Microbes, Siderophores, and Iron Deficiency with Dr. Tarah Sullivan

Relevant Citations:

Lewis, R., Islam, A., Dilla-Ermita, J.C., Hulbert, S.H., and T. S. Sullivan*, 2019. High-throughput siderophore screening from environmental samples: plant tissues, bulk soils, and rhizosphere soils. Journal of Visualized Experiments Issue 144 (DOI: 10.3791/59137, https://www.jove.com/video/59137/high-throughput-siderophore-screening-from-environmental-samples)

Lewis, R., Opdahl, L., Islam, A., Davenport, J., and T. S. Sullivan*, 2019. Comparative genomics, siderophore production, and iron scavenging potential of root zone soil bacteria isolated from ‘Concord’ grape vineyards. Microbial Ecology (DOI: 10.1007/s00248-019-01324-8).

Sullivan, T.S.*, and G.M. Gadd, 2019.  Metal bioavailability and the soil microbiome. Advances in Agronomy, Volume 155 (https://www.elsevier.com/books/advances-in-agronomy/sparks/978-0-12-817408-1).

“How soil microbiology might help your PB&J” May 15, 2019, WSU Insider   (https://news.wsu.edu/2019/05/15/soil-microbiology-might-help-pbj/)

“Digging into soil bacteria and chlorosis,” February 14, 2019, Good Fruit Grower (https://www.goodfruit.com/digging-into-soil-bacteria-and-chlorosis/).

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Episode Transcription:

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Drew Lyon: Hello. Welcome to the WSU Wheat Beat podcast. I’m your host, Drew Lyon, and I want to thank you for joining me as we explore the world of small grains production and research at Washington State University. In each episode, I speak with researchers WSU and the USDA-ARS to provide you with insights into the latest research on wheat and barley production. If you enjoy the WSU Wheat Beat podcast do us a favor and subscribe on iTunes or your favorite podcasting app and leave us a review while you’re there so others can find the show too.

[ Music ]

Drew Lyon: My guest today is Dr. Tarah Sullivan. Dr. Sullivan is a soil microbiologist from the Department of Crop and Soil Sciences at Washington State University. She originally trained as a soil scientist at Colorado State University, and then Cornell. Her current research at WSU addresses different aspects of how soil microbial communities assist in nutrient cycling and availability to plants. She runs an active research lab, teaches both undergraduates and graduate courses and engages in outreach locally to increase understanding of these highly complex systems that she studies. Hello, Tarah.

Dr. Tarah Sullivan: Hi, Drew.

Drew Lyon: So, the last time you were on my podcast, we spoke about the wide array of projects you’re working on. Most of these projects focused on microbial soil health and agricultural sustainability. But today, we’re going to focus in a little bit on your work on iron deficiency. Can you tell us why you’re interested in studying iron deficiency?

Dr. Tarah Sullivan: Well, Drew, when you think about something like soil health, and agricultural sustainability, those as I study them through the lens of soil microbiology, are key functions in services and processes that the microbial community actually provides to the system. And, nutrient availability is a really key process involved in soil health and agricultural sustainability. And when you think about iron, we realize that iron is all around us; in soils all over the place for the most part. The problem is that iron is not readily bioavailable. Which means plants and microbes can’t actually use it in their cellular metabolism in the form that it’s found in soils and in the environment. And so, iron is a very important element and nutrient that has to be changed biochemically in order to be used by plants and microbes. And so, this is a key function that my lab has really targeted to look at. In microbial communities, we not only look at the entire community, who’s there, basically the taxa, the genus and species of all the community, but we’re really interested in those functions that contribute to soil health and sustainability. So, iron deficiency, in any type of plant, allows us to really examine the microbial contribution to the bioavailability of that particular metal.

Drew Lyon: Okay. So, because it’s deficient, then you can see what impact the microbes have. If it’s not deficient, you can’t really get at that? Is what you’re telling me?

