Land and Food Systems, Faculty of
DSpace
UBCV
Damin, Sebastian
Master of Science - MSc
University of British Columbia
Strategies to promote capture and sequestration of CO₂ are necessary for achieving our climate mitigation goals in agriculture. Management of the orchard soil under crop trees to maximize storage of soil organic carbon (SOC), using diverse practices such as rootstock selection, application of organic mulches and cultivation of cover crops, have been proposed as low barrier approaches that remain under-studied. Increasing frequency of extreme drought events has also raised questions regarding irrigation costs/benefits associated with more intensive orchard soil management. To effectively evaluate use of orchard soil for long-term carbon storage we must also understand the resilience of newly stored SOC to future climate warming. My research investigated changes in SOC in the crop rows of a newly planted Okanagan apple (Malus domestica) orchard under diverse orchard soil management practices. I found that after two seasons, rootstock Malling 9 (M9) or Budogovski 9 (B9) did not affect total SOC content or concentration; woodchip mulch and a green mulch of woolly thyme (Thymus pseudolanuginosus) had limited effects on total SOC compared to conventionally managed bare soil, but particulate organic carbon was significantly higher under woolly thyme. I also evaluated tree water status and performance (as indicated by photosynthesis rates, and stomatal conductance) under each orchard soil management treatment during well-watered and simulated drought conditions. I found that in well-watered conditions, tree performance was similar among bare soil, woolly thyme and woodchip mulch treatments. During a week-long simulated drought, however, tree performance was better maintained under woodchip mulch but significantly reduced under woolly thyme compared to bare soil. Rootstock did not affect tree performance in either well-watered or drought conditions. Finally, I used Open Top Chambers (OTC) over two growing seasons to evaluate changes in the concentration of SOC in orchard drive rows under a simulated climate-warmed environment. I found that, while significantly altering the soil microclimate, OTC had minimal effect on total SOC pools or organic matter decay. Overall, this work has contributed to an improved understanding of orchard soil management as a sustainable climate change mitigation strategy, and the dynamics of, and methodologies used to estimate, SOC in orchard soils.
SOIL CARBON MANAGEMENT IN OKANAGAN APPLE (MALUS DOMESTICA) ORCHARDS by Sebastian Damin B.Sc., Simon Fraser University, 2021 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Soil Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2025 © Sebastian Damin, 2025 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Soil carbon management in Okanagan apple (Malus domestica) orchards Submitted by Sebastian Damin in partial fulfillment of the requirements for the degree of Master of Science in Soil Science. Dr. Sean Smukler, Associate Professor, Department of Land and Food Systems, UBC Supervisor Dr. Kirsten Hannam, Systems Agro-Ecologist, Agriculture and Agri-Food Canada Co-supervisor Dr. Mark Johnson, Professor, Department of Science, UBC Additional Examiner Additional Committee Members include: Dr. Thorsten Knipfer, Associate Professor, Department of Land and Food Systems, UBC Supervisory Committee Member Dr. David Ensing, Plant Community Ecologist, Agriculture and Agri-Food Canada Supervisory Committee Member iii Abstract Strategies to promote capture and sequestration of CO2 are necessary for achieving our climate mitigation goals in agriculture. Management of the orchard soil under crop trees to maximize storage of soil organic carbon (SOC), using diverse practices such as rootstock selection, application of organic mulches and cultivation of cover crops, have been proposed as low barrier approaches that remain under-studied. Increasing frequency of extreme drought events has also raised questions regarding irrigation costs/benefits associated with more intensive orchard soil management. To effectively evaluate use of orchard soil for long-term carbon storage we must also understand the resilience of newly stored SOC to future climate warming. My research investigated changes in SOC in the crop rows of a newly planted Okanagan apple (Malus domestica) orchard under diverse orchard soil management practices. I found that after two seasons, rootstock Malling 9 (M9) or Budogovski 9 (B9) did not affect total SOC content or concentration; woodchip mulch and a green mulch of woolly thyme (Thymus pseudolanuginosus) had limited effects on total SOC compared to conventionally managed bare soil, but particulate organic carbon was significantly higher under woolly thyme. I also evaluated tree water status and performance (as indicated by photosynthesis rates, and stomatal conductance) under each orchard soil management treatment during well-watered and simulated drought conditions. I found that in well-watered conditions, tree performance was similar among bare soil, woolly thyme and woodchip mulch treatments. During a week-long simulated drought, however, tree performance was better maintained under woodchip mulch but significantly reduced under woolly thyme compared to bare soil. Rootstock did not affect tree performance in either well-watered or drought conditions. Finally, I used Open Top Chambers (OTC) over two growing seasons to evaluate changes in the concentration of SOC in orchard drive rows under a iv simulated climate-warmed environment. I found that, while significantly altering the soil microclimate, OTC had minimal effect on total SOC pools or organic matter decay. Overall, this work has contributed to an improved understanding of orchard soil management as a sustainable climate change mitigation strategy, and the dynamics of, and methodologies used to estimate, SOC in orchard soils. v Lay Summary Conventional orchard management relies on herbicides to maintain bare soil under trees, maximizing water and nutrient availability for crop trees but leads to depletion of soil organic carbon (SOC). Organic mulches and cover crops offer low-barrier alternatives, yet their adoption remains limited, and their effects on irrigation practices are poorly understood. I investigated short-term impacts of woodchip mulch and a woolly thyme cover crop on SOC and water use in a young apple orchard. Woodchip mulch reduced tree water consumption but had no effect on SOC, whereas woolly thyme slightly increased both SOC and water use. I also evaluated SOC resilience under projected climate warming, using clear plastic mini greenhouses in drive rows of both drip and microsprinkler-irrigated orchards. After two growing seasons, warming had no measurable impact on SOC. Results suggest that surface management enhances SOC and reduces water use and SOC loss is not accelerated under short-term warming. vi Preface All projects were conducted in collaboration with other researchers, graduate students, and/or industry partners. In the following sections, I outline the contributions of those involved. Generative AI was not used in any aspects of this work. Chapter 2: The effects of orchard floor management on soil organic carbon and plant water relations in a young Okanagan apple (Malus domestica) orchard The orchard floor management and drought trials were established in a purpose-built apple (cv. ‘Ambrosia’) orchard located at the Agriculture and Agri-Food Canada Summerland Research and Development Centre (AAFC SuRDC). Kirsten Hannam (System Agro-Ecologist, AAFC SuRDC), and Shawn Kuchta (Research Technician) were responsible for site selection as well as the initial experimental design. Final study protocols were determined by the aforementioned individuals, Dr. Thorsten Knipfer (Professor, UBC), and myself. I collected data on soil organic carbon, tree biomass, midday stem water potential, photosynthesis, soil volumetric water content and irrigation volume from 2022 to 2023. Undergraduate students Stephanie Slamka, Jared Brown, Ivy Hiebert, Sarah O’Brien, also helped assist with data collection for this project. Chapter 3: The effects of warming on soil organic carbon dynamics in the drive rows of two Okanagan apple (Malus domestica cv. ‘Ambrosia’ and ‘Fuji’) orchards The open top chamber (OTC) climate warming trials were established in two apple (cv. ‘Ambrosia’, ‘Fuji’) orchards located at the Agriculture and Agri-Food Canada Summerland vii Research and Development Centre (AAFC SuRDC). Dr. Kirsten Hannam (System Agro-Ecologist, AAFC SuRDC), and Shawn Kuchta (Research Technician) were responsible for site selection as well as the initial experimental design. Final study protocols were determined by the aforementioned individuals, and Dr. David Ensing (Plant and Vegetation Ecologist, AAFC SuRDC), and myself. I collected data on soil organic carbon, organic matter decay, soil volumetric water content, and surface air temperature and humidity from 2022 to 2023. Undergraduate student Stephanie Slamka also helped assist with data collection for this project. viii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ................................................................................................................................ xi List of Figures ............................................................................................................................ xvii List of Abbreviations ............................................................................................................... xxiii Acknowledgements .................................................................................................................. xxiv Dedication ...................................................................................................................................xxv Chapter 1: Introduction ................................................................................................................1 1.1 Background ...................................................................................................................... 1 1.1.1 Soil Organic Carbon in Okanagan orchards ............................................................. 1 1.1.2 Orchard water use efficiency and plant water relations ............................................ 2 1.1.3 Soil Organic Carbon resilience to climate change .................................................... 4 1.2 Objectives ......................................................................................................................... 5 1.2.1 Objective 1: Effects of diverse orchard floor management on soil organic carbon and plant water relations in orchard crop rows. ...................................................................... 6 1.2.2 Objective 2: Effects of short-term soil warming, on soil organic carbon in orchard drive rows................................................................................................................................ 7 Chapter 2: The effects of orchard floor management on soil organic carbon and plant water relations in a young Okanagan apple (Malus domestica) orchard ..................................8 2.1 Introduction ...................................................................................................................... 8 ix 2.2 Methods .......................................................................................................................... 11 2.2.1 Study site ................................................................................................................. 11 2.2.2 Experimental design and treatments ....................................................................... 12 2.2.3 Horticultural management ...................................................................................... 15 2.2.4 Soil organic matter and fractionation ...................................................................... 15 2.2.5 Tree biomass and root excavations ......................................................................... 16 2.2.6 Plant water status and leaf gas exchange ................................................................ 17 2.2.7 Statistical approach ................................................................................................. 20 2.3 Results ............................................................................................................................ 22 2.3.1 The effects of ground cover and rootstock on soil organic carbon ......................... 22 2.3.2 The effect of ground cover and rootstock on particulate and mineral associated organic carbon ....................................................................................................................... 25 2.3.3 The effect of ground cover and rootstock on above and belowground woody biomass carbon...................................................................................................................... 29 2.3.4 The effects of ground cover treatment and rootstock on soil moisture, plant water status and leaf gas exchange ................................................................................................. 30 2.3.5 The effects of ground cover treatment on volumetric soil moisture during imposed drought 43 2.3.6 The effects of ground cover treatment and rootstock on plant water status and leaf gas exchange during imposed drought .................................................................................. 44 2.4 Discussion: ..................................................................................................................... 50 2.5 Conclusion:..................................................................................................................... 57 Chapter 3: The effects of warming on soil organic carbon dynamics in the drive rows of two Okanagan apple (Malus domestica cv. ‘Ambrosia’ and ‘Fuji’) orchards ........................59 3.1 Introduction .................................................................................................................... 59 3.2 Methods .......................................................................................................................... 62 3.2.1 Study site ................................................................................................................. 62 3.2.2 Soil microclimate .................................................................................................... 62 3.2.3 Soil organic carbon ................................................................................................. 63 3.2.4 Litter decay dynamics ............................................................................................. 64 3.2.5 Statistical approach ........................................................................................................ 66 x 3.3 Results ............................................................................................................................ 68 3.3.1 The effects of OTCs on air surface temperature and humidity ............................... 68 3.3.2 The effects of OTCs on soil volumetric water content ........................................... 77 3.3.3 The effects of OTCs on leaf litter decay ................................................................. 81 3.3.4 The effects of OTCs on soil organic carbon ........................................................... 85 3.3.5 The effects of OTCs on particulate and mineral associated soil organic carbon .... 87 3.4 Discussion: ..................................................................................................................... 93 3.5 Conclusion ...................................................................................................................... 98 Chapter 4: General Conclusions.................................................................................................99 Bibliography ...............................................................................................................................105 Appendix A .................................................................................................................................112 xi List of Tables Table 2.1. Irrigation water (mm) applied via drip emitters from May to September 2022 and 2023 in a young Okanagan apple (Malus domestica) orchard. ..................................................... 14 Table 2-2. Concentration (g kg-1) of soil organic carbon (0-15 cm and 15-30 cm depth) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch or woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Soil samples were collected in June 2022 and October 2023. ............. 22 Table 2-3. Two-way analysis of variance of concentration of soil organic carbon (g C kg-1; 0-15 cm and 15-30 cm depth) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. ............ 22 Table 2-4. Two-way analysis of variance of soil organic carbon content (SOC; Mg ha-1) of surface soils (0-30 cm) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. ............ 24 Table 2-5. Concentrations (g kg-1) of particulate organic carbon (POC) and mineral-associated organic carbon (MAOC) at the 0-15 cm soil depth in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. ................................................................................................................................ 26 Table 2-6. Two-way analysis of variance of the concentrations of particulate organic carbon (POC) and mineral associated organic carbon (MAOC) > 50 µm and MAOC < 50 µm at the 0-15 cm soil depth in the crop rows of a young apple (Malus domestica var. Ambrosia orchard) on M9 xii or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. Bold-faced type indicates a significant treatment difference (P < 0.05). ................................................................ 28 Table 2-7. Above and belowground biomass C (g tree-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Bench-grafted saplings were planted in November 2020 and samples were collected in October 2023. ................................... 29 Table 2-8. Two-way analysis of variance of above and belowground biomass C (g tree-1) of young apple trees (Malus domestica var. Ambrosia) planted to M9 or B9 rootstocks under bare soil control, woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments. ..................................................................................................................................... 29 Table 2-9. Volumetric soil moisture content (%; cm3 cm-3 *100) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground cover treatments, from May to September 2022 and 2023. .............................................................................................................................. 30 Table 2-11. One-way analyses of variance, by rootstock, of the effects of bare soil (control), woodchip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments on the stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1) and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under well-watered conditions, on August 3 and August 17 2022. One-way analyses were performed when significant ground cover treatment × rootstock effects were observed. .............................................................................................................................. 36 xiii Table 2-12. Two-way analysis of variance of stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground cover on six dates in 2022 under well watered soil conditions. .............................................................................................. 36 Table 2-13. Two-way analysis of variance of stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground covers on three dates in 2023 under well watered soil conditions. ..................................................................................... 42 Table 2-14. Analysis of variance of linear mixed effects model of the change in soil volumetric water content (%) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (black), woodchip mulch (brown) and woolly thyme (Thymus pseudolanuginosus) (green) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours) in 2023. The model included time (hr) since irrigation was shut off (T), ground cover treatment (G), and T x G as fixed effects, and plot and block as random effects. ............................................... 43 Table 2-15. Analyses of variance of linear mixed effects model of the change in 𝜑𝑠𝑡𝑒𝑚 (kPa), stomatal conductance (gs, mol H2O m-2 s-1), and photosynthesis rate (An, μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks and under bare soil, woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. The model included time (hr) since irrigation was shut xiv off (T), ground cover treatment (G), rootstock (R) and their interactions as fixed effects and plot and block as random effects. ......................................................................................................... 49 Table 3-1. Results of paired two-sample t-tests of mean (n=4) accumulated growing degree days (GDD; °C) in orchard drive row locations with (OTC) and without open-topped chambers (No-OTC) over the 2022 and 2023 growing seasons. T-tests were conducted on total accumulated GDD on the final day of OTC deployment in both years (October 31). ...................................... 69 Table 3-2. Two-way analysis of variance of mean (n=4) daily maximum temperature (°C) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. ......................................................................................................................................... 72 Table 3-3. Results of paired two-sample t-tests of mean (n=4) daily maximum temperature (°C) by month in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard.. .................................................................................................................. 73 Table 3-4. Two-way analysis of variance of mean (n=4) daily average humidity (%) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. ......... 76 Table 3-5. Results of paired two-sample t-tests of mean (n=4) daily average humidity (%) by month in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. ........................................................................................................................... 77 Table 3-6. Two-way analysis of variance of mean daily soil volumetric water content (%) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the xv 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). ............................................................................................................................. 80 Table 3-8. Results of paired two-sample t-tests of percent leaf litter mass remaining (%) after incubation in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8)............................................................................................... 