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Khellin and , from visnaga (L.) Lam., as Potential Bioherbicides † ⊥ ⊥ † ‡ Maria L. Travaini,*, , Gustavo M. Sosa, Eduardo A. Ceccarelli, Helmut Walter, § † § § Charles L. Cantrell, Nestor J. Carrillo, Franck E. Dayan, Kumudini M. Meepagala, § and Stephen O. Duke † Instituto de Biologıá Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias Bioquımicaś y Farmaceuticas,́ Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina ‡ AgroField Consulting, Gruenstadter Strasse 82, 67283 Obrigheim, Germany ⊥ Investigaciones Biologicaś en Agroquímicos Rosario S.A. (INBIOAR S.A.), Cordoba 1437, Fifth FloorOffice 2, 2000 Rosario, Argentina § Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 1848, University, Mississippi 38677, United States

*S Supporting Information

ABSTRACT: constitute a source of novel phytotoxic compounds to be explored in searching for effective and environmentally safe herbicides. From a previous screening of extracts for their phytotoxicity, a dichloromethane extract of (L.) Lam. was selected for further study. Phytotoxicity-guided fractionation of this extract yielded two furanochromones, and visnagin, for which herbicidal activity had not been described before. Khellin and visnagin were phytotoxic to model species lettuce (Lactuca sativa) and duckweed (Lemna paucicostata), with IC50 values ranging from 110 to 175 μM. These compounds also inhibited the growth and germination of a diverse group of weeds at 0.5 and 1 mM. These weeds included five grasses [ryegrass (Lolium multiflorum), barnyardgrass (Echinocloa crus-galli), crabgrass (Digitaria sanguinalis), foxtail (Setaria italica), and millet (Panicum sp.)] and two broadleaf species [morningglory (Ipomea sp.) and velvetleaf (Abutilon theophrasti)]. During greenhouse studies visnagin was the most active and showed significant contact postemergence herbicidal activity on velvetleaf and crabgrass at 2 kg active ingredient (ai) ha−1. Moreover, its effect at 4 kg ai ha−1 was comparable to the bioherbicide pelargonic acid at the same rate. The mode of action of khellin and visnagin was not a light-dependent process. Both compounds caused membrane destabilization, photosynthetic efficiency reduction, inhibition of cell division, and cell death. These results support the potential of visnagin and, possibly, khellin as bioherbicides or lead molecules for the development of new herbicides. KEYWORDS: Ammi visnaga, bioherbicide, , herbicide, khellin, phytotoxin, visnagin

■ INTRODUCTION Our group has developed a systematic process to search for, Billions of tons of agricultural production are lost annually due evaluate, and select plant extracts with promising phytotoxic to weeds. Herbicides are the most important method of weed activity. Active extracts could then be used to discover new management, as this technology has been much more effective herbicidal molecules. As a result of this screening process of than previous weed management approaches. After more than nearly 2400 plant extracts for their herbicidal activity, an extract 70 years of the dominance of synthetic herbicides for weed from Ammi visnaga (L.) Lam. was selected for further studies. control, evolved resistance to herbicides has become a major A. visnaga, also known as toothpick weed, visnaga, or khella, − problem.1 3 This problem is exacerbated by the fact that there is an annual or biennial herb belonging to the have been no herbicides with new modes of action introduced (Umbelliferae) family, growing to about 1 m height. The stem in more than 30 years to help manage evolved herbicide is erect and highly branched, and leaves are dissected into many 4 small linear to lance-shaped segments up to 20 cm long. The resistance. Natural phytotoxins are a source of compounds fl with new modes of action, which has fueled interest in their in orescence of A. visnaga is a compound of white 5−7 flowers, and fruits are compressed oval-shaped structures discoveries. Furthermore, the biggest pest management need 10,11 of organic farmers is economical and efficient natural herbicides around 3 mm in length. This herb is native to the approved for use in the organic marketplace.8 Mediterranean region of Europe, Asia, and North Africa, and it Plant natural products provide an attractive alternative in can be found as an introduced species in Argentina, Brazil, finding effective and environmentally safe phytotoxic com- pounds, with high structural diversity and novel modes of Received: June 1, 2016 action.9 Such compounds may be formulated and directly used Revised: October 26, 2016 as bioherbicides or used as lead structures for the development Accepted: November 28, 2016 of new products by chemical modifications. Published: November 28, 2016

