Episode 88: The Relationship Between Fungi, Endophytes, and Native Soil Biology with Dr. Mary Lucero

Hi, friends, this is John. Welcome back to the Regenerative Agriculture podcast where we talk about agronomic sciences and cultural management practices and all the fun stuff that are useful to know to help us understand how to regenerate soil health and plant health and public health and farm economics. You know, one of the pieces that I've come to really appreciate is how much valuable historical knowledge has been lost or has been ignored. When we look back at the domain of understanding disease suppressive soils, understanding soil microbiology, there's research going back all the way to the early 1900s, postulating plant absorption of nutrients through a process called endocytosis, and their research describing all of the plant potential impacts that soil biology and biology can have on plant development. For many years, a lot of that research was ignored. We prioritized chemistry and looking at life through and living processes through a lens of chemistry rather than through a lens of biology. And recently, with the work of Dr. James White and David Johnson and Christine Jones and many, Aline Ingham, many other pioneers in this space, we've come to appreciate the critical importance of the role of biology in living systems and fundamentally regeneration and regenerative agriculture is about understanding and facilitating and regenerating these relationships, whether there's relationships between livestock and the landscape or between plants and soils, soil microbes. In our conversation today, I'm really delighted to have, really, a pioneering scientist who helped to define and first discover some of these important relationships between microbes and plants. Mary Lucero is someone whose work I've admired from a distance for quite a few years, and her work has the, to some degree has been ignored or forgotten for lots of interesting reasons. But I wanted to have this conversation to help all of us remember some of the pioneering work that has been done and give honor and credit where it is due. So, Mary, I'm really excited to have you here on the podcast today and to talk about the work that you did decades ago, as well as more recently. Welcome.

Well, thank you, John, and thanks for having me on. I do want to say, before we get into it, that a lot of what you are doing in sharing information and informing the public is perhaps a larger contribution than the science and technology that we have put into a lot of this. And I know when I left conventional research, a big part of my motive for stepping back was recognizing that we invest billions in technology development in new research, learning new ideas, making new discoveries. And an abundance of that research sits on shelves in published journals and libraries and never works to advance human civilization. It gets published and Forgotten. And so without efforts like yours to do outreach, people are led to absorb the newest technology that's being marketed to them without really looking at all the facts and figures and information that maybe shows the weaknesses in some of those technologies.

Well, it takes both. I mean, in order to popularize and to make accessible the incredible research that has been done first. The research needs to be done. So it's certainly complementary. It takes both of them. But Mary, I think I'd like for, perhaps as a starting place for us to have this conversation, why don't you tell us a bit about your personal story, your background and some of the scope of the work that you were doing in the research that you had been working on decades ago and some of the memorable moments that led you to where you are today.

