Development of a new green technology for the revegetation of abandoned gold mine tailings using specific symbionts associated with Picea glauca Mémoire Martin Beaudoin Nadeau Maîtrise en agroforesterie Maître ès sciences (M.Sc.) Québec, Canada © Martin Beaudoin Nadeau, 2015 Résumé Le rôle et l'importance des micro-organismes telluriques (PGPR et champignons ECM) à favoriser la santé, la croissance et la nutrition de Picea glauca ont été étudiés sur stériles et résidus miniers fins riches en quartz-biotite de la mine d’or Sigma-Lamaque située dans la région de l'Abitibi au Canada. L’étude a été divisée en trois composantes. Premièrement, la structure des communautés de champignons ECM associés aux racines de Picea glauca a été analysée sur quatre sites différents à proximité du site minier. Deuxièmement, une expérience en laboratoire a été effectuée afin de sélectionner in vitro des champignons ECM prometteurs qui démontrent une excellente croissance sur résidus miniers. Troisièmement, une expérience en serre impliquant la croissance de semis de Picea glauca sur stériles et résidus miniers fins a été réalisée et la performance de différents traitements de champignons ECM et PGPR a été évaluée. Les résultats suggèrent que les champignons ECM et PGPR adaptés aux conditions du site jouent un rôle très important au niveau de la santé et de la croissance de Picea glauca sur résidus miniers riches en quartz- biotite. III Abstract The role and importance of soil microorganisms (PGPR and ECM fungi) in promoting the health, growth, and nutrition of Picea glauca were investigated on biotite- quartz-rich waste rocks and fine tailings of Sigma-Lamaque gold mine located in the Abitibi region of Canada. The study was divided into three components. Firstly, the community structure of ECM fungi associated with Picea glauca was analyzed on four locations near the mining site. Secondly, a laboratory experiment was conducted in order to in vitro select promising ECM fungi that were growing well on mine tailings. Thirdly, a glasshouse experiment involving the growth of Picea glauca seedlings on waste rocks and fine tailings was conducted and the performance of different treatments of ECM fungi and PGPR was evaluated. Results suggested that site-adapted ECM fungi and PGPR play a very important role in the health and growth of Picea glauca on biotite-quartz-rich waste rocks and fine tailings. V Table of contents Résumé ................................................................................................................................ III Abstract ................................................................................................................................. V Table of contents ............................................................................................................... VII List of tables .................................................................................................................... XIII List of figures ..................................................................................................................... XV List of abbreviations and acronyms .............................................................................. XXI Acknowledgements ........................................................................................................ XXV Foreword ...................................................................................................................... XXVII 1. General introduction ........................................................................................................ 1 1.1 Literature review ..................................................................................................................... 2 1.1.1 Principles of ecological restoration and bioremediation ............................................... 2 1.1.1.1 Ecological restoration ......................................................................................................... 2 1.1.1.2 Bioremediation ..................................................................................................................... 3 1.1.2 Traditional use of agricultural practices, trees, and organic amendments................. 5 1.1.3 Sigma-Lamaque mine and the Abitibi-Temiscamingue’s boreal forest ...................... 8 1.1.3.1 Vegetation zone and bioclimatic domain ..................................................................... 10 1.1.3.2 White spruce as a candidate for ecological restoration ............................................ 10 1.1.3.3 Other boreal tree species potentially useful in ecological restoration of mining sites ..................................................................................................................................................... 11 1.1.3.3.1 Jack pine ...................................................................................................................... 11 1.1.3.3.2 Speckled alder and Green alder ............................................................................. 12 1.1.4 Woody plant nutrient uptake in mineral soils ............................................................. 13 1.1.5 Ecology of ectomycorrhizal fungi and their role in plant nutrition ........................... 18 1.1.6 Role of ectomycorrhizal fungi in weathering ............................................................... 21 1.1.7 Ectomycorrhizal fungal community response to heavy metal in soil ........................ 23 1.1.8 Ectomycorrhizal diversity in boreal forest .................................................................. 23 1.1.9 Use of trees and their symbionts in mine reclamation: previous studies in the Canadian boreal forest ........................................................................................................... 25 1.1.10 Role of free-living soil bacteria in plant growth and nutrient .................................. 29 1.2 Research objectives and hypotheses .................................................................................... 31 VII 1.3 References .............................................................................................................................. 32 2. Chapter 1: Edaphic selection pressures as drivers of contrasting white spruce ectomycorrhizal fungal diversity and community structure in the Canadian boreal forest of Abitibi-Temiscamingue region ........................................................................... 37 2.1 Résumé ................................................................................................................................... 38 2.2 Summary ................................................................................................................................ 38 2.3 Introduction ........................................................................................................................... 39 2.4 Materials and methods .......................................................................................................... 41 2.4.1 Study area ....................................................................................................................... 41 2.4.2 Sampling of ECM fungal communities ......................................................................... 42 2.4.3 Identification of ECM fungi .......................................................................................... 42 2.4.3.1 Morphotyping ..................................................................................................................... 42 2.4.3.2 DNA extraction and sequencing ..................................................................................... 43 2.4.4 Bulk soil analyses ............................................................................................................ 43 2.4.5 Numerical analyses ......................................................................................................... 44 2.4.5.1 Phylogenetic analyses ........................................................................................................ 44 2.4.5.2 Diversity and colonization analyses ............................................................................... 44 2.4.5.3 Multivariate analyses ........................................................................................................ 45 2.5 Results .................................................................................................................................... 45 2.5.1 ECM fungal community and phylogenetic trees ......................................................... 45 2.5.2 Species diversity .............................................................................................................. 47 2.5.2.1 Relative abundance and frequency ................................................................................ 