<%BANNER%>

Characterization of a Suspected Herbicide Tolerant Hybrid Watermilfoil (Myriophyllum Spicatum X M. Sibiricum)

Permanent Link: http://ufdc.ufl.edu/UFE0043556/00001

Material Information

Title: Characterization of a Suspected Herbicide Tolerant Hybrid Watermilfoil (Myriophyllum Spicatum X M. Sibiricum)
Physical Description: 1 online resource (92 p.)
Language: english
Creator: Berger, Sarah T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: biology -- herbicide -- myriophyllum -- resistance -- tolerance
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: An invasive watermilfoil population from a lake in western Michigan was documented as a hybrid between the native northern watermilfoil and the exotic Eurasian watermilfoil (Myriophyllum sibiricum x M. spicatum). This population survived normally lethal concentrations of fluridone herbicide; therefore studies were conducted to elucidate the possibility and level of tolerance. Mesocosm studies showed a differential response in biomass and fluorescence yield, measure by Pulse Amplitude Modulated (PAM) fluorometery, to fluridone and this population was tolerant of the maximum permitted concentration of fluridone in Michigan (6 µg L-1). Further laboratory studies refined the use of the PAM fluorometer to document tolerance to fluridone while comparing the tolerant population to 12 invasive watermilfoils from across the United States. Simultaneous comparative pigment analysis confirmed the accuracy of the PAM fluorometer in detecting the responses of fluridone to watermilfoil at concentrations up to 48 µg L-1. The tolerant population was also found to respond similarly to norflurazon and topramezone herbicides when compared to susceptible populations. This common response to herbicides with differing mechanisms of action suggests the mechanism of tolerance is fluridone in watermilfoil is not at the active enzymatic site of fluridone. Collectively these studies confirmed herbicide tolerance and cross-tolerance in this population of hybrid watermilfoil and also demonstrated the utility of PAM fluorometery as a quick and reliable method to document fluridone plant responses.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah T Berger.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Macdonald, Greg.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043556:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043556/00001

Material Information

Title: Characterization of a Suspected Herbicide Tolerant Hybrid Watermilfoil (Myriophyllum Spicatum X M. Sibiricum)
Physical Description: 1 online resource (92 p.)
Language: english
Creator: Berger, Sarah T
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: biology -- herbicide -- myriophyllum -- resistance -- tolerance
Agronomy -- Dissertations, Academic -- UF
Genre: Agronomy thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: An invasive watermilfoil population from a lake in western Michigan was documented as a hybrid between the native northern watermilfoil and the exotic Eurasian watermilfoil (Myriophyllum sibiricum x M. spicatum). This population survived normally lethal concentrations of fluridone herbicide; therefore studies were conducted to elucidate the possibility and level of tolerance. Mesocosm studies showed a differential response in biomass and fluorescence yield, measure by Pulse Amplitude Modulated (PAM) fluorometery, to fluridone and this population was tolerant of the maximum permitted concentration of fluridone in Michigan (6 µg L-1). Further laboratory studies refined the use of the PAM fluorometer to document tolerance to fluridone while comparing the tolerant population to 12 invasive watermilfoils from across the United States. Simultaneous comparative pigment analysis confirmed the accuracy of the PAM fluorometer in detecting the responses of fluridone to watermilfoil at concentrations up to 48 µg L-1. The tolerant population was also found to respond similarly to norflurazon and topramezone herbicides when compared to susceptible populations. This common response to herbicides with differing mechanisms of action suggests the mechanism of tolerance is fluridone in watermilfoil is not at the active enzymatic site of fluridone. Collectively these studies confirmed herbicide tolerance and cross-tolerance in this population of hybrid watermilfoil and also demonstrated the utility of PAM fluorometery as a quick and reliable method to document fluridone plant responses.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Sarah T Berger.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Macdonald, Greg.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043556:00001


This item has the following downloads:


Full Text

PAGE 1

1 CHARACTERIZATION OF A SUSPECTED HERBICIDE TOLERANT HYBRID WATERMILFOIL ( Myriophyllum spicatum x M. sibiricum ) By SARAH BERGER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DE GREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

PAGE 2

2 2011 Sarah Berger

PAGE 3

3 To my friends and fa mily

PAGE 4

4 ACKNOWLEDGMENTS For their support and input throughout my time at the University of Florida I would like to thank my committee: Dr. Greg MacDonald, Dr. Mike Netherland, Dr. Bill Haller, and Dr. Michael Kane. S pecifically I would like to extend my appreciation to Dr. Mike Netherland for additional assistance and guidance he offered. I appreciate Dr. MacDonald for his advice and encouragement from the beginning In addition I would like to thank Dr. Ferrell for sharing his thoughts and guidance throughout my tenure as a Fellow Weed Science graduate students have made this experience enjoyabl e while also providing invaluable help. I am grateful for the funding for this research provided by Florida Fish and W ildlife Conservation Commission and support from the Center for Aquatic and Invasive Plants. ement to further my education and appreciate all they have done to encourage me throughout the years. Finally, I would like to thank Brandon Theisen for his support

PAGE 5

5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 9 ABSTRACT ................................ ................................ ................................ ................... 10 C HAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Myriophyllum Biology ................................ ................................ .............................. 13 Impacts and Management of Invasive Watermilfoil S pecies ................................ ... 17 Fluridone Herbicide ................................ ................................ ................................ 20 PAM Fluorometery ................................ ................................ ................................ .. 25 2 EVALUATION O F SUSPECTED FLURIDONE TOLERANT HYBRID WATERMILFOIL UNDER STATIC MESOCOSM CONDITIONS ............................ 29 Materials and Methods ................................ ................................ ............................ 31 Experiment 1 ................................ ................................ ................................ .... 31 Experiment 2 ................................ ................................ ................................ .... 32 Experiment 3 ................................ ................................ ................................ .... 34 Results and Discussion ................................ ................................ ........................... 35 Experiment 1 ................................ ................................ ................................ .... 35 Experiment 2 ................................ ................................ ................................ .... 36 Experiment 3 ................................ ................................ ................................ .... 36 3 A COMPARISON OF METHODS FOR CHARACTERIZING HERBICIDAL EFFECTS ON SUBMERSED AQUATIC VASCULAR PLANTS ............................. 43 Materials and Methods ................................ ................................ ............................ 47 Pigment Extraction Incubation Time ................................ ................................ 47 PAM Fluorometery ................................ ................................ ........................... 48 Shoot Length Response ................................ ................................ ................... 49 Results and Discussion ................................ ................................ ........................... 50 Pigment Extraction Incubation Time ................................ ................................ 50 PAM Fluorometry ................................ ................................ ............................. 51 Shoot Length Response ................................ ................................ ................... 52 4 CHARACTERIZATION OF FLURIDONE TOLERANT HYBRID WATERMILFOIL ULTILIZING PIGMENT ANALYSIS AND PAM FLUOROM ETERY ......................... 58

PAGE 6

6 Materials and Methods ................................ ................................ ............................ 63 Experiment 1 ................................ ................................ ................................ .... 63 Experiment 2 ................................ ................................ ................................ .... 64 Results and Discussion ................................ ................................ ........................... 65 Experiment 1 ................................ ................................ ................................ .... 65 Experiment 2 ................................ ................................ ................................ .... 67 5 EVALUATION OF A HYBRID WATERMILFOIL RESPONSE TO PIGMENT SYNTHESIS INHIBITING HERBICIDES ................................ ................................ 73 Materials and Methods ................................ ................................ ............................ 75 Results and Discussion ................................ ................................ ........................... 76 Fluridone ................................ ................................ ................................ .......... 76 Norflurazon ................................ ................................ ................................ ....... 77 Topramezone ................................ ................................ ................................ ... 77 6 CONCLUSIONS ................................ ................................ ................................ ..... 83 LIST OF REFERENCES ................................ ................................ ............................... 85 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 92

PAGE 7

7 LIST OF TABLES Table page 2 1 Invasive watermilfoil (Myriophyllum spp.) populations used in Experiment 2. .... 39 2 2 Invasive watermilfoil ( Myriophyllum spp.) populations used in Experiment 3. .... 39 2 3 Fluorescence yield (F v /F m ) in susceptible and Townline populations of invasive watermilfoils as a function of fluridone concentration 7 and 11 weeks after treatment (WAT). ................................ ................................ ........................ 39 2 4 Biomass of susceptible and Townline populations of invasive watermilfoils as a function of fluridone concentration 7 and 11 weeks after treatment (WAT). .... 39 3 1 Total chlorophyll and total carotenoids extracted from hydrilla and Eurasian watermilfoil (E WM) tissue in dimethyl sulfoxide as a function of 6 hour incubation time. ................................ ................................ ................................ .. 55 3 2 Total chlorophyll and total carotenoids extracted from hydrilla and Eurasian watermilfoil (EWM) tissue in dimeth yl sulfoxide as a function of 1 hour incubation time. ................................ ................................ ................................ .. 55 3 3 EC 50 chlorophyll, and total carotenoids for suspected fluridone tolerant and fluridone susceptible invasive watermilfoil populations. ................................ ...... 55 3 4 Fluorescence Yield (F v /F m ) in Eurasian watermilfoil shoot lengths as a function of fluridone concentration 2 days after treatment. ................................ 56 3 5 Fluorescence Yield (F v /F m ) in Eurasian wa termilfoil shoot lengths as a function of fluridone concentration 6 days after treatment. ................................ 56 3 6 Fluorescence Yield (F v /F m ) in Eurasian watermilfoil shoot lengths as a function of fluridone con centration 10 days after treatment. ............................... 56 3 7 Total chlorophyll in Eurasian watermilfoil shoot lengths as a function of fluridone concentration 10 days after treatment. ................................ ................. 57 3 8 Total carotenoids in Eurasian watermilfoil shoot lengths as a function of fluridone concentration 10 days after treatment. ................................ ................. 57 4 1 Populations of invas ive watermilfoils used in Experiment 1. ............................... 68 4 2 Fluorescence yield (F v /F m ) in Townline plants and 3 different populations of invasive watermilfoils after exposure to fluridone 3 days after treat ment. ........... 68

PAGE 8

8 4 3 Fluorescence yield (F v /F m ) in Townline plants and 3 different populations of invasive watermilfoils after exposure to fluridone 5 days after treatment. ........... 68 4 4 Fluorescence yield (F v /F m ) in Townline plants and 3 different populations of invasive watermilf oils after exposure to fluridone 7 days after treatment. ........... 69 4 5 Total chlorophyll in Townline plants and 3 different populations of invasive watermilf oils after exposure to fluridone 7 days after treatment. ........................ 69 4 6 Total carotenoids in Townline plant s and 3 different populations of invasive watermilf oils after exposure to fluridone 7 days after treatment. ........................ 69

PAGE 9

9 LIST OF FIGURES Figure page 1 1 An ex ample of two fluorescence induction cur ves measured by PAM fluorometer ................................ ................................ ................................ ......... 28 2 1 Above ground biomass in susceptible and Townline invasive watermilfoil plants as a function of fluridone concentrat ion 7 weeks after treatment. ............ 40 2 2 Fluorescence yield (F v /F m ) and dry bi omass of suspected fluridone tolerant hybrid watermilfoil and susceptible populations of invasive watermilfoils 6 weeks after treatment with fluridone. ................................ ................................ .. 41 2 3 Dry biomass of suspected fluridone tolerant hybrid watermilfoil and 4 combined populations of susceptible invasive watermilfoils 8 weeks after treatment w ith fluridone ................................ ................................ ..................... 42 4 1 Fluorescence Yield (F v /F m ) in Townline plants and nine different populations of susceptible invasive watermilfoils 7 days after treatment with fluridone. ........ 70 4 2 To tal chlorophyll in Townline plants and nine different populations of susceptible invasive watermilfoils 7 days after treatment with fluridone. .......... 71 4 3 Total carotenoids in Townline plants and nine different populations of susceptible invasive watermilfoils 7 days after treatment with fluridone. ............ 72 5 1 Fluorescence yield (F v /F m )( a), total chlorophyll ( b), and total carotenoids (c), in susceptible and Townline watermilfoil plants as a function of fluridone concentration 10 days after treatment. ................................ ............................... 80 5 2 Fluorescence yield (F v /F m )(a), total chlorophyll (b), and total carotenoids (c), in su sceptible and Townline watermilfoil plants as a function of norflurazon concentration 10 days after treatment. ................................ ............................... 81 5 3 Fluores cence yield (Fv/Fm)(a), total chlorophyll (b), and tota l carotenoids (c), in susceptible and Townline watermilfoil plants as a function of topramezone concentration 10 days after treatment. ................................ ............................... 82

PAGE 10

10 Abstract of Thesis Presente d to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHARACTERIZATION OF A SUSPECTED HERBICIDE TOLERANT HYBRID WATERMILFOIL ( Myriophyllum spicatum x M. sibiric um ) By Sarah Berger December 2011 Chair: Gregory MacDonald Major: Agronomy An invasive watermilfoil population from a lake in western Michigan was documented as a hybrid between the native northern watermilfoil and the exotic Eurasian watermilfoil ( Myr iophyllum sibiricum x M. spicatum ). This population survived normally lethal concentration s of fluridone herbicide; therefore studies were conducted to elucidate the possibility and level of tolerance. Mesocosm studies showed a differential response in bio mass and fluorescence yield, measure by Pulse Amplitude Modulated (PAM) fluorometery, to fluridone and this population was tolerant of the maximum permitted concentration 1 ). Further laboratory studies refined the use of the PAM fluorometer to document tolerance to fluridone while comparing the tolerant population to 12 invasive watermilfoils from across the United States. Simultaneous comp arative pigment analysis confirmed the accuracy of the PAM fluorometer in detecting the responses of fluridone to watermilfoil at concentrations up 1 The tolerant population was also found to respond similarly to norflurazon and topramezone her bicides when compared to susceptible populations. This common response to herbicides with differing mechanisms of action suggests the mechanism of tolerance is fluridone in watermilfoil is not at the active enzymatic site of fluridone.

PAGE 11

11 Collectively these s tudies confirmed herbicide tolerance and cross tolerance in this population of hybrid watermilfoil and also demonstrated the utility of PAM fluorometery as a quick and reliable method to document fluridone plant responses.

PAGE 12

12 CHAPTER 1 INTRODUCTION Submerse d aquatic vascular macrophytes are a unique group of plants that display a wide range of physiological adaptations to life under water. These plants comprise less than 1% of angiosperms, and most trace line age to terrestrial ancestors. Many have vestigial features such as a thin cuticle, poorly developed stomata and xylem elements which are essentially functi onless (Sculthorpe 1967). Submersed plants are often desirable and beneficial in waterb odies for a number of reasons. Increased diversity and abundance of aquatic flora directly correlates to an increase o f aquatic fauna (Rosine 1955) and b eds of submersed aquatic plants have been found to provide a rich foraging habitat for fish and greater protection from predators (Rozas and Odum 1988). In addition to biotic benefits, aquatic vegetation can also benefit abiotic factors such as stabilization of sediment and improved water clarity (Madsen 2009a). Although native submersed aquatic plants are desirable in aquatic ecosystems, invasive submersed species have the potential to overtake native plant communities and produce detrimental ecological effects. In Lake George, New York, an invasion of Eurasian watermilfoil ( Myriophyllum spicatum L. ) caused a rapid decline in the number of native aquatic plant species o ver a three yea r period (Madsen et al. 1988). Schmitz and Osborne (1984) found that beneficial zooplankton abundance decreased with increased hydrilla ( Hydrilla verticillata [L.f] Royle) growth. Anthropogenic uses of waterbodies are also impact ed by invasi ve aquatic plants. Dense, often monotypic infestations with surface canopies can impair commercial and recreational navigation, disrupt flood control, and provide a habitat for vectors of disease. Rapidly growing

PAGE 13

13 invasive aquatic plants are often managed t o maintain the ecological balance and recreational use of infested waters Management of aquatic plants differs from traditional weed control. Non target organisms such as native plants and wildlife will likely be exposed to submersed herbicide application s and their response must be taken into consideration when herbicide applicat ions are made to water bodies. Whereas chemical terrestrial weed control is focused on d efined areas of land, water dynamics such as flow rate and dilution complicate aqu atic plan t management efforts. Furthermore, large water bodies that contain invasive plants requiring mana gement are often public waters. Public waters have many different stakeholders including resource managers, federal and state agencies, residents with high val ue real estate, recreational end users, and other special interest groups. he vast and varying opinions of these stakeholders must be considered when formulating treatment plans for aquatic weeds in these types of water bodies. Myriophyllum Biology The wa termilfoils, contained in the genus Myriophyllum are dicotyledonous aquatic plants and membe rs of the Haloragaceae family. Approximately 68 species make up this genus, which are present on all continents excluding Antarctica with at least seven species co mmon between continents (Moody and Les 2010). Fourteen watermilfoils are present in North America and include bot h native and invasive species. Eurasian watermilfoil and parrotfeather ( M. aquaticum [Vell.] Verd. ) are invasive and listed as no xious weeds in several states. Variable leaf watermilfoil ( M heterophyllum Michx. ) is considered invasive outside of its original range of the Southeast and has also invaded Europe and Asia (Les and M ehrhoff 1999; Yu et al. 2002). Two

