would like to thank my advisors Dr. Paul Voroney and Dr. Derek Lynch for ...... Liang, Z., Drijber, R.A., Lee, D.J., Dwiekat, I.M., Harris, S.D., Wedin, D.A., 2008.
Understanding Biological Contributions to Phosphorus Availability in Organic Dairy Farm Soils by Kimberley D. Schneider
A Thesis presented to The University of Guelph
In partial fulfillment of requirements for the degree of Doctor of Philosophy in Environmental Science
Guelph, Ontario, Canada © Kimberley D. Schneider, September, 2014
ABSTRACT
UNDERSTANDING BIOLOGICAL CONTRIBUTIONS TO PHOSPHORUS AVAILABILITY IN ORGANIC DAIRY FARM SOILS
Kimberley D. Schneider University of Guelph, 2014
Advisors: Dr. Paul Voroney Dr. Derek Lynch
Recent studies exploring soil phosphorus (P) fertility on dairy farms under organic management in Canada have reported low concentrations of Olsen soil test P (STP), yet the impact on crop productivity is not clear. In this thesis, soil biological and biochemical properties relating to plant P availability in perennial forage fields (Medicago sativa / Phleum pratense) of three paired organic and conventional dairy farms in southwestern Ontario, Canada were compared. Molecular approaches (PCR-DGGE and quantitative real-time PCR) were used to explore arbuscular mycorrhizal fungi (AMF) communities in these soils. Finally, an incubation experiment using the radioisotope 33P was conducted to assess biological P fluxes and microbial P uptake in select forage soils along a gradient of STP concentrations. Results of the field study found no significant differences between forage yields on organically versus conventionally managed fields, and forage yield was not correlated with STP. Plant tissue P concentrations supported that plant-available P was greater under conventional management; however organic tissue P concentrations were not lower than critical concentrations. Solution
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P NMR-spectroscopy revealed a greater organic P (Po): inorganic P
(Pi) ratio and greater proportions of orthophosphate diesters in NaOH-EDTA soil extracts from
organically managed systems. Mean AMF root colonization of alfalfa was also greater under organic management (37%) compared with conventional management (23%). AMF community composition was found to differ between management systems; the data indicated that organic systems may support AMF communities that promote greater host-plant P-use efficiencies. In the
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P incubation experiment gross Po mineralization contributed a mean of 35% of
the total isotopically-exchangeable P after 8 days. Significant uptake of solution Pi by the microbial biomass occurred in 8 days, with 11/16 soils having greater than 20% of the initially added
33
P in the microbial biomass. The proportion of
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P found in the microbial P pool was
negatively related to solution Pi concentrations. This research supports the importance biological processes to soil P dynamics in organic farming systems. These relevant processes need to be considered when assessing P availability in agricultural systems, which would ultimately aid in the greater goal of achieving P-use efficiency in agriculture.
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For my parents, who always encouraged my academic choices and never ‘told me what to do’ (even when I wanted them to give me an answer!)
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Acknowledgements There are several organizations that I would like to acknowledge for providing financial and/or in-kind support throughout my doctoral research program; these include the Natural Sciences and Engineering Research Council (NSERC) for providing me with a Canada Graduate Scholarship for three years and also a Michael Smith Foreign Study Supplement, which allowed me to study abroad for four months, Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA), Agriculture and Agri-Food Canada (AAFC), the Canada Research Chairs Program, The Swiss Federal Institute of Technology (ETH Zurich), and the Organic Meadow Dairy Cooperative (Guelph, ON). Completing a PhD is a journey that takes a lot of time, work, dedication and perseverance. Although independently directed, it really is not an individual effort and I would not be finishing up my degree today if it were not for the assistance of many people. Firstly, I would like to thank my advisors Dr. Paul Voroney and Dr. Derek Lynch for their guidance and support throughout my degree program. I enjoyed working with you both and appreciated your open-minded approach to research (and my ideas!). I would also like to thank the other members of my advisory committee, Dr. Kari Dunfield, whose lab I worked in for about one year, Dr. Ivan O’Halloran, and Dr. Chantal Hamel, for their helpful and valuable comments throughout my PhD program. Thanks are also extended to Dr. Ralph Martin and Dr. Leo Condron for serving on my thesis defense examination committee and for their insightful comments on my research. During my PhD, I had the opportunity to study abroad for four months and I would like to acknowledge the Group of Plant Nutrition of ETH Zurich in Switzerland for hosting me as an academic guest and for being open and willing to share laboratory methods and techniques with me. In particular, I am very grateful for the adademic guidance and assistance of Dr. Else
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Bünemann, Dr. Astrid Oberson and Dr. Jan Jansa during my time abroad and ongoing advice thereafter. In addition, I am very grateful for the open and willing assistance of Dr. Barbara CadeMenun, of AAFC (Swift Current, SK), who guided me with the NMR sample preparation, interpreted the output into workable data and was available for questions when I was working with the data. I would also like to thank Dr. Michelle Edwards and Dr. Stephen Bowley for their willingness to provide statistical analysis advice throughout my degree program. This body of research would not have been possible without the willingness and much appreciated cooperation of the landowners who allowed us to sample their fields, and thanks are extended to these anonymous six farmers and their families. Michael Main and Joel Aitken were both a big help in getting me started with the field work by openly sharing with me their experiences with similar forage field sampling. During the busy field and laboratory components of my research, I was fortunate to have the assistance of two summer students, Ismena Bystron and Siobhan Dunets, who were both great workers and a huge help. In addition, there were several temporary part-time employees who assisted with field sampling and other labour intensive tasks (i.e. plant-grinding); thank you all for your hard work! I would like to extend a general heartfelt thank you to the faculty, staff and graduate students of the School of Environmental Sciences at the University of Guelph for the many times I was need of assistance and was greeted by individuals that were glad to help (too numerous to name you all). Special thanks to Kamini Khosla, Ranee Pararajasingham, Peter Smith and Peter von Bertoldi for technical assistance with different aspects of my research; you certainly made my life a lot easier! In addition, thank you to my office mates and the administrative staff for their support, kindness, and assistance over the years.
