Journal of Virological Methods 132 (2006) 174–180
A simple wax-embedding method for isolation of aphid
hemolymph for detection of luteoviruses in the hemocoel
Sijun Liu a,∗ , Bryony C. Bonning a , W. Allen Miller b
a
b
418 Science II, Department of Entomology, Iowa State University, Ames, IA 50011, USA
351 Bessey Hall, Department of Plant Pathology, Iowa State University, Ames, IA 50011, USA
Received 19 May 2005; received in revised form 16 September 2005; accepted 3 October 2005
Available online 22 November 2005
Abstract
A protocol for isolating hemolymph from viruliferous aphids has been developed. This method uses warm melted wax to immobilize the aphid.
Following removal of a hind leg, the hemolymph can be collected readily. Flushing with RNase-free water allows for collection of sufficient
hemolymph for RNA extraction from individual aphids. The extracted RNA was successfully used for detection of barley yellow dwarf virus
(BYDV) and pea enation mosaic virus (PEMV) from individual viruliferous Rhopalosiphum padi and Acyrthosiphon pisum aphids, respectively.
A TaqMan real-time RT-PCR protocol for quantitation of PEMV in the hemolymph of individual aphids was developed. The wax-embedding
hemolymph collection technique provides a useful tool for studying molecular interactions between persistent and circulative plant viruses and
their insect vectors.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Isolation of aphid hemolymph; Luteoviruses; Quantitative RT-PCR; RT-PCR; Wax-embedding methods
1. Introduction
Members of the Luteoviridae, such as barley yellow dwarf
virus (BYDV), potato leaf roll virus (PLRV) and beet western
yellows virus (BWYV) are crop pathogens of global economic
importance (Sylvester, 1989; Blackman, 2000). Luteoviruses are
transmitted exclusively by aphids in a persistent or circulative
manner. Circulative transmission of luteoviruses involves complex interactions between the virus and the aphid vector. During
aphid feeding, the virions pass with the sap through the maxillary food canal of the aphid stylet into the gut. From the gut
(midgut or hindgut) the virions penetrate the gut epithelium by
endocytosis (Gildow, 1993; Gray, 1996). The virus-containing
vesicle fuses with the plasmalemma, thereby releasing the virus,
which readily passes through the basement membrane into the
hemocoel. The virus is then transported to the accessory salivary gland. Upon aphid feeding, the luteovirus is injected into
the plant with saliva via the salivary canal in the maxillary stylets.
On passage through the aphid vector, the virus overcomes two
∗
Corresponding author. Tel.: +1 515 294 3963; fax: +1 515 294 5957.
E-mail address: sliu@iastate.edu (S. Liu).
0166-0934/$ – see front matter © 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2005.10.015
membrane barriers (gut and accessory salivary gland) and may
be protected from proteolytic degradation within the hemocoel
by association with a GroEL-like protein called symbionin (van
den Heuvel et al., 1997; Filichkin et al., 1997).
The transmission of luteoviruses by aphids is influenced by
multiple factors including the host plant, environmental conditions, as well as the aphid vector and virion surface structures
(Gray and Gildow, 2003). On acquisition of a virus by an insect
vector, transmission of the virus depends primarily on molecular
interactions between the virions and the vector. These interactions determine whether or not the virus is transmitted, and the
efficiency of transmission. For example, failure to transmit, or
low efficiency of aphid transmission, can result from mutations
that disrupt interaction between the viral capsid and the gut or
accessory salivary gland membrane. Hence, movement of the
virus from the gut to hemocoel or from the hemocoel to the
accessory salivary gland may be blocked. Mutations in the viral
capsid may also destabilize the virus, resulting in degradation of
virions in the gut or in the hemocoel. Virus transmission may also
be blocked by failure of virus release from the accessory salivary gland (Power and Gray, 1997; Gildow and Gray, 1993). To
understand why an isolate is not transmissible, it is important to
determine the stage at which virus transmission is blocked (gut,
S. Liu et al. / Journal of Virological Methods 132 (2006) 174–180
hemocoel or accessory salivary gland). Isolation of viral RNA
from hemolymph and quantitation of virus by real-time RT-PCR
can delineate the cause of attenuated virus transmission. Understanding the molecular interactions between luteoviruses and
their aphid vectors may facilitate development of measures to
prevent plant virus transmission by insect vectors.