PAGE 14

14 hybridizations are known between s pecies of this family. M. spicatum x M. sibiricum and M. heterophyllum x M. laxum (Shuttlw. ex Chapm.) hybridization were first docum ented by Moody and Les (2002). Species of interest in this document are Eurasian watermilfoil, northern watermilfoil ( M. sib iricum Kom. ), and the hybrid from the genetic cross of Eurasian and northern watermilfoil. Northern watermilfoil is a native plant whose range stretches from California north to the northern Canadian provinces and east to Maine and the eastern seaboard It is not found in the southeast ern United States (USDA 2011). Eurasian watermilfoil has been documented in every state in the U.S. and several Canadian provinces (Jacono and Richerson 2011). This species is the most widely managed aquatic weed in the countr y (Smith an d Barko 1990). It was first documented in North America in the Chesapeake river area in 1890 (Reed 1977) and introduction is speculated to be the result of the aquarium trade or the shipping industry via ship ballasts (Madsen 2009b). The hybrid been documented from Michigan to Washington in the northern part of the country (Moody and Les 2002, 2007). Northern watermilfoil, along with most Myriophyllum species, is a rooted submersed aquatic plant that has pectinate leaves in whorls around a white or pink colored stem. Leaves consist of 6 to 11 p airs of leaflets (Aiken 1980). This species reproduces through a variety of ways such as stem fragmentation, runners from root crow ns, seed, and axial turions, or winter buds, provide a dormancy mechanism in the winter months (Aiken 1980). Northern watermilfoil is not considered to be a nuisance species as it does not form dense canopies at the water surface (Aiken 19 80 ). This

PAGE 15

15 native species is considered excellent habitat for aquatic wildlife, specifically providing necessary aquatic plant architecture for fisheries success (Valley et al. 2004). Eurasian watermilfoil is a rooted submersed aquatic plant with pectinate leaves in whorls of four around a submersed stem. Leaves consist of 14 to 21 pairs of leaflets. It is closely related to the native northern watermilfoil although the two species rarely coexist for extended durations due to the rapid growth rate and subsequent displacement by the Eurasian species (Aiken 19 80 ; Nichols 1992). The plant also reproduces in a variety of ways including runners from root crowns, stem fragmenta tion, and seed (Madsen 2009b). Vegetative means are considered the major mechanism of wide spre ad distribu tion (Madsen et al. 1988). Eurasian watermilfoil has been shown to autofragment primarily after flowering and at the end of the growing season (Madsen et al. 1988). This species, when compared with other invasive aquatic plants, does not produce high level s of b iomass (Grace and Wetzel 1978). While biomass production is not excessive Eurasian watermilfoil has been documented to form dense canopies (>80% cover) that shade out native vegetation, reducing species diversity in as little as 2 years (Madsen et a l. 1991). Eurasian watermilfoil is photosynthetically a C 3 plant. Van et al. (1976) found that the ratio of Rubisco to Phosphophenylpyruvate ( PEP ) carboxylase was similar to that found in spinach ( Spinacia oleracea L. ) a C 3 plant. Another determinant of C 3 photosynthesis is the photorespiration pathway, documented by glycolate levels in the plant. Eurasian watermilfoil was found to have glycolate levels lower than most C 3 plants, but higher than observed in C 4 plants (Stan ley and Naylor 1972). Interestingl y, this plant does exhibit some form of Kranz anatomy that is usually associated with C 4

PAGE 16

16 pla nts (Stanley and Naylor 1972). Several authors detail the preference of free CO 2 as a carbon source, but the plant has the ability to use bicarbonate at high pH lev els (Steeman Nielsen 1947; Stanl ey 1970; and Van et al. 1976). The ability of some aquatic plants to use bicarbonate as a carbon source is advantageous in that free carbon dioxide is usually limited in the freshwater en vironment. With an alternative carbon source, such as bicarbonate, this species can continue to photosynthesize under low carbon dioxide conditions (Prins and Elzenga 1987). While present in Florida, Eurasian watermilfoil has never grown to problematic levels of infestation. An article publis hed in 1967 (1967) described the potential invasiveness of watermilfoil as the plant had invaded thousands of acres in the Chesapeake Bay and Tennessee Valley Authority reserv oirs Although the plant remains present in Florida, it has never reached nuisance levels requiring management Hybrid watermilfoil was first docume nted in 2002 by Moody and Les. Populations previously thought to be the invasive Eurasian watermilfoil were found, through nuclear ribosomal DNA analysis, to be hybrid populations from the parental species Eurasian watermilfoil and northern wate rmilfoil (Moody and Les 2002). It is suspected that hybrids went unnoticed for some time due to the morphological simil arities between pa rental species and the hybrid. The two parental species are visibly distinguished by number of leaflet pairs (northern 6 to 11 pairs, Eurasian 14 to 21 pairs), stem diameter, and presence of axial turions (Coffey and McNabb 1974; Crow and Helquist 2002 ). However, the hybrid plant can exhibit a range of leaflet pairs similar to either parent and have other characteri stics similar to each parent. Hybrid watermilfoils present unique

PAGE 17

17 challenges for management due to inherited traits such as in vasiveness and rapid growth rate from the Eurasian parent and the potential for turion formation in some hybrid populat ions from the northern parent. There are differing reports as to the dominance of a hybrid population co mpared to the Eurasian parent. Mo ody and Les (2007) found that hybrids tend to overtake lakes so that the parenta l species are no longer found. However, a more recent study by Sturtevant et al. (2009) found that hybrids and Eurasian watermilfoil parents often co existed in the same bodies of water. It is important to note that numerous hybrid watermilfoil populations have arisen independently and therefore traits associated with hybrids from one lake may be quite different when compared with those from a nother lake (Sturtevant et al. 2009) These genotypic and phenotypic differences that exist between populations of the hybrid plants preclude the use of generalities regarding specific growth or management traits. Repeated hybridization as well as back crossing has also been documented in th e field (Moody and Les 2002). Therefore, it is important to refer to each hybrid population independently. Impacts and Management of Invasive Waterm ilfoil S pecies Invasive water milfoils (Eurasian and hybrid) are problemat ic weeds in many water bodies. Pla nts grow to form dense canopies displacing native vegetation, inhibiting flood control and obstructing r ecreational uses of waterways. Madsen et al. (1991) found that Eurasian watermilfoil formed dense canopies on the water surface that shad e out desirable native plants. Dense canopy formation has been shown to negatively impact water quality by reducing dissolved oxygen in water below the mat and increasing surface temperatur es and pH (Bowes et al. 1979). Submersed aquatic weeds have also been shown to har bor algal species harmful to both wildlife and human health (Wilde et

PAGE 18

18 al. 2005). When water milfoil densities result in unfavorable conditions for wildlife and fisheries, displace desirable native vegetation, and impact recreational access or aesthetics, ma nagement of these plants is often required. Preventative management is necessary to limit spread of invasive pla nts to uninfested waterbodies. Practices such as removing fragments from boat trailers are helpful in reducing human vectored spread of the plan ts. Several states have highly visible public education and outreach programs to educate the public on pre ventative methods (UF/CAIP 2011, Cal IPC 2011, ISDA 2011). Mechanical control of invasive water milfoils, like many submersed plants, is not always an ef fective choice for management. Mechanical harvesters segment the plants, increasing fragmentation and possibly assisting in the spread and intensity of the invasion. This method is also non selective and has the potential to also damage native plant comm unities. Hand harvesting does limit fragmentation but requires a large financial investm ent (Kelting and Laxson 2010). Despite the problems associated with mechanical harvesting, there are numerous operational programs that continue to rely on mechanical harvesters as a primary means of control. Several biological controls are available for submersed Eura sian and hybrid watermilfoils. Triploid grasscarp ( Ctenopharyngodon idella Val. ) are a common choice to manage many submersed plant infestations Althou gh grasscarp are an attractive choice in some situations, invasive water milfoil management is not recommended because the fish prefer native plants over the invasive water milfoils (Stroganov 1963). A native weevil, Euhrychiopsis lecontei ( Dietz) does prefe r the invasive watermilfoil s to native submersed plants and is sold commercially (Alwin et al. 2010). The use of this

PAGE 19

19 biocontrol has been associated with seasonal declines but has not been shown to provide sufficient control to eliminate an invasion (Newma n et al. 2001). While there is a large body of literature on Eurychiopsis and water milfoil control, predictable and consistent control remains a problem (Alwin et al. 2010). Chemical control methods are also available for invasive water milfoils. Herbicides effective for control compromise several modes of action including plant gr owth regulators such as 2,4 D ([ 2,4 dichlorophenox y] acetic acid) and triclopyr ([3,5,6 trichloro 2 pryidinyl] oxy acetic acid), the carotenoid biosynthesis inhibitor fluridone (1 m ethyl 3 phenyl 5 [3 (trifluoromethyl)phenyl] 4(1 H ) pyridinone), and cell membrane disrupters such as diquat (6,7 dihydrodipyrido[1,2 c ] pyrazinediium ion) and endothall (7 oxabicyclo[2.2.1] he ptanes 2,3 dicarboxylic acid). Diquat and endothall are contact materials that do not translocate within the plant and typically provide short term control of infestations. The systemic herbicides generally provide long term, season long or even multiple season control of invasive water milfoils. Triclopyr and 2,4 D have been used and are documented to provide control as long as exposure times are sufficient (Netherland and Getsinger 199 2; Green and Westerdahl 1990). Fluridone is an attractive choice for water managers due to low use concentrations 1 ), native plant selectivity, ability to target water milfoil in the entire lake, cost effectiveness, and potential for multiple years of co ntrol from a single treatment. This herbicide is used frequently by water managers to control Eura sian and hybrid watermilfoils on a whole lake or whole system basis. There have been numerous claims of reduced herbicide response by hybrid water milfoils, however there is limited published in formation on this topic. Triclopyr and 2,4 D amine were found to inhibit growth of both Eurasian and

PAGE 20

20 hybrid watermilfoil accessions in a similar manner following exposure to labeled u se concentrations (Poovey et al. 2007). Recently, differences between Eurasian and hybrid watermilfoil populations were noted following exposure to low continuous concentrations of 2,4 D ( Glomski and Netherland 2010). This shows the potential for increased tolerance to fluridone. Fluridone Herbicide Fluridone was first described as having herbicidal effects in 1976. The compound was discovered and evaluated by Eli Lilly & Company during greenhouse screening and investigated as a selective herbicide for pre emergence use in cot ton (Waldrep and Taylor 1976). It was found to have activity against both monocotyledonous and dicotyledonous weeds such as r edroot pigweed ( Amaranthus retroflexus L. ) sickelpod ( Cassia obtusifolia L. ) johnsongrass ( Sorghum halepense Pers. ) and large crabgrass ( Digitaria sanguinalis [L.] Scop. ), among oth ers (Waldrep and Taylor 1976). Fluridone was then evaluated for use on a quatic vascular plants and was found to provide excellent contro l at low use concentrations on hydrilla, Eurasian watermilfoil, duckweed ( Lemna spp.), cabomba ( Cabomba caroliniana Gray ) and several other problematic aquatic species (McCowen et al. 1979). Fluridone was registered in the United States in 1986 for use in aq uatic environments by the U.S. E n vironmental Protection Agency. Several liquid and granular formulations are available for use in both lotic and lentic waters to control a variety of submer sed and emergent aquatic vegetation. Fluridone belongs to the substituted tetrahydropyrimidinone class of herbicides which are commonly referred to as the bleacher herbicides. Fluridone is an inhibitor of the carotenoid biosynthesis pathway. Specifically, it is a noncompetitive inhibitor of the

PAGE 21

21 phytoene desaturase (PDS) enzyme. The PDS enzyme catalyzes the desaturation of phytoene in the rate limiting step of this pa thway (Chamovitz et al. 1993). When PDS is inhibited, phytoene levels increase and carotenoi d pro duction in the cell is limited. Carotenoids function to shield chlorophyll from excess light and help dissipate the oxid ative energy of singlet oxygen. In fluridone treated plants, carotenoids are not present to quench the energy of oxygen radicals, a llowing for the formation of lipid radicals in chloroph yll molecules (Senseman 2007). This results in degradation of chlorophyll, bleaching of new tissue and subseq uent necrosis and plant death. Fluridone is an attractive management tool in aquatic e cosyst ems for several reasons. Use rates of fl uridone in lentic waters are 5 20 1 and 10 40 1 in lotic waters, which is significantly lower than the mg L 1 range typical of several other aquatic herbicides. Large scale treatments of fluridone have the potential to provide multiple years o f control of submersed plants. Flurid one is also a fairly selective herbicide that does not adversely impac t many desirable native plants. 1 fluridone was found to reduce the biomass of Eurasian watermilfoil while increasing the biomass of native species such as va llisneria ( Vallisneria Americana Michx. ) and two pondweed species ( Potamogeton nodosus Poir. and P. pectinatus L. ) over untreated con trols (Netherland et al. 1997). Field studies have shown that low use rates (5 1 ) of fluridone applied on a whole l ake scale have controlled invasive water milfoil populations while leaving desirable native plant communities intact (Getsinger et al. 2001, Getsinger et al. 20 02a, Getsinger et al. 2002b). For these reasons, resource managers often use this herbicide to c ontrol undesirable aquatic vegetation while preserving native vegetation in a wi de range of aquatic ecosystems.

PAGE 22

22 In addition to control of Eurasian watermilfoil, fluridone has been used to control hydrilla since the late 1980s. Schmitz et al. (1987) describ e using fluridone to control hydrilla in a central Florida lake such that the plant could no longe r be found in the water body. Haller et al. (1990) successfully used fluridone to control hydrilla for on e year in the St. Johns River. As such, fluridone was heavily used for hydrilla control in public til the early 2000s. Between 1999 and 2001, formerly susceptible hydrilla populations in several major lakes were not exhibiting the level of control previously associate d with fluridone use. Subsequent laboratory testing documented that several populations of hydrilla had developed resistance to fluridone (Michel et al. 2004, Arias e t al. 2005, Puri et al. 2006). Specifically, an amino acid substitution in the phytoene de saturase enzyme conferre d 2 to 5 fold resistance to fluridone in hydr illa (Michel et al. 2004). Previous to this discovery, herbicide resistance in hydrilla was thought to be unlikely due to the strictly vegetative reproduction exhibited by dioecio us femal e hydrilla in Florida. However, low use rates, extended exposure times, and repeated use of fluridone led to tremendous selection pressure in these water bodies. Repeated fluridone applications selected for plants that were resistant to typic al use rates o f the herbicide. Since this time, resistance has developed in many water bodies throughout the state and fluridone is no longer a widespread tool for hydrilla manageme nt in To date, hydrilla is the only plant that has been confirme d to have deve loped resistance to fluridone.

PAGE 23

23 (WSSA 1998). that there was no selection or genetic discussion has occurred debating whether the fluridone resistance in hydrilla is act ually resistance or tolerance. Since resistant plants were selected following low use rates of the herbicide, fluridone resistance is the proper term. Fluridone has been used for the past 20 years for inv asive watermilfoil management. Waterm ilfoil control requires similar use pattern s to that of hydril la control. 1 have been shown to provide water milfoil control given that sufficient exposure time is also met (N etherland and Getsinger 1995). Using fluridone as a chemical tool to control Eurasian watermilfoil did meet some opposit ion as concerns arose as to the effect of the herbicide on native plants and wildlife habitat at a whole lake level Getsinger et al. (2001) documented that after whole lake fluridone treatments in Michigan, species diversity increased 1.5 to 2.3 fold fr om pre treatment measurements. Native plant cover was also not negatively impacted. Concerns over n ative plant selectivity and economic factors continued to drive the use of lower rates. Currently, use patterns in several states consist of an initial flurido ne treatment between 1 followed by a subsequent application to return the fluridone concentration to these levels. Although low use rates may be cost effective and increase native plant selectivity, these practices could have implications for resistance as well. In terrestrial systems low use rates of selective herbicides have been shown to cause rapid development of resistance to those herbicides, as well as cross resistance, in annual ryegrass ( Lolium rigidum L. ) (Neve and Powles 2005).

