How Plants Absorb Living Microbes and Convert Soil Pathogens into Beneficials with James White

Hi, friends, this is John and this is the Regenerative Agriculture podcast. This interview is a must listen for anyone interested in biological agriculture. James White describes how plants farm the soil microbes much in the same way that we farm livestock. Plants absorb microbes whole and extract nutrients from their cells, while the same microbes change the behavior of potential pathogens to become beneficials. The science described in this conversation is going to revolutionize our understanding of how plants absorb nutrients from living soils. Hi friends, welcome back to the Regenerative Agriculture Podcast. Today I'm delighted to be here with Dr. James White from Rutgers University, plant pathologist and a researcher whose work is going to give us a very different perspective on plant pathology, susceptibility to soil borne pathogens, and to plant absorption of nutrients. James, I'm very happy to have you here and to be able to have this conversation. I was first exposed to your work when some colleagues pointed me to some of the white papers and the work that you've done on endophytes and seed vectored endophytes. What are the impacts of endophytes on plant health and performance?

Thank you, John. I'm very happy to be here. Endophytes are really in all plants and you know, we haven't recognized until very recently and many people still don't. But the traditional view has been that if you have a microbe, a bacterium or a fungus associated with the plant, it's usually a pathogen, okay. But in fact, healthy plants contain bacterial endophytes in the tissues of the roots especially, but also going into the leaves and the shoots, and also fungal endophytes in all of those tissues as well. It just so happens that bacterial endophytes are much more common in plants than fungal endophytes, and they actually are a feature of a healthy plant. If plants lose their endophytes, they no longer will grow properly, seed germination rates will go down and the seedlings will be susceptible to disease and stress and all kinds of other problems and nutrient deficiencies as well. So healthy plants need their endophytes.

So you're describing that endophytes also play a role in nutrient absorption. But I guess perhaps before we go there, what exactly is an endophyte?

Okay, an endophyte is any microbe that will enter into tissues of plants. And in some cases those microbes they'll be, well, many cases they'll be soil microbes, but they'll go into the, into the plant tissues and they will not cause any types of any type of disease or any outward symptoms. Plants may in fact appear healthier when those endophytes are there, and they'll be healthier when the endophytes are there. So the important thing about an endophyte is that it does not cause disease.

So essentially, endophytes are organisms that have a symbiotic relationship with a plant that live inside plant vascular tissue. I think many of us as growers and agronomists, perhaps the extent of us thinking about endophytes is in the context of producing endophyte fruits. Fescue. Do all plants have these endophytes?

All plants have endophytes. And it's not just the fescue grasses and the rye grasses and so forth. All plants have endophytes. And it's not just the vascular tissues. It really is. All of the tissues of the plant can get endophytes, can have endophytes in them, even to the extent that they go inside the plant cells. So there's all kinds of endophytes, and they tend to be distributed all over the plant. Plant tissues.

Where do they originally come from? How does the plant first attract these symbiotic organisms? And how do they develop their symbiosis with the plant over the course of a growing season?

