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Edizioni ETS Pisa, 2010

679

MODE OF TRANSMISSION OF PARIETARIA MOTTLE VIRUS

J. Aramburu1, L. Galipienso1, F. Aparicio2, S. Soler3 and C. Lpez3
1Institut

de Recerca i Tecnologia Agroalimentaries (IRTA), Ctra. de Cabrils Km 2, 08348 Cabrils, Barcelona, Spain
de Biologa Molecular y Celular de Plantas, Universidad Politcnica de Valencia (UPV-CSIC),
Avenida de los Naranjos s/n, 46022 Valencia, Spain
3Instituto de Conservacin y Mejora de la Agrodiversidad Valenciana, Universidad Politcnica de Valencia (COMAV-UPV),
Camino de Vera s/n, 46022 Valencia, Spain
2Instituto

SUMMARY

Parietaria mottle virus (PMoV) typically occurs at the

edge of tomato and pepper crops in north-eastern
Spain. Studies were conducted on PMoV transmission
both by pollen and by seven insect species of the orders
Hemiptera and Thysanoptera. The presence of PMoV
was detected by indirect ELISA (I-ELISA) in symptomatic tomato and pepper plants collected from commercial fields. All weed species collected in the area surrounding these crops were symptomless. However, the
virus was detected by I-ELISA in pollen extracts from
Parietaria officinalis plants and transmitted mechanically
to other species, including tomato and pepper. PMoV
was transmitted to other hosts using several insect
species and P. officinalis plants as a pollen source. Transmission was non-persistent, not very efficient, and it was
rare if flowers of infected P. officinalis plants had previously been removed or when alternative hosts that produced smaller quantities of pollen were used. In addition, 36% of the seedlings derived from seed of infected
P. officinalis plants were shown to be infected with
PMoV. Overall, our results suggest that eliminating
PMoV-infected P. officinalis plants that surround tomato and pepper crops could help restraining virus spread.
Key words: PMoV, Parietaria officinalis, insects,
mirids, pollen, epidemiology.

INTRODUCTION

Parietaria mottle virus (PMoV), a member of the

genus Ilarvirus, family Bromoviridae, was first found in
plants of pellitory-of-the-wall (Parietaria officinalis L.)
showing a bright yellow mosaic or mottling (Caciagli et
al., 1989). A tomato strain of this virus (PMoV-T) was
detected in various regions of Italy (Lisa et al., 1998;
Roggero et al., 2000) and, subsequently, in different areas of other countries, including southern France,
Corresponding author: J. Aramburu
Fax: +34.93.7533954
E-mail: josemaria.aramburu@irta.es

Greece, and the Mediterranean coast of Spain (Marchoux et al., 1999; Roggero et al., 2000; Aramburu,
2001; Janssen et al., 2005). Tomato plants infected with
PMoV-T exhibit a mosaic and necrosis of the apical
leaves, which progresses to the stem, causing top necrosis. Infected plants eventually recover from these symptoms and new symptomless shoots appear 15-30 days
later. Fruit on infected plants show rings and brown
patches with necrotic scars around the edges (Galipienso et al., 2005). Similar symptoms were observed in infected pepper plants (Jansen et al., 2005).
Transmission by pollen and seeds has been reported
for some Ilarviruses (Aparicio et al., 1999; Mink et al.,
1993). However, there was no evidence of seed transmission of PMoV-T in tomato (Ramasso et al., 1997)
nor has transmission of the virus by infected pollen or
viruliferous insects been proven.
In epidemiological and economic terms insects are one
of the most important factors in the transmission of plant
virus diseases. The most abundant groups of insects present in Mediterranean crops belong to the orders
Hemiptera and Thysanoptera, the most common representative species being: (i) the whitefly Bemisia tabaci
(Gennadius) (Hemiptera: Aleyrodidae), which transmits
virus species from several genera, Begomovirus in particular (Jones, 2003); (ii) the aphid Myzus persicae (Sulzer)
(Hemiptera: Aphididae), a vector of viruses from different genera, including Potyvirus, Cucumovirus and Luteovirus (Van Emden and Harrington, 2007); and (iii) the
western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae), which transmits viruses from the genera Tospovirus (German et al., 1992) and
Ilarvirus (Greber et al., 1992; Sdoodee and Teakle, 1993).
Some native polyphagous predators, including Nesidiocoris tenuis Reuter, Macrolophus caliginosus (Wagner),
Dicyphus tamaninii (Wagner) (Hemiptera: Miridae), and
Orius majusculus (Reuter) (Hemiptera: Anthocoridae),
which are frequently used in biological controls (Alomar
et al., 1994; Riudavets and Castae, 1998), are also present in weeds and horticultural crops in the Mediterranean basin. Tomato plants infected by PMoV tend to
be located either in the vicinity of P. officinalis plants
growing at the margins of the fields or in proximity of
greenhouse entrances (Ramasso et al., 1997). P. officinalis

