QUANTIFICATION OF DNA FROM THE ASTER YELLOWS MYCOPLASMALIKE ORGANISM IN ASTER LEAFHOPPERS (MACROSTELES FASCIFRONS STAL) BY A COMPETITIVE POLYMERASE CHAIN-REACTION

A rapid and accurate assay was developed for the quantification of the aster yellows plant pathogenic mycoplasmalike organism (MLO). The ass ay was based on the competitive polymerase chain reaction (PCR) techni que using the MLO 23S ribosomal gene and adjacent sequence as target t emplate. 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 st ringency. Coamplification of MLO and internal standard DNA provided ac curate quantification of 1 to 10(7) copies of MLO target DNA. The assa y was used to quantify the aster yellows MLO in aster leafhoppers (Mac rosteles fascifrons Stal) after the insects had fed on MLO-infected pl ants. Female leafhoppers accumulated MLOs much faster than males durin g the first 15 days following feeding. From 20 to 39 days after initia l exposure, the concentration of MLOs in male leafhoppers remained app roximately half that of females. Initially, the difference in MLO conc entration between male and female leafhoppers may have been due to a g reater amount of feeding, and therefore MLO uptake, by female leafhopp ers. Later, the difference may have been related to the larger body we ight of female leafhoppers. By 39 days after infection, the MLO popula tion 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 l eafhoppers.