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Allelopathic influence of tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea… Bainard, Luke D. 2007

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A L L E L O P A T H I C INFLUENCE OF T A L L HEDGE M U S T A R D (SISYMBRIUM LOESELIIL.) A N D SPOTTED K N A P W E E D (CENTAUREA MACULOSA L A M . ) ON A R B U S C U L A R M Y C O R R H I Z A L FUNGI by L U K E D. B A I N A R D B.Sc , Trinity Western University, 2003 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Plant Science) THE UNIVERSITY OF BRITISH C O L U M B I A February 2007 © Luke D. Bainard, 2007 Abstract Many exotic weeds interfere with other species by releasing allelochemicals into the environment that have a negative effect on their growth and distribution. Allelochemicals can have a direct influence on plant growth and/or indirect influence by disrupting interactions between plants and soil organisms, such as arbuscular mycorrhizal (AM) fungi. The goal of this research was to explore the allelopathic influences of the exotic weeds tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea maculosa Lam.). The allelopathic potential of tall hedge mustard was assessed using aqueous root and shoot extracts in seed germination and seedling growth bioassays. Aqueous tall hedge mustard root and shoot extracts strongly inhibited germination and growth of bluebunch wheatgrass, Idaho fescue, and spotted knapweed. Glucosinolate (GSL) analysis of tall hedge mustard tissues revealed the presence of two major GSLs (isopropyl GSL and sec-butyl GSL) and four indolylic GSLs. The degradation products of the two major GSLs (isopropyl isothiocyanate [ITC] and sec-butyl ITC) were identified in tall hedge mustard aqueous root and shoot extracts. Commercially available isopropyl ITC and sec-butyl ITC inhibited seed germination and seedling growth, suggesting their role in the allelopathic influence of tall hedge mustard. Tall hedge mustard aqueous extracts and ITCs incorporated into an agar medium inhibited Glomus intraradices Shenck & Smith spore germination and hyphal growth. Tall hedge mustard aqueous extracts strongly inhibited spore germination and hyphal growth of G. intraradices. Isopropyl ITC and sec-butyl ITC inhibited spore germination and hyphal growth, with the former exhibiting a stronger effect. Tall hedge mustard infestations were also found to reduce the A M inoculum potential of soil. The A M colonization and total biomass was reduced in bluebunch wheatgrass and spotted knapweed plants growing in tall hedge mustard infested compared to noninfested soil. Spotted knapweed is known to produce two major allelochemicals, (±)-catechin and cnicin. Both allelochemicals inhibited Glomus intraradices spore germination, and cnicin also inhibited the hyphal growth, suggesting that these allelochemicals may be involved in the inhibitory effect spotted knapweed has on A M fungi. Results of this study show that tall hedge mustard and spotted knapweed both produce allelochemicals that have the potential to directly and/or indirectly inhibit the growth of neighboring species and their A M fungal associates. Table of Contents Abstract H Table of Contents iii List of Tables vi List of Figures vii Abbreviations , ix Acknowledgements x Co-Authorship Statement xi Chapter 1. General Introduction and Literature Review 1 General Introduction 1 Literature Review 5 1.1 Plant species used in this study. 5 1.1.1 Tall hedge mustard (Sisymbrium loeseliih.) 5 1.1.2 Spotted knapweed (Centaurea maculosa Lam.) 5 1.1.3 Bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love) 6 1.1.4 Idaho fescue (Festuca idahoensis Elmer.) 7 1.2 Allelopathy 7 1.3 Allelochemicals of the exotic weeds used in this study 8 1.3.1 Allellochemicals of Brassicaceae 8 1.3.2 Spotted knapweed allelochemicals 9 1.4 Arbuscular mycorrhizal fungi 10 1.4.1 Glomus intraradices Shenck & Smith 12 1.5 Literature Cited ..13 Chapter 2. Role of Glucosinolate Degradation Products in the Allelopathic Potential of Tall Hedge Mustard (Sisymbrium loeselii L.) .21 2.1 Introduction 21 iii 2.2 Materials & Methods. .... 25 2.2.1 Seed sources 25 2.2.2 Tall hedge mustard extracts and whole plant leachate 25 2.2.2.1 Plant material and aqueous extract and leachate preparation 25 2.2.2.2 Seed germination bioassay , 26 2.2.2.3 Seedling growth bioassay 26 2.2.2.4 Soil bioassay 27 2.2.3 Glucosinolate analysis 28 2.2.4 Glucosinolate degradation product analysis 29 2.2.5 Phytotoxicity of glucosinolate degradation products 31 2.2.5.1 Isothiocyanates - seed germination bioassay 31 2.2.5.2 Isothiocyanates - seedling growth bioassay 32 2.2.6 Data analysis 32 2.3 Results , , 33 2.3.1 Effect of aqueous extracts and leachate on seed germination 33 2.3.2 Effect of aqueous extracts and leachate on seedling growth 35 2.3.3 Effect of aqueous extracts and leachate in soil 38 2.3.4 Glucosinolate analysis 41 2.3.5 Glucosinolate degradation product analysis. 41 2.3.6 Effect of isothiocyanates on seed germination 41 2.3.7 Effect of isothiocyanates on seedling growth 47 2.4 Discussion ..47 2.5 Literature Cited 56 Chapter 3. Inhibitory Effects of Tall Hedge Mustard (Sisymbrium loeselii L.) Allelochemicals on Arbuscular Mycorrhizal Fungi 60 3.1 Introduction 60 3.2 Materials & Methods 63 3.2.1 Seed and spore sources 63 3.2.2 Tall hedge mustard extracts and whole plant leachate preparation 63 3.2.3 Spore germination and hyphal growth bioassays 64 iv 3.2.3.1 Aqueous extracts and leachate. 64 3.2.3.2 Isothiocyanates ....65 3.2.4 Arbuscular mycorrhizal inoculum potential of tall hedge mustard infested soil 67 3.2.5 Data analysis 69 3.3 Results 70 3.3.1 Effect of aqueous extracts and leachate on spore germination. 70 3.3.2 Effect of isothiocyanates on spore germination,... 70 3.3.3 Effect of aqueous extracts and leachate on hyphal growth 70 3.3.4 Effect of isothiocyanates on hyphal growth 74 3.3.5 Arbuscular mycorrhizal inoculum potential of tall hedge mustard infested soil 74 3.4 Discussion : 74 3.5 Literature Cited , .80 CHAPTER 4. Effect of (±)-Catechin and Cnicin, Two Possible Allelochemicals of Spotted Knapweed (Centaurea maculosa), on Spore Germination and Hyphal Growth of Glomus intraradices 84 4.1 Introduction 84 4.2 Materials & Methods .....87 4.2.1 Allelochemicals ; 87 4.2.3 Spore germination and hyphal growth bioassay 87 4.2.4 Data analysis 88 4.3 Results ..! 88 4.3.1 Spore germination ..88 4.3.2 Hyphal growth '.. 90 4.4 Discussion 90 4.5 Literature Cited. .., 97 Chapter 5. General Discussion 101 5.1 Literature Cited 104 List of Tables Table 2.1a. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in experiment #1 36 Table 2.1b. Effect of tall hedge mustard root extract, shoot extract, and. whole plant leachate seedling growth in,experiment #2 37 Table 2.2. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seed germination in soil 39 Table 2.3a. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in soil in experiment #1 40 Table 2.3b. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in soil in experiment #2 40 Table 2.4. Glucosinolate content of tall hedge mustard root and shoot tissues 43 Table 3.1. Characteristics of soils used in A M inoculum potential experiment 68 Table 3.2. Effect of tall hedge mustard aqueous root extract, shoot extract, and whole plant leachate on spore germination of Glomus intraradices 71 Table 3.3. Effect of tall hedge mustard aqueous root extract on hyphal growth of Glomus intraradices 73 vi List of Figures Figure 2.1. Dense stand of tall hedge mustard in southern British Columbia 24 Figure 2.2. U V absorption spectra of desulfo-GSLs extracted from tall hedge mustard root and shoot tissues 30 Figure 2.3. Effect Of tall hedge mustard root extracts, shoot extracts, and plant leachate on germination of (A) bluebunch wheatgrass, (B) Idaho fescue, (C) spotted knapweed, and (D) tall hedge mustard 34 Figure 2.4. HPLC chromatogram of desulfo-GSLs extracted from tall hedge mustard shoot tissues collected from the field 42 Figure 2.5. HPLC chromatogram of desulfo-GSLs extracted from tall hedge mustard root tissues collected from the field 42 Figure 2.6. GC-MS chromatogram of dichloromethane fraction of tall hedge mustard aqueous shoot extract 44 Figure 2.7. GC-MS chromatogram of dichloromethane fraction of tall hedge mustard aqueous root extract 44 Figure 2.8. Effect of isopropyl ITC on seed germination of (a) bluebunch wheatgrass, (b) Idaho fescue, (c) spotted knapweed, and (d) tall hedge mustard 45 Figure 2.9. Effect of sec-butyl ITC on seed germination of (a) bluebunch wheatgrass, (b) Idaho fescue, (c) spotted knapweed, and (d) tall hedge mustard 46 Figure 2.10. Effect of isopropyl ITC on radicle and coleoptile growth of bluebunch wheatgrass 48 Figure 2.11. Effect of isopropyl ITC on radicle and coleoptile growth of Idaho fescue 48 Figure 2.12. Effect of isopropyl ITC on radicle and coleoptile growth of spotted knapweed 48 Figure 2.13. Effect of isopropyl ITC on radicle and coleoptile growth of tall hedge mustard 48 Figure 2.14. Effect of sec-butyl ITC on radicle and coleoptile growth of bluebunch wheatgrass 49 Figure 2.15. Effect of sec-butyl ITC on radicle and coleoptile growth of Idaho fescue 49 Figure 2.16. Effect of sec-butyl ITC on radicle and coleoptile growth of spotted knapweed. ....49 Figure 2.17. Effect of sec-butyl ITC on radicle and coleoptile growth of tall hedge mustard '.. 49 Figure 3.1. (A) Ungerminated Glomus intraradices spore, and (B) germinated Glomus intraradices spore 66 Figure 3.2. Effect of (A) isopropyl ITC and (B) sec-butyl ITC on spore germination of Glomus intraradices 72 Figure 3.3. Effect of (A) isopropyl ITC and (B) sec-butyl ITC on hyphal growth of Glomus intraradices 75 Figure 3.4. Influence of tall hedge mustard infested or noninfested soils on (A) arbuscular mycorrhizal (AM) colonization, and (B) total biomass of bluebunch wheatgrass and spotted knapweed 76 Figure 4.1. Effect of (±)-catechin on spore germination of Glomus intraradices 89 Figure 4.2. Effect of cnicin on spore germination of Glomus intraradices 91 Figure 4.3. Effect of (±)-catechin on hyphal growth of Glomus intraradices 92 Figure 4.2. Effect of cnicin on hyphal growth of Glomus intraradices 93 viii Abbreviations A M arbuscular mycorrhizal A N O V A analysis of variance EICA evolution of increased competitive ability ERH enemy release hypothesis GC-MS gas chromatography-mass spectrometry GSL glucosinolate HPLC high performance liquid chromatography ITC. isothiocyanate ix Acknowledgements I would like to thank my supervisor Dr. Mahesh Upadhyaya for giving me the opportunity to do my Master's, and for all of his guidance and advice. I would also like to thank Dr. Paul Brown for assisting with and providing the facilities for the glucosinolate analysis in this research. Finally, I would like to thank my family for their support and encouragement, and most importantly my wife, whose love and support were essential to the completion of this degree. x Co-Authorship Statement I designed the research project, performed all of the experiments and data analysis, and prepared each of the chapters in this thesis. Dr. Mahesh Upadhyaya was involved with the project design, and reviewing and editing all of the chapters in this thesis. Dr. Paul Brown was directly involved in the glucosinolate analysis in chapter 2, and reviewed the manuscripts for chapter 2 and 3. Each of the chapters (2, 3, and 4) will be submitted for publication in the future. Chapter 1. General Introduction and Literature Review General Introduction Exotic weeds are a serious threat as they are ecologically and economically destructive to natural and managed habitats. They have the ability to invade natural communities and threaten biological diversity and ecosystem function (Lonsdale, 1999). Invasive weeds are considered one of the main factors responsible for the extinction of native species and loss of biodiversity (Levine et al., 2003). Economically, exotic weeds cause major losses in agriculture and forestry due to direct production losses and management costs (Pimentel et al., 2005). Several hypotheses have been suggested for why exotic plants are more aggressive invaders outside their native range, such as the enemy release hypothesis (ERH) and evolution of increased competitive ability hypothesis (EICA). The E R H proposes that the increased competitive ability of exotic species is related to the lack of natural enemies in the introduced range compared with the native range (Colautti et al, 2004). The EICA hypothesis suggests exotic species have evolved to allocate less resources to traits that confer resistance to their specialist enemies, which are absent in the introduced range, to traits that provide greater competitive advantage (Callaway and Ridenour, 2004). Recently, Callaway and Ridenour (2004) have proposed a new theory to explain the success of some exotic weeds - the "novel weapons hyphothesis" (Callaway and Ridenour, 2004). This hypothesis proposes that some weeds produce allelopathic biochemicals that are highly inhibitory to plants and soil microbes encountered in the introduced range, but are relatively ineffective against these organisms in their native range (Callaway and Ridenour, 2004). 1 In general, the negative effect of chemicals released by one plant on the growth and distribution of neighboring plants is referred to as allelopathy (Inderjit and Callaway, 2003). Allelochemicals consist of a wide variety of plant secondary metabolites that can have a direct influence on plant growth. Allelochemicals could also influence plant growth indirectly by disrupting interactions between native plants and soil organisms, such as arbuscular mycorrhizal (AM) fungi (Wolfe and Klironomos, 2005). A M fungi are symbiotic fungi that colonize the roots of most vascular plant species and improve nutrient uptake, particularly that of phosphorus (Bucking and Shachar-Hill, 2005). Many of the plants that form mycorrhizal associations with A M fungi are dependent on this association for survival (Stinson et al., 2006). As a result, disruption of these mutualistic associations can be detrimental, resulting in long-term effects on the dynamics of plant species. In southern British Columbia, tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea maculosa Lam.) are two exotic weeds that threaten native plant communities. Tall hedge mustard is a non-mycorrhizal annual herb that is commonly found throughout southern British Columbia (Harley and Harley, 1987; Parish et ah, 1996). Although not classified as a noxious weed in British Columbia, tall hedge mustard is problematic because it covers fields and roadsides, forming relatively pure stands when well established (Parish et al, 1996). The ability to form monospecific stands suggests the use of unusually potent mechanisms, such as allelopathy (Hierro and Callaway, 2003). Little information is available regarding the allelopathic potential of tall hedge mustard. However, being a member of the Brassicaceae, tall hedge mustard contains glucosinolates (GSLs) (Fahey et al, 2001). GSLs are a class of secondary 2 metabolites that produce biologically active compounds upon enzymatic degradation that have been shown to exhibit allelopathic and anti-fungal properties (Brown and Morra, 1997). Whether tall hedge mustard utilizes allelopathy as a strategy to interfere with neighboring species either directly (i.e. by inhibition of germination and growth of plants) or indirectly (i.e. by inhibition of A M fungi) is not known. Spotted knapweed is a biennial or short-lived perennial and is one of the most destructive exotic weeds in western North America, where it has invaded millions of hectares of native grasslands (Walling and Zabinski, 2006). Invasion by spotted knapweed often results in complete competitive exclusion of native plants and formation of dense monospecific stands (Ridenour and Callaway, 2001). Recent evidence has shown that the competitive and invasive success of spotted knapweed may be linked at least in part to allelopathy (Ridenour and Callaway, 2001; Bais et al., 2002; Weir et al., 2003). The primary allelochemicals produced by spotted knapweed include the root-exuded flavanol (±)-catechin, and to a lesser degree, the sesquiterpene lactone cnicin. Despite the mycorrhizal status of spotted knapweed, recent studies have shown that spotted knapweed infestations may alter A M fungal communities by reducing A M fungal diversity and abundance, and decreasing extraradical hyphae in soil (Mummey and Rillig, 2006). In addition to their phytotoxic properties, both (±)-catechin and cnicin exhibit anti-microbial and anti-fungal properties (Veluri et al, 2004; Bruno et al., 2003; Skaltsa et al, 2000) and may be involved in spotted knapweed inhibition of A M fungi. However, the effect of these allelochemicals on A M fungi has not been investigated. The overall goal of this research was to increase our understanding of allelopathic influences of the exotic weeds, tall hedge mustard and spotted knapweed. 3 The specific objectives for this study were: 1. to determine the allelopathic potential of tall hedge mustard and identify the allelochemicals involved. 2. to determine if tall hedge mustard allelochemicals and infestations influence soil biology by influencing A M fungi 3. to determine i f spotted knapweed allelochemicals, (±)-catechin and cnicin, influence spore germination and hyphal growth of the A M fungi, Glomus intraradices. This thesis is written in manuscript format, with each chapter containing its own introduction, materials and methods, results, and discussion sections. Chapter 2 deals with the allelopathic potential of tall hedge mustard and identifies the allelochemicals involved (Objective 1). The effects of tall hedge mustard allelochemicals and infestations on A M fungi are examined in Chapter 3 (Objective 2). Chapter 4 investigates the effects of spotted knapweed allelochemicals on spore germination and hyphal growth of the A M fungus, G. intraradices (Objective 3). The general discussion, conclusions, and literature cited are presented in Chapter 5. 4 Literature Review This section briefly reviews relevant literature on the plant species used in this study, general concepts of allelopathy and allelochemicals, and A M fungi. 