© 2016 American Chemical Society 9475 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Chile, Uruguay, North America, Southwest Asia, and some gram of plant material. This procedure provided the following extracts: 10−12 fl fl Atlantic islands. It grows preferentially under high sun owersDCM (1.74 g), owersEtOH (3.57 g), leavesDCM (1.2 g), and exposure in clay soils, which are well drained and quickly leavesEtOH (2.5 g). desiccated on the surface, in the semiarid superior and Germination and Plant Growth Bioassays with Lettuce 13 (Lactuca sativa), Creeping Bentgrass (Agrostis stolonifera), and subhumid bioclimatic zones. In some regions, this plant has Ryegrass (Lolium multiflorum). 14−16 Extracts of A. visnaga and column become an invasive weed of cultivated fields. chromatography fractions were evaluated for their phytotoxic activity Fruits of A. visnaga have been described in pharmacopoeias on lettuce and creeping bentgrass bioassay as described by Dayan et as an antispasmodic, muscle relaxant, and vasodilator.11 Other al.36 Briefly, a filter paper (Whatman no. 1) and five lettuce seeds uses in traditional medicine include treatment of mild (L. sativa L. cv. iceberg A from Burpee Seeds) or 10 mg of creeping symptoms, supportive treatment of mild obstruction of the bentgrass (A. stolonifera var. Penncross from Turf-Seed Inc.) seeds were placed in each well of a 24-well plate. Stock solutions (10 mg respiratory tract in asthma or spastic bronchitis, and post- −1 operative treatment of conditions associated with the presence mL ) of test extracts or fractions were prepared in acetone. Distilled water (180 μL) was added to each well together with 20 μL of the of urinary stones. This herb has also been used as treatment for stock solution or acetone in solvent control. The final concentration gastrointestinal cramps, as a diuretic, and for treatment of −1 11,17 per well was 1 mg mL for extracts or fractions and 10% v/v for , diabetes, and kidney stones. acetone. This percentage of acetone was used only for these Aqueous and ethanolic extracts as well as the essential oil of − miniaturized assays, and two controls were done, with and without A. visnaga have antibacterial activity.18 20 Also, different solvent, to verify that germination and growth of seedlings were extracts of this species and their constituents have antioxidant comparable between control in solvent and control in water. Plates activity21 and prevented renal crystal deposition and cell were sealed with Parafilm and incubated at 26 °C in a Conviron μ −2 −1 damage caused by oxalate.22,23 (Winnipeg, Canada) growth chamber set at 173 mol photons m s With regard to its pesticide uses, alcoholic and aqueous continuous photosynthetically active radiation. Phytotoxic activity was extracts and essential oil of A. visnaga have insecticidal qualitatively evaluated by visually comparing germination and growth ff 24−32 in each well with solvent control at 7 days. A qualitative estimation of properties on di erent insect species. Previous work on phytotoxicity was obtained by using a visual rating scale from 0 to 5, the allelopathic potential of A. visnaga crude extracts reported where 0 was no effect (control), 1 was <50% seedlings shorter than some phytotoxicity toward legumes and maize and toward control, 2 was >50% seedlings shorter than control, 3 was <50% seeds 33−35 weeds associated with wheat cultivation. However, the germinated (only radicles observed), 4 was <50% seeds germinated compounds responsible for these activities of the crude extracts (only radicle tips observed), and 5 was no germination of seeds. This were not isolated and identified. procedure was also used for testing the phytotoxicity of pure The objectives of the present work were to isolate and compounds and for the dose−response assay on lettuce (L. sativa var. Waldmann’s green, Guasch SRL Argentina) and ryegrass identify the phytotoxic compounds from a crude extract of fl A. visnaga and to evaluate their herbicidal effects on different (L. multi orum provided by Dr. Ricardo Pavon, BASF Argentina), instead of creeping bentgrass, because ryegrass is a weed species with a model and weed species in laboratory and greenhouse tests. higher agronomical impact. The experiment with this grass was carried The possible modes of action of the isolated compounds were out in 12-well plates. Each well contained 10 seeds and 300 μL of test also explored. solution. Eight concentrations of the pure compounds (from 0 to 1 mM) were tested. Germination percentage and plant growth (length ■ MATERIALS AND METHODS of plants) were measured at 7 days to determine the concentration Chemicals. Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6- required for 50% germination and growth inhibition (IC50) with methylphenyl)acetamide] (90%) and glyphosate [N- respect to control. All experiments were performed in triplicate. fl (phosphonomethyl)glycine] (48%) were provided by Dr. Ricardo Bioassay-Guided Fractionation. The owersDCM extract of Pavon from BASF, San Jeronimo, Argentina. 4,9-Dimethoxy-7-methyl- A. visnaga (1.6 g) was divided into two 800 mg portions, and each 5H-furo[3,2-g]chromen-5-one, visnagin (khellin) (CAS Registry No. was subjected to column chromatography using an Isolera One system 82-02-0), 4-methoxy-7-methyl-5H-furo[3,2-g]chromen-5-one (visna- (Biotage, Uppsala, Sweden), equipped with a UV detector (302 and gin) (CAS Registry No. 82-57-5), and five structural analogues, 365 nm) and an automatic fraction collector. Separation was khelloside (CAS Registry No. 17226-75-4), (CAS Registry performed by a normal-phase chromatography column using a × μ No. 491-38-3), 4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-7-car- SNAP Cartridge KP-Sil (37 157 mm, 50 m irregular silica, 100 boxylic acid (MDL MFCD00410875), 4-hydroxy-9-methoxy-7-meth- g, Biotage) and a prepackaged SNAP Samplet Cartridge KP-Sil (37 × yl-5H-furo[3,2-g]chromen-5-one (MDL MFCD01533690), and 4,9- 17 mm, Biotage). A full gradient of hexane/ethyl acetate was used for dihydroxy-7-methyl-5H-furo[3,2-g]chromen-5-one (MDL elution, from 100:0 to 0:100 over 3000 mL. The flow rate was 40 mL −1 MFCD18432237), were purchased from Sigma-Aldrich (St. Louis, min , and 25 mL fractions were collected. Equal fractions were MO, USA). The nonionic surfactant AGRI-DEX (Helena Chemical recombined on the basis of similarities in their TLC and chromato- Co., Collierville, TN, USA), consisting of heavy range paraffinic oil, gram profiles, providing 19 fractions named I−XIX. All fractions were polyol fatty acid esters, and polyethoxylated derivatives (99%), was evaluated for their phytotoxicity on the germination bioassay of lettuce purchased from Oxford Farm and Ranch (Oxford, MS, USA). Solvents and creeping bentgrass. were of HPLC grade, and all other chemical supplies were purchased Chemical Analysis and Compound Identification. The crude fl from Sigma-Aldrich. owersDCM extract of A. visnaga and fractions XIV and XVII were Plant Material and Extraction Procedures. A. visnaga (L.) Lam. analyzed by GC-MSD on an Agilent Technologies 7890A GC system (Figure S1) was collected near Los Telares, Salavina Department, coupled to a 5975C Inert XL MSD. The GC was equipped with a DB- Santiago del Estero Province, Argentina, in January 2014. A voucher 5 fused silica capillary column (30 m × 0.25 mm, film thickness of 0.25 specimen, no. SI-209406, has been deposited in the herbarium of the μm) operated under the following conditions: injector temperature, Darwinion Botanical Institute, Buenos Aires, Argentina. Ground dried 240 °C; column temperature, 60−240 °Cat3°C/min then held at flowers (39 g) and leaves (27 g) of A. visnaga were consecutively 240 °C for 5 min; carrier gas, He; injection volume, 1 μL (splitless). extracted by maceration at 25 °C with two solvents: dichloromethane MS mass range was from m/z 40 to 650; filament delay, 3 min; target (DCM) first and then ethanol (EtOH). Plant material was extracted TIC, 20,000; prescan ionization, 100 μs; ion trap temperature, 150 °C; twice (24 h each) with each solvent, followed by Buchner funnel manifold temperature, 60 °C; and transfer line temperature, 170 °C. 1 13 filtration and concentration under reduced pressure in a rotary H and C NMR spectra were recorded in CDCl3 on a Bruker 500 evaporator. For each extraction step, 10 mL of solvent was added per MHz spectrometer (Billerica, MA, USA). High-resolution mass (ESI-

