 Hello, everyone, I am Chantelle Mertz, like she said, and I will be talking to you a little bit today about salinity and how plants respond to it. So first of all, first of all, where does the salinity come from, especially out here out west, a lot of our salinity is often a result of brine spills. So brine is a byproduct of the oil and gas industry, specifically for when we use hydraulic fracking, we use water that we pump down into the oil and gas deposits to break it up to get that oil and gas to the surface. And so when we do that, we actually hit marine shale deposits so thousands of years ago. North Dakota used to be more of a marine oceanic environment and so because of that, the deposits are very highly salinized with sodium and chloride. So when this water comes back up with the oil and gas, it's extremely salinized it can have ECs which is the electrical conductivity which is how salty the water is greater than 200 desiccements per meter. And so this water is usually stored in storage tanks next to the pumps, or it can also be in pipelines. But occasionally these things spill and so when this happens this can have detrimental effects to both the soil that's there, as well as any organisms that might be living there as well. So we know that salinity is not a new problem but it is a growing problem for example like Tom alluded to, out of the 45 million acres in North Dakota that we have around 6 million of them are salinized, just with natural agricultural salinity so this is a result of salts that come up with the groundwater, as that water evaporates it leaves the salts, and this causes a lot of agricultural salinity that we have. And so it's not a new problem but it is a growing problem so how do we deal with these brine spills and this agricultural salinity that's beginning to be such a big problem. Well the first part of that is understanding how it affects plants. So I know this kind of seems a little bit complicated and we're going to get into the plant physiology of things. It's my hair. We're going to get into the plant physiology of things but I'm going to try to make it as simple as possible. So there's two main effects that salinity or saline sodic soils like Tom was talking about have on the plants. The first is ion toxicity. So normally with any kind of nutrient you can have a deficiency which means you're not going to get enough growth of that plant because you don't have the nutrient to perform those functions. If you have a sufficient amount of that nutrient you can probably get the best yield possible you're going to be able to hit that sufficiency range but you can have too much of a good thing, or as we say you need to have everything in moderation. So with salinity comes sodium and chloride or other salts such as magnesium or calcium sulfates that become too much for the plant. So this is called ion toxicity. Basically it just builds up in the leaves and the plant is no longer able to function. It's a little bit more common that you like to think of as osmotic stress. This is just a fancy way of saying that the plant can't get enough water or it's drought stressed. And so, you can't really see the colors on there but there is the equation for photosynthesis and as you can see we need carbon dioxide and water to be able to make sugars to perform the functions that we need to and grow as a plant. And so if we don't have enough water, we can't make those sugars which means we can't grow as a plant. And so, osmotic stress is usually seen more rapidly because just like if we didn't have enough water, we would start to feel sick pretty quickly plants have a rapid response as well if they can't get enough water. And so this causes that decrease shoot growth because they can't make those sugars that they need to build up the plant material. Now ion toxicity isn't seen quite as much just because in a normal agricultural salinity situation you don't have that concentrated like sodium and chloride as you do in a brine spill so you don't see ion toxicity quite as much. But in brine spill situations you really see it because we have so much sodium and so much chloride concentrated in those soils. And so this is a little bit of a slower onset that onset than osmotic stress. And this kind of all those ions start to build up in the older leaves because those were the ones that are around the longest and so those ions build up in those mature leaves and they start to senesce which means that they're kind of starting to decompose or become necrotic and necrotic. So those are kind of the two main things that happen to plants, when they're exposed to salinity we also have a couple secondary effects. One of them is we can get like, we can get other nutrient deficiencies because if we can't bring water into the plant. That means we also can't bring the nutrients that the water brings into the plant as well. And we can't transfer those nutrients from the shoots to the roots and back again. So we have a lot of nutrient deficiencies such as calcium or magnesium and then we can also get something called reactive octan species. So basically if you think about it, if the sun's beating down on you, normally in photosynthesis you have electrons that are going to receive this energy and go where they need to go to be able to make their sugars. Well if the sun's beating down on them, and they can't perform photosynthesis because they don't have enough water, those electrons are going to take that energy and they're going to start to ping around like a pinball machine kind of thing. And what they do is it actually destroys their membranes. And so they can't keep all their organs and all everything that's supposed to be in certain places in certain places anymore. And so it starts to really damage these plants. And that's what ends up leading to death and the stunting that you will see in salinity. So those are kind of the main effects. So where does that lead us? I want to show you guys exactly what happens to a plant when it's under that