243: Microbial Communities in the Grapevine

Sustainable Winegrowing with Vineyard Team

Microbial communities vary widely from plant to plant, even from rootstock to rootstock! Philippe Rolshausen, Professor of Cooperative Extension for Subtropical Horticulture in the Department of Botany and Plant Sciences at the University of California Riverside studies the phytobiome. This includes all organisms associated with the vine including bacteria, fungi, insects, and animals. Learn the impact these communities have on your vineyard from young vine decline under extreme stress to the wine’s terrior.

Resources:

• 78: Biological Control for Grape Vine Trunk Diseases
• A Method to Detect and Quantify Eutypa lata and Diplodia seriata-Complex DNA in Grapevine Pruning Wounds
• Endophytic microbial assemblage in grapevine
• Grapevine pruning strategy affects trunk disease symptoms, wood pathobiome and mycobiome
• Philippe Rolshausen
• Phylogenomics of Plant-Associated Botryosphaeriaceae Species
• Rolshausen Lab
• Soil Health Playlist
• Temporal Dynamics of the Sap Microbiome of Grapevine Under High Pierce’s Disease Pressure

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Transcript

[00:02:07] Craig Macmillan: Our guest today is Philippe Roshausen. He is Professor of Cooperative Extension at University of California, Riverside. And today we're going to be talking about the soil microbiome. Thanks for being on the podcast, Philippe.

[00:02:19] Philippe Rolshausen: Thank you for having me.

[00:02:20] Craig Macmillan: Well, when we do these topics, like to start from the very basics. And then we can go very deep from there. What is your definition of the soil microbiome?

[00:02:31] Philippe Rolshausen: Well, a microbiome is the collection of microbes. The soil microbiome is the collection of microbes associated with the soil. I have to say we work on the soil microbiome, but we also work on the phytobiome, which is the collection of organisms. associated with the, the, vine, and that can be fungi, bacteria, viruses, even insects. So we are more interested in fungi and bacteria in my lab. And especially those that live inside the vine and how it's connected to the roots and the soil.

[00:03:12] Craig Macmillan: Yeah, I'm very interested in that. Just for our listeners, other aspects of the soil microbiome could include things like microarthropods,

uh, nematodes, etc. It's a very complex Complicated

ecosystem.

[00:03:26] Philippe Rolshausen: I and I only focus on one part of it.

[00:03:29] Craig Macmillan: The work that I've done with soil microbiome in a very primitive way. We just focused on total bacteria, total biomass, things like that.

You just mentioned this and this is a good jumping off point actually. Because plants are an active part of this ecosystem, you will find different soil microbiomes around different crops. What is the soil microbial community like around grapevines in particular?

[00:03:50] Philippe Rolshausen: Well compared to, to what is the question. Let's give you an example. I work on citrus and grapes in the same soil environment, you would see differences between a grapevine and the citrus plant just because of the property of the host. Within grapevine, you know. Depending on the variety or the rootstocks, I should say then you would also have different microbial communities associated with the rootstock.

And this is like a plant effect because plant recruits their microbes. So, so you have sort of a template of microbial or a core microbiome, which is you would find across a different rootstock or varieties of grapes or even plants. But then. Some are very variety specific or host specific, and so you would tend to see some varietal specificity and specific microbe associated with some of those rootstock operants.

[00:04:47] Craig Macmillan: That's news to me. That's fascinating.

there is some commonality, but you will find some differences just based on things like rootstock?

[00:04:56] Philippe Rolshausen: Yes.

[00:04:57] Craig Macmillan: Oh, that is interesting. We may come back to that. one of the things about your work that I was really intrigued by, which I was unaware of, is the different microorganisms that one might find inside the grapevine, and what roles they play in the functioning of the plant. I mean, I think most of familiar with Our gut microflora. Right, where we know that we have organisms inside us. We have organisms on our skin and all that. I didn't realize that was true for plants as well. Tell me more about that.

[00:05:26] Philippe Rolshausen: You can make the parallel between the roots and the gut, actually.

