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Plant growth promotion and nitrogen fixation by Paenibacillus polymyxa in corn and canola Puri, Akshit 2015

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Plant Growth Promotion and Nitrogen Fixation by Paenibacillus polymyxa in Corn and Canola by Akshit Puri 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) July 2015  © Akshit Puri, 2015  ii Abstract Availability of nitrogen is the most yield-limiting mineral factor in crop production. Several Paenibacillus bacterial strains that were able to fix nitrogen from atmosphere were isolated from extracts of surface-sterilized lodgepole pine seedling and tree tissues. One strain, Paenibacillus polymyxa P2b-2R, was found to derive high amounts of nitrogen from the atmosphere when introduced into gymnosperm species, namely lodgepole pine and western red cedar. I wanted to determine if Paenibacillus polymyxa P2b-2R could colonize, fix nitrogen and promote the growth of important agricultural crops such as corn and canola. For this I inoculated corn and canola seeds with P. polymyxa P2b-2R and grew them for 30 and 60 days, respectively. Corn seedlings were harvested 10, 20 and 30 days after inoculation and canola seedlings were harvested 20, 40 and 60 days after inoculation for evaluation of rhizospheric and internal tissue (endophytic) colonization (cfu). Seedlings were also evaluated for biological nitrogen fixation (15N dilution) and growth promotion (length and biomass) at these harvest intervals. The entire experiment was repeated to confirm the treatment effects. P2b-2R successfully colonized the corn rhizosphere with 5.52 x 106 cfu/g dry root and the canola rhizosphere with 1.08 x 108 cfu/g dry root. P2b-2R also colonized internal tissues of corn and canola root with population densities of 4.54 x 105 cfu/g fresh weight and 2.36 x 103 cfu/g fresh weight, respectively. Corn seedling growth was promoted significantly by inoculation with P2b-2R with an increase of 35% in length and 31% in biomass, 30 days after inoculation. Similarly, canola seedling length was promoted by 25% and biomass by 30%, 60 days after inoculation. Corn seedlings inoculated with P2b-2R were able to derive about 17% of foliar nitrogen from the atmosphere, 30 days after inoculation. Similarly canola seedlings derived nearly 20% of foliar nitrogen from atmosphere, 60 days after inoculation. These results clearly suggest that Paenibacillus polymyxa P2b-2R might have a broad range host capability and is able to fix nitrogen and promote the growth of at least certain important agricultural crops – corn and canola.  iii Preface This thesis represents original, unpublished work by the author, Akshit Puri. I was the lead investigator for the projects described in Chapter 2 and 3 where I was responsible for all major areas of research question formation, research work, data collection, data analysis, and thesis composition. Dr. Maja Kržić, Dr. Sue Grayston and Dr. James Kronstad 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 and canola 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 ....................................................................................................... vi  List of Figures ..................................................................................................... vii  List of Abbreviations ............................................................................................ ix  Acknowledgements .............................................................................................. x Dedication……………………………………………………………………………….xi Chapter 1 – General Introduction ......................................................................... 1      1.1. Plant growth promoting bacteria……………………………………..……...1  1.1.1. Plant growth promoting rhizobacteria (PGPR)……………………...2      1.2. Bacterial endophytes…………………………………………………………..3  1.2.1. Diazotrophic endophytes……………………………………………….5      1.3. Paenibacillaceae (formerly known as Bacillaceae)..……………………….7  1.3.1. Paenibacillus polymyxa…………………………………………………9       1.3.1.1. Paenibacillus polymyxa P2b-2R: a diazotrophic endophyte..10      1.4. Corn and canola……………………………………………………………...12      1.5. Research objectives and hypotheses..……………………………………14  1.5.1. Research objectives…………………………………………………...14  1.5.2. Hypotheses………………………………………………………..……14 Chapter 2 – Evidence of Nitrogen Fixation and Growth Promotion in Corn Inoculated with Diazotrophic P. polymyxa P2b-2R………………………………...15      2.1. Introduction…………………………………………………………………….15      2.2. Materials and methods………………………………………...……….…….18  2.2.1. Seed and bacteria……………………………………………………...18  2.2.2. Seed inoculation and plant growth……………………………………18  2.2.3. Evaluation of endophytic colonization…………………………..……20  2.2.4. Evaluation of rhizospheric colonization………………………………20  2.2.5. Nitrogen analysis and seedling biomass…………………………….21  v  2.2.6. Statistical analysis……………………..………………………………22      2.3. Results………………………………………..……………………………..…22  2.3.1. Endophytic and rhizospheric Colonization………………………….22  2.3.2. Plant growth promotion……………………………………………….23  2.3.3. Nitrogen fixation……………………………………………………….25      2.4. Discussion…………………….…...……………….………………………….28      2.5. Conclusions………………………....……………..………………………….33 Chapter 3 – Growth Promotion and N2-Fixation in Canola (Brassica napus L.) Colonized by an Endophytic Diazotroph Paenibacillus polymyxa P2b-2R………35      3.1. Introduction…………………………………………………………………….35      3.2. Materials and methods………………………………………...……….…….38  3.2.1. Seed and microorganism……………………………………………...38  3.2.2. Seedling growth and inoculation……………………………………...38  3.2.3. Quantification of rhizospheric colonization……………………..…...40  3.2.4. Quantification of endophytic colonization……………………….…...40  3.2.5. 15N analysis and seedling growth promotion…………………........41  3.2.6. Statistical analysis……………………………………………………..42      3.3. Results…………………………………………………………………………42  3.3.1. Rhizospheric and endophytic colonization by P2b-2R……………..42  3.3.2. Growth promotion and N- fixation…………………………………....43      3.4. Discussion……………………...……………………………………………...49      3.5. Conclusions…………………………………………………………………...54 Chapter 4 – General Summary and Conclusions…………………………………..56 References……………………………………………………………………………..61 Appendix A – Plant Nutrient Solution………………………………………………..74  Appendix B – Steps to Prepare Combined Carbon Medium (CCM)……………..75 Appendix C – Recipe to Make Phosphate Buffered Saline……………………….76 Appendix D – Additional Figures……………………………………………………..77   vi List of Tables  Table 1.1 Studies documenting plant growth promotion by Paenibacillaceae  isolates……………………………………………………………………………………8  Table 2.1 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and phosphate buffered saline (control) measured over 3 harvests in growth trial 1…………………………………………………..…………………….26  Table 2.2 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and phosphate buffered saline (control) measured over 3 harvests in growth trial 2…………………………………………………..…………………….27  Table 3.1 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa P2b-2R and phosphate buffered saline (control) measured 20, 40 & 60 days after inoculation (dai) in growth trial 1…………………………………………………..…47  Table 3.2 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa P2b-2R and phosphate buffered saline (control) measured 20, 40 & 60 days after inoculation (dai) in growth trial 2…………………………………………………..…48  vii  List of Figures  Figure 2.1 Shoot, root and seedling length of corn seedlings harvested thrice at 10, 20 and 30 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control)…...……………………………..……………………………………………....23  Figure 2.2 Shoot, root and seedling length of corn seedlings harvested thrice at 10, 20 and 30 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).………………………………………………………………………………....24  Figure 2.3 Shoot, root and seedling dry weight of corn seedlings harvested 10, 20 and 30 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).………………………………………………...............................................24  Figure 2.4 Shoot, root and seedling dry weight of corn seedlings harvested 10, 20 and 30 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).…………………………………………………………………………………25  Figure 3.1 Seedling, root and shoot length of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).…………………………………………………………………………….…...44  Figure 3.2 Seedling, root and shoot length of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).………………………………………………………………………………....45  Figure 3.3 Seedling, root and shoot dry weight of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control)…………………………………….…………………………..........................45  Figure 3.4 Seedling, root and shoot dry weight of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).………………………………………………………………………………....46  Figure 3.5 Number of canola seedlings having floral buds (reached reproductive stage) of each treatment measured 40 and 60 days after inoculation (dai) in two  growth trials (n=14)…………………………………………………………………….46   viii  Figure D.1 P2b-2R-inoculated (left) and control (right) seedling of corn plant harvested 10 days after inoculation (dai) showing clear difference in length,  biomass and plant health……………………………………………………………..77  Figure D.2 P2b-2R-inoculated (right) and control (left) seedling of corn plant harvested 20 days after inoculation (dai) showing clear difference in length,  biomass and plant health……………………………………………………………..77  Figure D.3 P2b-2R-inoculated (left) and control (right) seedling of corn plant harvested 30 days after inoculation (dai) showing clear difference in length,  biomass and plant health……………………………………………………………..78  Figure D.4 P2b-2R-inoculated (left) and control (right) seedling of canola plant harvested 20 days after inoculation (dai) showing clear difference in length,  biomass and plant health……………………………………………………………..78  Figure D.5 P2b-2R-inoculated (left) and control (right) seedling of canola plant harvested 40 days after inoculation (dai) showing clear difference in length,  biomass and plant health……………………………………………………………..79  Figure D.6 P2b-2R-inoculated (right) and control (left) seedling of canola plant harvested 60 days after inoculation (dai) showing clear difference in length, biomass and plant health……………………………………………………………..79  ix  List of Abbreviations ANOVA Analysis of Variance BNF  Biological Nitrogen Fixation C  Carbon CCM  Combined Carbon Medium CCMA Combined Carbon Medium Agar cfu  colony forming units dai  days after inoculation gfp  green fluorescent protein ISR  Induced Systemic Resistance N/N2  Nitrogen Ndfa  Nitrogen derived from atmosphere NF  Nitrogen Fixation PBS  Phosphate Buffered Saline PGPB  Plant Growth Promoting Bacteria PGPR  Plant Growth Promoting Rhizobacteria Pi  inorganic Phosphorous rrs  Ribosomal RNA 16S SAR  Systemic Acquired Resistance SBPS  Sub-boreal Pine Spruce TSA  Tryptic Soy Agar UBC  University of British Columbia  x  Acknowledgments I would like to start by acknowledging my research supervisor, Dr. Chris Chanway, for providing financial support and able guidance. I would also like to extend my thanks 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 owe special thanks to Dr. Alice Chang, for her contribution in analysis of 15N plant samples and providing great support in the lab work. A huge and sincere thanks to Henry Yang for mentoring me and teaching me microbiological techniques. I am also thankful to Dr. Les Lavkulich and Dr. Sandra Brown for helpful conversations and insights along the way. I am also grateful 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 would also like to appreciate the support provided by Dr. Pia Smets in making growth chamber facilities available for this research work. I owe special thanks to my dear friend Kiran Preet Padda for inspiration, ideas, encouragement and endless support. I would like to thank my parents for believing in my capabilities and supporting me in this undertaking. Last but not the least, I would like to appreciate the contribution provided by my friends and those who kept me in their prayers.  