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Evaluating the comprehensive role of endophytic nitrogen-fixing bacteria in sustaining the growth of… Puri, Akshit 2020

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Evaluating the comprehensive role of endophytic nitrogen-fixing bacteria in sustaining the growth of boreal forest trees on nutrient-poor soils by Akshit Puri M.Sc., The University of British Columbia, 2015 B.Tech., Punjab Agricultural University, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Soil Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) June 2020 © Akshit Puri, 2020   ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:  Evaluating the comprehensive role of endophytic nitrogen-fixing bacteria in sustaining the growth of boreal forest trees on nutrient-poor soils  submitted by Akshit Puri  in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Soil Science Examining Committee: Dr. Chris P Chanway, Professor, Faculty of Land and Food Systems & Faculty of Forestry, UBC Supervisor  Dr. Sue J Grayston, Professor, Faculty of Land and Food Systems & Faculty of Forestry, UBC Supervisory Committee Member  Dr. Maja Kržić, Associate Professor, Faculty of Land and Food Systems & Faculty of Forestry, UBC Supervisory Committee Member Dr. Louise Nelson, Professor Emeritus, Department of Biology, UBC Okanagan University Examiner Dr. Les Lavkulich, Professor Emeritus, Faculty of Land and Food Systems, UBC University Examiner Dr. Manish Raizada, Professor, Department of Plant Agriculture, University of Guelph External Examiner     iii Abstract West Chilcotin region located in the Sub-Boreal Pine-Spruce xeric-cold biogeoclimatic zone of British Columbia is characterized by cold climate, low annual precipitation and weakly developed soils. Lodgepole pine and hybrid white spruce comprise the most common tree stands in this region. Soils examined from pine and spruce stands revealed that they have poor physico-chemical health and limited plant-available nutrients, particularly nitrogen. Despite the severely nitrogen-limited soils, the continued, vigorous growth of pine and spruce trees is a major conundrum. A rarely evaluated but possible nitrogen-input could be through endophytic diazotrophic bacteria – nitrogen-fixing bacteria living inside plant tissues. Examining this possibility, 55 and 48 bacterial strains were isolated on nitrogen-free media from internal tissues of pine and spruce, respectively, of which 23 and 18 strains showed positive nitrogenase activity. Six strains each from pine and spruce that showed highest nitrogenase activity were evaluated for their effects on their original host as well as the other tree species (i.e., spruce bacteria tested with pine and pine bacteria tested with spruce) in greenhouse trials. All strains colonized both pine and spruce and performed similarly well in both hosts, contributing 15-56% of host’s foliar nitrogen from the atmosphere after one year and significantly enhancing seedling length and biomass. These strains also possess several other plant-growth-promoting abilities such as inorganic and organic phosphate solubilization, IAA production, ACC deaminase activity, siderophore production and lytic enzyme activity. Notably, four strains closely related to the genus Burkholderia – Caballeronia sordidicola LS-S2r and HP-S1r, Caballeronia udeis LP-R2r and Paraburkholderia phytofirmans LP-R1r – provided up to 5.5mg nitrogen per kg tissue to their host   iv after one year, enhanced seedling biomass by up to 7-fold in 18 months and showed significant potential in the aforementioned plant-growth-promoting mechanisms. Therefore, the ability of such highly effective endophytic diazotrophic bacteria to form beneficial ecological associations with pine and spruce trees could explain their sustained growth on extremely nutrient-limited soils of the West Chilcotin region. Furthermore, their effectiveness with a foreign host indicates the lack of plant x microbe specificity, raising the possibility of their utilization as potential biofertilizers to regenerate trees in disturbed and nutrient-poor ecosystems.    v Lay Summary In natural ecosystems, plants associate with beneficial microbes that play a crucial role in enhancing their growth and health, particularly on disturbed soils. Although mostly unexplored, beneficial bacteria living inside the plant tissues could be an important source of nutrition and development for plants growing in such conditions. In this thesis, the presence and possible role(s) of such endophytic bacteria were explored in lodgepole pine and hybrid white spruce trees growing on extremely nutrient-limited soils in central interior of British Columbia. These bacteria were able to enhance tree growth under nutrient-stress conditions by potentially employing various mechanisms to acquire plant-inaccessible nutrients, modulate plant hormone levels and control plant pathogen populations. Most importantly, these bacteria were observed to provide nitrogen nutrition to trees by directly fixing N2 from the atmosphere. In future, these bacteria could potentially be used as biofertilizers for trees, thereby representing a low-cost, environment-friendly alternative to chemical fertilizers.    vi Preface This thesis represents original research work conducted by me with valuable input from the supervisory committee including Drs. Christopher P Chanway, Sue J Grayston and Maja Kržić. Chapter 1 is an amalgamation of information from three invited book chapters I published as the lead author in association with Kiran Preet Padda and Dr. Chanway. I was the lead investigator, responsible for all major areas of concept formation, field sampling, experimentations, data collection and analysis, and manuscript composition for the studies described in chapters 2, 3, 4, 5 and 6. Kiran Preet Padda was involved in field sampling, experimentations, data analysis and manuscript editing for all these studies. Dr. Chanway was the supervisory author of these studies and was involved in concept formation and manuscript editing throughout. Dr. William K Chapman played a central role in sampling design, plant and soil sample collection and other fieldwork described in chapter 2. Clive Dawson and Andre Bindon from the Analytical Chemistry Services Laboratory, BC Ministry of Environment & Climate Change Strategy, Victoria were involved in soil analyses and acetylene reduction assays for the studies described in chapters 2, 3 and 5. The Sequencing and Bioinformatics Consortium at the University of British Columbia, Vancouver performed the genetic sequencing work described in chapter 2. Drs. Mina Momayyezi and Shannon Guichon from the UBC Stable Isotope Facility in the Department of Forest and Conservation Sciences were involved in analyzing the foliar 15N samples of pine and spruce seedlings for studies described in Chapter 3 and 5.    vii PUBLICATIONS Chapter 1 contains information published in the following book chapters: - Puri A, Padda KP, Chanway CP (2017) Beneficial effects of bacterial endophytes on forest tree species. In: Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 111-132. https://doi.org/10.1007/978-3-319-66544-3_6 - Puri A, Padda KP, Chanway CP (2017) Plant growth promotion by endophytic bacteria in nonnative crop hosts. In: Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 11-45. https://doi.org/10.1007/978-3-319-66544-3_2 - Puri A, Padda KP, Chanway CP (2018) Nitrogen-fixation by endophytic bacteria in agricultural crops: recent advances. In: Khan A, Fahad S (eds) Nitrogen in Agriculture – Updates. IntechOpen, London, pp 73-94. https://doi.org/10.5772/intechopen.71988  Chapter 2 has been published in the Forest Ecology and Management journal: Puri A, Padda KP, Chanway CP (2018) Evidence of endophytic diazotrophic bacteria in lodgepole pine and hybrid white spruce trees growing in soils with different nutrient statuses in the West Chilcotin region of British Columbia, Canada. Forest Ecology and Management 430: 558-565. https://doi.org/10.1016/j.foreco.2018.08.049 Chapter 3 has been published in the Soil Biology and Biochemistry journal: Puri A, Padda KP, Chanway CP (2020) Can naturally-occurring endophytic nitrogen-fixing bacteria of hybrid white spruce sustain boreal forest tree growth on extremely nutrient-poor soils? Soil Biology and Biochemistry 140: 107642. https://doi.org/10.1016/j.soilbio.2019.107642 Chapter 4 has been published in the Applied Soil Ecology Journal: Puri A, Padda KP, Chanway CP (2020) In vitro and in vivo analyses of plant-growth-promoting potential of bacteria naturally associated with spruce trees growing on nutrient-poor soils. Applied Soil Ecology 149: 103538. https://doi.org/10.1016/j.apsoil.2020.103538  Chapter 5 is under review for publication: Puri A, Padda KP, Chanway CP (2020) Evaluating lodgepole pine endophytes for their ability to fix nitrogen and support tree growth under nitrogen-limited conditions. Chapter 6 is under review for publication: Puri A, Padda KP, Chanway CP (2020) Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria.   viii PRESENTATIONS Puri A, Padda KP, Chanway CP (11 November 2019) Nitrogen-fixing bacteria: probiotic for boreal forest trees? 5-minute Rapid Oral and Poster Presentation. Soil Science Society of America International Annual Meeting, San Antonio, USA Puri A, Padda KP, Chanway CP (12 July 2019) Sustaining boreal forest tree growth via nitrogen fixing bacteria. Oral Presentation. Canadian Society of Soil Science Annual Meeting, Saskatoon, Canada Puri A, Padda KP, Chanway CP (9 July 2019) Evaluating the ability of endophytic bacteria to support boreal forest tree growth. Poster Presentation. Plant Canada 2019, Guelph, Canada Puri A, Padda KP, Chanway CP (8 January 2019) Biofertilizers: a sustainable approach to support growth of boreal forest trees. 5-Minute Rapid Oral and Poster Presentation. 2018-19 International Soils Meeting, San Diego, USA [First-Place Best Presentation Award] Puri A, Padda KP, Chanway CP (17 July 2018) Symbiosis between trees and nitrogen-fixing bacteria: can this relationship sustain the growth of boreal forests? Poster Presentation. 9th International Symbiosis Society Congress, Corvallis, USA Puri A, Padda KP, Chanway CP (21 June 2018) Isolation and identification of endophytic diazotrophic bacteria from the internal tissues of lodgepole pine and hybrid spruce trees. 3-minute Oral Flash Talk and Poster Presentation. 11th International Plant Growth-Promoting Rhizobacteria Workshop, Victoria, Canada Puri A, Padda KP, Chanway CP (11 June 2018) Can nitrogen-fixing bacteria sustain the growth of boreal forest trees in West Chilcotin region of BC? Poster Presentation. Canadian Society of Soil Science Annual Meeting, Niagara Falls, Canada [President’s Award: Third-Place Best Poster Presentation Award]    ix Table of Contents Abstract .................................................................................................................................iii Lay Summary……………………………………………………………………………………………………………….…….….v Preface ..................................................................................................................................vi Table of Contents ...................................................................................................................ix List of Tables .........................................................................................................................xii List of Figures .......................................................................................................................xiv List of Abbreviations………………………………………………………………………………………………………….xviii Acknowledgements ...............................................................................................................xx Dedication…………………………………………………………………………………………………………………………xxiii Chapter 1 – Introduction.………………………………………………………………………………….....………………..1 1.1. Plant-growth-promoting-bacteria (PGPB)…………………………………………………………....…………1 1.1.1. Plant growth promoting bacteria (PGPB) in forest ecosystems……………………………….2 1.2. Endophytic bacteria…………………………………………………………………………………………………..……3 1.2.1. Endophytic bacteria in forest ecosystems……………………………………………………………….4 1.2.1.1. Picea………………………………………………………………………………………………………………..6 1.2.1.2. Pinus………………………………………………………………………………………………………………..9 1.3. Endophytic diazotrophic bacteria………………………………………………………………………………….12 1.3.1. Endophytic diazotrophic bacteria in forest ecosystems…………………………………………13 1.4. Study area and rationale…..………………………………………………………………………………………….17 1.5. Thesis objectives and hypotheses…………………………………………………………………………………20 Chapter 2 - Evidence of endophytic diazotrophic bacteria in lodgepole pine and hybrid white spruce trees growing in soils with different nutrient statuses in the West Chilcotin region of British Columbia……………………………………………………………………………………………………….…….……28 2.1. Introduction………………………………………………………………………………………………………………….28 2.2. Materials and methods…………………………………………………………………………………………………30 2.2.1. Site description……………………………………………………………………………………………………..30 2.2.2. Soil and plant sampling…………………………………………………………………………………………31 2.2.3. Soil analyses………………………………………………………………………………………………………….32 2.2.4. Isolation of potential endophytic diazotrophic bacteria………………………………………..32 2.2.5. Evaluation of nitrogenase activity…………………………………………………………………………33 2.2.6. Identification of endophytic diazotrophic bacteria………………………………………………..34 2.2.7. Statistical analyses………………………………………………………………………………………………..35 2.3. Results………………………………………………………………………………………………………………………….36   x 2.3.1. Soil analyses………………………………………………………………………………………………………….36 2.3.2. Bacterial isolation and identification and nitrogenase activity evaluation……………..37 2.4. Discussion…………………………………………………………………………………………………………………….38 Chapter 3 – Can naturally-occurring endophytic nitrogen-fixing bacteria of hybrid white spruce sustain boreal forest tree growth on extremely nutrient-poor soils?.......................................51 3.1. Introduction………………………………………………………………………………………………………………….51 3.2. Materials and methods…………………………………………………………………………………………………54 3.2.1. Bacterial strains…………………………………………………………………………………………………….54 3.2.2. NifH gene analysis…………………………………………………………………………………………………55 3.2.3. Greenhouse experiments……………………………………………………………………………………..55 3.2.3.1. Seed acquisition and preparation………………………………………………………………….56 3.2.3.2. Plant inoculation and growth conditions……………………………………………………….56 3.2.3.3. Investigation of endophytic colonization……………………………………………….........58 3.2.3.4. Analysis of seedling growth promotion and foliar 15N……………………………………58 3.2.4. Statistical analyses………………………………………………………………………………………………..59 3.3. Results………………………………………………………………………………………………………………………….60 3.3.1. Nitrogenase activity and nifH gene……………………………………………………………………….60 3.3.2. Greenhouse experiments………………………………………………………………………………………60 3.3.2.1. Endophytic diazotrophic strains in original host (hybrid white spruce)……….…60 3.3.2.2. Endophytic diazotrophic strains in foreign host (lodgepole pine)…………………..62 3.4. Discussion…….………………………………………………………………………………………………………………64 Chapter 4 - In vitro and in vivo analyses of plant-growth-promoting potential of bacteria naturally associated with spruce trees growing on nutrient-poor soils…………………………………..80 4.1. Introduction………………………………………………………………………………………………………………….80 4.2. Materials and methods…………………………………………………………………………………………………84 4.2.1. Bacterial strains…………………………………………………………………………………………………….84 4.2.2. Evaluating plant-growth-promotion mechanisms………………………………………………….85 4.2.2.1. Inorganic and organic phosphate solubilization……………………………………………..85 4.2.2.2. IAA production………………………….…………………………………………………………………..87 4.2.2.3. ACC deaminase activity and gnotobiotic root elongation assay……………………..87 4.2.2.4. Cellulase activity………….………………………………………………………………………………..89 4.2.2.5. Protease activity…………………………………………………………………………………………….90 4.2.2.6. Chitinase activity……………………………………………………………………………………………91 4.2.2.7. b-1,3-glucanase activity….……………………………………………………………………………..91 4.2.2.8. Siderophore production…………………………………………………………………………………93 4.2.2.9. Catalase activity…………………………………………………………………………………………….93 4.2.2.10. Ammonia production…………………………………………………………………………………..93 4.2.3. Long-term greenhouse experiment……………………………………………………………………….94 4.2.4. Statistical analyses…………………………………………………………………….………………………….97 4.3. Results………..………………………………………………………………….…………………………………………….97 4.3.1. Plant growth promoting mechanisms…………………………………….……………………………..97 4.3.2. Long-term greenhouse experiment……………………….……………………………………………100   xi 4.4. Discussion……….……………………………………………………….…………………..…………………………….101 Chapter 5 - Evaluating lodgepole pine endophytes for their ability to fix nitrogen and support tree growth under nitrogen-limited conditions.……………………………………………………….…………120 5.1. Introduction……………………………………………….………………………………………………………………120 5.2. Materials and Methods…………………………………….………………………………………………………..124 5.2.1. Antibiotic-resistant derivative strains…………………………………….……………………………124 5.2.2. Acetylene reduction assay and nifH gene amplification………………………………………124 5.2.3. Greenhouse trials……….……………………………………………………….……….…………………….125 5.2.3.1. Bacterial inoculum……………………………………………….……….…………………………….126 5.2.3.2. Pre-treatment of seeds……………………………………………….……….……………………..126 5.2.3.3. Growth trial set-up……………………………………….……….…………………………………….127 5.2.3.4. Seedling harvest and analyses……………………….……….……………………………………128 5.2.3.5. Statistical analyses.……….…………………………………..……….……………………………….129 5.3. Results……………………….………………………………………………………..……………………….…………….130 5.4. Discussion………………….………………………………………………………..……………………….…………….133 Chapter 6 - Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria…………….………………………………………………………..……………………….148 6.1. Introduction……………………………………………….………………………………………………………………148 6.2. Materials and Methods…………………………………….………………………………………………………..152 6.2.1. Greenhouse growth trials. ………………………….………………………………………………………152 6.2.2. Direct mechanisms of plant-growth-promotion………….………………………………………155 6.2.2.1. Nutrient acquisition…….…………………………………………….………………………………..155 6.2.2.2. Plant growth hormone modulation………………………………………….………………….157 6.2.3. Indirect mechanisms of plant-growth-promotion………………………………………………..159 6.2.3.1. Phytopathogen suppression via cell wall degradation………………………………….159 6.2.3.2. Ammonia production…………………………………………..………………………………………163 6.2.3.3. Catalase enzyme activity…………………………………………..…………………………………163 6.2.4. Statistical analyses…………………………………..………………………………………………………….163 6.3. Results…………………………….……………………………………………………….…………………………………164 6.3.1. Long-term greenhouse growth trials………………………………………….……….………………164 6.3.2. Direct mechanisms of plant-growth-promotion………………………………….……….……..165 6.3.3. Indirect mechanisms of plant-growth-promotion…………………………….……….…………167 6.4. Discussion………………………….……………………………………………………….………………………………168 Chapter 7 - General Summary and Conclusions………………………...………………………………………..185 References…………………………………………………………………………………………………………………………194 Appendices….…………………………………………………………………………………………………………………….220 Appendix A………………………………………………………………………………………………………………………..220 Appendix B………………………………………………………………..………………………………………………………221 Appendix C………………………………………………………………..………………………………………………………222    xii List of Tables Table 1.1 List of endophytic bacteria isolated from important forest tree species and their beneficial effects on host trees……………………………………………………………………………………………..….25 Table 2.1 Soil characteristics and concentrations of macro- and micro-nutrients present in the forest floor and top mineral layer (0–10 cm) from high-elevation and low-elevation sites in the hybrid white spruce stand……………………………………………………………………………………………..………..43 Table 2.2 Soil characteristics and concentrations of macro- and micro-nutrients present in the forest floor and top mineral layer (0–10 cm) from high-elevation and low-elevation sites in the lodgepole pine stand………………………………………………………………………………………………..……………..44 Table 2.3 List of endophytic diazotrophic strains isolated from hybrid white spruce trees growing on high-elevation and low-elevation sites with their most closely related genus/species and the amount of ethylene produced in the acetylene reduction assay………………………….…………………….45 Table 2.4 List of endophytic diazotrophic strains isolated from lodgepole pine trees growing on high-elevation and low-elevation sites with their most closely related genus/species and the amount of ethylene produced in the acetylene reduction assay……………………………….……………….46 Table 3.1 List of endophytic diazotrophic bacterial strains selected from Chapter 2 (Puri et al. 2018a) and their antibiotic-resistant derivative strains used in this study along with the amount of ethylene produced in the acetylene reduction assay and the presence of the nifH gene in these derivative strains.………………………………………………………………………………………………………………..…..70 Table 3.2 Percent N derived from the atmosphere (% Ndfa) by each of the six bacteria in hybrid white spruce seedlings, determined 12 months after sowing using atom percent 15N excess in foliage values. N-fixation rate (mg of fixed N per kg of tissue per day) was determined using %Ndfa and foliar N concentration. Values are mean ± standard error for atom % 15N excess in foliage (n = 10 seedlings per treatment). Values followed by different letters are significantly different (P < 0.05)..………………………………………………………………………………………………………………………………………70 Table 3.3 Percent N derived from the atmosphere (% Ndfa) by each of the six bacteria in lodgepole pine seedlings, determined 12 months after sowing using atom percent 15N excess in foliage values. N-fixation rate (mg of fixed N per kg of tissue per day) was determined using %Ndfa and foliar N concentration. Values are mean ± standard error for atom % 15N excess in foliage (n = 10 seedlings per treatment). Values followed by different letters are significantly different (P < 0.05).……………………………………………………………………………………………………………………………………….71   xiii Table 4.1 Inorganic Ca-phosphate solubilization and phytate hydrolyzation measured both qualitatively and quantitatively, and ACC deaminase enzyme activity and indole-3-acetic acid (IAA) production measured quantitatively for the six bacterial strains…………………………………....112 Table 4.2 Activity of cell wall degrading enzymes – cellulase, protease and chitinase – measured for the six bacterial strains……………………………………………………………………………………………………..113 Table 4.3 Catalase activity, ammonia production and siderophore production (area of orange halo) measured for the six bacterial strains…………………………………………………………………………….114 Table 5.1 Nitrogen-fixing endophytic bacterial strains selected from Chapter 2 (Puri et al. 2018a) and their antibiotic-resistant derivatives along with the assessment of their nitrogen-fixing ability demonstrated via acetylene reduction activity and the presence of the nifH gene. Acetylene reduction activity has been expressed as nanomoles of ethylene produced per mL of culture tube headspace (mean ± standard error; n = 5)……………………………………………………………………………….140 Table 5.2 Foliar N concentration (mg N per g tissue), atom percent 15N excess in foliage, percent N derived from the atmosphere (% Ndfa) and rate of N-fixation (mg of fixed N per kg of tissue per day) of lodgepole pine and hybrid white spruce seedlings treated with six endophytic diazotrophic strains and a non-inoculated control, measured 12 months after sowing. Percent foliar N and atom percent 15N excess in foliage values are mean ± standard error (n = 10 seedlings per treatment). Values followed by different letters are significantly different at P < 0.05.………………………………………………………………………………………………………………………………………141 Table 6.1 Qualitative evaluation of major direct and indirect plant-growth-promoting mechanisms of the six bacterial strains using in vitro plate-based enzyme assays…………………...178 Table 6.2 Quantitative evaluation of major direct and indirect plant-growth-promoting mechanisms of the six bacterial strains using in vitro broth-based enzyme assays……………………179 Table 7.1 Concluding assessment and overall ranking of endophytic bacterial strains isolated from hybrid white spruce and lodgepole pine trees (Chapter 2) growing in the West Chilcotin region of BC. The assessment and ranking were generated using the bonitur scale (Krechel et al. 2002) based on the 12 plant-growth-promoting mechanisms analyzed in chapters 3, 4, 5 and 6…………………………………………………………………………………………………………………………………………….193    xiv List of Figures Figure 1.1 (a) Location of the Sub-Boreal Pine-Spruce biogeoclimatic zone represented on the map of British Columbia; (b) four subzones of the Sub-Boreal Pine-Spruce zone represented by different colours and codes, with the sampling area indicated by star shape in the xc subzone……………………………………………………………………………………………………………………………………27 Figure 2.1 (a) Location of sampling area in the West Chilcotin region, 250 km west of Williams Lake, BC on the Chilcotin-Bella Coola Highway (BC Highway 20); (b) Detailed view of the sampling area, showing the two sampling sites each in the hybrid white spruce and lodgepole pine stands that were selected at different elevations…………………………………………………………………………………47 Figure 2.2 Soil conditions at sampling sites in the West Chilcotin region. (a) very thin or negligible organic forest floor denoted by arrows; (b) soil sample collected using an Oakfield probe………………………..……………………………………………………………………………………………………………..48 Figure 2.3 Mean values (n=10) of: (a) Carbon to nitrogen ratio, (b) mineralizable nitrogen, (c) available nitrate, and (d) available ammonia present in forest floor and top mineral layer (0–10 cm) soil samples collected from the low-elevation (LE) site and high-elevation (HE) site in the hybrid white spruce stand. Error bars represent standard errors of mean; *P < 0.05 (significantly different from low-elevation site)…………………………………………………………………………………………….49 Figure 2.4 Mean values (n=10) of: (a) Carbon to nitrogen ratio, (b) mineralizable nitrogen, (c) available nitrate, and (d) available ammonia present in forest floor and top mineral layer (0-10 cm) soil samples collected from the low-elevation (LE) site and high-elevation (HE) site in the lodgepole pine stand. Error bars represent standard errors of mean; *P < 0.05 (significantly different from low-elevation site)…………………………………………………………………………………………….50 Figure 3.1 Endophytic population size of each of the six bacterial strains inside (a) needle, (b) stem, and (c) root tissues of hybrid white spruce seedlings evaluated 4, 8 and 12 months after sowing (n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted, and data were log-transformed. Strains that did not colonize a tissue type have not been included in the figure…………………………………………………………………………………………………………………………………72 Figure 3.2 Mean values of (a) length and (b) biomass of hybrid white spruce seedlings subjected to six bacterial treatments and a non-inoculated control harvested 4, 8 and 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05)…………………………………………………………………73 Figure 3.3 Bacteria-inoculated and non-inoculated control seedlings of hybrid white spruce harvested (a) 4 months, (b) 8 months, and (c) 12 months after sowing, showing clear differences in length and biomass……………………………………………………….….………………………………………………….74   xv Figure 3.4 Nitrogen (N) concentration (mg N per g tissue) in the foliage of hybrid white spruce seedlings subjected to six bacterial treatments and a non-inoculated control measured 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05)……………………………….75 Figure 3.5 Endophytic population size of each of the six bacterial strains inside (a) needle, (b) stem, and (c) root tissues of lodgepole pine seedlings evaluated 4, 8 and 12 months after sowing (n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted, and data were log-transformed. Strains that did not colonize a tissue type have not been included in the figure…………………………………………………………………………………………………………………………………….…76 Figure 3.6 Mean values of (a) length and (b) biomass of lodgepole pine seedlings subjected to six bacterial treatments and a non-inoculated control harvested 4, 8 and 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05).……………………………………………………………………………….77 Figure 3.7 Bacteria-inoculated and non-inoculated control seedlings of lodgepole pine harvested (a) 4 months, (b) 8 months, and (c) 12 months after sowing, showing clear differences in length and biomass………………………………………………………….………………………………………………………………...78 Figure 3.8 Nitrogen (N) concentration (mg N per g tissue) in the foliage of lodgepole pine seedlings subjected to six bacterial treatments and a non-inoculated control measured 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05).………………………..…….79 Figure 4.1 Canola seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. (a) Mean values of primary root length of canola seedlings evaluated after 5 days of growth. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). (b) Five-day old canola seedlings showing differences in root length between treatments.………………………………………………………………………………………..115 Figure 4.2 Tomato seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. (a) Mean values of primary root length of tomato seedlings evaluated after 5 days of growth. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). (b) Five-day old tomato seedlings showing differences in root length between treatments………………………………………………………..…………….116 Figure 4.3 Population size of each of the six bacterial strains in the internal tissues (needle, stem and root) and the rhizosphere of hybrid white spruce seedlings evaluated 18 months after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment each for rhizospheric and endophytic evaluations).………………………………………………………………………………………………………………………….117   xvi Figure 4.4 Population size of each of the six bacterial strains in the internal tissues (needle, stem and root) and the rhizosphere of lodgepole pine seedlings evaluated 18 months after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment each for rhizospheric and endophytic evaluations).………………………………………………………………………………………………………………………….117 Figure 4.5 (a) Seedling length and (b) seedling biomass of hybrid white spruce subjected to six bacteria-inoculated and one non-inoculated control treatments, harvested 18 months after inoculation. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05)….……………………………………………..…….118 Figure 4.6 (a) Seedling length and (b) seedling biomass of lodgepole pine subjected to six bacteria-inoculated and one non-inoculated control treatments, harvested 18 months after inoculation. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05).……………………………………………….…..…119 Figure 5.1 Lodgepole pine seedlings corresponding to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment harvested (a) 4-month, (b) 8-month, and (c) 12-month after sowing and inoculation.……..………………………………….……………………………….….….142 Figure 5.2 Hybrid spruce seedlings corresponding to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment harvested (a) 4-month, (b) 8-month, and (c) 12-month after sowing and inoculation.………………………………….……………………………………….…….143 Figure 5.3 (a) Length and (b) biomass of lodgepole pine seedlings subjected to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment, determined 4, 8 and 12 months after sowing and inoculation (means and standard errors; n = 10 seedlings per treatment). Error bars with different letters are significantly different (P<0.05)…………………..….144 Figure 5.4 (a) Length and (b) biomass of hybrid white spruce seedlings subjected to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment, determined 4, 8 and 12 months after sowing and inoculation (means and standard errors; n = 10 seedlings per treatment). Error bars with different letters are significantly different (P<0.05).……………………………….…………………………………………………………………….…………………………145 Figure 5.5 Endophytic colonization by the six endophytic diazotrophic bacterial strains in lodgepole pine (a) needle, (b) stem, and (c) root tissues, determined 4, 8 and 12 months after sowing and inoculation (expressed as colony forming units per gram fresh tissue; n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted, and data were log-transformed.……………….…………………………………………………………………….…………………………………..146 Figure 5.6 Endophytic colonization by the six endophytic diazotrophic bacterial strains in hybrid white spruce (a) needle, (b) stem, and (c) root tissues, determined 4, 8 and 12 months after sowing and inoculation (expressed as colony forming units per gram fresh tissue; n = 5 seedlings   xvii per treatment). For clarity of presentation, error bars were omitted, and data were log-transformed.……………….…………………………………………………………………….…………………………………..147 Figure 6.1 Mean values of (a) length and (b) biomass of 540-day old lodgepole pine seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Error bars represent standard errors of mean (n = 10 seedlings per treatment) and bars with different letters are significantly different (P < 0.05).….…………………………………………………………………………….……..180 Figure 6.2 Mean values of (a) length and (b) biomass of 540-day old hybrid white spruce seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Error bars represent standard errors of mean (n = 10 seedlings per treatment) and bars with different letters are significantly different (P < 0.05).…………………………………………………………………………….…………181 Figure 6.3 Population density of each of the six bacterial strains inside the endophytic tissues (needle, stem and root) and in the rhizosphere of (a) lodgepole pine and (b) hybrid white spruce seedlings evaluated 540 days after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment for endophytic colonization and 5 seedlings per treatment for rhizospheric colonization)…………….182 Figure 6.4 Mean values of primary root length of (a) canola and (b) tomato seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Seedlings were evaluated five days after germination in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05)……………………………………..…..183 Figure 6.5 Five-day old (a) canola and (b) tomato seedlings showing differences in root length between treatments. Seedlings were subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity..……………………………………………………………………………………………………………….184    xviii List of Abbreviations AAB Acetic acid bacteria ACC 1-aminocyclopopane-1-carboxylate ANOVA Analysis of Variance ARA Acetylene reduction assay BC British Columbia BLAST Basic Local Alignment Search Tool BNF Biological nitrogen fixation bp Base pairs CAS Chrome azurol S CCM Combined carbon medium CEC Cation exchange capacity cfu Colony forming units CLSM Confocal laser scanning microscopy CYE Casein yeast extract DNA Deoxyribonucleic acid GFP Green fluorescent protein IAA Indole-3-acetic acid IFAS Immunofluorescent antibody straining  Ndfa Nitrogen derived from atmosphere OM Organic matter PABA Para amino benzoic acid PBS Phosphate buffered saline PCR Polymerase chain reaction   xix PGP Plant growth promoting PGPB Plant growth promoting bacteria PSM Phytase screening medium PVK Pikovskaya qRT-PCR Quantitative reverse transcription polymerase chain reaction RGR Relative growth rate ROS Reactive oxygen species rRNA Ribosomal ribonucleic acid SBPS Sub-boreal pine-spruce SI Solubilization index TSA Tryptic soy agar U Unit UBC University of British Columbia xc Xeric cold     xx Acknowledgments First of all, I would like to pay my deepest gratitude to my research supervisor, Dr. Chris Chanway, for his guidance, motivation and unwavering support throughout my PhD and MSc degrees, and most importantly for the academic independence he gave me during my graduate career. Thank you so very much for saying “I am interested in taking you on as my MSc student” on 20 September 2012, it actually transformed the entire life of an aspiring young man from India. I am highly obliged! I am also tremendously grateful to my second mentor, Dr. Maja Kržić, who went above and beyond to support my teaching endeavours and give career advice at every stage of my grad school. Thank you very much for believing in me and for giving me a chance to explore my teaching potential. I never would have realized that I can be a teacher, but you saw it in me! I will be enormously grateful to you for igniting the passion for ‘Soils’ in me – I promise to keep it alive for the rest of my life. You totally deserve the title of “World’s Best Mentor”! I would also like to acknowledge Dr. Sue Grayston for her insightful comments and constructive feedback at various stages of the project and broadening my horizons. This thesis project would not have come this far without the invaluable and unparalleled contributions of Dr. William Chapman who was the central figure in the initial phase of the project. Thank you very much for providing scientific direction, helping me conceive research ideas, chauffeuring me to extremely remote sampling locations in the central interior of BC (driving 500 km daily), and assisting me in collecting soil and plant samples. I am also extremely grateful to Dr. Les Lavkulich for always keeping his door open, giving life and career advice, supporting me when I was down and making me realize my true potential – Thank you for being there!    xxi I am highly grateful to the University of British Columbia (UBC) for supporting my PhD studies via the Four-Year Doctoral Fellowship, Li Tze Fong Memorial Fellowship, and Mary and David Macaree Fellowships. In addition, I am highly thankful to UBC, International Symbiosis Society, Canadian Society of Soil Science and Canadian Society of Plant Biologists for funding my travel to present this research at several conferences. This research would not have been possible without the financial support provided by the National Science and Engineering Research Council (NSERC) through Discovery Grant to Dr. Chris Chanway. I would like to extend my sincerest thanks to Henry Yang for being a great research mentor in the lab. Thank you very much for keeping an open mind and always answering my questions, even after completing your grad school – I know you will always respond to my text/email. Special thanks to Clive Dawson and Andre Bindon for their contributions to the initial phases of the project – you were extremely accommodating! I am grateful to BC Ministry of Forests Tree Seed Centre in Surrey, BC for generously providing lodgepole pine and hybrid white spruce seeds. I would also like to thank Drs. Alice Chang, Mina Momayyezi and Shannon Guichon for their involvement in the analysis of 15N isotope samples and providing huge support in the lab work. I would also like to appreciate the support provided by UBC Plant Care Services and Dr. Pia Smets for making the greenhouse and growth chamber facilities available for this research. A big thank you to my undergraduate assistants and fellow graduate students, particularly, Jonathan Zajonc, Kelsey Marzotto, Lewis Fausak and Dixi Modi. I am also grateful to all members of the APBI 200 course (Introduction to Soil Science) over the years (2015–2020) with whom I spent a significant amount of time during my grad school. To all my teaching mentors in the Faculty of Land and Food Systems – Thank you for believing in my teaching capabilities! I would also like to thank Lia Maria Dragan and Shelly Small at the Faculty   xxii of Land and Food Systems, and Natasha Thompson at the Department of Forest and Conservation Sciences, for providing great support in all administrative matters and making my grad school journey at UBC a smooth ride. I am highly grateful to the editors and anonymous reviewers of the manuscripts published from this project – Thank you for keeping me on my toes and providing critical feedback and insightful comments to improve this research! This undertaking would not have been possible without the most inspirational figure in my life – my father. Without his moral support and blessings, this journey would not have even started. I will always be indebted to you for encouraging me to pursue my dream of getting a doctorate. I know I wasn’t able to thank you enough when we met for the last time – only if I knew. Not being there at the time of your last breath will remain my life’s biggest regret! I am also extremely grateful to my mother for believing in my capabilities, keeping me in her prayers, supporting me in this undertaking and being the foundation in our family.  Last but never the least, to my bestest-friend, love-of-my-life and partner-in-crime, Dr. Kiran Padda. Without your immense contributions, this PhD research would have been far from complete. My unending thanks to you for always standing by my side, supporting me in each and every phase of my personal and professional life, encouraging me to go big and cheering me up in this long and hard journey. I was damn lucky to find a partner like you! No acknowledgement will do justice to your efforts in making me a better and responsible individual!    xxiii    To My Father  Late Mr. Darshan K Puri (Dec 25, 1956 – Nov 28, 2014) From carrying me on your shoulders when I was a kid  to making me capable enough that I have reached here in my life.  Hope I have done you proud Papa…    1 Chapter 1 – Introduction 1.1. Plant-growth-promoting-bacteria (PGPB) The plant microbiome includes a diverse range of microbes living in their endosphere (plant interior), phyllosphere (plant exterior – aboveground) and rhizosphere (plant exterior – belowground) with important roles in sustaining the growth of the host plant (Schlaeppi and Bulgarell 2015). Studies have suggested that plants either sustain members from the seed microbiome or carefully recruit microbes from the surrounding environment in order to maintain their growth and health (Compant et al. 2019). Bacteria that play a key role in various growth and development processes throughout the plant’s life cycle are commonly known as plant growth promoting bacteria (PGPB) (Bashan and Holguin 1998). Although certain PGPB such as symbiotic nitrogen (N) fixing bacteria have been studied extensively for more than a century (McCosh 1984) and used in several biofertilizer products, other PGPB have received greater attention only recently (Mitter et al. 2019). PGPB can benefit the plant through one or more mechanisms leading to enhanced nutrient acquisition or abiotic/biotic stress tolerance (Glick 2012). Bacteria in the families Bacillaceae, Paenibacillaceae, Burkholderiaceae and Pseudomonadaceae have been isolated from diverse ecosystems including natural boreal forests, intensively cultivated agricultural farms and highly degraded mining sites, with notable abilities to promote the growth and health of both gymnosperms and angiosperms (Puri et al. 2017a, b; Padda et al. 2018). Due to their considerable success in field conditions, such PGPB are increasingly being commercialized as bioinoculants (reviewed by Glick 2015). However, it is also crucial to continue exploring PGPB   2 in local ecosystems as they may be better suited for native plant ecotypes (O’Neill et al. 1992; Shishido and Chanway 1999; Santoyo et al. 2016; Iyer and Rajkumar 2017). 1.1.1. Plant growth promoting bacteria (PGPB) in forest ecosystems Since mycorrhizal fungi have been the primary focus of research on beneficial interaction of microbes with trees (Perry et al. 1987; Thapar 1989; Brundrett 2009; Domínguez-Núñez and Albanesi 2019), PGPB other than symbiotic Frankia did not receive adequate attention, until recently (Chanway 1997; Anand et al. 2006; Chanway et al. 2014; Puri et al. 2017a). Although the first documented report of tree growth enhancement by associative PGPB came out in 1958 (Akhromeiko and Shestakova 1958), it was not until the 1980s that significant work on this subject began. Angiosperm trees such as oak, ash, beech, citrus, apple and Eucalyptus were reported to associate with PGPB from genera Azotobacter, Azosprillum, Bacillus and Pseudomonas, which were responsible for promoting tree seedling growth in greenhouse and field conditions (reviewed by Chanway 1997; Shishido 1997). Since the late 1980s, early research on beneficial interactions of PGPB with gymnosperm trees was performed primarily by Chanway and colleagues. Initially, they reported that rhizospheric strains of genera Bacillus, Pseudomonas and Arthrobacter significantly enhance the seedling biomass, seedling length, root proliferation and branch number of lodgepole pine (Pinus contorta), Douglas-fir (Pseudotsuga menziesii), hybrid white spruce (Picea glauca x engelmannii) and western hemlock (Tsuga heterophylla) seedlings in greenhouse, nursery, phytotron and field trials (Chanway and Holl 1994; Chanway and Holl 1992; Shishido and Chanway 2000; Chanway 1995). Subsequently, they reported that some of these strains can colonize internal root tissues of lodgepole pine and hybrid spruce (Shishido et   3 al. 1995; Shishido et al. 1999; Shishido and Chanway 2000), which consequently led to the exploration of novel PGPB from interior root, stem and needle tissues of conifers in later studies (Bal et al. 2012; Padda et al. 2018). In the last decade or so, several studies have focused on exploring culturable and non-culturable bacteria living in the internal tissues of conifers, such as Engelmann spruce, limber pine, lodgepole pine, Douglas-fir, hybrid white spruce, western red cedar and Scots pine, with a strong potential to enhance tree growth (Pirttilä et al. 2000; Kaewkla and Franco 2010; Bal et al. 2012; Carrell and Frank 2014; Moyes et al. 2016; Padda et al. 2018). PGPB, whether rhizospheric or endophytic (internal tissue), have been reported to provide benefits to conifers directly via fixing atmospheric N, solubilizing phosphate and modulating phytohormones as well as indirectly via suppressing pathogens and facilitating tree-mycorrhizal symbiosis (Chanway et al. 2014; Pirttilä and Frank 2011, 2018). 1.2. Endophytic bacteria By definition, an endophyte is an organism which lives inside a plant since "endo" is derived from the Greek word "endon" meaning within, and "phyte" is derived from the Greek word "phyton" meaning plant) (Chanway 1996). The term “endophyte” was first coined more than 150 years ago by de Bary (1866) for pathogenic fungi that were able to enter leaf tissues. Later, Galippe (1887) postulated that various vegetable plants host microbes within their internal tissues and that these microbes are originally derived from the soil. This hypothesis was also confirmed by di Vestea (1888), but well-known scientists at that time such as Pasteur, Chamberland, Fernbach, Laurent, and others claimed that plants are normally free of microbes. They indeed demonstrated contradictory results to disprove Galippe’s hypothesis (Compant et al. 2010).   4 However, it is now well accepted that plants host a wide range of phylogenetically distinct endophytes in various organs (Bacon and White 2000), which are either derived from the soil environment or the parent tree in the form of the seed microbiome (Rosenblueth and Martínez-Romero 2006; Hardoim et al. 2008; Ryan et al. 2008; Compant et al. 2010; Frank et al. 2017). Although the presence of endophytic bacteria was first reported several decades ago in the internal tissues of healthy potato plants (Trevet and Hollis 1948), the literature is still dominated by studies related to endophytic fungi (Doty 2011). In addition, most studies concerning endophytic bacteria have focused on evaluating their benefits in agricultural and horticultural plants (reviewed by Hallmann et al. 1997; Kobayashi and Palumbo 2000; Sturz et al. 2000; Suman et al. 2016). However limited the studies maybe, the role of endophytic bacteria in forest ecosystems should not be underrated (Chanway 1996; Puri et al. 2017a; Kandel et al. 2017a). To describe endophytic bacteria in this thesis, the term defined by Chanway et al. (2014) will be used – “bacteria that can be detected at a particular moment within the tissue of apparently healthy plant hosts without inducing disease or organogenesis.” 1.2.1. Endophytic bacteria in forest ecosystems Forest trees, due to their larger biomass and longer life in comparison to agricultural and horticultural plants, may provide unique ecological conditions for endophytic bacteria to thrive (Izumi 2011). Although limited, studies conducted until now have revealed that a considerable diversity of endophytic bacterial species exist in forest ecosystems (Izumi 2011), with the potential to enhance tree growth via multifarious mechanisms including the production of phytohormones [such as cytokinins (Pirttilä 2011), auxins (Taghavi et al. 2005; Madmony et al.   5 2005), gibberellins (Bottini et al. 2004)], N-fixation (Bal et al. 2012; Anand et al. 2013; Doty et al. 2016), phosphate solubilization (Germaine et al. 2004; Khan et al. 2015), siderophore production (Kandel et al. 2017a; Padda et al. 2017a), phytopathogen suppression (Brooks et al. 1994; Terhonen et al. 2018),  production of adenine derivatives and vitamin B12 (Pirttilä 2018) and improvement of the mutualistic relationship of mycorrhizae and the host plant (Anand et al. 2006). The initial notion in endophyte research indicated that most interactions between plants and endophytic bacteria occur in the root tissues of the host plant, however, it is now widely accepted that above-ground parts of the tree also harbour diverse endophytic bacteria that can carry out similar plant-beneficial activities as root endophytic bacteria. Highly effective endophytic bacteria have also been isolated from shoot tips, flowers, pollens, cones and seeds of trees (Pirttilä 2011). Common inhabitants of the internal tissues of major coniferous and deciduous tree species of the genera Picea, Pinus, Populus, Pseudotsuga, Quercus, Salix and Thuja include bacteria belonging to genera Acinetobacter, Arthrobacter, Burkholderia, Bacillus, Enterobacter, Methylobacterium, Microbacterium, Paenibacillus, Paraburkholderia, Pseudomonas, Rahnella, Sphingomonas, and Xanthomonas (Izumi 2011; Pirttilä 2011, 2018; Puri et al. 2017a; Aghai et al. 2019). A detailed list of endophytic bacteria isolated from these tree species and their plant growth promoting (PGP) properties have been summarized in Table 1.1.  Pinaceae is the largest and most well-known family of conifers in the world, including Picea and Pinus trees that cover more than half of the forests in the northern hemisphere (Burns and Honkala 1990). According to Statistics Canada (2018), Picea and Pinus trees, covering around 63% of Canadian forests, are the backbone of the timber industry. Primary research related to the association of endophytic bacteria with Picea and Pinus trees has been elaborated below.   6 1.2.1.1. Picea Picea, commonly known as spruce, is generally found in the northern temperate and boreal regions of the World including North America, North Europe and Eurasia. Commercially important species of spruce include black spruce (Picea mariana), Engelmann spruce (P. engelmannii), Sitka spruce (P. sitchensis), white spruce (P. glauca), Norway spruce (P. alpestris and P. abies), and Siberian spruce (P. obovata and P. omorika) (Parish and Thomson 1994).  