-: Thank you very much for the opportunity
to speak today to this group.
I'll be talking about finding and optimizing microbes.
This is a pointer?
No.
Voiceover: Turn it on.
-: Okay.
Great.
So first I want to start by thanking
the people in my lab before I forget.
And I'll try to keep to 30 minutes.
I want to thank Ellen Marsh, who's my lab manager.
I've been at UNL for a little over a year and a half,
and she's been critical in helping me set up my lab.
I have a graduate student, Morgan McPherson.
An undergrad from Malaysia, who's just
an amazing person to have in the lab, Yen-Ning Chai.
And then two post-docs, Peng Wang and Ying Yang.
And then, as Tom mentioned,
I like to do team science,
and so I have a long list of collaborators
that I've worked with since I've come here.
Aaron Lorenz and Greg Kruger on a project on corn.
Lisa Ainsworth and Andrew Leaky,
who are at the University of Illinois.
Ismail Dweikat, who I work with on some sorghum stuff.
Tom Clemente, we worked together
in the Center for Biotechnology,
as well as we have sorghum project together.
And then I'll mention two people
from the University of Minnesota,
Daryl Gohl and Kenny Beckmann.
We've gotten into sequencing,
and they've really helped us kind of kick-start this work.
And then finally, more recent collaborators
are George Graef and Brian Waters.
And I'll mention a project that we're doing with them.
Soils are some of the most complex
communities on the planet.
And they're really a challenge to work with.
One teaspoon of soil has maybe
a billion bacterial cells in it.
You may have heard a lot about the gut microbiome.
And so when you compare soils to the gut microbiome,
there's some interesting comparisons.
I won't belabor the point too much,
but in the gut we probably have more higher density
of microbes in our gut than you'd find in soils.
But what's really interesting in soils
is that the diversity of the bacterial
and fungal species in soils is just huge.
You know, it's 5,000 to 50,000 different species
in a soil sample whereas in our gut
there's probably only 500 to a thousand species of microbes.
And so this diversity in soil
makes it a really complex problem.
And so I kinda liken our work to an astronomer.
You know the astronomers look out into the universe
and they're constantly discovering new stars
and new planets and it just seems like
it's an endless type of discovery process.
And so we're doing the same kind of thing with soils.
We're discovering and looking for
all these unique different bacterial
and fungal species that are out there.
Our tools for the most part are fairly simple.
So we go out and we use shovels,
microscopes and Petri plates.
And then we are using a little bit more complex tool
in the area of sequencing.
Today I just want to briefly outline
some of the research questions
that we're asking and some of the tools
that we're using for this research.
I wanna just talk about exploring Nebraska soils.
And then I'll give you an example of some of the projects,
and a little tiny bit of the results,
and then finally I'll talk about
establishing our culture collection.
These are some of the questions we're answering.
They're pretty simple questions
and hopefully these simple questions
will lead to applications for agriculture in the long term.
We wanna know who's out there,
how do communities change,
these are microbial communities in soil, how do they change?
And then really what I wanna get to eventually
is what are these microbes doing in soils?
You know, how are they helping plants grow?
How are they helping plants acquire nutrients?
How are they protecting plant roots
against parasitic microbes?
One main question that we're asking
is how do plants alter their microbiome?
One of the projects that we've started,
this is in collaboration with Tom Clemente.
Tom has the sorghum lines.
Sorghum roots exude a compound called sorgoleone.
Tom's used transgenic methods
to knock out the production of sorgoleone.
And so we're testing how this root exudate
affects the microbiome and also
the nitrogen use efficiency of the system
through using this knockout line
and comparing it to the wild type.
We're also interested in how
the environmental stress alters microbiomes.
So there we have projects on drought.
We're gonna get more into drought
in the next five years with this DOE project,
and then also how low nitrogen stress affects microbiomes.
And then in addition to these questions,
there's a discovery aspect to the work,
so what I'd really like to find,
you know in addition to the basic research,
find some applications to agriculture.
