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Panel of real‐time PCR s for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species
Author(s) -
Brunschot S. L.,
Gambley C. F.,
Barro P. J.,
Grams R.,
Thomas J. E.,
Henderson J.,
Drenth A.,
Geering A. D. W.
Publication year - 2013
Publication title -
plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.928
H-Index - 85
eISSN - 1365-3059
pISSN - 0032-0862
DOI - 10.1111/ppa.12033
Subject(s) - whitefly , biology , begomovirus , tomato yellow leaf curl virus , leaf curl , virology , geminiviridae , dna extraction , polymerase chain reaction , real time polymerase chain reaction , vector (molecular biology) , plant virus , virus , horticulture , gene , genetics , recombinant dna
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.