47 2.5.2.2 Percentage of colonized roots, richness and diversity index.................................... 48 2.5.3 Shaping groups with similarities ................................................................................... 51 2.5.4 Looking for linear patterns............................................................................................ 53 2.6 Discussion ............................................................................................................................... 55 2.6.1 Genetic divergence within communities ....................................................................... 55 2.6.2 ECM fungal species diversity ........................................................................................ 56 2.6.3 Difference in species composition among sites ............................................................. 58 2.6.4 ECM fungal communities and soil chemical properties ............................................. 59 2.6.5 Edaphic selection pressures – drivers of ECM fungal community structure ........... 60 2.7 Conclusion .............................................................................................................................. 61 VIII 2.8 Acknowledgements ................................................................................................................ 61 2.9 References .............................................................................................................................. 62 3. Chapter 2: In vitro selection of ecologically adapted ectomycorrhizal fungi through production of fungal biomass and metabolites for use in reclamation of gold mine tailings .................................................................................................................................. 67 3.1 Résumé ................................................................................................................................... 68 3.2 Abstract .................................................................................................................................. 68 3.3 Introduction ........................................................................................................................... 69 3.4 Materials and methods ......................................................................................................... 71 3.4.1 ECM fungal isolation techniques and identification ................................................... 71 3.4.2 In vitro ECM fungal growth in gold mine tailings using solid medium ..................... 72 3.4.3 In vitro ECM fungal growth in gold mine tailings using liquid medium ................... 75 3.5 Results .................................................................................................................................... 77 3.5.1 In vitro ECM fungal growth on solid medium ............................................................. 77 3.5.2 In vitro ECM fungal growth on liquid medium ........................................................... 79 3.6 Discussion ............................................................................................................................... 85 3.6.1 In vitro selection of ECM fungi on solid medium ........................................................ 85 3.6.2 In vitro selection of ECM fungi on liquid medium ...................................................... 88 3.6.3 Methods for ECM fungal selection ............................................................................... 91 3.7 Conclusion.............................................................................................................................. 92 3.8 Acknowledgements ................................................................................................................ 92 3.9 References .............................................................................................................................. 93 4. Chapter 3: Ectomycorrhizal fungi and bacterial PGPR improve white spruce seedling health, growth, and nutrition on Precambrian rocky gold mine tailings ....... 97 4.1 Résumé ................................................................................................................................... 98 4.2 Abstract .................................................................................................................................. 98 4.3 Introduction ........................................................................................................................... 99 4.4 Materials and methods ....................................................................................................... 103 4.4.1 White spruce seed germination and seedling growth................................................ 103 4.4.2 Bacterial and ECM fungal inoculation of white spruce seedlings ........................... 103 4.4.2.1 Bacteria .............................................................................................................................. 103 IX 4.4.2.2 Ectomycorrhizal fungi .................................................................................................... 104 4.4.3 Experimental design and treatments .......................................................................... 104 4.4.4 Measurements of seedling survival, health, growth, and nutrition .......................... 105 4.4.5 Statistical analyses ........................................................................................................ 106 4.4.5.1 Differences among treatments ...................................................................................... 106 4.4.5.2 Correlation analyses ........................................................................................................ 107 4.5 Results .................................................................................................................................. 107 4.5.1 Seedling health .............................................................................................................. 107 4.5.2 Seedling growth ............................................................................................................ 111 4.5.3 Seedling nutrition ......................................................................................................... 112 4.5.4 Seedling mycorrhization rate ...................................................................................... 119 4.5.5 Correlation among variables ....................................................................................... 120 4.5.5.1 Percentage of root tips colonized by ECM fungi ...................................................... 120 4.5.5.2 Health and growth of individual seedlings ................................................................ 121 4.6 Discussion ............................................................................................................................. 124 4.6.1 Selection of ectomycorrhizal fungi, essential for seedling health ............................. 124 4.6.2 Selection of PGPR – fundamental for seedling growth ............................................. 126 4.6.3 Soil microorganisms play a huge role in seedling nutrition ...................................... 127 4.6.4 Relationships between health, growth, nutrition of individual seedlings and ECM fungal root colonization ........................................................................................................ 130 4.6.5 Understanding how microorganisms improved seedling health and growth .......... 132 4.7 Conclusion ............................................................................................................................ 133 4.8 Acknowledgements .............................................................................................................. 133 4.9 References ............................................................................................................................ 134 5. General conclusion and future prospects ................................................................... 139 5.1 Summary of the results ....................................................................................................... 139 5.2 Scientific and industrial significance of the research ....................................................... 141 5.3 Future studies ...................................................................................................................... 145 5.4 References ............................................................................................................................ 148 APPENDIX ....................................................................................................................... 149 Appendix I: Localization of the ECM fungal community field study .................................. 150 X