9476 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

MS) spectra of isolated compounds in MeOH were acquired by direct expressed as percent with respect to the solvent control. This − injection of 20 μL of sample (approximately 0.1 mg mL 1) in a JEOL procedure was also used to compare the phytotoxicity of pure USA, Inc. (Peabody, MA, USA) AccuTOF (JMS-T100LC). compounds in light and dark. In these experiments, 5 cm diameter Khellin (fraction XIV): high-resolution ESI-MS m/z 521.14458 [2M Petri dishes were used, with a final volume of 2 mL in each one, and + ff +H] calculated for C28H25O10 521.14477, mass di erence (mmu) the herbicide acetochlor was used at 1 mM. For treatment in the dark, − + 0.19; m/z 543.12622 [2M + Na] calculated for C28H24NaO10 Petri dishes were kept in a dark chamber for 7 days. ff − 1 δ 543.12672, mass di erence (mmu) 0.5; H NMR (in CDCl3) Glyphosate and acetochlor concentrations used were based on the 7.55, 6.92, 5.96, 4.09, 3.96, 2.30, in agreement with published values;37 recommended application rates, considering the surface area of the 13 δ C NMR (in CDCl3) 178.02, 163.84, 148.6, 147.09, 146.86, 145.35, Petri dishes used. All experiments were carried out in triplicate, and 129.66, 119.15, 113.44, 110.38, 104.95, 62.09, 61.27, 19.8, in results are expressed as the mean ± SD. agreement with published values.37,38 Greenhouse Studies. Herbicidal activity of pure khellin and Visnagin (fraction XVII): high-resolution ESI-MS m/z 231.06653 visnagin from Sigma-Aldrich was evaluated according to previously + ff 42 [M + H] calculated for C13H11O4 231.06573, mass di erence (mmu) described methods with some modifications. Compounds were + 0.8; m/z 461.12647 [2M + H] calculated for C26H21O8 461.12364, tested on a broadleaf weed velvetleaf (A. theophrasti) and two grasses, mass difference (mmu) 2.83; m/z 483.11048 [2M + Na]+ calculated crabgrass (D. sanguinalis) and barnyardgrass (E. crus-galli). ff 1 for C26H20NaO8 483.10559, mass di erence (mmu) 4.89; H NMR For all experiments, 10 cm diameter plastic pots were used. Pots δ (in CDCl3) 7.49, 7.09, 6.92, 5.93, 4.07, 2.21, in agreement with were filled with silty clay loam soil collected in a field that has never 39 13 δ published values; C NMR (in CDCl3) 177.98, 163.68, 157.51, been treated with herbicides near the USDA Jamie Whitten Research 155.67, 153.25, 144.94, 116.67, 112.09, 110.53, 105.07, 94.87, 61.49, Center in Stoneville, MS, USA. After seeding, pots were placed in trays 38,39 19.72, in agreement with published values. without drainage holes and watered from the bottom. Pots were Duckweed (Lemna paucicostata (L.) Hegelm.) Bioassay. subsequently moved to trays with drainage holes and watered as Phytotoxic activities of khellin and visnagin were evaluated on needed. Plants were grown in the greenhouse of the NPURU-USDA- 40 fl duckweed by using a previously described method. Brie y, duckweed ARS, Oxford, MS, USA, during May and June of 2015 at 25 °C stocks were grown from a single colony consisting of a mother and two without supplemental illumination. fi daughter fronds in a beaker on modi ed Hoagland medium. The Test compound and herbicide applications were performed fi medium was adjusted to pH 5.5 with 1 M NaOH and ltered through simulating field applications with a Generation III Spray Booth a 0.2 μm filter (no. 431118, Corning Inc.). Each well of nonpyrogenic fi (Devries Manufacturing, Hollandale, MN, USA) equipped with a polystyrene sterile 6-well plates (CoStar 3506, Corning Inc.) was lled model TeeJet EZ 8002 nozzle (Springfield, IL, USA) with conical with 4950 μL of Hoagland medium and 50 μL of double-distilled ° μ μ pattern and 80 spray angle. The distance from the nozzle to soil level water (ddH2O) or 50 L of acetone in solvent control or 50 Lof was 40 cm for all experiments. The spray head was set to move over acetone containing the appropriate concentration of test compound. the plants at 1.3 km h−1, and the apparatus was calibrated to deliver the The final concentration of acetone was 1% v/v. Two, three-frond −1 − equivalent of 400 L ha at a pressure of 207 kPa with agitation. To colonies from 4 5-day-old stock cultures were placed in each well. reach an application rate of 1 kg active ingredient (ai) ha−1, solutions Plates were kept in an incubator (no. CU-36L model, Percival −1 fi μ −2 −1 of test compounds were prepared at 2.5 mg mL . Final rates of 2 and Scienti c) with white light (94.2 mol photons m s ). Total frond 4kgaiha−1 were achieved by spraying two or four times, with a gap of area per well was recorded by the Scanalyzer (LemnaTec, Würselen, 20−40 min between sprays. Pure compounds were dissolved in Germany) image analysis system from days 0 to 7. Percentage of acetone and then in distilled water with surfactant (1% v/v) using increase between days 1 and 7 was determined relative to baseline area agitation. The final concentration of acetone in solution was 1% v/v. at day zero. Experiments were done in triplicate, and nine Three pots of each species were sprayed with each treatment or concentrations (from 0 to 1 mM) were tested. control. The height of the aerial part of the plants was measured 11 Bioassays with Weed Species. Phytotoxicities of pure natural compounds were tested in 9 cm diameter Petri dishes on a diverse days after treatment (DAT). Plants were subsequently removed, fi washed with distilled water, and air-dried on absorbent paper towels group of weeds, including ve grasses [barnyardgrass (Echinochloa ° crus-galli), crabgrass (Digitaria sanguinalis), foxtail (Setaria italica), for 7 days at 35 C for determination of dry weight. millet (Panicum sp.), and ryegrass (L. multiflorum)] and two broadleaf Pre-emergence Activity Assays with Pure Compounds. Selected species [morningglory (Ipomea sp.) and velvetleaf (Abutilon weeds were seeded 1 day before treatment. Ten seeds of velvetleaf and theophrasti)]. For these assays, stock solutions of test compounds 20 of crabgrass were seeded per pot. The soil was watered after (100×) were prepared in acetone, and an aliquot of 40 μL was diluted seeding, and then pots were moved to trays with drainage holes for 24 μ ff h. The following day, pots were sprayed with khellin and visnagin at 2 in 3960 L of distilled water prior to application. The e ect on −1 germination and growth was evaluated. kg ai ha . Tween 20 was used as a surfactant at 1% v/v. An aqueous Postemergence Assay. The assay described by Sampietro et al.41 solution with acetone and surfactant, without compounds, was sprayed was used. Seeds of ryegrass were pre-incubated in 9 cm diameter Petri in control pots. dishes on a moistened filter paper disk with 4 mL of distilled water for Postemergence Activity Assays with Pure Compounds. Seeds of 2 days at 22 °C. After that period, seedlings were moved to another velvetleaf, crabgrass, and barnyardgrass were planted to determine fi Petri dish (20 seedlings per dish) with a filter paper disk. Four germination rates and speci c times required for each species to milliliters of the aqueous solution containing test compound (500 μM) emerge. Once established, pots of each weed species were initiated at was added to each plate. The herbicide glyphosate (750 μM) was used staggered intervals to provide seedlings that would be at a similar as a positive control, and water with acetone (1% v/v) was used as developmental stage at the same time. Velvetleaf plants were thinned solvent control. Lids were sealed with Parafilm, and Petri dishes were to 5 and grasses to 10 seedlings per pot before treatment. incubated at 22 °C for and additional 4 days. Each treatment was For one of the experiments, seedlings of velvetleaf were 6 days old and those of crabgrass 8 days old at treatment. Khellin and visnagin performed in triplicaten and the plant growth (length of plants) was − measured for each condition at 6 days (4 days of treatment). were tested at 2 kg ai ha 1. Tween 20 (1% v/v) was added to the Pre-emergence Assay. Twenty seeds of the species of interest were solution as a surfactant, and the control consisted of water with placed in a 9 cm diameter Petri dish over a filter paper disk. Four acetone and surfactant. milliliters of the aqueous solution containing test compounds (0.5 or 1 Another experiment was conducted with plants at the two to three mM) was added to each plate or the herbicide acetochlor (0.5 mM) as true-leaf stage. Visnagin was sprayed on 13-day-old velvetleaf and 16- − a positive control or water with acetone (1% v/v) as solvent control. day-old crabgrass and barnyardgrass at 4 kg ha 1. The postemergence Lids were sealed with Parafilm, and Petri dishes were incubated at 22 activity of visnagin was compared with the bioherbicide pelargonic acid °C. Percentages of germination and plant growth were measured at 7 at the same rate. AGRI-DEX (1% v/v) was used as a surfactant, and days. For a more direct comparison among different species, results are the control consisted of water with acetone and surfactant.

