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Biology of the rangeland weed hoary alyssum (Berteroa incana (L.) DC.) Stopps, Gregory James 2012

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BIOLOGY OF THE RANGELAND WEED HOARY ALYSSUM (Berteroa incana (L.) DC.) by Gregory James Stopps B.Sc. Trinity Western University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Plant Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2012 © Gregory James Stopps, 2012  Abstract Hoary alyssum (Berteroa incana (L.) DC) has become a serious weed of rangelands, pastures, and forage crops in British Columbia. In order to develop effective management strategies for this weed, a sound knowledge of its biology and ecology is essential. The goal of this research is to improve our understanding of the biology of hoary alyssum and to identify vulnerable links in its persistence strategy that may be exploited to develop effective management strategies for this weed. The distribution of hoary alyssum seeds in the soil profile; the size and persistence of its seed bank; the effect of burial depth on the dynamics of seed dormancy and germination; the response to mechanical removal of flowering shoots (mowing); the potential allelopathic influence on seed germination and seedling growth of associated forage grasses; and the effect of nitrogen fertilization on the growth of hoary alyssum and associated forage grasses were investigated. Results show: (1) 93-95% of hoary alyssum seeds were present in the top 4 cm of the soil profile at infested sites in the Morrissey Creek, BC area; (2) the size of soil seed banks ranged between 0 to 132.4 million seeds ha-1; (3) seeds on the soil surface showed little primary dormancy, but buried seeds showed some enforced and some induced dormancy; (4) mowing of flowering shoots resulted in the release of apical dominance and the regeneration of shoots; (5) delaying mowing until later stages of flower and seed development increased plant mortality, but some plants were still able to reproduce shoots and seed; (6) leachates of hoary alyssum leaves strongly inhibited the seed germination and seedling growth of grassy species in Petri dish assays, but this allelopathic influence was not observed consistently in soil assays; (7) hoary alyssum biomass increased in response to nitrogen (urea) fertilization, but forage grasses showed no response due to leaching of this nutrient; and (8) nitrogen fertilization promoted bolting and  ii  flowering in hoary alyssum. These results improve our understanding of hoary alyssum biology, and may aid in the development of effective management strategies for this weed.  iii  Table of Contents Abstract .......................................................................................................................................... ii Table of Contents ......................................................................................................................... iv List of Tables...............................................................................................................................viii List of Figures ............................................................................................................................... ix Acknowledgements.....................................................................................................................xiii Chapter 1. General Introduction, Literature Review, & Field Site Description..................... 1 1.1 General Introduction ............................................................................................................. 1 1.2 Literature Review.................................................................................................................. 5 1.2.1 Plant species used in this study ...................................................................................... 5 1.2.1.1 Hoary alyssum (Berteroa incana (L.) DC) ............................................................. 5 1.2.1.2 Bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love) .......................... 6 1.2.1.3 Idaho fescue (Festuca idahoensis Elmer.) .............................................................. 7 1.2.1.4 Prairie junegrass (Koeleria macrantha (Ledeb.) J.A. Schultes) ............................. 8 1.2.1.5 Cheatgrass (Bromus tectorum L.)............................................................................ 8 1.2.2 Soil seed banks ............................................................................................................... 9 1.2.2.1 Hoary alyssum seed banks .................................................................................... 10 1.2.3 Mechanical removal of flowering shoots (mowing) .................................................... 10 1.2.3.1 Mowing hoary alyssum ......................................................................................... 11 1.2.4 Allelopathy ................................................................................................................... 12 1.2.4.1 Allelochemicals in hoary alyssum ........................................................................ 12 1.2.5 Nitrogen fertilization .................................................................................................... 12 1.2.5.1 The response of hoary alyssum to nitrogen fertilization ....................................... 13  iv  1.3 Field Site Description.......................................................................................................... 14 1.3.1 Field site selection and location ................................................................................... 14 1.3.2 Field site vegetation and topography ........................................................................... 15 1.3.3 Field site land usage ..................................................................................................... 16 Chapter 2. Hoary Alyssum Seed Bank Dynamics .................................................................... 17 2.1 Introduction ......................................................................................................................... 17 2.2 Materials and Methods ........................................................................................................ 18 2.2.1 Seed sources ................................................................................................................. 18 2.2.2 Field experiments ......................................................................................................... 18 2.2.2.1 Soil seed bank size and distribution ...................................................................... 19 2.2.2.2 Artificial soil seed banks ....................................................................................... 19 2.2.3 Data analysis ................................................................................................................ 23 2.3 Results ................................................................................................................................. 23 2.3.1 Soil seed bank size and distribution ............................................................................. 23 2.3.2 Artificial soil seed banks .............................................................................................. 25 2.4. Discussion .......................................................................................................................... 29 Chapter 3: The Response of Hoary Alyssum to Mowing ........................................................ 33 3.1 Introduction ......................................................................................................................... 33 3.2 Materials and Methods ........................................................................................................ 34 3.2.1 Seed source................................................................................................................... 34 3.2.2 Soil selection ................................................................................................................ 35 3.2.3 Mowing experiments.................................................................................................... 35 3.2.4 Data analysis ............................................................................................................... 37  v  3.3 Results ................................................................................................................................. 37 3.4 Discussion ........................................................................................................................... 39 Chapter 4: Allelopathic Influence of Hoary Alyssum on Native Forages.............................. 42 4.1 Introduction ......................................................................................................................... 42 4.2 Materials and Methods ........................................................................................................ 43 4.2.1 Seed sources ................................................................................................................. 43 4.2.2 Source of hoary alyssum leaves ................................................................................... 44 4.2.3 Leachate preparation .................................................................................................... 44 4.2.4 Seed germination assays............................................................................................... 45 4.2.5 Seedling growth assays ................................................................................................ 46 4.2.6 Allelopathic effects in soil............................................................................................ 47 4.2.6.1 Seedling emergence in soil with leaf leachate ...................................................... 47 4.2.6.2 Seedling emergence in soil with added hoary alyssum leaf biomass.................... 49 4.2.7 Data analysis ................................................................................................................ 49 4.3 Results ................................................................................................................................. 50 4.3.1 Seed germination assays............................................................................................... 50 4.3.2. Seedling growth assays ............................................................................................... 50 4.3.3 Seedling emergence in soil with leaf leachate ............................................................. 55 4.3.4 Seedling emergence in soil with added hoary alyssum leaf material........................... 58 4.4 Discussion ........................................................................................................................... 61 Chapter 5: The Effect of Nitrogen Fertilization on Hoary Alyssum and Associated Forage Species ............................................................................................................................. 64 5.1 Introduction ......................................................................................................................... 64  vi  5.2 Materials and Methods ........................................................................................................ 66 5.2.1 Field experiment........................................................................................................... 66 5.2.1.1 Field site ................................................................................................................ 66 5.2.1.2 Soil analysis and fertilizer treatment..................................................................... 66 5.2.1.3 Herbicide treatment ............................................................................................... 66 5.2.1.4 Nitrogen fertilization field experiment.................................................................. 67 5.2.2 Pot-culture experiment ................................................................................................. 69 5.2.2.1 Seed sources .......................................................................................................... 69 5.2.2.2 Soil selection ......................................................................................................... 69 5.2.2.3 Nitrogen fertilization pot-culture experiment ....................................................... 69 5.2.3 Data analysis ................................................................................................................ 72 5.3 Results ................................................................................................................................. 72 5.3.1 Nitrogen fertilization field experiment......................................................................... 72 5.3.2 Nitrogen fertilization pot-culture experiment .............................................................. 73 5.4 Discussion ........................................................................................................................... 77 Chapter 6. General Discussion................................................................................................... 81 6.1 Hoary alyssum seed bank dynamics.................................................................................... 82 6.2 The response of hoary alyssum to mowing......................................................................... 82 6.3 Allelopathic influence of hoary alyssum on native forages ................................................ 83 6.4 The effect of nitrogen fertilization on hoary alyssum and associated forage species ......... 84 6.5 General conclusion.............................................................................................................. 85 Literature Cited........................................................................................................................... 86  vii  List of Tables Table 3.1. Response of hoary alyssum plants to mowing at different stages of flowering shoot development......................................................................................................................... 38 Table 5.1. Available nitrogen in the upper 10 cm of soil at the conclusion of the pot-culture experiment..................................................................................................................................... 76 Table 5.2. Leaching of nitrogen due to watering in pot-culture experiments. ............................. 76  viii  List of Figures Figure 2.1. Soil cores (10 cm diameter x 10 cm depth) before (A), and after (B) extraction from the hoary alyssum infested study site near Morrissey Creek, Grand Forks BC. Soil cores (15) were taken randomly from the site in July 2008 and again in July 2009..................... 20 Figure 2.2. An artificial soil seed bank (A) utilizing mesh seed bags (B). Seed bags were buried at depths of 0, 2, and 5 cm. Four seed banks were established in August 2008, and again in August 2009..................................................................................................................... 21 Figure 2.3. Distribution of hoary alyssum seeds in soil profile in 2008 (■) and 2009 (□) in Grand Forks, British Colombia. Each value is mean ± SE of 15 cores. ....................................... 24 Figure 2.4. Fate of hoary alyssum seeds buried in August 2008 at depths of 0, 2, and 5 cm. Fates were: seedlings (S), empty seeds (E), enforced dormant (ED), induced dormant (ID), and non-viable (NV). Values are a mean % ± SE of 4 (or available) replicates........................... 26 Figure 2.5. Fate of hoary alyssum seeds buried in August 2009 at depths of 0, 2, and 5 cm. Fates were: seedlings (S), empty seeds (E), enforced dormant (ED), induced dormant (ID), and non-viable (NV). Values are a mean % ± SE of 4 replicates. ................................................ 27 Figure 2.6. Hoary alyssum seeds imbibed in 0.1% tetrazolium solution for 24 hours at 30°C. A viable seed with healthy (red) embryonic tissue (A), viable seeds showing partially decaying tissue (B and C), and a non-viable seed with dead embryonic tissue (D). .................... 32 Figure 4.1. Set up of assays testing seedling emergence in soil treated with 4% w/v hoary alyssum leachate. Assays were performed starting on July 12 and 19, 2010. .............................. 48 Figure 4.2. Effect of hoary alyssum leachate on the germination of select forage grasses in Petri dish assays (July 4, 2009). Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), and cheatgrass (D). Values are means of 5 replicates ± SE with 20 seeds per plate  ix  (10 for cheatgrass). Different letters above the histograms represent significantly different means (P ≤ 0.05). Leachate was made with leaf material collected in 2008. ............................... 51 Figure 4.3. Effect of hoary alyssum leachate on the germination of select forage grasses in Petri dish assays (July 15, 2009). Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), and cheatgrass (D). Values are means of 5 replicates ± SE with 20 seeds per plate (10 for cheatgrass). Different letters above the histograms represent significantly different means (P ≤ 0.05). Leachate was made with leaf material collected in 2008. .............................. 