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Research Project: New Weed Management Tools from Natural Product-Based Discoveries

Location: Natural Products Utilization Research

2016 Annual Report

1a. Objectives (from AD-416):
The overall goal of this project is to discover and develop natural product-based weed management solutions. The research is divided into discovery and development of biochemicial bioherbicides and creation of crops that are resistant to weeds by transgenically imparting or improving plant/plant allelopathy. This research should provide new avenues for the development of affordable and effective, yet sustainable, weed control strategies. Objective 1: Discover and develop natural product-based bioherbicides that provide environmentally safe and toxicologically benign tools for weed management, with novel modes of action, to address current problems associated with herbicide resistant weeds. Subobjective 1.1: Discover new and existing natural products for potential use as herbicides and bioherbicides. Subobjective 1.2: Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. Subobjective 1.3: Develop natural products as new weed management tools. Objective 2: Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals. Subobjective 2.1: Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. Subobjective 2.2: The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone. Subobjective 2.3: Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. Subobjective 2.4: Engineering de novo sorgoleone biosynthesis in non-producing host plants.

1b. Approach (from AD-416):
Bioassay-directed isolation of phytotoxin will be followed by their evaluation of their potential as bioherbicides and determination of their modes of action. Genes of the sorgoleone synthesis pathway with root hair-specific promoters will be inserted into plants with the intent to impart or improve allelopathic capacity for enhanced weed management.

3. Progress Report:
Towards achieving Objective 1, the following research was performed in FY 2016 and is continuing in FY 2017. 1. From the extract of toothpickweed (Ammi visnaga) two phytotoxic compounds (khellin and visnagin), whose herbicidal activity had not been described before, were isolated. The inhibitory activities of these compounds were shown to be similar to those of commercial herbicides acetochlor and glyphosate in bioassays conducted. A provisional patent has been filed for discoveries made from this study. 2. A plant pathogenic fungus, Curvularia intermedia, was isolated from the pandan plant (Pandanus amaryllifolius) and cultured in the laboratory. From the culture broth two phytotoxic constituents (curvularin and a,ß-dehydrocurvularin) were isolated. a,ß-Dehydrocurvularin was the more phytotoxic according to laboratory bioassays. This compound caused cell membrane disruption in cucumber cotyledons, suggesting that is has effects on plant plasma membranes. 3. Three new compounds (saponins) isolated from the Mediterranean plant Bellis longifolia were isolated, identified, and found to be highly phytotoxic. 4. Several new diterpenes from the Brazilian plant Vellozia gigantea were isolated, identified and found to be strong phytotoxins. Towards achieving Objective 2, the following research was performed in FY 2016 and is continuing in FY 2017. 1. Characterization of cytochrome P450 enzymes that are involved in the biosynthetic pathway was completed. Preparation of the manuscript is in progress and the paper is expected to be submitted for publication in 2016. 2. Multi-gene vectors containing genes coding for Sorghum sorgoleone biosynthetic enzymes for transient expression analysis were constructed. The activities of all these sorghum genes in a model plant Nicotiana benthamiana were confirmed. 3. Screening of Sorghum bicolor (S. bicolor) Tx430 transformation events harboring vectors designed for the overexpression of key sorgoleone biosynthetic enzymes was completed. 4. Characterization S. bicolor Tx430 transformation events, harboring vectors designed for RNAi-mediated downregulation of cytochrome P450 enzymes involved in sorgoleone biosythesis, was completed.

4. Accomplishments
1. Potent pytotoxins from toothpickweed identified. Plants constitute a rich source of novel and structurally diverse phytotoxic compounds to be explored in searching for effective and environmentally safe herbicides. Toothpickweed (Ammi visnaga) was subjected to phytotoxicity-guided fractionation. Two compounds, khellin and visnagin, whose herbicidal activity had not been described before were found to be highly phytotoxic. In laboratory assays, khellin and visnagin inhibited the growth of lettuce and duckweed. Also, both compounds reduced the growth of the weeds ryegrass, morningglory, foxtail and millet. The inhibitory activities of these compounds were similar to those of the commercial herbicides acetochlor and glyphosate in the lab bioassays. During greenhouse studies visnagin was more active, with significant contact post-emergence herbicidal activity on the weeds velvetleaf and crabgrass 2 kg ai ha-1. Moreover, its effect on velvetleaf, crabgrass and barnyardgrass (Echinochloa crus-galli) at 4 kg ai ha-1 was comparable to the bioherbicide pelargonic acid at the same rate. These results support the potential of visnagin, and possibly khellin, as bioherbicides or lead molecules for the development of new herbicides. Mode of action studies suggested that the compounds have a unique mode of action.

