Naphthoquinones from Walnut Husk Residues Show Strong

This work aimed to assess the effects of pure naphthoquinones (juglone; 1,4-naphthoquinone; plumbagin) on the mortality of the root-knot nematode Melo...
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Naphthoquinones from walnut husk residues show strong nematicidal activities against the root-knot nematode Meloidogyne hispanica Carla Maleita, Ivânia Esteves, Rita Chim, Luís Fonseca, Mara Elga Medeiros Braga, Isabel Abrantes, and Herminio C. C. de Sousa ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b00039 • Publication Date (Web): 10 Mar 2017 Downloaded from http://pubs.acs.org on March 12, 2017

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Naphthoquinones from walnut husk residues show strong nematicidal

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activities against the root-knot nematode Meloidogyne hispanica

3 4

Carla Maleita,*,†,‡ Ivânia Esteves,‡ Rita Chim,† Luís Fonseca,‡ Mara E. M. Braga,†

5

Isabel Abrantes,*,‡ Hermínio C. de Sousa*,†

6 7 8



9

Chemical Engineering Department, University of Coimbra, Rua Sílvio Lima, Pólo II,

CIEPQPF – Chemical Process Engineering and Forest Products Research Centre,

10

Pinhal de Marrocos, 3030-790 Coimbra, Portugal.

11



12

Coimbra, Calçada Martim de Freitas, 3000-456, Coimbra, Portugal.

CFE- Centre for Functional Ecology, Department of Life Sciences, University of

13 14 15 16

*Corresponding authors:

17

E-mail: [email protected] (C.M.); [email protected] (I.A.); [email protected]

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(H.C.d.S.).Telephone: +351239798749. Fax: +351239798703.

19 20

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ABSTRACT: Naphthoquinones exhibit important biological activities and are

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present in walnut husks residues in significant amounts. However, their potential as

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alternatives to synthetic nematicides has not been fully explored. This work aimed to

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assess the effects of pure naphthoquinones (juglone; 1,4-naphthoquinone; plumbagin)

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on the root-knot nematode Meloidogyne hispanica second-stage juveniles (J2)

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mortality. Extracts from Juglans spp. walnut husks were characterised and the effects of

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J. nigra extracts on attraction and life cycle of M. hispanica were evaluated. 1,4-

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naphthoquinone was the most effective compound causing 42% J2 mortality at 50 ppm.

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The extract from in natura J. nigra walnut husks presented similar effects on J2

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mortality to those observed for pure 1,4-naphthoquinone. The extract from dried husks

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was repellent and reduced nematode root penetration, but did not affect reproduction.

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Therefore, walnut residues can be valorised as renewable sources of naphthoquinone-

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based products and potentially employed as bio-nematicides against Meloidogyne spp.

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KEYWORDS: 1,4-naphthoquinone, Extracts, Juglans spp., Juglone, Naphthoquinone-

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based bio-nematicides, Plumbagin.

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INTRODUCTION

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The worldwide crop losses caused by plant-parasitic nematodes (PPN) were estimated

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to be higher than US$ 80 billion/year and root-knot nematodes (RKN, Meloidogyne

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spp.) are in the top 10 PPN responsible for the major ecological and economic impacts

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caused worldwide.1 Meloidogyne hispanica is a polyphagous RKN for several

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economically relevant crops, such as bean, corn, potato and tomato, and a species of

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emerging importance. Since is suited to soil temperatures around 25ºC, with the

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predicted future climate changes, there is a risk of spread to all over Southern Europe,

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and northwards.2,3 So far, it has been recorded worldwide, in countries such as

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Australia, Brazil, Burkina Faso, Cape Verde Islands, Fiji Islands, France, Korea,

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Malawi, Martinique, Puerto Rico, Spain, South Africa, The Netherlands, and United

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States (as mentioned in Maleita et al.2), and is able to infect and reproduce in a wide

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range of crops.2

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Once RKN are established in soil their eradication become difficult and current

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management strategies are mostly focused in reducing nematode population densities to

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limit damage to an economically acceptable level.4 In the past century, synthetic-origin

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nematicides were widely used to minimise crop losses caused by PPN. However, their