9477 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Electrolyte Leakage Assay. The effects of pure furanochromones tallying the cells in various stages of mitosis. At least 1000 cells slide−1 (Sigma-Aldrich) on membrane stability were studied as described by and in triplicate (3000 cells per treatment) were counted for suitable Dayan and Watson.43 Cucumber cotyledon disks were exposed to each statistical analysis of data. An Olympus BX60 microscope (Olympus, furanochromone at 100 and 300 μM. Control tissues were exposed to Center Valley, PA, USA) was used, and cells with abnormal mitotic the same solvent as treated tissues but without the compounds. configurations were counted as a separate class. Conductivity measurements were carried out at the beginning of the This procedure was slightly modified to evaluate if cell division dark incubation period, a second measurement was made after 16 h, at inhibition caused by furanochromones was reversible. For this which time the samples were placed under high light intensity, and experiment onion seeds were incubated with solutions containing final measurements were made after 8 and 26 h of light exposure. Each khellin or visnagin for 3 days. After this period, all seeds were washed experiment consisted of three replicates. Maximum conductivity was three times with distilled water and placed in new Petri dishes on fi measured by boiling three samples of each treatment for 20 min. To moistened lter paper disks with ddH2O (wash + treatment). Seeds study if electrolyte leakage caused by khellin and visnagin was light- were then incubated for an additional 4 days, after which a mitotic dependent, two sets of Petri plates were prepared with cucumber index analysis was performed. As a reference to compare results, cotyledon disks exposed to test compounds and control in both. One another set of onion seeds was kept with test compounds or control set was treated as described before (dark 16 h/light 26 h), whereas the for 7 days (wash − treatment) before analysis. other one was kept in darkness for 42 h. Conductivity measurements Cell Death Determination. To evaluate cell death in roots, onion were done at the indicated times. seeds were germinated and treated as described for the postemergence Effect of Compounds on Photosynthetic Efficiency. The assay with weed species. Onion seedlings were exposed to different effect of pure khellin and visnagin was evaluated by chlorophyll doses of each furanochromone (0, 100, and 300 μM) during 4 days. fluorescence measurements according to the procedure of Dayan and For the experiment with leaf disks 3-week cucumber plants were used. Zaccaro.44 Cucumber cotyledon disks were exposed to different One disk (1 cm) was placed in each well of a 12-well plate together dilutions of each furanochromone (10, 30, 100, and 300 μM). Control with 1 mL of a 2% w/v sucrose/1 mM MES, pH 6.5, solution tissues were exposed to the same solvent as treated tissues but without containing each of the compounds tested at the appropriate the test compounds. The cotyledon disks were incubated in darkness concentration (0, 100, or 300 μM) and acetone (1% v/v). Each for 18 h before exposure to light for 24 h. Photosynthetic quantum assay was performed with leaf disks from different plants. Plates were yield and electron transport rate (ETR) were measured. ETR values sealed with Parafilm and incubated in a growth chamber at 21−27 °C were expressed as percent of the ETR average values observed in with a 16/8 h light/dark cycle for 7 days. 47 control treatments. A time course experiment was performed by Cell death was determined by Evans blue staining. Root tips (5 measuring induced fluorescence of cotyledon disks after treatment at 3 mm sections) and leaf disks were incubated for 30 min in 0.25% w/v h in darkness (start) and after 18 h in darkness, after which the Evans blue aqueous solution at 25 °C on a rotary shaker. After samples were placed in the light, and further measurements were made staining, unbound dye was removed by extensive washing with after 6 and 24 h of light exposure. Three replicates were performed for deionized water. Three root tips (three replicates) or one leaf disk each experiment. (four replicates) were ground in a tissue grind tube with 500 μLof1% Detection and Measurement of Reactive Species w/v dodecyl sulfate (SDS). The resulting suspension was (ROS). ROS cellular localization was determined by confocal centrifuged for 20 min at 20000g, and the supernatant was used for dye microscopy using the fluorescent probe 2′,7′-dichlorofluorescein quantification by monitoring the absorbance at 600 and 680 nm. diacetate (DCFDA). Cucumber cotyledon disks (1 cm) were treated Relative cell death was expressed as A600 for root tips and A600/A680 as described for the electrolyte leakage assay. Five disks were placed in ratios for leaf disks.48 5 cm Petri plates and exposed to different dilutions of khellin and Statistical Analysis. Data from dose−response experiments were 40,49 visnagin (0, 100, and 300 μM). Disks were incubated in darkness for analyzed by a log−logistic model using the dose−response curve 50 51 16 h before exposure to high light intensity for 5 h or in darkness for module of R software version 2.2.1. Concentrations required for 30 h. As positive controls, disks were exposed to the same solvent as 50% germination or growth inhibition relative to control (IC50 values) treated tissues but with 10 mM peroxide for 30 min in the were obtained from estimated parameters in the regression curves. The light. After each treatment, they were vacuum-infiltrated in the dark standard error of each estimation is provided. Data from phytotoxicity with 50 μM DCFDA in 10 mM Tris-HCl pH 7.5, and ROS were bioassays in the laboratory and greenhouse, ROS, and cell death visualized in an Eclipse TE-2000-E2 Nikon confocal microscope with quantifications were analyzed by ANOVA using InfoStat statistical 52 excitation at 488 nm and emission at 515/530 nm. Green fluorescence software version 2015, and Scheffe’s test was employed to compare intensities were quantified using the image processing package Fiji of the means at α = 0.05. ImageJ software.45 Effect on Cell Division. Onion seed (Allium cepa L. Evergreen ■ RESULTS AND DISCUSSION Longwhite Bunching, Burpee & Co., 2012, EE.UU) germination was Phytotoxicity Bioassay-Guided Isolation and Identi- carried out for 7 days with a 14 h photoperiod in 9 cm diameter Petri fication of Khellin and Visnagin. Different extracts of dishes on a filter paper disk that was moistened with a dilution (2.5 mL) of test compound or control. Stock solutions of test compounds A. visnaga were phytotoxic to lettuce (L. sativa) and creeping × bentgrass (A. stolonifera)(Table 1). The dichloromethane (100 ) were prepared in acetone, and aliquots were diluted in ddH2O to get the final concentration. The control consisted of water with the same proportion of acetone (1% v/v) applied in the treatments. Table 1. Phytotoxic Activity of Extracts Prepared with At 7 days of incubation, samples were processed for mitotic index Flowers and Leaves of A. visnaga Using Two Different 46 analysis according to the method of Armbruster et al. Twenty root Solvents tips (1 cm sections) were fixed in glacial acetic acid/absolute ethanol (1:3 v/v) for 30 min. The segments were incubated with 5 N HCl at phytotoxicity at 7 daysa 25 °C for 1 h and washed several times with distilled water. After that, −1 ff’ extracts (1 mg mL ) lettuce creeping bentgrass segments were stained with Schi s reagent for 45 min in the dark at fl 25 °C. Stained meristematic regions were identified as purple tips. The owersDCM 35 root segments were transferred with tweezers to a drop of 45% acetic leavesDCM 24 fl acid in water on a microscope slide. The tips were cut with a razor owersEtOH 21 blade, and a coverslip was carefully placed over the tips and gently leavesEtOH 11 squashed by applying slight and constant pressure directly over the tissues. The edge of the coverslip was sealed with nail polish to delay aBioassay rating based on scale of 0−5: 0 = no effect and 5 = no the evaporation of acetic acid. The mitotic index was calculated by growth or germination.

9478 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article fl fl extract of owers ( owersDCM) exhibited the highest inhibitory effect on seed germination and growth. Therefore, its bioassay- guided fractionation was carried out to isolate the phytotoxic compounds. fl The owersDCM crude extract was subjected to normal-phase flash column chromatography, providing 19 fractions, I−XIX. All fractions were evaluated for their phytotoxicity on lettuce and creeping bentgrass at 1 mg mL−1. Four fractions, XIV− XVII, were the most active, causing complete germination inhibition of both species at 7 days (Table 2). The 1H NMR Figure 1. Structures of phytotoxic furanochromones khellin and Table 2. Phytotoxic Activity of Fractions Obtained by Flash visnagin isolated from A. visnaga and the structural analogues tested for Column Chromatography from the FlowersDCM Extract of phytotoxicity. A. visnaga

phytotoxicity at 7 daysa phytotoxic to lettuce and creeping bentgrass as the two b furanochromones isolated from A. visnaga (Table ST1). Five fraction mass (mg) lettuce creeping bentgrass structural analogues commercially available (Figure 1) were I 46.7 0 0 also tested (Table ST1). Interestingly, benzo-γ-pyrone II 62.5 0 0 (chromone), the structure of which is smaller and simpler III 57.6 0 3 than those of khellin and visnagin, showed a similar level of IV 10.7 0 3 activity to them. This may indicate that the rest of the structure V 28.1 0 2 contributes to activity, but it would not be essential for the VI 15.8 0 2 phytotoxicity. Also, the replacement of the methyl group in C-7 VII 58.5 1 3 of khellin by a carboxyl group (as in 4,9-dimethoxy-5-oxo-5H- VIII 28.4 0 3 furo[3,2-g]chromene-7-carboxylic acid) did not increase the IX 38 0 4 phytotoxicity, although it did improve the aqueous of X 45.6 0 4 the molecule. Except for these two compounds, the effects of XI 16.2 0 4 which were close to those of khellin and visnagin, none of the XII 32.8 0 4 other analogues was as phytotoxic as these furanochromones. XIII 28.5 3 4 Accordingly, only the herbicidal activities of khellin and XIV 296.4 5 5 visnagin were studied in more detail. XV 88.7 5 5 Phytotoxic Effects of Khellin and Visnagin on Differ- XVI 76.6 4 5 ent Species, Including Weeds. During dose−response XVII 144.2 4 5 bioassays in the laboratory, both furanochromones inhibited XVIII 18.2 3 5 the growth of duckweed (L. paucicostata) and germination and XIX (MeOH wash) 412.2 0 4 growth of lettuce and ryegrass (L. multiflorum)(Table 3) aBioassay rating based on scale of 0 to 5: 0 = no effect and 5 = no (Figures S8, S10, and S11). For dose−response curves with b − growth or germination. Each fraction was tested at 1 mg mL 1. lettuce and ryegrass, germination and growth of seedlings were comparable between solvent control (acetone 10% v/v) and spectra of these four active fractions revealed that fractions XIV control in water (Figure S12). and XVII were two different pure compounds, whereas the These compounds caused almost complete necrosis of intermediate fractions XV and XVI contained a mixture of duckweed tissue and total inhibition of growth at 333 and different compounds (Figures S2, S4, S5, and S6). The pure 1000 μM(Figures S8 and S9). This model genus (Lemna)is compounds in fractions XIV and XVII were identified by GC- routinely used to study the action of phytotoxins and herbicides MS, high-resolution mass spectrometry, and 1H and 13C NMR by the pesticide industry, environmental toxicologists, and data as the furanochromones khellin (4,9-dimethoxy-7-methyl- others.40,62,63 Image analysis allows for very accurate determi- 5H-furo[3,2-g]chromen-5-one) and visnagin (4-methoxy-7- nation of growth of the same plants over time. The Lemna methyl-5H-furo[3,2-g]chromen-5-one), respectively (Figure species used in this study, L. paucicostata, is smaller than 1). For both compounds, the 1H and 13C chemical shifts L. minor and L. gibba, facilitating miniaturization. Because a matched spectral data reported for khellin (Figures S2 and S3) wide range of herbicides have been previously tested by this − and visnagin (Figures S6 and S7).37 39 method,40,64 it was possible to compare khellin and visnagin These furanochromones have been previously isolated from with those products. The IC values for growth inhibition of − 50 A. visnaga,10,13,53 56 and different medicinal properties and duckweed were 162 ± 29 and 122 ± 28 μM for khellin and biological activities have been described for visnagin, respectively. Comparatively, in a study in which 26 − them.17,22,23,32,37,57 61 However, the phytotoxic or herbicidal different herbicides were tested on duckweed, there was a large fi activities of pure khellin and visnagin have not been reported range of responses with IC50 gures ranging from 0.003 to 407 μ 40 before. M. Therefore, the IC50 values of the isolated furanochro- We therefore assayed the activity of commercial chemical mones are in the middle range of commercial herbicides in this μ standards of khellin and visnagin. We used a standard bioassay, similar to clomazone (IC50 = 126 M) and naphtalam ff μ concentration of 1 mM to compare e ects of compounds of (IC50 = 128 M). unknown phytotoxicity. This concentration of almost all Ryegrass is a weed with high economic impact, which has commercial herbicides provides an effect for comparison. At evolved resistance to herbicides with several modes of action.1 the same concentration, the technical standards were equally Khellin and visnagin also showed a dose-dependent phytotoxic

