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N2-fixation and seedling growth promotion of lodgepole pine by wild-type and GFP-labeled Paenibacillus… Tang, Qian 2016

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N2-FIXATION AND SEEDLING GROWTH PROMOTION OF LODGEPOLE PINE BY WILD-TYPE AND GFP-LABELED PAENIBACILLUS POLYMYXA by  Qian Tang  B.Sc., Nanjing Agricultural University, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES  (Soil Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2016  © Qian Tang, 2016 ii  Abstract Paenibacillus polymyxa P2b-2R is a bacterium that originated from the internal stem tissues of lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) seedlings growing in the interior of British Columbia. Several studies have suggested that P. polymyxa P2b-2R can fix nitrogen in association with lodgepole pine and can promote plant growth. To further evaluate this strain, P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, was generated and used in a long term inoculation study. To confirm its ability to fix N2 and to contribute to the N nutrition of pine, as well as comparing the similarities or differences between GFP-labeled and wild-type P2b-2R, a one-year study on bacterial colonization and lodgepole pine seedling growth responses to inoculation with wild-type or GFP-labeled P. polymyxa P2b-2R, was performed. Surface-sterilized lodgepole pine seeds were sown in glass tubes containing an autoclaved sand/montmorillonite clay mixture that contained a nutrient solution with a small amount of nitrogen labeled with 15N as Ca(15NO3)2 (5 % 15N label). After sowing, seeds were inoculated with either P. polymyxa P2b-2R or P. polymyxa P2b-2Rgfp. Non-inoculated controls received phosphate-buffered saline.  The results indicated that both P2b-2R and P2b-2Rgfp were: (i) able to form persistent rhizospheric and endophytic populations, (ii) capable of in situ N fixation and (iii) enhancing seedling growth continuously after four months. Seedlings inoculated with P2b-2R derived 13%-40 % of foliar N from the atmosphere, while those treated with P2b-2Rgfp derived 18%-47 % of foliar N from the atmosphere during seedling growth period. There was a temporary seedling growth reduction in P2b-2R treatments two months after sowing, but seedlings recovered from the growth reduction thereafter. However, P2b-2Rgfp treatments did not display such a decline. Seedlings treated with P2b-2Rgfp had generally higher endophytic populations, foliar N iii  concentrations, %N derived from atmosphere and dry weights, but lower rhizospheric populations than those treated with P2b-2R. However, these differences were not significant.  GFP did not affect N-fixation and growth promotion of wild-type P2b-2R in general, however, GFP-labeled P2b-2R might have short-term advantages in early pine growth stages but these are not significant in the longer term. iv  Preface This dissertation is original, unpublished, independent work by the author, Qian Tang.  v  Table of contents Abstract .......................................................................................................................................... ii Preface ........................................................................................................................................... iv Table of contents ............................................................................................................................v List of tables.................................................................................................................................. vi List of figures ............................................................................................................................... vii List of abbreviations .................................................................................................................. viii Acknowledgements ...................................................................................................................... ix Chapter 1: Introduction ................................................................................................................1 Chapter 2: Materials and methods ...............................................................................................9 2.1 Seed and microorganisms ............................................................................................... 9 2.2 Seedling inoculation and growth .................................................................................... 9 2.3 Quantification of rhizospheric and endophytic colonization ........................................ 11 2.4 Nitrogen analysis and seedling growth response .......................................................... 12 2.5 Statistical analysis ......................................................................................................... 13 Chapter 3: Results........................................................................................................................14 3.1 Rhizospheric and endophytic colonization ................................................................... 14 3.2 Nitrogen analysis and seedling growth response .......................................................... 15 Chapter 4: Discussion ..................................................................................................................20 4.1 Rhizospheric and endophytic colonization ................................................................... 20 4.2 Nitrogen analysis and seedling growth response .......................................................... 22 Chapter 5: Conclusions ...............................................................................................................28 References .....................................................................................................................................30 vi  List of tables Table 1    P2b-2R and P2b-2Rgfp rhizosphere population size (log CFU g-1 root (dry weight) ± standard error; n = 3) of lodgepole pine seedlings two, four, eight and twelve months after sowing and inoculation ..................................................................................................................14 Table 2    P2b-2R and P2b-2Rgfp endophytic population size (log CFU g-1 (fresh tissue) ± standard error; n = 3) of lodgepole pine seedlings two, four, eight months after sowing and inoculation......................................................................................................................................15 Table 3    Foliar % N (mg ± standard error; n =12) of pine seedlings two, four, eight and twelve months after sowing and inoculation .............................................................................................16 Table 4    Atom % 15N excess in foliage (Mean ± standard error; n =12) of lodgepole pine seedlings two, four, eight and twelve months after sowing and inoculation .................................16 Table 5    %N derived from the atmosphere (Ndfa), of lodgepole pine seedlings inoculated with SPB (control), or P. polymyxa P2b-2R (P2b-2R) two, four, eight and twelve months after sowing ............................................................................................................................................16  vii  List of figures Figure 1    Root and shoot length (cm ± standard error; n = 12) two, four, eight and twelve months after sowing and inoculation ............................................................................................ 