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

Location: Natural Products Utilization Research

2024 Annual Report

Objectives
1. Discover and develop natural product-based bioherbicides with novel modes of action that are safe and effective tools for weed management. [C2, PS2A] 1.1. Discover uses of new and existing natural products for potential use as herbicides and bioherbicides for weed management. 1.2. Discovery of the mechanisms of action for newly discovered phytotoxins using chemical structure clues, physiological evaluations, and molecular genetics approaches. 2. Develop plant-incorporated bioherbicide technologies for weed management based on known or newly discovered allelochemicals and determine the role of allelopathy in the success of invasive weeds. 2.1. Identification of transporters required for the extracellular secretion of sorgoleone in Sorghum bicolor root hair cells. 2.2. Manipulation of sorgoleone levels in vivo to generate enhanced S. bicolor germplasm. 2.3. Generation of transgenic maize, wheat and soybean plants containing the complete sorgoleone biosynthetic pathway.

Approach
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.

Progress Report
A new scientist was hired in November 2023 for the position of Research Plant Physiologist. The new scientist has been assigned the lead scientist position for the project. In our research, we focus on implementing diverse strategies that improve pest management. Our goal is to prevent/delay, the selection process of herbicide-resistant weed species induced by persistent herbicide use. The idea is to discover natural product compounds that affects multiple plant targets simultaneously. We have submitted an Invention Disclosure entitled Dual Mode for Action Natural Product-based Proherbicies (Docket Number 0093.23). A draft for the patent application based on the invention disclosure is currently being prepared. We conducted an extensive evaluation of the phytotoxic activity of hundreds of extracts and pure compounds. Our approach involved initial bioassays to assess the phytotoxicity of these extracts and compounds using bentgrass (Agrostis stolonifera L.), lettuce (Lactuca sativa L.), and Arabidopsis (Arabidopsis thaliana) as test species. Subsequently, we performed dose-dependent response analyses to determine the concentration required for both half and complete inhibition of seed germination, using Arabidopsis seedlings or duckweed (Lemna paucicostata). The most promising compounds were selected for further characterization, including the determination of their mode of action (MoA) when possible. The compounds currently under investigation include momilactone B, small lactones, mevalocidin, pogostone, and ß-triketones such as leptospermone. Manuka oil from the Manuka tree (Leptospermum scoparium) contains ß-triketones that have been shown to inhibit a key enzyme, p-hydroxyphenylpyruvate dioxygenase (HPPD), in plants. Our scientists have refined a simple water soluble ß-triketone enriched extraction of Manuka oil that contains up to 30% ß-triketones that can be diluted to a powerful bioherbicide. Field and greenhouse experiments found that applying the mixture at 4% ß-triketones reduced growth in noxious weeds such as Amaranthus palmeri, Digitaria sanguinalis, and Cyperus esculentus. In a follow up greenhouse study, it was found that increasing the concentration from 0 to 6% significantly reduced the growth of Amaranthus retroflexus, Abutilon theophrasti, and Lolium multiforum. Additional studies are being conducted to determine a novel formulation for the enriched extraction of the ß-triketones that increases the efficacy of the mixture at the lowest possible concentration. Rice cultivars release a range of allelochemicals into the rhizospheres, such as momilactones. Research has focused on diterpenoid phytoalexins, momilactones A and B, confirming allelopathic properties and inhibiting the growth of plants and fungal phytopathogens. We explored the broad-spectrum antifungal potential of momilactone B (MOMB) against various phytopathogens. Initial screening showed that four fungal pathogens Colletotrichum fragariae (isolate Cf63), Phomopsis obscurans, Botrytis cinerea and Fusarium oxysporum were susceptible to MOMB at a concentration of 100 ug/spot. We then focused on Colletotrichum fragariae, an agriculturally significant pathogen from the ascomycete fungi family Glomerellaceae. Using the Microbroth Susceptibility Assay, a dose-response bioassay, we determined that the half maximal inhibitory concentration (IC50) for MOMB was 1.1 µg/ml at 48 hours, which is comparable to the commercial fungicides captan and cyproconazole having IC50 of 0.51 and 5.0 µg/ml, respectively. Currently, we are employing multi-omics methods to identify the impact of momilactone. Also, multi-omics techniques are being used to elucidate the mechanism of action of momilactone B in planta. The half-maximal inhibitory concentration of MOMB was calculated by assessing the root length of Arabidopsis seedlings exposed to various concentrations of the compound, yielding a very satisfactory value of 1.3 µM. One approach to elucidating the MoA for bioactive compounds involves utilizing forward genetics