Today we are going to be hearing from Dr. James White out of Rutgers University and Dr. White's fame in the ag sector has exploded over the past five years with his research on rhizophagy and a lot of the soil health nerds realizing the implication that this research has in determining the connection between the microbial health of the soil and the health and nutrition of plants and crops.

So I'll let Dr. White give the introduction and share his presentation. I'll probably have a few questions at the end, but thanks very much for joining us today.

Thank you, Jesse. I'm very happy to be here and to be able to talk about this, a very interesting soil and plant biology.

What I'm gonna cover is the highlights of basically answering the question, why is it that plants need soil microbiology? Why do we have to keep living microbial populations, living diverse microbial populations and soils? So that's what I'm gonna try to show, some of the processes that are going on and why those microbes are important to plants.

There are two big properties of microbes in plants that at least that we've talked about, this rhizophagy cycle or rhizophagy process. And this is a process that I'll go over in fair detail that shows how plants will absorb microbes from the soils and then extract nutrients from them. But also that those microbes once absorbed really will benefit plants in many ways. I mean, for example, they grow much better when they have the microbes and their growth is repressed when they don't have those microbes. They're also much more stress tolerant and resistant to all kinds of diseases and environmental stresses, abiotic stressors like excessive heat or salt in the soil. So these microbes are important for having healthy, well-nourished plants.

Okay, so I'm gonna go over, okay. So I'll contrast the biological and conventional agriculture. Biological I define as agriculture where microbes are involved and where microbes are considered conventional is more of a chemical approach to agriculture.

I'm gonna talk about endophytes. And endophytes are microbes that plants take in to their tissues and that generally these endophytes come from soils and that's where they get them from. And they just absorb them into the roots and then vector them through the tissues of the plant.

I may talk about seed, I'll talk about seed vectoring a little bit because some of these microbes are seed vectored and so they go on the seed. And so when the seed falls on the ground and germinates, the bacteria are already there that were working well with the mother plant.

I'll talk about a process called habitat adaptive symbiosis where plants will use microbes in particular environments in order to adapt themselves to the environment. They use microbes for stress. And so if the environment is highly stressful, they'll take microbes out of that environment and fortify their own stress tolerance.

Microbes in plants also are important in developing new cultivars of plants. And I probably won't talk about much of that, but that's a new area that we're working in. And exploring that area. And then I will mention how these microbes, soil microbes are important controllers of fungal diseases. And it's like part of the immune system of the plant.

Okay, so just to contrast the two types of agriculture, chemical agriculture and in chemical agriculture, we have chemical agriculture. We have chemical inputs, lots of chemical inputs, fertilizers, chemical fertilizers, fungicides, insecticides. It's basically the mainstay. It's how it works. It's how people thought plants work is you just put chemicals on them and they absorb them. That's how they believed or that they work. But actually they don't work that way. They work with microbes and yeah, they can absorb chemistry, but they use this microbiology in the soil. And so that actually makes the healthiest plant. And I call that using microbes, biological agriculture. And if the chemical agriculture is minimized, maybe not totally eliminated, but at least minimized to emphasize the biology component and the benefits that come. This biological agriculture using microbes. And when crops are produced with biology, they tend to be richer in nutrients and richer in antioxidants, human compounds that are helpful for humans. Yeah. Right? Also chemicals that the plants need, they need that. And these biological agriculture produced plants are stress-hardy. They're more resistant to diseases. They're more resistant to heat or any kind of stresses. All kinds of stresses, oxidative stress. And they're really highly oxidative stress resistant.

Okay, so another factor is this, these are just a couple of diagrams. One to the left is chemical agriculture where you try to put liquid chemical nutrients onto the plant and most of it actually is lost and it goes into the environment.

It's only really about a 30 to 40% efficient. So that means 60 to 70% is lost completely into the soil and for rain off and having non-target effects all over the farm, all over the environment. Whereas biological agriculture, it works differently. These microbes will enter into plant tissues and then the plant will use them as sources of nutrients. I included in this, not just bacteria, but fungi like mycorrhizae and any kind of fungal associated plants, fungal endophytes as well as bacterial endophytes.

But this biological agriculture is nearly 100% efficient. There's no loss into the environment. There's no degradation of the environment based on it. In fact, what happens instead is that the soils are built up, the more biological agriculture is used and the healthier soils become.

So when that plants absorb microbes from the environment, from the soils into their tissues, they become endophytes in the plants. Endo is for internal, phyte is for plant ,and it refers to a microbe of some sort, typically bacteria or fungi, or it could be algae. They get absorbed into the plant tissues and into the cells. And so to the left, you see a fungal endophyte and this is from a grass. You can see the height of the fungus. You see the cells in the background, that's the plant tissue. The endophyte is inside the plant tissue and does not cause any disease, as opposed to a pathogen. A pathogen would damage the plant but this is more of a beneficial symbiosist. Oftentimes there's positive impacts in development and growth and defense against herbivory and so forth, antioxidant, defense against oxidative stress.

Okay, so that's a fungal endophyte. This is what a bacterial endophyte looks like. This is actually near the root tip of a plant and we put a fungus, I mean a bacterium, not a fungus, it's a bacterium. We put a bacterium into this plant that makes little clusters. If you see my black arrow to the left, you can see over there, those little dots and there those little blue dots. Those bacteria that we put in, we stuck them in there. We put them on the seed, the root came out and it absorbed the bacteria into the cells. So you can actually see them. This is one called micrococcus and you can see the little clusters of spherical cells there.

