Ecologically Sound Soil Management of Tree Fruit Replant Disease

IDENTIFICATION OF PHOSPHATE SOLUBILIZING BACTERIA AND EVALUATION OF THEIR APPLICATION WITH INSOLUBLE PHOSPHORUS FERTILIZERS TO SOILS FROM CERTIFIED ORGANIC ORCHARDS AFFECTED BY REPLANT DISEASE

MOLLY ADAIR THURSTON

B.Sc., The University of Guelph, 2004

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

THE COLLEGE OF GRADUATE STUDIES

(Biology)

THE UNIVERSITY OF BRITISH COLUMBIA

(Okanagan)

August 2013

ABSTRACT

Replant disease (RD) affects the growth and establishment of young fruit trees in old

orchard soils. Organic management strategies are needed as an alternative to chemical controls.

Improved phosphorus (P) nutrition to stimulate root growth and improve tree establishment is a

well-established strategy; however, only insoluble Rock Phosphate (RP) and Bone Meal (BM)

inputs are available to Canadian organic growers. The ability of specific plant growth promoting

rhizobacteria to solubilize phosphate may improve P availability and its uptake in young apple trees, replanted into inoculated orchards. In this study, 101 bacteria isolated from the roots of legumes from Saskatchewan soils were screened for P solubilization. Thirty-four of these bacteria were positive for P solubilization as measured by halo diameter production on calcium phosphate medium. Twelve isolates showing the largest halo diameters and three known P solubilizing bacteria (PSB) were compared on three media: calcium phosphate, Pikovskaya

(PVK) and PVK with bromophenol blue. All twelve isolates previously identified as

Pseudomonas, Rhanella, Serratia and Klebsiella spp. solubilized P on all media, although the

halo diameters varied among media. The isolates were tested in liquid culture, where a marked

decrease in the pH of the solution was observed and six isolates were identified for further testing in growth pouch assays in the presence of insoluble P. The root growth of apple seedlings inoculated with one of the six bacterial isolates, showed significant increases in total root length, surface area and the number of root tips compared to the control after four weeks of incubation.

Three isolates were selected for greenhouse bioassays using five RD-affected soils collected from organic orchards. These isolates were inoculated onto apple trees, alone or in combination with RP or BM. Two field trials were simultaneously conducted in organic apple orchards, using

the same treatments; however there were no significant effects of the isolate treatments in either set of experiments. Although the strongest P solubilizers did not enhance tree growth in the greenhouse and orchard trials, the in vitro work showed the potential of PSB as a tool to mitigate the impact of RD.

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TABLE OF CONTENTS

ABSTRACT ...... ii TABLE OF CONTENTS ...... iv LIST OF TABLES ...... vii LIST OF FIGURES ...... xii LIST OF ILLUSTRATIONS ...... xiii LIST OF ABBREVIATIONS ...... xiv ACKNOWLEDGMENTS ...... xv DEDICATION...... xvi CHAPTER 1: INTRODUCTION ...... 0 1.1 Objectives ...... 4 CHAPTER 2: LITERATURE REVIEW ...... 5 2.1 Causal factors of replant disease ...... 5 2.1.1 Spatial distribution of RD ...... 7 2.2 Methods of control ...... 7 2.2.1 Chemical control ...... 7 2.2.2 Cultural control and alternatives to fumigation ...... 8 2.2.3 Phosphorus and problem replant soils ...... 10 2.3 Soil fertility and fertilization ...... 11 2.3.1 P distribution in the soil ...... 11 2.3.2. Root development in tree fruit and soil P ...... 13 2.3.3 P uptake by plants ...... 15 2.3.4 P nutrition from green manure, cover crops and composts ...... 16 2.3.5 Rock phosphate and bone meal for P nutrition ...... 17 2.4 Role of soil microorganisms in soil P nutrition...... 18 2.4.1 Plant growth promoting rhizobacteria ...... 18 2.4.2 Phytohormone-producing bacteria ...... 19 2.4.3 Antibiosis by PGPR ...... 20 2.4.4 Phosphate solubilizing bacteria...... 20 2.5 Using PSB as a strategy to enhance P acquisition ...... 21 2.5.1 Organic P mineralization by root exudates and enzymes ...... 22 2.5.2 Phosphatase ...... 22 2.5.3 Phytase ...... 23 2.5.4 Pi solubilization through organic acid production ...... 23 2.5.5 Rock phosphate and PSB activity ...... 25

2.6 Microbial inoculants for improving fertilizer P efficiency ...... 25 CHAPTER 3: MATERIALS AND METHODS ...... 28 3.1 Bacterial strains ...... 28 3.2 Phosphate plate assays ...... 28 3.3 Liquid phosphate assays ...... 30 3.4 QuantiChrom phosphate assays ...... 32 3.5 Growth pouch assays ...... 33 3.6 Greenhouse bioassay 1 – Organic soil amendments ...... 35 3.6.1 Site selection and soil sampling ...... 35 3.6.2 Pot preparation and treatment selection ...... 37 3.6.3 Broth preparation ...... 39 3.6.4 Experimental design and seedling germination ...... 40 3.7 Greenhouse bioassay 2 – PSB in potting mix ...... 41 3.7.1 Bacterial Isolate Identification ...... 44 3.7.2 Standard Curve Preparation ...... 44 3.8 Greenhouse bioassay 3 – PSB with field trial soils ...... 45 3.9 Orchard Field Trials ...... 47 3.9.1 Site selection ...... 48 3.9.2 Field trial set-up and fertilizer treatments ...... 49 3.9.3 Bacterial mixture preparation and treatment ...... 50 3.9.4 Field trial measurements and soil sampling ...... 51 3.9.5 Nematode sampling ...... 51 3.9.6 Year one fall field activities ...... 52 3.9.7 Year two field activities and sampling...... 52 3.10 Statistical Analysis ...... 53 CHAPTER 4: RESULTS ...... 54 4.1 Phosphate plate assays ...... 54 4.2 Liquid culture assays ...... 61 4.2.1 pH change for NBRIP with 5 g P /L ...... 61 4.2.2 Soluble Pi released by bacterial isolates for NBRIP with 5 g/L CaHPO4 ...... 63 4.2.3 pH change for NBRIP with 1 g P /L ...... 63 4.2.4 Soluble Pi released by bacterial isolates for NBRIP with 1 g/L CaHPO4 ...... 64 4.3. Growth pouch assays ...... 67 4.4 Greenhouse bioassay 1- Organic soil amendments ...... 70 4.5 Greenhouse bioassay 2- PSB in potting mix ...... 73 4.6 Greenhouse bioassay 3- PSB with field trial soils ...... 75 4.7 Field trial data...... 76 4.7.1 Nematode data ...... 80

CHAPTER 5: DISCUSSION ...... 82 5.1 Identification of PSB candidates from in vitro assays ...... 82 5.2 Behaviour and characteristics of isolates tested in liquid medium ...... 84 5.3 Performance of PSB in growth pouches ...... 86 5.4 Performance of PSB and PSB-fertilizer combinations in greenhouse bioassays ..... 89 5.5 Application of PSB and PSB-fertilizer combinations to orchards ...... 91 CHAPTER 6: CONCLUSIONS ...... 97 REFERENCES ...... 99 APPENDICES ...... 109 Appendix A. Supplementary Data ...... 110 A.1. List of Tables ...... 110 A.2. List of Figures ...... 156 A.3. List of Illustrations ...... 160 Appendix B. P-free Hoagland’s Nutrient Solution Recipe ...... 162 Appendix C. Soil amendment calculations for Fish Fertilizer and P amendments. .... 163

LIST OF TABLES

Table 3.1. A general description of the soils (E, F, G, H, I) obtained from Okanagan orchards for the greenhouse bioassays at AAFC-PARC and their characteristics...... 37

Table 3.2. A general description of the soil treatments used in the first series of greenhouse bioassays (Soils E, F, G, H, I) at AAFC-PARC...... 39

Table 3.3. A general description of the soil treatments used for Greenhouse bioassay 2- PSB in potting soil...... 43

Table 3. 4. A general description of the soil treatments used in greenhouse bioassay- 3 (McCoubrey and Reiger soils)...... 46

Table 3.5. A general description of the soil treatments used in the orchard field trials (McCoubrey and Reiger orchards)...... 48

Table 4.1. P solubilizing ability of PGPR isolates on CaHPO4 plates...... 54

Table 4.2. Solubilization index values for twelve bacteria isolates, following growth on CaHPO4 plates at 28°C for 7 days...... 58

Table 4.3. Zone of clearing (halo diameter) values for 15 bacterial isolates, on three different P media, after incubation at 28°C for 7 days (n=3)...... 60

Table 4.4. Effect of PSB isolates (1-18, 2-28, 6-8 and 4-42) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 62

Table 4.5. Effect of PSB isolates (5-24, 2-96, 3-32, 6-114 and 6-63) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 62

Table 4.6. Effect of PSB isolates (1-8, 2-23, 2-106 and 4-15) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 62

Table 4.7. Effect of PSB isolates (2-18, 2-9, 2-57 and 1-132) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 63

Table 4.8. Effect of PSB isolates (1-18, 2-96, 4-15 and 5-24) on pH of NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 64

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Table 4.9. Effect of PSB isolates (2-18, 2-106 and 2-23) on pH of NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 64

Table 4.10. Effect of PSB isolates (1-18, 2-18, 2-23, 2-96, 2-106 and 4-15) and RP on pH of liquid medium in growth pouch bioassay with apple seedlings grown for four weeks at 18°C with shaking at 100 rpm...... 68

Table 4.11. Effect of PSB and RP in liquid medium in growth pouch bioassay with apple seedlings grown for four weeks...... 69

Table 4.12. Root analysis data showing the effect of PSB and RP on apple seedling growth in a growth pouch bioassay...... 70

Table 4.13. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil E over a 9-week growth period...... 71

Table 4.14. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil F over a 9-week growth period...... 72

Table 4.15. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil G over a 9-week growth period...... 72

Table 4.16. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil H over a 9-week growth period...... 73

Table 4.17. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil I over a 9-week growth period...... 73

Table 4.18. Plant growth data for greenhouse bioassay 2- PSB in potting mix...... 74

Table 4.19. Plant growth data for greenhouse bioassay 3- PSB with Reiger field soil...... 75

Table 4.20. Plant growth data for greenhouse bioassay 3- PSB with McCoubrey field soil...... 76

Table 4.21. Trunk cross sectional area (cm2) for the Reiger orchard replant, measured at three time points and the total change in trunk cross sectional area (cm2) from planting (May 2011) to the end of the field trial (September 2012)...... 78

Table 4.22. Trunk cross sectional area (cm2) for the McCoubrey orchard replant, measured at three time points and the total change in trunk cross sectional area (cm2) from planting (May 2011) to the end of the field trial (September 2012)...... 78

Table 4.23. Total shoot length (cm) for the Reiger orchard replant, measured at three time points and the change in tree total shoot length (cm) from planting (May 2011) to the end of the field trial (September 2012)...... 79

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Table 4.24. Total shoot length (cm) for the McCoubrey orchard replant, measured at three time points and the change in the total shoot length (cm) from planting (May 2011) to the end of the field trial (September 2012)...... 80

Table 4.25. Number of nematodes (Pratylenchus sp.) present per 50 ml subsample of soil for the Reiger orchard by date sampled...... 80

Table 4.26. Number of nematodes (Pratylenchus sp.) present per 50 ml subsample of soil for the McCoubrey orchard by date sampled...... 81

Table A.1 Media prepared for in-vitro testing of phosphate solubilization...... 110

Table A.2 Gravimetric moisture content of dairy manure compost ...... 111

Table A.3 Gravimetric moisture content of soil E ...... 112

Table A.4 Gravimetric moisture content of soil F ...... 112

Table A.5 Gravimetric moisture content of Sunshine Organic and Natural Potting Mix # 3 ...... 113

Table A.6 Soil test report values for the McCoubrey and Reiger field sites, testing performed by A and L Laboratories, London ON...... 114

Table A.7 Key data extracted from the ANOVA tables for the mean halo diameter measured on Blue PVK medium, inoculated with PSB after a 7 day incubation period at 28°C...... 115

Table A.8 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4...... 115

Table A.9 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4 ...... 116

Table A.10 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4...... 116

Table A.11 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4...... 117

Table A.12 Key data extracted from the ANOVA tables for the mean pH measurements of 1 g/L of CaHPO4...... 117

Table A.13 Key data extracted from the ANOVA tables for the mean pH measurements of 1 g/L of CaHPO4...... 118

Table A.14 Effect of PSB isolates (1-18, 2-96, 4-15 and 5-24) on inorganic phosphate concentration (mg/ 10 mL) in NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 118

Table A.15 Key data extracted from the ANOVA tables for the mean Pi measurements of 1 g/L of CaHPO4...... 119

Table A.16 Effect of PSB isolates (2-18, 2-106 and 2-23) on inorganic phosphate concentration (mg/ 10 mL) in NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm...... 119

Table A.17 Key data extracted from the ANOVA tables for the mean Pi measurements of 1 g/L of CaHPO4...... 120

Table A.18 Key data extracted from the ANOVA tables for the mean pH measurements of NBRIP solution with RP in the growth pouches ...... 120

Table A.19 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters of apple seedlings ...... 121

Table A.20 Key data extracted from the one-way ANOVA tables for the root measurements and root architecture analysis of apple seedlings ...... 121

Table A.21 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment E. .... 122

Table A.22 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment F...... 122

Table A.23 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment G. .... 123

Table A.24 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment H. .... 123

Table A.25 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment I...... 124

Table A.26 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-2, PSB in potting mix...... 124

Table A.27 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-3, PSB with Reiger field soil...... 125

Table A.28 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-3, PSB with McCoubrey field soil...... 125

Table A.29 Key data extracted from the one-way ANOVA tables for the mean measurement of trunk cross sectional area of trees planted in the Reiger orchard ...... 126

Table A.30 Key data extracted from the one-way ANOVA tables for the mean measurement of the total shoot length for trees planted in the Reiger orchard ..... 126

Table A.31 Key data extracted from the one-way ANOVA tables for the mean measurement of trunk cross sectional area of trees planted in the McCoubrey orchard ...... 127

Table A.32 Key data extracted from the one-way ANOVA tables for the mean measurement of the total shoot length for trees planted in the McCoubrey orchard ...... 127

Table A.33 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, July 2011...... 127

Table A.34 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, September 2011...... 128

Table A.35 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, June 2012...... 128

Table A.36 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the McCoubrey orchard, September 2011...... 128

Table A.37 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the McCoubrey orchard, June 2012...... 128

Table A.38 Descriptive statistics showing variance for data contained in Tables 8- 31 and Figures 4 and 5...... 129

LIST OF FIGURES

Figure 1. P solubilization assay on CaHPO4 medium showing single colonies of five different bacterial isolates ...... 57

Figure 2. Ranking of the thirty-four bacterial isolates showing P solubilizing potential, by the size of the zone of clearing, as measured on CaHPO4 plates after incubation at 28° C for 7 days...... 57

Figure 3. Three media used to compare P solubilization on plate assays ...... 59

Figure 4. Effect of PSB isolates (1-18, 2-96, 4-15 and 5-24) on inorganic phosphate concentration (mg/ 10 mL) ...... 65

Figure 5. Effect of PSB isolates (2-18, 2-106 and 2-23) on inorganic phosphate concentration (mg/ 10 mL) ...... 66

Figure 6. Apple seedlings inoculated with PSB in growth pouches with RP fertilizer added to NBRIP medium...... 68

Figure A.2.1. Standard curve prepared for bacterial isolate 2-18...... 156

Figure A.2.2. Standard curve prepared for bacterial isolate 2-106...... 157

Figure A.2.3. Standard curve prepared for bacterial isolate 4-15...... 158

Figure A.2.4. Standard curve prepared for QuantiChrom phosphate assays...... 159

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LIST OF ILLUSTRATIONS

Illustration A.3.1 Map of treatment blocks for field trial at Orchard Corners/Reiger...... 160

Illustration A.3.2 Map of treatment blocks for field trial at McCoubrey Farms...... 161

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LIST OF ABBREVIATIONS

* (one asterisk) – pvalue = 0.05 – 0.01 ** (two asterisks) – pvalue = 0.01 – 0.001 *** (three asterisks) – pvalue = < 0.001 ½ TSA – half-strength tryptic soy agar ½ TSB – half-strength tryptic soy broth 16S rRNA- 16S ribosomal ribonucleic acid AAFC – Agriculture and Agrifood Canada ADP- adenosine diphosphate AMF- arbuscular mycorrhizal fungi ATP- adenosine triphosphate BC – British Columbia BCMAL - British Columbia Ministry of Agriculture and Lands Blue PVK- Pikovskaya medium with bromophenol blue added BM- bone meal CEC- cation exchange capacity CFU – colony forming units COR- Canadian Organic Regime HD- halo diameter log10 – logarithmic to the base 10 LSD – least significant difference MAP- monoammonium phosphate MOA – mode of action NBRIP- National Botanical Research Institute’s minimal phosphate growth medium OD600 – optical density at 600 nm P- phosphorus Po- organic phosphorus Pi- inorganic phosphorus PARC- Pacific Agri-Food Research Centre PBS – phosphate buffered saline PGPR- plant growth promoting rhizobacteria PSB- phosphate solubilizing bacteria PVK- Pikovskaya medium RD- replant disease RO- reverse osmosis RP- rock phosphate rRNA- ribosomal ribonucleic acid ΔSL- change in SL from time of planting to the end of project TCSA- trunk cross sectional area ΔTCSA- change in TCA from time of planting to the end of project TSA/B – trypic soy agar TSB- tryptic soy broth

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ACKNOWLEDGMENTS

I would like to extend my sincere appreciation to my supervisors, Dr. Louise M. Nelson and Dr. Gerry Nielsen for their support and guidance throughout my graduate studies. Thank you for your keen interest and active participation throughout the project. Your flexibility, patience and trust in me to work independently were much appreciated as I tried to juggle many commitments over the past several years.

I would also like to express my thanks to my committee members, Drs. John Klironomos and Melanie Jones. I am particularly grateful to my supervisors, course professors and committee members for their constructive feedback on my project and challenging questions which have broadened my thinking and served to deepen my understanding of the scientific process.

The completion of this project would not have been possible without the assistance of many individuals! Many thanks to Dr. Tom Forge for his nematode expertise and to his technicians at AAFC-Agassiz for their identification and analysis work. Thank you to the team in the Nelson Lab – Natashia Bose-Roberts, Daylin Mantyka, Morgan Roosenmaallen, Kersti Ojaama, Chris Milner, Kelly Berringer, Geet Hans and Tanja Voegel. Thank you to Bill Rabie for assistance with the greenhouse trials. Warmest thanks to Ms. Hiltrud Vogler and Barb Lucente for their administrative assistance.

I am so grateful for the support and encouragement of my many colleagues in the Field Service and Tree Fruit Industry, for your interest in my work and flexibility during my leave! In particular, to Dr. Bill McPhee whose enthusiasm for helping growers and practical approach to research sparked my interest in studying Phosphorus and Replant Disease and to Dr. Hilary Sampson for her encouragement to pursue more studies and sage advice, never stop learning.

To my dear friends and family – near and far, who have offered words of encouragement over a coffee, a long run or while helping out on the farm- Thank you! You are amazing!

Finally, an enormous thank you to my partner, Matt - for encouraging my dreams, tempering my manic tendencies and for always being my rock… maybe it’s time for a holiday!

DEDICATION

To my family

Welcome Angus Rex

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CHAPTER 1: INTRODUCTION

Pome and soft fruits are important horticultural crops in Canada. Canadian apple production in 2008 exceeded $177 million dollars in farm gate sales (Agriculture Agri-Food

Canada 2008). The British Columbia tree fruit industry is currently comprised of over 1200 growers and 17, 500 planted acres and leads Canada in sales, averaging over $60 million dollars in average farm gate sales (BCMAL 2010a). Since the inception of the Orchard Renovation

Program in 1991, growers have replanted over 9000 acres of aging orchards to new varieties, at

higher planting densities (Ference Weicker & Company 2007). This replant initiative has served

to revitalize the tree fruit industry and to increase average yields to growers.

Traditionally orchards were planted at low tree densities using standard or seedling

rootstocks that produced large, vigorous trees that were slow to bear fruit. In an effort to create

pedestrian-style orchards, most tree fruit growing regions of the world have converted to high-

density planting systems. These orchards, which may be trellised to create fruiting walls, make

use of dwarfing rootstocks such as the Malling 9 (M9), which was developed at the East Malling

Research Station in Kent, England (Westwood 1993). The use of dwarfing rootstocks is an effort

to create more pedestrian-style work areas, for ease of picking and performance of other orchard

activities. High-density orchards with M9 rootstocks also allow for earlier cropping and

increased fruit yields per land area (Westwood 1993). The drawback of planting dwarf trees at

high densities is that the trees have less natural vigour and shallower roots that provide poor

anchorage; in marginal soils, growers may be required to rely heavily on chemical fertilizers to

encourage tree growth in order to establish the root system of a young rootstock tree (Westwood

1993). Chemical fertilizers are expensive and when used in excess quantities may result in the

contamination of surrounding lands and waterways.

In addition to their need for adequate nutrition, young dwarf trees must be protected from

the many biotic factors that may negatively influence their early growth. Competition from

weeds, injury due to rodents, plant-parasitic nematodes and plant pathogens can seriously affect

fruit tree establishment (Westwood 1993). Of these factors, plant pathogens in particular are of

serious concern to tree fruit growers when replanting (Hoestra 1968; O’Kennedy and Kavanagh

1980). All of these factors combined form the complex disorder, commonly known as tree fruit replant disease.

Replant disease (RD) has been recognized for over 200 years in all major agricultural

regions of the world; it is characterized by a decline in the health and vigour of newly planted

seedlings and is often associated with soil pathogens (Yadava and Doud 1980). RD affects both

pome and stone fruits leading to reductions in tree growth, crop yield and the lifespan of the

orchard (Mai and Abawi 1981; Traquair 1984; Neilsen et al. 1991; Utkehde and Smith 1993;

Bent et al. 2009). In areas such as the Okanagan Valley of British Columbia, where irrigated

orchard land is valued at between $85,000 - $125,000 dollars per acre and orchard establishment

costs can be in the tens of thousands of dollars per acre, RD can lead to serious economic losses

for growers establishing new orchards (Farm Credit Canada 2010).

The symptoms of RD may present themselves in the orchard as highly variable tree

growth and reduced tree vigour. Newly planted trees may demonstrate an initial growth spurt,

followed by a rapid decline in tree health and vigour. If the young trees survive the first season

after planting in replant soils, they are generally not uniform in size and have little to no productive capacity; resulting in a failure to achieve a full crop load in an acceptable timeframe.

There are many possible contributing factors to RD. Infrequent or poor irrigation practices can contribute to the problem of stunted trees with poorly developed root systems.

Damage caused by rodents and other small animals feeding on the tree roots will slow the growth of the trees and leave them vulnerable to pathogen attack (BCMAL 2010b). Fungal pathogens and nematodes undoubtedly also play a very important role in the RD complex. Pythium,

Rhizoctonia, Phytopthora and Cylindrocarpon have been found by apple RD researcher, Mark

Mazzola to be responsible for seedling decline in numerous studies (Mazzola 1998; Mazzola and

Mullinix 2005). Finally, soil structure, soil pH and low nutrient status are also important factors

contributing to the overall health, rooting and establishment of young trees (BCMAL 2010b).

For over sixty years conventional agriculture has used methyl bromide and other

chemical fumigants to manage RD in agricultural soils. By controlling various fungi, bacteria,

soil viruses, nematodes, insects and killing weed seeds (Ristaino and Thomas 1997; Duniway

2002), methyl bromide presented an essentially sterile soil, in which to plant seedlings. Soil

sterilization has been reported in numerous studies to confer an advantage to seedlings, allowing

the young plants to establish their root system with very little chance of attack (Hoestra 1968;

O’Kennedy and Kavanagh 1980; Slykhuis and Li 1985). However, soil sterilization has negative

long-term environmental costs as indicated by Ristaino and Thomas (1997) and has been highly

criticized by proponents of sustainable agriculture who do not support the use of fumigants for

the purpose of controlling a small number of plant-pathogenic soil micro-organisms at the cost of severe negative effects on non-pathogenic soil microbes (Vyas 1988).

In the past three decades, safety and environmental concerns have led to the banning of methyl bromide and restrictions on several other chemical fumigants. In 1992, methyl bromide was listed under the UN’s Montreal Protocol as a controlled substance for its role in ozone depletion (UNEP 1992). Currently, only a few chemical fumigants are available for use by conventional tree fruit growers in North America and Europe. The problem is particularly acute for certified organic tree fruit producers in Canada who are prohibited from using chemical fumigants and currently have no access to biological fumigants to combat replant disease.

As public pressure to reduce chemical use in agriculture increases, an urgent need for alternatives to fumigation and chemical fertilizers to combat replant problems such as RD has developed. Recent research has found that the use of soluble phosphorus (P) fertilizers in non- fumigated orchards (Neilsen and Yorston 1991) and the application of organic matter with high organic phosphorus content to the root zone of newly planted trees can be used in order to stimulate root growth (Neilsen et al. 2004) and mitigate some of the effects of RD in new orchards. Other recent successes in pathogen control through the manipulation of the bacterial communities present in the rhizosphere suggest that bacterial inoculants created from plant growth promoting rhizobacteria may also serve as a viable option to mitigate the effects of RD

(Mazzola 2009).

1.1 Objectives

My thesis will build upon previous research to address the following objectives to: 1) identify a range of phosphate solubilizing bacteria (PSB) suitable for field testing in tree fruit orchards; 2) identify combinations of PSB and P-fertilizer amendments with stimulatory plant growth effects in soils from certified organic orchards which exhibit symptoms of RD.

I hypothesize that combinations of soil amendments and bacterial inoculation treatments will overcome orchard replant problems in certified organic production systems. Specifically, I hypothesize that phosphorus-solubilizing bacteria in combination with P amendments allowed by the Canadian Organic Regime (COR) for certified organic production, including compost, mineral rock phosphate (RP) and bone meal (BM) will promote improved root and shoot growth in apple seedlings.

Ultimately, this study aims to develop methods to mitigate the effects of RD in certified organic production systems by manipulating the biology of the rhizosphere in affected tree fruit orchards. The end goal of the project is to identify bacterial treatments for RD with the potential for practical use in the certified organic production systems.

CHAPTER 2: LITERATURE REVIEW

Replant disease is characterized by poor growth in the initial three years of orchard establishment, as well as reduced fruit yields over the lifetime of the orchard due to factors not addressed by good orchard management practices (Utkhede and Smith 1993). Symptoms of RD include poor growth and severe stunting of young trees, including fewer branches and underdeveloped leaves (Mazzola 1998). Trees affected by replant disease generally have under- developed root systems with few feeder roots (Savory 1966). Existing roots are often damaged and discoloured, with lesions or decay present (Hoestra 1968). Replant disease severely affects the growth and establishment of the young tree, resulting in delayed fruit bearing and poor yields

(Traquair 1984). If left uncontrolled, RD leads to the overall decline of the young tree and in severe cases, to tree death.

