Nematicidal Activities of 4-Quinolone Alkaloids Isolated from the Aerial

Article pubs.acs.org/JAFC

Nematicidal Activities of 4‑Quinolone Alkaloids Isolated from the Aerial Part of Triumfetta grandidens against Meloidogyne incognita Ja Yeong Jang,†,‡ Quang Le Dang,§ Yong Ho Choi,∥ Gyung Ja Choi,∥ Kyoung Soo Jang,∥ Byeongjin Cha,‡ Ngoc Hoang Luu,⊥ and Jin-Cheol Kim*,† †

Division of Applied Bioscience and Biotechnology, Institute of Environmentally Friendly Agriculture, College of Agriculture and Life Sciences, Chonnam National University, 77 Yongbong-Ro, Buk-Gu, Gwangju 500-757, Republic of Korea ‡ Department of Plant Medicine, Chungbuk National University, 52 Naesudong-Ro, Heungdeok-Gu, Cheongju, Chungbuk 361-763, Republic of Korea § Department of Phytochemistry, Vietnam Institute of Industrial Chemistry, 2 Pham Ngu Lao, Hoan Kiem District, Hanoi 10999, Vietnam ∥ Eco-friendly New Materials Research Group, Korea Research Institute of Chemical Technology, Yuseong-Gu, Post Office Box 107, Daejeon 305-600, Republic of Korea ⊥ Vietnam Chemicals Agency, Ministry of Industry and Trade, 91 Dinh Tien Hoang Street, Hanoi 10000, Vietnam S Supporting Information *

ABSTRACT: The methanol extract of the aerial part of Triumfetta grandidens (Tiliaceae) was highly active against Meloidogyne incognita, with second-stage juveniles (J2s) mortality of 100% at 500 μg/mL at 48 h post-exposure. Two 4-quinolone alkaloids, waltherione E (1), a new alkaloid, and waltherione A (2), were isolated and identified as nematicidal compounds through bioassay-guided fractionation and instrumental analysis. The nematicidal activities of the isolated compounds against M. incognita were evaluated on the basis of mortality and effect on egg hatching. Compounds 1 and 2 exhibited high mortalities against J2s of M. incognita, with EC50 values of 0.09 and 0.27 μg/mL at 48 h, respectively. Compounds 1 and 2 also exhibited a considerable inhibitory effect on egg hatching, which inhibited 91.9 and 87.4% of egg hatching, respectively, after 7 days of exposure at a concentration of 1.25 μg/mL. The biological activities of the two 4-quinolone alkaloids were comparable to those of abamectin. In addition, pot experiments using the crude extract of the aerial part of T. grandidens showed that it completely suppressed the formation of gall on roots of plants at a concentration of 1000 μg/mL. These results suggest that T. grandidens and its bioactive 4quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture. KEYWORDS: Meloidogyne incognita, nematicidal activity, Triumfetta grandidens, 4-quinolone alkaloids

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INTRODUCTION Nematode infestation is one of the major stresses affecting crop production worldwide. Plant parasitic nematodes, the most devastating pest groups responsible for insidious disease symptoms in different crops, are causing significant economic losses. Estimated annual yield losses in the world’s major crops because of plant parasitic nematodes is about 12.3%.1 Root knot nematodes (RKNs; Meloidogyne spp.), plant parasitic nematodes, have caused an estimated annual loss to world crop yields of U.S. $118 billion.2 Meloidogyne incognita (Kofoid and White) Chitwood is regarded as one of the most important species in RKNs.3 They cause plants to wither through inducing the formation of giant cells in roots of infected plants and taking nutrients from host plant roots. In addition, they cause physiological plant disorders by aiding infection of pathogenic microorganisms. It is difficult to control RKNs because they spend their lives in the soil or plant roots. The nematode cuticle and other surface organizations make it difficult for many organic molecules to pass through.4 Even though many synthetic nematicides, such as methyl bromide, aldicarb, and oxamyl, can be used to control RKNs, most of them are considerably toxic or volatile. Methyl bromide, the most widely used fumigant, © XXXX American Chemical Society

