Ann. appl. Biol. (2002), 140:215-231
Printed in Great Britain
215
The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci
— insights from studies with Tomato yellow leaf curl virus
By HENRYK CZOSNEK*, MIRIAM GHANIM and MURAD GHANIM
Institute of Plant Sciences and Genetics, Faculty of Agriculture, The Hebrew University of Jerusalem,
Rehovot 76100, Israel
(Accepted 1 May 2002; Received 23 October 2001)
Summary
Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia
tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex
and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from
the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In
this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses
by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent
unpublished data.
We describe the cells and organs encountered by begomoviruses in B. tabaci. We show
immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the
descending midgut, and TYLCV-specific labelling was also associated with food in the lumen. The
microvilli and electron-dense material in the epithelial cells of the gut wall were also labelled by the
anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a
putative site of long-term virus storage. We describe the path of begomoviruses in their vector B.
tabaci and in the non-vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus
translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating,
probably by exchange of haemolymph. We show that following a short acquisition access to infected
tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is
undetectable after a few hours in the non-vector T. vaporariorum. The implications of the long-term
association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors
are discussed.
Key words: Begomovirus, tomato, virus acquisition, virus retention, circulative transmission, Bemisia
tabaci, Trialeurodes vaporariorum
Whitefly-Transmitted Geminiviruses
Geminiviruses are sm all plant viruses
characterised by a 22 nm ´ 38 nm geminate particle
consisting of two joined incomplete icosahedra
encapsidating circular single-stranded (ss) DNA
genome molecules of about 2700 nucleotides
(Goodman, 1977; Harrison et al., 1977; Francki et
al., 1980; Zhang et al., 2001). Gem iniviruses
transmitted by the whitefly Bemisia tabaci are
assigned to the genus Begomovirus within the family
Geminiviridae (van Regenmortel et al., 2000).
Begomoviruses infect many important agricultural
plants worldwide including bean, cassava, cotton,
melon, pepper, potato, squash, tobacco, tomato and
watermelon. Begomoviruses originating from the
New World have a bipartite genome organisation
consisting of two approx. 2.8 kb circular ssDNA
genomic molecules named DNA A and DNA B.
Begomoviruses from the Old World have either a
*Corresponding Author E-mail: [email protected]
© 2002 Association of Applied Biologists
DNA A-like monopartite genome or a bipartite
genome genetically sim ilar to that of the
begomoviruses from the New World. The genomes
of monopartite viruses encode six genes, two on the
virion genome strand (V1 and V2) and four on the
complementary genome strand (C1 to C4). V1
encodes the coat protein (CP) and V2 may control
symptoms and movement. C1 encodes the Rep
protein necessary for virus replication, C2 C3 a
replication enhancer protein, and C4 may affect hostrange, symptom severity and movement (Jupin et
al., 1994; Laufs et al., 1995; Wartig et al., 1997).
The DNA A of bipartite viruses is similar in
arrangem en t to the genome of m onopartite
begomoviruses. For New World begomoviruses, the
DNA A component lacks the V2 gene. The DNA B
component encodes BV1 and BC1, proteins that are
essential for cell-to-cell and systemic movement
(Noueiry et al., 1994; Sanderfoot et al., 1996), and
can influence host range (von Arnim & Stanley,
216
HENRYK CZOSNEK ET AL.
1992; Ingham et al., 1995). Although not directly
involved in interaction with the whitefly vector,
DNA B sequences affect the efficiency of virus
acquisition by the insect by determining the location
of begomoviruses in plant tissues (Liu et al., 1997).
Co-Evolution of the Begomovirus-Whitefly
Complex
The begomovirus vector B. tabaci is an insect
species complex that has geographically distinct
phenotypic and genotypic variants (Bird &
Maramorosch, 1978; Perring et al., 1993; Bedford
et al., 1994; Brown et al., 1995; Frohlich et al.,
1999). The CP is the only begomoviral gene product
that directly interacts with whitefly factors during
the circulative transmission of the virus.
Phylogenetic analysis of begomovirus CP sequences
resulted in the grouping of the viruses according to
their geographical origin: 1) New World, 2) Western
Mediterranean basin, 3) Middle East, 4) Indian
subcontinent, 5) East and Southeast Asia and
Australia (Rybicki, 1994; Padidam et al., 1995).
Similarly, the B. tabaci complex could be resolved
into five major groups based on mitochondrial DNA
markers, essentially coinciding with the
geographical distribution of the begomoviruses
(Frohlich et al., 1999; Brown, 2001). This virusvector co-adaptation is likely to be the result of coevolution processes taking place in geographically
isolated locations.
Independent but converging information suggests
that the whitefly-begomovirus interaction may be
long-standing. 1) Geminiviral DNA sequences seem
to have integrated into the genome of some tobacco
ancestors by illegitimate recombination during
Nicotiana speciation, about 25 million years ago
(Bejarano et al., 1996). 2) The endosymbiotic
bacteria that produce the GroEL homologue
necessary for the survival of begomoviruses in their
insect vector (Morin et al., 1999), have been
associated with whiteflies for the last 200 million
years (Baumann et al., 1993). With the drift of
continen ts, the initial whitefly-begomovirus
complex(es) has developed with tim e into
geographically separated and co-adapted virus-insect
combinations (Bradeen et al., 1997).
It is inevitable that during this long-lasting virusvector relationship the virus has evolved to ensure
both its survival and efficient transmission by the
whitefly vector, and the insect also has evolved
strategies to safeguard it from possible deleterious
effects of the virus. Studying the interactions of
transmissible and non-transmissible begomoviruses
with vector and non-vector whitefly species may
help to identify the viral and cellular determinants
involved in transmission and shed some light on the
evolutionary history of the begomovirus-whitefly
complex. To this end, we will discuss the association
of B. tabaci with begomoviruses, in particular
TYLCV and related tomato infecting
begomoviruses. The nomenclature described by
Fauquet et al. (2000) has been used to distinguish
distinct begomovirus species.
Whitefly Cells and Organs Involved in the
Circulative Transmission of Begomoviruses
In order to identify the position of receptors that
are likely to mediate circulative transmission of
begomoviruses in their whitefly vector, it is
necessary to describe in some detail the insect cells
and tissues involved. The extensive anatomical
analysis of the begomovirus non-vector whitefly
Trialeurodes vaporariorum performed in the 1930s
(Weber, 1935) still serves as a reference for analysing
the internal anatomy of whitefly species. Several
recent publications have focussed on the anatomy
of B. tabaci mouthparts (Rosell et al., 1995), anterior
alimentary canal (Hunter et al., 1996), and digestive
tract, filter chamber and salivary glands (Cicero et
al., 1995; Harris et al., 1995, 1996; Ghanim et al.,
2001a). Molecular studies have helped define the
pathway of begomoviruses in their insect vector
(Hunter et al., 1998; Rosell et al., 1999; Ghanim et
al., 2001b). Virus particles ingested through the B.
tabaci stylets enter the oesophagus and the digestive
tract, penetrate the gut membran es into the
haemolymph, reach the salivary glands and finally
enter the salivary duct from where they are egested
with the saliva. A schematic drawing can be found
in Ghanim et al. (2001b).
B. tabaci feeds on phloem sap by inserting its
stylets into plant tissue and locating the vascular
tissue (Pollard, 1955). The stylet bundle is composed
of three stylets: the maxillary stylet, which contains
the food canal (through which phloem is acquired)
and the lateral salivary canal (through which saliva
is injected into the plant), and two mandibulary
stylets (Rosell et al., 1995). The stylet food canal
extends into the cibarium and oesophagus, which
runs along the dorsal side of the thorax before
entering the filter chamber. The internal oesophagus
expands within the filter chamber where it is united
with the continuous lumen that extends into the
connecting chamber, caecae, descending and
ascending midguts. Leaving the filter chamber, the
descending midgut is composed of thick epithelial
cells with large nuclei and microvilli extending into
a large lumen. It is prolonged by the ascending
midgut, which narrows until it enters the filter
chamber. The ascending midgut is formed by very
thick epithelial cells with an extensive brush border
of microvilli surrounding a rather small lumen. The
hindgut terminates with the rectal sac (Ghanim et
al., 2001b).
Whitefly transmission of begomoviruses
The epithelial cells of the whitefly digestive tract
separate the gut lumen and the hemocoel, which
occupies the entire body cavity. The hemocoel
contains the haemolymph, or primitive blood system,
which circulates around the body cavity between the
various insect organs, bathing them directly. It
consists of plasma in which are suspended several
types of nucleated cells or haematocytes, and
contains various inorganic ions, organic substances
and proteins. An important function of the
haematocytes is phagocytosis of foreign proteins,
microorganisms and tissue debris, constituting a nonspecific primitive immune system (Chapman, 1991).
Hence, virions face a particularly hostile
environment in the haemolymph.
Endosymbiotic bacteria housed in the whitefly
mycetocy tes seem to have a cardinal role in
safeguarding begomoviruses in the haemolymph. B.
tabaci (biotype B) mycetomes contain two types of
endosymbionts: the highly pleiomorphic P-type that
constitutes approximately 80% of the total
population, and the coccoid C-type (Costa et al.,
1995). The C-type endosymbionts produce a GroEL
homologue that is released into the haemolymph,
but not into the digestive tract (Morin et al., 2000).
As demonstrated for TYLCV, the GroEL homologue
seems to bind to and protect begomoviruses from
degradation in the haemolymph (Morin et al., 1999,
2000).
