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Isolation of endophytic bacteria from Lodgepole Pine (Pinus contorta var. latifolia (Dougl.) Engelm.)… Bal, Amandeep S. 2003

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ISOLATION OF ENDOPHYTIC BACTERIA FROM LODGEPOLE PINE (PINUS CONTORT A VAR. LATIFOLIA (DOUGL.) ENGELM.) AND WESTERN RED CEDAR (THUJA PLICATA DONN.) AND DETERMINATION OF THEIR NITROGEN FIXING ABILITY by AMANDEEP S. BAL B.Sc. (Agr), The University of British Columbia, 1998 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Faculty of Forestry (Department of Forest Sciences) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January, 2003 © Amandeep Singh Bal, 2003 UBC Rare Books and Special Collections - Thesis Authorisation Form Page 1 of 1 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the Un i v e r s i t y of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis f o r sch o l a r l y purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Faculty of Forestry Department of Fore-if $ci&h<&S The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada http://www.library.ubc.ca/spcoll/thesauth.html 1/27/03 ABSTRACT The diversity of endophytic bacteria in lodgepole pine and western red cedar tissues and whether endophytic diazotrophs isolated from these tissues were capable of supplying biologically significant amounts of fixed nitrogen to these two species were examined. Plant samples were collected from stands in central and southern British Columbia and endophytic bacteria were isolated primarily from stem tissues using a surface sterilization-trituration-plating technique. Most of the endophytic bacteria isolated from pine and cedar tissues belonged to the genera Bacillus and Paenibacillus. The N-fixing ability of isolates was tested using an acetylene reduction assay. Four isolates capable of reducing acetylene were selected to determine their ability to provide biologically significant amounts of fixed N to pine and cedar seedlings in two separate controlled environment experiments. This included Paenibacillus amylolyticus C3b, rifamycin-resistant derivatives of P. peoriae/polymyxa P2b and P18b, and a rifamycin-resistant derivative of strain P19a, which was closely related to Dyadobacter fermentans. Surface sterilized pine and cedar seeds were sown separately in glass tubes containing an autoclaved mixture of sand and montmorillonite clay treated with a nutrient solution having a 1 5 N label. Each tube was then inoculated with ca. 107 cfu of one of strains P2b-2R, P18b-2R, P19a-2R, orC3b. The only treatment in which seedlings had significantly lower atom % 1 5 N excess values in foliage than in control seedlings in both plant growth trials for cedar and pine was the P2b-2R treatment. Inoculation of seed with strain P2b-2R resulted in cedar and pine seedlings harvested 35 and 42 weeks, respectively, after sowing, deriving as much as 56% i i and 66%, respectively, of their foliar N from the atmosphere. Pine seedlings from the C3b and P18b-2R treatments from only the first trial derived less significant quantities of foliar N from the atmosphere. Strain P19a-2R did not provide significant amounts of fixed N to cedar and pine seedlings in either trial. Despite cedar and pine seedlings from the P2b-2R treatments deriving significant amounts of fixed N, increases in foliar N content were not common and growth of seedlings of both species was depressed. Strain P2b-2R could be readily isolated from rhizosphere samples following both seedling harvests for cedar and pine. However endophytic colonization of tissues by this strain could not be confirmed due to persistent contamination on imprint plates used to determine the effectiveness of the tissue surface sterilization techniques employed. iii TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi Acknowledgements viii SECTION 1 Introduction 1 SECTION 2 Materials and Methods 9 2.1 Plant sample and soil collection 9 2.2 Isolation of endophytic bacteria 10 2.3 Bacterial identification 11 2.4 Evaluation of nitrogenase activity. 11 2.5 Raising of antibiotic-resistant derivatives 12 2.6 Controlled environment experiments 13 2.7 Seedling harvest 16 2.8 Sampling for rhizosphere colonization 16 2.9 Sampling for endophytic colonization 17 2.10 Nitrogen analysis and growth response 18 2.11 Statistical analysis 19 SECTION 3 Results 21 3.1 Identification of endophytic bacteria and acetylene reduction activity 21 3.2 Rhizosphere colonization 24 3.3 Endophytic colonization 25 3.4 Identification of contaminant bacteria 26 3.5 Foliar atom % 1 5 N excess and foliar N content 26 3.6 Seedling growth response 30 SECTION 4 Discussion 32 4.1 Identification of endophytic bacteria and acetylene reduction activity 32 4.2 Rhizosphere colonization 34 4.3 Antibiotic masking 34 4.4 Endophytic colonization 36 4.5 Foliar atom % 1 5 N excess 37 4.6 Foliar N content 42 4.7 Seedling growth response 43 IV SECTION 5 Conclusions 49 LITERATURE CITED 52 APPENDIX 1 Chemical properties of soils collected from Williams Lake pine, Chilliwack Lake pine, and Boston Bar cedar stands 62 APPENDIX 2 Statistical analysis 64 v LIST OF TABLES Page Table 1 Genera and species of endophytic bacteria isolated from Williams Lake pine on TSA and CCM 22 Table 2 Genera and species of endophytic bacteria isolated from Chilliwack Lake pine on TSA and CCM 22 Table 3 Genera and species of endophytic bacteria isolated from Boston Bar cedar on TSA and CCM 23 Table 4 Rhizosphere colony counts for strains P2b-2R and C3b 25 Table 5 Associative nitrogen fixation, nitrogen content, and growth of lodgepole pine seedlings inoculated in first controlled environment experiment with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 27 Table 6 Associative nitrogen fixation, nitrogen content, and growth of lodgepole pine seedlings inoculated in second controlled environment experiment with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 27 Table 7 Associative nitrogen fixation, nitrogen content, and growth of western red cedar seedlings inoculated in first controlled environment experiment with strains P2b-2R, heat-killed P2b-2R, P18b-2R, and P19a-2R 28 Table 8 Associative nitrogen fixation, nitrogen content, and growth of western red cedar seedlings inoculated in second controlled environment experiment with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 28 Table A1 Chemical properties of soils collected from Williams Lake pine, Chilliwack Lake pine, and Boston Bar cedar stands 63 Table A2 Analysis of variance for effects on pine seedlings from first growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 65 Table A3 Analysis of variance for effects on pine seedlings from second growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 67 VI Table A4 Analysis of variance for effects on cedar seedlings from first growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, and P19a-2R 69 Table A5 Analysis of variance for effects on cedar seedlings from second growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b 71 vii ACKNOWLEDGEMENTS I would like to express my gratitude to my supervisor, Dr. Chanway, for his guidance, support, and encouragement over the course of this project. I would also like to thank my other committee members, Dr. Ballard and Dr. Holl, for their assistance. Thank you also to my family for all of the support they have provided me. Others who deserve much credit for providing me with assistance include Dr. Copeman, Dr. Guy, Dr. Upadhyaya, Dr. Klinka, Dr. Xiao, Dr. Mclnroy, Odile Berge, Dave Harris, Jamie Nairn, Jagdeep Khun Khun, Minette, Les Paul, Elizabeth Bent, Peter Garnett, Martin Hilmer, Gilles Galzi, Sylvia Leung, Pauline Tan, Jane Li, and Susan Ratson. Financial assistance was provided by Dr. Chanway through his NSERC and Global Forest (catalogue # GF-18-2000-68) research grants and by the Science Council of British Columbia and Brinkman and Associates Reforestation. viii SECTION 1: Introduction Lodgepole pine (Pinus contoiia var. latifolia (Dougl.) Engelm.) and western red cedar (Thuja plicata Donn.) are two commercially very important tree species in British Columbia. Lodgepole pine is a highly adaptable species found throughout most of BC. Some of its uses include lumber and paneling and for making doors and furniture (Parish 1994). Cedar occurs along the coast and the wet belt of the interior of the province. This species is known for its natural resistance to decay and insect damage and is an excellent source of house siding, decking, roofing, fencing, outdoor furniture, and interior paneling (Parish 1994). Lodgepole pine is capable of growing under a variety of conditions, including those which are nutrient poor (Weetman et al. 1988). Nitrogen (N) is the most growth-limiting nutrient for tree growth and is particularly susceptible to loss through volatilization, which may occur due to fire disturbances. Nitrogen is at a high risk of loss during fires since it has a low vaporization temperature and most of it is present in surface organic materials. The ability of lodgepole pine to grow under N-deficient conditions is interesting considering it is not known to fix N (Koch 1996). In addition, fertilization of pine with N on sites where this nutrient is limiting has resulted in quite variable growth responses (Weetman and Fournier 1982, Weetman et al. 1988, Brockley 1989, 1990, 1991, 1995, Yole et al. 1991, Marshall et al. 1992). Thus, it is unclear how this species satisfies its N requirements. Traditionally, the mineralization of N by free-living soil microorganisms has been considered the primary mechanism by which plant available N is released into the soil. Therefore, this has been cited as the step which limits the accumulation of 1 N in forest ecosystems (Chapin 1995). In addition, the pathway just mentioned may be bypassed by certain mycorrhizal fungi which release enzymes that render N, which is tied up in forest floor organic materials, available (Read 1991, Xiao 1994). This process may be important for making N available to pine under some nutrient-poor conditions (Northup et al. 1995). However, in light of the ability of pine to grow on nutrient-poor mineral soil with little or no forest floor organic N, it is unlikely that this process fully explains the N requirements of this species. The most obvious source of N for plants growing on N-poor soils would be the atmosphere via biological nitrogen fixation. Bacteria capable of fixing N are referred to as diazotrophs. Diazotrophs have been found in the rhizosphere and the mycorrhizosphere (i.e., the rhizosphere of mycorrhizae) of gymnosperms (Li et al. 1992). These bacteria are capable of supplying fixed N to pine, but the amount they have been shown to provide is not biologically significant (Chanway and Holl 1991, Binkley 1995). Cedar can also grow under conditions of low nutrient supply and it is also not entirely understood how its nitrogen needs are met. Gains in growth response have only been modest following fertilization of cedar with N (Prescott et al. 1996). As well, forest floors of stands containing cedar have low rates of N mineralization (Prescott and Preston 1994, Prescott et al. 1995b, Prescott et al. 1996). Low N mineralization rates lead to low rates of N availability which could be partly due to cedar litter having a slow decomposition rate relative to that of other gymnosperms (Harmon et al. 1990, Keenan et al. 1995, Prescott et al. 1995a). Reports of N 2 fixation