faces prohibition of use in 2015 because of its ozone depletion and human health concern in most countries.5,6 The demand for environmentally acceptable nematicides that can be applied in organic farm is increasing.7 Thus, a search for alternatives, such as botanical nematicides, has recently received much attention, even though their toxic effects should be carefully evaluated before commercialization. Plants are capable of resisting the invasion of plant parasitic nematodes by producing active substances because they live in the soil as stationary organisms.8 These compounds can be directly used as botanical nematicides or served as templates for chemically synthesized derivatives to enhance their activity and reduce their environmental impact.4 Plant-derived nematicidal metabolites were chemically classified into aldehydes, ketones, alkaloids, glycosides, glucosinolates, isothiocyanates, limonoids, quassinoids, saponins, phenolics, flavonoids, quinones, piperamides, polyacetylenes, polythienyls, and terpenes.8 Research on developing phytochemical-based nematicides was attempted Received: September 23, 2014 Revised: December 5, 2014 Accepted: December 12, 2014

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dx.doi.org/10.1021/jf504572h | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

reversed-phase column (Atlantis T3, 5 μm, OBD 19 × 250 mm, Waters, Co., Ireland). The solvent system was isocratic of MeOH/ 0.05% trifluoroacetic acid in water (55:45, v/v) at a flow rate of 3.5 mL/min. Peaks were collected for nematicidal activity bioassays. Eventually, two pure compounds 1 (8.5 mg) and 2 (9.0 mg) were isolated as nematicidal metabolites. Structure Determination of Nematicidal Metabolites. Chemical structures of the isolated compounds were determined by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization mass spectrometry (ESI−MS) analyses. 1H and 13C NMR spectra were measured in CDCl3 (Cambridge Isotope Laboratories, Inc., Woburn, MA) with a Bruker AMX-500 spectrometer (Bruker Analytiche Messtechnik Gmbh, Rheinstetten, Germany) at 500 MHz for 1H NMR spectra and 125 MHz for 13C NMR spectra. Chemical shifts were calculated using tetramethylsilane (TMS) as the internal standard. 1H and 13C NMR assignments were supported by 1H−1H correlation spectroscopy (COSY), heteronuclear multiple-quantum coherence (HMQC), nuclear Overhauser effect spectrometry (NOESY), and heteronuclear multiple-bond correlation (HMBC) experiments. ESI−MS analyses were performed on a MSD1100 single-quadruple mass spectrometer equipped with ESI (Hewlett−Packard Co., Palo Alto, CA). The high-resolution molecular mass values of the two compounds were determined by a Synapt G2 HDMS quadrupole time-of-flight (QTOF) mass spectrometer equipped with an electrospray ion source (Waters, Milford, MA). Nematode Juveniles. M. incognita was reared on tomatoes (Lycopersicum esculentum Mill. cv. Seokwang) for 2 months in a greenhouse at 25 ± 3 °C. Eggs were collected from infected tomato roots and extracted with 1% NaOCl solutions. The nematode eggs were obtained by passing through a 45 μm sieve. Eggs were collected on a 25 μm sieve. Surface-sterilized eggs were allowed to hatch in modified Bearmann funnels18 at 28 °C within 5 days to obtain J2s. Mortality Bioassay. Stock solutions of test materials obtained from aerial parts of T. grandidens were prepared using MeOH or acetone as the solvent. The final concentration of organic solvents did not exceed 1% of the volume. In all cases, working solutions were prepared 2 times higher than the test concentration. Nematicidal activities of the MeOH extract and solvent layers were tested at a concentration range of 7.8−500 μg/mL. Purified compounds were tested at a concentration range of 0.02−10 μg/mL. Distilled water containing MeOH or acetone alone was used as a negative control. Abamectin, a natural nematicide, was used as a positive control. Freshly hatched J2s were used within 24 h for the bioassay using 96well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ). Aliquots of 25 μL of J2s suspensions (about 50) were placed in each well. Working solutions were then added at a ratio of 1:1 (v/v). Plates were smoothly shaken and exposed to 100% of humidity in a plastic box to avoid evaporation of each well. Plates were incubated in the dark at 28 °C. Juveniles were observed under a light microscope after 24, 48, and 72 h after treatment. Nematodes were judged as dead if their bodies were straight with no movement even if physically stimulated with a fine needle. The experiment using pure compound was conducted twice with five replicates, and the other experiments using solvent layers and the fractions obtained during isolation were performed once with three replicates. The value was presented as a percentage of corrected mortality (±standard deviation). Mortality values were corrected according to Abbott’s formula.19