9479 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Table 3. Mean Inhibitory Concentrations (IC50) of Khellin and Visnagin Required To Reduce Growth and Germination of Three Different Species after 7 Days of Exposure

duckweed lettuce ryegrass ± μ ± μ ± μ ± μ ± μ compound IC50 growth SE ( M) IC50 germination SE ( M) IC50 growth SE ( M) IC50 germination SE ( M) IC50 growth SE ( M) khellin 162 ± 29 701 ± 93 110 ± 11 637 ± 112 244 ± 37 visnagin 122 ± 28 740 ± 98 175 ± 16 502 ± 115 214 ± 23 activity when they were applied to ryegrass seeds (Figure S11). potential, and the effect was compared with the pre-emergent Additionally, the postemergence application of khellin and herbicide acetochlor. visnagin at 500 μM on 2-day-old seedlings of ryegrass in Petri Khellin and visnagin showed a pre-emergence, nonselective dishes significantly reduced the growth of this weed (Figure 2). effect because they significantly reduced the growth of the various weed species tested by application on the seeds (Figure 3A). Germination of the grasses but not the broadleaf species

Figure 2. Phytotoxicity of khellin and visnagin on ryegrass compared to that of glyphosate after postemergence treatment: (A) ryegrass plant growth at 6 days (4 days of treatment); (B) photographs of ryegrass plants exposed to different treatments during 4 days. Each data point represents the mean of three experiments ± SD. Different letters above the bars indicate significant differences between Figure 3. Phytotoxicity of khellin and visnagin on six different weed treatments (p < 0.05). species compared to acetochlor after pre-emergence treatment: (A) plant growth as percent of control at 7 days; (B) germination as percent of control at 7 days; (C) weeds after different treatments. Bars represent, from left to right in each grouping, morningglory, velvetleaf, The plant growth at 6 days was much lower after treatment barnyardgrass, foxtail, millet , and crabgrass. Each data point represents with visnagin than with khellin. The growth inhibitory effect the mean of three experiments ± SD. Different letters above the bars caused by visnagin at 500 μM on ryegrass was similar to the one indicate significant differences between treatments (p < 0.05). caused by the postemergence herbicide glyphosate at 750 μM (Figure 2A). was affected by these treatments (Figure 3B). Interestingly, at Moreover, the phytotoxic activity of khellin and visnagin was 0.5 mM, visnagin caused a reduction in the growth and observed on other problematic weeds. To evaluate if this effect germination of weeds comparable to acetochlor at the same was selective, these compounds were tested on a diverse group dose. Foxtail and crabgrass were shorter after treatment with of target weeds, which included grasses such as barnyardgrass visnagin than with acetochlor. By increasing the concentration (E. crus-galli), crabgrass (D. sanguinalis), foxtail (S. italica), and of both furanochromones to 1 mM, germination of grasses and millet (Panicum sp.), and broadleaf species such as morning- growth of all species were significantly more affected than at 0.5 glory (Ipomea sp.) and velvetleaf (A. theophrasti). As it was mM (Figure 3), and the natural compounds were more observed that these molecules caused the highest reduction in phytotoxic to weeds than 0.5 mM acetochlor. plant growth at 500 μM or more (Figures S8, S10, and S11), To evaluate if khellin and visnagin were also effective on they were used at 0.5 and 1 mM to determine their herbicidal weeds in soil, their herbicidal activity was tested in greenhouse

9480 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article studies. As these compounds showed a generally nonselective The postemergence effect of visnagin at 11 DAT was effect, two weed species were selected for these assays: comparable to the one caused by pelargonic acid at the same crabgrass (D. sanguinalis) as representative of grasses and dose (Figure 5). The dry weight of the three weeds sprayed velvetleaf (A. theophrasti) as representative of broadleaf species. No significant pre-emergence activity 11 DAT was observed when these compounds were sprayed at 2 kg ai ha−1 (data not shown). This result is in contrast to what was detected in laboratory assays in Petri dishes. Many natural phytotoxins may decrease their activities in soil due to several factors.65,66 There are physical, chemical, and microbiological factors that can reduce herbicide activity by making them biologically unavailable or by degrading them by chemical or biological processes.67 On the other hand, during this greenhouse assay, both furanochromones produced a significant postemergence effect 11 DAT at 2 kg ai ha−1 (Figure 4). Visnagin was the most active

Figure 5. Postemergence herbicidal effect of visnagin on three weeds compared to the bioherbicide pelargonic acid during greenhouse assays: (A) plant dry weight 11 DAT; (B) plant height 11 DAT; (C) representative photos of velvetleaf 11 DAT. Bars represent, from left to right in each grouping, barnyardgrass, crabgrass, and velvetleaf. Visnagin and pelargonic acid were tested at 4 kg ai ha−1 on 13−16-day- old (2−3 true leaf stage) plants. Data represent means of three replicates ± SD. Different letters above the bars indicate significant Figure 4. Postemergence effects of khellin and visnagin on velvetleaf differences between treatments (p < 0.05). and crabgrass during greenhouse assays: (A) plant dry weight 11 DAT; (B) plant height 11 DAT; (C) photographs of plants 11 DAT. Bars fi represent, from left to right in each grouping, crabgrass and velvetleaf. with these two compounds was signi cantly smaller than the Furanochromones were tested at 2 kg ai ha−1 on 6−8-day-old plants. control (Figure 5A). A similar reduction was observed on weed Data represent means of three replicates ± SD. Different letters above plant height, except for barnyardgrass, the height of which was the bars indicate significant differences between treatments (p < 0.05). not affected by visnagin or pelargonic acid (Figure 5B). As in the previous greenhouse study, necrotic lesions of leaves after in these conditions, causing a biomass reduction of >50% in treatment with visnagin at 4 kg ai ha−1 indicated it can act as a crabgrass and velvetleaf with respect to control (Figure 4A). It contact herbicide. This was observed with both visnagin and also reduced significantly the height of both weeds, affecting pelargonic acid (Figure 5C). The oldest leaves, which received crabgrass more severely (Figure 4B). Plant growth reduction the spray with these compounds, were significantly more was observed with both compounds but was greater with injured than newer leaves. visnagin. Necrosis was observed on the leaf edges of both Pelargonic acid is a natural fatty acid, which is sold as a weeds sprayed with khellin and visnagin (Figure 4C). Necrotic broad-spectrum bioherbicide for nonselective vegetative burn- lesions were not found in parts of the plants not directly wetted down in many situations. It disrupts plant cell membranes, by the spray, such as new leaves, indicating a contact effect. causing rapid loss of cellular functions.68 This fatty acid is more As visnagin was more active than khellin as a postemergence effective than other bioherbicides such as acetic acid or corn treatment, a second greenhouse study was carried out to gluten meal that must be applied at rates of tons per hectare. evaluate its postemergence herbicidal activity at 4 kg ai ha−1 on Pelargonic acid is recommended at rates of 10−15 kg ai ha−1.69 velvetleaf, crabgrass, and barnyardgrass (E. crus-galli). Because The potential of visnagin as a bioherbicide seems comparable to crabgrass was more sensitive to visnagin, a second grass was that of pelargonic acid according to the results of the included in this assay. As a reference, treatment with pelargonic greenhouse studies. Both compounds caused a significant and acid (n-nonanoic acid) at the same rate was used, because it is a comparable reduction in biomass and height of weeds at commercial herbicide, but also is a natural product. equivalent doses.

