Impacts on the seagrass, Zostera nigricaulis, from the herbicide Fusilade Forte® used in the management of Spartina anglica infestations
Introduction
Seagrasses provide essential and valuable ecosystem services (Duarte, 2002, Nordlund et al., 2016). Their meadows provide nursery, migration and reproductive areas, and are a food source for commercially and ecologically important marine and water-bird species (Seitz et al., 2014). As they grow, seagrasses are highly efficient at sequestering and storing carbon (Macreadie et al., 2014). They perform essential roles in nutrient cycling, pathogen removal (Lamb, 2017), and stabilization of coastlines (van Katwijk et al., 1999).
Worldwide, seagrass habitat has decreased by greater than 29% since the 1980s (Waycott et al., 2009). The rate of loss is accelerating largely due to increased destruction and modification of habitat from anthropological development in coastal zones (Orth et al., 2006). Seagrasses are highly vulnerable to localised stressors such as invasive species (Williams, 2007), sedimentation (Ralph et al., 2007b, Wooldridge, 2017), eutrophication (Burkholder et al., 2007), decreases in light availability (Ralph et al., 2007a), exposure to toxicants (Devault and Pascaline, 2013, Flores et al., 2013), and disease (Bull et al., 2012).
Spartina anglica (family Poaceae) is a noxious weedy grass that was introduced to saltmarshes and estuaries in Australia, China, Germany, France, England, and North America (Ayers et al., 2004, Roberts and Pulling, 2008). If left uncontrolled, S. anglica forms expansive swards in the inter-tidal and re-engineers the ecosystem (Bouma et al., 2009, Cutajar et al., 2012, Shimeta et al., 2016). In some locations, Spartina spp. infestations overlap and displace seagrass meadows (Cottet, 2007, Ferraro and Cole, 2007).
In Australia, S. anglica infestations are managed primarily by application of the herbicide Fusilade Forte® (FF) (Palmer et al., 1995, Shimeta et al., 2016). FF is in the aryloxyphenoxypropionate (AOPP) class of Acetyl-CoA Carboxylase (ACCase) inhibiting herbicides (Burton et al., 1989, Page et al., 1994). Its active ingredient is fluazifop-P acid (FPA; (R)-2-[4-(5-trifluoromethyl-2-pyridyloxy) phenoxy] propionic acid). There are numerous commercially available FPA-formulated herbicides marketed under trade names such as Fusilade Forte®, Ezycrop, Novagaurd, Fusilade DX, 4Farmers, Resilience, Genfarm, Ornamec, Fusilade II, Fusion II, Apparent Salvo, and Chemisco Grass and Weed Killer. In most of these, FPA is present as the butyl ester, fluazifop-P-butyl (FPB), along with a hydrocarbon solvent.
Once absorbed through the leaf's cuticle, FPB hydrolyses to FPA, which translocates in the phloem and the xylem to accumulate at active growing sites (Tu et al., 2001). In aqueous environments, up to 50% of FPB may be hydrolysed to FPA in 0.3–3 days (Badawi et al., 2015, European Food Safety Authority, 2012, Durkin, 2014). FPA can persist in the environment for more than 100 days (Negre et al., 1988, Durkin, 2014). Consequently, transport of FPA to the marine environment may result from its use within the surrounding watershed.
FPA is selective for species in the family Poaceae (Burton et al., 1989, Page et al., 1994). The primary mode of action (MOA) of FPA is the inhibition of poaceous ACCase, which is achieved by binding to the ACCase carboxyl transferase domain (Nikolskaya et al., 1999). Plants contain two forms of ACCase that differ structurally and are located in different subcellular compartments, plastidic and extra-plastidic. Plastidic ACCase is the key rate-limiting enzyme in de novo fatty acid synthesis and therefore lipid biosynthesis, whereas extraplastidic ACCase is involved in the synthesis of flavonoids (Walker et al., 1988).
Poaceous grasses are unique in that they possess two homomeric multi-domain ACCase enzymes that have only subtle differences in their structures. This difference confers their vulnerability to inhibition by AOPP herbicides (Burton et al., 1989). Poaceous plastidic ACCase is significantly more sensitive to inhibition by FPA than the extraplastidic form. For example, in maize the plastidic form is approximately 150-fold more sensitive towards inhibition by FPA than the extraplastidic form (Herbert et al., 1996).
The secondary MOA of FPA involves the breakdown of chlorophyll (Chandrasena and Sagar, 1987) coupled with the production of reactive oxygen species (ROS) such as the hydroxyl radical (−OH), hydrogen peroxide (H2O2) and super oxide radicals (O2−), which are implicated in the peroxidation of cellular membranes (Luo et al., 2016).
