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Nematicidal Activities of 4‑Quinolone Alkaloids Isolated from theAerial Part of Triumfetta grandidens against Meloidogyne incognita

Ja Yeong Jang,† ,‡ Q uang 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 LifeSciences, 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 Meloidogyneincognita , 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 identied 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 eff ect 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 considerableinhibitory eff ect on egg hatching, which inhibited 91.9 and 87.4% of egg hatching, respectively, after 7 days of exposure at aconcentration 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 theformation of gall on roots of plants at a concentration of 1000 μg/mL. These results suggest that T. grandidens and its bioactive 4-quinolone alkaloids can be used as a potent botanical nematicide in organic agriculture.

KEYWORDS: Meloidogyne incognita, nematicidal activity, Triumfetta grandidens, 4-quinolone alkaloids

■ INTRODUCTION

Nematode infestation is one of the major stresses aff ecting cropproduction worldwide. Plant parasitic nematodes, the mostdevastating pest groups responsible for insidious diseasesymptoms in diff erent crops, are causing signicant economiclosses. Estimated annual yield losses in the world’s major crops

because of plant parasitic nematodes is about 12.3%.1 Rootknot nematodes (RKNs; Meloidogyne spp.), plant parasiticnematodes, 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 importantspecies in RKNs.3 They cause plants to wither throughinducing the formation of giant cells in roots of infected plantsand 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 livesin the soil or plant roots. The nematode cuticle and othersurface organizations make it difficult for many organicmolecules to pass through.4 Even though many syntheticnematicides, such as methyl bromide, aldicarb, and oxamyl, can

be used to control RKNs, most of them are considerably toxicor volatile. Methyl bromide, the most widely used fumigant,

faces prohibition of use in 2015 because of its ozone depletionand human health concern in most countries.5,6 The demandfor environmentally acceptable nematicides that can be appliedin organic farm is increasing.7 Thus, a search for alternatives,such as botanical nematicides, has recently received muchattention, even though their toxic eff ects should be carefully evaluated before commercialization.

Plants are capable of resisting the invasion of plant parasiticnematodes by producing active su bstances because they live inthe soil as stationary organisms.8 These compounds can bedirectly used as botanical nematicides or served as templates forchemically synthesized derivatives to enhance their activity andreduce their environmental impact.4 Plant-derived nematicidalmetabolites were chemically classied into aldehydes, ketones,alkaloids, glycosides, glucosinolates, isothiocyanates, limonoids,quassinoids, saponins, phenolics, avonoids, quinones, piper-amides, polyacetylenes, polythienyls, and terpenes.8 Researchon developing phytochemical-based nematicides was attempted

Received: September 23, 2014Revised: December 5, 2014 Accepted: December 12, 2014

Article

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for control of nematodes. Recently, bionematicides derivedfrom plants, such as Sincocin and NemaKILL, w ere developedusing plant extracts and essential oils, respectively.9 Sincocin, anenvironmentally friendly pesticide, is able to control citrusnematode, reniform nematode, and cyst nematode. However, ithas weak activities against M. incognita.9 NemaKILL, an organicnematicide for the control of soil nematodes of crops, is highly active to RKN and root lesion nematode.

In the search for botanical nematicides from Vietnameseplants, a methanol (MeOH) extract of the aerial part of Triumfetta grandidens Hance (Tiliaceae) showed strong activity against M. incognita. Triumfetta species, widespread acrosstropical regions, is reported to produce biologically activemetabolites, such as triterpenoids, ceramides, alkaloids,triterpenes, polyols, steroids, lupeol, tormentic and oleanolicacids, heptadecanoic acid, β -carotene, and glycosides.10−17

However, whether they have nematicidal compounds was notclear. Therefore, the purpose of this study was to isolate andidentify nematicidal compounds from T. grandidens andevaluate their in vitro and in vivo activities against M. incognita.

■ 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, ethylacetate (EtOAc), n-butanol (BuOH), chloroform (CHCl3), andacetone, were of analytical grade. They are commercially availablefrom 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 IndustrialChemistry, and dried. Plant species was identied, and voucherspecimens were deposited in the laboratory.