The first reported spruce endophyte, Pseudomonas sp. Ss2, was isolated from the roots of hybrid white spruce trees naturally regenerating near Salmon Arm, British Columbia (BC), Canada (51° 04ʹ N, 119° 26ʹ W, 1250 m elevation) (Shishido and Chanway 1999). The antibiotic-resistant derivative of this strain, Ss2-RN, was generated to evaluate plant colonization upon re-inoculation into spruce seedlings in a greenhouse growth trial (Shishido and Chanway 1999). In the 15-week long greenhouse trial, Ss2-RN-inoculated spruce seedlings had 19% greater root weight, 10% greater shoot weight and 6% greater seedling length in comparison to the non-inoculated control spruce seedlings (Shishido et al. 1996b). It was also reported that bacterial inoculation did not affect the mycorrhizal status of spruce seedlings and growth promotion achieved by bacterial inoculation was similar in mycorrhizal and non-mycorrhizal spruce seedlings (Shishido et al. 1996b). Interestingly, plant-ecotype specificity was observed for strain Ss2-RN, since spruce ecotypes that originated from the same geographical area as this bacterial strain performed significantly better than ecotypes from other areas (Shishido and Chanway 1999). In further experimentation, a combination of greenhouse and field trials were designed to further assess the growth-promoting effects of Ss2-RN (Shishido and Chanway 2000). Hybrid   7 white spruce seedlings were first grown in the greenhouse for 4 months and were then outplanted at predetermined field sites. At the end of the greenhouse growth period, strain Ss2-RN had significantly increased the shoot and root biomass (up to 17% and 19%, respectively) of spruce seedlings. Subsequently, the relative growth rate (RGR) (Hunt 1978) of outplanted seedlings was determined 4 months after outplanting in the field. Root and shoot RGR of inoculated seedlings were up to 234% higher than controls, thus establishing the fact that endophytic strain Ss2-RN can perform significantly well in field conditions. Although isolated from the rhizosphere of hybrid white spruce seedlings growing in a Sub-Boreal Spruce forest region near Mackenzie, BC, Canada (55°11ʹN, 122°58ʹW, 780 m elevation), Pseudomonas sp. Sm3-RN (Shishido and Chanway 1999) was reported to endophytically colonize the interior tissues of spruce seedlings in both greenhouse and field conditions. This strain colonized the internal root tissues of spruce seedlings with a population density of 102 – 104 cfu/g root tissue (Shishido and Chanway 2000; Chanway et al. 2000). Using immunofluorescent antibody staining (IFAS), strain Sm3-RN was also detected in root hairs, cortical cells, and stem vascular tissues of spruce seedlings 4 months after inoculation (Shishido et al. 1999). These studies indicate that the rhizospheric strain Sm3-RN could potentially enter into spruce seedlings through various root openings and form detectable endophytic colonies inside root and stem tissues. By colonizing the plant tissues, this strain not only enhanced the root and shoot weight (18% and 12%, respectively) of spruce seedlings in the greenhouse trial (Shishido et al. 1996b) but also increased the shoot RGR (up to 97%) of outplanted spruce seedlings under field conditions (Shishido and Chanway 2000).    8 An endophytic bacterium, Paenibacillus polymyxa Pw-2R, isolated from the internal root tissues of a young lodgepole pine tree growing near Williams Lake, BC, Canada (52"N, 122"W) (Shishido et al. 1995) was observed to colonize internal root and stem tissues of hybrid white spruce seedlings 5 months after inoculation with population sizes of 104 – 105 cfu/g root tissue (Chanway et al. 2000; Shishido and Chanway 2000). When evaluated using IFAS, this strain was observed to colonize stem vascular and root cortical tissues of spruce seedlings 4 months after inoculation, thereby further establishing the endophytic association of P. polymyxa Pw-2R with spruce (Shishido et al. 1999). Along with colonizing the plant interior, this strain promoted spruce seedling biomass by 57% in field trials 17 months after inoculation (Chanway et al. 2000). In another study, inoculation with strain Pw-2R increased shoot RGR by up to 82% in outplanted spruce seedlings 8 months after inoculation (Shishido and Chanway 2000). Endophytic bacteria have also been isolated from seeds of Norway spruce trees growing in various locations within a 36 km2 area in Pokljuka, Slovenia (1200–1400 m elevation) (Cankar et al. 2005). In that study, Pseudomonas and Rahnella strains were isolated from the seed coat, endosperm and embryonic tissues, with the potential to perform N-fixation and promote plant growth (Geric et al. 2000). In another study, N-fixing acetic acid bacteria (AAB) closely related to Gluconacetobacter diazotrophicus were found to be a major taxon in the needle microbiome of Engelmann spruce trees growing in a subalpine, nutrient-limited environment of Niwot Ridge, Colorado, USA (Carrell and Frank 2014). The authors postulated that the consistency with which AAB dominated the needle microbiota of Engelmann spruce trees in this region indicates that such bacteria may have been specifically harboured by spruce to perform the specialized beneficial function of fixing atmospheric N. In a recent study, an endophytic strain,   9 Paraburkholderia sp. PSSN1, was isolated from Sitka spruce trees growing in the riparian zones of the Snoqualmie (47°31ʹ14.30ʺ N, 121°46ʹ28.32ʺ W) and Skykomish (47°46ʹ48.3240ʺ N, 121°30ʹ01.3320ʺ W) rivers in Washington, USA (Aghai et al. 2019). When this strain was inoculated as part of a consortium of conifer endophytes into Douglas-fir and western red cedar seedlings, an improved resistance to drought and a significant increase in seedling length were observed. 1.2.1.2. Pinus Pinus is one of the largest and, certainly, the most important genera among conifers. Pines are widely distributed in the northern hemisphere, ranging from Alaska to Nicaragua, Scandinavia to North Africa and Siberia to Sumatra (Krugman and Jenkinson 1974). Lodgepole pine is one of the most common Pinus species found in western North America and has a wide ecological amplitude as it can be found in a range of environments, from water-logged bogs to bare gravel (Parish and Thomson 1994). It is a commercially important gymnosperm species that grows throughout the Rocky Mountain and Pacific Coast regions, extending from Yukon Territory, Canada in the north to Baja California, Mexico in the south and from the Pacific Ocean in the west to South Dakota, USA in the east (Lotan and Critchfield 1990). The first evidence related to the presence of endophytic bacteria in lodgepole pine was reported by Shishido et al. (1995). They isolated Bacillus polymyxa (now known as Paenibacillus polymyxa) strain Pw-2 from root tissues of a lodgepole pine seedling (<3 years old) naturally regenerating near Williams Lake, BC, Canada (52°N, 122°W). Inoculation with strain Pw-2 significantly increased shoot biomass (24%), root biomass (27%) and length (18%) of lodgepole pine seedlings as compared to the non-inoculated   10 controls in a 9-week greenhouse trial (Shishido et al. 1995). To confirm the ability of this strain to endophytically colonize internal tissues upon re-inoculation, an antibiotic-resistant strain, Pw-2R, was developed. The endophytic population size of 105 cfu/g was observed for strain Pw-2R in the fresh root tissues of lodgepole pine seedlings, 4 weeks after inoculation (Shishido et al. 1995). In a subsequent study, it was observed that strain Pw-2R similarly enhances the growth of mycorrhizal and non-mycorrhizal lodgepole pine seedlings, thereby ruling out the possibility of mycorrhizal growth promotion in field and nursery trials (Shishido et al. 1996a). The tree growth promotion observed for strain Pw-2R could be related to its ability to elevate the levels of phytohormones such as indole-3-acetic acid (IAA) and dihydrozeatin riboside (a form of cytokinin) (Bent et al. 2001). More than a decade later, Bal et al. (2012) isolated 99 distinct endophytic bacterial strains from stem and needle tissues of lodgepole pine trees (20 years old) and seedlings (2-4 years old) growing at sites near Williams Lake, BC (52°05ʹN, 122°54ʹW, elevation 1300 m; dry cool Sub-boreal Pine Spruce zone) and Chilliwack Lake, BC (49°03ʹN, 121°25ʹW, elevation 625 m; dry mild Coastal Western Hemlock zone). In subsequent studies, one of these strains, Paenibacillus polymyxa P2b-2R, was reported to fix significant amounts of N from the atmosphere and considerably enhance lodgepole pine shoot height (up to 33%), shoot biomass (up to 78%), and root biomass (165%), 13 months after inoculation (Anand et al. 2013; Bal and Chanway 2012a). In addition, strain P2b-2R colonized root, stem and needle tissues of lodgepole pine seedlings with population densities of 103 – 106 cfu/g fresh tissue (Anand et al. 2013; Bal and Chanway 2012a). To view the sites of endophytic colonization, green fluorescent protein (GFP) tagging in conjunction with confocal laser scanning microscopy (CLSM) was used (Anand and Chanway   11 2013a). Strain P2b-2R was tagged with a plasmid-borne GFP (pBSGV104) and designated as P2b-2Rgfp. Using CLSM, it was observed that P2b-2Rgfp had completely engulfed the root surface of lodgepole pine along with colonizing its stem cortical cells. These results indicated that the perceived growth promotion and N-fixation in P2b-2R-inoculated lodgepole pine seedlings was bacteria-driven (Anand et al. 2013; Anand and Chanway 2013a). In a recent study, endophytic bacteria were also isolated from lodgepole pine trees growing on gravel mining sites in central-interior BC (Padda et al. 2018). These strains were characterized for their role in supporting the growth of lodgepole pine trees on such degraded sites and it was found that some of these strains possessed several PGP abilities including N-fixation, phosphate solubilization, siderophore production, IAA production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity and biocontrol activity (Padda et al. 2019, 2020). Apart from lodgepole pine, endophytic bacteria have also been isolated from buds of mature Scots pine (a Pinus species native to Eurasia) trees growing in a natural stand in northern Finland (Pirttilä et al. 2000). One of the isolates DSM 13060, identified as Methylobacterium extorquens, was reported to produce adenine derivatives, which are regarded as precursors in cytokinin biosynthesis (Pirttilä et al. 2004). The strain DSM 13060 was reported to significantly enhance shoot and root biomass of Scots pine seedlings by colonizing the root epidermis as well as root parenchymatic and xylem tissues (Pohjanen et al. 2014). Additionally, this endophytic strain in association with ectomycorrhizal fungi (Suillus variegatus and/or Pisolithus tinctorius) was able to promote the growth of Scots pine seedlings even more than just ECM fungi inoculation alone. In an interesting set of studies, foliar endophytic bacterial communities of mature limber pine trees growing in alpine forests at different sites (Horseshoe Meadows,   12 California, USA and Niwot Ridge, Colorado, USA) were found to be mainly comprised of AAB with a major role in providing N nutrition to pine trees through N-fixation (Carrell and Frank 2014; Moyes et al. 2016; Carrell et al. 2016; Carper et al. 2018). 1.3. Endophytic diazotrophic bacteria Nitrogen is one of the most essential nutrients necessary for the survival of plants (Robertson and Vitousek 2009). Although N is found abundantly in the atmosphere, its availability for plants in the terrestrial ecosystem is often a concern in both agricultural and forest environments (Vitousek and Howarth 1991). Biological N-fixation is a well-known mechanism employed by certain bacteria to convert inert atmospheric N2 to plant-available form NH3 using the nitrogenase enzyme (Postgate 1998). Such N-fixing bacteria are known as ‘diazotrophic bacteria’. The association of diazotrophic bacteria with nodule-forming leguminous and actinorhizal plants has been widely studied for more than a century (Galloway and Cowling 2002). However, the interactions of diazotrophic bacteria with non-nodule forming plants such as grasses and conifers have received limited attention. The presence of diazotrophic bacteria in non-leguminous plants was first detected by Brazilian researchers in the rhizosphere of a perennial grass – sugarcane (Saccharum officinarum) (Döbereiner and Alvahydo 1959; Döbereiner 1961). Despite the isolation of a variety of diazotrophic bacteria from the rhizosphere of sugarcane plants in subsequent studies (Rennie et al. 1982; Magalhaes et al. 1983; Seldin et al. 1984; Baldani et al. 1986), it was concluded that the contribution of rhizospheric N to the sugarcane plants was limited, which led to the hypothesis that bacteria naturally occurring in the internal tissues of plants could be involved in the nitrogen fixation process (Cavalcante and Döbereiner 1988;   13 Boddey et al. 1991; Stephan et al. 1991). Saccharobacter nitrocaptans – renamed initially to Acetobacter diazotrophicus (Gillis et al. 1989) and then to Gluconacetobacter diazotrophicus (Yamada et al. 1997) – was the first diazotrophic bacteria isolated from the internal tissues of sugarcane (Cavalcante and Döbereiner 1988). Such bacteria that can fix N by primarily colonizing interior tissues of the plant were later designated as ‘endophytic diazotrophic bacteria’ (Döbereiner 1992). They have now been detected in a wide array of non-nodulating cereal (Rosenblueth et al. 2018) and oilseed (de Freitas and Germida 1998) crops as well as deciduous and coniferous trees (Oses et al. 2018). 1.3.1. Endophytic diazotrophic bacteria in forest ecosystems Endophytic diazotrophic bacteria have been reported to play a crucial role in supporting fast-growing, pioneering deciduous tree species – poplar and willow – in nutrient-poor environments such as gravel substrate beside riverbanks (Doty et al. 2009, 2016). Several endophytic diazotrophic strains were isolated on N-free media from young poplar and willow trees growing in Three Forks Park alongside the Snoqualmie River in Western Washington state, USA (Doty et al. 2009). Isolates belonging to the genera Acinetobacter, Burkholderia, Herbaspirillum, Pseudomonas, Rahnella and Sphingomonas possessed the nifH gene and showed positive nitrogenase enzyme activity in acetylene reduction assay (ARA). These endophytic diazotrophic strains were evaluated individually as well as collectively as part of a consortium in a greenhouse and a field experiment on poplar clones (Knoth et al. 2014). Inoculated poplar seedlings fixed up to 65% of N directly from the atmosphere in this study and accumulated significantly higher biomass than non-inoculated controls. Notably, the growth promotion was more pronounced   14 with the consortia than with single-strain inoculation, thereby indicating that, in certain cases, the combined action of the endophytic diazotrophic community could be significantly beneficial for the plant. In a subsequent study, Doty et al. (2016) reported the first direct evidence of N-fixation in aboveground poplar cuttings sampled from its native riparian N-limited habitat using a combination of ARA and 15N labelling assay. Poplar cuttings accumulated up to 20.6 mg of fixed N per kg plant tissue per day in an 15N labelling assay primarily due to the presence of endophytic diazotrophic communities. Building on the initial work done with endophytic bacteria from lodgepole pine trees and their potential growth-enhancing benefits to trees in phytotron-, greenhouse- and field-based studies (Shishido and Chanway 1999, 2000; Shishido et al. 1995, 1996a, b, 1999; Chanway et al. 2000; Bent et al. 2001), Chanway and colleagues searched specifically for endophytic diazotrophic bacteria potentially responsible for supporting the growth of lodgepole pine trees on N-limited soils. Although diazotrophic bacteria have been previously isolated from the rhizosphere of lodgepole pine trees growing on N-limited soils, the contribution of N to pine seedlings through rhizospheric N-fixation was reported to be low or negligible (Chanway and Holl 1991), which led them to explore the possibility of endophytic N-fixation (Bal et al. 2012). Of the 99 endophytic strains isolated from the internal root, stem and needle tissues of mature and young lodgepole pine trees, 14 strains demonstrated the potential characteristics of a diazotroph – they grew on N-free combined carbon medium (CCM) agar (Rennie 1981) and showed positive nitrogenase enzyme activity by reducing acetylene to ethylene in the ARA (Bal et al. 2012). However, only four strains consistently reduced significant amounts of acetylene to ethylene in multiple ARAs, two of which were identified as Paenibacillus polymyxa, one was identified as   15 Dyadobacter fermentans and one was identified as Paenibacillus amylolyticus (Bal et al. 2012). Bal and Chanway (2012a) tested these strains in planta to quantify the amount of fixed N provided by these strains to lodgepole pine seedlings using a 15N isotope dilution assay and found that only one of these four strains (Paenibacillus polymyxa P2b-2R) could fix significant amounts of N from the atmosphere (30% of foliar N after 27 weeks and 66% after 35 weeks). In a subsequent 13-month long study, P. polymyxa P2b-2R was observed to fix 79% of N from the atmosphere in lodgepole pine seedlings grown under N-limited conditions, which is regarded as highly significant (Anand et al. 2013). The considerable amount of N fixed by this strain was directly linked to the significant enhancement of length and biomass of pine seedlings observed in this study, which was conducted under severely N-poor conditions. Since N-fixation is an energy-intensive process, it has been theorized that plants rely on associative diazotrophs only when other significant sources of N are not present (Danso et al. 1987; Vargas et al. 2000; Reed et al. 2011; Bal and Chanway 2012a). This theory was also confirmed for diazotrophic strain P2b-2R by Yang et al. (2016), who reported that when pine seedlings were grown in sufficient N conditions, strain P2b-2R did not fix N from the atmosphere and its inoculation had negligible effects on seedling growth, thereby suggesting that low soil N levels are required to trigger N-fixation mechanism of strain P2b-2R. The molecular N-fixation mechanism of strain P2b-2R was reported by Anand and Chanway (2013c), who characterized its nif gene structure and described the full nifH gene sequence as well as partial nifB and nifD gene sequences of strain P2b-2R. In addition to lodgepole pine, P. polymyxa P2b-2R has also been reported to colonize western red cedar seedlings and fix 56% of N from the atmosphere in a 35-week growth trial (Bal and Chanway 2012b), signifying its ability to provide benefits to multiple hosts.   16 In a recent study, 77 potential endophytic diazotrophic strains were isolated from internal tissues of young lodgepole pine trees growing on extremely N-poor gravel mining pits, of which 32 strains showed positive nitrogenase enzyme activity in ARAs (Padda et al. 2018). Of these 14 strains were tested for in planta N-fixation and presence of the nifH gene in a subsequent study (Padda et al. 2019). All strains possessed the nifH gene and fixed significant amounts of N from the atmosphere in lodgepole pine seedlings in a year long greenhouse study. In particular, strains of the genus Pseudomonas performed extremely well in this study by fixing up to 50% of N and enhancing pine seedling length and biomass by up to 64% and 311%, respectively. It was postulated that such highly effective endophytic diazotrophic strains could be responsible for supporting pine tree growth on degraded gravel pits (Padda et al. 2019). The needle endophytic microbiome of limber pine and Engelmann spruce trees growing in a subalpine region with N-limited edaphic conditions was reported to comprise largely of taxa closely related to Gluconacetobacter diazotrophicus and other diazotrophic AAB (Carrell and Frank 2014). Interestingly, similar phyla were also isolated from pine and spruce trees growing in a different geographical region, suggesting a strong selection for a specific diazotrophic community under N-limited conditions (Carrell et al. 2016). Furthermore, the ability of these endophytic diazotrophic communities to fix N was assessed using an ARA on twigs sampled from mature limber pine trees (Moyes et al. 2016). The authors also used an innovative approach to measure N-fixation by exposing the twig samples to a 13N2-enriched atmosphere and then mapping the distribution of radioisotope in the needles. The results of this experiment suggested that foliar endophytes could provide 1–2 mg of N per square meter to limber pine stands in one year (Moyes et al. 2016).   17 Studies summarized in this section suggest that endophytic N-fixation in forests may be more widespread than previously thought. For long-lived Pinaceae trees, endophytic diazotrophic bacteria may represent an evolutionarily stable N-fixing strategy to grow on severely N-limited soil as indicated by studies with lodgepole pine, limber pine and Engelmann spruce. Since our understanding of the ecological importance and temporal-spatial dynamics of endophytic diazotrophic bacteria in conifers is still in its infancy, further studies are needed to fully understand how certain boreal and temperate forest ecosystems accumulate more N than can be accounted for by known nitrogen input pathways (Binkley et al. 2000; Bormann et al. 2002; Chapman and Paul 2012). Such studies could also encourage the use of endophytic diazotrophic bacteria in forestry for future plantation efforts particularly in nutrient-poor and disturbed ecosystems. 1.4. Study area and rationale British Columbia (BC) has some of the most diverse terrestrial ecosystems in North America. Its fourteen different biogeoclimatic zones support a variety of biota both aboveground and belowground. A biogeoclimatic zone is a geographic area that has similar patterns of energy flow, vegetation and soils due to a broadly homogeneous macroclimate (Pojar and Meidinger 1991). The Sub-Boreal Pine-Spruce (SBPS) biogeoclimatic zone is a montane zone in the west central-interior of BC (Figure 1.1a). It occupies the gently rolling landscape of the Fraser Plateau and the southern-most portions of the Nechako Plateau. Elevations in the northern part of the zone are mostly 850-1300m, while in the southern and western parts (near the Coast Mountains) elevations range from 1100m to 1500m (Steen and Demarchi 1991). The climate of the SBPS zone   18 is continental and characterized by cold, dry winters and cool, dry summers due to its position in the rain-shadow of the Coast Mountains and relatively high elevation.  It is regarded as one of the coldest and driest zones in BC based on annual climatic means – mean annual temperature: 0.3–3.2°C and mean annual precipitation: 335–580 mm (of which 30-50% falls as snow) (Steen and Coupé 1997). Based on the environmental conditions, the SBPS has been divided into four subzones (Figure 1.1b). From driest to wettest they are: (i) very dry cold SBPS subzone (SBPSxc), (ii) dry cold SBPS subzone (SBPSdc), (iii) moist cold SBPS subzone (SBPSmc), and (iv) moist cool SBPS subzone (SBPSmk) (Steen and Coupé 1997). The SBPSxc subzone is located in the southern and western parts of the SBPS zone (Figure 1.1b), along the leeward side of the Coast Mountains where the rainshadow effect of the mountains is most pronounced among the four subzones. Considerable water deficits occur in the middle and later parts of the growing season from June to September. Daily temperature extremes range from +40°C in the summertime to -30°C in the wintertime, with an annual mean of 1.9°C (Paul 2002). Soils in this subzone developed primarily on morainal deposits derived from granitic and metasedimentary rocks which are responsible for its coarse texture – predominantly sandy loam and often gravelly (Steen and Coupé 1997). Due to the cold, very dry climate, soils are typically thin and weakly developed and mainly belong to the Brunisolic soil order according to the Canadian System of Soil Classification (Cambisols in the World Reference Base for Soil Resources and Inceptisols in the US Soil Taxonomy). Frequent wildfires in the past have reduced the levels of organic matter in the soil and probably hindered their productivity for tree growth. The surface organic layer is typically very thin (<3cm) and has very slow rates of decomposition. To summarize, factors such as cold and dry climatic conditions, poorly developed, coarse and   19 parched soils, and thin surface organic layer have resulted in limited plant-available nutrients, particularly N. Due to these reasons, the SBPSxc subzone has been regarded as the least productive biogeoclimatic zone in BC where conifers have been observed to grow (Steen and Demarchi 1991; Steen and Coupé 1997; Paul 2002). The SBPSxc landscape is dominated by lodgepole pine as it is highly adaptable to diverse environmental conditions. In fact, it is the only tree species thriving in several forest stands at high and very dry parts of the subzone. Hybrid white x Engelmann spruce (Picea glauca x engelmannii) is the only other tree species common in this subzone, growing on relatively wetter and lower elevation sites (Steen and Demarchi 1991; Steen and Coupé 1997).  Despite such extreme climatic and edaphic conditions, the sustained growth of lodgepole pine and hybrid white spruce trees in this subzone is a conundrum. Since N is a major growth-limiting nutrient, the possible imbalance between the soil N and tissue N raises crucial questions regarding the N-input pathways for trees growing in this area (Paul 2002; Bal et al. 2012). Wet and dry deposition of N from the atmosphere is unlikely in this region due to lack of precipitation and absence of industries/agricultural farms nearby (Steen and Demarchi 1991; Steen and Coupé 1997). Therefore, biological N-fixation may potentially be an important pathway responsible for providing N nutrition to trees in this area. The lack of symbiotic N-fixing alders in this region and potentially unfavourable conditions (dry and nutrient-poor) for the survival of free-living diazotrophic bacteria in soils (Paul 2002; Chapman and Paul 2012) led me to hypothesize that endophytic diazotrophic bacteria may be a significant and novel source of biological N-fixation in this region. The survival and growth of some coniferous species on N-limited soils have been linked to endophytic diazotrophic bacteria in previous studies (summarized in section 1.3.1),   20 which further supports my postulation. Although endophytic diazotrophic bacteria have been studied in lodgepole pine trees growing in the SBPSdc subzone (Bal et al. 2012; Padda et al. 2018), there are no studies to date concerning these bacteria in the SBPSxc subzone. In addition, to the best of my knowledge, endophytic diazotrophic bacteria have never been studied in hybrid white spruce trees in BC, which represents a unique opportunity to expand our knowledge. 1.5. Thesis objectives and hypotheses This study was conducted in the West Chilcotin region of the SBPSxc subzone in BC, Canada with the overarching objective of isolating potential endophytic diazotrophic bacteria from lodgepole pine and hybrid white spruce trees and characterizing their roles in sustaining tree growth. The West Chilcotin region is located in the Cariboo forest region, around 250 km west of Williams Lake, BC (Figure 1.1b) and consists mainly of natural hybrid white spruce and lodgepole pine forest stands. A sampling area was selected (52°00ʹ N, 125°00ʹ W) to collect soil and plant samples from a predominantly hybrid white spruce stand and a predominantly lodgepole pine stand. The following specific objectives and hypotheses were addressed in chapters 2, 3, 4, 5 and 6: Chapter 2: Isolation of endophytic diazotrophic bacteria from spruce and pine Objectives: 1. To evaluate the nutrient contents and various physico-chemical properties of soils in which lodgepole pine and hybrid white spruce trees are growing in the West Chilcotin region of BC   21 2. To isolate and identify potential endophytic diazotrophic bacteria from internal root, stem and needle tissues of pine and spruce trees  3. To evaluate the isolated bacteria for in vitro N-fixing ability (nitrogenase enzyme activity) Hypotheses: 1. Soils in the West Chilcotin region have poor physical and chemical characteristics for plant growth and contain limited quantities of nitrogen and other nutrients to support tree growth 2. Topographical differences directly affect the nutrient contents and physical and chemical properties of soils in this region 3. Lodgepole pine and hybrid white spruce trees growing on these soils harbour endophytic diazotrophic bacteria with a potent ability to fix atmospheric N Chapter 3: Evaluation of spruce bacteria for N-fixation Objectives: 1. To investigate the presence of nifH genes in selected endophytic diazotrophic bacteria isolated from spruce trees 2. To evaluate the ability of these bacteria to colonize their original host (hybrid white spruce) and a foreign host (lodgepole pine) originating from the West Chilcotin region 3. To quantify the amount of N fixed by these bacteria in spruce and pine seedlings in a year long greenhouse study conducted in an extremely N-poor plant growth medium 4. To evaluate spruce and pine seedling growth (length and biomass) enhanced by these bacteria in the greenhouse study   22 Hypotheses: 1. Endophytic diazotrophic bacteria isolated from spruce trees of the West Chilcotin region are capable of gaining re-entry and colonizing internal tissue of their original host 2. These bacteria have the potential to fix significant quantities of N from the atmosphere in association with spruce seedlings along with considerably enhancing their growth 3. These bacteria have the potential to colonize the internal tissues and provide similar benefits of N-fixation and plant-growth-promotion to a foreign host (lodgepole pine) Chapter 4: Multifarious PGP abilities of spruce bacteria Objectives: 1. To investigate the potential of endophytic diazotrophic bacteria isolated from spruce trees to exhibit PGP traits including phosphate solubilization, phytate hydrolyzation, siderophore production, IAA production, ACC deaminase activity and lytic enzyme activity 2. To determine the ability of these spruce bacteria to colonize and enhance the growth of both spruce and pine seedlings under nutrient-limited edaphic conditions in an 18-month greenhouse growth trial Hypotheses: 1. Endophytic diazotrophic bacteria from spruce enhance the growth and health of their plant host by employing a wide array of mechanisms other than N fixation including enhanced phosphorus and iron acquisition, phytohormone modulation and biocontrol of phytopathogens   23 2. These bacteria promote the growth of their original host (hybrid white spruce) and a foreign host (lodgepole pine) 18 months after inoculation under nutrient-limited conditions by potentially employing diverse PGP mechanisms Chapter 5: Evaluation of pine bacteria for N-fixation Objectives: 1. To investigate the presence of nifH genes in selected endophytic diazotrophic bacteria isolated from pine trees 2. To evaluate the ability of these bacteria to colonize their original host (lodgepole pine) and a foreign host (hybrid white spruce) originating from the West Chilcotin region 3. To quantify the amount of N fixed by these bacteria in pine and spruce seedlings in a year long greenhouse study conducted in an extremely N-poor plant growth medium 4. To evaluate pine and spruce seedling growth (length and biomass) enhanced by these bacteria in the greenhouse study Hypotheses: 1. Endophytic diazotrophic bacteria isolated from pine trees of the West Chilcotin region are capable of gaining re-entry and colonizing internal tissue of their original host 2. These bacteria have the potential to fix significant quantities of N from the atmosphere in association with pine seedlings along with considerably enhancing their growth 3. These bacteria have the potential to colonize the internal tissues and provide similar benefits of N-fixation and plant-growth-promotion to a foreign host (hybrid white spruce)   24 Chapter 6: Multifarious PGP abilities of pine bacteria Objectives: 1. To investigate the potential of endophytic diazotrophic bacteria isolated from pine trees to exhibit PGP traits including phosphate solubilization, phytate hydrolyzation, siderophore production, IAA production, ACC deaminase activity and lytic enzyme activity 2. To determine the ability of these pine bacteria to colonize and enhance the growth of both pine and spruce seedlings under nutrient-limited edaphic conditions in an 18-month greenhouse growth trial Hypotheses: 1. Endophytic diazotrophic bacteria from pine enhance the growth and health of their plant host by employing a wide array of mechanisms other than N fixation including enhanced phosphorus and iron acquisition, phytohormone modulation and biocontrol of phytopathogens 2. These bacteria promote the growth of their original host (lodgepole pine) and a foreign host (hybrid white spruce) 18 months after inoculation under nutrient-limited conditions by potentially employing diverse PGP mechanisms   25 Table 1.1 List of endophytic bacteria isolated from important forest tree species and their beneficial effects on host trees.  Plant species Endophytic bacteria Beneficial effects References Picea Engelmann spruce (P. engelmannii) Uncultured bacteria Nitrogen fixation Carrell and Frank 2014 Norway spruce (P. abies) Pseudomonas spp.; Rahnella spp. - Cankar et al. 2005 Hybrid spruce (P. glauca x engelmannii) Bacillus polymyxa strain Pw-2R; Pseudomonas fluorescens Sm3-RN and Ss2-RN Enhance seedling biomass and length and produce IAA and cytokinins Bent et al. 2001; Chanway et al. 2000; Shishido et al. 1996a, b, 1999; Shishido and Chanway 1999, 2000 Sitka spruce (P. sitchensis) Paraburkholderia sp. PSSN1 - Aghai et al. 2019 Pinus Limber pine (P. flexilis) Uncultured bacteria Nitrogen fixation and antifungal activity Moyes et al. 2016; Carrell et al. 2016; Carper et al. 2018 Lodgepole pine (P. contorta var. latifolia)  Paenibacillus polymyxa Pw-2R; Pseudomonas fluorescens strains Sm3-RN and Ss2-RN; Paenibacillus polymyxa P2b-2R; Pseudomonas graminis AN1r; Pseudomonas migulae AR1r; Pseudomonas lini SN1r Enhance seedling biomass and length, nitrogen fixation, produce siderophores, IAA and cytokinins, solubilize phosphate Bent et al. 2001; Shishido et al. 1995, 1996a; Bal and Chanway 2012a; Bal et al. 2012; Anand et al. 2013; Tang et al. 2016; Yang et al. 2016; Padda et al. 2018, 2019, 2020 Scots pine (P. sylvestris) Methylobacterium extorquens DSM13060 Enhances root and shoot dry weight Pirttilä et al. 2000; Pohjanen et al. 2014 Pseudotsuga Douglas-fir (Pseudotsuga menziesii) Burkholderia vietnamiensis WPB; Rhodotorula graminis WP1; Rahnella sp. WP5, PMPF3 and PMSK6; Burkholderia sp. WP9 and TPSN7; Acinetobacter calcoaceticus WP19; Rhizobium tropici PTD1; Enterobacter sp. PDN3; Sphingomonas yanoikuyae WW5; Pseudomonas putida WW6; Sphingomonas sp. WW7; Paraburkholderia sp. TPSK3 and PSSN1; Herbaspirillum sp. TPSK5  Enhance biomass, root length and shoot height, solubilize phosphate, produce siderophore Khan et al. 2015; Aghai et al. 2019   26  Plant species Endophytic bacteria Beneficial effects References Populus Poplar (Populus trichocarpa, Populus trichocarpa x deltoides, Populus deltoides x nigra) Burkholderia vietnamiensis WPB; Rhizobium tropici PTD1; Rahnella sp. WP5; Enterobacter sp. WP7; Pseudomonas graminis WP8; Acinetobacter calcoaceticus WP19; Burkholderia sp. WP4-2-2, WP 4-3-2, WP40, WP41 and WP42; Curtobacterium sp. WW7, WP 4-3-3 and WP 4-10-4; Rahnella aquatilis WP 4-4-2 and WP 4-5-3; Pseudomonas sp. WP 4-4-6; Enterobacter sp. strain 638; Pseudomonas putida W619 and WW6; Pseudomonas sp. PopHV4, PopHV6 and PopHV9; Herbaspirillum sp. WW2; Sphingomonas yanoikuyae WW5 and WW1 Enhance seedling biomass, nitrogen fixation, produce IAA and siderophore, solubilize phosphate, suppress phytopathogens, synthesizes plant growth promoting compound acetoin, and remediates contaminated soils Germaine et al. 2004; Doty et al. 2005, 2009; Taghavi et al. 2005, 2009; Weyens et al. 2009, 2010, 2012; Knoth et al. 2014; Xin et al. 2009; Khan et al. 2016; Kandel et al. 2017a; Quercus Live oaks (Q. fusiformis) Pseudomonas denitrificans 1-15; Pseudomonas putida 5-48 Suppress phytopathogens Brooks et al. 1994 Salix Willow (S. sitchensis) Acinetobacter sp. WW1; Herbaspirillum sp. WW2; Stenotrophomonas sp. WW4; Sphingomonas yanoikuyae WW5 and WW11; Pseudomonas putida WW6; Sphingomonas sp. WW7 and WW12; Pseudomonas sp. WW8 and WW13 Enhance seedling biomass, nitrogen fixation, produce IAA and siderophore, solubilize phosphate, suppress phytopathogens Doty et al. 2009 Thuja western red cedar (T. plicata) Paenibacillus polymyxa P2b-2R; Burkholderia vietnamiensis WPB; Rhizobium tropici PTD1; Rahnella sp. WP5, PMPF3 and PMSK6; Enterobacter sp. WP7; Paraburkholderia sp. WP9, TPSK3 and PSSN1; Acinetobacter calcoaceticus WP19; Enterobacter sp. PDN3; Sphingomonas yanoikuyae WW5; Pseudomonas putida WW6; Sphingomonas sp. WW7; Burkholderia sp. TPSN7; Herbaspirillum sp. TPSK5 Enhance seedling biomass and length, improve drought resistance and Nitrogen fixation Bal and Chanway 2012b; Bal et al. 2012; Anand and Chanway 2013b; Aghai et al. 2019   27  Source: BC Ministry of Forests – Research Branch (1998)  Source: Centre for Forest Conservation Genetics, Faculty of Forestry, UBC Vancouver Figure 1.1 (a) Location of the Sub-Boreal Pine-Spruce biogeoclimatic zone represented on the map of British Columbia; (b) four subzones of the Sub-Boreal Pine-Spruce zone represented by different colours and codes, with the sampling area indicated by star shape in the xc subzone Sub-Boreal Pine-Spruce biogeoclimatic zoneBritish Columbia(a)Sub-Boreal Pine-Spruce subzones Williams Lake(b)West Chilcotin  28 Chapter 2 – Evidence of endophytic diazotrophic bacteria in lodgepole pine and hybrid white spruce trees growing in soils with different nutrient statuses in the West Chilcotin region of British Columbia 2.1. Introduction The importance of microbes for plant health and growth promotion has been known for a long time but internal tissue colonization was largely perceived as being related to the spread of disease. However, now it is widely accepted that microbes can colonize internal tissues of plants and establish beneficial symbiotic associations with the host plant (Puri et al. 2017b). Such microbes are known as ‘endophytes’. Although there is considerable literature available regarding endophytic fungi in the forest ecosystem (Doty 2011), studies of endophytic bacteria of forest tree species are rather limited. However, these limited studies have indicated that the importance of endophytic bacteria should not be underestimated in the forest ecosystem (Puri et al. 2017a). Principal mechanisms through which these endophytic bacteria can enhance the growth of trees include N-fixation (Padda et al. 2017a), production of phytohormones such as cytokinins (Pirttilä 2011), auxins (Taghavi et al. 2005) and gibberellins (Bottini et al. 2004), and suppression of pathogens, in addition to improving the mutualistic relationship of mycorrhizae with roots of the host tree (Anand et al. 2006). Endophytic bacteria that possess the ability to fix N while living inside the tissues of a plant are known as ‘endophytic diazotrophic bacteria’ (Döbereiner 1992). Although such bacteria have been widely studied in agricultural crops such as canola (Brassica napus) (Puri et al. 2016a), corn (Zea mays) (Puri et al. 2015), rice (Oryza sativa)   29 (Baldani et al. 2000), sugarcane (Saccharum officinarum) (Boddey et al. 1991), and tomato (Solanum lycopersicum) (Padda et al. 2016a), very few studies have reported their presence in forest trees including poplar (Populus trichocarpa) (Doty et al. 2009), willow (Salix sitchensis) (Doty et al. 2009), lodgepole pine (Bal et al. 2012), Engelmann spruce (Picea engelmannii) (Carrell and Frank 2014) and limber pine (Pinus flexilis) (Moyes et al. 2016). However, their existence and function in tree species require further evaluation.  Due to its geological history, the province of British Columbia (BC) is uniquely positioned to contain assorted terrestrial ecosystems. Based on the biological, geological and environmental characteristics, BC is divided into 14 different biogeoclimatic zones. The SBPS biogeoclimatic zone is a montane zone in the west-central interior of BC characterized by cold, dry winters and cool, dry summers due to its location in the strong rain-shadow of the Coast Mountains (Steen and Demarchi, 1991). It is divided into four subzones. The driest and coldest one is the SBPSxc subzone. West Chilcotin is a remote region in this subzone, located about 200 km west of Williams Lake. Soils in most parts of BC are young and started developing only 10,000 years ago after the last glacial period. This coupled with the harsh climate (cold and dry with very low annual precipitation) of the West Chilcotin region has led to weakly developed soils and typically, very thin or even no surface organic layer with very slow rates of decomposition (Steen and Coupé 1997). All these factors have severely limited the productivity of forests in this region. Lodgepole pine is the only tree species present in many extensive forest stands in this region, with hybrid white spruce occurring mostly on relatively moist sites (Steen and Coupé 1997). Most of the stands in the SBPSxc subzone are naturally occurring secondary forests that are >60 years old including one-third of the stands that are >120 years old (Coupé 2012). Considering the   30 sustained growth of pine and spruce trees on such nutrient-poor soils, N inputs in forests of this region have been a long-standing conundrum (Steen and Demarchi 1991; Steen and Coupé 1997). Bal et al. (2012) isolated endophytic diazotrophic bacteria from lodgepole pine trees in the SBPS zone and reported that they could be involved in fulfilling a significant portion of N requirements of pine trees. Subsequently, one of the isolates, Paenibacillus polymyxa P2b-2R, was reported to fulfil as much as 79% of N requirements of young pine seedlings (Anand et al. 2013). However, to the best of our knowledge, there are no reports in the literature regarding the presence of endophytic diazotrophic bacteria in hybrid white spruce trees.  In this study, the first objective was to obtain a comprehensive estimate of the physico-chemical properties and nutrient content of soils on which lodgepole pine and hybrid white spruce trees are growing in the West Chilcotin region. The soils were sampled from two distinct sites (a low elevation site and a high elevation site) underneath each tree species to capture a range of topographical and edaphic variability. The second objective was to isolate and identify potential endophytic diazotrophic bacteria from pine and spruce trees growing at these high-elevation and low-elevation sites. The third objective was to evaluate the N-fixing ability (nitrogenase activity) of isolated bacteria using acetylene reduction assay (ARA). 2.2. Materials and methods 2.2.1. Site description A sampling area (52°00ʹ N, 125°00ʹ W) was selected in the West Chilcotin region with a granitic type of parent material, which is generally regarded as a coarse-textured parent material with   31 very low nutrient content. This area was located about 250 km west of Williams Lake, BC, Canada on the Chilcotin-Bella Coola highway (Figure 2.1a). In this area, two sites were selected to sample lodgepole pine trees in a primarily pine stand, one at a lower elevation (52° 00ʹ 04.2ʺ N, 124° 59ʹ 44.7ʺ W, 1003 m a.s.l.) and the other at a higher elevation (52° 00ʹ 09.1ʺ N, 124° 59ʹ 25.2ʺ W, 1035 m a.s.l.) (Figure 2.1b). Similarly, two sites were selected to sample hybrid white spruce trees in a primarily spruce stand at a lower elevation (52° 00ʹ 23.8ʺ N, 125° 00ʹ 17.4ʺ W, 966 m a.s.l.) and a higher elevation (52° 00ʹ 19.5ʺ N, 125° 00ʹ 17.8ʺ W, 993 m a.s.l.) (Figure 2.1b). 2.2.2. Soil and plant sampling Soil samples were collected from the aforementioned four sites by using the following sampling design to cover spatial variability. At each site, a mature tree was chosen, and soil samples were collected in the four cardinal directions both inside and outside its dripline. Samples were collected from the forest floor and the top mineral layer (0–10 cm). Samples collected in all four cardinal directions from both inside and outside the dripline were pooled to obtain one forest floor sample and one mineral layer sample near each tree. In this way, soil samples were collected near 10 trees at each of the four sites. Subsequently, a young tree (height:<25 cm and age:<5 years) growing near each mature tree (around which soil samples were collected) was uprooted and collected, which resulted in 10 spruce tree samples from each of the two sites at spruce stand and 10 pine tree samples from each of the two sites at pine stand.     32 2.2.3. Soil analyses To determine the overall nutrient status of the low-elevation site and the high-elevation site at each stand, soil samples were analyzed for total C; total N; Available N (NH4+ and NO3−); Mineralizable N; Available P; total S; Available S; pH in H2O; cation exchange capacity (CEC); base saturation; organic matter (OM); percent sand, silt and clay; and macro- and micro-nutrients (Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S, and Zn). These analyses were performed at the Analytical Chemistry Services Laboratory, BC Ministry of Environment and Climate Change Strategy, Victoria, BC, Canada. 2.2.4. Isolation of potential endophytic diazotrophic bacteria Potential endophytic diazotrophic bacteria were isolated from stem, needle and root tissues of each seedling by using a surface sterilization-trituration-plating technique (Bal et al. 2012). Briefly, about 0.5 g (fresh mass) of each tissue was surface sterilized by immersion in 2.5% (w/v) sodium hypochlorite for 2 min, followed by three 30-s rinses in 10 mmol/L phosphate buffered saline (PBS) (pH 7) (Appendix A). To check for surface contamination, tissue samples were imprinted on tryptic soy agar (TSA) and incubated for 24 h. Tissues found to be free of surface contamination were triturated in 1 mL PBS using a sterile mortar and pestle. Triturated tissue suspensions were serially diluted, and 0.1 mL of each dilution was plated on an N-free combined carbon medium (CCM) (Rennie 1981; Appendix B) supplemented with 100 mg/L cycloheximide to suppress fungal growth. Following incubation for 3 days at 30°C, representative bacterial colonies were selected from dilution plates based on colony size, shape, morphology, and colour and were purified by streaking onto fresh CCM plates amended with cycloheximide (100 mg/L).   33 Purified isolates were grown in CCM broth amended with cycloheximide (100 mg/L) until turbid and stored frozen at −80°C in cryovials containing 2 mL CCM amended with 20% (v/v) glycerol. 2.2.5. Evaluation of nitrogenase activity Isolates were tested for nitrogenase enzyme activity using the acetylene reduction assay (ARA) described by Holl et al. (1988) with some modifications. In this assay, each isolate was inoculated into a 22 mL crimp top vial (fitted with a pressure release aluminum seal containing PTFE/Silicone Septa) containing 9 mL of CCM broth. Vials were placed on a rotary shaker at 30°C to allow bacterial growth until broths appeared turbid. This was followed by the injection of acetylene gas (Praxair Canada Inc., Mississauga, ON, Canada) into the vials to a final concentration of 10% (v/v). After two days, 1 mL gas sample from each vial was collected to analyze the ethylene content by using flame ionization gas chromatography at the Analytical Chemistry Services Laboratory, BC Ministry of Environment and Climate Change Strategy, Victoria, BC, Canada. Ethylene production in each vial was quantified, using a gas chromatograph (Perkin Elmer Clarus 580, Shelton, CT, USA) equipped with a flame ionization detector and a capillary column (Rt-Alumina BOND/Na2SO4, 30-m long, 0.53-mm internal diameter, with a 0.01-mm-thick film for affinity separation of elements, the flow rate of 80 mL/min). The inlet temperature was 200°C with an inlet pressure of 6.1 psi. The injector and detector temperatures were 200°C. The column temperature was 45°C at the time of injection, held for 1 min and then increased to 120°C at the rate of 10°C/min and after that increased to 200°C at the rate of 45°C/min and held for 1.5 min. The obtained chromatograms were used to integrate the area under the curve of ethylene to estimate ethylene production. Three replicate vials were used for each strain inoculated in CCM   34 and injected with acetylene (b+CCM+acetylene) to determine the mean amount of ethylene produced. Corresponding to each strain, three types of controls were also used namely, bacteria-inoculated CCM broth without acetylene (b+CCM; n=3), uninoculated CCM broth without acetylene (CCM; n=3), and uninoculated CCM broth with 10% acetylene (CCM+acetylene; n=3). To calculate the actual amount of acetylene converted to ethylene by each strain, the following formula was used: Acetylene converted to ethylene = (b+CCM+acetylene) – (b+CCM) – (CCM) – (CCM+acetylene) The whole experiment was duplicated to check for consistency of the acetylene reduction activity of isolates and to avoid any one-time experimental artifacts. 2.2.6. Identification of endophytic diazotrophic bacteria Isolates showing positive acetylene reduction activity were designated as endophytic diazotrophic bacteria. The 16S rRNA gene identification technique described by Paul et al. (2013), was used to identify these strains. Each frozen strain was thawed and streaked onto CCM plates and then grown in CCM broth on a rotary shaker for 2 days at 30°C. Bacterial cells were harvested by centrifugation and the genomic DNA of each strain was extracted by using the DNeasy UltraClean Microbial Kit (Qiagen Inc., Valencia, CA, USA) following the steps outlined by the manufacturer. The concentration of the isolated DNA was evaluated by using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the quality of the DNA was confirmed by electrophoresis on a 0.8% agarose gel. The 16S rRNA gene from each isolate was amplified using the primers 16S rRNA For [5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ] and 16S rRNA Rev   35 [5ʹ-ACGGCTACCTTGTTACGACTT-3ʹ] (Integrated DNA Technologies Inc., Coralville, IA, USA) corresponding to positions 8–27 and 1492–1512, respectively, on the Escherichia coli rrs sequence. This resulted in a PCR product of approximately 1500 base pairs (bp). PCR amplification was performed in 25 μL reaction volumes containing 2.5 μL of 10x PCR buffer (without MgCl2), 2.5 μL of MgCl2 (50 mM), 0.5 μL of Taq DNA Polymerase (5 U/μl), 1.5 μL of dNTP mix (consisting of four nucleotides: dATP, dCTP, dGTP, dTTP), 0.5 μL of each primer, and 3 μL of template DNA. The final volume was adjusted to 25 μL with nuclease-free water. Amplifications were performed on MJ Mini gradient thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) using the following program: initial cell lysis and denaturation for 3 min at 95°C; followed by 30 cycles of denaturation (30 s at 94°C), annealing (1 min at 55°C), and extension (2 min at 72°C); and a final extension for 10 min at 72°C. Quantity and quality of PCR products were evaluated on a 1% agarose gel. The PCR products were purified using the QIAquick PCR purification kit (Qiagen Inc., Valencia, California, USA) using the protocol provided by the manufacturer. Purified PCR products were sent for sequencing to the Sequencing + Bioinformatics Consortium Facility at the University of British Columbia, Vancouver, BC, Canada. Obtained sequences were compared to known sequences in the ‘16S ribosomal RNA sequences (Bacteria and Archaea)’ database of the BLAST search tool (NCBI, Bethesda, MD, USA). 2.2.7. Statistical analyses The nutrient status and soil properties of the high-elevation site and the low-elevation site at each stand were statistically compared using the statistical package SAS University Edition (SAS Institute Inc., Cary, NC, USA) and performing t-test to separate the means.   36 2.3. Results 2.3.1. Soil analyses Soils in the West Chilcotin were observed to be generally dry, coarse-textured and poorly developed with limited organic forest floor on top and evidence of strong eluviation (Figure 2.2). In the spruce stand, it was observed that the total N (%) of both forest floor and top mineral layer samples were significantly lower at the high-elevation site than the low-elevation site, resulting in significantly higher C:N ratio of soils at the high-elevation site (Table 2.1 and Figure 2.3a). Similar observations occurred for the pine stand where the C:N ratios were relatively higher at the high-elevation site (Table 2.2 and Figure 2.4a). The amount of available NO3− in forest floor and mineral layer was 41% and 241% lower at the high-elevation site than the low-elevation site, respectively in the spruce stand (Figure 2.3c), and 287% and 240% lower at the high-elevation site, respectively in the pine stand (Figure 2.4c). Similarly, available NH4+ levels in the forest floor and mineral layer samples were 95% and 58% lower at the high-elevation site, respectively in the spruce stand (Figure 2.3d), and 639% and 24% lower at the high-elevation site, respectively in the pine stand (Figure 2.4d). The amount of mineralizable N in the forest floor and the mineral layer was 47% and 84% lower at the high-elevation site than the low-elevation site, respectively in the spruce stand (Figure 2.3b). Likewise, the high-elevation site in the pine stand had 86% and 49% lower mineralizable N in the forest floor and mineral layer, respectively (Figure 2.4b). In addition, concentrations of several macro- and micro-nutrients were significantly lower in soils at the high-elevation site than the low-elevation site in both pine and spruce stands (Tables 2.1 and 2.2). It was also observed that the CEC and % OM of forest floor and mineral layer samples   37 from the high-elevation site were significantly lower than the low-elevation site at both stands. It was also noted that the thickness of the forest floor at high-elevation sites in each stand was significantly lower than the low-elevation sites (Tables 2.1 and 2.2). 