Application of competitive enzyme-linked immunosorbent assay for the quantification of imidacloprid titers in xylem fluid extracted from grapevines.

Abstract

A competitive enzyme-linked immunosorbent assay (ELISA) technique was evaluated for quantifying titers of imidacloprid in xylem fluid extracted from Vitis vinifera L. grapevines that were treated with systemic applications of the neonicotinoid insecticide Admire. Evidence of matrix effects, factors that compromise the precision and accuracy of the ELISA, was present in assays with undiluted xylem fluid. These effects could be eliminated by dilution of extracts in water, resulting in a lower sensitivity of the assay of 4 microg liter(-1). In a field trial conducted in a commercial vineyard, there was an excellent correlation between Admire application rates and xylem fluid concentrations of imidacloprid. At an Admire application rate of 1.17 liter ha(-1) (16 fl oz per acre), uptake of imidacloprid into vines was rapid. Imidacloprid was consistently detected in the xylem for up to 3 mo after application at concentrations known to be effective at managing populations of the sharpshooter Homalodisca coagulata Say, an important vector of Xylella fastidiosa Wells in California vineyards. The ELISA is a sensitive technique that can be used to study the behavior of systemic insecticides within crop systems and their impact on pest populations.

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INSECTICIDE RESISTANCE AND RESISTANCE MANAGEMENT
Application of Competitive Enzyme-Linked Immunosorbent Assay for
the Quantification of Imidacloprid Titers in Xylem Fluid
Extracted from Grapevines
FRANK J. BYRNE, STEVEN J. CASTLE,1JIAN LONG BI, AND NICK C. TOSCANO
Department of Entomology, University of California, Riverside, CA 92521
J. Econ. Entomol. 98(1): 182Ð187 (2005)
Acompetitiveenzyme-linkedimmunosorbentassay(ELISA)techniquewasevaluated
forquantifyingtitersofimidaclopridinxylemßuidextractedfromVitisviniferaL.grapevinesthatwere
treatedwithsystemicapplicationsoftheneonicotinoidinsecticideAdmire.Evidenceofmatrixeffects,
factorsthatcompromisetheprecisionandaccuracyoftheELISA,waspresentinassayswithundiluted
xylem ßuid. These effects could be eliminated by dilution of extracts in water, resulting in a lower
sensitivity of the assay of 4 ?g liter?1. In a Þeld trial conducted in a commercial vineyard, there was
an excellent correlation between Admire application rates and xylem ßuid concentrations of imida-
cloprid.AtanAdmireapplicationrateof1.17literha?1(16ßozperacre),uptakeofimidaclopridinto
vines was rapid. Imidacloprid was consistently detected in the xylem for up to 3 mo after application
at concentrations known to be effective at managing populations of the sharpshooter Homalodisca
coagulata Say, an important vector of Xylella fastidiosa Wells in California vineyards. The ELISA is
a sensitive technique that can be used to study the behavior of systemic insecticides within crop
systems and their impact on pest populations.
ABSTRACT
KEY WORDS
imidacloprid, ELISA, xylem, grapevine, Homalodisca coagulata
IMMUNOASSAYS ARE WIDELY USED for qualitative and
quantitativeassessmentsofenvironmentalsamplesfor
the presence of contaminants such as pesticides and
are often favored over other methods such as gas
chromatography, high-performance liquid chroma-
tography, and mass spectrometry because of their
speed, sensitivity, and cost-effectiveness (Hammock
andMumma1980).Althoughmanyimmunoassaysare
designed speciÞcally for environmental monitoring
purposes, there is the additional possibility that estab-
lished immunoassays can be used to provide a better
understanding of the behavior of an insecticide, in
terms of its uptake and persistence, after its applica-
tion to a crop. This information would greatly beneÞt
pest management if it enables the establishment of
threshold levels of insecticide required to protect
plants from pest infestation. Studies to better under-
stand the behavior of insecticides within agricultural
environments are restricted by the lack of simple, yet
effective, analytical tools. Immunoassays are a very
attractiveoptioninpartbecauseoftheirabilitytodeal
withlargesamplesizesgeneratedfromextensivemon-
itoringprograms.Thisadvantageofimmunoassayswas
demonstrated most emphatically in a recent study
conductedoncitrustreesinsouthernCalifornia(Cas-
tle et al. 2005), in which a competitive enzyme-linked
immunosorbentassay(ELISA)techniquewasusedto
monitor titers of the neonicotinoid insecticide 1-(6-
chloro-3-pyridinylmethyl)-N-nitro-2-imidazolidinimine
(imidacloprid)intreesthatweretreatedwithAdmire,
the systemic formulation of imidacloprid. The use of
the ELISA helped to establish the dynamics of imi-
dacloprid uptake and its distribution within citrus
trees and the titers of imidacloprid that were neces-
sary to effectively suppress densities of the recently
introduced pest, the sharpshooter Homalodisca co-
agulata Say. H. coagulata has become the principal
vector of the plant pathogenic, xylem-limited bacte-
rium Xylella fastidiosa Wells in southern California
(Costaetal.2000)sincetheintroductionoftheinsect
into the state in the early 1990s (Blua et al. 1999).
