Inhibition of acetylcholinesterase and glutathione S-transferase of the pinewood nematode (Bursaphelenchus xylophilus) by aliphatic compounds

Pesticide Biochemistry and Physiology 105 (2013) 184–188

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Inhibition of acetylcholinesterase and glutathione S-transferase of the pinewood nematode (Bursaphelenchus xylophilus) by aliphatic compounds Jae Soon Kang a, Yil-Sung Moon a, Si Hyeock Lee b, Il-Kwon Park a,⇑ a b

Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Republic of Korea Department of Agricultural Biotechnology, Seoul National University, Seoul 151–921, Republic of Korea

a r t i c l e

i n f o

Article history: Received 29 November 2012 Accepted 4 February 2013 Available online 16 February 2013 Keywords: Pinewood nematode Bursaphelenchus xylophilus Aliphatic compounds Acetylcholinesterase inhibition Glutathione S-transferase inhibition

a b s t r a c t To determine the nematicidal mode of action of aliphatic compounds against the pinewood nematode (Bursaphelenchus xylophilus), we evaluated the inhibition activity of 63 aliphatic compounds on B. xylophilus acetylcholinesterases (BxACEs) and glutathione S-transferase. In the primary inhibition assay using B. xylophilus crude proteins, more than 65% of BxACE inhibition activity was observed for C6, C9, C10, and C12 2E-alkenals. Other compounds showed moderate or weak inhibition activity. The inhibition activity against 3 recombinant BxACEs was subsequently evaluated using active compounds in a primary inhibition assay. C12 2E-alkenal showed the strongest inhibition activity against BxACE-1, followed by C9, C6, and C10 2E-alkenals. The IC50 values of C12, C6, C10, and C9 2E-alkenal against BxACE-2 were 0.0059, 0.57, 0.86, and 0.99 mg/ml, respectively. C12 2E-alkenal showed the strongest inhibition activity against BxACE-3 followed by C6 2E-alkenal. In an inhibition activity test using glutathione S-transferase from the pinewood nematode, C10, C9, and C6 2E-alkenals and C12 alkanoic acid showed >45% inhibition activity. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Pine wilt disease caused by the pinewood nematode Bursaphelenchus xylophilus is one of the most serious tree diseases in Asia and Europe, including South Korea, Japan, China, Taiwan, and Portugal [1–3]. This disease was first found in Gumjung Mt., Busan City in 1988 [4], and has become a serious threat to Korea’s pine forests [5]. The total damaged area along the Korean peninsula includes about 5123 ha, and the number of infected trees covered about 7644 ha in 2011 [6]. Several different methods have been introduced to control this disease. Examples include felling and fumigation of diseaseinfected trees using metham-sodium, aerial spraying of synthetic pesticides, application of thiacloprid to control the insect vector Monochamus alternatus, felling and crushing of trees to prevent the survival of the M. alternatus larvae, and injection of nematicides (such as abamectin and emamectin benzoate) into trunks [3,7,8]. The total budget for the control of pine wilt disease was about US$ 29.2 million in 2012 [9]. However, conventional pesticides or nematicides have many side effects, such as environmental pollution and toxicity to non-target organisms. Therefore, it is necessary to identify naturally occurring toxicants from plants that can be used for disease control, which in turn avoids the side effects of synthetic pesticides and nematicides.

⇑ Corresponding author. Fax: +82 2 961 2679. E-mail address: [email protected] (I.-K. Park). 0048-3575/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pestbp.2013.02.001