2.3.2. Bacterial isolation and identification and nitrogenase activity evaluation In total, 55 potential endophytic diazotrophic bacteria were isolated from the root, stem and needle tissues of spruce trees on N-free CCM agar plates, 27 from the high-elevation site (stem: 12, needle: 3, root: 12) and 28 from the low-elevation site (stem: 9, needle: 11, root: 8). Likewise, 48 potential endophytic diazotrophic bacteria were isolated from tissues of pine trees, 20 from the high-elevation site (stem: 3, needle: 4, root: 13) and 28 from the low-elevation site (stem: 8, needle: 7, root: 13). These isolates were evaluated for their ability to fix N by examining their nitrogenase activity using ARA and it was found that 18 out of the 55 bacteria isolated from spruce trees possessed nitrogenase activity, 10 from the high-elevation site and 8 from the low-elevation site (Table 2.3). Similarly, 23 out of the 48 bacteria isolated from the pine trees displayed nitrogenase activity, 13 from the high-elevation site and 10 from the low-elevation site (Table 2.4). The amount of acetylene reduced to ethylene by bacteria isolated from either tree species ranged between 0.1 and 2.9 nmol/mL (Tables 2.3 and 2.4). Subsequently, all isolates showing positive nitrogenase activity in the ARA were identified using 16S rRNA gene sequencing technique and the obtained sequences were deposited under accession numbers from MG561767 to MG561784 (spruce isolates) (Table 2.3) and from MG561788 to MG561807 (pine isolates) (Table 2.4) to the GenBank database. Isolates were identified based on the closest match (>98% sequence identity) in the BLAST search. Bacteria isolated from either spruce or pine trees   38 growing at any of the four sites belonged to the following phyla: Firmicutes, Proteobacteria and Actinobacteria. 2.4. Discussion In this study, soil samples collected from different sites in the West Chilcotin region of BC were analyzed and it was observed that, overall, soils in this region have poor physico-chemical health and low nutrient content (Figure 2.2). Comparing these results to other studies conducted in nearby regions, it was observed that soils in this region have relatively lower total and plant-available nutrients, % OM, and CEC (Driscoll et al. 1999; Sanborn et al. 2005; Kranabetter et al. 2006; Hope 2007). This could be explained by the cold and dry climate of the West Chilcotin region, which has led to poorly developed soils (Figure 2.2), generally lacking essential plant nutrients (Steen and Coupé 1997). In addition, it should also be noted that soils in our sampling area have been formed from granitic rocks that are coarse-textured (Figure 2.2), which is evident from the presence of high percent sand (loamy sand/sandy loam texture) in our soil samples (Tables 2.1 and 2.2). The results of the physico-chemical properties and nutrient content of soils collected from the high-elevation site and the low-elevation site in each forest stand were also compared. The forest floor and top mineral layer at the high-elevation sites in both spruce and pine stands had significantly lower available and mineralizable N, and a significantly higher C:N ratio as compared to the low-elevation sites (Figures 2.3 and 2.4). This is consistent with a previous report suggesting that elevation or topography of a site is inversely related to the soil moisture content which in turn directly influences the concentrations of available NO3− and NH4+ in the soil (Zhang and Wienhold 2002). In other words, a high-elevation site is expected to have   39 lower soil moisture content and lower concentrations of plant-available nutrients. In addition to having a statistically significant difference in soil N levels, soils at high-elevation sites in both stands had significantly lower levels of nutrients such as Ca, K, and Mg. Similarly, CEC of soils at high-elevation sites was also significantly lower which indicates that fewer nutrient cations are available on exchange sites of the soil for plants. The depth of the forest floor was also considerably thinner at high-elevation sites which in turn reflects the lower % OM observed at these sites as compared to low-elevation sites (Tables 2.1 and 2.2). These results are consistent with previous studies where site elevation was reported to be inversely related to the availability of water and nutrients for plants in soils (Blouin et al. 2008; Misra and Tyler 1999; Metwally and Pollard 1959; BC Ministry of Forests and Range & BC Ministry of Environment 2010). Overall, our results confirm that high-elevation sites at both pine and spruce stands were relatively nutrient-poor (particularly, N-poor) as compared to the low-elevation sites in the West Chilcotin region.  Several potential endophytic diazotrophic bacteria were isolated from pine and spruce trees growing at different sites in the West Chilcotin region. The number of bacteria isolated on N-free media is consistent with a previous study conducted on lodgepole pine in the SBPS zone (Bal et al. 2012). Most of our strains originated from aerial parts (stem and needle) of the trees with the only exception being pine trees at the high-elevation site. Isolation of strains from aerial parts suggests that they could have originated from the seed or are capable of systemic colonization of trees from the soil as observed in the case of strains of Paenibacillus polymyxa (Shishido et al. 1995; Bal et al. 2012). However, further work needs to be done to determine if the isolated endophytic diazotrophic bacteria colonize tree tissues via soil, seed or some other way (Frank et al. 2017). Using the well-established ARA, it was observed that 18 and 23 strains   40 isolated from spruce and pine trees respectively, successfully converted acetylene to ethylene (Tables 2.3 and 2.4). The values of ethylene produced by these strains were similar to those observed for isolates of lodgepole pine (Bal et al. 2012; Paul et al. 2013), limber pine (Moyes et al. 2016), poplar and Sitka willow (Doty et al. 2009), and western red cedar (Thuja Plicata) (Bal et al. 2012). It is important to note that the acetylene reduction activity of strains originating from low-elevation sites was similar to those originating from high-elevation sites for either tree species. A possible explanation could be that, due to the overall nitrogen-poor edaphic conditions that persist in this region, spruce and pine trees growing on both high-elevation and low-elevation sites may be dependent on endophytic diazotrophic bacteria to fulfill their N-requirements. Another important point to note is that most of the acetylene reducing strains originated from aerial parts of the plant especially in the case of spruce trees (Table 2.3). This could be related to the fact that plants perform photosynthesis in the aerial parts and most nutrients are also assimilated in these parts. Thus, these parts might be providing endophytic diazotrophic bacteria with better access to nutrients and energy to perform biological nitrogen fixation.  The endophytic diazotrophic bacteria that showed positive nitrogenase activity in the ARA represented several bacterial species, many of which were common to both tree species. Bacteria of genera Bacillus, Paenibacillus and Pseudomonas were commonly found at both sites in the spruce stand (Table 2.3). Similarly, Caballeronia, Paenibacillus and Pseudomonas genera were common at both sites in the pine stand (Table 2.4). However, differences in the genera of bacteria isolated from high-elevation site vs low-elevation site in both pine and spruce stands were also found (Tables 2.3 and 2.4). In the spruce stand, Frondihabitans, Herbiconiux,   41 Pedobacter and Pigmentiphaga species were only isolated from the low-elevation site and Caballeronia, Rhizobium, Sporolactobacillus and Subtercola were only isolated from high-elevation site. In a similar way, Frondihabitans, Methylobacterium and Phyllobacterium species were only isolated from pine trees at the low-elevation site and Bacillus, Sphingomonas, Paraburkholderia, Rhizobium, Caulobacter species were only isolated from pine trees at the high-elevation site. The plant-beneficial-environmental group of the Burkholderia genus was recently split to form two new genera, Caballeronia and Paraburkholderia (Dobritsa and Samadpour 2016; Sawana et al. 2014). In fact, Burkholderia is well-known to be a genus rich in plant-associated N-fixing species (Estradade los Santos et al. 2015). One of these endophytic strains, Paraburkholderia phytofirmans PsJN, first discovered by Frommel et al. (1991), has been studied and reported by several authors to endophytically colonize a wide range of plant hosts and promote their growth by one or more plant-growth-promoting (PGP) mechanisms (reviewed by Puri et al. 2017b). Strains of the genus Burkholderia have also been reported for their N-fixing and PGP ability in tree species including poplar, Sitka willow, and lodgepole pine (Xin et al. 2009; Bal et al. 2012; Knoth et al. 2014). Strains of Bacillus and Paenibacillus genera are also well-known for their ability to fix atmospheric N and improve plant growth through various mechanisms (reviewed by Padda et al. 2017b). Endophytic diazotrophic strains of P. polymyxa have been reported to fix considerable amounts of N from the atmosphere both in vitro (Bal et al. 2012) and in planta in tree species such as lodgepole pine (Anand et al. 2013) and western red cedar (Anand and Chanway 2013b), and crop species such as corn (Puri et al. 2016b) and canola (Padda et al. 2016b). Similarly, endophytic representatives of the genus Pseudomonas are renowned for their PGP and N-fixing properties (Cankar et al. 2005). Doty et al. (2009) isolated Pseudomonas strains   42 from young Sitka willow trees which were able to reduce acetylene to ethylene in an ARA and possessed nif genes necessary to encode nitrogenase enzymes. In a subsequent study, these strains endophytically colonized and promoted the growth of a distinct host, Douglas-fir (Pseudotsuga menziesii) in a greenhouse experiment (Khan et al. 2015). In addition, the first spruce endophytes were strains of Pseudomonas genus isolated from roots of hybrid spruce trees growing near Salmon Arm, BC, Canada (Shishido and Chanway 1999), which were later proved to colonize spruce endophytically and increase seedling biomass in field trials (Shishido and Chanway 2000).  Therefore, it can be postulated that strains isolated from pine and spruce trees in this study are potent N-fixers that could be promoting the growth of pine and spruce at different sites in the West Chilcotin region. However, more work – in particular, N-fixation analysis by inoculating seedlings with these strains – is needed to further prove this hypothesis. Actual quantification of the amount of N fixed by each strain when inoculated into their original host using robust methods such as the 15N foliar dilution assay would be ideal to demonstrate the effectiveness of these strains (Puri et al. 2018b). In the future, such effective N-fixers could be inoculated into seedlings and planted at sites that are particularly N-poor. Such bacteria may sustain the seedling growth for years and could act as an environment-friendly and cost-effective solution to offset/reduce the need to periodically apply chemical fertilizers to managed forest stands. This has been observed in some field studies where inoculation with endophytic bacteria significantly enhanced biomass of trees such as hybrid spruce and poplar through various mechanisms such as N-fixation (Chanway and Holl 1993; Chanway et al. 2000; Shishido and Chanway 2000; Knoth et al. 2014).    43 Table 2.1 Soil characteristics and concentrations of macro- and micro-nutrients present in the forest floor and top mineral layer (0–10 cm) from high-elevation and low-elevation sites in the hybrid white spruce stand.   Forest Floor  Mineral Layer (0–10 cm)  High elevation Low elevation  High elevation Low elevation Total Carbon (%)  47.75 ± 2.760a 48.56 ± 1.867  1.603* ± 0.338 4.196 ± 0.573 Total Nitrogen (%)  1.211* ± 0.100 1.707 ± 0.113  0.048* ± 0.009 0.152 ± 0.022 Total Sulfur (%)  0.100* ± 0.003 0.144 ± 0.011  n.d. n.d. Available Phosphorus as PO42- (mg/kg)  40.43 ± 4.529 56.26 ± 9.347  63.29 ± 28.93 146.9 ± 63.05 Available Sulfur as SO42- (mg/kg)  2.327 ± 1.827 6.056 ± 2.789  1.018 ± 0.228 2.898 ± 1.344 pH in water  5.480* ± 0.402 6.673 ± 0.152  5.853 ± 0.093 6.477 ± 0.534 Cation Exchange Capacity (cMol/kg)  74.93* ± 12.94 133.8 ± 10.41  9.407* ± 1.219 20.15 ± 2.361 Base Saturation (%)  96.12 ± 1.517 98.50 ± 0.399  95.96 ± 1.837 98.41 ± 0.253 Forest Floor depth (cm)  3.730* ± 0.597 9.688 ± 1.479  - - Organic Matter (%)  76.46* ± 1.440 86.82 ± 1.059  3.573* ± 0.476 8.128 ± 1.206 Sand (%)  - -  65.25 ± 1.548 64.39 ± 1.521 Silt (%)  - -  27.54 ± 1.697 28.61 ± 1.143 Clay (%)  - -  7.205 ± 0.435 6.996 ± 0.377 Soil Texture  - -  Sandy Loam Sandy Loam Bulk Density (g/cm3)  - -  1.077 ± 0.010 1.089 ± 0.014        Macro- and micro-nutrients (mg/kg)     Aluminum  261.9 ± 61.93 140.7 ± 88.37  1048 ± 282.8 584.0 ± 14.76 Boron  1.547 ± 0.698 2.601 ± 0.783  n.d. n.d. Calcium  8360* ± 1508 14650 ± 960.0  1336 ± 270.6 3616 ± 1043 Copper  1.392* ± 0.153 7.012 ± 2.013  0.373* ± 0.170 2.584 ± 0.684 Iron  184.5 ± 40.12 249.8 ± 88.64  481.5 ± 54.58 382.9 ± 66.84 Potassium  914.3* ± 115.3 1459 ± 10.38  166.1 ± 33.63 210.6 ± 44.26 Magnesium  828.8* ± 126.5 1865 ± 30.39  150.5 ± 34.37 412.1 ± 196.1 Manganese  214.6* ± 24.74 417.7 ± 59.06  22.25 ± 6.228 33.86 ± 6.238 Sodium  19.73 ± 2.403 39.67 ± 14.58  13.58 ± 3.758 23.82 ± 7.942 Phosphorus  132.7 ± 21.51 146.8 ± 9.005  53.83 ± 14.33 82.78 ± 26.53 Sulfur  26.08 ± 2.049 33.36 ± 4.581  4.792 ± 1.406 8.667 ± 0.607 Zinc  25.26 ± 3.532 25.94 ± 7.058  0.468 ± 0.014 0.887 ± 0.414 Molybdenum  n.d. n.d.  n.d. n.d. Nickel  0.821* ± 0.039 1.349 ± 0.158  n.d. n.d. n.d. = not detected; a Mean ± standard error (n=10); * P < 0.05 (significantly different from low-elevation site)   44 Table 2.2 Soil characteristics and concentrations of macro- and micro-nutrients present in the forest floor and top mineral layer (0–10 cm) from high-elevation and low-elevation sites in the lodgepole pine stand.   Forest Floor  Mineral Layer (0–10 cm)  High elevation Low elevation  High elevation Low elevation Total Carbon (%)  30.91 ± 1.771a 34.67 ± 2.791  0.785 ± 0.062 0.925 ± 0.080 Total Nitrogen (%)  0.779* ± 0.070 1.193 ± 0.089  0.027* ± 0.001 0.045 ± 0.006 Total Sulfur (%)  0.066* ± 0.005 0.091 ± 0.006  n.d. n.d. Available Phosphorus as PO42- (mg/kg)  38.95 ± 5.661 40.29 ± 7.164  90.63 ± 17.99 104.7 ± 9.450 Available Sulfur as SO42- (mg/kg)  1.307 ± 0.807 1.322 ± 0.822  n.d. n.d. pH in water  4.900 ± 0.112 5.023 ± 0.062  6.097 ± 0.038 5.870 ± 0.079 Cation Exchange Capacity (cMol/kg)  32.67* ± 2.862 52.49 ± 4.558  2.179* ± 0.059 3.863 ± 0.331 Base Saturation (%)  94.80 ± 0.828 96.24 ± 0.340  94.68* ± 0.170 95.72 ± 0.019 Forest Floor depth (cm)  1.650* ± 0.100 4.250 ± 1.072  –  – Organic Matter (%)  48.96* ± 3.710 67.63 ± 2.793  1.895* ± 0.020 2.221 ± 0.070 Sand (%)  – –  82.37 ± 1.930 74.39 ± 2.559 Silt (%)  – –  14.69 ± 2.105 21.41 ± 2.186 Clay (%)  – –  2.938 ± 0.419 4.199 ± 0.421 Soil Texture  – –  Loamy Sand Loamy Sand Bulk Density (g/cm3)  – –  1.357* ± 0.012 1.187 ± 0.020        Macro- and micro-nutrients (mg/kg)     Aluminum  902.8* ± 25.43 531.7 ± 97.32  1140* ± 91.75 681.2 ± 104.6 Boron  n.d. n.d.  n.d. n.d. Calcium  3685* ± 234.5 5974 ± 533.4  315.7* ± 19.15 479.4 ± 41.73 Copper  0.745* ± 0.040 1.302 ± 0.140  0.266* ± 0.014 0.323 ± 0.009 Iron  292.0 ± 42.90 300.5 ± 59.83  385.2 ± 12.03 405.2 ± 19.85 Potassium  366.5* ± 48.60 601.4 ± 18.82  77.61 ± 5.682 79.03 ± 3.744 Magnesium  373.7* ± 70.50 744.7 ± 34.93  30.66* ± 2.918 66.93 ± 7.212 Manganese  209.4* ± 7.432 370.5 ± 23.37  25.19* ± 1.751 35.16 ± 1.854 Sodium  9.572 ± 0.546 9.779 ± 1.261  2.435* ± 0.394 4.451 ± 0.480 Phosphorus  92.56 ± 3.987 92.81 ± 7.978  110.7 ± 21.60 120.7 ± 12.07 Sulfur  14.02* ± 0.925 25.40 ± 2.335  2.948 ± 0.137 2.993 ± 0.266 Zinc  19.81 ± 2.255 23.11 ± 2.575  0.406 ± 0.049 0.446 ± 0.083 Molybdenum  n.d. n.d.  n.d. n.d. Nickel  n.d. n.d.  n.d. n.d. n.d. = not detected; a Mean ± standard error (n=10); * P < 0.05 (significantly different from low-elevation site)   45 Table 2.3 List of endophytic diazotrophic strains isolated from hybrid white spruce trees growing on high-elevation and low-elevation sites with their most closely related genus/species (>98% sequence identity) and the amount of ethylene produced in the acetylene reduction assay. Strain Most closely related genus/species Acetylene reduction activity (nmol C2H4/mL)a GenBank Accession no. HS-S1 Pigmentiphaga litoralis 2.1 ± 0.31b MG561767 HS-S2 Herbiconiux solani 2.3 ± 0.12 MG561768 HS-S3 Pseudomonas migulae 2.3 ± 0.05 MG561769 HS-S4 Herbiconiux solani 1.7 ± 0.26 MG561770 HS-S5 Bacillus megaterium 0.1 ± 0.01 MG561771 HS-S6 Paenibacillus urinalis 0.9 ± 0.19 MG561772 HS-N1 Pedobacter sp. 2.0 ± 0.02 MG561773 HS-N2 Frondihabitans cladoniiphilus 0.8 ± 0.11 MG561774 LS-S1 Pseudomonas prosekii 2.6 ± 0.12 MG561775 LS-S2 Caballeronia sordidicola 2.8 ± 0.04 MG561776 LS-S3 Subtercola boreus 1.6 ± 0.25 MG561777 LS-S4 Paenibacillus provencensis 1.3 ± 0.14 MG561778 LS-S5 Sporolactobacillus laevolacticus 1.4 ± 0.06 MG561779 LS-S6 Caballeronia sordidicola 0.3 ± 0.01 MG561780 LS-N1 Paenibacillus provencensis 1.3 ± 0.14 MG561781 LS-N2 Bacillus oleronius 0.8 ± 0.10 MG561782 LS-R1 Caballeronia udeis 1.9 ± 0.18 MG561783 LS-R2 Rhizobium rosettiformans 1.1 ± 0.28 MG561784 a Nanomoles of ethylene produced per mL of culture tube headspace; b Mean ±  standard error (duplicate assays with n = 3 in each assay) Note: Strain names were given according to the site, tree type, and tissue type to which they belonged. For example, HS-S1: high-nutrient (low-elevation) spruce stem strain 1.   46 Table 2.4 List of endophytic diazotrophic strains isolated from lodgepole pine trees growing on high-elevation and low-elevation sites with their most closely related genus/species (>98% sequence identity) and the amount of ethylene produced in the acetylene reduction assay. Strain Most closely related genus/species Acetylene reduction activity (nmol C2H4/mL)a GenBank Accession no. HP-S1 Caballeronia sordidicola 2.4 ± 0.05b MG561788 HP-S2 Frondihabitans sucicola 0.2 ± 0.01 MG561789 HP-S3 Methylobacterium sp. 0.3 ± 0.01 MG561790 HP-S4 Caballeronia sordidicola 0.8 ± 0.01 MG561791 HP-N1 Pseudomonas frederiksbergensis 1.0 ± 0.34 MG561792 HP-N2 Caballeronia sordidicola 0.2 ± 0.01 MG561793 HP-R1 Phyllobacterium myrsinacearum 1.2 ± 0.27 MG561794 HP-R2 Caballeronia sordidicola 0.1 ± 0.01 MG561795 HP-R3 Paenibacillus tundrae 0.1 ± 0.01 MG561796 HP-R4 Paenibacillus provencensis 0.2 ± 0.01 MG561797 LP-S1 Pseudomonas mandelii 1.2 ± 0.12 MG561798 LP-S2 Paenibacillus alginolyticus 0.1 ± 0.01 MG561799 LP-S3 Paenibacillus massiliensis 0.4 ± 0.01 MG561800 LP-N1 Bacillus sp. 0.1 ± 0.01 MG561801 LP-N2 Sphingomonas faeni 0.8 ± 0.01 MG561802 LP-R1 Paraburkholderia phytofirmans 2.9 ± 0.36 MG561803 LP-R2 Caballeronia udeis 1.2 ± 0.15 MG561804 LP-R3 Rhizobium lusitanum 0.7 ± 0.02 MG561805 LP-R4 Caulobacter henricii 0.9 ± 0.06 MG561806 LP-R5 Paenibacillus endophyticus 0.1 ± 0.01 MG561807 a Nanomoles of ethylene produced per mL of culture tube headspace; b Mean ±  standard error (duplicate assays with n = 3 in each assay) Note: Strain names were given according to the site, tree type, and tissue type to which they belonged. For example, HP-S1: high-nutrient (low-elevation) pine stem strain 1.   47          Source: Google Earth (2017)          Source: Google Earth (2017) Figure 2.1 (a) Location of sampling area in the West Chilcotin region, 250 km west of Williams Lake, BC on the Chilcotin-Bella Coola Highway (BC Highway 20); (b) Detailed view of the sampling area, showing the two sampling sites each in the hybrid white spruce and lodgepole pine stands that were selected at different elevations. (a)Pine - high elevationPine - low elevationSpruce - low elevationSpruce - high elevation (b)  48        Figure 2.2 Soil conditions at sampling sites in the West Chilcotin region. (a) very thin or negligible organic forest floor denoted by arrows; (b) soil sample collected using an Oakfield probe.  (a) (b)  49             Figure 2.3 Mean values (n=10) of: (a) Carbon to nitrogen ratio, (b) mineralizable nitrogen, (c) available nitrate, and (d) available ammonia present in forest floor and top mineral layer (0–10 cm) soil samples collected from the low-elevation (LE) site and high-elevation (HE) site in the hybrid white spruce stand. Error bars represent standard errors of mean; *P < 0.05 (significantly different from low-elevation site).  01020304050HE LE HE LEForest Floor Mineral Layer (0-10 cm)C:N ratio(a)**02004006008001000HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgMineralizable N**(b)0.00.10.20.30.40.50.6HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgAvailable NO3-(c)**05101520253035HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgAvailable NH4+**(d)  50           Figure 2.4 Mean values (n=10) of: (a) Carbon to nitrogen ratio, (b) mineralizable nitrogen, (c) available nitrate, and (d) available ammonia present in forest floor and top mineral layer (0-10 cm) soil samples collected from the low-elevation (LE) site and high-elevation (HE) site in the lodgepole pine stand. Error bars represent standard errors of mean; *P < 0.05 (significantly different from low-elevation site).  01020304050HE LE HE LEForest Floor Mineral Layer (0-10 cm)C:N ratio(a)**0100200300400500600HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgMineralizable N(b)**0.00.10.20.30.40.50.60.7HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgAvailable NO3-(c)**0510152025303540HE LE HE LEForest Floor Mineral Layer (0-10 cm)mg/kgAvailable NH4+(d)**  51 Chapter 3 – Can naturally-occurring endophytic nitrogen-fixing bacteria of hybrid white spruce sustain boreal forest tree growth on extremely nutrient-poor soils? 3.1. Introduction Plants rely heavily on their microbiome for sustained growth and health. Interactions between a plant and its microbiome are highly diverse, and multiple factors can shape the community assembly and functioning. Members of the microbiome are actively recruited by plants from the surrounding environment including bulk soil, rhizosphere and leaf surfaces (horizontal transfer) (Hardoim et al. 2015). Microbes can also be transferred vertically via seed and become part of the plant microbiome when the seed germinates (Frank et al. 2017). Endophytic bacteria are extremely valuable members of the plant microbiome because of their role in nutrient acquisition, phytohormone modulation and biocontrol of phytopathogens. By definition, endophytic bacteria are those bacteria that can colonize the interior tissues of plants without causing any disease (Puri et al. 2017b). Endophytic bacteria have been reported to colonize and provide benefits to a myriad of agricultural hosts (Doty 2017). However, there is little evidence about their existence in natural ecosystems such as forests. Since forest trees exist for a much longer period of time in the terrestrial ecosystems and have larger biomass than the herbaceous plants, they can provide unique ecological conditions for the endophytic bacteria (Puri et al. 2017a).   52 One of the many ways in which endophytic bacteria have been reported to promote tree growth in natural ecosystems is via biological nitrogen fixation (BNF). Such endophytic bacteria, capable of fixing nitrogen (N) from the atmosphere (known as ‘endophytic diazotrophic bacteria’), have been reported to associate with coniferous trees including lodgepole pine (Pinus contorta var. latifolia) (Anand et al. 2013; Tang et al. 2017), limber pine (Pinus flexilis) (Moyes et al. 2016) and western red cedar (Thuja Plicata) (Anand and Chanway 2013b) as well as deciduous trees including black cottonwood (Populus trichocarpa) and willow (Salix sitchensis) (Doty et al. 2009). However, the presence of such naturally-occurring endophytic diazotrophic bacteria in other tree species and their ability to sustain tree growth, especially on highly disturbed and nutrient-poor sites, needs further evaluation.  The West Chilcotin is a montane region located in the west central-interior of British Columbia (BC), Canada. It lies in the Sub-Boreal biogeoclimatic zone and is characterized by a cold, dry climate with extremely low annual precipitation (~335 mm per year) due to the rain-shadow effect of the Coast Mountains. The mean annual temperature in this region ranges from 0.3 to 2.7°C. This region is frequently affected by wildfires, logging activity and attack by insect pests such as mountain pine beetle. These conditions have resulted in dry and weakly developed soils having limited organic matter levels. As reported in Chapter 2 (Puri et al. 2018a), soils in this region are mainly coarse-textured with sandy loam or loamy sand texture belonging to the Brunisolic soil order according to the Canadian System of Soil Classification (‘Cambisols’ as per the World Reference Base for Soil Resources and ‘Inceptisols’ as per the US Soil Taxonomy) with mainly acidic pH, ranging between 4 and 6 (Steen and Demarchi 1991; Steen and Coupé 1997). Hybrid white spruce (Picea glauca x engelmannii) is one of the most common tree species   53 growing in this region. Soils under spruce tree stands in this region have poor physico-chemical health, lacking several essential plant nutrients (Puri et al. 2018a; Chapter 2). Notably, these soils have extremely low amounts of mineralizable and available forms of N (Puri et al. 2018a; Chapter 2). The ability of spruce trees to grow on such nutrient-poor (particularly, N-limited) soils raises a crucial question regarding their N-sources. In the previous chapter, it was reported that spruce trees in this region harbour endophytic diazotrophic bacteria in their tissues, which may provide them with fixed N from the atmosphere to sustain their growth on such N-limited soils (Puri et al. 2018a). Such bacteria have also been observed in Engelmann spruce (Picea engelmannii) (Carrell and Frank 2014) and Norway Spruce (Picea abies) (Cankar et al. 2005) trees growing in the subalpine forests of Colorado, USA and Slovenia, respectively. However, to the best of our knowledge, no studies have examined the endophytic diazotrophic bacteria originating from spruce species in planta to quantify the amount of N-fixed and the impact of this fixed N on tree growth.  Fifty-five endophytic bacterial strains were isolated from the tissues of hybrid white spruce trees growing at N-limited sites in the West Chilcotin region, of which 18 strains showed N-fixing ability in vitro (i.e. nitrogenase enzyme activity) when analyzed using the acetylene reduction assay (ARA) (Puri et al. 2018a; Chapter 2). Six bacterial strains that showed the best performance in ARA were selected for further evaluation (listed in Table 3.1) to investigate how trees are able to thrive naturally in N-poor soils of this region. In this study, each of these six bacterial strains were evaluated in two greenhouse studies, one with hybrid white spruce (the original host of the bacteria) and another with lodgepole pine (a foreign host). The main motive behind using multiple hosts was to examine the plant x microbe specificity of these endophytic   54 strains. Lodgepole pine was chosen as the foreign host since it is also commonly found in the West Chilcotin region in addition to hybrid white spruce. If shown to be effective in multiple hosts, these bacterial species could potentially be used in the future as biofertilizers in the West Chilcotin region.  Therefore, the overarching questions addressed in this study were: (i) can each of the six selected bacterial strains re-colonize their original host (hybrid white spruce), fulfil its N-requirements via BNF and promote its growth? (ii) can these bacterial strains colonize and provide similar benefits to a foreign host (lodgepole pine)? 3.2. Materials and methods 3.2.1. Bacterial strains As stated above, the six selected endophytic diazotrophic strains originated from hybrid white spruce trees growing in the West Chilcotin region of BC and showed positive nitrogenase enzyme activity when analyzed using ARA (Puri et al. 2018a; Chapter 2). To facilitate the evaluation of endophytic colonization of seedlings by these strains in the greenhouse trials, an antibiotic-resistant derivative of each strain was raised by streaking multiple times on N-free combined carbon medium (CCM) agar (Appendix B) amended with antibiotic compound (200 mg/L rifamycin), as outlined by Bal and Chanway (2012a) (Table 3.1). Nitrogenase enzyme activity of antibiotic-resistant strains was reconfirmed using ARA (Table 3.1) as outlined in Chapter 2 (Puri et al. 2018a). The strains were stored in CCM amended with 200 mg/L rifamycin and 20% (v/v)   55 glycerol at – 80°C until further evaluation. These antibiotic-resistant strains were used for nifH gene analysis and greenhouse growth trials as outlined below. 3.2.2. NifH gene analysis The presence of the nifH gene in each strain was evaluated to further confirm the nitrogenase activity of the six selected bacterial strains. The extraction of the genomic DNA of each strain has been explained in Chapter 2 (Puri et al. 2018a). The nifH gene of each strain was amplified using the primers nifH1 forward primer [50-TGY GAY CCN AAR GCN GA-30] and nifH2 reverse primer [50-ADN GCC ATC ATY TCN CC-30] (Zehr and McReynolds 1989). PCR amplification was performed in 25 μL reaction volumes containing 2.5 μL of 1x PCR buffer (without MgCl2), 1.25 μL of MgCl2 (2.5 mM), 0.5 μL of Taq DNA Polymerase (5 U/μl), 0.5 μL of dNTP mix (consisting of four nucleotides: dATP, dCTP, dGTP, dTTP), 2.5 μL of each primer (1 μM), and 2.5 μL of template DNA (1 ng/μL) (Gaby and Buckley 2012). The final volume was adjusted to 25 μL with nuclease-free water. Amplifications were performed on MJ Mini gradient thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) using the following program: initial cell lysis and denaturation for 5 min at 95°C ; followed by 35 cycles of denaturation (1 min at 94°C), annealing (1 min at 55°C), and extension (1 min at 72°C); and a final extension for 10 min at 72°C. Amplification of PCR products was confirmed by electrophoresis on 1% agarose gel for 1 h at 100 V. 3.2.3. Greenhouse experiments Two separate year-long greenhouse growth trials were set up, one involving the inoculation of each selected strain into hybrid white spruce (original host) and the other involving inoculation   56 of each strain into lodgepole pine (foreign host). Thus, six bacteria-inoculated and one non-inoculated (control) treatments were evaluated in each trial. For each treatment, 55 seedlings were raised per tree species. 3.2.3.1. Seed acquisition and preparation Hybrid white spruce and lodgepole pine seeds were obtained from the BC Ministry of Forests Tree Seed Centre, Surrey, BC, Canada and originated from sites located in the West Chilcotin region (Hybrid white spruce – 51° 57’ N lat., 124° 59’ W long., elevation 1350 m, SBPSxc biogeoclimatic zone; Lodgepole pine – 51° 54’ N lat., 124° 51’ long., elevation 1320 m, SBPSxc biogeoclimatic zone). The spruce and pine seeds were surface sterilized by immersion in 30% (v/v) hydrogen peroxide for 1.5 min, and then rinsed (three times for 30 s) in sterile distilled water. The effectiveness of surface sterilization was confirmed by imprinting 10 randomly selected sterilized seeds on tryptic soy agar (TSA) plates and incubating the plates at 30°C for 2 days to check for microbial contamination. Once surface sterilized, the seeds were placed in sterile cheesecloth bags containing moist autoclaved sand and stored at 4°C for 28 days for stratification. After the stratification period, 10 seeds were selected randomly from cheesecloth bags, crushed, and imprinted on TSA plates supplemented with 200 mg/L rifamycin for 2 days to confirm the absence of internal seed contamination. 3.2.3.2. Plant inoculation and growth conditions Seedlings were grown in Ray Leach Cone-tainers (height: 210 mm; diameter: 38 mm) that were filled to 67% capacity with an autoclaved sand–Turface (montmorillonite clay, Applied Industrial   57 Materials Corporation, Deerfield, IL, USA) mixture (69% w/w silica sand; 29% w/w Turface; 2% w/w CaCO3). Each Cone-tainer was fertilized with 20 mL of a nutrient solution containing Ca(15NO3)2 (5% 15N label) (Appendix C). Three surface-sterilized seeds of either hybrid white spruce or lodgepole pine were sown aseptically in each Cone-tainer and covered with 5 mm of autoclaved silica sand. Inoculum of each bacterial strain was prepared by thawing a frozen culture of the strain, streaking a loopful onto CCM agar amended with 200 mg/L rifamycin, and incubating at 30°C for 2 days. A 1 L flask containing 500 mL of CCM broth amended with rifamycin was then inoculated with a loopful of bacterial growth from the agar, secured on a rotary shaker, and agitated (150 rpm) at room temperature for 2 days. Bacterial cells were harvested by centrifugation (3000 xg; 30 min), washed twice in sterile phosphate buffered saline (PBS) (pH 7.4) (Appendix A), and resuspended in the same buffer to a density of ca. 106 colony forming units (cfu) per mL. Immediately before covering the seeds with silica sand, a 5 mL bacterial suspension of each strain was pipetted directly over the seeds in each Cone-tainer designated for that strain. This process was repeated for each of the six bacterial strains. Non-inoculated control seeds received 5 mL of sterile PBS without any bacteria. The Cone-tainers were placed in trays (98 cells) in the greenhouse with a 16 h photoperiod (6 am–10 pm) having photosynthetically active radiation at the canopy level of 300 μmol/m2/s achieved through a combination of natural and artificial light in the greenhouse. Seedlings were watered as required with sterile distilled water and thinned to the largest single germinant per Cone-tainer, 2 weeks after sowing. Seedlings received 20 mL of the nutrient solution without Ca(15NO3)2 once per month during the 12-month growth trial. Tray positions were randomized weekly to reduce the positional effects during the experiment.   58 3.2.3.3. Investigation of endophytic colonization For each tree species, five randomly selected replicate seedlings per treatment were harvested 4, 8 and 12 months after sowing to evaluate endophytic colonization. At each harvest, seedlings were surface-sterilized with 1.3% sodium hypochlorite for 5 min and washed thrice with sterile distilled water. Seedlings were imprinted on TSA plates for 24 h to check for surface contamination. Root, stem, and needle tissue samples were then triturated separately in 1 mL PBS using a mortar and pestle. Triturated tissue suspensions were serially diluted, and 0.1 mL of each dilution was plated onto CCM supplemented with cycloheximide (100 mg/L) to inhibit fungal growth and rifamycin (200 mg/L) to inhibit the growth of other bacteria. Since the six strains used in the greenhouse trials were rifamycin-resistant, their growth on these plates was not inhibited. The numbers of cfu of each strain per gram of fresh tissue were evaluated 7 days after incubation at 30°C. 3.2.3.4. Analysis of seedling growth promotion and foliar 15N For each tree species, 10 randomly selected replicate seedlings per treatment were harvested destructively 4, 8 and 12 months after sowing to evaluate the seedling length and biomass (dry weight). Seedlings were removed from Cone-tainers, roots were washed under running water and the seedling length was measured. Seedlings were then oven-dried for 2 days at 65°C to determine their biomass. Ten randomly selected replicate seedlings from each treatment and tree species were harvested 12 months after sowing for foliar 15N analysis (Puri et al. 2018b). Seedlings were removed from the Cone-tainer, needles were separated from the stem and oven-dried for 2 days at 65°C. The oven-dried needles of each seedling were ground to a particle size   59 of <2 mm using a mortar and pestle and a 2.5 mg sample of this ground foliage of each seedling was sent to the Stable Isotope Facility located in the Faculty of Forestry, University of British Columbia, Vancouver, Canada to determine the foliar N concentration and atom % 15N excess in foliage. The amount of fixed N accumulated in the foliage of each bacteria-inoculated seedling was estimated by calculating the percent N derived from the atmosphere (%Ndfa) (Knoth et al. 2014): %Ndfa = '	1 −	atom	%15N	excess	(inoculated	plant)atom	%15N	excess	(control	plant) 	; 	x	100% The rate of N-fixation in bacteria-inoculated hybrid white spruce and lodgepole pine seedlings was calculated using the foliar N concentration and %Ndfa. 3.2.4. Statistical analyses Statistical analysis of each greenhouse trial (one involving hybrid white spruce and the other involving lodgepole pine) was performed separately. Since the two tree species have variable growth and soil N uptake patterns, the growth parameters cannot be compared between tree species (von Wirén et al. 1997). For each trial, a completely randomized experimental design was used to assess the treatment effects of each of the six bacterial strains on the growth of seedlings. The statistical package, SAS University Edition (SAS Institute Inc., Cary, NC, USA), was used to perform statistical analyses. Analysis of variance (ANOVA), P < 0.05, was performed (F-test and Student’s t-test) to determine significant differences between treatment means for seedling length, seedling biomass, atom %15N excess in foliage and foliar N concentration.   60 3.3. Results 3.3.1. Nitrogenase activity and nifH gene Nitrogenase enzyme activity of the six endophytic diazotrophic strains (antibiotic-resistant derivatives) used in this study was tested using the ARA. All strains reduced acetylene to ethylene in this assay thereby showing putative positive results for the presence of the nitrogenase enzyme. The amount of ethylene produced ranged between 2 and 3 nmol per mL of culture tube headspace with no statistically significant differences between bacterial strains (Table 3.1). In addition, the nifH gene of each strain was successfully amplified, further confirming their N-fixing ability (Table 3.1). 3.3.2. Greenhouse experiments 3.3.2.1. Endophytic diazotrophic strains in the original host (hybrid white spruce) Endophytic needle colonization in hybrid white spruce seedlings was observed for strains HS-S1r, LS-S1r, LS-S2r and LS-R1r at all harvest intervals, except LS-S1r strain which was only observed at the 12-month harvest (Figure 3.1a). The population density of these strains ranged from 102 to 105 cfu/g fresh tissue. All six strains were observed in the stem tissues of spruce seedlings at all harvest intervals (103–104 cfu/g fresh tissue), except strains HS-S2r, LS-S1r and LS-R1r which were not observed at the 4-month harvest (Figure 3.1b). Endophytic root colonization of spruce seedlings was observed for all bacterial strains at each harvest interval with a population size range of 104 –107 cfu/g fresh tissue (Figure 3.1c). Endophytic colonization was not observed in non-inoculated control seedlings of spruce.   61 Spruce seedlings inoculated with bacteria were significantly longer than non-inoculated control seedlings during the growth trial. At the 4-month harvest, seedlings inoculated with strains HS-S1r, HS-S3r, LS-S2r and LS-R1r were significantly longer (>35%) than control seedlings (Figures 3.2a and 3.3a). All bacteria-inoculated seedlings were significantly longer than control seedlings at the 8- and 12-month harvest (Figures 3.2a, 3.3b and 3.3c). Additionally, strains HS-S1r, LS-S1r and LS-S2r promoted seedling length by >40% as compared to control at the 12-month harvest. No statistically significant difference was observed for the seedling length between bacterial treatments at any harvest interval. It was also observed that bacteria-inoculated spruce seedlings had accumulated significantly higher biomass than non-inoculated control seedlings. At the 4-month harvest, all bacteria-inoculated seedlings had significantly higher biomass (>100%) than control seedlings with no significant difference between bacterial treatments (Figures 3.2b and 3.3a). All bacterial treatments except strain HS-S2r had significantly higher seedling biomass than controls at the 8-month harvest (Figures 3.2b and 3.3b). Particularly, strains HS-S1r, LS-S1r and LS-S2r promoted seedling biomass by >130% as compared to control, even outperforming other bacterial treatments. At the 12-month harvest, all bacteria-inoculated seedlings were significantly greater than controls in terms of biomass (Figures 3.2b and 3.3c). Notably, LS-S1r-inoculated seedlings and LS-S2r-inoculated seedlings had accumulated 218% and 475% higher biomass than control, respectively. The results of atom % 15N excess in foliage revealed that all bacterial strains were capable of fixing significant amounts of atmospheric N when inoculated into their original host (hybrid   62 white spruce) (Table 3.2). Notably, strain LS-S1r fulfilled 56% of the N-requirement of spruce seedlings, one year after inoculation, which was significantly higher than HS-S2r and LS-R1r bacterial treatments. Additionally, strains HS-S1r, HS-S3r, LS-S1r also fixed significantly higher amounts of N from the atmosphere than strain HS-S2r. Twelve months after inoculation, the concentration of N in the foliage of spruce seedlings inoculated with strain LS-S2r was also significantly greater than the control (71%) and other bacterial treatments except for LS-S1r (Figure 3.4). No significant difference was observed between the control treatment and bacterial treatments HS-S1r, HS-S2r, HS-S3r and LS-R1r for foliar N concentration. 3.3.2.2. Endophytic diazotrophic strains in the foreign host (lodgepole pine) In the case of lodgepole pine seedlings, needle colonization was not observed for any endophytic diazotrophic strain initially (4-month harvest) but strains HS-S1r, LS-S1r and LS-S2r were observed at the 8- and 12-month harvests with a population size range of 103–104 cfu/g fresh tissue (Figure 3.5a). These three strains also colonized stem tissues at all harvest intervals (102–105 cfu/g fresh tissue) along with strain LS-R1r, which colonized internal stem tissues at the 8- and 12-month harvests and strain HS-S3r that colonized stem tissues at the 12-month harvest only (Figure 3.5b). Endophytic root colonization was observed for all strains at all harvest intervals in pine seedlings with a population size range of 105–106 cfu/g fresh tissue (Figure 3.5c). No evidence of endophytic colonization was observed in the control seedlings of pine.  All strains except LS-R1r significantly increased pine seedling length as compared to the control at the 4-month harvest (Figures 3.6a and 3.7a). Seedlings inoculated with strains HS-S1r, HS-S3r, LS-S1r and LS-S2r were >25% longer than controls at this harvest time. These strains also   63 increased the seedling length by > 30% at the 8-month harvest (Figures 3.6a and 3.7b). All bacteria-inoculated seedlings were significantly longer than controls at both the 8- and 12-month harvests (Figures 3.6a, 3.7b and 3.7c). Particularly, strains LS-S1r and LS-S2r increased the seedling length by >55% when analyzed at the 12-month harvest. At all harvest intervals, no significant difference was observed for pine seedling length between bacterial treatments.  At the 4-month harvest, all bacteria-inoculated pine seedlings had accumulated significantly greater biomass than controls except strain LS-R1r (Figures 3.6b and 3.7a). Notably, seedlings inoculated with strains HS-S1r and LS-S1r had >125% higher biomass than controls. After 8 months of inoculation, only the seedlings inoculated with strains HS-S1r, LS-S1r and LS-S2r had significantly greater biomass than controls (Figures 3.6b and 3.7b). At this harvest time, strain LS-S2r promoted seedling biomass by 133% compared to the control. All bacterial treatments were significantly better than controls in terms of seedling biomass at the end of the growth trial (Figures 3.6b and 3.7c). Notably, inoculation with strains LS-S1r and LS-S2r increased pine seedling biomass by 229% and 363% compared to controls, respectively, at the 12-month harvest. Their biomass was also significantly higher than the biomass of pine seedlings inoculated with strains HS-S2r, HS-S3r and LS-R1r.  Similar to the seedling biomass results at the 12-month harvest, pine seedlings inoculated with strains LS-S1r and LS-S2r had a significantly higher concentration of N in their foliage compared to controls and the HS-S2r, HS-S3r and LS-R1r treatments (Figure 3.8). Particularly, seedlings inoculated with strain LS-S2r had 59% greater foliar N than control seedlings. All strains were successful in fixing significant amounts of N from the atmosphere in a foreign host, i.e.   64 lodgepole pine (Table 3.3). Results of the atom % 15N excess in foliage analysis indicated that strains LS-S1r and LS-S2r fulfilled 48% and 52% of the N-requirements of pine seedlings, respectively, through BNF. The amount of N fixed by these two strains was significantly higher than the amount of N fixed by strains HS-S2r and HS-S3r in pine seedlings. 3.4. Discussion On the basis of strong N-fixation potential in ARA experiments (Puri et al. 2018a; Chapter 2), six endophytic strains originally isolated from spruce trees growing in the West Chilcotin region were selected for inoculation into spruce and pine seeds in this study. To quantify the number of endophytic colonies formed by these strains in spruce or pine tissues after inoculation, antibiotic-resistant derivatives of these strains were raised. It was observed that these derivatives had similar nitrogenase enzyme activity (Table 3.1) as their wild-type strains (Table 2.3) when tested using ARA, therefore confirming that antibiotic-resistance mutation did not affect the N-fixing capability of endophytic bacteria, which is consistent with previous reports (Shishido et al. 1995; Bal and Chanway 2012a). The ARA is a very sensitive technique widely used to confirm the presence and activity of the nitrogenase enzyme of bacterial strains (Doty et al. 2009), however, some studies have reported that ARA can occasionally give false-positive results (Achouak et al. 1999). Therefore, we further confirmed the potential of these antibiotic-resistant strains to fix atmospheric N by amplifying their nifH genes and observed positive results for all bacterial strains (Table 3.1). The nifH gene is widely regarded as the marker gene to confirm the ability of a bacterium to possess the nitrogenase enzyme required to carry out BNF since this gene is found only in N-fixing bacteria (Zehr et al. 2003; Gaby and Buckley 2012).   65  When each of the six bacterial strains was inoculated into spruce and pine seedlings and assessed 4, 8 and 12 months after inoculation, a similar trend was observed in terms of endophytic root colonization for both spruce and pine (Figures 3.1 and 3.5). All bacteria successfully colonized internal root tissues of spruce and pine and generally, an increasing trend in population size was observed over the period of the growth trial. Similarly, an upward trend in population size was observed for strains that colonized internal stem and needle tissues of spruce and pine. This trend reflects the effective establishment and multiplication of colonies in the internal tissues of spruce and pine seedlings, thus confirming that all strains were capable of forming and sustaining endophytic colonies not only in their original host but in a foreign host as well. This aligns with some previous studies where conifer endophytes have shown recolonization abilities in a variety of host species, even agricultural crops (Puri et al. 2015; Padda et al. 2016a). Overall, the population sizes observed in root, stem and needle tissues of spruce and pine fall within the range observed previously for endophytic strains in coniferous trees (Chanway et al. 2000; Yang et al. 2016, 2017). Notably, Caballeronia sordidicola (previously known as Burkholderia sordidicola) strain LS-S2r established the highest number of colonies in tissues of spruce and pine (except root tissues of pine), which was followed by Pseudomonas prosekii strain LS-S1r and Pigmentiphaga litoralis strain HS-S1r. To the best of our knowledge, none of the six bacterial species tested in this study has previously shown the ability to form endophytic colonies in the internal tissues of plants. However, several other species of genera Pseudomonas and Burkholderia are known for their ability to form endophytic colonies in tree species such as interior spruce, live oak and poplar (Brooks et al. 1994; Germaine et al. 2004).   66  In this study, one of the most robust methods – the 15N isotope dilution method – was used to quantify the N-requirements of the plant fulfilled by each strain via BNF (Knoth et al. 2014). It was found that all endophytic strains provided atmospherically fixed N (17–56%) to spruce and pine seedlings (Tables 3.2 and 3.3), validating the hypothesis that these endophytic strains not only have the ‘ability’ to perform BNF (as evidenced through ARA and nifH gene amplification), but they can ‘actually’ fix N from the atmosphere in planta. Since spruce and pine seedlings were grown under N-poor conditions (N was provided only once in the form of a marginal amount of 15N at the onset of the growth trials), BNF appears to have been an important component of N nutrition for bacteria-inoculated plants (Urquiaga et al. 1992). To the best of our knowledge, this is the first study to quantify and report N-fixation by each of these six endophytic bacterial species. Previous studies have only reported their ability to perform BNF in vitro, specifically for Herbiconiux solani, Pseudomonas migulae, Caballeronia sordidicola and Caballeronia udeis (Suyal et al. 2014; Padda et al. 2018). Assuming that N-fixation occurred uniformly throughout the one-year growth period, the N-fixation rate for bacteria-inoculated spruce and pine seedlings ranged from 4.18 to 15.5 mg N/kg tissue/day and 2.88 to 14.2 mg N/kg tissue/day, respectively (Tables 3.2 and 3.3), which are consistent with/exceed the estimates reported for endophytic diazotrophic bacteria in poplar and limber pine (Moyes et al. 2016; Doty et al. 2016). If these bacterial strains continue to fix N at similar rates, then the highest N-fixing strain, C. sordidicola LS-S2r, would be able to provide 5.66 g and 5.18 g of N per kg tissue to spruce and pine seedlings, respectively, per year. These estimates of N-fixation rate, although lower than the N-fixation rates observed in legumes, could still be biologically significant for conifers, particularly when they are growing on extremely N-poor soils such as those found in the West   67 Chilcotin region. However, these N-fixation rates are presumptive estimates that need to be confirmed through a careful and comprehensive evaluation of the N cycle of forest stands in this region.  Endophytic colonization by each bacterial strain led to a significant enhancement of seedling length (>35%) and biomass (>100%) of spruce and pine seedlings by the end of the growth trial (Figures 3.2 and 3.6). Since the soil was fertilized with N only once at the beginning, with no further additions during the growth trial, it is logical to assume that the growth of a seedling will be affected unless it is accessing a source other than soil N. Results of 15N isotope dilution assays indicate that this other N-source is BNF performed by each bacterial strain in association with spruce and pine. Enhanced N nutrition from BNF might have been the reason for a significant incremental trend observed for length and biomass of bacteria-inoculated seedlings. In addition to fixing N, endophytic diazotrophic bacteria can also increase seedling growth through other plant-growth-promoting (PGP) mechanisms including solubilization of phosphate and modulation of phytohormones (Padda et al. 2017a, b). Therefore, further studies need to be done to elucidate any other PGP mechanism employed by these bacteria to promote seedling growth.  