Okay, that's a good question. For example, if you consider the fungal endophytes of grasses, they actually will, at least many of them will come with the seed. They're already in the seed. They come from the mother maternal plant, and then they'll colonize the plant, grow into the aerial parts of the plant in the fungus. Well, these endophytes are called epicloa. Endophytes used to be Neotyphoidium, but they will grow in between the cells and the aerial parts and the leaves, and into combs and into the seeds, where those seeds develop from ovaries. But all plants will have bacterial endophytes and in some cases yeast endophytes that will come from the soil and they will colonize if a seed germinates. In some cases, they're actually seed transmitted. So they go in the seed and at the same time, these seedlings, once they germinate, they'll have the ones that are seed transmitted. Then they'll attract some others from the soil, and then they'll cultivate. The plants actually will cultivate those microbes at the root tip meristem. Right around that meristem, there's a zone in which the plant is secreting exudates and these are carbohydrates, mostly carbohydrates, but also some organic acids and some other amino acids in some cases right in that exudate zone. So they'll cultivate those microbes there, attracting some from the soil. And then they will actually internalize the microbes at the root tip, meristem. And what I'm describing is the rhizophagy cycle and rhizophagy, root, rhizo for root facie, for eating. Root eating. So these plants actually will internalize those microbes into the root cells just underneath that exudate zone at the root tip. And then those microbes being internalized will actually convert into protoplast stages, protoplast, naked protoplasts. They'll lose their cell walls. And that actually is in response to the root cell secreting superoxide or reactive oxygen onto those microbes. And that, we know that causes them to lose their shell walls, because we can actually paralyze the rhizophage recycle by suppressing superoxide formation using carbon dioxide. And then. And then those bacteria that are internalized into the cells don't convert into protoplasts. So but once they convert into protoplasts, they are actually circulated around those root cells, around the periphery. And they don't go into the cytoplasm, but instead they go into what's called the paraplasm. That is the space between the cell wall and the root cell plasma membrane. So they're really outside, technically outside the cell cell. But their membrane, the plasma membrane of the microbe and the plasma membrane of the root cell are butt up against each other, next to each other. And we think that is an important element in establishing the ability for nutrients to flow between the microbe and the plant and the plant and the microbe. And what happens is we think of cells, root cells, as somewhat static, but actually they undergo a lot of what's called cyclosis, cyclosis. And this is streaming, and that is the cytoplasm circulates around, and that causes the microbes actually to circulate around the cell. So the plant cell is moving those microbes at a certain set rate as it obtains nutrients from the microbes. But the plant also propagates those microbes. A few microbe cells enter, but then they make a lot, many more cells in the process. And what happens then is the microbe will trigger root hair development. And as those root hairs elongate, the microbe protoplasts will be ejected out into the soil again, where they'll reform their cell walls and go out and acquire soil nutrients and then be attracted back again to the root tip meristem and then enter again. So that's why we call it the rhizophages cycle, because there's a cycle between the soil and the plant. And we think that's important for nutrient transfer to the plant.

Wow, James, you've just said so much that needs to be unwrapped. One of the topics that I'm really passionate about is seeking to understand how plants absorb microbial metabolites and how they absorb nutrients. Because I'm persuaded from a lot of field experience that the historical model of plants simply absorbing primarily simple ions is at the very least incomplete, if not largely incorrect. So what you're describing is that plants are actually absorbing entire microbes into the roots. How do they absorb the entire microbes cell at once?

So that's a, that's a good question. There are a couple hypotheses. We don't really know the actual explanation. We haven't proven either. But this process was actually discovered by an Australian group, Queensland Australian group of investigators. And they, they actually gave it the name rhizophagy, the root eating name. But they proposed that what's happening is that the plant cells themselves are engulfing the microbes and bringing them in. We posed an alternate hypothesis, but again, which is not neither of which are proved, but that the plants are actually manipulating the bacteria, causing the bacteria to degrade their way into the cell. In other words, to use their cellulases or gluconases or other enzymes to eat through the wall. And then once the microbes get through the wall, then they're susceptible to the plant superoxide. And then the plant supports superoxide on them, which causes the looser cell wall, but also is a way in which the microbe can be controlled by the plant so that the plant can't. I mean, so the microbe cannot cause any damage to the plant cell itself or the root cell.

The next important question becomes, once plants have absorbed these microbes, and you now have this entire microbe inside the cell, it's lost its cell membrane because of exposure to superoxide. How do those nutrients contain within those naked cells or those membraneless cells then move into the plant cells themselves?

Well, it's. We have a model for what's going on, and it is diffusion of those nutrients into the root cell. In other words, the movement of the microbes around the periphery of the root cell, circulating those microbes around will actually break down concentration gradients of nutrient concentration gradient, so that those nutrients can flow more readily and flow faster into the plant cell. So we think it's diffusion across those two membranes. But, you know, there also likely is some active transport. We know, for example, that when we put the microbes into plants, one thing that we, that we see is that nitrate transporters get upregulated. And the nitrate transporters, nitrate transporters in the, in the plant cell, in the root, they're actively, through the expense of ATP energy, they're actively transporting nitrate into the. So it's a combination of diffusion and active transport mechanisms.

