Powdery Mildew Resistance and Susceptibility with David Joly, PhD

Good afternoon, David. Thanks for joining us. Thank you. All right, so you and your team recently published a pretty important paper on powdery mildew susceptibility, which we are going to get into. But first, I wanted to lay some groundwork. Our audience here on the podcast, includes people from all facets of the cannabis industry, and not

everyone’s going to be intimately familiar with PM. So let’s start by outlining the powdery mildew problem. So I was wondering if you could explain why is powdery mildew a threat to cannabis cultivation and really what is at stake here?

Yeah, well, first of all, I would say that powdery mildew is a threat because it’s very common. First of all, so it’s not something rare that people are likely not to expect. So it certainly will happen at some point.

So it’s very common and it can spread very quickly. So when once a crop is infected, so usually the recovery options are often limited. So we don’t want growers, of course, to use those nasty fungicide that we see some time on other crops.

So we still have access to a few environmentally friendly options, but those are not always very efficient or they are not easy to apply on larger scale. So now about its impact on the plant itself, of course, it will rarely kill it because it’s what we call an obligate biotroph.

So it needs living tissues to propagate. But it can severely reduce photosynthesis when it gets out of control, especially. And this it will in the end affect yield. So something that no grower want. Another impact I can think of is, of course, it will affect the smell of the plant.

So when you have a plant infected by powdery mildew, people will notice it has a different smell. And so we know that consumers are more and more interested in the flavors and the scent, the aromas of their cannabis.

So certainly it’s a problem that people will want to avoid. Sure. And now it’s called powdery mildew, because it has sort of a powdery white look on the leaves when a plant is infected, and I’m familiar with the fact that it will show up on the leaves.

Does it also infect the flower itself? Will it sort of affect the bud that eventually would get to the consumer? Yeah, well, we often say that that powdery mildew can affect all aerial parts of the plants, which will include stems.

As long as they’re non-woody, it can infect the leaves and it can infect some part of the flowers, not all but like when we refer to cannabis flowers. So before we have the cracks and those can be infected by powdery mildew.

But, we need to understand that powdery mildew. So, you know, you will hear a lot of things about powdery mildew. People will say it’s systemic and stuff like that. So, if I can describe rapidly how powdery mildew works.

So usually like powdery mildew first, it’s a fungus, right? So it will propagate mainly through its asexuals spores that we call canadia. So those spores will land on a leaf and then they will germinate and produce those kind of tiny projections into the cells from the epidermis and only the epidermis.

So powdery mildew will only affect one layer of cells, which is the first layer of cells. It won’t get further down than that compared with other fungi. So it is certainly not systemic. It is very restricted to the epidermis.

Now, we have epidermis on leaves, but we also have epidermis on the stem. So that’s why you can see powdery mildew elsewhere. So those tiny projections I was talking about. So, that gets into the plant cells.

So they are needed by the fungus to absorb its nutrients from the plant. But it will also be a way for the fungus to suppress the defense response from the plant. But then why do people think it’s systemic?

Well, there’s many reasons for that. So in other plants, that survives winter, for example. So people talk it was systemic because very rapidly in the spring, you could see powdery mildew appear. And so the reason for that is that it can stay dormant within buds.

And I’m not talking about cannabis buds here, but like any axillary buds that you can find on a plant, and it can also stay dormant on plant debris. And mostly, I think the big reason is that it can be present, but at a very low level.

So people will think that the plant is healthy. They will then propagate it or share it with friends, and then suddenly the conditions become favorable and it will be a big boom for the fungus. But it was there.

It can stay, you know, dormant at very low levels. Yeah, so important thing you mentioned there, so environmental conditions can sort of bring on an infection. Yeah, for sure. And I mean, so that’s why usually so, you know, people will have their mother plants.

And so the mother plants are in a setting where usually the humidity is not as high as the cuttings will be once the ones they start propagating that plant. So you have a plan that was maybe not in the ideal conditions for the fungus.

So you don’t see it, but it’s still there. It’s still somewhere. And then when you do the cuttings, you put them, you know, into a higher humidity level and then the fungus likes it. It will start to propagate. And then when you.

