Abstract
The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.
Highlights
The whitefly Bemisia tabaci (Gennadius) is an invasive insect pest affecting the yield of many economically important crops including ornamental plants, vegetables, grain legumes, and cotton[1,2]
The specimens originated from eight different countries (Canary Islands, Dominican Republic, Israel, Malaysia, Morocco, Singapore, Thailand, and Vietnam) and reflect the genetic diversity of B. tabaci found at European POEs8
The correct-negative results originated from two Trialeurodes vaporariorum specimens, a non-regulated species at high risk to be confused with B. tabaci at POEs for plant products[8]
Summary
The whitefly Bemisia tabaci (Gennadius) is an invasive insect pest affecting the yield of many economically important crops including ornamental plants, vegetables, grain legumes, and cotton[1,2]. The novel B. tabaci LAMP assay is designed in such a way that the primers target a fragment at the 3' end of the mitochondrial COI gene[8]. This gene presents a suitable target for animal diagnostic assays because it harbors regions conserved enough to ensure diagnostic sensitivity for a specific species, while discriminating enough between closely related organisms[19,20]. Due to the accuracy of the method, its speed (
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