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. By the second period of SharCo, over 700 PPV isolates have been sequenced over the two most variable genomic regions of the virus and PPV diversity was estimated. PPV isolates were sampled mostly from European member states and associated countries as well as outside of Europe. Those isolates are covering the host and geographical diversity of PPV outbreaks. The PPV SharCo database that was setup during the first period of the project was upgraded during the second period by adding datasheet information and sequence analysis from the above mentioned >700 PPV isolates. This new and larger nucleotide diversity data has been used to generate the second generation of mini-oligo arrays. After the enrichment of the second generation of mini-oligo arrays with sequences generated in the first period, the technology is being 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 speed up through the development of those PPV isolate high throughput typing tools. They will thus be part of the SharCo propositions for PPV Early Warning System in the third period.
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. For this purpose, plum and apricot orchards have been established during the first period in different countries and epidimicity of PPV-D and -Rec isolates in those orchards has been scored in the second period. The construction of recombinant infectious clones of the PPV-C and –Rec was finalized; their host properties and biological behaviour were evaluated in the meanwhile and compared with other strains. The two geo-referenced databases built in the first period and surveying PPV outbreaks at a local (orchard) and regional scale were used to model i) chains of transmission through the development of adapted statistical and modelling methods, ii) epidemic development at a larger scale. It is expected that all those epidemic data and modelisation 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, mostly in the second period, 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, molecular tools (named markers) developed in the first period were transferred to the other SharCo partners for validation. In the meantime, new markers were generated; they are tightly linked with the genomic region explaining most of the resistance to PPV in apricot and will be used in the third period for marker assisted selection in view of improving resistance of plants cultivated in orchards. We also developed molecular tools in peach and European plums that will be validated in the next period. Breeding programmes aiming at introducing resistance to PPV in future cultivars were continued both in Western and Eastern European countries as well as in Turkey. Large populations of apricot and plum F1 individuals were completed and set up to implement marker assisted screening of PPV resistant pre-breeding material, in the third period of the project.
In the field of biology, innovative biotechnological approaches were assessed to broaden the spectrum of available resistance strategies against PPV in different fruit tree species, distinct from the one described above. Host and viral factors involved in plant-PPV interactions were identified and validated over the second period. A significant effort was produced in order to transfer these new mechanisms of resistance to stone fruit trees by screening for rare alleles of resistance in Prunus germplasm collections or by silencing host factors indispensable to the virus. A new host genetic determinants leading to recessive resistance to PPV infection was also identified and a patent was submitted. The identification of resistance genes activated in plant species showing hypersensitivity in response to PPV infection is pursued. Other resistance mechanisms based on the virus sequence and biotechnological approaches have also been 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. After consultation with national plant health organisations in the previous period, we focused on the preparation of Cultivation Guidelines for the production and the management of stone fruit trees that would allow reducing the impact and spread of sharka disease.
All knowledge and tools developed by this research programme have been widely disseminated all over Europe with special attention made to PPV endemic countries and, more broadly, to all interested parties dealing with the difficult problems associated with sharka disease. Training workshops targeting end-users and stakeholders took place all along the second period of the project, in five European and associated countries. A Sharco Research workshop was organised concomitantly with the ISHS International Plum Pox virus Symposium, in September 2011, in Bulgaria. End-users and stakeholders joined the SharCo advisory board during the half-yearly project meetings and could thus 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.