VIRUSES RECORDED IN PORTUGAL IN TOMATO PROTECTED CROPS. J. C. Sequeira Estaçâo Agronómica Nacional INIA 2780 OEIRAS Portugal

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1 VIRUSES RECORDED IN PORTUGAL IN TOMATO PROTECTED CROPS M. Lourdes V. Borges Estaçâo Agronómica Nacional INIA 2780 OEIRAS Portugal J. C. Sequeira Estaçâo Agronómica Nacional INIA 2780 OEIRAS Portugal Abstract Tobacco mosaic virus is the more common virus detected in tomato protected crops in Portugal. Tomato bushy stunt virus was detected only once in a greenhouse in the Algarve region. A geminivirus, named tomato leafroll virus, recently detected in tomato field crops in a Southern area of the Country, is considered potentially important for protected crops as it may be transmitted by fresh seed and the symptoms in plants in the glasshouse are different from the symptoms shown by field plants. This virus is thought to be transmitted by leafhoppers. Tobacco mosaic virus has been identified on the basis of its virion morphology and by the symptoms caused in test plants like Niootzana glutinosa L. and N. tabacum L. Tomato bushy stunt virus was identified by the symptoms caused in several test plants, virion morphology, host ultrastructure and serological tests. Tomato leafroll virus was purified from infected plants in the field and anti-serum was obtained by rabbit injection. Gel-diffusion tests proved that the virus is closely related to beet curly top virus and enzyme-1inked immunosorbent assay enabled the virus antigen to be detected in tomato and pepper and at lower concentration in potato, in some weeds and in leafhopper (Empoasaa sp.) extracts. Plants grown from fresh seed collected in infected fields showed symptoms of the disease. The identification of the virus was further confirmed by abnormalities in the nuclei of infected cells and the results obtained, mainly in serological tests, led to the conclusion that the virus is probably a strain of beet curly top virus. Some protection measures are mentioned in order to avoid the diseases caused by the viruses described. 1. Introduction Survey work carried out since many years ago in tomato field crops has been showing a decrease in the incidence of tobacco mosaic virus, which became very rare, and some increase of cucumber mosaic virus which, however, has never caused serious problems. Other viruses have been occasionally detected such as potato viruses X and Y. Some rhabdovirus particles have also been observed in tomato cells under the electron microscope. In the last few years, in a very limited area of Alentejo (Southern part of Portugal) a virus disease named tomato leaf roll has been detected in tomato fields. It was found to be caused by a geminivirus, closely related to beet curly top virus, and affecting also pepper plant s. As the symptoms in plants kept in the greenhouse are different from the symptoms in the fields and the virus seems to be transmitted by fresh seed, it is potentially dangerous for protected crops. Acta'Horticulturae, 191, Solanacea in Mild Winter

2 In tomato protected crops the more frequent virus is tobacco mosaic virus although tomato bushy stunt virus was also detected once in a greenhouse in the Algarve. Besides presenting the results that led to the identification of the viruses described, we point out aspects concerning epidemiology and protection measures. 2. Material and methods Tomato plants (Lyoopersioon esoulentwn Mill.) with symptoms of leafroll have been collected from fields in some confined areas of Alentejo, 20 to 30 Km from the coast. Tomato plants in protected crops were collected in greenhouses and glasshouses mainly in the centre and South of Portugal but sometimes in northern areas. Plants for inoculation experiments were grown from seed and kept in a glasshouse. Purification of tomato leafroll virus was done by using the method described by Larsen & Duffus (1984) for beet curly top virus with some minor modifications. The suspension obtained was used for electron microscopy, for anti-serum preparation and for centrifugation in density gradients. Virus particles in purified preparations or in sap were observed in the electron microscope Philips EM 300 at 80 KV. The grids coated with Formvar were stained with ammonium molibdate or with uranyl acetate after keeping a droplet of the virus preparation on a grid for a few seconds and draining with filter paper; the stain was also drained with filter paper. The preparation of thin sections for ultrastructure studies was done as described by Borges et at.(1981). 3. Results 3.1. Tobacco_mosaic_virus This virus has been frequently detected all over the Country in tomato plants, in protected crops, were it may cause different kinds of symptoms according to the strain of the virus and the environment. The symptoms can vary from mild to strong mosaic and from mild to severe leaf distortion, sometimes with strong reduction in the growth of the leaf tissue (Fig.l). It is a very infectious virus readily transmitted by mechanical means such as manipulation of plants or instruments. The identification of tobacco mosaic virus was based on the morphology of the virions (Fig.2) and on the symptoms in Niaotiana gtutinosa (local necrotic lesions about 48 h after inoculation), and in N.tabaeum (systemic mosaic about one week after inoculation). Due to the high concentrations of virus in the host plants, the virus particles could be seen in preparations of sap and, therefore, purification or concentration of the virus was not usually needed for identification Tomato_bushY_stunt_virus This virus was detected only once in protected crops in the Algarve region. The affected tomato plants showed mainly yellow leaf discolouration and stunting (Fig.3); in some leaves, necrotic spots surrounded by yellow areas were also noticed. 294

