Research Note Thrips tabaci (Lind.) (Thysanoptera, Thripidae), another vector for tomato spotted wilt virus in Finland Anne Lemmetty and Isa Lindqvist Lemmetty, A. & Lindqvist, 1.1993.Thrips tabaci (Lind.) (Thysanoptera, Thripidae), another vector for tomato spotted wilt virus in Finland. Agric. Sci. Finl. 2: 189-194. (Agric. Res. Centre of Finland, Inst. PI. Protect., FIN-31600 Jokioinen, Finland.) Populations of Thrips tabaci transmitted the isolate of tomato spotted wilt virus (TSWV) from infected china asters to healthy china asters in laboratory experiments. Great variation was observed among absorbance values between thrips infested china asters. The highest absorbance values were obtained from stems of thrips inoculated plants. According to our results, T. tabaci seems to be a noteworthy TSWV vector in Finland, where it is the only naturally occurring TSWV vector species. It is also one of the main pests on greenhouse crops in addition to Frankliniella occidentalis, the primary vector ofTSWV. Key words: TSWV, china aster, thrips, transmission, detection, ELISA Introduction Tomato spotted wilt vims (TSWV), a member of the tospovirus group, is vectored by seven thrips species; Frankliniella occidentalis (Perg.), F. schultzei (Trybom), F.fusca (Hinds), Thrips tabaci (Lind.), T. setosus (Moulton), T. palmi (Kamy) and Scirtothrips dorsalis (Hood) (GERMAN et al. 1992). The specificity between TSWV and these thrips species among 5000 known thrips species (Zur Strassen 1960) is not yet understood. According to Sakimura (1962), the thrips must acquire TSWV as nymphs in order to transmit the vims. F. occidentalis is considered to be the primary vector of TSWV in many countries (Allen and Broad- bent 1986, Cho et al. 1987). Only one of the above mentioned species, T. tabaci, is known to occur naturally in Finland, where it is one of the main pests of greenhouse vegetables and ornamentals. In recent years, also F. occidentalis has become a serious and rather com- mon pest in many Finnish greenhouses. Although T. tabaci has been reported to be a vector of TSWV (Best 1968), some studies indic- ate that not all insect populations or vims isolates are equally acquired (Mau et al. 1991)or transmit- ted (Paliwal 1974, 1976) by this species. In Finland, TSWV has been isolated from tomato and chrysanthemum in 1989 (Lemmetty 1991) and from cineraria in 1992. In both cases there were sig- nificant populations ofF. occidentalisand T. tabaciin the greenhouses where the vims was isolated. The object of this study was to find out whether TSWV can be vectored by T. tabaci alone. Material and methods The vims isolate used in this study was ob- tained from naturally infected cineraria (Senecio x 189 Agric. Sei. Fin l. 2 (1993) https://www.c-info.fi/en/info/?token=MIurUEcWzOgTx5pe.FhQzC2UMGBiVg_30H3JMQQ.oLcDwe2H1k_KWfkVderMaFi5bbm5cb3qCzxdxsiGBcvcImNRlfS8Llpwa01AXtlcAmC4oLivGQZnyGmARbPOD3mZx_Z-jh2a5LkPAhNa1oeruTB1qScekMq7uB9FUFZee6TmvXp6peKeOhZYOdSktQ2OJywW1kjeRZSY0NRjfEoXSQSVow4ZT75wOEE5bOy9YH7Xh4XxN9YQ2lHagnVGaWAldhgzIRR4u03Xqmx0PXRYsNnuNHdCVzS9mtVLK1d2z2rmK4st5mTrRSaVcRos6Ng_M66tJw Research Note hybridus) by sap transmission. Identification of TSWV was based on symptoms on host plants, enzyme-linked immunosorbent assay (ELISA) and electron microscopy (Fig. 1). To test thrips transmission, young and healthy china asters (Callistephus chinensis Nees.) cv. Remo original, planted in 10 cm plastic pots, were sap inoculated (0.05 M phosphate buffer, pH 7.0) with TSWV isolate. Inoculated asters, which showed necrotic local lesions and a yellow-green systemic mosaic, were tested by ELISA two weeks after inoculation. Asters ofwhich sap gave absorb- ance values (A405 nm) between 1 and 2 were chosen for the experiments. Three experiments were conducted during May to August 1992. Metal framed hard plastic cages, 60 x 60 cm in size, were used. The top of the cage was covered with a cloth, and on one side there was a narrow door. The cages were standing on a metal plate with a 5 cm layer of peat on each to allow pupation of thrips. Approximately 50 thrips were transferred one by one on each of the six TSWV inoculated asters, using a fine-tipped moistened brush. Mainly second instar larvae, but also some adults, were transferred on two or three leaves above