 So our next speaker is Dr. John Hendrickson. I read his bio yesterday. I'm not gonna read it today. You should all have heard it yesterday. It's a great bio though. But John today is gonna talk about using auxiliary buds to assess the impacts of management strategies on the abundance of invasive coalescing grasses. Thanks, Kevin. And I'd like to start by acknowledging my co-authors, Andrew Carlson, David Toledo and Chantel Kobolansky from the Northern Great Plains Research Lab. And also, and I forgot to add these co-authors on there, but I should also add on Sean DeKaiser, Brianna Kabila and Levi Benstock from NDSU who had a portion of another project on here. So I apologize for that oversight, but I'm gonna start off this talk by talking a little bit about population ecology. And I kind of hesitated on whether I should do this or not, but then I talked to Holly Johnson and Andrew Carlson who do all the heavy lifting in my research program. And they said, John, you've made us miserable with this multiple times, why not spread it around? And so I said, okay. So, and this was kind of mentioned yesterday, but if you look at our grasslands in the Northern Plains, they're very much developed under disturbance. If you look at our native grasses, they've had to do fire grazing drought. And even our introduced grasses have had to undergo grazing and understand how they respond to defoliation. And so when they do this, grasses have a tendency to develop coping mechanisms. And one of these coping mechanisms or persistence mechanisms is axillary buds. Now to put these in context a little bit, and this is where we talk a little bit about population ecology is if you think, if you think about our communities out there, you know, when you look and you measure grassland community, you see, oh, we have all these different species, you know, but really what that community is is a collection of populations of different species, okay? And the idea with population ecology is we can understand something that's happening here at the population, we can maybe get an idea on why our communities are changing. And further there, populations are made up of individuals and these usually when we talk about this with our rhizomenous plants, usually these are stems. If it's more of a cespitose where we know exactly what the plant is, we can kind of lump it in to say a plant. But at the base of those individuals are axillary buds. And axillary buds are very important in our grasslands. If you look at our native grasses, for example, and you look across there, about 99% of the grass shoots you see are going to be originally arise from axillary buds. So they make a big contribution to the persistence of these grasslands over time. They're kind of part of a modular growth form. And if you look here, you see, typically we call this a phytomer, it's where the node is and then there's a leaf, there's an axillary bud right at the axis of that leaf. And then there's an inner node. And if we go over here and look at this, and this is shrunk down, so it's the crown, but you kind of see the same thing. There's a node there and there's an inner node and there's an axillary bud. And this will kind of turn out to be a leaf. And the other thing, and I'm gonna talk about this later in the talk is that there's not just one of these on a crown, there's multiple ones. So we actually have multiple positions that can develop on that crown. And that's important when I'm gonna talk about what those different positions can turn out to be. Just a kind of a side note there, when you look at most of our tillers, they actually come from the upper portion of this crown, usually the ones down here that the older tillers that are developed down below usually don't grow out to be tillers. They're these, I'm sorry, these axillary, these crown positions lower don't turn out to be tillers. So this just kind of shows a little bit about what these are and I'm gonna give a little bit better explanation of this later. But an important thing to remember about these axillary buds is that they can be a really big part of the meristematic potential we have out there. They can be larger than seed banks. And I was trying to do some numbers on this and the numbers got really big, so I gave up on it. But basically Kentucky bluegrass, we ran some rough stuff and we had about 9,000 of these axillary buds per meter squared. And then if you wanted to get that up to a hectare, you take that times 10,000 and the numbers become really big and I'm just left it at that. But they can be really, there's a lot of these out there. Therefore, we need to understand how our management impacts these because then it can help us understand how effective our treatments may be. The one thing to say is that I think they're really a good way to evaluate treatments. We do have to be careful when we say we can use these to predict the future. And there's a lot of slippage that can occur there. And so it's just one thing that you need to think about. One other thing I wanna bring up before I start talking about this is a little bit about phenology. And Sean had talked about this yesterday about how there are different phenological aspects to smooth brome, for example, and both smooth brome and Kentucky bluegrass have our C3 grasses. Most of our C3 grasses have a tendency to produce two cohorts and new tillers every year. One in the spring, one in the fall. But to C4 grass, just the tendency is to have a more consistent tillering pattern across the season. These two final points here, this one about Kentucky bluegrass axillary buds forming in the fall. And the smooth brome grass axillary buds becoming more active after the reproductive phase. I want you to remember that because it kind of helps explain some of the things that we're talking about later in the talk. So with that, I'm gonna talk about two projects we have. One looks at