That's what the bacterial endophytes look like once they get into the plant. Where do these microbes come from? They all come from the soil actually. They're all soil absorbed. We can sterilize seedlings and you grow them in a rich soil environment and they can get their microbes back. Take the microbes off the seeds, we can put them in. So plants can actually, if we have very healthy soils, if you develop soil health, grow plants in it, they can get the microbes that they need, but it really the key is building that soil for a healthy soil environment. So these are soil microbes and they take it into the plant and the plant puts the best ones on the seeds and then the seeds when they are vectored, they're spread from the plant. They could fall in an area where there's no microbes around and they already have a set of microbes that help them develop and protect them from disease. So that seed transmission, seed vectoring is also important.

And I should say the ones with microbes that tend to be more bacteria on seeds, those are the ones that grow better and those are the ones that are defended from disease. The other is if you remove the microbes, especially if you don't have it in the soil and you don't have it in the seed, then what happens is damping off pathogens will come up and kill the plant. So it really is, I mean, these microbes are really important for plant growth and development and protection, protection from disease, part of the immune system. This is actually a grass seed germinating. You can see the root coming out here and you see a bit of yellow where the blue arrows are, that's where bacteria are coming out. So the seed carries on it bacteria on the surface and they come off of the seed and they'll colonize this root out here. But there, we think of a seed, what is a seed? And a seed is a plant, a baby plant, in a little case, right? But with its food, with some food in there. But actually we have to add the microbiome, the microbiome and that microbiome in that seed is important for the development and protection of that seed during its early, early seedling growth, no matter where it lands.

I guess that's another argument for saving seed if you're growing it in healthy soil.

Absolutely right. You grow it in healthy soil, you get good fruits coming from that, you save your seed and especially it's gonna build up on in the plant, the more you can plant that same seed in the environment, in the same soils, right?

So I mean, that was a grass seed. Here's another, this is tomato. We think of inside a fruit, it's sterile, but in fact, that's far from true. Around that, those ovules or those seeds in there, there were lots of microbes and there's microbes on the seed, tomato seed. And if you look inside the seed, there also are biofilms of bacteria that one can visualize in there. So these plants are vectoring these microbes in and on seeds. It's natural, it's how they do it.

So one of the first processes that we started studying is this process called, that's come to be called the rhizophagy cycle.

And this rhizophage process was actually discovered by some Australians. We didn't discover it here. It was their idea. We just studied it after they did. But this is actually from their article, from their paper. And you can see what it shows is what they did is they took bacteria, they took an E. coli and they took a yeast and they labeled it with green fluorescent protein, right? So they could look at it with a fluorescent microscope and then they put it on the seedlings and the plants took them in and then they studied them and they saw that they were internalized and they saw that they were degraded.

These are a couple of the investigators from that study. They're from Queensland, Australia.

This is figures from their paper. And I don't, it may be hard to see but there are little green dots in this long thing. That long thing is a root here. Little green dots are yeasts that are fluorescing from this green fluorescent protein tag that they put on it. So it shows, these images show that these plants absorb these microbes back both E. coli and yeast. And they call that rhizophage. Rhizo, so it was their concept, you see. Rhizophor root, phagee for eating. Okay, so that was the rhizophagee, rhizophagee. And it, oops.

So what we, one of the things that we found is that it's not just bacteria and yeasts. And you can show that here, bacteria here, yeast. You see them inside cells. You see them inside the root hairs. These are yeasts inside the root hairs, believe it. Believe it or not, those little tiny little dots those are the yeast and they're coming out there. And you can see here the bacteria and you can see them in here. This is this brown staining are these reactive oxygen stains that we use to visualize these bacteria. You can see it because they're interacting with the, the plant is interacting with the microbes using reactive oxygen. And that means it's high reactive oxygen around where the microbes are. So using these stains that appear when there's reactive oxygen there, we're able to see the bacteria. And so you can see them there with that brown around them. But we also showed that algae like chlorella can also be absorbed into the roots. And you can see here, root hair and see this little kind of bright structure. That's an alga. And what's more interesting to me at least, you look around it, you see little dots. Those actually are bacteria that are jumping out of that alga going into the plant. So they're actually, these algae carry their own bacterial endophytes. So when a plant gets the alga, it gets the bacterial endophytes that are in that alga. So it's kind of a very interesting sort of a predatory process.

Are algae single cell as well, pardon the ignorance?

There are single celled algae. The ones that chlorella is maybe have four cells in it. But they break apart when they divide and then you get single cells. And we think that they're probably being absorbed when they're the smaller single cell structures. That is significant because they're pretty large. And you can see the hair there and you can see how big it is. But it's not like these yeasts that go down to little dots of tiny little structures. These microbes inside, by the way, don't have their cell walls on them. So they can become very, very tiny without, they don't have the need to have this big wall. They replicate very fast inside. And anyways, we'll show that.

Okay, so we also showed that there's a cyclic pattern to some of the way that these microbes, at least the ones that survive inside the root, there's a cyclic pattern to it. And that is that there's a period of time that, well, I'll say that microbes living in the soil, they live in the soil, they're attracted to the root tip through exudates. Exudates are sugars and organic acids and maybe some other nutrients that the plant secretes at the root tip secretes it out. Those are attractants for the microbes. So these microbes out here, soils are attracted. They're grown at the root tip, they're absorbed into the cells at the surface of the root tip. And then it's in that area that the plant will hit them with reactive oxygen, superoxide actually, a very potent form of reactive oxygen. And that will oxidize off the walls, the cell walls off the microbes. And also because when they came in from the soil, there was nutrients hanging on those cell walls. And so when they were taken in and then oxidized those nutrients, metals mostly, or also some cases proteins in the cell walls of the bacteria, they're released into the plant. Okay, so we think that's one way that plants get nutrients through oxidizing those microbes.

And some microbes will be fully degraded, but some will survive. And the survivors will be replicated in those root cells and they'll trigger root hairs to grow. Without those bacteria there will have no root hairs.

