"Forestry, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Nairn, James D."@en . "2009-07-13T19:31:58Z"@en . "2000"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Soil microcosms may prove to be a useful tool in the development of\r\nstandardized methods for evaluating survival and persistence of soil organisms,\r\nparticularly genetically modified microorganisms, before field release. In this study, a\r\nrelatively simple soil microcosm, previously shown to be useful for predicting survival\r\nof a genetically modified pseudomonad in bulk soil and the rhizosphere of wheat,\r\nwas tested using two plant growth promoting rhizobacteria (PGPR) strains which\r\nwere naturally resistant to antibiotics, and spruce seedlings as the test plant. Bacillus\r\npolymyxa Pw-2R and Pseudomonas chloroaphis Sw5-RN were each inoculated onto\r\nbare soil and into the rhizosphere of spruce seedlings in field plots as well as in\r\nintact soil core microcosms that were incubated under controlled environmental\r\nconditions. Survival data collected over a two-year period were used to generate\r\npolynomial regressions that modeled the persistence of these strains in the field as\r\nwell as in the soil microcosms. Comparison of the slopes and intercepts of these\r\nregressions indicated that the intact soil core microcosm closely predicted the\r\nsurvival of both PGPR strains in bulk soil and in the spruce rhizosphere. These\r\nresults demonstrate that this small and inexpensive intact soil core microcosm may\r\nbe appropriate for general use in assessing field persistence of diverse soil\r\nmicroorganisms before environmental release.\r\nDuring this study, a temporary loss of antibiotic resistance was observed in\r\nboth PGPR strains, as they failed to grow on primary isolation agar media with\r\nantibiotics. However, they thrived on agar media with antibiotics if they were first\r\n\r\nisolated on agar without antibiotics. These results suggest that when using antibiotic\r\nresistance as a method to monitor rhizosphere microorganisms, the apparent\r\nmasking of antibiotic resistance should be evaluated thoroughly.\r\nBacillus polymyxa Pw-2R and Pseudomonas chloroaphis Sw5-RN are both\r\nplant growth promoting rhizobacteria. Pw-2R has previously been shown to be\r\ncapable of colonizing internal root and shoot tissues of hybrid spruce. There were no\r\nendophytic Pw-2R detected when attempts were made to isolate Pw-2R from\r\ninternal tissue in this study. Results further indicated spruce seedling growth was not\r\nsignificantly enhanced by the inoculation with the PGPR strains. These results are\r\nconsistent with the theory that positive results from PGPR seem to be linked with\r\nharsh growing sites and interaction with indigenous microorganisms from the\r\nprimary site of PGPR isolation."@en . "https://circle.library.ubc.ca/rest/handle/2429/10715?expand=metadata"@en . "4188949 bytes"@en . "application/pdf"@en . "VALIDATION OF A M I C R O C O S M DESIGNED FOR P R E - R E L E A S E RISK A S S E S S M E N T OF SOIL MICROORGANISMS USING PLANT G R O W T H PROMOTING RHIZOBACTERIA by J A M E S D.NAIRN B . S c , The University of Waterloo, 1989 B.Sc. (Agr), The University of Guelph, 1994 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE R E Q U I R E M E N T S FOR THE D E G R E E OF M A S T E R OF S C I E N C E in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Forest Science, Faculty of Forestry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA June, 2000 \u00C2\u00A9 James D. Nairn, 2000 In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of rdOLSt The University of British Columbia Vancouver, Canada ABSTRACT Soil microcosms may prove to be a useful tool in the development of standardized methods for evaluating survival and persistence of soil organisms, particularly genetically modified microorganisms, before field release. In this study, a relatively simple soil microcosm, previously shown to be useful for predicting survival of a genetically modified pseudomonad in bulk soil and the rhizosphere of wheat, was tested using two plant growth promoting rhizobacteria (PGPR) strains which were naturally resistant to antibiotics, and spruce seedlings as the test plant. Bacillus polymyxa Pw-2R and Pseudomonas chloroaphis Sw5-RN were each inoculated onto bare soil and into the rhizosphere of spruce seedlings in field plots as well as in intact soil core microcosms that were incubated under controlled environmental conditions. Survival data collected over a two-year period were used to generate polynomial regressions that modeled the persistence of these strains in the field as well as in the soil microcosms. Comparison of the slopes and intercepts of these regressions indicated that the intact soil core microcosm closely predicted the survival of both P G P R strains in bulk soil and in the spruce rhizosphere. These results demonstrate that this small and inexpensive intact soil core microcosm may be appropriate for general use in assessing field persistence of diverse soil microorganisms before environmental release. During this study, a temporary loss of antibiotic resistance was observed in both P G P R strains, as they failed to grow on primary isolation agar media with antibiotics. However, they thrived on agar media with antibiotics if they were first ii isolated on agar without antibiotics. These results suggest that when using antibiotic resistance as a method to monitor rhizosphere microorganisms, the apparent masking of antibiotic resistance should be evaluated thoroughly. Bacillus polymyxa Pw-2R and Pseudomonas chloroaphis Sw5-RN are both plant growth promoting rhizobacteria. Pw-2R has previously been shown to be capable of colonizing internal root and shoot tissues of hybrid spruce. There were no endophytic Pw-2R detected when attempts were made to isolate Pw-2R from internal tissue in this study. Results further indicated spruce seedling growth was not significantly enhanced by the inoculation with the P G P R strains. These results are consistent with the theory that positive results from P G P R seem to be linked with harsh growing sites and interaction with indigenous microorganisms from the primary site of P G P R isolation. iii TABLE OF CONTENTS Page Abstract ii Table of Contents iv List of Tables vi List of Figures ix Acknowledgements xi SECTION 1 Introduction 1 SECTION 2 Materials and Methods 8 2.1 Microorganisms 8 2.2 Seedlings 9 2.3 Field plot design and planting 10 2.4 Microcosms 11 2.5 Inoculation 12 2.6 Sample collection 13 2.6.1 Field sample collection 13 2.6.2 Microcosm sample collection 14 2.7 Soil sample processing 14 2.8 Rhizosphere sample processing 15 2.9 Sample plating and enumeration 16 2.10 Sample plating to uncover antibiotic resistance 17 2.11 Sampling for internal plant colonization 17 2.12 Statistical analysis 19 2.12.1 Microcosm validation 19 2.12.2 Plant growth promotion 21 SECTION 3 Results 22 3.1 Evaluation of experimental design 22 3.2 Microcosm evaluation 22 3.2.1 Bare soil 22 3.2.2 Rhizosphere 26 3.3 External plant colonization assay 34 3.4 Plant growth promotion assay 34 SECTION 4 Discussion 41 4.1 Microcosm validation 41 4.1.1 Bare soil 41 iv 4.1.2 Possible reasons for bacterial population decline 43 4.1.3 Antibiotic masking 45 4.1.4 Rhizosphere 51 4.2 Internal plant colonization 53 4.3 P G P R efficacy 54 S E C T O N 5 Conclusions 57 L ITERATURE CITED 60 A P P E N D I X 1 Statistical Analysis 65 v LIST OF T A B L E S Page Table A1 Analysis of variance and F-ratio test for the rate of population decline of Bacillus polymyxa (Pw-2R) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots 66 Table A2 Analysis of variance and F-ratio test for the coincidence of population decline of Bacillus polymyxa (Pw-2R) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots 67 Table A3 Analysis of variance and F-ratio test for the rate of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots 68 Table A4 Analysis of variance and F-ratio test for the coincidence of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots 69 Table A5 Analysis of variance and F-ratio test for the rate of population decline of Bacillus polymyxa (Pw-2R) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots 70 Table A6 Analysis of variance and F-ratio test for the coincidence of population decline of Bacillus polymyxa (Pw-2R) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots 71 Table A7 Analysis of variance and F-ratio test for the rate of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots 72 Table A8 Analysis of variance and F-ratio test for the coincidence of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots 73 vi Table A9 Table A10 Table A11 Table A12 Table A13 Table A14 Table AT5 Table A16 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R) 74 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R) 77 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R) 76 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R) 77 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN) 78 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN) 79 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R) 80 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce planted in laboratory intact soil core microcosms and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R) 81 vii Table A17 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R) 82 Table A18 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R) .... 