Dr. James White describes how microbes are replicated inside plant root hairs, how plants with microbes in them grow larger, and how endophyte-laden plants are more resilient
Acres U.S.A. There’s so much to talk about. Let’s hit on some basics real quick, but then maybe go into other topics — nitrosomes, how endophytes can affect epigenetics, etc. But to set the scene, can you define what endophytes are? What types of microbes are we talking about?
White. Sure. “Endophyte” is just “endo” — internal — and “phyte” — plant. It’s any microbe that goes into a plant. They can go into the roots, they can go in leaves, they can be in the seeds, and they don’t cause disease. Most microbes go into plants and don’t have anything to do with disease. They cause no negative impacts. Instead, they are either directly causing benefit to the plant in some way or are riders in the plant, not doing much. Maybe they’re interacting in a consortium of microbes — they’re part of the community — but many of those that go into the plant are having positive benefits in the plant. They can be bacteria, they can be fungi, they can be algae.
Acres U.S.A. And they can be “facultative,” correct? Something like Fusarium, which can be beneficial most of the time, but if there’s too much of it, or different conditions are set, can be pathogenic?
White. Yes. Fusarium is interesting. We just did a study of some grasses, actually — in this case, Poa annua reptans, which is being selected for putting greens. They’re using it for a turf grass, but it’s a weedy plant, an annual bluegrass. Normally you grow the plant and it grows fine. But it has an endophyte in it that’s related to Fusarium. You don’t see the fungus in the plant because it’s in there with bacteria, with the community, and the plant has control over what the fungus is doing. But when we take the plant and grow it under conditions of high zinc, it causes stress in the plant, and that causes the plant to be unable to regulate this endophyte that it has. This Fusarium then comes out and causes disease all over the plant.
So, there are these microbes like Fusarium that can be pathogens in the plant but are under control. They’re under microbial control. Other microbes modulate it. Microbes will go into the hyphae and will cause the fungus to be less virulent. The plant is also controlling development of the whole thing. When the plant gets under so much stress, the system becomes unregulatable, and then you have disease.
Acres U.S.A. And can you talk about how these microbes get into plants in the first place? Via the seed, via leaves, via soil through rhizophagy, right?
White. Yes — plants mostly absorb them out of the soil. When the roots grow, they put exudates out into the soil, and that will attract microbes over to the root tip, and then they’ll take them into the cells. We used to think endophytes were between the plant cells — that they were intercellular — but now it’s pretty clear that many of them become intracellular. They actually go inside the cells of the plant.
In rhizophagy, what happens is that the plant will attract microbes over to the root with exudates — sugars and other nutrients are in the exudates — and then it will take them into the cells of the root and remove nutrients from them using reactive oxygen, and then cycle some of them out as they form root hairs. The bacteria that survive that oxidation process — the nutrient extraction process — get ejected out of the root-hair tips as the root hairs grow back into the soil. That’s how this is a cycle. Microbes are actually cycled through plant roots.
Acres U.S.A. And this cycle — we’re talking on the order of hours, not days or weeks, correct?
White. Yes, they go through pretty quickly. Now, some microbes are going to stay in those root cells as they grow. They are essentially replicated. A microbe gets taken in at the root tip, cell walls get removed, and then it gets replicated by the plant. There’s lots of little copies. Plants are taking in a few microbes and making a lot of microbes, and they use those microbes then for their own development to build root hairs. Root hairs don’t grow unless there are bacteria inside the plant. The plant is replicating these microbes, growing a little bit, and ejecting the microbes out of the tip back into the soil.
Acres U.S.A. Explain the replication part a bit more.
White. We don’t have a clear understanding of how the development happens. But it so happens that root hairs are not actually formed just by the use of plant genes. They involve the bacteria, which are absorbed into those root cells, and then those bacteria are replicated in root hairs. We think the microbes are producing hormones that trigger growth in the root hair.
We do simple experiments where we remove the microbes from seeds. The roots that grow will not develop hairs because there aren’t any microbes there. The microbes come from the surfaces of the seeds, and it takes a whole community — it’s generally not just one microbe that they have. They carry this community that helps them to develop their structures.
