Nematicidal Activity of the Volatilome of Eruca sativa on Meloidogyne

Jun 17, 2015 - (9) These species, well represented in the Mediterranean basin, are widely .... The extraction was carried out on a 1 cm DVB/CAR/PDMS 5...
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Nematicidal Activity of the Volatilome of Eruca sativa on Meloidogyne incognita Nadhem Aissani,† Pietro Paolo Urgeghe,§ Chrisostomos Oplos,# Marco Saba,† Graziella Tocco,† Giacomo Luigi Petretto,⊥ Kodjo Eloh,† Urania Menkissoglu-Spiroudi,# Nikoletta Ntalli,† and Pierluigi Caboni*,† †

Department of Life and Environmental Sciences, University of Cagliari, Via Ospedale 72, 09124 Cagliari, Italy Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, I-07100 Sassari, Italy # Pesticide Science Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece ⊥ Department of Chemistry and Pharmacy, University of Sassari, via F. Muroni 23/b, I-07100 Sassari, Italy §

ABSTRACT: Research on new pesticides based on plant extracts, aimed at the development of nontoxic formulates, has recently gained increased interest. This study investigated the use of the volatilome of rucola (Eruca sativa) as a powerful natural nematicidal agent against the root-knot nematode, Meloidogyne incognita. Analysis of the composition of the volatilome, using GC-MS-SPME, showed that the compound (Z)-3-hexenyl acetate was the most abundant, followed by (Z)-3-hexen-1-ol and erucin, with relative percentages of 22.7 ± 1.6, 15.9 ± 2.3, and 8.6 ± 1.3, respectively. Testing of the nematicidal activity of rucola volatile compounds revealed that erucin, pentyl isothiocyanate, hexyl isothiocyanate, (E)-2-hexenal, 2-ethylfuran, and methyl thiocyanate were the most active with EC50 values of 3.2 ± 1.7, 11.1 ± 5.0, 11.3 ± 2.6, 15.0 ± 3.3, 16.0 ± 5.0, and 18.1 ± 0.6 mg/ L, respectively, after 24 h of incubation. Moreover, the nematicidal activity of fresh rucola used as soil amendant in a containerized culture of tomato decreased the nematode infection in a dose-response manner (EC50 = 20.03 mg/g) and plant growth was improved. On the basis of these results, E. sativa can be considered as a promising companion plant in intercropping strategies for tomato growers to control root-knot nematodes. KEYWORDS: rocket salad, root-knot nematodes, plant secondary metabolites, isothiocyanate

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INTRODUCTION Root-knot nematodes (Meloidogyne spp.) are among a vast array of pests continually attacking plants and causing annually significant crop losses in fruit and vegetable production.1 Among strategies to control these pests, natural nematicides isolated from plants or microorganisms tend to be successfully used as biocontrol agents to reduce nontarget exposure to hazardous pesticides and to overcome resistance development.2,3 Plants can produce compounds that directly or indirectly affect their biological environment; these compounds have a dramatic influence on the life cycle of the surrounding living organisms.4 Many scientific studies have reported data on the biological activity of plant secondary metabolites on root-knot nematodes.5,6 With this purpose, we recently discovered that methyl isothiocyanate from Capparis spinosa and allyl isothiocyanate from Armoracia rusticana were active on Meloidogyne incognita.7,8 Consistently, the tetrahydo-3,5dimethyl-1,3,5-thiadiazine-2-thione, the main component of the commercial fumigant nematicide Dazomet, is converted to methylisothiocyanate in the soil. Eruca sativa Mill. (salad rocket), along with Diplotaxis erucoides (wall rocket), Diplotaxis. tenuifolia (wild rocket), and Bunias orientalis (Turkish rocket), is a member of the Brassicaceae family.9 These species, well represented in the Mediterranean basin, are widely used as edible vegetables and spices. E. sativa Mill. is known to contain flavonoids and Dthioglucosides, a class of glucosinolates.10 Importantly, when © XXXX American Chemical Society

