modo de transmision de parietaria

SUMMARY

Parietaria mottle virus (PMoV) typically occurs at theedge of tomato and pepper crops in north-easternSpain. Studies were conducted on PMoV transmissionboth by pollen and by seven insect species of the ordersHemiptera and Thysanoptera. The presence of PMoVwas detected by indirect ELISA (I-ELISA) in sympto-matic tomato and pepper plants collected from com-mercial fields. All weed species collected in the area sur-rounding these crops were symptomless. However, thevirus was detected by I-ELISA in pollen extracts fromParietaria officinalis plants and transmitted mechanicallyto other species, including tomato and pepper. PMoVwas transmitted to other hosts using several insectspecies and P. officinalis plants as a pollen source. Trans-mission was non-persistent, not very efficient, and it wasrare if flowers of infected P. officinalis plants had previ-ously been removed or when alternative hosts that pro-duced smaller quantities of pollen were used. In addi-tion, 36% of the seedlings derived from seed of infectedP. officinalis plants were shown to be infected withPMoV. Overall, our results suggest that eliminatingPMoV-infected P. officinalis plants that surround toma-to 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 thegenus Ilarvirus, family Bromoviridae, was first found inplants of pellitory-of-the-wall (Parietaria officinalis L.)showing a bright yellow mosaic or mottling (Caciagli etal., 1989). A tomato strain of this virus (PMoV-T) wasdetected in various regions of Italy (Lisa et al., 1998;Roggero et al., 2000) and, subsequently, in different ar-eas of other countries, including southern France,

Corresponding author: J. AramburuFax: +34.93.7533954E-mail: [email protected]

Greece, and the Mediterranean coast of Spain (Mar-choux et al., 1999; Roggero et al., 2000; Aramburu,2001; Janssen et al., 2005). Tomato plants infected withPMoV-T exhibit a mosaic and necrosis of the apicalleaves, which progresses to the stem, causing top necro-sis. Infected plants eventually recover from these symp-toms and new symptomless shoots appear 15-30 dayslater. Fruit on infected plants show rings and brownpatches with necrotic scars around the edges (Galipien-so et al., 2005). Similar symptoms were observed in in-fected pepper plants (Jansen et al., 2005).

Transmission by pollen and seeds has been reportedfor some Ilarviruses (Aparicio et al., 1999; Mink et al.,1993). However, there was no evidence of seed trans-mission of PMoV-T in tomato (Ramasso et al., 1997)nor has transmission of the virus by infected pollen orviruliferous insects been proven.

In epidemiological and economic terms insects are oneof the most important factors in the transmission of plantvirus diseases. The most abundant groups of insects pres-ent in Mediterranean crops belong to the ordersHemiptera and Thysanoptera, the most common repre-sentative species being: (i) the whitefly Bemisia tabaci(Gennadius) (Hemiptera: Aleyrodidae), which transmitsvirus species from several genera, Begomovirus in particu-lar (Jones, 2003); (ii) the aphid Myzus persicae (Sulzer)(Hemiptera: Aphididae), a vector of viruses from differ-ent genera, including Potyvirus, Cucumovirus and Lu-teovirus (Van Emden and Harrington, 2007); and (iii) thewestern flower thrips, Frankliniella occidentalis (Per-gande) (Thysanoptera: Thripidae), which transmits virus-es from the genera Tospovirus (German et al., 1992) andIlarvirus (Greber et al., 1992; Sdoodee and Teakle, 1993).

