Special Edition: The Relationship Between Fungi, Endophytes, and Native Soil Biology - Interview with John Kempf

This special edition episode was hosted and recorded by John Kempf, and originally aired on the Regenerative Agriculture Podcast, with the agreement that we would share the episode on both platforms. 

John Kempf is the founder of Advancing Eco Agriculture.  His Regenerative Agriculture Podcast brings experts from agriculture, ecology, and plant and soil sciences to the table for in depth conversations that challenge listeners with new ideas about growing food.

While the Cornerstones of Healthy Food Systems Podcast targets people from all walks of life, and strives to help all listeners think differently about how our food systems influence our health, economy, and environment, the Regenerative Agriculture Podcast zeros in on the nitty gritty of eco-agriculture and the science that supports it.

Your regular host, Mary Lucero, will serve as the guest in today's  interview, so you will gain insights into the research and experiences that led Mary toward her own mission in regenerative agriculture. 

The Cornerstones of Healthy Food Systems Podcast Introduction by David Lucero.
Theme music by Zakhar Valaha 

This version was updated in summer, 2023, when we decided to remove the cornerstones.endofite.com website reference.  The only change was to remove the word cornerstones from the website reference. 

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Transcript

Mary Lucero: 0:21

Hello and welcome to this special edition interview. Several weeks ago I was contacted by a gentleman many of you in Regenerative Ag have heard of. John Kemp asked me if I would do an interview on his podcast, and I'm actually a pretty big fan of the Regenerative Ag podcast. I think John brings some fascinating people to the table to talk about cutting-edge ideas, and I was honored to be considered on his platform. At the same time, I knew the discussion was going to cover a line of topics that would fit in very well with everything we've covered this season on the cornerstones of healthy food systems, so I suggested that we play the final recording on both our podcasts. I think you're going to enjoy listening to this episode as much as I enjoyed the conversation with John as we were recording. Look to the show notes at the end of the episode to find a link to John Kemp's podcast and to his company site, advancing Eco Agriculture. And with that I'll turn this episode over to John.

John Kempf: 1:34

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 there are research describing all of the potential impacts that soil biology and biology can have on plant development. And 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.

John Kempf: 2:45

And recently, with the work of Dr James White and David Johnson and Christine Jones and many Elaine 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 those relationships between livestock on the landscape or between plants and soils and soil microbes, and in our conversation today, I'm really delighted to have a really 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.

Mary Lucero: 4:10

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 and new research, learning new ideas, making new discoveries, and an abundance of that research sits on shelves and 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 and some of those technologies.

John Kempf: 5:27

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 a 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, your background and some of the scope of the work that you were doing, the research that you had been working on decades ago, and some of the memorable moments that led you to where you are today?

Mary Lucero: 6:06

Okay, so, so actually I should throw in there because I think in some ways it influenced where I'm at, where all the products of how we were raised and and I should say that I am a 16th generation, new Mexico, and 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 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 big effort to develop and genetically engineered green chili 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.

Mary Lucero: 8:00

I went to graduate school thinking I was going to engineer microbes to break down biochemical waste, speak, or a 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 is into practical approach, because when you take a microbe out of the environment and culture it long enough to engineer it, it has lost all the genes that give it the ability to function in the environment and you put it back out there and it's really not cleaning up much of anything. It's, it's getting eaten, it becomes food for other microbes and and so the effort in microbial ecology was to look at at that point, at how you create the environment that allows the native microbes to thrive. You know, do you change the nutrients in the soil, do you change the aeration? What do you have to do to create that environment? Because microbes are already there, they just need the right food to get them going.

Mary Lucero: 9:15

Now, I didn't share this and I should back up that my undergraduate work was in agriculture and and so I did have that and 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 to plant in microbe communities to break down environmental waste. And we actually found a plant in that common Jimson weed de Tura 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 two, 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.

