131: Virus Detection in Grapevines

With the prevalence of Leaf Roll Three, Red Blotch, and other viruses, accurate and timely detection of viruses in grapevines has never been more imperative. Alan Wei, Owner and Lab Manager at Agri-Analysis LLC in Davis California explains how his lab is using next generation sequencing (NGS) to find new viruses. Currently, polymerase chain reaction (PCR) is the widely accepted method for testing for viruses. This process tests for one gene at time. Next generation sequencing allows labs to test multiple genes at a time and get results much faster.


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Craig Macmillan  0:00 

My guest today is Alan Wei, who's owner and lab manager of Agri Analysis LLC in Davis, California. Alan, thanks for being on the show.

Alan Wei  0:10 

Thank you very much, Craig, for hosting me. And I'm very delighted to be here. And I want to use this opportunity to say hello to listeners as well.

Craig Macmillan  0:19 

So Alan, I want to have you on the show, because I want to talk about anything that's new and exciting in the world of grapevine virology, and a lot of research and a lot of development in industry with labs like your own. So, what's what's what's happening out there, what's going on with detection of viruses these days?

Alan Wei  0:36 

There is a International conference on grapevine viruses that's held every three years. So last time was 2018, in Chile. And the second, the following time was supposed to be 2021 in Greece, and unfortunately, that was canceled due to the obvious reasons, and then was supposed to be happening this year. And by the way, is not happening, and it's postponed until next year. So as a result, we have not, the researchers in this field have not been able to meet to report the latest grapevine viruses. Just to mention something that in the literature, for example, there are two or three new DNA viruses being discovered and reported. In fact, in grapevines, but their practical consequences are known. So we probably don't want to get in too much into them.

Craig Macmillan  1:33 

Maybe not, but I think this is an interesting thing because for instance, red blotch, caught everybody by surprise. And so how are these are these these new viruses, how are they found, if you are looking forward, you're looking for other things, what kind of technology they're using to find this new stuff?

Alan Wei  1:49 

Typically, they're found by deep sequencing, also known as NGS Next Generation Sequencing. Researchers are always trying to look for the frontiers of why viruses virology by applying these methods and find this new viruses, but their practical impact needs to be validated, study to further be before we alarm growers. And red blotch was found a similar way. With the exception that the red blotch phenomena, and the disease was known to growers for years without the assay and the way the branch was first reported, or discovered through NGS that was, you know, the "wow" moment to growers. Yeah, we do now know what is causing this read leaf in my vineyard.

Craig Macmillan  2:43 

Tell me a little bit more about NGS, it sounds like this is gonna be an important technology for us, this deep sequencing.

Alan Wei  2:48 

Yeah, definitely. Deep sequencing is very widely used in the research community. And, when was that, in December meeting hosted by FFPS, they reported that NGS is going to be accepted by regulators like APHIS as a alternative way of testing materials coming from overseas. Which means shortened time and rapid, faster deployment of foreign important materials in in this country, or practically to growers hands. Yeah, the technology is definitely upcoming, and we're looking to possibly deploy it for routine use. We need to hear more feedback before we really do it.

Craig Macmillan  3:41 

This is obviously a very complicated technology, but like in a sense, can you explain what it is?

Alan Wei  3:48 

PCR is the way that accepted method in testing viruses or microbial in general. Compared to PCR, which tests one gene at a time, NGS would allow you to test multiple genes at a time. Because through the use of small, small redundant primers, which amplify many sometimes millions of hundreds of millions of fragments of the gene, which can parallelize sequenced with that data, and coupled with information, analysis, informatics, you can extract new new information from your sample, including new viruses, new bacteria.

Craig Macmillan  4:38 

So essentially, I've got a sample of plant material. And I run it through this NGS process, and it comes back and says, hey, there's genetic material in here that doesn't belong here. This is not grapevine, or hey, visit genetic material that's associated with some virus or something like that. And that's the flag that I get. And I get it from the whole picture. I'm not doing it like like you say gene by looking at for specific genes, I'm getting a kickback, I'm saying hey, there's there's a variety of things or whatever genes we weren't, wouldn't even thought to look for.

Alan Wei  5:10 

Exactly, exactly. You're right. And then that gene can be not not only you find genes and not belong to the grapevine, which we considered as, you know, the background gene, by further analysis of that, that special gene, you can assign them to, to pathogens, basically, different types of pathogens.

Craig Macmillan  5:30 

Gotcha. Yeah, that definitely speeds up the process a lot and makes it possible to catch things in finer net than we ever would have been able to do before. So that's pretty exciting.

Alan Wei  5:38 

Yes, def definitely.