2.1 Causal factors of replant disease

The causal factors of RD are complex and the etiology of the disease is not well defined; however, it is believed that there exist both abiotic and biotic factors that contribute to the disease (Mazzola 1998; Granatstein and Mazzola 2001). Many studies have observed positive tree growth responses after soil fumigation, providing strong evidence that RD is partially caused by biotic factors, such as fungal pathogens and plant parasitic nematodes (Mai and Abawi 1981;

Slykhuis and Li 1985; Granatstein and Mazzola 2001). Some of the abiotic factors believed to contribute to RD include: extremes in soil pH, poor soil structure (Granatstein and Mazzola

2001), and drainage issues and other environmental stresses (Mai and Abawi 1981; Traquair

1984). Soil nutrient imbalances and limitations, particularly phosphorus, also contribute to the

RD complex (Merwin and Stiles 1989; Neilsen and Yorston 1991).

Replant disease appears to be particularly prevalent in newly-established high-density plantings on old orchard sites; however, it does not affect all of the trees. It has been observed that the number of trees affected by RD and the severity of the effects can vary significantly

(Van Schoor et al. 2009). This observed variability in the infectious nature of RD has led some researchers to further classify replant disease by two general types: non-specific RD and specific

RD (Bent et al. 2009). Non-specific RD is characterized by a patchy distribution of several affected trees positioned throughout the orchard, but not located adjacent to one another. It is believed that high numbers of plant parasitic nematodes may be the causal agent of non-specific

RD (Utkhede and Smith 1993; Bent et al. 2009). Conversely, specific RD exhibits a more uniform distribution throughout the orchard and generally affects one or several adjacent or other trees located in close proximity to one another (Bent et al. 2009). It is possible that a complex of fungal pathogens may be the most significant causal agent for specific RD. Studies of RD etiology have consistently identified the same fungal complex comprised of the genera

Cylindrocarpon, Phytophthora, Pythium and Rhizoctonia in orchard blocks suffering from decline symptoms in growing regions around the world (Dullahide et al. 1994; Braun 1995;

Mazzola 1998, Mancini et al. 2003; Bent et al. 2009; Van Schoor et al. 2009).

2.1.1 Spatial distribution of RD

Replant disease may progress rapidly after planting and is thought to have a spatial component (Mazzola 2009). Symptoms of RD have been prevalent in many high-density orchard systems that were planted at 1500 to 2200 trees per acre. In these high-density plantings, the majority of tree roots are confined to the top 30 cm of the soil, spreading out in the weed-free planting row to a maximum width of one to two meters. Detrimental effects of RD are observed to be greatest where new plantings intersect the old tree rows and symptoms have been observed to decline with greater distance from the location of the row (Mazzola 2009). Typically high- density orchards are planted as monocultures, using only clonal rootstocks that have been produced to be genetically identical to the parent tree. Packer and Clay (2000) found that seedling density affected the incidence of tree infection in the presence of the root pathogen

Pythium. Higher density plantings of the same tree species were found to be more susceptible to infection than blocks of the same or mixed species separated from one another by a greater distance.

2.2 Methods of control

2.2.1 Chemical control

Soil fumigation has long been the standard pre-plant treatment of the horticulture industry

for the prevention of RD (Mai and Abawi 1981; Slykhuis and Li 1985; Mazzola 1998; Csinos et al. 2000). Many plant pests and pathogens of tree fruits can be controlled or at least mitigated by soil fumigation prior to replanting (Koch et al. 1980). Soil fumigation has been found to improve

seedling survival in Pythium-infected soils as compared to unsterilized soils from the same

location (Packer and Clay 2000). Until recently, methyl bromide was one of the most broad

spectrum and widely used fumigants in the world. A gas with an activity over a wide range of

temperatures, methyl bromide rapidly penetrates the soil allowing for fast and effective soil

fumigation (Yeates et al. 1991). However, methyl bromide is no longer available to tree fruit

growers in North America and Europe. Although other chemical fumigants, such as dazomet,

1,3-dichloropropene and chloropicrin (tear gas) have been used as replacement products for

methyl bromide, there exist legitimate concerns over the safety and sustainability of these

products (Ristaino and Thomas 1997; USEPA 2008). With increasing health and environmental

concerns over chemical use, alternatives to soil fumigation for tree fruit growers such as

biofumigants and biofertilizers must be investigated.

2.2.2 Cultural control and alternatives to fumigation

Soils from previously planted orchard sites are likely to have been irrigated and treated with chemical fertilizers prior to replanting. These agricultural activities have been found to contribute to the degradation of agricultural soils (Neilsen et al. 2003; Melero et al. 2008).

Orchard soils are also subject to disturbances such as compaction, which has been observed to have a detrimental effect on soil structure and therefore on crop productivity (Lee et al. 1996).

Compaction can also negatively affect the soil microbiome; a recent study in Brazil, found that bacterial counts were significantly reduced in corn fields subjected to high levels of equipment traffic (Pupin et al. 2009).

Crop rotations are an ancient practice used to reduce soil-borne plant pathogens by breaking the host-pest disease cycle, while maintaining soil structure and organic matter (Janvier

et al. 2007). In orchard soils, where crop rotations are not practical, soils degrade over time.

Degraded soils suffer from low soil fertility as a result of the loss of soil organic matter (Melero

et al. 2008). Microbial biomass, although only a small portion of the total soil organic matter,

contributes significantly to the breakdown and incorporation of organic residues in agricultural

ecosystems (Lynch and Bragg 1985; Kennedy and Smith 1995). Soils low in organic matter lack

the substrate necessary to sustain a healthy population of soil microbes; therefore, they exhibit

low levels of the appropriate and beneficial micro-organisms that appear to encourage successful

plant establishment (Dick 1997; Melero et al. 2008).

In replanted orchards, strategies to increase and maintain soil organic matter and microbial biomass are necessary to improve overall soil fertility and to reduce the impact of RD.

Several studies have shown that the type and quality of the crop residue, compost or other form

of organic matter added to the soil can affect the diversity of the microbial population present in

that soil (Kennedy and Smith 1995; Bending et al. 2002; Mazzola and Mullinix 2005). Cover crops and green manures produced from various Brassica spp. have been found to suppress plant parasitic nematodes and some plant pathogens (Kirkegard and Sarwar 1998; Mazzola and

Mullinix 2005).

2.2.3 Phosphorus and problem replant soils

Research performed by Slykhuis and Li (1985) demonstrated in greenhouse trials that monoammonium phosphate (MAP) fertilizer applied as a pre-plant treatment could improve the growth of apple seedlings in RD-affected soils. Further work by Nielsen and Yorston (1991) found large increases in leaf P concentration in the first year, when MAP was applied as a planting hole treatment. The large increase in leaf P concentration in this study demonstrates the availability of P from MAP to the seedlings. High P availability is known to improve the establishment of apple trees by increasing the initial root length (Nielsen et al. 1991). The combination of MAP with various broad-spectrum soil fumigants resulted in significantly improved tree growth and establishment in comparison to the untreated control (Nielsen and

Yorston, 1991).

Results of the above-mentioned field trials and observations of replanted orchard blocks in the Okanagan Valley by Dr. W. McPhee (personal communication, September, 2010) confirm the beneficial effects of applying soluble P fertilizer such as 10-50-10 (Plant Products, Brampton

ON) on the establishment of young fruit trees. As such, biofertilizer has been suggested as an alternative to MAP and other synthetic P fertilizers in organic orchard systems. The combination of rock phosphate (RP) or other high-P soil amendments, such as bone meal (BM) and manure- based composts with PSB to increase P fertility in the soil and plant uptake is of growing interest to tree fruit growers.

2.3 Soil fertility and fertilization

Nitrogen and phosphorus are the most limiting nutrients for plant growth and for the

sustainability of agriculture on a global scale (Havlin et al. 1999). Degraded soils that suffer from low soil fertility status due to loss of soil organic matter often require supplemental fertilization in order to maintain soil nutrient levels (Melero et al. 2008). Nitrogen is associated with vigorous vegetative growth in tree fruits and provides the building blocks for the synthesis of proteins within the plant (Havlin et al. 1999). Nitrogen is a very mobile nutrient, which moves relatively freely in the soil solution; unlike P, which is highly immobile and often tightly bound in the soil to complexes of iron or aluminum. P is an essential macronutrient for the growth and development of plants. P is a critical nutrient for root growth and an important component of the molecules formed at the cellular level that are used for plant energy storage, as adenosine di- and triphosphates (ADP and ATP) (Marschner 1995).

2.3.1 P distribution in the soil

Most agricultural lands require additions of P fertilizer, even when the total P of the soil

is reported as high; this is because few unfertilized soils are able to release the amount of P that

is required to support the high growth rates of crop plants in a season (Schachtman et al. 1998).

The availability of P to plants depends upon soil type. Ramaekers et al. (2010) suggest that

major soil types can be distinguished from one another based on their total P content. Sandy soils

are an example of a soil with low total P, where calcareous soils are an example of a P-fixing soil

(Ramaekers et al. 2010). Some soils, particularly volcanic soils, contain active minerals that tie

up soil P. Andosol soils, for example contain active aluminum that binds large quantities of P,

causing P to accumulate in the soil in a complex form that is unavailable to the plant

(Wickramatilake et al. 2010). The result is that these soils have high levels of total P; however, they are deficient in available P, thus requiring annual P fertilizer applications to maintain their productivity (Borie and Rubio 2003).

Over eighty percent of P applied to the soil is quickly immobilized and unavailable for plant uptake (Holford 1997). P in the soil can be broadly classified as organic P (Po) and

inorganic P (Pi). Po accounts for approximately 50% of total soil P in cropping systems, existing

principally as inositol hexaphosphate, commonly known as phytic acid or phytate (Richardson

1994). Phytate is the most abundant phosphomonoester in the natural environment. Other

phosphomonoesters include phospholipids, nucleic acids, and sugar phosphates (Richardson

2001; Gyeneshwar et al. 2002). Phytate is also found in the seeds of plants and provides an

important source of P during seed germination (Russell 1988). Phytate can form strong

complexes in the soil by being adsorbed to clay particles (Jorquera et al. 2008). Phytate may

also precipitate to form insoluble salts such as iron and aluminum oxides or hydroxides in acidic

soils and insoluble calcium salts in alkaline soils (Jorquera et al. 2008) This is important from an

environmental point of view as phytate is prone to leaching, run-off and thus subsequent losses of P (Borie and Rubio 2003).

The remainder of the total P in the soil is found in the inorganic form, consisting of up to

170 mineral forms of Pi (Holford 1997). Plant available Pi is often bound to metals in the soil, 12

forming oxides of iron and aluminum, aluminum silicates or carbonates with calcium

(Richardson 2001). The amount of Pi rarely exceeds 10 µM in the soil solution (Bieleski 1973).

Pi is principally moved by diffusion to the plant through the soil solution; this is a very slow

process occurring at a rate of 10-12 to 10-15 m2/ s (Schachtman et al. 1998). This process occurs so

slowly that it often results in depletion of the available P in the rhizosphere, which needs to be

replenished regularly with additions of P fertilizer to the root zone or supplied by a network of

mycorrhizal fungi that extend the absorptive surface of the root system and can reach P deposits

outside of the depletion zone (Morin and Fortin 1994).

2.3.2. Root development in tree fruit and soil P

The roots of fruit trees interact with the soil, playing a critical role in water and nutrient

uptake while providing support and anchorage for the fruit-bearing tree. Fruit trees affected with

RD generally have under-developed root systems with few feeder roots (Savory 1966). In tree

fruits, growth and development are influenced by the genetic control of the rootstock over the

aerial portion of the tree (Rom 1996). The M9 root system is relatively confined and shallow

rooted, making this root stock particularly vulnerable to RD. The fine feeder roots of the M9 root

system are important in water uptake and, if under-developed, can render the tree extremely

drought prone (Rom 1996).

Root geometry and morphology are important to maximizing P uptake. The growth and

proliferation of the roots into new regions of the soil, allow the plant to exploit soil nutrients in

various forms. The plant’s main strategies for P acquisition are to maximize its capacity to explore the soil through the proliferation and extension of all root types (Ramaekers et al. 2010) and to enhance P uptake by associating with specific PGPRs and mycorrhizal fungi (Koch et al.

1982). To increase access to available P in the soil, plants have developed strategies to change their root morphology by increasing the rate of root growth, total root length, root branching and amount/distribution of root hairs to better access soil P (Richardson 2001). The roots of plants interact with a diverse population of microorganisms and their root exudates can modify the physio-chemical properties and biological composition of the rhizosphere (Richardson et al.

2009). Acidification of the rhizosphere is one such modification, where microbial exudates produce a pH change in the soil surrounding the root tips that can influence the availability and uptake of nutrients by the plant (Grayston et al. 1996; Richardson et al. 2009).

In P-deficient soils, plants allocate more assimilates to root growth, leading to finer roots

with smaller diameters and increased surface areas (Schenk and Barber 1979; Snapp et al. 1995).

This, combined with an increased number of root hairs, is very effective in scavenging P from

the soil (Itoh and Barber 1983). The stimulation of root hair growth in plants associated with

microorganisms has been found to increase the surface area and P uptake as roots may also

exude P solubilizing compounds such as organic acids that release Pi and phosphatase enzymes

required for the mineralization of Po (Richardson 1994; Richardson 2001; Ramaekers et al.

2010). Early work by Drew (1975) indicated that barley grown in areas of high soluble phosphorus applications showed prolific development of first and second order root laterals.

Lynch (1995) also found that increasing the surface area to volume ratio of plant roots, allows

root systems to explore larger volumes of soil, enabling increased P uptake by the plants. These

examples of increased root growth and the proliferation of roots into new regions of the soil

allow for improved access to P and emphasize what Russell (1988) suggested is the main effect

of P applications to the root zone: improved feedback between root and shoot development.

2.3.3 P uptake by plants

Adequate P levels are critical to obtain optimal crop yields, as P is essential for cell

2- division (Russell 1988). Plants can only absorb P from the soil as phosphate anions (HPO4 and

- H2PO4 ); the form present in the soil solution depends upon the soil’s physical and chemical

properties and the proportions of each change significantly at different soil pH levels (Black

- 1968). The monovalent form of P (H2PO4 ) is thought to be the predominant form of phosphate

ion in the soil solution and is most available in soils of low (pH 5- 6) (Bieleski 1973; Richardson

2- 1994). Conversely, in soils of high pH (pH 8) the divalent form (HPO4 ) of phosphate

predominates (Black 1968).

There is a need to balance the amount of available P in the soil with the amount of P

required by the trees for growth and crop maintenance (Havlin et al. 1999). Green manure, cover crops and compost applications have been suggested as means through which producers can increase their soil organic matter and thus, soil P fertility without resorting to chemical

fertilizers.

2.3.4 P nutrition from green manure, cover crops and composts

The slow release of P from green manure has been found to be more effective than the

release of fertilizer P from insoluble sources; however, this is true only if the green manure is

incorporated into the soil and the nutrient distributed through the soil layer where the root zone is

localized (Havlin et al. 1999). Using a green manure prior to planting may provide several

benefits, as previously discussed; however, annual green manure applications are not appropriate

for high density orchards where once the trees are established it is impractical to turn over a

green manure crop in the tree row. After the initial planting of the young orchard, it is more

practical to use soil amendments applied by hand or machine.

The application of compost to improve soil fertility, soil organic matter, texture and as a means of inoculating the soil with “good” microbes has also been suggested for agricultural soils suffering from RD (Hoitink and Boehm 1999; Neilsen et al. 2003). Increased turnover of P, the stimulation of PSB and the production of organic acids that dissolve insoluble RP have also been observed in soils amended with compost (Wickramatilake et al. 2010).

Although cover crops and compost will contribute to the goal of mitigating some of the abiotic causes of RD such as improving the overall soil structure of the orchard and addressing nutrient deficiencies in the soil, cover crops may negatively affect seedling growth by providing competition for plant nutrients and water. Moreover, thick layers of compost may create habitats for rodents and immature compost may upset the carbon to nitrogen balance of the soil. The

disadvantages of cover crops and compost described here, coupled with the high cost of application, have generally meant that tree fruit growers in organic production systems forgo their usage. With no chemical fertilizers available, organic growers who seek to improve the levels of their soil phosphorus must then look to apply other forms of P, generally as relatively insoluble RP or BM or a blended fertilizer product with one or both of these components.

However; as discussed herein, both of these soil amendments require specific environmental/soil conditions in order to increase their efficiency and successful uptake by the tree.

2.3.5 Rock phosphate and bone meal for P nutrition

Excellent root growth and root system development have been observed in conventional orchards using applications of soluble P fertilizers (phosphoric acid) to the root zone of replanted trees, as well as in established orchard blocks suffering from RD (McPhee, personal communication, September 2010). Additions of untreated RP and BM may be used to increase P nutrition in certified organic orchards; however, both of these forms of P are insoluble and of limited value to plants in their natural states.

Unprocessed RP is less effective in soils with a low cation exchange capacity and high pH, as well as in soils with low organic matter content and therefore, low microbial activity

(Wickramatilake et al. 2010). Warm, moist soils favour RP dissolution and uptake, making the irrigated orchard soils of the Okanagan a viable location for RP applications. Thorough mixing of RP in soils also increases efficacy, as does the use of the apatite form of RP on acidic soils

(Havlin et al. 1999). Plant uptake of P from RP is strongly related to microbial biomass P and the

population density of PSB in the soil (Wickramatilake et al. 2010).

2.4 Role of soil microorganisms in soil P nutrition

In order to maximize soil P availability from organic and inorganic sources, it is essential

that soil microorganisms be present for the decomposition process to occur. Enzymatic release

of P from organic compounds occurs in the presence of soil microorganisms (Vessey 2003);

while the P provided by the vegetal matter from green manure, cover crops and composts when decomposed has increased availability in the presence of organic acids (Havlin et al. 1999).

Similarly, the production of organic acids by soil microorganisms increases the release of P from

inorganic phosphate compounds such as RP and BM (Rodriguez and Fraga 1999). The use of

naturally-occurring soil microorganisms, such as plant growth promoting rhizobacteria (PGPR)

or arbuscular mycorrhizae, is also considered to be more environmentally friendly than chemical

treatments of RP or BM and may enhance root growth and improve soil nutrient uptake by crop

plants (Rodriguez and Fraga 1999; Richardson 2001; Vessey 2003).

2.4.1 Plant growth promoting rhizobacteria

PGPR are naturally-occurring soil microorganisms. These root-colonizing bacteria have a

positive effect on the growth and development of a plant. PGPR have also demonstrated the

ability to colonize and perform important biological control functions in soils that have been

sterilized by fumigation or other experimental sterilization treatments (Bending et al. 2002;

Aslantas et al. 2007). PGPR have been researched and used extensively in agriculture, horticulture, forestry and for phytoremediation of contaminated sites (Schenck zu Schweinsberg-

Mickan and Muller 2009). There are three main functions of interest to researchers of PGPR: phytohormone production, fungal suppression and nutrient acquisition (Glick 1995; Lucy et al.

2004). It is also possible that mixtures of multiple microorganisms may be used to alter microbial

competition in the rhizosphere of plants leading to improved seedling growth in RD-affected soils (Avis et al. 2008).

2.4.2 Phytohormone-producing bacteria

PGPR have been observed to produce phytohormones important to plant development, including auxins and cytokinins (Garcia de Salamone et al. 2001). For example, indole acetic acid; IAA, is a phytohormone which induces cell division and root elongation. Cytokinins are another phytohormone which influences many physiological processes in the plant, including the production of additional root hairs (Garcia de Salamone et al. 2001). Increasing the volume of root hairs through the inoculation of roots with PGPR can promote the release of increasing amounts of root exudates, of which organic acids form part of the complex mixture of substances that comprise these exudates, and organic acids contribute to the solubilization of unavailable soil phosphates (Richardson 2001; Lucy et al. 2004; Aslantas et al. 2007).

2.4.3 Antibiosis by PGPR

PGPR can be antagonistic to fungal pathogens by competing for space and nutrients and

by producing antibiotic compounds that have the potential to suppress the growth of fungal

pathogens. For example, the compound pyrrolnitrin has been found to suppress fungal infections

of apples and pears and is produced by isolates of Serratia plymuthica and Pseudomonas cepacia

(Janisiewicz et al. 1991; Kamensky et al. 2003). PGPR also have the potential to assist the young

trees to overcome orchard replant disorder by conferring antifungal properties to the roots of the

plants with which they associate (Biro et al. 1998; Utkhede 2006).

2.4.4 Phosphate solubilizing bacteria

PGPR can also perform an important role as solubilizers of vital plant nutrients such as

phosphorus (Bending et al. 2002). In sustainable agro-ecosystems, PSB are used to improve the

bioavailability of accumulated P in soils and to increase the efficiency of the soil amendments

used. PSB are not intended to replace fertilizers that are added to the agro-ecosystem. Integrated

nutrient management is founded upon the use of locally available soil amendments to replace the

nutrients removed from the soil by the growing crop (Crowley and Rengel 1999). In order to

improve agronomic practices to utilize P more efficiently in the agro-ecosystem, an increased understanding of soil P dynamics and the effect of PSB on the existing P in the soil system and on other natural P sources, such as RP and BM is needed. With a better understanding of such factors, the efficiency of P uptake by crop plants could be improved, resulting in minimal losses of this important nutrient and providing beneficial economic and environmental impacts for farmers.

Neilsen and Yorston (1991) have previously established the importance of P to root growth and fruit precocity. Recent greenhouse studies using soil from an existing orchard demonstrating symptoms of tree decline, identified a P solubilizing bacterium and an auxin- producing bacterium (Sergeeva et al. 2007) as two treatments which stimulated growth of test seedlings (Nelson and Nielsen, unpublished data). These findings suggest that P solubilization and root length stimulation are two possible means of improving seedling establishment and performance in soils affected by RD.

2.5 Using PSB as a strategy to enhance P acquisition

PSB can be used as a means of enhancing P acquisition in the rhizosphere of plants by encouraging the production of root exudates, which modify the physical, chemical and biological properties of the rhizosphere. For example, exudates may cause acidification of the soil, thus decreasing the pH of the soil surrounding the root tips and therefore influencing the availability and uptake of nutrients by plants (Richardson et al. 2009). Roots may also exude enzymes useful in the mineralization of organic matter, which leads to the release of nutrients previously bound to the soil (Jorquera et al. 2008). Work by Malboobi et al. (2009) indicated that PSB are fairly dependent on root exudates, potentially for attachment to the root. Rhizodeposition and root turnover encourages microorganism growth by providing up to 40% of the carbon input into the soil (Grayston et al. 1996). Organic acids secreted by roots have an important role in solubilizing mineral nutrients and in creating a selective medium for the growth of microorganisms (Jones et al. 1996).

2.5.1 Organic P mineralization by root exudates and enzymes

Organic forms of P can be mineralized by enzymes and converted into usable forms of P

for plants. There are three groups of enzymes identified by Rodriguez et al. (2006) and by

Lugtenberg and Kamilova (2009) in their reviews of PGPR that help to mineralize P from organic compounds in the soil: non-specific phosphatases, phytases, phosphonatases and C-P

lyases. Phosphonatases and C-P lyases are enzymes that cleave carbon-phosphate bonds in organophosphonates but play a minor role in the mineralization of organic P as compared to the significant activity of acid phosphatases and phytases (Rodriguez et al. 2006). All of these enzymes may be produced by the plant itself or through an association of the plant and select microorganisms inhabiting the rhizosphere.

2.5.2 Phosphatase

Phosphatases are responsible for the dephosphorylation of phosphoric ester and

phosphoric anhydride bonds. The apical meristem of plant roots is known to exude phosphatases

into the rhizosphere in response to P deficiency (Crowley and Rengel 1999). Increased amounts

of phosphatase exudates have been produced under low P conditions (Crowley and Rengel 1999)

and in specific pH ranges (Rodriguez et al. 2006). Molecular work with Francisella tularensis

revealed that this bacterium expresses acid phosphatase activity at an optimal level in soil of pH

6 with a broad range of soil substrates (Reilly et al. 1996). Acid phosphatases work only to

release phosphate from Po; however, they are ineffective on phytin (Crowley and Rengel 1999).

2.5.3 Phytase

Phytin is broken down by the enzyme phytase; however, phytase is not excreted from

plant roots only by soil microbes themselves (Crowley and Rengel 1999). Phytase causes P

release in the form of free PO4 from phytic acid, Phytases can generally be classified in two major classes: acid and alkaline phytases (Jorquera et al. 2008). Recent work has further

identified phytase structure and catalytic mechanisms resulting, for example, in a distinction

between cysteine, beta-propeller and histidine acid phytases (Jorquera et al. 2008). Many studies

have isolated and identified bacteria that have been associated with a particular class of phytase

production. Members of gamma-proteobacteria have been most commonly reported to exhibit

phytase production, including species of the genera Bacillus, Enterobacter, Klebsiella and

Pseudomonas (Jorquera et al. 2008). Furthermore, a study by Richardson and Hadobas (1997)

identified species of the genera Enterobacter, Pantoea and Pseudomonas that exhibited the

ability to release inorganic orthophosphate from inositol hexaphosphate. It is evident from

research completed in this area that phytase-producing bacteria play a key part in the cycling of

Po in soils and are therefore of great agronomic value.

2.5.4 Pi solubilization through organic acid production

Pi binds easily to metal ions such as aluminum and iron to form insoluble mineral

complexes and metal-ion derivatives, such as iron or aluminum phosphates, which are known to accumulate in the soil. Researchers have found that Pi from these mineral complexes can be

solubilized by the organic acids produced by soil microorganisms (Rodriguez and Fraga 1999).

The mechanism of inorganic P solubilization relies primarily on the modification of the pH of 23

the rhizosphere soil and the extent to which the soil pH can be modified depends upon the buffering capacity of that soil (Rodriguez and Fraga 1999) To illustrate this mechanism, consider the example of a release of organic acids by PSB, which results in the acidification of the growing medium immediately surrounding the plant root system from which bound Pi is released

into the soil solution for plant uptake (Yadav et al. 2010). Goldstein (1996) proposed that the

oxidation of glucose to gluconic acid is the major mechanism of inorganic P solubilization.

However, it is now believed that many other organic acids are also produced by PSB and

contribute to the solubilization of Pi. Some examples of the most commonly identified organic

acids in the recent literature include oxalic, lactic, succinic and citric acids (Franklem et al. 2006;

Vyas and Gulati 2009; Wickramatilake et al. 2010).

The type of organic acid produced for Pi solubilization often depends on the organism

studied, as roots, soil bacteria and fungi can all release organic acids into the rhizosphere. Vyas

and Gulati (2009) examined organic acid production by P-solubilizing fluorescent pseudomonads

and found that they produced varying levels of gluconic, formic, malic and 2-ketogluconic acids.

