Defining the toxicological profile of 4-hydroxyphenylpyruvate dioxygenase-directed herbicides to Aedes aegypti and Amblyomma americanum.

Inhibitors targeting the 4-hydroxyphenyl pyruvate dioxygenase (HPPD) enzyme are well established herbicides and HPPD is also a primary enzyme within the tyrosine metabolism pathway in hematophagous arthropods, which is an essential metaboilic pathway post-blood feeding to prevent tyrosine-mediated toxicity. The objective of this study was to characterize the toxicity of triketone, pyrazole, pyrazolone, isoxazole, and triazole herbicides that inhibit HPPD to blood-fed mosquitoes and ticks. Topical exposure of nitisinone to blood-fed Aedes aegypti yielded high toxicity with an LD50 of 3.81 ng/insect (95% CI: 3.09 to 4.67 ng; Hillslope: 0.97, r2: 0.99), yet was non-toxic to non-blood fed (NBF) mosquitoes. The rank order of toxicity was nitisinone > tembotrione > pyrazoxyfen > tebuconazole > mesotrione against blood-fed Ae. Aegypti, but nitisinone was approximately 30-fold more toxic than other chemicals tested. We also assessed the toxicity of HPPD-inhibiting herbicides to the lone star tick, Amblyomma americanum and similarly, nitisinone was toxic to Am. americanum with a lethal time to kill 50% of subjects (LT50) of 23 h at 10 µM. Knockdown of the gene encoding the HPPD enzyme was performed through RNA-interference led to significant mortality after blood feeding in both, Ae. aegypti and Am. americanum. Lastly, a fluorescence assay was developed to determine relative quantities of L-tyrosine in Ae. aegypti and Am. americanum treated with HPPD inhibitors. L-tyrosine levels correlated with toxicity with nitisinone exposure leading to increased tyrosine concentrations post-blood feeding. Taken together, these data support previous work suggesting HPPD-inhibitors represent a novel mode of toxicity to mosquitoes and ticks and may represent base scaffolds for development of novel insecticides specific for hematophagous arthropods.

Molecular targets of insecticides and herbicides - Are there useful overlaps?

New insecticide modes of action are needed for insecticide resistance management strategies. The number of molecular targets of commercial herbicides and insecticides are fewer than 35 for both. Few commercial insecticide targets are found in plants, but ten targets of commercial herbicides are found in insects. For several of these commonly held targets, some compounds kill both plants and insects. For example, herbicidal inhibitors of p-hydroxyphenylpyruvate dioxygenase are effective insecticides on blood-fed insects. The glutamine synthetase-inhibiting herbicide glufosinate is insecticidal by the same mechanism of action, inhibition of glutamine synthetase. These and other examples of shared activities of commercial herbicides with insecticides through the same target site are discussed. Compounds with novel herbicide targets shared by insects that are not commercialized as pesticides (such as statins) are also discussed. Compounds that are both herbicidal and insecticidal can be used for insect pests not associated with crops or with crops made resistant to the compounds.

Antifungal and Phytotoxic Activities of Isolated Compounds from Helietta parvifolia Stems.

The identification of natural and environmentally friendly pesticides is a key area of interest for the agrochemical industry, with many potentially active compounds being sourced from numerous plant species. In this study, we report the bioassay-guided isolation and identification of phytotoxic and antifungal compounds from the ethyl acetate extract of Helietta parvifolia stems. We identified eight compounds, consisting of two coumarins and six alkaloids. Among these, a new alkaloid, 2-hydroxy-3,6,7-trimethoxyquinoline-4-carbaldehyde (6), was elucidated, along with seven known compounds. The phytotoxicity of purified compounds was evaluated, and chalepin (4) was active against Agrostis stolonifera at 1 mM with 50% inhibition of seed germination and it reduced Lemna pausicotata (duckweed) growth by 50% (IC50) at 168 µM. Additionally, we evaluated the antifungal activity against the fungal plant pathogen Colletotrichum fragariae using a thin-layer chromatography bioautography assay, which revealed that three isolated furoquinoline alkaloids (flindersiamine (3), kokusagenine (7), and maculine (8)) among the isolated compounds had the strongest inhibitory effects on the growth of C. fragariae at all tested concentrations. Our results indicate that these active natural compounds, i.e., (3), (4), (7), and (8), could be scaffolds for the production of more active pesticides with better physicochemical properties.

