Location: Natural Products Utilization Research2013 Annual Report
1a. Objectives (from AD-416):
Identify and evaluate new natural product-based agrochemicals for US agriculture and generate new fundamental knowledge of plant protection agrochemicals. 1) These collaborative studies will determine potential lead compounds that show promise for further research and possible eventual development by industry partners in either USA or China. Through cooperations between Hunan University of Chinese Medicine (HUCM) and the U.S. Department of Agriculture laboratories to discover and develop new agrochemical fungicides and biopesticides from natural products we can increase the opportunity for success. Technology transfer and scientific exchange through learning our advanced bioassay techniques, including various in vitro assays for establishing fungicide and agrochemical efficacy, and 2) apply HUCM experience, knowledge, and processing of plants used in Traditional Chinese Medicine will improved the evaluation process for new agrochemical and biopesticide discovery, and 3) work to establish a collaborative research program between HUCM and USDA to search for natural products with low mammalian and environmental toxicity for use as plant/human protectants important to USA and China. Bioactive plant extracts and partially purified compounds will be subjected to bioassay guided fractionation and purification towards determination and identification of the active compound(s)and marker compounds that guide the development of a standardized active plant extract at HUCM under the direction of HUCM faculty. Initial screening studies will take place in USA or CHINA then move to The NCNPR, CMAVE, or other ARS locations in the USA where further purification and structural elucidation and detailed biochemical and physiological studies can be conducted in collaboration between ARS and HUCM. Ideally this research project will provide an opportunity for international exchange of post-doctoral scientists, graduate students, or new career scientists to come to the USDA on their own funding from CHINA. The agreement will also provide for the opportunity for USDA scientists to conduct short term visits to HUCM to conduct research, meetings, coordinate research projects, and present lectures on agrochemical and discovery research. Some studies could also be conducted in China in collaboration with faculty in the College of Plant Protection of the Agricultural Universities or research institutions in China. The use of natural product-based agrochemicals provides an opportunity for better management of our natural resources by reducing dependence on synthetic and often more toxic chemicals and provide alternatives to other agrochemicals scheduled for market removal.
1b. Approach (from AD-416):
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.
3. Progress Report:
Three Chinese Visiting Scholars came to the USDA, Natural Products Utilization Research Unit in Oxford, MS, to work and evaluate natural compounds and their analogs from plants use in traditional Chinese medicinal for activity against plant pathogens, insecticides, adult mosquito deterrents and toxicants, mosquito larvicides, and natural herbicides. Essential oils from Acorus tatarinowii and Acorus calamus were obtained by hydrodistillation and bioassay guided fractionation. Isolation, identification, and characterization of compounds with antifungal activity was conducted. The plant pathogenic fungus, Colletotrichum fragariae, was used in bioautography of pure compounds from Acorus species to identify antifungal activity in five compounds: calamusenone, (-)-epi-Isocalamusenone, cis-Methyl isoeugenol, a-Asarone, and ß-Asarone. The most active molecules were (-)-epi-Isocalamusenone and ß-Asarone which caused ca. 40% growth inhibition of the plant pathogens Botrytis cinerea and Fusarium oxysporium. Some compounds also showed significant adult mosquito lethality and moderate mosquito larvidical activity. TLC-bioautography with Colletotrichum species as the antifungal detection agents demonstrated strong antifungal activity by compounds from Acorus dahurica. Six pure compounds were obtained from the ethyl acetate extract under bioassay-guided fractionation performed and the structures of the pure compounds were identified according to 1H and 13C NMR data. Four of the compounds, suberosin, bergapten, alloimperatorin, and xanthotoxol, were active against C. fragariae. The activity of these compounds was further evaluated using a microtitter assay with seven plant pathogenic fungal species. Four compounds were active against Phomopsis obscurans and P. viticola, fungi that cause serious leaf spot diseases worldwide. A. dahurica extracts also showed weak mosquito larvicidal activity. Extracts of Pileostegia viburnoides Hook. var. glabrescens were separated by chromatography. Four compounds from the n-butanol extract were identified as umbelliferone, skimmi, fraxin, and 7-O-ß-D-glucopyranosyl-8-methoxybenzo pyranone. These compounds were phytotoxic at various levels against seeds of a dicot, Iceberg lettuce, and a monocot, Agrostis grass. Umbelliferone and skimmin was highly phytotoxic against the monocot and moderately phytotoxic against the dicot. 7-O-ß-D-glucopyranosyl-8-methoxybenzopyranone inhibited seed germination of Agrostis. These three projects demonstrated the success of Chinese students in conducting part of their research as USDA Visiting Scholars. They received training to discover new applications for plants used in Traditional Chinese Medicine as agrochemicals and bio-pesticides.