17 Figure 2    Root and shoot biomass (mg ± standard error; n = 12) two, four, eight and twelve months after sowing and inoculation ............................................................................................ 19 viii   List of abbreviations ANOVA    Analysis of variance BNF           Biological nitrogen fixation CCM          Combined carbon medium CFU           Colony forming units GFP/gfp     Green fluorescent protein N/N2          Nitrogen Ndfa           Nitrogen derived from atmosphere PBS            Phosphate buffered saline PAR           Photosynthetically active radiation  PGPR         Plant growth promoting rhizobacteria P                 Phosphorous TSA           Tryptic soy agar UBC          University of British Columbia ix  Acknowledgements I would like to thank my supervisor, Dr. Chris Chanway, for financial support and guidance and also for enlarging my vision of science and providing coherent answers to my questions. I would also like to extend my thanks to my committee members, Dr. Grayston and Dr. Anand, for their constructive feedback on my work.   I owe particular thanks to Dr. Alice Chang, who gave me lots of advice in lab operations, and helped me in nitrogen analysis. Thanks also to the faculty, staff and my fellow students at UBC, who have inspired me to continue my work in this field.  Special thanks are owed to my parents, whose have supported me throughout my years of education, both morally and financially, and to all my friends, for their warm company and encouragement.  1  Chapter 1: Introduction Nitrogen is the mineral nutrient most in demand by plants and the fourth most common element in their composition. It is a major component of chlorophyll, the most important pigment needed for photosynthesis, as well as an important component of amino acids, the key building blocks of proteins. It is also found in other important biomolecules, such as ATP and nucleic acids (Wagner 2012).  Nitrogen is one of the most abundant elements on earth, however, it is predominately found in the form of nitrogen gas or dinitrogen (N2) in the Earth’s atmosphere. Plants are unable to utilize nitrogen gas and rely on reduced forms of nitrogen, which they acquire by: 1) the addition of ammonia and nitrate fertilizer (from the Haber-Bosch process) or manure to soil, 2) the release of these compounds during organic matter decomposition, 3) the conversion of atmospheric nitrogen into compounds by natural processes, such as lightning, and 4) biological nitrogen fixation (Vance 2001). The reduction of atmospheric nitrogen is a complex process that requires a large input of energy to proceed (Postgate 1982). The nitrogen molecule is composed of two atoms joined by a triple covalent bond, thus making the molecule highly inert and nonreactive. This extremely strong bond dominates N2 chemistry, causing difficulty in the conversion of dinitrogen to useful compounds. Nitrogen, therefore, is a crucial limiting element for plant growth and production. Nitrogenases are enzymes used by some organisms to fix atmospheric nitrogen gas, i.e. to reduce dinitrogen to ammonia (NH3), by catalyzing the breaking of the triple bond and the addition of three hydrogen atoms to each N atom. Plants can then easily assimilate NH3 to produce the aforementioned nitrogenous biomolecules.  2  Biological nitrogen fixation (BNF) occurs when atmospheric N2 is converted to NH3 by the enzyme nitrogenase. It was discovered by the German agronomist Hermann Hellriegel and Dutch microbiologist Martinus Beijerinck (Beijerinck 1901). A diverse group of prokaryotes contain nitrogenase responsible for the fixation of N2 and are referred to as diazotrophs. This group includes organotropic bacteria, phototrophic sulfur bacteria, and cyanobacteria (formerly known as blue-green algae) as well as some Archaea.  These diazotrophic microorganisms might be autotrophic or heterotrophic, hence, different strategies for gaining energy have evolved in both free-living and symbiotic N2 fixers. Free-living diazotrophs are self-sustaining and tend to fix just enough nitrogen to support their own growth. However, in terms of generating nitrogen available for other organisms, symbiotic associations greatly exceed the free-living species with the exception of cyanobacteria. Well known examples of symbiotic associations include nodule forming Rhizobium and legumes (Stacey 2007), Frankia and actinorhizal plants such as Alnus spp., which also results in root nodule formation (Pawlowski and Bergman 2007) and cyanobacterial symbioses with cycads, Gunnera or algae. The symbiosis between cyanobacteria and fungi results in lichen formation (Haselkorn 2009).  The importance of diazotrophic bacteria for the nitrogen nutrition of plants generally depends on nitrogen availability in soils and nitrogen requirements of the plant species growing there. High soil nitrogen levels are known to suppress BNF. Plant-growth-promoting rhizobacteria (PGPR) such as Arthrobacter, Azotobacter, Bacillus, and Rhizobium (Burdman et al. 2000) exert positive effects on plants by various mechanisms, including the secretion of plant growth regulators such as auxins, gibberellins and cytokinins that stimulate plant metabolic 3  activities in the roots and also by supplying plant growth-limiting nutrients such as biologically-fixed nitrogen (Cocking 2003). Nodulated legumes in symbiosis with rhizobia are usually considered to be amongst the most outstanding nitrogen-fixing systems. However, the study for inoculation of non-legumes with a variety of non-rhizobial diazotrophic bacteria generally underestimated intercellular colonization of bacteria within the plant and fixation of nitrogen endophytically, which provides fixed nitrogen for plant growth. More recently, N2 fixing endophytic bacteria have been studied in this regard. Endophytic bacteria are defined as bacteria that can be detected inside or isolated from internal tissues of plants that have no visible symptoms of disease (Hallmann et al. 1997).  The propagation of endophytes may be intercellular between adjacent cells, or by crossing walls or entering plant cells, probably as a consequence of their ability to secrete cellulases and pectinases (Cocking et al. 1994). When bacteria are confined by host cell walls, they may also form infection threads to enter plant cells (Sprent and de Faria 1987). By occupying the plant interior rather than the surface of the plant tissues, endophytic bacteria are thought to have a competitive advantage over rhizosphere-colonizing bacteria since they have better access to continuous supplies of nutrients as well as extra protection for competition from other rhizosphere microorganisms (Hallmann et al. 1997). Some of these bacteria may in turn stimulate plant growth by contributing growth-limiting nutrients such as biologically fixed N to their host plant. As those endophytes are often located within roots or dense plant tissue with high aerobic respiration rates, the bacteria are likely to be growing within a low O2 environment, which is necessary for the expression and operation of nitrogenase (Patriquin et al. 1983; Gallon, 1992; Baldani et al. 1997). 