plants of rucola sustain tissue damage, vacuole glucosinolates are hydrolyzed by myrosinase (EC 3.2.1.147), a thioglucosidase enzyme, to yield an aglycone that undergoes, depending on various physiological conditions such as the pH and the presence of certain cofactors, nonenzymatic rearrangements to produce volatile isothiocyanates, thiocyanates, indoles, and nitriles.11 Bennet et al.9 found that leaves of Eruca and Diplotaxis contained high amounts of 4-mercaptobutyl glucosinolate with lower levels of 4-methylthiobutyl glucosinolate and 4-methylsulfinylbutyl glucosinolate. Apart from having nematicidal activity, isothiocyanates and thiocyanates are also general biocides, the activity of which is based on irreversible interactions acting as electrophiles that are subject to nucleophilic attack by cysteine residues in biologically critical proteins.12,13 Moreover, being glucosinolate volatile breakdown products, they could represent good candidates for the control of nematodes as safe fumigants in integrated pest management procedures. Many of these volatiles have been shown to act as attractants for certain insects seeking food or egg-laying sites rather than to possess a direct insecticidal activity.14−18 In the past, fumigants such as methyl bromide have been identified as responsible of producing volatile organic Received: February 26, 2015 Revised: June 15, 2015 Accepted: June 17, 2015

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DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

optimization. After the extraction, the fiber was desorbed for 2 min into a Gerstel CIS6 PTV injector operating at 250 °C in a splitless mode. Compound separation and identification was first performed on an Agilent 7890 GC equipped with a Gerstel MPS autosampler, coupled with an Agilent 7000C MSD detector. The chromatographic separation was performed on a VF-Wax 60 m × 0.25 mm i.d., 0.5 μm (J&W Scientific, Folsom, CA, USA), with the following temperature program: 40 °C for 4 min, then increased to 150 °C at a rate of 5.0 °C/min, held for 3 min, then increased to 240 °C at a rate of 10 °C/min, and held for 12 min. Helium was used as the carrier gas at a constant flow of 1 mL/min. The MS detector was set as follows: EI ion source operating at 70 eV, full scan acquisition range between m/z 40 and 650, scan rate of 7.1 cycles/s. Source and quadrupole temperatures were respectively 230 and 150 °C; He and N2 were used respectively as quenching and collision gas at flows of 2.5 and 1 mL/ min, respectively. Further injections were performed, as a confirmation, on a 3800 gas chromatograph directly coupled with a 2000 ion trap mass detector (Varian, Milano, Italy). The separation was achieved on an HP-5MS column of 30 m, 0.25 mm i.d., 0.25 μm (J&W Scientific), with the following temperature program: 50 °C for 5 min, then increased to 250 at a rate of 3.0 °C/min, held for 10 min, and then increased to 300 °C at a rate of 10.0 °C/min. The MS detector was programmed as follows: EI ion source operating at 70 eV, acquisition range between m/z 40 and 650, scan rate of 1 scan/s. The trap, manifold, and transfer line temperature were set to 200, 80, and 200 °C, respectively. Compounds were identified using their MS data (Mass Spectral Library NIST08 MS), the linear retention indices, and comparison with authentic standard (when available). The data were processed using a MassHunter Workstation B.06.00 SP1. Nematode Paralysis Bioassays. A population of M. incognita of Italian origin was reared on tomato (Solanum lycopersicum Mill.) cv. Roma VF for 2 months in a glasshouse at 25 ± 5 °C. Batches of 30 egg masses (averaging 4500 eggs/batch) were collected from infected tomato roots and placed on 2 cm sieves (215 μm diameter), positioned on a 3.5 cm Petri dish. Distilled water was used as a natural hatching agent.23The eggs were then incubated in a growth chamber at 25 ± 2 °C, in the dark. First-emerging second-stage juveniles (J2) were discarded, and only second-stage juveniles collected after 2 days were used for the experiments. Compounds identified in rucola, including carbon disulfide and erucin, were tested on nematodes in a dose range of 1−1000 mg/L using fosthiazate as a chemical control. As part of this study, we also tested a series of structurally related isothiocyanates such as methyl isothiocyanate, butyl isothiocyanate, isobutyl isothiocyanate, pentyl isothiocyanate, hexyl isothiocyanate, benzyl isothiocyanate, phenyl isothiocyanate, and allyl isothiocyanate. Stock solutions of pure compounds were prepared in methanol to overcome insolubility, using aqueous Tween 20 (0.3% v/v) for further dilutions. The final concentration of methanol in each well never exceeded 1% (v/v) because preliminary experiments showed that this concentration was not toxic to the nematodes.23 Distilled water as well as a mixture of methanol and aqueous Tween 20 at 0.3% (v/v) served as controls. In all cases, working solutions were prepared containing twice the test concentration and mixed in 96-well cell culture plates at a ratio of 1:1 (v/v) with a suspension of 25 nematodes added to each well. Multiwell plates were covered to avoid evaporation and maintained in the dark at 20 °C. Juveniles were observed with the aid of an inverted microscope (Zeiss, Gottingen, Germany) at 20× after 1 and 3 days for pure compounds and extracts of E. sativa, respectively. The nematodes were at this point moved to plain water after washing through a 20 μm sieve to remove the excess test compounds. Numbers of motile and paralyzed nematodes were assessed by pricking the juvenile body with a needle, and they were counted. Paralysis experiments were performed three times, and every treatment was replicated six times. Experiment with Containerized Plants. For this experiment we used a clay loam soil, free of root-knot nematodes, with 1.3% organic matter and a pH of 7.8 that was collected from a noncultivated field at the Aristotle University of Thessaloniki Farm (Greece). The soil was sieved through a 3 mm sieve and partially air-dried overnight. The soil was mixed with sand at a ratio of 2:1 to obtain the mixture used for the