52 Figure 4.4. Effect of hoary alyssum leachate on the germination of its own seeds (autotoxicity) in Petri dish assays. July 25, 2009 assay (A) with leachate from leaf material collected in 2008; February 19, 2010 assay (B) with leachate from leaf material collected in 2009. Values are means of five replicates ± SE with 20 seeds per plate. Different letters above the histograms represent significantly different means (P ≤ 0.05). .................................... 53 Figure 4.5. Effect of hoary alyssum leachate on shoot (  ) and root (  ) growth of  forage grasses and itself in Petri dish assays (July 15, 2010). Idaho fescue (A), bluebunch wheatgrass (B), cheatgrass (C), hoary alyssum (D). Seedlings had 4 to 6 mm long radicles to start (3 to 5 mm in hoary alyssum). Leachate was made with leaf material collected in 2009. Values are means of five replicates ± SE with 10 seedlings per plate. Different letters above the data points represent significantly different means (P ≤ 0.05)................................................ 54 Figure 4.6. Hoary alyssum seedlings after 10 days of exposure to 0% leachate (distilled water control) (A), and 4% hoary alyssum leaf leachate (B). ....................................................... 56 Figure 4.7. Effect of hoary alyssum leaf leachate (4%) on germination of forage grasses and itself in soil. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil + Water (SW); Soil + Leachate (SL);  x  Soil/Charcoal + Water (CW); Soil/Charcoal + Leachate (CL). Leachate was made from leaf material collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05).............................................. 57 Figure 4.8. Effect of hoary alyssum leaf material (0.4 g mixed into soil) on germination of forage grasses and itself. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil control (S); Leaf biomass (L); Leached Leaf Residue (LR). Leaf biomass was collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05)............................................................................................................................ 59 Figure 4.9. Effect of hoary alyssum leaf material (0.2 g on the soil surface) on germination of forage grasses and itself. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil control (S); Leaf biomass (L); Leached Leaf Residue (LR). Leaf biomass was collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05)............................................................................................................................ 60 Figure 5.1. Nitrogen fertilization field plots (2 m x 2 m) at the hoary alyssum infested Morrissey Creek study site near Grand Forks, BC.; Field plots (A), and close up of a plot (B). . 68 Figure 5.2. Pot-culture experiment to study the effect of N on hoary alyssum and associated grasses. Hoary alyssum and Idaho fescue with 16 kg ha-1 N (A); hoary alyssum and bluebunch wheatgrass with 128 kg ha-1 N (B and C).................................................................... 70 Figure 5.3. Effect of nitrogen (urea 46-0-0) fertilization on dry biomass of hoary alyssum (HA), Idaho fescue (IF) and bluebunch wheatgrass (BB). Hoary alyssum + Idaho fescue (A);  xi  hoary alyssum + bluebunch wheatgrass (B); hoary alyssum + Idaho fescue + bluebunch wheatgrass (C). Values are means ± SE of 8 replications............................................................. 74 Figure 5.4. Effect of nitrogen (urea 46-0-0) fertilization on bolting of hoary alyssum rosettes. Idaho fescue (IF), bluebunch wheatgrass (BB), and Idaho fescue + bluebunch wheatgrass (Mix) mixtures. Values are means ± SE of 8 replicates................................................................ 75  xii  Acknowledgements I would like to thank my supervisor, Dr. Mahesh Upadhyaya, for his extensive advice and guidance throughout this experience. I also wish to thank my committee members Dr. David Clements and Dr. Edwardo Jovel for their assistance. Financial and/or in-kind support for this research was provided in part by: the Investment Agriculture Foundation of BC, the Alberta Agriculture and Food Council (through Agriculture and Agri-Food Canada’s Advancing Canadian Agriculture and Agri-Food program), FortisBC, the Boundary Weed Management Committee, the Regional District of Kootenay Boundary, and the Grand Forks Stockbreeders Association. I wish to specifically thank Mrs. Coreen Moroziuk (Investment Agriculture Foundation of BC), Mr. Doug Pickard (Right-of-Way Land Superintendent, Fortis BC), and Mr. Al Wait (Grand Forks Stockbreeders Association) for their help with this project. I also thank Mr. John Mehmal for allowing me to use his land as a field site. A very special thanks is reserved for Barb Stewart of the Boundary Weed Management Committee, without whom this thesis would not have been possible. Further, I wish to acknowledge Drs. Art Bomke, Maja Krzic, and Les Lavkulich for their help in soil selection and interpretation of soil analyses. I am indebted to Dr. Tony Kozac for his gracious assistance with the statistical analysis of data. I would like to thank all members of the M. K. Upadhyaya lab for their assistance, especially Jenny Hong and Eamonn Watson. Finally, I wish to thank my parents, family, and friends for their unending love, support, and prayers during this time.  xiii  Chapter 1. General Introduction, Literature Review, & Field Site Description 1.1 General Introduction Exotic weed species pose significant challenges to the health and sustainability of natural and agricultural ecosystems. They invade and establish quickly threatening the biodiversity and ecological function of natural systems (Lonsdale 1999) as well as the sustainability and economic value of agricultural systems (Pimentel et al. 2005). One such weed is hoary alyssum (Berteroa incana (L.) DC). Hoary alyssum has rapidly established and is becoming increasingly problematic in rangelands (uncultivated semi-natural ecosystems, used and managed for their forage value (Horton 1998; Friedel et al. 2000)) of southern British Columbia. An annual, winter annual, or short lived perennial (Darbyshire 2003) hoary alyssum, native to Europe, is thought to have been introduced to North America as a contaminant of forage and clover seeds in the late 19th or early 20th century (Mulligan 2002; Warwick and Francis 2006). Following its introduction, hoary alyssum has continued to spread, becoming a serious weed in rangelands, pastures, and forage crops across North America. To date, hoary alyssum has been reported in all Canadian provinces with the exception of Newfoundland and Prince Edward Island (Warwick and Francis 2006). It has become most prevalent and problematic in southern Ontario (Alex 1992) and south central/southeastern British Columbia (Douglas et al. 1998), particularly in the Okanagan, Thompson, and Kootenay Boundary regions. The BC Weed Control Act and the Forest and Range Practices Act now classify hoary alyssum as a noxious/invasive plant species in the Kootenay Boundary Regional District (BC Government 2001; BC Ministry of Forests 2004). In the United States, hoary alyssum has been reported from Maine to Washington State and as far south as New Mexico  1  (USDA, NRCS 2005). It is most problematic in Michigan, Minnesota, and Washington (Invaders Database System 2011). While often observed in meadows, pastures, and waste places, hoary alyssum appears to have spread almost unchecked by weed managers. Early research even ignored its weedy nature, suggesting that it has potential to be cultivated as an oil seed crop and would not likely become a weed in agricultural systems (Goering et al. 1965). Public awareness of the threat posed by hoary alyssum was not piqued until the early 1990s when veterinary researchers identified it as toxic to horses (Ellison 1992; Geor et al. 1992; Hovda and Rose 1993). Today, an increased awareness of conservational ecology and the potential ecological and economic impacts associated with increasing weed problems in BC rangelands has heightened interest in hoary alyssum (Warwick and Francis 2006). With an ability to grow in poor soils, a deep taproot, woody stem, absence of natural enemies, and prolific seed production hoary alyssum is a strong competitor establishing quickly to out-compete native forbs and grasses. Hoary alyssum may also reduce the biodiversity and species richness of native pollinators as it is not particularly attractive to insect pollinators (Reed 1993). Aside from its toxicity, once established in forage crops, hoary alyssum also reduces the yield, protein content, and palatability of economically valuable forages (Hastings and Kust 1970). Various broadleaf herbicides such as atrazine (2-chloro-4-(ethylamino)-6(isopropylamino)-s-triazine), simazine (2-chloro-4,6-bis(ethylamino)-s-triazine), 2,4-D (2,4dichlorophenoxyacetic acid) or a mixture of 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) are effective in killing hoary alyssum (Kust 1969; Warwick and Francis 2006; Barb Stewart 2008, personal comm.). However, the use of such herbicides in rangelands and pastures is neither  2  economical nor environmentally friendly. In order to control the spread of hoary alyssum and reduce its impact, alternative methods of weed management must be sought. Effective management of any invasive weed requires a sound knowledge and understanding of its biology and ecophysiology to identify weak links in its persistence strategy. Unfortunately, little information regarding the persistence and survival strategies of hoary alyssum is available. Thus it is pertinent to first develop a better understanding of hoary alyssum’s biological and ecophysiological characteristics before developing effective management strategies for this weed. The goal of this research was to increase our understanding of hoary alyssum’s biology and identify weak-links in its persistence strategy that can be exploited for effective management of the species.  The specific objectives of this research were to determine: 1. the distribution of hoary alyssum seed in the soil profile, the size and persistence of its seed bank, and the effect of seed burial depth on the dynamics of seed dormancy, germination, and/or survival, 2. if mechanical removal of hoary alyssum flowering stems (mowing) affects its seed production and survival, 3. if water soluble chemicals released from the leaves of hoary alyssum exert allelopathic influence on associated forage grasses by inhibiting their seed germination and seedling growth,  3  4. how the application of nitrogen fertilizer to a mixed population of forage grasses and hoary alyssum affects their growth and respective competitive abilities, and whether this practice could be used to control or limit hoary alyssum.  This thesis is presented in manuscript format, with each chapter containing its own introduction, materials and methods, results, and discussion sections. Chapter 2 deals with the dynamics of hoary alyssum’s soil seed banks (Objective 1). Chapter 3 examines the effect of mowing (mechanically removing the flowering shoots) on hoary alyssum (Objective 2). The allelopathic influence of hoary alyssum on associated forage species (Objective 3) is examined in Chapter 4. Chapter 5 investigates the effects of nitrogen fertilization on mixed populations of hoary alyssum and associated forage species. General discussion and conclusions from this research are presented in Chapter 6.  4  1.2 Literature Review This section briefly reviews relevant literature on the plant species used in this study, and the general concepts of soil seed banks, mechanical removal of flowering shoots (mowing), allelopathy, and the effects of nitrogen fertilization on growth and competitive ability.  1.2.1 Plant species used in this study 1.2.1.1 Hoary alyssum (Berteroa incana (L.) DC) Hoary alyssum, a member of the brassicaceae family, is an annual to short lived perennial that reproduces only from seed (Alex 1992). The common name comes from the grayish-green ‘hoary’ appearance of the plant, which is caused by a mix of star-shaped and simple hairs that cover the stems, leaves, sepals, and seedpods (Warwick and Francis 2006). The weed forms a basal rosette with a slender taproot that gives rise to one or more erect to semi-erect shoots. Shoots are usually green to purplish in colour, highly branched and rarely simple, growing to heights in excess of 1 m in extreme cases (Warwick and Francis 2006). Leaves are alternate and entire, appearing oval-lanceolate with long stalks and broad near the tips in the basal rosette. Leaves appearing on the lower portion of shoots are similar to the rosette leaves, while leaves on the upper part of shoots are stalkless and broad near the base tapering to a long narrow point (Warwick and Francis 2006). Flowers are small (3 mm across), with four white petals (each with a deep cleft), appearing clustered near the tips of shoots and their branches in simple to compound racemes (Warwick and Francis 2006). Flowers give rise to ellipsoid or ovoid seedpods that are held close to the stem. Seedpods are stalked with a beak like tip. They typically contain 4-12 seeds each (Warwick and Francis 2006). Seeds are lens-shaped and small  5  (1-1.5 mm across) appearing dark brown to reddish-brown in colour (Warwick and Francis 2006). Native to Europe, hoary alyssum was first collected in Canada near Wallbridge, ON in 1893 (Mulligan 2002). It has since spread across much of the country and many areas of the United States. It appears most commonly on poor sandy or gravelly soils in uncultivated areas, along roadsides, waste places and rangelands (Alex 1992; Mulligan 2002; Warwick and Francis 2006). It thrives under the temperate continental climate conditions of North America, able to survive cold winters and tolerate relatively hot dry summers (Warwick and Francis 2006). Seedlings that establish in the fall are resistant to winterkill while established flowering plants are considered to be xerophytes, able to withstand summer droughts because of their relatively low water requirements (Warwick and Francis 2006). Once established in an ecosystem, hoary alyssum stands can become quite dense (Warwick and Francis 2006), dominating native vegetation. Hoary alyssum most commonly appears in British Columbia across the southern interior, specifically in the Okanagan, Thompson, and Kootenay-Boundary regions. Infestations are common in high traffic areas, and populations have now spread by seed to relatively remote rangeland environments. Dissemination of seeds occurs primarily as a contaminant on service vehicles that utilize right-of-way access roads to maintain hydro and telecommunication lines (Boundary Weed Management Committee 2008, personal comm.).  1.2.1.2 Bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love) Bluebunch wheatgrass is perennial native bunchgrass reproducing from seed, and occasionally by short rhizomes in high rainfall areas (Parish et al. 1996; Ogle et al. 2010a). It  6  grows in large clumps up to 150 cm in diameter (Parish et al. 1996; Douglas et al. 1998) and up to a height of 40 to 130 cm (Douglas et al. 2001; Ogle et al. 2010a). Bluebunch wheatgrass commonly appears at low to mid elevations in grasslands, and dry open forests of the Fraser, Thompson, Okanagan, and Kootenay regions (Parish et al. 1996). It is a dominant grass species in the Interior Douglas-fir and Ponderosa Pine biogeoclimatic zones that spread throughout the interior of southern British Columbia (Hope et al. 1991a and b). Bluebunch wheatgrass performs well on dry soils with medium to course textures and on sandy sites. It is an important native forage species, grazed by both domestic livestock and wildlife (Parish et al. 1996; Ogle et al. 2010a). The species is generally unable to tolerate heavy and continuous grazing (Ogle et al. 2010a), which may leave its populations susceptible to competition from weedy species such as hoary alyssum. Bluebunch wheatgrass was used in this study because it commonly occurs in areas where hoary alyssum is spreading, and is observed as a dominant native species at the field site utilized for this research.  1.2.1.3 Idaho fescue (Festuca idahoensis Elmer.) Idaho fescue is another densely tufted native perennial bunchgrass (Parish et al. 1996) common to co-dominant with bluebunch wheatgrass in the Interior Douglas-fir and Ponderosa Pine biogeoclimatic zones of BC (Hope et al. 1991a and b; Olson and Wallander 2002). It appears bluish-green to green in colour, growing to heights of 30 to 100 cm (Ogle et al. 2008). A widely spread species in western North America, it is able to thrive under a variety of soil and environmental conditions. Idaho fescue is considered to be an important native forage species that is able to withstand heavy grazing (to an extent) (Ogle et al. 2008). It was selected for this  7  study because like bluebunch wheatgrass it commonly occurs as a dominant native species in areas of BC that are prone to hoary alyssum infestation.  1.2.1.4 Prairie junegrass (Koeleria macrantha (Ledeb.) J.A. Schultes) Prairie junegrass is yet another native perennial bunchgrass, which grows 20 to 50 cm tall (Parish et al. 1996). It is a common native species in western rangelands and throughout the Interior Douglas-fir biogeoclimatic zone (Hope et al. 1991b), often associated with bluebunch wheatgrass communities (Ogle et al. 2010b). Prairie junegrass has a moderate to high tolerance to grazing and is considered a good forage species in the spring months, becoming less palatable as it matures (Ogle et al. 2010b). This species was selected for use in this study because of its prevalence in the rangeland ecosystems currently being threatened by hoary alyssum infestation.  