4  One of the best known examples of this is endophyte-containing sugarcane (Saccharum officinarum), which has been shown to derive substantial amounts of nitrogen from a pool other than the soil. Using an acetylene reduction assay, Boddey et al. (1995) showed one endophyte of sugarcane, Gluconacetobacter diazotrophicus, to be a competent nitrogen fixer. Other agriculturally important grasses such as rice (Oryza sativa), wheat (Triticum aestivum), sorghum (Sorghum bicolor), maize (Zea mays), Panicum maximum, Brachiaria spp., and Pennisetum purpureum also contain numerous diazotrophic bacteria including G. diazotrophicus, Herbaspirillum spp. (Pimentel et al. 1991; Olivares et al. 1996), and Azospirillum spp. (James 1997).  Endophytic bacteria capable of BNF have been studied, primarily in crops of agricultural importance (Doty 2011). However, forest tree species are also known to harbor a wide range of bacterial endophytes (Izumi 2011), including diazotrophs, but very little is known about these microorganisms (Doty 2011).  Paenibacillus polymyxa P2b-2R is a nitrogen-fixing bacterium that was isolated from internal stem tissue of a naturally regenerating lodgepole pine seedling (Pinus contorta var. latifolia (Dougl.) Engelm.) (Bal and Chanway 2012).  P. polymyxa (Ash et al. 1993) is a Gram-positive, rod-shaped, motile bacterium capable of endospore formation (Zengguo 2007; Huo et al. 2010). It is non-pathogenic and found in environments such as plant roots, in soil and in marine sediments (Timmusk 2005; Ravi 2007). P. polymyxa is known to have a broad host plant range including wheat and barley, corn, white clover, sorghum, sugarcane and Douglas-fir (Lindberg et al.1984; Holl et al.1998; Shishido et al.1996). P. polymyxa is one of the most often studied plant growth promoting rhizobacteria (PGPR), and has been reported to possess a wide range of capabilities, including soil phosphorus 5  solubilization (Duff et al. 1963; Singh and Singh 1993); production of hormones that promote plant growth; production of antibiotics (Davis et al. 1968; Rosado and Seldin 1993; Lal and Tabacchioni 2009), chitinase (Mavingui and Heulin 1994) and other hydrolytic enzymes (Nielsen and Sorensen 1997); enhancement of soil porosity (Gouzou et al. 1993; Timmusk 1999) as well as nitrogen fixation (Lindberg et al. 1985; Heulin et al. 1994).  Lodgepole pine is one of the most widespread pines in western North America (Critchfield and Little 1966) and is a highly adaptable species that is known to thrive on severely N-deficient sites. When one of the strains of P. polymyxa, P2b-2R, was reintroduced to lodgepole pine seedlings which were grown in N-deficient soil, it was found to be capable of forming persistent rhizospheric (Bal et al. 2012) and endophytic (Anand et al. 2013) populations, and was able to enhance lodgepole pine seedling growth significantly in 13-month growth trials. The results are highly suggestive of a long-term mutualism between P2b-2R and lodgepole pine, where the pine host provides the carbon substrates to the endophytic diazotrophs in exchange for fixed nitrogen (Bal et al. 2012). Several lines of indirect evidence such as consistent growth on N-free media, in vitro acetylene reduction and the presence of the nifH gene suggested that it is nitrogen-fixing, and possibly plant growth-promoting (Bal et al. 2012). Based on calculations from a foliar 15N dilution assay, P2b-2R-inoculated lodgepole pine seedlings grown in N-deficient soil derived up to 66% (Bal and Chanway 2012) and 78% (Anand et al. 2013) of foliar N from the atmosphere by the end of 9- and 13-month growth trials, respectively.  Most seedling growth research with P. polymyxa P2b-2R has been performed in glass tubes filled with a sand-montmorillonite clay mixture. However, a recent experiment with P. polymyxa P2b-2R-treated pine seedlings grown in the greenhouse using seedling containers (Ray 6  Leach Cone-tainers 3.8 cm diameter) filled with greenhouse soil yielded inconsistent seedling growth promotion and no evidence of BNF (Yang 2015). Any one of several modifications to the glass-tube-based seedling growth protocol may have contributed to these variable findings but the use of greenhouse soil that was fertilized monthly with a small amount of N in the most recent work (Yang 2015) seems the most likely causative factor that inhibited biological N2 fixation. To avoid seedling growth response variability, the work in this thesis was performed according to the original protocol for pine described by Bal and Chanway (2012) and later by Anand et al. (2013) using a sand-montmorillonite clay mixture under a controlled environment. Culture-dependent surface-disinfestation dilution plate assays suggest that P2b-2R colonizes internal pine root, stem and needle tissues (Anand et al. 2013).  To further evaluate endophytic colonization by this strain, P. polymyxa P2b-2Rgfp, a green fluorescent protein (GFP)-labeled derivative of P2b-2R, was generated (Anand and Chanway 2013) and confocal laser scanning microscopy results with it confirmed that P. polymyxa P2b-2R is capable of endophytic colonization of pine seedlings with specific colonization sites that include seedling stem cortex cells.  The green fluorescent protein (GFP) was first isolated from the jellyfish Aequorea victoria and its properties were studied by Japanese researcher Osamu Shimomura (1962). It is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The biggest advantage of GFP is that it is heritable. In addition, visualizing GFP is noninvasive, and the operation is quite simply that the protein would be detected just by shining light on it under fluorescent microscopy. Furthermore, GFP is a relatively small and inert molecule that usually does not interfere with any biological processes being studied. Moreover, if used with a monomer it is able to diffuse readily throughout cells (Chalfie 2009). With 7  numerous advantages over existing marker systems, GFP has become one of the most convenient and effective tools for microbiologists to study microorganisms in complex biological systems (Errampalli 1999). Work with P. polymyxa P2b-2Rgfp confirmed endophytic colonization and demonstrated at least some of the sites it can colonize on and in pine (Anand and Chanway 2013). However, modification by GFP may alter bacterial characteristics from the wild type. Weyens (2012) and his team reported the harmful impact on plant health and growth by GFP-labelled P. putida W619 while the wild-type P. putida W619 resulted in significant growth promotion in hybrid poplar. Other studies have also shown that the GFP gene might result in positive or negative effects on the growth of plant hosts (Padda et al. 2015; Haseloff et al. 1997; Petri et al. 2008).  