SUMMARYAn indirect ELISA is described in which (i) virus is trapped by F(ab')2 fragments of specific IgG immobilized on a solid phase support, (ii) trapped virus is detected by intact IgG (from the same or a different antiserum) and (iii) positive reactions are identified using an enzyme conjugate specific for the Fc portion of IgG. Pepsin digestion of the Fc portion of the trapping antibody permits the use of a general purpose enzyme conjugate to discriminate between trapping and detecting antibody. Consequently, the assay requires only a single virus-specific antiserum which is often all that is available to the plant virologist. The assay was at least as sensitive for detecting small amounts of antigen as the standard double-antibody sandwich procedure and, for some viruses, two-to fourfold more sensitive. The improvement in performance resulted largely from lower and more consistent background reactions. Both assays were equally effective in revealing heterologous reactions when optimized for detecting homologous antigen. However, increased cross-reactions were obtained in the F(ab')2 procedure by the use of higher concentrations of detecting antibody. The assay is considered particularly suited for comparing antisera from different sources or of different bleeds from the same source, and for investigations involving so few tests that the effort or expense of preparing individual virus-specific conjugates is not justified.

Three nurseries produced apple rootstocks (M9) and budwood (cv. Royal Gala), which they exchanged at the end of the first year. Each nursery then budded its own budwood onto the rootstocks it had produced and that from the other two nurseries. Budded trees were grown on for a further year before being planted at HRI, East Malling in southern England; NIHPBS, Loughgall in Northern Ireland; and ADAS, Rosemaund in the West Midlands of England. Canker development was monitored twice a year. The position of the infected trees within the orchard was recorded, as was the position of the canker on each tree (main-stem or peripheral). Nectria galligena was isolated from representative cankers and analysed using molecular techniques. At the sites in Northern Ireland and HRI there was a strong positional effect, especially of peripheral cankers, indicating that most of the inoculum was external and had been spread from neighbouring orchards. There was little or no positional effect on main-stem cankers at any of the three sites. The proportions of different isolates taken from peripheral cankers was different in Northern Ireland from that in England, suggesting different populations associated with the geographic areas. In contrast, the populations of N. galligena obtained from main-stem cankers were very similar in England and Northern Ireland. It was concluded that a small proportion of trees developing canker were infected during propagation, with no symptom development until after planting. In a second trial it was demonstrated that trees infected during the propagation phase, and particularly at budding and heading back, could develop canker up to 3 years later. While it is clear that some canker developing in the orchard can be associated with the nursery of production, in climatic conditions conducive to the formation and dissemination of conidia, inoculum from surrounding infected orchards is the primary source of the pathogen. Aerial spread is therefore an essential element of the epidemiology of N. galligena , and its control is a crucial part of any canker-control programme.

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The mechanisms by which phytoplasmas interact with their hosts are not understood. Mollicute membrane proteins may play a role in such interactions and therefore the amp genes encoding immunodominant proteins from two phytoplasmas, aster yellows and clover phyllody, which fall within the largest taxonomic subclade of the phytoplasmas, have been cloned and characterized. The putative translation products, antigenic membrane proteins (Amps), of these genes have properties which are typical for bacterial membrane proteins, and which suggest that each has a single large extracellular hydrophilic domain held by a transmembrane region near the C-terminus, with only a short C-terminal intracellular sequence. Both of the Amps characterized here have bacterial leader sequences which are cleaved during maturation. Whilst the signal peptide and transmembrane regions of the two proteins are very similar, the major hydrophilic domains are highly divergent in both size and sequence. The Amps from the two phytoplasmas are also different in structure and sequence from the immunodominant membrane proteins of three other phytoplasmas whose genes have been cloned previously.

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