Extraction and Isolation of Nematicidal Metabolites. Thedried material of T. grandidens (100 g) was chopped and then

extracted twice with 70% MeOH (2 × 3 L) for 24 h at roomtemperature. The extracts were ltered through a Whatman No. 2lter paper and concentrated using a rotary evaporator under vacuumto yield crude extracts (11.26 g). The MeOH extract was dissolved in500 mL of distilled water and then successively partitioned twice withEtOAc and BuOH. The three layers were assayed for nematicidalactivity against second-stage juveniles (J2s) of M. incognita. Of thethree layers, the EtOAc layer showed the strongest nematicidal activity against M. incognita. Therefore, the EtOAc layer was used for furtherisolation of active compounds.

The EtOAc fraction (3.19 g) was subjected to chromatography on asilica 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 11fractions, F1−F11. The fractions were monitored using thin-layerchromatography (TLC) with the developing solvent CHCl3/MeOH(9:1, v/v). The TLC plate used was a Kieselgel 60GF 254 with 0.25mm layer thickness (E. Merck). The nematicidal activity of thefractions 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 R f value of 0.4. Five fractions (F4−F8) containing theactive spot were combined. The combined F4−F8 fractions (454.5mg) were subjected to Sephadex LH 20 column chromatography. Thecolumn used was a 62 × 2.8 cm inner diameter, glass column, which was packed with 70 g of Sephadex LH20 resin (70−100 μm, Sigma- Aldrich, Vienna, Austria) and eluted with methylene chloride/n-hexane/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 intoShimadzu 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 C 18

reversed-phase column (Atlantis T3, 5 μm, OBD 19 × 250 mm, Waters, Co., Ireland). The solvent system was isocratic of MeOH/0.05% triuoroacetic acid in water (55:45, v/v) at a ow rate of 3.5mL/min. Peaks were collected for nematicidal activity bioassays.Eventually, two pure compounds 1 (8.5 mg) and 2 (9.0 mg) wereisolated as nematicidal metabolites.

Structure Determination of Nematicidal Metabolites. Chem-ical 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. 1Hand 13C NMR spectra were measured in CDCl3 (Cambridge IsotopeLaboratories, Inc. , Woburn, MA) with a Bruker AMX-500spectrometer (Bruker Analytiche Messtechnik Gmbh, Rheinstetten,Germany) at 500 MHz for 1H NMR spectra and 125 MHz for 13CNMR spectra. Chemical shifts were calculated using tetramethylsilane(TMS) as the internal standard. 1H and 13C NMR assignments weresupported by 1H−1H correlation spectroscopy (COSY), heteronuclearmultiple-quantum coherence (HMQC), nuclear Overhauser eff ectspectrometry (NOESY), and heteronuclear multiple-bond correlation(HMBC) experiments. ESI−MS analyses were performed on aMSD1100 single-quadruple mass spectrometer equipped with ESI(Hewlett−Packard Co., Palo Alto, CA). The high-resolution molecularmass values of the two compounds were determined by a Synapt G2HDMS quadrupole time-of-ight (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 agreenhouse at 25 ± 3 °C. Eggs were collected from infected tomatoroots and extracted with 1% NaOCl solutions. The nematode eggs were obtained by passing through a 45 μm sieve. Eggs were collectedon a 25 μm sieve. Surface-sterilized eggs were allowed to hatch inmodied Bearmann funnels18 at 28 °C within 5 days to obtain J2s.

Mortality Bioassay. Stock solutions of test materials obtainedfrom aerial parts of T. grandidens were prepared using MeOH oracetone as the solvent. The nal concentration of organic solvents didnot exceed 1% of the volume. In all cases, working solutions wereprepared 2 times higher than the test concentration. Nematicidalactivities of the MeOH extract and solvent layers were tested at aconcentration range of 7.8−500 μg/mL. Puried compounds weretested 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 96- well 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 thedark at 28 °C. Juveniles were observed under a light microscope after24, 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 ne needle. The experiment using pure compound was conducted twice with ve replicates, and the other experimentsusing solvent layers and the fractions obtained during isolation wereperformed once with three replicates. The value was presented as apercentage of corrected mortality (±standard deviation). Mortality

values were corrected according to Abbott’s formula.19

=

−

− ×

mortality (%) [(mortality percentage in treatment

mortality percentage in control)

/(100 mortality percentage in control)] 100

Hatching Bioassay. Egg suspension was obtained using a sievecombination of 45 and 25 μm mesh sizes. Eggs on the 25 μm sieve were collected in a beaker using distilled water. Approximately 150eggs 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 puriedcompounds at a ratio of 1:1. Plates were gently mixed and covered with a lm to prevent evaporation. The plates were incubated in the

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dark at 28 °C. Nematode egg hatch inhibition rate was assessed withthe aid of a light microscope at 3, 7, and 14 days after treatment.Experiments were conducted twice with ve replicates. Hatchinhibition (HI) was calculated using the following formula:20

= − ×C T CHI (%) [( )/ ] 100

where C and T represented the percentages of egg hatch in the controland treatment, respectively.