So we're looking for what I call the good guys out there.
In particular we're interested in
nitrogen fixing endophytes that may improve
the nutrition for crop plants that are grown
in environments with less than optimal nitrogen.
And then we're also working on finding microbes
that will enhance a plant's ability to acquire iron.
So these are the tools, I'll talk a little bit
about the tools that we're working with.
One is the shovels, coolers, and tubes.
Those are pretty low tech.
I'll talk about how we identify
who's out there using this 16S DNA.
And then I'll also talk a little bit about
this next generation sequencing.
I get really excited about some of the new technologies
and I wanted to try to kind of give you an idea
of this new really cool technology of sequencing.
This just kinda reminds you what we're doing
when we're trying to figure out who's out there.
We're trying to either study how
the phylum level of a microbe community changes
all the way down to the species level.
This is the shovels, coolers, and tubes.
And this slide just kinda shows you
how we approach our field work.
We go out with shovels, a lot of tubes,
buckets, coolers, and we dig up plant roots.
This is the transgenic sorghum field
that we've been working in this past summer.
You can see it's a beautiful, well managed field.
And this facility that Tom Clemente really spearheaded.
So we're one of the few public universities
that can do this kind of transgenic research in the field,
and so it's very exciting to be able to
answer questions about these transgenic plants.
These are our shovels that we use.
And then we dig the plants up, we shake off the roots,
and then we partition the roots,
what we dig up into what we're calling bulk soil,
the root, and then the rhizosphere.
So we take the roots, cut them,
and we put them in tubes.
We shake 'em and with that comes the rhizosphere soil,
and then we're analyzing rhizosphere,
what we're calling the endosphere,
which are the microbes inside the roots,
and then the bulk soil.
And this is just kind of a cool picture.
We went down to South Carolina
to dig up some sorghum roots, this is energy sorghum,
and you can just see these massive, massive roots
of these energy sorghum varieties
growing in very sandy soils.
So that's the field work part of it.
And then we bring things into the lab,
and then we try to identify the microbes
that are in all these different samples.
And the way that we do that, answering the question is,
who's in the sample, is that we use
this 16S gene as our marker to figure out who's there.
So basically it's a tag
for a particular microbial species.
This is just showing you the anatomy of the 16S gene.
It's kind of a cool gene because it's very abundant,
so it's very easy to pick up
in many different types of samples.
You can see these regions here
in this yellowish green color,
these are highly variable regions.
And then you have these darker colors
that are the very conserved regions.
So with these conserved regions,
we can go in and we can make what's called PCR primers,
and we can amplify, let's say there's a V4 region,
and then we can sequence this V4 region.
And this is kind of our way of telling
which bacterial or fungal species is present in this soil.
So we sequence these pieces of DNA
and then there's these really good databases
that we can check them against
and then identify either the genus, the phylum,
or the species that are present in these samples.
Essentially what happens with this process
is that there's a glass slide
and five to 200 million pieces of DNA
are laid across this glass slide.
From that you synthesize
pieces of DNA from the existing DNA that's on the slide
and there are fluorescent tags,
so as the tags are added,
there's a beam of light that's emitted
and there's very high power microscopes or cameras
that capture these images.
And so what you can see here in the slide are
let's say a blue dot
or a blue light is captured
and then that's recorded as a T,
and a green here would be a G,
an orange would be a C.
And so it's continually captured
and then you have this array
of maybe five or 10 million pieces of DNA,
and then with software the computer's
able to figure out the sequence
of each strand of DNA.
So what you end up with is a huge amount of data
that luckily in this field, in the microbiome field,
there's some pioneers who developed open
source software that allows us to analyze the DNA.
The next thing I wanna briefly talk about, there we go,
is the field sites that we use.
We're using both agricultural fields and also rangeland.
Nebraska is really a fabulous place.
It's been discussed, you know we have this
great gradient of weather across the state,
and then we also have all sorts of soils,
and so we've been looking at a number of areas,
but kinda two of my favorites are highlighted here.