When you get your plants outside, outside of the rooting period there, it will add time to get established and then people will often also see it getting more important during flowering. So is it because it had time to mature or because the canopy is maybe more dense or also because I

don’t know if the light can affect I’m not sure it has been studied, but maybe like because we changed the light regime during flowering, maybe it also helps the powdery mildew to propagate more easily. Okay, so I know you briefly touched on it, but I mean, a lot of your paper is is about trying to identify these

genes that may be responsible for susceptibility to powdery mildew. And then there’s also the opportunity of finding powdery mildew resistant genes, which I’m sure we’ll get into. But in the absence of sort of breeding for resistance, What steps do cultivators currently have to either prevent pm infection or once it’s happened, sort of remediated?

Yeah, so I mean, right now, as I said at the very beginning, so there are a few options that exist to kind of reduce the incidence of the incidence of the disease. So and about those products, so I know there was there was a study conducted by the group of the group of Zamir Punj at Simon

Fraser University, who you also had on your podcast a year ago. So, they tested a bunch of different products. So some are based on the use of bacteria that will attack the fungus. So we can talk about the products like actinovate or rhapsody, but they also look at products that are based on plant extracts, such as

neem oil and also some other products that are well known in the organic farming, such as potassium bicarbonate, or that we know as milstaff is the name of the product that we often see for cannabis use or hydrogen peroxide also, which is another product.

So there are different products. And people will talk also about silicon, because in other crops, it has been shown to be very useful to reduce powdery mildew, UVC lights that are getting also popularity. So those are different methods that can be used to reduce the incidence.

But I guess before we have to use those products, I think the key is to try to keep your plants as clean as possible. So as soon as you detect infection, remove the infected plant parts. Try to not have dense canopies, as I said earlier, try to keep the humidity maybe a little lower.

Of course, it is a fungus that is airborne. So those spores are very easily transported by any air movement. So, you know, outside it’s easy to understand that I’m talking about wind, but inside, you know, just people moving around or so you can get spores on your on your clothes and then you get into another another

room and then those spores can infect the plants. And so, yeah, so trying to reduce or if you have a room that is completely infected, of course, don’t go in that room before going into your cleaning room. So, you know, just simple steps like that, trying to reduce the inoculum levels.

Excellent. Excellent. And so that brings us to the prospect of actually breeding for resistance to powdery mildew, which is what your paper is about. So first of all, powdery mildew doesn’t just affect cannabis and affects a whole range of other crops, so I was wondering if you could maybe explain how breeders have successfully bred for

resistance in other crops and how we might use that information to achieve it in cannabis. Yeah. So as you said, exactly. So powdery mildew is a big family of fungal pathogens. So we have different species. Some will be very host specific and others have a broader host range.

And it is the case for the one we have. And before we talk about resistance, this is something we can just talk for a few seconds. So it’s a problem in itself. So when you have something that’s very host specific, as long as you don’t have any of those plants nearby, so, you know, you’re less likely to

get that disease. So let’s say you have a cannabis field. But, you know, there is no cannabis field around. If the fungus was specific to cannabis, then it’s less likely it will get into your field. But the problem is that the species we have is not specific to only to cannabis.

So it can also infect a lot of different plants from the aster family. So we have a lot of flowers, like when we think about dahlias and sunflowers so they can get infected by that same species. And also some weeds like ragweed can also be infected.

So that’s something also to consider when we’re growing outside. But let’s get into the resistance, at least what we learned from other crops. So, I think some of the first things that were discovered, so it was on barley more than 50 years ago, was so they discovered some accessions from africa that were completely resistant

to powdery mildew. And then that was the first point. And so they started to investigate, trying to understand what was going on. And they realized at some point that it is a form of resistance, but we prefer to call it like a loss of susceptibility.

So, resistance and susceptibility, you know, we kind of, we often think that it is the same thing. So one is, you know, the plant is either resistant or susceptible, but in terms of breeding, So when you breed a resistant plant, we can see it as we’re bringing something new to the plant.

We bring new genes that will confer resistance. But there is another way to achieve resistance is to reduce susceptibility. So, you know, you have maybe something in the plant that confers susceptibility and you want to remove that from the plant.

So, of course, people will think, well, susceptibility, if there was something in the plan that would cause susceptibility, it would have been eliminated by years of natural selection. But the problem is that maybe those genes are very useful for the plan.

