Progress 10/26/15 to 10/25/20
Outputs
Progress Report 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 biochemical 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. 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. 1.1. Discover new and existing natural products for potential use as herbicides and bioherbicides. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. 1.3. Develop natural products as new weed management tools. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals. 2.1. Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. 2.2. The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone. 2.3. Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. 2.4. Engineering de novo sorgoleone biosynthesis in non-producing host plants. 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. This is the final report for this project; therefore, it includes highlights from the entire 5 year period as well as information for the current year. This project will be replaced with a new project, "Natural Product-Based Bioherbicides" pending completion of the National Program 304 research review. Significant progress on discoveries of natural products as potential herbicides were made over the past five years. A large number of natural products from plants, insects, and fungi were characterized. Many of these compounds are new compounds or are known natural compounds that had not been previously determined to be phytotoxic. The major natural compounds or derivatives discovered to have significant phytotoxicity include khellin, visnagin, curvularin, alfa,beta-dehydrocurvularin, saponins, pyrichalasin, cleistanthane terpenoids, triterpene novel lignans, new cassane diterpenoids, cytochalasins, confertin, etc. Khellin and visnagin are the subject of a patent filed on the use of these compounds as natural herbicides. Omics methods were used to probe the mode of action of serval phytotoxins. For example, proteomics was used to study the mode of action of the potent insect-derived phytotoxin cantharidin. RNA-seq was used to probe the mode of action of t-chalcone and citral. In silico binding studies were also used to determine that citral inhibits single-stranded DNA binding proteins. The mode of action of romidepsin and spliceostatin C were determined to be inhibition of histone deacetylase and spliceosome function. The roles of three amino acids (histidine, tryptophan, and cysteine) were implicated in the mode of action of the allelochemical 2- benzoxazolinone. Several contributions to the understanding of plant/ plant allelopathy were made. For example, the chemistry and biological activity of triketone allelochemicals were further explored. The transcriptional responses to chemical cues from a weed (barnyard grass) of genes of the gene cluster for synthesis of the allelochemical momilactone B in rice were characterized. Three genes in the cluster were upregulated in the presence of barnyard grass. With the successful characterization and identification of a cytochrome P450 enzyme (CYP71AM1) from sorghum, all of the genes required for sorgoleone biosynthesis beginning with the C16:1 fatty acid now been identified by ARS researchers in Oxford, Mississippi. Thus, the primary tools required to engineer the production of sorgoleone in transgenic crops are now in hand for creating the potential to generate novel varieties with reduced reliance on synthetic herbicides. As a proof of concept, a multi-gene vector was constructed which contains the complete open reading frames for desaturases (DES2, DES3), alkylresorcinol synthase (ARS2), O-methyltransferase (OMT3), and cytochrome P450 enzyme (CYP71AM1) driven by the constitutive promoters to direct the production of sorgoleone. When tested in a Nicotiana benthamiana transient assay system using this vector, sorgoleone was detected on the leaves expressing these genes, indicating that the expression of these sorghum genes simultaneously is capable of producing sorgoleone. One of the major goals has been the development of technologies that could confer the ability to conduct de novo biosynthesis of sorgoleone to root hair cells of non-producing crop species. Toward this aim, four different promoters with 3⿿-flanking region combinations capable of directing high-level root hair-specific expression were successfully characterized, with the potential to deliver recombinant gene products to root hair cells at significantly higher levels. The tissue specificity and activity of these transcriptional elements were confirmed by extensive promoter:reporter quantitative fluorimetric and histochemical analyses of beta- glucuronidase. These root hair-specific promoter/3⿿-flanking region combinations were subsequently used for the assembly of second-generation transgene cassettes for high-level expression of sorgoleone biosynthetic enzymes in root hairs. Preliminary experiments have also been recently initiated to analyze the ability of these root hair-specific transgene cassettes to direct the synthesis of sorgoleone in stably transformed rice plants, and these experiments are ongoing and will be continued in the new project cycle. Towards achieving Objective 1 during FY 2020, 130 of extracts and pure compounds were tested. Diplopyrone is a phytotoxin originated from the fungus Diplodia mutila. The herbicidal activity of diplopyrone analogues derived from a pyranopyran core was evaluated using a primary ranking bioassay, a commonly used method for preemergence test, against bentgrass and lettuce. Two compounds (pyranopyran nitrile and pyranopyran alkyne) demonstrated significant phytotoxic potential against lettuce. All tested pyranopyrans have a wide range of activity against monocot species. Although pyranopyran nitrile is less active than pyranopyran alkyne as a preemergence agent against the monocot bentgrass, its herbicidal activity is greater than pyranopyran alkyne using postemergence bioassay on the monocot duckweed. Among the tested compounds, the phytotoxicity of pyranopyran amide, desmethyldiplopyrone and desmethyldiplopyrone acetate is insignificant, similar to the previously reported results on diplopyrone derivatives. However, pyranopyran alkyne analog showed potent activity against bentgrass with IC50 = 38.1 µM, which offers a promising potential for further development. To evaluate the diplopyrone analog pyranopyran nitrile further, experiments