Methodology

Methodology. Materials & Reagents. Potato Dextrose Agar (PDA) Petri dishes Ethanol Nutrient Agar Conical flasks Lactophenol cotton blue stain Cotton wool Sterilized knife Streptomycin Syringes Test tubes Distilled water Glass rod 2 mm sized cork borer Petroleum jelly Aluminum foil Filter paper Rotten fruits Light microscope Slide and cover slip Fresh fruits.

Methodology. 1. Specimen Collection Two different fruits : 15 sweet oranges and 15 bananas purchased at Kara Market Each fruit type were purchased from three different points: Banana: B1 – B3 2. Sterilization of Glassware Disinfect working surface with ethanol Sweet orange: A1 – A3 Transported in ethanol sterile polyethene bag Soaked and washed thoroughly with tap water and detergent Rinsed with distilled water Sterilize fruit surface in 90% ethyl alcohol (1 min) and 1% sodium hypochlorite (3 min). Rinse with distilled water. Incubate at 28oC for 5 days under 12 hour photoperiod. 3. Isolation of Fungi Place onto PDA media containing streptomycin Segments (3 – 5 cm) of tissues from margin of rotted area Total of 30 fresh and rotten fruits Sterilized in steam oven at 121°C for 15 minutes.

4 . Identification of Fungi Lactophenol Cotton Blue Staining (LCBS) method on mycelium from the fungal culture (reference: Fawole and Oso) 5. Pathogenicity of Isolated Fungi (Decay Test) Morphological features of fungi: type of hyphae and asexual reproductive structure Healthy fruits were sterilized with ethanol Cylindrical plug tissues were cut out forming holes 1 week old fungal culture were placed in the holes and sealed off by petroleum jelly ii. Pathogenicity test result by measuring the diameter of spoilage. Data analysis by means of descriptive statistics. i . Frequency of occurrence of fungal pathogens associated with the spoilt fruits. Observed under microscope at 400 magnification 6. Data Collection Recorded parameters: Inoculated samples and control were incubated at 30 oC for 5 days The point of inoculation of each type of fungus was examined.