We've been doing some work in the Sandhills,
where we're looking for microbes
that may help plants grow under alkaline conditions.
This is just kinda the area, the Sandhills,
that we're collecting in.
We're looking at some of these alkaline lakes
that are found in the Sandhills.
This is an aerial view of the Sandhills.
And then this is just showing you one of these lakes
that we found on Rex Ranch.
Thanks to Don Adams, we were able
to get onto this ranch and do some sampling.
The pH of this water is about nine,
and what we're doing is digging up some of the plant roots
and taking some of the soil from around this soil,
looking at the microbes and trying to culture them as well.
Another favorite place of mine is Brule, Nebraska.
This in near North Platte, about an hour outside.
This is the project with Greg Kruger.
Brule's a really gorgeous place.
And the sky here is just never-ending.
And one of the great capabilities in Brule
that was developed by the university
is this variable rate irrigator.
So we can do really these high precision drought experiments
where we can dial in exactly how much water we're adding,
we can do a number of drought treatments.
And with Greg's help, we're doing
a corn project out at Brule.
I'll go into a little bit more depth
on a few different projects.
I want to just tell you about the work we're doing
on the changes in the root microbiome
in response to ozone and high CO2.
That's at the University of Illinois-Champaign.
I want to just tell you about the project
we started on the microbiome,
and how that relates to iron deficiency chlorosis.
And then just mention this new project
that was funded from the DOE.
At the University of Illinois they have this
really unique capacity to pump gases into a field.
Basically they have these rings here
that they can elevate either the CO2 or the ozone
and we're asking really simple questions like,
what happens to the root microbial communities
when we increase the CO2 for soybean,
or the ozone levels for corn?
And then this is just a short kind of bit of data.
You know the lab's fairly new.
We've been able to develop the methods
for DNA extraction and sequencing pretty well,
and then our next challenge is data analysis.
As I mentioned, you get a ton of data with this research,
and so this just kinda shows you some
of the methods that have been developed.
This is a large data set.
And this is principal coordinate analysis,
and all it's showing you here,
we haven't gone into huge depth
with this data to really understand it,
but what it's showing you here
is these are the root samples
that are coming out of the soil for soybean.
There's not a huge differentiation
between ambient and elevated CO2 here.
These are the rhizosphere samples,
and then these are the soil samples.
So you can see you can separate out
the different types of samples,
but really there's no differentiation in this data set
between ambient and elevated.
Another way you can look at this
is a lot more complicated.
So this is just showing you kind of all the
bacterial phyla that these DNA sequences let you identify.
This is showing the ambient conditions,
and then the elevated.
And here we have rhizosphere, root, soil,
rhizosphere, root, and soil.
What we aim to do is to figure out
what the differences are, what's going on,
and eventually figure out what
these differences will be,
you know why these differences occur
and how they'll affect plant growth and development.
So if you look kinda carefully here,
you can see this orange bar is
pretty much absent from this sample.
This is bacteroidetes that's the phylum here.
And you can see it's present,
roots growing under ambient CO2 conditions,
and here it's absent from the root
growing under the elevated CO2.
So as we dig into this data,
we'll be trying to figure out
if this has any functional significance.
Another way to look at the data
is just looking at distances between
different types of samples.
And what's kind of interesting here
is you can see these are the root microbes
are pretty far away from the soil microbes.
So roots somehow,
what the endophytes that are inside the root
are selected probably by the plant,
and so the overall community looks a lot different
in the root than in the soil.
And what's interesting here is the rhizospheres
somewhat intermediate between the root and the soil,
and so the outside of the root
is a place where the root is exuding some compounds,
so it may influence the microbes that are growing there,
but yet the bulk soil is a huge influence
on what's growing there.
So no conclusions yet,
but this is kinda what the data looks like.
So then the soybean project that we started
and the work on induced deficiency chlorosis
with George and Brian Waters.
We're doing a couple things here.
We're trying to understand
does or can the root microbiome
play a role in alleviating stress due to alkalinity.