Although no significant differences in spruce and pine seedling length were observed among the bacterial treatments (Figures 3.2a and 3.6a), C. sordidicola strain LS-S2r significantly outperformed all other bacterial strains by promoting spruce and pine seedling biomass by nearly 6-fold and 4.5-fold, respectively (Figures 3.2b and 3.6b). In addition, strain LS-S2r also provided the highest amount of N for spruce and pine seedlings through BNF (>50%) thereby significantly   68 increasing their foliar N concentration (>1.5-fold as compared to the control) (Figures 3.4 and 3.8). The considerable growth promotion and BNF observed for strain LS-S2r could be related to the extensive internal colonization of needle, stem and root tissues by this strain (Figures 3.1 and 3.5). This aligns with previous studies where significant colonization and establishment of endophytic colonies were directly correlated to considerable growth promotion (Shishido et al. 1995) and BNF (Anand et al. 2013). Although this is the first study where seedling growth promotion and BNF by a C. sordidicola strain has been quantified, previous reports have suggested that strains of C. sordidicola possess PGP abilities. Caballeronia sordidicola strains BLN 14 and BLN 20 were reported to have the ability to solubilize phosphate, produce siderophores to acquire iron and suppress phytopathogens, and modulate plant hormone levels through 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity (Palaniappan et al. 2010). In another study, the genome sequencing of C. sordidicola strain S170 revealed that it possesses the required genes for ACC deaminase activity, phosphate solubilization and metabolization, and siderophore production (Lladó et al. 2014). Although this strain was isolated from the topsoil of a natural coniferous forest, interestingly, it possessed genes to metabolize various carbon sources and therefore have the potential to colonize different plant niches. The presence of a C. sordidicola strain has also been reported recently in root tissues of lodgepole pine trees growing at gravel mining sites, with the ability to potentially perform BNF (Padda et al. 2019). In addition, several studies to date have regarded that the Burkholderia genus (to which the Caballeronia genus previously belonged) is rich in associated and symbiotic nitrogen fixers and plant-growth-promoters (Estrada-De Los Santos et al. 2001).   69  In conclusion, these results indicate that endophytic diazotrophic bacteria could be vital for spruce seedling survival on highly disturbed soils, such as those of the West Chilcotin region by forming a natural associative relationship. These results also provide the first evidence of significant endophytic N-fixation in hybrid white spruce trees and support the possibility of a novel and ecologically significant interaction between hybrid spruce trees and endophytic diazotrophic bacteria in boreal forests. In addition, these endophytic diazotrophic bacteria can successfully associate with at least one non-native host and help it thrive under N-limited conditions indicating that these bacteria are generalists with little plant x microbe specificity. Noteworthily, the large increase in seedling growth after C. sordidicola LS-S2r inoculation is highly significant, particularly from a silvicultural point of view. Looking at the success shown by C. sordidicola LS-S2r in both its original host and a foreign host, it can be concluded that this bacterium could potentially be utilized as a biofertilizer in the West Chilcotin region for both hybrid white spruce and lodgepole pine trees. However, long-term field studies need to be performed to observe the performance of seedlings inoculated with this bacterium in ‘real-world’ conditions before any reliable recommendation can be made about this bacterium. If shown to be effective in field studies, this bacterium may help reduce the application of chemical fertilizers and enhance tree growth in boreal forest stands in a low-cost, ecologically-friendly manner.    70 Table 3.1 List of endophytic diazotrophic bacterial strains selected from Chapter 2 (Puri et al. 2018a) and their antibiotic-resistant derivative strains used in this study along with the amount of ethylene produced in the acetylene reduction assay and the presence of the nifH gene in these derivative strains. Bacterial species Strain Antibiotic-resistant strain Acetylene reduction activity (nmol C2H4/mL)a nifH gene Pigmentiphaga litoralis HS-S1 HS-S1r 2.0 ± 0.11b + Herbiconiux solani HS-S2 HS-S2r 2.1 ± 0.08 + Pseudomonas migulae HS-S3 HS-S3r 2.3 ± 0.19 + Pseudomonas prosekii LS-S1 LS-S1r 2.5 ± 0.15 + Caballeronia sordidicola LS-S2 LS-S2r 3.0 ± 0.21 + Caballeronia udeis LS-R1 LS-R1r 2.1 ± 0.06 + a Nanomoles of ethylene produced per mL of culture tube headspace; b Mean ± standard error (n = 5)  Table 3.2 Percent N derived from the atmosphere (% Ndfa) by each of the six bacteria in hybrid white spruce seedlings, determined 12 months after sowing using atom percent 15N excess in foliage values. N-fixation rate (mg of fixed N per kg of tissue per day) was determined using %Ndfa and foliar N concentration. Values are mean ± standard error for atom % 15N excess in foliage (n = 10 seedlings per treatment). Values followed by different letters are significantly different (P < 0.05). Treatment Atom % 15N excess in foliage % Ndfa N-fixation rate (mg/kg/day) Pigmentiphaga litoralis HS-S1r 0.37 ± 0.02cd 46 8.22 Herbiconiux solani HS-S2r 0.51 ± 0.02b 25 4.18 Pseudomonas migulae HS-S3r 0.39 ± 0.02cd 44 7.59 Pseudomonas prosekii LS-S1r 0.36 ± 0.02cd 47 10.3 Caballeronia sordidicola LS-S2r 0.30 ± 0.02c 56 15.5 Caballeronia udeis LS-R1r 0.44 ± 0.01bd 36 5.92 Control 0.69 ± 0.01a - -   71 Table 3.3 Percent N derived from the atmosphere (% Ndfa) by each of the six bacteria in lodgepole pine seedlings, determined 12 months after sowing using atom percent 15N excess in foliage values. N-fixation rate (mg of fixed N per kg of tissue per day) was determined using %Ndfa and foliar N concentration. Values are mean ± standard error for atom % 15N excess in foliage (n = 10 seedlings per treatment). Values followed by different letters are significantly different (P < 0.05). Treatment Atom % 15N excess in foliage % Ndfa N-fixation rate (mg/kg/day) Pigmentiphaga litoralis HS-S1r 0.46 ± 0.03cd 41 8.56 Herbiconiux solani HS-S2r 0.64 ± 0.02b 17 2.88 Pseudomonas migulae HS-S3r 0.56 ± 0.02bc 28 4.96 Pseudomonas prosekii LS-S1r 0.41 ± 0.03d 48 11.8 Caballeronia sordidicola LS-S2r 0.37 ± 0.03d 52 14.2 Caballeronia udeis LS-R1r 0.47 ± 0.01cd 39 7.09 Control 0.78 ± 0.03a - -     72      Figure 3.1 Endophytic population size of each of the six bacterial strains inside (a) needle, (b) stem, and (c) root tissues of hybrid white spruce seedlings evaluated 4, 8 and 12 months after sowing (n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted, and data wer log-transformed. Strains that did not colonize a tissue type have not been included in the figure. 0123456Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueNeedle(a)0123456Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueStem(b)3456789Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueRootHS-S1r HS-S2r HS-S3rLS-S1r LS-S2r LS-R1r(c)  73    Figure 3.2 Mean values of (a) length and (b) biomass of hybrid white spruce seedlings subjected to six bacterial treatments and a non-inoculated control harvested 4, 8 and 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05). 05101520253035HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling length (cm)(a)b babb baabbbbbbabbbbbbab0102030405060708090100HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling biomass (mg)(b)b bbb babcbabbcacbbbbbac  74    Figure 3.3 Bacteria-inoculated and non-inoculated control seedlings of hybrid white spruce harvested (a) 4 months, (b) 8 months, and (c) 12 months after sowing showing clear differences in length and biomass. HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 1(a)HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 2(b)HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 3(c)  75  Figure 3.4 Nitrogen (N) concentration (mg N per g tissue) in the foliage of hybrid white spruce seedlings subjected to six bacterial treatments and a non-inoculated control measured 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05).  024681012HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlFoliar N (mg/g tissue)aa a abba  76      Figure 3.5 Endophytic population size of each of the six bacterial strains inside (a) needle, (b) stem, and (c) root tissues of lodgepole pine seedlings evaluated 4, 8 and 12 months after sowing (n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted, and data were log-transformed. Strains that did not colonize a tissue type have not been included in the figure.  012345Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueNeedle(a)0123456Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueStem(b)4567Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueRootHS-S1r HS-S2r HS-S3rLS-S1r LS-S2r LS-R1r(c)  77    Figure 3.6 Mean values of (a) length and (b) biomass of lodgepole pine seedlings subjected to six bacterial treatments and a non-inoculated control harvested 4, 8 and 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05). 0510152025303540HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling length (cm)(a)bb b bbababbb b bbabbbb bba020406080100120140160180HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling biomass (mg)(b)cbbbcab acbcababdaabcbcbbc bac  78    Figure 3.7 Bacteria-inoculated and non-inoculated control seedlings of lodgepole pine harvested (a) 4 months, (b) 8 months, and (c) 12 months after sowing showing clear differences in length and biomass. HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 1(a)HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 2(b)HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlHarvest 3(c)  79  Figure 3.8 Nitrogen (N) concentration (mg N per g tissue) in the foliage of lodgepole pine seedlings subjected to six bacterial treatments and a non-inoculated control measured 12 months after sowing. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05).  024681012HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlFoliar N (mg/g tissue) aba abccaa  80 Chapter 4 - In vitro and in vivo analyses of plant-growth-promoting potential of bacteria naturally associated with spruce trees growing on nutrient-poor soils 4.1. Introduction Each plant has a complex micro-ecosystem that harbours a range of microbes both in its internal tissues and on its external surfaces (Compant et al. 2019). The interaction of these microbes with their host plant can be harmful, beneficial, or neutral (Glick 2012). Bacteria that interact positively and provide benefits to the plant are known as plant growth promoting bacteria (PGPB). The most widely studied PGPB are free-living, associative and symbiotic bacteria living in the plant rhizosphere, however, recent literature also stresses the importance of endophytic PGPB (bacteria living in the internal tissues of plants) in sustaining plant growth (Puri et al. 2017b). This could be in part because endophytic bacteria might be better protected from abiotic stresses such as variations in temperature, pH, nutrient and water availability as well as biotic stresses such as microbial competition, as compared to rhizospheric bacteria (Chanway et al. 2014). Nevertheless, the rhizospheric and endophytic PGPB comprise the most critical components of the plant microbiome since they can promote plant growth through one or more of the following mechanisms: improved nutrient acquisition, phytohormone modulation, and phytopathogen suppression (Bashan and Holguin 1998). Nutrient acquisition mainly includes biological nitrogen fixation, siderophore production to accumulate metal micronutrients, and phosphate solubilization, whereas phytohormone modulation largely involves indole-3-acetic   81 acid (IAA) production and modulation of ethylene levels via 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase enzyme (Puri et al. 2016a; Kandel et al. 2017a). Both nutrient acquisition and phytohormone modulation are regarded as direct mechanisms of plant growth promotion. An indirect albeit crucial mechanism is the suppression of plant pathogens, which include the production of lytic enzymes such as cellulase, protease, chitinase and b-1,3-glucanase to degrade cell walls of pathogens, production of toxic ammonia gas, and production of siderophore to sequester metallic micro-nutrients thereby rendering them unavailable to pathogenic microbes (Chaiharn and Lumyong 2009).  The reports of PGPB isolation from agroecosystems around the world have been on the rise since symbiotic nitrogen fixation in leguminous plants was first documented (Hellriegel and Wilfarth 1888). In addition, their commercial application as potent inoculants to enhance crop growth has already been established on the farm scale (reviewed by Padda et al. 2017b). In contrast, our knowledge about PGPB in forest ecosystems, particularly boreal and temperate forests, is still elementary. Coniferous tree species, especially those belonging to the Pinaceae family constitute the largest proportion of trees found in the boreal and temperate forests of the Northern hemisphere (Farjon 1998). Considering the long life-span of these trees, PGPB have an even more important role in preparing these trees for extreme abiotic and biotic conditions prevailing in the boreal and temperate forests including cold climate, weakly-developed and nutrient-poor soils, slow mineralization rates of organic matter, and invasive pathogen/pest attack (Pirttilä and Frank 2018). Although limited, the reports of growth enhancement of Pinaceae trees including Scots pine (Pinus sylvestris), Douglas-fir (Pseudotsuga menziesii), lodgepole pine (Pinus contorta var. latifolia), hybrid white spruce (Picea glauca x engelmannii)   82 and Engelmann spruce (Picea engelmannii) by PGPB indicate that their role in natural forests may be crucial and their future potential as inoculants is promising (reviewed by Puri et al. 2017a, 2018b). An endophytic PGPB, Methylobacterium extorquens DSM 13060, isolated from natural forest stands of Scots Pine in northern Finland was able to significantly enhance the shoot and root biomass of Scots pine seedlings by colonizing their internal tissues along with forming beneficial associations with ectomycorrhizal fungi (Suillus variegatus and Pisolithus tinctorius) to promote tree growth (Pohjanen et al. 2014). A consortium developed from PGPB isolated from poplar (Populus trichocarpa) and willow (Salix sitchensis) trees growing on nutrient-poor gravel substrate alongside riverbanks was found to increase the growth of 15-month old Douglas-fir seedlings by accumulating 48% more biomass than the control in a greenhouse study (Khan et al. 2015). Each bacterium used in this consortium was confirmed positive for their ability to solubilize phosphate and produce siderophores to acquire iron in the in vitro analysis (Khan et al. 2015). In another study, root-associated PGPB of hybrid white spruce showed significant potential to enhance the growth rate of seedlings in natural field conditions when inoculated into hybrid white spruce and lodgepole pine (Shishido and Chanway 2000). Similarly, nitrogen-fixing endophytes of lodgepole pine and Engelmann spruce were reported to promote the growth of their host tree by fixing significant amounts of nitrogen from the atmosphere (Anand et al. 2013; Moyes et al. 2016).  The West Chilcotin region, located in the west central-interior of British Columbia (BC) province in Canada, is regarded as one of the most nutrient-poor regions in the province. The cold climate and extremely low annual precipitation in this region have resulted in poorly-developed soils with thin or no surface organic layer (Steen and Demarchi 1991). Frequent   83 disturbances including forest fire, logging activity and insect invasion (e.g., mountain pine beetle attack) have further eroded nutrients from soils in this region (Steen and Coupé 1997). Soil analyses from different areas in this region revealed that they have a limited supply of macro- and micro-nutrients including N, K, S, Ca, Mg, Mn, Cu and Ni as well as low cation exchange capacity and organic matter levels (Puri et al. 2018a; Chapter 2). Despite such highly disturbed and nutrient-poor edaphic conditions, hybrid white spruce trees can be seen thriving in this region, which intrigued us to look for the potential role of endophytic PGPB in supporting the continuous growth of spruce trees in this region. Fifty-five potential endophytic PGPB were isolated from the internal tissues of spruce trees and tested for their nitrogen-fixing ability since nitrogen was one of the most limiting soil nutrients in this region (Puri et al. 2018a; Chapter 2). Of these, 18 bacteria showed positive nitrogenase enzyme activity in the acetylene reduction assay indicating their potential to fix atmospheric nitrogen (Puri et al. 2018a; Chapter 2). Six bacteria that showed the highest nitrogenase enzyme activity were selected for further in planta analyses and I found that these bacteria were able to fix up to 56% of nitrogen from the atmosphere in one-year-old spruce seedlings (using 15N isotope dilution assay) and increase the tissue nitrogen content of needles by up to 70% (Puri et al. 2020a; Chapter 3). Furthermore, molecular analysis revealed the presence of nifH genes required to fix atmospheric nitrogen in these bacteria (Puri et al. 2020a; Chapter 3). Based on the results of the acetylene reduction assay, nif gene analysis and plant 15N isotope dilution assay, it was established that these six bacteria had significant nitrogen-fixing ability. Interestingly, several reports suggest that nitrogen-fixing bacteria have the ability to enhance plant growth through a variety of other PGP mechanisms as well (Okon and Labandera-Gonzalez 1994; Piccoli et al. 2011; Padda et al. 2017a;   84 Rho et al. 2018). This led us to hypothesize that these nitrogen-fixing endophytes might be sustaining the growth of spruce trees at the highly nutrient-poor West Chilcotin region through multiple PGP mechanisms. To evaluate this hypothesis, we first analyzed various PGP mechanisms of the six endophytic bacteria using in vitro qualitative and quantitative assays and then confirmed the PGP ability of these bacteria in vivo using long-term greenhouse growth trials (18 months) by inoculating them into their original host (hybrid white spruce) and a potentially foreign host (lodgepole pine). 4.2. Materials and Methods 4.2.1. Bacterial strains The six bacterial strains were originally isolated from internal tissues of young spruce trees (< 5 years old) growing in the West Chilcotin region of BC (52°00ʹ23.8ʺ N, 125°00ʹ17.4ʺ W, 966m a.s.l.; 52°00ʹ19.5ʺ N, 125°00ʹ17.8ʺ W, 993m a.s.l.) (Puri et al. 2018a; Chapter 2). Antibiotic-resistant derivatives of these strains were developed so as to enumerate the endophytic and rhizospheric colonies formed by these strains in hybrid white spruce and lodgepole pine seedlings in the greenhouse experiment. The antibiotic-resistant derivative of each strain was raised by streaking several times on combined carbon medium (CCM) (Rennie 1981; Appendix B) agar amended with 200 mg/L rifamycin (antibiotic compound) (Bal and Chanway 2012a). The antibiotic-resistant strains were stored in CCM broth amended with 200 mg/L rifamycin and 20% (v/v) glycerol at –80°C.    85 4.2.2. Evaluating plant-growth-promotion mechanisms 4.2.2.1. Inorganic and organic phosphate solubilization Each of the six bacterial strains was evaluated for their ability to solubilize a common form of inorganic phosphorus, i.e. tri-Ca phosphate. For qualitative evaluation, each strain was spot-inoculated onto triplicate Petri dishes containing Pikovskaya’s (PVK) agar medium (Pikovskaya 1948) containing 0.5% tri-Ca phosphate as the inorganic phosphate source. Plates were incubated at 30 °C for 14 days and inorganic phosphate solubilization was characterized by a clear halo around the bacterial colony. The ability of strains to solubilize tri-Ca phosphate on plates was estimated by using the solubilization index (Khan et al. 2015). Solubilization index (SI) = (halo + colony diameter) / colony diameter. For quantitative evaluation, each bacterial strain was grown in the PVK liquid medium (Pikovskaya 1948). For each strain, triplicate Erlenmeyer flasks containing the PVK medium were inoculated with 1 mL of the bacterial suspension (106 colony forming units (cfu)/mL). Autoclaved non-inoculated medium served as the control. The flasks were incubated at 30 °C for 72 h in a shaking incubator at 180 rpm. Culture supernatant was then harvested by centrifugation (8000x g; 10 min). Soluble phosphate in the culture supernatant was determined using the method outlined by Fiske and Subbarow (1925). The culture supernatant (1 mL) was mixed with 10% (w/v) trichloroacetic acid (500 µL) in a test tube to which 4 mL of the colour reagent (1:1:1:2 ratio of 3M H2SO4 | 2.5% (w/v) ammonium molybdate | 10% (w/v) ascorbic acid | distilled water) was added. The test tubes were incubated at room temperature for 15 min and the absorbance of the developed blue colour was measured at 820 nm using a spectrophotometer. The standard curve of KH2PO4 was used to determine the amount of soluble   86 phosphates produced by each bacterium per mL of the PVK medium (µg/mL) (Chaiharn and Lumyong 2009).  Each bacterial strain was also evaluated for its ability to hydrolyze the organic form of phosphorus (phytate). For qualitative assessment, each strain was spot-inoculated onto triplicate plates containing phytase screening medium (PSM) agar containing sodium phytate as the organic phosphorus source (Kerovuo et al. 1998). Plates were incubated at 30 °C for 14 days and phytate hydrolyzation was characterized by a clear halo around the bacterial colony. Similar to Ca-phosphate solubilization, phytate hydrolyzation by the bacterial strains on plates was also expressed as SI. For quantitative assessment, triplicate Erlenmeyer flasks containing PSM were inoculated with 1 mL of the bacterial suspension (106 cfu/mL) for each strain. Following incubation for 72 h at 30 °C in a shaking incubator at 180 rpm, culture supernatant was harvested by centrifugation (8000x g; 15 min). Autoclaved non-inoculated PSM served as the control. The culture supernatant (150 µL) was mixed with 600 µL of a solution containing 0.1M tris-HCl, 2mM sodium phytate and 2mM CaCl2, and incubated at 37 °C for 30 min. After that, 5% (w/v) trichloroacetic acid (750 µL) was added to stop the reaction and subsequently 750 µL of the colour reagent (4:1 ratio of 1.5% (w/v) ammonium molybdate in 5.5% (v/v) H2SO4 | 2.7% (w/v) ferrous sulfate solution) was added. The absorbance was measured at 700 nm after 5 min of incubation. The amount of inorganic phosphorus released by each bacterium was detected using the standard curve of KH2PO4. One unit (U) of phytase was defined as the amount of enzyme required to liberate 1 nmol of inorganic phosphorus per minute under the given assay conditions and was expressed per mL of the PSM culture (Yanke et al. 1998).   87 4.2.2.2. IAA production The production of an important plant-growth hormone, IAA, was evaluated using the method outlined by Bric et al. (1991). Briefly, for each strain, Luria Bertani broth amended with 5 mM L-tryptophan was inoculated with 1 mL of the bacterial suspension (106 cfu/mL) and incubated at 28 °C for 72 h in a shaking incubator at 150 rpm. Triplicate samples per bacterial strain were evaluated. Culture supernatant (1 mL) was extracted by centrifugation (8000x g; 15 min), to which 100 µL of 10mM orthophosphoric acid was added. Subsequently, 2 mL of the Salkowski reagent (1:30:50 ratio of 0.5M FeCl3 | 95% (w/w) sulfuric acid | distilled water) was added followed by incubation for 15 min at room temperature (Glickmann and Dessaux 1995). The absorbance of the pink colour that developed was measured at 530 nm. The standard curve developed using pure IAA was used to determine the amount of IAA produced by each bacterium per mL of the growth medium (µg/mL) (Gordon and Weber 1951). 4.2.2.3. ACC deaminase activity and gnotobiotic root elongation assay The ACC deaminase activity of each bacterial strain was evaluated using the methodology outlined by Penrose and Glick (2003). Briefly, each strain was first grown in a rich medium (Tryptic soy broth) at 30 °C until it reached the stationary phase. Bacterial cells were harvested by centrifugation (8000x g) and suspended in a minimal medium (DF salts minimal medium) amended with 3mM ACC as the sole source of nitrogen. Bacterial cells were then grown at 30 °C for 24 h at 200 rpm to induce the ACC deaminase activity. The bacterial cells were harvested by centrifugation (8000x g), washed and suspended in either 0.1M Tris-HCl to quantify the ACC deaminase activity or 0.03M MgSO4 to inoculate the seeds in a gnotobiotic root elongation assay.   88 The ACC deaminase activity was quantified as the amount of a-ketobutyrate produced per mg protein per hour. Bacterial cells suspended in 0.1M Tris-HCl were mixed with toluene and a portion of the toluene-treated cells was mixed with 0.5M ACC followed by incubation at 30 °C for 15 min. After the addition of 0.56M HCl, the mixture was centrifuged (16000x g) to collect the supernatant, which was again mixed with 0.56M HCl followed by the addition of the 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenylhydrazine in 2M HCl) and incubation at 30 °C for 30 min. The absorbance of the mixture was measured at 540 nm after the addition of 2M NaOH. The standard curve developed using pure a-ketobutyrate was used to determine the ACC deaminase activity of each strain. Triplicate samples were used to assess the ACC deaminase activity of each bacterial strain. A gnotobiotic root elongation assay was used to assess the in situ ACC deaminase activity of each bacterial strain. For this assay, the root growth of ethylene-sensitive plant species recommended by Penrose and Glick (2003), namely canola (Brassica napus) and tomato (Solanum lycopersicum) was evaluated. The ACC-induced bacterial cells of each strain suspended in 0.03M MgSO4 (OD600 = 0.15) were used. Canola seeds (var. Rugby Roundup ready) were obtained from the SeCan Association’s Alberta branch (Lamont, Alberta, Canada) and tomato seeds (var. Celebrity) were obtained from the West Coast Seed Company (Delta, BC, Canada). Seeds were surface sterilized by immersion in 30% H2O2 for 90 s, followed by three 30-s rinses in sterile distilled water. Canola and tomato seeds were incubated in Petri dishes for 1 h with the appropriate treatments: sterile 0.03M MgSO4 (control) or bacterial suspensions of each of the six strains in sterile 0.03M MgSO4. After incubation, 7 seeds of each plant species were aseptically placed in sterile CYG™ seed germination pouches (Mega International, Newport, Minnesota,   89 USA) designated for each of the 7 treatments, containing 15 mL sterile distilled water. The pouches were incubated in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, Manitoba, Canada) maintained at 20 °C with a day/night cycle beginning with 12 h of dark followed by 12 h of light and light intensity of 18 µmol/m2/s.  The primary root lengths were measured on the fifth day of growth. 4.2.2.4. Cellulase activity  The cellulase enzyme activity of each bacterial strain was evaluated qualitatively by spot-inoculation on casein–yeast extract (CYE) agar amended with 1% sodium carboxymethylcellulose, followed by incubation at 30 °C for 48 h. After the incubation period, plates were flooded with Congo red solution (0.5% w/v) for 30 min, drained and rinsed with 1 mol/L NaCl (Yang et al. 2017). The clear zone around the bacterial colony indicated cellulose degradation (i.e. positive cellulase activity). The width of clearance zone was calculated as = (halo + colony diameter) – (colony diameter). Triplicate plates were used to characterize the cellulase activity for each strain. For quantitative evaluation of cellulase activity, triplicate Erlenmeyer flasks containing CYE liquid medium amended with 1% sodium carboxymethylcellulose were inoculated with 1 mL of the bacterial suspension (106 cfu/mL) of each bacterial strain. The flasks were incubated at 30 °C for 5 days in a shaking incubator at 150 rpm. The culture supernatant was extracted by centrifugation (8000x g; 15 min). The supernatant (500 µL) was mixed with 1M citrate buffer (500 µL) and 1% carboxymethylcellulose (500 µL) and incubated at 37 °C for 30 min. The reaction was stopped by adding 2 mL of dinitrosalicylic acid and heating the reaction mixtures for 5 min in a boiling water bath. The absorbance was measured at 500 nm (Sahoo et al. 1999). The cellulase activity was   90 determined by measuring the release of reducing sugars from carboxymethylcellulose using glucose as the standard. One unit (U) of cellulase activity was defined as the amount of enzyme resulting in the release of 1 µmol of glucose from carboxymethylcellulose per minute (Chaiharn and Lumyong 2009). 4.2.2.5. Protease activity For qualitative evaluation of the protease enzyme activity, each bacterial strain was spot-inoculated on triplicate CYE agar plates amended with 7% skimmed milk powder, followed by incubation for 7 days at 30 °C. The clear zone surrounding the bacterial colony indicated positive protease activity and the width of the clearance zone was calculated (Padda et al. 2017a). Protease activity was assessed quantitatively using CYE liquid medium amended with 7% skimmed milk powder inoculated with 1 mL of the bacterial suspension (106 cfu/mL) of each bacterial strain. Triplicate samples were used for each bacterial strain. After incubation at 30 °C for 5 days in a shaking incubator at 150 rpm, the culture supernatant was extracted by centrifugation (8000x g; 15 min). The supernatant (500 µL) was mixed with 0.2 M phosphate buffer (500 µL) and 1% azocasein (500 µL) and incubated at 37 °C for 30 min. Subsequently, 2 mL of 10% (w/v) trichloroacetic acid was added followed by incubation at room temperature for 5 min. The absorbance was measured at 440 nm after the addition of 1 mL of 1M NaOH. The activity of protease was determined by measuring the release of reducing sugars from azocasein using tyrosine as the standard. One unit of protease enzyme activity was defined as the amount of the enzyme resulting in the release of 1 µmol of tyrosine equivalent from azocasein per minute (Chaiharn and Lumyong 2009).   91 4.2.2.6. Chitinase activity Chitinase activity of the bacterial strain was qualitatively tested using a chitin agar plate containing 1.62 g nutrient broth (Sigma-Aldrich, USA), 0.5 g NaCl, 6 g M 9 salts (Difco, USA), 8 g colloidal chitin and 15 g agar per litre (Sahoo et al. 1999). Each bacterial strain was spot-inoculated on triplicate chitin agar plates and incubated for 7 days at 30 °C. The formation of clear halos surrounding the bacterial colony indicated positive chitinase activity and the width of the clearance zone was calculated. For quantitative analysis, triplicate flasks for each bacterial strain containing the chitin medium mentioned above (except agar) were inoculated with 1 mL of the bacterial suspension (106 cfu/mL). Following the incubation period of 5 days at 30 °C in a shaking incubator (150 rpm), the culture supernatant was extracted by centrifugation (8000x g; 15 min). The supernatant (500 µL) was mixed with 1 M phosphate buffer (500 µL) and colloidal chitin (500 µL containing 10 mg chitin). After 30 min incubation at 37 °C, the mixture was centrifuged (8000x g; 3 min). The supernatant (1 mL) was pipetted into a test tube and 2 mL of dinitrosalicylic acid was added, followed by heating for 5 min in a boiling water bath. The absorbance of the resulting solution was measured at 575 nm (Chaiharn and Lumyong 2009). The chitinase activity was determined by measuring the release of reducing sugars from chitin using glucose as the standard. One unit (U) of chitinase activity was defined as the amount of enzyme resulting in the release of 1 µmol of glucose from colloidal chitin per minute. 4.2.2.7. b-1,3-glucanase activity Qualitative estimation of b-1,3-glucanase activity of the six bacterial strains was performed using the methodology outlined by Renwick et al. (1991). Each bacterial strain was spot-inoculated on   92 plates containing NH4NO3 (0.78 g/L),  K2HPO4 (0.80g/L), KH2PO4 (0.20 g/L), MgSO4.7H2O (0.20 g/L), CaCl2 (0.06 g/L), NaCl (0.10 g/L), Na2MoO4.2H2O (0.002 g/L), ZnSO4.7H2O (0.00024 g/L), CuSO4.5H2O (0.00004 g/L), CoSO4.7H2O (0.010 g/L), MnSO4.4H2O (0.003 g/L), Na2FeEDTA (0.028 g/L), H3BO3 (0.005 g/L), Biotin (0.000005 g/L), p-aminobenzoic acid (0.00001g/L), agar (15 g/L) and b-1,3-glucan as the carbon source (laminarin; 5 g/L). After incubation at 30 °C for 3 days, plates were stained with Congo Red (0.6 g/L) and left at room temperature for 90 min. Plates were observed for hydrolysis of glucan (glucanase activity) by a yellow/orange zone around the bacterial growth. Quantitative estimation of b-1,3-glucanase activity was conducted using the above mentioned medium without agar and inoculated with 1 mL of the bacterial suspension (106 cfu/mL) of each bacterial strain. Triplicate samples were used for each bacterial strain. Samples were incubated at 30 °C for 5 days in a shaking incubator at 150 rpm. The culture supernatant was extracted by centrifugation (8000x g; 15 min). The supernatant (500 µL) was mixed with 1 M citrate buffer (500 µL; pH 5.0) and 4% laminarin (500 µL) and incubated at 37 °C for 30 min. The reaction was stopped by adding 2 mL of dinitrosalicylic acid and heating for 5 min in a boiling water bath. The absorbance of the resulting solution was measured at 500 nm (Chaiharn and Lumyong 2009). The b-1,3-glucanase activity was determined by measuring the release of reducing sugars from laminarin using glucose as the standard. One unit (U) of b-1,3-glucanase activity was defined as the amount of enzyme resulting in the release of 1 µmol of glucose from laminarin per minute.     93 4.2.2.8. Siderophore production Siderophore production was evaluated using the blue agar chrome azurol S (CAS) assay (Louden et al. 2011). Each bacterial strain was spot-inoculated onto triplicate CAS agar plates. A colour change in the CAS agar from blue to orange/deep yellow indicated the production of siderophore by bacteria. The area of colour conversion around the bacterial colony was measured after 7 days of incubation at 30 °C (Kandel et al. 2017a). 4.2.2.9. Catalase activity The catalase production by each bacterial strain was evaluated by mixing a loopful of fresh bacterial culture with 50 µL of 3% (v/v) hydrogen peroxide on a sterile glass slide and incubating at room temperature for 1 min (Padda et al. 2017a). The evolution of oxygen (development of gas bubbles) was recorded as a positive catalase reaction. Triplicate samples per bacterial strains were used to confirm catalase activity. 4.2.2.10. Ammonia production The production of ammonia by each bacterial strain was tested by inoculating 100 µL of the bacterial suspension (106 cfu/mL) into triplicate test tubes containing peptone water (10 mL). The tubes were incubated for 72 h at 30 °C before adding 500 µL of Nessler’s reagent (Dey et al. 2004). The development of a brown-yellow colour after the addition of Nessler’s reagent indicated ammonia production by the bacterial strain.    94 4.2.3. Long-term greenhouse experiment The six bacterial strains were evaluated in two different 18-month long greenhouse growth trials: one with hybrid white spruce and another with lodgepole pine. In each trial, 7 treatments were evaluated (6 bacteria-inoculated and 1 non-inoculated control).  A frozen culture of each bacterial strain was thawed and streaked on CCM agar amended with 200 mg/L of rifamycin and incubated at 30 °C for 48 h. A loopful of bacterial growth was inoculated into CCM broth amended with 200 mg/L of rifamycin and stirred at 150 rpm for 48 h at 30 °C in a shaking incubator. Bacterial cells were harvested by centrifugation (3000x g; 30 min), washed twice in sterile phosphate buffered saline (PBS) (pH 7.4) (Appendix A) and resuspended in the same buffer to a density of ca. 106 cfu/mL (Puri et al. 2016b).  Hybrid white spruce and lodgepole pine seeds were procured from the Tree Seed Centre of the BC Ministry of Forests, Surrey, BC, Canada and originated from forest stands in the West Chilcotin region of BC (hybrid white spruce – 51° 57’ N lat., 124° 59’ long., elevation 1350 m, SBPSxc zone; lodgepole pine – 51° 54’ N lat., 124° 51’ long., elevation 1320 m, SBPSxc zone). Seeds were immersed in 30% (v/v) H2O2 for 90 s for surface sterilization and then rinsed three times for 30 s in sterile distilled water. Ten randomly selected surface-sterilized seeds were imprinted on tryptic soy agar (TSA) plates and incubated at 30 °C for 48 h to check for surface-contamination (i.e. to determine the effectiveness of surface sterilization). The surface-sterilized seeds were placed in sterile cheesecloth bags containing moist autoclaved silica sand and stored at 4 °C for 28 days for stratification. Ten seeds were selected randomly from cheesecloth bags after the stratification period, crushed and imprinted on CCM plates amended with 200 mg/L of   95 rifamycin and were examined after 48 h to confirm the absence of internal seed contamination by any of the six bacterial strains before inoculation. Hybrid white spruce and lodgepole pine seedlings were grown in Ray Leach Cone-tainers (height: 21 cm; diameter: 3.8 cm). Each Cone-tainer was filled to two-thirds capacity with sterile soil growth medium (69% w/w silica sand; 29% w/w Turface; 2% w/w CaCO3). Each Cone-tainer was fertilized with 20 mL of a nutrient solution containing Ca(NO3)2 (Appendix C). Three surface-sterilized seeds were sown aseptically in each Cone-tainer. The bacterial suspension (5 mL) of each strain was pipetted directly over the seeds in each Cone-tainer designated for that strain. This process was repeated for each of the six bacterial strains in both trials. Non-inoculated control seeds received 5 mL of sterile PBS without any bacteria in each trial. The Cone-tainers were placed in trays (98 Cone-tainers per tray) in the University of British Columbia Plant Care Services’ greenhouse with photosynthetically active radiation at the canopy level of 300 µmol/m2/s for a 16 h photoperiod (6 am to 10 pm). Seedlings were watered as required with sterile distilled water. Two weeks after sowing, seedlings were thinned to the largest single germinant per Cone-tainer. Seedlings received nutrient solution (20 mL) without Ca(NO3)2 once per month during the 18-month long growth period. Tray positions were randomized weekly to reduce the positional effects. To evaluate if the bacterial strains were able to colonize the rhizosphere of hybrid white spruce and lodgepole pine, five seedlings from each treatment were harvested at the end of the growth trials (18 months after sowing). Seedlings were removed from Cone-tainers and loosely adhering soil particles were removed from roots with gentle shaking. Roots were then separated   96 from shoots, placed in sterile Falcon tubes (50 mL; BD Biosciences, CA, USA) containing 10 mL of sterile PBS and vortexed at 1000 rpm for 1 min. After performing serial dilutions, a 0.1 mL aliquot of each dilution was plated on CCM agar amended with rifamycin (200 mg/L). Plates were incubated at 30 °C for 7 days, after which the number of bacterial colonies was counted (Padda et al., 2016a). Roots were oven-dried at 65 °C for 48 h before weighing. Rhizospheric bacterial populations were then calculated as cfu per gram of dry root tissue. Endophytic colonization by bacteria in needle, stem and root tissues of spruce and pine seedlings was evaluated at the end of the growth trials by randomly selecting five seedlings from each treatment. Seedlings were surface-sterilized with bleach (1.3 % NaOCl) for 5 min and washed thrice with sterile distilled water. Surface sterilization of seedlings was confirmed by imprinting seedlings on TSA plates, which were incubated at 30 °C and checked after 24 h for contamination. A sample from each of the root, stem, and needle tissues was then triturated separately in 1 mL of sterile PBS using a sterile mortar and pestle. Serial dilutions of the triturated tissue suspensions were performed, and 0.1 mL aliquot of each dilution was plated onto CCM agar amended with rifamycin (200 mg/L). Ideally, rifamycin should inhibit the growth of all bacteria except the six bacteria used in this study since they had acquired antibiotic resistance. Plates were incubated at 30 °C for 7 days, after which the number of bacterial colonies was counted (Padda et al. 2016b). Endophytic bacterial populations were then calculated as cfu per gram of fresh tissue. Hybrid white spruce and lodgepole pine seedling growth were evaluated at the end of the growth trials by randomly selecting 10 seedlings of each tree species from each treatment. Seedlings were removed from Cone-tainers and their total lengths were measured. Each seedling was then oven-dried at 65 °C for 48 h to determine biomass (dry weight).   97 4.2.4. Statistical analyses Statistical analysis of each greenhouse growth trial (one involving hybrid white spruce and the other involving lodgepole pine) was performed separately since each tree species has a different growth pattern, not comparable to one another (von Wirén et al. 1997). To evaluate the treatment effects of each bacterial strain on the growth of seedlings, a completely randomized experimental design was used. Analysis of variance (ANOVA) was performed (F-test and Student’s t-test) to determine significant differences between treatment means for seedling length and seedling biomass. Similarly, ANOVA was also performed to determine significant differences between treatment means of each bacterial strain for the quantitative evaluations of phosphate solubilization, phytate hydrolyzation, ACC deaminase activity, IAA production, cellulase activity, protease activity, chitinase activity and b-1,3-glucanase activity as well as siderophore production and root elongation in the gnotobiotic assay. Three replicates per treatment were evaluated for all quantitative evaluations and siderophore production, and 7 replicates per treatment were evaluated for the gnotobiotic root elongation assay. The statistical package, SAS University Edition (SAS Institute Inc., Cary, NC, USA), was used to perform all statistical analyses. The confidence level, a, was set to 0.05. 4.3. Results 4.3.1. Plant growth promoting mechanisms Inorganic and organic phosphate solubilization was confirmed for four of the six bacterial strains using qualitative plate tests (Table 4.1). The solubilization of inorganic tri-Ca phosphate and   98 hydrolyzation of organic phytate on agar plates was observed for strains HS-S3r, LS-S1r, LS-S2r and LS-R1r, with the SI ranging from 1.1 to 3.0. These results were supported by the quantitative assessment of the amount of tri-Ca solubilized and phytate hydrolyzed by these strains into soluble phosphates (Table 4.1). Strain LS-S2r solubilized the highest amounts of tri-Ca phosphate, significantly greater (up to 75%) than all other bacteria. This strain also hydrolyzed significantly higher amounts of organic phytate into soluble phosphates than the other bacterial strains (up to 44%). Notably, the SI of phosphate solubilization and phytate hydrolyzation observed for this strain was also the highest (2.7 and 3.0, respectively) among all bacterial strains (Table 4.1).  All six strains were confirmed to produce a crucial plant growth hormone (IAA) in vitro, with amounts ranging from 25 to 35 µg per mL of the growth medium (Table 4.1). Particularly, strains HS-S1r, LS-S1r and LS-S2r produced significantly higher amounts of IAA compared to the other bacterial strains. Similar to IAA production, the activity of the ACC deaminase enzyme, which inhibits the production of excess ethylene levels in plants, was also confirmed positive for all six strains. The amount of a-ketobutyrate produced by the strains, due to their ACC deaminase enzyme activity, ranged between 29 and 121 nmol per mg protein per hour (Table 4.1). Strain LS-S2r produced the highest amounts of a-ketobutyrate among all bacterial strains, followed by strains LS-S1r and HS-S1r. When each of the six strains was inoculated into canola and tomato plants in the gnotobiotic assay for ACC deaminase activity, significant root elongation was observed for inoculated plants as compared to the non-inoculated control plants (Figures 4.1 and 4.2). Notably, strain LS-S2r enhanced root length of canola by 291% and tomato by 479%, which was significantly higher than any other bacterial treatment. Strains LS-S1r and HS-S1r also   99 promoted canola root length by >125% and tomato root length by >300%, significantly higher than other bacterial strains except for LS-S2r (Figures 4.1 and 4.2). The qualitative assessment of cell wall degrading enzymes in plate assays revealed that all six strains had positive cellulase enzyme activity, with strains HS-S1r and LS-S1r showing the largest clearance zone on plates (15-25 mm) (Table 4.2). Protease enzyme activity was only found in three strains, with strains LS-S1r and LS-S2r having a larger zone of clearance than strains HS-S3r on plates (Table 4.2). Chitinase enzyme activity was observed in four strains (HS-S1r, HS-S3r, LS-S1r and LS-S2r). Strain LS-S2r had the largest zone of clearance (15-25 mm) on plates compared to other strains (Table 4.2). Three bacterial strains (HS-S1r, LS-S1r, LS-S2r) showed positive b-1,3-glucanase activity, with strain LS-S2r showing significantly larger zone of yellow-orange colour on plates (Table 4.2). These observations were also confirmed in the quantitative assays of these four enzymes. The amount of cellulose dissolved by the bacterial strains, due to their cellulase enzyme activity, ranged between 0.18 and 0.67 U/mL, with strains HS-S1r, LS-S1r and LS-S2r producing significantly higher amounts than other bacterial strains (Table 4.2). The protease enzyme activity, measured as the amount of azocasein dissolved by the bacterial strains, was highest for strain LS-S2r (Table 4.2). The amount of colloidal chitin dissolved due to chitinase activity of the four bacterial strains ranged from 0.35 to 0.72, with strain LS-S2r showing significantly higher chitinase enzyme activity compared to other strains (Table 4.2). Similarly, b-1,3-glucanase activity of three bacterial strains ranged from 0.47 to 0.81, where strain LS-S2r hydrolyzed a significantly higher amount of b-1,3-glucan compared to the other bacterial strains (Table 4.2). The production of siderophore (as indicated by orange halo development on blue CAS agar plates) was confirmed for four bacterial strains, of which LS-S1r and LS-S2r had   100 significantly larger orange halo area compared to the other bacterial strains (Table 4.3). The catalase enzyme activity was confirmed for all six strains, with strain HS-S1r showing the greatest activity, followed by strains HS-S2r and LS-S2r (Table 4.3). All strains except HS-S2r tested positive for the production of ammonia in vitro (Table 4.3). Particularly, strains LS-S1r, LS-S2r and LS-R1r produced the highest amounts of ammonia as observed via the development of darker brown-yellow colour compared to the other strains. 4.3.2. Long-term greenhouse experiment Each of the six bacterial strains colonized the rhizosphere of both spruce and pine seedlings, 18 months after inoculation, with population sizes ranging from 104 to 107 cfu/g dry root (Figures 4.3 and 4.4). Strain LS-S2r was the highest rhizosphere colonizer in both tree species (107 cfu/g dry root) closely followed by strains HS-S1r and LS-S1r (106 cfu/g dry root). Endophytic colonization by each bacterial strain was confirmed for internal stem and root tissues of both spruce and pine seedlings. Population sizes observed in the internal root tissues were similar in magnitude to the external populations (rhizosphere), ranging between 104 and 108 cfu/g fresh tissue (Figures 4.3 and 4.4). Stem colonization by all bacterial strains (102 – 105 cfu/g fresh tissue) further indicated their endophytic capabilities (Figures 4.3 and 4.4). Similar to rhizosphere colonization, strains LS-S2r, HS-S1r and LS-S1r were also the highest colonizers of the internal stem and root tissues of pine and spruce seedlings. The colonization of needle tissues of spruce was observed for all strains except HS-S2r (Figure 4.3) and needle tissues of pine seedlings were colonized by all but two strains (HS-S2r and HS-S3r) (Figure 4.4). The endophytic population sizes in the needle tissues ranged between 102 and 105 cfu/g fresh tissue, with strains LS-S2r and HS-  101 S1r being the highest colonizers 18 months after inoculation. No bacterial colonies were observed in the rhizospheric or internal tissues of non-inoculated control spruce and pine seedlings.  Inoculation with each of the six bacterial strains had a significant positive effect on the length and biomass of spruce and pine seedlings after 18 months. The seedling length of all bacteria-inoculated spruce seedlings was significantly greater (>40%) than controls (Figure 4.5a). In addition, all bacteria-inoculated spruce seedlings had significantly higher seedling biomass than control seedlings (>120%), with LS-S2r-inoculated seedlings having the highest seedling biomass (~600%), which was significantly greater than all other bacterial treatments (Figure 4.5b). Spruce seedlings inoculated with strain LS-S1r also accumulated considerably greater seedling biomass (>280%). For pine, the length of seedlings inoculated with any of the six bacterial strains was significantly greater than controls (>40%) (Figure 4.6a). Similar to spruce, the seedling biomass of pine trees subjected to any of the six bacterial treatments was significantly higher than the controls (Figure 4.6b). Strain LS-S2r considerably enhanced the seedling biomass of pine seedlings (>300%), significantly greater than all other treatments. Similarly, strain LS-S1r also significantly enhanced the biomass of pine seedlings (>200%) than all bacterial strains except LS-S2r. All in all, strain LS-S2r performed the best in both greenhouse trials via enhancing the spruce seedling length by 66% and biomass by 607% as well as pine seedling length by 73% and biomass by 328% (Figures 4.5 and 4.6). 