Egg hatch was calculated as follows:

= × +percentage of egg hatch 100 juveniles/[eggs juveniles]

In Vivo Experiment. Pot experiments were performed in agreenhouse at 25 °C. A total of 400 g of air-dried and steam-sterilizedsoil (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 wereobtained from the 2 month old tomato roots. About 5000 eggs wereapplied to the roots of tomato seedlings using a micropipette. TheMeOH extract obtained from the aerial part of T. grandidens wasdissolved 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) withdistilled water and served as a positive control. Negative controls were

treated with 0.025% (v/v) Tween 20 containing 5% MeOH. Eachsample was treated at a rate of 20 mL/pot by root drench applicationafter inoculating eggs of M. incognita. Experiments were performedtwice with ve replicates. Pots were treated after 2 weeks with 50 mLof a 0.1% solution of 20−20−20 (N−P−K) fertilizer. Experiments were terminated 6 weeks after inoculation. Galling index (GI) wasassessed, and a 0−5 galling scale was used, where 0 represents no gallson 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, and5 indicates more than 100 galls per root.21 On the other hand,phytotoxicity, such as deformation, discoloration, and reduced growthof the plant treated, was observed.

Statistical Analysis. One-way analysis of variation (ANOVA) withTurkey ’s honest signicant diff erence (HSD) test was used formultiple comparisons ( p = 0.05). The median eff ective concentration(EC50) values were calculated using Microsoft Excel (version 2010

software). Regression analyses were conducted using a linearregression model implemented in Excel. Statistical analyses wereperformed using SAS software (version 12.0, SAS Institute, Cary, NC).Statistical diff erence was considered when a p value was less than 0.05.

■ RESULTS AND DISCUSSION

Isolation and Identication 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. Inthis study, we report nematicidal activity of the MeOH extractfrom the aerial part of T. grandidens against M. incognita.

To isolate active nematicidal compounds, the MeOH extract was sequentially fractionated with EtOAc and BuOH. Twoorganic layers and one aqueous layer were tested for theirnematicidal activity against M. incognita together with MeOHextract. Their activities against J2s of M. incognita aresummarized in Figure 1. The MeOH extract and the EtOAcand BuOH layers showed dose-dependent activity against J2s.However, the water layer was virtually inactive. The EtOAclayer showed the strongest activity against J2s of M. incognita.The EC50 values for the EtOAc layer, MeOH extract, andBuOH layer were 8.35, 56.62, and 120.18 μg/mL, respectively.

Bioassay-guided column chromatography with instrumentalanalyses led to the isolation of two nematicidal compounds

(waltherione A and a new analogue named wlatherione E) fromthe EtOAc layer of the aerial part of T. grandidens.

Compound 1 was obtained as a white amorphous powder.[α ]D

25−29.0 (c 0.003, CHCl3). ESI−MS m/z: 422.06 [M −

H]−. 1H and 13C NMR data are summarized in Table 1 , whichare similar to those of waltherione A and its analogues.22−24

Compound 2 was obtained as a white amorphous powder.[α ]D

25−25.1 (c 0.004, CHCl3). ESI−MS m/z: 394.26 [M + H]

+.1H and 13C NMR spectra are listed in Table 1. These data werein agreemen t with those of waltherione A reportedpreviously.22 ,23

13C and 1H NMR, COSY, HSQC, and HMBC spectra andthe specic rotation value of compound 2 , w altherione A, wereconsistent with its reported literature values.19 High-resolutionESI−QTOF mass spectrometry gave the molecular formulaC23H23NO5 ([M + H]

+ , calcd., m/z 394.1654; found, m/z394.1652). 1H and 13C NMR data of isolated compounds 1 and2 are summarized in Table 1.