So the plan cannot get rid of them. But, there is a caveat of having those genes is that you know, because susceptibility to some to some fungal pathogens and this is exactly what some genes are and people are getting interest into those genes.

But MLO is one of those examples. So it’s not a resistant gene. It’s a susceptibility gene. So we need to find cannabis strains that have either an inactive version of that gene or mutated version of that gene that will make it more difficult for the pathogen to infect.

And so one interesting thing also when we’re talking about resistance, as you said, so I published a paper recently about the MLO family, but almost at the same time. So there’s another group that published a paper about PM1, which is a resistant gene.

So we can talk a little bit more about those two sides of the coin. So resistant gene, the way that I can describe it from a genetic point of view is what we call a gene for gene interaction.

So you have one resistant gene in the plant that is able to recognize one gene from the pathogen. So it’s a little bit like a, you know, like our own immune system. So it’s not working the same way, but it’s easier to understand if we compare it to that.

So we have something that is more of a recognition system so that gene can recognize the pathogen and triggers defense responses that will kill the pathogen. And the problem with that types of resistance is that it’s usually isolate specific.

So it will recognize only a few isolate or, you know, one population of the pathogen, or it can for a while. Looks like if it’s recognized the whole the whole species, but after a while, the pathogen will rapidly evolve to overcome that resistance.

So leading us to what we call a core evolutionary arms race. And so we constantly need to find new genes of that kind. So new resistant genes, and we can try to breed a lot of them together. So that would mean maybe it will be more durable.

But usually if you have only one, it will likely not be durable. And now when we talk about susceptibility, then it’s different. So susceptibility is usually more durable. And the reason for that is that you’re removing something that the fungus needs to enter or to infect the plant.

So it’s very difficult for the pathogen to adapt to that kind of situation. Something is missing, so it cannot just create it. So, that kind of resistant resistance. It’s loss of susceptibility will become a form of resistance.

So in that case it is way more durable. But the problem is, it’s also more difficult to breed because it’s a form of recessive resistance. A little bit like if you have two people with brown eyes, you know, it’s easy.

It’s easy to get a child with brown eyes, but to get a child with blue eyes, then it depends if there are some blue genes hidden somewhere. So it’s the same thing here. You need to find, to bring that level of having the two mutated copies or the two different genes must be inactivated at the same time.

So it’s a little bit harder to read. But if I come back to that example of barley, I was talking about 50 years later. So it’s still working. So we can see that it is still very durable.

So, yeah, and that was something that I needed to kind of wrap my head around when I was reading the paper, was that. You actually want less MLO for to really kind of simplify things, you want less MLO, to have more resistance.

And one of the quotes that I pulled out from the paper that really hammers this home is that you said expression of MLO is necessary for the successful invasion of PM. So really what you’re trying to do is eliminate that MLO so they can’t successfully invade. Correct?

Exactly. But then, as I said, it’s not that easy because If that gene is still there, it’s because it plays another role, and that’s usually what people are seeing, is that if you inactivate a susceptibility gene, you can have some pleiotropic effects.

So in the case of MLO’s, it’s very variable. So in some crops where they did that, so you have some, they will see that they have either an increased susceptibility toward other diseases. So it’s more resistant to powdery mildew, but it becomes more susceptible to other diseases, or you will have early leaves, in essence and stuff like that.

But in other crops, there doesn’t seem to be any effects or maybe under some conditions it would cause something bad. But under the conditions that we keep our plants into, you know, we take care of our plants, we fertilize them and so on.

So maybe. It will never show any bad effects, so we don’t know for cannabis if it will show some of those pleiotropic effects. But if not, then it would become something very interesting, to breed..

Sure. And now, do we understand what MLO does to sort of help PM invade the plant, like what the mechanism is there? No, even though people have been studying it for a while, so they consider MLO as a negative regulator of plant immunity.

But apart from that, we have no clear idea of what it does. There are other MLOs that are not involved in powdery mildew susceptibility. So we know a little bit of the functions that those genes are doing. But currently, it is not an indication that the one we’re talking about here that dictates powdery mildew susceptibility would have

a similar function. Sure. Yeah, and that’s a good point. So there are more than one MLO genes in the plant, and not all of them are sort of implicated in this PM infection, correct? Yeah, exactly. So in some plants, you can have, I don’t know, five, maybe six.