Their work also concluded that the type and quantity of organic acids produced differs depending on the form of P supplied to the system; more specifically, differences were found in the organic acids produced to solubilize tri-calcium P and RP. Few isolates produced oxalic acid to solubilize tricalcium phosphate; however, most produced malic and succinic acids and all

Pseudomonas isolates tested produced gluconic and 2-ketogluconic acids in the presence of tricalcium P (Vyas and Gulati 2009). In the presence of RP, the Pseudomonas isolates all produced oxalic and gluconic acids (Vyas and Gulati 2009).

2.5.5 Rock phosphate and PSB activity

Early work by Azcon et al. (1976) demonstrated that PSB significantly increased the total

P content of non-mycorrhizal plants compared to an untreated control. The researchers also

noted a significant increase in root to shoot ratios of PSB treated lavender plants, indicating that

the addition of PSB to plants treated with RP resulted in significantly more root growth (Azcon

et al. 1976).

Successful uptake of P from RP is strongly related to microbial biomass P and the

population density of PSB in the soil (Wickramatilake et al. 2010). The use of soil

microorganisms to solubilize RP is considered by many to be a more environmentally friendly

alternative to chemical treatments for the solubilization of RP (Wickramatilake et al. 2010).

Unprocessed RP is less effective in soils with low cation exchange capacity (CEC), high pH and

low organic matter content. In soils, where organic matter content is higher and thus the

microbial activity is also higher, there exists a high P sorption capacity in the soil, meaning that

the Pi component of mined RP can be used most effectively (Wickramatilake et al. 2010). This

work suggests that inoculation with PSB could be a practical solution for tree fruit growers to

improve the efficiency of their insoluble P fertilizers, such as RP and BM.

2.6 Microbial inoculants for improving fertilizer P efficiency

Several commercial microbial inoculants are currently available to enhance P

solubilization and uptake in agricultural systems; these include P-solubilizing fungal seed inoculants (JumpStart®) developed by Novozymes Biologicals and mycorrhizal products such as 25

MYKE® PRO, produced by Premier Tech. There are no commercial formulations of PSB

available to tree fruit growers in Canada. The bacterial collection of Dr. Louise Nelson at the

University of British Columbia Okanagan has several isolates that exhibit potential for stimulating root growth and other health promoting benefits in plants (Nelson 2004; Hynes et al.

2008) and may provide new strains for future bacterial inoculants. The Nelson collection contains a number of Pseudomonas isolates, the most efficient P solubilizing genera of bacteria

(Nautiyal 1999; Tilak et al. 2005; Vyas and Gulati 2009; Koch et al. 2012). Pseudomonas

species are ubiquitous in the local environment, commonly found in British Columbia’s forest

soils (Axelrood et al. 2002) and are not considered to be pathogenic to tree fruit. In fact, several

isolates of Pseudomonas are currently being studied for their biological control properties against

fungal pathogens (Mikani et al. 2008). This study will focus on examining isolates of

Pseudomonas and other potential species from the Nelson collection for use as biofertilizers in

organic tree fruit production.

There is growing interest among tree fruit producers in the use of PSB to enhance P

uptake in organic orchards; however, the use of biofertilizers by organic growers is not without

uncertainty. Questions of longevity and survivorship of the microorganisms used as biofertilizers

have been raised by many researchers. Bashan et al. (1995) suggested that many inoculated

organisms may be lost with the removal of the crop. This may be so where annual crops are

concerned and the entire plant is removed along with the root system; however, in a perennial

cropping system where the root system remains intact, the chances of survivorship may be

increased. A second concern with the efficient use of biofertilizer is the ability to obtain an even

distribution of the product during the inoculation of the seedlings. Crowley and Rengel (1999) 26

suggest that multiple applications of the biofertilizer may be beneficial to breaking the niches of

indigenous bacteria and allowing an improved opportunity for the introduced bacteria to

establish a long-term presence in the rhizosphere.

Naturally-occurring soil microorganisms have an ever-increasing role in assisting plants to adapt to soils with low productivity, where nutrient availability is poor. Although there are concerns over the longevity of organisms used as biofertilizers in the soil, future work to improve plant inoculation techniques and molecular advances to enhance the organisms’ efficacy could lead to significant improvements in the performance of PGPR-based biofertilizers to enhance the availability of soil P to plant seedlings. The use of PSB has great potential to improve the efficiency of P uptake in high-density apple production in the Okanagan Valley of

British Columbia.

CHAPTER 3: MATERIALS AND METHODS

3.1 Bacterial strains

One-hundred and one isolates of plant growth promoting rhizobacteria (PGPR) that were previously identified by Hynes et al. (2008) were screened for P solubilizing ability. The isolates were obtained from Dr. Louise Nelson’s stock collection at the University of British Columbia,

Okanagan Campus. The collection had been preserved in glycerol and stored at -80° C from

2002 and re-cultured in 2011.

3.2 Phosphate plate assays

P plate assays for each of the PGPR isolates in Dr. Nelson’s collection were performed under controlled laboratory conditions using the halo method of Mehta and Nautiyal (2001). The isolates for the P solubilization assays were grown individually, in a test tube filled with 20 mL of TSB. Each tube was inoculated with one loopful of the stock bacterial culture, which had been removed from storage at -80° C just prior to inoculation. The inoculated tubes were then incubated at 28° C with shaking at 200 rpm overnight.

The initial P solubilizing plate (YEDP agar) assays were performed using 10 μL of bacterial broth inoculated onto agar plates with insoluble calcium hydrogen phosphate (CaHPO4) added (Appendix A.1, Table A.1) (Mehta and Nautiyal 2001). Five isolates were assessed per plate and triplicate plates of each set were made. The plates were incubated for 7 days at 28° C.

After 7 days, the plates were removed from the incubator and halo measurements were taken

using calipers, to determine the amount of P solubilization observed. Using the methods of

Browne et al. (2009), the diameter of the colony was measured and recorded for each isolate,

this measurement was then subtracted from the total zone of clearing (halo diameter) produced

on the plate by the bacterial isolates to give a numeric value for each zone of clearing in

millimeters (mm).

From the results obtained during the initial assessment of the PSB using CaHPO4 plates,

the twelve isolates (2-57, 2-23, 2-9, 6-114, 2-106, 5-24, 1-8, 1-18, 2-18, 1-132, 2-96 and 3-32) exhibiting the largest zones of clearing were chosen for further study. In addition to these twelve isolates, three isolates (6-8, 2-28, 6-63) of known P solubilization ability currently being used in our research program were selected for a comparative in vitro assay using three different media with insoluble P added. The media used for the comparative plate assays included: CaHPO4

medium (P-medium) (Mehta and Nautiyal 2001), Pikovskaya medium (PVK) (Pikovskaya 1948) and modified PVK medium (Blue PVK) (Gupta et al. 1994) (Appendix A.1, Table A.1). The P- medium is a minimal medium, containing only dextrose, yeast extract and CaHPO4 whereas the

PVK medium includes additions of several micronutrients. The modified PVK medium contains

0.025 g/L of bromophenol blue which changes colour from blue to yellow as the pH of the

medium decreases. Triplicate plates for each medium and bacterial combination were made.

Isolates were inoculated onto plates of the three media types listed above, at a rate of 10

μL of bacterial broth per plate. Each plate was inoculated in triplicate and incubated for 7 days at 29

28° C. After the incubation period, the halo diameters were measured in the laboratory using

handheld calipers (mm) and the bacterial isolates were ranked according to their ability to

solubilize P using a calculated solubilization index, as per Kumar and Narula (1999). The

solubilization index is equal to A/B, where A= diameter (colony + zone of clearing/halo) and B=

diameter of colony.

3.3 Liquid phosphate assays

P assays in liquid culture were performed on a total of seventeen bacterial isolates. These

included the twelve bacterial isolates identified as strong PSB from the initial plate assays, three

isolates identified as PSB that were already being used in related greenhouse experiments and the

addition of two other isolates identified from the initial pre-screening plate assays as good candidates for further study (4-15, 4-42). All bacterial isolates were first plated onto tryptic soy agar (TSA) (Difco, VWR, Mississauga ON) using the stock bacterial cultures from the -80°C freezer and grown for 7 days at 28°C. A single colony from the prepared plates of each isolate was used to inoculate 20 mL of half-strength TSB. The inoculated test tubes were incubated for

24 hours at 28° C with shaking at 200 rpm; optical density (OD600) was determined for each

isolate and compared to the prepared standard curves in order to determine the CFU/ml of each

isolate.

The National Botanical Research Institute’s minimal phosphate growth medium (NBRIP)

was prepared, without agar, according to the methods of Nautiyal (1999). NBRIP medium was

chosen as it has similar chemical composition to the PVK medium, but differs in mineral nutrient content and does not contain yeast extract (Appendix A.1, Table A.1). The NBRIP medium has a more complex mineral nutrient composition than PVK, with less Mg present and none of the Fe and Mn sources that are found in the PVK medium. Most importantly, Nautiyal (1999) found that yeast extract is a non-essential component of P medium and inhibits P solubilization by the bacterial strains; as such the NBRIP medium was created and chosen for use in the liquid culture experiments.

Up to five isolates and a non-inoculated control were tested individually in liquid culture in each experiment. Three replicate 250-mL Erlenmeyer flasks were filled with 100 mL of a suspension of 5 g per L of CaHPO4 added to liquid NBRIP medium. Each flask of sterilized medium was inoculated with 100 μL of prepared bacterial broth. The control flasks were inoculated with 100 μL of sterile TSB. The initial pH of the inoculated medium was measured at the time of inoculation (Time = 0). The flasks were then incubated for a total of 36 hours at 28°

C with shaking at 200 rpm.

The pH of the flasks was sampled using an electronic pH meter (Fisher Scientific, Ottawa

ON) at time 0, 12, 24 and 36 hours. At each time point, a 2-mL aliquot of the NBRIP medium from each flask was aseptically transferred into a test tube and the pH of the medium was measured. A reduction in pH of the liquid medium was considered to be evidence of P solubilization. A separate 1-mL aliquot of the inoculated medium was taken from each flask and serially diluted in RO water to obtain a 4000-fold dilution. This sample was transferred into a sterile, plastic cuvette for storage at -80°C for later use in the QuantiChrom phosphate assay.

Precipitates in the medium prevented the reading of OD600 at each time point. Seven isolates

most effective at reducing pH were re-tested in liquid culture with a lower concentration of

CaHPO4 (1 g per L). All other procedures were identical to those described above.

3.4 QuantiChrom phosphate assays

The QuantiChrom phosphate assay kit (BioAssay Systems, Hayward CA) was used to

measure the concentration of phosphate in the supernatant of the samples. The QuantiChrom kit

is a quantitative 96-well colorimetric plate assay that uses malachite green dye and molybdate to

form a stable colored complex with inorganic phosphate. The optical density was read at 620 nm

on a spectrophotometer (Spectronic 21, Bausch and Lomb to determine the intensity of the

coloured complex that is formed by the Malachite Green method.

Three replicates of 50-µL aliquots of each 1-mL stored sample were transferred to a 96-

well plate. Following the directions given in the QuantiChrom kit, a 50-µL aliquot of the blank

was added to three replicate wells. The kit standard was also plated in 50, 25, 10 and 5-µL

aliquots to obtain a standard curve (Appendix, Figure A.2.4). Distilled water was added to each

of the standard wells to make a total volume of 50 µL per well. QuantiChrom reagent (100 µL)

was added to each well and the plate was tapped gently to mix. The plate was let stand for 20

minutes at room temperature before being read on a spectrophotometer (Spectronic 21, Bausch

and Lomb set at 620 nm. Once the OD620 values were obtained, the phosphate concentration

(mg/mL) was calculated using the calculation provided by the kit, where: Pi = ((ODsample -

ODblank)/(ODblank – ODstandard))* 0.28

3.5 Growth pouch assays

The purpose of the growth pouch assays was to determine the effect of PSB on the growth of apple seedlings in the presence of RP. Based on the results of the PSB plate and liquid culture assays, six bacterial isolates were selected for further study in growth pouch assays using the methods of Lifshitz et al. (1987). The six isolates used in the growth pouch assay were 1-18,

2-96, 2-18, 2-106, 4-15 and 2-23.

Apple seedlings at the two-leaf stage served as the model plant. The seedlings were grown from seed extracted from mature apples (var. Gala) after a vernalization period of several months in commercial cold storage. The apple seeds were sterilized with 95% (v/v) ethanol for

20 seconds, followed by a 10 minute soaking in 20% (v/v) bleach. Seeds were then washed with sterile, distilled water for 5 minutes. Seeds were air-dried, in a petri dish placed in the laminar flow hood for 24 hours. To check for contamination, surface sterilized seeds were chosen at random to be placed on half-strength TSA plates and incubated at 28°C. The seeds were considered to be free of contamination, as no bacterial or fungal growth was present on the incubated plates after 24 hours.

The surface-sterilized seeds were pre-germinated in a mixture of moistened, sterilized vermiculite and perlite (50:50) and grown at ambient temperature to the two-leaf stage, prior to being inoculated with the bacterial isolates and placed in the growth pouches. To ensure sterile conditions for growth, the growth pouches were wrapped in tin foil and autoclaved for 30

minutes at 121°C prior to seedling addition. The seedling roots were dipped for 30 seconds into

20 mL of bacterial broth in a sterile petri dish. After inoculation, three seedlings were transferred

to the fold of each growth pouch using surface-sterilized forceps. Three replicate pouches of

each bacterial treatment were made to create a set of pouches per phosphorus treatment.

One set of pouches was filled with a total volume of 20 mL of P-free Hoagland’s solution

(adapted from Hoagland and Boyer 1936) (Appendix B). A second set of pouches was filled with

20 mL of P-free Hoagland’s solution and 0.1 g per pouch of RP. A third positive control of

known soluble phosphorus in the form of potassium phosphate (K2HPO4) was prepared using 20

mL of P-free Hoagland’s solution and 400 µL of K2HPO4 stock solution. The K2HPO4 (1M)

stock solution was prepared, using 68.05 g K2HPO4 in 500 mL of water.

The pouches were placed standing in a box, wrapped in Saran wrap in order to prevent

desiccation of the seedlings and incubated in a growth chamber at 18°C, shaken at 100 rpm and a

light: dark cycle of 16:8 hours over the course of four weeks. Two-millilitre samples of the

nutrient solution were removed at 0, 7, 14, 21 and 28 days to test for pH change in the solution,

which was used as an indicator of P solubilization.

On day 28, the seedlings were harvested from the pouches and the health of the seedling

root systems was scored visually, on a qualitative scale of 0 to 5, where 0 was a root that showed

poor, stunted growth. A root rating of 3 was considered to be a root system of average growth 34

and development. A root rating of 5 was assessed as a root system with excellent growth, good root branching and healthy root tips. The roots of the plants harvested from the growth pouches were scanned as photographic images and analyzed using the WinRhizo software (Regent

Instruments, Quebec). The software calculated the number of root tips, forks in roots and crossings of roots. The software also measured and calculated the total root length (mm), average root diameter (mm), total root surface area (mm2) and total root volume (mm3).

3.6 Greenhouse bioassay 1 – Organic soil amendments

The pot assays followed the methods of Slykhuis and Li (1985) and were designed to assess the potential of soil amendments readily available to organic tree fruit growers to mitigate the effects of RD. A second objective was to collect initial data on the potential use of bacterial inoculants on apple seedlings in RD affected soils. The compost was selected for use in the greenhouse seedling bioassay based upon its (i) compositional suitability for organic use (i.e. no prohibited components such as biosolids, (ii) high P availability and (iii) availability to local organic growers. The compost used was a dairy manure based product, sourced from AAFC-

Agassiz.

3.6.1 Site selection and soil sampling

The greenhouse bioassays were performed on soils collected from five organic orchards throughout the Okanagan Valley in which poor growth and/or decline symptoms characteristic of

RD had been observed by the grower or area horticulturist. Composite soil samples of four (10

kg) bins were collected from the 0-30 cm soil layer in the tree rows covering one-hectare of each

organic orchard and following an ‘X’ pattern that required samples be taken from trees located in the four corners and center of the orchard block. The soil samples were collected between the

fall of 2010 and spring of 2011. Field data, including the grower name, site location, cropping

history and soil type were collected at the time of soil collection. A subsample of each soil was

sent to A and L Laboratories (2136 Jetstream Rd, London, Ontario) for analysis of nutrient

status, soil pH and organic matter content (Table 3.1).

The samples were sifted through a 5 mm sieve; coarse soil particles and root fragments

were removed. The soils were then bagged and stored in the cold room at Agriculture-Agri Food

Canada’s Pacific Agriculture Research Centre (AAFC-PARC) in Summerland, BC at 4°C until

required for the greenhouse assay. Before potting, the gravimetric moisture content of the soils

was calculated by taking the soil wet weight measurement and then drying the soil for 24 hours

in a soil drying oven at 110°C. Once dry, the soil was again weighed to determine the soil dry

weight. Soil moisture calculations, using an average of six tins of soil, were performed for each

of the collected soils to determine an equivalent of 1.25 kg dry weight of soil to fill each 0.5 L

pot (Appendix A1, Tables A.2-A.4).

Table 3.1. A general description of the soils (E, F, G, H, I) obtained from Okanagan orchards for the greenhouse bioassays at AAFC-PARC and their characteristics.

Grower Location Cropping Soil pH1 N2 P3 K4 OM CEC History Texture (ppm) (ppm) (ppm) (%) (meq/100g)

E Vernon Apple- Sandy 6.1 20 31 259 3.4 12.8 transitional loam

F Lake Apple- Sandy 6.4 20 24 187 2.7 12.8 Country organic loam

G East Apple- Sandy 6.4 20 61 281 6.5 15.6 Kelowna transitional loam

H Naramata Apple- Sandy 6.6 20 89 362 2.6 15.6 organic loam

I Naramata Apple- Sandy 6.9 20 40 237 2.8 15.7 organic loam

1pH testing performed using a probe on a 1:1 basis, recommended for mineral soils. 2Nitrate nitrogen obtained using an auto analyzer, by cadmium reduction method. 3Phosphorus obtained by sodium bicarbonate extraction method. 4 Potassium obtained using an ammonium acetate extraction method.

3.6.2 Pot preparation and treatment selection

The 0.5-L plastic greenhouse pots were washed in hot, soapy water and rinsed before being left to soak in a 10% bleach solution for two hours to sterilize the plastic. The pots were then dried overnight and filled with sieved and weighed orchard soil.

Seven different soil amendments or combinations of amendments were used as the

treatments in the greenhouse bioassays and a control treatment consisting of non-amended and

non-sterilized orchard soil was also included (Table 3.2). In order to determine the effect of P on

seedling growth, three phosphorus fertilizer treatments (10-50-10, compost and BM) were included. An untreated control with no P added and N amended to 100 mg N/kg dry weight soil was used in comparison to a highly soluble, conventional phosphorus fertilizer (10-50-10), BM

(2-14-0) alone and BM in combination with the PSB strains. All P applications were applied at a rate equivalent to 200 mg P per kg of dry weight soil and N was amended to 100 mg N/kg dry weight soil in all treatments except the conventional NPK fertilizer, which supplied 100 mg N/kg dry weight soil. The exact amount of amendment used per treatment was determined based on the gravimetric moisture content of each individual soil (Appendix C). The amendments were measured into individual weigh boats and applied to individual pots of soil. The amendments were thoroughly mixed into each pre-weighed pot of orchard soil prior to planting each pot with an apple seedling.

Table 3.2. A general description of the soil treatments used in the first series of greenhouse bioassays (Soils E, F, G, H, I) at AAFC-PARC.

Treatment Application Additional Fertilizer (2-3-0)8 1 Control n/a 6 mL/pot

2 10-50-101 1.15 g n/a 3 Dairy manure 52.25 g 6 mL/pot compost2 4 Bacterial ‘A’ mixture3 Root dip + 10 mL/pot 6 mL/pot

5 Bacterial ‘B’ mixture4 Root dip + 10 mL/pot 6 mL/pot

6 Bacterial ‘C’ mixture5 Root dip + 10 mL/pot 6 mL/pot

7 BM6 4.09 g 6 mL/pot

8 BM + Bacterial ‘C’ 4.09 g 6 mL/pot mixture7 Root dip + 10 mL/pot 1Water soluble fertilizer 10-50-10 (Plant Products, Brampton, ON) was used as a conventional grower standard . 2 Dairy manure compost 2-0.5-1.5 (Agassiz, BC). 3Bacterial mixture ‘A’ was composed of equal parts of three bacterial strains that demonstrated phytohormone production, all of which were identified as Pantoea agglomerans (3-117, 4-20, and 5-51). 4Bacterial mixture ‘B’ was composed of equal parts of three bacterial strains with fungus-suppressing properties which were identified as Pseudomonas fluorescens (2-28 and 1-112) and Serratia plymuthica (6-25). 5Bacterial mixture ‘C’ was composed of equal parts of three bacterial strains with phosphate solubilizing properties, which were identified as Pseudomonas fluorescens (2-28, 6-8, and 6-63). 6Bone meal 2-14-0 (Groundskeeper, Rocky View Country, AB). 7 A combination of 4.09 g per pot of BM and bacterial mixture ‘C’. 8Pacific Natural Fish Fertilizer 2-3-0 (Great Pacific BioProducts Ltd., Delta, BC).

3.6.3 Broth preparation

In order to test bacteria from Dr. Nelson’s collection for growth promoting and antifungal

properties, three separate bacterial mixtures were grown in the laboratory at UBC Okanagan. The

first was a mixture of three strains of phytohormone-producing bacteria, Mixture A (3-117, 4-20,

5-51), the second was a mixture of bacteria with fungal-suppressing characteristics, Mixture B

(1-112, 2-28, 6-25) and the third was a mixture of PSB, Mixture C (2-28, 6-8, 6-63). The

phytohormone-producing bacteria and the bacteria with fungal-suppressing characteristics were previously identified and described by Hynes et al. (2008).

To make the bacterial mixtures, 20 μL of each of the individually prepared liquid cultures were transferred to individual flasks with 200 mL of sterile, half strength TSB. Flasks were incubated for 48 hours at 28°C, with shaking at 200 rpm. For each mixture, one flask of each isolate was combined under sterile conditions to make a total bacterial mixture of 600 mL.

For inoculation with the bacterial treatments, the roots of the apple seedlings were removed from the growth medium and immersed in one of the bacterial broths (Mixtures A, B or

C) for one minute then air-dried for an additional one minute prior to being transplanted into the soil. Immediately after planting, an additional 10 mL of the bacterial mixture were applied with a pipette to the surface of the non-amended and non-sterilized orchard soil surrounding the seedling.

3.6.4 Experimental design and seedling germination

Apple seedlings were grown from seed extracted from mature apples (var. Golden

Delicious) after a vernalization period of several months in cold storage at AAFC-PARC and surface-sterilized as previously described in section 3.5.

The surface-sterilized seeds were pre-germinated in a mixture of moistened, sterilized

vermiculite and perlite (1:1) and grown in the greenhouse with supplemental lighting (16 hour

days) at a temperature of 24°C (day) and 18°C (night). The seedlings were transplanted at the

two leaf stage. Eight replicate pots were used per treatment. The pots were arranged in

randomized complete blocks by grower lot in the greenhouse. The randomization was derived

from the SAS computer application (SAS Institute Inc., Cary, North Carolina).

Seedlings were grown in the greenhouse chambers 14 and 15 at AAFC-PARC with supplemental lighting (16 hour days) at a temperature of 24°C (day) and 18°C (night) for 9

weeks. Plants were watered regularly and monitored for pest and disease issues with control

treatments, such as sulphur (Kumulus, BASF) applied twice for mildew. Weekly plant height

measurements were recorded. At harvest, plant tops and roots were harvested, the top fresh

weight (g) was recorded and the samples preserved for drying. The number of fully developed

leaves and final height of the plant (cm) were recorded. After one week of air drying at room

temperature, the plant top and root dry weights (g) were also recorded.

3.7 Greenhouse bioassay 2 – PSB in potting mix

The second greenhouse bioassay was designed as a completely randomized block

experiment, using a sterilized organically-approved potting mix as the growth medium in order to control for the differences in soil texture and effects of RD observed in the five soils tested

during the first series of greenhouse bioassays. The second greenhouse bioassay also followed

the methods of Slykhuis and Li (1985) in order to compare the effects of three bacterial isolates

with the most consistent PSB abilities, as observed in the laboratory and growth pouch assays.

RP and BM were also added as treatments to complement the laboratory work and to investigate the effects of these organically acceptable P fertilizers on the growth of apple seedlings under greenhouse conditions in a sterile potting mix (Table 3.3).

A maximum weight of 400 g of the wet potting mix would fit into each of the 0.5 L plastic greenhouse pots; therefore the experimental design for the second greenhouse bioassay was amended to be based on a dry weight of 250 g of potting mix. Pots were prepared as previously described in section 3.6.3 and filled with Sunshine Organic and Natural Potting Mix #

3 (Sun Gro Horticulture, Vancouver BC) (Appendix A.1, Table A.5). Thirteen treatments were applied in this bioassay (Table 3) and replicated eight times. All amendment application rates

were determined based on the dry weight of the potting mix, in order to satisfy the experimental

design that required nutrient inputs to meet 100 mg N/kg of dry weight soil and 200 mg P/kg of

dry weight soil (Appendix A.1, Table A.5).

Table 3.3. A general description of the soil treatments used for Greenhouse bioassay 2- PSB in potting soil.

Treatment Application Rate Additional Fertilizer (2-3-0)7 1 Control n/a 1.2 mL/pot

2 Isolate 4-151 Root dip + 10 mL/pot 1.2 mL/pot

3 Isolate 2-182 Root dip + 10 mL/pot 1.2 mL/pot

4 Isolate 2-1063 Root dip + 10 mL/pot 1.2 mL/pot

5 RP4 0.95 g/pot 1.2 mL/pot

6 RP + 4-15 0.95 g/pot, root dip + 1.2 mL/pot 10 mL/pot 7 RP + 2-18 0.95 g/pot, root dip + 1.2 mL/pot 10 mL/pot 8 RP + 2-106 0.95 g/pot, root dip + 1.2 mL/pot 10 mL/pot 9 BM5 0.82 g/pot 1.2 mL/pot

10 BM + 4-15 0.82 g/pot, root dip + 10 1.2 mL/pot mL/pot 11 BM + 2-18 0.82 g/pot, root dip + 10 1.2 mL/pot mL/pot 12 BM + 2-106 0.82 g/pot, root dip + 10 1.2 mL/pot mL/pot 13 10-50-10 fertilizer6 0.23 g/pot n/a

1 Pseudomonas fluorescens. 2 Pseudomonas fluorescens. 3 Pseudomonas marginalis. 4Rock phosphate 0-12-0+20Ca (GardenPro, Terralink, Abbotsford BC). 5 Bone meal 2-14-0 (Groundskeeper, Rocky View Country AB). 6 Water soluble fertilizer 10-50-10 (Plant Products, Brampton ON) was used as a conventional grower standard. 7 Pacific Natural Fish Fertilizer 2-3-0 (Great Pacific BioProducts Ltd., Delta BC).