compounds (VOCs) contributing to the reduction of the ozone layer and overall leading to poor air quality. These facts led to new agricultural practices such as anaerobic soil disinfestation and biofumigation. Both techniques involve incorporating in the soil large quantities of fresh organic material. To qualify as a good cover crop for the management of plant-parasitic nematodes, the crop should be a poor host for the nematodes and lower the population after incorporation into the soil.19 E. sativa cv. Nemat was found to be a poor host for M. incognita, Meloidogyne javanica, and Meloidogyne hapla when tested as a cover crop in a greenhouse treatment.20 Much of the scientific research on rucola extracts deals with their biological activities, but to the best of our knowledge, there are no reports on the nematicidal activity of E. sativa and its volatile phytochemicals on M. incognita. In the present study we investigated (1) the chemical characterization of the volatilome of E. sativa by means of solid phase microextraction (SPME) followed by GC-MS analysis, (2) the synthesis of the most abundant compound present in volatilome and the nematocidal activity (EC50) of the secondary metabolites of rucola, (3) the disruption of the parasites’ biological cycle in host roots treated with fresh rucola powder, and (4) the efficacy of field-grown rucola, incorporated in situ, on root-knot nematodes.

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MATERIALS AND METHODS

Chemicals. Carbon disulfide, erucin, iberin, and sulforaphane were obtained from Santa Cruz Biotechnology (Dallas, TX, USA); butyl isothiocyanate, isobutyl isothiocyanate, 2-ethylfuran, (E)-2-hexenal, pentyl isothiocyanate, hexyl isothiocyanate, (E)-3-hexen-1-ol, (Z)-3hexen-1-ol, 2-furoic acid, ethyl 2-furoate, 2-furanacetic acid, tetrahydrothiophene, 3-pentanone, dimethyl sulfoxide, and β-cyclocitral were obtained from Sigma-Aldrich (Milano, Italy). Methanol was of highperformance liquid chromatography grade. General Procedure for the Synthesis of (E)- and (Z)-3Hexenyl Acetate. (Z)-3-Hexenyl acetate and (E)-3-hexenyl acetate were prepared as reported in the literature, with slight modifications.21 (Z)- or (E)-3-hexen-1-ol (0.3 g, 2.99 mmol) and 4-(N,Ndimethylamino)pyridine (DMAP) (0.44 g, 3.59 mmol) were dissolved in 7.5 mL of diethyl ether. Acetic anhydride (0.37 g, 3.59 mmol) was then added dropwise with stirring, rapidly producing a white precipitate. The reaction mixture was warmed to 40 °C and stirred for 4 h. The crude mixture was then diluted with diethyl ether and washed with water (10 mL), 10% HCl (3 × 8 mL) and saturated NaHCO3 (3 × 10 mL). The combined organic phase was dried over Na2SO4 and then concentrated under vacuum to provide the desired acetate as a pale yellow oil.1H spectra, recorded on a UnitInova 500 MHz spectrometer (Varian, Walnut Creek, CA, USA), are consistent with those reported in the literature.21 Plant Material. Commercial E. sativa was obtained from a local market in Cagliari (Sardinia, Italy) on March 2014. Samples were used fresh and a voucher specimen was deposited at the Department of Life and Environmental Sciences, University of Cagliari, Cagliari, Italy, for species identification. GC-MS Analysis. SPME analysis was performed following the method proposed by Jirovetz et al.22 with minor modifications. Prior to analysis, the aerial parts of a sample of E. sativa were first ground to a paste in a mortar; 3 g of this paste was then placed into a 20 mL SPME vial, 75.5 × 22.5 mm, which was tightly closed with a septum and allowed to equilibrate for 5 min at 60 °C, and then the fiber was exposed to the headspace. The extraction was carried out on a 1 cm DVB/CAR/PDMS 50/30 Stableflex (Supelco, Milano, Italy) SPME fiber. The fiber was preconditioned at 270 °C for 1 h in a Gerstel MPS bake-out station, according to the manufacturer’s instructions. Prior to and after each analysis, the fiber underwent a further bake-out step for 5 min at 250 °C. The extraction time was fixed to 30 min, based on a previous B

DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 1. Percentage Composition (n = 6) of Rucola Volatile Phytochemicals Determined by SPME/GC-MS Analysis, Listed in Order of Elution DB-WAX peak

volatile compounda

trb (min)

LRIc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34

carbon disulfide 2-ethylfuran tetrahydrothiophene 2-pentenal 1-penten-3-ol 3-ethylthiophene D-limonene (E)-2-hexenal methyl isothiocyanate hexyl acetate methyl thiocyanate (E)-3-hexenyl acetate (Z)-2-penten-1-ol (Z)-3-hexenyl acetate (E)-2-hexenyl acetate 1-hexanol (E)-3-hexen-1-ol (Z)-3-hexen-1-ol nonanal unknown (Z)-3-hexen-1-yl butyrate 1-butene 4-isothiocyanate coeluting with (Z)-3-hexenyl isopentanoate (Z)-3-hexenyl pentanoate 1-pentyl isothiocyanate 4-methylpentyl isothiocyanate hexyl isothiocyanate β-cyclocitral (Z)-3-hexenyl hexanoate (Z)-3-hexenyl lactate 4-ethylbenzaldehyde unknown β-ionone 1-isothiocyanate-3-(methylthio)propane erucin

6.12 11.50 16.70 17.48 18.26 18.77 19.37 20.36 21.06 21.63 22.23 22.72 22.87 23.08 23.50 23.97 24.36 25.01 25.40 26.85 27.15 27.50 27.95 28.44 30.12 31.54 32.53 32.68 34.13 34.47 36.6 38.15 39.01 41.23

744 933 1127 1151 1175 1190 1209 1241 1265 1283 1302 1319 1324 1331 1346 1362 1376 1398 1412 1463 1473 1485 1501 1517 1572 1624 1665 1672 1740 1757 1881 1989 2051 2208

HP-5MS trb (min)

LRIc

IDd

1.72 2.61

613 702

3.35 2.42

747 683

13.50 5.85 3.18

1027 849 736

2.75

711

3.63 12.58 13.09

765 1008 1019

6.18 17.33

853 858 1105

21.34 11.39, 23.51 23.67 16.99 20.16 21.89 22.80

1180 983, 1234 1238 1098 1163 1199 1219

30.34

1388

34.37 26.94 32.17

1485 1310 1432

MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

RI RI, STD STD RI RI RI RI, STD RI, STD RI, STD RI RI, STD RI, STD RI RI, STD RI RI RI, STD RI, STD RI

MS, MS, MS, MS, MS, MS, MS, MS, MS, MS,

RI RI RI RI, STD RI RI, STD RI, STD RI RI RI

MS, RI MS, RI MS, RI, STD

area %e 3.6 0.8 2.4 0.2 0.8 0.3 1.6 2.2 0.5 1.2 1.2 1.7 0.2 22.7 1.0 0.7 0.7 15.9 6.2 0.9 4.8 6.1 1.4 1.0 4.5 2.3 1.2 0.7 1.2 0.4 0.3 1.7 1.0 8.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 0.2 0.1 0.1 0.1 0.1 0.7 0.7 0.1 0.2 0.1 0.4 0.1 1.6 0.1 0.2 0.2 2.3 0.3 0.1 0.8 2.3 0.5 0.1 0.4 0.1 0.1 0.1 0.8 0.2 0.3 0.1 0.1 1.3

a Compounds in order of linear retention index on DB-WAX column. bRetention time on DB-WAX column in GC-MS or HP5-MS column. cLinear retention index (LRI) from a linear equation between each pair of straight-chain alkanes (C7−C23). dIdentification method: LRI agrees with LRIs in the literature; MS, compared with NIST 08 Mass Spectral Database; STD, compared with authentic strandard solutions. ePeak area of each compound/sum of total peak area.