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is a perennial weed plant, commonly present next to vegetable crops, where it may act as a host or winter refuge
for different mirid species (Alomar et al., 1994; Goula
and Alomar, 1994), some of which have been implicated
in cases of viral transmission (Gibb and Randles, 1990).
The objective of this study was to investigate different aspects of the natural mode of transmission of
PMoV, taking into account the possible implications of
different insects acting as vectors. This knowledge could
be used to develop strategies to prevent or control
PMoV epidemics in tomato and pepper crops.

MATERIAL AND METHODS

Field surveys and PMoV detection. Samples of

tomato, pepper and weed species were collected in
open fields of Barcelona province (north-eastern Spain)
during the 2007 and 2008 growing seasons. Samples
from tomato and pepper crops consisted of leaves
showing symptoms similar to those previously reported
for PMoV-T (Aramburu, 2001; Galipienso et al., 2005,
Janssen et al., 2005). Leaf samples from 17 Amaranthus
retroflexus, 2 Convulvulus arvensis, 4 Chenopodium spp,
3 C. murale, 3 C. album, 9 Diplotaxis erucoides, 35
Erigeron sp., 3 Mirabilis jalapa, 72 Parietaria officinalis,
4 Solanum nigrum and 21 Urtica dioica were collected at
random. All these weeds grew around the three tomato
fields where the presence of PMoV had been previously
established. Flowers were also collected from symptomless P. officinalis, and from tomato and pepper plants
showing apparent PMoV infection. PMoV was detected
by ELISA (Clark and Adams, 1977), as modified for the
indirect method (I) by Galipienso et al. (2005), using a
polyclonal antiserum raised against the recombinant
coat protein of PMoV-T (Aparicio et al., 2009). No detection of PMoV by I-ELISA in leaf extracts of P. officinalis was possible and inconsistent results were obtained using one-step RT-PCR or molecular hybridization methods previously described (Galipienso et al.,
2005). Thus, the presence of PMoV in these plants
could only be confirmed indirectly by mechanical transmission to C. quinoa plants and subsequent detection in
that host by I- ELISA.
PMoV-infected hosts. C. quinoa, Nicotiana benthamiana, P. officinalis and tomato plants infected with PMoV
were used as inoculum sources for testing different
modes of PMoV transmission. With the exception of P.
officinalis, plants were mechanically inoculated with a
previously characterized PMoV-T isolate (Galipienso et
al., 2008), kept in a greenhouse, and then tested for the
presence of PMoV by I- ELISA. P. officinalis plants
were collected from the area surrounding the PMoV-infected tomato crop, transplanted in the greenhouse, and
tested for the presence of PMoV, as described above.