OK, you know, let’s start wiring that. So most plants will have, you know, around 15 or more MLOs. But within those MLOs, you know, you have different subfamilies. If I can say. And there is only one that is involved in powdery mildew susceptibility.

So the other ones, they play other roles, as I was explaining earlier on, but so and even within that subfamilies. So then it depends between crops. So for some plants, if you have, for example, three, three genes in that subfamily, maybe you will need to inactivate all those three to get resistance.

But in other crops, there seems to be maybe one that is the leader or more important. And then if you inactivate that one it should be enough. So in cannabis, we found two genes in that in that subfamily, but we don’t know yet if both are required for susceptibility or if maybe it’s only one of them

Okay. And now, to your knowledge, are there PM resistant strains of cannabis out there currently? Do they exist? Resistant strains, I would say, yes. Resistant strains that are resistant because of MLO inactivation, I’m not sure, maybe. So, because as I said, so we have those other examples like the strains that have the pm1 gene.

And I’m aware of other people that have those trends that will stay completely clean of powdery mildew and even though they are surrounded by plants that are covered with it. So clarity there. There are probably a lot of those genes that are similar to PM1.

So it’s just a matter now of identifying a lot of those genes and trying to bring them all together to have a very resistant plant that will resist to, you know, a lot of different powdery mildew populations.

MLO, I’m not sure. The problem right now is that if we look at what was done for barley and it was the same for peas and for tomatoes. So the plans, the accessions, they found that add that MLO resistance.

It was always, you know, in ancient landraces and stuff like that, something that, you know, we haven’t explored that much for cannabis yet. So and even if we consider going back to wild populations, then it will be a matter of trying to find those wild populations because it’s not clear if they still exist.

Right. Yeah, and so going back to sort of that PM1 side of the coin, as you said, so. And if I’m correct, that group that published on the PM1 gene, they were looking at one snp to sort of indicate whether or not a cultivar was resistant.

Is that enough information to really make that call, it seems? Well, I mean, it will not be a first for sure. So, if we look into systems where a lot of different genes like that have been identified and studied so we can think about wheat with regards to rust, which is a big problem affecting wheat or

even potato in its relation with LED light, which is also another big, big problem. So in those two systems, there is a lot of genes like that, that have been identified. And sometimes that’s the only thing that will be different between a susceptible and a resistant plant.

You have one snp in that resistant gene that will cause an amino acid change, and that’s enough to gain resistance or lose it, depending on how you want to see it. But then the same is true on the pathogen side.

So, that gene that is recognized, only one snp can make it not being recognized anymore. So that’s that arms race I was referring to. So it’s always difficult to predict how long it will be useful. Sure.

And then I guess another point that you had made is that there can be a lot of different ways to skin the cat here. It could be a combination of different proteins that are inferring the resistance or the lack of susceptibility.

So it could be that, you know, a snp that’s identified in a certain population might not necessarily apply to a wider a wider population, correct? Yeah, maybe we don’t know enough about those genes as of now. And then there is also that idea.

So for now, I talked about susceptibility and resistance, but within resistance or what I was talking about mostly is what we call qualitative resistance. So it’s either your resistance or you are not. But then there is also what we call quantitative resistance, which is a lot different, harder to breed as well, because it’s, we don’t know exactly

how it works. There is a lot of genes that are involved, each one, you know, playing a minor role. And it’s together that they bring resistance, resistance to a strain. And so, trying to find an example for cannabis would be maybe the genes that have been identified by the Medicinal Genomics group.

So they identified chitinases and thaumatin that seem to correlate with the levels of powdery mildew resistance. So in that case, I would tend to believe that maybe they are involved in something more quantitative. And so those are very interesting, because quantitative resistance, as I said, is harder to breed.

But as anything that is harder to breed, it also means that it’s harder for the pathogen to defeat. Hmm. Interesting. And another thing that I picked up in the paper that sort of maybe talks to maybe more of a quantitative type of lack of susceptibility, was that, if I understood correctly, hemp actually has fewer MLO

genes, in which case it might be less susceptible to PM, did I get that right? Yeah. Well, I mean, it’s I would say it’s an hypothesis that we put forward, of course, in our paper.