analysis. MOMB has demonstrated high phytotoxicity against other plant species, including weed species. The IC50 value for inhibiting root elongation in Arabidopsis is < 2 µM, suggesting its potential as a weed control agent, but the MoA is undetermined. In a screening of Arabidopsis T-DNA mutant lines against MOMB, a forward genetics approach, we identified 47 putative resistant lines. Subsequent secondary screening using MOMB concentrations up to 4 µM led to the selection of 9 MOMB resistant/tolerant lines for further analysis. We are investigating the mechanisms underlying this resistance by employing thermal asymmetric interlaced PCR (TAIL-PCR) and/or plasmid rescue techniques to identify the genes responsible for MOMB resistance. We report for the first time a natural lactone, known as menthalactone, that is derived from Mentha piperita L. The phytotoxic activity was assessed against bentgrass, and lettuce, with outstanding activity against bentgrass. The germination of bentgrass seeds was significantly inhibited and an IC50 value of 4.9 ± 1.2 µM. Duckweed plants were less responsive to menthalactone treatment with an IC50 of 293.4 ± 70.6 µM. The results suggest that menthalactone might have effects on seed germination but not on the metabolism in green tissues. The susceptibility to menthalactone of three common, obnoxious weed species i.e., ryegrass (Lilium perenne), barnyard grass (Echinochloa crusgalli), and crabgrass (Digitaria sanguinalis) was assessed. Menthalactone at 1000 µM completely inhibited the germination of all three species, while 330 µM inhibited germination by less than 50%. Post-emergence application of menthalactone at 1% did not produce a significant inhibitory effect against the weed spps. Herbicide resistance has rapidly evolved due to selection pressure exerted in weed populations. In many cases, the resistance involves gene to metabolize herbicide chemistries catalyzed by cytochrome P450 monooxygenases (P450). This type of resistance is often referred to as non-target site resistance. To explore this further, we generated transgenic Arabidopsis plants (T2) expressing CYP81A, an enzyme known to metabolize several classes of herbicides. A pure line (T3) is being generated for our unit to serve as a tool for identifying new compounds structurally related to known herbicides but resistant to metabolism in plant cells. The allelochemical sorgoleone plays a major role in sorghum’s natural ability to inhibit weed and represents a promising natural product-based alternative to synthetic herbicides. Our primary goal involves transferring the ability to synthesize and secrete sorgoleone to other crops, as a plant-incorporated pesticide (PIP). PIPs, pesticides produced by plants via genetic modification, are widely adopted by growers for insect management (e.g., Bt toxin-producing crops), reducing insecticide use substantially. Currently, no PIP herbicides are available for weed management. The incorporation of the identified transporter into existing technologies will facilitate the use of the potent phytotoxin sorgoleone as a PIP, and it is likely this technology will be as well-received as Bt toxin-producing crops. Given that sorgoleone targets multiple cellular activities, weed resistance is less likely to emerge. Our research unit successfully completed the isolation and characterization of all genes required for the biosynthesis of sorgoleone from the ubiquitous precursor palmitoleoyl-CoA. Elucidation of the cellular apparatus involved in the secretion of sorgoleone is also critical, as the efflux pumps associated with this process likely provide a mechanism for autotoxicity avoidance to the host plant Previously, we reported the identification of an ABC subtype G transporter required for the rhizosecretion of the allelochemical sorgoleone by utilizing a two-tier transcriptomics-based strategy, combined with a reverse genetics approach screening isolates obtained from a S. bicolor mutant population. We have generated knockouts of this sequence via CRISPR/Cas-mediated gene editing through our collaboration with the Donald Danforth Plant Science Center. To further explore the mechanistic details underlying sorgoleone rhizosecretion, we have initiated a collaboration with a researcher at Michigan State University, who has pioneered the use of atomic simulation tools to create accurate molecular-scale models for biological phenomena at the nanoscale level. An additional technical goal for our group is the transfer of the complete sorgoleone biosynthetic pathway into major crop species via agrobacterium-mediated transformation. Toward this end, we have recently generated multiple independent transformation events containing the complete sorgoleone biosynthetic pathway in corn (genotype Hi-II) as well as wheat (genotype Fielder J). The rationale for this multi-crop approach is that different crop species could vary significantly in their innate tolerance towards sorgoleone, thus entirely different outcomes could result from our efforts to synthesize sorgoleone in planta in different crops. Sorgoleone is produced exclusively by members of the genus Sorghum and has not been found in any other plant species examined to date.

