Land and Food Systems, Faculty of
DSpace
UBCV
Jones, Vanessa L.
Master of Science - MSc
University of British Columbia
The management of knotweed species (Reynoutria spp.), aggressive clonal invasive plants of riparian areas, present unique management challenges, particularly within the province of British Columbia (BC) where a knowledge gap exists between field practitioners who work to control knotweed infestations and academic researchers who study them. Academic research on invasive plant management tends to apply a target-based lens, which limits our knowledge to effectively control invasive plants long-term. The primary objective of this research project was to shift this focus from a target-based approach to other often overlooked important relationships, such as plant-soil-management interactions. These are relationships that may reveal much needed solutions to effective long-term control and subsequent ecological restoration. Accomplishing this required a broad set of research questions: 1. Examining the soil microbial ecology of knotweed infestations and native salmonberry (Rubus spectabilis) dominated sites. We found that knotweed infestation altered the soil bacterial community, decreased arbuscular mycorrhizal fungi (AMF) abundance and increased phosphorus compared to soil from salmonberry; 2. Testing the efficacy of a non-chemical control method that could be used near waterways where herbicide use is restricted, wire mesh girdling. It was determined that wire mesh significantly reduced stem and patch height but did not eradicate knotweed infestations in a field setting after two years; and 3. Attempting to conduct a dose-response assay to test for resistance to glyphosate in knotweed. Invasive Plant Managers have been reporting cases of epinastic knotweed patches in BC for several years that seem unaffected by repeated annual herbicide treatment. Rhizomes were collected from two such epinastic populations in Port Alice and North Vancouver, BC. However, a series of climate events impacted the ability to conduct the necessary experiments, and resistance to glyphosate could not be confirmed. While climate events had a significant impact on investigation of research questions, our primary research objective to focus on plant-soil-management interactions was accomplished and demonstrates that bridging the practitioner-researcher divide is critical to meeting long-term ecological restoration goals. The research also illuminates the importance of further understanding the legacy of knotweed invasion on the soil to inform restoration of native ecosystems in the post-control phase.
UNDERSTANDING PLANT-SOIL-MANAGEMENT INTERACTIONS OF REYNOUTRIA SPP. (KNOTWEEDS) TO INFORM ECOLOGICAL RESTORATION by Vanessa L. Jones B.Sc., Trinity Western University, 2019 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Plant Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) November 2022 © Vanessa L. Jones, 2022 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the thesis entitled: Understanding plant-soil-management interactions of Reynoutria spp (knotweeds) to inform ecological restoration submitted by Vanessa L. Jones in partial fulfilment of the requirements for the degree of Master of Science in Plant Science Examining Committee: Jennifer Grenz, Assistant Professor, Department of Forest Resources Management and Applied Biology, Faculty of Forestry and Faculty of Land and Food Systems, UBC Co-supervisor David R. Clements, Professor, Biology, Trinity Western University Co-supervisor Jichul Bae, Agriculture and Agri-Food Canada Supervisory Committee Member Tara Martin, Professor, Department of Forest and Conservation Science, Faculty of Forestry, UBC Additional Examiner Additional Supervisory Committee Members: Mahesh K. Upadhyaya, Professor Emeritus, Department of Plant Science, Faculty of Land and Food Systems Supervisory Committee Member iii Abstract The management of knotweed species (Reynoutria spp.), aggressive clonal invasive plants of riparian areas, present unique management challenges, particularly within the province of British Columbia (BC) where a knowledge gap exists between field practitioners who work to control knotweed infestations and academic researchers who study them. Academic research on invasive plant management tends to apply a target-based lens, which limits our knowledge to effectively control invasive plants long-term. The primary objective of this research project was to shift this focus from a target-based approach to other often overlooked important relationships, such as plant-soil-management interactions. These are relationships that may reveal much needed solutions to effective long-term control and subsequent ecological restoration. Accomplishing this required a broad set of research questions: 1. Examining the soil microbial ecology of knotweed infestations and native salmonberry (Rubus spectabilis) dominated sites. We found that knotweed infestation altered the soil bacterial community, decreased arbuscular mycorrhizal fungi (AMF) abundance and increased phosphorus compared to soil from salmonberry; 2. Testing the efficacy of a non-chemical control method that could be used near waterways where herbicide use is restricted, wire mesh girdling. It was determined that wire mesh significantly reduced stem and patch height but did not eradicate knotweed infestations in a field setting after two years; and 3. Attempting to conduct a dose-response assay to test for resistance to glyphosate in knotweed. Invasive Plant Managers have been reporting cases of epinastic knotweed patches in BC for several years that seem unaffected by repeated annual herbicide treatment. Rhizomes were collected from two such epinastic populations in Port Alice and North Vancouver, BC. However, a series of climate events impacted the ability to conduct the necessary experiments, and resistance to glyphosate could not be confirmed. While climate events had a significant iv impact on investigation of research questions, our primary research objective to focus on plant-soil-management interactions was accomplished and demonstrates that bridging the practitioner-researcher divide is critical to meeting long-term ecological restoration goals. The research also illuminates the importance of further understanding the legacy of knotweed invasion on the soil to inform restoration of native ecosystems in the post-control phase. v Lay Summary Knotweeds are invasive plants that spread throughout North America after being introduced from Asia. Knotweeds are tall, dense, and have a large underground root system; they cause damage to native ecosystems and infrastructure, making them difficult to control. We wanted to understand the relationship between herbicide management, knotweed plants, and soil. We studied the impacts of knotweed on soil quality and found that it altered phosphorus levels, abundance of certain fungal species, and bacterial community composition. To overcome legislation that regulates where herbicides can be used, we studied a non-chemical control method, wire mesh, to manage knotweed; this decreased height but did not kill the plant. Finally, we investigated deformed knotweed growth after herbicide treatment; these plants may be herbicide resistant. This research emphasizes that bridging the practitioner-researcher gap is critical for meeting long-term goals and can be used to inform future restoration of an area after knotweed is removed. vi Preface This thesis is original, unpublished work by the author, Vanessa L. Jones. Original project proposal and design was initiated by Dr. Jennifer Grenz (University of British Columbia, Plant Science and Forest Resources Management) and Dr. David R. Clements (Trinity Western University, Biology), while further guidance and supervision for this work was provided by Dr. Mahesh Upadhyaya (University of British Columbia, Plant Science), Dr. David R. Clements and Dr. Jennifer Grenz. The project evolved from Dr. Jennifer Grenz’s previous experiences as a field practitioner managing knotweed in BC for almost two decades. Assessing the interactions between invasive plants, the management techniques used to control them, and the impacts to the soil has not been well studied, despite observations in the field by both Vanessa Jones, and Dr. Jennifer Grenz that suggest native plants are not re-colonizing previously infested areas after knotweed eradication. Field data collection was done with the assistance of Aidan Anderson and Hannah Merritt, Undergraduate Research Assistants (Trinity Western University), Greener This Side Invasive Plant Management, host company of my Mitacs Accelerate Internship, and Dr. Jennifer Grenz. All data used in Chapter 2 were collected by Vanessa Jones, Virginia Oeggerli, and Dr. Jennifer Grenz in 2022. Chapter 2 will be published as a standalone paper. The data collection and paper structure followed Modi et al. (2021). Vanessa Jones was responsible for all the first draft of this paper. Dr. Jennifer Grenz aided in supervising, structuring, and editing from the conception of the research and Dr. Tara Martin provided additional guidance on structure and editing all of which contributed to the completion of the paper as presented. vii Table of Contents Abstract……………………………………………………………………………………………………. ii Lay Summary……………………………………………………………………………………………... v Preface ……………………………………………………………………………………………………..vi Table of Contents ………………………………………………………………………………………...vii List of Tables……………………………………………………………………………………………... ix List of Figures……………………………………………………………………………………………... x Acknowledgements……………………………………………………………………………………... xii Chapter 1: Introduction and Literature Review………………………………………………………... 1 Introduction……………………………………………………………………………………………. 1 Invasive Weed Species …………………………………………………………………………………2 Clonal Weed Growth…………………………………………………………………………………... 2 Overview of Knotweeds……………………………………………………………………………….. 3 Knotweed Management: Chemical Control…………………………………………………………… 5 Knotweed Management: Non-Chemical Control……………………………………………………… 7 Potential for Herbicide Resistance…………………………………………………………………….. 8 Knotweed Impacts …………………………………………………………………………………….10 Chapter 2: Impact of knotweed infestations on soil quality and soil microbial communities..……. 14 2.1 Introduction………………………………………………………………………………………. 14 2.2 Methods…………………………………………………………………………………………... 17 2.2.1 Site Selection………………………………………………………………………………. 17 2.2.2 Nutrient and pH Sampling…………………………………………………………………. 19 2.2.3 Bacterial and Fungal Diversity Sampling…………………………………………………. 20 2.2.4 Bacterial and Fungal Diversity Data Analysis…………………………………………….. 21 2.3 Results……………………………………………………………………………………………. 22 2.3.1 pH and Nutrient Analysis………………………………………………………………….. 22 2.3.2 Fungal Diversity Analysis…………………………………………………………………. 25 2.3.3 Bacterial Diversity Analysis………………………………………………………………. 30 2.4 Discussion………………………………………………………………………………………… 35 2.5 Conclusions………………………………………………………………………………………. 39 Chapter 3: Knotweed management using wire mesh treatments……………………………………. 41 3.1 Introduction…………………………………………………………………………………... 41 3.2 Methods and Experimental Design………………………………………………………….. 41 3.3 Results……………………………………………………………………………………….. 45 3.4 Discussion…………………………………………………………………………………… 49 viii Chapter 4: Knotweed management with herbicides and epinastic growth .………………………….52 4.1 Background…………………………………………………………………………………... 52 4.2 Methods……………………………………………………………………………………… 53 4.3 Results……………………………………………………………………………………….. 60 4.4 Discussion…………………………………………………………………………………… 71 Chapter 5: Climate event related research impacts…………………………………………………... 74 5.1 Experiment Ceased Due to Major Climate Event #1: Wire Mesh Pot Experiments: June-August 2021…………………………………………………………………………………………………... 74 5.2 Experiment Ceased Due to Major Climate Event #2: Dose-Response Pot Experiments: June - August 2021…………………………………………………………………………………………... 77 5.3 Experiment Ceased Due to Major Climate Event #3: Second Attempt at Dose Response Assay November 2021 - January 2022……………………………………………………………………… 79 5.4 Experiment Ceased Due to Major Climate Event #4: Third Attempt at Dose Response Assay May - August 2022………………………………………………………………………………………… 81 5.5 Experiment Ceased Due to Major Climate Event #5: Fourth Attempt at Dose Response Assay and DNA Analysis September - November 2022………………………………………………………… 82 Chapter 6: Conclusions…………………………………………………………………………………. 85 Knotweed management issues……………………………………………………………………….. 85 Impact of knotweed on soil nutrients and microbial diversity……………………………………….. 86 Wire mesh as an alternative control method ………………………………………………………….87 Herbicide use and knotweed epinasty………………………………………………………………... 88 References……………………………………………………………………………………………….. 90 ix List of Tables Table 2.1: Grevell soil layer characteristics ................................................................................................ 19 Table 2.2: T-test comparing Phosphorus levels between sites. ................................................................... 24 Table 2.3: ANOVA analysis of Shannon diversity index among groups (salmonberry and knotweed). .... 28 Table 2.4: ANOVA analysis of Shannon diversity index among groups. ................................................... 33 Table 2.5: Tukey post-hoc test .................................................................................................................... 33 Table 3.1: Field site locations for monitoring wire mesh control method. ................................................. 42 Table 3.2: Tukey HSD test for 2022 stem height ........................................................................................ 49 Table 4.1: Glyphosate herbicide treatments of one knotweed site in North Vancouver, BC showing epinastic growth. .......................................................................................................................................... 61 Table 4.2: Glyphosate herbicide treatments of one knotweed site in Port Alice, BC showing epinastic growth. ......................................................................................................................................................... 61 x List of Figures Figure 2.1: Knotweed (K) and Salmonberry (S) sites selected for nutrient and microbial community analysis. ....................................................................................................................................................... 18 Figure 2.2: Mean pH of 4 salmonberry (S) and 4 knotweed (K) sites showing standard error. .................. 22 Figure 2.3: Mean abundance of nutrients in sampled salmonberry and knotweed soils showing standard error. ............................................................................................................................................................ 23 Figure 2.4: Abundance of available phosphorus in each knotweed (K) and salmonberry (S) sample. ....... 24 Figure 2.5: Box-and-whisker plot illustrating the total number of quality reads filtered per sample. ........ 25 Figure 2.6: Taxonomic composition by Phylum comparing values across the Seabird Island sites described in section 2.2.1. ........................................................................................................................... 26 Figure 2.7: Shannon diversity comparing values across the Seabird Island sites described in section 2.2.1. ..................................................................................................................................................................... 27 Figure 2.8: PCoA ordination showing microbiome composition for all specimens. .................................. 28 Figure 2.9: Differential abundance of Dominikia aurea amongst treatments. ............................................ 29 Figure 2.10: Box-and-whisker plot illustrating the total number of quality filtered reads per sample. ...... 30 Figure 2.11: Taxonomic composition by Phylum comparing values across the Seabird Island sites described in section 2.2.1. ........................................................................................................................... 31 Figure 2.12: Shannon diversity comparing values across the Seabird Island sites described in section 2.2.1. ............................................................................................................................................................ 