So we looked at five different genomes. Those genomes are certainly not all perfect. And there was only one that was really from hemp. So Finola Genome, CBDRx is considered as having some hemp ancestry. So, those two were the one that only had one.

Well, for one of the copies, for one of the two genes, they only had one copy. So is this an indication that hemp would be more resistant? And it’s also hard to talk about hemp because what we call hemp in Canada is sometimes different than hemp in the U.S. So hemp

in Canada, it’s often what we call industrial hemp. While the U.S. hemp is more like CBD producing plants, but they often have the same ancestry as THC producing plants. Sure. Yeah, and so you touched on the fact that sort of the process that you went through to investigate these genes as you used a variety of different

reference genomes. Sort of, how important is it to have a really strong reference to do this type of work and sort of what was the variation in quality that you encountered? Yeah, so I think I mean, any people doing research is always looking for one reference genome, so usually that’s what they have in other crops.

So when we started working, we were looking for which one we should use as a reference, because it was not clear. Like, you know, a lot of people were considering Purple Kush as the reference just because, you know, there was a first draft of it in 2011.

But we were aware that Purple Kush was not the best one. So some people were considering CBDRx as the new reference genome, because, you know, it has a very high quality. So we decided to first decide on which one we would call our reference genome.

So we had to look at first Jamaica Lion, CBDRx, and Purple Kush. And then only looking at those three, it was not easy to decide which one was the best. So, you know, each one has its benefits, I would say so, CBDRx was, you know, very clean.

I would say so, maybe too clean. We’ll talk about that maybe later. So, you know, very less repetitive compared to the other genomes. And it adds a chromosome level assembly, which was very interesting. So we kind of you know, we were considering maybe that one is the best one.

Jamaican lion was also very good, but it was maybe a little bit more repetitive. And also, there was no chromosome assembly at least available to the public. Purple Kush, but it was the worst of the three. So we can skip this one for now.

But so it was, you know, deciding between Jamaican Lion and CBDRx. And from what I said, CBDRx, you know, might have looked better. But as I said, it was maybe too clean because when we started looking at Origin Family, so we realized that one gene that we found in the other genomes was absent from CBDRx.

So is it something real? Maybe CBDRx is missing that gene? Well, we haven’t looked into that or has it been, you know, cleaned a little bit too much. Would they try to remove, you know, repetitive elements from it?

And by accident, they removed that gene. So it’s not clear. So we kind of decided to move forward with more than one genome. And then we decided, okay, so we’re just going to add the other two that were available at the time.

So Finola, which was also interesting because it was hemp so you know, it was maybe a little bit different. And then Jamaican Lion, there was also a male version. So we decided to add that as well, because then we had Finola and Jamaican Lion males.

So two males, three females. So just to see if there could be differences as well with the Y chromosome and the X chromosome. And did you find any difference there? Oh, well, there were some differences. At this stage, is it, you know, real biological differences or are they differences that are caused by, you know,

technical aspect of some genomes being better than others? So, for sure. I would say that Purple Kush, Finola as well. So, you know, they were showing a lot of errors in the sequences when we were looking at the genes, like if we would try to transcribe those genes, they would not transcribe well into proteins because they had,

you know, some extra letters or some missing letters. And again, we talk well, maybe it’s a reality, maybe those genes that have been inactivated in those plants. But then when we looked at other data from the same plant, we had to look at some transcriptomic data from those plants.

And we see that those differences were not there. So clearly it was an error during the assembly process. So those two had more problems. I would say they were more fragmented as well. So for those two, it was easier to say that we would prefer not to work with those two.

Maybe they will get better at some point. I don’t know if they’re still trying to, you know, to make them cleaner or to make them better. But for the other two, it is really the difference that we talk.

So maybe CBDRx is being too clean, but Jamaican Lion, I don’t know, maybe still too repetitive. You know, it’s hard to come up with the perfect genome. And so how does the quality of reference genome that we were working with in cannabis compare to some of these other crops?

Are we still very far behind? We’re not that far behind, I mean, I think we’re certainly getting there, it’s just a matter of the community deciding on which one is the reference, and then we start building on that one.

I think one of the problems we had is that, you know, and it’s not only for I mean, it’s the same for all crops, and I would say for all organisms, you know, people it’s so easy right now to sequence things, to sequence complete genomes.

