Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against Tetranychus urticae

Article abstract of DOI:10.1021/acs.jafc.8b03806

Bioassay-guided fractionation of the supernatant of the biocontrol strain Bacillus amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the active fractions by column chromatography separation and HPLC purification. The chemi

Full text of DOI:10.1021/acs.jafc.8b03806

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Agricultural and Environmental Chemistry
Acaricidal Activity of Cyclodipeptides from Bacillus
amyloliquefaciens W1 against Tetranychus urticae
Xing-Yu Li, Yue-Hu Wang, Jun Yang, Wen-Yan Cui, Peng-
Jie He, Shahzad Munir, Peng-Fei He, Yi-Xin Wu, and Yue-Qiu He
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03806 • Publication Date (Web): 11 Sep 2018
Downloaded from http://pubs.acs.org on September 11, 2018
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Acaricidal Activity of Cyclodipeptides from Bacillus amyloliquefaciens W1 against
Tetranychus urticae
†
‡
§
§
†
†
XingꢀYu Li , YueꢀHu Wang *, Jun Yang , WenꢀYan Cui , PengꢀJie He , Shahzad
†
†‡
†‡
†‡
Munir , PengꢀFei He , YiꢀXin Wu , YueꢀQiu He *
†
Yunnan Agricultural University, Kunming 650201, China.
‡
Microorganism Fermentation Engineer Research Center of Yunnan, Kunming 650217,
China
§
Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory
for Wild Plant Resources, Chinese Academy of Sciences, Kunming 650201, China
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ABSTRACT:
Bioassayꢀguided fractionation of the supernatant of the biocontrol strain Bacillus
amyloliquefaciens W1 led to the isolation of eight acaricidal cyclodipeptides from the
active fractions by column chromatography separation and HPLC purification. The
chemical structures of these compounds were identified as cycloꢀ(Glyꢀ
L
ꢀPhe), 2,
ꢀPro), 5,
ꢀPro), 8, and
ꢀPro), 9. Those cyclodipeptides displayed significant
with values of 13.85 98.24 µM.
ꢀTyrꢀtransꢀ4ꢀOHꢀ ꢀPro) (LC50 13.85 µM) was five times more effective than
cycloꢀ(
cycloꢀ(
cycloꢀ(
L
L
L
ꢀPheꢀtransꢀ4ꢀOHꢀ
ꢀProꢀtransꢀ4ꢀOHꢀ
ꢀTyrꢀtransꢀ4ꢀOHꢀ
activities
L
ꢀPro), 3, cycloꢀ(Glyꢀ
L
ꢀTyr), 4, cycloꢀ(
LꢀAlaꢀL
LꢀPro), 6, cycloꢀ(Glyꢀ ꢀPro), 7, cycloꢀ(
L
LꢀProꢀL
L
acaricidal
Cycloꢀ(
LC50
–
L
L
the positive control abamectin (LC50 72.06 µM). The results indicated that the
hydroxyl group is an important component. This is the first report on the acaricidal
capabilities of cyclodipeptides against Tetranychus urticae. The results revealed that
the acaricidal activity of the biocontrol strain B. amyloliquefaciens W1 was dependent
on its constituent cyclodipeptides, which have the potential to be safe and
environmentally friendly acaricides.
KEYWORDS:
Cyclic dipeptides; Diketopiperazine; Bioassayꢀguided fractionation; Twoꢀspotted
spider mite; Acaricidal activity; Biological control
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INTRODUCTION
The twoꢀspotted spider mite, Tetranychus urticae Koch, is one of the most
polyphagous arthropod herbivores and feeds on more than 1,100 plant species
belonging to more than 140 different plant families. It is a key pest in field crops and in
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greenhouses, devastating both annual and perennial crops. The direct damage caused
by the pest is owing to feeding punctures, leading to spotting of the leaves, which can
significantly reduce the photosynthesis of plants. The chemical pesticides is major
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,3
control method against T. urticae for long time.
The mites has been developing
resistance to the most of chemical insecticides and acaricides owing to frequent and
4
long term use of pesticides. Additionally, chemical pesticides can have unintended
consequences, impacting environmental quality, food safety, human health, and
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biodiversity including natural enemy abundance.
Scientists have given much attention to biological control of spider mites, for
example, the predators or parasites is widely used to regulate the miters, particularly
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the use of phytoseiid mites, which are currently sold worldwide. Entomopathogenic
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fungi or entomogenous fungi such as Hirsutella thompsonii Fisher, Neozygites
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floridana Weiser and Muma, Beauveria bassiana (Balsamo) Vuillemin, Verticillium
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lecanii (Zimm.), and Isaria cateniannulata also attract a large number of
researchers' eyeballs. Few bacteria, however, have been reported as to their acaricide
activities owing to the piercingꢀsucking mouthparts of the mites, which make it
difficult for bacteria to infect them. In recent years, researchers have concentrated on
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intracellular organisms such as Wolbachia that may cause distorted sex ratios in the
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mite offspring, thereby impacting populations,
and toxinꢀproducing bacteria such
as Bacillus thuringiensis that can produce crystal proteins called δꢀendotoxins that are
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commonly used as a biological acaricides. Additional research has been carried out
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on and potential acaricide producing strains such as Pseudomonas putida.
Bacillus amyloliquefaciens W1 (CGMCC No. 11949), which was isolated from
twoꢀspotted spider mites that had died naturally, is a patented strain that has a strong
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capability to cause death of T. urticae. The B. amyloliquefaciens W1 strain has been
used for the prevention and treatment plant spider mites with a greater than 80%
success rate in corn, strawberry and tomato greenhouses. However, its exact
mechanism of action is not clear. In the present study, the secondary metabolites of B.
amyloliquefaciens W1 were investigated for active compounds because the supernatant
of this culture was found to have the highest acaricidal activity among the fermentation
liquid, supernatant and cells of this strain.