84 Table 3-9. Analyses of variance of linear mixed effects model of the percent leaf litter mass remaining (%) after incubation in orchard drive row locations (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8).. ................................................................................. 84 Table 3-10. Results of paired two-sample t-tests of concentration of soil organic carbon (g C kg-1; 0-15 cm and 15-30 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023......................................................................................................... 87 Table 3-11. Results of paired two-sample t-tests of concentration of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (0-15 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023............................................................................................................................................... 90 Table 3-12. Results of paired two-sample t-tests of concentration of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (15-30 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in xvi locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023............................................................................................................................................... 93 xvii List of Figures Figure 2-1. Linear model used to estimate baseline stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) of young apple (Malus domestica var. Ambrosia) trees under well watered conditions at a given atmospheric vapor pressure deficit (VPD; kPa). Points are mean (n=4) with error bars to indicate one standard error. ........................................................................................................................ 19 Figure 2-2. Soil organic carbon content (SOC; Mg ha-1) of surface soils (0-30 cm) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control; grey boxes), woodchip mulch (brown boxes), or woolly thyme (Thymus pseudolanuginosus) (green boxes) ground cover treatments in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. .................................................................................................... 24 Figure 2-3. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm in the crop row soils (0-15 cm) of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (control; grey boxes), woodchip mulch (brown boxes), and woolly thyme (green boxes) ground cover treatments in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). ............................................. 27 Figure 2-4. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; umol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees under bare soil (black square), woodchip mulch (brown circle), and woolly thyme (Thymus pseudolanuginosus) (green triangle) ground cover treatments on six dates in 2022 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different xviii letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. Treatment differences are not shown when interactive effects were statistically significant (See Table 2-10 below). ...................................................... 34 Figure 2-6. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; umol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees under bare soil (black square), woodchip mulch (brown circle), and woolly thyme (Thymus pseudolanuginosus) (green triangle) ground cover treatments on three dates in 2023 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. .................................................................................. 40 Figure 2-7. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees on M9 (grey circle) or B9 (blue square) rootstocks on three dates in 2023 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. ....................................................................................................... 41 Figure 2-8. Mean soil volumetric water content (%; 0-30 cm) in the crop rows of a young apple (Malus domestica) orchard under bare soil (black), woodchip mulch (brown) and woolly thyme (Thymus pseudolanuginosus) (green) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Values along the y-axis are means (n = 6); data were collected using Campbell Scientific CS616 time domain reflectometry probes (0-30 cm) connected to a datalogger that logged measurements every ten minutes. ..................................................................................... 44 xix Figure 2-9. Change in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) , stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees under bare soil (control – black square), woodchip mulch (brown circle) and woolly thyme (Thymus pseudolanuginosus) (green triangle) treatments (n = 4) during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Error bars indicate one standard error of the mean. Values of 𝜑𝑠𝑡𝑒𝑚 are expressed as the difference between measured and expected values (kPa); the black hashed horizontal line indicates no difference between measured and expected (baseline) values............................................................................................................................ 47 Figure 2-10. Change in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) , stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 (grey circle) and B9 (blue square) rootstocks (n = 4) during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Error bars indicate one standard error of the mean. Values of 𝜑𝑠𝑡𝑒𝑚 are expressed as the difference between measured and expected values (kPa); the black hashed horizontal line indicates no difference between measured and expected (baseline) values. ........ 48 Figure 3-1 Dimensions of open-topped chambers. Design from Marion 1996. ........................... 65 Figure 3-2 Fully assembled Kestrel data logger unit housed in custom-made solar shield and suspended on a steel peg. .............................................................................................................. 65 Figure 3-3 Litter bag (5 cm x 5 cm internal space) constructed with 0.53µm nylon mesh and filled with dried apple leaf tissue, prior to incubation in the field. ............................................... 66 Figure 3-4. Mean (n=4) accumulated growing degree days (GDD; °C) in orchard drive row locations with (OTC; red datapoints) and without (No-OTC; grey datapoints) open-topped xx chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left panels) and a drip-irrigated orchard (right panels). * indicates significant treatment differences in accumulated GDD between OTC and No-OTC locations (P < 0.05) at the end of each growing season. ........................................................................................................................................... 69 Figure 3-5. Mean (n=4) daily maximum temperature (°C) (calculated by month) in orchard drive row locations with (OTC; red circles) and without (No-OTC; grey squares) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left) and a drip-irrigated orchard (right). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). ................................................................ 71 Figure 3-6. Mean (n=4) daily average humidity (%) per month in orchard drive rows locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left) and a drip-irrigated orchard (right). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). .................................................................................................. 75 Figure 3-7. Mean daily soil volumetric moisture content (%) per month in orchard drive row locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). .................................................................................. 79 Figure 3-8. Mean percent leaf litter mass remaining (%) after incubation in orchard drive row locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip- xxi irrigated orchard (n = 8). Error bars indicate standard error of the mean. * Indicates significant treatment differences (P < 0.05). .................................................................................................. 83 Figure 3-9. Concentration (g kg-1) of soil organic carbon (0-15 cm and 15-30 cm depth) in the drive rows of a microsprinkler-irrigated (top row; n=6) and a drip-irrigated (bottom row; n=8) apple (Malus domestica) orchard in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). ..... 86 Figure 3-10. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (0-15 cm depth) in the drive rows of mature microsprinkler-irrigated (top row; n=6) and drip-irrigated (bottom row; n=8) apple (Malus domestica) orchards in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023.. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). ..... 89 Figure 3-13. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (15-30 cm depth) in the drive rows of a microsprinkler-irrigated (top row; n=6) and a drip-irrigated (bottom row; n=8) apple (Malus domestica) orchard in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). ..................... 92 xxii Figure A1. Mean (n=6) percent change in fraction weight as a proportion of total soil mass of each soil size fraction isolated (Particulate Organic Matter; POM, Sand; F > 50 µm, Silt & Clay; F < 50 µm) between samples collected in June 2022 and October 2023 in soils under woolly thyme (green triangles), woodchip mulch (brown circles), and bare soil control (black squares)...................................................................................................................................................... 112 Figure A2. Mean (n=6) percent change in C concentration of each soil size fraction isolated (Particulate Organic Matter; POM, Sand; F > 50 µm, Silt & Clay; F < 50 µm) between samples collected in June 2022 and October 2023 in soils under woolly thyme (green triangles), woodchip mulch (brown circles), and bare soil control (black squares). ................................... 113 xxiii List of Abbreviations AAFC Agriculture and Agri-Food Canada ANOVA analysis of variance An Photosynthesis Rate E Transpiration Rate ET Evapotranspiration F > 50 µm Fraction greater than 50 microns – Sand F < 50 µm Fraction less than 50 microns – Silt & Clay GDD Daily Growing Degrees gs Stomatal Conductance MAOC Mineral Associated Organic Carbon OTC Open Top Chamber 𝜑𝑠𝑡𝑒𝑚 Midday Stem Water Potential POC Particulate Organic Carbon POM Particulate Organic Matter SOC Soil Organic Carbon Su-RDC Summerland Research and Development Centre TDR Time Domain Reflectometry VPD Vapor Pressure Deficit VWC Volumetric Water Content xxiv Acknowledgements This thesis resulted from the collaboration between many wonderful people for whom without their help, this work would not have been possible. Firstly, I would like to thank my supervisor and co-supervisor, Drs. Sean Smukler and Kirsten Hannam, for their continual support and mentorship throughout these years of hard work. Your kindness, and enthusiasm has fostered my passion for research. To my committee: Drs. Thorsten Knipfer, and David Ensing, thank you for your guidance and time during these past years. I am also thankful for the assistance from many undergraduate students including: Stephanie Slamka, Jared Brown, Ivy Hiebert, and Sarah O’Brien. I am grateful to the technical staff and researchers at Agriculture and Agri-Food Canada (Su-RDC), particularly Shawn Kuchta, and Paige Munro as well as Drs. Mehdi Sharifi, and Thomas Forge. I am appreciative of the support from fellow graduate students in the faculty of Land and Food Systems and the Sustainable Agricultural Landscapes Lab at the University of British Columbia. xxv Dedication To my parents, who’s unconditional support and devotion has enabled me to pursue my research endeavours. 1 Chapter 1: Introduction 1.1 Background 1.1.1 Soil Organic Carbon in Okanagan orchards Agricultural strategies to promote the accumulation of soil organic matter and thus the sequestration of carbon dioxide (CO2) as soil organic carbon (SOC) are becoming increasingly popular for their potential to improve soil health and support climate change mitigation. Agricultural soils are an important potential carbon (C) sink that could remove sizeable amounts of CO2 from the atmosphere and store it, inhibiting its ability to contribute to climate warming. Therefore, collective action toward maximizing storage of CO2 as SOC may play a critical role in our fight against climate change. Proposals such as the “4 per 1000” initiative, which claim that an increase in global agricultural SOC stocks by 0.4% per year would effectively halt current increases in CO2 levels (Rumpel et al. 2019) have helped catalyzed a global interest in SOC. Increasing soil organic matter also has numerous benefits for soil quality and crop productivity (Neilsen et al. 2003). Soils high in organic matter have improved water holding capacity and nutrient retention which can reduce irrigation demand and dependency on synthetic fertilizers and improve yields, thereby reducing the environmental impact of agriculture (Neilsen et al. 2003, Hannam et al. 2016, Fu et al. 2021). In typical orchard management, the use of herbicides and/or surface tillage to maintain a strip of bare soil under the crop tree are detrimental to SOC accumulation (Scandellari et al. 2016, Midwood et al. 2020; Kan et al. 2022) and, as a result, orchard crop row soils tend to be depleted in SOC. The width of herbicide strips of bare soil can range from 1 to 3 meters, accounting for 25% to 50% of total orchard land area (Atlantic Committee on Tree Fruit 2013). 2 This represents a sizeable amount of bare soil that is currently not being utilized to capture and store atmospheric CO2 as SOC. By contrast, the ‘drive’ rows between the crop rows usually support a continuous and permanent cover of various weeds and grasses; a recent survey of orchards in the Okanagan Valley, BC (Canada) found that SOC tends to be significantly higher in drive rows than in crop rows (Midwood et al. 2020). Several methods for increasing SOC in orchard crop rows have been proposed, including applications of organic mulch (Neilsen et al 2003, Granatstein & Mullinix 2008, Neilsen et al. 2014, Hannam et al. 2016) and cover crops (Kemper et al. 2020; Saleem et al. 2020, Jansson et al. 2021, Marks et al. 2022), or selection of rootstocks with more abundant root biomass or more rapid root turnover (Bintarti et al. 2020). 1.1.2 Orchard water use efficiency and plant water relations In semi-arid regions such as the Okanagan Valley, which are increasingly experiencing periods of extreme drought and emergency water restrictions, methods for increasing SOC must also consider irrigation water demand. While organic surface mulches have been shown reduce irrigation demand by suppressing evaporation from the soil surface (Eissenstat et al. 2018), the effect of cover crops on soil moisture is not well understood: cover crops may increase soil moisture loss overall by promoting transpiration of water through cover crop leaf surfaces but may also simultaneously reduce soil moisture loss by suppressing evaporation from the soil surface (Blanco-Canqui & Ruis 2020). Plant water status indicators, such as midday stem water potential (𝜑𝑠𝑡𝑒𝑚) and stomatal conductance (gs) are commonly used to assess orchard tree performance under alternative irrigation regimes. Stem water potential is the direct measure of water tension within the plant; stem water potential (expressed as kPa of pressure), will increase when the soil is dry because 3 there is less water available for plant roots to extract from the soil (Jeanguenin et al. 2017). Stomatal conductance (gs) is a measure of the rate of H2O vapor lost through plant stomata, which is directly affected by the degree of plant stomatal opening and the availability of water to the plant. During periods of water stress, plant stomata close in an attempt to conserve water and prevent desiccation (Jeanguenin et al. 2017). Measures of photosynthesis rate (An) are also important for evaluating overall crop performance because An can increase or decrease with stomatal opening and because the rate of photosynthesis has a direct effect on the amount of newly fixed plant C that could contribute to SOC accumulation. Trees planted under applications of surface mulches have been shown to maintain higher 𝜑𝑠𝑡𝑒𝑚, gs and An values compared to bare soil under deficit irrigation (Eissenstat et al. 2018, Sharifi et al. 2022). To our knowledge, studies evaluating the effect of under-tree cover crops on plant water relations have not been conducted in Okanagan Valley orchards, although, there has been research conducted under grapevines (Abad et al. 2020). Given that rootstock genotypes have been shown to affect the performance of apple cultivars to water limitations (Valverdi & Kalcsits 2021, Tworkoski et al. 2016), the effect of ground cover management practices (e.g., applications of surface mulch or cover cropping in the tree row) on plant water relations may also be mediated by rootstock. While previous studies have focussed on instantaneous measures of plant water relations under alternative ground cover management practices or rootstocks, none have evaluated the effect of ground cover management practices or rootstock type on changes in plant water relations in response to changes in soil water availability over time. 4 1.1.3 Soil Organic Carbon resilience to climate change Recent climate change projections indicate that average summer temperatures in the Okanagan Valley are expected to rise by approximately 3-6 °C by 2050 and by 5-9 °C by 2080 (RDCO Climate Report 2020). As stated earlier, SOC in the drive rows of Okanagan orchards is usually higher than in tree rows (Midwood et al. 2020). However, SOC can be vulnerable to degradation under climate warming as increased temperatures can enhance microbial decomposition (Lal 2012, Qafoku 2015, Hartley et al. 2021, Verbrigghe et al. 2022,); the resilience of newly captured SOC in orchard drive rows to a warming climate is unknown. Open-topped chambers (OTC) have been used to explore the effect of increased surface temperatures (e.g., by 2-3°C) on vegetation and soil in previous studies (Marion et al. 1997, Hollister et al. 2023), but they have been used most prominently in arctic and alpine regions through the International Tundra Experiment Network (ITEX) (ITEX Manual; Center & Copenhagen 1996) OTC have yet to be widely deployed in agricultural settings. OTC essentially function as small greenhouses; their clear walls allow light energy to freely enter while preventing heat from radiating away, and their open top facilitates some ventilation. The greatest effect on temperature occurs during the daytime, warming air temperatures above ambient whilst sunlight is not obstructed from reaching OTC; little to no effect on air temperature is expected during overnight hours or when sunlight is absent (Marion et al. 1997). These structures can, therefore, be used to explore how increased warming might impact SOC dynamics. While there has been much work on understanding SOC dynamics, recent efforts have shown that recalcitrance is related to C fraction size or density. SOC is often divided into two fractions: Particulate organic C (POC) and mineral-associated organic C (MAOC) (Lavallee et al. 2020). POC is composed of large, complex organic molecules derived from the physical 5 breakdown of plant and animal biomass (Villarino et al. 2021). Organic materials in POC are readily available to decomposers and, with no theoretical limit to the amount of POC that the soil can hold, the size of the POC pool is only restricted by the volume of freshly available biomass and the presence of microbes and detritivores able to degrade it (Cotrufo et al. 2015). The problem with using POC for long-term C storage is that it is susceptible to degradation (Lavallee et al. 2020). In contrast, MAOC is composed of microscopic organic molecules that are closely associated with mineral particles in the soil and are derived from direct cellular deposition; formation of MAOC is limited by the presence of silt and clay surfaces in the soil that are not already saturated with C (Lavallee et al. 2020, Villarino et al. 2021). MAOC has been labelled as the “stable fraction” because it tends to be resistant to degradation (Lavallee et al. 2020). Storing C in the stable MAOC fraction is considered preferable because POC is believed to be more susceptible to accelerated degradation from future climate warming (Lavallee et al. 2020, Villarino et al. 2021). That being said, management strategies that promote MAOC versus POC formation in soil are not currently well understood, particularly in the context of warming temperature, though they may prove critical to long-term SOC sequestration efforts. 1.2 Objectives The main objectives for the research included in the thesis were to: 1. Examine the effects of diverse orchard floor management strategies (including mulching, cover cropping, and rootstock selection) on soil organic carbon and plant water status in the crop rows of a young Okanagan apple orchard. (Chapter 2) 6 2. Examine the effects of short-term soil warming, using open-top chambers, on soil organic carbon in the drive rows of two Okanagan apple orchards. (Chapter 3) 1.2.1 Objective 1: Effects of diverse orchard floor management on soil organic carbon and plant water relations in orchard crop rows. The first objective focussed on determining and comparing the effects of diverse orchard floor management including mulching, cover cropping, and rootstock selection on soil organic carbon, plant water status and leaf gas exchange. To accomplish this, soil organic carbon including: POC and, MAOC, soil moisture, midday stem water potential, stomatal conductance and photosynthesis rate were measured. The following questions were formulated to accomplish this objective: Question 1.1: Over two growing seasons, will mulching with wood chips or cover cropping with woolly thyme increase SOC in the tree rows of an Okanagan orchard relative to a control treatment in which soils are kept bare using herbicide? Question 1.2: Over two growing seasons, will SOC differ in the tree rows of an apple orchard planted to Malling 9 (M9) or Budogovski (B9) rootstocks? Question 1.3: Over two growing seasons, will mulching with wood chips or cover cropping with woolly thyme affect tree water status and leaf gas exchange, relative to a control treatment in which soils are kept bare using herbicide? Question 1.4: How does tree water status and leaf gas exchange differ between rootstocks M9 and B9? 