The root hair is actually a structure in which those microbes are replicated. Once the bacteria enter a root cell, the plant oxidizes off the microbe’s cell wall and creates a protoplast, and that protoplast is easily replicated. In other words, a bacterium without cell walls doesn’t have the same constraints on growth as one that has a cell wall. When they have a cell wall, they have to produce the wall — they make a wall in the middle of the cell, and they divide apart like that. But when they don’t have a cell wall, they’re just a protoplast, and they form little “buds” very fast — tiny little bodies with DNA in them. They can replicate really fast.
So, the plant, in the root hair cell, because it has this cyclosis process, bounces these protoplasts around. It causes them to break apart into little protoplasts. It grows these bacterial protoplasts. And those bacterial protoplasts will go to the tip of the hair and accumulate. They look like little ghosts at the hair tip. And when they get to a certain point, they produce enough hormones that they cause that hair to grow right at the tip. When it grows, the microbes that were there at the tip get pushed out. But there are still microbes that remain in the root hair cells. Those are replicated again by that cyclosis until you get a bunch in the tip, and then it ejects again.
So, root hairs actually grow in spurts, and it has to do with the replication of these microbes in the root hair cells.
Acres U.S.A. That’s incredible. Do you have any estimate of the numbers — for every one bacterium cell that goes into the root, how many can or does that replicate? And by replicate, you really mean reproduce, right?
White. Reproduce, yes. They break apart with a little bit of DNA in there. The bacteria have these little chromosomes in them, and they make a bunch of them. And then when they replicate, a little “bleb” that comes out has at least one of those chromosomes in it. I don’t know very much about bacterial replication, so I can’t tell you how that’s regulated, but essentially that’s what happens. A few go in, and maybe thousands representing come out — the progeny.
The plant is just using these microbes as part of its growth process. Root hairs and trichomes and other cells that become laden with bacteria also tend to develop more — they tend to be a little bit bigger. It triggers development. All over the plant, you have cells that elongate and grow in size, and microbes tend to be more represented in those cells. We think that there are cells all over the plant, just like in the root hair, whose development is in part determined by the presence of these microbes.
That also tells you the importance of a healthy soil, because proper plant development relies on having healthy soil. That’s where the plant gets these microbes from — the microbes it needs to adapt to any changing environment, that it needs to respond to drought or whatever is happening. Stress tolerance is regulated by microbes — by bacteria that go into the cells of the plant.
Acres U.S.A. So, the plant is not only “eating,” in a sense, microbes — it’s also producing them. It is multiplying them.
White. It is multiplying the microbes. Now, the ones that are going to be multiplied are the ones that work best inside the plant. The plant is hitting them with reactive oxygen, and some are so sensitive that they’re just going to oxidize completely, and those cells are going to die. Some will be so resistant to oxidation that they’re not going to lose their cell walls — the plant’s not going to be able to manipulate them. So, there’s a sweet spot in the middle, in terms of resistance to plant oxidative processes — mostly superoxide and hydrogen peroxide.
Those are the microbes the plant is able to use. The plant will take them in, they’ll lose their walls, they’ll replicate inside the plant, and then the plant can eject them back out. Then they’ll reform their walls and flagella, which they need to survive in the soil.
So, there are some microbes that work with plants, and some won’t.
Acres U.S.A. The implications for soil management practices seem obvious, then —you’re not going to have microbes unless you have living roots in the soil.
White. Another factor is stress tolerance. And by stress tolerance, I mean herbicide resistance, heavy metal tolerance, salt tolerance, and so forth. These are all factors that are really affected by microbes.
We use the term “modulated.” Microbes have a role in affecting stress tolerance. Plants are modulated, or influenced by, the microbes that go into the plant — by the endophytes. For example, we did a study with Poa annua where we removed the microbes, and then we put microbes back on some of them. Then we checked the stress tolerance of the plant when grown in high-salt soil, in high–zinc sulfate soil, and in soil with applied herbicide. We found that when the endophytes are there, the plants have an increased measure of resistance to those stresses.
