Podcast Short: Exploring Endophytes for Healthier Plants with Maizie Koentopp

Hi, friends, this is John. Welcome back to the Regenerative Agriculture podcast, where we have all kinds of fun conversations related to soil health and plant health. And in this fascinating world, as we are learning that plants have brains and they have the ability to think and to make informed decisions, they make very conscious, specific, informed decisions about how to interact with their environment, how to respond to different stimulus, how to associate and support their microbiome or not.

It's, we develop this, we're developing a.

Very different perspective than what we have generally collectively held historically on the way that plants interact with their environment and the way that they interact with their microbiome. And of course, the way we move these boundaries forward is constantly by researching, by studying more and trying to understand how interactions are happening. And some of these, some of these experiments can be very sophisticated, but often some of the best discoveries come from the simple experiments or simple observations out in the field. So for our conversation today, I'm joined by Maisie Cane Top, who ran an experiment that caught our team's attention here at aea, and they were delighted by the simplicity of the experiment, the elegance of the experiment, and the results that came out of it. So, Maisie, thank you for being willing to join me today. Tell us a little bit about yourself, your background and the experiment, and what inspired you to run this experiment.

For the past three years at my middle school, we have been required to do a Stanford project every year. And my dad is, he works in, like an urban farm and works to promote farm and like the, like agriculture. He like, works in that area. So I have, like, grown up around, like always hearing about that in my house. And I knew I wanted to explore something similar to that and something like a huge problem that I think, like, impacts a lot of young people is climate change and the impact on the environment that humans are having. So I wanted to connect those two areas. So I decided with the help of two soil scientists, Dr. Akilah Martin and Dr. Israel, that I wanted to research how we could exchange the use of harmful agrochemicals that are depleting soils and exchange those for endophytes, a type of bacteria which, instead of depleting soils, helps regenerate them and promotes ecosystem health as well as plant health.

And so what did your experiment end up looking like?

So the way I started, I took buckwheat seeds and I also took soil and I sterilized that soil because I was only testing one type of bacteria. So I needed to control the environment. And I inoculated some seeds and I Didn't inoculate the other seeds. And I had four different groups. I had one group that was the control, which was the non inoculated seeds grown in sterilized soil. I had one group that was inoculated seeds grown in sterilized soil. I had one group that was inoculated seeds grown in non sterilized soil. So soil that was just collected from my garden. And then I had a non sterilized, sorry, non inoculated seeds grown in non sterilized soil. I proceeded to grow those plants for different periods of time. So I had plants growing for two weeks, four weeks, and then three months. And after that period of time, I would collect plant samples and clean them with like a mesh to get all the soil off the roots. And originally my plan was to use a software that called Riso Vision that analyzes plant root hairs. But unfortunately I didn't have access to like the proper, like technology to use that software. So I ended up having to like re engineer my plan and I decided to use a microscope to take pictures of the roots and then count the root hairs myself instead of using the software to do it for me, which ended up taking a little more time than I originally planned, but it ended up, it ended up okay. And I discovered after collecting samples from all my data from all four groups that the plants that were inoculated overall, despite being in non sterilized soil versus sterilized soil overall, had the most number of root hairs. So root hairs are on like a root. They have little shoots that you can't see with your eyes. They're microscopic and they allow bacteria to cycle through the plant. So by measuring root hairs, it was a good way to see how the endophytes were actually impacting the plant. And I also measured brix, which is a way to measure the sugar level of a plant or of any liquid. And I found that. Well, originally I thought that the plants that were inoculated would have higher brix levels, but it turned out they didn't, which I realized was because they were using their sugars to flower, while the plants that weren't inoculated still were using their sugars to produce cotyledons and grow new leaves, which meant that the sugars were more concentrated, which in turn supported my original hypothesis that the inoculated plants and that the endophytic bacteria would benefit the plants.

So this is an important point. Did you observe the plants to be at different developmental stages or to develop at different speeds based on whether they.

Were inoculated or the inoculated plants, not only did they develop faster, but they also had a higher survival rate. So the plants that weren't inoculated, more of them died and didn't survive. The plants that were inoculated had healthier, fuller leaves and began to flower faster, while the plants that weren't inoculated hadn't started to flower yet by the time my experiment ended.

So even over the course of a 90 day experiment, they were not yet at the flowering stage, which is quite slow for buckwheat.

Yeah, I think that's also due to the fact that it was in sterile soil. So it was like very little microbial activity, which wasn't great for the buckwheat plants.

How significant were the differences that you observed in the root hair development? And I'm also curious just about the overall root biomass. What did the root biomass and the root hair development, how was it different? And I'm also curious about how it was different on the sterilized versus the non sterilized soil.

Yeah, so I didn't take the biomass of the roots. I only looked at root hair growth. And originally I wanted to measure like the length of root hairs, but I wasn't able to do that without like the software that I wanted to use, so I couldn't do that in my experiment. So the plants grown that were inoculated in sterile soil versus non, like the non related ones in sterile soil, they had pretty, they had like a pretty similar numbers. The ones that were inoculated did have more overall, but they were real closely followed by the ones that weren't inoculated. The biggest difference, even though they had similar root hairs, the biggest difference was in the like the growth that I observed, like how quickly they developed and.