32 Figure 2.13: PCoA ordination showing microbiome composition for all specimens. ................................ 34 Figure 3.1: Field site locations for monitoring wire mesh control method. ................................................ 43 Figure 3.2: Wire mesh installation at the Mission Library site on April 23, 2021; ¾” mesh (left); ½” mesh (centre); control, no mesh (right). ................................................................................................................ 43 Figure 3.3: Wire mesh installation at the Harrison site on April 23, 2021; ¾” mesh (left); ½” mesh (centre); control, no mesh (right). ................................................................................................................ 44 Figure 3.4: Knotweed at Majuba Hill site on May 13, 2021; mesh was installed in spring of 2019; ½” mesh (left); control, no mesh (right). ........................................................................................................... 44 Figure 3.5: Average stem height at each site over the 2021 growing season. ............................................. 46 Figure 3.6: Average stem height at each site over the 2022 growing season. ............................................. 46 Figure 3.7: Girdled stems at Harrison site, July 2021. ................................................................................ 47 Figure 3.8: Average stem and patch height at end of season, with ANOVA P values displayed. .............. 48 Figure 3.9: (Top): Progression of Bohemian knotweed patch treated with ½” x ½” wire mesh; (Bottom): Progression of untreated Bohemian knotweed control patch (Miami River, Harrison, BC, 2021). ............ 50 Figure 4.1: A photo of me working with Greener This Side applying herbicide to a knotweed stand. ...... 54 Figure 4.2: A typical knotweed patch, June 21, 2022, on a roadside in Coquitlam, BC. ............................ 55 xi Figure 4.3: Epinastic knotweed patch July 6, 2021; this is when plants elsewhere are mature, at least 1.5m in height (see Figure 4.2). ............................................................................................................................ 56 Figure 4.4: Closeup image of epinastic knotweed plants. ........................................................................... 58 Figure 4.5: Epinastic knotweed site in North Vancouver, BC, ................................................................... 59 Figure 4.6: Epinastic knotweed site in Port Alice, BC; epinastic knotweed plants circles in red. .............. 60 Figure 4.7: Epinastic knotweed plant observed pre-treatment at North Vancouver site on June 23, 2022. 63 Figure 4.8: Significant epinastic growth surrounding dead canes, North Vancouver, September 20, 2022. ..................................................................................................................................................................... 63 Figure 4.9: Epinastic population in Port Alice; small growth visible, but mostly old canes, June 5, 2020; living plants are circled in red. .................................................................................................................... 64 Figure 4.10: Extensive knotweed growth among dead canes at Port Alice site, July 7, 2020. ................... 65 Figure 4.11: Excavating knotweed rhizomes, May 7, 2021, at the Port Alice site. .................................... 66 Figure 4.12: Small, epinastic growth observed at the Port Alice site, June 6, 2021. .................................. 67 Figure 4.13: Epinastic knotweed plants observed at the Port Alice site, May 11, 2022. ............................ 67 Figure 4.14: Control knotweed shoots emerging from the soil, March 15, 2022. ....................................... 69 Figure 4.15: Epinastic plants growing on March 29, 2022. ........................................................................ 70 Figure 4.16: A large control knotweed plant (center) next to small epinastic plants (left). ........................ 70 Figure 5.1: (A) Pots containing knotweed rhizome fragments collected from Harrison and Mission Library; Control pots (left two rows), ½” mesh (middle two rows) and ¾” (right two rows). (B) Example of a knotweed fragment before planting. ..................................................................................................... 74 Figure 5.2: Daily temperature for the month of June in Surrey, BC (Environment and Climate Change Canada 2021). .............................................................................................................................................. 75 Figure 5.3: Few living browned, wilting control plants on August 1 2021. ................................................ 76 Figure 5.4: Plants that grew large enough to push through wire mesh, June 15, 2021. .............................. 77 Figure 5.5: Pots containing knotweed rhizome fragments collected from (A) Port Alice epinastic population and (B) control population. ........................................................................................................ 78 Figure 5.6: (A) Control plant on June 1, 2021; (B) the same plant wilting on August 1, 2021. ................. 79 Figure 5.7: Two epinastic plants grow from rhizomes collected from the epinastic population in Port Alice, BC, April 23, 2022. ........................................................................................................................... 81 Figure 5.8: Epinastic knotweed plants on July 25, 2022; August 21, 2022 showing chlorosis. ................. 82 Figure 5.9: Plant growth from rhizomes collected from epinastic population in North Vancouver (left) and from untreated population at Mission Library (right). ................................................................................ 83 xii Acknowledgements First, I would like to acknowledge that this thesis was researched and written on the traditional, ancestral, unceded territories of the Musqueam and Quatsino people as well as the Sto:lo, Kwantlen, Katzie, and Seabird Island First Nations. Second, I would like to acknowledge the team of mentors and supporters who willingly spent their time helping me design, troubleshoot, write, and edit this ever-changing project. From my time as an undergraduate with dreams of graduate-level research to the beginnings of this project, Dr. David Clements, my co-supervisor, has encouraged and helped me ceaselessly, offering both intellectual and emotional support for which I am exceptionally grateful. I would like to thank my initial co-supervisor, Dr. Mahesh Upadhyaya, for always pushing me to be the best writer and researcher I can be. Dr. Jennifer Grenz, who took over as my co-supervisor, has gifted me with hands-on, practical experience that I never could have expected. I am grateful for her helping shape my views on ecology and restoration in addition to her wealth of expertise in all things weed management, and teaching me to ask the questions that no one else is asking. I am very grateful for the wisdom imparted on me from my supervisors. Additionally, I would like to thank committee member Dr. Jichul Bae, and the other members of the Agriculture and Agri-Food Canada Weed Science lab, Ryan Critchley and Nathan Young, for their assistance in general troubleshooting and growing plants. I would also like to acknowledge the support from my field assistant undergraduate workers from Trinity Western University, Aidan Anderson and Hannah Merritt, for their dedication to tediously measuring knotweed plants biweekly, and to Aidan especially for diligently watering my potted plants despite the record-breaking heat. I would also like to thank Virginia Oeggerli for her assistance with soil sample collection. I would like to thank AAFC as well as my funding partner, MITACS, and Greener This Side for their funding which made this research possible. xiii Last but not least, I would like to thank my friends and family for their constant support and encouragement which helped me make it through this experience. 1 Chapter 1: Introduction and Literature Review Introduction The management of knotweeds (Reynoutria spp.), which are aggressive clonal invasive plant species, is made more challenging due to the knowledge gap that exists between field practitioners who work to contain and control knotweed infestations and academic researchers who study these species. Knotweed managers have voiced issues with current control practices, but academic research often tends to apply a target-based lens to controlling invasive plants, limiting the potential to expand our knowledge beyond just the plant and the efficacy of control methods. It was therefore the primary objective of this study to shift the focus away from this narrative, and toward other important relationships, such as plant-soil-management interactions, as they may inform long-term restoration post-knotweed invasion. To accomplish this, we carried out the following research objectives: 1. Examining the soil microbial community of knotweed infestations and native salmonberry (Rubus spectabilis) dominated sites 2. Testing the efficacy of a non-chemical control method that could be used near waterways where herbicide use is restricted, wire mesh girdling. 3. Attempting to conduct a dose-response assay to test for resistance to glyphosate in knotweed given recent observations. Following is a literature review on invasive weed species in general as well as knotweed species, their management issues and their impacts to the soil microbial community, in order to contextualize the conducted research within the current literature. 2 Invasive Weed Species Alien plant invasions are increasingly recognized as a global issue resulting in significant landscape-level changes (IPBES 2019). These changes may include biodiversity loss, altered ecosystem function and productivity, changes to soil composition, and economic impacts, which are often considerable (Lavoie 2017; Wilson et al. 2017). Recent policies in the EU, Canada, and other countries have prioritized the management of alien invasive species as a means of protecting biodiversity (Government of BC 2021; IPBES 2019). However, the control of invasive plants is a challenging task for land managers, as there are many considerations to be made regarding the plant and its surrounding environment. As a result, understanding specific plant traits and other external factors involved in the spread and propagation of invasive weeds is essential for effective management. Clonal Weed Growth Clonal growth, which is defined as the vegetative production of individual ramets that are connected below ground by a rhizome system, is a trait that is often associated with invasion success in plants (Roiloa et al. 2010). In fact, 67% of the world’s worst global invaders are clonal plant species (Wang et al. 2020). Despite being generally associated with lower genetic diversity, clonality provides unique advantages to invasive plants, such as resource-acquiring growth dynamics (Roiloa et al. 2010). In clonal species, each individual in the clone is known as a ramet, on which the resource-acquiring plant parts like leaves and roots are located. Ramets are fixed in one place, growing from a rhizome below ground, which act as spacers between ramets. Although the ramets are sessile, the whole clone is mobile; by changing the number, length, or 3 direction of spacers, the clone can “move” toward environmental stimuli, forage for resources, and likewise can escape unfavourable conditions (Martin et al. 2020). Furthermore, clonal species can also produce specialized ramets for specific purposes, such as only producing roots to acquire below ground resources, and divide the labour between them, as all ramets remain connected through physiological integration and therefore water, nutrients, and photosynthates can be shared throughout the plant (Wang et al. 2020). Despite these advantages, the role of clonal growth patterns in invasion dynamics is still not well characterized and studying these patterns will help advance the understanding of clonal reproduction in rhizomatous perennial invasives (Martin et al. 2020). Overview of Knotweeds An excellent example of the invasion success of clonal species is seen in Japanese knotweed (Reynoutria japonica), which is considered one of the worst global weeds present in over forty countries in nearly all temperate zones (CABI 2021). Japanese knotweed was introduced to North America sometime in the 19th century; it was intentionally planted due to its ability for erosion control, for fodder, and for ornamental use (Barney et al., 2006). It is widely recognised that environments that have been exposed to anthropogenic disturbance such as mine sites, railway banks, wastelands and quarries are particularly prone to invasion by R. japonica (Beerling et al. 1994). Some research has studied the potential for using knotweed to remediate degraded land sites such as those contaminated with heavy metals or mining waste; beyond this, the impacts of knotweed in its invasive range are primarily negative (Kovarova et al. 2010; Rahomonov et al. 2013). 4 Existing in its invasive range as a sterile clone, the spread of Japanese knotweed is primarily vegetative, though it can hybridize with closely related Giant knotweed (Reynoutria sachalinensis) creating Bohemian knotweed (Reynoutria ×bohemica) which produces large numbers of viable seeds (Gillies et al. 2016). Both Japanese and Bohemian knotweed in particular exhibit vigorous rhizome growth, as a significant portion of the plant biomass is below ground. Giant knotweed, as with Bohemian knotweed, exhibits both vegetative and sexual reproduction through viable seeds but has a less extensive rhizome system and is considered easier to control. Rhizomes of Japanese knotweed are highly efficient, as up to 35% of their dry weight consists of energy-storing compounds including soluble carbohydrates, crude fat, and starch (Beerling et al. 1994). As perennial species, carbohydrates are translocated to the rhizome at the end of the growing season and the rhizome remains active underground until the following spring, when new growth emerges. Rhizomes of knotweed species are extensive, growing up to 4.5 meters deep and up to 20 meters laterally (Beerling et al. 1994). During the growing season, Reynoutria spp. go through rapid shoot growth and leaf development, growing up to 10 cm per day (Tokarska-Guzik et al. 2006). Despite the evidence of strong clonal growth abilities of Japanese and Bohemian knotweed, research on rhizome growth of these species is limited, especially in regard to rhizome responses to different environmental conditions and management techniques. The majority of studies of Japanese and Bohemian knotweed look exclusively at the rhizome structure, the source-sink relationships and translocation of compounds within the plant, or the responses of the above-ground plant parts (Martin et al. 2020). In order to have a comprehensive understanding of a clonal, perennial species like knotweed, both the above-ground and below-ground responses to control methods and the relationship between the two must be studied. Additionally, plant-soil interactions need to be studied, as both invasive plants 5 and the methods used to control them can have impacts on soil microbial communities (Lavoie 2017). These impacts could have implications for re-invasion, secondary invasion by other non-native species, and restoration after eradication. Knotweed Management: Chemical Control Currently, application of herbicides is the most common practice for knotweed control as it is most effective, efficient, and cost effective (Clements et al. 2016). In British Columbia (BC), herbicides containing glyphosate, aminopyralid, and triclopyr are used to control knotweed (Metro Vancouver 2019). There are several different methods of foliar application that can be used when treating knotweed depending on the label for each herbicide product, applicable regulations, and the specific context of each treatment site: spray-on application, wipe-on application, and knock down and spray application. Spray-on application uses a backpack, handheld sprayer, or wick applicator to cover