Microbiome Research

Dr. Daniel Schachtman Author

01/07/2016 Added

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Microbiome Research presented by Dr. Daniel Schachtman

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[00:00:02.373]Thank you very much for the opportunity
[00:00:03.910]to speak today to this group.
[00:00:07.892]I'll be talking about finding and optimizing microbes.
[00:00:13.858]This is a pointer?
[00:00:16.487]No.
[00:00:19.205]Turn it on.
[00:00:23.466]Okay.
[00:00:27.633]Great.
[00:00:29.085]So first I want to start by thanking
[00:00:31.543]the people in my lab before I forget.
[00:00:34.062]And I'll try to keep to 30 minutes.
[00:00:36.722]I want to thank Ellen Marsh, who's my lab manager.
[00:00:39.489]I've been at UNL for a little over a year and a half,
[00:00:42.449]and she's been critical in helping me set up my lab.
[00:00:47.132]I have a graduate student, Morgan McPherson.
[00:00:50.092]An undergrad from Malaysia, who's just
[00:00:53.348]an amazing person to have in the lab, Yen-Ning Chai.
[00:00:57.237]And then two post-docs, Peng Wang and Ying Yang.
[00:01:00.399]And then, as Tom mentioned,
[00:01:03.161]I like to do team science,
[00:01:04.723]and so I have a long list of collaborators
[00:01:07.544]that I've worked with since I've come here.
[00:01:09.645]Aaron Lorenz and Greg Kruger on a project on corn.
[00:01:15.546]Lisa Ainsworth and Andrew Leaky,
[00:01:17.253]who are at the University of Illinois.
[00:01:19.156]Ismail Dweikat, who I work with on some sorghum stuff.
[00:01:23.033]Tom Clemente, we worked together
[00:01:26.067]in the Center for Biotechnology,
[00:01:28.343]as well as we have sorghum project together.
[00:01:31.803]And then I'll mention two people
[00:01:33.625]from the University of Minnesota,
[00:01:36.261]Daryl Gohl and Kenny Beckmann.
[00:01:38.245]We've gotten into sequencing,
[00:01:40.126]and they've really helped us kind of kick-start this work.
[00:01:43.311]And then finally, more recent collaborators
[00:01:46.016]are George Graef and Brian Waters.
[00:01:48.547]And I'll mention a project that we're doing with them.
[00:01:53.086]Soils are some of the most complex
[00:01:55.900]communities on the planet.
[00:01:58.535]And they're really a challenge to work with.
[00:02:01.785]One teaspoon of soil has maybe
[00:02:03.678]a billion bacterial cells in it.
[00:02:07.056]You may have heard a lot about the gut microbiome.
[00:02:10.237]And so when you compare soils to the gut microbiome,
[00:02:13.418]there's some interesting comparisons.
[00:02:15.101]I won't belabor the point too much,
[00:02:16.819]but in the gut we probably have more higher density
[00:02:21.242]of microbes in our gut than you'd find in soils.
[00:02:25.200]But what's really interesting in soils
[00:02:26.977]is that the diversity of the bacterial
[00:02:30.019]and fungal species in soils is just huge.
[00:02:33.861]You know, it's 5,000 to 50,000 different species
[00:02:38.598]in a soil sample whereas in our gut
[00:02:41.453]there's probably only 500 to a thousand species of microbes.
[00:02:46.299]And so this diversity in soil
[00:02:49.394]makes it a really complex problem.
[00:02:52.387]And so I kinda liken our work to an astronomer.
[00:02:56.903]You know the astronomers look out into the universe
[00:02:59.886]and they're constantly discovering new stars
[00:03:02.649]and new planets and it just seems like
[00:03:05.367]it's an endless type of discovery process.
[00:03:10.425]And so we're doing the same kind of thing with soils.
[00:03:13.373]We're discovering and looking for
[00:03:15.924]all these unique different bacterial
[00:03:17.792]and fungal species that are out there.
[00:03:20.626]Our tools for the most part are fairly simple.
[00:03:24.073]So we go out and we use shovels,
[00:03:26.813]microscopes and Petri plates.
[00:03:29.970]And then we are using a little bit more complex tool
[00:03:33.453]in the area of sequencing.
