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Impact of GFP-modification of Paenibacillus polymyxa on its ability to enhance growth of corn, canola… Padda, Kiran Preet 2015

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Impact of GFP-modification of Paenibacillus polymyxa on its ability to enhance growth of corn, canola and tomato seedlings by Kiran Preet Padda B.Tech, Punjab 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) October 2015  © Kiran Preet Padda, 2015   ii Abstract Green fluorescent protein (GFP) as a marker gene has facilitated biological research in plant-microbe interactions. However, our knowledge about the effects of introduction of this marker protein in a microbe is very limited. I analyzed the effect of GFP-tagging of a plant growth-promoting bacterium, Paenibacillus polymyxa P2b-2R, on its ability to fix nitrogen and promote growth of important crop species: corn, canola and tomato. To evaluate this, corn, canola and tomato seeds were inoculated with P2b-2R and P2b-2Rgfp and grown for 40 days. Seedlings were harvested 20, 30 and 40 days after inoculation for evaluation of rhizospheric and endophytic colonization (cfu). Biological nitrogen fixation (15N dilution) and growth response (length and biomass) of P2b-2Rgfp inoculated seedlings was also compared with control and P2b-2R inoculated seedlings. The entire experiment was repeated to confirm the treatment effects. P2b-2Rgfp inoculated seedlings (i) accumulated upto 55% more biomass than non-inoculated controls and 17% more biomass than P2b-2R treated seedlings; (ii) were upto 41% longer than controls and 17% longer than P2b-2R treated seedlings; and (iii) fixed 18% of nitrogen from atmosphere (5% higher than P2b-2R). Canola seedlings inoculated with P2b-2Rgfp (i) had 118% more biomass than controls and 69% more biomass than P2b-2R treated seedlings; (ii) were 69% longer than controls and 37% longer than P2b-2R treated seedlings; and (iii) derived 22% of nitrogen from atmosphere (6% more than P2b-2R). But in case of tomato, P2b-2Rgfp and P2b-2R inoculated seedlings were analogous in terms of growth promotion and nitrogen fixation. Seedlings inoculated with either P2b-2Rgfp or P2b-2R were nearly 40% longer than controls, assimilated nearly 90% more biomass than controls, and fixed nearly 17% of nitrogen from atmosphere. P2b-2Rgfp and P2b-2R strains formed consistent rhizospheric and endophytic populations in corn, canola and tomato roots. To the best of my knowledge, these results represent the first evidence that transformation of a bacterial strain by GFP can significantly enhance its plant growth-promoting efficacy in corn and canola. Since the results of tomato were contrary to corn and canola, it can also be concluded that effect of GFP-tagging of P2b-2R might vary depending on the host plant.   iii Preface This thesis represents original, unpublished work by the author, Kiran Preet Padda. I was the lead investigator for the projects described in Chapter 2, 3 and 4 where I was responsible for all major areas of research question formation, research work, data collection, data analysis, and thesis composition. Dr. Maja Kržić, and Dr. Sue Grayston contributed to thesis edits. Dr. Alice Chang from UBC Stable Isotope Facility in the Department of Forest and Conservation Sciences, Faculty of Forestry was involved in analyzing foliar 15N samples of corn, canola and tomato seedlings. Dr. Chris Chanway was the supervisory author on this project and was involved throughout the project, from research question formation through to thesis edits.  iv Table of contents Abstract ...............................................................................................................................ii Preface ...............................................................................................................................iii Table of contents ................................................................................................................iv List of tables .....................................................................................................................vii List of figures ...................................................................................................................viii List of abbreviations ...........................................................................................................x Acknowledgements ............................................................................................................xi Chapter 1: General introduction ..........................................................................................1      1.1 Nitrogen....................................................................................................................1  1.1.1. Nitrogen in agriculture...................................................................................1  1.1.2. Nitrogen fertilizers.........................................................................................2  1.1.3. Biological nitrogen fixation...........................................................................3      1.2. Plant growth promoting bacteria..............................................................................4  1.2.1. Plant growth promoting mechanisms.............................................................5       1.2.1.1. Biofertilization.......................................................................................5       1.2.1.2. Rhizoremediation and stress control......................................................6       1.2.1.3. Phytostimulation....................................................................................6       1.2.1.4. Biocontrol..............................................................................................7  1.2.2. Endophytes.....................................................................................................8       1.2.2.1. Effects of endophytic bacteria and benefits to the plant........................8      1.3. Paenibacillus polymyxa...........................................................................................9  1.3.1. Paenibacillus polymyxa P2b-2R..................................................................11      1.4. Green fluorescent protein......................................................................................12  1.4.1. GFP-tagging of P. polymyxa P2b-2R...........................................................14      1.5. Research objectives and hypotheses......................................................................15  1.5.1. Research objectives......................................................................................15  1.5.2. Hypotheses...................................................................................................15 Chapter 2: Does green fluorescent protein (GFP) tagging affect the efficacy of plant growth promoting bacteria when inoculated in corn?....……................................…........16  v      2.1. Introduction……………………….......……………………........……………….16      2.2. Materials and methods……......................………………………...……….…….19  2.2.1. Bacterial strains….............…....................………………………………...19  2.2.2. Seed inoculation and plant growth……………...............…………………20  2.2.3. Evaluation of endophytic colonization……..............................…......……22  2.2.4. Evaluation of rhizospheric colonization……..............................…………22  2.2.5. Nitrogen analysis and seedling growth response........................………….23  2.2.6. Statistical analysis…………..........…..............................…………………23      2.3. Results………………….......………………….........…..…....………………..…24  2.3.1. Endophytic and rhizospheric colonization…..........…............…………….24  2.3.2. Growth response……………….........................……............…………….24  2.3.3. Nitrogen fixation……………………….....…............…………………….25      2.4. Discussion………….........………….…...….......…………….………....……….29      2.5. Conclusions………………………...........……......…..………………………….32 Chapter 3: Effect of GFP modification of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola seedlings......................................................................………34      3.1. Introduction……………………………………………...……………………….34      3.2. Materials and methods………......………………………………...……….…….37      3.3. Results………………………………………………....…………………………39  3.3.1. Rhizospheric and endophytic colonization............................……………..39  3.3.2. Seedling growth promotion..................…………………………………....39  3.3.3. Nitrogen fixation..................................…………………………………....40      3.4. Discussion……………………...……....………………………………………...44      3.5. Conclusions……………………………………….....…………………………...48 Chapter 4: Growth promotion of tomato by an endophytic strain of Paenibacillus polymyxa and its GFP-derivative...........................................................................………49      4.1. Introduction……………………………………………...……………………….49      4.2. Materials and methods………......………………………………...……….…….53      4.3. Results………………………………………………....…………………………54  4.3.1. Endophytic and rhizospheric colonization.............................……………..54  4.3.2. Growth response..................................…………………………………....55  vi  4.3.3. Nitrogen fixation..................................…………………………………....55      4.4. Discussion……………………...……....………………………………………...60      4.5. Conclusions……………………………………….....…………………………...63 Chapter 5: General conclusions……….…….......................………………...........……..65 References…....………………………....………………………………………………..69 Appendix A: Plant nutrient solution……….….....……..………………………………..86 Appendix B: Steps to prepare combined carbon medium (CCM)……...............………..87 Appendix C: Additional figures…………………………......…………….……………..88  vii  List of tables  Table 2.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) in growth trial 1 and 2..........…………..……….......…………….28  Table 3.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation in Growth Trial 1 and 2..........................................................................43  Table 4.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from tomato seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) in growth trial 1 and 2………………......................…………..…59   viii  List of figures  Figure 2.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of corn seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)......26  Figure 2.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of corn seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)......................................................................................................................................27  Figure 3.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of canola seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)......41  Figure 3.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of canola seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)......................................................................................................................................42  Figure 4.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of tomato seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).….57  Figure 4.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of tomato seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)......................................................................................................................................58  Figure C.1 Control (left), P2b-2R-inoculated (centre) and P2b-2Rgfp-inoculated (right) seedlings of corn plant harvested 40 days after inoculation (dai) showing clear difference in length, biomass and plant health....................................................................................88    ix  Figure C.2 Control (left), P2b-2R-inoculated (centre) and P2b-2Rgfp-inoculated (right) seedlings of canola plant harvested 40 days after inoculation (dai) showing clear difference in length, biomass and plant health...................................................................89  Figure C.3 P2b-2R-inoculated (left), P2b-2Rgfp-inoculated (centre) and Control (right) seedlings of tomato plant harvested 40 days after inoculation (dai) showing clear difference in length, biomass and plant health...................................................................90    x  List of abbreviations ACC   1-aminocyclopropane-1-carboxylate ANOVA Analysis of Variance AFP  Auto fluorescent protein BNF  Biological Nitrogen Fixation CCM  Combined Carbon Medium cfu  colony forming units dai  days after inoculation gfp/GFP Green fluorescent protein IAA  Indole-3-acetic acid N/N2  Nitrogen Ndfa  Nitrogen derived from atmosphere PBS  Phosphate Buffered Saline PGPB  Plant Growth Promoting Bacteria PGPR  Plant Growth Promoting Rhizobacteria P  Phosphorous rrs  Ribosomal RNA 16S SBPS  Sub-boreal Pine Spruce TSA  Tryptic Soy Agar UBC  University of British Columbia  xi  Acknowledgments Huge and sincere thanks to my supervisor, Dr. Chris Chanway, for his invaluable guidance and support throughout this project. I am thankful to my committee members, Dr. Maja Krzic and Dr. Sue Grayston for their constructive feedback on my work. I would like to give special thanks to my former committee member, Dr. Richa Anand, for her suggestions in crop selection and experimental design. I also wish to thank Dr. Alice Chang, for her contribution in 15N analysis of plant samples and providing great support in the lab work. I am grateful to Dr. Les Lavkulich and Dr. Sandra Brown for their efforts to encourage me to develop many skills and explore learning opportunities beyond my thesis. I extend my sincere gratitude to NSERC for financially supporting this work. I am also thankful to Trent Whiting and SeCan Association for generously providing Canola seeds for this study. I would also like to thank Natasha Thompson at Department of Forest and Conservation Sciences, and Lia Maria Dragan and Shelly Small at Faculty of Land and Food Systems for providing great support in all administrative matters and making me feel comfortable at UBC. I owe special thanks to my dear friend Akshit Puri for his ideas, encouragement, inspiration and endless support. I thank my parents for believing in my capabilities and supporting me in this undertaking. A special thank you goes to my sister for her unconditional support. Last but not the least, I would like to thank God for everything in my life. Kiran Preet Padda 1 Chapter 1: General introduction 1.1. Nitrogen 1.1.1. Nitrogen in agriculture Nitrogen (N) is central to living systems, and its addition to agricultural cropping systems is an essential facet of modern crop management and one of the major reasons that crop production has kept pace with human population growth (Robertson & Vitousek, 2009). The world population is rapidly increasing and is expected to reach 9 billion in the middle of the 21st century with projected associated effects on all terrestrial ecosystems (Barnosky et al., 2012). To feed an increasingly large world population and enhance agricultural production, a parallel increase in N consumption has been observed. Environmental and economic issues have increased the need to better understand the role and fate of N in crop production systems.  