Akshit Puri xi  Dedication  To My Father  Late Mr. Darshan K. Puri (Dec 25, 1956 – Nov 28, 2014)   From carrying me on your shoulders when I was a kid to making me capable enough that I have reached here in my life.  Hope I have done you proud Papa… 1  Chapter 1 – General Introduction 1.1. Plant growth promoting bacteria   Soil is replete with microscopic life forms including bacteria, fungi, protozoa, and algae. Of these different microorganisms, bacteria are by far the most common (i.e., 95%) (Glick, 2012). Both the number and the type of bacteria that are found in different soils are influenced by the soil conditions including temperature, moisture, and the presence of salt and other chemicals as well as by the number and types of plants found in those soils (Glick et al., 1999). The interaction between soil bacteria and plants may be (from the perspective of the plant) beneficial, harmful, or neutral (Lynch, 1990). However, the effect that a particular bacterium has on a plant may change as the conditions change. For example, a bacterium that facilitates plant growth by providing either fixed nitrogen or phosphorus compounds that are often present in only limited amounts in many soils, is unlikely to provide any benefit to plants when significant amounts of chemical fertilizer is added to the soil. The bacteria that can promote plant growth, that is plant growth promoting bacteria (PGPB), include those that are free-living, those that form specific symbiotic relationships with plants (e.g., Rhizobia spp. and Frankia spp.), bacterial endophytes that can colonize some or a portion of a plant’s interior tissues, and cyanobacteria (formerly called blue-green algae) (Glick, 2012). The use of microorganisms with the aim of improving nutrient availability for plants is an important practice and necessary for agriculture (Freitas et al., 2007).   2  1.1.1. Plant growth promoting rhizobacteria (PGPR)  Hiltner (1904) discovered that the rhizosphere, i.e., the layer of soil influenced by the root, is much richer in bacteria than the surrounding bulk soil. These rhizosphere microbes benefit because plant roots secrete metabolites that can be utilized as nutrients. This rhizosphere effect is caused by the fact that a substantial amount of the carbon fixed by the plant, 5–21%, is secreted, mainly as root exudate (Marschner, 1995). The term plant growth promoting rhizobacteria (PGPR) was elaborated by Kloepper & Schroth (1978) and used to designate the rhizobacteria showing significant plant growth promotion, as shown with the substantial increases in fresh matter yield obtained with inoculated radishes. Plant growth promoting rhizobacteria (PGPR) represent a wide variety of soil bacteria which, when grown in association with a host plant, result in stimulation of growth of their host (Vessey, 2003). During the past couple of decades, the use of PGPR for sustainable agriculture has increased tremendously in various parts of the world. Significant increases in growth and yield of agronomically important crops in response to inoculation with PGPR have been repeatedly reported (Figueiredo et al., 2011). There are several PGPR inoculants currently commercialized that seem to promote growth through at least one mechanism: suppression of plant disease (bioprotectants), improved nutrient acquisition (biofertilizers), or phytohormone production (biostimulants). Bacteria in the genera Bacillus, Streptomyces, Pseudomonas, Burkholderia, and Agrobacterium are the biological control agents predominantly studied and increasingly marketed. They suppress plant disease through at least one  3  mechanism such as production of antibiotics or induction of systemic resistance (Tenuta, 2003).   The means by which PGPR enhance the nutrient status of host plants can be categorized into five areas: (1) biological N2 fixation, (2) increasing the availability of nutrients in the rhizosphere, (3) inducing increases in the root surface area, (4) enhancing other beneficial symbioses of the host, and (5) a combination of the previously mentioned modes of action (Vessey, 2003). There is also renewed interest in biological N2 fixation. The most studied and longest exploited PGPR include the rhizobia, such as Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium for their ability to fix nitrogen in their legume hosts. However, PGPR usually refer to any of number of free-living bacteria, for example Azospirillum spp., that are also able to fix nitrogen and provide it to plants whose rhizosphere they have colonized (Bashan & Levanony, 1990). 1.2. Bacterial endophytes   Endophytic bacteria have been defined as ‘bacteria that live within living plant tissues without doing substantive harm or gaining benefit other than securing residency’. Endophytic bacteria colonize an ecological niche similar to that of plant pathogens, especially vascular wilt pathogens, which might favor them as potential candidates for biocontrol agents and plant growth promotion agents (Bressan & Borges, 2004). In contrast to free-living, rhizosphere or phyllosphere microorganisms, bacterial endophytes are better protected from  4  abiotic stresses such as extreme variations in temperature, pH, nutrient, and water availability as well as biotic stresses such as competition. In addition, bacterial endophytes colonize niches that are more conducive to forming mutualistic relationships with plants through NF, for example, as suggested in sugarcane, corn and other crops (Chanway et al., 2014). Many seeds carry a diversity of bacterial endophytes (Coombs & Franco, 2003; Hallmann et al., 1997). By being seedborne, endophytes assure their presence in new plants. Plants that propagate vegetatively (such as potatoes or sugarcane) can transmit their endophytes to the next generation and would not require the infection process (Rosenblueth & Martínez-Romero, 2006). In order to colonize the plant, some bacteria must find their way through cracks formed at the emergence of lateral roots or at the zone of elongation and differentiation of the root. Dong et al. (2003) showed that cells of Klebsiella sp/ strain Kp342 aggregate at lateral-root junctions of wheat and alfalfa. Similarly, Gluconacetobacter diazotrophicus and Herbaspirillum seropedicae also colonize lateral-root junctions in high numbers (James & Olivares, 1997).  Endophytic bacteria are able to lessen or prevent the deleterious effects of certain pathogenic organisms. The beneficial effects of bacterial endophytes on their host plant appear to occur through similar mechanisms as described for rhizosphere-associated bacteria. It is believed that certain endophytic bacteria trigger a phenomenon known as induced systemic resistance (ISR), which is phenotypically similar to systemic-acquired resistance (SAR). SAR develops when plants successfully activate their defense mechanisms in response to  5  primary infection by a pathogen, notably when the latter induces a hypersensitive reaction through which it becomes limited in a local necrotic lesion of brown desiccated tissue. ISR is effective against different types of pathogens but differs from SAR in that the inducing bacterium does not cause visible symptoms on the host plant (van Loon et al., 1998). 1.2.1. Diazotrophic endophytes  Diazotrophs are the bacteria and archaea that fix atmospheric nitrogen gas into a biologically usable form such as ammonia. In the 1980’s, Brazilian researchers were perplexed by the consistently high yields of field-grown sugarcane, an N-demanding crop, without exogenous N fertilizer application and looked for a microbiological explanation for this apparently anomalous observation. After it was determined that rhizospheric NF 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). It is a small, Gram-negative, aerobic rod like bacterium that can grow on very high concentrations of sugar and has the capability to fix N (>100 nmoles C2H2 ml-1 h-1) at a pH as low as 3.0 (Stephan et al., 1991) and oxygen levels of 4.0 kPa (Reis et al., 1990). Its ability to fix nitrogen is also not affected by the presence of high levels of nitrate (Boddey et al., 1991). The discovery of a bacterium possessing such unique properties adapted to fixing nitrogen under  6  conditions very specific to the interior of a sugarcane plant has encouraged researchers to believe that such systems might be in place for other non-legumes, essentially waiting to be discovered (Anand, 2011).  Döbereiner (1992) introduced the term "endophytic diazotrophic bacteria" in the area of BNF to designate all diazotrophs that survive very poorly in soil but colonize the root interior of graminaceous plants, and fix nitrogen in association with them (Baldani et al., 1998). Since the discovery of diazotrophic endophytes in sugarcane (Saccharum officinarum L.) (Ruschel et al., 1975), several other agriculturally important crop species including rice (Oryza sativa) (Shrestha & Ladha, 1996), maize (Zea mays L.) (Montañez et al., 2009), kallar grass (Leptochloa fusca L.) (Malik et al., 1997) and canola (Brassica napus L.) (DeFreitas & Germida, 1998) have been postulated to receive significant amounts of fixed N2 in this way. Inoculation of rice with the diazotrophic endophyte Azoarcus sp. strain BH72 significantly promoted plant growth (Hurek et al., 1994). In this particular case, growth promotion also occurred with Nif- mutants, indicating that N2 fixation by Azoarcus sp. was apparently not involved in plant growth promotion. Therefore, the authors speculated that the observed plant growth promotion might have been caused by enhanced plant mineral uptake and improved plant water relationship associated with the colonization by Azoarcus. 7  1.3. Paenibacillaceae (formerly known as Bacillaceae)  The genus Paenibacillus was created by Ash et al. (1993) to accommodate the former ‘group 3’ of the genus Bacillus. It comprises over 30 species of facultative anaerobes and endospore-forming, neutrophilic, low G+C Gram-positive bacilli (Lal & Tabachonni, 2009). The name Paenibacillaceae (like that of its type genus, Paenibacillus) is derived from the Latin paene and the Greek Bacillus: “almost” or “nearly” a Bacillus. The name is meant to indicate a similarity between these organisms and Bacillus sensu stricto (Zeigler, 2013). The role of Paenibacillaceae in the environment is an ongoing research focus. Spores are commonly found in soils of all types, as is also the case for the Bacillaceae. Certain species are obligate pathogens of honeybees (Genersch, 2010) or scarab beetles (Pettersson et al., 1999). Others may be adapted to colonize the vertebrate intestinal tract, including that of humans (Hoyles et al., 2012). Perhaps the most frequently reported isolation of Paenibacillaceae, however, is from endophytic and other plant-associated communities. Isolates have been obtained from the interiors of surface-sterilized fruits, seeds, stems, and roots of a variety of wild plants and cultivated crops. Evidence strongly suggests that many of these isolates are actually plant-growth promoting bacteria, and that certain characteristics of these species—including antibiotic production and biofilm formation—play an essential role in this activity (Timmusk et al., 2005). Members of the family Paenibacillaceae (Table 1 and references therein), especially P. polymyxa, are well studied and known for their ability to promote plant growth by different mechanisms.  