1.1 Plant species used in this study 1.1.1 Tall hedge mustard (Sisymbrium loeselii L.) Tall hedge mustard is an annual herb (Douglas et al., 1998). Similar to most other members of the Brassicaceae, tall hedge mustard is non-mycorrhizal (Harley and Harley, 1987). It is freely branched, with sparsely to densely hairy stems and bright flowers in loose clusters at branch tips (Douglas et al., 1998; Parish et al., 1996). Tall hedge mustard is an introduced species from Eurasia, and has become naturalized across North America. In Canada, it is found across the country from Quebec westward - in dry cultivated fields, prairies, rangelands, roadsides, and wasteplaces (Warwick et al, 2003). Tall hedge mustard is commonly found in southern British Columbia where it often appears like a yellow blanket covering fields and roadsides in early summer (Parish et al., 1996). Similar to other weedy members of the Brassicaceae, tall hedge mustard can invade native plant communities and often forms relatively pure stands when well established. 1.1.2 Spotted knapweed (Centaurea maculosa Lam.) Spotted knapweed is a deeply taprooted, rosette-forming plant of the Asteraceae (Sheley et al., 1998). It is a biennial or short-lived perennial that lives up to 9 yr and produces flowers that are purple to pink, and occasionally white (Sheley et al., 1998). 5 Spotted knapweed is native to the grassland steppes of southeastern Europe and Asia Minor (Sheley et al, 1998). In western North America, spotted knapweed is one of the most destructive exotic weeds, invading millions of hectares of native grasslands (Walling and Zabinski, 2006). Although disturbance facilitates rapid encroachment and spread of spotted knapweed, the weed is capable of invading undisturbed rangelands (Sheley et al., 1998). The dominance of spotted knapweed in exotic locations is attributed to its prolific seed production, high seed viability, germination characteristics, absence of natural enemies, drought tolerance, and its competitive ability (Lacey et al., 1990; Jacobs et al., 1996). Recent studies have shown that the competitive and invasive success of spotted knapweed may also be linked to allelopathy (Ridenour and Callaway, 2001; Bais et ah, 2002; Weir et al., 2003). Invasions by spotted knapweed often result in complete exclusion of native plants and formation of dense monospecific stands (Ridenour and Callaway, 2001). These invasions have a variety of negative effects including reduced livestock forage, wildlife habitat and species diversity, and increased bare ground, surface water run-off, and stream sedimentation (Herron et al., 2001). 1.1.3 Bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love) Bluebunch wheatgrass is a late-successional perennial bunchgrass that grows to a height of 60 to 130 cm (Blicker et al., 2002; Douglas et al, 1998). This native species commonly occurs with co-dominant Idaho fescue (Festuca idahoensis Elmer.) in semi-arid grasslands in the northwestern United States and Canada (Blicker et al, 2002; Olson and Wallander, 2002). In southern British Columbia, bluebunch wheatgrass is widespread in dry, open grasslands, shrublands, rocky slopes and forest openings at low 6 to mid elevations (Parish et al, 1996; Douglas et al, 1998). This native grass is an important forage for domestic animals and wildlife, but is susceptible to damage or local extinction from overgrazing in spring (Parish et al, 1996). Bluebunch wheatgrass was used in this study as it grows in similar regions of southern British Columbia as tall hedge mustard and may be susceptible to invasion by this weed. 1.1.4 Idaho fescue (Festuca idahoensis Elmer.) Idaho fescue is a densely tufted perennial bunchgrass that grows to a height of 10 to 20 cm (Parish et al., 1996). This native rangeland grass is an important component of shrub steppe and occurs in locations with summer moisture stress, such as headlands and slopes with shallow soil (Ewing, 2002). It is commonly found in southern British Columbia in cool grasslands at low to subalpine elevations (May et al., 2004; Parish et al., 1996). Idaho fescue is resilient to fire and can grow in a variety of temperature regimes (Ewing, 2002). However, it is out-competed by exotic species, such as spotted knapweed, and produces less seed under stress (Ewing, 2002). Idaho fescue was chosen for this study as it also grows in similar regions of southern British Columbia as tall hedge mustard and may be susceptible to invasion by this weed. 1.2 Allelopathy Allelopathy is the inhibitory effect of chemicals released by one plant on the growth and distribution of other plants (Inderjit and Callaway, 2003). These toxic compounds, more commonly referred to as allelochemicals, consist of a variety of secondary metabolites that are released into the environment in a variety of ways. These 7 include leaching and volatilization from plant foliage, exudation from plant roots, and release upon decomposition of or volatilization from plant litter (Kobayashi, 2004; Reigosa et al., 1999; Inderjit and Duke, 2003). Allelochemicals can have a direct effect on neighboring species, or can influence plant growth indirectly by interacting with various abiotic and biotic factors (Inderjit and Nilsen, 2003). 1.3 Allelochemicals of the weedy species used in this study 1.3.1 Allelochemicals of Brassicaceae Tall hedge mustard, a member of the Brassicaceae, produces several glucosinolates (GSLs) that may be involved in the allelopathic influence of this weed. GSLs are sulfur-containing organic anions with a B-D-thioglucose moiety and various differentiating side-groups (Brown and Morra, 1995). More than 120 different GSLs have been identified and differentiated on the basis of their side-groups (Fahey et al., 2001). Most individual species and closely-related taxonomic groups tend to have a small number of GSLs (Fahey et al., 2001). The qualitative and quantitative aspects of GSL composition of plants can vary depending on developmental stage and tissue type (roots, leaves, flowers, etc.), and environmental factors such as soil fertility, moisture regime, pathogen challenge, and plant growth regulators (Brown and Morra, 1997; Fahey et al, 2001; Kiddle et al, 2001). The allelopathic properties associated with glucosinolates are not due to the GSLs themselves, but to their biologically active degradation products. Upon tissue disruption, GSLs are hydrolyzed by the enzyme, myrosinase (B-D-thioglucoside glucohydrolase E.C. 3.2.3.1.), into their bioactive products such as isothiocyanates (ITCs), organic cyanides, 8 oxazolidinethiones, and ionic thiocyanate (Brown and Morra, 1997). ITCs, the most common breakdown product of GSLs (Bialy et al, 1990), are a group of potentially toxic compounds whose primary activity is in the vapour phase as they are relatively insoluble in water (Fenwick et al., 1983; Vaughn and Boydston, 1997). ITCs are general biocides as they interact nonspecifically and irreversibly with proteins and amino acids, and are known to inactivate enzymes in vitro (Brown and Morra, 1997; Morra and Kirkegaard, 2002). In addition to their phytotoxic properties, ITCs exhibit anti-microbial, anti-fungal, nematicidal, and insecticidal properties (Brown and Morra, 1997). 1.3.2 Spotted knapweed allelochemicals Spotted knapweed produces two allelochemicals, (±)-catechin (trans-2-(3,4-dihydroxyphenyl)-3,4-dihydro-l(2H)-benzopyran-3,5,7-triol) and cnicin (3,4-dihydroxy-2-methylenebutanoic acid), which are involved in the allelopathic influence of this weed. Catechin is a flavanoid found in many plants (Veluri et al, 2004). (+)-Catechin is a more widespread bioflavanoid compared to (-)-catechin as it is found in many plant species, including the tea plant (Camellia sinensis L.). The racemic form (±) of catechin, which is produced by the roots of spotted knapweed, is only found occasionally (Bais et al, 2002; Veluri et al, 2004). Both (+)- and (-)-catechin exhibit phytotoxic acitivity, but (+)-catechin is 1.5- to 2.0-fold less active than (-)-catechin (Thelen et al, 2005). (-)-Catechin has broad-spectrum phytotoxicity (Bais et al, 2002). It triggers a wave of reactive oxygen species (ROS)-related signaling" that leads to rhizotoxicity in susceptible plants (Bais et al, 2003). In addition to the phytotoxic properties, (+)-catechin also exhibits anti-microbial and anti-fungal properties (Veluri et al, 2004). 9 Cnicin is a sesquiterpene lactone, or a germacranplide sesquiterpenoid (Landau et al., 1994). Sesquiterpene lactones are secondary metabolites characteristic of many members of the Asteraceae and show a variety of biologically activities (Landau et al., 1994). Cnicin is commonly found in the aerial tissues of many Centaurea species, including spotted knapweed. In spotted knapweed, cnicin occurs in the aerial tissues with the highest concentrations found in the leaves, where it is present in glandular trichomes on the epidermal surfaces (Locken and Kelsey, 1987). Cnicin has phytotoxic (Kelsey and Locken, 1987), anti-bacterial (Bruno et al., 2003), and anti-fungal properties (Skaltsa et al, 2000; Panagouleas et al., 2003). 1.4 Arbuscular mycorrhizal fungi The soil surrounding plant roots is generally referred to as the "rhizosphere"; it is a highly active zone where microorganisms interact with plant roots and soil constituents (Azcon-Aguilar and Barea, 1996). One of the most common groups of microorganisms in the rhizosphere is mycorrhizal fungi; approximately 90% of terrestrial plants form symbiotic associations with mycorrhizal fungi (Graham and Miller, 2005). The mycorrhizal association is a mutualistic interaction involving the transfer of carbon from the host plant to the fungus, and mineral nutrients from the fungus to the host plant (Graham and Miller, 2005). Mycorrhizal fungi can be grouped into two main types: ectomycorrhizal and endomycorrhizal fungi. Ectomycorrhizal fungi are characterized by dense mycelial sheaths around the roots, and intercellular hyphal invasion of the root cortex (Bolan, 1991). Endomycorrhizal fungi are characterized by fungi that form external hyphal networks in the soil and grow extensively within the cells of the cortex 10 (Bolan, 1991). There are several types of endomycorrhizal fungi including ericoid, arbutoid, orchid, and arbuscular mycorrhizal (AM) fungi.. A M fungi are the most common type of mycorrhizal fungi as they are associated with more than 80% of all terrestrial plant species, including angiosperms, gymnosperms, pteridophytes, lycophytes, and bryophytes (Bonfante and Perotto, 1995; Hause and Fester, 2005). The fungal partners involved in A M fungal symbiosis are obligate biotrophs. These fungi reproduce asexually as no known sexual form of the fungi has been found (Hause and Fester, 2005). There are approximately 150 different species of A M fungi and all are members of the order Glomales (Harrison, 1999). The A M fungi symbiosis is a mutualistic relationship, where both symbionts benefit from bidirectional nutrient exchange (Ferrol et al, 2002). The plant provides organic carbon derived from photosynthesis for the fungus; carbon is essential to the formation and functioning of A M fungi and completion of the fungal lifecycle. As a result, A M fungi can cause a carbohydrate drain of up to 20% of total photoassimilate production from the host plant (Ferrol et al, 2002). The host plant can tolerate this carbohydrate drain because A M fungi improve the plant's access to limiting soil resources by transferring mineral nutrients to the host plant. Along with increased access to mineral nutrients, A M fungi provide several other benefits to the host plant, including improved water uptake (Marulanda et al, 2003), tolerance to soil toxins (Joner et al, 2000; Khan et al, 2000), and resistance to pathogens (Borowicz, 2001) and herbivores (Gange and West, 1994). As a result, A M fungi are essential for sustained growth and competitive ability of many plants (van der Heijden et al, 1998). They are important components of natural ecosystems, influencing plant community structure, productivity, 11 and succession (Bever et al, 2001). Altering A M fungi communities via allelopathic inhibition may be an effective strategy utilized by exotic weeds to interfere with neighboring species. 1.4.1 Glomus intraradices Shenck & Smith Glomus intraradices is an A M fungus, widely used as a soil inoculant in agriculture and horticulture, and in scientific research involving A M fungi. Unlike most arbusuclar mycorrhizal species that produce spores externally, G. intraradices forms yellow or brown spores inside the host root (Maia and Kimbrough, 1994). Germination of G. intraradices spores occurs when the germ tube emerges from the subtending hyphae (Figure 3.1) (Maia and Kimbrough, 1994). 12 1.5 Literature Cited Azcon-Aguilar, C. and J .M. Barea. 1996. Arbuscular mycorrhizas and biological control of soil-borne plant pathogens - an overview of the mechanisms involved. Mycorrhiza 6:457-464. Bever, J.D., P.A. Schultz, A . Pringle and J.B. Morton. 2001. Arbuscular mycorrhizal fungi: more diverse than meets the eye, and the ecological tale of why. Bioscience 51:923-932. Bais, H.P., T.S. Walker, F.R. Stermitz, R.A. Hufbauer and J .M. Vivanco. 2002. Enantiomeric-dependent phytotoxic and antimicrobial activity of (±)-catechin. A rhizosecreted racemic mixture from spotted knapweed. Plant Physiology 128:1173-1179. Bais, H.P. R. Vepachedu, S. Gilroy, R . M . Callaway and J .M. Vivanco. 2003. Allelopathy and exotic plant invasion: From molecules and genes to species interactions. Science 301:1377-1380. Bialy, Z., W. Oleszek, J. Lewis and G.R. Fenwick. 1990. Allelopathic potential of glucosinolates (mustard oil glycosides) and their degradation products against wheat. Plant and Soil 129:277-281. Blicker, P.S., B.E. Olson and R. Engel. 2002. Traits of the invasive Centaurea maculosa and two native grasses: effect of N supply. Plant and Soil 247:261-269 Bolan, N.S. 1991. 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The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5-51. Fenwick, G.R., R.K. Heaney, W.J. Mullin. 1983. Glucosinolates and their breakdown products in food and food plants. Critical Reviews in Food Science and Nutrition 18:123-301. Ferrol, N . , J .M. Barea and C. Azcon-Aguilar. 2002. Mechanisms of nutrient transport across interfaces in arbuscular mycorrhizas. Plant and Soil 244: 231-237. Gange, A .C . and H . M . West. 1994. Interactions between arbuscular mycorrhizal fungi and foliar-feeding insects in Plantago lanceolata L. New Phytologist 128:79-87. Graham, J.H. and R . M . Miller. 2005. Mycorrhizas: Gene to function. Plant and Soil 274: 79-100. Harley, J.L. and E.L. Harley. 1987. A check-list of mycorrhiza in the British flora. New Phytologist 105:1-102. Harrison, M.J. 1999. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annual Review of Plant Physiology 50: 361-89. Hause, B. and T. Fester. 2005. Molecular and cell biology of arbuscular mycorrhizal symbiosis. Planta221: 184-196. Herron, G.J., R.L. Sheley, B.D. Maxwell and J.S. Jacobsen. 2001. Influence of nutrient availability on the interaction between spotted knapweed and bluebunch wheatgrass. Restoration Ecology 9:326-331. Inderjit and E.T. Nilsen. 2003. Bioassays and field studies for allelopathy in terrestrial plants: progress and problems. Critical Reviews in Plant Sciences 22:221-238. 15 Inderjit and R . M . Callaway. 2003. Experimental designs for the study of allelopathy. Plant and Soil 256:1-11. Jacobs, J.S., R.L. Sheley and B.D. Maxwell. 1996. Effect of Sclerotinia sclerotiorum on the interference between Bluebunch wheatgrass (Agropyron spicatum) and spotted knapweed (Centaurea maculosa). Weed Technology 10:13-21. Joner, E.J., R. Briones and C. Leyval. 2000. Metal-binding capacity of arbuscular mycorrhizal mycelium. Plant and Soil 226:227-234. Kahn, A .G . , T .M. Kuek, T .M. Chaudry, C S . Khoo and W.J. Hayes. 2000. Role of plants, mycorrhizae and phytochelators in heavy metal contaminated land remediation. Chemosphere 41:197-207. Kelsey, R.G. and L.J . Locken. 1987. Phytotoxic properties of cnicin, a sesquiterpene lactone from Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 13:19-33. Kiddle, G., R.N. Bennett, N.P. Botting, N.E. Davidson, A . A . B . Robertson and R . M . Wallsgrove. 2001. High-performance liquid chromatographic separation of natural and synthetic desulfoglucosinolates and their validation by U V , N M R and chemical ionization-MS methods. Phytochemical Analysis 12:226-242. Kobayashi, K. 2004. Factors affecting phytotoxic activity of allelochemicals in soil. Weed Biology and Management 4:1 -7. Lacey, J.P., P. Husby and G. Handl. 1990. Observations on spotted and diffuse knapweed invasion into ungrazed bunchgrass communities in western Montana. Rangelands 12: 30-33. 16 Landau, I. H . Muller-Scharer and P.I. Ward. 1993. Influence of cnicin, a sesquiterpene lactone of Centaurea maculosa (Asteraceae), on specialist and generalist insect herbivores. Journal of Chemical Ecology 20:929-942. Levine, J .M., M . Vila, C M . D'Antonio, J.S. Dukes, K . Grigulis and S. Lavorel. 2003. Mechanisms underlying the impacts of exotic plant invasions. Proceedings of the Royal Society B: Biological Sciences 270:775-781. Locken, L.J. and R.G. Kelsey. Cnicin concentrations in Centaurea maculosa, spotted knapweed. Biochemical Systematics and Ecology 15:313-320: Lonsdale, W . M . 1999. Global patterns of plant invasions and the concept of invisibility. Ecology 80:1522-1536. Maia, L.C. and J.W. Kimbrough. 1994. Ultrastructural studies on spores of Glomus intraradices. International Journal of Plant Science 155:689-698. Marulanda, A. , R. Azcon and J .M. Ruiz-Lozano. 2003. Contribution of six arbuscular mycorrhizal fungal isolates to water uptake by Lactuca sativa plants under drought stress. Physiologia Plantarum 119:526-533. May, K.W., W.D. Willms, Z. Mengli and T.J. Lysyk. 2004. A n assessment of variation in Idaho fescue [Festuca idahoensis (Elmer)] in southern Alberta. Canadian Journal of Plant Science 84:1077-1084. -Morra, M.J. and J.A. Kirkegaard. 2002. Isothiocyanate release from soil-incorporated Brassica tissues. Soil Biology and Biochemistry 34:1683-1690. Mummey, D.L- and M.C . Rillig. 2006. The invasive plant species Centaurea maculosa alters arbuscular mycorrhizal fungal communities in the field. Plant and Soil 288:81-90. 17 Olson, B.E. and R.T. Wallander. 