Craig Macmillan  5:40 

Coming to red blotch, this is continues to be, you know, a very hot topic, obviously, it continues to be an issue in the field and continues to be an issue in other places. Is there anything new that we've learned regarding the Red Blotch Virus in any realm, anything about how it moves, its symptomology, new means of detection, anything like that?

Alan Wei  6:08 

I have a list of articles that just simply published during the past a couple of years, and researchers from you know, several major universities have really dived deep into the physiology, the virology, their impact on wine quality, in aspect of, of a rather large virus. They're really fascinating. From a practical standpoint, though, the progress has been less because what was reported to us few years ago remain the same, which which you know, very well. Which means rogueing, you know, rogueing your infected vines as aggressively as possible. Sourcing for clean materials as diligently as possible to prevent any viruses infecting material being planted. And once they do present in your vineyard take them out as quickly as possible. And also, although we know the Threecornered Alfalfa Hopper is the vector for red blotch. And folks don't recommend you spray against this particular insect because it is not a very efficient transmitter of the virus. Grapevine is not its preferred host. So those information were already known through talks by various speakers in the past.

Craig Macmillan  7:33 

We were talking about spread. And this is something that is absolutely puzzling to me, in years of field checking, I had never once seen this Threecornered Alfalfa Hopper. But I have talked to people who have. And they apparently are very reclusive, they will move away from you, the signs of the damage and very subtle, they do this little kind of girdling thing in the leaves. I just feel like there's just kind of be another vector. I mean, just I just feel to kind of be another vector. I mean, is there is there anything new in that world? I mean, we've identified the one but it seems kind of mysterious. And I'm thinking about the spread at the Russell Ranch, that finish and plant services ranch where we've not only identified it, but they were able to see that was spreading, attributed to the Threecornered Alfalfa Hopper, correct me?

Alan Wei  8:19 

I completely agree with you. Yeah, we don't see too many of our tree hoppers in the field. Yet spread in Russell Ranch has been phenomenal. It's more like, more exponential increase year after year, since 2018. So it is a mystery. If some of you, listeners, went to the seminar by UC Davis in early December, particularly the presentation by Professor Kent Daane, then the entomologists have been looking at a number of potential hosts. But unfortunately, they either have not been proven yet or, most of them were disproven to be a potential host. So we're still in that regard., virtually in the context of Russell Ranch, it is a complete mystery. You would think through the very aggressive management by FPS, you know, any presence over vectors were eliminated. And any source of vectors were eliminated. We have but yet they see this exponential growth in terms of infected vines, which, which stopped Russell Ranch from operation, basically.

Craig Macmillan  9:40 

Yeah, exactly. And I was actually looking at a table for one of your publications earlier today, and it looks like it went from zero to exponential like there was no nothing was detected for a couple of years or two. Then blammo! And I've been thinking about the same thing happening in other vineyards, I'm familiar with. And obviously just underlines how big of a threat red blotch is because we don't understand, you know, a lot, there's a lot we don't understand about it. So that means you got to sample. That you should rogue vines when you see them. But also you got to be sampling. Are there any is there anything new in the way of sampling protocols? Because I know that the distribution of the virus varies quite a bit between different parts of the plant different times of year and whatnot. So it's easy to miss it. What's the what's the best recommendation these days, if I wanted to test some lines, asymptomatic vines for red watch?

Alan Wei  10:31 

Yeah, we still recommend growers to sample cane materials, because in our analysis, you know, relative concentration in different parts of the vine, the cane materials has highest concentration of virus tighter level. And we also suggest growers to consider combine cuttings from different vines to make a what's called a composite sample. Therefore, they can you know, cost, testing costs can be reduced, and their testing budget can be maximized. And of course, you know, the testing objectives dictate how high your sample. Sometimes growers want to test the individual vines to really zoom in to which vine is exactly is infected, that you can only do that by testing individual vines. But the composite testing gave you the first level of screening. To see if you composite ten vines into one sample, and the entire sample would be positive. But if you want to zoom in which vine, but you're gonna want to use positive so that you can take it out, then you will do individual testing after the first round over the course screen, if you will.

Craig Macmillan  11:44 

Yeah, so the strategy would be basically like test the vineyard. And then if you find that something, then you drill down, and you can get it down to decide kind of what area where the individual vines are. That's a very, very smart technique. It's a really great idea. How sensitive are the detection techniques these days? So like, if I've got a if I have 10 vines out of 1000, and I sample and I sampled 10 vines, and I hit one of them, one of the infected ones, is that enough to show up in in the in the analysis?

Alan Wei  12:15 

Yeah, definitely. So if there's only one out of 10 cuttings is positive, and that means practically you're diluting the by 10 times, it is very much detectable.