3 The identification of the virus was based on symptoms in indicator plants, morphology of the virions, ultrastructural aspects of infected cells and serology (Borges et at., 1979). Sap inoculation tests, showed that the virus causes local necrotic lesions in Oaimum basilioum L., Niootiana glutinosa L., Tetragonia expansa Thunb. and Gomphrena globosa L.. In pepper (Capsicum annuum L.) and tomato, systemic lesions were obtained whereas datura tatula L. showed both local and systemic lesions. The symptoms were found to be highly dependent on environment conditions as they varied considerably all over the year. Virions in purified preparations or in sap showed isometric morphology with c. 27 nm in diametre (Fig.4). Ultrastructure of pepper infected cells revealed the accumulation of orderly arranged virions in the cytoplasm, usually near the nuclei (Fig.5). In serological tests, the virus, in purified preparations or in sap, reacted both with the homologous anti-serum and with anti-serum to B-3 strain (Kindly supplied by Dr. 0. Lovisolo) Tomato leafroll_virus This virus has been identified recently (Sequeira & Borges,1985) as a geminivirus related to beet curly top and affects several vegetable crops. It is confined to some areas in Alentejo and the symptoms in the field plants (Fig.6), consist of rolling of the leaflets, mainly the younger ones, which become brittle and get a yellowish colour. The plants show some stunting which can be severe in early infections. Enzyme-linked immunosorbent assay ELISA) enabled the ' virus to be detected not only in tomato but also in pepper, potato and two weeds (Physalis sp. and Solanum nigrum L.). The vector is thought to be a leafhopper as the virus is closely related to beet curly top virus which has leafhoppers as vectors. Some preliminary results indicate that leafhoppers of the genus Empoasoa might be vectors of the virus. In fact, extracts of Empoasoa sp., collected in infected pepper plants in the field, were found to contain some virus antigen, as shown by enzyme-linked immunosorbent assay (ELISA) (Table 1), and these leafhoppers kept on healthy pepper plants induced the appearance of symptoms in some of them. In tomato plants kept in a greenhouse the symptoms induced by this virus were quite different from the symptoms in field plants.a few days after grafting infected material on healthy tomato plants some leaflets began to bend upwards like a hook. Later, the leaflets became twisted, and some of them had a shape of a corkscrew (Fig. 7). The difference in symptoms observed gives to this virus particular importance as it may be present in protected crops with mild expression of symptoms and attention should be drawn for this aspect. The identification of the virus was based mainly on the results of electron microscopy. In fact, the presence of the typical geminate particles (Fig.8) in purified preparations and some abnormalites in the nuclei of infected cells (Fig.9), led to the conclusion that a geminivirus was present. Serological tests of gel diffusion (Fig. 10), using anti-serum to a strain of beet curly top virus (kindly supplied by Dr. J. Duffus), showed that tomato leafroll virus is closely related to beet curly top virus, possibly a strain of this virus. Anti-serum to tomato leafroll virus was obtained by injecting a 295

