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Since the first detection of Xylella fastidiosa (Xf) in the Balearic Islands and Alicante province (mainland Spain), research has provided new insights on genetic diversity and host range of the strains present in the different Spanish outbreaks. The high genetic diversity of Xf in Spain (three subspecies and four STs) and the high host range (over 30) in total implies a risk for several host of agronomic interest if these genotypes spread to areas where they are not present yet. The main goal of this study was to evaluate the pathogenicity and the systemic colonization of four Spanish strains on three olive cultivars representative of the most widely grown in Spain. Two experiments are being conducted in a confined growth chamber (26ºC day/24ºC night), where 10-month-old self-rooted micro-propagated olive cultivars (Arbequina, Hojiblanca and Picual) were inoculated with the strains XYL1961 of Xf subsp. pauca ST80 and the Italian strain De Donno of Xf subsp.pauca ST53 (Control), strain XYL1966 of Xf subsp. multiplex ST81 isolated from olive trees in the Balearic Islands, and the strains IVIA5901 and ESVL of Xf subsp. multiplex ST6 isolated from almond trees in the Guadalest valley (Alicante). Leaf samplings were carried out at the inoculation point (IP), and at 5, 10, 15 and 20 cm above the IP, at 1, 3, 6, 12 and 18 months post-inoculation (mpi), respectively. Furthermore, destructive sampling of different sections of the main stem was performed at 9 mpi at the same levels of leaf sampling. qPCR tests were used to determine the presence and systemic colonization of the bacterium. After 2 years, no clear or specific symptoms associated to Xf infection has been observed. Our results show that all the strains of Xf subsp. multiplex were detected in the leaf petioles at the IP and 5 cm above it although at low frequency ( 34) indicating that the strains had some trouble colonizing the different olive genotypes. On the contrary, Xf subsp. pauca strains could be detected on all leaf petiole samples even up to 20 cm over the IP, although the frequency of positive samples and bacterial inoculum decreased drastically from the IP. When sampling the main stem, results indicated that the Spanish strain of Xf subsp. pauca ST80 colonized a higher number of plants and multiplied better than the Italian strain of Xf subsp. pauca ST53 which indicated the risk that this strain may represent for olive. No clear differences could be observed among the patterns of infection of the different Xf strains on the three olive cultivars tested. Acknowledgement This work has received funding from 727987 XF-ACTORS (H2020-UE), E-RTA2017-00004-C06-02 (AEI-INIA Spain and FEDER), and the Spanish Olive Oil Interprofessional.