Okay, so actually I should throw in there because I think in some ways it influenced where I'm at were all the products of how we were raised. And, and I should say that I am a 16th generation New Mexican. And so, so my family history goes back as far as there is recorded history in New Mexico and probably farther. And because of that there's this tie to the environment and the land that is maybe less on the forefront of the minds of people who have not been in the same place as long. But in any case, I, I started my career in sciences looking at my, my degree was in molecular biology. So I was very much in a biotech environment. I was, my goal was to apply this to environmental restoration. And because we didn't really, for family reasons, I stayed in New Mexico to do that. There really wasn't a degree program that focused on environmental use applications of molecular biology. And so I was interacting all the time with the plant molecular biologists who were doing genetic engineering for agricultural purposes. In fact, there was a big effort to develop and genetically engineer green Chile at the time because that's our, our biggest crop in the, in the state pretty much alfalfa and green chili and some others. But, but this was kind of the influence going into my career was, was looking at the plant biotechnology and the microbial biotechnology. And of course I was in graduate school maybe six months when I figured out I, I went to graduate school thinking I was going to engineer microbes to break down biochemical waste or chemical waste, because I had heard about this, but I think you're in there. A few months before the consensus had been reached among the scientists that, that this really isn't a practical approach because when you take a microbe out of the environment and culture, it Long enough to engineer has lost all the genes that give it the ability to function in the environment. And so you put it back out there and it's really not cleaning up much of anything. It's getting eaten. It becomes food for other microbes. And so the effort in microbial ecology was to look at that point, at how you create the environment that allows the native microbes to thrive. You know, do you. Do you change the nutrients in the soil? Do you change the aeration? What do you have to do to create that environment? Because the microbes are already there. They just need the right food to get them going. Now, I didn't share this, and I should back up that my undergraduate work was in agriculture. And so I did have that. And of course, I married into a family that was ranching at the time, and. And there was all this agricultural influence. It just wasn't a good time to get into agriculture. And that was probably what motivated my interest in environmental science. So my doctoral research really focused on using, I guess the new horizon was the plant microbe interface. And we were looking at using plants, plant and microbe communities to break down environmental waste. And we actually found a plant, in fact, common jimson weed. Datura could chew up TNT faster than any microbe that had ever been tested. And so in New Mexico, where we are the site of the first atomic bomb During World War II, there was a lot of weapons testing. And as they were developing the atomic bomb, they manufactured a lot of TNT that they were using to simulate atomic explosions. While they were developing all this, the rumor coming out of the laboratories at that time was that they had so much TNT waste pumped into dirt tanks and canyons up behind Los Alamos, New Mexico, that they were afraid a bolt of lightning was going to start a fire. And so they would artificially pump water into these canyons to keep it wet. I thought of that often years later when the area caught fire. But that wasn't public knowledge and it wasn't anything that I heard in a way I could confirm. I know there was a lot of interest in remediating tnt. And so we looked at using datura to break down the TNT waste. And it was working very well, very quickly. And the question came up, is it really the plant that's breaking down the tnt, or is it the microbes associated with the plant? And so at the time, the perception was that if you grew a plant in micropropagation in tissue culture, you were dealing with an exenic system. There were no microbes associated with, with it. And so we decided to grow the plants in tissue culture, feed them TNT and see if they could break it down. And so that's basically what we did. And yes, it broke the TNT down very quickly. And maybe three weeks before I defended my dissertation, I was kind of cleaning up my cell cultures in the lab. And I had done a final contamination check to make sure there were no microbes growing on the plant. Because we would routinely plate these things out, look for microorganisms, and see if, if there was anything that grew on the plate, you know, would put them on a richer microbial, a richer medium designed to feed common microorganisms that grew on plants. And I had finished the contamination test. Everything came out clean. And I kind of said something like, yay, now I can defend. And one of the technicians that was working in the lab with me kind of chuckled, and he says, you know, before you decide that those plants are exenic, you might want to go talk to Jerry Barrow. And I kind of looked at him, I said, well, who's Jerry Barrow? And he says, oh, he's some old USDA guy who thinks that all plants are high order lichens. And I'm the kind of personality that when I hear something really out of the box, I need to go listen and get the whole story. And so about two days later, I'm knocking on the door of Jerry Barrow's office, and he invites me in to chat about microbes. And he starts showing me micrographs he had taken of what he claimed were fungal structures inside plant micro propagated plant tissues. He was working in a range ecology unit. The Hornada Experimental Range in, in near Las Cruces is a large USDA AG Research Service facility. And he was looking at how microbes were influencing the advancement of woody shrubs. But he had hundreds of images of stained cells. He would use Tripan Blue and Sudan 4 Stain to target the fungal cell walls and the fungal lipids inside the plant. He had thousands of these micrographs showing fungal structures in every single tissue of the plant. I remember looking at his pictures, getting goosebumps, thinking, if this man's data is correct, pretty much everything I've learned in plant biotechnology is skewed. And the, the weakness to Jerry's work at the time was that he really didn't have, you need to look at things with many kinds of data. And all he really had was the microscopy and many efforts to isolate these microbes from the plant in tissue culture. And the problem is he was looking at endophytes that by Nature are optimized to live inside the plant. And so we know that 98% of the microbes in the environment can't be cultured on plates. And he was getting very inconsistent results trying to separate these microbes from the plant. And so of course I, you know, I jumped in. I was finishing my dissertation anyway, I needed a job and I'm like.

You.

