Location: Natural Products Utilization Research
2019 Annual Report
Objectives
The main objective of this project is to isolate and identify compounds from natural sources with pesticidal activity or have properties that are beneficial for human health. The overall goal is to be able to provide compound(s) amenable for commercial development as a pesticide or identify a “high value” plant with unique bioactive compounds. Over the next 5 years, we will focus on the following objectives:
Objective 1: Enable, from a technological standpoint, new commercial biopesticides; and identify optimum production practices for the plants from which these biopesticides are derived.
Subobjective 1.1: Identify nematicidal compounds from tall fescue.
Subobjective 1.2. Discover natural product based fungicides from plant extract collections or other useful sources for US agriculture.
Subobjective 1.3: Investigation of cashew nut shell liquid for insecticide activity and synthetic modification of the isolated compounds to gain insights into structure-activity relationship.
Sub-objective 1.4: Discover natural product based herbicidal and insecticidal compounds from crude plants and plant endophyte extract collections.
Objective 2: Identify human bioactive compounds in select plants and herbs, and determine plant growth conditions to enhance or optimize bioactive compound concentrations.
Subobjective 2.1: Identify anti-adipocyte compound(s) in Scutellaria ocmulgee and determine the effect of various growth conditions on the bioactive compound(s).
Approach
An “activity-guided” isolation approach will be employed in efforts to discover novel bioactive compounds. Focus will be on isolating single compounds from active fractions. The che mical structure of bioactive compounds isolated will be elucidated using a combination of spectroscopic techniques such as ultraviolet, infrared, mass spectrometry and nuclear magnetic resonance spectroscopy. Simple structure modification of the bioactive constituent(s) and synthesis of analogs will be performed for activity optimization. In general, four projects are included in the plan, employing specific approaches. These include: 1) Identification of nematotoxic compound(s) from tall fescue cultivar Jesup (Max-Q). Isolation will be guided by an in vitro assay on inhibition of nematodes. The activity of the isolated nematotoxic compound will be tested in soil. 2) Identification of fungicidal compound(s) from select plants from China. Isolation will be guided using in vitro assays against Botrytis cinerea, Colletotrichum species, Fusarium species, and Phomopsis species. The activity of isolated compounds will be tested in detached leaf assays. 3) Identification of compound(s) from cashew nut shell liquid with insecticidal activity. Isolation will be performed using assays to determine activity against mosquito (Aedis egypti) larvae and adult. Analogs of the mosquito larvicidal/adulticdal compound(s) will be synthesized following standard synthetic procedures such as Friedel-Crafts acylation reaction. 4) Identification of anti-obesity compound from Scutellaria ocmulgee. Isolation of compounds will be performed using inhibition of adipocyte differentiation as acidity indicator. Anti-adipocyte compounds isolated will be used as chemical markers in associated study determining the appropriate agronomic practices to generate highest amount of anti-adipocyte compound(s) and biomass.
Progress Report
This is the fourth full year of research for this project. For Objective 1, enable, from a technological standpoint, new commercial biopesticides; and identify optimum production practices for the plants from which these biopesticides are derived, milestones have been met and significant progress has been made.
Pertaining to Subobjective 1.1, our research chemist has been in touch with the collaborators with which a now vacant scientist had been working with on this fescue project and the collaborators have made it clear that they do not wish to continue this project. Specifically, many of the fractions and extracts produced by this now vacant scientist shortly before her passing are not usable in their current form to carryout isolation of active principle/s. Furthermore, due to a limited amount of plant material provided by the collaborator, these extracts and fractions could not be reproduced in quantities sufficient for bioassay guided fractionation. Milestone table was adjusted and the adjustment approved earlier this year to reflect this change.