2002. Effects of invasive forb litter on seed germination, seedling growth and survival. Basic and Applied Ecology 3:309-317 Panagouleas, C , H . Skaltsa, D. Lazari, A . L . Skaltsounis and M . Sokovic. 2003. Antifungal activity of secondary metabolites of Centaurea raphanina ssp. mixta, growing wild in Greece. Pharmaceutical Biology 14:266-270. Parish, R., R. Coupe and D. Lloyd (eds). 1996. Plants of Southern Interior British Columbia and the Inland Northwest. Vancouver: B.C. Ministry of Forests and Lone Pine Publishing, 461 p. Pimentel, D., R. Zuniga and D. Morrison. 2005. Update on the environmental and economic costs associated with alien-invasive species in the United States. Ecological Economics 52:273-288. Ridenour, W . M . and R . M . Callaway. 2001. The relative importance of allelopathy in interference: the effects of an invasive weed on a native bunchgrass. Oecologia 126:444-450. Sheley, R.L., J.S. Jacobs and M.F. Carpinelli. 1998. Distribution, biology, and management of diffuse knapweed (Centaurea diffusa) and spotted knapweed (Centaurea maculosa). Weed Technology 12:353-362. Skaltsa, H. , Lazari, D., C. Panagouleas, E. Georgiadou, B. Garcia and M . Sokovic. 2000. Sesquiterpene lactones from Centaurea thessala and Centaurea attica. Antifungal activity. Phytochemistry 55:903-908. Stinson, K .A . , S.A. Campbell, J.R. Powell, B.E. Wolfe, R . M . Callaway, G.C. Thelen, S.G. Hallett, D. Prati and J.N. Klironomos. 2006. Invasive plant suppresses the 18 growth of native tree seedlings by disrupting belowground mutualisms. Public Library of Science 4:727-731. Thelen, G.C., J .M. Vivanco, B. Newingham, W. Good, H.P. Bais, P. Landres, A . Caesar and R . M . Callaway. 2005. Insect herbivory stimulates allelopathic exudation by an invasive plant and the suppression of natives. Ecology Letters 8:209-217. van der Heijden, M.G.A.', J.N. Klironomos, M . Ursic, P. Moutoglis, R. Streitwolf-Engel, T. Boiler, A . Wiemken and I.R. Sanders. 1998. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396:69-72. Vaughn, S.F. and R.A. Boydston. 1997. Volatile allelochemicals released by crucifer green manures. Journal of Chemical Ecology 23:2107-2116. Veluri, R., T.L. Weir, H.P. Bais, F.R. Stermitz and J.M. Vivanco. 2004. Phytotoxic and antimicrobial activities of catechin derivatives. Journal of Agricultural and Food Chemistry 52:1077-1082. Walling, S.Z. and C.A. Zabinski. 2006. Defoliation effects on arbuscular mycorrhizae and plant growth of two native bunchgrasses and an invasive forb. Applied Soil Ecology 32:111-117. Warwick, S.I., A . Francis and G.A. Mulligan. 2003. Brassicaceae of Canada. Online Database. U R L : http://www.cbif.gc.ca/sppjpages/brass/index_e.php Weir, T.L., H.P. Bais and J .M. Vivanco. 2003. Intraspecific and interspecific interactions mediated by a phytotoxin, (-)-catechin, secreted by the roots of Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 29:2397-2412. 19 Wolfe, B.E. and J.N. Klironomos. 2005. Breaking new ground: soil communities and exotic plant invasion. Bioscience 55:477-488. - 20 Chapter 2. Role of Glucosinolate Degradation Products in the Allelopathic Potential of Tall Hedge Mustard (Sisymbrium loeselii) 2.1 Introduction The success of many exotic weeds has been attributed to a chemical-mediated plant-plant interaction known as allelopathy (Inderjit and Nilsen, 2003). Allelopathy is generally defined as the negative effect of chemicals released by one plant on the growth and distribution of other plants (Inderjit and Callaway, 2003). Allelochemicals consist of a wide variety of secondary metabolites that may be produced and released by plants into the environment in a variety of ways. These include leaching and volatilization from plant foliage, exudation from plant roots, and release upon decomposition or volatilization from plant litter (Kobayashi, 2004; Reigosa et al, 1999; Inderjit and Duke, 2003). Allelopathy is suspected to be one of the primary mechanisms used by many exotic mustard species to invade plant communities and form dense monocultures (Weston and Duke, 2003). Black mustard (Brassica nigra [L.] Koch.), an exotic member of the annual grassland vegetation of coastal California, uses allelopathy as one of the primary mechanisms to invade and form pure stands in these regions (Bell and Muller, 1973). It was determined that black mustard forms pure stands because of the inhibitory activity of water-soluble compounds leached from its dead stems or leaves (Bell and Muller 1973). The success of garlic mustard [Alliaria petiolata (Bieb.) Cavara & Grande], an invader of North American forests, may also be attributed to allelopathy as its root exudates reduced the germination of two perennial woodland herbs (Prati and Bossdorf, 2004). In addition, Vaughn and Berhow (1999) isolated several phytotoxic . chemicals from garlic mustard tissues that were inhibitory to seedling growth of two 21 different species. Another invasive mustard species, hoary cress (Cardaria draba L.), invades cultivated fields and rangelands. Root extracts of hoary cress contained phytochemicals that inhibit germination and seedling growth of several species (Kiemnec and Mclnnis, 2002). The allelopathic properties of these mustard plants and most members of the Brassicaceae are due to the presence of glucosinolates (GSLs). GSLs are a class of secondary metabolites found in eleven different plant families, but most commonly associated with the Brassicaceae (Brown et al., 1994). There have been at least 120 different GSLs identified (Fahey et al, 2001), and these can be distinguished from one another by different side-groups (Gardiner et al., 1999). Allelopathic properties associated with GSLs arise from their biologically active degradation products, such as isothiocyanates (ITCs), organic cyanides, oxazolidinethiones, and ionic thiocyanate which are released upon enzymatic degradation by myrosinase (thioglucosidase; EC 3.2.3.1.) (Brown and Morra, 1997). The formation of GSL degradation products depends on the side-group of the parent GSL and on reaction conditions such as pH, presence of Fe 2 + , and epithiospecifier proteins (Borek et al., 1995; Fahey et al., 2001). ITCs are the most common of the diverse breakdown products of GSLs (Bialy et al., 1990). They are potentially toxic compounds that tend to be volatile and are produced in high proportions at neutral pH (Gardiner et al., 1999). Several studies have identified the allelopathic properties of GSL degradation products produced by mustard plants. For example, turnip-rape mulch released high amounts of ITCs that were thought to be responsible for the suppression of weed germination after incorporation of green turnip-rape mulch into soil (Petersen et al., 2001). It was determined that low ITC concentrations can induce secondary seed 22 dormancy, and higher ITC concentrations can penetrate seeds and react with enzymes, causing the seeds to lose their viability as the reaction with the enzymes is irreversible (Petersen et al., 2001). Vaughn and Berhow (1999) found that dichloromethane extracts of garlic mustard tissues contained several phytotoxic hydrolysis products of GSLs, including allyl ITC and benzyl ITC. Using commercially available sources of these ITCs and their parent GSLs, it was determined that the ITCs were much more phytotoxic than their respective parent GSLs (Vaughn and Berhow, 1999). Tall hedge mustard (Sisymbrium loeselii L.), a member of the Brassicaceae, has become naturalized across North America. In Canada, this exotic weed is found across the country from Quebec westward in dry cultivated fields, prairies, rangelands, roadsides, and wasteplaces (Warwick et al., 2003). Tall hedge mustard is commonly found in southern British Columbia where it often appears like a yellow blanket covering fields and roadsides in early summer (Parish et al, 1996). Similar to other weedy members of the Brassicaceae family, tall hedge mustard often forms relatively pure stands (Figure 2.1) when well established. The overall goal of this study was to advance our understanding of the allelopathic potential of tall hedge mustard and the role of GSLs and their degradation products in the allelopathic influence of this weed. The specific objectives of the research described in this chapter were to (1) determine if aqueous tall hedge mustard extracts have an inhibitory effect on seed germination and seedling growth, (2) analyze the GSL content and identify the GSL degradation products of tall hedge mustard, and (3) determine if the GSL degradation products have an inhibitory effect on seed germination and seedling growth. 23 Figure 2.1. Dense stand of tall hedge mustard in southern British Columbia 2.2 Materials & Methods 2.2.1 Seed sources Bluebunch wheatgrass (Pseudoroegneria spicata [Pursh.] Love), Idaho fescue (Festuca idahoensis Elmer.), and spotted knapweed (Centaurea maculosa Lam.) are commonly found in southern British Columbia growing in similar regions as tall hedge mustard (Sisymbrium loeselii L.). Bluebunch wheatgrass seeds were obtained from Dawson Seed Company (Surrey, BC), and Idaho fescue seeds from Grassland West Seed Company (Clarkston, WA). Spotted knapweed and tall hedge mustard seeds were collected from natural populations in southern British Columbia. 2.2.2 Tall hedge mustard extracts and whole plant leachate Aqueous root and shoot extracts were prepared from tall hedge mustard plants collected in the field to determine if this weed contains allelochemicals inhibitory to the germination and growth of neighboring species. An aqueous whole plant leachate was also prepared from intact tall hedge mustard tissues to represent a more natural release of allelochemicals from tall hedge mustard tissues. 2.2.2.1 Aqueous extracts and leachate preparation Tall hedge mustard root and shoot (rosette leaves, stems, stem leaves, flowers, and seed pods) tissues were collected from natural populations in southern British Columbia on July 12, 2006. The plant tissues were air-dried at room temperature (22 to 26°C) for 5 d. Dried tissues were ground in a blender and stored in a freezer at -24°C 25 until they were used. Extracts of ground root and shoot tissues were prepared by incubating in distilled water. (4% w/v) on a rotary shaker (90 rpm) for 24 h. The whole plant leachate was prepared by incubating intact plants in distilled water (4% w/v) for 24 h without shaking. The incubation mediums were centrifuged at 1,015 g for 10 min and filter-sterilized using a surfactant-free cellulose acetate filter (0.45 um pores) (Nalgene, Rochester, NY) . These full strength solutions (4% w/v) were diluted with distilled water to prepare 1% and 2% solutions. 2.2.2.2 Seed germination bioassay Seeds were surface sterilized in 0.5% sodium hypochlorite (v/v in distilled water) for 10 min and rinsed several times with sterile distilled water. Fifteen bluebunch wheatgrass, Idaho fescue and tall hedge mustard seeds, and.ten spotted knapweed seeds were placed in 60-mm petri dishes lined with two Whatman No. 1 filter discs. Each petri dish received 3 ml of distilled water (control), or 1, 2 or 4% root extract, shoot extract, or whole plant leachate and sealed with parafilm. Petri dishes were incubated in darkness at 23°C. Percent seed germination was recorded after 10 d. The criterion for germination was a radicle length of ~4 mm. Petri dishes were arranged in a completely randomized design with four replicates for each treatment and species, and the experiment was repeated/ 2.2.2.3 Seedling growth bioassay Surface sterilized bluebunch wheatgrass, Idaho fescue, and spotted knapweed seeds were germinated by incubating in darkness at 23°C in 90-mm petri dishes lined 26 with filter discs (Whatman No. 1) wetted with 5 ml of distilled water. Six germinated seeds with -3-4 mm long radicles were transferred to 60-mm petri dishes lined with two Whatman No. 1 filter discs. Due to the thin and fragile radicles of tall hedge mustard, 10 seeds of this species were germinated directly in the 60-mm petri dishes lined with two Whatman No. 1 filter discs wetted with 1 ml of distilled water to minimize their handling. After 3 d, the number of seedlings was reduced to six per petri dish. Each petri dish received 3 ml of distilled water (control), or 1, 2, or 4% root extract, shoot extract, or whole plant leachate and sealed with parafilm. Petri dishes were incubated at room temperature (22 to 26°C) for 5 d. After 5 d, the seedling radicle length, and coleoptile or hypocotyl length were measured to the nearest millimeter using a ruler. Petri dishes were arranged in a completely randomized design with three replicates for each treatment and species, and the experiment was repeated. 2.2.2.4 Soil bioassay The effect of tall hedge mustard extracts and leachate on seed germination and seedling growth in soil were investigated because the effect of chemicals in soil can be altered compared to filter discs. Field soil was collected from a site in southern British Columbia occupied primarily by grasses, including bluebunch wheatgrass. The soil was collected to a depth of 10 cm, sieved (2-mm) to remove any large roots and debris, and analyzed to determine the texture, pH, and organic matter content (Martin Hilmer, Soil Science, University of British Columbia, pers. comm.). The soil had a loamy sand texture, pH 6.2, and contained 4% organic matter. Ten surface sterilized seeds of bluebunch wheatgrass, Idaho fescue, spotted knapweed, and tall hedge mustard were 27 placed in 60-mm petri dishes containing 15 g of field soil. Each petri dish received 5 ml of distilled water (control), or 4% root extract, shoot extract, or whole plant leachate and sealed with parafilm. Petri dishes were incubated at room temperature (22 to 26°C) for 10 d. After 10 d, the number of germinated seeds and seedling growth (radicle length, and coleoptile or hypocotyl length) was recorded. The criterion for germination was a radicle length of ~4 mm. Petri dishes were arranged in a completely randomized design with each treatment and species replicated four times and the experiment was repeated. 2.2.3 Glucosinolate analysis Tall hedge mustard root and shoot (rosette leaves, stems, stem leaves, flowers, and seed pods) tissues were collected from natural populations in southern British Columbia on July 12, 2006. For each sample, 1.4 ml of methanol was added to 0.05 g of dried plant material to inactivate the myrosinase enzyme and prevent GSL degradation. Al ly l (2-propenyl) GSL (Sigma Aldrich, Oakville, ON) was added (50 pi of 1 mM) to the samples prior to GSL extraction as the standard. Each sample received 50 pi of 0.3 M lead-barium acetate and incubated on a reciprocal shaker for 45 min before being centrifuged at 1,133 g for 10 min. The supernatant was removed and added to columns containing D E A E Sephadex A25 (anion exchange resin) (GE Healthcare Bio-Sciences Ltd., Piscataway, NJ). Columns were rinsed twice with 0.9 ml of 67% methanol, and 0.9 ml of distilled water. Each column received 1 ml of 0.02 M sodium acetate buffer (pH 5.2 - 5.5), and desulfated with 50 pi sulfatase (Sigma Aldrich, Oakville, ON) for 16 h. This step removes the negatively charged sulfate group leaving uncharged desulfo-GSL molecules. Desulfo-GSLs could then be eluted from the columns into vials by rinsing 28 twice with 0.8 ml of 60% methanol. Contents of the vials were dried under gentle air flow at 40°C, and dried contents were re-dissolved in 1 ml distilled water for high performance liquid chromatography (HPLC). An Agilent 1100 Series H P L C (Agilent Technologies Inc., Santa Clara, CA) equipped with an autosampler and diode array detector was used in combination with an ODS Hypersil (Keystone Scientific Inc., Bellfonte, PA) column (reverse phase CI8, 4.6 x 250 mm, 5 pm particle size). The desulfo-GSL peaks were detected with the diode array detector set at 229 nm. A n elution gradient was used starting at 2% methanol - 98% distilled water and ending with 5% methanol - 89% acetonitrile (6% distilled water). The run was 47 min, with 34 min for chromatography and 13 min for cleanup and equilibration. Identification of desulfo-GSLs was based on retention time, U V spectra (Figure 2.2), and mass spectrometry of GSL degradation products. 2.2.4 Glucosinolate degradation product analysis Dried root and shoot tissue samples (0.5 g) of tall hedge mustard were wetted with 3.0 ml distilled water and incubated for 15 min to allow GSL degradation. GSL degradation products were extracted by shaking for 15 min with dichloromethane. The samples were centrifuged at 1,133 g for 5 min, and the dichloromethane layer removed and dried with 0.4 g Na2S04 for lh . The root and shoot samples were filtered through a 0.45 pm PTFE syringe filter (Gelman, Ann Arbor, MI) and analyzed by gas chromatography-mass spectrometry (GC-MS). Samples were analyzed using a Hewlett Packard 5890 Series II equipped with a 5971 mass selective detector (Agilent Technologies Inc., Santa Clara, CA) , in combination with a 30 m x 0.25 mm 5% phenyl 29 Wavelength ' Figure 2.2. U V absorption spectra of desulfo-GSLs extracted from tall hedge mustard root and shoot tissues: (a) isopropyl GSL, (b) 4-hydroxy-3-indolylmethyl GSL, (c) .sec-butyl GSL, (d) 3-indolylmethyl GSL, (e) l-methoxy-3-indolylmethyl GSL, and (f) 4-methoxy-3-indolylmethyl GSL. 30 substituted methylpolysiloxane column (Phenomenex, Torrance, CA). Conditions for GSL degradation product analysis included an injection volume of 1 ul, injector temperature of 200°C, and splitless injection for 0.6 min. The detector temperature was 260°C, with the initial temperature set at 36°C, ramped to 96°C at 12°C per minute, then at 18°C per minute to 240°C and held for 6 minutes. Total run time was 22 minutes. Identification of GSL degradation products was based on mass spectral data. 2.2.5 Phytotoxicity of glucosinolate degradation products In order to determine if the GSL degradation products found in the root and shoot extracts of tall hedge mustard exhibit phytotoxic or allelopathic properties, seed germination and seedling growth were bioassayed using the commercially available compounds. 