Mealybug Transmission of Grapevine Leafroll Viruses:
An Analysis of Virus–Vector Specificity
Chi-Wei Tsai, Adib Rowhani, Deborah A. Golino, Kent M. Daane, and Rodrigo P. P. Almeida
First author: Department of Entomology, National Taiwan University, Taipei 106, Taiwan; second and third authors: Department of Plant
Pathology, University of California, Davis 95616; and first, fourth, and fifth authors: Department of Environmental Science, Policy and
Management, University of California, Berkeley 94720.
Accepted for publication 3 April 2010.
Tsai, C.-W., Rowhani, A., Golino, D. A., Daane, K. M., and Almeida, R.
P. P. 2010. Mealybug transmission of grapevine leafroll viruses: An
analysis of virus–vector specificity. Phytopathology 100:830-834.
To understand ecological factors mediating the spread of insect-borne
plant pathogens, vector species for these pathogens need to be identified.
Grapevine leafroll disease is caused by a complex of phylogenetically
related closteroviruses, some of which are transmitted by insect vectors;
however, the specificities of these complex virus–vector interactions are
poorly understood thus far. Through biological assays and phylogenetic
analyses, we studied the role of vector-pathogen specificity in the
transmission of several grapevine leafroll-associated viruses (GLRaVs)
by their mealybug vectors. Using plants with multiple virus infections,
several virus species were screened for vector transmission by the mealy-
bug species Planococcus ficus and Pseudococcus longispinus. We report
that two GLRaVs (-4 and -9), for which no vector transmission evidence
was available, are mealybug-borne. The analyses performed indicated no
evidence of mealybug–GLRaV specificity; for example, different vector
species transmitted GLRaV-3 and one vector species, Planococcus ficus,
transmitted five GLRaVs. Based on available data, there is no compelling
evidence of vector–virus specificity in the mealybug transmission of
GLRaVs. However, more studies aimed at increasing the number of
mealybug species tested as vectors of different GLRaVs are necessary.
This is especially important given the increasing importance of grapevine
leafroll disease spread by mealybugs in vineyards worldwide.
Additional keywords: Ampelovirus, Closteroviridae, semipersistent.
The majority of plant viruses are vector-borne. Arthropods,
nematodes, and fungi transmit 76% of plant viruses; among these,
the most important group of vectors are sap-sucking insects such
as aphids which, altogether, transmit 55% of described plant
viruses (16,23). The modes of pathogen transmission are generally
classified based on characteristics of virus–vector interactions;
viruses that replicate within insects are transmitted in a propaga-
tive manner and those that do not replicate are nonpersistently,
semipersistently, or circulatively (persistently) transmitted (16,24).
Virus–vector specificity, the ability of an insect species to trans-
mit a specific virus, varies substantially among vector-borne plant
viruses. However, a genus of plant viruses is usually transmitted
by vectors from one family of insects and has the same trans-
mission mode, suggesting long-term evolutionary relationships
between virus and vector (23). This characteristic of insect-borne
plant viruses is consistent across taxa, so much so that virus
taxonomy may be partially proposed based on the type of vector
The virus family Closteroviridae is composed of several insect-
borne plant viruses of economic importance, including Citrus
tristeza virus, Grapevine leafroll-associated virus-3 (GLRaV-3),
and Tomato chlorosis virus, among others. The family Clostero-
viridae is subdivided into three genera: Closterovirus, Ampelo-
virus, and Crinivirus (21). These are large, positive-sense, single-
stranded RNA viruses with genomes of 15.3 to 19.5 kb in size and
virions of 650 to 2,200 nm in length; in addition, the overall
structure of the linear genome of closteroviruses is similar (6,21).
Among shared molecular characteristics, viruses in the family
Closteroviridae have a unique heat-shock protein homologue that
is useful for across-taxa phylogenetic analysis due to its con-
served nature (6). Vectors of many closteroviruses have been
identified. Members of the genus Closterovirus have been shown
to be aphid-borne, while ampeloviruses are mealybug and soft
scale-borne and criniviruses are transmitted by whiteflies (18,21).
