Integrating Selective Herbicide and Native Plant Restoration to Control Alternanthera Philoxeroides (Alligator Weed)

Home , Alternanthera, Sagittaria platyphylla

INTEGRATING SELECTIVE HERBICIDE AND NATIVE PLANT RESTORATION

TO CONTROL Alternanthera philoxeroides (ALLIGATOR WEED)

Justin Adams, B.S.

Thesis Prepared for the Degree of

MASTER OF SCIENCE

UNIVERSITY OF NORTH TEXAS

December 2011

APPROVED:

Kevin Stevens, Major Professor Barney Venables, Committee Member Aaron Roberts, Committee Member Arthur J. Goven, Chair of the Department of Biological Sciences James D. Meernik, Acting Dean of the Toulouse Graduate School Adams, Justin. Integrating Selective Herbicide and Native Plant Restoration to Control

Alternanthera philoxeroides (Alligator Weed). Master of Science (Environmental Science),

December 2011, 39 pp., 7 tables, 19 figures, bibliography , 67 titles.

Exotic invasive aquatic weeds such as alligator weed (Alternanthera philoxeroides) threaten native ecosystems by interfering with native plant communities, disrupting hydrology, and diminishing water quality. Development of new tools to combat invaders is important for the well being of these sensitive areas. Integrated pest management offers managers an approach that combines multiple control methods for better control than any one method used exclusively. In a greenhouse and field study, we tested the effects of selective herbicide application frequency, native competitor plant introduction, and their integration on alligator weed. In the greenhouse study, alligator weed shoot, root, and total biomass were reduced with one herbicide application, and further reduced with two. Alligator weed cumulative stem length and shoot/root ratio was only reduced after two applications. In the greenhouse, introduction of competitors did not affect alligator weed biomass, but did affect shoot/root ratio. The interaction of competitor introduction and herbicide did not significantly influence alligator weed growth in the greenhouse study. In the field, alligator weed cover was reduced after one herbicide application, but not significantly more after a second. Introduction of competitor species had no effect on alligator weed cover, nor did the interaction of competitor species and herbicide application. This study demonstrates that triclopyr amine herbicide can reduce alligator weed biomass and cover, and that two applications are more effective than one. To integrate selective herbicides and native plant introduction successfully for alligator weed control, more research is needed on the influence competition can potentially have on alligator weed growth, and the timing of herbicide application and subsequent introduction of plants. Copyright 2011

Justin Adams

ii TABLE OF CONTENTS

Page

LIST OF TABLES ...... iv

LIST OF FIGURES ...... v

Chapters

INTRODUCTION ...... 1

METHODOLOGY ...... 8

RESULTS ...... 19

DISCUSSION ...... 29

WORKS CITED ...... 35

iii LIST OF TABLES

Page

Table 1. Greenhouse Experiment Table of Treatments ...... 8

Table 2. Field Experiment Table of Treatments ...... 14

Table 3. Cover Classes of Daubenmire Method ...... 17

Table 4. Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide

Applications and Four Competitor Species Introductions on A. philoxeroides ...... 20

Table 5. Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide

Applications and Competitive Species (Monocots and Dicots Grouped) Introduction on

A. philoxeroides ...... 21

Table 6. Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide

Applications and Competitor Introduction on A. philoxeroides Cover ...... 26

Table 7. Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide

Applications and Competitor Introduction (Monocots and Dicots Grouped) on A.

philoxeroides Cover ...... 27

iv LIST OF FIGURES

Page

Figure 1. A. philoxeroides in treatment containers prior to herbicide treatments or introduction of

other plants...... 9

Figure 2. Treatment containers randomly selected for the first application of triclopyr herbicide. ..

...... 10

Figure 3. Plant species used for competition treatments...... 10

Figure 4. Introduction of a competitor species into pots containing herbicide treated A.

philoxeroides...... 11

Figure 5. Satellite image of field study site in Grand Prairie, Texas...... 13

Figure 6. Example of one row of a block at the Grand Prairie field site...... 15

Figure 7. Planting of Carex hyalinolepis following the first herbicide application...... 15

Figure 8. The first triclopyr application on A. philoxeroides at the Grand Prairie field site...... 16

Figure 9. Carex brevior and A. philoxeroides in a greenhouse treatment container that received

two herbicide applications...... 19

Figure 10. Greenhouse treatment containers on day of harvest...... 22

Figure 11. Justicia american roots (top), A. philoxeroides roots (bottom)...... 23

Figure 12. Effect of herbicide application frequency on A. philoxeroides total, root, and shoot dry

weight. n = 15 for each treatment...... 23

Figure 13. Effect of herbicide frequency on A. philoxeroides cumulative stem heights. n = 15 for

each treatment...... 24

Figure 14. Effect of herbicide frequency on A. philoxeroides shoot/root ratio. n = 15 for each

treatment...... 25

v Figure 15. Effect of competitor introduction on A. philoxeroides shoot/root ratio. n = 9 for each

treatment...... 25

Figure 16. 1m x 1m PVC quadrat to aid in estimating A. philoxeroides cover within field

treatment plots...... 26

Figure 17. Mean ± standard error A. philoxeroides cover exposed to three herbicide application

frequencies. n = 25 for each treatment...... 27

Figure 18. Field experiment block four. A. philoxeroides after three levels of herbicide

application...... 28

Figure 19. Carex hyalinolepis and A. philoxeroides after two herbicide applications...... 28

