Client ID: Agencija za raziskovalno dejavnost RS (L4-5525)
Project type: Basic Research and Application Project
Project duration: 2013 - 2016
Due to the globalization-powered free movement of goods and services, we are exposed to an intensified exchange (import/export) of plants and plant origin products. With them new microorganisms and insects or other vectors for microbial plant pathogens, are travelling, which, can survive in new warmer areas. New emerging diseases constitute a major threat to agricultural production and forestry, but also present a threat to natural ecosystems and biodiversity. In addition to the emerging of new diseases, which are often difficult to identify, another problem is the genetic variation of already known pathogens. In response to this, the diagnosis of new pathogens or new genetic variants of already known microbes has become an extremely hot topic. New methods for determining the nucleotide sequence (next generation sequencing - NGS) provide new opportunities for pathogens identification.
Viruses occur in the plant as a set of different mutants (quasispecies). After entering a new host, the viral genome can change, or the ratio between the different mutants of the same virus can be altered. Quick mutations arising from replication of viruses give rise to more aggressive isolates that can spread faster and infect a wider range of hosts. Such examples are found in potato with the infection with different Potato virus Y (PVY) virus variants, that threatens the production of potato. Apart from PVY, we will focus on pathogens that infect grapevine. Pathogens of woody plants are problematic as they represent a permanent infection on plants that are grown for many years, and therefore their presence should be detected sooner in order to remove infected plants. Phytoplasma Flavescence Dorée is quarantine pathogen with great economic impact on grapevine production and Raspberry Bushy Dwarf Virus (RBDV) is a newly discovered virus infecting grapevine and its diversity and the importance for the grapevine and original host is still unknown. Determination of genetic traits by NGS and development of new diagnostic methods, such as isothermal amplification (LAMP), high resolution melting analysis (HRM) and immune real-time PCR (immuno-qPCR), which will be used and developed during the project, will allow rapid detection of plant pathogens. It will enable faster and more reliable discovery of new microbes/pathogens and will contribute to understanding the diversity of quarantine and harmful already identified microbes. The new approach to microbe identification and detection will enable tracking of the route of pathogen introduction and distribution.
Determination of genetic diversity that NGS allows will help to reveal the differences that are important for pathogenicity. In the case of plant viruses one of the bottleneck is a sample preparation in order to make easier the analysis of the huge amount of data generated by the sequencing, which is non selective. We will introduce a completely original way of preparing samples of virus infected plants for the sequencing of viruses using new generation monolithic chromatographic polymers (CIM).
The involvement of excellent national and international partners, using modern facilities allows us to obtain relevant results, useful both for co-financers (Ministry of Agriculture and environment and the company Omega) and for crop production improvements. The proposed studies belong to the European strategy, which emphasizes the genomics and biotechnology for health, food quality and sustainable development. The project is fully harmonized with the priorities of this public call for proposals – Plant Production.