties) 18 University of Ghana http://ugspace.ug.edu.gh H. Influence of Metabolites of Three Paecilomyces species on Germination and Radicle Development of Pepper (Capsicum annuum) and tomato (Lycopersicon esculentum Mill). 18 I. Influence of Metabolites of Three Paecilomyces species on Vegetative Growth and Dry Matter Accumulation and Chlorophyll Content of Abeleehi and Obaatanpa Varieties 18 J. Rhizosphere and Non-Rhizosphere Fungi Associated with Seedlings of Abeleehi and Obaatanpa Varieties Treated with M ycelium /M etabolites of Paecilomyces species 19 K. Preliminary Studies on the Anatomy of Maize seedling Growing under the Influence of the Metabolites of Paecilomyces spe-ci'es 20 RESULTS 21 A. Mycoflora of Zea Mays Var Abeleehi and Obaatanpa Incubated at ERH 55-95% for 36 days at 28 - 31°C 21 B. Radial Growth of Three Paecilomyces species Isolated from Abeleehi and Obaatanpa Varieties on Five Different M edia 25 C. Radial Growth of Three Aspergillus species Isolated from Abeleehi and Obaatanpa on Five Different Media 29 D. Radial Growth of Penicillium digitatum and Fusanum m onilifonne Isolated from Abeleehi and Obaatanpa Varieties on Five Different Media 33 E. In Vitro Studies on the Effects of M etabolites of Three Paecilomyces species on Germination and Radicle Development of Maize (Abeleehi and Obaatanpa Vars.) 36 F. In Vitro Studies on the Effect of Metabolites of Aspergillus alutaceus (A. ochraceus) on Germination and Radicle Development of M aize (Abeleehi and Obaatanpa Vars.) 54 G. In Vitro Studies on the Effect of the M etabolites of Fusaiium monilifonne and Penicillium digitatum on Germination and Radicle Development of Maize (Abeleehi and Obaatanpa Varieties) 58 University of Ghana http://ugspace.ug.edu.gh H. Influence of Metabolites of Three Paecilomyces species on Germination and Radicle Development of Pepper (Capsicum annuuml) and tomato (Lycopersicon esculentum Mill) 61 I. Influence of Metabolites of Three Peacilomyces species on Vegetative Growth and Dry Matter Accumulation and Chlorophyll Content of M aize Var Abeleehi and Obaatanpa 63 J. Rhizosphere and Non-Rhizosphere Fungi Associated with Seedlings of Abeleehi and Obaatanpa Varieties Treated with M ycelium / Metabolites of Paecilomyces species 74 K. Preliminary Studies on the Root Anatomy of Maize Seedling (Abeleehi and Obaatanpa Varieties) Growing under the Influence of Paecilomyces species 95 GENERAL DISCUSSION SUMMARY ACKNOWLEDGEMENT LITERATURE CITED APPENDIX University of Ghana http://ugspace.ug.edu.gh DECLARATION I, the undersigned, ANDREW AMEGBEDZI M INAM OR, author o f this thesis, do hereby declare that the work presented in this thesis: INFLUENCE OF THE METABOLITES OF THREE PAECILOM YCES SPECIES ON THE GERMINATION AND SEEDLING DEVELOPM ENT OF TWO GHANAIAN MAIZE VARIETIES ABELEEHI AND OBAATANPA was done entirely by me in the Department of Botany, University of Ghana, Legon from September 1993 to October 1995. This work has never been presented either in whole or part for any other degree of this University or elsewhere. Andrew Amegbedzi Minamor BSc (HONS) UNIVERSITY OF GHANA - LEGON (Professor G .T. Odamtten) SUPERVISOR University of Ghana http://ugspace.ug.edu.gh DEDICATION To Audrey and Wilhemina that they grow to fear the Lord, for the fear o f the Lord is the beginning o f Wisdom. University of Ghana http://ugspace.ug.edu.gh ABSTRACT The mycoflora of two recently-developed maize (Zea mays L) varieties Abeleehi and Obaatanpa have been studied under varying ambient equilibrium relative humidities E R H ’s (55, 60, 65, 70, 75, 80, 85, 90 and 95%) representative of the Ghanaian ambient conditions. The potential pathogenicity o f selected contaminating fungal species (A. alutaceus, = A. ochraceus, Fusarium, moniliforme, Penicillium digitatum, Peacilomyces carneus, P. puntoni and P. varioti) was also tested under laboratory, field and greenhouse conditions. Finally, the fungal succession or phenology o f the species encountered in the rhizosphere and non-rhizosphere soil containing treated maize grains (Abeleehi and Obaatanpa varieties) treated with conidia/mycelium or culture filtrate o f the three Paecilomyces species (P. carneus, P. puntoni and P. varioti) was studied. About thirty (30) and twenty-eight (28) species of fungi belonging to the genera Aspergillus, Penicillium, Curvularia, Chaetomium, Cladosporium, Emericella, Eurotium, Fusarium, Paecilomyces, Mucor, Neurospora and Rhizopus were isolated from Abeleehi and Obaatanpa varieties respectively at ERH ’s 55-95%. Aspergillus species {Aspergillus candidus, A. effusus, A. fum igatus, A. giganteus, A. niger, A. ochraceus, (= A. alutaceus), A. sulphureus, A. tamarii, A. ustus, A. versicolor, A. wentii and Aspergillus sp) predominated over the others followed by Penicillium (Penicillium brevi-compactum P. critinum, P. verrucosum, P. digitatum, P. expansum, P. funiculosum, P. glabrum and P. nigricans). Fungi belonging to the other genera encountered were Curvularia, Paecilomyces, Chaetomium, Cladosporium, Emericella, Eurotium, Fusarium, Mucor. The species diversity was influenced by grain variety and the ERH at which the grains were stored. Aspergillus flavus was ubiquitous and was encountered in all University of Ghana http://ugspace.ug.edu.gh grains stored at 55-95% ERH. Fusarium monilifonne was isolated from some grains incubated at 65-95% ERH. Xerophilic or xerotolerant fungal species like Aspergillus fum igatus, A. alutaceus ( = A. ochraceus), A. gigunteus, Paecilomyces carneus, P. puntoni and P. varioti were isolated at 55-65% ERH in both grain varieties. The best vegetative growth (radial) of selected species was influenced by both the medium and temperature of incubation. Paecilomyces carneus grew best at 30°C, P. puntoni at 30-35°C and P. varioti at 30°C. All the Paecilomyces species, however, could grow well at 40°C. Aspergillus species tested (A. flavus, A. giganteus, A. alutaceus) grew best at 30°C and remained depressed in growth at 40°C; so did Penicillium digitatum and Fusarium moniliforme. The three Paecilomyces species produced their toxic metabolites in 2 days and their undiluted culture filtrates depressed seed germination of ’Abeleehi’ and ’O baatanpa’ by 10-75% (depending on fungal species and period o f incubation). This inhibitory effect was gradually removed with increasing dilution (up to 1 :10v/v). There were varietal differences in the response of the germinating grains to the toxic metabolites of P. carneus, P. puntoni, and P. varioti. Undiluted culture filtrate of the listed three Paecilomyces species also severely depressed length of the emerging radicles of ‘Abeleehi’ and ‘Obaatanpa’ by 45-90% but this inhibition was gradually removed by increasing dilution of the culture filtrates (up to 1:10V/V dilution). The inhibition of seed germination and radicle development by culture filtrate o f the three Paecilomyces species was not confined to maize only as their adverse effect on seed germination and radicle development was reproduced in vitro using tomato (Lycopersicon esculentum Mill var. Owusu-Bio and Wosowoso) and pepper (Capsicum annuum L). In this instance, the inhibitory principle was still potent even at 1:10V/V dilution level. University of Ghana http://ugspace.ug.edu.gh Culture filtrate of Aspergillus alutaceus ( = A. ochraceus) at the highest concentration depressed seed germination of Abeleehi and Obaatanpa varieties by 50-70% and reduced radicle length by 60-90%. The inhibitory effect was gradually removed by increasing dilution (up to l:10v/v). Similarly, F. moniliforme and P. digitatum had the same deleterious effect on germination and radicle development of ’Abeleehi’ and ’Obaatanpa’ maize varieties. Seed germination was depressed 50-70% and radicle length by 40-90% when the undiluted culture filtrate of F. moniliforme and P. digitatum were applied to the grains. In all instances, the inhibitory effect was gradually removed by increasing dilution o f the culture filtrates. There were varietal differences between the three Paecilomyces species in their effect on vegetative growth and dry matter accumulation of Abeleehi and Obaatanpa maize varieties. Metabolites of P. carneus, P. puntoni and P. varioti variably depressed plant height, leaf width, leaf length, dry matter accumulation, (dry weight o f root and shoot systems) as well as chlorophyll a and b contents o f Abeleehi and Obaatanpa varieties cultivated in the field and under green house conditions. The maize cobs obtained from the field plants infected with Paecilomyces species were diminutive with fewer and smaller grains in the cob as compared to the control. Culture metabolites of P. carneus, P. puntoni and P. varioti reduced by 2-3 times diameter of roots of the seedlings of Abeleehi and Obaatanpa although the endodermis and pericycle were clearly formed and demarcated in both the control and treated seedlings. The pith parenchyma was thinly lignified and 2-3 times narrower in diameter in the treated plants exposed to the three Paecilomyces species; pro - and metaxylem vessels were about 2 times V University of Ghana http://ugspace.ug.edu.gh wider in the control seedlings and the phloem and xylem regions o f the roots o f the treated plants were reduced in number and