Michael D. Netherland

Somatic mutation‐mediated evolution of herbicide resistance in the nonindigenous invasive plant hydrilla (Hydrilla verticillata)

Hydrilla (Hydrilla verticillata L.f. Royle) was introduced to the surface water of Florida in the 1950s and is today one of the most serious aquatic weed problems in the USA. As a result of concerns associated with the applications of pesticides to aquatic systems, fluridone is the only USEPA‐approved chemical that provides systemic control of hydrilla. After a decrease in fluridones efficacy at controlling hydrilla, 200 Florida water bodies were sampled to determine the extent of the problem and the biological basis for the reduced efficacy. Our studies revealed that hydrilla phenotypes with two‐ to six‐fold higher fluridone resistance were present in 20 water bodies. Since fluridone is an inhibitor of the enzyme phytoene desaturase (PDS), the gene for PDS (pds) was cloned from herbicide‐susceptible and ‐resistant hydrilla plants. We report for the first time in higher plants three independent herbicide‐resistant hydrilla biotypes arising from the selection of somatic mutations at the arginine 304 codon of pds. The three PDS variants had specific activities similar to the wild‐type enzyme but were two to five times less sensitive to fluridone. In vitro activity levels of the enzymes correlated with in vivo resistance of the corresponding biotypes. As hydrilla spread rapidly to lakes across the southern United States in the past, the expansion of resistant biotypes is likely to pose significant environmental challenges in the future.

Hybrid watermilfoil lineages are more invasive and less sensitive to a commonly used herbicide than their exotic parent (Eurasian watermilfoil)

Hybridization may stimulate the evolution of invasiveness in human‐impacted habitats if unique hybrid genotypes have higher fitness than parental genotypes. Human efforts to control invasive taxa frequently involve the intentional alteration of habitats, but few studies have considered whether hybridization can result in decreased sensitivity to control measures. Here, we investigate whether interspecific hybrids between introduced Eurasian watermilfoil (Myriophyllum spicatum) and native northern watermilfoil (M. sibiricum) are more invasive than parental Eurasian watermilfoil, especially in regard to their relative responses to an herbicide commonly applied for their control (2,4‐dichlorophenoxyacetic acid; 2,4‐D). In two separate laboratory experiments, hybrids on average grew faster and were less sensitive to 2,4‐D compared with parental Eurasian watermilfoil. These two invasive traits appear to be common in hybrid watermilfoils, as opposed to being restricted to a few unique lineages, because they were found in a diversity of hybrid genotypes from several independent hybridization events. In addition, we found that hybrids occurred more frequently than parental species in natural lakes previously treated with 2,4‐D. Our results provide compelling empirical evidence that hybridization is associated with the evolution of increased invasiveness in watermilfoils, and have important implications for their management.

Whole Lake Fluridone Treatments For Selective Control of Eurasian Watermilfoil: I. Application Strategy and Herbicide Residues

ABSTRACT The herbicide fluridone is being used in northern lakes and reservoirs to control the exotic species Eurasian watermilfoil (Myriophyllumspicatum L.). Since quantitative information linking changes in plant communities following fluridone applications is limited, particularly with respect to water residue records, a study was conducted to investigate the effect of low-dose treatments on the submersed plant communities in four Michigan lakes. The overall study objective was to determine whether plant species diversity and frequency of occurrence were affected by low-dose fluridone applications in the year of treatment. The primary objective of this portion of the overall study was to provide an application strategy that would maintain a threshold dose of fluridone, 5 μg·L−1 declining to 2 μg·L−1, in the treated lakes to selectively control Eurasian watermilfoil. Study lakes were 55 to 220 ha in size and contained an average of nine species of submersed plants. Big Crooked, Camp, Lobdell, and Wolverine lakes were treated in mid-May 1997 with the formulation Sonar® AS, to yield an initial concentration of 5 μg·L−1 fluridone in the upper 3.05 m of each lake. Asequential application of Sonar® AS was conducted on each lake at 16 to 21 days after initial treatment (DAIT), intended to reestablish a fluridone concentration of 5 μg·L−1 in the upper 3.05 m of each lake. Bass, Big Seven, Clear, and Heron lakes received no fluridone applications and served as untreated reference sites. Water residue samples were collected at prescribed intervals on each fluridone-treated lake from pretreatment up to 81 DAIT. Samples were collected from six littoral stations and from two deep locations throughout each lake, and temperature profiles were measured at the deep stations. Fluridone residues were analyzed using two separate techniques, the newly developed enzyme-linked immunosorbent assay and the standard high performance liquid chromatography method. Fluridone levels on three of the treated lakes met the laboratory-derived criteria for achieving good control of Eurasian watermilfoil by providing a peak concentration of approximately 5 μg·L−1 during the first 2 weeks posttreatment, and by main taininga concentration >2 μg·L−1 through 60 DAIT. Residues became well mixed in the water column under isothermal conditions, and thermal stratification prevented mixing of fluridone into deeper and colder waters. Residue data indicated that thermal stratification, or the lack thereof, at the time of herbicide application can affect target herbicide concentrations. Using the volume of a pre-selected depth zones to calculate the amount of fluridone needed to achieve a particular target concentration can result in an over- or under-dosing of a water body.