Synthesis, Herbicidal Activity, Mode of Action, and In Silico Analysis of Novel Pyrido[2,3-d]pyrimidine Compounds.

Natural products are a main source of new chemical entities for use in drug and pesticide discovery. In order to discover lead compounds with high herbicidal activity, a series of new pyrido[2,3-d] pyrimidine derivatives were designed and synthesized using 2-chloronicotinic acid as the starting material. Their structures were characterized with 1H NMR, 13C NMR and HRMS, and the herbicidal activities against dicotyledonous lettuce (Lactuca sativa), field mustard (Brassica campestris), monocotyledonous bentgrass (Agrostis stolonifera) and wheat (Triticum aestivum) were determined. The results indicated that most of the pyrido[2,3-d] pyrimidine derivatives had no marked inhibitory effect on lettuce at 1 mM. However, most of the pyrido[2,3-d] pyrimidine derivatives possessed good activity against bentgrass at 1 mM. Among them, the most active compound, 3-methyl-1-(2,3,4-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-dione (2o), was as active as the positive controls, the commercial herbicides clomazone and flumioxazin. Molecular simulation was performed with molecular docking and DFT calculations. The docking studies provided strong evidence that 2o acts as an herbicide by inhibition of protoporphyrinogen oxidase. However, the physiological results indicate that it does not act on this target in vivo, implying that it could be metabolically converted to a compound with a different molecular target.

Synthesis, crystal structure, herbicidal activity and mode of action of new cyclopropane-1,1-dicarboxylic acid analogues.

A new series of cyclopropane-1,1-dicarboxylic (CPD) acid analogues were designed and synthesized. CPD is an inhibitor of ketol-acid reductoisomerase (KARI), an enzyme of the branched chain amino acid pathway in plants. The structures of CPD analogues were characterized by 1H NMR and HRMS. The structure of N,N'-bis(4-(tert-butyl)phenyl)cyclopropane-1,1-dicarboxamide was further elucidated by X-ray diffraction. The herbicidal activities of these compounds were tested against lettuce (Lactuca sativa) and bentgrass (Agrostis stolonifera). Most of these compounds exhibited low herbicidal activity against both plant species. Among them, N,N'-bis(2-ethylphenyl)cyclopropane-1,1-dicarboxamide displayed moderate activity against bentgrass. Inhibition of KARI activity by the CPD analogues was also assessed experimentally and by molecular docking simulation with results supporting inhibition of KARI as their mode of action. These results provide the basis for design of more effective KARI inhibitors.

Synthesis and Pesticidal Activity of New Niacinamide Derivatives Containing a Flexible, Chiral Chain.

Natural products are a source for pesticide or drug discovery. In order to discover lead compounds with high fungicidal or herbicidal activity, new niacinamide derivatives derived from the natural product niacinamide, containing chiral flexible chains, were designed and synthesized. Their structures were confirmed by 1H NMR, 13C NMR and HRMS analysis. The fungicidal and herbicidal activities of these compounds were tested. The fungicidal activity results demonstrated that the compound (S)-2-(2-chloronicotinamido)propyl-2-methylbenzoate (3i) exhibited good fungicidal activity (92.3% inhibition) against the plant pathogen Botryosphaeria berengriana at 50 µg/mL and with an EC50 of 6.68 ± 0.72 µg/mL, which is the same as the positive control (fluxapyroxad). Compound 3i was not phytotoxic and could therefore be used as a fungicide on crops. Structure-activity relationships (SAR) were studied by molecular docking simulations with the succinate dehydrogenase of the fungal mitochondrial respiratory chain.

Mechanisms of evolved herbicide resistance.

