1. business and industrial
2. agriculture and forestry
3. forestry

 The establishment of apple orchards as temperate forest garden systems and their impact on indigenous bacterial and fungal population abundance in Southern Ontario, Canada by Paul Wartman A Thesis Presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Plant Agriculture Guelph, Ontario, Canada © Paul Wartman, May, 2015 ABSTRACT THE ESTABLISHMENT OF APPLE ORCHARDS AS TEMPERATE FOREST GARDEN SYSTEMS AND THEIR IMPACT ON INDIGENOUS BACTERIAL AND FUNGAL POPULATION ABUNDANCE IN SOUTHERN ONTARIO, CANADA Paul Wartman Co-­‐Advisors: University of Guelph, 2015 Rene Van Acker Ralph Martin This thesis investigated soil microbial abundances affected by different ground management systems in establishing apple (Malus domestica cv. Idared, M9) orchards in Ontario. Four treatments including forest garden systems with and without compost (FGSC and FGS) and grass understory systems with and without compost (GC and G) were established, sampled and analyzed over the establishing two years for gene copy abundance of soil arbuscular mycorrhizal (AM) fungi, total fungi, and total bacteria using quantitative real-­‐time polymerase chain reactions. From Spring2013 to Fall2014 soil bacterial abundance decreased by -­‐0.78, -­‐0.84, -­‐
0.86, and -­‐0.88 ± 0.08 Log 16S gene copies g-­‐1 dry soil, total soil fungal abundance increased by 2.12, 1.86, 1.82, and 1.78 ± 0.15 Log ITS gene sequence copies g-­‐1 dry soil, and AM fungal abundance decreased by -­‐1.73, -­‐2.15, -­‐2.23, -­‐2.04 ± 0.55 Log AML gene sequence copies g-­‐1 dry soil within respective treatments FGSC, FGS, GC, and G. iii Acknowledgements I would like to acknowledge that this research was conducted on the traditional territory of the Attawandaron and the Mississaugas of the New Credit First Nations peoples, and offer respect to them and their ancestors. I hope that the food systems that support us can contribute to the reconciliation of the harm caused to your communities and our shared environment. Ecological farmers in southern Ontario area who are practicing regenerative food production on the land that they are stewarding inspired this work. Thank you all and I hope this work can support you. I am extremely grateful to my co-­‐advisors, Rene Van Acker and Ralph Martin, for their initial interest in this topic, and their generous financial, technical, and emotional support throughout this process. Your commitment to regenerative agriculture is inspiring! Thank you so much to the amazingly supportive folks at the School for Environmental Sciences: Kari Dunfield, who sat on my committee and greatly assisted in the whole process, Kamini Khosla, who provided so much lab support and laughter, Crystall McCall, who showed me “the ropes” of molecular analysis, and John Drummelsmith, who analyzed soil microbes with great dexterity! Armfuls of thanks to Ecosource in Mississauga for their donation of land and logistical support, to Martha and Martin from the Guelph Centre for Urban Organic Farming in Guelph for their donation of land and logistical support, and to Ignatius Farm for logistical support! So much love and thanks to my Mum, Dad, Sister, Brother, and Ilana for emotional and physical support over the two years. I give boundless appreciation for my housemates and friends who joined me on countless conversations, outdoor adventures, and board games when I felt challenged. To all iv of you who helped to set up and maintain this project, my heartfelt thanks! Thanks, also, to Arthur D. Latornell and family for their offering of a graduate scholarship in support of conservation and remediation. Finally, I acknowledge myself for committing to this project and following through with what I wanted to do. It took a lot of asking for help and self-­‐care, and I did it! v TABLE OF CONTENTS Acknowledgements iii Table of Contents v 1.0 Introduction 1 1.1 Introduction 1 1.2 Literature Review 4 1.2.1 Introduction 4 1.3 Land Acknowledgement of Southern Ontario 5 1.4 Development of Temperate Forest Garden Systems 8 1.4.1 Beginning and History 8 1.4.2 Present and Future Growth Opportunities in Academia 12 1.5 Holistic Conceptual and Practical Design Frameworks 13 1.6 Orchards as a Base for FGS 14 1.6.1 Soil Microbe-­‐Plant Communities and Interactions 17 1.6.2 Apple Orchard Plant-­‐Microbe Interactions 20 1.6.3 Agroforestry Plant-­‐Microbe Interactions 22 1.7 Perennial Contributions to Agricultural Systems 25 1.7.1 Internal System Inputs 26 1.7.2 Biodiversity and Balance 32 1.8 Interactions Between Plants and Management 36 1.9 Future Work 38 2.0 Materials and Methods 39 2.1 Site Description 39 2.2 Experimental Design 41 2.3 Sampling and Laboratory Analysis 50 2.4 Statistical Analysis 53 3.0 Results 54 3.1 Abundance of soil bacteria and fungi 54 3.2 Tree Growth 58 3.3 Soil Nutrients 58 4.0 Discussion 63 5.0 Conclusions 73 6.0 Summary and Final Thoughts 75 7.0 Literature Cited 78 8.0 Appendix A 86 1 1.1 Introduction Agriculture is going through a transition on a global scale. External input-­‐
intensive practices have led to great accomplishments in some regards but now, with reflection, effects of these practices on the environment and on some groups in society demonstrate a need to evolve these practices or look for alternative practices (Bainard et al., 2011A; Foley et al., 2005; FAO, 2013; Tomich et al., 2011; Tsonkova, et al., 2012; Van Acker, 2008). In temperate areas of North America, such as Southern Ontario, our landscapes have largely been transformed from forest and grassland to urban development and cultivation agriculture (Ontario, 2014). Consequences of such a large-­‐scale change in landscape include loss of biodiversity, soil erosion, contributions to climate change, water contamination, hydrological imbalances, and dependence on fossil fuels for management practices (Graves et al., 2014; Tomich et al., 2011). Academics have been researching these issues for many years and governments have created policies that support the transition towards reconciling the issues of the 21st century, but the actions are slow and, comparatively, under-­‐supported (FAO, 2014; Wotherspoon 2014; UNCTD, 2013). The shift away from late 20th century agricultural practices towards systems, such as forest garden systems (FGS), which are regenerative, locally appropriate, holistic in design, and resilient, is occurring (Ferguson and Lovell, 2014; Tomich et al., 2011). Indigenous communities, grass roots organizations, private and public research institutions, and ecological farmers are the leading practitioners of forest garden systems. They are applying indigenous knowledge, western scientific knowledge, and nature-­‐mimicked frameworks of system design to achieve this. 2 Academics have a great opportunity to support the development of resilient, regionally appropriate, community-­‐based food systems by providing research and data on system performance. A forest garden system (FGS) can be described simply as “a perennial polyculture of multipurpose plants” (Jacke and Toensmeier, 2005). Similar to some agroforestry concepts, FGS support ecosystem services, provide environmental benefits, and diversify economic products as a multi-­‐functional landscape (Jose, 2009). Agroforestry has been defined as a land-­‐use system where trees and/or shrubs are introduced into agricultural cropping and livestock systems or where crops are planted into forest systems. FGS expands this approach to land-­‐use and production systems designed primarily with diverse, multi-­‐strata perennials and self-­‐sowing annuals that mimic the structure of natural forest, woodland, and savannah ecosystems. The goals of FGS are to achieve a state of abundant diverse yields, self-­‐fertilization, self-­‐maintenance, and self-­‐renewal (Jacke and Toensmeier, 2005; Wiersum, 2004). In temperate climates, FGS are increasing in popularity for the production of food, medicine, fodder, fuel, fibre, and recreation, and the practice of these systems is increasing on smallholder farms in North America and the UK (M. Crawford, personal communication, August 27 2014; E. Toensmeier, personal communication, August 29 2014). FGS are noted as an area of great opportunity in agroforestry research and, although current scientific literature is lacking in temperate FGS, it is being practiced around the world. There is novel, larger-­‐scale, participatory research on FGS occurring at the University of Illinois (Savana Institute, 2013; Wiersum, 2004). 3 The purpose of this study was to initiate research in temperate forest garden systems with goals of measuring the effects that establishing an apple-­‐based FGS with varying amounts of compost has on the abundance of soil bacterial and fungal communities (integral components of a self-­‐sufficient ecosystem) in Southern Ontario, Canada. The overall null hypothesis is that newly established apple-­‐based FGS and newly established apple trees with mixed-­‐grass understories, both with an without compost, will have the same shift in soil bacterial and fungal population abundance, SOM, tree growth and soil chemistry over two years. The alternative hypothesis is that newly established apple-­‐based FGS with compost will have different soil fungal and bacterial abundance, different tree growth, and different soil nutrients compared to newly established apple trees with mixed-­‐grass understories after the first two establishing years. The specific objectives of this study were to measure and compare 1) the abundance of indigenous total bacterial, total fungal, and arbuscular mycorrhizal fungi populations in the soil, 2) soil organic matter, 3) apple tree growth, and, 4) chemical properties (i.e., the nutrients K, Mg, P), between newly established apple-­‐based FGS plots and grass understory managed apple plots, both with and without compost amendments. 4 1.2 Literature Review 1.2.1 Introduction In the tropics, FGSs, which are characterized as forest-­‐analogous agroforests (i.e., a cross between natural forests and specialized tree crop plantations) that have been established by indigenous peoples (Wiersum, 2004). FGSs are currently practiced to a large extent using systems developed by local communities with a goal of conserving the forests on which they depend for a livelihood (Wiersum, 2004). In temperate regions, forest ecosystems have been and still are an integral component of indigenous livelihood and they produce products for the global market (Uprety et al., 2012). Some forest functions provide direct value to people (e.g. food and medicine) while others are more indirect such as what we may now refer to as ecosystem services, such as water filtration (Kimmins, 2004; Uprety et al., 2012). There are many agroforestry systems that domesticate specific components of forest ecosystems for particular yields, for example, the use of trees in silvopasture, intercropping with alley crops, and orchards (Nerlich et al., 2013). The land-­‐use practice of FGS in temperate climate regions is relatively new but it mimics aspects from regional ecosystem models that have developed over centuries. FGSs provide opportunities for producing multiple human-­‐use yields, such as food, fibres, medicines, and fuel, while simultaneously preserving, or regenerating aspects and processes prevailing in undisturbed forests (Wiersum, 2004). Choosing to study our regional environments and the benefits of implementing FGS is a vital decision. With more understanding of these systems, and how farmers might adopt the practices to 5 establish and manage them, we can begin to transition away from systems of low diversity that require high external inputs towards holistic production systems. In this review of the literature I first acknowledge the history and ecology of the land in Ontario and present the development of FGS as a practice and science. Second, I present conceptual aspects of permaculture and agroecology from which FGS can be designed. The main focus of the latter section is on the relationships between diverse perennial plants and soil microorganism communities which informs questions around how FGS function and where research is most required. 1.3 Land Acknowledgement of Southern Ontario The temperate regions of the world have different conditions that support diverse ranges of ecosystems which can be mimicked to create systems that provide for humans, non-­‐human species, and the environment. For example, Canada’s temperate land mass is approximately 50% forest cover, containing, among others, the deciduous forest biome (McCartney, 2011). Southern Ontario’s landscape is made up of the mixed-­‐wood plains ecozone in the north and stretching up from the south is the Carolinian ecozone, which is the most tree species-­‐diverse zone in Canada with more than 1600 plant species (McCartney, 2011). Figure 1 shows the 19th century land cover in S. Ontario: large areas of forest types including maple (Acer spp. L.) and beech (Fagus grandifoloia L.) which covers most of S. Ontario, with larger patches of black ash (Fraxinus nigra Marshall) swamps, oak (Quercus spp. L.) forest, and interspersed patches of hemlock (Tsuga spp. Carriere), willow (Salix spp. L), tamarack (Larix spp. (Du Roi) K. Koch), chestnut (Castanea spp. L.), birch (Betula spp. L.), cedar (Cedrus spp. L.), spruce (Picea spp. Mill.), pine (Pinus spp. L.), poplar 6 (Populus spp. L.), elm (Ulmus spp. L.), and briar thickets. These forest type classifications are indicative of the major canopy species and contain within them multiple understory species of plants, animals, and microorganisms. Figure 1. Land cover classification by major tree canopy in southern Ontario prior to European settlement (1700s) (Butt et al., 2012). Thousands of years of environmental pressure from glacier-­‐formed topography, climate, and soil parent materials, have created landscape-­‐scale successional frameworks that eventually included these species in dynamic ecosystems (Kimmins, 2004). Within the influence of the landscape framework, 7 internal ecosystem mechanisms, such as plant-­‐affected soil compositional changes (e.g., decrease in pH or increase in nutrients) and diverse symbiotic relationships among diverse species, support ecological succession; the series of biological stages and transitions over a period of time that occur after disturbance (e.g., forest fires, extreme weather, and biological control including large mammal browsing, insect herbivory, and disease) (Kimmins, 2004). These internal mechanisms contribute to ecosystem productivity and resiliency, which is the capacity of a system to recover to a pre-­‐existing state after disturbance (Kimmins, 2004). Prior to European settlement in the 18th and 19th centuries, S. Ontario had over 80% forest cover (Butt et al., 2012). Based on settlers’ observations of the indigenous peoples, which are limited to the depth of their relationships with the indigenous peoples of the area, Butt et al. (2012) shared many examples of forest disturbance. The indigenous peoples used fire for campground and trail maintenance, to prepare agricultural land and to care for wild game habitat. They allowed the majority of forest and wetlands to remain intact for use in their physical, cultural, spiritual, and material activities, as well as preserving the intrinsic services of the natural environment upon which they were interdependent (Butt et al., 2012; Uprety et al., 2012; Wiersum, 2004). Compared to the minimal disturbance pre-­‐settlement, there is now only approximately 10% forest cover remaining in S. Ontario, although it varies in different counties, primarily due to increasing agricultural development and urbanization (McCartney, 2011; Ontario, 2014; Butt et al., 2005). Agriculture has 8 transformed ecosystems in S. Ontario from a state of high species and structural diversity to a state of very low species diversity and structure (Kulasekera, 2013). This shift in ecological state has resulted in ecosystems that lack resiliency, in that they are susceptible to disease and degradation, and are heavily dependent on continual inputs of fossil fuels demonstrated by the global annual use of 2.6 million tons of pesticides (Altieri and Nicholls, 2012; Tomich et al., 2011; Van Acker, 2008). 