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United States Department of Agriculture

Agricultural Research Service

Title: Fast, Easy and Efficient DNA Extraction and One-Step Polymerase Chain Reaction for the Detection of Xylella Fastidiosa in Potential Insect Vectors

Authors
item HUANG, QI
item Bentz, Jo Ann
item Sherald, J - NAT'L PK SERV., WASH, DC

Submitted to: Journal of Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 5, 2005
Publication Date: January 6, 2006
Citation: Huang, Q., Bentz, J. and Sherald, J.L. 2006. Fast, easy and effecient DNA extraction and one-step PCR for the detection of Xyllela fastidiosa in potential insect vectors. 88:77-81.

Interpretive Summary: X. fastidiosa is a slow growing, xylem inhabiting and nutritionally fastidious bacterium, and transmitted by insects such as sharpshooter leafhoppers and spittlebugs. The bacterium is associated with bacterial leaf scorch and decline in many economically important landscape trees and shrubs including oak, elm, sycamore, maple, and oleander. Currently, no effective therapy for infected plants or a strategy for prevention of infection is available. One of the limited control strategies for diseases caused by X. fastidiosa is identification and control of insect vectors. Identification of insect vectors, however, is hampered by the lack of fast, easy and sensitive detection methods. In this study, we describe a rapid, technically easy and convenient method that employs a commercially-available kit for the extraction of high-quality DNA from insects, followed by a one-step, DNA-based detection protocol for efficient detection of X. fastidiosa in potential insect vectors. The procedure does not require the use of organic solvents or precipitation of nucleic acids with alcohol. Also it does not need additional purification or enrichment steps, and can be completed in less than a day. The procedure was used successfully in detecting X. fastidiosa in two potentially important leafhopper species, Graphocephala versuta and G. coccinea, and in a treehopper species Entilia concisa, collected from a nursery where bacterial leaf scorch disease caused by X. fastidiosa occurs. Our work will be of value primarily to plant pathologists, entomologists and clinicians interested in diseases caused by X. fastidiosa.

Technical Abstract: A quick, simple and efficient procedure for detecting Xylella fastidiosa in potential insect vectors is described. The procedure employs a commercially available DNeasy tissue kit for the extraction of high-quality DNA from the insect, followed by one-step polymerase chain reaction amplification using previously published, highly efficient oligonucleotide primers specific to X. fastidiosa. The procedure does not require the use of phenol, chloroform or alcohol for the precipitation of nucleic acids. Also it does not need additional purification or enrichment steps, and can be completed in less than a day. The procedure was used successfully in detecting X. fastidiosa in two potentially important leafhopper species, Graphocephala versuta and G. coccinea, and in a treehopper species Entilia concisa, collected from a nursery where bacterial leaf scorch disease caused by X. fastidiosa occurs.

Last Modified: 8/19/2014
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