Nematicidal activity of benzyloxyalkanols against pine wood nematode

Article abstract of DOI:10.3390/biom11030384

Pine wilt disease (PWD) is caused by the pine wood nematode (PWN; Bursaphelenchus xylophilus) and causes severe environmental damage to global pine forest ecosystems. The current strategies used to control PWN are mainly chemical treatments. However, the continuous use of these reagents could result in the development of pesticide-resistant nematodes. Therefore, the present study was undertaken to find potential alternatives to the currently used PWN control agents abamectin and emamectin. Benzyloxyalkanols (BzOROH; R = C₂–C₉ ) were synthesized and the nematicidal activity of the synthetic compounds was investigated. Enzymatic inhibitory assays (acetylcholinesterase (AChE) and glutathione S-transferase (GST)) were performed with BzOC8OH and BzOC9OH to understand their mode of action. The benzyloxyalkanols showed higher nematicidal activity than did benzyl alcohol. Among the tested BzOROHs, BzC8OH and BzC9OH showed the strongest nematicidal activity. The LD₅₀ values of BzC8OH and BzC9OH were 246.1 and 158.0 ppm, respectively. No enzyme inhibitory activity was observed for BzC8OH and BzC9OH. The results suggested that benzyloxyalcohols could be an alternative nematicidal agent.

Full text of DOI:10.3390/biom11030384

biomolecules
Article
Nematicidal Activity of Benzyloxyalkanols against Pine
Wood Nematode
Junheon Kim ,*,† , Su Jin Lee 1,†,‡ , Joon Oh Park 1,§ and Kyungjae Andrew Yoon 2
1
1
Forest Insect Pests and Disease Division, National Institute of Forest Science, Seoul 02455, Korea;
sujin0316@inu.ac.kr (S.J.L.); joonoh6@gmail.com (J.O.P.)
Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Korea;
2
kongbob89@snu.ac.kr
*
†
‡
Correspondence: junheonkim@korea.kr; Tel.: +82-2-961-2672
These authors contributed equally to this study.
Present address: Division of Life Sciences & Convergence Research Center for Insect Vectors, College of Life
Science and Bioengineering, Incheon National University, Incheon 22012, Korea.
Present address: Urban Forest Clinic, Boryeong 33455, Korea.
§
Abstract: Pine wilt disease (PWD) is caused by the pine wood nematode (PWN; Bursaphelenchus
xylophilus) and causes severe environmental damage to global pine forest ecosystems. The current
strategies used to control PWN are mainly chemical treatments. However, the continuous use of
these reagents could result in the development of pesticide-resistant nematodes. Therefore, the
present study was undertaken to find potential alternatives to the currently used PWN control
agents abamectin and emamectin. Benzyloxyalkanols (BzOROH; R = C –C ) were synthesized
2
9
and the nematicidal activity of the synthetic compounds was investigated. Enzymatic inhibitory
assays (acetylcholinesterase (AChE) and glutathione S-transferase (GST)) were performed with
BzOC8OH and BzOC9OH to understand their mode of action. The benzyloxyalkanols showed higher
nematicidal activity than did benzyl alcohol. Among the tested BzOROHs, BzC8OH and BzC9OH
showed the strongest nematicidal activity. The LD50 values of BzC8OH and BzC9OH were 246.1 and
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Kim, J.; Lee, S.J.; Park, J.O.;
Yoon, K.A. Nematicidal Activity of
Benzyloxyalkanols against Pine
158.0 ppm, respectively. No enzyme inhibitory activity was observed for BzC8OH and BzC9OH. The
Wood Nematode. Biomolecules 2021,
results suggested that benzyloxyalcohols could be an alternative nematicidal agent.