PAGE 24

24 In M ay 2010, a 220 acre lake in western Michigan (Townline Lake) was treated 1 of fluridone, followed by a subsequent treatment to return the concentration to the original level, which was permitted by the Michigan De partment of Natural Resources. The population of documented hybrid watermilfoil in the water body was not controlled w ith this rate of the herbicide. Initial mesocosm studies found that the Townline population of water milfoil did not experience declines in biomass like that of susceptib le water milfoil populations when exposed to several rates of fluridone ( Thum et al. submitted ). The observation that a hybrid watermilfoil may show increased tolerance to fluridone has fueled more speculation on the nature of water milfoil hybrids and their response to herbicides. Pigment analysis has been used traditionally to document the biochemical res ponse of a plant to fluridone. Fluridone inhibits the phytoene desaturase enzyme of the carotenoid biosynthesis pathway and by analyzing pigment levels in this pathway e to fluridone can be studied. Sprecher et al. (1998) developed an extraction method that measures absorbance spectrophotometrically to quantify ph carotene levels. I n fluridone susceptible plants, phytoene levels carotene levels would be lower than control plants. carotene levels are similar to thos e of untreated control plants. Puri et al. (2006) used this technique to demonstrate fluridone resistance in several hydrilla populations collected throughout Florida. Chlorophyll analysis is an indirect method of determi ning fluridone resistance. Although f luridone does not directly affect chlorophyll biosynthesis, the absence or carotene leads to the destruc tion of chlorophyll molecules. Therefore, in a

PAGE 25

25 susceptible plant exposed to fluridone, chlorophyll levels in new tissue will be lower tha n those in control plants. Several extraction methods have been evaluated and utilized for pigment analysis using various solvents (Iriyama et al. 1974, Hiscox and Isrealstam 1979, Moran and Porath 1979 ) but the non macerated method of Wellborn (1994) usin g dimethyl sulfoxide (DMSO) proved to be the most useful in these studies. This method provides pigment analysis that is comprehensive in the study of fluridone response in plants in that two pigments affected by fluridone can be quantified. Although pigme nt analysis provides a method for determining herbicide activity in the plant, the methods employ a destr uctive harvest of plant tissue. There is a need for non destructive and repeatable methods of analysis. PAM Fluorometery In functioning plants, energy from light, in the form of photons, comes into contact with the various pigments of the plants in t he light harvesting complexes. These pigments, such as chlorophyll a chlorophyll b and the carotenoids, have many double bonds which are capable of absorbi ng this energy and performing photochemistry to pass an excited electron to the electron trans port chain of photosynthesis. The carotenoids function to absorb excess light energy in the light harvesting complexes thereby protecting chlorophyll against this energy and conversion to radical oxygen. When excess light is absorbed by chlorophyll in the light harvesting comp lex, several events can occur. Energy can be reradiated as heat, energy can be transferred to adjacent molecules via inductive resonance, pho tochemistry or charge separation can pass an excited electron to the electron transport chain, or lastly energy can be reradiated as fluorescence. Chlorophyll a and b molecules drive photosynthesis by absorbing light energy, which causes excitation of elec trons in those molecules, and

PAGE 26

26 transfer this energy to adjacent mole cules via inductive resonance. When chlorophyll is damaged or not functioning properly due to a variety of reasons such as stress or herbicidal effects, the chlorophyll will often emit the excess ene rgy as reradiated light. Measuring this reradiated light can give insight as to the efficiency and functionality of chlorophyll and photosynthesis in the plant (Papageorgiou 1975). Pulse amplitude modulated (PAM) fluorometery is used to measure c hlorophyll fluorescence and works by focusing a saturating beam of light on the desired region of the plant. Yield ratio is calculated by the instrument. Higher fluorescence yield ratio indicates highly functioning chlorophyll whereas lower yield ratios in dicate damaged o r non functioning chlorophyll. Yield is a ratio of F v /F max F max is equal to the fluorescence when the saturating p ulse is applied to the tissue. F v is equal to F max F where F is the fluorescence of the tissu e with no light pulse applied. The plant fluoresces more when chlorophyll is damaged, which indicates a higher F max value. However, the yield output ratio is lower because damaged chlorophyll fluoresces more under ambient light conditions (Figure 1 1 ). Therefore, a higher Y ratio value indicates chlorophyll that is functioning normally where a lower Y value indicates damaged chlorophyll (Bo lhar Nordenkampf et al. 1989). PAM fluorometery has been used to study irradiance stress (Ralph et al. 1998), salinity stress (Kamermans et al. 1999), and shoot to landscape differences in photosynthesis in sea grasses (Durako and Kunzelman 2002). In situ measurement of photosynthetic activity of Red Sea faviid corals has also been measured (Beer et al 1998). This technique is useful because it is a no n destructive method of evaluating the activity of chlorophyll and has also been used to evaluate herbicidal effects on plants.

PAGE 27

27 Ireland et al. (1986) used fluorometery to document decreased fluorescence in wheat ( Triticum spp.) 30 minutes after exposure to glyphosate ( N [phosphonomethyl] glycine) herbicide. The herbicide diuron ( N [3,4 dichlorophenyl] N N dimethylurea) a photosystem II inhibitor, was shown to reduce fluorescence yield ratio in sea grasses two hours after exposure as measured with a d iving PAM (Haynes et al. 2000). Junea et al. (2001) evaluated the effects of mercury and metolachlor (2 chloro N [2 ethyl 6 methylphenyl] N [2 methoxy 1 methylethyl] acetamide) a mitosis inhibiting her bicide, on six algal species. This research investigates th e suspected fluridone toleranc e in a hybrid watermilfoil population in Michigan and methods used to document response to fluridone. Chapter 2 focuses on mesocosm scale studies used to evaluate the response several populations of invasive watermilfoil throu gh PAM fluorom etery and biomass evaluations. While these studies are informative, they are limited by the significant inpu ts of time and space required. Laboratory methods are needed to circumvent these limitations, but specific methods to evaluate submers done have not been documented. Refinement of methods including pigment extraction incubation time, using PAM fluorometery to evaluate fluridone response, and determining optimal shoot length for these studi es are discussed in Ch apter 3. Chapter 4 employs these methods in small scale laboratory evaluations of a number of invasive water milfoil po pulations from several states. Chapter 5 evaluates the potential cross tolerance of the fluridone tolerant Townline population to pigment synthesis inhibiting herbicides with differing modes of action.

PAGE 28

28 F igure 1 1 An example of two f luorescence induction curve s measured by PAM fluorometer. The upper curve indicates damaged chlorophyll and the lower curve indi cates functioning chlorophyll Equations for Y ratio depicted in figure

PAGE 29

29 CHAPTER 2 EVALUATION OF SUSPECTED FLURIDONE TOLERANT HYBRID WATERMILFOIL UNDER STATIC MESOCOSM CONDITIONS Eurasian watermilfoil ( Myriophyllum spicatum L. ) and hybrid watermilfoil ( M. spicatum x M. sibiricum ), ar e problematic invasive weeds in many water bodies throughout the northern tier of the United States Plants grow to form dense surface canopies displacing native vegetation, altering water quality, and obstructing r ecreational uses of waterways. Madsen et al. (1991) found that Eurasian watermilfoil formed dense which can shade out desirable n ative plants in the ecosystem. Dense canopy formation has been shown to negatively impact water quality by reducing dissolved oxygen in water below the mat and increasi ng surface temperatur es and pH (Bowes et al. 1979). Submersed aquatic weeds have also been shown to harbor algae species harmful to both wildlife and hum an health (Wilde et al. 2005). When water milfoils spread within waterbodies and result in unfavorable c onditions for wildlife and fisheries, displacement of desirable native vegetation, and impacts on recreational access or aesthetics, management of these plants is often required. Hybrid populations of watermilfoil were first docume nted in 2002 by Moody and Les. Populations previously thought to be the invasive Eurasian watermilfoil were found, through nuclear ribosomal DNA analysis, to be hybrid s from the parental species Eurasian watermilfoil and northern watermilfoil (Moody and Les 2002). Hybrid watermilf oils may present unique challenges for management due to inherited traits such as increased invasiveness, or hybrid vigor, and the potential to acquire a trait such as turion formation from the northern parent that could confound management efforts It is important to note that numerous hybrid watermilfoil populations have arisen independently and therefore traits associated with hybrids from one lake may be quite

PAGE 30

30 different when compared with those from another lake ( Sturtevant et al. 2009 ) The extent of h ybridization in natural lakes is unclear and currently being investigated. Genotypic and phenotypic differences exist between populations of the hybrid plants and generalities cannot be made regarding specific growth or management traits. Repeated hybridiz ation as well as back crossing has been documented in the field (Moody and Les 2002). Therefore, it is important to refer to each hybrid population independently. Chemical control methods are commonly used to manage invasive water milfoils. Fluridone (1 me thyl 3 phenyl 5 [3 (trifluoromethyl)phenyl] 4(1 H ) pyridinone) is an attractive choice for water managers due to its low use rates, native plant selectivity, ability to target water milfoil in the entire lake, and potential for multiple years of co ntrol from a single treatment. This herbicide is used frequently by water managers to control Euras ian and hybrid watermilfoils. There have been numerous claims of reduced herbicide response by hybrid water milfoils, however there is limited publi shed information on this topic. Eurasian and northern parents are both highly susceptible to low use rates typical of fluridone tre atments (Crowell et al. 2006). Triclopyr ([3,5,6 trichloro 2 pryidinyl]oxy acetic acid) and 2,4 D amine ([2,4 dichlorophenoxy] acetic acid) were found to inhibit growth of both Eurasian and hybrid water milfoil accessions in a similar manner following exposure to labeled u se rates (Poovey et al. 2007). Recently, differences between Eurasian and hybrid watermilfoil biotypes were noted following expos ure to low continuous concentrations of 2,4 D ( Glomski and Netherland 2010). In May 2010, a 220 acre lake in western Michigan (Townline Lake) was treated with a concentration of fluridone permitted by the Michigan De partment of Natural

PAGE 31

31 Resources. The popul ation of documented hybrid watermilfoil in the water body was not controlled with th e legal rate of the herbicide. The observation that a hybrid watermilfoil may show increased tolerance to fluridone has fueled more speculation on the nature of hybrids and their response to herbicides. Mesocosm studies are needed to compare the Townline population of water milfoil to other water milfoil populations when exposed to fluridone. As fluridone is typically used to treat an entire lake, a failure to perform is parti cularly notable due to costs, exposure of native plants (some quite sensitive to fluridone) through the entire system, and subsequent requests to provide additional herbicide treatments for summer relief from the water milfoil infestation In order to deter mine if the hybrid watermilfoil population from Townline Lake shows a unique response to fluridone compared to other watermilfoil accessions, a series of m esocosm studies was conducted. The objective of these studies was to evaluate the response of several different water milfoil populations to a range of fluridone concentrations to determine variation across a spectrum of water milfoil s (both Eurasian and hybrid) against Townline, which is suspected to have increased tolerance to fluridone. Materials and Met hods Experiment 1 An initial screening study was conducted in a greenhouse at the Center for Aquatic and In vasive Plants in Gainesville, Florida during the summer of 2010. The suspected susceptible hybrid watermilfoil originating from Otter Lake, MN (herea fter referred to as and temperature. This population was confirmed as a hybrid watermilfoil through ITS analysis (Thum et al. submitted ) Shoot tips were harvested from thi s stock cult ure for

PAGE 32

32 the study. Apical shoot tips of the suspected tolerant hybrid watermilfoil, a population from Townline Lake, were obtaine d from Michigan for the study. Apical shoot tips 10 15 cm in length of Townline hybrid watermilfoil or Susceptible hybrid water milfoil were planted in 4.25 inch square pots containing top so il amended with osmocot e (15 9 12) fertilizer at a rate of 1g kg 1 (soil). Pots were capped with approximately 1 cm of sand prior to planting. Each 95 L container contained one pot of Townline water milfoil and one pot of susceptible water milfoil. Artificial light in the greenhouse provided a photoperiod o f 14 hour light: 10 hour dark. Plants were allowed to establish and grow for 2 weeks prior to treatment. After this initial time, each 95 L tan k was treated with 0, 2, 4, 1 of fluridone (Sonar AS S ePro Corporation, Carmel, IN). Each treatment was replicated 5 times (5 95 L tanks per fluridone concentration ) using a completely ra ndomized design. Plants were allowed to grow a nd respond to treatment for 7 weeks until a destructive harvest of al l shoot biomass was conducted. Harvested biomass was dried for 3 days in a 70C drying oven. Samples were weighed and results analyzed using analysis of variance ( ANOVA ) and ected LSD determine differences between populations at each concentration Experiment 2 Experiment 2 was conducted to compare the tolerant Townline population to a greater geographical range of invasive watermilfoils that were collected from several sta tes. This study was conducted in an enclosed greenhouse at the Center for Aquatic and Invasive Plants in Gainesville, Florida durin g the winter of 2010 and 2011. Townline plants were harvested from outdoor stock cultures for the study. Plant material colle cted from 3 populations of suspected fluridone susceptible watermilfoils was obtained for the study (Table 2 1). One susceptible population was collected at Frog Lake in Wisconsin

PAGE 33

33 and is a hybrid water milfoil. The second population was obtained from Auburn Lake, Minnesota and is Eurasian watermilfoil (EWM). T he final population i s an EWM population from Texas. onfirmed as hybrid or EWM through ITS analysis (Thum et al. submitted ). As in the previous study, 10 15 cm apical sh oo t tips were used for planting. A single apical shoot tip was planted in a 164 mL cone tainer c ontaining topsoil amended with O smocote and capped with sand. One cone tainer of each population was placed in a 4.5 inch square pot containing topsoil amended with slow release fertilizer and again cappe d with sand prior to planting. Each pot contained one c ontainer from each population. Two square pots were placed in each 95 L tank so that biomass could be sampled at each of two harvest inter vals by removing a single pot. Plants were allowed to establish and grow f or 2 weeks prior to treatment. At this time, 6 replications of each treat 1 of fluridone were added to the appropriate ta nks. This study was conducted using a completely randomized design. The first set of plants was harvested at 7 weeks after treatment (WAT) The fluorescence of s hoot tips were mea sured with a PAM fluorometer and above ground biomass of each population was destructively har vested. Biomass samples were dried for 3 days at 70C. The second harvest occurred 11 WAT and the same parameters were measured. Both fluorescence yield data and biomass data were combined across all susceptible populations at each harvest since no significant differences were found between these populations when data wer e subjected Non linear regression was fitted to the data. Data from each harvest were analyzed with ANOVA and

PAGE 34

34 significant differences between the combined susceptible populations and the Townl ine population. Experiment 3 Experiment 3 was designed to examine the response over time to fluridone by several invasive water milf oils as compared to Townline. This study was conducted in outdoor mesocosms at the Center for Aquatic and Invasive Plants in Gainesville, Florida during the spring and summer of 2011. Four populations of water milfoils were harvested from outdoor stock cultures of plants on site (Table 2 2). These populations were Townline, Indian, Auburn and Texas. Townline, Auburn and Texas p opulations are identical to those in Experiment 2. The Indian population is from Indian Lake in Michigan and has geographical and genetic s imilarities to Townline Lake. I t is also a hybrid and suspecte d to be tolerant to fluridone. One additional populati on of known susceptible EWM was obtained from North Carolina. Populations were again confirmed genetically as hybrid or EWM through ITS analysis ( Thum pers. comm. 1 ). Apical shoots were planted in an identical m anner to that of Experiment 2. The study was designed to allow for 3 harvesting dates, so 3 pots, each containing all populations were added to each mesocosm. Plants were allowed to grow f or 2 weeks prior to treatment. Treatments for this study included an untreated control, 3, 6, 9, 12, 18, and 36 1 with 4 replications each in a completely randomized design. Due to the potential photodegradation of fluridone herbicide in outdoor mesocosms, water samples were collected every 2 days to determine the half life of t he herbicide in the mesocosms. Sa mples were analyzed using ELISA Quanti Plate kits (QuantiPlate Kit for Fluridone, 1 R. Thum, Grand Valley State University, Annis Water Resourc es Institute Muskegon, MI 49441.

PAGE 35

35 Envirologix, Portland, ME) and half life was determined to be 10 days. Therefore, every ten days for the duration of the study each mesocosm was treated with a half concentr ation of the appropriate initial treatment. The first and second harvests occurred at 3 and 6 weeks after treatment, respectively. PAM yield and above ground biomass data w ere collected at each harvest. The final harvest occurred 8 weeks after treatment wi th only above ground biomass data being collected. All biomass data from this experiment was dried and weighed in the same manner as previous experiments. All susceptible populations were again combined due to no treatment by experiment significance as fou nd with ANOVA. Non linear regr ession was fitted to the data. Fluorescence yield data and biomass data from each harvest were analyzed using ANOVA and and the combined susceptible population s within each concentration Results and Discussion Experiment 1 The initial study determined that the Townline hybrid watermilfoil had a different response to flurido ne herbicide at all concentrations used in the experiment (Figure 2 1). The susceptible population had biomass reduced to less than 40% of untreated 1 fluridone while Townline did not exhibit that level of reduc tion at even the highest co ncentration For reference, the maximum permitted concentration to treat using fluridone for control of invasive water 1 There was very limited reduction in biomass of Townline plants at the essentially highest t reatment it cou ld be exposed to in the field. This initial screening study indicated that Townline exhibits an increased tolerance or possibly resistance to the herbicide fluridone and warrants further investigation.

