8 injured and r2, the radius of the circumferential discoloration r2 ), where r1 of new vascular tissue/cm2 IMA-jet (5% imidacloprid) was micro-infused into two trees on September 29, 2005. Two more trees received the same micro- infusion treatment on May 21, 2008. All four trees received 0.4 g AI/2.54 cm DBH. A second product containing imidacloprid, Merit Tree Injection (200 SL, 200 g ai/l), was injected into two trees on October 17, 2005, and two more trees on May 21, 2008. Injections were made with the Arborjet Air Hydraulic Device at a rate of 0.4 g AI/2.54 cm DBH. Each injection was made through a plastic septum (7 mm diameter, #3 Arborplug) at 1379 kPa. Two untreated control trees were included in the study for the purpose of collecting data on the growth rates of trees se- verely compromised by EAB. Water-injected controls were not included in this study because the study authors an- ticipated that all trees that were not treated with insecti- cide would decline rapidly due to EAB and would not yield any useful data to compare with insecticide-injected trees. All 16 trees (14 trunk-injected and two untreated control trees) were felled in July 2009 by cutting the trunks at a height of 1.5 m above the ground, and again at ground level. The 1.5 m trunk bolts were transported to the Michigan State University Entomology Research Farm in East Lansing, where cross-sec- tions and longitudinal-sections were cut to facilitate measure- ment of annual radial growth, area of vascular discoloration, area of wounding due to trunk injection, and annual closure of wounds from new growth of vascular tissue and woundwood. This was done by cutting the bolts again at 15 cm and 60 cm above and 15 cm below the injection sites. Each cut disc was planed (Dewalt model # DW680) smooth and photographed in transverse section. The age of the trees was determined by counting the number of growth increments from the pith to the most current growth ring. These sections were then quartered with a log splitter (Huskee 34 ton log splitter). Each quarter section was then cut through the injection site using a table saw (Craftsman 25 cm table saw with a Freud Avanti Tico high den- sity carbide blade) and Ryobi miter saw (25 cm Skil 60-tooth crosscut blade). Digital photos were taken of each radial sec- tion. Calculations for wound closure were based on the area of an ellipse (πr1 equals the radius of cambium measured. The percent area of wound closure was calculated for each growing year following injection using the formula cm2 of injured lateral cambium. Radial sapwood growth was measured by micrometer (Mod- el No. CD-6”GS, Mitsutoyo Corp., Japan) for two years pri- or to and the two years after the injection holes were made. Data Analysis The closure of wounds around each of four or five trunk injection sites per tree was measured each year. Percent wound closure was then determined based on the size of the original wound. Data for the four or five injection sites per tree were then averaged to give mean values for each tree. Because the 14 study trees were injected in different years, annual ring growth and wound closure data were recorded and analyzed in relation to the year of trunk injection. Year 1 or Year 2 refer to the amount of annual ring growth and wound closure occurring in the first or second (respectively) growing season following autumn or spring trunk injections. Therefore, for trunk injections made in October 2005 and in May 2006, ring growth and wound response in Year 1 were ©2011 International Society of Arboriculture Doccola et al.: Tree Wound Responses to Trunk Injection determined by tree growth in spring, summer, and autumn 2006; and ring growth and wound response in Year 2 were determined by tree growth in spring, summer, and autumn 2007. The rela- tionship of canopy thinning and dieback to ring growth in the same year was determined by regression analysis. Percent data were arcsine transformed using the formula: arcsin (sqrt (x/100), prior to analysis. Graphs of these relationships were made using nontransformed percent data. The polynomial regression for the relationship of annual ring growth to canopy thinning and die- back ratings was made using StatView (Abacus Concepts 1992). Linear regressions for the relationships of annual ring growth to canopy thinning, and for annual ring growth to percent wound closure, were made using SuperAnova (Abacus Concepts 1991). RESULTS All 16 study trees were felled and sectioned in July 2009, 1.1 to 3.8 years following trunk injection treatments. Mean DBH of the felled green ash trees in this study was 27.6 cm, and ranged from 18.8 to 36.3 cm. Growth-ring counts indicated that the trees ranged from 17- to 35-years-old (mean age 26.3). A total sixty- three trunk injection wound sites were evaluated by sectioning trunk bolts through the drilled injection holes, which were 16- to 52-mm-deep. No signs of cracking, oozing, or decayed tis- sues were found associated with any of the 63 injection sites. A total of 76.2% of the injection sites were completely closed by new wound growth, 12.7% of the injection sites were closed except the presence of a partially extruded plug, and 11.1% of the injection sites did not completely close. Discolored ar- eas associated with the injection sites were still visible four years after the injections were made, but the discolored areas were firm with no signs of infection or deterioration (Figure 2). Ring growth measurements indicate that two trees (tree #1 and tree #2) increased their growth rates in the first or second year after trunk injection, while eight trees continued to grow at a similar rate, and four trees decreased their growth rate in the first or second year after trunk injection (trees #6, #10, #11, and #12; Table 1). Regression analysis indicates that ring growth was strongly dependent on the level of EAB infestation as measured by canopy thinning and dieback ratings in July of the same year (Figure 3). EAB larvae actively tunnel in the cambial tissue from August to October. Therefore, canopy ratings in July reflect the amount of injury caused by EAB the previous autumn. Although many other factors can cause the same canopy thinning and die- back symptoms, in this case the study authors knew the dieback was caused by EAB because of a more extensive study being conducted at the same time in the same neighborhoods. In that study, 19 of the green ash street trees that were trunk injected with emamectin benzoate, the most effective insecticide treat- ment, had a mean canopy thinning and dieback rating of 13.5 ± 14.1% in July of 2009, compared with a rating of 58.1 ± 33.2% in July of 2009 for 10 untreated control trees (Smitley et al. 2010). Branch samples from the same trees revealed that the emamec- tin-injected trees had no detectable EAB larvae, while the con- trol trees averaged an infestation level of 28.7 ± 21.5 (mean ± SD) EAB larvae per m2 (Smitley et al. 2010). It can therefore be concluded that extensive canopy thinning and dieback caused by EAB resulted in a reduced rate of radial ring growth (width) in unprotected green ash trees. Regression analysis of this re- lationship indicates 53% of the variation in ring width can be
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