Dr. Tarah Sullivan: It doesn’t have to be deficient, but it’s provided us with an opportunity, in collaboration with Joan Davenport’s lab, we’ve been able to identify some vineyards, down in the Yakima Valley where the grapevines are displaying, primarily juice grapes, are displaying what they call “iron chlorosis”. So this is an iron deficiency and it’s classically treated with foliar applications of iron chelates. The interesting thing about it is that we’ve identified whole vineyards where there are huge areas of very, very healthy vines, interspersed with a few very chlorotic or iron deficient vines. And yet, the soils are all the same; the climate is the same; the management is the same. So this allows us to look very specifically at the iron deficiency and see what’s different in the microbial community of those plants that are iron deficient versus those plants that healthy.

Drew Lyon: Okay. So, how do you do that? Do you have a certain methodology you use to figure out what’s going on in there?

Dr. Tarah Sullivan: Yes. We’ve actually been pretty excited in my lab. Just in the last few months, we were able to publish a paper; I’m not going to give you the full, long title, but we’re studying specifically siderophore production. And what siderophores are is actually these small molecules that allow microbes to chelate iron, in the environment. So, like I said, it’s all around us, but it’s not bioavailable. Siderophores are molecules that certain types of plants can secrete, but also microbes can secrete it into the soil, and it actually chelates onto the iron and reduces it into a bioavailable form that plants and microbes can then use in their cellular metabolism. And so, the method that we actually have been working on, and we’re really excited to get out in the Journal of Visualized Experiments just recently, is a method that allows us to test a high number of either soil samples or plant tissue samples because we also know that microbes live inside plants and help them with nutrient absorption as well; but the method allows us to screen many, many samples simultaneously for this siderophore production. And it’s based on an older method, using a dye called Chrome Azurol S; I know that sounds complicated, we usually just call it CAS, so that’s C-A-S. And the key here is that the dye is a lot like a chelating molecule. And when it’s bound up with the iron, it shows as a bright blue. And we can actually find this on a spectrometer or look for the 420-nanometer wavelength, which is the bright blue. And so, we know that as that dye starts to give up the iron to another molecule, such as a siderophore, that dye changes color. And the dye will change color according to what type of siderophore is being produced. So, we’ve based this, you know, utilized this existing technology, but set it up in a way that we can do many, many samples all at once. So, we could take a whole vineyard, sample the soils from each grapevine and basically innoculate them into this media, with this dye, and incubate it over a period of time. Basically taking pictures of the plate, with all the different samples in the dye, and look for changes from the blue. And we can actually quantify that using a plate reader. So, this is, for us, it’s really instrumental in wide surveys of large areas, but also, it allows us to cultivate the organisms that are actually secreting the siderophores. So, what I mean by that is, we can literally take a single well that has changed color and get the microbes out of that well and put them onto another petri dish and cultivate it. We can then test it for other biochemical reactions. We can look and see if it has plant growth promoting characteristics. There’s a whole host of things that we can actually do with either the organism or extract the DNA and look for the genes that are actually responsible for siderophore production.

Drew Lyon: Okay. So, what’s the ultimate goal there; if to understand, you mentioned that some of these plants are iron deficient and some aren’t; so see whether there’s a different microbial presence in one set of roots than another set of roots? And therefore, be able to hopefully, what? Maybe transfer some of those microbes from one healthy rhizosphere into an unhealthy one?

Dr. Tarah Sullivan: Potentially. There’s a whole host of things we could actually do, but that would be ideal is if we know that these organisms are adapted to this environment, and we know under the healthy vines we have the siderophore producers that are chelating the iron in such a way that makes those vines healthy, then potentially we could take those organisms and try to inoculate them onto the sick vines. The other things that we’re looking at are, basically, is the function in siderophore production even different between the vines? If there’s no difference, then maybe they aren’t actually impacting iron availability to the plant at all. So there was that hypothesis was one: are they, can we actually link this activity to the health and status of the vine? And so, this is something we at first did through just chlorsis and over the next couple of years, we’re going to start looking at grape quality indicators as well to see how many– what the yield is, what the acidity of the juice is, the color, different things like this and see if any of this might be linked to the microbial activity specifically having to do with iron chelation.