In an earlier podcast interview I hosted, Jerry Pollack from Washington University, who's done some fascinating work on the types of water formation that are formed inside cells and what he defines as the fourth phase of water. And I believe there's some significant overlap between, in his work he described, or his foundational research describes a possible mechanism and a means for cellular absorption of macromolecules, which I think is something that would be worth digging into and trying to understand a bit better. And I don't fully understand all of Jerry's work myself, so I'm not really able to describe it as well as I would wish. But I think important question to be asked here is what proportion of a plant's total nutritional requirements can be supplied through this process of rhizophagy. If plants have the capacity to absorb entire microbes and then utilize their nutrients, how much nutrition can be supplied to a plant by this mechanism?

It's a really good question. We know that plants can absorb nutrients straight out of solubilized nutrients and soil water, for example, but there are plants that actually grow without soil. And those plants, for example, one of them is resurrection fern, for example, that would grow on the surface of bark. And so there's no soil there. So whatever nutrients. And those resurrection ferns have roots that are just loaded with these bacteria. So presumably most of the nutrients from the resurrection fern it's actually getting with the help of the bacteria that are undergoing rhizophagy cycle in the roots of the resurrection fern. Also, we did some studies on some sedges that grow in the island of Bonaire, the Dutch Antilles. And these sedges will actually grow into the little rock crevices, little craters in the rock where the sedge will grow. And so there's no soil there. So whatever these sedges are consuming, they're not getting from a fertile soil environment. They're living off the surface of the rocks. And These sedges are again loaded with rhizophagy microbes. So the microbes themselves have to be aiding. And of course, we know other plants, a lot of wild, non cultivated plants do perfectly well with no fertilizer of any sort. They survive. So presumably they are surviving on whatever is solubilized in the soil, but also through the rhizophagy cycle and the, and the plant cultivation of these microbes and extraction of nutrients from the microbes. So there's some kind of anecdotal then, evidence that some plants can rely very, very heavily on obtaining nutrients from their microbes. But we don't have detailed measurements and comparisons. I could indicate that in experiments that we've done where we grew grasses on substrate that contains isotopic nitrogen, 15N nitrogen, when we have the microbes there, the plants can obtain about 30% more of the nitrogen than when the microbes are not present. So potentially the rhizophagy cycle, at least in some plants, is 30% of their nutrients in general, or at least nitrogen. And in other plants it may be nearly 100% of nutrients. So it could be quite variable.

The example of the sedges that you provided reminded me of a phenomena that I've observed a number of times when I'm out hiking in the woods and we see trees and large plants growing out of a bare rock face out of a cliff. And I've always wondered about that phenomena. What sustains those trees and how are they able to derive nutrition in the almost complete absence of soil? In fact, I think you could say the complete absence of soil in some of these environments.

So, yeah, no, just a comment on that, John. The plants like willows or poplars. Sharon Dotty has done work where she looks at. They've studied poplars and willows growing in those types of habitats. And their conclusion and the evidence, what they've gathered is that there's nitrogen fixation occurring with microbes that are going into the tissues of the plant.

So just to make sure I understood that correctly, is there actually nitrogen fixation happening from endophytes or epiphytes on the leaf surface?

Yeah, they're endophytes. They're endophytes.

Is this process happening inside the plant tissue where the plant is absorbing N2 gas from the air?

That's the proposal. There is good evidence that is happening. You know, besides having, in the rhizophagy cycle, besides having nutrients that are mobilized from the soil going into the plant, you also may in some plants have some nitrogen Fixing microbes that are entering into the tissues and perhaps the cells themselves of the plants and are able to then fix nitrogen and transfer that nitrogen to the plant. We don't know exactly how that's happening. You know, there are some ideas about it, we're working on that ourselves, but there's nothing firm to really describe the mechanism at this point.

So what are the implications of this research for agriculture and crop production in the future?