Craig Macmillan  12:27 

Is there a lower detection limit?

Alan Wei  12:28 

Yeah, when there's a theoretical detection limit, and then there's a practical detection limit. If we do a back of the envelope calculation, a PCR method would allow you to detect one copy without the problem. But then of course, practically, there are other considerations such as whether you know, the one copy, you can sample that one copy into your PCR tube to begin with, because you know, if there's a one copy per microliter, and the way you want to use a two microliter in a PCR mix, you may or may not be able to transfer that one copy from a sample to the PCR tube to begin with. And even if you do, there may be potential inhibitors that present in grapevine material that could potentially influence your sensitivity as well. So there's a practical detection limit, and there's their theoretical detection limit issues. But overall, you know, we have found the red blotch detection to be not a problem, because typically the virus titer is high enough to be detected, even if you compounded multiple vines or cuttings into one sample.

Craig Macmillan  13:33 

That's good. That's very, very useful, very, very useful. I would love to move on to kind of other viruses because it's red blotch is not the only game in town these days. Leaf Roll complexes and Leaf Roll viruses, there's still a problem correct?

Alan Wei  13:44 

Especially Leaf Roll Three is very much that the top of our problems still and because you know the vaccine is very well known. It's very prevalent. Inoculum widespread to the percentage of vines tested positive for Leaf Roll Three that are coming through our lab is roughly about 15 percent. So Leaf Roll Three is very much prevalent. There have been some really nice talks, organized before the pandemic was by the Lodi growers group. There are some talks from including from South Africa. Recently from Red Blotch symposium where there's some presentations on Leaf Roll Three as well. So Leaf Roll Three is very much a serious problem. And growers need to be very vigilant against the Leaf Roll Three from from new planting materials to management of existing vineyards.

Craig Macmillan  14:37 

So let's say I've got a vineyard and I'm seeing some symptoms. I'm seeing some red leaves or I'm seeing some bronzing or I'm seeing something, and I've looked at the nutritional situation, I've ruled out either toxicity or deficiency. So I'm not thinking hey, you know, maybe this is a virus issue. Can you take samples of vines and just bring them to a lab and say, please help me? Can you tell me what this might be? I know we just talked about the deep sequencing. Is that, I'm not gonna say that technology. But like if I brought you some material and I said this has got a problem, how would you go about diagnosing it?

Alan Wei  15:14 

Oh, definitely. That's what we do every day. Most of our work is focused on helping growers find out what is possible cause of a programmatic vine in their vineyard. They will send in the samples, either individual vines or composite samples. We have a panel, what's called a combo panel that covers the 11 viruses, 11 major viruses. Leaf Roll Roll 1, 2, 3, 4, and two or three viruses, and of course, Red Blotch, and Fan Leaf, Pierce's. And then also Pinot Gris virus. That is the most frequently requested a panel. And by doing that panel, we typically find out if it's a virus issue.

Craig Macmillan  15:58 

That's very useful. It's very, very good to know. In relation to grapevine viruses, or just diseases overall, what is the one thing you would recommend to the listeners that they should keep in mind?

Alan Wei  16:08 

I think that you already touched on this earlier. You know, one thing is, if they see problems in the vineyard, they should consider the sample and test to validate whether they're viruses or not. And if they're considering to plant new materials, they should be very vigilant to to ask questions of the nurseries, and also do their own independent homework. And the you will be interviewing Dr. James Samp in another session. He can tell you more about how he go about sourcing for cleaning materials for his clients, which are very quality conscious.

Craig Macmillan  16:47 

Yeah, we're really looking forward to that conversation, Much like I was looking forward to this one. You know, this reminds me of something. You hear the word tighter a lot. And I don't think I fully understand what it means. I know that it's important and seems to be coming up a lot. Can you explain the concept and why it's important and what it means for us practically?