for control of nematodes. Recently, bionematicides derived from plants, such as Sincocin and NemaKILL, were developed using plant extracts and essential oils, respectively.9 Sincocin, an environmentally friendly pesticide, is able to control citrus nematode, reniform nematode, and cyst nematode. However, it has weak activities against M. incognita.9 NemaKILL, an organic nematicide for the control of soil nematodes of crops, is highly active to RKN and root lesion nematode. In the search for botanical nematicides from Vietnamese plants, a methanol (MeOH) extract of the aerial part of Triumfetta grandidens Hance (Tiliaceae) showed strong activity against M. incognita. Triumfetta species, widespread across tropical regions, is reported to produce biologically active metabolites, such as triterpenoids, ceramides, alkaloids, triterpenes, polyols, steroids, lupeol, tormentic and oleanolic acids, heptadecanoic acid, β-carotene, and glycosides.10−17 However, whether they have nematicidal compounds was not clear. Therefore, the purpose of this study was to isolate and identify nematicidal compounds from T. grandidens and evaluate their in vitro and in vivo activities against M. incognita.

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MATERIALS AND METHODS

Chemicals. Abamectin was purchased from Supelco (Bellefonate, PA). Tween 20 was obtained from Sigma-Aldrich (St. Louis, MO). Sunchungtan 150EC (active ingredients: 30% fosthiazate and 70% surfactant) was purchased from Dongbu Farm Hannong (Daejeon, Korea). All organic solvents used in the study, such as MeOH, ethyl acetate (EtOAc), n-butanol (BuOH), chloroform (CHCl3), and acetone, were of analytical grade. They are commercially available from E. Merck (Darmstadt, Germany) or Daejung Chemicals (Siheung, Korea). Plant Materials. The aerial part of T. grandidens was collected by the Department of Phytochemistry, Vietnam Institute of Industrial Chemistry, and dried. Plant species was identified, and voucher specimens were deposited in the laboratory. Extraction and Isolation of Nematicidal Metabolites. The dried material of T. grandidens (100 g) was chopped and then extracted twice with 70% MeOH (2 × 3 L) for 24 h at room temperature. The extracts were filtered through a Whatman No. 2 filter paper and concentrated using a rotary evaporator under vacuum to yield crude extracts (11.26 g). The MeOH extract was dissolved in 500 mL of distilled water and then successively partitioned twice with EtOAc and BuOH. The three layers were assayed for nematicidal activity against second-stage juveniles (J2s) of M. incognita. Of the three layers, the EtOAc layer showed the strongest nematicidal activity against M. incognita. Therefore, the EtOAc layer was used for further isolation of active compounds. The EtOAc fraction (3.19 g) was subjected to chromatography on a silica gel column (3.5 × 60 cm, Kieselgel 60, 200 g, 70−230 mesh, E. Merck) with elution with CHCl3/MeOH (95:5, v/v), yielding 11 fractions, F1−F11. The fractions were monitored using thin-layer chromatography (TLC) with the developing solvent CHCl3/MeOH (9:1, v/v). The TLC plate used was a Kieselgel 60GF 254 with 0.25 mm layer thickness (E. Merck). The nematicidal activity of the fractions were also performed using the second-stage juvenile (J2s) of M. incognita. The active spot was detected by ultraviolet (UV) light (254 nm) at a Rf value of 0.4. Five fractions (F4−F8) containing the active spot were combined. The combined F4−F8 fractions (454.5 mg) were subjected to Sephadex LH 20 column chromatography. The column used was a 62 × 2.8 cm inner diameter, glass column, which was packed with 70 g of Sephadex LH20 resin (70−100 μm, SigmaAldrich, Vienna, Austria) and eluted with methylene chloride/nhexane/MeOH (5:5:1, v/v/v). It yielded four fractions named F4-1− F4-4. The four fractions were combined (49.3 mg) and injected into Shimadzu LC-6AD prep-high-performance liquid chromatography (HPLC) equipped with a SPD-M10AVP photodiode array detector (Shimadzu, Tokyo, Japan). The column for prep-HPLC was a C18

mortality (%) = [(mortality percentage in treatment − mortality percentage in control) /(100 − mortality percentage in control)] × 100 Hatching Bioassay. Egg suspension was obtained using a sieve combination of 45 and 25 μm mesh sizes. Eggs on the 25 μm sieve were collected in a beaker using distilled water. Approximately 150 eggs in 25 μL of egg suspension were transferred to each well of a 96 well plate, followed by the addition of the working solutions of purified compounds at a ratio of 1:1. Plates were gently mixed and covered with a film to prevent evaporation. The plates were incubated in the B