Well, I think the main point is that these microbes are present in all plants. They're important in the plants. We need to conduct agriculture, to manage soils and to manage plant seeds, for example, so that we maintain those microbes in the soil and also in the plant. So I mean, in the past we thought, well, it's not so critical, you know, we could do some deep churning of the soil and this works, some nitrogen fertilizer on it, and that's what it takes to grow a plant. But in fact, what happens when we do that is we destroy soil communities. And also by removing the opportunity for the plants to actually cultivate microbes to get nutrients, we also weaken the plant. Because what happens, the plants will, when they internalize the microbe, they will also use oxidative interaction, squirt superoxide onto the microbe and that causes the plant itself to up regulate antioxidants and to become more oxidatively stress tolerant, you know, oxidative tolerance to stresses. And so the plants that are cultivating microbes are more stress tolerant than plants that, where we're squirting nitrogen and other fertilizers on them. If they've got to get their own nutrients, they're hardier than if we just give them liquid fertilizer. So an example of we've got to be mindful of what we're doing to the plants is for example, cotton. With cotton, we plant cotton typically by removing, we prepare the seed by removing the hairs on the seed or the cotton fibers on the seed by dipping this cotton in acid. So it's called acid delinting. And the acid actually will destroy all the fibers off the cotton seed. And it so happens that many of the, of the microbial endophytes are actually vectored in those cotton fibers and on those cotton fibers and on the surface and crevices on the surface. So when we put acid there, we're killing all those microbes. And then when you, when we germinate the cotton seed, what we get is a seedling that is weak. It doesn't have its microbiome, doesn't have Its defensive microbiome, no microbes are on the surface and then they're susceptible, the seedlings are susceptible to disease. And so with cotton, what we have to do in its cultivation is put lots of pesticides and fungicides and nutrients to grow that cotton. So it's been considered, it's considered a dirty crop because of everything that we need to put on it in order to cultivate it, which is contaminating the environment. So, you know, if we, if we change the way we grow cotton, which would be, for example, to consider the microbes, then we may, instead of removing all the fibers with acid, we might use some other process that's less destructive of the microbiome and that should provide a healthier cotton crop that requires fewer fertilizers and protective pesticides and so forth to grow it. Same thing with some certain seeds, some grass seeds, for example, bermudagrass, the way it's treated is the husk is removed off the bermudagrass seed and then the husk actually cover carries all the microbes, carries most of them microbes on the seed. So it's removed and then they coat the seed with fungicides and other kinds of, you know, some fertilizers in some cases in order to protect the seed from disease. And the reason all that disease protection is necessary is because we've removed the protective microbes and the protective microbiome from the surface of the seed.

James, you raise so many questions in different directions. I'd like to take this conversation in, but I guess perhaps a starting and opening question is you described the implications of the seed vectored endophytes for cotton. What other crops suffer from the challenge of having their endophytes being destroyed? You mentioned Bermuda grass. Immediately my thoughts go to our small grains where we also have the husk removed from the seeds that we're planting as well. Are there any similar implications for small grains?

Believe it or not, for example, like wheat and rice, the microbes that vector on the seeds are pretty hardy. Like for example, with wheat, the seed actually has this invagination in it and those microbes can get in there and resist, they can survive in there. So they're really, with wheat, it's a really, it's hard to get rid of the surface microbes. But there is this tendency now to put coatings on fertilizer coatings and fungicide coatings on seeds. And those coatings may be, and in many cases are impacting the native microbiome. That's probably not, probably not good for the seed vector microbiome, and it may not be good for the soil microbes either, especially if they're inhibitors of microbes. Those seedlings actually need to recruit some microbes from the environment. And if there's inhibitors on the seed itself, they may restrict those microbes from colonizing the seedling. So any kind of a seed treatment needs to be examined closely to make sure it's not inhibiting microbes. In fact, it may be better to be adding microbes to seeds in order to improve that microbial association with the seedling and improve attraction of new microbes to the seedling.

Even without fully knowing and being aware of the impacts of these endophytes just in the conversation, mycorrhizal fungi inoculation has become quite popular recently. And it never did seem to make much sense to consider adding a mycorrhizal fungi and at the same time to add a fungicide that just seemed rather oxymoronic. And what you're describing is that it's even more important or equally as important with the endophytes. What are the impacts of non fungicidal treatments, such as soaking seeds with hot water to try to remove bacterial pathogens, such as bacterial speck, on some of the vegetable crops?