1.2.1.5 Cheatgrass (Bromus tectorum L.) Cheatgrass is a tufted annual to winter annual which grows 20 to 60 cm tall (Parish et al. 1996; Skinner et al. 2008). It is a wide spread non-native species, considered invasive to noxious across may areas of North America (Skinner et al. 2008). Cheatgrass provides good quality forage early in the growing season but becomes unpalatable as it matures (Skinner et al. 2008). While cheatgrass is a weedy species, it was selected for use in this study because of its common occurrence throughout BC’s rangelands, and its presence amongst hoary alyssum communities at the field site used for this research.  8  1.2.2 Soil seed banks Plant life is ultimately perpetuated in two ways: by seed and/or by vegetative propagation (Simpson et al. 1989). Soil seed banks are reservoirs of viable seeds (seeds with the potential to produce new plants (Baker 1989)), buried in the soil or present on/in the soil surface and associated litter (Thompson and Grime 1979; Simpson et al. 1989). These seed banks are highly complex and extremely variable but essential in the persistence strategy of short-lived species (annuals to short lived perennials) that do not reproduce vegetatively (Christoffoleti and Castano 1998). Seed banks are in a constant state of flux, as new viable seeds are added and the number of those already present in the bank continuously declines because of germination or seed death (Cavers and Benoit 1989). Seed banks also have both spatial and temporal dimensions as they disperse seeds physically through space (into and through the soil) and through time (Sampson et al. 1989). Soil seed banks can be classified into two groups, transient and persistent. Transient soil seed banks are those in which few seeds, if any, persist as viable for more than 1 year (without germinating) (Thompson and Grime 1979; Simpson et al. 1989). Persistent soil seed banks are those where a significant portion of seeds remain viable in soil for more than one year (without germinating) (Thompson and grime 1979; Simpson et al. 1989). Seeds in transient seed banks are generally non-dormant (Garwood 1989) although various forms of short term dormancy may be observed. Seeds in persistent seed banks exploit various forms of primary and secondary, short and long-term dormancy. Regardless of dormancy, over time all seeds eventually lose viability. However, species vary greatly in their seed longevity (Radosevich et al. 1997). The longevity of seeds in the soil may be affected by numerous factors such as: seed characteristics (size, shape, seed coat), burial  9  depth, environmental conditions (temperature, moisture), soil type, seed chemistry, exhaustion of reserves through respiration, fungal or bacterial pathogens, and predation (Garwood 1989; Radosevich et al. 1997; Mohler 2001). From a weed management perspective it is imperative to understand the seed bank dynamics and factors that affect them in species that reproduce only by seed (e.g. hoary alyssum). With a better understanding of seed bank dynamics, it may be possible to develop effective control strategies for such species by targeting specific weak-links in their main mode of persistence.  1.2.2.1 Hoary alyssum seed banks Little is known about the soil seed banks of hoary alyssum. Hoary alyssum is known for its prolific seed production, producing upwards of 2500 seeds per plant (Stevens 1932; Reichman 1988). This combined with the persistence of its populations in various ecosystems, has lead to the common belief that the species maintains a large persistent soil seed bank (Warwick and Francis 2006). However, no studies on the soil seed banks of this weed are available to substantiate this belief.  1.2.3 Mechanical removal of flowering shoots (mowing) Physical removal of flowering shoots by mechanical means (mowing) is a common technique used to control monocarpic (plants that die after producing only one crop of seed (Cousins and Mortimer 1995)) weeds (Monaco et al. 2002; Donald 2006). Mowing is often used to encourage native plant establishment and discourage weed growth in rangelands, pastures,  10  waste places, and along roadsides (Donald 2006) because it reduces or prevents weed seed production (Anderson 1983; Donald 2006). Donald (2006) suggests that weeds can either be susceptible, tolerant, or resistant to mowing. Susceptible and tolerant weeds can be killed by one or more properly timed mowings, while resistant weeds are defined as those that can survive multiple mowings and still set seed (Donald 2006). Timing of mowing is important to reduce output of viable seeds; it must be performed before seed set. However, depending on the species, if mowing is performed too early in the lifecycle of the plant, apical dominance may be released (Ross and Lembi 1985) and subsequent shoots (regenerated from carbohydrate storage reserves in the roots (DiTomaso 2000)) may set seed.  1.2.3.1 Mowing hoary alyssum Mowing is commonly used in the attempt to control hoary alyssum in lawns and along roadsides in residential areas (Boundary Weed Management Committee 2008, personal comm.). However, no information is available in the literature on the effectiveness of mowing to prevent or reduce seed production in hoary alyssum. Reports from land managers (Boundary Weed Management Committee 2008, personal comm.) suggest that ill-timed mowing may release apical dominance and lower the plant’s profile, allowing it to set seed and escape subsequent mowings.  11  1.2.4 Allelopathy Allelopathy is defined as any direct or indirect effect of one plant on another through the release of chemicals (allelochemicals) into the environment (Rice 1984; Singh et al. 2001). In weed science, allelopathy commonly refers to the inhibitory effect of allelochemicals released from one plant on the growth and reproduction of another plant (Inderjit and Callaway 2003). Allelochemicals take many forms and are most often secondary metabolites (Einhellig 2002). They are produced, stored, and released into the soil (Einhellig 2002), where they elicit different effects on other plants directly or indirectly either through their byproducts or their interactions with other abiotic and biotic factors (Inderjit and Nilsen 2003).  1.2.4.1 Allelochemicals in hoary alyssum Hoary alyssum has been reported as a species with potential allelopathic properties (Bhowmic and Doll 1979; Foy and Inderjit 2001). Many members of the brassicaceae family contain allelochemicals most notably glucosinolates (Müller 2009). To date, the potential allelopathic ability of hoary alyssum remains unknown.  1.2.5 Nitrogen fertilization Nitrogen is essential for plant growth. It plays a central role in plant life by stimulating photosynthesis, amino-acid synthesis, and protein synthesis (Novoa and Loomis 1981; Whitehead 2000). Most nitrogen fertilizers, especially urea based fertilizers, are relatively inexpensive and commonly used to amend poor soils where stimulation of plant growth is desired (Rawluk et al. 2001).  12  Different plant species vary in their ability to compete for resources such as nitrogen (Grime 1977). The competitive ability of an individual species is influenced by both the availability of resources and the environmental conditions under which it is grown (Grime 1977). Amending resources through fertilization can increase a plant’s growth and competitive ability (Hautier et al. 2009) depending on the species’ ability to utilize the amended resources. Because different species vary in their ability to utilize resources, fertilization alters the competitive interactions between associated species. Weeds like hoary alyssum that invade and dominate native communities often do so because of their ability to survive on relatively poor soils with limited resource availability (Warwick and Francis 2006). By altering resources such as nitrogen it may be possible to alter competitive interactions and establish a competitive advantage for native species over that of introduced weeds. Identifying the ability to establish such a competitive advantage for native species may provide valuable information for better weed management practices.  1.2.5.1 The response of hoary alyssum to nitrogen fertilization Little information exists in the literature regarding the response of hoary alyssum to nitrogen fertilization. Tilman (1984) showed that hoary alyssum grown under field conditions with an imposed nitrogen:magnesium nutrient gradient responded favorably, in terms of biomass production, to high nitrogen inputs. Yet, Tilman (1987) showed that hoary alyssum’s relative abundance decreased with the addition of nitrogen under old field conditions where total nitrogen exceeded 500 mg kg-1 of soil. Studies examining the effects of nitrogen fertilization on the growth and relative competitive ability between native forage species, such as Idaho fescue or bluebunch wheatgrass, and hoary alyssum have not been conducted. Determining whether  13  nitrogen fertilization alters competitive interactions in favor of native forages may provide important information for the development of effective management practices for hoary alyssum.  1.3 Field Site Description This section provides a brief description of the Morrissey Creek field site utilized for the field experiments reported in this thesis.  1.3.1 Field site selection and location The field site was chosen in consultation with Mrs. Barb Stewart (Boundary Weed Management Committee), Mr. Doug Pickard (Right-of-Way Land Superintendent, Fortis BC), and Mr. John Mehmal (the land owner). The site chosen is a hoary alyssum infested rangeland site located just outside the city limits of Grand Forks, BC (at approximately N 49° 01.840’, W 118° 24.100’) just off the end of Morrissey Creek road. It can be accessed via a Fortis BC rightof-way/service road for transmission line number 11 between poles 221 and 223 (11L 221 to 11L 223). While much of the Grand Forks area experiences some hoary alyssum infestation, efforts to control the species via herbicides and other means made it difficult to find a relatively natural, undisturbed, uniform population for study. The Morrissey Creek study site was chosen based on the presence of a relatively healthy and uniform infestation of hoary alyssum that according to the land owner has not been chemically treated or exposed to other control efforts. The site was also chosen in part because of its close association with the right-of-way access road of our funding partner FortisBC, whose existing Integrated Pest Management program recognizes their  14  responsibility to prevent and manage the spread of invasive species and noxious weeds (FortisBC 2012).  1.3.2 Field site vegetation and topography The field site is located on the edge of the Ponderosa Pine biogeoclimatic zone of BC that surrounds Grand Forks (British Columbia Ministry of Forests and Range 2008). It closely borders/overlaps with the Interior Douglas-fir zone (British Columbia Ministry of Forests and Range 2008). In addition to the infestation of hoary alyssum, vegetation on the site is predominantly range grasses, most notably bluebunch wheatgrass which was the dominant native vegetation on the site. Idaho fescue, prairie junegrass, and cheatgrass were also observed, although they did not appear dominant on the site. Diffuse knapweed (Centaurea diffusa Lam.) was present on the site in small numbers but Urophora sp. were observed acting as a biocontrol to the population. Patches of brittle prickly pear (Opuntia fragilis (Nutt.) Haw.) were also common on the site. The site has a gentle south facing slope with an elevation of 640-665 m above sea level. Soils in the area were surveyed in early 1960s as part of the Soil Survey of the Kettle River Valley. Soils on the site are classified as a McCoy gravelly sandy loam belonging to the larger orthic dark brown subgroup of the dark brown soils (Sprout and Kelley 1964). A small creek bed (dry in the summer) and riparian area with small trees and shrubs divides the site. The site is further broken up by the presence of the right-of-way service road that winds its way through the area. The topography of the site gives rise to three distinct areas of roughly the same size and level of hoary alyssum infestation that were utilized as blocks in some of the experiments.  15  1.3.3 Field site land usage The study site is periodically accessed by Fortis BC when performing necessary maintenance on their transmission lines. However, this maintenance is localized to particular areas of the site, leaving the majority of the site undisturbed. Land owner John Mehmal has historically used the site as rangeland for his cattle, but during the course of the experiments presented in this thesis it was arranged that cattle would not be grazed in the area for any length of time apart from early spring and late fall when it was necessary to drive the cattle through the area from one range to another (Barb Stewart 2008, personal comm.).  16  Chapter 2. Hoary Alyssum Seed Bank Dynamics 2.1 Introduction Soil-borne seed banks and dormancy play an important role in the persistence of species that reproduce solely by seed (Christoffoleti and Castano 1998), allowing them to distribute seed germination over time. Soil seed banks can be transient or persistent. Transient seed banks contain seeds that are either non-dormant or exhibit short term dormancy; they do not persist from one year to the next (Thompson and Grime 1979; Garwood 1989; Simpson et al. 1989). Persistent seed banks contain dormant seeds that remain viable (without germinating) for more than a year (Thompson and Grime 1979; Simpson et al. 1989). Persistent seed banks present a major hurdle in weed control because they require long-term management that prevents recruitment of new seeds and promotes exhaustion of viable seeds already present in the seed bank (Cohen and Ruben 2007). Dynamics of seed dormancy, germination, and viability vary with time and space in soil seed banks. Burial depth and seasonal fluctuations in environmental conditions affect seed germination behavior and seed viability (Christoffoleti and Castano 1998) influencing long term persistence of weeds. Knowledge of the size, distribution, dormancy, germination, and viability dynamics of soil seed banks is essential to understanding the persistence strategy of weeds that reproduce solely by seed. Hoary alyssum (Berteroa incana (L.) DC.) is an annual to short lived perennial weed, known for its copious production of small seeds. It has been reported to produce up to 300 fruits per plant containing over 2500 seeds in total (Stevens 1932; Reichman 1988). The spatial and temporal dynamics of hoary alyssum soil seed banks and the factors affecting them are poorly understood. It is believed that hoary alyssum seeds show some dormancy when buried in soil  17  (Kust 1969) but dynamics of its seed dormancy and characteristics of its soil seed banks have not been studied. The objectives of this research were to utilize soil seed cores and artificial seed banks to: 1) establish the size of soil seed banks for of hoary alyssum at an infested rangeland site near Grand Forks BC, 2) determine the vertical distribution of seeds in the soil profile, 3) improve the understanding of dormancy, germination, and viability of hoary alyssum seeds buried in soil seed banks. This information will provide a better understanding of hoary alyssum’s persistence strategy and may help in developing effective management practices for this weed.  2.2 Materials and Methods 2.2.1 Seed sources Mature hoary alyssum seeds were collected (July 2008, 2009, and Sept. 2009) from a natural population at the Morrissey Creek study site outside Grand Forks, B.C., Canada. The seeds were stored at -20°C in darkness until use.  2.2.2 Field experiments All field experiments were carried out at a hoary alyssum infested site near Morrissey Creek road, outside the city limits of Grand Forks BC (Approx. N 49° 01.840’, W 118° 24.100’; elevation 640 – 665 m). (See sect. 1.3)  18  2.2.2.1 Soil seed bank size and distribution In order to determine the size of hoary alyssum soil seed banks and the vertical distribution of seeds in the soil profile, soil cores were taken from the Morrissey Creek study site at the end of July 2008 and again at the end of July 2009 (Fig. 2.1). Seed core extraction methods were based on those described in Qi (1993). Fifteen (10 cm diam., 10 cm deep) cores were taken randomly from the hoary alyssum infested area each year. Cores were transported to the laboratory at UBC where they were stored at -20°C until examination. Each core was sectioned into five layers (0-1, 1-2, 2-4, 4-6, 6-10 cm from the soil surface). Hoary alyssum seeds were separated from the soil by hand using a fine sieve (mesh No. 40), and counted. In order to account for any viable hoary alyssum seeds missed by this process, the remaining soil from each section was spread to a uniform depth of 0.5 cm in plastic trays, and watered. Seeds were allowed to germinate for 4 weeks in the UBC Plant Science greenhouse mist chamber. Hoary alyssum seedlings were counted. The size of the hoary alyssum seed bank (seeds ha-1) was calculated from the number of seeds and seedlings observed in each core.  2.2.2.2 Artificial soil seed banks Based on initial assessment of the vertical distribution of hoary alyssum seeds in the soil profile (see sect. 2.2.2.1), four artificial seed banks were established in August 2008 and in August 2009 at the Morrissey Creek study site (Fig 2.2). Seeds collected from the same site in July 2008 and 2009, respectively were used. Seeds were enclosed in packets made of “no-SeeUm-Netting” (Outdoor Innovations, Vancouver, BC), which has a mesh size small enough to prevent seed loss but allows free movement of water and oxygen (Qi 1993). Each packet (~5 cm x 5 cm) was filled with 200 seeds mixed with 10 g sterile soil (soil was sterilized in an  19  A  B  Figure 2.1. Soil cores (10 cm diameter x 10 cm depth) before (A), and after (B) extraction from the hoary alyssum infested study site near Morrissey Creek, Grand Forks BC. Soil cores (15) were taken randomly from the site in July 2008 and again in July 2009.  