Strains of X. fastidiosa cause several economically im-
portant plant diseases, including PierceÕs disease in
Vitis vinifera L. grapevines. In the Temecula Valley
viticultural region, which consists of ?700 ha of wine
grapes, a severe PierceÕs disease epidemic that began
in1996resultedinsomewineriesreportinglossesof20
to 30% of their vines (Hix 2001). There is no cure for
PierceÕs disease and infected vines must be removed
from the vineyard to reduce the risk of H. coagulata
becoming infective from feeding on them and trans-
ferringthepathogentohealthyvines.Insecticidesare
currently the most effective means of managing
H. coagulata populations in vineyards. In particular,
the systemic behavior of imidacloprid is well suited to
1USDAÐARSWesternCottonResearchLaboratory,4135E.Broad-
way Rd., Phoenix, AZ 85040.
0022-0493/05/0182Ð0187$04.00/0 ? 2005 Entomological Society of America

Page 2

exploit the xylophagous feeding behavior of H. coagu-
lata, and this insecticide is now widely used in vine-
yards (Castle et al. 2005).
The purpose of this study was to evaluate the ef-
fectiveness of a competitive ELISA technique, avail-
ablecommerciallyforresidueanalysisofenvironmen-
talwatersamples,forquantifyingimidaclopridtitersin
xylem ßuid extracted from grapevines treated with
Admire.Matrixeffectscanbeparticularlyproblematic
in ELISAs. These effects occur when components in
the test matrix (the xylem ßuid) disrupt the interac-
tion between the antibody and the target molecule
(imidacloprid). Experiments were initially designed
to evaluate potential matrix effects attributable to xy-
lem ßuid and the degree of sensitivity of the assay.
SubsequentÞeldexperimentswerethenconductedin
acommercialvineyard,byusingtheELISAtoquantify
imidacloprid titers in grapevines, with the aim of es-
tablishing its rate of uptake and temporal persistence
within treated vines.
Materials and Methods
Vineyard Study Site. Field studies were conducted
during the 2002 and 2003 grape seasons at a commer-
cial vineyard in the Temecula Valley of southern Cal-
ifornia. The vineyard was newly established in 2002
and consisted of 2 ha of Sirah vines. The soil compo-
sition, determined from 10 randomly selected soil
cores (to a 15-cm depth) within the study area, was
characterized by the University of CaliforniaÕs Divi-
sion of Agricultural and Natural Resources Analytical
Laboratory as a loamy sand (86% sand, 7% silt, and 7%
clay) with a pH of 6.5 and an average organic matter
contentof0.7%.Duringourstudy,vineswerewatered
once weekly for 4 h by drip irrigation that adminis-
tered water directly to the root zone of each vine.
Insecticide Applications. Admire (240 g liter?1
Flowable Insecticide; Bayer CropScience, Research
Triangle Park, NC) was applied by chemigation, by
usingthedripirrigationsystemestablishedatthevine-
yard. In accordance with label recommendations,
vines were pre-irrigated for at least 1 h before the
application of insecticide to ensure adequate wetting
of the soil. In 2002, Admire was applied at 1.17, 1.46,
and 2.34 liter ha?1to three separate 0.08-ha blocks of
vines (comprising 150 vines each) by injection into
the irrigation lines. After injection, the irrigation lines
were ßushed with water for 2 h. Thereafter, the irri-
gation was run weekly in accordance with the grow-
erÕs established agronomic practices. In 2003, Admire
wasappliedtothesamesiteatasinglerateof1.17liter
ha?1.