size. Maize grains (Abeleehi and Obaatanpa varieties) inoculated with three Paecilomyces species influenced the rhizosphere mycoflora and their succession profile. Generally, the species of fungi that were stimulated, depressed or eliminated varied from one grain variety to another growing either in the field or under greenhouse conditions. Aspergillus flavus, A. niger, A. alutaceus, ( = A. ochraceus) and A. versicolor remained viable in the rhizosphere soil inspite of the presence of the inhibitory principles exuding from the Paecilomyces species; Penicillium citrinum could tolerate the same metabolites while P. digitatum, P. brevi - compactum did not grow very well in competition with the three Paecilomijces species. Population o f other fungi encountered belonging to the genera Cladosporium, Fusarium, Mucor, Scopulariopsis, Trichoderma and Yeast declined with time. Cladosporium herbarum, F. moniliforme, M ucor sp, Rhizopus otyzae and T. viride survived in the treated soil in competition with the metabolites of the Paecilomyces species. The practical implication of these findings are discussed and future studies suggested. University of Ghana http://ugspace.ug.edu.gh I. INTRODUCTION AND LITERATURE REVIEW Maize (Zea mays. L) is an important cereal grains in Africa ranking as high as rice as a staple food. In Ghana, maize is an important crop cultivated throughout the country with varying degrees of success depending on edaphic and climatic factors. The areas o f maize cultivation in Ghana include the whole of Southern Ghana, Ashanti, Brong Ahafo, the Northern regionSand parts of the Upper Region. Intensive commercial production o f maize is however found in the (a) Somanya District o f the Eastern Region, (b) the midland maize belt o f Ashanti and Brong Ahafo Regions, (c) The Ho-Kpandu District of Volta Region and Central Region. Elsewhere substantial amounts may be produced but are mainly used for home consumption. Being a seasonal crop, especially in West Africa, maize is stored as dry grains and forms an enormous reserve of food. The Ghana Food Distribution Corporation has two warehouse in the Greater Accra Region Kaneshie warehouse and Supreme warehouse at Tema. Balduzzi warehouse at Kumasi, G .N .T .C . warehouse at Sunyani, Galvanised I warehouse at Koforidua, Office warehouse at Cape Coast. The rest are Air Force W arehouse at Takoradi, Office warehouse at Ho, GRPC warehouse at Balgatanga and office warehouses at W a and Tamale respectively. However, considerable amount o f grains in storage are attacked by a variety o f insects, fungi and other bioderioagents. Losses in storage due to insects and fungi is estimated at 25 - 30% of the annual harvest (Rawnsley, 1969 Adams 1977) The role of fungi in quality deterioration of grains is well - documented in developed countries Bothast et al. 1979; Christensen and Kaufmann, 1965; 1969) but there is limited information regarding fungal flora of maize in West Africa and the role such fungi play. Broad- 1 University of Ghana http://ugspace.ug.edu.gh bent, et al (1969) provided an extensive list o f fungi associated with maize stored in Nigeria. Later, Oyeniran (1973 a,b) extended the list by eleven fungal species belonging to the genera Aspergillus and Penicillium. Two ecological categories o f fungi that invade seedsare FIELD FUNGI and STORAGE FUNGI.Field fungi are those that invade seeds on the developing plants in the field. They may be saprophytes (eg Alternaria tenuis, Cladosporium herbarum, Epicoccum nigrum) or in some seed pathogens fungi (eg Fusarium moniliforme and Verticillium alboatrum). Storage fungi are those that contaminate stored products. M ost o f them are able to grow without free water. Most of the storage flora are species o f Aspergillus and Penicillium. Some fungi {Aspergillus, Penicilliun etc can survive in seeds as long as 4-8years (Neergard, 1983). In Ghana, 42 fungal species belonging to 12 genera.(Aspergillus, Chaetomium, Cladosporium, Curvularia, Emericella, Eurotium, Fusarium, Mucor, Neurospora, Paecilomyces, Penicillium and Rhizopus.) have been isolated from different local maize varieties. (Danquah, 1973; Odamtten, 1986). Aspergillus species predominated followed by Penicillium. M islevic and Tuite (1962) also found that the predominant fungi in store dent maize were Penicillium species (P. brevi-compactum, P. cyclopium and P. viridicatum). Several isolates obtained by Richard, Tiffany and Pier (1969) from mouldy maize samples collected in Central Iowa, U.S. A were mainly species of Aspergillus and Penicillium in lesser quantities species of Chaetomium, Fusarium, Nigrospora, Rhizopus and Trichothecium. Most of the seed-transmitted pathogens are fungi. Some are easily detected, others occur but cannot be revealed by conventional testing procedures. To survive in seeds most fungi must be able to withstand dehydration. Xerophilic or xerotolerant fungi are characteristically capable 2 University of Ghana http://ugspace.ug.edu.gh of producing abundant xerotolerant propagules such as chlamydospores conidia or other dormant structures, including dormant mycelium, sclerotia etc. Examples include species of Paecilomyces, Alternaria Cercospora, Curvularia, Dreschlera and Stemphylium (Neergaard 1983). Metabolites o f these fungi may have beneficial or adverse effects on plant growth including suppression of seed germination, malformation and retardation o f growth o f seedlings (Leelavathy, 1969; Narain and Prakash, 1968); (Odamtten and Clerk, 1985, 1988) root growth promoters (Kimura et al, 1992 a,b). Fungi involved are members o f the Aspergillus, Penicillium, Alternaria etc. The association o f such seed-borne fungi with stored grains may result in serious reduction in crop yield in the field by seeds infected with pathogenic seed-borne fungal species. The increased attempt by man to cultivate new varieties o f crops more suited to his climate has necessitated breeding programmes that require crossing o f local varieties with imported grain varieties which are not indigenous to Africa. The attendant problem is the production of new varieties whose versatility in terms o f drought tolerance, yield and susceptibility to local indigenous diseases have not been thoroughly investigated p rior to introduction of the crop to the farmers. Recently, a new storage problem o f maize grains has been reported in certain parts o f Africa (Tyler, 1992) called Stackburn. Maize grains stored in woven polypropylene bags in the warehouse heated (up to > 40°C) and caused discolouration of the seed coat and germ region turning from white to varying shades of brown. These grains are subsequently downgraded and 3 University of Ghana http://ugspace.ug.edu.gh disposed of cheaply. This constitute a great loss to the farmer and the warehousing agent and above all a great threat to food security in Africa. The European Community is funding a collaborative research (EC Contract TSD3 CT 920097) to establish the causes and prevention o f stackburn in sub-saharan Africa including Ghana, Zimbabwe, Mozambique, Angola and Tanzania with inputs from two European Countries, Britain (Natural Resources Institute) and Portugal (IICT, CEFA). As part o f the ECDGXII Maize Stackburn Project, in Ghana, we looked at the resident mycoflora of two newly developed maize varieties on the market, Abeleehi and Obaatanpa and their influence on the storage potential o f the two maize varieties. An exhaustive search through the pertinent literature revealed that no detailed study has ever been made in West Africa to elucidate the adverse effect o f the Paecilomyces species associated with stored maize grains not excepting the two-newly developed varieties. Furthermore, the effect of the metabolites of these species on seeds and other economic crops such as pepper and tomato earmarked for long term storage has hitherto received limited attention. This thesis reports the effect o f some of the seed-borne fungi (Paecilomyces carneus, P. puntoni, P. varioti) isolated from Abeleehi and Obaatanpa on the seed germination, radicle development and field and greenhouse performance of Abeleehi and Obaatanpa. The influence of the Paecilomyces species on seed germination of pepper and tomato as well as phenology of the rhizosphere mycoflora of Abeleehi and Obaatanpa were also studied. 4 University of Ghana http://ugspace.ug.edu.gh II. MATERIALS AND METHODS (i) MATERIALS The fungal species, Aspergillus f la w s , A, giganteus, A. ochraceus, (= A , alutaceus) Fusarium monilifonne, Paecilomyces carneus, P. varioti, Penicillium digitatum and P. expansum used in these investigations were isolated from maize grains and the air at Kaneshie W arehouse and Supreme Warehouse at Tema. The maize varieties used Abeleehi and Obaatanpa were purchased from Aglow Seed Company Accra. The soil used for greenhouse experiment was obtained from the Botanical gardens. Black polythene bags (35cm x 20.3 cm) were used for sowing the maize. A plot of land in front o f the Carpenter’s W orkshop in Botany Department, University o f Ghana, Legon was ploughed for planting maize for the field work. (ii) GENERAL METHODS Mvcoflora of maize grains Abeleehi and Obaatanpa varieties incubated at 55-95% ERH for 36 days at 28-31°C (a) Maize samples kept under humidity chamber Maize samples of Abeleehi and Obaatanpa varieties were kept at 55 ,65,75,85 and 95% Equilibrium Relative Humidity (ERH) provided by glycerol; water mixtures and at temperature of 28-31°C for 36 days. b Direct - Plating Method The maize grains were surface - sterilized by washing in M ilton’s reagent (1% sodium hypochorite + 16.5% sodium chloride) for 5 min. and then rinsed with three changes o f sterile water. Sodium hypochlorite treatment was used with the aim o f reducing or removing completely external saprophytes which compete with pathogens. Ten surfaced - sterilized grains were placed on either Sabouraud Dextrose Agar (Oxoid CM41) Dichloran Glycerol Agar, DG18 (Oxoid CM 727) in Petri plates without further treatment. The plates containing Sabouraud’s Agar and DG18 were incubated until fungi grew. There were 25 replicates for each grain variety. 