1
1, 384. https://doi.org/10.3390/
biom11030384
Keywords: pine wood nematode; pine wilt disease; benzyloxyalkanol; mode of action
Academic Editor: Manuel Galvão de
Melo e Mota
1
. Introduction
Pine wilt disease (PWD), caused by the pine wood nematode (PWN; Bursaphelenchus
xylophilus), causes severe environmental damage to global pine forest ecosystems [ ]. PWD
has become and is becoming a serious concern over multiple continents including Asia and
Europe [ ]. Since its first reported sighting in 1988 in Korea [ ], it has spread nationwide
and ca. 490,000 trees are affected by PWD [ ]. The majority of pine species affected by
PWD are red pine (Pinus densiflora), black pine (P. thunbergii) and P. koraiensis in Asia, while
P. pinaster, P. nigra and P. radiata are affected in Europe [ 10]. As Pinus species are the
Received: 15 January 2021
Accepted: 2 March 2021
Published: 5 March 2021
1
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
2–
7
8
9
1,3,
predominant tree species in Korean forests and highly affected by PWD, the ecological and
economic damage is substantial.
The current strategies used to control PWN are mainly chemical treatments, which in-
Copyright: © 2021 by the authors.
clude nematicide injection in trunks for PWD prevention and fumigation of wood material.
Licensee MDPI, Basel, Switzerland. The most frequently used nematicides in Korea are abamectin and emamectin benzoate,
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
which belong to the avermectin family [11,12]. These agents are known to be highly effec-
tive against PWN and are also considered environmentally safe [13 14]. However, it could
be inferred that the continuous use of these nematicides will induce selection pressure and
almost certainly produce resistant PWNs, as has been reported previously for nematodes
that developed resistance to avermectins [15–17].
,
4
.0/).
Biomolecules 2021, 11, 384. https://doi.org/10.3390/biom11030384
https://www.mdpi.com/journal/biomolecules

Biomolecules 2021, 11, 384
2 of 9
As nematicides against PWN are applied in closed systems, such as trunk injection,
the possibility of developing resistance against avermectins is low and opportunities for
selected nematode strains to spread in natural populations is limited. However, because
the nematicidal effect of avermectins is not always successful, the possibility of developing
resistance and spreading by insect vectors in natural populations remains. Although PWN
has not yet been reported to show resistance to abamectin, the alternative use of diverse
agents is recommended to avoid the development of resistance and to achieve efficient
control of PWN. In recent decades, as PWN control agents, the use of microorganism
extracts, plant extracts, essential oils and volatiles has been suggested [18–23].
It has been reported that metabolic resistance in insects involves some detoxifying
enzymes which are responsible for breaking down toxins entering their bodies [24]. Among
those enzymes, acetylcholinesterase (AChE) and glutathione S-transferase (GST) have been
assessed to obtain an understanding of the mode of action and the extent of its detoxification
in the target species [24–26]. Inhibition of AChE causes overstimulation of the neurons,
twitching of muscles and insect death [27] and GST is involved in resistance development
in insects through detoxifying of toxins [28].
Recently, the nematicidal effect of alkyloxyethanols (ROR’OH, R’ = ethyl), which mod-
ified hydroxy groups (-OH) to
against PWN was investigated [29]. The effect of modification of the modified hydroxy
group to the -hydroxyalkyl group in aromatic compounds has not been investigated
in PWN. In the present study, we synthesized benzyloxyalcohol (BzOROH; R = C –C ),
ɷ
-hydroxyalkyl groups (-OROH) in aliphatic compounds,
ɷ
2
9
evaluated the nematicidal effects of 8 benzyloxyalcohols and investigated their mode of
action using AChE and GST inhibition assays.
2. Materials and Methods
2.1. Nematodes
Bursaphelenchus xylophilus was provided by the National Institute of Forest Science,
South Korea. The identification of B. xylophilus was confirmed using morphological char-
acteristics and genetic differences, which were confirmed by the RFLP method [30]. The
B. xylophilus were reared on a fungal mat of Botrytis cinerea on potato dextrose agar (PDA)
◦
medium at 25 ± 1 C and 40% humidity for several generations.