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dark at 28 °C. Nematode egg hatch inhibition rate was assessed with the aid of a light microscope at 3, 7, and 14 days after treatment. Experiments were conducted twice with five replicates. Hatch inhibition (HI) was calculated using the following formula:20 HI (%) = [(C − T )/C ] × 100 where C and T represented the percentages of egg hatch in the control and treatment, respectively. Egg hatch was calculated as follows: percentage of egg hatch = 100 × juveniles/[eggs + juveniles] In Vivo Experiment. Pot experiments were performed in a greenhouse at 25 °C. A total of 400 g of air-dried and steam-sterilized soil (sand/nursery soil, 1:1, v/v) was added in 9 cm plastic pots. Tomato seedlings grown on clay−loam soil in a greenhouse for 2 weeks were transplanted into the pot. Eggs of M. incognita were obtained from the 2 month old tomato roots. About 5000 eggs were applied to the roots of tomato seedlings using a micropipette. The MeOH extract obtained from the aerial part of T. grandidens was dissolved in MeOH and then diluted with 0.025% (v/v) Tween 20. Sunchungtan 150EC (active ingredients: 30% fosthiazate and 70% surfactant) was diluted 2000-fold (150 μg/mL fosthiazate) with distilled water and served as a positive control. Negative controls were treated with 0.025% (v/v) Tween 20 containing 5% MeOH. Each sample was treated at a rate of 20 mL/pot by root drench application after inoculating eggs of M. incognita. Experiments were performed twice with five replicates. Pots were treated after 2 weeks with 50 mL of a 0.1% solution of 20−20−20 (N−P−K) fertilizer. Experiments were terminated 6 weeks after inoculation. Galling index (GI) was assessed, and a 0−5 galling scale was used, where 0 represents no galls on roots, 1 refers to 1−2 galls per root, 2 indicates 3−10 galls per root, 3 indicates 11−30 galls per root, 4 indicates 31−100 galls per root, and 5 indicates more than 100 galls per root.21 On the other hand, phytotoxicity, such as deformation, discoloration, and reduced growth of the plant treated, was observed. Statistical Analysis. One-way analysis of variation (ANOVA) with Turkey’s honest significant difference (HSD) test was used for multiple comparisons (p = 0.05). The median effective concentration (EC50) values were calculated using Microsoft Excel (version 2010 software). Regression analyses were conducted using a linear regression model implemented in Excel. Statistical analyses were performed using SAS software (version 12.0, SAS Institute, Cary, NC). Statistical difference was considered when a p value was less than 0.05.

Figure 1. Nematicidal activities of the MeOH extract of the aerial part of T. grandidens and its three solvent extracts against the J2s of M. incognita. The mortalities were measured 48 h after treatment. Each value represented the mean ± standard deviation from three replicates.