Because they have similar functions. Especially with respect to nutrient assimilation Defense against disease, for instance. Now, the gut is inside, the root is outside. Someone made the comment that the plant where there are guts on the outside, just because of that parallel that between the rhizosphere and the gut.

there is a connection between what's going on inside the plant and what's going on outside the plant. We are interested to understand what the organisms that live inside the plant. Where they're coming from. in terms of diversity the endosphere, which is the the region of the, the organism living inside the plant, the endosphere is not very diverse.

There's only a few of thousand fold less microbe capable of living inside the plant just because it's a more. Stringent environment I work on the xylem a lot and, you know, there's a negative pressure for instance, there are, there's not a lot of food available, for instance.

And so microbes have to adapt to those environments, right? And it's not every microbe is capable of doing that. there's only A certain type of microbe that can live inside them. we're interested in those because we work specifically on vascular disease of grapevines, so fungi, bacteria especially.

And we try to understand how those pathogens interact with the plant, but also really understand those pathogen within the, context of the microbial community and how those pathogen interact with the microbes living inside the plants. to go back to your question, it's like, where are those pathogen coming from?

How did they get there? How do they interact with the plant and how do they interact with the microbes? within those interaction, what would be the, the disease outcome in certain type of interaction.

[00:07:35] Craig Macmillan: So what we're talking about is we have these pathogenic organisms Which could be in the form of like grapevine trunk disease Things like that or Peirce's disease. In the case of bacteria and what you're getting at is it that there's other types and populations of microbes that may be antagonistic to the pathogen?

[00:07:56] Philippe Rolshausen: Yes. So that was our hypothesis. Traditionally, you know, microbiologists were looking for biological control agent against diseases and they were culturing those.

That was the traditional approach. So you take a plant tissue, You put it in the culture media and you see what's growing and then you test if those organism are able to inhibit the growth of your pathogen. That would be the traditional way of doing it. Now, with the metagenomics , the development of those technology, we're able to kind of look, at the entire community of those organisms living inside the plant and using those tools, we could actually select those that have some.

That might be antagonistic to certain pathogens. the beauty of this is that you can actually look at the entire community. When you look at a traditional microbial approach, you kind of zoom in on, to those that can grow in culture and those that can grow fast in culture and sort of overpower the other ones.

when you use those molecular approaches, you look at the entire community and you can And sort of a broader view of really what's going on and who does what. and so that's the approach that we've been doing to try to identify biological control agents or beneficial organism to plants and to grapes in particular.

what we do is we select plants with different phenotypes. So we will select. Plants that, you know, that range from very healthy to poor health. And we're able to build those sort of correlations with organisms that associate specifically with healthy plants and those that associate with sick plants.

and from that, once we have identified the organism, we can go back and try to culture it and to reintroduce him in the system to prove that Actually, they are beneficial in some capacity.

[00:09:52] Craig Macmillan: does this mean that I could have plant material, and one of those vines would be healthy and one would be weak and showing disease, but they both would have the pathogen, it's just that the other one has the microbial community to help fight it.

[00:10:07] Philippe Rolshausen: That's right.

[00:10:08] Craig Macmillan: Wow.

[00:10:09] Philippe Rolshausen: there's a environmental factor that comes into play as well. because abiotic stresses also so like, you know, drought or heat play a factor into the plant, weaken the plant in some capacity and will change the disease outcome and the interaction between the pathogen and the microbe.

There are more layers than, you know, the simple pathogen, microbe. plant interaction There's also the environment in which those components are part of that really plays a big factor.

[00:10:43] Craig Macmillan: So, are you finding or pursuing how those abiotic factors can be manipulated?

[00:10:50] Philippe Rolshausen: A little bit. I'm really interested in those aspects of, especially in the eras of climate change.

plants are going to be subjected to a lot more stress than they used to be. I think we need to understand how this is going to affect the, the microbiome at large and because I'm a pathologist, how this is going to affect disease outcome. we are starting to only scratch the surface of that.

