Messa a punto di tecniche diagnostiche per patogeni di quarantena/Development of diagnostics techniques for studying quarantine plant pathogens

The aim of this thesis was to optimize and improving reliable, fast, sensitive and specific field-deployable tools for the early detection of quarantine plant pathogens. In the first part of the thesis the work was concentrated in developing a field-applicable LAMP-based assay for the detection of Xylella fastidiosa, Phytophthora ramorum and Ceratocystis platani. Each assay, optimized on the portable instrument Genie II ® (Optigene, UK), was based on the conventional LAMP reaction and showed the capability to detect X. fastidiosa, C. platani and P. ramorum with high specificity and sensitivity in only 30 minutes also on plant samples for which a rapid kit method for in field-DNA extraction was also utilized. However, the assay targeting C. platani and P. ramorum were able to detect also C. fimbriata and P. lateralis, having also many cross reactions with other Phythophtora species. Even if the specificity was assessed by results obtained from melting analyses, that gave different temperature between target and non-target species, improving the specificity of a LAMP assay was needed. The second part of the thesis was hence concentrated in improving the chemistry and the specificity of a LAMP assay. The use of sequence-specific LAMP probes was analyzed by the development of a conventional and FRET-assimilating probe-based LAMP method targeting Fusarium circinatum, a pine pathogen for which specificity is a very important requirement concerning diagnostics. The capability of increasing the specificity using this novel LAMP chemistry was assessed by comparing LAMP results of conventional and probe-based LAMP reaction developed for F. circinatum: with conventional reaction many cross reactions were obtained with phylogenetically closest Fusaria while with the probe-based method only F. temperatum was amplified as cross reaction. Due to positive results obtained applying the probe-based method on wood samples DNA extracted with the field method the suitability for using it into the field was also assessed. The same probe-based LAMP chemistry was then implemented for multiplex application concerning pine needles pathogens Dothistroma septosporum, Dothistroma pini and Lecanosticta acicola, obtaining as preliminary results that of having a multiplex specific reaction directly in the field in about 10 minutes. Concerning this third part of this work, the possibility to apply the described method on crude samples was investigated concerning pine needles for which preliminary test to optimize a field suitable crude extraction method were carried out with promising results. As in the last part of this work was assessed that in Italy the distribution of Dothistroma septosporum is widespread by applying a TaqMan-based qPCR method while L. acicola was reported only in restricted places and D. pini was never reported, the developed LAMP method could be useful to prevent and monitoring their spread and introduction.