3.7.1 Bacterial Isolate Identification

Bacterial isolates (2-18, 2-106 and 4-15) from the -80° C were further identified using

16S rRNA. The DNA extraction, sequencing and isolate identification using the 16S rRNA

technique were performed in the laboratory at UBC-Okanagan by Dr. Tanja Voegel. Isolates 2-

18 and 4-15 were identified as Pseudomonas fluorescens and isolate 2-106 as Pseudomonas

marginalis which is a member of the Pseudomonas fluorescens group (Anzai et al. 2000).

3.7.2 Standard Curve Preparation

Standard curves of optical density versus colony forming units were prepared for the

three bacterial isolates used in greenhouse bioassay 2 and the field trials (Appendix A.2, Figures

A.2.1-A.2.3). Pseudomonas fluorescens (isolates 2-18 and 4-15) and Pseudomonas marginalis

(isolate 2-106) were inoculated from the -80° C stocks into 50 mL of half strength tryptic soy broth (TSB) (Difco, VWR, Mississauga ON) and grown overnight at 28° C with shaking at 200 rpm. Optical density was measured using a spectrophotometer (Spectronic 21, Bausch and

Lomb) at 600 nm within a range (OD600) of 0.05 to 0.99 by transferring aliquots into sterile

phosphate buffered saline (PBS) to make a number of solutions at different concentrations

(Undiluted, 3/4 strength, 1/2 strength, 1/4 strength, 1/10 strength, 1/50 strength and 1/100

strength). Serial dilutions were performed using each of the solutions to make triplicate plates at

concentrations ranging from 10-5 to 10-8. The plates were incubated at 28° C for 48 hours. Plates

with between 30-300 colonies were counted and related to the OD600 to give a standard curve of

OD600 vs. CFU/mL. 44

3.8 Greenhouse bioassay 3 – PSB with field trial soils

The third series of greenhouse bioassays were designed to assess the efficacy of the soil

amendments selected for the field trial under controlled greenhouse conditions. The purpose of

these bioassays was to test the effects of the application of organically approved phosphorus

fertilizer alone and in combination with three of the isolates observed to have the most consistent

P solubilizing activity, as identified in the laboratory assays on plant growth responses. Again,

all P applications were applied at a rate equivalent to 200 mg P per kg dry weight of soil and N

amended to 100mg N/kg dry weight of soil using fish fertilizer.

Composite soil samples were collected from the 0-30 cm soil layer in the tree rows and

following an ‘X’ pattern that required samples be taken from trees located in the four corners and

center of the orchard block at the two organic orchards (McCoubrey and Reiger) identified for field trial work (Appendix A.1, Table A.6). These orchards were both planted with young organic apple blocks suffering from poor growth and development thought to be linked to RP disorder. The soils were collected in the described blocks (Appendix A.3, Illustrations A.3.1 and

A.3.2) and transported to AAFC-PARC. There, the samples were sieved through a 5 mm sieve

and stored in the cold room at 4°C until planting.

The soil and pots were prepared as described in section 3.6.3. The pots were filled with a

2 parts (based on w:w ratio between the potting mix and soil) of Sunshine Organic and Natural

Potting Mix #3 (Sun Gro Horticulture, Vancouver) and 1 part of the respective orchard soil to the

equivalent weight of 0.400 kg of dry soil per pot. Nine treatments, with eight replicate pots per treatment were applied to the soils in this bioassay, as described in Table 3.4. The amendments were measured into individual weigh boats and thoroughly mixed into each pre-weighed pot of orchard soil prior to planting the pot with an apple seedling.

Table 3.4. A general description of the soil treatments used in greenhouse bioassay- 3 (McCoubrey and Reiger soils).

Treatment Application Rate Additional Fertilizer (2-3-0)9 1 Control n/a 2.2 mL /pot

2 RP1 0.77 g/pot 2.2 mL/pot

3 RP + 4-152 0.77 g/pot, root dip + 10 mL/pot 2.2 mL/pot

4 RP + 2-183 0.77 g/pot, root dip + 10 mL/pot 2.2 mL/pot

5 RP + 2-1064 0.77 g/pot, root dip + 10 mL/pot 2.2 mL/pot

6 BM5 0.66 g/pot 2.2 mL/pot

7 BM + 4-156 0.66 g/pot, root dip + 10 mL/pot 2.2 mL/pot

8 BM + 2-187 0.66 g/pot, root dip + 10 mL/pot 2.2 mL/pot

9 BM + 2-1068 0.66 g/pot, root dip + 10 mL/pot 2.2 mL/pot

1Rock phosphate 0-12-0-20Ca (GardenPro, TerraLink, Abbotsford BC). 2 RP and Isolate 4-15 (Pseudomonas fluorescens). 3 RP and Isolate 2-18 (Pseudomonas fluorescens). 4 RP and Isolate 2-106 (Pseudomonas marginalis). 5Bone meal 2-14-0 (Groundskeeper, Rocky View Country AB). 6BM and Isolate 4-15(Pseudomonas fluorescens). 7BM and Isolate 2-18 (Pseudomonas fluorescens). 8BM was and Isolate 2-106 (Pseudomonas marginalis). 9Pacific Natural Fish Fertilizer 2-3-0 (Great Pacific BioProducts Ltd., Delta BC).

All of the preparations of the bacterial isolates, soil amendments, the experimental design

and set-up, including the plant growth assessments performed at harvest followed the same protocols as with the greenhouse bioassays 1 and 2 (as described in sections 3.6 and 3.7).

3.9 Orchard Field Trials

Based on the results of the greenhouse bioassays, six treatments demonstrating

commercial potential were chosen for further testing in the field. In collaboration with two local

organic apple growers, two replicated field trials were established in their organic orchards

during the week of May 23, 2011. The field trials were designed as a fully replicated and

randomized complete block design, including five planting hole treatments and an untreated

control (Table 3.5). The treatments were applied to blocks of four trees in an orchard row and

replicated six times. A computer-generated randomization was performed on May 18, 2011

(SAS Institute Inc., Cary NC) and used to lay out the treatment blocks in the field.

Table 3.5. A general description of the soil treatments used in the orchard field trials (McCoubrey and Reiger orchards).

Treatment Application Rate 1 Control n/a

2 RP1 420 g/ planting hole

3 BM2 320 g/ planting hole

4 RP + PSB 420 g/hole, root dip + 250 mL

Mixture drench 5 BM + PSB 320 g/hole, root dip + 250 mL

Mixture drench 6 PSB3 Mixture root dip + 250 mL drench

1Rock phosphate 0-12-0-20Ca (GardenPro, TerraLink, Abbotsford BC). 2 Bone meal 2-14-0 (Groundskeeper, Rocky View Country AB). 3 PSB Mixture (Isolates 4-15 (Pseudomonas fluorescens), 2-18(Pseudomonas fluorescens) and 2-106 (Pseudomonas marginalis)).

3.9.1 Site selection

Two organic orchard sites were selected for the trial. The first field site, Orchard Corners

Emu Farm (Reiger) was established as an organic apple orchard in 2006. The Reiger site has a

sandy loam soil with overhead irrigation and is located in the Belgo area of Kelowna, BC

(49°51'37.70"N, 119°22'16.19"W). The second field site, McCoubrey Farm (McCoubrey) is an

organic apple and pear orchard that was established over twenty years ago in Lake Country, BC

(50° 0'45.12"N, 119°24'12.85"W). The soil is sandy loam with micro-sprinkler irrigation in

place. The respective growers were responsible for irrigating and maintaining the replant trial

sites over two years. Both sites have full grass cover in and between the planted tree rows and

mowing was performed three times per season by the growers in order to manage the grass

covering the orchard floor. No other weed control was performed, other than hand weeding on 48

two occasions during the season by summer students, once in July and again at the end of

August.

3.9.2 Field trial set-up and fertilizer treatments

At each of the field sites, two-year old ‘Nicola’ trees on M9 rootstock were planted 60 cm apart within the rows, and in rows 3.66 meters (12’) apart (Appendix A.3, Illustrations A.3.1 and A.3.2). One hundred and forty-four trees (6 replicates X 6 treatments x 4 trees per plot) were planted at each site into planting holes of 40 cm diameter and a depth of 60 cm that were dug with a mechanical auger, mounted on the back of the grower’s tractor. RP was added at a rate of

420 g per planting hole and BM was added at a rate of 320 g per planting hole. The P rates for

BM and RP were based on the recommendations of Dr. John Slykhuis when using the relatively soluble monoammonium phosphate (MAP; 11-55 -0 or equivalent). The recommended rate of application was 1.5 g of MAP per L of soil and therefore about 150 g of MAP per 100-L planting hole (Slykhuis and Li, 1985). Although MAP is more soluble than the two P forms chosen for this experiment, we have based our application rates on equivalent amounts of P. For example, if

1.5 g MAP contains 55 % P205 or approximately 24% P, Slykhuis applied 0.36 g of P per L of soil. In our experiment, the planting hole volume was calculated to be 59.3 L or approximately

60 L per planting hole. Realizing variation in hole size, we used 21.6 g of P per hole. Therefore the total amount of product used in each treatment was calculated based on the dimensions of the drilled planting and on the recommendations of Dr. Slykhuis, as given above. The RP and BM were incorporated into loose soil in the planting hole and thoroughly mixed by hand, prior to planting the young trees. 49

3.9.3 Bacterial mixture preparation and treatment

For treatments 3, 4 and 5, the trees were inoculated with the bacterial treatment in the field, the PSB mixture contained equal parts of three bacterial isolates: 4-15, 2-106 and 2-18. The mixture was prepared prior to the planting, in the laboratory of UBCO from the preserved isolates in Dr. Nelson’s collection. The isolates were plated and grown in liquid culture (1/2 strength TSB) following the same protocols used for the preparation of bacterial broths in the greenhouse experiments. One half litre of broth was made using each of the three bacterial isolates. The combined, 1.5 litres of broth was diluted at a 1:3 ratio with phosphate buffered saline solution (PBS) for a total volume of six liters of inoculant mixture per bucket. A final concentration of 1 X 107 CFU was used for the bacterial inoculants. For each field site, three 20 litre buckets of PSB mixture were prepared for the field inoculations.

Immediately prior to planting, the root system of the young trees was submerged into a 6-

L bucket of the bacterial mixture for one minute. The trees were then dried for one minute before being planted into the prepared hole. The soil was dug back around the trees and packed firmly into place around the root system. In the PSB treatments, an additional 250 mL of the bacterial broth was drenched onto the soil surface, near to the seedling tree after the soil was packed into place.

3.9.4 Field trial measurements and soil sampling

Initial measurements of total shoot length and trunk cross-sectional area (TCSA) were taken on May 30, 2011 at both sites. Soil samples were taken for nutrient analysis and nematode counts on June 7, 2011. Subsamples of soil were taken from five random plot locations throughout each of the two orchard blocks, using a 20 cm long and 2.5 cm diameter stainless steel soil probe. The subsamples were then combined to form a composite soil sample weighing approximately 0.5 kg that was sent for nutrient analysis to A&L Laboratories in London, Ontario

(Appendix A.1, Table A.6). Soil samples for nematode analysis weighing approximately 200 g each were taken by combining four subsamples of soil, taken from a depth of 15 cm from each of the four trees present in a respective treatment block. Each block and their six respective replicates were sampled for a total of thirty-six samples from each of the two field sites. The soils were sent for analysis to AAFC in Agassiz, BC.

3.9.5 Nematode sampling

All soil-nematode analyses were performed by Dr. Tom Forge of AAFC, Agassiz station.

Baermann pans (16 cm diameter) were used to extract nematodes from 50 ml subsamples of soil over seven days (Forge and Kimpinski 2007). Resulting nematode suspensions were poured into gridded counting dishes placed on an inverted microscope, and the Pratylenchus sp. nematodes in each sample were counted at 40X magnification.

3.9.6 Year one fall field activities

Fall measurements of total shoot length (SL) and trunk cross-sectional area (TCSA) were taken at a marked height above the graft union of each tree, from both sites on October 21, 2011.

Dormant pruning was performed on February 9, 2012; between zero to three branches were

removed per tree based on the overall growth and vigour exhibited in the October 21, 2011 data

and on field observations of the branching and leader growth made at the time of the pruning.

3.9.7 Year two field activities and sampling

Spring nematode samples were collected from all plots between May 8 -10, 2012 following

the protocols previously described; these were sent to Dr. Forge for analysis. The flower clusters

were removed from all of the trial trees at both sites on May 18-19, 2012. This activity is a

standard grower practice in young plantings in order to reduce the energy partitioned to fruit

production and instead to produce more vegetative growth throughout the season. Re-inoculation of the two field sites with the PSB mixture for the appropriate treatments occurred on May 26,

2012, following the same preparation protocols for the bacterial inoculants mixture as outlined for the 2011 inoculations. The bacterial broths were prepared at 1 X 108 CFU in the laboratory at

UBCO, diluted to a final concentration of 1 X 107 CFU and transported to the field sites in

sterilized buckets. Two hundred and fifty millilitres of the bacterial mixture were drenched onto

the soil surface, near the base of the trees.

A third set of measurements for SL and TCSA was taken on June 14, 2012, at both sites.

Additional soil samples for nematode analysis were taken at two time points during the second

field season, once mid-season during the week of July 9, 2012 and the second sample during the

week of September 17, 2012. The final growth measurements (SL and TCSA) were also taken at

this time.

3.10 Statistical Analysis

The data from the plate assays, growth pouches and greenhouse bioassays were analyzed

by a one-way analysis of variance (ANOVA) using the general linear model (GLM) function

and least squares method in the SAS 9.2 program (SAS Institute Inc., Cary, North Carolina). A

block component was included for the orchard field trials, where the experiments were a

randomized block design. In order to correct for variance heterogeneity, root data from the

growth pouch assays were log10 (n+0.5) transformed. All statistical tests were performed at α=

0.05, where p-values less than 0.05 were considered statistically significant. Differences among means were determined by using Duncan’s Multiple Range Test.

CHAPTER 4: RESULTS

4.1 Phosphate plate assays

The initial screening of 101 bacterial isolates from Dr. Nelson’s collection was conducted

on CaHPO4 medium (Mehta and Nautiyal, 2001) (Table 4.1). Phosphate solubilization by the

bacterial isolate was indicated by the zone of clearing or halo diameter observed around the

bacterial colony after seven days of incubation at 28°C (Figure 1). Thirty-four isolates exhibited some degree of phosphate solubilization (Table 4.1, Figures 1 and 2). Twelve isolates showing

the greatest P solubilizing ability, as indicated by the halo diameter, were then selected for

additional testing (Figure 2).

Table 4.1. P solubilizing ability of PGPR isolates on CaHPO4 plates.

Isolate Isolate Identification 1 P solubilization2

1-8 Pseudomonas chlororaphis + 1-18 Klebseilla pneumoniae + 1-20 Pseudomonas putida - 1-29 Bacillus pumilus -

1-39 Pseudomonas corrugata - 1-44 Pseudomonas putida - 1-51 Klebseilla pneumoniae +

1-72 Pantoea agglomerans - 1-73 Pseudomonas syringae + 1-74 Rahnella aquatilis - 1-88 Serratia grimesii + 1-89 Paenibacillus gordonae - 1-90 Rahnella aquatilis - 1-93 Kocuria kristinae + 1-101 Pseudomonas fluorescens + 1-107 Pseudomonas putida - 1-112 Pseudomonas veronii -

1-114 Pseudomonas corrugata + 1-132 Pseudomonas syringae +

1-134 Pseudomonas syringae + 1-135 Serratia grimesii + 2-9 Rahnella aquatilis + 2-12 Nocardia brasilienses - 2-13 Pseudomonas putida - 2-18 Pseudomonas putida +

2-20 Exiguobacterium acetylicum +

2-23 Serratia grimesii + 2-27 Pseudomonas syringae + 2-28 Pseudomonas syringae + 2-32 Erwinia persicina + 2-39 Pseudomonas chlororaphis - 2-45 Brevibacterium linens - 2-47 Bacillus licheniformis - 2-52 Rhodococcus fascians - 2-54 Erwinia persicina - 2-57 Rahnella aquatilis + 2-64 Flavobacterium johnsoniae - 2-68 Enterobacter agglomerans - 2-70 Enterobacter agglomerans - 2-96 Pseudomonas putida + 2-106 Pseudomonas putida +

3-10 Pseudomonas veronii -

3-13 Arthrobacter polychromogenes - 3-19 Bacillus licheniformis - 3-31 Bacillus pumilus - 3-32 Pseudomonas corrugata + 3-67 Pseudomonas veronii + 3-76 Pseudomonas syringae - 3-89 Nocardia asteroides - 3-109 Pseudomonas corrugata + 3-117 Erwinia persicina - 4-2 Pseudomonas veronii - 4-6 Pseudomonas veronii + 4-8 Pantoea agglomerans - 4-9 Hafnia alvei - 4-15 Pseudomonas veronii + 4-19 Erwinia persicina - 4-20 Erwinia persicina - 4-31 Serratia proteamaculans - 4-42 Pseudomonas syringae + 4-46 Pseudomonas corrugata + 4-61 Streptoverticillium recticulum - 4-62 Flavobacterium johnsoniae -

4-65 No match - 5-1 Pseudomonas veronii - 5-3 Microbacterium liquefaciens - 5-4 Pseudomonas chlororaphis - 5-6 Serratia plymuthica - 5-21 Rahnella aquatilis + 5-24 Rahnella aquatilis + 5-28 Pseudomonas putida + 5-37 Klebsiella terrigena -

5-48 Enterobacter agglomerans + 5-51 Erwinia persicina + 5-58 Nocardia globerula - 5-80 Pseudomonas putida - 5-105 Erwinia persicina +

5-109 Subtercola pratensis - 6-2 Alcaligenes piechaudii - 6-4 Enterobacter agglomerans - 6-5 Comamonas acidovorans - 6-7 Enterobacter intermedius - 6-8 Pseudomonas veronii - 6-9 Microbacterium esteraromaticum - 6-18 Enterobacter agglomerans - 6-20 Enterobacter agglomerans - 6-25 Hafnia alvei - 6-34 Enterobacter agglomerans - 6-50 Enterobacter agglomerans - 6-51 Enterobacter intermedius - 6-55 Pseudomonas putida - 6-57 Erwinia persicina -

6-63 Pseudomonas putida - 6-76 Enterobacter intermedius -

6-87 Pseudomonas putida - 6-96 Xanthomonas axonopodis -

6-98 Hafnia alvei - 6-99 Pseudomonas putida - 6-114 Pseudomonas chlororaphis +

6-117 Enterobacter agglomerans - 1 Isolate Identification by FAME analysis (Hynes et al. 2008). 2 +, indicates that the isolate produced a zone of clearing on CaHPO4 medium, indicating P solubilizing ability; -, indicates no zone of clearance was produced by the isolate on CaHPO4 medium.

Figure 1. P solubilization assay on CaHPO4 medium showing single colonies of five different bacterial isolates. Halo diameter as seen in ‘A’ after seven days of incubation at 28°C. No P solubilization was observed in isolates B-E.

Figure 2. Ranking of the thirty-four bacterial isolates showing P solubilizing potential, by the size of the

zone of clearing, as measured on CaHPO4 plates after incubation at 28° C for 7 days. Bars represent the mean of three replicates +/- the standard deviation. Isolates displaying no zone of clearing have been omitted from the figure. The small box highlights the twelve isolates selected for further study.

In order to confirm the rank of the selected bacterial isolates, the experiment was

repeated by plating the twelve selected isolates, individually on CaHPO4 plates and the solubilization index and the halo diameters were measured (Table 4.2). The ranking of the isolates varied somewhat from the initial ranking based on halo diameter (Figure 2).

Table 4.2. Solubilization index values for twelve bacteria isolates, following growth on CaHPO4 plates at 28°C for 7 days.

Bacterial Isolate Solubilization Index1 Halo diameter (mm)

2-57 7.7173 47.46

2-9 5.839 37.70

2-23 5.781 39.60

2-106 4.466 25.80

2-18 3.631 19.25

5-24 3.58 24.21

1-8 3.139 22.94

3-32 3.048 18.14

1-132 2.978 19.00

6-114 2.614 28.27

1-18 2.304 20.18

2-96 2.096 18.51

1Solubilization Index = A/B, where A = diameter (colony + zone of clearing) and B= diameter of colony.

The twelve isolates from Table 4.2 and three other isolates (2-28, 6-8 and 6-63) previously identified as having good P solubilizing qualities (Pallai 2005) were studied on three different solid media, with added insoluble P in the form of CaHPO4 (Figure 3 and Table 4.3).

Figure 3. Three media used to compare P solubilization on plate assays, including PVK (bottom left), P-medium (CaHPO4) (top) and Blue PVK (bottom right) as represented by bacterial isolate (2-9), after seven days incubation at 28°C.

Table 4.3. Zone of clearing (halo diameter) values for 15 bacterial isolates, on three different P media, after incubation at 28°C for 7 days (n=3).

Blue PVK Halo PVK Halo P-medium Halo Isolate diameter (mm) diameter (mm) diameter (mm) 2-9 23.24a1 5.37b 4.84cde 2-57 22.78ab 6.90b 11.45b 5-24 22.11ab 3.40b 7.17cd 2-23 21.79abc 5.66b 8.12bc 1-18 21.30abc 7.05b 4.63cde 2-106 20.50abc 4.48b --2 2-18 17.36abcd 3.39b 3.57de 2-96 16.44bcde 5.43b 4.04de 1-132 16.39bcde 4.64b 4.11de 3-32 15.53cde 5.01b 5.71cde 6-114 13.75def 15.79a 18.32a 1-8 12.09def 4.44b 4.90cde 6-63 10.97def 4.15b 2.58e 2-28 10.82ef 16.10a 17.50a 6-8 7.64f 3.59b 2.25e 1 Mean of three replicate plates. Means followed by the same letter within a column are not significantly different (p=0.05). 2--, indicates that no halo was produced by the isolate on the specified medium.

The isolates showed differences in the relative strength of their P solubilizing abilities on different media. Six bacterial isolates were identified as having relatively strong P solubilizing abilities across a range of media. Four of the isolates (2-9, 2-57, 5-24 and 2-23) showed high P solubilizing activity on the blue PVK medium and formed relatively large halos on the other two media. Two isolates (2-28 and 6-114) showed strong halo formation on the PVK and P-media, while also performing well on the Blue PVK medium. Three other isolates of interest were identified from the plate assays, including 1-18, 2-18, 3-32; all of which ranked well in the modified Blue PVK plate assays; however, showed smaller halos when plated on the PVK and P- medium. Isolate 2-106 showed good halo formation on the Blue PVK and PVK media; however failed to form a halo on the P-medium (Table 4.3; Appendix A.1, Table A.7).

4.2 Liquid culture assays

The liquid culture assays were used to monitor the change in pH caused by the bacteria in

the presence of insoluble P, over a 36-hour time period. In the first series of liquid culture assays, four experiments were performed to test a total of seventeen bacterial isolates, using NBRIP medium and 5 g/L of CaHPO4. The second series of liquid culture assays was performed to test

seven bacterial isolates, using NBRIP medium and 1 g/L of CaHPO4.

4.2.1 pH change for NBRIP with 5 g P /L

All of the isolates identified in Table 8 were tested in the liquid culture assays and two additional isolates with P solubilizing potential were added to the liquid culture experiments, 4-

15 (Pseudomonas fluorescens) and 4-42 (Pseudomonas syringae) in four separate experiments.

Isolates 4-15 and 4-42 were chosen for additional testing because both isolates are Pseudomonas spp., which are indicated in the literature to be among the most common PSB (Richardson 2001).

Isolates 4-15 and 4-42 also showed positive results for P solubilization in the initial screening tests (Table 4.1). A non-inoculated control was included with each set of experiments (Tables 4.4

– 4.7; Appendix A.1, Tables A.8-A.11). In all cases, a decrease in the pH of the solutions was observed after 12 hours of incubation compared to the non-inoculated controls which remained at a relatively constant pH over the 36-hour period. Isolates 6-63, 6-8 and 6-114 did not decrease the pH below the threshold of pH 4, as was observed with the other isolates. Isolates 1-18, 5-24,

2-96, 4-15 and 1-132 had the lowest final pH in solution relative to the control and other isolates

within each trial, after 36 hours.

Table 4.4. Effect of PSB isolates (1-18, 2-28, 6-8 and 4-42) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 1 6.56a 6.56a 6.55a 6.57a 1-18 6.42b 4.85d 4.00d 3.61d 2-28 6.42b 5.36b 4.11c 3.89c 6-8 6.42b 5.03c 4.64b 4.49b 4-42 6.43b 5.25b 4.17c 3.88c aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Table 4.5. Effect of PSB isolates (5-24, 2-96, 3-32, 6-114 and 6-63) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 2 6.44a 6.44a 6.47a 6.49a 5-24 6.44a 4.65c 3.83e 3.65e 2-96 6.44a 4.61c 3.83e 3.62e 3-32 6.40a 5.13b 4.20d 3.93d 6-114 6.43a 5.00b 4.41c 4.20c 6-63 6.36b 5.04b 4.82b 4.55b aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Table 4.6. Effect of PSB isolates (1-8, 2-23, 2-106 and 4-15) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 3 6.66a 6.61a 6.61a 6.62a 1-8 6.81a 4.63b 3.95b 3.84b 2-23 6.73a 4.5bc 3.77c 3.71b 2-106 6.57a 4.37c 3.85bc 3.70b 4-15 6.61a 4.36c 3.87bc 3.68b aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Table 4.7. Effect of PSB isolates (2-18, 2-9, 2-57 and 1-132) on pH of NBRIP solution containing 5 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 4 6.39b 6.56a 6.56a 6.53a 2-18 6.41b 4.63c 3.94b 3.91b 2-9 6.53a 4.69c 3.90b 3.94b 2-57 6.51a 4.75bc 3.91b 3.93b 1-132 6.57a 4.84b 3.98b 3.8b aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.2.2 Soluble Pi released by bacterial isolates for NBRIP with 5 g/L CaHPO4

No results were obtained from the QuantiChrom test for the solubilisation of Pi for the

samples of NBRIP medium amended with 5 g/L CaHPO4. The readings of the samples, diluted

2000-fold, were consistently found to fall outside of the linear range of the test and there was

insufficient sample remaining to test higher dilutions.

4.2.3 pH change for NBRIP with 1 g P /L

Seven isolates which exhibited the greatest decrease in pH in the initial series of liquid

culture assays were selected for the second series of liquid culture assays, including: 1-18, 2-18,

2-23, 2-96, 2-106, 4-15 and 5-24, and were inoculated into NBRIP medium with 1 g/L of

CaHPO4 added. A non-inoculated control was included with each set of experiments (Tables 4.8 and 4.9; Appendix A.1, Tables A.12 and A.13). Isolates 2-96 and 5-24 had the lowest final pH in solution after 36 hours, relative to the control and other isolates within each of the two trials. A

greater overall decrease in pH was observed at 24 and 36 hours in the experiment which used 1

g/L of CaHPO4 versus the experiments that used 5 g/L of CaHPO4.