different defoliation at different phenological stages in smooth brome. This kind of came up because I was looking at some, we had some long-term exposures at our location. I was looking at the amount of Kentucky, or a smooth brome in those exposures compared to outside. There's a lot less outside where it was being grazed outside the exposure. So it's suggested maybe there's something that's occurring there. The other is with drought and fire impact on axillary buds and Kentucky bluegrass. So these are kind of two distinct projects, but they both revolve around axillary buds. So the first one is with the defoliation effects on smooth brome. And we basically had four different defoliation treatments and a control on this, the defoliation treatments revolved around the phenological stage. So they could be defolated once when it was vegetative or twice in the vegetative stage. This elongation stage, that's when the grass plant starts to elongate. And you can tell that Sean mentioned this yesterday. You can kind of feel a little node there. When you run your hand up and down the stem, you can feel a little bit of a node there. And then also in the reproductive stage, which was, we call that roughly around the boot stage with that. And then we left an undefoliated control. We had 10 of these, each tillers in each block. And then there were three blocks and they were sort of placed in some pastures on loamy sites that we had at our location. These were inside exposures on the pasture. So there was more of them and they're excluded from grazing not only before we were tagging them, but also afterwards. You can see down here, when we mark these tillers, we put a little wire around the base of it. That's, we use telephone wire, but you can also use any type of wire there that's as long as it's mandible and colored and stays. But that helps us identify that for later on that we defoliated this specific tiller. So, we did that at each stage and you can roughly think some of it was in May and maybe when it started elongating might've been June and reproductive too. But then in the fall, we came back and we collected these and you can see when we collected them, oops, when we collected them, the other thing we did was we marked them with nail polish. And we mark them with nail polish there because that allows us to make sure that we're on the right tiller or chute. Because when you start taking these up, sometimes they're fragile, they can break. Having that nail polish there lets us make sure that we're on the right. You know, we're still getting the right one that we originally defoliated. When we brought them back to the lab, we cleaned them up and then we did what we call a double staining technique on that, basically we put them in tetrazoleum for 24 hours. Tetrazoleum basically, if the cell is active it takes up the tetrazoleum. We put them in Evans blue. Evans blue is the type of stain that if the cell wall is compromised it will go through the cell wall. And so we could tell if they were live or if the axillary buds were live or dead based off of that. Remember I'd said that it was important, remember that they could have multiple, you know, different positions on a stem. And we kind of can split these into categories. You know, we have our bud category here which is active and dead, but also dormant which are unstained. There is a little controversy about the dormancy using this technique to be fair but this is a pretty rapid technique that we use. We also looked at outgrowth. You can see here with tillers and rhizomes and then this leaf scar here and I'll talk about that a little bit later but sometimes you'd see these positions and there should have been something there but you could not see anything. And so we would call that a leaf scar and I'll give an example I think on the next slide here. Yeah. So you can kind of see here, here's an axillary bud that's what they look like. They're small, they're kind of conical. This is one that is active. You can see how that's stained red with the tetrazolium there. Here's one over here and this isn't showing up quite as good unfortunately but it's a very, very dark blue when it stains with the Evans blue in there. And up here is a dormant one and those kind of have a tendency there's like a blue tinge but basically it's on the outside. It's not the deep blue stain that you see when it's truly dead. Leaf scars are like these areas here. You can see there's a discoloration there. So you kind of say, okay, there's something there that should have been attached there and we're not seeing it. So those are kind of the categories along with either a rhizome or a tiller that grew out. So we call this, when we looked at outgrowth here we said, okay, it could either be a rhizome or a tiller on here. And there were differences between years which is kind of what you would expect but what I found kind of interesting between years also is there was differences in years between rhizomes and tillers. So certain years had a tendency to produce more rhizomes certain years had a tendency to produce more tillers. But over here with the treatment I really found this interesting if you look at our total outgrowth here if we clipped in the reproductive stage we ended up getting more outgrowth than we did in any other stage with this. We got the least outgrowth when we defoliated twice in the vegetative stage. And if you remember, I said that axillary bud tillers could become more active after the reproductive stage and this may be one sign of that. There's other things that could be active working on this but this could be one of the things that increases the activity there. The other thing too is that if you notice over here that there were differences in the proportions of tillers and rhizomes contributing to that total outgrowth again it was based off of the defoliation or the stage the phenological stage