Under Subobjective 1.2, we describe the bioassay-guided isolation of antifungal and phytotoxic compounds from the ethyl acetate extract of Ambrosia salsola twigs and leaves. With this methodology, we isolated and identified twelve compounds (four chalcones, six flavonols and two pseudoguaianolide sesquiterpene lactones). The phytotoxicity of purified compounds was evaluated, and neoambrosim was active against Agrostis stolonifera at 1 mM, while confertin was active against both, Lactuca sativa and A. stolonifera at 1 mM and 100 µM, respectively. Confertin and salsolol A and B had IC50 values of 261, 275, and 251 µM, respectively, against Lemna pausicotata (duckweed). Confertin showed a higher activity than that for neoambrosin.
Non-funded cooperative agreement between the USDA and Chinese Academy of Tropical Agricultural Sciences (CATAS) titled: Technology for discovery of agrochemicals, biopesticides, and nutraceuticals from United States of America and Chinese Agriculture was completed in May, 2019. The aim of this project was to identify new natural product-based pesticides and biopesticides for use as plant protectants or phytochemicals in plant protection and production. We focused on unique actinomycete species not previously studied as a source of antifungal constituents. Four bioactive organisms were isolated that can effectively control banana fusarium wilt disease caused by the soil-borne Fusarium oxysporum f. sp. cubense. These organisms were subsequently identified by sequence analyses of the ITS region of the rRNA genes as belonging to the species Streptomyces yunnanensis. The butanol extract of the culture filtrate from Streptomyces yunnanensis was analyzed by innovative technology and potent bioactive preparations were evaluated by various in-house rapid bioassays using an activity-directed fractionation approach to determine potential inhibitory effects towards phytopathogenic fungi included Colletotrichum fragariae, C. acutatum, and C. gloeosporioides. Further studies toward the identification of specific bioactive compounds and their detailed biochemical pathways and physiological studies are currently underway.
We have evaluated the culture filtrate of the plant pathogenic fungus Pyricularia grisea, that infects Brachiaria eruciformis (signal grass) for phytotoxins and fungitoxins. Pyrichalasin H was isolated and identified as the toxin that affects plant growth and for the first time identified as a fungitoxin against Colletotrichum species. Pyrichalasin H was also found to be the major metabolite of the culture filtrate.
Under Subobjective 1.3, the larvicidal constituents from cashew nut shell liquid were isolated and we still have to isolate adulticide constituents from a more polar fraction. Synthetic analogs have also been produced and are awaiting insecticidal evaluations. We are still carrying out synthetic modifications to some of minor constituents in the extract.
Under Subobjective 1.4, members of the highly polyphagous Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) species complex cause major crop damage by feeding and by transmitting plant viruses. The Middle East-Asia Minor 1 (MEAM1) of the B. tabaci complex is by far the most problematic whitefly affecting crops including the cultivated watermelon (Citrullus lanatus). Watermelon cultivars share a narrow genetic base and are highly susceptible to whiteflies. However, there is wide genetic diversity among the species of Citrullus. A wild relative of the cultivated watermelon is C. ecirrhosus; it is a perennial desert species. We conducted a study to assess the potential of C. ecirrhosus as a source of whitefly resistance for cultivated watermelon. This study is the first report to establish pest resistance in C. ecirrhosus. This wild species offers a source of resistance against whiteflies for cultivated watermelon.
We have been working on plant pathogenic fungi in search for herbicidal, insecticidal and antifungal constituents. We were able to isolate and identify three fungi and a bacterium from the leaves of dollar weed (Hydrocotyle spp.). Dollar weed is a highly invasive species that is affecting lawns and golf courses in the U.S. These microbes were isolated from a leaf of dollar weed that showed severe necrotic lesions. The fungi Diaporthe ceratozamiae, Alternaria alternate and Epicoccum sorghi were isolated and identified as the fungi affecting the dollar weed leaves together with the bacterium Pantoea ananatis. Isolation and identification of the active metabolites from these microbes are in progress. We have shown that Pantoea ananatis is a plant growth promotor. The main constituent in the culture medium has been isolated as indole and the column chromatography fractions showed phytotoxicity. We are carrying out experiments to see if this bacterium can be utilized as a seed and root treatment in soil to make enhance crop growth. We have isolated azaphilone type antifungal compounds from the fungus Diaporthe ceratozamiae. Currently we are isolating minor constituents and structure activity studies of these compounds.