The widely successful use of synthetic herbicides over the past 70 years has imposed strong and widespread selection pressure, leading to the evolution of herbicide resistance in hundreds of weed species. Both target-site resistance (TSR) and nontarget-site resistance (NTSR) mechanisms have evolved to most herbicide classes. TSR often involves mutations in genes encoding the protein targets of herbicides, affecting the binding of the herbicide either at or near catalytic domains or in regions affecting access to them. Most of these mutations are nonsynonymous SNPs, but polymorphisms in more than one codon or entire codon deletions have also evolved. Some herbicides bind multiple proteins, making the evolution of TSR mechanisms more difficult. Increased amounts of protein target, by increased gene expression or by gene duplication, are an important, albeit less common, TSR mechanism. NTSR mechanisms include reduced absorption or translocation and increased sequestration or metabolic degradation. The mechanisms that can contribute to NTSR are complex and often involve genes that are members of large gene families. For example, enzymes involved in herbicide metabolism-based resistances include cytochromes P450, GSH S-transferases, glucosyl and other transferases, aryl acylamidase, and others. Both TSR and NTSR mechanisms can combine at the individual level to produce higher resistance levels. The vast array of herbicide-resistance mechanisms for generalist (NTSR) and specialist (TSR and some NTSR) adaptations that have evolved over a few decades illustrate the evolutionary resilience of weed populations to extreme selection pressures. These evolutionary processes drive herbicide and herbicide-resistant crop development and resistance management strategies.

In vivo assembly of the sorgoleone biosynthetic pathway and its impact on agroinfiltrated leaves of Nicotiana benthamiana.

Sorgoleone, a hydrophobic compound exuded from root hair cells of Sorghum spp., accounts for much of the allelopathic activity of the genus. The enzymes involved in the biosynthesis of this compound have been identified and functionally characterized. Here, we report the successful assembly of the biosynthetic pathway and the significant impact of in vivo synthesized sorgoleone on the heterologous host Nicotiana benthamiana. A multigene DNA construct was prepared for the expression of genes required for sorgoleone biosynthesis in planta and deployed in N. benthamiana leaf tissues via Agrobacterium-mediated transient expression. RNA-sequencing was conducted to investigate the effects of sorgoleone, via expression of its biosynthesis pathway, on host gene expression. The production of sorgoleone in agroinfiltrated leaves as detected by gas chromatography/mass spectrometry (GC/MS) resulted in the formation of necrotic lesions, indicating that the compound caused severe phytotoxicity to these tissues. RNA-sequencing profiling revealed significant changes in gene expression in the leaf tissues expressing the pathway during the formation of sorgoleone-induced necrotic lesions. Transcriptome analysis suggested that the compound produced in vivo impaired the photosynthetic system as a result of downregulated gene expression for the photosynthesis apparatus and elevated expression of proteasomal genes which may play a major role in the phytotoxicity of sorgoleone.

Glyphosate: Uses Other Than in Glyphosate-Resistant Crops, Mode of Action, Degradation in Plants, and Effects on Non-target Plants and Agricultural Microbes.

Glyphosate is the most used herbicide globally. It is a unique non-selective herbicide with a mode of action that is ideal for vegetation management in both agricultural and non-agricultural settings. Its use was more than doubled by the introduction of transgenic, glyphosate-resistant (GR) crops. All of its phytotoxic effects are the result of inhibition of only 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), but inhibition of this single enzyme of the shikimate pathway results in multiple phytotoxicity effects, both upstream and downstream from EPSPS, including loss of plant defenses against pathogens. Degradation of glyphosate in plants and microbes is predominantly by a glyphosate oxidoreductase to produce aminomethylphosphonic acid and glyoxylate and to a lesser extent by a C-P lyase to produce sarcosine and phosphate. Its effects on non-target plant species are generally less than that of many other herbicides, as it is not volatile and is generally sprayed in larger droplet sizes with a relatively low propensity to drift and is inactivated by tight binding to most soils. Some microbes, including fungal plant pathogens, have glyphosate-sensitive EPSPS. Thus, glyphosate can benefit GR crops by its activity on some plant pathogens. On the other hand, glyphosate can adversely affect some microbes that are beneficial to agriculture, such as Bradyrhizobium species, although GR crop yield data indicate that such an effect has been minor. Effects of glyphosate on microbes of agricultural soils are generally minor and transient, with other agricultural practices having much stronger effects.

Ecotoxicology of Glyphosate, Its Formulants, and Environmental Degradation Products.