I think a lot of people are as well. It's a lot more complicated when you work with. Woody perennial than when you work with an annual plant, because you have the approaches and the logistics are more challenging, I would say. drought is the number one just because it's been on the radar of every grower in California, but so we, you know, we are interested to see how those factors really affect the Have an effect on disease.

Yes.

[00:11:44] Craig Macmillan: Are you doing that in the laboratory?

[00:11:46] Philippe Rolshausen: Well, yes. You have to sort of work in more controlled conditions and sort of dissect it one stress at a time. so it is in more greenhouse conditions or gross chamber conditions where you can, semi controlled environment where you can really control heat or, you know, water or, and see how that affects the microbiome.

[00:12:07] Craig Macmillan: I think this is fascinating. can you tell me more about what that actually looks like, like how do you set it up? How are you controlling it? What kind of ranges are you using? I love the details.

[00:12:17] Philippe Rolshausen: So we've done experiments mostly with irrigation so far. And we are only starting to tie irrigation to microbiome, but we have done the.

How irrigation affects severity of disease and impact on the plant. this is pretty straightforward, you have plants that are fully irrigated that are not on the deficit irrigation, and then you calculate what a deficit irrigation like a 25 percent deficit irrigation look like, a 50 percent deficit irrigation look like, measuring, you know, how much water.

How much irrigation you put in on your plants, you inoculate your tree or vine with a pathogen, and you let it incubate for, you know, weeks, because you know, those are slow pathosystems, they take time, and you see how that affects for trunk disease in particular lesion of the wood necrotic lesion, for instance.

And then we look at factors on the plant. We measure biomass, for instance, of the roots, the shoots. We look at gas exchange, photosynthesis, and so on and so forth. going down to the microbiome, you know, we would be sampling the tissue, extracting the DNA, and then sequencing, you know, all the microbial community, bacterial and fungal, to see how those communities have changed over time in a well watered versus a deficit irrigated plants.

[00:13:49] Craig Macmillan: And is that quantitative analysis, or is it simply the complexity of the diversity of the microbiome

[00:13:56] Philippe Rolshausen: it's semi quantitative so let's say, you know, like you're looking at a presence of a specific taxa.

It's a relative abundance of the taxa in comparison to the others. So when you see an increase of the taxa. in your community, it's either because it does increase or because the other taxa within that community decreased. So it increases in, in relation to that. So it's not really an absolute measure of abundance.

It is a relative measure of abundance, but you could still use it and make some correlation with that.

[00:14:34] Craig Macmillan: Can you explain, metagenomics?

[00:14:37] Philippe Rolshausen: That's not my field. To be honest with you, but the principle is that you're using primers that are universal primers and that allow you to, so there are.

Meaning that you can sequence conserved region. So those primers are aligned with conserved region across several taxonomic groups. Okay, so fungi or bacteria. And then you sequence a length of nucleotide. In our case, it's about 200, 250 nucleotides in length. In between, and this is valuable between those two.

Primers, that region is valuable. And so you could start making you can do a micro real fingerprinting and identify which types are, are present, you know, but it's the community of organism. So it's like all the fungal communities or all the bacterial communities. So some primers are better than others for special taxonomy group.

So for instance. I work with Dario Cantu at UC Davis, CBT Controlled Neurology. Davis uses field of knowledge, and so he developed primers for group causing grapevine trunk diseases. And so it's mostly what's called the Ascomycota phyla. his primers are mostly focusing on this group of, fungi, but it doesn't give any information on The basidiomycota, which is another group, or the glomerulomycota, which is the mycorrhizae fungi.

if you wanted to get information about this group, you would need to get a different set of primers, right? so it's really up to primer that you're using that gets you the right information. and also the database that you're using that gives you the right information because sometimes the database are not curated or they're not accurate, and those are getting better as we're getting more and more knowledge about the taxonomy and, and the biology of those organisms.

we're able to make more accurate prediction because we're gaining more knowledge about those organisms. So for instance, there's a lot more information about bacteria than, They are about fungi just because there are not a lot of reference genome for fungi compared to bacteria. so it's a lot more difficult to predict. Sometimes for fungi than it is for bacteria.