7 1.2.2 Objective 2: Effects of short-term soil warming, on soil organic carbon in orchard drive rows. The second objective focussed on determining the effects of short-term soil warming, using open-top chambers, on soil organic carbon in the drive rows of two mature Okanagan apple orchards. To accomplish this, soil moisture, surface air temperature and humidity, as well as soil organic carbon including POC and MAOC, and organic matter decay were measured. The following questions were formulated to accomplish this objective: Question 2.1a: Can the microclimate of orchard drive row soils be altered through the use of OTC placed on the soil surface? Question 2.1b: Will changes in microclimate induced by OTC differ in an orchard irrigated by micro-sprinklers and an orchard irrigated by drip emitters? Question 2.2a: Can changes in the microclimate of orchard drive row soils, imposed using OTCs over two growing seasons, cause changes in SOC dynamics, as indicated by the rate of litter decay and the concentrations of SOC, POC and MAOC (0-15 cm and 15-30 cm)? Question 2.2b: Will changes in SOC dynamics cause by OTCs, as indicated by the rate of litter decay and the concentrations of SOC, POC and MAOC (0-15 cm and 15-30 cm) differ in an orchard irrigated by micro-sprinklers and in an orchard irrigated by drip emitters? 8 Chapter 2: The effects of orchard floor management on soil organic carbon and plant water relations in a young Okanagan apple (Malus domestica) orchard 2.1 Introduction Large amounts of carbon (C) can be added to the surface of the soil in the form of organic-rich mulches such as woodchips, paper pulp or straw. The decomposition of mulches returns organic C to the soil over time (Neilsen et al 2003, Neilsen et al 2014). This method has been shown to be somewhat inefficient, however, because less than 30% of total mulch biomass is retained as soil C, with the majority being lost through microbial respiration as CO2 (Castellano et al. 2015). Most of the organic C retained in the soil through the application of organic mulches is stored as soil organic C (SOC) in the particulate organic C (POC) fraction (Villarino et al. 2021), which is believed to be more susceptible to decay than mineral-associated organic C (MAOC), which is bound within aggregates or on mineral particle surfaces (Cotrufo et al. 2020). Though mulch-derived C slowly becomes incorporated into the soil via the action of soil biota and farming activities, it tends to remain concentrated near the soil surface, where it is more likely to be lost to erosion or decay (Dungait et al. 2012, Lavallee et al. 2020). The use of cover crops could be a more effective method of increasing SOC (Marks et al. 2022, Villarino et al. 2021, Kemper et al. 2020; Saleem et al. 2020) because each year, small amounts of cover crop biomass C is added to surface soils through the decomposition of aboveground leaves and stems and belowground root turnover and rhizodeposition (Oburger and Jones 2018, Jacobs et al. 2020). Unlike decomposition of larger pieces of plant debris, the simple organic molecules derived from rhizodeposition are primarily stored in the stable MAOC 9 fraction, increasing their potential for retention in the soil (Villarino et al. 2021). Due to the long establishment time for some cover crops and the slow rhizodeposition process, however, accumulation of MAOC via cover cropping may be slow (Villarino et al. 2021). Common orchard cover crops include clover (Trifolium species), ryegrass (Lolium species), lentils (Lens species) and fescue (Festuca species) (Magdoff et al. 2021). Woolly thyme (Thymus pseudolanuginosus) is not common as a cover crop in orchards but is widely used as a ground cover in public gardens due to its ability to supress weed growth, and resist foot traffic. This perennial evergreen species grows in dense mats with a maximum height of 5 centimeters (Klett et al. 2007). The use of woolly thyme as an orchard cover crop has been proposed in previous studies because of its low canopy height and production of volatile secondary metabolites which discourage colonization by rodent pests (Hansen et al. 2015, Sullivan et al. 2018). It is of particular interest to us because of its low water needs. Woolly thyme also produces small pink flowers in the spring which are highly attractive to honeybees and other important pollinators (Wiese et al. 2018). Although some growers are experimenting with the use of cover crops in the orchard crop row, there remains considerable resistance to this practice because of the need for higher maintenance and the potential for competition between the cover crop species and the crop trees. Instead, the use of more vigorous rootstocks or rootstocks that preferentially produce greater root biomass could, like cover crops, be used to increase SOC via rhizodeposition from living root tissue and decomposition of root biomass. Rootstocks also have the ability to affect above ground inputs of C through their influence on cultivar vigor. Modern commercial apple (Malus domestica) production uses grafted trees composed of a scion (the varietal of choice) and a rootstock to dwarf scion vigor. Most research on apple rootstock properties has focused on plant 10 health attributes and water-use efficiency with little to no consideration of soil C storage potential (Bintarti et al. 2020, Valverdi & Kalcsits 2021). The dominant apple rootstock in the Okanagan Valley, Malling 9 (M9), has been chosen for its high yield efficiency and ability to withstand cold winter temperatures. However, it may not be the best rootstock for C sequestration (Bintarti et al. 2020). Rootstock Budagovsky 9 (B9) is slightly more vigorous than M9 and, thus, could be expected to increase SOC via greater biomass production (Zhou et al. 2020). Direct comparisons of root biomass have not been made between these rootstocks. To our knowledge there have been no studies that have directly compared the effects of a surface mulch and a cover crop on SOC accumulation, particularly in combination with different rootstocks. There is also limited understanding of how groundcover management, rootstocks or their interactions affect orchard water dynamics. The strength of midday 𝜑𝑠𝑡𝑒𝑚 in determining plant water stress and informing plant-based irrigation management practices for perennial woody crops has increased with the use of reference baselines (Shakel et al. 2021). 𝜑𝑠𝑡𝑒𝑚 reference baselines account for the changing air vapor pressure deficit (VPD), whose influence in regulating plant water relations is as significant as changing soil water conditions (Mencuccini et al. 2024). The use of a reference baseline allows for comparisons of 𝜑𝑠𝑡𝑒𝑚 across dates which has not been possible with instantaneous measures without VPD. Currently no refence baseline for midday 𝜑𝑠𝑡𝑒𝑚 exists for apple orchards in the Okanagan Valley, which is necessary for evaluating changing soil water availability under alternative ground cover management practices or rootstock varieties. To address these research gaps, I conducted an experiment over two production seasons to examine the effects of diverse orchard floor management strategies (including mulching, cover cropping, and rootstock selection) on SOC and plant water status in the crop rows of a 11 young Okanagan apple orchard. I hypothesized that both ground cover treatments would increase SOC relative to the control treatment. However, due to the large amount of biomass being added immediately, I expected that wood chip mulch would increase overall SOC more strongly than a cover crop of woolly thyme which requires a long establishment time, particularly in the POC fraction. I expected that treatment differences would be apparent by the end of the second growing season at the 0-15 cm soil depth. Because B9 rootstocks are expected to produce more root biomass, I expected that soils planted to B9 rootstock would increase SOC more rapidly than those planted to M9 rootstockand that treatment effects would be most apparent in the POC fraction. Finally, I hypothesized that ground cover treatments and rootstock selection would not affect 𝜑𝑠𝑡𝑒𝑚, An, E and gs as long as irrigation was managed to maintain equivalent soil volumetric water content across treatments. However, if irrigation was cut off for a period of time, I expected that 𝜑𝑠𝑡𝑒𝑚 would increase (indicating lower water availability), and rates of An, E, and gs would decrease most quickly in the woolly thyme treatment, followed by the control treatment relative to the wood chip mulch treatment; similarly, I expected that 𝜑𝑠𝑡𝑒𝑚 would increase and An, E, and gs would decrease more quickly in trees on M9 than on B9 rootstock. 2.2 Methods 2.2.1 Study site This work was conducted at Agriculture and Agri-Food Canada’s (AAFC) Summerland Research and Development Centre (SuRDC), in the Okanagan Valley, BC (Canada) (49°34′N, 119°39′W; 450 m above sea level). Between 1981 and 2010, this region experienced cool winters (December to February temperature: -0.5 °C), warm summers (June to August mean 12 temperature: 20 °C), and low annual precipitation (300mm annualy) (Environment Canada 2022).The research orchard was planted in a Skaha loamy sand, characterised as a brown chernozemic soil derived from fluvio-glacial material, with a thin veneer (up to 50 cm) of sand over gravel (BC Ministry of Environment 1986). 2.2.2 Experimental design and treatments The research orchard was planted in mid-November 2020 and consisted of eight rows of trees with 60 trees per row: the two outer rows were designated as ‘guard’ rows and the remaining inner six rows were designated as measurement rows. The experiment was designed as a split-plot experiment, replicated six times (each measurement row was considered one block), with ‘ground cover treatment’ as main plots and apple rootstock as split-plots. Each row was divided into three ‘ground cover treatment’ plots (18 m wide x 1.5 m deep), to which one treatment was randomly assigned. Within each ‘ground cover treatment’ plot, four 10-tree split plots (eight measurement trees plus one guard tree at each end of the plot) were randomly assigned to one of two factorial combinations of apple rootstock (M9 or B9) planted to the Ambrosia variety. The ground cover treatments were as follows: (1) a control treatment, in which the soil surface was managed using the typical grower practice of regular applications of herbicide (usually glyphosate); (2) a wood chip mulch treatment, to which herbicide was periodically spot-applied to augment hand-pulling of weeds; (3) a cover crop treatment planted with plugs of woolly thyme (Thymus pseudolanuginosus), in which weed control was managed by hand-pulling. The wood chip mulch consisted of shredded bark and wood chips (primarily Pinus contorta and Picea glauca) purchased from a local garden supply centre. Mulch was manually applied on April 11, 2022 (1,200 L plot-1) to a depth of approximately 10 cm over a 1.5 m wide x 13 18 m long strip centred on the tree row within each mulched plot. The woolly thyme was purchased from a local nursery and planted on April 27, 2022 (31 one-inch plugs plot-1); thyme was allowed to grow over a 1.5 m wide x 18 m long strip centred on the tree row within each woolly thyme plot. Mulch was reapplied (1,000 L plot-1) to a depth of 10 cm and thyme was fill-planted to cover exposed soil in May 2023. Trees were irrigated with a single line of drip emitters running parallel to the tree row down the centre of each plot and suspended on wires approximately 30 cm above the soil surface. One 4 L hr-1 emitter was positioned mid-way between each tree plus an additional emitter was place near the outer boundary of each plot for a total of eleven emitters per ten-tree plot. Due to the physical constraints of orchard irrigation management, it was impractical to individually control irrigation on a plot level; instead, irrigation was managed the same for each ground cover treatment. Control treatment plots were irrigated to replace 100% of the water lost to evapotranspiration (ET), which was measured as described in Neilsen et al. (2008). Briefly, daily ET was estimated using a Model E ETgage electronic atmometer with a No. 54 canvas cover (designed for agricultural crops; ETGage Co., Loveland, Colorado), linked to irrigation valves via a CR1000 datalogger (Campbell Scientific, Logan, Utah) (Parchomchuk et al. 2000). Plant water demand was calculated from the daily ET measured by the atmometer until midnight each day (minus any precipitation > 5 mm) using a daily crop coefficient calculation for apple (van der Gulik et al. 2011, derived from Allen et al. 1998), and an adjustment for overestimation of ET as described in Neilsen et al. (2008). The actual quantity of applied irrigation water (in mm) was calculated based on a 0.675 m2 area around each tree (0.45 m between trees and 0.75 m on each side of the tree row) (Table 2.1). Irrigation was applied once every second day, as required, starting at 4 AM. 14 Volumetric water content (VWC) (0-30 cm) was measured in 18 of the 60 plots using 30-cm time domain reflectometry (TDR) probes (CS616; Campbell Scientific; Logan, Utah) connected to a CR 1000 Campbell Scientific datalogger (Campbell Scientific; Logan, Utah) (Table 2.1). Six TDR probes were positioned in control plots (one per experimental block), six were positioned in woodchip mulch plots (one per experimental block) and six were positioned in woolly thyme (one per experimental block). Given that the TDR probe cables were only 30 m long, plots were selected for instrumentation, in part, based on their proximity to the datalogger and, as such, TDR probes were not distributed evenly between rootstock varieties. Soil moisture data were used to schedule irrigation in treatments (2) and (3) to maintain the same VWC as measured in the control treatment plots. Irrigation scheduling was initiated in May 2021 but was supplemented by 20-30 minutes of overhead sprinkler irrigation approximately once per week from early June to late August 2021 (to facilitate the establishment of drive row vegetation). In 2022 and 2023, all irrigation water was applied via drip emitter and scheduled as described above, except for an initial ‘wet-up’ in early May 2022 and 2023 (to flush irrigation lines and wet the soil for the start of the growing season) and immediately after application of broadcast fertilizers in June 2022 and May 2023 (to wash nutrients off the soil surface and into the soil). Table 2.1. Irrigation water (mm) applied via drip emitters from May to September 2022 and 2023 in a young Okanagan apple (Malus domestica) orchard. 2022 2023 Control Woodchip mulch Woolly thyme Control Woodchip mulch Woolly thyme Irrigation water applied (mm) May 22.7 21.4 19.9 15.5 4.4 3.3 June 24.1 10.2 20.6 37.6 10.2 31.4 July 37.4 13.5 28.8 45.1 19.4 42.8 August 64.2 26.5 33.4 51.6 22.3 42.4 September 44.0 16.5 29.6 34.7 13.5 21.9 Total 192.4 76.1 132.3 184.5 69.8 141.8 15 2.2.3 Horticultural management Tree nutrition, pests and diseases were managed according to industry standards for a newly planted apple orchard (BC Fruit Grower’s Association 2024). In 2022 and 2023, several trees in blocks 1 and 2 were severely affected by fire blight (Erwinia amylovora); affected trees were either heavily pruned or replaced to contain the fire blight infestation, but sufficient healthy trees remained to continue the experiment. 2.2.4 Soil organic matter and fractionation Composite soil samples were collected at two depths (0-15 cm and 15-30 cm) from each of the 36 plots at the beginning of the growing season (June 17) in 2022 and at the end of the growing season (October 6) in 2023. By Autumn 2023, some of the wood chip mulch had become incorporated into the soil; particular care was taken to scrape the mulch away from the mineral soil surface during sampling. Similarly, woolly thyme covered approximately 80% (measured with the Canopeo © canopy cover measurement tool; Oklahoma State University Department of Plant and Soil Sciences, Stillwater, Oklahoma) of the ground surface within the woolly thyme plots but care was taken to peel the living biomass away from the soil during sample collection. Each composite soil sample was comprised of six individual samples: two samples each were collected from the west and east sides of the tree row (four samples in total) and two additional samples were collected from the centre of the tree row. Composite soil samples were mixed by hand, air-dried in the lab for one week and then sieved (2 mm) to remove coarse fragments and large plant debris. In the spring of 2024 soils were sampled for bulk density at both the 0-15 cm and 15-30 cm depth via the core method. Brass cylinders of known volume (~ 135 ml) were driven into the soil to extract undisturbed soil cores. The weights of the 16 soil in the cores after coarse fragments were removed were determined after drying in an oven for at least 24 hours at 65 °C to calculate bulk density. A 50 g subsample of soil (0-15 cm depth only) from all 36 plots was separated into 3 fractions: (1) POC between 0.05 and 2 mm (POC > 50 µm), (2) MAOC between 0.05 and 2 mm (sand-sized; MAOC > 50 µm), and (3) MAOC < 0.05 cm (silt + clay-sized; MAOC < 50 µm) using a method derived from the Par+Den5 procedure described in Poeplau et al. (2018) except that only three fractions (rather than five) were obtained. A 1.7 g cm-3 solution of sodium iodide (NaI) was employed to facilitate separation of the less dense POC > 50 µm fraction from the MAOC > 50 µm. The total C content of each soil fraction plus unfractionated soils was measured using a LECO© CHN 628 series combustion analyzer (LECO Corporation, St. Joseph, Michigan) after grinding in a ball mill. 2.2.5 Tree biomass and root excavations In October 2023 one tree in 24 of the 36 plots was randomly selected for determination of above and belowground biomass. Measurements were not conducted in the two blocks that had been heavily affected by fireblight. A 60 cm x 60 cm square of soil was excavated to 30 cm depth around the base of each selected tree. All excavated soil was laid on a clean tarp and roots were retrieved by hand sieving (2 mm). Aboveground tree roots were sometimes observed in the mulch (i.e., between the mineral soil surface and the mulch surface above). These roots were carefully separated from the mulch by hand. Belowground roots were carefully rinsed with tap water to remove mineral soil. Aboveground biomass from the rootstock (extending approximately 15 cm above the soil surface) was counted as aboveground biomass. Above and belowground biomass samples were oven-dried at 65°C to constant mass and weighed. Samples were then chopped into small pieces with pruners; an aliquot of each sample was ground to a fine 17 powder in a ball mill prior to determination of organic C and N content using a LECO© CHN 628 series combustion analyzer (LECO Corporation, St. Joseph, Michigan). 2.2.6 Plant water status and leaf gas exchange Plant water status (𝜑𝑠𝑡𝑒𝑚) and leaf gas exchange (An, gs) were measured once per month in each of the 36 plots in June, July and August 2022 and 2023. Leaf gas exchange measurements are weather dependent, so measurements were only completed on clear, sunny days, between 9 AM and noon. It was not possible to complete these measurements on 36 plots within this three-hour period so, in order to ensure that all measurements were taken before noon in 2022, plant water status was measured in three of the six blocks (18 plots) every two weeks (block 1, 3 and 5 on one measurement day each month and blocks 2, 4 and 6 two weeks later). In 2023, two of the blocks had been heavily affected by fireblight so measurements were conducted on the remaining four blocks (24 plots) on a single measurement day every month. On each measurement day, two trees were randomly selected for 𝜑𝑠𝑡𝑒𝑚 measurements from each plot. Two fully formed leaves from the middle third of new extension growth were chosen from each of the two selected trees. Selected leaves (a total of four leaves per plot) were wrapped with black plastic and aluminum foil. After 20 minutes, wrapped leaves were removed from the tree and compressed using N2 gas in a PMS 610 (PMS Instrument Company, Albany, Oregon) pressure chamber, until fluid began to emerge from stem. The pressure at which this fluid became visible was recorded (kPa). On each measurement day, photosynthesis (An), and stomatal conductance (gs) were measured on the same trees used for 𝜑𝑠𝑡𝑒𝑚 monitoring. Only fully formed leaves in the middle third of new extension growth and in direct sunlight were selected. Analyses were conducted using an LCi-SD Leaf Respiration Analysis System (ADC BioScientific Ltd, Hoddesdon, United 18 Kingdom). During measurements, the leaves were clamped in the sample chamber and held steady in their natural orientation for approximately 2 minutes until substomatal CO2 (Ci) stabilized (i.e., fluctuating ±5 μmol mol-1). As mentioned above, irrigation was managed with the goal of maintaining the same volumetric moisture content across all ground cover treatments (Table 2.1). As such, the effect of ground cover treatment on plant water relations was not expected to be significant during regular irrigation function. To determine how ground cover treatment and/or scion/rootstock combination affected trees’ response to changes in water availability (e.g., as might occur during emergency drought restrictions), irrigation was shut off to all trees for approximately one week in July and in August 2023. Stem water potential and leaf gas exchange were measured as described above three times per week during the imposed drought period; monitoring was conducted between 9AM and 12PM in four of the six blocks. Because blocks 2 and 3 were more heavily affected by the fire blight outbreak, blocks 1, 4, 5 and 6 were monitored for this part of the experiment. I used a novel approach to correct stem water potential (𝜑𝑠𝑡𝑒𝑚) for the influence of changing atmospheric vapor pressure deficit (VPD) on individual measurement days during the imposed drought period in 2023: the availability of water to trees was evaluated by calculating the difference in stem water potential measured during the drought and that measured during well-watered conditions under the same vapor pressure deficit conditions (considered the baseline (expected) stem water potential). The stem water potential baseline provides an estimate of the stem water potential that would be expected in well-watered trees under similar conditions (i.e., air vapor pressure deficit (VPD; temperature and relative humidity). Stem water potential baseline data were generated from 𝜑𝑠𝑡𝑒𝑚 measurements in trees planted to the bare soil (control) 19 treatment on both rootstock varieties (M9, B9) during regular irrigation functioning in the summer of 2023. Average air temperature and relative humidity data from Environment and Climate Change Canada’s Summerland weather station (49°57’N, 119°65’W) were used to calculate VPD (Environment and Climate Change Canada 2023). In order to prevent long-term damage to the trees, irrigation was turned back on when stomatal conductance in at least some of the trees had declined by 50% relative to that measured in well-watered conditions (Dr. Thorsten Knipfer personal communication; June 2023). Only the data from the second drought period imposed in August will be presented because 6 mm of rain fell during the drought period imposed in July and, as a result, the trees showed no change in plant water status. That being said, wildfire smoke encountered on August 16, 2023, influenced the first few days of the second imposed drought period; by the last measurement day, the skies were again clear. Figure 2-1. Linear model used to estimate baseline stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) of young apple (Malus domestica var. Ambrosia) trees under well watered conditions at a given 20 atmospheric vapor pressure deficit (VPD; kPa). Points are mean (n=4) with error bars to indicate one standard error. 