INTRODUCTION

Spread and invasion by non-indigenous species is an expanding global problem. In the

U.S. alone, expensive prevention and eradication techniques costing hundreds of billions of dollars annually are employed to stop the environmental, agricultural, and health damages these invaders can cause (Bertness 1984, Vitousek et al. 1987, Mack et al. 2000, Pimentel et al. 2005,

Lodge et al. 2006). Invasive non-indigenous aquatic plants are particularly difficult to control due to their ability to move easily through aquatic and riparian corridors, invade and dominate disturbed habitat, and reproduce vegetatively from fragments carried downstream (Hobbs and

Huenneke 1992, Madsen and Smith 1997, Boose and Holt 1999, Hood and Naiman 2000). Alien aquatic weeds often out-compete native species, consequently altering important ecosystem processes (Madsen et al. 1991, Cheruvelil et al. 2001, Urgenson et al. 2009). As the movement to protect wetlands and other aquatic ecosystems continues to grow, so too does the need to establish new tools for combating invasion of non-indigenous aquatic plants.

Integrated pest management (IPM) strategies began to develop in the 1950’s in response to concerns over “undisciplined” and sometimes “unrestrained” use of chemical pesticides in agriculture (Castle et al. 2009). Modern IPM combines multiple control methods and integration of pest biology knowledge in an attempt to more efficiently manage target species (Buhler et al.

2000). Many IPM strategies are employed to increase effectiveness of natural enemies, therefore reducing dependence on chemical control. For example, modern insecticides tailored to specific insect pests that do not adversely affect natural predators can be used to keep natural predation viable (Gentz et al. 2010). Another strategy involves installation of “banker plants” amongst crops that, through carrying a non-pest food source, aid in attracting, retaining, or dispersal of natural enemies for biological control (Pineda et al. 2008). Some IPM strategies integrate

cultural methods. Examples include practices that interfere with pest lifecycles, such as crop rotation or tillage (Meissle et al. 2011). Increasingly, crops are being grown with genetic enhancements, such as introduction of genetic material from the natural insecticide producing bacterium Bacillus thuringiensis (Meissle et al. 2011).

IPM management programs uniquely tuned for weeds have grown as attention to weed biology and ecology has increased (Appleby 2005). As with insect pests, IPM for weeds attempts to improve control without solely relying on chemical herbicides. Agricultural examples of cultural IPM practices for weeds include; using alternative cultivars, adjusting fertilizer rates, crop rotations, and smothering of weeds through the use of “cover crops” (Valenti and Wicks

1992, Wyse 1994). As with insect pest, biological control of weeds is possible. Introducing herbivory from exotic insects or bolstered native populations are common tools (Cofrancesco

1998).

Integrated management techniques for aquatic weeds do exist, but integration of available tools is less intensely researched then their terrestrial counterparts. Aquatic herbicides are available for chemical control of aquatic weeds, but have a special set of problems associated with them. Available herbicides are limited because, due to the sensitive nature of aquatic habitats, many terrestrial herbicides cannot clear regulatory hurdles to cross over into aquatics.

Herbicides that can be used are often limited by application restrictions. Applications of aquatic herbicides can result in dissolved oxygen crashes and subsequent fish and invertebrate mortality if too much vegetation is eliminated at once. Control of invasive aquatic weeds with traditional herbicide applications can lead to further invasions and impede progress if native plant community restoration is the intended goal. Herbicides can create a disturbance that may rapidly fill with new invasive species or be re-invaded by the original target weed if high efficacy is not

achieved (Morrison 2002, Ogden and Rejmanek 2005). Development of new IPM strategies for aquatic weeds that replace, reduce, or improve effectiveness of herbicides is of particular importance due to a federal regulatory environment that may soon favor tighter controls on aquatic herbicide application practices (Nat’l Cotton Council v. EPA, 553 F.3d 927 6th Cir.

2009).

Biological and cultural control options for aquatics do exist. Well known insect biocontrol agents for invasive aquatics include the giant salvinia weevil (Flores and Carlson

2006), alligator weed flea-beetle (Sainty et al. 1998) and waterhyacinth weevils (Haag and

Habeck 1991). Fish as well as insects are used in aquatics plant management. Examples include introduction of tilapia for giant salvinia control (McIntosh et al. 2008), and triploid grass carp for hydrilla control (Cuda et al 2008). Current cultural methods for aquatic weed control involve watershed level techniques to reduce nutrient loads and improve water quality. Examples include wetland reestablishment, installation of vegetated buffer strips between agricultural and drainages, phosphate free detergents, and efficient agricultural fertilizer plans (Jeppesen et al.