Journal of Plant Pathology (2010), 92 (3), 679-684

Pollen transmission tests. Pollen from P. officinalis,

pepper and tomato plants collected in field surveys or
from mechanically infected C. quinoa and N. benthamiana plants were used as a source of PMoV. Pollen grains
were separated from anthers after air-drying, using a
small paint-brush under a stereo-microscope at a magnification of 100-500X. Extracts prepared from 50 mg of
pollen per plant were tested for PMoV by I- ELISA.
Pollen-transmission trials were carried out following
two strategies: (i) PMoV-infected C. quinoa or N. benthamiana plants at the flowering stage surrounded by
lots of six flowering-stage healthy plants of C. quinoa or
N. benthamiana, respectively, were used for testing
cross-pollination. For favouring a non forced pollination, plants were kept for one week in a chamber at
25C with a photoperiod of 16:8 h (light:dark) into
80x80x80 cm cages covered with anti-thrip 049-mesh
wire netting. Tomato plants were not used in these assays, because the apical necrosis caused by PMoV-infection prevented timely flowering under our greenhouse
conditions; (ii) 60 mg of pollen from flowers of PMoVinfected P. officinalis plants plus carborundum (w:w)
were directly dusted using a small brush onto one leaf
of lots of six N. benthamiana or C. quinoa plants at the
4-6 leaf stage, then rubbed with a pestle wetted with
0.05 M phosphate buffer, pH 7.2. Plants used in both
pollen transmission assays were transferred to an insectproof greenhouse, kept for 20 days at 20-27C (nightday) and tested for PMoV by I-ELISA.
Seed transmission tests. For ascertaining the presence of virus within seeds, 80 seeds from PMoV-infected P. officinalis and C. quinoa plants were collected,
washed three times in distilled water (to remove any
pollen sticking to them), then individually tested by IELISA.
Lots of 75 seeds from PMoV-infected P. officinalis
and C. quinoa plants were sown to test PMoV transmissibility through seeds. The presence of PMoV in emerging seedlings of C. quinoa was determined by I-ELISA.
The presence of PMoV in seedlings of P. officinalis was
confirmed by mechanical transmission to plants of C.
quinoa and subsequent detection by I-ELISA.
Insect transmission tests. The aphid M. persicae,
thrips F. occidentalis, whitefly B. tabaci, predator mirids
D. tamaninii, M. caliginosus and N. tenuis and predator
anthocorid O. majusculus were all tested as potential
PMoV vectors. Virus-free colonies of these insects were
reared in growth chambers at 27C and with a photoperiod of 12:12 h (light : dark). F. occidentalis and O. majusculus were maintained on detached green bean pods
and B. tabaci, M. persicae and the mirids were kept on
tobacco plants. Ephestia kuehniella (Zeller) (Lepidoptera: Pyralidae) eggs were placed on tobacco plants
as additional feed for predators. Insect transmission ex-

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periments were conducted under the conditions described above in cages kept inside growth chambers
separated from those used for rearing the insects.
Groups of 25 adult insects were placed for 48 h inside
individual cages containing one PMoV-infected plant
placed in the centre on a support of 15 cm and surrounded at the base without contact by lots of 6 healthy
plants. In each transmission assay 256 events of possible transmission were tested. The infected plants used
as source of PMoV infection were at their flowering
stage, except in the case of the tomato plants (for the
reasons mentioned before), while C. quinoa, N. benthamiana, N. tabacum cv. Xanthi, tomato and pepper
plants used as recepient hosts were at their 6-8 leaf
stage. PMoV-infected P. officinalis at the flowering stage
surrounded by tomato or N. benthamiana healthy plants
without the intervention of insects were used as control.
In order to determine whether PMoV transmission
was due to transportation of infected pollen by insects or
to insects feeding on infected plants, two additional assays were also done: (i) P. officinalis plant with removed
flowers were used as the source for virus acquisition in
transmission assays as described above in the presence of
25 F. occidentalis, M. persicae or N. tenuis insects; (ii)
pollen from PMoV-infected P. officinalis plants was dusted as previously described onto the leaves of lots of six
C. quinoa or N. benthamiana plants at the 4-6 leaf stage,
which were then transferred to the cages containing
groups of F. occidentalis or N. tenuis insects.
Finally, to determine virus persistence in insects, two
different assays were designed: (i) groups of fifty N.
tenuis individuals were tested by I-ELISA immediately
after transmission assays, to detect any possible retention of PMoV in their stylets. Only head extracts from
decapitated insects were analyzed to reduce competition for fixation to the ELISA plates in the indirect
method; (ii) groups of M. caliginosus and N. tenuis collected from previous transmission assays in the presence
of flowering infected P. officinalis plants were immediately transferred to another cage containing a new lot of
healthy plants of the same species and were kept there
for 48 h or more. Only lots of insects that afforded
transmission of the virus in previous assays were considered in these trials.
In order to validate data on PMoV transmission by
mirid insects, parallel transmission experiments using
different viruses were conducted. The capability of N.

