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United States Department of Agriculture

Agricultural Research Service

Research Project: Discovery and Development of Natural Product-based Weed Management Methods

Location: Natural Products Utilization Research

2013 Annual Report

1a. Objectives (from AD-416):
Objective 1: Discover natural product-based materials and technologies for weed and other pest management that would be accepted by organic farmers and/or farmers who desire more environmentally and toxicologically benign weed and other pest management tools. Subobjective 1.1: Discover uses of new and existing natural products for weed management in conventional and organic farming. Subobjective 1.2: Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. Subobjective 1.3: Discovery of the mechanisms of action for newly discovered phytotoxins using a genetics approach with resistant mutants. Subobjective 1.4: Discovery of the mechanisms of action for newly discovered phytotoxins using transcriptome analysis. Objective 2: Identify and characterize the biochemical pathways of phenolic lipid-type allelochemicals and fungicides from cereals, and manipulate these pathways to produce enhanced allelopathic and disease-resistant crops with reduced requirements for synthetic herbicides and/or fungicides. Subobjective 2.1: Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. Subobjective 2.2: Functional analysis of putative sorgoleone pathway enzymes by genetically engineering sorghum to either increase or reduce expression of the corresponding genes, and the use of these transformants to investigate the ecological role of sorgoleone. Subobjective 2.3: Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. Subobjective 2.4: Manipulation of phenolic lipid biosynthesis using rice and Arabidopsis models.

1b. Approach (from AD-416):
Conduct bioassays in collaboration with research chemists during bioassay-directed isolation of new phytotoxins. Molecular sites of action will be determined with genomic and biochemical approaches. Genes controlling synthesis of useful plant secondary products involved in plant defenses to pests will be identified, cloned, and manipulated.

3. Progress Report:
Two putative cytochrome P450 clones from sorghum were identified and functionally characterized in previous research. In order to further evaluate the potential role of these P450s in biosynthesis of the allelochemical sorgoleone, binary vectors for targeting the downregulation of these genes [Ribonucleic Acid (RNA) interference] were constructed. Generating transgenic sorghum plants using these vectors are underway. The photolytic degradation of the natural herbicide leptospermone was tested in collaboration with a French research group. The degradation of leptospermone is more rapid at acidic pH 3 than at neutral pH. The degradation involves oxidation, isomerization, and Norrish cleavage. Following our previous work on the bioavailability of leptospermone in soil, the binding of this natural herbicide to various soil components (kd) was tested. This works provides insight to better understand the differences in herbicidal activity observed when leptospermone is applied preermergence. The soil activity of leptospermone is negatively affected by the presence of clay. Bioassays were developed to measure the early steps of carotenoid synthesis. This work provides assays to test large number of natural herbicides for their effect on this pathway. Aplotaxene, a phytotoxin from several invasive species, was isolated and its effect on various physiological functions was investigated. This compound may be involved in the interference of these species with native plant species. Further research to determine its role is in progress. Studies on transcriptome responses of plants to two natural phytotoxins, citrol and chalcone, were continued. Analyses of Illumina sequences of transcriptome reponses to these two phytotoxins are in progress. The hormesis (stimulatory effect of a subtoxic level of a toxin) effect of a phytotoxin was found to be strongly dependent on the growth stage of the treated plant and also dependent on previous exposure to the phytotoxin. Phomalactone was found to be the phytotoxin produced by a fungus infecting Zinnia leaves. This highly phototoxic compound produces symptoms of the infection. Further study of its mode of action is in progress.

4. Accomplishments
1. Role of a glucosyltranferase in detoxification of an allelochemical. Manipulation of allelochemical biosynthesis in transgenic plants requires the ability of the host/producing plant to tolerate significant exposure levels of the allelochemical of interest. Thus, the identification of genes associated with allelochemical detoxification pathways represents a key component of our project. Previously, ARS scientists at the Natural Products Utilization Research Unit in Oxford, MS, performed transcriptional profiling experiments using Arabidopsis thaliana as a model receiver plant to examine whole-transcriptome responses to the allelochemical benzoxazolin-2(3H)-one (BOA), and also identified BOA-6-OH and BOA-O-glucoside as BOA detoxification intermediates in A. thaliana. To identify candidate cytochrome P450 and UDP-glucosyltransferases associated with this detoxification pathway, hypersusceptibility screens were performed using sequence-tagged A. thaliana T-DNA insertional mutants. The disrupted loci for the mutants tested corresponded to P450 and glucosyltransferase sequences previously shown to be significantly up-regulated in response to BOA exposure. Various mutant lines and wild-type plants were screened at concentrations of BOA causing 50% growth inhibition and control treatment exposure levels using vertical plate seedling assays. One glucosyltransferase insertion mutant in particular exhibited more than a 50-fold increase in sensitivity to the allelochemical. Chemical analyses revealed a dramatic reduction in the accumulation of the metabolite BOA-6-O-glucoside in root tissues of the mutant compared with wild type plants, indicating a deficiency in the ability to detoxify BOA via O-glycosylation. These results strongly suggest an involvement of the corresponding glucosyltransferase enzyme in BOA detoxification. Thus, a gene for this enzyme could be used to reduce adverse crop responses to benzoxinoid allelochemicals, thereby improving crop growth and yield.