Table 4.8. Effect of PSB isolates (1-18, 2-96, 4-15 and 5-24) on pH of NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 1 6.38a 6.26a 6.35a 6.43a 1-18 6.19a 4.35c 2.99b 2.88b 2-96 6.21a 4.61b 2.56c 2.41d 4-15 6.48a 4.21c 2.86c 2.87b 5-24 6.41a 4.63b 2.77c 2.75c aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Table 4.9. Effect of PSB isolates (2-18, 2-106 and 2-23) on pH of NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm.

pH Isolate 0h 12h 24h 36h Control 2 6.59a 6.68a 6.56a 6.93a 2-18 6.64a 4.14b 2.93b 3.08b 2-106 6.49ab 4.21b 2.96b 3.08b 2-23 6.26b 4.17b 2.84b 2.94b aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.2.4 Soluble Pi released by bacterial isolates for NBRIP with 1 g/L CaHPO4

The seven isolates selected for the second series of liquid culture assays in NBRIP

medium with 1 g/L of CaHPO4 added were tested for Pi concentration following the growth of the bacteria over 36 hours (Figures 4 and 5; Appendix A.1, Tables A.14-A.17).

Isolates 2-96 and 4-15 and 5-24 showed increases in Pi after 12 hours of inoculation.

Isolate 5-24 showed the highest concentration of Pi at 24 hours and demonstrated significantly greater P solubilization than the other isolates at both 12 and 24 hours (Figure 4). At 36 hours, isolate 1-18 showed a significantly higher concentration of Pi than the other isolates, during the

same time period. The control remained relatively constant throughout the experiment.

a a

ab b

bc b c

bc c bc d

Figure 4. Effect of PSB isolates (1-18, 2-96, 4-15 and 5-24) on inorganic phosphate concentration (mg/ 10 mL) in NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm. Means labeled with different letters within a series are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Isolates 2-18 and 2-106 followed a similar trend with Pi increasing over 36 hours (Figure

5). Isolate 2-106 solubilized significantly more P than the other isolates at both 12 and 24 hours.

Although all three isolates solubilized the greatest amounts of Pi at 36 hours, isolate 2-23 had the highest concentration of Pi at 36 hours.

a b a

b b

bc c c c c

Figure 5. Effect of PSB isolates (2-18, 2-106 and 2-23) on inorganic phosphate concentration (mg/ 10 mL) in NBRIP solution containing 1 g/L of CaHPO4 over a 36 hour incubation period at 28°C with shaking at 200 rpm. Means labeled with different letters within a series are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.3. Growth pouch assays

Six isolates (4-15, 2-18, 2-106, 2-23, 2-96 and 1-18) were selected for the growth pouch assays based on the results of the liquid culture assays and the potential for future field use of the isolates (Figure 6). The pH of the liquid medium generally fluctuated from week to week for each of the treatments, the final pH measurements (day 28) for all treatments except the 2-18 +

RP treatments increased slightly over the initial pH of the medium (day 1) (Table 4.10, Appendix

A.1, Table A.18). On days 1, 7 and 14 the pH of medium treated with Control + K2HPO4 was significantly less than all other treatments. On day 21, the pH of media treated with both the

Control + K2HPO4 and the Control treatments were significantly less than all other treatments; however not significantly different from each other. On day 28, the pH of medium treated with

Control + K2HPO4 was again significantly less than all other treatments. The bacteria and RP treatments did not differ significantly from the Control + RP treatment on days 7 and 21 and 24.

However, at 14 days a significant decrease was observed in the pH of solutions containing 2-106

+RP and 4-15 + RP relative to the control + RP treatment.

pH Treatment 1d 7d 14 d 21 d 28 d Control 5.53e 6.49b 6.34c 5.70b 6.17c f c d b d Control + K2HPO4 5.11 5.27 5.21 5.50 5.76 Control + RP 6.62d 7.14a 7.22a 6.90a 7.14ab 1-18 + RP 6.97bc 7.20a 7.14a 7.28a 7.39a 2-18 + RP 7.15ab 7.03a 7.12a 7.10a 7.02b 2-23 + RP 6.85cd 7.09a 7.10a 7.35a 7.21ab 2-96 + RP 7.26a 7.16a 7.06ab 7.23a 7.33a 2-106 + RP 6.92bc 6.99a 6.85b 7.14a 7.27ab 4-15 + RP 6.73cd 7.15a 6.86b 7.38a 7.13ab aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Figure 6. Apple seedlings inoculated with PSB in growth pouches with RP fertilizer added to NBRIP medium.

After 4 weeks of incubation no significant differences were observed between the means

of the treatments with respect to the top fresh weight, root fresh weight, shoot length, the number

of true leaves and the root health rating (Table 4.11; Appendix A.1, Table A.19).

Table 4.11. Effect of PSB and RP in liquid medium in growth pouch bioassay with apple seedlings grown for four weeks.

Treatment n Top Fresh Leaves Shoot Length Root Fresh Root Health

Weight (g) (mm) Weight (g) Control 3 0.074 2 19.85 0.370 0.55

Control + K2HPO4 2 0.098 1.78 24.15 0.014 1.22

Control + RP 3 0.076 2.11 17.79 0.027 1.67 1-18 + RP 3 0.086 1.56 11.66 0.010 1.45

2-18 + RP 3 0.138 2.56 26.50 0.021 1.11

2-23 + RP 3 0.094 2.44 20.36 0.014 1.89 2-96 + RP 3 0.054 1.56 13.68 0.005 0.33

2-106 + RP 3 0.118 1.89 24.81 0.118 1.89

4-15 + RP 1 0.047 1.33 27.76 0.013 1.33 ANOVA NS NS NS NS NS

No significant differences were observed between the means of the treatments with

respect to the number of forks or crossings produced by the root systems of the apple seedlings

(Table 4.12; Appendix A.1, Table A.20). Plants inoculated with isolates 1-18, 2-96 and 4-15

produced significantly fewer roots tips than the control + K2HPO4 and isolate 2-106 treatments.

Plants inoculated with the isolates 1-18, 2-96 and 4-15 had significantly reduced root length

compared to the control + K2HPO4, control + RP and isolates 2-18 and 2-106. Plants inoculated

with isolate 2-106 had greater total root surface area, than the control and those inoculated with

1-18, 2-23, 2-96 and 4-15. Plants inoculated with isolate 2-96 had significantly greater root

diameter than those of the non-inoculated control, control + K2HPO4, and plants inoculated with

2-18, 2-23 and 2-106. The control + RP treatment had significantly larger total root volume than

those of plants inoculated with isolates 4-15 and 2-23 and the control + K2HPO4 and the non- inoculated control treatments. Seedlings treated with RP and isolate 2-106 produced a significantly greater total projected root area than those treated with 1-18 + RP, 2-23 + RP, 2-96

+ RP and 4-15+ RP. In summary, it was observed that seedlings treated with RP and isolate 2- 69

106 demonstrated similar overall growth characteristics with respect to root development,

branching, projected root area and the volume of root produced as did the seedlings treated with

K2HPO4 in the 4-week growth cycle. K2HPO4 is a soluble P source and therefore readily

available to the plant. Plants inoculated with isolates 2-96, 1-18 and 4-15 performed poorly overall, showing growth characteristics that were not significantly different from the non- inoculated control.

Table 4.12: Root analysis data showing the effect of PSB and RP on apple seedling growth in a growth pouch bioassay.

Total Total Average Total Total Treatment n Tips Forks Crossings Length Surface Diameter Volume Area (mm) Area (mm) (mm3) (mm2) (mm2) Control 3 9.33ab 8.33 0 6.19bc 1.02cde 0.53cd 0.014b 0.32cde a ab abcd d b abcd Control + K2HPO4 2 13.78 18.22 1 8.78 1.24 0.47 0.014 0.39 Control + RP 3 9.57ab 8.43 0.43 7.09abc 1.53ab 0.72ab 0.029a 0.49ab 1-18+ RP 3 4.80b 4 0 4.25c 0.91de 0.69abc 0.016b 0.29de 2-18+ RP 3 10.40ab 12 0.8 10.44a 1.42abc 0.47d 0.016b 0.45abc 2-23+ RP 3 10.00ab 12 1.8 7.55abc 1.13bcde 0.52cd 0.014b 0.36bcde 2-96+ RP 3 5.33b 5.67 0.67 3.44c 0.83de 0.81a 0.017b 0.27de 2-106+ RP 3 15.57a 18.14 0.29 9.62a 1.57a 0.57bcd 0.022ab 0.50a 4-15+ RP 1 4.33b 4.33 0.33 3.57c 0.67e 0.59abcd 0.01b 0.21e ANOVA p-value 0.030 NS NS 0.005 0.002 0.011 0.025 0.00 aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.4 Greenhouse bioassay 1- Organic soil amendments

Harvest of the potted seedlings from greenhouse bioassay 1 (for soils E, F, G, H and I)

using organic soil amendments, took place after 9 weeks of growth (Tables 4.13-4.17). No significant differences were found among the treatments for any of the growth parameters

(Tables 4.13, 4.14, 4.16 and 4.17; Appendix A.1, Tables A.21, A.22, A.24 and A.25). Soil F had

the greatest number of dead plants at the end of the experiment and therefore the greatest overall

loss of replicates among the trials. This may have reduced my ability to distinguish among treatments. Only soil G showed some significant differences among specific growth parameters

(Table 4.15; Appendix A.1, Table A.23). The shoot to root dry weight ratio for the 10-50-10 fertilizer treatment was significantly higher than that of the control, compost and bacterial mixture ‘C’ treatments.

Table 4.13. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil E over a 9-week growth period.

Shoot Shoot Shoot Root Dry Shoot : Root Treatment n Leaves Length Fresh Wt Dry Wt 1 Wt (g) Dry Wt (cm) (g) (g)

Control 8 18.13 10.69 2.09 0.64 0.17 3.77 10-50-10 3 14.33 9.50 1.44 0.41 0.10 4.08 Compost 5 16.00 8.70 2.02 0.56 0.27 2.81 Bacterial Mix A2 4 15.25 8.75 1.67 0.48 0.12 3.70 Bacterial Mix B3 3 14.67 8.33 1.58 0.46 0.13 2.86 4 Bacterial Mix C 5 18.40 12.30 2.38 0.74 0.27 2.98

BM 3 13.33 7.17 1.13 0.33 0.16 2.16

BM+ Bacterial C 4 17.75 10.13 2.05 0.59 0.28 2.15 ANOVA NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units. 2Bacterial mixture A was composed of equal parts of three bacterial strains that demonstrated phytohormone production, all of which were identified as Pantoea agglomerans (3-117, 4-20, and 5-51). 3Bacterial mixture B was composed of equal parts of three bacterial strains with fungus-suppressing properties which were identified as Pseudomonas fluorescens (2-28 and 1-112) and Serratia plymuthica (6-25). 4Bacterial mixture C was composed of equal parts of three bacterial strains with phosphate solubilizing properties, which were identified as Pseudomonas fluorescens (2-28, 6-8, and 6-63).

Table 4.14. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil F over a 9-week growth period.

Shoot Shoot Shoot Root Dry Shoot : Root Treatment n Leaves Length Fresh Wt Dry Wt Wt (g) Dry Wt1 (cm) (g) (g) Control 2 20.50 17.00 3.06 1.38 0.47 2.73 10-50-10 5 19.40 16.30 3.42 1.63 0.45 4.11 Compost 5 18.80 9.25 2.38 0.89 0.32 3.63

Bacterial Mix A 6 19.00 14.00 3.27 1.27 0.33 3.90 Bacterial Mix B 3 13.67 10.67 2.31 0.98 0.37 2.72

Bacterial Mix C 3 18.67 14.50 3.79 1.59 0.56 2.94

BM 4 19.25 16.50 3.59 1.54 0.39 4.02

BM+ Bacterial C 5 17.60 10.80 3.05 1.16 0.41 2.82 ANOVA NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units.

Table 4.15. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil G over a 9-week growth period.

Shoot Shoot Shoot Root Shoot : Root Treatment n Leaves Length Fresh Wt Dry Wt Dry Dry Wt1 (cm) (g) (g) Wt (g) Control 5 14.80 9.00 1.77 0.68 0.25 3.14b 10-50-10 6 13.33 7.00 1.22 0.45 0.10 4.78a Compost 5 15.80 7.50 2.28 0.85 0.33 2.71b Bacterial Mix A 6 17.33 10.50 2.67 1.07 0.31 3.79ab Bacterial Mix B 7 16.71 11.29 2.61 1.03 0.28 4.32ab b Bacterial Mix C 6 18.83 8.17 2.05 0.82 0.33 2.85 ab BM 5 17.80 11.10 2.54 1.04 0.26 3.81 ab BM+ Bacterial C 4 18.50 9.13 2.17 0.82 0.20 4.17 ANOVA p value NS NS NS NS NS 0.0321 1Ratio of shoot to root dry weight, no units. aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Table 4.16. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil H over a 9-week growth period.

# True Shoot Shoot Shoot Root Shoot: Root Treatment n Leaves Length Fresh Wt Dry Wt Dry Dry Wt1 (cm) (g) (g) Wt (g) Control 8 15.00 18.15 4.52 0.47 0.53 0.89 10-50-10 7 15.29 18.87 5.07 0.46 0.70 0.81 Compost 3 13.33 8.87 2.33 0.29 0.31 1.11 Bacterial Mix A 6 15.50 20.08 5.83 0.58 0.63 1.22 Bacterial Mix B 7 13.00 15.53 3.80 0.54 0.50 1.32

Bacterial Mix C 7 15.57 21.43 6.10 0.60 0.68 0.92

BM 8 13.38 17.35 4.62 0.33 0.44 0.85

BM+ Bacterial C 8 16.13 22.29 5.53 0.60 0.43 1.51 ANOVA NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units.

Table 4.17. Effect of PSB, compost and BM on plant growth parameters in a greenhouse bioassay with Soil I over a 9-week growth period.

Shoot Shoot Shoot Root Shoot: Root Treatment n Leaves Length Fresh Wt Dry Wt Dry Dry Wt1 (cm) (g) (g) Wt (g) Control 8 13.75 12.70 2.76 0.30 0.42 0.65 10-50-10 8 13.88 12.46 2.71 0.41 0.33 1.37 Compost 6 10.50 8.55 1.63 0.11 0.21 0.46 Bacterial Mix A 6 9.83 8.85 1.91 0.25 0.23 1.54 Bacterial Mix B 8 9.88 8.41 1.58 0.13 0.26 0.87

Bacterial Mix C 8 9.13 8.14 1.51 0.16 0.30 0.61

BM 7 11.43 9.17 1.82 0.20 0.27 3.75

BM+ Bacterial C 7 10.29 7.90 1.55 0.18 0.29 0.66 ANOVA NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units.

4.5 Greenhouse bioassay 2- PSB in potting mix

The results of greenhouse bioassay 2 using PSB in potting mix are provided in Table 4.18

(Appendix A.1, Table A.26). The potted seedlings were harvested after 9 weeks of growth. No

significant differences were found among the treatments with respect to number of leaves, shoot length, root fresh and dry weights and shoot to root dry weight. The shoot fresh weights of RP,

RP + 2-18, BM + 2-18, BM + 2-106 and 10-50-10 fertilizer treatments were significantly greater

than the shoot fresh weight of the control, indicating that isolate 2-106 in combination with BM and isolate 2-18 when combined with both RP and BM were as effective at stimulating the growth of shoots and leaves as the soluble P fertilizer, 10-50-10.

Table 4.18. Plant growth data for greenhouse bioassay 2- PSB in potting mix.

Shoot Shoot Shoot Root Shoot : Root Dry Treatment n Leaves Length Fresh Wt Dry Wt Fresh Wt Root Wt (g) (cm) (g) (g) (g) Dry Wt1

Control 8 11.38 25.06 4.64b 1.92 3.06 0.62 3.22 Isolate 4-15 7 13.86 28.86 6.57ab 3.20 3.22 0.66 6.63 Isolate 2-18 8 13.63 27.38 7.29ab 3.99 3.81 0.74 7.16

Isolate 2-106 7 14.43 25.14 6.27ab 3.20 3.75 0.82 5.22 RP 8 14.38 33.06 8.39a 4.34 4.18 0.88 5.23 RP + 4-15 8 14.50 29.19 7.19ab 3.55 3.65 0.72 5.04 RP + 2-18 8 15.38 33.81 8.65a 3.96 4.30 0.90 4.64 RP + 2-106 8 13.75 24.44 5.90ab 2.91 2.97 0.66 6.19 BM 8 13.38 27.38 6.67ab 3.14 3.07 0.64 5.44 BM + 4-15 8 12.75 23.81 6.37ab 3.15 3.07 0.63 5.22 BM + 2-18 7 16.57 29.43 8.04a 3.96 4.18 0.86 4.72 BM + 2-106 7 15.43 29.14 7.78a 3.49 3.48 0.75 5.14 10-50-10 7 15.00 30.79 7.64a 3.76 4.17 0.67 6.91 ANOVA p-value NS NS 0.05 NS NS NS NS 1Ratio of shoot to root dry weight, no units. aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.6 Greenhouse bioassay 3- PSB with field trial soils

The results of greenhouse bioassay 3 with PSB and the field trial soils (Reiger and

McCoubrey) are provided in Tables 4.19 and 4.20, respectively. No significant differences were found among the treatments for any of the growth parameters assessed for the Reiger or the

McCoubrey field soils (Table 4.19 and 4.20; Appendix A.1, Tables A.27 and A.28). A two-way analysis of variance was also run for the data obtained from greenhouse bioassay 3. No significant treatment effects were found for any of the variables measured, but there were significant differences between the two soils in the greenhouse trials. Shoot height, shoot fresh and dry weight were higher overall in the Reiger soil and root fresh weight was higher overall in the McCoubrey soil (data not shown).

Table 4.19. Plant growth data for greenhouse bioassay 3- PSB with Reiger field soil.

Treatment n Leaves Shoot Shoot Shoot Root Root Shoot: Length Fresh Wt Dry Wt Fresh Wt Dry Wt Root (cm) (g) (g) (g) (g) Fresh Wt1 Control 8 20.75 29.13 8.41 2.87 4.59 0.64 4.93 RP 8 22.63 27.54 7.54 2.52 4.74 0.62 4.11 RP+ 4-15 7 22.13 27.09 8.56 2.86 4.81 0.60 4.87 RP+ 2-18 8 20.50 23.19 6.56 2.11 3.72 0.43 6.85 RP+ 2-106 8 20.25 21.24 7.52 2.65 4.05 0.63 4.45 BM 8 21.88 28.60 8.68 3.04 4.97 0.68 5.01 BM+4-15 8 20.63 27.26 7.90 2.64 3.77 0.46 6.71 BM+ 2-18 7 23.71 26.69 8.79 2.93 4.41 0.55 6.91 BM+2-106 8 20.63 28.89 8.61 2.67 4.23 0.59 4.92 ANOVA NS NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units.

Table 4.20. Plant growth data for greenhouse bioassay 3- PSB with McCoubrey field soil.

Shoot Shoot Shoot Root Root Shoot: Treatment n Leaves Length Fresh Wt Dry Wt Fresh Wt Dry Wt Root (cm) (g) (g) (g) (g) Fresh Wt1 Control 8 21.50 22.10 6.70 1.94 7.24 0.64 3.07 RP 8 20.00 22.64 6.10 1.97 6.71 0.63 4.04

RP+ 4-15 8 19.38 20.18 5.01 1.54 4.50 0.37 4.63 RP+ 2-18 8 19.50 22.84 6.04 1.92 4.79 0.43 4.56 RP+ 2-106 7 21.00 27.64 7.70 2.51 5.90 0.58 4.85 BM 6 21.83 26.60 8.28 2.67 13.36 0.69 4.12 BM+4-15 8 19.50 27.58 5.70 1.88 5.20 0.47 4.21 BM+ 2-18 8 21.50 22.76 5.46 1.71 4.75 0.44 4.13 BM+2-106 8 21.25 23.45 7.36 2.32 5.69 0.51 4.96 ANOVA NS NS NS NS NS NS NS 1Ratio of shoot to root dry weight, no units.

4.7 Field trial data

The tree measurements taken of the trees treated with P fertilizers, alone and in combination with PSB, at three time points (May 2011 (M11), October 2011 (O11) and

September 2012 (S12)) over two growing seasons include the trunk cross sectional area (TCSA)

(Table 4.21, Appendix, Table A.29) and total shoot length (Table 4.23, Appendix A.1, Table

A.30) of the two-year old trees planted in the field trials at the Reiger orchard and McCoubrey orchard (Tables 4.22 and 4.24; Appendix A.1, Tables A.31 and A.32).

There was no significant difference in the calculated change in TCSA (ΔTCSA = TCSA

S12 – TCSA M11) for any of the treatments at either of the orchards (Tables 4.21 and 4.22;

Appendix A.1, Tables A.29 and A.31). However, at the Reiger site the initial TCSA measurements for RP + PSB and BM + PSB treatments, taken at the time of planting, were 76

significantly different from the initial measurements for the control and BM treatments, indicating that the seedlings had not been properly randomized among treatments (Table 4.21;

Appendix A.1, Table A.29). No significant differences amongst the treatments were observed at the McCoubrey site (Table 4.22; Appendix A.1, Table A.31).

Although the trees were on average similar in size at the time of planting with an average

TCSA of 9.335 cm2 at the Reiger site and an average TCSA of 9.063 cm at the McCoubrey site, the trees planted at the McCoubrey site grew larger than those at Reiger’s. In comparing the

TCSA of the trees at both sites, it was observed that the overall change in TCSA at the Reiger site was less than one centimeter over two growing seasons for each of the treatments (range,

0.21 – 0.84 cm2); whereas, the overall range in the changes in TCSA observed at the McCoubrey site was between three and five centimeters over two growing seasons (range, 3.14 - 4.69 cm2).

A two-way analysis of variance was also run for the data obtained from the field trials.

No significant effects of treatment were found except for the initial TCSA and shoot length measurements (May 2011), where RP + PSB was greater than the control (again suggesting that rootstocks were not sufficiently randomized). There was a significant difference overall between the two field sites in the data obtained from October 2011 and September 2012, with

McCoubrey trees having significantly higher TCSA than those at Reiger. Trees at the

McCoubrey site (September 2012) had significantly greater overall tree height than those at

Reiger (data not shown).

Treatment n TCSA M11 n TCSA O11 TCSA S12 ΔTCSA (cm2) (cm2) (cm2) (cm2) Control 2 8.56b 23 8.63 9.20 0.75 ab RP 2 9.11 20 9.21 9.51 0.21 BM 2 8.86b 17 9.00 9.57 0.84 RP + PSB 2 10.06a 13 9.97 10.18 0.24 BM + PSB 2 10.09a 20 10.17 10.86 0.55 PSB 2 9.33ab 24 9.66 10.22 0.89 ANOVA p-value 0.0248 NS NS NS aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Treatment n TCSA M11 n TCSA O11 n TCSA S12 ΔTCSA (cm2) (cm2) (cm2) (cm2) Control 24 8.91 24 9.86 24 13.50 4.59 RP 24 9.14 24 10.10 24 13.82 4.69 BM 24 9.02 24 9.93 23 12.28 3.36 RP + PSB 24 9.82 24 10.68 20 13.07 3.14 BM + PSB 24 9.17 23 9.62 23 12.79 3.52 PSB 24 8.32 22 9.44 22 12.64 4.23 ANOVA p-value NS NS NS NS

There was no significant difference in the calculated change in total shoot length (Δ shoot

length = shoot length S12 – shoot length M11) for any of the treatments at either of the orchards

(Tables 4.23 and 4.24; Appendix A.1, Tables A.30 and A.32). At the Reiger site, the initial

height measurements for the BM + PSB and PSB treatments were significantly different from the

initial height measurements for the other four treatments. In the September 2012 measurements,

the shoot length of the BM + PSB treatment was significantly greater than the RP + PSB and BM

treatments (Table 4.23; Appendix A.1, Table A.30). At the McCoubrey site, the initial shoot length of the RP + PSB and BM + PSB treatments were significantly greater than the initial shoot length measured for the control and PSB treatments; however, there was no significant difference amongst any of the treatments in the second and third measurements (Table 4.24;

Appendix A.1, Table A.32).

The trees planted at each of the orchard sites were of similar shoot length at the time of

planting (Reiger site: 79.35 cm; McCoubrey site: 79.45 cm). The change in shoot length for

each of the treatments at the Reiger site (range, 11.93 – 21.43 cm) was almost half of the shoot

length change observed at the McCoubrey site (range, 29.46 - 40.10 cm) over the same two growing seasons. At both sites, the greatest final total shoot length measurement was observed in the BM + PSB treatment; however, total shoot length was only significant at the Reiger site and did not differ significantly from the control or PSB alone treatments at the Reiger site.

Treatment n SL M11 n SL O11 (cm) SL S12 ΔSL (cm) (cm) (cm) Control 24 77.68b 23 97.51 99.39ab 21.43 RP 24 78.21b 20 93.08 95.25abc 17.78 BM 24 74.88b 17 97.41 90.59bc 17.97 RP + PSB 24 75.46b 13 94.76 87.15c 11.93 BM + PSB 24 87.31a 20 105.66 104.35a 15.96 PSB 24 82.55a 24 102.45 102.33ab 19.78 ANOVA p-value 0.0259 NS 0.0192 NS aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

Treatment n SL M11 (cm) n SL O11 (cm) n SL S12 (cm) ΔSL (cm)

Control 24 73.27b 24 98.43 24 113.33 40.10 RP 24 78.95ab 24 102.09 24 117.50 38.55 BM 24 82.44ab 24 98.53 23 111.74 29.46 RP + PSB 24 84.03a 24 103.72 20 117.75 35.58 BM + PSB 24 83.82a 23 102.15 23 120.00 35.52 PSB 24 74.19b 22 96.41 22 109.14 33.86 ANOVA p-value 0.0455 NS NS NS aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

4.7.1 Nematode data

The results of the nematode sampling performed at the Reiger and McCoubrey sites are

presented in Tables 4.25 and 4.26 (Appendix A.1, Tables A.33-A.37). No significant difference in the numbers of Pratylenchus sp. nematodes were found between the treatments.

Table 4.25. Number of nematodes (Pratylenchus sp.) present per 50 ml subsample of soil for the Reiger orchard by date sampled.

Treatment n July 2011 September 2011 June 2012

Control 6 54.17 57.80 10.87 RP 6 39.83 10.00 11.48 BM 6 39.00 34.83 8.82 RP+ PSB 6 36.17 32.67 7.59 BM + PSB 6 39.00 32.00 4.51 PSB 6 24.67 11.33 4.51 ANOVA NS NS NS

Table 4.26. Number of nematodes (Pratylenchus sp.) present per 50 ml subsample of soil for the McCoubrey orchard by date sampled.