1999). Reservoir level cultural techniques include temporary manipulation of water level to suppress vegetation (Poovey and Kay 1998), or reduction of available light through aquatic dyes or floating shade mats (Schooler 2008). Manipulating the availability of the resources required for weed growth at the reservoir level is a possible management tool uniquely tuned for weeds

(Buhler et al. 2000). The availability of necessary resources to any particular plant is often limited by competition for the same resources from another plant (Harris 1967). Re-introduction of native plants to compete with aquatic weeds is a possible management tool, and has been explored with some success in terrestrial applications. In a prairie restoration project, Bakker and

Wilson (2004) found that plots restored with native grasses reduced invasion of A. cristatum by

one-third as compared to un-restored plots. In situations where re-introduction alone is not enough to stop an invasion, manipulation of the competitive arena before re-introduction of native species may be necessary to give native competitors a head start or competitive edge

(Perry and Galatowitsch 2003). Integrating herbicide treatments targeting an invasive weed prior to, and after native plant reintroduction is a management option that may result in more effective control than either technique used exclusively. Hubbard (1975) reported a 30% increase in native grass dry matter yield in knapweed infested plots treated with herbicide previous to seeding as compared to plots exposed to seeding only. Little information is available for applying this type of IPM technique to invasive aquatic weeds.

Alligator weed, Alternanthera philoxeroides, is a stoloniferous and rhizomatous perennial weed native to South America (Sainty et al. 1998). The plant can grow under aquatic, semi- aquatic, and terrestrial conditions. In water, hollow stems allow the plant to extend across deep water to form dense floating mats (Julien et al. 1995). A. philoxeroides was first reported in the

United States in 1894, and although it may have reached the maximum extent of its range in the

US, A. philoxeroides has invaded many waterways, wetlands, irrigation canals, and low-lying areas in California and much of the southeastern US (Maddox et al. 1971, Coulson 1977). A. philoxeroides is classified as a noxious plant in seven states including Texas (USDA, NRCS

2010). In the United States, A. philoxeroides does not produce viable seeds; reproduction is achieved vegetatively (Quimby and Kay 1977). Sections of stem containing a node are capable of establishing a new colony (Vogt et al. 1992), and fragmentation readily occurs as a result of mechanical or environmental disturbances such as harvest attempts or flooding. A. philoxeroides invasions can reduce the economic and ecological value of a waterway. A. philoxeroides can impede pump intakes for irrigation, restrict flow in small waterways leading to blockages and

sedimentation, and impede navigation and recreation (Holm et al. 1997). Like many exotic invasive weeds, A. philoxeroides is often able to out-compete and replace native plant communities. Vogt et al. (1992) reported un-contested spread of A. philoxeroides mats in the southern Mississippi valley prior to introduction of biocontrol agents. Floating mats of A. philoxeroides can restrict light penetration to the water below, effectively starving other submerged plants and reducing oxygen levels (Quimby and Kay 1976).

Effective management options for A. philoxeroides invasions are limited. Mechanical harvesting or tilling can be used for temporary control, but the plant is capable of rapid re-growth after disturbance, and dislodged fragments may increase the extent of invasion if allowed to disperse and colonize new territory (Vogt et al. 1992, Wilson et al. 2007). Biological control options exist, but effectiveness is dependent on target area climate and A. philoxeroides growth form. The alligator weed flea-beetle, Agasicles hygrophila, and the alligator weed stem borer,

Vogtia mallo, were first released in the southern United States by the USDA in 1964 and 1971 respectively (Sainty et al. 1998). These insects can cause significant damage to floating A. philoxeroides mats, but do not populate terrestrial infestations and are not tolerant of the cooler climate associated with the northern range of A. philoxeroides in the United States (Julien et al.

1995). Research into other possible biological control candidates is ongoing (Julien et al. 2004).

A. philoxeroides is a difficult plant to control chemically; numerous attempts at control with various herbicides, application frequencies, and applications timings have been attempted.

Typically, only partial control is achieved. Leaves and stems are damaged, but re-growth generally occurs. Bowmer et al. (1993) reported that only 7% of glyphosate herbicide applied to

A. philoxeroides via foliar treatment reached underground organs. Tucker et al. (1994) found better herbicide transport to roots with imazapyr than glyphosate, but field studies involving

imzapyr lack evidence for complete control (Allen et al. 2007, Schooler et al. 2008). Kay (1992) found increased resistance to quinclorac herbicide in a broad stemmed A. philoxeroides biotype compared to a slender stemmed biotype. Kay (1992) was able to reduce A. philoxeroides biomass after foliar applications applied to both biotypes, but was unable to completely kill the plant.

Recent field experiments involving relatively new selective aquatic herbicides offer promising results that can be applied to the development of new integrated A. philoxeroides control techniques. In a field study at a managed marsh in southeastern Alabama, Allen et al.

(2007) altered A. philoxeroides above ground biomass after applications and interactions of two herbicides (selective and non-selective), two application dates, and three application rates. Allen et al. (2007) included effects on native species into the design and found, in addition to a decrease in A. philoxeroides over controls, an increase of native plant biomass within a year of

July application using selective herbicides over non-selective herbicides. Schooler et al. (2008) reported presence of A. philoxeroides in all study plots seven months after treatment with glyphosate, metsulfuron and triclopyr at four applications each and at the highest label recommended rate. Although not directly tested, Schooler et al. (2008) credits increased competition from monocots for elimination of A. philoxeroides from 29% of plots sampled one year after treatment with dicot-selective herbicides (metsulfuron and triclopyr) versus 0% elimination with non selective herbicide (glyphosate). These studies hint at the possibility of integrating native plant community involvement with the use of selective herbicides for A. philoxeroides control.