Even though N is among the most abundant elements on Earth, it is the critical limiting element for growth of most plants due to its unavailability (Smil, 1999; Socolow, 1999; Graham & Vance, 2000). Production of high quality, protein-rich food is extremely dependent upon availability of sufficient N. Plants acquire N from two principal sources: (a) the soil, through commercial fertilizer, manure, and/or mineralization of organic matter; and (b) the atmosphere through symbiotic N2 fixation. The entire nutritional N required by humans is obtained either directly or indirectly from plants. However, since the 1970s, management of N inputs into agricultural systems has become a contentious issue (Vance, 2001).  2  For plants N is necessary as a primary constituent of nucleotides, proteins, and chlorophyll (Robertson & Vitousek, 2009). However, plants can only assimilate certain forms of N, including ammonium, nitrates, and organic compounds (urea). The availability of fixed N (nitrate or ammonium converted from dinitrogen) is the most yield-limiting factor related to the agricultural production of crops such as corn (Muthukumarasamy et al., 2002). 1.1.2. Nitrogen fertilizers Throughout history (pre-industrial), three main methods of adding N to fields have been used: 1) organic wastes (human and animal waste, and crop residue), 2) crop rotations (nitrogen-fixing legumes), and 3) leguminous cover plants which were ploughed under as green manure (alfalfa and clover) (Smil, 2002). A way to attain increased crop yield was achieved with the supplementation of N fertilizers by Fritz Haber and Carl Bosch (Erisman et al., 2008). In 1908, Haber successfully synthesized ammonium, and in 1913 Bosch was able to use Haber’s discovery and commercialize it in the large-scale production of ammonium (Smil, 2002; Erisman et al., 2008). The Haber-Bosch process synthesizes ammonium by reacting atmospheric dinitrogen with hydrogen at high pressures and temperatures in the presence of iron (Erisman et al., 2008). Since its commercialization, synthetic N fertilizer use has constantly been increasing. In 1950, approximately 2.75 million tons of synthetic fertilizer was used. This number increased to 63.75 million tons in 2000 and increased again to 100 million tons in 2008 (Smil, 2011). Assimilation of applied N fertilizer by crops such as corn is typically less than 50%, meaning that more than half of the applied fertilizer remains unutilized (Cassman et al.,  3 2002). Adding to this, the fact that N is mobile, reactive, and hard to contain makes it very vulnerable to losses due to denitrification, volatilization, and leaching (Smil, 1999; Cassman et al., 2002; Robertson & Vitousek, 2009). Some of the main detrimental effects to the environment due to the vast increase in the addition of synthetic N include: the acidification of soils, lakes, and streams, the eutrophication and hypoxia of coastal ecosystems, and the loss of biodiversity within both terrestrial and aquatic ecosystems (Vitousek et al., 1997; Galloway et al., 2003; Robertson & Vitousek, 2009). 1.1.3. Biological nitrogen fixation Along with having a large impact on the surrounding environment, N fertilizers are very expensive to a farming operation. With costs of fertilizers doubling or tripling over the last decade, farmers have seen N fertilizers account for up to 15% of all production costs (Duffy, 2009). Biological Nitrogen Fixation (BNF), the reduction of atmospheric dinitrogen to ammonia, carried out by a large and diverse group of free-living and symbiotic microorganisms, presents an inexpensive and environmentally sound, sustainable approach to crop production and constitutes one of the most important mechanisms of plant growth promotion (de Bruijn, 2015). BNF occurs when atmospheric di-nitrogen is converted to ammonia by an enzyme called nitrogenase (Postgate, 1998). Approximately 80% of BNF is accomplished through the symbiotic interaction between legumes (diverse angiosperms consisting of over 18,000 species) and α- proteobacteria in the order Rhizobiales, family Rhizobiaceae (Geetanjali, 2006). Non-specific N-fixing bacteria also exist and have opened up the possibility of symbiotic N fixation in a wide array of monocot crops, including corn (Peoples et al., 1995; Muthukumarasamy et al.,  4 2002). The majority of non-specific N-fixing bacteria are free-living, as saprobes (living on plant residues), endophytes (living within plants), and rhizobacteria (living in close association with plant roots) (Gothwal et al., 2008). The two main types, that require associations with host plants are endophytes and rhizobacteria, can be classified as plant growth promoting bacteria (PGPB) because they are beneficial to their host plants (Saharan & Nehra, 2011). 1.2. Plant growth promoting bacteria Legumes have the advantage that in symbiosis with soil rhizobial bacteria, they can obtain N through BNF. However, most agricultural plants, especially grasses, lack this ability and hence there has been sustained interest in transferring the ability to fix N into grass crops such as corn (Charpentier & Oldroyd, 2010). PGPB colonize roots and engage in associative symbiosis with various host plants (Santi et al., 2013). In most cases the mechanism by which plant growth is promoted is unknown. In selected cases, promotion of plant growth is attributed to antagonism toward phytopathogens (Raaijmakers et al., 2009) and/or the induction of plant resistance to pathogens (Verhagen et al., 2004). Other PGPB may act mostly by phytostimulation (e.g. release of phytohormones; Richardson et al., 2009). Several N fixing PGPB have been identified as endophytes of grass species, including Azoarcus spp. in kallar grass and rice (Reinhold-Hurek et al., 1986; Hurek et al., 1994a,b), Herbaspirillum seropedicae in sugarcane (James & Olivares, 1998) and sorghum (James et al., 1997) and Gluconacetobacter diazotrophicus in sugarcane (James et al., 1994).   5  1.2.1. Plant growth promoting mechanisms 1.2.1.1. Biofertilization Rhizobacteria that promote plant growth by improving the nutrient uptake of plants are termed biofertilizers. These bacteria have a role of improving the nutrient status of host plants by means of N fixation, increasing the availability of nutrients in the rhizosphere, promoting the root surface area, or enhancing beneficial symbiosis of the host (Pérez-Montaño et al., 2014). Usually, growth promotion is due to a combination of these modes of action. Atmospheric N-fixing bacteria such as Rhizobium and Bradyrhizobium can establish symbiosis-forming nodules on roots of leguminous plants (Murray, 2011). However, this process appears to be limited to legume crops. On the other hand, several non-symbiotic bacteria have been identified as free-living N2-fixers (Azospirillum, Azoarcus, Azotobacter, Bacillus polymyxa, Burkholderia, Gluconoacetobacter or Herbaspirillum). These potential plant-growth promoting rhizobacteria (PGPR) can fertilize several important agronomic plants such as wheat (Boddey et al., 1986), sorghum (Stein et al., 1997), corn (Garcia de Salamone et al., 1996), rice (Malik et al., 1997) or sugarcane (Boddey et al., 2001). Inoculation of these PGPR species usually increases the host plant’s dry weight, amount of flowering, grain production and root development, which allows better rates of water and mineral uptake (Okon et al., 1998). Another essential plant nutrient is phosphorus (P). Although there is a large reserve of P is in soils, most of it is in the form of non-soluble compounds, which cannot then be absorbed by plants, therefore limiting plant growth. Certain PGPR are able to solubilize those P forms through acidification (Richardson et al., 2009), chelation or enzymatically  6 (Hameeda et al., 2008). In particular bacteria such as Azospirillum, Bacillus, Burkholderia, Pseudomonas or Rhizobium have been reported as phosphate solubilizing bacteria (Sudhakar et al., 2000; Mehnaz & Lazarovits, 2006). 1.2.1.2. Rhizoremediation and stress control A key step during rhizoremediation consists of the selection of pollutant-degrading rhizobacteria that live in the rhizosphere and use root exudates as an energy source (Kuiper et al., 2001). Besides degrading the pollutant compounds, these bacteria often directly assist rhizoremediation by producing hormones, fixing atmospheric N, solubilizing P or secreting siderophores (Denton, 2007). Many PGPR destroy 1-aminocyclopropane-1-carboxylate (ACC), a precursor of the ethylene, via production of the enzyme ACC deaminase, which in turn facilitates plant growth and development by decreasing plant ethylene levels. In addition, several forms of stress are relieved by ACC deaminase producers, such as effects on phytopathogenic bacteria and resistance to stress from salt and drought (Glick et al., 2007). 1.2.1.3. Phytostimulation Diverse PGPR can alter root architecture and promote plant development due to their ability to synthesize and secrete plant hormones like indole-3-acetic acid (IAA), gibberellins (GAs), cytokinins and certain volatiles; hence they are termed phytostimulators (Bloemberg & Lugtenberg, 2001). This capacity is bacterial strain specific (Boiero et al., 2007). The PGPR stimulatory effect comes from a manipulation of the complex and balanced network of plant hormones that are directly involved in growth  7 promotion or stimulation of root formation. For instance, the biosynthesis of IAA by various PGPR has been demonstrated to enhance root proliferation (Dobbelaere et al., 1999; Khalid et al., 2004). Involvement of PGPR produced cytokinins was observed in root initiation, cell division, cell enlargement and increase in root surface area of crop plants through enhanced formation of lateral and adventitious roots (Salamone et al., 2005; Werner et al., 2003). Some strains of Azotobacter spp., Rhizobium spp., Pantoea agglomerans, Rhodospirillum rubrum, Pseudomonas fluorescens, Bacillus subtilis and Paenibacillus polymyxa have been reported to produce cytokinins (Salamone et al., 2001; Glick, 2012). 1.2.1.4. Biocontrol Plant growth promotion can be achieved indirectly through biocontrol activity of plant pathogens. Members of the bacterial genera Bacillus, Pseudomonas, Serratia, Stenotrophomonas, and Streptomyces and the fungal genera Ampelomyces, Coniothyrium, and Trichoderma are well-studied microorganisms with proven microbial influence on plant health. When testing microbial isolates from plant-associated habitats, up to 35% of microbes showed antagonistic capacity that inhibits the growth of pathogens in vitro (Berg, 2009). Mechanisms responsible for antagonistic activity include inhibition of the pathogen by antibiotic production, toxins and surface-active compounds (biosurfactants); competition for minerals, nutrients, and colonization sites; and a mechanism that develops production of extracellular cell wall degrading enzymes such as chitinase and β-1, 3-glucanase (Whipps, 2001; Compant et al., 2005; Haas & Défago, 2005).   8 1.2.2. Endophytes Endophytic bacteria can be defined as those bacteria that colonize the internal tissue of plants while showing no external sign of infection or negative effects on the plant (Holliday, 1989; Schulz & Boyle, 2006). Of the nearly 300 000 plant species that exist on the earth, each plant is host to one or more endophytes (Strobel et al., 2004). Bacterial endophytes colonize an ecological niche similar to that of phytopathogens, which makes them suitable as biocontrol agents (Berg et al., 2005a). Indeed, numerous reports have shown that endophytic microorganisms can have the capacity to control plant pathogens (Sturz & Matheson, 1996; Duijff et al., 1997), insects (Azevedo et al., 2000) and nematodes (Hallmann et al., 1997, 1998). In some cases, they can also accelerate seedling emergence, promote plant establishment under adverse conditions (Chanway, 1997) and enhance plant growth (Bent & Chanway, 1998). 1.2.2.1. Effects of endophytic bacteria and benefits to the plant The growth stimulation by the microorganisms can be a consequence of N-fixation (Hurek et al., 2002; Iniguez et al., 2004; Sevilla et al., 2001) or the production of phytohormones, biocontrol of phytopathogens in the root zone (through production of antifungal or antibacterial agents, siderophore production, nutrient competition and induction of systematic acquired host resistance, or immunity) or by enhancing availability of minerals (Sturz et al., 2000).  Volatile substances such as 2-3 butanediol and aceotin produced by bacteria seem to be a newly discovered mechanism responsible for plant-growth promotion (Ryu et al., 2003). The frequent isolation of Curtobacterium flaccumfaciens as endophytes from asymptomatic citrus plants infected with the pathogen  9 Xylella fastidiosa suggests that these endophytic bacteria may help citrus plants to better resist the pathogenic infection (Araujo et al., 2002). Endophytes from potato plants showed antagonistic activity against fungi (Berg et al., 2005b; Sessitsch et al., 2004) and also inhibited bacterial pathogens belonging to the genera Erwinia and Xanthomonas (Sessitsch et al., 2004). However after it was determined that rhizospheric N-fixation did not occur at sufficient rates to facilitate high sugarcane yields, Cavalcante & Döbereiner (1988) looked for microorganisms within sugarcane tissues that might be involved and isolated a diazotrophic bacterium, Gluconoacetobacter diazotrophicus, previously known as Acetobacter diazotrophicus (Chanway et al., 2014).  1.3. Paenibacillus polymyxa Paenibacillus polymyxa was originally assigned to the genus Bacillus by Mace in 1889 because of its rod-shaped cells, ability to form endospores, and other similarities to members of the genus Bacillus (Montefusco et al., 1993). Later, Ash et al. (1991) grouped this organism into a phylogenetically distinct cluster, referred to as “group 3,” and eventually reclassified it into a separate family Paenibacilliaceae and genus Paenibacillus (Priest, 2009). The genus Paenibacillus can be differentiated from members of the Bacillaceae by polymerase chain reaction (PCR) amplification of 16S rRNA gene fragments, using either the original diagnostic probe that led to reclassification of P. polymyxa and several other members into the genus Paenibacillus (Ash et al., 1993).   P. polymyxa, a gram-positive facultative anaerobe, can be active under anaerobic conditions and thrives in semi-anaerobic environments, and has a broad host range as a  10 PGPB. P. polymyxa is present in many environments: it occurs naturally in marine sediments (Lal & Tabacchioni, 2009), and has also been isolated from the rhizosphere and roots of many forest trees and crop plants such as wheat, barley, white clover, perennial ryegrass, crested wheatgrass, green bean, garlic (Raza et al., 2008), corn, sorghum and sugarcane (Lal & Tabacchioni, 2009).   