Well, that's a good question. Hot water depends on how extensive of an exposure it is, is probably not significant. Many of these endophytes that I'm talking about on the seed are pretty resistant. And so they're in, they're in a dormant condition in the seed. And you can actually hit them with, you know, Clorox treatments that you would think would sterilize them. And many of them, some of them at least, can survive. So hot water, I think is not going to be that problematic. I think. I wouldn't worry about that. It would be some more potent inhibitors that might inhibit the microbes from becoming vegetative once the seed begins to germinate. That's. I would worry about more the water washing or even using hot water. That's not too, you know, that's not such a sterile, you know, if it works, it depends on how long it is. You can actually do heat treatments that, for example, if you put seeds in a 50 degrees centigrade oven and you left them for a couple of days, the embryo wheat seeds, for example, the embryos will survive, but you will kill some of the microbes. And so that would, that would actually, that consume considerably weakens your seedling. But hot water washes for brief periods of time. That should be okay.

There's been this growing trend, just very recently, the last year or two, where more and more growers are considering planting what they term as planting seeds naked without any seed treatments at all and observing very nice responses rather than treating them with fungicides. It seems to me that what you're describing is an explanation or a mechanism for why they might be getting a crop response without treating with fungicides.

Yes, I actually think that's probably what's going on there. What we see with many of these microbes is that the microbes themselves will actually, besides going into the seedling and stimulating seedling growth and stress tolerance, providing nutrients, going to the soil and going back to the seedling when they go to the soil. One of the things that these bacteria will do, for example, so Pseudomonas or Bacillus will actually go out into the soil and colonize fungi there. Potential pathogens like Fusarium, for example, damping off disease, fungus, they'll colonize them and cause them to have reduced virulence. Okay, so they'll take some of the nutrients from those fungi and then the fungus will not be virulent and it won't cause disease. So they don't kill the fungus, but instead they will colonize the fungus and weaken the fungus, reduce its virulence so that it cannot cause disease or doesn't cause disease. And in fact, in some cases, those soil fungi will actually become endophytic fungi in the plant, things like Fusarium oxysporum, for example, that once it's colonized by the bacteria, it grows slower and it will grow onto the plant. And so long as the bacteria are there, there's no disease caused by the Fusarium. So you have this disease protection phenomenon that happens with the bacteria. So if you have a healthy plant microbiome, seed microbiomes, seedling microbiome, and you pair that with a healthy soil, then you should have much reduced disease in those seedlings, much reduced damping off and so forth. So the healthy, the microbiome itself is protective. If you remove that microbiome, if we take the seeds and we sterilize them to remove the surface microbes, then the Fusarium will just eat up the seedling. They're highly susceptible without their microbes on them. So it's very important in terms of disease protection.

Wow, this is really incredible. Another topic, Gary, that I'm really passionate about and interested in learning more is this development of disease suppressive soils. And what you just described seems like a fundamental of disease suppression. Is it possible for these organisms to develop resistance to the process that you're describing.

You know, I don't think so. And the reason why is it's not like a fungicide. It's not like you put the fungicide on and that will. That actually will kill the bacteria. But with these. I'm not the bacteria, but the fungus, the fungal pathogens, that'll kill the pathogen. And then any pathogen that isn't killed, it will then begin to grow. And then you have the development of resistance to your fungicide. But with these microbes, they don't kill the fungus. Instead, what they do is they'll colonize the fungus and they will use. Actually, in some cases, like, for example, Bacillus will use lipopeptides. These produce secondary metabolites that will then lipopeptide will go into the fungus membrane and it will cause nutrient leakage out of that, out of those fungal hyphae through the lipopeptide pores, through the pores that the lipopeptide forms in the membranes of the fungus. And then the bacterium can get those nutrients and then actually take the nutrients and take them back to the plant where they could provide bacillus, can provide it to the plant. But you don't have this killing phenomenon where the microbe is forced to. The fungus is forced to develop resistance. So you don't have that resistance. Instead, you just have essentially now a fungus that has reduced virulence and it doesn't cause disease. It becomes essentially part of the soil microbial community that benefits the plant itself. So you should not have resistance to bacteria that are not killing the fungi themselves.