osmotic stress. So here's a good diagram that shows typically our water moves from high water potential which means it moves from places with low salts to places with high or to low water potential with high salts. And so as you can see here, this is a soil that's at negative 0.3 bar. So you really need to know what that means. Basically the soil's at field capacity. We have a pretty good amount of water in that soil. So what the plant does is it's able to form kind of a straw through capillary action, using some adhesion cohesion. Basically, at the top of the leaf it opens up its stomata and because the atmosphere is so negative, it sucks up that water like a straw. And so the plant under normal conditions when there's no salt in the soil, the water moves from high to low. It moves from negative to even more negative. So under normal conditions, plants are able to uptake that water. And because of that, their cells are pretty happy. If you think of a tire, you have that inner tube inside a tire, you want that tube to be inflated, right? To have a pretty good tire that rolls around. If you have a plant cell that has enough water in it, it's turgor pressure is what it's called. It's going to be good. And it's going to be able to stand up straight. It's not wilting. It's going to be a healthy plant. So what happens if you have salinity? So now we have our sodium and our chloride in our plant, or in our soil. And so what this does is that sodium, that chloride, just like Dr. Sutter was talking about before, can become hydrated with water. They're almost stealing that water, and they're causing the soil water potential to become more negative. So as you can see, we're at now at negative 12 bar, which is getting kind of close to permanent wilting point. And because the leaf can only go to negative 15 bar, this is stopping that soil from having as much when it sucks through that straw. So now that plant can't get all that water into itself. And so what does that happen for our plant cells? Our plant cells start to lose water because the soil has more ions and is more saline than the cell itself. So the water wants to move to where those salts are. So this causes our plant cells to desiccate or dehydrate. And when this happens, our plant starts to wilt because it no longer has that trigger pressure that's keeping it healthy, letting it stand up. All right, so now that you guys know how salinity affects the plants, this can get us starting to thinking about the different kinds of plants that we use in remediation and reclamation and things like that. So we like to classify plants in two different ways. The first being glycophytes. So glycophytes are most of our agricultural crops, our wheat, our soybeans, our corn that we've domesticated for thousands of years. These crops are not very salinity tolerant. They don't have a lot of stress tolerance in general. They can only tolerate ECs, which is our electrical conductivities of around up to 10 deciements per meter. And even at 10 deciements per meter they're not doing too hot at that point. So, because we've been domesticating these crops for years, we've been making sure that we've been going for high yield, ripening at the same times, things like that. And so because of that we often lost those stress tolerances that they normally would have. And then the halifites. So halifites are defined as plants that can fulfill their life cycle, even under really saline conditions. So this is not a very broad group of species only about 1200 species. That's about 0.5% of all angiosperms so angiosperms, just a fancy way of saying plants that produce seeds. So it's not very broad, but what they can do is they can tolerate that salt and so these are plants that grew up in pretty extreme environments so this is your deserts this is your coastal areas with a lot of salt your marshes things like that. And so these plants used to have to be defined as plants that could tolerate seawater which is 58 deciements per meter, but now they kind of lowered it and said plants that can tolerate at least 20 deciements per meter of the water of the solution can be considered as plants. So here's a chart up here and I know it's not very big, but I just want you to look at the curve. So on the y axis, we have basically biomass. And then we have increasing salinity on the x axis and so as you can see we have Durham, we have bread wheat, we have barley, and they as soon as we get any kind of salinity in there we increase our salts, their biomass goes straight down, they're like I said they are just glycophytes that don't have a lot of stress tolerance. However, if you look at that blue line right there that is salt bush salt bush is a halophite and as you can see, it even increased in biomass a little bit when we had some increasing salts so it's able to tolerate those salts and in turn keep growing even when we have really high salinities and fulfill its life cycle. So here are the ways that different plants glycophytes and halophytes included can tolerate these different kinds of salts. Well I like to classify them by four different mechanisms. So we have exclusion. We have storage, we have utilization, and we have secretion. So the first one I will start with is exclusion. So almost all plants can exclude ions from coming inside to a point. They can exclude ions by opening and closing their stomata to different points and they have special things in their roots and transporters that they can use but that's all a little complicated. So the best way to explain it is even if there's salts in the soil, there's different plants that can stop those soils from or stop those salts from coming into their roots. Examples of this include mangroves which can exclude up to 90% of salts from coming in. For example, the barley that we grow here in North Dakota, a cousin of barley is sea barley. Sea barley can do the same thing and exclude salts from coming into the plant. The next thing that plants can do to make sure that the sodium and