83 Table A19 Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN) 84 Table A20 Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN) 85 viii LIST OF F IGURES Page Figure 1 Regression analysis for the survival of Bacillus polymyxa (Pw-2R) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. Populations are measured in colony forming units (CFU) remaining. Analysis of variance revealed the slopes, and the separation of the lines, are not significantly different at p<0.05. Each circle represents a single sample. 0 Represents overlapping data points 24 Figure 2 Regression analysis for the survival of Pseudomonas fluorescens (Sw5-RN) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. Populations are measured in colony forming units (CFU) remaining. Analysis of variance revealed the slopes, and the separation of the lines, are not significantly different at p<0.05. Each circle represents a single sample, > CO Q a) 2 T5 \u00E2\u0080\u00A2D ( B p m O J .\u00C2\u00ABi CD 5 I O CD CO \u00C2\u00A3 CO 3 _>. CO CD C O ^ co Si O) Jo id JO n o o c co o E ?2 CD CO \u00C2\u00BB- _>\u00C2\u00BB i r - co co 9 F \u00C2\u00B0 5. v 3 CL cn O sz o CO LU CO CD c CD c CD CD E c o 2 4- CD 8 ^ s i * 8 TJ CD CD CO 0 CO > CO o CD JOOJ US9JJ 6 / n J O 6o-| C L co J5 co >> CO TJ co o .2 CD CO >. CO \u00E2\u0080\u009E. CD 0) v - C O) o CD . a: o to CD 3 G) ii E c CD S> co co w g 8. o o + 3 CO J9 a, ^ x : CL D_ a) \u00E2\u0080\u00A2 co \u00C2\u00AB a) o > CD X> , CO - -\u00C2\u00BB\u00E2\u0080\u0094\u00C2\u00AB 5 F. O R a. i5 ^ co C T3 o \u00E2\u0080\u009E co .E ex C L co co CL CD \u00E2\u0080\u00A2 CO CD > o CO \u00C2\u00A3 c C CD CD CO CO 2 c CO o CO CL CD CO Q\u00C2\u00A3 CD \u00C2\u00A9 \u00C2\u00AB 0) += C L ^ \u00C2\u00AB i= co 30 microcosms was greater than the survival of the Pw-2R in the rhizospheres of spruce seedlings in the field plots. Testing for separation of regression slopes showed a significant difference (p<0.01) in the separation of the regression lines (Appendix-Table A6). Therefore the regression lines for the population decline of Bacillus polymyxa Pw-2R inoculated into field plot rhizospheres versus laboratory microcosm rhizospheres were parallel but were not coincidental. The populations of P. fluorescens Sw5-RN in spruce seedling rhizospheres in field plots and laboratory microcosms followed a similar pattern to that of B. polymyxa Pw-2R (Figures 3 and 4). The inoculation density of Sw5-RN was approximately 1.0 x 10 7 colony forming units (CFU)mL' 1 . Each seedling was inoculated by applying 5 mL of culture to the centre of the plug using a 10cc syringe and 18-gauge needle. The populations of Sw5-RN continued to decline and were below the level of detection by day 143 in the field rhizospheres and by day 165 in the laboratory rhizospheres. The limit of detection varied with the amount of root weight sampled. A lower limit of detection was possible with larger root samples. The minimum level of detection was 1.4 C F U g \" 1 fresh root weight for the largest (63.31 g) rhizosphere sampled. Time of sampling for the Sw5-RN rhizospheres was identical to that of the Pw-2R rhizospheres. Sampling stopped on day 186 and there were no target organisms (Sw5-RN) identified on this day. All of the samples were originally plated on tryptic soy agarwithlOO mg-L\"1 each of nalidixic acid and rifamycin along with the antifungal agents cycloheximide and nystatin (TSA**). Sampling resumed again on 31 CD ! o i CO \u00C2\u00A9 \u00E2\u0080\u00A2 o o m o! o o D or \u00E2\u0080\u00A2 5 CO or a: W d 2 II u. a: o o CO o o CM c o o o c 0) CO CO >\u00C2\u00BB CO Q C D #0 p 3 / / 0 o jo* \u00E2\u0080\u00A2 \u00E2\u0080\u00A2 C D * O / / o d e * o cot V 9 tx C O \u00C2\u00AB 5 i . \u00E2\u0080\u0094r~ CO o o CM JOOJ qseji 6/fidO Bon o c CD 8L L o J2 c c o CO CO CO 3 -Ti O a: c 3 CD L\u00E2\u0080\u0094 a. CD .E a) o \u00C2\u00A3 co 2 s i ! CD O O \u00C2\u00A3 ^ CO c 2 . E - c ^ JO fjj ^ CD i -C =3 CO \u00E2\u0080\u0094 CO co \"o co *Z. *i\ /1\ | a: co \u00C2\u00A9 \u00E2\u0080\u00A2 - ~ aJ H\u00E2\u0080\u0094 \u00E2\u0080\u0094 o a. c E O CO \u00E2\u0080\u00A2^3 CO CO ro\u00C2\u00A3 o CD CD . E CO CO CD _ O CD CD CD \" E ^3 O =3 3 CO # g> CO CD ir 5 >>5 3 CO CD CO a. o 2 0_ -o 42 c c CO 0) CO co\" g CD cl 8-2 w \u00C2\u00A3 o a: \u00C2\u00A3 E k. 3 O) \u00E2\u0080\u009E, i n 8 q c o CO V c a. co ^ \u00E2\u0084\u00A2 J2 r \u00E2\u0080\u0094 #n co ^ Q . >\u00C2\u00BB CO . E g -\u00E2\u0080\u00A2= u= CO CD = -c E u 10 O 32 day 333 and continued until day 489. Sampling for Pseudomonas fluorescens Sw5-RN on day 333 did not employ the use of the antibiotic plating method and no target colonies were isolated. On day 354, samples from the original dilutions were plated onto both TSA and TSA withlOO mg-L\"1 each of nalidixic acid and rifamycin along with the antifungal agents cycloheximide and nystatin (TSA**) to test for antibiotic masking. Target Sw5-RN colonies only grew on the TSA plates. When the colonies from the TSA plates were replicated onto TSA**, the same colonies were able to thrive. The same method of sampling continued until the final sampling day. Populations of Sw5-RN in the laboratory microcosm rhizospheres dropped below the limit of detection (3.2 CFU-g\" 1 fresh root weight) after sampling on day 375 and were never again recovered. Populations of Sw5-RN in the field plot rhizospheres dropped below the limit of detection (1.4 CFU-g\" 1 fresh root weight) after sampling on day 417 and were never again recovered. The line of best fit for the population decline for both the field and laboratory microcosms followed a cubic polynomial regression (Figure 4). Statistical testing by analysis of variance using the F-ratio test showed no significant difference (p<0.05) in the slopes of the two Sw5-RN regression lines (Appendix-Table A7). Therefore, there was no significant difference between the rates of population decline of Pseudomonas fluorescens Sw5-RN inoculated into field and laboratory rhizospheres. Further analysis revealed no significant difference (p<0.05) in the separation of the cubic regression lines (Appendix-Table A8). Therefore the regression lines for the population decline oi Pseudomonas fluorescens Sw5-RN 33 inoculated into field plot rhizospheres versus laboratory microcosm rhizospheres were not only parallel but also coincidental. 3.3 Internal Plant Colonization Assay: When internal root and shoot colonization was evaluated using the surface sterilization-dilution plating assay there were no target organisms identified. No Bacillus polymyxa Pw-2R were detected inside the roots or shoots of the spruce seedlings. Prior to the use of the antibiotic resistance plating method, the initial 50% strength tryptic soy agar (TSA) plates amended with cycloheximide (100 mg-L\"1) and nystatin (50 mg-L\"1) displayed growth of what looked morphologically to be Pw-2R. After replica plating onto 50% strength TSA amended with 200 mg-L\"1 rifamycin, 100 mg-L\"1 cycloheximide and 50 mg-L\"1 nystatin, no Bacillus polymyxa Pw-2R colonies grew. 3.4 Plant Growth Promotion Assay: The result of inoculation with either Bacillus polymyxa Pw-2R or Pseudomonas fluorescens Sw5-RN on seedling growth was minimal. The field inoculation data was plotted using linear regression (Figure 5) and analyzed using analysis of variance (ANOVA) with an F-ratio test. The results indicated there was no significant difference (p<0.05) in the rate of total biomass accumulation (Appendix-Tables A9, A11, and A13), shoot biomass accumulation, or root biomass accumulation between seedlings inoculated with Pw-2R, Sw5-RN, or left uninoculated. 