the inoculated leaves. The plants were spaced apart from each other in the cage and the larvae were allowed free access to feed on leaf surfaces for acquisition of the virus. The thrips completed their development and approximately three weeks later, after adult emergence, the TSWV inoculated asters were cut and left on one side of the cage for two to three days. Six young and healthy asters, cv. Carmen, 10-15 cm high, with six to ten leaves on each, were placed on the other side of the cage. Two ofthe cages (1 and 2) were placed on a table in the laboratory and the third cage (3) into a grow- ing chamber. The plants and thrips were maintained at 24±I°C with a 16 h light and 8 h dark cycle. All Fig. 1. Electron micrograph of spherical particles of TSWV on a thin section of Senecio x hybridus leaf tissue. Bar represents 250 nm. (Photo Anne Lemmetty). 190 Agric. Sei. Finl. 2(1993) Agric. Sd. Fint. 2 (1993) the thrips used in the experiments were laboratory reared and originated from colonies maintained on healthy china asters. The development of feeding scars was observed and thrips populations were monitored. Two weeks after healthy asters had been placed into the cages, additional healthy plants were placed in the cages to minimize the feeding damage. ELISA tests were done on leaf samples from healthy asters approximately three weeks after they had been exposed to T. tabaci. Two to three leaves, depending on the size and feeding damage, were removed from each plant for ELISA. Both old and young leaves were sampled. All plants were as- sayed for TSWV infection twice after the healthy asters had been placed in the cages. Samples for the second ELISA were taken from leaves of the six asters in cage 1, from stems of the six asters in cage 2 and from leaves and stems of the six asters in cage 3. A commercial diagnostic kit was purchased from Loewe Biochemica GmbH, Germany. The antise- rum used in all ELISA tests was against the nu- cleocapsid protein of the strain CNPH (de Avila et al. 1990). All samples were tested by direct double antibody ELISA according to the instructions of the kit, using a 1 to 50 dilutionof pressed sap in sample buffer. Absorbance values (A405 nm) were measured after one hour of substrate incubation at room tem- perature. Each value of one plant was the mean of four wells. A sample was considered TSWV posit- ive if the absorbance value was greater than three times the mean value of a healthy control. The healthy control asters originated from the same lot as those exposed to TSWV. Results Populations of T. tabaci transmitted the TSWV isolate from infected china asters to healthy china asters. The results of the three experiments repeated in successive weeks were consistent (Fig. 2). After pupation, there were numerous adult T. tabaci in each cage. Severe feeding damage, browning and scorching of foliage, was rapidly obvious. Indeed, two china asters (cage 2) became so desiccated that only their stem tissue could be tested. TSWV was readily detected by ELISA after a three weeks’ exposure period of the healthy plants to T. tabaci. The first ELISA test yielded generally low absorbance values. However, leaf samples of one plant gave an exceptionally high absorbance value compared to the others (Fig. 3). In general, the longer the exposure time between transferring the healthy plants into the cage and Fig. 2. TSWV absorbance values of china asters after exposure to TSWV transmission by T. tabaci for 25 days (cage 1) and 24 days (cages 2 and 3). Mean absorbance values of leaves of six china asters (cages 1 and 3) and four china asters (cage 2) 191 Research Note testing the plants, the higher the absorbance values (Figs. 3 and 4). The first maximum absorbance value (>2) was obtained from the stem of a china aster in cage 2 when the plants had been exposed to TSWV for 24 days. Maximum absorbance values were also measured from the stems of five china asters in cage 3 after the plants had been exposed to TSWV transmissionby T. tahaci for 32 days (Fig. 4). The absorbance values ofhealthy controls in six ELISA tests ranged from 0.003 to 0.008. TSWV was detected by ELISA in extracts from leaves or stems of china asters. However, as shown in Fig. 4, the stem tissue gave higher absorbance values. Discussion Our laboratory