Treatment n September 2011 June 2012

Control 6 20.33 26.50 RP 6 16.17 16.67 BM 6 11.67 19.67 RP+ PSB 6 12.50 22.50 BM + PSB 6 18.67 29.00 PSB 6 25.00 22.33 ANOVA NS NS

CHAPTER 5: DISCUSSION

5.1 Identification of PSB candidates from in vitro assays

The research objective accomplished by the in vitro plate assays performed during this study was to identify bacterial isolates with P solubilizing properties suitable for field testing in tree fruit orchards. This study reports that thirty-four of the 101 isolates (34%) from Dr. Nelson’s

bacterial collection showed positive P solubilization on CaHPO4 medium, although the size of

the halos formed was variable. These findings are supported by the evidence in the literature, that

P medium is a good initial screening medium but can sometimes fail to identify the strongest P

solubilizers when the halo formed on the CaHPO4 medium is weak or absent (Gupta et al. 1994).

Sometimes an inconspicuous halo is produced on CaHPO4 due to a variable rate of organic acid

diffusion into the medium and is therefore missed; whereas improved media such as the PVK or

blue PVK have been shown to produce more prominent and clear halos for many of the isolates

tested (Gupta et al. 1994), including those tested in this study.

A wide range of PSB have been identified in the literature, including Bacillus and

Pseudomonas spp., which are the most commonly reported bacterial species involved with P

solubilization (Richardson 2001). In our study, seven isolates of the twelve bacterial isolates

(58%) demonstrating the greatest overall P solubilizing ability were identified as Pseudomonas

spp. These isolates performed well on all three media, but were not the strongest solubilizers on

the blue PVK medium. A reason for this is that these isolates may exhibit a different mechanism

of action than organic acid production, for example they may use the assimilation of ammonium 82

without a release of organic anions (Illmer and Schinner 1995) or may release phosphatases to

perform the P solubilization function (Crowley and Rengel 1999). Another theory that may

explain the differences in the performance of our bacterial isolates on the different media is that

products such as exopolysaccharides may be excreted by a select number of these bacteria

resulting in increased P solubilization on calcium hydrogen phosphate medium (Yi and Huang

2008).

Three of the twelve isolates were identified as Rahnella aquatis (25%) and one each of

the isolates as Serratia sp. (8%) and Klebsiella sp. (8%). The last three isolates produced the greatest halos on the blue PVK medium, indicating a strong probability that the release of organic acids is the P solubilizing mechanism employed by these bacterial isolates. The halos observed on the blue PVK medium in this experiment could be explained by bacterial isolates exhibiting a release of organic acids such as citric or oxalic acid (Gupta et al. 1994). The release of organic acids causes the chelation of metal cations (Al3+, Fe3+, Ca2+ and Mg2+) to which

phosphate is bound (Yu et al. 2012; Rodriguez and Fraga 1999). The release of protons decreases

the pH of the blue PVK medium and results in the observed colour change which forms the

prominent halo we observed on the medium as reported by Gupta et al. (1994).

The great variability observed in the size of the halo diameters makes a comparison of the mechanisms of P solubilization across the different solid media difficult to identify; thus the liquid culture experiment was designed to look more closely for organic acid release by the twelve selected isolates. Work by Nautiyal (1999) found that NBRIP liquid medium was three 83

times more efficient at identifying PSB than the PVK medium, which has yeast extract as an

ingredient. Yeast extract was found to be a non-essential component of the medium, which inhibits P solubilization by the bacterial isolates, resulting in a decrease in halo formation on the

PVK media. Nautiyal’s work (1999) concluded that plate assays are not infallible and that liquid culture with the NBRIP medium is a better overall determinant of an isolate’s ability to solubilize P. Xie et al. (2009) also observed that there was no correlation between pH decrease and P solubilization for the PVK medium; however, a strong correlation was found for the

NBRIP medium.

5.2 Behaviour and characteristics of isolates tested in liquid medium

Decreases in the pH of the liquid medium were observed for all bacterial treatments;

however, a greater overall acidification of the NBRIP medium was observed in the experiments

using only 1 g/L of CaHPO4 versus the experiments which used 5 g/L of CaHPO4. Specifically,

these differences were characterized by a sharp decrease in pH of the 1 g/L of CaHPO4 broths at

24 and 36 hours during the experiment. It is possible that the greater amount of CaHPO4 (5 g/L) created a buffering effect in the medium, resulting in lower acidification than was seen in the

NBRIP medium containing 1 g/L CaHPO4.

The results of the QuantiChrom assays for P solubilization produced some unexpected

results. In the first set of isolates tested, an interesting trend of rapidly increasing P solubilization

over the first 12 hour period was observed, which was followed by a leveling off or decrease in

the amount of P solubilized. The observed trend may correspond to a release of P during the rapid and exponential growth of the bacteria, followed by the P being taken up into biomass during the stationary phase of the bacterial growth curve. This trend was not the same for the second set of isolates sampled, which showed the greatest release of P at the end of the 36-hour period.

In both experiments the amount of P released, as calculated from the QuantiChrom assay,

was in several cases more than the initial amount of P added to the NBRIP medium. The initial

amount of P added to the medium was equivalent to 1 mg/mL of CaHPO4 and it was expected

that a portion of this insoluble P would be released by the PSB. In several cases much more than

1 mg P/mL was found to be released. The CaHPO4 was added to the NBRIP medium, mixed and

dispensed as 100 mL quantities into Erlenmeyer flasks. It is possible that this resulted in uneven

amounts of P being dispensed into the flasks and that the final concentration of CaHPO4 in the flasks differed from the intended 1 mg P/mL. To minimize this risk in the future, it is recommended that the medium be dispensed into smaller flasks first prior to the CaHPO4 being

added, in order to ensure that the appropriate concentration is present in each flask. Similar

errors can also be made during sampling from the flasks. Although the flasks were shaken during

the experiments, the CaHPO4 was not always suspended in a uniform manner within the flasks

and it is also possible that uneven amounts of P could have been sampled from the flasks at each

time point.

The QuantiChrom test is a very sensitive assay. Several attempts were made to achieve

the correct dilution for the samples and to recreate the standard curve provided in the assay kit

instructions. Each attempt failed to replicate the standard curve provided in the testing protocol.

As a result the Pi values for our samples were calculated from the equation given in the

QuantiChrom kit. Even with the 2000 fold dilution, the sensitivity of the assay was so great that some of our samples may still have failed to fall within the test parameters, producing increased

variability in our results. Although the assay was designed to measure P concentration in

biological and environmental systems, including water and soil, it is possible that the kit was not

designed to be used for suspensions of P and perhaps the suspended particles interfered with the

test. More experience is needed with the QuantiChrom assay to determine the best procedures for its use with bacterial isolates inoculated in a P suspension.

5.3 Performance of PSB in growth pouches

During the growth cycle, the pH of the pouches was monitored weekly; however, no pH

drop was observed in the growth pouches, contrary to what was hypothesized. The pH of the

solution in the growth pouches increased in all cases. The growth pouches were open on one side

and with their exposure to heat and shaking; they tended to dry out frequently requiring more of

the liquid medium to be added at regular intervals. The pH of the medium was a constant 7 and frequent addition of fresh medium could have obscured any changes brought about by the PSB.

Another possibility is that there was no carbon source in the medium; therefore, the only carbon source available to the bacteria would have been that which was produced by root exudates from

the young apple seedlings and this may not have provided enough carbon to support the active

growth of the inoculated bacteria in the growth pouches.

The key finding of the growth pouch experiment was the difference observed in the

architecture of the root systems under the different fertilizer and bacterial treatments. The

- K2HPO4 treatment and bacterial isolate 2-106 treatment produced the most root tips and forks;

while the bacterial isolates 2-18 & 2-106 produced high total surface area and total length

measurements. Also in the growth pouches, RP alone significantly increased root surface area,

average root diameter, total root volume and total root area relative to the control. These

increases in root characteristics may be an effect of the low available P influencing plant

architecture as the root tries to maximize its ability to find P.

The plant’s main strategy for P acquisition is to maximize its capacity to explore the soil

through the proliferation and extension of all root types (Ramaekers et al. 2010). Associations made between the roots and soil organisms like PGPR and arbuscular mycorrhizal fungi (AMF)

are also beneficial to P acquisition. PGPR have been demonstrated to improve P uptake by

changing the conditions of soil in the rhizosphere (Rodriguez and Fraga 1999). AMF increase

the volume of soil explored by the root system, allowing for P uptake to occur in greater volumes

of soil, while also increasing the surface area of roots for greater nutrient absorption (Bolan et al.

1987). Increased numbers of forks or branches is an important characteristic, as most soils are

heterogeneous and root growth can be restricted in some areas due to unfavorable soil conditions

or the soil may present an uneven distribution of nutrients and branching can therefore be 87

increased where more of the desired nutrients are concentrated to take full advantage of their availability (Russell 1988). The increased number of root tips and the expected increased number of corresponding root hairs present are therefore also desirable changes in root morphology as they allow the seedling to increase water and nutrient uptake (Rom 1996).

Increased surface area is particularly valuable with respect to P uptake, as the low mobility of this nutrient is well known and its uptake is very dependent on the area of absorptive surface in contact with the soil solution (Russell 1988). Increases in root branching, surface area and total root length as demonstrated in the growth pouch experiments, would allow the seedlings to grow into new regions of the soil and better access P (Richardson 2001).

The three pseudomonads (2-18, 2-106 and 4-15) chosen for further experimentation in the greenhouse bioassays and orchard field trials were based on their identification as

Pseudomonas sp. and the results of the laboratory studies. Our findings are supported in the literature where a link between the Pseudomonas genotype and superior P solubilizing ability was proven under various conditions, in several laboratory and greenhouse studies (e.g. Browne et al. 2009; Gulati et al. 2008; Morrisey et al. 2002). Pseudomonas sp. was also chosen for our trial work for the characteristics which they exhibit such as their ability to be produced in large quantities with relative ease and low potential pathogenicity; all of which are desirable for their application as agricultural inoculants.

5.4 Performance of PSB and PSB-fertilizer combinations in greenhouse bioassays

The second research objective was addressed in the greenhouse bioassays: to identify combinations of PSB and P-fertilizers with stimulatory plant growth effects in soils from certified organic orchards which exhibit symptoms of RD. The proposed hypothesis was that combinations of soil amendments and bacterial inoculation treatments would overcome orchard replant problems in certified organic production systems. The choice of amendments and processes used in this work was dictated by the products and practices allowed under the production standards of the Canadian Organic Regime (COR) (Canadian General Standards

Board 2011). Although sterilization and biocide treatments have been proven to suppress the effects of RD (Slykhuis and Li 1985; Dullahide et al. 1994; Neilsen et al. 1991; Mazzola 1998; van Schoor et al. 2009); we specifically did not include these treatments in our bioassays as they are not allowable or applicable to organic farming practices. Instead, our work in the greenhouse bioassays focused specifically on the hypothesis that the combination of PSB and P amendments such as compost, RP and BM would make P more available in the soil and therefore promote improved root system establishment in young trees, reducing the negative effects of RD. The focus on treating RD with P amendments allowed under the COR is an extension of the greenhouse bioassay work performed by Slykhuis and Li (1985) with apple seedlings, in which they demonstrated significant improvement in seedling growth with the application of P to RD affected soils. According to the findings of Slykhuis and Li (1985) seedlings grew fastest and in more soils, with an application of MAP than in soils that were only pasteurized. A combination of soil pasteurization and MAP showed an additive effect and resulted in the best overall seedling growth in all soils (Slykhuis and Li 1985). Although the soils selected for the

greenhouse trials demonstrated symptoms of RD in the field and the nutritional aspect of RD is indicated in the response to P found by Slykhuis and Li (1985), the results of our greenhouse bioassay studies showed no significant effects from the organic P amendments on seedling growth and thus, did not support the hypothesis.

Although no differences in the treatments were observed among the tested soils, high variability in seedling growth parameters may have obscured the results. Conditions in the greenhouse, such as mildew incidence, thrips damage and water stress during the summer months may have contributed to the differences observed in seedling growth. No treatment effect was observed for the soils sampled from organic orchards; therefore, no one treatment could be generally recommended over another for the management of RD. The results of the greenhouse bioassays echo the conclusion made by Slykhuis and Li (1985) that no one treatment is best in all

RD-affected soils.

It was difficult to grow seedlings in many of the orchard soils sampled. It appears that the biological component of RD was strong in several of the soils and in others, soil texture proved to be a challenge. As a result, we endeavoured to reduce the total amount of RD-affected soil in the greenhouse bioassays, by mixing the field soil with potting mix. Similar difficulties were observed by other researchers, including Van Schoor et al. (2009) who agreed with work published by Hoestra (1968) that the stunting and root discolouration observed in RD affected soils could not be reduced to a non-damaging level by diluting specific soils. Also, Neilsen and

Yorston (1991) found that applications of MAP in their greenhouse bioassays were only

effective when used in combination with soil sterilization, indicating that the removal of the

biological component of the RD complex allowed for observable effects from P fertilization.

Another possible explanation for the lack of significant effects from P fertilization is that,

while the greenhouse trials were designed to be run under conditions of low available P, for

some of the treatments the soils added small amounts of insoluble P fertilizer. This fertilizer (RP,

BM or compost) may have had enough available P to mask the effects of a low P situation in the

short growing period. The availability of P in the soil is a biologically-mediated process that

takes time to occur. Positive results have been observed in compost and PSB amended trials

using grasses as the model plant over an eight week growing period. The results of these trials

have indicated improved efficiency of uptake from RP, resulting in increased plant biomass

production (Wickramatilake et al. 2010). By comparison grasses are a relatively fast growing

plant and have a higher root density than apple seedlings. Our experience was that apple

seedlings are a very slow growing model plant and might need a longer time period in the

greenhouse in order to demonstrate the effects of the P fertilizer treatments on seedling growth.

5.5 Application of PSB and PSB-fertilizer combinations to orchards

High density orchards have a life span of approximately 20 years (BCMAL 2010b);

therefore, the majority of orchardists will be faced with an orchard replant project during their

lifetime. RD delays production and reduces the total growth and yield of the orchard; therefore, it

is important for orchardists to identify and control RD early in the life of the new orchard, in

order to minimize potential economic effects. Although not all soils transmit RD and the severity 91

of RD can be highly variable between soils (as seen in the greenhouse studies), the increased use of dwarfing rootstocks puts newly planted orchards at the greatest risk of deleterious effects due to RD (Savory 1966).

The hypothesis was that combinations of soil amendments (BM and RP) and bacterial inoculation treatments would overcome orchard replant problems in certified organic production systems by promoting more growth and better establishment of the young trees. Although it was hypothesized that pot tests could be used for the identification of soil treatments that would improve first year tree growth, the combined results of the pot tests and field studies did not support the hypothesis. The failure of the bacterial isolates to perform in any of the RD-affected soils indicates that soil fertility issues, including P levels in the soil may not be the primary cause of RD. Many of the possible contributing factors to the etiology of the RD complex have been identified in the literature review and are categorized as abiotic or biotic in nature (Traquair

1984). Currently, RD is not well documented in organic orchards. It is therefore important that organic growers consider all of the possible factors when planning an orchard renovation.

Replant pot tests with orchard soil, conducted prior to planting, are a tool that may be useful to growers in identifying or eliminating possible contributing factors to RD and may guide the grower in their preparation and treatment decisions.

The two-year old Nicola trees on M9 rootstock that were used in the research plots were dug in the spring of 2010. Prior to planting, the trees were root pruned and cut to a similar height at the beginning of the project; the trees differed in calliper from the beginning of the project. In

particular, the trees in the Reiger orchard showed significant differences in the TCSA at the time

of planting, the implication of which is that the trees were not appropriately randomized during

the set-up of the trial blocks. This difference in initial tree vigour would have introduced

variability into the trial results. The results of the TCSA measurements demonstrate the slow growth rate at which tree calliper changes over time. Too much variation in the caliper of the tree cannot be overcome in two years’ worth of growth. Blocking the trees by original size before planting is a method that could have been used to reduce this variability. This would be accomplished by sorting the trees into similar calliper sizes, then choosing trees at random from the sorted groupings to be planted into each of the experimental blocks. This would ensure that the trees planted were truly randomized and that no block received all large or small calliper trees, as was the case in the Reiger orchard trial.

Other differences in grower management strategies, particularly the irrigation systems could have contributed to variability in the results. At the Reiger site, the overhead irrigation left shadows where water was poorly distributed to the young trees. Infrequent or poor irrigation practices can further contribute to the RD complex, leaving young trees stunted, with poorly developed root systems (BCMAL 2010b). One replicate was lost in the Reiger orchard due to an

irrigation shadow and several other trees in blocks further away from the overhead sprinklers

also died due to lack of irrigation during the hot summer months. Other trees planted in areas of

the Reiger orchard, out of the reach of the overhead sprinklers, also exhibited water stress during the summer of 2010.

In the McCoubrey site, the micro-sprinkler irrigation provided better coverage of the trees and did not result in differential growth limitations of the young trees. Irrigation is critical to orchard establishment in the dry Okanagan/Similkameen Valley climate (BCMAL 2010b) and

has been proven to enhance tree growth under many climatic conditions. In their experiments,

Dencker and Hansen (1995) showed that one and two year old apple trees planted into fresh soil

(not previously planted with orchard) and/or peat moss, with drip irrigation demonstrated very

high growth responses. The drip irrigation was applied to the root zone immediately after

planting and maintained all season, increasing the survival of the trees and new root growth,

especially in M9 rootstocks which are vulnerable to dry soils. Irrigation applied in short, frequent

bursts in the first year proved to be beneficial to the establishment of young trees and to reduce

the effects of RD (Dencker and Hansen 1995).

On both farms the soil was tested at different times in the season for the presence of

damaging levels of plant parasitic nematodes. The root lesion nematode Pratylenchus penetrans

has been identified as a potential causal agent of RD in numerous studies of the etiology of RD in apples (Utkhede et al. 1992; Dullahide et al. 1994; Mazzola 1998). However, the results of the nematode counts in this study revealed only a low-level presence of Pratylenchus sp., below

damaging levels (Forge and Kimpinski 2007). Furthermore, no significant difference in the

number of nematodes present was found between the soil treatments in this study. These findings

agree with work done in Washington State by Mazzola (1998) and the work performed by Van

Schoor et al. (2009) in South Africa, both of whom found only low levels of Pratylenchus sp. in

their samples and concluded that nematodes were not the primary causal agent of RD in apple

orchards and simply a complicating factor in replanted orchards. 94

Although efforts were made to account for potential biotic factors such as the presence of nematodes, more care could have been taken to standardize and account for the potential effects of other abiotic factors that contribute to the RD complex. To reduce the number of variables that could potentially affect the results obtained for the treated two year old trees, future research could be performed using seedlings grown from tissue culture or on clonal rootstock grown in a controlled nursery setting with an established irrigation system where water received by the trees could be better regulated. Such a site could also be more effectively monitored for rodents and damage controlled in a more timely fashion.

The greatest obstacle to achieving good tree growth and root establishment in PSB inoculated trees is the heterogeneous nature of soil, even on a small scale (van Elsas and van

Overbeek 1993). In order for PSB to survive in the soil, the inoculated organism must have a competitive edge over the native soil bacteria or an empty niche must be found (Bashan 1998).

Mazzola (1999) studied bacterial communities in soil prior to and after planting apple trees and found that significant shifts in the bacterial community of the apple rhizosphere could be correlated with increases in replant disease. Our field sites were planted into historical orchards, with young apple trees being planted into areas that had previously grown apples. No assessment of the natural P solubilizers and their potential for natural soil suppression of RD was conducted in this study to determine the presence or absence of naturally occurring rhizobacteria in the orchard soils. However, it is probable that other bacteria and possibly other fungi are already acting in a capacity as P solubilizers in the orchard soils that we tested. Soil fungi, such as AMF are likely to be found in orchard soils, especially those that receive minimal P inputs from fertilizer (Browne et al. 2009). Villegas and Fortin (2001) also found that AMF and 95

Pseudomonas spp. may interact in a positive manner to promote P solubilization. However,

research findings from apple tree inoculations in Brazil indicate that AMF inoculation of M9

rootstock had a negative impact on tree height and shoot and root fresh weight and dry matter

(Locatelli and Lovato 2002). Work detailing the root architecture of M9 rootstock inoculated

with AMF also found reduced root number and length in the M9 rootstock when mycorrhizal

inoculation occurred after a 21-day rooting period (Locatelli et al. 2002). More studies could be conducted to determine the biological makeup of soils in organic orchards in order to assess the potential for the soils to solubilize P. This information could also be useful in order to identify the dominant microorganisms in organically managed soils and to compare these populations of microorganisms with those existing under conventional orchard management systems.

Access to additional information about root growth and root architecture of young trees

in orchard soils would also be useful for modelling the efficacy of the inoculation with bacteria.

Knowledge about the distribution of roots with respect to bacteria over time could give useful information for drench applications, such as if roots were growing out of the inoculation zone or in the case of root dips and drenches, if the tree roots were accessing different pockets of soil and therefore different amounts of P over time. Using a rhizotron or similar experimental set-up for the bacterial inoculation of the young apple trees could give researchers more insight into the growth of the root system and associated bacterial/P dynamics.

CHAPTER 6: CONCLUSIONS

This thesis was built upon previous research and addressed the following objectives: 1) identify a range of PSB suitable for field testing in tree fruit orchards; 2) identify combinations of PSB and P-fertilizers with stimulatory plant growth effects in soils from certified organic orchards which exhibit symptoms of RD. It was hypothesized that combinations of PSB and P amendments, including compost, RP and BM would mitigate the effects of RD by promoting improved root and shoot growth in apple seedlings in the laboratory. In the field, it was hypothesized that seedling establishment would be improved in RD affected orchards by manipulating the biology of the rhizosphere in affected certified organic tree fruit orchards using

PSB in combination with P amendments.

Although the strongest P solubilizers did not enhance seedling growth or improve fertilizer efficiency in the greenhouse and orchard trials as hypothesized, the in vitro work showed good potential and could be extended to further investigate the specific genes or genetic mechanisms behind their P solubilising abilities. Perhaps with a better understanding of the genes involved in P solubilisation, the bacteria could be manipulated to enhance P solubilisation.

A marker could also be developed for selected PSB to aid in the application of PSB to highly variable soil conditions. The preparation of bacterial inoculants and the application of PSB to agricultural soils could also be refined to give more consistent results and a marker used to track the survivorship of the inoculated bacteria in the soil. This information would assist producers to assess the efficacy of the inoculant and to determine if there is a need to inoculate the soil again in subsequent years.

In conclusion, there is future potential for the use of PSB in organic replant blocks. We

have identified through this study, three isolates of Pseudomonas spp. that show strong P

solubilizing abilities and demonstrate the ability to influence plant growth by producing

significant effects on root growth when these bacterial isolates are applied in combination with

insoluble RP fertilizer. In particular, changes to root architecture by PSB in combination with RP

were observed as increased total root length, surface area and an increase in the number of root

tips produced under gnotobiotic conditions (growth pouches). Further laboratory and field testing will be needed in order to advance these isolates towards commercialization as bacterial inoculants for general grower use in certified organic production systems.

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APPENDICES

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Appendix A. Supplementary Data

A.1. List of Tables

Appendix A.1. Media prepared for in-vitro testing of phosphate solubilization.

Ingredient PVK (g/L) NBRIP (g/L) YEDP (g/L) Dextrose - - 10 Yeast Extract 0.5 - 5 CaHPO4 5 5 2 Glucose 10 10 - (NH4)2SO4 0.5 0.1 - NaCl 0.2 0.2 - MgSO4•7H20 0.1 0.25 - KCl 0.2 0.2 - MnSO4•H20 0.002 - - FeSO4•7H20 0.002 - - MgCl2•6H20 - 5 -

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Table A.2 Gravimetric moisture content of dairy manure compost (Agassiz, BC) as sampled on July 16, 2010.

% Empty Wet tin weight Dry tin weight Wet Weight Dry Weight Moisture Tin weight(g) (g) (g) (g) (g) (Dry/Wet)*100 Average 1 21.65 85.64 32.38 63.99 40.73 63.65 2 21.28 84.56 61.92 63.28 40.64 64.22

3 21.34 85.23 62.21 63.89 40.87 63.97 63.84

4 21.88 85.89 62.74 64.01 40.86 63.83 5 22 84.32 61.86 63.32 39.86 63.96 6 21.64 89.32 64.54 67.68 42.9 63.39 Require 100 mg N/kg of Dry weight soil

1.25 kg dry weight soil to be measured per pot

Dairy manure compost contains 1.87% N (from compost analysis) 18, 700 ppm thus 1 g contains 18, 700 µg N or 18.7 mg N (18.7 mg)(x)= 100 mg, thus x = 5.35 g dry compost

Using 1.25 kg dry wt soil/pot, then (1.25 kg)(5.35)= 6.69 g dry compost per pot.

To calculate for wet compost: 6.69g dry compost/ 0.64 moisture compost= 10.45 g Assume only 20% of the N will be mineralized and available thus, we need 10.45 g/0.2 = 52.25 g compost per pot

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Table A.3 Gravimetric moisture content of soil E (Gayle), as sampled on July 16, 2010.

Empty Wet tin weight Dry tin weight Wet Weight Dry Weight (Dry/Wet)*10 Averag % Tin weight(g) (g) (g) (g) (g) 0 e Moisture 1 21.85 88.21 78.24 66.36 56.39 84.98 2 21.64 84.67 75 63.03 53.36 84.66 3 21.73 87.23 77.12 65.5 55.39 84.56 84.64 15 4 21.45 88.11 77.81 66.66 56.36 84.55 5 21.82 84.67 74.75 62.85 52.93 84.22 6 21.86 82.35 73.19 60.49 51.33 84.86

Amount of wet soil calculated and required per pot = 1.47 kg, from 1.47 kg – (1.47 kg*0.15) = 1.2495 kg dry weight of soil per pot.

Table A.4 Gravimetric moisture content of soil F (McCoubrey), as sampled on July 16, 2010.

Empty Wet tin weight Dry tin weight Wet Weight Dry Weight (Dry/Wet)*10 Averag % Tin weight(g) (g) (g) (g) (g) 0 e Moisture 1 21.68 75.64 70.24 53.96 48.56 89.99 2 21.45 76.21 70.86 54.76 49.41 90.23 3 21.69 78.43 72.79 56.74 51.1 90.06 90.03 10 4 21.88 70.25 65.39 48.37 43.51 89.95 5 21.74 79.99 74.19 58.25 52.45 90.04 6 22.01 82.22 76.15 60.21 54.14 89.92

Amount of wet soil calculated and required per pot = 1.39 kg, from 1.39 kg – (1.39 kg*0.10) = 1.251 kg dry weight of soil per pot.

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Table A.5 Gravimetric moisture content of Sunshine Organic and Natural Potting Mix # 3 (Sun Gro Horticulture, Vancouver) as sampled on December 5, 2011 at AAFC-PARC.

Empty Wet tin weight Dry tin weight Wet Weight Dry Weight (Dry/Wet)*10 Averag % Tin weight(g) (g) (g) (g) (g) 0 e Moisture 1 22.5 58.5 39 36 16.5 45.83 2 22.3 60 39.6 37.7 17.3 45.89 46.03 54 3 18.2 55.5 35.5 37.3 17.3 46.38

Amount of wet potting mix calculated and required per pot = 0.250 kg, from 0.463 kg – (0.463 kg*0.5397) = 0.250 kg dry weight of potting mix per pot.

