Detection and Variability of Aster Yellows Phytoplasma Titer in Its Insect Vector, Macrosteles quadrilineatus (Hemiptera: Cicadellidae)

Abstract

The aster yellows phytoplasma (AYp) is transmitted by the aster leafhopper, Macrosteles quadrilineatus Forbes, in a persistent and propagative manner. To study AYp replication and examine the variability of AYp titer in individual aster leafhoppers, we developed a quantitative real-time polymerase chain reaction 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 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 aster leafhopper from 0 to 9 d after a 48 h acquisition access period on an AYp-infected plant. Average AYp titer was measured in 107 aster leafhoppers and, expressed as Log10 (copies/insect), ranged from 3.53 (+/- 0.07) to 6.26 (+/- 0.11) occurring at one and 7 d after the acquisition access period. AYp titers per insect and relative to an aster leafhopper chromosomal reference gene, cp6 wingless (cp6), increased approximately 100-fold in insects that acquired the AYp. High quantification cycle values obtained for aster leafhoppers not exposed to an AYp-infected plant were interpreted as background and used to define a limit of detection for the quantitative real-time polymerase chain reaction assay. This method will improve our ability to study biological factors governing AYp replication in the aster leafhopper and determine if AYp titer is associated with frequency of transmission.

Interaction of Phytophagous Insects with Salmonella enterica on Plants and Enhanced Persistence of the Pathogen with Macrosteles quadrilineatus Infestation or Frankliniella occidentalis Feeding

“…F. occidentalis colonies were maintained in plastic deli cups under ambient temperature and a 16:8 (L:D) photoperiod as previously described [29]. A colony of Macrosteles quadrilineatus Forbes (Hemiptera: Cicadellidae) was maintained on oat ( Avena sativa L.) seedlings in a controlled environment with a 16:8 (L:D) photoperiod (24°C light; 19°C dark) [30]. A colony of Myzus persicae  Sulzer (Hemiptera: Aphididae) was kindly provided by Dawn M. Smith (Cornell University), and established and maintained on Chinese cabbage ( Brassica rapa ) under similar controlled conditions as the M. quadrilineatus colony on the campus of the University of Wisconsin, Madison, Wisconsin.…”

Small, Smaller, Smallest: The Origins and Evolution of Ancient Dual Symbioses in a Phloem-Feeding Insect

“…This species is a widespread pest of North American agriculture (Frost et al 2011). As found in other members of Deltocephalinae, it harbors Sulcia and the betaproteobacterial co-primary symbiont, Nasuia (Noda et al 2012; Koga et al 2013).…”

The complete mitochondrial genome ofMacrosteles quadrilineatus(Hemiptera: Cicadellidae)

“…One such species is the aster leafhopper, Macrosteles quadrilineatus, that is widespread throughout the North American continent. This species causes millions of dollars in agricultural and ornamental crop losses annually by vectoring the Aster Yellows phytoplasma that can infect hundreds of plant species (Hoy et al 1992;Frost et al 2011;Frost et al 2013). Macrosteles quadrilineatus relies on bacterial symbionts for the provisioning of essential amino acids that are limited in their phloem diets, which also helped the host exploit novel niches (Moran 2007).…”