(waltherione A and a new analogue named wlatherione E) from the EtOAc layer of the aerial part of T. grandidens. Compound 1 was obtained as a white amorphous powder. [α]25 D −29.0 (c 0.003, CHCl3). ESI−MS m/z: 422.06 [M − H]−. 1H and 13C NMR data are summarized in Table 1, which are similar to those of waltherione A and its analogues.22−24 Compound 2 was obtained as a white amorphous powder. + [α]25 D −25.1 (c 0.004, CHCl3). ESI−MS m/z: 394.26 [M + H] . 1 13 H and C NMR spectra are listed in Table 1. These data were in agreement with those of waltherione A reported previously.22,23 13 C and 1H NMR, COSY, HSQC, and HMBC spectra and the specific rotation value of compound 2, waltherione A, were consistent with its reported literature values.19 High-resolution ESI−QTOF mass spectrometry gave the molecular formula C23H23NO5 ([M + H]+, calcd., m/z 394.1654; found, m/z 394.1652). 1H and 13C NMR data of isolated compounds 1 and 2 are summarized in Table 1. Compound 1, waltherione E, was obtained as an off-white solid. High-resolution ESI−QTOF mass spectrometry gave the molecular formula C24H25NO6 ([M + H]+, calcd., m/z 424.1760; found, m/z 424.1756). 13C NMR, distortionless enhancement by polarization transfer (DEPT), and HMQC spectra indicated the presence of 14 aromatic carbons between δ 111.43 and 153.06, carbonyl carbon at δ 171.06, two oxygenated methine carbons at δ 75.62 and 80.11, an oxygenated quaternary carbon at δ 78.06, two methylene carbons at δ 22.22 and 34.06, and three methoxy groups at δ 55.42, 55.86, and 60.46. The 1H NMR spectrum of compound 1 revealed the presence of five aromatic hydrogens between 5.89 and 7.66 ppm. The 1H NMR spectrum also revealed four methyl singlets [three at δ 3.95, 3.78, and 3.54 (3C−OMe, 2′C−OMe, and 5′C−OMe) and one at δ 2.46 (2C−Me)], two diastereotopic methylene groups at δ 2.03/2.43 and 2.01/2.33, and two methine hydrogens at δ 4.69 and 6.61. The 1H NMR spectrum allowed for the assignment of one hydroxyl group at δ 5.12. There was no signal as a result of an amino group, probably because of rapid proton exchange rates. The 1H and 13 C NMR data of compounds 1 and 2 were very similar to each other. However, compound 1 had an additional methoxy group attached to C-5′ (Table 1). 1H NMR spectra of compound 1 indicated the presence of a trisubstituted benzene spin system, H-3′, H-4′, and H-6′, showing a doublet of a doublet, doublet, and doublet, respectively. In comparison, compound 2 showed a disubstituted benzene spin system. The homonuclear 1H−1H

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RESULTS AND DISCUSSION Isolation and Identification of Compounds from T. grandidens. The MeOH extract of the aerial part of T. grandidens exhibited strong nematicidal activity against J2s of M. incognita, with 100% mortality at a concentration of 500 μg/ mL after 48 h of exposure (Figure 1). To our knowledge, the biological activity of T. grandidens has not yet been reported. In this study, we report nematicidal activity of the MeOH extract from the aerial part of T. grandidens against M. incognita. To isolate active nematicidal compounds, the MeOH extract was sequentially fractionated with EtOAc and BuOH. Two organic layers and one aqueous layer were tested for their nematicidal activity against M. incognita together with MeOH extract. Their activities against J2s of M. incognita are summarized in Figure 1. The MeOH extract and the EtOAc and BuOH layers showed dose-dependent activity against J2s. However, the water layer was virtually inactive. The EtOAc layer showed the strongest activity against J2s of M. incognita. The EC50 values for the EtOAc layer, MeOH extract, and BuOH layer were 8.35, 56.62, and 120.18 μg/mL, respectively. Bioassay-guided column chromatography with instrumental analyses led to the isolation of two nematicidal compounds C



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Table 1. 1H and 13C NMR Data for Waltherione E and Waltherione A in CDCl3 (J in Hz) waltherione E position

δC

2 3 4 4a 5 6 7 8 8a 9 10 11 12 13 1′ 2′ 3′ 4′ 5′ 6′ 2C−CH3 3C−OMe 2′C−OMe 5′C−OMe

140.96, C 144.09, C 171.06, C 118.50, C 141.33, C 132.35, C 133.17, CH 117.63, CH 138.52, C 77.95, C 80.05, CH 22.22, CH2 34.05, CH2 75.61, CH 135.53, C 150.48, C 111.43, CH 111.43, CH 153.06, C 119.37, CH 14.67, CH3 60.46 CH3 55.86, CH3 55.42, CH3

waltherione A

δH (mult; J, Hz)

δC

7.66, d (7.5) 7.38, d (7.0)

4.69, d (7.5) a (2.03, m), b (2.43, m) a (2.01, m), b (2.33, m) 6.61, br s

6.70, dd (9.0, 2.5) 6.88, d (8.5) 5.89, 2.46, 3.78, 3.95, 3.54,

d (2.0) s s s s

141.16. 143.41, 171.97, 118.83, 141.54, 132.24, 133.06, 117.52, 138.09, 78.06, 80.11, 22.22, 34.06, 75.62, 134.21, 156.19, 110.89, 128.71, 120.67, 131.64, 14.68, 60.29, 55.51,