Fish Fertilizer: If we need 25 mg N/pot, we will need: (25 mg N)/(21mg N/mL)= 1.19 mL fish fertilizer per pot 1.2 mL of Pacific Natural Fish Fertilizer was used per pot.

Bone Meal: 0.250 kg of soil and 200 mg P per kg (0.25)(200)= 50 mg P/ pot

(50 mg P per pot)(2.29) = 114.5 mg P2O5 per pot 114.5 mg (100/14%) = 817.86 mg or 0.81786 g of BM per pot 0.82 g of Bone Meal was used per pot.

Rock Phosphate: (50 mg P per pot)(2.29)= 114.5 mg P2O5 per pot 114.5 mg (100/12%) = 954.17 mg or 0.95417 g of RP 0.95 g of Rock Phosphate was used per pot.

10-50-10 Fertilizer: (50 mg P per pot)(2.29)= 114.5 mg P2O5 per pot 114.5 mg (100/50%)= 229.0 mg or 0.229 g of 10-50-10 0.23 g of 10-50-10 was used per pot.

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Table A.6 Soil test report values for the McCoubrey and Reiger field sites, testing performed by A and L Laboratories, London ON.

N P K Mg Ca CEC Field pH OM (%) (ppm) (ppm) (ppm) (ppm) (ppm) (meq/100g) McCoubrey 6 3.3 8 19 253 255 1160 11 Reiger 7.4 4.8 20 69 1005 460 2300 18.3

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Table A.7 Key data extracted from the ANOVA tables for the mean halo diameter measured on Blue PVK medium, inoculated with PSB after a 7 day incubation period at 28°C.

Main Effect

P solubilization, halo formation over time.

Category DF Type III SS F-value p-value

Blue PVK 14 1045.785090 6.44 <.0001

PVK 14 647.9958878 4.19 0.0010

P Media 13 1025.058207 18.60 <.0001

Table A.8 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (1-18, 2-28, 6-8 and 4-42), over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 4 0.04390667 19.72 0.0003

pH time = 12h 4 5.41889333 268.88 <.0001

pH time = 24h 4 13.65090667 980.20 <.0001

pH time = 36h 4 17.44490667 3226.55 <.0001

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Table A.9 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (2-96, 3-32, 5-24, 6-63 and 6-114), over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 5 0.01678333 5.92 0.0084

pH time = 12h 5 6.71149444 174.65 <.0001

pH time = 24h 5 14.81102778 983.40 <.0001

pH time = 36h 5 17.42009444 452.54 <.0001

Table A.10 Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (1-8, 2-23, 2-106 and 4-15), over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 4 0.11137333 0.36 0.8291

pH time = 12h 4 11.19586667 384.91 <.0001

pH time = 24h 4 18.17564000 1028.03 <.0001

pH time = 36h 4 20.00976000 288.24 <.0001

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Table A.11Key data extracted from the ANOVA tables for the mean pH measurements of 5 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (2-9, 2-18, 2-57and 1-132), over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 4 0.07522667 14.73 0.0009

pH time = 12h 4 8.15350667 409.18 <.0001

pH time = 24h 4 16.56970667 561.94 <.0001

pH time = 36h 4 16.64930667 412.04 <.0001

Table A.12 Key data extracted from the ANOVA tables for the mean pH measurements of 1 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (1-18, 2-96, 4-15 and 5-24) over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 4 0.2020667 1.66 0.2519

pH time = 12h 4 8.2641333 209.15 <.0001

pH time = 24h 4 30.6677600 3469.20 <.0001

pH time = 36h 4 33.3969733 2527.52 <.0001

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Table A.13 Key data extracted from the ANOVA tables for the mean pH measurements of 1 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (2-18, 2-106, 2-23) over a 36 hour incubation period. Main Effect

pH change over time.

Category DF Type III SS F-value p-value

pH time = 0 h 3 0.2531333 5.48 0.0325

pH time = 12h 3 14.1574917 426.54 <.0001

pH time = 24h 3 30.0907667 1750.31 <.0001

pH time = 36h 4 34.2460250 741.66 <.0001

Pi Isolate 0h 12h 24h 36h Control 1 1.68 4.16c 2.20c 3.12d 1-18 14.94 3.61c 7.49b 26.81a 2-96 0.97 12.54bc 5.21bc 19.01b

4-15 2.57 21.52ab 3.61c 2.72d

5-24 5.76 34.90a 39.72a 9.71c aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

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Table A.15 Key data extracted from the ANOVA tables for the mean Pi measurements of 1 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (1-18, 2-96, 4-15 and 5-24) over a 36 hour incubation period. Main Effect

Pi solubilization over time.

Category DF Type III SS F-value p-value

Pi time = 0h 4 3.9733658 1.49 0.2776

Pi time= 12h 4 20.7316680 7.51 0.0046

Pi time = 24h 4 30.0120025 201.97 0.0000

Pi time =36h 4 13.1474599 1059.73 0.0000

Pi Isolate 0h 12h 24h 36h Control 2 4.16 10.89bc 1.27c 1.99c 2-18 3.80 20.89b 28.98b 59.85b 2-106 4.84 52.70a 58.67a 82.81ab 2-23 4.42 4.63c 6.41c 100.38a aMeans followed by different letters within a column are significantly different (p≤ 0.05) according to Duncan’s Multiple Range test.

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Table A.17 Key data extracted from the ANOVA tables for the mean Pi measurements of 1 g/L of CaHPO4 in NBRIP solution, inoculated with PSB (2-18, 2-23 and 2-106) over a 36 hour incubation period. Main Effect

Pi solubilization over time.

Category DF Type III SS F-value p-value

Pi time = 0h 3 0.0174985 0.41 0.7501

Pi time= 12h 3 41.0541322 44.59 .0000

Pi time = 24h 3 56.4387991 171.95 .0000

Pi time =36h 3 164.9037862 28.45 .0001

Table A.18 Key data extracted from the ANOVA tables for the mean pH measurements of NBRIP solution with RP in the growth pouches, inoculated with PSB (1-18, 2-18, 2-96, 2-106, 2- 23 and 4-15) over a 28 day incubation period. Main Effect pH change over time.

Category DF Type III SS F-value p-value pH time = 1 d 8 13.2140074 77.55 0.0000 pH time = 7 d 8 9.3557852 75.00 0.0000 pH time = 14 d 8 9.8677333 82.43 0.0000 pH time = 21 d 8 12.4302519 20.07 0.0000 pH time = 28 d 8 7.7769185 33.50 0.0000

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Table A.19 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters of apple seedlings grown in growth pouches with NBRIP medium and RP, inoculated with PSB (1-18, 2-18, 2-96, 2-106, 2-23 and 4-15) over a 28 day incubation period.. Main Effect

Treatment

Category DF Type III SS F-value p-value

Shoot length 8 634.2702667 1.11 0.4100

Number of leaves 8 5.042274074 0.4494338 0.8752

Top Fresh Weight 8 0.0781028 0.75 0.6475

Root Fresh Weight 8 0.3327470 1.08 0.4260

Root Health 8 7.134318519 0.78 0.6290

Table A.20 Key data extracted from the one-way ANOVA tables for the root measurements and root architecture analysis of apple seedlings grown in growth pouches with NBRIP medium and RP, inoculated with PSB (1-18, 2-18, 2-96, 2-106, 2-23 and 4-15) over a 28 day incubation period. Main Effect

Treatment

Category DF Type III SS F-value p-value

Tips 8 644.9292063 2.4167442 0.0308

Forks 8 1381.206349 1.6092327 0.1521

Crossings 8 13.92380952 0.8290274 0.5822

Total Root Length 8 247.7322131 3.3275613 0.0051

Total Root Surface Area 8 3.875908559 3.814552 0.0020

Average Root Diameter 8 0.551564286 2.9337865 0.0110

Total Root Volume 8 0.001462828 2.522836 0.0249

Total Projected Root Area 8 0.392712443 3.814511 0.0020

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Table A.21 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment E.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 7 113.8231448 0.88 0.5392

Shoot length 7 112.4823078 1.17 0.3643

Top Fresh Weight 7 6.07342554 0.99 0.4663

Top Dry Weight 7 0.72818628 1.18 0.3582

Root Dry Weight 7 0.16979705 1.70 0.1671

Top: Root Dry Weight 7 6.25650627 1.35 0.2800

Table A.22 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment F.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 7 123.510050 1.10 0.4050

Shoot length 7 337.0623798 1.19 0.3610

Top Fresh Weight 7 7.04381925 0.50 0.8192

Top Dry Weight 7 2.11658687 0.78 0.6099

Root Dry Weight 7 0.18635430 0.61 0.7395

Top: Root Dry Weight 7 8.18739565 0.84 0.5714

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Table A.23 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment G.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 7 98.45832619 1.33 0.2739

Shoot length 7 88.81063514 1.15 0.3625

Top Fresh Weight 7 7.29077227 1.20 0.3323

Top Dry Weight 7 1.31740934 1.43 0.2329

Root Dry Weight 7 0.2454749 1.96 0.0964

Top: Root Dry Weight 7 23.87071079 2.61 0.0321

Table A.24 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment H.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 7 58.17022320 1.01 0.4377

Shoot length 7 438.3450073 1.15 0.3539

Top Fresh Weight 7 37.24727384 1.03 0.4247

Top Dry Weight 7 0.50215442 0.76 0.6265

Root Dry Weight 7 0.57198940 0.54 0.7963

Top: Root Dry Weight 7 3.44850640 1.15 0.3507

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Table A.25 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-1, Experiment I.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 7 186.2156479 1.25 0.2986

Shoot length 7 200.7654613 0.67 0.6978

Top Fresh Weight 7 14.31967305 0.73 0.6508

Top Dry Weight 7 0.49056708 1.25 0.2952

Root Dry Weight 7 0.23682017 0.56 0.7829

Top: Root Dry Weight 7 59.03107811 1.00 0.4473

Table A.26 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-2, PSB in potting mix.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 12 159.7468392 0.96 0.4901

Shoot length 12 914.4986577 1.38 0.1937

Top Fresh Weight 12 113.8754195 1.88 0.0500

Top Dry Weight 12 37.03061400 1.75 0.0709

Root Dry Weight 12 24.03315451 0.86 0.5934

Top: Root Dry Weight 12 104.8140094 1.67 0.0905

124

Table A.27 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-3, PSB with Reiger field soil.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 8 84.48086124 0.59 0.7847

Shoot length 8 459.6634432 1.14 0.3515

Top Fresh Weight 8 35.23483583 0.59 0.7804

Top Dry Weight 8 4.78351121 0.72 0.6722

Root Dry Weight 8 0.45733068 0.98 0.4627

Top: Root Dry Weight 8 76.59103771 1.60 0.1457

Table A.28 Key data extracted from the one-way ANOVA tables for the mean measurement of growth parameters in greenhouse bioassay-3, PSB with McCoubrey field soil.

Main Effect

Treatment

Category DF Type III SS F-value p-value

Number of leaves 8 54.2571461 0.74 0.6521

Shoot length 8 259.5352438 0.90 0.5254

Top Fresh Weight 8 58.8859913 1.46 0.1944

Top Dry Weight 8 6.76896420 1.90 0.0795

Root Dry Weight 8 0.70267279 1.69 0.1214

Top: Root Dry Weight 8 19.67639422 1.25 0.2890

125

Table A.29 Key data extracted from the one-way ANOVA tables for the mean measurement of trunk cross sectional area of trees planted in the Reiger orchard and treated with P fertilizers alone and in combination with PSB. Main Effect

Treatment

Category DF Type III SS F-value p-value

May 2011 5 47.28306181 2.67 0.0248

October 2011 5 34.55158932 1.91 0.0984

September 2012 5 40.86568628 2.05 0.0771

Total change in TCSA 5 8.31375222 0.63 0.6779

Table A.30 Key data extracted from the one-way ANOVA tables for the mean measurement of the total shoot length for trees planted in the Reiger orchard and treated with P fertilizers alone and in combination with PSB. Main Effect

Treatment

Category DF Type III SS F-value p-value

May 2011 5 2669.254683 2.64 0.0259

October 2011 5 2097.527985 2.00 0.0851

September 2012 5 4257.996190 2.84 0.0192

Total change in Height 5 1053.053899 1.08 0.3785

126

Table A.31 Key data extracted from the one-way ANOVA tables for the mean measurement of trunk cross sectional area of trees planted in the McCoubrey orchard and treated with P fertilizers alone and in combination with PSB. Main Effect

Treatment

Category DF Type III SS F-value p-value

May 2011 5 27.92652847 1.49 0.1986

October 2011 5 20.38807718 1.12 0.3506

September 2012 5 36.13322886 1.16 0.3312

Total change in TCSA 5 48.05520134 1.45 0.2117

Table A.32 Key data extracted from the one-way ANOVA tables for the mean measurement of the total shoot length for trees planted in the McCoubrey orchard and treated with P fertilizers alone and in combination with PSB. Main Effect

Treatment

Category DF Type III SS F-value p-value

May 2011 5 2773.378147 2.33 0.0455

October 2011 5 912.641889 0.76 0.5824

September 2012 5 1883.891116 0.62 0.6883

Total change in Height 5 1483.547481 0.39 0.8561

Table A.33 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, July 2011.

Main Effects DF Type III SS F-value p-value

Treatment 5 2663.8056 1.19 0.3430

Block 5 24215.8056 10.80 <0.0001

127

Table A.34 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, September 2011.

Main Effects DF Type III SS F-value p-value

Treatment 5 9173.0933 1.10 0.3855

Block 5 216450.8600 1.98 0.1189

Table A.35 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the Reiger orchard, June 2012.

Main Effects DF Type III SS F-value p-value

Treatment 5 287.8056 1.11 0.3818

Block 5 391.1389 1.50 0.2242

Table A.36 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the McCoubrey orchard, September 2011.

Main Effects DF Type III SS F-value p-value

Treatment 5 758.2222 0.88 0.5089

Block 5 2495.8889 2.90 0.0339

Table A.37 Key data extracted from the two-way ANOVA table for the mean nematode (Pratylenchus sp.) count in the McCoubrey orchard, June 2012.

Main Effects DF Type III SS F-value p-value

Treatment 5 599.2222 0.50 0.7725

Block 5 3189.5556 2.67 0.0459

128

Table A.38 Descriptive statistics showing variance for data contained in Tables 8- 31 and Figures 4 and 5.

Table 8. Zone of clearing (halo diameter) values for 15 bacterial isolates, on three different P media. Halo Diameter Treatment Mean Standard Dev. Blue PVK 1-132 16.39 1.90 1-18 21.30 4.56 1-8 12.09 7.84 2-106 20.50 2.79 2-18 17.36 1.44 2-23 21.79 0.41 2-28 10.67 0.40 2-57 22.78 1.59 2-9 23.24 1.58 2-96 16.44 0.24 3-32 15.53 5.62 5-24 22.11 2.33 6-114 13.75 2.34 6-63 10.97 4.65 6-8 7.64 1.83

Halo Diameter Treatment Mean Standard Dev. PVK 1-132 4.64 0.75 1-18 7.05 0.12 1-8 4.44 0.52 2-106 4.48 0.78 2-18 3.39 - 2-23 5.66 3.60 2-28 16.10 8.53 2-57 6.90 3.29 2-9 5.64 1.34 2-96 5.43 2.81 3-32 5.01 0.98 5-24 3.40 0.79 6-114 15.79 0.98 6-63 4.15 0.20 6-8 3.59 0.09

129

Halo Diameter Treatment Mean Standard Dev. P medium 1-132 4.11 1.51 1-18 4.63 0.07 1-8 4.90 1.44 2-106 - - 2-18 3.57 1.08 2-23 8.12 3.05 2-28 17.50 4.92 2-57 11.45 0.11 2-9 4.84 2.90 2-96 4.04 0.11 3-32 5.71 1.62 5-24 7.17 0.73 6-114 18.32 1.60 6-63 2.58 0.47 6-8 2.25 0.72

Tables 9-12. Effect of PSB isolates on pH in NBRIP solution containing 5 g/L of CaHPO4. Time Treatment Mean Standard Dev. T=0 1-132 6.57 0.03 1-18 6.42 0.02 1-8 6.81 0.53 2-106 6.57 0.02 2-18 6.41 0.05 2-23 6.73 0.27 2-28 6.42 0.02 2-57 6.51 0.03 2-9 6.53 0.04 2-96 6.44 0.01 3-32 6.40 0.04 4-15 6.61 0.13 4-42 6.43 0.02 5-24 6.44 0.02 6-114 6.43 0.01 6-63 6.36 0.04 6-8 6.42 0.03 Control 1 6.56 0.03 Control 2 6.44 0.03 Control 3 6.66 0.09 Control 4 6.39 0.02

130

Time Treatment Mean Standard Dev. T=12 1-132 4.84 0.10 1-18 4.85 0.02 1-8 4.63 0.03 2-106 4.37 0.10 2-18 4.63 0.07 2-23 4.50 0.06 2-28 5.36 0.14 2-57 4.75 0.09 2-9 4.69 0.05 2-96 4.61 0.04 3-32 5.13 0.02 4-15 4.36 0.09 4-42 5.25 0.10 5-24 4.65 0.21 6-114 5.00 0.05 6-63 5.04 0.05 6-8 2.03 0.02 Control 1 6.56 0.04 Control 2 6.44 0.03 Control 3 6.61 0.09 Control 4 6.56 0.03

Time Treatment Mean Standard Dev. T=24 1-132 3.98 0.02 1-18 4.00 0.07 1-8 3.95 0.05 2-106 3.85 0.06 2-18 3.94 0.06 2-23 3.77 0.04 2-28 4.11 0.05 2-57 3.91 0.11 2-9 3.90 0.12 2-96 3.83 0.03 3-32 4.20 0.08 4-15 3.87 0.11 4-42 4.17 0.06 5-24 3.83 0.08 6-114 4.41 0.10 6-63 4.82 0.04 6-8 4.64 0.06 Control 1 6.55 0.04 131

Time Treatment Mean Standard Dev. Control 2 6.47 0.03 Control 3 6.61 0.06 Control 4 6.56 0.03

Time Treatment Mean Standard Dev. T=36 1-132 3.80 0.17 1-18 3.61 0.03 1-8 3.84 0.10 2-106 3.70 0.09 2-18 3.91 0.03 2-23 3.71 0.09 2-28 3.89 0.05 2-57 3.93 0.10 2-9 3.94 0.11 2-96 3.62 0.21 3-32 3.93 0.05 4-15 3.68 0.24 4-42 3.88 0.04 5-24 3.65 0.10 6-114 4.20 0.06 6-63 4.55 0.07 6-8 4.49 0.07 Control 1 6.57 0.02 Control 2 6.49 0.02 Control 3 6.62 0.05 Control 4 6.53 0.01

Tables 13 and 14. Effect of PSB isolates on pH in NBRIP solution containing 1 g/L of CaHPO4. Time Treatment Mean Standard Dev. T=0 1-18 6.19 0.10 2-106 6.49 0.18 2-18 6.64 0.15 2-23 6.26 0.05 2-96 6.21 0.07 4-15 6.48 0.21 5-24 6.41 0.22 Control 1 6.38 0.16 Control 2 6.59 0.01

132

Time Treatment Mean Standard Dev. T=12 1-18 4.35 0.08 2-106 4.21 0.08 2-18 4.14 0.16 2-23 4.17 0.02 2-96 4.62 0.11 4-15 4.21 0.10 5-24 4.63 0.02 Control 1 6.26 0.15 Control 2 6.68 0.08

Time Treatment Mean Standard Dev. T=24 1-18 2.99 0.01 2-106 2.96 0.03 2-18 2.93 0.04 2-23 2.84 0.06 2-96 2.56 0.03 4-15 2.86 0.11 5-24 2.77 0.06 Control 1 6.35 0.06 Control 2 6.56 0.11

Time Treatment Mean Standard Dev. T=36 1-18 2.88 0.06 2-106 3.08 0.02 2-18 3.08 0.01 2-23 2.94 0.07 2-96 2.41 0.03 4-15 2.87 0.07 5-24 2.75 0.08 Control 1 6.43 0.08 Control 2 6.93 0.21

133

Figures 4 and 5. Effect of PSB on Pi concentration in NBRIP solution containing 1 g/L CaHPO4. Time Treatment Mean Standard Dev. T=0 1-18 1.49 1.81 2-106 0.48 0.22 2-18 0.38 0.01 2-23 0.44 0.05 2-96 0.10 0.04 4-15 0.26 0.03 5-24 0.58 0.25 Control 1 0.17 0.10 Control 2 0.42 0.09

Time Treatment Mean Standard Dev. T=12 1-18 0.36 0.09 2-106 5.27 1.06 2-18 2.09 0.13 2-23 0.46 0.06 2-96 1.25 0.40 4-15 2.15 0.81 5-24 3.49 1.49 Control 1 0.42 0.65 Control 2 1.09 0.28

Time Treatment Mean Standard Dev. T=24 1-18 0.75 0.07 2-106 5.87 0.28 2-18 2.90 0.53 2-23 0.64 0.06 2-96 0.52 0.10 4-15 0.36 0.17 5-24 3.97 0.37 Control 1 0.22 0.09 Control 2 0.53 0.28

Time Treatment Mean Standard Dev. T=36 1-18 2.68 0.03 2-106 8.28 0.26 2-18 5.98 1.25 2-23 10.04 2.47

134

Time Treatment Mean Standard Dev. 2-96 1.90 0.03 4-15 0.27 0.08 5-24 0.97 0.03 Control 1 0.31 0.08 Control 2 0.21 0.06

Table 15. Effect of PSB isolates and RP on pH of liquid medium in growth pouch bioassay. Time Treatment Mean Standard Dev. T= 1 d 1-18 + RP 6.97 0.08 2-106 + RP 6.92 0.21 2-18 + RP 7.15 0.07 2-23 + RP 6.85 0.27 2-96 + RP 7.26 0.07 4-15 + RP 6.72 0.15 Control + K2HPO4 5.11 0.07 Control + RP 6.62 0.05 Control 5.53 0.16

Time Treatment Mean Standard Dev. T= 7 d 1-18 + RP 7.2 0.1 2-106 + RP 6.99 0.09 2-18 + RP 7.03 0.09 2-23 + RP 7.09 0.06 2-96 + RP 7.16 0.20 4-15 + RP 7.15 0.21 Control + K2HPO4 5.27 0.06 Control + RP 7.14 0.08 Control 6.49 0.13

Time Treatment Mean Standard Dev. T= 14 d 1-18 + RP 7.14 0.17 2-106 + RP 6.85 0.09 2-18 + RP 7.12 0.06 2-23 + RP 7.10 0.08 2-96 + RP 7.06 0.07 4-15 + RP 6.86 0.06 Control + K2HPO4 5.21 0.16 Control + RP 7.22 0.22 Control 6.34 0.10

135

Time Treatment Mean Standard Dev. T= 21 d 1-18 + RP 7.28 0.07 2-106 + RP 7.14 0.19 2-18 + RP 7.10 0.09 2-23 + RP 7.35 0.25 2-96 + RP 7.23 0.06 4-15 + RP 7.38 0.19 Control + K2HPO4 5.50 0.13 Control + RP 6.90 0.69 Control 5.70 0.28

Time Treatment Mean Standard Dev. T= 28 d 1-18 + RP 7.39 0.08 2-106 + RP 7.27 0.13 2-18 + RP 7.02 0.12 2-23 + RP 7.21 0.15 2-96 + RP 7.33 0.08 4-15 + RP 7.13 0.04 Control + K2HPO4 5.76 0.26 Control + RP 7.14 0.15 Control 6.17 0.33

Table 16. Effect of PSB and RP in liquid medium in growth pouch bioassay. Observation Treatment Mean Standard Dev. # Leaves 1-18 + RP 1.56 1.54 2-106 + RP 1.44 1.02 2-18 + RP 2.56 0.20 2-23 + RP 2.44 1.64 2-96 + RP 1.56 0.51 4-15 + RP 1.22 1.17 Control + K2HPO4 1.78 1.84 Control + RP 2.11 0.70 Control 2.00 1.00

Observation Treatment Mean Standard Dev. Shoot Length 1-18 + RP 11.66 4.83 2-106 + RP 24.81 13.83 2-18 + RP 26.50 9.49 2-23 + RP 20.36 11.49 2-96 + RP 13.68 2.17 4-15 + RP 27.76 - 136

Observation Treatment Mean Standard Dev. Shoot Length Control + K2HPO4 24.15 8.82 Control + RP 17.79 7.40 Control 19.85 1.12

Observation Treatment Mean Standard Dev. Top Fresh Wt 1-18 + RP 0.09 0.06 2-106 + RP 0.12 0.01 2-18 + RP 0.14 0.05 2-23 + RP 0.09 0.08 2-96 + RP 0.05 0.01 4-15 + RP 0.14 - Control + K2HPO4 0.15 0.03 Control + RP 0.25 0.29 Control 0.07 0.06

Observation Treatment Mean Standard Dev. Root Fresh Wt 1-18 + RP 0.01 0.01 2-106 + RP 0.12 0.01 2-18 + RP 0.02 0.00 2-23 + RP 0.01 0.02 2-96 + RP 0.01 0.00 4-15 + RP 0.01 - Control + K2HPO4 0.01 0.00 Control + RP 0.03 0.01 Control 0.37 0.54

Observation Treatmen t Mean Standard Dev. Root Health 1-18 + RP 1.45 1.07 2-106 + RP 1.89 1.39 2-18 + RP 1.11 0.70 2-23 + RP 1.89 1.90 2-96 + RP 0.33 0.00 4-15 + RP 1.33 1.20 Control + K2HPO4 1.22 1.07 Control + RP 1.67 0.67 Control 0.55 0.39

137

Table 17. Root analysis data showing the effect of PSB and RP in a growth pouch bioassay. Observation Treatment Mean Standard Dev. Tips 1-18 + RP 4.80 1.48 2-106 + RP 15.57 7.32 2-18 + RP 10.40 5.55 2-23 + RP 10.00 8.25 2-96 + RP 5.33 3.06 4-15 + RP 4.33 1.53 Control + K2HPO4 13.78 6.83 Control + RP 9.33 3.98 Control 9.57 5.26

Observation Treatment Mean Standard Dev. Forks 1-18 + RP 4.00 1.41 2-106 + RP 18.14 14.02 2-18 + RP 12.00 8.72 2-23 + RP 12.00 14.00 2-96 + RP 5.67 5.13 4-15 + RP 4.33 3.51 Control + K2HPO4 18.22 14.44 Control + RP 8.33 4.93 Control 8.43 6.55

Observation Treatment Mean Standard Dev. Crossings 1-18 + RP 0.00 0.00 2-106 + RP 0.29 0.49 2-18 + RP 0.80 0.84 2-23 + RP 1.80 3.49 2-96 + RP 0.67 1.15 4-15 + RP 0.33 0.58 Control + K2HPO4 1.00 1.66 Control + RP 0.00 0.00 Control 0.43 1.13