PAGE 36

36 Experiment 2 The second experiment resu lted in differences in biomass and fluorescence yield L 1 between Townline and the combined susceptible populations (Tables 2 3 and 2 4). At the first harvest fluorescence yield decreased by more than half in the susceptible populations at 5 1 while Townline showed essentially no decrease in fluorescence (Table 2 3). At 11 WAT, fluorescence yield was decreased to less than 1 in susceptible populations while Townline actually showed increased fluorescenc e from untreated controls (Table 2 3). Dry biomass of susceptible populations had decreased to less than 10 % by 7 WAT at the lowest concentration and decreased further to less than 5 % at 11 WAT (Table 2 4). Townline did not exhibit a decline lower than 80% 1 at either harvest. This experiment confirmed that the Townline population does not respond to fluridone herbicide at the concentrations that would be legally permitted for control in Michigan. Plants originati ng from locations throughout the country showed little variat ion and results were combined. This speaks to the success and applicability of fluridone as a tool to treat invasive watermilfoi ls at low use rates. Experiment 3 The outdoor mesocosm experiment a gain demonstrated the response to fluridone by the different populations of watermilfoils. Due to the slow acting nature of fluridone, significant differences in fluorescence yield and biomass between susceptible and Townline populations were limi ted at 3 WAT (data not shown). The second harvest was conducted at 6 WAT and d ata from susceptible populations was combined since no significant differences in response to fluridone were found between populations. By this time, fluorescence yield differences were found at

PAGE 37

37 all concentrations 6 1 and above (Figure 2 2a ). Biomass differences existed at 3, 6, 1 for the susceptible populations when compared to Townline (Figure 2 2b ). Of note is the 200% increase in biomass of Townline plants from control to even the lowes t concen tration of fluridone herbicide. It appears from this data that Townline did not successfully compete with the other populations when no herbicide was present, however when fluridone decreased biomass of other populations the Townline population b iomass inc reased dramatically. By 8 WAT apical shoot tips were not present in many of the mesocosms so PAM yield data was not collected. Biomass data from susceptible populations was combined since no significant differences w ere found between populations. Townline plant biomass again continued to increase to 400% of untreated mesocosms at the lowest concentrations of fluridone (Figure 2 3). The combined susceptible populations showed significant differences from Townline at all concentrations (Figure 2 3). This stud y verified that Townline does not respond to fluridone herbicide in a similar manner to any of t he other analyzed populations. While the hybrid Indian Lake plants are genetically similar to Townline, their response was sig nificantly different by 8 WAT. Eve n though hybrid watermilfoils were geographically and genetically similar, as with Townline and Indian Lake populations, herbicide response was distinct. Little variation was found between all susceptible populations used in this study, which a llowed resul ts to be combined. This limited variation in response and sensitivity to fluridone is important to the success of fluridone as a tool to treat invasive watermilfoils across the country.

PAGE 38

38 o survive and (WSSA 1998). manipulation to make the plant tolerant; it is na 98). Since populations, specifically Townline population, cannot be referr ed to as herbicide resistance. These populations of plants do, however, exhibit an inc rease d tolerance to fluridone. It is unknown if the Townline population was selected for by previous use of fluridone herbicide or if this population developed tolerance for the h erbicide during hybridization. Now that a fluridone tolerant population of hybrid watermilfoil has been confirmed, it is important that resource managers take steps to prevent the spr ead of this unique po pulation to neighboring lakes. Townline Lake is located in central Michigan in the near vicinity of numerous other bodies of water. Th e potential for spread to other water bodies is highest in close proximity to the originally infested lake (Roley and Newman 2008). Resource managers should consider limiting public access to Townline Lake until the pop ulation of plants is controlled and a lso monitor neighboring lakes to detect any possible invasions of this unique hybrid watermilfoil

PAGE 39

39 Table 2 1. Invasive watermilfoil (Myriophyllum spp.) populations used in Experiment 2. Population Species Location Tolerance Townline Hybrid Michigan pr obable Frog hybrid Wisconsin susceptible Auburn EWM Minnesota susceptible Texas EWM Texas susceptible Note: Hybrid watermilfoil is a cross between northern watermilfoil (M. sibiricum) and Eurasian watermilfoil (EWM) (M. spicatum Table 2 2 Invasive watermilfoil ( Myriophyllum spp.) populations used in Experiment 3. Population Species Location Tolerance Townline hybrid Michigan probable Indian hybrid Michigan unknown Auburn EWM Minnesota susceptible Texas EWM Texas susceptible North Carolina EWM North Carolina susceptible Note: Hybrid watermilfoil is a cross between northern watermilfoil (M. sibiricum) and Eurasian watermilfoil (EWM) (M. spicatum). Table 2 3. Fluorescence yield (F v /F m ), represented as percent of the untreated control, in suscep tible and Townline populations of invasive watermilfoils as a function of fluridone concentration 7 and 11 weeks after treatment (WAT) 7 WAT 11 WAT fluridone concentration 1 ) Susceptible Townlin e Susceptible Townline 5 44.910.0 98.1 2.2 15.45.8 124.6 9.1 10 13.9 6.9 98.4 4.0 2.7 2.7 119.213.7 20 5.3 3.9 23.5 14.9 0 0 Note: Values indicate means with standard error (n=6). Concentrations with signific ant differences Table 2 4. Biomass represented as percent of the untreated control, in susceptible and Townline populations of invasive watermilfoils as a fu nct ion of fluridone concentration 7 and 11 weeks after treatment (WAT) 7 WAT 11 WAT fluridone concentration 1 ) Susceptible Townline Susceptible Townline 5 9.3 3.3 111.2 11.3 2.3 0.9 91.1 4.4 10 2.0 1.6 92.3 16.1 0.08 0.07 81.4 5.9 20 2.7 2.6 10.3 7.3 0 0 Note: Values indicate means with standard error (n=6). Concentrations with significant differences

PAGE 40

40 Figure 2 1. Above ground biomass, represented as percent of the untreated control, in susceptible and Townline invasive watermilfoil plants as a function of fluridone concentration 7 weeks after treatment Symbols represent means and error bars indicate standard error (n=5). Curves represent nonlinear regression. Concentrations with significant differences as found using Protected LSD

PAGE 41

41 Figure 2 2 Fluorescence yield (F v /F m ) (a) and dry biomass (b ) of suspected fluridone tolerant Towline hybrid watermilfoil and 4 combined susceptible populations of invasive water mil foils 6 weeks after treatment with fluridone, represented as per cent of the untreated control. Symbols represent means and error bars indicate standard error (n=4). Curves indicate nonlinear regression. Significant differences within each concentration bet ween Townline and susceptible population s as found using ( are marked with an asterisk a b

PAGE 42

42 Figure 2 3 Dry biomass of suspected fluridone tolerant Townline hybrid watermilfoil and 4 combined populations of susceptible invasive water milfoils 8 weeks after treatment with fluridone, represented as per cent of the untreated control. Symbols represent means and error bars indicate standard error (n=6). Curves r epresent nonlinear regression. Significant differences within each concentration between Townline and the combined susceptible populations, as found using ( are marked with an asterisk

PAGE 43

43 CHAPTER 3 A COMPARISON OF METHODS FOR CHARACTERIZING HERBICIDAL EFFECTS ON SUBMERSED AQUATIC VASCULAR PLANTS Although native submersed aquatic plants are often desirable in certain ecosystems, invasive plants have the potential to overtake desirable plant communities and have m any other detrimental effects. In Lake George, New York, an invasion of Eurasian watermilfoil ( Myriophyllum spicatum L.) caused a rapid decline in the number of aquatic plant species ove r a three yea r period (Madsen et al. 1988). Anthropogenic uses of waterbodies are also negatively impact ed by invasive aquatic plants. Heavy infestations and dense surface canopies can impair commercial and recreational navigation, disrupt flood control, a nd become a habitat for disease vectors. For this reason, invasive aquatic plants are often managed to maintain a non harmful level or eradicated from a water body. Many submersed aquatic plants have vestigial features from their terrestrial heritage such as a thin cuticle, poorly developed stomata and xylem elements which are essentially functionless (Sculthorpe 1967). Since submersed plants greatly differ from their terrestrial counterparts, research methods to characterize these plants are often vastly d ifferent than those dev eloped for terrestrial plants. Research concerning invasive submersed aquatic macrophytes has been ongoing for several decades both in the field and in the lab (Haller and Sutton, 1973, Van et al. 1976, Bowes et al. 1977). Laboratory techniques have been used to evaluate many aspects of these plants including physiological characteristics, herbicide response, and metabolic activity (Holladay and Bowes 1980, Kane and Gilman 1991, MacDonald et al. 1993). These evaluations are advantageo us due to the controlled nature of the laboratory

PAGE 44

44 environment, smaller area needs compared to field or mesocosm studies, and speed of determining outcomes. Pigment analysis has been used traditionally to characterize the biochemical response of a plant to fluridone (1 methyl 3 phenyl 5 [3 (trifluoromethyl)phenyl] 4(1 H ) pyridinone) Fluridone inhibits the phytoene desaturase enzyme of the carotenoid fluridone can be el ucidated Sprecher et al. (1998) developed an extraction method that measures absorbance spectrophotometrically to quantify p carotene levels. In fluridone susceptible plants, phytoene levels increase after fluridone carotene levels w ould be lower than control plants. In fluridone carotene levels are similar to thos e of untreated control plants. Puri et al. (2006) used this technique to determine and document fluridone resistance in several hydrilla ( Hy drilla verticillata [ L.f. ] Royle) populations in Florida. Chlorophyll analysis is an indirect method of det ermining fluridone resistance. Although fluridone does not directly affect chlorophyll biosynthesis, the absence or carotene leads to t he destruction of chlorophyll. Therefore, in a susceptible plant exposed to fluridone, chlorophyll levels in new tissue will be lower than those in control plants. Hiscox and Israelstam (1979) developed a method of chlorophyll extraction without maceratio n us ing dimethyl sulfoxide (DMSO). This study documented that chlorophyll extracted in DMSO is a simple process that produce s stable chlorophyll extracts. A more recent study determined that chlorophyll a chlorophyll b and total carotenoids could all be quantified by the same extract in DMSO and analyzed with a spectrophotometer at differen t absorbances (Wellburn 1994). This method provides

PAGE 45

45 pigment analysis that is comprehensive in the study of fluridone resistance or tolerance and less complex than the p reviou sly described Sprecher method. Hiscox and Israelstam (1979 ) used an incubation period of 6 hours in DMSO and a 65C water bath for chlorophyll extraction in terrestrial pl ants with a developed cuticle. This method has also been applied to submersed a quatic plants by several authors (Netherland et al 1993, Netherland and Get singer 1995, Bultemeier 2008). However, since the thickness of the cuticle in the plant sample is determinant of the incubation time, it is thought that incubation time for submerse d plants could be significantly less than 6 hours due to the poorly developed cuticle. Although pigment analysis provides a method for determining herbicide activity in the plant, the methods require a destructive harvest of plan t tissue. There is a need f or non destructive and repeatable methods of analy sis on the same tissue source. Pulse amplitude modulated (PAM) fluorometery can give information as to chlorophyll functionality by measu ring chlorophyll fluorescence. A PAM fluorometer works by focusing a saturating beam of light on the desired region of the plant. By measuring the re radiation, or fluorescence, a yield ratio is calculated by the instrument. Higher fluorescence yield ratio indicates highly functioning chlorophyll whereas lower yield ratios indicate damaged o r non functioning chlorophyll. Yield is a ratio of F v /F max where F max is equal to the fluorescence when the saturating p ulse is applied to the tissue. F v is equal to F max F where F is the fluorescence of the tissu e with no light pulse a pplied. The plant fluoresces more when chlorophyll is damaged, which indicates a higher F max value. However, the yield output ratio is lower because damaged chlorophyll also fluoresces more under ambient light condition s. Therefore, a higher Y ratio value

PAGE 46

46 indicates chlorophyll that is functioning normally where a lower Y value indicates damaged chlorophyll (Bo lhar Nordenkampf et al. 1989). PAM fluorometery has been used to study irradiance stress (Ralph et al. 1998), salinity stress (Kamermans et al. 1999), and shoot to landscape differences in photosynthesis in sea grasses (Durako and Kunzelman 2002). In situ measurement of photosynthetic activity of Red Sea faviid corals has also been measured (Beer et al 1998). This technique is useful because it is a no n destructive method of evaluating the activity of chlorophyll and has also been used to evaluate herbicidal effects on plants. Ireland et al. (1986) used fluorometery to document decreased fluorescence in wheat ( Triticum spp.) 30 minutes after exposure to glyphosate ( N [phosphonomethyl] glycine) herbicide. The herbicide diuron ( N [3,4 dichlorophenyl] N N dimethylurea) a photosystem II inhibitor, was shown to reduce fluorescence yield ratio in sea grasses two hours after exposure as measured with a di vi ng PAM (Haynes et al. 2000). Junea et al. (2001) evaluated the effects of mercury and metolachlor (2 chloro N [2 ethyl 6 methylphenyl] N [2 methoxy 1 methylethyl] acetamide) a mitosis inhibiting herb icide, on six algal species. Using a PAM fluorometer to detect the effects of pigment synthesis inhibiting herbicides such as fluridone has not been documented in aquatic plants, but would potentially be a non destructive method for evaluating fluridone a ctivity in the plant. Several authors have studied invasi ve water milfoils in the laboratory, but different methods were used t o evaluate herbicide response. Netherland (1995) used apical shoot tips 10 15cm in length, while Forney and Davis (1981) documented using ap ical shoot tips 7 cm in length. Shorter shoot s egment s are not expected to have the

PAGE 47

47 carbohydrate reserves necessary to thriv e for long periods in culture. However, these shoot segments are thought to respond more quickly to herbicide applic ation for this same reason. On the other hand, longer shoots ha ve the carbohydrate reserves to thrive in culture, but it is speculated that they also can use these same reserves to initially withst and the herbicide application. A multitude of methods have been well published using small scale laboratory experiments to study aquatic plants, however, no studies report on pigment analysis incubation periods, optimal shoot length for determining herbicide response in invasive water milfoils, or using pulse a mplit ude m odulated (PAM) fluorometry to evaluate herbicide resistan ce or tolerance in aquatic plants. Materials and Methods Pigment Extraction Incubation Time Stock plants of hydrilla and Eurasian watermilfoil (EWM) were maintained in 950 L outdoor mesocosms at the Center for Aquatic and Invasive Plants in Gainesville, F lorid a throughout the study period. These plants were exposed to ambien t temperature and photoperiod. Actively growing shoots were harvested for use in the study and washed thoroughly with tap water to re move algae and microorganisms. Apical shoot tips wei ghing 0.1 g were placed into 5 mL dimethyl sulfoxide (DMSO) in HDPE centrifuge tubes and incubated in a 65C water bath for 1 hour, 3 hours, or 6 hours. A second experiment utilized shorter time intervals of 15 minutes, 30 minutes, and 1 hour. Both experi ments were conducted using a completely randomized design in a factorial arrangement where each plant species received each incubation interval with 4 replications. At the designated time interval, a 2 mL aliquot of extract was analyzed using a BioMate spe ctrophotometer (Thermo Scientific, Pittsburgh, PA) at 480, 649, and

PAGE 48

48 665 nm to determine total carotenoids, chlorophyll a and chlorophyll b respectively. DMSO was used as a blank. Total chlorophyll was calculated with the following equation: (12.19*absorba nce at 665nm 3.45*absorbance at 649nm)+(21.99*absorbance at 64 9nm 5.32*absorbance at 665nm). Total carotenoids were calculated as (1000*absorbance at 480nm 2.14*chlorophyll a 70.12*chlorophyll b )/200. Experiments were repeated. Data were converted to perce nt of longest incubation time and subjected to analysis of variance ( ANOVA ) There was not a significant treatment by run interaction for either experiment, therefore results wer e combined. Means were separated using Fi PAM Fluorometery Stock plants of EWM were maintained in 950 L outdoor mesocosms at the Center for Aquatic and Invasive Plants in Gainesville, Florida throughout the study period. These plants were exposed to ambien t temperat ure and photoperiod. The first population used in this study, a hybrid watermilfoil, originated from Townline Lake in western Michigan, which is thought to have increased tolera nce to the herbicide fluridone. The second population used originated from Texa s and is confirmed EWM suspected t o be susceptible to fluridone. This susceptible population is hereafter Actively growing shoots of each population were harvested for use in the study and washed thoroughly with tap water to r emove algae an d microorganisms. Apical shoots 6 cm in length were placed in glass culture tubes with 4 g L 1 sodium bicarbonate. Plants were treated with 0, 2.5, 5, 10, or 2 1 fluridone herbicide (Sonar AS, S ePRO Corporation, Carmel, IN), with 4 replications per treatment in a completely randomized design. Plants were kept in a climate

