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Applied T-Rflp Analyses for the Identification And APPLIED T-RFLP ANALYSES FOR THE IDENTIFICATION AND CHARACTERIZATION OF MICROBIAL POPULATIONS ASSOCIATED WITH DAMPING-OFF INCIDENCE IN A TRANSITIONAL ORGANIC CROPPING SYSTEM DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By María Soledad Benítez, M.S. * * * The Ohio State University 2008 Dissertation Committee: Approved by: Dr. Brian McSpadden Gardener, Adviser Dr. Terrence L. Graham ___________________________ Dr. Warren Dick Adviser Dr. Enrico Bonello Plant Pathology Graduate Program ABSTRACT Within agricultural ecosystems the abundance and activities of diverse microbial populations affecting plant health can be significantly influenced by farm management practices. This work describes the effects of transitional cropping strategies on damping- off incidence and on soil and rhizosphere bacterial, fungal and oomycete communities. Specifically, individual microbial populations were associated with damping-off incidence and the suppression thereof, and two novel bacterial species were characterized for their ability to reduce seedling disease severity. Soils from an organic transition experiment exhibited different levels of damping-off suppression, with a perennial mixed hay transition consistently suppressing damping-off of tomato and soybean. Microbial community analyses were performed from the rhizosphere and surrounding soil of tomato and soybean seedlings grown in soils previously exposed to different transition strategies. The abundance of Pseudomonas and DAPG-producing Pseudomonas populations, did not clearly associate with disease levels. However, the analysis of bacterial, fungal and oomycete community profiles (generated from terminal restriction fragment length polymorphism of 16S and ITS genes, respectively) revealed individual members of the community (expressed as a terminal restriction fragment, TRF) that statistically associated with disease suppression or disease. Bacterial TRF more abundant in the mixed hay suppressive treatment were negatively correlated with disease incidence. In ii silico analysis of bacterial database sequences indicated that species of known biocontrol agents produce a TRF size corresponding to those identified in this study. Fungal and oomycete TRF abundance and diversity tended to be lower in the mixed hay transition strategy. Fungal and oomycete isolates of the genus Fusarium, Chaetomium and Pythium, among others, generate TRF that match those associated with disease in the studied transition strategies. Sequence information obtained from the bacterial T-RFLP data was used to identify and isolate two novel bacterial species from the mixed hay soils. Mitsuaria and Burkholderia isolates were phylogenetically related to the TRF sequences associated with disease suppression, inhibited pathogen growth in vitro, and reduced seedling disease severity. This work establishes the power of T-RFLP based screening of microbial communities to identify and direct the recovery of functionally important microorganisms in soil. iii To my parents iv ACKNOWLEGMENTS I would like to acknowledge first my advisor, Brian McSpadden Gardener for his support and guidance during this process, and especially for believing in me and encouraging me hard enough so to convince me to move into the PhD program. In addition thanks for starting me into a project that resulted being not only very productive and full of learning experiences but more than anything very fun to work in. I would like to thank the members of my student advisory committee, Terrance Graham, Warren Dick and Enrico Bonello, for all your helpful comments and support. Special thanks to the Department of Plant Pathology, professors, staff and students, for allowing me to be part of this program and making my time at this department so enjoyable but at the same time intellectually challenging. Thanks to all the people that in these years have shared the lab with me: Seth, Sonia, Jeff, Alejandra, Carrie, Kelly, Gloria, Dario, Jessica, Fulya, Guleray, Raghu, Laura, Amara, Clara, Jackie, Ethan, with a very special thank you for Rosa, Sunny, Dorith and Pal. A very special thank you to all those with whom I shared very nice moments during my stat at Ohio State: Wirat, Angel, Jose, Santiago, Miguel, Alieta, Frances, Andrea, Carla, Julio, Audrey, Diego, Fulya, Sawsan, Xiulan, Kirk, Luciana, Juan Pablo, v Sandra, Rodrigo, Alex, Sanja, Hanbae, Vanessa, Maggie, Hehe, Zhifen, Joe, Jorunn, Jing, Vero, Kristhen, Jhonny, Freddy, Carmen, Loren, Lynn... Of course thanks to my friend Gaby, who from California was a great support during these times, also thanks to Nessy, David and Pancho. The greatest thanks of all to my family, for their patience and support. Thanks to my mom for