δH (mult; J, Hz) C C C C C C CH CH C C CH CH2 CH2 CH C C CH CH CH CH CH3 CH3 CH3

7.64, d (8.5) 7.52, d (8.0)

4.70, d (7.5) a (2.08, m), b (2.41, m) a (2.01, m), b (2.35, m) 6.67, d (6.0)

6.96, 7.21, 6.73, 6.33, 2.46, 3.77, 4.00,

d (8.0) dd (7.5,7.5) dd (7.5, 7.5) d (7.5) s s s

correlation of C-5′ was not observed. The relevant COSY and HMBC correlations in compounds 1 and 2 are shown in Figure 3. Thus, compound 1 was identified as 5-methoxywaltherione A and named waltherione E (Figure 2). It is reported for the first time in this study. Waltherione A, a 4-quinolone alkaloid compound previously isolated from Waltheria douradinha, Melochia chamaedrys, and Melochia odorata,23−26 has only been isolated from the Hermannieae tribe of the Sterculiaceae family. To our knowledge, the isolation of 4-quinolone alkaloids, such as waltherione A and E from T. grandidens of Tiliaceae, is reported for the first time in this study. J2s Mortality and Hatch Inhibition of Compounds 1 and 2. The time course effects of the purified nematicidal metabolites on J2s are summarized in Table 2. The two compounds isolated from the aerial part of T. grandidens exhibited very strong nematicidal activity at 48 h post-exposure, with EC50 values of 0.09 μg/mL for waltherione E and 0.27 μg/ mL for waltherione A, which were comparable to the abamectin positive control (EC50 = 0.13 μg/mL; Table 2). Nematicidal activities were increased when the exposure time increased to

COSY spectrum of compound 1 did not show a vicinal coupling between C-5′ and C-6′ (at δ 6.70/6.88 ppm), which was observed in compound 2 (Figure 2). Furthermore, HMBC

Figure 2. Chemical structures of nematicidal metabolites isolated from the aerial part of T. grandidens.

Figure 3. Significant correlation in COSY (solid lines) and HMBC (arrows) spectra of compounds 1 and 2. D



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Table 2. EC50 and R2 Values of Purified Nematicidal Metabolites of T. grandidnes and Abamectin against M. incognita at 24, 48, and 72 h after Treatment EC50 (μg/mL) waltherione E waltherione A abamectin

2

24 h

R

0.15 0.26 0.96

0.96 0.94 0.91

EC50 (μg/mL) 2

48 h

R

0.09 0.27 0.13

0.94 0.96 0.95

activities against Ditylenchus myceliophagus and Caenorhabditis elegans, with LD50 values of 210 and 240 μg/mL, respectively.29 The mode of action of 4-quinolone alkaloids on nematicidal activity is likely to involve acetylcholinesterase inhibition. Nematode locomotion depends upon an array of various neurons and interneurons employing the neurotransmitters acetylcholine and γ-aminobutyric acid. The effects of acetylcholine can be reduced by numerous acetylcholinesterase inhibitors, such as carbamate and organophosphate nematicides.30−32 Even though the degree of acetylcholinesterase inhibition and nematicidal activity of the carbamate and organophosphate pesticides does not always correlate,33,34 there is a general agreement that their toxic action upon nematodes is caused by their ability to inhibit acetylcholinesterase.31,35,36 However, other mechanisms of the two 4-quinolone alkaloids involved in their nematicidal activity against M. incognita should also be considered.33,34 In Vivo Experiments. Disease control efficacy of the MeOH extract of the aerial part of T. grandidens was evaluated in vivo using the GI index and compared to Sunchungtan, a commercial nematicide with fosthiazate as the active ingredient. The crude extract of T. grandidens at concentrations of 500, 1000, and 2000 μg/mL effectively reduced the formation of gall on roots of tomato plants (Figure 5). At concentrations of 1000 and 2000 μg/mL, the MeOH extract completely suppressed gall formation, which was comparable to the efficacy of Sunchungtan. In contrast, control plants had heavily galled roots (Figure 5). No phytotoxic effect of the crude extract to

