Detection of elm yellows phytoplasma in elms and insects using real-time PCR

Abstract

A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (C T) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma. © 2010 The American Phytopathological Society.