While strain P2b-2Rgfp was selected because its growth rate was similar to the parent strain and plasmid stability was high (>88 %) after ten passages through nonselective medium (Anand and Chanway 2013), its ability to fix N2 or to contribute to the N nutrition of pine has not yet been tested. Therefore, the primary objective of my research was to evaluate the similarities (and differences) of P2b-2Rgfp to the wild type strain P2b-2R, from which it was derived.  The specific objectives of my project were to: 1. further elucidate endophytic colonization of GFP-labeled P2b-2R inside pine tissues; 2. determine if P. polymyxa P2b-2Rgfp  is capable of N2-fixation in association with lodgepole pine;  3. evaluate the plant growth promoting capability of P. polymyxa P2b-2Rgfp  with lodgepole pine; 8  4. compare the N2-fixing efficacy of P. polymyxa P2b-2Rgfp with that of wild-type P2b-2R.  My specific hypotheses, corresponding to these objectives, are:  1. P2b-2Rgfp will form persistent rhizospheric and endophytic populations on and inside lodgepole pine seedlings;  2. lodgepole pine seedlings inoculated with P2b-2Rgfp and grown in N-limited soil will incorporate atmospheric N into their foliage; 3. growth of P2b-2Rgfp-treated pine seedlings will be enhanced compared with non-treated controls; 4. BNF and growth enhancement of pine seedlings treated with P2b-2Rgfp will not differ significantly from pine treated with wild type P2b-2R.  9  Chapter 2: Materials and methods 2.1 Seed and microorganisms Lodgepole pine seeds were obtained from the British Columbia Ministry of Forest, Lands and Natural Resource Operations Tree Seed Centre, Surrey, British Columbia, Canada, collected from naturally regenerating seedlings near Williams Lake, British Columbia, Canada (52°05′ N lat., 122°54′ W long., elevation 1,300 m, Sub-Boreal Pine Spruce, SBPSdc zone). P. polymyxa P2b-2R is a bacterium that was isolated from internal stem tissue of a naturally regenerating lodgepole pine, also near Williams Lake (Bal et al. 2012). P2b-2Rgfp was previously generated by Anand and Chanway (2013). Both strain P2b-2R and P2b-2Rgfp were stored at -80 °C on combined carbon medium (CCM) (N-free) (Rennie 1981) amended with 20 % (v/v) glycerol. P2b-2R is resistant to 200 mg L-1 rifamycin. 2.2 Seedling inoculation and growth Lodgepole pine seedlings were grown in glass tubes (150mm×25 mm in diameter) filled to 67 % capacity with a sand–Turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, IL) mixture (69%w/w silica sand; 29 % w/w Turface; 2 % w/w CaCO3).  Before sowing, each tube was fertilized to saturation with 17 mL of a nutrient solution which was modified by replacing KNO3 and Ca(NO3)-4H2O with Ca(15NO3)2 (5 % 15N label) (0.0576 gL−1) and Sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ontario) with Na2FeEDTA (0.02 gL−1). Other nutrients in the nutrient solution include (in grams per liter) the following: KH2PO4, 0.14; MgSO4, 0.49; H3BO3, 0.001; MnCI2-4H2O, 0.001; ZnSO4-7H2O, 0.001; CuSO4-5H2O, 0.0001; and NaMoO4-2H2O, 0.001. Fertilized tubes were then autoclaved for 1 h before use in experiments. 10  Pine seeds were surface sterilized by immersion in 30 % (v/v) hydrogen peroxide (H2O2) for 1.5 min, followed by three 30-s rinses in fresh sterile distilled water. The effectiveness of the surface sterilization was confirmed by imprinting sterilized seeds on tryptic soy agar (TSA) (BD Difco) and checking for microbial contamination two days later. Seeds found to be free of surface contamination were placed in sterile cheese cloth bags containing moist autoclaved sand. These bags were then placed in a loosely tied autoclavable plastic bag and stored at 4°C for 5 weeks to stratify seeds. Stratified seeds were then imprinted on TSA plates for 48 h to examine for surface contaminants. In addition, ten randomly selected surface-sterilized seeds were crushed and imprinted on TSA plates supplemented with 200 mg L−1 rifamycin for 48 h to confirm the absence of internal seed contamination. Three surface-sterilized seeds were then aseptically sown in each glass tube and covered with 5 mm of autoclaved silica sand.  Bacterial inocula were prepared by thawing frozen cultures of strain P2b-2R and c and streaking each onto CCM plates amended with 200 mg L−1 rifamycin for P2b-2R or 200 mg L−1 rifamycin plus 5 mg L−1 chloramphenicol for P2b-2Rgfp. After colonies grew, a loopful of each strain was inoculated into 1 L flasks containing 500 mL of fresh CCM broth amended with rifamycin or rifamycin plus chloramphenicol as described above. Inoculated flasks were secured on a rotary shaker and agitated (150 rpm) for 24 h at room temperature. Bacteria were then harvested by centrifugation (3000×g, 20 min) and re-suspended in sterile phosphate buffer (SPB) to a density of 106 colony forming unit (CFU) mL−1.  For the seedling growth assay, 5 mL of the P2b-2R-SPB or the P2b-2Rgfp-SPB suspension were pipetted into each of 100 replicate tubes containing lodgepole pine seeds immediately after sowing. Seeds in the 100 control tubes received 5.0 mL of SPB. All tubes were placed in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB) 11  with photosynthetically active radiation (PAR) at canopy level of 300 μmol photons (400–700-nm wavelengths) m−2s−1, an 18-h photoperiod, and a 20/14 °C day/night temperature cycle.  Seedlings were thinned to the largest single germinant per tube two weeks after sowing and were watered with sterile distilled water to constant weight daily and with 4 mL nutrient solution without Ca(NO3)2 once a week. Seedling mortality was monitored throughout the 12-month growth period. Tubes containing dead seedlings were replaced, as required, from a separate pool of replicate tubes prepared for each treatment, when the experiment was set up. 2.3 Quantification of rhizospheric and endophytic colonization Three randomly selected seedlings from each treatment were harvested destructively 2, 4, 8 and 12 months after sowing to evaluate rhizospheric colonization. To do so, seedlings were removed from tubes and gently shaken to remove the adhering sand/turface particles. Roots were then separated from shoots and placed in sterile polypropylene Falcon tubes (50 mL; BD Biosciences, CA, USA) filled with 10 mL of autoclaved PBS and shaken on a vortex mixer at 1000 rpm for one minute. Tenfold serial dilutions were performed, and 0.1 mL of each dilution were plated onto CCM supplemented with cycloheximide (100 mgL-1) to suppress fungal growth for P2b-2R and CCM plus cycloheximide (100 mgL-1) and rifamycin (200 mgL-1) for P2b-2Rgfp. The number of CFU was evaluated seven days after incubation at 30 °C. Roots were washed and oven-dried at 65°C for two days before weighing. Rhizospheric populations were then calculated as the number of CFU g-1 root (dry weight). For the evaluation of endophytic colonization, three randomly selected seedlings from each treatment were collected on the 2nd, 4th, 8th and 12th month after sowing. At each harvest, seedlings were washed thoroughly under running water for 15 minutes, surface-sterilized in 1.3 % sodium hypochlorite for 5 min, and washed three times with sterile distilled water. Seedlings 12  were imprinted on TSA plates for 24 h to check for surface contamination. Root, stem, and needle tissue samples (20 mg fresh weight) were then triturated separately in 1 mL SPB using a mortar and pestle. Triturated tissue suspensions were then diluted serially, and 0.1 mL of each dilution was plated onto CCM plates supplemented with cycloheximide (100 mgL-1) as well as CCM plus cycloheximide (100 mgL-1) and rifamycin (200 mgL-1). The plates were incubated at 30 °C for 72 h, after which colonies were counted. 