[00:03:38.092]Today I just want to briefly outline
[00:03:40.403]some of the research questions
[00:03:41.552]that we're asking and some of the tools
[00:03:44.234]that we're using for this research.
[00:03:47.450]I wanna just talk about exploring Nebraska soils.
[00:03:51.385]And then I'll give you an example of some of the projects,
[00:03:55.205]and a little tiny bit of the results,
[00:03:57.468]and then finally I'll talk about
[00:03:58.791]establishing our culture collection.
[00:04:04.131]These are some of the questions we're answering.
[00:04:06.048]They're pretty simple questions
[00:04:07.847]and hopefully these simple questions
[00:04:11.932]will lead to applications for agriculture in the long term.
[00:04:17.430]We wanna know who's out there,
[00:04:20.182]how do communities change,
[00:04:21.657]these are microbial communities in soil, how do they change?
[00:04:24.802]And then really what I wanna get to eventually
[00:04:27.091]is what are these microbes doing in soils?
[00:04:30.016]You know, how are they helping plants grow?
[00:04:32.953]How are they helping plants acquire nutrients?
[00:04:35.612]How are they protecting plant roots
[00:04:38.166]against parasitic microbes?
[00:04:47.589]One main question that we're asking
[00:04:49.067]is how do plants alter their microbiome?
[00:04:54.226]One of the projects that we've started,
[00:04:56.188]this is in collaboration with Tom Clemente.
[00:04:59.044]Tom has the sorghum lines.
[00:05:01.923]Sorghum roots exude a compound called sorgoleone.
[00:05:06.683]Tom's used transgenic methods
[00:05:08.703]to knock out the production of sorgoleone.
[00:05:12.081]And so we're testing how this root exudate
[00:05:16.174]affects the microbiome and also
[00:05:18.927]the nitrogen use efficiency of the system
[00:05:22.009]through using this knockout line
[00:05:24.297]and comparing it to the wild type.
[00:05:26.522]We're also interested in how
[00:05:28.264]the environmental stress alters microbiomes.
[00:05:32.731]So there we have projects on drought.
[00:05:36.046]We're gonna get more into drought
[00:05:37.949]in the next five years with this DOE project,
[00:05:40.598]and then also how low nitrogen stress affects microbiomes.
[00:05:46.908]And then in addition to these questions,
[00:05:48.627]there's a discovery aspect to the work,
[00:05:50.961]so what I'd really like to find,
[00:05:54.057]you know in addition to the basic research,
[00:05:56.963]find some applications to agriculture.
[00:05:59.274]So we're looking for what I call the good guys out there.
[00:06:03.441]In particular we're interested in
[00:06:04.822]nitrogen fixing endophytes that may improve
[00:06:08.073]the nutrition for crop plants that are grown
[00:06:12.541]in environments with less than optimal nitrogen.
[00:06:17.944]And then we're also working on finding microbes
[00:06:20.997]that will enhance a plant's ability to acquire iron.
[00:06:26.616]So these are the tools, I'll talk a little bit
[00:06:28.744]about the tools that we're working with.
[00:06:31.443]One is the shovels, coolers, and tubes.
[00:06:34.940]Those are pretty low tech.
[00:06:36.658]I'll talk about how we identify
[00:06:38.736]who's out there using this 16S DNA.
[00:06:42.404]And then I'll also talk a little bit about
[00:06:44.436]this next generation sequencing.
[00:06:46.479]I get really excited about some of the new technologies
[00:06:48.975]and I wanted to try to kind of give you an idea
[00:06:52.571]of this new really cool technology of sequencing.
[00:06:57.307]This just kinda reminds you what we're doing
[00:06:59.587]when we're trying to figure out who's out there.
[00:07:02.745]We're trying to either study how
[00:07:05.786]the phylum level of a microbe community changes
[00:07:10.267]all the way down to the species level.
[00:07:15.990]This is the shovels, coolers, and tubes.
[00:07:19.849]And this slide just kinda shows you
[00:07:22.368]how we approach our field work.
[00:07:24.353]We go out with shovels, a lot of tubes,
[00:07:28.045]buckets, coolers, and we dig up plant roots.