P. polymyxa promotes plant growth by increasing nutrient availability (fixing N and solubilizing P), improving soil porosity, and producing a number of phytohormones that promote plant growth. P. polymyxa can also fix atmospheric N under anaerobic conditions (Gaby & Buckley, 2012). P. polymyxa efficiently solubilizes inorganic P through the excretion of organic acids (Hesham & Hashem, 2011). Greenhouse experiments show that inoculating corn with P. polymyxa under different levels of P chemical fertilizer in calcareous soil caused significant increases in the shoot and root dry weights as well as in phosphorus uptake in the shoots and roots of the plant as compared to the uninoculated controls (Hesham & Hashem, 2011). P. polymyxa produces a wide variety of phytohormones that help regulate plant growth and development. These include auxins, especially IAA and cytokinins, including iso-pentenyladenine (iP). In a study involving stationary growth phase of P. polymyxa strain B2 isolated from the wheat rhizosphere, iso-pentenyladenine and another unknown cytokinin-like compound were extracted from the growth medium. Volatile organic compounds, including acetoin and 2,3-butanediol produced by P. polymyxa, also promote plant growth (Ryu et al., 2003). P. polymyxa produces acetoin in the presence of oxygen, and accumulates 2,3-butanediol in significant quantities as a product of fermentation under intermediate oxygen concentrations (De Mas et al., 1988; Mankad & Nauman, 1992). In another study, P.  11 polymyxa strain RC05 and RC14 isolated from the rhizosphere of wild berries, wheat and barley exhibited nitrogenase activity, phosphate solubilisation capability and promoted plant growth when inoculated into wheat and spinach plants (Çakmakçi et al., 2007). 1.3.1. Paenibacillus polymyxa P2b-2R Lodgepole pine grows successfully on nutrient poor sites and often scorched sites that are severely limited in N (Weetman et al., 1988). As a result, N inputs of lodgepole pine forests are of great interest both from ecological and management perspectives. Similarly, western red cedar forests are known to have limited N availability due to low rates of N mineralization (Prescott & Preston, 1994; Prescott et al., 1996). Earlier work with lodgepole pine suggested that rhizospheric BNF contributed only small amounts of N to seedlings (Chanway & Holl, 1991). Bal et al. (2012) isolated a number of potential endophytic diazotrophic bacteria from surface-sterilized tissues of naturally growing lodgepole pine seedlings in a range of nutrient-poor sites in the interior of British Columbia, Canada, close to Williams Lake (52°05’ N lat., 122°54’W long., elevation 1300 m, Sub-Boreal Pine Spruce, SBPSxc Zone). The identity of these diazotrophic strains was refined using 16S rRNA gene analysis. Strains were reintroduced to lodgepole pine and western red cedar to assess their colonization and nitrogen fixing ability in two different growth trials of 27 weeks and 35 weeks, respectively. Among the isolates, strain P2b-2R identified as Paenibacillus polymyxa showed a consistently higher level of acetylene-reduction activity (N fixation potential) in culture. In a long-term experiment (13-month growth trial), P2b-2R showed even better results as the inoculated lodgepole pine and western red cedar seedlings derived 79% and 36% of their foliar N  12 from BNF, respectively and doubled their biomass. In these trials, P. polymyxa P2b-2R successfully colonized internal root, stem and needle tissues of both lodgepole pine and western red cedar with population sizes ranging from 10-1 – 10-3 cfu/g of tissue in needles and 10-4 – 10-7 cfu/g of tissues in stems and roots (Anand et al., 2013; Anand & Chanway, 2013a). Anand & Chanway (2013c) characterized nif gene structure of Paenibacillus polymyxa strain P2b-2R to determine the arrangement and sequences of genes in the nif operon. This strain was found to possess a single copy of the nifH gene with nifB located directly upstream of nifH and nifD. Phylogenetic analyses of the full nifH, partial nifB and nifD, and 16s rDNA (rrs) gene sequences indicated that P2b-2R was part of a monophyletic cluster with other members of the genus Paenibacillus.  1.4. Green fluorescent protein Autofluorescent protein (AFP) methods are now a key tool for studying processes such as microbe–plant interactions and biofilm formation. These techniques have been utilized to detect and enumerate microorganisms in situ on plant surfaces and inside plants (Gage et al., 1996; Tombolini et al., 1997; Tombolini & Jansson, 1998). One of these AFP strategies uses a marker system, which encodes the green fluorescent protein (GFP). GFP is a polypeptide, which converts the blue chemiluminescence of the Ca sensitive photoprotein, aequorin, into green light (Chalfie, 1994; Cody et al., 1993). Inouye and Tsuji (1994a) showed the active chromophore is a tripeptide and is dependent on the presence of oxygen to maturate. Wild-type GFP absorbs blue light at 395 nm and emits green light at 510 nm in bioluminescent organisms and when purified in solution (Ward et al., 1980). In 1994, a GFP-based reporter system was developed in which visible  13 fluorescence was created by molecular biological techniques. Prashar et al. (1992) cloned gfp cDNA from Aequorea victoria and Chalfie et al. (1994) showed the expression of the cloned gfp gene produces green fluorescence in various organisms such as Drosophila and Escherichia coli.  The GFP can be used as a reporter for the visualization of gene expression and protein subcellular localization (Chalfie et al., 1994). Inouye and Tsuji (1994b) showed that the GFP could be introduced into cells and intact organelles within cells. The use of GFP has allowed us to visualize the process of attachment, entrance and nodule occupancy of Rhizobium meliloti in great detail making it even possible to determine the growth rate of the cells in the infection thread (Gage et al., 1996). Anand & Chanway (2013a) used a GFP-labeled derivative of P. polymyxa P2b-2R, P2b-2Rgfp, to evaluate endophytic colonization in lodgepole pine seedling. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly. However, there is one major limiting factor in the detection of GFP in living organisms whose cells or tissues emit background autofluorescence such that it becomes difficult to detect the GFP’s fluorescence. Some reports have indicated that the autofluorescence of chloroplasts, normally present in the upper parts of most plants, can provide counter fluorescence for GFP, such that even when using the brightest GFP variant, its expression within the cells or tissues may be unsatisfactory (Haseloff, 1998). Thus, the expression of GFP can be limited to particular cell types or tissues within a plant, as a means for visualizing GFP tagged bacterial cells.   14 1.4.1. GFP-tagging of P. polymyxa P2b-2R Anand & Chanway (2013a) used a shuttle plasmid capable of replicating in both E. coli and Bacillus subtilis to introduce the plasmid-borne gfp gene into P. polymya strain P2b-2R. Plasmid pBSGV104 (Itaya et al., 2001) was a low copy number plasmid that carries a 786-bp gfp fragment inserted between the HindIII and EcoRI restriction sites of shuttle vector pHY300PLK (Ishiwa & Shibahara, 1985) and confered resistance to tetracycline and chloramphenicol. Plasmid pBSGV104 was isolated from its carrier strain B. subtilis BEST 3156 using the QIAprep spin miniprep kit (Cat # 27104, Qiagen, USA), modified to isolate plasmid from Gram-positive bacteria by adding 1 mg lysozyme mL-1 to the P1 solution, and incubating at 37°C for 1 h before adding P2 solution. Electroporation of P. polymyxa P2b-2R was carried out with minor changes to a previously described method (Rosado et al., 1994) using a Gene Pulser apparatus (Bio-Rad Laboratories, Richmond, CA).   GFP-transformed P2b-2R was detected within stem tissues of pine seedlings indicating that, as suggested by its origin, P2b-2R is an endophyte. P. polymyxa P2b-2Rgfp colonized the pine root surface extensively, and might have entered the epidermis, but due to excessive tissue autofluorescence, it was not possible to localize specific colonization sites in root tissues (Anand & Chanway, 2013a). Others have demonstrated the ability of P. polymyxa to colonize external and internal root tissues (Shishido et al., 1999; Timmusk et al., 2009; Yegorenkova et al., 2010) and form biofilms on the root surface (Timmusk et al., 2005). Anand & Chanway (2013a) however did not test P2b- 15 2Rgfp for its ability to dilute foliar 15N or promote plant growth in ways similar to the wild-type P2b-2R strain (Anand & Chanway, 2013b; Anand et al., 2013). 1.5. Research objectives and hypotheses 1.5.1. Research objectives The main research objective of this thesis was to evaluate the effect of GFP tagging of a plant growth promoting bacterium, P. polymyxa P2b-2R, on its ability to promote plant growth and fix N when inoculated into an important (i) cereal crop species, corn, (ii) oilseed crop species, canola, and (iii) vegetable crop species, tomato. A secondary objective of this project was to determine if P. polymyxa P2b-2R is capable of forming rhizospheric and endophytic populations, deriving nitrogen from atmosphere, and promoting growth when inoculated into tomato. 1.5.2. Hypotheses I developed two main hypotheses: H1: GFP tagged P. polymyxa P2b-2R colonizes the rhizosphere and internal tissues of important crop species corn, canola and tomato in a similar way as the wild type P2b-2R H2: GFP labeling of strain P2b-2R does not significantly alter its efficacy to fix nitrogen and promote plant growth in comparison to the wild type strain when inoculated into corn, canola and tomato.   16 Chapter 2: Does green fluorescent protein (GFP) tagging affect the efficacy of plant growth promoting bacteria when inoculated in corn?1 2.1. Introduction The rhizosphere is home to a variety of root-associated bacteria commonly referred to as rhizobacteria. Beneficial rhizobacteria, also called plant growth-promoting rhizobacteria (PGPR), helps in plant growth promotion by providing resources/nutrients that plants may lack such as fixed nitrogen, iron, and phosphorus (Glick, 2012). In addition to providing nutrients, PGPR may also control plant pathogens, enhance the efficiency of fertilizers, and degrade xenobiotic compounds (Bakker et al., 2007). Of all known plant growth-promoting mechanisms, biological nitrogen fixation (BNF) is of particular interest because after carbon, nitrogen (N) is frequently the most limiting nutrient in terrestrial ecosystems (Dalton & Krammer, 2006). Hence, acquisition and assimilation of N is second in importance only to photosynthesis for plant growth and development (Sulieman, 2011). The limited bioavailability of N and the dependence of crop growth on this element have spawned a massive N-based fertilizer industry worldwide (Dobermann, 2007; Westhoff, 2009). BNF represents an economically beneficial and environmentally sound alternative to chemical fertilizers (Ladha et al., 1997). However, most agricultural plants, especially grasses, lack this ability and hence there has been sustained interest in transferring the ability to fix N into grass crops such as corn (Charpentier & Oldroyd, 2010; Pankievicz et al., 2015).                                                         1A version of this chapter has been submitted to Soil Biology and Biochemistry Journal for publication under the title ‘Does Green Fluorescent Protein (GFP) tagging effect the efficacy of plant growth promoting bacteria?’ Authors: Kiran Preet Padda, Akshit Puri and Chris P Chanway.  17  The bacteria responsible for N fixation are called diazotrophs; they encode the nitrogenase enzyme, an enzyme complex that catalyzes the conversion of N2 gas to ammonia (Santi et al., 2013). Diazotrophic PGPB are growth-promoting bacteria that may be able to promote plant growth via BNF (Keyeo et al., 2011). Endophytes are microbial symbionts that colonize the interior of plant tissues without causing any disease symptoms (Wilson, 1995; Kandel et al., 2015). They establish an association with a host plant that benefits its health in several ways including providing biotic and abiotic stress resistance and tolerance, enhancing nutrient availability, degrading toxic substances, and producing plant hormones (Doty, 2011). In terms of the quantity of fixed N being supplied to the host plant, the most significant discovery yet has been that of an association between the endophytic diazotroph Gluconoacetobacter diazotrophicus and sugarcane (Saccharum officinarum L.) in Brazil (Lima et al., 1987; Cavalante & Dobereiner, 1988; Boddey et al., 1991).  Paenibacillus polymyxa (formerly Bacillus polymyxa), a non-pathogenic and endospore-forming Bacillus, is one of the most industrially significant facultative anaerobic bacterium. P. polymyxa has a wide range of plant-beneficial properties, including N fixation, plant growth promotion, phosphorus solubilisation and production of exopolysaccharides, hydrolytic enzymes, antibiotics and cytokinins (Lal & Tabacchioni, 2009).  An endophytic diazotroph, Paenibacillus polymyxa P2b-2R, was discovered from internal stem tissues of naturally regenerating lodgepole pine seedlings in the N-deficient Sub-Boreal Pine-Spruce zone near Williams Lake, BC (Bal et al., 2012). Several lines of  18 indirect evidence such as consistent growth on N-free media, acetylene reduction and the presence of the nifH gene indicate the N-fixing, and possibly growth-promoting potential of this bacterial strain (Bal et al., 2012; Anand & Chanway, 2013c). 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 & Chanway, 2012b) and 79% (Anand et al., 2013) of foliar N from the atmosphere by the end of the 9 and 13-month growth trials, respectively. Anand & Chanway (2013a) used a GFP-labeled derivative of P. polymyxa P2b-2R, P2b-2Rgfp, to evaluate endophytic colonization in lodgepole pine seedling. P2b-2Rgfp colonized the root surface extensively and was detected inside the stem cortex, primarily intracellularly.  GFPs and their derivatives are widely used as markers in molecular and cell biology. Due to their ease of expression in vitro and in vivo and their lack of required exogenous substrates and cofactors for fluorescence, GFPs are often used to monitor gene expression and protein localization in living organisms (Chalfie et al., 1994; Davidson & Campbell, 2009). Sun et al. (2014) marked an endophytic bacterium Pseudomonas sp. Ph6 with the GFP gene to directly visualize the colonization and distribution of strain Ph6-gfp in roots, stems, and leaves of ryegrass (Lolium multiflorum Lam.). The strain Ph6-gfp not only actively and internally colonized plant roots and transferred vertically to the shoots but also aided in removal of polycyclic aromatic hydrocarbons (PAHs) from plant bodies. GFP has been extensively used in detecting internal tissue colonization by endophytic diazotrophs of various genera including Bacillus, Paenibacillus and Pseudomonas (Ji et al., 2014; Anand & Chanway, 2013a; Timmusk et al., 2005; Sun et al., 2014; Gotz et al., 2005). But the effects of tagging a plant growth promoting bacteria  19 (PGPB) with GFP on the PGPB are still not known. In other words, does GFP tagging of a PGPB enhance, undermine or have no effect on its plant growth promoting capabilities? In this study, I looked for an answer to this question by using an endophytic diazotroph P. polymyxa P2b-2R previously tagged with GFP (Anand & Chanway, 2013a). An agronomically important crop, corn, was used as the model plant species in this study. Corn has the highest production of all the cereals with world production of more than 1 billion metric tons in 2014-15 (USDA FAS, 2015). It is an important food staple in many countries, as well as being used in animal feed. It also has many industrial applications.  