Know, I can get the DNA for you, I can do the DNA sequencing and let's prove that they're there. Well, it took us a few years to, to work in the funding and create that position and build on that opportunity. In the meantime, I did some plant phytochemistry. We looked at chili metabolism and there was some interest in learning how the heat factors in Chile are regulated. And so I looked at the biosynthesis pathway for chili and then actually started at the Hornada experimental range with a different advisor. We were looking at, you know, as a postdoc advisor, we were looking at herbivory in livestock and the plant volatiles. And so I did a lot of essential oil extractions and profiles back before, back before people knew what DTERO was and, and how you can use essential oils therapeutically or at least I guess those are ancient knowledges. But it hadn't been modernized at the time. And little by little we worked into the opportunities to where I could start working on endophytes. And so I actually spent about 10 years with USDA working very closely with Jerry Barrow and others to isolate these endophytes from black gramma grass and from four winged saltbush. Those were kind of the two model systems we were looking at. And we ended up characterizing many endophytes that lived in the plant while it was so called, while it was in cultures that appeared exhibit by common standards. We published papers on these microbial communities that lived inside the Beautyloa species, which is a range grammagrass, black gramma grass and also in the forming saltbush. In fact, I believe the first microbiome paper, the first paper to describe plants in terms of their microbiome was our paper on four wing saltbush. On the microbiome endophyte microbiome and four wing saltbush.

When I hear you describe the hypothesis that plants are simply high ordered lichens as proposed by Jerry Barrow. And when I hear you describe the abundance of fungal endophytes inside plants, there's all kinds of questions that come to mind. Some of the questions include what is their value to plants? How, how critical are they, how essential are they? At its most fundamental level, what does this mean for us in thinking about plant propagation and thinking about selecting plant varieties? And from an agronomic management perspective, how do we need to begin thinking differently about managing agronomy given the presence of these organisms in such large amounts?

And those are questions that, in my mind, also evolved as the research came forward. I know early on our idea was that these endophytes are certainly contributing to the tolerance in harsh environments. In fact, Jerry felt like one reason we, we were finding these endophytes so easily in native plants that people in crop production had, had perhaps missed was that we were, we were working in these extreme environments. You know, the annual precipitation on the Hornada experimental range was somewhere around 12 to 13 inches a year. And it can get hot in summer and cold in the winter, and it's dry most of the year. And when the rain comes, it tends to come all at once. And so the organisms living in these plants, anything living on that environment, has to be capable of tolerating extremes. And the hypotheses we were testing were that the endophytes were contributing to some of the drought tolerance and heat tolerance and stress tolerance of the plants because we could not really separate the endophytes from the plant. These were very difficult hypotheses to test. And so actually, you know, it was Jerry who came up with the idea that maybe what we need to do is transfer the endophytes. If we can't grow them in, in isolated culture, if we can't get them out of the plant, maybe we can transfer them into a plant that's more scientifically defined. You know, you don't find a lot of scientific papers on black grandma. We don't know what the genome looks like. We don't know a lot about it. And so we decided to move it into tomato. In fact, one of the big problems I was having using the PCR primers developed by people like Jim White was that I couldn't amplify fungi out of our plant tissues because the, the PCR methods were hitting too much plant DNA. And so we could see them under the microscope. We knew they were in there, but we couldn't get them to. We couldn't get that DNA out. And when we did get DNA sequences that didn't belong to a host plant, often they would, they would map in the databases to other plant species. I remember there was a, a Baela desert marigold that I was always pulling up sequences from.

Intriguing.

And I concluded that whoever had sequenced.

That plant had they captured a lot of endophyte?

There you go. There you go. I was convinced that they were pulling the DNA out of this plant, thinking it was only a plant, and they were actually sequencing the fungal DNA that was associated with the plant.

That's fascinating.

And that same fungus was probably in my four wing saltbush. And that's why I was given the sequence. Now, I don't know in recent years how much has changed with AI and what they're able to do in these, in terms of curating these national global DNA databases. But at the time there were a lot of erroneous sequences in the database. And I guess at the, you know, I, I would, I would share what I was finding at conferences or even just going across to our, our lab was on the campus of New Mexico State University and they had a big plant and genetic engineering group and I knew many of the faculty that worked there and, and I'd go kind of sound out some of my ideas with them and they would often tell me that I had to be pulling up contaminants. And I'd look around and, and you know, I had a dedicated PCR hood, I had barrier tips on my pipettes, and I had UV sterilization of the area I was working with. So, yeah, you can get contaminated in that environment. You can never rule out contamination. But I'd go into their labs and they had none of these controls and they were getting good data. And I thought, why do we assume that these are contaminants when these other assumptions, these other hypotheses have not been tested? You know, nobody has tested, nobody has disproven the idea that these sequences are actual fungi coming out of the plant. And I guess the other factor I was running into is that when I did get fungal sequences or when we did succeed in isolating a fungus from these micropropagated plants, the species we were getting were very common. Molds would isolate an Aspergillus eustis or an Alternaria species, and you'd take this over to the mycologist and ask them their opinion. Oh, that's just a common mold. Those are everywhere. And it's a latent pathogen, maybe in the plant, but it's certainly not an endophyte. And I've come to the conclusion that fungi are great shape shifters. They can take on different forms, they can live indefinitely inside a plant, and then they can transform and come out of the plant. We actually got pretty successful at isolating these common molds by simply letting the healthy micropropagated plant die would leave it on the same nutrient medium without transferring because normally you, you know, every, every four weeks or so you move your plants onto fresh culture medium and would just starve them to death. And as the plants starved, the fungus would emerge and start looking for another home. And some of these fungi can, can break down the tissue culture media that can live on this agarose or agar based media for quite some time.

