Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (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 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 ÿ-triketones such as leptospermone. Manuka oil from the Manuka tree (Leptospermum scoparium) contains ÿ- triketones that have been shown to inhibit a key enzyme, p- hydroxyphenylpyruvate dioxygenase (HPPD), in plants. Our scientists have refined a simple water soluble ÿ-triketone enriched extraction of Manuka oil that contains up to 30% ÿ-triketones that can be diluted to a powerful bioherbicide. Field and greenhouse experiments found that applying the mixture at 4% ÿ-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 ÿ-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 01 Leptospermone (ÿ-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 ÿ-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 ÿ-triketone enriched extract is undergoing development to be combined with a novel formulation to increase it efficacy at lower concentrations.
Impacts
(N/A)
Publications
- 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
- 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.
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Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (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. In FY2023, we tested approximately 180 fungal and plant extracts and pure compounds for phytotoxic activities using primary and secondary bioassays. These extracts and pure compounds were provided by chemists in our research unit, scientists at the National Center for Natural Products Research (NCNPR), and international collaborators. For bioassays, bentgrass (Agrostis stolonifera L.), lettuce (Lactuca sativa L.) and Arabidopsis were used as model species that served as representatives of monocotyledonous and dicotyledonous plants. These efforts resulted in the discovery of multiple leading natural compounds that displayed strong herbicidal activity. Examples of leading compounds include fusaricidin, momilactone, menthalactone, disobutyrylphloroglucinol, novel HPPD inhibitors, etc. Khellin and visnagin produced from the plant Ammi visnaga were recently reported as potential new bioherbicides with phytotoxic activities comparable to some commercially available herbicides. In an attempt to produce more effective pesticides than the natural compounds, synthetic o- alkyl and o-arylalkyl analogs based on khellin and visnagin were evaluated for biological activity. At least one analog showed enhanced phytotoxic activity compared to the parent molecule visnagin. A manuscript reporting these results was submitted for publication. Fifty putative protoporphyrinogen oxidase (PPO) inhibitors were evaluated for phytotoxicity on Lemna paucicostata. The results showed that ten of them had IC50 values ranging from 0.7 to 3 µM, indicating a high level of herbicidal activity. Experiments designed to confirm their mode of action as PPO inhibitors are being performed. Fusaricidins A and B (FA and FB), isolated from endophytic bacteria Paenibacillus ottowii, belong to the class of lipodepsipeptides (LPD). We conducted experiments with FA and FB, provided by a researcher from NCNPR, to evaluate their effects on two plant species: bentgrass (Agrostis stolonifera) and lettuce (Lactuca sativa). The mixture of fusaricidins A and B (1:1 of FA and FB) did not exhibit a notable pre-emergence activity against these plant species. However, it showed strong post-emergence activity with IC50 of 2.8 µM. At the concentration of 8.2 µM, it strongly inhibited the growth of monocot Lemna paucicostata, a common duckweed, and dicot Arabidopsis seedlings. The pure form of fusaricidin A displayed significant phytotoxicity against Arabidopsis with an IC50 of 0.9 µM. As part of the mode of action studies, we tested the chlorophyll fluorescence emitted from Arabidopsis leaves. The results indicated that photosynthesis may not be the primary target of the compounds. However, electric conductivity tests indicated that these compounds caused a plasma-membrane disintegration, most likely a result of the presence of a lipophilic ÿ-hydroxy fatty acid chain in the compound structure. The RNA- seq data generated from the treatments with fusaricidin A revealed a strong induction of genes related to plant response to bacteria. Momilactones (A and B) are allelochemicals produced from the roots of rice plants and possess potent phytotoxic activity. The molecular biosynthetic pathway for momilactones has long been a focus because of its bioherbicide and plant incorporated protectant potential. Although many of the genes for momilactone biosynthesis have been characterized, the genes in the missing steps are yet to be determined. We identified a homolog of momilactone synthase which may be involved in the biosynthesis of momilactone A. We made five constructs for investigating the function of this gene in FY2022 and the generation of