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And finally, last but definitely not least, I would like to acknowledge my my family for their support and encouragement not just throughout my PhD program but over my many years of schooling. Your constant love, support, and faith in me was a source of strength, helping me persevere through difficult times. My sincerest thanks go to my husband, mother, and mother-in-
law for their flexibility and support of all kinds over the last couple years. It is hard to know what to say, but the sacrifices that you made so that I was able to pursue this endeavor will be remembered forever. And thank you to my little girl, Caleigh, who kept me from working at the computer for too long in a row, and also for her ability to share her mom with her work at such a young age.
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Table of Contents Chapter 1 - General Introduction .................................................................................................... 1 1.1 Introduction ........................................................................................................................... 1 1.2 Research Goal and Objectives .............................................................................................. 2 1.3 Thesis format ........................................................................................................................ 3 1.4 Literature Cited ..................................................................................................................... 5 Chapter 2 - Literature Review......................................................................................................... 7 2.1 Phosphorus – an essential plant nutrient ............................................................................... 7 2.2 Soil Phosphorus .................................................................................................................... 7 2.3 P Availability in Agricultural Soils....................................................................................... 8 2.4 Soil Organic Phosphorus..................................................................................................... 11 2.4.1 Mineralization of Soil Organic P ................................................................................ 12 2.4.2 Measuring biological P fluxes in soil.......................................................................... 17 2.4.3 Organic P mineralization in organically managed systems ......................................... 19 2.5 Arbuscular Mycorrhizal Fungi ............................................................................................ 20 2.5.1 AMF and Phosphorus Uptake ...................................................................................... 20 2.5.2 AMF genetic and functional diversity ......................................................................... 21 2.5.3 Determining AMF diversity and community structure ............................................... 23 2.5.4 AMF and Organic Agriculture ..................................................................................... 24 2.6 Summary ............................................................................................................................. 26 2.7 Literature Cited ................................................................................................................... 27
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Chapter 3 - Indicators of plant-available phosphorus on organic and conventional dairy farms in relation to forage quality and yield. .............................................................................................. 35 3.1 Abstract ............................................................................................................................... 36 3.2 Introduction ......................................................................................................................... 37 3.3 Materials and Methods ........................................................................................................ 41 3.3.1 Site selection and description....................................................................................... 41 3.3.2 Soil and plant sampling................................................................................................ 42 3.3.3 Soil physical and chemical analyses ............................................................................ 43 3.3.4 Soil biological and biochemical analyses .................................................................... 46 3.3.5 AMF root colonization ................................................................................................. 47 3.3.6 Plant analyses ............................................................................................................... 48 3.3.7 Statistics ....................................................................................................................... 48 3.4 Results ................................................................................................................................. 49 3.4.1 Soil phosphorus forms as determined by solution 31P NMR spectroscopy ................. 49 3.4.2 Soil biological and biochemical properties .................................................................. 52 3.4.3 Forage yield and composition, P concentration and uptake. ....................................... 53 3.5 Discussion ........................................................................................................................... 54 3.5.1 Site and Management characteristics ........................................................................... 54 3.5.2 Soil phosphorus forms determined by solution 31P NMR spectroscopy...................... 55 3.5.3 Soil Biological and Biochemical Properties ................................................................ 64 3.5.4 AMF root colonization ................................................................................................. 68
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3.5.5 Forage Yield, Composition, and P uptake ................................................................... 70 3.6 Conclusions ......................................................................................................................... 80 3.7 Literature Cited ................................................................................................................... 82 3.8 Tables and Figures .............................................................................................................. 92 Chapter 4 - Arbuscular mycorrhizal fungi communities from dairy farm soils under organic and conventional management .......................................................................................................... 108 4.1 Abstract ............................................................................................................................. 109 4.2 Introduction ....................................................................................................................... 110 4.3 Materials and Methods ...................................................................................................... 113 4.3.1 Site selection and description..................................................................................... 113 4.3.2 Soil Sampling ........................................................................................................... 114 4.3.3 Soil DNA extraction ................................................................................................. 114 4.3.4 AMF Community Structure ...................................................................................... 114 4.3.4.5 Statistical Analysis ................................................................................................. 118 4.4 Results ............................................................................................................................... 120 4.4.1 AMF community analysis based on PCR–DGGE ..................................................... 120 4.4.2 Real-time qPCR results .............................................................................................. 121 4.5 Discussion ......................................................................................................................... 122 4.5.1 Effect of geographical location on AMF communities.............................................. 122 4.5.2 Effect of farm system management on AMF communities ....................................... 123 4.5.3 Methodological Considerations ................................................................................. 132