20  A  B  Figure 2.2. An artificial soil seed bank (A) utilizing mesh seed bags (B). Seed bags were buried at depths of 0, 2, and 5 cm. Four seed banks were established in August 2008, and again in August 2009.  21  autoclave in order to kill any other viable seeds). Packets were buried at three depths (0, 2, and 5 cm). Five packets were buried (Fig. 2.2) at each of these depths in each of the seed bank sites; one bag per burial depth per bank for exhumation in October 2008, May 2009, July 2009, August 2009 and August 2010. The banks established in August 2009 contained 4 bags for extraction in October 2009, May 2010, July 2010, and August 2010. Packets at the 0 cm depth were covered with a single layer of “no-See-Um-Netting” that was pinned down using 6” galvanized nails to prevent disturbance of the seed packets by animals or other natural factors. Exhumed seed packets were packed on ice and transported to UBC, where they were stored at -20°C until further analysis. Seeds were carefully removed from the seed bags and classified into five defined categories (seedlings, empty seed coats, seeds with enforced dormancy, seeds with induced dormancy, and non-viable seeds). Empty seed coats may represent three possible seed fates: seedlings, decayed embryos, or embryos eaten by predators. However, all three fates may be easily differentiated by careful examination of the seed coats. When seeds of hoary alyssum begin to germinate, their seed coats split and hinge open to allow seedling emergence. Resulting seed coats do not split completely apart but remain intact, taking on a ‘Pacman-like’ appearance (◔) (personal observation). The majority of empty seed coats exhumed from the seed banks took on this open appearance, thus they presumably represented seedlings. At later stages of exhumation the inability of seedlings to escape seed bags led to their decay; the presence of empty seed coats was therefore used to assess the fate of seeds in these samples. Enforced seed dormancy was evaluated by imbibing the exhumed seeds in Petri dishes (9 cm diameter) lined with two moist Whatman No. 1 filter discs for 10 days at ~25°C. Seeds that germinated were considered to have enforced dormancy. Seeds that did not germinate after 10 days had their seed coats pierced and were incubated in 0.1% tetrazolium solution at 30°C for 24 hours (Moore  22  1973). After 24 hours of incubation, seed coats were removed to expose the embryos. Initially colourless in solution, tetrazolium accepts electrons from the mitochondrial electron transport chain and stains living tissue red, while leaving dead tissue unstained (Moore 1973). Embryos with at least partial positive staining (red/pink colouration) were considered to have induceddormancy. Seeds that did not show any tetrazolium staining after 24 hours of incubation were deemed non-viable.  2.2.3 Data analysis A randomized complete block design was employed for the soil core sampling, and the artificial seed bank studies. All experiments were subjected to Least Square Means analysis with multiple pairwise comparisons of the means (α = 0.05) using SAS 9.2 (SAS Institute, Inc., Cary, NC., USA.) software. Alpha values were corrected for individual comparison of the means using Bonferroni’s correction method (Holm 1979).  2.3 Results 2.3.1 Soil seed bank size and distribution Results from the soil core samples showed that 93-95% (2008-2009) of hoary alyssum seeds were present in the top 4 cm of the soil profile (Fig. 2.3); 58-66% were confined to the top 1 cm of soil, and 77-79% (2009-2008) were limited to the top 2 cm of the soil profile. Significantly more seeds were found in the top 1 cm of the soil compared to any other depth (P ≤ 0.05). Using the total number of seeds found in each individual soil core, the size of hoary alyssum’s seed bank in the top 10 cm of soil profile was calculated to range from 0 to 132.4×106 seeds ha-1 at the Morrissey Creek study site.  23  2008  Seeds (% of annual total)  100  2009  a 80  60  40  b  bc  20  bc  c  0 0-1  1-2  2-4  4-6  6-10  Depth (cm)  Figure 2.3. Distribution of hoary alyssum seeds in soil profile in 2008 (■) and 2009 (□) in Grand Forks, British Colombia. Each value is mean ± SE of 15 cores.  24  2.3.2 Artificial soil seed banks Of the 200 seeds in each packet, 95% were accounted for in the 2008 cohort and 98% in the 2009 cohort. The fates of seeds reported here are based on 4 samples (one seed bag per treatment from each of the 4 established banks in each year: 2008 and 2009 cohorts). With the 9 month exhumation (May 2009) of the 2008 cohort, one set of surface seed bags went missing; evidence of animal interference was observed. By August 2010 (2 years after establishment of the 2008 cohort) only one set of bags remained at the surface. The means for this portion of the cohort are calculated accordingly. Cohorts (2008 and 2009) did not show a significant treatment difference but the assumption of variance for the residual data could not be met; results for both 2008 and 2009 are shown separately (Fig. 2.4 and Fig. 2.5). The fate of seeds differed significantly (P ≤ 0.05) between the surface (0 cm) and burial depths of 2 and 5 cm, but did not differ between the 2 and 5 cm burial depths in both cohorts. Seeds placed on the surface (0 cm) germinated and established seedlings quickly. In the 2008 cohort a mean of 130 seedlings (80.4% of total seeds recovered) had established within two months time (Oct. 2008). Empty seed coats were also found accounting for 80.2% of the seeds which is similar to the number of seedlings found. In the 2009 cohort fewer seedlings (39.3%) were found after two months of burial (Oct. 2009), but a large number of empty seed coats were found (52.3%), presumably representing seedlings that had decayed due to their inability to escape the mesh seed bags. After one year on the surface, 85.6–87.5% (2008-2009 cohorts) of the seeds were accounted for as empty seed coats and were presumed to have germinated. Of the seeds that did not germinate on the surface in the first year, 0-6.3% showed enforced dormancy, 1.9-6.3% showed induced dormancy, and 8.1-11.5% were non-viable. In the 2008 cohort which  25  0 cm  100 75 50 25 0  Seeds Recovered (% of total)  Oct '08  May '09  Jul '09  Aug '09  Aug '10  2 cm  100 75 50 25 0 Oct '08  May '09  Jul '09  Aug '09  Aug '10  5 cm  100 75 50 25 0 Oct '08  May '09  Jul '09  Aug '09  Aug '10  Collection Time  S  E  ED  ID  NV  Figure 2.4. Fate of hoary alyssum seeds buried in August 2008 at depths of 0, 2, and 5 cm. Fates were: seedlings (S), empty seeds (E), enforced dormant (ED), induced dormant (ID), and non-viable (NV). Values are a mean % ± SE of 4 (or available) replicates.  26  0 cm 100 75 50 25 0  Seeds Recovered (% of total)  Oct '09  May '10  Jul '10  Aug '10  2 cm 100 75 50 25 0 Oct '09  May '10  Jul '10  Aug '10  5 cm 100 75 50 25 0 Oct '09  May '10  Jul '10  Aug '10  Collection Time  S  E  ED  ID  NV  Figure 2.5. Fate of hoary alyssum seeds buried in August 2009 at depths of 0, 2, and 5 cm. Fates were: seedlings (S), empty seeds (E), enforced dormant (ED), induced dormant (ID), and non-viable (NV). Values are a mean % ± SE of 4 replicates.  27  included a 2-year burial treatment (exhumed Aug. 2010), 92.3% of seeds were accounted for as empty seed coats (presumed germinated), 6.6% were enforced dormant, and 1.1% non-viable. No induced dormancy was observed after two years. At 2 and 5 cm depths only a few seedlings or empty seed coats were observed; the majority of seeds (96.0– 97.8%) were recovered intact. After 1 year, 50.1-86.7% (2008 – 2009 cohort) of seeds were enforced dormant when buried at 2 cm; 2.4-5.8% were induced dormant, and 7.9-40.1% non-viable. After 2 years buried at 2 cm, 54.6% of seeds were enforced dormant, 2.1% were induced dormant, and 31.7% non-viable. Seeds buried at 5 cm were 52.4-65.1% (2008 - 2009 cohort) enforced dormant, 5.5-7.6% induced dormant, and 27.3-37.4% non-viable after 1 year. After 2 years, seeds buried at 5 cm were 61.3% enforced dormant, 1.8% induced dormant, and 33.2% non-viable. A large percentage of seeds buried at 2 and 5 cm depths were found to be non-viable after 2, 9, and 11 months of burial (Oct., May, and July exhumations). These results were puzzling as bags exhumed later showed fewer non-viable seeds. It was observed that soil in the seed bags of the 2, 9, and 11 month exhumations was moist compared to those exhumed in August. Storage at -20°C after exhumation, without allowing the soil to dry, may have influenced seed viability in these seed bags. To test this possibility, a study was conducted in which seeds were placed in moist or dry soil, frozen at -20°C for one month and their subsequent germination/viability (tetrazolium staining) studied. Results showed that seeds in moist soil lost viability compared to those in dry soil (data not shown). Thus, higher soil moisture content during storage may have caused loss of viability in seeds recovered from the 2, 9, and 11 month exhumations (Oct., May, and July).  28  2.4. Discussion Results of this study showed that hoary alyssum’s soil seed bank was shallow and potentially large, but very patchy in its distribution. Seeds are largely confined to the top 4 cm of the soil profile in undisturbed (not tilled) rangeland at the Morrissey Creek study site. On arable cultivated land or in waste places where the soil has been disturbed, seeds could become buried deeper in the soil seed bank (Ball 1992; Cardina et al. 2002). The shallow nature of hoary alyssum seed banks in rangelands should be encouraging for weed managers since seed banks confined to the surface or upper layers of the soil profile are easier to exploit, treat, or exhaust than those buried deep. This observation may help weed managers in developing control strategies for hoary alyssum. The range of estimated size for hoary alyssum’s seed bank (0 - 132.4×106 seed ha-1) indicates a very patchy seed bank distribution at the study site. Patchiness in the soil seed bank is linked to distribution of adult plants given that hoary alyssum seeds do not disperse far from a mother plant (Warwick and Francis 2006). As populations of hoary alyssum establish, and its abundance increases, seed bank size is expected to increase due to increased seed input. Hoary alyssum seed fate in the soil seed banks was significantly affected by burial depth. Seeds located on the soil surface germinated readily, with as much as 87.5% germinating within a year. Transient seed banks are defined as those where seeds do not persist beyond one year (Thompson and Grime 1979; Garwood 1989; Simpson et al. 1989). In this study a large portion of hoary alyssum seeds on the soil surface exhibited characteristics of a transient seed bank rather than a persistent one. However, the observed enforced and induced dormancy at 2 and 5 cm depths suggests that buried hoary alyssum seeds banks are persistent, with seeds remaining  29  viable (without germinating) for longer than a year (Thompson and Grime 1979; Simpson et al. 1989). The high rate of enforced dormancy observed in buried hoary alyssum seeds may represent an opportunity for controlling this weed. Environmental conditions (i.e. light, moisture, temperature) conducive to seed germination will release enforced dormancy allowing seeds to germinate (Harper 1977; Baskin and Baskin 2004). Weed management practices (mechanical, chemical, or other integrated means (Dyer 1995; Caldwell and Mohler 2001)) that provide conditions suitable for germination could be used to induce a flush of seedling emergence from the enforced dormant state of seeds in the seed bank. Such practices may deplete the seed bank of enforced dormant seeds, leaving emerged seedlings exposed and susceptible to other weed control methods. While only a small fraction of seeds were found with induced dormancy (max 7.6%), this fraction may cause significant problems for weed managers. The estimates of seed bank size at the Morrissey Creek study site were as large as 132.4×106 seed ha-1. The fraction of induced dormant seeds in a seed bank that large could therefore be over 1 million seeds ha-1, more than what would be needed to re-establish a hoary alyssum population in a short period of time. The physiological basis of seed dormancy in hoary alyssum is not known. How long dormant hoary alyssum seed can remain viable in soil is also not known. Kust (1969) suggested buried hoary alyssum seeds may remain dormant for a several years (a specific estimate was not given), while other anecdotal reports suggest seeds can remain dormant for approximately nine years (Parkinson et al. 2010). The results of this study employing artificial seed banks indicated that seeds can remain viable for at least two years. Since seed viability after 2 years was not studied, reports of viability beyond 2 years cannot be substantiated. Tetrazolium staining of  30  seeds with induced dormancy revealed some seeds that (while still considered viable) had patches of dead embryonic tissue present (Fig. 2.6). This suggests that seed/seedling vigor (the ability to germinate and establish within a reasonable time) may have been reduced in seeds with induced dormancy (Moore 1973). Further research examining causes of dormancy, seed longevity, and the loss of seed vigor and its effect on seedling establishment over time is necessary to understand hoary alyssum’s persistence and develop effective management strategies for this weed. Long term viability of seeds is affected by temperature and moisture (Shafer and Chilcote 1970; Murdoch and Ellis 2000). Seeds buried in the soil are insulated from extreme temperatures, but may still be susceptible to freezing and thawing when the soil is relatively moist. This may result in loss of seed viability; loss of viability upon storage of moist seeds at -20°C was reported earlier (see sect. 2.3.2). Unlike buried seeds, seeds on the soil surface can be exposed to extreme temperatures. Soil surface temperatures at the Morrissey Creek study site were measured using a hand-held Nexxtech infrared thermometer between 1:30 and 2 pm (on July 28, 2009; air temperature of 38.7°C) under sunny skies. A total of 30 random readings were taken (data not shown) and soil surface temperatures were found to reach up to 83ºC. Collaborative research with Dr. Hamid Madani (a visiting scholar in the M. K. Upadhyaya lab) revealed that while dry hoary alyssum seeds exposed to high temperatures remain viable, seeds that are pre-imbibed for an hour are killed by 45-60 minute exposure to temperatures in excess of 60°C (Madani et. al. unpublished data). Increased knowledge of the effects of temperature and moisture on hoary alyssum seed could potentially assist in the development of control strategies for this weed.  31  A  B  C  D  Figure 2.6. Hoary alyssum seeds imbibed in 0.1% tetrazolium solution for 24 hours at 30°C. A viable seed with healthy (red) embryonic tissue (A), viable seeds showing partially decaying tissue (B and C), and a non-viable seed with dead embryonic tissue (D).  32  Chapter 3: The Response of Hoary Alyssum to Mowing 3.1 Introduction Many annual weeds show the phenomenon of monocarpic senescence, where plants die after producing only one crop of seed (Cousins and Mortimer 1995). In these species senescence commonly results from nutrient starvation in the vegetative meristems as organic nutrients are diverted to developing seeds (Noodén 1988; Wilson 1997; Booth et al. 2010). The mechanism that triggers senescence in these species is not well understood. The most prominent theory, the ‘death hormone’ or ‘suicide signal’ hypothesis, postulates that senescence is directly linked to the release of hormones that are emitted at a species-specific time during reproductive development (Engvild 1989; Wilson 1997). After release of the ‘death hormone’, plants begin to senesce and seeds enter the final phases of maturation and dehydration. In cases where there is a delay between seed development and the release of ‘death hormones’ it may be possible to exploit that window of time to limit seed production by removing shoots mechanically. Mechanical control of monocarpic weeds by mowing (the physical excision of flowering shoots) can be a valuable component of integrated weed management programs (Monaco et al. 2002; Sheley et al. 2003; Donald 2006). Mowing alone has limited value for weed managers, but if properly timed, its ability to reduce viable seed production in some species can significantly alleviate the pressures of copious seed production exhibited by many annual weeds in lawns, meadows, pastures, waste places, and along roadsides (Anderson 1983; Donald 2006). Improperly timed, or applied to species that present a narrow window between ‘death hormone’ release and the final phases of viable seed development, mowing may prove to further exacerbate weed infestations (DiTomaso 2000). Mowing before the suicide signal has been sent, can release apical dominance (the suppression of lateral bud growth in the presence of an apical bud),  33  stimulating the growth of axillary buds below the point of mowing (Ross and Lembi 1985). The resulting branches could bolt (produce elongated flowering shoots), flower, and produce seeds. Repeated mowing may either deplete carbohydrate storage reserves, preventing further regeneration (DiTomaso 2000), or encourage prostrate growth forms, allowing plants to escape subsequent mowing treatments (Ross and Lembi 1985). Mowing after viable seeds have been produced will allow the weed to add seeds to the seed bank and possibly distribute seeds farther than normal (Bakker et al. 1996; Strykstra et al. 1997; Coulson et al. 2001; Donald 2006). Hoary alyssum (Berteroa incana L. DC) is a monocarpic species. However when mowed before flower development, it can persist as a short-lived perennial, regenerate from its carbohydrate storage reserves, and set seed (Kust 1969). It is unknown if properly timed mowing after flowering can be exploited to reduce seed production in this weed. The objective of this research was to examine how timing of mowing at different stages of flower and seed development affects plant survival and seed production. Given that this weed propagates by seed only (Warwick and Francis 2006), and is capable of producing in excess of 2500 seeds per plant (Stevens 1932; Reichman 1988), mowing may prove to be a valuable tool for weed managers if it can reduce seed production.  