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adverse impact on the environment and human/animal health has urged the development

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of safer and sustainable alternatives such as the use of natural-origin nematicides,

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obtained/derived from plant extracts.5 A large number of plant secondary metabolites

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have been extracted, identified and demonstrated to present moderate-to-strong

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nematicidal activity.5-8

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According to FAO9, the world production of shelled walnuts was estimated to be around

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3,420,000 tonnes with China being the largest producer (1,700,000 tonnes). Europe

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accounted with 175,000 tonnes, and France was the largest European producer (37,000

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tonnes). Walnut, Juglans spp., is a highly nutritious food, rich in bioactive natural

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products and is an important component of the Mediterranean diet.10

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Walnut husks and leaves are abundant agro-industrial residues obtained after walnut

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farming and processing activities.11 Several bioactive substances, including phenolic

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compounds and naphthoquinones (NTQ), such as chlorogenic, p-coumaric, ellagic,

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ferulic, gallic, protocatechuic, sinapic, syringic and vanillic acids, (+)-catechin,

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myricetin, juglone (5-hydroxy-1,4-NTQ) and 1,4-naphthoquinone (1,4-NTQ), were

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already identified in these residues.12,13 Moreover, moderate-to-strong nematistatic

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and/or nematicidal effects have also been reported for some of these compounds.6-8

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NTQ, an important class of quinones and a group of highly reactive phenolic

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compounds, are widespread in nature as products of micro-organisms, fungal and plants

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secondary metabolism.14 Among NTQ, 1,4-NTQ and two of its derivatives, juglone and

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plumbagin, are arousing great research and application interests due to their broad-range

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of potential biological activities. In plants, juglone is stored in vacuoles as

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hydrojuglone-β-D-glucopyranoside

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hydrojuglone-β-d-glucopyranoside-β-glucosidase. The release of juglone into the soil

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occurs by exudation from roots, leaching from leaves and through decomposition of

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plant materials.15 The persistence of juglone in the soil varies from moderate to low and

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it is particularly short-lived in soils supporting microbial activity.15,16 Juglone may

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accumulate in subsurface soils, due to its reduced microbial degradation, and be

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available for uptake by deep-rooting plants.16 Plumbagin is usually obtained from

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Plumbago species roots17 but it has also been reported, at smaller amounts than juglone

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and 1,4-NTQ, in J. nigra (black walnut), J. regia and J. cinerea roots, bark, xylem and

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leaves.18

and

can

be

enzymatically

degraded

by

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The objectives of this work were: i) to determine the in vitro toxicity of pure juglone,

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1,4-NTQ (alone or combined) and plumbagin, against M. hispanica infective second-

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stage juveniles (J2); ii) to obtain, characterise and evaluate the effects of J. nigra and J.

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regia walnut husks extracts on M. hispanica J2 mortality, attraction/repellence,

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penetration and reproduction; and iii) to evaluate the potential of these NTQ-based

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natural products/extracts towards the development of novel, safer and environmentally

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friendly RKN management strategies.

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EXPERIMENTAL SECTION

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Nematode Isolate. The M. hispanica isolate was maintained on tomato, Solanum

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lycopersicum, cv. Coração de Boi, in pots containing sterilised sandy loam soil and sand

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(1:1 v/v), at 25±2ºC and its identification was confirmed by esterase phenotype

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analysis.19

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Juglans regia and J. nigra Extracts. Walnuts husks of J. nigra and J. regia, cv.

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Franquette, collected in two localities of Portugal, Alcobaça (September 2014) and

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Arraiolos (October 2014), respectively, were transported and surface-sterilised with a

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1% (v/v) NaOCl (reagent grade, 10-15% chlorine) solution for 10 min, and rinsed 3

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times with distilled water. J. nigra raw material was used in the in natura form

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(comminuted after surface sterilisation/rinsing in a knife-mill, for 4 min) or dried, while

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the J. regia raw material was used only in a dried form. Due to the quick oxidation after

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harvest and hulling, it was not possible to obtain sufficient in natura form J. regia

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material for extraction.