2.2.7 Statistical approach All statistical analyses were performed using the R programming language in RStudio® 2024.04.2+764 (R Core Team 2024). Gaussian model and ANOVA assumptions of normality and homoscedasticity were visually verified using residual plots. Statistical analyses were performed using the ‘lmerTest’ (Kuznetsova et al. 2017), and ‘emmeans’ (v1.10.3, Lenth et al. 2024) packages. P-values of ≤0.05 were considered significant. Soil organic carbon A two-way analysis of variance (ANOVA) for a randomized complete block design replicated six times with split-plots (rootstock) nested within main plots (ground cover treatment) was used to assess the effect of ground cover treatment, rootstock and their interactive effects on total soil C concentration (0-15 cm and 15-30 cm; g kg-1) and content (0-30 cm; Mg ha-1), and the concentration of C in the individual soil fractions of POC, MAOC > 50 um, and MAOC < 50 um (0-15 cm and 15-30 cm; g kg-1). Two-way analysis of variance (ANOVA) was also used to assess the effect of ground cover treatment, rootstock and their interactive effects on above and belowground biomass C (g tree-1). When treatment effects were statistically significant (p ≤ 0.05), Tukey’s HSD post-hoc tests were used to conduct pairwise comparisons between individual treatments. When significant interaction effects between groundcover and rootstock treatments were present, the effect of groundcover was analyzed separately by rootstock. Plant water status & leaf gas exchange 21 For measurements conducted during regular irrigation function, a two-way ANOVA for a randomized complete block design replicated three times (2022) or four times (2023) with split-plots (rootstock) nested within main plots (ground cover treatment) was used to assess the effect of ground cover treatment, rootstock and their interactive effects on stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1) and photosynthesis rate (An; μmol CO2 m-2 s-1); separate analyses were conducted for data collected on individual days over the growing season. When treatment effects were statistically significant (p ≤ 0.05), Tukey’s HSD post-hoc test were used to conduct pairwise comparisons between individual treatments. When significant interactive effects between groundcover and rootstock treatments were present, the effect of groundcover was analyzed separately by rootstock. For data collected during the imposed drought periods in 2023, linear mixed-effects models (‘lmerTest’ package) for a randomized complete block design replicated four times with split-plots (rootstock) nested within main plots (ground cover treatment) were used to evaluate the effects of ground cover treatment, apple rootstock and their interactive effects on changes in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1) and photosynthesis rate (An; μmol CO2 m-2 s-1) over time. An additional linear mixed effects model (‘lmerTest’ package) was used to evaluate the effects of ground cover treatment on change in volumetric water content (VWC; %). ‘lstrends’ (‘emmeans’ package) were used to compare regression slopes for the changes in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1) and photosynthesis rate (An; μmol CO2 m-2 s-1) among ground cover treatments and between rootstocks. 22 2.3 Results 2.3.1 The effects of ground cover and rootstock on soil organic carbon In June 2022, the mean concentration of SOC in the orchard crop rows was 6.3 ± 0.2 g C kg-1 at the 0-15 cm soil depth and 3.9 ± 0.1 g C kg-1 at the 15-30 cm soil depth (Table 2-2); there were no significant differences in SOC among ground cover treatments or rootstocks at either soil depth (Table 2-3). By October 2023, the mean concentrations of SOC at both the 0-15 cm and 15-30 cm soil depths were slightly higher than in June 2022 (6.6 ± 0.2 g C kg-1 and 4.5 ± 0.2 g Ckg-1, respectively; Table 2-2) but, again, there were no significant differences in SOC among ground cover treatments or rootstocks at either depth (Table 2-3). Table 2-2. Concentration (g kg-1) of soil organic carbon (0-15 cm and 15-30 cm depth) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch or woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Soil samples were collected in June 2022 and October 2023. Control Woodchip mulch Woolly thyme M9 B9 M9 B9 M9 B9 June 2022 0 – 15 cm 6.3 (0.2) 6.4 (0.4) 5.9 (0.5) 6.2 (0.6) 6.5 (0.4) 6.7 (0.4) 15 – 30 cm 3.7 (0.2) 3.7 (0.2) 3.9 (0.3) 3.8 (0.2) 4.0 (0.3) 4.0 (0.3) October 2023 0 – 15 cm 6.1 (0.4) 6.8 (0.5) 6.2 (0.7) 6.8 (0.6) 6.5 (0.5) 7.0 (0.4) 15 – 30 cm 3.9 (0.3) 4.4 (0.3) 4.5 (0.6) 4.0 (0.1) 5.6 (0.7) 4.5 (0.4) Note: Values are means (n = 6) with standard error in parentheses. Table 2-3. Two-way analysis of variance of concentration of soil organic carbon (g C kg-1; 0-15 cm and 15-30 cm depth) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. Source of variation df F-value P June 2022 0 – 15 cm Ground Cover (G) 2 0.81 0.45 Rootstock (R) 1 0.060 0.81 G × R 2 0.22 0.81 23 Residual 30 15 – 30 cm Ground Cover (G) 2 0.56 0.58 Rootstock (R) 1 0.003 0.95 G × R 2 0.004 0.99 Residual 30 October 2023 0 – 15 cm Ground Cover (G) 2 0.26 0.78 Rootstock (R) 1 1.80 0.19 G × R 2 0.022 0.98 Residual 30 15 – 30 cm Ground Cover (G) 2 2.55 0.10 Rootstock (R) 1 1.11 0.30 G × R 2 1.50 0.24 Residual 30 In 2023, the mean fine fraction bulk density was 1.55 ± 0.004 g cm-3 and 1.54 ± 0.004 g cm-3 at the 0-15 cm and 15-30 cm soil depths, respectively; bulk density did not differ by ground cover treatment or rootstock (P > 0.05; data not shown). Given that there were no differences in bulk density or concentration of SOC among treatments in 2022 or 2023, corrections for equivalent soil mass were not warranted (Ellert and Bettany 1995; Fowler et al. 2023) when SOC concentration data were converted to SOC content (Mg ha-1) by correcting for bulk density and sampling depth. In June 2022, the mean SOC content of soil (0-30 cm) in the orchard crop rows was 23.7 ± 0.1 Mg C ha-1 across the whole orchard (Figure 2-2). There were no statistically significant differences in SOC content among ground cover treatments or rootstocks (Table 2-4). By October 2023, the SOC content of soil (0-30 cm) in the orchard crop rows had increased to 24.7 ± 0.3 Mg C ha-1 in the control, 24.8 ± 0.5 Mg C ha-1 under the wood chip mulch, and 27.2 ± 0.4 Mg C ha-1 under the woolly thyme (Figure 2-2), while the SOC content of soil (0-30 cm) planted to M9 and B9 rootstocks had increased to 25.2 ± 0.3 Mg C ha-1 and 26.1 ± 0.2 Mg C ha-1, respectively. There were no significant differences in SOC content among ground cover treatments or rootstocks (Table 2-4). 24 Figure 2-2. Soil organic carbon content (SOC; Mg ha-1) of surface soils (0-30 cm) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control; grey boxes), woodchip mulch (brown boxes), or woolly thyme (Thymus pseudolanuginosus) (green boxes) ground cover treatments in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Table 2-4. Two-way analysis of variance of soil organic carbon content (SOC; Mg ha-1) of surface soils (0-30 cm) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. Source of variation df F-value P June 2022 Ground Cover (G) 2 0.47 0.63 Rootstock (R) 1 0.34 0.56 G × R 2 0.09 0.91 Residual 30 October 2023 Ground Cover (G) 2 1.09 0.35 Rootstock (R) 1 0.25 0.62 G × R 2 0.43 0.66 Residual 30 25 2.3.2 The effect of ground cover and rootstock on particulate and mineral associated organic carbon On average, 121% of SOC was recovered for all June 2022 and October 2023 samples after soil fractionation when compared to unfractionated soils. There was a small tendency to overestimate C recovery when the mass of the C in each of the fractions was added up and compared with the mass of C in the unfractionated soil. In June 2022, the mean concentration of POC in the orchard crop row soils (0-15 cm depth) was 2.2 ± 0.19 g C kg soil-1, while the mean concentrations of MAOC were 0.8 ± 0.03 g C kg soil-1 and 4.1 ± 0.12 g C kg soil-1 for the >50 µm and <50 µm fractions, respectively (Table 2-5; Figure 2-3). POC accounted for approximately 30.5% of the SOC while MAOC accounted for approximately 69.5%. There were no significant differences in POC or MAOC among ground cover treatments or rootstocks in June 2022 (Table 2-6). By October 2023, the mean concentration of POC was 4.3 ± 0.25 g C kg soil-1, while the mean concentrations of MAOC were 0.75 ± 0.03 g C kg soil-1 and 3.5 ± 0.07 g C kg soil -1 in the >50 µm fraction and <50 µm fractions, respectively (Table 2-5; Figure 2-3). POC accounted for approximately 48.5% of the SOC while MAOC accounted for approximately 51.5%. There were no significant differences in MAOC among ground cover treatments or rootstocks but the effect of ground cover treatment (but not rootstock) on POC was statistically significant (P = 0.035; Table 2-6). Tukey’s HSD tests indicated that the concentration of POC was significantly greater in the woolly thyme treatment (5.1 ± 0.42 g C kg soil-1) than in the control (bare soil) treatment (3.6 ± 0.25 g C kg soil-1; P = 0.034). Concentration of C within each soil fraction were also tested but significant treatment effects were not observed (POC; P = 0.3, MAOC > 50 µm; P = 0.5, MAOC < 50 µm; P =0.4 , data are not shown). 26 Table 2-5. Concentrations (g kg-1) of particulate organic carbon (POC) and mineral-associated organic carbon (MAOC) at the 0-15 cm soil depth in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard on M9 or B9 rootstocks under bare soil (control), wood chip mulch or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. Control Woodchip mulch Woolly thyme M9 B9 M9 B9 M9 B9 POC > 50 µm June 2022 1.96 (0.39) 2.25 (0.23) 2.44 (0.67) 2.20 (0.62) 2.40 (0.36) 2.19 (0.53) October 2023 3.53 (0.23) 3.62 (0.52) 3.64 (0.60) 4.37 (0.76) 4.95 (0.43) 5.31 (0.76) MAOC > 50 µm June 2022 0.81 (0.08) 0.76 (0.09) 0.70 (0.03) 0.82 (0.09) 0.89 (0.05) 0.82 (0.04) October 2023 0.75 (0.02) 0.81 (0.07) 0.72 (0.11) 0.73 (0.05) 0.76 (0.06) 0.73 (0.05) MAOC < 50 µm June 2022 4.31 (0.29) 3.92 (0.37) 3.94 (0.22) 4.08 (0.28) 4.40 (0.28) 4.23 (0.35) October 2023 3.51 (0.18) 3.53 (0.11) 3.32 (0.18) 3.43 (0.17) 3.58 (0.19) 3.71 (0.19) Note: Values are means (n=6) with standard error in parentheses 27 Figure 2-3. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm in the crop row soils (0-15 cm) of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (control; grey boxes), woodchip mulch (brown boxes), and woolly thyme (green boxes) ground cover treatments in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). 28 Table 2-6. Two-way analysis of variance of the concentrations of particulate organic carbon (POC) and mineral associated organic carbon (MAOC) > 50 µm and MAOC < 50 µm at the 0-15 cm soil depth in the crop rows of a young apple (Malus domestica var. Ambrosia orchard) on M9 or B9 rootstocks under bare soil (control), wood chip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments in June 2022 and October 2023. Bold-faced type indicates a significant treatment difference (P < 0.05). Source of variation df F-value P POC > 50 µm June 2022 Ground Cover (G) 2 0.12 0.89 Rootstock (R) 1 0.020 0.89 G × R 2 0.19 0.83 Residual 30 October 2023 Ground Cover (G) 2 3.76 0.035 Rootstock (R) 1 0.69 0.41 G × R 2 0.15 0.87 Residual 30 MAOC > 50 µm June 2022 Ground Cover (G) 2 1.07 0.36 Rootstock (R) 1 0.003 0.95 G × R 2 1.16 0.33 Residual 30 October 2023 Ground Cover (G) 2 0.31 0.73 Rootstock (R) 1 0.038 0.85 G × R 2 0.21 0.81 Residual 30 MAOC < 50 µm June 2023 Ground Cover (G) 2 0.53 0.60 Rootstock (R) 1 0.33 0.57 G × R 2 0.39 0.68 Residual 30 October 2023 Ground Cover (G) 2 1.21 0.31 Rootstock (R) 1 0.35 0.56 G × R 2 0.062 0.94 Residual 30 29 2.3.3 The effect of ground cover and rootstock on above and belowground woody biomass carbon In October 2023, the mean mass of above and belowground biomass C was 152.8 ± 1.7 g C tree-1 and 142.8 ± 1.8 g tree-1, respectively (Table 2-7). On average, 3.2 ± 0.5 g C tree-1 (M9 rootstock) and 6.0 ± 0.7 g C tree-1 (B9 rootstock) consisted of C in roots growing above the mineral soil surface in the woodchip mulch itself; these roots were counted as aboveground biomass. There were no statistically significant differences in belowground biomass among ground cover treatments or rootstocks (Table 2-8). However, there was a significant effect of ground cover treatment on aboveground biomass (Table 2-7); Tukey’s HSD tests indicated that aboveground biomass in the woolly thyme treatment (123.3 ± 3.8 g C tree-1) was lower than in the woodchip mulch treatment (174.8 ± 6.5 g C tree-1) (P = 0.026). Table 2-7. Above and belowground biomass C (g tree-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Bench-grafted saplings were planted in November 2020 and samples were collected in October 2023. Control Woodchip mulch Woolly thyme M9 B9 M9 B9 M9 B9 Aboveground 161.8 (24.9) 158.9 (17.3) 198.8 (63.7) 150.8 (26.1) 117.7 (30.9) 129.0 (33.3) Belowground 160.2 (41.3) 178.1 (65.1) 148.1 (27.5) 126.8 (46.9) 120.6 (36.7) 123.0 (30.3) Note: Values are means (n=4) with standard error in parentheses. Table 2-8. Two-way analysis of variance of above and belowground biomass C (g tree-1) of young apple trees (Malus domestica var. Ambrosia) planted to M9 or B9 rootstocks under bare soil control, woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Source of variation df F-value P Aboveground Ground Cover (G) 2 4.37 0.028 Rootstock (R) 1 0.82 0.38 G × R 2 1.49 0.25 Residual 18 30 Belowground Ground Cover (G) 2 2.50 0.11 Rootstock (R) 1 0.000 0.98 G × R 2 0.42 0.66 Residual 18 2.3.4 The effects of ground cover treatment and rootstock on soil moisture, plant water status and leaf gas exchange Across all three ground cover treatments, average volumetric soil water content was 17.0 ± 0.011 % from May to September 2022 and 13.9 ± 0.009 % from May to September 2023 (Table 2-9). Two-way ANOVA to evaluate differences in soil moisture content among ground cover treatments and rootstock were not conducted because only half of the soil moisture probes were located in plots planted to Ambrosia and, of these, rootstock was sometimes not replicated. According to one-way ANOVA, the effects of ground cover treatment on volumetric moisture were not significant for any month measured (Table 2-10). Table 2-9. Volumetric soil moisture content (%; cm3 cm-3 *100) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground cover treatments, from May to September 2022 and 2023. 2022 2023 Control Woodchip mulch Woolly thyme Control Woodchip mulch Woolly thyme May 16.2 (0.008) 16.7 (0.006) 16.7 (0.007) 15.7 (0.008) 15.9 (0.012) 17.7 (0.019) June 16.4 (0.007) 17.2 (0.012) 17.1 (0.008) 14.0 (0.008) 13.7 (0.009) 14.3 (0.006) July 17.1 (0.004) 17.8 (0.016) 17.9 (0.011) 12.8 (0.009) 13.0 (0.009) 12.5 (0.007) August 16.8 (0.007) 17.7 (0.016) 17.6 (0.013) 13.1 (0.009) 13.3 (0.010) 12.3 (0.010) September 15.8 (0.005) 16.7 (0.013) 17.4 (0.012) 13.6 (0.009) 13.6 (0.010) 13.3 (0.012) 31 Volumetric moisture content was measured using Campbell Scientific CS616 time domain reflectometry probes (0-30 cm). Values are means (n = 6) with standard error in parentheses. Table 2-10. One-way analysis of variance of Volumetric soil moisture content (%; cm3 cm-3 *100) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground cover treatments. Year/ Month Source of variation df F-value P 2022 May Ground Cover (G) 2 0.2131 0.81 Residual 15 June Ground Cover (G) 2 0.2370 0.79 Residual 15 July Ground Cover (G) 2 0.1534 0.86 Residual 15 August Ground Cover (G) 2 0.1695 0.85 Residual 15 September Ground Cover (G) 2 0.5691 0.56 Residual 15 2023 May Ground Cover (G) 2 0.6349 0.54 Residual 15 June Ground Cover (G) 2 0.1545 0.86 Residual 15 July Ground Cover (G) 2 0.0782 0.93 Residual 15 August Ground Cover (G) 2 0.3195 0.73 Residual 15 September Ground Cover (G) 2 0.0197 0.98 Residual 15 In 2022, stem water potential (𝜑𝑠𝑡𝑒𝑚), stomatal conductance (gs) and photosynthesis (An) were measured during regular irrigation function (i.e., under well-watered conditions) on June 7, June 22, July 14, July 21, August 3 and August 17. Across measurement dates, mean 𝜑𝑠𝑡𝑒𝑚 varied between -836.1 ± 4.1 kPa and -1394.3 ± 16.3 kPa. There were no significant differences in 𝜑𝑠𝑡𝑒𝑚 among ground cover or rootstock treatments on June 7, June 21, July 14 or July 21 (Figure 2-3, Figure 2-5, Table 2-12). On August 3, however, there was a significant 32 effect of ground cover treatment (P = 0.003), rootstock (P = 0.013) and ground cover × rootstock (P = 0.002) on 𝜑𝑠𝑡𝑒𝑚. One-way of analyses of variance by rootstock showed that there were no effects of ground cover treatment on the 𝜑𝑠𝑡𝑒𝑚 of trees on M9 rootstock but the 𝜑𝑠𝑡𝑒𝑚 of trees on B9 rootstock was lower under woodchip mulch than in either the bare soil control (P = 0.001) or the woolly thyme (P = 0.008) treatments (Table 2-11). On August 17, 2022, only the effect of ground cover treatment on 𝜑𝑠𝑡𝑒𝑚 was statistically significant (P = 0.032); Tukey’s HSD test showed that 𝜑𝑠𝑡𝑒𝑚 was more negative in trees under woodchip mulch than in the woolly thyme treatment (P = 0.031) (Figure 2-4). Across measurement dates in 2022, mean gs varied between 0.078 ± 0.000 mol H2O m-2 s-1 and 0.176 ± 0.001 mol H2O m-2 s-1. There were no significant differences in gs among ground cover or rootstock treatments on June 7, July 14 or July 21. 2022 (Figure 2-4, Figure 2-5, Table 2-12). On June 22 and August 17, however, the effect of ground cover treatment was statistically significant (P = 0.035 and P < 0.001, respectively): on June 22, the gs of trees in the woolly thyme treatment was higher than in the bare soil control treatment (P = 0.038), while on August 17, the gs of trees in the bare soil control treatment was higher than in trees under both woodchip mulch (P < 0.001) and woolly thyme (P < 0.001). On August 3, there was a significant effect of ground cover (P < 0.001) and ground cover × rootstock (P = 0.018). One-way of analyses of variance by rootstock showed that there were no effects of ground cover treatment on the gs of trees on M9 rootstock but the gs of trees on B9 rootstock was lower in the woodchip mulch treatment than in the bare soil treatment (P < 0.001; Table 2-12). Across measurements dates in 2022, mean An varied between 7.69 ± 0.04 μmol CO2 m-2 s-1 and 13.47 ± 0.05 μmol CO2 m-2 s-1. There were no significant differences in An among ground cover or rootstock treatments on June 7 or July 21 (Figure 2-4, Figure 2-5, Table 2-12). On June 33 22, however, the effect of ground cover treatment was statistically significant (P < 0.001); Tukey’s HSD test showed that An was higher in trees under woolly thyme than under both the bare soil (control) (P < 0.001) and woodchip mulch (P = 0.019) treatments. On July 14, the effect of ground cover treatment was also statistically significant (P = 0.047); however, Tukey’s HSD test failed to show significant differences among ground cover treatments (P < 0.05). On August 3 and 17, however, the effect of both ground cover (P = 0.023, and P < 0.001, respectively) and ground cover x rootstock (P = 0.015 and P = 0.036, respectively) were statistically significant. One-way analyses of variance by rootstock showed that on August 3, there were no effects of ground cover treatment on the An of trees on M9 rootstock but the An of trees on B9 rootstock was lower under woodchip mulch than in the bare soil (control) treatment (P = 0.0041; Table 2-12). In contrast, on August 17, there were no effects of ground cover treatment on the An of trees on B9 rootstock but the An of trees on M9 rootstock was lower in trees under woodchip mulch then in the bare soil (control) treatment (P < 0.001; Table 2-11, Table 2-12). 34 Figure 2-4. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; umol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees under bare soil (black square), woodchip mulch (brown circle), and woolly thyme (Thymus pseudolanuginosus) (green triangle) ground cover treatments on six dates in 2022 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. Treatment differences are not shown when interactive effects were statistically significant (See Table 2-10 below). 35 Figure 2-5. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 (grey circle) or B9 (blue square) rootstocks on six dates in 2022 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. Treatment differences are not shown when interactive effects were statistically significant (See Table 2-11 below). 36 Table 2-11. One-way analyses of variance, by rootstock, of the effects of bare soil (control), woodchip mulch, or woolly thyme (Thymus pseudolanuginosus) ground cover treatments on the stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1) and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under well-watered conditions, on August 3 and August 17 2022. One-way analyses were performed when significant ground cover treatment × rootstock effects were observed. M9 B9 Control Woodchip Mulch Woolly Thyme Control Woodchip Mulch Woolly Thyme August 3 2022 𝜑𝑠𝑡𝑒𝑚 -1047 (29) -1085 (55) -1203 (125) -1062a (169) -1526b (110) -1159a (9) gs 0.08 (0.03) 0.07 (0.02) 0.08 (0.02) 0.11 b (0.03) 0.06a (0.02) 0.07ab (0.03) An 7.6 (2.53) 7.4 (1.97) 8.5 (1.70) 10.4b (2.20) 6.9a (2.27) 7.9ab (2.55) August 17 2022 An 10.2b (1.64) 5.9a (2.32) 7.7ab (2.54) 9.3 (1.11) 8.0 (1.92) 7.2 (2.70) Different letters indicate statistically significant treatment differences according to Tukey’s HSD post-hoc test. Table 2-12. Two-way analysis of variance of stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground cover on six dates in 2022 under well watered soil conditions. Source of variation df F-value P June 7 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 1.70 0.22 Rootstock (R) 1 0.32 0.58 G × R 2 0.010 0.99 Residual 12 gs Ground Cover (G) 2 0.39 0.68 Rootstock (R) 1 2.75 0.10 G × R 2 0.056 0.95 Residual 12 An Ground Cover (G) 2 0.062 0.94 Rootstock (R) 1 3.24 0.077 G × R 2 0.085 0.92 Residual 12 June 22 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 1.93 0.19 Rootstock (R) 1 1.99 0.18 G × R 2 0.052 0.95 37 Residual 12 gs Ground Cover (G) 2 3.53 0.035 Rootstock (R) 1 3.79 0.056 G × R 2 1.28 0.29 Residual 12 An Ground Cover (G) 2 9.89 <0.001 Rootstock (R) 1 0.12 0.73 G × R 2 2.17 0.12 Residual 12 July 14 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 0.25 0.78 Rootstock (R) 1 1.79 0.21 G × R 2 1.35 0.30 Residual 12 gs Ground Cover (G) 2 1.41 0.25 Rootstock (R) 1 2.32 0.13 G × R 2 1.30 0.28 Residual 12 An Ground Cover (G) 2 3.21 0.047 Rootstock (R) 1 1.021 0.32 G × R 2 1.61 0.21 Residual 12 July 21 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 0.54 0.60 Rootstock (R) 1 0.12 0.74 G × R 2 0.26 0.78 Residual 12 gs Ground Cover (G) 2 2.69 0.075 Rootstock (R) 1 0.29 0.59 G × R 2 1.13 0.33 Residual 12 An Ground Cover (G) 2 2.26 0.11 Rootstock (R) 1 0.029 0.87 G × R 2 2.046 0.14 Residual 12 Aug 3 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 9.46 0.003 Rootstock (R) 1 8.46 0.013 G × R 2 10.49 0.002 Residual 12 gs Ground Cover (G) 2 7.99 <0.001 Rootstock (R) 1 0.25 0.62 G × R 2 4.27 0.018 Residual 12 An Ground Cover (G) 2 3.99 0.023 Rootstock (R) 1 1.18 0.28 38 G × R 2 4.49 0.015 Residual 12 Aug 17 2022 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 4.81 0.032 Rootstock (R) 1 0.11 0.75 G × R 2 0.87 0.45 Residual 12 gs Ground Cover (G) 2 13.40 <0.001 Rootstock (R) 1 0.088 0.77 G × R 2 2.92 0.061 Residual 12 An Ground Cover (G) 2 11.79 <0.001 Rootstock (R) 1 0.25 0.62 G × R 2 3.50 0.036 Residual 12 In 2023, 𝜑𝑠𝑡𝑒𝑚, gs and An were measured during regular irrigation function (i.e., under well-watered conditions) on July 7, July 24 and August 11. Across measurement dates, mean 𝜑𝑠𝑡𝑒𝑚 varied between -794.6 ± 23.4 kPa and -1126.4 ± 23.9 kPa. The effect of both ground cover treatment and rootstock on 𝜑𝑠𝑡𝑒𝑚 were statistically significant on July 7 and July 24 (Table 2-13). Tukey’s HSD test showed that on both July 7 and July 24, 𝜑𝑠𝑡𝑒𝑚 was more negative in trees planted under woodchip mulch than in the bare soil (control) (P < 0.001 and P = 0.008, respectively) and woolly thyme treatments (P = 0.050, P = 0.032, respectively). There was no effect of ground cover treatment on 𝜑𝑠𝑡𝑒𝑚 on August 11. However, 𝜑𝑠𝑡𝑒𝑚 was more negative in trees on B9 rootstock than on M9 on all measurement dates (Table 2-13). Across measurement dates, in 2023, mean gs varied between 0.089 ± 0.005 mol H2O m-2 s-1 and 0.125 mol ± 0.005 H2O m-2 s-1. The effect of ground cover treatment on gs was statistically significant on July 7 and July 24 (Table 2-13). Tukey’s HSD test showed that on both dates, gs was lower in trees under woodchip mulch than in the bare soil (control) treatment 39 (P = 0.029 and P = 0.034, respectively). There was no effect of ground cover treatment on gs on August 11, and no effect of rootstock on gs on any measurement date. Across measurement dates, in 2023, mean An varied between 7.67 ± 0.45 μmol CO2 m-2 s-1 and 11.3 ± 0.38 μmol CO2 m-2 s-1. There were no significant effects of ground cover treatment or rootstock on any measurement date (Table 2-13). 