3.2 Materials and Methods 3.2.1 Seed source Hoary alyssum plants were grown from seed. Seeds were collected in July 2008 from populations at the Morrissey Creek study site (outside the city limits of Grand Forks B.C.; approx. N 49° 01.840’, W 118° 24.100’; elevation 640 – 665 m). (See sect. 1.3)  34  3.2.2 Soil selection Plants were grown on commercial man-made double sieved topsoil purchased from Kutny Richmond Soils (Richmond, BC). The soil was analyzed for nutrient content by the Analytical Chemistry Services Laboratory (B.C. Ministry of Environment’s Environmental Sustainability Division: Knowledge Management Branch, Victoria BC.).  3.2.3 Mowing experiments In order to closely monitor bolting and flowering following shoot excision, experiments were performed at the UBC greenhouse rather than at the Morrissey Creek field site located over 540 km east of the UBC campus. Initially, a total of 240 pots (15 cm diam. x 18 cm deep) were filled with the double sieved topsoil and hoary alyssum seeds were sown into them (Feb. 2009). The resulting plants did not exhibit the synchronous bolting required to study the effects of timing of mowing. Bolting was not uniformly distributed in time, and the total number of plants that bolted was far below the numbers required to apply the necessary treatments. Attempts were made to induce bolting by exposing plants to long days (16.5 hr day length) as well as by applying the plant hormone gibberellic acid (GA3). Desired levels of bolting were still not achieved. Furthermore, the plants that flowered did not produce seed, presumably because their pollination requirements were not met under greenhouse conditions. Exposure to low temperatures (vernalization) is known to promote flowering in many species (Simpson and Dean 2002). Given the latitude at which hoary alyssum occurs in BC, and the fact that it can survive as a winter annual (Warwick and Francis 2006), vernalization was thought to be essential for inducing the level of bolting required for the experiment. A second population of hoary alyssum plants was grown in pots (10 x 10 x 10 cm) filled with the same  35  double sieved topsoil (December 16, 2009). After 23 days the established rosettes were transferred to an outdoor space in the greenhouse courtyard for vernalization (January 8, 2010). The plants were left in the courtyard for the remainder of the experiment. By May 2010, the population was healthy and showed the uniform bolting required to apply the necessary mowing treatments. A randomized complete block experimental design with 5 blocks was used for the mowing experiment. There were 5 mowing treatments with 5 plants per treatment in each block. The experiment was repeated simultaneously in another area of the courtyard. While bolting (elongation of flowering shoots) was initially uniform, flowering was not. Blocks were therefore established at different dates in order to group plants at similar stages of bolting/flowering. The treatments used were: 1. no mowing (Control), 2. mowing at the appearance of the first flower (First Flower), 3. mowing when plants had developed a full flower (Full Flower), 4. mowing when plants were still flowering but had set approx. 50% of their seed (50% Flower/Seed), and 5. mowing when flowering had ceased and a full seed set had been achieved (Full Seed Set). Plants were excised with scissors just above the ground level to simulate mowing in this study. The number of shoots at the time of mowing was recorded to evaluate additional shoot production due to the release of apical dominance. Mowed plants were allowed to grow outdoors and were watered as necessary. The number of regenerated flowering shoots, the stage of flowering, and plant senescence were recorded periodically until the experiment was terminated (Oct. 17-19, 2010).  36  3.2.4 Data analysis The data was analyzed as a randomized complete block split-plot design to test treatment effects and the interaction between simultaneous replicates of the experiment. All experiments were subjected to Least Square Means analysis with multiple pairwise comparisons of the means (α = 0.05) using SAS 9.2 (SAS Institute, Inc., Cary, NC., USA.) software. Alpha values were corrected for individual comparison of the means using Bonferroni’s method (Holm 1979).  3.3 Results All mowing treatments had at least 4% of plants form new shoots, flowers, and seed following mowing. Since the two experiments (Expt. 1 and 2) showed a statistically significant difference (P ≤ 0.05) in response to mowing; both experiments are reported (Table 3.1). Control plants (not mown) had an average of 2.72 ± 0.28 (Expt. 1) and 2.28 ± 0.23 (Expt. 2) shoots per plant. The number of regenerated shoots was not significantly different from the number of shoots present before mowing in both the first flower and full flower treatments. In Expt. 1, the 50% Flower/Seed treatment produced significantly less (P ≤ 0.05) shoots after mowing; in Expt. 2, the Full Seed treatment produced significantly more (P ≤ 0.05) shoots after mowing. When analyzing the difference in shoot number before and after mowing, the assumption of normality could not be met for the residuals of the data. The significant differences reported represent conservative calculations of significance, despite the inability of the data to meet the assumption of normality (Dr. Tony Kozac 2012, personal comm.).  37  Table 3.1. Response of hoary alyssum plants to mowing at different stages of flowering shoot development. Mowing at  Experiment 1 Control First Flower Full Flower 50% Flower/Seed Full Seed Set Experiment 2 Control First Flower Full Flower 50% Flower/Seed Full Seed Set  Plants diedA  Response of plants to mowing New shoots did New shoots Treatment Change in shoot numberC produced rankingB not produce seedsA (P ≤ 0.05) seedsA  4.0 4.0 8.3 54.2 80.0  0.0 16.0 12.5 16.7 8.0  96.0 80.0 79.2 29.2 12.0  a a a b b  -D 0.00 ± 0.52 0.70 ± 0.57 -0.90 ± 0.46* 0.00 ± 0.58  8.0 20.0 32.0 47.8 88.0  0.0 8.0 12.0 17.4 8.0  92.0 72.0 56.0 34.8 4.0  a ab bc cd d  -D 0.40 ± 0.33 0.75 ± 0.38 0.91 ± 0.60 1.00 ± 0.00*  A  Percent of plants per treatment. Values are means of two experiments, each with 5 blocks of 5 plants per treatment.  B  Overall treatment ranking based on final response (including plants that died, produced new shoots but not seed, and produced shoots with seed). (P ≤ 0.05).  C  Mean change in the number of shoots per plant, in plants that produced new shoots following mowing, compared to the control (no mowing).  D  Control (unmowed) plants had an average of 2.72 ± 0.28 (Expt. 1) and 2.28 ± 0.23 (Expt. 2) shoots per plant.  *  Significant difference (P ≤ 0.05) between initial number of shoots (before mowing) and final number of shoots produced (after mowing) in plants that generated new shoots.  38  Delayed mowing significantly increased (P ≤ 0.05) mortality in hoary alyssum plants (Table 3.1). When plants were mowed just after the onset of flowering (First Flower), 4-20% of plants died. Conversely, 80-88% of plants died when mowing was applied at the full seed set stage (Table 3.1). When mowed at the First Flower stage 72-80% of plants produced new shoots and seed, compared to only 4-12% in plants mowed at the Full Seed stage.  3.4 Discussion The results of this study showed that if hoary alyssum is mowed while in the early stages of flowering (First or Full Flower) it will easily regenerate, with 56-80% of plants producing new shoots, flowers, and seed. This suggests that mowing is not a useful control method for reducing seed production when applied during early stages of flower development. The number of regenerated shoots did not differ significantly from the number present prior to mowing at these early stages. This indicates that mowing, while ineffective at reducing seed production, will not further exacerbate the weed problem by stimulating increased shoot, flower, and seed production. Delaying the application of mowing until later stages of flower and seed development (50% Flower/Seed and Full Seed) did increase plant mortality and reduce the number of plants capable of generating new shoots. However, 4-34.8% of these plants were still able to produce new shoots, flowers, and seed before the end of the experiment. A further 8-17.4% of plants were unable to produce new shoots before the end of the experiment, but they survived vegetatively, and may have continued to persist as short-lived perennials as has been reported by Kust (1969). These surviving plants while few in number, will continue to facilitate the persistence of this weed. Furthermore, delaying mowing until the late stages of flower and seed  39  development could allow plants to produce some seed before they are mowed. Generally when mowing is applied to plants once seeds have set it only serves to disseminate seeds into the seed bank (Strykstra et al. 1997; Coulson et al. 2001; Donald 2006). Even immature seeds, as long as they have begun to ripen, may continue to mature into viable seeds after their stalks have been mowed (Monaco et al. 2002). It is unknown whether seeds of hoary alyssum can continue to mature in such a way after their stalks have been mowed, but other mustards such as shepherd’spurse (Capsella bursa-pastoris (L.) Medik.) do show this ability (Gill 1938). A single mowing treatment regardless of when it is applied does not appear to be an effective weed control method for hoary alyssum. Plants mowed at early flowering stages do not senesce, but regenerate. Plants mowed at later flowering/seed stages are more likely to senesce, but mowing at these stages serves to disseminate seeds that have already matured. While a single mowing, may not serve to reduce seed production or limit seed dispersal, the effectiveness of repeated mowings should be evaluated as it may prove useful for weed managers. Repeated mowings can lead to the depletion of nutrient and carbohydrate reserves (DiTomaso 2000), eventually influencing plant survival by limiting flower production, reducing competitive ability, and exposing injured plants to harsh climate conditions. Plants that survived mowing in these experiments did not regenerate prostrate shoots. However if a prostrate growth form can occur in hoary alyssum, as has been reported by weed managers and land owners (Boundary Weed Management Committee 2008, personal comm.), those plants may manage to persist even through repeated mowings. Regardless of whether mowing is applied once or repetitively, there are significant practical issues that may limit the usefulness of mowing in areas infested by hoary alyssum. While lawns and roadsides are relatively easy to access with mechanical mowers, the vast  40  rangeland areas occupied by hoary alyssum are often not accessible to, or suited for the use of mowing machinery. Furthermore, any mowing that is performed in hoary alyssum infested areas needs to avoid damaging associated native plant species. Both bluebunch wheatgrass and Idaho fescue (associated native forage grasses) are slow to recover from heavy mowing or grazing (Mueggler 1975). Further damage to native species populations must be avoided, as it will only provide further opportunity for weeds such as hoary alyssum to invade, establish, and persist. In conclusion, a single mowing treatment cannot be recommended and should likely be avoided as a mechanical control for hoary alyssum. While repetitive mowing may hold some value, its effects on hoary alyssum have yet to be evaluated. A single mowing during early stages of flower development does not promote senescence. Instead apical dominance is released and new shoots, flowers, and seeds are formed. Mowing during later stages of flower/seed development does promote increased senescence in a large number of plants, but the high risk of disseminating seeds that have already developed is a significant problem. This study provides valuable information about the response of hoary alyssum to a single mowing treatment. It is hoped that in the future weed managers will consider these results and the risks associated with mowing while developing weed management programs for hoary alyssum.  41  Chapter 4: Allelopathic Influence of Hoary Alyssum on Native Forages 4.1 Introduction Plants produce an array of chemicals that impact neighboring plants (Qasem and Foy 2001; Singh et al. 2001), affecting plant-plant interactions. This phenomenon, the release of chemicals into the environment and the direct or indirect, positive or negative, effects of one plant on another, is known as allelopathy (Rice 1984). Many weeds utilize allelochemicals (chemicals that exert allelopathic influence) to establish a competitive advantage in interactions with other plant species (Weston and Duke 2003). Allelochemicals act by inhibiting growth, establishment, and or reproduction of associated plants (Inderjit and Callaway 2003). They aid in the establishment of weed monocultures, and may influence species composition in an ecosystem (Foy and Inderjit 2001; Weston and Duke 2003). A vast range of chemicals have been implicated in allelopathic interactions (Rice 1984; Putnam 1988; Quasem and Foy 2001; Weston and Duke 2003). These include: alkaloids, phenolic acids, and glucosinolates (Putnam 1988; Müller 2009). Many of these water soluble compounds enter the soil as a ‘leachate’ (a solution of water soluble chemicals leached from leaf material as leaves decay) (Olson and Wallander 2002). Glucosinolates in particular have been implicated for their allelopathic role in the invasive establishment of many mustard species (Brassicaceae family) (Müller 2009). Little is known about the allelopathic ability of hoary alyssum. Hoary alyssum, a member of the mustard family, well known for its complex chemistry and toxic effects on horses (Ellison 1992; Geor et al. 1992; Hovda and Rose 1993), has long been suspected of utilizing allelochemicals in its interactions with other plants (Bhowmik and Doll 1979; Foy and Indergit  42  2001). However, Bhowmik and Doll (1979) provide the only scientific observation of allelopathy in hoary alyssum to date. They reported that a 1 % (w/w) extract from hoary alyssum leaves had a stimulatory effect on the growth of radicles and coleoptiles in corn (Zea mays L.) and hypocotyl growth in soybeans (Glycine max (L.) Merr.). However, reports of the ability of hoary alyssum to establish dense stands and outcompete native forage grasses (Warwick and Francis 2006) would suggest the ability to inhibit the growth, establishment, and or reproduction of associated species. The objective of this research is to examine the ability of water soluble chemicals extracted from the leaves of hoary alyssum to exert an allelopathic influence on associated forage grasses by inhibiting their seed germination and seedling growth. This research will improve our understanding of hoary alyssum’s interactions with other species.  4.2 Materials and Methods  4.2.1 Seed sources Idaho fescue (Festuca idahoensis Elmer.), bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love), and prairie junegrass (Koeleria macrantha (Ledeb.) J.A. Schultes) are native rangeland grasses commonly found in hoary alyssum infested areas. Seeds of Idaho fescue, bluebunch wheatgrass, and prairie junegrass used for seed germination assays in Petri dishes were obtained from Grassland West Seed Co. (Clarkston, WA, USA). Seeds for all other experiments were purchased from Premier Pacific Seeds Ltd. (Surrey, BC). Even though cheatgrass (Bromus tectorum L.) is itself a weedy species in some ecosystems, it commonly occurs as an associated forage grass in hoary alyssum infested rangelands around Grand Forks,  43  BC. Cheatgrass seeds were obtained from the Agriculture and Agri-Food Canada Research Centre in Lethbridge, AB. Since these seeds showed a low germination percentage, they were only used in some preliminary studies. Seeds of cheatgrass from a population on the UBC campus were collected (June 16, 2009) for use in subsequent experiments. Hoary alyssum seeds used in this study were collected in July 2008 from populations at the Morrissey Creek field site near Grand Forks BC (approx. N 49° 01.840’, W 118° 24.100’; elevation 640 – 665 m) (see sect. 1.3).  4.2.2 Source of hoary alyssum leaves Leaves of hoary alyssum rosettes that had started to bolt and/or flower were collected on August 1, 2008, July 30, 2009, and July 2, 2010 from natural populations at the Morrissey Creek study site, dried for 4 days at 60°C, and stored at -20°C until use. Leaves from different collection dates were used as indicated.  