that we defoliated in. This, and I hesitated a little bit to show this because I'm not using this information the way it truly should be used because I don't have the information for this but it does give us a little bit of an example and what that is is basically if you think of a tiller out there what that needs to do is it needs to produce one live tiller or one live rhizome over its lifetime in order to maintain the population, okay? And so we could look at live tillers and rhizomes per crown when we got these and the reason I say this wasn't total is that we don't know what happened to some of the dead ones. They may have not have really totally they may have emerged and died really quickly. We don't know if the original tiller was dead here but still this gives us a little bit of an idea on if that population is growing or not. So anything above one here it's a population that's growing and you can see regardless that our population was growing but if we look here at defoliating it twice in the vegetative stage we were reducing that population growth and in fact you look at the reproductive stage here and it was producing 62% more live outgrowth than when we defoliated it twice in the vegetative stage and looking at that here too if you'll notice vegetative stage had the least outgrowth here reproductive stage had the most. The other thing and I just noticed this when I was going over my talk this morning is that I found kind of interesting is that probably what's again if you look at active buds and total live buds which is the active buds plus the dormant buds because they have the potential to grow out also we're seeing the most here in the reproductive stage which you would expect but there's also quite a few here in the twice defoliated twice in the vegetative stage and to me that says there is something happening between that bud being active in that rhizome growing out there's something that's kind of short circuiting that ability to actually produce a new tiller in there and I found that really kind of interesting with this and we need to think about it more which brings us to some takeaways from this project which is defoliation in the reproductive stage increases outgrowth but what we really need here is we need to have a better understanding of the tiller demographics and Zach Johnson I know has a poster on this that he's actually looking at the tiller demographics in this so I think that'll be kind of interesting to see what he's finding on that The next project I wanna talk about is looking at axillary buds in Kentucky bluegrass and when we looked at this we were looking at two different treatments on here one was burning and the reason for that is as you know fire has the potential to reduce the abundance of Kentucky bluegrass but socially there's been some constraints on us using fire across the landscape The other thing too is that if you look and this is data from our location in Mandan there if you look here between 1916 and 1990 we had about 15 inches of rainfall a year in the 90s that shot up to almost 20 so and we're seeing that increase in precipitation throughout the decades after that which says that not only did fire removal but we also have this large increase in precipitation in David Toledo and I when we were talking about this project you know some people that we knew said okay you know if we have a good drought Kentucky bluegrass is going to go away and so we said okay let's see if that's going to actually occur so when we looked at this we looked at this using kind of three different or you know three different drought treatments one was an ambient treatment we use these rain intercept shelters and you can see the more of these transparent plexiglass gutters we had on there the more rainfall we intercepted so we had a 0% which just got the ambient rainfall we had a 60% which got 60% of the rainfall that would fall on the plot in a 30% and we had those oh one other thing is then each plot half of it was burned and half of it was left unburied we use this firebox in order to do that so each of these was replicated three times within these exposures that we put up and then each exposure was replicated three times we had three replicates within a block and then each block was replicated then we had three blocks of this in addition we had outside here we had three additional plots where we wanted to look at grazing we did not add the rainfall intercept on these plots because the livestock having access to them but we did burn and not burn them so they could be grazed and burned, grazed and unburned so we went out there and we sampled within these plots we used a sampling probe and it was about five centimeters we went down two or three centimeters we brought the samples in you can see how they were kind of with Kentucky bluegrass you see a lot of this root and thatch there we cleaned them up put them in tetrazoleum so we could just kind of see the activity on these and then we analyzed this using a randomized complete block so what did we find on this and I found this kind of interesting one is we had a time sampling time by burning interaction burning had a tendency to reduce the number of axillary buds you can see this across time but there was a little bit delay and I was thinking that we might see the maximum impact here because these treatments have been in place since 2017 it would burn right the year before and we didn't instead we saw the only significant time we difference we saw between burning and unburning with number of axillary buds was here in November of 2020 and there's a couple reasons that that could be one was that September of 2019 was very wet if you remember I said that Kentucky bluegrass has a tendency to form its tillers in the fall and if it had increased moisture during that time or not tillers but form its axillary buds in the fall if it had increased moisture during