390 Tanis and McCullough: Evaluation of Xylem Discoloration in Ash Trees Potential wounds or related injuries from trunk injections of systemic insecticides can be a concern for tree care professionals, municipal arborists, and homeowners. Systemic insecticides are applied to trees either as microinjections, with holes ≤0.5 cm in diameter, or as macroin- jections, which require larger holes (Costonis 1981). Microinjections generally deliver 1 to 3 ml of product per injection site and have been used for many years (Costonis 1981). Macroinjection devices enable a greater volume of product to be applied, facilitating higher application rates and potentially improving within-tree distribution of the insecticide (Doccola and Wild 2012). Drill- ing through the outer bark and into the sapwood, however, could potentially sever conductive tis- sues and may provide an entry point for pathogens (Shigo 1977; Lawson and Dahlsten 2003; Smith and Lewis 2005). Tree damage can also occur if products are injected under high pressure. For example, a microinjection system (Wedgle Direct- Inject®, ArborSystems, Omaha, Nebraska, U.S.) caused bark on maple (Acer saccharum) and ash (Fraxinus americana) trees to physically separate from cambium, damaging tissue at and around the injection point (Smith and Lewis 2005). In addition to wounds made by drilling, trunk-injected insecticides have also reportedly caused discoloration of internal tissues in mul- tiple tree genera, including Fraxinus (Shigo et al. 1977; Smith and Lewis 2005; Mota-Sanchez et al. 2009; Doccola et al. 2011; Tanis et al. 2012). When maple (Acer spp.) and ash trees were injected with imidacloprid, internal discolor- ation around the injection points was assumed to be a wound compartmentalization response (Smith and Lewis 2005). If tissue discoloration is indicative of compartmentalization, insec- ticide distribution throughout the tree could be compromised because barrier zone tissues rarely translocate (Shigo 1977). In a study where 14 C-imidacloprid (Imicide™ 10%, J.J. Mauget Co., Arcadia, California, U.S.) was injected into small (≈ 6 cm diameter) ash trees, discolored sapwood adjacent to injection sites had 75 to 300 times higher imidacloprid equivalent concentrations than adjacent unstained tissues (Mota-Sanchez et al. 2009; Tanis et al. 2012). Whether discol- oration is indicative of a wound response or of ©2016 International Society of Arboriculture non-viable cells, however, remains unknown. Discoloration was previously observed in green ash (Fraxinus pennsylvanica) trees (21.5 to 36.3 cm DBH) treated with a formulation of 4% ema- mectin benzoate (TREE-äge®, Arborjet, Inc., Woburn, Massachusetts, U.S.), but the discolored areas remained “firm,” and no symptoms of infec- tion or deterioration were observed four years post-injection (Doccola et al. 2011). This study, however, included only 16 injection sites on four trees injected with emamectin benzoate, and the integrity of xylem cells were not evaluated. A systemic insecticide with the active ingredi- ent emamectin benzoate, sold as TREE-äge (4.0%, ArborJet, Inc., Woburn, Massachusetts, U.S.), is commonly used in many areas to protect valuable landscape trees from EAB (Smitley et al. 2010a; Doccola et al. 2011; McCullough et al. 2011; Herms et al. 2014) and in some areas, to slow EAB population growth (Mercader et al. 2011; McCullough et al. 2015; Mercader et al. 2015). Large-scale studies have shown trunk injections of TREE-äge in spring or early summer provide >99% EAB control for at least two years (Smitley et al. 2010a; McCullough et al. 2011; Lewis and Turcotte 2015). Systemic products with the active ingredients imidacloprid or dinotefuran can also be used for EAB control (Herms et al. 2014). These products can be applied as soil drenches, basal trunk sprays, or trunk injection, but require annual application and were less effective than the emamectin benzoate insecticide (TREE- äge) in side-by-side studies (Smitley et al. 2010a; Smitley et al. 2010b; McCullough et al. 2011). Trunk injection of TREE-äge typically begins by inserting a plastic plug (Arborplugs®, Arbor- Jet, Inc., Woburn, Massachusetts, U.S.) into a hole drilled with a 0.95 cm drill bit. The emamectin benzoate product is injected with a needle that penetrates a membrane in the plug at approxi- mately 310 kPa or 1,379 kPa of pressure for the TREE IV® or QUIK-jet®, respectively (Doccola et al. 2011). Because TREE-äge does not require annual application, protecting ash trees requires fewer injection sites compared to other products that must be injected annually (Smitley et al. 2010a; Doccola et al. 2011; McCullough et al. 2011). Although trunk injection systems and insec- ticide formulations continue to be refined, tree
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