[00:07:33.211]This is the transgenic sorghum field
[00:07:35.839]that we've been working in this past summer.
[00:07:38.323]You can see it's a beautiful, well managed field.
[00:07:41.539]And this facility that Tom Clemente really spearheaded.
[00:07:46.043]So we're one of the few public universities
[00:07:49.039]that can do this kind of transgenic research in the field,
[00:07:52.694]and so it's very exciting to be able to
[00:07:56.301]answer questions about these transgenic plants.
[00:08:00.844]These are our shovels that we use.
[00:08:02.609]And then we dig the plants up, we shake off the roots,
[00:08:05.952]and then we partition the roots,
[00:08:07.890]what we dig up into what we're calling bulk soil,
[00:08:11.855]the root, and then the rhizosphere.
[00:08:14.961]So we take the roots, cut them,
[00:08:16.946]and we put them in tubes.
[00:08:18.363]We shake 'em and with that comes the rhizosphere soil,
[00:08:22.506]and then we're analyzing rhizosphere,
[00:08:25.886]what we're calling the endosphere,
[00:08:27.073]which are the microbes inside the roots,
[00:08:29.686]and then the bulk soil.
[00:08:32.008]And this is just kind of a cool picture.
[00:08:33.731]We went down to South Carolina
[00:08:38.747]to dig up some sorghum roots, this is energy sorghum,
[00:08:41.481]and you can just see these massive, massive roots
[00:08:45.057]of these energy sorghum varieties
[00:08:47.413]growing in very sandy soils.
[00:08:50.663]So that's the field work part of it.
[00:08:52.804]And then we bring things into the lab,
[00:08:54.941]and then we try to identify the microbes
[00:08:58.017]that are in all these different samples.
[00:09:00.037]And the way that we do that, answering the question is,
[00:09:02.717]who's in the sample, is that we use
[00:09:05.236]this 16S gene as our marker to figure out who's there.
[00:09:09.961]So basically it's a tag
[00:09:12.259]for a particular microbial species.
[00:09:15.463]This is just showing you the anatomy of the 16S gene.
[00:09:20.177]It's kind of a cool gene because it's very abundant,
[00:09:23.439]so it's very easy to pick up
[00:09:24.820]in many different types of samples.
[00:09:27.298]You can see these regions here
[00:09:29.113]in this yellowish green color,
[00:09:32.666]these are highly variable regions.
[00:09:34.570]And then you have these darker colors
[00:09:36.532]that are the very conserved regions.
[00:09:38.807]So with these conserved regions,
[00:09:40.688]we can go in and we can make what's called PCR primers,
[00:09:44.851]and we can amplify, let's say there's a V4 region,
[00:09:49.598]and then we can sequence this V4 region.
[00:09:52.344]And this is kind of our way of telling
[00:09:55.889]which bacterial or fungal species is present in this soil.
[00:10:00.661]So we sequence these pieces of DNA
[00:10:04.527]and then there's these really good databases
[00:10:06.528]that we can check them against
[00:10:09.348]and then identify either the genus, the phylum,
[00:10:13.748]or the species that are present in these samples.
[00:10:18.931]Essentially what happens with this process
[00:10:21.891]is that there's a glass slide
[00:10:25.060]and five to 200 million pieces of DNA
[00:10:29.649]are laid across this glass slide.
[00:10:34.665]From that you synthesize
[00:10:39.075]pieces of DNA from the existing DNA that's on the slide
[00:10:43.509]and there are fluorescent tags,
[00:10:45.854]so as the tags are added,
[00:10:48.895]there's a beam of light that's emitted
[00:10:51.671]and there's very high power microscopes or cameras
[00:10:56.268]that capture these images.
[00:10:58.148]And so what you can see here in the slide are
[00:11:02.224]let's say a blue dot
[00:11:07.412]or a blue light is captured
[00:11:10.177]and then that's recorded as a T,
[00:11:12.673]and a green here would be a G,
[00:11:15.030]an orange would be a C.
[00:11:16.910]And so it's continually captured
[00:11:19.534]and then you have this array
[00:11:21.171]of maybe five or 10 million pieces of DNA,
[00:11:25.234]and then with software the computer's
[00:11:28.833]able to figure out the sequence
[00:11:33.065]of each strand of DNA.