Detection of plant viruses in their respective insect vectors
is also an important component of integrated pest management
for virus control (Boonham et al., 2002). Several methods for
isolation and detection of plant viruses in insect vectors have
been described. These methods are based on detection of viral
genomic DNA or RNA by PCR or RT-PCR (Lopez-Moya et al.,
1992; Mehta et al., 1994; Minafra and Hadidi, 1994; Olmos et al.,
1997, 1999; Stevens et al., 1997; Naidu et al., 1998; Vercruysse
et al., 2000), real-time RT-PCR (Boonham et al., 2002; Fabre et
al., 2003), or on detection of either viral structural proteins by
ELISA (Gera et al., 1978; Ahoonmanesh et al., 1990; Stevens et
al., 1997) or non-structural proteins (Chomchan et al., 2002).
Methods used for isolation and detection of luteoviruses in
aphids have also been reported for BYDV (Rochow and Brakke,
1964; Lister and Rochow, 1979; Torrance, 1987; Robertson et
al., 1991; Canning et al., 1996; Chay et al., 1996; Fabre et al.,
2003), PLRV (Singh et al., 1995, 1996, 2004; Singh, 1998, 1999;
Nie and Singh, 2001), BWYV (Veidt et al., 1992; Reinbold et
al., 2001) and pea enation mosaic virus (PEMV) (French et al.,
1973).
To understand the mechanisms of transmission (or lack
thereof), it is important to distinguish virus in the hemocoel,
from virus that remains only in the gut. Yet, in most examples above, the methods describe use of whole insects, rather
than insect hemolymph for isolation and detection of viruses.
One exception is Chay et al. (1996) who used a microinjector to inject RNase-free water into the aphid hemocoel. Diluted
hemolymph was then collected on removal of a hind-leg from
the aphid. However, this approach is technically challenging and
relatively little hemolymph can be collected. A simpler method
for collection of hemolymph from individual aphids in sufficient
quantities to allow extraction of viral RNA is required.
The goal of this study of the mechanisms of luteovirus
transmission was to determine why some BYDV and PEMV
mutants were not aphid transmissible. To this end, a reproducible method for isolation and detection of viruses in the aphid
hemocoel was critical. In this paper a rapid and simple approach
for isolating hemolymph from individual viruliferous aphids is
described. The isolation method is reliable and robust. Sufficient hemolymph can be acquired for extraction of viral RNA for
detection of luteoviruses in the hemocoel of individual aphids by
using reverse transcription polymerase chain reaction (RT-PCR)
and high throughput quantitative RT-PCR (QRT-PCR).
175
was used for inoculation and aphid feeding. Infectious PEMV1 and PEMV-2 clones (Demler et al., 1997) were generously
provided by Dr. G.A. de Zoeten. A non-transmissible PEMV
mutant (RTDY114N) was also used in all assays. The infectious
Brome mosaic virus (BMV) clones (pT7B1, pT7B2 and pT7B3)
were obtained from Dr. A.L.N. Rao, University of California at
Riverside.
2.2. Aphids
The bird cherry-oat aphid, Rhopalosiphum padi and pea
aphid, Acyrthosiphon pisum were reared on oat and pea, respectively (22–24 ◦ C, L:D 12:12 h). The non-viruliferous aphids
were given a 48 h acquisition period on BYDV, PEMV or BMVinfected plants before hemolymph was extracted. Total RNA
was also extracted from whole aphids.
2.3. In vitro transcription of viral RNA and inoculation of
viral transcripts
Methods used for in vitro transcription of PAV6-129 and inoculation of the transcripts to oat protoplasts were as described
previously (Dinesh-Kumar and Miller, 1993; Koev et al., 2002).
The transcripts of PEMV and BMV genomes were synthesized by using the mMessage mMachineTM T7 kit (Ambion,
Austin, TX) according to the manufacturer’s instructions. Equal
amounts (approximately 1 !g of RNA for each plant) of PEMV
RNA-1 and RNA-2 were mixed and rubbed onto 7-day-old pea
seedlings (Pisum sativum cv. Progress No. 9). A similar method
was also used for inoculation of BMV; the mixed RNAs 1, 2 and
3 were mechanically inoculated on to 7-day-old barley seedlings
(cv. Manchuria).
2.4. Virus purification and aphid transmission of BYDV
2. Materials and methods
The method used for semi-purification of BYDV from
infected oat protoplasts was modified from Chay et al. (1996).
