Naphthoquinones from Walnut Husk Residues Show Strong

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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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ACS Sustainable Chemistry & Engineering

1

Naphthoquinones from walnut husk residues show strong nematicidal

2

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]

18

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

19 20

1

ACS Paragon Plus Environment


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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

23

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

24

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

27

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

30

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.

32

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

33

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

34 35 36

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

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based bio-nematicides, Plumbagin.

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2


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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,

47

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

50

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

51

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

3



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

65

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

67

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

68

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

69

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

70

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

71

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

72

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

74

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

75

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

76

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

77

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

78

hydrojuglone-β-D-glucopyranoside

79

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

80

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

81

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

84

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

85

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

86

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

87

leaves.18

and

can

be

enzymatically

degraded

by

4


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

89

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

90

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

91

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

92

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

93

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

94

friendly RKN management strategies.

95 96

EXPERIMENTAL SECTION

97

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

98

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

99

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

100

analysis.19

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

material for extraction.

110

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

111

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

112

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

5



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materials were stored in sealed recipients and protected from light.

114

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

115

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

116

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

117

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

118

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

119

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

120

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

121

light.

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

(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.

6


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

138

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

hispanica mortality studied.

153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

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

7



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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

213

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

214

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

215

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

216

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

217

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

261

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

11



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Page 12 of 35

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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

264

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

265

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

266

controls (p1,4-

304

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,

306

the

307

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

651

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

655

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

656

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

657

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

658

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.

661

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

662 663

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

664

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

28


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665

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

666

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

667

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

668

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

669

(according to the Fisher’s LSD test).

670

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671 672

Page 30 of 35

TABLES

673

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

30


Page 31 of 35

FIGURES

679

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


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


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


III

b 24

48

72


682 683 684

31



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685

Page 32 of 35

Figure 2.

686 687 688

32


Page 33 of 35

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


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

33



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

Page 34 of 35

90 80 70 60 50 40 30 20 10 0

b)

a a,b b

Water

Triton X-100

Walnut extract

695 696

34


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697


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

35


Naphthoquinones from Walnut Husk Residues Show Strong

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