2.4 Nitrogen analysis and seedling growth response Twelve seedlings from each treatment were randomly harvested 2, 4, 8 and 12 months after sowing for evaluation of seedling biomass as well as foliar N and 15N content.  At each harvest, selected seedlings were removed from tubes, separated into roots and shoots, and the roots were washed. After measuring the root and shoot length, all the samples were dried for 2 days at 65 °C before weighing. Needles were then separated from stems and roots for nitrogen analysis. Evaluation of seedling growth was based on comparison of the growth parameters between seedlings inoculated with wild-type P2b-2R, P2b-2Rgfp and controls.  Treatment effects were expressed using the percentage difference from the appropriate controls and were calculated as follows: (Gi-Gc)/Gc x 100%, where Gi and Gc are growth parameter values of the inoculated (P2b-2R or P2b-2Rgfp) (Gi) and control seedlings (Gc), respectively. A 15N isotope dilution assay was conducted with the objective to estimate the amount of nitrogen gained through biological fixation within lodgepole pine inoculated with P2b-2R and P2b-2Rgfp. The 15N method, as one of the most commonly used methods for measuring BNF, is operationally and mathematically simple, and also considered to be the most accurate approach 13  (Danso 1995). In this experiment, the 15N isotope dilution method was used for measuring the amount of N gained through N-fixation. Needles (from each seedling sampled as described above) were ground to a particle size <2 mm using a mortar and pestle, and 1 mg subsample from each seedling were sent to the Stable Isotope Facility at the University of British Columbia for determination of % 15N using an elemental analyzer interfaced with an isotope ratio mass spectrometer (Europa Scientific Integra). The amount of fixed foliar N in each N treatment was then calculated according to Rennie et al. (1978) as follows: %Ndfa (N derived from the atmosphere)  = [1 − atom % 15N excess (inoculated plant )atom % 15N excess (control plant) ] × 100% where SPB-treated controls are the non-inoculated plants. 2.5 Statistical Analysis A completely randomized experimental design (n=100) was used to assess the inoculation effects on growth of lodgepole pine seedlings. Analysis of variance (ANOVA) was performed to determine treatment effects on seedling dry weight, atom percent 15N excess, and foliar N concentration using SAS 9.3 software (SAS Institute Inc., Carey, NC). Bacterial colonization was logarithmically-transformed for analysis in order to meet the statistical assumptions of normally-distributed data and equality of variance Treatment means were separated using Fisher’s protected least significant difference. Standard errors were calculated according to Steele and Torrie (1980) (P<0.05).   14  Chapter 3: Results 3.1 Rhizospheric and endophytic colonization  The rhizosphere colonization densities of P2b-2R and P2b-2Rgfp were very high reaching 8.9 and 8.1 log CFU g-1 root (dry weight) by the first harvest, 2 months after sowing and inoculation (Table 1). Population sizes of both strains declined each month thereafter, and eventually reached a stable level of approximately 106 for P2b-2R and 105 for P2b-2Rgfp (P<0.05) one year after the growth trial was initiated.  The results suggest that both strains are able to colonize rhizosphere, which is in agreement with previous findings of Bal and Chanway (2012). The densities of wild-type P2b-2R tended to be higher than GFP-labeled P2b-2R in all harvests, however, the differences were not significant. Table 1    P2b-2R and P2b-2Rgfp rhizosphere population size (log CFU g-1 root (dry weight) ± standard error; n = 3) of lodgepole pine seedlings two, four, eight and twelve months after sowing and inoculation. Treatment 2 months 4 months 8 months 12 months P2b-2R 8.9* ± 0.3 7.5* ± 0.1 6.4* ± 0.1 6.3* ± 0.0 P2b-2Rgfp 8.1* ± 0.2 7.4* ± 0.1 5.2* ± 0.1 5.7* ± 0.2 *P<0.05 (significantly different from control); No P2b-2R and P2b-2Rgfp strains were detected in controls  No endophytic colonization was detected in the surface-sterilized tissue extracts of seedlings treated with bacteria when first assessed 2 months after inoculation. However, approximately 4.7 and 5.7 log CFU g-1 (fresh tissue) P. polymyxa strain P2b-2R and P2b-2Rgfp were detected respectively in four-month-old seedling root tissues (Table 2), when the surface-sterilization protocol was improved by decreasing the concentration of surface sterilant (sodium hypochlorite) from 1.3% to 1.0%. The population sizes increased slightly inside roots two months later and both P2b-2R and P2b-2Rgfp colonization was also detected inside stem tissues 15  at this time; i.e., 4 months after sowing and inoculation. Endophytic population densities decreased during the last days of the growth trial, and ranged from 5.2-4.1 and 4.8-4.1 log CFU g-1 (fresh tissue) in stem and root tissues in P2b-2R treatments, and 5.9-4.3 and 5.5-4.5 log CFU g-1 (fresh tissue) in stem and root tissues in P2b-2Rgfp treatments. Such a decline of endophytic bacterial population sizes in root and stem tissues has also been reported by Anand and Chanway (2013).   No endophytes were detected in needles during the experiment, possibly due to too high a surface sterilant concentration. The endophytic colonization by GFP-labeled P2b-2R was generally higher than the wild-type strain, even though the differences were not statistically significant. Table 2    P2b-2R and P2b-2Rgfp endophytic population size (log CFU g-1 (fresh tissue) ± standard error; n = 3) of lodgepole pine seedlings four, eight and twelve months after sowing and inoculation. Inoculation 4 months   8 months  12 months stem   stem root  stem root P2b-2R 4.7* ± 0.1  5.2* ± 0.1 4.8* ± 0.1  4.1* ± 0.1 4.1* ± 0.0 P2b-2Rgfp 5.7* ± 0.1   5.9* ± 0.0 5.5* ± 0.2  4.3* ± 0.1 4.5* ± 0.2 *P<0.05 (significantly different from control); No P2b-2R and P2b-2Rgfp strains were detected in controls 3.2 Nitrogen analysis and seedling growth response  Inoculation started to have significant positive effects on foliar nitrogen concentration four months after sowing and inoculation (P<0.05) (Table 3). Foliar N concentrations of seedlings inoculated by P2b-2Rgfp were higher than seedlings treated with the wild type P2b-2R. Percentage foliar N of P2b-2R treatments was lower than the two other treatments at the first harvest, but then