James, this is really incredible. My understanding in the past had been that when you have this beneficial soil microbiome, you can prevent infection of Fusarium, let's say, for example, and it will change the Fusarium from being a potential pathogen to being a saprophyte, possible decomposer. But what you're describing is actually it's not a saprophyte, but it's changing the behavior to actually be a beneficial and to be an endophyte and enhance the plant. So you're saying that these quote unquote pathogens, given the right microbial environment, can actually support and benefit plants?

Yes, no doubt about it.

That's incredible.

No doubt about it. Yeah. No, we see that in experiments, in laboratory experiments, you know, those are much less complicated than soil. With soil, you have, you know, huge diversity of microbes out there interacting. But in simpler experiments, more controlled experiments, where we have, say, a fungus and we have one or two bacteria. We see exactly. What we see is that rather than being pathogenic, having the fungus there increases the benefit to the plant. What we think is going on is that the fungus that would be pathogenic is then acquiring from the soil, perhaps, like you said, through saprophytic activity, some nutrients that then the bacterium can get and carry back to the fungus. I mean, carry back to the plant. Okay. But at the same time, we have that fungus that would have been a pathogen. Instead it's not. And some of the mycelium will actually go into the tissues of the plant, but not cause disease. And so that establishes a situation where nutrients can flow into the plant more readily. So the plant does benefit. So that healthy microbiome in the soil, but also in the. In the seed, and the seedling will improve the performance, the ability of the plant itself to grow, develop, survive, become hardy and healthy. And I want to. You know, I didn't mention one thing I want to mention now, and that is that the microbes actually are necessary for the seedling to develop properly. In other words, if we take all the microbes away from a seed, for example, and grass seed or any kind of crop plant, and then we germinate that seed, what we see is that the seedlings don't develop properly frequently. What we see is that the roots no longer show a gravitropic response where the roots normally go down into the soil. Instead, the roots will stay on the surface of the soil or stay on the surface if we do agar, these agar media or agarose media, where the roots will just stay on the surface. And the other thing we see is roots don't form root hairs without these bacteria in them. In other words, to form a root hair, those microbes need to be inside those root cells. If they're not there, you have no hairs, you have hairless roots. So that what we think is going on is that the microbes themselves are actually participating in the process of root development. And what we think is going on is that the microbes are secreting. We think it's nitric oxide, which is one of the signaling molecules the plant responds to. But the other possibility is it's ethylene. And we're trying to determine right now what exactly the microbes are secreting that cause that root hair elongation. So we're leaning towards nitric oxide because that's also an antioxidant that the microbes, because they're inside the cells and they're getting bombarded with superoxide that they may be secreting nitric oxide as an antioxidant to degrade or to react with, to neutralize the superoxide produced by the plant. But the plant perceives that superoxide, or it could be ethylene.

We don't know.

We're still trying to determine. The plant perceives it as a signal to elongate for the hair elongation. So we think that the microbes are secreting this signal that causes root hairs to elongate. So when we remove all the microbes, we get no root hair elongation.

This raises a question about the impact of these symbiotic microbes and these endophytes on. On the development of the overall root system architecture and structure and strength. There's been a recurring conversation about the differences between, I'll just generically refer to them as older varieties or even older hybrids and more modern genetics. And this has been a conversation in fruit and vegetable production as well as in grain production. And the general dialogue is something along the lines of that older varieties used to have much larger, more robust root systems. They appeared to absorb more nutrients. The fruit itself appeared to have a higher nutritional content. The plants appeared to be better at absorbing some of these trace minerals such as cobalt and nickel, etc. Than many of the more modern varieties and more modern hybrids. And I understand that I'm making some very broad generalizations here, but what do you think would be the impacts are the impacts of these endophytic organisms in developing the structure of the overall root system and also the overall nutritional absorption capacity of the crop itself?

