Download Expected and achieved results

Microbial Ecology of Rhizosphere
Bacteria in Brazilian Maize as
Influenced by Plant Genotype and Soil
Environment
David Morris Johnston Monje
Plant Agriculture, University of Guelph
Advisor: Manish Raizada
Brazilian Partner: Dra. Veronica Reis (EMBRAPA Agrobiologica)
Funding: Manish Raizada and IICA Canada
Maize has been important all over the
Americas for thousands of years...
Mayan maize god
Yumil Kaxob
Zapotec maize god
Pitao Cozobi
Mitchell Corn Palace, USA
Moche culture
Peru
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Where does maize come from?
Developed
9,000 years ago
In Oaxaca, Mexico
Already
researched
genotypes
and soils
(by Dr.
Johnston)
Distinct
genotypes
and soils, yet
unresearched
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Dramatic differences in soil may have
forced maize to develop novel
bacterial surface populations
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Dramatic Physiological Change in Plants May
Have also Altered Bacterial Populations
Domestication,
breeding, and migration
have altered plant and
seed architecture while
forcing maize to grow in
different environments
and to be completely
dependent on humans.
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Obvious structural or environmental differences
may coincide with changes to the microbial
ecology
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What are plant associated bacteria and
why study their ecology in maize plants?
• Plants have been shown to have large numbers of bacteria
inside their bodies and on their surfaces
• Plant root surfaces interact with soil, absorbing all the
water and nutrients needed for growth and metabolism.
Bacteria living on this surface or inside the plant are able to
influence both absorption and metabolism of these
compounds.
• Discovering new rare bacteria with capacity to improve
plant growth, nutrient acquisition and metabolism posses
great potential for productive, environmental agriculture
• Previous work done by Dr. David Johnston Monje suggests
that bacteria living inside the plant are not rare or
influenced by the external environment. Are root or leaf
surface bacteria rare or influenced by external
environment? What are these microbes and can we use
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them in agriculture?
Rare New Strains May Help Agriculture
• Rhizobial bacteria first patented and
sold as ‘Nitragin’ in the United
States in 1895
• Burkholderia phytofirmans PsJN
from rotting onions can promote
plant growth.
• High levels of nitrogen fixation found
in Brazilian sugarcane thanks to
Gluconacetobacter diazotrophicus.
• Nitrogen fixing Klebsiella pneumonii
isolated from wild maize shown to
improve wheat growth.
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(Dalton and Kramer 2006)
Previous Research on Maize Bacteria?
• 99% microbes in the world are believed to have not
yet been cultured.
• Chelius and Triplett (2001) studied 16S rDNA (a
bacterial gene used for barcoding) from in Wisconsin
grown maize roots of genotype Pioneer 3751.
• 74 phylotypes in 6 bacterial divisions including were
found by culture-independent analysis, as opposed
to 27 cultured phylotypes in 4 divisions.
It is impossible to accurately use only
culturing to study bacteria in corn!!!
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How can DNA identify bacteria?
• All life contains ribosomes: protein/RNA complexes
responsible for production of protein using
information coded in DNA
• A ribosomal gene called 16S is found in all bacteria,
although it has accumulated diagnostic mutations in
different species over evolutionary time
• Each sequence can be compared bioinformatically to
reference sequences to predict what species it is from
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TRFLP is one way to comprehensively view
a microbial community using DNA
Outline of the terminal restriction fragment length polymorphism method
Extract DNA
from the
community
PCR label with a
fluorescently labelled rRNA
forward primer
Relative fluorescence
Restriction Digest of PCR
Product
Recognition of labelled
fragments
Fragment separation in
sequencing gel
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How can a DNA fingerprint like TRFLP
be interpreted statistically?
Principle Component 2
Based on 99 microsatellites
Principle Component 1
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Matsuoka et al. (2002)
Objectives
• Objective 1: Isolate novel microbes from Brazilian corn
grown on diverse and stressful soils such as
contaminated mine tailings. These would be transported
back to Canada for further testing and eventually
developed as biofertilizers for corn agriculture.
• Objective 2: Establish whether different soils do
dramatically alter the surface bacterial populations of
maize plants. This is a basic research question with
implications for future sampling strategies.
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Methodology Employed
Growth of an ancient and one
modern Brazilian maize genotype
on a sterile sand, a mining residue,
and black amazonian soil
Molecular fingerprinting (TRFLP)
Ancient
Modern
Surface sterilization:
bleach and ethanol
DNA extraction
Maceration
in buffer
Multivariate
Statistics
Isolating novel bacteria
Collect washings
from roots and
shoots
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Significance of the internship to
recipient and home country/region
• Dr. Johnston Monje completed a PhD at the
University of Guelph (microbial ecology of maize
in Mexico and Canada) just before the internship
took place in Brazil, so the proposed research was
a way to immediately continue the research
started during the doctorate
• Maize is a tropical plant which made it to tropical
South America thousands of years ago, where it
may have maintained or developed unique and
beneficial microbial relationships no longer
present in temperate climates like Canada
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Significance of the internship to
recipient and home country/region
• Brazilian ecological agricultural research is world famous, so
experience and networking within EMBRAPA is invaluable for
future collaboration or even career opportunities
• Specifically important, the lab of Dr. Reis is the same lab Dr.