have indicated only very small amounts of N fixation occurring in the forest floor and mineral soil of stands containing cedar (Cushon and Feller 1989). Microorganisms can also colonize the internal tissues of healthy plants and those which can do so without causing symptoms of disease are referred to as endophytes (Wilson 1995). The internal tissues of plants could be considered a favourable site for microorganisms to colonize as it is buffered against environmental extremes and is relatively rich in nutrients (Chanway 1998). Much of the work with endophytes has been with plants of agronomic importance and the microorganisms most widely studied have been fungi. Much less is known about endophytic bacteria. Bacteria belonging to several different genera have been found to occur naturally inside the tissues of plants. Large populations of endophytic bacteria (i.e. up to 107 colony forming units (cfu)/g of plant matter) have been reported in red clover (Trifolium pratense L.) (Sturz et al. 1997). Endophytes have been found to promote plant growth using a variety of mechanisms. These include biological control of pathogens, either by direct antagonism of disease-causing organisms or by inducing systemic resistance to pathogens (Pleban et al.1995, Nowak et al. 1995, Wei et al. 1996), as well as plant parasitic nematodes and insects (Hallmann et al. 1995). Other mechanisms include improvement of nutrient and water uptake, phytohormone production, and N fixation (Triplett 1996, Hallmann et al. 1997, Lazarovits and Nowak 1997, Sturz et al. 2000). Diazotrophic bacteria have been found inside a number of plants of agronomic importance. However, in terms of the quantity of fixed N being supplied to the host plant, the most significant discovery yet has been that of an association 3 between N-fixing bacteria and sugar cane (Saccharum officinarum L.) in Brazil (Lima et al. 1987, Cavalcante and Dobereiner 1988, Boddey et al. 1991, Urquiaga et al. 1992), in which the diazotroph, Gluconoacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) has been isolated from surface sterilized root, stem, and leaf tissue. Through 1 5 N isotope dilution and N balance experiments, it has been demonstrated that N fixation provides 50% - 80% of the N requirements of sugar cane (Lima et al. 1987, Boddey et al. 1991). Recently, in a study using both wild-type and Mr" mutants of G. diazotrophicus, sugar cane plants inoculated with the wild-type strain grew better and had higher N contents than plants inoculated with the Nif mutant or uninoculated plants (Sevilla et al. 2001). In this same study, 1 5 N 2 incorporation experiments showed that wild-type strains were capable of actively fixing N in sugar cane. G. diazotrophicus populations of 104 - 106 cells/g fresh weight of sugar cane tissue have been reported inside sugar cane (Reis et al. 1994, Baldani et al. 1997). Prior to the discovery of G. diazotrophicus inside the tissues of sugar cane in Brazil, it was not clear how this plant was capable of growing in soils that had no N fertilizer added to them (Cavalcante and Dobereiner 1988). Nitrogen-fixing bacteria belonging to the genus Beijerinckia had earlier been isolated from the rhizosphere of sugar cane (Baldani et al. 1997). However, this association has not been shown to provide a significant quantity of N to sugar cane (Baldani etal. 1997). G. diazotrophicus possesses a number of interesting physiological properties, that may relate to its apparent ability to fix N. These include acid production and the ability to grow and fix N at pH's as low as 2.5 (Gillis et al. 1989, Stephan et al. 1991). 4 It can grow in vitro at pH's as high as 7.5, but grows best at a pH of 5.5 (Gillis et al. 1989, Stephan et al. 1991). This bacterium can tolerate high sucrose concentrations (10-30%) and is also quite tolerant of O2 (Cavalcante and Dobereiner 1988). G. diazotrophicus has also been found in other plants having a high sugar content, including sweet potato (Ipomoea batatas L. Lam), Cameroon grass (Pennisetum purpureum Schumach), coffee (Coffea arabica L.), and pineapple (Ananas comosus L. Merr.) (Paula et al. 1991, James and Olivares 1997, Jimenez-Salgado et al. 1997, Tapia-Hernandez et al. 2000). It can fix N at dissolved 0 2 levels as high as 4.0 kPa, but the optimal level is 0.2 kPa when growing on 10% sucrose (Boddey and Dobereiner 1995). Another interesting property of G. diazotrophicus is its ability to fix N in the presence of mineral N (Boddey et al. 1991). Evidence to support this includes its lack of nitrate reductase, which allows it to fix N in the presence of up to 80 mM N0 3 " (Li and MacRae 1991). As well, Boddey et al. (1991) found the presence of NH 4 + and amino acids only partially suppresses the nitrogenase activity of G. diazotrophicus, particularly when grown on 10% sucrose. In addition, the bacterium can release fixed N for other organisms to utilize (Cojho et al. 1993). Where G. diazotrophicus persists in the environment and how it enters sugar cane tissues has also been investigated. Interestingly, G. diazotrophicus has not been found in soil (Baldani et al. 1997, James and Olivares 1997, Sevilla et al. 2001), but it has been detected in the rhizosphere and particularly in sugar cane trash (Li and MacRae 1992, Reis et al. 1994). For transmission into the internal tissues of sugar cane, the rhizosphere and trash may be an important source of inoculum (Chanway 1998). One theory as to how sugar cane infection occurs is that 5 G. diazotrophicus enters through young root tips and where lateral roots emerge (James et al. 1994, Sevilla et al. 2001). Another theory is that infection occurs through infection threads in root hairs (Bellone et al. 1997). G. diazotrophicus might also enter sugar cane via mycorrhizal fungi (Paula et al. 1991,1992). Insects may be responsible for transmission between plants. G. diazotrophicus has been found in the pink sugar cane mealy bug [Saccharococcus sacchari), which feeds on sugar cane (Ashbolt and Inkerman 1990). Another diazotroph belonging to the genus Herbaspirillum has been isolated from inside sugar cane. However, it was found to possess nitrate reductase and the presence of fixed N prevents its nitrogenase from being active (Baldani et al. 1992). Thus, based on the evidence to date, G. diazotrophicus is most likely responsible for N fixation in sugar cane (Triplett 1996, Baldani et al. 1997). Compared to plants in agriculture, studies of endophytic bacteria in forest tree species, particularly gymnosperms, have been minimal. Endophytes have been isolated from the roots of white x Engelmann hybrid spruce (P/'cea glauca x Picea engelmannii) seedlings (O'Neill et al. 1992a). Of the 22 strains isolated in the greenhouse trial, five promoted seedling growth, three were inhibitory, and the rest had no effect on seedling growth. The two strains responsible for the greatest and most reproducible seedling growth promotion were chosen for a field trial at three different reforestation sites in which two ecotypes of one-year-old spruce seedlings were planted (Chanway and Holl 1993). One of the strains, which was identified as Hydrogenophaga pseudoflava, promoted seedling root growth or branch number in four of the six spruce ecotype x planting site combinations. The second strain, 6 which was identified as Pseudomonas putida, stimulated seedling growth of only one of the ecotypes planted at two of the reforestation sites. However, in three of the ecotype x site combinations, seedling growth was inhibited. In a separate greenhouse assay, three Bacillus strains, three actinomycetes strains (most likely Streptomyces), one Phyllobacterium strain, and one unidentified strain which were originally isolated from the internal root tissues of white x Engelmann hybrid spruce seedlings consistently increased the biomass of two-month-old spruce seedlings by as much as 36%, following inoculation of seed (Chanway et al. 1994). One of the Bacillus strains (N4) and one of the actinomycetes strains (N1) only enhanced the growth of seedlings when trials were performed using a small amount (2% v/v) of forest soil containing minor pathogenic organisms that were known inhibit seedling growth. This indicated that growth promotion by these strains was due to biocontrol of minor pathogens. In contrast, Phyllobacterium strain W3 and actinomycetes strain W2 only promoted the growth of spruce seedlings in the absence of forest soil, suggesting these strains did not act through a biocontrol mechanism. Endophytic bacteria have also been isolated from lodgepole pine. One Paenibacillus strain (Pw2) which was originally isolated from surface sterilized root tissues of a naturally-regenerating 2-3-year-old pine seedling was shown to enhance seedling growth in greenhouse experiments (Shishido et al. 1995). Following root or seed inoculation, strain Pw2 has been found to colonize the external and internal root tissues of both pine and spruce. Bacterial populations as large as 105 cfu/g root tissue have been found inside roots (Shishido et al. 1995, Chanway 1997, Shishido 1997) and colonization of this region may depend on the formation of lateral roots 7 (Shishido et al. 1995). As well, 5 months following inoculation of seed, a rifamycin-resistant derivative (Pw-2R) of strain Pw2 was reported inside the root and stem tissues of white x Engelmann hybrid spruce using surface sterilization-dilution plating and immunofluorescent antibody staining assays (Shishido et al. 1999). Thus, it appears that strain Pw-2R can be transported systemically. The most interesting property of this strain is that it possesses nitrogenase activity (Shishido and Chanway, unpublished data). These observations lead to the possibility that, similar to the sugar cane example, N-fixing bacteria may reside inside pine and cedar tissues, and assist them in meeting their N requirements when growing under N deficient conditions. The following hypotheses were examined: 1. Endophytic bacteria, including diazotrophs, exist in the internal tissues of lodgepole pine and western red cedar. 2. Diazotrophic bacteria isolated from the internal tissues of lodgepole pine and western red cedar are capable of supplying a biologically significant amount of fixed N to "host" seedlings grown under N-poor conditions. 