2. Generation of a panel of metabolomic responses to seven herbicides with diverse modes of action. At a given dose of an applied phytotoxin at a specific time point after exposure, changes to a plant's transcriptome and metabolome specific for phytotoxins sharing a common molecular target site can, in theory, be identified. A library of transcriptomic and metabolomic profiles generated for phytotoxins representing different molecular target sites can therefore be useful in the determination of the molecular targets of phytotoxins with unknown sites of action. For example, more labor intensive follow-up experiments such as biochemical approaches can be more efficiently targeted towards compounds possessing novel modes of action with the availability of such reference data sets. Obtaining detailed characterizations of the transcriptomic and metabolomic response profiles for herbicides representing diverse modes of action therefore represents a key component of our project. With a collaborator at the University of Aarhus in Denmark, we have completed the characterization of metabolomic responses in Arabidopsis to herbicides with seven different modes of action. Treatments resulting in 50% and 99% reduction in fresh weight accumulation during a 4 day post-treatment period were performed for the seven herbicides. Metabolome data sets which have been generated from this work will be used to guide our efforts to identify and characterize the mode of action of natural product-based pesticides for agriculture featuring novel modes of action.

5. Significant Activities that Support Special Target Populations:

Review Publications
Parveen, I., Wang, M., Zhao, J., Chittiboyina, A.G., Tabanca, N., Ali, A., Baerson, S.R., Techen, N., Chappell, J., Khan, I.A., Pan, Z. 2015. Investigating sesquiterpene biosynthesis in Ginkgo biloba: molecular cloning and functional characterization of (E,E)-farnesol and a-bisabolene synthases. Plant Molecular Biology. 89:451-462.

Wang, R., Zhu-Salzman, K., Baerson, S.R., Xin, X., Li, J., Su, Y., Zeng, R. 2016. Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm moth (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides. Insect Science. DOI 10.1111/1744-7917.12315.

Duke, S.O., Dayan, F.E. 2015. Discovery of new herbicide modes of action with natural phytotoxins. American Chemical Society Symposium Series. 1204:79-92.

Salas, R.A., Scott, R.C., Dayan, F.E., Burgos, N.R. 2015. EPSPS gene amplification in glyphosate-resistant in Italian ryegrass (Lolium perenne ssp. multiflorum) populations from Arkansas, USA. Journal of Agricultural and Food Chemistry. 63:5885-5893. DOI:10.1021/acs.jafc5b00018

Maroli, A.S., Nandula, V.K., Dayan, F.E., Duke, S.O., Gerard, P., Tharayil, N. 2015. Metabolic profiling and enzyme analyses indicate a potential role of antioxidant systems in complementing glyphosate resistance in an Amaranthus palmeri biotype. Journal of Agricultural and Food Chemistry. 63:9199-9209.

Carbonari, C., Latorre, D.O., Gomes, G., Velini, E., Owens, D.K., Pan, Z., Dayan, F.E. 2016. Resistance to glufosinate is proportional to phosphinothricin acetyltransferase expression and activity in LibertyLink® and WideStrike® Cotton. Planta. 243:925-933. DOI 10.1007/s00425-015-2451-3

Correa, E.A., Dayan, F.E., Owens, D.K., Rimando, A.M., Duke, S.O. 2016. Glyphosate-resistant and conventional canola (Brassica napus L.) responses to glyphosate and Aminomethylphosphonic Acid (AMPA) treatment. Journal of Agricultural and Food Chemistry. 64:3508-3513.

Silva, F.M., Duke, S.O., Dayan, F.E., Velini, E.D. 2015. Low doses of glyphosate change the response of soybean to later glyphosate exposures. Weed Research. 56:124-136. DOI: 10.1111/wre.12189

Reddy, K.N., Duke, S.O. 2014. Soybean mineral composition and glyphosate use. In: Processing and Impact on Active Food Components in Food, V.R. Preedy, Ed.,Elsevier, Inc., London. p. 369-376.