Alan Wei  17:09 

Yeah, I'm so glad you brought this topic up. You know, you and I have been serving on AVF committee, Grant Review Committee for a number of years. And last week, we had our review meeting for this year, and the subject came up. You know, we can talk about different aspects of Red Blotch impact in wine quality, wine physiology, you know, readily and so on and so forth. If we want to contribute one single factor of all of this different symptomology, it would be the virus tighter level. The virus tighter means the number of particles in the vine. If the vine is only infected with a smaller number of particles, its response to the virus is going to be different than the vines that are infected with larger or large number of particles. In our experience, the virus level in different vines can be very much different. I'm not talking about different by you know, 50 percent, or two or three fold. I'm talking about several orders of magnitude. There is a poster right behind me, which you cannot see. But we did a measurement of three infected vines. One, with clean, non effective. Another one is chosen for medium Red Leaf symptom. Another one, it's very heavily Red Leaf symptom. And virus level, the obviously the non infected vine was zero. And then the mediumly infected vine was about one or 200. And then heavily infected vine, was one hundred thousand in relative copy numbers. So this tells you that you know, this virus kinda level concentration level in the vine, really affect symptomology as well as the vine performance and the barrel quality and obviously, eventually, wine  quality. If we read the scientific literature, lots of studies report Red Blotch positive, Red Blotch negative, they did not talk about the virus tighter level. That's why we were so glad to see last week one of the research proposes to study the virus tighter level on different aspects of vine physiology and berry quality. I just think it was so so so important because the virus tighter will make it make a huge difference.

Craig Macmillan  19:39 

So we may be moving from a world of infected or not, to not, to more sick, less sick.

Alan Wei  19:47 

Yes, absolutely right. That is actually how we protect ourselves against the human viruses as well. You know, our vaccine does not completely protect us from infection. But it does protect us from viruses being propagated in high numbers in our body. Therefore, our symptoms of the infection in the individual is much less. And the ability for that individual to infect others are much less. Simply because of the lower virus tighter level in an infected person and similarly is true in grapevines.

Craig Macmillan  20:24 

So plants and animals are obviously very different organisms and where an animal has an immune system plants do not, they do not have an immune defense system. Is that correct?

Alan Wei  20:35 

Yes, you're right, correct. But they do have basic defense system against foreign organisms. One of them is the RNAi system. So speaking of that, you know, the simple symptomology in response to Red Blotch, and most of it is a total response, as a result of virus infection. The RNAi defense system gets activated. For example, the accumulation of the sugar of the raisin should be gradually, in a normal process, will be gradually moving towards the berries. But in Red Blotch, in fact, in vines, they are accumulated in leaves. Not moving toward the sugars. And the same for anthocyanins. That's why we see this red leaf. And those red color should be you know, in the berries, but they're not. They get stuck together, accumulating in leaves. It's fascinating. Unfortunately, we are still at the beginning of understanding all of this. Some reports are gradually coming out.

Craig Macmillan  21:40 

And so I want to make sure that I understand kind of how this works. So there's a grapevine that becomes infected. However way. The virus is very, very tiny bits of genetic material. Unlike, unlike a bacteria, which has a cell wall. Viruses don't have that they're just genetic material. The plant recognizes that somehow. And then RNA is the material that is produced from genes, the genes or have a have a sequence and then when that is reproduced that goes out into the world as RNA. Is that right?

Alan Wei  22:17 

Yes, the RNA is inside the host. And in response to a virus infection. And the defense mechanism get activated, which involves what is called enzymes. These RNA into smaller pieces, typically 20 nucleotide long. And they are, they are the what's called the interference RNA, or RNAi which inhibit the host from propagation inside the plant.

Craig Macmillan  22:48 

This is just, we need to wrap up for time, but I just have been thinking about this for years. And that is, where do these viruses come from? Where, how do they, how do they show up? What are these plant viruses? What? Are they jumping from other plants as a mutation of one into another? we know? Do I have any idea where these things come from? Because it seems like it's not just a question of finding it. Seems it's got to come from someplace.

Alan Wei  23:14 

Yeah, that's that's a really good question. I you know, you have biology, you have a load viruses, and obviously, RNA. Some viruses are readier to evolve, to change, to mutate. And that's why we see so many different mutants in the COVID virus family. And this is Red Blotch, is a DNA base virus, which have shown less mutation. And so far, we only seen two mutants, two clay types. And they practically they don't have much difference. As far as the, you know, the origin and the evolution. We need to have folks like Mark Fuchs to answer that.

Craig Macmillan  23:55 

At Cornell. Well, that's fantastic. That's that's our future. That's where, that's where we're going. Well, I think we've covered everything. Where can people find out more about you?

Alan Wei  24:03 

We have a website, And then they could call us or email us anytime. We're here to help growers to build a better and clean vineyard so that they can make the best wine possible for the for their clients. Yes, sorry, Craig for the background noise. I think folks who are preparing samples as we speak.

Craig Macmillan  24:26 

I want to thank you Alan, our guest today has been Alan Wei, Owner and Lab Manager at Agri-analysis, David California. Thanks so much. This is really fascinating conversation.

Alan Wei  24:35 

Thank you very much Craig for hosting me. Continue to the great job. I'm so glad you're back at the Vineyard Team. You guys. You guys are wonderful team and doing great job. I'm very pleased to be here.

Craig Macmillan  24:44 

Thank you. I appreciate that.


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