8 SECTION 2: Materials and Methods 2.1 Plant Sample and Soil Collection Entire lodgepole pine seedlings, as well as root, stem, and needle samples from trees were collected from stands near Williams Lake, British Columbia (52°05' N lat., 122°54'W long., elevation 1300 m) and Chilliwack Lake, British Columbia (49° 06'N lat., 121°26'W long., elevation 600 m). The Williams Lake stand was within the very dry cold subzone of Sub-Boreal Pine-Spruce biogeoclimatic zone (Steen and Demarchi 1991). Mean annual temperature is about 2°C and mean annual precipitation is around 400 mm. The Chilliwack Lake stand was located in the dry submaritime subzone of the Coastal Western Hemlock biogeoclimatic zone (Pojar et al. 1991). Mean annual temperature is about 10°C and mean annual precipitation is approximately 1500 mm. Stem samples from trees were obtained by taking cores with an increment borer. Prior to extracting each core, the borer interior was cleaned with a pipe cleaner after which the borer was disinfected by dipping it in 6% (w/v) sodium hypochlorite (NaOCI) for 2 minutes 30 seconds, then 70% ethanol for 2 minutes, followed by three 30 second rinses in sterile distilled water. Then, a scalpel cleansed with 6% NaOCI was used to shave off a thin layer of bark from the stem sampling site in order to minimize epiphytic contamination. Stem cores were placed in sterile plastic bags, sealed, and transported on ice back to the lab. Needle samples from trees were obtained by clipping off branches close to the ground and roots were only sampled from seedlings. Cedar samples were collected from a stand near Boston Bar, British Columbia (49°50'N lat., 121°31'W long., elevation 600 m). The stand was within the wet warm subzone of the Interior Douglas-fir 9 biogeoclimatic zone (Hope et al. 1991). Mean annual temperature is about 9°C and mean annual precipitation is around 1200 mm. The only difference between the cedar and pine sampling procedures was cedar stem samples were taken from trees by cutting small wedges from stems using a pruning knife which was disinfested with 6% NaOCI prior to each sampling. Soil was collected from each of the sampling sites to a depth of approximately 30 cm. Chemical analysis of the mineral horizons of soil from the Williams Lake site was performed at the Ministry of Forests Glynn Road Research Station, Victoria, BC. according to methodology described by Carter (1993). Mineral horizons of soil from the Chilliwack Lake and Boston Bar sites were analyzed at Pacific Soil Analysis Incorporated, Richmond, BC. according to methodology described by McKeague (1978). 2.2 Isolation of Endophytic Bacteria Endophytic bacteria were isolated primarily from stems, but root and needle tissues also yielded endophytic bacteria when a surface sterilization-trituration-plating technique was used within five days of collection. The technique involved immersing tissue samples in 2.5% (w/v) NaOCI for 2 minutes, followed by three 30 second rinses in 10 mM sterile phosphate buffer (SPB) (pH 7). Samples were imprinted on triplicate plates of both tryptic soy agar (TSA) and combined carbon medium (CCM) (Rennie 1981), which is a N-deficient medium, to check for surface contamination. Both media were supplemented with 100 mg/L cycloheximide to suppress fungal growth. Tissues were then triturated by hand using a mortar and 10 pestle in a small volume of SPB. Triturated tissues were diluted with SPB and triplicate plated onto TSA and CCM. TSA was used to assess endophytic diversity and CCM was used to select for N fixing bacteria. Following aerobic incubation at room temperature, representative bacterial colonies were selected from dilution plates based on colony size, shape, morphology, and colour, and purified by restreaking onto fresh plates of the same medium used for primary isolations. Purified isolates were placed on a rotary shaker at 175 rpm in separate 50 mL Erlenmeyer flasks containing either tryptic soy broth or CCM broth until broths appeared turbid. Isolates were then stored frozen at -80°C in cryovials containing 2.0 mL isolating medium amended with 20% (v/v) glycerol. 2.3 Bacterial Identification Frozen isolates were thawed, streaked onto TSA and sent to Auburn University for identification by gas chromatographic analysis of bacterial fatty acids (as methyl esters) (GC-FAME) using the MIDI (Microbial ID, Inc.) Microbial Identification System (Kloepper et al. 1992). Selected isolates including those used in the seedling assays were also identified using 16S rDNA analysis by Odile Berge at Univ-Mediterranee in St. Paul-Lez-Durance, France. 2.4 Evaluation of Nitrogenase Activity Those bacterial isolates which grew on CCM and those isolates which grew on TSA that were identified by GC-FAME as being Paenibacillus polymyxa, which is a bacterial species known to fix N (Postgate 1998), were tested in vitro for the 11 presence of N-fixing activity using an acetylene reduction assay (ARA) described by Holl et al. (1988) with some modifications. This assay involved inoculating each isolate in a 5 ml culture vial fitted with a teflon seal that contained 2 mL of CCM broth. Vials were placed on a rotary shaker (175 rpm) and bacterial growth was allowed to occur at room temperature until broths appeared turbid, which was followed by the injection of acetylene into the culture vials to a final concentration of 10% (v/v). Two to five days later, each vial had 1 mL samples of gas removed to analyze for ethylene content by flame-ionization gas chromatography following separation in a stainless steel column (0.3 x 180 cm) containing Porapak N (80-100 mesh) at 50°C with N 2 carrier gas at a flow rate of 40 mL/min. Controls included uninoculated culture medium with and without 10% acetylene as well as inoculated culture medium without 10% acetylene. The latter control was used to determine if any isolates were capable of endogenous ethylene formation. 2.5 Raising of Antibiotic-Resistant Derivatives Antibiotic-resistant derivatives of those bacterial isolates originally isolated from CCM plates which were capable of reducing acetylene were raised in order to detect isolates in controlled environment experiments. Strains capable of growing on CCM plates containing 200 mg/L rifamycin were selected, i.e., P2b-2R, P18b-2R, and P19a-2R, and stored frozen at -80°C in cryovials containing 2.0 mL of isolating medium amended with 20% glycerol. Strains P2b-2R, P18b-2R, and P19a-2R were rifamycin-resistant derivatives of strains P2b, P18b, and P19a, respectively. All three wild-type strains were originally isolated from pine tissues from the Williams 12 Lake site. Strain P2b was isolated from within the surface sterilized stem of a pine seedling, strain P18b was isolated from within surface sterilized needles of another pine seedling, and strain P19a was isolated from the internal stem tissue of a third pine seedling. An ARA described earlier was performed on rifamycin-resistant derivatives to confirm their acetylene reducing activity. A rifamycin-resistant derivative of the fourth acetylene-reducing bacterial strain (C3b) selected for the seedling growth assays could not be raised. Therefore, detection of this strain in controlled environment experiments was determined by visual assessment on CCM plates. Strain C3b was originally isolated from within the surface sterilized stem of a cedar tree at the Boston Bar site. 2.6 Controlled Environment Experiments For the growth chamber experiments, glass tubes (150 mm x 25 mm in diameter) were 2/3 filled with a sand-Turface (montmorillonite clay, Applied Industrial Materials Corporation, Deerfield, IL) mixture (69% w/w silica sand; 29% w/w Turface; 2% w/w CaC0 3 ) . Each tube was fertilized to saturation with 17 mL of a nutrient solution (Chanway et al. 1988) which was modified by replacing K N 0 3 and Ca(N03)24H20 with Ca( 1 5 N0 3 ) 2 (5% 1 5 N label) (0.0576 g/L) (Chanway and Holl 1991) and Sequestrene 330 Fe (CIBA-GEIGY, Mississauga, Ont.) with Na 2FeEDTA (0.02 g/L). Other nutrients in the nutrient solution included (g/L): K H 2 P 0 4 , 0.14; MgS0 4 , 0.49; H 3 B0 3 , 0.001; MnCI24H20, 0.001; ZnS0 4 7H 2 0, 0.001; CuS0 4 5H 2 0 , 0.0001; and NaMo0 4 2H 2 0, 0.001 (Chanway and Holl 1988). Tubes were then autoclaved for 1 hour. 13 Pine and cedar seeds which were used in the assays were obtained from the British Columbia Ministry of Forests Tree Seed Centre, Surrey, B.C. Pine and cedar seed originated from provenances of similar location and elevation as the Williams Lake pine site and the Boston Bar cedar site, respectively. Both seed types were surface sterilized in separate batches by immersion in 30% hydrogen peroxide (H2O2) for 1 minute 30 seconds, followed by three 30 second rinses in sterile distilled water. The effectiveness of the surface sterilization was confirmed by imprinting sterilized seed on TSA and checking for microbial contamination two days later. Three surface sterilized seeds of either pine or cedar were then aseptically sown in each tube and covered with ca. 5 mm of autoclaved silica sand. Bacterial inoculum was prepared by thawing and streaking frozen cultures onto plates of the appropriate isolating medium. Strains P2b-2R, P18b-2R, and P19a-2R were cultured on CCM amended with 200 mg/L rifamycin and strain C3b was cultured on CCM. Following growth on plates, a loopful of each strain was separately inoculated into 1 L flasks containing 500 mL of the corresponding CCM broth. Flasks were then secured on a rotary shaker (150 rpm; room temperature) and agitated for a maximum of two days. A heat-killed suspension of strain P2b-2R was selected as another treatment in order to determine if P2b-2R mediated growth was due to factors such as production of phytohormones. Heat-killed P2b-2R was prepared by autoclaving broth containing strain P2b-2R for 1 hour. All bacterial cultures were harvested by centrifugation (10000 x g for 30 minutes), and resuspended in SPB. Strains P2b-2R and C3b were resuspended to a density of ca. 107 cfu/mL and strains P18b-2R and P19a-2R were resuspended to a density of ca. 14 10 6 cfu/mL. For inoculation, 5.0 mL of each bacterial suspension was pipetted into separate tubes. Control seeds received 5.0 mL of SPB. Tubes were placed in a growth chamber (Conviron CMP3244, Conviron Products Company, Winnipeg, MB). Photosynthetically active radiation (PAR) at canopy level was ca. 300 mols 'W 2 during an 18-h photoperiod, and 20°C/14°C day/night temperature cycle. Pine seedlings were thinned to the largest single germinant per tube 2 weeks after sowing. Cedar seedlings emerged at a slower rate than pine seedlings and were thinned to the largest single germinant per tube 3 weeks after sowing. Seedlings were watered as required with sterile distilled water until 6 weeks following sowing, after which seedlings were watered with the nutrient solution initially used to fertilize the tubes, but without Ca(15NC>3)2. The controlled environment experiment was repeated with the following exceptions: strains P2b-2R and C3b were resuspended in SPB to a density of ca. 106 cfu/mL and strains P18b-2R and P19a-2R were resuspended to a density of ca. 107 cfu/mL; seeds were inoculated with 4.0 mL of each bacterial suspension and control seeds received 4.0 mL of SPB before tubes were saturated; pine and cedar seedlings were thinned to the largest single germinant per tube 3 and 4 weeks after sowing, respectively, and sterile distilled water was used to water seedlings up to 3 weeks after sowing, after which seedlings were water with the nutrient solution not containing Ca(1 5NC>3)2. 15 2.7 Seedling Harvest Cedar and pine seedlings from the first growth trial were harvested 27 and 35 weeks after sowing, respectively. For the second assay, cedar and pine seedlings were harvested 35 and 42 weeks after sowing, respectively. All of the cedar seedlings from the strain C3b treatment in the first growth trial were used in surface sterilization tests and thus, it could not be determined if this strain was capable of providing fixed N to seedlings and/or promoting seedling growth. Prior to each harvest, seedling shoot heights from the surface of the sand in tubes were recorded. Bacterial colonization of seedlings was assessed for each treatment using five samples consisting of three randomly selected seedlings each. 2.8 Sampling for Rhizosphere Colonization Seedlings were removed from tubes and loosely adhering growth medium was shaken off of seedlings under aseptic conditions. Roots were then separated from shoots and the fresh weight of roots along with tightly adhering growth medium was recorded. Roots were then placed in SPB and shaken on a vortex mixer (1000 rpm) for 1 minute. Serial dilutions were made in 9 mL of SPB using 1 mL pipetted from the original solution. 