[00:17:01] Craig Macmillan: But it sounds like that would be coming down the road.

[00:17:04] Philippe Rolshausen: Oh yeah, yeah, it's a matter of time. I mean, really, you know, this technology is going so fast, so quickly that, in five years from now, I can't even predict what it's going to look like,

[00:17:15] Craig Macmillan: This thought just occurred. your work is obviously, I don't want to say infancy, but it's, it's pretty early work. You know, we're, we're working with tools that are still in development, basically. at this point, would you say that there are certain conditions or practices or manipulations that a grower could use to promote the most beneficial endo, um, microbiome in their plants?

[00:17:40] Philippe Rolshausen: In principle, yes, and I believe that this is true. We just don't have the knowledge yet to make recommendations that are solid. There we go. So, when you apply something to your soil, you know, if you're a grower and you apply fertilizers, or this is going to, or even if you if you till your soil or it will have some impact on the soil microbiology.

That we know. How that reflects to what's going on inside the plant, we are, we are not there yet. We, we don't have that knowledge yet just because like I've said before, only a few of those organisms move inside the plant. There's always a a gap between you know, what's going on outside the plant and what's going on inside the plant.

The inside the plant it's a buffered environment that changes very slowly compared to the root of, a grapevine. what we've done is we've looked at the microbiome of vines that we planted in the field and look at the rhizosphere microbiome, or the, the The macro bill community associated with the root and the macro bill, community associated with the trunk, the graft union, and so forth.

And we see right after planting that the microbial communities associated with the root changes really quickly, whereas the the microbiome in the trunk are the graph union. changes very slowly. whatever growers do and cultural practices that they implement in the vineyard will have a limited effect inside the vine or it will take years for this to see the effect.

the rhizosphere microbiome drive a lot of, of biological function, you know, they, they, they fix nitrogen metabolize phosphorus detoxify compounds and so on and so forth. So there's a lot of, benefits of, using some cultural practices, beneficial practices and how they affect microbial communities associated with the roots.

[00:19:48] Craig Macmillan: We know that for like the pathogenic organisms, like the fungal ones, you know, they come in through wounds, whether that's a grafting or whether that's a pruning, we know that bacteria are introduced through things like piercing sucking insects. Like Leaf Hoppers and Peirce's disease. what is the, pathway for the bacteria and fungi that you find inside of a vine that are not the pathogens?

[00:20:10] Philippe Rolshausen: Well, you said it. the pathway is like from inside the vine, it's first of all inherited from the nursery. That's, that's the main pathway.

And then. Some of those organisms come from the soil, there's a fraction that comes from the soil and move throughout the plant systemically through the sap, the plant sap. So that's another pathway one of the major pathway. But again, that takes time. Right. And then, you have fewer introduction that comes through, you know, the plant natural openings, stomatas on the leaves, for instance.

Right. Or pruning wounds. Some organism when vines are pruned some organism common pruning wounds and are able to colonize down into the vascular system. That's another path. Or introduction through insects. And, you know, OSA is, you know, one of the best example of that being introduced with shop shooters. during feeding So, but I would say those, are minor introduction compared to what's already there when the vine is being planted and secondary to movement you know, sap movement throughout the plant, from the root to the upper part of the plant.

[00:21:29] Craig Macmillan: One thing that I saw mentioned is that these variations in The microbiome inside the plant may also have kind of a role in our concept of terroir in terms of how different areas have different characteristics. Is there, something to that idea?

[00:21:46] Philippe Rolshausen: the characteristic of wine region or the terroir is not only linked to the soil, the variety or the weather condition.

. It's also linked to the microbial communities associated with those vines. There has been a study that's very famous that was done 10 years ago at UC Davis that showed that. But it's mostly true for organisms that live on the plant surfaces the surface of the berries or the surface of the leaves.

