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Assessing the Effect of Siderophore Producing Bacteria for the Iron Nutrition in Lentil And

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Assessing the Effect of Siderophore Producing Bacteria for the Iron Nutrition in Lentil And ASSESSING THE EFFECT OF SIDEROPHORE PRODUCING BACTERIA FOR THE IRON NUTRITION IN LENTIL AND PEA A Thesis Submitted to the College of Graduate and Postdoctoral Studies in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Department of Soil Science University of Saskatchewan Saskatoon, SK, Canada By Md Mortuza Reza © Copyright Md Mortuza Reza, August 2017. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department of Soil Science or the Dean of the College of Agriculture and Bioresources. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use that may be made of any material in my thesis. Requests for permission to copy or to make other uses of materials in this thesis, in whole or part, should be addressed to: Head, Department of Soil Science University of Saskatchewan Saskatoon, Saskatchewan Canada, S7N 5A8 Or Dean, College of Graduate Studies and Research University of Saskatchewan Saskatoon, Saskatchewan Canada S7N 5A2 i DISCLAIMER Reference in this dissertation to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favouring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. ii ABSTRACT Lentil (Lens culinaris Medik., cv. CDC Milestone) was grown for 45 days in a growth chamber using eight Saskatchewan agricultural soils to isolate root associated siderophore- producing and total heterotrophic bacteria. Results indicated that siderophore-producing bacteria and total heterotroph populations ranged between 4.17–6.30 log cfu·g-1 and 7.16–8.08 log cfu·g- 1, respectively. The in vitro siderophore production assay of 491 siderophore-producing isolates using SideroTec® kit revealed that siderophore production ranged from 0.04 to 119.27 µg∙mL-1. Isolates that exhibited high siderophore producing potential (n=68) were characterized using Sanger sequencing and were identified as: Bacillaceae, Burkholderiaceae, Enterobacteriaceae, Oxalobacteraceae, Pseudomonadaceae, Rhizobiaceae and Xanthomonadaceae. Next, isolates that exhibited higher siderophore production (n=40) were inoculated on lentil to enhance plant Fe uptake when grown in a sand-soil mixture. Results revealed that 33 isolates significantly increased total Fe uptake in lentil plants, but the ability to increase total Fe uptake in lentil was not associated with the ability of in vitro siderophore production of individual isolates. The most effective five isolates of this study were subsequently tested to enhance Fe uptake in four pea cultivars in a growth chamber study. Results indicated that inoculation significantly increased total Fe uptake in 75% treatments. A study involving phytosiderophore production by four lentil and four pea cultivars was conducted to assess the potential to chelate Fe from nutrient medium during germination. Results of this study, indicated that all lentil and one pea, cultivar CDC Dakota, exhibited positive phytosiderophore activity. However, results from the pea growth chamber study indicated that the pea cultivar that exhibited in vitro phytosiderophore production did not significantly enhance Fe uptake when compared to the remaining non phytosiderophore- iii producing cultivars. This difference might be attributed to the differences between plant growth conditions and/or plant physiological stages. Overall, significant increases in total Fe uptake by lentil and pea inoculated with siderophore-producing bacteria were observed. Moreover, the significant interactions between bacterial isolates and pea cultivars suggesting that plant species and cultivars might be dependent on their associated siderophore-producing isolates or vice versa for Fe nutrition and plant cultivars were more responsive when inoculated with their suitable siderophore-producing isolates. iv ACKNOWLEDGEMENTS To begin with, I would like to acknowledge my co-supervisors, Dr. Fran Walley and Dr. Renato de Freitas for every support and guidance they have given throughout this study. The incredible level of effort and enthusiasm you put into a student are highly appreciated. I would like to express sincere gratitude to my advisory committee members, Professors, Drs. Jim Germida and Albert Vandenberg for their valuable advice, useful suggestions and feedbacks throughout my school and research project. I would thank Dr. Russell Hynes for reviewing my thesis as an external examiner and for his time and comments to improve the quality of this thesis. This thesis work would not have been possible without the help of the members of Soil Agronomy (5C29) and Soil Microbiology (5E25) Labs. Special thanks to Jorge Cordero for his support in Microbiology Lab is media preparation and isolation. I am very grateful to Dr. Jeff Schoenau for providing me soils from his farm. Special thanks to Brent Barlow and Thiago Prado at the Crop Development Centre for providing me soils and quality seeds for my growth chamber studies. I would also like to thank Cory Fatteicher to Ranjan Kar for guidance regarding plant digestion processing for analysis. Thanks to all the friends and personnel at College of Agriculture and Bioresources for making the days in the university so pleasant. I couldn’t have done this without the inspiration and supports of my family and friends, I am very thankful to all of you who provided much needed support in completing work. Thanks to my parents, Nural Islam and Morsheda Begum for encouraging and supporting me in all my undertakings. Thanks to the University of Saskatchewan and the Government of Saskatchewan for their financial support to carryout this research project. v DEDICATION I would also like to dedicate this work to my father Nural Islam for his amazing encouragement. Finally, to my supervisors Dr. Fran Walley, Dr. Renato de Freitas and all the teachers around the world who help students to become successful. vi TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................... i DISCLAIMER ................................................................................................................................ ii ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS .............................................................................................................v TABLE OF CONTENTS .............................................................................................................. vii LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS ....................................................................................................... xii 1. GENERAL INTRODUCTION ....................................................................................................1 2. LITERATURE REVIEW ............................................................................................................5 2.1 Importance of iron in human animal and plant .....................................................................5 2.2 Availability of Fe for the plants in soil .................................................................................7 2.3 Mechanisms of Fe-chelation by crop associated bacteria in soil ..........................................8 2.4 Siderophores ..........................................................................................................................9 2.5 Effect of siderophore producing bacteria on Fe-nutrition and growth promotion of crop ............................................................................................................. 10 2.6 Isolation of siderophore-producing microorganisms ..........................................................12 2.7 Methods to measure siderophore production ......................................................................12 2.8 Methods for identification of the microbes .........................................................................14 3. ISOLATION AND IDENTIFICATION OF SIDEROPHORE PRODUCING BACTERIA FROM RHIZOSPHERE AND ENDORHIZOSPHERE OF LENTIL PLANTS GROWN IN DIFFERENT SASKATCHEWAN SOILS ...........................17 3.1 Preface .................................................................................................................................17 3.2 Abstract ...............................................................................................................................17
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