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.

ANSES Laboratoire de la Santé des Végétaux Unité de Mycologie Domaine de Pixérécourt Bât E 54220 Malzéville France

Bioforest S A Camino a Coronel km 15S N 4030000 Concepción Chile

Çankırı Karatekin University Faculty of Forestry 18200 Çankırı Turkey

Cardinal Stefan Wyszynski University in Warsaw Faculty of Biology and Environmental Sciences Wóycickiego 1 3 Street 01 938 Warsaw Poland

Departamento de Biologia CESAM Universidade de Aveiro 3810 193 Aveiro Portugal

Department for Innovation in Biological Agro food and Forest Systems University of Tuscia via S Camillo de Lellis snc 01100 Viterbo Italy

Department of Agricultural Sciences Biotechnology and Food Science Cyprus University of Technology 3036 Limassol Cyprus

Department of Agriculture Food and Environment University of Catania Via Santa Sofia 100 Catania 95123 Italy

Department of Agriculture Food and Environment University of Catania Via Santa Sofia 100 Catania 95123 Italy Dipartimento di Scienze Agrarie Alimentari e Forestali Università degli Studi di Palermo Viale delle Scienze 90128 Palermo Italy

Department of Biochemistry Genetics and Microbiology Forestry and Agricultural Biotechnology Institute University of Pretoria 0028 Hatfield Pretoria South Africa

Department of Food Environmental and Nutritional Sciences University of Milan via Celoria 2 1 20133 Milano Italy

Dipartimento di Scienze delle Produzioni Agroalimentari e dell'Ambiente University of Florence 50144 Florence Italy

Forest Health Center of Calabazanos Regional Government of Castilla y León JCyL Poligono Industrial de Villamuriel S N 30190 Villamuriel de Cerrato Palencia Spain

Forest Research Alice Holt Lodge Farnham Surrey GU10 4LH United Kingdom Phytophthora Research Center Department of Forest Protection and Wildlife Management Faculty of Forestry and Wood Technology Mendel University in Brno Zemědělská 3 613 00 Brno Czech Republic

Institute for Sustainable Plant Protection National Research Council Via Madonna del Piano 10 1 50019 Sesto Fiorentino Florence Italy

Institute of Forestry and Rural Engineering Estonian University of Life Sciences 51006 Tartu Estonia

Instituto Agroforestal Mediterráneo Universitat Politècnica de València Camino de Vera s n 46022 Valencia Spain

Instituto Nacional de Investigação Agrária e Veterinária 1 P Quinta do Marquês 2780 159 Oeiras Portugal

Julius Kühn Institut Institute for National and International Plant Health Messeweg 11 12 38104 Braunschweig Germany

Lithuanian Research Centre for Agriculture and Forestry Vokė Branch Zalioji Sq 2 02232 Vilnius Lithuania

Phytophthora Research Center Department of Forest Protection and Wildlife Management Faculty of Forestry and Wood Technology Mendel University in Brno Zemědělská 3 613 00 Brno Czech Republic

Plant Health and Environment Laboratory Diagnostic and Surveillance Services Biosecurity New Zealand Ministry for Primary Industries PO Box 2095 Auckland 1140 New Zealand

Slovenian Forestry Institute Department of Forest Protection Večna pot 2 SI 1000 Ljubljana Slovenia

Sustainable Forest Management Research Institute University of Valladolid INIA Department of Vegetal Production and Forest Resources University of Valladolid 47011 Palencia Spain

Swiss Federal Institute for Forest Snow and Landscape Research WSL Zuercherstrasse 111 8903 Birmensdorf Switzerland

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