The main objective of this study was to evaluate the effect of GFP tagging of a plant growth promoting bacterium, P. polymyxa P2b-2R, on its efficacy to promote plant growth and fix N in an agronomically important crop, corn. 2.2. Materials and methods 2.2.1. Bacterial strains P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp were used in this study. P. polymyxa strain P2b was isolated from surface-sterilized stem tissues of a lodgepole pine seedling naturally regenerating near Williams Lake, British Columbia, Canada (52°05’ N lat., 122°54’W long. elevation 1300 m, Sub-Boreal Pine Spruce, SBPSdc Zone) (Bal & Chanway, 2012a). P. polymyxa P2b-2R is a spontaneous mutant derived from strain P2b that is capable of growing on combined carbon medium (CCM) agar (Rennie, 1981) amended with 200mg/L rifamycin (Bal et al., 2012). Transformation of P. polymyxa strain P2b-2R with GFP was carried out by the use of a shuttle plasmid  20 (pBSGV104) that introduced the plasmid-borne GFP into P. polymyxa strain P2b-2R. To constitutively drive the expression of GFP, a bsr promoter was used (Anand & Chanway, 2013a). Strain P2b-2R is resistant to rifamycin while P2b-2Rgfp strain confers resistance to rifamycin, tetracyline and chloramphenicol. Both P. polymyxa strain P2b-2R and P2b-2Rgfp were stored at -80°C in CCM amended with 20% (v/v) glycerol. 2.2.2. Seed inoculation and plant growth Corn seeds (var. Golden Bantam) were obtained from West Coast Seeds (Delta, British Columbia, Canada). Seeds were surface sterilized by immersion in 30% hydrogen peroxide for 90 seconds, followed by three 30-second rinses in sterile distilled water. The effectiveness of the surface sterilization was confirmed by imprinting sterilized seed on tryptic soy agar (TSA) and then checking for microbial contamination 24h later.   Seedling growth assays were performed in small pots (12cmx8cmx4cm) filled to 67% capacity with sterile sand-turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, Ill., USA) mixture (69% w/w silica sand; 29% w/w Turface; 2% w/w CaCO3). Each pot was fertilized with 50 mL of a nutrient solution (Chanway et al., 1988) modified by replacing KNO3 and Ca (NO3)-4H2O with Ca (15NO3) 2 (5 % 15N label) (0.0576g/L) and Sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ontario) with Na2FeEDTA (0.02g/L). Other nutrients in the nutrient solution included (in grams per liter): 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.  21  Corn seeds were inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative; P2b-2Rgfp. Non-inoculated seeds were treated with sterile phosphate buffer saline (PBS) and were used as control. This resulted in three treatment levels (P2b-2R, P2b-2Rgfp and control), each replicated 36 times and arranged in a completely randomized design. Bacterial inoculum was prepared by thawing frozen cultures of strains P2b-2R and P2b-2Rgfp and streaking a loopful onto CCM plates amended with 200mg/L rifamycin and 200mg/L rifamycin plus 5mg/L chloramphenicol, respectively and incubating at 30oC for 2 days. After the colonies grew, a loop full of each strain was inoculated into 1L flasks containing 500 mL of fresh CCM broth amended with rifamycin or rifamycin plus chloramphenicol. Flasks were then secured on a rotary shaker (150 rpm) and agitated for 48 h at room temperature. Bacterial cells were harvested by centrifugation (3000x g, 30 min), washed twice in sterile PBS (pH 7.4) and resuspended in the same buffer to a density of ca. 106 cfu/mL. Immediately after sowing the seed, 5 mL of the P2b-2R – PBS suspension and 5 mL of the P2B-2Rgfp – PBS suspension was pipetted directly into each replicate pot designated for P2b-2R and P2B-2Rgfp respectively. Non-inoculated control seeds received 5.0 mL of sterile PBS. Pots were placed in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB, Canada) under an 18-h photoperiod with an intensity of at least 300 μmol·s-1·m-2 and a 25/18oC day/night temperature cycle. Seedlings were thinned to contain the single largest germinant per pot once emergence was complete. Seedlings received modified nutrient solution without Ca(15NO3)2 20 days after sowing and were watered with sterile distilled water as required. The entire experiment was repeated to confirm treatment effects.   22 2.2.3. Evaluation of endophytic colonization Two randomly selected seedlings from each treatment were harvested destructively 20, 30 and 40 days after inoculation (dai) to evaluate endophytic colonization. Seedlings were rinsed in a 2L flask containing 1L sterile distilled water for removal of loosely adhering growth media. Seedlings were surface-sterilized in 0.6% (w/v) sodium hypochlorite for 5 minutes, rinsed three times with sterile distilled water and imprinted on TSA plates for 24-h to check for surface contamination. Samples of root, stem and leaf tissues were triturated separately in 1mL of sterile PBS using a mortar and pestle. Triturated tissue suspensions of control and P2b-2R inoculated seedlings were diluted serially and 0.1 mL of each dilution was plated onto CCM supplemented with 100mg/L cycloheximide and 200mg/L rifamycin. Similarly, tissue suspensions of P2b-2Rgfp inoculated seedlings were plated onto CCM supplemented with 100mg/L cycloheximide, 200mg/L rifamycin and 5mg/L chloramphenicol. Plates were incubated at room temperature for 7 days and numbers of colony forming units (cfu) were evaluated. Data from seedlings that showed contamination after surface sterilization were excluded from further analysis. 2.2.4. Evaluation of rhizospheric colonization For evaluation of rhizosphere colonization, 2 randomly chosen seedlings of each treatment were harvested destructively 20, 30 and 40 dai. Seedlings were removed from pots and loosely adhering soil particles were removed from roots with gentle shaking. Roots were then separated from shoots, placed in sterile Falcon tubes (50mL; BD Biosciences, CA, USA) filled with 10mL of sterile PBS and shaken on a vortex mixer at  23 1000 rpm for 1 minute. Serial dilutions were performed, and aliquots of 0.1 mL were plated on CCM amended with rifamycin (200mg/L) and cycloheximide (100mg/L). Plates were incubated at room temperature for 7 days and colonies were counted after incubation. Roots were oven-dried at 65oC for 2 days before weighing. Rhizospheric bacterial populations were then calculated as cfu per gram of dry root tissue. 2.2.5. Nitrogen analysis and seedling growth response Corn seedlings of each treatment were harvested destructively 20, 30 and 40 dai, separated into roots and shoots, and the roots were washed. Shoot and root lengths were measured for each seedling. Stem, leaves and roots of each seedling were then oven dried at 65oC for 2 days before being weighed. For foliar N analysis, ground and oven dried foliage of each seedling from each treatment was mixed thoroughly and 1mg sample was sent to the UBC Stable Isotope Facility for determination of foliar N content and %15N excess with an elemental analyzer interfaced with an isotope ratio mass spectrometer (Europa Scientific Integra). The amount of fixed N in foliage was estimated by calculating the percent N derived from the atmosphere (%Ndfa) (Rennie et al., 1978): % Ndfa = [1 −atom % 15N excess (inoculated plant)atom % 15N excess (uninoculated plant)]  x 100 2.2.6. Statistical analysis A completely randomized experimental design with 36 replicates per treatment was used to assess the treatment effects on growth of corn seedlings. Analysis of variance (ANOVA) was performed to determine treatment effects on seedling dry weight, seedling length, atom percent 15N excess and foliar N concentration. The statistical package, SAS  24 v9.4 (Copyright © 2014, SAS Institute Inc., Cary, NC, USA.), was used to perform statistical analyses.. The confidence level, α, was set to 0.05 to determine the significance of the model and treatment effects. 2.3. Results 2.3.1.Endophytic and rhizospheric colonization P. polymyxa strain P2b-2R and P2b-2Rgfp colonized the corn roots endophytically with population densities of 1.18 x 106 cfu/g fresh weight and 6.57× 106 cfu/g fresh weight, respectively. P2b-2R and P2b-2Rgfp colonies were also observed on both CCM and TSA root, stem and leaf imprint plates after these tissues were surface sterilized. But there was no evidence of endophytic colonization in stem and leaf tissues by either P2b-2R or P2b-2Rgfp.  P2b-2R and P2b-2Rgfp colonized corn rhizosphere with population densities of 8.54 x 105 cfu/g dry root and 1.42 x 106 cfu/g dry root, respectively. No evidence of rhizospheric or endophytic colonization was found in control plants.  2.3.2. Growth response Seedling growth was promoted significantly by inoculation with P2b-2Rgfp and P2b-2R in comparison to control plants. P2b-2Rgfp inoculation caused seedling length enhancement of up to 36% in the 1st growth trial and 41% in the 2nd growth trial compared to controls (Figure 2.1). Similarly, P2b-2R inoculation led to seedling length promotion of up to 24% in the 1st trial and 28% in the 2nd trial (Figure 2.1). Interestingly, it was also observed that growth response of P2b-2Rgfp inoculated seedlings was more prominent than P2b-2R inoculated seedlings. P2b-2Rgfp inoculated seedlings were up to  25 14% and 17% longer than P2b-2R inoculated seedlings in 1st and 2nd growth trials respectively (Figure 2.1).  P2b-2Rgfp inoculation promoted seedling dry weight (biomass) by 55% in the 1st growth trial and 48% in the 2nd growth trial as compared to the control (Figure 2.2). In a similar way, P2b-2R inoculation led to an increase of up to 34% in 1st trial and 28% in the 2nd trial in seedling biomass (Figure 2.2). Intriguingly, P2b-2Rgfp inoculated seedlings accumulated greater biomass than P2b-2R inoculated seedlings (up to 17% in 1st trial and 16% in 2nd trial) (Figure 2.2). 2.3.3. Nitrogen fixation In the 1st growth trial, foliar N concentration of seedlings inoculated with P2b-2Rgfp was up to 25% higher than the control (Table 2.1). Similarly in the 2nd growth trial, %foliar N of P2b-2Rgfp inoculated seedlings was up to 27% higher than the control (Table 2.1). P2b-2R inoculated seedlings had 15% and 17% higher foliar N than the control in 1st trial and 2nd trial, respectively (Table 2.1). As observed in the case of seedling length and biomass, the foliar N concentration of seedlings inoculated with P2b-2Rgfp was nearly 8-11% higher than P2b-2R inoculated seedlings in either growth trials (Table 2.1). Based on 15N foliar dilution assay, seedlings inoculated with P2b-2Rgfp derived up to 16% of foliar N from the atmosphere (Ndfa) in the 1st growth trial and 18% in the 2nd growth trial (Table 2.1). P2b-2R inoculated seedlings were found to derive up to 12% and 15% of foliar N from atmosphere (Ndfa) in 1st and 2nd growth trials, respectively (Table 2.1). An interesting finding was that P2b-2Rgfp inoculated seedlings derived more N from atmosphere (nearly 5%) than their wild type counterpart and the difference in atom %15N 26   Figure 2.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of corn seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).  02040608010012014020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 1P2b-2RgfpP2b-2RControlababababababababab aaaaaaaaa(a)02040608010012014020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 2P2b-2RgfpP2b-2RControlababababababababaaaaaaaaaa(b) 27   Figure 2.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of corn seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).  03006009001200150020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 1P2b-2RgfpP2b-2RControlabaaaaaaaabababababaabab(a)03006009001200150020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 2P2b-2RgfpP2b-2RControlababababababaaaaaaaaaaaa(b) 28 Table 2.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) in Growth Trial 1 and 2   Atom %15N excess in foliage  % Foliar N  %Ndfa Growth trial Treatment 20 dai 30 dai 40 dai  20 dai 30 dai 40 dai  20 dai 30 dai 40 dai 1 P2b-2Rgfp 0.41ab±0.006* 0.40ab±0.005 0.40ab±0.006  1.35ab±0.03 1.26ab±0.02 1.32ab±0.03  10.86 13.04 16.67 P2b-2R 0.43a±0.006 0.42a±0.006 0.42a±0.004  1.21a±0.02 1.18a±0.03 1.22a±0.02  6.52 8.69 12.5 Control 0.46±0.009 0.46±0.009 0.48±0.016  1.08±0.05 1.07±0.04 1.06±0.03  - - - 2 P2b-2Rgfp 0.41ab±0.005 0.40ab±0.006 0.39ab±0.009  1.27ab±0.04 1.30ab±0.03 1.34ab±0.03  9.23 14.11 18.03 P2b-2R 0.42a±0.003 0.42a±0.005 0.40a±0.006  1.17a±0.02 1.21a±0.02 1.23a±0.02  6.01 10.93 15.67 Control 0.45±0.006 0.47±0.007 0.48±0.010  1.03±0.03 1.06±0.04 1.05±0.03  - - - *Mean ± standard error; n=8 for atom % 15N excess & % Foliar N aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)  29 excess values of both these treatments was statistically significant (Table 2.1). 2.4. Discussion This study has shown for the first time that GFP tagging of a PGPB enhances its growth promotion efficacy. In this study, a lodgepole pine endophyte, P. polymyxa P2b-2R, labeled with GFP performed better than the wild type strain in terms of seedling growth promotion and N-fixation. The entire experiment was duplicated, so as to confirm that increased plant growth promotion efficacy of P2b-2R after GFP-labeling is in fact repeatable and not a one-time experimental artifact. Since corn ears didn’t emerge by harvest time, plant length and biomass were used to assess the growth stimulation facilitated by P2b-2Rgfp and P2b-2R. It was observed that P2b-2R inoculated seedlings were significantly longer and accumulated greater biomass than the control (Figure 2.1 and Figure 2.2), which is consistent with previous reports about this bacterial strain (Puri et al., 2015a; Anand et al., 2013; Anand & Chanway, 2013b; Bal & Chanway, 2012a). Similarly, P2b-2Rgfp inoculated seedlings were significantly longer and accumulated greater biomass than the control (Figure 2.1 and 2.2). An important and interesting finding was that the P2b-2Rgfp inoculated seedlings were considerably longer and amassed greater biomass than P2b-2R inoculated seedlings at each harvest interval in each growth trial. This difference in length and biomass between seedlings treated with the two inoculants was statistically significant. It was also observed that P2b-2Rgfp inoculated seedlings grew longer and accumulated more biomass aboveground than belowground, if compared with both P2b-2R inoculated and control seedlings (Figure 2.1 and 2.2). The increased growth observed in the P2b-2Rgfp and P2b-2R inoculated plants  30 under N-limiting conditions, is presumed to be due to multiple beneficial traits of P. polymyxa. Commonly reported plant growth promoting mechanisms of Paenibacillus include the production of substances like auxins, cytokinins and gibberellins (Talboys et al., 2014; Gutierrez-Manero et al., 2001), increasing nutrient availability for plants, for e.g. N fixation by deriving N directly from atmosphere, phosphate mineralization by producing enzymes that cleave phosphate groups and sulfur oxidation (Chanway, 2008; Singh & Singh, 1993; Chanway et al., 2014), and synthesizing many antibacterial and antifungal secondary metabolites (Borriss, 2015; Rosado & Seldin, 1993; Choi et al., 2008). I suspect that transformation of P2b-2R strain with GFP might have positively affected one or more above mentioned plant growth promoting mechanism(s) of P2b-2R.  