Herbicide resistance in some Poaceae populations has been observed. Target-site mutations that change ACCase structure can result in resistance to FPA's primary MOA (Cha et al., 2014, Kaundun, 2014), while non-target-site mutations can enhance FPA and ROS metabolisation, conferring resistance to FPA's secondary MOA (Hidayat and Preston, 1997, Matthews et al., 2000).
Non-poaceous monocotyledons and all dicotyledons are typically resistant to FPA's primary MOA since they contain structurally resistant forms of ACCase (Podkowiński and Tworak, 2011). They also have more effective FPA and ROS metabolisation pathways. Some non-poaceous plants, however, are vulnerable to FPA's secondary MOA. In lentils (Aksoy and Dane, 2011), bristly starbur (Luo et al., 2016), maize (Horbowicz et al., 2013), and peanuts (Fayez et al., 2014), exposure to FPA has been shown to induce changes in the ultra-structure of cells and organelles, reduce growth, decrease chlorophyll concentrations, and increase levels of lipid peroxidation. Exposure to FPA in some native Australian plants species has resulted in restricted germination and growth (Rokich et al., 2009).
Studies investigating the impact of FF, and similar FPA herbicide formulations, in seagrass are limited and generally report no effects. For instance, Wahedally et al. (2012) found no impact of FF exposure in the tropical seagrass, Thalassodendron ciliatum. However, their study only tested for impacts on photo-system II (PSII) photochemistry, yet FF is not a PSII-inhibiting herbicide. Palmer et al. (1995) conducted field spraying of intertidal Zostera muelleri seagrass meadows with FF at low tide and found no impact on biomass after four weeks, or seagrass ‘health’ after 5 months. No previous studies have investigated whether FPA acts on seagrass ACCase.
In Australia, some meadows of the endemic seagrass, Zostera nigricaulis, have declined in areas adjacent to Spartina infestations controlled with FF (Ford et al., 2016). We therefore investigated whether Z. nigricaulis is susceptible to FF's primary MOA (inhibition of ACCase), and if short-term exposure to FF has adverse physiological effects associated with FPA's secondary MOA (photosynthetic pigment content, PSII performance and lipid peroxidation). We exposed Z. nigricaulis plants to a commercial formulation product, Fusillade Forte® (containing FPA as the active ingredient) under laboratory conditions and assessed impacts to ACCase activity, photosynthetic pigment content, PSII performance, and lipid peroxidation.
Section snippets
Chemical reagents
Fusilade Forte® was obtained from Syngenta Australia Pty Ltd as an emulsified concentrate of 128 g L−1 FPA (equivalent to 0.39 M FPA) present as the butyl ester, fluazifop-P-butyl (FPB), and 156 g L−1 hydrocarbon solvent (Syngenta, 2016). All other chemical reagents, including analytical grade FPA ((R)-2-[4-(5-trifluoromethyl-2-pyridyloxy) phenoxy] propionic acid, CAS 83066-88-0) and Reserpine (CAS 50-55-5) were purchased from Sigma–Aldrich. Individual FPA solutions used in the ACCase activity
Herbicide in exposure experiment aquaria water
The majority of FPB had hydrolysed to FPA as treatments approached their respective nominal FPA concentration after 7 d exposure (Table 1). LCS and matrix spike recoveries were within acceptable limits (70–100%). Control aquaria as well as QA/QC blanks contained no FPA.
Acetyl CoA Carboxylase assay
FPA did not significantly inhibit the activity of ACCase isolated from Z. nigricaulis, measured as Pi production (Fig. 1), since none of the 95% confidence intervals for Z. nigricaulis falls outside a value of 0% inhibition. In
Discussion
To our knowledge, this is the first study to test if a seagrass is susceptible to the primary mode of action (MOA) of an arylophenoxypropionate (AOPP) group herbicide designed to target poaceous grasses. The findings from this study demonstrate that the seagrass Z. nigricaulis, which is in the family Zosteraceae, is not susceptible to the primary MOA (inhibition of plastidic ACCase) of the AOPP herbicide, Fusilade Forte® (FF). However, it is susceptible to secondary impacts as indicated by
Acknowledgement
The West Gippsland Catchment Management Authority and RMIT University funded this research. We thank Associate Professor R. Ritchie for advice on the PAM fluorometery method, Dr. B. Cromer, J. O’Brien and F. Carve Luzardo for chromatography and laboratory assistance.
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