8  Table 1.1 Studies documenting plant growth promotion by Paenibacillaceae isolates. Bacterium Plant Stress Proposed Mechanism References P. polymyxa  Leptosphaeria maculans Fusaricidin-like cyclic depsipeptides (Beatty & Jensen, 2002) Brevibacillus Sugarcane Curvularia and Fusarium  (Raton et al., 2012) P. polymyxa  Fusarium oxyporum Direct interaction (Dijksterhuis et al., 1999) P. polymyxa Peanut Aspergillus niger Biofilm formation, niche exclusion (Haggag & Timmusk, 2008) P. polymyxa Wheat Fusarium graminearum Enzymatic or antibiotic activity (He et al., 2009) P. polymyxa Wheatgrass, Clover  IAA production (Holl et al., 1988) P. polymyxa Wheat  Cytokinin production (Timmusk et al., 1999) P. polymyxa Tomato Root-knot nematode  (Khan et al., 2008) P. polymyxa Tomato Phytophthora infestans  (Lamsal & Kim, 2013) P. polymyxa Arabidopsis Pseudomonas syringae Volatile compounds (Lee et al., 2012) P. polymyxa  Xanthomonas campestris 1.3 kDa lipopeptide (Mageshwaran et al., 2012) P. polymyxa Wheat Erwinia spp. Polymyxin P (Niu et al., 2013) P. polymyxa Pepper Xanthomonas campestris Volatile compounds (Phi et al., 2010) Paenibacillus sp. Soybean Rhizoctonia bataticola  (Senthilkumar et al., 2009) P. polymyxa, P. lentimorbus Tomato root-knot nematode and Fusarium  (Son et al., 2009) P. polymyxa Arabidopsis Erwinia, drought Induction of plant stress genes (Timmusk et al., 2005) P. polymyxa Arabidopsis Phytophthora palmivora, Pythium aphanidermatum Biofilm formation, niche exclusion (Timmusk et al., 2009) Brevibacillus sp. White clover Zn-toxicity Zn accumulation; IAA stimulation (Vivas et al., 2006) Paenibacillus sp. Locust Penicillium expansum Heat stable 4.5 kDa protein (Zhou et al., 2008) P. polymyxa Sesame Nine fungal pathogens  (Ryu et al., 2006)  9  1.3.1. Paenibacillus polymyxa  Paenibacillus polymyxa (formerly Bacillus polymyxa), a non-pathogenic and endospore-forming Bacillus, is one of the most industrially significant facultative anaerobic bacterium. It occurs naturally in soil, the rhizosphere and roots of crop plants and in marine sediments. During the last two decades, there has been a growing interest in the ecological and biotechnological importance of this genus, particularly due to their limited genomic information. von der Weid et al. (2000) investigated the influence of plant development both at the phenotypic and genotypic levels by P. polymyxa populations naturally occurring in the maize rhizosphere. Their investigation(s) suggested that a more homogeneous P. polymyxa population was present during the middle stages of maize growth (30 and 60 days after sowing) than in the first stage (10 days) and after 90 days of maize growth. The effect of plant cultivar on the degree of genetic diversity of 67 P. polymyxa isolates recovered from the root system of maize planted in a tropical Brazilian soil was evaluated by da Mota et al. (2002). Results revealed a high level of genetic polymorphism among isolates recovered from different cultivars, yielding a total of 54 distinct groups. Guemori-Athmani et al. (2000) demonstrated the nitrogen fixing ability by P. polymyxa. These authors measured nitrogenase activity of some representative isolates of P. polymyxa recovered from Algerian soil by acetylene reduction assay (ARA). Results showed that only 14 of the 23 strains tested were able to reduce acetylene. In another study, P. polymyxa strain RC05 and RC14 isolated from rhizosphere of wild berries, wheat  10  and barley exhibited nitrogenase activity, phosphate solubilisation capability and promoted plant growth when inoculated into wheat and spinach plants (Çakmakçi et al., 2007). Antagonistic activity of P. polymyxa was demonstrated against the nematode Meloidogyne javanica. The inoculation of P. polymyxa alone or together with Rhizobium increased lentil plant growth both in M. javanica-inoculated and non-inoculated plants (Siddiqui et al., 2007).  1.3.1.1 Paenibacillus polymyxa P2b-2R: a diazotrophic endophyte  Lodgepole pine (Pinus contorta var. latifolia (Dougl.) Engelm.) and western red cedar (Thuja plicata Donn.) are common, commercially important gymnosperms in western North America with a wide ecological range from Alaska to California. Lodgepole pine grows successfully on nutrient poor sites and often scorched sites that are severely limited in nitrogen (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 nitrogen availability due to low rates of nitrogen mineralization in cedar forests (Prescott & Preston, 1994; Prescott et al., 1996). Based on earlier work with lodgepole pine suggesting that rhizospheric BNF contributed only small amounts of N to seedlings (Chanway & Holl, 1991), Bal et al. (2012) searched for endophytic diazotrophs in lodgepole pine and western red cedar as a possible explanation for the ability of this species to grow on N-deficient substrates.   11   Bal et al. (2012) isolated several bacterial strains from plant tissues collected from three sampling sites. But only four isolates were able to consistently reduce acetylene and grow on N-free Combined Carbon Medium Agar (CCMA). These diazotrophic strains were 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. One of the strains, Paenibacillus polymyxa strain P2b-2R (GU132543), showed consistent N-fixation with each tree species in each growth trial (Bal & Chanway, 2012a,b). 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 from BNF, respectively and doubled their biomass. In these trials, P. polymyxa P2b-2R successfully colonized internal tissues of root, stem and needle 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. These findings confirm the ability of P. polymyxa P2b-2R to  12  colonize and fix nitrogen in at least certain gymnosperms, lodgepole pine and western red cedar. But can this bacterial strain fix nitrogen if introduced into agricultural crops? The answer is not known and determining it formed the main objective of my thesis project. 1.4. Corn and canola  In Canada, canola production is very important to the agricultural industry. According to Statistics Canada (n.d.), the total seeded area under canola for the year 2014 was 8,225,074 hectares in Canada, which was the second highest just after wheat. The total production of canola in Canada for the year 2013 was also the second highest with 15,555,271 metric tonnes. A study released in 2013 shows that Canadian-grown canola contributes $19.3 billion to the Canadian economy each year, and contributes to more than 249,000 Canadian jobs and $12.5 billion in wages (Canola Council of Canada, n.d. a). Canola is a very popular crop In British Columbia as well. The total seeded area of canola in 2014 was 42,492 ha which was the highest among all crops grown in BC (Statistics Canada, n.d.).   When it comes to cereal crop production around the world, corn is the king with a world production of 990,690,000 metric tonnes in the year 2013-14. Corn is Canada’s third most valuable crop with a booming annual production of 14,190,000 metric tonnes in year 2013-14. Canada annually produces approximately 1.2% of the corn produced globally and is ranked 11th in the world (Hamel & Dorff, 2014; United States Department of Agriculture, 2015).   13   In addition, corn and canola crops differ physiologically as well as botanically. The main physiological difference between corn and canola is that corn belongs to the C4 category of plant species whereas canola belongs to the C3 category. The most important difference between C3 and C4 species is that C3 species continue to increase photosynthesis with rising CO2 level whereas in C4 species, photosynthesis does not. So, C3 plants respond readily to higher CO2 levels, and C4 plants make only limited responses. The main botanical difference between corn and canola is that corn is a monocot whereas canola is a dicot. The main reason for choosing corn and canola for this study was that these crops are agronomically important and very different physiologically and botanically.   Several different studies have observed the ability of endophytic diazotrophs to colonize corn and canola plants under field, greenhouse, and aseptic conditions. Studies have shown that a bacterial endophyte found in sugarcane, G. diazotrophicus is capable of inhabiting several corn genotypes when different means of inoculation were used: seed coating, applications to the base of stems, and root dipping (Riggs et al., 2001; Cocking et al., 2006; Tian et al., 2009). A study found that a Paenibacillus polymyxa strain (PKB1) produces fusaricidin-type antifungal antibiotics which are very active against Leptosphaeria maculans (the causative agent of blackleg disease of canola) thus enhancing canola plant growth (Beatty & Jensen, 2002). Lifshitz et al. (1987) and Kloepper et al. (1988) also found that many PGPR like Pseudomonas putida strains are capable of growth promotion of canola.  14  1.5. Research objectives and hypotheses 1.5.1. Research objectives  The main research objectives of this project were to determine if Paenibacillus polymyxa strain P2b-2R is capable of: (i) colonizing rhizosphere and internal tissues of Zea mays (corn) and Brassica napus L. (canola), (ii) deriving nitrogen from atmosphere in association with these crop plants and, (iii) increasing biomass production and growth rate of Zea mays and Brassica napus L. 1.5.2. Hypotheses I developed three main hypotheses: H1: Paenibacillus polymyxa P2b-2R is capable of colonizing rhizosphere and internal tissues (endophytically) of the important agricultural crops, corn and canola. H2: P. polymyxa P2b-2R is capable of supplying a biologically significant amount of fixed nitrogen to colonized plants grown under nitrogen poor conditions. H3: P2b-2R inoculation enhances biomass production (dry weight) and growth rate of corn and canola seedlings.  15  Chapter 2 – Evidence of Nitrogen Fixation & Growth Promotion in Corn Inoculated with Diazotrophic P. polymyxa P2b-2R1 2.1. Introduction  For plants, nitrogen is necessary as a primary constituent of nucleotides, proteins, and chlorophyll (Robertson & Vitousek, 2009). Many agricultural scientists see the availability of fixed nitrogen (nitrate or ammonium converted from dinitrogen) as the most yield-limiting factor related to the crop production (Muthukumarasamy et al., 2002). An inexpensive and natural way of providing plants with fixed nitrogen is by biological nitrogen fixation (BNF). Approximately 80% of all BNF is accomplished through the symbiotic interaction between legumes and α-proteobacteria in the order Rhizobiales, family Rhizobiaceae (Garg & Geetanjali, 2007). However, non-specific nitrogen-fixing bacteria also exist and their discovery has opened up the possibility of symbiotic nitrogen fixation in a wide array of agricultural crops like corn.  Endophytic bacteria have been defined as ‘bacteria that live within living plant tissues without doing substantive harm or gaining benefit other than securing residency’ (Bressan & Borges, 2004). In contrast to free-living, rhizosphere or phyllosphere-dwelling microorganisms, bacterial endophytes are better protected from abiotic stresses such as extreme variations in temperature,                                                         1 A version of this chapter has been submitted to Soil Biology and Biochemistry Journal for publication under the title ‘Can a diazotrophic endophyte originally isolated from lodgepole pine colonize an agricultural crop (corn) and promote its growth?’ Authors: Akshit Puri, Kiran Preet Padda and Chris P Chanway and it is currently under review.  16  pH, nutrient, and water availability as well as biotic stresses such as competition (Chanway et al., 2014). Diazotrophs comprise certain bacteria and archaea that fix atmospheric nitrogen into a biologically usable form such as ammonia. The term "endophytic diazotrophic bacteria" was introduced in the area of BNF by Döbereiner (1992) to designate all diazotrophs able to colonize primarily the root interior of graminaceous plants, survive very poorly in soil and fix nitrogen in association with these plants. Since the discovery of diazotrophic endophytes in sugarcane (Saccharum officinarum L.) (Ruschel et al., 1975), several other agriculturally important crop species including rice (Oryza sativa) (Shrestha & Ladha, 1996) and maize (Zea mays L.) (Montañez et al., 2009) have been postulated to receive significant amounts of fixed N2 in this way.  Paenibacillus polymyxa is a Gram-positive, rod-shaped, endospore-forming bacterium that is non-pathogenic and found in environments such as plant roots in soil and marine sediments (Timmusk et al., 2005; Ravi et al., 2007; He et al., 2007). The wide range of potential plant growth promoting characteristics this bacterium possess includes the ability to fix nitrogen and produce hormones that promote plant growth as well as hydrolytic enzymes and antibiotics that protect against harmful microorganisms (Lal & Tabacchioni, 2009). Górska et al. (2015) isolated two nitrogen-fixing microorganisms from agricultural soil and identified them as Paenibacillus polymyxa [laboratory names EG2 and EG14] based on 16S rRNA sequence. The genome of these bacterial strains was found to carry nif genes coding the individual components of the  17  nitrogenase complex. Their nitrogen fixing ability was confirmed by studying nitrogenase activity in cultures of the studied bacteria in N-free medium.  