The ability of P. polymyxa P2b-2R and its GFP derivative to increase the availability of important nutrient, nitrogen, for corn seedlings was also tested. It was demonstrated that corn seedlings inoculated with P2b-2R derive a significant amount of foliar N from the atmosphere, which is consistent with previous reports about this bacterial strain (Puri et al., 2015a). Nitrogenase activity of P. polymyxa P2b-2R was confirmed when this strain demonstrated consistently high acetylene-reduction activity (Bal et al., 2012). nif gene structure of P2b-2R was also analyzed and found to possess a single copy of the nifH gene (Anand & Chanway, 2013c). These findings along with growth chamber studies (Bal & Chanway, 2012a,b; Anand et al., 2013; Anand & Chanway, 2013b; Puri et al., 2015b) have confirmed the ability of P2b-2R to fix N. It was also found that P2b-2Rgfp inoculated seedlings derived significant amounts of N from atmosphere. But an important finding of this assay was that, P2b-2Rgfp inoculated seedlings derived a greater amount of N from the atmosphere (nearly 5%) than P2b-2R  31 inoculated seedlings. It is noteworthy that the percentage of foliar N derived from the atmosphere by P2b-2Rgfp and P2b-2R increased with time over the duration of the either growth trial (Table 2.1). This trend may be due to the decline in available N in the planting medium, or the establishment of the N fixation process and increasing populations of the bacteria in the foliage. But the difference observed in the amount of N derived from atmosphere by P2b-2Rgfp inoculated and P2b-2R inoculated seedlings is still a mystery. I suspect that the genetic modification of P2b-2R by introducing GFP might have caused a positive shift in its ability to associate with a plant species.  Due to high autofluorescence of corn tissues and low emissivity of GFP, detection of endophytic colonies of P. polymyxa P2b-2Rgfp by confocal laser scanning microscopy was not possible. To overcome this difficulty, I used the direct plating technique of surface-sterilized tissue extracts on a selective growth media (CCM) amended with antibacterial (rifamycin or rifamycin + chloramphenicol) and antifungal (cycloheximide) compounds. Since the main objective of this study was to compare P2b-2R and its GFP derivative in terms of plant growth promotion, just an indication was needed that P2b-2R and P2b-2Rgfp colonize corn seedlings endophytically. P2b-2R and P2b-2Rgfp successfully formed consistent endophytic populations in corn roots confirming that the growth promotion and BNF detected in inoculated seedlings was bacteria driven. P2b-2R and P2b-2Rgfp were not detected in aerial parts of the plant suggesting that these bacteria were not able to move up to aerial plant parts after inoculation in this short time span (40 day growth trial). P2b-2R and P2b-2Rgfp strains were also re-isolated from the rhizosphere of inoculated corn seedlings further suggesting that the both rhizospheric and endophytic bacterial populations were involved in growth promotion.  32  A hypothesis can be proposed to explain the observed enhancement of plant growth promotion of P. polymyxa P2b-2R by GFP tagging. Genetic modification of P. polymyxa carried out by introducing a marker protein (GFP) positively affects the bacterial strain’s plant growth promoting ability by helping in the release of certain proteins responsible for nutrient uptake, BNF, enhanced resistance against pathogenic microbes and/or mineralization of important inorganic compounds. German-Retana et al. (2000) observed that GFP tagging of Lettuce mosaic virus has significant negative effects on the biology of the virus, abolishing its resistance-breaking properties and reducing its pathogenicity in susceptible lettuce cultivars. Thus, this study shows that introduction of a marker protein like GFP in a virus affects its pathogen inducing ability, thereby helping in plant growth promotion. To the best of my knowledge, these results represents the first evidence that transformation of a bacterial strain by GFP significantly enhanced its ability to promote plant growth. Further work is clearly needed to gain a better understanding of the mechanisms involved and to test the presented hypothesis. 2.5. Conclusions Since its discovery, GFP has been used extensively in research involving interaction of microbes with host plant. But our knowledge about its effect on the functioning of a microbe is very limited. In this study, it is shown for the first time that GFP tagging of a PGPB enhances its growth promotion efficacy. I compared the growth response of a GFP labeled PGPB, P. polymyxa P2b-2Rgfp, with wild type P2b-2R and control when inoculated into an important agricultural crop, corn (Zea Mays L.). Seedlings inoculated with either P2b-2Rgfp or P2b-2R demonstrated better plant growth (length and biomass)  33 and fixed significant amounts of N from atmosphere when grown in N-limited conditions. But the most interesting finding was that P2b-2Rgfp inoculated corn seedlings were significantly longer, accumulated greater biomass and fixed more atmospheric N than P2b-2R inoculated seedlings. This clearly suggests that genetic modification of a PGPB, P. polymyxa P2b-2R, by introduction of a marker protein (GFP) positively affects the bacterium’s growth promotion efficacy. To the best of my knowledge, this is the first of its kind finding in the field of plant-microbe interactions.   34 Chapter 3: Effect of GFP modification of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola seedlings2 3.1. Introduction The maintenance of a high agricultural productivity, combined with increasing global demand for food by a growing population and the depletion of natural resources, has become a major challenge in both developed and developing countries (Matson et al., 1997; Cassman, 1999; Tilman et al., 2002). Up to now, traditional nutrient management for preserving high crop productivity has been mainly based on external chemical fertilizer inputs (Zhang et al., 2010). However, such an approach will certainly be counterproductive in the longer term. Thus, instead of the increased use of chemicals, it is essential that the use of transgenic plants and the widespread application of plant growth-promoting microorganisms, both bacteria and fungi, be embraced and practiced on a large scale (Glick, 2015). Plant growth-promoting bacteria (PGPB) (Bashan & Holguin, 1998), a subset of root colonizing plant growth-promoting rhizobacteria (PGPR) (Kloepper & Schroth, 1978) are bacteria that, via numerous independent or linked mechanisms, are capable of positively affecting plant growth for sustainable agriculture (Compant et al., 2010), counteracting many stress effects in plants (Kang et al., 2010; Kim et al., 2012), and assisting in the recovery of damaged or degraded environments (de-Bashan et al., 2012). This PGPR can play an important role in promoting nutrient acquisition by plants,                                                         2A version of this chapter has been submitted to FEMS Microbiology Ecology Journal for publication under the title ‘Effect of GFP modification of Paenibacillus polymyxa P2b-2R on its ability to promote growth of canola seedlings’ Authors: Kiran Preet Padda, Akshit Puri and Chris P Chanway.  35 favouring factors that induce root biomass accumulation and/or hindering those that could have detrimental effects on root system development (Pii et al., 2015).   One of the most often reported PGPR is Bacillus polymyxa, now named P. polymyxa (Ash et al., 1993). P. polymyxa inhabits different niches such as soils, roots, rhizosphere and internal tissues of various plant species including crops such as wheat, corn, sorghum, sugarcane, canola and barley (Guemouri-Athmani et al., 2000; von der Weid et al., 2000; Puri et al., 2015a, b), forest trees such as lodgepole pine (Anand et al., 2013), Douglas fir (Shishido et al., 1996) and marine sediments (Ravi et al., 2007). P. polymyxa is well known for its plant growth promoting traits, such as nitrogen fixation (Lindberg et al., 1985; Heulin et al., 1994), soil phosphorus solubilisation (Singh & Singh, 1993) and production of antibiotics (Rosado & Seldin, 1993; Choi et al., 2007; He et al., 2007). One such novel PGPB strain, P. polymyxa P2b-2R, was isolated from internal tissues of a lodgepole pine tree (Bal et al., 2012). This strain formed consistent endophytic and rhizospheric colonies, promoted plant growth and fixed significant amounts of N from the atmosphere when introduced into lodgepole pine (Anand et al., 2013), western red cedar (Anand & Chanway, 2013b), corn (Puri et al., 2015a) and canola (Puri et al., 2015b). The nif gene structure of P. polymyxa P2b-2R was characterized by Anand & Chanway (2013c) and was found to possess a single copy of the nifH gene. P. polymyxa P2b-2R was transformed with green fluorescent protein (GFP) to detect the sites of colonization in the internal tissues of lodgepole pine seedlings (Anand & Chanway, 2013a).   36  The GFP gene found in the jellyfish Aequorea victoria is the most popular autofluorescent protein system used for localization of endophytic bacteria because the chromophore requires only oxygen and water to fluoresce and marked cells can be detected easily using fluorescence microscopy (Chalife et al., 1994; Zimmer, 2002). Germaine et al. (2004) found that after a period of inoculation of poplar trees, several gfp-labeled poplar endophytes were detected in all of the interior tissues of the poplar trees. Ferreira et al. (2008) documented that the endophytic bacterium Pantoea agglomerans 33.1, tagged with the GFP gene, colonized Eucalyptus roots, mainly in intercellular spaces, stems, and xylem vessels. In a study to evaluate the colonization of plant roots of Arabidopsis thaliana by a natural isolate of P. polymyxa tagged with GFP gene, it was found that GFP-tagged P. polymyxa colonized predominantly the root tip, where it formed biofilms (Timmusk et al., 2005). These bacteria also accumulated in intercellular spaces outside the vascular cylinder. Thus, GFP is widely used to visualize and track the colonization patterns of bacterial strains within inoculated host plants; however, to date, no study has reported the effect of GFP tagging on the functioning of a bacterial strain. Following up on the study conducted with corn, reported in Chapter 2, a similar study was conducted with canola (Brassica napus L.) to elucidate the effect of GFP-tagging of strain P. polymyxa P2b-2R on its nitrogen fixing and plant growth promotion efficacy.   Canola is a significantly different crop species from corn, due to major physiological and botanical differences. Another reason for choosing canola as the model crop species for this study was its agronomic importance to Canada and the world. In just a few decades, canola has become one of the world's most important oilseed crops and  37 the most profitable crop commodity for Canadian farmers. Canada's canola industry adds $19.3 billion in economic activity to the Canadian economy and more than 43,000 Canadian farmers grow canola – largely as full-time farmers and in family farm businesses. They depend on canola to generate between one third and one half of their revenues (Canola Council of Canada, n.d.). Islam et al. (2009) studied the effects of a free-living diazotrophic bacteria, Paenibacillus sp. strain RFNB4, on canola plant’s growth and found that under pot culture conditions this bacterial strain shows significant increase in plant height and biomass production. In a report submitted to the Canola Council of Canada, de Freitas & Germida (1998) documented that Bacillus polymyxa (now known as “Paenibacillus polymyxa”) was the most common N2-fixing species isolated from the rhizosphere, root surface and interior of canola plants growing at three field sites in Saskatchewan, Canada. Subsequent re-introduction of Bacillus polymyxa strains in canola showed that they enhance shoot-N content, plant height and shoot biomass.  The objective of this study was to determine the effect of GFP-tagging of P. polymyxa P2b-2R on its plant growth promoting and nitrogen fixing ability when inoculated into an important oilseed crop species, canola. 3.2. Materials and methods P. polymyxa strain P2b-2R, its GFP-tagged derivative P2b-2Rgfp and the assay system have been described in Chapter 2. Briefly, the canola (Brassica Napus L.) seeds (var. Rugby Roundup ready) were obtained from SeCan Association’s Alberta branch (Lamont, Alberta, Canada). Seeds were surface sterilized by immersion in 30% hydrogen  38 peroxide for 90 seconds, followed by three 30-second rinses in sterile distilled water.     Seedling growth assays were performed in small pots (12cmx8cmx4cm) filled to 67% capacity with sterile sand-turface mixture. Canola seeds were inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative; P2b-2Rgfp. Non-inoculated seeds were treated with sterile phosphate buffer saline (PBS) and were used as control. This resulted in three treatment levels (P2b-2R, P2b-2Rgfp and control), each replicated 36 times and arranged in a completely randomized design. Two randomly selected seedlings from each treatment were harvested destructively 20, 30 and 40 days after inoculation (dai) to evaluate endophytic colonization. Samples of root, stem and leaf tissues were triturated separately in 1mL of sterile PBS using a mortar and pestle. Tissue suspensions of P2b-2Rgfp inoculated seedlings were plated onto CCM supplemented with 100mg/L cycloheximide, 200mg/L rifamycin and 5mg/L chloramphenicol. Triturated tissue suspensions of control and P2b-2R inoculated seedlings were diluted serially and plated onto CCM supplemented with 100mg/L cycloheximide and 200mg/L rifamycin. For evaluation of rhizosphere colonization, 2 randomly chosen seedlings from each treatment were harvested destructively 20, 30 and 40 dai. After gentle shaking, roots were then separated from shoots, placed in sterile Falcon tubes (50mL; BD Biosciences, CA, USA) filled with 10mL of sterile PBS and shaken on a vortex mixer. Serial dilutions were performed, and aliquots of 0.1 mL of each dilution were plated. For both endophytic and rhizospheric colonization, plates were incubated at room temperature for 7 days and numbers of colony forming units (cfu) were evaluated. Roots were oven-dried at 65oC for 2 days and rhizospheric bacterial populations were then calculated as cfu per gram dry root. For foliar N analysis, ground and oven dried foliage of each seedling from each  39 treatment was mixed thoroughly and 1mg sample was sent to the UBC Stable Isotope Facility for determination of foliar N content and %15N excess with an elemental analyzer interfaced with an isotope ratio mass spectrometer (Europa Scientific Integra). 