[00:11:34.745]So what you end up with is a huge amount of data
[00:11:39.212]that luckily in this field, in the microbiome field,
[00:11:42.515]there's some pioneers who developed open
[00:11:45.928]source software that allows us to analyze the DNA.
[00:11:55.256]The next thing I wanna briefly talk about, there we go,
[00:11:58.987]is the field sites that we use.
[00:12:02.203]We're using both agricultural fields and also rangeland.
[00:12:07.741]Nebraska is really a fabulous place.
[00:12:10.624]It's been discussed, you know we have this
[00:12:12.350]great gradient of weather across the state,
[00:12:16.030]and then we also have all sorts of soils,
[00:12:17.980]and so we've been looking at a number of areas,
[00:12:22.671]but kinda two of my favorites are highlighted here.
[00:12:26.660]We've been doing some work in the Sandhills,
[00:12:29.060]where we're looking for microbes
[00:12:31.184]that may help plants grow under alkaline conditions.
[00:12:35.619]This is just kinda the area, the Sandhills,
[00:12:38.085]that we're collecting in.
[00:12:39.710]We're looking at some of these alkaline lakes
[00:12:41.834]that are found in the Sandhills.
[00:12:44.852]This is an aerial view of the Sandhills.
[00:12:46.826]And then this is just showing you one of these lakes
[00:12:48.626]that we found on Rex Ranch.
[00:12:52.967]Thanks to Don Adams, we were able
[00:12:54.353]to get onto this ranch and do some sampling.
[00:12:57.627]The pH of this water is about nine,
[00:13:00.494]and what we're doing is digging up some of the plant roots
[00:13:04.012]and taking some of the soil from around this soil,
[00:13:06.786]looking at the microbes and trying to culture them as well.
[00:13:11.151]Another favorite place of mine is Brule, Nebraska.
[00:13:15.307]This in near North Platte, about an hour outside.
[00:13:18.848]This is the project with Greg Kruger.
[00:13:22.609]Brule's a really gorgeous place.
[00:13:26.107]And the sky here is just never-ending.
[00:13:28.874]And one of the great capabilities in Brule
[00:13:30.894]that was developed by the university
[00:13:34.324]is this variable rate irrigator.
[00:13:36.301]So we can do really these high precision drought experiments
[00:13:40.665]where we can dial in exactly how much water we're adding,
[00:13:44.160]we can do a number of drought treatments.
[00:13:46.144]And with Greg's help, we're doing
[00:13:47.898]a corn project out at Brule.
[00:13:54.736]I'll go into a little bit more depth
[00:13:56.160]on a few different projects.
[00:13:59.039]I want to just tell you about the work we're doing
[00:14:01.145]on the changes in the root microbiome
[00:14:03.803]in response to ozone and high CO2.
[00:14:06.822]That's at the University of Illinois-Champaign.
[00:14:09.875]I want to just tell you about the project
[00:14:11.500]we started on the microbiome,
[00:14:14.015]and how that relates to iron deficiency chlorosis.
[00:14:17.963]And then just mention this new project
[00:14:19.866]that was funded from the DOE.
[00:14:26.012]At the University of Illinois they have this
[00:14:27.639]really unique capacity to pump gases into a field.
[00:14:32.815]Basically they have these rings here
[00:14:35.333]that they can elevate either the CO2 or the ozone
[00:14:39.431]and we're asking really simple questions like,
[00:14:42.568]what happens to the root microbial communities
[00:14:46.092]when we increase the CO2 for soybean,
[00:14:49.970]or the ozone levels for corn?
[00:14:54.625]And then this is just a short kind of bit of data.
[00:14:58.905]You know the lab's fairly new.
[00:15:00.786]We've been able to develop the methods
[00:15:02.991]for DNA extraction and sequencing pretty well,
[00:15:06.405]and then our next challenge is data analysis.
[00:15:10.050]As I mentioned, you get a ton of data with this research,
[00:15:13.270]and so this just kinda shows you some
[00:15:15.626]of the methods that have been developed.
[00:15:19.745]This is a large data set.