You know, when we think about the microbiome, I've certainly read the information, but I forget the, the exact numbers. But it's communicated that for our own bodies, our own cells, our, our cells, the cells that we call human, are exceeded in number by the number of cells that are, that constitute our microbiome, by, I forget exactly an order of magnitude or a couple orders of magnitude. In other words, we are more bacteria than we are human. And that raises the question of what you were finding inside plants. What was the relative balance of microbial cells versus plant cells?

We once again, these fungi are shape shifters and so they're very hard to get a good count on, but easily similar. You see reports of 10 microbes to one human cell. And I think that kind of mimics what we would see with electron microscopy or with the staining techniques when we looked at our plant cells. There are some fascinating papers and I don't know if he's still publishing. I know he, he was never a prolific publisher, but there was a professor emeritus out of UC Irvine named Peter at SAT who I had several conversations with over the years. And he published what he called the mycosome hypothesis. And it was that fungi could create these very small propagules that could exist inside other cells. And he would isolate these by filtering through a 0.2 micron filter. Now that's usually a filter size that you use to keep solutions sterile because that's pretty small even for a bacterium to pass through. But he would create these filtrates from plant tissues and I know he did some with eggs and he was interested in looking at meats and other tissues and he could grow them in a low nutrient acidic medium and pretty soon they'd start developing hyphae and evolving into fungi. In fact, I sequenced one for him. I think it was a Penicillium species, if I remember correctly. Correctly, I don't think he ever published that data because he was waiting for some other components that he couldn't pull together. He was. This kind of work was not well funded. It was very difficult to get, you know, no grants, no Requests for proposal were available to look for this kind of fungal characterization. And it was just a difficult area to advance the research in, especially when you'd have this perception that what you were looking at was simply contaminants. In fact, one of the more amusing anecdotes from those days was that I had a technician sequence some isolates that we had. And in those days, you know, the sequencing technology was a little bit slower and you'd sequence a 500 base pair segment of an indicator gene like the ITS sequence and the ribosomes. And that's what you would match against the databases today. You can sequence a genome for, with less effort than it took us to sequence all that in the past. But any case, she came up with, so we call it a clone because you cut off this little fragment of the sequence that you're going to target and you stick it into an E. Coli to replicate so that you have enough DNA to sequence it and then you sequence it out of the bacterium, out of the bacterial plasmid that you've cloned it into. And so she had a clone that we sequenced and ran across, ran through the GenBank database and this sequence aligned with 90 species of whale. And that's when I went back to some of the people at the university who kept telling me we had contaminants and acknowledged that they were correct. I really had to find a way to get the graduate students to stop dropping whales in my laboratory. But at the same time I went back and looked at this paper where the group had done an entire phylogeny of whales. And they were collecting the DNA from whales by following the pods of whales in their boats and collecting the skin that sloughs off the surface of the whale. And that's what they would sequence. And I thought they sequenced, they sequenced a phylogeny of whale associated fungi. And these fungi tolerate salt. And that's why I was finding them in my four wing salt bush.

That's fascinating.

Now again, you know, we never had enough data on this to publish it. It was something we didn't want to go off on a complete new tangent with because we had to stay focused on our land restoration mission. But you always leave wondering, is this really what happened? And I think it was when you.

