Submitted to: Journal of Economic Entomology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2011
Publication Date: 12/1/2011
Citation: Frost, K., Willis, D.K., Groves, R. 2011. Detection and variability of Aster Yellows Phytoplasma Titer in its insect vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae). Journal of Economic Entomology. 104(6):1800-1816. Interpretive Summary: Aster yellows is an economically important disease of carrot in Wisconsin and other carrot growing areas of the U. S. The aster yellows disease organism is transmitted by a leafhooper that feeds on carrots. Current control measures consist of testing leafhoppers trapped in the field for the presence of the aster yellows pathogen using non-quantitative PCR. This is a time consuming method that does not address the relationship between the amount of pathogen within an insect and its ability to transmit the disease. We developed a quantitative PCR method to quantify the amount of pathogen within each insect. We established that the aster yellows pathogen replicates to a maximum level within 7 days of acquiring the pathogen and that male and female leafhoppers contain approximately the same amount of pathogen. The ultimate goal of this research is to establish the relationship between the amount of pathogen within an insect and it ability to transmit the disease. This information will be used to improve the current model of disease detection that will improve the current control strategies for the aster yellows disease.
Technical Abstract: The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper (ALH), Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual ALHs, we developed a quantitative, real-time PCR (qPCR) assay to measure AYp concentration in insect DNA extracts. Absolute quantification of AYp DNA was achieved by comparing the amplification of unknown amounts of an AYp target gene sequence, elongation factor TU (tuf), from whole insect DNA extractions, to the amplification of a dilution series containing known starting quantities of the tuf gene sequence cloned into a plasmid. The capabilities and limitations of this method were assessed by conducting time course experiments that varied the incubation time of AYp in the ALH from 0 to 9 days following a 48 hour acquisition access period (AAP) on an AYp source plant. AYp concentration was measured in 107 ALHs and the average titer, expressed as Log10 (copies/insect), ranged from 3.53 (±0.07) to 6.26 (± 0.11) occurring at 1 and 7 days post acquisition access. AYp titers per insect and relative to an ALH chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle (Cq) values obtained for individual ALHs not exposed to an AYp-infected plant were interpreted as background and used to define a diagnostic limit of detection for the qPCR assay. AYp titers measured using the tuf target sequence were significantly correlated (R = 0.95; p < 0.001) to titers measured using a second AYp gene target, lysyl-tRNA synthetase (lysS). This method will improve our ability to study the biological factors governing AYp replication in the ALH and determine if AYp titer is associated with frequency of transmission.