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Drying procedures started after sterilisation in an air-circulated oven, at ~35°C, and at

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atmospheric pressure, for 6 days. Dried materials were comminuted and sieved. Only

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the solid fractions, collected at the mesh 60 sieve (Tyler series), were selected. All raw

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materials were stored in sealed recipients and protected from light.

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A solid-liquid low pressure solvent extraction was used to obtain 3 walnut husks

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extracts: in natura and dried J. nigra, and dried J. regia walnut husks. Extraction of the

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in natura J. nigra raw material was performed 1 day after the comminution, and from

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dried raw materials 1-2 days after drying, being the extractions conducted at 70°C, for 3

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h, using a mixture of ethanol (p.a., purity ≥ 99.9% w/w) and ethyl acetate (p.a.,

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purity>99.9%) (1:1 v/v) and a solid-to-solvent ratio of 1:50 (m/v). Extracts were

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vacuum-dried (BÜCHI Rotavapor R-114) at 50ºC, stored at 4°C, and kept away from

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light.

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Chromatographic analyses were carried out in a HPLC system (Shimadzu, UFLC, pump

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LC-20AD coupled to Diode array detector SPDM20A) and using a Eurospher column

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(100-C18 RP, 250×4 mm i.d., 5 mm). Employed chromatographic assays were

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performed according to Jakopic et al.12 with some modifications in elution profile [0-24

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min, 5-20% (v/v) B; 24-25 min, 30% B; 25-65 min, 35% B; 35-75 min, 80% B; 75-80

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min, 85% B; and an equilibration time of 10 min]. Diode array detector was set to 254

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nm. Extract samples were filtered (0.2 µm) before injection. The concentrations of 1,4-

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NTQ (purity≥97% w/w), juglone (purity≥95%, w/w), and of ellagic (HPLC grade,

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purity≥95%, w/w), p-coumaric (HPLC grade, purity≥98%, w/w), and syringic

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(purity≥95%, w/w) acids were calculated based on previously obtained calibration

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curves. The standards from Sigma-Aldrich were used without purification.

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In vitro Mortality Bioassays Using Pure NTQ. Pure bioactive compounds, juglone,

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1,4-NTQ and plumbagin (purity≥95% w/w) were solubilised in Triton X-100

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(laboratory grade) aqueous solutions (5000 ppm) to obtain final NTQ concentrations of

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500, 250, 175, 100 and 50 ppm. Water and Triton X-100 were used as controls.

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Solutions were stirred for 3 days, at 37ºC, away from light. Each treatment consisted of

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5 replicates and each mortality experiment was repeated 3 times.

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Twenty four hours RKN J2 hatched from egg masses were collected and 20 nematodes

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placed on a glass-staining block containing 1 mL of each pre-prepared NTQ

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solution/water/Triton X-100. Glass-staining blocks were maintained in a moist chamber,

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in the dark, at room temperature (20-22ºC) and nematode mortality monitored at 3, 6,

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12, 24, 48 and 72 h after exposure (HAE). NTQ solutions were not replaced as it was

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assumed that NTQ activity was preserved during the tested period. Nematodes not

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showing movements when touched with a bristle were transferred to water and

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considered dead if they still fail to react. Lower NTQ concentrations (100 and 50 ppm)

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were only tested when the mortality at 150 ppm reached at least 70%.

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Additional experiments were performed using mixtures of 1,4-NTQ and juglone (1:2

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w/w), since these substances were found at this relative composition in J. regia walnut

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husks extracts.12 These mixtures were prepared in order to achieve final solution

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concentrations of 500, 250 and 150 ppm (1,4-NTQ+juglone) and the effects on M.

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hispanica mortality studied.

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Bioassays Using J. nigra Extracts. J. nigra extracts were solubilised in Triton X-100

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5000 ppm, and stirred for 3 days at 37ºC in the dark. The amounts of extracts to be

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added to the test solutions were calculated taking into consideration the relative

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composition of the extracts in 1,4-NTQ (Table 2). The J. nigra extract from in natura

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walnut husks was only tested on M. hispanica mortality, due to the insufficient amount

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of the extract. The J. regia dried extract was not employed on any bioassay, due to their

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low contents of 1,4-NTQ and juglone (Table 2).