[00:15:22.241]And this is principal coordinate analysis,
[00:15:25.898]and all it's showing you here,
[00:15:28.127]we haven't gone into huge depth
[00:15:29.601]with this data to really understand it,
[00:15:32.539]but what it's showing you here
[00:15:34.105]is these are the root samples
[00:15:38.006]that are coming out of the soil for soybean.
[00:15:41.222]There's not a huge differentiation
[00:15:43.845]between ambient and elevated CO2 here.
[00:15:47.816]These are the rhizosphere samples,
[00:15:51.152]and then these are the soil samples.
[00:15:52.685]So you can see you can separate out
[00:15:54.450]the different types of samples,
[00:15:56.145]but really there's no differentiation in this data set
[00:16:00.532]between ambient and elevated.
[00:16:04.321]Another way you can look at this
[00:16:06.039]is a lot more complicated.
[00:16:09.185]So this is just showing you kind of all the
[00:16:12.736]bacterial phyla that these DNA sequences let you identify.
[00:16:18.569]This is showing the ambient conditions,
[00:16:22.830]and then the elevated.
[00:16:24.305]And here we have rhizosphere, root, soil,
[00:16:30.759]rhizosphere, root, and soil.
[00:16:35.033]What we aim to do is to figure out
[00:16:37.737]what the differences are, what's going on,
[00:16:40.095]and eventually figure out what
[00:16:42.998]these differences will be,
[00:16:45.442]you know why these differences occur
[00:16:47.788]and how they'll affect plant growth and development.
[00:16:52.582]So if you look kinda carefully here,
[00:16:55.256]you can see this orange bar is
[00:16:57.834]pretty much absent from this sample.
[00:17:01.560]This is bacteroidetes that's the phylum here.
[00:17:06.323]And you can see it's present,
[00:17:09.779]roots growing under ambient CO2 conditions,
[00:17:12.721]and here it's absent from the root
[00:17:15.268]growing under the elevated CO2.
[00:17:18.170]So as we dig into this data,
[00:17:20.642]we'll be trying to figure out
[00:17:21.939]if this has any functional significance.
[00:17:25.102]Another way to look at the data
[00:17:26.813]is just looking at distances between
[00:17:29.975]different types of samples.
[00:17:31.774]And what's kind of interesting here
[00:17:33.400]is you can see these are the root microbes
[00:17:38.241]are pretty far away from the soil microbes.
[00:17:43.284]So roots somehow,
[00:17:46.824]what the endophytes that are inside the root
[00:17:49.596]are selected probably by the plant,
[00:17:52.271]and so the overall community looks a lot different
[00:17:55.731]in the root than in the soil.
[00:17:57.844]And what's interesting here is the rhizospheres
[00:18:00.386]somewhat intermediate between the root and the soil,
[00:18:04.589]and so the outside of the root
[00:18:07.850]is a place where the root is exuding some compounds,
[00:18:12.982]so it may influence the microbes that are growing there,
[00:18:15.938]but yet the bulk soil is a huge influence
[00:18:19.236]on what's growing there.
[00:18:20.989]So no conclusions yet,
[00:18:22.497]but this is kinda what the data looks like.
[00:18:28.929]So then the soybean project that we started
[00:18:31.356]and the work on induced deficiency chlorosis
[00:18:35.349]with George and Brian Waters.
[00:18:38.612]We're doing a couple things here.
[00:18:40.532]We're trying to understand
[00:18:43.634]does or can the root microbiome
[00:18:46.031]play a role in alleviating stress due to alkalinity.
[00:18:50.476]This is the agricultural part of the problem,
[00:18:52.937]so these are some lines that George is working with,
[00:18:56.096]and you can see some of them are very chlorotic.
[00:19:01.087]Some of them look great.
[00:19:02.531]And so we want to understand are there differences
[00:19:06.153]in the microbiomes in these sensitive
[00:19:10.460]varieties versus the tolerant varieties.
[00:19:14.349]So it's an exploratory project.
[00:19:16.474]And then what we're doing in the Sandhills
[00:19:18.458]is we're prospecting for microbes
[00:19:22.269]in the roots of these plants growing in alkaline conditions
[00:19:25.904]to find microbes that may help plants
[00:19:29.003]acquire iron more efficiently.