Given the potential economic and ecological damages that can ensue from the spread of

A. philoxeroides and the paucity of information on effective control measures for this plant, the objective of this study is to determine the effects of integrating selective herbicide applications

and native plant restoration on A. philoxeroides growth. I will test the hypothesis that introduction of native monocot species between dicot-selective herbicide treatment intervals may provide stronger control than selective herbicide treatments or native plant restoration alone. The results of this study will lead to the availability of an effective tool and management option for A. philoxeroides control.

METHODOLOGY

A greenhouse component and a field component were included in this project.

Similar treatment regimes were applied to both components.

Greenhouse Experiment

The greenhouse component of this project took place at the Water Research Field Station greenhouse at the University of North Texas. The experiment utilized a 3X5 factorial design with three replicates per treatment level (Table 1). Treatments applied were “herbicide application frequency” and “competitor species.” Effects of herbicide application frequency were tested using three levels; application frequencies of zero, one, and two. Effects of competitor species were tested using five levels; no competitors, two native monocot species, and two native dicot species. The four plant species were selected based on data from previous field surveys and seed bank surveys taken within the field study site.

Table 1

Greenhouse Experiment Table of Treatments

Competitor Species

Justica Polygonum Sagittaria

No Competitor americana densiflorum platyphylla Carex brevior

Herbicide 0 n=3 n=3 n=3 n=3 n=3

Application 1 n=3 n=3 n=3 n=3 n=3

Frequency 2 n=3 n=3 n=3 n=3 n=3

Note. Three replicates per treatment were used.

Greenhouse Experiment Materials and Methods

A. philoxeroides was propagated vegetatively from similarly sized, single node, clippings collected from greenhouse stock plants originally collected from a retention pond at Six Flags

over Texas in Arlington, Texas. On August 6th 2010, clippings were planted in 45- 6.59L plastic treatment containers filled with 4.0L of a (Premier Tech Horticulture Ltd, Quakertown, PA)

50/50 mixture of sand and PRO-MIX™ potting soil (Figure 1). Holes drilled near the top of each container allowed irrigation water to drain, creating a shallow layer of surface water (< 1.0 cm) for plants to grow in. Before A. philoxeroides clippings were planted, empty 0.95L pots were placed in the soil of 36 randomly selected treatment containers to allow space for introduction of competitor species with minimal A. philoxeroides root disturbance (Figure 4). Two competitor dicot species; Justica americana and Polygonum densiflorum, and two competitor monocot species; Sagittaria platyphylla and Carex brevior, were propagated at the University of North

Texas aquatic plant nursery in 0.95L pots containing a 50/50 mixture of PRO-MIX™ (Premier

Tech Horticulture Ltd, Quakertown, PA), and sand (Figure 3). All plants were drip irrigated with

1.89 L/Day filtered well water.

Figure 3. A. philoxeroides in treatment containers prior to herbicide treatments or introduction of other plants.

Figure 4. Treatment containers randomly selected for the first application of triclopyr herbicide.

Figure 3. Plant species used for competition treatments. Left to Right: Juncus sp. (not used),

Carex brevior, Sagittaria platyphylla, Justica americana, Polygonum densiflorum.

Figure 4. Introduction of a competitor species into pots containing herbicide treated A. philoxeroides.

Greenhouse temperature was maintained at 22°C. Sufficient lighting to keep plants from entering senescence was not available until January 10th 2011, after which plants were grown under a

14/10 light/dark cycle. Triclopyr amine, a dicot-selective herbicide under the trade name

Renovate®3 (Renovate, SePRO, Carmel, IN 46032) was used for herbicide treatments. Triclopyr was applied at a rate of 3.42 kg ae ha¯¹ using a Solo® 425® 15.14L pressurized back-pack sprayer. AQUA-KING®Plus (Estes Inc., Wichita Falls, TX 76301) a non-ionic surfactant was added at a rate of 0.25% of spray solution. Forty-three days after A. philoxeroides was planted, the first round of herbicide application was administered. To prevent cross contamination from herbicide drift, treatment containers randomly selected for herbicide treatment were removed from the greenhouse and sprayed outside (Figure 2). Spraying took approximately 15 minutes.

To eliminate the possibility of herbicide transferring to neighboring plants through plant to plant contact, treated plants remained outside for one hour to allow the spray solution to evaporate.

Competitor species were introduced into randomly selected treatment and control containers after 54 days. On day 85, a subset of previously sprayed treatment containers was randomly selected for a second spray application. After 239 days, all vegetation was removed from treatment containers and separated into root and shoot sections. Fresh cumulative stem heights were obtained to the nearest millimeter during processing. Above ground, below ground, and total dry biomass was to the nearest mg obtained after plant material was placed in drying ovens at 40°C for five days.

Statistical Analysis

After harvesting, a two-way analysis of variance (ANOVA)(SAS Institute 2003) was used to test effects of competitor species, herbicide application frequency, and all interactions on total A. philoxeroides dry biomass, above ground dry biomass, below ground dry biomass, fresh cumulative stem length, and dry shoot/root ratio. A duplicate analysis was run with individual species combined as monocots or dicots. Data met requirements for normality and homoscedasticity. Means were considered significant at p < 0.05. When significant main effects or interaction effects were detected, multiple comparisons were conducted using the Bonferonni correction (Zar 2009).