5 University of Ghana http://ugspace.ug.edu.gh C. Identification of Fungi Fungi encounted in these investigations were identifi^by their colour, culture and morphological characteristics using the conventional identification manuals of fungi by; Barnett and Hunter (1972), Ellis and Ellis (1988), Funder (1953), Gilman (1957), Ramirez (1982), Samson and Reenen-Hoeskstra (1988) and Smith (1960). Where necessary, the identification o f the fungi were confirmed by my supervisor. D. Serial-Dilution Method A lOg sample of the grains was weighed and transferred aseptically into 100ml 0.1% Peptone in 250ml Erlenmeyer flasks and then shaken in Gallenkamp M odel Orbital shaker at 140rev/min for 30 mins. From this stock suspension serial dilution method was employed up to 1:10v/v and spores were raised either in Sabouraund’s Agar (Oxoid CM 41) or Oxytetracycline GlucoseYeast Extract Agar (Oxoid CM 545). The objective of using two media is to recover a wider range of fungal species from the incubated grain. The plates were incubated at 28-31°C until fungi grew (7-14 days). E. Maintenance of Stock Cultures Stock cultures of A. flavus, A. giganteus, A. ochraceus, (= A. alutaceus) F. moniliforme, P. carneus, P. varioti, Penicillum digitatum and P. expansion were maintained on slopes o f Potato Dextrose Agar, slants in MaCartney tubes and sub-culturdevery two weeks. F. Preparation of Media The composition of media used were as follows: (i) Czapek-Dox Agar NaNo3 - 20g; KCI - 0.5g; M „P04 - 0.5g FeS04 + 7H20-01g; K2S 0 4, 7H20 - 0.35g; Sucros - 30.Og, Agar - (12-15)g PH 6.3, Sterile distilled water - 100ml 6 University of Ghana http://ugspace.ug.edu.gh 7 (ii) Potato Dextrose Agar 200g of peeled potato were boiled in 500ml of water, strained and made up to 1000ml; 20g glucose and 20g Agar were added. (iii) AND (iv) MAIZE MEAL AGAR PREPARED FROM EITHER ABELEEHI OR OBAATANPA 200g maize blended and 500ml o f distilled water added. This was heated for a few minutes. The suspension was filtered through Buchner funnel to obtain a near clear solution. 20g of glucose and 20g Agar were added and made up to 1 litre with sterile distilled water. (v) MALT EXTRACT AGAR Maltose 52.2% Dextrose 19.1% Sucrose 1.8% Dextrin 15.0% Other carbohydrate 3.8% Protein 4.6% Ash 1.5% Water 2.0% Agar (15 - 20)g. (vi) MODIFIED COOK’S MEDIUM (AFTER COOKE, 1954) Dextrose - lOg; Peptone, - 5g, KH2Po4 - lg M gS04 - 7H20 - 0.5g; Rose bengal - 0.035g; Streptomycin - 0.35g; Agar - (10 - 15)g Sterile distilled water - 1000ml. University of Ghana http://ugspace.ug.edu.gh G Method of Inoculation Two diameters at right angles to each other were drawn at the bottom of the Petri dishes (9.0cm) with grease pencil after the agar medium has set. Each plate was held in inverted position, the lid was removed and the plate inoculated at the intersection o f the two diameters with conidia on 2mm Agar disks at the tip of a flamed - sterilized innoculation pin. The lid was placed back and the plates incubated in the inverted position. This method o f inoculation completely obviated the usually sprinkling of powdery spores o f Penicillium and Aspergillus species on plates inoculated in the upright position. In the case o f Paecilomyces and Fusarium species, the agar disks bearing the inoculation was placed directly at the centre o f the plate. The plates were inoculated in triplicate for each species and were incubated at 18°, 30°, 35° and 40°C. H. Assessment of Growth Fungal Cultures Vegetation growth in liquid medium was assessed by estimating the dry weight of harvested mycelium at the end of the incubated period. Mycelium collected on a previously weighed and dried Whatman N o.l filter paper was dried at 80°C for 24h. The filter paper carrying the dried mycelium was then weighed after it has been allowed to cool in a desiccator. Growth of cultures on solid media in Petri dishes was assessed by measuring width of culture along two diameters for 7 days. I Seed Viability Test Maize seeds completely free from fungal attacks were used in the viability test. Fifty seeds each of Abeleehi and Obaatanpa varieties were cut longitutdinally to expose the germ region and then placed in sterile Petri dishes containing Tetrazolium Chloride solution. There were five replicates for each maize variety. The plates were incubated in total darkness for at least three hours. Thereafter the number of seeds showing characteristics pinkish colour in the germ region were counted and percentage viability calculated. University of Ghana http://ugspace.ug.edu.gh J frti vitro studies on the effect of Fungal M etabolites on Germination and Radicle Development Liquid static culture filtrate of the local isolates o f Aspergillus flavus, A giganteus, A. OChcraceus, (A. alutaceus), Fusarium moniliforme, Paecilomyces carneus, P. puntoni, P. varioti, Penicillium expansum, P. digitatum were obtained by raising the listed fungi (aliquot o f 1.2 - 1.8 x 105 spores/ml per flask) in either 30ml of Potato Dextrose Broth (PDB), M aize Meal Broth (MMB) prepared from both Abeleehi and Obaatanpa varieties. The mycelium was harvested after 2,4 and 8 days at 28 - 31°C. Vegetative growth o f the fungi was assessed by the convetional dry weight method and the cultural filtrates stored separately in 500ml Erlenm eyer flasks covered with black polythene bags for immediate use. The pH of the filtrates were taken before and after each pre-determined incubation period using TOA pH meter HM - 60s (TOA Company Japan). The culture filtrates were used either undiluted or diluted (1:1. 1:2, 1:5 and 1:10 v/v). About 10 grains of either Abeleehi O f Obaatanpa varieties were placed on sterile filter paper in 9.0cm petri dishes moistened with 10 ml distilled water (control) or with 10 ml of culture filtrate of the listed fungi diluted (1:1. 1:2, 1:5 and 1:10 7 V) as above. There were 250 grains for each dilution level of culture filtrates and period of growth (2,4, 8 days) of the repective fungi. Percentage germination was calculated after 5 days incubation at 28 - 31°C and the length of radicle noted. The length o f the radicles (hypocotyl) are given as ratio (°/0) to those of the control seedlings in distilled water (Kimura et al 1992a). The above experiment was repeated this time using only culture filtrate o f the three Paecilomyces species. Seeds of pepper (Capsicum annuum L) and tomato (Lycopersicon 9 University of Ghana http://ugspace.ug.edu.gh (!( )'l esculentum var Wosowoso and Var. Owusu Bio) were used for the bioassay on the effect o f fice culture filtrate on seedlings in distilled water (Kimura et al 1992 a). o j V / ̂ n S V ' K. Influence of Fungal Metabolites on Vegetative Growth and Dry M atter Accumulation by Maize Seedlings (i) Field Studies Healthy surface - sterilized grains o f Abeleehi and Obaatanpa were inoculated with mycelium/spore suspension of the respective 7 days old culture o f P. carneus, P. puntoni and P. varioti. A small superficial slit was made in the germ region o f the grain and the inoculum (about 1.8 - 2.8 x pores/ml) applied directly into the slit. The inoculated grains were incubated in Petri dishes for 24h at 30°C to allow the grains to take th fungus. The grains were then sown in the field plot at the recommended spacing o f holes 25 cm apart in rows 90cm apart. There were 50 replicates per treatment. Records o f plant height, leaf width (at the broadest point) and leaf length were taken after 7, 21, 35, 42 and 56 days growth. (II) Greenhouse Studies Black polythene bags (35cm x 20.3cm) served as pot for soil which were seeded with five grains of either Abeleehi and Obaatanpa varietiesand then thinned to two per bag after germination. The soil in each bag was moistened with either 20-30ml undiluted culture filtrate of either P. carneus, P puntoni or P. varioti initially at two days intervals for 2 weeks and there^ter at weekly intervals. There were 50 replicates per treatment. The germinating seedlings were kept in the greenhouse exposed to the normal day/night regime at 28-31°C. Measurement of stem height, leaf width, leaf length dry,weight o f shoot, leaf and root systems University of Ghana http://ugspace.ug.edu.gh were made after 7, 21 35, 42 and 56 days. The dry weight o f plant parts were determined by keeping them at 80°C for 48h and then weighed after cooling. L Chlorophyll content About 100ml of 80% acetone extract of maize leaf (2g wt) was filtered through W hatman No. 1 filter paper in Buchner funnel. The colour of the resultant liquid was read on Shimadzu Spectrophotometer at 663nm and 645nm using 80% acetone as blank. Chlorophyll concentration was calculated as follows: Chlorophyll a = 12.7 x Absorption at 663nm - 2.69 x Absorption at 645 (mg/1). Chlorophyll b = 22.2 x Absorption at 645nm - 4.67 x Absorption at 663 (mg/1). Total Chlorophyll = 20.2 x Absorption at 6 45nm + 8.02 x Absorption at 663 (mg/1). M Anatomical Studies The structure of the root of seedlings growing under the influence o f the metabolites of the three Paecilomyces species in the field was studied. Sliding microtome (Reichert Nr. 15917, Austria) sections were stained temporarily in aniline chloride and permanently with safranin and fast green following the procedure of Purvis et al (1966). N Effect of Culture Filtrates on Germination and Radicle Development of Maize Grains (BLOTTER TEST METHOD) Ten surface-sterilized grains were placed on sterile W hatm an’s No. 1 filter paper in 9.0cm Petri dishes. The filter papers were moistened with 5ml of appropriate dilutions of the cultured filtrate. There were 25 replicates (250 grains) for each treatment. Sterile distilled water served as control. 