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Comparing herbicide application methods for controlling Ilex aquifolium (English holly) in Pacific Spirit… Law, Alyssia; Chookolingo, Brenton; Soria, Carlos; Nathania, Laurentia Apr 24, 2017

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i  Comparing herbicide application methods for controlling Ilex aquifolium (English holly) in Pacific Spirit Regional Park     Prepared by: Alyssia Law, Brenton Chookolingo, Carlos Soria, Laurentia Nathania Faculty Advisor: Sara Harris    ENVR 400: Research Project in Environmental Science Date: April 24, 2017   ii   Abstract Ilex aquifolium (English holly) is an invasive species that has been identified as a species of concern in Pacific Spirit Regional Park by the Metro Vancouver Regional Parks. Invasive plants can greatly affect biodiversity by altering the fire regime, nutrient cycling, hydrology, and energy budgets in a native ecosystem, as well as greatly diminish the abundance or survival of native species. Previous manual control methods have shown to be ineffective in providing successful long term control of English holly in Pacific Spirit Regional Park. Herbicide application was suggested by Metro Vancouver as an alternate long-term pest management strategy. This study compared two methods of glyphosate application (EZJect and Paint method) used by Metro Vancouver at the site to verify each method’s effectiveness in killing the target English holly trees. The results of this analyses suggests that the Paint method has, thus far, been the most effective at reducing English holly survival. Further monitoring of the study area is recommended to gain more insight on the herbicide’s effect in the spring and summer seasons.              iii  Team Biographies Members of this team are environmental science undergraduate students at the University of British Columbia in their fourth and final year of study. All members have focussed their degree on the Land, Air, and Water Area of Concentration. Alyssia Law Alyssia has worked with the Department of National Defence in developing an integrated pest management program to encourage the ecological health of regional range and training areas. Her relevant professional experience has included conducting biological field surveys, creating maps from field data utilizing ArcGIS, and invasive plant species identification. Laurentia Nathania Laurentia’s academic interests include in a wide range of environmental topics such as the effects of climate change on crops, ecology, and water contamination. Her previous experiences with statistical programming and field work applied from her undergraduate biology courses were valuable in successfully completing this project Carlos Soria Carlos’s main areas of academic focus include ecology, biology, and oceanography. Previous courses in these subject areas have gained him experience including experimental design, ecological sampling methods and data analysis. Brenton Chookolingo Brenton has focussed his academic experience on climate change, marine pollution, and GIS. Through academic and work experiences he has knowledge of biological field sampling techniques, map creation using GIS and coding in R.   iv  Contents 1 Introduction ........................................................................................................................................... 1 2 Methods................................................................................................................................................. 1 2.1 Area of Study ................................................................................................................................ 2 2.2 Herbicide Treatment Application .................................................................................................. 3 2.3 Post-Herbicide Treatment Data Collection and Analysis ............................................................. 4 3 Results ................................................................................................................................................... 5 4 Discussion ............................................................................................................................................. 8 5 Conclusion .......................................................................................................................................... 10 6 Recommendations ............................................................................................................................... 10 6.1 Longer Monitoring ...................................................................................................................... 