S14 - Session O6 - High throughput sequencing testing of certified apple M9 rootstocks

Authors: Josef Spak *, Igor Kolonuk, Jaroslava Pribylova, Ondrej Lenz, Jiri Sedlak, Frantisek Paprstejn

A project on High Throughput Sequencing (HTS) testing of certified rootstocks for virus-like agents (virus, viroid, phytoplasma) started in 2019. The aim is to acquire virus-like agent-free apple propagation material with qualitatively new knowledge about health status using HTS. Twenty-eight M9 rootstock plants certified as Standard, Elite, and Basis material purchased from a Czech and two international producers, respectively, were HTS tested. The HTS libraries were prepared from enriched double-stranded RNA and processed with NovaSeq 6000. All HTS tested plants were free of Apple mosaic virus, (ApMV), ASGV, Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Candidatus Phytoplasma mali (Apple proliferation phytoplasma), and Apple scar skin viroid and complied with the EPPO certification standard (Anonym 1999). The results were verified using conventional RT-PCR. Three of eight Basis quality rootstocks were found free of any virus or viroid-like sequence while in those remaining Apple hammerhead viroid-like RNA (AHVd) and Citrus concave gum-associated virus (CCGaV) were found by HTS and confirmed by RT-PCR. All Elite rootstocks were free of CCGaV and AHVd. In the Standard and Basis rootstocks, we detected CCGaV, Solanum nigrum ilarvirus 1, and several sequences of three tentative novel viruses, which are subjects of further studies. As CCGaV has not yet been reported to occur in the Czech Republic, we RT-PCR screened several commercial orchards, old apple alleys, and home garden trees, and found trees of several cultivars infected. CCGaV and AHVd mixed infection was reported in apple also from Hungary (Jahan et al., 2019) and CCGaV from the USA (Wright et al., 2018). Our results suggest the application of more rigorous HTS testing of rootstock source plants to determine their health status. Granted by the project QK1910065 of the Czech Ministry of Agriculture.