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  • EFFECT OF PLANT GROWTH-PROMOTING RHIZOBACTERIA ON

    CANOLA (Brassica napus. L) AND LENTIL (Lens culinaris. Medik) PLANTS

    A Thesis Submitted to the College of

    Graduate Studies and Research

    In Partial Fulfillment of the Requirements

    For the Degree of Master of Science

    In the Department of Applied Microbiology and Food Science

    University of Saskatchewan

    Saskatoon

    by

    Rajash Pallai

    Copyright Rajash Pallai, April 2005. All rights reserved.

  • i

    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 or the Dean of the College in which my thesis work was done.

    It is understood that copying or publication or use of this thesis or parts thereof for

    financial gains shall not be allowed without my written permission. It is also understood

    that due recognition shall be given to me and the University of Saskatchewan in any

    scholarly use which may be made of any material in my thesis.

    Requests for permission to copy or make other use of material in this thesis in

    whole or part should be addressed to:

    Head of the Department, Applied Microbiology and Food Science

    University of Saskatchewan

    Saskatoon, Saskatchewan, S7N5A8.

  • ii

    ABSTRACT

    Plant growth-promoting rhizobacteria (PGPR) are free-living, soil-borne bacteria

    that colonize the rhizosphere and, when applied to crops, enhance the growth of plants.

    Plant growth-promoting rhizobacteria may enhance plant growth either by direct or

    indirect mechanisms. The direct mechanisms of action include nitrogen fixation,

    production of phytohormones and lowering of ethylene concentrations. The objective of

    this study was to determine whether Pseudomonas putida strain 6-8 isolated from the

    rhizosphere of legume crops grown in Saskatchewan fields was able to promote the

    growth of canola cv. Smart and lentil cv. Milestone plants by direct mechanisms.

    Initial studies determined the effect of strain 6-8 and other known phytohormone-

    producing PGPR strains on the growth of canola and lentil plants both in gnotobiotic and

    growth chamber conditions. Variations in the results were observed, as there were

    significant differences among trials. Strain 6-8 enhanced the growth of canola cv. Smart

    in growth pouches but not in pots in growth chamber studies. In the case of lentil cv.

    Milestone, strain 6-8 had no significant effect in growth pouches, but it significantly

    increased root dry weight, shoot dry weight and root surface area in pots in growth

    chamber studies. A similar effect was observed with wild-type strains GR12-2 and G20-

    18. Strain GR12-2 was consistent in promoting the growth of lentil cv. Milestone both in

    growth pouches and in pots in growth chambers when compared to other strains and the

    control.

    The ability of the PGPR strains to produce auxin and cytokinin phytohomones in

    pure culture and in the canola rhizosphere was tested using the enzyme linked

    immunosorbent assay (ELISA). All the PGPR strains produced indole compounds and

    the concentration of the indoles produced increased with increasing concentrations of the

  • iii

    precursor tryptophan. There were no significant differences among PGPR strains in

    production of indole-3-acetic acid (IAA) when assayed using ELISA. The

    concentrations of IAA secreted by PGPR strains were extremely low (0.19 g/ml 9.80

    g/ml). Strain 6-8 produced the cytokinins, isopentenyl adenosine (IPA), zeatin riboside

    (ZR) and dihydroxyzeatin riboside (DHZR) in pure culture. Indole-3-acetic acid was

    detected in supernatants obtained from canola growth pouches inoculated with PGPR

    strains, but there were no significant differences in the concentrations of IAA secreted

    among PGPR strains. Significantly higher concentrations of IPA and ZR were observed

    in the rhizosphere of canola inoculated with strain 6-8 than in the non-inoculated

    control.

    Strain 6-8 produced siderophores, solubilized inorganic phosphate and used

    1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, as sole

    nitrogen source. These traits are considered to be alternative mechanisms for direct plant

    growth promotion.

    A qualitative and quantitative study of root colonization by strain 6-8 was

    conducted by tagging the strain with green fluorescent protein in conjunction with

    confocal laser scanning microscopy and by conventional plating. The populations of

    strain 6-8 were higher on canola roots than on lentil roots by conventional plating.

    Similar results were also observed in confocal laser scanning microscopy (CLSM)

    studies after 5, 7 and 9 days for canola and 3, 6 and 9 days for lentil.

