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Plant material
Seeds of two cowpea cultivars, “Bechwana White” (BW) obtained from the Grain Crops Institute – Agricultural Research Council, Potchefstroom (South Africa) and “Kpodjiguégué” (Kpod) collected from a market in Cotonou, Benin (West Africa) were planted under greenhouse conditions. The plants were harvested after ± two months of growth.
Preparation of extracts
Two solvents, namely acetone and ethanol were used for the extractions of both the cultivars. Air dried plant material (100 g) was homogenised with 250 ml of the solvent for 1 min and then filtered.
This process was repeated three times. The filtrate was concentrated to dryness at reduced pressure with a rotary evaporator (Büchi Laboratoriums, Technik AG, Germany). The resultant residues were later dissolved with the respective solvent to 100 mg/ml. In the case of the antibacterial tests, the ethanol extract was re-dissolved using dimethyl sulphoxide since prior investigations showed ethanol to be toxic to the bacteria.
Micro-organisms
The fungal pathogens used in this investigation were Alternaria alternata (Fr: Fr.), Aspergillus flavus Link ex. Fries, Fusarium equiseti (Corda) Sacc., F. proliferatum (Matsushima) Nirenberg and Penicillium chrysogenum Thom. The fungal cultures were maintained on potato dextrose agar (PDA) at ± 25°C. The bacteria used in this study to determine antibacterial activity of the extracts included five Gram-positive bacteria: Bacillus cereus Frankland and Frankland, B. pumilus Meyer and Gottheil, B. subtilis (Ehrenberg) Cohn, Staphylococcus aureus Rosenbach, Enterococcus faecalis (Andrews and Horder) Schleifer and Kilpper-Balz and five Gram-negative bacteria: Enterobacter cloacae (Jordan) Hormaeche and Edwards, Escherichia coli (Migula) Castellani and Chalmers, Klebsiella pneumoniae (Schroeter) Trevisan, Pseudomonas aeruginosa (Schroeter) Migula and Serratia marcescence Bizio.
All the bacteria were obtained from the bacterial collection at the Department of Microbiology and Plant Pathology, University of Pretoria. Bacterial cultures were recovered for testing by culturing in nutrient broth for 24 hr at 37 ºC.
Antimicrobial tests
For the antifungal assay, the required amount of extract was added to sterile PDA in 65 mm Petridishes before congealing to yield final concentrations of 0.5, 1.0, 2.5, and 5.0 mg/ml. Unamended PDA plates served as controls. Once the agar had solidified, a 5 mm plug of a 7-d-old fungal culture was placed in the centre of the Petri-dish containing the extract-amended and unamended PDA plates. The plates were sealed with Parafilm and placed in an incubator at 25 °C. Fungal growth was measured on two preset diametral lines after 3, 6, and 9 d of growth. Each treatment was analysed in triplicate. The results of the 6-d growth was statistically analysed using two-way analysis of variance (ANOVA) and least significant differences (P=0.05) were determined according to the students t test.
Prior to streaking, each bacterial culture was diluted 1:100 with fresh sterile nutrient broth. The minimum inhibitory concentration (MIC) of the extracts was determined by incorporating various amounts (0.5 – 5.0 mg/ml) of the extract into 65 mm Petri-dishes containing sterile nutrient agar (NA). Petri-dishes containing only the culture medium served as controls. The bacteria were streaked out in radial patterns onto the extract-amended NA and unamended NA plates. The Petri dishes were sealed with Parafilm and incubated for 24 hrs at ± 37 °C. The MIC was regarded as the lowest concentration of an extract where no growth of a bacterium was visible. Each treatment was replicated three times.
Results and Discussion
The results pertaining to the antifungal investigations revealed that both the acetone and ethanolic extracts of the leaves of BW and Kpod cultivars, with the exception of F. equiseti, significantly inhibited growth of the fungal pathogens at 5.0 mg/ml [Figures 1(a-d)]. Only the BW ethanolic extract inhibited the growth of F. equiseti at the same concentration when compared to the control. Alternaria alternata was significantly reduced by both BW extracts at 2.5 mg/ml whereas only the ethanolic extract exhibited antifungal activity against F. proliferatum at the same concentration.
The acetone extract from Kpod also inhibited the growth of A. alternata at 2.5 mg/ml when compared to the control. The acetone and ethanolic extracts of both cultivars showed no inhibitory activity at 1.0 mg/ml.
CHAPTER 1 GENERAL INTRODUCTION
1.1. BACKGROUND AND MOTIVATION OF THE STUDY
1.2. OBJECTIVES OF THE STUDY
1.3. STRUCTURE OF THESIS
1.4. LITERATURE CITED
CHAPTER 2 LITERATURE REVIEW
2.1. AN INTRODUCTION TO COWPEA: VIGNA UNGUICULATA (L.) WALP
2.2. FUNGI AND MYCOTOXINS ASSOCIATED WITH COWPEA SEED
2.3. THE FUMONISIN MYCOTOXINS
2.4. ANTIMICROBIAL EFFECTS OF PLANT EXTRACTS ON BACTERIAL AND FUNGAL PATHOGENS
2.5. SECONDARY METABOLITES ASSOCIATED WITH COWPEA
2.6. THE INFLUENCE OF PHYSICAL FACTORS ON THE ULTRASTRUCTURE OF COWPEA SEEDS
2.7. LITERATURE CITED
CHAPTER 3 SURVEY ON THE IMPORTANCE AND UTILISATION OF COWPEA BY RURAL COMMUNITIES IN THE MPUMALANGA PROVINCE OF SOUTH AFRICA
3.1. INTRODUCTION
3.2. METHODOLOGY
3.3. RESULTS AND DISCUSSION
3.4. CONCLUSION
3.5. ACKNOWLEDGEMENTS
3.6. LITERATURE CITED
CHAPTER 4 Mycoflora and fumonisins associated with cowpea (Vigna unguiculata (L.) Walp) seeds
Abstract
University of Pretoria etd – Kritzinger, Q (2005)
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
ACKNOWLEDGEMENTS
LITERATURE CITED
CHAPTER 5 Phytotoxic effects of fumonisin B1 on cowpea seed
ABSTRACT
MATERIALS AND METHODS
RESULTS AND DISCUSSION
Effect on seed germination and root and shoot elongation
Effect on ultrastructure
LITERATURE CITED
CHAPTER 6 Antimicrobial activity of cowpea (Vigna unguiculata (L.) Walp) leaf extracts
Abstract
Introduction
Materials and methods
Results and discussion
References
CHAPTER 7 GENERAL DISCUSSION
7.1. LITERATURE CITED
SUMMARY
APPENDIX A SURVEY OF COWPEAS
APPENDIX B LIST OF PUBLICATIONS AND PRESENTATIONS
