Project : USDA ARS

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

Location: Natural Products Utilization Research

2025 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
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. A Research Molecular Biologist on this project retired in June of 2025. In 2020 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, and confertin. Khellin and visnagin are the subject of a patent filed on the use of these compounds as natural herbicides. Successful characterization and identification of a cytochrome P450 enzyme (CYP71AM1) from sorghum, all the genes required for sorgoleone biosynthesis beginning with the C16:1 fatty acid have now been identified by ARS researchers in Oxford, Mississippi. In 2021 compounds related to inhibitors of sterol 14a-demethylase (30 putatives and 2 knowns) were evaluated for phytotoxicity in a secondary bioassay against Lemna paucicostata. Many of these 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. Streptomycetes 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. 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. 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. In 2022 a set of analogs based on the natural products khellin and visnagin were also evaluated for herbicidal activity. Fusaricidins A and B, secondary metabolites isolated from bacterium Paenibacillus polymyxa, significantly inhibit the growth of Arabidopsis L. paucicosata with IC50 values of 2.3 µM (fusaricidin A) and 1 µM fusaricidin. 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. 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. We have completed 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. 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. 2023: Efforts resulted in the discovery of multiple leading natural compounds including fusaricidin, momilactone, menthalactone, disobutyrylphloroglucinol, novel HPPD inhibitors. 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. 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). 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. 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.We now report on the identification of an ATP Binding Cassette subfamily G (ABCG) type transporter, designated SbABCG2, which is required for the rhizosecretion of the allelochemical sorgoleone produced in root hair cells of members of the genus Sorghum. The in vivo role of SbABCG2 was confirmed using two independent loss-of-function ems-generated S. bicolor mutants. Disruption of SbABCG2 transport activity in the abcg2-1 or abcg2-2 mutant lines resulted in the near total loss of sorgoleone extracellular secretion. 2024: 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. 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. 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% ßtriketones reduced growth in noxious weeds such as Amaranthus palmeri, Digitaria sanguinalis, and Cyperus esculentus. The halfmaximal 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. The IC50 value for inhibiting root elongation in Arabidopsis are < 2 µM. We report for the first time a natural lactone, known as menthalactone, that is derived from Mentha piperita L with 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. Our research unit successfully completed the isolation and characterization of all genes required for the biosynthesis of sorgoleone from the ubiquitous precursor palmitoleoyl-CoA. Wehave recently generated multiple independent transformation events containing the complete sorgoleone biosynthetic pathway in corn (genotype Hi-II) as well as wheat (genotype Fielder J). 2025: Pogostone isolated from Pogostemon cablin exhibited herbicidal activity against Lemna paucicostata (IC50 = 0.9 µM) comparable to atrazine (IC50 = 1.1 ± 0.23 µM), and Amaranthus palmeri in postemergence trials. Secondary metabolites isolated from a Curvularia spp., with tyrosol and 4-hydroxybenzaldehyde identified as the active compounds. Application for patent “Dual mode of action natural product-based proherbicides and uses thereof” was filed on 12/30/2024. Secondary metabolites isolated from a Westerdykella multisporan identified eight compounds. 6-methylsalicyclic acid showed phytotoxic activity against L. paucicostata with an IC50 value of 38.7 µM. Secondary metabolites isolated from a Botryosphaeria rhodina identified two compounds, 4-hydroxyphenethyl alcohol and indole-3-carboxylic acid, with indole-3-carboxylic acid identified as the active compound. A pre-emergence study found a ß-triketones mixture from 0.03125 mg ml-1 to 2.5 mg ml-1 ß-triketones inhibited germination of the noxious weeds Abutilon theophrasti and Lolium multiflorum. Complete inhibition of germination from both weed species were as low as 0.625 mg ml-1. During this FY we generated knockouts of the S. bicolor SbABCG-2 sorgoleone transporter sequence via CRISPR/Cas-mediated gene editing through a collaboration with the Donald Danforth Plant Science Center and Texas A&M. Vectors designed with two independent guide sequences targeting SbABCG-2 to increase the probability of successfully obtaining genome edits. Approximately 40 Ro events were selected based on initial QC screening for follow up analyses. Illumina libraries were prepared for the selected SbABCG-2 events, and samples were subjected to targeted next-gen sequence analysis and compared with the publicly-available Tx430 genome. We detected a high frequency of edits among the events analyzed, indicating extensive disruption within the SbABCG-2 coding region and likely loss of transport function. The sorgoleone transporting activity within the R1 progeny of the putative SbABCG-2 knockout lines is currently under evaluation.

Accomplishments
1. 4A. Lipodepsipeptides and Paenibacillus ottowii preparations as novel bioherbicides or microbial bioherbicides. The invention is two structurally related lipodepsipeptides (LPD) fusaricidins A and B, isolated from endophytic bacteria Paenibacillus ottowii with potential herbicidal activity. Alternatively, this invention could be preparation of Paenibacillus ottowii as potential microbial bioherbicides. Fusaricidin A (FA) consists of neutral peptide sequences with six amino acids from position 1 to 6 L-Thr D-Val L-Val D-allo-Thr D-Asn D-Ala and like the majority of fusaricidins is linked to a beta-hydroxy 15 carbon fatty acid chain ended with a single positively charged guanidinium group. Fusaricidin B is a structural analog with D-Gln replacing D-Asn at the respective position. Microbial-derived natural products are broadly utilized by diverse industries such as food, medicine, agriculture, and cosmetics. Currently, several bacteria-originating bioherbicides are utilized in numerous countries. Treatment of weed species with herbicides, which is a conventional method of weed management employed through decades became insufficient. The main caveat is the development of herbicide resistant weed species that drastically impact crop yields. Nowadays, this phenomenon occurs more frequently due to the limited number of molecular targets against plants offered by commercial herbicides. Bioherbicides carrying a new mode of action that allow farmers to control herbicide resistant weeds would be highly desired products. There is no available commercial herbicide with similar structure to the chemical entities disclosed in the current invention.

2. 4B. Dual mode of action natural product-based proherbicides. The invention is a series of structurally related triketone 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides linked to the natural herbicide, pelargonic acid. These molecules are designed to break down into pelargonic acid and the HPPD inhibitor once systemically absorbed by the plant. Herbicides that are not in the active form until chemically altered in the plant are termed proherbicides. There are fourteen commercial HPPD inhibitor herbicides, and six of these are proherbicides. However, all of these are activated to only one herbicide, a HPPD inhibitor. The new chemical entities disclosed in the current invention breakdown into two types of herbicides, a HPPD inhibitor and a membrane disruptor in the plant. These proherbicide compounds are not in scientific or patent literature. These new chemical entities, including keto-diesters linked to pelargonic acid, provide a new class of herbicides to effectively manage weed control in commercial crops. New herbicide classes are badly needed because of the rapid evolution of both target-site and non-target-site based herbicide resistance in weeds. These new compounds could be used for both weed and herbicide resistance management and the breakdown product, pelargonic acid, might enhance these novel proherbicides absorption, permeability, and other physicochemical properties. Their dual mode of action capability makes evolution of resistance in weeds less likely. Another possible use is as insecticides on blood-feeding insects (e.g., ticks, mosquitos, and bed bugs), as HPPD inhibitors have recently been found to kill these insects after a blood meal because the insects’ HPPD is needed to detoxify tyrosine from blood.

U.S. DEPARTMENT OF AGRICULTURE

Natural Products Utilization Research: Oxford, MS