Bal et al. (2012) isolated several Paenibacillus strains that possessed significant acetylene reduction activity from extracts of surface-sterilized lodgepole pine seedling and tree tissues (Bal & Chanway, 2012a,b). When one of the strains, Paenibacillus polymyxa strain P2b-2R, was reintroduced to lodgepole pine and grown in a very N-limited soil for 35 weeks, seedlings were found to derive more than half (66%) of their foliar N from biological nitrogen fixation (BNF) (Bal & Chanway, 2012a). In a long-term experiment (13-month growth trial), P2b-2R showed even better results as the inoculated lodgepole pine seedlings derived 79% of their foliar N from BNF and doubled their biomass (Anand et al., 2013). These results provided confirmation of the ability of P. polymyxa strain P2b-2R to colonize gymnosperms (lodgepole pine and western red cedar) and fix nitrogen when associated with them. However, we were interested in determining if this bacterial strain could also fix nitrogen and promote plant growth if introduced into an agricultural crop. I chose corn for our study, as it is an important agricultural crop grown on a large scale worldwide.  In this study, I test the hypothesis that the endophytic diazotroph P. polymyxa P2b-2R isolated from lodgepole pine is capable of colonizing rhizosphere and internal tissues of an important agricultural crop, corn (Zea Mays), and stimulating biomass production through BNF.    18  2.2. Materials and methods 2.2.1. Seed and bacteria  Corn seeds (var. Golden Bantam) were obtained from West Coast Seeds (Delta, British Columbia, Canada). Paenibacillus 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 et al., 2012). Paenibacillus polymyxa P2b-2R (GU132543) was a spontaneous antibiotic-resistant derivative of strain P2b, capable of growing on combined carbon medium (CCM) (Rennie, 1981) agar amended with 200mg/L rifamycin (Bal & Chanway, 2012a). Strain P2b-2R was stored at -80oC in CCM amended with 20% (v/v) glycerol. 2.2.2. Seed inoculation and plant growth  Seedling growth assays were performed in small pots (12cmx8cmx4cm) filled to 67% capacity with sterile Sand-Turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, IL, USA) mixture (69% w/w silica sand; 29% w/w Turface; 2% w/w CaCO3). Each pot was fertilized with 50mL of a nutrient solution (Appendix A) (Chanway et al., 1988), which was 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, ON, Canada) with Na2-FeEDTA (0.02g/L).  19   Corn seeds were surface-sterilized by immersion in 30% hydrogen peroxide for 90 seconds, followed by three 30-second rinses in sterile distilled water. To confirm the effectiveness of surface sterilization, seeds were imprinted on tryptic soy agar (TSA) and checked for microbial contamination 24h later. Two surface sterilized seeds were aseptically sown in each pot.  Two seed inoculation treatments – live P2b-2R and sterile PBS (Control) were evaluated using corn seedlings, replicated 60 times. Bacterial inoculum was prepared by thawing a frozen culture of strain P2b-2R and streaking a loopful onto combined carbon medium (CCM) agar amended with 200mg/L rifamycin, and incubating at 30oC for 2 days. A 1 L flask containing 500mL of CCM broth amended with rifamycin was then inoculated with a loopful of bacterial growth from the agar plate, secured on a rotary shaker and agitated (150 rpm) at room temperature for 2 days. Bacterial cells were harvested by centrifugation (3000x g, 30 min), washed twice in sterile phosphate buffered saline (PBS) (pH 7.4) and resuspended in the same buffer to a density of 106cfu/mL. Immediately after sowing the seeds, 5mL of the P2b-2R – PBS suspension was pipetted directly into each pot designated for live P2b-2R. This process was repeated using 5mL of sterile PBS without bacteria for uninoculated (control) pots. 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 300µmol s–1 m–2 and a 25/18oC day/night temperature cycle. Seedlings were thinned to the largest single germinant per pot 2 days after sowing and were watered as required with sterile distilled water. Seedlings received modified  20  nutrient solution without Ca(15NO3)2, 20 days after inoculation. The entire experiment was repeated to confirm treatment effects. 2.2.3. Evaluation of endophytic colonization  To evaluate endophytic colonization by P2b-2R in corn, 3 randomly chosen seedlings of each treatment were harvested destructively 10, 20 and 30 days after inoculation. Seedlings were rinsed in a 2L flask containing 1L sterile distilled water for removal of loosely adhering growth media. In the first growth trial, seedlings were surface-sterilized in 1.3% (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 stem, root and leaf tissues were triturated separately in 1mL of sterile PBS using a mortar and pestle. Triturated tissue suspensions were serially diluted on CCM plates supplemented with 100mg/L cycloheximide and 200mg/L rifamycin. Plates were incubated at room temperature for 7 days and colonies were counted after incubation. 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 by P2b-2R, 3 randomly chosen seedlings of each treatment were harvested destructively 10, 20 and 30 days after inoculation. Seedlings were removed from pots and loosely adhering soil particles were removed from roots with gentle shaking. Roots were then  21  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 1000 rpm for 1 minute. Serial dilutions were performed, and aliquots of 100μL were plated on CCM plates 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 dry root. 2.2.5. Nitrogen analysis and seedling biomass  Fourteen corn seedlings of each treatment were harvested destructively 10, 20 and 30 days after inoculation for evaluation of seedling biomass as well as foliar N and 15N content. Roots were separated from shoots, and shoot and root lengths were measured. Shoots and roots of each seedling were then oven dried at 65 oC 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 a 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%  22  2.2.6. Statistical analysis  A completely randomized experimental design with 60 replicates per treatment was used to assess the treatment effects on growth of corn seedlings. The statistical package, SAS v9.4 (Copyright © 2014, SAS Institute Inc., Cary, NC, USA.), was used to perform statistical analyses. A linear generalized model was used to analyze the correlations between the dependent variables viz. plant growth parameters, number of CFUs, and atom percent excess of 15N and independent variable, N. 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  Paenibacillus polymyxa strain P2b-2R colonized the corn rhizosphere in both growth trials with 2.18 x 106 cfu/g dry root and 8.85× 106 cfu/g dry root, respectively. P2b-2R colonies were also observed on both CCM and TSA stem and leaf imprint plates after these tissues were surface sterilized. P2b-2R colonized corn root endophytically with 4.54 x 105 cfu/g fresh weight in the 2nd growth trial but there was no evidence of endophytic colonization in stem and leaf tissues by P2b-2R strain in either growth trial. No evidence of rhizospheric or endophytic colonization was found in control plants.  23  2.3.2. Plant growth promotion  Seedling growth was promoted significantly by inoculation with P2b-2R. P2b-2R inoculation increased shoot length by 19-40% in the 1st growth trial (Figure 2.1) and 21-45% in the 2nd growth trial (Figure 2.2). Seedling length was promoted by 6-25% in the 1st trial (Figure 2.1) and 10-35% in the 2nd trial due to P2b-2R inoculation (Figure 2.2). P2b-2R inoculation significantly increased shoot dry weight (24-33% in the 1st trial (Figure 2.3) and 27-38% in the 2nd trial (Figure 2.4)) and seedling biomass (18-26% in the 1st trial (Figure 2.3) and 20-30% in the 2nd trial (Figure 2.4)).  Figure 2.1 Shoot, root and seedling length of corn seedlings harvested thrice at 10, 20 and 30 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control). 020406080100120SeedlingRootShootSeedlingRootShootSeedlingRootShoot10 dai 20 dai 30 daiLength (cm)P2b-2R Control****** 24   Figure 2.2 Shoot, root and seedling length of corn seedlings harvested thrice at 10, 20 and 30 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).   Figure 2.3 Shoot, root and seedling dry weight of corn seedlings harvested 10, 20 and 30 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control). 020406080100120SeedlingRootShootSeedlingRootShootSeedlingRootShoot10 dai 20 dai 30 daiLength (cm)P2b-2R Control******01002003004005006007008009001000SeedlingRootShootSeedlingRootShootSeedlingRootShoot10 dai 20 dai 30 daiDry weight (mg)P2b-2R Control****** 25   Figure 2.4 Shoot, root and seedling dry weight of corn seedlings harvested 10, 20 and 30 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control). 2.3.3. Nitrogen fixation  In the first growth trial, % foliar N of seedlings inoculated with P2b-2R was 5, 8, and 10% higher than the control seedlings in the 1st, 2nd and 3rd harvests, respectively (Table 2.1). Similarly in the second growth trial, % foliar N of inoculated seedlings was 9, 14, and 19% higher than the control in the 1st, 2nd and 3rd harvest, respectively (Table 2.2). Based on % foliar 15N atom excess data, it was found that inoculated seedlings derived 5% of the foliar N from atmosphere (Ndfa) in the 1st harvest, which increased to 8% in the 2nd harvest and 15% in the 3rd harvest in first growth trial (Table 2.1). A similar trend was observed in the second growth trial, as inoculated seedlings derived 6% of the foliar N from atmosphere in the 1st harvest, which increased to 10% in the 2nd harvest and 19% in the 3rd harvest (Table 2.2). 01002003004005006007008009001000SeedlingRootShootSeedlingRootShootSeedlingRootShoot10 dai 20 dai 30 daiDry weight (mg)P2b-2R Control******* 26  Table 2.1 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and phosphate buffered saline (control) measured over 3 harvests in growth trial 1   Harvest 1   Harvest 2   Harvest 3   Atom %15N Excess in Foliage %Foliar N %Ndfa   Atom %15N Excess in Foliage %Foliar N %Ndfa   Atom %15N Excess in Foliage %Foliar N %Ndfa P2b-2R 0.41*±0.006a 1.60*±0.05 5.18  0.39*±0.003 1.78*±0.02 8.25  0.38*±0.006 1.97*±0.01 15.35 Control 0.43±0.005 1.52±0.06 -   0.42±0.009 1.65±0.05 -   0.45±0.007  1.80±0.05 - a Mean ± standard error; n=14 for atom %15N excess & %foliar N *P<0.05 (significantly different from control)   27  Table 2.2 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from corn seeds inoculated with P. polymyxa strain P2b-2R and phosphate buffered saline (control) measured over 3 harvests in growth trial 2   Harvest 1   Harvest 2   Harvest 3   Atom %15N Excess in Foliage %Foliar N %Ndfa   Atom %15N Excess in Foliage %Foliar N %Ndfa   Atom %15N Excess in Foliage %Foliar N %Ndfa P2b-2R 0.40*±0.005a 1.75*±0.05 6.65  0.39*±0.014 1.84*±0.04 10.78  0.33*±0.005 2.06*±0.02 19.62 Control 0.43±0.006  1.66±0.05 -   0.43±0.006  1.70±0.05 -   0.41±0.006  1.87±0.01 - a Mean ± standard error; n=14 for atom %15N excess & %foliar N *P<0.05 (significantly different from control)  28  2.4. Discussion  The objective of this study was to determine if an endophytic diazotroph P. polymyxa P2b-2R isolated from internal tissues of lodgepole pine is capable of colonizing rhizosphere and internal tissues of corn. We were also interested to see if it is capable of promoting growth and fixing nitrogen in association with this crop plant. P2b-2R colonized the corn rhizosphere with population sizes similar to those observed previously in gymnosperms (Holl & Chanway, 1992; Bal & Chanway, 2012a,b). This shows that P2b-2R possess the necessary adaptations for rhizosphere colonization of this agricultural crop despite being isolated from internal tissues of a tree species. Internal tissue (endophytic) colonization of corn roots was observed in the second growth trial with population densities similar to those observed in other published reports of this bacterial strain (Anand et al., 2013; Anand & Chanway, 2013a). This strain was also present on CCM and TSA, stem and leaf imprint plates and thus we feel it would be premature to rule out endophytic colonization of above ground parts of corn by this strain. The native environment of P. polymyxa P2b-2R is the plant interior (Bal et al., 2012) and it was previously reported to colonize stem and leaf tissues of the plant with higher population densities than the root tissues (Anand et al., 2013; Anand & Chanway, 2013a). Thus, we expected this strain to colonize extracts of surface sterilized stem and leaf tissues of corn with high population densities, but this was not the case. The comparatively harsh protocol that was required to ensure complete surface sterilization of seedling samples may have disinfected internal  29  cortical tissues and precluded the detection of this bacterial strain inside stem and leaf tissues. Future studies utilizing a direct detection technique such as gfp- labeled bacteria (Gage et al., 1996) with confocal scanning laser microscopy will facilitate more sensitive studies of endophytic colonization by this strain (Anand & Chanway, 2013b).  