11 University of Ghana http://ugspace.ug.edu.gh The plates were incubated in darkness for 5 days at 28-31°C. Two maize varieties Abeleehi and Obaatanpa were used for the germination tests. The lengths o f the emerging radicles were measured and dry weight determined at 80°C for 24h for each treatment. Tables of values shown at the appropriate places in the text present the mean values obtained from the readings taken. O Preparation of Polythene Bags and Field for Growing of Seeds The soil used in this investigation is a sandy loam (C, 0 .8% , N, 0 .1% , pH (H20 ) 5,2). The soil was mixed thoroughly, air-dried,sieved (< 2 m m ) to collect unwanted particles before transferring into polythene bags (35 x 20.3 cm) provided with holes for drainage. Forty polythene bags were filled to about 3/4 full making sure that the same amount o f soil was found in each polythene bag. Washed coarse sand was spread over the surface o f the soil to prevent compaction of the soil surface during watering. Thereafter, the soil was moistened daily with tap water before planting and placed in the greenhouse. A plot o f land (80 x 50m) in front o f the carpenter’s workshop in Botany Department University of Ghana was weeded and ploughed. The plot was left for three (3) days before sowing the grains at the recommended spacing for maize grains (seed holes 25cm apart in rows 90cm apart). 12 University of Ghana http://ugspace.ug.edu.gh P Isolation of Rhizosphere and Non-Rhizosphere Fungi Rhizosphere and non-rhizosphere fungi were isolated initially and after 14,28,42 and 56 days growth of seedlings. The conventional serial dilution plate method (Waksman 1922) was used to isolate the fungi from both the rhizosphere and non-rhizosphere soil using Cooke’s medium (Cooke, 1954) Samples of rhizosphere soil (lg ) from two plants were collected at each pre-determ ined harvest of 14,28,42 and 56 days. The samples were selected according to the table o f random numbers set out for the potted seedlings. A quantity of root (roots o f two seedlings) material was used after shaking off excess soil to allow only an adequately rich suspension adhering to the roots for serial dilutions up to 1:105. Samples of non-rhizosphere soil were collected by removing 2.5cm core o f the top soil with sterile N o.6 Cork borer (1cm in diameter). Q Methods of Sterilization Maize grains were surface sterilized with 2% sodium hypochlorite for 5 min. The grains were then rinsed in three changes or more sterile distilled water. All media, conical flasks, MaCartney tubes pipettes were sterilized by autoclaving for (1.5 mins at 121°C steam pressure of l.lk g t/cm 2). Cotton wool plugs, filter papeiSwere temporarily covered with grease paper to prevent penetration by any condensed water during autoclaving. 13 University of Ghana http://ugspace.ug.edu.gh Slides cleaned with detergent and thoroughly rinsed under running water and then in distilled water were stored in 90% ethyl alcohol and sterilized by flaming just before use. Forceps and inoculating needles and loops were flamed - sterilized by heating in an electric-oven at 165°C for 6h. The inoculation room was sterilized by spraying with 5% dettol solution immediately before use. R Experiment Precautions 1. Glassware was scrupulously kept clean. Glassware which had already been cleaned with water and detergents was rinsed several times with tap water and distilled water and allowed to drain before use. 2. Petri dishes were sterilized by putting them in canisters and then put in the oven at 105°C for 8h, removed and allowed to cool before use. 3. All the media including filter papers were sterilzed at steam pressure o f l.lk g t/c m 2 in the autoclave (121°C for 15 min.). 4. Inoculation room was sprayed with 5% dettol. All tables were wiped with 95% ethanol, loop, scapel were all flamed on spirit burner lamp before use. 14 University of Ghana http://ugspace.ug.edu.gh III. EXPERIMENTAL PROCEDURE A. Mycoflora of Maize Grains Abeleehi and Obaatanpa Varieties Incubated at 55-95% ERH for 36 days at 28-31"C Seeds of cereal grains and legumes harvested from the field harbour both field fungi (mycoflora contaminating seeds in the field before harvest) and storage fungi (those fungi that become resident during post-harvest storage of seeds). These two categories o f fungi have been known to shorten the shelf-life o f stored seeds; reducing seed viability, nutrient content and imparting toxic metabolites into the seed to mention but a few. In this chapter mycoflora of two newly developed maize varieties Abeleehi and Obaatanpa was assessed under environmental conditions representative o f the local Ghanaian tropic condition (ERH’s 55, 65, 75, 85 and 95% and temperature 28-31°C) provided by glycerol; water mixtures. The methods employed are described under the Materials and General M ethods section (ii) (a) & (b). Results obtained are presented in Table 1. B. Radial Growth of Three Paecilomyces species Isolated from Abeleehi and Obaatanpa Varieties on Five Different Media. Many fungal species were isolated from both maize varieties in chapter A. O f interest, however, were three Xerophilic Paecilomyces species (P. carneus P. puntoni, P. varioti) which are being recorded for the first time on stored Ghanaian maize varieties. These fungi could be of pathological importance if their metabolites affect the viability and germination capacity of grains of Abeleehi and Obaatanpa used as seed maize in the next planting season. There is hardly any information in the pertinent literature on the physiological condition for optimal growth of these species isolated in Ghana. In this Chapter five different natural and 15 University of Ghana http://ugspace.ug.edu.gh synthetic mycological media (Czapek-Dox Agar, Maize Meal Agar (Abeleehi), M aize Meal Agar (Obaatanpa), Malt Extract Agar and Potato-Dextrose Agar) were tested for ability to support optimal growth of the Paecilomyces species at different temperature (18°, 30°, 40° and 45°C). Radial growth in 7 days on agar was measured along two diameters marked at the bottom o f the Petri plates (see Materials and General methods, section (ii) (h). Results obtained are presented in figs. 1 to 3 and Appendices la to If. C . Radial Growth of Three Aspergillus spp Isolated From Abeleehi and Obaatanpa Varieties on Five Different Media The experiments in Chapter B were repeated this time using three Aspergillus species (A. flavus, A. giganteus, A alutaceus (= A. ocliraceus) which were also isolated from the two maize varieties. The pathological importance of fungi in stored seeds cannot be confined to the genus Paecilomyces only. Aspergillus species are ubiquitous affecting the viability o f stored seeds of several unrelated general of crops (Christensen, 1972, Neergaard, 1983). The experimental methods used were same as used in chapter B. Results are summarised in figs 4 to 6 and Appendices la to le. D. Radial Growth of Penicillium digitatum and Fusarium m onilifonne Isolated from Abeleehi and Obaatanpa Varieties on Five Different M edia. Radial growth on agar as a measure of vegetative growth on five different natural and synthetic media was used to determine near optimal conditions for growth of Paecilomyces and Aspergillus species which were encountered as storage fungi in Abeleehi and Obaatanpa maize varieties. In order to augment the information obtained in chapter B, C and D, experiments carried out in chapter B,C, and D were repeated using Penicillium (P. digitatum, P. expansion) 16 University of Ghana http://ugspace.ug.edu.gh and Fusarium (F. moniliforme) species equally of pathological importance in seed storage. Results obtained are presented in figs. 7 to 8 and in Appendices la to If. E. In Vitro Studies on the Effect of Metabolites of Three Paecilomyces Species on Germination and Radicle Development of Abeleehi and Obaatanpa Varieties Metabolite of fungi may have either beneficial or adverse effect on the germination and radicle development of seedlings. In this chapter, the effect of the metabolites o f three Paecilomyces species were used to prepare varying dilutions (undiluted - 1 :1 0 \} o f germinating mediumjfor testing germination capacity of grain of Abeleehi and Obaatanpa varieties. The method of production of metabolites, the blotter test method, and the assessment o f the effect of the metabolites on radicle development are spelled out under the M aterials and General Methods section ii (n). Results obtained are presented in figures 9 to 16 and in plates 5 to 10. F. IN Vitro Studies on the Effect of Metabolites of some Aspergillus species on Germination and Radicle Development of Abeleehi and Obaatanpa Varieties. Experiments carried out in chapter E were repeated using metabolites o f Aspergillus species (A. flavus, A. giganteus and A. alutaceus (= A. ochraceus) prepared according to the methods described in the Materials and General methods section (ii) (n). The percentage germination and length of radicle obtained (measured as % of the control) are presented in fig 20 and in Tables 2 to 3. 