had reached full growth, it was mechanically incorporated in the soil. A week later 7-week-old, at the six-leaf stage, tomato plants, cv. Belladonna, were transplanted. No other treatments were performed for nematode control during the culture period, and the efficacy was assessed 73 days post transplanting by uprooting the plants and categorizing with root index for RKN infection. Successively, the weight of roots and aerial parts was recorded. Statistical Analysis. For containerized bioassays, data were expressed as a percentage decrease in the number of females and galls per gram of root corrected for the control, according to Abbott’s formula:

bioassays. It was separated into seven plastic bags, which received appropriate amounts of fresh rucola paste to achieve concentrations from 4 to 100 mg/g, whereas a treatment with plain water was used as control. Each soil sample was then inoculated with 2500 nematodes of M. incognita and was used for transplanting 7-week-old, at the six-leaf stage, tomato plants, cv. Belladonna, into plastic pots containing 200 g of soil, which were maintained at 27 °C and 60% relative humidity for a 16 h photoperiod; each pot received 20 mL of water every 3 days. Forty days later, root samples were stained with acid fuchsin,24 and the following variables were assessed: fresh root and shoot weight and total number of females and galls of nematodes per gram of root at 10×, under uniform illumination by transparent light within tissue sample. The experiment was conducted twice, and the treatments were always arranged in a completely randomized design with five replicates. Field Experiment. To verify the efficacy under field conditions E. sativa was planted in the field and, after having reached full growth, it was incorporated in soil that subsequently was planted with tomatoes. Specifically, the experiment consisted of two treatments, namely (1) rucola seeds planted in full field surface at a concentration of 5 g/m2 and (2) an untreated control. Each treatment consisted of four replicates, and each plot area was 10 m2. One month later when rucola

⎛ no. of females in treated plot ⎞ corrected % = 100 × ⎜1 − ⎟ no. of females in control pot ⎠ ⎝ It was fitted in the log−logistic model25 to estimate the concentration causing a 50% decrease in females per gram of root (EC50 value). In this regression equation, the rucola paste concentration (mg/g) was the independent variable (x) and the number of females and galls of nematodes (percentage decrease over water control) was the dependent variable (y). Additionally, Tuckey’s test was used to C

DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry separate treatment differences and root or shoot fresh weight, P ≤ 0.05. Because ANOVA indicated no significant treatment by time interaction (between runs of experiments), means were averaged over experiments. Because paralysis in solvent (methanol, Tween-20) did not significantly differ from that observed in distilled water, the percentages of paralyzed nematodes were corrected by eliminating the natural paralysis in the water control (0−5% of total number of nematodes) according to the Schneider−Orelli formula26

Table 2. EC50 Values (n = 4) of Tested Compounds against M. incognita Calculated after 1 Day of Nematode Immersion in Test Solutions

corrected % = [(% mortality in treatment − % mortality in control) /(100 − % mortality in control)] × 100 and they were analyzed (ANOVA) combined over time. Because ANOVA indicated no significant treatment by time interaction, means were averaged over experiments. Corrected percentages of paralyzed nematodes treated with tested compounds were subjected to nonlinear regression analysis using the log−logistic equation proposed by Seefeldt et al.:25

Y = C + (D − C)/{1 + exp[b(log(x) − log(EC50))]} C = lower limit, D = upper limit, b = slope at EC50, and EC50 = test substances’ concentration required for 50% death/paralysis of nematodes after elimination of the control (natural death/paralysis). In the regression equation, the test substances’ concentration was the independent variable (x) and the paralyzed nematodes (percentage increase over water control) was the dependent variable (y). The mean value of the six replicates per test substance concentration and immersion period was used to calculate the EC50 value. Mean data values were reported with the respective standard deviations.