the chloride doesn't come into their system to prevent that ion toxicity is they try to retain the sodium in their roots. We want to retain something in our roots. Because if it gets to the leaves, if that sodium or that chloride starts to accumulate in the plant and get to the leaves, we cannot perform photosynthesis. Our membranes are going to start to get broken up by those reactive oxygen species, and we're just going to have a lot of problems. So what the plant does is it tries to make sure that it retains that sodium and that chloride in its roots. And then the last thing a couple of plants can do is let's say the concentration of salts in the soil is so high, we can no longer prevent it from coming in the plant. We can no longer stop it from going past the roots, and it's starting to go into the xylem well there's certain plants that can grab that sodium grab that chloride and shove it back down to the roots. And so this is called ion retrieval it's still pretty new. But there are plants that are able to do that to make sure that it doesn't hit those leaves where we're performing photosynthesis. All right, the next one storage now this has a lot to do with what we just talked about. So here's an example of a plant cell you have your cell wall that gives the plant structure, you have your cell membrane that's what regulates what water and ions go into it, you have the chondria that's where respiration occurs, your nucleus is where your DNA is your chloroplast that's the most important that's where photosynthesis occurs. And so what plants do just like I said they store that sodium in the roots, well they're actually storing it inside the vacuole which is the storage mechanism, organelle of the plant cells. And so you will be storing that sodium or that chloride in the vacuoles in the plant cells in the roots first. And so, basically plants are storing sodium and chloride as much as possible in their membranes to prevent it from getting into this cytoplasm, where all the organelles are hanging out. We want to keep that salt is far away from the chloroplast where photosynthesis is occurring as possible. Different membranes and different plants have the ability to store these membranes to a point. So, for example, broadleaf plants are more leaky, they can only hold so much sodium and chloride before those salts will start to leak into the cell, versus grasses have a lot greater ability to keep that sodium and chloride inside just because of what their membranes are made out of. So the next one is called utilization. This is when plants can actually use sodium or use chloride for different functions within their plant cells, so that it doesn't become toxic or so that it can prevent that osmotic stress from hurting them. So the main way that I can explain this is that sodium can be used as a substitute for potassium. So I don't know about you guys but if I'm thirsty or if I just ran a lot maybe I'm going to drink a Gatorade because a Gatorade has electrolytes in it. Well the principle electrolyte that humans use is sodium. We use sodium to regulate our water in our body if we need to sweat it out we do, but plants don't use sodium like that. Plants actually use potassium to regulate their water. And so, that being said, sodium and potassium are actually very similar in their size when they're hydrated meaning when they have water near them. And they also are both monovalent cations which means they each have a plus one charge. And so, if there's a function that a plant might need potassium for such as that osmotic regulation, they might be able to use sodium instead because they're so alike. And so an example of plants that can do that is castor bean. So what they do is once castor bean has mature roots. It says okay, I'm going to take this sodium. I'm going to take this chloride. I'm going to put it in the vacuole so it can't hurt my other organs, and then I'm going to use that sodium and chloride that's in the vacuole to make the cell of that plant more negative, so that I can get even more water into the plant. Because what they're trying to do is out compete the soil. So if they're able to keep the plant and get the plant more negative, they can suck water up that straw. And so this is a way that plants can utilize sodium instead of using potassium. Another thing is C4 species versus C3 species. C4 species examples are like corn and sedan grass, things like that. And so C4 species actually needs sodium to function. They cannot, they need it as an essential micronutrient and without it they can't perform their life cycle. So for example, the plant like this is wild proso millet. It actually needs it to make chlorophyll to do photosynthesis, and it needs it to make certain enzymes for C4 photosynthesis. And then the last one is sodium can actually act as a beneficial nutrient, so it's not needed for their life cycle and most things do not need sodium, but if we can find some way for them to utilize it, some of them can do a lot better. So examples of plants that can use sodium, and it actually increases their photosynthetic capability, which means they can grow more is beets. So beets, turnips, marigolds, things like that actually need sodium. It helps them grow better, and it helps them use less potassium, which in the end helps them do a little bit better because they need less of that nutrient that they usually need so much of. So here is an example of a paper and I know it's a little bit hard to see but basically they did a study where they gave plants enough potassium, as well as they added sodium. And there was plants that even though they had enough potassium, when they got sodium they did better. And so that was like sugar beet Swiss chart red beet and turnips so it's pretty interesting to see how some of these plants can utilize some of these ions we think are so bad. So this is my favorite one so excretion is the last method that plants can use to either get rid of those ions that might become toxic. And so, just like we