Some native polyphagous predators, including Nesid-iocoris tenuis Reuter, Macrolophus caliginosus (Wagner),Dicyphus tamaninii (Wagner) (Hemiptera: Miridae), andOrius majusculus (Reuter) (Hemiptera: Anthocoridae),which are frequently used in biological controls (Alomaret al., 1994; Riudavets and Castae, 1998), are also pres-ent in weeds and horticultural crops in the Mediter-ranean basin. Tomato plants infected by PMoV tend tobe located either in the vicinity of P. officinalis plantsgrowing at the margins of the fields or in proximity ofgreenhouse entrances (Ramasso et al., 1997). P. officinalis

Journal of Plant Pathology (2010), 92 (3), 679-684 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, Spain2Instituto de Biologa Molecular y Celular de Plantas, Universidad Politcnica de Valencia (UPV-CSIC),

Avenida de los Naranjos s/n, 46022 Valencia, Spain3Instituto de Conservacin y Mejora de la Agrodiversidad Valenciana, Universidad Politcnica de Valencia (COMAV-UPV),

Camino de Vera s/n, 46022 Valencia, Spain

012_JPP210RP(Arambu)_679 16-11-2010 15:26 Pagina 679

680 Parietaria mottle virus transmission Journal of Plant Pathology (2010), 92 (3), 679-684

is a perennial weed plant, commonly present next to veg-etable crops, where it may act as a host or winter refugefor different mirid species (Alomar et al., 1994; Goulaand Alomar, 1994), some of which have been implicatedin cases of viral transmission (Gibb and Randles, 1990).

The objective of this study was to investigate differ-ent aspects of the natural mode of transmission ofPMoV, taking into account the possible implications ofdifferent insects acting as vectors. This knowledge couldbe used to develop strategies to prevent or controlPMoV epidemics in tomato and pepper crops.

MATERIAL AND METHODS

Field surveys and PMoV detection. Samples oftomato, pepper and weed species were collected inopen fields of Barcelona province (north-eastern Spain)during the 2007 and 2008 growing seasons. Samplesfrom tomato and pepper crops consisted of leavesshowing symptoms similar to those previously reportedfor PMoV-T (Aramburu, 2001; Galipienso et al., 2005,Janssen et al., 2005). Leaf samples from 17 Amaranthusretroflexus, 2 Convulvulus arvensis, 4 Chenopodium spp,3 C. murale, 3 C. album, 9 Diplotaxis erucoides, 35Erigeron sp., 3 Mirabilis jalapa, 72 Parietaria officinalis,4 Solanum nigrum and 21 Urtica dioica were collected atrandom. All these weeds grew around the three tomatofields where the presence of PMoV had been previouslyestablished. Flowers were also collected from symptom-less P. officinalis, and from tomato and pepper plantsshowing apparent PMoV infection. PMoV was detectedby ELISA (Clark and Adams, 1977), as modified for theindirect method (I) by Galipienso et al. (2005), using apolyclonal antiserum raised against the recombinantcoat protein of PMoV-T (Aparicio et al., 2009). No de-tection of PMoV by I-ELISA in leaf extracts of P. offici-nalis was possible and inconsistent results were ob-tained using one-step RT-PCR or molecular hybridiza-tion methods previously described (Galipienso et al.,2005). Thus, the presence of PMoV in these plantscould only be confirmed indirectly by mechanical trans-mission to C. quinoa plants and subsequent detection inthat host by I- ELISA.

PMoV-infected hosts. C. quinoa, Nicotiana benthami-ana, P. officinalis and tomato plants infected with PMoVwere used as inoculum sources for testing differentmodes of PMoV transmission. With the exception of P.officinalis, plants were mechanically inoculated with apreviously characterized PMoV-T isolate (Galipienso etal., 2008), kept in a greenhouse, and then tested for thepresence of PMoV by I- ELISA. P. officinalis plantswere collected from the area surrounding the PMoV-in-fected tomato crop, transplanted in the greenhouse, andtested for the presence of PMoV, as described above.

