We were able to mechanically transmit a small isometric virus from field tomato samples showing severe necrotic symptoms, collected in the Culiacan area of Sinaloa state (Mexico). After gradient purification and three rounds of single-lesion passage on Chenopodium quinoa, the virus was back-inoculated to tomato plants and reproduced the original apical necrosis symptoms. The virus could be transmitted to a wide range of experimental hosts, including a number of solanaceous plants. Purified virus was used to produce specific polyclonal rabbit antibodies and serological tests such as enzyme-linked immunosorbent assay, Western blot analysis, and an immunochromatographic lateral flow assay. Such assays confirmed the wide distribution of this virus in symptomatic field plants in the area of the epidemic. Purified particles contained two genomic RNA molecules of ca. 7 kb (RNA1) and 5 kb (RNA2) estimated length. Analysis of clones from a cDNA library provided 6.5 and 3.0 kb of sequence for RNA1 and RNA2, respectively. Sequence analysis of the encoded replicase showed greatest similarity with members of the Sequiviridae family, and indicated that the virus we isolated is a new virus species, provisionally named Tomato apex necrosis virus.
Four different viral species were isolated from diseased Ranunculus asiaticus plants growing in Imperia Province (Italian Riviera-Liguria Region). Infected plants exhibited mosaic symptoms and growth abnormalities. The viruses were mechanically inoculated to a range of herbaceous hosts and differentiated biologically. Long flexuous particles were present in leaf dip extracts observed by electron microscopy. A general protocol for the amplification of potyvirus genome fragments through reverse transcription-polymerase chain reaction generated products that were cloned and sequenced. Sequence and phylogenetic analysis suggested that three of these isolates could be considered new viral species belonging to the genus Potyvirus. The fourth isolate is a new member of the genus Macluravirus. Purified virus was used as antigen to produce a specific polyclonal antiserum in rabbit; serological features were established through double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), antigen coated plate (ACP)-ELISA, and western blot analysis. DAS-ELISA was highly specific for each virus isolate, whereas some cross-reactivity was shown in ACP-ELISA and western blot analysis. Aphid transmission by Myzus persicae was demonstrated in a controlled environment for each of the four viral isolates, whereas no transmission through seed was observed.
Methods are described for pre-and post-embedding immunogold labeling of mycoplasmalike organisms (MLOs) in thin sections of infected plants. Antisera against primula yellows (PY), tomato big bud (TBB) and bermudagrass white leaf (BGWL) MLOs, and a monoclonal antibody (mab) against PY were tested with the three serologically unrelated MLOs. Labeling was specific for each MLO and was localized to the outer surface of the MLOs. The antisera performed well in both pre-and post-embedding experiments; the mab reacted well in pre-embedding conditions but gave no labeling with post-embedding. Glutaraldehyde fixation reduced levels of labeling in post-embedding conditions. The results show that these techniques can be used to differentiate MLOs reliably, and extend the usefulness of electron microscopy in this area.
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