Location: Biological Control of Pests Research
Project Number: 6066-42000-006-05-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Apr 1, 2018
End Date: Sep 30, 2019
We propose to better understand the mechanism of root infection of soybeans from the soil reservoir by Macrophomina (M.) phaseolina and other fungi that infect the roots of soybean plants from soil reservoirs. These fungi access the vascular system of soybean plants through the roots by using a mycotoxin to induce localized root cell killing, which creates necrotic or tissue-free zones through which fungal hyphae can readily penetrate. The objective of the proposed research is to better understand the range of mycotoxin types used by M. phaseolina isolates from Mississippi soybean plants to facilitate root infection in soybeans. The responses will be compared to those of a limited selection of other fungal pathogens that cause losses in Mississippi soybean production by infecting plants from the soil through the roots.
Macrophomina (M.) phaseolina isolates will be screened for the three responses observed in culture with previously studied isolates exposed to (i) locust bean gum added to the agar surface, and (ii) high levels of sucrose added to a well cut in the agar layer. The studies will be carried out by placing 4 mm diameter plugs of complete Czapek-Dox medium with an actively growing fungal isolate on a 10 cm plate of media lacking sucrose but containing ß-alanine, which reacts with the mycotoxin (-)-botryodiplodin to produce a red pigment. Specifically, the responses to the inducers that will be scored are (i) production of the mycotoxin (-)-botryodiplodin, (ii) stimulation of intense branching of fungal hyphae visible under the microscope, and (iii) production of microsclerotia. In addition, six to eight isolates of the following fungi will be obtained from other sources and tested in vitro under the same conditions. Fungal isolates that do not produce detectable (-)-botryodiplodin in plate assay, but respond to locust bean gum with intensive branching and/or microsclerotia formation, will be studied further by growing them in potato dextrose broth in the presence of sterile locust bean gum with reciprocal shaking for 7 days at 28°C, filtering off the mycelium from each with cheese cloth and filter-sterilizing the filtrates through 0.22 micron membrane filters. Sterilized filtrates will be mixed 1:1 with hydroponic culture medium and tested for prevention of lateral root growth of soybean seedlings in unstirred hydroponic culture. If the lateral root growth is blocked by a filtrate, but not in controls, it may indicate that the fungus has been producing a toxin targeting meristematic tissue. Any effect on root tips will be confirmed by transmission electron microscopy. Isolates that produce culture filtrates that block lateral root growth of soybean seedlings in hydroponic culture will be examined for mycotoxin production by growing on 10 plates of medium with added sterile locust bean gum until they cover the agar surface, then extracting the agar medium 5 times with ethyl acetate and once with 50% ethanol. The extracts will be evaporated and assayed (i) for antibacterial activity against a Bacillus subtilis strain sensitive to all known antibiotics; (ii) for phytotoxicity with soybean leaf discs and in Lemna paucicostata axenic cultures; and for cytotoxicity against dividing mammalian cells in culture, particularly the mouse fibroblast line NIH3T3. Bioassay-guided fractionation will be carried out for active extracts using preparative thin layer chromatography, preferably using antibacterial activity because it is by far the easiest and fastest assay to use. Purified active agents can then be tested for other activities. Structural identification of active agents will be undertaken using analysis by liquid chromatography with mass spectrometric detection. Timeline: Beginning studies in April will be directed toward setting up assays and examining cultures of isolates from the laboratory collection. Structural elucidation studies will be conducted at facilities in Minnesota.