Accomplishments
1. Leptospermone (ß-triketones) enriched extract found to reduce weed growth significantly more than commercial organic herbicides. The demand for organic foods throughout the developed world is substantially increasing year over year. From 2000 to 2019, the worldwide sales of organic food increased from $16.5 to $116 billion, and the North American organic food and drink market expanded 16.7 percent in the same period. However, weed management on organic farms is the single largest expense and has been linked to production loses. Thus, new bioherbicide technologies that are highly efficacious and economical are needed to improve yields, quality, and nutritional values of organic foods. ARS researchers at Oxford, Mississippi, demonstrated that a mixture of ß-triketones (2% and 4%), a natural systemic (only known) bioherbicide, extracted from Manuka oil are significantly more effective than commercially available bioherbicides such as D-limonene (12%) and vinegar (20% acetic acid) and as effective as the commercial herbicide glyphosate in reducing palmer amaranth and large crabgrass growth. The ß-triketone enriched extract is undergoing development to be combined with a novel formulation to increase it efficacy at lower concentrations.

Review Publications
Soltani, A., Ospanov, M., Ibrahim, Z.M., Bajsa Hirschel, J.N., Cantrell, C.L., Cizdziel, J.V., Khan, I.A., Ibrahim, M.A. 2024. Menthalactone from Mentha piperita L., a Monocot-Selective Bioherbicide. International Journal of Plant Biology. 15:293-303. https://doi.org/10.3390/ijpb15020025.
Gonçalves, V.N., Carvalho, C.R., Martins, L.M., Barreto, D.L., Queiroz, S.C., Tamang, P., Bajsa Hirschel, J.N., Cantrell, C.L., Duke, S.O., Rosa, L.H. 2024. Bioactive metabolites produced by fungi present in Antarctic, Arctic and alpine ecosystems. In: Abdel-Azeem, A.M., Yadav, A.N., Yadav, N., Sharma, M., editors. Bioactive Metabolites from Fungi in Pharmaceutical Research and Development: Prospects & Avenues. p. 537-563. https://doi.org/10.1007/978-981-99-5696-8_17.
Cantrell, C.L., Travaini, M., Bajsa Hirschel, J.N., Svendsen, L.D., Reichley, A.C., Sosa, G.M., Kim, S., Tamang, P., Meepagala, K.M., Duke, S.O. 2023. Synthesis, Herbicidal Activity, and Structure-Activity Relationships of O-Alkyl Analogues of Khellin and Visnagin. Journal of Agriculture and Food Chemistry. 71:14593-14603. https://doi.org/10.1021/acs.jafc.3c03254.
Sun, N., Min, L., Sun, Z., Zhai, Z., Bajsa Hirschel, J.N., Wei, Z., Hua, X., Cantrell, C.L., Xu, H., Duke, S.O., Liu, X. 2024. Novel pyrazole acyl(thio)urea derivatives containing a biphenyl scaffold as potential succinate dehydrogenase inhibitors: Design, synthesis, fungicidal activity and SAR. Journal of Agricultural and Food Chemistry. 72:2512-2525. https://doi.org/10.1021/acs.jafc.3c07735.
Simionato Bidóa, V., Dos Santos Neto, J.C., De Goes Maciel, C.D., Tropaldi, L., Carbonari, C.A., Duke, S.O., De Carvalho, L.B. 2023. Lack of significant effects of glyphosate on glyphosate-resistant maize in different field locations. Agronomy. 13(10. Article 13041071. https://doi.org/10.3390/agronomy13041071.
Ribeiro, V., Bajsa Hirschel, J.N., Tamang, P., Meepagala, K.M., Duke, S. 2023. Antifungal and phytotoxic activities of isolated compounds from Helietta parvifolia stems. Molecules. 28(23):7930. https://doi.org/10.3390/molecules28237930.
Liang, W., Wang, Q., Min, L., Han, L., Cantrell, C.L., Bajsa Hirschel, J.N., Duke, S.0., Ye, P., Liu, X. 2023. Synthesis, herbicidal activity and in silico analysis of novel pyrido[2,3-d]pyrimidine compounds. Molecules. https://doi.org/10.3390/molecules28217363.
Young, S.L., Anderson, J.V., Baerson, S.R., Bajsa Hirschel, J.N., Blumenthal, D.M., Boyd, C.S., Boyette, C.D., Brennan, E.B., Cantrell, C.L., Chao, W.S., Chee Sanford, J.C., Clements, D.D., Dray Jr, F.A., Duke, S.O., Porter, K.M., Fletcher, R.S., Fulcher, M.R., Gaskin, J., Grewell, B.J., Hamerlynck, E.P., Hoagland, R.E., Horvath, D.P., Law, E.P., Madsen, J., Martin, D.E., Mattox, C.M., Mirsky, S.B., Molin, W.T., Moran, P.J., Mueller, R.C., Nandula, V.K., Newingham, B.A., Pan, Z., Porensky, L.M., Pratt, P.D., Price, A.J., Rector, B.G., Reddy, K.N., Sheley, R.L., Smith, L., Smith, M., Snyder, K.A., Tancos, M.A., West, N.M., Wheeler, G.S., Williams, M., Wolf, J.E., Wonkka, C.L., Wright, A.A., Xi, J., Ziska, L.H. 2023. Agricultural Research Service weed science research: past, present, and future. Weed Science. 71(4):312-327. https://doi.org/10.1017/wsc.2023.31.
Machingura, M.C., Glover, S., Settles, A., Pan, Z., Bajsa Hirschel, J.N., Chitiyo, G., Weiland, M.H. 2024. Transcriptome and physiological analyses reveal the response of Arabidopsis thaliana to poly(aspartic acid). Plant Stress. 12:1-14. https://doi.org/10.1016/j.stress.2024.100478.
Duke, S.O., Pan, Z., Bajsa-Hirschel, J.N., Tamang, P., Hammerschmidt, R., Lorsbach, B.A, and Sparks, T.C. 2023. Molecular targets of herbicides and fungicides - are there useful overlaps for fungicide discovery? J. Agric. Food Chem. 71:20532-20548. https://doi.org/10.1021/acs.jafc.3c07166