Pollen transmission tests. Pollen from P. officinalis,pepper and tomato plants collected in field surveys orfrom mechanically infected C. quinoa and N. benthami-ana plants were used as a source of PMoV. Pollen grainswere separated from anthers after air-drying, using asmall paint-brush under a stereo-microscope at a magni-fication of 100-500X. Extracts prepared from 50 mg ofpollen per plant were tested for PMoV by I- ELISA.Pollen-transmission trials were carried out followingtwo strategies: (i) PMoV-infected C. quinoa or N. ben-thamiana plants at the flowering stage surrounded bylots of six flowering-stage healthy plants of C. quinoa orN. benthamiana, respectively, were used for testingcross-pollination. For favouring a non forced pollina-tion, plants were kept for one week in a chamber at25C with a photoperiod of 16:8 h (light:dark) into80x80x80 cm cages covered with anti-thrip 049-meshwire netting. Tomato plants were not used in these as-says, because the apical necrosis caused by PMoV-infec-tion prevented timely flowering under our greenhouseconditions; (ii) 60 mg of pollen from flowers of PMoV-infected P. officinalis plants plus carborundum (w:w)were directly dusted using a small brush onto one leafof lots of six N. benthamiana or C. quinoa plants at the4-6 leaf stage, then rubbed with a pestle wetted with0.05 M phosphate buffer, pH 7.2. Plants used in bothpollen transmission assays were transferred to an insect-proof greenhouse, kept for 20 days at 20-27C (night-day) and tested for PMoV by I-ELISA.

Seed transmission tests. For ascertaining the pres-ence of virus within seeds, 80 seeds from PMoV-infect-ed P. officinalis and C. quinoa plants were collected,washed three times in distilled water (to remove anypollen sticking to them), then individually tested by I-ELISA.

Lots of 75 seeds from PMoV-infected P. officinalisand C. quinoa plants were sown to test PMoV transmis-sibility through seeds. The presence of PMoV in emerg-ing seedlings of C. quinoa was determined by I-ELISA.The presence of PMoV in seedlings of P. officinalis wasconfirmed 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 miridsD. tamaninii, M. caliginosus and N. tenuis and predatoranthocorid O. majusculus were all tested as potentialPMoV vectors. Virus-free colonies of these insects werereared in growth chambers at 27C and with a photope-riod of 12:12 h (light : dark). F. occidentalis and O. ma-jusculus were maintained on detached green bean podsand B. tabaci, M. persicae and the mirids were kept ontobacco plants. Ephestia kuehniella (Zeller) (Lepi-doptera: Pyralidae) eggs were placed on tobacco plantsas additional feed for predators. Insect transmission ex-


Journal of Plant Pathology (2010), 92 (3), 679-684 Aramburu et al. 681

periments were conducted under the conditions de-scribed above in cages kept inside growth chambersseparated from those used for rearing the insects.Groups of 25 adult insects were placed for 48 h insideindividual cages containing one PMoV-infected plantplaced in the centre on a support of 15 cm and sur-rounded at the base without contact by lots of 6 healthyplants. In each transmission assay 256 events of possi-ble transmission were tested. The infected plants usedas source of PMoV infection were at their floweringstage, except in the case of the tomato plants (for thereasons mentioned before), while C. quinoa, N. ben-thamiana, N. tabacum cv. Xanthi, tomato and pepperplants used as recepient hosts were at their 6-8 leafstage. PMoV-infected P. officinalis at the flowering stagesurrounded by tomato or N. benthamiana healthy plantswithout the intervention of insects were used as control.

In order to determine whether PMoV transmissionwas due to transportation of infected pollen by insects orto insects feeding on infected plants, two additional as-says were also done: (i) P. officinalis plant with removedflowers were used as the source for virus acquisition intransmission assays as described above in the presence of25 F. occidentalis, M. persicae or N. tenuis insects; (ii)pollen from PMoV-infected P. officinalis plants was dust-ed as previously described onto the leaves of lots of sixC. quinoa or N. benthamiana plants at the 4-6 leaf stage,which were then transferred to the cages containinggroups of F. occidentalis or N. tenuis insects.

Finally, to determine virus persistence in insects, twodifferent assays were designed: (i) groups of fifty N.tenuis individuals were tested by I-ELISA immediatelyafter transmission assays, to detect any possible reten-tion of PMoV in their stylets. Only head extracts fromdecapitated insects were analyzed to reduce competi-tion for fixation to the ELISA plates in the indirectmethod; (ii) groups of M. caliginosus and N. tenuis col-lected from previous transmission assays in the presenceof flowering infected P. officinalis plants were immedi-ately transferred to another cage containing a new lot ofhealthy plants of the same species and were kept therefor 48 h or more. Only lots of insects that affordedtransmission of the virus in previous assays were consid-ered in these trials.