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RESULTS AND DISCUSSION SPME is a useful analytical technique for the semiquantitation of volatile compounds in different matrices, whereas GC-MS is used for compound identification. With these techniques, we identified 32 compounds in E. sativa paste; (Z)-3-hexenyl acetate was the most abundant, followed by (Z)-3-hexen-1-ol, with relative percentages of 22.7 ± 1.6 and 15.9 ± 2.3, respectively (Table 1). Stereoisomers of (E)- and (Z)-3-hexen1-ol are produced by plants when mechanical damage occurs and may have indirect plant defense signaling properties.27 The level of the isothiocyanate erucin in the E. sativa volatilome was 8.6 ± 1.3%, although Jirovetz et al.22 reported levels of approximately 14.2%. According to Cataldi et al., 28 the most abundant glucosinolates isolated from rucola leaves are glucoerucin, 4methylpentyl glucosinolate, 1-hexyl glucosinolate, glucoraphanin, 4-mercaptobutyl glucosinolate, progoitrin, sinigrin, and glucobrassicin.28 In fact, three of the isothiocyanates deriving from the three first mentioned glucosinolates reported above were identified in E. sativa after GC-MS analysis (Table 1). When we tested the nematicidal activity of rucola volatile compounds, we found that erucin, pentyl isothiocyanate, hexyl isothiocyanate, 2-(E)-hexenal, 2-ethylfuran, and methyl thiocyanate were the most active, with EC50 values of 3.2 ± 1.7, 11.1 ± 5.0, 11.3 ± 2.6, 15.0 ± 3.3, 16 ± 5.0, and 18.1 ± 0.6 mg/ L after 24 h of incubation with nematodes, whereas the EC50 for (Z)-3-hexen-1-ol was 323 ± 91 mg/L (Table 2). Figure 1 reports the chemical structures of the most active compounds tested. On the other hand, furan derivatives such as 2-furoic acid, ethyl 2-furoate, and 2-furanacetic acid were not active at 100 mg/L (Table 2). Furthermore, β-cyclocitral found in trace amounts in leaves of E. sativa29 was not active at 100 mg/L. Interestingly, acetates of (E)- and (Z)-3-hexen-1-ol were not

a

compound

EC50/1day (mg/L ± SD)

(1) erucin (2) methyl isothiocyanate (3) pentyl isothiocyanate (4) hexyl isothiocyanate (5) (E)-2-hexenal (6) 2-ethylfuran (7) methyl thiocyanate (8) (Z)-3-hexen-1-ol (9) 3-pentanone (10) tetrahydrothiophene (11) (Z)-3-hexenyl acetate (12) (E)-3-hexenyl acetate (13) carbon disulfide (14) (E)-3-hexen-1-ol (15) benzyl isothiocyanatea (16) allyl isothiocyanatea (17) butyl isothiocyanatea (18) sulforaphane (19) iberin (20) 2-furoic acid (21) ethyl 2-furoate (22) 2-furanacetic acid (23) β-cyclocitral (24) isobutyl isothiocyanate (25) phenyl isothiocyanatea (26) fosthiazatea

3.2 ± 1.7 7.9 ± 3.1 11.1 ± 5.0 11.3 ± 2.6 15.0 ± 3.3 16 ± 5.0 18.1 ± 0.6 323 ± 91 >100 >100 >400 >400 >500 >700 1.9 ± 0.3 6.6 ± 2.5 12 ± 8.0 152 ± 35 180 ± 21 >100 >100 >100 >100 >200 >1000 0.4 ± 0.3

Data published by Aissani et al.8

Figure 1. Chemical structures of most active nematicidal compounds.

D

DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry active at concentrations >400 mg/L. Among isothiocyanate derivatives, not found in the E. sativa volatilome, the most effective in paralyzing M. incognita was benzyl isothiocyanate, followed by allyl isothiocyanate and butyl isothiocyanate, exhibiting EC50/1day values of 1.9 ± 0.3, 6.6 ± 2.5, and 12 ± 8 mg/L, respectively. It is noteworthy that isobutyl isothiocyanate and phenyl isothiocyanate were not active at concentrations >200 and >1000 mg/L, respectively. Sulforaphane was 50 times less active if compared with its reduced isothiocyanate analogue erucin (EC50 = 152 ± 35 mg/L), whereas iberin showed an EC50 of 180 ± 21 mg/L. Finally, carbon disulfide and (E)-3-hexen-1-ol were not found active at concentrations >500 and >700 mg/L, respectively. Interestingly, in our in vitro conditions, butyl isothiocyanate showed a fumigant activity at 160 mg/L (data not shown). Slight structural differences among isothiocyanates conferred significantly different nematicidal effects. This suggests that the biological activity is not only a concentration-dependent function but also related to the alkyl chain characteristics. Notably, Caboni et al.7 observed that nematodes treated with different isothiocyanates were paralyzed in a straight shape with evident internal vacuolization (Figure 2). The same evidence

