Luchi et al.: Real-time PCR: an useful tool for disease survey of forest trees pathogens IUFRO Working Party 7.03.10 Proceedings of the Workshop 2006, Gmunden/Austria 154 REAL-TIME PCR - AN USEFUL TOOL FOR DISEASE SURVEY OF FOREST TREES PATHOGENS Nicola Luchi 1 , Paolo Capretti 1 , Pamela Pinzani 2 , Mario Pazzagli 2 1 University of Florence, Dept. of Agricultural Biotechnology, Plant Pathology Section. Piazzale delle Cascine 28, Florence, ITALY (e-mail:nluchi@unifi.it; paolo.capretti@unifi.it) 2 University of Florence, Dept. of Clinical Physiopathology, Clinical Biochemistry Unit. Viale Pieraccini 6, Florence, ITALY(e-mail: p.pinzani@dfc.unifi.it; m.pazzagli@dfc.unifi.it) Abstract Real-time PCR has been used to detect the occurrence of pathogenic fungi (Diplodia pinea, Biscogniauxia mediterranea, B. nummularia) from asymptomatic samples of pine oak and beech respectively. The initial colonization by those armful fungal parasites on the host trees has been detected more efficiently than with isolation on agar plates. Keywords: Biscogniauxia mediterranea, Biscogniauxia nummularia, Diplodia pinea, early detection, fungi, Real-time PCR. 1. Introduction Climate change that affects European forest ecosystems could enhance the possibilities that fungal plant pathogens would extend their range affecting plantations and stands subjected to difficult environmental conditions. Trees have been often debilitated by drought and fungal attacks that induced plant to “decline”. In this contest play an important role fungal microrganisms able to infect host tissue and spend long period of latency before to cause visible symptoms. In Europe, during the last few years, much effort and public resources have been dedicated to monitoring diseases of forest trees. A specific programme of insect and disease monitoring was supported by the Regional government of Tuscany (WWW.ARSIA.TOSCANA.IT/META ). Usually the fungal presence on trees were detected by isolation on agar media or the study of symptoms. Such work is often time consuming and expensive especially when the aim is to detect and identify latent pathogens in a-symptomatic trees (Catal et al. 2001). Therefore, the use of molecular approaches, revealed useful tool for early detection of fungal pathogens in asymptomatic plants (Mazzaglia et al. 2001). During last year a molecular tool the real-time PCR, by using TaqMan chemistry, were used in human and plant disease (Pinzani et al. 2001; Schena et al., 2004).This technique uses a fluorogenic probe, it detects the DNA of specific organisms inside complex matrices that also contain extraneous DNA (Bustin, 2000). In this study a real-time PCR assay was optimized for the fungal detection in symptomless tissues in three different pathosystems: Diplodia pinea-Pinus nigra, Biscogniauxia mediterranea-Quercus cerris, Q .ilex and Biscogniauxia nummularia- Fagus sylvatica. 2. Materials and Methods Fungal presence was detected in symptomless shoots, previously surface sterilised with 70% Ethanol, 1.05% NaOCl and Tween 80, and sterile water (Luchi et al. 2005a; 2005b).