transgenic rice using these constructs has extended into FY2023. The effects of momilactone A and B on fungal plant pathogens were evaluated, and the results indicated that they have a high inhibitory activity against Colletotrichum fragariea and Colletotrichum gloeosporioides, which is comparable to commercial fungicides cyproconazole and captan. Projects are ongoing to investigate the genomic response to momilactones by comparing the transcriptome of the fungal pathogens to that of Arabidopsis that has been exposed to momilactone B. The molecular mechanisms involved in the phytotoxicity of momilactone B (MB) are unknown. We utilized genetic approaches to identify the target(s) of this bioactive compound. Arabidopsis mutant lines which are publicly available were screened for the events that are MB resistant/tolerant. After screening approximately 65,000 T-DNA mutant lines, twenty-two putative resistant lines were obtained. The analysis of these Arabidopsis lines for the possible mechanisms involved in the resistance is underway. A Non Assistance Cooperative Agreement (NACA) with Texas State University, San Marcos, was initiated in June 2023 to identify the mode of action of phytotoxins using genetics approaches. The cooperator is making progress towards meeting the objectives of this agreement. 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. Our primary goal for this work involves transferring the ability to synthesize and secrete sorgoleone to other crops, as a plant- incorporated pesticide. Previously, 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. Our group has employed a two-tier transcriptomics-based strategy, combined with a reverse genetics approach for the identification of genes associated with sorgoleone rhizosecretion. Candidate transporter sequences identified are currently being subjected to CRISPR/Cas-mediated gene editing, and in parallel we are also screening isolates obtained from a S. bicolor mutant population. We now report on the identification of a transporter required for the rhizosecretion of the allelochemical sorgoleone produced in root hair cells of members of the genus Sorghum. The in vivo role of the transporter was confirmed using two independent loss-of-function S. bicolor mutants. Disruption of the corresponding gene sequence resulted in the near total loss of sorgoleone extracellular secretion in both mutant lines. Plant incorporated pesticides (PIP), pesticides produced by plants via genetic modification, have been widely adopted by growers for insect management (e.g., Bt toxin-producing crops), reducing synthetic insecticide use substantially. Currently, no PIP herbicides are available for weed management, despite the obvious economic and environmental benefits such technologies could offer. The incorporation of the recently- identified transporter into existing technologies will facilitate the use of the potent phytotoxin sorgoleone as a PIP, and it is likely that this technology will be similarly well-received as Bt toxin-producing crops. Moreover, given that sorgoleone targets multiple cellular activities, weeds resistant to its inhibitory effects are far less likely to emerge. Artificial Intelligence (AI)/Machine Learning (ML) No artificial intelligence or machine learning methods were used. ACCOMPLISHMENTS 01 Triketone derivatives in herbicide development for weed management. The triketone class of herbicides plays an important role in controlling weeds, especially in crops such as corn, soybean and wheat. The main herbicides in this class are mesotrione and sulcotrione which are analogs of the allelochemical leptospermone from the bottlebrush plant. However, the degradation products of mesotrione and sulcotrione can negatively affect aquatic plants and microorganisms. The benzoic rings resulting from the degradation of these herbicides are responsible for the toxic effects. To develop more environmentally friendly triketone herbicides, ARS researchers in Oxford, Mississippi, made a series of structurally related triketone analogs originating from malonic acid, a naturally occurring compound found in many fruits and vegetables. Bioassay results indicated that these newly synthesized compounds exhibited strong herbicidal activity. Unlike mesotrione and sulcotrione, these compounds lack benzoic rings. The new chemical entities (keto- diesters) provide a new class of herbicides and their mode of action is likely similar to that of mesotrione and sulcotrione. In addition, several keto diether-based compounds developed in this project can reduce potential leaching in plant leaves compared to commercial triketone herbicides. The ARS National Chemical Patent Committee approved the invention disclosure entitled ⿿Derivatives of natural triketones and their uses" in 2022. The patent application is being written and should be filed with the USPTO shortly. This project is part of the collaboration with the University of Mississippi.