Mary Lucero: 11:13

I know there was a lot of interest in remediating TNT and so we looked at using de Tura 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 micro propagation, 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.

Mary Lucero: 12:04

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 quite 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.

Mary Lucero: 13:30

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 micro-propagated plant tissues. He was working in a range ecology unit. The Hornata Experimental Range 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 tripam, blue and sedan four stain to target the fungal cell walls and the fungal lipids inside the plant, and he had thousands of these micrographs showing fungal structures in every single tissue of the plant, and I remember looking at his picture, getting goosebumps, thinking if this man's data is correct, pretty much everything I've learned in plant biotechnology is skewed, and 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 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 jumped in.

Mary Lucero: 15:41

I was finishing my dissertation anyway, I needed a job and I'm like 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 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 chili are regulated. We 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.

Mary Lucero: 16:27

We were looking at herbivory and livestock and the plant volatiles and so I did a lot of essential oil extractions and profiles back before people knew what doTERRA was 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-wing salt bush.

Mary Lucero: 17:13

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 exenic so-called exenic while it was in cultures that appeared exenic by common standards. We published papers on these microbial communities that lived inside the Bula Loa species, which is a range black gramma grass and ambelliflu in the four-wing salt bush. 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 salt bush, on the microbiome, endophyte, microbiome and four-wing salt bush.

John Kempf: 18:09

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 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?

Mary Lucero: 19:01

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 were finding these endophytes so easily in native plants that people in crop production had perhaps missed was that we were working in these extreme environments. 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 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.

Mary Lucero: 20:52

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 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 map in the databases to other plant species. I remember there was a Baylor Desert Marigold that I was always pulling up sequences from.

John Kempf: 21:52

Intriguing.

Mary Lucero: 21:53

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.

John Kempf: 22:17

That's fascinating.

Mary Lucero: 22:19

And that same fungus was probably in my four-winged salt push and that's why I was getting the sequence. Now I don't know in recent years how much has changed with AI and what they're able to do 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 that the I would share. What I was finding at conferences or even just going across to 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 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 out contaminants. And I'd look around and 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? Nobody has tested, Nobody has disproven the idea that these sequences are actual fungi coming out of the plant.

Mary Lucero: 24:02

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 the fungus from these micro propagated plants, the species we were getting were very common. Molds Would isolate an aspergillus oostus or an alternator species and you'd take this over to the mycologists and ask them their opinion. Oh, that's just a common mold. These are everywhere. It's a latent pathogen, maybe in the plant, but it's certainly not an endophyte.

Mary Lucero: 24:36

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 micro propagated plant die, Would leave it on the same nutrient medium without transferring, because normally you, you know, every four weeks or so you move your plants onto fresh culture medium and would just starve them to death. And as the plants starve the fungus would emerge and start looking for another home. And some of these fungi can break down the tissue culture media that can live on this augurose or augur based media for quite some time.

John Kempf: 25:34

You know, when we think about the microbiome. I've certainly read the information, but I forget the exact numbers. But it's communicated that for our own bodies, our own cells, our cells, the cells that we call human, are exceeded in number by the number of cells 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 quote unquote human. And that raises the question of what you were finding inside plants. What was the relative balance of microbial cells versus plant cells?

Mary Lucero: 26:22

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 the 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 was never a prolific publisher, but there was a professor emeritus out of UC Irvine named Peter Aksat 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. I don't think he ever published that data because he was waiting for some other components that he couldn't pull together.

Mary Lucero: 28:18

This kind of work was not well funded. It was very difficult to get. No grant, no request 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 sequenced some isolates that we had and in those days 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 with less effort than it took us to sequence all that in the past, in any case, she came up with.

Mary Lucero: 29:26

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.

Mary Lucero: 29:45

And so she had a clone that we sequenced and 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 had 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 sluffs off the surface of the whale and that's what they would sequence. And I thought 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.

John Kempf: 30:55

That's fascinating.