Yeah, that's a good question. I would, of course, you know, from my perspective, because I study endophytes, have for 40 years now propose that endophytes are being lost in many of our crops due to the way we're managing those crops and managing seeds and storing seeds and not managing to maintain the microbes. But I add to that that the way we use our fertilizers, for example, if we're cultivating a plant, cultivating plants using constant fertilizer applications, we make it so that the plant no longer needs these microbes. And, and in fact, it's entirely possible that some of these microbes are being lost over time in this process, and that as they're lost, we lose some of the functionality in terms of these micronutrient absorption capacity and content in plants. So that's one factor. There actually was an experiment published just a couple of years ago in the National Proceedings of the National Academy of Science where some investigators cultivated a wild. They went out and they collected a wild tobacco plant, and then they brought it into cultivation, and it was an annual tobacco. So every, every year they would cultivate it and then store the seed in the winter and then go back and cultivate it the next year and so forth. And so what they found was that after about seven years, the wild tobacco started seeing a disease, serious, actually, wilt disease, caused by some fungi that. That they didn't see in the wild populations. And so what they did was they went back to the wild populations of that tobacco, and then they started isolating microbes out, and they isolated some bacteria, a number of things, and then they started treating their plants, their seed that they'd been in cultivation for several years. And they found that when they treated them with their bacteria, put their bacteria back in, that their disease disappeared. And so what could happen in. What apparently happened in their process of cultivation is they lost these beneficial microbes into seeds, and that led to the disease. Disease that they were. That they were getting. So the same thing could be happening in our cultivation of some of our crops. The older, older varieties may have been more efficient because they had. Have those microbes there. And using our modern cultivation methods, we may have lost many of those microbes. Of course, you know, for any given situation, one would want to go in and do some experiments to see if that hypothesis, if that idea holds up, and that is that they're losing microbes. But that's a study for some other time.

Have we lost any of our microbes and any of the endophytes to some of the more modern breeding techniques? Is it possible that some of our more modern hybrids don't have the same?

Yeah, that's a good question. When you consider, for example, some of our modern corn, we know that some of our modern varieties of corn tend to be very dependent on nitrogen applications, nitrogen fertilizer and other fertilizers. And part of the way that those hybrids, those modern hybrids were developed is they have gone through tissue culture protocols that are very. That sterilize the microbes. And of course, they're cultivated using fertilizer applications. And so the microbes aren't really needed on the plant in order to acquire nutrients. And so when we've examined some of our modern corn varieties, we see that there's fewer microbes in those corn seedlings coming from modern corn. Whereas when we look at Indian corn and tropical corn, certain kinds of tropical corn, we see that they're loaded with these microbes. And we can also See that there's a certain amount of nitrogen thickness that's occurring in association with the corn seedlings itself and probably the mature corn plant. We are actually actively collaborating with Walter Goldstein at the Mandamin Institute in Wisconsin, where he actually is trying to put back into corn, tropical corn, other corn varieties by breeding the natural nitrogen use, if efficiency, nutrient use efficiency, and nitrogen fixation back into these wild or these corn varieties so that they no longer will be so dependent on liquid fertilizer applications.

Yes, I know, Walter, and the work that they've been doing at the Mondawmin Institute, some really incredible work on producing higher lysine, higher carotene, and methionine corn, in addition to corn varieties that require less nitrogen applications. So I would encourage anyone who's interested in that area, any of our listeners, to connect with Walter and see how you may be able to help and contribute to his efforts in breeding and developing some of those traits into our corn varieties. In reference to what you just said, James, about the modern varieties and seed produced by cell culture propagation, not vectoring these endophytes, if the endophytes are not on the seed, you mentioned earlier that they can also recruit some from the soil. Are there different species in the soil versus what is vectored on the seed? What impact does that have on the seedling's vigor and emergence and overall health over the course of the plant's entire life?

