Project overview

Production of agricultural plants to feed the world growing population is endangered by many pathogens and sometimes can even trigger large-scale migrations. Constant endeavors from the biotechnology professionals to fight the production losses caused by pathogens are often hampered with the inability to detect the cause of the disease. The introduction of HTS to plant virus discovery has led to a large increase in the number and frequency of novel viruses being discovered. The currently predominant HTS platform on the market, Illumina sequencing, requires expensive sequencers or few weeks time, if samples are sent to external sequencing providers. Although Illumina sequencing has become cost-effective, it often requires batch processing of hundreds of samples to achieve cost savings and therefore this approach offers less flexibility for urgent, small-scale sequencing studies often required in research of viruses or diagnostics of plant diseases. In contrast and complement, Oxford Nanopore Technologies offers a range of sequencing devices, which are suitable for low cost processing of a small number of samples and they obtain results rapidly. In addition, it is currently the only HTS approach, which shows a great potential for analysis of samples outside laboratories. Nanopore sequencing devices, e.g. MinION, can be powered through the USB port of laptop computers, such that sequencing can be conducted anywhere, even in the field.

Since the nanopore sequencing approach directly detects the input molecule without DNA amplification or synthesis, there is no apparent limit to the length of DNA that can be sequenced. The challenge in read length using nanopore sequencers therefore is not in the sequencing technology itself, but in the library preparation step, which needs to extract and load intact high molecular weight nucleic acids into the flow cell of the sequencer. The technology was already shown to be applicable for detection of a large array of pathogens, since longer read lengths provide additional benefit for classification of sequencing reads.

In many cases, plant samples sent to diagnostic labs have unusual disease symptoms that do not point to a specific pathogen. In these cases, diagnosticians, based on previous experience, make informed estimations of possible disease agents and choose the diagnostic procedures involving different methods accordingly. This process takes time and often the results are inconclusive so that the procedures are repeated for different possible pathogens. The possibility of using total nucleic acids and nanopore HTS sequencing could become a method for initial disease diagnosis resulting in discovery of different microbes in the same sample at the same time, from viroids, viruses to microorganisms such as bacteria.

The outcomes of the proposed project will surely serve to expand the present knowledge on plant viruses and phytoplasmas, their epidemiology and the burden they pose for important field crops such as tomato, grapevine etc. Researching the virome of tomato in different areas and countries (Croatia, Slovenia, Serbia), using powerful HTS based tools, such as long read based nanopore sequencing will help stablishing the baseline viral fingerprint of such important crop and distinguish pathogenic viruses from potentially harmless or even beneficial ones. It will also help to distinguish between strains that despite being genetically similar have different pathogenicity in their host and that are currently difficult to assembly with sufficient accuracy from short read based platforms. Assessing water as a route of transmission of plant viruses and providing methods and pipelines for HTS based analysis of the plant viruses present in such milieu will help confirming the relevance of water as a potentially important modulator of plant virus epidemiology. This is especially relevant for arid regions, where water is scarce and in many cases, treated wastewater is used for irrigation. Concerning phytoplasmas, developing HTS based tools that will enable an increase in the genomic data of this impossible-to-culture pathogen, will deeply benefit the field, and allow a more accurate classification of existing phytoplasmas and designing of molecular assays to distinguish between highly virulent and low virulent pathotypes. Finally, the optimization of wet lab workflows and dry lab algorithms and pipelines that will allow discovery and identification of complex mixtures of plant pathogens (Viruses, Phytoplasma) in different matrixes such as crop plants, weeds, and water, will serve to expand the knowledge on the epidemiology and behavior of such pathogens and to devise novel efficient strategies for plant protection through prevention and improved diagnostics.

Project is connected also to international projects such as European Marie Curie Innovative Network for Next Generation Training and Sequencing of Virome (INEXTVIR) and Euphresco projects Faster, cheaper identification of emerging virus problems (VIRFAST), Plant Health Bioinfromatics network (PHBN) and project Development of efficient methods and identification of barcodes for discriminating Grapevine flavescence dorée sensu-stricto from other related phytoplasmas and investigation of potential correlation between taxonomic identity and grapevine, alders and hazelnut plant hosts, which will start in 2021.