the top and bottom of all leaves with the herbicide. The knock down and spray method involves two people, where the first person knocks the knotweed stems over using a strong stick, taking care not to break the stem. The second person follows with a backpack or handheld sprayer and sprays the leaves of the bent stems. This technique is useful for tall knotweed stems, as it avoids spraying overhead and also makes it easier to spray both sides of the leaves; both sides of knotweed leaves contain stomata, so spraying both sides increases absorption of the product. Wipe-on application, which can be used for glyphosate-based products, uses a specialized handheld wipe-on applicator to directly apply the herbicide onto leaf surfaces. This method can be time consuming unless the stand is quite small. The various foliar application techniques are considered to be more effective than stem injection, in which the herbicide is directly injected into the stem using a stem injector, a needle-6 like device; this can also be used only with the glyphosate-based Roundup WEATHERMAX herbicide (Metro Vancouver 2019). Mechanical removal of knotweed is not recommended, as leaving behind stem or rhizome fragments as small as 0.7 g can regenerate a new plant (Tokarska et al. 2006). However, management of these species across all of their North American invaded range is complicated by the varying jurisdictions and their unique herbicide regulations (Clements et al. 2016). There are three selective herbicides that are recommended to control knotweed in BC: Milestone (aminopyralid), Clearview (aminopyralid and metsulfuron-methyl), and Garlon (triclopyr) (BC Ministry of Forests, Lands, Natural Resource Operations 2019). There are also two non-selective herbicides, Roundup (glyphosate) and Arsenal (imazapyr) (BC Ministry of Forests, Lands, Natural Resource Operations 2019). However, imazapyr is not an approved herbicide for use on knotweed species in BC, despite success with this herbicide in neighbouring Washington State where over 90% control of knotweed was recorded after foliar application of imazapyr (Miller 2005). Historically, the most common control method for all three knotweed species is the use of herbicides with the active ingredient glyphosate (Clements et al. 2016). However, other herbicides such as Clearview have recently become increasingly utilized (Jennifer Grenz, pers. Comm. 2022). In BC, it is recommended that glyphosate is applied using foliar application to actively growing plants at a volume spray mixture application rate of 400-800 L/ha at an application rate according to the product label (BC Ministry of Forests, Lands, Natural Resource Operations 2019). In BC, the use of herbicides is restricted near waterways, these areas are known as pesticide-free zones (PFZs) (Gaskin et al. 2014). According to BC’s Integrated Pest Management Act (2016), PFZs consist of a 10 meter buffer maintained around bodies of water, measured from the high water mark, where herbicides cannot be used. However, 7 when using glyphosate near a fish bearing water body, this distance can be reduced to 2 meters, or to 1 meter if it is not fish bearing. This results in glyphosate still being most commonly relied upon for use to control knotweed species in riparian areas, one of the most common places these species are found. This means that a large proportion of knotweed species are left untreated or only partially treated in BC, which is especially concerning as it has been confirmed that waterways are a major vector of spread and floating in water can even increase the duration of viability of seeds and rhizome fragments (Bimova et al. 2003; Rouifed et al. 2011). Knotweed Management: Non-Chemical Control In order to overcome this gap in permissible control methods within BC, a non-herbicide control practice was implemented by knotweed managers in Nanaimo, BC in 2016 and Chilliwack, BC in 2018 (Cunningham 2016; Bob Drinkwater, pers. Comm. 2020). This method involves girdling of the knotweed stems by placing a wire mesh on the ground in early spring before new stems have emerged. As the stems grow, they will push up through the holes in the mesh, but eventually will be too large for the space, and the wire mesh will girdle the stems, causing them to die. As more stems die, the plant would theoretically keep producing more new stems, putting a strain on rhizome resources. Currently, there is no literature available on the efficacy of this practice; but it could provide an option for control or suppression of knotweed patches in PFZs where other control methods cannot be used. A private company in the UK, Japanese Knotweed Solutions (2020), holds a patented technology called “Mesh Tech” which seems to use a similar method; however, no information is available online regarding the efficacy. It is only advertised as a non-chemical treatment that customers can choose in order to cause minimal non-target 8 damage to a site, as it has negligible impacts on surrounding soil and water, unlike control methods such as herbicides or digging (Japanese Knotweed Solutions 2020). Land managers in Vermont and New York have tried installing wire mesh recently, but again have not published any results from these trials (Vermont Invasives 2020; Scenic Hudson 2020). Potential for Herbicide Resistance The most common control practice for knotweeds in North America and Europe is the use of herbicides due to their cost-effectiveness as well as their ability to prevent further spread of the species and prevent non-target damage to the surrounding ecosystem (Lawson et al. 2021). However, widespread use of these herbicides over many years has led to concerns about whether these species could develop resistance, as repeated application of herbicides over time increases the probability of resistance developing (Brunharo et al. 2015). The widespread use of glyphosate in both agricultural and ecological settings has resulted in over 60 reported species of weeds that are glyphosate resistant in over 30 countries around the world, and this list continues to grow (Heap 2019). This is especially concerning when considering that an individual stand of knotweed usually takes several years of repeated herbicide applications to be eradicated. Both target-site and non-target-site resistance mechanisms of glyphosate resistance have been observed in various plant species. Glyphosate kills plants by stopping essential amino acid production, which it does by competitively inhibiting phosphoenolpyruvate (PEP) and binding to the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), halting the progression of the aromatic amino acid biosynthetic pathway (Steinrucken and Amrhein 1980). Target-site resistance has been conferred by altering the EPSPS gene, which can be done through single codon mutations, double amino acid substitution, or gene duplication (Kaundun et al. 2008; Yu 9 et al. 2015; Patterson et al. 2017). This allows the plant to either keep producing EPSPS at a competitive level to glyphosate or produce a slightly altered form of the protein that has higher affinity for the product, PEP, than the inhibitor glyphosate (Kaundun et al. 2008). Additionally, non-target-site resistance mechanisms such as preventing translocation of glyphosate through phloem or sequestration in vacuoles have also been identified in various plant species (Shaner et al. 2009; Ge et al. 2014). Given that glyphosate is the only approved non-selective herbicide in BC that can be used to control knotweed; if resistance were to develop, knotweed managers would have to turn to other herbicides to use as a replacement, which are not approved for use near waterways. Additionally, it has been observed that resistance to glyphosate can be transferred between hybrids in Conyza species (Zelaya et al. 2007); this could mean that resistance in one knotweed ecotype can be transferred to another through hybridization or further backcrossing. Backcrossing via sexual reproduction between Bohemian knotweed and both its Giant and Japanese parents is well documented; this may also increase the possibility of resistance due to the increased genetic diversity (Vanwallendael et al 2021; Gaskin et al. 2014). A large knotweed population located on Vancouver Island, BC that has been treated with glyphosate has been reported to display epinastic conditions (Jennifer Grenz, pers. Comm. 2021). Despite treatment over several years, this population persists, and the epinastic conditions may indicate that resistance to the herbicide is developing. Epinasty involves the downward or outward bending of leaves and stems induced by auxin compounds (Sandalio et al. 2016). The mode of action of glyphosate is through inhibition of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, which in turn reduces the formation of aromatic amino acids (phenylalanine, tyrosine, and tryptophan). Because tryptophan is a precursor to the 10 phytohormone indole-acetic acid (IAA) found in the meristem, IAA levels are also reduced when glyphosate is applied, leading to epinasty and loss of apical dominance (Piasecki et al. 2019). Though there is currently a lack of literature describing epinastic conditions in Reynoutria species, severely stunted stem growth of R. japonica 1 and 2 years after application of glyphosate and imazapyr has been documented in Washington and BC (Delbart et al. 2012; Jennifer Grenz, pers. Comm. 2021). Detection of herbicide resistance is extremely important in an aggressive clonal invader like knotweed, especially considering the lack of effective alternative non-herbicide control methods and the limited options for herbicides that can be used for controlling knotweed in BC, particularly in riparian areas. In order to make the most informed possible decisions regarding knotweed management, invasive plant program managers need to have comprehensive information about how the species respond to differing chemical control methods. In order to have a comprehensive understanding of response to herbicide application by a clonal, perennial species such as Reynoutria spp., a plant wherein herbicides can be translocated to the rhizome, rhizome responses also must be measured as the health of above-ground foliage throughout one growing season provides only a partial indicator of overall plant health (Jones et al. 2018). Knotweed Impacts Both Japanese and Bohemian knotweed species form dense, monotypic stands that grow rapidly early in the season and shade out most native vegetation and have been reported to have allelopathic chemicals including anthraquinones and polyphenols that can directly inhibit growth of nearby plants (Murrell et al. 2011; Gaskin et al. 2014; Mincheva et al. 2016). In addition to causing ecological disturbance, knotweed shoots can grow through asphalt and concrete, causing 11 damage to roads, parking lots, buildings, and drainage systems (Bashtanova et al. 2009). Knotweed spreads rapidly once introduced, especially in disturbed areas like roadsides and riparian areas. In addition to its well documented impact on native plant communities through allelopathic inhibition of growth and germination, recent studies have indicated that knotweed may also negatively impact soil microbial communities (Kato-Noguchi 2022). A review of eight studies by Lavoie (2017) found that knotweed infested sites had a higher abundance, biomass, and species richness of fungi compared to non-invaded sites; conversely, it was found that knotweed infested sites had a lower abundance, biomass, and species richness of bacteria compared to non-invaded sites. Authors generally attribute this disparity to the high abundance and slowly decomposing leaf litter of knotweed stands which favours fungal abundance (Lavoie 2017). Knotweeds are known to produce high amounts of phenols, which require enzymes produced by specific species of fungi in order to be broken down in the soil; this may also favour abundance of fungi in knotweed invaded soils (Suseela et al. 2016). However, when specifically looking at arbuscular mycorrhizal fungi (AMF) species, two studies have shown that knotweed (a non-mycorrhizal plant) is negatively correlated with AMF abundance when compared to non-invaded sites (Stefanowicz et al. 2016; Zubek et al. 2016). Furthermore, it was found that soils with lower microbiological properties were more likely to be invaded by knotweed compared to other invasive species. Knotweed infestation also resulted in differences in bacteria community composition compared to other native plant species (Stefanowicz et al. 2016). More recent studies indicated that both fungal and bacterial biomass as well as enzymatic activity are negatively impacted by knotweed invasion, likely due to the addition of phenolic compounds to the soil (Stefanowicz et al. 2021; 2022). As the authors conclude, these negative impacts on soil 12 bacterial and fungal biomass, community composition, and activity by knotweed invasion can have lasting impacts on soil quality, which can in turn affect future restoration efforts. However, more research is needed to provide land managers what is required to restore knotweed invaded areas, as restoration is an important part of weed management that is often overlooked. Very few studies have investigated the restoration of native species in areas previously infested with knotweed, as it is often implied that eradication is the “end” goal when it comes to weed management. Dommanget et al. (2014) demonstrated that restoration with willow and cottonwood species was negatively impacted by allelopathic chemicals from knotweed; however, this was a pot experiment in which knotweed exudate was used to water the willow and cottonwood cuttings, and is not necessarily representative of the amount of polyphenols found in previously knotweed-infested soils. A field study of riparian plant species recovery 3-6 years after knotweed treatment with herbicides in Washington state found that non-native species were more abundant than native species, and the size of the initial infestation was negatively correlated with final native tree cover (Claeson and Bisson 2013). Overall, it was found that herbicide application successfully reduced knotweed cover over time, but the effects on native plant communities were mixed and dependent on environmental conditions such as canopy cover and stream size (Claeson and Bisson 2013). One recent study has investigated the addition of soil carbon amendments to previously knotweed infested soils, as the carbon present in phenolic compounds produced by knotweed can negatively impact growth and germination; it was found that native species did not respond positively to carbon amendments, although some prairie grass species did demonstrate an increase in biomass (Zhang et al. 2021). The authors concur that the specific mechanisms responsible for how carbon amendments impact the microbiome as well as abiotic processes must be studied further. Elucidating the relationship between the legacy of 13 knotweed and the soil microbiome is crucial for efficient restoration of native plant communities following invasion. 