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Toxicity and Repellency of Hot Pepper

Extracts to Spider Mite, Tetranychus
urticae Koch
a a b
GEORGE F. ANTONIOUS , JANET E. MEYER & JOHN C. SNYDER
a
Department of Plant and Soil Science , Land Grant Program,
Kentucky State University , Frankfort, KY, USA
b
Department of Horticulture , University of Kentucky , Lexington,
KY, USA
Published online: 05 Dec 2006.

To cite this article: GEORGE F. ANTONIOUS , JANET E. MEYER & JOHN C. SNYDER (2006) Toxicity and
Repellency of Hot Pepper Extracts to Spider Mite, Tetranychus urticae Koch, Journal of Environmental
Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 41:8, 1383-1391

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 Journal of Environmental Science and Health Part B, 41:13831391, 2006
Copyright
C Taylor & Francis Group, LLC
ISSN: 0360-1234 (Print); 1532-4109 (Online)
DOI: 10.1080/0360123060096419

Toxicity and Repellency of Hot

Pepper Extracts to Spider Mite,
Tetranychus urticae Koch
Downloaded by [Central Michigan University] at 22:17 25 December 2014

George F. Antonious,1 Janet E. Meyer,1 and John C. Snyder2

1
Department of Plant and Soil Science, Land Grant Program, Kentucky State University,
Frankfort, KY, USA
2
University of Kentucky, Department of Horticulture, Lexington, KY, USA
Increasing concern about persistence and environmental impact of synthetic pesticide
residues require development of biodegradable and environmentally safe alternatives.
The potential of using fruit extracts of hot pepper as alternatives to synthetic acari-
cides for controlling the two-spotted spider mite, Tetranychus urticae Koch, is explored
in this study. Twenty-four Capsicum accessions (Solanaceae) were screened for their
toxicity and repellency to the spider mites. Crude extracts from fruits of C. chinense,
C. frutescens, C. baccatum, C. annuum, and C. pubescens were prepared in methanol
and tested for their acaricidal properties. Spider mite mortality was greatest (45%) when
fruit extract of accession Grif-9169 (C. annuum) was used. Results from diving board
bioassays indicated that mites avoided filter paper strips treated with hot pepper ex-
tracts from accessions PI-596057 (C. baccatum), PI-195299 (C. annuum), and Grif- 9270
(C. annuum). This investigation suggests that methanolic extracts of these three acces-
sions may have a great potential for repelling spider mites and should be field-tested on
a large-scale to assess their value in managing populations of spider mites, which could
reduce reliance on synthetic acaricides. An attempt was made to correlate repellency
with chemical constituents of fruit extracts of the most repellent accessions to identify
chemical sources of repellency. Capsaicin and dihydrocapsaicin, the pungent components
of pepper fruit, were not correlated with toxicity or repellency, indicating that these are
not likely related to the toxicity or repellency of the pepper fruit extracts. Other, uniden-
tified chemicals are likely responsible for toxicity and repellency to the two-spotted
spider mite

Key Words: Bioassay; Capsicum spp.; Mortality; Pepper extracts; Pepper chemical
composition; Capsaicinoids.

Received March 20, 2006.

Address correspondence to G. F. Antonious, Department of Plant and Soil Science, Land
Grant Program, 218 Atwood Research Facility, Kentucky State University, Frankfort,
KY 40601; E-mail: george.antonious@kysu.edu