4.2.3 Leachate preparation Water soluble chemicals from hoary alyssum leaf material were extracted into solution using the procedure described by Furness et al. (2008) (henceforth this extract is referred to as ‘leachate’). Hoary alyssum leaves were ground in a coffee grinder (Black & Decker Corp., Towson, MD, USA), screened through a fine sieve (Mesh No. 40), and the resulting powder stored in a freezer at -20°C until extraction. Four concentrations of water-soluble compounds were extracted from the leaf powder by stirring it in distilled water (0.5, 1, 2, and 4% w/v) in Erlenmeyer flasks for 4 hrs on a rotary shaker (80 rpm) at room temperature in darkness. Leaf material was separated from the leachates with two rounds of suction filtration using a vacuum  44  pump (Marathon Electric Manufacturing Corp. Wausau, WI, USA). Whatman No.1 filter paper was used for the first filtration to separate large particles of leaf material. Whatman No. 42 filter paper was used for the second filtration to extract all finer particles. Filtered leaf residue was collected and re-extracted using the same procedure at 4% w/v; the resulting filtrate was discarded. The extracted leaf residue (henceforth called leached leaf residue) was collected, dried, and stored at -20°C until use in soil assays (see sect. 4.2.6.2).  4.2.4 Seed germination assays To assess allelopathic effects of hoary alyssum leaf leachate on seed germination of associated forage grasses (Idaho fescue, bluebunch wheatgrass, prairie junegrass, and cheatgrass), and itself (‘autotoxicity’ (Singh et al. 1999)), germination assays as described by Furness et al. (2008) were performed. Four leachate treatments (0.5, 1, 2, and 4%) and a distilled water control (0%) were used. A randomized complete block design with 5 blocks (1 Petri dish per block) and 5 leachate concentrations was employed for each assay. Seeds (20) were placed in Petri dishes (10 cm diam.) lined with two layers of 9 cm Whatman No.1 filter discs and treated with 4 ml of leachate solution or distilled water (control). Only 10 seeds per Petri dish were used for cheatgrass due to a limited supply of seeds. Leachate was prepared before each assay and used immediately. Petri dishes (one per treatment) were placed randomly into 5 sealable Rubbermaid® Lock-its™ containers (a block) (37 cm length x 27 cm width, 5.6 L volume) lined with moist paper towels. The containers were sealed and placed in 5 separate growth chambers at ~25°C in darkness. Seed germination was recorded after 14 days. Seeds with ≥ 5 mm radicles were considered germinated. Two germination assays were performed on July 4, 2009, and July 25, 2009, using leachate made from leaf material collected in 2008. A third assay was performed  45  on February 19, 2010 using leachate made from leaf material collected in 2009. The effect on hoary alyssum seeds (autotoxicity) was studied in the assays performed on July 25, 2009, and February 19, 2010.  4.2.5 Seedling growth assays To assess the effect of hoary alyssum leaf leachate on seedling root and shoot elongation of forage grasses (Idaho fescue, bluebunch wheatgrass, and cheatgrass), as well as on itself (autotoxicity), seedling growth assays as described by Furness et al. (2008) were performed. Due to small seed size and a fragile radical vulnerable to damage during handling, prairie junegrass was not included in this study. Seeds of each species were pre-germinated on wet paper towel in plastic containers (see sect. 4.2.4) in darkness. Seedlings with 4-6 mm radicles (3-5 mm for hoary alyssum) were used. Four leachate treatments (0.5, 1, 2, and 4%) and a distilled water control (0%) were employed. A randomized complete block design with 5 blocks (1 Petri dish per block) and 5 leachate concentrations was used for each assay. Seedlings (10) were placed in Petri dishes (10 cm diam.) lined with two layers of 9 cm Whatman No.1 filter discs and treated with 4 ml of leachate solution or distilled water (control). Leachate was prepared before each assay and used immediately. Petri dishes (one per treatment) were placed randomly into 5 sealable containers (see sect. 4.2.4) (each a block) lined with moist paper towels. The containers were sealed and placed in 5 separate growth chambers at ~25°C in darkness. Root and shoot length were measured with a ruler after 10 days. An initial assay was performed by Dr. Hamid Madani (visiting scholar from the Department of Agriculture, Islamic Azad University, Arak, Iran) in February 2010 using leachate made from leaf material collected in 2009. This assay was repeated on July 15, 2010 and the results were compared.  46  4.2.6 Allelopathic effects in soil After observing some alleopathic inhibition of seed germination and seedling growth in Petri dish assays, a separate set of assays based on methods described by Furness et al. (2008) were performed to determine allelopathic influence in soil.  4.2.6.1 Seedling emergence in soil with leaf leachate Seeds of all four associated forage grasses (Idaho fescue, bluebunch wheatgrass, prairie junegrass, and cheatgrass) as well as that of hoary alyssum were used. A completely randomized design with 5 repetitions per treatment was employed. Seeds were sown into the surface of 20 g of soil in small plastic wells (excised from egg cartons) (10 seeds per well) (Fig. 4.1). Four treatments (water control; 4% hoary alyssum leaf leachate; charcoal + 4% leachate; and charcoal + water) were applied. A charcoal treatment (0.4 g mixed into soil) was used because activated charcoal is known to neutralize the effects of allelochemicals (Callaway and Aschehoug 2000; Furness et al. 2008). Soil used in this study was obtained from an area of the Morrissey Creek study site that has never been infested by hoary alyssum. Treatments were applied by saturating the soil with 8 ml of the 4% hoary alyssum leachate or distilled water (control). Distilled water (2-4 ml) was applied to each well when necessary to prevent soil drying. Seedling emergence was assessed after 14 days. Seedlings with ≥ 5 mm growth above the surface were considered emerged. An initial assay was performed on July 12, 2010 using leachate made from hoary alyssum leaves collected in 2009, and repeated on July 19, 2010 using leachate made from leaves collected in 2010.  47  Figure 4.1. Set up of assays testing seedling emergence in soil treated with 4% w/v hoary alyssum leachate. Assays were performed starting on July 12 and 19, 2010.  48  4.2.6.2 Seedling emergence in soil with added hoary alyssum leaf biomass The effect of dry unleached hoary alyssum leaf biomass (ground powder) on seedling emergence of all four associated forage grasses (Idaho fescue, bluebunch wheatgrass, prairie junegrass, and cheatgrass) and itself was studied. Seeds were sown into the surface of 20 g of soil in small plastic wells (10 seeds per well) (see sect. 4.2.6.1). The treatments employed were: 0.4 g of either ground unleached leaf biomass or leached leaf residue (no longer containing water soluble compounds) (see sect. 4.2.3.) mixed into 20 g of soil in plastic wells, or 0.2 g of either ground unleached leaf biomass or leached leaf residue spread on the top of the soil. A soil-only control was also used. At the beginning of each assay, each well was saturated with 8 ml distilled water. Distilled water (2-4 ml) was applied to each well when necessary to prevent soil drying. Seedling emergence was assessed after 14 days. Seedlings with ≥ 5 mm growth above the surface were considered emerged. An initial assay was performed on July 17, 2010 using unleached hoary alyssum leaf biomass collected in 2009, and repeated on July 24, 2010 using unleached leaf biomass collected in 2010. All leached leaf residue used in the assays was obtained from leaves collected in 2009.  4.2.7 Data analysis A randomized complete block design was employed for all seed germination and seedling growth assays. A completely randomized design was used for seedling emergence assays. All data was subjected to Least Square Means analysis with multiple pairwise comparisons of the means (α = 0.05) using SAS 9.2 (SAS Institute, Inc., Cary, NC., USA.) software. Alpha values were corrected for individual comparison of the means using Bonferroni’s method (Holm 1979).  49  4.3 Results 4.3.1 Seed germination assays In seed germination assays all four forage grasses showed reduced seed germination with increasing concentration of hoary alyssum leachate (Figs. 4.2 and 4.3). While this effect varied significantly (P ≤ 0.05) between species and between assays, in all species and assays seed germination was significantly (P ≤ 0.05) reduced by the highest leachate concentration (4%). Low seed germination percentages of the forage grasses in all treatments (including controls) prevented any inhibitory effect from appearing in the February 19, 2010 assay (data not show). New seeds with higher germination percentages were used in all subsequent experiments. Idaho fescue appeared to be the most susceptible species, followed by cheatgrass, bluebunch wheatgrass, and prairie junegrass. Idaho fescue seed germination was significantly reduced (P ≤ 0.05) compared to the control at all leachate concentrations; there was a 52-88% reduction of germination in the 4% leachate treatment. Cheatgrass seeds showed 80-86% reduction at the 4% leachate concentration, while bluebunch wheatgrass and prairie junegrass showed 65-77% and 53-57% reductions, respectively. Hoary alyssum leaf leachate inhibited its own seed germination (autotoxicity) (Fig. 4.4). Its seeds showed a significant reduction (P ≤ 0.05) in germination when exposed to 1% (Feb.19 assay) or higher leachate concentrations. The highest leachate concentration (4%) showed the most (33-100%) reduction of seed germination.  4.3.2. Seedling growth assays Root elongation in each of the three forage species tested decreased with increasing concentrations of hoary alyssum leachate (Fig. 4.5). Shoot elongation was not significantly  50  A  100 75  a  a  a  a  Germination (%)  a  a  75  50  B a  a  50  b  25  b  25  0  0  0.0  100  100  0.5  1.0  2.0  C  a b  75  4.0  bc  50  0.0  100  bc  c  0  0 1.0  2.0  4.0  2.0  4.0  D  ab ab  b  50 25  0.5  1.0  a  75  25 0.0  0.5  c  0.0  0.5  1.0  2.0  4.0  Leachate (% w/v) Figure 4.2. Effect of hoary alyssum leachate on the germination of select forage grasses in Petri dish assays (July 4, 2009). Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), and cheatgrass (D). Values are means of 5 replicates ± SE with 20 seeds per plate (10 for cheatgrass). Different letters above the histograms represent significantly different means (P ≤ 0.05). Leachate was made with leaf material collected in 2008.  51  A  100 75  ab  a  b  Germination (%)  25  b  b b  50  c  0  c  25 0  0.0  100  B  a  75  ab  50  100  0.5  1.0  2.0  4.0  C  a  75  0.0  100  b  b  25  1.0  c  0  4.0  D ab  b  50  b  2.0  a  75  50  0.5  c  25  d  0 0.0  0.5  1.0  2.0  4.0  0.0  0.5  1.0  2.0  4.0  Leachate (% w/v) Figure 4.3. Effect of hoary alyssum leachate on the germination of select forage grasses in Petri dish assays (July 15, 2009). Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), and cheatgrass (D). Values are means of 5 replicates ± SE with 20 seeds per plate (10 for cheatgrass). Different letters above the histograms represent significantly different means (P ≤ 0.05). Leachate was made with leaf material collected in 2008.  52  100  a  a  ab  b  A  75  Germination (%)  50 25 c  0  100  0.0  0.5  1.0  2.0  a  ab  b  b  75  4.0  B c  50 25 0 0.0  0.5  1.0  2.0  4.0  Leachate (% w/v)  Figure 4.4. Effect of hoary alyssum leachate on the germination of its own seeds (autotoxicity) in Petri dish assays. July 25, 2009 assay (A) with leachate from leaf material collected in 2008; February 19, 2010 assay (B) with leachate from leaf material collected in 2009. Values are means of five replicates ± SE with 20 seeds per plate. Different letters above the histograms represent significantly different means (P ≤ 0.05).  53  80  A  B  120  60 40  80 a  b  20  Length (mm)  a  a c  40  b  b  b c  d  0  0 0.0  0.5  1.0  2.0  0.0  4.0  0.5  1.0  2.0  4.0  40 30  120 80  D  C  160  a  a  a  a  20 b  40  10  0  0 0.0  0.5  1.0  2.0  4.0  a  a  a  b  a a  0.0  0.5  a  a  1.0  2.0  b b  4.0  Leachate (% w/v) Figure 4.5. Effect of hoary alyssum leachate on shoot ( ) and root ( ) growth of forage grasses and itself in Petri dish assays (July 15, 2010). Idaho fescue (A), bluebunch wheatgrass (B), cheatgrass (C), hoary alyssum (D). Seedlings had 4 to 6 mm long radicles to start (3 to 5 mm in hoary alyssum). Leachate was made with leaf material collected in 2009. Values are means of five replicates ± SE with 10 seedlings per plate. Different letters above the data points represent significantly different means (P ≤ 0.05).  54  affected after 10 days. While bluebunch wheatgrass root elongation was significantly inhibited (P ≤ 0.05) by all leachate concentrations, only leachate concentrations ≥ 1.0% significantly affected (P ≤ 0.05) root elongation in Idaho fescue. Cheatgrass root elongation was inhibited only at the 4% concentration. Self inhibition of hoary alyssum root and shoot elongation was observed (Fig. 4.5). All leachate concentrations significantly affected (P ≤ 0.05) hoary alyssum root elongation, with the most pronounced effect occurring at the 4% leachate concentration. Shoot growth was significantly inhibited (P ≤ 0.05) only at 2 and 4% concentrations. Hoary alyssum seedlings exposed to 4% leachate concentration were visibly stunted with a number of seedlings starting to decay before the assay was terminated at 10 days (Fig. 4.6).  4.3.3 Seedling emergence in soil with leaf leachate Unlike Petri dish assays, no clear and consistent effect of hoary alyssum leaf leachate on seed germination (and subsequent seedling emergence) was observed (Fig. 4.7). A significant (P ≤ 0.05) treatment effect between assays and species was observed; both assays are shown (Fig. 4.7). Bluebunch wheatgrass showed significant inhibition (52% reduction) of seedling emergence in the soil + leachate (4%) treatment when compared to the water control in the July 19 assay but not the July 12 assay. Similarly, hoary alyssum exposed to its own leachate (4%) in soil showed significant inhibition (P ≤ 0.05) (28% reduction) of emergence compared to the water control in the July 19 assay but not in the July 12 assay. Idaho fescue showed no significant differences in the July 19 assay, but the soil + leachate treatment in the July 12 assay showed a significant increase (34%) (P ≤ 0.05) in emergence.  55  A  B  Figure 4.6. Hoary alyssum seedlings after 10 days of exposure to 0% leachate (distilled water control) (A), and 4% hoary alyssum leaf leachate (B).  56  July 12 Assay  July 19 Assay A  100 75  75  50  50  25  25  0  0 SW  SL  CW  CL  SW  B  100  75  50  50  25  25  0  SL  CW  CL  B  a  ab  ab  CW  CL  b  0 SW  Germination (%)  100  75  SL  CW  CL  SW  C  100  a  75 50  A  100  ab  C  100 75  b  b  SL  50  25  25  0  0 SW  SL  CW  CL  SW  D  100  CW  CL  D  100  75  75  50  50  25  25  0  SL  0 SW  SL  CW  CL  SW  E  100  100  75  75  50  50  25  25  0  SL  CW  b  ab  SL  CW  a  CL  ab  E  0 SW  SL  CW  CL  SW  CL  Treatments Figure 4.7. Effect of hoary alyssum leaf leachate (4%) on germination of forage grasses and itself in soil. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil + Water (SW); Soil + Leachate (SL); Soil/Charcoal + Water (CW); Soil/Charcoal + Leachate (CL). Leachate was made from leaf material collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05).  57  Treatments with leachate and activated charcoal were not significantly different than the soil + water controls or the charcoal + water treatment for all species. Idaho fescue in the July 12 assay showed a significantly lower (P ≤ 0.05) seedling emergence compared to the soil + leachate treatment.  4.3.4 Seedling emergence in soil with added hoary alyssum leaf material Seedling emergence of all four forage grasses and hoary alyssum was not clearly affected when unleached hoary alyssum leaf biomass was mixed into soil or placed on the soil surface (Figs. 4.8 and 4.9). When unleached leaf biomass was mixed into the soil (0.4 g per well), only bluebunch wheatgrass in the July 17 assay showed any significant (P ≤ 0.05) treatment effect (Fig. 4.8). In that assay bluebunch wheatgrass exposed to the unleached leaf biomass showed significantly less (P ≤ 0.05) (20%) seedling emergence compared to treatments containing leached leaf residue (0.4 g per well). All other species showed no significant reduction in seedling emergence regardless of treatment. When unleached leaf biomass was placed on the soil surface (0.2 g per well), cheatgrass and hoary alyssum showed significant treatment effects (hoary alyssum only in the July 24 assay) (Fig. 4.9). In the July 17 assay, cheatgrass exposed to unleached leaf biomass had significantly lower (P ≤ 0.05) (22%) seedling emergence compared to the leached leaf residue treatment. In the July 24 assay, cheatgrass exposed to unleached leaf biomass had significantly lower (P ≤ 0.05) (30%) seedling emergence compared to the soil control. Hoary alyssum seeds exposed to unleached leaf biomass and leached leaf residue in the July 24 assay showed  58  July 17 Assay  July 24 Assay A  100 75 50 25 0 S  100 75 50 25 0  ab  LR  a b  S  Germination (%)  L  L  S  B  S  L  S  L  S  L  LR  D  S  E  L  100 75 50 25 0  LR  100 75 50 25 0  LR  C  S  D  L  100 75 50 25 0  LR  100 75 50 25 0  LR  B  S  C  L  100 75 50 25 0  LR  100 75 50 25 0  A  100 75 50 25 0  L  LR  E  100 75 50 25 0  LR  S  L  LR  Treatments Figure 4.8. Effect of hoary alyssum leaf material (0.4 g mixed into soil) on germination of forage grasses and itself. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil control (S); Leaf biomass (L); Leached Leaf Residue (LR). Leaf biomass was collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05).  59  July 17 Assay  July 24 Assay A  100 75 50 25 0 S  L  LR  Germination(%)  S  L  S  ab  S  L  a  L  L  100 75 50 25 0  LR  D ab b  L  LR  E  a  S  LR  L  a  S  E  S  100 75 50 25 0  LR  100 75 50 25 0  LR  C  S  D  L  100 75 50 25 0  LR  b  LR  B  S  C  L  100 75 50 25 0  LR  100 75 50 25 0  100 75 50 25 0  S  B  100 75 50 25 0  A  100 75 50 25 0  b  b  L  LR  Treatments Figure 4.9. Effect of hoary alyssum leaf material (0.2 g on the soil surface) on germination of forage grasses and itself. Prairie junegrass (A), bluebunch wheatgrass (B), Idaho fescue (C), cheatgrass (D), and hoary alyssum (E). Treatments: Soil control (S); Leaf biomass (L); Leached Leaf Residue (LR). Leaf biomass was collected in 2009. Values are means of 5 replicates with 10 seeds per well ± SE. Different letters represent significantly different means (P ≤ 0.05).  60  significantly reduced (P ≤ 0.05) (40 and 34%, respectively) seedling emergence compared to soil control. Significant inhibition of hoary alyssum was not seen in the July 17 assay.  4.4 Discussion Hoary alyssum leaf leachate significantly inhibited seed germination and seedling root growth of associated forage grasses in Petri dish assays. Inhibition of growth and establishment could have a significant impact on native forage grass population dynamics and species biodiversity (Foy and Inderjit 2001), allowing hoary alyssum to produce dense monoculture stands. An ability to inhibit growth and establishment of neighboring plants would further emphasize the invasiveness (Foy and Inderjit 2001; Weston and Duke 2003; Müller 2009) of hoary alyssum. Results also showed self-inhibition (autotoxicity) of hoary alyssum seed germination and seedling growth (both root and shoot) when exposed to high leachate concentrations. Such autotoxicity, also observed in other weed species (Singh et al. 1999), may provide a mechanism for regulation of hoary alyssum populations (Picman and Picman 1984). Autotoxic effects on seed germination and seedling growth could prevent establishment of hoary alyssum seedlings close to the mother plant, which would reduce intraspecific competition between the mother plant and its seedlings, and promote a wider spatial distribution of the population (Edwards et al. 1988; Singh et al. 1999). While Petri dish assays suggested that hoary alyssum has allelopathic potential, the application of both leaf leachate and unleached leaf biomass in soil assays showed no clear effects on seedling emergence of forage grasses or hoary alyssum itself. Further research is  61  needed to confirm whether an allelopathic influence is ever exerted by hoary alyssum under natural conditions. One of the biggest problems with current allelopathic studies (and a potential problem in this study) is that their methods often fail to replicate appropriate allelochemical inputs as well as other biotic and abiotic factors that occur in natural ecosystems (Foy and Inderjit 2001; Inderjit and Callaway 2003; Inderjit and Nilsen 2003). In order to properly assess the allelopathic ability of a plant, natural levels of potential allelochemical input need to be determined. Natural levels of potential allelochemical inputs from hoary alyssum were not established prior to these experiments. Whether leachate concentrations and unleached leaf biomass were used in appropriated amounts needs to be confirmed. Personal anecdotal field observations of average rosette size (~17 cm diam.) and dry weight (1.14 g) combined with average annual precipitation (50.98 cm over 12 months) (Environment Canada 2012a) were used to generate a rough estimate of hoary alyssum water soluble leachate and unleached leaf biomass inputs in soil under natural conditions. The estimate suggests that the natural input of hoary alyssum leaf leachate is close to ~ 0.01% w/v, far lower than the concentrations used in the assays of this study. The amount of unleached leaf biomass used in soil assays was estimated to be 2.5-5x (soil surface vs mixed) greater than what would occur under natural conditions. Natural leachate inputs may be even lower given that leachate extraction occurs slowly over time (not all at once as assumed in the given estimate). Variations in biomass and population density may also increase or decrease natural levels of allelochemical input. A detailed study of potential allelochemical exposure under natural conditions would provide important information about hoary alyssum.  62  While Petri dish assays showed that hoary alyssum exerts allelopathic influence on the germination and seedling growth of associated forage species as well as itself, soil assays did not show clear allelopathic effects. Concentrations of leachate and inputs of unleached leaf biomass used in this study appear to be far higher than what would occur in nature; further research is needed to confirm this. Without further investigation of hoary alyssum allelopathy under appropriate field conditions, it is impossible to confirm or deny that hoary alyssum utilizes allelopathic influence to gain an advantage over associated forage grasses.  .  63  Chapter 5: The Effect of Nitrogen Fertilization on Hoary Alyssum and Associated Forage Species 5.1 Introduction Plant species differ in their ability to compete for the resources required for growth and survival (Grime 1977). Environmental conditions further influence the competitive ability of individual plants (Grime 1977). Altering environmental conditions by amending resources (e.g. soil nutrients) can increase a plant’s growth and competitive ability (Hautier et al. 2009) depending on that species’ ability to utilize the altered resource. By altering the competitive ability of individual plants, resource amendments may in turn influence competitive interactions among associated species. Invasive weeds like hoary alyssum (Berteroa incana L. DC) have been able to establish in and dominate native plant communities because of their ability to thrive on nutrient poor soils (Warwick and Francis 2006). Altering nutrient availability, and competitive interactions between species, may establish competitive advantages for native species over that of introduced weeds. Improving our understanding of how species respond to increased nutrient availability (fertilization) may provide grounds for improving weed management practices and restoring native plant communities. Nitrogen is essential to plant growth. It plays a central role in the formation of proteins and nucleic acids (Novoa and Loomis 1981; Whitehead 2000). It stimulates photosynthesis, amino-acid synthesis and protein synthesis (Novoa and Loomis 1981), thus stimulating biomass production (growth) in plants. Literature examining the response of hoary alyssum to increased levels of soil nitrogen is limited. Tilman (1984) showed that hoary alyssum growth (biomass) responded favorably to 64  high inputs of nitrogen under field conditions with an imposed nitrogen:magnesium gradient. Tilman (1987) observed a decrease in the relative abundance of hoary alyssum when nitrogen was added to an old-field system in Minnesota, where total nitrogen already exceeded 500 mg kg-1 of soil. Besides Tilman’s observations (Tilman 1984; 1987), the response of hoary alyssum to added soil nitrogen has not been studied in detail. Idaho fescue (Festuca idahoensis Elmer) and bluebunch wheatgrass (Pseudoroegneria spicata (Pursh) A. Löve) are native rangeland grasses commonly found in areas infested by hoary alyssum. Little information on their response to nitrogen fertilization is available. Bluebunch wheatgrass has been shown to increase protein content and biomass when provided with additional nitrogen (Mason and Miltmore 1959). However, added nitrogen may not provide a competitive edge over associated weed species. For example: Herron et al. (2001) compared the responses of bluebunch wheatgrass and the invasive spotted knapweed (Centaurea maculosa (Lam.)) to added nitrogen and found that competitive interactions between the two did not lend bluebunch wheatgrass an advantage. Effects of nitrogen fertilization on the competitive interaction between hoary alyssum and these associated native forage species has not been studied. The objective of this research was to asses the response of hoary alyssum and selected native forage grasses to nitrogen fertilization. Improving our understanding of how fertilization affects each species, and assessing whether an advantage can be given to native forages, could provide valuable information to weed managers attempting to control hoary alyssum.  65  5.2 Materials and Methods 5.2.1 Field experiment 5.2.1.1 Field site Field experiments were conducted on a hoary alyssum infested site near Morrissey Creek road, located outside the city limits of Grand Forks BC. (Approx. N 49° 01.840’, W 118° 24.100’; elevation 640 – 665 m) (see sect. 1.3).  5.2.1.2 Soil analysis and fertilizer treatment Three soil samples were randomly taken from the field site (Aug. 1, 2008), and sent to the Analytical Chemistry Services Laboratory (BC Ministry of Environment’s Environmental Sustainability Division: Knowledge Management Branch, in Victoria BC) for nutrient analysis. The results indicated that the availability of nitrogen was most likely to be the major limiting factor for plant growth at the Morrissey Creek study site (Drs. A. Bomke and M. Krzic 2008, personal comm.). Based on fertilization recommendations for native rangelands on similar Alberta soils (Alberta Agriculture 2000), five nitrogen fertilizer (urea 46:0:0) treatment rates were chosen to study the effect of fertilization on the growth, development, and competitive ability of hoary alyssum and associated forage grasses.  5.2.1.3 Herbicide treatment In order to determine the response of grasses to added nitrogen in the absence of hoary alyssum, broadleaf herbicide treatments were included to selectively control hoary alyssum in some treatment plots. In consultation with Barb Steward and the Boundary Weed Management  66  Committee, a mixture of the broadleaf herbicides 2,4-D amine 600 (Nufarm Agriculture Inc, Calgary AB.) (dimethylamine salt of 2,4-D: 564 g a.e. L-1) and dicamba (Oracle®, Gharda Chemicals Limited, Newtown, PA.) (dimethylamine salt of dicamba: 480 g a.e. L-1) was chosen to control hoary alyssum. Both 2,4-D and dicamba are selective herbicides that control broad leaf species without affecting grasses. Herbicides were mixed in a ratio of 46% 2,4-D amine 600 to 54% dicamba. Two liters of the mixture was prepared and applied to 15 plots at a rate of approx. 325 L ha-1 (each plot received ~0.13 L of the herbicide mixture).  5.2.1.4 Nitrogen fertilization field experiment Field plots (2 m x 2 m) (Fig. 5.1) were established in a randomized complete block design (3 blocks with 10 plots per block) at the Morrissey Creek study site. Five fertilization levels (0 (control), 15, 30, 60, 120 lbs ac-1– or approx. 0, 16.8, 33.6, 67.2, and 134.4 kg ha-1) with or without herbicide treatment (see section 5.2.1.3), were employed. Pre-weighed amounts of urea pellets (46-0-0) were evenly spread in each plot on the same day that the herbicide treatment was applied. Herbicides were applied by the Boundary Weed Management team (May 29, 2009). Over the two days prior to treatment application (May 27 and 28, 2009), two biomass samples (50 cm x 50 cm plots) were randomly taken from each plot, sorted into three groups (hoary alyssum, grass, or other broadleaf species), and weighed (pre-treatment biomass). Each sample plot was marked so that the area directly adjacent to it could be sampled for assessing the treatment effect at the end of the experiment (Oct. 21-23, 2009). All samples were placed in paper bags, transported to UBC, dried at 60°C for 7 days, and weighed.  67  A  B  Figure 5.1. Nitrogen fertilization field plots (2 m x 2 m) at the hoary alyssum infested Morrissey Creek study site near Grand Forks, BC.; Field plots (A), and close up of a plot (B).  68  5.2.2 Pot-culture experiment Since hoary alyssum and associated forage species showed no response to nitrogen fertilization (see sect. 5.3.1 and 5.4), a pot-culture experiment at the UBC Botanical Garden Nursery’s poly-house was established in the summer of 2010. The effect of nitrogen fertilization on the growth, and development of hoary alyssum and associated forage grasses (viz. Idaho fescue and bluebunch wheatgrass) was studied.  5.2.2.1 Seed sources Idaho fescue and bluebunch wheatgrass seeds were obtained from Premier Pacific Seeds Ltd. (Surrey, BC). Hoary alyssum seeds used in this experiment were collected from populations at the Morrissey Creek site in July 2008.  5.2.2.2 Soil selection With the help of Dr. Les Lavkulich, professor of Soil Science at UBC, a natural soil from the Fraser Valley BC was selected for use in the pot-culture experiments. The soil was similar in texture, nutrient content, and organic matter content when compared to the soil at the Morrissey Creek study site. The soil was obtained from the Port Kell’s Nursery (Surrey, BC).  5.2.2.3 Nitrogen fertilization pot-culture experiment Pots (27 cm diameter x 30 cm depth) were filled with soil (sect. 5.2.2.2). Mixtures of hoary alyssum with either Idaho fescue, bluebunch wheatgrass (Fig. 5.2), or both, were grown at five nitrogen levels similar to those used in the field study (0, 16, 32, 64, 128 kg ha-1) described above (section 5.2.1.4) (June 1 to Aug 31, 2010). The fertilization treatment rates in this  69  A  B  A  C  Figure 5.2. Pot-culture experiment to study the effect of N on hoary alyssum and associated grasses. Hoary alyssum and Idaho fescue with 16 kg ha-1 N (A); hoary alyssum and bluebunch wheatgrass with 128 kg ha-1 N (B and C).  70  experiment were based on published fertilization regimes for grasses in the Kootenay-Boundary Region (BC Ministry of Agriculture 1978). Urea pellets (46-0-0), ground to a fine powder in order to achieve better soil surface cover, were used as fertilizer. A randomized complete block experimental design with 8 blocks was used. The experiment was repeated concurrently in the summer of 2010. Pots were watered and weeded for two weeks prior to seeding (or transplanting) and fertilizer application. Weeding continued throughout the experiment. Excess grass seed was sown in each pot and the seedlings thinned to maintain the set numbers (20 seedlings of each grass species when grown individually, 15 of each when grown together). Hoary alyssum seeds were germinated in the laboratory and the seedlings transplanted into the pots. Ten hoary alyssum seedlings were transplanted in an asterisk pattern into each pot. Hoary alyssum seedlings were thinned to 7 after they had begun to establish. All pots were lightly watered twice a day (mid-morning and early evening). A mister was used for watering in order to minimize damage to fragile seedlings during the first few weeks of growth and to avoid leaching of nitrogen. At the end of the experiment (92 days) the number of plants in each pot was counted, the plants excised at the ground level, dried for 7 days at 60°C, and weighed. Visual observation showed that flowering of hoary alyssum plants appeared to be affected by nitrogen fertilization. The number of plants that flowered in each pot was therefore recorded. Soil samples from each treatment were taken to a depth of 10 cm at the end of the experiment and tested for available nitrogen (Analytical Chemistry Services Laboratory, BC Ministry of Environment, Victoria BC).  71  5.2.3 Data analysis A randomized complete block design was employed for all experiments. Pot-culture experiments were repeated at the same time, and the data was analyzed as a randomized complete block split-plot design in to test treatment x experiment interactions. All experiments were subjected to Least Square Means analysis with multiple pairwise comparisons of the means (α = 0.05) using SAS 9.2 (SAS Institute, Inc., Cary, NC., USA.) software. Alpha values were corrected for individual comparison of the means using Bonferroni’s method (Holm 1979).  5.3 Results 5.3.1 Nitrogen fertilization field experiment Nitrogen application did not influence hoary alyssum or associated forage grasses (biomass) when compared to respective controls (data not shown). Variability in the biomass samples was very high preventing proper statistical assessment of treatment effects. Visual observations suggest that herbicide treatments effectively controlled hoary alyssum and other broadleaf species. Due to the failure of both hoary alyssum and the associated forages to respond to urea under field conditions, it was decided that the study would be repeated in a pot-culture experiment where growth conditions could be controlled and the experiment monitored more closely.  