A pair of primary salivary glands is located in the
prothorax. The paired accessory glands are much
smaller and slightly anterior to the primary glands.
The primary salivary glands comprise at least 13
nearly symmetrical large cells surrounding a central
lumen lined with microvilli, which empties into a
duct at the base of the gland. This duct joins the
accessory salivary gland duct and the medial duct.
Each accessory gland is composed of four, similar,
large cells that encircle a central lumen lined with
extensive microvilli (Ghanim et al., 2001b). The
primary and accessory gland ducts on either side
fuse to form the lateral salivary ducts. The two lateral
ducts fuse above the hypopharynx to form a single,
dual-channelled, medial salivary duct (Harris et al.,
1996). The salivary canal is contained almost entirely
within one stylet, while the food canal is centrally
located and is formed by the apposition of the food
grooves in both stylets. The food and salivary canals
end at the stylet tip (Rosell et al., 1995).
Parameters of Acquisition and Transmission of
TYLCV by the Whitefly Bemisia tabaci
Tomato yellow leaf curl virus
TYLCV from Israel was one of the first
begomoviruses characterised in terms of its
relationship with its vector, the B biotype of B.
tabaci, and its host range (Cohen & Harpaz, 1964;
217
Cohen & Nitzany, 1966). TYLCV has an immense
economical impact worldwide (Picó et al., 1996;
Nakhla & Maxwell, 1998). Molecular comparisons
of virus isolates from distinct geographical regions
have revealed that leaf curl disease of tomato is
caused by closely- as well as distantly-related
monopartite or bipartite begomoviruses (Czosnek &
Laterrot, 1997).
Minimum time needed for efficient acquisition and
inoculation of TYLCV by B. tabaci
Whiteflies develop from an egg, through four
nymphal stages (also called instars), to an adult. B.
tabaci instars are able to ingest and transmit
begomoviruses such as TYLCV (Cohen & Nitzany,
1966) and Tomato yellow leaf curl Sardinia virus
(TYLCSV) (Caciagli et al., 1995). However the
disease is spread in the field by flying adults.
Whitefly-mediated transmission of TYLCV to
tomato plants and observation of disease symptoms
have indicated that the minimum acquisition access
period (AAP) and inoculation access period (IAP)
were 15-30 min. Moreover, similar values were
obtained with TYLCV isolates from the Middle East
(Ioannou, 1985; Mansour & Al-Musa, 1992; Mehta
et al., 1994) and from Italy (Caciagli et al., 1995),
and with Tomato lea f curl Bangalore virus
(ToLCBV) isolates from India (Reddy &
Yarag untaiah, 1981; Muniyappa et al., 2000).
However, using PCR TYLCV DNA can be detected
in a single insect as early as 5-10 min after the
beginning of the AAP (Atzmon et al., 1998; Ghanim
et al., 2001a; Navot et al., 1992). Similarly, the viral
DNA can be detected at the site of inoculation in
tomato after a 5 min IAP (Atzmon et al., 1998).
A single insect is able to infect a tomato plant with
TYLCV following a 24 h AAP, although not all
plants inoculated in this way will become infected.
The efficiency of transmission reaches 100% when
five to 15 insects are used (Cohen & Nitzany, 1966;
Mansour & Al-Musa, 1992; Mehta et al., 1994). A
similar number of insects are necessary to achieve
100% transmission of the New World bipartite
geminivirus, Squash leaf curl virus (SLCV) (Cohen
et al., 1983).
Changes in acquisition and transmission
efficiency as a function of the age and the gender
of the insect vector
B. tabaci reproduces by arrhenotoky. Unfertilised
eggs give rise to haploid males while fertilised eggs
develop into diploid females. Mated females can
regulate the sex of their progeny by selectively
fertilising some of their eggs (reviewed by Byrne &
Bellows, 1991). The efficiency of TYLCV
acquisition and transmission changes with the gender
and age of B. tabaci. It has been reported previously
that fem ale whiteflies transm it TYLCV and
218
HENRYK CZOSNEK ET AL.
ToLCBV with higher efficiency than males (Cohen
& Nitzany, 1966; Muniyappa et al., 2000). In these
studies, the effect of age was not determined. We
have studied the effect of the gender and age of
synchronised populations of adult B. tabaci on the
efficiency of transmission of TYLCV acquired
following a 48 h AAP (Czosnek et al., 2001). Nearly
all of the 1-2 wk-old adult females were able to cause
an infection in tomato plants following a 48 h IAP.
In comparison, only about 20% of the males of the
same age were able to produce infected plants.
Inoculation capacity decreased with the age of the
insects; 60% of the 3 wk-old females were able to
cause an infection in plants, whereas no infected
plants were obtained following inoculation by males
of the same age. Only 20% of the 6 wk-old females
were able to infect tomato plants. Although the rate
of TYLCV translocation is similar in males and
females, it is possible that different amounts of virus
translocate in the two genders (Ghanim et al.,
2001a), and the putative begomovirus receptors in
males and females may differ. In contrast, female
and male B. tabaci transmitted SLCV with the same
efficiency (Polston et al., 1990). The reason for these
differences is unclear.
The decreased inoculation capability of ageing
female whiteflies has been correlated with a
diminution of the amount of TYLCV they acquire
during a 48 h AAP (Rubinstein & Czosnek, 1997).
At the age of 17 days, the insects acquired less than
half the virus acquired by 10 day-old insects and at
24 days the amount was only about 10%. At the age
of 28 days and thereafter, the viral DNA associated
with the insects was undetectable by Southern blot
hybridisation although the insects retained about
20% of their initial capacity to produce infected
plants. It is likely that being less active than young
whiteflies in probing and feeding on infected plants,
older insects acquire fewer virus particles.
Field and laboratory populations of B. tabaci
comprise males and females of various ages, which
have different abilities to acquire and transmit
begomoviruses. The ratio of males to females
changes throughout the course of the year in the field
as well as in the laboratory (Horowitz & Gerling,
1992). Hence, in our studies we generally use female
B. tabaci 1-2 wk after eclosion. For practical
purposes, we suggest the use of synchronised
populations of insects of the same sex for studies
aimed at comparing parameters of acquisition and
transmission of B. tabaci populations. This is
particularly pertinent when whitefly-mediated
inoculation is the sole experimental tool to select
tomato genotypes with resistance, or tolerance to
TYLCV.
Path of TYLCV in B. tabaci and Speed of Virus
Translocation
Visualisation of TYLCV in sections of B. tabaci
Visualisation of begomoviruses in sections of
whiteflies may shed some light on the cells involved
in the translocation of virions, and on the pathways
that have evolved to allow the crossing of the gut/
haemolymph and haem olymph/salivary gland
barriers. Two bipartite begomoviruses (Tomato
mottle virus, ToMoV, and Cabbage leaf curl virus,
CaLCuV) have been immunolocalised in the B.
tabaci filter chamber and in the anterior part of the
midgut, with ToMoV also detected in the salivary
glands (Hunter et al., 1998).
We have initiated an extensive study of the
localisation of TYLCV in anatomical sections of
viruliferous female B. tabaci. We have focused our
attention on those cells and organs involved in the
circulative transmission of this virus. Using TYLCVspecific antiserum, immunogold label was present
in the stylets (Fig. 1) and was associated mainly with
the lumen of the food canal. Label was detected in
the proximal part of the descending midgut (Fig. 2)
associated with food in the lumen and with electrondense material in the microvilli-rich gut wall
epithelial cells. In another study, we reported the
immunolocalisation of TYLCV to the filter chamber
and the distal part of the descending midgut (Brown
& Czosnek, 2002). These results suggest that the
microvilli may constitute one of the sites rich in
begomoviral receptors and may serve as the primary
site allowing internalisation of viral particles. Hence
these cells may constitute a transit site for the virus
on its way to the haemocoel, or may serve as a virus
long-term storage site. In another study, we have
used in situ hybridisation to detect TYLCV in the
nucleus of three of 14 of the cells of the B. tabaci
primary salivary glands (Brown & Czosnek, 2002).
Locating the virus in nuclei may suggest, but does
not prove, replication of the virus within the insect.
Speed of TYLCV translocation in B. tabaci
The stylets of B. tabaci must pass between the
epidermal and parenchymal cells before penetrating
the vascular tissues to allow the whitefly to feed in
the phloem (Costa, 1969; Pollard, 1955). Analysis
of the electronic waveforms produced during feeding
of B. tabaci on Lima bean (Phaseolus lunatus) has
indicated that it took an average of 16 min (as early
as 10 min for some of the insects, as late as 45 min
for others) from initiation of leaf penetration to
phloem ingestion (Walker & Perring, 1994). The
minimum phloem contact threshold period observed
for successful inoculation of TYLCV by B. tabaci
was 1.8 min (Jiang et al., 2000). PCR-based studies
of TYLCV ingestion and transmission have shown
that whiteflies may reach the phloem of tomato
Whitefly transmission of begomoviruses
219
Fig. 1. Immunodetection of TYLCV in longitudinal sections through the stylets of a B. tabaci female after a 24 hacquisition access period on a TYLCV-infected tomato plant. Insect heads were separated from the body and fixed in
0.25% glutaraldehyde, 4% paraformaldehyde in PBS for 3 h. Following ethanol dehydration, the tissues were infiltrated
with LR white resin and embedded in capsules (essentially as described by Wescot et al., 1993). Sections of 60-90
nm were deposited on 200 mesh formvar-coated nickel grids, incubated for 3 h with a polyclonal antibody raised
against the TYLCV coat protein expressed in Escherichia coli (diluted 1:1000), followed by 1 h with a goat anti
rabbit IgG gold (15 nm diameter) conjugate, stained with uranyl acetate and lead citrate and observed in a transmission
electron microscope. A: section showing the food canal (FC) and the salivary canal (SC) (bar = 2 mm). B: section
showing the food canal and the presence of TYLCV-specific labeling in the lumen (bar = 0. 5 mm). C: enlargement
of boxed image in B showing the label in the lumen of the food canal.
plants within minutes after landing (Atzmon et al.,
1998). Phloem probing occurs more quickly in
TYLCV-infected tomato plants compared with noninfected plants. Microscopic examination of infected
tomato leaves revealed that, even before the
appearance of symptoms, spongy mesophyll cells
collapse, leading to the displacement of the veins
towards the abaxial epidermis, within closer reach
of the whitefly stylets (Michelson et al., 1997).