14 Chapter 2: Impact of knotweed infestations on soil quality and soil microbial communities 2.1 Introduction Currently, a gap exists in the literature regarding the lasting impacts of invasive plant species on soil quality, especially within the context of restoration ecology. In this study, we are examining knotweeds (Reynoutria species), Japanese knotweed (Reynoutria japonica), Bohemian knotweed (Reynoutria x bohemica), and possible backcrosses (ref our 2016 paper?), as they are identified as a priority for management in the province of British Columbia (BC Inter-Ministry Invasive Species Working Group 2021). The BC Inter-Ministry Invasive Species Working Group characterizes knotweeds as high-risk, well established species with high potential for spread, and emphasizes prevention of further expansion into new areas through regional containment. In the field, knotweed continues to challenge invasive species management specialists as it takes years to gain control and eradication can be evasive on landscape level infestations (Jones et al. 2018). Field practitioners who control knotweed species have also observed that even after an invasion appears to be eradicated, sites appear devoid of native plants for years and are often subjected to secondary invasion by other invasive plant species such as foxglove (Digitalis purpurea), Himalayan blackberry (Rubus armeniacus), and Policeman’s helmet (Impatiens glandulifera) (Jennifer Grenz, pers. Comm., 2022). The field of invasive plant management often focuses on two distinct areas of study: the physiology of the plant itself; and the management approaches themselves. Other possibly important relationships, such as plant-soil interactions, are largely neglected in the literature within the context of invasive plant management in restoration ecology. Therefore, the primary goal of this study is to determine the impact of knotweed 15 invasion on the soil microbiome in order to inform future ecological restoration efforts beyond just eradication of target invasive plant species. Several studies have indicated that knotweed alters soil bacterial and fungal communities, though the impacts of these changes are complex and not well understood as they are not strictly positive or negative (Stefanowicz et al. 2016; Suseela et al. 2016). Literature emphasizes the lasting negative impact that knotweed can have on soil quality but does not bridge the gap and discuss the potential for remediation of these soils within the context of restoring native plant species (Stefanowicz et al. 2021). To date, only a few studies have investigated the relationship between knotweed infestation and soil fungal communities (Mincheva et al. 2014; Tamura and Tharayil 2014; Stefanowicz et al. 2016; Garnica et al. 2022). Generally, it has been found that knotweed is associated with higher abundance of fungi compared to native species, likely due to the large proportion of leaf litter to be decomposed (Lavoie 2017). In fact, it has been determined that breakdown of knotweed leaves requires fungi that produce enzymes specifically for aiding in the breakdown of polyphenols (Suseela et al. 2016). Garnica et al. (2022) also recently determined that the fungal root endophyte Serendipita herbamans colonizes knotweed roots and results in increased growth, but only in certain environmental conditions. However, two studies investigating the impacts of invasive plants, including knotweed, on the soil microbiome found that a specific type of fungi, arbuscular mycorrhizal fungi (AMF), was significantly decreased with invasion by knotweed (Zubek et al. 2016; Stefanowicz et al. 2016). Several studies have investigated the relationship between knotweed invasion and soil bacterial community composition to date; generally, it is considered that knotweed decreases abundance and diversity of soil bacteria, in addition to decreasing enzymatic activity of bacteria (Lavoie 2017; Stefanowicz et al. 2016; Dassonville et al. 2011). This makes sense considering that 16 researchers have demonstrated that knotweeds produce antimicrobial compounds against some pathogenic bacteria species (Kawai et al. 2004; Nawrot-Hadzik et al. 2019). However, there is a lack of literature identifying the potential relationship between bacterial community diversity and knotweed infestation. Knowing that knotweed eradication does not often lead to re-establishment of native plant communities, it may be the case that soil remediation, such as simple inoculation, is a necessary step in the recovery of ecosystems in the post-invasion phase. To begin the exploration of the need for soil remediation in the post-control phase of knotweed invasion to assist with the successful establishment of desired native plant species, this chapter will investigate the impact of knotweed species on soil quality, fungal and bacterial community diversity, and fungal and bacterial abundance. We utilize sites with the species salmonberry, Rubus spectabilis, a plant endemic to the Pacific Northwest region, as controls. Salmonberry was chosen as the species for comparison as it occupies a similar ecological niche, being found in moist forests, riparian areas, bogs, and shorelines, as well as disturbed areas like roadsides and woodlot edges (Delmatier 2018). It also forms large, dense patches like knotweed, growing 1-4 meters in height and 9 meters wide, storing 30-40% of its biomass below ground (California Native Plant Society 2022). Like invasive plant species, the soil microbial community requirements of native plant species, such as Salmonberry, have not been well studied to date. In this study, the nutrient composition and pH, as well as the diversity and abundance of fungal and bacterial communities in the soils of knotweed invaded sites, will be compared to non-invaded sites composed mainly of salmonberry within close proximity. We know that changes in pH and plant available nutrients directly impact the soil microbiome: pH determines nutrient availability in soils; and bacteria and fungi transform nutrients from organic to inorganic forms which can be taken up by plants 17 (Brady and Weil 2017). All interactions that will inform our understanding of the legacy of knotweed invasion on soils. We predict that by applying a plant-soil interactions lens to better understand knotweed invasion, we will be able to better inform best management practices in the post-control phase to assist with the re-establishment of native plant species. 2.2 Methods 2.2.1 Site Selection Soil sampling was conducted at eight different sample sites split evenly into two groups, four knotweed (Reynoutria spp.) (K1-4) and four salmonberry (Rubus spectabilis) (S1-4 ). Limits in time and funding constrained the number of samples that could be analyzed, as the cost of nutrient analysis and fungal and bacterial diversity analysis was high. The salmonberry sites are acting as a control, as they were free from any invasive species, were populated in greatest abundance by salmonberry (>75% species composition), and a minimum distance of 20 m and a maximum distance of 200 m from any knotweed plants or infestations. The eight sites were chosen in collaboration with, and were located on, Seabird Island First Nation. The sites can be seen in Figure 2.1 below; Seabird Island First Nation is outlined in red and white, and the selected sites are shown as insets A and B. 18 A B Figure 2.1: Knotweed (K) and Salmonberry (S) sites selected for nutrient and microbial community analysis. 19 Using the BC Soil Information Finder Tool, it was determined that soils in both areas A and B in Figure 2.1 are orthic regosols belonging to the Grevell series (Comar et al. 1962). These are well draining soils primarily composed of mineral material and are in their native state, never having been used for agriculture (Comar et al. 1962). As fluvial soils, they consist mainly of gravel and sand resulting in a coarse texture and a medium acidic to neutral pH (CanSIS 2022). They are young soils that lack a developed A and B horizon, instead transitioning from the forest floor layer which contains litter, humic and fermented material to the C horizon, which is usually a sandy loam (Comar et al. 1962). Table 2.1: Grevell soil layer characteristics Layer Upper depth (cm) Lower depth (cm) Horizon Description 1 -3 0 LFH Litter/forest floor/humic horizon 2 0 22 C1 Mineral horizon 1 3 22 47 C2 Mineral horizon 2 4 47 60 Cgj1 C horizon with faint to distinct gleying/mottling 1 5 60 120 Cgj2 C horizon with faint to distinct gleying/mottling 2 2.2.2 Nutrient and pH Sampling At each site, three pits were dug until the C2 horizon was reached (at least 30cm); the centre of each site was identified based on the species distribution and the three pits were dug approximately 5 meters apart equidistant from the centre, as plant growth allowed. The soil from these three pits was combined to form a composite sample that was then sent to CARO Analytical Services for essential soil nutrient and pH testing. The following nutrient abundances 20 were measured: Ca, Mg, K, Na, NH3, NO4, and P. Available Ca, Mg, K, and Na were tested using 1N ammonium acetate extraction and atomic spectroscopy. Available NH3 and NO4 were tested using nitrate and exchangeable ammonium nitrogen technique. Available P was tested using colorimetric Bray extraction. The pH of the soil was tested by electrometry using a 1:2 soil to water slurry. 2.2.3 Bacterial and Fungal Diversity Sampling At the same sites as shown in Figure 2.1 used for essential nutrient and pH analysis, soil samples were collected for fungal and bacterial diversity analysis. First, the leaf litter was removed before digging a pit with a disinfected shovel. Samples were then collected using designated disinfected trowels from the following soil layers: 1) Fermentation and humus layer 2) The C1 (0 to 10 cm) soil horizon 3) The C2 (10 to 20cm) mineral soil horizon Following methods developed by Modi et al. (2021), samples were collected in triplicate from each horizon and combined for a composite sample. This resulted in one sample for each horizon at each site. A photo was then taken of the pit and each layer depth recorded. As previously mentioned, it was determined that this is Grevell soil lacking a developed A and B horizon; meaning we sampled the fermentation, C1 and C2 horizons. However, as this was determined after data analysis, samples are still labelled “F,” “A,” and “B.” Trowels and shovels were disinfected between each sample pit using a 1:1 solution of water and bleach. After collection and composition, soil was sieved through a 2mm sieve and 1.00g was weighed into a 21 microcentrifuge tube. Samples were stored at -70℃ in a liquid nitrogen solution during collection and transport to the lab. 2.2.4 Bacterial and Fungal Diversity Data Analysis For bacterial and fungal diversity, samples were submitted to Microbiome Insights in Richmond, BC for analysis. Deep amplicon sequencing, combined with the principles of statistical ecology can be used to survey microbiome communities. Here, deep sequencing of the Internal Transcribed Spacer (ITS) region, situated between the small- and large-subunit of the rRNA genes was used to survey the fungal community and gain insights on the population dynamics (Microbiome Insights 2022). Deep sequencing of the small ribosomal rRNA subunit (16S rRNA) gene was used to survey the bacterial community. Broadly, this was achieved by taking thousands of copy variants of the ITS region and 16S rRNA gene and clustering the sequence data into count data in the form of Operational Taxonomic Units (OTUs) (Microbiome Insights 2022). The count data, or OTUs, were then classified into taxonomic assignments. ITS2 and 16sv4 amplicons were sequenced from soil sample samples on a MiSeq (Microbiome Insights 2022). MiSeq-generated Fastq files were quality-filtered and clustered into 97% similarity operational taxonomic unit (OTUs) using the mothur software package [http://www.mothur.org]. The resulting dataset had 5022 OTUs (not including those occurring total count < 3). An average of 68485 quality-filtered reads were generated per bacterial sample and an average of 28499 quality-filtered reads were generated per fungal sample. 22 2.3 Results 2.3.1 pH and Nutrient Analysis On average, soil pH was similar among the sampled sites; a t-test revealed no significant difference between the four salmonberry and four knotweed sites. Mean pH of salmonberry soils was 5.39 ± 0.14 and mean pH of knotweed soils was slightly more alkaline at 5.87 ± 0.28 (Figure 2.2). Both of these values are within the expected range of a Grevell soil (5.3-7.4) (CanSIS 2022). Figure 2.2: Mean pH of 4 salmonberry (S) and 4 knotweed (K) sites showing standard error. An average of the four knotweed sites and four salmonberry sites was taken and the resulting average abundance of nutrients in salmonberry and knotweed infested soils is shown in Figure 2.3. Overall, both the salmonberry and knotweed sites were abundant in Ca, with over 2000 mg/kg dry soil. This is typical of Grevell soils which are high in sand and gravel containing calcium carbonates. Mg abundance was similar between knotweed and salmonberry sites, as was 23 Na, NH3 and NO4. T-tests performed for each of these nutrients revealed no significant difference in nutrient abundance when comparing salmonberry and knotweed infested soils. Abundance of K appeared slightly higher in knotweed infested areas than salmonberry; however, this was not found to be statistically significant based on a t-test. The abundance of P was found to be significantly higher in knotweed infested soils than salmonberry after comparison with a t-test, with a mean of 33.5 mg/kg in knotweed soils three times higher than the average of 10.75 mg/kg in salmonberry soils. Figure 2.3: Mean abundance of nutrients in sampled salmonberry and knotweed soils showing standard error. 24 The breakdown of phosphorus abundance by site (Figure 2.4) clearly shows the difference between salmonberry and knotweed infested soils. Figure 2.4: Abundance of available phosphorus in each knotweed (K) and salmonberry (S) sample. A t-test (Table 2.2) was conducted to determine whether there was a significant difference in the average phosphorus levels when comparing the four salmonberry sample sites to the knotweed sample sites. The t-test determined the mean phosphorus levels at the two sites to be significantly higher at the knotweed samples sites than the salmonberry sites (P <0.02). Table 2.2: T-test comparing Phosphorus levels between sites. Salmonberry Knotweed Mean (mg/kg) 10.75 33.50 Standard Deviation 6.75 12.82 P (two-tailed) 0.020068 P critical 0.05 25 2.3.2 Fungal Diversity Analysis The sequencing quality (Figure 2.5) indicates the number of usable reads generated per sample; each OTU is assigned a taxonomic classification to the finest level possible. As seen in Figure 2.5, the salmonberry soils generated slightly more reads per sample, though they also seemed to have more variation compared to the knotweed soils. Figure 2.5: Box-and-whisker plot illustrating the total number of quality reads filtered per sample. 26 Based on the ITS2 amplicon sequencing, it was determined that relative abundances of the most abundant phyla and genera did not appear to be different across groups. The aggregated taxa are displayed at each taxonomic rank in the taxonomic bar plot that was produced (Figure 2.6). Figure 2.6: Taxonomic composition by Phylum comparing values across the Seabird Island sites described in section 2.2.1. 27 Alpha diversity was calculated using the Shannon diversity index and displayed in Figure 2.7. This was done in order to compare the richness and evenness of each sample. ANOVA was also carried out in order to compare the Shannon diversity index between groups (Table 2.3). This analysis determined that there were no significant differences in the Shannon diversity index and is similar among groups. Figure 2.7: Shannon diversity comparing values across the Seabird Island sites described in section 2.2.1. 28 Table 2.3: ANOVA analysis of Shannon diversity index among groups (salmonberry and knotweed). Df F value Pr (>F) Groups 5 2.18 0.102 Residuals 18 NA NA Beta diversity among groups was also analyzed to observe similarly among microbiomes. This was done by summarizing the taxon abundances into Bray-Curtis dissimilarities and performing a PCoA ordination. This microbiome composition is displayed in Figure 2.8. The overlapping clusters indicate that the fungal communities do not differ among groups. The beta-diversity among groups was also determined using permutational analysis of variance and likewise there was no significant difference. Figure 2.8: PCoA ordination showing microbiome composition for all specimens. 29 Differential abundance testing was also carried out to identify differentially abundant taxa among groups. There was one differentially abundant species of note; Dominikia aurea, an arbuscular mycorrhizal fungi (AMF) (Figure 2.9). Abundance of this fungus was significantly higher in the salmonberry samples compared to the knotweed samples (Padj=0.00098). Figure 2.9: Differential abundance of Dominikia aurea amongst treatments. 30 2.3.3 Bacterial Diversity Analysis The sequencing quality shown below (Figure 2.10) indicates the number of usable reads generated per sample; each OTU is assigned a taxonomic classification to the finest level possible. As seen in Figure 2.10, the number of reads per sample does not seem different between horizons or between knotweed and salmonberry. Figure 2.10: Box-and-whisker plot illustrating the total number of quality filtered reads per sample. 