Drew Lyon: So, I suppose there might be a little bit of this chicken and egg thing, right? Because it’s an interaction between the two: is the plant affecting the microbes? Or is the microbes affecting the plant? How do you tease that out?

Dr. Tarah Sullivan: That’s a really good question. I would love to explore that more through looking– first taking a healthy vine and following the microbial community and how it changes as the vine gets sicker. Because you’re exactly right. Did the vine actually lose a certain amount of functionality in the rooting zone and then the microbial community changed? Which we have definitely shown in our data, that the communities are completely different.

Drew Lyon: Okay.

Dr. Tarah Sullivan: Or is it something where the healthy vines are secreting very specific molecules to recruit the plant beneficial organisms into the rhizosphere. And so, we don’t actually know which happens first, but we do know that the communities are different, and they do function differently. We’ve actually been able to identify organisms that we cultivated from healthy vines and from the chlorotic vines and find a whole host of different functionality of these different types of organisms. And, one thing that you’ll hear about in macroecology are some concepts like cheating, where there’s some community good and some organism may or may not contribute to that community good, but they are able to utilize it. And so, in the case of siderophores, we’ve actually found in some of the genetic data that we’re looking into that the, a lot of these organisms associated with the sick vines could receive a huge array of siderophores because they’re very specific membrane receptors for these large molecules by the time they’ve found the iron. And yet, they were not producing the same number or the same types of siderophores. So, this was some indication, it’s not proof, but it’s some indication that they can actually sequester iron from not only other microbes in the environment but potentially from what the grape roots could actually sequester themselves. We know that grapes actually secrete hydrogen ions into their rhizosphere to try to acidify.

Drew Lyon: Okay.

Dr. Tarah Sullivan: And therefore, try to reduce the iron and make it more bioavailable. And the microbes that we found, under the sick vines, were actually able to take advantage of that iron, through their different pathways that we found in their genomes. So, that might indicate that the, even though they’re producing siderophores, when we get them into the lab, they could actually be detrimental to grape health. They’re not pathogens, they’re just cheaters.

Drew Lyon: Okay. So, a grapevine’s a perennial plant; has its root system that develops over a long period of time. Wheat’s an annual.

Dr. Tarah Sullivan: Right.

Drew Lyon: The root system doesn’t exist as long; of some of these same concepts, could they be applied to wheat? An annual crop like that?

Dr. Tarah Sullivan: Yes, in fact, wheat is really interesting because it falls into the category of strategy 2 plants. So, when it comes to siderophore production, there are strategy 1 plants and strategy 2 plants. Strategy 2 plants are mostly grasseous type species; wheat is one of those that actually secretes its own phytosiderophores into their rhizosphere.

Drew Lyon: Oh, okay.

Dr. Tarah Sullivan: So, there’s a lot of cross-talk, if you will, between the microbes in the rhizosphere of wheat, when it comes to siderophores. So the microbes can secrete the siderophores, but they can also take up the siderophores and wheat can do the same thing. So, wheat can secrete its own phytosiderophores, and it can also take up microbial siderophores. And so, we’ve been looking into how different genotypes or isolines of wheat can impact not only the structure of the microbial community in the rhizosphere, which we talked about a little bit last time, but now we’re also applying this method of siderophore detection to these different wheat genotypes to see if anything that’s happening in the rhizosphere is affecting or being affected by these siderophore producing microorganisms. And, what we found is really fascinating. We had these two different isogenic wheat lines, basically, I’ll call them 2-5 and 2-7, where we knew that 2-5 was tolerant to aluminum. And we’ve talked about how acidification in the Palouse soils is really causing a lot of aluminum to come into solution and it’s causing a lot of yield loss and some other problems. So, we have this isogenic line, 2-5, that’s aluminum tolerant, and it’s been assumed that this is because of an active gene that allows the wheat to secrete malate into the rhizosphere. So, we know malate is another chelating molecule, and it supposedly protects the rooting system from aluminum. And then we have the isogenic lines to that, that’s 2-7, but doesn’t have that active gene that actually transports malate or produces malate in the rhizosphere. And so, we wanted to compare, is the microbial community actually different between the aluminum tolerant and the aluminum susceptible? And what we found was not only is the genetic makeup of the community completely different, but the siderophore production is also completely different.