Impacts
(N/A)
Publications
- Bajsa Hirschel, J.N., Pan, Z., Padney, P., Asolkar, R., Gopal Chittiboyina, A., Boddy, L., Machingura, M., Duke, S.O. 2023. Spliceostatin C, a component of a microbial bioherbicide, is a potent phytotoxin that inhibits the spliceosome. Frontiers in Plant Science. https://doi.org/10. 3389/fpls.2022.1012939.
- Wei, Z., Wang, Q., Min, L., Bajsa Hirschel, J.N., Cantrell, C.L., Han, L., Tan, C., Weng, J., Liu, X., Duke, S.O. 2022. Synthesis and pesticidal activity of new niacinamide derivatives containing a flexible, chiral chain. Molecules. https://doi.org/10.3390/molecules28010047.
- Duke, S., Pan, Z., Chittiboyina, A., Swale, R., Sparks, T. 2023. Molecular targets of insecticides and herbicides ⿿ are there useful overlaps. Pesticide Biochemistry and Physiology. https://doi.org/10.1016/j.pestbp. 2023.105340.
- Min, L., Shen, Z., Bajsa Hirschel, J.N., Cantrell, C.L., Han, L., Hua, X., Liu, X., Duke, S.O. 2022. Synthesis, crystal structure, herbicidal activity and mode of action of new cyclopropane-1,1-dicarboxylic acid analogues. Pesticide Biochemistry and Physiology. https://doi.org/10.1016/ j.pestbp.2022.105228.
- Shi, H., Zhai, Z., Min, L., Han, L., Sun, N., Cantrell, C.L., Bajsa Hirschel, J.N., Duke, S.O., Liu, X. 2022. Synthesis and pesticidal activity of new 1,3,4-oxadiazole thioether compounds containing a trifluoro-methylpyrazoyl moiety. Research on Chemical Intermediates. https://doi.org/10.1007/s11164-022-04839-x.
- Förster, B., Rourke, L., Weerasooriya, H.N., Pabuayon, I.C., Au, E., Bala, S., Bajsa Hirschel, J.N., Kaines, S., Kasili, R., Laplace, L., Machingura, M.C., Massey, B., Rosati, V.C., Stuart-Williams, H., Badger, M.R., Price, G., Moroney, J.V. 2023. The Chlamydomonas reinhardtii chloroplast envelope protein LCIA transports bicarbonate in planta. Journal of Experimental Botany. https://doi.org/10.1093/jxb/erad116.
- Sparks, T.C., Sparks, J.M., Duke, S.O. 2023. Natural product-based crop protection compounds - origins and future prospects. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs.jafc.2c06938.
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Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (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 Fiscal Year (FY) 2022 and is continuing in FY2023: In 2022, we tested about 150 fungal and plant extracts and pure compounds for phytotoxicity using bioassays with bentgrass (Agrostis stolonifera L.), lettuce (Lactuca sativa L.) and Arabidopsis as model species that served as representatives of monocotyledonous and dicotyledonous plants. These extracts and pure compounds were provided by chemists in our research unit and scientists at the National Center for Natural Products Research. Some compounds were provided by international collaborators. A set of analogs based on the natural products khellin and visnagin were also evaluated for herbicidal activity. The analogs/ compounds that demonstrated very satisfactory herbicidal activity are subjected to further investigation. Greenhouse studies are in progress to determine the efficacy of the most active analog as both a post- and a pre-emergent herbicide. Forty bipartite synthetic conjugates of plant growth regulators tested against the model plant Arabidopsis did not exhibit noteworthy post- emerge herbicidal activity. While ten compounds from another pool of chemicals (fifty putative PPO inhibitors) displayed strong phytotoxicity in the primary bioassays and have been selected for further assessment. Fusaricidins A and B, secondary metabolites isolated from bacterium Paenibacillus polymyxa, possess strong antifungal (plant pathogens) and antibacterial (gram-positive bacteria) properties. We tested these compounds for phytotoxic activity. Bioassays showed that they significantly inhibit the growth of Arabidopsis thaliana and Lemna paucicosata with IC50 values of 2.3 µM (fusaricidin A) and 1 µM fusaricidin AB mixture (Pure fusaricidin B was not available for testing since fusaricidin A was associated B during isolation due to the high degree of structural similarity). The investigation of the mode of action of these compounds suggests that the membrane disintegration may be the result of the interaction of fusaricidins with the plasma membrane. The measurements of chlorophyll fluorescence indicated that these compounds do not directly impact photosystems I or II. In collaboration with the National Center for Natural Products Research, we tested a series of synthesized triketone derivatives for putative herbicidal activity. These synthetic compounds exhibit promising herbicidal activity revealed by phytotoxicity assays against Arabidopsis thaliana and Lemna paucicostata. The mechanism of action of these compounds is to inhibit hydroxyphenylpyruvate dioxygenase (HPPD). Hence they have the potential to be developed as herbicides against herbicide- resistant weeds. An invention