Mary Lucero: 30:59

Now again, 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.

John Kempf: 31:18

When you think about. Earlier in our conversation you mentioned that you had started thinking on 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 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 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.

Mary Lucero: 32:40

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 four wing, salt, bush and black gram of grass. And sometimes I think you know I was kind of the big talker and Jerry Barrow was really the one who had a lot of the great ideas. But I remember he asked me because I kept getting these, I kept having such a hard time sorting out what was 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 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.

Mary Lucero: 34:14

Three days later he shows me some some culture plates where they had taken the the callus culture from from the four wing salt bush and placed a bunch of tomato seeds on it. And about a week later he shows me some little tomato plants and you know, he had the control plates that didn't get the salt bush 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 we started repeating this and adding more control and eventually we took these plants out and cultured them in the greenhouse and 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 salt bush or black grandma cultures 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 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.

John Kempf: 35:40

That's the idea that occurred to me.

Mary Lucero: 35:42

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 salt bush. We had detour cultures and we were transferring these to to different crop plants and we were writing up a patent on the technology. And what the patent had turned, we had told us, is that to submit a patent, you don't need to have a lot of background data. It's going to have, 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. And so I had a lot of questions. You know, part of my training was in toxicology and kind of environmental health and I wanted to make sure this was a safe technology. And they kind of reassured me. Well, no, you'll have a lot of time to do this research and 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 that were transferred by co-culturing with any part of that plant, because we were using callus 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 endophytes to transfer.

Mary Lucero: 37:50

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 went from this tedious process of isolating and cloning individual gene fragments to, for the same cost, time and effort, sequencing home the 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 genes 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 it meant, while some of these appeared to be classic endophytes that would only live in Black grandma, others were things like the aspergillus ustus or the alternaria alternata that could be anywhere but just happened to be residing inside our, cryptically inside our Black grandma grass. And that's when we really started wondering well gosh, did we just patent life itself? And in the meantime we did.

Mary Lucero: 39:25

By that time, jerry was approaching retirement and I was working more and more with a microbial ecologist named Adrian Unck, 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 US, you know, on the Hornado. We had sample sites up in the Four Corners region of New Mexico, and at each sample site we would look at four-winged salt bush and collect tissues. We'd 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 soil. Which ones are true endophytes and which are just only associated? Which ones can associate with anything?

John Kempf: 40:55

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 is? 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.

Mary Lucero: 41:22

Right and what we saw is that there, you know, indeed there's a lot of this horizontal organism transfer.

Mary Lucero: 41:28

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?

Mary Lucero: 41:41

Because many of these endophytes associate inside the seed. And what we were able to do with these salt-bush 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 forewing salt-bush seed. And then you have other species that only stay in the forewing salt-bush and these are what you might consider the true co-evolved forewing salt-bush endophytes. 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.

John Kempf: 42:55

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 research and his descriptions of the rhizophage cycle tend to focus on bacterial endophytes, and hearing you having a conversation focused 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?

Mary Lucero: 43:42

And I think that goes back to first remember that under the microscope a fungal cell is much, much bigger than a bacterial cell. It's like comparing mice and elephants in the in the pasture. Which one's gonna 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 it.

Mary Lucero: 44:26

And going back to the conversations I would have with Peter Atzat, one of the questions I asked him once when he was getting these filtrates, these 0.2 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 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 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 multi-nucleate. 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? A bacterium might be bigger. You'd have a hard time seeing just the nuclei.

John Kempf: 45:37

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.

Mary Lucero: 45:46

When you start thinking about bacterial endophytes colonizing fungi and the fungi or fungal endophytes colonizing plants, it becomes a really a highly fractalized picture really quickly and inevitably, 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 on our ability to examine things change.

John Kempf: 46:31

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. And 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 dated to prove this or 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 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 technology that you're describing, or the approach that you're describing of being able to transfer the associated stress tolerance.