31 Based on the ITS2 amplicon sequencing, it was determined that relative abundances of the most abundant phyla and genera did not appear to be different across groups. The aggregated taxa were displayed at each taxonomic rank in a taxonomic bar plot (Figure 2.11). Figure 2.11: Taxonomic composition by Phylum comparing values across the Seabird Island sites described in section 2.2.1. 32 Alpha diversity was calculated using the Shannon diversity index and displayed in Figure 2.12. This was done in order to compare the richness and evenness of each sample. ANOVA was also carried out in order to compare the Shannon diversity index between groups (Table 2.4). This analysis determined there were significant differences in the Shannon diversity index among groups. Figure 2.12: Shannon diversity comparing values across the Seabird Island sites described in section 2.2.1. 33 Table 2.4: ANOVA analysis of Shannon diversity index among groups. Df F value Pr (>F) Groups 5 3.25 0.029 Residuals 18 NA NA Tukey post-hoc test determined significant pairwise differences in Shannon diversity for groups. The pair of interest is reported in bold in Table 2.5. Table 2.5: Tukey post-hoc test Pairing Diff P.adj Knotweed_F-Knotweed_B 0.118 0.836 Knotweed_F-Knotweed_A 0.036 0.999 Knotweed_F-berry soil_F 0.131 0.773 Knotweed_F-berry soil_B 0.347 0.027 Knotweed_F-berry soil_A 0.221 0.275 Knotweed_B-Knotweed_A -0.083 0.958 Knotweed_B-berry soil_F 0.013 1.000 Knotweed_B-berry soil_B 0.229 0.245 Knotweed_B-berry soil_A 0.103 0.900 Knotweed_A-berry soil_F 0.095 0.925 Knotweed_A-berry soil_B 0.311 0.055 Knotweed_A-berry soil_A 0.186 0.453 berry soil_F-berry soil_B 0.216 0.298 berry soil_F-berry soil_A 0.090 0.940 berry soil_B-berry soil_A -0.126 0.800 34 Beta diversity among groups was also analyzed to observe similarly among microbiomes. This was done by summarizing the taxon abundances into Bray-Curtis dissimilarities and performing a PCoA ordination. This microbiome composition is displayed in Figure 2.13 below. The overlapping clusters indicate that the fungal communities do not differ among groups. The beta-diversity among groups was also determined using permutational analysis of variance and found there was no significant difference. However, when looking at this Figure compared to the fungal diversity PCoA analysis in Figure 2.7, it is evident that there is more variability in bacterial community composition than was observed in fungal communities. Figure 2.13: PCoA ordination showing microbiome composition for all specimens. 35 2.4 Discussion It has been previously documented that when phosphorus levels are too high in the soil, it leads to plants with underdeveloped roots and stunted growth; total P in surface soils ranges from 0.005 to 0.15%, decreasing with increased weathering (Brady and Weil 2017). Under moderate P limitation, plants will be forced to adapt and develop new roots and associations with mycorrhizae to search for new sources of nutrients (Brady and Weil 2017). It makes sense that knotweed infested soils would exhibit higher levels of P than salmonberry, considering that plant available (inorganic) P increases when organic matter is added to the soil, such as the large quantity of leaf litter produced by knotweed stands (Brady and Weil 2017). Kovarova et al. (2010) demonstrated a positive correlation between P present in the soil and knotweed biomass; in this study, soil P was much higher than what was found at the salmonberry and knotweed sample sites, ranging from 500-1500 mg/kg. However, the knotweed sites selected for this study had no chemical control history, meaning they had never been treated with glyphosate. A follow up study that also includes a sample group of previously glyphosate-treated sites would confirm that glyphosate raises P levels in soil even more. Not only this, but the authors also found that P is taken up from the soil by knotweed and stored in the rhizomes, and the P content of the rhizomes was positively correlated with P content of the soil as well as above and below-ground biomass; interestingly, this trend was not seen with nitrogen (Kovarova et al. 2010). This indicates that high P was a positive input for knotweed growth, but not all plant species thrive with high levels of P. However, this took place in the context of studying the potential for growing knotweed as a remediation method for spoil banks, which are piles of excavated mining waste, where the knotweed was grown in different substrates with or without white melilot (Melilotus alba), a plant found in spoil banks (Kovarova et al. 2010). 36 Furthermore, glyphosate, one of the most commonly used active ingredients used in herbicides for knotweed control in BC, is rich in P and also acts as a Phosphorus addition to the soil when applied. This could mean that application of glyphosate exacerbates the potential problems associated with high Phosphorus levels in knotweed infested soils, even after the knotweed has been eradicated. By adding P to the soil through glyphosate herbicides, native plants may not be able to develop strong enough root systems to compete with the vast underground network of knotweed rhizomes. Additionally, high P additions are also a risk to be washed away into waterways, especially with heavy rainfall and flooding (Brady and Weil 2017). Considering that knotweed is a riparian species with many incidences of the plant occurring within a few metres of waterways, this is an important risk to consider. Runoff of P into waterways has been shown to increase the growth of aquatic plants and algae which in turn decreases oxygen levels in the water; this process is known as eutrophication and can have detrimental impacts on aquatic organisms including salmon due to the decrease in available oxygen as well as the possibility of toxic algae blooms (Prasad and Chakraborty 2019). Looking at Figure 2.5, more reads were found in the salmonberry soils than knotweed, correlating to more species found. Although this difference is not statistically significant, it is contradictory to current literature which often attributes knotweed invasion to higher abundance of fungi due to its high amounts of leaf litter available for decomposition (Suseela et al. 2016). Despite a lack of statistical significance, looking at Figure 2.7 it seems that there is a trend toward higher fungal diversity in knotweed samples. This is an expected trend, as other studies of fungal communities have indicated that knotweed invasion increases fungal diversity and abundance in the soil (Lavoie 2017). Although it may seem contradictory to have a higher number of reads in the salmonberry samples but higher diversity in the knotweed samples, this 37 indicates that the number of different species (richness) is much greater in the knotweed samples than salmonberry. In summary, there was no significant difference in fungal diversity and abundance when comparing knotweed and salmonberry infested soils, although a more robust sample set may have changed the result, as there was a visual trend in increased diversity of fungi with knotweed infestation (Figure 2.7, 2.8). The next step in determining the impact of knotweed invasion on the fungal microbiome is to determine which types of fungi are present/absent or more abundant in the samples. Looking specifically at an arbuscular mycorrhizal fungal (AMF) species, Dominikia aurea, it was determined that knotweed infestation decreased the abundance of this species in the soil (Figure 2.9). This result was expected considering that other studies of specifically AMF species found lower abundance and diversity when associated with knotweed infested soils (Zubek et al. 2016; Stefanowicz et al. 2016). Considering that some AMF species are specific to the plant host they infect, and knotweed is a non-arbuscular species, it makes sense that AMF species would be lower in abundance in knotweed infested soils compared to salmonberry, which is known to be associated with arbuscular mycorrhizae (Begum et al. 2019; Siemens et al. 2020). This relationship between knotweed infestation and AMF diversity and abundance will need to be studied further in order to determine whether it applies to more species of AMF at more knotweed-degraded sites. This would involve determination of abundance of all AMF species present in the soil of knotweed compared to salmonberry, which was beyond the scope of this study. AMF species are known to form symbiotic relationships with certain arbuscular plant species, helping increase rates of photosynthesis and gas exchange (Begum et al. 2019). This relationship between knotweed infestations and lower abundance of AMF could have a negative impact on the soil quality and could result in lower rates of reestablishment of native plant 38 species in the context of ecological restoration, such as salmonberry, even after the knotweed is eradicated. This would likely increase the probability of non-mycorrhizal plant species invading the area after knotweed infestation, as they do not need associations with fungi to thrive. Most vascular plants (~90%) are considered to have some kind of association with mycorrhizae, leaving only about 10% of species non-mycorrhizal (NM) (Wang et al. 2022). Generally, plants belonging to the families Proteaceae, Chenopodiaceae, Brassicaceae, Fabaceae and Cactaceae, including several major agricultural crops and weeds are considered to be NM plants (Wang et al. 2022). Invasion by species in Fabaceae and Brassicaceae in previously knotweed infested areas has already been documented (Claeson and Bisson 2013). Knowing that these species may be more likely to invade knotweed degraded soils can help inform land managers of the risks associated with restoring these areas, as the lack of AMF could result in the lack of recruitment of AMF species like salmonberry. The temporal and spatial effects of diminished AMF species should be studied in order to confirm this. Investigation into whether soil inoculation could be an important step in ecological restoration for successful re-establishment of native AMF species should also be examined in the future. ANOVA determined that there was a significant difference in the Shannon diversity index of bacterial communities between salmonberry and knotweed infested soils. Although this difference was statistically significant, it was contrary to what was expected. The Shannon diversity of bacterial communities of knotweed infested soils was significantly higher than that of salmonberry. Based on the review by Lavoie (2017), it was expected that knotweed infested soils would exhibit a lower bacterial diversity than salmonberry. Stefanowicz et al. (2021) showed that knotweed polyphenols negatively impact bacterial abundance and biomass; however, this was not reflected in the reads tabulated (which corresponds to the number of 39 species) found in Figure 2.10, which showed no difference between salmonberry and knotweed soils. The PCoA ordination showed more variability in bacterial community composition (Figure 2.13) compared to that of fungal community composition, made evident by fewer overlapping points (Figure 2.8). This indicates that knotweed impacts bacterial community composition more than it does fungal community composition, which makes sense considering the significant difference in bacterial diversity. In terms of management, this seems to indicate that restoring diverse bacterial communities will be a necessary step in the restoration of knotweed-infested soils. There could be a potential link between the high levels of P and altered bacteria community found in knotweed infested soils, as bacteria make plant available P by converting it from organic P which is sorbed to the soil surface with Fe, Ca, and Al oxides to inorganic P that can be taken up by plants (Prasad and Chakraborty 2019). However, investigating this connection would require a more in-depth experimental design, where the proportions of organic and inorganic P are measured as well as bacterial community composition and enzymatic activity. Ultimately, studying this phenomenon will help inform management decisions about amendments to soil during native plant restoration of knotweed degraded soils; i.e., bacteria inoculation may be more beneficial than traditional inorganic fertilizer additions. 2.5 Conclusions Current invasive plant management practices often focus solely on elimination of the target species without considering management within the context of successful ecological restoration. Invasive plant management research has largely neglected the post-control phase of management 40 and ignored the possible importance of plant-soil-management interactions. Much of invasive plant control and research is left to field practitioners and volunteers who do not have the time or resources to investigate commonly made observations regarding lack of efficacy in knotweed treatments, secondary invasion, and a lack of recovery of eradicated sites. This has likely contributed to both the gap in the literature on these topics, and the perpetuation of poor long-term ecological restoration outcomes As both researchers and invasive plant field practitioners, we had hypothesized that plant-soil interactions hold potential for improving both the management of invasive plants (achieving long-term control) and successful establishment of native plants species in their place. Our findings indicate that knotweed significantly alters soil bacterial community diversity, with a more diverse soil community than salmonberry. However, the specific composition of bacterial species needs to be studied further, as there may be a connection between the type of bacteria present and the increased phosphorus levels found in knotweed-infested soil compared to salmonberry. We also found that the AMF species Dominikia aurea was significantly decreased in the knotweed-infested soil, which may discourage native plants from re-establishing after knotweed eradication. Abundance of other AMF species in knotweed-infested soil should be studied in order to confirm this trend. These findings indicate that soil remediation could be a necessary step for ensuring that future invasive plant management follows through on management going beyond a target-based approach to ensuring both eradication and desired recovery of native species. 41 Chapter 3: Knotweed management using wire mesh treatments 3.1 Introduction A gap exists in the control of knotweeds within the 1m riparian buffer where herbicides cannot be used in British Columbia (BC Integrated Pest Management Act 2016). As a riparian species whose growth and reproductive ability is increased by proximity to waterways, the lack of control in these areas is likely contributing to further spread of this invasive within BC as floating in water has been shown to increase the viability of knotweed seeds and rhizome fragments (Rouifed et al. 2011). One non-chemical control method that has recently been developed to control knotweeds is girdling via wire mesh. As the knotweed stems grow larger, through the openings in the mesh, they will become too large for the openings, girdling themselves. Theoretically, this would eventually cause the stems to die, and as the plant keeps producing more stems, the energy stores in the rhizome would be depleted leading to eventual death of the plant. In order to overcome the lack of literature investigating this method, a field experiment was designed to investigate the viability of wire mesh as a non-chemical control method. 