Allelochemicals Biologica... - Name

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DAVID E. WEDGE
AND BARBARA J. SMITH
by the fungus C. acutatum and stem end rot caused by the fungus Gnomonia comari.
Stem end rot lesions were often invaded by secondary pathogens such as Botrytis and
Colletotrichum. The combination fungicides such as Elevate® + Captan®, Pristine®,
and Switch® were effective in controlling this disease complex. The untreated control
plants and those treated with Emerald® had the most berries affected with stem
end rot. The incidence of gray mold and anthracnose fruit rot was extremely low.
Gray mold was controlled by the fungicides Scala®, Elevate® + Captan®, Pristine®,
Switch®, Emerald®, and Elevate®.
4. CONCLUSIONS
Information gained from the examination of thousands of extracts and their associated
pure compounds have culminated in the development of a variety of standardized
operating protocols for natural product discovery that are currently being used in our
laboratory. Successful discovery, evaluation, and development of natural product
fungicides are totally dependent upon the availability of high quality miniaturized
antifungal bioassays. Bioassay-directed screening of compounds and extracts is the
initial step in the discovery process for new agrochemicals and pesticides.
Standardization of inoculum allows for meaningful comparison of growth
inhibition between different fungal pathogens, test compounds, and experiments
repeated in time. Bioautography provides a simple technique to visually follow
antifungal components through the separation process. The 96-well microbioassay
allows for the evaluation of microgram quantities, determination of dose-response
relationships, and comparison of antifungal activity with fungicides with a known
mode of action. Coupling bioautography techniques with the 96-well microbioassay
provides us with a discovery protocol that combines the simple and visual nature of
direct bioautography with the rapid, sensitive, and high throughput capabilities of a
microtiter system.
The 96-well microbioassay is accurate and sensitive; as little as 0.1 µM amounts
of test compound permit discrimination between germination and mycelial growth
inhibitors and identification of fungicide resistant pathogens. The microbioassay
utilizes a chemically defined liquid medium with a zwitterion buffer that limits chemical
interaction with test compounds and controls for pH variations. This new standardized
method provides high-throughput capability and the capacity to study chemical
compounds in detail, to perform mode of action studies, and to determine fungicide
resistance profiles for specific fungal pathogens.
Detached leaf assays are critical for establishing ‘real world’ activity prior to the
field testing that agrochemical companies require before investing millions of dollars
needed to develop a agrochemical. Subsequent efficacy testing in the greenhouse
ultimately helps determine the potential usefulness of compounds as pest control agents.
To maximize the detection of natural products, high-throughput bioassay techniques
must target significant agricultural pests, include relevant commercial pesticide
standards, and adhere to sound statistical principles.