2.2. Preparation of Benzyloxyalcohols
Benzyloxyalkanols ( ) were synthesized by modifying the method of Tanabe and
1–8
Peters [31] (Scheme 1). The general procedure was as follows: a 3-necked round-bottom
flask was equipped with a magnetic stirring bar, a pressure-equalizing dropping funnel,
a reflux condenser, and an inlet for nitrogen. The flask was charged with silver (I) oxide
(
15 mmol, Alfa Aesar, Heysam, UK) and alkanediol (15 mmol; ethanediol, propanediol,
butanediol, pentanediol, hexanediol, heptanediol, octanediol and nonanediol) in 30 mL
of CH Cl (Daejung, Hwaseong, Korea), while benzyl bromide (11 mmol, Alfa Aesar) in
2
2
1
0 mL of CH Cl was added over ca. 10 min. The mixture was stirred for 15 h at room
2 2
temperature. The suspension was filtered through Celite (Alfa Aesar), and the filter cake
was washed with two portions of 50 mL of diethyl ether. The ethereal phase was washed
with water and brine and dried over MgSO . After filtration and evaporation, the residue
4
was subjected to silica gel column chromatography to obtain the desired compounds (35%
diethyl ether in hexane fraction). Their structure was confirmed by NMR. Their purity was
determined by gas chromatography-mass spectrometry (GC-MS).
Scheme 1. Synthetic scheme of benzyloxyalcohols.

Biomolecules 2021, 11, 384
3 of 9
2
-(Benzyloxy)-1-ethanol (BzOC2OH, 1)
1
Yield 51.2%. Purity 97.1%. H NMR (500 MHz, CDCl )
δ
(ppm) 7.386–7.320 (m, 4H),
3
1
3
7
.314–7.233 (m, 1H), 4.558 (s, 2H), 3.749 (q, J = 4.9 Hz, 2H), 3.592 (t, J = 4.9 Hz, 2H).
C
NMR (126 MHz, CDCl3) (ppm) 137.97 (C), 128.47 CH 2), 127.81 (CH 3), 73.30 (CH2),
δ
×
×
71.39 (CH ), 61.89 (CH ).
2 2
3
-(Benzyloxy)-1-propanol (BzOC3OH, 2)
1
Yield 93.8. Purity 95.6%. H NMR (500 MHz, CDCl )
δ (ppm) 7.384–7.309 (m, 4H),
3
7
.309–7.237 (m, 1H), 4.521 (s, 2H), 3.786 (t, J = 5.0 Hz, 2H), 3.662 (t, J = 5.8 Hz, 2H), 1.867 (p,
13
J = 5.8 Hz, 2H). C NMR (126 MHz, CDCl3)
δ
(ppm) 138.09(C), 128.47 (CH
×
2), 127.73
(
CH × 2), 127.66 (CH), 73.29 (CH ), 69.40 (CH ), 61.91(CH ), 32.12(CH ).
2
2
2
2
4
-(Benzyloxy)-1-butanol (BzOC4OH, 3)
1
Yield 67.2%. Purity 99.5%. H NMR (500 MHz, CDCl )
δ
(ppm) 7.379–7.238 (m, 5H),
3
1
3
4
.519 (s, 2H), 3.675–3.611 (m, 2H), 3.520 (t, J = 5.8 Hz, 2H), 1.766–1.626 (m, 4H). C NMR
(ppm) 138.16 (C), 128.43 (CH 2), 127.73 (CH 2), 127.68 (CH), 73.07
CH ), 70.34 (CH ), 62.72 (CH ), 30.16 (CH ), 26.70 (CH ).
(
126 MHz, CDCl3)
δ
×
×
(
2
2
2
2
2
5
-(Benzyloxy)-1-pentanol (BzOC5OH 4)
Yield 67.7%. Purity 99.5%. 1H NMR (500 MHz, CDCl3)
H), 4.498 (s, 2H), 3.621 (t, J = 6.5 Hz, 2H), 3.479 (t, J = 6.5 Hz, 2H), 1.692–1.514 (m, 4H),
(ppm) 138.55 (C), 128.36 (CH 2),
2), 127.53 (CH), 72.93 (CH ), 70.30 (CH ), 62.77 (CH ), 32.49 (CH ), 29.45
δ
(ppm) 7.387–7.230 (m,
5
1
1
.494–1.394 (m, 2H). 13C NMR (126 MHz, CDCl3)
27.65 (CH
δ
×
×
2
2
2
2
(
CH ), 22.43 (CH ).