Current evidence indicates that the level of vector–pathogen
specificity for different virus species is variable for the family
Closteroviridae; some viruses have a wide vector range and
others have been demonstrated to be transmitted by only one
insect species (18).
The identification of vector species for insect-borne plant
viruses and the level of vector specificity among these inter-
actions are of epidemiological importance (23). The recognition
of trends in transmission biology provides information that drives
research in poorly characterized systems because transmission
characteristics (not efficiency) are generally shared among viruses
belonging to the same family, genus, or species. Among clostero-
viruses, species in the genus Ampelovirus are especially under-
studied in relation to transmission biology, with the exception of
pineapple mealybug wilt-associated viruses and GLRaV-3, for
which some information is available (4,8,28,29,31).
GLRaVs cause grapevine leafroll disease as a virus complex,
with several viruses sequentially named GLRaV-1, GLRaV-2,
GLRaV-3, and so on. All members of this virus complex belong
to the genus Ampelovirus, with the exception of GLRaV-2 (Clos-
terovirus) and GLRaV-7 (unassigned genus). Leafroll disease has
become an emerging problem in different grape-growing regions
of the world, with awareness about the disease increasing after
Corresponding author: R. P. P. Almeida;
E-mail address: firstname.lastname@example.org
*The e-Xtra logo stands for “electronic extra” and indicates that the online version
contains two supplemental articles and one table not included in the print version.
Figure 2 appears in color online.
© 2010 The American Phytopathological Society
Vol. 100, No. 8, 2010 831
this virus complex, once thought to be only graft transmissible,
was found to be spreading within vineyards (3,11,13,14). Mealy-
bugs were first shown to transmit Ampelovirus spp. in 1990 (8).
Since then, some mealybug (Pseudococcidae) and soft-scale
(Coccidae) species have been shown to transmit different GLRaVs
(1,4,8,28,30). Transmission of GLRaVs, based on a limited
number of studies, seems to occur in a semipersistent manner
In California, where evidence of leafroll disease spread is
recent (11), four mealybug species have been associated with
grapevines for decades, including Pseudococcus longispinus; while
one, Planococcus ficus, was introduced into the state in 1994 (5).
Direct damage to grapevines due to these mealybug species is
primarily associated with infestation of the fruit clusters and
growth of sooty molds as a consequence of honeydew excretion.
Populations of these species are often kept low due to insecticide
applications and natural enemies. However, these approaches are
unlikely to be successful in reducing mealybug-vector popula-
tions low enough to limit GLRaVs spread. The species used in
this study, include Planococcus ficus, the sole Planococcus sp.
important in California vineyards, and Pseudococcus longispinus,
which is representative of three species in the Pseudococcus–
maritimus complex. Planococcus ficus is present in most grape-
growing regions and Pseudococcus longispinus is found in cooler
coastal regions where GLRaVs may cause greater damage.
Despite growing interest in the biology, ecology, and transmis-
sion of closteroviruses associated with grapevine leafroll disease,
the degree of vector specificity has not been analyzed for this
virus complex. Here, we present data showing that two species of
mealybugs transmit different GLRaV species; two of these
ampeloviruses have not been previously reported to be vector-
borne. In addition, we discuss the degree of vector specificity
among mealybugs and GLRaVs in the genus Ampelovirus.
MATERIALS AND METHODS
Screening for Planococcus ficus-transmissible viruses.
Planococcus ficus colonies were established on butternut squash
(Cucurbita moschata) from single females collected in vineyards
near Del Rey, CA. Colonies were maintained in a growth chamber
at 22 ± 2°C, with a 12:12-h photoperiod. First instars were used in
all experiments because this life stage was shown previously to be
the most efficient vector of GLRaV-3 (31). Virus source vines
used for this study and their infection status are listed in Table 1.
We collected the source vines from a virus collection at the
University of California (UC) Davis (9) in the fall and brought
them to the UC Berkeley for transmission tests. The source vines
were rinsed with water to remove dust and blotted dry with Kim-
wipe papers. Healthy test plants used for mealybug transmission
assays were rooted from dormant cuttings of grapevine cv.