Drew Lyon: Oh, that’s interesting.

Dr. Tarah Sullivan: We found that the tolerant line actually had much– the community associated with that tolerant line had a much higher capability to produce siderophores. So, it’s definitely possible that that malate production or something else in the rhizosphere there, is recruiting these organisms that are able to chelate the aluminum because siderophores don’t just chelate iron; they also chelate a number of other metals.

Drew Lyon: Okay.

Dr. Tarah Sullivan: It’s possible that they’re chelating the aluminum, and that’s contributing to protecting the rooting system from these aluminum ions blocking the calcium channels.

Drew Lyon: That is really fascinating. And it opens up a whole new avenue of research and sounds like this new technique may be kind of key to helping you get at what’s going on there.

Dr. Tarah Sullivan: I certainly think so. It allows us to look at individual genes, whole genomes, metagenomics, as well as, like you said, being able to cultivate the organisms and if they work well for a particular species of crop in one location, potentially they could work where we’re seeing yield decline in other aluminum impacted areas as well. So, I think it has a lot of potential to move things forward, in terms of understating the function of microbes in metal biogeochemistry and how we can use that in agricultural sustainability, for sure.

Drew Lyon: Okay, how do you see taking this technique and moving– moving forward with it in your future research?

Dr. Tarah Sullivan: Well, the first thing we’re doing right now, Scott Holbert and I are co-advising a student who has actually planted over 30, maybe over 35 different parental lines of wheat, in a couple of the different research farms around Pullman. And we’re going to examine all their above-ground characteristics and look for everything from yield to grain quality to even the micronutrient content of the grain.

Drew Lyon: Okay.

Dr. Tarah Sullivan: And see if we can tie that to the microbial community siderophore production. And if we can, that’s really huge in terms of micronutrient nutrition, for not only the plant but also the consumers, in the long run.

Drew Lyon: Very, very interesting. So, if our listeners want to learn a little bit more about your work, is there someplace they can go to find that?

Dr. Tarah Sullivan: They can definitely go to the Crop and Soils WSU website, which is css.wsu.edu and look for my faculty page. But hopefully also, by the time this podcast is out, we will have a link to all of these articles that I’ve recently been putting out on the method and on things we’re able to do with the method in the notes for the show.

Drew Lyon: Okay, we’ll make sure we get that web address in the show notes, as well as any links that you have to papers. Well, Tarah, this has been fascinating. I’m really looking forward to finding out what you learn in the coming years. Thank you for sharing this with our listeners today.

Dr. Tarah Sullivan: Thank you, Drew.

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Drew Lyon: Thanks for joining us and listening to the WSU Wheat Beat podcast. If you like what you hear don’t forget to subscribe and leave a review on iTunes or your favorite podcasting app. If you have questions or topics, you’d like to hear on future episodes please email me at drew.lyon That’s lyon@wsu.edu (drew.lyon@wsu.edu). You can find us online at smallgrains.wsu.edu and on Facebook and Twitter @WSUSmallGrains. The WSU Wheat Beat podcast is a production of CAHNRS Communications and the College of Agricultural, Human and Natural Resource Sciences at Washington State University. I’m Drew Lyon, we’ll see you next time.

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