In order to validate data on PMoV transmission bymirid insects, parallel transmission experiments usingdifferent viruses were conducted. The capability of N.

tenuis as a potential vector was analyzed using Cucum-ber mosaic virus (CMV) or Tomato spotted wilt virus(TSWV)-infected N. benthamiana plants as the sourceof inoculum and healthy N. benthamiana plants as re-cepient hosts.

After removing the insects, all plants used in thetransmission assays were transferred to an insect-proofgreenhouse and kept there for 20 days under controlledconditions. Plants were monitored for the appearanceand development of PMoV symptoms at five-day inter-vals and finally leaf samples were tested for PMoV by I-ELISA. CMV or TSWV infection was tested by DAS-ELISA using commercial kits and following the manu-facturer`s instructions (Loewe Biochemical, Germany).

RESULTS

Field survey. PMoV was detected by I-ELISA insymptomatic tomato and pepper plants. No symptomswere observed in the leaves from weed species collectedaround tomato and pepper fields and in no case PMoVwas found in them. However, PMoV was transmitted toC. quinoa by mechanical inoculation from 27 of 72 P. of-ficinalis plants, but not from other weed species collect-ed in the surveys.

Pollen and seed transmission. PMoV was detectedin pollen from infected P. officinalis, pepper and tomatoplants collected in the survey and also in pollen frommechanically inoculated plants of C. quinoa and N. ben-thamiana. PMoV was transmitted by mechanical inocu-lation to N. benthamiana and C. quinoa, when pollenfrom infected P. officinalis plants was directly dustedonto the leaves. In contrast, transmission by cross-polli-nation 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. quinoaand P. officinalis plants. Seeds of both species germinat-ed at a rate of approximately 98%. PMoV was not de-tected in any of the C. quinoa seedlings originating frominfected mother plants, but it was transmitted to C.quinoa from 36% of the P. officinalis seedlings originat-ing 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-in-fected P. officinalis on leaves of receiver-host and favouring its entrance by mechanical inoculation (MI) orwith the help of two vector insect species.

Recipient host CRP MI F. occidentalis N. tenuisC. quinoa 0/12 a 1/6 0/6 0/6N. benthamiana 0/12 3/6 6/6 5/6a Number of infected plants/ number of plants tested.



ciently to N. benthamiana plants that had been previ-ously dusted with pollen from infected P. officinalis inthe presence of F. occidentalis (6/6) or N. tenuis (5/6) in-dividuals. In contrast, there was no transmission to C.quinoa (Table 1). PMoV was also transmitted to C.quinoa, N. benthamiana, N. tabacum cv. Xanthi, tomatoand pepper when PMoV-infected P. officinalis plants atthe flowering stage were used as the source of inoculumin the presence of the seven insect species tested (Table2). No transmission from PMoV-infected P. officinalis totomato and N. benthamiana was obtained in control as-says conducted in the absence of insects.

PMoV was rarely transmitted when flowers of infect-ed P. officinalis plants had been removed (Table 3).Moreover, no transmission of PMoV by insects was ob-served when infected P. officinalis plants were replacedby PMoV-infected tomato plants. Only 2 of 60 and 5 of90 plants, respectively, were infected when N. benthami-ana and C. quinoa plants at the flowering stage were

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 wasnot detected by I-ELISA in head extracts from 50 decap-itated N. tenuis individuals used in transmission assays.M. caliginosus and N. tenuis recovered from previoustransmission assays in the presence of flowering PMoV-infected P. officinalis plants and those immediately trans-ferred to another cage and kept for 48 h on new lots ofhealthy plants were unable to transmit the virus.

DISCUSSION

Symptomatic tomato and pepper plants collectedfrom commercial fields in north-eastern Spain proved tobe infected by PMoV. All weed species collected in thearea surrounding the surveyed tomato fields were symp-

Table 2. Proportion of PMoV-infected plants obtained in transmission tests using PMoV-infected P. offici-nalis plants at the flowering stage as source of inoculum. Five plant species were used as hosts and seven in-sect species as transmission vectors.