Cabernet Franc obtained from the Foundation Plant Services
(FPS) at UC Davis, and were grown in a greenhouse until they
reached ≈20 cm tall with approximately six expanded leaves. We
used Cabernet Franc because it is a good biological indicator for
leafroll disease (27). All experiments had greenhouse negative
controls, grapevines from the same batch of test plants that were
not exposed to mealybug vectors.
In the first experiment conducted, mealybugs were allowed to
move onto source vine cuttings (20 cm) laid on mealybug
colonies. After 2 h, the cuttings were removed from the mealybug
colonies and maintained in flasks of water. After an acquisition
access period (AAP) of 24 h, insects were gently shaken off the
source tissue onto paper disks (0.5 cm in diameter). Potentially
viruliferous mealybugs were transferred on paper disks to healthy
test plants by caging the insects on leaf blades using clip cages
previously described (31). For each treatment, 10 plants were
inoculated with groups of 5 mealybugs and another 10 plants with
groups of 20 individuals. Therefore, each accession number (i.e.,
virus source tissue) was used as source material for 20 insect-
group inoculation events. After a 24-h inoculation access period
(IAP), mealybugs were removed from the test plants with a fine
brush. These plants were then treated with an insecticide. The test
plants were maintained in a greenhouse and sprayed with
insecticide and fungicide until tested for viruses. We pruned all
plants periodically to avoid overgrowth. The plants in this experi-
ment were inoculated by mealybugs in October 2007 and then
went through the winter in a light-supplemented greenhouse.
Petiole samples were harvested in April and August 2008 for
reverse-transcription polymerase chain reaction (RT-PCR)-based
detection of grapevine viruses (described below).
Following results from the first experiment, all nontrans-
missible virus accessions were tested again; a similar protocol
was used to screen these virus sources. In this experiment, the
number of mealybugs per group was increased and longer AAP
and IAP were used to maximize transmission rates. Fifty mealy-
bugs were transferred to each of 10 healthy test plants for an AAP
and IAP of 48 h each. Test plant maintenance and pesticide
spraying were the same as described above. The plants from this
TABLE 1. Summary of transmission experiments performed with two mealybug species, Planococcus ficus and Pseudococcus longispinus
Source planta Viruses in source plantsb Virus detectedc 5 insects 20 insects 50 insects
GLRaV-1, -2, -5, RSP, GVB
GLRaV-2, -3, RSP, GFkV
GLRaV-2, -4, GFkV
GLRaV-1, -2, -7, GFkV, GVB
GLRaV-2, -4, -5, RSP, GFkV, GVB
GLRaV-1, -5, GVA, GFkV
a Source plant material was from a virus collection at the University of California, Davis.
associated virus; GFkV, Grapevine fleck virus. Plants were not tested for RSP and GFkV.
c Virus detected in test plants. Infection of viruses in the test plants was assayed by reverse-transcription polymerase chain reaction.
d Transmission rate is based on the proportion of plants inoculated with GLRaV by groups of 5, 20, or 50 individual mealybugs. Number of positive plants/number
of test plants for groups of 5, 20, and 50 mealybugs used as vectors during the inoculation access period. Ten to twenty mealybugs were used for the Pseudo-
coccus longispinus trials.
GLRaV, Grapevine leafroll-associated virus; GVA, Grapevine virus A; GVB, Grapevine virus B; RSP, Grapevine rupestris stem pitting-
trial were inoculated by mealybugs in September 2008 and then
went through the winter in a light-supplemented greenhouse.
Petiole samples were harvested in April 2009 for virus detection.
In both trials, mealybug transmission from healthy grapevines to
healthy test plants and test plants with no mealybug inoculations
were used as controls, ensuring that our mealybug colonies were
not naturally infected and that virus spread had not occurred
within the greenhouse during experimental periods.
viruses. Two sources of Pseudococcus longispinus were used for
this experiment: one from a vineyard in San Luis Obispo, CA and
the other from a natural infestation in a greenhouse on the UC
Davis campus. Colonies were established with single adult
females; mealybugs were maintained on sprouted potato tubers
(Solanum tuberosum). GLRaV sources were also obtained from a
virus collection at UC Davis (9). Virus accessions LR100 and
LR118, which are infected with GLRaV-5 and GLRaV-9, respec-
tively, were used as sources. The vines were rooted and trans-
planted to 4-liter pots in a greenhouse until they were ≈1.5 m tall.
