‘Colouration, distortion, death’ – we must be talking plant virus symptoms!
We were delighted to welcome around 50 attendees from at least seven countries to our 10th CONNECTED webinar, with two great talks from Dr Adrian Fox and Dr Ian Adams of Fera Science Ltd.
Here you can find a round-up of the talks, and we will release the video to members when it has been edited. But first, some handy resources that are free to download:
Facilitating the adoption of high-throughput sequencing technologies as a plant pest diagnostic test in laboratories: A step-by-step description
Next Generation Sequencing for Detection and Discovery of Plant Viruses and Viroids: Comparison of Two Approaches
Virus symptoms and diagnostics
Dr Adrian Fox, Fera Science Ltd, UK
Adrian told us that it is generally preferable to use a targeted approach to virus diagnostics, depending on what you suspect the pathogen is. However, a targeted approach, searching for a known pathogen, does make it more difficult to uncover novel viruses.
If you need a more multi-target approach, you can use generic ELISA or PCR to test for a wider range of viruses. Non-targeted methods involve symptom recognition, bioassay, electron microscopy, dsRNA isolation and high throughout sequencing (HTS). A workflow might look as follows:
- Assess host and any symptoms
- Consider likely viruses
- Prepare sample and undertake a selection of appropriate tests
- Interpret the results
- Then either: get a secondary confirmation… Or: undertake further, non-targeted diagnostics.
It can be challenging to relate the symptoms to the virus – sometimes it is not even obvious that it is a virus (rather than something bacterial or fungal). There is a huge range of viral symptoms, including mottles, stripes, colouration changes, vein effects, leaf/flower/fruit distortion, or necrosis. Symptoms can be a reaction to a wide range of things, and what you observe is likely to be far more subtle than the pictures shown in textbooks.
HTS is a useful method of confirming virus identification, and has been used for outbreaks across the UK. It can be used for wide-ranging surveys, to see what viruses are present in the field in different areas.
It is really important to validate all your tests so you can trust the results. You also need to ensure you test the right parts of a plant. This is something that can make a big difference – for example, when trying to diagnose Tomato Brown Rugose Fruit Virus (ToBRFV).
Validating your tests can help to ensure that you are really detecting a particular virus in a plant, rather than your results being caused by environmental contamination. Virus detection tests were once performed as an experiment on different surfaces in a glasshouse – the panels, the fan, phones, gloves and so on, and there were strong results indicating the presence of viruses, all from contamination.
Adrian’s talk was followed by several questions, one of which was: “How can you diagnose a virus in your field which is affecting several different species of crops?” The answer was that, as well as using HTS or bioassay, you can use tests that indicate the presence of certain virus families. These will help narrow it down, so you can then use more targeted testing.
High throughout sequencing (HTS) and library preparation
Dr Ian Adams, Fera Science Ltd, UK
Ian gave an excellent and very specialist talk on HTS and how best to prepare your samples. There are several different machines used for HTS. In terms of the larger ones, most people tend to use MiSeq, but the most sequencing worldwide is done by Novaseq (Illumina). These large machines can be expensive to purchase, but luckily there are several smaller and cheaper options available. For instance, the MinION Flongle adapter, which allows use of £75 flowcells, or the Illumina iSeq100 and nano flowcells.
There are several approaches you can take to sequencing in terms of sample types. These include:
- Using Microbial genomes and metagenomics
- Metabarcoding (PCR and Illumina indexing)
- RNA (Viral)
To begin your library preparation, it is important to chop your DNA down to an appropriate size for sequencing. You can fragment the DNA molecules in several ways – there are machines that use ultrasound or spin columns to break it up, or you can squirt it through a syringe. The process is then as follows:
- Clean up your DNA – end repair and phosphorylate
- Ligate index adaptors
- Denature the molecule and amplify the final product.
If you are doing RNA sequencing, begin the process above by making double-stranded cDNA.
For metabarcoding, Fera uses a two-stage PCR process. It starts with target primers, followed by the addition of indexed primers.
Sample pooling can be used –– enabling you to combine multiple samples in the same run. Bulking up samples can save money, since a library preparation kit costs about $100.
Take a look at the video below to learn more about Illumina sequencing.
It was great to have so many questions for speakers, and they have kindly agreed to answer those which we did not have time for by email. We will post these remaining questions and answers on our Watch CONNNECTED Webinars page, along with the video of these talks.
Huge thanks to Ian and Adrian for such a great event!