1383
 1384 Antonious, Meyer, and Snyder

INTRODUCTION
The development of resistance to existing classes of pesticides and the increas-
ing public concern over environmental pollution and health hazards created by
synthetic pesticides generate a great need for new classes of pest control agents
with higher activity against the target pests, and lower impact on humans and
environmental quality.
The two-spotted spider mite, T. urticae Koch, is a well-known herbivorus
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pest of cultivated crops. Many crops must be protected with synthetic acaricides
during the hot and dry seasons that favor severe outbreaks of spider mites. Basic
and applied research to provide new and effective pest control that do not rely
upon synthetic pesticides is needed. Many studies have indicated the potential
ecological damage due to the widespread use of synthetic pesticides.[16] The
U.S. Food Quality Protection Act (FQPA) in 1996 initiated a systematic effort to
identify and reduce potential risks posed by synthetic pesticides to safeguard
public health. Among the provisions of the FQPA is a requirement for the EPA
to reassess all synthetic pesticide tolerances (9,700+) within ten years of pas-
sage of the act. Among those that are significant to varying degrees to Kentucky
growers are azinphos-methyl (Guthion), chlopyrifos (Lorsban), phosmet (Imi-
dan), diazinon and malathion (Cythion).[3,7] Accordingly, there is a need to find
an effective pesticide with low mammalian toxicity to control vegetable insects
and spider mites.
The use of plant products for pest control may impart a selective advan-
tage to plants by inhibiting, repulsing, and even killing non-adapted organisms
that feed upon, or compete with the plant. Repellency, which involves push-
ing pests away from growing plants, has three advantages: 1) reduced reliance
on synthetic pesticides; 2) reduced chance for pesticide adverse environmental
impacts; and 3) reduced pesticide residues on crops reaching the consumers.
Plant-derived products have a broad spectrum of activity against
insects[811] and mites.[12,13] Dried plants or their extracts have been used by
farmers in many developing countries to protect food and fiber from insects.[14]
Cowles et al.[15] reported that chili pepper powder deterred oviposition of the
onion fly, Delia antiqua. Capsaicin in hot pepper has been reported to reduce
larval growth of the spiny bollworm, Earias insulana[16] and the use of oleoresin
from Capsicum as a repellent against cotton pests has been reported.[17]
The potential of using hot pepper extracts for controlling spider mites is
explored in this study. This investigation is a continuation of our previous work
on natural products for pest control and was designed to: 1) test the repellency
and toxicity of 24 pepper fruit extracts to adult spider mites, T. urticae; 2) deter-
mine the concentration of hot pepper extracts that repels 50% and 75% of spider
mites; and 3) characterize the biochemical composition of hot pepper fruit ex-
tracts having acaricidal performance for potential identification of compounds
responsible for toxicity and/or repellency.
 Toxicity of Hot Pepper Extracts to Spider Mite 1385
MATERIALS AND METHODS

Seeds of twenty-four Capsicum accessions were obtained from the U.S. Depart-
ment of Agriculture/Agricultural Research Service USDA/ARS Plant Introduc-
tion Station, Tifton, GA and planted in the greenhouse in the spring of 2004 and
transplanted to the field in June. Eight C. chinense Jacq. (PI-224424; PI-257059;
PI-593925; PI-438622; PI-585253; Grif-9117; Grif-9273; and Grif-9317); 7
C. frutescens L (PI-238057; PI-224416; PI-439522; PI-555644; PI-159261; Grif-
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9320; and Grif-9324); 4 C. baccatum L (PI-633758; PI-596057; PI-497985; and

Grif-9217); 4 C. annuum L (PI-195299; Grif-9149; Grif-9169; and Grif-9270);
and one C. pubescens Ruiz & Pav (PI-387838) were selected to represent the
five pepper cultivated species, and a cross-section of the geographic range of
origin. Fruits from each of 24 accessions were harvested at random from field-
grown plants and their crude extracts were prepared in methanol for testing
their toxicity and repellency to spider mites.
Crude extracts were prepared by collecting ten fresh fruits of compara-
ble size from each accession, and blending them with 250 mL of methanol for
one min. The solvent extracts were decanted through 55 mm Whatman 934-
AH glass microfiber filter discs (Fisher Scientific, Pittsburgh, PA) and concen-
trated in a rotary vacuum evaporator (Buchi Rotovapor, Model 461, Flawil,
Switzerland) at 35 C, chased with nitrogen gas (N2 ), and reconstituted in
10 mL of methanol. Each extract was subsequently passed through a 0.45 m
GD/X Fisher disposable syringe filter. A portion of these extracts was used
for bioassays.
A laboratory strain of the two spotted spider mite (Tetranychus urticae
Koch) was reared in the laboratory under fluorescent lighting on snap bean
leaves, Phaseolus vulgaris L. For mortality test, 100 L of each fruit extract was
evenly distributed onto a Whatman No.1 filter paper of 4.25 cm diameter. Filter
papers treated with solvent (methanol) only were used as negative control,
while 100 L of 2-tridecanone (0.1 mg mL1 ) was used as a positive control.
The solvent was allowed to evaporate and the filter paper was placed into a
5.0 cm Petri dish.
Four replicates of 10 gravid female adult spider mites (a total of 40 mites
per treatment) were placed into the Petri dishes and a tight-fitting lid was
placed on each dish. The dishes were then sealed with parafilm and incu-
bated at 27 C in the dark for 6 h with no food. Dishes were removed from
the incubator, the number of mites in each dish was recounted, and their
movement was checked. One-hundred L of distilled water and a bean leaf
disk (1-cm in diameter) were added to each dish to provide moisture and
nutrients. Dishes were returned to the incubator for an additional 18 h.
Live mites were counted at 6 and 24 h. Mites were considered alive if they
were able to move any appendage when gently probed with a soft brush.
The acaricidal efficacy of each extract was expressed as percent mortality.
 1386 Antonious, Meyer, and Snyder