So the bacteria are critical for that root hair elongation. And then the bacteria accumulate in those hairs and they're ejected as those hairs grow, actually happens in spurts, grow spurts and every growth spurt, there's an injection of bacteria into the soil. And then once they're ejected, they reform their cell walls and they go back and do what bacteria do in the soil, swimming around and going to other organisms and degrading stuff and eventually to be attracted back to the plant where the plant can absorb them. So we turn that the rhizophage cycle. So there's an alteration or alternation in which the microbes spend part of their time in the soil acquiring nutrients. And then part of the time in the plant where the plant extracts those nutrients from them. And it's just, and that happens, it's really interesting. Happens involves the root hairs and the exudates and everything. But so that's just a diagram of that. You can see the microbe have gone in, attracted. Here's another one coming in and it goes there then it's hit with reactive oxygen and those were nutrients coming out. And eventually it ends up, they form in these, they go into the root hairs. And then as they're in the root hairs, the plant will actually replicate them. And I'll show you that. So it increases them and it's pretty cool process. So this is, so where this rhizophage cycle happens is it's only on root tips. So this is a diagram or drawing of a complex root for corn. And these root branches, these little branches form all over these, these little. And so all these little branches, all these little lines sticking out, those are root tips. That's where rhizophage is happening. So plants that have a lot of branching roots, grasses and other plants that have those branching roots are doing a lot of rhizophage cycle. They're cultivating microbes and are extracting nutrients all over those root tips.

And this just, yeah, Jesse. Sorry.

No, the plants themselves are actually multiplying the microbes. That's something I never realized.

They're multiplying the microbes inside of them. And I think I'm gonna show some pictures of that. So you kind of still see that. But this, what we're seeing now is this is a root, branch root, you can see the big root there. She's just coming out. And you can see now it's stained for the bacteria. It's stained using aniline blue. So we can see all these little blue haze out here. That's all the bacteria you can see. And they're actually taken in right here. This is the meristem. They're taken into the cells right at the zone. And why can they be taken in? Because that's where the cells are dividing and the cell walls are soft there. And so the plant can either pull them through or absorb them into it. There's still a debate exactly how they're getting in, how they're getting into the cells there. But there are a few hypotheses which I won't get into. But they're getting in because we can see them going in there. We know they're going in there because they end up in there. And this actually shows, okay, so this is a,

this shows the bacteria, the dots, these little, this is where they first go in at the root tip. This is stained for the cell wall, staining the cell wall using crystal violet, which is a common microbiological stain that stains these bacterial cell walls tends to stain them. Especially if they have lots of protein in them, it stains them in a dark color. And you can see the dark there. But in this area, it's not as, they're not round. What's happened in this zone is they've lost their cell walls because this is the area where the plant

puts the superoxide on them, starts. So they go in, they seem to be losing their shape a little bit here, but where they really fall apart is right here behind this. This is in the elongation zone of the root, just behind that root tip.

So it's, they're extracting the walls off of them. So you can kind of see that a close up here. You can see the dark, and then you can see purple here every now and then, a little bit of purple. Whoops, little purple spherical things. It's hard to see it right here, but right there, there's one, I see one in. So those are the little light purple things are the protoplasts, because when they lose their cell wall, they form these protoplasts.

Okay.

And it, yeah, let's see. Okay, this shows it too. This shows a different kind of an image though. This shows, you know, on the surface of the root, you can see a, you can actually see a parenchyma cell, and then you see these brown things. Those are the little protoplasts of the bacteria inside. And you can see also, it's in a chain. See that lower area, you can see big one, then a one in the middle, and then a tiny one. They're in a chain, and this is stained for protein. The little tiny one is very dark blue. The old, the bigger one is older and it's light blue. What happens is, the plant is putting this superoxide into the bacterial cell, and that's degrading the protein contents. And so these cells are swelling as there is. So they're dying. So the microbes are having to replicate very rapidly in there to stay alive. It's kind of an interesting balance there. Wow.

Okay, this just shows, this is where one of my graduate students labeled the bacteria with this, you know, we had to do this, we have to do this tracking thing too. It's a basic technique where you tag with the green fluorescent protein or a different kind of fluorescent protein, and then you track it, right? So my graduate students so last year did it, and you can see them in, actually here, the orange, she used something called mCherry, and these are the root cap cells. And the blue is the cell wall of the root cap cell. That's the cell that peels off of the wall as the root grows. And the orange there is the bacterium labeled with this mCherry, and you can see that it just scans through that. So you kind of see it inside the cell, and that kind of illustrates this intracellular invasion that you see. And nothing happens to those cells, the cells are fine. They're essentially going, this is a diagram of one of these cells, and you can see the little where I labeled bacteria. They go through that wall, and again, right there at the meristem, right there just behind the root tip, the wall is plastic, it's very soft. When it, because it's growing, right? When it gets older, it's very hard. The other place where the cell wall is soft is at the root hair tip, the root hair tip, because that's a growing structure. And so the cell walls of the plant are very, very soft in that area. So they go in at the root tip, and they go out at the root hair tip.

But those are the only two places where the wall is not developed.

Okay, so this reactive oxygen actually comes from this, comes from oxygen. And the enzyme, there's an enzyme in the plant, in the plant root, it's called a NOx enzyme. But what it does is it'll take oxygen out of the air, and it'll convert it into superoxide. And that's the oxygen that the plant, that the plant will use to oxidize nutrients out of these bacteria.