2 2
6
-(Benzyloxy)-1-hexanol (BzOC6OH, 5)
Yield 64.7%. Purity 99.5%. 1H NMR (500 MHz, CDCl3)
H), 7.316–7.244 (m, 1H), 4.499 (s, 2H), 3.623 (t, J = 6.6 Hz, 2H), 3.470 (t, J = 6.5 Hz, 2H),
(ppm) 138.64 (C),
2), 127.50 (CH), 72.89 (CH ), 70.34 (CH ), 62.92 (CH ), 32.71
δ
(ppm) 7.376–7. 314 (m,
4
1
1
.662–1.521 (m, 4H), 1.455–1.316 (m, 4H). 13C NMR (126 MHz, CDCl3)
28.35 (CH 2), 127.64 (CH
δ
×
×
2
2
2
(
CH ), 29.71 (CH ), 26.01 (CH ), 25.58 (CH ).
2 2 2 2
7
-(Benzyloxy)-1-heptanol (BzOC7OH, 6)
Yield 69.0%. Purity 99.5%. 1H NMR (500 MHz, CDCl3)
H), 4.500 (s, 2H), 3.628 (t, J = 6.7 Hz, 2H), 3.465 (t, J = 6.6 Hz, 2H), 1.645–1.587 (m, 2H),
(ppm) 138.68 (C),
2), 127.48 (CH), 72.87 (CH ), 70.43 (CH ), 63.02 (CH ), 32.72
δ
(ppm) 7.399–7.245 (m,
5
1
1
.586–1.525 (m, 2H), 1.439–1.286 (m, 6H). 13C NMR (126 MHz, cdcl3)
28.34 (CH 2), 127.62 (CH
δ
×
×
2
2
2
(
CH ), 29.69 (CH ), 29.24 (CH ), 26.17 (CH ), 25.68 (CH ).
2 2 2 2 2
8
-(Benzyloxy)-1-octanol (BzOC8OH, 7)
1
Yield 61.0%. Purity 99.5%. H NMR (500 MHz, CDCl )
δ 7.396–7.230 (m, 5H), 4.499 (s,
3
2
8
H), 3.623 (t, J = 6.7 Hz, 2H), 3.461 (t, J = 6.6 Hz, 2H), 1.673–1.507 (m, 4H), 1.412–1.276 (m,
13
H). C NMR (126 MHz, CDCl3)
δ
(ppm) 138.70 (C), 128.34 (CH
×
2), 127.63 (CH
×
2),
1
27.47 (CH), 72.87 (CH ), 70.48 (CH ), 63.04 (CH ), 32.74 (CH ), 29.74 (CH ), 29.41 (CH ),
2 2 2 2 2 2
2
9.35 (CH ), 26.12 (CH ), 25.67 (CH ).
2
2
2
9
-(Benzyloxy)-1-nonanol (BzOC9OH, 8)
Yield 25.4%. Purity 99.5%. 1H NMR (500 MHz, CDCl3)
H), 4.500 (s, 2H), 3.626 (t, J = 6.7 Hz, 2H), 3.461 (t, J = 6.7 Hz, 2H), 1.666–1.564 (m, 2H),
δ
(ppm) 7.375–7.225 (m,
5
1
1
13
.564–1.509 (m, 2H), 1.404–1.203 (m, 10H). C NMR (126 MHz, CDCl )
δ
(ppm) 138.71 (C),
3
28.34 (CH
×
2), 127.62 (CH
×
2), 127.47 (CH), 72.86, (CH ) 70.50 (CH ), 63.06 (CH ), 32.79
2
2
2
(
CH ), 29.76 (CH ), 29.53 (CH ), 29.39 (CH ), 29.35 (CH ), 26.17 (CH ), 25.71 (CH ).