[00:19:34.088]And then finally, the last project I'll mention briefly
[00:19:36.885]is this DOE funded sustainable biofuel project.
[00:19:42.961]This is just showing you a grain sorghum,
[00:19:45.387]what that looks like.
[00:19:46.908]These are sweet sorghums.
[00:19:48.324]And then you have these giant biomass sorghums
[00:19:50.925]or energy sorghums.
[00:19:52.422]You can see that this is what they look like.
[00:19:55.301]It's really quite amazing to,
[00:19:57.623]you know if you know sorghum,
[00:19:59.248]it's a little tiny seed, you plant it in the ground,
[00:20:02.197]and in about three months or four months
[00:20:04.519]you get these just tremendously giant plants.
[00:20:07.247]So it has huge potential to be
[00:20:09.639]an important biofuel feedstock.
[00:20:17.556]The project is pretty complicated.
[00:20:19.483]It will be starting with some field surveys,
[00:20:23.082]as well as some laboratory work.
[00:20:25.566]In the fields we'll be applying stresses of nitrogen.
[00:20:29.699]And water stress, we'll be doing phenomics,
[00:20:32.844]with Art Zygielbaum and a physiologist at Washington State,
[00:20:37.964]we'll be looking at how the plants grow in the field.
[00:20:41.099]We'll be doing microbial census,
[00:20:43.316]so we'll be digging up roots and looking at
[00:20:46.902]how the microbe component of the root changes
[00:20:49.482]with climate, and stress,
[00:20:53.777]low nitrogen, and low water,
[00:20:55.600]and how these also change with the genotypes
[00:20:58.061]of sorghum that we're using.
[00:21:00.998]We have wide diversity of sorghum,
[00:21:03.197]so we have sorghum from three different breeders
[00:21:06.924]in the United States, including Ismail Dweikat.
[00:21:10.023]And then we'll be doing physiology on these plants.
[00:21:13.680]We'll be doing laboratory studies
[00:21:15.761]on these plants at the Danforth Center,
[00:21:17.923]trying to understand the plant genotype,
[00:21:21.185]by microbial genotype, by environment interactions.
[00:21:25.975]And then we'll be testing some more controlled environments
[00:21:29.849]and eventually we hope to identify the best genetics.
[00:21:34.087]The plants that grow best under marginal conditions,
[00:21:39.021]where water and nitrogen may be limiting
[00:21:41.761]and then also look for microbial cocktails
[00:21:45.510]that may give these plants an advantage
[00:21:47.705]in these marginal lands.
[00:21:52.213]Again, this is a map of Nebraska.
[00:21:55.161]And we'll be capitalizing on the moisture
[00:21:58.273]gradient across the state.
[00:22:04.275]In western Nebraska we'll be working with Cody Creech
[00:22:07.537]and we'll be doing the drought experiments there,
[00:22:09.812]and then we'll be doing the nitrogen experiments
[00:22:12.715]more in central to eastern Nebraska.
[00:22:18.090]And then these are just the stats on the project.
[00:22:20.713]It's a big project.
[00:22:21.734]I get to lead 15 PIs at eight different universities,
[00:22:25.693]plus one DOE institute.
[00:22:28.212]All told, it's about a 14.5 million dollar project,
[00:22:32.671]so I keep making it bigger it sounds like.
[00:22:35.689]I think Roch's started at 13.5, then it went to 14.
[00:22:40.368]But really it's about 14.5 million.
[00:22:42.937]It's five years.
[00:22:44.192]We'll be using four field sites.
[00:22:47.616]Two with low nitrogen, two with drought.
[00:22:50.719]We'll be collecting about 16,000 samples
[00:22:53.309]for this microbiome analysis.
[00:22:55.618]And then in the project we'll be sequencing
[00:22:58.394]about a thousand microbial genomes.
[00:23:02.550]So part of the work that we're doing
[00:23:04.372]is building these culture collections.
[00:23:06.148]So as we find these associations in the field
[00:23:08.876]between a certain microbe and drought conditions,
[00:23:12.429]what we want to be able to do
[00:23:13.717]is to go back into this collection,
[00:23:16.771]pull out a microbe that's either the same or similar
[00:23:20.253]and then test it in the greenhouse
[00:23:23.205]to determine if this microbe increases
[00:23:25.881]the drought tolerance or nitrogen use efficiency.