Think about earlier in our conversation, you mentioned that you had started thinking along the lines of transferring these endophyte populations or these fungal populations from one plant species to another. And I find that concept intriguing because as we develop our understanding of epigenetics and genetic expression. We know that we have different varieties or different plants, plant species who are much more stress tolerant than others. And we now understand that that stress tolerance is not just due to genetics necessarily, but also to the associated microorganisms that are in that plant microbiome. And so the technology that you're describing or the idea that you're describing holds a lot of potential and a lot of promise for enhancing the epigenetic expression of other varieties and other species. You know, it strikes me that this, this is a pathway that arguably would have a bigger promise and holds more potential for developing stress tolerant species than the going rage which is genetic engineering or CRISPR Cas9 engineering.

So John, it was probably might have been 20 years ago that I gave a presentation to a, there was a small southwest plant consortium, but we had just begun some of this transfer of endophytes and we chose a tomato plant because they grow quickly and because they already had a published genome. And we started taking some of these endophytes out of the, that we had isolated from 4 Wing Saltbush and black gramma grass. And sometimes I think, you know, I, I was kind of the big talker and Jerry Barrow was, was really the one who had a lot of the great ideas. But I remember he, he asked me because I kept getting these, I kept having such a hard time sorting out what was fun fungal and what was plant in the DNA I was isolating from these plants. And today that would be a simple problem. But back then it, it was a lot easier talked about than done. And he told me, well, what if you could get the endophytes into another plant that we already know the genome of and then you can, you know, you can prove which are the tomato genes and so what's not tomato must be fungal. And yeah, that'd be great. But these endophytes have co evolved with the plant. How are you going to get it to go into a different host plant? And he said, well, I don't know. Three days later he shows me some, some culture plates where they had taken the, the callus culture from, from the four wing saltbush and placed a bunch of tomato seeds on it. And about a week later he shows me some little tomato plants and he had the control plates that didn't get the saltbush culture on them. And these things are growing way bigger. And I thought, oh, that's interesting. And it could have been hormones from the culture media, it could have been a lot of things. But we started repeating this and adding more controls and eventually we took these plants out and cultured them in the greenhouse and carried them out for a couple of generations. And over and over again the plants that had been co cultured with the endophyte laden saltbush or black grammacultures were growing more robustly than the plants that had not. And so then we probably got a little carried away with this idea because and, and what I presented at the Southwest Plant Consortium 20 years ago was that we think we're looking at a technology that can make genetic engineering obsolete.

That's the idea that occurred to me.

Yeah, yeah. And, and so we got a little carried away with it. Pretty soon we were generating cultures of all the, you know, several of the major desert range plants. We had creosote bush and tar bush and four wing saltbush, we had datura cultures and we were transferring these to different crop plants and we were writing up a patent on the technology. And what the patent attorney had told us is that to submit a patent you don't need to have a lot of background data. It's going to sit in the patent office for five or six years before it actually gets reviewed and you can be accumulating your data in the meantime. I had a lot of questions. Part of my training was in toxicology and environmental health and I wanted to make sure this was a safe technology. They reassured me, well, no, you'll have a lot of time to do this research and just put forward what you know and make the patent as broad as you can. Because one way that big companies can get around a patent is to just find some other slight way to do it. And so you want to use language that's very broad. And we basically claimed in our patent that we, we claimed any plant that contained endophytes from a non host plant plant that were transferred by co culturing with any part of that plant because we were using callous cultures, these are undifferentiated cells. And so basically we were saying, if you can't touch my plant and the endophytes got on it, well, it becomes my plant. And this sounded okay when we thought we were dealing with endophytes that were very plant dependent symbionts. You know, we thought it would be kind of a rare event for the endophyte transfer. But in the years that followed, while we were waiting for the patent to, while we were doing additional research and waiting for the patent to be reviewed, we really hit an explosion in genomic technology. And that's when we read from this Tedious process of isolating and cloning individual gene fragments to, for the same cost, time and effort, sequencing whole genomes and metagenomes. And the first metagenomic study I did. Now, even those early metagenome, what we were calling metagenomics then, was still based on sequencing its sequences, ribosomal genesis from many species, but at least we were looking at whole populations instead of individuals. And the first time we did a metagenomic analysis of the Black grandma cultures we had, we realized we're dealing with dozens of endophytes, dozens of species, and that men, while some of these are appeared to be classic endophytes that would only live in Black grandma, others were things like the Asperger's Eustis or the Alternaria alternata that could be anywhere, but just happened to be residing inside our cryptically inside our black grammagrass. And that's when we really started wondering, well, gosh, did we just patent life itself? And in the meantime, we did. By that time, Jerry was approaching retirement, and I was working more and more with a microbial ecologist named Adrian Unk, and he brought in a bunch of community profiling techniques. And so we started working with the metagenomics and also looking at the migration of these endophytes across plant communities. And so this turned into a study that went. We had sample sites in Jordan, sample sites in the U.S. you know, on the Hornada, we had sample sites up in, in the Four Corners region of New Mexico. And at each sample site, we would look at four wing saltbush and collect tissues, would look at the rhizosphere, the soil around the roots, the soil farther away from the plant. And also we collected samples from all the plants that were growing near the canopy of the shrub. And we compared the microorganisms in all of these. So what we were looking for was how these microbes are moving across species and in and out of the soil, which ones are true endophytes and which are just only associated, which ones can associate with anything.