Aramburu et al.

tenuis as a potential vector was analyzed using Cucumber mosaic virus (CMV) or Tomato spotted wilt virus
(TSWV)-infected N. benthamiana plants as the source
of inoculum and healthy N. benthamiana plants as recepient hosts.
After removing the insects, all plants used in the
transmission assays were transferred to an insect-proof
greenhouse and kept there for 20 days under controlled
conditions. Plants were monitored for the appearance
and development of PMoV symptoms at five-day intervals and finally leaf samples were tested for PMoV by IELISA. CMV or TSWV infection was tested by DASELISA using commercial kits and following the manufacturer`s instructions (Loewe Biochemical, Germany).

RESULTS

Field survey. PMoV was detected by I-ELISA in

symptomatic tomato and pepper plants. No symptoms
were observed in the leaves from weed species collected
around tomato and pepper fields and in no case PMoV
was found in them. However, PMoV was transmitted to
C. quinoa by mechanical inoculation from 27 of 72 P. officinalis plants, but not from other weed species collected in the surveys.
Pollen and seed transmission. PMoV was detected
in pollen from infected P. officinalis, pepper and tomato
plants collected in the survey and also in pollen from
mechanically inoculated plants of C. quinoa and N. benthamiana. PMoV was transmitted by mechanical inoculation to N. benthamiana and C. quinoa, when pollen
from infected P. officinalis plants was directly dusted
onto the leaves. In contrast, transmission by cross-pollination did not occur with either healthy C. quinoa or N.
benthamiana plants (Table 1).
Virus presence was ascertained in 95% and 81.25%,
respectively, of the seeds from PMoV-infected C. quinoa
and P. officinalis plants. Seeds of both species germinated at a rate of approximately 98%. PMoV was not detected in any of the C. quinoa seedlings originating from
infected mother plants, but it was transmitted to C.
quinoa from 36% of the P. officinalis seedlings originating from infected plants.
Insect transmission. PMoV was transmitted very effi-

Table 1. Proportion of PMoV-infected plants obtained by cross-pollination (CRP) from PMoV-infected C.

quinoa or N. benthamiana plants to receiver-host at the flowering stage or dusting pollen from PMoV-infected P. officinalis on leaves of receiver-host and favouring its entrance by mechanical inoculation (MI) or
with the help of two vector insect species.
Recipient host
CRP
MI
C. quinoa
0/12 a
1/6
N. benthamiana
0/12
3/6
a
Number of infected plants/ number of plants tested.

681

F. occidentalis
0/6
6/6

N. tenuis
0/6
5/6

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Table 2. Proportion of PMoV-infected plants obtained in transmission tests using PMoV-infected P. officinalis plants at the flowering stage as source of inoculum. Five plant species were used as hosts and seven insect species as transmission vectors.
Receiver-hosts
NT
DT
MC
OM
BT
MP
FO
Tomato
8/24a
6/30
4/12
1/6
1/6
3/6
12/36
C. quinoa
0/6
5/12
0/6
0/6
0/6
2/6
0/6
N. benthamiana
5/6
2/6
3/6
6/6
5/6
2/6
6/6
N. tabacum
1/6
3/6
0/6
4/6
0/6
2/6
4/6
Pepper
0/6
0/12
0/6
2/18
0/6
0/6
3/12
a
Number of infected plants/ number of plants tested.
NT: Nesidiocoris tenuis, DT: Dicyphus tamaninii, MC: Macrolophus caliginosus, OM: Orius majusculus, BT:
Bemisia tabaci, MP: Myzus persicae and FO: Frankliniella occidentalis.

Table 3. Proportion of PMoV-infected plants obtained in transmission tests using as inoculum sources
PMoV-infected P. officinalis plants whose flowers had been removed. Five plant species were used as hosts
and three insect species as vectors.
Receiver-hosts
N. tenuis
Tomato
0/6a
C. quinoa
0/6
N. benthamiana
0/6
N. tabacum
0/6
Pepper
0/6
a
Number of infected plants/ number of plant tested.