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CD ^\u00C2\u00A3 s \u00C2\u00B0 ~\u00E2\u0080\u0094' CD r- P C C CO U CD TJ x : ro * o \u00E2\u0080\u00A2 CD CO CO b P c 3 CD E ro > \u00C2\u00A3 o x- CO p Lo u . ro _ c CD < \u00C2\u00AB CD co w \u00E2\u0080\u00A2O T3 a> CD _ r o ro 3 3 o o o o c c CO CD CD >* \u00C2\u00BB C CO o c CD 'co CO TJ CO x : CO CD C T 3 CD CD CO 00 CD (6) JU6I9M Ajp avi lejoj 6o~| 39 biomass than the control. The same results occurred for the increase in shoot and root biomass with the Pw-2R and Sw5-RN inoculated seedlings having a significantly greater (p<0.05) increase. These were the only two sampling days to reveal any significant differences in biomass accumulation. 40 Section 4. Discussion: Social implications and public confidence in the rapid advancement of molecular biology are crucial areas of concern. Recombinant DNA techniques have been developed to improve crop production and to enhance degradation of environmental pollutants (Halvorson et ai. 1985). Evidence suggests that the public perception of the risks associated with genetically engineered microorganisms (GEM's) is very volatile (Stewart-Tull et al. 1992). Public attitudes towards risk suggest the need for standardization of microcosm design for risk assessment of new products that result from genetic engineering. Microcosms are intended to provide a standardized model ecosystem to simulate the extremely complex interaction of physical, chemical and biological components of terrestrial environments. Prerelease microcosm studies may be used to determine if genetic modifications introduced into a microorganism are likely to create any unacceptable risks before that organism is released into the environment (Krimsky et al. 1995). Soil microcosms can be an invaluable tool for the study of microorganisms in natural soil conditions. However, in order to become a standard assessment tool, a microcosm must be proven to accurately simulate field conditions. 4.1 Microcosm Validation: 4.1.1 Bare Soil: One of the objectives of this research was to determine how accurately results obtained in microcosms reflected those obtained in the field. This was 41 accomplished by assessing the fate of Bacillus polymyxa strain Pw-2R as well as Pseudomonas fluorescens strain Sw5-RN, in bare soil and in the rhizosphere of spruce seedlings, in both field plots and in intact soil core microcosms. Pw-2R as well as Sw5-RN have been shown to induce growth stimulation in conifer species and have been classified as plant growth promoting rhizobacteria (PGPR) (Chanway and Holl 1993a, Chanway and Holl 1994). The initial inoculation density for Pw-2R was approximately 2.0 x 10 6 colony forming units (CFU)g\" 1 fresh soil. The population of Pw-2R continued to decline for the first two sampling days. This was followed by an increase in capture size on sampling day 24 in both field plots and laboratory microcosms and then a rapid decline until population levels dropped below the limit of detection (Figure 1). Analysis of variance using Fisher's LSD revealed that the increase in capture size of Pw-2R on day 24 was not significant. A similar trial was performed using Pseudomonas fluorescens Sw5-RN inoculated onto bare soil in field plots and laboratory microcosms and similar results from the Pw-2R trial were obtained. The population of Sw5-RN continued to decline immediately after inoculation until population levels were below the limit of detection (Figure 2). The initial recovery rate of Sw5-RN on the first sampling day was slightly lower than the expected level. This initial recovery rate was not significantly lower, (p<0.05), than the expected recovery level. To compare the population decline of Pw-2R inoculated onto bare soil field plots with bare soil microcosms, regression analysis was used to compare the population decline overtime (Kozak 1964). From both a biological and statistical 42 perspective, the line of best fit for both field and microcosm data sets was a cubic polynomial regression. In general, the survival trend for Pw-2R inoculated onto bare soil was a slight increase followed by a steep decrease until population levels dropped below the limit of detection. Using an F-ratio test, the analysis of variance (ANOVA) allowed an accurate comparison between the slope and position of the regression lines for Pw-2R inoculated field plots and laboratory microcosms. The results indicate that the bare soil laboratory microcosms can be used to accurately predict the survival of Bacillus polymyxa Pw-2R inoculated onto bare soil in the field at this location. Regression analysis was further used to compare the results of population decline between Sw5-RN inoculated bare soil field plots and laboratory inoculated bare soil microcosms. The line of best fit for both field and microcosm data sets was a cubic polynomial regression. Using an F-ratio test, the analysis of variance (ANOVA) revealed no significant difference between the slopes or the separation of the two regression lines. These results indicate that the bare soil microcosms can be used to accurately predict the survival of Pseudomonas fluorescens Sw5-RN inoculated onto bare soil in the field at this location. 4.1.2 Possible Reasons for Bacterial Population Decline; Bacillus polymyxa Pw-2R was originally isolated from internal root tissue of a naturally regenerating pine seedling (Shishido et al. 1995), and is a plant growth promoting endophyte that is able to live inside plant tissue. It is known that the number of soil organisms at successive distances from a root surface is inversely correlated with increased distance (Paul and Clark 1989). The region under direct 43 influence from plant roots, the rhizosphere, contains large amounts of easily oxidizable nutrients that support bacterial growth. Without the availability of nutrients it would be expected that population levels would decrease. Pw-2R is likely adapted to colonize plant tissue, therefore with the lack of a suitable host, the rapid decrease in population of Pw-2R inoculated onto bare soil was not surprising. Prior to taking the intact soil core microcosms from the field, and prior to inoculation, a cultivator was used to plough under any remaining plant material in the field. Being placed under the black polyvinyl sheeting had previously killed most of the plant material. The cultivating would have chopped up and randomly distributed any remaining plant material, including root systems. The brief increase in Bacillus polymyxa Pw-2R population may have been caused in part by the Pw-2R becoming established in microsites that contained easily oxidizable nutrients. These nutrients may have become accessible as a result of chopping up the root systems of the original plant matter in the field. The possibility exists that once this nutrient store had been depleted the population levels of Pw-2R rapidly decreased. The field plots and laboratory microcosms were all weeded by hand to keep them free of any plant growth through the duration of the experiment; therefore new root systems were kept to a minimum. Without the root systems, the level of available nutrients for these rhizobacterial populations would be extremely low. The possibility also exists that competition from other microorganisms caused the overall rapid decline in Pw-2R population numbers. A recent study has shown the partial inhibition of growth-promoting effects of Pw-2R due to the presence of other rhizobacteria (Bent and Chanway 1998). Recent studies have shown P G P R 44 efficacy on interior spruce seedling growth was greatest when the seedlings were grown in forest soil and inoculated with rhizosphere bacteria that originated from the same site as the spruce seed (O'Neill et al. 1992a). Further studies with P G P R have illustrated significant soil community responses to P G P R inoculation (Shishido and Chanway 1998). Shishido (1998) demonstrated that soil community responses depended on the origin of the soil and the presence of seedlings. The indigenous soil community present at the University of British Columbia may have caused a negative impact on the Pw-2R population levels. The rapid decline was seen in both inoculated field and laboratory microcosms, therefore the soil conditions were likely the same in both. As is true with the Bacillus polymyxa Pw-2R, Pseudomonas fluorescens Sw5-RN is likely adapted to colonize plant tissue, therefore with the lack of a suitable host the rapid decrease in population of Sw5-RN inoculated onto bare soil was not surprising. The survival trend of the Sw5-RN populations in the field and microcosms did not show any increase after inoculation. Therefore, any nutrients available may not have been as readily accessible as they were for the Pw-2R, or may not have been the nutrients necessary for the survival of the Sw5-RN. Competition from other microorganisms may also have been a cause for the overall rapid decline in Sw5-RN population numbers. 