Short chain aldehydes and corresponding alcohols are major constituents of volatile organic chemicals produced by plants when wounded and in response to insect attack [10,11]. Further, the antimicrobial and insecticidal activity of short chains aliphatic compounds has been reported [12]. Furthermore, Seo et al. [13] reported the nematicidal activity of aliphatic compounds against the pinewood nematode. However, studies on the primary modes of action of phytochemicals with nematicidal activity against pinewood nematode have not been conducted. To understand the primary modes of actions of phytochemicals, it is important to efficiently screen the effective nematicidal agents. In this study, we estimated the inhibition activity of short chain aliphatic compounds against acetylcholinesterase (ACE) and glutathione S-transferase (GST) of B. xylophilus to learn the nematicidal mode of action of aliphatic compounds. 2. Materials and methods 2.1. Pinewood nematode B. xylophilus specimens from infected pine trees in the Donghae area of Gangwon province, Korea were separated by Baermann method [14], and confirmed by real-time species specific PCR [15]. The fungus Botrytis cinerea was cultured on potato dextrose agar (PDA) for rearing pinewood nematodes, which was separated by the Baermann method. Separated B. xylophilus organisms were washed with M9 buffer (Wormbook, http://www.wormbook.org) to remove any surface bacterial or fungal contaminants.

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2.2. Chemicals Authentic compounds used for bioassays were commercially obtained or synthesized as shown in Table 1. A detailed description of the synthesis of test compounds is shown in our previous study [13]. 2.3. Extraction of crude protein Crude proteins from pinewood nematode were extracted using a Bullet Blender (Next Advance, Averill Park, NY). B. xylophilus (ca. 300 ll) specimens were transferred to a 1.5-ml tube containing 500 ll of protein extraction buffer (0.1 M Tris–HCl buffer, containing 20 mM NaCl and 0.5% Triton X-100; pH 7.8) and the metal beads (half of the total volume) and vigorously shaken for 1 min. To avoid protein degradation by protease, a protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO) was added to the extract. The extract was centrifuged at 17,000g for 15 min at 4 °C, and crude protein was separated from the cell debris. The concentration of crude protein isolated from pinewood nematodes was estimated with Bradford reagent method by using a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA). Bovine serum albumin (BSA) was serially diluted in 0.1 M Tris–HCl buffer (pH 7.8), containing 20 mM NaCl and 0.5% Triton X-100, and was used as the standard protein for the quantification. 2.4. Inhibition assay against B. xylophilus crude protein extract Aliphatic compounds were diluted in acetone to 100 mg/ml concentration. The protein solution (79 ll), containing 30 lg protein in 0.1 M Tris–HCl buffer (pH 7.8) mixed with 20 mM NaCl and 0.5% Triton X-100, was combined with 1 ll of the chemical (final concentration of 1 mg/ml) and pre-incubated at room temperature for 10 min. The control reaction contained a solution of the protein and 1 ll acetone without any chemical. The acetone concentration of all reactions was 1%. Then, 10 ll of 10 mM acetylthiocholine iodide (ASChI, Sigma–Aldrich) as substrate and 10 ll of

4 mM 5,50 -dithiobis (2-nitro-benzoic acid) (DTNB, Sigma–Aldrich) as the colorimetric assay reagent dissolved in 0.1 M Tris–HCl (pH 7.8) containing 20 mM NaCl and 0.5% Triton X-100, were added to a pre-incubated blend of proteins and chemicals (with the final concentration of ASChI and DTNB being 1 mM and 0.4 mM). The residual activity of ACE, along with Vmax (max velocity), was measured by following the reaction at 412 nm at 30 s intervals for 20 min at room temperature, by using the VersaMax microplate reader (Molecular Devices). The inhibition rate was calculated as a percentage with respect to the control by the following formula: % Inhibition = 100 – (Enzyme Activity of Treatment/Enzyme Activity of Control  100) All experiments were performed three times to determine the primary inhibition rate of ACE, which was then converted to the arcsine square root value for analysis of variance. The mean values of treatments were compared and analyzed using Scheffe’s test [16].

2.5. Recombinant BxACEs expression About 0.2 mg of pinewood nematodes was soaked in 200 ll TRI reagent (MRC, Cincinnati, OH), and vigorously ground for 1 min using a Bullet Blender (Next Advance) and 0.5 mm metal beads. Total RNA layer was separated from genomic DNA or the protein layer by the BCP reagent (MRC, Cincinnati, OH), and precipitated using isopropanol (Sigma–Aldrich). The total RNA finally obtained was purified by rinsing with 70% ethanol. This purified total RNA was treated using DNaseI (Takara, Shiga, Japan) to remove all remaining genomic DNA, which had been used for the synthesis of single-stranded cDNA with the SuperScript first-strand cDNA synthesis system (Invitrogen, Carlsbad, CA). Three recombinant BxACEs (BxACE-1, -2, and -3) were expressed using the bEasyBac baculovirus expression system [17]. Each open reading frame (ORF) was amplified from the cDNA by PCR-amplification. All 3 ORFs included the signal peptide sequence and the His-tag sequence, but excluded the 3 cleavage site region to increase the expression efficiency. The amplified ORFs were cloned into the