Briefly, the infected protoplasts were harvested 48 h post inoculation by centrifugation at 700 × g for 5 min. The cells were
resuspended in 300 !l of 10 mM sodium phosphate buffer pH
7.0, sonicated for 2–3 s, and centrifuged at 8000 × g for 5 min.
The supernatants were then centrifuged at 50k rpm for 30 min
(Sorval Discovery M150 centrifuge, S150AT rotor). The pellets were resuspended in 100 !l of 10 mM sodium phosphate
buffer pH 7.0 and diluted 1:1 in 50% sucrose for feeding to
virus-free R. padi through parafilm membranes. Following a
16–18 h acquisition period, 15–20 aphids were placed on individual oat seedlings (Avena sativa cv. Clinton64). The aphids
were later killed. The infected plants were used for aphid feeding tests. Hundred to 200 aphids were fed on the infected plants
for 24–48 h before they are used for isolation of RNA.
2.1. Virus isolates
2.5. Isolation of hemolymph
Two luteoviruses, barley yellow dwarf virus (BYDV) and
pea enation mosaic virus (PEMV) were used in this study. An
infectious BYDV cDNA clone, pPAV6-129 (Koev et al., 2002)
The first step is to immobilize the aphid on a thin layer of
melted wax. Wax (Paraseal® , W & F products Inc.) was melted
and dropped onto a piece of Parafilm® (American National
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S. Liu et al. / Journal of Virological Methods 132 (2006) 174–180
CanTM ) to produce a thin layer (∼1–2 mm). The wax on the
parafilm was then remelted on a warm hot plate (50–60 ◦ C) and
an aphid placed on to the melted wax. Once the melted wax
completely covered the body of the aphid, the parafilm containing the embedded aphid was removed from the hot plate to allow
the wax to harden around the aphid (Fig. 1A). Under a binocular
microscope, a hind leg was removed from the embedded aphid
using a needle (26 3/8 Gauge Beckin Dickinson) (Fig. 1B and
C). RNase-free water was flushed on to the wound three or four
times using a 10 !l pipette. The diluted hemolymph was then
collected by using a Pasteur pipette (Fig. 1D). The hemolymph
samples were then used for total RNA extraction for RT-PCR
and qRT-PCR detection of viral RNA.
2.6. Extraction of total RNA from aphids
Total RNA was prepared by using a StrataPrep® Total RNA
Microprep Kit (Stratagene® ). Hemolymph isolated from the
hemocoel of individual or multiple R. padi or A. pisum was
resuspended in 50 !l of the lysis buffer for individual aphids, or
100 !l for two to three aphids. Total RNA from whole aphids
was extracted by grinding one aphid per 100 !l and two to
three aphids in 200 !l of lysis buffer. The following extraction procedures were performed according to the manufacturer’s
instructions.
2.7. RT-PCR and primers
The 1st strand viral cDNA was synthesized from the
purified total RNA using SuperScriptTM II RNase H− Reverse
Transcriptase (Invitrogen® ), according to the manufacturer’s
directions. Briefly, 4 !l of RNA extraction, 2 !l of 2.5 mM dNTP
mixture, 1 !l of 20 pmol reverse primers, and 5 !l of RNase-free
water were mixed and incubated at 65 ◦ C for 5 min; 4 !l of 5×
reaction buffer, 1 !l of RNasin (Promega) and 2 !l of 0.1 M
DTT were then added into the tubes and incubated at 42 ◦ C for
2 min before adding 1 !l of Super Script II reverse transcriptase.