For Objective 2, identify human bioactive compounds in select plants and herbs, and determine plant growth conditions to enhance or optimize bioactive compound concentrations, milestones have been met and significant progress has been made.
Under Subobjective 2.1, pertaining to the optimization of the bioactive constituents, in vitro propagation of S. ocmulgee during first half of the year has been completed. We have changed the extraction and fractionation methods that have been done in the past. Dried, ground S. ocmulgee leaves were extracted with hexane, ethyl acetate, methanol, and 80% acetone/water. Portions of the extracts were sent for bioassay at Wayne State University. We are fractionating each extract and the fractions will be tested for bioactive compounds. This approach is different to what the now vacant scientist carried out.
The remaining progress made thus far falls more broadly under Objective 2. Following the unexpected passing of the now vacant scientist in 2018, another scientist on this project has been transitioning into a role originally filled by the now vacant scientist as it pertains to an antidiabetes basil project. Ocimum is a genus of aromatic annual, and perennial herbs and shrubs in the family Lamiaceae native to Asia, Africa, South, and Central America. Ocimum includes about 150 species; of which the most well-known and widely used for culinary purposes is O. basilicum (basil). Ocimum species have been reported to exhibit anti-diabetic properties. Sixteen Ocimum accessions were analyzed for antidiabetic properties with the objective of identifying compound(s) that may be best associated with its antihyperglycemic effects. Organic extracts of the five basil species O. basilicum (Brazil), O. gratissimum (Taiwan), O. gratissimum (Zambia), O. tenuiflorum (Cuba), and O. tenuiflorum (India) were discovered to be the most bioactive extracts at inhibition of alpha-glucosidase. O.tenuiflorum demonstrated comparable IC50 inhibitory concentrations as acarbose, a widely-prescribed anti-diabetic medication.
Also more broadly under Objective 2, capsinoids (capsiate and derivitives) are non-pungent analogs of capsaicinoids that were first isolated from the sweet pepper cv. CH-19. Capsinoids offer similar types of biological activity as capsaicinoids. Capsiate can be chemically synthesized - though the process is lengthy, expensive, and the yield is low. A Capsicum annum mutant that produces high levels of capsiate was reported in 2014. We filed a patent that involves a method for the extraction and purification of capsiate from this mutant and is applicable to the isolation of capsiate (capsinoids) from other Capsicum annuum cvs. and species.
Accomplishments
1. New phytotoxic diterpenes, the neocassanes, with a never before reported carbon skeleton. Eragrostis plana (Nees) is a problematic invasive plant with reported allelopathic potential. To isolate and identify phytotoxic compounds from leaves and roots of E. plana, a bioassay-directed isolation of the bioactive constituents was performed by ARS scientists at Oxford, Mississippi. This is the first report on a new diterpene carbon skeleton, the neocassanes, and of three new neocassane diterpenes, neocassa-1,12(13),15-triene-3,14-dione, 1, 19-norneocassa-1,12(13),15-triene-3,14-dione, 2, and 14-hydroxyneocassa-1,12(17),15-triene-3-one, 3, identified from the roots. Compounds 1, 2, and 3 inhibited the growth of duckweed 50% at concentrations of 109 ± 28, 200 ± 37, and 59 ± 15 µM, respectively. The compounds identified here could explain the allelopathy of E. plana. The description of the newly discovered compounds, besides contributing to the chemical characterization of the species, may be the first step in the study of the potential of these compounds as bioherbicides.
Review Publications
Favaretto, A., Cantrell, C.L., Fronczek, F.R., Duke, S.O., Wedge, D.E., Abbas, A., Scheffer-Basso, S.M. 2019. New phytotoxic Cassane-like diterpenoids from Eragrostis plana (Nees). Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.8b06832.