4.4. Discussion The first report of isolation of endophytic bacteria from hybrid white spruce trees growing under highly disturbed and nutrient-poor edaphic conditions of the West Chilcotin region of BC (Puri et   102 al. 2018a; Chapter 2) inspired this study, where we investigated the potential of these bacteria to sustain tree growth under such harsh conditions by employing various PGP mechanisms. Although a large number of culturable endophytic bacteria (55) were isolated from spruce trees in this region (Puri et al. 2018a; Chapter 2), it was important to focus the research on those bacteria that have the greatest potential to provide maximum benefits to the plant. Therefore, six bacteria that showed the highest nitrogen-fixing ability under in vitro and in planta conditions were chosen (Puri et al. 2018a, 2020a; Chapters 2 and 3) to evaluate what other PGP abilities these bacteria possess and if they can potentially act as multi-mechanism biofertilizers for trees. It should be noted that nitrogen-fixing endophytes of poplar, willow and lodgepole pine have been reported to possess a variety of PGP traits, which may have a synergistic effect on plant growth and development (Xin et al. 2009; Khan et al. 2015; Padda et al. 2017a; Rho et al. 2018). A variety of in vitro microbiological techniques were used to examine key phenotypic traits associated with plant growth promotion for the six endophytic bacteria. An important nutrient acquisition trait of PGPB is the solubilization of complex inorganic and organic phosphate compounds. It was observed that four bacterial strains (HS-S3r, LS-S1r, LS-S2r and LS-R1r) were able to solubilize a major form of insoluble inorganic phosphorus (tri-Ca phosphate) in plate assays (Table 4.1). Three strains solubilized double or more amount of tri-Ca phosphate relative to their colony size on plates as indicated by their SI. The results of quantitative analysis of the tri-Ca phosphate solubilized by these bacteria in the broth echoed the plate assay results since the same four strains produced soluble phosphates in the broth (Table 4.1). The amount of soluble phosphates produced by these strains is consistent with previous studies conducted with known phosphate solubilizing bacteria (Anandham et al. 2008; Chaiharn and Lumyong 2009;   103 Oliveira et al. 2009; Dutta and Thakur 2017). The main mechanism of phosphate solubilization by PGPB involves the production of organic acids such as gluconic, acetic, citric, fumaric, glycolic, lactic, malonic, oxalic, propionic and succinic acids (Ahmad and Shahab 2011). These organic acids either drop the pH so as to bring phosphorus into the soil solution or chelate the attached mineral ion (e.g., Ca) thereby liberating the complexed phosphate for plant uptake (Walia et al. 2017). Plants also obtain phosphates from the organic pool via phytate degradation, which is mainly carried out by microbes. Phytase enzyme, which hydrolyzes phytate compounds, was detected in the same four strains for which tri-Ca phosphate solubilization was detected (Table 4.1). It established the fact that these strains are comprehensive phosphate solubilizing bacteria that have the capability to convert both inorganic and organic forms of insoluble phosphorus into plant-available forms. As suggested in previous studies, the amount of phytate hydrolyzed by these strains may compensate for the inability of plants to acquire phosphorus directly from phytate (Yanke et al. 1998; Kumar et al. 2013; Richardson and Simpson 2011). Notably, Caballeronia sordidicola LS-S2r solubilized significantly higher amounts of inorganic and organic phosphates in both plate and enzyme assays compared to other strains (Table 4.1), which indicates that this bacterial strain could be highly effective in providing soluble phosphorus resources to plants for their growth and development. Another key method of nutrient acquisition employed by PGPB includes the production of siderophores, which are iron-chelating compounds produced by microbes to reduce iron deficiency in plants (Saha et al. 2016). In addition to providing the plant with chelated iron, siderophores produced by PGPB may serve a dual purpose in plant disease protection by – (i) eliciting induced systemic resistance in the host plant (van Loon et al. 2008); and (ii) scavenging   104 and sequestering iron from phytopathogens (which need iron to grow and establish) thereby outcompeting them (Schippers et al. 1987). Four strains produced siderophores as indicated by the presence of orange halo on CAS blue agar plates (Table 4.3). The area of orange halo observed for our bacterial strains was comparable to that reported for endophytic strains of poplar, willow, rice (Oryza sativa), corn (Zea mays) and potato (Solanum tuberosum) (Sessitch et al. 2004; Loaces et al. 2011; Khan et al. 2015; Kandel et al. 2017a; Padda et al. 2017a). In addition, genomic analyses have also indicated that various tree and crop endophytes possess siderophore-producing genes (reviewed by Frank 2018), further supporting our results. Along with nutrient acquisition, PGPB can also play a crucial role in affecting the physiology of a plant by altering their hormonal balance (Ping and Boland 2004). Auxin is a crucial phytohormone that regulates plant development and growth. PGPB can produce a naturally-occurring auxin, IAA, that has a major role in cell proliferation leading to root and stem elongation. In this study, it was observed that all six strains can synthesize IAA from tryptophan, which is a complex amino acid that is costly to produce for the bacteria but commonly found inside the plant and in plant exudates. PGPB present in both the rhizosphere and endophytic tissues, capable of converting plant-produced tryptophan to IAA, can significantly promote the growth of their host plant (Kamilova et al. 2006; Madmony et al. 2005). Therefore, seedling length and biomass enhancement observed for all of our bacterial strains (Figures 4.5 and 4.6) may have resulted from their ability to produce IAA. In particular, Pigmentiphaga litoralis HS-S1r, Pseudomonas prosekii LS-S1r and C. sordidicola LS-S2r produced significantly higher amounts of IAA than the other bacterial strains which could be related not only to their superior plant growth promoting ability (Figures 4.5 and 4.6) but also to their efficient colonization ability (Figures 4.3   105 and 4.4), since IAA production has been linked to the colonization strategy of a bacterium to form an associative or mutualistic relationship with the plant, whether inside or at the surface (Suzuki et al. 2003; Frank 2018). In addition to growth, IAA can also be associated with various developmental processes in plants such as apical dominance, tropic responses and reproduction, making it one of the most crucial plant growth hormones (Spaepen 2015).  Another critical growth hormone that alters plant physiology is ethylene. Overproduction of ethylene, which happens during abiotic and biotic stresses such as drought, salinity, nutrient-limitations, and phytopathogen attack, can be deleterious for the plant (Glick 2012). However, certain PGPB have the capability to cleave ACC (the precursor of plant-ethylene) by producing the ACC deaminase enzyme, thereby lowering plant-ethylene levels and promoting plant growth under stress conditions (Glick et al. 1998). Our results from the in vitro analysis and the in vivo greenhouse experiment were consistent with this theory since all strains produced ACC deaminase – as indicated by the production of a-ketobutyrate after cleaving ACC (Table 4.1) – and enhanced seedling growth of pine and spruce under nutrient-limited conditions in the 18-month long greenhouse trial (Figures 4.5 and 4.6). The ACC deaminase enzyme is also extremely important during seed germination. Although the overproduction of ethylene is required to break the seed dormancy, persistently high amounts of ethylene after germination can hinder root elongation (Esashi 1991; Jackson 1991). Coating the seeds with PGPB can lower plant-ethylene levels after germination and lead to greater root extension, which is what we observed in the gnotobiotic root elongation assay for our bacterial strains with canola and tomato (ethylene-sensitive plants) (Figures 4.1 and 4.2). In theory, this could give the PGPB-inoculated plants an initial advantage over the non-inoculated plants, which may be reflected in the consistently   106 better growth of inoculated plants throughout their lifecycle. Although, according to Penrose and Glick (2003), the amount of a-ketobutyrate produced in vitro cannot be directly related to the extent of root elongation observed in the gnotobiotic assay, for our strains this relation was uniform. Caballeronia sordidicola LS-S2r produced a significantly higher amount of a-ketobutyrate than all other strains (Table 4.1), which was consistent with the significantly greater primary root length observed for canola and tomato plants inoculated with this strain (Figures 4.1a and 4.2a). In addition, P. litoralis HS-S1r and P. prosekii LS-S1r also produced considerable amounts of a-ketobutyrate, which was similarly reflected for canola and tomato root lengths in the gnotobiotic assay (Table 4.1; Figures 4.1a and 4.2a). The six bacterial strains were able to produce at least one of the major lytic enzymes – cellulase, protease, chitinase and b-1,3-glucanase. Cellulase activity was observed for all strains in both plate and broth assays, with P. litoralis HS-S1r, P. prosekii LS-S1r and C. sordidicola LS-S2r showing significantly higher activity than the other strains (Table 4.2). However, protease activity was only observed for three strains (HS-S3r, LS-S1r and LS-S2r), with strain LS-S2r converting significantly higher amounts of azocasein to tyrosine using protease enzyme in the broth assay (Table 4.2). Both cellulase and protease activities could be linked to the ability of endophytic bacteria to actively colonize plant tissues since these enzymes can macerate plant cell wall polymers and metabolize organic compounds in the apoplast (Hurek et al. 1994). Such a mode of entry has been proposed for endophytic strains not only in agricultural crops including rice and sugarcane (Saccharum officinarum) (Reinhold-Hurek et al. 2006; Bertalan et al. 2009) but also in forest trees including lodgepole pine and poplar (Shishido et al. 1995; Taghavi et al. 2010; Kandel et al. 2017a; Yang et al. 2017). This may be true for our endophytic strains as well since they were   107 able to colonize the internal tissues of spruce and pine seedlings in biologically significant numbers (Figures 4.1 and 4.2). Four strains (HS-S1r, HS-S3r, LS-S1r and LS-S2r) showed positive chitinase activity and three strains (HS-S1r, LS-S1r and LS-S2r) showed positive b-1,3-glucanase activity in plate and broth assays, with a uniform correlation observed between both kinds of assay (Table 4.2). Caballeronia sordidicola LS-S2r showed significantly higher chitinase and b-1,3-glucanase enzyme activities compared to the other strains (Table 4.2). Bacterial chitinase and b-1,3-glucanase can play a crucial role in protecting the plant from fungal pathogens by lysing fungal cell walls and triggering plant defence mechanisms (Ryan et al. 2008), which have been reported in a variety of plant species such as Kentucky bluegrass (Poa pratensis), tea (Camellia sinensis), sugarcane, wheat (Triticum aestivum), cotton (Gossypium barbardense), cucumber (Cucumis sativus), bean (Phaseolus vulgaris), melon (Cucumis melo) and Scots pine (Renwick et al. 1991; Fridlender et al. 1993; Kobayashi et al. 2002; Pirttilä et al. 2002; Kruasuwan et al. 2017; Shan et al. 2017). Cellulase and protease production by PGPB have also been reported to control phytopathogen populations by degrading their cell walls (Elad and Kapat 1999; Kandel et al. 2017a; Padda et al. 2017a). It should be noted that the amount (units) of cellulase, protease, chitinase and b-1,3-glucanase enzymes produced by our bacterial strains in the quantitative analyses align with previous studies (Farah et al. 2006; Chaiharn and Lumyong 2009). However, the amount of enzyme produced in vitro under controlled conditions cannot be directly related to the in situ production of these enzymes; therefore, further studies need to be performed to evaluate their expression and activity under natural conditions with biotic stress factors.  Another mechanism by which PGPB can defend plants from pathogen attack is by using phenylalanine ammonia-lyase enzyme to convert L-phenylalanine to ammonia (Anandham et al.   108 2008; Ahmad et al. 2008; Latha et al. 2009). This conversion is the first step of the reaction that subsequently leads to the synthesis of a variety of polyphenols such as flavonoids, phenylpropanoids and lignins to protect against pathogens (Hahlbrock and Grisebach 1979; Babalola 2010). In addition, the ammonia gas produced by PGPB is a toxic compound for phytopathogens (Weise et al. 2013). Ammonia production was observed for five of our strains (Table 4.3), with three strains (LS-S1r, LS-S2r and LS-R1r) showing highest production of ammonia as indicated by their darker brown/yellow colour in vitro compared to the other strains, thereby potentially playing a greater role in plant defence (Chaiharn and Lumyong 2009). Catalase enzyme production was observed for all strains as indicated by the release of oxygen following the addition of hydrogen peroxide to the bacterial growth (Table 4.3). This enzyme can help the bacterium to protect itself from the overproduction of reactive oxygen species (ROS) under stress conditions, thereby promoting plant growth indirectly (Bumunang and Babalola 2014). Therefore, survival and multiplication of our bacterial strains under nutrient-stress conditions in the greenhouse experiment (Figures 4.3 and 4.4) could be linked to the ability of these bacteria to secrete the catalase enzyme. In addition, since these strains were originally isolated from spruce trees growing at highly disturbed and nutrient-poor sites (Chapter 2; Puri et al. 2018a), catalase activity may be an evolutionary approach for these bacteria to survive in such conditions. In addition, living in endophytic niches of the plant may also require the bacteria to protect themselves from plant ROS (released by plants as a defence compound), therefore catalase can also potentially help the bacteria to thrive in internal tissues (Padda et al. 2017a; Frank 2018). The long-term greenhouse trials established to quantify plant-growth-promotion by the six bacterial strains in vivo under nutrient-poor conditions revealed highly positive results. Since   109 nitrogen was the major limiting nutrient in soils of the West Chilcotin region (Puri et al. 2018a; Chapter 2), nitrogen fertilizer was provided only once to our seedlings at the onset of the growth trial. In addition, seedlings were subjected to other nutrient stresses such as phosphorus since the amount of phosphorus provided to the seedlings was lower than the available phosphorus in the soils of the West Chilcotin region (Chapter 2 – Puri et al. 2018a; Steen and Demarchi 1991; Steen and Coupé 1997) as well as other natural forest stands (Zavišić et al. 2016; Zavišić and Polle 2017; Zavišić et al. 2018). Each strain performed similarly well in both hybrid white spruce (the natural host) and lodgepole pine (a foreign host) (Figures 4.5 and 4.6). Inoculation with any of the six strains stimulated spruce and pine seedling length by > 40% and biomass by > 90% compared to the non-inoculated controls (Figures 4.5 and 4.6). Each strain was able to colonize the rhizosphere and root tissues as well as the stem tissues of pine and spruce (Figures 4.3 and 4.4). In addition, needle colonization was observed for all strains except HS-S2r (in both spruce and pine) and HS-S3r (in pine only), suggesting that either these bacterial strains prefer to thrive in certain plant-niches or plants deliberately confine them to specific tissues only (Frank et al. 2017). Overall, the population sizes observed in the rhizosphere as well as in the interior tissues of spruce and pine seedlings are consistent with previous studies in coniferous trees (Shishido et al. 1995; Anand and Chanway 2013b; Yang et al. 2016; Tang et al. 2017).  Notably, C. sordidicola LS-S2r, P. litorallis HS-S1r and P. prosekii LS-S1r extensively colonized pine and spruce seedlings in the greenhouse trials (Figures 4.3 and 4.4). The Proteobacteria phylum, in particular, the Burkholderiales order (containing Pigmentiphaga and Caballeronia genera) and the Pseudomonadales order (containing Pseudomonas genus) are well-known to closely associate with plants and potentially provide a variety of benefits to their host   110 (Estrada-De Los Santos et al. 2001; Bulgarelli et al. 2012; Padda et al. 2018, 2019). Studies have reported extensive endophytic colonization by bacteria belonging to these orders in tree species such as interior spruce, live oak and poplar (Brooks et al. 1994; Germaine et al. 2004). In addition to significantly colonizing the pine and spruce seedlings, C. sordidicola LS-S2r greatly enhanced seedling growth by accumulating 607% and 328% more biomass than control in spruce and pine, respectively, which was also significantly greater than all other bacterial strains (Figures 4.5 and 4.6). In the in vitro assays, this bacterial strain also showed the highest PGP abilities among all strains including phosphate and phytate solubilization, IAA production, ACC deaminase activity, cell wall degrading enzyme activity and ammonia production. Studies have shown that C. sordidicola strains possess such abilities to enhance plant growth. For example, Palaniappan et al. (2010) reported that C. sordidicola strains BLN 14 and BLN 20 have the ability to solubilize phosphate, produce siderophores and modulate plant hormone levels through ACC deaminase activity. In another study, C. sordidicola strain S170, isolated from coniferous forest soil, possesses the required genes for ACC deaminase activity, phosphate solubilization and metabolization, and siderophore production as well as the ability to metabolize various C sources so as to colonize internal niches of the plant (Lladó et al. 2014). Pine and spruce seedlings inoculated with P. prosekii LS-S1r also accumulated considerably more biomass than controls and the other bacterial strains in our greenhouse trial, which is consistent with significant PGP traits observed for this strain in vitro. Pseudomonas spp. have been extensively reported to increase the growth of coniferous and deciduous trees in both greenhouse- and field-based studies. One of the first reported spruce endophytes, Pseudomonas sp. Ss2-RN, significantly increased weight and length of interior spruce seedlings along with enhancing their mycorrhizal-root network in a   111 15-week greenhouse study (Shishido et al. 1996). The success of this endophytic bacterium was replicated in field studies, where the growth of spruce seedlings was enhanced by up to 234% as compared to controls (Shishido and Chanway 2000). Similarly, poplar and willow endophytes of Pseudomonas genus (strains WW6 and WP8) showed significant phosphate solubilization, IAA production and nitrogen-fixation abilities in vitro along with substantially increasing plant growth in greenhouse and field studies (Knoth et al. 2014; Khan et al. 2015; Kandel et al. 2017a). Our study illustrates that endophytic bacteria, isolated from hybrid white spruce trees growing on highly disturbed and nutrient-poor soils, have the ability to promote plant growth. These bacteria showed significant PGP potential in lab-based in vitro assays including phosphate solubilization, phytate hydrolyzation, siderophore production, IAA production, ACC deaminase activity, cell wall degrading enzymes (cellulase, protease, chitinase and b-1,3-glucanase) activity, ammonia production, and catalase activity. They showed strong potential to colonize the rhizosphere and internal tissues and enhance the length and biomass of their native host (hybrid white spruce) as well as a foreign host (lodgepole pine). This ability to form ecological associations with multiple boreal forest trees is an indication that these bacteria are generalists. This ability should be further tested in field-based studies and potentially harnessed to increase the growth and survival rates of tree saplings planted in nutrient-limited edaphic conditions. In particular, C. sordidicola LS-S2r that accumulated 7-fold and 4.25-fold more biomass in spruce and pine seedlings than controls and showed the highest PGP ability in lab-based in vitro assays may potentially be utilized as a comprehensive biofertilizer not only in natural ecosystems but also in intensively-managed ecosystems.  112 Table 4.1 Inorganic Ca-phosphate solubilization and phytate hydrolyzation measured both qualitatively and quantitatively, and ACC deaminase enzyme activity and indole-3-acetic acid (IAA) production measured quantitatively for the six bacterial strains. Bacterial strains  Ca-phosphate  Phytate  IAA production (µg/mL)‡  ACC deaminase activity (nmol a-ketobutyrate/mg/h)‡  Solubilization index (SI)† Phosphate solubilization (µg/ml)‡  Solubilization index (SI)† Phytate hydrolyzation (U/ml)‡, §    Pigmentiphaga litoralis HS-S1r  – –  – –  33.1 ± 0.31c  96.6 ± 0.86c Herbiconiux solani HS-S2r  – –  – –  25.4 ± 0.54a  29.6 ± 0.78a Pseudomonas migulae HS-S3r  2.0 76.0 ± 1.15a  1.4 52.2 ± 0.09a  25.0 ± 0.20a  51.0 ± 0.72b Pseudomonas prosekii LS-S1r  1.1 70.7 ± 1.20a  1.8 58.5 ± 0.24b   32.5 ± 0.29c  99.9 ± 0.78c Caballeronia sordidicola LS-S2r  2.7 125 ± 1.45b  3.0 75.3 ± 0.77c  34.8 ± 0.40c  121.8 ± 1.28d Caballeronia udeis LS-R1r  2.2 77.3 ± 0.88a  2.1 54.1 ± 0.47a  28.4 ± 0.21b  55.13 ± 0.96b † solubilization index represents the zone of solubilization relative to bacterial growth on triplicate plates. ‡ values are mean ± standard error (n = 3) and values followed by different letters are significantly different (P < 0.05). § One unit (U) of phytase hydrolyzation was defined as the amount of enzyme required to liberate 1 nmol of inorganic phosphorus from sodium phytate per minute.    113 Table 4.2 Activity of cell wall degrading enzymes – cellulase, protease and chitinase – measured for the six bacterial strains. Bacteria  Cellulase activity  Protease activity  Chitinase activity  b-1,3-glucanase activity  Plate assay† Quantitative assay (U/mL)‡, §  Plate assay† Quantitative assay (U/mL)‡, §  Plate assay† Quantitative assay (U/mL)‡, §  Plate assay† Quantitative assay (U/mL)‡, § Pigmentiphaga litoralis HS-S1r  +++ 0.67 ± 0.02c  – –  ++ 0.40 ± 0.01ab  ++ 0.60 ± 0.02b Herbiconiux solani HS-S2r  + 0.18 ± 0.01a  – –  – –  – – Pseudomonas migulae HS-S3r  ++ 0.47 ± 0.01b  + 22.7 ± 1.45a  ++ 0.45 ± 0.02b  – – Pseudomonas prosekii LS-S1r  +++ 0.63 ± 0.02c  ++ 40.7 ± 1.20b  + 0.35 ± 0.02a  ++ 0.47 ± 0.01a Caballeronia sordidicola LS-S2r  ++ 0.60 ± 0.01c  ++ 75.7 ± 1.20c  +++ 0.72 ± 0.01c  +++ 0.81 ± 0.01c Caballeronia udeis LS-R1r  ++ 0.51 ± 0.01b  – –  – –  – – † zone of clearance/yellow-orange zone for each bacterium on triplicate plates, where ‘–’ means no observed zone, ‘+’ means 0 – 5 mm zone diameter, ‘++’ means 5 – 15 mm zone diameter, and ‘+++’ means 15 – 25 mm zone diameter. ‡ values are mean ± standard error (n = 3) and values followed by different letters are significantly different (P < 0.05).  § One unit (U) of cellulase, chitinase and b-1,3-glucanase activity represents the amount of enzyme resulting in the release of 1 µmol of glucose from carboxymethylcellulose, colloidal chitin and laminarin, respectively, per minute. One unit (U) of protease activity represents the amount of enzyme resulting in the release of 1 µmol of tyrosine from azocasein per minute.   114 Table 4.3 Catalase activity, ammonia production and siderophore production (area of orange halo) measured for the six bacterial strains. Bacteria  Catalase activity†  Ammonia production†  Siderophore production –  area of orange halo‡ (cm2) Pigmentiphaga litoralis HS-S1r  +++  +  1.83 ± 0.2a Herbiconiux solani HS-S2r  ++  –  – Pseudomonas migulae HS-S3r  +  ++  1.56 ± 0.3a Pseudomonas prosekii LS-S1r  +  +++  2.58 ± 0.3b Caballeronia sordidicola LS-S2r  ++  +++  2.22 ± 0.5b Caballeronia udeis LS-R1r  +  +++  – † evaluated using triplicate samples per bacteria, where ‘–’ means no activity/production, ‘+’ means low activity/production, ‘++’ means medium activity/production and ‘+++’ means high activity/production. ‡ values are mean ± standard error (n = 3) and values followed by different letters are significantly different (P < 0.05).    115   Figure 4.1 Canola seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. (a) Mean values of primary root length of canola seedlings evaluated after 5 days of growth. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). (b) Five-day old canola seedlings showing differences in root length between treatments.   024681012HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlPrimary root length (cm)(a)abedbbcc  116   Figure 4.2 Tomato seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. (a) Mean values of primary root length of tomato seedlings evaluated after 5 days of growth. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). (b) Five-day old tomato seedlings showing differences in root length between treatments.   02468101214HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlPrimary root length (cm)(a)abecbbcd  117  Figure 4.3 Population size of each of the six bacterial strains in the internal tissues (needle, stem and root) and the rhizosphere of hybrid white spruce seedlings evaluated 18 months after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment each for rhizospheric and endophytic evaluations).  Figure 4.4 Population size of each of the six bacterial strains in the internal tissues (needle, stem and root) and the rhizosphere of lodgepole pine seedlings evaluated 18 months after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment each for rhizospheric and endophytic evaluations). 012345678910HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rNEEDLE STEM ROOT RHIZOSPHEREColonization (log cfu/g)012345678HS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rHS-S1rHS-S2rHS-S3rLS-S1rLS-S2rLS-R1rNEEDLE STEM ROOT RHIZOSPHEREColonization (log cfu/g)  118   Figure 4.5 (a) Seedling length and (b) seedling biomass of hybrid white spruce subjected to six bacteria-inoculated and one non-inoculated control treatments, harvested 18 months after inoculation. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05). 0510152025303540HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlSeedling Length (cm)(a)abbbbbb020406080100120140HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlSeedling Biomass (mg)(b)abbcbdb  119   Figure 4.6 (a) Seedling length and (b) seedling biomass of lodgepole pine subjected to six bacteria-inoculated and one non-inoculated control treatments, harvested 18 months after inoculation. Error bars represent standard errors of mean (n = 10 seedlings per treatment). Bars with different letters are significantly different (P < 0.05). 05101520253035404550HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlSeedling Length (cm)(a)abbbbbb020406080100120140160180HS-S1r HS-S2r HS-S3r LS-S1r LS-S2r LS-R1r ControlSeedling Biomass (mg)(b)abbcbdb  120 Chapter 5 - Evaluating lodgepole pine endophytes for their ability to fix nitrogen and support tree growth under nitrogen-limited conditions 5.1. Introduction Lodgepole pine (Pinus contorta var. latifolia) is regarded as a ubiquitous species in Western North America with an extensive ecological amplitude. It has one of the widest ranges of environmental tolerance among any coniferous species in North America since it thrives on an array of soil, moisture and topographical conditions (Lotan and Critchfield 1996). It is unique among conifer species in its ability to thrive on nutrient-poor and fire-affected sites that are severely limited in nitrogen (N) (Weetman et al. 1988). Lodgepole pine stands can be found on rough and rocky terrain as well as steep slopes and ridges, including bare gravel that contains very minimal amounts of plant-essential nutrients, particularly N (Chapman and Paul 2012) British Columbia (BC), Canada has some of the most diverse terrestrial ecosystems in North America and is divided into 14 biogeoclimatic zones (Pojar and Meidinger 1991). The Sub-Boreal Pine-Spruce (SBPS) biogeoclimatic zone is a montane region that lies in the central interior of BC. One of the subzones of SPBS – xeric cold (xc) – is an extremely cold and dry region because of its position in the rain-shadow of the Coast Mountains, which has resulted in thin and weakly developed soils comprised of mostly sand or sandy loam texture (Puri et al. 2018a; Chapter 2). The organic forest floor is either very thin or completely absent on these soils due to frequent wildfire activity (Steen and Coupé 1997). In addition, analyses of various physico-chemical soil properties such as pH, C:N ratio, cation exchange capacity, base saturation, organic matter,   121 texture and bulk density have revealed that soil health at various sites in this subzone is relatively poor (Puri et al. 2018a; Chapter 2). These soils lack several essential macro- and micro-nutrients and, most importantly, they are poor in available as well as mineralizable N. Lodgepole pine are the most common forest stands in this region, which aligns with the ability of this tree species to grow on highly disturbed sites. But the fundamental question is, how can lodgepole pine thrive on such nutrient-poor, N-limited soils of this region? Since soil N is scarce in the SBPSxc subzone, N-fixing bacteria (diazotrophs) can be viewed as an important pathway by which lodgepole pine trees could be fulfilling their N-requirements. Although associative N-fixation in the rhizosphere is widely recognized for plants, rhizospheric N-fixation in lodgepole pine has been reported to be insignificant (Chanway and Holl 1991). In addition, the harsh environmental and soil conditions in this zone may not be suitable for rhizospheric diazotrophs (Bal et al. 2012; Chapman and Paul 2012). Interestingly, potential diazotrophs have been observed inside the tubercles of a tuberculate ectomycorrhizae species (Suillus tomentosus), but neither their N-fixation marker gene (nifH) was successfully identified nor their ecological role was demonstrated in planta (Paul et al. 2007, 2013). Endophytic diazotrophs (N-fixing bacteria living inside the plant tissues) could provide a crucial source of N for lodgepole pine trees in this region since they are thought to be better protected from abiotic and biotic stresses inside the tissues (Chanway et al. 2014). For instance, an endophytic diazotrophic strain, Paenibacillus polymyxa P2b-2R, has shown significant potential in promoting growth and fulfilling the N-requirements of lodgepole pine seedlings in growth chamber studies (Anand et al. 2013). In addition, endophytic diazotrophs have been speculated to provide significant amounts of fixed N to limber pine (Pinus flexilis) and Engelmann spruce (Picea   122 engelmannii) trees growing in a subalpine forest in Colorado, USA (Carell and Frank 2014; Moyes et al. 2016). These studies led us to hypothesize that lodgepole pine trees growing in this SBPSxc subzone harbour endophytic diazotrophs in their internal tissues, which have the potential to provide pine trees with significant amounts of fixed N from the atmosphere. Endophytic bacteria were previously isolated from lodgepole pine trees growing at two different sites in the West Chilcotin region of the SBPSxc subzone (52°00ʹ04.2ʺ N, 124°59ʹ44.7ʺ W and 52°00ʹ09.1ʺ N, 124°59ʹ25.2ʺ W) and were tested for their ability to fix N by using an acetylene reduction assay (ARA) (Puri et al. 2018a; Chapter 2). This assay indirectly tests the activity of nitrogenase enzyme responsible for the process of biological N-fixation. Nitrogenase enzyme breaks the triple bonded atmospheric N2 and converts it to plant-available ammonia (NH3). This ability to break triple bonded atoms is indirectly tested in ARA by evaluating the amount of acetylene (CH º CH) converted to ethylene (CH2 = CH2), which can be easily detected using gas chromatography (Holl et al. 1988). Forty-eight endophytic bacteria were isolated on N-free culture media, of which 23 bacteria showed positive nitrogenase activity in an ARA (Puri et al. 2018a; Chapter 2). In an effort to understand the potential role of these endophytic diazotrophs in fulfilling the N-requirements of lodgepole pine trees and sustaining their growth on N-limited soils, we selected six endophytic diazotrophic strains for this study (Table 5.1) that showed the best performance in ARA, i.e. they converted highest amounts of acetylene to ethylene (Puri et al. 2018a; Chapter 2). These strains were examined in a year long greenhouse experiment by inoculating each strain into lodgepole pine. In an additional greenhouse experiment, each of these strains was tested with another tree species native to the West Chilcotin region – hybrid white spruce (Picea glauca x engelmannii). The motive behind testing   123 these strains with another host was to observe their ecological functioning in a different plant niche. This study was part of the research project exploring the natural ecological association and plant x microbe specificity of endophytes to support tree (lodgepole pine and hybrid white spruce) growth at highly disturbed and nutrient-poor sites. In chapter 3 (Puri et al. 2020a), endophytic N-fixing bacteria were isolated from hybrid white spruce trees growing in this West Chilcotin region and evaluated for their ability to fix N and promote tree growth. In contrast to previous suggestions regarding how specific the endophyte x tree species association could be (O’Neill et al. 1992; James and Olivares 1998; Shishido et al. 1995; Chanway et al. 2000), no evidence of such plant x microbe specificity was observed in that study.  Our primary objective in this study was to evaluate pine endophytes for their potential to form beneficial associations with different tree species and support their growth under extremely N-poor conditions by supplementing their N-requirements through biological N-fixation. The specific objectives were: (i) to evaluate if each of the selected endophytic diazotrophic strains can colonize the internal tissues of lodgepole pine (native host) and hybrid white spruce (foreign host) following inoculation; (ii) to quantify the proportion of N-requirements of pine and spruce fulfilled by each strain; and (iii) to determine the potential of each strain to enhance pine and spruce seedling growth (biomass and length) upon in planta inoculation.     124 5.2. Materials and Methods 5.2.1. Antibiotic-resistant derivative strains Six strains originating from the internal root, stem and needle tissues of lodgepole pine trees growing at highly-disturbed, nutrient-poor sites of the West Chilcotin region in BC were used in this study. These strains reduced the highest amounts of acetylene to ethylene in ARA (Puri et al. 2018a; Chapter 2), indicating the possibility of high in planta nitrogenase enzyme activity. Antibiotic-resistant mutants of these strains were derived so that endophytic colonies formed by each strain when inoculated into lodgepole pine and hybrid white spruce seedlings could be quantified in the greenhouse experiments. The antibiotic-resistant derivative of each strain was raised (Table 5.1) by streaking multiple times on N-free combined carbon medium (CCM) agar (Appendix B) amended with the antibiotic rifamycin (200 mg/L) (Bal and Chanway 2012a). The antibiotic-resistant strains were stored in CCM amended with 200 mg/L rifamycin and 20% (v/v) glycerol at –80 °C until further evaluation. 5.2.2. Acetylene reduction assay and nifH gene amplification To confirm the N-fixing ability of the antibiotic-resistant derivative strains, their nitrogenase enzyme activity was analyzed using ARA as outlined in chapter 2 (Puri et al. 2018a). Subsequently, the nifH gene of each antibiotic-resistant strain was also amplified. Each frozen strain was thawed and streaked onto CCM agar plates amended with rifamycin (200 mg/L) and then grown in CCM broth amended with rifamycin on a rotary shaker for 2 days at 30 °C. Bacterial cells were harvested by centrifugation and the genomic DNA of each strain was extracted by using the   125 DNeasy UltraClean Microbial Kit (Qiagen Inc., Valencia, CA, USA) following the steps outlined by the manufacturer. The concentration of the isolated DNA was evaluated by using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and the quality of the DNA was confirmed by electrophoresis on a 0.8% agarose gel. The nifH gene of each isolate was amplified using the primers nifH1forward primer [5ʹ-TGY GAY CCN AAR GCN GA-3ʹ] and nifH2 reverse primer [5ʹ-ADN GCC ATC ATY TCN CC-3ʹ] (Zehr and McReynolds 1989). PCR amplification was performed in 25 μL reaction volumes containing 2.5 μL of 1x PCR buffer (without MgCl2), 1.25 μL of MgCl2 (2.5 mM), 0.5 μL of Taq DNA Polymerase (5 U/μl), 0.5 μL of dNTP mix (consisting of four nucleotides: dATP, dCTP, dGTP, dTTP), 2.5 μL of each primer (1 μM), and 2.5 μL of template DNA (1 ng/μL) (Gaby and Buckley 2012; Paul et al. 2013). The final volume was adjusted to 25 μL with nuclease-free water. Amplifications were performed using the MJ Mini gradient thermal cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) with the following program: initial cell lysis and denaturation for 5 min at 95 °C; followed by 35 cycles of denaturation (1 min at 94 °C), annealing (1 min at 55 °C), and extension (1 min at 72 °C); and a final extension for 10 min at 72 °C. Amplification of PCR products was confirmed by electrophoresis on 1% agarose gel for 1 h at 100 volts. 5.2.3. Greenhouse trials Two separate year long greenhouse growth trials – the first involving the inoculation of each selected strain into lodgepole pine (native host) and the second involving the inoculation into hybrid white spruce (foreign host) – were conducted. Therefore, six bacteria-inoculated and one non-inoculated control treatments were evaluated in each growth trial.   126 5.2.3.1. Bacterial inoculum Inoculum of each antibiotic-resistant strain was prepared by thawing a frozen culture of the strain, streaking a loopful onto CCM agar amended with 200 mg/L rifamycin, and incubating at 30 °C for 48 h. A loopful of bacterial growth from the agar was introduced into CCM broth amended with rifamycin (200 mg/L) and agitated at 150 rpm for 48 h at 30 °C in a rotary shaker. Bacterial cells were harvested by centrifugation (3000×g; 30 min), washed twice in sterile phosphate buffered saline (PBS) (pH 7.4) (Appendix A), and resuspended in the same buffer to a density of ca. 106 cfu/mL. 5.2.3.2. Pre-treatment of seeds Lodgepole pine and hybrid white spruce seeds were acquired from the BC Ministry of Forests Tree Seed Centre, Surrey, BC and originated from forest stands in the West Chilcotin region (Lodgepole pine – 51° 54’ N lat., 124° 51’ long., elevation 1320 m, SBPSxc zone; hybrid white spruce – 51° 57’ N lat., 124° 59’ long., elevation 1350 m, SBPSxc zone). Seeds were surface sterilized by immersion in 30% (v/v) H2O2 for 90 s, and then rinsed three times for 30 s in sterile distilled water. Ten randomly selected sterilized seeds were imprinted on tryptic soy agar (TSA) plates to determine the effectiveness of surface sterilization. TSA plates were then incubated at 30 °C for 48 h and checked for microbial contamination. The surface-sterilized seeds were placed in sterile cheesecloth bags containing moist autoclaved silica sand and stored at 4 °C for 28 days for stratification. Seed stratification is done to simulate the natural environmental conditions (cold and high moisture) that seeds must experience for a certain period of time, also known as the dormancy period, before germination. The combination of cold and moist conditions triggers   127 biochemical changes that transform complex food substances into simpler forms that are utilized by the embryo when it germinates (Willan 1986; Kolotelo et al. 2001). Ten seeds were selected randomly from cheesecloth bags after the stratification period, crushed and imprinted on TSA plates amended with 200 mg/L rifamycin and were examined after 48 h to confirm the absence of internal seed contamination by any of the selected bacterial strains before inoculation. It was assumed that all seeds used in this study harbour similar communities of endophytic bacteria, therefore their effect would be pronounced similarly in all treatments (bacteria-inoculated and non-inoculated control). 5.2.3.3. Growth trial set-up Ray Leach Cone-tainers (height: 210 mm; diameter: 38 mm) were used to grow seedlings in each greenhouse trial. Each Cone-tainer was filled to two-third capacity with an autoclaved sand–Turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, IL, USA) mixture (69% w/w silica sand; 29% w/w Turface; 2% w/w CaCO3). Each Cone-tainer was fertilized with 20 mL of a nutrient solution having Ca(15NO3)2 (5% 15N label) (Appendix C). Three surface-sterilized seeds were sown aseptically in each Cone-tainer. The bacterial suspension (5 mL) of each strain (106 cfu/mL) was pipetted directly over the seeds in each Cone-tainer designated for that strain and then covered with 5 mm of autoclaved silica sand. This process was repeated for each of the six bacterial strains in both trials. Non-inoculated control seeds received 5 mL of sterile PBS without any bacteria in each trial. The Cone-tainers were placed in trays (98 Cone-tainers per tray) in the University of British Columbia Plant Care Services’ greenhouse with photosynthetically active radiation at the   128 canopy level of 300 µmol/m2/s in a 16 h photoperiod (6 am to 10 pm). Seedlings were watered as required with sterile distilled water. Two weeks after sowing, seedlings were thinned to the largest single germinant per Cone-tainer. Seedlings received 20 mL of the nutrient solution without Ca(15NO3)2 once per month during the year long growth period. Tray positions were randomized weekly to reduce the positional effects. 5.2.3.4. Seedling harvest and analyses To evaluate seedling growth promotion, lodgepole pine and hybrid white spruce seedlings were randomly selected (n = 10) from each treatment and harvested destructively 4, 8 and 12 months after sowing. Seedlings were removed from Cone-tainers, measured for length and oven-dried for 48 h at 65 °C to determine biomass (dry weight). The foliar 15N isotope dilution assay was used to quantify the amount of N fixed from the atmosphere by each strain in planta. For this assay, N was provided in the form of 15N (i.e., Ca(15NO3)2) to all seedlings once at the beginning of the growth trial. Pine and spruce seedlings were randomly selected (n = 10) from each treatment 12 months after sowing. Needles of each selected seedling were oven-dried for 48 h at 65 °C and ground to a particle size < 2 mm using a mortar and pestle. A 2.5 mg sample of ground foliage of each seedling was sent to the Stable Isotope Facility in the Faculty of Forestry, University of British Columbia, Vancouver, Canada to determine the foliar N concentration and atom % 15N excess in foliage using an elemental analyzer (vario EL cube, Elementar Analysensysteme GmbH) interfaced with an isotope ratio mass spectrometer (IsoPrime PrecisION, Elementar Analysensysteme GmbH). The amount of fixed N accumulated in the foliage of each bacteria-inoculated seedling was estimated (Rennie et al. 1978) by calculating the percent N derived from   129 the atmosphere (%Ndfa) = {atom % 15N excess (control plant) – atom % 15N excess (inoculated plant)} ÷ atom % 15N excess (control plant). The rate of N-fixation in bacteria-inoculated lodgepole pine and hybrid spruce trees was calculated using the %Ndfa and foliar N content. To evaluate if the bacterial strains were able to colonize the internal tissues of lodgepole pine and hybrid white spruce (i.e. endophytic colonization), seedlings from each treatment (n = 5) were harvested 4, 8 and 12 months after sowing. Seedlings were surface-sterilized with bleach (1.3 % NaOCl) for 5 min and washed thrice with sterile distilled water. Surface sterilization of seedlings was confirmed by imprinting on TSA plates and growing them for 24 h. A sample of each of the root, stem, and needle tissues was then triturated separately in 1 mL PBS using a sterile mortar and pestle. Serial dilutions of the triturated tissue suspensions were performed, and 0.1 mL of each dilution was plated onto CCM agar amended with cycloheximide (100 mg/L) to inhibit fungal growth and rifamycin (200 mg/L) to inhibit the growth of other bacteria. The growth of the six strains used in this trial was not inhibited by rifamycin since they had acquired antibiotic resistance. The number of colonies formed by each strain (cfu) was evaluated a week after incubation at 30°C. 5.2.3.5. Statistical analyses Statistical analysis of each greenhouse growth trial was performed separately. To evaluate the treatment effects of each bacterial strain on the growth of seedlings in each growth trial, a completely randomized experimental design with 55 replicates per treatment was used. The statistical package, SAS University Edition (SAS Institute Inc., Cary, NC, USA), was used to perform statistical analyses. Analysis of variance (ANOVA) was done (F-test and Student’s t-test) to   130 determine significant differences between treatment means for seedling length, seedling biomass, atom % 15N excess in foliage and foliar N concentration. The confidence level, a, was set to 0.05. 5.3. Results All antibiotic-resistant derivative strains successfully reduced acetylene to ethylene in ARA, producing 1 to 2.7 nmol of ethylene per mL capacity of the vial (Table 5.1). It was also found that each of these strains likely possesses the nifH gene required to form the nitrogenase enzyme to fix atmospheric N (Table 5.1). When each strain was inoculated into their native host (lodgepole pine) and foreign host (hybrid white spruce) in the year long greenhouse trial, it was observed that all strains were able to derive significant amounts of N from the atmosphere and provide it to their host. This was evident from the considerably lower atom % 15N excess values for inoculated seedlings as compared to the controls (Table 5.2). In particular, seedlings inoculated with strains HP-S1r and LP-R2r had significantly lower atom % 15N excess in foliage than other bacterial treatments. These strains fulfilled 46–50% of the N requirement of both pine and spruce seedlings from the atmosphere. If it is assumed that N-fixation occurred uniformly throughout the one-year growth period, then the rate of N-fixation for bacteria-inoculated pine and spruce seedlings ranged from 3.06 to 14.9 mg N/kg tissue/day and 2.47 to 13.2 mg N/kg tissue/day, respectively (Table 5.2). The N content (%) in foliar tissues of pine and spruce seedlings inoculated with these strains was also significantly higher than controls and other bacterial treatments (Tables 5.2). Notably, HP-S1r-inoculated pine and spruce seedlings had accumulated 73% and 56% higher N in their foliage, respectively, as compared to the control seedlings.   131  Apart from fixing N, all six bacterial strains successfully promoted seedling length and biomass of lodgepole pine and hybrid white spruce seedlings (Figures 5.1 and 5.2). At the 4-month harvest, pine seedling length of HP-S1r, LP-S1r, LP-R1r and LP-R2r treatments was significantly greater than control, HP-N1r and HP-R1r treatments (Figure 5.3a). However, at the 8- and 12-month harvest, all six bacterial treatments were significantly better than the control treatment in terms of pine seedling length (Figure 5.3a). With regard to pine seedling biomass, strains HP-S1r, LP-S1r and LP-R2r very effectively increased the biomass as compared to the control and all other bacterial treatments at the 4- and 8-month harvests (Figure 5.3b). Similar to seedling length, all bacteria-inoculated pine seedlings had accumulated significantly higher biomass (> 100%) than controls at the end of the growth trial (12 months) (Figure 5.3b). Notably, inoculation with strain HP-S1r increased pine seedling length by 53% and biomass by 278% as compared to the control. Additionally, strain LP-R2r also performed considerably well by increasing length and biomass of pine seedlings by 40% and 200%, respectively, in comparison to the control. In the case of spruce seedlings, all bacterial treatments except LP-S1r significantly increased the seedling length as compared to the control (> 30%) at the 4-month harvest (Figure 5.4a). Later in the growth trial, all bacteria-inoculated spruce seedlings were significantly longer than the controls at the 8- and 12-month harvests (Figure 5.4a). Particularly, strain LP-R2r had increased seedling length by nearly 50% in comparison to the control at the end of the growth trial. All bacterial treatments accumulated significantly greater biomass (nearly 100%) than control in spruce seedlings at the 4-month harvest (Figure 5.4b); whereas at the 8-month harvest, only HP-S1r, LP-S1r and LP-R1r and LP-R2r treatments had significantly higher biomass than   132 control (Figure 5.4b), with LP-R2r-inoculated spruce seedlings acquiring 175% more biomass than controls. At the last harvest, all bacteria-inoculated spruce seedlings were significantly greater than controls in terms of biomass (Figure 5.4b). Notably, strains HP-S1r, LP-R1r and LP-R2r increased spruce seedling biomass by > 200% as compared to the controls. The significant plant-growth-promotion and N-fixation observed for bacteria-inoculated pine and spruce seedlings could be related to the colonization of internal tissues by these bacterial strains. Although needle colonization by any of the six strains was not observed for pine and spruce seedlings initially at the 4-month harvest, strains HP-S1r and LP-R2r were observed in both pine and spruce needle tissues (pine – 103 to 105 cfu/g fresh tissue & spruce – 102 to 103 cfu/g fresh tissue) at the 8- and 12-month harvests (Figures 5.5a and 5.6a). Colonies of strain LP-R1r were also observed in both pine and spruce needles at the 12-month harvest, whereas strain HP-R1r was only observed in pine needles at this harvest. All strains successfully colonized the stem tissues of pine and spruce seedlings at the 8- and 12-month harvests with population densities ranging from 102 to 104 cfu/g fresh tissue (Figures 5.5b and 5.6b). Initially, at the 4-month harvest, only three strains (HP-S1r, LP-S1r and LP-R2r) were able to colonize pine stem tissues and two strains (HP-S1r and LP-R2r) were able to colonize spruce stem tissues. In the case of root tissues, endophytic colonization was observed for each bacterial strain at all harvest intervals in both pine and spruce seedlings with the population densities ranging between 103 and 107 cfu/g fresh tissue (Figures 5.5c and 5.6c). No evidence of endophytic colonization was observed in control seedlings of pine and spruce at any harvest interval.    133 5.4. Discussion Notwithstanding the poorly developed, nutrient-poor soils that characterize the SBPSxc biogeoclimatic region in BC (Steen and Demarchi 1991; Puri et al. 2018a; Chapter 2), lodgepole pine forest stands thrive in this region. We suspected that N-fixation by endophytic diazotrophs could be one of the possible ways through which pine trees are fulfilling their N-requirements (Puri et al. 2018a; Chapter 2). However, the crucial questions were: (i) how much N can these endophytic diazotrophs provide to lodgepole pine trees via biological N-fixation, and (ii) is there a plant x microbe specificity in this association?  Six top-performing endophytic diazotrophs – originally isolated from the internal tissues of lodgepole pine trees growing in the West Chilcotin region – were selected based on their nitrogenase enzyme activity (Puri et al. 2018a; Chapter 2). Antibiotic-resistant mutants of each strain were derived in order to quantify the population sizes of these endophytes in plant tissues and rhizosphere (Table 5.1). Although previous reports suggest that the spontaneous antibiotic-resistance mutation to rifamycin does not affect the nitrogenase activity of endophytic bacteria (Shishido et al. 1995; Bal et al. 2012), to be sure, the antibiotic-resistant strains were evaluated for nitrogenase enzyme activity using ARA (Table 5.1). Our results support the previous findings regarding the spontaneous mutation to rifamycin resistance since the amounts of ethylene produced in ARA by antibiotic-resistant strains were similar to the wild-type strain reported in Chapter 2 (Puri et al. 2018a) (Tables 2.4 and 5.1). The ability of these strains to fix N was further confirmed by amplifying their nifH gene, which is regarded as the marker gene for N-fixation and is believed to be conserved in diazotrophs (Gaby and Buckley 2012). The amplified nifH gene of   134 each strain was detected on agarose gel (Table 5.1), emphasizing the likeliness of these strains to have the ‘ability’ to fix N. However, we looked for further evidence to determine the ‘actual’ proportion of the N-requirement of plants fulfilled by these strains via N-fixation using the 15N isotope dilution method. The results of the 15N isotope dilution assay revealed that each endophytic diazotrophic strain fixed significant amounts of N from the atmosphere to supplement the N-requirements of pine and spruce seedlings (Table 5.2). This method works on the principle that bacteria-inoculated seedlings obtain fixed 14N from the atmosphere, thereby resulting in diluted 15N levels in their tissues as compared to the control seedlings (Hardarson and Danso 1993). This is clear from the atom % 15N excess values observed for bacteria-inoculated and control seedlings of pine and spruce (Table 5.2). Irrespective of the plant host, Caballeronia sordidicola HP-S1r and Caballeronia udeis LP-R2r emerged as potent N-fixing bacteria as they fulfilled 46-50% of the N-requirements of pine and spruce seedlings through biological N-fixation. Certain tree endophytes have shown similar abilities to fulfill such a considerable proportion of N-requirements of their host via N-fixation (Anand et al. 2013; Knoth et al. 2014; Moyes et al. 2016). The concentration of N in the foliage of pine and spruce seedlings inoculated with these two strains was also significantly higher than the control seedlings (Table 5.2). Since the seedling growth medium was fertilized with a very limited amount of N at the onset of the experiment, it is reasonable to conclude that higher N concentration in seedlings inoculated with strains HP-S1r and LP-R2r was due to the accumulation of fixed N in their foliage from endophytic diazotrophs. However, it is still unclear how such endophytic diazotrophs provide fixed N to the plant. Two possibilities are that (i) there could be an active transfer of fixed N compounds from the bacteria to the host   135 plant, similar to the rhizobium-legume model, or (ii) bacteria fix N in planta for their own metabolism and provide fixed N indirectly to the plant after they die and decompose in the plant (Doty 2017). Assuming that N-fixation occurred uniformly throughout the growth trials, the rate of N-fixation in bacteria-inoculated pine and spruce seedlings per day during the growth trial ranged from 2 to 15 mg N/kg tissue (Table 5.2), which are consistent with estimates reported in poplar and limber pine tissues (Moyes et al. 2016; Doty et al. 2016). If calculated on a per-year basis, C. sordidicola HP-S1r fixed 5.5 g of N per kg pine tissues and 4.8 g of N per kg of spruce tissues while C. udeis LP-R2r fixed 3.8 g of N per kg tissue for both pine and spruce. Although these N-fixation rates are generally lower than those fixed in the nodules of leguminous plants, this N-accumulation pathway could still be biologically significant for pine trees growing in the West Chilcotin region due to the severely limited soil N (Steen and Coupé 1997; Puri et al. 2018a; Chapter 2). However, a complete accounting of the N cycle in these forest stands should be performed to further explore the importance of this endophytic N-fixation pathway.  