NCPN-Roses Meeting JUly 22, 2020


This series of slides has to do with 2019-2020 virus testing of selected rose collections. The testing was done by Texas A and M scientists.

This series of slides has to do with a comparison of how the accuracy of the previous type of tests compares with high throughput sequencing (HTS). Please note that even PCR misses some infections.

I feel that HTS is considered the present state of the art method for plant virus detection. HTS is also called “Next Generation Sequencing”.

I’m glad you participated in the virtual meeting Henry. I’m excited we’ll just delay the meeting plan and have what was planned in person in the Twin Cities for this year in the Twin Cities next year. Hopefully people would consider coming to attend.

HTS is sure a great tool. As Maher shared, it is expensive (at least relatively and and this point)- ~$1500 per sample I remember him saying. It captures massive amounts of nucleic acid sequence and then Maher and his team search through it to see if there are sequences common to families/groups of known viruses versus plant DNA. It is a great way, as you mentioned Henry, to assess material in that it is more sensitive than PCR.

The current goals, as I understand them with HTS at Foundation Plant Services, is to especially help much more quickly and accurately assess a variety for viruses to streamline quarantine issues to get new varieties moved around the world and released to growers. This is particularly important it sounds like for the grape work they are doing because of the high value associated with grapes. The tool is also especially valuable to help recognize/find a virus we didn’t realize was in a host plant and study it. The current ELISA (protein) and PCR (nucleic acid) tests are very specific as we look for something associated with a specific virus (unique protein or nucleic acid). From the ELISA or PCR tests all we can say is if that particular virus is present or absent (and as Maher presented there are false positives and negatives at times). Looking for basically everything in a sample with HTS we can actually find something we weren’t specifically looking for with ELISA or PCR and then characterize a novel virus- determine the symptoms it produces in roses in this case, if it is something of serious concern, how it is transmitted, etc.

One of the main NCPN-R goals is to help come us develop effective, specific (and affordable) diagnositic tools that are accurate for the different viruses impacting roses. We can then standardize and recommend tests for particular viruses to plant disease clinics. Even though HTS is the “gold standard” right now, it is being used from my understanding to basically help assess the accuracy of other more specific and affordable tests to recommend the best of them. It will be those other optimized tests likely that plant disease clinics will be using (most likely a variation of a PCR test) to hopefully confidently assess samples growers send in to have tested.

Maybe with enough funding and interest, HTS may be able to become the standard way a new rose gets into the clean stock block at Foundation Plant Services to have propagation material for industry, even though it likely won’t be practical for routine testing of viruses from nursery growers or homeowners wanting to know what their material may have. The overall NCPN has a strong focus on helping industry get and use clean plant material to help secure US agriculture. The critical key is generating and having clean stock to make available, but then also have tools to help industry and the clean stock locations to know their stock remains clean over time. If we had to use HTS to retest the 800+ roses in the clean stock block every couple to few years to double check they remain clean at $1500 per sample, that would be too expensive. The goal would be to test for just the key viruses recognized to be of higher importance.

I’m excited for all the work possible with the NCPN-R. I think in the years to come a key is for more industry members recognizing how they can use this resource to get their key varieties into the program so they can always have clean stock to get back out as needed. Also, having reasonable protocols for nurseries to manage their stock to help keep it clean and test it is important too. Foundation Plant Services has a limited amount of plant material of any single variety and can’t be the direct commercial source for all the propagation material needed by industry. The industry needs to get clean stock and bulk it up at their facility and manage it.

The NCPN 2019 meeting estimated the cost of HTS as $350. slide 27 of 30

Having a reliable testing method is a primary necessity for developing a clean plant program.
However, it is not the only necessity. Having a reliable method to clean an infected rose is also a necessity.

Does a reliable cleaning method exist? The following February 2019 link appears to be an admission that we did not have a reliable method at that point in time.

The NCPN 2019 meeting contained similar information: slide 17 of 26.

The NCPN 2020 information showed the slides presented, but did not include the speaker’s comments. I do not recall any comment(s) that a method was developed that is successful.

Dave, can you add anything on this point?