Observation Treatment Mean Standard Dev. Root Length 1-18 + RP 4.25 0.77 2-106 + RP 9.62 3.80 2-18 + RP 10.44 4.24 2-23 + RP 7.56 4.38 2-96 + RP 3.44 1.21 4-15 + RP 3.57 0.15 Control + K2HPO4 8.78 3.40 138

Observation Treatment Mean Standard Dev. Root Length Control + RP 6.19 2.24 Control 7.09 1.98

Observation Treatment Mean Standard Dev. Surface Area 1-18 + RP 0.91 0.23 2-106 + RP 1.57 0.30 2-18 + RP 1.42 0.53 2-23 + RP 1.13 0.47 2-96 + RP 0.83 0.20 4-15 + RP 0.67 0.10 Control + K2HPO4 1.24 0.31 Control + RP 1.02 0.33 Control 1.53 0.41

Observation Treatment Mean Standard Dev. Root Diameter 1-18 + RP 0.69 0.15 2-106 + RP 0.57 0.15 2-18 + RP 0.47 0.16 2-23 + RP 0.52 0.14 2-96 + RP 0.81 0.18 4-15 + RP 0.59 0.07 Control + K2HPO4 0.47 0.06 Control + RP 0.53 0.08 Control 0.72 0.26

Observation Treatment Mean Standard Dev. Root Volume 1-18 + RP 0.02 0.01 2-106 + RP 0.02 0.00 2-18 + RP 0.02 0.01 2-23 + RP 0.10 0.00 2-96 + RP 0.02 0.00 4-15 + RP 0.01 0.00 Control + K2HPO4 0.01 0.00 Control + RP 0.01 0.01 Control 0.03 0.02

Observation Treatment Mean Standard Dev. Root Area 1-18 + RP 0.29 0.07 2-106 + RP 0.50 0.10 2-18 + RP 0.45 0.17

139

Observation Treatment Mean Standard Dev. Root Area 2-23 + RP 0.36 0.15 2-96 + RP 0.27 0.06 4-15 + RP 0.21 0.03 Control + K2HPO4 0.39 0.10 Control + RP 0.32 0.10 Control 0.49 0.13

Table 18. Plant growth data for greenhouse bioassay 1- Soil E. Observation Treatment Mean Standard Dev. # Leaves Control 18.13 2.17 10-50-10 14.33 2.89 Compost 16.00 4.85 Bacteria 'A' 15.25 5.06 Bacteria 'B' 14.67 8.08 Bacteria 'C' 18.40 4.34 BM 13.33 4.04 BM + Bact 'C' 17.75 3.30

Observation Treatment Mean Standard Dev. Shoot Length Control 10.69 3.01 10-50-10 9.50 3.61 Compost 8.70 3.09 Bacteria 'A' 8.75 5.27 Bacteria 'B' 8.33 5.01 Bacteria 'C' 12.30 3.70 BM 7.17 2.89 BM + Bact 'C' 10.13 5.31

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 2.09 0.63 10-50-10 1.44 0.31 Compost 2.02 1.09 Bacteria 'A' 1.67 1.20 Bacteria 'B' 1.58 1.37 Bacteria 'C' 2.38 1.04 BM 1.13 0.70 BM + Bact 'C' 2.05 1.04

140

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 0.64 0.29 10-50-10 0.41 0.10 Compost 0.56 0.30 Bacteria 'A' 0.48 0.38 Bacteria 'B' 0.46 0.41 Bacteria 'C' 0.74 0.36 BM 0.33 0.23 BM + Bact 'C' 0.59 0.31 Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.17 0.06 10-50-10 0.10 0.02 Compost 0.27 0.21 Bacteria 'A' 0.12 0.07 Bacteria 'B' 0.13 0.11 Bacteria 'C' 0.27 0.16 BM 0.16 0.11 BM + Bact 'C' 0.28 0.12

Table 19. Plant growth data for greenhouse bioassay 1- Soil F. Observation Treatment Mean Standard Dev. # Leaves Control 20.50 6.36 10-50-10 19.40 2.88 Compost 18.80 3.35 Bacteria 'A' 19.00 4.34 Bacteria 'B' 13.67 5.77 Bacteria 'C' 18.67 1.53 BM 19.25 4.92 BM + Bact 'C' 17.60 3.44

Observation Treatment Mean Standard Dev. Shoot Length Control 17.00 8.49 10-50-10 16.30 7.09 Compost 9.25 3.07 Bacteria 'A' 14.00 6.78 Bacteria 'B' 10.67 6.45 Bacteria 'C' 14.50 3.04 BM 16.50 8.40 BM + Bact 'C' 10.80 5.03

141

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 3.60 1.58 10-50-10 3.42 1.17 Compost 2.38 1.25 Bacteria 'A' 3.27 1.39 Bacteria 'B' 2.31 1.73 Bacteria 'C' 3.79 1.21 BM 3.59 1.83 BM + Bact 'C' 3.05 0.81

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 1.38 0.98 10-50-10 1.63 0.62 Compost 0.89 0.48 Bacteria 'A' 1.27 0.55 Bacteria 'B' 0.98 0.80 Bacteria 'C' 1.59 0.31 BM 1.54 0.89 BM + Bact 'C' 1.16 0.37 Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.47 0.18 10-50-10 0.45 0.22 Compost 0.32 0.23 Bacteria 'A' 0.33 0.15 Bacteria 'B' 0.37 0.34 Bacteria 'C' 0.56 0.19 BM 0.39 0.21 BM + Bact 'C' 0.41 0.11

Table 20. Plant growth data for greenhouse bioassay 1- Soil G. Observation Treatment Mean Standard Dev. # Leaves Control 14.80 2.59 10-50-10 13.33 3.61 Compost 15.80 2.59 Bacteria 'A' 17.33 1.51 Bacteria 'B' 16.71 2.21 Bacteria 'C' 18.83 3.06 BM 17.80 5.17 BM + Bact 'C' 18.50 3.87

142

Observation Treatment Mean Standard Dev. Shoot Length Control 8.83 1.63 10-50-10 7.00 2.43 Compost 7.50 2.76 Bacteria 'A' 10.50 2.14 Bacteria 'B' 11.29 2.78 Bacteria 'C' 8.17 2.56 BM 11.10 7.05 BM + Bact 'C' 9.13 3.86

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 1.77 0.68 10-50-10 1.22 0.55 Compost 2.28 1.24 Bacteria 'A' 2.67 0.68 Bacteria 'B' 2.61 1.01 Bacteria 'C' 2.05 0.56 BM 2.54 1.40 BM + Bact 'C' 2.17 1.20

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 0.68 0.28 10-50-10 0.45 0.20 Compost 0.85 0.45 Bacteria 'A' 1.07 0.31 Bacteria 'B' 1.03 0.42 Bacteria 'C' 0.82 0.23 BM 1.04 0.59 BM + Bact 'C' 0.82 0.45 Observation Treatment Mean Standard Dev. Root Fresh Wt Control 0.25 0.17 10-50-10 0.10 0.05 Compost 0.33 0.16 Bacteria 'A' 0.31 0.15 Bacteria 'B' 0.28 0.19 Bacteria 'C' 0.33 0.11 BM 0.26 0.06 BM + Bact 'C' 0.20 0.06

143

Table 21. Plant growth data for greenhouse bioassay 1- Soil H. Observation Treatment Mean Standard Dev. # Leaves Control 15.00 4.38 10-50-10 15.29 1.98 Compost 13.33 2.89 Bacteria 'A' 15.50 1.52 Bacteria 'B' 13.00 2.16 Bacteria 'C' 15.60 2.94 BM 13.38 2.67 BM + Bact 'C' 16.13 3.00

Observation Treatment Mean Standard Dev. Shoot Length Control 18.15 8.95 10-50-10 18.87 7.51 Compost 8.87 3.73 Bacteria 'A' 20.08 4.58 Bacteria 'B' 15.53 4.99 Bacteria 'C' 21.43 8.57 BM 17.35 7.21 BM + Bact 'C' 22.29 8.34

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 4.52 2.31 10-50-10 5.07 1.85 Compost 2.33 1.47 Bacteria 'A' 5.83 1.96 Bacteria 'B' 3.80 1.69 Bacteria 'C' 6.10 2.88 BM 4.62 2.73 BM + Bact 'C' 5.53 2.23

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 0.47 0.27 10-50-10 0.46 0.30 Compost 0.29 0.09 Bacteria 'A' 0.58 0.32 Bacteria 'B' 0.54 0.32 Bacteria 'C' 0.60 0.37 BM 0.33 0.26 BM + Bact 'C' 0.60 0.42 Observation Treatment Mean Standard Dev.

144

Root Dry Wt Control 0.53 0.25 10-50-10 0.70 0.60 Compost 0.31 0.19 Bacteria 'A' 0.63 0.49 Bacteria 'B' 0.50 0.31 Bacteria 'C' 0.68 0.40 BM 0.44 0.39 BM + Bact 'C' 0.43 0.28

Table 22. Plant growth data for greenhouse bioassay 1- Soil I. Observation Treatment Mean Standard Dev. # Leaves Control 13.75 4.06 10-50-10 13.88 2.17 Compost 10.50 5.32 Bacteria 'A' 9.83 5.64 Bacteria 'B' 9.88 4.58 Bacteria 'C' 9.13 5.99 BM 11.43 4.69 BM + Bact 'C' 10.29 3.68

Observation Treatment Mean Standard Dev. Shoot Length Control 12.70 7.15 10-50-10 12.46 8.33 Compost 8.55 6.85 Bacteria 'A' 8.85 4.69 Bacteria 'B' 8.41 5.04 Bacteria 'C' 8.14 4.98 BM 9.17 7.48 BM + Bact 'C' 7.90 4.02

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 2.76 1.46 10-50-10 2.71 2.31 Compost 1.63 1.60 Bacteria 'A' 1.91 1.81 Bacteria 'B' 1.58 1.20 Bacteria 'C' 1.51 1.59 BM 1.82 1.56 BM + Bact 'C' 1.55 1.00

145

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 0.30 0.24 10-50-10 0.41 0.43 Compost 0.11 0.12 Bacteria 'A' 0.25 0.23 Bacteria 'B' 0.13 0.13 Bacteria 'C' 0.16 0.18 BM 0.20 0.15 BM + Bact 'C' 0.18 0.14 Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.42 0.27 10-50-10 0.33 0.19 Compost 0.21 0.14 Bacteria 'A' 0.23 0.26 Bacteria 'B' 0.26 0.19 Bacteria 'C' 0.30 0.41 BM 0.27 0.22 BM + Bact 'C' 0.29 0.17

Table 23. Plant growth data for greenhouse bioassay 2- PSB in potting mix. Observation Treatment Mean Standard Dev. # Leaves Control 11.38 2.00 4-15 13.86 1.77 2-18 13.63 2.26 2-106 14.43 3.41 RP 14.38 2.39 RP + 4-15 14.50 2.33 RP + 2-18 15.38 3.81 RP + 2-106 13.75 5.95 BM 13.38 1.60 BM + 4-15 12.75 3.24 BM +2-18 16.50 5.81 BM + 2-106 15.43 6.11 10-50-10 15.00 3.51

Observation Treatment Mean Standard Dev. Shoot Length Control 25.07 7.47 4-15 28.86 5.73 2-18 27.38 9.72 2-106 25.14 7.06 RP 33.06 6.81 146

Observation Treatment Mean Standard Dev. Shoot Length RP + 4-15 29.19 6.63 RP + 2-18 33.81 5.99 RP + 2-106 24.44 12.19 BM 27.38 6.74 BM + 4-15 23.81 9.57 BM +2-18 30.25 6.53 BM + 2-106 29.14 5.27 10-50-10 30.79 7.64

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 4.64 1.81 4-15 6.57 1.80 2-18 7.29 3.04 2-106 6.27 2.29 RP 8.39 2.60 RP + 4-15 7.19 2.10 RP + 2-18 8.65 2.15 RP + 2-106 5.90 3.21 BM 6.67 2.20 BM + 4-15 6.37 3.82 BM +2-18 8.04 0.96 BM + 2-106 7.78 1.97 10-50-10 7.64 2.49

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 1.93 0.72 4-15 3.20 1.24 2-18 3.99 1.79 2-106 3.20 1.35 RP 4.34 1.91 RP + 4-15 3.55 1.27 RP + 2-18 3.96 1.28 RP + 2-106 2.91 1.51 BM 3.14 1.29 BM + 4-15 3.15 2.32 BM +2-18 3.83 1.27 BM + 2-106 3.49 1.41 10-50-10 3.76 1.60

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Observation Treatment Mean Standard Dev. Root Fresh Wt Control 3.06 1.01 4-15 3.22 2.08 2-18 3.82 2.15 2-106 3.75 1.53 RP 4.18 1.46 RP + 4-15 3.65 0.88 RP + 2-18 4.30 1.28 RP + 2-106 2.97 1.96 BM 3.07 1.42 BM + 4-15 3.07 1.75 BM +2-18 4.05 1.61 BM + 2-106 3.48 1.51 10-50-10 4.17 2.71

Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.62 0.22 4-15 0.66 0.54 2-18 0.74 0.54 2-106 0.82 0.44 RP 0.88 0.39 RP + 4-15 0.72 0.23 RP + 2-18 0.90 0.35 RP + 2-106 0.66 0.46 BM 0.64 0.46 BM + 4-15 0.64 0.25 BM +2-18 0.82 0.27 BM + 2-106 0.75 0.33 10-50-10 0.67 0.36

Table 24. Plant growth data for greenhouse bioassay 3- Reiger. Observation Treatment Mean Standard Dev. # Leaves Control 20.75 2.05 RP 22.63 4.75 RP + 4-15 22.13 1.96 RP + 2-18 20.50 3.85 RP + 2-106 20.25 3.69 BM 21.88 1.96 BM + 4-15 20.63 5.71 BM +2-18 23.71 7.72 BM + 2-106 20.63 2.39

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Observation Treatment Mean Standard Dev. Shoot Length Control 29.13 6.44 RP 27.54 4.79 RP + 4-15 27.09 4.54 RP + 2-18 23.19 8.63 RP + 2-106 21.24 6.21 BM 28.60 4.21 BM + 4-15 27.26 8.37 BM +2-18 26.69 10.51 BM + 2-106 28.89 6.19

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 8.41 2.66 RP 7.54 1.81 RP + 4-15 8.58 1.23 RP + 2-18 6.56 2.60 RP + 2-106 7.52 2.51 BM 8.68 2.39 BM + 4-15 7.90 3.29 BM +2-18 8.79 4.51 BM + 2-106 8.61 2.42

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 2.87 0.97 RP 2.52 0.58 RP + 4-15 2.72 0.61 RP + 2-18 2.11 0.93 RP + 2-106 2.65 0.69 BM 3.04 0.83 BM + 4-15 2.64 1.13 BM +2-18 2.93 1.50 BM + 2-106 2.67 0.59

Observation Treatment Mean Standard Dev. Root Fresh Wt Control 4.59 2.42 RP 4.74 1.61 RP + 4-15 4.68 1.38 RP + 2-18 3.72 2.07 RP + 2-106 4.05 1.99 BM 4.97 1.80 BM + 4-15 3.77 1.75 BM +2-18 4.41 2.99 149

Observation Treatment Mean Standard Dev. Root Fresh Wt BM + 2-106 4.23 1.37

Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.64 0.32 RP 0.62 0.15 RP + 4-15 0.54 0.19 RP + 2-18 0.43 0.23 RP + 2-106 0.63 0.22 BM 0.68 0.30 BM + 4-15 0.46 0.24 BM +2-18 0.55 0.32 BM + 2-106 0.59 0.21

Table 25. Plant growth data for greenhouse bioassay 3- McCoubrey. Observation Treatment Mean Standard Dev. # Leaves Control 21.25 4.40 RP 20.00 2.67 RP + 4-15 19.38 3.25 RP + 2-18 19.50 4.28 RP + 2-106 21.00 1.83 BM 21.83 1.60 BM + 4-15 19.50 2.56 BM +2-18 21.50 4.31 BM + 2-106 21.30 1.75

Observation Treatment Mean Standard Dev. Shoot Length Control 22.10 8.27 RP 22.60 7.07 RP + 4-15 20.18 7.45 RP + 2-18 22.84 7.60 RP + 2-106 27.64 4.02 BM 26.60 7.75 BM + 4-15 22.58 5.73 BM +2-18 22.76 7.62 BM + 2-106 23.45 3.26

Observation Treatment Mean Standard Dev. Shoot Fresh Wt Control 6.70 4.81 RP 6.10 2.95 RP + 4-15 5.01 2.51 150

Observation Treatment Mean Standard Dev. Shoot Fresh Wt RP + 2-18 6.04 2.12 RP + 2-106 7.70 1.66 BM 8.28 2.75 BM + 4-15 5.70 2.33 BM +2-18 5.46 1.74 BM + 2-106 7.36 1.56

Observation Treatment Mean Standard Dev. Shoot Dry Wt Control 1.94 1.10 RP 1.97 0.98 RP + 4-15 1.54 0.79 RP + 2-18 1.93 0.74 RP + 2-106 2.51 0.59 BM 2.67 0.85 BM + 4-15 1.88 0.73 BM +2-18 1.71 0.63 BM + 2-106 2.32 0.50

Observation Treatment Mean Standard Dev. Root Fresh Wt Control 7.24 2.56 RP 6.71 3.43 RP + 4-15 4.50 2.22 RP + 2-18 4.79 1.26 RP + 2-106 5.90 1.96 BM 13.36 17.47 BM + 4-15 5.20 2.09 BM +2-18 4.75 2.03 BM + 2-106 5.69 2.19

Observation Treatment Mean Standard Dev. Root Dry Wt Control 0.64 0.29 RP 0.63 0.38 RP + 4-15 0.37 0.20 RP + 2-18 0.43 0.14 RP + 2-106 0.58 0.26 BM 0.69 0.29 BM + 4-15 0.47 0.18 BM +2-18 0.44 0.18 BM + 2-106 0.51 0.19

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Table 26. Trunk cross sectional area (cm2) for the Reiger orchard. Date Sampled Treatment Mean Standard Dev. May-11 Control 8.56 2.14 RP 9.11 1.91 BM 8.86 1.65 RP + PSB 10.06 1.95 BM + PSB 10.09 1.80 PSB 9.33 1.78

Date Sampled Treatment Mean Standard Dev. Oct-11 Control 8.63 2.17 RP 9.21 1.58 BM 9.00 2.15 RP + PSB 9.97 1.96 BM + PSB 10.17 1.84 PSB 9.66 1.85

Date Sampled Treatment Mean Standard Dev. Sep-12 Control 9.2 2.57 RP 9.51 1.73 BM 9.57 2.15 RP + PSB 10.18 2.19 BM + PSB 10.86 2.33 PSB 10.22 1.48

Table 27. Trunk cross sectional area (cm2) for the McCoubrey orchard. Date Sampled Treatment Mean Standard Dev. May-11 Control 8.91 2.05 RP 9.14 1.40 BM 9.02 1.72 RP + PSB 9.82 2.28 BM + PSB 9.17 2.14 PSB 8.32 1.80

Date Sampled Treatment Mean Standard Dev. Oct-11 Control 13.50 2.46 RP 13.82 2.50 BM 12.28 2.19 RP + PSB 13.07 2.30 BM + PSB 12.79 2.40 152

Date Sampled Treatment Mean Standard Dev. Oct-11 PSB 12.64 2.98

Date Sampled Treatment Mean Standard Dev. Sep-12 Control 9.2 2.57 RP 9.51 1.73 BM 9.57 2.15 RP + PSB 10.18 2.19 BM + PSB 10.86 2.33 PSB 10.22 1.48

Table 28. Total shoot length (cm) for the Reiger orchard. Date Sampled Treatment Mean Standard Dev. May-11 Control 77.68 19.30 RP 78.21 11.72 BM 74.87 12.65 RP + PSB 75.46 8.69 BM + PSB 87.31 15.29 PSB 82.55 14.73

Date Sampled Treatment Mean Standard Dev. Oct-11 Control 97.51 18.77 RP 97.41 9.66 BM 93.08 13.76 RP + PSB 94.76 7.43 BM + PSB 105.66 16.37 PSB 102.45 14.88

Date Sampled Treatment Mean Standard Dev. Sep-12 Control 99.39 24.97 RP 95.25 12.16 BM 90.59 14.56 RP + PSB 87.15 15.58 BM + PSB 104.35 18.89 PSB 102.33 18.70

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Table 29. Total shoot length (cm) for the McCoubrey orchard. Date Sampled Treatment Mean Standard Dev. May-11 Control 73.24 15.69 RP 78.95 12.73 BM 82.44 10.78 RP + PSB 84.03 15.15 BM + PSB 83.82 18.19 PSB 74.19 18.91

Date Sampled Treatment Mean Standard Dev. Oct-11 Control 98.43 12.51 RP 102.09 13.14 BM 98.53 16.32 RP + PSB 103.72 15.23 BM + PSB 102.15 19.05 PSB 96.40 17.80

Date Sampled Treatment Mean Standard Dev. Sep-12 Control 113.33 28.88 RP 117.5 28.51 BM 111.74 21.67 RP + PSB 117.75 26.48 BM + PSB 120 24.17 PSB 109.14 16.48

Table 30. Number of nematodes for the Reiger orchard by date sampled. Date Sampled Treatment Mean Standard Dev. Jul-11 Control 54.17 54.84 RP 39.83 34.89 BM 39.00 29.70 RP + PSB 36.17 30.71 BM + PSB 39.00 27.50 PSB 24.67 16.71

Date Sampled Treatment Mean Standard Dev. Sep-11 Control 57.80 98.84 RP 10.00 4.90 BM 34.83 39.09 RP + PSB 32.67 29.38 BM + PSB 32.00 30.74 154

Date Sampled Treatment Mean Standard Dev. Sep-11 PSB 11.33 10.42

Date Sampled Treatment Mean Standard Dev. Jun-12 Control 10.87 13.82 RP 11.48 6.45 BM 8.82 6.15 RP + PSB 7.59 4.5 BM + PSB 4.51 6.45 PSB 4.51 3.08

Table 31. Number of nematodes for the McCoubrey orchard by date sampled. Date Sampled Treatment Mean Standard Dev. Sep-11 Control 20.33 17.27 RP 16.17 9.50 BM 11.67 11.60 RP + PSB 12.50 7.92 BM + PSB 18.67 17.82 PSB 25.00 21.39

Date Sampled Treatment Mean Standard Dev. Jun-12 Control 26.5 22.98 RP 16.67 10.42 BM 19.67 13.85 RP + PSB 22.5 18.49 BM + PSB 29 19.97 PSB 22.33 16.24

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A.2. List of Figures

Figure A.2.1. Standard curve prepared for bacterial isolate 2-18.

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Figure A.2.2. Standard curve prepared for bacterial isolate 2-106.

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Figure A.2.3. Standard curve prepared for bacterial isolate 4-15.

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Figure A.2.4. Standard curve prepared for QuantiChrom phosphate assays.

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A.3. List of Illustrations

Rep 1 Rep 2 Rep 3 xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx Rep 4 Rep 5 Rep 6

Treatments xxxx Control xxxx RP xxxx BM xxxx RP + PSB xxxx BM + PSB xxxx PSB

Illustration A.3.1. Map of treatment blocks for field trial at Reiger/Orchard Corners.

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Rep 1 Rep 2 xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx Rep 3 Rep 4 xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx Rep 5 Rep 6 xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx xxxx

Treatments xxxx Control xxxx RP xxxx BM xxxx RP + PSB xxxx BM + PSB xxxx PSB

Illustration A.3.2. Map of treatment blocks for field trial at McCoubrey Farms.

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Appendix B. P-free Hoagland’s Nutrient Solution Recipe

Stock solutions were prepared as follows:

Macronutrients Stock Solutions -1 K2SO4 (0.5M) 87.135 g l -1 MgSO4.7H2O (1M) 246.48 g l -1 KNO3 (1M) 101.11 g l -1 Ca(NO3)2.4H2O (1M) 236.2 g l

Micronutrients (Make 1 L total) -1 Boric acid 1.00g l (H3BO3) -1 Manganous chloride 1.00 g l (MnCl2.4H2O) -1 Zinc sulfate 0.58 g l (ZnSO4.7H2O) -1 Cupric sulfate 0.13 g l (CuSO4.5H2O) -1 Sodium molybdate 0.10 g l (Na2MoO4.2H2O)

Iron stock solution 20 g l-1 Fe EDTA

Final Medium contained (1 L total): -1 2.5 ml l KNO3 stock solution -1 2.5 ml l Ca(NO3)2 stock solution -1 4 ml l K2SO4 stock solution -1 1 ml l MgSO4 stock solution 1 ml l-1 Mictonutrient stock solution 1 ml l-1 Iron stock solution

The pH of the final solution was adjusted to 7.0 using 0.5 M KOH and sterilized in the autoclave for 20 minutes at 121°C.

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Appendix C. Soil amendment calculations for Fish Fertilizer and P amendments.

1. Fish Fertilizer - Pacific Natural brand (2-3-0) Fish fertilizer was applied by volume to meet the required 100 mg N per kg of dry weight soil, as outlined in the methods.

If each pot contains 1.25 kg of dry weight soil, then we require 125 mg N per pot. The density of the fish fertilizer is 21 kg (mass) per 20 L (volume), thus 21/20 = 1.05 The material has an analysis of 2-3-0 (N-P-K) according to the manufacturer. We have assumed that all of the N present in the fish fertilizer is mineralized and therefore available to the plant. (20% N)(1.05)= 210 g N/L or 21 mg N/mL If we need 125 mg N/pot, we will need: (125 mg N)(21mg N/mL)= 5.95 mL fish fertilizer per pot 6.0 mL of Pacific Natural Fish Fertilizer was used per pot of wet soil.

2. Bone Meal- Groundskeeper brand (2-14-0) Bone meal was applied by weight per pot to meet the required 200 mg P per kg of dry weight soil, as outlined in the methods. If each pot contains 1.25 kg of dry weight soil, then we require

250 mg P per pot. According to the manufacturer, 14% of the bone meal is in the P2O5 form.

To calculate the amount of P in the product, first we calculate the atomic weight of P2O5 from the periodic table of the elements is equal to 141.94, rounded up to 142. P =61.94 (atomic weight), rounded up to 62. 142/62 = 2.29

(250 mg P per pot)(2.29) = 572.5 mg P2O5 per pot 572.5 mg (100/14%) = 4089.3 mg or 4.09 g of Bone Meal per pot 4.09 g of Bone Meal was used per pot of wet soil.

3. 10-50-10 – Plant Products brand of Fertilizer

10-50-10 fertilizer was applied by weight per pot to meet the required 200 mg P per kg of dry weight soil, as outlined in the methods. If each pot contains 1.25 kg of dry weight soil, then we

require 250 mg P per pot. According to the manufacturer, 50% is in the P2O5 form.

(250 mg P per pot)(100/21.83) = 1146.8 mg P2O5 per pot or 1.146 g of 10-50-10 fertilizer per pot

1.15 g of soluble 10-50-10 fertilizer was used per pot of wet soil.

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