John Kempf: 48:34

Inducing or inducing is not the quite word that I'm looking for, but these collaborative endophytes with other plant species. It reminds me of a story 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 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.

Mary Lucero: 49:40

I think as as we looked at this salt bush dynamic and the continuum of microbes extending from the seed through the risosphere 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 what 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 epicoe endophytes 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 it as that in some environments the epicoe 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 it made me think.

Mary Lucero: 52:03

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.

Mary Lucero: 52:15

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 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, and so 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 stand soil. But we couldn't really contain that.

John Kempf: 53:45

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 useful 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 were 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.

Mary Lucero: 54:40

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 area. I was born in a little town called Thiru, which is very much in what we refer to as Indian country, was surrounded by Navajo and Zuni communities. I was the first child in my family not born in Zuni and we moved away.

Mary Lucero: 55:07

When I was real young, my dad went to work for the Bureau of Indian Affairs and did this nationwide study on boarding schools.

Mary Lucero: 55:16

He was actually 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 away from our own culture.

Mary Lucero: 55:30

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 but somehow for me 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.

John Kempf: 57:04

When I have conversations with people about there is this phrase that keeps coming up of 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 cultures have a 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 and allow these systems that they are stewarding to really flourish.

John Kempf: 58:07

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.

Mary Lucero: 59:19

Yeah, and I agree with you. My friend, james Skeet talks about this a 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 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.

Mary Lucero: 1:00:21

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 Hagueman, 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 found a treasure.

John Kempf: 1:01:18

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 speculated, these speculations by pioneers in the space, have later been validated or that have been ignored and have never been validated or invalidated.

Mary Lucero: 1:02:13

Yes, yes, 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 was destroyed that was essential knowledge for maintaining our ecosystems and our sustainability?

John Kempf: 1:02:38

Yeah, it's not the first or the only incident of that happening.

Mary Lucero: 1:02:41

No, no, in every part of the world that story has been repeated.

John Kempf: 1:02:45

When we think back, coming back to your journey, your path, where you've described the research, and then you eventually left academia and you started looking at how do you transfer this into rather than looking at individual plants and transferring end of fights between plant species how do you improve the soil microbiome and how do you produce really healthy soil? And one of the topics that I'm really interested in and understanding better is how do we produce a disease-suppressive soil? What defines a disease-suppressive soil? So I'd love to hear about the next steps. Where did you go from once you started looking at soil health? Where did that take you?

Mary Lucero: 1:03:32

A lot of things happened in my life in a very short period of time and so I actually stepped away from all of this for a while because I had some major family and health and other issues that kind of put a, I should say. My career exploded and I spent several years picking up the pieces again. But one of the problems that was really growing as I progressed with my research was that I was starting to see how impossible it was to move forward and advance any finding from within USDA. And it was kind of ironic because we had this phenomenal research budget. I had resources that ran circles around my colleagues at the universities, but you also have all these political forces, all these every year that you hear on the news about budget disputes in Congress. Remember that there are government agencies all over the country waiting for that budget so they can make decisions about what they're going to do that year. And we were told that our positions were that the role of an ARS scientist was to be somewhat like an endowed chair at a university. You were to have this continuous research budget that gave you the flexibility to explore in depth long-term, high-risk questions that were not being addressed by universities through the five-year grant cycle and that was what was said on paper and in discussions.

Mary Lucero: 1:05:24

But in reality, every time the administration changed, our focus would change and would be to throw in a curveball and need to change. For example, I remember I guess that wasn't the focus changing but I remember working with some growers to set up some field trials. When we did this end-of-fight transfer and, oh my gosh, ars came down on us. Bad, because we were doing agronomic trials and our mission was range science and we explained we're trying to find out what these range microbes are doing and we haven't been able to explore that in the range plans. But it was kind of like, ok, you can't play with field trials anymore. So it just got more and more frustrating and I started watching the older scientists. In fact we had a gentleman on site who had developed the virtual fence concept and he was using this radio technology to herd cattle, which would really be a fantastic thing to have functioning and it finally is coming to be. But I remember he would just get held back. His patent sat in the patent office for years and the scientists aren't allowed to market these patents but nothing really moves out of the patent office once the patent comes to play.