10 6.2 Root Differentiation .................................................................................................................... 10 6.3 Cut Tree Removal ....................................................................................................................... 10 7 Acknowledgements ............................................................................................................................. 11 8 References ........................................................................................................................................... 11 9 Appendix I: Site Photographs ............................................................................................................. 14 10 Appendix II: Weather Data ............................................................................................................. 20 11 Appendix III: ANOVA Summary ................................................................................................... 21  Figures Figure 1: Figure of Site Area. ....................................................................................................................... 3 Figure 2: Graphical Representation of ANOVA. .......................................................................................... 6 Figure 3: Total survivorship of sprouts within a 60 to 90 cm radius of tree stumps ..................................... 7 Figure 4: Total survivorship of all sprouts within a 90 cm radius of tree stumps ......................................... 7 Figure 5: Total survivorship of sprouts within a 30 cm radius of tree stumps .............................................. 8 Figure 6: Total survivorship of sprouts within a 30 to 60 cm radius of tree stumps ..................................... 8 Figure 7: Photo Appendix ........................................................................................................................... 14 Figure 8: Photo Appendix  .......................................................................................................................... 14 Figure 9: Photo Appendix ........................................................................................................................... 15 Figure 10: Photo Appendix ......................................................................................................................... 15 Figure 11: Photo Appendix ......................................................................................................................... 16 Figure 12: Photo Appendix ......................................................................................................................... 16 Figure 13: Photo Appendix ......................................................................................................................... 17 Figure 14: Photo Appendix ......................................................................................................................... 17 v  Figure 15: Photo Appendix ......................................................................................................................... 18 Figure 16: Photo Appendix ......................................................................................................................... 18 Figure 17: Photo Appendix. ........................................................................................................................ 19 Figure 18: Photo Appendix ......................................................................................................................... 19 Figure 19: Daily mean temperatures (℃) for the months of December, January and February. ................ 20 Figure 20: Daily snowfall (mm) for the months of December, January and February. .............................. 20   Tables Table 1: Number of trees studied within each treatment plot………………………………………………4 Table 2: Summary statistics of results…………………………………………...…………………………5 Table 3: ANOVA Summary………………………………………………………………………………21 1  1 Introduction Ilex aquifolium (English holly) is an evergreen tree commonly found in the BC lower mainland and its removal is of interest to stakeholders such as Metro Vancouver and the Pacific Spirit Park Society. Native to Eurasia, this tree is capable of surviving temperatures up to -4.6°C (Peterken and Lloyd, 1967), making the mild winters of British Columbia’s lower mainland an ideal climate for its proliferation. It can reproduce through both seeds and vegetative clones, and responds to ground level cutting and pruning by producing more leading shoots (Peterken and Lloyd, 1967). Due to the aggressive nature of its reproduction