MATERIALS AND METHODS
Microbial strain and culture conditions
B. amyloliquefaciens W1 was isolated from the twoꢀspotted spider mite that had
died naturally and was collected in Anning, Yunnan Province, China. It was identified
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based on the analysis of 16 S rDNA sequence. The voucher specimen of W1 was
preserved in the China General Microbiological Culture Collection Center (CGMCC
No. 11949). The bacterium was inoculated into LuriaꢀBertani (LB) medium in an
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Erlenmeyer flask and incubated for 72 h with 170 rpm at 35 °C and pH 8.
Plants and mites
The spider mite was collected from infested tomato plants in fields near the city of
Anning (102° 19' E, 25° 01' N, 1835 m a.s.l.), Yunnan Province, China. It was not
required specific permission for the collection because it is a harmful agricultural
insect and is distributed extensively. We confirmed that the mites
impact endangered or protected wild.
’ collection did not
The mites used in the research were feeding on tomato and corn in greenhouse that
had been maintained for more than five years without the use of acaricides and
pesticides.
Chemicals and reagents
All chemical reagents were of analytical purity or the highest one commercially
available. The solvents (CHCl , MeOH, EtOH, EtOAc and Me CO) were bought from
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Sinopharm Chemical Reagent Co. Ltd. (Shanghai, P.R. China), and were used directly
without further purification. TFA, HCOOH, MeCN, and MeOH were purchased from
Merck (Darmstadt, Germany) and used for HPLC. Abamectin was provided by Sigma
(St Louis, MO), and
L amino acid standards and Marfey's reagent FDAA were also
purchased from Sigma. Analytical grade water was obtained by ultrafiltration.
Instrumentation
The NMR spectra were detected by DRXꢀ500, DRXꢀ600 and DRXꢀ800 NMR
spectroscopic instruments (Bruker, Bremerhaven, Germany) with TMS as an internal
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standard. The data of MS were collected LCMSꢀITꢀTOF apparatus (Shimadzu, Kyoto,
Japan). IR and UV spectra were measured on VECTOR22 FTꢀIR instrument (Bruker)
and UVꢀ2450 UVꢀVIS spectrophotometer (Shimadzu, Japan) respectively. Column
chromatography was performed using a D101ꢀtype macroporous adsorption resin,
silica gel and Preꢀcoated TLC sheets of silica gel 60 GF254 (Qingdao Haiyang
Chemical, Inc. Qingdao, China), Sephadex LHꢀ20 (GE Healthcare BioꢀSciences AB,
Sweden), and silica gel C18ꢀRP (50 µm) (YMC, Tokyo, Japan). The TLC sheets spots
were observed with UV light instrument (Gongyi, Zhengzhou, China). A series 1200
HPLC equipped with an Eclipse XDBꢀC18 column 250 mm × 4.6 mm i.d., 5 µm, was
made use of compositional analysis, and an Agilent ZORBAX SBꢀC18 column 250
mm × 9.4 mm, 5 µm was applied to purify compound (Agilent, Santa Clara, CA). A
SZX10 stereomicroscope (Olympus Corp., Tokyo, Japan) was also used. Images were
captured with an AxioCam (Carl Zeiss, Thornwood, NY). A CR22G centrifuge
(Hitachi, Tokyo, Japan) was used to remove the bacteria cells from the fermentation
liquid.
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Extraction, isolation and purification
The culture broth (10 L) of the B. amyloliquefaciens W1 strain was centrifuged for
15 min at 12000 rpm (14570 × g) to remove the bacteria cells. The supernatant was
absorbed by D101ꢀtype macroporous adsorption resin column chromatography (CC)
using 95% EtOH as the eluent to obtain a crude extract (29.6 g) after removing the
EtOH with a vacuum rotary evaporator at 55 °C. The extract was suspended in water
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(400 mL, 50 °C), and then successively extracted with EtOAc (3 × 400 mL) and
nꢀBuOH (3 × 400 mL) to obtain fraction E (2.2 g) and fraction B (5.9 g), respectively.
Although the aqueous residue provided the majority of the weight from fraction W
(21.2 g), this material had no effect on the spider mites. Fraction E was separated with
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silica gel CC using a gradient system of CHCl /EtOAc (20:1ꢀ1:1) as the eluent to
obtain 9 fractions, E1ꢀE9, in which only fractions E6 and E8 showed acaricidal activity.
These two fractions were then analysed by preparative HPLC (mobile phase
MeCN/H O, 40:60; UV 230 nm; the temperature of column 30 ºC; the velocity of flow
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1 mL/min) to gained compound 1 (2.4 mg) from E6, compounds 2 (5.6 mg) and 3
(36.5 mg) from E8. Fraction B was severed into 12 fractions using C18 silica gel CC
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with a H O/MeOH gradient in which only B1, B2 and B3 had acaricidal activity.
Fraction B1 was subjected to Sephadex LHꢀ20 CC using methanol as the mobile phase
to get compounds 4 (2.1 mg), 5 (1.5 mg), 6 (9.8 mg) and 7 (6.9 mg), which were
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further purified by preparative HPLC (mobile phase MeCN/H O, 55:45; UV 230 nm;
the temperature of column 30 ºC; the velocity of flow 1 mL/min). Fraction B2 was
purified with Sephadex LHꢀ20 CC using methanol as the mobile phase to afford
compounds 8 (2.6 mg), 9 (3.4 mg) and 10 (2.1 mg), which were further sublimated by
HPLC (mobile phase MeCN/H O, 55:45; UV 230 nm; the temperature of column 30
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ºC; the velocity of flow 1 mL/min).