Dobreiner ran for many years, arguably the most famous
grass-bacterial research lab in the world
Dr. Dobreiner
Dr. Reis
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Expected and achieved results
Can I acquire interesting, diverse Brazilian soils?
After loosing support from the Canadian mine partner in Brazil and having
negotiated with EMBRAPA Soils for 1.5 months, I got deep iron mine soil
and Amazonian dark earth for my experiment. The sand was heat sterilized
and was included as a negative control. (note the pre-Colombian pottery
shard I found in the Amazonian soil)
Sterile sand
Iron mine subsoil
Amazonian black earth
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of the Indians
Expected and achieved results
Are there bacterial differences in different Brazilian soils?
Based on simple plate culturing, you can see there is.
Sterile sand
Iron mine subsoil
Amazonian black earth
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of the Indians
Expected and achieved results
Can I acquire interesting maize genotypes to study?
Lehna
Although Dr. Reis told me there was too much red-tape to
get the exact types I wanted, there was a relevant preColombian variety in their collection called Lehna. This
was contrasted to a modern variety found to be highly
responsive to bacterial innoculation.
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Expected and achieved results
Can I isolate bacteria and DNA to ship to Canada for
further study?
I was able to sign a material transfer agreement between
the University of Guelph and EMBRAPA, allowing me to
transport DNA back to Canada. I was unable to buy DNA
extraction kits with EMBRAPA (importation fees and times
were prohibitive) so my supervisor in Guelph bought the
MoBio kit and mailed it so that it arrived 2 weeks before I
left Brazil.
Export of live bacteria, seeds or soil were absolutely out of
the question Dr. Reis told me, as there would be years of
paperwork involved. I never isolated any bacterial strains
since I could not do any further analysis.
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Expected and achieved results
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Expected and achieved results
X120
Extracted DNA from leaf and root surfaces, and root
interiors from 5 plants per genotype per soil treatment.
Also extracted DNA from soil samples, water in empty
cups in the greenhouse, and washes from seed surfaces.
These 120 tubes were put in my lugagge and flown back
to Canada for analysis.
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Lessons Learnt and Next Steps
• I had big intentions to conduct a experiment in
Brazil, sourcing biological materials from all over
the world, sending biological materials back to
Canada, and discovering biological novelty for
international agriculture and my own personal
glory – I learnt that Brazilians are very paranoid
about biopiracy and have implemented many
rules to protect and restrict access of biological
materials. The prehistoric Amazonian soil I
selected to use is world famous, and very highly
regulated for transportation and research
purposes.
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Lessons Learnt and Next Steps
• Beurocracy, taxation, and uncertainty figure
prominently in the mindset and reality of
scientific research in Brazil (as they might in
many developing countries). Research products
are often imported with over 100% import
taxes, huge wait times, and the need for
lying/cheating to expedite delivery.
• Only Brazilians can currently apply for jobs at
the Brazilian ministry of agriculture (this might
be changing so as to lure more international
talent to Brazil)
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Lessons Learnt and Next Steps
• A huge competitive advantage exists for
companies to develop scientific products for
sale within Brazil – importation taxes and
delivery times are very effective tariffs.
• EMBRAPA is not very international. Few
people speak English (although all claim to
want to) and Brazilians have to leave the
country to gain exposure to international
research.
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Next Steps
• I am in the process of doing analysis of DNA at my
NSERC Funded Post-doc at the start up soil
microbiology company, A&L Biologicals in London,
Ontario.
• I have made a partnership with Dr. Jeff Dangl at the
University of North Carolina, Chapel Hill, who will
generate additional data with the DNA using
pyrosequencing.
• Great opportunities to partner A&L Biologicals with
EMBRAPA to research soil borne plant diseases such as
yellow death of oil palm, which would be a boon to
many countries all over the world. We’ll just need to
find money and time to do this...
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Future budget and future
requirements
• This internship/project would not have been possible
without IICA funding. I was to have recieved additional
funding from two other sources, but neither came
through and I had to cover some personal and
profesional expenses using pocket money
• Additional experiments are planned, but these are to
be funded by my current employer and my collaborator
at the University of North Carolina. I hope IICA will
consider funding future students who may wish to visit
the University of Guelph or A&L Biologicals to learn
about Canadian research on soil microbiology.
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Thank you!
• IICA Canada for funding agricultural
cooperation and this project!
• Dr. Manish Raizada for co-writing
the grants and purchasing DNA
extraction kits
• Dr. Veronica Reis for hosting me and
helping me conduct this experiment
in Brazil
• All my new colleagues and friends
in Seropedica, Rio de Janeiro,
Brasilia, Salvador and Sao Paolo
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