and shade tolerance that allows for it to outcompete native species, the Invasive Species Council of British Columbia has designated English holly as an invasive species (Invasive Species Council of BC, n.d.). Due to the importance of its removal to native biodiversity, Metro Vancouver, the regional park service that manages parks, ecological reserves, and greenways in the Greater Vancouver Region, has proposed a pilot project to investigate the potential use of herbicides to control English holly population in Pacific Spirit Regional Park. The use of herbicide was considered after a 2016 study by ENVR 400 students at the University of British Columbia suggested that one-off manual removal of English holly was not an effective long-term strategy for its control (Haines, Cameron, & Hughes, 2016). This study investigated the long-term growth effects of this removal strategy and inferred from their results that cutting down the English holly trees without follow up treatment of sprouts resulted in further stimulation of the plant’s growth. In order to provide an effective long-term management plan to reduce English holly growth in Pacific Spirit Regional Park, a low impact herbicide application that does not spill into the surrounding environment was suggested.  To determine the most suitable herbicide application technique for controlling English holly in Pacific Spirit Regional Park, our project investigated which method of glyphosate application was the most effective at controlling the English holly population. To determine this, our research questions were:  1. Which method of the two compared glyphosate applications is the most effective at killing English holly?  2. How does the survival of sprouts found adjacent or on the tree itself differ between treatments?  3. Which method is the most effective at controlling the growth of sprouts that are connected to the English holly trees? The results of the study are aimed to assist Metro Vancouver in determining an appropriate long-term strategy plan for the removal of English holly throughout Pacific Spirit Regional Park. 2 Methods In order to answer the research questions detailed in Section 1, this project investigated the survival of sprouts surrounding English holly trees after two glyphosate based herbicide application methods were applied, EZJect application and paint application. These application methods were selected based on the 2  findings of a similar study conducted by Salisbury in 2013. It was therefore hypothesized that using a glyphosate solution with the EZJect method would result in the highest percentage of sprout death. 2.1 Area of Study The area of study was located within Pacific Spirit Regional Park, a community park located on the University of British Columbia endowment lands. The forest is a mixture of deciduous and coniferous trees, and English holly infestations were generally observed to be in areas of deciduous growth. Our study area was located within one of the deciduous forest plots on the western side of the park (Figure 1). English holly trees that were included within the study were geotagged using a GPS and were assigned a unique ID for easy identification. A map of the tree locations was later generated using ArcGIS to ease the distribution of treatments. This map was then supplied to an outside herbicide application contractor who was assigned to perform the different applications according to the assigned treatment. The treatment assignment was dependent on the location of the tree, and which plot it was situated within (Section 3.2). 3   Figure 1: Area of study designated by treatment plots and the location of the sample trees. 2.2 Herbicide Treatment Application After geotagging each study tree, the area was then divided into three plots that contained roughly an equal amount of English holly trees per plot. Each plot was assigned its own application treatment to compare against, and English holly trees were cut to the stump in all three plots. Two of the three treatments involved different methods of glyphosate application, while the third did not utilize any form of herbicide application. This plot was used as a control used for comparison (“Control” method).  Herbicide application occurred on December 1st, 2016; weather conditions for this day were dry during application, but began raining approximately 2 to 3 hours after application. Both methods of glyphosate 4  application used in this study were applied after cutting the trees to the stump. The two treatment methods were as follows: 1. “EZJect” Method: A long lance was used to inject the herbicide shells directly into the tree at stump level. The active ingredient in each shell was glyphosate, N-(phosphonomethyl) glycine, in the form of isopropylamine salt at 83.5% concentration (EZject, n.d.). The number of shells used per stump varied depending on the tree circumference. One pellet was injected per every 7.5 centimetres of circumference, as per the product’s application instructions. 