0.1 mL aliquots of the serial dilutions were spread plated in triplicate onto plates of CCM, CCM amended with 200 mg/L rifamycin, and TSA and incubated for up to 1 week at room temperature and then checked for microbial growth. Samples were plated onto TSA to determine the type of bacterial contamination present. 16 Growth was seen on some CCM plates, but no bacterial growth was seen on any CCM plates amended with 200 mg/L rifamycin. Thus, bacteria could not grow when initially plated on media containing rifamycin. These results suggested that antibiotic masking may have been present, which is the temporary loss of an antibiotic resistant strain to grow in the presence of specific levels of antibiotics (Mclnroy et al. 1996). Replica plating techniques must be performed in order to test for antibiotic masking. Therefore, selected CCM plates which had bacterial growth were replica plated onto CCM plates supplemented with 200 mg/L rifamycin using a Replica Plating Tool (VWR Canlab, West Chester, PA). Plates were checked for microbial growth 1 week later. Bacterial contamination found on plates was identified using GC-FAME for the first and second controlled environment experiments for both pine and cedar. In addition, 16S rDNA analysis was used to identify contaminants found in the rhizosphere of seedlings from the first cedar growth trial. 2.9 Sampling for Endophytic Colonization Following plating of rhizosphere samples, endophytic colonization of stem and root tissues of seedlings was assessed. Stems were separated from foliage and then stems and roots were separately washed and their fresh weights were recorded. For the first cedar growth trial, roots and stems were separately immersed in 70% ethanol for 1 minute 30 seconds, then in 30% H2O2 for 13 minutes, followed by three 30 second rinses in sterile distilled water. Root and stem tissues were then imprinted on triplicate plates of TSA, CCM, and CCM amended with 200 mg/L 17 rifamycin to check for surface contamination of tissues. Due to the presence of bacterial growth on some imprint plates in the first cedar trial, pine tissues from the first assay were surface sterilized for 2 minutes in 70% ethanol, followed by 16 minutes in 30% H2O2, and then rinsed three times for 30 seconds in sterile distilled water. Assessment of imprint plates from this assay indicated that surface sterilization was still incomplete. Therefore, for the next sampling, which was with cedar seedlings from the second growth trial, root and stem tissues were immersed in 70% ethanol for 2 minutes, then in 30% H 2 0 2 for 20 minutes, followed by three 30 second rinses in sterile distilled water. Contamination was still seen on some imprint plates. Thus, pine tissues from the second growth trial, which was the final sampling, were surface sterilized for 2 minutes in 70% ethanol, followed by 21 minutes in 30% H2C_, and then rinsed three times for 30 seconds in sterile distilled water. Following surface sterilization, root and stem tissues were triturated using a mortar and pestle in a small volume of SPB. Triturated tissues were then serially diluted and 0.1 mL aliquots were spread plated in triplicate onto TSA, CCM, and CCM supplemented with 200 mg/L rifamycin and then checked for microbial growth 1 week later. For the first cedar trial, heavy fungal growth was seen on many plates. Therefore, plates were supplemented with 100 mg/L cycloheximide for all subsequent assays to assess both rhizosphere and endophytic colonization. 2.10 Nitrogen Analysis and Growth Response Seedlings not assessed for bacterial colonization were separated into roots and shoots and dried for 2 days at 65°C, after which dry weights were recorded. 18 Foliage from each treatment was pooled into six samples and then ground to a particle size <2 mm. Each sample was measured to 5.0 +/- 0.5 mg and sent to the Stable Isotope Facility at the University of California at Davis for determination of percent 1 5 N levels using a stable isotope mass spectrometer (Europa Scientific Integra). The N content of foliage (dry weight basis) was also assessed. The amount of fixed N in foliage was calculated using the formula provided by Rennie et al. (1978) which involves determining the percent N derived from the atmosphere (% Ndfa) as follows: % Ndfa = 1 - atom % 1 5 N excess (inoculated plant) atom % 1 5 N excess (uninoculated plant) x 100 2.11 Statistical Analysis Treatments were arranged in a completely randomized design with each treatment consisting of 40 tubes. However, because surface sterilization tests were performed on seedlings from different treatments prior to final harvests, the number of observations for shoot height, shoot dry weight, and root dry weight for each treatment varied. Analysis of variance (ANOVA) at« < 0.05 was performed to determine significant differences between treatments of plant growth responses, atom percent 1 5 N excess, foliar N percent, and foliar N'content. Since residuals were well distributed, data were left untransformed. Due to variable growth responses being reported in earlier studies investigating the effects of endophytic bacteria on gymnosperm growth (O'Neill et al. 1992a, Chanway and Holl 1993, Chanway et al. 1997, Chanway et al. 2000), treatment means for root dry weight, 19 shoot dry weight, and shoot height were separated using two-tailed Fisher's Protected Least Significant Difference (LSD). Treatment means for atom percent 1 5 N excess, foliar N percent and foliar N content were separated using one-tailed Fisher's protected LSD since seed inoculation with putative N-fixing bacteria would only result in a gain in N incorporation in seedlings. 20 SECTION 3: Results 3.1 Identification of Endophytic Bacteria and Acetylene Reduction Activity Tables 1 to 3 comprise the GC-FAME identities of 99 different isolates of endophytic bacteria isolated from pine and cedar at the three different sites. Both genera and species are listed, as many of the isolates could only be identified to the genus level. Isolates without superscripts were isolated from the stem tissue of trees. The genera Paenibacillus and Brevibacillus formerly belonged to the genus Bacillus and the genus Burkholderia originally belonged to the genus Pseudomonas. Many of the identities of the bacteria at the three different sites were similar, including those between the pine and cedar sites. The majority of endophytic bacteria isolated from the three sites belonged to the genera Bacillus and Paenibacillus. Bacterial species common to both pine and cedar included Paenibacillus polymyxa, P. gordonae, P. pabuli, Bacillus megaterium, B. mycoides, and B. pumilus. As well, a number of the bacteria isolated on both TSA and CCM at each of the sites had similar identities. Many of the strains identified by GC-FAME as being P. polymyxa could not reduce acetylene. In addition, although numerous isolates were capable of growing on CCM, which is a selective medium for N fixing bacteria, only four isolates which were originally isolated on CCM were capable of reducing acetylene. These strains were P2b, P18b, P19a, and C3b. The rifamycin-resistant strains P2b-2R, P18b-2R, and P19a-2R were also able to reduce acetylene. Strains P19b and P20b, which were originally isolated from the stem tissue of pine seedlings from the Williams Lake site on TSA, also exhibited acetylene reduction. Strain C3b was the only strain 21 Table 1. Genera and species of endophytic bacteria isolated from Williams Lake pine on TSA and CCM. TSA CCM Bacillus^235 Brevibacillus Paenibacillus 1 Bacillus mycoides Bacillus pumilus 2,5 Kocuria rosea Paenibacillus polymyxa 1 Bacillus Paenibacillus 1 Bacillus longisporus Bacillus megaterium 1,2 Bacillus mycoides Brevundimonas vesicularis Cellulomonas biazotea Kocuria kristinae Paenibacillus pabuli3 Paenibacillus peoriae 1 Paenibacillus polymyxa 1,3 isolated from stem of seedling isolated from stem of tree isolated from needles of seedling isolated from roots of seedling Table 2. Genera and species of endophytic bacteria isolated from Chilliwack Lake pine on TSA and CCM. TSA CCM Bacillus 1,2 Brevibacillus 1,2 Paenibacillus 4 Pseudomonas Bacillus licheniformis Bacillus megaterium Bacillus pumilus 4 Kocuria rosea Paenibacillus gordonae 3 Paenibacillus polymyxa 2,4 Bacillus Bacillus megaterium Bacillus pumilus Burkholderia pyrrocinia Paenibacillus gordonae 4 Paenibacillus polymyxa isolated from stem of seedling isolated from stem of tree isolated from needles of seedling isolated from needles of tree 22 Table 3. Genera and species of endophytic bacteria isolated from Boston Bar cedar on TSA and CCM. TSA CCM Bacillus 2,3 Brevibacillus Paenibacillus Pseudomonas 1,2 Arthrobacter agilis Bacillus megaterium Bacillus mycoides Bacillus pumilus 1,2 Bacillus sphaericus 1,2 Cellulomonas turbata 4 Paenibacillus gordonae Paenibacillus pabuli1 Paenibacillus polymyxa 1,2 Bacillus 2 4 Brevibacillus Paenibacillus Pseudomonas Burkholderia Bacillus halodenitrificans 4 Bacillus megaterium Bacillus mycoides Bacillus pumilus 1,2 Bacillus subtilis Paenibacillus gordonae 1,2 Streptoverticillium reticulum isolated from stem of seedling isolated from stem of tree isolated from needles of seedling isolated from needles of tree capable of reducing acetylene which was not isolated from the internal tissues of pine seedlings from the Williams Lake site. Using GC-FAME, strains P2b, P19b, and C3b could only be identified to the genus level as Paenibacillus and strains P18b, P19a, and P20b were identified as P. polymyxa. Strains P2b, P2b-2R, P18b, P19a, and C3b were also identified using more powerful 16S rDNA analysis. Strains P2b, P2b-2R and P18b showed high levels of homology with the two closely related species P. peoriae and P. polymyxa. Both of these strains are closely related to P. polymyxa strains isolated from the rhizosphere CF43 (AJ223989) and PMD230 (AJ223988) (Achouak et al. 1999). Strain C3b was phylogenetically affiliated to P. 23 amylolyticus and strain P19a belonged to the Flexibacter group and was closely related to Dyadobacter fermentans. 