The pure compounds was analyzed with TLC and analytical HPLC (mobile phase
MeCN/H
2
O, 55:45; injection volume 1 µL; UV 230 nm, the temperature of column 30
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ºC; the velocity of flow 1 mL/min; run time 15 min).
Absolute configuration evaluation of cyclodipeptides
The experiment to determinate absolute configuration of compounds was carried
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out according to the Marfey’s method with some modifications. Each cyclodipeptide
(1.5 mg) was mixed with 6 M HCl (1.0 mL), reacted for 24 h at 125 ºC. The products
2 3
were added in H O (100 µL), 1% FDAA acetone solution (200 µL) and 1 M NaHCO
(40 µL), and then heated for 1 h at 40 ºC with shaking. The reaction products was
quenched using 2 M HCl (20 µL). The mixture was allow to cool, dried, and dissolved
in MeOH for further analysis. Each kind of standard of
L amino acid (1 mg) was used
to react with Marfey’s regent in the same way. An Agilent 1200 series machine
equipped with an Agilent eclipse XDBꢀC18 column 250 mm × 4.6 mm i.d., 5 µm was
used for HPLC analysis at 30 ºC using two solvent systems: solvent A, 0.1% TFA
aqueous solution; solvent B, MeCN; linear gradient: 0 min, 25% B; 40 min, 60 % B;
45 min, 100 % B; UV 340 nm.
Acaricidal activity assay
The acaricidal activity assay, used to test the effects of contact toxicity against T.
urticae of B. amyloliquefaciens W1 and the different fractions of its secondary
metabolites, was composed of three methods, including the slide dip assay, the leaf
spray assay, and the microscale dip assay. The acaricidal activity components were
obtained from the fermentation liquid through bioassayꢀguided procedures using the
first two methods. The last method was carried out to evaluate the acaricidal ability of
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the compounds that were purified from the acaricidal activity fractions.
Slide dip assay
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The slide dip bioassay (Figure 1) was used to assess the death rate of T. urticae
treated with the isolations from the fermentation products of B. amyloliquefaciens W1.
The different fractions dissolved in water containing 1% azone (to increase the
compounds permeability in aqueous medium) were diluted to 0.5 mg/mL as test
solutions. Adult mites were stuck to doubleꢀsided adhesive tape that was placed at the
end of a glass slide (10 cm × 2 cm) using a brush. The energetic mites selected were
stuck on the glass slide and took count of starting number under a stereomicroscope,
and then the slide was dipped into the agent for 5 s, allowed to be upright until it dried
using the absorbent paper to remove the agent, and observed under a stereomicroscope
2 h after treatment, similarly, 1% azone was served as the control. The experiment was
carried out in triplicate. The dead mites' appendages will not move when brushed
carefully.
Leaf spray assay
The acaricidal activity of all fractions, was similarly evaluated by the leaf spray
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assay (Figure 1). Adult spider mites (50) were move to a fresh tomato leaf from the
source leaves infested by T. urticae. They were then sprayed individually with various
test agents that consisted of the different fractions of the fermentation products of B.
amyloliquefaciens W1, which were dissolved in water containing 1% azone and diluted
to 0.5 mg/mL. The leaves were placed onto cotton soaked with water to make the leaf
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alive for 2 h, and then observed under a stereomicroscope. Water with 1% azone used
as the control. The experiment was performed in triplicate. The dead mites' appendages
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will not move when brushed carefully. The half leaf spray assay was also used to
detect the acaricidal activity, with the same procedures as in the leaf spray assay. One
half leaf was sprayed with test solution, and the other half was sprayed with water in 1%
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azone to serve as the control. The Abbot’s formula was used to calculate the corrected
mortality rate (%).
Microscale dip assay
2
2
The microscale dip assay (Figure 1), designed especially for microcomponents,
was used to evaluate the acaricidal ability of the compounds isolated from the
acaricidal activity fractions. The compounds were dissolved in water containing 1%
azone and were diluted gradually to 50.00 µg/mL, 25.00 µg/mL, 12.50 µg/mL, 6.26
µg/mL, 3.13 µg/mL, 1.56 µg/mL, and 0.78 µg/mL as test solutions. Adult mites,
moved from the leaves to smooth white paper using a soft brush, were affixed to a
needle pinpoint with adhesive and inspected under a stereomicroscope to take away
any dead or immobile one, and maintain only the active one as the starting number.
The needle was immersed into the compound solutions (contained in a centrifuge tube)
for 5 s, and then observed and counted under a stereomicroscope 2 h after treatment.
Similarly, 1% azone aqueous used as the control. The experiment was triplicate. The
dead mites' appendages will not move when brushed carefully.
Statistical analysis
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The mortalities were corrected using Abbot’s formula .
ꢀ
ꢁ = ꢂꢃꢀꢄ − ꢀꢅꢆ⁄ꢃ100 − ꢀꢅꢆꢇ × 100
1
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2
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Where Pt represents the means of corrected mortality (%), Po represents the means of
the treated mortality (%) and Pc represents the means of the control mortality (%).
Data analysis was done using descriptive statistics software MINITAB version 14.
Concentrationꢀmortality data were conducted to probit analysis for compounds lethal
concentation (LC50) and toxicity regression equation. The LC50 values for each
acaricidal preparation and theit treatments were consdered to be significantly different
from one another when their 95% confidence limits did not overlap.