2 2 2 2 2 2 2
2
.3. Instrumental Analysis
GC-MS analysis was performed on an Agilent 5975C mass selective detector coupled
with an Agilent 7890A (Agilent Technologies, Santa Clara, CA, USA) equipped with an
HP-Innowax (30 m
×
0.25 mm i.d., 0.25
µ
m film thickness; Agilent Technologies, Santa
◦
Clara, CA, USA). The oven temperature was programmed as 40 C for 1 min and then
◦
◦
1
13
raised to 250 C at 6 C/min, and the temperature was held for 4 min. H and C NMR
500 and 125 MHz, respectively) analysis was performed with a Varian UI500 (Agilent
(

Biomolecules 2021, 11, 384
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Technologies, Santa Clara, CA, USA) spectrometer using TMS in CDCl as an internal
3
standard at the Korea Basic Science Institute, Seoul, Korea.
2.4. Nematicidal Activities of Benzyloxyalkanols against B. xylophilus In Vitro
Benzyloxyalkanols ( ) were solubilized in 1% DMSO solution (laboratory grade,
1–8
Daejung, Hwaseong, Korea) to obtain final concentrations of 1000, 500, 100, and 50 ppm
using serial dilutions, and 1% DMSO solution was used as the control (mortality with
the 1% DMSO solution was not different from that in the untreated groups using water).
As a negative control, abamectin (Sigma–Aldrich, St. Louis, MO, USA) solution was
prepared as above and final concentrations of 100, 50, 10, 5, and 1 ppm solution were tested.
Benzyl alcohol in 1% DMSO solution was prepared at a final concentration of 2000 ppm, as
above. The working solutions were prepared and used on the day of the experiment. Each
treatment comprised three replicates, and each mortality experiment was repeated five
times. Approximately 1000 nematodes were placed on each well of a 96-well cell culture
plate containing 100
the 96-well cell culture plates were maintained at 25
and nematode mortality was observed at 24 h after treatment. Nematodes were considered
dead if their bodies were straight and they did not move, even after transfer to clean water.
µ
L of each prepared solution and 1% DMSO solution (control). Then,
◦
±
1 C and 40% humidity in the dark,
2.5. Extraction of Crude Protein
Crude protein of pine wood nematodes was extracted by following the method of
Kang et al. [32]. Briefly, B. xylophilus (ca. 300
tube containing 500 L of protein extraction buffer (0.1 M Tris–HCl buffer containing
0 mM NaCl and 0.5% Triton X-100; pH 7.8) with metal beads (half of the total volume)
µL) specimens were transferred to a 1.5-mL
µ
2
and vigorously shaken for 3 min. To avoid protein degradation by protease, a protease
inhibitor cocktail (Sigma–Aldrich) was added to the extract. The extract was centrifuged
◦
at 17,000× g for 15 min at 4 C, and crude protein was separated from the cell debris
and filtered through glass wool to remove the excess lipids. The concentration of crude
protein isolated from the PWN was estimated with the Bradford reagent method by using
a VersaMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). 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 quantification.
2
.6. Inhibitory Activity of Benzyloxyalkanols against Acetylcholinesterase (AChE) and Glutathione
S-Transferase (GST)
The AChE and GST inhibitory activities of the compounds were analyzed by the
methods of Ellman et al. [33] and Kang et al. [32], respectively, with modifications. Chemi-
cals (BzOC8OH and BzOC9OH) were prepared by dilution in DMSO. One microliter of
chemical and 79 µL of crude protein were mixed in a 96-well microplate. DMSO without
any chemical was treated as a positive control. The concentrations of the test chemicals
were 1, 0.5, 0.1 and 0.05 mg/mL. For AChE inhibition, after 10 min of preincubation,
0
1
0
µL of 10 mM acetylthiocholine iodide (ASChI) and 10
µ
L of 4 mM 5,5 -dithiobis (2-
nitrobenzoic acid) (DTNB) were added. For GST inhibition, the substrate solution, which
included 10 µL of 20 mM reduced glutathione (Sigma Aldrich) and 10 L of 10 mM 1-
chloro-2,4-dinitrobenzene (CDNB, Sigma Aldrich) diluted in 0.1 M Tris HCl (pH 7.8),
−
µ
−
−
was added to the preincubated mixtures of proteins and the synthetic compounds. The
AChE and GST inhibitory activity was estimated by measuring the Vmax for 30 min at 30 s
intervals at 412 nm and 340 nm, respectively, at room temperature by using the VersaMax
ELISA microplate reader. The inhibitory activity (%) was estimated as 100 ((Vmax of
treatment/Vmax of control) × 100). These assays were performed in triplicate.