Paenibacillus polymyxa P2b-2R showed consistent and significant effect on corn foliar 15N abundance. Based on atom % 15N excess in foliage data, seedlings treated with live P2b-2R derived 15% of foliar N at the end of the 1st growth trial (Table 2.1) and 19% at the end of the 2nd growth trial (Table 2.2) from a source other than soil N, likely the atmosphere. The proportion of fixed N assimilated in inoculated corn seedlings was lower than that observed in sugar cane (Saccharum officinarum L.) (Lima et al., 1987; Boddey et al., 1991), kallar grass (Leptochloa fusca) (Malik et al., 1987), and rice (Oryza sativa L.) (Malik et al., 1997), but were consistent with estimates from wheat (Triticum aestivum) (Rennie & Larson, 1979; Rennie et al., 1983) and western red cedar (Thuja plicata) (Bal & Chanway, 2012b). The amount of nitrogen fixed in P2b-2R inoculated plants was significant considering the fact that this was a short growth trial. The percent nitrogen derived from atmosphere increased at each harvest in both the growth trials (Table 2.1 and 2.2) suggesting that BNF is an important component of N nutrition of corn growing in a nitrogen limited soil medium. But the source of this BNF is still unknown and it could be speculated that it is induced by either rhizospheric P2b-2R or endophytic P2b-2R found in root tissues. Significant foliar 15N dilution with no concomitant increase in foliar N  30  concentration during nitrogen fixation has been observed previously and is thought to be due to a compensatory mechanism where less N is taken up from soil when fixed N is assimilated by plants (Rennie et al., 1983; Rennie & Thomas, 1987; Kucey, 1988). Uriquaga et al. (1992) previously observed an increasing reliance on BNF with seedling age and concomitant decreasing dependence on soil N in sugarcane, as %Ndfa rose from 6 to 55 % during the interval of 100-250 days after emergence.   Werner et al. (2014) recently reported that Zea mays (Corn) does not belong to a phylogenetic clade of angiosperms with an N-fixing precursor. They also reported that species that are in the precursor state are roughly 100 times more likely to evolve a functioning N2-fixation state than a non-precursor is to evolve a precursor. But interestingly, Paenibacillus polymyxa and other diazotrophs have been reported to form a symbiotic relation with corn in many reports. Crops like corn and sugarcane have high sucrose content and sucrose is postulated as an important factor to the colonization success of many diazotrophic species (Muthukumarasamy et al., 2002). High sucrose concentrations (10%) are the best source of carbon for the bacterium’s growth (Cavalcante & Dobereiner, 1988). Additionally, when looking specifically at the process of nitrogen fixation, sucrose levels play an invaluable role, by providing the bacterium with a sufficient source of energy for the process of nitrogen fixation (Galar & Boiardi, 1995). It was recently reported that P. polymyxa P2b-2R could readily oxidize about 39 different carbon substrates, out of which ‘carbohydrates’ group represent the largest source of utilizable carbon. These  31  carbon sources are found naturally in plants as freely available forms such as sucrose, raffinose and maltose (Yang, 2015). Thus, it is possible that P. polymyxa strain P2b-2R interacts mutualistically with corn by supplying biologically significant amounts of fixed N to its plant host in return for nutrient rich colonization sites on or in the plant.  P2b-2R inoculation of corn seedlings increased shoot length by 40% in the 1st growth trial and 45% in the 2nd growth trial, 30 days after inoculation. Seedling length was also increased significantly (25% in the 1st trial and 35% in the 2nd trial), 30 days after inoculation. This indicates that P2b-2R inoculation enhances the vertical growth of corn seedlings especially above ground, which is consistent with a previous report of this bacterial strain (Anand et al., 2013). P2b-2R also enhanced biomass production in corn seedlings, particularly the above ground biomass. Above ground biomass of P2b-2R inoculated seedlings was 33-38% greater than the control seedlings. The significant difference in shoot length and shoot dry weight between P2b-2R inoculated and control seedlings suggests that P2b-2R promotes plant growth both vertically (length) as well as horizontally (biomass), unlike some previous reports about this bacterial strain using lodgepole pine and western red cedar seedlings (Bal & Chanway, 2012a,b). While P. polymyxa possess several characteristics that can result in plant growth promotion (Chanway, 2008), foliar N data (Table 2.1 and 2.2) suggests that the enhanced growth promotion was caused primarily by an increase in the amount of N derived from atmosphere. The concentration of foliar N increased  32  consistently during both growth trials (Table 2.1 and 2.2) leading to a consistent increase in biomass and length of seedlings.  There are many ways in which Paenibacillus polymyxa P2b-2R might have promoted plant growth in corn. One of the most commonly reported plant growth promoting mechanism of Paenibacillus is the production of substances like auxins, cytokinins and/or gibberellins by this bacterium. Bacillus amyloliquefaciens FZB42, a model strain for scientific research was reported to be involved in auxin biosynthesis, leading to the re-modeling of Pi transporter expression as well as elevating organic C exudation in wheat (Triticum aestivum). This shows the importance of rhizobacterial-derived auxin following colonization of root surfaces by a PGPR (Talboys et al., 2014). Another way by which PGPR bacteria can stimulate plant growth is by suppressing endogenous ethylene production in the host plant’s rhizosphere and root system. Various Paenibacillus species are also responsible for increased nutrient availability in the rhizosphere. For example, phosphorous is often abundant in soils, but in forms mostly unavailable to plants, as part of insoluble or poorly soluble inorganic or organic phosphate pools. Studies have shown that Peanibacillus sp. is capable of phosphate mineralization by producing enzymes, which cleave phosphate groups (Chanway, 2008). Paenibacillus polymyxa is also capable of synthesizing many antibacterial and antifungal secondary metabolites (Borriss, 2015). A study was conducted with Paenibacillus polymyxa HT16 bacterial strain to evaluate its ability to reduce white rot disease caused by the fungus Coniella diplodiella on table grapes. HT16 strain showed significant inhibitory effects on C.  33  diplodiella in vitro, reducing white rot disease in ‘Kyoho’ and ‘Thompson Seedless’ table grape varieties by more than 40% after inoculation (Han, 2015). In another study, P. polymyxa strain Sb3-1 (syn. Paenibacillus kribbensis Sb3-1) isolated from agricultural, organically managed soil in Egypt showed antifungal and antibacterial characteristics in vitro (Köbrel et al., 2013). The genome of this strain revealed several genes that potentially contribute to its antagonistic and plant growth promotion activity (Rybakova et al., 2015). Lei et al. (2015) reported the complete genome sequence of P. polymyxa CF05 strain isolated from the interior of an ancient tree, Cryptomeria fortunei, in China. This bacterial strain displayed potent biocontrol effects against certain soil-borne diseases and the elicitation of induced systemic resistance in tomatoes. 2.5. Conclusions  Paenibacillus polymyxa P2b-2R, a bacterial strain originally isolated from a lodgepole pine tree (gymnosperm) is capable of colonizing rhizosphere and internal tissues of root (endophytically) of an important cereal crop corn (angiosperm). I think that this is an important finding in terms of this bacterial strain’s broad range host capability. This bacterial strain might possess necessary adaptations for root colonization of corn plant but the mechanisms involved in these adaptations are still unknown. P2b-2R was not successful in endophytically colonizing above ground parts of the corn in this study for unknown reasons. Two possibilities are: either the surface sterilization protocol used was too harsh or the bacterium was not able to reach the above ground  34  parts of the plant in such short duration. Corn seedlings inoculated with P2b-2R were successful in deriving nitrogen from atmospheric N pool. The amount of nitrogen fixed was significant considering the fact that this was a short growth trial. But the question is, does this BNF originate from endophytic or rhizospheric P2b-2R? Our results also suggest that P2b-2R inoculation promotes growth (length and biomass) of corn seedlings. A consistent increase in foliar N of seedlings inoculated with P2b-2R is a major contributing reason for growth promotion. Other possibilities include production of plant growth promoting substances, nutrient mineralization and assistance in nutrient uptake by this bacterial strain. Future studies might involve an in-depth investigation of the abilities of P. polymyxa to endophytically colonize above ground parts of corn, especially in a long-term growth trial and to look for possible role of this bacterial strain in nutrient mineralization and uptake.  35  Chapter 3 – Growth Promotion and N2-Fixation in Canola (Brassica napus L.) Colonized by an Endophytic Diazotroph Paenibacillus polymyxa P2b-2R2 3.1. Introduction  Nitrogen is one of the most common nutrients required for plant growth and productivity as it forms an integral part of proteins, nucleic acids and other essential biomolecules (Bøckman, 1997). Almost 80% of our atmosphere is nitrogen, but plants can’t use it. It needs to be converted into ammonia, a form available to plants. Atmospheric nitrogen is converted into forms utilized by plants by three different processes- i) conversion of atmospheric nitrogen into oxides of nitrogen in the atmosphere by lightning, ii) industrial nitrogen fixation using catalysts and high temperature (300-500ºC) to convert nitrogen to ammonia, and iii) biological nitrogen fixation involving the conversion of nitrogen to ammonia by microorganisms using a complex enzyme system identified as nitrogenase (Kim & Rees, 1994). Biological nitrogen fixation represents an economically beneficial and environmentally sound alternative to chemical fertilizers (Ladha et al., 1997). The rhizobia-legume symbiosis is perhaps the most common N2 fixing agent in agricultural systems (Herridge et al., 2008). But unlike nodule-forming bacteria there is another group of bacteria that fixes nitrogen even in non-leguminous plant species. Plant growth-promoting                                                         2  A version of this chapter has been submitted to Biology and Fertility of Soils Journal for publication under the title ‘Plant Growth Promotion and N2-Fixation in Canola (Brassica napus L.) by an Endophytic Diazotroph Paenibacillus polymyxa P2b-2R’ Authors: Akshit Puri, Kiran Preet Padda and Christopher P Chanway and it is currently under review.  