It is not so true for organisms that live Inside the vasculature of the vine, just because of what we've talked about, just because it takes time for those communities to change over time. really those the microbial composition of the trunk is, from what we understand today, mostly inherited from what's coming from the nurseries.

you know, nurseries have a huge impact in shaping. the microbial communities of the trunk and the vines and you know, can you talk about terroir when it comes to the endosphere of the vine? I don't think so. I think it's, it's a debatable question. I think it's mostly true on epiphytic organism, organism that live on the vine surface just because they are more subject to the environment. Whereas it's not so true for trunk organisms,

[00:23:11] Craig Macmillan: , Even though we've been talking about the endophytic side, what's some of the things that the other parts do you were just talking about, we have a different community in the environment that's on the surface of the plant and that, that has a role in, you know, differences between regions in terroir. What impact are those kinds of organisms having on the physiology of the vine that contributes to those differences?

[00:23:35] Philippe Rolshausen: Well, that I don't know because, you know, that's not the field I've studied. But they, could influence the fermentation process, but I'm more interested in the one that live inside.

Do they have an impact on the wine quality itself, perhaps, you know, that's a question we're interested in. they have an impact on disease. We know that much because they, they affect disease outcome and we've shown that.

The question also, I think, from the standpoint of A grape grower or a winemaker is like, well, do they also affect winemaking?

we found some bacterium in the sap of grape vines that are present during the fermentation process. So lactobacillus, for instance we found those in the sap of grapes and some bacteria that also that spoil the wine fermentation process will also. Found them in the sap. So the question is like, are those, are those coming from the soil?

You know, where are they coming from? We find them in the sap and then can they go inside? Can they be moved to the berry of the grapes and then participate in the fermentation process later on? We don't know the answer to that question. The only thing we know so far is that we can detect them inside the plant stem.

And so. What is the impact? That's the question mark, but those are really relevant questions questions we're interested in because, if you can connect the root system to the cluster, and then if you can influence the root system or the microbial community of the root system, then perhaps you can influence the communities living in the cluster and then, you know, Later on, influence your fermentation process.

So that's the idea behind it. It will take time to decipher those questions.

[00:25:27] Craig Macmillan: Coming back to the pathology side. we have testing programs for virus in the nursery system. And we have the ability to test for fungal diseases, of course.

Do you see a role for testing for bacteria and fungi other than the pathogens at the nursery stage?

[00:25:48] Philippe Rolshausen: I don't think it makes sense. So we've been working with nurseries for several years now. And we are looking for where diseases are coming from prior to vineyard establishment. there's no certified program for fungal diseases and some bacterial disease like Crown gall.

there is a fraction of those pathogen that is coming from the nurseries. That's a fact and this is true for California, but this is true for everywhere else in the world. So if you get any. any vines from a nursery, you are going to find fungi causing grapevine trunk disease. And the reason is that there are over a hundred taxa of fungi causing grapevine trunk disease you will likely find one of them and not only that, but they are able to live on a different environment. And so they can live in soil, they can live in water, they can live in plant debris for some of them. And so you cannot get rid of them. I think that growers have to have the mindset that you are not going to be able to have plants free of pathogens causing fungal, grapevine, and trunk disease.

That's just not possible. Viruses, you can get rid of them and there is a certified program that works for that. This program would not work for fungi. It's just not realistic. Right. It would be too costly to do it. Right. Right. Right. Right. The question is like, can you live with it, right? Do pathogen causing grapevine trunk disease will cause the vine to die?

If that were the case, you would have no grape production in California because virtually all the vines are infected with one fungus causing grapevine trunk disease. that's not the case. It happens, it's called young vine decline, you know, when sometimes growers plant their vine. And the vine dies within five years.

But this is because what we understand now is that it's related to the stress factor that I was talking about earlier. there is something going on with the environmental stress that caused the vine to decline after five years. what we've done is to monitor the microbiome and the vine, like I've told before, following planting.