2. “Paint” Method: This is a common technique called cut and paint (Heiligmann, n.d). The trees that were assigned for this treatment were cut down to the stump and were then immediately coated with a quick-dry glyphosate solution at the wound. Herbicide concentration for the cut and paint method was 480 g/L of glyphosate present as a dimethylamine salt. According to the product manufacturer, this product should be absorbed within 30 minutes of application under dry conditions (Dow AgroScience, n.d.). The amount of solution used per tree stump was directly proportional to the surface area of the stump in order to maintain a constant concentration of herbicide. All the treatments were carried out by a third-party contractor that was authorized to use herbicides in a public space. A summary table of the plots is provided below (Table 1). Control plot trees were not cut to the stump until December 15th, 2016, and no herbicide treatment was applied to these trees. Locations of the plots are identified in Figure 1. Table 1: Number of trees studied within each treatment plot Plot Type EZJect Paint Control Number of Trees 19 18 13  2.3 Post-Herbicide Treatment Data Collection and Analysis Several surveying sessions were carried out in the three treatment plots after the herbicide was applied. For the EZject and Paint method, data was collected a total of five times. However, due to the later date in which the Control plot was cut, data for this plot was only collected four separate times. Data for all three treatment plots were collected approximately every three weeks. To determine if the health of the English holly tree stumps were declining, sprout survival within a 90 cm radius of each stump was analysed. The following steps were completed as such:  1. Prior to the first sampling session, all sprouts within 90cm of the stump were tagged using flagging tape and were given a unique identifier (Appendix I, Figure 10).  5  2. During each data collection, each tagged sprout was recorded as either alive or dead, as per Arrieta and Suarez’s 2005 study. The sprout was deemed alive or dead according to the overall appearance of the sprout and by using a simple method known as the scratch test, in which a sharp object is used (e.g. a key or knife) to scratch the surface of the sprout to determine if the inner structure contained healthy tissue which is generally green in colour (Appendix I, Figure 13).  3. The delimited area around each stump was divided into three concentric areas relating to distance from the stump. Each sprout within the 90 cm radius of the stump was categorized according to these three areas. The area closest to the stump covered sprouts between 0 and 30 cm from the stump, the middle area included all the sprouts between 30 and 60 cm, and the outer area covered all sprouts found between 60 and 90 cm. This was done to determine if proximity to the stump affected herbicide absorption by the sprouts, as it was assumed that the sprouts closest to the stump would be directly connected to the root system of the stump.  4. Observations were made of discoloration present in the sprouts as a sign of declining health or potential death.  5. After the data was collected, a one-way analysis of variance test (ANOVA) was used to determine if the mean sprout survival, between each application method (EZject and Paint), was significantly different from control (Salisbury, 2013 and Hutchinson and Langeland, 2012).  6. A one-way analysis of variance (ANOVA) test was also used to determine if there was a significant difference in mean sprout survival between EZject and Paint methods.  7. Lastly, sprout survivorship curves were plotted as the percentages of total surviving sprouts per treatment after herbicide application. 3 Results The results from this study suggest that the paint method resulted in the lowest mean percentage of survival overall (Table 2). As expected, the control plot resulted in the largest mean percentage survival of the three plots.  