in greenhouse setting were carried out. Four plant species, velvetleaf, field bindweed, crabgrass, and barnyardgrass were selected for spray experiments. Leaf tissues of both monocot species (crabgrass and barnyardgrass) developed signs of injures within 24 hours after application (1 day after treatment - DAT), which then turned into necrotic brown lesions at 4 DAT. Leaves of dicot species (velvetleaf and field bindweed) showed little or no signs of damage. However, necrosis spots were observed, possibly a result of direct contact of plant tissue with droplets of sprayed solution. This finding suggests that pyranopyran nitrile may act in a similar manner as a contact herbicide. Azoles are a group of five-membered heterocyclic compounds wildly used in agriculture and medicine. Thirty compounds that belong to the class of azoles were tested and almost all of them exhibited very satisfying herbicidal activity by inhibiting the germination of bentgrass (Agrostis stolonifera) and lettuce (Lactuca sativa). As a highly effective group of fungicides, azoles target a class of cytochrome P450 enzymes (CYP51), for example, lanosterol 14a-demethylase. It has been reported that azoles also inhibit plant CYP51 enzymes leading to the reduction of plant sterol hormones brassinosteroids (BR). Seeds of transgenic Arabidopsis overexpressing CYP51 from Fragaria vesca were obtained for further studying the mode of action of azole compounds. For Objective 2, characterization of transformed rice plants expressing the sorgoleone biosynthetic pathway was conducted. These transgenic rice plants were generated previously using a multi-gene vector constructed in our unit via agrobacterium-mediated plant transformation performed at the Iowa State University transformation laboratory in Ames, Iowa. In collaboration with ARS researchers in Oxford, Mississippi, an high- performance liquid chromatography-ultraviolet (HPLC-UV) based detection method was developed for the detection of sorgoleone in transgenic seedling root samples, and in addition, a novel soil-free in vitro propagation system was developed and successfully utilized which optimized root hair production in seedling root tissues. In addition to the transformation work performed at Iowa State, a second multi-gene vector was designed and is currently being utilized by the University of Wisconsin (UW) Crop Innovation Center for the generation of transgenic corn, wheat, and soybean plants. The UW Crop Innovation center is also actively working on the generation of transgenic sorghum plants with enhanced sorgoleone content, utilizing vectors containing root hair- specific gene promoters driving the expression of ARS1, a rate-limiting enzyme which catalyzes the formation of the 5-pentadecatrienyl resorcinol sorgoleone pathway intermediate. The root hair-specific gene promoters used for the above-described transformation experiments were isolated from S. bicolor by our research unit and were mentioned in a previous annual report. The transgenic plant screens performed to date have utilized total root tissues from seedlings grown in vitro under conditions which optimize root hair production, as the direct analysis of isolated root hairs is not amenable to high-throughput approaches, and the number of seeds obtained from individual R0 transformants is generally not sufficient for performing established methods for root hair isolation from sorghum. To address this problem, we have recently developed a novel small-scale root hair isolation procedure which we have successfully tested with both sorghum and rice 10-day old seedlings grown in vitro. The new method enables biochemical and molecular analyses of highly-purified root hair tissues, starting with as few as 100 R1 transgenic seedlings. This small- scale root hair isolation procedure will dramatically improve the sensitivity of our assays, and greatly enhances our ability to detect sorgoleone accumulation specifically occurring in root hairs of transgenic seedlings. We anticipate that positive outcomes from these efforts will represent major breakthroughs in the plant-produced biopesticide field, resulting in the generation of novel germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests.
Impacts
(N/A)
Publications
- Duke, S.O. 2019. Enhanced metabolic degradation: The last evolved Glyphosate resistance mechanism of weeds? Plant Physiology. 181:1401-1403.
- Yu, W., Zhai, Z., Min, L., Wedge, D.E., Duke, S.O., Wu, H., Weng, J., Tan, C., Zhang, Y., Liu, X. 2019. Synthesis and biological activity of novel 1, 3,4-oxadiazole derivatives containing a pyrazole moiety. Research on Chemical Intermediates. 45:5989-6001.
- Gu, C., Xia, X., Lv, J., Tan, J., Baerson, S.R., Pan, Z., Song, Y., Zeng, R. 2019. Diterpenoids with herbicidal and antifungal activities from hulls of rice (Oryza sativa). Fitoterapia. 136:104183.
- Duke, S.O. 2020. Glyphosate: Environmental fate and impact. Weed Science. 68(3):201-207.
- Dayan, F.E., Duke, S.O. 2020. Discovery for new herbicide sites of action by quantification of plant primary metabolite and enzyme pools. Engineering. 6:509-514.
- 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.
- Grana, E., Diaz-Tielas, C., Sanchez-Moreiras, A.M., Reigosa, M.J., Celeirao, M., Abagyan, R., Teijeira, M., Duke, M.V., Clerk, T., Pan, Z., Duke, S.O. 2019. Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins. Physiologia Plantarum. 169(1) :99-109.
- Bajsa Hirschel, J.N., Pan, Z., Duke, S.O. 2020. Rice momilactone gene cluster: Transcriptional response to Barnyard grass (Echinochloa crus- galli). Molecular Biology Reports. 47:1507-1512.
- Cerveira, W.R., Santos Da Costa, Y.K., Carbonari, C., Duke, S.O., Aguiar Alves, P., Bianco De Carvalho, L. 2020. Growth, morphological, metabolic, and photosynthetic responses of clones of eucalyptus to glyphosate. Forest Ecology and Management.
- Kepler, R., Epp Schmidt, D.J., Yarwood, S.A., Cavigelli, M.A., Buyer, J.S., Duke, S.O., Reddy, K.N., Williams, M., Bradley, C.A., Maul, J.E. 2020. Soil microbial communities in diverse agroecosystems exposed to glyphosate. Applied and Environmental Microbiology.
- Owens, D.K., Bajsa Hirschel, J.N., Duke, S.O., Carbonari, C.A., Gomes, G.L. , Asolkar, R., Boddy, L., Dayan, F.E. 2020. The contribution of Romidepsin to the herbicidal activity of Burkholderia rinojensis biopesticide. Journal of Natural Products. 83(4):843-851.