disclosure was submitted and approved for the preparation of a patent application. Momilactones A and B are diterpenoid phytoalexins with antimicrobial, allelopathic activity and strong phytotoxicity. Despite the fact that most of the genes for momilactone biosynthesis found in gene clusters in rice genome have been reported, the candidates for the missing steps are yet to be characterized. We identified a homolog of momilactone synthase (designated OsMAS2, also known as short-chain alcohol dehydrogenase/ reductase) which may be involved in the synthesis of momilactone A. We made five constructs for investigating the function of this gene, which include promoter-driven reporter gene beta-glucuronidase gene (GUS), CRISPR knockout constructs, and overexpression construct for rice transformation. The generation of transgenic rice is in progress. We tested several phytoalexins (momilactones, juglone, aminophenoxazin, protocatechualdehyde, khellin, visnagin) using FluorCam, an instrument for chlorophyll fluorescence imaging of plants, plant tissues and algae. Our results suggested that photosystems I and II are not the primary targets of these phytoalexins. This method was established for detecting an early event in plant response to natural compounds for mode of action studies. Towards achieving Objective 2, the following research was performed in FY 2022 and is continuing in FY2023: 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. Our goals for this work have included transferring the ability to synthesize and secrete sorgoleone to other crops, as well as the development of enhanced sorghum germplasm with increased sorgoleone content. Previously, 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. Our group has employed a strategy involving the analysis of genes differentially expressed in sorgoleone-deficient transgenic sorghum, relative to wild-type sorghum and null segregant cohorts, for the identification of genes associated with sorgoleone rhizosecretion. The rationale for this approach is the likelihood that within a deficient background, genes directly involved in the biosynthesis and transport of sorgoleone will exhibit expression profiles which differ from those of wild-type plants, thus facilitating their identification. We have now completed these analyses and have identified a candidate list of sequences for follow up tests in transgenic sorghum plants. These candidate sequences will be subjected to CRISPR/Cas-mediated gene editing, and the design of the required guide sequences have also been completed by our group. We will also employ a recently-developed technological approach involving the addition of a morphogenetic marker (Wuschel2 from A. thaliana), which has been demonstrated to dramatically improve the efficiency of both S. bicolor transformation and CRISPR/Cas-mediated gene editing. To facilitate this work, a Non-Assistance Cooperative Agreement is being established with the director of the Donald Danforth Plant Science Center Transformation Laboratory, who has extensive experience performing CRISPR/Cas-mediated gene editing in sorghum. This collaboration will also provide the ARS with access to the proprietary Wuschel2-enhanced CRISPR/Cas technology developed by Corteva Agriscience (Indianapolis, IN). Additionally, the Collaborator's lab has developed a rapid in vitro assay using isolated S. bicolor embryos, which we will use to evaluate the efficiency of various guide RNA sequences prior to their use in stable transformation experiments. Ideally, these experiments will lead to the identification of key cellular components required for the transport of sorgoleone from its site of synthesis in root hairs to the root system-soil interface. ACCOMPLISHMENTS 01 Determination of the mode of action of spliceostatin C.. Spliceostatin C (spC), one of the bioactive components produced from the soil bacterium Burkholderia rinojensis, displayed a high phytotoxic activity at low doses against several dicot weeds. The spliceosome is generally thought to be the target of this compound. However, due to the complexity of the spliceosome which contains a large number of proteins/ subunits, the precise mechanism of action of spC is yet to be elucidated. ARS researchers in Oxford, Mississippi, analyzed the genomic sequences retrieved from the genome database for 13 spC susceptible and resistant weeds revealed that there could be mutations in the nucleotide sequence encoding two spliceosome-specific proteins (SF3b14b and SF3b155). Molecular docking showed that spC formed H- bonding with two amino acids which are critical residues for herbicidal activity. These findings provide important information for further investigation into the possibility of generating spC-resistant crops through genetic engineering and breeding and develop spC as a bioherbicide for weed management targeting herbicide-resistant weeds.
