Preventive measures for infectious diseases caused by the harmful plant pathogenic bacterium Xylella fastidiosa include inspections and diagnostic tests on imported consignments of plants and in nurseries. Currently, mandatory checks on plant propagating materials are enforced in Europe (EU regulation 2021/1201) for the most susceptible species found in the European outbreaks, and prior to move propagating materials of the “specified plants” from nurseries located in the so-called “demarcated areas”. These requirements imply sampling and laboratory manipulation of a large number of samples, nevertheless plants to be sampled are often small size potted plants. While statistically based methods for inspections and sampling are available, namely the International Standards for Phytosanitary Measures n. 31, validated laboratory procedures to test large volumes of plant materials are lacking. In this work, we optimized two distinct protocols to detect X. fastidiosa in pooled plant materials collected from lots of plants for planting. The first protocol was designed to test in pool few samples (up to 8), the second to process through a single diagnostic test plant material from a high number of samples (up to 225). Accuracy of the newly developed protocols was assessed by pooling at different ratio tissues collected from healthy and infected Polygala myrtifolia, Nerium oleander, Olea europaea, Lavandula stoechas and Prunus avium. Moreover, tests included pools of plantlets of Brassicaceae and Solanaceae artificially inoculated with stem portions of infected periwinkle. Using both protocols, high diagnostic sensitivity values were generated using serological and molecular tests, with qPCR consistently yielding the highest performance values, regardless the host species tested.

The olive tree (Olea europaea subsp. europaea var. europaea) represents the cornerstone crop of Apulian agriculture, which is based on the production of oil and table olives. The high genetic variability of the Apulian olive germplasm is at risk of genetic erosion due to social, economic, and climatic changes. Furthermore, since 2013, the spread of the Gram-negative bacterium Xylella fastidiosa subsp. pauca responsible for the olive quick decline syndrome (OQDS) has been threatening olive biodiversity in Apulia, damaging the regional economy and landscape heritage. The aim of this study was to investigate the differential response to X. fastidiosa infection in a collection of 100 autochthonous Apulian olive genotypes, including minor varieties, F1 genotypes, and reference cultivars. They were genotyped using 10 SSR markers and grown for 5 years in an experimental field; then, they were inoculated with the bacterium. Symptom assessments and the quantification of bacterium using a qPCR assay and colony forming units (CFUs) were carried out three and five years after inoculation. The study allowed the identification of nine putatively resistant genotypes that represent a first panel of olive germplasm resources that are useful both for studying the mechanisms of response to the pathogen and as a reserve for replanting in infected areas.

Phytosanitary legislation involves government laws that are essential to minimize the risk of the introduction and diffusion of pests, especially invasive non-native species, as a consequence of the international exchange of plant material, thus allowing us to safeguard agricultural production and biodiversity of a territory. These measures ensure compliance with adequate requirements relating to the absence of pests, especially of harmful quarantine organisms through inspections and diagnosis tests of the consignments to ascertain the presence of the pests concerned. They also regulate the eradication and containment measures that are implemented in the eventuality of an unintentional introduction of these organisms. In the present contribution, the current plant protection legislation for the exchange of plants or propagation material within the European Union or for export to foreign countries, represented by Regulation (EU) 2016/2031, has been reviewed, with a particular focus on the olive tree (Olea europaea L.). Furthermore, a brief summary of the main olive tree pests transmissible with the propagation material is also reported, indicating their current categorization with respect to the relative quarantine status.

The high biodiversity of the olive tree is an important opportunity to develop sustainable plans to control Xylella fastidiosa (Xf) through breeding programs. Olive tree breeding activities have been limited due to various features of this species including the long time required for seed germination caused by the inhibition effect of the woody endocarp, the seed integument, and the endosperm. Starting from F₁ seeds by cross-breeding, the embryo culture was compared with traditional seed germination, evaluating the effectiveness of in vitro multiplication of the plantlets for large-scale production. The isolated embryos were established on a new medium based on Rugini ‘84 macroelements, Murashige & Skoog ‘62 microelements, with Nitsch J. P. & Nitsch C. ‘69 vitamine and subcultured on Leva MSM modified. The results obtained confirmed that in vitro culture of olive embryos is a valid tool for increasing the percentage and speed of germination, helping to reduce the time of the olive breeding programs, offering the possibility to effectively propagate plantlets for further experiments.

The objective of this work was to evaluate the effectiveness of thermotherapy alone or combined with either shoot tip culture or in vitro micrografting treatments and shoot tip culture alone in eliminating olive leaf yellowing-associated virus (OLYaV) and strawberry latent ringspot virus (SLRSV) from naturally infected olive (Olea europaea L.) cultivars to obtain virus-free stock plants. Reverse-transcription polymerase chain reaction was used to determine the efficiency of virus elimination in the in vitro regenerated plants at 6 months following the acclimation to ex vitro conditions. All treatments proved effective in obtaining virus-free plants for all selected cultivars except for the heat therapy used alone which did not completely remove the virus from infected propagative material. The establishment of in vitro cultures of shoot tips from plants exposed to an alternating day/night temperature of 38/30°C or from vitroplants maintained at a constant temperature of 38°C resulted in OLYaV elimination rates of 67 to 100% and 60 to 100%, respectively. The combination of in vitro thermotherapy with shoot-tip grafting yielded up to 100% of OLYaV-negative plants, although grafting success rates were relatively low (29 to 33%). Instead, in vitro culture of shoot tip after eight consecutive cycles of subculturing without heat therapy led to the elimination of OLYaV/SLRSV from 17% of coinfected plants and OLYaV from 20% of single-infected plants. These results indicate that the combined application of treatments is an effective method for virus elimination in olive trees and could be used to ensure the production of healthy nursery stock plants.