2.7. Statistics
The mortality was corrected using Abbott’s formula [34], and the corrected mortality
was arcsine square-root transformed for one-way ANOVA. The means were compared and
separated by the Tukey–Kramer HSD test. The LD (lethal dose) values were estimated by

Biomolecules 2021, 11, 384
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probit analysis with dose-response data. Statistical analysis was performed using JMP ver.
9
.0.2 (SAS Institute Inc., Cary, NC, USA). Mean (
±
SE) values of untransformed data are
reported. Untransformed data are shown.
3. Results and Discussion
3.1. Nematicidal Activities of Benzyloxyalkanols against B. xylophilus In Vitro
The nematicidal activities of benzyloxyalkanols (BzOROH) and monochamol are
shown in Table 1. The mortality results of benzyloxyalkanols showed dose-dependent
responses and was significantly different according to chain length. The mortality of all
tested benzyloxyalkanols was higher than that of benzyl alcohol even at half concentration.
BzOC8OH (7) and BzOC9OH (8) showed 100% mortality at a concentration of 1000 ppm.
The mortality of benzyl alcohol was 31.0% at a concentration of 2000 ppm. As the chain
length of the aliphatic portion was shortened, the mortality was weakened at all tested
concentrations. At a concentration of 500 ppm, this trend was clearly shown. BzOC9OH (
9)
)
showed 85.1% mortality. BzOROH showed a C8–C6 carbon length in the alkyl group (
6–8
and resulted in relatively mild mortality (45.4–56.0% mortality), and BzOROH showed a
shorter than C6 carbon length ( ) and exhibited weaker mortality (37.7–13.9% mortality).
1–4
Table 1. Nematicidal activity of benzyloxyalkanols.
Mortality at Each Concentration (%, Mean ± SE)
Treatment
2
000 1
1000
500
100
50
-
Benzyl alcohol
BzOC2OH (1)
BzOC3OH (2)
BzOC4OH (3)
BzOC5OH (4)
31.0 ± 3.9
-
-
-
30.6 ± 4.2 d
13.9 ± 6.3
e,f
e
e
-
-
-
-
2.5 ± 0.6
4.9 ± 0.7
-
2
5.9 ± 2.5
d,e
c,d
43.3
±
4.6
-
c
4.0 c,d
d,e
53.2 ± 4.2
33.0
37.7
±
±
5.3 ± 0.4
-
8
.8 ± 1.1
c,d,e
b,c
3.0 c,d
57.4
±
4.7
-
b
b,c
c,d
c
BzOC6OH (5)
BzOC7OH (6)
BzOC8OH (7)
BzOC9OH (8)
Control
-
-
-
-
-
71.5 ± 7.5
45.4
47.6
±
3.0
3.2
12.8
±
1.2
-
-
b,c
b,c
60.2
±
5.3
±
16.2 ± 4.3
28.9 ± 2.9
39.2 ± 2.4
a
b
b
a
a
a
100
100
56.0 ± 4.3
5.2 ± 0.5
a
a
85.1 ± 1.9
5.0 ± 0.5
e
f
g
b
0
0
0
0
F8,36 = 159.1
p < 0.001
F8,36 = 67.0
p < 0.001
F8,36 = 42.43
p < 0.001
F2,12 = 48.83
p < 0.001
Statistical value
-
1
ppm. Within the same column, different uppercase letters indicate statistical differences among the treatment, as
measured by one-way ANOVA analysis followed by Tukey’s test (p < 0.05).