You'Re preempting the question that was in my mind, which is there's these conversations today, as we understand of horizontal gene transfer between organisms. And that made me think of what's the horizontal organism transfer when we think about symbiotic crop species or cover crop cocktails, as they're called, how much of the associated benefits that are happening there is really a horizontal organism transfer.

Right? And what we saw is that there, you know, indeed there's a lot of this horizontal organism transfer. So we had already been looking at the seed borne endophyte question how many of these endophytes are vertically transmitted through the seed to the next generation? Because many of these endophytes associate inside the seed. And what we were able to do with these saltbush studies is look at which ones were in the seed, which were in the leaf, which were in the root, which were in the rhizosphere and which were in the neighboring plants. And we concluded that there are many species that are kind of promiscuous and they jump around between the different plants and between the soil and the plant, but they're still carried in the, in the four wing saltbush seed. And then you have other species that only stay in the four wing saltbush. And these are what you might consider the true co evolved four wing saltbush endophytes. But, but we really need to kind of think of the plant in terms of this whole dynamic community of epiphytes that live on the surface of the plant, endophytes that live inside the plant, and then of course your soil and rhizosphere microbes that might associate with the plant and also act endophytically. Because the word endophyte really just means inside the plant.

One of the things that I find intriguing, James White tends to, he certainly speaks a bit to the fungal component, but my, my perception, my understanding is that his, his research and his descriptions of the rhizophagy cycle tend to focus on bacterial endophytes and hearing you having a conversation, focus largely on the fungal endophytes. So my question is, and please correct me if I'm not understanding this accurately, what is the relationship or perhaps what is the relative community presence of bacterial versus fungal species or fungal endophytes inside a plant? And how might that be useful information?

And I think that goes back to first, remember that under the microscope, a fungal cell is much, much bigger than a bacterial cell. You know, it's like comparing mice and elephants in the, in the pasture. Which one's going to create the bigger impact? It still depends. You can have millions of mice and have quite an impact. But I don't know that there's a logical way to really measure that question because you have, you have bacteria that act endophytically, so to speak, within fungi. They colonize the fungi and live inside of them. And going back to the conversations I would have with Peter Atsat, one of the questions I asked him once when he was Getting these filtrates, these 02 micron filtrates the only thing I could imagine that would pass through something that small would be an organelle, like a nucleus. And I asked him, I said, well, are you transferring? Are these like free living nuclei? And he just kind of chuckled because I know he was thinking the same thing. But he didn't, he could, he couldn't prove it. He didn't have the data to prove it with. And fungi are just kind of weird organisms. They can be multinucleate. A single fungal cell can have many nuclei inside it. But we were wondering if the nuclei could be pushed out and could survive independently. Now this creates a whole different. How do you tell a fungal nuclei under the microscope from a bacterium? Bacterium might be bigger. You'd have a hard time seeing just the nuclei.

This strikes me as so fascinating that we're. This is such a multi dimensional, layers within layers, you could say a fractal, such a fractal conversation. When you start thinking about bacterial endophytes colonizing fungi and the fungi or fungal endophytes colonizing plants, it becomes a really and admittedly a highly fractalized picture really quickly.

Admittedly, I have to admit some of these are hypotheses that are out in the twilight zone and that we really don't have the hard data or the techniques to measure them. And so it's not. I certainly haven't proven any of this, but I think they are valid questions that in time will reveal themselves. As technology advances and are ability to examine things change.