4.1.3 Ant ibiot ic Mask ing: Previous studies have been completed using the same microcosm with the genetically engineered microorganism (GEM) Pseudomonas chloroaphis 3732RN-L11 and Canadian hard red spring wheat; Triticum aestivum; cultivar A .C . Karma 45 (Angle et al. 1995). One of the goals of this research was to validate the microcosm for use with other organisms and other plants. As a general trend, population levels of Pw-2R declined following inoculation until the population size fell below the limit of detection (Figure 3). The first rhizosphere sampling day occurred on May 13,1998 and continued every third week until the final rhizosphere sampling day for that year, November 4 (day 186). Population levels declined from the initial sampling day until levels were below the limit of detection on day 122 in both field plot rhizospheres and laboratory microcosm rhizospheres. During the sampling period, between day 122 and day 186, no target organisms (Pw-2R) were detected and all samples were assumed to be below the limit of detection. A standard method to recover specific bacterial strains after environmental release is the use of spontaneously generated antibiotic resistant strains. It is an inexpensive and effective method for marking bacteria for easy recovery using cultural methods (Kloepperand Beauchamp 1992). Bacteria become resistant to antibiotics in different ways. Resistance mechanisms include bypassing the metabolic step affected by the antibiotic, overproducing an enzyme or product to detoxify the antibiotic, altering the structure of a target enzyme, and developing an uptake system for destruction or modification of the antibiotic (Dawes and Sutherland 1991). It is assumed in these methods that the antibiotic resistant microorganism retains its ability to thrive in the presence of specific levels of the antibiotic. 46 Recent studies on internal plant colonization by endophytic bacteria marked with antibiotic resistance have discovered what seems to be the temporary loss of the ability to thrive in the presence of specific levels of antibiotics. This loss has been termed antibiotic masking (Mclnroy et al. 1996). Mclnroy (1996) treated plants with rifampicin-resistant endophytic bacteria (rif+ mutants), and attempted reisolation of these bacteria after 3-14 days on agar amended with rifampicin (RTSA) with no success. However, colonies initially isolated from the same sample on agar with no antibiotics and then transferred to RTSA grew within 18 hours. Mclnroy (1996) does not attempt to explain the causes for the observed antibiotic masking other than the possibility that internal plant extracts may be affecting the ability of the endophyte to grow on RTSA. Mclnroy (1996) does go on to state that antibiotic masking was not encountered when isolating bacteria from external root surfaces. From day 10 until day 186 the diluted samples of Bacillus polymyxa Pw-2R were all plated on tryptic soy agar containing 200 mg-L\"1 rifamycin, 100 mg-L\"1 cycloheximide and 50 mg-L\"1 of nystatin (TSA*). Pw-2R population counts from sampling days 122 to 186, apparently dropped below the limit of detection. However, with the use of sample plating to uncover antibiotic resistance, viable populations were detected on sampling days 354 and 389 in the field rhizospheres and days 354 through to the final sampling day (489) for laboratory rhizospheres. Bacillus polymyxa Pw-2R may have been present during sampling days when population counts of zero occurred in the field rhizospheres. The organisms may have just been undetectable, as they could have been below the limit of detection but above zero. Other studies have.shown that large bacterial (PGPR) population 47 levels may not be needed to result in a plant growth response (Holl and Chanway 1992). On the other hand, the populations may have just completely died out in the second sampling season in most rhizospheres. Similar results were obtained when Pseudomonas fluorescens Sw5-RN was inoculated into the root plugs of the spruce seedlings in the laboratory microcosms and in the field. Sampling of Pseudomonas fluorescens Sw5-RN began on day 10 and continued until day 489. The first rhizosphere sampling day was May 13,1998 and the final rhizosphere sampling day for that year was November 4 (day 186). Population levels of Sw5-RN increased slightly following the inoculation until day 38, then continued to decline until population levels were below the limit of detection (Figure 4). Pseudomonas fluorescens Sw5-RN was originally isolated from the rhizosphere of hybrid spruce (Picea glauca x engelmannii) seedlings (Shishido et al. 1996b). The seedlings used in this experiment were hybrid spruce and the microenvironment that the inoculum was first presented with may have caused the initial population levels to increase slightly. An analysis of variance concluded that the initial rise in population was not significant in either the laboratory microcosm rhizospheres or the field plot rhizospheres. The slight increase in Sw5-RN population may be explained by the fact that Pseudomonas species are much more vigorous root colonizers than are Bacillus species (Kloepper et al. 1989). Kloepper (1989) explains that the rhizosphere is preferentially inhabited by pseudomonads, therefore Pseudomonas fluorescens Sw5-RN inoculated into the rhizosphere of the spruce seedlings may have had an advantage in survival. 48 As with the Pw-2R, the Sw5-RN inoculum was injected into the middle of the root plugs. Microsites where exudates and root derived organic material will be available are favourable sites in which microbial growth and competition will occur (Metting 1993). Thus the lack of nutrients or competition from indigenous microorganisms in the rhizosphere may have caused the decline in both the Pw-2R and Sw5-RN populations. The first time the Sw5-RN population levels in the field and laboratory rhizospheres dropped below the limit of detection was on day 143. At the beginning of sampling in the second season (day 333), testing for antibiotic masking occurred. The population counts between sampling days 143 to 186 that were assumed to be below the limit of detection might have just been displaying antibiotic masking. With the use of antibiotic resistance plating methods, viable populations were detected on sampling days 354 and 417 in the field rhizospheres and days 354 and 375 in laboratory rhizospheres. Populations in the field and laboratory rhizospheres may have been present after these sampling days but undetectable as they may have been below the limit of detection but above zero, or they may have dropped to zero in the second sampling season. The Bacillus polymyxa Pw-2R cells were originally cultured for inoculation in broth amended with 200 mg-L\"1 rifamycin. The mechanism(s) that allowed the cells to thrive in this level of antibiotic may have been suppressed after the Pw-2R was inoculated into the rhizosphere of the spruce seedlings. Plating of all samples from the original dilutions onto TSA and TSA amended with antibiotics, (TSA*), showed that the only Pw-2R populations able to grow initially were the colonies plated onto 49 the TSA containing no antibiotics. When the TSA plates were replicated onto the TSA* plates containing the antibiotics, Pw-2R colonies were able to grow. The Pseudomonas fluorescens Sw5-RN cells were originally cultured for inoculation in tryptic soy broth amended and 100 mg-L\"1 each of nalidixic acid and rifamycin. The mechanism(s) that allowed the cells to thrive in this level of antibiotic may have been suppressed similarly to the Pw-2R mechanism(s). One explanation for the antibiotic masking may be that there was a decrease in the necessity of the Pw-2R and Sw5-RN to overproduce inhibitory enzymes or products, as there were no antibiotics present in the rhizosphere of the seedlings. Rifamycin acts by inhibiting bacterial RNA synthesis by binding strongly to the beta subunit of DNA-dependant RNA polymerase, preventing the attachment of the enzyme to DNA and thus blocking initiation of RNA transcription (McEvoy 1995). Nalidixic acid appears to act by inhibiting bacterial DNA synthesis, probably by interfering with DNA polymerization by inhibiting DNA gyrase (McEvoy 1995). Bacillus polymyxa Pw-2R may produce compounds that bind or break down the rifamycin before it is able to block RNA transcription. The resistance of Pw-2R to rifamycin may not have been lost but only suppressed. Pseudomonas fluorescens Sw5-RN may similarly produce compounds enabling resistance to both rifamycin and nalidixic acid. The colonies of both Pw-2R and Sw5-RN that were replicated onto TSA plates containing antibiotics would contain a logarithmic population of cells. This may allow the colonies to grow on the TSA plates containing antibiotics, as some cells would be initially physically shielded from the high levels of antibiotics in the plates. 