PAGE 49

49 controlled growth chamber for the duration of the experiment (23C: 21C day:night t 2 light). Fluorescence was measured at the growing point 7 days after treatment (DAT) using a PAM fluorometer (Mini PAM, Walz, E ffetrich Germany). New apical tissue was harvested for pigment an alysis as described previously. The experiment was repeated. There was not a significant treatment by run interaction for either experiment, the refore results were combined. Data were converted to percent of the untreated control and subj ected to nonlinear regres sion. the effective concent ration to reduce pigments by 50% (EC 50 ) for fluorescence yield, total chlorophyll, and total carotenoids for each population Shoot Length Response Stock plants of flurid one susceptible EWM were maintained in 950 L outdoor mesocosms at the Center for Aquatic and Invasive Plants in Gainesville, Florida throughout the study period. Established and thriving plants were harvested for use in the study and washed thoroughly with tap water to re move algae and microorganisms. The study was repeated using EWM plants harvested from the Chassahowi tzka River in Central Florida. Apical shoot tips were excised into 2, 4, 6, or 8 cm segments and washed thoroughly in tap water to remove al gae and microorganisms. These segments with 4 g L 1 sodium bicarbonate. Each shoot tip size was then treated with 5, 10, or 20 1 fluridone herbicide. An untreated cont rol of each shoot length was also included. Plants were kept in a climate controlled growth chamber for the duration of the experiment (23C: 21C day:night temperature, 14 light:8 dark photoperiod, and 350 2 light). Fluorescence was measured at the growing point 2, 6, and 10 DAT using

PAGE 50

50 a PAM fluorometer. At 10 DAT, 0.1 g of new growth from each shoot was h arvested for pigment analysis. The previously described method of total carotenoid and total chloro phyll quantification was used. The study was cond ucted using a completely randomiz ed design with 4 replications. Data were converted to percent of the untreated control, co mbined and subjected to ANOVA. There was not a significant treatment by run interaction for either experiment, therefore results were combined. Means were between treatm ents within each shoot length. Results and Discussion Pigment Extraction Incubation Time In the first experiment there were no significant differences in the levels of total chlorophyll or carotenoids for either species, with the exception of slightly lower levels at the 6 hour incubation time (Table 3 1). In fact, lower levels were measured at the longer incu bation times of 3 and 6 hours. C hlorophyll and carotenoids are known to degrade when exposed to light ( MacKinney 1941), and it is speculated that the longer incubation times showed a loss of these pi gments due to photodegradation. In the second experiment, the total incubation time was 1 hour since Experiment 1 had previously shown that this interval was sufficient incubation time. However, in this experiment a significantly lower percentage of total chlorophyll was extracted for both hydrilla and EWM at 15 and 30 minutes (Table 3 2). For total carotenoids, no significant differences were shown for hydrilla at 30 minutes and 1 hour incubation time, however 15 m inutes was significantly lower. EWM exhibited significantly decreased total carotenoids at both 15 and 30 minutes compared to 1 hou r.

PAGE 51

51 These results indicated that the 6 hour incubation time period as suggested by Hiscox and Israelstam and employed in aquatic pl ant research is not necessary. A 1 hour incubation time is sufficient, but shorter extraction periods are not long enough to o btai n complete pigment extraction. These results do not correspond to those of Spencer and Ksander (1987) who found that a 20 minute incubation time at 65C was sufficient for chlorophyll analysis in hydrilla and EWM. However, due to the lag period after e xtraction and spectrophotometric analysis some pigment degradation could have occurred, thus compromisi ng the actual pigment content. It is also possible that the pigment concentration in these shoots was significantly lower, thus requiring a shorter incub ation time. PAM Fluorometry This study was conducted to evaluate the accuracy of three different methods of determining fluri done response in watermilfoil. More specifically, PAM fluorometery was compared to more traditional chemical analysis of pigment co ntent, which is laborious, expensive, time consuming and requires destructive harvest of the tissue Regression analysis was used to interpret the response of the plants to fluridone a s a function of concentration. From this analysis, the effective concen tration that would cause a predicted reduction of 50% was calculated (EC 50 ) To statistically compare each method, 95% confidence intervals of the mean EC 50 values were generated. Fluorescence yield EC 50 values for both the tolerant and susceptible populat ions overlapped with EC 50 values of total carotenoids and total chlorophyll (Table 3 3). These results indicate that measuring fluorescence yield gives similar and statistically equivalent results as that of the more complex and destructive pigment analysi s. As expected, fluorescence yield, total carotenoids, and total chlorophyll EC 50 values of the

PAGE 52

52 tolerant population were all significantly higher than those of the susceptible population of Eurasian watermilfoil (Table 3 3). Within each population, the EC 5 0 values of fluorescence yield, chlorophyll, or carotenoid content wer e not significantly different. This indicates that each method provides a similar measure of fluridone response, and the ability to elucidate tolerance or resistance, as shown by the dif ferences bet ween populations, is reliable. This finding will greatly aid resource managers because PAM fluorometery determines results in a m uch more time sensitive fashion without a requirement for destructive harvest of plant tissue. Shoot Length Respons e One of the most challenging aspects of laboratory experimentation with aquatic plants, especially submersed aquatic macrophytes, is the select ion of uniform plant material. Herbicide response studies further complicate this situation, because many herb ic ides work only on new growth. Therefore, the growth status of the plant tissue and the ability to have fairly uniform growth potential fr om plant material is critical. A key factor for growth in these types of plants is thought to be initial shoot length. Therefore, these studies were conducted to determine the optimal shoot length to provide accurate results of fluridone response. PAM fluorometery was shown to provide an accurate assessment of fluridone in the previous experiment and was thus used in this study to measure response over time as a function of shoot length. At 2 DAT, differences were observed at the lowest concentration 1 ) for 3 4). At concentrations 1 or higher, differences were o bserved in all segment lengths. By 6 DAT, differences between the control and all concentrations were observed regardless of shoot length (Table 3 5). In 2 cm and 4

PAGE 53

53 cm shoots, differences were also observed between concentrations 1 and concentrations 1 Differences were also observed at the lowest concentration in all shoot lengths by 10 DAT (Table 3 6). This demonstrates the abil ity to determine 1 of fluridone within 2 DAT if 4 cm or longer shoots are utilized. To determine differences between lower concentrations over a longer period of time, 2 cm shoot lengths provide the most consistent results of lengths studied Resource managers concerned with potentially fluridone tolerant or resistant EWM populations would benefit from obtaining this knowledge as quickly as possible, therefore 4 cm or 6 cm shoot segments lengths would be recommended in such studies. Total chlorophyll content was significantly lower at the highest concentration of 1 ) in all shoot lengths while shoots 2 cm in length displayed no 1 fluridone (Table 3 7). Shoots 4 cm, 6 cm, and 8 cm in length showed differences between the control and each conce ntration of fluridone. Total carotenoids were also significantly lower at the highest concentration in all shoot lengths (Table 3 8). Shoots 6 cm in length produced the most logical differences between concentrations and would likely be the best choice of shoot length to determine pigment responses to fluridone in EWM. Taking into consideration fluorescence yield data and pigment data, 6 cm shoot length segments would provide the most accurate determination of fl uridone response in EWM. At all data collect ion intervals after treatment, 6 cm shoot tips produced significant responses between th e control and different concentrations Pigment analysis was also most informative with 6 cm shoot tips in that differences were

PAGE 54

54 observed between the control and each c oncentration and differences were al so observed between some concentrations Based on visual observations from the study, 2 cm and 4 cm shoot lengths do not have enough carbohydrate reserves to produce the biomass needed for eff ective pigment analysis. Sh oots 8 cm in length did not exhibit logical differences between rates in fluoresc ence yield or pigment analysis. This is likely due to the excess reserves in the 8 cm shoot that allow it to initially withstand herbicide treatment. Refining and developing t hese methods are integral for herbicide response research with submersed aquatic plants, specifically Eura sian and hybrid watermilfoils. Decreasing incubation time during chlorophyll and carotenoid extraction not only is a time saving measure, but reduces the likelihood of pigment degradation dur ing extended incubation times. Utilizing PAM fluorometery to document herbicide re sponse is also more efficient. This method is an improvement from destructive pigment analysis in that pl ants remain intact. In turn, the analysis is able to be repeated over time to nthesis inhibiting herbicides. Choosing the optimal shoot length for laboratory experiments is essential to obtaini ng accurate and rapid r esults. These methods will help resource managers to efficiently evaluate potentially tolerant populations of invasive watermilfoils and allow for more rapid decision making to occur.

PAGE 55

55 Table 3 1. Total chlorophyll and total c arotenoids extracted from hy drilla and Eurasian watermilfoil (EWM) tissue in dimethyl sulfoxide as a function of 6 hour incubation time Total Chlorophyll Total Carotenoids Incubation Time Hydrilla EWM Hydrilla EWM 1 hr 109.8 ab 89.4 a 113.5 a 98.7 a 3hr 115.0 a 98.8 a 110.3 a 102.3 a 6hr 100 .0 b 100 .0 a 100 .0 a 100 .0 b LSD 12.3 11.4 9.2 6.3 Note: Values represent percentage of total (6 hour incubation) pigment extracted at each time interval (n=8). Letters indicate significant differences between incubation times within a column as found using F Table 3 2 Total chlorophyll and total carotenoids extracted from hydrilla and Eurasian watermilfoil (EWM) tissue in dimethyl sulfoxide as a function of 1 hour incubation time Total Chlorophyll Total Carotenoids Incuba tion Time Hydrilla EWM Hydrilla EWM 15 min 62.7 c 48.7 c 85.5 b 56.7 c 30 min 85.8 b 78.6 b 96.3 a 83.1 b 60 min 100 .0 a 100 .0 a 100 .0 a 100 .0 a LSD 8.3 17.3 5.8 15.0 Note: Values represent percentage of total (1 hour incubation) pigment extracted at each time interval ( n=8). Letters indicate significant differences between incubation times within each column as found using F Table 3 3 EC 50 values for fluorescence yield, total chlorophyll, and total carotenoids for suspected fluridone tolerant and fluridone susceptible invasive watermilfoil populations Tole rant Population Susceptible Population EC 50 95% Confidence Interval R 2 value EC 50 95% Confidence Interval R 2 value Fluorescence Yield 21.3 6.3 0.7376 3.7 0.5 0.8924 Carotenoids 25.4 10.7 0.6064 7.4 3.2 0.5531 Chlorophyll 24.9 11.8 0.5648 7.4 2.7 0.6772 Note: R 2 values indicate the fit of the exponential decay model (y=a*exp( b*fluridone concentration)) with n=8.

PAGE 56

56 Table 3 4. Fluorescence Yield (F v /F m ), as percent of the untreated control in Eurasian watermilfoil shoot lengths as a fun ction of fluridone concentration 2 days after treatment Fluridone Concentration 1 ) Shoot Length 2 cm 4 cm 6 cm 8 cm 0 100 .0 a 100 .0 a 100 .0 a 100 .0 a 5 98.1 a 89.2 b 92.9 bc 94.2 b 10 89.3 b 88.2 bc 95.9 ab 87.1 c 20 91.1 b 80.4 c 87.9 b 90.0 bc LSD 5.9 7.8 5.3 4.8 Note: Letters following percent of the untreated control values represent significant differences between Table 3 5. Fluorescence Yield (F v /F m ), as percent of the untreated control in Eurasian watermilfoil shoot lengths as a function of fluridone concentration 6 days after treatment Fluridone Concentration 1 ) Shoot Length 2 cm 4 cm 6 cm 8 cm 0 100 a 100 a 100 a 100 a 5 80.9 b 66.7 b 68.0 b 72.8 bc 10 62.1 c 70.1 b 73.6 b 86.4 ab 20 53.4 c 55.2 c 69.4 b 62.2 c LSD 14.1 10.1 12.2 19.3 Note: Letters following percent of the untreated control values represent significant differences between Table 3 6. Fluorescence Yield (F v /F m ), as percent of the untreated control in Eurasian watermilfoil shoot length s as a function of fluridone concentration 10 days after treatment Fluridone Concentration 1 ) Shoot Length 2 cm 4 cm 6 cm 8 cm 0 100 a 100 a 100 a 100 a 5 72.9 b 57.9 b 57.1 b 65.5 bc 10 60.9 c 51.6 b 59.0 b 75.5 b 20 53.7 c 53.1 b 53.2 b 47.1 c LSD 11.3 14.5 12.1 8.2 Note: Letters following percent of the untreated control values represent s ignificant differences between with n=8

PAGE 57

57 Table 3 7. Total chlorophyll, represented as percent of the untreated control in E urasian watermilfoil shoot lengths as a function of fluridone concentration 10 d ays after treatment Fluridone Concentration 1 ) Shoot Length 2 cm 4 cm 6 cm 8 cm 0 100 a 100 a 100 a 100 a 5 98.8a 70.6 b 79.1 b 52.8 bc 10 88.1 ab 76.0 b 63.1 bc 64.8 b 20 76.5 b 57.8 b 50.2 c 37.2 c LSD 19.9 21.0 17.2 18.3 Note: Letters following percent of the untreated control values represent significant differences between Table 3 8. Total carotenoids, rep resented as percent of the untreated control in E urasian watermilfoil shoot lengths as a function of fluridone concentration 10 days after treatment Fluridone Concentration 1 ) Shoot Length 2 cm 4 cm 6 cm 8 cm 0 100 a 100 a 100 a 100 a 5 78 .1 b 72.6 b 70.5 b 57.8 c 10 78.2 b 73.1 b 58.1 bc 73.2 b 20 66.3 b 61.9 b 50.1 c 41.4 d LSD 13.4 16.8 13.9 15.0 Note: Letters following percent of the untreated control values represent significant differences between concentrations in eac

PAGE 58

58 CHAPTER 4 CHARACTERIZATION OF FLURIDONE TOLERANT H YBRID WATERMILFOIL ULTILIZING PIGMENT A NALYSIS AND PAM FLUO ROMETERY Invasive water milfoils Eurasian watermilfoil ( Myriophyll um spicatum L. ) and hybrid watermilfoil ( M. spicatum x M. sibiricum ), are problematic weeds in many water bodies they inhabit. Plants grow to form dense canopies displacing native vegetation, altering water quality, and obstructing recreational uses of wat erways. Madsen et al. (1991) found that Eurasian watermilfoil formed dense canopies which can shade out desirable n ative plants in the ecosystem. Dense canopy formation has been shown to negatively impact water quality by reducing dissolved oxygen in water below the mat and increasing surface temperatur es and pH (Bowes et al. 1979). Submersed aquatic weeds have also been shown to harbor algae species harmful to both wildlife and hum an health (Wilde et al. 2005). When water milfoils spread within waterbodies and result in unfavorable conditions for wildlife and fisheries, displacement of desirable native vegetation, and impacts on recreational access or aesthetics, management of these plants is often required. Hybrid watermilfoil was first docume nted in 2002 b y Moody and Les. Populations previously thought to be the invasive Eurasian watermilfoil were found, through nuclear ribosomal DNA analysis, to be hybrid populations from the parental species Eurasian watermilfoil and northern wat ermilfoil (Moody and Les 2 002). Hybrid watermilfoils present unique challenges for management due to inherited traits such as invasiveness and rapid growth rate from the Eurasian parent and the potential for turion formation in some hybrid populat ions from the northern parent. Once a hybrid population is established in a water body, the gene combinations gained from parental species tend to become incorporated in the hybrid popula tion (Sturtevant et al. 2009). It is important

PAGE 59

59 to note that numerous hybrid watermilfoil populations hav e arisen independently and therefore traits associated with hybrids from one lake may be quite different when compared with those from another lake. Genotypic and phenotypic differences exist between populations of the hybrid plants and generalities cannot be made regarding specific growth or management traits. Repeated hybridization as well as back crossing has been do cumented (Moody and Les 2002). Therefore, it is important to refer to each hybrid population independently. Chemical control methods are co mmonly used to manage invasive water milfoils. Fluridone (1 methyl 3 phenyl 5 [3 (trifluoromethyl)phenyl] 4(1 H ) pyridinone) is an attractive choice for water managers due to its low use rates, native plant selectivity, ability to target water milfoil in the entire lake, and potential for multiple years of c ontrol from a single treatment. This herbicide is used frequently by water managers to control Euras ian and hybrid watermilfoils. There have been numerous claims of reduced herbicide response by hybrid wate r milfoils; however there is limited publis hed information on this topic. Triclopyr ([3,5,6 trichloro 2 pryidinyl]oxy acetic acid) and 2,4 D amine ([2,4 dichlorophenoxy] acetic acid) were found to inhibit growth of both Eurasian and hybrid watermilfoil acce ssions in a similar manner following exposure to labeled use rates (Poovey et al. 2007). Recently, differences between Eurasian and hybrid watermilfoil biotypes were noted following exposure to low continuous concentrations of 2,4 D ( Glomski and Netherland 2010). Pigment Analysis Fluridone is an inhibitor of the carotenoid biosynthe sis pathway; specifically a noncompetitive inhibitor of the phytoene desaturase (PDS) enzyme. The PDS enzyme