That just shows that. But another point here is that this oxygen has to come from the air spaces in the soil. So you have to have soil that's aerated, that's pretty good air oxygenation, and good air spaces and so forth, in order for rhizophage to happen. If the soil is flooded, and the plant can't get oxygen, it can't extract nutrients from these microbes. But it also, I mean, if you do that, plants also flooded soils, they're also much more vulnerable to diseases because this superoxide that the plant produces is also an important defense compound for the plant. And so, you have lots of organisms like pythium or phytophthora that can then get a hold, and any other pathogen really that can grow in those low oxygen conditions. And the plant doesn't have defense against it. So it's a, it's very, that's the aeration in the soil, it's important. And the compound superoxide is an important oxygenate and defense molecule for the plant. And what happens is, you can see the bacterium to the left, you can see how these bacteria are, they're rods, right? They have a cell wall, and then you see, after the superoxide, you see they lose their walls and they turn into these little blobs, these little spherical things, and they don't have a wall and they replicate very fast. So it's in this phase inside the plant that the plant can replicate these microbes a lot. So really a few microbes can go in a plant cell and many will go out because the plant is continually replicating these microbes. So in a sense, they're populating their environment with these microbes that work optimally with them. So they're engineering that environment, they're engineering, you know, they're favoring certain microbes and certain microbes that they're disfavoring, you know, the ones that can survive the process and help the plant survive, then those are the ones that they favor.

Yeah, so, but the further, it's very important that plants get microbes because plants use microbes in their development and structures like root hairs that just don't grow without microbes. And if you look, you look, if you consider, I mean, for example, we'll just look at that. Okay, this is a seedling root, seedling root, where we took the seed and we sterilized the seed to remove all the microbes. And what you see here is the root, but you see, there's no root hairs on it. And there's little bumps over here where my arrow is, but those are root hair initials, but they don't elongate unless there's bacteria in there. The bacteria are critical for this kind of growth. And these hair growth, also root structure, you know, more branching in roots, root architecture increases, plants produce more exudates and attract more microbes. If you remove the microbes, plants really, roots don't grow very well. They're oxidative Lee, not resistant, they're susceptible to all kinds of stresses, all kinds of pathogens. It's a night and day. If you really try to grow plants without microbiology, you really can't, you really can't. But to the left here, this is actually the grass that I showed before, the little seedling of a, this is a Bermuda grass where we removed all the microbes, like the one where I showed just before, but we put a microbe back on. And when you see, we put it back on, you see the root hairs immediately. See those hairs are there. And here's a little bit older tissue, you can see the brown dots in there. Those are all the bacteria in the hairs. And they're actually important for the growth of the root hairs. They won't grow. Developmentally, plants are not plants, unless they have those microbes there. They don't behave like plants. This is part of their behavior.

Plants use these microbes to trigger proper development. So they essentially, they become part of the developmental program of the plant. And all plants do this, they all need this. Yes, some will have other kinds of mycorrhizae that come along, but early on, they need these soil bacteria too, just like the other mycorrhizae, just like other plants without mycorrhizae. They all need these soil bacteria and soil yeast and soil fungi and the fights that go into their tissues and modulate their development. This is one of these hairs, close up.

And here, this is, you can see all these little brown dots. Those are all the bacterial protoplasts that are inside there, inside that.

And we look at another plant here. Those are, okay, here is tomato. And we took tomato and we use streptomycin to remove the bacteria. And you can see here, the root is steady there. And you see there's no hairs, same age where we did not use an antibiotic. And you see the hairs forming there.

It's critical for development, really. This shows where on tomato, actually where there's a root hair initial and this is without bacteria in it. And you see the initial didn't elongate. It's the throat and you don't see anything there, but to the left, you see now this hair with bacteria. You see the brown dots in there and you see that those hairs elongate. And when they do, the bacteria get pushed out here at the tip.

And you see here's even older and you can see the bacteria out here in the tip. Okay, but as it turns out, actually, this is a dynamic process. And I'll show you, these plants are actually processing these microbes. And here's an example. This is a sedge. This grows in coral reef formations. And this was in the Dutch Antilles.

Sometimes they get to go in the field and do work. And sometimes it's really cool places they get to go. And this is really cool place. But these things, these sedges grow in rocks and they don't have soil, they have, but they have lots of microbes and they get nutrients out of the rocks or whatever washes in. But they definitely use these microbes. And this is, if you look below, this is one of the root hairs of the sedge. And this is stained for reactive oxygen again. And you can see these bacteria where they are around the edges of the hair. Now, this is, top picture is actually a living view, a movie that shows actually what happens in these hairs. These hairs are active. They're living things. And this is not pulled out of the soil. If you pull a root out of the soil, you won't see any movement. This is called cyclosis or cytoplasmic streaming, but it's processing. It's where these root hairs are processing these microbes, these protoplasts and breaking them up. And you can see their shadows that move around. Those are the bacteria there that were there. They're going around and around. The plant is breaking them up as they divide, spreading them around and getting more and more of those by moving them like that.

Here, you can see that. This is a hair. You can see the clusters there. These probably came, the whole cluster of bacteria, protoplasts, they're all protoplasts, came from probably a single one. So you can see the plant is replicating these microbes. And it's a really, really a cool process. But here you can see that root hair again. You can see the bacteria going to the tip and you see them buckling around in the tip, the little white ghosts there. Those are the bacteria. Every now and then one will be caught and they'll be pulled back. But in the tip here is where they produce hormones. And that causes a growth spurt, a growth spurt to happen.

And actually here you can see one of these root hairs. You can see how fat it is. That fatness is because it's full of bacteria. They're full of these microbes. The actual cytoplasm in that root hair is in the middle of all those bacteria. So these hairs become fat with bacteria, full. And you can see them coming out here, right there.