40 Figure 2-6. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; umol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees under bare soil (black square), woodchip mulch (brown circle), and woolly thyme (Thymus pseudolanuginosus) (green triangle) ground cover treatments on three dates in 2023 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. 41 Figure 2-7. Stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees on M9 (grey circle) or B9 (blue square) rootstocks on three dates in 2023 under well-watered soil conditions. Error bars indicate one standard error of the mean (n = 3). Different letters indicate statistically significant treatment differences (p< 0.05) according to Tukey’s HSD post-hoc test for a given measurement day. 42 Table 2-13. Two-way analysis of variance of stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa), stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var Ambrosia) trees on M9 or B9 rootstocks under bare soil (control), woodchip mulch, woolly thyme (Thymus pseudolanuginosus) ground covers on three dates in 2023 under well watered soil conditions. Source of variation df F-value P July 7 2023 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 11.69 <0.001 Rootstock (R) 1 17.00 <0.001 G × R 2 1.25 0.31 Residual 18 gs Ground Cover (G) 2 4.51 0.026 Rootstock (R) 1 0.092 0.77 G × R 2 0.050 0.95 Residual 18 An Ground Cover (G) 2 0.14 0.87 Rootstock (R) 1 0.078 0.78 G × R 2 0.041 0.96 Residual 18 July 24 2023 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 6.63 0.007 Rootstock (R) 1 15.42 0.001 G × R 2 0.47 0.63 Residual 18 gs Ground Cover (G) 2 3.86 0.040 Rootstock (R) 1 0.25 0.62 G × R 2 0.093 0.91 Residual 18 An Ground Cover (G) 2 0.92 0.42 Rootstock (R) 1 0.27 0.61 G × R 2 0.058 0.94 Residual 18 August 11 2023 𝜑𝑠𝑡𝑒𝑚 Ground Cover (G) 2 2.46 0.11 Rootstock (R) 1 22.049 0.002 G × R 2 0.55 0.58 Residual 18 gs Ground Cover (G) 2 1.53 0.24 Rootstock (R) 1 0.28 0.6 G × R 2 0.027 0.974 Residual 18 An Ground Cover (G) 2 1.93 0.17 Rootstock (R) 1 0.44 0.51 G × R 2 0.27 0.77 Residual 18 43 2.3.5 The effects of ground cover treatment on volumetric soil moisture during imposed drought At the beginning of the imposed drought period, soil VWC was very similar in all three ground cover treatments (between 13.9 ± 1.0 % and 15.3 ± 1.1 %); by the end of the imposed drought period, mean soil moisture was 10.9 ± 0.6 % in the bare soil (control) treatment, 11.8 ± 0.9 % in the woodchip mulch treatment and 8.6 ± 0.3 % in the woolly thyme treatment (Figure 2-8). The linear mixed effects model showed that ground cover treatment had a significant effect on soil VWC over time (Table 2-14). The post hoc LSTRENDS test indicated that soils under woolly thyme lost water more rapidly than soils in the bare soil (control) (P < 0.001) and woodchip mulch (P < 0.001) treatments. Table 2-14. Analysis of variance of linear mixed effects model of the change in soil volumetric water content (%) in the crop rows of a young apple (Malus domestica var. Ambrosia) orchard under bare soil (black), woodchip mulch (brown) and woolly thyme (Thymus pseudolanuginosus) (green) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours) in 2023. The model included time (hr) since irrigation was shut off (T), ground cover treatment (G), and T x G as fixed effects, and plot and block as random effects. Response variable Fixed effects P Soil volumetric water content (%) Time (T) <0.001 Ground Cover (G) 0.51 T × G <0.001 44 Figure 2-8. Mean soil volumetric water content (%; 0-30 cm) in the crop rows of a young apple (Malus domestica) orchard under bare soil (black), woodchip mulch (brown) and woolly thyme (Thymus pseudolanuginosus) (green) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Values along the y-axis are means (n = 6); data were collected using Campbell Scientific CS616 time domain reflectometry probes (0-30 cm) connected to a datalogger that logged measurements every ten minutes. 2.3.6 The effects of ground cover treatment and rootstock on plant water status and leaf gas exchange during imposed drought On a given measurement day, 𝜑𝑠𝑡𝑒𝑚 can vary with both the availability of water to plant roots and the vapor pressure deficit of the atmosphere. In order to ensure that measurements of 𝜑𝑠𝑡𝑒𝑚 during the imposed drought period were not masked by day-to-day differences in vapor pressure deficit, changes in 𝜑𝑠𝑡𝑒𝑚 were expressed as the difference between baseline (expected) 𝜑𝑠𝑡𝑒𝑚 for trees planted to bare soil control at equivalent vapor pressure deficit but under well- 45 watered conditions (Figure 2-1). Using this approach, values of zero indicate no changes in 𝜑𝑠𝑡𝑒𝑚 while values below zero indicate that 𝜑𝑠𝑡𝑒𝑚 has become more negative (indicating a reduction in the availability of water; Figure 2-9 and Figure 2-10). At the beginning of the imposed drought period, 𝜑𝑠𝑡𝑒𝑚 was very similar to baseline (expected) values in all three ground cover treatments (Figure 2-9; Figure 2-10). By the end of the imposed drought period, 𝜑𝑠𝑡𝑒𝑚 differed from baseline (expected) values by a mean of -611.5 ± 88.2 kPa in the bare soil (control) treatment, -502 ± 48.6 kPa in the woodchip mulch treatment and -1192.1 ± 63.1 kPa in the woolly thyme treatment. The linear mixed effects model showed that ground cover treatment had a significant effect on the deviation of 𝜑𝑠𝑡𝑒𝑚 from baseline (expected) values over time (Table 2-15). Post hoc LSTRENDS tests indicated that the 𝜑𝑠𝑡𝑒𝑚 of trees in the woolly thyme treatment deviated from baseline (expected) values more rapidly than trees in the bare soil (control) (P < 0.001) and the woodchip mulch (P < 0.001) ground cover treatments, and that the 𝜑𝑠𝑡𝑒𝑚 of trees in the bare soil (control) treatment deviated from baseline (expected) values more rapidly than the woodchip mulch (P = 0.039) ground cover treatment (Figure 2-9). Rootstock had no effect on the deviation of 𝜑𝑠𝑡𝑒𝑚 from baseline (expected) values, but the 𝜑𝑠𝑡𝑒𝑚 of trees planted to B9 rootstock were more negative from baseline (expected) values than trees planted to the M9 rootstock at both the beginning and end of the imposed drought period. At the beginning of the imposed drought period, mean gs was 0.125 ± 0.003 mol H2O m-2 s-1 across ground cover treatments and rootstocks (Figure 2-9, Figure 2-10). By the end of the imposed drought period, mean gs was 0.077 ± 0.007 mol H2O m-2 s-1, 0.069 ± 0.004 mol H2O m-2 s-1 and 0.041± 0.002 mol H2O m-2 s-1 in the bare soil (control), woodchip mulch and woolly thyme ground cover treatments, respectively. The linear mixed effects model showed that ground 46 cover treatment, rootstock and their interactions had significant effects on the change in gs over time (Table 2-15). For trees on M9 rootstock, post hoc LSTRENDS tests indicated that the gs of trees under woolly thyme decreased more rapidly than in the woodchip mulch ground cover treatment (P < 0.001). For trees planted on B9 rootstock, post hoc LSTRENDS tests indicated that the gs of trees under woolly thyme decreased more rapidly than in both the bare soil control (P < 0.001) and the woodchip mulch (P < 0.001) ground cover treatments. Unlike 𝜑𝑠𝑡𝑒𝑚, average gs did not differ significantly between rootstock on any measurement day throughout the imposed drought period. At the beginning of the imposed drought period, mean An was 11.35 ± 0.23 μmol CO2 m-2 s-1 across ground cover treatments and rootstocks (Figure 2-9, Figure 2-10). By the end of the imposed drought period, mean An was 8.66 ± 0.58 μmol CO2 m-2 s-1, 8.35 ± 0.46 μmol CO2 m-2 s-1 and 5.30 ± 0.31 μmol CO2 m-2 s-1 in the bare soil (control), woodchip mulch and woolly thyme ground cover treatments, respectively. The linear mixed effects model showed that ground cover treatment had a significant effect on the change in An (Table 2-15) over time. Under the imposed drought period, An declined more rapidly in trees in the woolly thyme ground cover treatment than in the bare soil (control) (P < 0.001) and woodchip mulch (P < 0.001). The linear mixed effects model also showed that An declined more rapidly in trees on M9 rootstock than on B9 rootstock (P = 0.002). Like gs, average An did not differ significantly between rootstock on any measurement day throughout imposed drought period. 47 Figure 2-9. Change in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) , stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees under bare soil (control – black square), woodchip mulch (brown circle) and woolly thyme (Thymus pseudolanuginosus) (green triangle) treatments (n = 4) during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Error bars indicate one standard error of the mean. Values of 𝜑𝑠𝑡𝑒𝑚 are expressed as the difference between measured and expected values (kPa); the black hashed horizontal line indicates no difference between measured and expected (baseline) values. 48 Figure 2-10. Change in stem water potential (𝜑𝑠𝑡𝑒𝑚; kPa) , stomatal conductance (gs; mol H2O m-2 s-1), and photosynthesis rate (An; μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 (grey circle) and B9 (blue square) rootstocks (n = 4) during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. Error bars indicate one standard error of the mean. Values of 𝜑𝑠𝑡𝑒𝑚 are expressed as the difference between measured and expected values (kPa); the black hashed horizontal line indicates no difference between measured and expected (baseline) values. 49 Table 2-15. Analyses of variance of linear mixed effects model of the change in 𝜑𝑠𝑡𝑒𝑚 (kPa), stomatal conductance (gs, mol H2O m-2 s-1), and photosynthesis rate (An, μmol CO2 m-2 s-1) of young apple (Malus domestica var. Ambrosia) trees on M9 or B9 rootstocks and under bare soil, woodchip mulch and woolly thyme (Thymus pseudolanuginosus) ground cover treatments during an imposed drought period in which irrigation was shut off from August 11 at 9 AM (0 hours) to August 18 at 12 PM (171 hours), 2023. The model included time (hr) since irrigation was shut off (T), ground cover treatment (G), rootstock (R) and their interactions as fixed effects and plot and block as random effects. Response variable Fixed effects P Deviation of 𝜑𝑠𝑡𝑒𝑚 from baseline (expected) value Time (T) <0.001 Ground Cover (G) 0.21 Rootstock (R) 0.075 T × G <0.001 T × R 0.97 G × R 0.77 T × G × R 0.59 gs Time (T) <0.001 Ground Cover (G) 0.11 Rootstock (R) 0.63 T × G <0.001 T × R 0.011 G × R 0.37 T × G × R 0.019 An Time (T) <0.001 Ground Cover (G) 0.21 Rootstock (R) 0.34 T × G <0.001 T × R 0.002 G × R 0.71 T × G × R 0.21 50 2.4 Discussion: This study explored the effects of ground cover treatment (bare soil (control); woodchip surface mulch; woolly thyme cover crop) and rootstock (M9 or B9 rootstock) on the accumulation of soil C in the tree rows of a young apple orchard in the Okanagan Valley, BC, and the implications of these treatments for plant water relations. I compared treatment differences in SOC, POC and MAOC in June of 2022 (when the study began) and again in October 2023. I expected that the woodchip mulch and woolly thyme treatments would increase the concentration (0-15 cm and 15-30 cm) and content (0-30 cm) of SOC over the two growing seasons of this study, due to inputs of C from the surface-applied woodchip mulch and the living woolly thyme. Although there was a trend toward increasing SOC concentration and content between 2022 and 2023 across all treatments (including the bare soil control), there were no significant treatment differences (Table 2-2, Figure 2-2). The lack of treatment effects is likely due to the short study period; previous studies at Su-RDC reported significant increases in SOC under woodchip mulch after four years in an apple orchard (Neilsen et al. 2014) and after three years in a vineyard (Hannam et al. 2016). Soils in woody perennial production systems, such as orchards, tend to remain undisturbed for several years and, as such, are particularly conducive to the accumulation of SOC (Morande et al. 2017, Scandellari et al. 2016); given more time, it is likely that treatment effects would have been stronger. Contrary to my hypothesis, the concentration of POC at the 0-15 cm soil depth was not greater in the tree rows mulched with woodchips, by October 2023 (Table 2-5, Figure 2-3). Given the large quantity of C-rich, woody plant material added onto the soil surface in the mulch treatment, I expected that POC in the woodchip mulch plots would increase relative to the control and woolly thyme-treated plots. Instead, the concentration of POC was significantly 51 greater in plots planted with woolly thyme than in the bare soil control, while the concentration of POC in the woodchip mulch plots was intermediate between the two (Figure 2-3). Clearly, there was insufficient time for mixing of surface-applied mulch biomass into the mineral soil. By contrast, root biomass from the woolly thyme was directly added into the soil and presumably became POC. Being non-woody, the woolly thyme roots are likely more easily degradable than woody particles from the bark mulch and could be expected to augment POC pools over time. Contrary to my expectations, there were no effects of ground cover treatment on MAOC > 50 µm or MAOC < 50 µm. Between 2022 and 2023, the proportion of SOC in the POC fraction increased from 30.5% to 48.5% overall while the proportion of SOC in the two MAOC fractions fell from 69.5% to 51.5% overall (Figure 2-3). There are a few possible explanations for this trend. First, deep-ripping of the site in 2020, prior to planting, may have pulled to the surface finer soil particles from deeper soil layers. The combination of natural precipitation, periodic overhead irrigation, and drip irrigation in the crop rows may have facilitated downward translocation of these finer soil particles below 15 cm (Emde et al. 2021). Indeed, the concentration of MAOC < 50 µm fell by 0.6 g C kg-1 overall (from 4.1 g C kg-1 to 3.5 g C kg-1), while the concentration of MAOC > 50 µm fell by only 0.05 g C kg-1 overall (from 0.8 g C kg-1 to 0.75 g C kg-1) (Figure 2-3). This is confirmed by fractionation data showing a roughly 25% decrease in the mass of the silt and clay fraction as a proportion of total soil mass in all treatments between samplings in June 2022 and October 2023 (Appendix; Figure A1). It is interesting to note that the while the soil particles in the fine fraction were lost, the overall concentration of C in that fraction increased between June 2022 and October 2023 across all groundcover treatments (Appendix; Figure A2). This strongly indicates that mineral particles are not fully saturated with C and the MAOC limit has not been reached in these soils. Because I did 52 not conduct fractionation of the samples collected at the 15-30 cm depth, I am unable to confirm whether the MAOC < 50 µm showed a proportional increase in deeper soils. Experimental error could also play a role in the observed trend of decreasing MAOC between 2022 and 2023. However, all soil fractionations were performed by me and, due to the long process involved, only two months elapsed between completing the fractionations of the 2022 samples and starting the fractionations of the 2023 samples. Therefore, it is unlikely that the observed trend can be explained by differences in sample processing methods. Again, contrary to expectations, there were no effects of rootstock on the concentration or content of SOC or the concentration of POC or MAOC in 2022 or 2023 (Table 2-2, Table 2-5). The lack of treatment effects may be due to the young nature of this orchard. I found no differences in root biomass in 60 cm x 60 cm x 30 cm soil excavations centred around selected measurement trees; once trees mature, with more fully developed root systems, these differences may become more evident. Previous studies have shown that trees planted on B9 rootstocks produce more root biomass, which should contribute more SOC pools, than trees planted on M9 rootstocks (Bintarti et al. 2020, Valverdi & Kalcsits 2021). Separate analyses were also preformed comparing net change in SOC concentrations between June 2022 and October 2023 on a plot level. These analyses did not change our understanding of SOC dynamics in orchard crop row soil under alternative orchard floor groundcovers. The second goal of this study was to evaluate the effects of ground cover treatment and rootstock on plant water status and leaf gas exchange, as indicated by 𝜑𝑠𝑡𝑒𝑚, gs and An. During regular irrigation function, when all trees were watered to maintain equivalent soil moisture to a depth of 30 cm, I expected to observe no treatment differences in ground cover treatment or rootstock. Instead, I found that trees planted under the woodchip mulch showed a higher water 53 tension (more negative 𝜑𝑠𝑡𝑒𝑚 ) than both the woolly thyme and bare soil treatments on August 3, 2022 (B9 rootstock only), July 7, 2023 and July 24, 2023; treatment differences were only significant between the woodchip mulch and woolly thyme treatments on August 17, 2022 (Figure 2-4, Figure 2-6). Ground cover treatment effects on gs were less consistent: most frequently, gs was higher in the bare soil control than in the wood chip mulch treatment (August 3 2022, B9 rootstock only; August 17, 2022; July 7 2023; July 24, 2023); on occasion, however, gs was higher in the bare soil control than in the woolly thyme (August 17, 2022) or higher in the woolly thyme than in the bare soil control (June 22, 2022) (Figure 2-4, Table 2-11). There were few effects of ground cover treatment on An, although An was lowest in the woodchip mulch treatment on occasion (June 22, 2022; August 3, 2022 B9 only). Thus, the woolly thyme ground cover treatment did not show as strong of or a consistent negative effect on plant water status or leaf gas exchange as the woodchip mulch ground cover treatment. Periodic reductions in plant water status and leaf gas exchange under woodchip mulch relative to the other treatments suggests that mulch application is restricting access of water to tree roots, even under in well-watered conditions. This observation is contrary to previous studies evaluating 𝜑𝑠𝑡𝑒𝑚 under organic mulches in tree fruit (Eissenstat et al. 2018, Gebretsadikan 2019, Sharifi et al. 2021); however, these studies irrigated both bare soil control and woodchip mulch treatment with the same volume of water (rather than irrigating to maintain the same soil moisture as in my study). There may be several possible explanations for this observation. Extensive rooting was observed above the mineral soil surface in mulch-covered soils; tree roots that have not penetrated as deeply into the mineral soil may have restricted access to water, particularly as surface mulches dry out in warm conditions. Surface mulches may also interfere with the percolation of water into the mineral soil. However, I found no differences in soil moisture among ground cover 54 treatments to a depth of 30 cm, suggesting that irrigation water was making its way into the mineral soil even when covered with woodchip mulch or woolly thyme. Tree roots can extend beyond 30 cm, however, so reduced soil moisture under woodchip mulch below 30 cm depth may be a possible explanation; VWC was not measured beyond 30 cm. Significant water savings were achieved by irrigating each ground cover treatment to maintain the equivalent VWC (at least to 30 cm): relative to the bare soil control treatment, 31.3% and 23.1% less water was required to irrigate the woolly thyme treatments and 60.4% and 62.2% less water was required to irrigate the woodchip mulch treatments in 2022 and 2023, respectively. In practice, this resulted in less frequent applications of irrigation water in the woodchip mulch treatment, which may have resulted in more shallow percolation of irrigation water into the soil and, therefore, less available water at depth in the woodchip mulch treatments. Despite this, values of gs and An recorded in trees planted under woodchip mulch suggest trees are not experiencing any significant level of water stress (as defined by a 50 % reduction in gs) and are maintaining equivalent productivity when compared to trees planted under bare soil or woolly thyme. The effect of rootstock on plant water relations and leaf gas exchange is less clear. Contrary to my hypothesis, trees on B9 rootstock had significantly lower 𝜑𝑠𝑡𝑒𝑚 than M9 in 2023 (Figure 2-7), which implies that B9 rootstock somehow restricts root uptake of water or alters water transport through the tree relative to trees on M9 rootstock maintained under equivalent moisture conditions. At the same time, however, gs and An did not differ significantly between rootstocks. This suggests that although water tension within the cultivar is higher in trees on B9 rootstock, overall plant performance is relatively similar and, therefore, water access is not more restricted with B9 rootstock. The overall difference in 𝜑𝑠𝑡𝑒𝑚 may instead be explained by physiological differences among rootstocks, where B9 rootstock has been showed to have a more 55 negative 𝜑𝑠𝑡𝑒𝑚 under ideal conditions when compared to M9 (Valverdi & Kalcsits et al. 