Receiver-hosts NT DT MC OM BT MP FOTomato 8/24a 6/30 4/12 1/6 1/6 3/6 12/36C. quinoa 0/6 5/12 0/6 0/6 0/6 2/6 0/6N. benthamiana 5/6 2/6 3/6 6/6 5/6 2/6 6/6N. tabacum 1/6 3/6 0/6 4/6 0/6 2/6 4/6Pepper 0/6 0/12 0/6 2/18 0/6 0/6 3/12a 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 sourcesPMoV-infected P. officinalis plants whose flowers had been removed. Five plant species were used as hostsand three insect species as vectors.

Receiver-hosts N. tenuis M. persicae F. occidentalisTomato 0/6a 0/6 0/6C. quinoa 0/6 0/6 0/6N. benthamiana 0/6 1/6 2/6N. tabacum 0/6 0/6 0/6Pepper 0/6 0/6 1/6a Number of infected plants/ number of plant tested.

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 insectspecies as vectors.

N. benthamiana C. quinoa TomatoReceiver hosts NT MP NT MP FO NT FOTomato 0/12a 0/6 0/12 0/6 0/6 0/12 0/6C. quinoa 0/12 0/6 0/12 0/6 0/6 0/12 0/6N. benthamiana 1/6 0/6 0/6 1/6 0/6 0/6 0/6N. tabacum 0/6 0/6 0/6 0/6 0/6 0/6 0/6Pepper 0/6 1/6 0/6 3/6 1/6 0/6 0/6a Number of infected plants/ number of plants tested.NT: Nesidiocoris tenuis, MP: Myzus persicae and FO: Frankliniella occidentalis.


tomless and the virus was not detected by I-ELISA, one-step RT-PCR and molecular hybridization in leaf ex-tracts of P. officinalis. I-ELISA negative results could beexplained by the combination of two effects: (i) the pres-ence of inhibitory compounds in leaf extracts as de-scribed in other plant species (Saade et al., 2000), and(ii) the irregular distribution of PMoV in P. officinalisplants, which would result in a virus titre below the sen-sitivity threshold of the assay. Analysis by RT-PCR of 15leaves collected from a single PMoV-infected P. offici-nalis plant detected the presence of the virus in only twoof these leaves (data not shown). However, the virus wasdetected by I-ELISA in pollen extracts or after mechani-cal transmission from leaves of P. officinalis to C. quinoa.

The fact that 36% of the plants germinated fromseed of P. officinalis tested positive for PMoV and noneof the germinated seedlings of C. quinoa indicates a fail-ure of vertical transmission in C. quinoa. This may indi-cate that the virus occurs only in the seed coat and othertissues of C. quinoa seeds but enters the embryo of P. of-ficinalis. Similar failures have been previously reportedin tomato (Ramasso et al., 1997). All tested insectspecies, members of five different families, transmittedPMoV and it is possibile that the vector range of thisvirus may include other phytophagous insects. Thrips,aphids and whiteflies transmit a large number of virusesby different modes. However, the native polyphagouspredators tested in this work, which are frequently usedin biological controls, have never been described asvirus vectors. Most mirids are phytophagous causing afeeding damage by extra-oral digestion and less isknown about plant feeding activities of anthocorids, butfeeding does occur (Schaefer and Panizzi, 2000). How-ever, there is very little information available about therole of mirids in viral transmission.