This is the agricultural part of the problem,
so these are some lines that George is working with,
and you can see some of them are very chlorotic.
Some of them look great.
And so we want to understand are there differences
in the microbiomes in these sensitive
varieties versus the tolerant varieties.
So it's an exploratory project.
And then what we're doing in the Sandhills
is we're prospecting for microbes
in the roots of these plants growing in alkaline conditions
to find microbes that may help plants
acquire iron more efficiently.
And then finally, the last project I'll mention briefly
is this DOE funded sustainable biofuel project.
This is just showing you a grain sorghum,
what that looks like.
These are sweet sorghums.
And then you have these giant biomass sorghums
or energy sorghums.
You can see that this is what they look like.
It's really quite amazing to,
you know if you know sorghum,
it's a little tiny seed, you plant it in the ground,
and in about three months or four months
you get these just tremendously giant plants.
So it has huge potential to be
an important biofuel feedstock.
The project is pretty complicated.
It will be starting with some field surveys,
as well as some laboratory work.
In the fields we'll be applying stresses of nitrogen.
And water stress, we'll be doing phenomics,
with Art Zygielbaum and a physiologist at Washington State,
we'll be looking at how the plants grow in the field.
We'll be doing microbial census,
so we'll be digging up roots and looking at
how the microbe component of the root changes
with climate, and stress,
low nitrogen, and low water,
and how these also change with the genotypes
of sorghum that we're using.
We have wide diversity of sorghum,
so we have sorghum from three different breeders
in the United States, including Ismail Dweikat.
And then we'll be doing physiology on these plants.
We'll be doing laboratory studies
on these plants at the Danforth Center,
trying to understand the plant genotype,
by microbial genotype, by environment interactions.
And then we'll be testing some more controlled environments
and eventually we hope to identify the best genetics.
The plants that grow best under marginal conditions,
where water and nitrogen may be limiting
and then also look for microbial cocktails
that may give these plants an advantage
in these marginal lands.
Again, this is a map of Nebraska.
And we'll be capitalizing on the moisture
gradient across the state.
In western Nebraska we'll be working with Cody Creech
and we'll be doing the drought experiments there,
and then we'll be doing the nitrogen experiments
more in central to eastern Nebraska.
And then these are just the stats on the project.
It's a big project.
I get to lead 15 PIs at eight different universities,
plus one DOE institute.
All told, it's about a 14.5 million dollar project,
so I keep making it bigger it sounds like.
I think Roch's started at 13.5, then it went to 14.
But really it's about 14.5 million.
It's five years.
We'll be using four field sites.
Two with low nitrogen, two with drought.
We'll be collecting about 16,000 samples
for this microbiome analysis.
And then in the project we'll be sequencing
about a thousand microbial genomes.
So part of the work that we're doing
is building these culture collections.
So as we find these associations in the field
between a certain microbe and drought conditions,
what we want to be able to do
is to go back into this collection,
pull out a microbe that's either the same or similar
and then test it in the greenhouse
to determine if this microbe increases
the drought tolerance or nitrogen use efficiency.
And so the way that we're doing this
is we're collecting both endophytes,
those are from the roots,
and then the soil microbes either from soils or rhizosphere.
We're kinda prospecting widely.
Looking at these alkaline soils.
The soils in Brule are yielding some interesting microbes.
We've collected microbes from sandy soils in South Carolina.
From Mead and from various plant species,
including corn, sorghum, switchgrass,
sideoats and soybean.
And so we're using standard culturing methods.
How am I doing on time?
Okay, I've got about four more minutes.
Standard culturing methods
with seven different types of media.
So for example, for the nitrogen fixing endophytes,
we are growing the microbes
on nitrogen free media, to pick those up.
And then we're also using a more
innovative method called an iCHIP.
Basically what this is is it's like a 96 well format.
It has membranes on both sides
and then agar plugs
inside these individual wells.
