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Title: Transferability of PCR-based diagnostic protocols: An international collaborative case study assessing protocols targeting the quarantine pine pathogen Fusarium circinatum
Authors: Nowakowska, Justyna Anna 
Ioos, Renaud 
Mullett, Martin 
Vettraino, Anna Maria 
Douanla-Meli, Clovis 
Cornejo, Carolina 
Alves, Artur 
Diez, Julio J. 
Ahumada, Rodrigo 
Kanetis, Loukas 
Berbegal, Mónica 
Luchi, Nicola 
Pasquali, Matias 
Adamson, Kalev 
Aloi, Francesco 
Guinet, Cécile 
Kačergius, Audrius 
Dvořák, Miloň 
Baskarathevan, Jeyaseelan 
Fourie, Gerda 
Bragança, Helena 
Cacciola, Santa Olga 
Piškur, Barbara 
Martínez-Álvarez, Pablo 
Ghelardini, Luisa 
Sanz-Ros, Antonio 
Oskay, Funda 
Aguayo, Jaime 
Major Field of Science: Agricultural Sciences
Field Category: AGRICULTURAL SCIENCES;Agricultural Biotechnology;Other Agricultural Sciences
Keywords: Gibberella circinata;Pinus radiata;Pitch canker
Issue Date: 3-Jun-2019
Source: Scientific Reports, 2019, vol. 9, no.1
Volume: 9
Issue: 1
Journal: Scientific Reports 
Abstract: Fusarium circinatum is a harmful pathogenic fungus mostly attacking Pinus species and also Pseudotsuga menziesii, causing cankers in trees of all ages, damping-off in seedlings, and mortality in cuttings and mother plants for clonal production. This fungus is listed as a quarantine pest in several parts of the world and the trade of potentially contaminated pine material such as cuttings, seedlings or seeds is restricted in order to prevent its spread to disease-free areas. Inspection of plant material often relies on DNA testing and several conventional or real-time PCR based tests targeting F. circinatum are available in the literature. In this work, an international collaborative study joined 23 partners to assess the transferability and the performance of nine molecular protocols, using a wide panel of DNA from 71 representative strains of F. circinatum and related Fusarium species. Diagnostic sensitivity, specificity and accuracy of the nine protocols all reached values >80%, and the diagnostic specificity was the only parameter differing significantly between protocols. The rates of false positives and of false negatives were computed and only the false positive rates differed significantly, ranging from 3.0% to 17.3%. The difference between protocols for some of the performance values were mainly due to cross-reactions with DNA from non-target species, which were either not tested or documented in the original articles. Considering that participating laboratories were free to use their own reagents and equipment, this study demonstrated that the diagnostic protocols for F. circinatum were not easily transferable to end-users. More generally, our results suggest that the use of protocols using conventional or real-time PCR outside their initial development and validation conditions should require careful characterization of the performance data prior to use under modified conditions (i.e. reagents and equipment). Suggestions to improve the transfer are proposed.
ISSN: 2045-2322
DOI: 10.1038/s41598-019-44672-8
Rights: © The Author(s).
Type: Article
Affiliation : Unité de Mycologie 
University of Catania 
Slovenian Forestry Institute 
Forest Research 
Universitat Politècnica de València 
Instituto Nacional de Investigação Agrária e Veterinária 
Çankırı Karatekin University 
Swiss Federal Institute for Forest 
Estonian University of Life Sciences 
Institute for National and International Plant Health 
Lithuanian Research Centre for Agriculture and Forestry 
University of Valladolid 
Cardinal Stefan Wyszynski University in Warsaw 
Institute for Sustainable Plant Protection 
University of Tuscia 
University of Milan 
University of Pretoria 
Cyprus University of Technology 
Universidade de Aveiro 
University of Florence 
Mendel University in Brno 
Forest Health Center of Calabazanos 
Università degli Studi di Palermo 
Ministry for Primary Industries 
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