2.2.5.1 Isothiocyanates - seed germination bioassay Isopropyl ITC (Sigma Aldrich, Oakville, ON) and sec-butyl ITC (Fisher Scientific, Ottawa, ON) solutions (0.01, 0.1, 0.5, 1.0 mM) were prepared. The chemicals were first dissolved in methanol and diluted to the appropriate concentrations with distilled water. The final concentration of methanol was 0.5% for all solutions including the control. Fifteen surface sterilized bluebunch wheatgrass, Idaho fescue and tall hedge mustard seeds, and ten spotted knapweed seeds were placed in 60-mm petri dishes lined with two Whatman No. 1 filter discs. Each petri dish received 3 ml of distilled water (control), or isopropyl ITC (0.01, 0.1, 0.5, 1.0 mM), or sec-butyl ITC (0.01, 0.1, 0.5, 1.0 mM) solutions and sealed with parafilm. Petri dishes were incubated in darkness at 23°C 31 and percent seed germination recorded after 10 d. The criterion for germination was a radicle length of ~4mm. Petri dishes were arranged in a completely randomized design with four replicates for each treatment and species, and the experiment was repeated. 2.2.5.2 Isothiocyanates - seedling growth bioassay Surface sterilized bluebunch wheatgrass, Idaho fescue, and spotted knapweed seeds were germinated by incubating in darkness at 23°C in 90-mm petri dishes lined with Whatman No. 1 filter discs wetted with 5 ml distilled water. Six germinated seeds with -3-4 mm long radicles were transferred to 60-mm petri dishes lined with two Whatman No. 1 filter discs. Due to the thin and fragile radicles of tall hedge mustard, 10 seeds of this species were germinated directly in the 60-mm petri dishes lined with two Whatman no. 1 filter discs wetted with 1 ml distilled water. After 3 d, the number of tall hedge mustard seedlings was reduced to six per petri dish. Each petri dish received 3 ml of distilled water (control), or isopropyl ITC (0.01, 0.1, 0.5, 1.0 mM), or sec-butyl ITC (0.01, 0.1, 0.5, 1.0 mM) solutions and sealed with parafilm. Petri dishes were incubated at room temperature (22 to 26°C) for 5 d. After 5 d, the seedling radicle length, and coleoptile or hypocotyl length were measured to the nearest millimeter using a ruler. Petri dishes were arranged in a completely randomized design with three replicates for each treatment and species, and the experiment was repeated. 2.2.6 Data Analysis Seed germination data were arcsine-transformed prior to analysis. The seedling growth data (2.2.2.3) and soil bioassay data (2.2.2.4) were subjected to analysis of 32 variance (ANOVA) and treatment means separated from the control by Dunnett's test (P = 0.05) using SPSS 15.0 (SPSS Inc., Chicago, IL). Seed germination and seedling growth data from ITC dose-response experiments (2.2.5.2 and 2.2.5.3) were subjected to regression analysis using Sigma Plot 10 (Systat Software, Inc., San Jose, CA). Data from repeated experiments were pooled when there was no significant (P = 0.05) experiment by treatment interaction; otherwise the data from both experiments are presented. 2.3 Results 2.3.1 Effect of aqueous extracts and leachate on seed germination Both of tall hedge mustard aqueous root and shoot extracts strongly inhibited the germination of bluebunch wheatgrass, Idaho fescue and spotted knapweed seeds, but not that of tall hedge mustard seeds compared to the control (Figure 2.3). The shoot extract had a much stronger inhibitory effect compared to the root extract at all concentrations for all species tested except tall hedge mustard. At the highest concentration (4% w/v), the shoot extract reduced seed germination of bluebunch wheatgrass by 95.2%, Idaho fescue by 97.6%, spotted knapweed by 90.6%, and tall hedge mustard by 10.5% compared to the control, whereas the root extract only reduced germination by 72.4%, 67.1%o, 47.7%o, and 0.0%, respectively. Tall hedge mustard whole plant leachate exhibited a similar inhibitory effect on seed germination as the shoot extract. 33 Concentration (% w/v) Concentration (% w/v) Figure 2.3. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on germination of (A) bluebunch wheatgrass, (B) Idaho fescue, (C) spotted knapweed, and (D) tall hedge mustard. Values are means ± standard error of two experiments of four replicates. 2.3.2 Effect of aqueous extracts and leachate on seedling growth The results from both experiments (Table 2.1a and Table 2.1b) are presented because there was a significant (P = 0.05) experiment by treatment interaction. However, the overall trends are quite similar and the remainder of the discussion will focus on the first experiment (Table 2.1a). Tall hedge mustard aqueous root and shoot extracts had a stronger inhibitory effect on radicle length compared to coleoptile or hypocotyl length. The root extract did not have a significant effect on coleoptile growth of bluebunch wheatgrass and Idaho fescue, or spotted knapweed hypocotyl growth, but increased the hypocotyl growth of tall hedge mustard seedlings. Only the 4% shoot extract had a significant inhibitory effect on coleoptile or hypocotyl length, reducing bluebunch wheatgrass coleoptile length by 56.5% and spotted knapweed hypocotyl length by 51.0% compared to the control. The shoot extract exhibited a stronger inhibitory effect on radicle growth of all species tested compared to the root extract. The shoot extracts strongly inhibited radicle growth for all species tested. The 4% shoot extract inhibited radicle length of bluebunch wheatgrass by 82.3%, Idaho fescue by 80.0%, spotted knapweed by 70.8%, and tall hedge mustard by 69.3% compared to the control. On the other hand, 4% root extract inhibited the radicle length of bluebunch wheatgrass by 30.2%, Idaho fescue by 59.2%, and spotted knapweed by 39.2% compared to the control, but the lowest concentration (1%) had no effect. Tall hedge mustard radicle length was increased by 1% (18.2%), 2% (59.6%o), and 4% (66.7%) root extract compared to the control. Tall hedge mustard whole plant leachate had a similar effect on radicle and coleoptile or hypocotyl length of all species tested as the shoot extract. 35 Table 2.1a. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in experiment #1 Treatment Bluebunch wheatgrass Idaho fescue Spotted knapweed Tall hedge mustard [w/v] Radicle3 Coleoptile2 Radicle3 Coleoptile3 Radicle3 Hypocotyl3 Radicle3 Hypocotyl3 Root extract 1% 96.5 ±5.4 104.5 ±5.9 92.0 ±8.5 111.1 ± 12.0 106.1 ±7.8 119.8 ±6.3 118.2 ±9.3 147.1 ±7.2* 2% 79.2 ±6.5* 87.1 ±6.8 79.6 ±9.0 85.8 ± 16.1 91.5 ±9.3 117.9 ±5.3 159.6 ±9.6* 150.2 ±8.3* 4% 69.8 ± 6.4* 81.2 ± 10.6 40.8 ±5.8* 64.3 ± 12.3 60.8 ±5.8* 96.2 ± 8.5 166.7 ±10.8* 148.6 ±8.5* Shoot extract 1% 77.2 ±5.4* 93.7 ± 11.6 83.7 ±8.4 108.4 ± 14.6 65.9 ±5.6* 91.2 ±6.9 113.5 ±5.7 159.2 ±7.7* 2% 27.8 ± 2.9* 88.4 ± 10.9 23.6 ± 1.7* 86.6 ± 15.7 40.2 ±3.0* 79.8 ±5.0 55.6 ±4.6* 150.9 ±7.7* 4% 17.7 ±2.1* 43.5 ±9.6* 20.0 ± 1.8* 70.6 ± 13.6 29.2 ±2.6* 49.0 ± 6.4* 30.7 ±2.6* 110.4 ±6.2 Whole Plant 1% 94.2 ± 4.2 125.2 ±7.8 71.8 ±8.8* 105.9 ± 16,9 52.9 ± 5.4* 95.9 ±8.3 150.1 ±9.8* 153.2 ±4.8* leachate 2% 21.8 ±2.2* 81.6 ± 11.2 25.0 ±2.3* 76.1 ± 15.3 37.6 ±4.5* 93.4 ±6.8 48.5 ±5.1* 144.9 ±6.1* 4% 20.2 ±2.0* 76.2 ± 10.3 19.1 ± 1.3* 72.1 ±11.0 25.9 ±2.2* 41.3 ±4.5* 24.8 ± 2.6* 103.6 ±5.8 "Mean radicle, coleoptile, or hypocotyl length calculated as percentage of control ± standard error of one experiment of three replicates. *Means significantly different from control according to Dunnett's test (P = 0.05). Table 2.1b. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in experiment #2 Treatment [w/v] Bluebunch wheatgrass Idaho fescue Spotted knapweed Tall hedge mustard Radicle3 Coleoptile3 Radicle3 Coleoptile3 Radicle3 Hypocotyl3 Radicle3 Hypocotyl3 Root extract 1% 90.9 ± 6.9 105.2 ±8.2 91.4 ± 11.8 85.3 ± 11.3 112.0 ± 12.6 114.6 ±9.2 223.8 ± 15.0* 159.5 ±5.3* 2% 87.3 ± 7.5 99.1 ±11.8 69.0 ±7.8* 74.5 ± 9.2 . 92.8 ±8.1 114.0 ± 10.7 308.6 ± 22.4* 145.7 ±4.0* 4% 71.0 ±7.7* 87.4 ± 12.5 52.9 ±4.9* 80.5 ± 7.9 78.8 ±5.7 111.7 ±8.8 199.1 ± 19.5* 142.1 ±6.6* Shoot extract 1% 77.6 ±7.1 ' 90.8 ± 11.9 95.0 ±9.5 95.1 ±9.1 64.1 ±6.4* 92.6 ± 10.0 112.7 ±9.4 159.5 ±4.6* 2% 52.6 ±4.9* 92.9 ±9.7 53.2 ±6.8* 73.9 ±7.7 42.7 ±4.7* 90.0 ±6.7 49.4 ±4.8* 140.7 ±4.5* 4% 19.5 ± 1.9* 82.7 ± 12.1 28.7 ±3.8* 42.9 ±10.1* 34.1 ±3.3* 58.1 ±7.2* 32.4 ±2.5* 83.7 ±4.4 Whole Plant 1% 80.4 ±7.8 104.7 ±11.4 84.8 ± 10.1 106.7 ± 12.6 69.9 ± 6.5 106.3 ±5.8 191.4 ±14.4* 140.0 ±5.8* leachate 2% 38.4 ±3.6* 75.7 ±75.7 41.8 ±5.0* 83.6 ±12.9 37.5 ±4.7* 66.1 ±9.8 91.0 ±6.3 158.7 ±6.0* 4% 24.7 ± 2.4* 59.5 ± 9.8 24.8 ±3.2* 67.6 ± 9.7 34.1 ±3.1* 49.5 ± 5.4* 32.4 ±2.5* 79.4 ±5.5 "Mean radicle, coleoptile, or hypocotyl length calculated as percentage of control ± standard error of one experiment of three replicates. *Means significantly different from control according to Dunnett's test (P = 0.05). 2.3.3 Effect of aqueous extracts and leachate in soil Tall hedge mustard germination was not significantly affected by its root and shoot aqueous extracts or the whole plant leachate compared to the control (Table 2.2). The root extract inhibited the germination of bluebunch wheatgrass and Idaho fescue seeds, but not that of spotted knapweed. The shoot extract and whole plant leachate had a stronger inhibitory effect compared to the root extract on germination of bluebunch wheatgrass, Idaho fescue and spotted knapweed seeds. Overall, tall hedge mustard aqueous root and shoot extracts and whole plant leachate showed a similar but reduced inhibitory effect on seed germination in soil compared to bioassays that used filter disc lined petri dishes (2.3.1). The seedling growth results from both experiments (Table 2.3a and Table 2.3b) are presented because there was a significant (P = 0.05) experiment by treatment interaction. There are some notable difference between the experiments (i.e. whole plant leachate reduced the radicle length of Idaho fescue more than the shoot extract in experiment 2, but not in experiment 1); however, the overall trends are similar and the remainder of the discussion will focus on the first experiment (Table 2.3a). The radicle length of all species was significantly reduced by all treatment solutions. The tall hedge mustard shoot extract and whole plant leachate had a stronger inhibitory effect on seedling growth compared to the root extract on all species. For example, the radicle length of Idaho fescue was reduced 52.8% by the shoot extract, 52.5% by the whole plant leachate, but only 29.5% by the root extract compared to the control. The aqueous root and shoot extracts and whole plant leachate had a similar but slightly reduced effect on 38 Table 2.2. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seed germination in soil Treatment Germination (% of control)3 [w/v] Bluebunch wheatgrass Idaho fescue Spotted knapweed Tall hedge mustard Root extract Shoot extract Whole plant leachate 4 % 4 % 4 % 66.1 ±3 . 1 * 35.6 ± 6 . 2 * 40.7 ± 7.2* 54.8 ±9 . 1 * 26.2 ± 8 . 8 * 26.2 ± 6 . 2 * 83.6 ± 4 . 2 44.2 ± 6 . 5 * 57.3 ± 6 . 5 * 97.2 ± 2.5 86.3 ± 5.3 89.0 ± 3 . 8 "Mean seed germination calculated as percentage of control ± standard error of two experiments of four replicates. *Means significantly different from control according to Dunnett's test (P = 0.05) Table 2.3a. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in soil in experiment #1 Treatment Bluebunch wheatgrass Idaho fescue Spotted knapweed Tall hedge mustard [w/v] Radicle" Coleoptile3 Radicle8 Coleoptile3 Radicle3 Hypocotyl3 Radicle3 Hypocotyl1 Root extract 4% 81.3 ± 6 . 3 * 76.4 ± 5 . 7 * 70.5 ± 1 2 . 4 * Shoot extract 4% 55.8 ± 7 . 7 * 51.7 ± 5 . 2 * 47.2 ± 1 4 . 3 * Whole plant 4% 60.3 ± 7.3* 65.0 ± 5 . 5 * 47.5 ± 5.2* leachate "Mean radicle, coleoptile, or hypocotyl length calculated as percentage of control ± standard error of one experiment with four replicates per treatment. *Means significantly different from control according to Dunnett's test (P = 0.05). 76.2 ± 12.4 65.3 ± 4 . 4 * 85.5 ± J.S* 8 Z . 0 ± J . J T a/.i±z.<t-7 0 . 4 ± 1 3 . 9 21.2 ± 3.6* 6 0 . 6 ± 4 . 7 * 54.1 ± 2.7* 8 6 . 2 ± 2 . 4 * 72.2 ± 8 . 5 * 24.8 ± 3 . 3 * 61.0 ± 5 . 6 * 51.2 ± 2 . 6 * 82.5 ± 2.5* Table 2.3b. Effect of tall hedge mustard root extract, shoot extract, and whole plant leachate on seedling growth in soil in experiment #2 Treatment [w/v] Bluebunch wheatgrass Radicle3 Coleoptile3 Idaho fescue Spotted knapweed Radicle3 Coleoptile3 Radicle3 Hypocotyl3 Tall hedge mustard Radicle3 Hypocotyl3 Root extract Shoot extract Whole plant leachate 4% 80.4 ± 7 . 6 * 87.1 ± 9 . 1 4% 39.1 ± 9 . 6 * 51.1 ± 10.4* 4% 50.2 ± 5.7* 56.0 ± 8.8* 81.5 ± 10.0 79.0 ± 1 1 . 0 52.9 ± 8 . 5 * 65.4 ± 1 2 . 4 36.8 ± 1 1 . 2 * 50.6 ± 2 0 . 5 58.3 ± 4 . 2 * 81.5 ± 4 . 7 * 18.0 ± 2 . 6 * 55.1 ± 4 . 5 * 24.0 ± 4 . 1 * 70.7 ± 4 . 4 * 67.7 ± 7 . 5 * 84.0 ± 4 . 9 * 38.9 ± 4 . 9 * 55.0 ± 4 . 4 * 62.1 ± 4 . 4 * 71.4 ± 3 . 3 * "Mean radicle, coleoptile, or hypocotyl length calculated as percentage of control ± standard error of one experiment with four replicates per treatment. •Means significantly different from control according to Dunnett's test (P = 0.05). seedling growth in soil compared to bioassays that used filter disc lined petri dishes (2.3.2). 2.3.4 Glucosinolate analysis HPLC analysis of tall hedge mustard tissues revealed the presence of four GSLs in the shoots (Figure 2.4), and six GSLs in the roots (Figure 2.5). Isopropyl (1-methylethyl) GSL was found in the highest concentration in both the root and shoot tissues (Table 2.4). Tall hedge mustard shoot tissues had a higher total GSL content compared to the root tissues, primarily due to the high concentration of isopropyl GSL and significant quantities of sec-butyl (1-methylpropyl) GSL. Tall hedge mustard tissues also contained relatively low concentrations of four indolylic GSLs. 2.3.5 Glucosinolate degradation product analysis The major GSL degradation product found in aqueous extracts of tall hedge mustard shoots (Figure 2.6) and roots (Figure 2.7) was isopropyl ITC. A smaller peak identified as sec-butyl ITC was also observed in both extracts. 2.3.6 Effect of isothiocyanates on seed germination Isopropyl ITC and sec-butyl ITC significantly (P < 0.0001) inhibited seed germination of bluebunch wheatgrass, Idaho fescue, and spotted knapweed (Figure 2.8 and Figure 2.9). Isopropyl ITC had a significant (P < 0.0001) effect on seed germination of tall hedge mustard, but the inhibitory effect was much lower compared to bluebunch wheatgrass, Idaho fescue, and spotted knapweed. sec-Butyl ITC did not have a 41 b 1 e A . 10 20 - i — 30 40 Time (min) Figure 2.4. HPLC chromatogram of desulfo-GSLs extracted from tall hedge mustard shoot tissues collected from the field, (a) allyl GSL (standard), (b) isopropyl GSL, (c) 4-hydroxy-3-indolylmethyl GSL, (d) .vec-butyl GSL, and (e) 3-indolylmethyl GSL. g T — 1 1 r -10 20 30 40 Time (min) Figure 2.5. HPLC chromatogram of desulfo-GSLs extracted from tall hedge mustard root tissues collected from the field; (a) allyl GSL (standard),.(b) isopropyl GSL, (c) 4-hydroxy-3-indolylmethyl GSL, (d) sec-butyl GSL, (e) 3-indolylmethyl GSL, (f) 4-methoxy-3-indolylmethyl GSL, and (g) l-methoxy-3-indolylmethyl GSL. 42 Table 2.4 Glucosinolate content of tall hedge mustard root and shoot tissues Glucosinolate Retention time Root tissues Shoot tissues (min) (umol g"1)* (umol g"1)* Isopropyl 12.72 7.93 80.54 4-Hydroxy-3-indolylmethyl 16.36 0.37 2.82 sec-Butyl 17.08 0.51 4.82 3-Indolylmethyl 21.51 0.49 1.09 4-Methoxy-3 -indolylmethyl 23.55 0.41 0.00 1 -Methoxy-3 -indolylmethyl 26.69 3.76 0.00 Values are means (pmol g"1 dry weight) of four replicates. 43 a 1 ___JU—•J^-i-*^ ~5 10 15 20 Time (min) Figure 2.6. GC-MS chromatogram of dichloromethane fraction of tall hedge mustard aqueous shoot extract, (a) isopropyl ITC, and (b) sec-butyl ITC. a Time (min) Figure 2.7. GC-MS chromatogram of dichloromethane fraction of tall hedge mustard aqueous root extract, (a) isopropyl ITC, and (b) sec-butyl ITC. 44 100 0 J 1 1 r - 1 1 =!= 1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Isopropyl ITC (mM) Figure 2.8. Effect of isopropyl ITC on seed germination of (a) bluebunch wheatgrass (y = 80.7446"1 9 3 7 x , R 2 = 0.87, P < 0.0001), (b) Idaho fescue (y = 64.056e"3 0 5 3 7 x , R 2 = 0.89, P < 0.0001), (c) spotted knapweed (y = 78.651c" 1 5 1 0 x, R 2 = 0.86, P < 0.0001), and (d) tall hedge mustard (y = 93.578 + 6.392x - 22.364x2, R 2 = 0.51, P < 0.0001). Values are means ± standard error of two experiments of six replicates. o 'is c O 100 80 A 60 A 40 A 20 4 0 Bluebunch wheatgrass Idaho fescue Spotted knapweed Tall hedge mustard 0.0 0.2 0.4 0.6 0.8 sec-Butyl ITC (mM) 1.0 1.2 Figure 2.9. Effect of sec-butyl ITC on seed germination of (a) bluebunch wheatgrass (y = 83.001e_1 2 9 9 x , R 2 = 0.87, P < 0.0001), (b) Idaho fescue (y = 65.178 - 59.197x + 13.423x2, R 2 = 0.85, P < 0.0001), (c) spotted knapweed (y = 75.482e-' 2 1 5 x, R 2 = 0.77, P < 0.0001), and (d) tall hedge mustard (y = 95.520 + 2.474x -3.241x2, R 2 = 0.004, P = 0.93). Values are means ± standard error of two experiments of six replicates. as significant (P = 0.93) effect on tall hedge mustard seed germination. Isopropyl ITC had a slightly stronger inhibitory effect on seed germination compared to sec-butyl ITC. At the highest concentration (1.0 mM), isopropyl ITC reduced the germination of bluebunch wheatgrass by 89.1%, Idaho fescue by 95.2%, and spotted knapweed by 76.9%, whereas sec-butyl ITC only reduced the germination of these species by 71.3%, 72.3%, and 69.2% respectively. 2.3.7 Effect of isothiocyanates on seedling growth Isopropyl ITC and sec-butyl ITC significantly (P < 0.0001) reduced seedling growth of bluebunch wheatgrass, Idaho fescue, spotted knapweed, and tall hedge mustard Figure 2.10 - Figure 2.17). Differing from the tall hedge mustard aqueous root and shoot extracts, the ITCs had a strong inhibitory effect on coleoptile and hypocotyl growth of all species tested. 2.4 Discussion The results from this study indicate that tall hedge mustard produces allelochemicals that strongly inhibit seed germination and seedling growth in petri dishes. Tall hedge mustard root and shoot extracts strongly inhibited the germination of bluebunch wheatgrass, Idaho fescue, and spotted knapweed seeds, but had little effect on tall hedge mustard itself. The mechanism that tall hedge mustard uses to avoid inhibiting its own germination is not known, but this ability may explain the high densities that it can have in the field. A number of mechanisms have been suggested for the prevention of autotoxicity by allelochemicals, including resistance at the molecular target site, and 47 0.0 0.2 . 