| dc.description.abstracteng | The overall aim of this dissertation was the implementation of high-throughput sequencing (HTS) technologies for plant viruses’ identification and characterisation. Different viral enrichment approaches were tested to optimise a tool that would detect a broad range of different viruses and that can be used as a generic, reliable and cost-effective strategy for virus diagnostics. Moreover, the interactions of aphid vectors of nanoviruses with infected plants were investigated and the effect of NSP on aphid feeding behaviour were closely observed.
Chapter 1 gives a general introduction to the concept of this research, and it also outlines the aims of this work. Currently used plant virus diagnostics methods are described with a focus on HTS for virus detection using different enrichment and platforms. It also covers the virus-vector-host interactions with focus on the model system nanoviruses-aphid-host used in this research.
In addition, this thesis is divided into two parts (part one and part two). Part one focuses on the plant virus identification and describes the discovery and characterisation of new viruses, divergent strains and isolates from new virus hosts or geographical locations. The virome of peas was investigated over a period of three growing seasons in six different German regions. Furthermore, three viral enrichment approaches for plant viruses and viroids detection by HTS were compared. Part one contains three chapters (2, 3 and 4). In Chapter 2, the discovery and characterisation of two new viruses i.e., alfalfa associated nucleorhabdovirus from Medicago sativa (Austria) and caraway yellows virus from Carum carvi (Germany)are described.
Moreover, the reports on the identification and characterisation of the causal agent of several diseases in legumes and vegetables. HTS in combination with conventional diagnostic methods (ELISA, EM, PCR-based methods and Sanger sequencing) were applied to various plant virus samples in the JKI selection. The following viruses were described:
• Divergent strains of:
1. Melon chlorotic spot virus from M. lupulina from Austria
2. Carrot torradovirus 1 from Apium graveolens from Germany
3. Turnip crinkle virus from Alliaria petiolata from Germany
• Divergent isolates of turnip yellows virus (TuYV) from pea and rapeseed oil, in addition to a first report of turnip yellows associated RNA from Germany
• First reports of:
1. Beet soil borne virus and beet cryptic virus 2 from Beta vulgaris subsp. vulgaris
2. Physostegia chlorotic mottle virus from Solanum lycopersicum from Germany
3. Southern tomato virus from S. lycopersicum from Germany (new MinION approach was described)
4. Pea necrotic yellow dwarf virus (PNYDV) was detected for the first time infecting Vicia faba, V. sativa and Lens culinaris in Austria and Germany, and for the first time in Denmark and the Netherlands.
The focus in chapter 3 is set on the identification and characterisation of viruses in German pea fields. For three successive seasons, pea fields in six different regions were surveyed. Samples were taken from symptomatic pea and asymptomatic pea plants, in addition, samples from surrounding non-crop legume and non-legumes weeds were taken. The samples were analysed using HTS and the presence of all detected viruses was confirmed by RT-PCR. Thirty-five viruses in total were detected during this survey, including nine new viruses, thirteen viruses new for Germany. In addition to plant viruses, virus associated nucleic acids were also detected. Pea enation mosaic virus 1 and 2, TuYV, and PNYDV were the most common viruses in the German pea fields. Interestingly a new emaravirus was detected for two successive seasons in the same region, tentatively called pea associated emaravirus. Additionally, several new virus sequences and divergent sequences were detected in the surrounding legumes and non-legumes. By analysing the data spatially and temporally, no differences were observed.
Chapter 4 describes the comparison of three different viral enrichment approaches for virus discovery by HTS. Double stranded (dsRNA), ribosomal RNA depleted total RNA (ribo-depleted totRNA) and small RNA (sRNA) enrichment approaches were compared for the detection of viruses and a viroid representing different genomes i.e., ssRNA [(+ve) and (-ve) senses], DNA and a viroid. The dsRNA approach in this study was better compared to the other two approaches as it is faster, cheaper and all the known and unknown viruses/viroid in the study were detected. However, the number of reads from dsRNA approach were lower when compared to the other two methods. Interestingly, two additional unknown viruses were discovered with the dsRNA enrichment approach i.e., Vicia cryptic virus and Wuhan aphid virus 2 (WHAV2) but only one of them was discovered with sRNA or ribo-depleted totRNA, respectively. To our knowledge this is the first detection of WHAV2, a recently discovered virus from aphids in China, from plant tissues.
In the second part of this thesis (part two), the interactions of nanoviruses with their insect vectors and their hosts were studied. Chapter 5 reviews the current knowledge about nanovirus-vector interactions. In chapter 6, the probing and feeding behaviours of Acyrthosiphon pisum on nanoviruses-infected faba beans were investigated using electrical penetration graph. The probing and feeding behaviours of A. pisum changed on faba bean necrotic yellows virus and PNYDV-infected plants. Additionally, the effect of the absence of the nuclear shuttle protein of FBNSV on the feeding and probing behaviours of A. pisum was tested. Additionally, few significant differences were detected, however no clear differences could be concluded.
A general discussion of the findings of this thesis is laid out in the final chapter (7), and a prospect for future research is given. | de |