17 University of Ghana http://ugspace.ug.edu.gh G. In Vitro Studies on the Effect of Metabolites of Penicillium digitatum and Fusarium moniliforme on Germination and Radicle Development of Abeleehi and Obaatanpa Varieties This chapter was a logical sequel to studies already carried out in chapters E& F to complete the effect of metabolites of selected fungal species (isolated from Abeleehi and Obaatanpa varieties) on the germination and radicle development o f maize. The procedure and methods used were exactly as stated in chapters E and F. Results obtained are presented in Tables 2 and 3. H. Influence of Metabolites of Paecilomyces species on Germination and Radicle Development of Capsicum annum and Lycopersicon esculentum. In chapter E the culture metabolites of the three Paecilomyces species variably depressed germination and radicle development of Abeleehi and Obaatanpa varieties. In this chapter the experiments in chapter E were repeated this time using seeds o f two vegetables, pepper (Capsicum annuumL) and tomato (Lycopersicon esculentum Mill) to see if the depressive effect of the metabolites of the three Paecilomyces spp would have same or similar effects on the germination and radicle development of the selected vegetable seeds. Results obtained are presented in Table 4. I. Influence of Metabolites of Paecilomyces species on Vegetative Growth and Dry Matter Accumulation and Chlorophyll content of Abeleehi and Obaatanpa Varieties In vitro studies in chapter E-H have demonstrated that metabolites o f Paecilomyces species have indeed variable adverse effect on the germination and radicle development of Abeleehi and Obaatanpa maize varieties. In this chapter studies were carried out in the field and 18 University of Ghana http://ugspace.ug.edu.gh under greenhouse conditions to ascertain the effect o f the metabolites and the mycelium o f the three Paecilomyces spp on vegetative growth and dry matter accumulation by maize seedlings. The grains sown in the field were inoculated with mycelia/conidia o f the test fungi before seeding in soil, (see Materials and General Methods sectionl), whereas those kept in the greenhouse were provided with pre-determined volumes (20-30ml) o f metabolites o f the fungi at varying intervals (see Materials and General\M ethods section K(ii)). The dry weight o f the root and shoot systems of the seedlings were determined at varying intervals (7 ,21,35,42 and 56 days) by the oven-dry method (see Material and General Methods section K(i) & (ii). In the case of the field experiment the seedlings were left long enough for three months to bear cobs and the yield and dimensions of the cobs determined. The Chlorophyll content o f the leaves of the seedling was also estimated (see Materials and General Methods section J). Results obtained are presented in Figs. 23 to 25 and in Table 5. J. Rhizosphere and Non-Rhizosphere Fungi Associated with Seedlings of Abeleehi and Obaatanpa Varieties Treated with M ycelium/M etabolites of Paecilomyces species Phenology of rhizosphere fungi is characteristics of seedlings o f a particular species and may vary from one crop to another. Would the rhizosphere mycoflora of maize grains inoculated with Paecilomyces species influence the vegetative growth and dry matter accumulation by Abeleehi and Obaatanpa varieties in soil? The phenology of rhizosphere and non-rhizosphere mycoflora of Abeleehi and Obaatanpa treated with Paecilomyces spp was followed for up to 56 days using the decimal serial dilution method on Cookes medium (Cooke, 1954). Results obtained are presented in Tables 6 to 21. 19 University of Ghana http://ugspace.ug.edu.gh K. Preliminary Studies on the Anatomy of Maize seedling Growing under the Influence of the Metabolites of Paecilomyces spp In chapter 1 seedlings growing under the influence o f Paecilomyces spp became diminutive and had a slower dry matter accumulation rates. Anatomy o f transverse sections of the roots were studied to provide clues as to the nature o f effect o f the metabolites on the growing seedlings. Transverse sections of the root tissues were cut by a sliding microtome (Reichert Nr 15917 Austria) and double stained using the method o f Purvis et al (1966). Plates 15 a,b and 16 a,b shows results obtained. 20 University of Ghana http://ugspace.ug.edu.gh IV RESULTS A. Mycoflora of Zea Mays Var Abeleehi and Obaatanpa Incubated at ERH 55-95% for 36 days at 28 - 3 1°C The list of seed-borne fungi isolated from the two maize varieties is presented in Table 1. About thirty different seed-borne fungal species were isolated from Abeleehi and twenty eight from Obaatanpa variety at the varying equilibrium relative humidities (55,60,65,70,75,80,85,90,95% ) at which the grains were stored. Aspergillus species (A. Candidas, A. ejfusus, A. fum igatus,A. n iger^alutaceus (= A. ochraceus), A. sulphureus, A tamarii, A. ustus, A. versicolor, A. wentii and Aspergillus sp 1) predominated over other species encountered followed by Penicillium (P. brevi compactum, P. citrinum, P. verrucosum, P. digitatum, P. expansum, P. funiculosum , P. glabrum, P. nigricans and Penicillium sp). Fungi of other genera (Curvularia, Paecilomyces, Chaetomium Cladosporium, Emericella, Eurotium, Fusarium, Mucor, Neurospora and Rhizopus) were also isolated. The species diversity was influenced by grain variety and the ERH at which they were incubated. A. flavus was ubiquitous and was isolated from both Abeleehi and Obaatanpa stored at ERH 55-95%; Fusarium moniliforme was encountered at ERH 65-95% . Xerophilic species like A. fumigatus, A. giganteus, A. alutaceus, (= A. ochraceus), Paecilomyces carneus, P. puntoni and P. varioti were isolated at ERH ’s 55-65% in both grain varieties. Plates 3-4 show some of the fungi isolated from the maize varieties. 21 University of Ghana http://ugspace.ug.edu.gh 2 2 LIST OF SEED-BORNE FUNGI ISOLATED FROM M AIZE VARIETIES ABELEEHI AND OBAATANPA INCUBATED AT 55-95% EQUILIBRIUM RELATIVE HUM IDITY FOR 36 DAYS AT 28-31°C. TABLE 1 lergilhis •ffusus Tiradoschi2 lavus Link ex Fr. 1,2 umigatus Fresenius1,2 ’iganteus W ehmer1 tiger Van Tieghem1,2 Khraceus W ilhelm1,2 ulphureus1 (Fresenius) Thom and Church. amarii Kita1,2 erreus Thom1 Geerlings1,2 istus Bainier Thom and Church1 ’ersicolor (Vuillemin) Tiraboschi2 ventii Wehmer1 lergillus indet2 iicillium brevicompactum Dierckx1,2 'itrium Thom1,2 ’errucosum Dierckx1'2 ligitatum Sacc1,2 •xpansum Link1,2 uniculosum Thom2 ’labrum (Wehmer) Westling1’2 ligricans Bainier nicillium indet1,2 Paecilomyces carneus (Duche et Heim) A .H . Brown G. Smith 1,2 P.puntonii (Vuillemin) Nannizzi1,2 P.varioti Bainier1,2 Chaetomium globosum Kunze : Fries2 Cladosporium herbarum (Persoon : Fries) Link1,2 Curvularia lunata Boedji12 Emericella nidulans (Eidam) V uill.1 Eurotium sp. 1,2 Fusarium moniliforme Sheldon1,2 Mucor haemalis W elm er f .hiemalis1 Rhizopus oryzae W ent and Pri Scopulariopsis brevicaulis (Sacc) Bain2 Neurospora sitophila Shear and D odge1,2 1 - Abeleehi variety 2 - Obaatanpa variety candidus Link ex. F r.1,2 University of Ghana http://ugspace.ug.edu.gh 23 Plate 3. Photograph of 6 days old Paecilomyces varioti isolated from ’Abeleehi’ and ’Obaatanpa’ maize varieties growing on Potato Dextrose Agar at 29°C. (X 3/4) University of Ghana http://ugspace.ug.edu.gh 24 Plate 4. Photograph of 6 days old Fusarium moniliforme isolated from ’A beleehi’ and ’Obaatanpa’ maize varieties growing on Potato Dextrose Agar at 29°C for 6 days, (x 3/4) University of Ghana http://ugspace.ug.edu.gh B. Radial Growth of Three Paecilomyces species Isolated from Abeleehi and Obaatanpa Varieties on Five Different Media Radial growth of Paecilomyces species on agar was influenced by the media and temperature of incubation. Each Paecilomyces species behaved differently. Optimum growth of P. carneus on all media tested (Czapek-Dox Agar; M aize meal Agar- Abeleehi; Maize Meal Agar - Obaatanpa; Potato Dextrose Agar) was attained at30°C ; 35°C and 40°C were clearly unsutable for growth in some instances. Although the fungus grew at 35°C and 40°C, it remianed static after 2-4 days (Fig. 1) except on (Capek-Dox and M aize Meal Agar (prepared from Obaatanpa variety ) where growth at 35°C nearly approximated that obtained at30°C. Growth at a temperature of 18°C was intermediate between 30°C and 40°C whereas radial growth was slowest on Potato dextrose agar diameter 60mm at 30°C for 7 days (Fig. 1). The best radial growth of P. puntoni was attained between 30-35°C (Fig. 2). Growth at 18°C initially lagged behind that of cultures incubated at 30-40pC but approximated their radial growth after 6-7 days incubation period. There were no significant statistical differences (P < 0 .0 5 student t-test) in the growth of P. puntoni incubated in all the media tested at all temperatures ie all the media tested equally supported radial growth of P. puntoni (Fig. 2). Temperature of 35°C and 40°C depressed radial growth o f P. varioti in all the media tested (Fig. 3). Although the best temperature for growth of P. varioti was 30°C, 18°C was almost as suitable as 30°C in supporting vegetative growth of this fungus in most instances (Fig. 3). Radial growth of P. varioti was slowest on Potato dextrose agar (Fig. 3). 