- Perera, W.H., Meepagala, K.M., Wedge, D.E., Duke, S.O. 2020. Sesquiterpenoids from culture of the fungus Stereum complicatum (Steraceae) : Structural diversity, antifungal, and phytotoxic activities. Phytochemistry Letters. 37:51-58.
- Nocera, P., Bajsa Hirschel, J.N., Masi, M., Ross, S.A., Cantrell, C.L., Duke, S.O., Surico, G., Evidente, A. 2020. Secondary metabolites of Thymelaea hirsuta, a plant collected from the Sicilian Island of Lampedusa. Natural Product Research.
- Liu, X., Yu, W., Min, L., Wedge, D., Tan, C., Weng, J., Wu, H., Cantrell, C.L., Bajsa Hirschel, J.N., Hua, X., Duke, S.O. 2020. Synthesis and pesticidal activities of new Quinoxalines. Journal of Agricultural and Food Chemistry. 68:7324-7332.
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Progress 10/01/18 to 09/30/19
Outputs
Progress Report 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 biochemical 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. 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. 1.1. Discover new and existing natural products for potential use as herbicides and bioherbicides. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. 1.3. Develop natural products as new weed management tools. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals. 2.1. Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. 2.2. The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone. 2.3. Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. 2.4. Engineering de novo sorgoleone biosynthesis in non-producing host plants. 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. Towards achieving Objective 1, the following research was performed in FY 2019 and milestones have been met: Determination of the mode of action of spliceostatin C. Spliceostatin C (sp C) is an active component of bioherbicide MBI-014 isolated from soil bacterium Burkholderia rinojensis. The chemical structure of this natural product is related to spliceostatin A (sp A) which was characterized as an anticancer agent and splicing inhibitor. The comprehensive study included phytotoxic activity and mode of action analysis of this natural product. Spliceostatin C also significantly inhibited the growth of Arabidopsis thaliana seedlings and triggered leaf bleaching, with growth IC50 of 2.2 µM. To elucidate the inhibitory functions of sp C in planta, 7-day-old Arabidopsis seedlings were treated with sp C at the concentration of IC50, and RNAs were extracted for semiquantitative RT- PCR (RT-sqPCR) analysis. Among 20 genes that were selected for the assays, five transcripts (tubulin alpha-5, mRNA splicing factor, SF3b14b, flowering focus M and circadian clock associated 1) underwent intron rearrangements such as intron retention and alternative 5⿿ or 3⿿ splicing side upon exposure to sp C. The results obtained from RT-sqPCR confirmed that spliceostatin C inhibits plant spliceosome catalysis. To investigate the impact of sp C on gene expression further, global proteome profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed. After exposure to sp C for 6 h, 145 protein isoforms were identified with fold changes greater than 1.5 (p = 0.05), and, among them, 134 were decreased and 11 increased. KEGG pathway analysis revealed that these proteins are associated with metabolic pathways, carbon metabolism, the ribosome, and secondary metabolite pathways. Further analysis of these proteins could provide insight into the mechanisms of phytotoxicy of sp C to the plant cell. Identification of a Natural Product-Based Potentiator of Echinocandin Class Fungicides. Cell wall-targeting echinocandin fungicides such as caspofungin are highly potent, yet lack efficacy against some fungal species due to their propensity to induce pathogen resistance. One potential strategy to address this limitation is to combine echinocandins with a potentiating compound that improves their activity by disrupting cellular adaptation pathways. In collaboration with University of Mississippi investigators, we identified the marine sponge-derived sesquiterpene quinone puupehenone (PUUP) as a potentiator of the echinocandin fungicide caspofungin (CAS) in CAS-resistant fungal pathogens. Using RNA-Seq analysis, coupled with genetic and molecular studies in the model organism S. cerevisiae, we determined that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2, the terminal MAP kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its co-chaperone Cdc37. Through these experiments we⿿ve demonstrated that PUUP likely potentiates echinocandin fungicide activity via the disruption of Hsp90 activity and the CWI pathway. The identification of a novel natural product-based potentiator of echinocandin fungicides has important implications for the development of novel approaches for the control of pathogenic fungi. Bioassay-guided isolation of phytotoxic compounds was accomplished from several sources. For example, confertin and salsolol A and B from Ambrosia salsola had IC50 values of 261, 275, and 251 µM, respectively, against duckweed. New sesquiterpenoids were isolated from the fungus Stereum complicatum and some of these compounds were moderately toxic to duckweed. The microbial broth of Burkholderia rinojensis strain A396 is herbicidal to a number of weed species with greater observed efficacy against broadleaf than grass weeds. A portion of this activity is attributed to romidepsin, a 16-membered cyclic depsipeptide bridged by a 15-membered macrocyclic disulfide. Romidepsin, which is present in small amounts in the microbial broth, was isolated and purified using standard chromatographic techniques. We established that pure romidepsin is a natural proherbicide that targets the activity of plant histone deacetylases (HDAC). Molecular dynamic simulation of the binding of romidepsin to atHDAC19 