Table 2: Summary statistics illustrating the number of individuals per sample, the mean percentage of survival of English holly sprouts, and the standard deviation percentage of survival of English holly sprouts Treatment Distance from stump (cm) Number of stumps Number of initial sprouts Mean Percentage Survival (%) SD Percentage Survival (%) Paint 0-90 (all sprouts) 16 190 73.5 34.9 Paint 0-30 14 83 71.1 36.6 6  Paint 30-60 11 53 76.5 39.6 Paint 60-90 11 54 94.1 13.6 EZJect 0-90 (all sprouts) 19 214 89.6 23.3 EZJect 0-30 14 54 76.8 38.7 EZJect 30-60 15 88 95.0 10.4 EZJect 60-90 13 72 92.8 17.6 Control 0-90 (all sprouts) 12 105 94.9 9.4 Control 0-30 10 50 96.9 6.9 Control 30-60 9 25 93.3 14.1 Control 60-90 8 38 91.7 23.6  Appendix III provides a summary of the one-way ANOVA that was calculated on percentage survival of sprouts after herbicide treatment. Both treatments (Paint and EZJect) were compared against the control and against each other. Treatments were further grouped into categories based on distance. A graphical representation of this ANOVA is presented in Figure 2.   Figure 2: A graphical representation that indicates that the ANOVA results are statistically significant. Bars with the same letter label are statistically similar, whereas bars with different labels are statistically different. .  There was a statistically significant difference between the Paint and Control methods, (ANOVA, F(1,26) = 4.26, p = 0.049). However, there was no statistically significant difference between the EZJect and the Control methods (ANOVA, F(1,29) = 0.577, p = 0.454), or between the Paint and EZJect methods (ANOVA, F (1,33) = 2.64, p = 0.114). Within the Paint and Control methods, sprouts survival within 0 to 7  30 cm from the stump were statistically significantly different (ANOVA, F(1,22) = 4.79, p = 0.040) while sprouts survival within 30 to 60 cm and 60 to 90 cm were not. Figures 3 to 6 graphically illustrate the average English holly survival with associated error bars. These figures show the same statistically significant differences, except for differences between EZJect and Paint between all sprouts and between 30-60 cm. However, it should be noted that although these two treatments may significantly differ on this graph, EZJect sprout survival was not found to be significantly different from Control sprout survival.   60657075808590951000 20 40 60 80 100Average English Holly Sprout Survival (%)Days after Treatment Application60-90 cmEZJect Paint Control60657075808590951000 20 40 60 80 100Average English Holly Sprout Survival (%)Days after TreatmentAll sproutsControl EZJect PaintFigure 4: Total survivorship of all sprouts within a 90 cm radius of tree stumps, represented as percentage of total sprouts surviving per treatment type after treatment application. Error bars represent Standard Error with a 95% confidence interval Figure 3: Total survivorship of sprouts within a 60 to 90 cm radius of tree stumps, represented as percentage of total sprouts surviving per treatment type after treatment application. Error bars represent Standard Error with a 95% confidence interval 8   4 Discussion The results indicated that the Paint method showed a statistically significant difference in percentage sprout survival when compared to the control method while the EZJect method did not. Interestingly, the EZJect and Paint methods do not show a statistically significant difference when looking at percentage sprout survival after 3 months; however, when comparing the EZJect and Paint after 3 weeks (the first resample) there is a statistically significant difference (Figure 3). This would suggest that the Paint method is more effective right after that herbicide is applied while the EZJect method requires several weeks to manifest substantial sprout death.  It is also important to note that within a single treatment, there is variation in percentage sprout survival between the three concentric areas relating to distance from the stump (0-30cm, 30-60cm and 60-90cm). The Paint method results show that the sprouts within 0-30cm and 30-60cm have higher sprout death than the sprouts within 60-90cm. It is the sprouts that are closer to the stump that are dying and contributing to the Paint method being the quicker way to reduce the percentage of sprouts around the stump.  Plants have a vascular system that is essential for transporting nutrients to different parts of the plants (Achard and Lacombe, 2016). The vascular system consists of two conductive tissues: the xylem and the phloem. The xylem transports water and nutrients from the roots to the shoots while the phloem distributes 60657075808590951000 20 40 60 80 100Average English Holly Sprout Survival (%)Days after Treatment Application30-60 cmEZJect Paint Control60657075808590951000 50 100Average English Holly Sprout Survival (%)Days after treatment application<30 cmEZJect Paint ControlFigure 6: Total survivorship of sprouts within a 30 to 60 cm radius of tree stumps, represented as percentage of total sprouts surviving per treatment type after treatment application. Error bars represent Standard Error with a 95% confidence interval Figure 5: Total survivorship of sprouts within a 30 cm radius of tree stumps, represented as percentage of total sprouts surviving per treatment type after treatment application. Error bars represent Standard Error with a 95% confidence interval. 