Panel of real‐time PCR s for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease ( TYLCD ) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of T omato yellow leaf curl virus ‐Israel ( TYLCV ‐ IL ), T omato leaf curl virus (To LCV ), B emisia tabaci Middle East Asia Minor 1 species ( MEAM 1, B biotype) and B . tabaci M editerranean species ( MED , Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B . tabaci DNA . All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV ‐ IL , 100 plasmid copies of To LCV , 500 fg B . tabaci MEAM 1 and 300 fg B . tabaci MED DNA . Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B . tabaci captured on yellow sticky traps and for bulking of multiple B . tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B . tabaci vector species in A ustralia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in A ustralia and worldwide.