The reactions were carried out at 42 ◦ C for 50 min and the reactions were stopped by incubating at 95 ◦ C for 5 min. The cDNA
was stored at −80 ◦ C. PCR was subsequently performed in a
20 !l volume, which contained 2 !l of 1st strand viral cDNA,
and 1 unit of rTaq DNA polymerase (Takara). Amplification was
carried out using a 1 min hot start of 94 ◦ C, 40 cycles of 94 ◦ C for
30 s, 55 ◦ C for 45 s, 72 ◦ C for 1.5 min and a final elongation step
of 72 ◦ C for 7 min. The primer used for 1st strand viral cDNA
synthesis of PAV6-Ill was 5& -GAGAAGCCCGGCATCCC-3& ,
which corresponds to the nucleotide (nt) positions 5681 to the 3&
end of the viral RNA. RT-PCR of BYDV RNA was performed
using the 3& end reverse transcription (RT) primer along with the
5& end primer: 5& -GCTCAGTGAGGGGATTAA (corresponding
to nts 5096–5114 of BYDV RNA). For PEMV-1 RNA, the 3&
end RT primer and 5& primer were: 5& -GAACCAGTGCAATTCATGGTG-3& (nts 3849–3870) and 5& -CCATATTGGAGGAAATGCG-3& (corresponding to nts 3726–3744), respectively. The 3& end RT primer used for BMV first strand
cDNA synthesis was 5& -GGCGATTAAGTTGGGTAACG-3&
(nts 2227–2207) of BMV RNA 3. The 5& end PCR primer
was 5& -GTTGCAGACTCCTCGAAAGA-3& (nts 1638–
1658).
Fig. 1. Isolation of hemolymph from aphids. (A) Individual aphid was embedded in a thin layer of melted wax on a piece of parafilm. (B) A hind leg (indicated by
arrow) was dissected to release hemolymph. (C) The hind leg was detached from the aphid body and hemolymph released on to the wax surface. One to 2 !l of
RNase-free water was added to the hemolymph (arrow). (D) The diluted blood was collected using a pipette (arrow). (C) and (D) were repeated several times.
S. Liu et al. / Journal of Virological Methods 132 (2006) 174–180
2.8. Quantitative RT-PCR (QRT-PCR)
Real-time RT-PCR was designed for quantitative detection of PEMV-1 viral RNA from the hemolymph of viruliferous A. pisum. The primers used for QRT-PCR were the
same as those used for regular RT-PCR. A TaqMan probe,
5& -ACATGCTTATAGAGCATCTCGG-3& (nts 3771–3792), was
designed within the conserved ORF2 region of PEMV RNA-1.
The 5& end of the probe was labeled with 6-FAM (synthesized by
IDT) and the 3& end was labeled with the Blackhole Quencher 1
(synthesized by IDT). A pair of primers for A. pisum 18S rRNA
was also designed and used as a control for RNA samples: 5& CTGGTTGATCCTGCCAGTAG-3& (18S RNA nts 4–23) and
5& -GGTCGGAACTCTGTCGGC-3& (18S nts 190–174). PCR
products were detected using Sybr-Green labeling (Molecular
Probes, Whitham et al., 2003) for quantitative detection of 18S
rRNA.
The QRT-PCR was performed in two steps. The RT reaction
was carried out as described in Section 2.7. TaqMan reactions
were set up in 96-well reaction plates. Each reaction (25 !l)
contained 2 !l of the 1st strand DNA RT synthesis, 7.5 pmol
each of the forward and reverse primers, 5 pmol of 6-FAM
probe, 0.3 mM dNTP, 3 mM MgCl2 and 1 unit Platimum® Taq
DNA polymerase (Invitrogen). For the control QRT-PCR of 18S
rRNA, 100 pmol of Sybr-Green and 10 nmol of fluorescent Calibration Dye were added into the 25 !l reactions (Whitham et al.,
2003). PCR reactions were performed on a BIO-RAD iCyclerTM
iQ system using the thermal cycle conditions: 95 ◦ C for 1 min,
followed by 94 ◦ C for 45 s, 58 ◦ C for 1 min and 72 ◦ C for 1 min
for 40 cycles. A standard curve consisting of serial 1:10 dilutions was prepared with cDNA concentrations of 100, 10, 1,
0.1, and 0.01 ng/!l. PCR reactions were performed in triplicate
and analyzed on a BIO-RAD iCyclerTM iQ Optical system using
Software Version 3.0a.
3. Results
3.1. Wax-embedding technique for isolation of aphid
hemolymph
Ideally, isolation of aphid hemolymph from the hemocoel
should provide sufficient viral RNA from an individual aphid for
quantitative detection of virus. However isolation of hemolymph
uncontaminated by gut contents presents a number of technical
challenges. It is difficult to remove an aphid leg and inject water
to collect hemolymph without damaging the aphid to the extent
that hemolymph becomes contaminated with other aphid fluids
or tissues. To overcome this problem, Aphids were immobilized in melted wax. As the wax cools and hardens, a hind leg
was removed from the embedded aphid using a needle (26 3/8
Gauge Beckin Dickinson) (Fig. 1B and C). RNase-free water
was flushed on to the wound three or four times using a 10 !l
pipette. The diluted hemolymph was then collected by using a
Pasteur pipette (Fig. 1D). Details of this technique are described
in Section 2. Two luteoviruses, BYDV and PEMV, and their
respective vectors R. padi and A. pisum were used to evaluate
the versatility of this method. The body size of an A. pisum indi-
177
vidual is approximately 5–10 times larger than that of R. padi.