Comparison of Immunoassay and HPLC for Analyzing Fluridone Concentrations: New Applications for Immunoassay Techniques

ABSTRACT High performance liquid chromatography (HPLC) and an enzyme-linked immunosorbent assay (ELISA) technique were used to analyze concentrations of the aquatic herbicide fluridone (l-methyl-3 phenyl-5-[-3 (trifluromethyl) phenyl]-4 (IH) pyridinone) in 488 surface water samples collected from two lakes in Michigan treated in 1997, 2 Michigan lakes treated in 1998, 1 lake in Florida treated in 1996, and a series of research ponds treated in Florida in 1997. Samples were collected following application of fluridone associated with operational treatment programs that targeted the exotic submersed plants Myriophyllum spicatum L. (Michigan) and Hydrilla verticillata (L.f) Royle (Florida). ELISA and HPLC results compared well (r2 = 0.84 to 0.98) across a broad range of initial fluridone treatment rates (5 to 150 μg L−1). The potential use of ELISA to monitor fluridone residues in the water in near real-time, and to use this residue data for lake specific herbicide treatment recommendations represents a unique use of ELISA technology.

Efficacy, selectivity, and herbicide concentrations following a whole-lake 2,4-D application targeting Eurasian watermilfoil in two adjacent northern Wisconsin lakes

Abstract The herbicide 2,4-D (2,4-dichlorophenoxy acetic acid) has been used to control the nonnative aquatic plant Eurasian watermilfoil (Myriophyllum spicatum; EWM) since the 1950s. Although published research evaluates the herbicides predicted and observed concentration and exposure times in both laboratory and field settings, few data are available evaluating selectivity and long-term efficacy as well as herbicide concentration behavior following large-scale, whole-lake applications. A controlled study was conducted on 2 adjacent oligo-mesotrophic northern Wisconsin lakes to determine the potential efficacy and selectivity of large-scale and low-dose 2,4-D applications. Initial 2,4-D concentrations in both treated lakes were approximately 100 μg/L higher than the nominal lakewide targets of 500 and 275 μg/L, respectively, and the herbicide dissipated and degraded more slowly than predicted. A lakewide regression model relating 2,4-D concentration at monitoring sites to days after treatment (DAT) found the mean half-life of 2,4-D to be 34–41 DAT, and the threshold for irrigation of plants not labeled for direct treatment with 2,4-D (<100 μg/L) was not met until 50–93 DAT. In the lake treated at the higher 2,4-D rate, EWM was not detected for 3 consecutive years posttreatment. Additionally, several native monocotyledon and dicotyledon species also showed sustained significant declines posttreatment. This study is the first to link field-collected 2,4-D concentration measurements to selectivity and long-term efficacy in EWM control following whole-lake management efforts. Although multiyear EWM control was achieved with these single low-dose applications, longer than expected herbicide persistence and impacts to native plants demonstrate the challenges facing aquatic plant managers and the need for additional field studies.