50 Colony formation did take longer after the colonies had been replicated onto TSA plates containing antibiotics. This lag in time may have indicated a slow response of the cells due to the levels of antibiotics. Being subjected to the lower levels of antibiotics by being physically shielded in the colonies may have allowed some organisms time to produce the necessary compounds required for antibiotic resistance. This is only speculation and further studies need to be undertaken in order to determine the reasons for the antibiotic masking. The fact that these two different antibiotic resistant organisms, Pw-2R and Sw5-RN, displayed antibiotic masking properties shows that care should be taken when attempting to recover antibiotic resistant organisms after release. Further studies should be undertaken to better understand the reasons the antibiotic masking. 4.1.4 Rhizosphere: Using an F-ratio test, the analysis of variance (ANOVA) allowed an accurate comparison between the slope and position of the regression lines for the population decline of Pw-2R inoculated field plot rhizospheres and laboratory microcosm rhizospheres. There was no significant difference (p<0.01) between the slopes of the regression lines. The statistical analysis indicated that the lines were not coincidental. The length of survival of the Pw-2R inoculated into the rhizospheres of laboratory microcosms was greater than the survival of the Pw-2R inoculated into the field rhizospheres. The results indicate that the laboratory microcosms containing Pw-2R inoculated spruce seedlings can be used to accurately predict the rate of population 51 decline of the Pw-2R inoculated into the rhizosphere of the spruce seedlings in the field at this location. The results also indicate that the microcosms can not be used to predict the final day when the Bacillus polymyxa Pw-2R population reaches zero in the field rhizospheres. Therefore the microcosms have the ability to conservatively predict the survival rate of the Bacillus polymyxa Pw-2R inoculated into the field rhizospheres. Similarly to Pw-2R, in order to analyze the results of population decline between Sw5-RN inoculated field rhizospheres and laboratory inoculated microcosm rhizospheres, regression analysis was used to compare slopes. The analysis of variance (ANOVA) using an F-ratio test allowed an accurate comparison between the slope and position of the regression lines for Sw5-RN inoculated field plot rhizospheres and laboratory microcosm rhizospheres. The rate of population decline between the laboratory microcosm rhizospheres and the field plot rhizospheres was not significantly different. The A N O V A further revealed that the regression lines are coincidental. The results indicate that the laboratory microcosms containing Pseudomonas fluorescens Sw5-RN inoculated into the rhizosphere of the spruce seedlings can be used to accurately predict the survival of the Sw5-RN inoculated into the rhizosphere of the spruce seedlings in the field at this location. The results further revealed that the laboratory microcosms could be used to predict the final day when the Pseudomonas fluorescens Sw5-RN population reaches zero in the field rhizospheres. 52 Therefore, the trials with Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN inoculated onto this bare soil and into the rhizosphere of these spruce seedlings validate the use of this intact soil core microcosm to predict the survival of microorganisms that may be released to the environment. 4.2 Internal Plant Colonization: Bacillus polymyxa Pw-2R is a plant growth promoting rhizobacteria that has been detected inside roots of hybrid spruce seedlings using a surface sterilization-dilution plating assay (Shishido et al. 1999). Shishido et al (1999) demonstrated the ability of Pw-2R to enter spruce root tissues, establishing endophytic populations after seed inoculation without causing visible symptoms of disease. The results presented here indicate that the Pw-2R did not colonize the internal root or shoot tissues of the spruce seedlings. Soil conditions and indigenous soil organisms may have reduced or eliminated the ability of the Pw-2R populations to enter the root and travel to the shoot tissues. Rhizobacteria present at this site may have inhibited the ability of the Pw-2R to enter the roots, in turn inhibiting the growth promoting effects (Bent and Chanway 1998). This may have also been caused by predation, or other negative interactions occurring before the Pw-2R had a chance to establish sustainable internal populations (Shishido et al. 1999). Chanway (1998) suggested that a period of growth in a controlled environment to establish endophytic populations may have great potential for reforestation. The results presented here indicate that the Pw-2R were not able to establish an endophytic population when inoculated after being planted. The conception of pre-inoculating seedlings with 53 growth promoting rhizobacteria in a controlled environment, such as a nursery, before use in reforestation, should be investigated further. 4.3 PGPR Efficacy: Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN are both plant growth promoting rhizobacteria (PGPR) (Chanway and Holl 1993a, Chanway and Holl 1994). The results from the current trial indicate that P G P R strains Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN inoculated into the rhizosphere of spruce seedlings did not increase the biomass accumulation rate when the seedlings were in a relatively stress free environment. The only difference in the field plots occurred on the final sampling day; the mean for the dry biomass of the control seedlings was significantly greater than either of the inoculated seedling treatments. Results for the rate of seedling biomass accumulation for Pw-2R or Sw5-RN inoculated seedlings in the laboratory microcosms also displayed no significant difference. When individual sampling days were examined, only two days showed differences in treatment means. Each showed a significantly greater shoot and root biomass accumulation in the Pw-2R inoculated seedlings. Several theories have been put forth to attempt to explain the mechanisms for P G P R activity. Chanway (1997) has reviewed these mechanisms and divides them into direct and indirect. The review suggests that direct mechanisms occur when bacteria produce a metabolite or compound that stimulates plants to grow independently of other soil organisms. Lack of direct mechanisms may be the 54 reason for the lack of growth promotion in the seedlings inoculated with either Pw-2R or Sw5-RN. In order to induce growth promotion, seedlings may need one or a set of precursors produced by the P G P R in order to create a growth-promoting hormone. Recent studies have shown specific plant hormones (auxins) can be produced by the microbial transformation of an available precursor (Arshad and Frankenberger 1991). If Pw-2R or Sw5-RN promote growth in seedlings by synthesizing growth hormones, the precursor molecule(s) that may be necessary may not have been present in the soil used for these trials. Another suggestion would be that plant growth promotion may require a balance of different growth hormones and the entire set of hormones necessary may not have been present in the soil used for these trials. Chanway's (1997) review suggests plant growth promotion can also be caused by indirect methods involving bacteria which affect other factors that in turn stimulate plant growth. Indirect methods include increased mycorrhizal infection and suppression of deleterious bacteria. However, mycorrhizal interactions or the lack thereof were not considered in these trials. Recent work suggests P G P R strains may inhibit the activity or growth of indigenous, plant growth inhibiting rhizosphere microorganisms (Kloepper 1993). The presence of the P G P R would therefore decrease the negative impact of the deleterious organisms and therefore allow increased growth of the seedlings. There may have been no deleterious organisms present in the soils used for these trials; therefore there may have been no negative effects to overcome. 