experiments demonsrated that T. tabaci is capable of obtaining and transmitting TSWV as also reported by Sakimura (1963), even though Paliwal (1976) and Reddy et al. (1983) could not confirm Sakimura’s report. Some as- sumptions have been presented to elucidate the nontransmissibility of TSWV by T. tabaci. Cho et al. (1991) found out thatTSWV was not transstadi- ally passed from larvae to adults in T. tahaci al- though that happened in F. occidentalis, the prim- ary vector of TSWV. Zawirska (1976) explained that failure to spread TSWV by T. tahaci correlated with the absence ofmales in Polish T. tabaci popu- lations. Two factors may have served as preconditions for successful transmission of TSWV by T. tabaci in our experiment. Firstly, the TSWV isolate in- volved was detected with the antiserum raised against the CNPH strain. It was very likely that our TSWV isolate belonged to a common serogroup because some exceptional strains seem to require special thrips species for transmission (German et al. 1992). Secondly, the TSWV isolate found in cineraria was only once transferred by sap inocula- tion from cineraria to asters. It has been observed that TSWV can lose its vector transmissibility after prolonged culture by sap transmission (Best 1968). Ie (1982) also suggested that it is unlikely that the Fig. 3. TSWV absorbance values of six china asters in cage 1. Values were recorded after plants had been exposed to TSWV transmission by T. tabaci for 19 and 25 days. Fig. 4. TSWV absorbance values of six china aster leaves and stems in cage 3. Values were recorded after plants had been exposed to TSWV transmission by T. tabaci for 24 and 32 days. 192 Research NoteAgric. Sei. Fin!. 2(1993) Research Note defective form of TSWV would be transmitted by thrips. Undoubtedly there are differences in transmis- sion efficiency of TSWV isolates by different spe- cies of thrips as demonstrated by Paliwal (1976). In our trial the density of T. tabaci was high; even if the transmission efficiency could have been low, the thrips transmittedTSWV. The number of larval thrips was unnecessarily high as our assumption that the larvae would suffer from being transferred with a brush turned out to be incorrect. We found asters a very suitable source plant for T. tabaci-TSWV transmission assays. Firstly, 7. tabaci fed readily on asters and, secondly, asters provided systemic infection of TSWV with relat- ively high virus titer for a prolonged period. In some host plants it is typical ofTSWV that thevims titer is maintained high for only one to two days before a dramatic decrease (Best 1968). The stage of larval thrips may affect the trans- mission efficiency of TSWV. In our trial, most of the thrips were transferred on the infected asters as second instars. Sakimura (1961) found that the feeding activity ofT. tabaci larvae on Emilia leaves increased during the second instar stage. Our results showed great variation in absorbance values of the asters inoculatedby thrips. This might be due to the fact that TSWV is unevenly distrib- uted in host plants. It was interesting to find out that the TSWV concentration was highest in stems of asters. Also Allen et al. (1990) detectedTSWV in stems of chrysanthemum by ELISA. There are still open questions concerning the transmissibility of TSWV by T. tabaci, but this study provided information that T. tabaci popula- tions in greenhouses can be a link between TSWV infected and healthy plants. So far, the spread of TSWV infected plant material has been restricted in Finland and no epidemic has yet become prevalent. References Allen, W. R. & Broadbent, A. B. 1986. Transmission of tomato spotted wilt virus in Ontario greenhouses by the western flower thrips Frankliniella occidentalis (Per- gande). Can. J. PI. Path. 8: 33-38. —, Matteoni, J. A. & Broadbent, A. B. 1990.Susceptibility ofcultivars of florist’s chrysanthemum to tomato spotted wilt virus. Can. J. PI. Path. 12: 417-423. Avila, A. C. de, Huguenot,C„ Resende, R. 0., Kitajima, E. W., Goldbach, R. W. & Peters, D. 1990. Serological differentiation of20 isolates of tomato spotted will virus. J. Gen. Virol. 