3.2 Methods and Experimental Design In order to test the viability of a non-chemical wire mesh control method for knotweed stands, three field sites where no herbicides had previously been used were selected in the Fraser Valley: Mission Library, Harrison, and Majuba Hill (Figure 3.1, Table 3.1). An approximate 20mx5m area surrounding a ditch on Majuba Hill Road in Chilliwack, BC had ½” x ½” opening steel wire mesh installed by Drinkwater Environmental Services in 2019, and so installation of mesh at the 42 other two sites followed a similar method. Sites 1 and 2 were divided into three equal sections (10m x 6m): one section of ½” x ½” mesh, another of ¾” x ¾”, and no mesh to act as a control. These sizes were selected by Drinkwater Environmental Services, based on their observations that a 1” x 1” opening was too large, and smaller than ½” x ½” would be too small for the knotweed shoots to initially push through. At the Mission Library and Harrison sites, mesh was installed on April 24, 2021. Any shoots that were beginning to emerge were cut down, then the mesh was laid on the ground and secured with landscaping staples (Figures 3.2-3.4). The following observations of above-ground shoots were taken approximately every 2 weeks from emergence to the end of the growing season: percent coverage of shoots, proportion of living and dead shoots, presence or absence of flowers, average stem height, and degree of girdling. Table 3.1: Field site locations for monitoring wire mesh control method. Label Number Site Wire Mesh Size(s) Site Description 1 Mission Library (ML) ¾”, ½” Large, dense infestation approximately 150m x 30m along both sides of Lane Creek, Mission, BC; extends up slope until dense blackberry cover on the east, up to walking path on the west. Mixed in with Policeman’s helmet and horsetail in some areas. 2 Harrison (H) ¾”, ½” Dense monoculture on north side of Miami River, Harrison, BC; approximately 30m x 10m, extends up to landscaped property of Kitami Sushi Restaurant; mixed in with grasses. 3 Majuba Hill (MH) ½” Approximately 30m x 10m infestation on the corner of Majuba Hill Rd and Wilson Rd, Chilliwack, BC on both sides of ditch; mixed in with blackberry. 43 Figure 3.1: Field site locations for monitoring wire mesh control method. Figure 3.2: Wire mesh installation at the Mission Library site on April 23, 2021; ¾” mesh (left); ½” mesh (centre); control, no mesh (right). 44 Figure 3.3: Wire mesh installation at the Harrison site on April 23, 2021; ¾” mesh (left); ½” mesh (centre); control, no mesh (right). Figure 3.4: Knotweed at Majuba Hill site on May 13, 2021; mesh was installed in spring of 2019; ½” mesh (left); control, no mesh (right). 45 3.3 Results Of the previously mentioned observations of above ground shoots, only stem height produced relevant data. Percent coverage and proportion of live to dead shoots did not change significantly over the growing season, as the mesh did not cause substantial enough girdling damage to affect stem density. Presence or absence of flowers was monitored in order to observe any potential differences between control and mesh-treated plants; however, both mesh-treated and control plants seemed to flower around the same time regardless of plant height. In 2021, stem height was quite variable between sites, and so they are shown separately in Figure 3.5. Overall, no clear trend is seen comparing the mesh (orange and blue) to the control (green) throughout the season. It is worth noting that on July 21, 2021, the knotweed at the Majuba Hill (MH) site was knocked over due to its impedance onto the roadway. A phone call to the City of Chilliwack determined that this was done by city staff, as there is a tight turn in the road where the knotweed stand is located, and it was hanging in the road causing an obstruction to drivers and pedestrians. As a result, the stems were pushed back off the road into the patch; no biomass was removed from the site. 46 Figure 3.5: Average stem height at each site over the 2021 growing season. Figure 3.6: Average stem height at each site over the 2022 growing season. 47 After the 2021 season, it was determined that pulling each stem straight to measure its height was not the most accurate representation of the overall height of the actual patch. This is because as the mesh girdles the stems over the growing season, they begin to fall over, reducing stem height (Figure 3.7). Therefore, during the 2022 season we measured both stem height, where the stem was pulled straight and its full length measured, as well as patch height, where the height of the same stem was measured without pulling it straight. The average height across all sites at the end of the growing season (August 5, 2021 and 2022) is displayed on Figure 3.8. A better way to compare the results of the mesh trials to the controls would be through an estimation of aboveground biomass; however, both time and budgetary constraints did not allow for this. This project was designed in Fall 2020 during the COVID pandemic, which made access to professors and lab equipment more difficult. As a result, I did not investigate ways to estimate knotweed biomass until the 2022 growing season, and at that point there was no room in the budget for additions to the mesh project, and by all indications it was unlikely that comparing biomass would yield significant results. Figure 3.7: Girdled stems at Harrison site, July 2021. 48 Figure 3.8: Average stem and patch height at end of season, with ANOVA P values displayed. An ANOVA was also carried out for the average stem and patch height at the end of the season. For 2021, the ANOVA between treatments determined there was no significant difference with a P value >0.05. However, in 2022 it was observed that there was a significant difference between treatments in both stem and patch heights, with P values of 0.0021 and 0.0005, respectively. A Tukey HSD test (Table 3.2) was then executed for the 2022 end of season data; for both stem and patch height, there was no significant difference in mean height when comparing the two sizes of mesh to one another; however, there was a significant difference in mean height when comparing either the ¾” mesh to the control (P<0.01) and when comparing the ½” mesh to the control (P<0.05). 49 Table 3.2: Tukey HSD test for 2022 stem height Mean of Sample 2022 Stem Height 2022 Patch Height ¾” vs ½” Nonsignificant Nonsignificant ¾” vs control P<0.01 P<0.01 ½” vs control P<0.05 P<0.05 3.4 Discussion Despite the difference in height between the control patch and the mesh treatments, wire mesh control methods are lacking in efficacy needed to achieve eradication. After two years, only a few stems across the three sites were fully girdled to the point of death (Figure 3.7). The difference in both stem and patch height when comparing the wire mesh to the control (Table 3.2) demonstrates the ability of this method to significantly decrease knotweed height over the growing season. This may be useful in a variety of settings; such as where knotweed impedes access to waterways, blocks signage, or hangs into roadways. Looking at Figure 3.8, it is seen that the control knotweed stem height increases significantly from 2021 to 2022, while the mesh treated knotweed stays more constant over the two years. These results indicate that long term application of mesh, for more than two years, can control knotweed height but is highly unlikely to ever result in complete eradication due to the vigorous rhizomes that can “search” for resources and send up shoots as far as 20 m laterally from the main plant (Beerling et al. 1994). 50 Figure 3.9: (Top): Progression of Bohemian knotweed patch treated with ½” x ½” wire mesh; (Bottom): Progression of untreated Bohemian knotweed control patch (Miami River, Harrison, BC, 2021). Because the wire mesh has not demonstrated the ability to eradicate knotweed, applications of this method are very limited; as previously mentioned, it is safe to use near water where herbicides cannot be applied. In a management setting, the goal is often eradication; whether it be for ecological restoration, construction, or other safety reasons. For those purposes, wire mesh may not be a feasible mechanical control method, even if herbicides also cannot be used. However, wire mesh is a low-cost, low-maintenance option for private landowners who do not want to use herbicides; all the materials used can be purchased at a hardware or garden supply store. As there was no significant difference in stem height when comparing the two sizes of mesh, it seems that ½” or ¾” mesh are both appropriate for this purpose. However, users of this method must be aware that the knotweed patch has the ability to send out new shoots beyond the 51 mesh, and may require additional mesh installation in future growing seasons. It also may require yearly maintenance such as re-securing mesh close to the ground if knotweed begins to heave it upward. Other mechanical control options are available and used in various settings to control knotweed; these include digging, repeated cutting, and tarping (Clements et al. 2016). Tarping or smothering involves using heavy, light blocking material to cover the knotweed stand; however, sites in BC where this control method has been used for 10 years has not successfully eradicated the infestations (Metro Vancouver 2019). This control method presents a similar problem to that of wire mesh, as the knotweed may send out shoots to search for available light outside of the tarp (Martin et al. 2020). Similarly, repeated cutting will result in additional lateral growth; this method may also leave behind stem fragments that can regenerate, and is not recommended unless necessary (Metro Vancouver 2019). Digging may be feasible for small infestations where every piece of rhizome can be removed, as any small fragments left in the soil can regenerate a new plant (Beerling et al. 1994). Excavation with heavy equipment may be an option for larger infestations, but again requires that all rhizome material must be removed, which may mean that a buffer of additional soil must also be excavated around the infestation (BC Ministry of Forests, Lands, Natural Resource Operations 2019). 52 Chapter 4: Knotweed management with herbicides and epinastic growth 4.1 Background The current best management practice for knotweed control is treatment with herbicides; however, this is not without its challenges. Studies of knotweed in both field and pot settings often show good levels of control with one application of glyphosate per season (Kadlecova et al. 2022). However, after spending two seasons as a herbicide applicator spraying glyphosate on knotweed infestations myself, I know that this is not the case. Full eradication of a site is often not reached for several years; this is well documented in BC’s Invasive Alien Plant Program (IAPP) database, which contains invasive plant surveys, treatments, and activity plans for the entire province of BC. Based on previous research, we know that repeated application of glyphosate increases the potential for herbicide resistance and also results in plants that display epinasty, even in the beginning of the year following treatment (Brunharo et al. 2015; Delbart et al. 2012). Therefore, the connection between epinasty and the potential for herbicide resistance should be investigated, as potentially glyphosate resistant knotweed would require new management strategies. Several ecological weeds have been reported as being resistant to glyphosate including kochia (Kochia scoparia), perennial ryegrass (Lolium perenne), rigid ryegrass (Lolium rigidum), and sourgrass (Digitaria insularis) in addition to problematic agricultural weeds such as palmer amaranth (Amaranthus palmeri) and Canada fleabane (Conyza canadensis) (Heap 2019). Currently in BC, glyphosate-based products are used most often as they are the only approved herbicide for use within 10m of waterway, where knotweed often occurs as riparian species (BC Integrated Pest Management Act 2016). This leads to two 53 significant issues when managing knotweed: partial treatments and lack of herbicide rotation. Partially treating knotweed patches (ie. only spraying the foliage of an infestation up to 1 m of the waterway and leaving the rest of the foliage untreated) can lead to an insufficient amount of herbicide reaching the rhizome to kill the plant, this is known as sublethal dosage (Jones et al. 2018). Repeated sublethal dosing has been shown to increase the selective pressure for the development of herbicide resistance mechanisms in other glyphosate resistant species such as Junglerice (Echinochloa colona) (Benedetti et al. 2020). Additionally, in agricultural settings, it is standard practice to implement herbicide rotation, where at least two different herbicides with different modes of action are used in alternate years; this decreases the selective pressure for weeds to develop resistance to one herbicide (Darmency et al. 2017). Knowing that these factors increase the probability of glyphosate resistant knotweed developing, the Province of BC must do the necessary regulatory and permitting work at both the provincial and federal levels to prevent this from happening by approving additional herbicides such as Imazapyr for controlling knotweed near waterways. 4.2 Methods I worked in the summers of 2021 and 2022 as a herbicide applicator through a “Mitacs Accelerate Internship” for “Greener This Side”, a landscape-level, invasive plant management and ecological restoration company partially funding my Master’s research. During my experience in this capacity, I often noticed in the IAPP database that the knotweed sites we were spraying had been treated with glyphosate-based herbicides for multiple years in a row, sometimes 2 or 3 times per year. Knotweed sites that had been surveyed and entered into the 54 database in the early 2000s, were still growing strong in 2021 and 2022. Despite this, we continued to spray the sites with glyphosate as no other control methods were available to us. Figure 4.1: A photo of me working with Greener This Side applying herbicide to a knotweed stand. Several sites that had herbicide applied for many years in a row no longer resembled a typical knotweed infestation, nor typical knotweed plant characteristics. Rather than the large, bamboo-like canes and giant, heart-shaped leaves towering overhead (Figure 4.2), these plants were small, sometimes only a few centimetres above the ground, with twisted red leaves and stalks (Figure 4.3). 55 Figure 4.2: A typical knotweed patch, June 21, 2022, on a roadside in Coquitlam, BC. 56 Figure 4.3: Epinastic knotweed patch July 6, 2021; this is when plants elsewhere are mature, at least 1.5m in height (see Figure 4.2). This described growth pattern is known as epinasty, a disturbance in regular plant growth due to the imbalance in plant hormone levels. It is known that glyphosate and other herbicides can cause this reaction in plants (Foster et al. 2019). However, this effect has been observed in these populations for several years in a row at the beginning of each field season (spring) before any herbicide has been applied (Jennifer Grenz, pers. Comm. 2021). This is especially worth noting because glyphosate, like all systemic herbicides, requires significant leaf surface area in order for sufficient uptake of the chemical; this is because the herbicide moves directly through the stomata on the surface of the leaf before translocation to the below ground rhizome system 57 (Hartzler 2022). Epinastic plants often exhibit greatly reduced leaf surface area, and the impact of this on herbicide uptake and translocation to the rhizome system has yet to be studied (Figure 4.4). It may be the case that the reduction in surface area results in a sublethal amount of herbicide reaching the rhizome, and this must be investigated in order to determine whether continued herbicide treatment of epinastic knotweed is effective. 58 Figure 4.4: Closeup image of epinastic knotweed plants. 