increased steadily thereafter. For seedlings inoculated by P2b-2Rgfp, foliar N concentration fluctuated during the experiment however it generally increased with time. There 16  was a peak (1.20%, P< 0.05) at month four and a slight decline between four and eight months after sowing and inoculation. Table 3    Foliar % N (mg ± standard error; n =12) of pine seedlings two, four, eight and twelve months after sowing and inoculation. Treatment 2 months 4 months 8 months 12 months Control 0.83b ± 0.02 0.76a ± 0.02 0.74a ± 0.02 0.80a ± 0.01 P2b-2R 0.67a ± 0.01 0.90b ± 0.02 0.97b ± 0.02 1.11b ± 0.02 P2b-2Rgfp 0.84b ± 0.02 1.20c ± 0.03 1.12b ± 0.04 1.19b ± 0.01 Within the same harvest group, means with different letters are significantly different at P<0.05 Foliar 15N atom percent excess was affected significantly by inoculation (P<0.05) and decreased over time in all treatments (Table 4). %Ndfa was calculated based on 15N foliar dilution, and both of the inoculation treatments were found to have derived N from sources other than the plant growth medium (Table 5). Seedlings inoculated with P2b-2R derived 13%-40 % of foliar N from the atmosphere, while those treated with P2b-2Rgfp derived 18%-47 % of foliar N from the atmosphere between month two and twelve of the seedling growth period. Table 4    Atom % 15N excess in foliage (Mean ± standard error; n =12) of lodgepole pine seedlings two, four, eight and twelve months after sowing and inoculation.  Treatment 2 months 4 months 8 months 12 months Control 0.82b ± 0.01 0.78c ± 0.01 0.75b ± 0.02 0.70b ± 0.05 P2b-2R 0.71a ± 0.02 0.66b ± 0.03 0.50a ± 0.04 0.42a ± 0.02 P2b-2Rgfp 0.67a ± 0.00 0.61a ± 0.04 0.45a ± 0.02 0.37a ± 0.03 Within the same harvest group, means with different letters are significantly different at P<0.05 Table 5    %N derived from the atmosphere (Ndfa), of lodgepole pine seedlings inoculated with P. polymyxa strain P2b-2R and P2b-2Rgfp two, four, eight and twelve months after sowing. Treatment 2 months 4 months 8 months 12 months P2b-2R 13 15 33 40 P2b-2Rgfp 18 22 40 47  17  Seedlings inoculated with P. polymyxa P2b-2R and P2b-2Rgfp generally increased root and shoot length compared to controls four months after sowing and inoculation, however, seedling growth was inhibited slightly by bacterial inoculation till month two (Figure 1). The seedling growth enhancement by bacterium appeared to be greatest at month 8, where seedlings treated with P2b-2R were 12.2% and 27.2 % higher than non-inoculated controls in roots and shoots, respectively (P<0.05), and P2b-2Rgfp treatments were 19.4% and 28.9% taller in roots and shoots, respectively (P<0.05).  When comparing the two bacterial inoculation treatments, the results suggest no apparent difference, except root length at month eight where seedlings from P2b-2Rgfp treatments were significantly longer than P2b-2R (P<0.05).   Figure 1    Root and shoot length (cm ± standard error; n = 12) two, four, eight and twelve months after sowing and inoculation. Within the same group, bars with different letters are significantly different at P<0.05 Seedling biomass of P2b-2R- and P2b-2Rgfp- treated seedlings was enhanced greatly compared to non-inoculated controls four months after sowing and inoculation (Figure 2). P2b-18  2R treatments decreased seedling dry weights slightly at month two, which coincides with the results from seedling lengths. The growth reduction by P2b-2R was also reported in previous studies by Bal and Chanway (2012), where seedling growth was inhibited by bacterial inoculation 7 and 9 months after treatment. Seedling biomass of P2b-2R treatment increased progressively after two months, and growth rate was steady during growth time. Seedlings inoculated with P2b-2R were 13% and 33% greater in root and shoot weights than non-inoculated controls twelve months after sowing and inoculation (P<0.05, Figure 2). GFP-labeled P2b-2R did not appear to have such a decline as did wild-type P2b-2R at the beginning of the growth period, and seedling growth promotion was significant throughout entire experimental time. Seedlings inoculated by P2b-2Rgfp tended to have fatter roots than other treatments, therefore, the root biomass was high even though root length was reduced in the first harvest.  The growth promotion of seedling roots slowed down after four months, and by month twelve, P2b-2Rgfp increased root and shoot dry weights by 14% and 42%, respectively. The increase in root biomass by P2b-2Rgfp treatments was significant in the first three harvests compared with P2b-2R treatments (P<0.05), however, the gap narrowed in each month, and eventually decreased to be negligible by month twelve (P2b-2R root biomass: 21.0 mg, P2b-2Rgfp root biomass: 21.2 mg). As for shoot dry weight, P2b-2Rgfp treatments were generally better than P2b-2R, however, the tendency was not significant. When considering the whole seedling biomass, P2b-2Rgfp treatments were slightly better than P2b-2R, although the differences were not significant. 19   Figure 2    Root and shoot biomass (mg ± standard error; n = 12) two, four, eight and twelve months after sowing and inoculation. Within the same group, bars with different letters are significantly different at P<0.05 20  Chapter 4: Discussion The primary objective of this thesis is to evaluate the similarities (and differences) between the colonization, plant growth promotion and nitrogen fixing abilities of P. polymyxa P2b-2Rgfp and the wild-type strain P2b-2R, from which it was derived.  A secondary objective was to determine N2-fixing efficacy and plant growth promoting capability of P2b-2Rgfp with lodgepole pine. 4.1 Rhizospheric and endophytic colonization The rhizospheric bacterial population densities of P2b-2R and P2b-2Rgfp ranged from 8.9 to 6.3 and 8.1 to 5.7 log CFU g-1 root (dry weight) respectively in my one year growth trial (Table 1). There was a decline in population size in both bacterial treatments between month two and month eight, and then a steady state was achieved by month twelve. According to the protocol, a density of 106 CFU mL−1 inoculum of each bacterium were used for inoculation at the beginning of the experiment, therefore, notwithstanding the variation,  both and P2b-2Rgfp recovered to approximately the original level after one year growth. This suggests that both wild-type and GFP-labeled P2b-2R are competent rhizosphere colonizers, and population sizes were close to other reports for strains of P. polymyxa on conifers, e.g., 1.65*105 CFU·g–1 root tissue on lodgepole pine (Bal and Chanway 2012). The bacterial population reduction over time has also been reported for Bacillus strains colonizing the rhizosphere of lodgepole pine (Holl and Chanway 1992). The densities of the wild-type were higher than GFP-labeled P2b-2R at all harvests; however, the differences were small and not significant. To form endophytic colonies, bacteria usually enter into roots from the rhizosphere, particularly at the base of emerging lateral roots (Cocking 2003). In my experiment, tissue surface sterilization and dilution plating of triturated extracts indicated that P. polymyxa strain P2b-2R and P2b-2Rgfp colonized internal root and stem tissues of lodgepole pine during the 21  seedling growth period, ranging from 5.2-4.1and 4.8-4.1 log CFU g-1 (fresh tissue) in stem and root tissues from P2b-2R treatments, and 5.9-4.3 and 5.5-4.5 log CFU g-1 (fresh tissue) in stem and root tissues from P2b-2Rgfp treatments (Table 2). No endophytic colonization was detected when first assessed 2 months after inoculation, and no endophytes were detected in needles during the growth period. The reason for these might be too high a concentration of surface sterilant, which is known to significantly reduce microbial populations (Nakagawara et al. 1998). Another possible reason for the latter could be that the growth trial was not long enough for the bacteria to form quantifiable colonies in needles.  