PAGE 60

60 catalyzes the desaturation of phytoene in the rate limiting step of t his p athway (Chamovitz et al. 1993). When PDS is inhibited, phytoene levels increase and carotenoid prod uction in the cell is limited. Carotenoids function to shield chlorophyll from excess light and help dissipate the oxidat ive energy of singlet oxygen. I n fluridone treated plants, carotenoids are not synthesized in new tissue. Therefore, they are not present to quench the energy of oxygen radicals, allowing for the formation of lipid radicals in chloroph yll molecules (Senseman 2007). This results in degra dation of chlorophyll, bleaching of new tissue and subsequent necrosis a nd plant death. By e to fluridone can be studied. Sprecher et al. (1998) developed an extraction method that utilizes absorb ance spectroscopy to quantify p carotene levels. In fluridone susceptible carotene levels are less than control plants. carotene levels are similar to thos e of un treated control plants. Puri et al. (2006) used this technique to demonstrate resistance in several hydrilla ( Hydrilla verticillata [L.f.] Royle) populations collected throughout Florida. Chlorophyll analysis is an indirect method of det ermining fluridone resistance. Although fluridone does not directly affect chlorophyll biosynthesis, the absence or carotene leads to the destruc tion of chlorophyll molecules. Therefore, in a susceptible plant exposed to fluridone, chlorophyll levels in new tissue will be lower than those in control plants. Several extraction methods have been evaluated and utilized for pigment analysis using various solvents (Iriyama et al. 1974, Hiscox and Isrealstam

PAGE 61

61 1979, Moran and Porath 1979 ) but the non macerated method of Wellborn ( 1994) using dimethyl sulfoxide (DMSO) proved to be the m ost useful in these studies. Although pigment analysis provides a method for determining herbicide activity in the plant, the methods employ a destr uctive harvest of plant tissue. Due to the transitio nal nature of fluridone response, there is a need for non destructive and r epeatable methods of analysis. Pulse amplitude modulated (PAM) fluorometery can give information as to chlorophyll functionality as a measur e of chlorophyll fluorescence. PAM fluoro metery has been used to study irradiance stress (Ralph et al. 1998), salinity stress (Kamermans et al. 1999), and shoot to landscape differences in photosynthesis in sea grasses (Durako and Kunzelman 2002). In situ measurement of photosynthetic activity of Red Sea faviid corals has also been measured (Beer et al 1998). This technique is useful because it is a non destructive method of evaluating the activity of chlorophyll and has also been used to evaluate herbicidal effects on plants. Ireland et al. (198 6) used fluorometery to document decreased fluorescence in wheat ( Triticum spp.) 30 minutes after exposure to glyphosate ( N [phosphonomethyl] glycine) herbicide. The herbicide diuron ( N [3,4 dichlorophenyl] N N dimethylurea) a photosystem II inhibitor, w as shown to reduce fluorescence yield ratio in sea grasses two hours after exposure as measured with a di ving PAM (Haynes et al. 2000). Junea et al. (2001) evaluated the effects of mercury and metolachlor (2 chloro N [2 ethyl 6 methylphenyl] N [2 methoxy 1 methylethyl] acetamide) a mitosis inhibiting her bicide, on six algal species. A PAM fluorometer works by focusing a saturating beam of light on the desired region of the plant. Yield ratio is calculated by the instrument. Higher fluorescence yield

PAGE 62

62 ratio indicates highly functioning chlorophyll whereas lower yield ratios indicate damaged o r non functioning chlorophyll. Yield is a ratio of F v /F max F max is equal to the fluorescence when the saturating p ulse is applied to the tissue. F v is equal to F max F w here F is the fluorescence of the tissu e with no light pulse applied. The plant fluoresces more when chlorophyll is damaged, which indicates a higher F max value. However, the yield output ratio is lower because damaged chlorophyll fluoresces more under amb ient light conditions which is noted in a higher F value. Therefore, a higher Y ratio value indicates chlorophyll that is functioning normally where a lower Y value indicates damaged chlorophyll (Bo lhar Nordenkampf et al. 1989). In May 2010, a 220 acre lak e in western Michigan (Townline Lake) was treated with a rate of fluridone permitted by the Michigan De partment of Natural Resources. The population of documented hybrid watermilfoil in the water body was not controlled with the legal rate of the her bicide The observation that a hybrid watermilfoil may show increased tolerance to fluridone has fueled more speculation on the nature of hybrids and their response to herbicides. The use of a PAM fluorometer to detect the effects of pigment synthesis inhibiting herbicides such as fluridone has been documented in aquatic plants, but with limit ed results (unpublished data). Fluorometery would potentially be a non destructive method for evaluating fluridone activity in specific plant tissues over a period o f time. This method will be compared to traditional pigment analysis when determining differential responses of Townline and several susceptible invasive watermilfoil populations in laboratory experiments. Although mesocosm studies have been used previously to eva luate invasive water r intensive

PAGE 63

63 and time consuming. Laboratory methods could potentially reduce the time needed to document response from several weeks to several days. This reduction in lag time could quickly provide resource managers with valuable information to efficiently treat water milfo il infestations in the field. This study focuses on further evaluating Townline hybrid watermilfoil and implementing laboratory methods when making comparisons with water milfoil populations obtained from several states. Materials and Methods Experiment 1 Apical shoot tips 6 cm in length were harvested from outdoor stock cultures of water milfoil populations at the Center for Aquatic and Invasive Plant s in Gainesville, Florida. Populations used in this study included Townline, Indian, Auburn, and North Carolina (Table 4 1). Townline is a hybrid watermilfoil suspected to be tolerant to fluridone. The Indian population is also a hybrid and is closely related to Townline d ue to similar geography and genetics (T hum, pers comm. 1 ). Both Auburn and North Carolina populations a re confirmed EWM populations. Apical shoot tips were thoroughly washed with tap water and a single tip was placed in a glass culture tube containing Andr (1989), supplemented with 4 g L 1 of sodium bicarbonate a s an additional carbon source. Plants were immediately treated with 0, 3, 6, 9, 12, 24, or 1 of fluridone (Sonar AS, S ePro Corporation, Carmel, IN). C ulture tubes were maintained in a climate controlled growth chamber for the duration of the experiment (23C: 21C day:night 2 light). 1 R. Thum, Grand Valley State University, Annis Water Resources Institute Muskegon, MI 49441.

PAGE 64

64 Shoot tips were removed 3, 5, and 7 days after treatment (DAT) an d new apical growth was measured with a PAM fluorometer (Mini PAM, Walz, Effetrich Germany) to determine fluorescence yield. After measurement, shoot tips were returned to t heir respective culture tubes. At 7 DAT, 0.2 g of new apical shoot growth was harv ested from each shoot for pigment analysis. Total chlorophyll and total carotenoids were quantified using methods descr ibed by Wellburn et al (1994). Harvested samples were placed in plastic tubes with 5mL of dimethyl sulfoxide ( DMSO ) and incubated fo r 1 h our in a 65C water bath. Extract from each sample was analyzed spectrophotometrically (BioMate 5, Thermo Scientific, Pittsburgh, PA) at 480nm for total carotenoids, 649 nm for chlorophyll b and 665 nm for chlorophyll a DMSO was used as a blank. Total chl orophyll was calculated with the following equation: (12.19*absorbance at 665nm 3.45*absorbance at 649nm) + (21.99*absorbance at 649nm 5.32*absorbance at 665nm). Total carotenoids were calculated as (1000*absorbance at 480nm 2.14*chlorophyll a 70. 12*chlorophyll b ) / 200. Each treatment was replicated four times and the entire study was repeated. Analysis of variance (ANOVA) was used to determine mean effe cts and possible interactions. There was no experiment by treatment interaction so the results from b oth experiments were combined. Protected LSD each individual population and Townline population. Experiment 2 Plants with an unknown response to fluridone were obtained from 5 lakes in Minnetonka, Minnesota to be compared to the re sponse of Townline population. The Michigan lakes tested were Missaukee, Cadillac, Shamrock, Ryerson, and Round

PAGE 65

65 Lakes. All of these popul ations were confirmed as EWM. The study wa s identical to Experiment 1 in methodology except that PAM yield was onl y measured at harvest (7 DAT). No significant differences were found between the 8 populations tested. Data was combined and compared to Townline using ANOVA and D differences within each concentration Nonlinear regression was fitted to the data. Results and Discussion Experiment 1 Chlorophyll fluorescence data showed limited differences at 3 DAT (Table 4 2 ). However, by 5 DAT diffe rences were detected between Townline and each population at each concentration (Table 4 3 ). By 7 DAT, significant differences were still apparent at most concentrations (Table 4 4 ). Total chlorophyll data did not exhibit the same level of differences, whi le total carotenoids also showed fewer differences than fluorescence data as well (Table s 4 5 and 4 6 ). The lack of differences found using pigment analysis is most likely due to possible varia tion when using these methods. The PAM fluorometer resulted in less variation in re sults at 5 and 7 DAT. These results suggest that using a PAM fluorometer to measure fluorescence provides more distinguishable results than typical pigment analysis. Indian Lake is suspected to have a similar response to fluridone as th at of Townline Lake due to geographical proximity and genetic sim ilarities between populations. However, the fluorescence yield response differed at several concentrations 3 DAT, all concentrations at 5 DAT, and all concentrations but the highest concentra tion at 7 DAT (Table s 4 2 4 3, and 4 4 ). Chlorophyll analysis showed 1 while total carotenoids were

PAGE 66

66 significantly different at all concentrations but the highest (Table s 4 5 and 4 6 ). Although repor ted as being genetically similar to Townline Lake, the response of the hybrid Indian Lake population to fluridone was different to that of Townline. Fluorescence yield of Auburn differed from Townline at only the lowest and highest concentrations 3 DAT, bu t differences were found at all concentrations at 5 and 7 DAT (Table s 4 2 4 3, and 4 4 ). This suggests that the PAM fluorometer does not detect a quantifiable response to fluridone until at least 5 DAT. Chlorophyll and carotenoid data did not exhibit the same distinguishable trends as fluorescence yield. Pigment data was 1 1 for carotenoids (Table s 4 5 and 4 6 ). This is again evidence that the PAM fluorometer provides mor e precise measurement when compared to pigment analysis. As evidenced by the fluorescence yield results, Auburn and Townline plants had a different response to fluridone, with Townline being less affected at each concentration The population of water milfo il from North Carolina began to show significant differences at sev eral concentrations beginning at 3 DAT. At this time, differences were found at 3, 6, 12 and 48 1 (Table 4 2). By 5 DAT, differences were found at each concentration and this trend continued at 7 DAT (Table s 4 3 and 4 4 ). Differences were found in total chlorophyll at all concentrations but the highest (Table 4 5 ). Differences were found in tota l carotenoids at all concentrations tested (Table 4 6 ). North Carolina plants also had a different response than Townline plants. These results show the different responses of invasive water milfoils to fluridone. Although the product is labeled for identic al use concentrations for all invasive water milfoils, differences in response could account for the varying levels of treatment

PAGE 67

67 success observed in the field. Townline population has an increased tolerance for the herbicide fluridone when compared to other known susceptible populations. Experiment 2 Plants collected from 5 Michigan lakes and 3 bays in Lake Minnetonka, Minnesota showed varying responses to fluridone w hen compared to Townline Lake. These populations differ in h istory of fluridone exposure. F also fluorescence yield, was different to that of Townline at all concentrations tested (Figure 4 1). Both total chlorophyll and total carotenoids were diff erent between Townline and susceptible at all concentrations but the highest (Figures 4 2 and 4 3). This study demonstrates that Townline plants have an increased tolerance to fluridone when compared to other invasive water milfoils from several states. Use of a PAM fluorometer was less variable than traditional pigment analysis and is a l ess complex method to utilize. The limited variability between susceptible watermilfoils collected across the country demonstrates the high level of sensitivity these spec i es often exhibit to fluridone. Fluridone has traditionally been a successful tool in controlling watermilfoil invasions due in part to the widesp read response of these plants. Townline Lake is the first documented population of invasive watermilfoils that does not show susceptibility to fluridone. This population should be carefully monitored by resource managers to preven t spread to neighboring lakes. Lakes in close proximity need to be monitored as well in order to rapidly detect any invasion of fluridone tolerant plants. The use of PAM fluorometery in lab scale studies is a promising tool for quickly evaluating the response to fluridone of other populati ons of invasive watermilfoils.

PAGE 68

68 Table 4 1. Popula tions of invasive watermilfoils used in Experiment 1. Population Species Location Tolerance Townline hybrid Michigan probable Indian hybrid Michigan suspected Auburn EWM Minnesota susceptible North Carolina EWM North Carolina susceptible Note: Hybrid watermilfoil is a cross between northern watermilfoil ( Myriophyllum sibiricum ) and Eurasian watermilfoil (EWM) ( M. spicatum ). Table 4 2. Fluorescence yield (F v /F m ), represented as percent untreated, in Townline plants and 3 different populations of invasive watermilfoils collected from three states after e xposure to fluridone 3 days after treatment fluridone concentration 1 ) Townline Indian Auburn North Carolina 3 102.1 98.3* 99.8* 95.5* 6 97.9 91.7 97.4 94.3* 9 96.6 91.9* 90.6 95.4* 12 93.4 91.2 88.8 85.2 24 87.7 88.3 87.2 86.0 48 91.1 84.3* 83.7* 81.2* Note: Differences between each population compared to Townline within each concentration, found using Table 4 3. Fluorescence yield (F v /F m ), represented as percent untre ated, in Townline plants and 3 different populations of invasive watermilfoils collected from three states after exp osure to fluridone 5 days after treatment fluridone concentration 1 ) Townline Indian Auburn North Carolina 3 104.4 96.5* 101.1* 96.9 6 102.4 85.7* 93.8* 88.7* 9 101.8 84.7* 81.4* 89.7* 12 97.7 88.8* 82.3* 83.8* 24 91.2 75.9* 76.0* 73.9* 48 86.7 69.4* 72.1* 63.5* Note: Differences between each population compared to Townline within each concentration, found using

PAGE 69

69 Table 4 4. Fluorescence yield (F v /F m ), represented as percent untreated, in Townline plants and 3 different populations of invasive watermilfoils collected from three states after exp osure to flurid one 7 days after treatment fluridone concentration 1 ) Townline Indian Auburn North Carolina 3 101.6 100.6 95.7* 98.2 6 99.2 85.8* 87.7* 82.4* 9 97.0 80.6* 67.7* 86.5* 12 97.5 80.3* 78.7* 77.2* 24 84.0 62.3* 69.5* 65.7* 48 70.0 59.1* 59.1* 41.3* Note: Differences between each population compared to Townline within each concentration, found using Table 4 5 Total chlorophy ll represented as percent untreated, in Townline plants and 3 different populations of invasive watermilfoils collected from three states after exposure to fluridone 7 days after treatment fluridone concentration 1 ) Townline Indian Auburn North Car olina 3 112.9 101.3 96.8 83.3* 6 106.5 98.0 85.7 75.3* 9 90.7 62.4* 78.2* 67.1* 12 93.1 72.0* 77.6 57.7* 24 79.1 60.1 64.1 48.5* 48 54.4 63.7 55.3 41.2 Note: Differences between each population compared to Townline within each concentration found using denoted with an asterisk with n=8. Table 4 6. Total carotenoids, represented as percent untreated, in Townline plants and 3 different populations of invasive watermilfoils collected from three states after exposure to fluridone 7 days aft er treatment. fluridone concentration 1 ) Townline Indian Auburn North Carolina 3 107.3 93.2* 89.1 74.2* 6 98.4 76.7* 72.1* 65.2* 9 80.4 54.0* 68.9* 53.4* 12 81.0 62.0* 79.2 47.7* 24 69.6 51.3* 56.1 39.2* 48 51.2 62.9 56.8 39.4* Note: Differences between each population compared to Townline within each concentration, found using

PAGE 70

70 Figure 4 1. Fluorescence Yield (F v /F m ) represented as percent of the untreated control in Townline plants and nine different populations of susceptible invasive watermilfoil s collected from two states 7 days after treatment with fluridone. Symbols represent means at each concentration and er ror bars represent standard error (Townline n=8, Susceptible n=32) Curves indicate nonlinear regression. Concentration s with significant differences as found using Protected LSD are marked with an asterisk

PAGE 71

71 Figure 4 2. Total chlorophyll represented as percent of the untreated control in Townline plants and nine different populations of susceptible invasive water milfoils collected from two states 7 days aft er treatment with fluridone. Symbols represent means at each concentration and error bars represent standard error (Townline n=8, Susceptible n=32) Curves indicate nonlinear regression. Concentration s with significant differences as found using Protected LSD are marked with an asterisk

PAGE 72

72 Figure 4 3. Total carotenoids represented as percent of the untreated control in Townline plants and nine different populations of susceptible invasive water milfoils collected from two states 7 days aft er treatment with fluridone. Symbols represent means at each Concentration and error bars represent standard error (Townline n=8, Susceptible n=32 ). Curves indicate nonlinear regression. Concentrations with significant differences as found using P rotected LSD are marked with an asterisk.