Pseudococcus longispinus individuals (mixed-life stages) were
allowed a 2-week AAP on virus source plants. One-node rooted
cuttings of healthy grapevines (cv. Cabernet Franc) were used as
test plants. After a 2-week AAP, leaves of the mealybug-infected
source plants were cut into sections and arranged on healthy test
plants to allow insects to crawl off as the leaf dried. In all, ≈10 to
20 mealybugs were observed feeding on each test plant; a total of
74 plants was infested with potentially viruliferous insects. After
a 2-week IAP, the test plants were sprayed with an insecticide to
kill all mealybugs. Pseudococcus longispinus individuals from
healthy grapevines and test plants with no mealybug inoculation
were used as controls. Test plant maintenance and pesticide spray-
ing were the same as described above. All plants were assayed at
9 months postinoculation using RT-PCR (see below for methods).
Virus detection. All samples were sent to the Whitter Labora-
tory in the Department of Veterinary Medicine at UC Davis for
total RNA extraction by the ABI 9600 automated RNA extraction
system. Before testing the samples for grapevine viruses, a
housekeeping control (NADH dehydrogenase subunit 5 gene) was
used to evaluate the quality of extracted RNA (22). If samples did
not pass this housekeeping test (i.e., NADH amplicon was not
amplified), we again extracted RNA from those plants using the
RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. All plants were tested for nine grape-
vine viruses by RT-PCR as previously described (26). Briefly,
viruses screened in this project included GLRaV-1, -2, -3, -4, -5,
-7, and -9, Grapevine virus A (GVA; genus Vitivirus), and Grape-
vine virus B (GVB; genus Vitivirus). The enzymes and reagents
used for RT-PCR were obtained from Invitrogen Life Technol-
ogies (Carlsbad, CA). The sequences for primers used for RT-
PCR have been published (26). The RT reaction was performed at
52°C for 30 min, followed by a PCR activation step at 94°C for
2 min; amplification of 35 cycles at 94°C for 30 s, 58°C for 45 s,
and 72°C for 1 min; and a final extension step at 72°C for 7 min.
Amplification products were analyzed by electrophoresis on 1.5%
agarose gels and visualized on a UV transilluminator following
ethidium bromide staining.
Virus transmission by Planococcus ficus. The results of virus
transmission experiments by Planococcus ficus are summarized
in Table 1. As expected, increasing the number of vectors during
the IAP increased transmission efficiency. GLRaV-1, -3, -4, -5,
and -9 were transmitted by Planococcus ficus; GLRaV-4 and -9
had not been shown to be vector-transmitted before. GLRaVs
from both single-infection accession numbers LR106 and LR118
(GLRaV-4 and GLRaV-9, respectively) were successfully trans-
mitted to healthy test vines (Table 1). Although accession number
LR109 was infected with multiple grapevine viruses, only
GLRaV-3 was transmitted from source vines to test plants. Acces-
sion numbers LV92-02 and LV92-04 were infected with multiple
grapevine viruses but not with GLRaV-3; however, the test plants
were positive for GLRaV-3. The reason for this inconsistency is
not clear. It might be due to in-greenhouse mealybug transmis-
sion, contaminated mealybug colonies, or contaminated test
plants. However, all of our greenhouse controls were negative for
all viruses tested. In addition, other transmission tests resulted
only in the transmission of viruses present in their respective
source plants. Therefore, we believe these source plants were
infected with GLRaV-3 but virus detection failed, possibly due to
a population level within plants that was too low for standard RT-
PCR detection. Accession numbers LR102, LR110, LR135, and
LV93-09 were also infected with multiple grapevine viruses but
none of these viruses was transmitted from source vines to test
plants with 5 or 20 Planococcus ficus individuals per plant (Table
1). When the number of vectors was increased to 50 individuals
per plant, Planococcus ficus transmitted GLRaV-1 and -5 from
LR102 to healthy test plants, and one test plant was infected with
both viruses (Table 1). In addition, Planococcus ficus also
transmitted GVA from LR135 to test plants (Table 1). Engelbrecht
and Kasdorf (8) showed that GVA was dependent on GLRaV-3
for its transmission by Planococcus ficus but we found that
Planococcus ficus transmitted GVA independently of GLRaV-3.