Percentages of mortality were calculated using Abbotts formula[18] for each ac-
cession and transformed to arcsine square root of the value prior to analysis of
variance.
For repellency tests, the methanol extracts were tested against gravid fe-
male spider mites using a diving board bioassay that was developed by Guo
et al.[19] The diving board bioassay assembly was prepared using paper clamps
and filter paper strips of 0.5 cm 1.5 cm and 0.2 cm 1.5 cm. One end of each
strip was clamped by small binder clip with two strips per clip. Two binder clips
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were then clamped side by side by a larger binder clip. This allowed simulta-
neous tests of two mites. Ten L of a crude extract was applied onto one of the
0.5 1.5 cm paper strips using micropipette. The adjacent strip was treated
with solvent only (methanol). Accordingly, each extract covered a 0.75 cm2 (sur-
face area of filter paper strip). Methanol was allowed to evaporate completely
from the treated and untreated paper strips. Then, the 0.2 1 cm filter paper
strip was placed as a bridge connecting a control and treated strips.[19] One
gravid female mite (n = 30) was placed in the center of each bridge and given
the freedom to exit over the treatment or control strip. A small mirror placed
under the assembly allowed simultaneous observation of the upper and lower
surfaces. Exits over treatment or control strips were recorded and exit ratios
were determined (treated: control) for each of thirty mites per dose. Exit ratios
were tested for a significant departure from the expected 1:1 exit ratio by 2 .
Eight of the most repellent accessions were chosen for additional characteriza-
tion of the repellency of their fruit extracts. Using the diving board bioassays,
extracts of all 8 accessions were tested at 50 g/cm2 (surface area of filter pa-
per strip). Serial five-fold dilutions were then tested until two serial dilutions
produced an exit ratio that indicated no repellency (15:15, or 16:14). EC50 (the
concentration at which 50% of the mites are repelled) and EC75 values (75% of
mites are repelled) and their fiducial limits were then calculated using Probit
Analysis.[20]
Attempts were also made to correlate mortality and repellency of pepper ex-
tracts to spider mite with capsaicin constituents of fruit extracts, in an attempt
to identify the cause of mortality and/or repellency. One L of each fruit filtrate
(n = 3) was injected into a HP gas chromatograph (GC) model 5890A equipped
with a mass selective detector (GC/MS) operated in total ion monitoring with
electron impact ionization (EI) mode and 70 eV electron energy for identifica-
tion and confirmation of individual peaks. The instrument was auto-tuned with
perfluorotributylamine (PFTBA) at m/z 69, 210, and 502. GC/MS separations
were accomplished using a 25 m 0.20 mm ID capillary column with 0.33
m film thickness (HP-1). Operating conditions were 230 C, 250 C, and 280 C
for injector, oven, and detector, respectively with a carrier gas (He) flow
rate of 5.2 mL min1 . Quantifications of individual peak was based on av-
erage peak areas of 1 L injections of fruit crude extracts prepared in
methanol.
 Toxicity of Hot Pepper Extracts to Spider Mite 1387
Under these conditions, three capsaicinoids were identified in some pepper
accessions. Retention times (Rt ) were 9.06, 11.50, 11.75 min, for nordihydro-
capsaicin, capsaicin, and dihydrocapsaicin, respectively. Peak identities were
confirmed by GC/MS under the conditions described above. Purified standards
of capsaicin (N-vanillyl-8-methyl-6-noneamide), dihydrocapsaicin, and nordi-
hydrocapsaicin from pepper oil were obtained from Sigma-Aldrich Inc. (Saint
Louis, MO 63103, USA) and used to prepare calibration curves. Minimum de-
tectable levels of the three capsaicinoids found in the fruit extracts averaged
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0.02 to 0.005 g g1 fruit. To determine the recovery of the extraction, clean-up,