What was, how would you describe the differences? How much more rapidly did the plants develop that were inoculated? What were the. If you can describe that.

They were, they were very different. At the end of my experiment I have, I took photos of the control and the plants that were the seeds that weren't inoculated and compared them to the ones that were. And the plants that weren't inoculated, they only had like, like five leaves and they still had potted as I said, and they weren't flowering and they were pretty short, while the plants that were, they were tall like they were like spilling out of the containers and they had many leaves and the leaves were fully developed and they were flowering.

And so that was on the sterilized soil was a similar pattern also true on the non Sterile soil?

Yeah, they were less developed than the plants that were inoculated in the non sterilized soil, but they, they were kind of like in the middle ground. So they had developed leaves and didn't have cotyledons anymore, but weren't as far along as the plants that were inoculated.

So the, the central thesis or hypothesis that you were, that your testing method is based on is you're, you're extrapolating the presence or the activity of associated endophytes based on root hair development in essence. And I'm sorry I kind of distracted you because I asked about overall root biomass, but what did you discover in terms of the, the root hair development itself? Did you have substantially more root hair? How much more?

So at the first like point, like the first time that I collected the root hairs, the inoculated plants had like a huge amount more. Like it was very like apparent they had like triple the amount of root hairs. And then as time passed, like the other groups slowly started to catch up. But the gap, they were never able to close the gap. So during the second like time area, like the second.

So you were collecting at two weeks and four weeks and then three months.

I think you said three months. Yeah, yeah. So at the second time they had like about like 300 for the, that I counted like the native microbes and inoculated and then this native microbes and then the inoculated plants had 600. So it was almost triple. And then during, after three months, the plants that were inoculated and in sterilized soil versus the plants that were inoculated in native soil had almost 300 more. So over time the gap slowly started to close, but the inoculated plants always had more root.

I think this is a common phenomena that we observe in practical application out in the field is that when seeds are inoculated there is often this early and rapid proliferation of biology. And if you have soils that have, where the microbiome is compromised, they do have the ability to recruit microbes from the soil microbiome. But it just, it takes time to build up that soil, that root microbiome population and they never fully catch up to what. So your, your experimental evidence matches very well with what we observe out in the field. So the, what was the, how difficult was it to set up this experiment and to conduct it?

I had. So when I started my experiment, I started it like months, more than three months before, like the deadline. And I had to repeat the experiment three times because in the beginning my soil was the Ph wasn't like just. It wasn't the right PH for the buckwheat, and many of them died, and I didn't have enough to collect the number of samples that I needed. So then I started over and I repeated the same process again, and the same thing happened where they didn't survive. So I did it a third time, and I changed the PH by adding sulfur because buckwheat prefers more acidic soils. And this time I did have enough samples. I also think that it was difficult for the buckwheat to survive and completely sterilized soil, which contributed, I think, to the higher, like, death rate of the buckwheat plants. So it was. It was difficult to have to redo my whole experiment multiple times to get the results that I wanted. And it was another challenge for me was taking all the pictures of all the root samples because I had. I had a lot of groups, I had a lot of root samples, and I was taking. I had to take like, like 70, between like 50 and 70 photos of each sample and then count the number of root hairs for every single photo for every single route. And that. That took a lot of time. So that. That was challenging, but I. It worked out in the end.

So that sounds like a lot of counting anyway. Yeah.

Yeah, it was a lot of counting.

So you mentioned the. The unexpected results in regards to the. The variation in BRIX response versus the plants that were flowering and those that were not. Were there any other results that surprised you or that were unexpected?

It surprised me that the plants that were grown in native microbes were like how you described earlier, like they started with a lower number of root hairs, and it surprised me how they were able to catch up over time. But my hypothesis in the end was proven correct. So although the brix numbers surprised me and the initial to final numbers of plants grown without the inoculant surprised me, the overall results supported what I initially thought.

I think it's worth noting for. For the benefit of our listeners, we're describing this experiment in terms of the. The endophytic microbes. And obviously you're measuring root hair development with, I think, based on Dr. James White's work and other people's work, those certainly are associated with the presence of endophytes within the plant and their. Their activity. And also the product that you were using was not just endophytic microbes alone. It was also mycorrhizal fungi and other things.

And this year I'm building off of my project from last year, and I'm Taking individual strains of endophytes, specifically Bacillus species like Bacillus subtilis and thuringiensis, and inoculating buckwheat to see what. How the specific strains impact the root hairs. And then I'm also growing buckwheat on a farm to see how the inoculant will work in a actual agricultural environment instead of in a lab. Laboratory environment.

Yeah, it would be interesting. Of course, it would add another layer of complexity, but it would be interesting to again compare, to see what if there is a contrast between the individual species and the combination of different species that also includes the mycorrhizae.

Yeah.

Yeah. Well, Maisie, thank you. Thank you for running the experiment. Oh, our. Our team would not be happy if I didn't mention that the product that Maisie was using was actually biocode Gold. So, Maisie, thank you for that experiment. Thank you for the work that you're doing and being willing to come on here onto the show and to talk about it. Thanks for all that you do.

Thank you. Thanks for having me.

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We test.

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