RESULTS AND DISCUSSION
Identification of compounds
Ten compounds were obtained from the culture broth of B. amyloliquefaciens W1
by bioassayꢀguided fractionation for acaricidal activity, with CC separation and HPLC
purification, and conducted to various spectroscopic analyses, such as NMR and MS.
The structures identified for eight compounds 2–9 were classified as diketopiperazines
or cyclodipeptides, as shown in Figure 2, and an additional two compounds
corresponded to macrolactin A, 1 and bacimethrin, 10. by analysing and comparing for
the NMR spectra with that reported in the literature, they were identified as the
2
3
24
following: macrolactin A, 1, cycloꢀ(Glyꢀ
L
ꢀPhe), 2, cycloꢀ(
L
ꢀPheꢀtransꢀ4ꢀOHꢀ
ꢀProꢀtransꢀ4ꢀOHꢀ
ꢀTyrꢀtransꢀ4ꢀOHꢀ
LꢀPro),
LꢀPro),
LꢀPro),
2
5
26
27
3, cycloꢀ(Glyꢀ
L
ꢀTyr), 4, cycloꢀ(
L
ꢀAlaꢀ
L
ꢀPro), 5, cycloꢀ(
L
2
8
29
27
6, cycloꢀ(Glyꢀ
L
ꢀPro), 7, cycloꢀ(
L
ꢀProꢀ
L
ꢀPro), 8, cycloꢀ(
L
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9, and bacimethrin, 10. All compounds purities was greater than 95% according to
the peak area in HPLC analysis.
Analysis of Marfey’s derivatives of cyclodipeptides
The absolute stereochemistry of the eight cyclodipeptides were determined with
the modified Marfey’s method. The results showed that eight cyclodipeptides contain L
amino acids, apart from the glycine, which is achiral.
Acaricidal activity for bioassay-guided fractionation
The acaricidal activity components were isolated from B. amyloliquefaciens W1
through bioassayꢀguided fractionation. The corrected mortality rate of T. urticae
treated with each fraction was recorded (Figure 3). The culture broth, supernatant and
its extract showed effects on the spider mites, and the extract was then subjected to
chemical analysis to produce different the fractions. Fractions E6, E8, B1, B2 and B3,
which were derived from activity fractions E and B, were more effective (corrected
mortality rate ranging from 86.0% – 93.0% for slide dip assay and 86.0% – 91.2% for
the leaf spray assay) than the others, including the positive control abamectin
(corrected mortality rate of 80.7% for the slide dip assay and 78.9% for the leaf spray
assay). All of the tested fraction concentrations were standardized at 0.5 mg/mL in
aqueous solution containing 1% azone, except for the culture broth, supernatant and
strain samples.
Acaricidal activity of pure compounds
All isolates 1–10 were evaluated for acaricidal activity with a microscale dip assay
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against T. urticae and were compared with the positive control abamectin under the
same conditions. Among the compounds, the cyclodipeptides 2–9 exhibited a potent
lethality, with LC values ranging from 13.85 – 98.24 µM (Table 1), which is similar
5
0
to or higher than the activity of abamectin (LC50 72.06 µM). Macrolactin A, 1 and
bacimethrin, 10 displayed only a weak activity (data not shown). The LC50 values of
cyclodipeptides 2–9 against T. urticae were 84.60, 25.19, 43.98, 98.24, 43.71, 95.96,
63.90 and 13.85 µM, respectively, with the isolated compound 9 (LC50 13.85 µM)
being approximately five times more effective than the positive control (LC50 72.06
µM).
These indicate
results
that
cyclodipeptides
2–9,
particularly
cycloꢀ(
LꢀTyrꢀtransꢀ4ꢀOHꢀLꢀPro), 9, have potential for the development of new
acaricidal agents for the control of T. urticae.
Structure-activity relationship
Natural peptides are well known as a kind of significant resources of medicinal
key composition. Among all cyclic peptides, the cyclodipeptide is the simplest one,
which compose a great family of metabolites biosynthesized by bacteria, fungi, flora,
and fauna. They display extensive bioactivities, for instance bioherbicide, algicidal,
antibacterial, antimicrobial, anticancer, antitumour, antiviral, antifungal, and
31ꢀ36
antihyperglycaemic activities.
In this study, eight cyclodipeptides were isolated
from B. amyloliquefaciens W1 and were tested for acaricidal agents against the
twoꢀspotted spider mite. The isolated cyclodipeptides exhibited distinctive acaricidal
activities against T. urticae, with LC50 values of 13.85 – 98.24 µM. We demonstrated
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that the acaricidal efficacy of B. amyloliquefaciens W1 was dependent on the various
cyclic dipeptide components. Furthermore, this is the first report on the acaricidal
capabilities of cyclodipeptides against T. urticae.
Interestingly, the acaricidal activities of the diketopiperazines change with a
change in the amino acid, and amino acids with hydroxyl groups, such as
hydroxyproline and tyrosine, could greatly improve the activity. A comparison of the
structure (Figure 2) and LC50 (Table 1) data of the diketopiperazines reveals that
compounds that are cyclized by two amino acids without a hydroxyl group, such as
cycloꢀ(Glyꢀ
cycloꢀ( ꢀProꢀ
with hydroxyl group, such as cycloꢀ(
cycloꢀ( ꢀProꢀtransꢀ4ꢀOHꢀ ꢀPro), 6, and cycloꢀ(
L
ꢀPhe), 2, cycloꢀ(
ꢀPro), 8, have lower acaricidal activities than other diketopiperazines
ꢀPheꢀtransꢀ4ꢀOHꢀ ꢀPro), 3, cycloꢀ(GlyꢀTyr), 4,
ꢀTyrꢀtransꢀ4ꢀOHꢀ ꢀPro), 9. The
LꢀAlaꢀLꢀPro), 5, cycloꢀ(LꢀGlyꢀLꢀPro), 7, and
L
L
D
D
L
L
L
L
compound 9 with two hydroxyl groups which is cyclized by tyrosine and
hydroxyproline demonstrated the best acaricidal activity among eight isolates.