Table 1 Aliphatic compounds tested in this study.a

Carbon length (Manufactory, purity)

Hydrocarbon R

Alkanol OH

R

2E-Alkenol OH

R

Alkanal O

R

2E-Alkenal O

R

Alkyl acetate R

O

O

Alkanoic acid OH

R

C6

C7

C8

C9

C10

C11

C12

C13

C14

Merck

Aldrich

Aldrich

Aldrich

Wako

Wako

Wako

Aldrich

Wako

96%

99%

98%

97%

99%

99%

99%

99%

99%

Wako

TCI

TCI

Aldrich

Aldrich

TCI

Aldrich

Aldrich

Aldrich

97%

98%

98%

98%

99%

98%

98%

97%

97%

Wako

Synthetic

Synthetic

Synthetic

Aldrich

Synthetic

Synthetic

Synthetic

Synthetic

95%

99%

99%

99%

97%

98%

97%

99%

95%

TCI

Wako

Aldrich

Aldrich

Aldrich

Aldrich

Aldrich

Aldrich

Synthetic

98%

95%

99%

95%

99%

97%

92%

90%

92%

Aldrich

Aldrich

Aldrich

Aldrich

Synthetic

Synthetic

Synthetic

Synthetic

Synthetic

98%

94%

94%

97%

98%

98%

97%

99%

95%

TCI

TCI

Aldrich

Synthetic

Synthetic

Synthetic

Aldrich

TCI

Synthetic

98%

98%

99%

99%

99%

99%

97%

98%

99%

TCI

TCI

TCI

TCI

TCI

TCI

Aldrich

TCI

Aldrich

98%

96%

98%

>90%

98%

98%

98%

98%

99%

O

a

Aliphatic compounds test in this study was well describe by Seo et al. [13].