All of those stresses essentially cause damage through the generation of reactive oxygen in the plant — superoxide and other kinds of reactive oxygen that damage cells. But when plants have bacteria in their roots — they’re getting longer root hairs, and the root hairs are longer because they have more microbes — they are upregulating their reactive oxygen genes — the antioxidants, for example, and the phenolics and whatever they need to cope with stress. Because of the microbes, they upregulate those compounds, and then they’re more resistant to other kinds of stresses.
One big problem with chemical agriculture is resistance of weeds to herbicides. We think that the beginnings of that resistance actually have to do with the microbiome. Because weeds have a rich microbiome, they have the capacity to develop tolerance to herbicides and other kinds of toxins. A lot of herbicides generate oxidative stress — they work through increasing reactive oxygen and damaging the plant that way. The microbiome is the first level of anti-stress defense that a plant has. In addition to bringing in nutrients and all the other benefits, oxidative stress tolerance is a base-level benefit of having these microbes inside plants. Microbes make stronger plants.
Acres U.S.A. Why do weeds have such a strong microbiome?
White. Because they are essentially environmentally selected. Our crop plants — we select them, we control the soil, and we control seed production, and we’re losing microbes in that process. Most of our selection is done without any consideration of microbes. Now, not all of them are lost, but a lot of the good ones are.
But a weed is very different because the development of a weed seed is very different. The seed will mature outside. It’ll be open to colonization. Some microbes will colonize it as it matures. Maybe the seed will fall on the ground, or it’ll be swallowed by an animal and go through the gut. There’s a natural process in which the microbiome develops. And that microbiome, because we’re talking about a particular place, is being selected by environmental pressures. For example, if there’s a disease in the area that’s wiping out a lot of the plants, the microbes that the weeds will take in are the ones that will make it resistant to that microbe, because those are the seedlings that’ll survive. The others will simply be selected against. They won’t survive. So, the microbiome is the first level of selection — before you even get to genetics from the plant.
There’s actually more we’re seeing with microbes and plant genetics, and this is really interesting. We can see that microbes are being incorporated into cells that are dividing, and they’re actually being incorporated in the nucleus or the nuclear envelope.
The other thing is that it’s been shown that microbes will cause plant genes to be expressed. When plants want to turn off genes, they methylate them. They put methionine groups on them that’ll turn genes off. The microbes reverse that, and they turn genes on. What that means is that when microbes are used to grow plants, those plants tend to be richer in metabolites and enzymes and certain other internal chemicals. It’s thought that that is a direct effect of the microbes on the genes of the plant, causing that expression. That’s epigenetic again.
And then there’s another level here, and this comes out of this work that I’m doing with Walter Goldstein in Wisconsin looking at high-microbial corn. We’re seeing that the corn that’s selected for high microbial rates also is showing higher transposon activity. These transposons are the jumping genes that Barbara McClinton discovered many years ago — the genes will pop out and move around, and they’ll move little bits of DNA with them. They’ll turn genes on, and they’ll turn genes off. And we could tell this from Walter Goldstein’s corn because there are more of these different-colored kernels. That’s a microbial effect. So, microbes also increase mutation rates.
Another process that may be happening there is endopolyploidy — causing polyploidy. Some of the growth that we see in the roots, for example, and in maybe hairs and other cells that get big, is that the microbes are causing polyploidy in the plant. They may be diploid, but they may then become tetraploid and hexaploid. When they increase their polyploid level, the cells get bigger. That’s likely what’s correlating with bigger cells. Some of the growth we’re seeing is likely polyploid in origin.
Acres U.S.A. More variation doesn’t necessarily sound attractive to a grower. But that has to be balanced out with all the positive benefits — disease protection and nutrient delivery and resilience.
White. Well, typically it’s a no-go for plant breeders. This is a problem with the highly microbial corn — the plants become more variable. If you’re using a machine to pick corn, for example, and you have big plants and little plants, you’re going to miss some. It’s going to screw things up for your machinery. We like everything in modern agriculture to be perfectly uniform. But then you don’t get the variability in the plant.