have an epidermis our skin or top layer of our skin plants also have that it's the epidermal or epidermal layer of their cells so right on top of the leaf. So what happens is plants have two different organelles that can help excrete salt from the plant so that it doesn't accumulate so much that they get that it gets toxic to them. And so the first one of those is assault gland assault gland is like a multi cells structure, and what it works like is like a pump to take all of that salt that might be accumulating in the plant and shoot it out to the outside so that it doesn't accumulate inside their leaves because like I said, we want to keep doing photosynthesis and so examples of plants that can do this are gray and white mangroves, as well as inland salt grass which is people out West here is probably pretty familiar with. The second structure I like a lot it's a called assault bladder. This one's a little bit different it's only one cell. And what it is it's kind of like a stock and on that stock is a balloon. And so in that balloon there's a vacuum and they shove all of the sodium and chloride and other ions that they don't want in their system. And it keeps getting bigger and bigger until it bursts and when it bursts it shoots all of that sodium and chloride outside of the plant. And so examples of things that do this are quinoa and lambs quarters. Now there's still a lot of science being done with these kinds of things. And they don't always develop these glands or these bladders and only certain kinds of plants have them, but it is pretty interesting to see how plants can protect themselves like this. And then here's an example they use an electron microscope so this is what they look like in real life. Here's an example of assault plan, as well as assault bladder. And this is a picture of a mangrove where you can actually see the salt crystals on the outside of the leaf because the plant was able to expel them. This is what's going to protect them in the end from that high salinity soil. All right so now that we know where salinity comes from how the plants are not like how the plants do with salinity and ways that they can combat it how does this come into my research. Now that we know that we have saline plants or that we have plants that we know that we need plants that can tolerate salinity first. And so we need something that can tolerate that ion toxicity can tolerate that osmotic stress and still live in that environment. Well this caused me and Dr. de Sutter to look through a couple different plant species which I will talk about later in the presentation. However, we know that when we get a plant like this that can tolerate salinity can tolerate the ionic stress and the ion toxicity and the osmotic stress. Then we know that it will be able to form roots that go down into the soil. If it's able to form roots that's going to allow us to infiltrate with precipitation hopefully leech salts down, as well as have roots that are there using up the water to make sure that more salts don't come up with the water table so that's like the first step in remediation. The first step is finding a plant that might be able to utilize or store those salts above ground so that we can remove whatever biomass that is to take the salt out of the soil and get it out of that area to try to remediate it. And then should we find a plant that can do both of these things or at least one of them, then hopefully we can do a field trial and do remediation. So here is the first step and this is the research that I've done so far. So I have done a girl chamber experiment utilizing five different plants, more alternative species that are Halifites so examples of these Halifites are Oratch Swiss Charred common lambs quarters which is a weed, as well as a weed, as well as curns out which is intermediate wheat grass it's actually a perennial. And then I also tested iron corn which is an ancient grain, because we thought if we were testing. We know that weeds relatively tolerant if we could go back to weeds roots perhaps it would still have those those stress tolerance genes that maybe got bred out. And then I also tested black turtle beans as well as hard red spring because we know hard red spring we is tolerant and black turtle beans are relatively susceptible to salinity. And so what we did, it was we grew them in containers with half soil half sand. Once they emerged, we had eight different increasing sodium chloride treatments to mimic a brine spill. And then we also had a control with no salts. So we let them grow for 30 days. And then after that we took the above ground dry mass and we weighed it. And then we also took the EC or the electrical conductivity of the soil to see how salty it was. And so here's an example of my eight different sodium chloride treatments as well as my control. And the EC one to one here which is half water half soil we had almost ECs of up to four, but most of the time we do things in saturated pace which is a conversion of 2.2. So we got up to around eight desicciments per meter in our soil. And so that's relatively anything greater than four saline usually. And then I typically went a lot higher than most normal salt tolerance experiments. Here are the photos from that experiment. Here are the black turtle beans as you can see most of the black turtle beans are either dead or dying, unless they were part of the control, or the very beginning salt treatment so they were not very high. However, lamb's quarter over here was growing pretty good. It was putting on a decent amount of biomass and it was actually starting to pollinate within that 30 day period. So here's an example of what we found. We compared all of the crops to each other. And so this might look a little confusing but what we did is we assumed that the control treatment with no salt had 100% biomass those plants didn't have any stresses so we assume their biomass was at 100% so we could compare them all to each other because they all grow at different rates. And