2021). That being said, as described above, trees on B9 rootstocks appeared to be more sensitive to ground cover treatments than those on M9 rootstocks. The results of the imposed drought trial are consistent with my hypothesis that changes in deviation of 𝜑𝑠𝑡𝑒𝑚 from baseline, gs and An would be most rapid in the woolly thyme treatments, followed by the control, and slowest in the woodchip mulch treatments as the drought progressed (Figure 2-9). At the start of the imposed drought on August 11, 2023, VWC was approximately 14.4 ± 1.7 % and there were no differences in VWC, 𝜑𝑠𝑡𝑒𝑚, gs or An among ground cover treatments. Over the imposed drought period (from August 11, 2023 to August 18, 2023), trees in the woolly thyme ground cover treatment deviated from the 𝜑𝑠𝑡𝑒𝑚 baseline most rapidly and experienced a more rapid decrease in gs and An than the bare soil (control) or woodchip mulch treatments, consistent with a more rapid loss of water from the soil and increasing water stress in the trees. In the woolly thyme plots, this relatively rapid loss of water is probably attributed to increased leaf transpiration (due to the living thyme). Given that the woolly thyme had grown to cover most of the soil surface within the crop row by 2023, evaporative loss of water was probably reduced relative to the bare soil control treatment; apparently, the increase in transpiration caused by the woolly thyme relative to bare soil plots far outweighs the decrease in evaporation from the soil surface. As expected, trees in the woodchip mulch ground cover treatment deviated from 𝜑𝑠𝑡𝑒𝑚 baseline least rapidly and experienced a slower decrease in gs and An than trees planted to the bare soil control. In this case, the reduction in evaporative loss caused by covering the soil with a dense mat of woodchips improved soil moisture retention, although, the difference was not as strong as anticipated. Interestingly, the soil surface in the bare soil control treatments appeared to have formed a surface crust that may have acted as a barrier 56 to evaporation. Despite the fact that trees planted to the woodchip mulch ground cover treatment had the largest (though not statistically significant) 𝜑𝑠𝑡𝑒𝑚 deviation from baseline and the lowest gs at the start of the imposed drought trial compared with the bare soil (control) or woolly thyme treatments, woodchip mulch was more effective at maintaining plant water status and leaf gas exchange in the absence of irrigation than the other two treatments. Contrary to my expectations, rootstock did not significantly affect deviation from 𝜑𝑠𝑡𝑒𝑚 baseline over the imposed drought period. Since rootstock genotypes have been shown to affect the response of apple cultivars to water limitations (Tworkoski et al. 2016), I anticipated that the B9 rootstocks would be more effective at maintaining water uptake under drought conditions due to their more vigorous root systems (Valverdi & Kalcsits 2021). Although trees on B9 rootstock had the greatest 𝜑𝑠𝑡𝑒𝑚 deviation from baseline at the start of the imposed drought trial (consistent with observations during well-watered conditions), the rate of change in 𝜑𝑠𝑡𝑒𝑚 from baseline did not differ between rootstocks (Figure 2-10). Given the observed physiological differences between rootstock 𝜑𝑠𝑡𝑒𝑚, a separate baseline should be generated for trees planted to the B9 rootstock. Meanwhile, the gs and An of trees on B9 rootstock decreased at a slower rate than those on M9 rootstock (Figure 2-10). From a physiological perspective, this suggests that trees on M9 rootstock are experiencing greater limitations in water access than trees on B9 rootstock and are less resilient to drought conditions. Although the magnitude of the difference in gs and An at the end of the drought trial between rootstock varieties is relatively small, the B9 rootstock could be valuable in protecting yields and preventing tree mortality in more extreme cases of water insecurity. Cases of extreme water insecurity are expected to become more common in the Okanagan Valley over the coming decades (Mote et al. 2005, Neilsen et al. 2016). Recognizing that these trees are still quite juvenile, differences in drought response 57 between rootstocks may become more apparent as trees fully develop their root systems and reach maximum crown height. It should be noted that heavy forest fire smoke was encountered on August 16, 2023 (the third measurement date of the imposed drought trial). Light intensity and photosynthetically active radiation are significantly reduced in the presence of extreme forest fire smoke (Hemes et al. 2018). When stomates constrict in response to low light (low gs), trees are unable to transpire efficiently, which reduces the flow of water through plant tissue, thereby lowering internal water tension (Jeanguenin et al. 2017). This resulted in 𝜑𝑠𝑡𝑒𝑚 values at or near baseline despite limited water availability mid-way through the imposed drought period (Table 2-9). Significantly reduced rates of photosynthesis were also observed, which is consistent with other studies evaluating ecosystem productivity under forest fire smoke (Hemes et al. 2018). If conditions were clear, it is likely that 𝜑𝑠𝑡𝑒𝑚 would be much more negative, which would have resulted in both a steeper model slope. However, the overall interpretation of the relationships observed would remain unchanged. This had the additional effect of greatly exaggerating the appearance of non-linearity in the relationship of 𝜑𝑠𝑡𝑒𝑚, gs, and An over time. 2.5 Conclusion: Aside from the contributions to POC in the woolly thyme treatment, two years of surface mulching with woodchips or cover cropping with woolly thyme had minimal impact on the concentration or content of SOC in the top 30 cm of soil in the tree rows of a young Ambrosia apple orchard relative to conventionally managed bare soils maintained with herbicide. Similarly, two years is insufficient to determine if strategic selection of rootstocks is effective for promoting SOC sequestration in orchard soils. SOC in the crop row should instead be evaluated 58 over the entire lifetime of an orchard. More work needs to be done to monitor the rate of change in SOC over time, to determine whether SOC values in these soils will eventually reach a plateau and to evaluate whether ground cover management can impact the rate at which a plateau is reached. Although this short-term study did not find that ground cover management increased SOC, there are numerous other economic and environmental reasons to consider implementing ground cover management practices in orchards. This research showed that during regular irrigation function, surface mulching can reduce irrigation water requirements by ~60% relative to bare soils with trees irrigated to replace water lost to evapotranspiration and surface mulching can also protect trees from short-term droughts. Similarly, cover cropping with woolly thyme can reduce irrigation water requirements by ~25% relative to bare soils with trees irrigated to replace water lost to evapotranspiration, although they may leave trees more susceptible to short-term droughts Further research should be conducted to compare the effects of these treatments on tree nutrition, crop yield and quality, pollination, weed control, and resilience to climatic stresses. 59 Chapter 3: The effects of warming on soil organic carbon dynamics in the drive rows of two Okanagan apple (Malus domestica cv. ‘Ambrosia’ and ‘Fuji’) orchards 3.1 Introduction Two of the most common irrigation methods for apple orchards in the Okanagan are drip irrigation and micro-sprinkler irrigation. Drip irrigation applies water in concentrated spots along the tree row; as a consequence, drive row vegetation in drip irrigated orchards does not receive direct inputs of irrigation water and relies primarily on precipitation or overhead sprinklers for moisture. Drip irrigation is very efficient because it applies water in a targeted way, delivering water only to the crop roots themselves (Fallahi et al. 2010) and because little water is lost to evaporation. On the other hand, microsprinklers typically throw irrigation water across both orchard tree rows and drive rows. The less targeted application of water using microsprinklers can support a robust cover of vegetation in the drive row, but this comes at a cost to increased water consumption (Fallahi et al. 2010). Due to the more vigorous vegetation in drive rows of micro-sprinkler-irrigated orchards, they tend to store more soil C than in drip-irrigated orchards (Midwood et al. 2020). Understanding soil C decay dynamics in response to climate warming of drip- and microsprinkler-irrigated orchards may be instructive in evaluating the trade-offs between the resilience of soil C stores and water usage in the future. The main climatic factors controlling SOC concentration are temperature and moisture (Lal 2012, Qafoku 2015). Soil microbes need heat energy to metabolize C efficiently, and water to facilitate movement of nutrients (Cotrufo et al. 2015). At the same time, moisture and temperature control the rate at which plant biomass (the main source of SOC) is produced but also the rate at which SOC 60 decays (Cotrufo et al. 2015, Neilsen et al. 2014, Villarino et al. 2021). Together, these processes contribute to fluctuations in the soil C balance. The main goal of this study was to understand how increasing temperatures due to climate change might alter the balance of these processes. Open-topped chambers (OTCs) are used to warm the soil surface within the chamber. They have been used most prominently in arctic and alpine regions through the International Tundra Experiment Network (ITEX) (Center & Copenhagen 1996). By imposing higher surface temperatures, both evaporation from the soil surface and transpiration from foliage are expected to increase. In orchard rows, this effect is dependent on the density of plant coverage, and the availability of soil moisture (Fallahi et al. 2010). In micro-sprinkler irrigated systems, where the soil surface tends to be covered with dense vegetation, evaporative losses of water from the soil surface are already low while transpiration is high (Fallahi et al. 2010). In drip irrigated systems, where plant coverage is less dense, soil moisture content can fall below the permanent wilting point. In these conditions, very little water evaporates from the soil surface and drive row vegetation tends to go dormant in the heat of the summer (Fallahi et al. 2010). Surface-level humidity is directly correlated with evapotranspiration losses from the soil. In micro-sprinkler orchards, relative humidity in the drive row tends to be higher than in drip irrigated orchards (Fallahi et al. 2010). Therefore, changes in soil microclimate will be key to understanding how SOC decay dynamics may respond to warming within the OTC. The two fractions of SOC - POC and MAOC - have been shown to respond differently to changing climactic conditions, with POC being more susceptible to degradation (Lavallee et al. 2020). As there is an increased investment to promote the use of agricultural soil to sequester and store CO2 as SOC as a means of climate change mitigation (Neilsen et al. 2014), it is vital to understand the resilience of both SOC fractions to a warming climate. 61 To address these research gaps, I conducted an experiment over two growing seasons to examine the effects of OTCs on soil surface microclimate (accumulated growing degree days (GDD; °C); maximum air temperature (°C), average humidity (%), and soil volumetric water content (cm3*cm-3)), organic matter decay (g) and SOC (g kg-1) in the drive rows of two mature Okanagan apple orchards. I hypothesized that the microclimate of orchard drive row soils, as indicated by the accumulated growing degree days (from the date of sensor installation in spring until the end of October each year), and the maximum daily daytime near-surface air temperature, mean daily daytime near-surface relative humidity and mean daily surface (0-10 cm) soil moisture (calculated by month) would all be altered through the use of OTCs placed on the soil surface. I expected that OTCs would increase the accumulated growing degree days and the maximum daily daytime near-surface air temperature in both the microsprinkler and drip-irrigated orchards. I also expected that in the drip-irrigated orchard, OTCs would have no effect on mean daily daytime near-surface relative humidity but in the micro-sprinkler-irrigated orchard, OTC would increase mean daily daytime near-surface relative humidity, and that in the drip irrigated orchard, OTC would increase the mean daily surface (0-10 cm) soil moisture but in the micro-sprinkler irrigated orchard, OTCs would decrease the mean daily surface (0-10 cm) soil moisture. I hypothesized that in the relatively dry soils of the drip-irrigated orchard, warmer conditions within the OTCs would promote organic matter decay and have no effect on C inputs from plant biomass. As a consequence, I expected that OTCs in both the drip-irrigated and micro-sprinkler irrigated orchards would promote litter decay and decrease SOC, and that the loss of SOC would be more evident in the POC fractions than in the MAOC fraction. 62 3.2 Methods 3.2.1 Study site This work was conducted at Agriculture and Agri-Food Canada’s (AAFC) Summerland Research and Development Centre (SuRDC), in the Okanagan Valley, BC (Canada) (49°34′N, 119°39′W; 450 m above sea level). Between 1981 and 2010, this region experienced cool winters (December to February temperature: -0.5 °C), warm summers (June to August temperature: 20 °C), and low annual precipitation (Environment Canada 2022). The study orchards used for this project are planted on an Osoyoos loamy sand, which is characterised as a brown chernozemic soil derived from deep (up to 2 m), stone-free, fluvio-glacial deposits (BC, Ministry of Environment 1986). Two established apple orchards were re-purposed for this study: a drip-irrigated Ambrosia (M9 rootstock) orchard, established in 2003 and consisting of eight rows of 72 trees; and a microsprinkler-irrigated Fuji (M9 rootstock) orchard, established in 2001 and consisting of five rows of 20 trees. The two orchards are located directly adjacent to each other. 3.2.2 Soil microclimate Open-topped chambers were constructed from 1.5 mm thick clear polycarbonate plexiglass (Lexan) according to a design derived from Marion (1996; Figure 3.1). Each OTC had a base diameter of 1 m, an open top diameter of 60 cm, and stood 35 cm tall. Eight pairs of OTC and No-OTC locations were set up in the larger Ambrosia block and six pairs of OTC and No-OTC locations were set up in the smaller Fuji block in May 2022 (Figure 3.2). The footprint of each OTC and No-OTC location was marked with a circle of black lawn edging and OTCs were secured to the ground with four steel pegs. OTCs were kept in place between May and 63 November 2022 and 2023 but were removed from the field over the winter months (November 2022 to May 2023) to avoid damage from wind, snow and wildlife. Kestrel Drop D2© climate data loggers were placed in four OTC, and four No-OTC locations within each of the two orchards (16 loggers in total) to record air temperature and relative humidity every thirty minutes. Custom-made opaque solar shields were constructed from plastic sample vials (sprayed with white latex paint and drilled with four holes to facilitate air flow) to shield the loggers from direct sunlight (Figure 3.3). Each shielded logger was suspended 15 cm above the soil surface on a metal stake that was placed approximately 20 cm toward the centre of each designated OTC or No-OTC circle. Data were retrieved from the Kestrel loggers on a weekly basis from June to November 2022 and from May to November 2023. Soil volumetric water content was measured within every OTC and No-OTC location, twice per week over the same period using a Theta probe (Delta-T Devices Ltd. Cambridge, UK). 3.2.3 Soil organic carbon Composite soil samples were collected from each of the 28 OTC and No-OTC locations at two depths (0-15 cm and 15-30 cm) on June 17, 2022 and approximately 16 months later, on October 6, 2023. The composite samples were made up of six individual samples collected in an evenly distributed pattern across the soil surface within each circular OTC and No-OTC location. Soil samples were mixed by hand, air-dried for one week and then sieved (<2 mm) to remove coarse fragments and large plant debris. A 50 g subsample of soil (0-15 cm and 15-30 cm depths) from all 28 OTC and No-OTC locations was separated into three fractions: (1) POC between 0.05 and 2 mm (POC > 50 µm), (2) MAOC between 0.05 and 2 mm (sand-sized; MAOC > 50 µm), and (3) MAOC < 0.05 cm (silt + clay-sized; MAOC < 50 µm) using a method derived from the Par+Den5 procedure described in Poeplau et al. (2018) except that only three fractions 64 (rather than five) were obtained. A 1.7 g/cm3 solution of sodium iodide (NaI) was employed to facilitate separation of the less dense POC > 50 µm fraction from the MAOC > 50 µm. The total C content of each soil fraction plus unfractionated soils was measured using a LECO© CHN 628 series combustion analyzer (LECO Corporation, St. Joseph, Michigan) after grinding in a ball mill. 3.2.4 Litter decay dynamics Unblemished apple leaves were collected in May 2022 from suckers produced in the drip- irrigated apple orchard. Leaves were oven-dried at 65 °C for 48 hours. Litter bags were constructed from 7 cm x 7 cm pieces of 53 µm non-degradable nylon mesh sewn into a “sachet” with a 25 cm2 internal space. In total, 140 litter bags filled with ~0.45 g dried apple leaf tissue and stapled shut (Figure 3.3) were installed within each of the 28 OTC and No-OTC locations on June 6, 2022. One of the five sachets from each OTC and No-OTC location was randomly selected and removed on August 30, 2022, November 7, 2022, June 9, 2023, August 3, 2023 and November 1, 2023. After collection from the field, litter bags were gently rinsed clean of loose dirt using tap water, dried at 65 °C and weighed to determine mass loss. 65 Figure 3-1 Dimensions of open-topped chambers. Design from Marion 1996. Figure 3-2 Fully assembled Kestrel data logger unit housed in custom-made solar shield and suspended on a steel peg. 66 Figure 3-3 Litter bag (5 cm x 5 cm internal space) constructed with 0.53µm nylon mesh and filled with dried apple leaf tissue, prior to incubation in the field. 3.2.5 Statistical approach All statistical analyses were performed using the R programming language in RStudio® 2024.04.2+764 (R Core Team 2024). Gaussian model and t-test assumptions of independence, normality and homoscedasticity were visually verified using residual plots. Statistical analyses were performed using the ‘lmerTest’ (Kuznetsova et al. 2017), and the ’emmeans’ test (v1.10.3, Lenth et al. 2024) packages in RStudio. P values ≤ 0.05 were considered statistically significant. Drive row microclimate Growing degrees were calculated for every temperature measurement (every 30 min; growing degree hours (GDH) with bounds being set at 5°C (base) and 30°C (limit). Daily growing degrees (GDD) were then calculated by averaging GDH above base temperature for 67 each location individually. Accumulated GDD was calculated by taking the sum of GDD for each location. Daily maximum temperature was identified and average humidity was calculated on each day. Monthly average maximum temperature and humidity were also calculated. Microclimate data for the microsprinkler-irrigated orchard and the drip-irrigated orchard were analysed separately. One-way analyses of variance (ANOVA) for a randomized block design replicated four times (both microsprinkler-irrigated and drip-irrigated orchards) were used to compare accumulated GDD (°C) from the date of sensor installation in spring until the end of October each year. Two-way ANOVA replicated four times (both microsprinkler-irrigated and drip-irrigated orchard) was used to assess the effect of OTC on daily maximum temperature (°C), daily average humidity (%), and soil volumetric water content (%) with month as an interaction 2022 and 2023. Paired two-sample t-tests for individual months were then used to evaluate treatment effects when OTC X month interactions were statistically significant. Soil organic carbon dynamics SOC data for the microsprinkler-irrigated orchard and the drip-irrigated orchard were analysed separately. Paired two-sample t-tests for a randomized block design replicated six times (microsprinkler-irrigated orchard) or eight times (drip-irrigated orchard) were used to assess the effect of OTC on SOC concentration (g Kg-1), POC (g kg-1), MAOC > 50 µm (g kg-1), and MAOC < 50 µm (g kg-1) in 2022 and 2023. Leaf litter decay Paired two-sample t-tests were also used to assess the effect of OTC on percent mass remaining (%) of leaf litter samples collected on the five collection dates throughout the two-year study period. A linear mixed effects model (‘lmerTest’ package) for a randomized complete 68 block design replicated six times (microsprinkler-irrigated) or eight times (drip-irrigated) was also used to evaluate the effects of OTC on change in percent mass remaining (%) of leaf litter buried 5-10 cm underneath orchard drive row soil. Separate models were applied to litter samples collected in 2022 and 2023 due to the large over winter gap between the final sample collected in 2022 (November 7, 2022) and the first sample collected in 2023 (June 9, 2023) with little to no change in litter mass. 3.3 Results 3.3.1 The effects of OTCs on air surface temperature and humidity By the end of the 2022 growing season, average accumulated GDD in the microsprinkler-irrigated orchard was 1,856 ± 26 °C in OTC locations and 1,832 ± 38 °C in No-OTC locations (Figure 3-4). By the end of the 2023 growing season, average accumulated GDD in the microsprinkler-irrigated orchard was 2,466 ± 61 °C in OTC locations and 2,364 ± 59 °C in No-OTC locations (Figure 3-4). There were no significant differences in accumulated GDD between OTC and No-OTC locations in either 2022 or 2023 in the microsprinkler-irrigated orchard (Table 3-1). By the end of the 2022 growing season, average accumulated GDD in the drip-irrigated orchard was 2,219 ± 18 °C in OTC locations and 2,054 ± 9 °C in No-OTC locations (Figure 3-4). By the end of the 2023 growing season, average accumulated GDD in the drip-irrigated orchard was 2,924 ± 26 °C in OTC locations and 2,707 ± 15 °C in No-OTC locations (Figure 3-4). In both years, the effect of OTC on GDD was statistically significant (P = 0.0015 and P = 0.033, respectively; Table 3-1). 