Detection of plant by next-generation sequencing (NGS) Nataša Mehle, Denis Kutnjak, Anja Pecman, Maja Ravnikar Department of biotechnology and Systems Biology, National Institute of Biology, Ljubljana, Slovenia

INTRODUCTION IDENTIFICATION METHODS Climate changes around the globe and increased international trade are ELISA, PCR, pushing plant pathogens and their vectors into unexpected new areas, real time PCR, NGS

raising many new risks for agriculture. These not only increase the chance PAST etc.

of introduction of new pathogens into the new area, but also increase the NOWADAYS chance of the pathogen persisting in a new host species. The accurate identification of plant pathogens, e.g., virus species, is crucial for planning Methods designed to identify one Universal technology for an effective prevention of new disease spreading and its eradication. or few viruses at the same time virus identification

WETLAB & AUTOMATIC TECHNICAL WORKFLOW OF NGS ANALYSIS Sample —> RNA isolation —> small RNA deep sequencing —> user-friendly automatic bioinformatics pipeline in CLC Genomics Workbench developed at NIB —> confirmation (e.g., by electron microscopy (EM), ELISA, PCR-based methods) and study of biological impact

EXAMPLES: D566/16 (parsley) D159/15 (tomato) ELISA (PVM): pos

ELISA (PVY, WMV,

ZYMV): neg potyvirus Potato virus M (PVM)

carlavirus (,

or or ther ther

o Carlavirus) (note: observed symptoms were NGS: not typical of PVM infection!) PVM & confirmed by ELISA, PCR Southern tomato virus (STV; PVM and HMV confirmed by NGS: and mechanical unassigned plant virus) & mechanical inoculation of test Apium virus Y (ApVY) & inoculation of test plants Henbane mosaic virus (HMV; plants and PCR or ELISA; Carrot thin leaf virus (CTLV) , Potyvirus) STV confirmed by PCR (both: Potyviridae, Potyvirus) ApVY was found also in asymptomatic parsley —> Is responsible for symptomatology CTLV Further study of biological impact confirmed alone or in mixed infection with ApVY? that HMV was responsible for symptomatology D193/15 (eggplant) mechanical inoculation of D64/16 (petunia) ELISA and PCR-based methods test plants: severe (CMV, CSNV, INSV, PVY, TMV, symptoms on several test ToMV, ToRSV, TRSV, TSWV): neg plants

EM & mechanical inoculation NEGATIVE EM: neg of test plants: neg

ELISA and PCR-based pathogen other

unidentified virus or or virus unidentified NGS: methods for several Petunia vein clearing virus (PVCV) viruses: neg (, Petuvirus) NGS: Cucumber mosaic virus (CMV) confirmed by ELISA on sample and test plants Additional research is needed to evaluate whether (, the detected viral sequences exist in an episomal Cucumovirus) form and not only integrated in the plant genome

CONCLUSIONS  With NGS analyses of ornamental and vegetable samples we discovered new plant viruses and new hosts in Slovenia  Automatic bioinformatics pipeline in CLC Genomics Workbench developed at NIB was shown to be fast and reliable tool for the official detection of plant pathogens, as a support for the Phytosanitary Administration of the Republic of Slovenia and the Phytosanitary Inspection Service  NGS was introduced as regular diagnostic method at NIB

ACKNOWLEDGEMENT This study was supported by the Slovenian Research Agency (grant number L4-5525), by the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia and by the COST FA 1407. EM analyses were carried out by dr. Magda Tušek Žnidaršič. The authors thank Larisa Gregur for excellent technical help.