0.4 0.6 0.8 1.0 1.2 Isopropyl ITC (mM) Figure 2.10. Effect of isopropyl ITC on radicle (y = 24.602e"2331x, R2 = 0.64, P < 0.0001) and coleoptile (y = 47.096e"2 5 6 3 \ R2 = 0.71, P < 0.0001) growth of bluebunch wheatgrass. Values are means ± standard error of two experiments of six replicates. . •' . 0 J-, i , , • . 1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Isopropyl ITC (mM) Figure 2.12. Effect of isopropyl ITC on radicle (y = 24.204e2 2 2 0 x , R2 = 0.56, P < 0.0001) and hypocotyl (y = 15.539e' 6 8 5 x , R2 = 0.63, P < 0.0001) growth of spotted knapweed. Values are means ± standard error of two experiments of six replicates. 0.0 0.2 0.4 0.6 0.8, 1.0 1.2 Isopropyl ITC (mM) Figure 2.11. Effect of isopropyl ITC on radicle (y =30.963e"3 2 9 0 x , R2 = 0.67, P < 0.0001) and coleoptile (y = 28.513e"3 4 9 5 x , R2 = 0.60, P < 0.0001) growth of Idaho fescue. Values are means ± standard error of two experiments of six replicates. 0 J-, • . • , • 1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Isopropyl ITC (mM) Figure 2.13. Effect of isopropyl ITC on radicle (y = 4.164 - 6.516x + 3.640x2, R2 0.66, P < 0.0001) and hypocotyl (y = 6.257 - 5.673x + 2.899x2, R2 = 0.62, P < 0.0001) growth of tall hedge mustard. Values are means ± standard error of two experiments of six replicates. 50 40 So c <o -J 30 20 I 10 0 o Radicle • Coleoptile SI 0.0 0.2 0.4 0.6 0.8 1.0 1.2 sec-Butyl ITC (mM) Figure 2.14. Effect of sec-butyl ITC on radicle (y = 22.74e-L888x, R2 F= 0.67, P < 0.0001) and coleoptile (y = 45.75 - 53.29x + 16.65x2, R2 = 0.63, P < 0.0001) growth of bluebunch wheatgrass. Values are means ± standard error of two experiments of six replicates. 00 c <o 30 25 20 15 10 Radicle Hypocotyl . 0 , —-, , , , 1 0.0 0.2 0.4 0.6 0.8 1.0 1.2 sec-Butyl ITC (mM) Figure 2.16. Effect of sec-butyl ITC on radicle (y = 25.383e' 0 5 4 x , R2 = 0.46, P < 0.0001) and hypocotyl (y = 15.243e-°571x, R2 = 0.30, P < 0.0001) growth of spotted knapweed. Values are means ± standard error of two experiments of six replicates. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 sec-Butyl ITC (mM) Figure 2.15. Effect of sec-butyl ITC on radicle (y = 28.988e"2 2 4 9 x , R2 = 0.65, P < 0.0001) and coleoptile (y = 29.011 - 31.745x + 4.540x2, R2 = 0.63, P < 0.0001) growth of Idaho fescue. Values are means ± standard error of two experiments of six replicates. 00 c Radicle Hypocotyl 5 . 0.0 0.2 0.4 0.6 0.8 1.0 1.2 sec-Butyl ITC (mM) Figure 2.17. Effect of sec-butyl ITC on radicle (y = 4.089 - 4.189x + 1.537x2, R2 = 0.60, P < 0.0001) and hypocotyl (y = 6.310 - 4.949x + 2.315x2, R2 = 0.57, P < 0.0001) growth of tall hedge mustard. Values are means ± standard error of two experiments of six replicates. preventing the allelochemicals from encountering the molecular target site (Inderjit and Duke, 2003). In the seedling growth experiment, tall hedge mustard root and shoot extracts had a stronger effect on radicle length compared to coleoptile or hypocotyl length. In general, radicle growth tends to be more susceptible to phytotoxins or allelochemicals compared to coleoptile or hypocotyl growth (Dudai et al, 1999; Oueslati et al, 2005). The radicle length of all species including tall hedge mustard was significantly reduced by the 2% and 4% shoot extract. Overall, the shoot extract had a stronger inhibitory effect on seed germination and seedling growth compared to the root extract, which could be due to a greater concentration or variation of allelochemicals in tall hedge mustard shoot than root tissues. The aqueous extract and leachate seed germination and seedling growth experiments were done in petri dishes lined with filter discs. This type of experiment is valuable as it can evaluate whether certain compounds or extracts have an effect on germination or growth of certain species. However, it is important to determine if the effect seen in the filter discs bioassays also occurs in a more natural medium, such as soil. The results from the soil experiment showed that tall hedge mustard aqueous root and shoot extracts also inhibit the germination of bluebunch wheatgrass, Idaho fescue, and spotted knapweed seeds, but to a lesser extent compared to the filter disc experiment. Similar to the filter disc experiment, tall hedge mustard seed germination was not significantly affected by the root or shoot extracts. The radicle length of all species was inhibited by the shoot extract, but not as strongly as in the filter disc experiment. These results indicate that the allelochemicals in the root and shoot extract of tall hedge mustard 50 have a similar but lower inhibitory effect on germination and seedling growth in soil compared to filter discs. The lower inhibitory effect of allelochemicals in soil can be due to a number of factors that can affect the concentration and behaviour in soil, including soil texture, organic and inorganic matter, moisture and microorganisms (Kobayashi, 2004). The root and shoot extracts were prepared using ground tissues and would not represent a natural release of allelochemicals in nature. However, the whole plant leachate which was prepared from intact tissues represented a more natural release or leaching of allelochemicals from tall hedge mustard tissues. In both the filter disc and soil-based experiments, the whole plant leachate had a similar but slightly lower effect on seed germination and seedling growth as the shoot extract. From these results it can be concluded that tall hedge mustard produces allelochemicals in both the root and shoot tissues that can potentially inhibit the germination and seedling growth of neighboring species. Further investigation would be required to determine the primary mode of release of these allelochemicals, but it does appear that leaching from tall hedge mustard litter and foliage could result in the release of potent allelochemicals into the environment. The allelopathic properties associated with mustard plants are typically due to GSLs and their degradation products (Brown and Morra, 1997). Chemical analysis of tall hedge mustard tissue samples taken from field populations in southern British Columbia revealed the presence of six GSLs. The shoot tissues contained the highest quantity of GSLs compared to the roots. Isopropyl GSL was the major GSL found in both the roots and shoots, with a concentration greater than ten fold more than any other GSL in the 51 shoots and two fold more in the roots. The GSL profile of tall hedge mustard samples from this study (Table 2.4) differ from what has previously been reported for this species (Cole, 1976; Daxenbichler et al, 1991). In an analysis of the hydrolysis products of greenhouse grown tall hedge mustard, Cole (1976) found 4-methyl-2-oxazolidinethione and allyl ITC, which are the hydrolysis products of l-methyl-2-hydroxyethyl GSL and allyl GSL. In another study, 4-methyl-2-oxazolidinethione and 4-ethyl-2-oxazolidinethione, the hydrolysis products of l-methyl-2-hydroxyethyl GSL and 1-ethyl-2-hydroxyethyl GSL, were identified in tall hedge mustard seeds (Daxenbichler et al., 1991). However, within the Sisymbrium genus there have been several reports of both isopropyl GSL and sec-butyl GSL (Fahey et al., 2001; Vaughn et al., 2006). The reason for the variation in GSL composition reported for tall hedge mustard is unknown, but the qualitative and quantitative GSL composition of plants can vary based on developmental stage and tissue type, and environmental factors such as soil fertility, moisture regime, pathogen challenge, or plant growth regulators (Brown and Morra, 1997; Fahey et al., 2001; Kiddle et al, 2001). Further investigation of the temporal and spatial variation of the GSL content of tall hedge mustard would be beneficial to gain a better insight into the GSL composition, and in turn allelopathic capabilities of this weed. The allelopathic properties associated with GSLs are primarily due to their biologically active degradation products. Chemical analysis of the GSL degradation products in the dichloromethane layer of aqueous tall hedge mustard,extracts revealed the presence of isopropyl ITC and sec-butyl ITC. These compounds are the degradation products of isopropyl GSL and sec-butyl GSL (Daxenbichler et al., 1991), which were found in the highest concentration in tall hedge mustard tissues. Previous experiments 52 have shown that dichloromethane extracts of mustard plants contain the most active phytotoxins, which includes many of the GSL degradation products. For example, Vaughn and Berhow (1999) found that methanol, hexane, and water garlic mustard extracts had minimal activity, while the dichloromethane extract was highly phytotoxic. GC-MS of the dichloromethane extract revealed the presence of two major allelochemicals, allyl ITC and benzyl ITC (Vaughn and Berhow, 1999). Seed germination and seedling growth experiments in the current study revealed that both isopropyl ITC and sec-butyl ITC exhibit strong phytotoxic properties. Like tall hedge mustard extracts, ITCs strongly inhibited bluebunch wheatgrass, Idaho fescue, and spotted knapweed seed germination, with minimal effect on tall hedge mustard. Tall hedge mustard extracts and ITCs also strongly inhibited radicle growth of all species tested. However, the ITCs differed in that they also strongly inhibited coleoptile and hypocotyl growth, whereas the tall hedge mustard extracts had little or no effect. Overall, Isopropyl ITC had a slightly greater effect than sec-butyl ITC on seed germination and seedling growth. The decreased inhibitory effect of sec-butyl ITC compared to isopropyl ITC could be due to the longer side-chain as it is commonly observed that aliphatic ITCs with shorter side-chains have greater biological activity (Vaughn et al, 2006). The results from this study showed that tall hedge mustard produces allelochemicals that have the ability to inhibit seed germination and seedling growth, but how ecologically significant are the tall hedge mustard root and shoot extracts and ITC concentrations? The 1%, 2%, and 4% tall hedge mustard extracts were used to represent a range of concentrations that could be naturally produced by tall hedge mustard. Since tall hedge mustard grows in dense stands and is an annual species, a thick layer of litter 53 can accumulate on the ground. Due to the semi-arid climate in southern British Columbia and low precipitation (Anon., 2006), leachates from tall hedge mustard litter would be concentrated in the top layer of the soil. There are many factors that could influence the amount of allelochemicals that reach the soil, including density of tall hedge mustard stands, amount of rainfall, and mode of release. Further investigation of these factors may provide a better assessment of the allelopathic influence of tall hedge mustard. Glucosinolate analysis revealed that tall hedge mustard shoot and root tissues contained 80.54 umol g"1 and 7.93 umol g"1 isopropyl GSL, respectively. If the isopropyl GSL content in the 4% shoot extract and 4% root extract had a complete (100%) breakdown (or release efficiency) to isopropyl ITC then these extracts would have a concentration equivalent to ~3.2 m M and 0.3 m M , respectively. However, the release efficiency is likely to be lower. For example, Vaughn et al. (2006) found that money plant (Lunaria annua L.) seedmeal (defatted ground seeds), whose primary GSL was isopropyl GSL, had a release efficiency of 44.1% in soil. As a result, the ITC concentrations used in this study represent a range of ITC concentrations that may be present in the tall hedge mustard extracts used in this study. Further research investigating the concentration of isopropyl ITC and other potential allelochemicals in tall hedge mustard extracts, and in and around the rhizosphere of tall hedge mustard plants in the field may provide better insight into the allelopathic influence of this exotic weed. Overall, it appears that isopropyl ITC is one of the major allelochemicals produced by tall hedge mustard. Isopropyl ITC was the prominent peak in the 54 dichloromethane layer of tall hedge mustard root and shoot extracts, and its parent GSL was found in much greater concentration compared to any other GSL. In addition, the phytotoxic activity of tall hedge mustard aqueous extracts appears to be associated with the isopropyl GSL concentration. The shoot tissues contained a much greater concentration of isopropyl GSL compared to the root tissues, and in turn had a higher inhibitory effect on both seed germination and seedling growth. However, the role of other glucosinolate degradation products, as well as other secondary metabolites in the allelopathic influence of this weed cannot be ruled out. 55 2.5 Literature Cited Bell, D.T. and C.H. Muller. 1973. Dominance of California annual grasslands by Brassica nigra. American Midland Naturalist 90:277-299. Bialy, Z., W. Oleszek, J. Lewis and G.R. Fenwick. 1990. Allelopathic potential of glucosinolates (mustard oil glycosides) and their degradation products against wheat. Plant and Soil 129:277-281. Borek, V. , M.J . Morra, P.D. Brown and J.P. McCaffrey. 1995. Transformation of the glucosinolate-derived allelochemicals allyl isothiocyanate and allylnitrile in soil. Journal of Agricultural Food Chemistry 43:1935-1940. Brown, P.D., M.J . Morra and V . Borek. 1994. Gas chromatography of allelochemicals produced during glucosinolate degradation in soil. Journal of Agricultural Food Chemistry 42:2029-2034. Brown, P.D. and M.J . Morra. 1997. Control of soil-borne plant pests using glucosinolate-containing plants. Advances in Agronomy 61:167-231. Cole, R.A. 1976. ITCs, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phytochemistry 15:759-762. Daxenbichler, M.E. , G.F. Spencer, D.G. Carlson, G.B. Rose, A . M . Brinker and R.G. Powell. 1991. Glucosinolate composition of seeds from 297 species of wild plants. Phytochemistry 30:2623-2638. Dudai, N . , A . Poljakoff-Mayber, A . M . Mayer, E. Putievsky and H.R. Lerner. 1999. Essential oils as allelochemicals and their potential use as bioherbicides. Journal of Chemical Ecology 25:1079-1089. 56 Fahey, J.W., A.T. Zalcmann and P. Talalay. 2001. The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5-51. Gardiner, J.B., M.J . Morra, C.V. Eberlein, P.D. Brown, and V, Borek. 1999. Allelochemicals released in soil following incorporation of rapeseed (Brassica napus) green manures. Journal of Agricultural Food Chemistry 47:3837-3842. Hierro, J.L. and R . M . Callaway. 2003. Allelopathy and exotic plant invasion. Plant and Soil 256:29-39. Inderjit and R . M . Callaway. 2003. Experimental designs for the study of allelopathy. Plant and Soil 256:1-11. Inderjit and S.O. Duke. 2003. Ecophysiological aspects of allelopathy. Planta 217:529-539. - Inderjit and E.T. Nilsen. 2003. Bioassays and field studies for allelopathy in terrestrial plants: progress and problems. Critical Reviews in Plant Science 22:221-238. Kiddle, G., R.N. Bennett, N.P. Botting, N.E. Davidson, A . A . B . Robertson and R . M . Wallsgrove. 2001. High-performance liquid chromatographic separation of natural and synthetic desulfoglucosinolates and their validation by U V , N M R and chemical ionization-MS methods. Phytochemical Analysis 12:226-242. Kiemnec, G.L. and M . L . Mclnnis. 2002. Hoary cress (Cardaria draba) root extract reduces germination and root growth of five plant species. Weed Technology 16:231-234. Kobayashi, K. 2004. Factors affecting phytotoxic activity of allelochemicals in soil. Weed Biology and Management 4:1-7. 57 Oueslati, 0., M . Ben-Hammouda, M . H . Ghorbal, M . Guezzah, and R.J. Kremer. 2005. Barley autotoxicity as influenced by varietal and seasonal variation. Journal of Agronomy and Crop Science 191:249-254. Parish, R., R. Coupe and D. Lloyd (eds). 1996. Plants of Southern Interior British Columbia and the Inland Northwest. Vancouver: B.C. Ministry of Forests and Lone Pine Publishing. 461 p. Petersen, J., R. Belz, F. Walker and K . Hurle. 2001. Weed suppression by release of isothiocyanates from turnip-rape mulch. Agronomy Journal 93:37-43. Prati, D. and O. Bossdorf. 2004. Allelopathic inhibition of germination by Alliaria petiolata (Brassicaceae). American Journal of Botany 91:285-288. Reigosa, M.J. , A . Sanchez-Moreiras and L. Gonzalez. 1999. Ecophysiological approach in allelopathy. Critical Reviews in Plant Science 18:577-608. Roberts, K.J . and R.C. Anderson. 2001. Effect of garlic mustard [Alliaria petiolata (Bieb. Cavara & Grande)] extracts on plants and arbuscular mycorrhizal (AM) fungi. American Midland Naturalist 146:146-152. Vaughn, S.F. and M . A . Berhow. 1999. Allelochemicals isolated from tissues of the invasive weed garlic mustard {Alliaria petiolata). Journal of Chemical Ecology 25:2495-2504. Vaughn, S.F., D.E. Palmquist, S.M. Duval and M . A . Berhow. 2006. Herbicidal activity of glucosinolate-containing seedmeals. Weed Science 54:743-748. Warwick, S.I., A . Francis and G.A. Mulligan. 2003. Brassicaceae of Canada. Online Database. U R L : http://www.cbif.gc.ca/spp_pages/brass/index_e.php 58 Weston, L .A. and S.O. Duke. 2003. Weed and Crop Allelopathy. Critical Reviews in Plant Science 22:367-389. Yemane, A. , J. Fujikura, H. Ogawa and J. Mizutani. 1992. Isothiocyanates as allelopathic compounds from Rorippa indica Hiern. (Cruciferae) roots. Journal of Chemical Ecology 18:1941-1954. 