2. Manuka oil, a green weed management product with preemergence systemic herbicidal activity. Unlike the management of other pests, the control of weeds with green products is very problematic because farmers have few green products available. Most of these are essential oils with burn-down effects that require multiple applications of relatively concentrated solutions (10% or more). Nonetheless, the rationale for a natural products approach to herbicide discovery rests on the fact that there is generally little overlap between the mode of action of natural and synthetic phytotoxins. ARS scientists at Oxford, MS, have discovered that manuka oil, a triketone-rich essential oil, has both pre- and post-emergent herbicidal activity, which is particularly potent against grass weeds. This oil is rich in natural ß-triketones that inhibit the enzyme p-hydrophyphenylpyruvate dioxygenase, a key enzyme in plastoquinone synthesis. This oil can be applied as a foliar herbicide or to soils and cause growth inhibition via inhibition of this target site. Quantitative structure activity relationship analysis and molecular modeling highlighted the importance of a lipophilic side chain for herbicidal activity. The primary active ß-triketone in the oil is leptospermone, and its structure enables a systemic effect, being absorbed through the roots and translocated to the apical meristems where it exerts its herbicidal action. This research has received the interest of industry and the work has been expanded under the terms of a CRADA with a leader in the production and commercialization of green pest management products. The company is evaluating the potential synergism of the natural essential oil in combination with their commercial products.

Review Publications
Sumiyanto, J., Dayan, F.E., Cerdeira, A., Wang, Y., Khan, I.A., Moraes, R.M. 2012. Oligofructans content and yield of yacon (Smallanthus sonchifolius) cultivated in Mississippi. Scientia Horticulturae. 148:83-88.

Owens, D.K., Nanayakkara, D.N., Dayan, F.E. 2013. In planta mechanism of action of leptospermone: Impact of its physico-chemical properties on uptake, translocation, and metabolism. Journal of Chemical Ecology. 39:262-270.

Duke, S.O. 2012. Glyphosate metabolism in plants. Book Chapter. pp.17-38.

Carvalho, L., Alves, P., Duke, S.O. 2013. Hormesis with glyphosate depends on coffee growth stage. Annals of the Brazilian Academy of Science. 85(2):813-821.

Duke, S.O., Bajsa, J., Pan, Z. 2013. Omics methods for probing the mode of action of natural phytotoxins. Journal of Chemical Ecology. 39:333-347.

Weston, L.A., Alsaadawi, I.S., Baerson, S.R. 2013. Sorghum allelopathy – from ecosystem to molecule. Journal of Chemical Ecology. 39:142-153.

Franco, D.A., Almeida, S., Cerdeira, A.L., Duke, S.O., Moraes, R.M., Lacerda, A., Matallo, M.B. 2012. Evaluation of glyphosate application on transgenic soybean and its relationship with shikimic acid. Planta. 30(3):659-666.

Salas, R.A., Dayan, F.E., Pan, Z., Watson, S.B., Dickson, J.W., Scott, R.C., Burgos, N.R. 2012. ESPS gene amplification endows resistance to glyphosate in Italian ryegrass (Lolium perene ssp multiflorum) from Arkansas, USA. Pest Management Science. 68:1223-1230.

Wang, R., Staehelin, C., Dayan, F.E., Song, Y., Si, Y., Zeng, R. 2012. Simulated acid rain alters litter decomposition and enhances the allelopathic potential of the invasive plant Wedelia trilobata (Creeping Daisy). Weed Science. 60:462-467.

Duke, S.O., Lydon, J., Koskinen, W.C., Moorman, T.B., Chaney, R.L., Hanmerschmidt, R. 2012. Glyphosate effects on plant mineral nutrition, crop rhizosphere microbiota, and plant disease in glyphosate-resistant crops. Journal of Agricultural and Food Chemistry. 60:10375-10397.

Sondhia, S., Duke, S.O., Green III., S., Gemejiyeva, N.G., Mamonov, L.K., Cantrell, C.L. 2012. Phytotoxic furanocoumarins from the shoots of Semenovia transiliensis Regel & Herder. Natural Product Communications. 7(10):1327-1330.

Nandula, V.K., Ray, J.D., Ribeiro, D.N., Pan, Z., Reddy, K.N. 2013. Glyphosate resistance in tall waterhemp (Amaranthus tuberculatus) from Mississippi is due to both altered target-site and nontarget-site mechanisms. Weed Science. 61:374-383.

Li, S., Liu, L., Zhuang, X., Yu, Y., Liu, X., Cui, X., Ji, L., Pan, Z., Cao, X., Mo, B., Zhang, F., Raikhel, N., Jiang, L., Chen, X. 2013. MicroRNAs inhibit the translation of target mRNAs on the endoplasmic reticulum in Arabidopsis. Cell. 153(3):562-574.

Last Modified: 05/24/2017
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