PCR has been a useful tool to determine the speed
of begomovirus translocation in the whitefly vector
(Caciagli & Bosco, 1997; Atzmon et al., 1998; Rosell
et al., 1999; Ghanim et al., 2001a). Temporal PCR
analysis of the translocation of the New World SLCV
and the Old World TYLCV in tissues and organs
involved in circulative transmission has shown that
the timing of translocation is independent of the
identity of the virus (as long as it is transmissible)
and of the geographical origin of the B. tabaci vector.
W hen DNA from B. tabaci and saliva,
haemolymph and honeydew were used as substrates
for PCR, SLCV DNA was detected in extracts of B.
tabaci after a 30 min AAP on infected pumpkin. After
2 h, viral DNA was present in the haemolymph
although it was detected in the saliva and honeydew
only after a further 6 h (Rosell et al., 1999). We have
investigated the translocation of TYLCV DNA and
CP using whitefly stylets, head , midgut,
haemolymph and salivary glands dissected from a
single insect (Ghanim et al., 2001a). The organs and
tissues were used directly as substrate for PCR and
homogenates were used in immunocapture-PCR (ICPCR). TYLCV was detected in the head of whiteflies
as early as 10 min after the beginning of the AAP
and in the midgut after approximately 40 min. The
crossing of TYLCV from the m idgut to the
haemolymph was surprisingly fast; virus reached the
haemolymph 30 min after it was first detected in the
midgut, just 90 min after the beginning of the AAP.
TYLCV was detected in the salivary glands
approximately 5.5 h after it was first detected in the
haemolymph, 7 h after the beginning of the AAP,
and approximately 1 h before the insects were able
to infect tomato plants. The results obtained by PCR
and by IC-PCR overlapped, suggesting that the viral
DNA is within virions. These results showed that
once acquired from infected plants, begomoviruses
transit in the body of B. tabaci according to an
invariab le sequential path: head-midguthaemolymph-salivary glands (Ghanim et al., 2001a).
Translocation of TYLCV in T. vaporariorum, a
whitefly species able to acquire but not to transmit
begomoviruses
The whitefly species T. vaporariorum, which also
feeds in the phloem and has a host range similar to
B. tabaci, is capable of ingesting but not transmitting
begomoviruses such as SLCV (Polston et al., 1990)
and TYLCV (Antignus et al., 1993). SLCV was
detectable by PCR in whole body homogenates and
honeydew of T. vaporariorum but not in the
haemolymph or saliva (Rosell et al., 1999),
suggesting that the gut wall of the non-vector
whitefly constitutes a barrier that begomoviruses are
unable to cross. To test this hypothesis, we have
followed concomitantly the translocation of TYLCV
220
HENRYK CZOSNEK ET AL.
Fig. 2. Immunodetection of TYLCV in longitudinal sections through the descending midgut of B. tabaci females
after a 24 h acquisition access period on a TYLCV-infected tomato plant. Dissected digestive tracts were washed
with PBS, fixed, and sections were processed as described in Fig. 1. A: Section through the descending midgut. (Mv:
microvilli, F: food intake, Lu: lumen); note the label associated with food in the lumen (bar = 1 mm). B: Section
through the gut wall (Ec: epithelial cell, Gw: gut wall). Insert: enlargement of image boxed; note the label associated
with electron-dense material in the epithelial cell.
221
Whitefly transmission of begomoviruses
in B. tabaci and T. vaporariorum from the time the
insects accessed infected tomato plants, using
whitefly head, midgut, haemolymph and salivary
glands as substrate for PCR (Ghanim et al., 2001a).
Analyses of the PCR products (Fig. 3) showed that
TYLCV had reached the head of B. tabaci and T.
vaporariorum 10 min after the beginning of the AAP.
After 1 h, the virus was found in the midgut of both
whitefly species. TYLCV DNA was detected in the
haemolymph of B. tabaci after 2 h and in the salivary
glands after 8 h. In contrast, the virus was not found
in the haemolymph or the salivary glands of T.
vaporariorum even after a 24 h AAP. Interestingly
TYLCV was detected in a small number of midgut
samples. These experiments indicate that TYLCV
does not cross the gut/haemolymph barrier of T.
vaporariorum. They may also suggest that most of
the virus is destroyed in the digestive tract. Hence
Bemisia tabaci
Acquisition access
Min
M 10 30 1
Hours
2 4
8 12 24 0
Trialeurodes
vaporariorum
Acquisition access
Min
M 10 30 1
Hours
2 4
8 12 24 0
Head
Midgut
Haemolymph
Salivary glands
Fig. 3. Comparative analysis of translocation of TYLCV in the vector B. tabaci and in the non-vector T. vaporariorum.
B. tabaci and T. vaporariorum were caged with infected tomato plants. After the acquisition access periods indicated,
groups of five insects were collected, dissected and the pooled heads, midgut, haemolymph and salivary glands were
subjected to PCR, using TYLCV-specific primers (Ghanim et al., 2001a). The PCR products were subjected to
agarose gel electrophoresis and stained with ethidium bromide. Note that viral DNA was not found in the haemolymph
and the salivary glands of the begomovirus non-vector T. vaporariorum. 0: non-viruliferous whitefly. M: 1 kbp
ladder molecular weight marker.
222
HENRYK CZOSNEK ET AL.
the inability of T. vaporariorum to inoculate tomato
plants is correlated with the inability of TYLCV to
transit from the digestive tract to the haemolymph.
Transmission during mating: another route of
acquisition of TYLCV by B. tabaci
TYLCV from Israel can be transmitted between
whiteflies in a sex-dependent manner, in the absence
of any other source of virus (Ghanim & Czosnek,
2000). TYLCV was transmitted from viruliferous
males to females, and from viruliferous females to
males, but not between insects of the same sex.
Transmission took place when insects were caged
in groups or in couples, either in a feeding chamber
or on TYLCV non-host cotton plants. All evidence
indicates that TYLCV is transferred during sexual
contact. Non-viruliferous whiteflies were unable to
ingest detectable amounts of TYLCV from artificial
medium used to feed viruliferous whiteflies, ruling
out the possibility that the virus was acquired from
the diet. Transmission of TYLCV was observed only
when males and females were caged together, not
when whiteflies were of the same gender. TYLCV
was detected first in the haemolymph of the recipient
insects, later in their head, but never in their digestive
tract (Ghanim et al., 2001a).
Two conditions have to be met in order to observe
virus transmission amongst whiteflies. First the
insects need to mate; second virus needs to be present
in the haemolymph of the donor insect. The key role
of the haemolymph was demonstrated by caging
whiteflies previously fed on Abutilon mosaic virus
(AbMV)-infected abutilon plants. AbMV is a nontransmittable begomovirus; it can be ingested by B.
tabaci but it does not cross the gut wall into the
haemolymph (Morin et al., 2000). When nonviruliferous B. tabaci males were caged with females
fed on AbMV-infected abutilon plants, AbMV DNA
was not detected in the males. Identical results were
obtained in the reciprocal mating scheme (H
Czosnek and M Ghanim, unpublished). These results
suggest that sexual transmission of TYLCV occurs
by exchange of haemolymph during intercourse.
Acquisition and Long-Term Storage of TYLCV
in B. tabaci and T. vaporariorum
Latent period of TYLCV in B. tabaci
Once ingested, beg omoviruses are not
immediately available for infection. They need to
translocate from the digestive tract to the salivary
glands from which they are excreted with the saliva
during feeding. The time it takes for a geminivirus
to complete this path and to infect susceptible plants
is called the latent period. The latent period may not
only reflect the speed of virus translocation but also
the time it takes for an insect to accumulate enough
virions (the number is undetermined) to be able to
efficiently transmit the disease to plants. For some
begomoviruses this threshold may be reached much
earlier than for others. For example, SLCV has been
detected by PCR in the saliva 8 h after the beginning
of the AAP (Rosell et al., 1999) while the minimal
latent period was reported to be approximately 19 h
(Cohen et al., 1983). In contrast, TYLCV has been
detected in the salivary glands of B. tabaci 7 h after
the beginning of the AAP, only 1 h before the insects
were able to transmit virus to produce infected
tomato plants (Ghanim et al., 2001b). The estimated
latent period for a given virus may vary due to the
experimental conditions or to changes in virus and/
or vector with time. For example, the latent period
of TYLCV from Israel was reported to be 21 h in
the early 1960s (Cohen & Nitzany, 1966) while it
was found to be 8 h 35 years later (Ghanim et al.,
2001b). Clearly, care is needed when making such
comparisons.