59 Chapter 3. Inhibitory Effects of Tall Hedge Mustard (Sisymbrium loeselii) Allelochemicals on Arbuscular Mycorrhizal Fungi 3.1 Introduction Allelopathy is defined as the negative effect of chemicals released by one plant on the growth and distribution of other plants (Inderjit and Callaway, 2003). This direct plant-plant chemical interaction has been widely used as an explanation for the success of some exotic plants. Recently it has been shown that allelochemicals could also affect interactions between native plants and soil organisms, such as arbuscular mycorrhizal (AM) fungi (Wolfe and Klironomos, 2005). A M fungi are symbiotic fungi that colonize the roots of most vascular plant species and improve nutrient uptake, particularly phosphorus (Bucking and Shachar-Hill, 2005). Many of the plants that form mycorrhizal associations with A M fungi are dependent on this association for survival (Stinson et al., 2006). Disruption of these mutualistic associations can have long-term effects on the dynamics of the plant species. Recent studies have shown that allelochemicals have a negative effect on A M fungi. For example, garlic mustard (Alliaria petiolata [Bieb.] Cavara & Grande) water leachates inhibited spore germination and A M colonization of tomato roots, and garlic mustard infestations reduced the A M inoculum potential of field soil (Roberts and Anderson, 2001). Phytochemicals produced by garlic mustard suppressed growth of native tree seedlings by disrupting mutualistic associations with A M fungi (Stinson et al., 2006). Similar to other members of the Brassicaceae, garlic mustard is non-mycorrhizal and produces glucosinolates (GSLs). GSLs are a class of secondary metabolites that produce biologically active compounds upon enzymatic degradation, including 60 isothiocyanates (ITCs), organic cyanides, oxazolidinethiones, and ionic thiocyanate (Brown and Morra, 1997). The enzyme myrosinase (thioglucosidase; EC 3.2.3.1.), found in the tissues of GSL-containing species, hydrolyses GSLs to these compounds which have allelopathic and antifungal activities (Brown and Morra, 1997; Smolinska et al., 1997; Ludwig-Muller et al, 2002). GSL degradation products have been found to be inhibitory to A M fungi (Vierheilig and Ocampo, 1990; Schreiner and Koide, 1993). For example, aqueous cabbage {Brassica oleracea L.) root extract and volatiles from the root extract inhibited spore germination of Glomus mosseae Nicol. & Gerd. (Vierheilig and Ocampo, 1990). Autoclaving the cabbage extracts and thereby denaturing myrosinase and preventing GSL degradation, caused the inhibitory effect to disappear (Vierheilig and Ocampo, 1990). The inhibitory effect of the GSL degradation products was further confirmed by adding myrosinase to the autoclaved extract and finding a return of the inhibitory effect; and finding that neither allyl GSL, the primary GSL in cabbage, or myrosinase had an effect on spore germination, but when combined they were highly inhibitory (Vierheilig and Ocampo, 1990). In another study, Schreiner and Koide (1993) provided evidence that ITCs were responsible for the inhibitory effect of intact mustard plants by adding ITC scavengers (e.g. lysine, arginine, and glutathione) to ameliorate the effects of the ITCs. The addition of these ITC scavengers restored the germination of Glomus intraradices Shenck & Smith and Glomus etunicatum Becker & Gerd. to control levels (Schreiner and Koide, 1993). Tall hedge mustard {Sisymbrium loeselli L.), a non-mycorrhizal (Harley and Harley, 1987) member of the Brassicaceae family, has become naturalized across North 61 America (Warwick et al, 2002). It is commonly found on disturbed soils, cultivated fields, rangeland, and waste places in southern.British Columbia and often appears like a yellow blanket covering fields and roadsides in early summer (Douglas et al., 1998; Parish et al., 1999). Like many weedy mustards, when well established it forms dense , monocultures allowing few other plant species to grow. The establishment of mono-specific stands suggests use of unusually potent mechanisms, such as allelopathy (Hierro and Callaway, 2003). Tall hedge mustard has been shown to exhibit allelopathic properties as the aqueous extracts of its root and shoot tissues are highly inhibitory to seed germination and radicle elongation of two rangeland grasses, and a broadleaved species (Bainard et al, in press; Chapter 2, Figure 2.2, Table 2.1a and Table 2.1b). The allelopathic properties of tall hedge mustard are believed to be due to GSLs or more importantly their degradation products. GSL analysis of tall hedge mustard root and shoot tissues revealed the presence of several GSLs including the two most prominent GSLs, isopropyl (1-methylethyl) GSL and sec-butyl (1-methylpropyl) GSL (Bainard et al., in press; Chapter 2, Table 2.4). The degradation products of these two GSLs (isopropyl ITC and sec-butyl ITC) were identified in hydrolyzed tall hedge mustard tissues. Both ITCs exhibited phytotoxic properties by inhibiting seed germination and radicle elongation of several species. Tall hedge mustard allelochemicals may inhibit A M fungi and this may provide another strategy for tall hedge mustard to invade neighboring mycorrhizal species. The effect of these allelochemicals on A M fungi is not known. This study investigated the allelopathic potential of tall hedge mustard allelochemicals on A M fungi. The specific 62 objectives of this research were to (1) determine the effect of tall hedge mustard allelochemicals on A M fungal spore germination and hyphal growth, and (2) determine the effect of tall hedge mustard infestations on A M inoculum potential of field soil. 3.2 Materials & Methods 3.2.1 Seed and spore sources Bluebunch wheatgrass {Pseudoroegneria spicata [Pursh.] Love) and spotted knapweed {Centaurea maculosa Lam.) are mycorrhizal species (Marler and Callaway, 1999) that grow and compete with tall hedge mustard {Sisymbrium loeselii L.) in southern British Columbia. Bluebunch wheatgrass seeds were obtained from Dawson Seed Company (Surrey, B C , Canada), and spotted knapweed seeds were collected from natural populations in southern British Columbia. Sterile spores of Glomus intraradices Shenck & Smith used for the spore germination and hyphal growth bioassays in this study were obtained from Premier Technologies (Riviere-Du-Loop, Quebec, Canada). 3.2.2 Tall hedge mustard extracts and whole plant leachate preparation Tall hedge mustard roots and shoots (rosette leaves, stems, stem leaves, flowers, and seed pods) were collected from natural populations in southern British Columbia and air-dried at room temperature (22 to 26°C) for 5 d. Dried biomass was ground in a blender and stored at -24°C until their extraction. The root and shoot extracts were prepared by incubating respective ground tissues in distilled water (4% w/v) on a rotary shaker (90 rpm) for 24 h at room temperature. The whole plant leachate was prepared by 63 incubating intact plants in distilled water (4% w/v) for 24 h without shaking. The incubation media were centrifuged at 1,015 g for 10 min, and passed through a 0.20 um Millipore syringe filter (Millipore Corporation, Billerica, M A ) to sterilize the solutions. 3.2.3 Spore germination and hyphal growth bioassays Two separate experiments were conducted to study the effect of tall hedge mustard allelochemicals on spore germination and hyphal growth. The first experiment investigated the effect of tall hedge mustard aqueous root and shoot extracts and a whole plant leachate, and the second experiment investigated the effect of the commercially available ITCs (isopropyl and sec-butyl), also found in tall hedge mustard extracts (Chapter 2, Figure 2.6 and 2.7), on spore germination and hyphal growth of G. intraradices. 3.2.3.1 Aqueous extracts and leachate The effect of tall hedge mustard aqueous root and shoot extracts, and whole plant leachate on A M spore germination and hyphal growth was investigated by incorporating these solutions into 1% agar media (1% bacto-agar in distilled water; Difco Laboratories, Detroit, MI) in 60-mm petri dishes (10 ml/dish). The four treatment solutions were: 1% agar (control), and 1% agar amended (1:1) with tall hedge mustard root extract, shoot extract, or whole plant leachate. Five G. intraradices spores were aseptically transferred using a sterile blade (Juge et al., 2002) to petri dishes, sealed with parafilm and incubated in the dark at 21°C. Percent spore germination was recorded after 10 d. Spores were considered germinated when the germ tube was at least twice the diameter of the spore 64 (Elias and Safir, 1987). Germ tubes emerge from the subtending hyphae of G. intraradices spores (Figure 3.1). After 14 d, the hyphal length of spores that germinated was measured under a light microscope using an ocular micrometer at 40x magnification (Kirk et al., 2005). The petri dishes were arranged in a completely randomized design with six replicates (petri dishes) per treatment, and the experiment was repeated. 3.2.3.2 Isothiocyanates The effect of the major allelochemicals identified in the aqueous extracts of tall hedge mustard tissues (Section 2.3.5) on A M spore germination was investigated by incorporating isopropyl ITC (Sigma-Aldrich, Oakville, ON) and sec-butyl ITC (Fisher Scientific, Ottawa, ON) into 1% agar media (1% bacto-agar in distilled water) in 60-mm petri dishes (10 ml/dish). Both isopropyl ITC and sec-butyl ITC were first dissolved in methanol, and then diluted with distilled water; all treatment solutions including the control contained 0.5% methanol. Agar media was autoclaved (121°C at 16 psi) for 20 min and allowed to cool (approx. 40°C) prior to addition of isopropyl ITC and sec-butyl ITC. The treatments were: 1% agar containing 0.5% methanol (control), and 1%> agar containing 0.5% methanol and 0.001, 0.01, 0.1, 0.5, or 1.0 m M isopropyl ITC, or 0.001, 0.01, 0.1, or 1.0 m M sec-butyl ITC. Five G. intraradices spores were aseptically transferred using a sterile blade to petri dishes, sealed with parafilm and incubated in the dark at 21°C. Percent spore germination was recorded after 10 d. After 14 d, the hyphal length of spores that germinated was measured as described above. The petri dishes were arranged in a completely randomized design with six replicates (petri dishes) per treatment, and the experiment was repeated. 65 Figure 3.1. (A) Ungerminated Glomus intraradices spore, and (B) germinated Glomus intraradices spore. The subtending hyphae from which the germ tube emerges following germination is shown by arrows. 66 3.2.4 Arbuscular mycorrhizal inoculum potential of tall hedge mustard infested soil In order to investigate the effect of tall hedge mustard infestations on A M inoculum potential of soil, field soil was collected in July 2006 from a location heavily infested by tall hedge mustard in southern British Columbia. Soil samples were taken from two randomly selected sites within tall hedge mustard stands and classified as infested soil. Directly adjacent to each of the infested sites, soil samples were collected and classified as noninfested soil. The noninfested sites lacked tall hedge mustard and were occupied primarily by grasses including bluebunch wheatgrass. Soil samples were collected from 5 to 15 cm depth and kept in cold storage (4°C) until the soil was used (Requena et al, 1996). The soil samples were sieved (< 2 mm) to remove any coarse roots and debris and then analyzed to determine the pH, texture and organic matter (Table 3.1). Plastic tubes (200 ml, 3.8 mm diam) were filled with a 1:1 mixture of field soil from each of the sites and silica sand. Five seeds of either spotted knapweed or bluebunch wheatgrass were sown in the soil mixture, watered, and placed on a bench in the greenhouse. After emergence, seedlings were thinned to one per tube. The plants were placed in a completely randomized design on the greenhouse bench with eight subsamples for each treatment soil and species and watered regularly with tap water without fertilizer. After 7 weeks, the roots and shoots were harvested, dried at 60°C for 48 h and weighed to determined total biomass. A root sample was taken from each plant at 7 weeks to determine the amount of A M colonization. The roots from each replicate were cut into 1 cm segments and cleared for 1 hr in hot 10% K O H (Sigma Aldrich, Oakville, ON) and washed several times with 67 Table 3.1. Characteristics of soils used in A M inoculum potential experiment Soil Site pH Texture Organic matter Noninfested 1 6.7 Loamy sand 3% Noninfested 2 6:5 Sandy loam 5% Infested 1 6.6 Loamy sand 5% Infested 2 6.9 Sandy loam 4% distilled water. The root samples were acidified for 1 h with 3% HC1 (Fisher Scientific, Ottawa, ON) and then stained for 1 h in hot 0.05% trypan blue in lactoglycerol (Sigma Aldrich, Oakville, ON). The root segments were mounted on slides and examined using a light microscope (200x) to determine the percentage of root colonization using the modified line intersect method (McGonigle et al., 1991). 3.2.5 Data analysis Spore germination and A M colonization data were arcsine-transformed prior to analysis. Spore germination and hyphal growth data from the tall hedge mustard aqueous extracts and leachate experiment (3.2.3.1) were subjected to analysis of variance (ANOVA), and treatment means were separated by Tukey's HSD test (P = 0.05) using SPSS 15.0 (SPSS Inc., Chicago, IL). Spore germination and hyphal growth data from the ITC dose^response experiment (3.2.3.2) were subjected to regression analysis using Sigma Plot 10 (Systat Software, Inc., San Jose, CA). A M colonization and total biomass from the A M inoculum potential experiment (3.2.4) were subjected to A N O V A for two fixed factors (species and soil type). Data from repeated experiments were pooled because there was no significant (P = 0.05) experiment by treatment interactions. 69 3.3 Results 3.3.1 Effect of aqueous extracts and leachate on spore germination The tall hedge mustard aqueous root and shoot extracts and whole plant leachate significantly inhibited G. intraradices spore germination compared to the control (Table 3.2) . The root extract inhibited spore germination by 86.7% compared to control, and the shoot extract and whole plant leachate caused complete inhibition. Effects of root extract, shoot extract, and whole plant leachate on G. intraradices spore germination were not significantly different (P = 0.05). 3.3.2 Effect of isothiocyanates on spore germination Isopropyl ITC and sec-butyl ITC significantly (P < 0.0001) inhibited spore germination (Figure 3.2). However, isopropyl ITC was more effective at inhibiting G. intraradices spore germination compared to sec-butyl ITC. This difference was most apparent at 0.1 and 0.5 m M concentrations. 3.3.3 Effect of aqueous extracts and leachate on hyphal growth The tall hedge mustard root and shoot extracts and whole plant leachate significantly reduced the hyphal length of G. intraradices compared to the control (Table 3.3) . The root extract had a strong effect on G. intraradices hyphal growth reducing the hyphal length by 74.6%» compared to the control. Since no spores germinated in the shoot extract or whole plant leachate, there was no hyphal growth. 70 Table 3.2. Effect of tall hedge mustard aqueous root extract, shoot extract, and whole plant leachate on spore germination of Glomus intraradices Treatment Germination (%)* Control 88.3 ±4 .6 (a) Root extract 11.7 ± 4.6 (b) Shoot extract 0.0 ± 0.0 (b) Whole plant leachate 0.0 ± 0.0 (b) * Values are the mean ± standard error of two experiments of six replicates. *Values with different letters are significantly different according to Tukey's HSD test (P = 0.05) 71 Figure 3.2. Effect of (A) isopropyl ITC (y = 80.389e"8 2 6 6 x , R 2 = 0.85, P < 0.0001) and (B) sec-butyl ITC (y = 83.282 - 133.576x + 53.301x2, R 2 = 0.76, P < 0.0001) on spore germination of Glomus intraradices. Values are means ± standard error of two experiments of six replicates. 72 Table 3.3. Effect of tall hedge mustard aqueous root extract on hyphal growth of Glomus intraradices Treatment Hyphal length (mm)* Control 4.49 ± 0.25 (a) Root extract 1.14 ±0.24 (b) •Values are the mean ± standard error of two experiments of six replicates. No spores germinated in shoot extract or whole plant leachate and therefore there was no hyphal growth. *Values with different letters are significantly different according to Tukey's HSD test (P = 0.05) 3.3.4 Effect of isothiocyanates on hyphal growth Hyphal growth was significantly (P < 0.0001) inhibited by isopropyl ITC and sec-butyl ITC (Figure 3.3). Similar to spore germination, isopropyl ITC had a stronger effect on hyphal growth compared to sec-butyl ITC. This effect was most apparent at 0.1 mM concentration. Since none of the spores germinated at 0.5 and 1.0 m M isopropyl ITC, there was no hyphal growth. 3.3.5 Arbuscular mycorrhizal inoculum potential of tall hedge mustard infested soil Tall hedge mustard infestations reduced the A M inoculum potential of the soil from both infested sites compared to the noninfested soils for bluebunch wheatgrass and spotted knapweed (Figure 3.4). Tall hedge mustard infested soils significantly (P < 0.001) decreased the percent A M colonization compared to noninfested soils for spotted knapweed (43.6%) and bluebunch wheatgrass (51.2%). Tall hedge mustard infested soils also significantly reduced the total biomass of spotted knapweed by 34.6% and bluebunch wheatgrass by 24.7%, compared to noninfested soils. 3.4 Discussion It has been shown in previous experiments (Chapter 2) that tall hedge mustard may utilize allelopathy as a strategy to directly (i.e. inhibition of germination and growth of plants) compete with neighboring species. The objective of this study was to determine i f tall hedge mustard potentially utilizes allelopathy as a strategy to indirectly 74 0.00 0.02 0.04 0.06 0.08 Isopropyl ITC (mM) 0.10 0.12 0.0 0.2 0.4 0.6 0.8 sec-Butyl ITC (mM) 1.0 Figure 3.3. Effect of (A) isopropyl ITC (y = 4.122e-" 9 0 x, R 2 = 0.15, P < 0.0001) and (B) sec-butyl ITC (y = 4.163 - 3.901x + 0.914x 2,R 2= 0.063, P = 0.0011) on hyphal growth of Glomus intraradices. Notice that the scale on the x-axis for isopropyl ITC is only l/10 t h of the sec-butyl ITC x-axis. This is because no spores germinated at 0.5 or 1.0 m M isopropyl ITC and therefore there was no hyphal growth. Values are means ± standard error of two experiments of six replicates. 