9481 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Formulation and chemical modification of lead molecules can death induced by these furanochromones would explain the − greatly improve herbicide efficacy.70 72 The analogues tested in necrosis observed in plants sprayed with these compounds in the course of this study were less active than visnagin or khellin, greenhouse assays. but different analogues could have improved activity. However, At 42 h of incubation, either in the dark or after 26 h of high their identification and study go beyond the objectives of the light intensity exposure, both furanochromones triggered present work. significant electrolyte leakage on cucumber cotyledon disks Studies on the Possible Mode of Action of Khellin and (Figure 6). Ion leakage caused by 100 and 300 μM khellin and Visnagin. The herbicidal activity or phytotoxicity of these two visnagin in the dark (Figure 6B,D) indicates that the mode of furanochromones has not been described before. Therefore, we action of these compounds is not light-dependent and involves conducted several assays that can provide information about cellular leakage. This was also shown when the phytotoxicity of their mode(s) of action. Because the IC50 values of these khellin and visnagin was compared under light and dark compounds to inhibit growth of duckweed, lettuce, and conditions. There were no significant differences in their ryegrass were in the range from 110 ± 11 to 244 ± 37 μM, phytotoxic effects on lettuce in light and darkness (Figure 8). doses in the range of the IC50 values were used for these assays. However, the most intense electrolyte leakage was observed The integrity of the plant plasma membrane is a good after incubation of cucumber cotyledon disks with khellin and biomarker to help identify modes of action of herbicides and visnagin plus 26 h of high light intensity (Figure 6A,C). Under their dependence on light.43 Stress conditions are often these conditions, the effect of these compounds at 100 and 300 accompanied by the accumulation of high levels of ROS μM was comparable to the positive control obtained by boiling exceeding the detoxification mechanisms of plant cells. This can the cotyledon disks and bleaching was also observed (Figure lead to membrane lipid peroxidation resulting in the S13). This may be a consequence of the higher level of ROS uncontrolled release of cellular electrolytes.73 Khellin and produced in light, combined with the stress caused by the visnagin produced a destabilization of the cell membranes at furanochromones. 100 and 300 μM, leading to significant electrolyte leakage Most biocides cause ROS generation as a side effect before (Figure 6). The stress caused by these furanochromones may cell death occurs. To evaluate if there was an increase in ROS trigger this phenomenon through direct or indirect effects. levels after treatment with khellin and visnagin, cucumber cotyledon disks were studied under presymptomatic conditions (before the detection of high levels of electrolyte leakage). As for cell death and electrolyte leakage assays, each compound was tested at 100 and 300 μM. The tissues were incubated in darkness (16 h) before exposure to high light intensity for 5 h. Cucumber cotyledon disks were subsequently treated with the ROS-dependent fluorescent probe DCFDA for visualization by confocal microscopy. In control disks and disks treated with the compounds at 100 μM, most of the label was recovered in chloroplasts as expected in light conditions, co-localizing with chlorophyll autofluor- escence (Figure 9A). Image analysis indicates that ROS levels in cotyledon disks exposed to 300 μM khellin or visnagin were significantly higher than in the control (Figure 9B) and comparable to the treatment with 10 mM H2O2. Under these conditions, additional green fluorescence was detected in other cellular compartments and membranes (Figure 9A), indicating increased peroxidation. According to these results, the treatment with these Figure 6. Electrolyte leakage induced by khellin (A, B) and visnagin furanochromones at 300 μM, together with high light intensity, (C, D) under different conditions of illumination: (A, C) 16 h dark + caused an increase in cellular ROS levels prior to the plasma 26 h light (arrows indicate the start of light exposure). Bars represent, membrane destabilization. However, no significantly higher from left to right in each grouping, start, 16 h dark, 8 h light, and 26 h generation of ROS was detected after treatment with light. (B, D) 42 h dark. Bars represent, from left to right in each compounds at 100 μM plus 5 h of high light intensity (Figure grouping, start, 16 h, 24 h, and 42 h. Data represent means of three replications ± SD. The dotted line represents maximum leakage 9), even though visnagin caused electrolyte leakage at this dose obtained by boiling the cotyledon disks. after 8 h in the light. A longer exposure to light is probably required to detect ROS generation. Thus, the increase in ROS may not be directly associated with the molecular target site of It has been recently suggested that electrolyte leakage, which these compounds, but is more likely to be a secondary or stimulates proteases and endonucleases, and programmed cell tertiary effect of what is causing cellular leakage. Moreover, after death are often linked to each other when plant cells are incubation in complete darkness for 30 h, ROS generation was severely stressed.74 In cucumber leaf disks exposed to 100 μM not detected in cucumber cotyledon disks with either khellin or khellin or visnagin, an increase (∼35%) in relative cell death visnagin at 300 μM(Figure S14). This observation suggests was detected with respect to the control, as estimated by the that the main source of ROS in furanochromones-treated plants Evans blue staining procedure47 (Figure 7A,B). This rose to a is associated with photosynthetic activities. Moreover, whereas 3.5-fold increase when the concentration was 300 μM, ROS propagation may contribute to damage in the light, the indicating severe damage to leaf tissue at the higher dose. basic mechanism of visnagin and khellin toxicity would be Such processes of plasma membrane destabilization and cell ROS-independent.

9482 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Figure 7. Cell death induced by khellin and visnagin in cucumber leaf disks and onion roots: (A) cucumber leaf disks stained with Evans blue after exposure to different doses of khellin and visnagin at 7 days; (B) relative cell death in cucumber leaf disks estimated by Evans blue staining; (C) onion root tips stained with Evans blue after exposure to different doses of khellin and visnagin at 4 days; (D) relative cell death in onion root tips estimated by Evans blue staining. Bars reprsent, from left to right in each grouping, khellin and visnagin. Data are means of three replicates ± SD. Different letters above the bars indicate significant differences between treatments (p < 0.05).