    Pseudomonas putida strain 6-8 produced cytokinins and also possessed other direct

    growth promoting characteristics. The ability of strain 6-8 to promote the growth of

    canola cv. Smart in growth pouches and lentil cv. Milestone in growth chamber studies

  • iv

    may be related to these direct growth promoting characteristics. Strain 6-8 may have

    potential for development as a plant growth-promoting rhizobacterial inoculant.

  • v

    ACKNOWLEDGMENT

    I wish to extend my appreciation and sincere thanks to my research supervisor,

    Professor Louise M. Nelson, for her support and guidance throughout the research

    project. I also extend my sincere appreciation to Dr. George G. Khachatourians, Dr.

    Darren R. Korber and Dr. Russell K. Hynes for their suggestions and critical comments

    as members of my advisory committee. Special thanks to Dr. Brij Verma for his

    technical support in handling the confocal laser scanning microscopy. I would also like

    to thank Dr. Harry Deneer for his critical comments and also serving as my external

    examiner.

    Special thanks to my laboratory co-workers, Mr. Grant Leung, Mr. Edwin Pensaert

    and Ms. Marilyn Gould. I would also like to thank the staff members of the Department

    of Applied Microbiology and Food Science for their congenial atmosphere.

    Finally, I am grateful for the personal support, understanding and encouragement I

    received from my parents Mr. Madhava Rao and Mrs. Hemalath Madhava Rao

    throughout this years. I would also thank my close friends who have taken care of my

    parents in my absence during this period.

    The funding for the project was provided by the Natural Sciences and Engineering

    Research Council of Canada.

  • vi

    TABLE OF CONTENTS PERMISSION TO USE i

    ABSTRACT ii

    ACKNOWLEDGMENTS v

    TABLE OF CONTENTS vi

    LIST OF TABLES ix

    LIST OF FIGURES xi

    LIST OF ABBREVIATIONS xiv

    1.0 INTRODUCTION 1

    2.0 LITERATURE REVIEW 4

    2.1 Plant growth-promoting rhizobacteria 4

    2.2 Direct mechanisms of action 6 2.2.1 Nitrogen-fixation 6 2.2.2 Phytohormones 8

    2.2.2.1 Auxins 9 2.2.2.2 Cytokinins 12

    2.2.3 Lowering of ethylene concentration, phosphate 14 solubilization and siderophore production

    2.3 Effect of PGPR on plant growth 18

    2.4 Root colonization 22

    2.5 Green fluorescent protein 26

    3.0 MATERIALS AND METHODS 29

    3.1 Bacterial strains 29

    3.2 Plant cultivars 31

    3.3 Gnotobiotic assay 31

    3.4 Growth chamber study 33

    3.5 Identification and quantification of phytohormones 34

  • vii

    3.6 Detection of siderophores 36

    3.7 Solubilization of inorganic phosphate 36

    3.8 Characterization of PGPR strains for ability to use 37 ACC as sole nitrogen source

    3.9 Root colonization of canola and lentil roots by strain 6-8 38

    3.9.1 Bacterial strains and culture conditions 38 3.9.2 Transfer of gfp gene present in pAG408 into 38

    strain 6-8 by conjugation 3.9.3 Gnotobiotic root colonization studies 39 3.9.4 Confocal laser scanning microscopy 40

    3.10 Statistical analysis 41

    4.0 RESULTS 42

    4.1 Strain identification 42

    4.2 Gnotobiotic studies 42 4.2.1 Canola gnotobiotic study 42

    4.2.2 Lentil gnotobiotic study 43

    4.3 Growth chamber studies 46 4.3.1 Canola growth chamber studies 46

    4.3.2 Lentil growth chamber studies 53

    4.4 Indole production 61 4.4.1 Identification and quantification of indole-3- 64 acetic acid (IAA)

    4.5 Identification and quantification of the cytokinin 67

    Phytohormones isopentenyl adenosine (IPA), dihydroxy- zeatin riboside (DHZR), zeatin riboside (ZR)

    4.6 Phytohormone production in the rhizosphere of 69 canola cv. Smart inoculated with PGPR strains in growth pouches

    4.7 Production of siderophores, phosphate solubilization 69 and ability to use ACC as sole nitrogen source

    4.8 Root colonization studies 74

    4.8.1 Recovery of strain 6-8-GFP-Rif+ from roots of canola 74

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