Impacts
(N/A)
Publications
- Da Cruz-Silva, C.T., Cantrell, C.L., Nobrega, L.P., Ali, A., Duke, S. 2021. Bioassay-guided isolation of phytotoxins from three salvia species. Allelopathy Journal. 54(1):13-24. https://doi.org/10.26651/allelo.j/2021- 54-1-1344.
- Cárdenas, D.M., Bajsa Hirschel, J.N., Cantrell, C.L., Rial, C., Varela, R. M., Molinillo, J.G., Macías, F.A. 2022. Evaluation of the phytotoxic and antifungal activity of C17-sesquiterpenoids as potential biopesticides. Pest Management Science. https://doi.org/10.1002/ps.7042.
- Min, L., Wang, H., Bajsa Hirschel, J.N., Yu, C., Wang, B., Yao, M., Han, L. , Cantrell, C.L., Duke, S.O., Sun, N., Liu, X. 2022. Novel dioxolane ring compounds for the management of phytopathogen diseases as ergosterol biosynthesis inhibitors: synthesis, biological activities and molecular docking. Journal of Agricultural and Food Chemistry. https://doi.org/10. 1021/acs.jafc.2c00541.
- Rajvi, D., Rodriguez, A., Bajsa Hirschel, J.N., Pan, Z., Machingura, M.C. 2021. Differential gene expression patterns in Sorghum bicolor genotypes in response to high vapor pressure deficit. Journal of Crop Improvement. https://doi.org/10.1080/15427528.2021.2009077.
- Duke, S.O., Pan, Z., Bajsa Hirschel, J.N., Boyette, C.D. 2022. The potential future roles of natural compounds and microbial bioherbicides in weed management in crops. Advances in Weed Science. 40(spe1):e020210054. https://doi.org/10.51694/AdvWeedSci/2022;40:seventy-five003.
- Ding, C., Lin, X., Zuo, Y., Yu, Z., Lu, Y., Chen, X., Baerson, S.R., Pan, Z., Zeng, R., Song, Y. 2021. Transcription factor OsbZIP49 controls tiller angle and plant architecture through the activation of indole-3-acetic acid-amido synthetases in rice. The Plant Journal. https://doi.org/10.1111/ tpj.15515.
- Belz, R.G., Duke, S.0. 2022. Modelling biphasic hormetic dose responses to predict sub-NOAEL effects using plant biology as an example. Current Opinion in Toxicology. https://doi.org/10.1016/j.cotox.2022.01.003.
- Belz, R.G., Carbonari, C.A., Duke, S.O. 2022. The potential influence of hormesis on evolution of resistance to herbicides. Current Opinion in Environmental Science & Health. https://doi.org/10.1016/j.coesh.2022. 100360.
- Duke, S.O., Belz, R.G. 2022. Stepping beyond hormesis modelling and sub- NOAEL predictions in plant biology. Current Opinion in Environmental Science & Health. https://doi.org/10.1016/j.coesh.2022.100366.
- Nazish, T., Huang, Y., Zhang, J., Xia, J., Alfatih, A., Chao, L., Cai, X., Xi, J., Xu, P., Xiang, C. 2022. Understanding paraquat resistance mechanisms in Arabidopsis thaliana to facilitate developing paraquat- resistant crops. Plant Communications. https://doi.org/10.1016/j.xplc.2022. 100321.