Xylem Fluid Extraction. Xylem ßuid was extracted
fromgrapestemsbyusingaplantwaterstressconsole
(pressure chamber), an instrument that is normally
used for measuring the water potential of plants
(Scholander et al. 1965). The Þrst samples of xylem
ßuid were extracted immediately before the applica-
tionofAdmire,andthereafteratleastonceevery2wk
overa4-mosamplingperiod.Samplingfrequencywas
most intensive immediately after the Admire applica-
tion, to establish the initial rate of uptake. Xylem ßuid
wasextractedfromcaneterminalscutfrom16treated
vinesoneachsamplingdate.Theouterring(2.5cmin
length) of phloem tissue was stripped from the stem
attheseveredendtominimizephloemcontentduring
extractions and to maximize xylem ßuid content. Xy-
lem ßuid extracts were obtained which were largely
devoid of phloem and other tissue contents, thereby
giving a better estimate of the mobile imidacloprid
component within the xylem vascular system. Once
the stem was inserted through the grommet in the
sealinglid,thepressurechamberwaspressurizedto35
kg/cm2(500 psi) delivered from a cylinder of com-
pressed air. At this pressure, the xylem ßuid was ex-
truded easily from the stem and was collected using a
1-ml pipette. Typically, a minimum of 200 ?l was
collectedfromeachstem,andtheßuidwasthenadded
to a 1.5-ml Eppendorf tube. Samples were immedi-
ately transferred to dry ice for storage.
Chemical Quantification. Concentrations of imida-
cloprid within xylem ßuid samples were determined
using a competitive ELISA technique, in which insec-
ticide residues in the xylem ßuid extracts compete
withenzyme(horseradishperoxidase)-labeledimida-
cloprid for a limited number of antibody binding sites
on the wells of a microplate. The levels of bound
conjugate are determined colorimetrically and are
inversely proportional to the levels of insecticide
present in the xylem ßuid. The ELISA kit is available
commercially (QuantiPlate kit for imidacloprid, cat-
alog no. EP 006, EnviroLogix Inc., Portland, ME) and
hasareported0.2Ð6?gimidaclopridliter?1sensitivity
range. The assay was calibrated in our laboratory be-
foreusetotestformatrixeffectsassociatedwithxylem
ßuid. To ensure that preliminary biochemical evalu-
ations of the ELISA were not compromised by imi-
dacloprid contamination, xylem ßuid was extracted
from vines in an organically managed vineyard in
Temecula that had never been treated with imidaclo-
prid.
TheÞrstseriesoftestsinvolvedthedilutionofa5?g
imidacloprid liter?1standard calibrator (supplied
withthekit)ineitherwateror100%xylemßuidtogive
concentrations of 0.2, 0.5, 1, and 2 ?g imidacloprid
liter?1. Under these conditions, the imidacloprid con-
centration was decreased as the proportion of xylem
ßuid within the mixtures was increased. Additional
tests were performed to measure the recovery of imi-
dacloprid from spiked xylem ßuid samples. For this, a
range of xylem ßuid concentrations was initially pre-
paredbydilutingseveralextractswithwater,andthen
mixing each with an equal volume of imidacloprid to
giveaÞnalconcentrationof0.2?gimidaclopridliter?1
(the sensitivity limit of the assay kit as supplied).
Based on results from these preliminary experiments,
standard curves for imidacloprid were then con-
structed for water and 5% xylem ßuid. A series of
imidacloprid concentrations were prepared in water,
andtoeachconcentrationwasaddedanequalvolume
of 10% xylem ßuid to give the desired imidacloprid
concentrationrangeinaconstant5%xylemßuidback-
ground.Allwaterstocksolutionsofimidaclopridwere
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183

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freshly prepared immediately before each experi-
ment.
StatisticalAnalysis.Allstatisticalanalyseswereper-
formed using the GraphPad Prism version 4 (Graph-
Pad Software, Inc., San Diego, CA). Analysis of vari-
ance(ANOVA)wasusedtotestforsigniÞcanteffects
of xylem ßuid concentration on imidacloprid recov-
ery, whereas pairwise comparisons of means were
analyzed by TukeyÕs honestly signiÞcant difference
(HSD) test. Linear regression was used to determine
the relationship between application rate and xylem
ßuid content. The signiÞcance level for all statistical
tests was set at P ? 0.05.
Results
Evidence of Matrix Effects. Matrix effects associ-
ated with grape xylem ßuid were detected using sev-
eral protocols (Figs. 1Ð3). Dilution of the 5 ?g imida-
cloprid liter?1calibrator with water and xylem ßuid
yielded signiÞcantly different responses (F ? 22.70;
df ? 1, 21; P ? 0.0001; Fig. 1). This indicated that
matrix effects occurred over the sensitivity range of
the assay when xylem ßuid concentrations were at
least 60%. Mild effects also were detected at lower
xylemßuidconcentrationsbasedonmeasurementsof
the recovery of 0.2 ?g imidacloprid liter?1(Fig. 2).
Although these mild effects were not signiÞcant (F ?