25 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 ) D IA M E T E R O F C O L O N Y (n u n ) PAECILOMYCES CARNEUS CZAPEK - DOX AGAR PAECILOMYCES CARNEUS MALT - EXTRACT AGAR INOCULATION PER IO D (IN d t y t ) I8*C + 30*C *-35«C * 4 0 « C IN O CU LA TIO N PE R IO D ON d l y t ) — lfl«C -4-30*C * 3 5 » C PAECILOMYCES CARNEUS MAIZE-MEAL AGAR, A beleeh i PAECTLIOMYCES CARNEUS MAIZE-MEAL AGAR, O b a a ta n p a ‘ 18*C + 30*C 35*C 40*C - 18*C + 30*C 35*C -» 4 0 * C PAECILONYCES CARNEUS POTATO - DEXTROSE AGAR — 18*C + 30*C * 36«C ♦ 40*C Fig. 1 i„dica?edaL £ a ° Wth °f carneus on £ive different University of Ghana http://ugspace.ug.edu.gh 2 7 PAECILOMYCES PUNTONI CZAPEK - DOX AGAR PAECILOMYCES PUNTONI MALI - EXTRACT AGAR — IB 'C + 30*C * 3 5 * C -» 4 0 "C - 1 8 ‘ C + 30«C * a s - c PAECTLIOMYCES PUNTONI MAIZH-MKAL AGAR, A beleeh i PAECILOMYCES PUNTONI HAIZE-MRAL AGAR, O b a a ta n p a INOCULATION P E fllO D ON d l r < ) - IB‘C + 30»C * 3 5 * C -»40*C INOCULATION PE R IO D (IN d i r t ) — I8"C + 3 0 * C * 3 5 * C '• ’■40'C PAECILOMYCES PUNTONI POTATO - DEXTROSE AGAR INOCULATION PE R IO D (IN d > r « ) — 1B"C + 3 0 " C * 3 B * C * 1 0 * C Fig. Radial growth of P_̂ puntoni on five different indicated media. note (The growth at 18°C initially lagging behind the remaining temperatures). 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 ) D IA M E T E R O F C O U 2M V (m m ) 28 PAECILOMYCES VARIOTI CZAPEIC - DOX AGAR INOCULATION PER IO D (IN d » jr t ) —- 1S*C + 3 0 - C * 3 5 » C 4 0 #C INOCULATION PE R IO D ON d ajr» ) 1 B*C + 3 0 * C -* 3 5 * C -» 4 0 * C PAECILOMYCES VARIOTI MALT - EXTRACT AGAR PAECILIOMYCES VARIOTI MAIZE-MEAL AGAR, O b a a ta n p a INOCULATION PE R IO D (IN d t y t ) — lfl»C + 3 0 - C * 3 5 * C •»40»C PAECILOMYCES VARIOTI M1AIZE-MEAL AGAR, A beleeh i PAECILOMYCES PUNTONI POTATO - DEXTROSE AGAR Fig. Radial growth (note The relative INOCULATION PE R IO D (IN d i r t ) — 18*C + 3 0 * C * -3 5 * C -» 4 0 * C INOCULATION PE R IO D (IN d f ty l) + 30«c -**35«C 40*C of varioti on five different indicated media. slow growth of fungus on PDA) . University of Ghana http://ugspace.ug.edu.gh C. Radial Growth of three Aspergillus species Isolated from Abeleehi and Obaatanpa on Five Different Media The mycological media as well as temperature of incubation influenced radial growth of Aspergillus species (A. flavus, A. giganteus and A. alutaceus (= A. ochraceus) tested. Generally, the optimum growth of A. flavus was obtained on all media at between 30°C and 35°C (Fig. 4). Growth was depressed at 40°C but not at 18°C (Fig. 4) except on Czapek- Dox Agar. The best medium for growth o f A. flavus was Maize Meal Agar prepared from Obaatanpa variety. Radial growth was poorest on Potato Dextrose Agar and Czapek-Dox Agar attaining a radius of 50mm after 7 days at 30°C as compared to 85.90mm after 7 days at 30°C on maize meal agar prepared from Obaatanpa variety (Fig. 4). A. gigianteus grew best at 30°C followed by 18°C on all media. Growth on Czapek-Dox Agar and Malt Extract Agar lagged behind the rest. A temperature o f 40°C was unsuitable as radial growth remained nearly static after 2-3 days (Fig. 5). A. alutaceus (= A.ochraceus) grew best at 35°C on Czapek-Dok Agar and M alt extract Agar and at 30°C on Maize Meal Agar. Radial growth of the fungus was considerably depressed at 40°C for all media tested except on Czapek-Dox Agar where at 40°C the fungus attained radius of about 30mm in 7 days as compared with 50mm on Czapek-Dox Agar (Fig 6). Interestingly, radial growth of A. alutaceus at 35°C was nearly the same as at 40°C on Maize Meal Agar prepared from Abeleehi and Obaatanpa varieties. The optimum growth for this fungus therefore was between 30 and 35°C. 29 University of Ghana http://ugspace.ug.edu.gh D IA M E T E R OF C O L O MY (I N n m ) D IA M E T E R O F C O L O N Y (n n * ) 30 ASPERGILLUS FLAVUS CZAPEK - DOX AGAR ASPERGILLUS FLAVUS MALT - EXTRACT AGAR - I8*C + 30*C * 3 5 ‘ C -*-40>C INO CU LA TIO N PE R IO D (IN d i r t ) — 1B*C + 30»C * 3 5 " C '•'■ iO 'C ASPERGILLUS FLAVUS (MAIZE-MEAL AGAR, A b e leeh i) ASPERGILLUS FLAVUS (MAIZE-MEAL AGAR, O b a a ta n p a ) " IB 'C + 3 0 ‘ C * 3 5 - C » 4 0 » C ~ 1 9 *C + 3 0 - C * 3 5 'C * 4 0 * C ASPERGILLUS FLAVUS POTATO - DEXTROSE AGAR — 1 8 'C + 30*C * 3 5 'C • 40*C Fig. 4 indicate^media °f O s v u a on five different 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 ) D IA M E T E R O F C O L O N Y (m m ) 31 ASPERGILLUS GIGANREU9 CZAPEK - DOX AGAR ASPERGILLUS GIGANTEUS MALT - EXTRACT AGAK — 1B-C + 30*C - * 3 5 'C » 4 0 'C INOCULATION PE R IO D (IH d * y » ) — IS*C + 3 0 * C * 3 5 > C ♦ 4 0 'C ASPERGILLUS GIGANTEUS (MAIZE—MEAL AC AH, A b e leeh i) ASPERGILLUS GIGANTEUS MAIZE—MEAL AGAR, O b a a ta n p a - 18*C + 30*C * 3 5 * C 40*C — 18*C + 30*C -* 3 5 » C 40*C ASPERGILLUS GIGANTEUS POTATO - DEXTROSE AGAR — 10«C -l"30»C * 3 6 * c •* 4 Fig . Radial growth of Aspergillus indicated media Tiganteus on five different 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 ) D IA M E T E R OF C O L O N Y (m m ) 32 ASPERGILLUS OCIIRACEUS CZAPEK - DOX AGAR ASPERGILLUS OCIIRACEUS MALT - EXTRACT AGAR — 1B*C + 30»C * 3 5 * C -»40»C - 18»C + 3 0 » C -^ 3 5 » C -»-40*C ASPERGILLUS OCHRACEUS MAIZE-MEAL AGAR, A beleeh i ASPERGILLUS OCHRACEUS MAIZE—MEAL AGAR, O b a a ta n p a — 18*C + 3 0 ‘ C -* 3 5 * C -»-40«C INOCULATION PE R IO D (IN d t y t ) • 18«C + 3 0 * C -*-35*C ♦ 4 0 » C ASPERGILLUS OCHRACEUS POTATO - DEXTROSE AGAR - 1Q»C + 30»C *• 35«C ♦ 40*C Fig. 6 f 9r°wth of Aspergillus. ochraceus(=A. alutaceus ) onfive different indicated media. US ' on University of Ghana http://ugspace.ug.edu.gh D. R adial G row th of Penicillium digitatum and Fusarium m oniliform e on Five D ifferen t M edia Vegetative growth of P. digitatum on all the five media was inferior to what existed for the Paecilomyces and Aspergillus species growing on the same media (Chapters B and C). The best temperature for optimum growth attained in this experiment was 30°C. Temperatures 35 - 40°C were unsuitable and 18°C was intermediate between the optimum (30°C) and minimum (40°C) Fig. 7). Radial growth of F. moniliforme followed characteristic sigmoid curves (Fig. 8). The best temperature for growth in all media was 30°C. Vegetative growth at 18°C was better than that of 35°C and 40UC. The fungus never grew significantly after 2 days at 40°C such that the diameter o f colonies of the fungus growing at 40°C on all media remained nearly the same for up to 7 days (Fig. 8). The best growth of the fungus was obtained on Maize Meal Agar and M alt Extract Agar for F. moniliforme while Czapek-Dox Agar was the best medium for P. digitatum. 33 University of Ghana http://ugspace.ug.edu.gh ER O F C O L O N Y (m m ) D IA M E T E R OF C O L O N Y (m m ) 34 PENICILIUM DIGITATUM CZAPEK - DOX AGAR PENICILLIUM DIGITATUM MALT - EXTRACT AGAR — 18*C + 30*C * 3 5 * C 4 0 #C - 18*C + 30*C * 3 5 * C 40*C PENICILLIUM DIGITATUM KtAIZE-MEAL AGAR, A beleeh i PENICILLIUM DIGITATUM MAIZE-MEAL AGAR, O b a a ta n p a ~ 1 B » C + 3 0 ‘ C * 3 5 * C » 4 0 * C INOCULATION P E R IO D (IN d l y t ) - 18*C + 3 0 -C ■*"35*C -» 4 0 * C PENICILLIUM DIGITATUM POTATO - DEXTROSE AGAR — 1B»C + 3 0 » C * '3 5 « C Fig. 7 indicated^edTa0"1" ° f £ s n i c i l l i u l "- - U a i t a t ™ on five different 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 ) D IA M E T E R OF C O L O N Y (m m ) - 3 5 FUSARIUM MONILIFORME F1JS' ^ J JMF™rCT'AGS!RME CZAPEK - DOX AGAR MALT “ EXTKALI AirAK INOCULATION PE R IO D ON d t j r t ) — 18»C + 3 0 - C * 3 5 * C -» 4 0 * C INOCULATION PE R IO D (IN d t y t ) ~ 1 8 » C + 3 0 * C * 3 5 * C * 4 0 * C INOCULATION PE R IO D (IN d t y « ) INOCULATION PE R IO D (IN d t y * ) - 18»C + 3 0 » C -^3 5 » C ■*" 4 0 - C — ia»C + 3 0 » C ^ 3 5 * C -* 4 0 * C FUSARIUM MONILIFORME POTATO - DEXTROSE AGAR FUSARIUM MONILIFORME MAIZE—MEAL AGAE, O b a a ta n p a FUSARIUM MONILIFORME MAIZE-MEAL AGAR, A beleeh i INOCULATION PE R IO D (IN d a y * ) - 18*C 4- 3 0 * c 35«C ♦ 40*C Fig. 8 indicatedaL d i raOWth °f moniliforme on five different University of Ghana http://ugspace.ug.edu.gh E. In Vitro Studies on the Effects of Metabolites of Three Paecilomyces species on Germination and Radicle