Acres U.S.A. Can we talk a little bit about nitrosomes as well? What could be happening there? This is new research, right?
White. It is new. The concepts came from work done by others in England and also in the United States. The investigators take the microbes and genetically modify them to put a tag on the promoter region of NIF — the nitrogen fixation gene. That means that when the NIF gene is turned on, it expresses the tag — a color, or a fluorescence — and the researchers can see it with a microscope.
They put these modified microbes into the plant and found them accumulating in the root cells, in what looked like vesicles — indicating that in those vesicles there was nitrogen fixation happening. NIF was being expressed. That means the microbes there were trying to make nitrogen — or were at least using that gene.
That’s where the concept came from. And we see them all the time, accumulating. Some microbes are broken down when they’re taken into the root cells. Other microbes will go into vesicles, and they’ll accumulate in those vesicles as big multicellular structures. And very likely there is nitrogen fixation happening inside those structures.
Now, it’s not the kind of process that would happen in a legume with rhizobia. Those root nodules are structures where oxygen is controlled. Nutrients are controlled. Oxidative interactions of the microbes are controlled. Some of the same things are happening — microbes are going into cells and are becoming intracellular in those nodules. But rhizobia are a longer-term functioning structure — the root nodule lasts much longer, so nitrogen comes out much more efficiently and for a longer period of time. The nodule is the ultimate in extraction of nitrogen from microbes.
But what we see with these nitrosomes is that they’re temporary. They’re only in these areas near or behind the root tip, or whatever tissues they might be in, and they are not present in older tissues. It’s a different way of bringing in nutrients. Plants like grasses and others are very successful — they obviously don’t need root nodules — whatever they’re doing works fine. And what they’re doing is this. They’re doing this — they’re absorbing nutrients from the soil, and they’re getting them from live microbes.
Acres U.S.A. When you say that nitrogen is being fixed in these nitrosomes, what form of nitrogen are they taking in and converting? N2 from the air?
White. Yes, N2. Don’t forget — around the roots in a good soil, with good structure, there are air spaces everywhere. They’re just getting the nitrogen out of the air and are taking it in. And very likely — we don’t know about this for sure — the plant is actively metabolically encouraging that process — not just by providing nutrients for the process, but possibly also manipulating it in some other ways that we don’t know about.
Acres U.S.A. It makes sense that N2 would be able to get to those nodules on legumes because of the air in the soil. Can N2 also flow through the outer wall of the plant because it’s an inert gas?
White. Yes, it can flow in there. And don’t forget — these are in the surface layers of the root — maybe a layer or two underneath. It’s close to the air.
Acres U.S.A. And this is only in the roots, not in other parts of the plant, like trichomes?
White. Oh, no — this happens all over the plant. In trichomes, in epidermal cells, in vascular tissues. Some will be even more deeply embedded. It really depends on how the microbe is distributed in the plant. If it’s coming off the surface of the seed, then, when the seed germinates, it’s colonizing from the surface and is just going to be a couple of layers. If it’s internal in the embryo, it may be throughout the column of tissue. It just depends.
Acres U.S.A. Is there a name for the type of bacteria that gets incorporated into these nitrosomes?
White. No. It could be many different things. It’s a difficult problem — nitrogen fixation. And this is not my area of research — other people are doing it. But most nitrogen fixation research is dominated by people who work on root nodules, and they’ve already dismissed associative nitrogen fixation — which is what they call other microbes on plants that may be fixing nitrogen — as largely irrelevant. It’s little studied — partly as a consequence of that.
I’ve found that the best thing to do is to study endophytes and how they function in plants and to have a broader perspective. There’s still a lot unknown about how endophytes work.
Acres U.S.A. Can you talk a little bit about the sequence, or hierarchy maybe, of what the plant extracts from microbes — that it extracts micronutrients first before getting to the primary macronutrients?