1.4 Development of Temperate Forest Garden Systems 1.4.1 Beginning and History The first known temperate-­‐climate FGS to be documented is Robert Hart’s garden in England (Jacke and Toensmeier, 2005). It was planted in 1979, has an area of 3200 sq ft, is in the USDA hardiness zone 8, and was consciously created as a perennial, self-­‐sufficient home garden for all his basic needs. Much like multi-­‐strata forest gardens in the tropics, on which Hart modelled his, the garden has three intentionally designed vertical layers, consisting of canopy trees, including plums (Prunus spp. L.), apples (Malus spp. Mill.), pears (Pyrus spp. L.), elder (Sambucus nirgra L.), elm, ash, and hawthorn (Crataegus spp. Tourn. ex L.), understory shrubs, including Ribes spp. L. (e.g., gooseberries and red, white, and black currants), Rubus spp. L. (e.g., raspberries, blackberries), hazelnuts, roses (Rosa spp. L.), and Siberian pea shrubs (Caragana arborescens Lam.), and over 25 herbaceous species, including chives (Allium schoenoprasum L.), comfrey (Symphytum officinale L.), good King Henry (Chenopodium bonus-­‐henricus L.), many mints (Mentha spp. L.), grasses, and nettles (Urtica dioica L.). Jacke and Toensmeier (2005) share other structural layers, 9 which contain within them diverse functional roles, including the following from tallest to shortest: canopy/tall trees, sub-­‐canopy/large shrubs, shurbs, herbaceous plants, ground covers/creepers, underground/root zone-­‐occupying plants, mushrooms, and vines. Inspired by Robert Hart and his text, Forest Gardening (Hart, 1996), and Russel J. Smith’s book, Tree Crops: A Permanent Agriculture (Smith, 1987), other institutions have expanded, both in scale and capacity, upon Hart's FGS model. Martin Crawford is the Trust Director of the Agroforestry Research Trust, which was registered as a charity in England in 1992; he formed a participatory Forest Garden Network with 1100 members so that practitioners can learn about and share their research on perennial polycultures (M. Crawford, personal communication, August 27, 2014). The Trust has published a textbook, Creating a Forest Garden (Crawford, 2010), publishes Agroforestry News (Agroforestry News, 2014) which is a quarterly newsletter that focuses on temperate plant projects, and in 1994 began the Forest Garden Project, which is 2.1 acres of land (with another 8 acres of perennial polyculture experimental grounds added in 1995) converted into a demonstration forest garden consisting of multi-­‐strata plants (140 different species) that coexist, producing fruits, nuts, edible leaves, spices, medicinal plants, poles, fibres, basketry materials, honey, fuelwood, fodder, mulches, game, mushrooms, and sap products. The purpose of the garden is to demonstrate how forest gardens can be self-­‐sustaining systems with multiple diverse yields and to be a location to test polyculture interactions of multi-­‐strata plants. 10 Eric Toensmeier is an author of multiple texts on perennial plants, co-­‐author with Dave Jacke of Edible Forest Gardens Vol I & II (Jacke and Toensmeier, 2005; E. Toensmeier, personal communication, Aug 28, 2014), and is a researcher and lecturer of permaculture and “carbon farming” across the globe (Perennial Solutions, 2012). He is an advocate of stabilizing the climate by shifting to permanent agriculture, arguing, based off existing research and anecdotal evidence, that permanent agriculture, and integrated design, not only outcompetes annuals for carbon sequestration but also restores the land for production of human-­‐use yields while reducing the need for machines and other fossil fuel-­‐based inputs. “Plants For A Future” is a 20 year old charity that focuses on researching and openly sharing information on ecologically sustainable food production systems and plants, specifically “woodland gardening”. Their research team consists of academic researchers including Ed Sears, Chris Warburton Brown, Tomas Remiarz, and Rafter Sass Ferguson, as well as practitioner association members. They partner with the Permaculture International Research Network and the Permaculture Association of Britain to work toward achieving their key goals of: 1) establishing a baseline of data for permaculture research with a focus on temperate-­‐climate perennial polycultures, 2) assessing permaculture research for future directions, and, 3) engaging people to get involved through educational outreach. Tomas Remiarz (2014) completed a survey of 114 temperate-­‐climate FGSs that included countries around the world (e.g. United Kingdom (UK), Italy, Canada, United States (US), France, Germany, New Zealand) with 46 and 27 of those gardens being in the UK and the US, respectively. Garden sizes ranged from a few square 11 meters to 30 hectares (the majority were under 1 ha), and forest garden uses varied from private garden use (59%), to community garden projects (24%), to commercial production (15%), the latter were not primarily based on sales of produce but rather education and tours. The earliest of these FGS were started in the 1980s but the majority were established after 2000. The results of this survey provide baseline numbers and guiding questions that aid in the direction of a 10-­‐
year food forest research project. The project is in its fourth year and consists of 10 practitioner participants who are practicing food forest systems on 1 hectare or less. The focus of the study is to determine 1) the ratio between energy input and yields (i.e., economic, social, and biodiversity), 2) whether FGS are economically viable, and, 3) the application of FGS in urban design (Remiarz, 2014). Dr. Naomi van der Velden, the lead researcher for the Cumbria Forest Food Network at the University of Cumbria, specializes in landscape ecology, permaculture, and forest ecology, and is also collaborating with the Permaculture Association to implement participatory trials and properly designed systems in the 10-­‐year food forest research project (University of Cumbria, 2015). Other commercial operations, such as the New Forest Farm and Badgersett Research Corporation, located in Wisconsin and Minnesota respectively, are beginning the transition from small, homestead or private gardens, to large scale FGS, and from annual staple crops to woody perennial staple crops (Badgersett Research Corporation, 2014; Forest Agriculture Enterprises, 2012). Both advocate for a shift in production of staple crops (i.e., carbohydrates, fats, and proteins) from annuals to perennials. Mark Shepard, the head farmer of family-­‐run New Forest 12 Farm and the CEO of Forest Agriculture Enterprises, has created an approach to perennial woody polyculture that mimics the ecosystems of natural oak savannah, successional brushland, and eastern woodlands. New Forest Farm in Southwest Wisconsin, consists of 110 acres that has been converted from row-­‐cropping to perennial agricultural ecosystem that incorporate permaculture principles and acts both as a demonstration site and a commercial farm for nuts, fruits, livestock, fibre, and biofuels. 1.4.2 Present and Future Growth Opportunities in Academia Increased interest from academics has sprouted from the success and profile of FGS practitioners, such as Mark Shepard. The newest quantitative academic research on temperate forest garden systems is emerging from the research team led by Dr. Sarah Taylor Lovell from the University of Illinois, Urbana-­‐Champaign, who are researching multiple factors in large-­‐scale savanna-­‐based agroecosystems (Woody Perennial Polyculture Research Site, 2013). Dr. Lovell was recently (2012) awarded $400,000.00 by the Institute for Sustainability, Energy, and Environment to develop a research infrastructure, which consists of 5 acres of intercropped chestnuts (Castanea spp.), hazelnuts, apples, grapes (Vitis vinifera L.), currants, raspberries, with alleys planted to perennial pasture mix of grasses and clover (Woody Perennial Polyculture Research Site, 2013). Factors measured in the study include aspects of biogeochemistry (e.g., carbon fluxes, nitrous oxide emissions, nitrate leaching, self-­‐fertility, water use efficiency, drought tolerance, runoff and erosion), ecology (e.g., biodiversity, phenology, resiliency), agronomy (e.g., 13 establishment, maintenance, harvesting and processing), and economics (e.g., diverse and total yield, nutritional composition, economic modelling, and inputs). Dr. Taylor Lovell is also partnered with the Savanna Institute, which has created a formal case study that involves 7 commercial-­‐scale farms in Wisconsin and Illinois where they are tracking ecological, economic and social impacts during transition from conventional farming to restorative agricultural practices, which includes FGSs and integrative design (Savanna Institute, 2013). The aforementioned examples are evidence that temperate climate FGS, and systems with similar design principles that make use of woody perennial polycultures, are growing in popularity with commercial producers and academics. This momentum creates opportunities for academics to cooperate with these farmers in appropriate ways and to contribute to this field of interest with research and data. Quantitative academic literature specific to temperate-­‐climate FGS is lacking, but research from other perennial-­‐based temperate production systems, such as agroforestry and orchard systems provides some research and data for the development of FGS academically. Reviewing the practices of these production systems may provide insight into how to develop integrated methodologies for future research into and practical applications of FGS. 1.5 Holistic Conceptual and Practical Design Frameworks Ecologists have commented on the history of agriculture and how major land disturbances, typically in the form of denuding land of diverse perennial vegetation cover, have led to extreme ecological shifts and, in some cases, societal catastrophe (Altieri and Nicholls, 2012; Hillel, 1991; Tomich et al., 2011). Globally, many 14 academic researchers, public institutions, community organizations, private corporations and citizens have demonstrated the importance of diverse perennial ecosystems in their respective regions. Wes Jackson (2002) provides a historical report demonstrating the importance of observing the structural patterns of local natural systems and to designing aspects of these systems into agricultural systems. Scientific disciplines such as landscape ecology and permaculture studies address the highly nonlinear dynamics of complex landscapes and place emphasis on understanding relationships between spatial patterns and temporal flows of energy within natural systems (Ferguson and Lovell, 2014; Wu and Hobbs, 2002). Agroecology is described by Tomich et al. (2011) as “the integrative study of the ecology of the entire food system, encompassing ecological, economic and social dimensions,” and provides a framework for the design and management of low input, ecological service-­‐based food systems. These are some examples of alternative frameworks in which agriculture is holistically designed with importance placed on diverse perennial vegetation to meet the needs of the existing human population while sustaining, and even restoring, natural systems. Utilizing the guiding principles (Table 1) crafted by these frameworks helps to discern components of complex systems and apply them so that newly designed systems can operate on reduced external inputs while conserving resources and regenerating environmental services. 15 1.6 Orchards as a Base for FGS The basic requirements for apple trees to grow in temperate North American undisturbed ecosystems are met without human input, as demonstrated by the suggested origin of the family (Rosaceae) in and distribution throughout North America (Potter et al., 2007). It has been suggested that wild apples originated in forest ecosystems of Turkestan, central Asia, and that some are indigenous to North America as well (Harris et al., 2002; Janick, 2003). This is helpful because it enables opportunities to observe undomesticated ecosystems as models for FGS design. Table 1. Agroecological principles for the design of biodiverse, energy efficient, resource-­‐conserving and resilient farming systems (Tomich et al., 2011). Enhance the recycling of biomass, with a view to optimizing organic matter decomposition and nutrient cycling over time. Strengthen the “immune system” of agricultural systems through enhancement of functional biodiversity -­‐ natural enemies, antagonists, etc. Provide the most favourable soil conditions for plant growth, particularly by managing organic matter and by enhancing soil biological activity. Minimize losses of energy, water, nutrients and genetic resources by enhancing conservation and regeneration of soil and water resources and agrobiodiversity. Diversify species and genetic resources in the agroecosystem over time and space at the field and landscape level. Enhance beneficial biological interaction and synergies among the components of agrobiodiversity, thereby promoting key ecological processes and services. An apple orchard ecosystem typically mimics aspects of the vegetational structure of a forest at a young successional stage or that of a forest edge; there are patches in the canopy that allow sun through to the understory, the canopy species is relatively short, and the understory consists of perennial herbaceous plants and 16 short woody plants (Kimmins, 2004). However, orchards are typically managed at low levels of diversity regarding families and species of plants, function of plants, architectural layout of plants (i.e., their height and shapes), and overall system biodiversity compared to forest ecosystems. Hoagland et al. (2008) demonstrated this in their study when they designed an organic apple orchard in the same space previously planted to cherries, with one variety of apple tree (cv. Pinata) planted on one type of rootstock (M7) at a spacing of 1.5 m in parallel rows that were separated by 4 m. Leinfelder and Merwin (2006) described how it is common for apple producers to replant old apple orchards with apples, sometimes continuously for more than a century, and they share a survey that states approximately 50% of New York, US, fruit growers experience replant disease, which results from degrading soil conditions over time. Apple replant disease, which presents symptoms of reduced tree vigour, delayed productivity, and even tree death, is