and quantification procedures, concentrations of capsaicinoids in the range of
20-200 g g1 fresh fruit were added to 20 g of bell pepper (C. annuum) fruits.
Recoveries of the added capsaicinoids averaged 95 to 98%.

RESULTS AND DISCUSSION

Of the 24 Capsicum accessions that we screened, only one accession (Grif-9169)
was highly toxic to spider mite after 24 h exposure (Fig. 1). Of the 24 Capsicum
accessions that were screened for spider mite repellency, 22 had repellency as

Figure 1: Mortality of two-spotted spider mite, Tetranychus urticae Koch, exposed to hot
pepper fruit extracts prepared in methanol from 24 pepper accessions. Methanol was
used a negative control, while 2-tirdecanone (0.1 mg mL1 ) was used as a positive control.
Bars accompanied by different letter(s) for each exposure period indicated significant
differences (P > 0.05; Duncans multiple range test.[20]
 1388 Antonious, Meyer, and Snyder

Table 1: Repellency of hot pepper fruit extracts from 24 accessions of 5 cultivated

Capsicum species as measured by the spider mite diving board bioassay.

Wt. of Fruit Tested, Exit ratio

Accession Taxon g/ 0.75 cm2 (treatment : control) X2 ratio
Grif-9217 C. baccatum 0.079 12:18 1.20
PI-387838 C. pubescens 0.069 11:19 2.13
PI-555644 C. frutescens 0.060 9:21 4.80
Grif-9324 C. frutescens 0.248 9:21 4.80
PI-593925 C. chinense 0.083 8:22 6.53
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PI-238057 C. frutesens 0.030 7:23 8.53

PI-585253 C. chinense 0.078 7:23 8.53
Grif-9169 C. annuum 0.260 7:23 8.53
PI-224424 C. chinense 0.180 6:24 10.80
PI-497985 C. baccatum 0.061 6:24 10.80
Grif-9149 C. annuum 0.211 6:24 10.80
Grif-9317 C. chinense 0.087 5:25 13.33
PI-633758 C. baccatum 0.096 5:25 13.33
PI-439522 C. frutesens 0.035 4:26 16.13
Grif-9117 C. chinense 0.140 4:26 16.13
PI-257059 C. chinense 0.120 3:27 19.20
Grif-9273 C chinense 0.051 3:27 19.20
PI-224416 C. frutescens 0.040 2:28 22.53
Grif-9320 C. fruescens 0.150 2:28 22.53
PI-438622 C. chinense 0.110 1:29 26.13
Grif-9270 C. annuum 0.170 0:30 30.00
PI-596057 C. baccatum 0.100 0:30 30.00
PI-159261 C. frutescens 0.025 0:30 30.00
PI-195299 C. annuum 0.009 0:30 30.00
Weight of fruit used to cover a filter paper strip of 0.5 1.5 cm for testing spider mite repel-
lency. The exit ratio is the ratio of the number of mites exiting over the hot pepper extract
and the number exiting over the methanol control. The expected exit ratio used for calcu-
lating 2 was 1:1. , significant (P < 0.05) and highly significant departure (P < 0.01) from an
expected 1:1 exit ratio, respectively as determined by 2 .

indicated by their significant 2 (chi-square) values (Table 1). However, 2 val-

ues were not uniform, ranging from 4.8 to 30, with 30 indicating that all mites
were repelled. The results suggested that crude extracts from pepper fruits can
be explored for developing natural products for use as biodegradable alterna-
tives to synthetic acaricides. Using repellent chemicals for crop protection is
a unique way to prevent insects and spider mites from laying eggs on target
plants and to prevent plant leaf and fruit damage. Results also indicated that
concentrated extracts provided increased repellency in all accessions (data not
shown).
The eight most repellent accessions were chosen for additional characteriza-
tion. A dose-response curve for each of the eight accessions was established and
the concentrations of the crude extract that repelled 50% and 75% (EC50 and
EC75 values, respectively) of spider mite, were estimated (Table 2). In Table 2,
there were differences in degree of repellency among extracts. The extracts of
the C. baccatum accession PI-596057 was most repellent, having the lowest
 Toxicity of Hot Pepper Extracts to Spider Mite 1389
Table 2: EC50 and EC75 values obtained from spider mite exposed to hot pepper
extracts using diving board bioassay.