White. Sure. Plants bring in a lot of different microbes. We’ve found from experiments in the lab that different microbes bring in different nutrients. If you put them on one at a time, you’ll see different nutrients going into the plant, depending on what microbe you use. So, we draw the conclusion that different microbes are effective at bringing in different nutrients.
Some nutrients can be obtained very easily in the soil, like nitrogen, phosphorus, and potassium. But some of the micronutrients are more difficult to obtain. They’re not in a soluble form plants can get — but plants can oxidize them out of microbes. And we think that micronutrients like manganese and maybe some of the others are critical for the rhizophagy cycle. Micronutrients may be the key.
Plants get all kinds of nutrients from microbes, based on the experiments we do. Plants cannot develop properly without microbes. They need them for development. They need them in order to be hardy. If you don’t have healthy soil, plants aren’t going to be hardy — they’re not going to develop properly.
Acres U.S.A. Can you talk a bit about how elevated CO2 levels seem to repress rhizophagy? I think of CO2 levels as good for a plant — for photosynthesis.
White. Yes, to a certain level. But in our experiments, we raised the CO2 level using dry ice to the point that the carbon dioxide in the air suppresses reactive oxygen formation by the plant. And because of that repression, because CO2 will essentially prevent the plant from producing any superoxide, it’ll compete for the binding sites. Essentially, the plant can’t make superoxide, and if it can’t make superoxide, it cannot process these microbes. It screws up development because the plant can’t make copies of the microbes inside it, and they don’t develop properly. The microbes that enter the root remain in their walled form and don’t replicate, and the plant can’t get nutrients out of them. You get this repression of development.
Acres U.S.A. Sometimes, people like you and us are criticized for engaging in what some people call “pop ecology.” These people say that rhizophagy is an intriguing idea, but there’s no evidence that it really works in large-scale agriculture, and we shouldn’t get too excited about it. How would you respond to that?
White. I would just say that what we’re seeing is that plants are using these microbes — to the extent that they even go into the cells of the plant. I mean, if plants are doing this naturally, why do we think that plants don’t need it? That’s kind of crazy, really. If we see, for example, that plants are taking on mycorrhiza — which everyone knows that they do — should we then say that plants don’t need to do that? That’s ridiculous. They do need this biology. So, I think it’s a narrow view of things. It’s kind of the idea that we are engineers of nature and that we know what’s best.
Acres U.S.A. It seems to me like a very nutrient-focused approach, while downplaying the role of microbes.
White. I mean, we’re humans — we’re in control of the universe — so we’d like to think! We can control everything!
But the fact is that there’s so much we don’t understand. We don’t know how the system is working. We know we can use our chemistry to grow crops. We know a lot about that. We also know that that’s causing problems all over the earth. We start having disease, and we start having to use pesticides and other chemicals. That’s affecting us in a big way.
We’re only now coming to understand the role of microbes in plant growth. And there are people who are applying that, using microbiology, and they’re meeting with success. So, while it’s true that we have a tried-and-true chemical approach to agriculture, that doesn’t mean it’s the best approach. That doesn’t mean it’s the approach that everybody has to use.
I think some criticism comes out of actual ignorance — people who don’t think that there’s another way agriculture can be done. It’s the view that what they have is solid — it explains everything, and anything that doesn’t fit needs to be ignored. That’s kind of where they’re coming from.
Acres U.S.A. What do you think the consensus is going to be ten years from now — on rhizophagy, on endophytes, on all of what we’ve been talking about? Will there be more acceptance of it?
White. I think so, but I think we’re always going to have chemical agriculture, because it’s easy. But it’s always going to be very expensive. It’s always going to have problems.
At the same time, our understanding of how endophytes work and their benefits, and development of products that use these microbes — the understanding of how to manage soils for microbiology, to maintain microbes in the soil and improve soil health — that’s going to increase. I think people will demand it more. People appreciate natural systems, and this is a natural system. Healthy soil, microbiology interface, rhizophagy — this is part of a natural system. The other way is just chemical manipulations. That’s not a natural system. That’s not treating the soil as something living and full of microbes that are essential for plant development.