thought to be caused by particular soil microorganisms as well as nutrient imbalances, soil compaction, and herbicide use (Leinfelder and Merwin, 2006). The use of pesticides and tillage are practices that, in this context, maintain vegetation, soil microbes, and animals in a state that best produces apples. The varying amounts of inputs are retarding ecological succession. For example, unwanted plants in an orchard understory have been killed via a combination of three herbicides (glyphosate, norflurazon and diuron) tank-­‐mixed at 2.0, 3.0, and 2.5 kg active ingredient ha-­‐1, respectively, applied in early spring each year (Yao et al., 2005), and soil tillage was performed “as needed” for weed control in another orchard (Hoagland et. al, 2008). These processes of maintenance 17 contribute to the issue and, perhaps, if observed in the larger context of the ecosystem there may be alternative management paths forward. FGSs are designed with the intention to serve the long-­‐term needs of the system and not just for one species. This is proposed through the implementation of ecosystem service-­‐based components mimicked from regionally appropriate ecosystem models, such as forests for apples (Janick, 2003). An influential aspect that encourages stability coinciding with productivity in forest ecosystems is plant species diversity (Kimmins, 2004). Microorganisms are also a major component of all natural system functions and recent research is providing insight on how agricultural practices can support microorganisms to in turn support ecosystem processes and potentially reduce the need for external inputs (Bonfante and Anca, 2009; Smith and Smith, 2011; Welbaum et al., 2004; Zak et al., 2003). A review of the literature relating to apple orchards and soil microorganisms in the context of management practices that support microbial ecosystem services brings up limited results. Literature that does exist typically involves production systems that use synthetic inputs for pest control and fertility management, which has effects on soil microorganism function and composition, and makes comparison to FGS difficult (Imfeld and Vuilleumier, 2012; Lisek, 2014; Tian et al, 2013). In this regard, agroforestry research offers data. 1.6.1 Soil Microbe-­‐Plant Communities and Interactions Soil microorganisms function as drivers of ecosystem productivity affecting nutrient cycling (e.g., nitrogen, phosphorus, and carbon), plant symbiotic relations, 18 food sources, and soil structure (Smith and Smith, 2011; Whiteside et al., 2012). Examples include rhizobial bacteria and arbuscular mycorrhizal (AM) fungi. Soil microorganisms are strongly influenced by plants and have a powerful effect on plants through multifaceted feedback exchanges of nutrients, secondary metabolites, carbon-­‐rich materials, sugars, and amino acids, which are considered, in all cases, forms of allelopathy that affect the soil (Welbaum et al., 2004). Welbaum et al. (2004) provide a review on exchanges that influence microbial and plant communities in terms of establishment, growth, diversity and abundance, competition, disease, activity, dormancy, nutrient economies, and frost damage. All of these effects may vary depending on external and internal factors (e.g., tillage practices, climate, and fertility inputs), and achieving desired functionality requires more than just pairing particular plants (Miras-­‐Avalos et al. 2011; Nair and Ngouajio, 2012). However, particular trees are known to have significant associations with specific microorganisms, for example: hardwoods associate with AM fungi, most conifers associate with only ecto-­‐mycorrhizal fungi, and some trees (e.g., poplars) associate with both types of mycorrhizal fungi (Bainard et al. 2011B). Historically, it was thought that plant community structure was mostly influenced by competition for scarce resources, and now, with increasing interest and technological accessibility, microbe communities’ effects on microbe-­‐plant and plant-­‐plant interactions are creating new conceptual frameworks for understanding the drivers of plant community structure (Bever et al., 2012). Understanding how plant 19 composition and soil microbe community structure interact may provide insights for designing effective FGS. Even though the composition of microbial communities and their interactions with plant communities are integral to ecosystem function, our ability to observe them, and therefore our application of practices that work with them, has been limited (Bing-­‐Ru et al., 2006). Now, novel and advanced methods in molecular studies have increased our ability to be and our accuracy in observing microorganisms in soil, the rhizosphere of plant roots, and plant roots. Standardized procedures have been created to sample bulk soil, extract soil nucleic acids of bacterial and fungal populations and proceed with further analysis (Gorzelak et al., 2012; Hirsch et al., 2010). Analytical molecular techniques, such as quantitative polymerase chain reaction provide a highly reproducible method that enables glimpses of the molecular structures of complex soil communities (Van Elsas et al., 2011). When utilized, these methods allow us to assess how certain production practices affect soil microbiota populations. Multiple studies use available technology to monitor the parallel fluctuations of microbial and plant communities in production systems providing insight to the above-­‐mentioned interactions (van Elsas and Boersma, 2011). For example, Lee et al. (2008) developed novel primer effectiveness in the molecular process polymerase chain reaction (PCR), which contributed to the successful amplification, classification, and survey of abundance and diversity of AM fungi populations associated with Miscanthus sinensis, Glycine max, and Panacx ginseng from the field. Another study used PCR to determine the influence of Bt and non-­‐Bt corn lines on 20 AM fungi (Tan et al., 2011). They mention that certain primers (e.g., 18S rDNA) are effective at sequencing certain AM fungi (e.g., Glomus) but further primer refinement is needed to increase the reliability of PCR-­‐based processes. In FGS design, perennial polycultures are grown in strips of diverse crops or patches of community support guilds with the intent of cycling diverse resources within the system and creating a plethora of microhabitats for microorganisms and animals. Organic residues and microclimate niches support diverse soil microbes and influence their function and composition. Orchard and agroforestry science provides the most recent and technologically accurate examples of perennial, higher-­‐level diversity production systems that resemble FGS components and insights on plant interactions with soil microorganisms, although the technology and application of it is still in its infancy. 1.6.2 Apple Orchard Plant-­‐Microbe Interactions Current research in holistic orchard management includes the development of practices to reduce N fertility inputs, mitigate nutrient loss, promote productivity, and manage weeds and disease through the implementation of practices that support ecosystem services (Brown, 2012; Granatstein and Sanchez, 2009; Hoagland et al., 2008). There are only a few studies focusing on soil microbe community composition and how they are affected by the abovementioned developments. Hoagland et al. (2008) studied multiple methods of organic orchard ground management systems ranging from cultivation with compost amendments, to wood chip and living mulches. They found that increased compost additions (5.4 and 8.1 kg) with cultivation as a form of short-­‐term nutrient supply did not change 21 the soil microbial activity (nor did it affect tree growth) compared to the control (2.7kg), whereas living mulch treatments (mix of broadleaf and grass species) measured significantly higher in all soil microbial indicators. Compost applied at a rate of 2.7kg was found to provide early growth needs of apple trees compared to increased amounts (Hoagland et al., 2008). Peck et al. (2011) compared integrated and organic fruit production systems, which were managed for weed control using 1) wood chips, mowing and pesticides, and 2) cultivation. Neither treatment intentionally used any plants other than the apple trees. In regards to soil microbe community composition and activity, they found no significant differences between plots due to treatments, but over time they noted small, non-­‐significant shifts, which they attributed to local environmental conditions. Another study showed that the number of culture forming units for fungi in grass (red fescue (Festuca rubra L.)) understory treatments, which grew into a mixture of about 25 herbaceous species, was significantly greater (approximately 200%) than treatments with just bark mulch or herbicide use (Yao et al., 2005). Bacteria numbers were similar over most treatments, except for the treatment in which pre-­‐emergent herbicides were used, where numbers were significantly lower. Although this study presented significant differences, it’s important to note that the use of cultured-­‐based methodologies for measuring microbe composition is out-­‐
dated because only a small portion (1 – 10%) of bacteria and fungi can be cultured in laboratory media reliably (Hirsch et al., 2010). As mentioned before, molecular 22 methods, such as PCR, are much more accurate and effective at capturing a larger portion of a microbe community. These studies demonstrate that living understories in orchards contribute to the increase in the abundance, the activity and the diversity of soil microorganisms while also contributing to production goals. Hoagland et al. (2008) discuss management succession strategies that implement living understories in a way that reduces competition to the main crop during sensitive establishing growth years and that matures into self-­‐sufficiency. This concept and these types of systems are similar to FGSs and tend to implement diversity regarding plant structures, species, functions, and interactions. Continued research into how diverse perennial orchard management systems, such as FGS, affect soil microorganism interactions over time is needed to increase our understanding of and to demonstrate practical models for self-­‐sufficiency. 1.6.3 Agroforestry Plant-­‐Microbe Interactions Current research on agroforestry plant-­‐microbe interactions lacks organic practices and the majority of studies focus on reducing synthetic inputs to row cropping systems. As noted above, this complicates plant-­‐microbe interactions. The available temperate climate agroforestry research on plant-­‐microbe interactions consists primarily of low diversity, tree-­‐based intercropping systems consisting of tree crops in rows with alleys planted to annual crops. The focus of these studies is demonstration of plant effects on soil microbes regarding their function, composition, and physical location in relation to the plant, as well as the effects that varying levels of diverse perennial plants mixed with annuals have on soil microbes. 23 Lacombe et al. (2009) showed that two respective sites practicing tree based intercropping systems consisting of 1) a mix of poplar hybrids and soybeans (Glycine max L. (Merrill)) in Quebec, and, 2) black walnut, silver maple, white ash intercropped with soybeans, winter wheat (Triticum aestivum L.), maize (Zea mays L.), and barley (Hordeum vulgare L.) in Ontario, had significantly higher (approximately 50% and 33% more, respectively) concentrations of AM fungi metabolites than nearby conventional monocropping systems growing the same respective annual alley crops. Locaombe and colleagues (2009) attributed the greater microorganism activity rates in the more diverse systems to the greater variety of tree-­‐based organic inputs. Also, bacteria community composition was significantly different between the Quebec sites, but not so in the Ontario sites, which they speculate was caused by the diverse plantings before and during the experiment in Ontario. Systems that make use of cover crops, such as cereal rye (Secale cereale L.) and hairy vetch (Vicia villosa Roth.), have shown varying amounts of plant biomass produced each year which contributes to changes in microbial biomass and diversity. Nair and Ngouajio (2012) measured significantly higher microbial biomass by 20-­‐30 ug g-­‐1 dry soil in treatments of rye only compared to a rye-­‐vetch mix from 2007 to 2009. Interestingly, the microbial diversity in the rye-­‐only treatment remained stagnant over the two years while in the rye-­‐vetch treatment it increased significantly over each year and eventually surpassed levels in the rye-­‐
only treatment in 2009, measuring 3 and 2.75 on the Shannon-­‐Wiener Diversity Index, respectively. The Shannon-­‐Wiener Diversity Index is used to characterize 24 species diversity in a community; (H’): H’= -­‐Ppi (ln pi) where pi = ni/N; N = total number of fungi sampled; ni = number of sampled fungi in a particular taxonomic group; ln = natural logarithm (Costa et al., 2012). Similarly, while total microbial biomass was similar between adjacent treatments in the agroforestry system (multiple oak species with diverse grass strips interplanted with soy-­‐corn rotation) versus the soy-­‐corn crop rotation alone, the composition of mycorrhizal fungi, gram-­‐negative bacteria, and anaerobic bacteria communities were significantly more diverse by 35%, 15%, 21%, respectively, in the agroforestry system (Unger, et al., 2013). Chifflot et al. (2009) show how higher diversity of plants in tree based intercropping systems results in a higher diversity of AM fungi. In plots of poplar rows with alley-­‐planted soy there were 13 AM fungal phylotypes, of which 7 were common to both plants’ roots, compared to 8 phylotypes in a poplar-­‐only plantation with black plastic mulch. An interesting observation is that poplar roots from both treatments were colonized primarily (53 – 76%) by the same single phylotype, which demonstrates host specificity of some AM fungi (Bever et al., 2012; Turner et al., 2013). Turner et al. (2013) demonstrated that host specificity could occur quite quickly. Specifically, in only four weeks of growth the rhizosphere of three crop plants (peas (Pisum sativa L.), oats (Avena sativa L.), and wheat) selected for significantly different bacterial and fungal communities. These studies demonstrate that tree and other plant species, when interplanted with other crops, increase the diversity of microorganisms with a great deal of specificity and commonality. The explanations as to why these interactions 25 form are not entirely known. In FGS, diverse perennial plantings are a major component, and understanding how they affect other components of the ecosystem is important for informing FGS design and practice. 1.7 Perennial Contributions to Agricultural Systems Some agricultural systems are based on the integration of trees and shrubs and other perennial plants for multiple uses (Table 2). Table 2. Major agroforestry practices and descriptions (compiled by Nerlich et al., 2013) Agroforestry practice Silvoarable systems Silvopastoral systems Orchard intercropping Forest farming Riparian buffer strips Windbreaks Description Trees are planted in single or multiple rows with arable or horticultural crops between the rows Trees are combined with forage and livestock production including high (forest or woodland grazing) and low density (open forest trees) stands Fruit tree systems on arable land or grassland mixed with grazing animals (special agroforestry system) Utilizing forested areas for producing or harvesting natural or cultivated specialty crops for medical, ornamental, or culinary uses Perennial vegetation (grass, shrubs, trees) are planted in strips between arable land or pastures to enhance and protect aquatic resources (streams, lakes) from negative effects of agricultural practices Rows of trees are planted around farms and fields to protect crops, animals and soil from wind. Perennial plants contribute to ecosystem services in multiple ways including; carbon sequestration, nutrient accumulation, water storage and filtration, 26 production of food (e.g., fruits, nuts, greens, fuel, lumber, sugars, fodder) medicine and other human-­‐use yields, habitat creation and shelter, and other mutually beneficial interdependencies (Jose, 2009; Nerlich et al., 2013; Thevathasan and Gordon, 2004; Uprety et al., 2012; Wotherspoon, 2014). The annual internal inputs from these plants, typically with no requirement of external inputs, contribute to soil microorganism function as noted above. Studying the types of inputs provided, whether flows of nutrients or microclimate creation, by different plants is integral to understanding how they might interact with soil microbes. 1.7.1 Internal System Inputs The impacts that trees and shrubs have on system fertility are well documented in tropical climates and are becoming more apparent in temperate climates in regards to agriculture. Similar to compost application, pathways of tree-­‐