indicated the reduced form of the compound could reach deep inside the catalytic domain and interact with an associated zinc atom required for enzyme activity. For Objective 2, milestones have been met and significant progress has been made. Manipulation of Sorgoleone Biosynthesis in Planta - The allelochemical sorgoleone likely plays a major role in the sorghum plant⿿s natural ability to fend off weed infestations, and also represents a promising natural product-based alternative to synthetic herbicides. Previously, our research unit successfully completed a long term effort to clone all of the genes required for the biosynthesis of sorgoleone from the ubiquitous precursor palmitoleoyl-CoA, thus providing us with the necessary tools for manipulating sorgoleone production in planta. Our goals for this work have included transferring the entire biosynthetic pathway to other crops, as well as the development of enhanced sorghum germplasm with increased sorgoleone content. We reported on the generation of transgenic rice plants containing the complete sorgoleone biosynthetic pathway in our 2018 annual report, and have now developed and utilized high-throughput methods to screen the transgenic rice events using qRT-PCR and GC-MS. It was also necessary for this screening work to devise an in vitro culture approach for increasing root hair production, which was accomplished by 1-aminocyclopropane-1-carboxylic acid (ACC) addition to the plant growth media, and the design of polypropylene mesh supports in the culture vessels to increase root biomass. We are currently nearing completion of our transgenic rice seedling screening efforts, and we will also be initiating similar experiments in other crops this FY, including corn, wheat, and soybean. We have also initiated experiments for generating transgenic sorghum with increased sorgoleone content this FY by using root hair-specific desaturase and glutathione-s-transferase gene promoters driving the expression of ARS1, which encodes an enzyme required for the production of the (rate limiting) 5-pentadecatrienyl resorcinol sorgoleone pathway intermediate. The root hair-specific desaturase and glutathione-s- transferase gene promoters we used were also isolated by our research unit and were mentioned in a previous annual report. We anticipate that positive outcomes from these efforts will represent major breakthroughs in the plant-produced biopesticide field, resulting in the generation of novel germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests. NP / Component Coding: 304 2 B 2008
Impacts
(N/A)
Publications
- Duke, S.O., Owens, D.K., Dayan, F.E. 2019. Natural product-based chemical herbicides. In: Weed Control: Sustainability, Hazards and Risks in Cropping Systems Worldwide. Korres, N.E, Burgos, N.R and Duke, S.O. Taylor and Francis. pp. 153-165.
- Duke, S.O., Reddy, K.N. 2018. Is mineral nutrition of Glyphosate-resistant crops altered by Glyphosate treatment? Outlooks on Pest Management. 29(5) :206-208.
- Duke, S.O., Dayan, F.E. 2018. Herbicides. In eLS. John Wiley & Sons, Ltd: Chichester.
- Mu, J., Zhai, Z., Tan, C., Weng, J., Wu, H., Duke, S.O., Zhang, Y., Liu, X. 2019. Synthesis and herbicidal activity of 1,2,4-triazole derivatives containing a pyrazole moiety. Journal of Heterocyclic Chemistry.
- Korres, N., Burgos, N., Travlos, I., Vurro, M., Gitsopoulos, T., Varanasi, V., Duke, S.O., Kudsk, P., Brabham, C., Rouse, C., Salas-Perez, R. 2019. New directions for integrated weed management: Modern technologies, tools and knowledge discovery. Advances in Agronomy.
- Duke, S.O., Powles, S.B., Sammons, R. 2018. Glyphosate ⿿ how it became a once in a hundred year herbicide and its future. Outlooks on Pest Management. 29:247-251.
- Tan, L., Wang, M., Kang, Y., Azeem, F., Zhou, Z., Tuo, D., Preciado-Rojo, L., Khan, I.A., Pan, Z. 2018. Biochemical and functional characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L. Molecules. 23(11) :2876-2896.
- Kumarihamy, M., Ferreira, D., Croom, E.M., Sahu, R., Tekwani, B.L., Duke, S.O., Khan, S., Techen, N., Nanayakkara, N.D. 2019. Antiplasmodial and cytotoxic cytochalasins from an endophytic fungus, Nemania sp. UM10M, isolated from a diseased torreya taxifolia leaf. Molecules. 24(4):777.
- Duke, S.O., Stidham, M.A., Dayan, F.E. 2018. A novel approach to herbicide and herbicide mode of action discovery. Pest Management Science.
- 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.
- Maroli, A.S., Gaines, T.A., Foley, M.E., Duke, S.O., Dogramaci, M., Anderson, J.V., Horvath, D.P., Chao, W.S., Tharayil, N. 2018. OMICS in weed science research: A perspective from genomics, transcriptomics and metabolomics approaches. Weed Science.
- Nandula, V.K., Riechers, D.E., Ferhatoglu, Y., Barrett, M., Duke, S.O., Dayan, F.E., Goldberg-Cavalleri, A., Tetard-Jones, C., Wortley, D.J., Onkokesugn, N., Brazier-Hicks, M., Edwards, R., Gaines, T., Iwakami, S., Jugulam, M., Ma, R. 2019. Herbicide metabolism: Crop selectivity, bioactivation, weed resistance mechanisms, and regulation. Weed Science. 67:149-175.
- 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.
- Costa, F.R., Rech, R., Duke, S.O., Carvalho, L.B. 2018. Lack of effects of glyphosate and glufosinate on growth, mineral content, and yield of glyphosate- and glufosinate-resistant maize. GM Crops & Food.
- Diaz-Tielas, C., Grana, E., Sanchez-Moreiras, A.M., Reigosa, M.J., Vaughn, J.N., Pan, Z., Bajsa Hirschel, J.N., Duke, S.O. 2019. Transcriptome responses to the phytotoxin t-Chalcone in Arabidopsis thaliana L. Pest Management Science.
- Reddy, K.N., Cizdziel, J.V., Williams, M., Maul, J.E., Rimando, A.M., Duke, S.O. 2018. Glyphosate resistance technology has minimal or no effect on maize mineral content and yield. Journal of Agricultural and Food Chemistry. 66:10139-10146.