PAGE 73

73 CHAPTER 5 EVALUATION OF A HYBR ID WATERMILFOIL RESP ONSE TO PIGMENT SYNTHESIS INHIBITING HERBICIDES Invasive water milfoils, Eurasian watermilfoil ( Myriophyllum spicatum L. ) and hybrid watermilfoil ( M. spicatum x M. sibi ricum ), are problematic weeds in m any water bodies they inhabit. Plants grow to form dense canopies displacing native vegetation, inhibiting flood control and obstructing recreational uses of wa terways. Madsen et al. (1991) found that Eurasian watermilfoil formed dense canopies on the water surface to shade out desirable n ative plants in the ecosystem. Dense canopy formation has been shown to negatively impact water quality by reducing dissolved oxygen in water below the mat and increasing surface temperatu r es and pH (Bowes et al. 1979). Submersed aquatic weeds have also been shown to harbor algae species harmful to both wildlife and hum an health (Wilde et al. 2005). When water milfoils spread within waterbodies and result in unfavorable conditions, managemen t of these plants is often required. F luridone herbicide (1 methyl 3 phenyl 5 [3 (trifluoromethyl)phenyl] 4(1 H ) pyridinone) has been used to control Eurasian watermilfoil and hydrilla ( Hydrilla verticillata [L.f.] Royle) since the late 1980s. Schmitz et al (1987) describe using fluridone to control hydrilla in a central Florida lake such that the plant could no long er be found in the water body. Haller et al. (1990) successfully used fluridone to control hydrilla for on e year in the St. Johns River. As suc h, fluridone was heavily used for hydrilla control in public waters of Florida from the late Between 1999 and 2001, formerly susceptible hydrilla populations in several Florida lakes were not exhibiting the level of control pr eviously associated with fluridone use. Subsequent laboratory testing documented that several populations of hydrilla had developed resistance to fluridone (Michel et al. 2004, Arias et al. 2005, Puri et al. 2006).

PAGE 74

74 Specifically, an amino acid substitution in the phytoene desaturase enzyme conferred 2 to 5 fold resistance to the herbicide fluridone in hydrilla (Michel et al. 2004). Previous to this discovery, herbicide resistance in hydrilla was thought to be unlikely due to the strictly vegetative reproduct ion exhibited by dioecious hydrilla in Florida. However, low use rates, extended exposure times, and repeated use of fluridone led to tremendous selection pressure on these waterbodies. Repeated fluridone applications selected for plants that were resistan t to typical use rat es of the herbicide fluridone. Since this time, resistance has developed in many water bodies throughout the state and fluridone is no longer a widespread tool for hydrilla manageme To date, hydrilla is the only plant that has been confirmed to show resistance to fluridone. Since the fluridone use patterns are almost identical for hydrilla and invasive watermilfoils, the potential for resistance in water milfoils is expected. In 2010, a population of hybrid w atermilfoil in Townline Lake, Michigan was treated with standar d rates of fluridone herbicide. After a lack of response, the plants were evaluated in controlled studies and found to be tolerant of fluridone rates exceeding the maximum rate permitted by the Michigan De partment of Natural Resources. Hydrilla resistance to fluridone was found to be a conformational change in the targe t enzyme, phytoene desaturase. Due to the specificity of this resistance, other pigment synthesis inhibitors that target alterna tive enzymatic reactions are effective on both suscepti ble and resistant populations. Moreover, studying the response of hydrilla to multiple herbicide mechanisms of action allowed researchers to elucidate the primary mechanism of resistance. Therefore, th e objective of this research was to determine the

PAGE 75

75 response of the tolerant Townline hybrid watermilfoil to phytoene desaturase inhibiting herbicides specifically norflurazon (4 chloro 5 [methylamino] 2 [3 (trifluoromethyl) phenyl] 3[ 2H ] pyridazinone), and a pigment synthesis inhibiting herbicide with a different mechanism of action topramezone ( [3 4,5 dihydro isoxazol 3 yl) 4 methylsulfonyl 2 methylphenyl](5 hydroxy 1 methyl 1H pyrazole 4 yl)methanone ) in an effort to elucidate the possible mechanism of resistance and determine if alternative herbicides would be effective in controlling this population. Materials and Methods Populations of hybrid and Eurasian watermilfoil were obtained and put into culture at the Center for Aquatic and Invasive Plants in Gainesville, F lorida outdoor mesocosm tanks. Stock cultures of plants were exposed to ambient water and air temperature throughout the culture period. For the first experiment, plants originating from Townline Lake in central Michigan were used and compare d to plants with a known susceptibility to fluridone from a Tex as population of watermilfoil. In the second study, the Townline population of plants was compared to a population of fluridone susceptible Eurasian watermilfoil originating from Auburn Lake in Minnesota. Apical shoot tips 6 cm in length were harvested from stock cultures cleaned thoroughly with flowing tap wate (Selim et al. 1989) supplemented with 4 g L 1 sodium bicarbonate. Plants were treated with fluridone (Sonar AS, SePRO Corporation Carmel, IN), topramezone (BAS 670, BASF, Raleigh, NC), or norflurazon (Solicam 80DF, Syngenta Crop Protection Inc., 1 Culture tubes were main tained in a climate controlled growth chamber for the duration of the experiment (23C: 21C 2 light).

PAGE 76

76 Chlorophyll fluorescence was measured at 10 days after treatment (DAT) with a PAM fluor ometer (Mini PAM, Walz, Effetrich Germany). At this time, 0.1g of new apical growth was harvested for total chlorophyll and total carotenoid analysis with methods similar to those described by Wellburn (1994). Pigment extract in dimethyl sulfoxide (DMSO) was analyzed at 480 nm, 649 nm, and 665 nm for quantification of total carotenoids, chlorophyll b and chlorophyll a respectively. DMSO was used as a blank. Total chlorophyll was calculated with the following equation: (12.19*absorbance at 665nm 3.45*ab sorbance at 649nm) + (21.99*absorbance at 649nm 5.32*absorbance at 665nm). Total carotenoids were calculated as (1000*absorbance at 480nm 2.14*chlorophyll a 70.12*chlorophyll b ) / 200. Extracts were analyzed using a spectrophotometer (Biomate 5, Ther mo Scientific, Pittsburgh, PA). Each treatment was replicated four times and the entire study was repeated. Analysis of variance (ANOVA) was used to determine mean effects and possible interactions. There was no experiment by treatment interaction so the r esults from both experiments were combined. significant differences in response between the susceptible population s and Townline population within each concentration Results and Discussion Fluridone Flu orescence yield showed differences in response to fluridone between Townline 1 (Figure 5 1 a ). Total chlorophyll had differences at 5, 10, and 40 1 while total carotenoids showed differences at 5, 20 and 40 1 (Figures 5 1 b and 5 1c ). Differences were not expected at the two highest concentrations since fluridone was previously documented to be lethal to

PAGE 77

77 Townline plants between 20 and 30 1 The legally permitted rate in Michigan, where Townl ine is located, is 6 1 Considering the differences between the two populations at 5 1 in both at harvest fluorescence yield and pigment analysis, it is logical to surmise that Townline would not be controlled at the legally permitted rate, while the known susceptible population would be controlled. Norflurazon 1 of norflurazon 10 DAT (Figure 5 2). The typically lethal concentration for norflurazon on water milfoil is unknown, hence the w id e range of concentrations in this study. Total chlorophyll varied in response at all concentrations but the highest and total carotenoids were different at all concentrations (Figure s 5 2 b and 5 2c ). It is possible that PAM fluorometry simply does not su fficiently evaluate response to norflura zon as it does with fluridone. P igment analysis suggests that the Townline population appears to be tolerant or resistant to norflurazon at concentrations 1 This would be understandable since norflurazon and fluridone work on the same enzyme, PDS, in the ca rotenoid biosynthesis pathway. Fluridone resistant hydrilla in Florida was also found to be cross resistant to norflurazon (Puri et al. 20 09). Topramezone Fluorescence yield was different between Townline and susceptible plants 10 DAT 1 (Figure 5 3). Total chlorophyll had differences at 5, 20, and 40 1 L 1 (Figure s 5 3 b and 5 3c ). The typically lethal rate for topramezone on water 1 It is apparent from these results that Townline is also tolerant of topramezone herbicide. This herbicide does not directly act on PDS enzyme, but b locks synthesis of a cofactor

PAGE 78

78 for this enzyme. The fluridone resistant hydrilla documented in Florida did not show cross resistance to HPPD inhibiting herbicides such as topramezone (Puri et al. 2009). Fluridone and norflurazon inhibit the phytoene desatur ase (PDS) enzyme in the ca rotenoid biosynthesis pathway. With reduction in carotenoid biosynthesis, chlorophyll molecules are not shielded from excess light and degrade. Topramezone is an HPPD inhibiting herbicide that blocks synthesis of a cofactor for th e PDS enzyme. These results suggest that the Townline population of hybrid watermilfoils have some form of enhanced tolerance to, at minimum, the pigment synthesis inhibiting herbicides tested in this trial. It is unlikely that a mutation in the phytoene d esaturase gene is a cause of this tolerance, as it was in hydrilla, since tolerance was also shown to a herbicide with a different mech anism of action (topramezone). The increased tolerance exhibited by Townline plants could be the result of several tolera nce mechanisms such as increased metabolism of the herbicides tested or decre ased uptake of the herbicides. reproduce following exposure to a dose of herbicide normally let hal (WSSA 1998). ). Some discussion has occurred debating whether the fluridone response of h ybrid water milfoil is act ually resistance or tolerance. Since hybrid plants were not documented to be at one time susceptible to fluridone, it is not appropriate to refer to them The Townline population of hybrid water milfoil displays incre ased toler ance to the herbicides tested. The results of this study confirm the observations of resource managers that

PAGE 79

79 some hybrid watermilfoil populations do not have the same response to herbicides as Eurasian watermilfoil.

PAGE 80

80 Figu re 5 1. Fluorescence yield (F v /F m ) (a) total chlorophyll (b) and total carotenoids (c) represented as percent of untreated control in susceptible and Townline invasive watermilfoil plants as a function of fluridone concentration 10 days after treatment Error bars indicate sta ndard error of the mean (n=8). Curves indicate nonlinear regression. Concentration s with significant differences as found using denoted with an asterisk. a b c

PAGE 81

81 Figure 5 2 Fluorescence yield (F v /F m ) (a) total chlorophyll (b) and total carotenoids (c) represented as percent of the untreated control in susceptible and Townline invasive watermilfo il plants as a function of norflurazon concentration 10 days after treatment Error bars indicate standard error of the mean (n=8). Curves indicate nonlinear regression. Concentration s with significant differences as found using .05) are denoted with an asterisk. a b c

PAGE 82

82 Figure 5 3. Fluorescence yield (Fv/Fm)(a), total chlorophyll (b), and total carotenoids (c), represented as percent of the untreated control in susceptible and Townline invasive watermilfoil pl ants as a function of topramezone concentration 10 days after treatment Error bars indicate sta ndard error of the mean (n=8). Curves indicate nonlinear regression. Concentration s with significant differences as found using are denoted with an asterisk. a b c

PAGE 83

83 CHAPTER 6 CONCLUSIONS Invasive watermilfoils grow rapidly and overtake water bodies through displacement of native vegetation, inhibition of recreational use and obst ruction of natural water flow. Within the past 10 15 years genetically verified hybrids of the native Northern watermilfoil (M. sibiricum Kom.) and the exotic invasive Eurasian watermilfoil (M. spicatum L.) have been documented in the Midwestern U nited States. The hybrid exhibits the invasive characteristics of E urasian watermilfoil, thus requiri ng a similar level of control. A number of herbicides have been used to control both the hybrid and Eurasian watermilfoils, with the herbicide fluridone being a common choice by water managers due to low use rates and mini mal dama ge to desirable native plants. Fluridone herbicide was also used extensively for hydrilla (Hydrilla verticillata [L.f.] Royle) management in Florida, but the development of wide spread resistance has eliminated effectiveness. I n May 2010, a lake c ontaining a documented hybrid watermilfoi l was treated with fluridone. The population of watermilfoil on this lake Townline, survived normally lethal rates of fluridone, raising serious concerns that fluridone resistance or tolerance has developed in ano t her submersed aquatic species. However, confirmation and characterization of this phenomenon is needed. To characterize the level of resistance or tolerance, suspected tolerant Townline plants and susceptible populations were evaluated over a range of flu ridone concentrations in both me socosm and laboratory studies. Mesocosm growth studies confirmed increased tolerance to fluridone as compared to susceptible populations. A pulse amplitude modulated (PAM) fluorometer was used to measure fluorescence of trea ted plants in an attempt to develop a less time

PAGE 84

84 consuming method of flur idone resistance confirmation. Pigment analysis of chlorophyll and carotenoids confirmed the results of the PAM fluorometer and results were comparable to those found util izing biomass experimentation. These studies demonstrate the utility of PAM fluorometery in detecting plant tolerance to fluridone herbicide, and this method may be useful for detecting differences with other herbicides. Confirmed fluridone tolerant hybrid watermilfoi l plants were found to also be tolerant to topramezone herbicide, which has a similar mode of activity but different mechanism of action than fluridone, and norflurazon herbicide, which acts on the same enzy me as fluridone. This indicates a different mecha nism of resistance or tolerance in this population of hybrid watermilfo il than was found in hydrilla. This research has confirmed, characterized, and documented a herbicide tolerant hybrid watermilfoil population from Townline Lake. This finding raises ser ious concerns with management of this species, therefore, it is imperative that resource managers actively limit the spread of this plant.

PAGE 85

85 LIST OF REFERENCES Aiken, S. G. 1980 A conspectus of Myriophyllum (Haloragaceae) in North America. Brittonia 33: 57 69. Alwin, T.G., M.G. Fox and K. S. Cheruvelil. 2010. Estimating lake wide watermilfoil weevil ( Euhrychiopsis lecontei ) density: the roles of quadrat size, sample size, and effort J. Aquat. Plant Manage. 48: 96 102. Arias, R.S., M.D. Neth erland, B.E. Sc heffler, A. Puri and F.E. Dayan. 2005. Molecular evolution of herbicide resistance to phytoene desaturase inhibitors in Hydrilla verticillata and its potential use to gener ate herbicide resistant crops. Pest Manag e Sci. 61: 258 268 Beer, S., M. Ilan, A. Eschel, A. Weil and I. Brickner. 1998. Use of pulse amplitdue modulated(PAM) fluoromtery for in situ measurements of photosynthesis in two Red Sea faviid corals. Mar. Biol. 131: 607 612. Bl ackburn, R.D. and L.W. Weldon. 1967. Eurasian watermilfoil Fl s new underwater menace. J. Aquat. Plant Manage. 6: 15 18. Bohlhar Nordenkampf, H.R., S.P. Long, N.R. Baker, G. Oquist, U. Schreibers and E.G. Lechner. 1989. Chlorophyll fluorescen ce as a probe of the photosynthe tic competence of leaves in the field: a rev i ew of current instrumentation. Func t Ecol. 3:497 514. Bowes. G, T.K. Van, L.A. Ga rrard and W.T. Haller. 1977. Adaptation to low light levels by hydrilla. J. Aquat. Plant Manage. 15: 32 35. Bowes. G., A.S. Holaday and W.T. Haller. 1979. Seasonal variation i n the biomass, tuber density, and photosynthetic metabolism of hydrilla i n three Florida lakes. J. Aquat. Plant Manage. 17: 61 65. Bultemeier, B. W. 2008. The response of three cabomba populations to herbicides and environmental parameters: implications for taxonomy and management [dissertation]. Gainesville, Florida. Uni versity of Florida. Cal IPC. 2011. Cali fornia Invasive Plant Council. (http://www.c al ipc.org, 15 February 2011). California Invasive Plant Council, Berkley, CA 94709 USA. Chamovitz, D., G Sandmann and J. Hirschberg. 1993. Molecular and biochemica l characterization of herbic ide resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate limiting st ep in carotenoid biosynthesis. J. Biol. Chem. 268:17348 17353. Coffey, B.T and C.D. McNabb. 1974. Eurasian watermilfoil in Michi gan. Mich. Bot. 13: 159 165.