M. persicae
0/6
0/6
1/6
0/6
0/6

F. occidentalis
0/6
0/6
2/6
0/6
1/6

Table 4. Proportion of PMoV-infected plants obtained in transmission tests using PMoV-infected tomato,
N. benthamiana and C. quinoa as inoculum sources. Five plant species were used as hosts and three insect
species as vectors.
N. benthamiana
C. quinoa
Receiver hosts
NT
MP
NT
MP
Tomato
0/12a
0/6
0/12
0/6
C. quinoa
0/12
0/6
0/12
0/6
N. benthamiana
1/6
0/6
0/6
1/6
N. tabacum
0/6
0/6
0/6
0/6
Pepper
0/6
1/6
0/6
3/6
a
Number of infected plants/ number of plants tested.
NT: Nesidiocoris tenuis, MP: Myzus persicae and FO: Frankliniella occidentalis.

ciently to N. benthamiana plants that had been previously dusted with pollen from infected P. officinalis in
the presence of F. occidentalis (6/6) or N. tenuis (5/6) individuals. In contrast, there was no transmission to C.
quinoa (Table 1). PMoV was also transmitted to C.
quinoa, N. benthamiana, N. tabacum cv. Xanthi, tomato
and pepper when PMoV-infected P. officinalis plants at
the flowering stage were used as the source of inoculum
in the presence of the seven insect species tested (Table
2). No transmission from PMoV-infected P. officinalis to
tomato and N. benthamiana was obtained in control assays conducted in the absence of insects.
PMoV was rarely transmitted when flowers of infected P. officinalis plants had been removed (Table 3).
Moreover, no transmission of PMoV by insects was observed when infected P. officinalis plants were replaced
by PMoV-infected tomato plants. Only 2 of 60 and 5 of
90 plants, respectively, were infected when N. benthamiana and C. quinoa plants at the flowering stage were

FO
0/6
0/6
0/6
0/6
1/6

Tomato
NT
FO
0/12
0/6
0/12
0/6
0/6
0/6
0/6
0/6
0/6
0/6

used as source of virus (Table 4).

No transmission of CMV or TSWV between infected
and healthy N. benthamiana plants in the presence of N.
tenuis was observed in two repeated assays. PMoV was
not detected by I-ELISA in head extracts from 50 decapitated N. tenuis individuals used in transmission assays.
M. caliginosus and N. tenuis recovered from previous
transmission assays in the presence of flowering PMoVinfected P. officinalis plants and those immediately transferred to another cage and kept for 48 h on new lots of
healthy plants were unable to transmit the virus.

DISCUSSION

Symptomatic tomato and pepper plants collected

from commercial fields in north-eastern Spain proved to
be infected by PMoV. All weed species collected in the
area surrounding the surveyed tomato fields were symp-

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tomless and the virus was not detected by I-ELISA, onestep RT-PCR and molecular hybridization in leaf extracts of P. officinalis. I-ELISA negative results could be
explained by the combination of two effects: (i) the presence of inhibitory compounds in leaf extracts as described in other plant species (Saade et al., 2000), and
(ii) the irregular distribution of PMoV in P. officinalis
plants, which would result in a virus titre below the sensitivity threshold of the assay. Analysis by RT-PCR of 15
leaves collected from a single PMoV-infected P. officinalis plant detected the presence of the virus in only two
of these leaves (data not shown). However, the virus was
detected by I-ELISA in pollen extracts or after mechanical transmission from leaves of P. officinalis to C. quinoa.
The fact that 36% of the plants germinated from
seed of P. officinalis tested positive for PMoV and none
of the germinated seedlings of C. quinoa indicates a failure of vertical transmission in C. quinoa. This may indicate that the virus occurs only in the seed coat and other
tissues of C. quinoa seeds but enters the embryo of P. officinalis. Similar failures have been previously reported
in tomato (Ramasso et al., 1997). All tested insect
species, members of five different families, transmitted
PMoV and it is possibile that the vector range of this
virus may include other phytophagous insects. Thrips,
aphids and whiteflies transmit a large number of viruses
by different modes. However, the native polyphagous
predators tested in this work, which are frequently used
in biological controls, have never been described as
virus vectors. Most mirids are phytophagous causing a
feeding damage by extra-oral digestion and less is
known about plant feeding activities of anthocorids, but
feeding does occur (Schaefer and Panizzi, 2000). However, there is very little information available about the
role of mirids in viral transmission.
The transmission of Velvet tobacco mottle virus (VTMoV) by the mirid Cyrtopeltis nicotianae in a semi-persistent manner following an ingestion-defecation mechanism has been described (Gibb and Randles, 1988,
1990). These authors detected the virus by ELISA in viruliferous mirids. However, the persistence of PMoV in
the stylets of N. tenuis was not ascertained in our assays,
since the virus was not detected by I- ELISA in head extracts from decapitated insects after feeding on PMoVinfected P. officinalis flowering plants. This result suggests that PMoV could not be transmitted by a similar
mechanism. If only a few virus particles were retained in
the stylet, I-ELISA might not be sensitive enough. However, this possibility seems unlikely because no transmission of PMoV was observed when groups of M. caliginosus and N. tenuis, which had been previously fed on
these same plants, were immediately transferred to cages
containing new lots of healthy plants. N. tenuis insects
did not transmit either CMV or TSWV from infected to
healthy N. benthamiana plants in similar assays.
PMoV was transmitted to five different plant species