Compound 1 , waltherione E, was obtained as an off -whitesolid. High-resolution ESI−QTOF mass spectrometry gave themolecular formula C24H25NO6 ([M + H]

+ , calcd., m/z424.1760; found, m/z 424.1756). 13C NMR, distortionlessenhancement by polarization transfer (DEPT), and HMQCspectra indicated the presence of 14 aromatic carbons betweenδ 111.43 and 153.06, carbonyl carbon at δ 171.06, twooxygenated methine carbons at δ 75.62 and 80.11, anoxygenated quaternary carbon at δ 78.06, two methylenecarbons 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 compound1 revealed the presence of ve aromatic hydrogens between5.89 and 7.66 ppm. The 1H NMR spectrum also revealed fourmethyl 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)], twodiastereotopic 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 and13C NMR data of compounds 1 and 2 were very similar to eachother. However, compound 1 had an additional methoxy groupattached to C-5′ (Table 1). 1H NMR spectra of compound 1indicated 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 showeda disubstituted benzene spin system. The homonuclear 1H−1H

Figure 1. Nematicidal activities of the MeOH extract of the aerial partof 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.


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COSY spectrum of compound 1 did not show a vicinalcoupling between C-5′ and C-6′ (at δ 6.70/6.88 ppm), which

was observed in compound 2 (Figure 2). Furthermore, HMBC

correlation of C-5′ was not observed. The relevant COSY andHMBC correlations in compounds 1 and 2 are shown in Figure3. Thus, compound 1 was identied as 5-methoxywaltherione A and named waltherione E (Figure 2). It is reported for the rsttime in this study.

Waltherione A, a 4-quinolone alkaloid compound previously isolated from Walther ia douradinha , Melochia chamaedrys , and

Melochia odorata ,23−26 has only been isolated from theHermannieae tribe of the Sterculiaceae family. To ourknowledge, the isolation of 4-quinolone alkaloids, such as

waltherione A and E from T. grandidens of Tiliaceae, is reportedfor the rst time in this study.

J2s Mortality and Hatch Inhibition of Compounds 1and 2. The time course eff ects of the puried nematicidalmetabolites on J2s are summarized in Table 2. The twocompounds isolated from the aerial part of T. grandidensexhibited 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 abamectinpositive control (EC50 = 0.13 μg/mL; Table 2). Nematicidal

activities were increased when the exposure time increased to

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

waltherione E waltherione A

position δ C δ H (mult; J , Hz) δ C δ H (mult; J , Hz)

2 140.96, C 141.16. C

3 144.09, C 143.41, C

4 171.06, C 171.97, C

4a 118.50, C 118.83, C

5 141.33, C 141.54, C

6 132.35, C 132.24, C

7 133.17, CH 7.66, d (7.5) 133.06, CH 7.64, d (8.5)

8 117.63, CH 7.38, d (7.0) 117.52, CH 7.52, d (8.0)

8a 138.52, C 138.09, C

9 77.95, C 78.06, C

10 80.05, CH 4.69, d (7.5) 80.11, CH 4.70, d (7.5)

11 22.22, CH2 a (2.03, m), b (2.43, m) 22.22, CH2 a (2.08, m), b (2.41, m)

12 34.05, CH2 a (2.01, m), b (2.33, m) 34.06, CH2 a (2.01, m), b (2.35, m)

13 75.61, CH 6.61, br s 75.62, CH 6.67, d (6.0)

1′ 135.53, C 134.21, C

2′ 150.48, C 156.19, C

3′ 111.43, CH 6.70, dd (9.0, 2.5) 110.89, CH 6.96, d (8.0)

4′ 111.43, CH 6.88, d (8.5) 128.71, CH 7.21, dd (7.5,7.5)

5′ 153.06, C 120.67, CH 6.73, dd (7.5, 7.5)6′ 119.37, CH 5.89, d (2.0) 131.64, CH 6.33, d (7.5)

2C−CH3 14.67, CH3 2.46, s 14.68, CH3 2.46, s

3C−OMe 60.46 CH3 3.78, s 60.29, CH3 3.77, s

2′C−OMe 55.86, CH3 3.95, s 55.51, CH3 4.00, s

5′C−OMe 55.42, CH3 3.54, s

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

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


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72 h, with EC50 values of 0.08 μg/mL for waltherione E and0.18 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 strongeractivity 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 plant-derived compounds based on data reported in the literature.Caboni et al.5 reported that phthalaldehyde, the most activealdehyde 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 v alues of 11± 1 and 12 ± 5 μg/mL, respectively. Le Dang et al.27 reportedthat squamosin G among various annonaceous acetogenninsfrom Annona squamosa seeds showed nematicidal activity against M. incognita , with a LC50 value of 0.287 μg/mL after 72h of exposure.

The two puried compounds in this study also displayed asignicant inhibitory eff ect on egg hatching, with an inhibitory eff ect of over 90% at a concentration of 1.25 μg/mL on day 7compared to the negative control (Figure 4), which was similar

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 aninhibitory eff ect on egg hatching of M. incognita.