3.3. Results 3.3.1. Rhizospheric and endophytic colonization P. polymyxa strain P2b-2R and P2b-2Rgfp colonized the canola rhizosphere with 7.76 x 106 cfu/g dry root and 2.67 x 107 cfu/g dry root, respectively. P2b-2R and P2b-2Rgfp were found to colonize canola roots endophytically with population density of 4.15 x 104 cfu/g fresh weight and 3.91× 105 cfu/g fresh weight, respectively. P2b-2R and P2b-2Rgfp colonies were also observed on CCM and TSA root, stem and leaf imprint plates after these tissues were surface sterilized, but there was no evidence of endophytic colonization in stem and leaf tissues by either P2b-2R or P2b-2Rgfp using the surface sterilization protocol. No evidence of rhizospheric or endophytic colonization was found in control plants.  3.3.2. Seedling growth promotion Seedling growth in terms of length and biomass was promoted significantly by inoculation with P2b-2Rgfp and P2b-2R. P2b-2Rgfp inoculation promoted seedling length up to 69% in the 1st growth trial and 50% in the 2nd growth trial as compared to control (Figure 3.1). Similarly, P2b-2R inoculated seedlings were up to 24% and 28% longer than the control in the 1st and 2nd growth trial respectively (Figure 3.1). Strikingly, the growth response of P2b-2Rgfp inoculated seedlings was more salient than P2b-2R  40 inoculated seedlings. In comparison to P2b-2R, P2b-2Rgfp inoculation increased seedling length up to 37% in the 1st and up to 18% in the 2nd trial (Figure 3.1).  Biomass (dry weight) of seedlings inoculated with P2b-2Rgfp was more than two-fold (118% in 1st trial and 108% in 2nd trial) greater than controls (Figure 3.2). P2b-2R inoculation led to an increase of up to 43% in 1st trial and 57% in the 2nd trial in seedling biomass (Figure 3.2). Intriguingly, P2b-2Rgfp inoculated seedlings accumulated significantly greater biomass than P2b-2R inoculated seedlings (up to 69% in 1st trial and 52% in 2nd trial) (Figure 3.2). 3.3.3. Nitrogen Fixation Foliar N concentration of seedlings inoculated with P2b-2Rgfp was up to 41% higher than the control in the 1st growth trial and up to 38% higher in the 2nd growth trial (Table 3.1). P2b-2R inoculated seedlings had upto 20% higher foliar N than the control in both growth trials (Table 3.1). Similar to seedling length and biomass results, the foliar N concentration of seedlings inoculated with P2b-2Rgfp was higher than P2b-2R inoculated seedlings (17% in 1st trial and 19% in 2nd trial) (Table 3.1). Based on 15N foliar dilution assay results, seedlings inoculated with P2b-2Rgfp derived up to 17% of their foliar N from the atmosphere (Ndfa) in the 1st growth trial and 22% in the 2nd growth trial (Table 3.1). P2b-2R inoculated seedlings were found to have derived up to 14% and 16% of foliar N from the atmosphere (Ndfa) in the 1st and 2nd growth trial, respectively (Table 3.1). P2b-2Rgfp inoculated seedlings derived more N from the atmosphere (nearly 4-6%) than their wild type counterpart and the difference in atom %15N excess values of both these treatments was statistically significant (Table 3.1).  41   Figure 3.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of canola seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).  01020304050607020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 1P2b-2RgfpP2b-2RControlabababababababababaaaaaaaaa(a)01020304050607020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 2P2b-2RgfpP2b-2RControlababababababababaaaaaaaaaa(b) 42   Figure 3.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of canola seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).05010015020025030020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 1P2b-2RgfpP2b-2RControlabaaaaaaaabababababaabab(a)05010015020025030020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 2P2b-2RgfpP2b-2RControlababababababaaaaaaaaaaaa(b) 43 Table 3.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation in Growth Trial 1 and 2   Atom %15N excess in foliage  % Foliar N  %Ndfa Growth trial Treatment 20 dai 30 dai 40 dai  20 dai 30 dai 40 dai  20 dai 30 dai 40 dai 1 P2b-2Rgfp 0.88a±0.007* 0.79ab±0.007 0.78ab±0.005  1.47ab±0.04 1.66ab±0.07 1.69ab±0.05  11.11 14.13 17.89 P2b-2R 0.91a±0.009 0.81a±0.006 0.81a±0.007  1.29a±0.06 1.44a±0.03 1.44a±0.05  8.08 11.95 14.73 Control 0.99±0.027 0.92±0.014 0.95±0.024  1.09±0.05 1.22±0.03 1.20±0.03  - - - 2 P2b-2Rgfp 0.84ab±0.005 0.80ab±0.006 0.73ab±0.004  1.53ab±0.07 1.71ab±0.07 1.74ab±0.04  13.02 15.14 22.07 P2b-2R 0.89a±0.007 0.82a±0.004 0.78a±0.008  1.33a±0.07 1.47a±0.04 1.47a±0.06  7.39 12.89 16.24 Control 0.96±0.010 0.94±0.011 0.93±0.010  1.11±0.05 1.31±0.07 1.26±0.03  - - - *Mean ± standard error; n=8 for atom % 15N excess & % Foliar N aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R)  44 3.4. Discussion This study clearly demonstrated that GFP tagging of a PGPB, P. polymyxa P2b-2R, increased its plant growth promotion efficiency when associated with an important oilseed crop, canola. Duplicate experiments confirmed this finding, thus, indicating that it was not just a one time experimental artifact. Seedling length and seedling biomass data were used to assess the growth stimulation of canola plants facilitated by P2b-2R and P2b-2Rgfp. Seedlings inoculated with P2b-2R grew significantly longer than the control seedlings in both growth trials with the % difference in length increasing at each harvest interval (Figure 3.1). P2b-2R inoculation also enhanced canola seedling biomass in both growth trials (Figure 3.2). These results are similar to those reported by Anand et al. (2013); Anand & Chanway (2013b); Puri et al. (2015); Puri (2015) with P2b-2R. In a similar way, P2b-2Rgfp inoculation significantly promoted canola seedling length (nearly 1.5 fold) at each harvest interval in both growth trials (Figure 3.1). Increased biomass accumulation (more than 2-fold) was also observed in P2b-2Rgfp inoculated seedlings as compared to the controls (Figure 3.2). These striking differences in seedling length and biomass between the P2b-2Rgfp inoculated seedlings and the controls were very large. This finding inclined me to compare the seedling length and biomass data of P2b-2Rgfp and P2b-2R inoculated seedlings statistically. The differences in seedling length and biomass values of the aforementioned treatments were statistically significant in both growth trials. These results provide a clear indication that P2b-2Rgfp inoculated seedlings performed better than P2b-2R inoculated seedlings in terms of growth promotion.  45  Since, P. polymyxa is well known for its plant growth promoting properties, including N fixation, soil phosphorus solubilisation and production of exopolysaccharides, hydrolytic enzymes, antibiotics, cytokinins (Lal & Tabacchioni, 2009), I think that the growth promotion observed in P2b-2Rgfp and P2b-2R inoculated seedlings might be due to one or more of the abovementioned traits. The production of plant growth promoting compounds by P. polymyxa similar in activity to indole-3-acetic acid has been suggested to stimulate growth in crested wheatgrass (Holl et al., 1988). It also releases iso-pentenyladenine and one unknown cytokinin-like compound during its stationary phase of growth which promotes seed germination, de novo bud formation, release of buds from apical dominance, stimulation of leaf expansion and reproductive development and retardation of senescence (Mok, 1994) in wheat (Lindberg et al., 1985; Lindberg & Granhall, 1986). Beatty & Jensen (2002) isolated and identified a potential biological control agent, P. polymyxa strain PKB1, against Leptosphaeria maculans, the causative agent of blackleg disease of canola. They reported that it is capable of inhibiting the growth of L. maculans by producing antifungal peptides.  P. polymyxa P2b-2R consistently reduced high amounts of acetylene when evaluated for nitrogenase activity (Bal et al., 2012). Acetylene reduction assay provides an indirect evidence of biological N fixation; therefore, I used a more robust 15N isotope dilution technique to evaluate the ability of P2b-2R and P2b-2Rgfp to provide colonized canola plants with fixed N in situ. 15N atom percent excess was used as a measure of nitrogen derived from the atmosphere. Seedlings inoculated with either P2b-2gfp or P2b-2R had significantly lower 15N atom percent excess as compared to non-inoculated controls (Table 3.1). This clearly indicates that inoculated seedlings derived a greater  46 proportion of N from the atmosphere than from the planting medium. The results obtained for P2b-2R inoculated seedlings are in agreement with studies of this bacterial strain’s association with agricultural crops (Puri et al., 2015; Puri, 2015) and gymnosperms (Anand et al., 2013; Anand & Chanway, 2013b). Interestingly, when 15N atom percent excess values of P2b-2Rgfp inoculated seedlings were compared with P2b-2R inoculated seedlings, a statistically significant difference was detected at each harvest interval in each growth trial (except 20 dai in 1st growth trial) (Table 3.1). Similar findings were reported for corn (Chapter 2), providing further evidence that GFP is responsible for enhancing the N-fixing efficacy of P2b-2R. My results also indicate that foliar N content of either P2b-2Rgfp or P2b-2R inoculated seedlings were significantly higher than the controls in both growth trials (Table 3.1). This suggests that growth promotion observed in inoculated canola seedlings could be due to a significantly higher N content as compared to the controls. Since N was only provided once to the seedlings at the onset of experiment, plant N levels are expected to decline during the course of experiment and this was observed in control plants. Thus, it is clear that inoculated seedlings fulfill their N requirements from a source other than plant growth medium, possibly if not likely the atmosphere. Significantly higher foliar N content observed in P2b-2Rgfp inoculated seedlings as compared to the P2b-2R inoculated seedlings (Table 3.1) is another indication that GFP-tagging enhances this bacterial strain’s ability to grasp more N from the atmosphere than seedlings inoculated with the wild type strain.  To check for rhizospheric and endophytic colonization by either P2b-2Rgfp or P2b-2R, I used the direct plating technique on a selective growth media (CCM) amended with antibacterial (rifamycin or rifamycin + chloramphenicol) and antifungal  47 (cycloheximide) compounds. Successful isolation of P2b-2Rgfp colonies from rhizospheric and internal canola root tissues confirms its colonization ability. Similarly, P2b-2R colonies were also observed in rhizosphere and internal root tissues of P2b-2R inoculated seedlings. The population density observed is comparable with earlier reports of this bacterial strain in corn and canola (Puri et al., 2015; Puri, 2015) and other endophytic diazotrophs in rice (Elbeltagy et al., 2001), sugarcane (Sevilla et al., 2001), and grape (Compant et al., 2005). There was no evidence of endophytic colonization in stem and leaf tissues of inoculated seedlings. A possible reason for this could be that bacteria was not able to move up to the aerial parts of the plant and form detectable colonies in only 40 days. Seedlings were grown for 40 days for the sake of axenic growth and ease of study. It is possible that a longer study would be necessary for more substantial shoot colonization.  The results of this study are in agreement with the previous study with corn (Chapter 2). Although a statistical comparison of the results obtained for corn and canola was not done, generally speaking P2b-2Rgfp performed better in association with canola. This study has shown that the role of GFP is not restricted to a marker protein and looks to be involved in enhancing this PGPB’s ability to promote growth. But how does GFP enhance a PGPB’s growth promotion efficacy? Further experimentation by using molecular and genetic tools is clearly needed to answer this question and to gain in-depth knowledge about its other potential roles.   48 3.5. Conclusions Since its discovery, GFP has been used extensively as a marker protein to tag a particular protein or a microbe of interest but the effects of this tagging on microbial behavior are lesser known. Through this study, I tried to investigate the unexplored effects of introducing a marker protein (GFP) into a PGPB (P. polymyxa P2b-2R). P. polymyxa P2b-2R tagged with a plasmid-borne GFP outperformed the wild type P2b-2R in enhancing the growth of an important oilseed crop, canola. P2b-2Rgfp inoculated canola seedlings grew longer (37%), assimilated greater biomass (69%) and fixed higher amount of N from atmosphere (5%) than the P2b-2R inoculated seedlings. These findings are in agreement with the study documented in corn (Chapter 2) and strengthens my claim that transformation of a PGPB, P. polymyxa P2b-2R, have a positive effect on its growth promotion efficacy. Although it’s too early to say, but this type of genetic modification of PGPB might have a potential in future to increase crop yield.   49 Chapter 4: Growth promotion of tomato by an endophytic strain of Paenibacillus polymyxa and its GFP-derivative3 4.1. Introduction Soil is an excellent niche for the growth of many microorganisms including protozoa, fungi, viruses, and bacteria. Some microorganisms are able to colonize soil surrounding plant roots, the rhizosphere, rendering them under the influence of plant roots (Hiltner, 1904; Kennedy, 2005). Kloepper & Schroth (1978) introduced the term ‘rhizobacteria’ to describe the soil bacterial community that competitively colonizes plant roots and stimulates growth often by reducing the incidence of plant diseases. Kloepper & Schroth (1981) termed these beneficial rhizobacteria ‘plant growth-promoting rhizobacteria (PGPR)’. PGPR can be defined as the indispensable part of rhizosphere biota that when grown in association with the host plants can stimulate the growth of the host (Bhattacharyya & Jha, 2012). The concept of PGPR has now been confined to the bacterial strains that can fulfil at least two of the three criteria such as aggressive colonization, plant growth stimulation and biocontrol (Weller et al., 2002; Vessey, 2003).   