that time that could have provided a larger pool of axillary buds this here was burned in September and then we sampled in November we do know that burning can sometimes reduce the amount of axillary buds on there as you can see from all this other data so that could have been an impact here the other thing and I forgot to mention this but it's important to know that 2020 growing season in 2020 was like and the Bismarck weather airport weather station was the third driest year since they started keeping records and 2021 was also really dry and in fact it was probably even drier than 2020 just because we didn't really get any moisture till later in the year and so what I think you're seeing here with a reduction in the number of the reduction in the number of axillary buds there is that that's an impact of drought that dryness was there and it was continued dry and so we're seeing some of that impact of drought there so what about drought? Well, when we look at this we see again that regardless of our drought level burning had a tendency to reduce the number of axillary buds but it was really only significant at that 30% drought level and so I think this interaction here between a moderate drought and burning is going to be important to think about in the future. Again, one thing to remember here is these rain intercept shelters intercepted the amount of precipitation that was falling and it was dry anyway, you know during the two years that we did this and you just need to think about that a little bit when we're thinking about these interpretations of what's going on here but that again could say why my thought would be at the 60% that we're seeing a suppression and axillary bud numbers that we're just not seeing the big difference between the burning and the not burning that we did see at the 30%. Finally at the grazing and with the grazing we did not see a big difference here. There was, I looked at this several times it looks to me like grazing adds variability to these axillary bud numbers but there's not a clear trend with the impact of grazing on it. The only time I saw that there might be some impact of grazing or might have interacted with burning was clear down here in November of 2021 when we're seeing a little bit lower numbers of Kentucky bluegrass axillary buds with grazing compared to burning. So there might have been a little interaction there but that did not show true through the rest of this of the rest of the project. The other thing here to remember too and I don't know if you kept an eye on this axis here which is a number of axillary buds per meter squared but again, there's a ton of them. Like I said, there's about 10,000 here per meter squared. If you scale that up you're seeing a lot of numbers there and it just shows how resilient these are. There's a lot of axillary meristematic potential out there that has the potential to turn into a tiller or something. So these are very resilient grasses and we can throw a lot at them and it's hard to really reduce them as much as we want to based off of this. So looking at what we found out with a summary here that we had from the burning and drought on with Kentucky bluegrass burning is important. And I think even though it wasn't always significant we did usually have a trend that there was less axillary buds when we burn than when there wasn't. So I think that's burning is an important tool to think about using here. One thing to keep in mind with the burning is that it may be linked as far as effectiveness with your weather patterns. And again, I'm thinking about that wet September we had in 2019 and we weren't seeing the impact of that burning out into the fall. So there may be something, I mean into the spring the following spring. Grazing, we really did not. Grazing, we could not see a clear story with that. It could be one there if we tease it out more but I really did not see a clear story with what the data we had. I found this one interesting here where probably the maximum or significant difference with between axillary bud numbers with bird and unburn came under moderate drought. To me, that's something as land managers and others I mean, that's kind of could be a hard sell to some people to say, okay, if you want to maximize the effect of your fire, you got to wait till you're going to burn up some of your forage when it's dry. And it's one of those things you just have to think about as you're talking about this. And the extreme drought can limit the effectiveness of burning, and again, if you look at work we're harvesting 60% of very limited rainfall there. Some of these plots were probably getting, I think we had eight inches for the whole year. 60% of that is, I mean, you're down to maybe they're getting two or three inches of rain on some of those plots. So overall, I do think that we can evaluate use these axillary buds as a way to kind of evaluate the effectiveness of strategies. I think we saw that with burning with the Kentucky bluegrass and we also saw that with clipping at the different phenological stages with the smooth brome. We do need to think a little bit about the management by environment action. I would also say thinking about adding on there the management by morphological stage or the phenology with looking at how these grasses and for example, on the smooth brome when we defoliate with the Kentucky bluegrass thinking about when we're going to apply the fire to maximize the effectiveness. And finally, like I said before there's a lot of meristematic potential out there with these looking at these axillary buds. While that can be overwhelming it does show us that these grasses are really resilient and that we as I said yesterday it's not a one and done type of thing. These type of management strategies have to be repeatedly placed on the landscape in order to maximize the effectiveness of them. So with that, thank you very much.