Travasarou, A., Vougogiannopoulou, K., Aligiannis, A., Cantrell, C.L., Fokialakis, N., Pratsinis, H. 2019. Bioactive metabolites of the stem bark of Strychnos aff. darienensis and evaluation of their antioxidant and UV protection activity in Human Skin Cell Cultures. Cosmetics. 6(7):1-10.
Semerdjieva, I.B., Shiwakoti, S., Cantrell, C.L., Zheljazkov, V.D., Astatkie, T., Schlegel, V., Radoukova, T. 2019. Hydrodistillation extraction kinetics regression models for essential oil yield and composition in Juniperus virginiana, J. excelsa, and J. sabina. Molecules. 24:1-17. https://doi.org/10.3390/molecules24050986.
Park, S., Scheffler, J.A., Scheffler, B.E., Cantrell, C.L., Pauli, C.S. 2019. Chemical defense responses of upland cotton, Gossypium hirsutum L. to physical wounding. Plant Direct. 3:5. https://doi.org/10.1002/pld3.141.
Schrader, K., Ibrahim, M.A., Abd-Alla, H.I., Cantrell, C.L., Pasco, D.S. 2018. Antibacterial activities of metabolites from Vitis rotundifolia (Muscadine) roots against fish pathogenic bacteria. Molecules. 23:2761-2768. https://doi:10.3390/molecules23112761.
Tsikolia, M., Bernier, U.R., Wedge, D.E., Tabanca, N., Abboud, K.A., Linthicum, K. 2019. Fungicidal properties of some novel trifluoromethylphenyl amides. Chemistry and Biodiversity. https://doi.org/10.1002/cbdv.201800618.
Rosa, L.H., Zani, C.L., Cantrell, C.L., Duke, S.O., Dijck, P.V., Desideri, A., Rosa, C.A. 2019. Fungi in Antarctica: diversity, ecology, effect of climate changes, and bioprospection for bioactive compounds. Book Chapter. https://doi.org/10.1007/978-3-030-18367-7_1.
Simmons, A.M., Jarret, R.L., Cantrell, C.L., Levi, A. 2019. Citrullus ecirrhosus: Wild source of resistance against Bemisia tabaci (Hemiptera: Aleyrodidae) for cultivated watermelon. Journal of Economic Entomology. https://doi.org/10.1093/jee/toz069.
Tabanca, N., Wedge, D.E., Li, X., Gao, Z., Ozek, T., Bernier, U.R., Epsky, N.D., Can Baser, K., Ozek, G. 2018. Biological evaluation, overpressured layer chromatography (OPLC) separation and isolation of a new acetylenic derivative compound from Prangos platychlaena ssp. platychlaena fruit essential oils. Planar Chromatography. 31(1):61-71.
Liu, X., Qiao, L., Zhai, Z., Cai, P., Cantrell, C.L., Tan, C., Weng, J., Han, L., Wu, H. 2019. Novel 4-pyrazole carboxamide derivatives containing flexible chain motif: Design, synthesis, and antifungal activity. Pest Management Science. https://doi.org/10.1002/ps.5463.
Perera, W.H., Meepagala, K.M., Fronczek, F.R., Cook, D., Wedge, D.E., Duke, S.O. 2019. Bioassay-guided isolation and structure elucidation of fungicidal and herbicidal compounds from Ambrosia salsola (Asteraceae). Molecules. 24:1-12. https://doi.org/10.3390/molecules24050835.
Meepagala, K.M., Clausen, B., Johnson, R.D., Wedge, D.E., Duke, S.O. 2019. A phytotoxic and antifungal metabolite (Pyrichalasin H) from a fungus infecting Brachiaria eruciformis (Signal Grass). Journal of Agricultural Chemistry and Environment. 8(3):115-128. https://doi.org/10.4236/jacen.2019.83010.