- Lazzara, N., Giovine, M., Vagadia, P., Rosano, R., Piro, N., Kassel, S., Boyko, W., Zubris, D., Schrader, K., Wedge, D.E., Duke, S.O., Giuliano, R. 2018. Synthesis and biological evaluation of 6-[(1R)-1-hydroxyethyl]-2, 4a(R),6(S),8a(R)-tetrahydropyrano-[3,2-b]-pyran-2-one and structural analogs of the putative structure of Diplopyrone. Journal of Organic Chemistry.
- 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.
- Wu, X., Lin, X., Baerson, S.R., Ding, C., Zhang, L., Wu, C., Song, Y., Zeng, R. 2018. The roles of jasmonate (JA) signaling in nitrogen uptake and allocation in rice (Oryza sativa L.). New Phytologist.
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Progress 10/01/17 to 09/30/18
Outputs
Progress Report 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 biochemical 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. 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. 1.1. Discover new and existing natural products for potential use as herbicides and bioherbicides. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues and physiological evaluations. 1.3. Develop natural products as new weed management tools. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals. 2.1. Complete the characterization of the gene products of putative genes for enzymes of the sorgoleone biosynthetic pathway. 2.2. The use of sorghum transformants possessing altered sorgoleone levels to investigate the ecophysiological role of sorgoleone. 2.3. Identification of plant promoters to facilitate root hair-specific metabolic engineering of sorgoleone biosynthesis. 2.4. Engineering de novo sorgoleone biosynthesis in non-producing host plants. 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. A series of compounds related to a natural phytotoxin were evaluated for potential as herbicides. One of these had unusually high phytotoxicity. An invention disclosure was filed. Results of these studies are confidential until a patent is filed. 2-Benzoxazolinone (BOA) is a phytotoxic compound that induces strong effects on plant metabolism. BOA effects include increased membrane permeability, degradation of proteins and pigments, increased lipid peroxidation, oxidative stress and senescence induction. In this work, the effects of amino acid supplementation on BOA action were analyzed. Roles of histidine, tryptophan and cysteine in the mode of action of BOA action were proposed from their protective effects. The mode of action of the terpenoid phytotoxin citral was probed in Arabidopsis thaliana with RNA-Seq transcriptome analysis. The concentration of citral that inhibited growth by 50% (202 �M) down regulated transcription of 9156 and 5541 genes in roots and shoots, respectively, at the p = 0.001 level after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the roots, these effects dissipated after 3 and 6 h, but similar effects began again at 12 and continued until 24 h. In the shoots, the down regulation increased at 3 h (6239 genes downregulated, vs. 66 upregulated) with the effect decreasing at from 6 to 24 h, with only 1 gene downregulated at 24 h. Of all genes affected in roots at 1 h and shoots at 3 h (times of greatest effect), 6.5 and 9.2 %, respectively, of affected genes were for DNA and RNA binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly down downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at 12, and 24 h. Citral docking calculations for WHY1, WHY2, and WHY3 crystal structures and the SSBP homology model predicted strong binding in the hydrophobic pockets of these proteins. Such an effect could account for the profound and unusual effects on downregulation of gene transcription. New cassane-like diterpenes from Eragrostis plana were found to be moderately phytotoxic. An invention disclosure was filed. A series of triazole derivatives containing a pyrazole moiety were synthesized and characterized by 1H nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). Most of the tested compounds were moderately herbicidal to lettuce and bentgrass. Indirect evidence was via transcriptome and biochemical methods to link the mode of action of the natural phytotoxin t-tchalcone to inhibition of tyrosine metabolism. Hymnoclea salsola, a weed species that is found in the arid regions of Utah, Texas and Arizona was investigated in search of phytotoxic compounds. Confertin and several analogs of confertin and two novel chalcones were found to be phytotoxic. These compounds are being evaluated further. Bioassay guided isolation of an ethyl acetate extract from an Amryis species led to isolation and identification of a novel phytotoxic furanocoumarin. Palmitoleoyl-CoA is found in virtually every plant cell type due to its central role in plant fatty acid and lipid metabolism. Therefore in principle, the transfer of the sorgoleone biosynthetic pathway genes DES2, DES3, ARS1/2, OMT3, and CYP71AM1 to any plant cell could result in the accumulation of dihydrosorgoleone, which upon exposure to oxygen will spontaneously form the benzoquinone sorgoleone. Furthermore, the expression of these genes specifically in root hair cells is likely prerequisite to the successful deployment of sorgoleone as a bioherbicide in other crop plants. Toward this end, our research unit previously identified and characterized highly specific gene promoters to direct the root hair-specific expression of transgenes in plants, and as mentioned we have also isolated all of the genes required for the biosynthesis of the potent bioherbicide sorgoleone starting from palmitoleoyl-CoA. Recently, we have constructed five transgene cassettes containing the complete open reading frames of S. bicolor DES2, DES3, ARS1, OMT3, and CYP71AM1, which also feature the root-hair specific gene promoters our research unit has identified. These five transgene cassettes were all positioned within a single binary transformation vector, which was then sent to the Iowa State University Plant Transformation Facility and used for the generation of transgenic rice events. R1 seed were produced in our research unit's greenhouse from the primary transformants generated by Iowa State University, and multiple tranformant lines are currently being evaluated by our research unit to determine if a functional sorgoleone biosynthetic pathway has been successfully transferred to rice. A positive outcome from these efforts will represent a major breakthrough in the plant-produced biopesticide field, and the generation of novel rice germplasm possessing enhanced resistance to weed infestations, and potentially other agronomic pests as well. Accomplishments 01 Completion of biochemical and genetic characterization of the sorgoleone biosynthetic pathway. ARS researchers at Oxford, Mississippi, goal of identifying and characterizing the genes and enzymes required for the biosynthesis of the potent bioherbicide sorgoleone is now completed with the characterization of a root-hair specific cytochrome P450 from S. bicolor genotype BTx623. The cytochrome P450 protein was determined to belong to the CYP71 enzyme subfamily and has been formally designated S. bicolor CYP71AM1. Biochemical characterization of recombinant CYP71AM1 using both yeast (S. cerevisiae) and a Nicotiana benthamiana transient expression system demonstrated that CYP71AM1 is capable of performing dihydroxylation on the proposed physiological substrate 3-methyl-5-pentadecatrienyl resorcinol, yielding dihydrosorgoleone. These results clearly demonstrate a role for CYP71AM1 in the biosynthesis of sorgoleone in Sorghum bicolor, and represent the first example of a cytochrome P450 enzyme capable of catalyzing a dihydroxylation reaction occurring on a resorcinolic ring structure. Previously, our research team successfully identified the fatty acid desaturases, alkylresorcinol synthases, and an O-methyltransferase participating in the sorgoleone biosynthetic pathway. The characterization of CYP71AM1 represents a highly significant milestone, as our research unit has now characterized the genes and enzymes required for all of the biosynthetic steps leading to the formation of dihydrosorgoleone, beginning with palmitoleoyl-CoA. With this accomplishment we now have the tools in hand to engineer the production of sorgoleone in other crop species, which could potentially lead to the generation of novel germplasm possessing enhanced resistance to weed competition, and potentially against other agronomic pests as well.