Mary Lucero: 1:06:57

And so I really started to question what was happening within USDA and whether or not I was a fit for the agency. And initially I thought, well, that's a lot of career to throw away, but maybe I can just transfer to a different unit. Maybe it's the local scenario that's not working out. And I began exploring that and actually ended up in a conversation with the national program officer about where I might be a better fit for the agency. And we talked about the work I was doing and the research and by that time I was confident that understanding the microbial community could have major impacts on agriculture.

Mary Lucero: 1:07:47

It could transform the way we grow food. The microbes could not only replace pretty much every action we rely on chemicals to perform, but it could also kind of revitalize local economies, because the better microbes for your farm are not going to be the same ones that are good in my community. To some extent There'll be a lot of local development of biofertilizers and this kind of thing. And as I explained all this in my discussion with the national program leader, he finally just kind of shook its head and told me that he didn't think there was a place for me in USDA and I was kind of puzzled by the way he put it and I said, well, what exactly is the problem? And he said, well, this agency is never going to support an effort that does not benefit the agrochemical companies. And that was an alarm to me. It hit me like cold water. But it also just brought back Wow, that's quite a transparent statement.

Mary Lucero: 1:09:00

It was.

Mary Lucero: 1:09:01

I think I had heard similar things at many levels from many people and washed them off as conspiracy theories.

Mary Lucero: 1:09:12

And when I heard it at this level and I think the poor guy I was talking to was actually just trying to be honest and kind of regretted what he was saying but it was such an eye-opener to me in terms of why the story of microbes had not been advanced in the 1800s and why it has always received low funding, because chemicals are.

Mary Lucero: 1:09:42

For the reasons I encounter, chemicals are much easier to own than patent and I haven't shared this yet in our discussion. But we did eventually abandon our patent. It was awarded and when it came up time to renew, we told them not to renew it, and the reason for that is by that time we had realized that the microbial community is so complex that what we were claiming was just too broad. It was like a patent on life itself and if you think there are headaches and legal issues and controversy over GMOs, what we had was would have come into 10 times the controversy and the ethical violations and other problems. It was a bad patent and I hope that by making it and letting it go, what we have accomplished is to leave this as public prior art, and because it's prior art, it cannot be patented by anybody, and so that makes microbial technologies a lot more accessible to the public.

John Kempf: 1:10:53

Well, yes and no, it makes them a lot more accessible to the public, but arguably because of our IP protection structure, it also means that large corporations will not seek to develop this because they can't patent and profit from it, which is kind of a double-edged sword. It's a positive and a negative simultaneous.

Mary Lucero: 1:11:13

I have scratched my head several times in the years that evolved and so actually, when I guess in terms of where I've come since then, because, like I say, I actually went through a major illness and a car accident that all hit within six months of each other and the downside of that is that it really forced me to step back and get off the radar and I lost a lot of my professional connections and this kind of thing. But I think I was also so disheartened by this whole discussion I'd had with USDA that I really wanted to do something different and I wanted to explore the entrepreneurial world. I wanted to work more with the public because I felt by that time like telling the story was probably a big part of this. There's a lot of information that's not getting to the public, and so I spent a few years doing a lot of speaking engagements and this kind of thing, and in the meantime, my husband actually started pushing. Well, you know, if you're going to talk the story and tell people how they should be treating their soil and doing things, you need to be putting your money where your mouth is.