Based on the dose-response data, the LC50 and LC90 values were calculated to compare
toxicity (Table 2). The LD50 and LD90 of BzO8OH were higher than those of BzO9OH.
The LD50 value of BzOC8OH was 1.6 times higher than that of BzOC9OH, and the LD90
value of BzOC8OH was approximately twice that of BzOC9OH. However, the toxicity of
BzO8OH and BzO9OH was much weaker than that of abamectin in the terms of LD values.
Table 2. LD50 and LD90 values of benzyloxyheptanol (7), bezyloxyoctanol (8), and abamectin.
Treatment
LD50 (95% CL) 1
LD90 (95% CL)
χ2 (df)
BzOC8OH (7)
BzOC9OH (8)
Abamectin
246.1 (228.5–265.2)
158.0 (81.4–168.9)
1.7 (1.51–1.94)
1004.6 (893.1–1145.6)
490.0 (444.9–545.3)
11.8 (10.2–13.9)
193.1 (18)
71.5 (18)
147.1 (18)
1
LC50 and LC90 values are in ppm and 95% CL are confidence limits.
Aromatic compounds in natural products are known to possess nematicidal activity.
Benzylic compounds, phenylpropanoids, such as cinnamyl alcohol and cinnamic acid ester,

Biomolecules 2021, 11, 384
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eugenol and its analogs, and terpenoids, such as thymol and carvacrol, from essential
oils (EOs) have been reported to have nematicidal activity against PWNs [18,23,35,36]
Suga et al. [37] reported that pinosylvin monomethyl ether ((E)-3-hydroxy-5-methoxystilbene)
from pine wood extracts has nematicidal activity. This compound possesses the partial
structure of phenylpropanoid and eugenol. Additionally, aromatic compounds from fungi,
such as 4-hydroxyphenylacetic acid and sparassol, showed nematicidal activity against
.
PWN [38,39]. Benzyl alcohol is also reported from plant EOs [35,40]. The mortality resulting
from the benzyloxyalkanols ranged from 42.3% to 100% at a concentration of 1000 ppm,
and they showed nematicidal activity.
Recently, Kim et al. [29] reported the nematicidal activity of the homologues of 2-(1-
undecyloxy)-1-ethanol against PWN, and modification of the hydroxy group of alkanols
(-OH) to the
ɷ
-hydroxyalkyloxy group (-OROH) increased their nematicidal activity against
PWN [29 41]. In this study, the same modification was performed with benzyl alcohol,
,
and their nematicidal activity against PWN was tested. Benzyloxyalkanols showed higher
nematicidal activity than benzyl alcohol even at a half concentration of benzyl alcohol.
From these results, it can be concluded that the modification of the hydroxy group to the
ɷ
-hydroxyalkyloxy group could increase nematicidal activity. There are many reports
about aromatic compounds showing higher nematicidal activity than benzyl alcohol. These
aromatic compounds possess phenolic hydroxy groups and phenylallyl groups [18 35 42].
,
,
The increase in nematicidal activity of such compounds could be expected by modification
of the hydroxy group to the ɷ-hydroxyalkyloxy group.
As the carbon chain length of the alkyl group in benzyloxyalkanols increased, the
nematicidal activity increased. Similar trends were also reported for alkyloxyethanols and
aliphatic alcohols [29
but also the chain length of
,
41]. Not only the chain length of alkyl groups in alkyloxyethanols
ɷ
-hydroxyalkyloxy groups in benzyloxyalkanols seemed
to be responsible for the nematicidal activity. The relationship between the structure
and activity of pine wood nematodes has been scarcely reported. The relationship of
the toxicity and linear carboxylic acids with C -C carbon atom chains suggested that
4
10
structural characteristics would allow for easy transfer of the compound through the insect
cuticle [43]. Li et al. [44] suggested that the steric hindrance of chain analogs affects the
toxicity of aliphatic isothiocyanate. However, the relationship between the nematicidal
activity and the structure of benzyloxyalkanols remains to be resolved.