There's a part of me that I'm a bit of a dreamer. I'm constantly trying to connect the dots and understand the vision of what might be possible. So when you start describing the ability to transfer endophytes between species, you know, we have clearly established that there are exceptional, powerful symbiosis that can occur when we grow certain plants in combination. And it seems very reasonable to expect, I don't know, data to prove this or to disprove this, but it seems very reasonable to expect that a large proportion of those supportive or symbiotic effects are a result of the associated microbiomes between those species. And the ways that these plants collaborate with each other rather than compete with each other is really mediated by their associated microbiomes. And when we transfer that thinking to the next step, which is where you and I both went, is how can this be used to think about plant breeding? And particularly, you know, we live in an environment on a planet today where arguably there are large parts of, of the planet which are very stressed. We have rapid desertification in the Southwest where you are. And when we think about the possibilities of adapting plant species to thrive in those environments and to help us regenerate those environments, there are two very different worldviews. The one is the worldview of potential domination of genetic engineering and engineering plants to thrive in those stress environments. And the other approach is an approach of collaboration and stewardship and associated with powerful science. And the. The technology that you're describing or the approach that you're describing of being able to. To transfer the associated stress tolerance inducing or. Inducing is not the right word that I'm looking for. But these collaborative endophytes with other plant species, it reminds me of a story that, that Jim White shared, which is a Native American ritual of soaking corn seeds in a pounded plant pulp of seven different species and soaking them there for a few days before planting them like that. That is a mechanism that's a pathway by which to transfer endophytes from some species to others. And so anyway, my mind is going all these different directions. Like in my mind, this idea holds so much potential and so much promise that it seems that the implications and the opportunity is far bigger and far richer than that of genetic engineering. And so anyway, I'm. I guess I feel like I'm rambling on here, but I'm just so. I'm inspired by the potential and I'd love to see some of this be developed further.

I think as. As we looked at this saltbush dynamic and the continuum of microbes extending from the seed through the rhizosphere and out to the soil, I had to kind of expand my outlook beyond how do we make this plant grow better. And I thought a lot about what I had learned early on in the microbial remediation world in that it's really about creating the environment where the natural microorganisms can thrive. And I think that's when I kind of shifted from the focus on how do we make better plants or better plant endophyte combinations to how do we create this environment where the soil is so alive that the plants we put in it thrive. It kind of took me out of the plant and into the soil and into more of the soil health realm, because there were some studies that came out and it's been a while now, John. I'm trying to remember the name of the author of these studies, but they were looking at probably the. Still today, the best characterized endophyte system is the fescue epicloan to fights that we see in our Cool season grasses. And there was a study, kind of an ecology group that looked at how these endophytes persist across habitats. And they took samples all the way from, you know, our North American fescue populated grazing lands all the way down into South America. And they found that as that in some environments the epicloa is actually leaving the plant and populating the soil and not acting as an endophyte. And I thought, well, that's kind of what we're seeing in tissue culture with these plants. When we starve the cell, they leave. And anything at first, that was one of the first alarms I had thinking about the patent we had filed was that the endophyte might not stay as an endophyte in the plant. And I remembered when I was in graduate school, I actually studied under one of the gentlemen who had developed the first BT construct. He taught my genetic engineering course. And I remember him talking a lot about the safety testing that they had done to prevent, to prove that this engineered gene would not get out into the environment. And by this time in my career, I already was seeing that those safety tests were inadequate to show that the gene was not entering the environment. But I also saw that in our own patent there was absolutely nothing to keep this endophyte that we had transferred from staying within the plant as it went into different environments. We really didn't have a way to contain what we had claimed on the patent. An organism may behave as an endophyte under some environmental conditions and then as the temperature changes or the moisture changes or the climate changes or something different happens, other microbes move into the system. It could migrate out and possibly infect other plants or possibly stay in the soil. But, but we couldn't really contain that.

Yeah, I think what I hear you saying is that while knowing and understanding this science is fascinating, I find it fascinating and valuable and use in reality, if we take a more macro perspective and we look at the principles, the foundational principles of improving soil health, of having a diverse species and constantly maintaining living root systems, perhaps we look at some of these indigenous practices of, as I described the Native American example of inoculating corn seed and so forth, that if we really to incorporate those fundamental principles, then in reality, while we may not know and understand all the mechanisms, we're already capturing much of what we need to capture to ensure that our ecosystem thrives.