so what we found is that lambs quarter over there it's not connected to any other rectangles but itself, meaning that lambs quarter was significantly different from all of these other ones that we tested. And so this comes into play here where it's a little bit better to see. So as you can see here we have the broad leaves, specifically lambs quarter Swiss chart or after all Halifites and tested against black turtle beans. So black turtle beans all of these started at 100% biomass and then went down black turtle beans. As soon as it saw any kind of salt, it decreased in biomass we knew that it was susceptible and it did exactly what we thought. Swiss chart and Orach, they did pretty good they either stayed about the same with increasing salinity levels, or they actually increased in biomass, a little bit which means they grew. However, the best one the one on top there is common lambs quarters, common lambs quarters did not care at all that it was in salt it kept growing it didn't have any decreases in biomass. Now this wasn't it didn't have a statistically significant linear relationship but it did have a biological significance which is really important and so that's why we are going to go with lambs quarter in the future which I will talk about later. So here is the results of our grasses so we have I'm corn and hard red spring we as well as Kernza I'm corn and hard red spring we did about the same. The point where they had 50% biomass which means they were at about 50% of what they could have been if they were in the control. They were at 50% EC of 1.9. And as Kernza did a lot better and the point when it was at 50% biomass was 2.7. And so, we know it decreased with increasing salts but the fact that Kernza is a perennial means that we could possibly skip that germination and early vegetative stage that might be more sensitive, because we don't have to keep replanting it year after year. And so that had us looking into Kernza for future experiments as well. So the advantages of these two plants that we kind of saw were pretty salt tolerant. So Kernza for one is perennial we don't we can get past those sensitive stages, as well as it can be harvested for grain or forage. It's actively being bred in both Minnesota as well as Kansas by the Land Institute, and it currently has about one third the yield of wheat. So it's a good resource for landowners if you want to start to possibly remediate that soil. Then we also have lambs quarters now lambs quarters might seem a little bit daunting because it is a weed. However, common lambs quarters is a little bit easier to control than Kosha, or water hemp that are more difficult weeds. So I mean lambs quarter does have some resistance to photo system to inhibitors as well as ALS inhibitors. But in general, we can control it a lot better than we can Kosha so it's a better option. It can be used as a forge for livestock, as well as it has a pretty wide emergence window meaning that we can plant it at different times we don't have to worry about getting in or if we haven't had a rain yet for any kind of oil and gas industry things because it has a really wide emergence window. It also produces a lot of seeds so should we be able to find that it is a really good fighter and mediator we potentially have the capabilities to get that out there to people who want to try it, and it also has heteromorphic seed production now what does this mean. Heteromorphic seed productions means that when it's under stress it can produce brown seeds that are non dormant that are really stress tolerant, because it realizes that it's going to die and it want to make sure that it has progeny to continue on versus if it's in ideal conditions and it's not under salt stress, it can produce black seeds that are dormant because it doesn't want any competition. So we have a lot of benefits of lamb's quarter and curns up that we are looking into to possibly use for remediation. So what does this mean for moving forward, moving forward, we want to do a greenhouse experiment where instead of just doing the early vegetative stage we're going to go from germination to maturity. And we're going to use a lot higher salts because we do want to find what the salinity tolerance of lamb's quarter is because it never did decrease in biomass. We want to test their capability as a fighter mediator so can they take up that salt can they utilize that salt in any way that we can potentially remove that biomass from that area to get the salt out of that system. And then we'd also like to do field trials in the future, possibly this summer in Carrington we're going to utilize some natural salinity gradients, where we will test curns in a multi year experiment since it is a perennial and see how it goes after year with salinity, as well as possibly testing lamb's quarter and calcium acetate which Dr said or talked about earlier, because not only does it have. Not only can it help the soil when under saline conditions but it also has been known to possibly help with that drought stress or that osmotic stress. So, these are my references, and thank you all take any questions. So lamb's quarter does have some natural resistance to glyphosate it's not like resistance it's just tolerant it's naturally a little bit tolerant to glyphosate so you can have a little bit of trouble if you want to just use roundup. There's a little bit of tolerance to liberty, which is glue fosnate but it still does pretty good on it, and that photosystem to resistance and that ALS resistance is not so much widely seen as North Dakota as it is in other states so right now it's still relatively low, but it's definitely something we want to watch and we can prevent that kind of stuff resistance from spreading just by mowing it down just like you would kosher to prevent seed production so it could be a nuisance but for now it's not and it's a better. A better idea looking forward than kosher potentially would be just because we don't have to worry about that quite as much.