71:2801-2807. Best, R. J. 1968.Tomato spotted wilt virus. In: Smith, K. M. & Lauffer, M. A. (eds.). Adv. Virus Res. 13: 65-146. Cho, J. J., Mitchell, W. C., Mau, R. F. L & Sakimura, K. 1987. Epidemiology of tomato spotted wilt virus disease on crisphead lettuce in Hawaii. PI. Dis. 71: 505-508. —, Mau, R. F. L., Ullman, D. E. & Custer, D. M. 1991, Detection of the tomato spotted wilt virus (TSWV) within thrips. In: Hsu, H. T. & Lawson, R. H. (eds.). Virus-thrips-plant interactions of tomato spotted wilt virus . Proc. USDA workshop. ARS-87. Beltsville. p. 144-152. German, T. L., Ullman, D. E. & Moyer, J. W. 1992. To- spoviruses: diagnosis, molecular biology, phylogeny, and vector relationships. Ann. Rev. Phytopath. 30: 315- 348. le, T. S. 1982. A sap-transmissible defective form oftomato spotted wilt virus. J. Gen. Virology. 59: 387-391, Lemmetty, A. 1991. First reported occurrence of tomato spotted wilt virus in greenhouse crops in Finland, Växtskyddsnotiser 55: 7-9. Mau, R. F. L„ Bautista, R., Cho, J. J., Ullman, D. E., Gusukuma-Minuto, L. & Custer, D. 1991. Factors af- fecting the epidemiology of TSWV in field crops: com- parative virus acquisition efficiency of vectors and suit- ability of alternate hosts to Frankliniella occidentalis (Pergande). In: Hsu, H. T. & Lawson, R. H. (eds.). Virus- thrips-plant interactions of tomato spotted wilt virus. Proc, USDA workshop. ARS-87. Beltsville. p. 21-27. Paliwal, Y. C. 1974. Some properties and thrip transmission of tomato spotted wilt virus in Canada. Can. J. Bot. 52: 1177-1182. 1976. Somecharacteristics of the thrip vector relationship of tomato spotted wilt virus in Canada. Can. J. Bot. 54: 402-405. Reddy, D. V. R„ Amin, P. W., Mc Donald, D. & Ghanekar, A. M. 1983. Epidemiology and control of groundnut bud necrosis and other diseases of legume crops in India caused by TSWV. In: Plumb, R. T. & Thresh, J. M. (eds.). Plant virus epidemiology. Oxford, p. 93-102. Sakimura, K. 1961.Techniques for handling thrips in trans- mission experiments with the tomato spotted wilt virus. PI. Dis. Rep. 45: 766-771. 1962. The present status of thrips-borne viruses. In: Ma- 193 Aghc. Sei. Fint. 2 (1993) Research Note ramorosch, K. (ed.). Biological transmission of disease agents. New York. p. 33-40. South African Thysanoptera. J, Ent. Soc. S. Afr. 23: 321-367. 1963. Frankliniella fusca, an additional vector for the tomato spotted wilt virus, with notes on Thrips labaci , another vector. Phytopath. 53: 412-415. Manuscript received February 1993 Zawirska, I. 1976. Untersuchungen liber zwei biologische typen von Thrips tabaciLind. (Thysanoptera, Thripidae) in der VR Polen. Arch. Phytopath. Pfl.schutz. Berlin. 12: 411-422. Anne Lemmetty Isa Lindqvist Agricultural Research Centre of Finland Institute of Plant Protection Zur Strassen, R. 1960. Cataloque of the known species of FIN-31600 Jokioinen,Finland SELOSTUS Tupakkaripsiäinen, Thrips tabaci (Lind.), tomaatin pronssilaikkuviruksen (TSWV) toinen vektori Suomessa Anne Lemmetit ja Isa Lindqvist Maatalouden tutkimuskeskus Tomaatin pronssilaikkuvirus on aiheuttanut suuria vahinkoja eri puolilla maailmaa vihannes- ja koristekasviviljelmillä. Suomessa virus löydettiin ensimmäisen kerran vuonna 1989 tomaatista jakrysanteemista sekä vuonna 1992 sineraariasta. Virusta levittävät aikuisetripsiäi set, jotkaovat toukkavaihees - sa syöneet viroottista kasvia. Tehokkaimpana viruksen levittäjänä pidetään kalifomian - ripsiäistä (Frankliniella occidentalis), mutta kuuden muun ripsiäislajin on myös todettu siirtävän virusta. Tupakkaripsiäi ■ sen siirrostuskyvystä on kuitenkin ristiriitaisia tietoja. Laboratoriokokeen tarkoituksena oli selvittää, siirtääkö tu pakkaripsiäinen TSWV:tä. Kokeessa käytetty ripsiäispopu ■ laatio kasvatettiin terveissä astereissa laboratorio-olosuhteis- sa. Tutkittu virusisolaatti oli eristetty sineraariasta. Häkkiko- keessa tupakkaripsiäistoukat siirrettiin TSWV;n infektoimille kasveille, joita ne saivat vapaasti syödä. Kun koteloituneista toukista alkoi kehittyä aikuisia, häkkeihin siirrettiin terveitä astereita syöttikasveiksi. Kasvien viroottisuus testattiin ELISA-menetelmää käyttäen. Kokeessa todettiin tupakkaripsiäisen siirtäneen TSWV:n infektoituneista kasveista terveisiin kasveihin. Tupakkaripsi- äinen on ainoa maassamme luontaisesti esiintyvä TSWV:n vektorilaji. Sen yleisyyden huomioon ottaen se on kokeen perusteella huomionarvoinen TSWV:n siirtäjä Suomessa. 194 Agric. Sei. Fin!. 2(1993)