Accession Taxon EC75 95% Fiducial limits EC50 95% Fiducial limits
PI-596057 C. baccatum 0.03 0.01 0.10 0.0040 0.0000 0.0020
PI-195299 C. annuum 0.20 0.07 0.60 0.0035 0.0006 0.0114
Grif-9270 C. annuum 0.40 0.21 0.83 0.0340 0.0160 0.0680
Grif-9320 C. frutescens 0.61 0.22 1.95 0.0090 0.0010 0.0310
PI-224416 C. frutescens 1.60 0.54 8.86 0.0961 0.0070 0.3087
PI-438622 C. chinense 1.75 0.51 9.59 0.0080 0.0004 0.0377
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PI-159261 C. frutescens 5.40 4.01 7.85 0.0044 2.2872 1.3567

PI-257059 C. chinense 5.69 2.18 31.96 0.1062 0.0164 0.2878

EC75 value. Other accessions from C. annuum were also repellent (PI-195299
and Grif-9270), but not as repellent as PI-596057. The two accessions PI-159261
(C. frutescens) and PI-257059 (C. chinense) were the least repellent accessions
among the 8 accessions chosen for additional characterization (Table 2).
We investigated chemical composition of fruits extracts that may explain
the observed differences in repellency among accessions. For the crude extracts
of the 24 accessions (Table 1), neither capsaicin concentration (r = 0.01, P =
0.94) nor dihydrocapsaicin concentration (r = 0.08, P = 0.71) was correlated
with 2 value from the diving board bioassays. The total concentration of cap-
saicinoids (capsaicin plus dihydrocapsaicin) was also not correlated with the
2 value (r = 0.03, P = 0.88). Likewise, for the eight accessions for which
EC75 values were obtained (Table 2), capsaicin did not correlate with repellency
(r = 0.28, P = 0.43), nor did dihydrocapsaicin correlate with repellency
(r = 0.25, P = 0.48) as estimated by the EC75 values. Accordingly, other
unidentified components of the fruit are likely responsible for repellency of
hot pepper extracts to spider mite.
Plants produce a vast array of volatiles that play important role in plant
defense.[21] Hot pepper accessions contain significant amounts of tannins.[22]
Breakdown products of tannins (phenols) behave as toxins and feeding
deterrents.[9,23] It could be concluded from this investigation that in most cases
unidentified components in the fruit extracts are playing a role in spider mite
repellency. However, the crude extracts from accession Grif-9169 which caused
45% mortality to spider mite and extracts of most repellent accessions (PI-
596057 and PI-195299) can be explored for developing natural products for use
as a biodegradable alternative to many synthetic acaricides, especially in small
acreage, high-value crops. One must recognize that even though complete con-
trol is most desirable, 45% spider mite mortality for each application would
delay pest development and reduce its attendant damage.
Based on their EC75 values, extracts from accessions PI-596057 (C. bac-
catum), PI-195299 (C. annuum), Grif-9270 (C. annuum), and Grif-9320
(C. frutescens) are potential candidates for use as repellents for spider mite
 1390 Antonious, Meyer, and Snyder

control. These accessions might be incorporated into plant breeding programs

to produce fruit with a high concentration of pest control agents. Future work
will be designed to identify the repellent compounds in hot pepper fruits. Field
studies for testing the selected accessions will be necessary to validate these
laboratory results under field conditions.

ACKNOWLEDGMENTS
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We thank Richard Thacker for his technical assistance in spider mite testing
and Zachary Ray for his kind assistance in preparing hot pepper crude ex-
tracts. This investigation was supported by a grant from U.S. Department of
Agriculture USDA/CSREES to Kentucky State University under agreement
No. KYX-2004-15102.

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