Impacts
(N/A)
Publications
- Heap, I., Duke, S.O. 2017. Overview of glyphosate-resistant weeds worldwide. Pest Management Science. 74:1040-1049. doi 10.1002/ps.4760.
- Pan, Z., Baerson, S.R., Wang, M., Bajsa Hirschel, J.N., Rimando, A.M., Wang, X., Nanayakkara, N., Noonan, B.P., Fromm, M.E., Dayan, F.E., Khan, I. , Duke, S.O. 2018. A cytochrome P450 CYP71 enzyme expressed in S. bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone. New Phytologist. doi: 10.1111/nph.15037.
- Belz, R.G., Duke, S.O. 2018. Predicting hormesis in mixtures of herbicidal compounds � where are we and how far can we go? Julius K�hn Archiv. 458:161-167.
- Labruzzo, A., Cantrell, C.L., Carrubba, A., Ali, A., Wedge, D.E., Duke, S. O. 2018. Phytotoxic Lignans from Artemisia arborescens. Natural Product Communications. 13(3):237-240.
- Lu, X., Zhang, J., Xu, M., Li, R., Liu, B., Rodriguez-Romero, J., Luo, D., Pan, Z., Baerson, S.R., Li, Z., Sesma, A., Yang, B., Peters, R.J. 2018. Inferring roles in defense from metabolic allocation of rice diterpenoids. The Plant Cell. 30:1119-1131.
- Morimoto, M., Cantrell, C.L., Khan, S., Tekwani, B.L., Duke, S.O. 2017. Antimalarial and antileishmanial activities of phytophenolics and their synthetic analogues. Chemistry and Biodiversity. DOI: 10.1002/cbdv. 201700324.
- Sanchez-Moreiras, A.M., Pedrol, N., Duke, S.O. 2018. Influence of amino acids on the phytotoxicity of 2-benzoxazolinone on Lemna paucicostata. Journal of Allelochemical Interactions. 4(1):33-39.
- Wang, R., Liu, S., Baerson, S.R., Qin, Z., Ma, Z., Su, Y., Zhang, J. 2018. Identification and functional analysis of a novel cytochrome P450 gene CYP9A105 associated with pyrethroid detoxification in Spondoptera exigua Hubner. International Journal of Molecular Sciences. 19:737.
- Westwood, J.H., Charudattan, R., Duke, S.O., Finnimore, S.A., Marrone, P., Slaughter, D.C., Swanton, C., Zollinger, R. 2018. Weed management in 2050: Perspective on the future of weed science. Weed Science. 66:275-285.
- Maroli, A.S., Nandula, V.K., Duke, S.O., Gerard, P., Tharayil, N. 2017. Comparative metabolomic analyses of two Ipomoea lacunosa biotypes with contrasting glyphosate tolerance elucidates glyphosate-induced differential perturbations in cellular physiology. Journal of Agricultural and Food Chemistry. (66):2027-2039.
- Meepagala, K.M., Briscoe, W.E., Techen, N., Johnson, R.D., Clausen, B.M., Duke, S.O. 2017. Isolation of a phytotoxic isocoumarin from Diaporthe eres- infected Hedera helix (English Ivy) and synthesis of its phytotoxic analogs. Pest Management Science. 74:37-45.
- Carvalho, L.B., Duke, S.O., Alves, P. 2018. Physiological responses of Eucalyptus x urograndis to glyphosate are dependent on the genotype. Scientia Forestalis. 46(118): 177-187.
- Duke, S.O. 2018. Interaction of chemical pesticides and their formulation ingredients with microbes associated with plants and plant pests. Journal of Agricultural and Food Chemistry. 66:7753-7561. DOI: 10.1021/acs.jafc. 8b02316.
- Duke, S.O., Pan, Z., Bajsa Hirschel, J.N., Sanchez-Moreiras, A.M., Vaughn, J.N. 2018. Use of omics methods to determine the mode of action of natural phytotoxins. American Chemical Society Symposium Series. 1294:33-46.