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pDualBac baculovirus vector, and then 3 BxACE-pDualBacs were homologously recombined into Autographa californica nuclear polyhedrosis virus (AcNPV) chromosome. Three recombinant BxACE-AcNPVs were infected into sf9 insect cells, which were incubated at 27 °C for 1 week in TC-100 insect cell media (Invitrogen) containing 10% fetal bovine serum (FBS). The medium was harvested by centrifugation at 1500g for 5 min, and the recombinant proteins were isolated on HiTrap affinity chromatography columns (GE Healthcare, Uppsala, Sweden) at 4 °C. The optimum imidazol concentration to separate the recombinant proteins from HiTrap affinity chromatography was 40 mM. The purified proteins were finally buffer-exchanged to 0.1 M Tris–HCl buffer (pH 7.8) containing 20 mM NaCl and 10% glycerol by using HiTrap Desalting columns (GE Healthcare) to remove imidazole. The recombinant proteins were stored at 80 °C. 2.6. Inhibition assay against recombinant BxACEs Four active chemicals, which showed more than 65% BxACE inhibitory activity at 1 mg/ml in the primary inhibition assay, we selected, including C6, C9, C10, and C12 2E-alkenal. These chemicals were serially diluted with acetone to obtain 4 different working concentrations; the final concentrations were 1, 0.5, 0.1, and 0.05 mg/ml for C6 2E-alkenal; 1, 0.5, 0.1, 0.05, and 0.01 mg/ml for C9 and C10 2E-alkenals; and 1, 0.5, 0.1, 0.05, 0.01, and 0.005 mg/ ml for C12 2E-alkenal. The inhibition assay was conducted as described in the above section. The IC50 was estimated using probit analysis [16]. 2.7. Inhibition assay against BxGST All chemical compounds were diluted to 100 mg/ml in acetone. The 79 ll protein solution containing 30 lg protein and 1 ll aliphatic compound mixture (the final concentration of compound was 1 mg/ml) was pre-incubated at room temperature for 10 min. The control reaction was mixed with protein and 1 ll acetone, without the addition of any compound. The substrate solution, which included 10 ll of 20 mM reduced glutathione (Sigma–Aldrich) and 10 ll of 10 mM 1-chloro-2,4-dinitrobenzene (CDNB, Sigma–Aldrich) diluted in 0.1 M Tris–HCl (pH 7.8), was added to the pre-incubated mixtures of proteins and aliphatic compounds. To estimate the remaining GST activity, the Vmax was measured at 340 nm at 30 s intervals for 20 min at room temperature using a VersaMax microplate reader (Molecular Devices). Inhibition activity was calculated by comparing the activity ratio of the chemical-treated reaction to the control reaction. All experiments were performed 3 times to determine the primary inhibition rate of ACE, which was then converted to the arcsine square root values for analysis of variance. The mean values of treatments were compared and analyzed using Scheffe’s test [16]. 3. Results and discussion 3.1. Inhibition assay against B. xylophilus crude protein extract In the primary BxACE inhibition assay, the activity of various aliphatic compounds was observed (Table 2). Among the 63 aliphatic compounds, the 2E-alkenal group exhibited high inhibitory activity. The inhibitory activities of C12, C6, C9, and C10 2E-alkenals were 88.5%, 87.4%, 71.4%, and 65.6% at 1 mg/ml concentration, respectively. Aliphatic compounds belonging to hydrocarbons, alkanol, and alkyl acetate showed less than 40% inhibitory activity against BxACE. C11 2E-alkenol, C14 alkanal, and C7 and C12 alkanoic acid exhibited 46.2%, 41.4%, 42.9% and 41.1% inhibitory activities, respectively; however, the inhibitory activities of other

compounds belonging to these functional groups were less than 40%. The nematicidal activities of aliphatic compounds against the pinewood nematode have been previously investigated by Seo et al. [13]. The authors reported a significant difference in nematicidal activity among functional groups. Compounds belonging to alkanol, 2E-alkenol, 2E-alkenal, and alkanoic acid showed strong nematicidal activity. Another factor that determines the nematicidal activity of aliphatic compounds is chain length. In this study, compounds belonging to 2E-alkenal with C6, C9, C10, and C12 chain lengths exhibited strong inhibitory activities against BxACE. Seo et al. [13] reported that 2E-alkenals with C6  C10 chain lengths showed strong nematicidal activity against pinewood nematode. Our results and previous study indicated that there was a direct correlation between nematicidal activity of C6, C9 and C10 2E-alkenal and BxACE inhibition. It is very interesting that C12 2E-alkenal showed the strongest inhibitory activity against BxACE, despite its nematicidal activity being very weak [13]. Similar results were observed in a previous study, where carbamate nematicides (including carbofuran, carbaryl, and aldicarb) or phytochemicals (a-pinene, b-pinenes, 3-carene, dihydrocarvone, o-anisaldehyde, coniferyl alcohol, and cis-nerolidol) from plant essential oils exhibited weak nematicidal activity against the pinewood nematode [18,19], despite showing strong inhibitory activity against BxACE. In a BxACE inhibitory study, C12 2E-alkenal directly interacted with acetylcholinesterase. However, C12 2E-alkenal must penetrate the cuticle or cell membrane of the pinewood nematode in the nematicidal activity test. A low penetration rate by C12 2E-alkenal might be one reason for the low nematicidal activity. Grodnitzky and Coats [20] studied the QSAR of monoterpenoids against house flies, Musca domestica. They reported that monoterpenoids must possess an optimum shape and size requirement to fit into a toxic action site. Further, Seo et al. [13] reported that aliphatic compounds must be of the correct chain length for nematicidal activity against the pinewood nematode. 3.2. Inhibition assay against recombinant BxACEs A summary of the inhibitory activity of 4 chemicals against 3 recombinant BxACEs is presented in Table 3. C12 2E-alkenal showed the strongest inhibitory activity against BxACE-1, followed by C9, C6, and C10 2E-alkenals. The IC50 values of C12, C9, C6, and C10 2E-alkenals were 0.02, 0.42, 0.6, and 0.72 mg/ml, respectively. In a test using BxACE-2, the strongest inhibitory activity was recorded for C12 2E-alkenal, followed by C6, C10, and C9 2E-alkenal. The IC50 values of C12, C6, C10, and C9 2E-alkenal against BxACE-2 were 0.0059, 0.57, 0.86, and 0.99 mg/ml, respectively. Only C12 and C6 2E-alkenals showed strong inhibition activity against BxACE-3. The IC50 values of C12 and C6 2E-alkenal were 0.038 and 0.41 mg/ ml, respectively. In this study, all 3 chemicals, including C6, C9, and C10 2E-alkenals strongly inhibited BxACE-1 and BxACE-2. Because BxACE-1 and BxACE-2 are considered important for post-synaptic neural transmission in pinewood nematodes, the nematicidal mode of action of C6, C9, and C10 2E-alkenals against the pinewood nematode might be strongly related to acetylcholinesterase inhibition. Only 2 compounds, including C6 and C12 2Ealkenals, strongly inhibited BxACE-3, which has been reported to be insensitive to most anti-ACE chemicals. Because BxACE-3 is speculated to be involved in the chemical defense mechanism of B. xylophilus against many xenobiotics, [19,21] our result indicated that C6 and C12 2E-alkenal may be used as nematicide synergist. 3.3. Inhibition assay against BxGST The inhibitory activities of aliphatic compounds against glutathione S-transferase of pinewood nematodes are shown in Table 4. Among the test compounds, 2E-alkenal and alkanoic acid