3.2 Rhizosphere Colonization When rhizosphere samples from the P2b-2R treatments from both the first and second trials for pine and cedar were assessed for colonization, strain P2b-2R was seen on CCM plates, but not on CCM plates supplemented with 200 mg/L rifamycin. CCM plates were then replica plated onto CCM plates amended with 200 mg/L rifamycin to test for antibiotic masking. Antibiotic masking was present as Pb-2R growth was seen on replica plates, which were then counted (Table 4). In the first and second pine trials, rhizosphere colony counts were obtained for strain C3b, which was visible on CCM plates following rhizosphere sampling of seedlings from the C3b treatments (Table 4). In the second cedar trial, strain C3b was observed on only some CCM dilution plates after the sampling of the rhizosphere of seedlings originating from C3b-inoculated seed. Therefore, a C3b rhizosphere colony count could not be obtained in the second cedar trial. In the first and second trials for pine and cedar, strains P18b-2R and P19a-2R could not be detected on plates of CCM or CCM amended with 200 mg/L rifamycin when rhizosphere samples from the P18b-2R and P19a-2R treatments, respectively, were sampled. Analysis of CCM plates and CCM plates supplemented with 200 mg/L rifamycin from all of the treatments from both trials for pine and cedar revealed no cross contamination of treatments. The identity of strain P2b-2R as P. peoriaelpolymyxa was confirmed using 16S 24 rDNA analysis upon re-isolation of the strain from P2b-2R-treated seedlings in the first cedar trial. Table 4 . Rhizosphere colony counts for strains P2b-2R and C3b (cfu/g fresh weight) (population means +/- standard error; n = 5) P2b-2R C3b First pine trial 1.5 +/- 0.5 x 105 3.9+/-0.5 x 10 5 Second pine trial 1.8 +/- 0.4 x 105 2.2 +/- 0.8 x 10 5 First cedar trial 2.0 +/- 0.5 x 105 Second cedar trial 1.7 +/- 0.7 x 105 3.3 Endophytic Colonization Endophytic colonization of pine and cedar seedlings from both trials was assessed using five root and five stem tissue samples from each treatment. For each treatment, five separate sets of triplicate CCM and TSA imprint plates for root and stem tissues were used to check for surface contamination. Bacterial growth was often seen on at least one root and one stem imprint plate from each treatment. No bacterial growth was observed on any of the CCM plates amended with 200 mg/L rifamycin. Whether or not contaminant bacteria were visible on CCM and/or TSA imprint plates, growth was often not seen on dilution plates. When bacterial growth was seen on dilution plates, it often only consisted of a few colonies. After attempts to surface sterilize root and stem tissue samples from the P2b-2R treatment in the first pine and cedar trials, P2b-2R colonies were seen on CCM root and stem imprint plates. Strain P2b-2R was not visible on any of the CCM root and stem imprint plates or dilution plates following the attempted surface sterilization of 25 root and stem tissue samples from the P2b-2R treatment in the second pine and cedar trials. C3b colonies were noted on CCM root and stem imprint plates from the first pine trial and on CCM root imprint plates from the second pine and cedar trials f following attempts to surface sterilize tissue samples from the C3b treatments. Growth of strains initially used to inoculate pine and cedar seed was not observed in any of the other treatments on imprint plates or dilution plates. 3.4 Identification of Contaminant Bacteria The types of bacterial contaminants found in the rhizosphere were similar in all of the treatments across all four seedling harvests. Variovorax paradoxus was the most prevalent contaminant and GC-FAME identities of other contaminants occurring to a much lesser extent included isolates belonging to the genera Methylobacterium and Sphingobacterium. 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CD 00 CO 00 oo o d d + + + co CD CO oo oo o d d CD co o o LO o o o co co JD o co co in oo -<t Tf co -rf o o o o o o d d <6 d <5 d + O + + + 1^ - CO CO CN 00 CN CN CN + + LO CN CO CO CN CN T J CD CD CD ^ or or o or or CN CN i i ^ ^ JD ro fZ JD JD 00 CD O CM CM r - T-O Q_ Q_ 0_ Q_ JD co O CD CO ZJ ro o CD JD Qi o ro CD E o c CD I —^< C CD O c CD CO O c CD i _ CD 'CD O , J O CD J= JD " I CD CD • i ° >1 CD C CO £ CD CD til CO CD CD CD JZ c CD JZ JZ ^ p to tz CD E ro CD v_ +-» CD E o co CL 3 T3 CD T3 C Zl 2 CD CD 5 H. cS ro oo > CN had quite similar atom % 1 5 N excess levels in foliage (Tables 7, 8). In contrast, foliar atom % 1 5 N excess values for pine and cedar seedlings treated with strain P2b-2R were lower in the second growth trials than in the first growth trials. This translated into seedlings from the P2b-2R treatments in the second pine and cedar trials deriving 66% and 56%, respectively, of their foliar N from the atmosphere compared to 30% and 23% in the first pine and cedar trials. For the first growth trials for both pine and cedar, atom % 1 5 N excess values in foliage were lowest for seedlings originating from heat-killed P2b-2R-inoculated seed. Heat-killed P2b-2R seedlings from the first pine and cedar trials derived 72% and 60%, respectively, of their foliar N from the atmosphere. However, by the time pine and cedar seedlings were harvested from the second growth trials, there was no significant difference in foliar atom % 1 5 N excess values between pine and cedar seedlings treated with heat-killed P2b-2R and controls. In the first pine trial, inoculation of seed with strains C3b and P18b-2R resulted in seedlings with significantly lower atom % 1 5 N excess levels in foliage than control seedlings, which translated to C3b and P18b-2R seedlings deriving 17% and 10%, respectively, of their foliar N from the atmosphere. However, by the time pine seedlings from the second growth trial were harvested, the C3b and P18b-2R treatments had no significant difference in foliar atom % 1 5 N excess values from the control treatment. Atom % 1 5 N excess values in foliage for seedlings originating from P19a-2R-inoculated seed were not significantly different from control seedlings in either the first or second pine growth trials. 29 Results from the first and second cedar trials indicated no significant difference in foliar atom % 1 5 N excess values between seedlings treated with either strain P18b-2R, P19a-2R, or C3b and control seedlings. The first cedar trial was the only trial in which foliar N contents were significantly different in control seedlings than in other treatments. Control seedlings had significantly less foliar N contents than all other treatments. 3.6 Seedling Growth Response Seedling growth response measurements included root dry weight, shoot dry weight, and shoot height. Results from the first pine trial indicated no significant difference in seedling root dry weights between controls and all other treatments (Table 5). However, shoot dry weights were significantly less in seedlings treated with the strains P2b-2R, C3b and heat-killed P2b-2R compared to control seedlings. The most depressed shoot growth was observed in the heat-killed P2b-2R treatment. The first pine trial was the only trial in which control seedlings had significantly greater shoot heights than seedlings from all other treatments. By the time seedlings from the second pine trial were harvested, the only treatment which yielded significantly different root and shoot dry weights than the control treatment was the P2b-2R treatment, which had the lowest root and shoot dry weights (Table 6). Also, the second pine trial was the only trial in which no significant difference in shoot height was observed between any of the treatments. Growth response data from the first cedar trial showed that both root and shoot dry weights were significantly different in seedlings originating from P2b-2R 30 and heat-killed P2b-2R-inoculated seed compared to control seedlings (Table 7). The heat-killed P2b-2R treatment had the lowest root and shoot dry weights, followed by the P2b-2R treatment. Seedlings treated with heat-killed P2b-2R were also the only seedlings which had significantly lower shoot heights than control seedlings. In the second cedar trial, seedlings from the P2b-2R treatment had significantly different root and shoot dry weights relative to control seedlings (Table 8). Root and shoot dry weights were lowest in the P2b-2R treatment, which was similar to what was seen in the second pine trial. Seedlings originating from C3b-inoculated seed had significantly lower root weights than control seedlings. Shoot heights were also significantly lower in both the P2b-2R and C3b treatments compared to the control treatment. When comparing the growth response data between the first and second pine trials and the first and second cedar trials, shoot dry weights of seedlings from the P2b-2R treatments were notably less in the second pine and cedar trials. 31 SECTION 4: Discussion 4.1 Identification of Endophytic Bacteria and Acetylene Reduction Activity The GC-FAME identities of bacteria isolated from plant tissue in the field indicated that many of the bacteria colonizing the internal tissues of pine and cedar were common to both tree species. The likely source of many of these bacteria is the soil, as many of the isolates identified are common soil bacteria such as P. polymyxa, P. gordonae, P. pabuli, B. megaterium, B. mycoides, B. pumilus, B. licheniformis, B. sphaericus, and B. subtilis (Slepecky 1992). A Paenibacillus strain (Pw2) has been isolated from the internal root tissues of lodgepole pine and has been shown to colonize seedlings systemically (Shishido et al. 1999). In the present study, since many of the bacteria isolated from aerial plant parts are common soil bacteria, these bacteria may have also been able to colonize plants systemically. Two of the strains which had an active nitrogenase enzyme based on acetylene reduction assay results (P2b and P18b) were identified using 16S rDNA analysis as being closely related to P. polymyxa strains isolated from the rhizosphere CF43 (AJ223989) and PMD230 (AJ223988) (Achouak et al. 1999). The pine endophytic Paenibacillus strain Pw-2R has also been shown to possess nitrogenase activity (Shishido and Chanway, unpublished data). Many of the strains identified as P. polymyxa using GC-FAME could not reduce acetylene, as not all strains of this species can fix N (Postgate 1998). 16S rDNA analysis revealed strain C3b was phylogenetically affiliated to P. amylolyticus, which is a species known to be able to degrade starch, and contains endophytic strains (Reiter et al. 2002, Sakiyama et al. 2001). In addition, strain P19a-2R was closely related to 32 Dyadobacter fermentans, which has recently been described as a Gram-negative endophytic bacterium (Chelius and Triplett 2000). The fact that very few strains which grew on CCM plates exhibited acetylene reduction may have been due to either one or a combination of two factors. Bacteria may not have been N fixers and/or the ARA used may have failed to activate the nitrogenase enzyme in N fixing bacteria. The results are in contrast to an earlier study in which most of the bacteria isolated from a soil on CCM were capable of reducing acetylene (Rennie 1981). If some bacteria growing on the N-deficient CCM were not capable of fixing N, their growth may have been facilitated by utilization of the small amount of N present in the yeast extract in the medium (Rennie 1981). Soil chemical analysis did not indicate many differences in N content and mineralizable N between the three sampling sites (Appendix 1-Table A1), but a possible reason as to why all but one of the strains capable of reducing acetylene were isolated from pine tissues from the Williams Lake pine site may be that the litter layer at the Williams Lake site was only 1 to 2 cm deep, whereas litter layers at the other two sites were thicker. The litter layer ranged from 5 to 12 cm at the Chilliwack Lake pine site and 15 to 18 cm at the Boston Bar cedar site. In addition to the litter layer being much smaller at the Williams Lake site, the colder and drier conditions at this site would likely contribute to this smaller litter layer decomposing at a slower rate than litter layers at the other two sites. This suggests that N availability was most limiting at the Williams Lake site and this may have increased the opportunity for diazotrophs to colonize pine tissues and provide plants with N. 33 4.2 Rhizosphere Colonization Despite the difference in time between the harvesting of pine and cedar seedlings from the first and second trials, results indicated that P2b-2R and C3b rhizosphere populations had stabilized at ca. 105 cfu/g fresh weight. Since no carbon source was added to the growth medium, it is likely the growth of strains P2b-2R and C3b was sustained by root exudates. Despite the fact that strain P2b was originally isolated from pine seedling tissue, it was shown that substantial populations of its rifamycin-resistant derivative could persist in the rhizosphere of not only pine seedlings, but also cedar seedlings. Interestingly, the largest rhizosphere populations of strain C3b, which was initially isolated from cedar stem tissue, were found on the pine seedlings. In both the first and second growth trials for pine and cedar, assessment of plates from the P18b-2R and P19a-2R treatments indicated that neither strain could be detected in the rhizosphere. Therefore, root exudates alone may not have provided enough nutrients for the survival of these two strains. Even though the surrounding growth medium was not sampled for bacterial colonization, it is unlikely that these two strains were alive in that region in any of the four samplings, as the number of soil organisms decreases with increasing distance from a root surface (Paul and Clark 1989). 