Whiteflies acquire a finite amount of TYLCV
during a feeding episode
Begomoviral DNA in B. tabaci accumulates with
increasing AAP on infected plants up to a peak at
approximately 12 h for TYLCV (Zeidan & Czosnek,
1991), 24 h for TYLCSV (Caciagli & Bosco, 1997),
and 48 h for SLCV (Polston et al., 1990). At the
peak the insects contained the equivalent of
approximately 600 million viral genomes (about 1
ng viral DNA). It seems therefore, that the amount
of virions an insect can acquire from an infected
plant during a single feeding event is finite, reaching
a steady state between ingestion and egestion after
12-48 h of AAP.
We have designed an experiment to determine
whether consecutive feedings lead to the
displacement of the acquired virus (Fig. 4).
Whiteflies were first caged with tomato plants
infected with TYLCV for 48 h. Then, the insects
were collected and caged with tomato plants infected
with TYLCSV for an additional 48 h. Quantification
of the viral DNAs showed that as TYLCSV
accumulated during the second feeding, the amount
of TYLCV remained approximately constant. At
the end of the two successive 48 h AAPs, the
whiteflies contained approximately similar amounts
of TYLCV and TYLCSV. These results showed that
the newly acquired virus did not chase the virus
already associated with the insect. At the end of the
successive AAP, the tomato plants infected by these
whiteflies contained similar amounts of TYLCV and
TYLCSV DNA. These results contrast with earlier
experiments showing that whiteflies that were fed
for 48 h on SLCV-infected squash then transferred
to Melon leaf curl virus (M LCuV)-infected
watermelon for 24 h exhibited a 35-90% reduction
in transmission of MLCuV during a 48 h IAP,
compared with those fed on MLCuV only (Cohen
223
Whitefly transmission of begomoviruses
TYLCSV
TYLCV
700
600
300
200
TYLCSV
400
TYLCV
Million genomes
500
100
0
0
10
20
30
40
50
60
70
80
90
Acquisition access feeding (h)
Fig. 4. Successive acquisition of TYLCV and TYLCSV by B. tabaci. Whiteflies were caged with a tomato plant
infected with TYLCV. Groups of 20 whiteflies were collected every 2-4 h. After 48 h the remaining insects were
collected and caged with a tomato plant infected with TYLCSV. Groups of 20 whiteflies were removed every 2-4 h.
Total DNA was extracted from all the groups of 20 insects and DNA equivalent to one whitefly per time point were
Southern blotted. The samples were hybridised with a radiolabelled TYLCV probe (Navot et al., 1991), washed at
high stringency (to discriminate between the two viruses) and exposed to X-ray film. The TYLCV probe was removed
and the samples were hybridised with a radiolabeled TYLCSV probe (Kheyr-Pour et al., 1991), washed at high
stringency and exposed to X-ray film. The autoradiograms were scanned and the DNA quantified using cloned viral
DNA standards (1 pg DNA is equivalent to 0.6 million genomes). Vertical arrows indicate the beginning of AAP of
the two viruses. Horizontal arrows point to the autoradiograms obtained after hybridisation with the virus-specific
probes. Note that during the second feeding, whiteflies acquired amounts of TYLCSV similar to the amounts of
TYLCV acquired during the first feeding. The quantities of TYLCV remained approximately constant during the
acquisition of TYLCSV.
et al., 1989). The results were interpreted as an
interference of transmission of MLCuV by SLCV.
However, the transmission of SLCV by whiteflies
harbouring the SLCV-MLCuV virus mixture was not
assessed and the amount of virus acquired during
the successive AAP was not measured.
Retention of TYLCV in B. tabaci and T.
vaporariorum
Following a 1-2 day AAP, begomoviruses may be
retained in their whitefly vector for several weeks
and sometimes for the entire life of the insect. SLCV
and TYLCV remain associated with B. tabaci during
the entire life of the vector (Cohen et al., 1989;
Rubinstein & Czosnek, 1997) while TYLCSV is
undetectable after approximately 20 days (Caciagli
& Bosco, 1997).
In most instan ces the viral DNA remained
associated with the insects for much longer than
transmission ability suggested. For example, while
TYLCSV DNA was detectable up to 20 days after
the end of the 48 h AAP, transmission could occur
only for up to 8 days (Caciagli et al., 1995). Detection
of viral DNA (by Southern blot hybridisation or
PCR) and CP (by Western blot immunodetection or
IC-PCR) suggests these are not retained in B. tabaci
for the same time periods. Following the end of the
48 h AAP, TYLCV DNA was detected throughout
the 5 wk life span of the insect while the amount of
TYLCV C P steadily decreased until it was
undetectable at day 12 (Rubinstein & Czosnek,
1997). The disappearance of the virus CP was
224
HENRYK CZOSNEK ET AL.
associated with a rapid decrease in the ability of the
whitefly to produce infected host plants, as shown
for TYLCV (Rubinstein & Czosnek, 1997) and
SLCV (Cohen et al., 1983). It is interesting to note
that a difference in the retention of viral DNA and
CP in B. tabaci was also observed with an Israeli
isolate of the non-transmissible bipartite
begomovirus AbMV (Morin et al., 2000). Following
a 4-day AAP on infected abutilon plants, the virus
DNA remained associated with B. tabaci throughout
the 15 days sampling period, while the CP was
detectable only for up to 7 days (Fig. 5).
TYLCV was retained for much shorter time in the
non-vector T. vaporariorum than in the B. tabaci
vector. We have compared the retention periods of
TYLCV DNA and CP in the two insect species
reared on cotton for 7 days following a 3 h AAP on
infected tomato plants (Fig. 6). TYLCV DNA was
detected in B. tabaci over the entire 7 days of the
experiment while the CP was detected during the
first 4 days only. In contrast, TYLCV DNA was
detected in T. vaporariorum only during the first 6
h that followed the end of the AAP, and the CP for
up to 4 h. Thus TYLCV vanished very quickly from
T. vaporariorum once acquisition feeding has
ceased, but nonetheless, the DNA appears to be
retained longer than the CP even in the non-vector.
Reduced longevity and fertility of viruliferous B.
tabaci and TYLCV invasion of the insect
reproductive system
The life-long association of TYLCV with B. tabaci
led to a significant decrease of the insect longevity.
Mortality curves of whiteflies reared on eggplants
Days after acquisition access
P
0
3
5
8
12
15
A
DNA
(Hybridisation)
B
P
14
4
0
1
2
1
2
7
1
2
10
1
2
1
2
CP
(IC-PCR)
Fig. 5. Retention of AbMV in B. tabaci. Whiteflies were transferred to cotton plants following a 4-day access to
infected abutilon plants. During the 15 day experiment, three groups each of 20 insects were collected at the time
points indicated. DNA was prepared from insects of the first group and divided into two equal portions, which were
Southern blot hybridised, respectively, with radiolabelled probes for AbMV DNA A (A) and AbMV DNA B (B)
(Frischmuth et al., 1990). Extracts from the second and third groups of 20 insects each (1 and 2) were incubated with
PCR tubes coated with an antiserum raised against the CP of Tomato golden mosaic virus (which recognises the CP
of AbMV); the DNA from the immunocaptured virions was detected by PCR with primers specific to AbMV DNA
A (Morin et al., 2000). The PCR products were subjected to agarose gel electrophoresis and stained with ethidium
bromide. P: infected abutilon plant. Note that DNA A and B were detected during the entire experiment while the CP
was detectable by immunocapture-PCR (IC-PCR) only up to 7 days after the end of the AAP.
225
Whitefly transmission of begomoviruses
(a TYLCV non-host plant) were established after 1
day-old adult female acquired TYLCV from infected
tomato for a 48 h AAP. The difference at the 50%
mortality point between viruliferous and nonviruliferous whiteflies was between 5 and 7 days.
Thus the association of TYLCV with B. tabaci led
to a reduction of 17-23% in the whitefly life
expectancy compared with insects that have not
acquired the virus (Rubinstein & Czosnek, 1997).
Similarly, the life span of female whiteflies fed for
24 h on SLCV-infected plants was on average 25%
shorter than that of whiteflies fed on the same virus
source for 4 h (Cohen et al., 1983). These
observations indicate that at least these two
begomoviruses have deleterious effects on their
insect vector.
The long-term association of B. tabaci with
TYLCV also affected the insect fertility (Rubinstein
& Czosnek, 1997). The effect was not immediate,
as if the virus had first to invade the reproductive
Time after acquisition access
Hours
M
P
1
2
4
6
Days
8
12
1
2
3
4
5
6
7
DNA
(PCR)
Bemisia
tabaci
CP
(IC-PCR)
Hours
M P
1
2
4
6
Days
8
12
1
2
3
4
5
6
7
DNA
(PCR)
Trialeurodes
vaporariorum
CP
(IC-PCR)
Fig. 6. Retention of TYLCV in B. tabaci and in T. vaporariorum. Whiteflies of the two species were caged with
infected tomato plants. After 3 h of access feeding, the insects were removed and transferred to cotton plants, a
TYLCV non-host plant. At the time points indicated, two groups of 10 insects each were collected; the first group
was used to assess the presence of TYLCV DNA by PCR, the second group was used to determine the presence of
the virus coat protein (CP) by immunocapture PCR (IC-PCR). The 10 whiteflies from the first group were pooled,
their DNA extracted and subjected to PCR with TYLCV-specific primers. The 10 whiteflies from the second group
were pooled and insect extracts were incubated with PCR tubes coated with an antiserum raised against the TYLCV
CP; the DNA from the immunocaptured virions was detected by PCR with TYLCV-specific primers (Ghanim &
Czosnek, 2000). The PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide.