My results suggest that both P2b-2R and P2b-2Rgfp colonized lodgepole pine seedling shoot and roots endophytically, and population densities were comparable to those observed in rice (Prasad 2001), poplar (Ren 2013), western red cedar and lodgepole pine (Anand and Chanway 2013).  A wide range of endophytic diazotrophs were reported to be able to colonize the root cortex cells, such as Azorhizobium caulinodans, Gluconacetobacter diazotrophicus, Azoarcus spp. Herbaspirillum spp. and some strains of Azospirillum (Dõbereiner et al. 1993; Cocking 2003). One of the most remarkable associations is that between Gluconacetobacter diazotrophicus and sugarcane, where the bacteria were found in the roots, stems, and aerial parts, and established G. diazotrophicus colonies are capable of growing up to 108 CFU per gram of root tissue within sugarcane (Reis et al. 1994).  My results also suggested an increasing trend in stem and root endophytic colonization between month four and month eight, although the tendency was not apparent afterwards because the densities of both bacteria decreased thereafter. Such a decline of endophytic bacterial population sizes in lodgepole pine tissues has been reported in an earlier paper from Anand and 22  Chanway (2013). The declining trend of both endophytic and rhizospheric colonies also happened in other experiments. In the research for growth promotion of maize by P. aurantiaca JD37 from Fang’s team (2013), a bacterial population of 107 CFU was observed, which was then maintained at about 106 CFU during the following period (8–28 days post-inoculation) per gram in rhizosphere. And in each gram of root tissue, the population density increased up to 4.2*104 CFU by 4 days post-inoculation and remained at 1*104 until 28 days post-inoculation.  In contrast to rhizospheric colonization, the endophytic colonization by GFP-labeled P2b-2R was generally higher than the wild-type, however the differences were not significant.  4.2 Nitrogen analysis and seedling growth response  Inoculation had significantly positive effects on foliar nitrogen concentration after four months, and foliar %N of seedlings inoculated by P2b-2Rgfp generally outperformed the ones by P2b-2R (Table 3). Seedlings treated with P2b-2R resulted in lower foliar N concentrations than other treatments at month two and then increased to 1.11% by the end of the one year growth trial. For seedlings inoculated by P2b-2Rgfp, foliar %N fluctuated over time, however, it demonstrated a rising tendency in general.  Foliar 15N atom percent excess was affected significantly by inoculation as well and decreased over time in all treatments (Table 4). Based on 15N foliar dilution, %Ndfa was calculated, and both of the inoculation treatments were found to have successfully derived N from sources other than the plant growth medium (Table 5). Seedlings inoculated with P2b-2R derived 13%-40 % of foliar N from the atmosphere, while those treated with P2b-2Rgfp derived 18%-47 % of foliar N from the atmosphere between month two and twelve of the seedling growth period. Considered together with seedling biomass, the results are highly suggestive of 23  biological nitrogen fixation by both P2b-2R and P2b-2Rgfp, which was consistent with results reported by Anand & Chanway (2013). As the most promising result for N fixation by endophytic bacteria, the studies for the associations between Gluconacetobacter diazotrophicus and sugarcane have showed that the bacteria were capable of fixing up to 80% of their required N, under ideal conditions (Urquiaga et al. 1992). Research for other endophytic diazotrophs with agriculturally important grasses also have shown some notable results. Baldani et al. (1995) reported that H.seropedicae strain Z94 contributed over 50% fixed N with the cultivated rice (Oryza sativa L.). Boddey and Victoria (1986) have reported, using 15N isotope dilution, that two species (Brachiaria humidicola, Brachiaria decumbens) associated with Brachiaria and Paspalum grasses, were capable of deriving up to 40% of their N from the atmosphere. Besides, some explorations for endophytic bacteria in long-living forest trees have also shown positive results for N fixation. Research indicated nitrogen fixation by endophytic diazotrophs within Populus deltoides (Schink et al. 1981) and Populus trichocarpa (Kamp 1986). Knoth et al. (2014) reported 45% BNF of total nitrogen based on isotope-dilution values within poplar inoculated with endophyte consortium Poplar Mix B2.  In my experiment, root and shoot length increased significantly in seedlings inoculated with Paenibacillus polymyxa P2b-2R and P2b-2Rgfp compared to the controls four months after sowing and inoculation; however, seedling growth was inhibited slightly by bacterial inoculation before month two (Figure 1). The results suggest that no differences between P2b-2R and P2b-2Rgfp treatments exist, in general. P2b-2R- and P2b-2Rgfp- treated seedlings accumulated significantly more biomass than non-inoculated controls four months after sowing and inoculation (Figure 2). In accordance with 24  seedling length, P2b-2R treatments decreased seedling dry weights slightly at month two and then increased progressively over time, indicating that N2-fixing seedlings could recover from the growth reduction over time. The growth reduction and recovery in P2b-2R treatments was also reported in previous studies by Bal and Chanway (2012) and Anand et al. (2013), where seedling growth was inhibited by bacterial inoculation 7 and 9 months after treatment and promotion occurred by month 12. Seedlings treated with P2b-2R were 13% and 33% greater in root and shoot weights than non-inoculated controls 12 months after sowing and inoculation. The large significant seedling biomass enhancement by P2b-2R falls in line with previous findings (Anand et al. 2013), and supports the hypothesis that a beneficial mutualism exists between lodgepole pine and this bacterium. It also suggests that soil nitrogen depletion would restrict the growth of control seedlings to a certain point where they would be exceeded by N2-fixing seedlings, and that the full development of a beneficial N2-fixing bacterial population is an involved process that requires a certain period of time to occur, perhaps up to a year depending on the initial soil N level. However, GFP-labeled P2b-2R did not appear to have such an apparent decline in seedling biomass like wild-type P2b-2R had at early stage, and seedling growth promotion was significant throughout the whole experimental period. The greatest increase occurred after month four, where seedlings treated with P2b-2Rgfp were 77% and 40% greater in root and shoot biomass than controls, respectively. Afterwards, growth promotion in seedling roots slowed down, so that by month twelve, P2b-2Rgfp increased 14% in root and 42% in shoot dry weights.  