9  the products of photosynthesis from the leaves of the plant to the roots and shoot apices (Achard and Lacombe, 2016). Because English holly can reproduce through clonal sprouts and existing roots, herbicides can be transported throughout the tree system through the xylem or the phloem and translocated to the neighbouring sprouts that share a common root system (Schalau, n.d.).   The roots of an English holly can grow as long as 2 m with many clonal sprouts growing from a single root (Appendix I, Figure 16). Because our study observed sprouts that were within 90 cm of the treated stump, in was inferred that many of the surrounding sprouts were connected to the stump through the root system. However, English holly trees undergo a dormancy period during the winter despite being evergreen trees (Paul et al., 2014), but continue their basic metabolism activities at a significantly slower rate than the rest of the year (Drown, 2012 and Groom et al., 1991). It is possible that the translocation of glyphosate to the sprouts was hindered due to the slow circulation of sap throughout the plant and roots system. As a result, the full potential of the herbicide to reduce the percentage of English holly sprouts may not have been observed.  Temperatures during the sample period were also notably lower than the past five-year average, while snowfall was also significantly higher than the past five-year average (Appendix 2). This colder winter may have caused a more intense dormancy than previous years, resulting in less effective circulation of glyphosate throughout the plant’s system. Furthermore, the snow made locating and identifying the sprouts more difficult; several sprouts were lost or damaged while resampling in the snow.   Another concern raised on the efficacy of the glyphosate in reducing sprout survival, is that weather conditions were rainy the day that the herbicide was applied. Glyphosate requires a rain-free period of at least 30 minutes (Dow AgroSciences, n.d.) and is not recommended to be used before heavy rainfall. It is possible that some of the painted glyphosate may have been washed off due to the rain that occurred the afternoon of treatment application.  Dark discolouration on the stump and on the sprouts were observed in trees located within the EZJect and Paint plots (Appendix I, Figures 14 and 15). We hypothesize that this discolouration is a result of Phytophthora ilicis, a pathogen often found associated with Ilex aquifolium (Scanu et al., 2014). We recommend further study of this observation to confirm the presence of this disease; if it is present, then further investigation may be needed to discern its relationship to the glyphosate application.     10  5 Conclusion The results of our data have, thus far, indicated the Paint method to be the quickest and most effective method at killing English holly sprouts. Based off this information, we would recommend that future herbicide applications within Pacific Spirit Regional Park utilize this method to efficiently reduce English holly growth. However, due to this project’s time constraints, we were unable to complete a full monitoring plan that would take into considerations other factors potentially affecting the herbicide application efficacy, or English holly health. Therefore, we have created a list of recommendations for Metro Vancouver to review and consider prior to further herbicide application. 6 Recommendations 6.1 Longer Monitoring A longer study period is recommended to monitor the English Holly sprouts further and observe if the upcoming spring and summer months show a difference in sprout growth. Glyphosate is formulated to inhibit a synthase found primarily in plastids1, which in turn blocks the plant’s production of an aromatic amino acid, affecting the plant’s metabolism (Baylis, 2000). Therefore, circulation of the herbicide throughout the tree may be inhibited by the plant’s dormancy during the winter months. Another study completed in 2013 by Salisbury completed their monitoring over the course of a full year to observe the plant’s survival throughout the seasons of the year and found much more significant results in English holly death.   