Note that TYLCV DNA was detected in B. tabaci over 7 days while the CP was detected during the first 4 days only.
In contrast TYLCV DNA was detected in T. vaporariorum only during the first 6 h while the CP was found for up to
4 h.
226
HENRYK CZOSNEK ET AL.
system. During the 24 h following the AAP, the mean
number of eggs laid by viruliferous and nonviruliferous insects was similar (6.0 vs 5.1). The
mean number of eggs laid by 3 day-old viruliferous
insects during a 7 day or a 20 day period was
significantly lower than that laid by non-viruliferous
insects (22.7 vs 38.1 and 56.0 vs 33.4, respectively).
Eggs maturing in the ovaries of viruliferous B. tabaci
contained viral DNA detectable by PCR, indicating
that TYLCV has invaded the reproductive system.
Thus infection of the maturing egg may have led to
its abortion. Following oviposition, viral DNA was
detected in some, but not all, eggs, instars and adult
progeny of individual whiteflies. A relatively small
and variable proportion of insects that developed
from eggs of viruliferous whiteflies were able to
transmit the virus to tomato in biological tests
(Ghanim et al., 1998). In another study, using a
different B. tabaci colony and an almost identical
TYLCV isolate, viral DNA was detected in eggs and
up to the third instar, but not in the adult progeny of
viruliferous insects. In contrast, in the same
investigation, ToMoV was not found in eggs and
instars, and was not transmitted to progeny (Polston
et al., 2001).
Viral and insect Determinants of Begomovirus
Circulative Transmission
Viral determinants
It is believed, but not proven, that begomoviruses
are acquired, translocated and inoculated as virus
particles by the whitefly vector. It is therefore likely
that the capsid is the structure that is exposed to the
whitefly tissues and interacts with insect receptors
and chaperones. This hypothesis is supported by
experiments in which acquisition of Bean golden
mosaic virus by B. tabaci was lost following
mutagenesis of the CP gene (Azzam et al., 1994).
Furthermore, replacing the CP of AbMV with that
of the closely-related whitefly transmissible Sida
golden mosaic virus (SiGM V) produced a
transmissible chimeric AbMV (Höfer et al., 1997).
Vector specificity of geminiviruses is determined by
the CP and there is no evidence for the involvement
of other virus-encoded proteins in transmission.
Exchanging the CP gene of the whitefly-transmitted
African cassava mosaic virus (ACMV) with that of
the leafhopper-transmitted Beet curly top virus
(BCTV, genus Curtovirus) produced a leafhoppertransmitted ACMV chimera (Briddon et al., 1990).
Loss of transmission by B. tabaci can be caused
by a surprisingly small number of amino acid
replacem ents in the begomovirus CP. Natural
TYLCSV mutants have been isolated which are
acquired, but not transmitted, by B. tabaci, although
they are able to systemically infect tomato plants
following agroinoculation. Loss of transmission was
due to the replacement of two amino acids at
positions 129 and 134 in the TYLCSV CP (Noris et
al., 1998). This region of the CP is also implicated
in transmission of the bipartite Watermelon chlorotic
stunt virus (Kheyr-Pour et al., 2000). AbMV is
another begomovirus that has lost the ability to be
transmitted (Wu et al., 1996), probably because it
has been maintained and propagated by cuttings.
Mutagenesis of AbMV CP showed that exchange of
two amino acids at CP positions 124 and 149 were
sufficient to restore partial transmissibility by
whiteflies. Alteration of amino acid 174 in addition
to those at position 124 and 149 completely restored
transmission of AbMV (Höhnle et al., 2001).
Insect determinants
The mechanism of transmission of viruses of the
genera Tospovirus (fam ily Bunyaviridae) and
Luteovirus (Luteoviridae) show many similarities
to that of the geminiviruses (Nault, 1997). These
viruses are transmitted in a propagative manner by
thrips and a circulative manner by aphids,
respectively. Investigations into the transmission of
these viruses is more advanced than those on
geminiviruses and thus point the way for study of
geminivirus transmission. These systems have
indicated the involvement of two sites regulating
virus transmission: 1) the gut epithelia in the thrip/
tospovirus (Ullman et al., 1992) and aphid/luteovirus
(Gildow, 1993) systems; 2) the basal lamina and the
basal plasmalemma of the accessory salivary gland
in the aphid/luteovirus system (Peiffer et al., 1997;
Gildow et al., 2000). In the begomovirus/B. tabaci
system, the digestive tract was the only organ shown
to be a barrier to transmission of some viruses.
AbMV as well as some non-transmissible TYLCSV
mutants are unable to cross the midgut membranes
into the haemolymph. These mutants may have lost
the capsid structure enabling binding to putative
receptors in the vector midgut followed by
translocation to the haemolymph.
It is likely that begomovirus receptors may be
present in the midgut of B. tabaci. Indeed, the midgut
(within and outside the filter chamber) and internal
ileum epithelia have a brush border at the apical
membrane and the microvilli would provide an ideal
site for viral endocytosis (Fig. 2, Ghanim et al.,
2001b). In contrast, the oesophagus, caec um,
continuous lumen within the filter chamber and
rectum are lined with a cuticular intima making them
unlikely sites of virus uptake. In the tospovirus/thrip
system, a recept or has been isolated from the
plasmalemma of the thrip midgut, which may serve
in virus attachment (Bandla et al., 1998).
Comparison of vector and non-vector insects has
allowed the isolation of virus receptor candidates.
Two proteins isolated from head tissues of the aphid
vector, Sitobion avenae, but not from the non-vector
Whitefly transmission of begomoviruses
aphid, Rhopalosiphum maidis, have been identified
as potential receptors for Barley yellow dwarf
luteovirus (Li et al., 2001). Similarly, it might be
possible to isolate begomoviral receptors by
comparing protein profiles of the midgut from the
vector B. tabaci and the non-vector T. vaporariorum.
The recep tor properties may be confirmed by
protein-protein interaction studies using virus
overlay assays and the yeast two-hybrid system.
Endosymbionts appear to play a cardinal role in
the safe transit of begomoviruses within their insect
vectors. The role of chaperonins synthesised by
insect endosymbiotic bacteria was first demonstrated
in aphids, where an interaction between the
luteovirus and the endosymbiotic chaperone, GroEL,
was shown to be essential for virus retention (van
den Heuvel et al., 1994). The survival of TYLCV,
and probably other beg om oviruses, in the
haemolymph of B. tabaci is likely to be ensured by
a similar strategy. GroEL produced by the whitefly
coccoid bacterium was identified in the insect
haemolymph as a native 14-mer unit, each subunit
having a mass of 63 kDa (Morin et al., 2000).
TYLCV particles displayed affinity for the B. tabaci
GroEL homologue in a virus overlay assay and the
TYLCV CP and B. tabaci GroEL interacted in the
yeast two-hybrid system. Interestingly, B. tabaci
GroEL interacted as well with the CP of the nontransmissible AbMV (Morin et al., 2000), indicating
that mutations in the CP which prevented AbMV
from crossing into the insect haemolymph (Höhnle
et al., 2001), do not prevent binding to GroEL.
The function of GroEL in the circu lative
transmission of begomoviruses may not be limited
to protection in the haemolymph. We do not know
how begomoviruses penetrate and cross the gut
epithelial cells. We do not know whether the virus
translocates as a geminate particle, and whether it
changes its conformation in the process. In the latter
case, the role of GroEL might be to correctly refold
the viral particle once in the haemolymph.
Concluding Remarks
Begomovirus-whitefly co-adaptation
Plant viruses, and especially geminiviruses, have
much more specific relationships with their insect
vector than with plant hosts (reviewed by Power,
2000). The evolutionary constraints that narrowed
the gem inivirus-vector complex to a one
begomovirus-one insect species are not understood.
Capsid structure and insect receptors are likely to
be the key to this one-to-one relationship.
Evolution of begomoviruses might have been
towards a better adaptation of the capsid to putative
receptors of the local whitefly to ensure optimisation
of virus transmission. Hence a beg omovirus
infecting a given host tends to possess a CP with
227
epitopes more closely related to those of other
begomoviruses in the same geographical region than
to a virus infecting the same host in other regions.
For example, the CP of Indian cassava mosaic virus
is more similar to those of other geminiviruses of
the Indian subcontinent than to ACMV. Similarly
transmission of TLCV by an insect from the same
geographical region is more efficient than when the
virus and insect originate from two different regions
(McGrath & Harrison, 1995).
Recombination as a driving force in the rapid
evolution of geminiviruses has been appraised
recently (Padidam et al., 1999; Harrison & Robinson,
1999). Although it seems that recombination in the
CP gene is less frequent than in other parts of the
geminiviral genome, in those viruses where CP
recombination has occurred, the transmission
determinants, dictated by the amino acid stretches
that influence the structure of the virus CP, have been
preserved and possibly improved (Sanz et al., 1999).
As the begomoviral capsid evolved toward a better
adaptation of the virus to the insect, some whitefly
species may have developed receptors in their
digestive and salivary systems that facilitate
begomovirus translocation. The question remains
why whiteflies have evolved a system that allows
the circulative transmission of potentially harmful
begomoviruses, instead of confining the virus to the
stylet or destroying the virus in the digestive system.