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J.S. Kang et al. / Pesticide Biochemistry and Physiology 105 (2013) 184–188 Table 2 The inhibition of BxACE activity from B. xylophilus crude protein extract by aliphatic compounds.a Carbon length

Inhibition activity of each compound group (Mean ± S.D., N = 3) Hydrocarbon

a b c

Alkanol

2E-Alkenol

Alkanal

2E-Alkenal

Alkyl Acetate

Alkanoic acid

C6

1.8 ± 3.1 ab (b)c

0 b (b)

0 c (b)

87.4 ± 0.9 a (a)

33.5 ± 24.6 a (b)

36.4 ± 17.5 a (b)

C7

4.8 ± 2.2 a (c)

0 b (c)

0.8 ± 1.4 bc (c)

19.7 ± 16.3 bc (b) 63.7 ± 5.8 a (a)

38.9 ± 2.5 cd (b)

39.4 ± 4.9 a (b)

42.9 ± 10.1 a (b)

18.9 ± 10.8 bc (ab) 29.3 ± 7.4 a bc (b) 15.2 ± 13.3b c (b) 0 c (b)

28.0 ± 3.2 d (a)

33.8 ± 0.8a (a)

27.1 ± 7.6 a (a)

71.4 ± 5.2 ab (a)

36.0 ± 0.9 a (b)

35.1 ± 7.7 a (b)

65.6 ± 13.2 abc (a)

19.5 ± 15.5 a (b)

27.6 ± 17.8 a (ab)

47.9 ± 9.6 bcd (a)

26.8 ± 23.5 a (ab)

26.2 ± 4.9 a (ab)

0 c (b)

88.5 ± 2.8 a (a)

25.6 ± 14.9 a (b)

41.1 ± 32.2 a (ab)

0 c (c)

54.3 ± 3.1 bcd (a)

14.1 ± 12.2 a (bc)

35.2 ± 2.9 a (ab)

41.4 ± 17.5 ab (a) F8,18 = 106.3, p < 0.0001

44.1 ± 14.1 bcd (a)

6.3 ± 11.0 a (a)

36.7 ± 22.3 a (a)

C8

14.9 ± 13.0 a (ab)

0 b (b)

0 b (b)

C9

7.3 ± 12.0 a (c)

0 b (c)

0 b (c)

C10

5.4 ± 5.2 a (b)