- Bajracharya, A., Xi, J., Karlie, G., Bayer, E., Grant, C., Clutton, C., Baerson, S.R., Agarwal, A.K., Qiu, Y. 2022. PIF4/HEMERA-mediated daytime thermosensory growth requires the Mediator subunit MED14. bioRxiv. https:// doi.org/10.1101/2022.03.02.482654.
- Roireau, J.H., Rosano, R.J., Chen, T., Bajsa Hirschel, J.N., Schrader, K.K. , Duke, S.O., Wykoff, D., Giuliano, R.M. 2020. Synthesis of pyranopyrans related to diplopyrone and evaluation as antibacterials and herbicides. Journal of Agricultural and Food Chemistry. https://doi.org/10.1021/acs. jafc.0c02564.
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Progress 10/01/20 to 09/30/21
Outputs
Progress Report Objectives (from AD-416): 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 (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 2021 and is continuing in FY2022: A series of compounds related to inhibitors of sterol 14a-demethylase (30 putatives and 2 knowns) were evaluated for phytotoxicity in a secondary bioassay against Lemna paucicostata. Six of them displayed a high level of herbicidal activity with IC50 values between 8.7 and 26 �M. Five active compounds were selected for further study and were found to inhibit the growth of a dicot model plant Arabidopsis as shown on the half strength of Murashige-Skoog culture medium containing 5 �M of a compound. About 30 compounds/extracts from our research unit were tested in a primary bioassay and were found to have satisfactory herbicidal activity. Further testing in a secondary bioassay is in progress. Many of these include the analogs of khellin and visnagin, the lead compounds in development with INBIOAR and the subject of a pending patent application. Determination of the mode of action of spliceostatin C (spC) is a continuation of the collaborative project with Marrone Bio Innovations, Inc. It has been shown that spC significantly inhibits the growth of Arabidopsis seedlings. To better understand the impact of spC on the protein level, proteomics approaches - two-dimensional difference gel electrophoresis (2D DIGE) technique � were employed to identify proteins for alterations in spC treated Arabidopsis seedlings. Primary results indicated that 66 proteins, as determined on 2-DIGE, were significantly affected upon treatment with spC as compared to the control. Further analysis using MALDI/TOF (matrix-assisted laser desorption/ionization- time of flight) mass spectrometry and Mascot software identified that 40 of them are unique proteins and 26 isoforms, and of which two proteins - extra-large guanine nucleotide-binding protein and thylakoid lumen protein - were significantly affected with the average ratio (treated seedlings vs control) of 11.36 and 8.91, respectively, the highest up- regulated proteins. Ongoing work is in progress to further elucidate the roles of these proteins in response to spC. Mechanisms of herbicide resistance can be broadly divided into two categories, target-site resistance where an herbicide acts to disrupt a particular plant process or function (mode of action) and non-target-site resistance. It has been reported that a cytochrome P450 enzyme from cotton is responsible for the non-target site resistance against the herbicide trifloxysulfuron. To further characterize this enzyme, a binary vector containing the coding sequence of the enzyme was constructed, in which the expression of the cDNA is driven by a double 35S promoter for overexpression. The vector was transformed into Arabidopsis via Agrobacterium tumefaciens. Four homozygous lines (third generation) of transgenic plants were generated. These transgenic lines will be used for studying the possible conversion of the herbicide, as well as screening for natural products with a structure similar to trifloxysulfuron and analogs. Streptomycetes are known for their ability to synthesize many secondary metabolites, some of which possess pesticide activity. These secondary metabolites are normally encoded by large biosynthetic gene clusters. Two regulators (pathway-specific and global) were selected for metabolic engineering to improve the production of bioactive compounds. The constructs for overexpression of these regulators were made and are being tested in two Streptomyces strains that are currently used for the extraction of active compounds in our unit. Towards achieving Objective 2, the following research was performed