The effect of khellin and visnagin on cell division was evaluated by mitotic index analysis of root meristem cells of onion (A. cepa).36,75 Because the sensitivity of this plant species to these furanochromones was unknown, a broader range of concentrations was tested (0−1000 μM). Both compounds inhibited cell division in a dose-dependent manner (Figure 11A). Visnagin was more active than khellin and completely inhibited onion cell division at 300 μM. Inhibition was not associated with the arrest of a particular phase of mitosis. The percentage of cells observed in each mitotic phase after exposure to these furanochromones was smaller than in the control. However, when there was not a total inhibition, the Figure 8. Comparison of khellin and visnagin phytotoxicity on lettuce under different conditions of illumination. Bars represent, from left to relative proportion of cells in the mitotic phases after treatment right in each grouping, light and darkness. Acetochlor was included as with both compounds was comparable to the control (Figure reference of pre-emergent herbicide, and to get the highest herbicidal 12). A different situation was observed regarding cells with effect, all compounds were tested at 1 mM. Data represent means of abnormal configurations such as chromosome aberrations three replicates ± 1 SD. Different letters above the bars indicate (chromosome bridges, breaks, and losses), nuclear abnormal- significant differences between treatments (p < 0.05). ities (lobulated nuclei, nuclei carrying nuclear buds, polynuclear cells, etc.), or micronuclei. The proportion of these types of cells with abnormal configurations after treatment with khellin Chlorophyll fluorescence measurements showed that khellin or visnagin was higher than in the control (Figure 12). and visnagin significantly reduce the photosynthetic efficiency Inhibition of cellular division caused by khellin and visnagin μ of cucumber cotyledon disks. The ETR was reduced ∼35 and at 100 M was irreversible under the experimental conditions ∼50% after 24 h of incubation in the light (42 h total) with employed (Figure 11B). As in the dose−response assay, we these compounds at 100 and 300 μM, respectively (Figure 10). failed to detect alterations in the distribution of dividing cells These results suggest that photosynthesis is not a primary into the different mitotic phases with respect to the control, target of khellin or visnagin. Considering the long time period except for cells with abnormal configurations, the proportion of of incubation and irradiation required for a significant which after treatment with furanochromones was higher than in photosynthetic efficiency decline, khellin and visnagin probably the control (Figure S15). However, root meristem onion cells affect photosynthesis indirectly, most likely altering chlorophyll did not recover their normal division rate after washing the fluorescence as a consequence of the membrane peroxidation seeds at 3 days with distilled water. This effect might be and destabilization previously detected under these conditions associated with a cell death process induced by these (Figures 6 and 9). compounds. In onion roots exposed to 100 μM khellin or

9483 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Figure 11. Effect of khellin and visnagin on A. cepa root meristem cell division. (A) Amount of dividing cells after exposure to different concentrations of each furanochromone. Bars represent, from left to right in each grouping, khellin and visnagin. (B) Amount of dividing cells with and without washing A. cepa seeds at 3 days with distillated water after an exposure to khellin and visnagin. Bars represent, from left to right in each grouping, without wash and with wash. The amount of dividing cells is expressed as percent of total counted cells at 7 days. Data are means of three replicates.

Figure 9. ROS production in cucumber cotyledon disks exposed to khellin and visnagin: (A) ROS determined using DCFDA and detected by confocal laser scanning microscopy in cucumber cotyledon disks; (B) quantification of DAF fluorescence. Bars represent, from left to right in each grouping, khellin and visnagin. The dotted line represents the DAF fluorescence obtained by treatment of cotyledon ± disks with H2O2. Values are means of three replicates 1 SD. Different letters above the bars indicate significant differences between treatments (p < 0.05). AU, arbitrary units.

Figure 10. Photosynthetic electron transport rate (ETR) in cucumber cotyledon disks exposed to different concentrations of khellin (A) and visnagin (B). Bars represent, from left to right in each grouping, start, 18 h dark, 6 h light, and 24 h light. Data are means of three replications − ff ± SD. The dotted line represents ETR of untreated solvent control. Figure 12. Dose response e ect of khellin and visnagin on A. cepa root meristem cell division. (A) Amount of dividing cells in each phase of mitosis and with abnormal configurations. Bars represent, from left to righ in each grouping, 0 μM (control), 30 μM, 100 μM, 300 μM, visnagin, a 2-fold increase in relative cell death was detected and 1000 μM. (B) Photographs of meristem cells of A. cepa at 7 days: (Figure 7C,D). These furanochromones inhibited cell division, (1, 2) control; (3−7) 30 μM visnagin; (8) 100 μM visnagin; (9−11) μ μ but also caused cell death in onion roots, which was dependent 300 M khellin; (12) 1000 M khellin; P, prophase; M, metaphase; A, on the dose used. At 300 μM, both compounds induced a 2- anaphase; T, telophase; CA, chromosomal aberrations; MN, micro- fold increase in relative cell death compared to 100 μM and a nuclei. The amount of dividing cells is expressed as percent of total counted cells at 7 days. 3−4-fold increase relative to the control conditions (Figure 7C,D).

9484 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

Our results indicate that the mode of action of khellin and visnaga (L.) Lam. (Figure S1). 1H NMR data of fractions visnagin could be a complex process involving multiple targets. XIV (khellin) (Figure S2), XV (Figure S4), XVI (Figure The inhibition of cell division and the increased cell death S5), and XVII (visnagin) (Figure S6). 13C NMR data of caused by these furanochromones, together with cell membrane fractions XIV (khellin) (Figure S3) and XVII (visnagin) destabilization, would account for the reduction in plant (Figure S7). Dose−response curves of khellin and growth. In addition, these effects explain the development of visnagin on duckweed (Figure S8), lettuce (Figure necrosis and abnormal leaves observed after treatment with S10), and ryegrass (Figure S11). Effect of khellin and both compounds. visnagin on the tissue of duckweed (Figure S9) and Because membrane destabilization caused by these furano- cucumber cotyledon disks under different conditions of was intensified after a light irradiation period, and illumination (Figure S13). Comparison of germination considering their chemical nature, a phototoxic effect might be and growth of lettuce and ryegrass in water and solvent expected. Some biological activities caused by furanochro- control (Figure S12). Detection and quantification of mones, as well as their possible role in plant defense, have been − ROS in cucumber cotyledon disks in darkness (Figure associated with their photoactivity.57,58,60,61,76 78 However, S14). Effect of khellin and visnagin on root meristem cell their phytotoxicity was not light-dependent, because both division of A. cepa (Figure S15) (PDF) compounds induced electrolyte leakage in darkness, and the inhibitions of lettuce growth under light and darkness were ■ AUTHOR INFORMATION similar. Corresponding Author Despite the similarities in chemical structures and properties * that furanochromones share with , or psor- (M.L.T) E-mail: [email protected]. alens, they differ in their photochemical properties and in their ORCID ability to photodamage eukaryotic cells and form cross-links.79 Maria L. Travaini: 0000-0003-3675-5280 Visnagin is much less phototoxic and photomutagenic than Eduardo A. Ceccarelli: 0000-0001-5776-3306 when compared at equimolar concentrations and Franck E. Dayan: 0000-0001-6964-2499 equal UV-A doses on the green alga Chlamydomonas 58 80 Notes reinhardtii. According to Martelli et al. visnagin and khellin A provisional patent was filed with the results presented in this could react with DNA and generate activated oxygen species work: “Herbicidal composition comprising chromone deriva- upon UV irradiation. However, in later work, the extent of tives and a method for weed control.” (2015) Provisional photoaddition was low compared with most furanocoumarins, patent in the United States 62/272.880. N/ref.: 577 US PROV. and oxygen-dependent photo-oxidation of DNA was not The authors declare no competing financial interest. observed.81 The absence of DNA photo-oxidation after treatment with visnagin or khellin plus UV-A suggested that ■ ACKNOWLEDGMENTS furanochromones do not have any photodynamic effect on DNA. The phototoxicity of these molecules, albeit low when We are grateful to Robert Johnson, Susan Watson, Amber compared to furanocoumarins, might contribute to their Reichley, and Solomon Green for their excellent technical herbicidal activity. Under high irradiation conditions, ROS assistance. production increased after treatment with khellin and visnagin (Figure 9), resulting in higher oxidative damage to cell ■ ABBREVIATIONS USED membranes and other cellular components. However, the DCM, dichloromethane; EtOH, ethanol; PSI, photosystem I; potential phototoxic effect under high light would not explain PSII, photosystem II; ROS, reactive oxygen species; DCFDA, the phytotoxicity in the dark, indicating that other mechanisms 2′,7′-dichlorofluorescein diacetate are involved. In conclusion, the mode of action of these furanochromones ■ REFERENCES appears to be a complex process. It is not light-dependent and (1) Heap, I. The International Survey of Herbicide Resistant Weeds, involves effects on membrane stability, cell division, and cell http://weedscience.org/ (accessed Jan 30, 2016). viability in leaves and roots that may not be related. Both (2) Duke, S. O.; Heap, I. Evolution of weed resistance to herbicides: compounds also reduce photosynthetic efficiency through what have we learned after seventy years? In Biology, Physiology and indirect effects and induce oxidative damage under high light Molecular Biology of Weeds; Jugulam, M., Ed.; CRC Press: Boca Raton, intensity. Visnagin had the best contact postemergence FL, USA, in press. ff (3) Heap, I. Global perspective of herbicide-resistant weeds. Pest herbicidal activity in greenhouse assays. Its e ect was − comparable to that of the commercial bioherbicide pelargonic Manage. Sci. 2014, 70, 1306 1315. ’ (4) Duke, S. O. Why have no new herbicide modes of action acid at the same rate, indicating visnagin s potential as a appeared in recent years? Pest Manage. Sci. 2012, 68, 505−512. bioherbicide or lead molecule for the discovery of new (5) Dayan, F. E.; Owens, D. K.; Duke, S. O. Rationale for a natural synthetic herbicides. products approach to herbicide discovery. Pest Manage. Sci. 2012, 68, 519−528. ■ ASSOCIATED CONTENT (6) Duke, S. O.; Dayan, F. E. Discovery of new herbicide modes of − * action with natural phytotoxins. ACS Symp. Ser. 2015, No. 1204,79 S Supporting Information 92. The Supporting Information is available free of charge on the (7) Dayan, F. E.; Duke, S. O. Natural compounds as next generation ACS Publications website at DOI: 10.1021/acs.jafc.6b02462. herbicides. Plant Physiol. 2014, 166, 1090−1105. (8) Duke, S. O.; Owens, D. K.; Dayan, F. E. The growing need for Additional data as cited in the manuscript. Phytotoxicity biochemical bioherbicides. ACS Symp. Ser. 2014, No. 1172,31−43. of commercial standards of khellin and visnagin as well as (9) Duke, S. O.; Baerson, S. R.; Cantrell, C. L.; Wedge, D. 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9485 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Journal of Agricultural and Food Chemistry Article

