Host resistance is the most practical, long-term, and economically efficient disease control measure for Verticillium wilt in olive caused by the xylem-invading fungus Verticillium dahliae (Vd), and it is at the core of the integrated disease management. Plant’s microbiome at the site of infection may have an influence on the host reaction to pathogens; however, the role of xylem microbial communities in the olive resistance to Vd has been overlooked and remains unexplored to date. This research was focused on elucidating whether in vitro olive propagation may alter the diversity and composition of the xylem-inhabiting microbiome and if those changes may modify the resistance response that a wild olive clone shows to the highly virulent defoliating (D) pathotype of Vd. Results indicated that although there were differences in microbial communities among the different propagation methodologies, most substantial changes occurred when plants were inoculated with Vd, regardless of whether the infection process took place, with a significant increase in the diversity of bacterial communities when the pathogen was present in the soil. Furthermore, it was noticeable that olive plants multiplied under in vitro conditions developed a susceptible reaction to D Vd, characterized by severe wilting symptoms and 100% vascular infection. Moreover, those in vitro propagated plants showed an altered xylem microbiome with a decrease in total OTU numbers as compared to that of plants multiplied under non-aseptic conditions. Overall, 10 keystone bacterial genera were detected in olive xylem regardless of infection by Vd and the propagation procedure of plants (in vitro vs nursery), with Cutibacterium (36.85%), Pseudomonas (20.93%), Anoxybacillus (6.28%), Staphylococcus (4.95%), Methylobacterium-Methylorubrum (3.91%), and Bradyrhizobium (3.54%) being the most abundant. Pseudomonas spp. appeared as the most predominant bacterial group in micropropagated plants and Anoxybacillus appeared as a keystone bacterium in Vd-inoculated plants irrespective of their propagation process. Our results are the first to show a breakdown of resistance to Vd in a wild olive that potentially may be related to a modification of its xylem microbiome and will help to expand our knowledge of the role of indigenous xylem microbiome on host resistance, which can be of use to fight against main vascular diseases of olive.

Olive tree, Olea europaea L., is one of the most commercially important oil crops. A reliable protocol for the genetic transformation of this species has been developed. Embryogenic calli were infected with different Agrobacterium tumefaciens strains harboring pBINUbiGUSint or pGUSINT binary plasmids. These vectors contain the nos-nptII and the uidA gene driven by the maize polyubiquitin Ubi1 and CaMV35S promoter, respectively. Inoculated explants were cocultured for 2 days, and later selected in the presence of 200 mg l -1 paromomycin. The inclusion of a 3 weeks selection period in liquid medium supplemented with 50 mg l -1 paromomycin was critical for elimination of chimaeric calli. Agrobacterium strain AGL1 containing pBINUbiGUSint plasmid yielded higher transformation frequencies than EHA105 or LBA4404. Globular somatic embryos (SE), 1-2 mm diameter, cultured in the selection medium in groups of three, were the best explant for transformation. Using this protocol, transformation frequencies in the range of 20-45%, based on the number of infected explants proliferating in the selection medium, have been obtained. More than 100 independent transgenic lines were generated, and 16 of them converted to plants. Transgenic plants were acclimated and grown in the greenhouse, being phenotypically similar to wild type plants. The uidA gene was strongly expressed in transgenic material during the in vitro regeneration phase; however, b-glucuronidase (GUS) activity in pBINUbiGUSint transgenic plants was neither detected in shoots growing in vitro nor in acclimated plants. Transgenic leaves, however, contained high levels of NPTII protein. By contrast, plants transformed with the pGUSINT plasmid showed a strong GUS activity in leaves. The protocol here described will allow the genetic improvement of this traditional crop.