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by several insect species using PMoV-infected P. officinalis plants at the flowering stage as the source for virus
acquisition. However, when flowers of P. officinalis
plants had been removed prior to acquisition, PMoV
transmission to healthy plants was only possible in a few
cases. This could be due to the incomplete elimination
of pollen from leaf and stem surfaces.
Although the precise mechanism involved in PMoV
dissemination under field conditions is yet to be determined, our results indicate that pollen from PMoV-infected P. officinalis could serve as a means for PMoV
spreading to other plant species. The direct intervention
of insects would be necessary to cause lesions by feeding or otherwise so as to facilitate the exposure of cell
protoplasm to infected pollen. This model has been suggested in the transmission of some ilarviruses from cherry pollen to cucumber seedlings (Greber et al., 1992),
and for the transmission of Pelargonium zonate spot
virus (Vovlas et al., 1989), Prunus necrotic ringspot virus
(Hamilton et al., 1984) and Tobacco streak virus (Sdoodee and Teakle, 1993; Greber et al., 1991). Transmission
by this mechanism is supported by three lines of evidence: (i) PMoV was transmitted by mechanical inoculation with pollen from infected plants, (ii) PMoV transmission from infected plants failed to occur without the
intervention of insects when these plants were at the
flowering stage, and (iii) effciency of insect transmission
of PMoV decreased significantly when transmission was
attempted using infected plants with flowers removed
previous to acquisition or plants that exhibited a low
level of pollen production.
As previously mentioned, PMoV was detected in
pollen from different plant species. However, due to the
low efficiency shown by this particular mode of transmission only P. officinalis plants could be considered important sources of infection. The efficiency of PMoV
transmission was very low when plants such as C.
quinoa and N. benthamiana, which produced less pollen
than P. officinalis, were used as sources for virus acquisition. No virus transmission was observed in the case of
infected tomato plants, probably due to the fact that the
apical necrosis caused by PMoV infection prevented
normal flowering under our greenhouse conditions.
Thus, secondary spread within crops of tomato is unlikely to be high. This would explain both the relatively
low percentage of infection by PMoV observed in tomato crops and the much higher level of infection in tomato plants growing adjacent to P. officinalis plants, as reported by others (Ramasso et al., 1997).
In summary, results of the present study suggest that
the incidence of PMoV will be reduced by removing P.
officinalis plants around tomato or pepper crops. This
conclusion is reinforced by the fact that in a tomato plot
exhibiting an abnormally high rate of infection over two
consecutive years (24% and 9.2%), we found that the incidence of PMoV decreased to less than 1% after the re-

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moval of all P. officinalis plants in the surrounding area of

the crop (unpublished information). The application of
this cultural practice would avoid the indiscriminate control of insects that transmit PMoV, which is essential if we
consider that mirids are predator species frequently used
as biological control agents in Spain.
ACKNOWLEDGEMENTS

We would like to thank A. Barba for excellent technical assistance and P. Hernandez, V. Muz, and P.
Oliv for helping with the rearing of insects. This work
was supported by Project RTA2006-00024-C02 from
the Instituto Nacional de Investigacin y Tecnologa
Agraria y Alimentaria (INIA).
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Received March 4, 2010
Accepted July 7, 2010

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