The chemical and biological properties of glyphosate are key to understanding its fate in the environment and potential risks to non-target organisms. Glyphosate is polar and water soluble and therefore does not bioaccumulate, biomagnify, or accumulate to high levels in the environment. It sorbs strongly to particles in soil and sediments and this reduces bioavailability so that exposures to non-target organisms in the environment are acute and decrease with half-lives in the order of hours to a few days. The target site for glyphosate is not known to be expressed in animals, which reduces the probability of toxicity and small risks. Technical glyphosate (acid or salts) is of low to moderate toxicity; however, when mixed with some formulants such as polyoxyethylene amines (POEAs), toxicity to aquatic animals increases about 15-fold on average. However, glyphosate and the formulants have different fates in the environment and they do not necessarily co-occur. Therefore, toxicity tests on formulated products in scenarios where they would not be used are unrealistic and of limited use for assessment of risk. Concentrations of glyphosate in surface water are generally low with minimal risk to aquatic organisms, including plants. Toxicity and risks to non-target terrestrial organisms other than plants treated directly are low and risks to terrestrial invertebrates and microbial processes in soil are very small. Formulations containing POEAs are not labeled for use over water but, because POEA rapidly partitions into sediment, risks to aquatic organisms from accidental over-sprays are reduced in shallow water bodies. We conclude that use of formulations of glyphosate under good agricultural practices presents a de minimis risk of direct and indirect adverse effects in non-target organisms.

Soil Microbial Communities in Diverse Agroecosystems Exposed to the Herbicide Glyphosate.

Despite glyphosate's wide use for weed control in agriculture, questions remain about the herbicide's effect on soil microbial communities. The existing scientific literature contains conflicting results, from no observable effect of glyphosate to the enrichment of agricultural pathogens such as Fusarium spp. We conducted a comprehensive field-based study to compare the microbial communities on the roots of plants that received a foliar application of glyphosate to adjacent plants that did not. The 2-year study was conducted in Beltsville, MD, and Stoneville, MS, with corn and soybean crops grown in a variety of organic and conventional farming systems. By sequencing environmental metabarcode amplicons, the prokaryotic and fungal communities were described, along with chemical and physical properties of the soil. Sections of corn and soybean roots were plated to screen for the presence of plant pathogens. Geography, farming system, and season were significant factors determining the composition of fungal and prokaryotic communities. Plots treated with glyphosate did not differ from untreated plots in overall microbial community composition after controlling for other factors. We did not detect an effect of glyphosate treatment on the relative abundance of organisms such as Fusarium spp.IMPORTANCE Increasing the efficiency of food production systems while reducing negative environmental effects remains a key societal challenge to successfully meet the needs of a growing global population. The herbicide glyphosate has become a nearly ubiquitous component of agricultural production across the globe, enabling an increasing adoption of no-till agriculture. Despite this widespread use, there remains considerable debate on the consequences of glyphosate exposure. In this paper, we examine the effect of glyphosate on soil microbial communities associated with the roots of glyphosate-resistant crops. Using metabarcoding techniques, we evaluated prokaryotic and fungal communities from agricultural soil samples (n = 768). No effects of glyphosate were found on soil microbial communities associated with glyphosate-resistant corn and soybean varieties across diverse farming systems.

Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins.

The mechanism of phytotoxicity of citral was probed in Arabidopsis thaliana using RNA-Seq and in silico binding analyses. Inhibition of growth by 50% by citral downregulated transcription of 9156 and 5541 genes in roots and shoots, respectively, after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the shoots, the downregulation increased at 3 h (6239 genes downregulated, vs 66 upregulated). Of all genes affected in roots at 1 h (time of greatest effect), 7.69% of affected genes were for nucleic acid binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at just 1 h after the treatment and then at 12 and 24 h. Similar effects occurred with the transcription factors MYC-2, ANAC and SCR-SHR, which were also significantly downregulated for the first hour of treatment, and downregulation occurred again after 12 and 24 h treatment. Downregulation of ANAC in the first hour of treatment was significantly (P < 0.0001) decreased more than eight times compared to the control. In silico molecular docking analysis suggests binding of citral isomers to the SSBPs WHY1, WHY2, and WHY3, as well as with other transcription factors such as MYC-2, ANAC and SCR-SHR. Such effects could account for the profound and unusual effects of citral on downregulation of gene transcription.

The Contribution of Romidepsin to the Herbicidal Activity of Burkholderia rinojensis Biopesticide.