75 100 80 1 | I noninfested soil CD infested soil el o N '3 o o 60 40 20 H X Spotted knapweed Bluebunch wheatgrass 250 B o o Spotted knapweed Bluebunch wheatgrass Figure 3.4. Influence of tall hedge mustard infested or noninfested field soils on (A) arbuscular mycorrhizal (AM) colonization of spotted knapweed (P < 0.001) and bluebunch wheatgrass (P < 0.001); and (B) total biomass of spotted knapweed (P < 0.01) and bluebunch wheatgrass (P < 0.05). Values are means ± standard error of two sites with eight subsamples per site. (i.e. inhibition of A M fungi) compete with neighboring species. The results from this study showed that tall hedge mustard contains allelochemicals that are inhibitory to A M fungi. Tall hedge mustard aqueous extracts inhibited spore germination and hyphal growth of G. intraradices in petri dishes. The root extract inhibited spore germination by 86.7% and hyphal growth by 74.6% compared to the control. The shoot extract and whole plant leachate completely inhibited spore germination. Due to the lack of germinating spores in these treatments, the effect of tall hedge mustard shoot extract and whole plant leachate on hyphal growth could not be assessed. Tall hedge mustard root and shoot tissues contain several GSLs. The two most prevalent GSL degradation products (isopropyl ITC and sec-butyl ITC) found in tall hedge mustard extracts strongly inhibited the spore germination and hyphal growth of A M fungi. Isopropyl ITC had a stronger inhibitory effect than sec-butyl ITC on both spore germination and hyphal growth of G. intraradices. The lower level of inhibition by sec-butyl ITC compared to isopropyl ITC could be due to the longer side-chain as it is commonly observed that aliphatic ITCs with shorter side-chains have greater biological activity (Vaughn et al., 2006). Isopropyl GSL, the precursor to isopropyl ITC, was found in substantially higher concentrations in both the root (ca: 2 fold) and shoot (ca. 10 fold) . tissues (Bainard et al., in press; Chapter 2, Table 2.4). As a result, isopropyl ITC is most likely the primary ITC involved in the inhibitory effect of tall hedge mustard root and shoot extracts on A M fungi, although the role of sec-butyl ITC and other GSL degradation products cannot be ruled out. Isopropyl ITC and sec-butyl ITC had a similar inhibitory effect on spore germination as they did on hyphal growth. The germ tube is the most sensitive structure 77 of A M fungi as the walls of the germ tube are more permeable than the multistratified walls of the spores and hence, it would be expected that inhibitory chemicals would have a stronger effect on hyphal growth than spore germination (Chiocchio et al., 2000). For example, the fungicide benomyl can reduce the hyphal length at concentrations that are not inhibitory to spore germination (Chiocchio et al, 2000). Based on the results from this study it appears that the inhibitory effect of tall hedge mustard extracts and the ITCs is not greater on hyphal growth compared to spore germination. Nonetheless, tall hedge mustard produces allelochemicals that have the potential to inhibit the germination and hyphal growth of A M fungi. The results from this study also showed that tall hedge mustard infested soils had a lower A M inoculum potential compared to noninfested soils. The A M inoculum potential of the soil was based on the percentage of roots colonized by A M fungi growing in field collected soil. Bluebunch wheatgrass and spotted knapweed both showed a significant reduction in A M colonization in the tall hedge mustard infested compared to noninfested soils. Two separate studies on garlic mustard infestations found a similar trend. Roberts and Anderson (2001) found a correlation between high densities of garlic mustard in the field and low inoculum potential of A M fungi. In another study, Stinson et al. (2006) found that native hardwood tree species grown in soil that had been invaded by garlic mustard showed significantly less A M colonization of roots and slower growth compared to soils with no history of garlic mustard. The lower total biomass of bluebunch wheatgrass and spotted knapweed had when grown in tall hedge mustard infested soils could possibly be a direct allelopathic effect on plant growth and/or an indirect effect of a lower level of A M colonization. Stinson et al. (2006) linked the 78 reduced growth of the native hardwood tree species growing in garlic mustard invaded soil to the reduction of A M fungi and not a direct allelopathic influence. Further investigation would be required to clarify this. These results indicate that tall hedge mustard produces allelochemicals that are not only inhibitory to the germination and growth of neighboring species, but also inhibit A M fungal spore germination and hyphal growth. Tall hedge mustard infestations also reduced the A M inoculum potential of soil. The results suggest that tall hedge mustard produces allelochemicals that inhibit A M fungi and may be responsible for the reduced A M inoculum potential of tall hedge mustard infested soil, resulting in a disadvantage for species (e.g. bluebunch wheatgrass or spotted knapweed) that benefit from A M associations. 79 3.5 Literature Cited Bainard, L.D. , P.D. Brown and M . K . Upadhyaya. Inhibitory effect of tall hedge mustard allelopathic secondary metabolites on arbuscular mycorrhizal fungi. Proceedings of Canadian Weed Science Society Annual Meeting, Victoria, B C , November 27-30, 2006. In press. Brown, P.D. and M.J . Morra. 1997. Control of soil-borne plant pests using glucosinolate-containing plants. Advances in Agronomy 61:167-231. Bucking, H. and Shachar-Hill, Y . 2005. Phosphate uptake, transport and transfer by the arbuscular mycorrhizal fungus Glomus intraradices is stimulated by increased carbohydrate availability. New Phytologist 165:899-912. Cavagnaro, T.R., F.A. Smith, M.F. Lorimer, K . A . Haskard, S.M. Ayling and S.E. Smith. 2001. Quantitative development of P^m-type arbuscular mycorrhizas formed betweenAsphodelus fistulosus'and Glomus coronatum. New Phytologist 149:105-113. Chiocchio, V. , N . Venedikian, A .E . Martinez, A. Menendez, J.A. Ocampo and A . Godeas. 2000. International Microbiology 3:173-175. Douglas, G.W., G.B. Straley, D.V. Meidinger, and J. Pojar (eds). 1998. Illustrated Flora of British Columbia. Volume 2: Dicotyledons (Balsaminaceae Through Cucurbitaceae). Victoria: B.C. Ministry of Environment, Lands & Parks and B.C. Ministry of Forests, 401 p. Elias, K.S. and G.R. Safir. 1987. Hyphal elongation of Glomus fasciculatus in response to root exudates. Applied and Environmental Microbiology 53:1928-1933. 80 Hierro, J.L. and R . M . Callaway. 2003. Allelopathy and exotic plant invasion. Plant and Soil 256:29-39. Inderjit and R . M . Callaway. 2003. Experimental designs for the study of allelopathy. Plant and Soil 256:1-11. Inderjit and S.O. Duke. 2003. Ecophysiological aspects of allelopathy. Planta 217:529-539. Juge, C , J. Samson, C. Bastien, H. Vierheilig, A . Coughlan and Y . Piche. 2002. Breaking dormancy in spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period. Mycorrhiza 12:37-42. Kirk, J.L., P. Moutoglis, J. Klironomos, H . Lee and J.T. Trevors. 2005. Toxicity of diesel fuel to germination, growth and colonization of Glomus intraradices in soil and in vitro transformed carrot root cultures. Plant and Soil 270:23-30. Kobayashi, K . 2004. Factors affecting phytotoxic activity of allelochemicals in soil. Weed Biology and Management 4:1-7. Ludwig-Muller, J., R .N. Bennett, J .M. Garcia-Garrido, Y . Piche and H . Vierheilig. 2002. Reduced arbuscular mycorrhizal root colonization in Tropaeolum majus and Carica papaya after jasmonic acid application cannot be attributed to increased glucosinolate levels. Journal of Plant Physiology 159:517-523. Marler, M.J. , C A . Zabinski and R . M . Callaway. 1999. Mycorrhizae and fine root dynamics of Centaurea maculosa and native bunchgrass species in western Montana. Northwest Science 73:217-224. 81 McGonigle, T.P., M . H . Miller, D.G. Evans, G.L. Fairchild and J.A. Swan. 1990. A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi. New Phytologist 115:495-501. Parish, R., R. Coupe and D. Lloyd (eds). 1996. Plants of Southern Interior British Columbia and the Inland Northwest. Vancouver: B.C. Ministry of Forests and Lone Pine Publishing, 461 p. Reigosa, M.J. , A . Sanchez-Moreiras and L. Gonzalez. 1999. Ecophysiological approach in allelopathy. Critical Reviews in Plant Science 18:577-608. Requena, N . , P. Jeffries.and J .M. Barea. 1996. Assessment of natural mycorrhizal potential in a desertified semiarid ecosystems. Applied and Environmental Microbiology 62:842-847. Roberts, K.J . and R.C. Anderson. 2001. Effect of garlic mustard [Alliaria petiolata (Beib. Cavara & Grande)] extracts on plants and arbuscular mycorrhizal (AM) fungi. American Midland Naturalist 146:146-152. Schreiner, R.P. and R.T. Koide. 1993. Mustards, mustard oils, and mycorrhizas. New Phytologist 123:107-113. Smolinska, U . , M.J. Morra, G R . Knudsen and P.D. Brown. 1997. Toxicity of glucosinolate degradation products from Brassica napus seed meal toward Aphanomyces euteiches f. sp.pisi. Phytopathology 87:77-83. Stinson, K . A . , S.A. Campbell, J.R. Powell, B.E. Wolfe, R . M . Callaway, G.C. Thelen, S.G. Hallett, D. Prati, and J.N. Klironomos. 2006. Invasive plant suppresses the growth of native tree seedlings by disrupting belowground mutualisms. Public Library of Science 4:727-731 82 Vaughn, S.F. and M . A . Berhow. 1999. Allelochemicals isolated from tissues of the invasive weed garlic mustard (Alliaria petiolata). Journal of Chemical Ecology 25:2495-2504. Vaughn, S.F., D.E. Palmquist, S.M. Duval, M . A . Berhow. 2006. Herbicidal activity of glucosinolate-containing seedmeals. Weed Science 54:743-748. Vierheilig, H . and J.A. Ocampo. 1990. Effect of ITCs on germination of spores of G. mosseae. Soil Biology and Biochemistry 22:1161-1162. Warwick, S.I., L A . Al-Shehbaz, R.A. Price and C. Sauder. 2002. Phylogeny of Sisymbrium (Brassicaceae) based on ITS sequences of nuclear ribosomal DNA. Canadian Journal of Botany 80:1002-1017. Wolfe, B.E. and J.N. Klironomos. 2005. Breaking new ground: soil communities and exotic plant invasion. Bioscience 55:477-488. 83 Chapter 4. Effect of (±)-Catechin and Cnicin, Two Possible Allelochemicals of Spotted Knapweed (Centaurea maculosa), on Spore Germination and Hyphal Growth of Glomus intraradices 4.1 Introduction Spotted knapweed (Centaurea maculosa Lam.) is one of the most abundant noxious weeds in western North America, where it covers millions of acres in the United States and Canada (LeJeune and Seastedt, 2001; Bais et al., 2003; Zabinski et al., 2002). While spotted knapweed commonly invades disturbed rangelands, it can also invade undisturbed native grasslands (Zabinski et al., 2002). Numerous methods and mechanisms have been implicated in the invasive ability of exotic invaders (Blair et al., 2005). Callaway and Ridenour (2001) found that spotted knapweed roots allelopathically inhibited the elongation of a native grass (Festuca idahoensis Elmer.) and that the balance of competition shifted when allelopathy was ameliorated. This weed has been proposed to use a "novel weapons" approach in this regard by producing allelochemicals that are inhibitory to plants and soil microbes in an exotic location (i.e. North America), but are relatively ineffective in their native range (Callaway and Ridenour, 2004). Spotted knapweed roots exude the stable flavanol compound (±)-catechin, which is primarily responsible for the allelopathic capability of spotted knapweed (Bais et al, 2002). It begins to exude phytotoxic levels of (±)-catechin as early as 2-3 weeks after seedling emergence (Weir et al, 2003). The phytotoxicity of the root exudates of spotted knapweed is primarily due to the (-)-catechin enantiomer and the (+)-catechin enantiomer has shown anti-bacterial and anti-fungal properties (Veluri et al, 2004). However, (+)-catechin holds some level of phytotoxicity, approximately 1.5 to 2.0 times lower 84 compared to (-)-catechin (Thelen et al., 2005). The mechanism of action by which (-)-catechin inhibits the growth of target species is by triggering a wave of reactive oxygen species that initiate signalling events and leads to cell death in the roots (Bais et al., 2003). (±)-Catechin has been shown to inhibit the germination of a range of species, including spotted knapweed, possibly allowing this weed to avoid both inter- and intraspecific competition in spotted knapweed infested areas (Weir et al., 2003; Perry et al., 2005a). Another compound produced by spotted knapweed plants that has been shown to exhibit allelopathic properties is cnicin (Kelsey and Locken, 1987). Cnicin is a sesquiterpene lactone found in the glandular trichomes on the epidermal surfaces of spotted knapweed (Locken and Kelsey, 1987). The stem leaves contain the highest concentration of cnicin, with peak concentrations occurring in the fall when the dead tissue contains up to 2.8% cnicin (w/v) (Locken and Kelsey, 1987). Cnicin has little effect on seed germination, but it significantly inhibits seedling growth of several species, including spotted knapweed (Kelsey and Locken, 1987). In addition to its phytotoxicity, cnicin also exhibits antibacterial (Bruno et al., 2003) and antifungal properties. (Skaltsa et al., 2000; Panagouleas et al., 2003). Spotted knapweed is a heavily arbuscular mycorrhizal (AM) species that links into the existing native grass A M networks in recently invaded areas and in heavily infested areas (Zabinski et al., 2002). A M fungi can enhance the growth and invasiveness of spotted knapweed when growing with native grasses (Zabinski et al., 2002). Several studies have demonstrated that A M fungi enhance the competitive effect of spotted knapweed on native grasses (Marler et al., 1999; Zabinski et al., 2002; 85 Callaway et al, 2004; Carey et al, 2004). Zabinski et al (2002) showed that spotted knapweed can exploit its A M symbiosis more effectively than native grasses by excess consumption of phosphorus through efficient utilization of extra-radicle hyphae. Carey et al. (2004) found evidence for mycorrhizally-mediated transfer of carbon from native grasses to spotted knapweed. • In addition to using A M networks to their advantage, it appears that heavy infestations of spotted knapweed alter A M fungal communities. Lutgen and Rillig (2004) found that spotted knapweed infestations reduced glomalin concentrations in soil. Glomalin is a glycoprotein that is produced by A M fungi and correlated with soil aggregate stability (Lutgen and Rillig, 2004). They also reported that spotted knapweed reduced A M hyphal length in areas with high density of spotted knapweed compared to areas receiving various management treatments that resulted in low spotted knapweed density (Lutgen and Rillig, 2004). In another study, Mummey and Rillig (2006) found that spotted knapweed invasions alter A M fungi communities in native grasslands, resulting in decreased A M fungi diversity and abundance, and decreased extraradical hyphae in soil (Mummey and Rillig, 2006). It was postulated that spotted knapweed root exudates, such as (±)-catechin may be one potential mechanism used to influence A M fungi community structure (Mummey et al, 2005; Mummey and Rillig, 2006); however, this has yet to be determined. While spotted knapweed produces two major alleochemicals ([±]-catechin and cnicin) that have exhibited anti-fungal properties, the response of A M fungi to these allelochemicals is unknown. Therefore the objective of this study was to investigate the effects of (±)-catechin and cnicin on spore germination and hyphal growth of A M fungi. 86 4.2 Materials & Methods 4.2.1 Allelochemicals Cnicin was obtained from Rick Kelsey (USDA - PNW Research Station, Corvallis, OR). Cnicin was extracted by Rick Kelsey from the aerial tissues of field collected spotted knapweed and had a purity of 95%, determined by thin layer chromatography (Rick Kelsey, pers. comm.). (±)-Catechin used in this study was obtained from Sigma-Aldrich (Oakville, ON). 4.2.2 Spore germination and hyphal growth bioassay The purpose of this experiment was to determine the effect of spotted knapweed alellochemicals, (±)-catechin and cnicin, on spore germination and hyphal growth of the A M fungal species, Glomus intraradices. Sterile spores of G. intraradices were obtained from Premier Technologies (Riviere-Du-Loop, Quebec, Canada). Eleven treatment solutions were prepared in agar media (1% bacto-agar in distilled water; Difco Laboratories, Detroit, MI) and placed in 60-mm petri dishes. Both (±)-catechin and cnicin were first dissolved in methanol, and then diluted with distilled water; all treatment solutions including the control contained 0.5% methanol. Agar media was autoclaved (121°C) for 20 min and allowed to cool (approx. 40°C) prior to addition of (±)-catechin and cnicin. Petri dishes (60-mm diam) with 10 ml of 1% agar and 0.5% methanol (control), or 1% agar containing 5,10, 50, 100, or 1000 pg ml"1 (±)-catechin and 0.5% methanol, or 1% agar containing 1,10, 50, or 100 pg ml"1 cnicin and 0.5% methanol were prepared. Five G. intraradices spores were aseptically transferred using a sterile blade to 87 petri dishes (Juge et al., 2002), which were sealed with parafilm and incubated in the dark at 21°C. Percent spore germination was recorded after ten days. Spores were considered germinated when the germ tube was at least twice the diameter of the spore (Elias and Safir, 1987). After 14 d, the hyphal length of spores that germinated was measured under a light microscope using an ocular micrometer at 40x magnification (Kirk et al., 2005). The petri dishes were arranged in a completely randomized design with six replicates (petri dishes) per treatment, and the experiment was repeated. 4.2.3 Data analysis Spore germination data were arcsine-transformed prior to analysis. Spore germination and hyphal growth data were subjected to regression analysis using Sigma Plot 10 (Systat Software, Inc., San Jose, CA). Data from the two experiments were pooled as there was no significant (P = 0.05) experiment by treatment interaction. 4.3 Results 4.3.1 Spore germination (±)-Catechin significantly (P < 0.0001) inhibited spore germination of G. intraradices (Figure 4.1). At 50 ug ml"1 percent spore germination was reduced by 34% and at 100 ug ml"1 by 70% compared to the control. There was no spore germination at 500 and 1000 ug ml"1 (±)-catechin. 88 Figure 4.1. Effect of (db)-catechin on spore germination of Glomus intraradices (y = 80.6299e"° 0 1 0 3 x , R 2 = 0.85, P < 0.0001). Values are means ± standard error of two experiments of six replicates. Cnicin significantly (P < 0.0001) inhibited spore germination of G. intraradices (Figure 4.2). At 10 pg ml"1 percent spore germination was reduced by 24% and at 50 pg ml"1 by 72% compared to the control. Spores did not germinate at 100 pg ml"1 cnicin. 