- Seiber, J.N., Coats, J., Duke, S.O., Gross, A.D. 2018. Pest management with biopesticides. Frontiers of Agricultural Science and Engineering. 5(3) :205-300.
- Beck, J.J., Duke, S.O., Rering, C.C. 2018. Roles of natural products for biorational pesticides in agriculture. ACS Symposium Series. 1294:1-4. doi:10.1021/bk-2018-1294.ch001.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report 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. 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. Objective 1. Pyrichalasin H was identified as the phytotoxic compound from the culture broth of the fungus Pyricularia grisea that was isolated from infected Brachiaria (B.) eruciformis (signal grass), a common weed in the state of Mississippi. Brachiaria eruciformis commonly known as Signal grass is a common weed in lawns and turfs in the state of Mississippi. From infected leaves of B. eruciformis showing necrosis a fungus was isolated and identified as Pyricularia (P.) grisea. P. grisea, Magnaporthe (M.) gisea and P. oryzae are closely related fungi that are responsible for crop losses in rice and cereals worldwide. P. grisea was grown in culture broth. The ethyl acetate extract of the culture broth was found to be phytotoxic. The phytotoxic constituent, isolated by bioassay guided isolation, was identified as pyrichalasin H. We have also shown for the first time that pyrichalasin H has antifungal activity against Colletotrichum species. This study provides not only further evidence that plant pathogenic fungi are good sources of herbicidal compounds but also chemical basis for the necrosis in infected leaves. A phytotoxic isochromene analog was identified from the culture broth of a fungus determined to be a member of the Phoma genus that was isolated from infected leaf of Malabar spinach (Basella alba), a popular green leafy vegetable native to tropical Asia. The compound was found to be phytotoxic. Two phytotoxic compounds (tyrosol and a compound having an isocoumarin core structure) were identified from the Diaporthe eres-infected leaf of Hedera helix (English Ivy) exhibiting necrosis. Isocoumarin analogs were synthesized, and two of the analogs were two- to three-fold more phytotoxic than the naturally occurring compound in a Lemna paucicostata growth bioassay. About 100 semisynthetic compounds from a visiting scientist were bioassayed, and several were found to have good herbicidal activity and one has a novel mode of action. Analysis of RNA sequence data from treatment of Arabidopsis thaliana with the allelochemical chalcone found a massive effect on the genes for enzyme of the phenylpropanoid pathway and tyrosine metabolism. Bioassay-guided isolation of phytotoxins from Artemisia arborescens found the two lignans, sesamin and ashantin, to account for most of the phytotoxicity. Other materials from several collaborators were evaluated for phytotoxicity. Details cannot be provided here because of confidentiality agreements. Objective 2. The characterization of transgenic sorghum plants expressing hairpin RNA (hpRNA) for repression of CYP71AM1 (RNA interference studies) was completed. Suppression of the expression of CYP71AM1 significantly reduced the production of sorgoleone. Preparation of the multi-gene constructs for co-expression of sorgoleone biosynthetic enzymes driven by sorghum root hair promoters is completed. Accomplishments 01 Phytotoxic compounds isolated from a rare plant in Brazil. Vellozia (V. ) gigantean is a rare, ancient, and endemic neotropical plant present in the Brazilian Rupestrian grasslands. The dichloromethane extract of the adventitious roots of V. gigantea was phytotoxic against Lactuca sativa, Agrostis stolonifera, and Lemna paucicostata. Phytotoxicity assay-directed fractionation of the extract revealed one new isopimaradiene compound and three new cleistanthane diterpenoids. The new isopimaradiene compound was especially phytotoxic with activity comparable to those of the commercial herbicides clomazone, s-ethyl dipropylthiocarbamate (EPTC), and naptalam. With this study, ARS researchers in Oxford, Mississippi, show that ancient and unique plants, like the endangered narrowly endemic neotropical species V. gigantea present in the Rupestrian grasslands, should also be protected because they can be sources of new bioactive compounds. 02 Visualization of sorgoleone spatial distribution using matrix assisted laser desorption/ionazation-mass spectrometric imaging (MALDI-MSI) on whole sorghum root systems. Prior work performed by our research unit as well as other laboratories has conclusively demonstrated that biosynthesis of the natural herbicide sorgoleone occurs primarily or exclusively within root hair cells of sorghum plants. There is limited information however concerning the fate of this compound once it is extruded from these cells. For this reason ARS researchers in Oxford, Mississippi, investigated the use of an emerging analytical technique in plants referred to as mass spectrometry imaging (MSI), an adaptation of mass spectrometry which facilitates direct visualization of the spatial distribution of chemical compounds on tissue sample surfaces. Working in collaboration with Iowa State University, we completed an imaging study using matrix-assisted laser desorption ionization MALDI- MSI on 10-day-old sorghum seedlings. These studies demonstrated that sorgoleone accumulation is not localized to the root hair zone, but rather appears to form a continuous layer coating the entire root system of young seedlings. This would be consistent with the notion that this compound could function as a type of chemical barrier, serving as a defensive interface between the root system and surrounding soil environment.
Impacts
(N/A)
Publications
- Carvalho, L.B., Duke, S.O., Messa, J.R., Regina Da Costa, F., Bianco, S. 2016. Plant growth responses of apple and pear trees to doses of glyphosate. Planta Daninha. 34(4):815-822.
- Travaini, M.L., Sosa, G.M., Ceccarelli, E.A., Walter, H., Cantrell, C.L., Carrillo, N.J., Dayan, F.E., Meepagala, K.M., Duke, S.O. 2016. Khellin and visnagin, furanochromones from Amni visnaga (L.) Lam., as potential bioherbicides. Journal of Agricultural and Food Chemistry. 64:9475-9487.