Field Experiment

The field component of this project occurred within an oxbow of the Trinity River adjacent to the city landfill in Grand Prairie, Texas (96°56'35.793"W 32°46'27.035"N) (Figure

5). The oxbow covers an approximate area of 7.5 hectares and is subject to periodic flooding after heavy rains. A. philoxeroides was first reported within the oxbow area in 2008 and was widespread by 2010. A. philoxeroides growth in the oxbow may best be described as rooted emergent. As a follow up to the greenhouse component of this project, the field component used

a similar experimental design. The field experiment utilized a blocked 3X5 factorial design with

5 replicates per treatment level (Table 2). Treatments applied were “herbicide application frequency” and “competitor species”. Effects of herbicide application frequency were tested using three levels; frequencies of zero, one, and two applications. Effects of competitor species were tested

Figure 5. Satellite image of field study site in Grand Prairie, Texas.

using five levels; no re-established native competitors, two re-established native monocot species, and two re-established native dicot species. The four plant species were selected based on data from previous field surveys and seed bank surveys taken within the oxbow area.

Table 2

Field Experiment Table of Treatments

Competitor Species

No Justica Polygonum Sagittaria Carex

Competitor americana setaceum platyphylla hyalinolepis

Herbicide 0 n=5 n=5 n=5 n=5 n=5

Application 1 n=5 n=5 n=5 n=5 n=5

Frequency 2 n=5 n=5 n=5 n=5 n=5

Note. Five replicates for each treatment combination were used.

Field Experiment Materials and Methods

Five experimental blocks were established at separate sites within the oxbow. Each block exhibited relatively uniform hydrology and A. philoxeroides coverage, and was of sufficient size to house subplots. Study blocks consisted of fifteen 1m² treatment sub-plots divided into three columns (Figure 6). To simplify herbicide applications and reduce the risk of cross-plot herbicide drift, entire columns received the same level of herbicide application frequency. Columns had a

2m buffer zone between them to minimize contamination from drift. Within each column, all representatives of the “competitor” treatment were randomly assigned a subplot. Two competitor dicot species; Justica americana and Polygonum setaceum, and two competitor monocot species;

Sagittaria platyphylla and Carex hyalinolepis, were propagated at the University of North Texas

Figure 6. Example of one row of a block at the Grand Prairie field site. A. philoxeroides pictured has not been subjected to any treatments.

Figure 7. Planting of Carex hyalinolepis following the first herbicide application.

Figure 8. The first triclopyr application on A. philoxeroides at the Grand Prairie field site. aquatic plant nursery in 0.95L pots containing a 50/50 mixture PRO-MIX™ (Premier Tech

Horticulture Ltd, Quakertown, PA) potting soil and sand. On October 1st 2010, two columns from each study block were randomly selected to receive the initial herbicide application (Figure

8). Triclopyr amine, a dicot-selective herbicide under the trade name Renovate®3 (Renovate,

SePRO, Carmel, IN 46032) was used for herbicide treatments. Triclopyr was applied at a rate of

3.42 kg ae ha¯¹ using a Solo® 425® 15.14L pressurized back-pack sprayer. AQUA-KING®Plus

(Estes Inc., Wichita Falls, TX 76301) non-ionic surfactant was added at a rate of 0.25% of spray solution. Thirteen days after initial spraying, competitor plant species were randomly distributed amongst the subplots. To simulate a high density re-establishment, each subplot was issued four

individual plants of its assigned species. Individual plants were uniformly dispersed within subplots using a PVC planting “jig” (Figure 7). The “jig” was placed at each right angle corner of subplots and directed the planter to a specific planting location so plants were situated identically across all treatment plots. On day 28, one of the previously sprayed columns in each study block was randomly selected to receive the second herbicide treatment. Two-hundred thirty-seven days after the initial herbicide application, A. philoxeroides coverage was evaluated in the field with the Daubenmire method. The Daubenmire method allows a sampler to place percent cover estimates into one of six cover classes (Table 3) (Dabenmire 1959). Digital photos of each subplot were taken as well. This data has been archived for use in future projects where comparisons of the historic plant community in the subplots may be appropriate (Figure 16).

Table 3

Cover Classes of Daubenmire Method

Daubenmire Cover Class Range of Coverage Midpoint of Range

1 < 5% 2.5%

2 5-25% 15.0%

3 25-50% 37.5%

4 50-75% 62.5%

5 75-90% 85.0%

6 95-100% 97.5%

Statistical Analysis

A two-way randomized complete block analysis of variance (ANOVA)(SAS Institute

2003) was used to test effects of competitor species, herbicide application frequency, and all interactions on A. philoxeroides mean cover class midpoints. A duplicate analysis was run with individual species combined as monocots or dicots. Data met requirements for normality and

homoscedasticity. Means were considered significant at p < 0.05. When significant main effects or interaction effects were detected, multiple comparisons were conducted using the Bonferonni correction (Zar 2009)

RESULTS

Greenhouse Experiment

Regardless of treatments, there was no complete eradication of A. philoxeroides from the treatment containers (Figure 9). A. philoxeroides cumulative stem length, shoot, root, and total dry weight were affected by herbicide application, but not significantly influenced by competitor introduction or the interaction of competition and herbicide (Table 4). A. philoxeroides shoot/root ratio was affected by herbicide application and competitor introduction, but not influenced by their interaction (Table 4). Combining individual competitors into monocot or dicot groups did not significantly alter results (Table 5).