4.3 Antibiotic Masking An effective and inexpensive method for marking bacteria for recovery from plants following inoculation is the use of spontaneously-generated antibiotic-resistant 34 derivatives of parental bacterial strains (Kloepper and Beauchamp 1992). A number of mechanisms exist as to how bacteria become resistant to antibiotics. These include changing the structure of a target enzyme, producing inhibitory compounds, developing an uptake system for destroying or altering the antibiotic, and bypassing the metabolic step the antibiotic is affected by (Dawes and Sutherland 1991). Antibiotic masking, which is the temporary loss of antibiotic resistance, was reported in a study by Mclnroy et al. (1996), in which rifampicin-resistant bacteria did not grow on TSA supplemented with rifampicin following the attempted recovery of these bacteria from surface sterilized root tissues. Colonies which grew on TSA were transferred to TSA amended with rifampicin, upon which growth was seen. No antibiotic masking was observed when rifampicin-resistant bacteria were isolated from external root tissue surfaces. One explanation for the antibiotic masking observed in internal root tissues was growth of the endophyte on TSA plates amended with rifampicin may have been affected by internal plant extracts. It was recommended that future studies using antibiotic resistant bacteria to inoculate seeds or plants should test for antibiotic masking upon recovery of bacteria. In a later study (Nairn 2000), antibiotic masking was noted with two rifamycin-resistant bacterial strains in the rhizosphere of hybrid spruce seedlings. One year following root inoculation, Paenibacillus strain Pw-2R and Pseudomonas fluorescens strain Sw5-RN grew on TSA plates, but not on TSA plates supplemented with rifamycin. However, both strains were present on the rifamycin containing TSA plates following the replica plating of TSA plates onto rifamycin containing plates. It was reported that the mechanism(s) which allowed these rifamycin-resistant strains 35 to grow on a particular level of rifamycin may have been suppressed following root inoculation. One explanation for the antibiotic masking was strains may have decreased production of inhibitory compounds since antibiotics were not present in the rhizosphere of seedlings. Another theory which was stated was that a logarithmic population of cells would be present in colonies of the two strains which were replicate plated onto TSA plates containing antibiotics. Some of the cells would initially be exposed to lower levels of antibiotics by being physically shielded in the colonies and this may have allowed some of the cells enough time to produce compounds required for antibiotic resistance. In the present study, antibiotic masking was observed with strain P2b-2R in the rhizosphere of pine and cedar seedlings from both trials. Since strain P2b-2R was not exposed to rifamycin in the glass tubes, mechanism(s) which allowed this strain to become resistant to and grow in 200 mg/L rifamycin may have been suppressed. It is likely that the mechanism(s) for resistance to this level of rifamycin were reactivated only after repeat exposure to 200 mg/L rifamycin. Further investigation is required to determine why antibiotic masking occurs. 4.4 Endophytic Colonization It was surprising that bacterial growth was often seen on imprint plates, given that surface sterilization treatments incorporated a much higher concentration of chemical sterilant for longer periods of time than treatments used in previous studies to surface sterilize gymnosperm seedling tissues (Chanway et al. 1994, Shishido et 36 al. 1995, Shishido 1997). However, seedling tissues from these earlier studies were imprinted only on TSA plates, and not CCM. Despite the failure of the surface sterilization of root and stem tissue samples from the P2b-2R treatment in the first pine and cedar trials, endophytic colonization of root and stem tissues of pine and cedar in the first trials by strain P2b-2R cannot be ruled out. The presence of strain P2b-2R on CCM stem imprint plates at least suggests that this strain was capable of colonizing the surface of stem tissues of P2b-2R-treated seedlings from the first pine and cedar trials. Endophytic colonization by strain C3b of root and stem tissues in the first pine trial and of root tissues in the second pine and cedar trials cannot be ruled out following the unsuccessful surface sterilization of tissue samples from these treatments. In the first pine trial, strain C3b was at least able to colonize stem surface tissues of seedlings from the C3b treatment, as this strain was visible on CCM stem imprint plates. In light of the unsuccessful surface sterilization of tissues, future experiments should employ the use of immunological techniques such as immunofluorescent antibody staining in conjunction with microscopic techniques such as scanning confocal laser microscopy in order to determine endophytic colonization of root and stem tissues by strains P2b-2R and C3b. 4.5 Foliar Atom % 15N Excess Atom % 1 5 N excess values in pine and cedar foliage from the first and second growth trials indicated that strain P2b-2R, which was a rifamycin-resistant derivative of a strain originally isolated from the internal stem tissue of a pine seedling, was the 37 only strain capable of consistently fixing N for utilization by not only pine seedlings, but also cedar seedlings. In fact, relative to the length of time seedlings were grown, cedar seedlings from the P2b-2R treatment appeared to derive a greater amount of foliar N from the atmosphere than pine seedlings from the same treatment. P2b-2R-treated pine seedlings from the first trial, which were harvested 35 weeks after sowing, derived 30% of their foliar N from the atmosphere, whereas P2b-2R-treated cedar seedlings from the second trial, which were also harvested 35 weeks after sowing, derived 56% of their foliar N from the atmosphere. Nevertheless, by the time both pine and cedar seedlings originating from P2b-2R-inoculated seed from the second trials were harvested, both plant species derived very significant amounts of foliar N from the atmosphere. The amount of fixed N assimilated by pine and cedar seedlings associated with strain P2b-2R would be in the range of the N-fixing bacterial association found in sugar cane (Lima et al. 1987, Boddey et al. 1991). In another study, inoculation of kallar grass (Leptochloa fusca) with two N-fixing bacteria (Klebsiella pneumoniae strain NIAB-1 and Beijerinckia sp. strain lso-2) previously isolated from the rhizosphere of this plant resulted in 65% to 80% of plant N being derived from atmospheric N fixation (Malik et al. 1987). One variety of rice (Oryza sativa L.) derived nearly 70% of its N from the atmosphere following inoculation with an Azospirillum strain originally isolated from the roots of rice (Malik et al. 1997). A number of experiments performed on wheat (Triticum aestivum) found that N fixation by a Bacillus strain isolated from the wheat rhizosphere and Azospirillum was responsible for 14% to 38 63% of plant N (Larson and Neal 1978, Rennie and Larson 1979, 1981, Rennie et al. 1983). Foliar atom % 1 5 N excess levels for both pine and cedar seedlings from the P2b-2R treatment were lower in the second growth trials than in the first growth trials. Pine and cedar seedlings from the second growth trials were harvested 7 and 8 weeks, respectively, after pine and cedar seedlings from the first growth trials were harvested. During this time, strain P2b-2R continued to fix N which was assimilated by seedlings. This led to atom % 1 5 N excess values in foliage decreasing as seedlings treated with strain P2b-2R became increasingly dependent on fixed N as a source of N with time. It is likely that foliar atom % 1 5 N excess levels would have dropped even further if pine and cedar seedlings originating from P2b-2R-inoculated seed were allowed to grow. An explanation for seedlings treated with heat-killed P2b-2R yielding the lowest foliar atom % 1 5 N excess levels in the first pine and cedar trials could be that prior to seed inoculation, the broth containing strain P2b-2R, which was to be used for the heat-killed P2b-2R treatment, had an optical density (A64o) of 0.08, whereas the P2b-2R broth had an A ^ o of 0.04. This would indicate that the broth to be used for the heat-killed P2b-2R treatment contained a greater number of cells than the broth to be used for the P2b-2R treatment. This greater number of cells would be capable of fixing more N and when this broth was autoclaved for 1 hour, these cells would have lysed and released their fixed N. Following centrifugation of the heat-killed P2b-2R broth, some of the fixed N would have remained in the bacterial pellet. Thus, the SPB used to resuspend the pellet and inoculate seed with would contain 39 fixed N which would be readily available for plant uptake. This would be in contrast to the slower and more regulated release of fixed N from living cells, such as in the P2b-2R treatment. Unlike the first trials, foliar atom % 1 5 N excess values in the second pine and cedar trials were not significantly lower in seedlings from the heat-killed P2b-2R treatments than in control seedlings. This may have been due to P2b-2R cells fixing N at a less intense rate in the broth to be used for the heat-killed P2b-2R treatment in the second pine and cedar trials. In addition, the heat-killed P2b-2R broth used in the second trials had an A ^ o of 0.06 compared to an A 6 4o of 0.08 in the first pine and cedar trials. This indicates that pine and cedar seed in the second trials were inoculated with fewer heat-killed P2b-2R cells. Therefore, less fixed N would have been present in the SPB used to inoculate seed in the second trials. It is likely that by the time pine and cedar seedlings from the longer duration second growth trials were harvested, this smaller amount of fixed N present in the heat-killed P2b-2R treatment had been entirely used by seedlings and seedlings had to resort to only using N from the growth medium. This is in contrast to the P2b-2R treatment, in which living cells were continually fixing N for seedlings to utilize. Foliar atom % 1 5 N excess levels from the first pine trial indicated that strain C3b was fixing N which was being utilized by seedlings. However, despite strain C3b being found in the rhizosphere of