6.2 Root Identification Since English holly reproduces via seed and clonal sprout growth, sprout survival after herbicide application may differ depending on whether that sprout is attached to the treated tree. If a sprout has grown directly from seed, as opposed to clonally, the herbicide should not show signs of affecting it. Because sprouts can grow out at least 2-3 m from a single plant (Appendix I, Figure 16), it is difficult to identify which category a sprout falls into upon sight. Therefore, we recommend that after the year long monitoring has occurred, sprouts around the treated trees be dug up to identify which are clonal sprouts, and which have grown straight from seed.   6.3 Cut Tree Removal After cutting the holly trees and applying herbicide, the fallen trees and their berries were left on the ground within the treatment areas throughout the remainder of the study (Appendix I, Figure 17). English holly berries are commonly dispersed by birds such as thrushes which are common to British Columbia (Rendell and Ennos, 2003). Many of these trees were found after the fieldwork for the study was completed to have ripened berries that would be ideal for birds. Therefore, to prevent further dispersal of the English holly, it                                                           1 Plastids: an organelle found almost exclusively in plants and algae (Wise, 2006) 11  is highly recommended that these trees be removed from the study area, and that cut trees be removed immediately after any future herbicide applications.  6.4 Leave Canopy for Future EZJect Application In order eliminate any bias within this study, all trees, regardless of herbicide application method, were cut to the stump. This was done to assure that any signs of death were solely a result of herbicide application, not because of any side effects caused by cutting the English holly trees within one plot. However, if EZJect is to be applied in the future, we suggest that the trees remain standing in order to encourage further circulation of the herbicide through the trees and their connected sprouts. We hypothesize that leaving the canopy in tact may encourage tree sap circulation through increased photosynthesis, thus increasing the internal spread of herbicide.  Furthermore, if the canopy is left standing, additional physical effects of the herbicide may be observed that otherwise may not have been viewed with a single stump. 7 Acknowledgements We would like to thank Robyn Worcester for her help in this project by providing field equipment and the opportunity to work with Metro Vancouver and their partners. We would also like to extend our gratitude to Bernardo Ranieri who has provided us with invaluable technical guidance for completing this research.  8 References Achard, Patrick and Lacombe, Benoit (2016). Long-distance Transport of Phytohormones Through the Plant Vascular System. Current Opinion in Plant Biology, 34: 1-8. http://doi.org/10.1016/j.pbi.2016.06.007  Arrieta, S and Suarez, F (2005). Spatial patterns of seedling emergence and survival as a critical phase in holly (Ilex aquifolium L.) woodland recruitment in Central Spain. Forest Ecology and Management, 205 (1): 267-282. DOI: 10.1016/j.foreco.2004.10.009  Baylis, A. D. (2000). Why glyphosate is a global herbicide: strengths, weaknesses and prospects. Pest Management Science, 56(4), 299-308.  Dow AgroSciences. (n.d.). Vantage XRT Herbicide. Maximum Glyphosate Performance Plus the Support of Dow AgroSciences. Retrieved on March 12, 2017 from: http://msdssearch.dow.com/PublishedLiteratureDAS/dh_08d6/0901b803808d6fad.pdf?filepath=%2Fpdfs%2Fnoreg%2F010-22041.pdf&fromPage=GetDoc  Drown, W.D. (2012, October 26). Sleep Cycle of Trees. AmericanNews. Retrieved from: http://articles.aberdeennews.com/2012-10-26/farmforum/34754643_1_tree-species-cool-temperatures-sleep-cycle  12   EZJect. (n.d.). Diamondback Herbicide Shells (Fact Sheet). Retrieved on March 12, 2017 from: http://www.ezject.com/?page_id=547  Invasive Species Council of British Columbia. (n.d.). English Holly. Retrieved on March 10, 2017 from ISC: http://bcinvasives.ca/resources/photo-gallery/english-holly/  Groom, Q., Baker, N., & Long, S. (1991). Photoinhibition of holly (Ilex aquifolium) in the field during the winter. Physiologia Plantarum, 83, 585-590. DOI: 10.1111/j.1399-3054.1991.tb02472.x  Haines, M., Cameron, C., & Hughes, T. (2016). One-off manual removal of English holly (Ilex aquifolium) may contribute to its spread.  