Location: Biological Control of Pests Research
Project Number: 6066-42000-007-003-A
Project Type: Cooperative Agreement
Start Date: Apr 7, 2021
End Date: Apr 30, 2023
1. To determine if C. glycines isolates produce one or more mycotoxin/phytotoxins that it uses to kill adjacent soybean plant tissue to facilitate expansion of the lesion. 2. To isolate and identify any mycotoxin/phytotoxins produced by C. glycines isolates.
Scientist from Ft. Detrick will grow isolates of C. glycines from his collection in the containment facility. Preliminary data identified four general colony appearance categories among the single hyphal tip RLB isolates. It is proposed to examine one isolate from each of the first three categories, which will be grown on 10 culture plates of V8 agar medium with antibiotics for two to three weeks. In the proposed study, mycelium plus agar medium will be extracted with ethyl acetate by peeling the agar medium plus mycelium off all the dishes into one beaker, slicing into approximately 0.5-1 cm square pieces with a spatula or knife, covering agar pieces with ethyl acetate (~1:1 v/v), covering the top of the beaker with aluminum foil, and allowing to stand at room temperature overnight. The ethyl acetate is then decanted into a screw cap bottle and replaced with fresh ethyl acetate. The extraction process is repeated four more times combining extracts. The extracted residue will be autoclaved and disposed of. The combined extracts will be tested for viable C. glycines by streaking on plates of V8 agar medium with antibiotics in triplicate and culturing for a week. When the extracts pass this test, which they are expected to do, the bottles will be surface-swabbed with alcohol then couriered to the laboratory in Stoneville, where they will be evaporated on a rotary evaporator. Small samples of the extract residues will be submitted for LC-MS/MS analysis to determine quantitatively if known mycotoxins were produced by the C. glycines isolates. The extract residues will also be tested for phytotoxicity Phytotoxicity is identified as chlorosis, tissue death and stunting of growth. Chlorosis can be quantitated in cultures by extracting and measuring residual chlorophyll using spectrophotometry. If phytotoxicity is observed, a species specificity study will be conducted with leaves of tomato, corn and other legumes. Samples of extracts that exhibit phytotoxicity will be sent to the scientist at the University of Minnesota for the initial stages of bioassay-guided fractionation using preparative thin layer chromatography, reverse phase chromatography and other standard methods to yield samples for phytotoxicity testing in Stoneville. Possible pitfalls include C. glycines not producing a phytotoxin by or using a toxin not effectively extracted by ethyl acetate. Usually mycotoxins need to diffuse through the lipid membrane of plant cells to be active and that property requires mycotoxins to have structures that are readily extractable with ethyl acetate. The production and extraction conditions in the proposal were selected based on lack of equipment in the containment facility and are far from ideal. The amounts to be produced should provide plenty of material to demonstrate toxin production, and should allow identification of known toxins produced, but are very unlikely to allow structure determination of an unknown toxin, although they should allow defining parameters needed for larger scale production at another facility, possibly in Africa, where containment is not required.