Quantification of DNA from the Aster Yellows Mycoplasmalike Organism in Aster Leafhoppers ( Macrosteles fascifrons Stål) by a Competitive Polymerase Chain Reaction

Abstract

Summary A rapid and accurate assay was developed for the quantification of the aster yellows plant pathogenic mycoplasmalike organism (MLO). The assay was based on the competitive polymerase chain reaction (PCR) technique using the MLO 23S ribosomal gene and adjacent sequence as target template. Amplification from this region was MLO-specific, and DNA from several different aster yellows MLO strains yielded PCR products. For the internal standard, a PCR product was cloned from Bacillus cereus after amplification with the target primers under reduced annealing stringency. Coamplification of MLO and internal standard DNA provided accurate quantification of 1 to 10 7 copies of MLO target DNA. The assay was used to quantify the aster yellows MLO in aster leafhoppers ( Macrosteles fascifrons Stal) after the insects had fed on MLO-infected plants. Female leafhoppers accumulated MLOs much faster than males during the first 15 days following feeding. From 20 to 39 days after initial exposure, the concentration of MLOs in male leafhoppers remained approximately half that of females. Initially, the difference in MLO concentration between male and female leafhoppers may have been due to a greater amount of feeding, and therefore MLO uptake, by female leafhoppers. Later, the difference may have been related to the larger body weight of female leafhoppers. By 39 days after infection, the MLO population had reached stationary phase in the insect, and MLO DNA detected by the assay comprised 0.8–0.9% of the total DNA content of infected leafhoppers.