EC50 (μg/mL) 72 h

R2

0.08 0.18 0.11

0.96 0.95 0.96

72 h, with EC50 values of 0.08 μg/mL for waltherione E and 0.18 μg/mL for waltherione A. The EC50 value for abamectin was 0.11 μg/mL at 72 h. These results revealed that waltherione E isolated from T. grandidens had a stronger activity than abamectin against J2s of M. incognita. The two 4-quinolone alkaloids isolated from the aerial part of T. grandidens have stronger activities compared to other plantderived compounds based on data reported in the literature. Caboni et al.5 reported that phthalaldehyde, the most active aldehyde among selected aromatic aldehydes, showed a LC50 value of 11 ± 6 μg/mL against J2s of M. incognita, followed by salicylaldehyde and cinnamic aldehyde, with LC50 values of 11 ± 1 and 12 ± 5 μg/mL, respectively. Le Dang et al.27 reported that squamosin G among various annonaceous acetogennins from Annona squamosa seeds showed nematicidal activity against M. incognita, with a LC50 value of 0.287 μg/mL after 72 h of exposure. The two purified compounds in this study also displayed a significant inhibitory effect on egg hatching, with an inhibitory effect of over 90% at a concentration of 1.25 μg/mL on day 7 compared to the negative control (Figure 4), which was similar

Figure 4. Inhibitory effects of waltherione E and waltherione A on egg hatching of M. incognita 7 days after treatment. Each value represented the mean ± standard deviation of two runs with five replicates each.

to that of abamectin. In this study, we clearly demonstrated in vitro that the two 4-quinolone alkaloids produced by T. grandidens possessed strong killing activities against J2s and an inhibitory effect on egg hatching of M. incognita. Waltherione A was reported to possess antifungal activity against Candida albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae and acetylcholinesterase inhibitory activity.26,28 In addition, waltherione A and its analogue waltherione C displayed in vitro anti-human immunodeficiency virus (HIV) activity.24 The killing effect on J2s and inhibitory effect on egg hatching of waltherione A and E from T. grandidens are reported for the first time in this study. The nematicidal activity of quinolone alkaloids has not been previously reported. Only thiazolidinones based on fluoroquinolone is known to have

Figure 5. Effect of the MeOH extract from the aerial part of T. graindidens at concentrations of 500−2000 μg/mL on (A) gall formation on tomato plant roots by M. incgonita and (B) treated plants 6 weeks after inoculation. Sunchungtan (active ingredient 30% fosthiazate), a commercial nematicide, was diluted 2000-fold (150 μg/mL fosthiazate) as a positive control. Values were the mean ± standard deviation of combined results from two experiments with five replicates. Relationships among means were analyzed with one-way ANOVA and Turkey’s HSD test (p = 0.05). Means with the same letter were not significantly different. E



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the plants was observed. These results indicate that the crude extract of T. grandidens has potential as a botanical nematicide. For the first time, our research has provided potent nematicidal activities of the crude extract of the aerial part of T. grandidens and its compounds waltherione E and waltherione A against M. incognita. The crude extract of T. grandidens effectively suppressed gall formation on tomato plant roots. Thus, our results suggest that the solvent extract of the aerial part of T. grandidens and their 4-quinolone alkaloids could be used as botanical nematicides for the control of RKNs.

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ASSOCIATED CONTENT

* Supporting Information S

Typical HPLC chromatogram and UV spectra of waltherione E (1) and waltherione A (2) (Figure S1), high-resolution ESI− QTOF mass spectrum of waltherione E (1) (Figure S2), NMR spectra of waltherione E (1) (Figure S3), and contents of waltherione E (1) and waltherione A (2) in the MeOH and EtOAc extracts of T. grandidens (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +82-62-530-2132. Fax: +82-62-530-2139. E-mail: [email protected]. Funding

This study was performed with support from the Cooperative Research Program for Agricultural Science and Technology Development (Project PJ01020702), Rural Development Administration, Republic of Korea. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Young Hye Kim [Korea Basic Science Institute (KBSI)] for high-resolution ESI−QTOF mass spectrometry analysis.

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REFERENCES

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Nematicidal Activities of 4-Quinolone Alkaloids Isolated from the Aerial

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