1.72; df ? 5, 18; P ? 0.182), the noticeable increase in
absorbance at 10% xylem ßuid was of concern. All
evidence of matrix effects was eliminated when the
ßuid was diluted at least 20-fold in water. The ade-
quacy of this dilution step was conÞrmed by compar-
ing standard curves for imidacloprid prepared in 5%
xylem ßuid and water (F ? 0.57; df ? 1, 26; P ? 0.46;
Fig. 3). Our experiments thus established that the
Fig. 1.
was diluted with either water or xylem extract, resulting in an increased extract concentration with successive dilutions of
the standard. The numbers adjacent to each point on the xylem series indicate the percentage of xylem ßuid tested at that
speciÞc imidacloprid concentration. Each data point represents the mean ? SE of three independent assessments.
Effect of xylem ßuid concentration on the detection of imidacloprid by ELISA. A 5 ?g imidacloprid liter?1stock
Fig. 2.
eliminated when the extracts were diluted at 20-fold with water before assay. The undiluted xylem extract contained no
imidacloprid and was included to show the response in assays of unadulterated xylem ßuid. Each bar represents the mean ?
SE of four replicate extracts.
Impact of xylem ßuid concentration on the detection of 0.2 ?g imidacloprid liter?1by ELISA. Matrix effects were
184JOURNAL OF ECONOMIC ENTOMOLOGY
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bindingcharacteristicsofstandardspreparedinwater
were comparable with a series of standards prepared
directly in xylem ßuid, provided that the ßuid was
diluted at least 20-fold before conducting the assay
(Fig. 3). The linear range of detection in grape xylem
ßuid was thus 4Ð120 ?g liter?1for imidacloprid. For
sample analyses, extracts were routinely diluted 20-
foldbeforeassays,andwhenreadingsmeasuredabove
the detection limits of the assay, the samples were
diluted further in distilled water and the assay rerun.
Assays were conducted in accordance with the kit
recommendations.
Field Trials. The Þrst experiment to evaluate the
ELISA under Þeld conditions was conducted in a
newly established vineyard of Sirah grapes that was
treated with three rates of Admire. Due to the age of
the vines, and the destructive nature of the sampling
process, sampling during the Þrst season was limited
to just one date at two months post-application. The
Admire application rate had a signiÞcant impact upon
the titers of imidacloprid within the xylem ßuid (F ?
7.31; df ? 2, 27; P ? 0.003; Fig. 4). The two-fold
increase in application rate between the 1.17 and
2.34 liter ha?1rates resulted in a doubling of imida-
clopridtiters,whereasthe1.46literha?1rateresulted
in intermediate levels (Fig. 4).
During 2003, a more substantial sampling program
was conducted over a 4-mo period within the same
vineyard,usingasingleAdmireapplicationof1.17liter
ha?1(Fig. 5). Imidacloprid was detected within the
vines on 23 June, 3 d post-treatment, and quickly rose
to?40?gimidaclopridliter?1byday5.Peaklevelsof
imidacloprid were measured on 16 July, 26 d after
application. During the entire 15-wk sampling period,
the average titers on any sampling date remained
above 15 ?g imidacloprid liter?1.
Discussion
Xylem is the principal water-conducting tissue of
vascular plants and the primary means of distribu-
tion for soil-applied systemic insecticides such as
imidacloprid(Elbertetal.1990).Therefore,theavail-
ability of a tool for the detection and quantiÞcation of
residues within xylem ßuid could provide pest man-
agement specialists with valuable insights into the
dynamics of uptake and availability of systemic insec-
ticides. In this study, we have shown the utility of a
competitive ELISA technique for quantifying imida-
clopridtiterswithinthexylemßuidofgrapevinesafter
their systemic treatment with Admire. With appro-
priate calibration before sample analysis, the assay
proved to be very effective at determining insecticide
levels within the xylem, with a lower limit of quanti-
Þcation of 4 ?g imidacloprid liter?1.
In the initial Þeld assessment of the ELISA, there
was an excellent correlation between the rate of
Admire application and the resulting titers measured
within the xylem ßuid. This outcome provides strong
evidenceforthesuitabilityoftheELISAtechniquefor
monitoring imidacloprid titers within xylem ßuid and
Fig. 3.
ter,andxylemßuidthatwasdiluted20-foldwithwaterbefore
ELISA. Each point represents the mean ? SE of a minimum
of three independent determinations.
Standard curves for imidacloprid prepared in wa-
Fig.4.
concentration measured within xylem ßuid of grapevines.
Xylem ßuid was extracted from vines two months after the
treatment. Each bar represents the mean ? SE of 10 vines
chosen randomly within each treatment block.