69 Figure 3-4. Mean (n=4) accumulated growing degree days (GDD; °C) in orchard drive row locations with (OTC; red datapoints) and without (No-OTC; grey datapoints) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left panels) and a drip-irrigated orchard (right panels). * indicates significant treatment differences in accumulated GDD between OTC and No-OTC locations (P < 0.05) at the end of each growing season. Table 3-1. Results of paired two-sample t-tests of mean (n=4) accumulated growing degree days (GDD; °C) in orchard drive row locations with (OTC) and without open-topped chambers (No-OTC) over the 2022 and 2023 growing seasons. T-tests were conducted on total accumulated GDD on the final day of OTC deployment in both years (October 31). Irrigation Type/ Year Source of variation df t-statistic P 70 Microsprinkler-Irrigated 2022 OTC 3 -0.38 0.73 2023 OTC 3 -1.43 0.25 Drip-Irrigated 2022 OTC 3 -11.28 0.0015 2023 OTC 3 -5.39 0.033 Across OTC and No-OTC locations, mean daily maximum temperature in the microsprinkler-irrigated orchard ranged between 22.6 ± 0.4 °C (October) and 37.3 ± 0.4 °C (July) in 2022 and between 22.2 ± 0.6 °C (October) and 38.9 ± 0.7 °C (July) in 2023 (Figure 3-5). In 2022, mean daily maximum temperature were not significantly different between OTC and No-OTC locations for any measurement month (Table 3-3). In 2023, average daily maximum temperature was significantly higher in OTC locations compared to their corresponding No-OTC location in May (by 3.5 °C; P = 0.076) and June (by 3.4 °C; P = 0.017) (Table 3-3). Across OTC and No-OTC locations, mean daily maximum temperatures in the drip-irrigated orchard ranged between 26.5 ± 1.0 °C (October) and 42.0 ± 1.3 °C (August) in 2022 and between 24.4 ± 1.2 °C (October) and 46.4 ± 1.9 °C (July) in 2023 (Figure 3-5). In 2022, mean daily maximum temperature was significantly higher in OTC locations than their corresponding No-OTC location in June (by 3.8 °C; P < 0.001), July (by 4.0 °C; P = 0.025), August (by 4.6 °C; P = 0.002), September (by 5.7 °C; P = 0.002), and October (by 4.9 °C; P = 0.007) (Table 3-3). In 2023, average mean daily maximum temperature per month was significantly higher in OTC locations than No-OTC locations in May (by 5.7 °C; P < 0.001), June (by 6.3 °C; P < 0.001), July (by 9.3 °C; P = 0.011), August (by 7.5 °C; P = 0.012), September (by 7.4 °C; P = 0.020), and October (by 4.9 °C; P = 0.014) (Table 3-3). 71 Figure 3-5. Mean (n=4) daily maximum temperature (°C) (calculated by month) in orchard drive row locations with (OTC; red circles) and without (No-OTC; grey squares) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left) and a drip-irrigated orchard (right). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). 72 Table 3-2. Two-way analysis of variance of mean (n=4) daily maximum temperature (°C) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. Irrigation Type/ Year Source of variation df F-value P Microsprinkler-Irrigated 2022 OTC (O) 1 5.86 0.022 Month (M) 4 179.27 <0.001 O × M 4 1.57 0.21 Residual 30 2023 OTC (O) 1 4.16 <0.001 Month (M) 5 26.09 <0.001 O × M 5 0.31 0.66 Residual 36 Drip-Irrigated 2022 OTC (O) 1 307.04 <0.001 Month (M) 4 388.65 <0.001 O × M 4 4.91 0.004 Residual 30 2023 OTC (O) 1 22.81 <0.001 Month (M) 5 117.54 <0.001 O × M 5 1.97 0.11 Residual 36 73 Table 3-3. Results of paired two-sample t-tests of mean (n=4) daily maximum temperature (°C) by month in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard.. Irrigation Type/ Month Source of variation df t-statistic P Microsprinkler-Irrigated 2022 June OTC 3 -2.47 0.09 July OTC 3 -1.18 0.32 August OTC 3 -0.75 0.51 September OTC 3 0.33 0.76 October OTC 3 -1.27 0.29 2023 May OTC 3 -6.42 0.008 June OTC 3 -4.82 0.017 July OTC 3 -2.08 0.13 August OTC 3 -2.76 0.07 September OTC 3 -1.97 0.14 October OTC 3 -0.53 0.63 Drip-Irrigated 2022 June OTC 3 -18.30 <0.001 July OTC 3 -4.16 0.025 August OTC 3 -11.22 0.002 September OTC 3 -11.33 0.002 October OTC 3 -6.61 0.007 2023 May OTC 3 -21.38 <0.001 June OTC 3 -14.69 <0.001 July OTC 3 -5.66 0.011 August OTC 3 -5.43 0.012 September OTC 3 -4.57 0.020 October OTC 3 -5.13 0.014 Across OTC and No-OTC locations, mean daily humidity in the microsprinkler-irrigated orchard ranged between 75.9 ± 1.2 % (June) and 84.6 ± 2.6 % (October) in 2022 and between 76.8 ± 1.0 % (July) and 85.8 ± 1.5 % (October) in 2023 (Figure 3-6). Differences in mean daily humidity between OTC and No-OTC locations was dependent on measurement month (Table 3-4). In 2022, mean daily humidity was significantly higher in OTC locations than their 74 corresponding No-OTC location in July (OTC: 85.2 ± 1.4 %, No-OTC: 78.9 ± 0.3 %; P = 0.026), August (OTC: 88.7 ± 2.1 %, No-OTC: 81.8 ± 0.5 %; P = 0.038), September (OTC: 91.9 ± 2.3 %, No-OTC: 76.9 ± 2.8 %; P = 0.030), and October (OTC: 90.5 ± 1.6 %, No-OTC: 78.6 ± 2.5 %; P = 0.032) (Table 3-5). In 2023, mean daily humidity was significantly higher in OTC locations than their corresponding No-OTC location in June (OTC: 71.7 ± 0.9 %, No-OTC: 63.1 ± 0.8 %; P = 0.006), August (OTC: 76.7 ± 2.3 %, No-OTC: 72.0 ± 2.7 %; P = 0.025), and September (OTC: 84.9 ± 0.8 %, No-OTC: 79.6 ± 1.7 %; P = 0.046) (Table 3-5). Across OTC and No-OTC locations, average daily humidity in the drip-irrigated orchard ranged between 55.5 ± 0.9 % (August) and 69.1 ± 0.7 % (November) in 2022 and between 42.5 ± 0.8 % (July) and 74.4 ± 0.5 % (October) in 2023 (Figure 3-6). In 2022, average daily humidity was significantly higher in OTC locations than No-OTC locations in June (OTC: 70.2 ± 0.6 %, No-OTC: 66.4 ± 0.5 %; P = 0.017), and July (OTC: 62.5 ± 0.9 %, No-OTC: 57.5 ± 1.6 %; P = 0.016) in 2022 (Table 3-5). In 2023, average daily humidity was significantly higher in OTC locations than their corresponding No-OTC location in June (OTC: 51.5 ± 0.6 %, No-OTC: 50.0 ± 0.5 %; P = 0.037) 2023 (Table 3-5). 75 Figure 3-6. Mean (n=4) daily average humidity (%) per month in orchard drive rows locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (left) and a drip-irrigated orchard (right). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). 76 Table 3-4. Two-way analysis of variance of mean (n=4) daily average humidity (%) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. Irrigation Type/ Year Source of variation df F-value P Microsprinkler-Irrigated 2022 OTC (O) 1 66.76 <0.001 Month (M) 4 9.66 <0.001 O × M 4 2.89 0.039 Residual 30 2023 OTC (O) 1 21.22 <0.001 Month (M) 5 27.87 <0.001 O × M 5 0.88 0.50 Residual 36 Drip-Irrigated 2022 OTC (O) 1 12.08 0.002 Month (M) 4 73.66 <0.001 O × M 4 2.28 0.08 Residual 30 2023 OTC (O) 1 2.95 0.09 Month (M) 5 303.04 <0.001 O × M 5 0.096 0.99 Residual 36 77 Table 3-5. Results of paired two-sample t-tests of mean (n=4) daily average humidity (%) by month in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard and a drip-irrigated orchard. Irrigation Type / Month Source of variation df t-statistic P Microsprinkler-irrigated 2022 June OTC 3 -2.50 0.09 July OTC 3 -4.10 0.026 August OTC 3 -3.56 0.038 September OTC 3 -3.88 0.030 October OTC 3 -3.81 0.032 2023 May OTC 3 -2.37 0.10 June OTC 3 -6.92 0.006 July OTC 3 -1.21 0.31 August OTC 3 -4.19 0.025 September OTC 3 -3.29 0.046 October OTC 3 -1.15 0.33 Drip-irrigated 2022 June OTC 3 -4.85 0.017 July OTC 3 -4.96 0.016 August OTC 3 -1.35 0.27 September OTC 3 0.50 0.65 October OTC 3 -0.62 0.58 2023 May OTC 3 -1.90 0.15 June OTC 3 -3.59 0.037 July OTC 3 -1.62 0.20 August OTC 3 -0.78 0.49 September OTC 3 -0.44 0.69 October OTC 3 -0.30 0.79 3.3.2 The effects of OTCs on soil volumetric water content Across OTC and No-OTC locations, average daily VWC in the microsprinkler-orchard ranged between 21.8 ± 0.8 % (October) and 31.1 ± 0.6 % (June) in 2022 and between 20.0 ± 1.2 % (July) and 26.0 ± 0.7 % (September) in 2023 (Figure 3-7). In 2022, average daily VWC was 78 significantly higher in No-OTC locations than their corresponding OTC location in August (OTC: 22.5 ± 0.9 %, No-OTC: 27.1 ± 0.9 %; P = 0.020), and September (OTC: 23.7 ± 1.0 %, No-OTC: 26.9 ± 0.9 %; P = 0.038) (Table 3-7). In 2023 average daily VWC was significantly higher in No-OTC locations than their corresponding OTC location in May (OTC: 19.6 ± 0.7 %, No-OTC: 23.0 ± 0.7 %; P = 0.015), June (OTC: 17.4 ± 1.0 %, No-OTC: 23.9 ± 0.5 %; P = 0.0027), July (OTC: 17.2 ± 1.8 %, No-OTC: 22.7 ± 0.4 %; P = 0.026), August (OTC: 17.7 ± 1.3 %, No-OTC: 23.3 ± 0.6 %; P = 0.0035) and October (OTC: 23.6 ± 1.5 %, No-OTC: 26.4 ± 0.7 %; P = 0.050) (Table 3-7). Across OTC and No-OTC locations, average daily VWC in the drip-irrigated orchard ranged between 5.6 ± 0.2 % (August) and 16.4 ± 0.5 % (June) in 2022 and between 2.5 ± 0.1 % (August) and 11.3 ± 0.6 % (October) in 2023 (Figure 3-7). In 2022, average daily VWC was significantly higher in No-OTC locations than their corresponding OTC locations in June (OTC: 15.0 ± 0.5 %, No-OTC: 17.8 ± 0.5 %; P = 0.002) and July (OTC: 10.2 ± 0.2 %, No-OTC: 11.3 ± 0.3 %; P = 0.043) (Table 3-7). In 2023, average daily VWC was significantly higher in No-OTC locations than OTC locations in May (OTC: 10.5 ± 0.2 %, No-OTC: 11.9 ± 0.2 %; P < 0.001), September (OTC: 7.0 ± 0.5 %, No-OTC: 9.0 ± 0.6 %; P = 0.008), and October (OTC: 9.4 ± 0.4 %, No-OTC: 13.1 ± 0.7 %; P = 0.002) (Table 3-7). 79 Figure 3-7. Mean daily soil volumetric moisture content (%) per month in orchard drive row locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). Error bars indicate standard error of the mean. * Indicates significant treatment differences by month (P < 0.05). 80 Table 3-6. Two-way analysis of variance of mean daily soil volumetric water content (%) in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). Irrigation Type/ Year Source of variation df F-value P Microsprinkler-Irrigated 2022 OTC (O) 1 18.36 <0.001 Month (M) 4 26.40 <0.001 O × M 4 0.98 0.43 Residual 50 2023 OTC (O) 1 51.66 <0.001 Month (M) 5 12.82 <0.001 O × M 5 1.79 0.13 Residual 60 Drip-Irrigated 2022 OTC (O) 1 24.49 <0.001 Month (M) 4 345.41 <0.001 O × M 4 4.13 0.004 Residual 70 2023 OTC (O) 1 37.23 <0.001 Month (M) 5 215.32 <0.001 O × M 5 6.69 <0.001 Residual 84 81 Table 3-7. Results of paired two-sample t-tests of mean monthly soil volumetric moisture content (%) by month in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). Irrigation / Month Source of variation df t-statistic P Microsprinkler-irrigated 2022 June OTC 5 1.48 0.20 July OTC 5 1.67 0.16 August OTC 5 3.37 0.020 September OTC 5 2.80 0.038 October OTC 5 1.16 0.34 2023 May OTC 5 3.61 0.015 June OTC 5 5.52 0.003 July OTC 5 3.11 0.026 August OTC 5 5.19 0.004 September OTC 5 1.31 0.25 October OTC 5 2.58 0.050 Drip-irrigated 2022 June OTC 7 4.91 0.002 July OTC 7 2.46 0.043 August OTC 7 1.4427 0.19 September OTC 7 1.34 0.22 October OTC 7 0.90 0.40 2023 May OTC 7 8.00 <0.001 June OTC 7 2.33 0.053 July OTC 7 -0.26 0.80 August OTC 7 2.14 0.070 September OTC 7 3.63 0.008 October OTC 7 4.76 0.002 3.3.3 The effects of OTCs on leaf litter decay Over the 2022 growing season, leaf litter in the microsprinkler-irrigated orchard lost >75 % of its original mass (22.0 ± 2.7 % leaf litter mass remaining in November 2022) across OTC and No-OTC locations. Over the 2023 growing season, the mass of leaf litter remaining fell from 82 24.0 ± 1.8 % to 13.5 ± 2.2 % between June and November (Figure 3-8). The OTC had no effect on the mass of litter remaining on any removal date in either year (Table 3-8). According to the linear mixed effects models of litter mass loss over the 2022 and 2023 growing seasons, the presence of OTC had no effect on the rate of litter decay in the microsprinkler-irrigated orchard (Table 3-9). Over the 2022 growing season, leaf litter in the drip-irrigated orchard lost approximately 65% of its original mass (35.2 ± 1.7 % leaf litter mass remaining in November 2022) across OTC and No-OTC locations (Figure 3-8). In November 2022, there was greater litter mass remaining in the OTC than No-OTC locations (OTC: 37.9 ± 1.9 %, No-OTC: 32.5 ± 1.4 %; P = 0.020) (Figure 3-8). Over the 2023 growing season, the mass of leaf litter remaining fell from 40.6 ± 3.4 % to 34.7 ± 4.2 % between June and November (Figure 3-8). The OTC had no effect on the mass of litter remaining on any removal date in 2023 (Tabel 3-8). According to the linear mixed effects models of litter mass loss over the 2022 and 2023 growing seasons, the presence of OTC had no effect on the rate of litter decay in the drip-irrigated orchard (Table 3-9). 83 Figure 3-8. Mean percent leaf litter mass remaining (%) after incubation in orchard drive row locations with (OTC; red circle) or without (No-OTC; grey square) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8). Error bars indicate standard error of the mean. * Indicates significant treatment differences (P < 0.05). 84 Table 3-8. Results of paired two-sample t-tests of percent leaf litter mass remaining (%) after incubation in orchard drive row locations with (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8).. Irrigation Type/ Date Source of variation df t-statistic P Microsprinkler-irrigated August 30, 2022 OTC 5 1.50 0.19 November 7, 2022 OTC 5 -0.66 0.53 June 9, 2023 OTC 5 -1.12 0.32 August 31, 2023 OTC 5 -0.74 0.49 November 1, 2023 OTC 5 0.99 0.37 Drip-irrigated August 30, 2022 OTC 7 -0.73 0.49 November 7, 2022 OTC 7 -3.01 0.020 June 9, 2023 OTC 7 0.19 0.85 August 31, 2023 OTC 7 -2.12 0.072 November 1, 2023 OTC 7 -1.88 0.10 Table 3-9. Analyses of variance of linear mixed effects model of the percent leaf litter mass remaining (%) after incubation in orchard drive row locations (OTC) or without (No-OTC) open-topped chambers over the 2022 and 2023 growing seasons in a microsprinkler-irrigated orchard (n = 6) and a drip-irrigated orchard (n = 8).. Irrigation Type/ Date Response variable Fixed effects P Microsprinkler-irrigated 2022 Percent of litter mass remaining (%) Days in ground (T) OTC (O) <0.001 0.56 T × O 0.62 2023 Percent of litter mass remaining (%) Days in ground (T) OTC (O) <0.001 0.92 T × O 0.58 Drip-irrigated 2022 Percent of litter mass remaining (%) Days in ground (T) OTC (O) 0.003 0.49 T × O 0.55 2023 Percent of litter mass remaining (%) Days in ground (T) OTC (O) 0.021 0.19 T × O 0.13 85 3.3.4 The effects of OTCs on soil organic carbon In June 2022, the mean concentration of SOC in the drive rows of the microsprinkler-irrigated orchard was 16.4 ± 0.36 g C kg soil-1 (0-15 cm) and 4.78 ± 0.15 g C kg soil-1 (15-30 cm) across OTC and No-OTC locations. There were no significant differences in the concentration of SOC between OTC and No-OTC locations at either depth (Table 3-10). By October 2023, the mean concentration of SOC in the drive rows of the microsprinkler-irrigated orchard increased to 20.01 ± 0.60 g C kg soil-1 (0-15 cm) and 6.5 ± 0.48 g C kg soil-1 (15-30 cm) across OTC and No-OTC locations. Again, there were no significant differences in the concentration of SOC between OTC and No-OTC locations at either depth (Table 3-10). In June 2022, the mean concentration of SOC in the drive rows of the drip-irrigated orchard was 18.93 ± 1.25 g C kg soil-1 (0-15 cm) and 5.16 ± 0.12 g C kg soil-1 (15-30 cm) across OTC and No-OTC locations. There were no statistically significant differences in the concentration of SOC between OTC and No-OTC locations at either depth (Table 3-10). By October 2023, the mean concentration of SOC in the drive row soils of a drip-irrigated orchard was to 16.86 ± 0.48 g C kg soil-1 (0-15 cm) and 10.79 ± 0.47 g C kg soil-1 (15-30 cm) across OTC and No-OTC locations. Again, there were no statistically significant differences in the concentration of SOC between OTC and No-OTC locations at either depth (Table 3-10). 86 Figure 3-9. Concentration (g kg-1) of soil organic carbon (0-15 cm and 15-30 cm depth) in the drive rows of a microsprinkler-irrigated (top row; n=6) and a drip-irrigated (bottom row; n=8) apple (Malus domestica) orchard in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). 87 Table 3-10. Results of paired two-sample t-tests of concentration of soil organic carbon (g C kg-1; 0-15 cm and 15-30 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023. Irrigation Type/ Depth Source of variation df t-statistic P Microsprinkler-Irrigated June 2022 0 – 15 cm OTC 5 -1.15 0.30 15 – 30 cm OTC 5 0.26 0.81 October 2023 0 – 15 cm OTC 5 0.20 0.85 15 – 30 cm OTC 5 0.016 0.99 Drip-Irrigated June 2022 0 – 15 cm OTC 7 1.36 0.22 15 – 30 cm OTC 7 -0.42 0.69 October 2023 0 – 15 cm OTC 7 1.36 0.22 15 – 30 cm OTC 7 1.67 0.14 3.3.5 The effects of OTCs on particulate and mineral associated soil organic carbon In June 2022, the mean concentrations of POC, MAOC > 50 µm, and MAOC < 50 µm in the drive row soils of the microsprinkler-irrigated orchard (0-15 cm depth) were 5.87 ± 0.31 g C kg soil-1, 0.58 ± 0.07 g C kg soil-1 and 9.89 ± 0.24 g C kg soil-1, respectively, across OTC and No-OTC locations (Figure 3-10). Particulate organic C accounted for approximately 36 % of the SOC while MAOC accounted for approximately 64 %. There were no significant differences in the concentrations of the POC, MAOC > 50 µm or MAOC < 50 µm between OTC and No-OTC locations (Table 3-11). By October 2023, the mean concentration of POC, MAOC > 50 µm, and MAOC < 50 µm (0-15 cm depth) were 8.6 ± 0.47 g C kg soil-1, 0.83 ± 0.06 g C kg soil-1 and 10.39 ± 0.25 g C kg soil -1, respectively (Figure 3-10). Particulate organic C accounted for approximately 43 % of the SOC while MAOC accounted for approximately 57 %. Again, there 88 were no significant differences in the concentrations of POC, MAOC > 50 µm or MAOC < 50 µm between OTC and No-OTC location (Table 3-11). In June 2022, the mean concentration of POC, MAOC > 50 µm, and MAOC < 50 µm in the drive row soils of the drip-irrigated orchard (0-15 cm depth) were 9.21 g C kg soil-1, 0.78 g C kg soil-1 and 8.95 g C kg soil-1, respectively, across OTC and No-OTC locations (Figure 3-10). Particulate organic C accounted for approximately 47 % of the SOC while MAOC accounted for approximately 53 %. There were no significant differences in the concentrations of POC, MAOC > 50 µm or MAOC < 50 µm between OTC and No-OTC locations (Table 3-11). By October 2023, the mean concentration of POC, MAOC > 50 µm, and MAOC < 50 µm (0-15 cm depth) were 8.42 g C kg soil-1, 1.03 g C kg soil-1 and 7.42 g C kg soil -1, respectively, across OTC and No-OTC locations (Figure 3-10). Particulate organic C accounted for approximately 50 % of the SOC while MAOC accounted for approximately 50 %. There were no significant differences in the concentrations of POC or MAOC between OTC and No-OTC locations (Table 3-11). 89 Figure 3-10. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (0-15 cm depth) in the drive rows of mature microsprinkler-irrigated (top row; n=6) and drip-irrigated (bottom row; n=8) apple (Malus domestica) orchards in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023.. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). 90 Table 3-11. Results of paired two-sample t-tests of concentration of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (0-15 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023. Irrigation Type/ Fraction Source of variation df t-statistic P Microsprinkler-Irrigated POC > 50 µm June 2022 OTC 5 -1.21 0.28 October 2023 OTC 5 -0.52 0.63 MAOC > 50 µm June 2022 OTC 5 1.37 0.23 October 2023 OTC 5 -1.36 0.23 MAOC < 50 µm June 2023 OTC 5 -0.41 0.70 October 2023 OTC 5 0.90 0.41 Drip-Irrigated POC > 50 µm June 2022 OTC 7 -0.15 0.89 October 2023 OTC 7 -1.23 0.26 MAOC > 50 µm June 2022 OTC 7 1.50 0.18 October 2023 OTC 7 -0.31 0.77 MAOC < 50 µm June 2023 OTC 7 1.07 0.32 October 2023 OTC 7 1.27 0.25 In June 2022, the mean concentrations of POC, MAOC > 50 µm, and MAOC < 50 µm in the drive row soils of the microsprinkler-irrigated orchard (15-30 cm depth) were 0.55 ± 0.06 g C kg soil-1, 0.23 ± 0.03 g C kg soil-1 and 3.99 ± 0.17 g C kg soil-1, respectively, across OTC and No-OTC locations (Figure 3-11). Particulate organic C accounted for approximately 12 % of the SOC while MAOC accounted for approximately 88 % at the 15-30 cm depth. There were no significant differences in the concentrations of POC, MAOC > 50 µm or MAOC < 50 µm between OTC and No-OTC locations (Table 3-12). By October 2023, the mean concentration of POC, MAOC > 50 µm, and MAOC < 50 µm in drive row soils of the microsprinkler-irrigated orchard (15-30 cm depth) were 1.03 ± 0.10 g C kg soil-1, 0.66 ± 0.05 g C kg soil-1 and 4.80 ± 91 0.39 g C kg soil -1, respectively, across OTC and No-OTC locations (Figure 3-13). Particulate organic C accounted for approximately 16 % of the SOC while MAOC accounted for approximately 84 %. Again, there were no significant differences in the concentration of POC, MAOC > 50 µm, and MAOC < 50 µm between OTC and No-OTC location (Table 3-12). In June 2022, the mean concentrations of POC, MAOC > 50 µm and MAOC < 50 µm in the drive row soils of the drip-irrigated orchard (15-30 cm depth) were 0.87 ± 0.05 g C kg soil-1, 0.18 ± 0.02 g C kg soil-1 and 4.12 ± 0.08 g C kg soil-1, respectively, across OTC and No-OTC locations (Figure 3-13). Particulate organic C accounted for approximately 17 % of the SOC while MAOC accounted for approximately 83 %. There were no significant differences in the concentrations of POC, MAOC > 50 µm and MAOC < 50 µm between OTC and No-OTC locations (Table 3-12). By October 2023, the mean concentrations of POC, MAOC > 50 µm and MAOC < 50 µm in the drive row soils of the drip-irrigated orchard (15-30 cm depth) were 3.67 ± 0.31 g C kg soil-1, 0.66 ± 0.03 g C kg soil-1, and 6.61 ± 0.18 g C kg soil-1, respectively, across OTC and No-OTC locations (Figure 3-13). Particulate organic C accounted for approximately 34 % of the SOC while MAOC accounted for approximately 66 %. There were no significant differences in the concentration of POC, MAOC >50 µm, and MAOC <50 µm between OTC and No-OTC locations (Table 3-12). 92 Figure 3-13. Concentrations (g kg-1) of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (15-30 cm depth) in the drive rows of a microsprinkler-irrigated (top row; n=6) and a drip-irrigated (bottom row; n=8) apple (Malus domestica) orchard in locations with (OTC; red boxes) or without (No-OTC; grey boxes) open-topped chambers in June 2022 and October 2023. Vertical lines indicate median values, boxes indicate the interquartile zones, and error bars indicate one standard deviation. Different letters indicate significant treatment differences within sampling year (P < 0.05). 