3
.2. Inhibitory Activity of Benzyloxyalkanols against Acetylcholinesterase (AChE) and Glutathione
S-Transferase (GST)
The PWN AChE and GST inhibition rates were determined with the two compounds,
BzOC8OH ( ) and BzOC9OH ( ), which showed the highest mortality among the tested
7
8
compounds. In the AChE and GST inhibitory assays, two compounds did not show any
inhibitory activities at any tested concentration (Table 3).
Table 3. Acetylcholinesterase (AChE) and glutathione S-transferase (GST) inhibitory activity of
benzyloxyheptanol (7) and benzyloxyoctanol (8).
Inhibitory Activity (%)
Treatment
Concentration (ppm)
AChE
GST
BzOC8OH (7)
50
−0.22
−3.45
−2.86
−3.64
−5.24
0.01
1
5
00
00
1.37
2.57
0.19
0.17
1
000
50
BzOC9OH (8)
1
00
−0.24
−0.01
0.60
0.45
500
−2.60
1000
−6.74

Biomolecules 2021, 11, 384
7 of 9
The management of pests relies mainly on the use of insecticides, and the continuous
use of insecticides may result in the development of resistance in pests. To overcome or sup-
press the resistance of a pest against pesticides, it is recommended that active compounds
with different modes of action be used [45]. There are few reports on the modes of action of
aromatic compounds against PWN. Cha et al. [19] reported that naphthoquinones generate
reactive oxygen species that cause oxidative stress in B. xylophilus. Rajasekharan et al. [46
]
reported that 5-iodoindole, like abamectin, rigidly binds to the active sites of the GluCL
receptor by molecular docking assay and suggested that this binding is crucial for main-
taining the open pore structure of the GluCL complex. 5-Iodoindole also induced diverse
phenotypic deformities in nematodes, including abnormal organ disruption/shrinkage and
increased vacuolization. The AChE inhibitory activity of phenylpropanoid and eugenol
analogs was tested by Kang et al. [47]. Isoeugenol and o-anisaldehyde showed approxi-
mately 50% inhibitory activity against AChE at a concentration of 1000 ppm, while other
eugenol analogs showed less than 10% inhibitory activity. Although not aromatic, the
inhibitory activity of aliphatic compounds against PWN, AChE and GST were estimated to
correspond to the mode of action [32]; some aldehyde compounds showed >70% AChE
inhibition and alkanoic acid showed approximately 40% GST inhibition. However, the
compounds that showed enzyme inhibitory activity did not coincide with those with the
best nematicidal activity. In this study, BzOC8OH and BzOC9OH showed relatively higher
nematicidal activity than the other tested compounds; however, they showed no or little
inhibition of the AChE and GST of PWN. This result suggests that AChE and GST may
not be the targets of benzyloxyalkanols. For the safe practical use of benzyloxyalkanols as
nematicides, the other mode of action of benzyloxyalkanols should be investigated.
4
. Conclusions
In this study, benzyloxyalcohols were investigated for their nematicidal activities
against B. xylophilus for the first time. Our results indicated that benzyloxyalcohols could
be alternative nematicidal agents and that modification of the hydroxy group to the
ɷ
-
hydroxyalkyloxy group could increase their nematicidal activity. However, further stud-
ies, including elucidation of the mode of action, are necessary for developing nematici-
dal agents.
5. Patents
Kim J., Lee S., 2019. Composition for controlling pine wood nematodes containing
benzyloxyalcohol. Korea Patent Application Publication, Application No. 10-2019-0156768.
Author Contributions: Conceptualization, J.K.; methodology, J.K.; synthesis, J.K. and J.O.P.; in-
vestigation, J.K., S.J.L. and K.A.Y.; data curation, J.K.; writing—original draft preparation, J.K.;
writing—review and editing, J.K., S.J.L., J.O.P., and K.A.Y. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the National Institute of Forest Science, grant number FE0702-
2
016-11-2020.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Acknowledgments: The authors thank Si Hyeock Lee at Seoul National University for advice on
inhibitory activity experiments.
Conflicts of Interest: The authors declare no conflict of interest.
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