based nutrient deposition and crop availability have specific management practices depending on the type of tree and system design (Seiter and Horwath, 1999). There are a wide variety of trees currently under study in tree-­‐based systems with different applications. Examples of trees currently being researched in southern Ontario in this regard include: silver maple (Acer saccharinum L.), hazelnut (Corylus avellana L.), white ash (Fraxinus Americana L.), alder (Alnus spp. Mill.), black walnut (Juglans nigra L.), Norway spruce (Picea abies (L.) H. Karst.), poplar hybrids (Populus spp. L.), red oak (Quercus rubra L.), black locust (Robinia pseudoacacia L.), willow (Salix discolor L.), and white cedar (Thuja occidentalis L.), which are often intercropped with corn (Zea mays L.), soybean (Glycine max (L.) Merr.), and winter wheat (Triticum aestivum L.) (Thevathasan and Gordon, 2004). 27 These trees are chosen for how they would be functionally appropriate to a production system. For example, alder trees grow quickly fixing atmospheric nitrogen into above ground biomass at 197 kg ha-­‐1 at 10 years of age, 70% of which is in the leaves (Uri, 2011). Below ground sloughing of roots, especially at times of coppicing, accounts for a large addition of N to the soil pool. For example, root tissues in 1-­‐year alder trees contained 52 kg N ha-­‐1 and transferred 3.8% of the corn plants’ requirements, but only to those in close proximity to trees (i.e., 63 and 127 cm) (Sieter and Horwath, 1999). Uri et al. (2011) showed that older stands (i.e., 10 years) of alders increased the upper 10 cm soil N content by an average of 26.4 kg N ha-­‐1 year-­‐1. A study in Oregon injected 15N into red alders within hedgerows to determine the transfer between adjacent non-­‐injected trees and alley cropped sweet corn. On average, the trees (1.5 m in height), prior to coppicing, contained 79 kg N ha-­‐1 of which it contributed an average of 5.9 kg N ha-­‐1 to soil N pools over two years (5.4 kg N ha-­‐1 and 6.3 kg N ha-­‐1, in respective years) (Seiter, and Horwath, 1999). The sweet corn plants within the plots that had all above ground alder biomass chipped and equally incorporated, showed equal uptake of 15N compared to unequal (i.e., patchy) uptake in plots where the trees were unmanaged. Ntayombya, and Gordon, (1995) studied the effects that black locust had on nitrogen fertility in intercropped barley under laboratory conditions (i.e., in pots). They concluded that the tree crop not only improved the N content of barley by 11% compared to barley alone, in both loamy and sandy soils, but also reduced the 28 amount of N fertilizer (i.e., 150 to 75 kg N ha-­‐1) needed while maintaining crop yield (4.54 to 4.48 t ha-­‐1, at respective N fertilizer rates). In a ten-­‐year old alley-­‐cropping system, poplar leaf litter was measured at 80% within 2.5 m from the tree row and the remaining 20% dispersed out to 15 m from the tree row, which provided on average 73 and 34 μg N03 100 g−1 dry soil day−1, respectively (Thevathasan, and Gordon, 2004). However, two years later a similar biomass of leaf litter created a heavier skewed range of N03 in the soil at the aforementioned locations (109 and 5.7 μg 100 g−1 dry soil day−1, respectively) which means that litterfall was uneven and inconsistent over the years. In contrast, Bambrick et al. (2010) found that the soils of a 21-­‐year old poplar tree-­‐based intercrop system in Guelph, ON, had on average 5.08 Mg N ha-­‐1 (0 to 20 cm depth) with no significant differences as distance increased from the tree into the alley. Mungai et al. (2006) studied the spatial distribution of total nitrogen (TN) and the C:N ratio within an 11-­‐year, long-­‐term, established alley crop system with silver maple with a no-­‐till corn-­‐soy rotation. Results showed that TN was 0.02% higher at the tree row (0.18 %) compared to 9 m into the alley (0.16 %) and that it decreased by 0.04% and 0.05% with increasing depth at 0 to 10 cm and 20 to 30 cm, respective to the tree row and alley distances from tree. However, N fertilizer was applied in 2001 at 20 kg ha-­‐1 and in 2002 at 130 kg N ha-­‐1, and the alley crops were harvested throughout the years whereas the trees were not. This could have contributed to the higher TN levels within the tree row. Nitrogen fixed by trees is considered an on-­‐farm, financial benefit as it has the potential to reduce the amount of nitrogen fertilizer purchased and applied by farmers (Dyack et al., 1999). In my 29 literature search, there were no temperate climate studies that demonstrate the use of nitrogen fixing trees in orchard production systems. From 1981 to 2006 between 95% and 82% of Ontario agricultural land had decreasing soil organic carbon with the majority of land decreasing at a rate of 25 to greater than 90 kg ha-­‐1yr-­‐1, which is likely due to historically high rates of soil erosion from row crop cultivation and the conversion of perennials to annual crops (Eiler et al., 2010). There are many studies demonstrating the potential gain in soil organic carbon through the implementation of trees into food production systems. Bambrick et al. (2010) found that soil organic carbon is expected to increase over time after agricultural land is converted to agroforestry practices. For example, a significant gain (11.5% greater) in soil organic carbon was seen in a 21-­‐year old poplar intercropped plot (57.0 Mg C ha-­‐1) when compared to a same-­‐age conventionally managed farm nearby (50.8 Mg C ha-­‐1). In this same study, three other experimental locations, each containing both a tree-­‐based and conventional section, in Quebec were measured for soil organic carbon. Two of them, both 4 years old, had no significant increases between tree-­‐based and conventional sections (66.9 vs. 66.3 Mg C ha-­‐1, in St. Paulin, and 76.9 vs. 80.1 Mg C ha-­‐1, in St. Edouard). The third location, in St. Remi, was 8 years old and the tree-­‐based section had significantly more soil organic carbon than the conventional section at 77.1 and 43.5 Mg C ha-­‐1, respectively. A study of a 12-­‐year-­‐old silver maple alley cropping system reinforces the potential additions of soil carbon that are possible with agroforestry systems. Maple tree annual leaf litter inputs of 2942 kg ha-­‐1 showed that there is a significant 30 addition of carbon to the top 0 to 10 cm (1.45% to 1.95%) of the soil with no significant differences with distance from trees (Mungai et al., 2006). Oelbermann, and Voroney, (2007) found similar results in a 20-­‐year established hybrid poplar alley cropping system in Guelph, Ontario. They measured a mean carbon input from crop residues, including roots, of 134 g C m-­‐2 year -­‐1 with an additional 117 g C m-­‐2 year –1 from tree leaf litterfall. Wotherspoon (2014) demonstrated that poplars, oaks, walnuts, spruces, and cedars, when planted at 111 trees ha-­‐1 in Southern Ontario, sequestered in their total (above and below ground) biomass 27, 16, 15, 13, and 16 t C ha-­‐1, respectively, over the past 25 years. Annual sloughing of the roots and litterfall of these trees contributes to the soil organic carbon (SOC) stock. For example, the aforementioned trees contributed to a SOC stock of 86.86, 83.77, 83.23, 78.29, 76.84 t C ha-­‐1 year-­‐1, respectively, with the greatest percentage of SOC in the top 10 cm. Other agroforestry practices, such as riparian buffer zones and short-­‐rotation woody crops, have been shown to have great potential for sequestering carbon. Montagnini and Nair (2004) estimated that if 8 million km of 30 m wide (total of 59.3 million acres) forested riparian buffer were newly planted in place of annual crops in Canada, it would sequester 1.5 Tg C yr-­‐1 or exceed 30 Tg C in 20 years. They noted that there is 140.9 million acres of marginal or degraded land in riparian areas suitable for establishment of trees in Canada. Wotherspoon (2014) provides a more in depth review of the potential that trees in tree-­‐based intercropping systems contribute to sequestering carbon for climate change mitigation. Carbon stored in high value trees, such as black walnuts, can also contribute to on-­‐farm income via 31 the veneer, pallet, and saw log markets in Canada at lucrative rates of CDN $2020, CDN $1066, and CDN $300 per 1000 board feet, respectively (Dyack et al. 1999). Water erosion is the greatest contributor to soil erosion risk and in 2006 Ontario had 57% of cropland in unsustainable risk category, which is explained by the high proportion of land intensively cropped with corn and soybean (Eiler et al., 2010). Trees deposit organic matter, such as leaves, which contribute to the creation of water stable aggregates, which build soil structure (Tsonkova, 2012). In an experiment studying water stable soil aggregates on a slope in Missouri, Udawatta, et al. (2008) measured double the proportion of water stable soil aggregates (WSA) in the agroforestry systems with 4 m wide strips consisting of redtop (Agrostis gigantean Roth), brome grass (Bromus spp.), birdsfoot trefoil (Lotus corniculatus L.), and pin oak (Quercus palustris Muenchh.) compared to a no-­‐till, corn-­‐soy rotation planted on contour (measures of 15% and 7.6% of WSA, in the two systems, respectively). Measuring the percentage of WSA in soils indicates soil health in regard to water and air movement, and resistance to soil and mechanical disturbance (Udawatte et al., 2008). Soil carbon was deposited in significantly higher amounts from the top, to the middle and bottom sections of the slope within the row crops (1.2%, 1.7%, and 2.05% total soil carbon in surface soil, respectively) compared with the agroforestry (2.2%, 2.3%, and 2.7% total soil carbon in surface soil, respectively). The larger proportions at the bottom of the slope were assumed to be the result of increased soil moisture, slower decomposition, and additions from erosion. 32 Eutrophication of fresh water systems in Southern Quebec and leaching of N and P from agricultural fields have also become topics of research that is relevant to FGS. Bergeron, et al. (2011) demonstrated that trees, specifically hybrid poplar, black walnut, and red oak, can significantly reduce leaching of NO3-­‐ through root uptake in deeper soil layers. In 2006 and 2007, they measured significantly greater NO3-­‐ leaching rates in root-­‐pruned intercropped plots compared to unmanaged intercropped plots, especially during October and November, 2006, when NO3-­‐ leached at 7.5 kg ha-­‐1 and 0.3 kg ha-­‐1, respectively. This “safety-­‐net” effect is a benefit of deep-­‐rooted plants that re-­‐cycle leached nutrients back into the system. This can reduce application rates of N fertilizer and decrease the risk of non-­‐point pollution (Thevathasan and Gordon, 2004; Tsonkova et al., 2011) However, further research is needed to demonstrate design and management practices that provide the benefits of the “safety net” effect in addition to reducing competition for water. These studies demonstrate the ability of trees to contribute significant amounts of N and C to agricultural systems with minimal inputs. They also show the potential for trees with diverse structures and ecological functions to generate soil and enhance soil structure, which preserves and recycles carbon, nutrients, and water on site. Understanding where, how, why, and to what extent soil microorganisms facilitate these processes may help in the design of more intentional production systems. Implementing perennials into systems in ways that maximize contributions through interactions with soil microbes while limiting competition, can help reduce costs, both financial and environmental, of external 33 inputs and enable multiple, diverse income streams for producers, while also regenerating the soil, air, and water. 