Development of Maize (Abeleehi and O baatanpa Vars.) The three Paecilomyces species (P. carneus, P. puntoni and P. varioti) produced their inhibitory metabolites in 2 days because inhibition of germination o f both Abeleehi and Obaatanpa varieties could be observed when 2 days old culture metabolites produced in maize meal broth and Potato Dextrose Broth were used as germinating media for the grains (Figs 9- 16). Percentage germination of maize grains (Abeleehi and Obaatanpa) was depressed by 10- 75% by the undiluted 2,4 and 8 days old culture filtrate o f the three Paecilomyces species (Fig. 9-16). This inhibitory effect was gradually removed with increasing dilution (up to 1:10V/V) . There were varietal differences in the response of the germinating maize grains to the active ingredients in the culture metablolites of the three Paecilomyces spp. Cultural filtrate of P. carneus, P. puntoni and P. varioti severely depressed length of emerging radicles of the two maize varieties by 45-90% at the highest concentration applied (Fig. 17-19). There were marginal differences between the yield o f the active ingredients in the various media used (Maize Meal Broth and Potato Dextrose Broth). No significant differences (P <_ 0.05) were found in the depression of germination and radicle length obtained in the metabolites obtained from the three media used. As expected, the inhititory effect o f the metabolites was gradually removed with dilution such that radicle length o f the germinating grains in the presence of 1:10V/V dilution of the filtrates nearly approximated that of the control in most instances (Fig. 17-19). In the case o f P. carneus and P. varioti, the reduction in length of radicle was severer on Abeleehi variety as compared to Obaatanpa whereas the reverse was obtained in culture filtrate of P. puntoni. The three Paecilomyces species are therefore of pathological importance affecting the development of the seedling stage, at least in vitro. Plates 5 to 10 show the effect of the fungal metabolites on the radicle development in petri plates. 36 University of Ghana http://ugspace.ug.edu.gh U tt io uon'®nTcnjsQ W < Q l*r non^ti1u3J3o a8v iu?O J3 j w > CQ Q Q* a o n o u ju u a o aSa-juso-iad uonoaiuuoo 03V1U»DJ<*dl n o n » n \iu j3 o aB w Tasojsj 0 > Mh c 0o *H >. -p -Pn3 . u u -p c QJ rC O C o o • H c ^5 10<0 C•r-I 6 Q) O ' 3 Q) U £ ° c _ o C- c* .2 5 « °S CQ X5 ^ a) u 05 5 0u ^ X 0 5 [/] Q >- O +J E r a -p > u 3 t ; c u ^ a) 3 i/) rH 3 M-< Q) C Ci—l !m fO • o CT ■H Ci-f H3 a c ra . -p cul fO <4—1 £* O O University of Ghana http://ugspace.ug.edu.gh Fia 10 . In fl ue nc e of the aqe (d ay s of in cu ba ti on ) of cu lt ur e and co nc en tr at io n (V /V ) of the cu lt ur e fi lt ra te of P. ca rn eu s cu lt ur ed in Ma iz e Me al br ot h (p re pa re d fr om Ab el ee hi va ri et y at 30 °C ) on the ge rm in at io n ca pa ci ty of (T op ) Ab el ee hi and (B ot to m) , Ob aa ta np a va ri et y- gr ai ns . University of Ghana http://ugspace.ug.edu.gh 10 0 □onflairaaeo eaemooaed ao n » u « u ij» o s S v ia s o ja j Fig 11 . In fl ue nc e of the age (d ay s of in cu ba ti on ) of cu lt ur e and co nc en tr at io n (V /V ) of the cu lt ur e fi lt ra te of P. ca rn eu s cu lt ur ed in Ma iz e Me al Br ot h (p re pa re d fr om ob aa ta np a va ri et y at 30 °C ) on the ge rm in at io n ca pa ci ty of (T op ) Ab el ee hi and (B ot to m) , Ob aa ta np a va ri et y- gr ai ns . University of Ghana http://ugspace.ug.edu.gh CQ a i CO uonmiCDJJO w < Q I ao n su iu ijs-o aSviuaoj^d riB aiauao »8vTa30J»d a o T jo u iu jja o s S e iu a o ja j >-< p 3 -H P Oi—i fd3 a o Q) Cr> > 0) •C P C O C O *H -P 03 — ^ >i 4-> -P 0)a) o ■p e '—I o 3 UU M-J (4_j n ■ O w a u ^ c (0 -H CL <0 o <“ Uo M 01 a • ■*£ — o r v •9 P IM <0 (0 o ® a s c , <0 I p to w at>i po (0 -H T3 m JO ^ ^ XI w Q I uon«>aiaiJoo oao-ju&ojed u o n « a |m j> o a S v jaa o js j a) c pc-t ' “ 'O E n, ^ O ■c ^ ^•H 3 p P O ^ CQ ^ O -__ o u 111 c 3 ^ 3. 3 Qj<4-1 H ^ 111 M-l -5 o ^ a o B ■H <*-J M-l O University of Ghana http://ugspace.ug.edu.gh D A YS 4 -D A Y S 8 -D A Y S O O O O O o O <0 © N n o n « u im ja o a3«i,uac-J3H 1 o -p•H •H •P u 03 OJ VH a -p 03 c 0 (D O c C 0 O *rH O -P 03 T3 C C -H « £ Vh d) a)$CP -p i-P 03 0 Tl a, >—- i C ! 0) '■H -, u a -p 0) -P u o C -H Q) k 3 03i—I > <4-1 • C W ( D H X) OO Q) — • ■P E 03 0 rH Vh P -P P CF 'H 0 *H •H CQ Cl, IM ^ University of Ghana http://ugspace.ug.edu.gh -D A Y S 4 -D A Y S 8 -D .A Y S ao n o a iu x ja o aSw iaaojad aoneojiruso s8vqu»ojad > > - m aonBa|uiis») >B®iaaoa»d o o rj« a ? in j3 0 d9viu»3J3d uoiiboiuuoo o3viu«»j«d a o n 5 a |u t« o sS sn a ao a s j "arc S-l o P -H + J 4-> r-H (T3 P C U -He Q) !-l •s s.4J CP c o C 4-> O -p s -u 0) § « U Q, ■Q T3 C C «* e a) ° ' M iw i 3 4-> m 1,3 -I m U 3 2 01 o s • • » ! * o 2 .2a ̂ c r; >O +J •^0(0 -*-* s-i q,t0 CQ r •S «g r-i +Jy & 1 -p -p tn fU ■rH i—i 5̂ u -P rtf D i—1 a •H -H nea!UU90 d?eiusoj3d Fig 16 . In fl ue nc e of the age (d ay s of in cu ba ti on ) of cu lt ur e and co nc en tr at io n (v /v ) of the cu lt ur e fi lt ra te of P. va ri ot i cu lt ur ed in ma iz e me al Br ot h( pr ep ar ed fr om Ob aa ta np a va ri et y) on the ge rm in at io n ca pa ci ty of (T op ) Ab el ee hi and (B ot to m) , Ob aa ta np a va ri et y. gr ai ns . University of Ghana http://ugspace.ug.edu.gh I-J Jo a ■J o c ~I + 2? | ! t I ~ ! De ve lo pm en t of ra di cl e of tw o ma iz e va ri et ie s( Ab el ee hi an d Ob at aa np a) gr ow in g on fi lt er pa pe r mo is te ne d wi th cu lt ur e fi lt ra te of Pa ec il om yc es ca rn eu s University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh D A Y -O LD 4 D A Y -O LD 8 D A Y -O L D A ■P < P* Z < E-< < m 0 1 w B O PS H J -p 01 =H 0 6 M 8)a a u a) -P H •H fi o Cn a -h £ 0 o -H £ £ > a C 0) rtJ 0 t 0 2 >1g £ ° o Hi i *h ! o' ail id -H P*i •c ,II) >w a o; Ho Η1 0) PN U £ (d •H (TJ e 0 -p M-l < 9 - 0 ~xf- 0) 3'CD o O + •H TJ S (dLJi—J uU.' c M-J i 0 -p G Q) e Qj o\ O H H 0 Cr > •H H H Q N W H W <£ H 2 f t PS H M H fa O W a pi D H fa m rt QH fa Eh O < CJ H Qa H m a w o Eh H Eh a PI H H 2 fa 2 a w u Pi D Eh ! J I D O w w Eh fa O H CJa w D faa H u a) >i ■P fd <*h T3 fd CO IS) 2 tr» G 3 <4H T5 a) -p cd 0 • H T5 G *>H UH 0 w 0) >i -p fd -p rH •H *4H Q) Jh -P rH 3 0 C •H c 0 •H-P >1 aJ (dC T5 *H e — CM }H U) CU >1 O rd T5 o\o ^ 0 *H-P > rd >î a)v-r-' Jh c 3 a) 0 ■P -P i— I (d-P 3 Jh 0 -P rH rH •H MH •H Q 0 «4H O O ■O o o o o o o P4 if) lO h Ol H H C 0) O P -H <0 P > 1 p d) •H H-B- fd e jh 3 -p • H rH T5 -H 0) <4H g MH tP tP C o o o o o o in vo vo oo cp o o o o o o o o o (< VO [*■* CO CP H H o o 73 o O O O O O P_I VO VO 00 H rl o o g o o o o o o tin If) ID VO Is H H o o o o o o o o o o ID CO CP H rl rl T5 d. e o o o o o o o Is CO CO H H o o o o o o VO VO r* CP rl o o o o o o o o VO t''- CP CP H H T5 Q) -p 3 rH •H T5 G D H rl rl H 0 -H ^ -P -P If) G *H 0 Q CJ — > i 0 •H U) ,—, (0 eQ) QJ •H 3N a > T3■H *H >1 o QJ fd Eh W QJ2 '■w* a 3 e si p 0 __. X! B ■ O i— i cO (0 <4-1> 0) H ■H ffi QJ X! w OJ 1̂ Q) w N CO QJ •H CU rH w (0 QJ QJm s >H Ac a c O OO O O O O O ID VO CP rl rl o o o o o o vo vo co (P h o o o o o o vo co cp cp H o o o o o o o o VO VO Is CO H H o o o o o o o o ID VO CO H H o o o o o o o o LD VO VO CP rl rH o o o o o o o o VO h CO CO H H o o o o o o vo h CO CO (P H o o o o o o o o ^ VO h CO H H T5 Q) -p 3 T5 c ID a) -p fd £ V CD rH rH 0 rH p p P M C -H 0 Q U — sip0 __ Sh Si e0 10i—I u > rd >*H QJ (0 H B T3 ftffi QJ c H QJ COW N (0p•rH f t CO W (0 QJ 10m 2 ^ X!c '— a o o o o o o o in la vfl co oi o o o o o o o o CO CO rl rt o o o o o o o o o lO CO CO H H rl o o o o o o o o^ in U3 05 rl rl o o o o o o in >o > oi o o o o o o o o o o o o in in vo t-' o o o o vo vo r~ co o o o o vo r~ co co TS QJ P 3 rl CM in rl 0 -H P -P P W C - r l 0 Q U QJ W o u p X 0) T3 H K O w P — w (0 J3 l-q P P w 0 O Oh 1̂ < ' CQ P a) XI 6 3c o £ -P QJp <0a)c aj •C P CO ■U T3 • H OJ tn P ■ H U ■O OJ O P O O e p 0^ rl 3 O - H U rH M w O O ■ M QJ o o 0 P rl i— 1 ■ H 3 q cp Q) -rl a< fa. o o o o i i rl rl r t fa * W 3 0) 0 i -P 03 4-1 03 CO W P tn C P i CU 03 -P T3 03 U -P r—\ *H 4H CU u p -p rH P 0 £ • H c 0 • H -P >i 03 03 G TJ •H £ — CNJ M w CU >. O 05 T5 o\o ^ 0 •H■P > 03 > Jh <1) £ a a) 0 ■p -p •H rH rd-P 3 >H P 0 -P rH r—1 *H 1 •H rH •H -P n •rH e CU 0) 4-1 •H e 0) u tn tn 03 in C > ~* 0 ■ H m ,—̂ (0 e CU Q) •H 3 N a > 73 •H *H >i 0 (1) TS 05 Eh u w 0) a CU P e 03 < Q) a. g < cu E-* N-h T3 -H a) x : u cu 03 CU Q-l rH cu cu U A o o o o o o m ^ vo cn h o o o o o o o o ^ in VO CJ) H H o o o o o o in vo O'* h o o o o o o o o ^ r-' CO CT\ rH rH o o o o o o o o in in vo vo h h o o o o o o n in vo co cri h o o o o o o o o in vo vo h h h o o o o o o o o VO VD h CO H rl o o o o o o o o in vo h co h h T3a) 0 -H Ĥ -P -P U) C -H O Qu •— x: -p 0 ,—. u . sx e 0 rd1— 1 M > (d HH < a) td a e T3 a a H id Eh N Ifl -p 'S •H a 0} N -H rtf £ O £ -P 4-i o a)r~H o •rH T5 rtf 4-1 O -P C Q) £ Q. O ■—la) > 0) Q University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh H. Influence of Metabolites of Three Paecilomyces species on Germination and Radicle Development of Pepper (Capsicum annuuml) and tomato (Lycopersicon esculentum Mill) The inhibitory effect of the metabolites o f Paecilomyces carneus, P. puntoni and P. varioti on germination and radicle development cannot be confined to maize grains only. These same metabolites in the culture filtrates o f the respective Paecilomyces species significantly (P < 0.05) depressed seed germination and radicle development o f two tomato varieties (C V ’s Owusu-Bio and Wosowoso) and hot pepper (Var, Legon 18.). Each culture filtrate from the test fungi was unique in its effect on the germination and radicle development of the seedling of tomato and pepper. There were also varietal differences in the response of the tomato and pepper seed to the same metabolites (Table 4). The inhibitory effect of the metabolites was highest on var. Owusu-Bio than var. W osowoso. The inhibitory principle was still potent even at l:1 0 7 v dilution level. Clearly, tomato and pepper seeds are also adversely affected by the culture metabolites o f the Paecilomyces species. 