Diagnosis of Flame Chlorosis, a Viruslike Disease of Cereals, by Detection of Disease-Specific RNA with Digoxigenin-Labeled RNA Probes. S. Haber, Research Station, Agriculture Canada, Winnipeg, Manitoba R3T 2M9 Canada. D. A. Wakarchuk, S. Cvitkovitch, and G. Murray. Phero Tech Inc., Delta, British Columbia V4G 1E9 Canada; Research Station, Agriculture Canada, Winnipeg, Manitoba R3T 2M9 Canada; and Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2 Canada. Plant Dis. 76:590-594. Accepted for publication 22 January 1992. Copyright 1992 The American Phytopathological Society. DOI: 10.1094/PD-76-0590.

Flame chlorosis (FC) is a novel, viruslike disease of cereals for which FC-specific double-stranded RNAs (FCdsRNAs) have been identified. Visual symptoms of the disease are distinctive, but definitive diagnosis requires examination of cytopathology and/or the detection of FCdsRNA. FC-specific RNA (FC-RNA) was detected in 1.5-mm leaf disks using labeled RNA probes made from cDNA clones of the FCdsRNAs. Crude leaf extract was prepared in a citrate/formaldehyde buffer, heated at 95 C for 10 min, cooled on ice, and briefly centrifuged, and the supernatant was applied to a nylon or polyvinylidene fluoride membrane with a dot or slot blotting manifold. Blotted samples were fixed by being heated for 3 min in a microwave oven and hybridized with digoxigenin-labeled RNA probes made from cDNA clones. After incubation with antidigoxigenin-phosphatase and reaction with chromogenic substrate, the intensity of colored spot or band formation was relative to the level of FC-RNA in the original sample. The assay is useful as a research tool as well as for clinical diagnosis because the small sample size allows repeated multiple sampling from plants or comparison of adjacent chlorotic and green areas on the same leaf.