Therefore, the hydroxyl groups in diketopiperazines are important for acaricidal
capability.
In agricultural and horticultural cultivation, various chemical acaricides have been
greatly used to control spider mites because of their outstanding trait, such as having a
wide selection, quick mode of action and low cost. However, the frequent application
of chemical pesticides has led to various issues, such as pesticide resistance, pesticide
residues, concerns regarding food safety and human health, and impacts on
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biodiversity, including natural enemy abundance. However, some diketopiperazines
have acaricidal capabilities against T. urticae, are safe and ecoꢀfriendly, and many of
them have been reported to have other bioactivities or are used as medicinal
3
8
components. For example, cycloꢀ(
L
ꢀProꢀGly),
7
can enhance memory.
39
Cycloꢀ( ꢀProꢀ ꢀPro), 8 can be used for chemical selectivity oxidations of sulfides.
L
L
Cycloꢀ(GlyꢀTyr), 4 has been shown to inhibit bioluminescence and exhibit synergistic
4
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antimicrobial activity and has also been used as herbicide.
diketopiperazines 2–9, and in particular, compound, 9 cycloꢀ(
have the potential to be safe and environmentally friendly acaricides.
Therefore, the isolated
L
ꢀTyrꢀtransꢀ4ꢀOHꢀ ꢀPro),
L
Although the resurgence of spider mites, induced by chemical pesticides, is the
result of a combination of several factors from the homeostatic modulation of the pest
itself to the reduction of natural enemies, pesticide resistance is thought to contribute to
4
3
the resurgence phenomenon. T. urticae quickly progresses to acaricide resistance,
generally only a few years after the implementation of novel acaricides. Some
acaricides such as clofentezine, abamectin and hexythiazox used in greenhouses to
44
control T. urticae on roses and other economic crops, have lost most of their impact.
The T. urticae resistance have been arose to nearly one hundred ingredients of
45
acaricides. Therefore, the problem has become urgent, as T. urticae is presently
deemed as the most resistant species as far as the sum total of compounds to which
2
populations have turned resistant. It is significant to effectively manage insecticide
resistance to preserve the usefulness of insecticide whenever, and we have many
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options such as cultural methods, autocidal techniques, crop rotation, semiochemicals,
46
host plant resistance, chemical control, genetically modified plants, and biocontrol. In
this study, we used the biocontrol strain B. amyloliquefaciens W1 to control T. urticae
in the greenhouse, which is in accordance with insecticide resistance management
procedure. Ten natural products were obtained from the strain, of which eight
compounds displayed acaricidal activity against T. urticae. Natural compounds have
been used effectively as biocontrol agents not only due to their environmental safety,
and ease of degradation but also because they do not elicit drug resistance. Our
findings help to provide an alternative source of microbial acaricides that do not have
the unwanted effects of drug resistance, residual agrochemicals and environmental
pollution.
ASSOCIATED CONTENT
Supporting Information
This supporting information is available free of charge via the Internet at
http://pubs.acs.org.
1
13
Figures S1−S30: H NMR spectrum, C NMR and DEPT spectrum, and ESIꢀMS
spectrum of compounds; Figures S31ꢀ38: HPLC profile of FDAA derivatives of amino
acid standards and compounds; Figure S39: HPLC chromatogram of compounds;
Figure S40: The follow chart of compounds from the supernatant of B.
amyloliquefaciens W1; Physicochemical parameters of compounds.
AUTHOR INFORMATION
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Corresponding Authors
* Telephone/Fax: +86ꢀ871ꢀ65228532. Eꢀmail: ynfh2007@163.com (Y.ꢀQ. H.)
* Telephone/Fax: +86ꢀ871ꢀ65223318. Eꢀmail: wangyuehu@mail.kib.ac.cn (Y.ꢀH. W)
ORCID
XingꢀYu Li: 0000ꢀ0001ꢀ5801ꢀ7989
Funding
This work was supported by the National Natural Science Foundation of China
(31660536).
Notes
The authors declare no competing financial interests.
ABBREVIATIONS USED
HPLC, high performance liquid chromatography; CC, column chromatography;
TLC, thin layer chromatography; LB, LuriaꢀBertani; TFA, trifluoroacetic acid; FDAA,
1ꢀfluoroꢀ2ꢀ4ꢀdinitrophenylꢀ5ꢀLꢀalanine amide; NMR, nuclear magnetic resonance;
TMS, tetramethylsilane; MS, Mass spectrometry; LCMSꢀITꢀTOF, liquid
chromatographyꢀmass spectrometryꢀionꢀtrapꢀtime of flight instrument; EtOH, ethanol;
EtOAc, ethyl acetate; BuOH, butanol; MeCN, acetonitrile; MeOH, methanol; Me
acetone; LC , half maximal inhibitory concentration.