in FY 2021 and is continuing in FY 2022. One approach our group has pursued for the identification of genes associated with the extracellular transport of the allelochemical sorgoleone, is the analysis of genes differentially expressed in sorgoleone-deficient transgenic sorghum relative to wild-type sorghum, as well as null segregants derived from the same transformation events. The basis for this approach is the likelihood that within a deficient background, genes directly involved in the biosynthesis and transport of sorgoleone will exhibit expression profiles which differ from those of wild-type plants, thus providing additional clues facilitating their identification. Using a small-scale root hair isolation procedure devised by our team, root hair preparations were successfully prepared from homozygous positive and negative (null) transgenic seedlings representing multiple independent transgenic events, as well as from the nontransgenic parental Tx430 genotype. High quality total RNAs were obtained from these samples, and Illumina next-gen sequencing libraries were prepared from these RNAs. Sequence analysis experiments from this work are currently ongoing, and we anticipate that the results obtained will provide an important tool for identifying transporters and carrier proteins involved in sorgoleone rhizosecretion. Record of Any Impact of Maximized Teleworking Requirement: The COVID-19 pandemic has affected our research activity tremendously. Since the COVID-19 pandemic started and our research unit closed, the normal research activity in the labs and greenhouse has been interrupted. It has greatly impacted our research progress towards the objectives because all the projects require conducting experiments in the laboratory and greenhouse. For example, the experiments to evaluate the phytotoxicity of many compounds and extracts as we planned were limited due to the limited hands-on access to the labs and greenhouse. These include the use of equipment and greenhouse for investigating the modes of action of selected bioactive compounds. As a result, some experiments are delayed, which slows down the progress towards the objectives.
Impacts
(N/A)
Publications
- Duke, S., Pan, Z., Bajsa Hirschel, J.N. 2020. Proving the mode of action of phytotoxic phytochemicals. Plants. https://www.doi.org/10.3390/ plants9121756.
- Kumarihamy, M., Rosa, L.H., Techen, N., Ferreira, D., Croom, E.M., Duke, S. O., Tekwani, B.L., Khan, S., Nanayakkara, N. 2020. Antimalarials and phytotoxins from Botryosphaeria dothidea identified from a seed of diseased Torreya taxifolia. Molecules. https://doi.org/10.3390/ molecules26010059.
- Pan, Z., Bajsa Hirschel, J.N., Vaughn, J.N., Rimando, A.M., Baerson, S.R., Duke, S.O. 2021. In vivo assembly of sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana. New Phytologist. 230:683-697.
- Sun, Z., Li, Q., Lin, Y., Shi, Q., Baerson, S.R., Xu, C., Wang, R., Chen, L., Zeng, R., Song, Y. 2021. Olfactory perception of herbivore-induced plant volatiles elicits counter-defenses in larvae of tobacco cutworm, spodoptera litura. Functional Ecology. 2021;35:384�397. https://doi.org/10. 1111/1365-2435.13716.
- Hijano, N., Nepomuceno, M.P., Cantrell, C.L., Duke, S.O., Alves, P.L. 2021. Characterization of the allelopathic potential of sugarcane leaves and roots. Journal of Agricultural Chemistry and Environment. https://doi.org/ 10.4236/jacen.2021.103016.
- Barreto, D.L., Nogueira De Azevedo, R., Carvalho, C., Ferreira, M.C., Cantrell, C.L., Duke, S.O., Rosa, L.H. 2021. Bioactive compounds produced by Neotropical endophytic fungi applied to agriculture. In: Rosa L.H. editors. Neotropical Endophytic Fungi. Springer, Cham: Springer International Publishing. p. 257-295.
- Yu, C., Wang, Q., Min, L., Bajsa Hirschel, J.N., Hua, X., Cantrell, C.L., Duke, S., Liu, X. 2021. Synthesis, crystal structure, herbicidal activity, and SAR study of Novel N-(Arylmethoxy)-2-chloronicotinamides derived from nicotinic acid. Journal of Agricultural and Food Chemistry. https://doi. org/10.1021/acs.jafc.0c07538.
- Meepagala, K.M., Bracken, A.K., Fronczek, F.R., Johnson, R.D., Wedge, D.E., Duke, S.O. 2020. A novel furanocoumarin with phytotoxic activity from the leaves of Amyris elemifera (Rutaceae). Journal of Agricultural and Food Chemistry. https://www.doi.org/10.1021/acsomega.0c04778.
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