Morphological Identification / Evaluations Of Entomopathogenic Fungi

1628 Words Sep 18th, 2016 7 Pages
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 samples were centrifuged for 5 min with a mini centrifuge to form a pellet. 300 μl of Nuclei Lysis Solution (Promega) was added to the fungal pellet and vortexed gently. The samples were incubated at 65 °C for 15 min on the heat block. The lysates were mixed 2 or 3 times during incubation by inverting the tube and 100 μl of 10 M ammonium acetate solution was added. The samples were vortexed, incubated for 10 min on ice and centrifuged at13,000 rpm for 5 min and supernatants were transferred to clean…

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