The culture broth of Burkholderia rinojensis strain A396 is herbicidal to a number of weed species with greater observed efficacy against broadleaf than grass weeds. A portion of this activity is attributed to romidepsin, a 16-membered cyclic depsipeptide bridged by a 15-membered macrocyclic disulfide. Romidepsin, which is present in small amounts in the broth (18 to 25 µg mL-1), was isolated and purified using standard chromatographic techniques. It was established that romidepsin is a natural proherbicide that targets the activity of plant histone deacetylases (HDAC). Assays to measure plant HDAC activity were optimized by testing a number of HDAC substrates. The activity of romidepsin was greater when its macrocyclic-forming disulfide bridge was reduced to liberate a highly reactive free butenyl thiol side chain. Reduction was achieved using 200 mM tris(2-carboxyethyl)phosphine hydrochloride. A similar bioactivation of the proherbicide via reduction of the disulfide bridge of romidepsin was observed in plant-cell-free extracts. Molecular dynamic simulation of the binding of romidepsin to Arabidopsis thaliana HDAC19 indicated the reduced form of the compound could reach deep inside the catalytic domain and interact with an associated zinc atom required for enzyme activity.

Antimalarials and Phytotoxins from Botryosphaeria dothidea Identified from a Seed of Diseased Torreya taxifolia.

The metabolic pathways in the apicoplast organelle of Plasmodium parasites are similar to those in plastids in plant cells and are suitable targets for malaria drug discovery. Some phytotoxins released by plant pathogenic fungi have been known to target metabolic pathways of the plastid; thus, they may also serve as potential antimalarial drug leads. An EtOAc extract of the broth of the endophyte Botryosphaeria dothidea isolated from a seed collected from a Torreya taxifolia plant with disease symptoms, showed in vitro antimalarial and phytotoxic activities. Bioactivity-guided fractionation of the extract afforded a mixture of two known isomeric phytotoxins, FRT-A and flavipucine (or their enantiomers, sapinopyridione and (-)-flavipucine), and two new unstable γ-lactam alkaloids dothilactaenes A and B. The isomeric mixture of phytotoxins displayed strong phytotoxicity against both a dicot and a monocot and moderate cytotoxicity against a panel of cell lines. Dothilactaene A showed no activity. Dothilactaene B was isolated from the active fraction, which showed moderate in vitro antiplasmodial activity with high selectivity index. In spite of this activity, its instability and various other biological activities shown by related compounds would preclude it from being a viable antimalarial lead.

Synthesis and Biological Evaluation of 6-[(1 R)-1-Hydroxyethyl]-2,4a( R),6( S),8a( R)-tetrahydropyrano-[3,2- b]-pyran-2-one and Structural Analogues of the Putative Structure of Diplopyrone.

The phytotoxin diplopyrone is considered to be the main phytotoxin in a fungus that is responsible for cork oak decline. A carbohydrate-based synthesis of the enantiomer of the structure proposed for diplopyrone has been developed from a commercially available derivative of d-galactose. Key steps in the synthesis are a highly stereoselective pyranose chain-extension based on methyltitanium, preparation of a vinyl glycoside via Isobe C-alkynylation-rearrangement/reduction, and RCM-based pyranopyran construction. Crystallographic and NMR analysis confirms an earlier report that the structure originally proposed for diplopyrone may require revision. Structural analogues were prepared for biological evaluation, the most promising being a pyranopyran nitrile synthesized from tri- O-acetyl-d-galactal by Ferrier cyanoglycosidation, Wittig chain extension, and lactonization. Biological assays revealed potent antibacterial activity for the nitrile analogue against common bacterial pathogens Edwardsiella ictaluri and Flavobacterium columnare that cause enteric septicemia (ESC) and columnaris disease, respectively, in catfish. The IC50 value of 0.002 against E. ictaluri indicates approximately 100 times greater potency than the antibiotic florfenicol used commercially for this disease. Phytotoxic activity for all three target compounds against duckweed was also observed. The antibiotic and phytotoxic activities of the new pyranopyrans synthesized in this study demonstrate the potential of such compounds as antibiotics and herbicides.

Bioassay-Guided Isolation and Structure Elucidation of Fungicidal and Herbicidal Compounds from Ambrosia salsola (Asteraceae).