And I think that's a lot of what really drove me to change my career path is, I mean, I was born in the Four Corners, I was Born in a little town called Taru, which is very much in. In what we refer to as Indian country. We're surrounded by Navajo and Zuni communities. I was the first child in my family not born in Zuni, and we moved away when I was real young. My dad went to work for the Bureau of Indian affairs and did this nationwide study on boarding schools. He was actually in. Instrumental in bringing an end to the boarding school and. But it took us all over the country very young, at an early age and. And away from our own culture. And so it wasn't until years later that, you know, as an adult, little by little, I'd start to see that a lot of these things that. That my family had told me were actually indigenous ideas and concepts and principles that I just didn't know where they had come from. But somehow with the science, as I started seeing the connectivity of this, I started realizing that all these ancient teachings had a lot of wisdom in them because they focused a lot more on the balance and maintaining the community and community health more than individual health. And I think that putting some of those principles in place helps create the environment that lets these diverse soil communities thrive, and in turn, the plant populations thrive with them. I don't know if I went on too big a tangent there, but somehow for me, all these. All these old things that my relatives had said when I was very young just started coming to mind as I kept trying to assemble all the new information and figure out how we turn this into something practical that people can use to reduce the problems that we're seeing in agriculture.

When I have conversations with people about, there is this phrase that keeps coming up around traditional knowledge and other terms for traditional knowledge might be instinctive knowledge or instinctive knowing, or an intuitive knowing or heart knowing, like, you can call it many different things. But I find that for many different cultures, when. When cultures have a climate, close connection to life and living systems in whatever way that manifests itself, or not even just cultures, but individuals within those cultures, when they have this really close connection, they develop this instinctive knowing of how to foster ecosystems and how to cultivate or to propagate and allow these systems that they are stewarding to really flourish. And today we have modern, linear, mechanistic science that continues to uncover and reveal these truths, these scientific understandings, or these scientific foundations for why this intuitive, instinctive knowing is true and accurate. And it just, at least in the conversations that I have and in the research that I do, my personal experience has been that this intuitive knowing, this traditional knowledge is validated far more frequently than it is invalidated. I would say there's arguably a few instances where some of this traditional knowledge is either A, it's debunked, or B, we don't understand it well enough yet. Either one of those could be true. But a great deal of the time this traditional knowledge is validated as having been extremely valuable, useful, powerful, and it was debunked for a time or disbelieved for a time until the science developed by which we could validate it.

Yeah, and I agree with you. My friend James Skeet talks about this a lot, lot. And he calls it indigenous knowledge. But it really does come from inside us. And I think that science can be a great tool for explaining what's happening around you and measuring what's happening around you. Because science is so expensive to carry out, it takes such a big funding stream. It always turns into something that gets manipulated either by big corporations or by big governments, or by the handshaking that goes on between the two. And so if you really browse through the scientific literature, you find that people have been saying these things for years. You know, in the, in the years that I was so excited about all these fungi inside the plant because it was new to me, I hadn't been taught this in graduate school. Somewhere along the line I went back to, I found a paper from the 1800s talking about plant associated fungi and how they were helping the plant to survive. And it was like cold water in the face. I thought, my gosh, we've known this all along. And little by little, that stuff kept coming up. In fact, one of the, one of the professors from graduate school that I most admired was a man by the name of Jim Hagman. And I remember him telling us several times that we needed, no matter how busy we got in our studies, we needed to make time to read those old papers. He said, don't just focus on the latest research, go back and dig up some of the old ones. And every time I take that time, I feel like I've found a treasure.

The experience that you just described, I've probably experienced, I don't know, hundreds of times. There is fascinating research in the literature, in the peer review literature, even 150 years old. And it's like, you know, the scientific methods that they had available, the analytical methods they had available at that point in time are so primitive compared to what we have access to today. And yet they knew so much and they extrapolated and hypothesized much. I think this goes back to this intuitive knowing that when you're really deeply connected to something, it is remarkable how many of those hypothesis that were speaking speculated these speculations by pioneers in the space have later been validated or that have been ignored and have never been validated or invalidated.

Yes, yes. And. And they just kind of get set aside. I often wonder, with all the destruction that occurred when the Spanish came to the New World with their Inquisition mindset and their conquest mindset, how much knowledge.