Heiligmann, R. B. (n.d.). Extension Factsheet F-45-97, Controlling Undesirable Trees, Shrubs, And Vines in Your Woodland [Fact Sheet]. Retrieved from The Ohio State University: http://woodlandstewards.osu.edu/sites/woodlands/files/d6/files/pubfiles/0045m.pdf;  Hutchinson, J. T. and Langeland, K. A. (2012). Repeated Herbicide Application for Control of Old World Climbing Fern (Lygodium microphyllum) and the Effects on Nontarget Vegetation on Everglade Tree Islands. Invasive Plant Science and Management, 5(4), 477-486. DOI: 10.1614/IPSM-D-12-00015.1  Namuth, D., Nissen, S.J. and Sterling T.M. Foliar Absorption and Phloem Translocation. Plant & Soil Sciences eLibrary. Retrieved from: http://passel.unl.edu/pages/informationmodule.php?idinformationmodule=1056648673&topicorder=1&maxto=8&minto=1   Paul, A., et al. (2014). RNA-seq-mediated transcriptome analysis of actively growing & winter dormant shoots identifies non-deciduous habit of evergreen tree tea during winters. Scientific Reports, 4(5932). doi: 10.1038/srep05932  Peterken, G. & Lloyd, P. (1967). Ilex Aquifolium L. The Journal Of Ecology, 55(3), 841. doi: 10.2307/2258429    Rendell, S. & Ennos, R.A. (2003). Chloroplast DNA diversity of the dioecious European tree Ilex aquifolium L. (English holly). Molecular Ecology, 12(10), 2681-2688. Doi: https://www.doi.org/10.1046/j.1365-294X.2003.01934.x  Salisbury, N. (2013). English Holly (Ilex aquifolium) Herbicide Treatment Study. Seattle: Earth Corps. Retrieved from: http://www.earthcorps.org/ftp/ECScience/Projects/Holly_Research/Holly_Treatment_Study_Report_2013.pdf  13  Scanu, B., Linaldeddu, B. T., Perez-Sierra, A., Deidda A., & Franceschini, A. (2014). Phytophthora ilicis as a leaf and stem pathogen of Ilex aquifolium in Mediterranean islands. Phtypathologia Mediterranea, 53(3), 480-490. Doi: http://dx.doi.org/10.14601/Phytopathol_Mediterr-14048  Schalau, J. (n.d.). Application of Herbicides to Manage Woody Vegetation. The Univeristy of Arizona: College of Agriculture and Life Sciences. Retrieved from: https://cals.arizona.edu/extension/ornamentalhort/landscapemgmt/prunning/cutstump.pdf   Wise, R.R. (2006). The Diversity of Plastid Form and Function. Advances in Photosynthesis and Respiration, p 3-26.    14  9 Appendix I: Site Photographs   Figure 7: Team members Laurentia and Brenton collecting preliminary data on English holly trees in Pacific Spirit Regional Park prior to herbicide application  Figure 8: Plant species Ilex opaca (Yellow berried American holly) found within the control plot 15   Figure 9: English holly tree #33, tagged and cut to the stump prior to herbicide application  Figure 10: Glyphosate solution being applied to the cut holly tree stump via the Paint method 16   Figure 11: English holly sprouts tagged for future data collection  Figure 12: Applying glyphosate using an EZJect lance into English holly tree stump 17   Figure 13: EZJect pellets inserted into English holly tree stump  Figure 14: Scratch test method to determine if sprouts were alive or dead 18   Figure 15: Leaf discolouration observed after herbicide application on tree #31  Figure 16: Stump discolouration observed after herbicide application 19   Figure 17: A single English holly sprout colony reaching approximately 2-3 metres in length. Sprouts are attached clonally to each other.  Figure 18: Ripened berries present on the cut and fallen trees left on Site (left) vs. unripened berries still growing from the tree (right). 20  10 Appendix II: Weather Data  Figure 19: Daily mean temperatures (℃) for the months of December, January and February. Blue line represents five year daily average values (2012 -2016) and the orange line represents daily average values for 2016-2017. 2016-2017 data show colder temperatures than previous years. Data collected at the Vancouver International Airport weather Downloaded from Government of Canada historical climate data records.  Figure 20: Daily snowfall (mm) for the months of December, January and February. Blue line represents five year daily average values (2012 -2016) and the orange line represents daily values for 2016-2017. Data collected at the Vancouver International Airport weather station. Downloaded from Government of Canada historical climate data records.   21  11 Appendix III: ANOVA Summary  Table 3: ANOVA summary table illustrating the F ratio, df, and P-value calculated during the ANOVA. Significance level is 95%.  Treatment Plots F Ratio P-value Statistically Significant at 0.95 Paint and EZJect All Sprouts F (1,33) = 2.64 0.114 N Paint and EZJect 0cm-30cm F (1,26) = 0.16 0.692 N Paint and EZJect 30cm-60cm F (1,24) = 3.03 0.094 N Paint and EZJect 60cm-90cm F (1,22) = 0.04 0.844 N Paint and Control All Sprouts F (1,26) = 4.26 0.049 Y Paint and Control 0cm-30cm F (1,22) = 4.79 0.040 Y Paint and Control 30cm-60cm F (1,18) = 1.46 0.243 N Paint and Control 60cm-90cm F (1,17) = 0.08 0.780 N EZJect and Control All Sprouts F (1,29) = 0.577 0.454 N EZJect and Control 0cm-30cm F (1,29) = 0.58 0.454 N EZJect and Control 30cm-60cm F (1,22) = 2.59 0.122 N EZJect and Control 60cm-90cm F (1,19) = 0.02 0.902 N  

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