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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #157175
Title: DETECTION AND QUANTIFICATION OF PLUM POX POTYVIRUS IN APHID VECTORS BY REAL-TIME FLUORESCENT REVERSE TRANSCRIPTION-PCR

Author
item Schneider, William
item Sherman, Diana
item Stone, Andrew
item Damsteegt, Vernon
item Frederick, Reid

Submitted to: Acta Horticulture Proceedings
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/1/2004
Publication Date: 9/1/2004
Citation: Acta Horticulture 657:135-140

Interpretive Summary: Plum pox potyvirus (PPV) is a destructive pathogen of Prunus that is difficult to detect and control. A previously developed real-time PCR assay based not only improved PPV detection sensitivity and specificity, but also provided the ability to accurately quantify viral load in any given sample. This assay was used to compare virus acquisition in aphid vector species to virus acquisition in non-vectors. Both vectors and non-vectors acquired the virus, however, it was determined that vector species acquire 10-20 times more virus from infected tissue than aphids that are non-vectors. In addition, virus could occasionally be detected in single aphids, although not consistently. This discovery suggests that there are basic biological factors that determine whether or not an aphid species can vector PPV, and if these factors could be identified they may be exploited in combating the spread of viral diseases.

Technical Abstract: A real-time, fluorescent, reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of Plum pox potyvirus (PPV). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by visualization with ethidium-bromide. All strains of PPV were detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantification of viral template. The assay was used to measure the levels of virus acquired by several aphid species.