- Duke, S.O., Rimando, A.M., Reddy, K.N., Cizdziel, J.V., Bellaloui, N., Shaw, D.R., Williams, M., Maul, J.E. 2017. Lack of transgene and glyphosate effects on yield, and mineral and amino acid content of glyphosate-resistant soybean. Pest Management Science. doi:10.1002/ps.4625.
- Moreas, R.M, A.L. Cerdiera, S.O. Duke, F.E. Dayan, C.L. Cantrell, and S.C. N. Queiroz. 2016. Pesticidas Naturais Derivdos de Plantas: Descoberta et Usos (Natural Pesticides Derived from Plants: Discovery and Uses). In: Halfeld-Vieira, B.A, Marinho-Prado, J.S., Nechet, K.L., Morandi, M.A.B., Bettiol, W., editors. Defensivos Agr�colas Naturais: Uso e Perspectivas (Natural Agricultural Defenses: Use and Perspectives). Jaguari�a, Brazil: Emprapa Meio Ambiente. p. 505-541.
- Maroli, A.S., Nandula, V.K., Duke, S.O., Tharayil, N. 2016. Stable isotope resolved metabolomics reveals the role of anabolic and catabolic processes in glyphosate-induced amino acid accumulation in Amaranthus palmeri biotypes. Journal of Agricultural and Food Chemistry. 64:7040-7048.
- Nascentes, R.F., Carbonari, C., Simoes, P.S., Brunelli, M.C., Velini, E.D., Duke, S.O. 2017. Low doses of glyphosate enhance growth, CO2 assimilation, stomatal conductance and transpiration in sugarcane and eucalyptus. Pest Management Science. doi:10.1002/ps.4606.
- Duke, S.O. 2012. Why are there no new herbicide modes of action in recent years. Pest Management Science. 68:505-512.
- Bajsa, J.N., Pan, Z., Duke, S.O. 2011. Serine/threonine protein phosphatases: multi-purpose enzymes in control of defense mechanisms. Plant Signaling and Behavior. 6(12):1921-1925.
- Dayan, F.E., Owens, D.K., Duke, S.O. 2012. Rationale for a natural products approach to herbicide discovery. Pest Management Science. 68:519- 528.
- Bajsa, J.N., Pan, Z., Dayan, F.E., Owens, D.K., Duke, S.O. 2012. Validation of serine-threonine protein phosphatase as the herbicide target site of endothall. Journal of Pesticide Biochemistry and Physiology. 102(2) :38-44.
- Chen, Y., Li, J., Li, S., Zhao, J., Bernier, U.R., Becnel, J.J., Agramonte, N.M., Duke, S.O., Cantrell, C.L., Wedge, D.E. 2016. Identification and characterization of biopesticides from Acorus Tatarinowii and A. Calamus. American Chemical Society Symposium Series. 1218:121-143.
- Wu, X., Yu, Y., Liang, G., Baerson, S.R., Pan, Z., Song, Y., Ding, C., Wu, W., Niu, J., Zeng, R. 2017. Interactions between nitrogen and silicon in rice and their effects on resistance towards the brown planthopper Nilaparvata lugens. Frontiers in Plant Science. doi:10.3389/fpls.2017. 00028.
- Ye, M., Song, Y., Baerson, S.R., Long, J., Pan, Z., Lin, W., Zeng, R. 2017. Ratoon rice generated from primed parent plants exhibit enhanced herbivore resistance. Plant, Cell & Environment. 40:779-789. doi:10.1111/ poe.12897.
- Duke, S.O. 2017. The history and current status of glyphosate. Pest Management Science. doi:10.1002/ps.4652.
- La Hovary, C., Baerson, S.R., Williamson, J.D., Danehower, D.A., Ma, G., Pan, Z., Mask, M.M., Burton, J.D. 2016. Phytotoxicity and benzoxazinone concentration in field grown cereal rye (Secale cereale L.). International Journal of Agronomy. 2016:1-11. doi:10.1155/2016/6463826.
- Duke, S.O., Heap, I. 2017. Evolution of weed resistance to herbicides: What have we learned after 70 years? In: Jugulam, M., editor. Biology, Physiology and Molecular Biology of Weeds. Boca Raton, FL:CRC Press. p. 63- 86.
- Belz, R.G., Duke, S.O. 2017. Herbicide-mediated hormesis. In: American Chemical Society Symposium Series. 1249:135-148.
- Duke, S.O. 2017. Pesticide dose - a parameter with many implications. In: American Chemical Society Symposium Series. 1249:1-13.
- Ferreira, M.C., Cantrell, C.L., Duke, S.O., Ali, A., Rosa, L.H. 2017. New phytotoxic diterpenoids from Vellozia gigantea (Velloziaceae), an endemic neotropical plant living in the endangered Brazilian biome Rupestrian grasslands. Molecules. 22:175. doi:10.339/molecules22010175.
- Stavropoulou, M.I., Angelis, A., Aligiannis, N., Kalpoutzakis, E., Mitakou, S., Duke, S.O., Fokialakis, N. 2017. Phytotoxic triterpene saponis from Bellis longifolia, an endemic plant of Crete. Phytochemistry. 144:71-77. doi:10.1016/j.phytochem.2017.08.019.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. 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,�- dehydrocurvularin) were isolated. a,�-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. Accomplishments 01 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. 02 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.
Impacts
(N/A)
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. Journal of Agricultural and Food Chemistry. 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.
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