teaching me so early about the scientific method. Also for coming so many times to visit me, being such a good company and a great friend of my friends, and thanks to my dad, my sister and my nieces for letting her come. Thanks for my dad for being my scientific and work ethic role model. Thanks to my sister’s unconditional support. Thanks for Ale for her patience and understanding. Thanks to my brother who I know is proud of me. Finally thanks to all of you who will eventually be reading this thesis. Don’t wonder too much why I didn’t mention your names in it, it will have taken too much space. That includes my “witch” friends, my high school friends, my college friends, my cousins, aunts and grandma, and all other “unclassified” friends. iv VITA 1978 Born, Quito, Ecuador 1996-2002 Licenciatura, Biological Sciences, Pontificia Universidad Católica del Ecuador, Quito 2002-2004 Auxiliary Professor and Research Associate, Department of Biological Sciences, Pontificia Universidad Católica del Ecuador, Quito 2007 MSc, The Oho State University, obtained as a successful completion of the candidacy examination 2004 – present Graduate Research Associate, Department of Plant Pathology, The Ohio State University PUBLICATIONS 1. Baysal, F., Benítez, MS., Kleinhenz, M., Miller, S., McSpadden Gardener, B. 2008. Field Management Effects on Damping-off and Early Season Vigor of Crops in a Transitional Organic Cropping System. Phytopathology 98:562-570. 2. Benítez, MS., Baysal Tustas, F., Rotenberg, D., Kleinhenz, M.D., Cardina, J., Stinner, D., Miller, S.A., McSpadden Gardener, B. 2007. Multiple statistical approaches of community fingerprint data reveal bacterial populations associated with disease suppression arising from the application of different organic field management strategies. Soil Biology and Biochemistry 39: 2289-2301. 3. Rotenberg, D., Joshi, R., Benítez, MS., Gutierrez Chapin, L., Camp, A., Zumpetta, C., Osborne, A., Dick, W.A., McSpadden Gardener, B. B. 2007. Farm management effects on rhizosphere colonization by native populations of 2,4- diacetylphloroglucinol-producing Pseudomonas spp. and their contributions to crop health. Phytopathology 97: 756-766. 4. Pérez, O., Benítez, MS., Nath, K., Heasman, J., del Pino, E.M., Elinson, R. 2007. Comparative analysis of Xenopus VegT, the meso-endodermal determinant, identifies an unusual conserved sequence. Differentiation 75: 559-565. vii 1. McSpadden Gardener, B., Benítez, MS., Camp, A., Zumpetta, C. 2006. Evaluation of a seed treatment containing a phlD+ strain Pseudomonas fluorescens on organic soybeans, 2005. Biological and Cultural Tests 21:FC046. 2. del Pino, E.M., Avila, M-E., Pérez, O.D., Benítez, MS., Alarcón, I., Noboa, V., Moya, I. 2004. Development of the dendrobatid frog Colostethus machalilla. Int. J. Dev. Biol. 48: 663-670. 3. Benítez, MS. and del Pino, E.M. 2002. Expression of Brachyury During Development of the Dendrobatid Frog Colostethus machalilla. Dev. Dyn. 225:592–596. FIELDS OF STUDY Major Field: Plant Pathology viii TABLE OF CONTENTS Page Abstract ………………………………………………………………………... ii Dedication……………………………………………………………………… iv Acknowledgments……………………………………………………………... v Vita…………………………………………………………………………….. vii Table of Contents………………………………………………………………. ix List of Tables…………………………………………………………………... xi List of Figures………………………………………………………………….. xiv Chapters: 1. Setting up the stage: Identification of a disease suppression differential Introduction…………………………………………………………. 1 Identification of a disease suppressive differential: Transitional organic cropping system effects on soybean and tomato seedling damping-off and vigor……………………………………………… 6 Transition strategy and compost amendment effects on soil chemistry……………………………………………………………. 8 Transition strategy and compost amendment effects on soybean and tomato damping-off and seedling vigor…………………………….. 10 Transition strategy effects on the abundance and incidence of DAPG-producing Pseudomonas colonizing the rhizosphere of tomato and soybean………………………………………………… 13 Summary……………………………………………………………. 19 References…………………………………………………………... 21 2. Multiple statistical approaches of community fingerprint data reveal bacterial populations associated with general disease suppression arising from the application of different organic field management Abstract………………………………………………………….. 41 Introduction……………………………………………………… 42 Materials and Methods…………………………………………... 46 Results…………………………………………………………… 50 Discussion……………………………………………………….. 58 Acknowledgements……………………………………………… 64 References……………………………………………………….. 65 ix 3. Association of multiple fungal and oomycete populations with damping-off disease incidence in soils previously managed under different organic transition strategies Abstract………………………………………………………….
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