In the field of epidemiology, we aim at identifying factors driving PPV spread and diversification and developing novel and high throughput detection and warning systems for sharka outbreaks. In the first period of the project, several hundreds of PPV isolates were sampled from European member states and associated countries. Those isolates are covering the host and geographical diversity of PPV outbreaks. Samples were sequenced over the two most variable genomic regions of the virus and PPV diversity was estimated. A PPV database was setup and will gather all PPV sequences generated in SharCo or already present in the Genbank database. In the second period, we will target also the diversity of the virus in other European countries, where the data on PPV diversity are scarce or missing and outside Europe (Asia, South America). Moreover, this nucleotidic diversity data will be used to generate the second generation of mini-oligo arrays. The first generation, based on the PPV-D, -M and –Rec sequences, was elaborated. It allowed developing protocols for the use of those arrays by end-users such as the quarantine and plant protection services. After the enrichment of the second generation of mini-oligo arrays with sequences generated in the first period, the technology will be challenged against isolates of the lyophilised reference and the live PPV collections. It is expected that, in the future, the characterisation of the emergence of new PPV variants will be achieved and improved through the development of those PPV isolate high throughput typing tools.
However, after PPV introduction in an orchard, the installation of sharka disease varies according to the identity of the PPV strain and the Prunus species. Epidemic properties at a larger scale are under scrutiny within the SharCo project. Plum and apricot orchards were established, recombinant infectious clones of the PPV-C and –Rec were obtained or are under construction and the mapping of PPV outbreaks and diversity at a regional or national scale was achieved. Two geo-referenced databases were built, one at the tree level on a 450 ha area, the other one at the orchard level at a regional scale. All those data and tools will allow a better understanding about the risk of installation and further spreading of PPV variants after their accidental introduction into a new territory or given agro-system.
In order to develop novel and accurate systems for sampling, detecting, assessing and warning sharka outbreaks, especially in nurseries, we established in the first period new rootstock nursery blocks and estimated the following parameters : i) the susceptibility of selected Prunus rootstocks to PPV natural infection, ii) the number and species of PPV vectors landing in nursery blocks, depending on the environmental conditions, iii) methods of sampling and detecting PPV infected material in nursery blocks, iv) the efficiency of oil treatments in limiting natural PPV infection.
In the field of genetics, we developed in the first period new molecular tools (named markers) that will be validated in the second period for use in marker assisted selection in view of improving resistance of plants cultivated in orchards. We also refined the position of the genomic loci linked to PPV resistance in peach and apricot and initiated a physical map of the main apricot resistance locus. Breeding programmes aiming at introducing resistance to PPV in future cultivars were continued in Western European countries and initiated in Eastern European countries as well as in Turkey. Those plants and tools will allow implementing marker assisted selection of PPV resistant pre-breeding material, in the second period of the project. A similar approach was initiated in European plums with the objective to produce RNA or/and DNA markers linked to hypersensitivity when PPV infected.
In the field of biology, innovative biotechnological approaches are being assessed to broaden the spectrum of available resistance strategies against PPV in different fruit tree species. In the first period, we started to evaluate the potential of new and complementary resistant genetic mechanisms, distinct from the one described above. We are presently characterizing host and viral factors involved in plant-PPV interactions and screening for rare alleles of resistance in Prunus germplasm collections. New host genetic determinants leading to recessive resistance to PPV infection were also identified and are being cloned. The identification of resistance genes activated in plant species showing hypersensitivity in response to PPV infection is underway. Other resistance mechanisms based on the virus sequence and biotechnological approaches are also being assessed and their biosafety, evaluated.
SharCo is also designed to become a source of knowledge and expertise to support policy-makers, regulatory bodies and extension services. In the first step towards the production of cultivation guidelines and toward restructuring PPV management, we mapped out how European directives on sharka disease are implemented in EU and two associated countries. In consultation with national plant health organisations, we reviewed case-histories and identified ones most tractable for research on PPV management. This inventory included the practical strategies as used by end-users and the differences with the strategies recommended in the EU directive. For this purpose, we established operational linkages with plant protection services and initiated the process of Sharka containment by setting up a network that will facilitate two-way exchange between SharCo and these other players.
All knowledge and tools developed by this research programme will be widely disseminated all over Europe with special attention made to PPV endemic countries and, more broadly, will be made available to all interested parties dealing with the difficult problems associated with sharka disease. Training workshops targeting end-users and stakeholders will take place in the second period of the project. Contacts with neighbouring countries were taken; partners from European or non-European countries, end-users and stakeholders were proposed to join the SharCo advisory board in order to benefit rapidly from exploitable SharCo foregrounds. Indeed, this initiative addresses all concerned stakeholders, breeders, nursery gardeners, fruit producers, and plant protection services with relevant outcomes including respectively resistant varieties, management guidelines, cultivation guidelines, optimised survey and detection methods and tools.