Mary Lucero: 1:12:30

And we decided to buy a farm, so we are now growing apples and raising cattle and trying to set up a regenerative system, or like three years into this, and that has been an eye-opener also. You can make your mind up that you're going to grow regeneratively and you're going to build this healthy soil microbial community, and we do. We invest a lot in testing. I actually have a lab on site and we keep a pretty good pulse on what's happening in our plants and in our soil. But you realize that you're not a vacuum and what neighbors are doing impacts your land and what's happening around you Impacts your land in a big way, and so we've been dealing with a lot of herbicide drift issues.

Mary Lucero: 1:13:23

We've been dealing with just outside complications and, of course, labor and policy issues that I was well aware of. Getting into all this, that make the whole process more challenging than it looks on paper. I think dealing with the microbial community the way I had, I had this idea. You just get in there, you balance the nutrients, you plant a diverse plant community and things are going to happen and they are. Our soil health is improving and it's exciting, but I guess the learning curve getting out of books and onto the ground has been a big one.

John Kempf: 1:14:09

Well, there's a few thoughts that come to mind. Mary, I would say first of all, welcome, welcome to the joys of co-creation, and welcome. There's just a lot of joy that happens when you start engaging with ecosystems and with living processes, living systems. I'm sure you've experienced that. And then when you think about the obstacles you know Ryan Holiday authored a wonderful little book that is titled the Obstacle is the Way it's that when you embrace the obstacles and you work your way through them, then not only do you emerge stronger, but you learn things that can change the whole nature of reality for yourself and even for the world as a whole.

John Kempf: 1:14:57

You know what? What popped out to me when, when you were sharing that was at the very beginning of your career you were intrigued and you were working with micro plant symbiosis in the case of the Torah and its associate identifies that had the capacity to degrade TNT. And what if it's the case that you have the unique skill set and you're the right person in the right place at the right time, to identify similar symbiosis that can remediate pesticides and pesticide drift, or beside drift? Anyway, that just popped into my mind as a possible inspiration for you. I think you have a very unique skill set and a gift, and maybe the obstacle is the way. Maybe there's a reason you have a farm that is challenged, with her beside drift.

Mary Lucero: 1:15:49

Well, and that is that exactly what I am working on. It's looking at ways, first of all, to look for the early signs of drift in the soil. So I'm using the same community profiling techniques that we used on our four wing salt bush studies to do what we call substrate induced respiration. You can look at different how the soil responds, how the respiration in the soil changes with different substrates, and we are comparing them at a baseline level and then in the difference of in after exposure to different levels of pesticide. I like working at that level because I can keep the pesticides contained and in the lab and using micro leaders of it as opposed to ounces or gallons. But it provides some insights and so it's just been.

Mary Lucero: 1:16:46

Working on a lab in the middle of a small farm is a whole lot different than my UV and sterility controlled environment in USDA. It's slower work because you have a lot more to deal with just keeping dust out of the lab. I spent 10 minutes a day vacuuming and dust blowing and removing, trying to keep my instruments free of dust and of course, as an independent scientist, there are very limited sources of funding. But at the same time I think it, I think you're right. I think we can start. What I hope to do with these community profiles of the microbes is start coming up with very early signals that growers can use to show this product is improving your health, soil health this product is not, and that way you can start testing and measuring and demonstrating immediately while you're making the decision about what you want to buy and what you want to apply, and I think that in itself would would be valuable.

John Kempf: 1:17:57

Certainly well, mary. This has been a fascinating conversation. I really appreciate you sharing your wisdom and knowledge with all of our listeners. What is something important that we haven't touched on yet? If anything, what's the question you wish I would have asked?

Mary Lucero: 1:18:13

I think we have covered quite a bit really. I think I am looking forward to sharing this. As you know, I also do a podcast and I'm going to put this on share it with my community as well, because we covered some some good stuff here, I think.

John Kempf: 1:18:33

Wonderful. Well, thank you for being here. I've really enjoyed our conversation and I wish you all success in your endeavors, and let's stay connected and work together in the future.

Mary Lucero: 1:18:43

Sounds good. Thanks for reaching out.