Apart from PGPR, there are certain other plant growth promoting bacteria (PGPB) that live inside the plant tissues, known as plant growth-promoting bacterial endophytes. Endophytic bacteria colonize the host tissue internally, sometimes in high numbers, without damaging the host or eliciting symptoms of plant disease according to a widely used definition (Quispel, 1992). Prominent sites for active ingress by endophytic                                                         3A version of this chapter has been submitted to Biology and Fertility of Soils Journal for publication under the title ‘Plant growth promotion and N-fixation by an endophytic strain of Paenibacillus polymyxa and its GFP-derivative in tomato’ Authors: Kiran Preet Padda, Akshit Puri and Chris P Chanway.  50 bacteria are the emergence points of lateral roots and the zone of differentiation and elongation near the root tip (Reinhold-Hurek & Hurek, 2011). Major sites of endophytic colonization are intercellular spaces in the epidermal and cortical regions, vascular tissue, xylem cells and lysed plant cells (Chi et al., 2005; Compant et al., 2010). Endophytic bacteria may be of particular interest as they have the advantage of being relatively protected from the competitive, high-stress environment of the soil (Sturz et al., 2000; Sturz & Nowak, 2000). Moreover, plant growth promotion is often greater when it is induced by endophytes rather than by bacteria restricted to the rhizosphere and the root surface (Chanway et al., 2000; Conn et al., 1997). In a recent study, root-associated bacterial endophytes isolated from different cultivars of tomato were found to provide host resistance against the wilt pathogen Ralstonia solanacearum when inoculated into tomato seedlings (Upreti & Thomas, 2015). Khan et al. (2014) isolated an endophyte Sphingomonas sp. LK11 from the leaves of Tephrosia apollinea. Tomato plants inoculated with this endophytic strain showed significant increase in growth attributes (shoot length, chlorophyll contents, shoot and root dry weights) compared to controls. In vitro experiments revealed that this enhanced plant growth was due to the presence of physiologically active and inactive gibberellins, as well as production of indole acetic acid by this endophyte.   Several bacterial endophytes have also been reported to fix atmospheric N in association with crop species (Ruschel et al., 1975; Shrestha & Ladha, 1996; Malik et al., 1997; Montañez et al., 2009; Puri et al., 2015a,b). Döbereiner (1992) designated such microorganisms as ‘diazotrophic endophytic bacteria’. The association between endophytic diazotroph Gluconoacetobacter diazotrophicus (formerly Acetobacter  51 diazotrophicus) and sugarcane (Saccharum officinarum L.) in Brazil was the most significant discovery reported until present in terms of the quantity of fixed N being supplied to the host plant (Lima et al., 1987; Cavalcante & Döbereiner, 1988; Boddey et al., 1991). However, an endophytic diazotroph, Paenibacillus polymyxa P2b-2R, was isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues growing in nutrient poor sites near Williams Lake, BC, Canada (Bal et al., 2012). Re-introduction of this strain into gymnosperms, lodgepole pine and western red cedar, resulted in significant growth promotion (seedling height and biomass) and large amounts of N being derived from atmosphere when grown in a growth chamber (Anand et al., 2013; Anand & Chanway, 2013b). P. polymyxa is well known for its capability to thrive in a wide range of environmental conditions, which is likely due to its endospore-forming ability. In a mini-review, Lal & Tabacchioni (2009) highlighted a range of potential plant growth promoting characteristics this bacterium possesses including the ability to fix N and to produce hormones that promote plant growth.   When P. polymyxa P2b-2R was introduced into the agricultural crops- corn and canola, it was found that the bacterial strain significantly promoted plant growth, fixed N, and successfully colonized corn and canola seedlings (Puri et al., 2015a,b). These results were the very first evidence of this bacterial strain’s broad range host capability. The results reported in Chapter 2 and 3 of this thesis are in agreement with these results. Since P. polymyxa P2b-2R efficaciously associated with a cereal crop (corn) and an oilseed crop (canola), I was interested in evaluating its broad range host capability by also inoculating it into a berry fruit, tomato. While it is botanically a berry fruit, Food and Agricultural Organization of the United Nations and Statistics Canada consider tomatoes  52 under vegetable crops. Tomatoes are considered the most important vegetable crop in world with an estimated production of 160 million tonnes in 2011, which was about 15% of total global vegetable production (Garming, 2014). Marketed production of tomato was also the highest in Canada among vegetable crops, with an estimated production of more than 270,000 metric tonnes (Statistics Canada, n.d.).  The introduction of green fluorescence protein (GFP) as a marker of gene expression (Prasher et al., 1992; Chalfie et al., 1994) has facilitated research in localization and identification of specific genes in GFP tagged bacteria in the infected cells and plant tissues. The use of GFP has become one of the powerful and valuable tools for addressing most of the biological research in plant–microbe interactions in living systems (Chalfie et al., 1994). Bacterial cells tagged with GFP can be enumerated in situ, as samples do not need to be fixed, hybridized or stained (Tombolini et al., 1997). In addition, bacterial multiplication can be followed temporally and spatially (Valdivia et al., 1998; Compant et al., 2005). Anand & Chanway (2013a) tagged P. polymyxa P2b-2R with a plasmid borne GFP to evaluate endophytic colonization of lodgepole pine seedlings by a tagged derivative of the diazotrophic strain P2b-2R. But the effects of introducing the GFP gene into this bacterial strain were not known, until they were determined and reported in chapter 2 and 3 by using corn and canola as the model crop species, respectively. Since, the GFP gene positively affected the growth promoting capability of P. polymyxa P2b-2R when inoculated into corn and canola, I was very interested in evaluating its effect when inoculated into tomato.  53  The main objectives of this study were, (i) to determine if P. polymyxa P2b-2R can associate with a berry crop, tomato (Solanum lycopersicum), in ways similar to corn and canola, and (ii) to determine if GFP tagging of P2b-2R affects its growth promoting ability when evaluated on tomato seedlings. 4.2. Materials and methods P. polymyxa strain P2b-2R, its GFP-tagged derivative P2b-2Rgfp and the assay system for rhizospheric and endophytic colonization, growth response and N-analysis have been described previously (Chapter 2). Briefly, tomato (Solanum lycopersicum) seeds (var. Celebrity) were obtained from the West Coast Seed Company (Delta, British Columbia, Canada). Seeds were surface sterilized by immersion in 30% hydrogen peroxide for 90 seconds, followed by three 30-second rinses in sterile distilled water. Seedling growth assays were performed in small pots (12cmx8cmx4cm) filled to 67% capacity with sterile sand-turface mixture. Each pot was fertilized with 50 mL of a nutrient solution (Chanway et al., 1988) modified by replacing KNO3 and Ca (NO3)-4H2O with Ca (15NO3) 2 (5 % 15N label) (0.0576g/L) and Sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ontario) with Na2FeEDTA (0.02g/L). Other nutrients in the nutrient solution included (in grams per liter): 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. Tomato seeds were inoculated with P. polymyxa strain P2b-2R or its GFP-tagged derivative; P2b-2Rgfp. Non-inoculated seeds were treated with sterile phosphate buffer saline (PBS) and were used as control. This resulted in three treatment levels (P2b-2R, P2b-2Rgfp and control), each replicated 36 times and arranged in a completely randomized design. Seedlings  54 received modified nutrient solution without Ca(15NO3)2 10 days after sowing and were watered with sterile distilled water as required. Two randomly selected seedlings from each treatment were harvested destructively 20, 30 and 40 days after inoculation (dai) to evaluate endophytic and rhizospheric colonization. For foliar N analysis, ground and oven dried foliage of each seedling (n=8) from each treatment was mixed thoroughly and 1mg sample was sent to the UBC Stable Isotope Facility for determination of foliar N content and %15N excess with an elemental analyzer interfaced with an isotope ratio mass spectrometer (Europa Scientific Integra). The amount of fixed N in foliage was estimated by calculating the percent N derived from the atmosphere (%Ndfa) (Rennie et al., 1978): % Ndfa = [1 −atom % 15N excess (inoculated plant)atom % 15N excess (uninoculated plant)]  x 100 4.3. Results 4.3.1. Endophytic and rhizospheric colonization P. polymyxa strain P2b-2R and P2b-2Rgfp successfully colonized the tomato rhizosphere. P2b-2R colonized tomato rhizosphere with 4.95 x 106 cfu/g dry root in the 1st growth trial and 6.13 x 106 cfu/g dry root in the 2nd growth trial. Similarly, P2b-2Rgfp colonized tomato rhizosphere with 9.82 x 105 cfu/g dry root in the 1st growth trial and 3.54 x 106 cfu/g dry root in the 2nd growth trial. P2b-2R and P2b-2Rgfp were found to colonize tomato roots endophytically with population densities of 1.35 x 106 cfu/g fresh weight and 2.56 x 105 cfu/g fresh weight, respectively in the 1st growth trial. In the 2nd growth trial, P2b-2R and P2b-2Rgfp were found to colonize tomato roots endophytically with population densities of 3.02 x 106 cfu/g fresh weight and 8.83 x 105 cfu/g fresh weight,  55 respectively. Strain P2b-2R and P2b-2Rgfp colonies were also observed on both CCM and TSA root, stem and leaf imprint plates after these tissues were surface sterilized but there was no evidence of endophytic colonization by these strains using surface sterilization protocol. No evidence of rhizospheric or endophytic colonization was found in control plants.  4.3.2. Growth response Seedling growth (length and biomass) was promoted significantly by inoculation with P2b-2Rgfp and P2b-2R in comparison to non-inoculated control plants. P2b-2Rgfp inoculated seedlings were up to 48% and 36% longer than control in the 1st and 2nd growth trials respectively (Figure 4.1). Similarly, P2b-2R inoculation led to seedling length promotion of up to 33% in the 1st trial and 40% in the 2nd trial (Figure 4.1) P2b-2Rgfp inoculation promoted seedling dry weight (biomass) by 82% in the 1st growth trial and 73% in the 2nd growth trial as compared to the control (Figure 4.2). In a similar way, P2b-2R inoculation led to an increase of up to 93% in 1st trial and 85% in the 2nd trial in seedling biomass (Figure 4.2). Unlike corn and canola, growth (length and biomass) of seedlings inoculated with P2b-2Rgfp did not differ significantly from P2b-2R inoculated seedlings in both growth trials. 4.3.3. Nitrogen fixation In the 1st growth trial, foliar N concentration of seedlings inoculated with P2b-2R and P2b-2Rgfp was up to 33% and 25% higher than the control, respectively (Table 4.1). Similarly in the 2nd growth trial, P2b-2R inoculate seedlings had upto 29% higher foliar  56 N than the control and P2b-2Rgfp inoculated seedlings had up to 22% higher foliar N (Table 4.1). As observed in the case of seedling length and biomass, the foliar N concentration of seedlings inoculated with P2b-2Rgfp did not significantly differ from P2b-2R inoculated seedlings in both growth trials (Table 4.1). Based on 15N foliar dilution assay results, seedlings inoculated with P2b-2Rgfp derived up to 15% of their foliar N from atmosphere (Ndfa) in the 1st growth trial and 16% in the 2nd growth trial (Table 4.1). P2b-2R inoculated seedlings derived up to 17% and 18% of their foliar N from atmosphere in 1st and 2nd growth trial, respectively (Table 4.1).  57   Figure 4.1 Seedling length, root length and shoot length (mean and standard error; n = 8 seedlings per treatment) of tomato seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp harvested thrice at 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).  010203040506020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 1P2b-2RgfpP2b-2RControlaaaaabaaaaaaaaaaaaa(a)010203040506020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootLength (cm)Growth Trial 2P2b-2RgfpP2b-2RControlaaaaaaaabaaaaaaaaaa(b) 58   Figure 4.2 Seedling dry weight, root dry weight and shoot dry weight (mean and standard error; n = 8 seedlings per treatment) of tomato seedlings inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) (a) in the 1st growth trial and (b) in the 2nd growth trial. aP<0.05 (significantly different from control); bP<0.05 (significantly different from P2b-2R).  05010015020025030020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 1P2b-2RgfpP2b-2RControlaaaaaaaaaaaaaaaaa(a)05010015020025030020 dai 30 dai 40 dai 20 dai 30 dai 40 dai 20 dai 30 dai 40 daiSeedling Root ShootDry weight (mg)Growth Trial 2P2b-2RgfpP2b-2RControlaaaaaaaaaaaaaaaaaa(b) 59 Table 4.1 Atom percent 15N excess in foliage, percent foliar N and percent N derived from the atmosphere (Ndfa), developed from tomato seeds inoculated with P. polymyxa strain P2b-2R and its GFP-tagged derivative P2b-2Rgfp measured 20, 30 and 40 days after inoculation (dai) in growth trial 1 and 2   Atom %15N excess in foliage  % Foliar N  %Ndfa Growth trial Treatment 20 dai 30 dai 40 dai  20 dai 30 dai 40 dai  20 dai 30 dai 40 dai 1 P2b-2Rgfp 0.65a±0.013* 0.63a±0.009 0.56a±0.006  1.92a±0.06 1.38a±0.06 1.47a±0.05  7.34 9.39 15.55 P2b-2R 0.64a±0.010 0.62a±0.004 0.55a±0.013  2.04a±0.08 1.46a±0.05 1.56a±0.05  8.30 11.07 17.01 Control 0.70±0.007 0.69±0.005 0.66±0.019  1.53±0.07 1.12±0.03 1.20±0.05  - - - 2 P2b-2Rgfp 0.63a±0.017 0.61a±0.010 0.54a±0.007  2.00a±0.05 1.50a±0.05 1.55a±0.03  8.32 11.18 16.73 P2b-2R 0.62a±0.010 0.60a±0.007 0.53a±0.009  2.11a±0.06 1.54a±0.05 1.61a±0.04  10.02 12.31 18.11 Control 0.69±0.004 0.68±0.008 0.65±0.014  1.63±0.05 1.23±0.04 1.30±0.03  - - - *Mean ± standard error; n=8 for atom % 15N excess & % Foliar N aP<0.05 (significantly different from control)  60 4.4. Discussion The present study clearly demonstrated that P. polymyxa P2b-2R, a lodgepole pine endophyte, can colonize, fix N and promote overall growth of tomato (S. lycopersicum) seedlings. To the best of my knowledge, this study has reported for the first time that an endophytic diazotroph belonging to P. polymyxa, isolated from lodgepole pine trees, can associate with a berry fruit tomato. Another major finding was that GFP tagging of P2b-2R didn’t effected its growth promoting ability when evaluated with tomato as the model plant species. This was unusual, as the GFP tagged P2b-2R strain performed better than wild P2b-2R strain with corn and canola and I expected similar results with tomato as well. It can be postulated that GFP tagging spikes the growth promoting capabilities of P. polymyxa P2b-2R when associated with certain hosts. Studies contrasting the plant growth promoting capacity of wild type and GFP-tagged endophytes in Vitis vinifera (Compant et al., 2005) and Jerusalem artichoke (Meng et al., 2014) have reported similar results as were observed with tomato in this study. Meng et al. (2014) also compared the plant growth-promotion properties of wild type and GFP tagged endophyte and found no significant difference in nitrogenase activity, indole 3-acetic acid production, siderophore production and phosphate solubilization. On the contrary, Weyens et al. (2012) reported that GFP labeling negatively affects the growth promoting ability and colonization capacity of an endophyte, Pseudomonas putida W619, when inoculated into hybrid poplar. Thus, it is quite clear that the effects of GFP tagging of an endophytic bacterial strain might vary depending on the type of host plant. But there is definite need to further investigate these effects at genetic as well as molecular levels, so as to conclude more specifically about these observed effects.  