Figure 9. Carex brevior and A. philoxeroides in a greenhouse treatment container that received two herbicide applications.

Table 4

Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide Applications and Four Competitor Species Introductions on A. philoxeroides Biomass, Cumulative Stem

Length, and Shoot/Root Ratio

Competitive Species Herbicide Herbicide X Competitive Species Response Variable

F p Value F p Value F p Value

Shoot Dry Weight 1.90 0.1372 35.29 <.0001 1.21 0.3292

Root Dry Weight 0.66 0.6233 38.25 <.0001 1.59 0.1692

Total Dry Weight 1.16 0.3478 43.12 <.0001 1.47 0.2090

Cumulative Stem 1.51 0.2231 23.46 <.0001 1.04 0.4297

Length

Shoot/Root Ratio 3.10 0.0301 25.52 <.0001 0.77 0.6329

Table 5

Summary Table of Two-way ANOVA Assessing the Effects of Triclopyr Herbicide Applications and Competitive Species (Monocots and Dicots Grouped) Introduction on A. philoxeroides

Biomass, Cumulative Stem Length, and Shoot/Root Ratio

Competitive Species Herbicide X Competitive Species (Monocots and Dicots Herbicide Response Variable (Monocot and Dicot Combined) Combined )

F p Value F p Value F p Value

Shoot Dry Weight 2.02 0.1479 31.48 <.0001 1.43 0.2444

Root Dry Weight 0.54 0.5851 31.72 <.0001 1.38 0.2612

Total Dry Weight 1.09 0.3476 36.86 <.0001 1.52 0.2159

Cumulative Stem 2.65 0.0841 22.44 <.0001 1.04 0.2069

Length

Shoot/Root Ratio 5.87 0.0062 21.33 <.0001 0.41 0.8003

Mean A. philoxeroides shoot, root, and total dry weight were significantly reduced with increasing herbicide application frequency (Figure 12). Triclopyr applications reduced mean shoot weight below controls (3.722g ± 0.285) after one application (2.257g ± 0.285), and further after two applications (0.341g ± 0.285). Triclopyr applications reduced mean root weight below controls (8.004g ± 0.501) after one application (5.028g ± 0.501), and further after two applications (1.744g ± 0.501). Triclopyr applications reduced mean total weight below controls

(11.726g ± 0.735) after one application (7.284g ± 0.735), and further after two applications

(2.085g ± 0.735). A. philoxeroides mean cumulative stem length was significantly lower at the

two herbicide application level (23.660cm ± 15.893), but did not differ significantly between zero (175.67cm ± 15.893) and one application (120.85cm ± 15.893) (Figure 13). A. philoxeroides mean shoot/root dry weight ratio was significantly lower at the two herbicide application level (0.1486 ± 0.035), but did not differ significantly between zero (0.461 ± 0.035) and one application (0.4522 ± 0.035) (Figure 14). Shoot/root dry weight ratio was the only measured response affected by a treatment other than herbicide application (Figure 11).

Introduction of competitor species influenced A. philoxeroides mean shoot weight/ root dry weight ratio (Table 4). Of introduced competitors, Justicia sp was the only plant that significantly reduced A. philoxeroides shoot/root dry weight ratio lower than control (Figure 15).

Figure 10. Greenhouse treatment containers on day of harvest.

Figure 11. Justicia american roots (top), A. philoxeroides roots (bottom).

Figure 12. Effect of herbicide application frequency on A. philoxeroides total, root, and shoot dry weight. n = 15 for each treatment. Means are presented ± standard error. Different letters within each group of response variables indicate significantly different means (p < 0.05). 0 = no herbicide application. 1 = 1 herbicide applications. 2 = 2 herbicide applications.

Figure

13. Effect of herbicide frequency on A. philoxeroides cumulative stem heights. n = 15 for each treatment. Means are presented ± standard error. Different letters indicate significantly different means (p < 0.05).

Figure 14. Effect of herbicide frequency on A. philoxeroides shoot/root ratio. n = 15 for each treatment. Means are presented ± standard error. Different letters indicate significantly different means (p < 0.05).

Figure 15. Effect of competitor introduction on A. philoxeroides shoot/root ratio. n = 9 for each treatment. Means are presented ± standard error. Different letters indicate significantly different means (p < 0.05). No_comp = no competitor introduction. Sag_pla = Sagittari platyphylla.

Car_bre = Carex brevior. Pol_den = Polygonum densiflorum. Jus_ame = Justicia american.

Field Experiment

A. philoxeroides cover was affected by herbicide application, but not significantly influenced by competitor introduction or the interaction of competition and herbicide (Table 6)

(Figure 19). Combining individual competitors into monocot or dicot groups did not significantly alter results (Table 7). Triclopyr reduced A. philoxeroides mean cover significantly over controls

(31.00 ± 6.935) after both one application (16.70 ± 6.935) and two applications (6.90 ± 6.935), but there was no significant difference in cover between one and two herbicide applications

(Figure 17) (Figure 18). Eight months after initial treatments, A. philoxeroides above ground parts were not present in six plots that received two applications, two plots that received one application, and two plots that received zero applications.