The seedling length and biomass of lodgepole pine and hybrid white spruce were evaluated thrice during the greenhouse trials in order to track the growth of inoculated and non-inoculated seedlings. Length and biomass of pine and spruce seedlings increased with time during the growth trial for all bacterial treatments compared to the slower-growing control seedlings, thus indicating the growth-promoting effects of bacterial inoculation (Figures 5.3 and 5.4). This could be attributed to the limited N-source for control seedlings (i.e. soil N), which likely depleted after the single small N application at the beginning of the growth trial. However, bacteria-  136 inoculated seedlings had access to an additional N-source (atmospheric N) as evidenced by the results of the 15N isotope dilution assay. The length and biomass of all bacteria-inoculated pine and spruce seedlings were significantly higher than control seedlings at the end of the growth study (Figures 5.3a and 5.4a). It is notable to see that inoculation with each of the six endophytic diazotrophic strains increased biomass accumulation by >2-fold in both pine and spruce seedlings (Figures 5.3b and 5.4b). Such an increase in biomass accumulation could be ascribed to the increased accessibility of inoculated seedlings to fixed N from the atmosphere as postulated in previous studies (Bal and Chanway 2012a; Knoth et al. 2014). However, endophytic diazotrophs are also known for their ability to promote plant growth through other mechanisms such as phytohormone modulation, phosphate solubilization, and increased access to certain micronutrients including iron (Kandel et al. 2017a; Padda et al. 2017a). Therefore, it is certainly important to study such mechanisms to further explain the growth promotion observed for bacteria-inoculated pine and spruce seedlings. Regardless of the mechanism(s) at work, our results, including those presented in chapters 3 and 4 (Puri et al. 2020a, b), clearly indicate that plant x microbe specificity is unimportant in endophytic diazotroph x tree species interactions.  Among all bacterial treatments, C. sordidicola HP-S1r, C. udeis LP-R2r and Paraburkholderia phytofirmans LP-R1r enhanced the seedling length of pine and spruce the most (35-53%) by the end of the growth trials (Figures 5.3a and 5.4a). Pine seedlings inoculated with C. sordidicola HP-S1r accumulated 278% more biomass than the control at the end of the growth trial, which was significantly higher than all other bacterial treatments (Figure 5.3b). This strain performed equally well in spruce by accumulating 243% more biomass than the control by the end of the growth trial (Figure 5.4b). Such considerable biomass accumulation could be crucial to   137 sustain tree growth, especially under adverse conditions such as those found in the West Chilcotin region. Previous studies have suggested that C. sordidicola strains possess multifarious plant-growth-promoting traits such as phosphate solubilization, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, siderophore production and N-fixation (Palaniappan et al. 2010; Lladó et al. 2014; Puri et al. 2020a, b). Similarly, C. udeis LP-R2r strain considerably increased the biomass of pine and spruce seedlings by 200% and 295%, respectively, by the end of the growth trial. Another noteworthy strain was P. phytofirmans LP-R1r that enhanced the spruce seedling biomass by 224% compared to the control. P. phytofirmans species is widely-known, mainly due to the endophytic strain PsJn, which was initially isolated from onion roots and later reported in numerous studies to endophytically colonize and promote the growth of diverse host species ranging from monocots to dicots (reviewed by Puri et al. 2017b). It is important to mention that both Caballeronia and Paraburkholderia genera were previously part of the plant-beneficial-environmental group of the Burkholderia genus (Dobritsa and Samadpour 2016; Sawana et al. 2014), which is rich in symbiotic and associative N-fixing as well as plant-growth-promoting bacteria (Estrada-De Los Santos et al. 2001). Each of the six endophytic strains colonized one or more internal tissues of pine and spruce seedlings during the growth trial (Figures 5.5 and 5.6), indicating that survival and multiplication in biologically significant numbers are likely necessary for these bacteria to perform N-fixation and provide growth-promoting benefits to the host (Chanway et al. 2000; Germaine et al. 2004; Pohjanen et al. 2014). The endophytic bacterial populations observed in pine and spruce for our strains are comparable to previous studies where coniferous trees were artificially inoculated with endophytic bacteria (Shishido et al. 1999; Tang et al. 2017; Yang et al.   138 2016). Though all strains were able to colonize the internal root and stem tissues of pine and spruce seedlings, needle colonization was observed for certain strains only (Figures 5.5 and 5.6). This may be explained by the following postulations – either the plant drives the selection process regarding where certain endophytic bacteria would be harboured, or the endophytic bacteria have a preference for certain plant tissues types. However, neither of these hypotheses have been proven, so it is not possible to conclude how and why endophytic bacteria harbour certain plant tissues.   The endophytic colonization pattern observed for each strain was similar to the N-fixation and plant-growth-promotion patterns observed in the growth trial. For instance, strains C. sordidicola HP-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r, which demonstrated the highest N-fixation and seedling growth promotion during the growth trial, were also the most aggressive colonizers of needle, stem and root tissues of pine and spruce seedlings (Figures 5.5 and 5.6). This observation indicates that plant colonization size of these bacterial strains may have a direct role in the N-fixation and growth enhancement of pine and spruce seedlings. Similar observations have been reported in inoculation studies with interior spruce, lodgepole pine and poplar (Shishido et al. 1995; Germaine et al. 2004; Yang et al. 2016; Padda et al. 2019). In addition to lodgepole pine, Caballeronia strains with potential N-fixing ability were also isolated from stem and root tissues of hybrid white spruce trees growing in the West Chilcotin region (Puri et al. 2018a; Chapter 2), which suggests the possibility of the widespread presence of the genus Caballeronia in this region. In a similar study, a C. udeis strain provided 36-39% of host nitrogen and a C. sordidicola strain provided 52-56% of host nitrogen from the atmosphere   139 in spruce and pine while significantly enhancing their length and biomass (Puri et al. 2020a; Chapter 3). The N-fixation and plant-growth-promotion observed for C. sordidicola HP-S1r and C. udeis LP-R2r inoculated pine and spruce seedlings in this study provides further evidence that the genus Caballeronia may have a significant role in supporting the growth of lodgepole pine and hybrid white spruce trees in the West Chilcotin region. Interestingly, the non-specific nature of C. sordidicola and C. udeis strains observed in this study and chapter 3 (Puri et al. 2020a) has also been observed in other endophytic diazotrophic strains isolated from poplar, willow, lodgepole pine and western red cedar trees (Doty et al. 2009; Bal et al. 2012), as those strains have shown N-fixing capabilities not only in deciduous and coniferous trees but also in agricultural crops (Knoth et al. 2014; Khan et al. 2015; Kandel et al. 2015; Bal et al. 2012; Anand and Chanway 2013; Puri et al. 2015, 2016a). This study, as well as that described in chapter 3 (Puri et al. 2020a), represent a series of experiments designed to explore the ecological associations of pine and spruce endophytes with their natural hosts in the extremely N-poor West Chilcotin region. From these studies, It can be concluded that N-fixation and plant-growth-promotion by bacterial endophytes may play a crucial role in sustaining the growth of trees in this region. The lack of plant x microbe specificity observed in these studies suggests that plant growth-promoting endophytic diazotrophs could be generalists, which are able to enhance the N content and growth of gymnosperms naturally regenerating on nutrient-poor soils in the boreal forests of BC.    140 Table 5.1 Nitrogen-fixing endophytic bacterial strains selected from Chapter 2 (Puri et al. 2018a) and their antibiotic-resistant derivatives along with the assessment of their nitrogen-fixing ability demonstrated via acetylene reduction activity and the presence of the nifH gene. Acetylene reduction activity has been expressed as nanomoles of ethylene produced per mL of culture tube headspace (mean ± standard error; n = 5). Bacterial species Strain Antibiotic-resistant strain Acetylene reduction activity (nmol C2H4/mL) nifH gene Caballeronia sordidicola HP-S1 HP-S1r 2.7 ± 0.10 + Pseudomonas frederiksbergensis HP-N1 HP-N1r 1.5 ± 0.21 + Phyllobacterium myrsinacearum HP-R1 HP-R1r 1.4 ± 0.14 + Pseudomonas mandelii LP-S1 LP-S1r 1.0 ± 0.09 + Paraburkholderia phytofirmans LP-R1 LP-R1r 2.2 ± 0.25 + Caballeronia udeis LP-R2 LP-R2r 1.9 ± 0.03 +   141 Table 5.2 Foliar N concentration (mg N per g tissue), atom percent 15N excess in foliage, percent N derived from the atmosphere (% Ndfa) and rate of N-fixation (mg of fixed N per kg of tissue per day) of lodgepole pine and hybrid white spruce seedlings treated with six endophytic diazotrophic strains and a non-inoculated control, measured 12 months after sowing. Percent foliar N and atom percent 15N excess in foliage values are mean ± standard error (n = 10 seedlings per treatment). Values followed by different letters are significantly different at P < 0.05. Treatment Lodgepole Pine  Hybrid White Spruce Foliar N (mg/g) Atom % 15N excess in foliage % Ndfa N-fixation rate (mg/kg/day)  Foliar N (mg/g) Atom % 15N excess in foliage % Ndfa N-fixation rate (mg/kg/day) Caballeronia sordidicola HP-S1r 10.9 ± 0.05c 0.39 ± 0.03d 50 14.9  9.75 ± 0.04c 0.35 ± 0.03d 49 13.2 Pseudomonas frederiksbergensis HP-N1r 6.19 ± 0.02a 0.64 ± 0.02b 18 3.06  5.99 ± 0.03a 0.58 ± 0.02b 15 2.47 Phyllobacterium myrsinacearum HP-R1r 6.50 ± 0.04a 0.52 ± 0.01bc 34 6.05  6.14 ± 0.04a 0.53 ± 0.01bc 23 3.84 Pseudomonas mandelii LP-S1r 7.06 ± 0.02ab 0.54 ± 0.01b 31 6.03  7.19 ± 0.02ab 0.47 ± 0.02c 32 6.31 Paraburkholderia phytofirmans LP-R1r 6.22 ± 0.01a 0.58 ± 0.02b 26 4.42  7.19 ± 0.03ab 0.50 ± 0.02bc 27 5.33 Caballeronia udeis LP-R2r 8.24 ± 0.04b 0.42 ± 0.03c 46 10.3  8.03 ± 0.03b 0.36 ± 0.02d 48 10.5 Control 6.27 ± 0.03a 0.78 ± 0.03a - -  5.92 ± 0.02a 0.69 ± 0.01a - -  142    Figure 5.1 Lodgepole pine seedlings corresponding to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment harvested (a) 4-month, (b) 8-month, and (c) 12-month after sowing and inoculation. HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 1(a)HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 2(b)HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 3(c) 143    Figure 5.2 Hybrid spruce seedlings corresponding to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment harvested (a) 4-month, (b) 8-month, and (c) 12-month after sowing and inoculation. HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 1(a)HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 2(b)HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlHarvest 3(c) 144   Figure 5.3 (a) Length and (b) biomass of lodgepole pine seedlings subjected to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment, determined 4, 8 and 12 months after sowing and inoculation (means and standard errors; n = 10 seedlings per treatment). Error bars with different letters are significantly different (P<0.05). 0510152025303540HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling length (cm) b aab babbbbbcbcabbbbcbab(a)020406080100120140HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling biomass (mg)bbaababbaacbcab bbcbdbaa(b) 145   Figure 5.4 (a) Length and (b) biomass of hybrid white spruce seedlings subjected to six endophytic diazotrophic bacterial treatments and a non-inoculated control treatment, determined 4, 8 and 12 months after sowing and inoculation (means and standard errors; n = 10 seedlings per treatment). Error bars with different letters are significantly different (P<0.05). 05101520253035HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling length (cm)(a)b bbb baabbbcbcab bbbbcbab010203040506070HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rControlHarvest 1 (4 months) Harvest 2 (8 months) Harvest 3 (12 months)Seedling biomass (mg)b bbb babbcababccabcbbbccac(b) 146    Figure 5.5 Endophytic colonization by the six endophytic diazotrophic bacterial strains in lodgepole pine (a) needle, (b) stem, and (c) root tissues, determined 4, 8 and 12 months after sowing and inoculation (expressed as colony forming units per gram fresh tissue; n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted and data were log-transformed. 0123456Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueNeedle(a)01234567Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueStem(b)4567Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueRootHP-S1r HP-N1r HP-R1rLP-S1r LP-R1r LP-R2r(c) 147    Figure 5.6 Endophytic colonization by the six endophytic diazotrophic bacterial strains in hybrid white spruce (a) needle, (b) stem, and (c) root tissues, determined 4, 8 and 12 months after sowing and inoculation (expressed as colony forming units per gram fresh tissue; n = 5 seedlings per treatment). For clarity of presentation, error bars were omitted and data were log-transformed.  01234Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueNeedle(a)012345Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueStem(b)3456Harvest 1(4 months)Harvest 2(8 months)Harvest 3(12 months)log cfu/g fresh tissueRootHP-S1r HP-N1r HP-R1rLP-S1r LP-R1r LP-R2r(c) 148 Chapter 6 - Sustaining the growth of Pinaceae trees under nutrient-limited edaphic conditions via plant-beneficial bacteria 6.1. Introduction Boreal ecosystems across the northern hemisphere are home to a wide variety of coniferous trees, mainly belonging to the Pinaceae family such as spruce, pine, fir and cedar. In Canada, one of the largest forested countries in the world, boreal forests cover around 77% of the total forest area (Canadian Forest Service 2019). Soils in these forests are generally regarded as immature, coarse-textured and acidic with an extremely slow release rate of plant nutrients due to factors such as high carbon : nutrient ratio, harsh climate, reduced microbial activity, and acidic, recalcitrant plant-litter. In addition, the changing global climate and growing anthropogenic activities are causing more frequent and serious disturbances to the boreal forest ecosystems (Deluca and Boisvenue 2012). The Sub-Boreal Pine-Spruce xeric-cold (SBPSxc) zone is one such nutrient-poor, disturbed region located in the central-interior of British Columbia (BC), a Canadian province with the highest timber production rate (42%) (Canadian Forest Service 2019). The SBPSxc zone is an extremely dry, cold montane region with a mean annual temperature of 1.7 °C and mean annual precipitation of 389 mm (Steen and Coupé 1997). Soils in this region (as in the rest of BC) are relatively young (10,000 years old) and develop very slowly due to harsh climatic conditions. These soils belong to the Brunisolic soil order according to the Canadian System of Soil Classification (‘Cambisols’ in the World Reference Base for Soil Resources and ‘Inceptisols’ in the US Soil Taxonomy). They are sandy (often gravelly) in texture since they  149 develop from granitic rocks and have thin or no organic forest floor with slow mineralization rates (Steen and Demarchi 1991). Previously, we analyzed soils from the West Chilcotin region of SBPSxc and concluded that they typically have poor physico-chemical health with high carbon to nutrient ratio, low cation exchange capacity, acidic pH, limited organic matter, high bulk density and minimal amounts of plant-available macro- and micro-nutrients (Puri et al. 2018a; Chapter 2). In addition, this region is frequently disturbed by wildfires, logging activity and attack of pests such as the mountain pine beetle, pine blister rust, pine gall rust and pine root collar weevil (Steen and Coupé 1997). In spite of such highly disturbed and extreme climatic and edaphic conditions, lodgepole pine (Pinus contorta var. latifolia) – a prominent member of the Pinaceae family found ubiquitously in Western North America – thrives in the West Chilcotin region.   Lodgepole pine has possibly the widest range of environmental tolerance among any coniferous species in North America (Lotan and Critchfield 1996). It has been reported to thrive under severe soil, moisture and topographical conditions including road cuts, mining sites, fire-affected regions and extremely dry, nutrient-poor sites (Bal et al. 2012; Chapman and Paul 2012; Padda et al. 2018; Turner et al. 2019). In recent studies, lodgepole pine trees were observed to naturally regenerate on a bare gravel substrate without topsoil at highly disturbed mining sites with growth rates similar to lodgepole pine trees growing at nearby undisturbed sites with intact topsoil (Chapman and Paul 2012; Padda et al. 2018). Lodgepole pine trees have also been observed to regenerate in Yellowstone National Park, USA on soils affected by high-severity fires which had incinerated several plant-essential nutrients (Turner et al. 2019). These studies suggest that lodgepole pine trees form close associations with potent plant-growth-promoting bacteria  150 (PGPB) and reliance on such bacteria could be an evolutionary approach for pine trees to sustain their growth under such stressful environments.  Plant-growth-promoting bacteria have been studied widely in agricultural and horticultural crops for decades and several agro-tech companies have made PGPB inoculants commercially available for farmers as an alternative to chemical fertilizers and pesticides (Doty 2017). However, the use of such inoculants in natural boreal and temperate forest stands to promote regeneration of coniferous trees is less common, primarily because the association of conifers with PGPB and the underlying mechanisms of tree growth promotion are understudied (Pirttilä and Frank 2018). Although limited, studies conducted so far have suggested that PGPB, whether residing in the rhizosphere or internal plant tissues (endophytes), could play a crucial role in supporting the growth of conifers under a range of environmental conditions (reviewed by Pirttilä and Frank 2011, 2018; Puri et al. 2017a). The principal mechanisms by which PGPB can promote plant growth include assisting the plants in obtaining nutrients such as nitrogen, phosphorus and iron; modulating the levels of phytohormones such as indole-3-acetic acid and ethylene to enhance plant growth and overcome abiotic and biotic stresses; and suppressing phytopathogens by degrading their cell walls or by enhancing natural plant defence mechanisms (Glick 2012). Experimentations have revealed that both rhizospheric and endophytic PGPB can be effective in promoting the growth and health of conifers in the Pinaceae family, such as Douglas-fir (Pseudotsuga menziesii), hybrid spruce (Picea glauca × engelmannii), lodgepole pine (Pinus contorta), white spruce (Picea glauca), Engelmann spruce (Picea engelmannii), Scots pine (Pinus sylvestris) and limber pine (Pinus flexilis) (Chanway 1997; Puri et al. 2017a; Pirttilä and Frank 2018).  151 Taking this into account, we hypothesized that lodgepole pine trees growing in the West Chilcotin region form beneficial associations with PGPB in order to survive on highly nutrient-poor and disturbed soils under dry, cold environmental conditions. To test this hypothesis, 48 potential PGPB strains were isolated from needle, stem, and root tissues of lodgepole pine trees growing at two different sites in this region (52° 00ʹ 04.2ʺ N, 124° 59ʹ 44.7ʺ W, 1003m a.s.l. and 52° 00ʹ 09.1ʺ N, 124° 59ʹ 25.2ʺ W, 1035m a.s.l.) (Puri et al. 2018a; Chapter 2). The ability of these bacteria to fix atmospheric nitrogen (a key PGP trait) was evaluated using in vitro and in vivo techniques and six bacterial strains were recognized as potent nitrogen-fixers (Puri et al. 2018a, 2020c; Chapters 2 and 5). As evidenced in several reports, PGPB can also enhance plant growth via multiple mechanisms other than nitrogen fixation (Padda et al., 2017b; Khan et al. 2015; Kandel et al. 2017a; Puri et al. 2017b), which led us to hypothesize that the bacteria we isolated may stimulate lodgepole pine tree growth in the nutrient-limited West Chilcotin region through PGP mechanisms unrelated to nitrogen fixation. To test this hypothesis, in vitro assays were used to examine various PGP traits of the six bacterial strains via qualitative and quantitative enzyme assays. The PGP ability of these six bacteria was also evaluated in planta by inoculating them into their natural host (lodgepole pine) in a long-term greenhouse growth trial (18 months) under nutrient-poor edaphic conditions. In addition, a similar greenhouse trial with hybrid white spruce was established to test the potential of these bacteria to promote the growth of another prominent Pinaceae tree species native to the West Chilcotin region.    152 6.2. Materials and Methods The antibiotic-resistant mutants of the six bacterial strains evaluated in this study were derived by streaking them multiple times on combined carbon medium (CCM) (Rennie 1981; Appendix B) amended with an antibiotic compound (200 mg/L rifamycin) (Bal and Chanway 2012a). The reason for raising such mutants was to track the endophytic and rhizospheric colonization by these bacterial strains in lodgepole pine and hybrid white spruce seedlings during the greenhouse growth trials. Previous studies confirmed that this spontaneous mutation does not affect the PGP efficiency of bacteria (Shishido et al. 1995; Bal and Chanway 2012a, Padda et al. 2019).  The antibiotic-resistant strains were stored in CCM broth amended with 200 mg/L rifamycin and 20% (v/v) glycerol at –80 °C. Unless otherwise stated, a frozen culture of each strain was thawed and streaked on CCM agar amended with 200 mg/L rifamycin and incubated for 48 h at 30 °C before being used in assays.  6.2.1. Greenhouse growth trials Two growth trials (18-month long) – one with lodgepole pine and the other with hybrid white spruce – were established in the University of British Columbia Plant Care Services’ greenhouse facility. Photosynthetically active radiation of at least 300 µmol/m2/s at the canopy level was provided during a 16-hour photoperiod (0600 h – 2200 h). Since each tree species has a different growth pattern that is not comparable to one another (von Wirén et al. 1997), each trial was analyzed separately. In each trial, 6 bacteria-inoculated and 1 non-inoculated control treatments were evaluated.   153 Lodgepole pine and hybrid white spruce seeds that originated from forest stands in the SBPS xc zone in BC (lodgepole pine – 51° 54’ N lat., 124° 51’ W long., elevation 1320 m; hybrid white spruce – 51° 57’ N lat., 124° 59’ W long., elevation 1350 m) were obtained from the BC Forest Service Tree Seed Centre, Surrey, BC, Canada. Surface sterilization of seeds was conducted by immersion in 30% (v/v) hydrogen peroxide for 90 sec followed by rinsing seeds thrice in sterile distilled water. The effectiveness of surface sterilization was confirmed by imprinting ten randomly selected seeds on tryptic soy agar (TSA) plates which were then incubated for 48 h at 30 °C to check for surface contamination. Surface-sterilized seeds were stratified using the protocol outlined by the Tree Seed Centre, i.e. storing the seeds aseptically for 28 days at 4 °C in sterile cheesecloth bags containing sterile moist silica sand (sterile). After the stratification period, ten randomly selected seeds were crushed and imprinted on CCM plates amended with 200 mg/L rifamycin and incubated for 48 h at 30 °C. The plates were examined for internal seed contamination by any of the six bacterial strains used in this study before their inoculation. Three surface-sterilized and stratified seeds of either lodgepole pine or hybrid white spruce were sown aseptically in a Ray Leach Cone-tainer (dia: 38 mm and ht: 210 mm) containing sterile soil growth media (filled to 67 % capacity). The soil medium consisted of silica sand (69% w/w), Turface (29% w/w) and dolomite (2% w/w), and was fertilized with limited amounts of sterile nutrient solution (20 mL), containing Ca(NO3)2 (Appendix C) at the beginning of the growth trials and every 30 days thereafter (without Ca(NO3)2). Since the objective was to grow seedlings in the greenhouse under nutrient-poor conditions, similar to what were observed in the SBPSxc region, the content of major nutrients including nitrogen and phosphorus in the fertilizer was kept similar/lower than the nutrient content in soils of the SBPSxc region (Puri et al. 2018a; Chapter 2). A bacterial  154 suspension (5 mL) of each strain was applied directly over the seeds in each Cone-tainer designated for that strain. The bacterial suspension of each strain was prepared by inoculating a loopful of bacterial growth from CCM plates into CCM broth amended with 200 mg/L rifamycin and agitating the broth at 150 rpm for 48 h at 30 °C. Bacterial cells were harvested by centrifugation at 3000 xg for 30 min, washed twice in sterile phosphate buffered saline (PBS) (pH 7.4) (Appendix A) and resuspended in the same buffer to a density of ca. 106 cfu/mL. Non-inoculated control seeds received 5 mL of sterile PBS without any bacteria. Two weeks after sowing, seedlings were thinned to the single largest germinant per Cone-tainer and were provided with sterile distilled water as required. The effect of bacterial colonization on the growth of lodgepole pine and hybrid white spruce seedlings was evaluated 18 months after sowing. Ten randomly selected seedlings per tree species from each treatment were analyzed. Seedlings were removed from Cone-tainers and the total seedling length was measured. Seedlings were then oven-dried at 65 °C for 48 h to determine their dry weight biomass. Five seedlings per tree species from each treatment were randomly selected to evaluate rhizospheric colonization in lodgepole pine and hybrid white spruce 18 months after sowing. Seedlings were removed from Cone-tainers and gently shaken to remove loosely adhering soil particles from roots. Roots were separated from shoots, placed in Falcon tubes containing 10 mL of sterile PBS and vortexed at high setting for 1 min. Serial dilutions were performed and a 0.1 mL aliquot of each dilution was plated on CCM agar amended with 200 mg/L rifamycin. The number of bacterial colonies on each plate was counted after an incubation period of 7 days at 30 °C. Ideally, rifamycin should inhibit the growth of all bacteria except the six used in this study since they had acquired antibiotic resistance. Roots were oven- 155 dried for 48 h at 65 °C to determine their dry weight. Rhizospheric populations were calculated as colony forming units (cfu) per gram of dry root tissue. Similarly, five seedlings per tree species from each treatment were randomly selected to enumerate the endophytic colonies in internal needle, stem and root tissues of lodgepole pine and hybrid white spruce 18 months after sowing. Seedlings were surface-sterilized by immersing them in 1.3% NaOCl for 5 min and washing them thrice with sterile distilled water. The effectiveness of surface-sterilization was confirmed by imprinting the seedlings on TSA plates and incubating the plates for 24 h at 30 °C to check for surface contamination. Using a mortar and pestle, a sample from each tissue (needle, stem and root) was triturated separately in 1 mL of sterile PBS. Serial dilutions of triturated suspensions were performed before plating a 0.1 mL aliquot of each dilution on CCM agar amended with 200 mg/L of rifamycin. After incubating the plates for 7 days at 30 °C, the number of colonies was counted. The endophytic population in each tissue was calculated as cfu per gram of fresh tissue. 6.2.2. Direct mechanisms of plant-growth-promotion 6.2.2.1. Nutrient acquisition The potential to help the host plant in accessing unavailable nutrients, such as insoluble organic and inorganic phosphates and complexed iron compounds, was evaluated for the six bacterial strains using in vitro assays. The ability to convert inorganic tri-Ca phosphate into more soluble mono- and di-calcium phosphates was evaluated using qualitative plate-based and quantitative broth-based analyses. For the qualitative assay, each bacterial strain was spot-inoculated on Pikovskaya’s (PVK) agar medium plates containing 0.5% tri-Ca phosphate (Pikovskaya 1948). After incubating the plates for 14 days at 30 °C, phosphate solubilization was determined by the  156 occurrence of a clear halo around bacterial growth. The phosphate solubilization ability on plates was expressed using the solubilization index (Khan et al. 2015), where solubilization index (SI) = (halo + colony diameter) / colony diameter. To quantify the amount of phosphate solubilized, each bacterial strain was inoculated in PVK liquid broth to a concentration of 106 cfu/mL and incubated for 72 h at 30 °C in a shaking incubator (180 rpm). Subsequently, the culture supernatant was extracted via centrifugation for 10 min at 8000x g and 1 mL of this supernatant was mixed with 500 µL of 10% (w/v) trichloroacetic acid and 4 mL of the colour reagent (1 : 1 : 1 : 2 ratio of 3M H2SO4 : 2.5% (w/v) ammonium molybdate : 10% (w/v) ascorbic acid : distilled water). After incubation for 15 min at room temperature, the absorbance of the blue colour that developed in the resulting solution was measured at 820 nm. The amount of soluble phosphates produced by each strain per mL of the PVK medium (µg/mL) was estimated using a standard KH2PO4 curve (Chaiharn and Lumyong 2009).  The ability of each bacterial strain to hydrolyze phytate (an organic form of plant-unavailable phosphate) using the phytase enzyme was evaluated in both qualitative and quantitative assays. For qualitative evaluation, each strain was spot-inoculated onto phytase screening medium (PSM) agar plates containing sodium phytate (Kerovuo et al. 1998). After incubating the plates for 14 days at 30 °C, phytate hydrolyzation was determined by the development of a clear halo around bacterial growth and was expressed as SI. For quantitative evaluation, each bacterial strain was inoculated to a concentration of 106 cfu/mL in PSM broth and incubated at 30 °C for 72 h in a shaking incubator (180 rpm). The culture supernatant was then extracted via centrifugation for 15 min at 8000x g and 150 µL of the supernatant was mixed with 600 µL of a solution containing 0.1M tris-HCl, 2mM sodium phytate and 2mM CaCl2.  157 Following the incubation period of 30 min at 37 °C, 750 µL of 5% (w/v) trichloroacetic acid and 750 µL of the colour reagent (4 : 1 ratio of 1.5% (w/v) ammonium molybdate in 5.5% (v/v) H2SO4 : 2.7% (w/v) ferrous sulfate solution) were added. After 5 min of incubation, the absorbance of the resulting solution was measured at 700 nm. The standard curve of KH2PO4 was used to estimate the amount of soluble phosphorus released by each bacterium by hydrolyzing phytate. One unit (U) of phytase activity was defined as the amount of phytase enzyme required to liberate 1 nmol of soluble phosphorus per minute under the given assay conditions and is expressed per mL of PSM culture (Yanke et al. 1998).  To evaluate siderophore production by the six bacterial strains, each strain was spot-inoculated on chrome azurol S (CAS) agar plates and incubated for 7 days at 30 °C (Louden et al. 2011). The colour change from blue to orange/deep yellow around the bacterial growth on the CAS agar plates indicated the production of siderophores by bacteria. This area of the orange halo was measured and expressed as cm2 (Kandel et al. 2017a). 6.2.2.2. Plant growth hormone modulation The ability of the six bacterial strains to modulate vital plant hormones (IAA and ethylene) in order to enhance the growth and development of the host plant was analyzed. To evaluate the in vitro production of IAA, each bacterial strain (ca. 106 cfu/mL) was inoculated into Luria Bertani broth amended with 5 mM L-tryptophan and incubated for 72 h at 28 °C in a shaking incubator (150 rpm) (Bric et al. 1991). After centrifugation (8000x g; 15 min), 1 mL of culture supernatant was mixed with 100 µL of orthophosphoric acid (10mM) and then 2 mL of the Salkowski’s reagent (1 : 30 : 50 ratio of 0.5M FeCl3 : 95% (w/w) sulfuric acid : distilled water) was added. The resulting  158 solution was incubated for 15 min at room temperature and the absorbance of the pink colour that developed was measured at 530 nm (Glickmann and Dessaux 1995). A standard curve of pure IAA was used to estimate the amount of IAA produced by each strain per mL of the growth medium (Gordon and Weber 1951). The ACC deaminase activity of the six bacterial strains was examined using in vitro and in vivo techniques described by Penrose and Glick (2003). Each strain was grown to stationary phase in tryptic soy broth (nutrient-rich medium) at 30 °C. Bacterial cells of each strain were harvested via centrifugation at 8000x g. To induce the ACC deaminase activity, bacterial cells were suspended in DF salts minimal medium (nutrient-poor medium) amended with 3mM ACC as the sole source of nitrogen and grown for 24 h at 30 °C in a shaking incubator (200 rpm). The bacterial cells were harvested via centrifugation (8000x g), washed and suspended either in 0.1M Tris-HCl for in vitro analysis or 0.03M MgSO4 for in vivo analysis. For the in vitro assay, bacterial cells suspended in 0.1M Tris-HCl were mixed with toluene and a portion of the toluene-treated cells was mixed with 0.5M ACC and incubated for 15 min at 30 °C. After adding 0.56M HCl, the solution was mixed, and the supernatant was collected by centrifugation (16000x g). The supernatant was then mixed with 0.56M HCl and 2,4-dinitrophenylhydrazine reagent (0.2% 2,4-dinitrophenylhydrazine in 2M HCl) and incubated for 30 min at 30 °C. After adding 2M NaOH, the absorbance of the resulting solution was measured at 540 nm. ACC deaminase activity was quantified using pure a-ketobutyrate as the standard and expressed as the amount of a-ketobutyrate produced per mg protein per hour. To analyze ACC deaminase activity in vivo, a gnotobiotic root elongation assay was used involving ethylene-sensitive plants – canola (Brassica napus) and tomato (Solanum lycopersicum). Canola seeds (var. Rugby Roundup ready) were  159 obtained from the SeCan Association’s Alberta branch (Lamont, AB, Canada). Tomato seeds (var. Celebrity) were obtained from the West Coast Seed Company, Delta, BC, Canada. For surface-sterilization, seeds were immersed in 30% hydrogen peroxide for 90 sec and washed thrice in sterile distilled water. Effectiveness of the surface sterilization was confirmed by imprinting ten randomly selected canola and tomato seeds on tryptic soy agar (TSA) plates which were incubated for 48 h at 30 °C to check for surface contamination. The ACC-induced bacterial cells of each strain suspended in 0.03M MgSO4 (OD600 = 0.15) were used in this assay. Surface-sterilized canola and tomato seeds were incubated in Petri dishes for 1 hr with one of the following treatments: sterile 0.03M MgSO4 (control) or bacterial suspensions of each of the six strains. Following the incubation period, 7 seeds per plant species from each treatment were aseptically placed in sterile CYG™ germination pouches (Mega International, Newport, MN, USA) containing 15 mL of sterile distilled water. Subsequently, the pouches were incubated in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB, Canada) maintained at 20 °C with a day/night cycle beginning with 12 h of dark followed by 12 h of light, with light intensity set to 18 µmol/m2/s.  The primary root lengths of canola and tomato seedlings from each treatment were measured five days after germination. 6.2.3. Indirect mechanisms of plant-growth-promotion 6.2.3.1. Phytopathogen suppression via cell wall degradation The ability of the six bacterial strains to secrete key cell wall degrading enzymes (chitinase, b-1,3-glucanase, protease and cellulase) to suppress pathogens was evaluated in vitro. Qualitative evaluation of chitinase activity included spot-inoculating each strain on chitin agar plates (Sahoo  160 et al. 1999). After incubation for 7 days at 30 °C, a clear halo surrounding the bacterial growth indicated positive chitinase activity and the width of the clearance zone was calculated as = (halo + colony diameter) – (colony diameter). The amount of colloidal chitin converted to simple sugars due to the chitinase activity was quantified by inoculating each bacterial strain to a concentration of 106 cfu/mL in liquid chitin medium. After incubating for 5 days at 30 °C in a shaking incubator (150 rpm), the culture supernatant was extracted by centrifugation for 15 min at 8000x g and 500 µL of the supernatant was mixed with 500 µL of 1 M phosphate buffer and 500 µL of colloidal chitin solution containing 10 mg chitin. The resulting solution was incubated for 30 min at 37 °C and centrifuged for 3 min at 8000x g to collect the supernatant. The supernatant (1 mL) was mixed with dinitrosalicylic acid (2 mL) and heated for 5 min in a boiling water bath. The absorbance of the final solution was measured at 575 nm (Chaiharn and Lumyong 2009). Using glucose as the standard, the chitinase enzyme activity was estimated by measuring the release of reducing sugars from chitin. One unit (U) of chitinase activity was defined as the amount of chitinase enzyme that resulted in the release of 1 µmol of glucose from colloidal chitin per minute. The b-1,3-glucanase activity was evaluated qualitatively by spot-inoculating each bacterial strain on plates containing b-1,3-glucan (laminarin) as the sole carbon source (5 g/L) along with other essential nutrients outlined by Renwick et al. (1991). Following incubation for 3 days at 30 °C, plates were stained with Congo Red (0.6 g/L) and left at room temperature for 90 min. The hydrolysis of glucan (i.e. glucanase activity) was indicated by the development of a yellow/orange zone around the bacterial growth on plates and the width of this yellow/orange zone was measured. For the quantitative determination of b-1,3-glucanase activity, the  161 aforementioned laminarin medium (without agar) was inoculated with each bacterial strain (ca. 106 cfu/mL) and incubated for 5 days at 30 °C in a shaking incubator (150 rpm). The culture supernatant was extracted by centrifugation for 15 min at 8000x g and 500 µL of the supernatant was mixed with 500 µL of 1 M citrate buffer (pH 5.0) and 500 µL of 4% laminarin. After an incubation period of 30 min at 37 °C, 2 mL of dinitrosalicylic acid was added and the solution was heated for 5 min in a boiling water bath. The absorbance of the resulting solution was measured at 500 nm (Chaiharn and Lumyong 2009). The b-1,3-glucanase activity was estimated by measuring the release of reducing sugars from laminarin using glucose as the standard. One unit (U) of b-1,3-glucanase activity was defined as the amount of b-1,3-glucanase enzyme that resulted in the release of 1 µmol of glucose from laminarin per minute. To evaluate the protease enzyme activity qualitatively, each bacterial strain was spot-inoculated on casein–yeast extract (CYE) agar plates amended with 7% skimmed milk powder (Padda et al. 2017a). The plates were incubated for 7 days at 30 °C and the development of a clear zone surrounding the bacterial growth indicated protease activity. The width of the clear zone was measured. To quantify the protease enzyme activity, each bacterial strain was inoculated to a concentration of 106 cfu/mL in CYE liquid medium amended with 7% skimmed milk powder and incubated for 5 days at 30 °C in a shaking incubator (150 rpm). The culture supernatant was extracted by centrifugation for 15 min at 8000x g and 500 µL of the supernatant was mixed with 500 µL of 0.2 M phosphate buffer and 500 µL of 1% azocasein. After incubation for 30 min at 37°C, 2 mL of 10% (w/v) trichloroacetic acid was added and the solution was further incubated for 5 min at room temperature. The absorbance was measured at 440 nm after the addition of 1M NaOH (1mL). Protease activity was determined by measuring the release of  162 reducing sugars from azocasein using tyrosine as the standard. One unit of protease enzyme activity was defined as the amount of the protease enzyme that resulted in the release of 1 µmol of tyrosine from azocasein per minute (Chaiharn and Lumyong, 2009). The cellulase enzyme activity for each bacterial strain was assessed qualitatively by spot-inoculating on CYE agar plates amended with 1% sodium carboxymethylcellulose (Yang et al. 2017). After incubating for 48 h at 30 °C, the plates were flooded with Congo red solution (0.5% w/v) and left at room temperature for 30 min. Subsequently, plates were drained and rinsed with 1 mol/L of NaCl and the development of a clear zone around the bacterial growth indicated cellulase activity. The width of the clearance zone was measured. For quantitative evaluation of cellulase activity, each strain was inoculated to a concentration of 106 cfu/mL in CYE liquid medium amended with 1% sodium carboxymethylcellulose and incubated for 5 days at 30 °C in a shaking incubator (150 rpm). The culture supernatant was extracted via centrifugation for 15 min at 8000x g and 500 µL of the supernatant was mixed with 500 µL of 1M citrate buffer and 500 µL of 1% carboxymethylcellulose. After an incubation period of 30 min at 37 °C, dinitrosalicylic acid (2 mL) was added and the solution was heated for 5 min in a boiling water bath. The absorbance of the resulting mixture was measured at 500 nm (Sahoo et al. 1999). Using glucose as the standard, cellulase activity was estimated by measuring the release of reducing sugars from carboxymethylcellulose. One unit (U) of cellulase enzyme activity was defined as the amount of cellulase enzyme that resulted in the release of 1 µmol of glucose from carboxymethylcellulose per minute (Chaiharn and Lumyong 2009).   163 6.2.3.2. Ammonia production The six bacterial strains were analyzed for in vitro production of ammonia by inoculating each strain (ca. 106 cfu/mL) into 10 mL of peptone water and incubating for 72 h at 30 °C. After that, 500 µL of Nessler’s reagent was added and the development of a brown-yellow colour indicated ammonia production (Dey et al. 2004). 6.2.3.3. Catalase enzyme activity Catalase activity was evaluated for each bacterial strain by mixing a loopful of fresh bacterial culture with 50 µL of 3% (v/v) hydrogen peroxide on a sterile glass slide and incubating at room temperature for 1 min. The evolution of oxygen, i.e. development of gas bubbles indicated a positive catalase reaction (Padda et al. 2017a). 6.2.4. Statistical analyses Each plant growth trial was arranged in a completely randomized experimental design with 20 replicates per treatment (5 replicates to evaluate rhizospheric colonization, 5 replicates to evaluate endophytic colonization, and 10 replicates to evaluate seedling length and biomass). Statistical analysis for each greenhouse trial (one involving lodgepole pine and the other involving hybrid white spruce) was performed separately since each tree species has a different growth pattern, not comparable to one another (von Wirén et al. 1997). Analysis of variance (ANOVA) was performed (F-test and Student’s t-test) to determine significant differences between treatment means for seedling length and seedling biomass in each trial. ANOVA was also performed to determine differences between treatment means for the quantitative assays  164 evaluating phosphate solubilization, phytate hydrolyzation, ACC deaminase activity, IAA production, cellulase activity, protease activity, chitinase activity and b-1,3-glucanase activity as well as for siderophore production and root elongation in the gnotobiotic assay. Three replicates per treatment were evaluated for all quantitative assays as well as for siderophore production, and seven replicates per treatment were evaluated for the gnotobiotic root elongation assay. The statistical package, SAS University Edition (SAS Institute Inc., Cary, NC, USA), was used to perform all statistical analyses (a = 0.05). 6.3. Results 6.3.1. Long-term greenhouse growth trials Each of the six bacterial strains analyzed in this study had a significant positive effect on the growth of 18-month old lodgepole pine and hybrid white spruce seedlings. All bacterial treatments significantly promoted the length and biomass of pine and spruce seedlings as compared to the non-inoculated control treatment (Figures 6.1 and 6.2). Bacteria-inoculated pine seedlings had > 125% higher biomass and were > 30% longer than the non-inoculated control seedlings (Figure 6.1). Similarly, spruce seedlings inoculated with bacteria were also > 30% longer and had > 130% higher biomass than the control seedlings (Figure 6.2). Notably, the pine and spruce seedlings inoculated with strains HP-S1r, LP-R1r and LP-R2r were around 50% longer than controls (Figures 6.1a and 6.2a). Moreover, inoculation with these three strains enhanced pine seedling biomass by > 200% (Figure 6.1b) and spruce seedling biomass by > 275% (Figure 6.2b) as compared to the control.  165  Each of the six bacterial strains evaluated in this study was able to form rhizospheric and endophytic colonies in their original host (lodgepole pine) as well as the foreign host (hybrid white spruce). The bacterial strains, on average, had population sizes of 104 – 106 cfu/g dry root tissue in the rhizosphere of pine and spruce seedlings, 18 months after inoculation (Figure 6.3). Similar population sizes were observed in the internal root tissues of pine and spruce seedlings. All strains were able to colonize the stem tissues with population sizes ranging from 103 to 105 cfu/g tissue in pine and 103 to 104 cfu/g tissue in spruce (Figure 6.3). Needle colonization was observed for all strains except HP-N1r in pine seedlings, with population densities ranging between 101 and 105 cfu/g tissue (Figure 6.3a). Spruce needles were colonized by four bacterial strains only (101 – 103 cfu/g tissue) as no colonies were observed in the needle tissues of HP-N1r and HP-R1r inoculated spruce seedlings (Figure 6.3b). Strains HP-S1r and LP-R2r showed the highest colonization potential in stem and needle tissues of pine and spruce (up to 105 cfu/g tissue). Moreover, these two strains along with strain LP-R1r colonized the root tissues and rhizosphere of pine and spruce extensively (106 – 107 cfu/g tissue). No bacterial colonies were observed in the rhizosphere and internal tissues of non-inoculated control pine and spruce seedlings. 6.3.2. Direct mechanisms of plant-growth-promotion The ability of bacteria to convert inorganic tri-Ca phosphate and organic phytate into more soluble forms (i.e. plant-available) was evidenced for all strains except HP-N1r (Tables 6.1 and 6.2). The SI for phosphate solubilization ranged from 1.2 to 2.6 whereas for phytate hydrolyzation it ranged from 2.0 to 3.6 (Table 6.1). These results were consistent with the quantitative results  166 since the same bacterial strains solubilized phosphate and phytate in broth-based assays (Table 6.2). The amount of soluble phosphates detected in the tri-Ca phosphate solubilization test ranged between 66 and 112 µg/mL (Table 6.2). For the phytate hydrolyzation test, 42 – 86 units of phytase enzyme were detected per mL of the broth (Table 6.2). Strain LP-R2r solubilized > 2.5 times tri-Ca phosphate and > 3.5 times phytate relative to its growth on plates. In addition, this strain solubilized significantly higher amounts of phosphate (up to 68%) and secreted significantly greater amounts of phytase (up to 103%) than all other bacterial strains in the broth assays. Strains HP-S1r and LP-R1r also showed considerable phosphate solubilization and phytate hydrolyzation activities in both qualitative and quantitative evaluations (Tables 6.1 and 6.2). The ability to produce siderophores was observed for all strains except HP-R1r on the CAS blue agar. The area of the orange halo produced by the bacterial strains on CAS agar plates ranged from 1.63 to 2.73 cm2, with strain LP-R2r producing the largest orange halo closely followed by strain LP-R1r (Table 6.1). All bacterial strains were capable of producing IAA and ACC deaminase in vitro. The amount of IAA produced from L-tryptophan by bacterial strains ranged from 15 to 32 µg/mL of broth, with strains LP-R2r, HP-S1r and LP-R1r producing significantly higher IAA than other bacterial strains (Table 6.2). The amount of a-ketobutyrate produced (when ACC deaminase enzyme cleaves ACC) by strains ranged between 33 and 111 nmol per mg of protein in an hour (Table 6.2). Notably, strain LP-R2r produced a significantly higher amount of a-ketobutyrate than all other strains. In addition, strains HP-S1r and LP-R1r also produced considerable amounts of a-ketobutyrate in this assay. Similar ACC deaminase activity was observed in the plant-based gnotobiotic assay. All bacterial strains significantly enhanced the root length of canola and  167 tomato plants in the gnotobiotic assay by synthesizing ACC deaminase to suppress the overproduction of plant-produced ethylene after germination. The primary root length of five-day-old bacteria-inoculated canola and tomato plants was more than 2-fold greater compared to control plants (Figures 6.4 and 6.5). Notably, canola and tomato plants inoculated with strain LP-R2r had the longest primary roots of all treatments (487% and 237%, respectively greater than controls). In addition, strains HP-S1r and LP-R1r promoted canola root length by 340% and 432%, respectively, and tomato root length by 204% and 193%, respectively (Figures 6.4 and 6.5). 