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Mortality Bioassays. These bioassays were carried out as previously described, for 1,4-

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NTQ concentrations of 175 and 50 ppm (235 and 67 ppm of juglone, respectively) for

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the in natura black walnut husks extract, and of 175, 100 and 50 ppm (166, 95 and 47

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ppm of juglone, respectively) for the dried black walnut husks extract.

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Chemotaxis Assays. Plates (5 cm Ø) were filled with 1% water-agar (5 mL/dish). Two

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wells (0.5 cm Ø) made at opposite sides were filled with 50 µL of the dried black

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walnut husk extract solutions (175:166, 100:95 and 50:47 ppm, 1,4-NTQ:juglone) 4 h

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before the inoculation of 20 M. hispanica J2 at the centre. Water, Triton X-100 5000

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ppm, 1% v/v gacial acid acetic (repellent) and 50 µg/mL salicylic acid (purity≥95%,

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w/w, attractant) solutions were used as controls.7 Each treatment was replicated 3 times

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and assayed twice. Plates were kept in the dark at room temperature (20-22°C) and after

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2 h, nematode positions were recorded using a counting template divided into 16

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segments. The number of nematodes at the attractive and repellent zones of plates was

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registered. Results were presented as the number of nematodes on attractive zones

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divided by the number of nematodes on the repellent ones (chemotaxis factor, Cf).

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Extracts are classified as attractive (Cf>2), repellent (Cf90% mortality at 72 HAE (Figure 1). However, juglone induced

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100% mortality within 12 to 24 h at 500 and 250 ppm, respectively; 1,4-NTQ within 6 h

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at 500 ppm and 12 h at 250 ppm; and 1,4-NTQ:juglone and plumbagin induced 100%

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mortality within 48 h of exposure, at 500 ppm (Figure 1). Significant differences in M.

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hispanica mortality were found for juglone and 1,4-NTQ at 150, 100 and 50 ppm

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(p0.05, Figure 4a). At 30 DAI, M. hispanica reproduction was also influenced

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by the exposure to the extract (Figure 4b). The number of galls (47 to 88 galls/root

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system) found in tomato roots was not significantly different between all treatments

263

(data not shown), but a significant reduction of the Rf values was observed when J2

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were exposed to the extract, and when compared to the treatment with 5000 ppm Triton

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X-100 solution (p>0.05, Figure 4b). No significant differences were found between the

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controls (p1,4-

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NTQ>plumbagin. This trend was already found for other biological systems such as

305

epidermal human keratinocytes, protozoa and tobacco BY-2 cells.18,36,37 In this study,

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the

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NTQ≥juglone>plumbagin. At lower concentrations (0.05)

648

according to the Fisher LSD test or to the Kruskal-Wallis test.

649 650

Figure 2. HPLC chromatograms of the extracts from in natura or dried Juglans nigra

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and J. regia, cv. Franquette, walnut husks. Extracts were obtained by solid-liquid

652

ethanol:ethyl acetate (1:1 v/v) solvent extraction.

653 654

Figure 3. Corrected cumulative mortality (%) of Meloidogyne hispanica J2 exposed to

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solutions of extracts from (a) in natura and from (b) dried J. nigra walnut husks, at 175

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ppm (I), 100 ppm (II) and 50 ppm (II) of 1,4-naphthoquinone in the test solutions.

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Water and a 5000 ppm Triton X-100 aqueous solution were used as controls. Data are

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averages of 5 replicates and bars represent the corresponding standard errors. Presented

659

averages followed by the same lower case, at the same exposure time, do not differ

660

significantly (p>0.05) according to the Fisher LSD test or to the Kruskal-Wallis test.

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Please note that the corrected cumulative mortality axis is different in a and b.

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Figure 4. (a) Number of Meloidogyne hispanica J2 found inside tomato cv. Coração de

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Boi roots and (b) reproduction factor, 3 and 30 days after inoculation, respectively.

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Before inoculation, J2 were soaked for three days in water, in a 5000 ppm Triton X-100

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solution, and in a solution containing the extract from dried J. nigra walnut husks at 50

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ppm of 1,4-NTQ in the test solution. Each bar represents the average±standard deviation

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of 4 replicates and bars denoted by different letters differ significantly at p>0.05

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(according to the Fisher’s LSD test).

