Hybridization analysis of chrysanthemum stunt viroid with complementary DNA and the quantitation of viroid RNA sequences in extracts of infected plants.

In order to develop a method for the quantitation of viroids in extracts of infected plants by hybridization analysis with labeled complementary DNA (cDNA), the kinetics of hybridization of chrysanthemum stunt viroid (CSV) with [32P]cDNA were investigated. Two buffer systems were used, each with a total cation concentration of 0.19 M, with one containing no formamide (Buffer A) and the other 40% (v/v) formamide (Buffer B). Initial experiments with Buffer A at 60 degrees showed that the rate of hybridization of [32P]cDNA was slowest with the circular form of CSV, fastest with nuclease S(1)-treated CSV, while linear CSV gave an intermediate rate. This variation in rate was considered to reflect the variation in residual secondary structure of the three forms of CSV under the conditions used. By contrast, the three forms of CSV hybridized with [32P]cDNA at essentially the same rate in Buffer B at 50 degrees , conditions under which no viroid secondary structure remained. A linear relationship was observed between the temperature of hybridization and the log R (0)t (1 2 ) of cDNA:RNA hybrids formed in both buffer systems; the rate of hybridization decreased 50-fold as the hybridization temperature was decreased from 10 to 25 degrees below the T(m) of the hybrids. No such relationship was found when similar experiments were carried out using chrysanthemum 7 S RNA and the satellite RNA of cucumber mosaic virus. The optimal hybridization conditions of Buffer B at 50 degrees were chosen to quantitate the levels of CSV in CSV-infected chrysanthemums and Gynura aurantiaca; from the concentrations found in partially purified nucleic acid extracts of these plants, it was calculated that CSV sequences were present at 1.65 and 0.095 mg/kg of plant material, respectively.