59 Field Sites In order to investigate whether epinasty could be an indication of resistance or tolerance to glyphosate, two epinastic sites were selected for further study. The first site, located in North Vancouver, BC, borders Seymour Creek Golf Centre to the east and a ditch with flowing water to the left. Typical knotweed surrounds the ditch on the left, epinastic plants throughout the dead canes on the right (Figure 4.5). Figure 4.5: Epinastic knotweed site in North Vancouver, BC, 60 The second site, located in Port Alice, BC, borders Victoria Lake to the northeast. Small clumps of knotweed growth are visible among rocks and dead canes (Figure 4.6). Figure 4.6: Epinastic knotweed site in Port Alice, BC; epinastic knotweed plants circles in red. 4.3 Results Using the IAPP database, I downloaded the chemical treatment history of two known, well-established knotweed patches that had been treated with glyphosate for several years and displayed epinasty. This was done to confirm the extensive use of glyphosate over a multi-year time period at each site. The first site, located in North Vancouver, BC, has been sprayed with glyphosate-based products 10 times since 2014 (Table 4.1). The second site, located in Port Alice, BC, has been sprayed or stem injected with glyphosate-based products 12 times since 2016 (Table 4.2). I observed significant epinastic growth at both of these sites pre-treatment in 61 2022. Greener This Side began treatments of the North Vancouver site in 2022 and the Port Alice site in 2019 and was not the spray contractor for the entire treatment history. Table 4.1: Glyphosate herbicide treatments of one knotweed site in North Vancouver, BC showing epinastic growth. Date Herbicide Used Area Treated (ha) Amount of Spray Mix Used (L) 6/2/2014 Vantage XRT 0.0515 11 7/10/2018 VP480 Glyphosate 0.025 2.5 9/19/2018 VP480 Glyphosate 0.0012 0.4 7/9/2019 VP480 Glyphosate 0.014 1.4 8/29/2019 VP480 Glyphosate 0.0055 0.55 5/19/2020 VP480 Glyphosate 0.002 0.8 5/11/2021 Roundup Weathermax 0.4 40 7/5/2021 Roundup Weathermax 0.026 26 6/23/2022 Roundup Weathermax 0.005 5 9/20/2022 Roundup Weathermax 0.004 4 Table 4.2: Glyphosate herbicide treatments of one knotweed site in Port Alice, BC showing epinastic growth. Date Herbicide Used Area Treated (ha) Amount of Spray Mix Used (L) 62 7/21/2016 Roundup Weathermax 0.1 20 9/14/2016 Roundup Weathermax 0.15 30 9/15/2016 Vantage XRT 0.0225 9 6/20/2017 Vantage XRT 0.01 6 7/24/2018 Vantage XRT 0.02 6 8/9/2018 Vantage XRT 0.014 4.2 6/27/2019 Roundup Weathermax 0.06 3.1 8/14/2019 Roundup Weathermax 0.15 45 9/10/2019 Roundup Weathermax 0.0217 7.38 6/25/2020 Roundup Weathermax 0.02 3 7/7/2020 Roundup Weathermax 0.0002 0.2 7/6/2021 Roundup Weathermax 0.0053 1.5 The North Vancouver (NV) knotweed site was initially visited on June 23, 2022. Before any treatment, epinastic conditions of some of the plants were already observed (Figure 4.7). There was a section of the plants on the west side of the patch that seem to have been left untreated, as they hang over a water-filled ditch (Figure 4.6, left). On the east side of the patch, which had clearly been treated due to the abundance of dry, dead canes underfoot, the plants were stunted, with many stems and long leaves (Figure 4.7). The plants were treated with glyphosate at this time, up to 1 meter of the ditch. 63 Figure 4.7: Epinastic knotweed plant observed pre-treatment at North Vancouver site on June 23, 2022. Upon returning to the site for a second pass on September 20, 2022, even more epinastic growth was present. These plants were low to the ground, bushy, and the leaves were smaller and more elongated than typical knotweed leaves (Figure 4.8). Figure 4.8: Significant epinastic growth surrounding dead canes, North Vancouver, September 20, 2022. 64 Similarly, the Port Alice (PA) population, which has now been observed for several years in a row, also exhibits epinastic plants prior to any treatments in the beginning of the season (Figure 4.9). Similar to the trend observed in other areas of BC, it seemed as though the size of the infestation increased throughout the season, despite herbicide treatment (Figure 4.10) Figure 4.9: Epinastic population in Port Alice; small growth visible, but mostly old canes, June 5, 2020; living plants are circled in red. 65 Figure 4.10: Extensive knotweed growth among dead canes at Port Alice site, July 7, 2020. In May 2021, the epinastic population was observed again; at this time, there was very little growth, but actively growing rhizomes were found from tiny red shoots and excavated (Figure 4.11). Any plants that were growing were sprayed with glyphosate at this time, due to the proximity to the lake. 66 Figure 4.11: Excavating knotweed rhizomes, May 7, 2021, at the Port Alice site. During the second pass in June, new growth had occurred despite the treatment with glyphosate, and the plants seemed even more stunted than the previous year (Figure 4.12). To an untrained eye, it would be difficult to even identify these plants as a knotweed species. Once again, any growing plants were treated with glyphosate at this time. 67 Figure 4.12: Small, epinastic growth observed at the Port Alice site, June 6, 2021. Upon returning to the site in 2022, it was found once again that there was significant epinastic growth, and again it seemed even smaller and redder than the previous year (Figure 4.13). This year, the contract managers did not request that this area be treated, so no herbicides were applied during the 2022 season. Figure 4.13: Epinastic knotweed plants observed at the Port Alice site, May 11, 2022. 68 As mentioned, rhizomes from the PA population were excavated in May 2021, these were then stored in water in an 8℃ refrigerator until they could be used; initially the goal was to grow knotweed plants from epinastic rhizome fragments in order to analyze their rhizome structure in comparison to a typical knotweed plant. Additionally, the plants were to be used in a dose-response assay to determine potential glyphosate resistance. However, slow growth and low regeneration rates of rhizomes in combination with a variety of climate event related research delays impacted the ability for this research to be completed. The impacts of climate change on my research timeline will be discussed further in Chapter 5. Rhizomes collected from the PA site along with control rhizomes from the Harrison site (which has never been treated with herbicides) were planted in trays with potting soil on March 8, 2022 in a greenhouse at the Agriculture and Agri-Food Canada (AAFC) Research Station in Agassiz. The plants were watered daily, and within one week, the control plants were beginning to emerge; looking closely at Figure 4.14, three small shoots can be seen poking through the soil. At this time, these were the only plants growing, and no growth was seen from any of the epinastic samples. 69 Figure 4.14: Control knotweed shoots emerging from the soil, March 15, 2022. After an additional two weeks, on March 29, 2022, 4 epinastic plants had grown (Figure 4.15) compared to 26 control plants. Additionally, the control plants were much larger than the epinastic plants at this time (Figure 4.16). 70 Figure 4.15: Epinastic plants growing on March 29, 2022. Figure 4.16: A large control knotweed plant (center) next to small epinastic plants (left). 71 4.4 Discussion Based on the spray data presented in Tables 4.1 and 4.2, as well as my personal experience as a herbicide applicator, knotweed in BC seems to be glyphosate tolerant. Herbicide tolerance is defined as the inherent ability of a plant to survive and reproduce after herbicide treatment (WSSA 1998). This is distinguished from herbicide resistance, which is defined as the inherited ability of a plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type (WSSA 1998). Plants that are suspected to be resistant must therefore be compared to wild type plants in order to confirm or deny resistance. This is a necessary next step for confirming or denying resistance to glyphosate in BC. Based on our observations of the North Vancouver (NV) site, it seemed as though there was additional epinastic growth between first and second spray passes. We have observed this trend in other areas where we were treating knotweed species in Metro Vancouver, Vancouver Island and the Sunshine Coast during my two seasons working with Greener This Side, indicating that this is a widespread issue and not isolated to only a few knotweed infestations. During our first round of treatment, occurring from May to early July, epinastic plants were observed and then sprayed with either Roundup, which contains glyphosate (if within 10m of a waterway, up to 1m) or Clearview, which contains aminopyralid and metsulfuron-methyl (if greater than 10m from a waterway). During the second pass, from mid-July to September 2022, an overall pattern of increased epinastic growth was observed, regardless of the herbicide initially used. Because Clearview is only effective on actively growing plants, Roundup was primarily used during the second pass (Corteva 2022). There is currently a lack of research investigating the connection between epinasty and resistance in knotweed; however, the abundance of data available in IAPP should be used to further investigate this phenomenon. IAPP data on chemical treatments could 72 be used to correlate frequency of glyphosate application to epinastic appearance of plants. It may also be useful to go back in the IAPP database to look at sites where total eradication has been achieved and see whether those infestations displayed epinastic characteristics, what herbicides were used and how many times, etc. As Roundup requires significant leaf area for sufficient absorption, the current best management practice is to wait until knotweed canes are at least one metre high and the majority of canes have emerged (Metro Vancouver 2019; Hartzler 2022). This is because knotweed has a large, extensive underground rhizome system which makes up approximately 48% of total plant biomass (Price et al. 2002). As a result, knotweed plants need a high proportion of foliage available for the herbicide to contact in order to ensure there is enough herbicide taken up and translocated to the vast rhizome network. When an insufficient amount of herbicide is applied, ie. a sublethal dose, the ability of the plant to develop a resistance mechanism to overcome the herbicide increases (Benedetti et al. 2020). This presents a problem in the case of many epinastic knotweed infestations, as the plants may not reach 1 meter high even by the end of the growing season. These plants also exhibit significantly smaller leaves than a typical knotweed plant (Figure 4.13). It may be useful for future management efforts to study the stomatal structure of epinastic knotweed plants compared to typical plants, in order to determine whether than can effectively absorb herbicides in the same way as a normal knotweed plant. It would also be interesting to observe the Port Alice population over the 2023 growing season, having had a break from herbicide pressure during the 2022 season; it would be worth noting whether the severity of epinasty increases or decreases after a break in herbicide application. However, the process of growing epinastic knotweed plants is slow and unpredictable. When comparing the growth of the epinastic plants to the control plants, the control plants grew much 73 larger in a shorter amount of time, and more of the fragments regenerated. For the purposes of a dose-response assay, this means that both populations cannot be grown simultaneously, as they will reach the 3-4 leaf stage needed for spraying with glyphosate at different times. However, the 3-4 leaf stage needed for a typical knotweed plant may not apply to epinastic plants, and the relationship between their leaf structure and herbicide translocation should be studied in the future. It would also be useful to measure the regeneration capacity of rhizome fragments from epinastic populations compared to a control population. This was not measured in this experiment, as the number of fragments, their size, and greenhouse conditions were not always recorded or controlled. Knowing this information would help inform about the dynamics of epinastic populations, which would in turn impact management strategies such as herbicide timing. Furthermore, it is not known what happens to epinastic populations if they are left untreated, whether they remain epinastic or return to “normal”; monitoring populations such as the Port Alice site, which was not treated in 2022, will therefore also be important for informing our understanding regarding population dynamics of epinastic plants. 74 Chapter 5: Climate event related research impacts 5.1 Experiment Ceased Due to Major Climate Event #1: Wire Mesh Pot Experiments: June-August 2021 The wire mesh experiment (Chapter 3) was intended to include an additional experiment in 2021 that replicated the field experiment in potted plants. The objective of this was to measure impacts of girdling on the rhizomes as rhizomes would be more accessible for measurements in pots than in the field. Rhizomes were collected from the Harrison and Mission sites in early 2021 and refrigerated for 3 days to improve potential regeneration. The rhizomes were cut into fragments of uniform size, weighed, and placed in a 12” pot with potting soil on June 15, 2021. The pots were covered with either ¾” mesh, ½” mesh, or no mesh as a control. Each of the sample sites had 10 replicates resulting in 20 reps for each treatment. The plants were grown outdoors at A Rocha Canada, located in Surrey, BC and watered daily (Figure 5.1). (A) (B) Figure 5.1: (A) Pots containing knotweed rhizome fragments collected from Harrison and Mission Library; Control pots (left two rows), ½” mesh (middle two rows) and ¾” (right two rows). (B) Example of a knotweed fragment before planting. 75 A significant climate event caused the cessation of this experiment. Beginning in June of 2021, BC experienced an extreme heat wave, referred to as a heat dome, considered a once-in-1000-year weather event made 150 times more likely by climate change (Phillip et al. 2021). Lasting from June 25 and July 7, 2021, the heat wave increased temperatures anywhere from 11-19°C above normal across BC (O’Brien 2021). By June 29, 103 all-time high temperature records had been set in Western Canada, with temperatures reaching well over 40°C over June 27-29 for much of the province (Environment and Climate Change Canada 2021). Figure 5.2: Daily temperature for the month of June in Surrey, BC (Environment and Climate Change Canada 2021). The heat dome caused the death of nearly every potted plant in the experiment in spite of increased and frequent watering. The epinastic plants were at an earlier growth stage than the control plants; only 1 out of 20 plants had sprouted by June 28 compared to 20 out of 20 control plants. For the mesh trials, 9 out of 30 plants from Mission Library had sprouted, and only 1 out of 30 from Harrison. After June 28, no more plants sprouted. By August 4, those plants that were living began to decline in health, and the epinastic plant and Harrison plant both died. Those 76 plants that survived were browned and stunted (Figure 5.3). After the heat dome, only 6 plants grew large enough to push through the wire mesh and none of the stems were large enough to be girdled (Figure 5.4). After discussion with my supervisory committee, the decision was made to discontinue this experiment in 2022. Results from the field study (Chapter 3) provided sufficient results to indicate that control by girdling with wire mesh, would provide sufficient control leading to eradication but could provide limited suppression of plant growth where needed. We have noted the cessation of this experiment due to this extreme climate event in this chapter as we feel it an important acknowledgement of the impacts of climate change on ecological research, and what could be an important factor for consideration in experimental design in the future. Figure 5.3: Few living browned, wilting control plants on August 1 2021. 77 Figure 5.4: Plants that grew large enough to push through wire mesh, June 15, 2021. 