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TABLES

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Table 1. Estimated values of lethal concentration (ppm) necessary to result in 50% Meloidogyne hispanica J2 mortality (LC50), at 24 and 72 h after exposure to juglone, to 1,4-NTQ, to 1,4-NTQ:juglone (1:2 w/w), and to plumbagin lethal concentration (LC50, ppm)

hours after exposure

1,4-NTQ:juglone

juglone

1,4-NTQ

24

137.42

119.34

124.70

249.46

72

113.27

63.42

115.40

177.70

(1:2 w/w)

plumbagin

674 675 676

Table 2. Extraction yields (% g/g) and contents (mg/g) of some phenolic compounds identified at the extracts from in natura or dried Juglans nigra, and from J. regia, cv. Franquette, walnut husksa extraction raw material

identified/quantified substances (mg/g) ellagic

yield

p-coumaric syringic juglone

(% g/g)

acid

2.0 ± 0.1



49.4 ± 0.3

J. nigra(dried)

1.9 ± 0.2



J. regia (dried)

9.5 ±1.0

1,4-NTQ acid

acid

36.8 ± 0.3

3.5 ± 0.1



3.7 ± 0.1

3.9 ± 0.1

0.5 ± 0.1



0.3 ± 0.1 0.3 ± 0.1

0.7 ± 0.3

0.1 ± 0.1 5.7 ± 0.2

J. nigra (in natura)

a

Extracts were obtained by solid-liquid ethanol: ethyl acetate (1:1 v/v) solvent extraction.

677 678

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FIGURES

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Figure 1.

Corrected cumulative mortality (%)

680

100

I

a

a)

90

a

a

a

a

II

b

a,b

70

b

40

b

a

30 20

c

b

0

c

IV

b

a,b

IV b

40 30

c

b

b

3

6

d

c 12

24

V

d

d

III

10 0

48

0

72

c

V

c

c

c

20

a,b

10 0

c

b

II

50

III

a

a

I

60

50

a

b

80 70

b

60

a

a

b)

90

80

a

a

100

b

d

3

6

c d 12

24

48

72

a

a

b

a

Hours after exposure

Hours after exposure

681

Corrected cumulative mortality (%)

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 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100

a

c)

90

a

80 70 50

II

a

a

a b

b b

0

3

6

II b

a

c

10 0

12

24

48

72

b

b

20

a,b b

a

0

a,b

I

30

a

10

a

80

40

c

20

d)

90

50

III

30

a

60

b

40

a

100

70

I

60

a

0

b

c

c

3

6

12

Hours after exposure

III

b 24

48

72

Hours after exposure

682 683 684

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685

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Figure 2.

686 687 688

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689

Figure 3. 100 Corrected cumulative mortality (%)

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 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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90

20 18

80

16

70

14

60

12

50 40

b

30

a a

20 10

a

0

a 3

a

b

a

I

a

a a

8

a

a

a

a

a

a

a

III II

a

6

III

a

a

a

b)

10

I

0

690

a

a

a)

4

a

a

a

3

6 12 24 Hours after exposure

a

2 0

6 12 24 Hours after exposure

48

72

0

48

72

691 692

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Figure 4.

694

150 135 120 105 90 75 60 45 30 15 0

a)

a a b

Water

Triton X-100

Walnut extract

Reproduction factor

693

Number of J2 inside the roots

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 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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90 80 70 60 50 40 30 20 10 0

b)

a a,b b

Water

Triton X-100

Walnut extract

695 696

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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 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

697

ACS Sustainable Chemistry & Engineering

For Table of Contents Use Only

698 699 700

Naphthoquinones from walnut husk residues show strong nematicidal activities against the

701

root-knot nematode Meloidogyne hispanica

702 703

Carla Maleita,* Ivânia Esteves, Rita Chim, Luís Fonseca, Mara E. M. Braga, Isabel Abrantes,*

704

Hermínio C. de Sousa*

705 706

Synopsis:

707

Extract from J. nigra walnut husks residues was effective on root-knot nematodes J2 mortality and

708

prevented nematode root attraction and penetration.

709

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