Yeah, so we know that seeds vector microbes, and many of these microbes we can actually cultivate them. Things like Bacillus, Pseudomonas, Methylobacter, and some Pantoea, for example. Some other species, and in some cases yeasts, even Saccharomyces, can actually be vectored on seeds and be internalized into the roots. Most of those microbes that are vectored onto seed, at least the ones that are tightly held on the seed, actually participate in the rhizophagy cycle. So they go between the soil, and they go between the internal spaces of the root, the cells internal in the root, in that paraplasmic space between the plasma membrane of the root cell and the cell wall. So they'll actually go there and then cycle between the soils. And of course, the soil. Things like Bacillus and Pseudomonas, they're on the plant, but they're also out in the soil, and they can cycle between the two, probably at the root tips there continuously. But as the roots mature, other kinds of microbes will colonize the surface of the roots. But you don't tend to have the microbes going into the root cells, that happens only at the root tips themselves. But you do have microbes in the intercellular spaces of the roots and also leaves and the shoot, the upper. The aerial parts of the plant. You have the microbes colonizing the shoot tissue and being internalized into the shoots and the leaves themselves. We know that for one reason, because when the. Because we can isolate them from shoots and because we can visualize them in some cases in some plants in the shoot tissues. But. But when the seed is formed from the ovule, we can find them in those ovules and on those seeds as they develop. So essentially these microbes are being vectored throughout the plant. They may not be doing exactly the same thing in different parts of the plant. You don't have, for example, you don't have rhizophage happening in the shoot tissue. That only happens in the root tissue. But those microbes go into those tissues, and so they're definitely affecting those aerial shoot tissues as well. So the plants are really fully colonized, all parts of the plant, by certain microbes. Methylobacter is one you see in almost all plants.

You described the mechanisms by which these organisms can change potential pathogens to endophytes in the rhizosphere. Do you have any insights into whether this might also be a possible disease resistance mechanism on the leaf surface or in the upper aerial parts of the plant?

That's a good question. Yes. If Fusarium goes up, for example, into the shoot tissues, and we know that, for example, with grasses, Fusarium is everywhere. The microbes, the bacteria are there with them. And in many cases we can see the bacteria there in association with the Fusarium in the plant tissues. It is not as significant in at least the mature leaves, for example, as it is in the growing tissues. You would have most of your microbes down in the roots. You would have a subset of microbes that are active up in the shoot tissues going into growing. And part of that is because that's where the nutrients are. But as those tissues mature, the nutrients become greatly reduced. And those microbes, if, for example, it's bacillus, what they'll do in the older tissues is they will turn into. They will form as the nutrients diminish, they'll form endospores and they'll become dormant in those tissues. We actually see that in vanilla organs and vanilla organs. The orchid will actually cultivate, it's a vine vanilla orchid, and it'll cultivate bacteria down in the meristem shoot. The growing shoot of the plant is almost like a vase like structure. And it'll grow a little biofilm that will contain some bacteria, multiple species. One of them is bacillus, and it'll grow in that little biofilm. And as those leaves form from the shoot, the bacillus will grow into the tissues and actually go in the cells in the shoot, and they will then grow there as those tissues mature. And as the tissues, as the leaves mature, the bacteria will convert from the vegetative phase, where there's lots of nutrients in the growing shoot, to the endospore phase down in the boulder leaves. And you can actually just look with a microscope and you can see these endospores forming as those leaves mature. And it so happens that it actually is part of the biology of the vanilla vine, because what happens with vanilla vine is that vine may form some lateral shoots. And as those lateral shoots form from buds, the endospores will then wake up in the shoots and, and then that tissue will then contain the growing meristem shoot. Meristem will then contain the bacillus again in it, so that it is protected from disease. And development of the shoot is benefited by presence of the bacteria.

This reminds me of a question that I should have asked when you were describing your tobacco example. What was it about the efforts of collecting and saving seeds and propagating them, farming them over seven generations, that resulted in the loss of the beneficial organisms and the endophytes on the seed itself? Do we need to begin thinking differently about saving seeds? How can we prevent that from happening with our modern seed varieties?

Well, that's a really good question. It must. We don't know obviously at this point, but it must be due to the way the seed are stored over the winter period or since they were produced. What normally happens with seeds in nature is there. There's no dry storage. Instead, seeds will usually fall to the ground where they will be exposed to soil moisture, even.