Indeed, available data suggest that TYLCV as well
as SLCV are reminiscent of insect pathogens and
are deleterious to their whitefly vector (Cohen et
al., 1983; Rubinstein & Czosnek, 1997). The
whitefly host may have developed antiviral strategies
through the expression of factors preventing virus
replication as described in the case of TYLCV
(Cohen, 1967; Cohen & Marco, 1970).
Chaperonins produced by the vector
endosymbionts may be part of the overall strategy
devised to neutralise deleterious viruses. It has been
suggested that viruses belonging to unrelated
taxonom ic groups have taken advantage of
endosymbiotic bacterial proteins produced by their
insect vector to avoid degradation in the
haemolymph (Gibbs, 1999). However, it is possible
that the purpose of translocating viruses throughout
the insect body is ultimately to remove potentially
harmful particles. Accordingly, facing invasion by
progenitors of modern begomoviruses, whitefly
ancestors, like aphids (van den Heuvel et al., 1994),
m ay have taken advantage of chaperonins
synthesised by endosymbionts (Baumann et al.,
1993) to facilitate the transit of the virions until they
are expelled, instead of attempting to destroy them
by expressing enzymes or antiviral factors. This
strategy may not have been adopted by the whitefly
T. vaporariorum, which is able to ingest but not
transmit begomoviruses (Rosell et al., 1999). Once
228
HENRYK CZOSNEK ET AL.
ingested during feeding, the virus spreads in the
digestive tract of T. vaporariorum but is unable to
cross the gut epithelial cells into the haemolymph,
and is destroyed within hours (Fig. 6).
Long-term association of begomoviruses with the
whitefly vector
B. tabaci ingests increasing quantities of
begomovirus during feeding on an infected plant
until the amount of virions present in the insect body
reaches an ingestion-egestion steady state after 1248 h of AAP. Following the end of the AAP, the viral
DNA remains detectable for much longer than the
CP, sometimes during the entire life of the insect
(Rubinstein & Czosnek, 1997). Since only an
infinitesimal amount of virus is egested during
feeding and excreted with the honeydew, the
question as to the fate of the ingested virus is
intriguing. The finite num ber of putative
begomoviral receptors may become saturated during
the first hours of acquisition feeding. We postulate
that with longer AAP, the virions that do not interact
with the receptors leave the circulative pathway and
invade as yet unidentified cells and tissues where
they are stored. In the process, the virions
disassemble, the viral DNA binds to proteins that
protect it from degradation, and the CP is
progressively destroyed. Once acquisition access
ends, the supply of virion ceases. The particles bound
to the insect receptors progressively leave the
digestive tract, reach the haemolymph and the
salivary glands, and may be transmitted to plants.
This process may continue during the entire adult
life of B. tabaci, explaining the residual infectivity
of TYLCV observed 4 wk after the end of the AAP
(Rubinstein & Czosnek, 1997). In contrast, we
suppose that when the non-vector T. vaporariorum
accesses infected plants, the ingested virions do not
encounter begomoviral receptors in the insect gut
and therefore are not retained in the digestive tract
of this insect for more than a few hours (Fig. 6).
The invasion-storage hypothesis may be
substantiated by an experiment where B. tabaci
acquires successively two begomoviruses, TYLCV
followed by TYLCSV. W hile the kinetics of
TYLCSV acquisition was similar to that of TYLCV,
the amount of TYLCV acquired during the first
feeding remained approximately constant (Fig. 4).
If B. tabaci does not possess a different set of
receptors for TYLCV and TYLCSV, the results may
be explained by the progressive displacement of
TYLCV by TYLCSV. According to this hypothesis,
TYLCSV binds to the B. tabaci receptors previously
occupied by TYLCV, and most of the TYLCV
invades unidentified insect tissues and leaves the
circulative pathway, although enough TYLCV
remains in the digestive tract to allow co-infection
of tomato plants along with TYLCSV.
The pattern of long-term association of the nontransmissible AbMV with B. tabaci also may be
interpretated in the light of the invasion-storage
hypothesis. We speculate that following acquisition,
AbMV binds to the putative B. tabaci receptors
present in part of the digestive tract. However
because of a change in the structure of the capsid
due to mutations in the CP (Wu et al., 1996; Höhnle
et al., 2001), AbMV cannot be internalised in the
epithelial cells by the microvilli system and delivered
to the haemolymph. With time, the virions may leave
the receptors, invade insect tissues in which the viral
genome is protected and the CP progressively
destroyed (Fig. 6).
The persistence of begomoviruses in B. tabaci as
infective entities for longer than the latent period,
sometimes for the entire life of the insect, raises the
question of replication of the virus in the insect.
Currently, it is postulated that geminiviruses do not
replicate in their insect vectors (Harrison, 1985).
However, data showing accumulation of TYLCV
DNA in B. tabaci reared on a TYLCV non-host plant,
after first feeding on a TYLCV-infected plant,
suggested multiplication of TYLCV in its vector
(Mehta et al., 1994). Begomovirus DNA acquired
over 24-48 h remains associated with the insect for
several weeks, much longer than infectivity (Caciagli
& Bosco, 1997; Rubinstein & Czosnek, 1997;
Muniyappa et al., 2000). Persistence of the viral
DNA may suggest a certain level of replication or
some association beyond what is expected of a
circulative transmission model. Long-term retention
of TYLCV, negative effects on longevity and
fecundity of the host (Rubinstein & Czosnek, 1997),
and transmission to eggs (Ghanim et al., 1998;
Polston et al., 2001) supports this hypothesis since
deleterious effects on the insect and transovarial
transmission are all characteristics associated with
replication of a plant virus in its insect vector
(Sylvester & Richardson, 1969; Sylvester, 1973;
Gingery, 1988). The recently developed continuous
whitefly cell line originating from B. tabaci
embryonic tissues might provide a tool to study
putative begomovirus replication and expression
(Hunter & Polston, 2001).
References
Antignus Y, Perlsman M, Ben-Yoseph R, Cohen S. 1993. The
interaction of Tomato yellow leaf curl virus with its whitefly
vector, Bemisia tabaci. Phytoparasitica 21:174-175.
Atzmon G, van Hoss H, Czosnek H. 1998. PCR-amplification
of tomato yellow leaf curl virus (TYLCV) from squashes of
plants and insect vectors: application to the study of TYLCV
acquisition and transmission. European Journal of Plant
Pathology 104:189-194.
Azzam O, Frazer J, Delarosa D, Beaver J S, Ahlquist P,
Maxwell D P. 1994. Whitefly transmission and efficient
ssDNA accumulation of bean golden mosaic geminivirus
require functional coat protein. Virology 204:289-296.
Whitefly transmission of begomoviruses
Bandla M D, Campbell L R, Ullman D E, Sherwood J L.
1998. Interaction of Tomato spotted wilt tospovirus (TSWV)
glycoproteins with a thrips midgut protein, a potential cellular
receptor for TSWV. Phytopathology 88:98-104.
Baumann P, Munson M A, Lai C-Y, Clark M A, Baumann
L, Moran N A, Campbell B C. 1993. Origin and properties
of bacterial endosymbionts of aphids, whiteflies, and
mealybugs. ASM News 5:21-24.
Bedford I D, Briddon R W, Brown J K, Rosell R C,
Markham P G. 1994. Geminivirus transmission and
biological characterisation of Bemisia tabaci (Gennadius)
biotypes from different geographic regions. Annals of Applied
Biology 125:311-325.
Bejarano E R, Khashoggi A, Witty M, Lichtenstein C P. 1996.
Integration of multiple repeats of geminiviral DNA into the
nuclear genome of tobacco during evolution. Proceedings of
the National Academy of Sciences USA 93:759-764.
Bird J, Maramorosch K. 1978. Viruses and virus diseases
associated with whiteflies. Advances in Virus Research 22:55110.
Bradeen J M, Timmermans M C P, Messing J. 1997. Dynamic
genome organization and gene evolution by positive selection
in geminivirus (Geminiviridae). Molecular Biology and
Evolution 14:1114-1124.
Briddon R W, Pinner M S, Stanley J, Markham P G. 1990.
Geminivirus coat protein gene replacement alters insect
specificity. Virology 177:85-94.
Brown J K. 2001. Molecular markers for the identification and
global tracking of whitefly vector-begomovirus complexes.
Virus Research 71:233-260.
Brown J K, Czosnek H. 2002. Whitefly transmission of plant
viruses. Botanical Research 36:In press.
Brown J K, Frohlich D R, Rosell R C. 1995. The sweetpotato
or silverleaf whiteflies: Biotypes of Bemisia tabaci or a
species complex? Annual Review of Entomology 40:511-534.
Byrne D N, Bellows T S Jr. 1991. Whitefly biology. Annual
Review of Entomology 36:431-457.
Caciagli P, Bosco D. 1997. Quantitation over time of tomato
yellow leaf curl geminivirus DNA in its whitefly vector.
Phytopathology 87:610-613.
Caciagli P, Bosco D, Al-Bitar L. 1995. Relationships of the
Sardinian isolate of tomato yellow leaf curl geminivirus with
its whitefly vector Bemisia tabaci Gen. European Journal of
Plant Pathology 101:163-170.
Chapman R F. 1991. The Insects. Structure and Function, 3rd
Edn. London: Edward Arnold. 919 pp.