1.7.2 Biodiversity and Balance Plant diversity and disturbance regimes (i.e., cultivation and management) help to shape the biodiversity of the entire production site, which, if designed for complementary functionality, can result in the improvement of multiple ecosystem services including soil microbe functions. Landscape heterogeneity is a component of design that can be described as the complexity of the area of study and adjacent areas (Plecas et al., 2014). Landscape heterogeneity has been considered in studies with respect to determining the effects on predator and prey communities and their interactions (Fenoglio et al., 2013; Plecas et al., 2014). Multiple variables, including mean field size, percentage of arable land (crop) and wild forest or grassland (non-­‐crop), Shannon habitat diversity index, and human settlement, can be taken into consideration when determining the effects on biological control, habitat creation, formation of microclimates, and soil microbial population structure, which all contribute to ecosystem stability. For example, research by Thevathasan et al. (2004) in Guelph, Ontario, has shown 10 times the biomass of worms under three different tree species in intercropped systems compared to conventional corn crops with a majority of the worms located within the canopy drip line of the trees. Their study also showed that trees provided habitat for small mammals and birds; for example, 10 species of birds, including carnivorous raptors, foraged in the 34 intercropped fields whereas only 2 species were found in the conventional fields (Thevathasan et al., 2004). Stamps and Linit (1998) stated that arthropod diversity is much higher in more diverse, tree based systems, and this is supported by results from a study by Altieri and Schmidt (1986), which found 10 times greater numbers of the aphid predator Coccinella quinquepunctata L. in apple orchards surrounded by deciduous forest compared to orchards without forests nearby. A study in Beijing, China, tested how aromatic herbs affected pest beetle communities in pear orchards (Tang et al. 2013). They found that intercropping with aromatic plants, of which there were 9 respective species, significantly reduced the annual cumulative numbers of individual beetles, but had no significant influence on the parasitoids of beetles. Another study by Brown (2012) showed that interplanted extrafloral nectar-­‐bearing peach and cherry trees within apple and pear orchards increased biodiversity and contributed to reduced pest injury. Poch and Simonetti (2013) provided evidence of habitat creation via trees for predatory insect biological control, stating that it is profitable for both financial and environmental reasons. The presence of unmanaged, non-­‐crop land, such as hedgrows, has effects on biological control in nearby agricultural fields. A study in south-­‐eastern France on Codling moth (Cydia pomonella L.) distribution showed significantly lower abundances of larvae in orchard traps that were near a hedgerow. The physical structure and floral composition were measured for 205 hedgerows around 91 orchards (Ricci et al., 2011). They found that hedgerows affected the wind, shade, 35 and moisture levels within their microcosm, and that contributed to consistent spatial heterogeneity of codling moth larvae year-­‐to-­‐year. Maalouly et al. (2013) found that reducing pesticide application could result in increased codling moth parasitism (organic and conventional orchards had a calculated parasitism rate, “ratio of emerging parasitoids to the total number of adult individuals,” of 1.289 x 10-­‐3 and -­‐0.025 x 10-­‐3, respectively), but perhaps not to commercial industry standards. They also found that, much like the herbs in the previous mentioned study, hedgerows spontaneously placed throughout and around orchards provided alternative food sources and hosts for parasitoids, while also limiting codling moth dispersal. This is important because it provides an understanding of how microclimates influence where the pest species locate within an orchard and can help facilitate the management of these pests via landscape design. In Scotland, UK, it was demonstrated that characteristics of woodlands such as density, canopy cover, and tree diameter, have effects on insect communities (Fuentes-­‐Montemayor et al. 2013). The number of insects in the order Diptera and sub-­‐order Lepidoptera were higher in young successional stage trees in stands with high tree densities and abundant understory cover, but no full canopy. The study of woodland ecologies can help us to understand the dynamics and possible management regimes that encourage certain forms of pest control. Systems that incorporate trees or maintain naturally forested land create habitat for predatory-­‐prey community interactions. The results found show that with increased plant diversity comes increased animal diversity, both of which provide a variety of organic inputs to the soil as well as potential set back in 36 production goals. Future research could include determining the effects that hedgerows, neighbouring natural vegetation zones, and migrating animals have on soil microbial function and composition, as well as how they might be incorporated into production systems in a way that balances loss of production area with production. 1.8 Interactions Between Plants and Management Tree based systems are land use practices which incorporate trees in attempts to generate economic, ecological, and cultural benefits while minimizing the negative effects of competition (Reynolds et al. 2007). Dyack et al. (1999) concluded that the interactions between row crops and tree crops on a given land area, need to be more profitable than either of the two individual activities in order for landowners to adopt these practices. Thevathasan et al. (2004) identified common areas of competition between trees and crops for soil space, solar radiation, water and nutrients. Corn and soy plants intercropped with poplar and maple displayed symptoms of reduced growth up to 2 m from the tree row, but not 6 m away from trees or in control plots. On average, they found a 20 to 25% yield reduction per unit land-­‐base for corn intercropped with poplar. They attributed most of the yield loss to shading by the tree canopy, which significantly reduced photosynthetic radiation, net assimilation, and growth of the corn plants. Water deficit measurements did not significantly correlate with corn growth or net assimilation. Another study of corn and soy intercropped with separate rows of maples and poplars also showed significantly reduced yields within 2 m of the trees (soy yield = 1.04 t ha-­‐1 at 2 m from poplar 37 trees), but were similar to the control at 6 m from trees (2.59 and 1.97 t ha-­‐1, respectively) (Reynolds, et al. 2007). These results also suggest that yield reductions are not due to competition for water but for light and Reynolds and colleagues showed that the photosynthetically active radiation (PAR) levels were significantly lower within 2 m of poplar rows compared to control plots corn (952.0 and 1528.0 umol s-­‐1 m-­‐2, respectively) and soy (1133.0 and 1464.0 umol s-­‐1 m-­‐2, respectively) plots. There is potential to manage competition for light with coppicing regimes, especially with poplar, which coppices effectively (Tsonkova, et al., 2012). Alternatively, plants that function in shade, such as Rubus and Ribe species, could be planted in the lower production area between trees and field crops. Regarding water competition, Jose et al. (2000) found that black walnut tree roots compete with corn roots in the upper 30 cm of soil for water, which resulted in less absorption of N and reduced corn grain and stover biomass compared to no tree root competition (Grain yield = 3.6 and 7.0 Mg ha-­‐1, respectively, and stover biomass = 3.9 and 5.8 Mg ha-­‐1, respectively). They recommended tree root pruning and temporal spacing of alley crop planting to reduce the negative effects of root-­‐based competition. These studies provide evidence that trees have significant effects on alley crops with possible management practices to prevent and reduce competition. The effects of competition are thought to only be associated with plant structural and functional competition, but soil microorganisms play a role in nutrient transformation and transportation, as well as plant-­‐plant interactions, which could be confounding in these cases (Bever et al., 2012). Keeping these relations in mind 38 during system design could help to create complimentary perennial polycultures that take advantage of shaded or water scarce-­‐microclimates created by certain plants for optimal production in a given space. Future research studying the roles that soil microbes play throughout the growing season on plant-­‐plant interactions, and nutrient storage, can contribute to the design of functional plant communities for specific production goals. 1.9 Future Work The development of temperate climate forest garden systems is occurring at an increasing rate in practice but FGSs remain relatively unstudied. The environment is set for academics to support farmers in the refinement of FGS practices, through research and sharing. Currently, research exists in orchard science and agroforestry which can guide the practice of FGSs, and there are many areas open for future research. The University of Illinois is taking progressive novel research approaches in this regard and they are creating the model to lead other academic institutions (Woody Perennial Polyculture Research Site, 2013). Providing empirical evidence that shows the effects of these diverse perennial production practices, in parallel with education on holistic production frameworks, will allow potential FGS farmers, and academic researchers to regain power in their vocation. The impact on soils and the functionality that results from changes to the soil is a key area of interest not only from potential FGS practitioners but also from researchers. Providing clear evidence that shows how regionally appropriate planting systems affect the ecosystem services provided by soil microorganisms is a powerful base research area for FGSs. 39 Further data and the creation of practical examples are needed for 1) observation of regionally appropriate ecosystems, 2) applications of agroecology and permaculture frameworks in design, 3) measuring plant composition effects on soil microbe community structure and function, 4) how diverse FGS orchards are designed and how associated soil microorganism interactions mature over time, 5) multi-­‐functional perennial polycultures to produce diverse, marketable, human-­‐use yields, and 6) how adjacent undisturbed, natural areas and associated soil microbes affect crop production land. Results from this research would help to support producers in better designing and managing temperate FGSs and making the transition towards holistic, more ecosystem service-­‐based practices. This study provides the first ever, academic example of a temperate-­‐climate, apple-­‐based forest garden system. It will offer new data on the quantification and composition of soil bacteria and fungi in a temperate-­‐climate apple-­‐based forest garden system. This empirical data can support farmers using locally appropriate practices in FGSs and provide academics a base to further our scientific understanding of ecosystem-­‐based approaches to food production. 2.0 Materials and methods 2.1 Site Description This study was carried out in 2013 and 2014 at three sites in southern Ontario. 1) Guelph Centre for Urban Organic Farming (GCUOF) at the University of Guelph (43°32’18”, -­‐80°13’ 20”) Guelph, Ontario; 1 ha of urban agricultural land 40 established in 2008 and managed organically. The Guelph area had a mean annual temperature of 6.5°C and a mean annual precipitation of 923 mm (Government of Canada, 2015). This site has a Canadian Land Index of 3, soils that are Luvisols with a loam texture (40% sand, 40% silt, 20% clay), a pH of 7.2, and soil organic matter (OM) of 4.9%. This experimental site was previously planted to mixed perennial vegetables, which were managed organically, and was within 10 m of a mixed forest ecosystem as well as 200m away from multiple apple tree varieties. 2) Ignatius Farm (IFARM) (43°34’10”, -­‐80°17’24”), Guelph, Ontario; 242 ha of organic agricultural land and conservation area which has been managed organically for the past 100 years at least. The Guelph area had a mean annual temperature of 6.5°C and a mean annual precipitation of 923 mm (Government of Canada, 2015). This site has a Canadian Land Index of 3, soils that are Luvisols with a loam texture (40% sand, 40% silt, 20% clay), a pH of 7.5, and soil OM of 3.8%. This experimental site was previously planted to mixed annual vegetables, which were managed organically, and was within 10 m of a woody vegetated wetland as well as 10 m from crab apple trees and 150m from many varieties of apples. 3) Iceland Teaching Garden (ITG) at Ecosource’s Community Garden (43°37’32”, -­‐79°39’10”) Mississauga, Ontario; a quarter-­‐hectare urban garden that has been under organic production practices for the past 2 years. The greater Toronto area has a mean annual temperature of 9.4°C and a mean annual precipitation of 831 mm (Government of Canada, 2015). This site has a Canadian Land Index of 1, soils that are Luvisols with a loam texture (40% sand, 40% silt, 20% clay), a pH of 7.4, and soil OM of 3.7%. This experimental site was previously 41 planted to mixed annual vegetables, which were managed organically, and were within 10 m of a woody vegetated wetland as well as 50m of crab apple trees. All sites were tilled early spring 2013 (S13) in preparation for planting (Table 3). Apple trees (Malus domestica cv. Idared on M9 rootstocks) were planted as one-­‐year bare roots, pruned to 106 cm in height, in May 2013 at all three sites. The particular cultivar and rootstock were chosen as a result of availability at that time. Thirty-­‐two trees were planted between the sites: 8 at ITG, 12 at GCUOF, and, 12 at IFARM. Weather data for Guelph area and Toronto area for 2013 and 2014, as well as past averages available in Appendix A. 2.2 Experimental Design The experiments at each site were set up as a completely randomized design with two, three, and three replicates of four ground management system (GMS) treatments at the respective sites, ITG, GCUOF, IFARM (Figure 2), with goals to compare the effects of different levels of plant diversity and compost addition on soil microbial abundance, tree growth and soil properties. The experimental units (EU) consisted of respective 1.5 m by 4.5 m (6.75 m2) areas of soil with one apple tree and one GMS treatment in each unit. The four GMS treatments were as follows: 1) G: an equal mix of three grasses: Kentucky blue (Festuca arundinacea cv. Schreb.), creeping red fescue and perennial rye grass (Lolium perenne L.), was hand broadcast in spring each year for better establishment and a 15 cm layer of coarse softwood chip mulch was laid around the root zone of the apple tree (0.75 m2); 2) GC: same as G with 2.7 kg (Hoagland et al., 42 2008) of organically certified turkey litter compost (Cold Springs Farm, Guelph, ON, CAN; 1.02% N, 19.06% OM, 1.31% P, 0.72% K, and a C:N ratio of 10.6) applied around the root zone of the apple tree in the first year (Figure 3); 3) FGS: Forest garden system, holistically designed understory of diverse, perennial support plants (comfrey (Symphytum x uplandicum cv. Blocking 14), chives (Allium tuberosum cv. Rottler ex Spreng), sorrel (Rumex acetosa L.), lupines (Lupinus perennis L.), white clover (Trifolium repens L.), mint (Mentha x piperita L. cv. Chocolate Mint), bergamot (Monarda fistulosa L.), onion (Allium proliferum cv. (Moench) Schrad, ex Willd), and Siberian pea shrub (Caragana arborescens Lam.) (Shortt and Vamosi, 2012), and a 15 cm layer of coarse softwood chip mulch around all plants (2.25 m2) (Jacke and Toensmeier, 2005); and, 4) FGSC: same as FGS with 2.7 kg of organically certified turkey litter compost applied around the root zone of the apple tree in the first year (Figure 4). 43 Figure 2. Completely randomized design of three experimental sites presenting the layout of replicated treatments. At IFARM there was an area (3 m by 3 m) of organic annual veg production between two areas of the experiment. 44 Table 3. Main research activity timeline across all three experimental sites from May to November in 2013 and 2014. 2013 Nov. -­‐ -­‐ Planting and seeding of treatments 22 to 6 -­‐ -­‐ -­‐ -­‐ -­‐ Soil sampling 12 to 20 -­‐ -­‐ -­‐ -­‐ 29 to 5 Hand scything of G-­‐
treatments -­‐ -­‐ 15 to 30 -­‐ -­‐ 29 to 5 Hand weeding and mulching of FGS-­‐
