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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

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Maize has been important all over the Americas for thousands of years... Mayan maize god Yumil Kaxob Zapotec maize god Pitao Cozobi Moche culture Peru Mitchell Corn Palace, USA 2

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Where does maize come from? Developed 9,000 years ago In Oaxaca, Mexico 3 Distinct genotypes and soils, yet unresearched Already researched genotypes and soils (by Dr. Johnston)

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Dramatic differences in soil may have forced maize to develop novel bacterial surface populations 4

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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. 5

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Obvious structural or environmental differences may coincide with changes to the microbial ecology 6

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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 them in agriculture? What are plant associated bacteria and why study their ecology in maize plants? 7

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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. (Dalton and Kramer 2006) 8

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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!!! 9

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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 10

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TRFLP is one way to comprehensively view a microbial community using DNA Extract DNA from the community PCR label with a fluorescently labelled rRNA forward primer Restriction Digest of PCR Product Fragment separation in sequencing gel Recognition of labelled fragments Relative fluorescence Outline of the terminal restriction fragment length polymorphism method 11

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How can a DNA fingerprint like TRFLP be interpreted statistically? Based on 99 microsatellites Principle Component 1 Principle Component 2 Matsuoka et al. (2002) 12

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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. 13

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Methodology Employed Maceration in buffer DNA extraction Molecular fingerprinting (TRFLP) Modern Ancient Surface sterilization: bleach and ethanol Multivariate Statistics Collect washings from roots and shoots Growth of an ancient and one modern Brazilian maize genotype on a sterile sand, a mining residue, and black amazonian soil Isolating novel bacteria 14

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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 15

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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 16 Significance of the internship to recipient and home country/region Dr. Dobreiner Dr. Reis

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Expected and achieved results 17 Sterile sand Iron mine subsoil Amazonian black earth of the Indians 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)

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Expected and achieved results 18 Sterile sand Iron mine subsoil Amazonian black earth of the Indians Are there bacterial differences in different Brazilian soils? Based on simple plate culturing, you can see there is.

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Expected and achieved results 19 Can I acquire interesting maize genotypes to study? Although Dr. Reis told me there was too much red-tape to get the exact types I wanted, there was a relevant pre- Colombian variety in their collection called Lehna. This was contrasted to a modern variety found to be highly responsive to bacterial innoculation. Lehna

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20 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. Expected and achieved results

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21 Expected and achieved results

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22 Expected and achieved results 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. X120

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Lessons Learnt and Next Steps 23 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 24 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. 25

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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. Well just need to find money and time to do this... 26

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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. 27

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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 28