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Woody canker diseases caused by fungi of the Botryosphaeriaceae family are producing increasing losses in many economically important woody crops, including almond. To develop a molecular tool for the detection and quantification of the most aggressive and threatening species is of main importance. This will help to prevent the introduction of these pathogens in new orchards and to conveniently apply the appropriate control measures. Three reliable, sensitive and specific duplex qPCR assays using TaqMan probes have been designed for the detection and quantification of (a) Neofusicoccum parvum and the Neofusicoccum genus, (b) N. parvum and the Botryosphaeriaceae family and (c) Botryosphaeria dothidea and the Botryosphaeriaceae family. The multiplex qPCR protocols have been validated on artificially and naturally infected plants. Direct systems to process plant materials, without DNA purification, allowed high-throughput detection of Botryosphaeriaceae targets even in asymptomatic tissues. These results validate the qPCR using the direct sample preparation method as a valuable tool for Botryosphaeria dieback diagnosis allowing a large-scale analysis and the preventive detection of latent infection.

Resistance to the defoliating pathotype of Verticillium dahliae has been evaluated in a pool of 68 wild genotypes of olive belonging to the SILVOLIVE collection. Resistance was evaluated by assessing symptom severity using a 0-4 rating scale, estimating the relative area under the disease progress curve (RAUDPC), determining the percentage of dead plants (PDP), and measuring the evolution of morphological parameters in inoculated plants over time. In addition, the density levels of V. dahliae in the stem of root-inoculated genotypes have been quantified by means of quantitative real-time PCR at 35 and 120 days after inoculation (dai). Fifteen genotypes (22%) were cataloged as resistant to V. dahliae (i.e., disease parameters did not significantly differ from those of the resistant cultivar Frantoio, or were even lower). Resistant genotypes are characterized by presenting fewer symptoms and a lower amount of V. dahliae DNA at 120 dai than at 35 dai, indicating their ability to control the disease and reduce the density of the pathogen. The rest of the evaluated genotypes showed variable levels of susceptibility. Overall analysis of all genotypes showed high correlation between symptomatology and the amount of V. dahliae DNA in the stem of inoculated genotypes at 120 dai, rather than at 35 dai. However, correlation at 120 dai was not observed in the set of resistant genotypes, suggesting that resistance to defoliating V. dahliae in olive is based on the occurrence of different mechanisms such as avoidance or tolerance. These mechanisms are valuable for designing breeding programs and for the identification of target genes and resistant rootstocks to better control Verticillium wilt in the olive grove.

Olive cultivation has adopted intensive practices in high or super high plant densities (SHD). It has increased olive productivity, but has also led to new problems such as: new diseases, varietal impoverishment, and new forms of interaction with environmental factors. Fertigation has strongly enhanced the proliferation of soil-borne fungi like Verticillium dahliae causing verticillium wilt, currently the most threatening disease for olive crops. The increasing use of very few cultivars of reduced vigour in SHD plantations is leading to strong genetic impoverishment of the crop. SHD management in semi-arid regions requires good adaptation to deficit irrigation techniques and favours soil salinization, etc. Surprisingly olive is the only woody crop grown on its own roots. Incorporation of dwarfing rootstocks to the intensive plantation systems will allow: 1) to increase the number of cultivars compatible with the SHD system; 2) greater control of diseases such as verticillium wilt; 3) better control of abiotic disturbances. The SILVOLIVE collection consists of 149 genotypes belonging to all known subspecies of Olea europaea: europaea, laperrinei, cuspidata, cerasiformis, guanchica and maroccana. Seeds were prospected from world olive germplasm collections (Córdoba and Marrakech) and regions of Spain, Africa and the Macaronesian archipelago. The genotypes are being characterized in terms of anatomical parameters, branching habit, vigour, susceptibility to verticillium wilt and water use under full and deficit irrigation. We will take advantage of the high genetic variability of the collection as a new source of resistance genes for breeding programs and as dwarfing rootstocks that increase SHD performance

In recent years, the olive cultivation model has undergone a change from the traditional cropping model under rain-fed regime to intensive and super-intensive olive groves subjected to fertilization and irrigation. This has enhanced the proliferation of soilborne fungi like Verticillium dahliae causing verticillium wilt, currently the most threatening disease for olive crops in Spain due to the rapidity of its extension and the severity of its damage. Moreover, the most widely planted olive cultivars (Picual, Hojiblanca and Arbequina) are susceptible or very susceptible to the disease, especially under intensive conditions. We aim to take advantage of the genetic variability present in the wild subspecies of Olea europaea to identify and characterize wild olive genotypes with a high resistance to the verticillium wilt disease. The SILVOLIVE collection consists of 149 genotypes from all known subspecies of Olea europaea described so far, including the subspecies: europaea, laperrinei, cuspidata, cerasiformis, guanchica and maroccana. These genotypes were prospected from world olive germplasm collections (Córdoba and Marrakech) and different regions of Spain, continental Africa and the Macaronesian archipelago. Susceptibility to V. dahliae of 56 wild olive genotypes has been evaluated under controlled conditions inoculating six-month-old olive plants with a cotton defoliating isolate of V. dahliae (V117). After root inoculation of the fungus (107 conidia ml-1), quantification by qPCR at 35 and 120 days after inoculation, and analysis of symptoms in aerial organs, three genotypes showed high resistance/tolerance indexes. These genotypes can be used as high tolerance rootstocks as a control strategy for the growth of commercial and productive olive cultivars.