Despite the size difference between these two aphid species, the
viral RNAs were successfully detected in the hemolymph samples isolated from a single viruliferous aphid of both species.
3.2. Detection of BYDV in the hemocoel of single R. padi
Total RNA extracted from the hemolymph of single viruliferous R. padi was used as template for RT-PCR. The amount of
RNA used for the first strand synthesis of the viral cDNA was
equivalent to half of the total RNA (100 ng to 1 !g) extracted
from the aphid. Results of the RT-PCR are shown in Fig. 2.
BYDV cDNAs were amplified from the RNA samples, indicating that our hemolymph isolation method provided sufficient
viral RNA template for detection of BYDV in the hemocoel of
individual viruliferous aphids.
3.3. Detection of BMV in R. padi
The next experiment was to determine whether hemolymph
collected by our method may be contaminated by virus from the
alimentary canal. Contamination with gut contents could potentially occur if the gut was ruptured during the aphid embedding
or hemolymph collection process. It has been shown previously
that BMV ingested by R. padi, is unable to penetrate the gut
membrane to enter the hemocoel (Gildow, 1993). Therefore,
BMV was used as a control for contamination of hemolymph
with gut contents. The presence of BMV in the hemolymph
would be indicative of contamination from other sources. Aphids
(R. padi) were fed on BMV-infected barley, and total RNA was
extracted from either whole aphids or the hemolymph of individual or multiple aphids using the wax-embedding and hemolymph
collection method. The RNA samples were used subsequently
for RT-PCR (Fig. 3). As a positive control, BMV-specific DNA
fragments were readily amplified from RNA isolated from whole
aphids. In contrast, no DNA was amplified from hemolymph
samples of single or multiple aphids infected with BMV. Thus,
although BMV is abundant in total aphid extracts, presumably
from virus in the gut, the hemolymph samples were free of
detectable BMV and thus not contaminated by gut contents.
3.4. Detection of PEMV in individual A. pisum by RT-PCR
and QRT-PCR
To verify that the new hemolymph collection method can provide sufficient RNA for quantitative detection of a luteovirus in
Fig. 2. RT-PCR detection of BYDV in the hemolymph of individual R. padi.
M: mock, T: total RNA sample extracted from the whole body of a single aphid.
H1–H4: RNA samples extracted from the hemolymph of individual R. padi.
BYDV RNA was detected from all of the viruliferous aphids, but not from the
control aphid (M).
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S. Liu et al. / Journal of Virological Methods 132 (2006) 174–180
Fig. 3. Detection of BMV from R. padi. A faint but clear PCR product was
reverse transcribed and amplified from the RNA sample extracted from the
whole bodies of three R. padi that fed on BMV infected plants (lane 3). The
PCR band migrated at the same position as the RT-PCR product amplified from
the RNA3 transcripts of BMV (lane 1). A barely detectable band was also shown
in the sample of the individual aphid (lane 4). No viral RNA was detected from
the samples of control aphids (lane 2), or from the hemolymph samples of a
group of three aphids (lane 5) and one aphid (lane 6) that fed on BMV infected
plants.
Fig. 4. RT-PCR of PEMV from A. pisum. PEMV RNA was detected from total
RNA extracted from a whole viruliferous aphid (T) and from hemolymph samples of individual aphids (H1–H4), but not from the control aphid (M).
the aphid vector, PEMV and its vector A. pisum were used to perform RT-PCR and QRT-PCR. RNA extracted from hemolymph
samples from viruliferous A. pisum individuals as well as from
whole aphids was used for RT and QRT-PCR experiments. RNAs
of aphid transmissible PEMV (AT+ ) and a non-transmissible
mutant, RNA1RTDY114N (AT− ) (Liu, unpublished result) were
used for the RT-PCR assay. Both PEMV AT+ and AT− were
detected in hemolymph samples of single viruliferous A. pisum
aphids (Fig. 4). The QRT-PCR showed that both AT+ and
AT− viruses had similar viral RNA concentrations in the aphid
hemolymph (Fig. 5), 30 min after feeding on infected plants
for 48 h. These results suggest that the AT− virus entered the
hemocoel and attained titers comparable to those of wild type
PEMV. This result also indicates that the failure of aphid transmission of RNA1RTDY114N is not due to blockage of virus
entry into the hemocoel. This result illustrates the importance
of this method for detection of luteoviruses in the hemocoel, for
understanding the stage at which virus transmission is blocked in
the aphid.