[00:23:29.363]And so the way that we're doing this
[00:23:31.302]is we're collecting both endophytes,
[00:23:34.518]those are from the roots,
[00:23:35.935]and then the soil microbes either from soils or rhizosphere.
[00:23:39.975]We're kinda prospecting widely.
[00:23:42.575]Looking at these alkaline soils.
[00:23:45.257]The soils in Brule are yielding some interesting microbes.
[00:23:48.901]We've collected microbes from sandy soils in South Carolina.
[00:23:53.139]From Mead and from various plant species,
[00:23:56.773]including corn, sorghum, switchgrass,
[00:23:59.454]sideoats and soybean.
[00:24:01.532]And so we're using standard culturing methods.
[00:24:05.096]How am I doing on time?
[00:24:06.014]Okay, I've got about four more minutes.
[00:24:08.079]Standard culturing methods
[00:24:09.694]with seven different types of media.
[00:24:11.714]So for example, for the nitrogen fixing endophytes,
[00:24:15.231]we are growing the microbes
[00:24:18.133]on nitrogen free media, to pick those up.
[00:24:21.593]And then we're also using a more
[00:24:23.056]innovative method called an iCHIP.
[00:24:25.733]Basically what this is is it's like a 96 well format.
[00:24:30.930]It has membranes on both sides
[00:24:36.247]and then agar plugs
[00:24:38.508]inside these individual wells.
[00:24:41.735]And we take what we hope is like
[00:24:43.593]one bacterial cell will innoculate
[00:24:46.368]the individual wells in this plate
[00:24:50.373]and then we'll cover it with these membranes
[00:24:53.774]and then we'll bury it in soil and leave it
[00:24:55.852]for two or three months and this should allow
[00:24:57.895]the microbes to grow with the nutrients
[00:25:00.705]that they would normally be getting in soil.
[00:25:02.829]And it's been shown to be a method
[00:25:04.454]where the dogma in the field now
[00:25:07.705]is that about only 1% of soil microbes are culturable,
[00:25:11.768]but with this method people have been able
[00:25:13.811]to culture about 50% of soil microbes.
[00:25:19.047]So this is just showing you what some
[00:25:20.405]of the microbes look like that we've been able to culture.
[00:25:23.771]I think this one is actually from these alkaline lake areas.
[00:25:26.790]And it's kind of a cool one.
[00:25:28.102]So it basically turns the media blue.
[00:25:31.352]So this is kinda the normal color of the media.
[00:25:34.162]It exudes something that turns the media blue.
[00:25:37.064]And you can see when we look
[00:25:39.710]at the other side of the plate
[00:25:41.464]you can see that the bacteria themselves
[00:25:43.588]are actually a white color.
[00:25:45.248]So this looks like it'll be pretty interesting.
[00:25:48.325]This is just showing you kind of some of the diversity
[00:25:51.209]in color of these different microbes.
[00:25:54.176]And then what we do is we amplify this 16S gene,
[00:25:58.262]we sequence it to determine what
[00:26:01.001]genus or species the bacteria belongs to.
[00:26:05.116]And this is just kind of another cool microbe here.
[00:26:09.148]It turns the plate red.
[00:26:11.434]And this one itself is red.
[00:26:14.041]This one is one of our favorite ones.
[00:26:16.167]It's a purple bacteria that we found in soils.
[00:26:21.339]It's been characterized before,
[00:26:24.053]but this just kinda shows you the diversity
[00:26:26.348]of the microbes that are in the soil.
[00:26:29.037]So the status of the collection right now we have so far,
[00:26:33.518]it's really pretty quick and easy
[00:26:36.409]to build up these collections.
[00:26:38.255]It's almost overwhelming.
[00:26:39.671]So we already have about 1200 bacterial isolates.
[00:26:43.351]We have about a hundred fungal isolates.
[00:26:46.416]And we've identified about 140 of them so far.
[00:26:50.549]And it'll soon be 340 in the next week or two.
[00:26:54.450]And then the next steps will be to assay,
[00:26:56.887]to develop assay systems to test these on plants
[00:27:00.165]to see if they improve growth
[00:27:01.569]or provide nitrogen nutrition.