ImpactofAdmireapplicationrateonimidacloprid
Fig. 5.
vines treated systemically with Admire at a rate of 1.17 liter
ha?1(16 ß oz per acre) on 20 June 2003 (indicated by the
arrow).Eachpointrepresentsthemean?SEof16vines.The
same vines were sampled throughout the assessment period.
ProÞle of imidacloprid concentrations in grape-
February 2005BYRNE ET AL.: ELISA DETECTION OF IMIDACLOPRID IN XYLEM FLUID
185

Page 5

for assessing the impact of application rates on these
titers.Moreextensivesamplingwasundertakeninthe
2003 Þeld trial. Levels of imidacloprid measured
within the xylem of treated vines rose rapidly after
chemigation with 1.17 liter ha?1Admire and were
maintained above 15 ?g imidacloprid liter?1for at
least 3 mo. In a recent study, in which the ELISA was
used to monitor imidacloprid titers in xylem ßuid ex-
tracted from citrus trees treated with 2.34 liter ha?1
Admire, populations of H. coagulata began to decline
and remain at low levels when the imidacloprid con-
centrations were at least 10 ?g liter?1(Castle et al.
2005). In our study, we used 1.17 liter ha?1and still
maintained concentrations of imidacloprid above this
minimum effective level. This information is encour-
agingforgrowersintheireffortstoprotecttheirvines
from H. coagulata infestations. Our results highlight
the utility of the assay as a pest management tool that
is capable of monitoring pesticide titers in crops to
determine whether they are at acceptable levels for
effective pest control. With appropriate calibration,
the ELISA will be an ideal tool for assessing the be-
havior of imidacloprid within different plant systems.
It will be especially useful for establishing suitable
application rates on plants such as grapevines.
AfterapplicationofAdmire,imidaclopridiscontin-
uously taken into the roots over a prolonged period
and distributed through the xylem tissue to all vege-
tative parts of the plant (Elbert et al. 1990). The assay
is, therefore, ideally suited for measurements of in-
secticide concentrations within the rather innocuous
background of xylem ßuid. However, extra care is
required when using similar assays to quantify resi-
dues in extracts that are likely to contain the contents
of other plant tissues. One potential problem is the
likelihood that metabolites of the parent compound
will cross-react with the antibody. The metabolism of
imidaclopridwithinplantshasbeenwelldocumented
(Westwood et al. 1998; Nauen et al. 1998, 1999), and
several of the metabolites are known to cross-react
with antibodies that have been prepared for the de-
tectionofimidacloprid(LiandLi2000,Leeetal.2001,
Wanatabeetal.2001).Forthisreason,thecompetitive
ELISA approach may be best suited to imidacloprid
quantiÞcationduringitsmobilizationwithinthexylem
before it encounters plant detoxiÞcation systems. To
optimize xylem content in extracts, we recommend
using the pressure bomb as an extraction device. The
isolationofthexylembeforeßuidextractionprovided
for a more concentrated plant extract focused on the
xylem contents and eliminated the necessity for pos-
textraction cleanup. After extraction, samples were
ready for analysis and required nothing more than
simple dilution in water to mitigate relatively mild
matrix effects.
There is an increasing number of neonicotinoid in-
secticidesunderdevelopmentortestingforsystemicuse.
AlthoughtherelativeefÞcaciesofthesematerialsagainst
speciÞcinsectpestscanbeassessedusingbioassays,their
toxicologicalproÞlesmaynotreßecttheirefÞcacyunder
Þeld conditions, due to differences in uptake caused by
agronomic or abiotic factors (e.g., irrigation practices,
and soil type) that impact delivery to the pestÕs feeding
zone.WearecurrentlyusingtheELISAapproachinÞeld
studies that are aimed at evaluating the uptake and per-
sistenceoftoxicologicallyactivechemicalsundervarious
agronomicandenvironmentalconditions.Thisapproach
willcontributeimportantinformationforimprovingthe
deployment of insecticides within different cropping
systems.Althoughitisunlikelythatgrowerswillhavethe
facility to perform assays themselves, the ELISA tech-
nique could assist researchers in their studies on the
behavior of imidacloprid and other neonicotinoids for
which assays are available, within agricultural environ-
ments. The information obtained from such studies can
then be used to evaluate the suitability of systemic in-
secticide treatments across the diverse array of agro-
nomic and environmental conditions characteristic of
California agriculture.
Acknowledgments
We acknowledge Mac Learned (Bayer CropScience) for
technical assistance and expertise with Admire applications.
We thank Ben Drake for providing access to vineyards. We
thank Kristen Mello and Greg Ballmer for assistance with
samplecollection.Fundingforthisstudywasprovidedbythe
California Department of Food and AgricultureÕs Pierces
Disease Program Award # 001213-001.
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