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ARS Home Southeast Area Oxford, Mississippi Natural Products Utilization Research Research





- Pan, Zhiqiang - Peter
- Baerson, Scott
- Bajsa-Hirschel, Joanna
- Barickman, Thomas - Casey Barickman
- 2023
- 2022
- 2021
- Publications
- Crop Protection & Quarantine (304)

Research Project: Novel Weed Management Tools from Natural Product-Based Discoveries **Location:
                            Natural Products Utilization Research** 2023 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**
                                

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 ß-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. **Accomplishments**
                                

1. 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. **Review 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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