2
CO,
5
0
3
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3
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REFERENCES
(1) Grbic, M.; Van Leeuwen, T.; Clark, R. M.; Rombauts, S.; Rouze, P.; Grbic, V.;
Osborne, E. J.; Dermauw, W.; Thi Ngoc, P. C.; Ortego, F.; HernandezꢀCrespo, P.; Diaz,
17
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Journal of Agricultural and Food Chemistry
Page 18 of 31
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
I.; Martinez, M.; Navajas, M.; Sucena, E.; Magalhaes, S.; Nagy, L.; Pace, R. M.;
Djuranovic, S.; Smagghe, G.; Iga, M.; Christiaens, O.; Veenstra, J. A.; Ewer, J.;
Villalobos, R. M.; Hutter, J. L.; Hudson, S. D.; Velez, M.; Yi, S. V.; Zeng, J.;
PiresꢀdaSilva, A.; Roch, F.; Cazaux, M.; Navarro, M.; Zhurov, V.; Acevedo, G.; Bjelica,
A.; Fawcett, J. A.; Bonnet, E.; Martens, C.; Baele, G.; Wissler, L.; SanchezꢀRodriguez,
A.; Tirry, L.; Blais, C.; Demeestere, K.; Henz, S. R.; Gregory, T. R.; Mathieu, J.;
Verdon, L.; Farinelli, L.; Schmutz, J.; Lindquist, E.; Feyereisen, R.; Van de Peer, Y.,
The genome of Tetranychus urticae reveals herbivorous pest adaptations. Nature 2011,
479, 487−492.
(2) Van, L. T.; Tirry, L.; Yamamoto, A.; Nauen, R.; Dermauw, W., The economic
importance of acaricides in the control of phytophagous mites and an update on recent
acaricide mode of action research. Pestic. Biochem. Physiol. 2015, 121, 12−21.
(3) Jafari, S.; Fathipour, Y.; Faraji, F.; Bagheri, M., Demographic response to constant
temperatures in Neoseiulus barkeri (Phytoseiidae) fed on Tetranychus urticae
(Tetranychidae). Syst. Appl. Acarol. 2016, 15, 83−99.
(4) Minazzi Stocco, R. S.; Sato, M. E.; Santos, T. L., Stability and fitness costs
associated with etoxazole resistance in Tetranychus urticae (Acari: Tetranychidae). Exp.
Appl. Acarol. 2016, 69, 413−425.
(5) Yang, L.; Elbakidze, L.; Marsh, T.; McIntosh, C., Primary and secondary pest
management in agriculture: balancing pesticides and natural enemies in potato
production. Agric. Econ. 2016, 47, 609−619.
18
ACS Paragon Plus Environment

Page 19 of 31
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
(6) Funayama, K.; Komatus, M.; Sonoda, S.; Takahashi, I.; Hara, K., Management of
apple orchards to conserve generalist phytoseiid mites suppresses twoꢀspotted spider
mite, Tetranychus urticae (Acari: Tetranychidae). Exp. Appl. Acarol. 2015, 65, 43−54.
(7) ElꢀSharabasy, H. M., Laboratory evaluation of the effect of the entomopathogenic
fungi, Hirsutella thompsonii and Paecilomyces fumosoroseus, against the citrus brown
mite, Eutetranychus orientalis (Acari: Tetranychidae). Plant Prot. Sci. 2015, 51,
39−45.
(8) Klingen, I.; Waersted, G.; Westrum, K., Overwintering and prevalence of
Neozygites floridana (Zygomycetes: Neozygitaceae) in hibernating females of
Tetranychus urticae (Acari: Tetranychidae) under cold climatic conditions in
strawberries. Exp. Appl. Acarol. 2008, 46, 231−245.
(9) Ullah, M. S.; Lim, U. T., Laboratory bioassay of Beauveria bassiana against
Tetranychus urticae (Acari: Tetranychidae) on leaf discs and potted bean plants. Exp.
Appl. Acarol. 2015, 65, 307−318.
(10) Seiedy, M., Compatibility of Amblyseius swirskii (Acari: Phytoseiidae) and
Beauveria bassiana for biological control of Trialeurodes vaporariorum (Hemiptera:
Aleyrodidae). Syst. Appl. Acarol. 2015, 20, 731−738.
(11) Zhang, X.; Jin, D.; Zou, X.; Guo, J., Laboratory and field evaluation of an
entomopathogenic fungus, Isaria cateniannulata strain 08XSꢀ1, against Tetranychus
urticae (Koch). Pest Manage. Sci. 2016, 72, 1059−1066.
(12) Chen, Y. T.; Zhang, Y. K.; Du, W. X.; Jin, P. Y.; Hong, X. Y., Geography has a
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 31
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
greater effect than Wolbachia infection on population genetic structure in the spider
mite, Tetranychus pueraricola. Bull. Entomol. Res. 2016, 106, 685−694
(13) Morrow, J. L.; Frommer, M.; Shearman, D.; Riegler, M., Tropical tephritid fruit
fly community with high incidence of shared Wolbachia strains as platform for
horizontal transmission of endosymbionts. Environ. Microbiol. 2014, 16, 3622−3637.
(14) Neethu, K.; Priji, P.; Unni, K.; Sajith, S.; Sreedevi, S.; Ramani, N.; Anitha, K.;
Rosana, B.; Girish, M.; Benjamin, S., New Bacillus thuringiensis strain isolated from
the gut of Malabari goat is effective against Tetranychus macfarlanei. J. Appl.
Entomol. 2016, 140, 187−198.
(15) Aksoy, H. M.; OzmanꢀSullivan, S. K.; Ocal, H.; Celik, N.; Sullivan, G. T., The
effects of Pseudomonas putida biotype B on Tetranychus urticae (Acari:
Tetranychidae). Exp. Appl. Acarol. 2008, 46, 223−230.