36  rhizobacteria that fix nitrogen in non-leguminous plants are known as diazotrophs. They form a non-obligate interaction with the host (Glick et al., 1999).  Endophytic bacteria have been known for more than 120 years (Hardoim et al., 2008). In 1926, Perotti was the first to use the term endophytes to describe non-pathogenic bacteria that had been isolated from within plants, other than rhizobia (Hallmann et al., 1997; Hardoim et al., 2008). In comparison with rhizosphere and rhizoplane bacterial communities, endophytic bacteria are likely to interact more closely with their host plant as they are provided with a readily available source of nutrients and a secure residence inside intercellular spaces of plants (Rosenblueth & Martinez-Romero, 2006; Weyens et al., 2009). In return, the endophytic bacteria could enhance host plant growth and health via various direct and indirect mechanisms. The most important direct growth promotion mechanism is nitrogen fixation.   Döbereiner (1992) introduced the term "endophytic diazotrophic bacteria" in the area of BNF to designate all diazotrophs that survive very poorly in soil but colonize the interior tissues of roots of graminaceous plants, and fix nitrogen in association with them (Baldani et al., 1998). Some examples of endophytic diazotrophs of rice, maize and sugarcane are Azotobacter diazotrophicus, Herbaspirillum seropedicae, Azoarcus spp. and Enterobacter asburiae, which serve as nitrogen fixers when other available sources of nitrogen are absent or at low levels (Reinhold et al., 1986, Döbereniner et al., 1993; Kirchhof et al., 1997).  37   Canola was developed in the early 1970s using traditional plant breeding techniques by Canadian plant breeders to remove the anti-nutritional components (erucic acid and glucosinolates) from rapeseed to assure its safety for human and animal consumption (Soyatech, n.d.). Global canola production has grown rapidly over the past 40 years, rising from the sixth largest oil crop to the second largest. Canola oil, obtained from crushing canola seed, was the third-most-produced vegetable oil globally in 2008/09 (United States Department of Agriculture, n.d.). Canola seeds are 44% oil, more than double the oil content of soybeans. High-protein meal is produced from the other 56% of the canola seed, which is an excellent animal feed for cattle, poultry, swine and fish. When fed to dairy cows, it can increase milk production by one litre per day. Canola meal is the second largest feed meal after soybean meal (Canola Council of Canada, n.d. b).  P. polymyxa P2b-2R was reported to colonize and fix nitrogen in gymnosperms (lodgepole pine and western red cedar) in both short duration and long duration growth trials (Bal & Chanway, 2012a,b; Anand & Chanway, 2013a; Anand et al., 2013). But can this bacterial strain associate with Canola (Brassica napus L.), an important oil crop? The main objectives of this study were to determine if Paenibacillus polymyxa strain P2b-2R is capable of: (i) colonizing rhizosphere and internal tissues of Canola (Brassica napus L.), (ii) fixing N and stimulating growth rate in association with Canola.  38  3.2. Materials and methods 3.2.1. Seed and microorganism  Canola (Brassica Napus L.) seeds (var. Rugby Roundup ready) were obtained from SeCan Association’s Alberta branch (Lamont, Alberta, Canada). Paenibacillus polymyxa strain P2b was isolated from internal tissues of a lodgepole pine seedling stem and needles 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). Paenibacillus polymyxa P2b-2R is a spontaneous antibiotic-resistant mutant that was derived from strain P2b (Bal et al., 2012). P. polymyxa P2b-2R is resistant to 200 mg/L rifamycin and was stored at -80 oC on combined carbon medium (CCM) amended with 20% glycerol (Bal & Chanway, 2012a). 3.2.2. Seedling growth and inoculation  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 industrial sand; 29% w/w Turface; 2% w/w CaCO3). Each pot was fertilized with 50 mL of a sterile nutrient solution (Appendix A) (Chanway et al., 1988), which was modified by replacing KNO3 and Ca(NO3) 4H2O with Ca(15NO3)2 (5% 15N label) (0.0576 g/L) and sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ont., Canada) with Na2-FeEDTA (0.02 g·L–1).   39   Canola seeds were surface-sterilized by immersion in 30% hydrogen peroxide (H2O2) for 1.5 min, followed by three 30-second rinses in sterile distilled water. To confirm the effectiveness of surface sterilization, seeds were imprinted on tryptic soy agar (TSA) and checked for microbial contamination after 2 days. Three surface-sterilized seeds were then aseptically sown in each pot.  Two seed inoculation treatments – live P2b-2R and control (phosphate buffered saline) were evaluated using canola seedlings, with 60 replications per treatment. Bacterial inoculum was prepared by thawing a frozen culture of strain P2b-2R and streaking onto combined carbon medium (CCM) plates amended with 200 mg/L rifamycin. After colonies grew, a loopful of the strain was inoculated into a 1-L flask containing 500 mL of fresh CCM broth amended with rifamycin. Flasks were then secured on a rotary shaker (150 rpm) and agitated for 24 h at room temperature. Bacteria were harvested by centrifugation (3000x g, 30 min) and re-suspended in phosphate buffered saline (PBS) to a density of 106 cfu/mL. For inoculation, 5 mL of the P2b-2R-PBS suspension was pipetted directly into each of 60 replicate pots immediately after sowing canola seeds. This process was repeated using 5 mL of sterile phosphate buffered saline (PBS) without bacteria for control (noninoculated) pots. The pots were placed in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB, Canada) with photosenthetically active radiation at canopy level of 300 µmol s–1 m–2, an 18-h photoperiod and a 25/18 oC day/night temperature cycle. Seedlings were thinned to the largest single germinant per pot 5 days after sowing and were watered as required with sterile distilled water. A modified  40  nutrient solution was provided to each pot (without Ca(15NO3)2), 20 and 40 days after inoculation. The entire experiment was repeated again to confirm the treatment effects. 3.2.3. Quantification of rhizospheric colonization  To evaluate rhizosphere colonization, 3 randomly selected seedlings from each treatment were harvested destructively. Seedlings were removed from pots and gently shaken to remove loosely adhering soil. Roots were then separated from shoots, placed in falcon tubes (BD Biosciences, CA, USA) containing 10 mL of sterile PBS and shaken on a vortex mixer at 1000 rpm for 1 min. Serial dilutions were made in 9 mL of PBS using 1 mL pipetted from the original suspension. Aliquots of the serial dilutions (0.1 mL) were plated onto CCM amended with 200 mg/L rifamycin plates and incubated for 7 days at 30°C. Colonies were counted after 7days of incubation. Roots were oven-dried at 65oC for 2 days before weighing to determine the dry root weight. Rhizospheric bacterial populations were then calculated as cfu per gram dry root. 3.2.4. Quantification of endophytic colonization  Three randomly selected seedlings from each treatment were harvested destructively 20, 40 and 60 days after sowing to evaluate endophytic colonization. At each harvest, seedlings were surface-sterilized with 1.3% sodium hypochlorite for 5 min, and washed thrice with sterile distilled water. Seedlings were imprinted on TSA plates for 24 h to check for surface contamination. Root,  41  stem, and leaf tissue samples were then triturated separately in 1 mL PBS using a mortar and pestle. Triturated tissue suspensions were then diluted serially, and 0.1 mL of each dilution was plated onto CCM supplemented with cycloheximide (100 mg/L) to suppress fungal growth and rifamycin (200 mg/L) to suppress bacterial growth. The number of colony forming units (cfu) was evaluated 7 days after incubation at 30°C. 3.2.5. 15N analysis and seedling growth promotion  Fourteen canola seedlings were harvested destructively 20, 40 and 60 days after sowing for evaluation of seedling growth promotion as well as foliar N and 15N content. Seedling growth promotion was evaluated in three ways- lengths, dry weight and no. of seedlings with floral buds. Roots were separated from shoots, and shoot and root lengths were measured. Shoots and roots of each seedling were then oven dried at 65 oC for 2 days before being weighed to determine the dry weight. For foliar N analysis, oven dried and ground foliage of each seedling from each treatment was mixed thoroughly and a 1 mg sample was sent to the Stable Isotope Facility (Department of Forest and Conservation Sciences, Faculty of Forestry, The University of British Columbia, Vancouver, Canada) for determination of foliar N content and %15N excess with an elemental analyzer interfaced with an isotope ratio mass spectrometer (Isoprime, GV Instruments). The amount of fixed N in foliage was estimated by calculating the percent nitrogen derived from the atmosphere (%Ndfa) (Rennie et al., 1978):  42  % Ndfa = [1 – {atom % 15N excess (inoculated plant) / atom % 15N excess (control plant)}] x 100% 3.2.6. Statistical analysis  A completely randomized experimental design with 60 replicates per treatment was used to assess the treatment effects on growth of canola seedlings. The statistical package, SAS v9.4 (Copyright © 2014, SAS Institute Inc., Cary, NC, USA.), was used to perform statistical analyses. Analysis of variance (ANOVA), P < 0.05, was performed to determine significant differences between treatment means for atom percent 15N excess, foliar N content and all plant growth parameters. Since residuals were well distributed, data was left untransformed. Treatment means were separated using Fisher's protected least significant difference (LSD). 3.3. Results 3.3.1. Rhizospheric and endophytic colonization by P2b-2R  Paenibacillus polymyxa strain P2b-2R colonized canola rhizosphere with 1.41 x 108 cfu/g dry root in 1st growth trial and 7.49× 107 cfu/g dry root in 2nd growth trial. P2b-2R colonized internal tissues of canola root (endophytically) with 3.48 x 105 cfu/g fresh weight in the 1st growth trial and 2.36 x 103 cfu/g fresh weight in the 2nd growth trial. But there was no evidence of endophytic colonization in stem and leaf tissues in either growth trials. Although, P2b-2R colonies were observed on TSA stem and leaf imprint plates after these tissues  43  were surface sterilized. No evidence of rhizospheric or endophytic colonization was found in control plants. 3.3.2. Growth promotion and N- fixation  Seedling growth was enhanced significantly by inoculation with P. polymyxa P2b-2R. P2b-2R inoculation increased shoot length by 9%, 19%, and 31% in 1st, 2nd and 3rd harvest respectively in the 1st growth trial (Figure 3.1). Similarly in the 2nd growth trial, P2b-2R inoculation increased shoot length by 17%, 20%, and 30% at each harvest interval respectively (Figure 3.2). Seedling length of P2b-2R inoculated seedlings was promoted by 8%, 18%, and 25% at each harvest interval respectively in the 1st trial (Figure 3.1). Similarly in the 2nd growth trial, P2b-2R inoculation promoted seedling length by 14%, 15%, and 21% at each harvest interval respectively (Figure 3.2). P2b-2R inoculation significantly increased shoot dry weight (22%, 32%, and 38% at each harvest interval respectively in the 1st trial (Figure 3.3) and 18, 42 and 31% at each harvest interval respectively in the 2nd trial (Figure 3.4)) and seedling biomass (17, 26, 30% at each harvest interval respectively in the 1st trial (Figure 3.3) and 16, 32, 24% at each harvest interval respectively in the 2nd trial (Figure 3.4)). Canola plants reached reproductive stage after 40 days of inoculation, as the floral buds started blooming. It was observed that a greater number of inoculated canola seedlings reached reproductive stage than the control seedlings in both growth trials (Figure 3.5).  