They're coming out the tip. That's what they do. They eject them out the tip. This just shows diagram that shows that. You can see how that kind of happens. You see microbes accumulated at the tip and then a growth spurt happens in the hair and there's little pores there and they come out of the pores because they're protoplasts. They just squeeze out a little tiny holes. They don't have to have big holes to go out. They don't have to tear it up. They just whip out there as soon as that growth happens. And then the ones that are left in that are on the side, I painted them blue there. They start to replicate again. The plant moves them around and replicates them. And then they accumulate again little by little at the tip. And then in a bulk, right in mass, they produce these hormones. The hormones we think are ethylene and nitric oxide. Those are two substances that we can detect that have to do with growth.

So we think that's it, but there are also other factors that might be at play here. And some of these, usually growth like this has to do with polyploid cells becoming locally polyploid. So we think there could be some polyploid formation, local polyploid formation called endopolyploidy happening in these hairs and in other cells to get these bacteria in them because they get fatter and bigger. So I mean, this could be just part of them. It's interesting.

We think there's a basic exchange of nutrients in the process. I'm not gonna go into the details there, but there could be some nitrogen fixation that comes out of these microbes in an interaction. Also the production of ethylene, which is works as a growth hormone. And anyways, there's an exchange, right? That's the point of that.

And I want to show the actual ejection there. And you can see the, this is tomato and you see the arrows down there, especially that one labeled three right over there. You can see there's a little, three little pores there. It looks like the bacteria squirt, they're almost like ejected, being ejected out of that pore, out of those pores at the tip and they accumulate there. And what would happen now, once they're ejected, the plant actually puts a little bit of exudate there, little nutrient sugars and other nutrients and allows those microbes to reform their cell walls. And if they have flagella, they reform their flagella and then they can go back out and survive in the soil and get some more nutrients like that. So it's very much a farming process where plants are farming these microbes. At least that's what it looks like. That's what it looks like to me. And yeah, nutrient absorption function. And I don't want to go into too much detail here, so I'm not, I think we're, Jesse, how many questions do we want to do?

 Three or four, keep going.

Okay. So it's not gonna make sense. So we know that plants, we know that plants get nutrients as chemicals in the soil. That's why our fertilizers work. I mean, that's well-known. And the chemistry of it, that's pretty well-known. But one source is, you know, solubilized nutrients in water. Plants do that. That's a natural way that plants can absorb nutrients. But all plants in nature that are not, where humans are not attempting to manage every little aspect of plant growth, you know, using our chemistry and other factors, they use microorganisms. And we know they use mycorrhizae, and we know that mycorrhizae associate with trees, have a big effect. We know some plants will create rhizobia, and they'll fix nitrogen with rhizobia. And there's rhizophageocycle. And what do they get in rhizophageocycle? Well, what we, evidence is so far, they certainly get metals and nutrients that may be difficult to get in the soil by themselves, but these microbes can carry them in. And things like manganese and iron and calcium, magnesium and so forth, zinc, and nitrogen. And nitrogen because the cell walls contain nitrogen.

So some nitrogen comes from rhizophageocy. Okay,

so let me see, I wanna, okay, these are some other experiments where I'm not gonna go into the details. We've talked about it, that hormones may be responsible for some of the, the developmental effects of microbes on plants. Microbe produced hormones and signal molecules, even hydrogen peroxide, which is another form of reactive oxygen, is a signal molecule. So all of this stuff could be turning genes on, so forth in the plant. And we have some evidence of specific, some clues about specifically what is happening. But those are all, that's all developing science. And so it's not as certain yet. So I'm not gonna go into too much of that. But there is an interesting effect of endophytes on plants. And this is that endophytes, when they're present in plants, enable plants to develop some resistance to stresses, stress resistance.

And there has been a hypothesis put forward a few years ago by Rusty Rodriguez and Regina Redman, which they turned into a very interesting topic. Which they termed the Habitat Adaptive Symbiosis Hypothesis. And this is the idea that plants, when they enter an environment that is stressful, say for example, it has high salt in it, they'll attract microbes to them from that environment that give them tolerance and capacity to thrive or survive, survive and thrive ideally in that environment. And that's this idea. And they supported that experimentally. And we see the same thing. And we see exactly the same thing.

So plants can adapt, we could call that epigenetic adaptation, right? To the environment using the microbes, using microbiology. And this kind of plant, selection of microbes is really gonna be natural if you consider which plants, which microbes are plants gonna get? They're gonna get the ones that enable the plant to survive, right? And the plant can bury what microbes they take in by what nutrients they put out in their exudates. So they can kind of fish for specific types of microbes.

But there's further, there's the possibility of genetic change in these plants and with microbes. And that's kind of a new area that we're looking at, especially in corn, we're seeing that where we grow plants with microbes, ignore microbiology, those plants show a lot more genetic diversity. They start to change. We see more mutations, more transposon movement and so forth.

And we think the reason for that is very interesting because the microbes at some point, at least some of the microbes, affiliate or associate very closely with the nuclei of the plant. And you can see, this is a potato cell really here. And the nucleus is right in the middle and you can see these little dots around it. Those are bacteria. Those are bacteria.

And here's a hosta, right? The flower, this is a developing, this is from a developing meristematic or young leaf. And you can see the hosta cell there and you see the nucleus in the middle and you see these bacteria around it. The bacteria are actually coming out of the nucleus, but they're associating with it. That's the point. They're associating with it. So, meaning the nucleus behavior could be influenced by that.

