This is the report from a BSPP Undergraduate ‘Vacation’ Bursary.
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Barley yellow dwarf virus (BYDV) is considered the most damaging cereal virus, vectored by aphids, and reducing yields in affected wheat by 40-50%. Yield losses occur insidiously with plants presenting symptoms, such as leaf yellowing and plant stunting, many of which are inseparable from common stress. This means cereal crops may not be recognised as infected unless appropriate molecular diagnostics are employed. This can render the pathogen unchallenged whilst other mitigating factors limit effective disease control.
There are three prominent strains of BYDV in the UK; MAV, PAV, and PAS. The outcomes of mixed BYDV infections, where multiple strains coinfect a host, are poorly understood. This means that identifying and explaining ongoing changes in strain dominance proves challenging. In my time at Rothamsted Research, I studied mixed-strain infections using aphids carrying BYDV-MAV and -PAS and three varieties of wheat: Wolverine (the first commercial UK wheat variety with resistance/tolerance to BYDV), Cadenza and Solstice (two BYDV-susceptible cultivars).
Sitobion avenae carrying PAS were first used to challenge 18 plants of each variety with appropriate randomisation and controls employed over a 24-hour feeding interval before being removed. 5 days later, S. avenae aphids vectoring MAV were similarly applied to plants. Cuttings were taken 1 and 2 weeks’ post final inoculation. The interval between virus exposure served to study cross-protection where one established strain perhaps prevents a second challenger from colonising the host. This is thought to occur where the first established viral strain coat proteins bind to the second viral genome, preventing replication.
In the second scenario, 18 wheat plants were treated in the reverse, first challenged with MAV and then 5 days later with PAS. Finally, a simultaneous inoculation with both strains was performed on a third set of plants. To explore the reacquisition of virus strains from mixed infection scenarios, non-viruliferous S. avenae were allowed to feed on all plants then tested for BYDV. To simulate the spread of the virus in the field, aphids were also allowed to feed on dually infected (BYDV-MAV and -PAS) plants then transferred to healthy plants which were later tested for BYDV.
To test for and quantify BYDV strains, total RNA extractions were performed from plant and aphid samples, cDNA synthesised using random hexamers and various molecular assays undertaken. BYDV primers designed in silico to target all BYDV variants were first validated through optimising polymerase chain reaction conditions. These generic and other BYDV strain-specific primers were then used in a series of quantitative TaqMan assays with probes unique to the MAV or PAS coat protein encoding regions, fluorescing proportionally to viral titre in each sample.
Results showed consistent patterns in strain dominance across the infection scenarios and wheat varieties, but suitable reference genes must be employed in future testing to ensure viral titre quantifications are robust and not an indicator for quality of RNA extractions or cDNA synthesis reactions performed. Findings generated from the Wolverine variety may have implications for promoting effective deployment of this valuable source of genetic BYDV resistance. The operation of the Bdv2 gene responsible for the Wolverine mode-of-action is currently unknown. Therefore, it is possible that Wolverine plants could be reserves for BYDV whilst being unaffected themselves.
Future research into BYDV-mixed infection should explore PAS-PAV strain dynamics. Despite being serologically similar, PAV is preferentially vectored by Rhopalosiphum padi (a key aphid vector species in the UK alongside S. avenae) whilst PAS presents less preference, being vectored by a broader range of aphid species. It would be interesting to study whether, through PAS-PAV mixed infection, heterologous encapsidation could be observed where viral genomes get mis-packaged in the alternative strain coat proteins and what implications this may have for aphid vector transmission.
Through the BSPP studentship, I have learned to perform a wide range of molecular techniques, I have gained confidence in experimental planning, and adapting then optimising protocols to ensure experimental success. I became so much more passionate about plant pathology when presented with all the gaps in knowledge regarding BYDV and the challenges of investigating an insect-vectored virus. More broadly, learning about other research conducted across Rothamsted from genome editing through to gene mapping techniques in order to underpin mechanisms of disease resistance has encouraged me to look into PhD programmes within the field. I would like to thank my supervisor Lawrence Bramham for all the support, and BSPP for giving me this amazing opportunity.
Anna Dickinson
University of Bristol