Table 3. Weight Increase of Aerial Parts after Treatment with Rucola Paste in Nematode-Infested Tomato Plants E. sativa paste (mg/g) 0 4 8 16 32 64 128

aerial part weighta (g) 4.45 4.99 5.61 6.23 7.35 7.14 6.95

± ± ± ± ± ± ±

0.24c 0.15cb 0.05cbd 0.36cbd 0.26d 0.16d 0.15bd

Data are presented as means of five replicates with standard deviations. Means followed by the same letter are not significantly different according to Tukey’s test (P ≤ 0.05). a

In the literature reporting on the use of soil amendants in RKN management, the family Brassicaceae is probably the most cited due to the isothiocyanate levels observed in its members.32 This is the first study on the nematicidal properties of the volatilome of E. sativa on M. incognita, which shows the possibility of its potential use as an intercrop plant. Intercropping, being a cost-effective, environmentally friendly pest management method, can be easily used in developing countries. In our previous works, we largely stressed the importance of compound volatility in the nematicidal character of various botanical extracts.5 This feature is essential for delivering the active substance to the inner parts of the soil infested by nematodes. Herein we report the high activity in containerized and field experiments of rocket and attribute it to its isothiocyanates contents. Nevertheless, under field conditions the efficacy of the isothiocyanates decreases due to their short half-life, making mandatory the repetition of applications,8 a fact that should not be underestimated. The fate in soil of pure isothiocyanates is to be studied with regard to their half-life as ingredients in complex botanical soil amendments. This together with the cost effectiveness of respective treatments could establish a nematode control method suitable for integrated crop management.

Figure 2. M. incognita J2 before (A) and after (B) a 24 h immersion in erucin at 10 mg/L. After treatment, nematodes were paralyzed in a straight shape, and internal vacuoles were evident.

was observed when nematodes were treated with some VATPase inhibitors such as α,β,γ,δ-unsaturated aldehydes and pyocyanin.30,31 Conversely, nematodes treated with the organophosphorous fosthiazate were paralyzed in a coiling shape.7 When the methanolic extract of fresh rucola paste was tested against M. incognita, a clear dose−response relationship was established and significant paralysis/death of nematodes was evident after 3 days of exposure with a calculated EC50 value of 93 ± 59 mg/L (data not shown). According to the results of our experiment with containerized tomato plants infested with M. incognita, when E. sativa paste was incorporated to the growing medium, it decreased root infestation and increased plant growth in a dose-response manner. The EC50 values based on the numbers of females and galls were calculated at 20.02 ± 1.65 (95% CI, 16.64−23.41) and 20.75 ± 1.65 (95% CI, 16.99−24.53) mg/g, respectively. The treatment of tomato plants with rucola paste induced fresh shoot weight increase (Table 3), whereas no root weight increase was observed. Most interestingly, rucola used as a soil amendant fully controlled RKN under our field conditions. Specifically, on the basis of the galling index, all roots were severely knotted in control plants and the root system was absent (root index 10) due to secondary infections by soil pathogens, whereas in rucola-treated plants no knots were evident (root index 1).

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AUTHOR INFORMATION

Corresponding Author

*(P.C.) Phone: +39 070 675 8617. Fax: +39 070 675 8612. Email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Margherita Addis (AGRIS Sardegna, Italy) for helpful suggestions. We are particularly indebted to Prof. Renato Iori and Dr. Gina De Nicola (CRA-Bologna, Italy).

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ABBREVIATIONS USED SPME solid phase microextraction; GC-MS gas chromatography−mass spectrometry; ITC isothiocyanates; RKN rootknot nematodes

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DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.5b02425 J. Agric. Food Chem. XXXX, XXX, XXX−XXX