The advantage of P2b-2Rgfp treatments in root biomass was significant in the first eight months compared with P2b-2R treatments, however, the gap was narrowed in each month, and eventually decreased to negligible by month twelve (P2b-2R root biomass: 21.0 mg, P2b-2Rgfp 25  root biomass: 21.2 mg). As for shoot dry weight, P2b-2Rgfp treatments were generally better than P2b-2R, however, the tendency was not apparent. When considering the whole seedling biomass, P2b-2Rgfp treatments were slightly better than P2b-2R, and the differences were no-significant.  P. polymyxa is well known for its variety of plant growth-promoting properties including N fixation (Lindberg et al. 1985; Heulin et al. 1994), soil phosphorus solubilization (Duff et al. 1963; Singh and Singh 1993), production of hormones that enhance plant growth, production of different antibiotic substances (Davis et al. 1968; Rosado and Seldin 1993; Lal and Tabacchioni 2009), chitinase (Mavingui and Heulin 1994), and enhancement of soil porosity (Gouzou et al. 1993; Timmusk 1999). Kwon et al. (2016) reported a significant increase in shoot fresh and root dry weights, whereas root length was decreased in Arabidopsis thaliana by treatment with P. polymyxa E681, and the study indicated P. polymyxa E681 may promote plant growth by induced metabolism and activation of defense-related proteins against fungal pathogen. Ryu et al. (2006) also found that P. polymyxa E681 effectively controlled pre-emergence and post-emergence damping-off diseases on sesame plants as a biocontrol agent.  Therefore, the growth promotion observed in P. polymyxa P2b-2Rgfp- and P2b-2R-inoculated seedlings might also be due to one or more of the capabilities mentioned above. In my experiment, GFP-labeled P2b-2R behaved slightly different from the wild-type, especially in the earlier stage of growth trial. GFP-labeled P2b-2R outperformed the wild-type most in endophytic colonization and N-fixation ability which was demonstrated by higher %Ndfa and concomitant increase in foliar N concentration, however, GFP-modification did not affect the N-fixing and growth promoting of wild-type P2b-2R generally. Although P2b-2R had higher rhizosphere population sizes than P2b-2Rgfp, the higher endophytic densities of P2b-26  2Rgfp might be more revealing, concerning endosymbiotic nitrogen fixation might be more efficient (Cocking 2003). Weyens et al. (2012) suggested that plant growth promotion is usually not affected indirectly by GFP-insertion, and inoculation of GFP-labelled bacteria could result in different colonization patterns in comparison to the wild-type strains. Therefore, the GFP gene most likely affects the ability for endophytic colonization of wild-type P2b-2R, and consequently results in influencing the nitrogen fixing ability of this organism.  Different colonization capacity between wild type and GFP-labelled P2b-2R might be concerned with plant defense response pathways (Iniguez 2005), symbiotic signaling pathway (Ben J. Duijff 1997), which could possibly be related to GFP expression (Haseloff et al. 1997; Agbulut et al. 2006; Weyens et al. 2012).  Previous paper has suggest that the GFP gene might modify some characteristics from the wild-type microorganism. Weyens et al. (2012) reported the harmful impact on plant health and growth by GFP-labelled P. putida W619 while the wild-type P. putida W619 resulted in significant growth promotion in hybrid poplar. Padda et al. (2015) found that GFP tagging of P. polymyxa P2b-2R increases its plant growth-promoting efficacy when associated with canola significantly, but not with tomato. Other studies also have shown that the GFP gene might result in positive or negative effects on the growth of plant hosts (Meng et al. 2014; Petri et al. 2008). These may imply that the effects of GFP labelling on an endophytic bacteria might vary with the host plant or the bacteria. GFP labeling should be used carefully since it may alter the system that it is being used to study. On the basis of all the results, GFP-labeled P2b-2R may have little short-term advantages over wild type P2b-2R in early pine growth stages, but in long term, the advantage is not significant. Hence, the GFP labeling method could be used as a reporter in the colonization 27  studies for Paenibacillus polymyxa P2b-2R. To further investigate this in future work, knock out mutants of genes should be constructed and their effects on plant fitness should be evaluated.  28  Chapter 5: Conclusions Based on the experimentation described above, the following conclusions can be drawn: • Both wild-type and GFP-labeled P. polymyxa P2b-2R are able to form persistent rhizospheric and endophytic colonization inside lodegepole pine root and stem tissue • Both wild-type and GFP-labeled P. polymyxa P2b-2R are capable of N2-fixation in association with lodgepole pine after 12 months of coexistence, and P2b-2Rgfp tended to provide more N from the atmosphere than P2b-2R. • Compared to the controls, inoculation had significant effects on seedling length and biomass after four months, however, no significant differences were detected in the final results (after one year) between P2b-2R and P2b-2Rgfp treatments. • Temporary seedling growth reduction may characterize early stages in the development of effective BNF for P2b-2R treatments, and N2-fixing seedlings could recover from this growth reduction within one year.  • The reliance of pine on N fixed by P2b-2R and P2b-2Rgfp increases with seedling age. • GFP-labeled P2b-2R may have short-term advantages in early pine growth stages, but in long term, the advantage is not significant. The reason could due to modification of the ability of endophytic colonization of wild-type P2b-2R by GFP gene, consequently resulting in influencing the growth promoting and nitrogen fixing capacity.  The research suggests that both wild-type and GFP-labeled P. polymyxa P2b-2R could form beneficial mutualisms with lodgepole pine and enhance seedling growth significantly in the long term. GFP labeling method could be used as a reporter in the study for Paenibacillus polymyxa P2b-2R. However, the reason for different behaviors between wild-type and GFP-labeled P. polymyxa P2b-2R is unclear. According to previous studies, the differences in 29  colonization by the wild-type and GFP-labelled P2b-2R and their effects on the plant growth promotion could possibly be related to GFP expression, furthermore, the effects of GFP labelling on an endophytic bacterium might vary with host plant or bacteria. 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