Location: Natural Products Utilization Research
Project Number: 6060-41000-008-03
Start Date: May 01, 2012
End Date: Apr 30, 2017
We actively employ ethnobotanical, chemical ecology, anatomical or physiological clues to suggest candidate species. Our research focus is on unique plant species not previously studied as a source for agrochemicals, often these are medicinal and aromatic plants from China. Hunan University of Chinese Medicine (HUCM) has access to extensive ethnobotanical information regarding medicinal herbs used in traditional Chinese medicines and cultivated Good Agricultural Practices (GAP) certified Traditional Chinese Medicine (TCM) plant materials. HUCM has vast resources and plant materials associated with extraction and processing of plants used for TCM. HUCM also has the Ethnomedicine Innovation & Development Laboratory that is set up to purify active compounds for structural elucidation. The HUCM laboratories have well developed several standardization protocols for plant extracts used in TCM medical applications and the expertise will be used for plant-based fungicide or biopesticide development. The ARS scientist has developed and extensively validated two sensitive detection systems, which will be used for initial identification of analogs with antifungal activity. First, bioautography assays will be used to identify antifungal components from plant extracts, eliminate commonly occurring nuisance compounds, and characterize antifungal activity of pure compounds. Second, a microbioassay, using fungal conidia in a 96-well microtiter format, will be used to evaluate growth effects of active fractions or pure compounds identified by bioautography. These micro-dilution broth bioassays allow for the evaluation of microgram quantities, determine dose-response relationships; compare antifungal activity with fungicide standards of known mode of action. This coupled approach to natural product discovery combines the simple and visual nature of direct bioautography with the rapid, sensitive, and high throughput capabilities of a microtiter system. In vitro activity will be determined using a standardized 96-well plate dose-response format and antifungal activity is evaluated in comparison to commercial fungicide standards. Chemical isolation, structural elucidation and structure activity relationships will be conducted in collaboration with NCNPR chemists. Coupling planar bioautographic 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 96-well microtiter system. Using 24-Well Leaf Disc Assay developed by Wedge’s lab also evaluates new potential lead compounds, can be repeated quickly in time, and real on-the-leaf-surface activity can be evaluated in high throughput formats and published in a reasonable time frame. Compounds applied directly to the leaf surface can be evaluated in a dose-response for fungicidal activity and phytotoxicity. The assay is sensitive to microgram quantities, can determine chemical sensitivity between fungal isolates, and is adaptable to complex mixtures, lipophilic extracts, and non-polar compounds.