Application of Next Generation Sequencing (NGS) for detection of plant pathogens

Transcription

1 Application of Next Generation Sequencing (NGS) for detection of plant pathogens Igor Koloniuk Biology Centre CAS Institute of Plant Molecular Biology BIOTECHNOLOGY - the Phenomenon of the 21st Century 20th September, 2016 Česke Budějovice

2 Plant pathogens viruses viroids phytoplasmas bacteria fungi

3 Why diagnosticts is important? Practical purposes elucidate crop losses with unclear etiology/ new disease certification of pathogen-free seed material for growers testing imported/exported plant material to prevent spread of pathogens between countries and continents Research purposes searching for novel pathogen species study of pathogen s population at the level of a single host

4 Why diagnosticts is important? Diagnosis what caused the disease symptoms Monitoring looking for a specific pathogen Surveillance prediction for future risks

5 Why diagnosticts is important? Diseases with unknown ethiology: unknown pathogen complicated and inefficient sample preparation difficulties in a transfer to a new host Pathogens present at low titer phloem-limited viruses

6 Methods of detection and identification Visual inspection, electron microscopy Serological detection (ELISA, lateral tests) PCR, qpcr, hybridization methods

7 Traditional diagnostics vs NGS Biological characterization Virus purification Nucleic acid isolation Sequence determination low virustiter inefficient method or problematic matrix mixed infection bioinformatics parameters choice of platform NGS sequence determination

8 How? Nucleic acid isolation Preparation of sequencing libraries Sample Bioinformatic analysis, verification of the results Sequencing

9 Species diversity of viruses per cherry sample Project LD14004 COST CZ The analysis of cherry and sour cherry trees virome by sequencing new generation and the development of instruments for specific PCR virus detection

10 Species diversity of viruses per cherry sample; sorted by host type Project LD14004 COST CZ The analysis of cherry and sour cherry trees virome by sequencing new generation and the development of instruments for specific PCR virus detection

11 Species diversity of viruses per clover sample Project LD15035 COST CZ Implementation of NGS in plant virology unprecedented capacity with a risk of data overflow

12 Problematic sample with unclear ethiology Strawberry cv Čačanska rana A summer-fruiting cultivar of moderate vigor and marked production of runners. Early vegetation onset. Flowers and ripens early. Very good cropping. Ripens within 20 days. Resistant to fungal diseases of strawberry Project QJ Research and utilization of advanced technology systems in organic and integrated production

13 Problematic sample with unclear ethiology Strawberry cv Čačanska rana known host of: Strawberry crinkle cytorhadbovirus Strawberry mottle sadwavirus Project QJ Research and utilization of advanced technology systems in organic and integrated production

14 Problematic sample with unclear ethiology Strawberry cv Čačanska rana known was presence of: Strawberry crinkle cytorhadbovirus Strawberry mottle sadwavirus NGS revealed additionally: Cherry leaf roll nepovirus two genotypes of Strawberry mottle sadwavirus Project QJ Research and utilization of advanced technology systems in organic and integrated production

15 Perspectives/challenges of NGS vs going deeper at the scale of -omics versatility of application extra sequence information from a sample high analysis cost per sample complexity of the data analysis biases for low-titer viruses

16 Transfer of technology Verified NGS method for the detection of plant pathogens developed at IPMB License offered to private and government institutions for: Pathogen detection Seed certification (potato, fruit trees) Quarantine purposes TACR GAMA transfer of technology Diagnostics of viruses, viroids and phytoplasmas with Next Generation Sequencing

17 Acknowledgement Jana Fránová Jaroslava Přybilová Josef Špak Karel Petrzik Lenka Hrabáková Ondřej Lenz