Comparison of monoclonal antibodies, DNA probes, and PCR for detection of the grapevine yellows disease agent.
Two monoclonal antibodies (MAbs) specific for a grapevine yellows mycoplasma-like organism (GY-MLO) were produced by fusing mouse myeloma cell lines NS1/1 to spleen cells of mice immunized with partially purified GY-MLO from diseased periwinkles. Using ELISAs and indirect immunofluorescence (IF) staining, the MAbs were shown to be specific for the GY-MLO. Although both ELISA and IF tests were satisfactory with diseased periwinkles (Catharanthus roseus), serological detection of GY-MLO in diseased grapevines was not always reliable because of the extremely low titer of GY-MLO in the phloem and the interference of plant pigments in dot blot immunoassays. Specific DNA probes and primers were developed for use in polymerase chain reaction (PCR) for the detection of the GY-MLO. An enriched preparation of GY-MLO chromosomal DNA was obtained by bisbenzimide-CsCl buoyant density gradient centrifugation of the total DNA extracted from diseased periwinkles. The enriched DNA was cloned into pUC19 and used to transform Escherichia coli DH5α cells. Two recombinant plasmids (pGYD-1 and -2) reacted specifically to GY-MLO DNA and contained 9.0- and 1.6-kb inserts, respectively. Based on the partial sequence of the cloned genomic DNA fragment GYD-2, 3 oligonucleotides (oligos 1, 2 and 3) were designed and synthesized for use as primers in PCR. Using 3 specific DNA probes and dot hybridizations, positive results were observed with 10 ng of total DNA from GY-MLO-infected periwinkles. PCR could detect GY-MLO when only 10-2 pg of DNA from the same source was used as template. The labelled DNA probes were used to successfully detect GY-MLO in grapevine samples collected from Italy and the USA. Using oligos 1 and 2, a 550-bp DNA fragment was amplified from crude DNA extracts of infected periwinkles and grapevines. Employing oligos 1 and 3, a 600-bp DNA fragment was amplified only from infected periwinkles, not from diseased grapevines. No PCR products were detected when DNAs from healthy periwinkles or grapevines were used as templates. Results from PCR suggested that related but distinct strains of GY-MLO must exist, causing similar yellows symptoms in grapevines. An inverse relationship was also observed between symptoms of yellows and results from both dot hybridization and PCR. Almost all wild grapevine (Vitis riparia) samples collected near vineyards in New York showed a positive association with GY-MLO. Although none of the V. riparia samples showed GY symptoms, they may serve as an important alternative host for the GY causal agent.