(16) He, Y. Q.; Li, X. Y.; He, P. F.; Wu, Y. X.; He, P. B.; Wang, Z. Q.; Wang, Q.; Yang,
F. X. A strain of Bacillus amyloliquefaciens W1 for the control of phytophagous mite.
CN 105543151 A.05.04, 2016.
(17) Marfey, P., Determination of
Dꢀamino acids. II. Use of a bifunctional reagent,
1,5ꢀdifluoroꢀ2,4ꢀdinitrobenzene. Carlsberg Res. Commun. 1984, 49, 591.
(18) Zhang, Y.; Zhang, Q.; Luo, J.; Ding, W., Acaricidal active fractions from acetone
extract of Aloe vera L. against Tetranychus cinnabarinus and Panonychus citri. Acta
Physiol. Plant. 2017, 39, 195−202.
(19) Sparks, T. C.; Thompson, G. D.; Kirst, H. A.; Hertlein, M. B.; Larson, L. L.;
20
ACS Paragon Plus Environment

Page 21 of 31
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Worden, T. V.; Thibault, S. T., Biological activity of the spinosyns, new fermentation
derived insect control agents, on tobacco budworm (Lepidoptera: Noctuidae) larvae. J.
Econ. Entomol. 1998, 91, 1277−1283.
(20) Fatemikia, S.; Abbasipour, H.; Saeedizadeh, A., Phytochemical and acaricidal
study of the Galbanum, Ferula gumosa Boiss.(Apiaceae) essential oil against
Tetranychus urticae Koch (Tetranychidae). J. Essent. OilꢀBear. Plants 2017, 20,
185−195.
(21) Abbott, W. S., A method of computing the effectiveness of an Insecticide. J. Econ.
Entomol. 1925, 18, 265−267.
(22) Li, X. y.; Li, S. Q.; He, P. F.; Wu, Y. X.; He, P. B. A microscale dip assays for
small insect and its device. CN 106290736 B. 15.06, 2018.
(23) He, S.; Wang, H. Q.; Yan, X. J.; Zhu, P.; Chen, J. J.; Yang, R., Preparative
isolation and purification of macrolactin antibiotics from marine bacterium Bacillus
amyloliquefaciens using highꢀspeed counterꢀcurrent chromatography in stepwise
elution mode. J. Chromatogr. A. 2013, 1272, 15−19.
(24) Kumar, S. N.; Sreekala, S. R.; Chandrasekaran, D.; Nambisan, B.; Anto, R. J.,
Biocontrol of Aspergillus species on peanut kernels by antifungal diketopiperazine
producing Bacillus cereus associated with entomopathogenic nematode. Plos One
2014, 9, e106041.
(25)Fdhila, F.; Vazquez, V.; Sanchez, J. L.; Riguera, R., DDꢀdiketopiperazines:
Antibiotics active against Vibrio anguillarum isolated from marine bacteria associated
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 31
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
with cultures of Pecten maximus. J. Nat. Prod. 2003, 66, 1299−1301.
(26) Mitova, M.; Tommonaro, G.; Hentschel, U.; Muller, W. E. G.; De Rosa, S.,
Exocellular cyclic dipeptides from a Ruegeria strain associated with cell cultures of
Suberites domuncula. Mar. Biotechnol. 2004, 6, 95−103.
(27) Stark, T.; Hofmann, T., Structures, sensory activity, and dose/response functions
of 2,5ꢀdiketopiperazines in roasted cocoa nibs (Theobroma cacao). J. Agric. Food
Chem. 2005, 53, 7222−7231.
(28) Gajbhiye, M. H.; Kapadnis, B. P., Antifungalꢀactivityꢀproducing lactic acid
bacteria as biocontrol agents in plants. Biocontrol Sci. Techn. 2016, 26, 1451−1470.
(29) Oleinikova, G. K.; Afiyatullov, S. S.; Mikhailov, V. V.; Shevchenko, L. S.;
Menzorova, N. I.; Yurchenko, E. A., Diketopiperazines from marine isolate of
actinobacterium Nocardiopsis umidischolae KMM 7036. Chem. Nat. Compd. 2015, 51,
192−193.
(30) Li, S. Q.; Yang, Y. B.; Yang, X. Q.; Jiang, Y.; Li, Z. J.; Li, X. Z.; Chen, X.; Li, Q.
L.; Qin, S. H.; Ding, Z. T., Two new cyclic tetrapeptides of Streptomyces rutgersensis
T009 Isolated from Elaphodus davidianus excrement. Helv. Chim. Acta 2016, 99,
210−214.
(31) Mishra, A. K.; Choi, J.; Choi, S.ꢀJ.; Baek, K.ꢀH., Cyclodipeptides: an overview of
their biosynthesis and biological activity. Molecules 2017, 22, 1796−1089.
(32) Xue, J.; Wu, M., Cyclic dipeptides from Streptomyces michiganensis. Chem. Nat.
Compd. 2015, 51, 395−396.
22
ACS Paragon Plus Environment

Page 23 of 31
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
(33) Mollica, A.; Costante, R.; Fiorito, S.; Genovese, S.; Stefanucci, A.; Mathieu, V.;
Kiss, R.; Epifano, F., Synthesis and antiꢀcancer activity of naturally occurring
2,5ꢀdiketopiperazines. Fitoterapia 2014, 98, 91−97.
(34) Borthwick, A. D., 2,5ꢀDiketopiperazines: synthesis, reactions, medicinal
chemistry, and bioactive natural products. Chem. Rev. 2012, 112, 3641−3716.