treatments -­‐ -­‐ 15 to 30 -­‐ -­‐ 29 to 5 Apple tree trunk measurements 22 to 6 -­‐ -­‐ -­‐ -­‐ -­‐ Insect herbivory measurements 31 -­‐ -­‐ -­‐ 8 -­‐ -­‐ Large mammal herbivory measurements -­‐ -­‐ -­‐ -­‐ -­‐ 29 to 5 Small mammal herbivory measurements -­‐ -­‐ -­‐ -­‐ -­‐ 29 to 5 Nov. Oct. -­‐ Oct. Sept. -­‐ Sept. Aug. -­‐ Aug. July -­‐ July June 10 to 20 June May Soil tilled May Activity 2014 -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ 19 to 25 -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ 19 to 25 -­‐ -­‐ -­‐ 22 to 1 -­‐ 19 to 25 -­‐ 1 to 8 -­‐ 22 to 1 -­‐ 19 to 25 -­‐ 1 to 8 -­‐ 22 to 1 -­‐ -­‐ -­‐ -­‐ -­‐ 22 to 1 -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ -­‐ 22 to 1 -­‐ -­‐ -­‐ -­‐ -­‐ 22 to 1 45 The plants in the forest garden-­‐based treatments were chosen for their known functions, based on anecdotal and academic sources, and how these species might contribute to the ecology of this production system. Aggressive perennials rather than annuals were selected for purposes of reduced weed competition and to prevent the need for cultivation. Eight plants in the forest garden-­‐based treatments provide sources of nectar throughout the whole season, from April to October (Table 4). Nitrogen fixing plants, consisting of the Siberian pea shrub, white clover, and lupines, were planted to an area of 40% of the apple tree’s mature canopy area (Crawford, 2012) and were spaced accordingly in regard to plant growth, sun position, and shade development. 46 Figure 3. Perspective from above of experimental design of grass treatments with apple tree in centre and mix of three grass species as understory. 47 Figure 4. Perspective from above of experimental design of forest garden system treatments. Number in brackets represents the number of plants at initial establishment per EU (6.75 m2). Siberian pea shrubs are considered exotic, but not noxious, in North America. They provide multiple functions to a production system, such as: tolerance to extreme temperatures (-­‐38oC), drought, infertile soils, high winds, and saline conditions; production of inhibitory compounds that deter the growth of some grass species; 48 fixing nitrogen at low temperatures (3-­‐5oC); alternative herbivore browsing (i.e., deer eat it) and fodder production for livestock; ability to coppice for fuel, fiber, and dyes; medicinal applications for multiple diseases; soil stabilization; edible pods that can be cultivated as a vegetable; and, flowers for pollinators (Shortt and Vamosi, 2012). Table 4. Approximate flowering times over the growing season of eight plants in forest garden treatments and functional animals that they may support (compiled from Jacke and Toensmeier, 2005). Oct x Sept x x x x x x Aug x x Jul x Hummingbird Jun Generalist Nectary May Apple Siberian pea shrub White clover Lupine Chives Comfrey Bergamot Mint Apr Common Name Approximate Flowering Times Sorrel, lupine, and white clover, have been used as cover and catch crops to help fix nitrogen and reduce leaching of nutrients (Askegaard and Eriksen, 2007). They also provide potential secondary crops for culinary, medicinal and ornamental purposes, as well as nectar-­‐producing flowers for pollinators. 49 Mint and bergamot are used for: culinary and medicinal purposes, thick sprawling growth patterns, flowers that attract pollinators and aromatherapy (Jacke and Toensmeier, 2005). Chives and onions can act as possible secondary crops, flowering plants for pollinators, and weed exclusion with clumping and sprawling growth patterns, respectively (Jacke and Toensmeier, 2005). Comfrey is an aggressive species that can create barriers to keep grass and other unwanted plants out of certain areas. It can be coppiced multiple times in a season for mulch purposes and produces flowers for pollinators (Jacke and Toensmeier, 2015). Unwanted plants in G and GC EUs were manually mown bi-­‐monthly with a hand-­‐
held scythe and, in FGS and FGSC, unwanted plants were hand pulled in the first year and mulched with woodchips from within respective plot in the second year (Table 3). Woodchip mulch was only applied in spring 2013. No fertilizers, other than compost, nor pesticides were used prior to planting for bed maintenance or during management over the two years. Apple trees were measured for growth and herbivorous damage. Tree trunk circumferences 15 cm above grafting point were measured before planting and at the end of the second growing season, F14. Herbivore-­‐related damage was measured by rating the damage to foliage, including young branches, and trunk from 0 to 10 at the end of each season (November 2013 and 2014). Vole, rabbit, and other small mammal herbivore damage to apple trees, which mostly occurred as trunk bark damage, was deterred with reusable plastic spiral wraps. Deer herbivore 50 damage was deterred with bird netting and stakes around individual trees, to little avail, and larger permanent fencing where it already existed (E.g. the Mississauga site). Insect herbivore damage was measured and then the pest was stripped by hand if tree was at risk of death (only occurred once in first year). 2.3 Sampling and Laboratory Analysis Soils in the EUs were sampled spring (June) and fall (October) for two consecutive years (2013 and 2014), including a baseline sample, before anything was planted, in May 2013 (Table 3). All soil samples in this study were taken to a 10 cm depth and at consistent locations over the 4 sampling periods at least 30 cm in from the borders of each EU (Figure 4). Ten composite soil samples were collected per EU with a stainless steel corer with a 1.5 cm diameter. Rocks and surface debris were manually removed and the samples were each sealed in a ziplock bag, and transported in a cooler with ice packs to a 4oC fridge. They were stored there for max 48 hours until microbial analysis, and for up to one year until physical and chemical analysis. 51 Figure 4. Layout of soil samples for 10 composite samples of experimental units in 2013 and 2014. Within 48 hours of sample collection, 0.25 to 0.55 g subsamples of soil were taken from respective EU samples for microbial DNA extraction using the MoBio UltraClean Soil DNA extraction kit and following manufacturer’s protocols (Mo-­‐Bio, Carlsbad, CA, USA). Subsamples of DNA were then used for analysis and the stock DNA was labeled and stored in freezer (-­‐80oC). 52 The extracted DNA was analyzed for respective target gene sequence abundance of arbuscular mycorrhizal (AM) fungi, total fungi and total bacteria using the molecular technique real-­‐time quantitative polymerase chain reaction (qPCR) by Kari Dunfield’s Lab, School of Environmental Science, University of Guelph (Guelph, ON, CAN). Briefly, qPCR is a process of multiple cycles of denaturing DNA, annealing primers, and extension of gene sequences for the purpose of quantifying gene sequence copies. The primers AML 1F/ AML 2R (Lee et al., 2008), ITS 1F/ ITS 4R (Gardes et al., 1990), and 338F/ 518R 16S (Fierer et al., 2005) were used during the annealing phase to target specific gene sequences for AM fungi, total fungi, and total bacteria, in respective qPCR processes. A fluorescent dye, SsoFastTM Evagreen Supermix #172-­‐5201 (BioRad, Mississauga, ON, CAN), which linked to the target gene sequence, was included for the purpose of measuring amplification of target gene sequences between the annealing and extension phases of qPCR using a CFX Real-­‐Time PCR Detection System (BioRad, Mississauga, ON, CAN). The measurement of threshold amplification was then used to calculate gene copy per gram of dry soil for the respective soil microbial communities. In preparation for qPCR, a standard curve was created via a process of 1) cloning desirable gene sequences from environmental fungal samples of Gaeumannomyces incrustans and fungi removed from Flacourtia jangomas roots for total fungi and AM fungi, respectively, and a pure culture of Bacteroides fragilis for total bacteria using TOPO TA Cloning Kits (Invitrogen, Burlington, ON, CAN) according to manufacturer’s protocol, 2) amplifying plasmids containing desirable gene sequences using PCR in a Mastercycler Epgradient S thermocycler (Eppendorf, 53 Hamburg, Germany), 3) isolating plasmid DNA using QIAprep spin mini-­‐kit (Qiagen, Inc., Mississauga, ON, CAN), and, 4) determining the concentration of plasmid DNA by serial dilution using Nanodrop 8000 technology (Thermo Scientific, Mississauga, ON, CAN). The standard curve used as a reference in qPCR ranged between 101 and 108 gene copies and had a a PCR efficiency between 85% and 110%, a slope between -­‐3.1 and -­‐3.7, and a R2 value greater than or equal to 0.99. Samples were also analyzed for soil pH (1:1 method) (Carter and Gregorich, 2006), OM (Walkley-­‐Black method adapted to include dichromate as colour indicator) (Handson et al., 1996), phosphorus (sodium bicarbonate method) (Olsen et al., 1954), potassium, and magnesium (ammonium acetate reference method OMAFRA Cations) by Agri-­‐food laboratories (Guelph, ON, CAN) (Carter and Gregorich, 2006). 2.4 Statistical Analysis Analysis of variance (ANOVA) was performed using the PROC MIXED procedure in SAS (version 9.4 Statistical Analysis System, SAS Institute Inc., Cary, NC, USA, 2013) to assess the relationship within and between different treatments and sample times with AM fungal, total fungal, and bacterial abundances, and apple tree diameter as dependent variables. Using this model variance was partitioned into fixed effects (treatments and season), the fixed effects interaction (treatment*season), random effect (site), and random effect interactions (site*treatment site*season site*season*treatment). The major assumptions of ANOVA include: the model is a linear additive model, the variables are independent of each other, and errors are randomly and normally distributed around a mean of 54 zero and have a common variance. These assumptions were tested before analysis by examining patterns in errors using PROC PLOT (residual vs predicted plot), Shapiro-­‐Wilks normality test, and a comparison of covariance structures. A repeated measures analysis was used to account for the lack of variable independence which was due to repeated measurements over time on the experimental units and apple trees. The covariance structure compound symmetry was used. Lunds test was also conducted to test for outliers. Multiple means comparisons of soil microbial abundances, soil properties, and tree measurements were compared using a Tukey adjustment (α = 0.05). Significant differences of least-­‐squared means were determined statistically significant based on p-­‐values (P=0.05 to <0.05) and marginally significant (P=0.10 to 0.051). 3.0 Results 3.1 Abundance of soil bacteria and fungi With regards to soil bacteria abundance there were significant differences in all respective treatments over time but not between treatments at any one sampling period. In spring 2014 (s14) forest garden system with compost (FGSC), forest garden system (FGS), grass mix with compost (GC), and grass mix (G) treatments across all sites, were on average, all significantly less compared to levels in fall 2013 (f13) (Tukey P=<0.0001 for all). Specifically, from S13 to F14 bacteria abundance changed within FGSC, FGS, GC, and G by -­‐0.78, -­‐0.84, -­‐0.86, and -­‐0.88 ± 0.08 Log 16S gene copies g-­‐1 dry soil, respectively (Figure 5). 55 Total fungi abundance significantly changed for all treatments over time, but not between treatments, at any one sampling period. Averaged over all sites, FGSC, FGS, GC, and G, all respectively increased from f13 to s14 by 2.12, 1.86, 1.82, and 1.78 ± 0.15 Log ITS gene sequence copies g dry soil-­‐1 (Tukey P=<0.0001 for all), (Figure 6). Averaged over all sites, arbuscular mycorrhizal (AM) fungi abundance significantly lowered in all treatments (FGSC, FGS, GC, G) between F13 to S14 (P = 0.005) by -­‐1.73, -­‐2.15, -­‐2.23, -­‐2.04 ± 0.55 Log AML gene sequence copies g-­‐1 dry soil, respectively (Figure 7). 16S rRNA gene abundance, Log 16S gene copies / g dry soil 10.5 a a a a 10 a a a a b b b b 9.5 b b b b FGSC FGS 9 GC 8.5 G 8 Spring 13 Fall 13 Spring 14 Fall 14 Season and Year Figure 5. Average change in total soil bacterial abundance affected by four treatments over two years in three sites across Guelph and Mississauga, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. ITS gene abundance, Log DNA / g dry soil 56 6 b b b b 5 b b b b 4 3 a a a a FGSC a a a a FGS 2 GC 1 G 0 Spring 13 Fall 13 Spring 14 Fall 14 Season and Year Figure 6. Average change in total soil fungi abundance affected by four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. AML gene abundance, Log DNA / g dry soil 57 7 a a a a a a a a 6 b b b b 5 b b b b 4 FGSC 3 FGS 2 GC G 1 0 Spring 13 Fall 13 Spring 14 Fall 14 Season and Year Figure 7.1. Average change in soil arbuscular mycorrhizal fungi abundance affected by four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. Gene abundance, Log DNA / g dry soil 58 7 a a a a a a a a 6 5 b b b b b b b b 4 Spring 13 3 Fall 13 Spring 14 2 Fall 14 1 0 FGSC FGS GC G Season and Year Figure 7.2. Average change in soil arbuscular mycorrhizal fungi abundance over four sampling periods within four respective treatments averaged over three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. 3.2 Tree Growth Although all apple tree trunk circumferences showed growing trends, only FGSC treatments averaged over all sites had a significant effect on circumference over two years (Tukey P=0.04), growing from 2.31 ± 0.88 cm in S13 to 6.34 ± 0.88 cm in F14 (Figure 8). Trees in FGS treatments averaged across all sites marginally increased in circumference from S13 to F14 by 3.44 ± 0.88 cm (Tukey P=0.079). The only difference between treatments occurred in the last sampling period, F14, between the averages over all sites of FGSC and G treatments by 1.31 ± 0.3 cm (Tukey P=0.048) (6.34 ± 0.3 cm and 5.03 ± 0.3 cm, respectively). 59 Apple tree trunk circumference (cm) 8 Bb 7 ABb ABa 6 Aa Spring 13 5 4 3 Aa Aa Aa Aa Fall 14 2 1 0 FGSC FGS GC G Treatment Figure 8. Average growth of apple tree trunk circumference in each of four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments., and the letters (A, B) indicate significantly different means between treatments. 3.3 Soil Nutrients No significant changes to soil phosphorus, potassium or magnesium (Figure 9) occurred between treatments or over time. There are no clear trends specific to compost amendments or any particular treatment (Table 5). 60 330 Magnesium ppm 310 290 a
a
a
a
a a
a
a
270 250 FGSC 230 FGS 210 GC 190 G 170 150 Spring 13 Fall 14 Season and Year Figure 9. Average changes in soil magnesium concentration (ppm) affected by four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. Soil pH was not significantly affected over time nor significantly different between treatments. All are slightly basic ranging between 7.4 and 7.5 ± 0.06 (Figure 10). There were no significant changes in soil organic matter between treatments nor within treatments over time (Figure 11). 61 7.6 a
7.55 7.5 Soil pH 7.45 a
a
a
a
a
a
a
7.4 FGSC 7.35 FGS 7.3 GC 7.25 G 7.2 7.15 Spring 13 Fall 14 Season and Year Figure 10. Average changes in soil pH affected by four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. 62 Soil organic matter % 6 5 a
a
a
a
a
a
a
a
4 FGSC 3 FGS 2 GC 1 G 0 Spring 13 Fall 14 Season and Year Figure 11. Average changes in soil organic matter affected by four treatments over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b) above bars indicate significantly different means within respective treatments. 63 Table 5. Average apple tree measurements and soil chemical analysis under four ground management systems over two years in three sites across Mississauga and Guelph, Ontario, Canada. Different letters (a, b, c) indicate significantly different means within respective treatments.