People will sequence it. The quality is not good, but we’re still going to, you know, upload it onto NCBI. And so you end up having access to a lot of genomes, but the quality is not there. And then if you try to work with those, then it’s you start with a big, you know, a big bias

in your study because you try to predict genes. But if the genome is not complete, then you will miss genes, will lead you to erroneous conclusions. So it’s important to have a good reference genome. But I think we have maybe two right now.

So I think we’re getting there, yeah, for sure. Yeah, but to your point, we sort of need to choose a gold standard, so everyone’s working off the same reference, correct? Yeah. But then it leads us to maybe the other problem that we have is that usually when you have a reference genome for another crop, so that

plant that was used to create that reference genome is also accessible to scientists so people can share that plan toward that accession. And so they will conduct studies on that same plant from which the genome was obtained. And right now, I mean, it’s not a matter that people don’t want to share, that they cannot share the plan because it’s forbidden.

So a Jamaican lion, I mean, I’m sure that people would like to get their hands on it. But right now, especially between countries or from Canada, we cannot import from the US. And I’m not sure you guys could export anyway.

So it’s a very big problem. So even going further, so, you know, most crops, they have those germplasm repositories, you know, that scientists have access to. So they have all those different accessions, landraces, sometime mutants, or sometime mapping populations.

And that’s how the scientific community will move forward more rapidly. But we don’t we don’t have that for cannabis yet. Hmm. So, winding down here for the purposes of maybe breeding out some of these MLO genes that are causing susceptibility.

Where do we go from here? What are sort of the next steps? Well, what I just talked about, so if we could have access to more plants, you know. Because, right now we know that what’s available in the commercial space is still very limited from a genetic point of view.

So we know that there are huge. Yeah, there is a lot more variation elsewhere. So we need to get our hands on that and see if that MLO based resistance maybe already exists within that germplasm. If it doesn’t exist, then maybe we need to create it.

So we have access now to some biotechnological tools like CRISPR to do some genome editing. But those tools are not working for cannabis now. So I know a lot of people are trying to develop those tools, but once we get them, it will be and I’m not talking only about MLO here, but for any trade that we

will discover, it may be a link between a genotype and a phenotype. Let’s talk about, you know, Trichome formation or flowering or other types of disease resistance. So, you know, we see correlation between the genomic data and what we observe of the plant.

But we need to confirm those by, you know, inactivating genes or over expressing genes or some functional studies. But right now, we don’t have the tools to do that. So that’s where we’re lagging or where there is a bottleneck right now.

Do you worry about how the cannabis community might accept CRISPR? Well, you know, maybe so that’s where also we, that’s why I always like to, you know, have both words. So we develop the biotechnological tools, but at the same time, we try to explore as much as possible, you know, the natural variation that we have.

And then if the community really doesn’t want to hear about CRISPR, then maybe we can find some alternative that have been accepted by the organic farming world. So when we talk about traditional mutant mutagenesis, so, you know, we have the pink ruby grapefruit, which was created that way, but you can still grow it like organically.

So to me, CRISPR is not different from that. It’s even better, but we’ll go with what people accept. All right. Well, it seems like the regulations, though, are really an impediment to really making a dent in this and many other breeding breakthroughs that we could be making.

Yeah, for sure. But, you know, at the same time, you know, doing research on cannabis was not even possible 10 years ago. So things are moving fast or maybe those impediments will also fall in the near future.
Let’s hope so. So before I let you go, I wanted to give you the opportunity to share with the audience any additional resources you think might be of interest or any website or social media to stay in contact with you.

Share away. Well, I mean, I’m on Twitter @idjoly so that’s my username there, so I don’t tweet that often. But you know, when I tweet, it’s usually because we have a new paper or something. So that’s a good way to know what we’re doing.

So we published quite a few in the last few months. So we are also working on, you know, looking at beneficial bacteria or they can promote cannabis growth and stuff like that. So there is a you know, it’s not only very hard to understand genomics stuff, but we also do other type of stuff.

So and hopefully we will continue to look at that in cannabis. So, and not only disease resistance. So we’re interested in looking into other traits. So we’ll see what the future brings. All right, it will be the topic for another podcast, I’m sure.

Yeah. All right, well, thanks again, David, for joining us and hopefully we’ll see you out at CannMed. For sure. See you.