61  Re-isolation of P2b-2R and P2b-2Rgfp was carried out by the direct plating technique using a selective growth media (CCM) in addition to with antibiotic and antifungal agents. P. polymyxa P2b-2R successfully colonized rhizosphere of tomato seedlings with population sizes similar to those reported in previous studies about this bacterial strain (Bal & Chanway, 2012a,b; Anand & Chanway, 2013b, Anand et al., 2013; Puri et al., 2015a,b). P2b-2Rgfp was also successful in colonizing the tomato rhizosphere with population sizes similar to those observed in corn and canola (Chapter 2 and 3). This indicates that, apart from surviving, both P2b-2Rgfp and P2b-2R can multiply in numbers in the tomato rhizosphere. Survival and multiplication in rhizosphere is necessary since prominent sites for active ingress by bacteria, to form endophytic colonies, are the emergence points of lateral roots and the zone of differentiation and elongation near the root tip (Reinhold-Hurek & Hurek, 2011). These results suggest that both P2b-2R and P2b-2Rgfp colonized tomato roots endophytically with population densities comparable to those observed in crops like corn, canola and rice (Puri et al., 2015a,b; Kandel et al., 2015) and tree species like western red cedar and lodgepole pine (Anand & Chanway, 2013b; Anand et al., 2013). However, I was not able to detect endophytic colonies of P2b-2R and P2b-2Rgfp strains in stem and leaf tissues of the inoculated tomato seedlings. A possible reason could be that the time span of the experiment was not long enough for the bacteria to form quantifiable endophytic colonies in stem and leaf, as was observed in the case of lodgepole pine (Bal & Chanway 2012a) and western red cedar (Bal & Chanway, 2012b).   P. polymyxa P2b-2R and its GFP labeled derivative (P2b-2Rgfp) showed consistent and significant effects on corn foliar 15N abundance. Based on atom % 15N  62 excess in foliage data, seedlings treated with either P2b-2R or P2b-2Rgfp derived significant amount of N from a source other than the soil N, likely the atmosphere (Table 4.1). Similar amounts of N derived from the atmospheric pool have been observed in other plant species including corn (Zea mays L.) and canola (Brassica napus) (Puri et al., 2015a,b). It is also noteworthy that the percentage of foliar nitrogen derived from the atmosphere increased with time over the duration of the growth trial. A similar trend was reported in the sugarcane cultivar ‘Krakatau’ which obtained an increasing percentage (6.2 to 54.8 %) of its nitrogen from the atmosphere over 4 samplings between 100 and 250 days after emergence (Uriquaga et al., 1992). N was added to the soil mix only once at the onset of the experiment and in the absence of further additions N was expected to deplete with continued seedling growth and ultimately restrict the growth rate of seedling, which was observed in control tomato plants. But this was not the case with P2b-2R or P2b-2Rgfp inoculated seedlings as the N-fixing trait of this bacterial strain led to the accumulation of higher total foliar N than the controls, thus promoting the growth. GFP tagging didn’t effected the N-fixing property of P. polymyxa P2b-2R when inoculated in tomato seedlings as there wasn’t a significant difference in atom %15N excess values of P2b-2R and P2b-2Rgfp inoculated seedlings.  In addition to fixing N2, P. polymyxa possesses several characteristics that can result in plant growth promotion including the production of phytohormones and antibiotics as well as the ability to enhance N, P, and S availability in the rhizosphere (Chanway, 2008; Talboys et al., 2014; Borriss, 2015; Han et al., 2015). P2b-2R inoculated seedlings showed consistent increase in height during each growth trial and were much taller than the control at each harvest interval (Figure 4.1). There was a  63 consistent increase in inoculated seedling biomass during each growth trial. Seedling biomass of P2b-2R-inoculated seedlings was nearly 90% greater than the control after 40 days of inoculation (Figure 4.2). These results are consistent with previous reports about this bacterial strain, where P2b-2R inoculation significantly enhanced seedling height and biomass in lodgepole pine, western red cedar, corn and canola (Anand & Chanway, 2013b; Anand et al., 2013; Puri et al., 2015a,b). P2b-2Rgfp inoculation also enhanced height and biomass of tomato seedlings by a similar amount as wild type P2b-2R inoculation (Figure 4.1 and 4.2). No significant difference was found in height and biomass of seedlings inoculated with P2b-2R and P2b-2Rgfp, which was unusual since contrasting results were reported in chapter 2 and 3 for corn and canola respectively. 4.5. Conclusions P. polymyxa P2b-2R is an endophytic diazotroph originally isolated from internal tissues of lodgepole pine (Bal et al., 2012) and evaluated for its ability to enhance plant growth (height and biomass) and fix N in a cereal crop species, corn (Puri et al., 2015a), and an oilseed crop species, canola (Puri et al., 2015b). In this study, I evaluated P2b-2R’s ability to associate in a similar way with a berry fruit crop, tomato, and found that inoculation with this bacterial strain resulted in colonization of the rhizosphere and internal root tissues, fixed significant amounts of N from atmosphere (nearly 18%), and significantly promoted length (nearly 40%) and biomass (nearly 90%). These results are important in terms of this bacterial strain’s broad range host capability. Though, GFP-tagging of P2b-2R enhanced its growth promoting ability when inoculated into corn and canola, I suspected that a similar effect will be seen with tomato. But I did not observe a  64 statistically significant difference between P2b-2R inoculated and P2b-2Rgfp inoculated tomato seedlings in terms of growth promotion. This indicates that the effect of GFP-tagging of an endophytic diazotroph like P. polymyxa P2b-2R might vary depending on the host plant species and the mechanism by which growth promotion occurs.   65 Chapter 5 – General conclusions Sustaining high agricultural productivity is a major challenge for both developed and developing countries, mainly due to the increasing global demand for food for a growing global population and the depletion of natural resources (Matson et al., 1997; Cassman, 1999; Tilman et al., 2002). External chemical fertilizer input is the leading approach used by farmers to maintain high crop productivity (Zhang et al., 2010). But such an approach has many negative effects on our environment and human health. Thus, instead of the increased use of chemicals, it is essential that the use of transgenic plants and the widespread application of plant growth-promoting microorganisms, both bacteria and fungi, be embraced and practiced on a large scale (Glick, 2015).  PGPB are capable of positively affecting plant growth via numerous independent and linked mechanisms (Compant et al., 2010). P. polymyxa is one such PGPB, well known for its wide range of plant-beneficial properties, including N-fixation, plant growth promotion, phosphorus solubilisation and production of exopolysaccharides, hydrolytic enzymes, antibiotics and cytokinins (Lal & Tabacchioni, 2009). P. polymyxa P2b-2R, was isolated from internal tissues of lodgepole pine (Bal et al., 2012) and was found to promote plant growth and derive significant amounts of nitrogen from atmosphere when introduced into the gymnosperms, lodgepole pine (Bal & Chanway, 2012a; Anand et al., 2013) and western red cedar (Bal & Chanway, 2012b; Anand & Chanway, 2013b), and agricultural crops, corn (Puri et al., 2015a) and canola (Puri et al., 2015b). P. polymyxa  P2b-2R was tagged with GFP to visualize its endophytic colonization sites when inoculated into lodgepole pine (Anand & Chanway, 2013a).  66  Since its discovery, the GFP gene marker has been widely used to visualize and track the colonization patterns of bacterial endophytes within inoculated host plants (Chelius & Triplett, 2000; Knoth et al., 2013; Sun et al., 2014; Kandel et al., 2015). The use of GFP has become one of the powerful and valuable tools for addressing most of the biological research in plant–microbe interactions in the living systems (Chalfie et al., 1994). But very little is known about the effects of introduction of GFP on the functioning of a microbe. Some studies have reported that GFP tagging does not affect the growth promoting ability of an endophyte (Compant et al., 2005; Meng et al., 2014), whereas a study with an endophyte, Pseudomonas putida W619, reported negative effects of GFP tagging on growth promoting ability (Weyens et al., 2012). In this thesis project, I studied the effects of GFP tagging of an endophyte, P. polymyxa P2b-2R, on its growth promoting ability inside different hosts viz., corn, canola and tomato.  The effect of GFP tagging of P. polymyxa P2b-2R on its growth-promoting efficacy when inoculated into an agronomically important crop species, corn, is reported in Chapter 2. Growth promotion was evaluated based on factors including plant length and biomass. It was found that corn seedlings inoculated with either P2b-2R or P2b-2Rgfp performed better than the control seedlings in terms of length and biomass. Nitrogen derived from atmosphere by the inoculated seedlings was also significant at each harvest interval, which I think was the main reason for better growth of inoculated seedlings. Comparing length, biomass and amount of N-fixed of P2b-2R and P2b-2Rgfp inoculated corn seedlings, I found that P2b-2Rgfp inoculated corn seedlings were significantly longer, accumulated greater biomass and fixed more atmospheric N than P2b-2R inoculated seedlings. This clearly suggests that genetic modification of the  67 PGPB; P. polymyxa P2b-2R, by introduction of a marker protein (GFP) positively affected the bacterium’s growth promotion efficacy when inoculated into corn. In a similar way, effects of GFP tagging of P2b-2R was also evaluated inside an important oilseed crop, canola (Brassica napus L.) and are reported in chapter 3. P2b-2R tagged with a plasmid-borne GFP outperformed the wild P2b-2R in enhancing the growth of canola. P2b-2Rgfp inoculated canola seedlings grew 37% longer, assimilated 69% more biomass and fixed a greater amount of N from atmosphere (5%) than the P2b-2R inoculated seedlings. A statistical comparison between the results obtained for corn and canola was not done, but generally speaking P2b-2Rgfp performed better in association with canola. These studies with corn and canola have shown that the role of GFP is not restricted to a marker protein and it might be involved in enhancing a PGPB’s ability to promote growth. But how does GFP enhance a PGPB’s growth promotion efficacy? Further experimentation by using molecular and genetic tools is clearly needed to answer this question and to gain in-depth knowledge about its other potential roles.  In the study reported in chapter 4, I looked at the ability of P. polymyxa P2b-2R to colonize, promote growth and fix N when associated with a berry fruit, tomato. Continuing on my main objective of this thesis, I also compared the growth promotion and N-fixation by GFP tagged P2b-2R with wild P2b-2R. P. polymyxa P2b-2R successfully colonized rhizosphere and internal root tissues of tomato, promoted the growth of tomato seedlings (length and biomass) and fixed significant amounts of N from atmosphere. These results were comparable with previous studies performed with corn (Puri et al., 2015a) and canola (Puri et al., 2015b) and thus helped in further confirming this bacterial strain’s broad range host capability. After successful results with GFP  68 tagged P2b-2R in corn and canola, I suspected that P2b-2Rgfp would again outperform wild P2b-2R when inoculated in tomato. But, no statistically significant difference was observed between P2b-2R inoculated and P2b-2Rgfp inoculated tomato seedlings in terms of growth promotion (length and biomass) and N-fixation. This was unusual, especially after observing positive results in corn and canola. This lead me to conclude that the effect of GFP-tagging of an endophytic diazotroph like P. polymyxa P2b-2R might vary depending on the host plant species. But there is a definite need to further investigate these effects at genetic as well as molecular levels to conclude these observed effects more confidently.  In summary, my thesis provides insights into the possible effects of GFP tagging of a growth promoting bacteria, P. polymyxa P2b-2R, on its plant growth promoting ability. Genetic modification of P. polymyxa carried out by introducing a marker protein (GFP) positively affects the bacterial strain’s plant growth promoting ability when associated with two important agricultural crop species, corn and canola. Although it’s too early to say, this type of genetic modification of PGPB might have potential to increase crop yield in the future. But when evaluated in tomato, GFP tagging had no significant affect on the bacterial strain’s growth promoting capability. 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Academic Press, Volume 107, pp 1-32. Zimmer M. (2002). Green fluorescent protein (GFP): applications, structure, and related  photophysical behavior. Chem Rev 102:759–782.   86 Appendix A – Plant nutrient solution4 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 0.0576g/L                                                         4 Modified from Chanway et al. (1988)  87 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  88 Appendix C – Additional figures   Figure C.1 Control (left), P2b-2R-inoculated (centre) and P2b-2Rgfp-inoculated (right) seedlings of corn plant harvested 40 days after inoculation showing clear difference in length, biomass and plant health  Corn: 40 days after inoculation P2b-2Rgfp inoculated P2b-2R inoculated Control  89  Figure C.2 Control (left), P2b-2R-inoculated (centre) and P2b-2Rgfp-inoculated (right) seedlings of canola plant harvested 40 days after inoculation showing clear difference in length, biomass and plant health  Canola: 40 days after inoculation P2b-2Rgfp inoculated P2b-2R inoculated Control  90  Figure C.3 P2b-2R-inoculated (left), P2b-2Rgfp-inoculated (centre) and Control (right) seedlings of tomato plant harvested 40 days after inoculation showing clear difference in length, biomass and plant health Tomato: 40 days after inoculation P2b-2Rgfp inoculated P2b-2R inoculated Control 

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