The transmission of Velvet tobacco mottle virus (VT-MoV) by the mirid Cyrtopeltis nicotianae in a semi-per-sistent manner following an ingestion-defecation mecha-nism has been described (Gibb and Randles, 1988,1990). These authors detected the virus by ELISA in vir-uliferous mirids. However, the persistence of PMoV inthe stylets of N. tenuis was not ascertained in our assays,since the virus was not detected by I- ELISA in head ex-tracts from decapitated insects after feeding on PMoV-infected P. officinalis flowering plants. This result sug-gests that PMoV could not be transmitted by a similarmechanism. If only a few virus particles were retained inthe stylet, I-ELISA might not be sensitive enough. How-ever, this possibility seems unlikely because no transmis-sion of PMoV was observed when groups of M. caligi-nosus and N. tenuis, which had been previously fed onthese same plants, were immediately transferred to cagescontaining new lots of healthy plants. N. tenuis insectsdid not transmit either CMV or TSWV from infected tohealthy N. benthamiana plants in similar assays.

PMoV was transmitted to five different plant species

by several insect species using PMoV-infected P. offici-nalis plants at the flowering stage as the source for virusacquisition. However, when flowers of P. officinalisplants had been removed prior to acquisition, PMoVtransmission to healthy plants was only possible in a fewcases. This could be due to the incomplete eliminationof pollen from leaf and stem surfaces.

Although the precise mechanism involved in PMoVdissemination under field conditions is yet to be deter-mined, our results indicate that pollen from PMoV-in-fected P. officinalis could serve as a means for PMoVspreading to other plant species. The direct interventionof insects would be necessary to cause lesions by feed-ing or otherwise so as to facilitate the exposure of cellprotoplasm to infected pollen. This model has been sug-gested in the transmission of some ilarviruses from cher-ry pollen to cucumber seedlings (Greber et al., 1992),and for the transmission of Pelargonium zonate spotvirus (Vovlas et al., 1989), Prunus necrotic ringspot virus(Hamilton et al., 1984) and Tobacco streak virus (Sdood-ee and Teakle, 1993; Greber et al., 1991). Transmissionby this mechanism is supported by three lines of evi-dence: (i) PMoV was transmitted by mechanical inocu-lation with pollen from infected plants, (ii) PMoV trans-mission from infected plants failed to occur without theintervention of insects when these plants were at theflowering stage, and (iii) effciency of insect transmissionof PMoV decreased significantly when transmission wasattempted using infected plants with flowers removedprevious to acquisition or plants that exhibited a lowlevel of pollen production.

As previously mentioned, PMoV was detected inpollen from different plant species. However, due to thelow efficiency shown by this particular mode of trans-mission only P. officinalis plants could be considered im-portant sources of infection. The efficiency of PMoVtransmission was very low when plants such as C.quinoa and N. benthamiana, which produced less pollenthan P. officinalis, were used as sources for virus acquisi-tion. No virus transmission was observed in the case ofinfected tomato plants, probably due to the fact that theapical necrosis caused by PMoV infection preventednormal flowering under our greenhouse conditions.Thus, secondary spread within crops of tomato is un-likely to be high. This would explain both the relativelylow percentage of infection by PMoV observed in toma-to crops and the much higher level of infection in toma-to plants growing adjacent to P. officinalis plants, as re-ported by others (Ramasso et al., 1997).

In summary, results of the present study suggest thatthe incidence of PMoV will be reduced by removing P.officinalis plants around tomato or pepper crops. Thisconclusion is reinforced by the fact that in a tomato plotexhibiting an abnormally high rate of infection over twoconsecutive years (24% and 9.2%), we found that the in-cidence of PMoV decreased to less than 1% after the re-

Journal of Plant Pathology (2010), 92 (3), 679-684 Aramburu et al. 683


moval of all P. officinalis plants in the surrounding area ofthe crop (unpublished information). The application ofthis cultural practice would avoid the indiscriminate con-trol of insects that transmit PMoV, which is essential if weconsider that mirids are predator species frequently usedas biological control agents in Spain.

ACKNOWLEDGEMENTS

We would like to thank A. Barba for excellent tech-nical assistance and P. Hernandez, V. Muz, and P.Oliv for helping with the rearing of insects. This workwas supported by Project RTA2006-00024-C02 fromthe Instituto Nacional de Investigacin y TecnologaAgraria y Alimentaria (INIA).

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Received March 4, 2010Accepted July 7, 2010


modo de transmision de parietaria

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