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Ecologically Sound Soil Management of Tree Fruit Replant Disease

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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 by 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 in THE COLLEGE OF GRADUATE STUDIES (Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) August 2013 © Molly Adair Thurston, 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 ii 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. iii 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 iv 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 ...................................................................................................
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