European Diagnostic Protocols (DIAGPRO) for Citrus tristeza virus in Adult Trees

Abstract

DIAGPRO is a European Union (EU)-funded project to develop and validate European schemes for detection and diagnosis of a broad range of important organisms harmful to plants, according the EU Plant Health Directive (2000/29 as amended). Citrus tristeza virus (CTV) is included, and here are reported the results of a study to compare and standardize diagnostic methods for this virus. The most appropriate protocols were found to be graft inoculation of Mexican lime, sweet orange and grapefruit seedlings, DAS and DASI-ELISA, tissue print-ELISA, immunocapture RT-PCR and immunocapture nested RT-PCR in a single closed tube (conventional, print and squash capture formats). Samples of adult trees were tested during the different seasons (springtime gives the highest CTV titers) using five young shoots or fruit peduncles or 10 fullyexpanded leaves. Five flowers or fruits per tree, when available, were also found appropriate for reliable detection in recently infected adult trees. Different commercial polyclonal antibodies and the mixture of monoclonal antibodies (MCAs) 3DF1 and 3CA5 are recommended after calibration for conventional ELISA; the same MCAs are recommended for tissue print ELISA, and the primers of Olmos et al., 1999 (NAR 27, 1564-1565) for RT-PCR variants. A combination of two selected methods based on biological indexing, serological or molecular detection, will be required to officially declare a positive detection. Tissue print-ELISA using specific MCAs was found to be the most reliable, simple and economic method for routine analysis of plant material. The protocols and reagents have been ring tested and are published at http://www.csl.gov.uk/prodserve/know/ diagpro/ctv.pdf, an Internet website for comments before publication in the EU Official Bulletin.