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4.6 Conclusions ....................................................................................................................... 135 4.7 Literature Cited ................................................................................................................. 138 4.8 Tables and Figures ............................................................................................................ 145 Chapter 5 - Gross organic P mineralization and microbial P uptake rates in forage soils along a gradient of available inorganic P ................................................................................................ 150 5.1 Abstract ............................................................................................................................. 151 5.2 Introduction ....................................................................................................................... 152 5.3 Materials and Methods ...................................................................................................... 155 5.3.1 Soil Properties ............................................................................................................ 155 5.3.2 Experimental Design .................................................................................................. 156 5.3.3 Isotopic exchange kinetics (IEK) experiment ............................................................ 156 5.3.4 Incubation experiment ............................................................................................... 158 5.3.5 Soil Analyses /Analytical Methods ............................................................................ 159 5.3.6 Calculation of gross P fluxes ..................................................................................... 161 5.3.7 Statistics ..................................................................................................................... 162 5.4 Results ............................................................................................................................... 163 5.4.1 Isotopic exchange kinetics batch experiment ............................................................ 163 5.4.2 Incubation Experiment ............................................................................................... 164 5.5 Discussion ......................................................................................................................... 167 5.5.1 Isotopic Exchange Kinetics Parameters ..................................................................... 167 5.5.2 Incubation experiment ............................................................................................... 170
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5.5.3 P fluxes....................................................................................................................... 174 5.5.4 Implications of P fluxes for agroecosystems ............................................................. 178 5.5.5 The use of the isotopic dilution method in high P-fixing calcareous soils ............... 179 5.5.6 General methodological considerations and limitations of this study ....................... 181 5.6 Conclusions ....................................................................................................................... 182 5.7 Literature Cited ................................................................................................................. 184 5.8 Tables and Figures ............................................................................................................ 188 Chapter 6 – Conclusions ............................................................................................................. 195 Appendices .................................................................................................................................. 200
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List of Tables
Table 3.1. Description of select management practices and soil properties (0-15 cm) of three paired organic (Org) and conventional (Con) dairy farm forage fields. Within columns, means followed by the same letter are not significantly different (P < 0.05, REML test for fixed effects). ....................................................................................................................................................... 92 Table 3.2. Mean annual temperature, precipitation and growing season data for the three study site locations.................................................................................................................................. 93 Table 3.3. Distribution of P forms (relative % of total P) in 0.5 M NaOH-EDTA soil extracts from three paired organic (Org) and conventional (Con) dairy farm fields calculated from NMR spectra by integration. If differences between the two treatments were significant (P < 0.05), an asterisk (*) is shown on the mean organic percentage. ................................................................. 94 Table 3.4. Concentration of P forms in 0.5 M NaOH-EDTA soil extracts from three paired organic (Org) and conventional (Con) dairy farm fields. Values were determined by multiplying relative amounts of each P form (calculated from NMR spectra by integration) by total P in the extract. If differences between the two treatments were significant (P < 0.05), an asterisk (*) is shown on the mean organic concentration. ................................................................................... 95 Table 3.5. Soil biological and biochemical parameters including levels of Medicago sativa (alfalfa) arbuscular mycorryhzal fungal (AMF) root colonization, determined for three paired organic (Org) and conventional (Con) dairy farm forage fields (0-15 cm). Statistically significant differences (analysed using the Restricted Maximum Likelihood (REML) method test for fixed effects) between management type are shown. .............................................................. 96
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Table 3.6. Forage yield, composition, tissue P and P uptake data for three paired organic (Org) and conventional (Con) dairy farm forage fields in Ontario, Canada. Statistically significant differences (analysed using the Restricted Maximum Likelihood (REML) method test for fixed effects) between management type are shown.............................................................................. 97 Table 3.7. Pearson correlation coefficients and corresponding p values for soil and plant parameters and Olsen Soil Test Phosphorus for soils from three organic and three conventional dairy farm fields. A P value of 300), net immobilization tends to occur and as it decreases (20 y) organically-managed dairy farms with low Olsen STP (mean of 5.1 mg kg-1) were selected and compared with adjacent conventional dairy farm fields with long-term histories of synthetic P fertilizer application and higher STP levels (mean of 11.7 mg kg-1). Plant and soil (015 cm) samples were collected from each field in the 2009 growing season. Soil P forms were compared using solution 31P Nuclear Magnetic Resonance Spectroscopy (NMR). After correcting for orthophosphate diester degradation during NMR sample analysis, relative proportions of orthophosphate diesters, known to be readily mineralized forms of organic P, were greater in soils from organically managed systems (34% vs 26%), and were found to be correlated to legume content (r = 0.80, P