PAGE 86

86 Crow, G.E. and C.B. Helquist. 2002. Aquatic and wetland plants of northeastern North America. Volume one: pteridophytes, gymnosperms, and angiosperms: dicotyledons. Univ ersity of Wisconsin press, Madison, WI. 480 pages. C rowel, W.J., N.A. Proulx and C.H. Welling. 2006. Effects of repeated fluridone treatments over nine years to control Eurasian water milfoil in a mesotrophic lake. J Aquat. Plant Manage. 44: 133 136. Du ra ko, M.J. and J.I. Kunzelman. 2002. Photosynthetic characteristics of Thalassia testudinum measured in situ by pulse amplitude modulated (PAM) fluorometery: methodological a nd scale based considerations. J Aquat. Bot. 73: 173 185. Forney, D. R. and D. E. Da vis. 1981. Effects of low concentrations of herbicide s on submersed aquatic plants. Weed Sci. 29: 677 685. Getsinger, K. D., J. D. Madsen, T. J. Koschnick, M. D. Netherland, R. M. Stewart, D.R. Honnel A.G. Staddon and C.S. Owens. 2001. Whole lake applicati ons of Sonar for selective control o f Eurasian watermilfoil. Technical Report TR 07 1, U.S.Army Engineer Waterways Experiment Station, Vicksburg, M.S. 37pp. Getsinger, K.D., J.D. Madsen, T.J. Koschnick and M.D. Netherland. 2002a. Whole lake fluridone treat ments for selective control of Eu rasian watermilfoil: I. Application st rategy and herbicide residues. Lake Reserv. Manage. 18: 181 190. Getsinger, K.D., R.M. Stewart, J.D. Madsen, A. S. Way, C.S. Owens, H.A. Crosson and A.J. Burns. 2002b. Use of whole lake f luridone treatments to selectively control Eurasian watermilfoil in Burr Po nd and Lake Hortonia, Vermont. Technical Report TR 02 39, U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS 79pp. Glom ski, L.M. and M.D. Netherland. 2010. Response of E urasian and hybrid watermilfoil to low use rates and extended exposures of 2,4 D and tricl opyr. J. Aquat. Plant Manage. 48: 12 14. Grace, J.B. and R.G. Wetzel. 1978. The production biology of Eurasian watermilfoil ( Myriophyllum spicatum L.): A review. J. A quat. Plant Manage 16: 1 11. Gr een, W.R. and H.E. Westerdahl. 1990. Response of Eurasian watermilfoil to 2,4 D con centration and exposure times. J. Aquat. Plant Manage. 28: 27 32. Haller, W.T., A.M. Fox and D.G. Shilling. 1990. Hydrilla control program in t he upper St. Johns River, Florida, USA. Proc. Eur. Weed Res. Soc. Symp. Aquat. Weeds. 8:111 116. Haller, W.T. and D. L. Sutton. 1973. Factors affecting the uptake of endothall 14 C by hydrilla. Weed Sci. 21:446 448.

PAGE 87

87 Haynes, D., P. Ralph, J. Prange and B. De nnison. 2000. The impact of the herbicide diuron on photosynthesis in three species of tropical seagrass. Mar Pollut Bull 41: 288 293. His cox, J.D. and G.F. Israelstam. 1979. A method for the extraction of chlorophyll from leaf tissue without mac eration. Can. J. Bot. 57: 1332 1334. Holaday, A. S. and G. Bowes. 1980. C 4 acid metabolism and dark CO 2 fixation in a submersed aquatic macrophyte ( Hydrilla verticillata ). Plant Physiol. 65:331 335. Ireland, C.R., M.P. Percival and N.R. Baker. 1986. Modification of the in duction of photosynthesis in whe at by glyphosate, an inhib itor of amino acid metabolism. J. Ex. Bot. 37: 299 308. Iriyama, K., Ogura, N. and Takamiya, A. 1974. A simple method for extraction and partial purification of chlorophyll from plant material using dioxane. J. Biochem. 76: 901 904. ISDA, 2011. I daho Invasive Species Council. (http:// www. agri.state.id.us/Categories/Environment/InvasiveSpeciesCouncil/indexInvSp Council.php 15 February 2011). Idaho State Department of Agriculture, Boise, ID 83701 USA. Jacono, C. J. and M.M. Richerson. 2011. Myriophyllum spicatum U nited S tates G eological S ervice Nonindigenous Aquatic Spe cies Database. Gainesville, FL. Juneau, P., D. Dewez, S. Matsui, S. Kim and R. Popovic. 2001. Evaluation of different algal sp ecies sensitivity to mercury and m etolachlor by PAM fluorometry. Chemosphere 45: 589 598. Kamermans, P., M.A. Hemminga and D. J. deJong. 1999. Significance of salinity and silicon levels for growth of a formerly estuarine eelgrass ( Zostera marina ) populatio n. Mar. Biol. 133: 527 539. Kane, M.E. and E. F. Gilman. 1991. In vitro propagation and bioassay systems for evaluating growth regulator effects on Myriophyllum species. J. Aquat. Plant Manage. 29: 29 32. Kelting, D.L. and C.L. Laxson. 2010. Cost and effecti veness of hand harvesting to control the Eurasian watermilfoil population in Upper Saranac Lake, New York. J. Aquat. Plant Manage. 48: 1 5. Les, D. H. and L.J. Mehroff. 1999. Introduction of nonindigenous aquatic vascular plants in southern New Engl and: a h istorical perspective. Biological Invasions 1: 281 300. MacDonald, G.E., D.G. Shilling, R.L. Doong and W.T. Haller. 2003. Effects of fluridone on hyd rilla growth and reproduction. J. Aquat. Plant Manage. 31:195 198.

PAGE 88

88 MacKinney G. 1941. Absorption of l ight b y chlorophyll solutions. J. Biol. Chem. 140: 315 322. Madsen, J.D. 2009a. Chapter 1: Impact of invasive aquatic plants on aquatic biology, pp.1 8. In: Biology and control of aquatic plants: a best management practices handbook (Gettys, L.A., W. T. Haller an d M. Bellaud, eds.) Aquatic Ecosystem Restoration Foundation, Marietta, GA. 210 pages. Madsen, J. D. 2009b. Chapter 13.2: Eurasian watermilfoil, pp.95 98. In: Biology and control of aquatic plants: a best management practices handbook (Gettys, L.A., W. T. Haller and M. Bellaud, eds.) Aquatic Ecosystem Restoration Foundation, Marietta, GA. 210 pages. Madsen, J.D., L.W. Eichler and C.W. Boylen. 1988. Vegetative spread of Eurasian watermil foil in Lake George, New York. J. Aquat. Plant Manage. 26: 47 50. Madsen J.D., J.W. Sutherland J.A. Bloomfield, L.W. Eichler and C.W. Boylen. 1991. The decline of native vegetation under dense Eurasian watermilfoil canopies. J. Aquat. Plant Manage. 29: 94 99. McCowen, M.C., C. L. Young, S.D. West, S.J. Parka and W.R. Arnold. 1 979. Fluridone, a new herbicide for aquatic plant management. J. Aquat. Plant Manage. 17:27 30. Michel, A., R.S. Arias, B.E. Scheff ler, S.O. Duke, M.D. Netherland and F.E. Dayan. 2004. Somatic mutation mediated evolution of herbicide resistance in the noni ndigenous invasive plant hydrilla ( Hydrilla verticillata ). Mol. Ecol. 13:3229 3237. Mo ody, M. L. and D.H. Les. 2002. Evidence of hybridity in invasive watermilfoil ( Myriophyllum ) populations. P N atl. A cad. S ci. USA 99: 14867 14871. Mo ody, M.L. and D.H. Les 2007. Geographic distribution and genotypic composition of invasive hybrid watermilfoil ( Myriophyllum spicatum x M. sibiricum ) populations in North America. Biol. Invasions 9: 559 570. Mood y, M. L. and D. H. Les. 2010. Systematic of the aquatic angiospe rm genus Myriophyllum (Haloragaceae). Syst Bot 35: 121 139. M oran, R. and Porath, D. 1979. Chlorophyll determination in intact tissu e using N,N dimethylformamide. Plant Physiol. 65: 478 479. Nethe rland, M.D. and K.D. Getsinger. 1992. Efficacy of triclopyr on Eurasian watermilfoil: concentrat ion and exposure time effects. J. Aquat. Plant Manage. 30: 1 5. Nether land, M.D. and K.D. Getsinger. 1995. Laboratory evaluation of threshold fluridone concentrations under static conditions for controlling hydri lla and E urasian watermilfoil. J. Aquat. Plant Manage. 33:33 36.

PAGE 89

89 Netherland, M.D., K.D. Getsinger and J.D. Skogerboe. 1997. Mesocosm evaluation of the species sel ective potential of fluridone. J. Aquat. Plant Manage. 35: 41 50. Netherland, M.D., K.D. Getsinger and E G. Turner. 1993. Fluridone concentration and exposure time requirements for control of hydrilla and Eurasian watermilfoil. J. Aquat. Plant Manage. 32:189 194. Neve, P. and Powles, S. 2005. High survival frequencies at low herbicide use rates in populatio ns of Lolium rigidum result in rapid evol ution of herbicide resistance. Heredity 95: 485 492. Newman, R.M., D.W. Ragsdale, A. Mil les and C.Oien. 2001. Overwinter habit and the relationship of overwinter to in lake densities of the milfoil weevil, Euhrychiop sis lecontei a Eurasian watermil foil biological control agent. J. Aquat. Plant Manage. 39: 63 67. Nichols, S.A. 1992. Depth, substrate, and turbidity relationships of some Wisconsin lake plants. Trans. Wisc. Acad. Sci. Arts Lett. 80: 97 118. Papageorgiu, G. 1975. Chlorophyll fluorescence. pp 320 366 An intr insic probe of photosynthesis. In : Bioenergetics of p hotosynthesis (J. Amesz Govindjee ed. ) Academic Press, New York, NY. Poovey, A.G., J .G. Slade and M.D. Netherland. 2007. Susceptiblity of Eurasian watermilfoil ( Myriophyllum spicatum ) and a milfoil hybrid ( M. spicatum x M. sibiricum ) to triclopyr and 2, 4 D amine. J. Aquat. Plant Manage. 45: 111 115. Prins, H.B. and J.T. Elezenga. 1989. Bicarbonate utiliz ation: function and mechanism. Aquat Bot 34: 59 83. Puri, A. W. T. Haller, and M. D. Netherland. 2009. Cross resistance in fluridone resistant hydrilla to other bleaching herbicides. Weed Sci. 57:482 488. Puri, A., G.E. MacDonald, W.T. Haller and M.Singh. 2006. carotene response of flur idone susceptible and resistant hydrilla ( Hydrilla verticillata ) biotypes to fluridone. Weed Sci. 54: 995 999. Ralph, P. J., R. Gademann and W. C. Dennison. 1998. In situ seagrass photosynthesis measured using a submersible, pulse amplitude modulated fl uor ometer. Mar. Biol. 132: 367 373. Reed, C.F. 1977. History and distribution of Eurasian watermilfoil in the United Sta tes and Canada. Phytologia 36: 416 436. Roley, S.S. and R.M. Newman. 2008. Predicting Eurasian watermilfoil invasions in Minnesota. Lake Res erv M anage 24: 361 369.

PAGE 90

90 Rosine, W. N. 1955. The distribution of invertebrates on submerged aquatic plant surfaces in Muskee Lake, Colorado. Ecology 36:308 314. Rozas, L.P. and W.E. Odum. 1988. Occupation of submerged aquatic vegetation by fishes: testin g the roles of food and refuge. Oecologia 77: 101 106. Schmitz, D.C., A.J. Leslie, L.E. Nall and J. A. Osborne. 1987. Hydrosoil residues and Hydrilla verticillata control in a central Florida lake using fluridone. Pest. Sci. 21:73 82. Schmitz, D.C. and J.A. Osborne 1984 Zooplankton densities in a Hydrilla infes ted lake. Hydrobiologia. 111: 127 132. Sculthorpe, C. D. 1967. The biology of aquatic vascular plants. Edward Arnold Publishers, London, England. 610 pages. eal, M.A. Ross a nd C.A. Lembi. 1989. Bioassay of photosynthetic inhibitors in water and aqueous soil extracts with Eurasian watermilfoil ( Myriophyllum spicatum ). Weed Sci. 37: 810 814. Senseman, S.A. (ed.). 2007. Herbicide Handbook. 9 th e d. Lawrence, KS: Weed Science Socie ty of America. Smith, C.S. and J.W. Barko. 1990. Eco logy of Eurasian watermilfoil. J. Aquat. Plant Manage. 28: 55 64. Spe ncer, D. F. and G. G. Ksander. 1987. Comparison of three methods for extracting chlorophyll fr om aquatic macrophytes. J. Freshwater Ecol 4: 201 208. S precher, S. L., M.D. Netherland and A.B. Stewart. 1998. Phytoene and carotene response of aquatic plants to fluridon e under laboratory conditions. J. Aquat. Plant Manage. 36: 111 120. Stanley, R.A. 1970. Studies on nutrition, photosynthesis, a nd respiration in Myriophyllum spicatum L. Ph.D. Disserta tion, Duke Univ., N. Carolina. 128 pages. Stanley, R.A. and A.W. Naylor. 1972. Photosynthesis in Eurasian watermilfoil ( Myriophyllum spicatum L.). Plant Physiol. 50: 149 151. Steemann Nielsen, E. 1947. Photosynthesis of aquatic plants with speci al reference to carbon sources. Dansk Bot. Ark. 12(8). 77 pages. Stroganov, N.S. 1963. The food selectivity of the amur fishes. ppgs 181 191 I n : Conference on fishery exploitation of the phytophagus fishes of the USSR. Ashkhabad Academy of Science, Turkmen Soviet Socialist Republic.

PAGE 91

91 Sturtevant, A. P., N. Hatley, G.D. Pullman, R. Sheick, D. Shorez, A. Bordine, R. Mausolf, A. Lewis, R. Sutter and A. Mortimer. 2009. Molecular characterizati on of Eurasian watermilfoil, northern watermilfoil, and the invasive interspec ific hybrid in Michigan lakes. J. Aquat. Plant Manage. 47: 128 135. Thum, R.A., M. Heilman, P. Hausler, L. Huberty, P. Tyning, D. Wcisel, M. Zuellig, S. Berger and M. Netherland. Field and laboratory documentation of reduced fluridone sensitivity by a hybrid watermilfoil biotype. Submitted to J. Aquat. Plant Manage. UF, C AIP (University of Florida, Center for Aquatic and Invasive Plants) 2011. The Center for Aquatic and Inva sive Plants. (http://plants.if as.ufl.edu, 15 February 2011). Center for Aquatic and Invasive Plants, Gainesville, FL 32653 USA. USDA, NRCS (United States Department of Agriculture, Natural Resources Conservation Service) 2011. The PLANTS database. ( http://plants.usda.gov 2 February 2011). National Plant Data Center, Baton Rouge, LA 70874 4490 USA. Valley, R.D., T.K. Cross and P. Radmonski. 2004. The role of submersed aquatic vegetation as habitat for fish in Minnes ota lakes, including the implications of non native plant invasions and their management Minnesota De partment of Natural Resources. Special Publication 160. Van, T. K., W.T. Haller and G. Bowes. 1976. Comparison of the photosynthetic characteristics of t hr ee submersed aquatic plants. Plant Physiol. 58: 761 768. Waldrep, T.W. and H.M. Ta ylor. 1976. 1 Methyl 3 phenyl 5 [3 trifluoromethyl)phenyl] 4(1 H ) pyridinone, a new herbicide. J. Agr. Food Chem. 24 :1250 1251. Wellburn, A.R. 1994. The spectral determinatio n of chlorophylls a and b as well as total carotenoids, using various solvents with spectrophotom eters of different resolution. J. Plant Physiol. 144: 307 313. Wilde, S.B., T.M. Murphy, C.P. Hope, S.K. Habrun, J. Kempton, A. Birre nkott, F. Wiley, W. W. Bow erman and A.J. Lewitus. 2005. Avian vacuolar myelinopathy linked to exotic aquatic plants and a novel cyanobacterial species. Env iron Tox icol 20: 348 353. [WSSA] We ed Science Society of America. 1998. ed. Weed Technol. 12:789. Yu, D., W. Dong, L. Zhen Yu and M. M. Funston. 2002. Taxonomic revision of the genus Myriophyllum (Haloragacea) in China. Rhodora 104:396 421.

PAGE 92

92 BIOGRAPHICAL SKETCH Sarah Berger was born in Fremont, Ohio in 1984 to William and Pat ricia Berger. After graduating salutatorian from Fremont Ross High School in 2002 she attended Ohio Nor thern University in Ada, Ohio. Sarah received a Bachelor of Science degree majoring in biology and environmental studies in 2007. S he then pursued a m as degree beginning in the summer of 2009 at the University of Florida. Upon graduation in 2011 Sarah began to pursue a doctor of philosophy degree in Agronomy at the University of Florida.