61 University of Ghana http://ugspace.ug.edu.gh wJra C•H ,2 -P £ 4-1 CD O CD U M •H -p T3 i ^ R 2-P o> Q) &CD OT5 -H 0 3cd u 0 0 u(1) rd m cdw > ^ d) > CJ O0 m0 £ o*H £0]■P 0 U CJ inCD CD oQ̂r-H£0 30 0 u*X( (J) cdH CD > EiD0•H a, c; (t3 q O (Q co tr>Q) iJ +1 VO in o\o 4-4 O > r - CO CM CO in CO in in in > O c CD cu rH CO CO in in vo CO cn o CO rH CO in 0 -P •rH (0 -P Jh o td -P a in o O in r - in in CO CO o CO o o r* O CO CM CO c i—I 0 i rH CO in in VO CO V0 Is* CO G\ cn rH rH rH in■H•H e 4H CD C u O o in o o o in o > CO o CO f" U CO in r - CO cn O rH rH CO 'u 'u XT CD 0) CD 0 -p -p -P•H rd rd rd■p £ to CD u -P T5 T5 T5 rd T5 CD T5 CD T5 ac u CD i—1 CD rH CD rH 0 ■p -P i—1 rH -p rH i—1 ■P rH t—1•HH 3 0 •rH 3 0 •H 3 0 -H •p ■H r-l u -P rH u -P rH u -P 3 > *rH o -p U) •<—) o -p in *H o -p (01—1 rH CM in rH c TS rH CM in rH c •rH rH CM in rH c •H *r| 4-1 C • • •• • • 0 Q c • • • • • • • • 0 a G .. • • • • • • o QO 0 w D rH rH rH rH u — o 1— 1rH rH rH o '— D rH rH rH rH u uPU P ae ci lo m yc es ca rn eu s; Pp - P. pu nt on i ; Pv - P .v a ri o ti University of Ghana http://ugspace.ug.edu.gh I. Influence of Metabolites of Paecilomyces on Vegetative Growth and Dry M atter Accumulation and Chlorophyll Content of Maize (Var Abeleehi and Obaatanpa) Field Studies The height, leaf length and leaf width of Abeleehi and Obaatanpa varieties plants whose seeds were inoculated with the mycelium/conidia o f the three Paecilomyces species prior to sowing were significantly (student’s t test; P <. 0.05) depressed by the metabolites o f the growing Paecilomyces species (Fig. 23). There were differences between the efficacy o f three Paecilomyces species in depressing plant development in the field and each plant part responded differently to the inhibitory effect of the culture metabolities. Photographs o f the seedlings growing in the field are shown in the plates 11 and 12. The resulting cobs on the treated plants were also diminutive and carried fewer grains as compared to the control (Plate 13 and 14). Greenhouse Studies Reduction in plant height, leaf length and leaf width of Abeleehi and Obaatanpa in pots in the greenhouse by the culture filtrate o f P. carneus, P. puntoni and P. varioti can be described as marginal and did not differ significantly (P _< 0.05) from the control seedlings (Fig. 24) except in the case of Obaatanpa seedlings where the leaves were narrower than the untreated (control). Furthermore, P. carneus metabolites exerted greater inhibitory effect on leaf width than that of P. puntoni and P. varioti (in decreasing order) (Fig. 24). Each plant part of the respective maize variety behaved differently in its response to the inhibitory principle in the metabolites of the test Paecilomyces species. Fig. 25 shows result o f the dry matter accumulation by leaves, stem and root systems o f maize (Abeleehi and Obaatanpa) in pots moistened with culture metabolites of Paecilomyces species in the greenhouse. There were varietal differences in the response of the maize grains to dry matter accumulation by shoot and root systems in the presence of the metabolites of the three Peacilomyces species. Each plant part behaved differently. For example, whilst there was no statistical differences between the 63 University of Ghana http://ugspace.ug.edu.gh control and the dry weight o f leaves of Abeleehi treated with metabolites o f the three Peacilomyces species, there were clear statistical (P _< 0.05) differences between the dry weight obtained in Obaatanpa plants treated with P. carneus, P. puntoni and P. varioti (Fig. 25). The severity of the depression can be ranked as following in decreasing order: P. varioti > P. carneus > P. puntoni. The effect o f the Paecilomyces metabolites on the dry matter accumulation by stem and roots of Abeleehi and Obaatanpa seedlings were different and can be ranked as follows. P. puntoni > P. varioti > P. carneus. Data which was used in plotting Figures 23-25 are presented in the Appendices 2a - 2c Chlorophyll Content Interestingly, the metabolites of P. carneus, P. puntoni and P. varioti affected the chlorophyll content o f the leaves o f the growing seedlings o f Abeleehi and Obaatanpa varieties both in the field and in the greenhouse. Chlorophyll a and b contents were lowest in Abeleehi and Obaatanpa plants growing in the field whose seeds were inoculated with P. carneus followed by seeds treated with P. puntoni (Table 5). Duncian’s Multiple Range Test showed that the data recorded were significantly (P <. 0.05) different from the untreated (control). Total chlorophyll content o f Abeleehi seedlings was also significantly (P _< 0.05) lower in seedlings whose seeds were inoculated with P. carneus and P. puntoni but not with P. varioti. 64 University of Ghana http://ugspace.ug.edu.gh Seedlings o f Abeleehi and Obaatanpa in the greenhouse which were treated with metabolites of the three Paecilomyces species behaved differently. M etabolites o f P. carneus, P. puntoni and P. varioti severely reduced chlorophyll a and b contents o f both Abeleehi and Obaatanpa. P. puntoni metabolite was more potent. Total chlorophyll contents o f the leaves of both maize varieties treated with the metabolites o f the three Paecilomyces species were also lower (Table 5). Therefore the metabolites o f Paecilomyces species also affected the photosynthetic apparatus of the maize varieties tested. 65 University of Ghana http://ugspace.ug.edu.gh OF SE ED LI NG S OF IN DI CA TE D MA IZ E VA RI ET IE S GR OW IN G IN UN DE R NO RM AL DA Y/ NI GH T RE GI ME AT 28 - 32° C 56 DA YS . W Eh Eh S O W PU EhS a O H U « J O X Q B J PM W O H « Pho hj w B B O Eh - P C a)•pcoo >1£aoVi0 rH & C J > 1 X2aO rH Ĥ fd O -P O ^ H U X3 C J ,G C J T5 u c o 3 G Ph *h > 1 - p a) Q) N -H •H U rd fd S > - PoPH 13i—i a)•Hfn cn CO VO ID rH O CT\ rH O O co CM CM CO rH CO \—1 CT» in rH CO -P • • • • 0 CO in VOa. CM rH rH rH T3 CO in CO rH i— 1 CM rH CO VO 0) • • • • •H CT> in CO fa rH rH rH rH rH in c\ rH Is* in CO CO o o o VO CO -P • • • • • • • • 0 in CO cr\ rH VO as CO CO O. rH rH rH rH T3 CO 'sf rH VO CO OS CO CM i— 1 CM O VO OS Q> • • • • • • • • •H rH CO o rH vo a \ CO infa rH rH rH 1— 1 rH rH T50) ■P Cl) - pG£> OJh -PCOU UJ3a) 5 T5 n CJ fd Jh G fd 3 fd > -p 0 0 a ] G CJ • D '— o] University of Ghana http://ugspace.ug.edu.gh 67 ABELEEHI Multiple Range Tests: Duncan test with significance level .05 Indicates significant differences which are shown in the lower triangle G G G G r r r r P P P P 3 2 4 1 Mean ABPCARN 12.0800 Grp 3 20.2033 Grp 2 28.9417 Grp 4 47.8417 Grp 1 * * Homogeneous Subsets (highest and lowest means are not significantly different) Subset 1 Group Grp 3 Grp 2 Grp 4 Mean 12.0800 20.2033 28.9417 Subset 2 ANALYSIS OF VARIANCE Group Mean Grp 4 Grp 1 28.9417 47.8417 University of Ghana http://ugspace.ug.edu.gh 6 8 Indicates significant differences which are shown in the lower triangle G G G G r r r r P P P P 3 1 4 2 OBAATANPA Mean OBCODE 19.0517 Grp 3 26.1050 Grp 1 70.8200 Grp 4 81.3333 Grp 2 Homogeneous Subsets (highest and lowest means are not significantly different) Subset 1 Group Grp 3 Grp 1 Mean 19.0517 26.1050 Subset 2 Group Grp 4 Grp 2 Mean 70.8200 81.3333 University of Ghana http://ugspace.ug.edu.gh Plate 11. Photograph of ’Obaatanpa’ variety growing untreated (Control) in the field after 90 days at 29°C (X 1/4) Plate 12. Photograph showing the devastating effect of the three Paecilomyces species on the development of the maize plant in the field (Note the moribund plants at the centre of the field) (X 1/8) University of Ghana http://ugspace.ug.edu.gh 70 Plate 13. Photograph showing cobs form on the Abeleehi maize plant after 3 months (90 days) exposure to the indicated Paecilomyces in the field (X !4) (Top) From left Control; P. varioti, P. carneus, P. puntoni Plate 14. Photograph of plate 13 showing cobs with husks removed (X V4) University of Ghana http://ugspace.ug.edu.gh ni iH an x &5 S i R§ u 5 E s»! 3̂ * O " 0 Im a < ft z rf < rt m o f 0 .B.X * t— B Q D HU U li I 3I | U♦ XH*O as fto*> E- £in * u m 5 + c? I -O CO CM tJ •H En In fl ue nc e of me ta bo li te s of th e in di ca te d Pa ec il om yc es Sp ec ie s on he ig ht , le ng th an d wi dt h of le af of ma iz e va ri ti es in th e fi el d at 28° - 31 ° University of Ghana http://ugspace.ug.edu.gh University of Ghana http://ugspace.ug.edu.gh T. K A V K .q ST EM R O O TS | o * m v 0 jh to ma) S' XI 4)OJ N V - H (fl 5 a E 'O m a o •H to 01 4J XI ■U 0 Tl m