Articles
High throughput sequencing testing of certified apple M9 rootstocks
Article number
1378_51
Pages
391 – 396
Language
English
Abstract
A project on high throughput sequencing (HTS) screening of certified rootstocks for virus-like agents (virus, viroid, phytoplasma) started in 2019. The aim is to acquire apple propagation material free of the virus-like agents with qualitatively new knowledge about its health status using HTS. Twenty-eight M9 rootstock plants labelled as standard CZ, elite, and basis material were purchased from a Czech and two international producers and were HTS tested.
The HTS libraries were prepared from enriched double-stranded RNA extracts and processed with NovaSeq 6000. All HTS tested plants were free of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Candidatus Phytoplasma mali (Apple proliferation phytoplasma), and Apple scar skin viroid (ASSVd) and complied with the EPPO certification standard (Anonymous, 1999). The results were verified using conventional RT-PCR. All elite rootstocks were free of the virus-like agents.
In the standard CZ and basis rootstocks, we detected Apple hammerhead viroid-like RNA (AHVd), Citrus concave gum-associated virus (CCGaV) and several sequences of three tentative novel viruses, which are subjects of further studies.
CCGaV and AHVd mixed infections were reported in apple in Hungary (Jahan et al., 2019) and CCGaV in USA (Wright et al., 2018). Our results suggest the necessity for more rigorous HTS testing of rootstock source plants to determine their health status.
The HTS libraries were prepared from enriched double-stranded RNA extracts and processed with NovaSeq 6000. All HTS tested plants were free of Apple mosaic virus (ApMV), Apple stem grooving virus (ASGV), Apple stem pitting virus (ASPV), Apple chlorotic leaf spot virus (ACLSV), Candidatus Phytoplasma mali (Apple proliferation phytoplasma), and Apple scar skin viroid (ASSVd) and complied with the EPPO certification standard (Anonymous, 1999). The results were verified using conventional RT-PCR. All elite rootstocks were free of the virus-like agents.
In the standard CZ and basis rootstocks, we detected Apple hammerhead viroid-like RNA (AHVd), Citrus concave gum-associated virus (CCGaV) and several sequences of three tentative novel viruses, which are subjects of further studies.
CCGaV and AHVd mixed infections were reported in apple in Hungary (Jahan et al., 2019) and CCGaV in USA (Wright et al., 2018). Our results suggest the necessity for more rigorous HTS testing of rootstock source plants to determine their health status.
Authors
J. Špak, I. Koloniuk, J. Přibylová, O. Lenz, J. Sedlák, F. Paprštein
Keywords
viroid, virus, apple, rootstock, PCR, HTS, molecular detection
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