(35) Guo, X.; Liu, X.; Wu, L.; Pan, J.; Yang, H., The algicidal activity of Aeromonas sp.
strain GLYꢀ2107 against bloomꢀforming Microcystis aeruginosa is regulated by Nꢀacyl
homoserine lactoneꢀmediated quorum sensing. Environ. Microbiol. 2016, 18,
3867−3883.
(36) Li, L.; Yang, M.; Luo, J.; Qu, Q.; Chen, Y.; Liang, L.; Zhang, K.,
Nematodeꢀtrapping fungi and fungusꢀassociated bacteria interactions: the role of
bacterial diketopiperazines and biofilms on Arthrobotrys oligospora surface in hyphal
morphogenesis. Environ. Microbiol. 2016, 18, 3827−3839.
(37) Gigon, V.; Camps, C.; Le Corff, J., Biological control of Tetranychus urticae by
Phytoseiulus macropilis and Macrolophus pygmaeus in tomato greenhouses. Exp. Appl.
Acarol. 2016, 68, 55−70.
(38) Samonina, G.; Ashmarin, I.; Lyapina, L., Glyproline peptide family: review on
bioactivity and possible origins. Pathophysiology 2002, 8, 229−234.
(39) Kienle, M.; Argyrakis, W.; Baro, A.; Laschat, S., Diketopiperazineꢀderived
hydroperoxide for chemoselective oxidations of sulfides and enantioselective Weitz–
Scheffer epoxidations. Tetrahedron Lett. 2008, 49, 1971−1974.
23
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Journal of Agricultural and Food Chemistry
Page 24 of 31
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
(40) Stierle, A. C.; Cardellina, J. H.; Strobel, G. A., Maculosin, a hostꢀspecific
phytotoxin for spotted knapweed from Alternaria alternata. Proc. Natl. Acad. Sci. U. S.
A. 1988, 85, 8008−8011.
(41) Cain, C. C.; Lee, D.; Waldo, R. H.; Henry, A. T.; Casida, E. J.; Wani, M. C.; Wall,
M. E.; Oberlies, N. H.; Falkinham, J. O., Synergistic antimicrobial activity of
metabolites produced by a nonobligate bacterial predator. Antimicrob. Agents
Chemother. 2003, 47, 2113−2117.
(42) Teasdale, M. E.; Donovan, K. A.; ForschnerꢀDancause, S. R.; Rowley, D. C.,
Gramꢀpositive marine bacteria as a potential resource for the discovery of quorum
sensing inhibitors. Mar. biotechnol. 2011, 13, 722−732.
(43) Patil, C.; Udikeri, S.; Karabhantanal, S., A note on pesticide induced resurgence of
two spotted spider mite, Tetranychus urticae (Acari: Tetranychidae) on grape. Persian
J. Acarol. 2018, 7, 75−84.
(44) Pavlidi, N.; Khalighi, M.; Myridakis, A.; Dermauw, W.; Wybouw, N.; Tsakireli, D.;
Stephanou, E. G.; Labrou, N. E.; Vontas, J.; Van Leeuwen, T.,
A
glutathioneꢀSꢀtransferase (TuGSTd05) associated with acaricide resistance in
Tetranychus urticae directly metabolizes the complex II inhibitor cyflumetofen. Insect
Biochem. Mol. Biol. 2017, 80, 101−115.
(45) Kwon, D. H.; Clark, J. M.; Lee, S. H., Toxicodynamic mechanisms and
monitoring of acaricide resistance in the twoꢀspotted spider mite. Pestic. Biochem.
Physiol. 2015, 121, 97−101.
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Page 25 of 31
Journal of Agricultural and Food Chemistry
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4
90
91
(46) Sparks, T. C.; Nauen, R., IRAC: Mode of action classification and insecticide
resistance management. Pestic. Biochem. Physiol. 2015, 121, 122−128.
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FIGURE CAPTIONS
Figure 1. Scheme of acaricidal activity assay methods. (A): slide dip assay; (B): leaf
spray assay; (C): microscale dip assay
Figure 2. The structures of compounds 1–10
Figure 3. Corrected mortality rate of T. urticae treated with culture broth, bacteria cells,
supernatant, 0.5 mg/mL fractions from the supernatant, and 0.5 mg/mL abamectin in
the slide dip assays and leaf spray assays. Bars represent standard deviations of means.
(A): slide dip assays; (B): leaf spray assays.
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Table 1. LC Values of Isolated Compounds 2–9 and Abamectin against T. urticae.
50
b
LC50
b
b
Compound Regression equation
(µM) 95% Confidence interval
84.60 62.45 − 114.63
25.19 17.54 − 36.18
43.98 34.26 − 56.46
98.24 71.54 − 134.90
43.71 32.91 − 58.05
95.96 71.59 − 128.61
63.90 47.93 − 85.19
13.85 7.80 − 24.59
R
SE
2
3
4
5
6
7
8
9
PC
y = 2.31x + 0.55
y = 2.35x + 1.70
y = 3.19x − 0.24
y = 2.15x + 0.72
y = 2.76x + 0.48
y = 2.29x + 0.46
y = 2.30x + 0.85
y = 1.99x + 2.72
y = 2.51x + 1.86
0.96 0.05
0.99 0.02
0.99 0.02
0.98 0.05
0.95 0.03
0.98 0.04
0.95 0.04
0.99 0.02
0.99 0.07
a
72.06 52.43 − 99.05
a
positive control: abamectin
b
The regression equation, LC50 and 95% confidence limit were determined by probit
analysis.
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Figure 1
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Figure 2
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