Figure 16. 1m x 1m PVC quadrat to aid in estimating A. philoxeroides cover within field treatment plots.

Table 6

Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide Applications and Competitor Introduction on A. philoxeroides Cover

Competition Herbicide Herbicide X Competition Response Variable

F p Value F p Value F p Value

A. philoxeroides 0.13 0.9697 9.03 0.0004 0.14 0.9966 cover

Table 7

Summary Table of Two-Way ANOVA Assessing the Effects of Triclopyr Herbicide Applications and

Competitor Introduction (Monocots and Dicots Grouped) on A. philoxeroides Cover

Competition (Monocots Herbicide Herbicide X Competition Response Variable and Dicots Combined )

F p Value F p Value F p Value

A. philoxeroides 0.20 0.8167 8.01 0.0008 0.21 0.9333

Figure 17. Mean ± standard error A. philoxeroides cover exposed to three herbicide application frequencies. n = 25 for each treatment. Means are presented ± standard error. Different letters indicate significantly different means (p < 0.05)

Figure 18. Field experiment block four. A. philoxeroides after two herbicide applications (left),

Zero applications (middle), one application (right).

Carex hyalinolepis

A. philoxeroides

Figure 19. Carex hyalinolepis and A. philoxeroides after two herbicide applications

DISCUSSION

Greenhouse Experiment

There was no evidence in the greenhouse experiment of enhanced control of A. philoxeroides through integrating herbicide application and competitor introduction. When each component is considered separately, it maybe surmised that the herbicide fulfilled its role, but the competitive interaction needed was not present. The greenhouse experiment should have provided the opportunity to run the experiment at any photoperiod desired. Prior to early January

2011, there was inadequate lighting available to keep the introduced plants from falling into their seasonal senescence cycle. To take advantage of the window of competitive opportunity provided by the herbicide applications, introduced plants may have benefited from more vigorous growth associated with longer day lengths. Total biomass at harvest for most untreated competitors was quite low in comparison to A. philoxeroides. For example, final mean biomass for untreated Sagitaria was only 1.43g ± 0.42 as compared to 12.35g ± 2.86 for untreated A. philoxeroides sharing the same treatment containers. Size certainly plays a role in a plants ability to influence neighbors (Goldberg and Werner, 1983). In this experiment, competitors may not have been given adequate conditions during a critical time to achieve the growth rate and size required to take advantage of stressed A. philoxeroides.

A. philoxeroides biomass was reduced by triclopyr applications. This result was expected and reported in the literature (Schooler et al. 2008, Allen et al. 2007). A second application six weeks after the first did result in further biomass reduction than a single application. To our knowledge, this has not been reported in A. philoxeroides experiments using triclopyr. Schooler et al. (2008) reported no difference in A. philoxeroides biomass between three applications and four applications, but did not test a single or double application in his design. Allen et al. (2007)

tested single triclopyr applications. Two herbicide applications were required to reduce cumulative stem length beyond controls. After two applications, 50% of treatment pots had no stems present, whereas after one treatment, 100% of pots had stems present. These results suggest that although one application reduced stem density of above ground parts, elimination of stems and leaves required two applications.

Literature points to poor herbicide translocation to roots as a reason for low herbicide efficacy with A. philoxeroides treatments (Sainty et al. 1998, Julien et al. 2004). Bowmer et al.

(1993) reported that only 7% of glyphosate herbicide applied to A. philoxeroides via foliar treatment reached the root system. We observed significant root reduction after both herbicide applications frequencies, but this effect may not have been a direct result of root mortality due to triclopyr. The shoot/root ratio is a measure of how a plant is allocating resources. The ‘functional equilibrium’ theory suggests that a plant will shift resource allocation between roots and shoots to aid in acquisition of resources that are most limiting (Brouwer, 1962). Experiments involving pruning of roots and shoots have demonstrated that plants will grow toward a specific shoot/root ratio over time after a disturbance (Alexander and Maggs 1971, Poorter and Nagel 2000). The reduction of root biomass we observed could be attributed to a reallocation of plant resources from roots to shoots due to disturbance of above ground parts via herbicide. Only a snapshot of the plants' recovery and subsequent shoot/root partitioning was observed because the experimental design called for a single harvest of all treatments. After one application of triclopyr, the shoot/root ratio of treatment plants did not differ from controls. A. philoxeroides that was treated with two applications had significantly lower shoot/root ratios than controls. The plants exposed to a single treatment may have had enough time to reach functional equilibrium at the cost of some total biomass, whereas the plants exposed to two treatments did not.