44   Based on percent foliar 15N atom excess of P2b-2R inoculated and control seedlings, 7% of the foliar N of inoculated seedlings was derived from the atmosphere (Ndfa) at the time of 1st harvest, which increased to 11% at the time of 2nd harvest and 22% at the time of 3rd harvest in the first growth trial (Table 3.1). A similar trend was observed in the second growth trial, as inoculated seedlings derived 5% N from atmosphere in the 1st harvest, which increased to 8% in the 2nd harvest and 19% in the 3rd harvest (Table 3.2). In the 1st growth trial, percent foliar N of seedlings inoculated with P2b-2R was 14, 23, and 41% higher than the control seedlings at the time of 1st, 2nd and 3rd harvests respectively (Table 3.1). Similarly in the second growth trial, percent foliar N of inoculated seedlings was 23, 29, and 38% higher than the control at the time of 1st, 2nd and 3rd harvest respectively (Table 3.2).  Figure 3.1 Seedling, root and shoot length of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control). 05101520253035404550SeedlingRootShootSeedlingRootShootSeedlingRootShoot20 dai 40 dai 60 daiLength (cm)P2b-2R Control****** 45   Figure 3.2 Seedling, root and shoot length of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).   Figure 3.3 Seedling, root and shoot dry weight of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 1st growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control). 05101520253035404550SeedlingRootShootSeedlingRootShootSeedlingRootShoot20 dai 40 dai 60 daiLength (cm)P2b-2R Control******050100150200250SeedlingRootShootSeedlingRootShootSeedlingRootShoot20 dai 40 dai 60 daiDry weight (mg)P2b-2R Control****** 46   Figure 3.4 Seedling, root and shoot dry weight of canola seedlings harvested 20, 40 and 60 days after inoculation (dai) in 2nd growth trial. Error bars represent standard error of the mean (n=14). *P<0.05 (significantly different from control).  Figure 3.5 Number of canola seedlings having floral buds (reached reproductive stage) of each treatment measured 40 and 60 days after inoculation (dai) in two growth trials (n=14). 050100150200250SeedlingRootShootSeedlingRootShootSeedlingRootShoot20 dai 40 dai 60 daiDry weight (mg)P2b-2R Control******01234567891040 dai 60 dai 40 dai 60 daiGrowth Trial 1 Growth Trial 2No. of Seedlings with Floral BudsP2b-2R Control 47  Table 3.1 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa P2b-2R and phosphate buffered saline (control) measured 20, 40 & 60 days after inoculation (dai) in growth trial 1  20 dai   40 dai   60 dai   Atom %15N Excess in Foliage % Foliar N %Ndfa   Atom %15N Excess in Foliage % Foliar N %Ndfa   Atom %15N Excess in Foliage % Foliar N %Ndfa P2b-2R 0.72*±0.014a 1.48*±0.06 7.24  0.63*±0.011 1.16*±0.05 11.46  0.54*±0.006 0.94*±0.09 21.76 Control 0.77±0.016 1.21±0.06 -   0.71±0.017 0.94±0.05 -   0.69±0.010 0.67±0.03 - a Mean ± standard error; n=14 for atom % 15N excess & % foliar N *P<0.05 (significantly different from control) 48  Table 3.2 Atom % 15N excess in foliage, % foliar N and % N derived from the atmosphere (Ndfa), developed from canola seeds inoculated with P. polymyxa P2b-2R and phosphate buffered saline (control) measured 20, 40 & 60 days after inoculation (dai) in growth trial 2   20 dai   40 dai   60 dai   Atom % 15N Excess in Foliage % Foliar N %Ndfa   Atom % 15N Excess in Foliage % Foliar N %Ndfa   Atom % 15N Excess in Foliage % Foliar N %Ndfa P2b-2R 0.70*±0.011a 1.21*±0.06 5.38  0.59*±0.012 1.13*±0.05 8.48  0.52*±0.010 0.90*±0.08 18.63 Control 0.74±0.010  0.94±0.08 -   0.64±0.019  0.92±0.04 -   0.63±0.015  0.65±0.03 - a Mean ± standard error; n=14 for atom % 15N excess & % foliar N *P<0.05 (significantly different from control) 49  3.4. Discussion  The primary objective of my study was to determine if Paenibacillus polymyxa strain P2b-2R is capable of colonizing rhizosphere and internal tissues of canola (Brassica napus L.), fixing N and stimulating plant growth. P. polymyxa P2b-2R was able to colonize rhizosphere of canola seedlings with population sizes greater than those previously reported for this bacterial strain (Bal & Chanway, 2012a,b; Yang, 2015). The population size was also greater than other reported P. polymyxa strains (Holl & Chanway, 1992; Heulin et al., 1994; Defreitas & Germida, 1998). This shows that P2b-2R possesses necessary adaptations for rhizosphere colonization of this agricultural crop despite being isolated from internal tissues of a tree species. Timmusk et al. (2005) previously reported that colonization of P. polymyxa is initiated by bacterial accumulation at the root tip and subsequent formation of biofilms around the root tip. One of the most important factors that determine bacterial colonization on the plant root is root exudation (Walker et al., 2003; Weller & Thomashow, 1994). Paenibacillus polymyxa has been found to colonize rhizosphere of canola plant in some previous reports as well. Defreitas & Germida (1998) isolated more than 800 bacterial strains from rhizosphere of field-grown canola and found that about 5% of the isolates were diazotrophs. The most promising diazotrophic bacteria of the collection were two Bacillus polymyxa strains (ES600A and RSN17).   Endophytic colonization of canola roots by P2b-2R was observed in both growth trials with population sizes comparable to those observed in other  50  published reports of this bacterial strain (Anand & Chanway, 2013a; Anand et al., 2013). However, there was no evidence of endophytic colonization in stem and leaf tissues by P2b-2R strain in either growth trial. This was unexpected as P2b-2R previously colonized above ground parts of the plant in lodgepole pine and western red cedar (Anand & Chanway, 2013a; Anand et al., 2013). Possible reasons could be that, either the bacteria was not able to reach above ground parts of the plant in such short time duration or the comparatively harsh protocol that was required to ensure complete surface sterilization of tissue samples disinfected internal cortical tissues thereby precluding detection of this bacterial strain. Additional studies involving direct detection using confocal laser microscopy need to be done to confirm colonization in stem and leaf tissues as was done with lodgepole pine seedlings (Anand & Chanway, 2013b). According to our results, the preferred endophytic colonization region of canola was root tissue, which is consistent with reports about some other endophytic bacteria (Timmusk et al., 2005; Hurek et al., 1994). Quadt-Hallmann & Kloepper (1997) observed that hydrolytic enzymes synthesized by P. polymyxa aid entry into intact root tissue.  Paenibacillus polymyxa P2b-2R had consistent and significant effects on canola foliar 15N content. The magnitude of 15N foliar dilution suggests that canola seedlings derived 19-22% of foliar N from the activity of P2b-2R (Table 3.1 and 3.2). Similar amounts of N derived from the atmospheric pool have been observed in other plant species including sugar cane (Saccharum officinarum L.) (Lima et al., 1987; Boddey et al., 1991), kallar grass (Leptochloa fusca) (Malik et  51  al., 1987), rice (Oryza sativa L.) (Malik et al., 1997), wheat (Triticum aestivum) (Rennie & Larson, 1979; Rennie et al., 1983), western red cedar (Thuja plicata) (Bal & Chanway, 2012b; Anand & Chanway, 2013a), lodgepole pine (Pinus contorta) (Bal & Chanway, 2012a), and corn (Zea mays L.) (Araújo et al., 2015). The percent nitrogen derived by inoculated seedlings from atmosphere increased at each harvest in both growth trials (Table 3.1 and 3.2) suggesting that BNF is an important component of N nutrition of canola growing in a nitrogen limited soil medium. But the source of this BNF is still unknown and it could be speculated that it is either induced by the rhizospheric P2b-2R or endophytic P2b-2R present in root tissues. Nitrogen was added to the soil mix only once at the onset of this experiment. In the absence of further additions, N would be expected to deplete with continued seedling growth and ultimately restrict their growth rate. But it was observed that nitrogen fixation by P2b-2R contributed positively to plant’s performance as the inoculated seedlings were having higher foliar N content than the control seedlings (nearly 40%). These results are much better than what was found with corn (presented in Chapter 2), where this bacterial strain increased the foliar N content of corn seedlings by 20%.  Growth promotion of canola seedlings by P. polymyxa P2b-2R was inferred on the basis of two parameters- length and biomass. Inoculated seedlings were 25% taller than the control seedlings after 60 days of inoculation. This was primarily due to a significant difference of shoot length (about 30%) between inoculated and control seedlings (Figure 3.1 and 3.2). These results are consistent with previous reports about this bacterial strain, where P2b-2R  52  inoculation significantly enhanced shoot and seedling length in lodgepole pine and western red cedar (Anand & Chanway, 2013a; Anand et al., 2013). In another report, two strains of Bacillus polymyxa species, previously isolated from field-grown canola, were found to enhance plant growth when reintroduced into canola in growth chamber and field trials (Defreitas & Germida, 1998). Seedling biomass of P2b-2R inoculated seedlings was nearly 30% greater than control seedlings after 60 days of inoculation and I think that a significant difference in shoot dry weight (35%) between inoculated and control seedlings was the primary reason for this (Figure 3.3 and 3.4). The substantial differences of shoot length and shoot dry weight between inoculated and control seedlings suggest that P2b-2R promotes plant growth both vertically (length) and horizontally (biomass). The reason for this enhanced growth of canola seedlings is likely related to the nitrogen fixing ability of this bacterium, leading to a higher content of N especially in the above ground parts of the plant. Canola seedlings started blooming after 40 days of inoculation and it was observed that P2b-2R inoculation enhanced the ability of seedlings to reach reproductive stage by 2-2.5 times as compared to the control (Figure 3.5). This clearly indicates that P2b-2R inoculation boosts the ability of canola seedlings to self-pollinate and form floral buds. Thus, P2b-2R might play a significant role in increasing the yield of canola in a field trial. In a previous study, a plant growth promoting bacterium showed the ability to increase yield of canola crop by 57% in field trials (Kloepper et al., 1988).   53   Plant growth promotion can be achieved by the direct interaction between beneficial microbes and their host plant and also indirectly due to their antagonistic activity against plant pathogens (Prasad et al., 2015). One of the most commonly reported direct plant growth promoting mechanism by Bacillus and relatives is the production of plant growth substances such as auxins, cytokinins and giberellins (Chanway, 2008).  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). Timmusk & Wagner (1999) reported that natural isolates of P. polymyxa induce drought tolerance and antagonize pathogens in Arabidopsis thaliana. Studies have shown that bacteria belonging to Paenibacillus genus are capable of mineralizing phosphorous by producing enzymes, which cleave phosphate groups (Chanway, 2008). Paenibacillus polymyxa is also capable of synthesizing many antibacterial and antifungal secondary metabolites (Borriss, 2015). Beatty & Jensen (2002) isolated a P. polymyxa strain PKB1 capable of inhibiting the growth of Leptosphaeria maculans, the causative agent of blackleg disease of canola (Brassica napus L. and Brassica rapa L.). They found that P. polymyxa produces fusaricidin-type antifungal antibiotics, which are quite active against Leptosphaeria maculans.  