And I mean, they're actually, we know that certain bacteria will, when they affiliate with the nucleus, when they will affiliate, they will go into plants, they'll affiliate with the nuclei and they'll pause this polyploidy to happen. And they cause endopolyploidy. That is those cells, then the nucleus, the DNA divides, but it doesn't separate, right? It'll replicate, but not separate. So those cells become more and more polyploid, more and more nuclei in them, or more and more bigger nuclei, because more chromosomes in them and they get fat. And it so happens that those polyploids are highly stress resistant. So this changes the whole biology of the plant. And so, I mean, this doesn't, obviously doesn't change the DNA and the pollen ovules, but those, the pollen and ovules also carry bacteria. So genetic changes in those structures of the plant, those sexual structures could increase the variation in the plant and the gene, cause genes to be more varied. It's interesting thing that we're thinking about and trying to evaluate at the present time. But I mean, what I'm trying to show here is the with biologically grown crops, they tend to be more variable because of epigenetic changes and because of possible genetic changes that may be happening. So you have more variation, more like a wild population compared to chemically grown crops, chemical fertilizing, which tend to be more uniform. And that's an actual problem for crop production because for our machinery, it's easier to have plants that are all identical. Yeah, but this is one of the features of biological versus chemical agriculture.

Okay, nitrogen fixation. Okay, I'm not really gonna talk about this, but nitrogen fixation, we have good evidence that nitrogen is fixed in plants.

Okay, inside of, you know. Inside a plant, inside of the plants, there are places that nitrogen appears to be formed inside the plant, tiny bits, tiny bits here and there. And there's lots of other investigators that are doing this now to,

we're not at the point of saying yet that we have the clear mechanism, how nitrogen is happening, because it appears to be happening all over plants and not just in one place.

But I'm not gonna go into that.

But one place where we see it is in actually trichomes on plants, in plant hairs. And this is from cannabis, this is what you're seeing here. And if we look at one of these heads on this cannabis trichome, right, this glandular trichomes, this is where all the, a lot of the terpenoids and stuff are located. But you look at that and you can see, now this stained for the bacteria, and you can see these little, those are bacteria in those trichomes. These trichomes, these heads are actually, they're microbial processing structures. And so as they age, and this is stained for superoxide, so the bacteria have superoxide all around them, and that strips the walls off, makes them protoplasts. And then you can see, now you see the little dots in here. Those are the bacterial protoplasts when the trichome head is a little bit older. And you could see the cells of the trichome in the middle there, and you can see the blue. The blue is actually the interface, that's superoxide now, the interface of the plant and the microbes. So these plant hairs, they're like making popcorn out of these microbes, removing the cell walls and essentially percolating them inside these heads of these trichomes. So we think that's a way that they're fixing getting nitrogen inside it. And we, you know, I mean, anyways, that's a model of it. We think some of the chemicals are to control oxygen around. So some of the cannabinoids and other substances and phenylolics that are inside these hairs, and, you know, there's a lot of oxidative interaction. So we think that a lot of those are to control that oxygen in order to manage those microbes, even through nitroficcation happening, or simply some other processes that are happening in those hairs, because those hairs also become polyploid, they're endopolyploid as well. So, I mean, that could just be part of, you know, this changes that are happening inside those hairs, but hops similar, and you see the bacteria in there. This is for nitrogen in these land-intertrichomes. You can see the purple there, that's the nitrogen that's being stained.

And finally, I want to say, which is not too much time left, but finally, the microbes on plants are really an immune system, an important part of the immune system of that plant. And to illustrate that,

I'll talk about an experiment where we used basil seeds and we sterilized the seeds to remove the bacteria into fights. And then we put a pathogen on the plant to see what happened, okay? To show what happens, okay? So, to the left here, I mean, to the left here, you have, it's grown on agarose, you see the little green, those are basil seedlings, they germinated, they look fine. Okay, to the right top, you see basil seedlings with their microbes, with their microbes, and then we added fusarium, fusariums of pathogen. Fusarium is damping off pathogen, it'll eat these seedlings up, but you see, it did not affect them. With the microbes there, if you look to the bottom, what we did is we took the seeds, we removed the microbes using surface sterilization and we germinated them. See, without seedlings without microbes, we germinate them, they're fine, they're smaller than the ones, they're not as healthy, you can see that, obviously, it's healthy, they're not even as dark green, but they're still okay, they're still okay. But then you add the fusarium, the pathogen, the fungal pathogen, and now you see they don't grow at all. You see some white there, that's the fusarium, fusarium eats those. In other words, without microbes there, there's no resistance to this pathogen. That is very important for part of the immune system and one of the processes here is that with the bacteria there on the fusarium, fusarium is not pathogenic, so its virulence goes down, so the behavior of the path changes, it's no longer, it tends to remain as a saprophite in the soil, okay, but when the bacteria are gone, the fusarium attacks the plant and kills the plant and we don't understand everything that's happening there but it's an interesting phenomenon. The microbes are key to the immune system of the plant. They also make the plant partially regulate up their own defenses and all these antioxidants and other compounds have to do with that and I'm not gonna go into, this just shows fungi with bacteria on them and that has an effect on the fungus, it changes the behavior. Here we can see a fungus and you see bacteria, this is actually a zygomycida common soil fungus, right? You can see the hypha, you can see this stuff coming out. Those are bacteria inside and you can see those little dots there, those are bacteria inside. The behavior of these organisms, these fungi, potential pathogens changes, even mushrooms carry these bacteria inside them and it affects their behavior. Here's the, this is the rishi mushroom, right? Common for health purposes and people use it but here you can see the hypha of the fungus there and you see the little dots in there, those are bacteria. Bacteria coming out, bacteria in the hyphae, they're all in here. The normal behavior of these fungi, of plants and fungi depends on soil microbiology and so healthy soils, you're gonna see changes in the pathogen community but you'll see changes in the plant community, changes overall system health really and I think that's pretty much all that I was gonna cover

I would say that Jeff Loebfels wrote a book, he's a popular, he's actually a garden writer, friend of mine and a garden writer and he wrote a book called "Teaming with Bacteria", the organic gardener's guide to endophytic bacteria in the rhizophage cycle, 25 bucks but he's an excellent writer and he simplifies the whole process, talks about endophytes and he talks about rhizophagy.