seedlings in the second pine trial, atom % 1 5 N excess values in foliage showed that fixed N was not being assimilated by foliage at the time of harvest. This would suggest that by the time pine seedlings from the second growth trial were harvested, strain C3b was not fixing N. 40 In the first pine trial, strain P18b-2R was not found on any of the serial dilution plates used to assess for rhizosphere colonization, but foliar atom % 1 5 N excess values indicated that this strain was capable of fixing N, which was being utilized by seedlings. These results indicate that strain P18b-2R had died some time prior to seedling harvest and that seedlings were assimilating fixed N from dead P18b-2R cells which were capable of fixing N when they were alive. Therefore, as was the case with heat-killed P2b-2R-treated seedlings from the second pine and cedar trials, seedlings would eventually use up the entire supply of fixed N provided by dead cells and would have to solely rely upon the N available in the growth medium as a N source. This may have occurred by the time seedlings from the second pine trial were harvested, as atom % 1 5 N excess levels in foliage for seedlings treated with strain P18b-2R were not significantly different from those of control seedlings. Foliar atom % 1 5 N excess values for seedlings treated with strain P19a-2R in the first and second trials for pine and cedar and for seedlings treated with strain C3b in the second pine and cedar trials indicated that these strains were not fixing N for utilization by seedlings. Strain P18b-2R, which is phylogenetically very similar to strain P2b-2R, was not detected in the rhizosphere of pine and cedar seedlings in both trials and seedlings from the first and second cedar trials and the second pine trial treated with this strain had foliar atom % 1 5 N excess levels which were not significantly different from control seedlings. Unlike pine and cedar seedlings treated with strain P2b-2R, growth depression was not observed in P18b-2R-treated pine and cedar seedlings in both trials relative to control seedlings. This indicates that minor genetic differences between strains can result in profoundly different 41 effects on the survival of bacteria, seedling growth, and foliar N derived from the atmosphere. Atom % 1 5 N excess values in foliage were similar for control seedlings between the first and second trials for pine and between the first and second trials for cedar. This suggests that the amount of foliar 1 5 N taken up by pine controls in both of the trials and by cedar controls in both of the trials remained similar despite the difference in time of seedling harvest between the two growth trials. 4.6 Foliar N Content It is not clear if the significant increase in foliar N contents in the first cedar trial in seedlings originating from P2b-2R and heat-killed P2b-2R-inoculated seed was due to fixed N being utilized by seedlings, as no significant increases in foliar N contents over controls were observed in any of the other growth trials. As well, foliar N contents in the first cedar trial were significantly greater in the P18b-2R and P19a-2R treatments than in the control treatment, even though these two strains were not detected in the rhizosphere and foliar atom % 1 5 N excess values indicated these two strains were not fixing N for utilization by seedlings. Although atom % 1 5 N excess values in foliage were significantly less in seedlings from the P2b-2R treatments in the first and second pine trials and the second cedar trial, the foliar N contents of seedlings from the P2b-2R treatments in these trials were not significantly different from control seedlings. Other experiments using a Bacillus and Azospirillum strain to inoculate wheat have reported no significant increase in N yield, in spite of inoculated treatments having significantly 42 less 1 5 N enrichment than controls (Rennie et al. 1983, Rennie and Thomas 1987, Kucey 1988). Rennie et al. (1983) and Rennie and Thomas (1987) believed the lack of increase in N content in inoculated treatments was due to a compensatory mechanism in which less N was taken up from soil when fixed N was assimilated by plants and wee versa. The compensatory mechanism may also explain why foliar N contents did not increase in P2b-2R-treated seedlings relative to control seedlings in three of the four harvests. However, it is possible that over a longer period of time, seedlings from the P2b-2R treatments would have significantly greater foliar N contents than those from the other treatments, in which fixed N was not being utilized by seedlings. The reason for this is that the N present in the growth medium would eventually be entirely used up by seedlings and only those seedlings associated with bacteria actively fixing N, such as strain P2b-2R, would continue to obtain N. 4.7 Seedling Growth Response Inoculation with strain P2b-2R was the only treatment in which growth inhibition was observed in seedlings relative to control seedlings in both the first and second trials for pine and cedar. In the first pine trial, seedlings from the P2b-2R treatment had significantly lower shoot dry weight and height than control seedlings. However, all of the treatments in the first pine trial had seedlings with significantly lower shoot height compared to control seedlings. In the second pine trial and the first and second cedar trials, both root and shoot dry weights were significantly lower in the P. peoriaelpolymyxa strain P2b-2R-treated seedlings than in control seedlings. 43 In the second cedar trial, seedlings from the P2b-2R treatment also had significantly lower shoot heights relative to control seedlings. A Paenibacillus strain (L6-16R) has previously been shown to inhibit lodgepole pine seedling growth compared to uninoculated controls (Holl and Chanway 1992, Chanway and Holl 1994). Holl and Chanway (1992) found shoot growth to be inhibited in seedlings from one of four provenances. The reason given for the depressed growth was seedlings from the four provenances may have had a differential sensitivity to the mechanism of growth promotion by strain L6-16R, which could have lead to a different growth response. Chanway and Holl (1994) discovered seedlings inoculated with strain L6-16R and outplanted at one site had significantly less shoot dry weights than noninoculated control seedlings. It was speculated the growth depression was due to the relatively large L6-16R rhizosphere population (ca. 104 cfu/g root tissue) using up seedling or soil resources. Two Paenibacillus strains (L5 and L6) have also been shown to inhibit growth of white spruce (Picea glauca Voss.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings (O'Neill et al. 1992b). In addition, strain L5 has been shown to lower total dry weights and heights in western hemlock (Tsuga heterophylla (Raf.) Sarg.) seedlings from one provenance (Chanway 1995). In the O'Neill et al. (1992b) study, strain L5 was responsible for causing white spruce seedlings to have significantly lower root dry weights 13 weeks after sowing and significantly less shoot dry weights 26 weeks after sowing, relative to uninoculated control seedlings. This strain also reduced root and shoot dry weights of Douglas-fir seedlings 13 weeks after sowing. Inoculation of white spruce seed with strain L6 significantly 44 reduced the root dry weights of seedlings 13 weeks after sowing. Douglas-fir seedlings originating from L6-inoculated seed had significantly less shoot dry weights 13 weeks after sowing and significantly lower root dry weights 26 weeks after sowing compared to uninoculated control seedlings. One explanation given for the growth depression was similar to that given in the Chanway and Holl (1994) study, whereby the presence of a relative large L5 and L6 population in the rhizosphere may have lead to nutrient immobilization. Another reason proposed for the seedling growth inhibition was the strains may have surpassed a threshold population size, beyond which phytohormone production occurred at levels which depressed seedling growth. A similar explanation was given for the growth response observed in maize (Zea mays) following inoculation with different concentrations of rhizobacteria (Fallik et al. 1988). Reduced root weights and shoot lengths seen in wheat seedlings inoculated with P. polymyxa may have also been a phytohormonal effect (Lindberg et al. 1985). Paenibacillus strain L6 has been shown to produce indoleacetic acid in vitro (Holl et al. 1988). A significant linear relationship was found correlating the production of indoleacetic acid in vitro by rhizobacteria with reduced sugar beet (Beta vulgaris convar. crassas (Alef.) J . Helm.) root elongation, and it was speculated that as a consequence of phytohormone production, the rhizobacteria may have acted as "minor pathogens" (Loper and Schroth 1986). In the present study, growth depression was also seen in seedlings from the heat-killed P2b-2R treatments. Shoot dry weights were significantly lower in the first pine trial and root and shoot dry weights and shoot heights were significantly lower in the first cedar trial compared to control seedlings. 45 These results suggest that the growth depression observed in seedlings originating from P2b-2R-inoculated seed may have also been due to phytohormone production by strain P2b-2R at levels which were inhibitory to seedling growth. However, there is one significant difference between the aforementioned studies of plant growth inhibition by Paenibacillus and the current work with diazotrophs, particularly strain P2b-2R. The substantial amount of foliar N derived from the atmosphere in P2b-2R-treated seedlings suggests that a hitherto unknown mutualism may be operating. Nitrogen fixation is an energetically "expensive" process, so seedlings deriving N from P2b-2R nitrogenase may have been providing carbon substrates to the bacterium to drive N fixation. In addition, if P2b-2R did colonize seedlings systematically, it is possible that seedling growth would be temporarily depressed while the symbiosis developed. This phenomenon has been repeatedly observed when ectomycorrhizal fungi infect plant roots and begin synthesizing a mature mycorrhizal root tip (Vozzo and Hacskaylo 1971, Harley and Smith 1983, Gagnon et al. 1987). If a similar situation exists with strain P2b-2R and gymnosperm seedlings, seedling growth would be expected to decline for a time and then accelerate once the mutualism is fully established. The reason for the heat-killed P2b-2R treatment yielding seedlings with the lowest shoot dry weights in the first pine trial and the lowest root and shoot dry weights in the first cedar trial was possibly due to seed from this treatment being inoculated with the greatest number of cells, which in turn may have been able to produce the greatest amount of phytohormone. The lysing of cells as a consequence of autoclaving the P2b-2R broth for the heat-killed treatment would 46 have resulted in the release of this large amount phytohormone from cells. Some of this phytohormone would have remained in the bacterial pellet following the centrifugation of cells. The SPB used to resuspend the pellet and inoculate seed with would therefore contain a large amount of readily available phytohormone for plant use. This would be in contrast to the slower and more regulated release of phytohormone from living cells, such as in the P2b-2R treatment. As a result of this, it is likely that the greater amount of phytohormone in the heat-killed P2b-2R treatment was responsible for inhibiting seedling growth more. No growth depression was observed in seedlings from the heat-killed P2b-2R treatment compared to control seedlings in the longer duration second pine and cedar trials. This may have been due to P2b-2R cells producing phytohormone at a less intense rate in the broth to be used for the heat-killed P2b-2R treatment in the second trials than in the first trials. In addition, pine and cedar seed were inoculated with fewer heat-killed P2b-2R cells in the second trials. Therefore, phytohormone may not have been present at levels which were inhibitory to seedling growth in the second trials. Although seedlings from the second pine and cedar trials were allowed to grow longer than seedlings from the first pine and cedar trials, seedlings from the P2b-2R treatment had notably lower shoot dry weights in the second pine and cedar trials. One explanation for this is phytohormone production may have been more active in P2b-2R cells in the second pine and cedar trials, which could have resulted in greater shoot growth inhibition. 