However, LR135 was co-infected with GLRaV-4, and we do not
know whether GLRaV-4 plays any role in GVA transmission. In
summary, Planococcus ficus transmitted GLRaV-1, -3, -4, -5, and
-9, and GVA.
Virus transmission by Pseudococcus longispinus. The results
of virus transmission by Pseudococcus longispinus are sum-
marized in Table 1. Nine months after inoculation, none of the
inoculated plants tested positive for GLRaV-5 and 18% of the
inoculated plants tested positive for GLRaV-9. As above, all
controls were negative for grape viruses tested.
Traditionally, studies aimed at the identification of vectors of
plant pathogens focus on a few insect species and strains of a
specific pathogen. These simple vector–pathogen combinations
are desirable because the research focus is often on an important
vector or pathogen. In addition, due to evolutionary constraints of
such systems, one insect is often more commonly found associ-
ated with a specific disease and the most likely vector of its
respective etiological agent (23). However, it is important to con-
sider that extensive within-vector and -virus species variation in
transmission efficiency has been documented for insect-borne
plant viruses (2,12,33). On the other hand, for complex systems
such as grapevine leafroll disease, which is caused by several
viruses transmitted by many insect species, screening all possible
individual combinations is technically challenging and labor
intensive (10). Here, an alternative approach for the identification
of new vector–virus relationships that resulted in pathogen trans-
mission to plants was employed by screening a large number of
viruses in multiply infected plants. We showed that GLRaV-4 and
GLRaV-9 are vector transmitted and mealybug-borne, which
represent new findings, with Planococcus ficus transmitting both
GLRaV-4 and -9 and Pseudococcus longispinus transmitting
GLRaV-9. Mealybug transmission of GVA was previously dem-
onstrated (8). These results suggest that this approach is helpful in
determining if viruses are vector-borne, and which of a complex
of viruses may be transmitted by a specific insect species. Con-
straints of this approach include the fact that multiple infections
must be available for studies and little information is generated in
addition to the identification of new virus–vector associations.
To visually evaluate the degree of mealybug–GLRaV specifi-
city, we reconstructed phylogenies for vector-transmitted GLRaVs
Vol. 100, No. 8, 2010 833
and its vector species (Supplementary Material). GLRaVs in the
genus Ampelovirus form two groups (Figs. 1 and 2), one com-
posed of GLRaV-1 and -3 and the other with all other species, as
previously reported (20). Despite the limited number of taxa used
in the mealybug phylogenetic tree, species were grouped similarly
to a more thorough analysis of this insect family (15). Impor-
tantly, overlaying experimental transmission data on these trees
highlighted a few aspects of virus–vector interactions in this
system. First, research seems biased toward GLRaV-3, potentially
because it has been shown to be the most important GLRaV in
many grape-growing regions. In this analysis, all mealybug
species transmitted GLRaV-3. In addition, there seems to be no
evidence of virus–vector specificity or co-evolution between virus
and vector (crossed, not parallel lines connecting taxa in both
trees). That is best evidenced by Planococcus ficus, which
transmits five GLRaVs. Finally, a few mealybug species tested
did not transmit GLRaV-1 (Supplementary Table 1); however, it is
not clear whether the lack of transmission was due to virus–vector
specificity or other factors, such as the use of nontransmissible
Many biological factors determine the successful vector trans-
mission of plant viruses. A given insect species proven to be a
vector of a given virus does not always transmit the virus.
Because of the nature of genetic variations among populations,
different biotypes of vector species and different isolates of a
given virus affect the results of transmission tests (16,24). The
virion concentration in diets has been shown to influence the
transmission efficiency of a closterovirus (25). Genetic variation
among insect or virus populations and low virus population with-
in source tissues may explain why some mealybug-transmissible
GLRaVs were not transmitted in our tests. In addition, the within-
plant distribution of GLRaVs in infected grapevines is uneven
(19), likely introducing variability in transmission studies. To-
gether, any of these factors could explain why, for example,
GLRaV-4 from only one virus source was transmitted when it was
present in other four sources. Furthermore, transmission of some
semipersistent viruses has been shown to depend on helper
viruses (24). For example, Parsnip yellow fleck virus is dependent
on Anthriscus yellows virus for its aphid transmission (7) and
Rice tungro badnavirus is dependent on Rice tungro waikavirus
for its leafhopper transmission (17). Although there is no evi-
dence that the transmission of ampeloviruses is dependent on
helper viruses, this may have been a factor in this study because
most source plants used were infected with multiple viruses.