Treatment FGSC FGS GC G Season and Year Spring 2013 Fall 2014 Spring 2013 Fall 2014 Spring 2013 Fall 2014 Spring 2013 Fall 2014 (Phosphorus ppm) Phosphorus Potassium Std. Error (ppm) (ppm) Potassium Std. Error (ppm) 19.97a 1.57 97.35a 8.37 20.28a 1.57 108.38a 8.37 19.49a 1.57 97.11a 8.37 14.00a 1.57 99.31a 8.37 20.58a 1.57 115.06a 8.37 23.69a 1.57 109.56a 8.37 17.87a 1.57 90.95a 8.37 15.23a 1.57 101.99a 8.37 4.0 Discussion All orchard floor management treatments contributed to significant shifts in abundance of all studied soil microbes throughout the first two establishing years, spring 2013 (S13) to fall 2014 (F14). All treatments respectively increased total fungi abundance, decreased total bacteria abundance without significant differences between treatments. This resulted in a higher total fungi: total bacteria ratio (F:B) for all treatments in 2014 compared to 2013. Continuous vegetative cover (e.g., permanent cover crop or living ground cover) without tillage and varied complex organic matter inputs that 64 contain high carbon concentrations have been acknowledged as factors contributing to a higher F:B. Unger et al., (2013) compared tree-­‐based intercropping systems (AIS) with vegetative strips of grass (VSG) and annual crop systems (CS), all of which were planted in 1991. They showed a similar lack of significant difference in overall soil microbial abundance (SMA) between treatments in 2009. With more analysis, significant shifts of specific bacteria and fungal communities were found. For example, the AIS soil had 23% more anaerobic bacteria and 35% more mycorrhizal fungi compared to CS, whereas AIS and VGS supported the same amounts of mycorrhizal fungi, total bacteria, and total fungi. This is very similar to the present study’s situation between different treatments. Differences between VGS, AIS, and CS were attributed to lack of tillage, carbon contributions from roots of continuous vegetative cover, and more complex and varied organic matter inputs. All of the sites in the present study were tilled in previous years as well as in S13 in preparation for orchard establishment. After planting in S13 there was no further soil disturbance other than soil sampling and hand pulling of unwanted plants. The lack of disturbance may have had a positive effect on soil structure and soil fungi communities (Unger et al., 2013). All treatments had continuous perennial vegetative cover, which continued growing from planting until the killing frost in late November 2013. This may have continued to support microbe growth and function beyond the f13 sampling period earlier in November. In s14, all the plants in all treatments emerged early-­‐ to mid-­‐April perhaps beginning to interact with soil microbes at that time. 65 Other studies also found that more diverse organic inputs, which had higher carbon: nitrogen ratios (C:N), resulted in a higher F:B. Cheng et al. (2013) studied 5 natural secondary forest (NSF) ecosystems that were at least 10 years old after logging disturbance in China and found that soil microbial biomass, measured as microbial carbon (Cmic) and microbial nitrogen (Nmic), varied greatly between different types of forest and had a significant positive correlation with soil organic carbon (SOC). Wilson spruce (Picea wilsonii Mast.) NSF contributed the greatest amount of SOC (48.89g kg-­‐1) and had the highest soil Cmic (~910μg g-­‐1) compared to the SOC and Cmic of Armand pine (Pinus armandii Franch.) (18.09 kg-­‐1 and ~500μg g-­‐1, respectively), Chinese pine (Pinus tabulaeformis Carr.) (37.08g kg-­‐1 and ~780μg g-­‐1, respectively), and, sharptooth oak (Quercus aliena Blume. var. acuteserrata) (26.44g kg-­‐1 and ~840μg g-­‐1, respectively) NSFs. They also found that a mixed NSF of sharptooth oak and Chinese pine had significantly lower Cmic (~480μg g-­‐1) and SOC (24.30g kg-­‐1) than all other NSFs except for Armand pine, due to higher N inputs. Regarding Nmic, sharptooth oak was significantly greater at ~125μg g-­‐1 than all other NSFs and had greater soil NH4+-­‐N (20.87mg kg-­‐1) and NO3-­‐N (2.65mg kg-­‐1) than all other NSFs. Higher ratios of Cmic/Nmic in the coniferous NSFs revealed predominant fungal populations over bacterial populations compared to the broadleaved and mixed NSFs (Cheng et al., 2013). Similarly, Fierer et al. (2005) suggested that higher C:N inputs in a coniferous forest caused a F:B of 3.6 compared to 0.2 and 0.6 for desert soil and prairie soil, respectively. Macdonald et al. (2009) also found that pine forest soils, ranging in age 66 from 5 to 20 years since planting into pasture, had at least 50% greater F:B than pasture soils. The aforementioned studies suggest that longer time periods (e.g., at least 5 years) and increasing carbon-­‐heavy internal inputs result in increased fungal population abundance and decreased bacterial population abundance. Conversely, as Cheng et al. (2013) demonstrated, forests with plant species that contribute larger amounts of N maintain a lower F:B. All of our experimental treatments may have contributed larger amounts of carbon compared to the previous land-­‐uses which resulted in increased F:B. Whether or not this trend continues beyond the first two years requires further research, but it could be expected that as the nitrogen-­‐fixing plants in forest garden-­‐based treatments continue to grow increased amounts of N might enter the system. Also, Yao et al. (2005) suggested that a thatch layer of grass, which resulted from mowing over multiple years, might contribute a consistent supply of C to the soil through aboveground litter fall and root exudates. Based on these studies and the fact that both treatment types, forest garden-­‐based and grass-­‐based, shifted SMA similarly it could be suggested that they are currently contributing similar amounts of C and N to the soil despite the differences in plant functional diversity (e.g., nitrogen fixing plants and biomass production) and species diversity. A study comparing 4 land-­‐uses, consisting of: 1) at least 40 years of annual crop rotation of corn, millet (unspecified), wheat, sorghum (Sorghum bicolor (L.) Moench), sunflowers (Helianthus annuus L.), and fallow with conventional tillage practices, 2) at least 40 years of pasture sites that were continuously grazed and 67 dominated by rye grass, 3) pine forest of 50 years, and, 4) hardwood mature oak-­‐
hickory (Carya spp. L.) stands of at least 75 years, found that there were no significant differences in soil C:N nor F:B between land-­‐uses. It was noted that both variables were typically lower in the annual cropping and pasture systems (Lauber et al., 2008). They also found that, similarly to Unger et al. (2013), within these ratios, abundances of specific soil microbe species ranged significantly, which was attributed to the plant composition and types of organic matter inputs. For example, acidobacterial abundances were significantly greater in forest soils (21% to 57% of total bacteria) compared to pasture and cropland soils (9.5% to 24% of total bacteria), and forest soils had greater abundances of basidiomycete Agaricales compared to agricultural soils (0% to 84%, and 0% to 27% of total fungi, respectively), which were relatively greater in Ascomycota (3% to 58%, and 38% to 74% of total fungi, respectively) (Lauber et al., 2008). This study measured similar soil C:N and F:B ratios between various land-­‐
uses, which mirrors our findings. Deeper analysis found significantly different diversity of fungi and bacteria species within similar F:B between different land-­‐
uses (Lauber et al., 2008). We did not perform molecular analysis to determine specific soil microbe diversity, but there is opportunity to explore further in future research with frozen (-­‐80oC) stock DNA from treatment samples. It would be expected that within the similar F:Bs that different treatments would contain varying species of bacteria and fungi due to host-­‐specific relationships and different composition of litterfall (Merwin, 2004; Bever et al., 2012). 68 It was probable that there was some overlap of microbial species between our different treatments as well as many host-­‐specific species within respective treatment types. Apple was the only species that were in all treatments and were likely hosts of specific soil microbes as demonstrated in similar research with poplars (Chifflot et al., 2009; Merbach et al., 1999). Otherwise, forest garden-­‐based treatments had 9 different plant species compared to 3 unique species in the grass-­‐
based treatments, all of which may have host-­‐specific and shared soil microbial species. Increased plant species diversity is known to increase soil microbe species diversity (Bever et al., 2012). Bonfante and Anca (2009) explained that plant communities have specific host effects that influence fungi composition, and that both ecto mycorrhizal and AM fungi host distinct bacteria. They also shared that bacteria community composition is affected more by fungi composition and soil pH than by plant species composition. Our treatments had no significant changes in pH over time within respective treatments or between treatments in F14, so pH may not have contributed to the measured shifts in the bacterial abundance shift. However, it is possible that the shifts in fungi composition affected the soil bacterial abundance in some capacity, the extent of which is beyond the scope of this experiment. Further molecular analysis would provide insights into these particular relationships. Currently, we have observed that all treatments resulted in decreased AM fungi abundance over the first two years, all of which occurred within an increase of total fungi abundance. As Bainard et al. (2011) mentioned in their review of AM 69 fungi, there are few studies on how AM fungi are influenced by temperate agroforestry systems, but they concluded that incorporating trees into agricultural practices has a positive effect on soil life. Chifflot et al. (2009) found that AM fungal spore abundance was similar between systems of only poplar and poplar intercropped with soybean (22.96 and 22.70 spores g-­‐1 soil, respectively). However, in the intercropped system, diversity of AM fungi was higher than the poplar only system (0.78 and 0.53 on the Shannon Weiner Index, respectively) and the total spore count increased with increasing distance from the tree row, which is where more diverse plant roots interacted. In another study across two sites (Saint-­‐Remi, Quebec and Guelph, Ontario), Lacombe et al. (2009) found higher concentrations of AM fungi in tree based intercropping systems compared with conventional monocropping in two sites, Saint-­‐Remi, Quebec (6.2 and 3.7 ng g-­‐1 AM fungi fatty acids, respectively) and Guelph, Ontario (4.8 and 3.3 ng g-­‐1 AM fungi fatty acids, respectively). Perhaps, in the present study, the level of plant diversity was greater prior to tillage and experimental treatment planting in spring 2013 which may have resulted in an initial higher read of AM fungi in 2013. It was also expected that increased diversity of plants in the forest garden-­‐based treatments compared to grass-­‐based treatments would cause higher AM fungi abundance, but it’s possible that the limited mowing maintenance in the grass treatments allowed for increased plant diversity, which may have increased the AM fungi abundance. Other studies have demonstrated the effects that organic amendments, such as compost and woodchips, can have on soil microbe composition. It seems as though the minimal compost additions (2.7kg per tree in S13) in this present study 70 created no significant change in SMA compared to treatments without compost over two years. Similarly, a study with three levels (2.7, 5.4, and 8.1 kg total) and four split applications (April, early May, mid May, June) of incorporated compost applications at the base of newly established apple trees resulted in no significant changes in soil microbial activity (CO2 respiration), which is indicative of microbe growth, or tree leaf nutrients between treatments (Hoagland et al., 2008). Conversely, a three-­‐year study measured annual significant increases in microbial soil respiration, and marginal increases in microbial biomass, when compost was added and incorporated (dairy compost at the rate of 25 t ha-­‐1 in spring 2008 and 12.5 t ha-­‐1 in 2009) in respective cover crop treatments of rye (280 and 195 μg CO2 g-­‐1 dry soil with and without compost, respectively, in 2008) and vetch-­‐rye mixture (180 and 138 μg CO2 g-­‐1 dry soil with and without compost, respectively, in 2008) (Nair and Ngouajio, 2012). Flieβbach et al. (2007) measured multiple farming systems of varying manure application rates for the effects on soil biology. Treatments that received manure at a higher application rate (1.2 and 1.4 livestock units per hectare in the first two and the third crop rotation periods, respectively) resulted in 13% more soil microbial biomass C, 19% more soil microbial biomass N, 23% higher dehydrogenase activity and 11% higher basal respiration than the lower application rate (0.6 and 0.7 livestock units per hectare in the first two and the third crop rotation periods, respectively). These studies demonstrate that compost application can affect soil microbe community function and composition, but further research would help clarify appropriate application rates and the different forms of 71 interactions that compost has with microbes (e.g., plant-­‐microbe interactions or microbe-­‐soil interactions). Woodchips have been shown to affect soil microbes and other system variables. Yao et al. (2005) compared different ground management systems in a 9-­‐