Hi Henry,

It is so true we need to keep improving methods to both accurately detect viruses of concern in our roses and to also optimize methods to clean infected roses of virus that can hopefully work on a wide range of genotypes. That is interesting about the discrepancy in costs between the 2019 slides and what I remember Maher verbalizing and writing down during our time together. Hopefully Maher misspoke or I misheard and it actually is $350 per sample as written on that 2019 slide. That would sure make getting samples through HTS much more attainable. Hopefully over time the costs will fall like many technology related things.

Virus clean up is such a struggle… They would definitely prefer to get material from breeders as early as possible before a genotype hopefully gets infected. They’ve emphasized over the years they’d rather get a few advanced selections before release and toss those that don’t get introduced in order to increase the probability to start with clean material of what is introduced, than waiting and eventually having a greater chance at receiving infected material of something of high value and have the long and unpredictable process of trying to clean it up.

I suspect therapy/clean up will continue to be a struggle and the tools found that can help will continue to be incremental. Hopefully I’m wrong and there will be something amazing found that allow us to leap forward. People have been working on optimization of the process for years with a number of crops using a number of tools (e.g. cryotherapy, modifying tissue culture conditions, using antivirals in culture, etc.). Hopefully we can keep trying the latest advances in other crops with roses. FPS shared heat therapy followed by grafting of axillary buds not being very effective for them in the past. That is disappointing and has pushed them to focusing on meristem isolation and tissue culture over the past several years and work to optimize the process in roses. They definitely have lots of expertise in all the fantastic clean up work they do with grapes, strawberries, sweet potatoes, etc. So many roses don’t respond well to the tissue culture environment. I can definitely attest to that. I have some polyantha roses doing okay in culture now that have a strong dose of R. wichurana in them. Most of the other roses I tried were much more finicky.

Even if there are shoots that grow from isolated meristems, the folks at FPS have shared many don’t root well even with auxins, lowered humidity, etc. I’m excited for the discussion we had about them moving towards trying micrografting. Hopefully as long as one can get meristems to survive and shoots to at least start to develop some, they can be micrografted to a clean rose growing in culture to help increase survival eventually get them successfully out of culture to test. Some viruses definitely sound easier to get rid of than others in roses. I think Prunus Necrotic Ringspot Virus was one of the harder ones to clean up from their past presentations. Definitely, having a number of plantlets from different meristem isolations is helpful to find at least one that is clean of the virus(es) in question. For the ageratum and hydrangea clean up I have done (tobacco streak virus, Hydrangea chlorotic mottle virus, Hydrangea ringspot virus, etc.), that has definitely increased the probability to find at least one clean sample. There is a large art component to it too. I’m grateful my hands are pretty steady still. It is challenging to effectively get down to just the growing point and eliminate as much of the unwanted material as possible. When I isolate meristems, I have a number of sterilized scalpels ready so I can keep switching to a clean one as I get closer to the meristem to try not to drag infected sap onto the meristem. Moving quickly after the meristem is isolated and on the scalpel is a challenge too so it doesn’t dry out too much and die before it is on the media. I’ve switched to Petri plates versus test tubes for the first media because I’ve found for me at least there is more control to quickly get the dish open and get the meristem onto the media at a controlled depth. That has definitely helped with hydrangea. Using sterilized vitamin C in the final sterile rinse water has helped reduced oxidation and led to a higher percent of hydrangea meristems surviving too. If I can make some time to commit to it, I should try some rose meristem isolations of something of value not in the collection yet that may be dirty. Hopefully if I can get something to survive through the process I can pass it along to FPS to test. If it goes okay, I should see if I can help the team more routinely to supplement their work. Kevin Ong at Texas A&M wants to set up a lab to begin to try. I already have the lab and expertise in other species, so hopefully I will be able to help too. It will be a nice opportunity to involve a student from time to time in the process.

Thanks David for the complete reply.

I am not familiar with Nanopore sequencing. It may be useful to the NCPN due to the lower cost:

“Nanopore sequencing has several advantages such as the relatively low cost compared to other HTS technologies, high mobility due to the sequencer small size and rapid sample processing, without the need for reverse transcription for RNA viruses (Kiselev et al., 2020).”

Frontiers | Detection of Plant Viruses and Disease Management: Relevance of Genetic Diversity and Evolution

Here is another group interested in rose viruses.