Cicero, J M, Hiebert E, Webb S E. 1995. The alimentary canal
of Bemisia tabaci and Trialeurodes abutilonea (Homoptera,
Sternorrhynchi): histology, ultrastructure and correlation to
function. Zoomorphology 115:31-39.
Cohen S. 1967. The occurrence in the body of Bemisia tabaci
of a factor apparently related to the phenomenon of “periodic
acquisition” of tomato yellow leaf curl virus. Virology 31:180183.
Cohen S, Harpaz I. 1964. Periodic, rather than continual
acquisition of a new tomato virus by its vector, the tobacco
w hitefly (Bemisia tabaci G ennadius). Entomologia
experimentalis et Applicata 7:155-166.
Cohen S, Marco S. 1970. Periodic occurrence of an anti-TMV
factor in the body of whiteflies carrying the tomato yellow
leaf curl virus (TYLCV). Virology 40:363-368.
Cohen S, Nitzany F E. 1966. Transmission and host range of
the tomato yellow leaf curl virus. Phytopathology 56:11271131.
Cohen S, Duffus J E, Liu H Y. 1989. Acquisition, interference,
and retention of cucurbit leaf curl viruses in whiteflies.
Phytopathology 79:109-113.
Cohen S, Duffus J E, Larsen R C, Liu H Y, Flock R A. 1983.
Purification, serology, and vector relationships of Squash leaf
curl virus, a whitefly-transmitted geminivirus.
229
Phytopathology 73:1669-1673.
Costa A S. 1969. Whiteflies as virus vectors. In Viruses, Vectors,
and Vegetation, pp. 95-119. Ed. K Maramorosch. New York:
Interscience Press.
Costa H S, Westcot D M, Ullman D E, Rosell R C, Brown J
K, Johnson M W. 1995. Morphological variation in Bemisia
endosymbionts. Protoplasma 189:194-202.
Czosnek H, Laterrot H. 1997. A worldwide survey of tomato
yellow leaf curl viruses. Archives of Virology 142:1391-1406.
Czosnek H, Ghanim H, Morin S, Rubinstein G, Fridman V,
Zeidan M. 2001. Whiteflies: vectors, and victims (?), of
geminiviruses. Advances in Virus Research 56:291-322.
Fauquet C M, Maxwell D P, Gronenborn B, Stanley J. 2000.
Revised proposal for naming geminiviruses. Archives of
Virology 145:1743-1761.
Francki R I B, Hatta T, Boccardo G, Randles J W. 1980. The
composition of chlorotis striate mosaic virus, a geminivirus.
Virology 101:233-241.
Frischmuth T, Zimmat G, Jeske H. 1990. The nucleotide
sequence of the Abutilon mosaic virus reveals prokaryotic as
well as eukaryotic features. Virology 178:461-468.
Frohlich D, Torres-Jerez I, Bedford I D, Markham P G,
Brown J K. 1999. A phylogeographic analysis of the Bemisia
tabaci species complex based on mitochondrial DNA
markers. Molecular Ecology 8:1593-1602.
Ghanim M, Czosnek H. 2000. Tomato yellow leaf curl
geminivirus (TYLCV-Is) is transmitted among whiteflies
(Bemisia tabaci) in a sex-related manner. Journal of Virology
74:4738-4745.
Ghanim M, Morin S, Czosnek H. 2001a. Rate of Tomato
yellow leaf curl virus (TYLCV) translocation in the circulative
transmission pathway of its vector, the whitefly Bemisia
tabaci. Phytopathology 91:188-196.
Ghanim M, Morin S, Zeidan M, Czosnek H. 1998. Evidence
for transovarial transmission of tomato yellow leaf curl virus
by its vector, the whitefly Bemisia tabaci. Virology 240:295303.
Ghanim M, Rosell R C, Campbell L R, Czosnek H, Brown J
K, Ullman D E. 2001b. Digestive, salivary and reproductive
organs of Bemisia tabaci (Gennadius) (Hem iptera:
Aleyrodidae) biotype B. Journal of Morphology 248:22-40.
Gibbs M. 1999. Chaperonin camouflage. Nature 399:415.
Gildow F E. 1993. Evidence for receptor-mediated endocytosis
regulating luteovirus acquisition by aphids. Phytopathology
83:270-277.
Gildow F E, Damsteegt V D, Stone A L, Smith O P, Gray S
M . 2000. Virus-vector cell interactions regulating
transmission specificity of Soybean dwarf luteoviruses.
Journal of Phytopathology 148:333-342.
Gingery R E. 1988. The rice stripe virus group. In The Plant
Viruses: The Filamentous Plant Viruses, Vol 4, pp. 297-329.
Ed. R G Milne. New York: Academic Press.
Goodman R M. 1977. Single-stranded DNA genome in a
whitefly-transmitted plant virus. Virology 83:171-179.
Harris K F, Pesic-Van Esbroeck Z, Duffus J E. 1995.
Anatomy of a virus vector. In Bemisia 1995: Taxonomy,
Biology, Damage, Control and Management, pp. 289-318.
Eds D Gerling and R Mayer. Andover, Bucks, UK: Intercept.
Harris K F, Pesic-Van Esbroeck Z, Duffus J E. 1996.
Morphology of the sweet potato whitefly, Bemisia tabaci
(Homoptera: Aleyrodidae) relative to virus transmission.
Zoomorphology 116:143-156.
Harrison B D. 1985. Advances in geminivirus research. Annual
Review of Phytopathology 23:55-82.
Harrison B D, Robinson, D J. 1999. Natural genomic and
antigenic variation in whitefly-transmitted geminiviruses
(begomoviruses). Annual Review of Phytopathology 37:369398.
Harrison B D, Barker H, Bock K R, Guthrie E J, Meredith
G, Atkinson M. 1977. Plant viruses with circular single-
230
HENRYK CZOSNEK ET AL.
stranded DNA. Nature 270:760-762.
Höfer P, Bedford I D, Markham P G, Jeske H, Frischmuth
T. 1997. Coat protein gene replacement results in whitefly
transmission of an insect non-transmissible geminivirus
isolate. Virology 236:288-295.
Höhnle M, Höfer P, Bedford I D, Briddon R W, Markham P
G, Frischmuth T. 2001. Exchange of three amino acids in
the coat protein results in efficient whitefly transmission of a
nontransmissible Abutilon mosaic virus isolate. Virology
290:164-171.
Horowitz A R, Gerling D. 1992. Seasonal variations of sex
ratio in Bemisia tabaci on cotton in Israel. Environmental
Entomology 21:556-559.
Hunter W, Hiebert E, Webb S E, Polston J E, Tsai H T. 1996.
Precibarial and cibarial chemosensilla in the whitefly, Bemisia
tabaci (Gennadius) (Homoptera: Aleyrodidae). International
Journal of Insect Morphology and Embryology 25:295-304.
Hunter W B, Polston J E. 2001. Development of a continuous
whitefly cell line [Homoptera: Aleyrodidae: Bemisia tabaci
(Gennadius)] for the study of begomovirus. Journal of
Invertebrate Pathology 77:33-36.
Hunter W B, Hiebert E, Webb S E, Tsai J H, Polston J E.
1998. Location of geminiviruses in the whitefly Bemisia
tabaci (Homoptera: Aleyrodidae). Plant Disease 82:11471151.
Ingham D J, Pascal E, Lazarowitz S G. 1995. Both bipartite
geminivirus movement proteins define viral host range, but
only BL1 determines viral pathogenicity. Virology 207:191204.
Ioannou N. 1985. Yellow leaf curl and other diseases of tomato
in Cyprus. Plant Pathology 345:428-434.
Jiang Y X, De Blas C, Barrios L, Fereres A. 2000. A correlation
between whitefly (Homoptera: Aleyrodidae) feeding behavior
and transmission of Tomato yellow leaf curl virus. Annals of
the Entomological Society of America 93:573-579.
Jupin I, De Kouchkovsky F, Jouanneau F, Gronenborn B.
1994. Movement of tomato yellow leaf curl geminivirus
(TYLCV): involvement of the protein encoded by ORF C4.
Virology 204:82-90.
Kheyr-Pour A, Bendahmane M, Matzeit V, Accotto G P,
Crespi S, Gronenborn B. 1991. Tomato yellow leaf curl
virus from Sardinia is a whitefly-transmitted monopartite
geminivirus. Nucleic Acids Research 19:6763-6769.
Kheyr-Pour A, Bananej K, Dafalla G A, Caciagli P, Noris E,
A hoonm anesh A, Lecoq H, G ronenborn B. 2000.
Watermelon chlorotic stunt virus from the Sudan and Iran:
Sequence comparisons and identification of a whiteflytransmission determinant. Phytopathology 90:629-635.
Laufs J, Traut W, Heyraud F, Matzeit V, Rogers S G, Schell
J, Gronenborn B. 1995. In vitro cleavage and joining at the
viral origin of replication by the replication initiator protein
of tomato yellow leaf curl virus. Proceedings of the National
Academy of Sciences USA 92:3879-3883.
Li C Y, Cox-Foster D, Gray S M, Gildow F. 2001. Vector
specificity of barley yellow dwarf virus (BY DV)
transmission: Identification of potential cellular receptors
binding BYDV-MAV in the aphid, Sitobion avenae. Virology
286:125-133.
Liu S, Bedford I D, Briddon R W, Markham P G. 1997.
Efficient whitefly transmission of African cassava mosaic
geminivirus requires sequences from both genomic
components. Journal of General Virology 78:1791-1794.