72  5.3.2 Nitrogen fertilization pot-culture experiment Biomass of hoary alyssum increased with increased rates of nitrogen application (Fig 5.3). Since a significant (P ≤ 0.05) difference between experiments was observed, results of the two experiments are reported separately. In all cases (except when hoary alyssum and both grasses were grown together, Fig. 5.3, Exp.1C), the highest nitrogen treatment (128 kg ha-1) elicited a significant increase in hoary alyssum biomass over that of the 0 (control) to 32 kg ha-1 treatments. Hoary alyssum biomass was not significantly different between the 64 and 128 kg ha-1 treatments in some cases (Fig.5.3. Exp.1B&C, Exp. 2C). Biomass of bluebunch wheatgrass and Idaho fescue showed no significant (P ≤ 0.05) response to nitrogen, with the exception of bluebunch wheatgrass at 64 kg ha-1 in Expt. 2 (Fig 5.3, Exp. 2C). Nitrogen fertilization significantly (P ≤ 0.05) increased bolting and flowering in hoary alyssum 92 days after planting, in all species mixtures (Fig. 5.4). Since a significant difference (P ≤ 0.05) between experiments was observed the results of the two experiments are presented separately (Fig. 5.4). In control, 64 kg ha-1 N, and 128 kg ha-1 N treatments, 0-14%, 7-27%, and 32-57% of plants respectively, either bolted or flowered; all 128 kg ha-1 N treatments were significantly different (P ≤ 0.05) from the 0-32 kg ha-1 treatments. Analysis of soil taken from the upper 10 cm of the pots at the end of the experiment (Table 5.1) showed little difference in either available (NH4N or NO3N) or total nitrogen (N) levels between nitrogen treatments. A separate pot-culture experiment was conducted with 0, 64, and 128 kg ha-1nitrogen treatments to examine the possibility of nitrogen leaching to the lower layers of the pots due to repeated watering. Soil nitrogen at depths of 0, 15, and 30 cm (surface, middle, and bottom of the pot) was analyzed. Results (Table 5.2) showed that over the course of  73  Experiment 1 250  Experiment 2 250  A  200  200  150  150  100  100  50  50  0  0  -50  -50 0  250  Dry Weight (% Increase)  A  64  128  0  250  B  200  200  150  150  100  100  50  50  0  0  64  128  64  128  64  128  B  -50  -50 0 250  64  0  128 250  C  C  200  200  Bi HA IF  150  150  BB  100  100  50  50  0  0 -50  -50 0  64  128  0  -1  Nitrogen Treatment (kg ha ) Figure 5.3. Effect of nitrogen (urea 46-0-0) fertilization on dry biomass of hoary alyssum (HA), Idaho fescue (IF) and bluebunch wheatgrass (BB). Hoary alyssum + Idaho fescue (A); hoary alyssum + bluebunch wheatgrass (B); hoary alyssum + Idaho fescue + bluebunch wheatgrass (C). Values are means ± SE of 8 replications.  74  Bolting or Flowering (%)  50  Experiment 1  70  Experiment 2  60  40  50  30  40  20  30 u  20  10 IF 0  BB MIX  10 0 -10  -10 0  64  128  0  64  128  Nitrogen (kg ha-1) Figure 5.4. Effect of nitrogen (urea 46-0-0) fertilization on bolting of hoary alyssum rosettes. Idaho fescue (IF), bluebunch wheatgrass (BB), and Idaho fescue + bluebunch wheatgrass (Mix) mixtures. Values are means ± SE of 8 replicates.  75  Table 5.1. Available nitrogen in the upper 10 cm of soil at the conclusion of the pot-culture experiment. Available N  Sample / Treatment  NH4N (mg kg-1)  Nitrogen Rate  Total C and N  NO3N (mg kg-1)  C (%)  N (%)  0 kg ha-1  0.36  1.48  1.61  0.098  16 kg ha-1  0.95  1.68  1.38  0.088  32 kg ha-1  0.23  1.44  1.36  0.085  64 kg ha-1  0.22  1.87  1.58  0.096  128 kg ha-1  0.14  1.80  1.48  0.093  Table 5.2. Leaching of nitrogen due to watering in pot-culture experiments. Treatment Nitrogen Rate Deptha  a  Available Nitrogen NH4N (mg kg-1) NO3N (mg kg-1)  Total C and N C (%) N (%)  0 kg ha-1  0 cma 15 cm 30 cm  < 0.01 < 0.01 < 0.01  1.05 3.28 3.56  1.52 1.39 1.27  0.096 0.089 0.082  64 kg ha-1  0 cm 15 cm 30 cm  < 0.01 < 0.01 < 0.01  1.51 7.86 20.27  1.30 1.46 1.65  0.083 0.088 0.101  128 kg ha-1  0 cm 15 cm 30 cm  < 0.01 < 0.01 < 0.01  1.21 4.57 18.65  1.62 1.59 1.69  0.098 0.098 0.101  Samples were taken from 0, 15, and 30 cm depths for soil analysis.  76  the experiment, available nitrogen, specifically in the form of nitrate (NO3N), leached to the bottom of the pot (30 cm depth).  5.4 Discussion The lack of response, in terms of increase in biomass, from plants in the field nitrogen experiment (data not shown), despite initial (pre-treatment) soil analysis indicating available nitrogen as the most likely limiting factor for growth, was puzzling. Since the highest nitrogen application rate was more than double the recommended rate (Alberta Agriculture 2000), the lack of effect could not be due to insufficient amounts of nitrogen being applied. Readily soluble in water (Gould et al. 1986; Smith et al. 2007), urea is known to rapidly wash off soil surfaces (Smith et al. 2007) or leach beyond the rooting depth of some species (Vlek et al. 1980; Campbell et al. 1993). However large volumes of water over a short period of time are required for such leaching (Singh et al. 1984). Environment Canada (2012b) daily data for the Billings weather station (located 30 km east of the Morrissey Creek study site, within the same Kettle River Valley system), showed no significant rainfall within a week after fertilizer application (May 29 – June 5, 2009); in fact only 122.4 mm of rain fall occurred in the first three months of the experiment (May 29 - Aug. 31). This suggests that the added nitrogen could not have leached or washed away from the experimental plots. Urea fertilizer is known for its volatility when applied to warm dry soils (Ernst and Massey 1960; Nelson 1982; Grant et al. 1996; Rawluk et al. 2001). Increasing amounts of urea loss has been reported with increasing temperatures up to ~45°C (Nelson 1982). Grant et al. (1996) observed that 40 and 88% (May and August, respectively) of granular urea fertilizer could be converted into and volatilized as ammonia (NH3) with 7 days of application in a no-till  77  system near Brandon Manitoba. In the week following fertilizer application at the Morrissey Creek site, maximum daily air temperatures in the area averaged in excess of 25°C with extreme temperatures exceeding 32°C (Environment Canada 2012b). More importantly, the soil surface temperatures at the Morrissey Creek site reach as high as 83°C (personal observations, see sect. 2.4). Thus it is possible that a significant amount of the added urea could have volatized in this study. The destructive sampling (Catchpole and Wheeler 1992) used in the experiment to compare treatment biomass, combined with patchy hoary alyssum cover (personal observation) over the field site likely influenced the high variability in this experiment (data not shown). In order to limit the variability of biomass between replications, the response of hoary alyssum and associated forage grasses to added nitrogen was studied in pot culture experiments, where more control over experimental conditions was possible. Results of the pot-culture experiment revealed a significant increase in hoary alyssum biomass upon addition of increasing amounts of nitrogen (Fig. 5.3). Idaho fescue and bluebunch wheatgrass however showed no response to the added nitrogen, regardless of the application rate (Fig. 5.3). Soil analysis (Tables 5.1 and 5.2) indicated that a significant amount of nitrogen likely leached through the soil profile in the experimental pots. While Idaho fescue, and bluebunch wheatgrass can both achieve significant rooting depths (in excess of 130 cm for bluebunch wheatgrass (Weaver 1915)), both perennial species are relatively slow growing (Nasri and Doescher 1995; Arredondo et al. 1998) taking 2-3 years to fully establish from seed (Ogle et al. 2008; Ogle et al. 2010a). In this study their roots were not observed below 10 cm soil depth in the pots. Hoary alyssum, a relatively fast growing annual with a deep taproot (Warwick and  78  Francis 2006) had its roots reach the bottom of the pots, enabling them to absorb the nitrogen that had leached downward. A favorable response of hoary alyssum to nitrogen in terms of increasing biomass has been reported under field conditions by Tilman (1984). The pot-culture experiments support this report. Bluebunch wheatgrass was also previously reported to respond favorably to nitrogen in terms of increased biomass (Mason and Miltmore 1959). However, the leaching of nitrogen in the pot-culture experiments prevented any conclusions regarding the effects of nitrogen on bluebunch wheatgrass or Idaho fescue. Subsequently, the effect of nitrogen fertilizer on the relative competitive ability of hoary alyssum and these associated native grasses cannot be inferred. Further research to examine the effect of nitrogen fertilization on competitive interactions between these species is needed. Perhaps the most interesting result of this study was the increase in flowering of hoary alyssum in response to nitrogen fertilization. Nitrogen is known to increase plant biomass, growth rate (Olson and Kurtz 1982), and even flower and seed production in some species (Benner 1988; Wright et al. 1988; Bi et al. 2008). The effects of nitrogen on bolting, flowering, and eventual seed production in hoary alyssum have not been reported to date. The results of the pot-culture study show an increase in biomass accumulation as well as flowering with increased nitrogen application. Thus nitrogen fertilization may increase the rate of hoary alyssum’s development. The majority of hoary alyssum plants flower in June, setting seed in July and August. These typically germinate, establish a rosette, and over-winter to produce flowers the following year (Warwick and Francis 2006). Seeds maturing before the middle of July are capable germinating, establishing, flowering, and producing seed by the fall (Warwick and Francis 2006). With appropriate conditions during the fall, those seeds may germinate and  79  establish to over-winter as rosettes. Increasing the rate of hoary alyssum’s development with nitrogen fertilization, may allow plants to flower and set seed earlier in the year. Earlier flower and seed development could enable hoary alyssum populations to produce two full seed sets in one year. Given hoary alyssum’s copious seed production (Stevens 1932; Reichman 1988; Warwick and Francis 2006) this could create a significant problem for weed managers. Further clarification regarding the effect of nitrogen on hoary alyssum development in the field is needed. Such information could have significant implications for the management of this weed.  80  Chapter 6. General Discussion Exotic (non-native) and invasive weed species are a significant threat to the health and sustainability of natural and agricultural ecosystems (Lonsdale 1999; Pimentel et al. 2005). Understanding what allows exotic weeds to invade and persist in an ecosystem is essential for developing effective weed management strategies. Hoary alyssum (Berteroa incana (L.) DC) is an exotic invasive (annual to short-lived perennial) weed across North America (Warwick and Francis 2006). In recent years public interest in hoary alyssum has increased in British Columbia due to its continual expansion in the south central/southeastern rangelands of the province, and the recent awareness of its toxicity to horses (Geor et al. 1992). Unfortunately for weed managers, very little is known about the biological and ecophysiological characteristics that allow hoary alyssum to persist. The research presented in this thesis examined four distinct and poorly understood aspects of hoary alyssum biology: 1. the size and dynamics of hoary alyssum seed banks, 2. the response of hoary alyssum to mowing (removal of flowering shoots), 3. the ability of hoary alyssum to exert an allelopathic influence on native forages, 4. the effect of nitrogen fertilization on the growth and development of hoary alyssum and native forages. The results from these studies improve our understanding of hoary alyssum persistence, and may aid weed managers in developing effective control strategies for this weed.  81  6.1 Hoary alyssum seed bank dynamics Studies examining hoary alyssum’s seed banks revealed that hoary alyssum maintains a large (132.4×106 seeds ha-1), but patchy, and shallow (0-4 cm) soil seed bank. Seeds on the soil surface exhibit characteristics of a transient seed bank (Thompson and Grime 1979; Garwood 1989; Simpson et al. 1989) with as much as 87.5% germination within one year. For weed managers, prevention of seed input and control of plants/emerging seedlings, may help exhaust seed banks within a relatively short period time. Seed burial depth significantly affected seed fate; buried seeds remained ungerminated but viable in the soil for up to 2 years. Seeds buried 2-5 cm deep showed significant enforced dormancy (as much as 86.7% of seeds after 1 year). These seeds would germinate should conditions become suitable for germination. Enforced dormancy can be broken with a variety of weed management practices (Dyer 1995; Caldwell and Mohler 2001) inducing a flush of seedlings vulnerable to controls. Only a small fraction of seeds (max 7.6%) showed induced dormancy when buried. However, in large seed banks even this small fraction may represent millions of seeds, more than what is needed to re-establish a population. When stained with tetrazolium some damage (and decay) of embryos with induced dormancy was observed. This suggests a possible loss of seed vigor over time (Moore 1973). Determining the causes of seed dormancy and identifying factors that effect lost of seed vigor and viability over time would provide valuable information to weed managers.  6.2 The response of hoary alyssum to mowing This study showed that hoary alyssum plants mowed during early stages of flower development are capable of regenerating new shoots and producing seed by the end of a growing  82  season. Apical dominance (Ross and Lembi 1985) is released, but mowing did not appear to stimulate increased shoot growth and seed production. When mowed at later stages of flower and seed development, fewer plants survived to reproduce new shoots. However some plants (at least 4%) did produce new shoots and seed by the end of the study. The results suggest that a single application of mowing is not an effective control for hoary alyssum. Mowed plants at all stages of development were able to regenerate and produce seed by the end of the study. Furthermore, mowing may disseminate seed (Bakker et al. 1996; Strykstra et al. 1997; Coulson et al. 2001; Donald 2006) if done after seeds have begun to mature. The effects of repeated mowings on hoary alyssum have not been determined. Further research on the effectiveness of repeated mowings to exhaust carbohydrate storage reserves and weaken this weed (DiTomaso 2000) may provide useful information for weed managers.  6.3 Allelopathic influence of hoary alyssum on native forages Petri dish assays performed in this study suggest that water soluble compounds extracted from the leaves of hoary alyssum are capable of exerting an allelopathic influence, inhibiting germination and root growth of associated forage species. Self-inhibition or autotoxicity (Singh et al. 1999) of germination and seedling growth (root and shoot) in hoary alyssum was also observed in Petri dishes. However, soil assays utilizing both leaf leachate and unleached leaf biomass did not consistently inhibit seedling emergence. Levels of hoary alyssum allelochemical exposure faced by associated species under field conditions have not been established. Based on rough estimates, it is believed that the leachate  83  concentrations and amounts of unleached leaf biomass used in this study far exceed what plants would naturally be exposed to under field conditions. At this point in time, it is not possible to conclude whether hoary alyssum utilizes an allelopathic influence to invade, establish, and compete with native forages in a rangeland environment. Research is needed to examine the potential allelopathic influence of hoary alyssum on associated forages under natural conditions.  6.4 The effect of nitrogen fertilization on hoary alyssum and associated forage species Pot-culture studies revealed that nitrogen fertilization increased biomass and stimulated shoot/flower production in the deep rooted hoary alyssum. Added nitrogen did not affect the growth of bluebunch wheatgrass or Idaho fescue, presumably because nitrogen had leached below their rooting depth over the course of the study. Because of this leaching, it was not possible to study the influence of nitrogen on the competitive interactions between hoary alyssum and the two grass species. The stimulation of growth (biomass) and flowering in hoary alyssum exposed to nitrogen fertilization should be of concern to weed managers. Stimulating the development of flowers (and subsequently of seeds) may alter the timing of flowering and seed production of hoary alyssum in nature, allowing its populations to complete more than one lifecycle in a given season. While the effects of nitrogen on the competitive interactions between hoary alyssum, bluebunch wheatgrass, and Idaho fescue could not be studied because of nutrient leaching, hoary alyssum was found to clearly benefit from the added nitrogen. Therefore weed managers and land owners should consider avoiding nitrogen fertilization in hoary alyssum infested areas,  84  particularly in areas where nutrients can leach below the rooting zone of forage grasses. In those areas added nitrogen will only serve to further exacerbate the weed problem as it may increase the growth and competitive ability of hoary alyssum and stimulate its flowering.  6.5 General conclusion The results presented in this thesis contribute important information to our understanding of persistence in hoary alyssum. The information that has been revealed about seed banks, the effects of a single mowing treatment, potential use of allelopathy, and response of hoary alyssum to nitrogen fertilization, may directly contribute to the development of weed management practices and/or provide a strong foundation upon which researchers may continue to build our understanding of this weedy species.  85  Literature Cited  Alberta Agriculture. 2000. Agri-Facts: Native Range Fertilizer Guide. Alberta Agriculture Food and Human Development. Agdex 130/541. Alex, J. F. 1992. Ontario weeds. Ontario Ministry of Agriculture and Food, Toronto ON. 304 pp. Anderson, W. P. 1983. 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