Nault (23) proposed that plant viruses in the same genus share
vectors; for the family Closteroviridae, Karasev (18) suggested
that three genera exist within the family and each genus is trans-
mitted by a specific family of insects. The literature review and
phylogenetic analysis conducted here confirms the proposals
made by both authors (see Supplementary Material). All viruses
in the genera Closterovirus, Crinivirus, and Ampelovirus are
transmitted only by aphids, whiteflies, and mealybugs, respec-
tively (Fig. 2). The phylogenetic placement of the aphid-borne
Fig. 1. Maximum parsimony trees of grapevine ampeloviruses (left) and all mealybug species shown to transmit grapevine leafroll-associated viruses; asterisks
indicate >70% branch support. Lines indicate which virus species were transmitted by each mealybug vector; nv = nonvector species. Supplementary material
provides more detail.
Fig. 2. Maximum parsimony tree of virus species in the family Clostero-
viridae; asterisks indicate >70% branch support. Colored branches indicate
experimental evidence for virus transmission by a specific family of insect
vectors. Blue = aphids, Aphididae; red = whiteflies, Aleyrodidae; and green =
mealybugs, Pseudococcidae. Vectors that have not been identified for taxa are
in black. Supplementary Material provides more detail.
834 PHYTOPATHOLOGY Download full-text
Mint vein banding-associated virus within the family Clostero-
viridae is interesting, and is discussed in further detail in relation
to its aphid transmission by Tzanetakis et al. (32). The trans-
mission of GLRaV-1 and -3 by soft scales (Hemiptera, Coccidae),
a sister taxon of mealybugs, was not addressed in our study
(1,30). Among the eight Ampelovirus spp. (taxonomically accepted,
in addition to proposed GLRaV-10 and -11) causing grapevine
leafroll disease, five have now been shown to be vector-trans-
mitted (4,8,10,28,30, and this work). There are no reports on
GLRaV-6, -10, and -11 transmission; however, it is expected that
these are also mealybug-borne. These viruses were not included
in this study. GLRaV-2 and -7 were not transmitted in our trials
despite multiple assays, suggesting that these viruses are not
mealybug-borne. These results are consistent with the phylo-
genetic placement of both species (21) and confirm previous
reports in the case of GLRaV-2 (8).
The data obtained in our study suggest a general lack of
transmission specificity between mealybugs and GLRaVs; how-
ever, this observation is supported by a limited number of data
points, and more research on this topic is necessary. Due to the
fact that no comparative transmission studies have been per-
formed for any GLRaVs, there is no information available regard-
ing the relative efficiency with which these viruses are transmitted
or what factors affect competence. Furthermore, existing data on
transmission biology of GLRaVs are strongly biased toward
GLRaV-3, limiting meaningful comparisons in this respect. Fu-
ture work should also explore the specificity of Ampelovirus
transmission by soft scales. Finally, all closteroviruses for which
studies have explored multiple aspects of transmission biology
were transmitted in a semipersistent manner (21,24). Limited in-
formation is available for GLRaVs in that regard but work con-
ducted support that hypothesis (4,31). Although vector transmis-
sion is essential to pathogen spread and GLRaVs are of growing
economic importance worldwide, this disease complex still lacks
fundamental knowledge regarding virus–mealybug interactions
required for the development of disease management practices.
This research was supported by the American Vineyard Foundation,
California Competitive Grants Program for Research in Viticulture and
Enology, and United States Department of Agriculture NIFA-SCRI
(award no. 2009-51181-06027). We thank M. Cooper, S. Sim, R.
Aldamrat, and A. Fong for technical support; and J. Wang, the editor, and
reviewers for helpful comments that improved this manuscript.
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