55 A similar answer for the lack of growth promotion may also involve the indigenous bacterial populations. If the Pw-2R or the Sw5-RN were producing compounds such as growth hormones, the indigenous populations may have been depleting those compounds as soon as they were being created. Recent studies with microcosms have shown soil bacterial populations can be significantly altered and large shifts in carbon substrate utilization profiles can be caused in response to P G P R inoculation (Shishido and Chanway 1998). In addition, plant growth promoting rhizobacteria may only cause significant increases in biomass accumulation when the seedlings are stressed. Recent studies have illustrated a greater P G P R effect shown in interior spruce and lodgepole pine seedlings that were grown at sites of lower productivity (Chanway and Holl 1993b, Chanway and Holl 1994). Chanway hypothesized that the P G P R inoculum may be more beneficial at sites that have harsh growing conditions. Further investigation into the relationships of Pw-2R and Sw5-RN with the indigenous soil community in the field is necessary to understand the effects of these plant growth promoting rhizobacteria. Many complicated interactions are involved in this system of microbial plant growth promotion. Further work must be undertaken in order to answer these questions. 56 5. CONCLUSIONS: The data generated in the experiments presented here confirm the hypothesis that the intact soil core microcosm can be used to accurately predict the fate of Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN inoculated onto a bare field soil. The data confirmed the intact soil core can be used to predict the rate of population decline in the field as well as the day at which the population drops to zero in field inoculated soil. The data also confirm that the Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN populations declined more gradually in the rhizosphere of the seedlings. The intact soil core microcosms were able to predict the rate of population decline of Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN inoculated into field rhizospheres. The microcosms were able to predict the day at which the population of Pseudomonas fluorescens Sw5-RN reached zero in the field plots but were not successful in predicting the same for the Bacillus polymyxa Pw-2R. However, the microcosms were able to conservatively predict the final day in which the field populations of both Bacillus polymyxa Pw-2R and Pseudomonas fluorescens Sw5-RN reached zero. The testing of the predictive ability of the intact soil core microcosm revealed that this small, inexpensive and simple tool may be appropriate for risk assessment of microorganisms that might eventually be introduced to a soil or rhizosphere environment. Validation with additional soils, microorganisms and plants should be undertaken for this microcosm to be accepted as a standard for risk assessment but these results show great promise. 57 Two spontaneously generated antibiotic resistant strains of plant growth promoting rhizobacteria (PGPR) were used in these trials. The technique of using antibiotic resistant strains has been commonly used as it allows easy recovery of inexpensively marked bacteria using cultural methods. Recent studies have discovered what seems to be the temporary loss of the ability of antibiotic resistant strains to thrive in the presence of those antibiotics. This same loss was discovered in the two P G P R strains used in these trials. These results further confirm previous observations that antibiotic resistant bacteria that fail to grow upon primary isolation on agar media with antibiotics will grow on those same plates if first isolated on the same agar media without antibiotics. Previous studies have only observed this when isolating bacteria from within roots or stems. Antibiotic masking has been observed here for the first time when isolating antibiotic resistant bacteria from external root surfaces. Future work using antibiotic resistance as a method of selective isolation for soil microorganisms should be designed to include methods for identifying the possibility of antibiotic masking. Since Bacillus polymyxa P w - 2 R is a plant growth promoting endophyte it was hypothesized that colonies would be isolated from internal shoot and root tissues. Using the surface sterilization-dilution plating assay there were no P w - 2 R organisms identified in any of the internal shoot or root tissues. The results of these trials indicate that the P w - 2 R were not able to establish an endophytic population when inoculated after the seedlings had been planted. Previous research suggests that endophytic P G P R may be able to establish internal populations more readily before being subjected to competition, predation, or other negative interactions (Shishido et 58 al. 1999). Therefore, the Bacillus polymyxa Pw-2R may have had a better opportunity to establish an endophytic population if it had been inoculated into the rhizosphere prior to the seedlings being planted. Finally, it was hypothesized that seedlings in the field or in the intact soil core microcosms inoculated with either Bacillus polymyxa Pw-2R or Pseudomonas fluorescens Sw5-RN would show an increase in biomass. Both Pw-2R and Sw5-RN have previously been shown to promote the growth of conifer seedlings and studies indicated that the positive effects of a single inoculation at planting can extend at least through the second year of growth (Chanway et al. 1997). The results from these trials indicated spruce seedling growth was not significantly enhanced by inoculation with Pw-2R or Sw5-RN in the field rhizospheres. Many complex interactions are undoubtedly involved in the mechanisms of plant growth promotion by rhizobacteria. 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Transport of a genetically engineered Pseudomonas fluorescens strain through a soil microcosm. Applied & Environmental Microbiology 56: 401-408. Verhoef, H., A. 1996. The role of soil microcosms in the study of ecosystem processes. Ecology 77: 685-690. Walter, M. V., L. A. Porteous, V. J . Prince, L. Ganio, and R. J . Seidler. 1991. A microcosm for measuring survival and conjugation of genetically engineered bacteria in rhizosphere environments. Current Microbiology 22: 117-121. 64 APPENDIX 1 Statistical Analys 65 Table A1. Analysis of variance and F-ratio test for the rate of population decline of Bacillus polymyxa (Pw-2R) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = uo + u-tDAY + u 2 DAY 2 + u 3 DAY 3 , where Y: Log CFU/g fresh soil, p. x: constants, and DAY: Days after inoculation. (a) Field Source of Variation DF S S Regression 3 46.76593 Residual 24 11.43754 Total 27 (b) Lab Source of Variation DF S S Regression 3 38.24962 Residual 22 7.498513 Total 25 SSRes(max) - SSRes(Field) + SSRes(Lab) - 18.93605 DFRes(max) = DFRes(Field) + DFRes(Lab) = 4 A N O V A MODEL: Y = uo + mDAY + f j 2 DAY 2 + u. 3DAY 3 + ^ D u m m y where Y: Log CFU /g fresh soil, u.x: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field +Lab + Dummy Source of Variation DF(Dar) SS(par) Regression 4 85.92189 Residual 49 19.30014 Total 53 Test: H 0 : the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. a ; : >Res(par) ~ ^Resdnax) FTeSt = DFRp^nari - DFR^Ima^ SSRpsfmaY^ DFRes(max) FT est= 0.29486 F cm = F0.o5 (3,46) = 2.79 F c n t \u00C2\u00BB F test: The lines A R E parallel at p<0.05. 66 Table A2. Analysis of variance and F-ratio test for the coincidence of population decline of Bacillus polymyxa (Pw-2R) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = u 0 + u-|DAY + u 2 DAY 2 + u 3 DAY 3 , where Y: Log CFU/g fresh soil, u*: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field +Lab Source of Variation DF(com) SS(com) Regression 3 85.36295 Residual 50 19.85907 Total 53 A N O V A MODEL: Y = no + mDAY + L I 2 D A Y 2 + p. 3DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, p x: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 85.92189 Residual 49 19.30014 Total 53 Test: H' 0 : the two surfaces coincide given that they are parallel. H V the two surfaces do not coincide given that they are parallel. QvSResfcorrO ~ QQRes(par) Fjest = 1 S S DFRes(par) FTest =1.4189 F cm = Fo.o5 (1,49) = 4.03 F c r i t \u00C2\u00BB F Test: The lines A R E coincidental at p<0.05. 67 Table A3. Analysis of variance and F-ratio test for the rate of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = uo + u.-iDAY + H2DAY2 + u 3 DAY 3 , where Y: Log CFU/g fresh soil, u.x: constants, and DAY: Days after inoculation. (a) Field Source of Variation D F S S Regression 3 46.58873 Residual 24 13.60805 Total 27 (b) Lab Source of Variation D F S S Regression 3 34.01674 Residual 21 7.23766 Total 24 SSRes(max) - SSR e s (Field) + SSRes(Lab) - 20.84571 DFRes(max) = DFRes(Field) + DFR e s (Lab) = 45 A N O V A MODEL: Y = uo + ^ D A Y + u. 2DAY 2 + a^DAY 3 + mDummy where Y: Log C F U / g fresh soil, a*: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 80.36804 Residual 48 21.61826 Total 52 Test: H 0 : the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSRes(par) ~ SSRes(max) F Tes t = D F l W n a r t - DFRwafrnmrt SSRftsrmay^ DFRes(max) F T e s t = 0.55591 F crit = Fo.o5 (3,45) = 2.81 F c r i t \u00C2\u00BB F Test: The lines A R E parallel at p<0.05. 