0 b (b)

0 c (b)

C11

19.4 ± 16.9 a (ab)

0 b (b)

C12

6.2 ± 1.2 a (b)

C13

23.3 ± 3.2 a (bc)

C14

22.9 ± 2.2 a (a)

13.1 ± 11.3 b (b) 28.6 ± 4.2 a (ab) 28.9 ± 3.1 a (a)

F8,18 = 73.6, p = 0.03

F8,18 = 17.2, p < 0.0001

46.2 ± 10.8 a (a) 12.0 ± 5.8 bc (b) 33.7 ± 15.0 ab (ab) 21.6 ± 18.7 abc (a) F8,18 = 81.1, p < 0.0001

F8,18 = 59.6, p < 0.0001

F8,18 = 213.6, p = 168

F6,14 = 188.0, p < 0.0001 F6,14 = 25.0, p < 0.0001 F6,14 = 50.9, p < 0.0001 F6,14 = 41.2, p < 0.0001 F6,14 = 134.2, p < 0.0001 F6,14 = 154.0, p < 0.0001 F6,14 = 204.4, p < 0.0001 F6,14 = 60.3, p < 0.0001 F6,14 = 213.5, p = 0.079

F8,18 = 267.8, p = 899

1 mg/ml concentration. Means within a column followed by same letters are not significantly different (Scheffe’s test). Means within a row followed by same letters are not significantly different (Scheffe’s test).

Table 3 The inhibition activities of four active compounds against three recombinant BxACEs. Compounds

BxACE-1

C6 2E-alkenal C9 2E-alkenal C10 2E-alkenal C12 2E-alkenal a

BxACE-2

BxACE-3

IC50 (mg/ml)

Slope

95% cla

v2

IC50 (mg/ml)

Slope

95% cl

v2

IC50 (mg/ml)

Slope

95% cl

v2

0.60 0.42 0.72 0.02

1.4 ± 4.7 0.48 ± 2.6 0.54 ± 2.72 0.45 ± 2.59

0.56–0.66 0.35–0.53 0.58–0.91 0.01–0.03

1.76 0.88 1.33 4.83

0.57 0.99 0.86 0.0059

1.22 ± 4.43 0.51 ± 2.73 0.52 ± 2.72 0.42 ± 2.10

0.52–0.63 0.78–1.30 0.69–1.12 0.0043–0.0077

1.22 6.52 9.95 3.63

0.41 >1 >1 0.038

1.18 ± 4.25 – – 0.68 ± 2.71

0.37–0.45 – – 0.033–0.044

2.31 – – 0.001

Confidence limit.

Table 4 The inhibition of BxGST activity from B. xylophilus crude protein extract by aliphatic compounds.1 Carbon length

1 2 3

Inhibition activity of each compound group (Mean ± S.D., N = 3) Hydrocarbon 2

C6

0 a (c)

C7

3

Alkanol

2E-Alkenol

Alkanal

2E-Alkenal

Alkyl Acetate

Alkanoic acid

0 b (c)

1.8 ± 2.3 c (c)

9.6 ± 7.5 a (c)

46.3 ± 5.6 a (a)

13.4 ± 3.2 a (bc)

26.6 ± 7.6 a (b)

0 a (c)

0 b (c)

2.0 ± 2.4 c (c)

11.5 ± 8.2 a (c)

43.1 ± 7.5 a (a)

8.6 ± 2.9 a (c)

28.1 ± 2.8 a (b)

C8

0.1 ± 0.3 a (a)

2.2 ± 3.1 ab (a)

7.0 ± 5.0 bc (a)

15.7 ± 13.0 a (a)

36.7 ± 25.7 a (a)

26.5 ± 18.6 a (a)

26.3 ± 18.4 a (a)

C9

0 a (d)

1.8 ± 1.3 ab (d)

4.3 ± 5.2 c (cd)

14.8 ± 3.7 a (c)

49.9 ± 5.3 a (a)

29.4 ± 4.4 a (b)

30.6 ± 4.2 a (b)

C10

0.4 ± 0.7 a (b)

3.1 ± 3.5 ab (b)