Competitive influence of introduced plants was limited to effects on A. philoxeroides shoot/root ratio. Only one species, Justicia american, reduced A. philoxeroides shoot/root significantly over controls. Competition between plants can affect biomass allocation. Gersani et al. (2001) found lower shoot/root ratios among soybeans that experienced intraspecific competition than those that did not. Shortly after transplanting at the end of September, existing above ground parts of both sprayed and un-sprayed Justicia withered away to no observable living tissue, before exhibiting minor regrowth at the time of harvest in April. Any competition between A. philoxeroides and Justicia must have occurred below ground. Resource availability in the absence of plant to plant interactions has the ability to effect plant biomass allocation

(Wilson, 1988). Plant species respond differently to competition, but these competitive responses are also altered by abiotic factors, particularly nutrients (Wilson and Tilman, 1995). Introduction of Justicia may have diminished nutrients in shared treatment pots enough to prompt a biomass re-allocation response from A. philoxeroides. In addition to resource mediated competition, plants can interfere with one another below ground through allelopathy (Mahall and Callaway,

1992).

Field Experiment

Results from the field component did not suggest any reduction in A. philoxeroides cover after the integration of introduced competitor plants and triclopyr applications. The herbicide applications alone resulted in reduction, but as in the greenhouse study, introduction of competitors resulted in little effect. Alternative herbicide timing may have resulted in different results. Allen et al. (2007) found that a high rate of herbicide applications in April for A. philoxeroides resulted in increased native plant biomass within the treatment year over July applications. Allen et al. (2007) proposes earlier applications provided a critical window of

opportunity for native plants to become established as A. philoxeroides cover diminished. In this study, A. philoxeroides was sprayed at maximum rate in October. Late season applications of herbicides tend to work better because plants tend to accumulate carbohydrates and other nutrients into roots and other storage structures in preparation for winter senescence (Madsen and Owens 1998). Because A. philoxeroides suppression through introduced native plants after herbicide application was the intended goal of this study, early season applications and plant introductions may have provided different results.

Herbicide applications effected A. philoxeroides cover in the field. There was no significant difference between one and two applications. Late season applications in general are more effective than early season applications. Allen et al. (2007) found improved reduction in A. philoxeroides biomass with July triclopyr applications as compared to April applications. In this experiment, the two triclopyr applications in the field occurred at the beginning and end of

October. The first application on October 1st may have resulted in maximum potential effect for the remaining growing season. If the project had occurred earlier in the spring, a second application may have provided improved control.

There was no evidence of introduced competition alone effecting A. philoxeroides cover in the field. Due to availability of viable plants, different species of Polygonum and Carex as competitors were substituted moving from the greenhouse to the field. This in effect broadens the range of potential competitive influence of different plants on A. philoxeroides examined in the context of this study by two species. Unlike the greenhouse component, native plants and seeds were present in all treatment plots during the entirety of the field experiment. Any effect caused by the introduced plants may have been overshadowed by effects of plants already present. Below ground aspects of A. philoxeroides were not measured in the field, and therefore

there was no data to measure competitive influence on roots or biomass partitioning. There was evidence in the greenhouse of influence by one competitor species on A. philoxeroides shoot/root ratio. There may have been interactions underground that could not be quantified due to constraints inherent by the chosen data collection method.

Comparing Greenhouse and Field Components

In general, results of the field experiment and greenhouse components were similar, with a few exceptions. As in the greenhouse component of this study, A. philoxeroides was reduced by triclopyr applications. In contrast to the greenhouse component, there was no difference between a single application and two applications in above ground metrics recorded. There was a reduction in cover after one application in the field, which contrast with no reduction in cumulative stem length after one application in the greenhouse. We must be cautious about making direct comparisons between the field and greenhouse components because of data collection techniques used. For example, we found cumulative stem length was only reduced after two applications in the greenhouse, but above ground biomass was reduced by one and further by two. In effect, the stems were still there, but the ratio of mass to length was reduced.

We used human observers to place A. philoxeroides into cover classes in the field. Cover class estimates could be somewhat inflated in comparison to above ground biomass simply because of the influence of the presence of stems on sampling estimates, regardless of stem density.

Conclusions and Suggestions for Future Work

This study contributes to evidence that triclopyr is effective at reducing alligator biomass and cover, but is not successful at completely eliminating the plant. Managers that wish to use triclopyr to control A. philoxeroides can expect improved results after a second application.

Schooler et al. (2008) attributed improved A. philoxeroides control with selective herbicides over

broad spectrum herbicides to the competition from existing monocot species. After two experiments, we found no advantage of integrating herbicide applications and introductions of six species of plants to compete with A. philoxeroides. To fully explore this theoretical integration of management tools completely, the individual components should be better understood. It is well reported that herbicides can reduce A. philoxeroides biomass, but further understanding of herbicide transport, or lack thereof, and effects throughout the entire plant are needed. Because competitive interactions underground are so important, it may be important to use herbicides that could directly damage A. philoxeroides roots to improve the magnitude of competitive responses from other plants. Trials with a larger pool of potential competitors and their subsequent influence on A. philoxeroides could have allowed for improved competitor plant selection for this study and resulted in a different outcome. We knew that herbicides would most likely damage A. philoxeroides, but we had little evidence of A. philoxeroides being directly suppressed by native plants or how the plant responds physiologically to competition. Further research is needed relating timing herbicide application and subsequent introduction of plants.

Herbicides chosen may control A. philoxeroides better after late season applications, but opening an earlier window for response from introduced competitors may improve results over either individual method. Because of the morphological plasticity of A. philoxeroides, any study attempting to characterize competitive effects from other plants would certainly have to take different hydrological regimes and the resulting A. philoxeroides growth forms into account.

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