And we do capture those pathogen from the nursery to the vineyard. And they are evolving in the vineyard just and we can detect them. But under no stress conditions the vines are doing just fine. So every year a vine will lay out new wood, right? It will grow out some wood. The trunk gets bigger and bigger.

the fight between the trunk disease pathogen and the vine is if the vine can grow wood faster than it loses some to trunk disease, then it's fine, you know, it will be able to survive. Plants and grapes are able to compartmentalize the infection. They lay out walls to compartmentalize infection, and if they do that successfully, you know, Then they will survive now when you have a stress factor that comes into play and here you have to Go back and define well define stress this is where we go back and go back to your question about what stress are we talking about?

Heat a drought I think overcropping can be one of them also, when growers take tend to push the vine early on after the establishment to get into production quickly after two years. I view this as a stress factor, so this will influence the microbiome of the vine, living of the microbiome living inside the vine, and as a result, it will influence disease outcome.

[00:29:34] Craig Macmillan: I will not look at grapevines the same way. , is there one thing, one takeaway from this conversation that you'd like growers to hear

[00:29:44] Philippe Rolshausen: what we're trying to do is to identify cultural practices that are beneficial to production.

I think you, you have to look at it from a probiotic or prebiotic. Standpoint, just like we do for humans, you know. when you look at the prebiotics, those are similar to the cultural practices that you implement in vineyards to support the presence of beneficial organisms. that can be, you know, adding compost to your soil or cover cropping because they provide some benefits to the microbes living in the soil, and as a result, the microbe associated with the vine.

that's one of them. The probiotic is the addition of single microbes to the system identify which one of those probiotics using the metagenomics approach. And we've made some some stride in, in, in that research, we've identified several organisms that are beneficial to the plant and that are antagonistic to some of the disease.

So for instance Pierce's disease we've identified several organism living inside the vines that are antagonistic to PD. And today we are testing those organism in field trials at UC Davis. Because we've demonstrated that they, they work on the greenhouse condition. And now we moved on to field trials.

And when we inoculate those beneficial organisms to, or those probiotics to vine, they are able to stimulate the vine health in some capacity, or are being antagonistic to the pathogen in some capacity. And the vines are able to sustain the disease. So, the takeaway message from that is that we are making progress, you know, understanding what those probiotics and prebiotics are for viticulture.

[00:31:39] Craig Macmillan: That is great. and I'm really happy, That you're doing this work. It takes time Oh yeah, It takes time. Absolutely, that's, and that's, part of what we do here is we, bring people kind of what the future is looking like and what's possible because if you understand it, then when it does come along, The learning curve is already hopefully down the road a little ways. doing your homework, basically. thank you in your lab. This is really exciting. interesting stuff. Where can people find out more about you?

[00:32:09] Philippe Rolshausen: Well, they can go to the University of California, Riverside Botanical and Plant Sciences website. I'm a faculty member, so they will find me there.

I have also my personal website. Rolshausen. Slash lab. com. The problem is like the spelling of my name, right? It's not easy to do.

[00:32:27] Craig Macmillan: We'll have a link to that show page plus A a number of your recent publications.

[00:32:33] Philippe Rolshausen: Yeah, we have several publications about what we've talked about today.

We have just had one released about how pruning practices affect also the microbiome and, and disease. So. You know, this is an active area of research. I'm not the only one doing this. Like I've said, I've collaborated with Dario Cantu at UC Davis. And I think he should be mentioned because he's doing some excellent work.

And we are a great team working together. As a cooperative extension specialist, I collaborate with others. I collaborate with other faculty. And this is a collaborative work that I'm talking about. I'm not the only one,

[00:33:11] Craig Macmillan: there's a network. want to thank our guest, Philippe rolshausen. He is professor of cooperative extension with the university of California riverside and doing some very exciting work. And thanks for being on the podcast, Philippe.

[00:33:24] Philippe Rolshausen: Well, that was great. Thank you for having me.

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