Waltherione A was reported to possess antifungal activity against Candida albicans , Cryptococcus neoformans , and Saccha-romyces cerevisiae and acetylcholinesterase inhibitory activ-ity.26 ,28 In addition, waltherione A and its analogue waltherioneC display ed in vitro anti-human immunodeciency virus (HIV)activity.24 The killing eff ect on J2s and inhibitory eff ect on egghatching of waltherione A and E from T. grandidens arereported for the rst time in this study. The nematicidal activity of quinolone alkaloids has not been previously reported. Only thiazolidinones based on uoroquinolone is known to have

activities against Ditylenchus myceliophagus and Caenorhabditiselegans , with LD50 values of 210 and 240 μg/mL, respectively.

29

The mode of action of 4-quinolone alkaloids on nematicidalactivity is likely to involve acetylcholinesterase inhibition.Nematode locomotion depends upon an array of variousneurons and interneurons employing the neurotransmittersacetylcholine and γ -aminobutyric acid. The eff ects of acetylcho-line can be reduced by numerous acetylcholinesteraseinhibitors, such as carbamate and organophosphate nemati-cides.30−32 Even though the degree of acetylcholinesteraseinhibition and nematicidal activity of the carbamate andorganophosphate pesticides does not always correlate,33 ,34

there is a general agreement that their toxic action uponnematodes is caused by their ability to inhibit acetylcholinester-ase.31,35 ,36 However, other mechanisms of the two 4-quinolonealkaloids involved in their nematicidal activity against M.incognita should also be considered.33 ,34

In Vivo Experiments. Disease control efficacy of theMeOH extract of the aerial part of T. grandidens was evaluatedin vivo using the GI index and compared to Sunchungtan, acommercial nematicide with fosthiazate as the active ingredient.The crude extract of T. grandidens at concentrations of 500,1000, and 2000 μg/mL eff ectively reduced the formation of gallon roots of tomato plants (Figure 5). At concentrations of 1000and 2000 μg/mL, the MeOH extract completely suppressedgall formation, which was comparable to the efficacy of Sunchungtan. In contrast, control plants had heavily galledroots (Figure 5). No phytotoxic eff ect of the crude extract to

Table 2. EC50 and R 2 Values of Puried Nematicidal

Metabolites of T. grandidnes and Abamectin against M.incognita at 24, 48, and 72 h after Treatment

EC50 ( μg/mL) EC50 ( μg/mL) EC50 ( μg/mL)

24 h R 2 48 h R 2 72 h R 2

waltherione E 0.15 0.96 0.09 0.94 0.08 0.96

waltherione A 0.26 0.94 0.27 0.96 0.18 0.95

abamectin 0.96 0.91 0.13 0.95 0.11 0.96

Figure 4. Inhibitory eff ects of waltherione E and waltherione A on egghatching of M. incognita 7 days after treatment. Each value representedthe mean ± standard deviation of two runs with ve replicates each.

Figure 5. Eff ect of the MeOH extract from the aerial part of T. graindidens at concentrations of 500−2000 μg/mL on (A) gallformation on tomato plant roots by M. incgonita and (B) treated plants6 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 vereplicates. Relationships among means were analyzed with one-way ANOVA and Turkey ’s HSD test ( p = 0.05). Means with the sameletter were not signicantly diff erent.


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the plants was observed. These results indicate that the crudeextract of T. grandidens has potential as a botanical nematicide.

For the rst time, our research has provided potentnematicidal 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. grandidenseff ectively suppressed gall formation on tomato plant roots.Thus, our results suggest that the solvent extract of the aerialpart of T. grandidens and their 4-quinolone alkaloids could beused as botanical nematicides for the control of RKNs.

■ ASSOCIATED CONTENT

*S Supporting Information

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 andEtOAc extracts of T. grandidens (Table S1). This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Telephone: +82-62-530-2132. Fax: +82-62-530-2139. E-mail:[email protected].

Funding

This study was performed with support from the CooperativeResearch Program for Agricultural Science and Technology Development (Project PJ01020702), Rural Development

Administration, Republic of Korea.

Notes

The authors declare no competing nancial interest.

■ ACKNOWLEDGMENTS

The authors thank Dr. Young Hye Kim [Korea Basic ScienceInstitute (KBSI)] for high-resolution ESI−QTOF massspectrometry analysis.

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