year-­‐old apple orchard for their effects on bacterial and fungal activity, as well as changes in soil characteristics and tree growth. The mulch treatment, consisting of biennial spring applications of 15 cm layer of shredded hardwood bark mulch, compared with a treatment of grass mowed monthly had significantly greater soil organic matter (SOM) (8.6% vs. 5.1%, respectively), soil phosphorus (P) (1.57 vs. 0.56 mg kg-­‐1, respectively), calcium availability (2630 vs. 1102 mg kg-­‐1, respectively), soil cation exchange capacity (22.5 vs. 16.8 cmol kg-­‐1, respectively), and soil pH (7.2 vs. 6.5, respectively). The mulch treatment compared to the grass treatment had similar bacterial colony forming units (CFU) (7.5e6 vs. 6.8e6 CFU/g soil DW, respectively) and significantly less fungal CFU (5.2e5 vs. 1.5e6 CFU/g soil DW, respectively) (Yao et al., 2005). Mulch treatments had more fine feeder-­‐root biomass, higher soil N content, higher leaf N content, 15% larger trunk cross sectional area (TCSA), and significantly higher fruit yields for 7 of 10 years (1994 to 2003) compared to grass treatments (Yao et al., 2005). These differences were attributed to increased nutrient cycling due to increased soil biological activity, large additions of N in bark mulch (162 kg m-­‐2 over the 12-­‐year experiment), N retention as a result of high C:N, and conserved soil moisture. Hoagland et al. (2008) also found that 15 cm of woodchip mulch in newly established apple orchards resulted in increased tree growth. Mulched (WC) 72 treatments caused a 298% increase in TCSA, compared to cultivated (CLT), living leguminous mulch (LML), and living non-­‐leguminous mulch (LMNL) treatments, which had 285%, 180%, 196% increases in TCSA, respectively, after one year (2005 to 2006). In 2006, P was highest in WC treatments at 38% compared to CLT, LML, and LMNL which had 0.21%, 0.24%, and 0.24%, respectively, whereas WC had the lowest N% of all the treatments, which might have been caused by the incorporation of the wood chips into the soil in the fall. In the present study forest garden-­‐based treatments, which had 15 cm of woodchip mulch applied unincorporated in S13, saw increased tree growth (269% and 240% increase in trunk circumference for FGSC and FGS, respectively) over the two years compared to no significant tree growth in the grass-­‐based treatments (196% and 190% increase in trunk circumference for GC and G, respectively) (Figure 8). Our results follow similar trends to the two aforementioned studies regarding positive tree growth with woodchip mulch application. Conversely, the forest garden-­‐based treatments, which had leguminous and non-­‐leguminous perennial cover crops, saw no comparable disadvantage to tree growth, as Hoagland et al. (2008) did. Perhaps the spacing of supportive plants outside of the establishing apple tree root zone (60 cm beyond roots at planting) and woodchip mulch application was an effective mix of understory management techniques that mitigated competition. There were no changes in soil chemical properties: P, potassium, magnesium, pH, or SOM. Although, FGSC treatments had on average the greatest non-­‐significant increase in SOM, followed by FGS treatments. Perhaps over time the unincorporated 73 woodchip mulch will contribute more to the soil system, as it did in Yao et al.’s (2005) study. 5.0 Conclusions The establishing two years of apple orchards designed as temperate forest garden systems and more common grass understory systems, both with and without compost amendments, saw similar increases in soil fungi abundance and similar decreases in soil bacteria abundance from spring 2013 to fall 2014.. FGSC treatments had the greatest apple tree growth. Forest garden system without compost treatments resulted in marginally significant tree growth. There were no significant differences between grass-­‐based treatments due to compost addition. Soil nutrients (P, K, Mg) and pH had no changes in the first two years in any of the treatments. All treatments had increasing trends in soil organic matter, but there were no significant increases. This study was the first to establish a temperate forest garden system. We did so in an apple orchard context and quantified the shifts in the soil microbial community in the beginning years of growth. Soil microbes are considered to be the driving force of ecosystem function, so understanding how they are affected by land-­‐
uses in addition to how they affect ecosystem functions will help in the design of more self-­‐sufficient production systems. Many factors influenced the communities of soil bacteria and fungi in both the forest garden-­‐based and grass-­‐based apple orchard systems including plant species diversity, litterfall nutrient composition, organic amendments, soil disturbance and management practices, and time. How these systems are designed 74 with these factors in mind is important. For example, woodchip mulch contributes multiple nutrients (e.g., P, N, C, and Ca) to the soil over time, conserves moisture, and prevents weeds. These contributions support both soil microbial growth and apple tree growth, which have mutually beneficial relations (Bever et al., 2012). However, incorporating woodchips into the soil may result in a nitrogen deficient soil and cause poor apple tree growth and economic loss, at which point soil microbial abundance is not as important. In addition to the appropriate use of woodchips and manure composts, both of which can be produced on site, the proper placement of diverse, multi-­‐functional support plants, such as nitrogen fixing woody shrubs with nectar producing flowers, can result in an orchard that matures into a more internally sufficient system (Hoagland et al. 2008). Diverse perennial polyculture systems create habitats for more diverse soil microbes that help to balance disease, cycle nutrients, and stabilize soil structure, all of which, if designed appropriately, can reduce the current dependencies on, and associated costs of, fossil fuel-­‐based external inputs. This study is in support of many ecological farmers across the globe whom are already practicing temperate forest gardening systems, and is a catalyst for future research. This management practice should be further studied as it relates to the importance of diversifying and strengthening our local markets, regenerating our shared environments, mitigating contributions to climate change, and shifting towards resilient, holistic systems. Specifically, future research could include developing and measuring these systems for variables such as individual crop and total yield, pest and disease damage, short and long term economics, appropriate 75 plant species diversity and composition for different land scales and internal nutrient production cycles. In southern Ontario there is opportunity for participatory research with farmers who are already practicing forest gardening systems on multiple scales. 6.0 Summary and Final Thoughts This thesis aimed to evaluate the changes of total soil bacterial and fungal abundances, as well as apple tree growth and soil chemical properties, under the effects of different orchard management systems. Comparisons across three sites were made between forest garden system-­‐based apple orchards and orchards with mixed grass understories over two years, 2013 to 2014. This field study was unique in that it was the first experiment on temperate forest garden systems. The establishing year, 2013, saw no significant changes within or between treatments for any of the measured variables. The second year, 2014, saw all treatments increase total fungi abundance, decrease total bacterial abundance, and decrease in arbuscular mycorrhizal fungi compared to 2013. A combination of woodchip mulch, compost, and plant diversity in the forest garden treatment with compost resulted in the most significant findings of the greatest increase in tree growth. Tree growth was marginally significant in forest garden treatments without compost, where as both grass-­‐based treatments saw growing trends but no significant differences. All treatments had no significant changes in soil nutrients, pH, or soil organic matter. 76 Soil microbial abundance measurements provide insight into some of the first ecological shifts under the effects of new land-­‐uses, but there is much more comprehensive insight to be had. Further molecular analysis, specifically around detection of soil microbe diversity, would help to understand the true depth of change in soil microbe conposition. For example, within similar total microbial shifts under different land-­‐uses, such as increasing fungal : bacterial ratios, there may exist unique changes of specific species of fungi and bacteria. Increasingly, genetic sequencing allows for deeper understanding of specific microbial species and this allows classification of function and role in particular ecosystems. With existing stock DNA this step to measure soil microbe species diversity is possible. In addition, future research on these sites could continue to measure the changes in soil microbe composition as the treatments continue to develop and interact with their environments. As development continues with time, research could expand to measure specific and overall yields, pest and disease damage, beneficial insect populations and overall biodiversity, soil structure and water holding capacity, and nutrient cycles. These present sites have their boundaries for effective research, which were subject to time, finances, and a desire to engage the local communities in the process. The boundaries to future research that exist are the small plot sizes and proximity of treatments to each other without much buffer space, as well as proximity to existing undisturbed forest and wetland ecosystems at all three sites. 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Data not available
Temp (oC) -­‐6.5 -­‐5.5 -­‐1.0 6.2 12.5 17.6 20.0 18.9 14.5 8.2 2.5 -­‐3.3 Precip. Temp (mm) (oC) 65.2 -­‐a 54.9 -­‐a 61.0 -­‐a 74.5 -­‐a 82.3 18.3 82.4 18.6 98.6 20.5 83.9 18.9 87.8 14.7 67.4 9.6 87.1 1.2 71.2 -­‐4.9 Precip. Temp (mm) (oC) -­‐a -­‐9.6 -­‐a -­‐9.1 a
-­‐ -­‐5.3 -­‐a 5.2 68.7 13.2 83.0 18.8 177.0 18.5 95.0 18.2 91.0 14.9 130.0 9.3 32.0 0.7 12.0 -­‐1.1 Precip. (mm) 16.0 15.0 6.0 85.0 57.0 59.0 130.0 121.0 176.0 72.0 45.0 23.0 88 Table 8. Average weather data from 1981-­‐2010, 2013 and 2014 gathered from Toronto Lester B. Pearson International Airport weather station for the Mississauga area (Government of Canada, 2015). Month 1981-­‐2010 average 2013 2014 Temp (oC) January -­‐5.5 February -­‐4.5 March 0.1 April 7.1 May 13.1 June 18.6 July 21.5 August 20.6 September 16.2 October 9.5 November 3.7 December -­‐2.2 a Precip. (mm) 51.8 47.7 49.8 68.5 74.3 71.5 75.7 78.1 74.5 61.1 75.1 57.9 Temp (oC) -­‐3.0 -­‐5.6 -­‐0.3 5.8 14.6 18.6 21.8 20.2 15.3 10.4 1.5 -­‐5.0 Precip. (mm) 68 93.4 21.4 99.0 80.9 166.6 102.4 98.6 79.2 102.0 43.6 71.6 Temp (oC) -­‐8.6 -­‐8.3 -­‐4.3 6.1 14.1 19.8 20.2 20.4 16.7 10.8 1.9 0.0 Precip. (mm) 46.0 54.4. 27.0 91.6 56.2 97.0 86.0 38.8 102.8 55.6 43.6 34.2