Mansour A, Al-Musa A. 1992. Tomato yellow leaf curl virus:
host range and vector-virus relationships. Plant Pathology
41:122-125.
McGrath P F, Harrison B D. 1995. Transmission of tomato
leaf curl geminiviruses by Bemisia tabaci - effects of virus
isolate and vector biotype. Annals of Applied Biology
126:307-316.
Mehta P, Wyman J A, Nakhla M K, Maxwell D P. 1994.
Transmission of tomato yellow leaf curl geminivirus by
Bemisia tabaci (Homoptera: Aleyrodidae). Journal of
Economical Entomology 87:1291-1297.
Michelson I, Zeidan M, Zamski E, Zamir D, Czosnek H.
1997. Localization of Tomato yellow leaf curl virus (TYLCV)
in susceptible and tolerant nearly isogenic tomato lines. Acta
Horticulturae 447:407-414.
Morin S, Ghanim M, Sobol I, Czosnek H. 2000. The GroEL
protein of the whitefly Bemisia tabaci interacts with the coat
protein of transmissible and non-transmissible begomoviruses
in the yeast two-hybrid system. Virology 276:404-416.
Morin S, Ghanim M, Zeidan M, Czosnek H, Verbeek M,
van den Heuvel J F J M. 1999. A GroEL homologue from
endosymbiotic bacteria of the whitefly Bemisia tabaci is
implicated in the circulative transmission of Tomato yellow
leaf curl virus. Virology 30:75-84.
Muniyappa V, Venkatesh H M, Ramappa H K, Kulkarni R
S, Zeidan M, Tarba C-Y, Ghanim M, Czosnek H. 2000.
Tomato leaf curl virus from Bangalore (ToLCV-Ban4):
sequence comparison with Indian ToLCV isolates, detection
in plants and insects, and vector relationships. Archives of
Virology 145:1583-1598.
Nakhla M K, Maxwell D P. 1998. Epidemiology and
management of tomato yellow leaf curl disease. In Plant Virus
Disease Control, pp. 565-583. Eds A Hadidi, R K Khetarpal
and H Koganezawa. St Paul, Minnesota: APS Press, The
American Phytopathological Society.
Nault L R. 1997. Arthropod transmission of plant viruses: a
new synthesis. Annals of the Entomological Society of
America 90:521-541.
Navot N, Pichersky E, Zeidan M, Zamir D, Czosnek H. 1991.
Tomato yellow leaf curl virus: a whitefly-transmitted
geminivirus with a single genomic component. Virology
185:151-161.
Navot N, Zeidan M, Pichersky E, Zamir D, Czosnek H. 1992.
Use of polymerase chain reaction to amplify tomato yellow
leaf curl virus DNA from infected plants and viruliferous
whiteflies. Phytopathology. 82:1199-1202.
Noris E, Vaira A M, Caciagli P, Masenga V, Gronenborn B,
Accotto G P. 1998. Amino acids in the capsid protein of
tomato yellow leaf curl virus that are crucial for systemic
infection, particle formation, and insect transmission. Journal
of Virology 72:10050-10057.
Noueiry A O, Lucas W J, Gilbertson R L. 1994. Two proteins
of a plant DNA virus coordinate nuclear and plasmodesmatal
transport. Cell 76:1-20.
Padidam M, Beachy R N, Fauquet C M. 1995. Classification
and identification of geminiviruses using sequence
comparisons. Journal of General Virology 76:249-263.
Padidam M, Sawyer S, Fauquet C M. 1999. Possible
emergence of new geminiviruses by frequent recombination.
Virology 265:218-225.
Peiffer M L, Gildow F E, Gray S M. 1997. Two distinct
mechanisms regulate luteovirus transmission efficiency and
specificity at the aphid salivary gland. Journal of General
Virology 78:495-503.
Perring T M, Cooper A D, Rodriguez R J, Farrar C A J,
Bellows T S J. 1993. Identification of a whitefly species by
genomic and behavioral studies. Science 259:74-77.
Picó B, Diez M J, Nuez F. 1996. Viral diseases causing the
greatest economic losses to tomato crop. II. The tomato yellow
leaf curl virus - a review. Scientia Horticulturae 67:151-196.
Pollard D G. 1955. Feeding habits of the cotton whitefly. Annals
of Applied Biology 43:664-671.
Polston J E, Al-Musa A, Perring T M, Dodds J A. 1990.
Association of the nucleic acid of squash leaf curl geminivirus
with the whitefly Bemisia tabaci. Phytopathology 80:850856.
Polston J E, Sherwood T, Rosell R, Nava A. 2001. Detection
of tomato yellow leaf curl and tomato mottle virus in
Whitefly transmission of begomoviruses
developmental stages of the whitefly vector, Bemisia tabaci.
Third International Geminivirus Symposium, John Innes
Centre, Norwich, UK, 24-28 July 2001, Abstract 81.
Power A G. 2000. Insect transmission of plant viruses: a
constraint on virus variability. Current Opinion in Plant
Biology 3:336-340.
Reddy K S, Yaraguntaiah R C. 1981. Virus-vector relationship
in leaf curl disease of tomato. Indian Phytopathology 34:310313.
Rosell R, Lichty J E, Brown J K. 1995. Ultrastructure of the
mouthparts of adult sweetpotato whitefly, Bemisia tabaci
(Gennadius) (Homoptera: Aleyrodidae). International
Journal of Insect Morphology and Embryology 24:297-306.
Rosell R C, Torres-Jerez I, Brown J K. 1999. Tracing the
geminivirus-whitefly transmission pathway by polymerase
chain reaction in whitefly extracts, saliva, hemolymph, and
honeydew. Phytopathology 89:239-246.
Rubinstein G, Czosnek H. 1997. Long-term association of
tomato yellow leaf curl virus (TYLCV) with its whitefly
vector Bemisia tabaci: effect on the insect transmission
capacity, longevity and fecundity. Journal of General Virology
78:2683-2689.
Rybicki E P. 1994. A phylogenetic and evolutionary justification
for three genera of Geminiviridae. Archives of Virology
139:49-77.
Sanderfoot A A, Ingham D J, Lazarowitz S G. 1996. A viral
movement protein as a nuclear shuttle: the geminivirus BR1
movement protein contains domains essential for interaction
with BL1 and nuclear localization. Plant Physiology 110:111.
Sanz A I, Fraile A, Gallego J M, Malpica J M, Garcia-Arenal
F. 1999. Genetic variability of natural populations of cotton
leaf curl geminivirus, a single-stranded DNA virus. Journal
of Molecular Evolution 49:672-681.
Sylvester E S. 1973. Reduction of excretion, reproduction, and
survival in Hyperomyzus lactucae fed on plants infected with
isolates of sowthistle yellow vein virus. Virology 56:632-635.
Sylvester E S, Richardson J. 1969. Additional evidence of
multiplication of the sowthistle yellow vein virus in an aphid
vector - serial passage. Virology 37:26-31.
Ullman D E, Cho J J, Mau R F L, Wescot D M, Custer D M.
1992. A midgut barrier to Tomato spotted wilt virus
acquisition by adult western flower thrips. Phytopathology
82:1333-1342.
231
van den Heuvel J, Verbeek M, van der Wilk F. 1994.
Endosymbiotic bacteria associated with circulative
transmission of potato leafroll virus by Myzus persicae.
Journal of General Virology 75:2559-2565.
van Regenmortel M H V, Fauquet C M, Bishop D H L,
Carstens E B, Estes M K, Lemon S M, Maniloff J, Mayo
M A, McGeoch D J, Pringle C R, Wickner R B. 2000.
Virus Taxonomy: The Classification and Nomenclature of
Viruses. The Seventh Report of the International Committee
on Taxonomy of Viruses. San Diego: Academic Press. 1167
pp.
von Arnim A, Stanley J. 1992. Determinants of tomato golden
mosaic virus symptom development located on DNA B.
Virology 186:286-293.
Walker G P, Perring T M. 1994. Feeding and oviposition
behavior of whiteflies (Homoptera: Aleyrodidae) interpreted
from AC electronic feeding monitor waveforms. Annals of
the Entomological Society of America 87:363-374.
Wartig L, Kheyr-Pour A, Noris E, de Kouchkovsky F,
Jouanneau F, Gronenborn B, Jupin I. 1997. Genetic
analysis of the monopartite tomato yellow leaf curl
geminivirus. Roles of V1, V2, and C2 ORFs in viral
pathogenesis. Virology 228:132-140.
Weber H. 1935. Der bau der imago der Aleurodinen. Zoologica
89:1-71.
Wescot D M, Ullman D E, Sherwood J L, Cantone F A,
German T L. 1993. Rapid fixation and embedding method
for immunochemical studies of tomato spotted wilt tospovirus
(TSWV) in plant and insect tissues. Microscopy Research
and Technique 24:514-520.
Wu Z C, Hu J S, Polston, J E, Ullman D E, Hiebert E. 1996.
Complete nucleotide sequence of a nonvector-transmissible
strain of Abutilon mosaic gem inivirus in Hawaii.
Phytopathology 86:608-613.
Zeidan M, Czosnek H. 1991. Acquisition of tomato yellow
leaf curl virus by the whitefly Bemisia tabaci. Journal of
General Virology 72:2607-2614.
Zhang W, Olson N H, Baker T S, Faulkner L, AgbandjeMcKenna M, Boulton M I, Davies J W, McKenna R. 2001.
Structure of the Maize streak virus geminate particle. Virology
279:471-477.