Cross-Resistance in Fluridone-Resistant Hydrilla to Other Bleaching Herbicides

Abstract The development of fluridone resistance by hydrilla has significantly impacted hydrilla management, and research is ongoing to develop alternate herbicides for effective hydrilla control. We determined the potential cross-resistance in fluridone-resistant hydrilla to other bleaching herbicides norflurazon, mesotrione, and topramezone-methyl. Phytoene, β-carotene, and chlorophyll contents as a function of hydrilla biotype and herbicide treatment were evaluated. Hydrilla shoot tips were collected from fluridone-susceptible (S) and -resistant (R) biotypes and exposed to 5, 25, 50, 75, and 100 µg L−1 of herbicide. The susceptible biotype showed an increase in phytoene and a decrease in β-carotene and chlorophyll contents when treated with 5 µg L−1 fluridone, whereas higher doses of fluridone were required to affect these pigments in the resistant biotype. There was no difference in response by S and R biotypes to mesotrione and topramezone-methyl, with both biotypes showing significant affects on pigment contents at 5 µg L−1. Higher doses of norflurazon were required to affect these pigments in the R compared to the S biotype. The S biotype had EC50 values of 11.7, 12.2, and 4.7 µg L−1, whereas the R biotype had EC50 values of 56.6, 41.1, and 41.7 µg L−1 fluridone for phytoene, β-carotene, and chlorophyll contents, respectively. There was no difference in EC50 for phytoene, β-carotene, and chlorophyll values between the hydrilla biotypes for mesotrione and topramezone-methyl herbicides. In fluridone-susceptible and -resistant hydrilla biotypes, EC50 values for phytoene, β-carotene, and chlorophyll were 12.4 to 11.8, 10.2 to 13.2, and 3.1 to 4.6 µg L−1 mesotrione and 12.6 to 13.5, 13.3 to 11.9, and 4.6 to 5.7 µg L−1 topramezone-methyl, respectively. For norflurazon, S and R biotypes had EC50 values of 33.1, 45.4, and 40.6 µg L−1 and 84.6, 81.0, and 92.7 µg L−1 for phytoene, β-carotene, and chlorophyll, respectively. These studies confirmed negative cross-resistance of fluridone-resistant hydrilla to mesotrione and topramezone-methyl and a positive cross-resistance to norflurazon. Nomenclature: Fluridone; mesotrione; norflurazon; topramezone-methyl; hydrilla, Hydrilla verticillata (L.f.) Royle HYLLI.

Differential Herbicide Response among Three Phenotypes of Cabomba caroliniana

Abstract Cabomba is a submersed aquatic plant native to the southeastern United States that is commonly sold worldwide through the aquarium trade. While infrequently managed in its native range, cabomba has recently been reported as invasive and tolerant to management efforts in the northern areas of the United States and in other countries. Invasive populations of cabomba are characterized by a phenotype that is bright green. In contrast, cabomba native to the southeastern United States is characterized by a red phenotype, while plants sold through the aquarium trade have intermediate characteristics of both the green and red phenotypes. The response of the three cabomba phenotypes to selected herbicides was evaluated by measuring photosynthetic response over the course of a static 144-hr exposure. Plants were exposed to the maximum recommended use-rates of 2,4-D, carfentrazone, copper, diquat, endothall (amine and dipotasium salt formulation), flumioxazin, quinclorac, triclopyr, and a combination of diquat and copper. A submersed plant species known to be sensitive to each of these herbicides was also included to compare photosynthetic response of the cabomba to a susceptible plant. The photosynthetic response of the red and green phenotypes differed following exposure to carfentrazone, diquat, 2,4-D, triclopyr, and flumioxazin. Diquat, diquat plus copper, endothall (amine salt), and flumioxazin were the only products that resulted in a greater than 50% reduction of photosynthesis in all three phenotypes of cabomba. A second experiment was conducted where all three phenotypes of cabomba were exposed to these four herbicides for 24 hr, and photosynthesis was evaluated. Following the 24-hr exposure, results further documented distinct response differences between the green and red phenotypes, with the green phenotype demonstrating a reduced sensitivity to the herbicides evaluated. Results demonstrate clear phenotypic differences in response to herbicide treatments and lack of susceptibility of cabomba to most herbicides. Nomenclature: Carfentrazone; copper; 2,4-D; diquat; endothall; flumioxazin; triclopyr; quinclorac; Cabomba, Cabomba caroliniana Gray CABCA.

Laboratory Documentation of Multiple-Herbicide Tolerance to Fluridone, Norflurazon, and Topramazone in a Hybrid Watermilfoil (Myriophyllum spicatum × M. sibiricum) Population

Abstract Invasive watermilfoils, specifically Eurasian watermilfoil and the interspecific hybrid of Eurasian watermilfoil × northern watermilfoil, continue to be problematic for water resource managers. Herbicides are often used to control these nuisance weeds and have been historically successful in controlling Eurasian watermilfoil. A population of hybrid watermilfoil from Townline Lake in Michigan has shown increased tolerance to the herbicide fluridone. The objective of this work is to determine if cross- and multiple tolerance have also developed in this population. Eurasian watermilfoil plants collected from multiple sites and plants from Townline Lake were treated with 0, 5, 10, 20, 40, or 80 µg L−1 of fluridone, norflurazon, or topramezone. Fluridone and norflurazon inhibit phytoene desaturase, whereas topramezone is a 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide. Chlorophyll fluorescence (Fv/Fm) and pigment content was measured at 10 d after treatment. Townline Lake plants responded differently from susceptible plants when treated with fluridone, norflurazon, and topramezone at 40 µg L−1. These results indicate that the Townline population of hybrid watermilfoil has inherent tolerance to multiple herbicide modes of action. These results are especially significant as topramezone has recently been labeled for aquatic use. Screening of additional herbicides to determine potential herbicide tolerance of the Townline Lake population is recommended. Nomenclature: Fluridone; norflurazon; topramezone; Eurasian watermilfoil, Myriophyllum spicatum L.; hybrid watermilfoil, M. spicatum × M. sibiricum; northern watermilfoil, M. sibiricum Komarov.

