Entomopathogenic Fungi Lab Report

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2.5. Morphological Identification/Evaluations of Entomopathogenic Fungi
The six isolates of fungal entomopathogens were provided by Department of Entomology, Penn State USA. Preliminary morphological identifications of fungal entomopathogens such as color of colony, mycelial characteristics and spores from all six isolates were done at microscopic level.
2.6. Molecular Characterizations of Entomopathogenic Fungi
Cultures of Metarhizium fungus were grown in nutrient broth (Bacto, Australia) in 100 mm x 15 mm Petri dishes incubated at 25 ± 2 ºC at 150 rpm. The media having mycelia and conidia were centrifuged in microcentrifuge tubes and approximately 40 mg of fungal material was collected for each isolate. The floating foam microcentrifuge rack with the microcentrifuge tubes (containing the fungal material) were put in the liquid nitrogen to thoroughly freeze the samples. GenoGrinder 2000 was used for grinding the fungal material into a fine powder without allowing it to thaw. The
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mays, var. B73) seeds were immersed in 0.5 % % sodium hypochlorite for surface-sterilization for two minutes, dipped in 70 % ethanol for two minutes and rinsed in sterile distilled water thrice. 100 μl of the last rinsing water was plated on potato dextrose agar (PDA) media, and incubated 10 days at 25 °C to ensure the success of sterilization. The experiment was continued on successful seeds sterilization. The seeds were inoculated with all the fungal cultures having to 1 X 108 conidia ml_1 and with 0.1 % Triton X-100 for control group for two hours. The seeds in groups of three in 6” x 6” pots containing a doubly autoclaved mixture of field soil and potting mix at a 1:1 ratio. The maize plants were allowed to grow in 16 h light and 8 h dark at 28 °C ±2 in a greenhouse. Two least vigorous maize seedlings were eliminated to minimize the competition. The plants with five biological replicates for each treatment were put on randomized block design and watered every 1-2

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