it's pretty much the same presentation sort of whenever you give it or do you add to the track depending on the--

It changes every time, it changes every time a little bit but the details of the rhizophage cycle, that's pretty much standard but the most recent stuff that we're looking at are the epigenetic and the genetic alteration, that's the most exciting thing, to think that plants rely on microbes to enhance genetic variability, that is wild, you think about what does that mean about our crop plants, we're destabilizing them, their ability to change and our ability to select new stress tolerance because we're not thinking about the, or new properties of the crop because we're not thinking about the microbiome too so we can have major ramifications I think for how we think about plant breeding, you have to breed the microbes too, we have to bring the microbes along so that seems exciting to me and philosophically just the idea that the microbes that we thought just remained in the soil actually affected genetics of the plant, that's wild.

I met you at the Acres Healthy Soil Summit in Sacramento in 2022 I think.

Yeah, I think I remember you.

I think I actually witnessed your first physical meeting with John Kempf.

Probably I think you did.

It was cool to see, I mean, John obviously, he's a walking encyclopedia with just the excitement to be able to begin to nderstand that level of connectivity between plants and microbes and the implications that that has for our ability to make kind of informed decisions about how we grow our food which is kind of really, that's who we interact with, right?

As growers who are trying to go more down the soil health wavelength, get more biology involved in their operation. And kind of one question I often ponder is to what extent can you make granular informed decisions, for instance, looking at the specific bacterial strains, what they do. Can you test the soil, see if they're there or not? Do you want to try to add them in if they're absent? Or is it better to try to just elevate the overall microbiology profile in your soils and then hope that nature takes care of the rest?

That's a really good question, Jesse. You know, my belief is that the best situation is where you create a very healthy, you know, living soil, right? With using cover crops to keep microbes alive all year round, you know, create a good environment.

And in terms of adding things to it, they would be short term, you know, maybe compost washes or ferments, you know, things like that, that would tend to give it the plants that you plant a bump, but the healthy soil is critical to begin with.

And, you know, if humans, you know, there's a lot of microbes, a lot of gamma-proteobacteria and beta-proteobacteria, mostly gamma-proteobacteria that plants can use for these processes and that they absorb. And those are probably the most abundant in the soils also.

In terms of picking one and adding it or picking a consortium, yeah, you can do that. But all of these are, you know, these are manmade mixes and they're not, there's a limited amount of work that people can do into trying to optimize, right, a mix. And you can't, it's hard to optimize for every soil. It's hard to optimize for, you know, so you can get something that works pretty good, but the best environment is gonna be where plants can fish out what they need. They can select what they need in that environment and get those microbes. So, you know, my feeling is that really, now this is, we know there's a limited amount we know and there's a lot of people working on it and trying to figure out what is a healthy plan and what is the microbial mix. And, you know, I can't say that we've done, this is just kind of my philosophy on it, right? My philosophy is that we can't approximate and we can't know precisely what that plant needs. And strains of microbes are different. So it's not like you can pick out one species and how do you know which strain it is, whether that's beneficial. It's really, I'm gonna hate to say it, but that there's so much uncertainty there and there's so much complexity in the soil environment in which microbes plants can use that I just don't think we can predict. I don't think we have enough understanding of it.

I tend to actually have a more of a look at the process that's going on as opposed to what microbes are specifically or to understand, for example, okay, what the question is really important to me would be like, why do plants use these microbes and not these microbes? Right, that kind of question or interesting. The taxonomic questions, you know, they can vary by strain and you could have the same species with totally different properties really. So that would be hard to, I think that's right. And the reality is that plants use communities of microbes. They don't use just one microbe. They take in a bunch of different microbes that help them and they use those for different things in the plant.

And so the plants are back there back to their farming soil microbiology. So it's our advantage to make that microbiology as healthful and diverse as possible for the plant to get. That would be my strategy for agriculture, biological agriculture. It's basically regenerative agriculture.

Yeah, I'm not looking for an endorsement necessarily of our approach, you know, as a company, obviously most of our farmers are, they're sourcing from a compost, worm castings, Johnson-Su compost, you know, and then making their extracts and putting them in the soil. But we do often, the question comes up, what's the difference between doing that versus, you know, like you're bug in a jug, biologicals.

And if, just let me know if you think I'm on the right track here, I think there's two differences. One is the diversity that you mentioned that you get from the worm castings versus trying to isolate different strains. And that that's actually beneficial, which you already kind of addressed. And the other one is just, when you have worm castings, you know, they've been produced almost in a soil like condition through a firm of composting, and they've gone through the worm. And our feeling is there, the microbes in those castings are hardier and more able to adapt to the soil than something that's maybe propagated in a lab. Do you think that's fair?

I would say very similar to what you said, and that is that they come through the worm, so they're really worm gut microbes. And a lot of these, you know, a lot of, well, I mean, they are being processed by the worm. They're in higher concentrations, being processed through the gut of the worm, right? They're in higher concentrations.

They're some of the same microbes that we actually see in plants, in soils and around plants, in rhizobacteria and different, you know, bacillus and so forth, other common bacteria that plants use. So we think that it's a natural sharing of microbes between the worms and worm compost and the plant.

You're right, they're adapted to using, they're being used by eukaryotes, right? So what does that mean? You know, eukaryotes use the same kind of a reactive oxygen, you know, innate defense system. And so they, they're the worm, I'm not an expert on worm biology, but they may be controlling microbes in a similar way, going through their bodies using reactive oxygen. So these microbes could be primed, you know, and in higher concentrations like that. It's well known that worm castings are rich source of plant promotional microbiology.