And we take what we hope is like
one bacterial cell will innoculate
the individual wells in this plate
and then we'll cover it with these membranes
and then we'll bury it in soil and leave it
for two or three months and this should allow
the microbes to grow with the nutrients
that they would normally be getting in soil.
And it's been shown to be a method
where the dogma in the field now
is that about only 1% of soil microbes are culturable,
but with this method people have been able
to culture about 50% of soil microbes.
So this is just showing you what some
of the microbes look like that we've been able to culture.
I think this one is actually from these alkaline lake areas.
And it's kind of a cool one.
So it basically turns the media blue.
So this is kinda the normal color of the media.
It exudes something that turns the media blue.
And you can see when we look
at the other side of the plate
you can see that the bacteria themselves
are actually a white color.
So this looks like it'll be pretty interesting.
This is just showing you kind of some of the diversity
in color of these different microbes.
And then what we do is we amplify this 16S gene,
we sequence it to determine what
genus or species the bacteria belongs to.
And this is just kind of another cool microbe here.
It turns the plate red.
And this one itself is red.
This one is one of our favorite ones.
It's a purple bacteria that we found in soils.
It's been characterized before,
but this just kinda shows you the diversity
of the microbes that are in the soil.
So the status of the collection right now we have so far,
it's really pretty quick and easy
to build up these collections.
It's almost overwhelming.
So we already have about 1200 bacterial isolates.
We have about a hundred fungal isolates.
And we've identified about 140 of them so far.
And it'll soon be 340 in the next week or two.
And then the next steps will be to assay,
to develop assay systems to test these on plants
to see if they improve growth
or provide nitrogen nutrition.
And then we'd also like to get
into some phenotyping of the actual bacteria.
So it's interesting when we'd have this 16S sequence,
you can sequence these isolates
and you can find two that have the same exact 16S sequence,
but then when you look at the deeper,
their genomes can be very different,
and their functional characteristics as well
that can be very different.
We'll eventually do some genome
sequencing of these microbes.
Okay, so just to recap,
I gave you an idea of some of the research questions
that we're asking, you know, who's out there?
How do plants influence?
How do the microbes influence the plants?
And then I told you about the tools that we're using.
The shovels and the coolers and the tubes,
as well as the 16S ribosomal RNA
that we're using as a tag.
And then the sequencing methodology.
And then I told you a little bit about
what we're doing exploring Nebraska soils.
I've only lived in Nebraska for about a year and a half,
and I'm very kind of taken by the beauty of this state
and the interesting diversity that we have here,
so it's really a great kinda natural lab to work in.
I talked a little bit about these projects
and some of the results.
And then I showed you what we're doing
with establishing a culture collection.
And so we're doing a lot of basic research,
but really I think a lot about agriculture
and how we can apply this research to agriculture.
There's some very difficult questions
that we need to really solve with this research.
One is how do we efficiently find the best microbes
that are enhancing growth and development?
There's thousands and thousands
and thousands of microbes out there,
so how do we pull these all out
and study them quickly and find the ones
that'll be most useful for agriculture?
And then the second part of the equation,
is how do we engineer stable relationships
between roots and specific microbes?
One of the things that I'm really
excited about to be at Nebraska,
is to have colleagues like Tom Clemente,
who have this unique capability to engineer crop plants,
and I really think that's where
the stable relationships are gonna be.
We're gonna need to engineer the plants
to really cultivate specific microbes
and make those stable when we go across
thousands and thousands of acres.
Okay, so that's pretty much the end.
I wanted to just point out
that today we have a talk at 3:30.
I organized the seminar series
and so I wanted to make you aware of this.
And so my collaborator from the University of Illinois
is actually here today visiting with faculty,
and she'll be giving a talk,
so come on over to Keim Hall at 3:30.
I was gonna advertise the popcorn,
but I heard that there's no popcorn today, Roch.
No.
Voiceover: So you point at me?
-: Yeah! (audience laughs)
Yep, you had to scoop the staff over here.
But anyways, we usually have popcorn,
and please come to the talk at 3:30.
And thank you very much for your attention today.
(audience applauds)