54  3.5. Conclusions  In conclusion, my results suggest that canola seedlings are able to access an N pool other than the soil, likely the atmospheric pool, when colonized by P. polymyxa P2b-2R and demonstrate that N2-fixing seedlings develop higher foliar N level and grow much better than non-fixing seedlings in N-limited soil. Paenibacillus polymyxa P2b-2R bacterial strain was originally isolated from internal stem tissues of lodgepole pine, a gymnosperm species. Successful association of this bacterial strain with canola, an important agricultural crop, indicates that it might be able to colonize broad range of hosts. No bacterial population was observed in stem and leaf tissues of canola, which was unexpected as this strain was originally isolated from stem tissues of lodgepole pine (Bal et al., 2012) and was found to colonize stem and leaf tissues of western red cedar and lodgepole pine (Anand & Chanway, 2013a,b; Anand et al., 2013). Either the surface sterilization protocol was too harsh for stem and leaf tissues or the bacterium was not able to form detectable population sizes. Direct detection techniques such as using confocal laser scanning microscopy with gfp-labelled bacteria might be more useful to detect endophytic colonization of stem and leaf tissues of canola. P2b-2R inoculated canola seedlings successfully derived significant amounts of N from atmosphere. But we don’t know if this BNF originated from rhizospheric or endophytic P2b-2R populations. P2b-2R inoculation promoted seedling growth by increasing length and biomass. Inoculated seedlings were directly assessing atmospheric N pool, thereby increasing foliar N content, which could be the reason for enhanced seedling  55  growth. Other possibilities could be that this bacterial strain was involved in nutrient mineralization and uptake, or pathogen suppression. P2b-2R inoculation also boosted the ability of canola seedlings to reach reproductive stage as greater numbers of inoculated seedlings were having floral buds than the control during 2nd and 3rd harvest. This finding reflects the ability of P2b-2R to increase yield of canola plant.  56  Chapter 4 – General Summary and Conclusions  Nitrogen is recognized as the most frequently limiting nutrient for plant growth in ecosystems (Vitousek & Howarth, 1991). Despite its abundance, nitrogen (N) is one of the most growth-limiting nutrients in terrestrial and aquatic ecosystems (Dalton & Krammer, 2006) because its gaseous form is inert and unusable by most living organisms except for nitrogen fixing microorganisms. For it to become biologically available, atmospheric nitrogen must be transformed or “fixed” from its inert gaseous form (N2) to ammonia (NH3).   Nitrogen is fixed naturally through energy-releasing abiotic processes such as lightening, forest fires and volcanic activity. Manufacturing of artificial nitrogen fertilizers became very common in the post-industrial revolution era. Fertilizer production using high temperatures and pressures in the Haber-Bosch process occurs widely and accounts for approximately 20% of annual global NF (Bezdicek & Kennedy, 1998). However, the process is fossil fuel intensive and consumes 3-5% of the world’s natural gas annually (Myrold & Bottomley, 2007). Alternatively, NF occurs through the normal metabolic activity of many diazotrophs, through a process commonly referred to as biological nitrogen fixation (BNF) (Chanway et al., 2014). Biological nitrogen fixation is an important component of the nitrogen cycle and has been proven to play a pivotal role in the productivity of agricultural and forest ecosystems dominated by leguminous plants. As a result, most previous research has been focused on the Rhizobium–legume symbiosis and the organisms involved. But since the discovery of  57  endophytic diazotroph Gluconoacetobacter diazotrophicus from internal tissues of sugarcane plant in Brazil, many researchers have shifted their focus on isolation and identification of bacterial endophytes capable of deriving nitrogen from atmosphere.   One such strain, Paenibacillus polymyxa P2b-2R, was isolated from internal tissues of lodgepole pine naturally regenerating at a site near Williams Lake, British Columbia, Canada (Bal et al., 2012). When this bacterial strain was reintroduced into lodgepole pine and grown in nitrogen limited environment, it was found to derive 79% of nitrogen from atmosphere (Anand et al., 2013). Inoculation of another gymnosperm species, western red cedar, with this bacterial strain showed significant results as the inoculated seedlings derived 56% of nitrogen from atmosphere (Bal & Chanway, 2012b). Endophytic and rhizospheric colonization of lodgepole pine and western red cedar by P2b-2R was also reported (Anand et al., 2013; Anand & Chanway, 2013a). Thus, P2b-2R possesses the ability to colonize rhizosphere and internal tissues of lodgepole pine and western red cedar (gymnosperm species) and fix nitrogen. But I was interested in investigating this bacterial strain’s ability to colonize a broad range of plant hosts, specifically agricultural crops. I chose two important agricultural crops, corn and canola, for my study. Corn is a principal cereal crop grown on a large scale worldwide and canola is an important oil crop generally grown in Canada and exported globally generating very significant revenues. Also, corn and canola crop species differ physiologically as well as botanically.  58   The ability of P2b-2R to colonize, fix nitrogen and promote plant growth in Corn is presented in Chapter 2. P2b-2R colonized the corn rhizosphere with population sizes consistent with previous reports about this bacterial strain (Bal & Chanway, 2012a,b). P2b-2R was also successful in colonizing internal tissues of corn root with population sizes similar to those observed in reports about lodgepole pine and western red cedar (Anand et al., 2013; Anand & Chanway, 2013a). Endophytic colonization of stem and leaf tissues was not observed, which was unexpected. However, it would be premature to completely rule out the ability of this bacterial strain to colonize aboveground parts of corn, as this strain was present on tissue imprint plates. A direct detection technique such as gfp-labeling with the use of confocal laser microscopy will provide more insight in this matter. Successful association of P2b-2R with corn seedlings was observed in terms of N-fixation and growth promotion. P2b-2R showed consistent and significant effect on corn foliar 15N abundance. According to atom % 15N excess in foliage data, P2b-2R inoculated corn seedling derived 20% of the foliar N from atmosphere and their foliar N content was also higher than the control. P2b-2R significantly enhanced plant growth by increasing length and biomass (dry matter content) of corn seedlings. Plant length was increased by 35% and biomass by 30% due to the inoculation of this bacterial strain. A consistent increase in foliar N content of inoculated seedlings could be a major contributing reason for growth promotion.  Growth promotion and N-fixation ability of P2b-2R in association with an important oil crop, canola, is reported in chapter 3. Growth promotion of canola  59  seedlings by P2b-2R was inferred on the basis of length and biomass. P2b-2R inoculated canola seedlings were taller and possessed greater biomass than the control. These results were consistent with previous reports about this bacterial strain (Anand & Chanway, 2013a; Anand et al., 2013). Two strains of Bacillus polymyxa species isolated from field-grown canola were also reported to enhance seedling growth in growth chamber and field trials (Defreitas & Germida, 1998). P2b-2R was also involved in increasing the reproductive ability of canola seedlings as greater number of inoculated seedlings were having flower buds as compared to the control, which indicates that this bacterial strain might play a significant role in increasing the yield of canola. A long-term growth trial could be designed to see the effects of this bacterial strain on yield of canola plant. P2b-2R inoculation showed consistent and significant effect on canola foliar 15N content as the seedlings were found to derive more than 20% of foliar N from atmosphere. Similar amounts of N derived from atmospheric pool have been observed in other plant species including sugar cane (Saccharum officinarum L.) (Lima et al., 1987; Boddey et al., 1991), rice (Oryza sativa L.) (Malik et al., 1997), wheat (Triticum aestivum) (Rennie & Larson, 1979; Rennie et al., 1983), western red cedar (Thuja plicata) (Bal & Chanway, 2012b; Anand & Chanway, 2013a), lodgepole pine (Pinus contorta) (Bal & Chanway, 2012a), and corn (Zea mays L.) (Araújo et al., 2015). Nitrogen fixation by P2b-2R contributed positively to plant’s performance by increasing the foliar N content of inoculated seedlings. Rhizospheric and endophytic colonization experiments revealed that P2b-2R successfully colonized canola rhizosphere and internal tissues of roots  60  with population sizes comparable to those observed in other published reports about this bacterial strain (Anand & Chanway, 2013a; Anand et al., 2013).  In summary, my thesis provides insight into the ability of a PGPB isolated from a gymnosperm species to colonize diverse plant hosts (agricultural crops). The ability of Paenibacillus polymyxa P2b-2R to colonize, fix N and promote growth of two agronomically important crops, corn and canola, is an effective finding in terms of sustainable agriculture. With an increasing interest of researchers worldwide towards diazotrophic endophytes as means of increasing plant growth, this PGPB promises to be an endophyte that could be commercialized in the future.   61  References Anand, R. (2011). Endophytic colonization and nitrogen fixation by Paenibacillus  polymyxa in association with lodgepole pine and western red cedar  (Doctoral Dissertation). Available from:  https://circle.ubc.ca/bitstream/handle/2429/30254/ubc_2011_spring_anan d_richa.pdf?sequence=6 (accessed on: July 21, 2015). Anand, R., Grayston, S., & Chanway, C. (2013). N2-fixation and seedling growth  promotion of lodgepole pine by endophytic Paenibacillus polymyxa.  Microbial Ecol 66(2): 369-374. Anand, R., & Chanway, C. (2013a). 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(2013). The Family Paenibacillaceae, Bacillus Genetic Stock  Center Catalog of Strains Part 5. Bacillus Genetic Stock Center.  Columbus, OH, USA. Available from:  http://bgsc.org/_catalogs/Catpart5.pdf (accessed on: July 21, 2015). Zhou, W. W., Huang, J. X., & Niu, T. G. (2008). Isolation of an antifungal  Paenibacillus strain HT16 from locusts and purification of its medium‐ dependent antagonistic component. J Appl Microbiol 105(3): 912-919.  74  Appendix A – Plant Nutrient Solution3 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                                                         3 Modified from Chanway et al. (1988)  75  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  76  Appendix C – Recipe to Make Phosphate Buffered Saline NaCl 8 g/L KH2PO4 0.24 g/L KCl 0.2 g/L Na2HPO4 1.44 g/L Distilled Water 1 L  77  Appendix D – Additional Figures  Figure D.1 P2b-2R-inoculated (left) and control (right) seedling of corn plant harvested 10 days after inoculation (dai) showing clear difference in length, biomass and plant health   Figure D.2 P2b-2R-inoculated (right) and control (left) seedling of corn plant harvested 20 days after inoculation (dai) showing clear difference in length, biomass and plant health Corn: 10 dai Corn: 20 dai  78   Figure D.3 P2b-2R-inoculated (left) and control (right) seedling of corn plant harvested 30 days after inoculation (dai) showing clear difference in length, biomass and plant health   Figure D.4 P2b-2R-inoculated (left) and control (right) seedling of canola plant harvested 20 days after inoculation (dai) showing clear difference in length, biomass and plant health Corn: 30 dai Canola: 20 dai  79   Figure D.5 P2b-2R-inoculated (left) and control (right) seedling of canola plant harvested 40 days after inoculation (dai) showing clear difference in length, biomass and plant health   Figure D.6 P2b-2R-inoculated (right) and control (left) seedling of canola plant harvested 60 days after inoculation (dai) showing clear difference in length, biomass and plant health* Canola: 40 dai Canola: 60 dai 

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