A high-throughput plate method for nucleic acid extraction from beet leafhopper (Hemiptera: Cicadellidae) and potato psyllid (Hemiptera: Triozidae) for pathogen detection

Abstract

AbstractPlant pathogens that are transmitted by insect vectors cause considerable damage to crops when pests or pathogens are not detected early in the season and populations are not controlled. Knowledge of pathogen prevalence in insect pest populations can aid growers in their insect pest management decisions but requires the timely dissemination of results. This process requires that specimen capture, identification, nucleic acid extraction, and molecular detection of a pathogen(s) occur alongside a platform for sharing results. The potato psyllid (Bactericera cockerelli, Sulc; Hemiptera: Triozidae) and beet leafhopper (Circulifer tenellus, Baker; Hemiptera: Cicadellidae) transmit pathogens to potato and other vegetable or seed crops each season in the northwestern United States. While the potato psyllid has been tested for pathogen occurrence for the past decade, testing of the beet leafhopper is a new endeavor and substantially increases the specimen number that must be tested by our laboratories each season. To aid in the rapid processing of individual insect specimens, we optimized and validated a new high-throughput 96-well plate nucleic acid extraction method for use in place of a standard 1.5-ml single-tube extraction method. Processing efficiency, in terms of total specimens processed over a 2-day period, improved 2.5-fold, and the cost associated with processing a single sample was nearly cut in half with this newly developed plate nucleic acid extraction method. Overall, this method has proven to be an excellent tool for the rapid testing of large numbers of small, individual insect vectors to enable timely dissemination of data on pathogen prevalence to growers.