68 Table A4. Analysis of variance and F-ratio test for the coincidence of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated onto bare soil in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = u 0 + u^DAY + u 2 DAY 2 + u 3 DAY 3 , where Y: Log CFU/g fresh soil, px: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field +Lab Source of Variation DF(com) SS(com) Regression 3 80.3293 Residual 49 21.657 Total 52 A N O V A MODEL: Y = uo + p iDAY + p 2 D A Y 2 + p.3 D A Y 3 + ^ D u m m y where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 80.36804 Residual 48 21.61826 Total 52 Test: H' 0 : the two surfaces coincide given that they are parallel. H V the two surfaces do not coincide given that they are parallel. SSResfcorrO ~ SSRes(par) FTest = 1 S S Rfis(par) DFRes(par) F T e s t =0.08602 F crit = F0.05 (1,44) = 4.02 F c r i t \u00C2\u00BB F Test: The lines A R E coincidental at p<0.05. 69 Table A5. Analysis of variance and F-ratio test for the rate of population decline of Bacillus polymyxa (Pw-2R) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = p 0 + m D A Y + p 2 D A Y 2 + p 3 DAY 3 , where Y: Log CFU/g fresh root, p x: constants, and DAY: Days after inoculation. (a) Field Source of Variation DF S S Regression 3 188.0498 Residual 81 44.00217 Total 84 (b) Lab Source of Variation DF S S Regression 3 127.130 Residual 77 72.58817 Total 80 SSRes(max) = SSRes(Field) + SSR e s (Lab) - 116.59 DFRes(max) = DFR e s (F ie ld) + DFRes(Lab) = 158 A N O V A M O D E L : Y = ix0 + m D A Y + M-2DAY2 + p 3 D A Y 3 + p^Dummy where Y: Log C F U / g fresh soil, u x : constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 315.579 Residual 161 123.077 Total 165 Test: H 0 : the regression equations describe parallel surfaces. H i : the regression equations do not describe parallel surfaces. ^>^Resfpar) ~ ^^>Res(max) FTest = DFRp C/ n art - DFlWmmrt SSRpsfmax^ DFRes(max) F T e s t = 2.78 F cnt = Fo.05 (3,158) = 2.66 F crit < F rest: The lines are NOT parallel at p<0.05. F Crit = Frj.01 (3,158) = 3.78 F crit > F Test: The lines A R E parallel at p<0.01. 70 Table A6. Analysis of variance and F-ratio test for the coincidence of population decline of Bacillus polymyxa (Pw-2R) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = no + in DAY + n 2 DAY 2 + ^ D A Y 3 , where Y: Log CFU/g fresh root, | i x : constants, and DAY: Days after inoculation. (a) Coincidental Model = Field +Lab Source of Variation DF(com) SS(com) Regression 3 307.369 Residual 162 131.2874 Total 165 A N O V A MODEL: Y = p 0 + mDAY + ^ 2 D A Y 2 + n 3 DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, jax: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 315.579 Residual 161 123.077 Total 165 Test: H' 0 : the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. SSRes(com) ~ SSRes(par) Fiest = 1 S S Ras(part DFRes(par) Frest = 10.74 F crit = Fo.o5 (1,162) = 3.9 F crit \u00C2\u00AB F Test: The lines are NOT coincidental at p<0.05. F crit = F0.01 (1,162) = 6.82 F crit \u00C2\u00AB F Test: The lines are NOT coincidental at p<0.01. 71 Table A7. Analysis of variance and F-ratio test for the rate of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = no + mDAY + n 2 DAY 2 + n 3 DAY 3 , where Y: Log CFU/g fresh root, \ix: constants, and DAY: Days after inoculation. (a) Field Source of Variation D F S S Regression 3 216.7127 Residual 82 64.03867 Total 85 (b) Lab Source of Variation D F S S Regression 3 199.4587 Residual 77 58.82832 Total 80 SSRes(max) - SSRes(Field) + S S R e S ( L a b ) = 122.86699 DFRes(max) = DFRes(Field) + DFR e s(Lab) = 159 A N O V A MODEL: Y = ^ 0 + niDAY + n 2 DAY 2 + ^ D A Y 3 + ^ D u m m y where Y: Log CFU/g fresh soil, y.x: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 411.809 Residual 167 127.40688 Total 171 Test: H 0 : the regression equations describe parallel surfaces. H i : the regression equations do not describe parallel surfaces. SSResfpar) ~ SSRes(max) Flest = DFRpsfnart - DFRBRfmax^ SSRpfifmav^ DFRes(max) Fiest =2.01991 F Crit = Frj.05 (3,164) = 2.60 F c r i t > F jest: The lines A R E parallel at p<0.05. 72 Table A8 . Analysis of variance and F-ratio test for the coincidence of population decline of Pseudomonas fluorescens (Sw5-RN) inoculated into the rhizosphere of one year old hybrid spruce seedlings in both laboratory intact soil core microcosms and field plots. A N O V A MODEL: Y = \iQ + u iDAY + i i 2 D A Y 2 + u. 3DAY 3, where Y: Log CFU/g fresh root, u.x: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field +Lab Source of Variation DF(Com) SS(com) Regression 3 411.47745 Residual 168 127.7387 Total 171 A N O V A MODEL: Y = |io + m D A Y + ^ D A Y 2 + |a 3 DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, \ix: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field +Lab + Dummy Source of Variation DF(par) SS(par) Regression 4 411.809 Residual 167 127.40688 Total 171 Test: H' 0 : the two surfaces coincide given that they are parallel. H'-i: the two surfaces do not coincide given that they are parallel. ^QRes(com) ~ ^^>Res(par) FTest = 1 S S RfiRfoar) DFRes(par) F T e s t =0.43494 F crit = Fo.o5 (1,167) = 3.84 F crit \u00C2\u00AB F Test: The lines A R E coincidental at p<0.05. 73 Table A9. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R). ANOVA MODEL: Y = no + mDAY, where Y: Log total dry weight(g), constants, and DAY: Days after inoculation. (a) Field Pw-2R Source of Variation DF SS Regression 1 16.26731 Residual 113 2.61954 Total 114 (b) Field Sw5-RN Source of Variation DF SS Regression 1 16.80608 Residual 111 2.22862 Total 112 SSRes(max) - SSRes(Field) + S S R e s ( Lab ) ~ 4.84816 DFRes(max) = DFR e s(Field) + D F R e s (Lab) = 224 ANOVA MODEL: Y = no + n iDAY + n2DAY2 + nsDAY 3 + ^Dummy where Y: Log CFU/g fresh soil, nx: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field Pw-2R + Field Sw5-RN \u00E2\u0080\u00A2 Dummy Source of Variation DF(Par) SS(Par) Regression 2 33.06628 Residual 225 4.85822 Total 227 Test: H0: the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSRes(par) ~ SSRes(max) F TeSt = D F R P S ^ - D F R p S / m a ^ DFRes(max) Frest =0.46480 F crit = F 0.o5 (1,224) = 3.87 F c r i t \u00C2\u00BB F Test: The lines ARE parallel at p<0.05. 74 Table A10. Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R). ANOVA MODEL: Y = ^ 0 + m^AY, where Y: Log total dry weight (g), nx: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field Pw-2R +Field Sw5-RN Source of Variation DF(com) SS(com) Regression 1 33.04919 Residual 226 4.87531 Total 227 ANOVA MODEL: Y = uo + mDAY + ^ 2 DAY 2 + i^DAY3 + ^Dummy where Y: Log CFU/g fresh soil, px: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field Pw-2R +Field Sw5-RN + Dummy Source of Variation DF(par) SS(par) Regression 2 33.06628 Residual 225 4.85822 Total 227 Test: H'0: the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. SSRes(com) ~ SSpfesi SS Ftofrw} DFRes(par) Ftest =0.79149 F cnt = Foes (1,225) = 3.87 F cm \u00C2\u00AB F T e st: The lines ARE coincidental at p<0.05. 75 Table A11. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R). ANOVA MODEL: Y = uo + uiDAY, where Y: Log total dry weight(g), \ix: constants, and DAY: Days after inoculation. (a) Field Pw-2R Source of Variation DF SS Regression 1 16.26731 Residual 113 2.61954 Total 114 (b) Field Control Source of Variation DF SS Regression 1 5.97272 Residual 46 1.38692 Total 47 SSRes (max) - SSRes(Field) + SSRes (Lab) - 4.00646 DFRes(max) = DFRes(Fieid) + DFRes(Lab) = 159 ANOVA MODEL: Y = u 0 + uiDAY + u 2DAY 2 + u 3DAY 3 + ^Dummy where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy. Dummy variable. (c) Parallel Model = Field Pw-2R + Field Control + Dummy Source of Variation DF(par) SS(par) Regression 2 22.6899 Residual 160 4.0077 Total 162 Test: H 0: the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSResfoart ~ SSRes(max) F T e s t = DFffrwrfnart - D F R p g f m a ^ SSRpafmay^ DFRes(max) F i e s t =0.04921 F crit = F 0 .o5 (1,159) = 3-90 F c r i t \u00C2\u00BB F Test: The lines ARE parallel at p<0.05. 76 Table A12. Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R). A N O V A MODEL: Y = uo + mDAY, where Y: Log total dry weight (g), ux: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field Pw-2R +Field Control Source of Variation DF(com) SS(com) Regression 1 22.68311 Residual 161 4.01499 Total 162 ANOVA MODEL: Y = u 0 + mDAY + u 2 DAY 2 + u 3 DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field Pw-2R + Field Control + Dummy Source of Variation DF(par) SS(par) Regression 2 22.6899 Residual 160 4.0077 Total 162 Test: H' 0 : the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. SSRes(com) ~ SSRes(par) FTest ~ 1 S S DFRes(par) F T e st =0.29104 F Crit = Fo.05 (1,160) = 3.90 F crit F Test: The lines A R E coincidental at p<0.05. 