Host range and searching behaviour of Cricotopus lebetis (Diptera: Chironomidae), a tip miner of Hydrilla verticillata (Hydrocharitaceae)

Abstract A chironomid midge, Cricotopus lebetis Sublette (Diptera: Chironomidae), was discovered feeding on Hydrilla verticillata (L.f.) Royle (Hydrocharitaceae) in Crystal River, Citrus, Co., Florida, in the 1990s. Larvae of the midge mine the apical meristems of hydrilla, causing terminal branching and stunting of the plant. We investigated the fundamental host range of the midge by conducting a series of no-choice and paired-choice tests. No-choice developmental tests with neonate larvae revealed that the fundamental host range of C. lebetis included not only on hydrilla but also several other aquatic plants in different families, suggesting that this insect is not a hydrilla specialist. In paired-choice bioassays, larval colonisation of Elodea canadensis Michx. (Hydrocharitaceae) and Najas guadalupensis (Spreng.) Magnus (Najadaceae) was greater than colonisation of H. verticillata. Behavioural bioassays in a Y-tube olfactometer and in Petri dishes suggested that neonate larvae were not able to locate host plant material, whereas older larvae were successful in finding hosts. In paired-choice oviposition tests, adult females discriminated between potential oviposition sites, with greater numbers of eggs laid on E. canadensis and N. guadalupensis than on H. verticillata. This study is the first detailed account of host searching and oviposition behaviour of a phytophagous chironomid midge. The results will be used to assess the potential value of C. lebetis as a biological control agent of hydrilla.

Sensitivity of freshwater molluscs to hydrilla-targeting herbicides: providing context for invasive aquatic weed control in diverse ecosystems

Hydrilla (Hydrilla verticillata) is an invasive aquatic weed that has spread rapidly throughout the USA, especially in the southeast. A common control method is the application of aquatic herbicides, such as fluridone and endothall. However, there is limited documentation on the effects of herbicides commonly used to control hydrilla and other aquatic weeds on many non-target freshwater species and no published information exists on the toxicity of these herbicides to freshwater molluscs. We exposed juveniles (96 h) and glochidia (48 h) of the unionid mussel Lampsilis siliquoidea and adults (28 d) of Lampsilis fullerkati to a formulation of fluridone (Sonar – PR®) in laboratory toxicity tests. The early life stages of L. siliquoidea were also exposed to a formulation of the dipotassium salt of endothall (Aquathol – K®) in separate tests. Juveniles of the freshwater gastropod snail, Somatogyrus viriginicus (Lithoglyphidae), were exposed (96 h) to the Sonar – Genesis® fluridone formulation. Endpoints were survival (all species and life stages) as well as siphoning behavior and foot protrusion (adult mussels). Median lethal fluridone concentrations (LC50s) were 865 μg/L (95% CI, 729–1,026 μg/L) for glochidia (24 h), 511 μg/L (309–843 μg/L) for juvenile L. siliquoidea (96 h), and 500 μg/L (452–553 μg/L) for juvenile S. viriginicus (96 h). No mortality occurred in the 28-d exposure of adult L. fullerkati and we found no statistically significant effect of fluridone concentration on foot protrusion (p = 0.06) or siphoning behavior (p = 0.08). The 24-h LC50 for glochidia exposed to the dipotassium salt of endothall was 31.2 mg/L (30.3–32.2 mg/L) and the 96-h LC50 for juvenile mussels was 34.4 mg/L (29.3–40.5 mg/L). Freshwater molluscs were more sensitive to fluridone and endothall than most other species previously tested. Fluridone and endothall concentrations typically recommended for hydrilla treatment (5–15 μg/L and 1–5 mg/L, respectively) were not acutely toxic to the molluscs we tested and a 28-d exposure to fluridone was not lethal to adult mussels even at the highest concentration (300 μg/L), indicating minimal risk of short-term exposure effects.