4. Discussion
A particular concern in isolating hemolymph from a viruliferous aphid vector is the risk of cross-contamination from the
aphid gut, honeydew or stylets during the hemolymph collection
process. Cross-contamination was shown not to occur, because
BMV was not detected in hemolymph extracts even though it
was easily detected in whole aphids (Fig. 3). The lower amount
of BMV than BYDV in whole aphids, as detected by QRT-PCR
Fig. 5. Quantitative detection of PEMV RNA from single viruliferous A. pisum.
(A) QRT-PCR amplification of viral RNA of wt PEMV (AT+ : line a) and the
non-transmissable mutant (AT− : line b) isolated from the hemolymph of single
viruliferous aphids. Similar amounts of viral RNA were present in the hemocoel
for the two viruses. (B) QRT-PCR of 18S rRNA, the internal control for samples
used in (A).
may be because the aphid is not a vector of BMV. BMV may
degrade in the aphid gut. These results resemble those of Chay
et al. (1996).
The advantages of the wax-embedding technique compared
to the published hemolymph isolation method (Chay et al., 1996)
are: (1) the aphid is immobilized, which expedites collection of
hemolymph; (2) cross-contamination from the stylets and the
other parts of the body is reduced because the aphid is submerged
in wax; (3) hemolymph collection is conducted outside of the
aphid body on the surface of the wax, which further alleviates
the possibility of gut cavity contamination; (4) the technique is
simple, reliable and robust.
Methods have been reported for isolation of virus from whole
aphids for detection by ELISA (Lister and Rochow, 1979). The
wax-embedding technique may also be applied for detection
of coat proteins or capsids from the hemocoel of viruliferous
aphids.
In summary, the wax-embedding method for hemolymph
isolation will facilitate research on the molecular interactions
between luteoviruses and their aphid vectors. This method will
be used to clarify the roles of virus structural proteins in virus
transmission via the aphid vector, and will provide the means to
identify capsid protein motifs involved in movement of the virus
within the aphid. This technique can also be applied for study of
interactions between other persistent and circulative viruses and
their respective insect vectors, and for investigation of passage
of West Nile virus from the mosquito to avian hosts. This procedure has also been modified for isolation of hemolymph from
adult Drosophila melanogaster. In this case, removal of a wing
S. Liu et al. / Journal of Virological Methods 132 (2006) 174–180
from the wax-embedded vinegar fly provides more hemolymph
than removal of a leg (Liu, unpublished result).
Transmission of luteoviruses is dependent on multiple factors. Identification of the precise mechanisms associated with
reduced virus transmission will facilitate understanding of the
molecular interactions required for aphid transmission. A sensitive method for detection of ingested virions in the aphid
hemocoel is particularly important for elucidation of virus transmission determinants. In this paper a relatively easy and cost
effective approach for isolating hemolymph and luteovirus RNA
from the aphid hemocoel was described. This novel and reproducible method provides sufficient viral RNA for detection of
virus in the hemocoel of both small and large, single aphids
(R. padi and A. pisum, respectively) by RT and QRT-PCR.
This method could easily be used to isolate hemolymph from
other important insect vectors of plant viruses, such as whitefly,
leafhoppers, planthoppers and mealybugs.
Acknowledgements
We thank Professor Gus de Zoeten for providing full length
clones of PEMV RNAs, Dr. Steven A. Whitham for assistance
with QRT-PCR and Dr. William R. Staplin for critical reading of
the manuscript. This journal paper of the Iowa Agriculture and
Home Economics Experiment Station, Ames, Iowa, Project No.
3559, was supported by the USDA North Central Biotechnology
Initiative grant number 97-34340-3987, Bayer Crop Science,
Syngenta, and by Hatch Act and State of Iowa funds.
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