6.7 ± 3.9 bc (b)

19.0 ± 38.1 a (ab)

51.0 ± 5.6 a (a)

27.6 ± 9.3 a (ab)

37.3 ± 4.3 a (ab)

C11

0 a (e)

38.3 ± 5.4 a (ab)

18.9 ± 8.2 a (cd)

37.0 ± 9.0 a (ab)

23.6 ± 6.1 a (bc)

44.4 ± 4.6 a (a)

C12

1.5 ± 3.1 a (d)

5.8 ± 3.4 ab (de) 8.3 ± 4.5 a (cd)

20.0 ± 3.9 b (bcd)

13.3 ± 13.7 a (cd)

33.7 ± 4.2 a (ab)

22.1 ± 5.2 a (bc)

45.3 ± 8.4 a (a)

C13

0.6 ± 1.1 a (b)

1.5 ± 2.5 ab (b)

7.9 ± 1.3 bc (ab)

14.1 ± 3.0 a (ab)

26.5 ± 19.3 a (a)

14.6 ± 1.6 a (ab)

26.2 ± 19.3 a (a)

C14

0 a (c)

1.4 ± 1.6 ab (c)

10.8 ± 8.1 bc (bc)

14.2 ± 3.6 a (b)

33.1 ± 5.7 a (a)

21.0 ± 2.2 a (ab)

21.9 ± 4.0 a (ab)

F8,27 = 1.2, p = 583

F8,27 = 7.1, p = 0.002

F8,27 = 81.1, p < 0.0001

F8,27 = 227.0, p = 0.994

F8,27 = 107.1, p = 0.30

F8,27 = 60.7, p = 0.009

F8,27 = 102.9, p = 0.022

1 mg/ml concentration. Means within a column followed by same letters are not significantly different (Scheffe’s test). Means within a row followed by same letters are not significantly different (Scheffe’s test).

F6,21 = 23.5, p < 0.0001 F6,21 = 20.9, p < 0.0001 F6,21 = 222.6, p = 0.014 F6,21 = 15.4, p < 0.0001 F6,21 = 231.5, p < 0.0001 F6,21 = 35.90, p < 0.0001 F6,21 = 50.2, p < 0.0001 F6,21 = 58.8, p < 0.0001 F6,21 = 19.4, p < 0.0001

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compounds showed >40% inhibitory activity against BxGST at 1 mg/ml concentration. The inhibitory activities of C10, C9, C6, and C7 2E-alkenals were 51%, 49.9%, 46.3%, and 43.1%, respectively. C12 and C11 alkanoic acids exhibited 45.3% and 44.4% inhibitory activities, respectively. The remaining compounds exhibited less than 40% inhibitory activity against BxGST. The glutathione Stransferases are a group of multifunctional enzymes that play an important role in the detoxification and elimination of toxic and undesirable foreign compounds [22,23]. Some allelochemicals have been known to inhibit insect glutathione S-transferases, including plant phenols (quercetin, ellagic acid, juglone, menadione, plumbagin, and dicumarol), ethacrynic acid, and a,b-unsaturated carbonyl compounds (trans-2-hexenal, benzaldehyde, and trans,trans-2,4-decadienal) [23–25]. A similar result was observed in the present study. a,b-Unsaturated aliphatic compounds (C6, C7, C9, and C10 2E-alkenals) inhibited BxGST. Conjugated aldehydes have been reported to inhibit enzyme with cysteine residues which are essential to their enzymatic or receptor activity [26–28]. Early and present study indicated that 2E-alkeanls might inhibit ACE or GST of pinewood nematode by high affinity for the critical cystein residue of those enzymes. In this study, we found that the nematicidal activity of 2E-alkenal is related to the inhibition of BxACE, with some 2E-alkenal compounds inhibiting BxGST. Another finding is that the inhibition of ACE or GST of the pinewood nematode, in addition to nematicidal activity [13,29] is strongly related to the presence of a double bond at the a,b-position of the carbonyl group. These findings provide new information toward improving our understanding about the nematicidal activity of aliphatic compounds, which will contribute towards the development of new effective nematicides.

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