6.3.3. Indirect mechanisms of plant-growth-promotion All six bacterial strains were able to produce at least one of the four major cell wall degrading enzymes (chitinase, b-1,3-glucanase, protease and cellulase). Chitinase enzyme activity was observed in four strains in both qualitative plate-based and quantitative broth-based assays, but strains HP-N1r and HP-R1r showed no activity (Tables 6.1 and 6.2). Strain HP-S1r showed the greatest chitinase activity by solubilizing around 15-25 mm of the colloidal chitin relative to its growth in the plate assay and by producing around 0.52 units of chitinase enzyme in the broth assay. The b-1,3-glucanase activity was detected in three strains only (HP-S1r, LP-R1r and LP-R2r) in the qualitative plate-based assay, which was consistent with the results of the quantitative broth-based assay (Tables 6.1 and 6.2). Strain LP-R2r showed the highest b-1,3-glucanase activity in both qualitative and quantitative assays and was significantly higher than all other bacterial strains. The ability to synthesize protease was observed in all strains except HP-R1r (Tables 6.1 and 6.2). Strain LP-R1r showed the greatest protease activity in the plate assay (15-25 mm clear zone) and broth assay (82 units of protease enzyme produced per mL of broth). Cellulase activity  168 was observed for all but one strain (HP-N1r) in plate-based and broth-based assays (Tables 6.1 and 6.2). Strains LP-R2r and LP-R1r showed the greatest abilities to degrade cellulose as indicated by the clearance zone size around bacterial growth on plates and by the amount of cellulase enzyme produced per mL of broth. It is interesting to note that strains HP-S1r, LP-R1r and LP-R2r showed positive activity for all 4 cell wall degrading enzymes.  All bacterial strains were able to produce ammonia in vitro (Table 6.1). Particularly, strain LP-R1r showed the highest ammonia production of all strains, followed by strain LP-S1r that showed medium ammonia production. Catalase enzyme activity was observed for all but one strain (HP-N1r), with strain LP-R2r showing the highest catalase activity (Table 6.1). 6.4. Discussion In this study, our main motive was to investigate the PGP abilities of endophytic bacteria, isolated from lodgepole pine trees growing in a nutrient-poor, disturbed ecosystem in the West Chilcotin region in BC. When the six selected endophytic bacteria were analyzed for their potential to enhance tree growth via inoculation studies with their original host (lodgepole pine) and a foreign host (hybrid white spruce), it was observed that all strains were effective in significantly increasing the length and biomass of both tree hosts. After 18 months of inoculation, the length and biomass of pine and spruce seedlings treated with bacteria had increased by 30-60% and 125-302%, respectively, compared to the non-inoculated controls (Figures 6.1 and 6.2). Comparable growth promotion has been observed in previous greenhouse studies, conducted for a similar time period, in which endophytic PGPB were inoculated into Pinaceae trees such as lodgepole pine, Douglas-fir, Scots pine, western red cedar and hybrid white spruce (Shishido and  169 Chanway 2000; Anand et al. 2013; Pohjanen et al. 2014; Khan et al. 2015; Aghai et al. 2019). Since pine and spruce seedlings were grown in soil media fertilized with limited quantities of nutrients in the greenhouse trials, the growth enhancement achieved by bacteria-inoculated seedlings in comparison to control seedlings under stress conditions indicates that all six bacteria possess one or more PGP abilities, which was observed in our in vitro enzyme-based assays.  Forming a close association with their host is a major strategy employed by PGPB to stimulate plant growth and health (Frank et al. 2017). This was true for our bacteria since all strains had formed ten thousand to ten million colonies per gram tissue in the rhizosphere and internal root tissues of spruce and pine seedlings, 18 months after inoculation (Figure 6.3). Internal stem colonization was also detected for all bacterial strains; however, needle colonization was observed for certain strains only, which indicates niche preferences exerted by either the bacterium or the tree host (Kandel et al. 2017b). The rhizospheric and endophytic population sizes observed in our study align with those observed for other PGPB in coniferous as well as deciduous tree species (Brooks et al. 1994; Germaine et al. 2004; Shishido et al. 1995; Anand and Chanway 2013; Khan et al. 2015; Tang et al. 2017). Plant colonization by all of our bacterial strains could be linked to their potential to synthesize IAA (Table 6.2) since studies have suggested that biosynthesis of IAA by bacteria can help circumvent host plant’s defence responses in order to colonize the rhizosphere and internal tissues (Suzuki et al. 2003; Spaepen et al. 2007). In addition, the ability of all bacterial strains except HP-N1r to secrete catalase enzyme (Table 6.1) could also be related to their potential to colonize and survive in the rhizosphere and internal tissues under nutrient-stress conditions. Catalase can neutralize the overproduction of reactive oxygen species (ROS) under stress conditions in bacteria. This enzyme  170 can also counteract the plant-secreted ROS, thus helping the bacteria to colonize internal plant tissues (Bumunang and Babalola 2014). Entry to and survival in internal plant tissues is also facilitated by the secretion of protease and cellulase enzymes since these enzymes can disintegrate and metabolize plant cell wall polymers, proteins and other organic compounds in the apoplast (Hurek et al. 1994; Reinhold-Hurek et al. 2006). Since all strains possess the ability to secrete at least one of these enzymes (Tables 6.1 and 6.2), it can be suggested that internal tissue colonization may have resulted due to the functioning of these enzymes. It is interesting to note that four strains that showed the presence of all three enzymes – catalase, cellulase and protease – were the only ones that were able to colonize the needle tissues of both pine and spruce (Figure 6.3), potentially indicating the combined role of these enzymes in helping the bacteria to enter, move, survive and multiply in plants, particularly in the needle tissues. Comparing the different bacterial treatments, it can be observed that the colonization density appears to have a similar trend as seedling growth (length and biomass), suggesting that the number of colonies formed by each bacterial strain may directly affect their efficacy to enhance the growth of pine and spruce seedlings. Similar observations have been reported in inoculation studies with interior spruce, lodgepole pine, poplar, corn, canola and tomato (Shishido et al. 1995; Germaine et al. 2004; Yang et al. 2016; Padda et al. 2016a; Puri et al. 2016b). In addition, the best plant colonizers in our greenhouse growth trials – Caballeronia sordidicola HP-S1r, Caballeronia udeis LP-R2r and Paraburkholderia phytofirmans LP-R1r – were the best plant growth promoters as they promoted seedling length by up to 60% and seedling biomass by up to 302% (Figure 6.1, 6.2 and 6.3). However, it is difficult to determine whether this plant-growth-promotion effect was due to the rhizospheric population or endophytic population, or  171 due to a synergistic effect of both populations, therefore further research focusing on this subject is necessary. Phytohormones such as IAA and ethylene play a crucial role in the growth and development processes of a plant such as seed germination, root development and proliferation, stem and root elongation, reproduction, and fruit ripening (Glick 2012). Modulation of these hormones by PGPB living in close association with the host plant is a well-known phenomenon to enhance the growth and health of the host plant in exchange for energy for the microbes (Kamilova et al. 2006). For instance, when the endogenous production of IAA by plants is insufficient to support their growth and development, reliance on exogenous IAA produced by associative PGPB can be a viable alternative (Xin et al. 2009; Kandel et al. 2017a). Such PGPB convert L-tryptophan, a metabolite commonly present in plant exudates, into IAA indicating the development of a mutually-beneficial relationship between the PGPB and the host plant. The ability to convert L-tryptophan to IAA was confirmed in all of our strains via in vitro broth assay (Table 6.2). Since all strains were also observed to significantly enhance the length and biomass of pine and spruce seedlings in the greenhouse trials (Figures 6.1 and 6.2), our results are consistent with the theory that IAA production leads to greater elongation, proliferation and development of the plant tissues (Glick 2012; Madmony et al. 2005; Kamilova et al. 2006). In particular, the highest IAA producing strains – C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r – were also the best performing strains in the long-term greenhouse which further support this theory. The significant plant growth promotion observed specifically under nutrient-stress conditions in the greenhouse can also be linked to the ability of all of our strains to modulate plant-ethylene levels by releasing ACC deaminase (Table 6.2). Plants produce excess  172 amounts of ethylene when subjected to stress conditions which can inhibit their growth and development (Morgan and Drew 1997). However, certain associative PGPB produce ACC deaminase to cleave ACC – the precursor of plant-ethylene – and convert it to α-ketobutyrate and ammonia, thereby reducing excess plant-ethylene levels (Penrose and Glick 2003; Glick 2012). ACC deaminase producing bacteria utilize the ammonia produced from this reaction for their own metabolism. A binary approach – in vitro enzyme assay and in vivo gnotobiotic assay – was used to evaluate the ACC deaminase activity of our bacterial strains. The amount of ACC converted to α-ketobutyrate by our bacterial strains (33 – 111 nmol/mg/h) in the in vitro enzyme assay was analogous to the typical range observed in previous studies with endophytic bacteria (Glick et al. 2007; Sun et al. 2009; Xing et al. 2012; Koskimäki et al. 2015). All bacteria also showed positive ACC deaminase activity when inoculated into ethylene-sensitive plants – canola and tomato – in the in vivo gnotobiotic assay (Figure 6.4). The primary root length of inoculated canola and tomato seedlings was 2-fold or higher than the non-inoculated control seedlings, which is consistent with the findings of Anandham et al. (2008) and Onofre-Lemus et al. (2009). Notably, strains C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r that produced the highest amounts of a-ketobutyrate in the in vitro assay, also showed highest root length enhancement for both canola (up to 6-fold) and tomato (up to 3-fold) in the in vivo assay. It has been postulated that alleviation of excess plant-ethylene levels after seed germination by ACC deaminase producing bacteria is a priming effect to significantly enhance the root length of inoculated seedlings after germination. Although excess ethylene levels are required to break seed dormancy, high levels after germination could lead to stunted growth. However, ACC deaminase producing bacteria could negate this effect (Penrose and Glick 2003; Glick 2012).  173  In addition to their own defence mechanisms, plants rely on closely-associated PGPB to suppress the population of phytopathogens by secreting secondary metabolites. Lytic enzymes, including cellulase, protease, chitinase and b-1,3-glucanase, represent a major category of secondary metabolites released by PGPB to lyse fungal and oomycete cell walls as well as cuticles and eggshells of pests such as nematodes (Chet and Inbar 1994; Khan et al. 2017; Marin-Bruzos and Grayston 2019). All of our strains tested positive for the presence of at least one of these four enzymes in both qualitative and quantitative assays, with comparable results observed for each strain in both assay types. The enzyme units of cellulase, protease, chitinase and b-1,3-glucanase produced by our strains fall within the usual range detected for rhizospheric and endophytic PGPB (Farah et al. 2006; Chaiharn and Lumyong 2009; Jadhav et al. 2017; Kubiak et al. 2018; Terhonen et al. 2018). However, the enzyme activity detected under in vitro conditions should be interpreted with caution because the ‘actual’ production of these enzymes may vary under in vivo conditions where a variety of external factors can affect the syntheses of these enzymes. Each lytic enzyme has a specific function to degrade particular compounds in the cell walls and cuticles of pathogens. For example, cellulase enzyme degrades the glycosidic linkages in cellulose chains that form intra- and inter-molecular hydrogen bonds, protease enzyme breaks the peptide bonds present in the protein matrix, chitinase enzyme disintegrates the rigid chitin polymer, and b-1,3-glucanase enzyme degrades the β-1,3-linked backbone of glucan, a cell wall polysaccharide (Jadhav et al. 2017). Notably, the activity of all four enzymes was detected only in C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r, which indicates that these antagonistic strains could be effective at controlling phytopathogens by possibly exerting a synergistic biocontrol mechanism. Another common biocontrol mechanism includes the  174 production of toxic compounds such as ammonia gas by PGPB (Weise et al. 2013). All of our strains possessed the ability to produce ammonia gas as observed by the formation of yellow/brown colour when Nessler’s reagent – a common ammonia-detecting compound – was added to the broth. In comparison to other strains, the development of darker brown colour for P. phytofirmans LP-R1r indicated the highest ammonia production by this strain. In addition to synthesizing this toxic ammonia gas, certain ammonia-producing enzymes of PGPB, including the phenylalanine ammonia lyase (PAL) enzyme, are also responsible for eliciting induced systemic resistance (ISR) in plants against biotic stresses (Yasmin et al. 2016). PAL catalyzes the deamination of L-phenylalanine (an a-amino acid) to yield ammonia gas (Camm and Towers 1973), thereby initiating the phenylpropanoid pathway that results in the production of phytoalexins, phenolic compounds, flavonoids and lignins to deter pathogens (Rais et al. 2017).   Siderophore production in combination with the ability to synthesize lytic enzymes and toxic gases is believed to be extremely lethal against phytopathogens, particularly fungi (Sindhu et al. 1999; Foyer and Noctor 2005; Rais et al. 2017). In addition to efficiently chelating the Fe3+ molecules present in soil and depriving the pathogens from taking up iron, siderophores produced by PGPB are also responsible for triggering ISR in plants (Schippers et al., 1987; Audenaert et al. 2002; van Loon et al., 2008; Dutta and Thakur 2017). All but one strain showed positive siderophore production in the range observed for PGPB isolated from natural and cultivated ecosystems (Chaiharn et al. 2009; Khan et al. 2015; Kandel et al. 2017a; Padda et al. 2017a; Rais et al. 2017; Dutta and Thakur 2017). Along with assisting the plant in mediating biotic stress, PGPB-produced siderophores can also facilitate the plant in acquiring ferric iron, a scarce micronutrient that mostly exists in the plant-unavailable form in soils (Loaces et al. 2011). PGPB  175 can also facilitate plant acquisition of macronutrients including phosphorus via two major mechanisms – solubilization of inorganic phosphorus and mineralization of organic phosphorus (Walia et al. 2017). Since the scarcity of plant-available phosphorus in soils is often an issue in both agriculture and forest ecosystems, the presence of PGPB that can effectively convert the unavailable forms of phosphorus in the soil to available forms is vital for sustainable productivity (Glick 2012). In vitro plate and broth assays revealed that all but one bacterial strain possessed both inorganic phosphate solubilizing and organic phytate hydrolyzing abilities (Tables 6.1 and 6.2). For inorganic phosphate solubilization, the SI observed in the plate assay (1.2 – 2.6) and the amount of soluble phosphates released in the broth assay (66 – 112 µg per mL broth) by our bacterial strains align with previous studies in which PGPB isolated from crop and tree hosts with strong phosphate solubilization abilities have been reported (Premono et al. 1997; Kumar and Narula 1999; Chaiharn and Lumyong 2009; Khan et al. 2015; Kandel et al. 2017a; Padda et al. 2017a; Rincón-Molina et al. 2020). The mechanisms of inorganic phosphate solubilization mainly include the production of organic acids, protons, siderophores and exopolysaccharides (Walia et al. 2017). Although several organic acids could be released by PGPB, gluconic acid represents one of the most common and effective forms of organic acid responsible for releasing phosphorous (Zeng et al. 2016). Interestingly, when the availability of soluble phosphates is low in the soil solution, phosphate-solubilizers belonging to the Pseudomonas and Burkholderia genera work more efficiently due to the upregulation of genes related to the phosphate solubilization pathway (Zeng et al. 2016, 2017). Therefore, under the nutrient stress conditions of the West Chilcotin region, our strains might be effectively fulfilling the phosphorus requirements of trees. Since the major source of phosphorus in forest ecosystems is organic litter, the ability to produce phytase  176 to mineralize certain forms of organically-bound phosphorus is crucial for the growth of trees. As plants are not known to directly take up phytate from the soil or mineralize it, phytase-secreting microbes have an important role to play in hydrolyzing phytate and making it available for plant uptake (Idriss et al. 2002; Walia et al. 2017). The five strains that hydrolyzed Na-phytate in both qualitative plate assay and quantitative broth assay (Tables 6.1 and 6.2) performed similarly well in comparison to previous studies (Kerovuo et al. 1998; Yanke et al. 1998; Idriss et al. 2002; Kumar et al. 2013). All in all, the five strains that possess the ability to make both inorganic and organic phosphorus available for plants could be referred to as comprehensive phosphate solubilizing bacteria. In particular, C. udeis LP-R2r which had significantly higher inorganic and organic phosphate solubilizing ability than all other strains should be further evaluated to quantify the amount of phosphorus made available by this strain in planta. In conclusion, the six bacterial strains evaluated in this study can colonize the rhizosphere and internal tissues of multiple Pinaceae trees – lodgepole pine and hybrid white spruce – and enhance their growth by potentially exerting diverse PGP mechanisms. Of the 11 mechanisms tested in this study, all bacterial strains tested positive for at least 5 different mechanisms involving key PGP traits such as nutrient acquisition, phytohormone modulation and biocontrol. Notably, three bacterial strains – C. sordidicola HS-S1r, P. phytofirmans LP-R1r and C. udeis LP-R2r – possess the highest potential to promote plant growth using all mechanisms tested in this study. This impressive suite of in vitro PGP capabilities could be related to the significant enhancement of pine and spruce length and biomass observed for these strains in the long-term greenhouse growth trials. Bacterial strains belonging to Caballeronia and Paraburkholderia genera possess multiple PGP abilities as reported in previous studies using lab-based enzyme  177 assays, genomic analyses, and in planta assays (Sessitsch et al. 2005; Palaniappan et al. 2010; Lladó et al. 2014; Padda et al. 2018, 2019). Intriguingly, these genera previously belonged to the plant-beneficial group of the Burkholderia genus (Sawan et al. 2014; Dobrista and Samadpour 2016), which is rich in potent plant-probiotics (Estrada-De Los Santos et al. 2001; Puri et al. 2017b). Considering the results of this study and those reported in chapters 2 and 5 where these bacteria showed considerable nitrogen-fixing ability (Puri et al. 2018a, 2020c), we can conclude that such bacteria with multifarious PGP abilities may be playing a significant role in sustaining the growth of Pinaceae trees under nutrient-limited, disturbed edaphic conditions of the West Chilcotin region. Henceforth, such bacteria with the inherent ability to enhance the growth of multiple tree hosts should be further evaluated in field conditions, since they have the potential to be used as comprehensive biofertilizers for long-term sustenance of trees under challenging environments. 178 Table 6.1 Qualitative evaluation of major direct and indirect plant-growth-promoting mechanisms of the six bacterial strains using in vitro plate-based enzyme assays. Bacterial strains Phosphate solubilization (SI) † Phytate hydrolyzation (SI) † Siderophore production (area of orange halo in cm2) ‡ Chitinase activity § b-1,3-glucanase activity § Protease activity § Cellulase activity § Ammonia production # Catalase activity # Caballeronia sordidicola HP-S1r 1.5 3.2 1.93 ± 0.09ab +++ ++ + + + + Pseudomonas frederiksbergensis HP-N1r – – 2.33 ± 0.12bc – – + – + – Phyllobacterium myrsinacearum HP-R1r 1.2 2 – – – – + + ++ Pseudomonas mandelii LP-S1r 1.3 2.2 1.63 ± 0.09a + – + ++ ++ ++ Paraburkholderia phytofirmans LP-R1r 1.9 2.9 2.43 ± 0.12bc ++ + +++ +++ +++ + Caballeronia udeis LP-R2r 2.6 3.6 2.73 ± 0.10c + +++ ++ +++ + +++ † solubilization index (SI) represents the zone of solubilization on triplicate plates relative to bacterial growth. ‡ values are mean ± standard error (n = 3), where values followed by different letters are significantly different at P < 0.05. § clearance zone for chitinase, protease and cellulase activities and yellow-orange zone for b-1,3-glucanase activity was evaluated for each bacterium on triplicate plates, where ‘–’ means no observed zone, ‘+’ means 0 – 5 mm zone, ‘++’ means 5 – 15 mm zone, and ‘+++’ means 15 – 25 mm zone. # assessed using triplicate samples per bacteria, where ‘–’ means no production/activity, ‘+’ means low production/activity, ‘++’ means medium production/activity and ‘+++’ means high production/activity.  179 Table 6.2 Quantitative evaluation of major direct and indirect plant-growth-promoting mechanisms of the six bacterial strains using in vitro broth-based enzyme assays. Bacterial strains Phosphate solubilization (µg/mL) † Phytate hydrolyzation (U/mL) † IAA production (µg/mL) † ACC deaminase activity (nmol a-ketobutyrate/mg/h) † Chitinase activity (U/mL) † b-1,3-glucanase activity (U/mL) † Protease activity (U/mL) † Cellulase activity (U/mL) † Caballeronia sordidicola HP-S1r 96.3 ± 0.88b 79.5 ± 0.45c 32.0 ± 0.83c 101 ± 1.17d 0.52 ± 0.02c 0.59 ± 0.02a 51.8 ± 0.42c 0.35 ± 0.02b Pseudomonas frederiksbergensis HP-N1r – – 21.0 ± 0.92b 33.3 ± 0.69a – – 37.3 ± 1.45b – Phyllobacterium myrsinacearum HP-R1r 66.7 ± 1.20a 43.0 ± 0.38a 15.5 ± 0.69a 65.8 ± 1.03c – – – 0.17 ± 0.01a Pseudomonas mandelii LP-S1r 70.3 ± 1.45a 42.7 ± 0.55a 23.5 ± 0.69b 54.4 ± 1.16b 0.28 ± 0.01a – 20.3 ± 1.45a 0.51 ± 0.02c Paraburkholderia phytofirmans LP-R1r 92.7 ± 1.76b 68.2 ± 0.42b 29.8 ± 0.91c 96.3 ± 1.26d 0.49 ± 0.02c 0.51 ± 0.02a 82.3 ± 1.45e 0.54 ± 0.01cd Caballeronia udeis LP-R2r 112 ± 1.45c 86.6 ± 0.23d 32.3 ± 0.55c 111 ± 1.41e 0.40 ± 0.01b 0.89 ± 0.01b 73.4 ± 0.33d 0.61 ± 0.02d † Values are mean ± standard error (n = 3), where values followed by different letters are significantly different (P < 0.05). 180   Figure 6.1 Mean values of (a) length and (b) biomass of 540-day old lodgepole pine seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Error bars represent standard errors of mean (n = 10 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). 05101520253035404550HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlSeedling length (cm)(a)abbbbbb020406080100120140160HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlSeedling biomass (mg)(b)ab b bccdd 181   Figure 6.2 Mean values of (a) length and (b) biomass of 540-day old hybrid white spruce seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Error bars represent standard errors of mean (n = 10 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). 0510152025303540HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlSeedling length (cm)(a)abbbbbb01020304050607080HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlSeedling biomass (mg)(b)abb bc cc 182   Figure 6.3 Population density of each of the six bacterial strains inside the endophytic tissues (needle, stem and root) and in the rhizosphere of (a) lodgepole pine and (b) hybrid white spruce seedlings evaluated 540 days after inoculation. For clarity of presentation, the data were log-transformed. Error bars represent standard errors of mean (n = 5 seedlings per treatment for endophytic colonization and 5 seedlings per treatment for rhizospheric colonization). 0123456789HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rNEEDLE STEM ROOT RHIZOSPHEREColonization (log cfu/g)Lodgepole Pine(a)012345678HP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rHP-S1rHP-N1rHP-R1rLP-S1rLP-R1rLP-R2rNEEDLE STEM ROOT RHIZOSPHEREColonization (log cfu/g)Hybrid White Spruce(b) 183   Figure 6.4 Mean values of primary root length of (a) canola and (b) tomato seedlings subjected to six bacteria-inoculated and one non-inoculated control treatments. Seedlings were evaluated five days after germination in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity. Error bars represent standard errors of mean (n = 7 seedlings per treatment) and bars with different letters are significantly different (P < 0.05). 024681012HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlPrimary root length (cm)Canola(a)abcbcdee024681012HP-S1r HP-N1r HP-R1r LP-S1r LP-R1r LP-R2r ControlPrimary root length (cm)Tomato(b)aabcbcdcdd 184 Canola  Tomato  Figure 6.5 Five-day old (a) canola and (b) tomato seedlings showing differences in root length between treatments. Seedlings were subjected to six bacteria-inoculated and one non-inoculated control treatments in the gnotobiotic root elongation assay to evaluate in situ ACC deaminase activity.   185 Chapter 7 - General Summary and Conclusions The main objective of this dissertation was to explore the possibility of nitrogen (N) fixation by endophytic bacteria in conifers growing under the severe climatic and edaphic conditions of the West Chilcotin region in the Sub-Boreal Pine-Spruce xeric-cold zone, located in the Central-Interior of British Columbia, Canada. An additional objective was to characterize these bacteria for their wider ecological role, besides N-fixation, in supporting tree growth on these nutrient-poor, disturbed soils. Lodgepole pine and hybrid white spruce, the most common tree species in this region, were chosen as the model plants for endophytic bacterial isolation and in planta evaluation of the effects of these bacteria in this research project. The major findings of each research chapter (chapters 2, 3, 4, 5 and 6) have been summarized below. In chapter 2, the first objective was to assess soil health in the West Chilcotin region by evaluating the physical and chemical properties as well as the nutrient status of soils. It was found that, relative to nearby biogeoclimatic zones (Driscoll et al. 1999; Sanborn et al. 2005; Kranabetter et al. 2006; Hope 2007), soils in this region are coarse-textured (loamy sand/sandy loam) with an acidic pH (due to the predominance of Al and Fe cations), low cation exchange capacity, high C:N ratio, thin forest floor and limited organic matter levels. Soil macronutrients such as N, P and S as well as micronutrients such as Mo, Ni and B are generally low. In particular, the quantities of available NO3- and NH4+ in these soils could be considered growth-limiting for plants. As expected, significant differences between soils sampled from high-elevation sites and  186 low-elevation sites were found, with soils at high-elevation sites having considerably lower nutrient contents and poorer physico-chemical characteristics. These extremely low levels of available N in soils led me to explore the possibility of endophytic diazotrophic bacteria in lodgepole pine and hybrid white spruce trees growing at different sites (low-elevation and high-elevation) in this region. Forty-eight and 55 potential endophytic diazotrophic bacteria were isolated on N-free culture medium from root, stem and needle tissues of pine and spruce trees, respectively. Results of multiple acetylene reduction assays revealed that 23 isolates from pine trees and 18 isolates from spruce trees possessed the nitrogenase enzyme. These isolates were identified using 16s rRNA gene sequencing and found to belong to phyla Firmicutes, Proteobacteria and Actinobacteria. The greatest number of isolates from both pine and spruce trees belonged to genera Caballeronia and Paenibacillus. Based on their nitrogenase enzyme activity, six endophytic diazotrophic strains of spruce trees and six endophytic diazotrophic strains of pine trees were selected for further analyses In chapter 3, the six selected strains of hybrid white spruce were evaluated with their original host and a foreign host (lodgepole pine) in year long greenhouse trials. Similarly, in chapter 5, the six selected strains of lodgepole pine were tested with their native host and a foreign host (hybrid white spruce) in year long greenhouse trials. The main objective of these studies was to quantify the amount of fixed N the bacteria can provide to their host using a 15N isotope dilution assay. An additional objective was to assess the plant x microbe specificity of these bacteria to their original host. It was observed that both spruce and pine endophytic diazotrophic strains can fix significant amounts of N from the atmosphere (15-56%) in both  187 original and foreign hosts while colonizing their internal tissues. In addition, inoculation with these strains significantly promoted the length and biomass of the host tree in comparison to the non-inoculated controls after one year of growth. Along with the acetylene reduction assay and the 15N isotope dilution assay, a molecular approach (nifH gene amplification) was used to further confirm the N-fixing ability of these 12 strains. Interestingly, all spruce and pine strains performed similarly well with their native host and the foreign host, indicating that these endophytic diazotrophic strains have no plant x microbe specificity. Caballeronia sordidicola LS-S2r (from spruce) and Caballeronia sordidicola HP-S1r (from pine) emerged as the most prominent N-fixers in these studies by fixing ≥50% of their host N from atmosphere, which is nearly equivalent to 5.5 g of N fixed by these strains per kg of plant tissue in one year. If similar rates of N-fixation could be sustained under field conditions, fixed N from these bacteria would constitute 50-60% of the total N uptake by lodgepole pine trees in a fully stocked forest in the SBPS region of BC (Kimmins et al., 1999; Anand 2010). This estimate is reasonably close to the total N uptake reported for lodgepole pine forests in south-eastern Wyoming, USA (Fahey et al. 1985).    The major objective of studies reported in chapters 4 and 6 was to investigate the wider ecological role, apart from N-fixation, of the selected strains from spruce and pine. For that, the various plant growth-promoting (PGP) mechanisms related to nutrient acquisition, phytohormone modulation and phytopathogen suppression of the six spruce endophytes (in chapter 4) and six pine endophytes (in chapter 6) were analyzed using in vitro tests. It was observed that all strains possessed several PGP abilities in addition to N-fixation, with two spruce strains and three pine strains showing positive results for every PGP mechanism tested in these studies, including phosphate solubilization, phytate hydrolyzation, siderophore production,  188 indole-3-acetic acid (IAA) production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, lytic enzymes (cellulase, protease, chitinase, b-1,3-glucanase) activity, ammonia production and catalase activity. In addition to showing positive ACC deaminase activity in vitro, all strains demonstrated significantly positive results for in situ activity of this enzyme to control stress ethylene levels in canola and tomato (ethylene-sensitive plants). In 18-month long greenhouse trials conducted under nutrient-poor soil conditions, spruce and pine bacteria significantly enhanced seedling growth when inoculated into their original host as well as a foreign host, raising the possibility that these bacteria employ one or more PGP mechanisms to support tree growth under nutrient stress. Along with showing the highest potential in most of the PGP mechanism assays, the spruce bacterium – C. sordidicola LS-S2r – promoted seedling length and biomass by up to 1.75-fold and 7-fold, respectively (Chapter 4). Similarly, pine bacteria – C. sordidicola HP-S1r, Caballeronia udeis LP-R2r and Paraburkholderia phytofirmans LP-R1r – enhanced seedling length and biomass by up to 1.5-fold and 4-fold, respectively, in addition to showing greatest potential in all PGP mechanism tests (Chapter 6).  In order to identify the endophytic diazotrophic strains with the greatest potential to promote plant growth among the 12 pine and spruce strains evaluated in this thesis, a bonitur scale (Krechel et al. 2002) was generated by awarding points for each PGP trait (Table 7.1). Up to three points were assigned for each of the 12 PGP traits tested in this thesis, so the maximum possible bonitur score was 36 points. The strains were ranked according to the total scores obtained for all PGP tests on this bonitur scale. With the highest bonitur score of 34, C. sordidicola LS-S2r emerged as the most effective plant-growth-promoting bacteria (PGPB) among all pine  189 and spruce strains, closely followed by C. udeis LP-R2r with a score of 33 (Table 7.1). Notably, 4 out of the top 5 strains on this bonitur scale originated from the high-elevation pine and spruce stands. Since both high-elevation stands had a significantly poorer nutrient content and physico-chemical soil health in comparison to the low-elevation sites, this finding supports the theory that plants tend to recruit PGPB with higher efficiency when subjected to relatively nutrient-poor conditions (Bal and Chanway 2012a; Yang et al. 2016). In addition, it should also be noted that the top 4 strains on the bonitur scale with the highest PGP potential belong to the genera Caballeronia and Paraburkholderia (Table 7.1). Both genera were previously part of the plant-beneficial group of the genus Burkholderia (Dobritsa and Samadpour 2016; Sawana 2014), which has been widely studied for its N-fixing and PGP bacteria (Estrada-De Los Santos et al. 2001).  Overall, this thesis represents an attempt to explore the comprehensive role of endophytic diazotrophic bacteria in promoting the growth of conifers on nutrient-poor soils. Based on the results, it can be suggested that the severe environmental and edaphic conditions found in the West Chilcotin region of BC have coerced hybrid white spruce and lodgepole pine trees to recruit highly efficient endophytes that can not only provide them with fixed nitrogen from the atmosphere but also enhance their growth and health by employing one or more of the various PGP mechanisms including hormone modulation, biocontrol and nutrient acquisition. Hence, harbouring such effective PGPB could be an evolutionary approach for spruce and pine trees to survive in the West Chilcotin region. As observed in this project, the ability of these endophytic diazotrophic bacteria to form similar beneficial associations with the original host as well as the foreign host, clearly indicate that these bacteria are ‘generalists’ with no plant x microbe specificity. A similar characteristic has been observed for endophytic diazotrophic  190 bacteria of poplar, willow and lodgepole pine trees (Doty et al. 2005, 2009; Bal et al. 2012), as they were also able to associate and provide significant benefits to not only tree species but also agricultural and bioenergy crops (Khan et al. 2015; Kandel et al. 2015; Xin et al. 2009; Knoth et al. 2013; Rho et al. 2018; Bal and Chanway 2013b; Puri et al. 2015; Padda et al. 2016a).  In future, the top PGPB strains such as C. sordidicola LS-S2r, C. udeis LP-R2r, C. sordidicola HP-S1r and P. phytofirmans LP-R1r with multifarious abilities to enhance tree growth should be evaluated in long-term field studies. BC’s forest industry is the largest supplier of Roundwood and other wood-based products made from pine and spruce in Canada. To sustain the supply chain, millions of pine and spruce seedlings are planted each year in BC (98 and 88 million respectively in 2017-18) (Source: Government of British Columbia). However, their long-term survival is always a concern due to limited nutrient availability, transplant shock after outplanting (restricted shoot growth and/or inadequate root development to extract water and nutrients), forest fires and pest attack. Although mechanical site preparation, frequent fertilization, and control of competing vegetation/pests can improve plantation survival and performance, the costs associated with these operations is prohibitive. Based on the results of this thesis, I suggest that an inexpensive and environmentally-benign solution for the long-term survival of pine and spruce trees may include inoculating the seedlings with these potent PGPB strains at the nursery-growth stage before outplanting in the field. In addition, I also propose that the strains described in this thesis should be further evaluated for their ‘generalist’ nature (providing benefits to diverse hosts) with other commercially important species of the Pinaceae family such as Douglas-fir (Pseudotsuga), hemlock (Tsuga), fir (Abies) and Larch (Larix) as well as the other varieties of spruce and pine found in western North America. As reported in several studies, endophytic  191 bacteria originally isolated from natural environments could successfully associate with domesticated species customized to grow in intensively cultivated ecosystems (Kandel et al. 2015; Xin et al. 2009; Knoth et al. 2013; Johnston-Monje et al. 2014; Mousa et al. 2015; Shehata et al. 2016). Therefore, strains tested in this study should also be evaluated with agricultural crops in future studies in order to discover their potential wide-ranging PGP capabilities as generalists. Since each strain was evaluated separately with the host plant in this project, another aspect that requires investigation is – how would these bacteria perform when inoculated together into the host as a multi-strain consortium? Whether or not these strains work synergistically to promote plant growth could help reveal how these strains co-exist and interact inside their host tree species in nature. If shown to be effective in such studies, these PGPB strains could be used as biofertilizers for a wide range of plants in both forest and agricultural ecosystems, potentially serving as an eco-friendly and cost-effective alternative to chemical fertilizers.  In addition to examining the beneficial associations of these bacterial strains with their host plants in natural field conditions, gathering more in-depth knowledge about their interactions, physiology and ecology is also necessary using lab-based studies. For instance, studying the sites and mode through which these bacteria gain entry into plant tissues remains to be determined. Several approaches could be employed to study the entry and colonization sites, including confocal laser microscopy, scanning electron microscopy and transmission electron microscopy. Along with that molecular-based identification using qRT-PCR could be conducted for a more reliable estimate of the population sizes of specific bacteria in the tissues and rhizosphere of plants after inoculation. Although a robust method (15N isotope dilution assay) was used to quantify the amount of fixed N provided by these bacteria to their host, a molecular  192 approach involving the evaluation of nifH gene expression in different sections of the plant needs to be performed to determine if the N-fixation is being performed by the endophytic population or rhizospheric population or both. Another method may include tagging the nifH gene of these bacterial strains with a reporter gene and determining the transcription of the tagged nifH gene in different sections of the plant after inoculation. This can also help us understand whether and under what conditions the transcription of the nifH genes is initiated in planta, which could be crucial in proving whether N-poor soil conditions are required to trigger N-fixation in these bacteria. There is also an extremely important, universal question regarding the N-fixation mechanism of endophytic diazotrophic bacteria that remains unanswered since they were first reported in the 1980s. How are low-oxygen/anoxic conditions created inside the plant tissues for endophytic diazotrophic bacteria to allow the functioning of their nitrogenase-enzyme complex, which is extremely oxygen labile? Several theories have been proposed to answer this question (Doty 2017; Doty et al. 2016; Chanway et al. 2014; Oses et al. 2018), but compelling evidence is still lacking to prove any of those theories. Another aspect that needs to be further explored includes the molecular and proteomic examination of the various PGP traits of these bacteria. Last but not the least, findings of this study indicate that there ought to be other beneficial endophytic diazotrophic bacteria (culturable and unculturable) in the microbiome of hybrid white spruce and lodgepole pine trees that may provide a significant contribution to the nitrogen budgets of forest stands in the West Chilcotin region, and other nutrient-poor sites that support tree growth. Therefore, an exhaustive community-based analysis should be conducted in order to understand the structure and functioning of all nitrogen-fixing bacteria present in the microbiome of trees growing in such extremely nutrient-poor, disturbed ecosystems.  193 Table 7.1 Concluding assessment and overall ranking of endophytic bacterial strains isolated from hybrid white spruce and lodgepole pine trees (Chapter 2) growing in the West Chilcotin region of BC. The assessment and ranking were generated using the bonitur scale (Krechel et al. 2002) based on the 12 plant-growth-promoting mechanisms analyzed in chapters 3, 4, 5 and 6. Bacterial strains Ndfaa Phob Phyc Sidd IAAe ACCf Celg Proh Chii b-1,3j Ammk Catl Total Ass. (36)m Rank Caballeronia sordidicola LS-S2r 3 3 3 2 3 3 3 3 3 3 3 2 34 1st Caballeronia udeis LP-R2r 3 3 3 3 3 3 3 3 2 3 1 3 33 2nd Caballeronia sordidicola HP-S1r 3 3 3 2 3 3 1 2 3 2 1 1 27 3rd Paraburkholderia phytofirmans LP-R1r 1 3 2 3 2 2 2 3 3 2 3 1 27 3rd Pseudomonas prosekii LS-S1r 3 1 2 3 3 3 3 2 1 1 3 1 26 5th Pigmentiphaga litoralis HS-S1r 3 0 0 1 3 2 3 0 2 2 1 3 20 6th Pseudomonas migulae HS-S3r 2 2 2 1 2 1 2 1 2 0 2 1 18 7th Caballeronia udeis LS-R1r 2 2 2 0 2 1 2 0 0 0 3 1 15 8th Pseudomonas mandelii LP-S1r 2 1 1 1 1 1 2 1 1 0 2 2 15 8th Phyllobacterium myrsinacearum HP-R1r 2 1 1 0 1 1 1 0 0 0 1 2 10 10th Herbiconiux solani HS-S2r 1 0 0 0 2 1 1 0 0 0 0 2 7 11th Pseudomonas frederiksbergensis HP-N1r 1 0 0 2 1 0 0 2 0 0 1 0 7 11th a Nitrogen derived from atmosphere (3 = 60-45%, 2 = 45-30%, 1 = 30-15%, 0 = 15-0%); b Phosphate solubilization (3 = 90-125 μg/mL, 2 = 90-75 μg/mL, 1 = 75-60 μg/mL, 0 = 60-0 μg/mL);  c Phytate hydrolyzation (3 = 100-75 U/mL, 2 = 75-50 U/mL, 1 = 50-25 U/mL, 0 = 25-0 U/mL); d Siderophore production (3 = 2.9-2.4 cm2, 2 = 2.4-1.9 cm2, 1 = 1.9-0 cm2);  e ACC deaminase activity (3 = 100-140 nmol/mg/h , 2 = 100-60 nmol/mg/h, 1 = 60-20 nmol/mg/h, 0 = 20-0 nmol/mg/h);  f Cellulase activity (3 = 0.75-0.60 U/mL, 2 = 0.60-0.45 U/mL, 1 = 0.45-0.30 U/mL, 0 = 0.30-0 U/mL); g Protease activity (3 = 105-70 U/mL, 2 = 70-35 U/mL, 1 = 35-20 U/mL, 0 = 20-0 U/mL);  h Chitinase activity (3 = 0.45-0.75 U/mL, 2 = 0.45-0.40 U/mL, 1 = 0.40-0.25 U/mL, 0 = 0.25-0 U/mL); i b-1,3-glucanase activity (3 = 1.00-0.75 U/mL, 2 = 0.75-0.50 U/mL, 1 = 0.50-0.25 U/mL, 0 = 0.25-0 U/mL);  j Ammonia production (3 = +++, 2 = ++, 1 = +, 0 = absent); k Catalase activity (3 = +++, 2 = ++, 1 = +, 0 = absent); m Total Assessment points. 194 References Achouak W, Normand P, Heulin T (1999) Comparative phylogeny of rrs and nifH genes in the Bacillaceae. Int J Syst Evol Microbiol 49:961–967. https://doi.org/10.1099/00207713-49-3-961 Aghai MM, Khan Z, Stoda AM, Sher AW, Ettl GJ, Doty SL (2019) The effect of microbial endophyte consortia on Pseudotsuga menziesii and Thuja plicata survival, growth, and physiology across edaphic gradients. Front Microbiol 10:1353. https://doi.org/10.3389/fmicb.2019.01353  Ahmad N, Shahab S (2011) Phosphate solubilization: their mechanism genetics and application. Inter J Microbiol 9:1-19 Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res 163:173–181. https://doi.org/10.1016/j.micres.2006.04.001  Akhromeiko AI, Shestakova VA (1958) The influence of rhizosphere microorganisms on the uptake and secretion of phosphorus and sulphur by the roots of arboreal seedlings. Mikrobiologiya 27:67–74 Anand R (2010) Endophytic colonization and nitrogen fixation by Paenibacillus polymyxa in association with lodgepole pine and western red cedar. Doctoral dissertation, University of British Columbia, Vancouver. https://doi.org/10.14288/1.0071481  Anand R, Paul L, Chanway C (2006) Research on endophytic bacteria: recent advances with forest trees. In: Schulz B, Boyle C, Sieber TN (eds) Microbial root endophytes, Part 1. Springer-Verlag, Berlin, pp 89–106. https://doi.org/10.1007/3-540-33526-9_6  Anand R, Chanway CP (2013a) Detection of GFP-labeled Paenibacillus polymyxa in auto fluorescing pine seedling tissues. Biol Fertil Soils 49:111–118. https://doi.org/10.1007/s00374-012-0727-9  Anand R, Chanway C (2013b) N2-fixation and growth promotion in cedar colonized by an endophytic strain of Paenibacillus polymyxa. Biol Fertil Soils 49:235–239. https://doi.org/10.1007/s00374-012-0735-9 Anand R, Chanway CP (2013c) nif gene sequence and arrangement in the endophytic diazotroph Paenibacillus polymyxa strain P2b-2R. Biol Fertil Soils 49:965–970. https://doi.org/10.1007/s00374-013-0793-7   195 Anand R, Paul L, Chanway C (2006) Research on endophytic bacteria: recent advances with forest trees. In: Schulz B, Boyle C, Sieber TN (eds) Microbial Root Endophytes. Springer-Verlag, Berlin, pp 89–106. https://doi.org/10.1007/3-540-33526-9_6  Anand R, Grayston S, Chanway CP (2013) N2-fixation and seedling growth promotion of lodgepole pine by endophytic Paenibacillus polymyxa. Microb Ecol 66:369–374. https://doi.org/10.1007/s00248-013-0196-1  Anandham R, Gandhi PI, Madhaiyan M, Sa T (2008) Potential plant growth promoting traits and bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants. J Basic Microbiol 48:439–447. https://doi.org/10.1002/jobm.200700380  Audenaert K, Pattery T, Cornelis P, Höfte M (2002) Induction of systemic resistance to Botrytis cinerea in tomato by Pseudomonas aeruginosa 7NSK2: role of salicylic acid, pyochelin, and pyocyanin. Mol Plant-Microbe Interact 15:1147-1156. https://doi.org/10.1094/MPMI.2002.15.11.1147 Babalola OO (2010) Beneficial bacteria of agricultural importance. Biotechnol Lett 32:1559–1570. https://doi.org/10.1007/s10529-010-0347-0  Bacon CW, White JF Jr (2000) Microbial endophytes. Marcel Dekker Inc., New York Bal A, Chanway CP (2012a) Evidence of nitrogen fixation in lodgepole pine inoculated with diazotrophic Paenibacillus polymyxa. Botany 90:891–896. https://doi.org/10.1139/b2012-044 Bal A, Chanway CP (2012b) 15N foliar dilution of western red cedar in response to seed inoculation with diazotrophic Paenibacillus polymyxa. Biol Fertil Soils 48:967–971. https://doi.org/10.1007/s00374-012-0699-9  Bal A, Anand R, Berge O, Chanway CP (2012) Isolation and identification of diazotrophic bacteria from internal tissues of Pinus contorta and Thuja plicata. Can J For Res 42:807–813. https://doi.org/10.1139/x2012-023  Baldani JI, Baldani VLD, Seldin L, Döbereiner J (1986) Characterization of Herbaspirillum seropedicae gen. nov., sp. nov., a root associated nitrogen fixing bacterium. Int J Syst Bacteriol 36:86–93. https://doi.org/10.1099/00207713-36-1-86  Baldani VLD, Baldani JI, Döbereiner J (2000) Inoculation of rice plants with the endophytic diazotrophs Herbaspirillum seropedicae and Burkholderia spp. Biol Fertil Soils 30:485–491. https://doi.org/10.1007/s003740050027  Bashan Y, Holguin G (1998) Proposal for the division of plant growth promoting rhizobacteria into two classifications: biocontrol-PGPB (plant growth-promoting bacteria) and PGPB. Soil Biol Biochem 30:1225–1228. https://doi.org/10.1016/S0038-0717(97)00187-9   196 BC Ministry of Forests - Research Branch (1998) The ecology of the sub-boreal pine-spruce zone. https://www.for.gov.bc.ca/hfd/pubs/docs/bro/bro59.htm. [accessed 08 June 2020] BC Ministry of Forests and Range and BC Ministry of Environment (2010) Field Manual for Describing Terrestrial Ecosystems - 2nd edition. https://www.for.gov.bc.ca/hfd/pubs/docs/lmh/Lmh25-2.htm. [accessed 08 June 2020] Bent E, Tuzun S, Chanway CP, Enebak SA (2001) Alterations in plant growth and in root hormone levels of lodgepole pines inoculated with rhizobacteria. Can J Microbiol 47:793–800. https://doi.org/10.1139/w01-080  Bertalan M, Albano R, de Padua V et al. (2009) Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5. BMC Genom 10:450. https://doi.org/10.1186/1471-2164-10-450  Binkley D, Son Y, Valentine D (2000) Do forest receive occult inputs of nitrogen? Ecosystems 3:321–331. https://doi.org/10.1007/s100210000029  Blouin VM, Schmidt MG, Bulmer CE, Krzic M (2008) Effects of compaction and water content on lodgepole pine sidling growth. For Ecol Manage 255:2444–2452. https://doi.org/10.1016/j.foreco.2008.01.008  Boddey RM, Urquiaga S, Reis V, Döbereiner J (1991) Biological nitrogen fixation associated with sugar cane. Plant Soil 137:111–117. https://doi.org/10.1007/BF02187441  Bormann B, Keller C, Wang D, Bormann H (2002) Lessons from the sandbox: is unexplained nitrogen real? Ecosystems 5:727–733. https://doi.org/10.1007/s10021-002-0189-2  Bottini R, Cassán F, Piccoli P (2004) Gibberellin production by bacteria and its involvement in plant growth promotion and yield increase. Appl Microbiol Biotechnol 65:497–503. https://doi.org/10.1007/s00253-004-1696-1  Bric JM, Bostock RM, Silverstone S (1991) Rapid in situ assay for indoleacetic acid production by bacteria immobilized on a nitrocellulose membrane. Appl Environ Microbiol 57:535–538 Brooks DS, Gonzalez CF, Appel DN, Filer TH (1994) Evaluation of endophytic bacteria as potential biological-control agents for Oak Wilt. Biol Control 4:373–381. https://doi.org/10.1006/bcon.1994.1047  Brundrett MC (2009) Mycorrhizal associations and other means of nutrition of vascular plants: understanding the global diversity of host plants by resolving conflicting information and developing reliable means of diagnosis. Plant Soil 320:37–77. https://doi.org/10.1007/s11104-008-9877-9   197 Bulgarelli D, Rott M, Schlaeppi K et al. (2012) Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488:91-95. https://doi.org/10.1038/nature11336  Bumunang EW, Babalola OO (2014) Characterization of rhizobacteria from field grown genetically modified (GM) and non-GM maizes. Braz Arch Biol Technol 57:1–8. https://dx.doi.org/10.1590/S1516-89132014000100001  Burns RM, Honkala BH (1990) Silvics of North America – Volume 1: Conifers. Agriculture Handbook 654. US Department of Agriculture, Forest Service, Washington DC. https://www.srs.fs.usda.gov/pubs/misc/ag_654_vol1.pdf. [accessed 08 June 2020] Camm EL, Towers GN (1973) Phenylalanine ammonia lyase. Phytochemistry 12:961-973. https://doi.org/10.1016/0031-9422(73)85001-0 Canadian Forest Service (2019) The state of Canada’s forests: Annual Report 2018. Natural Resources Canada, Ottawa. https://cfs.nrcan.gc.ca/pubwarehouse/pdfs/39336.pdf [accessed 08 June 2020] Cankar K, Kraigher H, Ravnikar M, Rupnik M (2005) Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst). FEMS Microbiol Lett 244:341–345. https://doi.org/10.1016/j.femsle.2005.02.008  Carper DL, Carrell AA, Kueppers LM, Frank AC (2018) Bacterial endophyte communities in Pinus flexilis are structured by host age, tissue type, and environmental factors. Plant Soil 428:335–352. https://doi.org/10.1007/s11104-018-3682-x  Carrell AA, Frank AC (2014) Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation. Front Microbiol 5:333. https://doi.org/10.3389/fmicb.2014.00333  Carrell AC, Carper DL, Frank AC (2016) Subalpine conifers in different geographical locations host highly similar foliar bacterial endophyte communities. FEMS Microbiol Ecol 92:fiw124. https://doi.org/10.1093/femsec/fiw124  Cavalcante VA, Döbereiner J (1988) A new acid tolerant nitrogen fixing bacterium associated with sugarcane. Plant Soil 108:23–31. https://doi.org/10.1007/BF02370096  Centre for Forest Conservation Genetics (n.d.) Sub-Boreal Pine – Spruce Zone maps. https://cfcg.forestry.ubc.ca/resources/cataloguing-in-situ-genetic-resources/sbps-zone/sbps-subzone-maps/. [accessed 08 June 2020]  198 Chaiharn M, Lumyong S (2009) Phosphate solubilization potential and stress tolerance of rhizobacteria from rice soil in Northern Thailand. World J Microbiol Biotechnol 25:305–314. https://doi.org/10.1007/s11274-008-9892-2  Chaiharn M, Chunhaleuchanon S, Lumyong S (2009) Screening siderophore producing bacteria as potential biological control agent for fungal rice pathogens in Thailand. World J Microbiol Biotechnol 25:1919-1928. https://doi.org/10.1007/s11274-009-0090-7 Chanway CP (1995) Differential response of western hemlock from low and high elevations to inoculation with plant growth-promoting Bacillus polymyxa. Soil Biol Biochem 27:767–775. https://doi.org/10.1016/0038-0717(94)00236-T  Chanway CP (1996) Endophytes: they’re not just fungi. Can J Bot 74:321–322. https://doi.org/10.1139/b96-040  Chanway CP (1997) Inoculation of tree roots with plant growth promoting soil bacteria: an emerging technology for reforestation. For Sci 43:99–112. https://doi.org/10.1093/forestscience/43.1.99  Chanway CP, Holl FB (1991) Biomass increase and associative nitrogen fixation of mycorrhizal Pinus contorta seedlings inoculated with a plant growth promoting Bacillus strain. Can J Bot 69:507–511. https://doi.org/10.1139/b91-069  Chanway CP, Holl FB (1992) Influence of soil biota on Douglas-fir (Pseudotsuga menziesii) seedling growth: the role of rhizosphere bacteria. Can J Bot 70:1025–1031. https://doi.org/10.1139/b92-127  Chanway CP, Holl FB (1993) First year field performance of spruce seedlings inoculated with plant growth promoting rhizobacteria. Can J Microbiol 39:1084–1088. https://doi.org/10.1139/m93-164  Chanway CP, Holl FB (1994) Growth of outplanted lodgepole pine seedlings one year after inoculation with plant growth promoting rhizobacteria. For Sci 40:238–246. https://doi.org/10.1093/forestscience/40.2.238  Chanway CP, Shishido M, Nairn J, Jungwirth S, Markham J, Xiao G, Holl F (2000) Endophytic colonization and field responses of hybrid spruce seedlings after inoculation with plant growth-promoting rhizobacteria. For Ecol Manage 133:81–88. https://doi.org/10.1016/S0378-1127(99)00300-X  Chanway CP, Anand R, Yang H (2014) Nitrogen fixation outside and inside plant tissues. In: Ohyama T (ed) Advances in Biology and Ecology of Nitrogen Fixation. InTechOpen, London, pp 3–23. https://doi.org/10.5772/57532   199 Chapman WK, Paul L (2012) Evidence that northern pioneering pines with tuberculate mycorrhizae are unaffected by varying soil nitrogen levels. Microb Ecol 64:964–972. https://doi.org/10.1007/s00248-012-0076-0  Chet I, Inbar J (1994) Biological control of fungal pathogens. Appl Biochem Biotechnol 48:37-43. https://doi.org/10.1007/BF02825358 Compant S, Clément C, Sessitsch A (2010) Plant growth-promoting bacteria in the rhizo- and endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization. Soil Biol Biochem 42:669–678. https://doi.org/10.1016/j.soilbio.2009.11.024  Compant S, Samad A, Faist H, Sessitsch A (2019) A review on the plant microbiome: Ecology, functions and emerging trends in microbial application. J Adv Res 19:29–37. https://doi.org/10.1016/j.jare.2019.03.004  Coupé R (2012) Tree Species Selection Tool: BEC ZONES: Sub-Boreal Pine Spruce – Very Dry Cold. British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development, Victoria, Canada. https://www.for.gov.bc.ca/hfp/silviculture/tss/bec_zones/SBPSxc.html. [accessed 08 June 2020] Danso SKA, Hera C, Douka C (1987) Nitrogen fixation in soybean as influenced by cultivar and Rhizobium strain. Plant Soil 99:163–174. https://doi.org/10.1007/BF02370163  de Bary A (1866) Morphologie und Physiologie Pilze, Flechten, und myxomyceten. Hofmeister’s Handbook of Physiological Botany, vol 2. Verlag Von Wilhelm Engelmann, Leipzig. https://babel.hathitrust.org/cgi/pt?id=hvd.32044053007316. [accessed 08 June 2020] Deluca TH, Boisvenue C (2012) Boreal forest soil carbon: distribution, function and modelling. Forestry 85:161-84. https://doi.org/10.1093/forestry/cps003 Dey R, Pal KK, Bhatt DM, Chauhan SM (2004) Growth promotion and yield enhancement of peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria. Microbiol Res 159:371–394. https://doi.org/10.1016/j.micres.2004.08.004  di Vestea A (1888) De l’absence des microbes dans les tissus végétaux. Annales de l’lnstitut Pasteur 670–671 Döbereiner J (1961) Nitrogen fixing bacteria of the genus Beijerinckia Drex. in the rhizosphere of sugarcane. Plant Soil 15:211–216. https://doi.org/10.1007/BF01400455  Döbereiner J (1992) Recent changes in concepts of plant bacteria interactions: endophytic N2 fixing bacteria. Ciênc Cult 44:310–313  200 Döbereiner J, Alvahydo R (1959) Sóbre a influénciada canade-acucar na occoréncia de “Beijerinckia” no solo II. Influéncia das diversas partes do vegetal. Rev Bras Biol 19:401–412 Dobritsa AP, Samadpour M (2016) Transfer of eleven Burkholderia species to the genus Paraburkholderia and proposal of Caballeronia gen. nov., a new genus to accommodate twelve species of Burkholderia and Paraburkholderia. Int J Syst Evol Microbiol 66:2836–2846. https://doi.org/10.1099/ijsem.0.001065  Domínguez-Núñez JA, Albanesi AS (2019) Ectomycorrhizal Fungi as Biofertilizers in Forestry. In: Mirmajlessi SM, Radhakrishnan R (eds) Biostimulants in Plant Science. IntechOpen, London, pp. https://doi.org/10.5772/intechopen.88585  Doty SL (2011) Growth-promoting endophytic fungi of forest trees. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees: Biology and Applications, 1st edn. Springer, Dordrecht, pp 151–156. https://doi.org/10.1007/978-94-007-1599-8_9  Doty SL (2017) Functional importance of the plant endophytic microbiome: implications for agriculture, forestry, and bioenergy. In: Doty SL (ed) Functional Importance of the Plant Microbiome. Springer, Cham, pp 1–5. https://doi.org/10.1007/978-3-319-65897-1_1  Doty SL, Dosher MR, Singleton GL, Moore AL, Van Aken B, Stettler RF, Strand SE, Gordon MP (2005) Identification of an endophytic Rhizobium in stems of Populus. Symbiosis 39:27–35 Doty SL, Oakley B, Xin G, Kang JW, Singleton G, Khan Z, Vajzovic A, Staley JT (2009) Diazotrophic endophytes of native black cottonwood and willow. Symbiosis 47:23–33. https://doi.org/10.1007/BF03179967  Doty SL, Sher AW, Fleck ND, Khorasani M, Bumgarner RE, Khan Z, Ko AWK, Kim S-H, DeLuca TH (2016) Variable nitrogen fixation in wild Populus. PLoS One 11:e0155979. https://doi.org/10.1371/journal.pone.0155979  Driscoll KG, Arocena JM, Massicotte HB (1999) Post-fire soil nitrogen content and vegetation composition in sub-boreal spruce forests in British Columbia’s central interior, Canada. For Ecol Manage 121:227–237. https://doi.org/10.1016/S0378-1127(99)00003-1  Dutta J, Thakur D (2017) Evaluation of multifarious plant growth promoting traits, antagonistic potential and phylogenetic affiliation of rhizobacteria associated with commercial tea plants grown in Darjeeling, India. PLoS ONE 12:e0182302. https://doi.org/10.1371/journal.pone.0182302  Elad Y, Kapat A (1999) The role of Trichoderma harzianum protease in the biocontrol of Botrytis cinerea. Eur J Plant Pathol 105:177–189. https://doi.org/10.1023/A:1008753629207   201 Esashi Y (1991) Ethylene and seed germination. In Matoo AK, Suttle JC (eds) The Plant Hormone Ethylene. CRC Press, Boca Raton, pp 133–157. https://doi.org/10.1201/9781351075763  Estrada-de los Santos P, Rojas-Rojas FU, Tapia-García EY, Vásquez-Murrieta MS, Hirsch AM (2015) To split or not to split: an opinion on dividing the genus Burkholderia. Ann Microbiol 66:1303–1314. https://doi.org/10.1007/s13213-015-1183-1  Fahey TJ, Yavitt JB, Pearson JA, Knight DH (1985) The nitrogen cycle in lodgepole pine forests, southeastern Wyoming. Biogeochemistry 1:257-275. https://doi.org/10.1007/BF02187202  Farah A, Iqbal A,Khan MS (2006) Screening of free-living rhizospheric bacteria for their multiple plant growth-promoting activity. Microbiol Res 63:11–19. https://doi.org/10.1016/j.micres.2006.04.001  Farjon A (1998) World Checklist and Bibliography of Conifers. Scientific Publications Department, Royal Botanic Gardens, Kew, Richmond, Surrey Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66:375–400 Foyer CH, Noctor G (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell 17:1866-1875. https://doi.org/10.1105/tpc.105.033589 Frank AC (2018) The genomes of endophytic bacteria. In Pirttilä A, Frank A (eds) Endophytes of Forest Trees: Biology and Applications. Springer, Cham, pp 141–176. https://doi.org/10.1007/978-3-319-89833-9_7  Frank A, Saldierna Guzmán J, Shay J (2017) Transmission of bacterial endophytes. Microorganisms 5:70. https://doi.org/10.3390/microorganisms5040070  Fridlender M, Inbar J, Chet I (1993) Biological control of soilborne plant pathogens by a β-1, 3 glucanase-producing Pseudomonas cepacia. Soil Biol Biochem 25:1211–1221. https://doi.org/10.1016/0038-0717(93)90217-Y  Frommel MI, Nowak J, Lazarovits G (1991) Growth enhancement and developmental modifications of in vitro growth potato (Solanum tuberosum spp. tuberosum) as affected by a non-fluorescent Pseudomonas sp. Plant Physiol 96:928–936. https://doi.org/10.1104/pp.96.3.928  Gaby JC, Buckley DH (2012) A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS One 7:e42149. https://doi.org/10.1371/journal.pone.0042149   202 Galippe V (1887) Note sur la présence de micro-organismes dans les tissus végétaux. C R Hebd Sci Mem Soc Biol 39:410–416 Galloway JN, Cowling EB (2002) Reactive nitrogen and the world: 200 Years of change. AMBIO: A journal of the human environment 31:64-71. https://doi.org/10.1579/0044-7447-31.2.64  Geric B, Rupnik M, Kraigher H (2000) Isolation and identification of mycorrhization helper bacteria in Norway spruce, Picea abies (L.) Karst. Phyton 40:65–70 Germaine K, Keogh E, Garcia-Cabellos G, Borremans B, Van Der Lelie D, Barac T, Oeyen L, Vangronsveld J, Moore FP, Moore ERB, Campbell CD, Ryan D, Dowling DN (2004) Colonisation of poplar trees by gfp expressing bacterial endophytes. FEMS Microbiol Ecol 48:109–118. https://doi.org/10.1016/j.femsec.2003.12.009  Germida J, de Freitas J (1998) Nitrogen fixing rhizobacteria as biofertilizers for canola. Saskatchewan Canola Development Commission (Project code: CARP 9513). https://www.saskcanola.com/quadrant/System/research/reports/report-Germida-nitrogenfixing-long.pdf. [accessed 08 June 2020] Gillis M, Kersters K, Hoste B, Janssens D, Kroppenstedt RM, Stephen MP (1989) Acetobacter diaztrophicus sp. nov., a nitrogen fixing acetic acid bacterium associated with sugarcane. Int J Syst Bacteriol 39:361–364. https://doi.org/10.1099/00207713-39-3-361  Glick BR (2012) Plant growth-promoting bacteria: mechanisms and applications. Scientifica 2012:963401. https://doi.org/10.6064/2012/963401  Glick BR (2015) Beneficial plant-bacterial interactions. Springer, Cham. https://doi.org/10.1007/978-3-319-13921-0 Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentrations by plant growth-promoting bacteria. J Theor Biol 190:63–68. https://doi.org/10.1006/jtbi.1997.0532  Glickmann E, Dessaux Y (1995) A critical examination of the specificity of  the  Salkowski  reagent  for  indolic  compounds  produced  by phytopathogenic bacteria. Appl Environ Microbiol 61:793–796 Gordon SA, Weber RP (1951) Colorimetric estimation of indoleacetic acid. Plant Physiol 26:192–195 Government of British Columbia (n.d.) Silviculture Statistics. https://www2.gov.bc.ca/gov/content/industry/forestry/managing-our-forest-resources/silviculture/silviculture-statistics. [accessed 08 June 2020]  203 Hahlbrock K, Grisebach H (1979) Enzymic controls in the biosynthesis of lignin and flavonoids. Annu Rev Plant Physiol 30:105–130. https://doi.org/10.1146/annurev.pp.30.060179.000541  Hallmann J, Quadt-Hallmann A, Mahaffee WF, Kloepper JW (1997) Bacterial endophytes in agricultural crops. Can J Microbiol 43:895–914. https://doi.org/10.1139/m97-131  Hardarson G, Danso SKA (1993) Methods for measuring biological nitrogen fixation in grain legumes. Plant Soil 152:19–23. https://doi.org/10.1007/BF00016330  Hardoim PR, van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16:463–471. https://doi.org/10.1016/j.tim.2008.07.008  Hardoim PR, Van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79:293–320. https://doi.org/10.1128/MMBR.00050-14  Hellriegel H, Wilfarth H (1888) Untersuchungen uber die Stickstoffnahrung der Gramineen und Leguminosen. Beilageheft zu der Zeitschrift des Vereins für die Rübenzuckerindustrie des Deutschen Reichs. https://doi.org/10.5962/bhl.title.27102  Holl FB, Chanway CP, Turkington R, Radley RA (1988) Response of crested wheatgrass (Agropyron cristatum L.), perennial ryegrass (Lolium perenne L.), and white clover (Trifolium repens L.) to inoculation with Bacillus polymyxa. Soil Biol Biochem 20:19–24. https://doi.org/10.1016/0038-0717(88)90121-6  Hope GD (2007) Changes in soil properties, tree growth, and nutrition over a period of a 10 years after stump removal and scarification on moderately coarse soils in interior British Columbia. For Ecol Manage 242:625–635. https://doi.org/10.1016/j.foreco.2007.01.072  Hurek T, Reinhold-Hurek B, Van Montagu M, Kellenberger E (1994) Root colonization and systemic spreading of Azoarcus sp. strain BH72 in grasses. J Bacteriol 176:1913–1923. https://doi.org/10.1128/jb.176.7.1913-1923.1994  Idriss EE, Makarewicz O, Farouk A, Rosner K, Greiner R, Bochow H, Richter T, Borriss R (2002) Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-promoting effect. Microbiology 148:2097-2109. https://doi.org/10.1099/00221287-148-7-2097 Iyer B, Rajkumar S (2017) Host specificity and plant growth promotion by bacterial endophytes. Curr Res Microbiol Biotechnol 5:1018–1030. http://crmb.aizeonpublishers.net/content/2017/2/crmb1018-1030.pdf. [accessed 08 June 2020]  204 Izumi H (2011) Diversity of endophytic bacteria in forest trees. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees, 1st edn. Springer, Dordrecht, pp 95–105. https://doi.org/10.1007/978-94-007-1599-8_6  Jadhav HP, Shaikh SS, Sayyed RZ (2017) Role of hydrolytic enzymes of rhizoflora in biocontrol of fungal phytopathogens: An overview. In Mehnaz S (ed) Rhizotrophs: Plant growth Promotion to Bioremediation. Springer, Singapore, pp 183-203. https://doi.org/10.1007/978-981-10-4862-3_9 James EK, Olivares F (1998) Infection and colonization of sugar cane and other graminaceous plants by endophytic diazotrophs. Crit Rev Plant Sci 17:77-119. https://doi.org/10.1080/07352689891304195  Jackson MB (1991) Ethylene in root growth and development. In Matoo AK, Suttle JC (eds) The Plant Hormone Ethylene. CRC Press, Boca Raton, pp 159–181. https://doi.org/10.1201/9781351075763  Johnston-Monje D, Mousa WK, Lazarovits G, Raizada MN (2014) Impact of swapping soils on the endophytic bacterial communities of pre-domesticated, ancient and modern maize. BMC Plant Biol 14:233. https://doi.org/10.1186/s12870-014-0233-3  Kaewkla O, Franco CMM (2010) Nocardia callitridis sp. nov., an endophytic actinobacterium isolated from a surface-sterilized root of an Australian native pine tree. Int J Syst Evol Microbiol 60:1532–1536. https://doi.org/10.1099/ijs.0.016337-0  Kamilova F, Kravchenko LV, Shaposhnikov AI, Azarova T, Makarova N, Lugtenberg B (2006) Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria. Mol Plant-Microbe Interact 19:250–256. https://doi.org/10.1094/MPMI-19-0250  Kandel SL, Herschberger N, Kim SH, Doty SL (2015) Diazotrophic endophytes of poplar and willow for growth promotion of rice plants in nitrogen-limited conditions. Crop Sci 55:1765-1772. https://doi.org/10.2135/cropsci2014.08.0570 Kandel SL, Firrincieli A, Joubert PM, Okubara PA, Leston ND, McGeorge KM, Mugnozza GS, Harfouche A, Kim S-H, Doty, S. L. (2017a). An in vitro study of bio-control and plant growth promotion potential of Salicaceae endophytes. Front Microbiol 8:386. https://doi.org/10.3389/fmicb.2017.00386  Kandel S, Joubert P, Doty S (2017b) Bacterial endophyte colonization and distribution within plants. Microorganisms 5:77. https://doi.org/10.3390/microorganisms5040077  205 Kerovuo J, Lauraeus M, Nurminen P, Kalkkinen N, Apajalahti J (1998) Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis. Appl Environ Microbiol 64:2079–2085 Khan Z, Kandel S, Ramos D, Ettl GJ, Kim S-H, Doty SL (2015) Increased biomass of nursery-grown Douglas-fir seedlings upon inoculation with diazotrophic endophytic consortia. Forests 6:3582–3593. https://doi.org/10.3390/f6103582  Khan Z, Rho H, Firrincieli A, Hung SH, Luna V, Masciarelli O, Kim S-H, Doty SL (2016) Growth enhancement and drought tolerance of hybrid poplar upon inoculation with endophyte consortia. Curr Plant Biol 6:38-47. http://dx.doi.org/10.1016/j.cpb.2016.08.001  Khan AL, Shahzad R, Al-Harrasi A, Lee IJ (2017) Endophytic microbes: a resource for producing extracellular enzymes. In Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 95-110. https://doi.org/10.1007/978-3-319-66544-3_5 Kimmins JP, Mailly D, Seely B (1999) Modelling forest ecosystem net primary production: the hybrid simulation approach used in FORECAST. Ecol Modell 122:195-224. https://doi.org/10.1016/S0304-3800(99)00138-6  Knoth JL, Kim SH, Ettl GJ, Doty SL (2013) Effects of cross host species inoculation of nitrogen-fixing endophytes on growth and leaf physiology of maize. GCB Bioenergy 5:408-418. https://doi.org/10.1111/gcbb.12006  Knoth JL, Kim SH, Ettl GJ, Doty SL (2014) Biological nitrogen fixation and biomass accumulation within poplar clones as a result of inoculations with diazotrophic endophyte consortia. New Phytol 201:599–609. https://doi.org/10.1111/nph.12536  Kobayashi D, Palumbo J (2000) Bacterial endophytes and their effects on plants and uses in agriculture. In: Bacon CW, White JF (eds) Microbial endophytes. Marcel Dekker, New York, pp 199–233 Kobayashi DY, Reedy RM, Bick J, Oudemans PV (2002) Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control. Appl Env Microbiol 68:1047–1054. https://doi.org/10.1128/AEM.68.3.1047-1054.2002  Kolotelo D, van Steenis E, Peterson M, Bennett R, Trotter D, Dennis J (2001) Seed handling guidebook. British Columbia Ministry of Forests, Tree Seed Centre. https://www2.gov.bc.ca/assets/gov/farming-natural-resources-and-industry/forestry/tree-seed/tree-seed-publications/seed_handling_guidebook_hi.pdf. [accessed 08 June 2020] Koskimäki JJ, Pirttilä AM, Ihantola EL, Halonen O, Frank AC (2015) The intracellular scots pine shoot symbiont Methylobacterium extorquens DSM13060 aggregates around the host nucleus  206 and encodes eukaryote-like proteins. MBio 6:e00039-15. https://doi.org/10.1128/mBio.00039-15 Kranabetter JM, Sanborn P, Chapman BK, Dube S (2006) The contrasting response to soil disturbance between lodgepole pine and hybrid white spruce in sub-boreal forests. Soil Sci Soc Am J 70:1591–1599. https://doi.org/10.2136/sssaj2006.0081  Krechel A, Faupel A, Hallmann J, Ulrich A, Berg G (2002) Potato-associated bacteria and their antagonistic potential towards plant-pathogenic fungi and the plant-parasitic nematode Meloidogyne incognita (Kofoid & White) Chitwood. Can J Microbiol 48:772-786. https://doi.org/10.1139/w02-071  Kruasuwan W, Hoskisson PA, Thamchaipenet A (2017) Draft genome sequence of root-associated sugarcane growth-promoting Microbispora sp. Strain GKU 823. Genome Announc 5:e00647-17. https://doi.org/10.1128/genomea.00647-17  Kubiak K, Wrzosek M, Przemieniecki S, Damszel M, Sierota Z (2018) Bacteria Inhabiting Wood of Roots and Stumps in Forest and Arable Soils. In Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees: Biology and Applications, 2nd edition. Springer, Cham, pp 319-342. https://doi.org/10.1007/978-3-319-89833-9_14 Kumar V, Narula N (1999) Solubilization of inorganic phosphates and growth emergence of wheat as affected by Azotobacter chroococcum mutants. Biol Fertil Soils 28:301-305. https://doi.org/10.1007/s003740050497 Kumar V, Singh P, Jorquera MA, Sangwan P, Kumar P, Verma AK, Agrawal S (2013) Isolation of phytase-producing bacteria from Himalayan soils and their effect on growth and phosphorus uptake of Indian mustard (Brassica juncea). World J Microbiol Biotechnol 29:1361–1369. https://doi.org/10.1007/s11274-013-1299-z  Latha P, Anand T, Rappathi N, Prakasam V, Samiyappan R (2009) Antimicrobial activity of plant extracts and induction of systemic resistance in tomato plants by mixtures of PGPR strains and Zimmu leaf extract against Alternaria solani. Biol Control 50:85–93. https://doi.org/10.1016/j.biocontrol.2009.03.002  Lladó S, Xu Z, Sørensen SJ, Baldrian P (2014) Draft genome sequence of Burkholderia sordidicola S170, a potential plant growth promoter isolated from coniferous forest soil in the Czech Republic. Genome Announc 2:e00810–14. https://doi.org/10.1128/genomeA.00810-14  Loaces I, Ferrando L, Scavino AF (2011) Dynamics, diversity and function of endophytic siderophore-producing bacteria in rice. Microb Ecol 61:606–618. https://doi.org/10.1007/s00248-010-9780-9   207 Lotan JE, Critchfield WB (1996) Lodgepole Pine. In: Burns RM, Honkala BH (Tech Coords) Silvics of North America – Volume 1: Conifers. Agriculture Handbook 654. US Department of Agriculture, Forest Service, Washington DC. https://www.srs.fs.usda.gov/pubs/misc/ag_654/volume_1/silvics_vol1.pdf. [accessed 08 June 2020] Louden BC, Haarmann D, Lynne AM (2011) Use of blue agar CAS assay for siderophore detection. J Microbiol Biol Educ 12:51–53. https://doi.org/10.1128/jmbe.v12i1.249  Madmony A, Chernin L, Pleban S, Peleg E, Riov J (2005) Enterobacter cloacae, an obligatory endophyte of pollen grains of Mediterranean pines. Folia Microbiol 50:209–216. https://doi.org/10.1007/BF02931568  Magalhaes FMM, Baldani JI, Souto SM, Kuykendal JR, Döbereiner J (1983) A new acid tolerant Azospirillum species. An Acad Bras Cien 55:417–430 Marin-Bruzos M, Grayston SJ (2019) Biological Control of Nematodes by Plant Growth Promoting Rhizobacteria: Secondary Metabolites Involved and Potential Applications. In Singh H, Keswani C, Reddy M, Sansinenea E, García-Estrada C (eds) Secondary Metabolites of Plant Growth Promoting Rhizomicroorganisms. Springer, Singapore, pp 253-264. https://doi.org/10.1007/978-981-13-5862-3_13 McCosh FWJ (1984) The plant and nitrogen. In: McCosh FWJ (ed) Boussingault. Springer, Dordrecht, pp 123–138. https://doi.org/10.1007/978-94-009-6297-2_10  Mitter B, Brader G, Pfaffenbichler N, Sessitsch A (2019) Next generation microbiome applications for crop production—limitations and the need of knowledge-based solutions. Curr Opin Microbiol 49:59–65. https://doi.org/10.1016/j.mib.2019.10.006  Morgan PW, Drew MC (1997) Ethylene and plant responses to stress. Physiol Plant 100:620-630. https://doi.org/10.1111/j.1399-3054.1997.tb03068.x Mousa WK, Shearer CR, Limay-Rios V, Zhou T, Raizada MN (2015) Bacterial endophytes from wild maize suppress Fusarium graminearum in modern maize and inhibit mycotoxin accumulation. Front Plant Sci 6:805. https://doi.org/10.3389/fpls.2015.00805  Moyes AB, Kueppers LM, Pett-Ridge J, Carper DL, Vandehey N, O’Neil J, Frank AC (2016) Evidence for foliar endophytic nitrogen fixation in a widely distributed subalpine conifer. New Phytol 210:657–668. https://doi.org/10.1111/nph.13850  Okon Y, Labandera-Gonzalez CA (1994) Agronomic applications of Azospirillum: an evaluation of 20 years worldwide field inoculation. Soil Biol Biochem 26:1591–1601. https://doi.org/10.1016/0038-0717(94)90311-5   208 Oliveira CA, Alves VMC, Marriel IE, Gomes EA, Scotti MR, Carneiro NP, Guimarães CT, Schaffert RE, Sá NMH (2009) Phosphate solubilizing microorganisms isolated from rhizosphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome. Soil Biol Biochem 41:1782–1787. https://doi.org/10.1016/j.soilbio.2008.01.012  O’Neill GA, Chanway CP, Axelrood PE, Radley RA, Holl FB (1992) Growth response specificity of spruce inoculated with coexistent rhizosphere bacteria. Can J Bot 70:2347–2353. https://doi.org/10.1139/b92-294  Onofre-Lemus J, Hernández-Lucas I, Girard L, Caballero-Mellado J (2009) ACC (1-aminocyclopropane-1-carboxylate) deaminase activity, a widespread trait in Burkholderia species, and its growth-promoting effect on tomato plants. Appl Environ Microbiol 75:6581-6590. https://doi.org/10.1128/AEM.01240-09 Oses R, Frank AC, Valenzuela S, Rodríguez J (2018) Nitrogen Fixing Endophytes in Forest Trees. In: Pirttilä A, Frank A (eds) Endophytes of Forest Trees, 2nd edn. Springer, Cham, pp. 191–204. https://doi.org/10.1007/978-3-319-89833-9_9  Padda KP, Puri A, Chanway CP (2016a) Effect of GFP tagging of Paenibacillus polymyxa P2b–2R on its ability to promote growth of canola and tomato seedlings. Biol Fertil Soils 52:377–387. https://doi.org/10.1007/s00374-015-1083-3  Padda KP, Puri A, Chanway CP (2016b) Plant growth promotion and nitrogen fixation in canola by an endophytic strain of Paenibacillus polymyxa and its GFP-tagged derivative in a long-term study. Botany 94:1209–1217. https://doi.org/10.1139/cjb-2016-0075  Padda KP, Puri A, Zeng Q, Chanway CP, Wu X (2017a) Effect of GFP-tagging on nitrogen fixation and plant growth promotion of an endophytic diazotrophic strain of Paenibacillus polymyxa. Botany 95:933–942. https://doi.org/10.1139/cjb-2017-0056  Padda KP, Puri A, Chanway CP (2017b) Paenibacillus polymyxa – a prominent biofertilizer and biocontrol agent for sustainable agriculture. In: Meena VS, Mishra P, Thakuria D, Bisht J, Pattanayak A (eds) Agriculturally Important Microbes for Sustainable Agriculture: Applications in Crop Production and Protection. Springer, Singapore, pp 165–191. https://doi.org/10.1007/978-981-10-5343-6_6  Padda KP, Puri A, Chanway CP (2018) Isolation and identification of endophytic diazotrophs from lodgepole pine trees growing at unreclaimed gravel mining pits in central interior British Columbia, Canada. Can J For Res 48:1601–1606. https://doi.org/10.1139/cjfr-2018-0347  Padda KP, Puri A, Chanway CP (2019) Endophytic nitrogen fixation – a possible ‘hidden’ source of nitrogen for lodgepole pine trees growing at unreclaimed gravel mining sites. FEMS Microbiol Ecol 95:fiz172. https://doi.org/10.1093/femsec/fiz172   209 Padda KP, Puri A, Chanway CP (2020) Characterizing the potential ecological role of Pinus contorta bacterial endophytes in sustaining host-tree growth on gravel mining pits. Submitted to Symbiosis journal Palaniappan P, Chauhan PS, Saravanan VS, Anandham R, Sa T (2010) Isolation and characterization of plant growth promoting endophytic bacterial isolates from root nodule of Lespedeza sp. Biol Fertil Soils 46:807–816. https://doi.org/10.1007/s00374-010-0485-5 Parish R, Thomson S (1994) Tree book: learning to recognize trees of British Columbia. Canadian Forest Service, Victoria, Canada. https://www.for.gov.bc.ca/hfd/library/documents/treebook/TreeBook.pdf. [accessed 08 June 2020] Paul LR (2002) Tuberculate ectomycorrhizae on lodgepole pine (Pinus contorta) and associated nitrogen fixation. Doctoral Dissertation, University of British Columbia, Vancouver. https://doi.org/10.14288/1.0090671  Paul LR, Chapman BK, Chanway CP (2007) Nitrogen fixation associated with Suillus tomentosus tuberculate ectomycorrhizae on Pinus contorta var. latifolia. Ann Bot 99:1101–1109. https://doi.org/10.1093/aob/mcm061  Paul LR, Chapman WK, Chanway CP (2013) Diazotrophic bacteria reside inside Suillus tomentosus/Pinus contorta tuberculate ectomycorrhizae. Botany 91:48–52. https://doi.org/10.1139/cjb-2012-0191  Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-containing plant growth promoting rhizobacteria. Physiol Plant 118:10–15. https://doi.org/10.1034/j.1399-3054.2003.00086.x  Perry DA, Molina R, Amaranthus MP (1987) Mycorrhizae, mycorrhizospheres, and reforestation: current knowledge and research needs. Can J For Res 17:929-940. https://doi.org/10.1139/x87-145  Piccoli P, Travaglia C, Cohen A, Sosa L, Cornejo P, Masuelli R, Bottini R (2011) An endophytic bacterium isolated from roots of the halophyte Prosopis strombulifera produces ABA, IAA, gibberellins A1 and A3 and jasmonic acid in chemically-defined culture medium. Plant Growth Regul 64:207-210. https://doi.org/10.1007/s10725-010-9536-z  Ping L, Boland W (2004) Signals from the underground: bacterial volatiles promote growth in Arabidopsis. Trends Plant Sci 9:263–266. https://doi.org/10.1016/j.tplants.2004.04.008  Pirttilä AM (2011) Endophytic bacteria in tree shoot tissues and their effects on host. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees, 1st edn. Springer, Dordrecht, pp 139–150. https://doi.org/10.1007/978-94-007-1599-8_8   210 Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with the vital activity of some microbial species. Mikrobiologiya 17:362–370 Pirttilä AM (2018) Endophytic bacteria in tree shoot tissues and their efects on host. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees, 2nd edn. Springer, Cham, pp 177–190. https://doi.org/10.1007/978-3-319-89833-9_8  Pirttilä AM, Frank AC (2011) Endophytes of Forest Trees: Biology and Applications, 1st edn. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-1599-8 Pirttilä AM, Frank AC (2018) Endophytes of Forest Trees: Biology and Applications, 2nd edn. Springer, Cham. https://doi.org/10.1007/978-3-319-89833-9  Pirttilä AM, Laukkanen H, Pospiech H, Myllylä R, Hohtola A (2000) Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization. Appl Environ Microbiol 66:3073–3077. https://doi.org/10.1128/AEM.66.7.3073-3077.2000  Pirttilä AM, Laukkanen H, Hohtola A (2002) Chitinase production in pine callus (Pinus sylvestris L.): a defense reaction against endophytes? Planta 214:848–852. https://doi.org/10.1007/s00425-001-0709-x  Pohjanen J, Koskimäki JJ, Sutela S, Ardanov P, Suorsa M, Niemi K, Sarjala T, Häggman H,Pirttilä AM (2014) Interaction with ectomycorrhizal fungi and endophytic Methylobacterium affects nutrient uptake and growth of pine seedlings in vitro. Tree Physiol 34:993–1005. https://doi.org/10.1093/treephys/tpu062  Pojar J, Meidinger DV (1991) Introduction. In: Meidinger DV, Pojar J (eds) Ecosystems of British Columbia. British Columbia Ministry of Forests, Lands, and Natural Resource Operations, Victoria, Canada, pp 195-207. https://www.for.gov.bc.ca/hfd/pubs/docs/Srs/Srs06/chap1.pdf. [accessed 08 June 2020] Postgate JR (1998) Nitrogen fixation, 3rd edn. Cambridge University Press, Cambridge Premono ME, Moawad AM, Vlek PLG (1996) Effect of phosphate-solubilizing Pseudomonas putida on the growth of maize and its survival in the rhizosphere. Indonesian Journal of Crop Science. 1996; 11: 13-23. Puri A, Padda KP, Chanway CP (2015) Can a diazotrophic endophyte originally isolated from lodgepole pine colonize an agricultural crop (corn) and promote its growth? Soil Biol Biochem 89:210–216. https://doi.org/10.1016/j.soilbio.2015.07.012  Puri A, Padda KP, Chanway CP (2016a) Evidence of nitrogen fixation and growth promotion in canola (Brassica napus L.) by an endophytic diazotroph Paenibacillus polymyxa P2b–2R. Biol Fertil Soils 52:119–125. https://doi.org/10.1007/s00374-015-1051-y   211 Puri A, Padda KP, Chanway CP (2016b) Seedling growth promotion and nitrogen fixation by a bacterial endophyte Paenibacillus polymyxa P2b–2R and its GFP derivative in corn in a long-term trial. Symbiosis 69:123–129. https://doi.org/10.1007/s13199-016-0385-z  Puri A, Padda KP, Chanway CP (2017a) Beneficial effects of bacterial endophytes on forest tree species. In: Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 111–132. https://doi.org/10.1007/978-3-319-66544-3_6  Puri A, Padda KP, Chanway CP (2017b) Plant growth promotion by endophytic bacteria in nonnative crop hosts. In: Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 11–45. https://doi.org/10.1007/978-3-319-66544-3_2  Puri A, Padda KP, Chanway CP (2018a) Evidence of endophytic diazotrophic bacteria in lodgepole pine and hybrid white spruce trees growing in soils with different nutrient statuses in the West Chilcotin region of British Columbia, Canada. For Ecol Manage 430:558–565. https://doi.org/10.1016/j.foreco.2018.08.049  Puri A, Padda KP, Chanway CP (2018b) Nitrogen-fixation by endophytic bacteria in agricultural crops: recent advances. In: Khan A, Fahad S (eds) Nitrogen in Agriculture – Updates. InTech, London, pp 73–94. https://doi.org/10.5772/intechopen.71988  Puri A, Padda KP, Chanway CP (2020a) Can naturally-occurring endophytic nitrogen-fixing bacteria of hybrid white spruce sustain boreal forest tree growth on extremely nutrient-poor soils? Soil Biol Biochem 140:107642. https://doi.org/10.1016/j.soilbio.2019.107642  Puri A, Padda KP, Chanway CP (2020b) In vitro and in vivo analyses of plant-growth-promoting potential of bacteria naturally associated with spruce trees growing on nutrient-poor soils. Appl Soil Ecol 149:103538. https://doi.org/10.1016/j.apsoil.2020.103538  Puri A, Padda KP, Chanway CP (2020c) Evaluating lodgepole pine endophytes for their ability to fix nitrogen and support tree growth under nitrogen-limited conditions. Submitted to Plant and Soil journal Qiu Y, Fu B, Wang J, Chen L (2001) Spatial variability of soil moisture content and its relation to environmental indices in a semi-arid gully catchment of the Loess Plateau, China. J Arid Environ 49:723–750. https://doi.org/10.1006/jare.2001.0828  Rais A, Jabeen Z, Shair F, Hafeez FY, Hassan MN (2017) Bacillus spp., a bio-control agent enhances the activity of antioxidant defense enzymes in rice against Pyricularia oryzae. PLoS One 12:e0187412. https://doi.org/10.1371/journal.pone.0187412 Reed SC, Cleveland CC, Townsend AR (2011) Functional ecology of free-living nitrogen fixation: a contemporary perspective. Annu Rev Ecol Evol Syst 42:489–512. https://doi.org/10.1146/annurev-ecolsys-102710-145034   212 Reinhold-Hurek B, Maes T, Gemmer S, Montagu MV, Hurek T (2006) An endoglucanase is involved in infection of rice roots by the not-cellulose-metabolizing endophyte Azoarcus sp. strain BH72. Mol Plant-Microbe Interact 19:181–188. https://doi.org/10.1094/MPMI-19-0181  Rennie RJ (1981) A single medium for the isolation of acetylene-reducing (dinitrogenfixing) bacteria from soils. Can J Microbiol 27:8–14. https://doi.org/10.1139/m81-002  Rennie RJ, Fried M, Rennie DA (1978) Concepts of 15N usage in dinitrogen fixation studies. In: Isotopes in Biological Dinitrogen Fixation, pp 107–131 Rennie RJ, de Freitas JR, Ruschel AP, Vose PB (1982) Isolation and identification of nitrogen fixing bacteria associated with sugarcane (Saccharum sp.). Can J Microbiol 28:462–467. https://doi.org/10.1139/m82-070  Renwick A, Campbell R, Coe S (1991) Assessment of in vivo screening systems for potential biocontrol agents of Gaeumannomyces graminis. Plant Pathol 40:524–532. https://doi.org/10.1111/j.1365-3059.1991.tb02415.x  Rho H, Van Epps V, Wegley N, Doty SL, Kim S-H (2018) Salicaceae endophytes modulate stomatal behavior and increase water use efficiency in rice. Front Plant Sci 9: 188. https://doi.org/10.3389/fpls.2018.00188  Richardson AE, Simpson RJ (2011) Soil microorganisms mediating phosphorus availability update on microbial phosphorus. Plant Physiol 156:989–996. https://doi.org/10.1104/pp.111.175448  Rincón-Molina CI, Martínez-Romero E, Ruiz-Valdiviezo VM, Velázquez E, Ruiz-Lau N, Rogel-Hernández MA, Villalobos-Maldonado JJ, Rincón-Rosales R (2020) Plant growth-promoting potential of bacteria associated to pioneer plants from an active volcanic site of Chiapas (Mexico). Appl Soil Ecol 146:103390. https://doi.org/10.1016/j.apsoil.2019.103390 Robertson GP, Vitousek PM (2009) Nitrogen in agriculture: Balancing the cost of an essential resource. Annu Rev Environ Resour 34:97–125. https://doi.org/10.1146/annurev.environ.032108.105046  Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interaction with hosts. Mol Plant-Microbe Interact 19:827–837. https://doi.org/10.1094/MPMI-19-0827  Rosenblueth M, Ormeño-Orrillo E, López-López A, Rogel MA, Reyes-Hernández BJ, Martínez-Romero JC, Reddy PM, Martinez-Romero E (2018) Nitrogen fixation in cereals. Front Microbiol 9:1794. https://doi.org/10.3389/fmicb.2018.01794   213 Ryan RP, Germaine K, Franks A, Ryan DJ, Dowling DN (2008) Bacterial endophytes: recent developments and applications. FEMS Microbiol Lett 278:1–9. https://doi.org/10.1111/j.1574-6968.2007.00918.x  Saha M, Sarkar S, Sarkar B, Sharma BK, Bhattacharjee S, Tribedi P (2016) Microbial siderophores and their potential applications: a review. Environ Sci Pollut Res 23:3984–3999. https://doi.org/10.1007/s11356-015-4294-0  Sahoo A, Agarwal N, Kamra DN, Chudhary LC, Pathak NN (1999) Influence of the level of molasses in de-oiled rice bran-based concentrate mixture on rumen fermentation pattern in crossbred cattle calves. Anim Feed Sci Technol 80:83–90. https://doi.org/10.1016/S0377-8401(99)00055-3  Sanborn PT, Priezel J, Brockley RP (2005) Soil and lodgepole pine foliar responses to two fertilizer sulphur forms in the sub-boreal spruce zone, central interior British Columbia. Can J For Res 35:2316–2322. https://doi.org/10.1139/x05-138  Santoyo G, Moreno-Hagelsieb G, del Carmen Orozco-Mosqueda M, Glick BR (2016) Plant growth-promoting bacterial endophytes. Microbiol Res 183:92-99. https://doi.org/10.1016/j.micres.2015.11.008  Sawana A, Adeolu M, Gupta RS (2014) Molecular signatures and phylogenomic analysis of the genus Burkholderia: proposal for division of this genus into the emended genus Burkholderia containing pathogenic organisms and a new genus Paraburkholderia gen. nov. harboring environmental species. Front Genet 5:1–22. https://doi.org/10.3389/fgene.2014.00429  Schippers B, Bakker AW, Bakker PA (1987) Interactions of deleterious and beneficial rhizosphere microorganisms and the effect of cropping practises. Ann Rev Phytopathol 25:339–358. https://doi.org/10.1146/annurev.py.25.090187.002011  Schlaeppi K, Bulgarelli D (2015) The plant microbiome at work. Mol Plant-Microbe Interact 28:212-217. https://doi.org/10.1094/MPMI-10-14-0334-FI Seldin L, van Elsas JD, Penido EGC (1984) Bacillus azotofixans sp. nov., a nitrogen-fixing species from Brazilian soils and grass roots. Int J Syst Bacteriol 34:451–456. https://doi.org/10.1099/00207713-34-4-451  Sessitsch A, Reiter B, Berg G (2004) Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities. Can J Microbiol 50:239–249. https://doi.org/10.1139/w03-118  Sessitsch A, Coenye T, Sturz AV, Vandamme P, Barka EA, Salles JF, Van Elsas JD, Faure D, Reiter B, Glick BR, Wang-Pruski G (2005) Burkholderia phytofirmans sp. nov., a novel plant-associated  214 bacterium with plant-beneficial properties. Int J Syst Evol Microbiol 55:1187-1192. https://doi.org/10.1099/ijs.0.63149-0 Shan W, Liu H, Zhou Y, Yu X (2017) Draft genome sequence of Streptomyces sp. XY006, an endophyte Isolated from Tea (Camellia sinensis). Genome Announc 5:e00971-17. https://doi.org/10.1128/genomea.00971-17  Shehata HR, Lyons EM, Jordan KS, Raizada MN (2016) Bacterial endophytes from wild and ancient maize are able to suppress the fungal pathogen Sclerotinia homoeocarpa. J Appl Microbiol 120:756-769. https://doi.org/10.1111/jam.13050  Shishido M (1997) Plant growth promoting rhizobacteria (PGPR) for interior spruce (Picea engelmannii x P. glauca) seedlings. Doctoral Dissertation, University of British Columbia, Vancouver. https://doi.org/10.14288/1.0088248  Shishido M, Chanway CP (1999) Spruce growth response specificity after treatment with plant growth-promoting Pseudomonads. Can J Bot 77:22–31. https://doi.org/10.1139/b98-197  Shishido M, Chanway CP (2000) Colonization and growth of outplanted spruce seedlings pre-inoculated with plant growth-promoting rhizobacteria in the greenhouse. Can J For Res 30:848–854. https://doi.org/10.1139/x00-010  Shishido M, Loeb BM, Chanway CP (1995) External and internal root colonization of lodgepole pine seedlings by two growth-promoting Bacillus strains originated from different root microsites. Can J Microbiol 41:707–713. https://doi.org/10.1139/m95-097  Shishido M, Massicotte HB, Chanway CP (1996a) Effect of plant growth promoting Bacillus strains on pine and spruce seedling growth and mycorrhizal infection. Ann Bot 77:433–441. https://doi.org/10.1006/anbo.1996.0053  Shishido M, Petersen DJ, Massicotte HB, Chanway CP (1996b) Pine and spruce seedling growth and mycorrhizal infection after inoculation with plant growth promoting Pseudomonas strains. FEMS Microbiol EcoI 21:109–119. https://doi.org/10.1111/j.1574-6941.1996.tb00338.x  Shishido M, Brevil C, Chanway CP (1999) Endophyic colonization of spruce by plant growth promoting rhizobacteria. FEMS Microbiol Ecol 29:191–196. https://doi.org/10.1111/j.1574-6941.1999.tb00610.x  Sindhu SS, Gupta SK, Dadarwal KR (1999) Antagonistic effect of Pseudomonas spp. on pathogenic fungi and enhancement of growth of green gram (Vigna radiata). Biol Fertil Soils 29:62-68. https://doi.org/10.1007/s003740050525  215 Spaepen S (2015) Plant hormones produced by microbes. In Lugtenberg B (ed) Principles of Plant-Microbe Interactions. Springer, Cham, pp 247–256. https://doi.org/10.1007/978-3-319-08575-3_26  Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 31:425-48. https://doi.org/10.1111/j.1574-6976.2007.00072.x Statistics Canada (2018) Human Activity and the Environment 2017: Forests in Canada. https://www150.statcan.gc.ca/n1/pub/16-201-x/16-201-x2018001-eng.pdf. [accessed 08 June 2020] Steen O, Demarchi DA (1991) Sub-Boreal Pine-spruce zone. In: Meidinger DV, Pojar J (eds) Ecosystems of British Columbia. British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development, Victoria, Canada, pp 195–207. https://www.for.gov.bc.ca/hfd/pubs/docs/Srs/Srs06/chap13.pdf. [accessed 08 June 2020] Steen OA, Coupé RA (1997) A field guide to forest site identification and interpretation for the Cariboo forest region. British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development, Victoria, Canada. https://www.for.gov.bc.ca/hfd/pubs/docs/lmh/lmh39.htm. [accessed 08 June 2020] Stephan MP, Oliveira M, Teixeira KRS, Martinez-Drets G, Döbereiner J (1991) Physiology and dinitrogen fixation of Acetobacter diazotrophicus. FEMS Microbiol Lett 77:67–72. https://doi.org/10.1111/j.1574-6968.1991.tb04323.x  Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing sustainable systems of crop production. Crit Rev Plant Sci 19:1–30. https://doi.org/10.1080/07352680091139169  Suman A, Yadav AN, Verma P (2016) Endophytic microbes in crops: Diversity and beneficial impact for sustainable agriculture. In: Singh DP, Abhilash PC, Prabha R (eds) Microbial inoculants in sustainable agricultural productivity, vol 1: Research perspectives. Springer, India, pp 117–143. https://doi.org/10.1007/978-81-322-2647-5_7  Sun Y, Cheng Z, Glick BR (2009) The presence of a 1-aminocyclopropane-1-carboxylate (ACC) deaminase deletion mutation alters the physiology of the endophytic plant growth-promoting bacterium PsJN. FEMS Microbiol Lett 296:131-136. https://doi.org/10.1111/j.1574-6968.2009.01625.x Suyal DC, Yadav A, Shouche Y, Goel R (2014) Differential Proteomics in Response to Low Temperature Diazotrophy of Himalayan Psychrophilic Nitrogen Fixing Pseudomonas migulae S10724 Strain. Curr Microbiol 68:543–550. https://doi.org/10.1007/s00284-013-0508-1   216 Suzuki S, He Y, Oyaizu H (2003) Indole-3-Acetic acid production in Pseudomonas fluorescens HP72 and its association with suppression of creeping bentgrass brown patch. Curr Microbiol 47:138–143. https://doi.org/10.1007/s00284-002-3968-2  Taghavi S, Barac T, Greenberg B, Borremans B, Vangronsveld J, van der Lelie D (2005) Horizontal gene transfer to endogenous endophytic bacteria from Poplar improves phytoremediation of toluene. Appl Environ Microb 71:8500–8505. https://doi.org/10.1128/AEM.71.12.8500-8505.2005  Taghavi A, Garafola C, Monchy S, Newman L, Hoffman A, Weyens N, Barac T, Vangronsveld J, van der Lelie D (2009) Genome survey and characterization of endophytic bacteria exhibiting a beneficial effect on growth and development of poplar trees. Appl Environ Microbiol 75:748–757. https://doi.org/10.1128/AEM.02239-08  Taghavi S, van der Lelie D, Hoffman A, Zhang Y-B, Walla MD, Vangronsveld J, Newman L, Monchy S, Burkholder WF (2010) Genome sequence of the plant growth promoting endophytic bacterium Enterobacter sp. 638. PLoS Genet 6:e1000943. https://doi.org/10.1371/journal.pgen.1000943  Tang Q, Puri A, Padda KP, Chanway CP (2017) Biological nitrogen fixation and plant growth promotion of lodgepole pine by an endophytic diazotroph Paenibacillus polymyxa and its GFP-tagged derivative. Botany 95:611–619. https://doi.org/10.1139/cjb-2016-0300  Terhonen E, Kovalchuk A, Zarsav A, Asiegbu FO (2018) Biocontrol potential of forest tree endophytes. In: Pirttilä AM, Frank AC (eds) Endophytes of Forest Trees, 2nd edn. Springer, Cham, pp 283–318. https://doi.org/10.1007/978-3-319-89833-9_13  Thapar HS (1989) Role of mycorrhizae in forestry. In: Dhawan V (eds) Applications of Biotechnology in Forestry and Horticulture. Springer, Boston, pp 297–307. https://doi.org/10.1007/978-1-4684-1321-2_24  Trevet IW, Hollis JP (1948) Bacteria in storage organs of healthy plants. Phytopathology 38:960–967 Turner MG, Whitby TG, Romme WH (2019) Feast not famine: Nitrogen pools recover rapidly in 25-yr-old postfire lodgepole pine. Ecology 100:e02626. https://doi.org/10.1002/ecy.2626 Urquiaga S, Cruz K, Boddey R (1992) Contribution of Nitrogen Fixation to Sugar Cane: Nitrogen-15 and Nitrogen-Balance Estimates. Soil Sci Soc Am J 56:105–114. https://doi.org/10.2136/sssaj1992.03615995005600010017x  Vargas MA, Mendes IC, Hungria M (2000) Response of field-grown bean (Phaseolus vulgaris L.) to Rhizobium inoculation and nitrogen fertilization in two Cerrados soils. Biol Fertil Soils 32:228–233. https://doi.org/10.1007/s003740000240   217 van Loon LC, Bakker PA, van der Heijdt WH, Wendehenne D, Pugin A (2008) Early responses of tobacco suspension cells to rhizobacterial elicitors of induced systemic resistance. Mol Plant-Microbe Interact 21:1609–1621. https://doi.org/10.1094/MPMI-21-12-1609  Vitousek PM, Howarth RW (1991) Nitrogen limitation on land and in the sea: How can it occur? Biogeochemistry 13:87-115. https://doi.org/10.1007/BF00002772  von Wirén N, Gazzarrini S, Frommer WB (1997) Regulation of mineral nitrogen uptake in plants. Plant Soil 196:191–199. https://doi.org/10.1023/A:1004241722172  Walia A, Guleria S, Chauhan A, Mehta P (2017) Endophytic bacteria: role in phosphate solubilization. In Maheshwari DK, Annapurna K (eds) Endophytes: Crop Productivity and Protection. Springer, Cham, pp 61–93. https://doi.org/10.1007/978-3-319-66544-3_4  Weetman GF, Fournier RM, Schnorbus E (1988) Lodgepole pine fertilization screening trials: four-year growth response following initial predictions. Soil Sci Soc Am J 52:833–839. https://doi.org/10.2136/sssaj1988.03615995005200030042x  Weise T, Kai M, Piechulla B (2013) Bacterial ammonia causes significant plant growth inhibition. PLoS One 8:e63538. https://doi.org/10.1371/journal.pone.0063538  Weyens N, van der Lelie D, Artois T, Smeets K, Taghavi S, Newman L, Carleer R, Vangronsveld J (2009) Bioaugmentation with engineered endophytic bacteria improves contaminant fate in phytoremediation. Environ Sci Technol 43:9413–9418. https://doi.org/10.1021/es901997z  Weyens N, Truyens S, Dupae J, Newman L, van der Lelie D, Carleer R, Vangronsveld J (2010) Potential of Pseudomonas putida W619-TCE to reduce TCE phytotoxicity and evapotranspiration in poplar cuttings. Environ Pollut 158:2915–2919. https://doi.org/10.1016/j.envpol.2010.06.004  Weyens N, Boulet J, Adriaensen D et al (2012) Contrasting colonization and plant growth promoting capacity between wild type and a gfp-derative of the endophyte Pseudomonas putida W619 in hybrid poplar. Plant Soil 356:217–230. https://doi.org/10.1007/s11104-011-0831-x  Willan RL (1985) A guide to forest seed handling. Food and Agricultural Organization of United Nations, Rome. http://www.fao.org/3/ad232e/ad232e00.htm. [accessed 08 June 2020] Xin G, Zhang GY, Kang JW, Staley JT, Doty SL (2009) A diazotrophic, indole-3-acetic acid-producing endophyte from wild cottonwood. Biol Fertil Soils 45:669–674. https://doi.org/10.1007/s00374-009-0377-8  Xing K, Bian GK, Qin S, Klenk HP, Yuan B, Zhang YJ, Li WJ, Jiang JH (2012) Kibdelosporangium phytohabitans sp. nov., a novel endophytic actinomycete isolated from oil-seed plant Jatropha  218 curcas L. containing 1-aminocyclopropane-1-carboxylic acid deaminase. Antonie Van Leeuwenhoek 101:433-441. https://doi.org/10.1007/s10482-011-9652-4 Yamada Y, Hoshino K, Ishkawa T (1997) The phylogeny of acetic acid bacteria based on the partial sequences of 16 S ribosomal RNA: the elevation of the subgenus Gluconacetobacter to the generic level. Biosci Biotechnol Biochem 61:1244–1251. https://doi.org/10.1271/bbb.61.1244  Yang H, Puri A, Padda KP, Chanway CP (2016) Effects of Paenibacillus polymyxa inoculation and different soil nitrogen treatments on lodgepole pine seedling growth. Can J For Res 46:816–821. https://doi.org/10.1139/cjfr-2015-0456  Yang H, Puri A, Padda KP, Chanway CP (2017) Substrate utilization by endophytic Paenibacillus polymyxa that may facilitate bacterial entrance and survival inside various host plants. FACETS 2:120–130. https://doi.org/10.1139/facets-2016-0031  Yanke LJ, Selinger LB, Cheng KJ (1998) Phytase activity of anaerobic ruminal bacteria. Microbiology 144:1565–1573. https://doi.org/10.1099/00221287-144-6-1565  Yasmin S, Zaka A, Imran A, Zahid MA, Yousaf S, Rasul G, Arif M, Mirza MS (2016) Plant growth promotion and suppression of bacterial leaf blight in rice by inoculated bacteria. PloS One 11:e0160688. https://doi.org/10.1371/journal.pone.0160688 Zavišić A, Polle A (2017) Dynamics of phosphorus nutrition, allocation and growth of young beech (Fagus sylvatica L.) trees in P-rich and P-poor forest soil. Tree Physiol 38:37-51. https://doi.org/10.1093/treephys/tpx146  Zavišić A, Nassal P, Yang N, Heuck C, Spohn M, Marhan S, Pena R, Kandeler E, Polle A (2016) Phosphorus availabilities in beech (Fagus sylvatica L.) forests impose habitat filtering on ectomycorrhizal communities and impact tree nutrition. Soil Biol Biochem 98:127-137. https://doi.org/10.1016/j.soilbio.2016.04.006  Zavišić A, Yang N, Marhan S, Kandeler E, Polle A (2018) Forest soil phosphorus resources and fertilization affect ectomycorrhizal community composition, beech P uptake efficiency, and photosynthesis. Front. Plant Sci 9:463. https://doi.org/10.3389/fpls.2018.00463  Zehr JP, McReynolds LA (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55:2522–2526 Zehr JP, Jenkins BD, Short SM, Steward GF (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ Microbiol 5:539–554. https://doi.org/10.1046/j.1462-2920.2003.00451.x   219 Zeng Q, Wu X, Wen X (2016) Effects of soluble phosphate on phosphate-solubilizing characteristics and expression of gcd gene in Pseudomonas frederiksbergensis JW-SD2. Curr Microbiol 72:198-206. https://doi.org/10.1007/s00284-015-0938-z Zeng Q, Wu X, Wang J, Ding X (2017) Phosphate solubilization and gene expression of phosphate-solubilizing bacterium Burkholderia multivorans WS-FJ9 under different levels of soluble phosphate. J Microbiol Biotechnol 27:844-855. https://doi.org/10.4014/jmb.1611.11057 Zhang R, Wienhold BJ (2002) The effect of soil moisture on mineral nitrogen, soil electrical conductivity, and pH. Nutr Cycling Agroecosyst 63:251–254. https://doi.org/10.1023/A:1021115227884    220 Appendices Appendix A Recipe to make phosphate buffered saline (PBS) NaCl 8 g/L KH2PO4 0.24 g/L KCl 0.2 g/L Na2HPO4 1.44 g/L Distilled Water 1 L     221 Appendix B Steps to prepare combined carbon medium (CCM)  Solution 1 Sucrose 5 g/L Mannitol 5 g/L Sodium Lactate (ml, 60%, v/v) 0.5 ml/L K2HPO4 0.80 g/L KH2PO4 0.20 g/L NaCl 0.10 g/L Na2MoO4.2H2O 0.025 g/L Na2FeEDTA 0.028 g/L Yeast Extract 0.1 g/L Distilled Water 900ml  Solution 2 MgSO4.7H2O 0.20 g/L CaCl2 0.06 g/L Distilled water 100 ml   Autoclave solution 1 and 2 separately, cool and mix Add filter sterilized Biotin: 5μg/L and Para Amino Benzoic Acid (PABA): 10μg/L  222 Appendix C Recipe to make plant nutrient solution KH2PO4 0.14g/L H3BO3 0.001g/L ZnSO4.7H2O 0.001g/L NaMoO4.2H2O 0.001g/L Na2Fe EDTA 0.025g/L MgSO4 0.49g/L MnCl2.4H2O 0.001g/l CuSO4.5H2O 0.0001g/L Ca15(NO3)2 / Ca(NO3)2 0.0576g/L  

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