4 rabbit with purified preparations. This anti-serum enabled several serological tests to be done. The results of ELISA (Table 1), show that antigen to the virus was present at high concentration in tomato and pepper. Lower concentrations were detected in potato, Physalis sp. and Solarium nigirum L., collected near infected tomato plants in the field. Fruits collected from infected tomato plants in the field were used for seed-transmission experiments. The seed was removed and used fresh or dry. Some of the plants grown from fresh seed showed symptoms typical of the disease. These results indicate that seed-transmission of tomato leafroll virus may occur when fresh seed is used. 4. Discussion The results of this work showed that tobacco mosaic virus is by far the more common virus in tomato protected crops, in Portugal. This can be explained taking into account the very high infectiousness of the virus, the efficient way of transmission by contact and the frequent manipulation of plants and instruments when a protected crop is set up. As this virus is not transmitted by aphids, its incidence could perhaps be strongly reduced by drawing attention of the people working with nursery plants for the means of transmission of the virus and for the need of desinfecting instruments and the hands during manipulation of the plants. Tomato bushy stunt virus has been reported as propagated possibly by fungi (Martelli et al., 1971) or by the water (Tomlinson, 1982). Therefore, transmission of the virus might occur during plant watering. We think that the only case of infection so far recorded in Portugal, in the Algarve region, does not give sufficient importance to this virus to consider it as dangerous for protected crops in the Country. Probably the low efficiency of the means of transmission is the cause of such a low incidence of the virus. Nevertheless, people should be aware of measures to be adopted once the virus is detected. Protection measures should involve mainly the elimination of infected plants as well as care during plant manipulation as the virus is mechanically transmitted. Although tomato leafroll virus has only been detected so far in field crops, we think that it is potentially important for protected crops for several resons: the virus is probably a strain of beet curly top virus and therefore should have numerous hosts among vegetables and weeds; it seems to be seed-transmitted when fresh seed is used and is thought to be transmitted by leafhoppers; symptom expression in the greenhouse is very different from symptom expression in the fields and this can be misleading in the identification of the virus. References Borges, M.L.V., Sequeira, J.C. & Louro, D., Aparecimento em Portugal do virus do emanjericado do tomateiro (tomato bushy stunt virus). Hospedeiros, morfologia e localiza^ao nas celulas de pimenteiro. Phytopathologia mediterranea 18: Borges, M.L.V., Sequeira, J.C. & Louro. D., Potyviruses recorded in Portugal. Purification, serology and host-virus ultrastructural relationships. Boletim da Sociedade Broteriana 53: Larsen, R.C. & Duffus, J.E., A simplified procedure for the purification of curly top virus and the isolation of its monomer and dimer particles. Phytopathology 74:

5 Martelli, G.P., Quacquarelli, A. & Russo, M., Tomato bushy stunt virus. CMI/AAB Descriptions of Plant Viruses Number 69. Sequeira, J.C. & Borges, M.L.V.,1985. A leafroll disease of tomato in Portugal. Etiology, virus isolation and characterization. Abstracts of the 5th Conference of the ISHS-Vegetable Virus Working Group, Bet Dagan, Israel, September Tomlinson, J.A., Studies on the ecology of tomato bushy stunt virus. Abstracts of the 4th Conference of the ISHS-Vegetable Virus Working Group, Wellesbourne, U.K., September Table 1 - Tomato leafroll virus antigen in leaf sap and in leafhopper extracts tested by ELISA \o5nm Tomato in the field 0.43 to 1.50 Tomato in the greenhouse With symptoms 0.60 With no symptoms 0.19 Other plants in the field Potato 0.25 to 0.60 Pepper 0.10 to 0.86 Solarium nigrum 0.23 Physalis sp Leafhoppers (Enrpoasoa sp.)

6 Fig.2 - Vir ions of tobacco mosaic virus. Fig.3 - Symptoms of tomato bushy stunt virus. 298

7 Fig.4 - Virions of tomato bushy stunt virus. Fig.5 - Ultrastructural aspect of'a cell with tomato bushy stunt virus. Virions are orderly arranged near the nucleus. 299

8 Fig.6 - Tomato plants with tomato leafroll symptoms in the field. Fig.7 - Tomato plants experimentally infected with tomato leafroll virus and kept in the glasshouse. Leaf with typical symptoms. 300

9 Fig.8 - Morphological aspects of tomato leafroll virus particles in a purified suspension. Fig.9 - Ultrastructural aspects of a cell with tomato leafroll virus. 301

10 Fig.10- Gel-diffusion test using tomato leafroll virus antiserum (A), beet curly top virus antiserum (B) and a purified suspension of tomato leafroll virus (V). 302