The discovery of potent natural and ecofriendly pesticides is one of the focuses of the agrochemical industry, and plant species are a source of many potentially active compounds. We describe the bioassay-guided isolation of antifungal and phytotoxic compounds from the ethyl acetate extract of Ambrosia salsola twigs and leaves. With this methodology, we isolated and identified twelve compounds (four chalcones, six flavonols and two pseudoguaianolide sesquiterpene lactones). Three new chalcones were elucidated as (S)-ß-Hydroxy-2',3,4,6'-tetrahydroxy-5-methoxydihydrochalcone (salsolol A), (S)-ß-Hydroxy-2',4,4',6'-tetrahydroxy-3-methoxydihydrochalcone (salsolol B), and (R)-α, (R)-ß-Dihydroxy-2',3,4,4',6'- pentahydroxydihydrochalcone (salsolol C) together with nine known compounds: balanochalcone, six quercetin derivatives, confertin, and neoambrosin. Chemical structures were determined based on comprehensive direct analysis in real time-high resolution mass spectrometry (HR-DART-MS), as well as 1D and 2D NMR experiments: Cosy Double Quantum Filter (DQFCOSY), Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Coherence (HMBC), and the absolute configurations of the chalcones were confirmed by CD spectra analysis. Crystal structure of confertin was determined by X-ray diffraction. The phytotoxicity of purified compounds was evaluated, and neoambrosim was active against Agrostis stolonifera at 1 mM, while confertin was active against both, Lactuca sativa and A. stolonifera at 1 mM and 100 µM, respectively. Confertin and salsolol A and B had IC50 values of 261, 275, and 251 µM, respectively, against Lemna pausicotata (duckweed). The antifungal activity was also tested against Colletotrichum fragariae Brooks using a thin layer chromatography bioautography assay. Both confertin and neoambrosin were antifungal at 100 µM, with a higher confertin activity than that of neoambrosin at this concentration.

Antiplasmodial and Cytotoxic Cytochalasins from an Endophytic Fungus, Nemania sp. UM10M, Isolated from a Diseased Torreya taxifolia Leaf.

Bioassay-guided fractionation of an EtOAc extract of the broth of the endophytic fungus Nemania sp. UM10M (Xylariaceae) isolated from a diseased Torreya taxifolia leaf afforded three known cytochalasins, 19,20-epoxycytochalasins C (1) and D (2), and 18-deoxy-19,20-epoxy-cytochalasin C (3). All three compounds showed potent in vitro antiplasmodial activity and phytotoxicity with no cytotoxicity to Vero cells. These compounds exhibited moderate to weak cytotoxicity to some of the cell lines of a panel of solid tumor (SK-MEL, KB, BT-549, and SK-OV-3) and kidney epithelial cells (LLC-PK11). Evaluation of in vivo antimalarial activity of 19,20-epoxycytochalasin C (1) in a mouse model at 100 mg/kg dose showed that this compound had weak suppressive antiplasmodial activity and was toxic to animals.

A cytochrome P450 CYP71 enzyme expressed in Sorghum bicolor root hair cells participates in the biosynthesis of the benzoquinone allelochemical sorgoleone.

Sorgoleone, a major component of the hydrophobic root exudates of Sorghum spp., is probably responsible for many of the allelopathic properties attributed to members of this genus. Much of the biosynthetic pathway for this compound has been elucidated, with the exception of the enzyme responsible for the catalysis of the addition of two hydroxyl groups to the resorcinol ring. A library prepared from isolated Sorghum bicolor root hair cells was first mined for P450-like sequences, which were then analyzed by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) to identify those preferentially expressed in root hairs. Full-length open reading frames for each candidate were generated, and then analyzed biochemically using both a yeast expression system and transient expression in Nicotiana benthamiana leaves. RNA interference (RNAi)-mediated repression in transgenic S. bicolor was used to confirm the roles of these candidates in the biosynthesis of sorgoleone in planta. A P450 enzyme, designated CYP71AM1, was found to be capable of catalyzing the formation of dihydrosorgoleone using 5-pentadecatrienyl resorcinol-3-methyl ether as substrate, as determined by gas chromatography-mass spectroscopy (GC-MS). RNAi-mediated repression of CYP71AM1 in S. bicolor resulted in decreased sorgoleone contents in multiple independent transformant events. Our results strongly suggest that CYP71AM1 participates in the biosynthetic pathway of the allelochemical sorgoleone.