4.3.2 Hyphal growth (±)-Catechin concentrations up to 100 pg ml"1 had no significant (P = 0.31) effect on G. intraradices hyphal growth (Figure 4.3). No spores germinated at 500 and 1000 pg ml"1 (±)-catechin and therefore there was no hyphal growth. Cnicin significantly (P < 0.0001) inhibited G. intraradices hyphal growth (Figure 4.4). Cnicin concentrations of 1 and 10 pg ml"1 slightly reduced the hyphal length by 9% and 20%), respectively compared to the control. However, at 50 pg ml"1 cnicin, the hyphal length of G. intraradices was reduced by 60% compared to the control. No spores germinated at 100 pg ml"1 cnicin and therefore there was no hyphal growth. 4.4 Discussion The results from this study indicate that two allelochemicals produced by spotted knapweed, (±)-catechin and cnicin, have a strong inhibitory effect on spore germination of G. intraradices. Both allelochemicals significantly (P < 0.0001) reduced spore germination, with complete inhibition occurring at 500 pg ml"1 (±)-catechin and 100 pg ml*1 cnicin. Cnicin strongly inhibited hyphal growth of G. intraradices at 50 pg ml" 1, and no hyphal growth occurred at 100 pg ml"1. In contrast, (±)-catechin,did not influence the 90 Figure 4.2. Effect of cnicin on spore germination of Glomus intraradices (y = 82.639 - 1.604x + 0.0078x2 R 2 = 0.85, P < 0.0001). Values are means ± standard error of two experiments of six replicates. 6 5 A l A 0 I I • I 1 ; 1 1 1 •0 20 40 60 80 100 120 (±)-Catechin (ug ml"1) Figure 4.3. Effect of (±)-catechin on hyphal growth of Glomus intraradices (y = 4.265 - 0.01061 x + 0.00004x2, R 2 = 0.013, P = 0.31)..No spores germinated at 500 and 1000 u.g ml"1 and therefore there was no hyphal growth/Values are means ± standard error of two experiments of six replicates. 6 5 H 1 1 0 1 ; 1 r 1 1 — ~ i 1 0 10 20 30 40 50 60 Cnicin (pg ml - 1) Figure 4.4. Effect of cnicin on hyphal growth of Glomus intraradices (y = 4.205 - 0.0834x + 0.0007x2, R 2 = 0.85, P < 0.0001). No spores germinated at 100 pg ml' 1 and therefore there was no hyphal growth. Values are means ± standard error of two experiments of six replicates. hyphal growth at any concentration in which spore germination occurred. It is possible that (±)-catechin may affect hyphal growth at higher concentrations, but since spores did not germinate at 500 and 1000 ug ml"1 this could not be varified. Further investigation is required to determine the effect of higher concentrations of (±)-catechin and cnicin on hyphal growth. The results showed that A M fungi can be inhibited by (±)-catechin and cnicin, but how biologically important are these concentrations? There have been several recent studies that have investigated the production of (±)-catechin by spotted knapweed, with differing results. The first report of (±)-catechin concentrations in soil infested with spotted knapweed found levels ranging between 291.6 to 389.8 \xg g"1 on average depending on proximity to the taproot (Bais et al, 2002). In another study, Perry et al. (2005b) found very high levels of (±)-catechin (mean = 1.55 mg g"1; high of 7.10 mg g"1) in field soil infested by spotted knapweed. However, Blair et al. (2005) detected no (±)-catechin in field soil collected from two different spotted knapweed infested sites. Following this study, soil samples from three separate spotted knapweed infested sites were collected and analyzed during the summer and fall of 2005 (Blair et al. 2006). They found (±)-catechin levels ranging from 0.0 to 1.3 ug g"1 on average depending on sampling date and site location (Blair et al, 2006). The secretion patterns of (±)-catechin by spotted knapweed may account for this difference as it can vary depending on season, age of plants, and presence of soil microbes (Jorge Vivanco, pers. comm.). The effect of (±)-catechin on A M fungi is difficult to determine as the production and stability of this allelochemical is not completely understood. If the level of (±)-catechin in soil occupied by spotted knapweed is similar to those published by Bais et al. 94 (2002) or Perry et al. (2005b) then it is likely that (±)-catechin plays a significant role in the altering of A M fungal communities by spotted knapweed. However, i f only trace amounts (< 5 ug g"1) are present in field soil, then (±)-catechin likely has little to no significant role in influencing A M fungi as this study showed that 5 pg g"1 of (±)-catechin has minimal affect on spore germination and hyphal growth of G. intraradices. Extensive investigation of (±)-catechin concentration in spotted knapweed infested soil is necessary to assess the impact this allelochemical has on A M fungi. In contrast to (±)-catechin, cnicin is not produced by spotted knapweed roots but is prevalent in the aerial tissues. Since cnicin is not volatile and is absent in the roots, it could reach soil due to leaching and/or decomposition of spotted knapweed litter (Kelsey and Locken, 1987). However, the low water solubility of cnicin may limit the amount that leaches into the soil. Cnicin concentrations in spotted knapweed tissues tend to peak in October when the tissue dies and dehydrates (Locken and Kelsey, 1987), and the litter accumulates (up to 5 cm thick) (Olson and Wallander, 2002). Only one study has measured the concentration of cnicin in spotted knapweed infested soils and found only trace amounts (up to 0.7 pg g"1) (Locken and Kelsey, 1987). The presence of such low amounts of cnicin in field soils is unlikely to affect A M fungi. Further investigation of cnicin levels and its stability in spotted knapweed infested soils would be valuable as results of this study shows that cnicin concentrations as low as 10 pg g"1 can inhibit A M fungi. Inhibitory effects of lower concentrations of cnicin, as well as (±)-catechin over longer duration also cannot be ruled out and must be explored. The results from this study indicate that (±)-catechin and cnicin inhibit A M fungi (G. intraradices) spore germination, and cnicin inhibits its hyphal growth. One or both 95 of these compounds may affect A M fungi in the field; however more information on the natural levels of these compounds in the field is necessary to verify this. Further investigation into how these allelochemicals affect other A M species in a more natural medium (i.e. soil) would be valuable. 96 4.5 Literature Cited Bais, H.P., T.S. Walker, F.R. Stermitz, R.A. Hufbauer and J .M. Vivanco. 2002. Enantiomeric-dependent phytotoxic and anti-microbial acitivity of (±)-catechin. A rhizosecrected racemic mixture from spotted knapweed. Plant Physiology 128:1173-1179. Bais, H.P., R. Vepachedu, S. Gilroy, R . M . Callaway and J .M. Vivanco. 2003. Allelopathy and exotic plant invasion: from molecules and genes to species interactions. Science 301:1377-1380. Blair, A .C . , B.D. Hanson, G.R. Brunk, R.A. Marrs, P. Westra, S.J. Nissen and R.A. Hufbauer. 2005. New techniques and findings in the study of a candidate allelochemical implicated in invasion success. Ecology Letters 8:1039-1047. Blair, A.C. , S.J. Nissen, G.R. Brunk and R.A. Hufbauer. 2006. A lack of evidence for an ecological role of the putative allelochemical (±)-catechin in spotted knapweed invasion success. Journal of Chemical Ecology 32:2327-2331. Bruno, M . , S. Rosselli, A . Maggio, R.A. Raccuglia, F. Napolitano and F. Senatore. 2003. Antibacterial evaluation of cnicin and some natural semisynthetic analogues. PlantaMedica 69:277-281. Callaway, R . M . and W . M . Ridenour. 2004. Novel weapons: invasive success and the evolution of increased competitive ability. Frontiers in Ecology and the Environment 2:436-443. Callaway, R . M . , G.C. Thelen, S. Barth, P.W. RAMsey and J.E. Gannon. 2004. Soil fungi alter interactions between the invader Centaurea maculosa and North American natives. Ecology 85:1062-1071. 97 Carey, E.V., M.J . Marler and R . M . Callaway. 2004. Mycorrhizae transfer carbon from a native grass to an invasive weed: evidence from stable isotopes and physiology. Plant Ecology 172:133-141. Elias, K.S. and G.R. Safir. 1987. Hyphal elongation of Glomus fasciculatus in response to root exudates. Applied and Environmental Microbiology 53:1928-1933. Juge, C , J. SAMson, C. Bastien, H. Vierheilig, A . Coughlan and Y . Piche. 2002. Breaking dormancy in spores of the arbuscular mycorrhizal fungus Glomus intraradices: a critical cold-storage period. Mycorrhiza 12:37-42. Kelsey, R.G. and L.J. Locken. 1987. Phytotoxic properties of cnicin, a sesquiterpene lactone from Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 13:19-33. Kirk, J.L., P. Moutoglis, J. Klironomos, H. Lee and J.T. Trevors. 2005. Toxicity of diesel fuel to germination, growth and colonization of Glomus intraradices in soil and in vitro transformed carrot root cultures. Plant and Soil 270:23-30. LeJeune, K.D. and T.R. Seastedt. 2001. Centaurea: the forb that won the west. Conservation Biology 15:1568-1574. Locken, L.J. and R.G. Kelsey. 1987. Cnicin concentrations in Centaurea maculosa, spotted knapweed. Biochemical Systematics and Ecology 15:313-320. Lutgen, E.R. and M . C . Rill ig. 2004. Influence of spotted knapweed (Centaurea maculosa) management treatments on arbuscular mycorrhizae and soil aggregation. Weed Science 52:172-177. 98 Marler, M.J. , C A . Zabinski and R . M . Callaway. 1999. Mycorrhizae indirectly enhance competitive effects of an invasive forb on a native bunchgrass. Ecology 80:1180-1186. Mummey, D.L., M . C Rillig and W.E. Holben. 2005. Neighboring plant influences on A M fungal community composition as assessed by T-RFLP analysis. Plant and Soil 271:83-90. Mummey, D.L. and M.C . Rillig. 2006. The invasive plant species Centaurea maculosa alters arbuscular mycorrhizal fungal communities in the field. Plant and Soil 288:81-90. Olson, B.E. and R.G. Kelsey. 1997. Effect Centaurea maculosa on sheep rumen microbial activity and mass in vitro. Journal of Chemical Ecology 23:1131 -1144. Olson, B.E. and R.T. Wallander. 2002. Effects of invasive forb litter on seed germination, seedling growth and survival. Basic and Applied Ecology 3:30^9-317. Panagouleas, C , H: Skaltsa, D. Lazari, A . L . Skaltsounis and M . Sokovic. 2003. Antifungal activity of secondary metabolites of Centaurea raphanina ssp. mixta, growing wild in Greece. Pharmaceutical Biology 14:266-270. Perry, L .G. , C. Johnson, E.R. Alford, J .M. Vivanco and M.W. Paschke. 2005a. Screening of grassland plants for restoration after spotted knapweed invasion. Restoration Ecology 13:725-735. Perry, L.G. , G . C Thelen, W . M . Ridenour, T.L. Weir, R . M . Callaway, M.W. Paschke and J.M. Vivanco. 2005b. Dual role for an allelochemical: (±)-catechin from Centaurea maculosa root exudates regulates conspecific establishment. Journal of Ecology 93:1126-1135. 99 Ridenour, W . M . and R . M . Callaway. 2001. The relative importance of allelopathy in interference: the effects of an invasive weed on a native bunchgrass. Oecologia 126:444-450. Skaltsa, H. , Lazari, D., C. Panagouleas, E. Georgiadou, B. Garcia and M . Sokovic. 2000. Sesquiterpene lactones from Centaurea thessala and Centaurea attica. Antifungal activity. Phytochemistry 55:903-908. Thelen, G.C., J .M. Vivanco, B. NewinghAM, W. Good, H.P. Bais, P. Landres, A . Caesar and R . M . Callaway. 2005. Insect herbivory stimulates allelopathic exudation by an invasive plant and the suppression of natives. Ecology Letters 8:209-217. Veluri, R., T.L. Weir, H.P. Bais, F.R. Stermitz and J .M. Vivanco. 2004. Phytotoxic and antimicrobial activities of catechin derivatives. Journal of Agricultural and Food Chemistry 52:1077-1082. Weir, T.L., H.P. Bais and J .M. Vivanco. 2003. Intraspecific and interspecific interactions mediated by a phytotoxin, (-)-catechin, secreted by the roots of Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 29:2397-2410. Zabinski, C.A., L . Quinn and R . M . Callaway. 2002. Phosphorus uptake, not carbon transfer, explains arbuscular mycorrhizal enhancement of Centaurea maculosa in the presence of native grassland species. Functional Ecology 16:758-765. 100 Chapter 5. General Discussion Many exotic weeds are a serious problem ecologically and economically to natural and managed habitats. This study investigated the allelopathic influence of two exotic weeds, tall hedge mustard (Sisymbrium loeselii L.) and spotted knapweed (Centaurea maculosa Lam.). Allelopathy is a strategy that several weed species use to influence the growth and distribution of competing species. The results of this research show that tall hedge mustard produces allelochemicals that have a direct effect on several competing species. Aqueous extracts prepared from tall hedge mustard root and shoot tissues inhibited seed germination and seedling growth of bluebunch wheatgrass, spotted knapweed, and Idaho fescue. One of the primary allelochemicals responsible for this inhibitory effect appears to be a glucosinolate (GSL) degradation product, isopropyl isothiocyanate (ITC). The parent GSL of isopropyl ITC (isopropyl GSL) was found in substantially higher concentrations in tall hedge mustard roots (ca. 2-fold) and shoots (ca. 10-fold) compared to other GSLs. Isopropyl ITC was the primary GSL degradation product identified in tall hedge mustard aqueous root and shoot extracts. It also exhibited an inhibitory effect on seed germination and seedling growth. The role of other glucosinolate degradation .products (e.g. sec-butyl ITC) and other secondary metabolites in the direct allelopathic influence of tall hedge mustard cannot be ruled out. The results showed that tall hedge mustard allelochemicals, which inhibited germination and growth of neighboring species, also inhibited the A M fungus, Glomus intraradices. Aqueous tall hedge mustard extracts and their two.primary glucosinolate degradation products (isopropyl ITC and sec-butyl ITC) strongly inhibited spore 101 germination and hyphal growth of Glomus intraradices. The results also showed that tall hedge mustard infestations reduce the A M inoculum potential of soil, supporting a negative effect of tall hedge mustard on A M fungal communities. The inhibitory effect of these allelochemicals could create an environment favouring only tall hedge mustard plants and a few other species capable of tolerating these chemicals. This would enable tall hedge mustard to outcompete neighboring species and form relatively pure stands. Further research investigating the presence of these allelochemicals in and around the rhizosphere of tall hedge mustard plants in the field may provide better insight into the allelopathic influence of this exotic weed. It is concluded from these results that tall hedge mustard produces allelochemicals that inhibit A M fungi and may be responsible for the reduced A M inoculum potential of tall hedge mustard infested soil, which could adversely affect the competitive ability of neighboring species benefiting from A M associations. Previous studies have revealed that spotted knapweed utilizes allelopathy as a strategy to directly compete with neighboring species (Ridenour and Callaway, 2001). The allelochemicals implicated in this effect include the root exuded flavanol (±)-catechin (Bais et al., 2002; Weir et al., 2003) and the sesquiterpene lactone cnicin (Kelsey and Locken, 1987). The results from this study have shown that these allelochemicals also have a strong inhibitory effect on A M fungi. Both allelochemicals inhibited the spore germination of Glomus intraradices, and cnicin inhibited the hyphal growth. It is possible that these compounds may be involved in the negative effect spotted knapweed infestations have on A M fungal communities. However, determining at what concentrations these allelochemicals are produced in the field will be required to 102 make an accurate assessment. The amount of (±)-catechin produced by spotted knapweed in the field is a controversial issue as a wide range of concentrations (0 to 7.1 mg g"1) has been found in soil occupied by spotted knapweed (Bais et al., 2002; Perry et al, 2005; Blair et al., 2006). The secretion patterns of (±)-catechin by spotted knapweed may account for this difference as it can vary depending on season, age of plants, and presence of soil microbes (Jorge Vivanco, pers. comm.). Further investigation is needed to determine if biologically active concentrations of these allelochemicals are produced by spotted knapweed, and their role in influencing mycorrhizal associations in nature. This research has increased our understanding of possible allelopathic strategies utilized by the exotic weeds tall hedge mustard and spotted knapweed in their interaction with neighboring species. Both species produce allelochemicals that directly inhibit germination and growth of competing species, and inhibit the spore germination and hyphal growth of A M fungi. Tall hedge mustard infestations also reduced A M inoculum potential of soils. These allelochemicals may be released into the environment to create a habitat not favorable for neighboring species thereby allowing these weeds to dominate and possibly establish monocultures. An understanding of the mechanisms used by exotic weeds may facilitate development of better strategies to reduce their negative ecological/economic impact in natural and managed habitats. 103 5.1 Literature Cited Bais, LLP., T.S. Walker, F.R. Stermitz, R.A. Hufbauer and J .M. Vivanco. 2002. Enantiomeric-dependent phytotoxic and anti-microbial acitivity of (±)-catechin. A rhizosecrected racemic mixture from spotted knapweed. Plant Physiology 128:1173-1179. Blair, A.C. , S.J. Nissen, G.R. Brunk and R.A. Hufbauer. 2006. A lack of evidence for an ecological role of the putative allelochemical (±)-catechin in spotted knapweed invasion success. Journal of Chemical Ecology 32:2327-2331. Kelsey, R.G. and L.J . Locken. 1987. Phytotoxic properties of cnicin, a sesquiterpene lactone from Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 13:19-33. Perry, L.G. , G.C. Thelen, W . M . Ridenour, T.L. Weir, R . M . Callaway, M.W. Paschke and J .M. Vivanco. 2005. Dual role for an allelochemical: (±)-catechin from Centaurea maculosa root exudates regulates conspecific establishment. Journal of Ecology 93:1126-1135. Ridenour, W . M . and R . M . Callaway. 2001. The relative importance of allelopathy in interference: the effects of an invasive weed on a native bunchgrass. Oecologia 126:44.4-450. Weir, T.L., H.P. Bais and J .M. Vivanco. 2003. Intraspecific and interspecific interactions mediated by a phytotoxin, (-)-catechin, secreted by the roots of Centaurea maculosa (spotted knapweed). Journal of Chemical Ecology 29:2397-2412. 104 

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