77 Table A13. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN). ANOVA MODEL: Y = u 0 + uiDAY, where Y: Log total dry weight(g), ux: constants, and DAY: Days after inoculation. (a) Field Sw5-RN Source of Variation DF SS Regression 1 16.80608 Residual 111 2.22862 Total 112 (b) Field Control Source of Variation DF SS Regression 1 5.97272 Residual 46 1.38692 Total 47 SSRes(max) = SSR e s (F iekJ) + SSR e s (Lab} - 3.61554 DFRes(max) = DFR e s (F ie ld) + DFRes(Lab) =157 ANOVA MODEL: Y = u 0 + mDAY + u 2DAY 2 + u 3DAY 3 + ^Durnrny where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Field SwS-RN + Field Control + Dummy Source of Variation DF(Par) SS(Par> Regression 2 23.28379 Residual 158 3.61698 Total 160 Test: H 0: the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSResfpar) ~ SSRes(max) Fjest = DFRfis/nart ~ DFRpsfmayY SSR R a (rmx^ DFRes(max) F T e s t =0.06253 F Crit = Fo.05 (1,157) = 3.90 F c r i t > ; > F Test: The lines ARE parallel at p<0.05. 78 Table A14. Analysis c-f variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in field plots and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN). A N O V A MODEL: Y = u 0 + u iDAY, where Y: Log total dry weight (g), a*.: constants, and DAY: Days after inoculation. (a) Coincidental Model = Field Sw5-RN +Field Control Source of Variation DF( C o m) SS(com) Regression 1 23.28378 Residual 159 3.616977 Total 160 A N O V A MODEL: Y = uo + u iDAY + u 2 DAY 2 + u 3 D A Y 3 + ^ D u m m y where Y: Log CFU/g fresh soil, u x : constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Field Sw5-RN + Field Control + Dummy Source of Variation DF(par) SS(par) Regression 2 23.28379 Residual 158 3.616975 Total 160 Test: H' 0 : the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. OORes(com) ~ oORes(par) FTest = 1 , SSR f i s(r)ar) DFRes(par) FTest =0.00009 F Crit = Fo.05 (1,158) = 3.90 F c r i t < < : F Test: The lines A R E coincidental at p<0.05. 79 Table A15. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa (Pw-2R). A N O V A MODEL: Y = u 0 + u iDAY, where Y: Log total dry weight(g), u x : constants, and DAY: Days after inoculation. (a) Lab Pw-2R Source of Variation DF S S Regression 1 4.17941 Residual 89 0.98631 Total 90 (b) Lab Sw5-RN Source of Variation DF S S Regression 1 5.64283 Residual 110 1.19738 Total 111 SSRes(max) - SSR e S (Field) + SSRes(Lab) = 2.18369 DFRes(max) = DFR e s(Field) + DFR e s(Lab) =199 A N O V A MODEL: Y = u 0 + mDAY + u 2 D A Y 2 + u 3 D A Y 3 + ^ D u m m y where Y: Log CFU/g fresh soil, u x : constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Lab Pw-2R + Lab SwS-RN + Dummy Source of Variation DF(par) SS(par) Regression 2 10.13279 Residual 200 2.18454 Total 202 Test: H 0 : the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSResfoart ~ SSRes(max) F T e s t = DFRpsfnart - DFR^mavY SSRpsfmavY DFRes(max) F T e s t = 0.07746 F crit = F 0 .o5 (1,199) = 3.90 F c r i t \u00C2\u00BB F Test: The lines A R E parallel at p<0.05. 80 Table A16. Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce planted in laboratory intact soil core microcosms and inoculated with Pseudomonas fluorescens (Sw5-RN) and Bacillus polymyxa(Pw-2R). ANOVA MODEL: Y = u 0 + uiDAY, where Y: Log total dry weight (g), a*: constants, and DAY: Days after inoculation. (a) Coincidental Model = Lab Pw-2R +Lab Sw5-RN Source of Variation DF(com) SS(Com) Regression 1 10.10438 Residual 201 2.21295 Total 202 ANOVA MODEL: Y = u 0 + uiDAY + u 2DAY 2 + u 3DAY 3 + ^Dummy where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Lab Pw-2R + Lab Sw5 -RN + Dummy Source of Variation DF(par) SS(par) Regression 2 10.13279 Residual 200 2.18454 Total 202 Test: H'0: the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. SSResfcorrO ~ SSRes(par) Fxest = 1 Rfisjnar.) DFRes(par) Fiest =2.60101 F Crit = Fo.05 (1,200) = 3.87 F c r i t < < F test: The lines ARE coincidental at p<0.05. 81 Table A17. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R). A N O V A MODEL: Y = u 0 + mDAY, where Y: Log total dry weight(g), ux: constants, and DAY: Days after inoculation. (a) Lab Pw-2R Source of Variation DF S S Regression 1 4.17941 Residual 89 0.98631 Total 90 (b) Lab Control Source of Variation DF S S Regression 1 1.41990 Residual 27 0.20837 Total 28 SSRes(max) ~ SSRes(Field) + SSRes(Lab) - 1.19468 DFRes(max) = DFR e S(Field) + DFRes(Lab) =116 ANOVA MODEL: Y = uo + u^DAY + u 2 DAY 2 + u 3 DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Lab Pw-2R + Lab Control + Dummy Source of Variation DF(par) SS(par) Regression 2 5.61536 Residual 117 1.19528 Total 119 Test: H 0 : the regression equations describe parallel surfaces. H i : the regression equations do not describe parallel surfaces. SSRes(par) ~ SSRes(max) FTest = DFRssfnart ~ DFRpsfmayY SSRRRCmflx) DFRes(max) FTest = 0.05826 F crit = F 0 .o5 (1,116) = 3.94 F c r i t \u00C2\u00BB F Test: The lines A R E parallel at p<0.05. 82 Table A18. Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Bacillus polymyxa (Pw-2R). A N O V A MODEL: Y = u 0 + uiDAY, where Y: Log total dry weight (g), ux: constants, and DAY: Days after inoculation. (a) Coincidental Model - Lab Pw-2R +Lab Control Source of Variation DF(com) SS(com) Regression 1 5.60369 Residual 118 1.20694 Total 119 A N O V A MODEL: Y = u 0 + uiDAY + u 2 DAY 2 + u 3 DAY 3 + mDummy where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Lab Pw-2R + Lab Control + Dummy Source of Variation DF(par) SS(par) Regression 2 5.61536 Residual 117 1.19528 Total 119 Test: H' 0 : the two surfaces coincide given that they are parallel. H'-i: the two surfaces do not coincide given that they are parallel. SSRes(com) ~ SSRes(par) FTest = 1 S S Resfoar) DFRes(par) F T e s t = 1.14232 F crit = Fo.o5 (1,117) = 3.92 F crit < F Test: The lines A R E coincidental at p<0.05. 83 Table A19. Analysis of variance and F-ratio test for the rate of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN). A N O V A MODEL: Y = uo + uiDAY, where Y: Log total dry weight(g), ux: constants, and DAY: Days after inoculation. (a) Lab Sw5-RN Source of Variation DF S S Regression 1 5.64283 Residual 110 1.19738 Total 111 (b) Lab Control Source of Variation DF S S Regression 1 1.41990 Residual 27 0.20837 Total 28 SSRes(max) ~ SSR e s (F ie ld) + SSRes(Lab) - 1.40574 DFRes(max) = DFR e s (F ie ld) + DFRes(Lab) = 137 A N O V A MODEL: Y = u 0 + mDAY + u 2 DAY 2 + u 3 DAY 3 + ^ D u m m y where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (c) Parallel Model = Lab Sw5-RN + Lab Control + Dummy Source of Variation DF(par) SS(par) Regression 2 7.32284 Residual 138 1.40577 Total 140 Test: H 0 : the regression equations describe parallel surfaces. H-i: the regression equations do not describe parallel surfaces. SSRes(par) ~ SSRes(max) FTest = DFRpsfnart - DFn\u00C2\u00BB\u00C2\u00AB/m\u00C2\u00AB\u00C2\u00ABrt SSRf\u00C2\u00BBs(max> DFRes(max) F T e s t =0.00292 F crit = F0.o5 (1,137) = 3.91 F crit \u00C2\u00BB F Test: The lines A R E parallel at p<0.05. 84 Table A20. Analysis of variance and F-ratio test for coincidence of total biomass accumulation of one year old hybrid spruce seedlings planted in laboratory intact soil core microcosms and left uninoculated (Control) compared with those inoculated with Pseudomonas fluorescens (Sw5-RN). A N O V A MODEL: Y = uo + u-iDAY, where Y: Log total dry weight (g), ux: constants, and DAY: Days after inoculation. (a) Coincidental Model = Lab Sw5-RN +Lab Control Source of Variation DF(com) SS(com) Regression 1 7.27134 Residual 139 1.45727 Total 140 A N O V A MODEL: Y = u 0 + mDAY + u 2 DAY 2 + u 3 DAY 3 + ^Durnrny where Y: Log CFU/g fresh soil, ux: constants, DAY: Days after inoculation, and Dummy: Dummy variable. (b) Parallel Model = Lab Sw5-RN + Lab Control + Dummy Source of Variation DF(par) SS(par) Regression 2 7.32284 Residual 138 1.40577 Total 140 Test: H ' 0 : the two surfaces coincide given that they are parallel. H'i: the two surfaces do not coincide given that they are parallel. SSRes(com) ~ SSRes(par) FTest = 1 S S Rpsfnart DFR e s (par) F T e s t =5.05559 F crit = F 0 . o5 (1,138) = 3.91 F c r i t \u00C2\u00AB F Test: The lines A R E NOT coincidental at p<0.05. F cnt = F0.01 (1,138) = 6.83 F crit > F Test: The lines A R E coincidental at p<0.01. 85 "@en . "Thesis/Dissertation"@en . "2000-11"@en . "10.14288/1.0089520"@en . "eng"@en . "Forestry"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "Validation of a microcosm designed for pre-release risk assessment of soil microorganisms using plant growth promoting rhizobacteria"@en . "Text"@en . "http://hdl.handle.net/2429/10715"@en .