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9487 DOI: 10.1021/acs.jafc.6b02462 J. Agric. Food Chem. 2016, 64, 9475−9487 Travaini et al. (SI)‐ 1

Supporting information

Khellin and Visnagin, Furanochromones from Ammi visnaga (L.) Lam., as

Potential Bioherbicides

Maria L. Travaini,*†┴ Gustavo M. Sosa,┴ Eduardo A. Ceccarelli,† Helmut Walter,‡ Charles L.

Cantrell,§ Nestor J. Carrillo,† Franck E. Dayan,§ Kumudini M. Meepagala,§ and Stephen O. Duke§

†Instituto de Biología Molecular y Celular de Rosario (IBR), CONICET, Facultad de Ciencias

Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario,

Argentina

‡AgroField Consulting, Gruenstadter Str.82, 67283 Obrigheim, Germany

┴Investigaciones Biológicas en Agroquímicos Rosario S.A. (INBIOAR S.A.), Cordoba 1437,

Fifth Floor-Office 2, 2000 Rosario, Argentina

§ Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 1848, University, Mississippi 38677, United States Travaini et al. (SI)‐ 2

Table ST1. Phytotoxicity of Commercial Standards of Khellin and Visnagin, and Five Structural Analogues.

Phytotoxicity at 7 days a Compounds tested at 1 mM Lettuce Creeping bentgrass Fraction XIV (khellin) 3 5 Khellin (technical standard from Sigma-Aldrich) 3 5 Fraction XVII (visnagin) 3 5 Visnagin (technical standard from Sigma-Aldrich) 3 5 Khelloside 1 2 Benzo-γ-pyrone 3 4 4,9-dimethoxy-5-oxo-5H-furo[3,2-g]chromene-7-carboxylic acid 3 3 4-hydroxy-9-methoxy-7-methyl-5H-furo[3,2-g]chromen-5-one 2 1 4,9-dihydroxy-7-methyl-5H-furo[3,2-g]chromen-5-one 3 1 Control 0 0 a Bioassay rating based on scale of 0 to 5: 0= no effect and 5= no growth or germination. Travaini et al. (SI)‐ 3

Figure S1. (A) Ammi visnaga (L.) Lam. growing in Santiago del Estero province, Argentina, where plant material was collected; (B) herbarium specimen prepared for identification of the species. Travaini et al. (SI)‐ 4

Figure S2. 1H NMR (500 MHz) data for fraction XIV (khellin). Travaini et al. (SI)‐ 5

Figure S3.13C NMR (125 MHz) data for fraction XIV (khellin). Travaini et al. (SI)‐ 6

Figure S4. 1H NMR (500 MHz) data for fraction XV. Travaini et al. (SI)‐ 7

Figure S5. 1H NMR (500 MHz) data for fraction XVI. Travaini et al. (SI)‐ 8

Figure S6. 1H NMR (500 MHz) data for fraction XVII (visnagin). Travaini et al. (SI)‐ 9

Figure S7.13C NMR (125 MHz) data for fraction XVII (visnagin) Travaini et al. (SI)‐ 10

Figure S8. Dose-response curves of khellin and visnagin on duckweed (Lemna paucicostata). Percentage of increase between days 1 and 7 was determined relative to baseline area at day zero. Data represents means of three replicates ± SD. Travaini et al. (SI)‐ 11

A

B

Figure S9. Effect of khellin and visnagin on the tissue of duckweed (L. paucicostata): (A) Tissue condition at 7 d; (B) Comparison between initial (day 0) and final conditions (day 7) of duckweed treated with the highest doses of khellin and visnagin. Travaini et al. (SI)‐ 12

Figure S10. Dose-response curves of khellin and visnagin on lettuce (Lactuca sativa). The percentage of germination and length of plants were determined at 7 days. Data represents means of three replicates ± SD. Travaini et al. (SI)‐ 13

Figure S11. Dose-response curves of khellin and visnagin on ryegrass (Lolium multiflorum). The percentage of germination and length of plants were determined at 7 days. Data represents means of three replicates ± SD. Travaini et al. (SI)‐ 14

Figure S12. Germination and plant growth at 7 d in water and solvent control with acetone 10% v/v. Black bars correspond to controls of curves with khellin and grey bars correspond to controls of curves with visnagin. Data are the means of three replicates ± SD. Travaini et al. (SI)‐ 15

Figure S13. Tissue condition of the cucumber cotyledon discs exposed to khellin and visnagin under different conditions of illumination. Travaini et al. (SI)‐ 16

Figure S14. Detection and quantification of ROS in cucumber cotyledon discs in darkness. The dotted line represents the DAF fluorescence obtained by treatment of cotyledon discs with 10 mM H2O2. Black bars correspond to khellin and grey bars to visnagin. Values are means of three replicates ± SD and different letters above the bars indicate significant differences between treatments (p < 0.05). AU, arbitrary units. Travaini et al. (SI)‐ 17

A

Control (0 µM) Control (0 µM) 6 Control (0 µM) + Wash 6 Control (0 µM) + Wash Khellin (100 µM) Visnagin (100 µM) Khellin (100 µM) + Wash Visnagin (100 µM) + Wash 4 4

2 2 Percent of total Percent of total 0 0

B

Figure S15. Evaluation of reversibility of cell division inhibition caused by khellin and visnagin. (A) Amount of dividing cells in each phase of mitosis and with abnormal configurations, without and with washing A. cepa seeds at 3 days with distillated water. (B) Photographs of meristem cells of onion at 7 days, after exposure to different treatments: (1-2) control (wash -); (3-4) control (wash +); (5-7) 100 µM khellin; (8-10) 100 µM khellin (wash +); (11) 100 µM Visnagin; (12-16) 100 µM visnagin (wash +). P, prophase; M, metaphase; A, anaphase; T, telophase; CA, chromosomal aberrations; MN, micronuclei; NA, nuclear abnormality. Amount of dividing cells is expressed as percent of total counted cells at 7 days.