1Universidade de Trás-os-Montes e Alto Douro, Portugal; 2Escola Superior Agrária de Ponte de Lima, Portugal; 3Wageningen Agricultural University, The Netherlands

Background and objectives
Nepoviruses have a wide host range of plant species including woody and herbaceous plants. The symptoms produced by these viruses consist of a mosaic or mottling usually forming concentric rings or line patterns on systemic infected leaves, and chlorotic and necrotic spots or rings on the inoculated leaves. The latter symptoms are often found on indicator plants such as Nicotiana sp. The nepoviruses [1] consist of isometric particles with a size of ca 28 nm, a coat protein with of Mr ca 60,000, and a genome divided over two RNA species. They are transmitted by dorylaimid nematodes. In north Portugal, lettuce plants with necrotic spots were found in greenhouse crops. Studies were made to characterize the virus, which appeared to be a nepovirus serologically related to arabis mosaic virus (ArMV).

Materials and methods
Extracts of infected lettuce plants were mechanically inoculated to several test plants. The virus was purified from infected Nicotiana rustica plants, showing ringspots, according to the procedure described by Rana et al. [2], with some modifications. Purified virus was used to produce polyclonal antiserum. Rabbits were injected three times, intradermally and subcutaneously, with an emulsion of purified virus and incomplete Freund´s adjuvant. Serological relationships of this virus with other nepoviruses were determined by dot-blot ELISA. The size of the coat protein was estimated by SDS-PAGE in a 12% polyacrylamide gel. The RNA was extracted from purified virus, isolated by 1% SDS, followed by phenol extraction and ethanol precipitation, and analysed on 1% agarose gels.

Results and conclusions
Observations made by EM on leaf-dip preparations of infected lettuce plants showed isometric particles with a size varying between 25 and 30 nm. Analyses of soil samples from the infected greenhouses revealed the presence of Xiphinema diversicaudatum and a few specimens of some other Xiphinema species (PAA Loof, Dept. Nematology, Wageningen Agricultural University). Dot-blot tests were made with antisera to 12 different nepoviruses (ToRSV, TRSV, TBRV, AYRSV, AILV, GTSV, PBRSV, CLRV, ArMV, RrSV, SLRSV, ToTNV) with infected leaf material, as well as purified virus. Except for ArMV, none of the antisera tested gave a positive response. A host range study was done on Chenopodium quinoa, Petunia hybrida, Nicotiana clevelandii, N. rustica, N. benthamiana, N. glutinosa, N. tabacum cvs White Burley and Samsun, and Datura stramonium. The symptoms obtained were compared with those produced by ArMV. They differed on D. stramonium, N. rustica and N. tabacum cv. Samsun. The virus from lettuce induced systemic infections on D. stramonium and local rings on the Nicotiana sp., while ArMV did not produce any reaction on these plants.

The virus was purified from N. rustica primary infected leaves. A protein with an estimated size of 58.5 kDa, presumably the coat protein, was found on the polyacrylamide gels. Analysis of the RNA on non-denaturating agarose gels showed that the virus contained two different RNAs, with estimated sizes of 3.5 and 6.7 kb. These RNAs are smaller than the ArMV RNAs, for which values of 4 and 9 kb have been reported.

We conclude that a thus far unknown nepovirus which is serologically related to ArMV has been found in lettuce cultivation in the north of Portugal. Studies are in progress to further compare the properties of ArMV and the virus isolated, as well as its molecular characterization. This virus is tentatively designated lettuce necrotic spot virus.

1. Mayo MA, Robinson DJ, 1996. In Harrison BD, Murant AF, eds, The Plant Viruses: Nepoviruses: Molecular Biology and Replication. Plenum, New York.
2. Rana GL, Galitelli D, Kyriakopoulou PE et al., 1980. Annals of Applied Biology 96, 177-185.