5.2 Experiment Ceased Due to Major Climate Event #2: Dose-Response Pot Experiments: June - August 2021 To determine whether resistance to glyphosate is present in epinastic knotweed populations, a dose-response assay was to be carried out using samples of rhizome collected from infestations exhibiting epinastic growth characteristics collected in Port Alice, British Columbia. Rhizomes were excavated from the soil with a garden shovel and cut into fragments of uniform sizes before soaking in water at 8℃ for 5 days. Following the period of soaking in water, rhizomes were planted in trays in potting mix and grown in the greenhouse at the Agriculture and Agri-Food Canada (AAFC) Research Centre in Agassiz, BC. Following methods developed by Strelau et al. (2018), the plants were to grow for one month after planting. After allowing one month of growth, the plants would be sprayed with varying concentrations of glyphosate based on equivalent mean biomass: 0x (no glyphosate, as a control), 0.25x, 0.5x, 1x, 2x, 4x, 8x and 16x the label recommended maximum dosage for perennials (4.32 kg a.e. L-1). After a 28-day period 78 after spraying, the plants would be determined to be either living or dead based on the observed plant health characteristics. This experiment was also ceased as a result of the June 2021 extreme climate event referred to as the “heat dome”. Both epinastic and control rhizomes were collected from Port Alice in May 2021 and refrigerated for several days before planting at A Rocha Canada in Surrey, BC along with the potted plants from the mesh experiment described in 5.1. Each treatment (epinastic and control) had 20 replicates (Figure 5.5). (A) (B) Figure 5.5: Pots containing knotweed rhizome fragments collected from (A) Port Alice epinastic population and (B) control population. As these were grown alongside the potted plants used in the mesh experiments, they were also subjected to the extreme heat event in June of 2021. The control plants exhibited similar issues to those in the mesh experiments, wilting and browning significantly by the end of July (Figure 79 5.6). As a result, not enough viable plants were grown to carry out the dose response assay and thus the experiment was ceased. Again, we have noted the cessation of this experiment due to this extreme climate event in this chapter as we feel it an important acknowledgement of the impacts of climate change on ecological research, and what could be an important factor for consideration in experimental design in the future. (A) (B) Figure 5.6: (A) Control plant on June 1, 2021; (B) the same plant wilting on August 1, 2021. 5.3 Experiment Ceased Due to Major Climate Event #3: Second Attempt at Dose Response Assay November 2021 - January 2022 I had planted several leftover rhizomes from the epinastic Port Alice population in early November at the Agriculture and Agri-Food Canada (AAFC) Centre in Agassiz, BC in a second attempt to grow potted plants for the glyphosate dose-response assay. After the climate-related research setbacks as a result of the heat dome of June 2021, another major climate event in the 80 Fall of 2021 occurred. Heavy flooding in the Eastern Fraser Valley of BC, caused by an atmospheric river, a narrow corridor of concentrated atmospheric moisture, shut down any movement to Agassiz from November to January 2021. Hope, a city just east of Agassiz, recorded 280mm of rain from November 14 to 15, exceeding the average rainfall level for the month in just two days (Environment and Climate Change Canada 2021). Not only did the area receive unprecedented levels of precipitation, but the catastrophic wildfire season (made worse by the heat dome) of summer 2021 increased the risk of severe flooding (BC Ministry of Forests 2011). This is because the heat from wildfires forms a hydrophobic crust on the soil from the waxy compounds found in the fermenting forest floor, which can no longer absorb rainfall in the same way as native forest soils, increasing risk of flooding during intense rainfall (BC Ministry of Forests 2011). In this case, the atmospheric river and severe rain, considered a one-in-100-year climate event, were enough to cause flooding prompting a state of emergency for the province of BC (CBC News 2021). The floods resulted in the closures of every major highway as well as CPR and CNR railway lines, cutting off the Lower Fraser Valley and Metro Vancouver from the rest of Canada (Evans 2021). On the north side of the Fraser River, Highway 7 was closed due to multiple mudslides, while on the south side, Highway 1 was completely submerged and would not reopen until December 2, 2021 (Little 2021). These highway closures along with restrictions due to the concern of the COVID variant Omicron resulted in restricted access to the AAFC Centre, where I was growing my plants; I was not able to return until January 2022. As a result of the closures, all plants growing at that time were not watered or cared for by greenhouse staff and as a result had to be discarded. 81 5.4 Experiment Ceased Due to Major Climate Event #4: Third Attempt at Dose Response Assay May - August 2022 I attempted to grow more plants from the remaining rhizomes I had collected and stored from the Port Alice population in early March 2022; By the end of April, only 3-4 plants had grown from the >100 rhizome fragments (Figure 5.7). The rhizomes had been stored in water in an 8°C refrigerator since they were collected the previous year. Once again, the number of plants was insufficient to begin the dose-response assay. Figure 5.7: Two epinastic plants grow from rhizomes collected from the epinastic population in Port Alice, BC, April 23, 2022. A second trip was made to Port Alice in May 2022, where I collected many more rhizomes and another round of planting utilizing the methods described in section 5.3 took place on June 6, 2022 at the AAFC Centre in Agassiz. This time, hundreds of pieces of rhizome were used in hopes that enough plants would grow for the dose response assay. By July 25, 2022, I had 82 approximately 40 epinastic plants grown (Figure 5.8). However, the next climate-related setback would arise when AAFC Agassiz was inaccessible for several weeks due to severe wildfires in the area; wildfires in Agassiz as well as nearby Hope, Harrison, and Chilliwack prompted various evacuation orders and the closure of Highway 7, one of two main routes into Agassiz. This was enough time for the plants to start browning and dying, and they were no longer viable for dose-response test (Figure 5.8). Figure 5.8: Epinastic knotweed plants on July 25, 2022; August 21, 2022 showing chlorosis. 5.5 Experiment Ceased Due to Major Climate Event #5: Fourth Attempt at Dose Response Assay and DNA Analysis September - November 2022 In a final attempt to salvage the dose response assay experiment, I went to a second known knotweed epinastic growth site in North Vancouver on September 24, 2022 to collect more rhizomes. Following the same procedures as sections 5.3 and 5.4, rhizome fragments were placed in trays and left in the greenhouse on October 3, 2022. Rhizomes from the Mission 83 Library site were also collected and planted at the same time. Unfortunately, as of October 31, 2022, there was insignificant epinastic growth to carry out the dose-response assay (Figure 5.9). If time and budget allowed, it would have been much more simple to send samples for a DNA-based assay to determine resistance. This would also be more informative as DNA analysis can also determine what resistance mechanism the plant is employing. However, we now know the challenges of confirming resistance using a classical method with an epinastic plant population. It may also be more useful in the future to find suspected resistant populations that do not display epinastic traits as well, as they may be easier to regenerate from rhizome fragments. Figure 5.9: Plant growth from rhizomes collected from epinastic population in North Vancouver (left) and from untreated population at Mission Library (right). As a student, experiencing these seemingly constant and unexpected climate-related events causing setbacks in my research was extremely frustrating. Ultimately, there were two chapters of my thesis that I was not able to investigate in the way I wanted, and I could see the direct 84 impact of climate change right before me. Knowing that these events were outside my control was difficult, but climate change is our reality, and researchers must be prepared for setbacks like this. It would have been difficult to predict whether events like flooding would impede my access to facilities; however, knowing that summer temperatures in the Pacific Northwest are increasing should have informed me not to attempt a potted plant experiment outside without a temperature mitigation plan in place. However, I was also dealing with limitations in access to indoor facilities due to the timing of when I was planning my project during the 2020 COVID pandemic. In the future, I would not advise researchers to attempt a potted plant experiment outside unless climate change is also a considered variable in the experimental design. Considering that the impacts of invasive species are only made worse by climate change, this type of research is important; even negative results due to the impacts of climate change are worth reporting. It was disheartening as a student to be researching restoration methods to potentially mitigate climate change impacts while simultaneously experiencing direct climate impacts that inhibited that research. However, I believe this goes to show how important this field of research is; climate change is having tangible impacts on our ecosystems and research methods right now, and it is vital to continue researching and reporting this. 85 Chapter 6: Conclusions Knotweed management issues Management of invasive species is not without its difficulties; however, knotweed species present unique challenges due to the lack of management options, the regulations around these options, and the large divide in knowledge that exists between academic researchers and on-the-ground practitioners. Many academic researchers have little or no experience managing the plant themselves in the field, while on the other hand, practitioners may not have access to the information that researchers have regarding efficacy of treatment options. This is evident in the vast expanses of knotweed that persist throughout BC, despite decades of management efforts. The issues of knotweed are unique, as it has a vast underground rhizome network capable of vigorous growth that is not well understood. It is known that knotweed roots produce exudates that can have allelopathic effects on other plant species, but little research has been conducted regarding their impacts on soil microbial communities (Lavoie 2017). Additionally, the relationship between above ground foliage, herbicide application and translocation to the rhizome is still not well understood, and may be highly dependent on species diversity and environmental conditions (Jones et al. 2018). It has recently been demonstrated that the rhizomes can alter its rhizome growth patterns to “move” toward or away from environmental stimuli; however, the impacts of these findings on chemical and non-chemical control methods has not yet been investigated (Martin et al. 2020). As a clonal, perennial invasive species, understanding the dynamics of the rhizome and plant-soil interactions that take place below ground is crucial for effective management of knotweed species. 86 Impact of knotweed on soil nutrients and microbial diversity Invasive plants have been known to degrade ecosystems and impact soil quality; this study discovered that knotweed infested soils demonstrate an altered bacterial community and nutrient availability compared to native salmonberry soils. We confirmed that knotweed infestation was associated with higher plant-available soil Phosphorus when compared to salmonberry which may be due to the large quantity of leaf litter produced by knotweed stands; high P content has been previously associated with increased knotweed biomass (Kovarova et al. 2010). Additionally, glyphosate acts as an addition of P to soils, which can persist in the soil even after knotweed has been eradicated, negatively impacting the establishment of native species root systems and potentially contributing to eutrophication of waterways. Shannon diversity of fungal species in the soil was not different when comparing knotweed infested soils to salmonberry; however, a trend can be seen of higher fungal diversity and abundance in knotweed-invaded soils. Differential abundance testing of an arbuscular mycorrhizal fungi (AMF) species, Dominikia aurea, indicated lower abundance of AMF with knotweed invasion. Knotweed is a non-arbuscular species, but the majority of plants (~90%) are, and reducing AMF in the soil in turn reduces the establishment of native plant species. Diversity and abundance of AMF species specifically should be studied, as reduction of these species by knotweed can have a lasting legacy in the soil and a negative impact on restoration of native species. We discovered a difference in Shannon diversity of bacterial species when comparing knotweed infested soil to salmonberry, with knotweed having a higher diversity. There may be a link between the altered community diversity of the knotweed soil microbiome and high phosphorus 87 levels, as bacteria convert organic P to inorganic, plant available P. These factors may also have implications for future restoration efforts and should not be ignored by researchers, as non-conventional restoration practices such as inoculation of beneficial bacterial or fungal species to the soil as amendments may be important for re-establishment of native plant species like salmonberry. Practitioners must learn about the impacts that treatment of knotweed has on the soil, and conversely researchers must learn from practitioners their important observations in the field that may indicate these phenomena. Wire mesh as an alternative control method The widespread infestation of knotweed in riparian areas in BC, known as Pesticide-Free Zones (PFZs) where glyphosate cannot be applied, necessitates new control methods. We conducted a field experiment to test the efficacy of a method not yet described in the literature for controlling knotweed, wire mesh girdling. This involved placing wire mesh with openings of ½” and ¾” on the ground in early spring and allowing the shoots to grow through the openings, girdling themselves as they grow larger. It was found that mesh was ineffective in eradicating the plant; however, it did significantly reduce stem and patch height. Wire mesh can therefore be used to reduce height of knotweed if other control options are not feasible, as it is a low cost and low maintenance control method that can be used in nearly any environmental setting without causing non-target damage. 88 Herbicide use and knotweed epinasty Currently, the legislation at the provincial and federal government levels limits the ability of herbicides to be used effectively. This means that control options for knotweed are limited, and as such could be contributing to a control context promoting the development of herbicide resistant (or tolerant) invasive plant species. Repeated application of glyphosate-based products has resulted in epinastic stands of knotweed that are persisting for up to 10 years and may be becoming more difficult to control due to their reduced leaf surface area, as evidenced by their lack of response to treatment. Weed management teaches the importance of herbicide active group rotation to prevent the resistance to one herbicide group that can arise when used repeatedly. However, with the case of knotweed, which often occurs close to waterways as a riparian species, only glyphosate can be used within 10 metres of a waterway. In order to overcome the gap in knotweed management in riparian areas in BC, new legislation needs to be put in place to allow for the use of chemicals like Imazapyr to be rotated with glyphosate to prevent resistance, and to allow for use of herbicides near waterways for knotweed so that partial treatments are not increasing selective pressure. Additionally, investments into future research regarding the future of control methods for epinastic patches must also be made. It is unknown whether application of glyphosate to epinastic patches is effective, as they do not seem to respond to treatment. On the other hand, it is unknown whether these plants would go back to resembling a typical knotweed stand if left untreated or whether they would remain epinastic. Ultimately, it is still uncertain whether knotweed in BC is resistant to glyphosate, and this must be elucidated in order to effectively manage this species; whether or not resistance is confirmed, management efforts must change in BC in order to make a difference in stopping the spread of knotweed species. 89 This research, meant to examine knotweed plant-soil-management relationships to inform ecological restoration, has shown us that knotweed does indeed alter soil microbial communities and essential nutrients. 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Thesis/Dissertation
eng
Plant Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Grenz, Jennifer
Clements, David (David R.)
Understanding plant-soil-management interactions of Reynoutria spp. (knotweeds) to inform ecological restoration
Text