47 Inoculation of pine seed with strain C3b resulted in seedlings with significantly lower shoot dry weights in the first pine trial and significantly lower root dry weights and shoot heights in the second cedar trial relative to control seedlings. However, seedlings from the C3b treatment in the second pine trial had no significant difference in any of the growth response measurements compared to control seedlings. Therefore, the relatively large rhizosphere population of strain C3b (105 cfu/g fresh weight) was not utilizing seedling resources to the extent that growth depression was occurring in the second pine trial. These results show that seedling growth depression in C3b-treated seedlings did not occur as consistently as in P2b-2R-treated seedlings. The reason for the growth inhibition by strain C3b in the first pine and second cedar trials is unknown. Since a heat-killed C3b treatment was not used in any of the assays, it is not known if the observed growth depression was a phytohormonal effect. 48 SECTION 5: Conclusions The results confirmed the presence of endophytic bacteria in the internal tissues of lodgepole pine and western red cedar. Many of the bacteria isolated from the internal tissues of both pine and cedar at the three different sites had similar identities. The most frequently occurring bacteria belonged to the genera Bacillus and Paenibacillus. The presence of diazotrophic bacteria within the surface sterilized tissues of pine and cedar was also confirmed. Four of the diazotrophic bacteria were selected for use in controlled environment experiments in order to determine their ability to provide biologically significant quantities of fixed N to pine and cedar seedlings under N deficient conditions. This included Paenibacillus amylolyticus C3b, rifamycin-resistant derivatives of P. peoriae/polymyxa P2b and P18b, and a rifamycin-resistant derivative of strain P19a, which was closely related to Dyadobacter fermentans. Results from the controlled environment experiments indicated that pine and cedar seedlings were capable of deriving biologically significant amounts of fixed N following inoculation with the rifamycin-resistant derivative (P2b-2R) of strain P2b. Although plants of agricultural importance have been reported to derive significant amounts of fixed N following inoculation with diazotrophic bacteria, this is the first report of such an occurrence in pine and cedar. Pine and cedar seedlings originating from P2b-2R-inoculated seed derived a greater amount of foliar N from the atmosphere the longer they were grown. Longer growth trials should be conducted to find out if P2b-2R-treated seedlings are capable of deriving even greater amounts of fixed N than in the present study. Further experimentation is 49 also required to determine if diazotrophic bacteria are able to supply significant amounts of fixed N to pine and cedar in the field. Inoculation of pine and cedar seed in the first trial with heat-killed P2b-2R also resulted in seedlings deriving a very significant amount of foliar N from the atmosphere. However, foliar atom % 1 5 N excess values in pine and cedar seedlings treated with heat-killed P2b-2R were not significantly different from those of control seedlings in the longer duration second trials. This was likely due to pine and cedar seedlings from the second trials having used up the entire supply of fixed N provided by heat-killed P2b-2R cells. As a result of this, seedlings had to resort to only using N from the growth medium as a N source. Strains C3b and P18b-2R provided less significant amounts of fixed N to only pine seedlings in the first trial. Pine and cedar seedlings originating from P19a-2R-inoculated seed did not derive significant quantities of foliar N from the atmosphere in either trial. Despite significant amounts of fixed N being provided to seedlings by strain P2b-2R, increases in foliar N content were not common. Similar to previous reports, the lack of increase in N content may have been due to less N being taken up from the growth medium when fixed N was incorporated by seedlings and wee versa. Growth depression was routinely seen in pine and cedar seedlings originating from P2b-2R-inoculated seed. Similar to reports in earlier studies, strain P2b-2R may have been producing phytohormones at levels which were inhibitory to seedling growth. Alternatively, the growth inhibition may have been a consequence of energy being diverted from seedlings due to the establishment of a symbiosis between 50 strain P2b-2R and seedlings. Further research is required to determine the exact cause of this growth depression. As had been observed previously, antibiotic masking was present when antibiotic-resistant bacteria were isolated from external root surfaces. Strain P2b-2R was only seen on agar media without antibiotics upon primary isolation from the external root tissues of pine and cedar seedlings from both growth trials. However, P2b-2R growth was observed on the same agar media with antibiotics after replica plating. Future studies using antibiotic-resistant bacteria to inoculate seeds or plants should incorporate techniques to test for antibiotic masking. Results also indicated that relatively large populations (105 cfu/g fresh weight) of strain P2b-2R could persist in the rhizosphere of both pine and cedar seedlings. However, due to persistent contamination on imprint plates used to test the effectiveness of the tissue surface sterilization techniques employed, endophytic colonization of pine and cedar seedlings by strain P2b-2R could not be verified. Immunological techniques should be used to confirm endophytic colonization. 51 LITERATURE CITED Achouak, W., Normand, P., and Heulin, T. 1999. 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Organic matter, Cu, and Zn content were not determined for Williams Lake soil. 63 APPENDIX 2 Statistical analysis 64 Table A2. Analysis of variance for effects on pine seedlings from the first growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b. ANOVA MODEL: Y = + INOC, where Y: effect, : constant, and INOC: inoculation with strains including uninoculated control. (a) Foliar atom % 1 5 N excess SOURCE SS DF MS F-ratio P INOC 5.855 5 1.171 77.322 0.000 ERROR 0.454 30 0.015 Fcrit - FQ.05 (1) (5,30) - 2.53 (b) Percent foliar N SOURCE SS DF MS F-ratio P INOC 0.067 5 0.013 2.207 0.080 ERROR 0.183 30 0.006 Fcrit - FQ.05(1)(5,30)- 2.53 (c) Total foliar N SOURCE SS DF MS F-ratio P INOC 150.172 5 30.034 1.942 0.117 ERROR 464.077 30 15.469 Fcrit - FQ.05 (1) (5,30) - 2.53 (d) Root weight SOURCE SS DF MS F-ratio P INOC 543.649 5 108.730 2.179 0.061 ERROR 5739.616 115 49.910 Fcrit - Fo.05(2)(5,115) _ 2.68 65 (e) Shoot weight SOURCE SS DF MS F-ratio P INOC 2074.463 5 414.893 9.675 0.000 ERROR 4888.735 114 42.884 Fcr i t - F Q . 0 5 ( 2 ) ( 5 , 1 1 4 ) - 2.68 (f) Shoot height SOURCE SS DF MS F-ratio P INOC 1620.357 5 324.071 7.223 0.000 ERROR 9197.065 205 44.864 Fcr i t - F r j . 0 5 ( 2 ) ( 5 , 2 0 5 ) - 2.63 66 Table A3. Analysis of variance for effects on pine seedlings from the second growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b. ANOVA MODEL: Y = + INOC, where Y: effect, : constant, and INOC: inoculation with strains including uninoculated control. (a) Foliar atom % 1 5 N excess SOURCE SS DF MS F-ratio P INOC 5.940 5 1.188 79.065 0.000 ERROR 0.451 30 0.015 Fcrit ~ FQ.05(1)(5,30)- 2.53 (b) Percent foliar N SOURCE SS DF MS F-ratio P INOC 0.010 5 0.002 0.419 0.832 ERROR 0.144 30 0.005 Fcrit - Fo.05(1)(5,30)~ 2.53 (c) Total foliar N SOURCE SS DF MS F-ratio P INOC 25.749 5 5.150 0.404 0.842 ERROR 382.107 30 12.737 Fcrit - Fo.05(1)(5,30)- 2.53 (d) Root weight SOURCE SS DF MS F-ratio P INOC 1447.493 5 289.499 2.890 0.017 ERROR 11319.523 113 100.173 Fcrit - FQ.05 (2) (5,113) ~ 2.68 67 (e) Shoot weight SOURCE SS DF MS F-ratio P INOC 3551.686 5 710.337 15.203 0.000 ERROR 5279.782 113 46.724 Fcrit - Fo.05(2)(5,113)~ 2.68 (f) Shoot height SOURCE SS DF MS F-ratio P INOC 276.394 5 55.279 1.969 0.085 ERROR 5669.836 202 28.068 Fcrit - FQ.05 (2) (5,202) - 2.63 68 Table A4. Analysis of variance for effects on cedar seedlings from first growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, and P19a-2R. ANOVA MODEL: Y = + INOC, where Y: effect, : constant, and INOC: inoculation with strains including uninoculated control. (a) Foliar atom % 1 5 N excess SOURCE SS DF MS F-ratio P INOC 9.144 4 2.286 114.553 0.000 ERROR 0.499 25 0.020 Fcrit - FQ.05 (1) (4,25) - 2.76 (b) Percent foliar N SOURCE SS DF MS F-ratio P INOC 0.038 4 0.010 12.767 0.000 ERROR 0.019 25 0.001 Fcrit - Frj.05 (1) (4,25) ~ 2.76 (c) Total foliar N SOURCE SS DF MS F-ratio P INOC 95.745 4 23.936 14.230 0.000 ERROR 42.053 25 1.682 Fcrit - Frj.05(1)(4,25)- 2.76 (d) Root weight SOURCE SS DF MS F-ratio P INOC 4314.244 4 1078.561 34.096 0.000 ERROR 2941.858 93 31.633 Fcrit - FQ.05 (2) (4,93) - 2.93 69 (e) Shoot weight SOURCE SS D F MS F-ratio P INOC 4833.790 4 1208.448 40.352 0.000 ERROR 2785.132 93 29.948 Fcrit - FQ.05 (2) (4,93) ~ 2.93 (f) Shoot height SOURCE SS DF MS F-ratio P INOC 1210.792 4 302.698 24.687 0.000 ERROR 2059.937 168 12.262 Fcrit - FQ.05 (2) (4,168) - 2.87 70 Table A5. Analysis of variance for effects on cedar seedlings from second growth trial inoculated with strains P2b-2R, heat-killed P2b-2R, P18b-2R, P19a-2R, and C3b. ANOVA MODEL: Y = + INOC, where Y: effect, : constant, and INOC: inoculation with strains including uninoculated control. (a) Foliar atom % 1 5 N excess SOURCE SS DF MS F-ratio P INOC 8.582 5 1.716 139.789 0.000 ERROR 0.368 30 0.012 Fcrit - FQ.05(1)(5,30)- 2.53 (b) Percent foliar N SOURCE SS DF MS F-ratio P INOC 0.004 5 0.001 1.184 0.340 ERROR 0.021 30 0.001 ns Fcrit ~ Fo.05(1)(5,30)- 2.53 (c) Total foliar N SOURCE SS DF MS F-ratio P INOC 7.293 5 1.459 0.745 0.596 ERROR 58.707 30 1.957 ns Fcrit - FQ.05 (1) (5.30) - 2.53 (d) Root weight SOURCE SS DF MS F-ratio P INOC 2045.538 5 409.108 10.400 0.000 ERROR 4484.371 114 39.337 Fcrit - FQ.05 (2) (5,114) - 2.68 71 (e) Shoot weight SOURCE SS DF MS F-ratio P INOC 2038.364 5 407.673 16.595 0.000 ERROR 2800.574 114 24.566 Fcrit ~ FQ.05 (2) (5,114) ~ 2.68 (f) Shoot height SOURCE SS DF MS F-ratio P INOC 216.612 5 43.322 3.387 0.006 ERROR 2571.108 201 12.792 Fcrit - FQ.05 (2) (5,201) ~ 2.63 72 

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