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Title: DEVELOPMENT OF A REAL-TIME, QUANTITATIVE PCR FOR DETECTION OF THE COLUMBIA BASIN POTATO PURPLE TOP PHYTOPLASMA IN PLANTS AND BEET LEAFHOPPERS

Author
item Crosslin, James
item Vandemark, George
item Munyaneza, Joseph - Joe

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/4/2006
Publication Date: 5/1/2006
Citation: Crosslin, J., Vandemark, G.J., Munyaneza, J.E. 2006. Development of a real-time, quantitative PCR for detection of the Columbia Basin potato purple top phytoplasma in plants and beet leafhoppers. Plant Disease. 90:663-667.

Interpretive Summary: The Columbia Basin potato purple top phytoplasma has been identified as a strain of the beet leafhopper transmitted virescence agent. This bacteria-like pathogen has caused disease outbreaks in the potato-producing regions of Washington and Oregon in the last several years. Detection and identification of the pathogen has traditionally required a molecular test called nested PCR. This procedure is quite reliable but requires a great deal of time and does not give information on how much of the pathogen is present in a sample. The research reported in this paper describes for the first time a real-time PCR test for detection of the pathogen in potato plants and the beet leafhoppers responsible for transmission of the phytoplasma. The test is relatively rapid and provides information on how much of the pathogen is present.

Technical Abstract: A quantitative, real-time TaqMan® polymerase chain reaction assay (qPCR) was developed which was capable of detecting and quantifying a group 16SrVI phytoplasma in DNA extracts prepared from infected tomatoes, potatoes, and beet leafhoppers (Circulifer tenellus Baker). Primers and probe were designed from the 16S-23S rRNA genes of the Columbia Basin potato purple top phytoplasma, which is closely related to the beet leafhopper transmitted virescence agent. The detection limit in phytoplasma-infected tomato DNA was approximately 50 pg. The detection limit of the assay with a plasmid clone of clover proliferation phytoplasma 16S-23S rDNA was approximately 500 ag. The concentration of phytoplasma varied considerably among potato plants showing symptoms of purple top. The pathogen was readily detected in extracts from single or groups of five beet leafhopper vectors. As with infected potatoes, the concentration of phytoplasma in individual leafhoppers was variable. The assay also detected aster yellows (group 16SrI) and pigeon pea witches-broom (group 16SrIX) phytoplasmas. The qPCR was at least as sensitive as the commonly used and more labor-intensive nested PCR for detection of the pathogen. To our knowledge this is the first report of qPCR of phytoplasma in potatoes and beet leafhoppers, and only the second report of qPCR of a group 16SrVI phytoplasma.