Extraction and Identification of Antimvcotic Componds From Acremonium Implicatum Infected Brachiaria Grasses

Plant samples will be obtained from endophyte-infected grasses from tropical South America provided by Dr. Segenet Kelemu (CIAT). These grasses are infected with the fungal endophyte, Acremonium implicatum. Plant samples will be dried and ground. Test fungi from the Abou-Jawdaw et al. (2002) paper were Botrytis cinerea, Alternaria solani, Penicillium sp., Cladosporium sp., Fusarium oxysporum f. sp. melonis, Verticillium dahlia, Phytoptora infestans, and Rhizoctonia solani. Since the Dreschleria complex was shown to be affected by the fungus, this would need to be included in our analysis as well as some of the listed ones from above. Thin layer chromatography will be carried out using Whatman K6F silica gel60A (250 urn thickness) 20 x 20 cm plates and solvents will be of analytical grade from Aldrich. Either GC-MS or LC-MS will be utilized to identify the isolated compounds. A sample of the dried plant material will be extracted with either petroleum ether or methanol in both basic and acidic conditions. These extracts will be concentrated and dissolved in methanol. Initial screening for fungicidal activity can be carried out using filter disks soaked in extract and placed on potato dextrose agar plates with spread spore suspensions of test fungi. Disks with extracts exhibiting inhibition will further looked at with TLC autobiography tests. For the TLC autobiography, aliquots of these extracts will be spotted onto a silica gel plate in duplicate and run using different solvent systems, initially chloroform-methanol (9:1) as reported in Abou-Jawdaw et al. (2002). The duplicate plate is for later isolation of compounds from the corresponding areas on test plates that exhibited inhibition of growth. These plates will be dried at 3TC for one hour to remove solvent residue before a suspension of spores of the different test organisms in potato dextrose broth is sprayed onto the plates and control plates (fresh silica gel plates and silica gel plates run in solvent). These plates are grown in a humidity chamber for several days to observe zones of inhibition. The corresponding areas on the unsprayed plates are then scraped and analyzed via GC-MS or LC-MC to identify compounds of interest.