[00:27:04.983]And then we'd also like to get
[00:27:06.805]into some phenotyping of the actual bacteria.
[00:27:09.487]So it's interesting when we'd have this 16S sequence,
[00:27:12.969]you can sequence these isolates
[00:27:15.303]and you can find two that have the same exact 16S sequence,
[00:27:19.645]but then when you look at the deeper,
[00:27:22.798]their genomes can be very different,
[00:27:25.081]and their functional characteristics as well
[00:27:27.542]that can be very different.
[00:27:30.567]We'll eventually do some genome
[00:27:32.041]sequencing of these microbes.
[00:27:34.948]Okay, so just to recap,
[00:27:36.538]I gave you an idea of some of the research questions
[00:27:39.278]that we're asking, you know, who's out there?
[00:27:42.332]How do plants influence?
[00:27:44.549]How do the microbes influence the plants?
[00:27:48.577]And then I told you about the tools that we're using.
[00:27:51.577]The shovels and the coolers and the tubes,
[00:27:55.223]as well as the 16S ribosomal RNA
[00:27:59.715]that we're using as a tag.
[00:28:01.759]And then the sequencing methodology.
[00:28:04.800]And then I told you a little bit about
[00:28:06.510]what we're doing exploring Nebraska soils.
[00:28:11.820]I've only lived in Nebraska for about a year and a half,
[00:28:14.214]and I'm very kind of taken by the beauty of this state
[00:28:18.346]and the interesting diversity that we have here,
[00:28:21.527]so it's really a great kinda natural lab to work in.
[00:28:26.135]I talked a little bit about these projects
[00:28:28.044]and some of the results.
[00:28:29.564]And then I showed you what we're doing
[00:28:31.179]with establishing a culture collection.
[00:28:33.523]And so we're doing a lot of basic research,
[00:28:35.880]but really I think a lot about agriculture
[00:28:38.875]and how we can apply this research to agriculture.
[00:28:42.764]There's some very difficult questions
[00:28:44.320]that we need to really solve with this research.
[00:28:47.303]One is how do we efficiently find the best microbes
[00:28:50.380]that are enhancing growth and development?
[00:28:52.643]There's thousands and thousands
[00:28:55.221]and thousands of microbes out there,
[00:28:57.101]so how do we pull these all out
[00:28:59.876]and study them quickly and find the ones
[00:29:02.419]that'll be most useful for agriculture?
[00:29:04.972]And then the second part of the equation,
[00:29:06.922]is how do we engineer stable relationships
[00:29:09.779]between roots and specific microbes?
[00:29:13.760]One of the things that I'm really
[00:29:14.763]excited about to be at Nebraska,
[00:29:18.303]is to have colleagues like Tom Clemente,
[00:29:20.816]who have this unique capability to engineer crop plants,
[00:29:25.901]and I really think that's where
[00:29:27.673]the stable relationships are gonna be.
[00:29:29.891]We're gonna need to engineer the plants
[00:29:31.644]to really cultivate specific microbes
[00:29:34.407]and make those stable when we go across
[00:29:38.133]thousands and thousands of acres.
[00:29:41.349]Okay, so that's pretty much the end.
[00:29:44.360]I wanted to just point out
[00:29:45.706]that today we have a talk at 3:30.
[00:29:48.283]I organized the seminar series
[00:29:50.106]and so I wanted to make you aware of this.
[00:29:52.323]And so my collaborator from the University of Illinois
[00:29:55.260]is actually here today visiting with faculty,
[00:29:58.186]and she'll be giving a talk,
[00:29:59.787]so come on over to Keim Hall at 3:30.
[00:30:02.458]I was gonna advertise the popcorn,
[00:30:04.547]but I heard that there's no popcorn today, Roch.
[00:30:09.282]No.
[00:30:10.106]So you point at me?
[00:30:11.208]Yeah! (audience laughs)
[00:30:14.115]Yep, you had to scoop the staff over here.
[00:30:16.517]But anyways, we usually have popcorn,
[00:30:18.194]and please come to the talk at 3:30.
[00:30:22.780]And thank you very much for your attention today.
[00:30:25.338](audience applauds)

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