Meepagala, K.M., Johnson, R.D., Techen, N., Wedge, D.E., Duke, S.O. 2015. Phomalactone from a phytopathogenic fungus infecting Zinnia elegans (Asteraceae) leaves. Journal of Chemical Ecology. 41:602-612.

Kaymakcioglu, B.K., Beyhan, N., Tabanca, N., Ali, A., Wedge, D.E., Duke, S.O., Bernier, U.R., Khan, I.A. 2015. Discovery and structure activity relationships of 2-pyrazolines derived from chalcones from a pest management perspective. Medicinal Chemistry Research. 24:3632-3644.

Williams, M.M. II, Bradley, C.A., Duke, S.O., Maul, J.E., Reddy, K.N. 2015. Goss’s wilt incidence in sweet corn is independent of transgenic traits and glyphosate. Horticultural Science. 50:1791-1794.

Xie, Q., Li, S., Jiang, J., Liao, D., Wang, W., Tekwani, B., Ali, A., Rehman, J., Schrader, K., Duke, S.O., Cantrell, C.L., Wedge, D.E. 2015. Bio-pesticidal and anti-microbial coumarins from Angelica dahurica (Fisch. Ex Hoffm). Records of Natural Products. 10(3):294-306.

Meepagala, K.M., Johnson, R.D., Duke, S.O. 2016. Curvularin and dehydrocurvularin as phytotoxic constituents from curvularia intermedia infecting pandanus amaryllifolius. Journal of Agricultural and Food Chemistry. 5:12-22.

Duke, S.O. 2016. Secondary effects of glyphosate on plants. In: A Era Glyhosate, Agricultura, Meio Ambiente e Homem (The Age of Glyphosate: Agriculture, Environment and Humans), D.K. Meschede and D.L.P. Gazziero, Eds., Midiograf II, Londrina, Brazil, pp. 87-96.

Trivella, A., Stawinoga, M., Dayan, F.E., Cantrell, C.L., Mazellier, P., Richard, C. 2015. Photolysis of natural B-triketonic herbicides in water. Water Research. 78:28-36.

Carvalho, C.R., Wedge, D.E., Cantrell, C.L., Silva-Hughes, A.F., Pan, Z., Moraes, R.M., Madoxx, V.L., Rosa, L.H. 2016. Molecular phylogeny, diversity and bioprospecting of endophytic fungi associated with wild ethnomedicinal North American plant Echinacea purpurea (Asteraceae). Chemistry and Biodiversity. 13:918-930 DOI: 10.1002/cbdv.201500299.

Killeen, D.P., Larsen, L., Dayan, F.E., Gordon, K.C., Perry, N.B., Van Klink, J.W. 2016. Nortriketones: antimicrobial trimethylated acylphloroglucinols from manuka (Leptospermum scoparium). Journal of Natural Products. doi: 10.1021/acs.jnatprod.5b00968.

Mcintosh, C., Owens, D.K. 2016. Advances in flavanoid glycosyltransferase research: integrating recent findings with long-term citrus studies. Phytochemistry Reviews. 15. 10.1007/s11101-016-9460-6.

Devaiah, S.P., Owens, D.K., Sibhatu, M.B., Sarkar, T.R., Strong, C.L., Mallampalli, V., Asiago, J., Cooke, J., Kiser, S., Lin, Z., Wamucho, A., Hayford, D., Williams, B.E., Loftis, P., Berhow, M.A., Pike, L.M., McIntosh, C.A. 2016. Identification, recombinant expression, and biochemical analysis of putative secondary product glucosyltransferases from Citrus paradisi. Journal of Agricultural and Food Chemistry. 64(9):1957-1969.

Chen, S., Yu, J., Li, Q., Zhao, J., Wedge, D.E., Duke, S.O., Liao, D., Wang, Y., Fronczek, F.R., Khan, I.A., Wang, W. 2016. 7a-hydroxfriedelan-3one-26-OL-29-OIC acid and other constituents from Pileostegia Viburnoids VAR. Glabrescens. Natural Product Communications. 11(7):931-934.