You know, I think that, and the for the Johnson-Su bioreactor, you got a diverse population of, probably when it's mature, when it's fully mature on the plant, probably bacillus and different species of bacillus and endosporiformers and microbes that are really resistant, you know, because it's an apex and the, it's an ultimate digestion phase where you get fewer swimmers and microbes in there. And you come down to, you know, a more concentrated set of microbes that can survive. And then those are the ones that you put on the seed and that play the role. And again, they're, you know, they're the ones that survive. They have the endosporous and they're gonna be a little bit tougher. And so, you know, in a sense, you may be putting more endophytes on the plant, more plant adapted ones that can form endosporous and wait around for the plant to come. And once the plant takes it in, form endosporous in the plant again, as that matures, you know, so it, yeah, so yeah, I think you're concentrating microbes in those systems that are adapted or that help the plant adapt and grow.

So you can make a compost tea where you're growing out the population of microbes before you put it in the soil or an extract where you're just kind of getting whatever's in the compost and putting that in the soil and, you know, adding foods for it to grow in the soil.

Given the life cycle of a plant, you know, especially an annual plant, do you think it makes a big difference if you're going to be adding, say you're dealing with soil that doesn't have a lot of microbes in and you want to add microbes into it. Do you think it makes a big difference to add kind of that big boost from tea right away? Or once you've got a smaller population established in the soil with the plant, will that naturally kind of grow out the way the plant wants it to?

It will, but when you put those microbes on, especially if they're optimized, you know, from the ferment or compost tea or something, they come from, you know, a rich source of microbiology and you've got microbes that are adapted to the plant. Then you should see an immediate jump and, you know, you're helping those plants get the microbes. There's nothing wrong with that.

Waiting, I mean, what you do, one thing you do is the more of those bacteria are there, the fewer diseases you're going to have. So right away by putting those microbes on the plant, you're going to see a protection of the plant and the plant is going to have more of those endophytic microbes that it can use around the roots and grow a little bit better and be a little bit stronger, develop a little bit better, you know, like that.

So yeah, I don't, and a lot of our soils are diminished. We've pulled through their structure, you know, they don't have enough, essentially habitats for microbes to grow, too sandy or, you know, something too something, too acid or something along those lines, you know, so microbes are suppressed. So adding microbiology that the plant can get right away could be a big benefit.

If you were going to try to optimize rhizophage in your soil, you kind of touched on this already, but your soil conditions, you know, is it better to have soil that's more moist soil that drains well?

I mean, I imagine that as the microbes build the aggregate structure around the plants, that is the optimal conditions, but kind of...

Drains well.

So you don't want a lot of water around it, but you probably also don't want it to dry out.

No, you kill the microbes. You kill the microbiology that dries out. That's what we do and it's a terrible practice. We turn our soils and we leave them to dry out over the winter, exposed to the sunlight and the dry air. And the microbes die then. So we're decimating our microbial population in the soil like that. Yeah, the way to do it is, you know, some other method to keep that soil moist and, you know, some kind of plants in it growing. If not, at least keep it moist so that the microbiology can stay alive. The ones that are able to grow on organic material, you know, so you need organic material in it for the microbes to live on.

Yeah, but better, much better to have air space. If it's flooded, the whole process shuts down. And it's worse, it's worse than that because the plant produces ethylene, the microbes are producing ethylene in the roots and around the roots. And the plant produces ethylene. And the plant actually, at least in our experience, uses some superoxide to remove some of the ethylene and essentially detoxify in the plant by removing that ethylene.

And yeah, it's a growth hormone, but it's also a stress hormone. So if the soils are flooded, not only can plants not get nutrients from the microbes that it takes in and rhizophagy, but ethylene builds up because that superoxide is normally what will react with the ethylene, it'll oxidize the ethylene. And then the stress goes down, but all that depends on having oxygen there. So the system doesn't work without having aeration and air spaces in the soil.

If you pivoted completely away from any kind of chemical inputs, I know John Kemp and others have opinions on this and just added biology to your soil or just fostered the biology in the soil. Would the plant be able to get everything it needed from just what's available in the soil or do you think there are still micro doses of chemicals that you'd need to, maybe your molybdenum and stuff like that?

 Well, you have to recognize that we're pushing plants for maximum yield, right? And that doesn't happen in nature. So we're really expecting a lot out of a plant. We're trying to turn a plant into a nutrient factory and the process we're actually reducing the nutrient quality in plants because we're removing the microbiology that they need to get the more optimal nutrients and to be healthier. Okay, that's true, we're pushing plants. And so in that quest to feed humanity, to feed as many people as possible, we've developed this chemical approach. And I personally think that we're never gonna totally eliminate chemistry, that we may evolve the kinds of chemicals that we use, but these are made for human interventions, right? And so if you have a disease, you need to stop that from destroying the whole crop. So we just hopefully in time, we'll have cleaner and cleaner and better and healthier ways to do that. But we're convinced that actually you can do it with microbiology, that we could use bacteria, for example, to control diseases so that we don't have to use fungicides. Instead of all the high doses of fertilizers, we could develop, improve our soils and improve our crop plants so that they can grow with very low input using soil microbiology predominantly, which includes a micro-rise in different bacteria and just having healthy soils. So yeah, I think chemistry is here to stay, but we can improve agriculture by making it closer to what happens in nature, what mother nature does, and help using the microbiology in the soils and on any kind of additives that we use to kind of consider the microbes and favor, essentially if we fertilize plants, intend to fertilize plants, we fertilize the microbes. We cultivate the microbes intentionally in order to have impacts on the plants. So that kind of a more biological vision for the future, but I don't think we ever get away from all kinds of human chemical interventions. I think we have to, I think that's with us for good, really is, we just don't want it to be destructive and minimize it as much as we can.