93 Table 3-12. Results of paired two-sample t-tests of concentration of particulate organic carbon (POC) > 50 µm, mineral associated organic carbon (MAOC) >50µm, and MAOC <50 µm (15-30 cm depth) in the drive rows of a microsprinkler-irrigated and a drip-irrigated apple orchard in locations with (OTC) or without (No-OTC) open-topped chambers in June 2022 and October 2023. Irrigation type/ Fraction Source of variation df t-statistic P Microsprinkler-Irrigated POC > 50 µm June 2022 OTC 5 0.50 0.64 October 2023 OTC 5 2.11 0.090 MAOC > 50 µm June 2022 OTC 5 -1.57 0.18 October 2023 OTC 5 0.74 0.49 MAOC < 50 µm June 2023 OTC 5 0.34 0.75 October 2023 OTC 5 -0.53 0.62 Drip-Irrigated POC > 50 µm June 2022 OTC 7 1.00 0.35 October 2023 OTC 7 1.10 0.31 MAOC > 50 µm June 2022 OTC 7 -1.21 0.26 October 2023 OTC 7 1.30 0.23 MAOC < 50 µm June 2023 OTC 7 -1.19 0.27 October 2023 OTC 7 2.26 0.059 3.4 Discussion: This study explored the use of OTC to alter microclimatic conditions in the drive rows of a mature microsprinkler-irrigated apple orchard and a mature drip-irrigated apple orchard in the Okanagan Valley, BC, and to evaluate the effects of altered microclimatic conditions on SOC. The OTC were more effective at altering temperatures in the drip-irrigated orchard than in the microsprinkler-irrigated orchard. In the drip-irrigated orchard, OTC increased accumulated GDD in both study years while monthly averages of maximum air temperature were consistently higher within OTC. In the microsprinkler-irrigated orchard, OTC had no effect on accumulated GDD, although monthly averages of maximum air temperature were higher within OTC in May 94 and June 2023. These observations were somewhat contrary to expectations - OTC were expected to increase accumulated GDD and daily maximum temperature in both orchards by preventing radiative heat loss. Differing temperature responses in the two orchards may be due, in part, to differences in orchard architecture and training systems: the microsprinkler-irrigated orchard had three meter row spacing and trees with larger canopies while the drip-irrigated orchard had four meter spaces between rows and were grown according to the ‘slender spindle’ training system; as a result, OTCs in the microsprinkler-irrigated orchard probably received fewer hours of direct sunlight because incoming radiation was blocked by foliage. As well, surface air temperatures in the micro-sprinkler orchard were probably reduced by the abundant ground vegetative growing inside and around OTCs, thereby preventing light from reaching the soil surface and also restricting air flow. Water transpired through plant tissue surfaces itself both cools the plant and absorbs heat from the surrounding area resulting in a cooling effect thereby reducing surface temperature (Winbourne et al. 2020). In 2023, daily maximum air temperatures in the microsprinkler-irrigated orchard were greater within the OTC early in the growing season, though this did not translate into significant differences in accumulated GDD. The effects of OTC on air temperature observed in this study are consistent with previous studies that have used OTC to manipulate surface temperature (Hollister et al. 2023). A study by Welshofer et al. (2018) found that OTCs deployed in both forests clearings and previous agricultural fields increased daytime temperatures by 1.8 °C. As expected, the OTC had a greater influence on humidity and soil moisture in the microsprinkler-irrigated orchard than in the drip-irrigated orchard. In the drip-irrigated orchard, monthly mean humidity was higher in May and June 2022 and in June 2023, and volumetric moisture content was lower in May and June 2022 and in May, September and October 2023 95 within OTC. In the microsprinkler-irrigated orchard, however, monthly mean humidity was higher in the months of June through October 2022 and in June, August and September 2023 while volumetric moisture content was lower in August and September 2022 and in May through August and in October 2023. It is likely that higher temperatures within the OTC promoted evaporation and plant transpiration which, in turn, drew more moisture from the soil and released it into the atmosphere within the OTC. In the drip irrigated orchard, the effects of OTC were restricted to late spring and fall only; thus, when soil moisture was higher (e.g., from snowmelt or rainfall), the effect of OTC was similar in the drip-irrigated orchard to that in the microsprinkler-irrigated orchard. In the summer months, however, when soil moisture in the drive row is low and ground vegetation is dead or dormant, little moisture is available to be drawn out of the soil through evaporation or transpiration, even as surface air temperatures rise within the OTC. These observations are consistent with other studies that have evaluated soil moisture conditions under OTCs in both wet and dry regions of sub arctic grasslands (Hollister et al 2023). While the conclusions of these studies are not directly transferable to this study, their findings lend credence to the conclusions that OTC are an effective method of imposing warmer conditions at the agricultural soil surface. Increased temperatures within the OTC were expected to promote soil microbial activity and accelerate the decay of leaf litter contained in the buried bags (Cotrufo et al. 2015). However, there were no effects of OTC on litter decay rates in 2022 or 2023 in either orchard, and only one date on which greater litter mass remained within OTC compared with No-OTC (November 2022, in the drip-irrigated orchard). In the drip-irrigated orchard, the lack of strong and consistent effects of OTC on litter decay may be related to low soil moisture contents within the OTC, coupled with very high temperatures (e.g., in July, 2023 average maximum air 96 temperatures were 51.0 ± 1.3 °C for OTC and 41.7 ± 0.5 °C for No-OTC). Soil microbes responsible for litter decay would already be limited by the lack of moisture in drive rows of the drip-irrigated orchard so the increase in temperature from the presence of the OTCs had little influence. Studies have shown that even slight increases in soil moisture from very dry conditions can considerably increase microbial activity (Orchard & Cook 1983, Schimel et al. 2018), and in dryland ecosystems, soil moisture is one of the most important factors governing litter decay rates (Zhao et al. 2025). It is possible that over very short time durations and with more frequent litter extractions such differences could be discerned, but over longer time frames this effect is hidden. The litter decay rate in the microsprinkler-irrigated orchard was also similar in OTC and No-OTC locations. This may be due to the competing effects of temperature and moisture: within the OTC, i.e.,. increased decay rates caused by warmer temperatures may have been offset by reduced soil moisture which then slowed microbial decay of organic matter. Given that OTCs in the microsprinkler-irrigated orchard rarely increased air temperatures, it is unlikely that the decrease in soil moisture observed in OTCs was sufficient to limit decay. Nevertheless, contrary to expectations, the effect of OTC on soil microclimate did not impact the decay rate of the apple leaves used in this study. Increased temperatures typically accelerate decay of SOC through greater microbial activity (Lal, R. 2012, Qafoku, N. P. 2015); consequently, OTC were expected to promote degradation of SOC in both orchard types. However, increased temperatures can also increase plant inputs of C into the soil by increasing photosynthesis rates and promoting plant growth (Cotrufo et al. 2015). Temperature increases in the early spring and late fall might also contribute to promoting inputs of C as plants would become more active earlier and stay active later. If 97 OTCs were deployed for longer each year, this relationship may have been more apparent. Given that the soil surface in the microsprinkler-irrigated orchard supported actively growing plants throughout the entire growing season of 2022 and 2023, inputs of plant-derived C were expected to exceed rates of SOC decay within OTC in the microsprinkler-irrigated orchard and, as a result, concentrations of SOC within OTC were expected to increase. By contrast, ground vegetation in the drip-irrigated orchards tended to go dormant by late June and, therefore, concentrations of SOC within OTC were expected to decline due to accelerated rates of decay. Contrary to expectations, after two seasons of warming, total concentration of SOC and POC in orchard drive rows soils remained similar within OTC and No-OTC locations in both orchards and at both soil depths (0-15 cm and 15-30 cm). However, in the drip irrigated orchard, there was a strong trend (though P > 0.05) toward a greater concentration of MAOC < 50 μm (g kg-1) in No-OTC at the 15-30 cm depth than within the OTC. This was a surprising result because POC (not MAOC) is considered more susceptible to environmental change (Lavallee et al. 2020; Rocci et al. 2021, Villarino et al. 2021) and, further, SOC decay is believed to be most active in surface soils (Verbrigghe et al. 2022). Nevertheless, the microclimatic changes caused by the OTC were either not enough to significantly impact C dynamics in the soil over this short-term study or were offset by increased in belowground C inputs. Longer-term studies are required to evaluate the effects of warming more effectively; previous research that monitored changes in SOC in response to soil warming have detected treatment responses in SOC after five years (Verbrigghe et al. 2022) although, this study was performed in subarctic grassland soils and was the result of natural geothermal warming. Separate analyses were also performed comparing change in SOC concentrations between June 2022 and October 2023 on a location level. These analyses did not change our understanding of SOC dynamics in orchard drive row soil. 98 3.5 Conclusion Over two growing seasons, OTC successfully altered orchard drive row soil surface microclimate in both a drip-irrigated and a microsprinkler-irrigated orchard. Open-topped chambers more consistently increased maximum surface temperature and accumulated GDD at the soil surface in the drip irrigated orchard, which had wider tree rows and trees with a smaller canopy size, allowing more light penetration to the soil surface. This study was effective in showing how changes in soil surface microclimate due to increased surface temperature are dependent on the availability of water and the presence of living surface vegetation (which was determined by orchard irrigation strategy); OTC more strongly reduced soil moisture and increased surface humidity in the micro-sprinkler irrigated orchards. As growing seasons become increasingly warm due to climate change, decisions about orchard architecture (row spacing, training system) and irrigation strategy may need to consider implications for resilience to extreme weather conditions. This short-term study did not find that the altered surface microclimate from the presence of OTC affected rates of litter decay or concentrations of SOC in the top 30 cm of orchard drive row soil in either a drip-irrigated or a microsprinkler-irrigated orchard. Two years is insufficient to determine if SOC storage in orchard soils is affected by changing surface microclimate. In future studies, the effects of warming on SOC in the drive row should be evaluated over the entire lifetime of an orchard. More work needs to be done to monitor the rate of change in SOC under altered climactic conditions, to quantify degradation of SOC in response to increasing surface temperatures caused by climate change and to determine whether using agricultural soil to sequester and store CO2 is an effective means of climate change mitigation. 99 Chapter 4: General Conclusions Soil organic carbon (SOC) sequestration in agriculture has been proposed as one means of approaching Canada’s climate mitigation goals. Okanagan orchards and perennial cropping systems with large barren herbicide strips present a potential zone for low barrier adoption of practices that increase SOC. Previous work has shown that Okanagan orchards tend to store more SOC than non-cultivated soils and that ‘drive row’ soils are more enriched in SOC than ‘crop row’ soils (Midwood et al. 2020). Organic mulches and cover crops in the crop rows are being promoted as beneficial for soil health but less research has evaluated their effects on SOC storage; rootstock selection for the purpose of SOC sequestration has been studied even less (Nielsen et al. 2014, Valverdi et al. 2021, Sharifi et al. 2023). Regardless of the strategy used to promote SOC storage in Okanagan orchards, there are concerns over the implications of these practices for irrigation water requirements, plant water stress, and plant function. These concerns will be exacerbated under future climate change scenarios as reduced snow packs, earlier snow melts and increased water demand are expected to threaten irrigation water supplies in a region that is already facing instances of emergency water restrictions and fish protection orders (Mote et al. 2005, Neilsen et al. 2016). The use of agricultural soil as a C sink is also dependent on the resilience of SOC stocks to climatic changes (Rumpel et al. 2020, Rocci et al. 2021). SOC is susceptible to decay and previous studies have suggested that future climate warming will promote SOC losses, but this has not been evaluated in orchard soils (Lal 2012, Qafoku 2015). The research presented in this thesis aimed to: (i) Directly evaluate the contributions of diverse orchard floor management strategies including mulching, cover crops, and rootstock selection to SOC pools in orchard crop rows and, address concerns regarding water use under diverse orchard floor management strategies by investigating the impacts of an imposed drought 100 treatment on midday 𝜑𝑠𝑡𝑒𝑚 and plant function (Chapter 2), and (ii) Investigate the resiliency of SOC in orchard drive rows subjected to climate warming using OTC that simulate a climate-warmed scenario over the short term (Chapter 3). Overall, the findings presented in Chapter 2 do not agree with my hypotheses that both wood chip mulch and woolly thyme would increase SOC relative to the bare soil control, that POC would increase the most in the woodchip mulch treatment, or that MAOC would increase the most in the woolly thyme ground cover treatment. My hypothesis that B9 rootstocks would promote greater SOC storage compared with M9 rootstocks was also not supported by my findings. Instead, there was no effect of woodchip mulch or woolly thyme ground cover and no effect of rootstock variety on the concentration or content of SOC in the crop rows of the experimental Ambrosia apple orchard over two growing seasons. After the first two growing seasons, however, the concentration of POC in the crop row soils under woolly thyme increased relative to the bare soil, which is in partial agreement with my hypothesis that ground cover treatments would promote the formation of POC relative to the bare soil control (although this trend was expected to be greater in the wood chip mulch treatment). The additional POC under the woolly thyme was probably derived from the abundant roots produced by the dense mat of vegetation. Contrary to expectations, however, no difference in root biomass was observed between the M9 and B9 rootstocks, which may explain the lack of treatment differences in SOC. Furthermore, applications of irrigation water directly under the crop row may have accelerated the microbial decay of organic material in and on the soil surface. That being said, the lack of treatment differences in this experiment is probably primarily due to the short duration of the trial; if the experiment was maintained for a longer period, treatment differences in SOC may 101 have been more easily detected due to greater incorporation of surface mulch material (wood chip mulch) and/or inputs of plant biomass, particularly roots (woolly thyme, B9 rootstocks). The finding presented in Chapter 2 did not agree with my hypotheses on the effects of the ground cover treatments on tree water status and leaf gas exchange under regular irrigation function but, did agree with my hypotheses for treatment effects during the imposed drought period, when irrigation was cut off for approximately one week. Contrary to expectations, trees planted under woodchip mulch sometimes exhibited more negative stem water potentials and slower rates of leaf gas exchange than the bare soil control treatment, despite irrigation treatments that maintained an equivalent soil VWC to a depth of 30 cm. Although experiencing a higher water deficit, trees planted under woodchip mulch are not considered to be water stressed according to our guideline of 50 percent reduction in stomatal conductance. Two possible (and inter-related) explanations for this pattern could be: i. applications of wood chip mulch impaired the percolation of water below the 30 cm soil depth relative to the bare soil control or woolly thyme treatments, and/or ii. the wood chip mulch discouraged root growth into deeper soils relative to the bare soil control or woolly thyme treatments. If both of these occurred in tandem, the tree roots in the wood chip mulch may have been more susceptible to water stress as surface soils dried out. Nevertheless, when the drought period was imposed, tree water status and leaf gas exchange was better maintained in trees under woodchip mulch compared to the bare soil and woolly thyme ground cover treatments, demonstrating that suppression of water loss via surface evaporation could be an important tool in improving the drought resilience of Okanagan orchards. Also contrary to my expectations, trees on B9 rootstocks exhibited more negative stem water potentials than trees on M9 rootstocks in 2023 (but not in 2022), despite showing no 102 differences in leaf gas exchange rates between rootstocks in either year. During the imposed drought trial, however, stem water potential and leaf gas exchange responded similarly in trees on both rootstocks, suggesting that, overall, tree performance is similar regardless of rootstock and that differences in the 𝜑𝑠𝑡𝑒𝑚 of trees on M9 and B9 rootstocks during regular irrigation function is probably due to physiological differences among rootstocks rather than differences in their ability to access water. Overall, the research presented in Chapter 2 has contributed to our understanding of the effect of orchard floor groundcovers and rootstock selection on irrigation water requirements. A particular strength of this research was the comparison of both organic surface mulch and a living cover crop treatment to a bare soil control treatment: typically, the effects of cover crops and mulches are not compared in the same study. Furthermore, evaluations of the deviation of midday 𝜑𝑠𝑡𝑒𝑚 from a reference baseline proved to be a very powerful tool to evaluate plant available water in drought conditions under alternative orchard groundcovers. Midday 𝜑𝑠𝑡𝑒𝑚 trends over the course of the drought trial without consideration of VPD did not show a strong relationship and treatment effects would have been hidden. The main limitations of the research presented in Chapter 2 is that it was only conducted over a period of 16 months, between June 2022 and October 2023. Future research should follow the effects of both groundcover management treatments and rootstock selection on SOC storage and plant water relations over a longer time period. Other studies that would contribute to our understanding of the costs and co-benefits of crop row floor management for the purpose of increasing SOC would be to expand simulated drought trials to include other commonly used cover crops (e.g., annual species) and mulch types. Evaluation of soil nutrient dynamics under cover crops and organic mulches are also necessary because of concerns about competition for 103 nutrients between trees and cover crops and/or reduced availability of N under mulching materials with a high C:N ratio. Adverse weather events (un-forecasted rain, wildfire smoke, etc.) severely limited success of the drought trials. Out of the three imposed drought trials attempted, only one was viable for analyses. Ideal weather conditions (clear sunny days) are necessary for the success of trials like these; this requires advanced planning and good fortune to achieve. Future studies should expand on the use of midday 𝜑𝑠𝑡𝑒𝑚 reference baselines for informing irrigation decisions, and reference baselines should be generated for other perennial woody crops like grape and cherry. As anticipated, OTC altered the air temperature, humidity and surface soil moisture in both the microsprinkler-irrigated and the drip-irrigated orchard, though the direction of change was not always consistent with my hypotheses. For example, OTC increased accumulated GDD in the drip-irrigated orchard but not the microsprinkler-irrigated orchard in both 2022 and 2023; at the same time, OTC increased maximum daily temperatures in the drip-irrigated orchard in every month, but treatment differences were rarely significant in the microsprinkler-irrigated orchard. As hypothesised, the OTC tended to increase humidity in the microsprinkler-irrigated orchard but, counter to expectations, humidity was also sometimes greater in the OTC in the drip-irrigated orchard. Similarly, as hypothesised, the OTC tended to reduce surface soil moisture in the microsprinkler-irrigated orchard but, counter to expectations, surface soil moisture was also sometimes reduced in the drip-irrigated orchard. It is likely that vegetation within the drive rows of the microsprinkler-irrigated orchard played an important (and unexpected) role in mediating the effect of the OTC on microclimatic conditions. Contrary to expectations, the OTC had no effect on the rates of litter decay or the concentrations of SOC, POC or MAOC in either orchard type. It appears that in this short-term study, differences in 104 surface microclimatic conditions attributed to OTC are insufficient to significantly effect litter decay rates and SOC dynamics. Overall, the research presented in Chapter 3 has contributed to our understanding of how orchard drive row climatic conditions could be manipulated experimentally using OTCs. A strength of this research was that the experiment was conducted in both a drip- and microsprinkler-irrigated orchard. This demonstrated the important role of water availability and living ground vegetation in mediating the effects of warmer conditions. Deployment of OTC was effective in imposing short term climate warming within orchard drive rows, though it is unclear how closely the climatic conditions within the OTC reflect future climate conditions. The main weakness of the research presented in Chapter 3 is that it was only conducted over a period of 17 months (May 2022 to November 2023), with OTC only being deployed for 11 of those months. Furthermore, the effects of the climate warming on plant growth and productivity (above and belowground), and species composition were not measured. Future experiments should deploy OTC for longer time periods and more fully characterise the effect of the warming experiments on nutrient dynamics and on C inputs and outputs. 105 Bibliography Abad, J., Marín, D., Santesteban, L. G., Cibriain, J. F., & Sagüés, A. (2020). Under-vine cover crops: Impact on weed development, yield and grape composition. 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Mean (n=6) percent change in fraction weight as a proportion of total soil mass of each soil size fraction isolated (Particulate Organic Matter; POM, Sand; F > 50 µm, Silt & Clay; F < 50 µm) between samples collected in June 2022 and October 2023 in soils under woolly thyme (green triangles), woodchip mulch (brown circles), and bare soil control (black squares). 113 Figure A2. Mean (n=6) percent change in C concentration of each soil size fraction isolated (Particulate Organic Matter; POM, Sand; F > 50 µm, Silt & Clay; F < 50 µm) between samples collected in June 2022 and October 2023 in soils under woolly thyme (green triangles), woodchip mulch (brown circles), and bare soil control (black squares).
Thesis/Dissertation
eng
Soil Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Smukler, Sean
Hannam, Kirsten
Soil carbon management in Okanagan apple (Malus domestica) orchards
Text