Arboriculture & Urban Forestry 42(6): November 2016 ined on each cross section, and the presence of plastic plugs, external bark cracks, internal xylem necrosis, evidence of pathogen infection, and presence of xylem (earlywood, latewood) grow- ing over injection sites was recorded (Figure 1). Evaluation of Discolored Xylem Tissue Transverse tissue samples (approximately 5 mm × 3 mm) were removed with a razor blade from the interface of the discolored and unstained xylem tissue associated with four randomly se- lected injection sites on 20 of the 46 trees (80 total injection sites) from the two-year study, and each of the 15 trees from the six-year study (60 total injection sites). Five to ten samples were removed from each injection site to ensure researchers would have quality samples. Samples were imme- diately placed into fixing solution (50% ethanol, glycerol, formaldehyde, 18:1:1) and were leſt un- disturbed for at least 72 hours. Aſter fixation, samples were placed in a solution of safranin orange dye (1% safranin, Carolina Biological Supply Company, Burlington, North Carolina, U.S.) for 15 minutes, dehydrated through 30%, 50%, 75%, 90%, and 100% ethanol (Decon Labs, Inc., King of Prussia, Pennsylvania, U.S.), and then rehydrated backward through the same solutions. Samples remained in each alcohol solution for 10 minutes. Solutions were changed multiple times during each cycle to remove excess dye. Aſter rehydrating, sam- ples were placed in deionized water for 15 minutes, then processed through 25%, 50%, and 75% glyc- erol solutions (1 hour each with frequent decant- ing), and finally leſt overnight in 100% glycerol. Confocal laser scanning microscopy (CLSM) and/or polarizing digital microscopy was used to evaluate sapwood integrity. Images were viewed and recorded the day aſter samples were soaked in 100% glycerol. Xylem samples from trees used in the two-year study were viewed with a confocal laser scanning microscope (Olympus FluoView™ 1000, Olympus Corporation, Tokyo, Japan), while xylem samples from trees used in the six-year study were viewed with a handheld polarizing digital microscope (Dino-Lite AM4113ZT®, AnMo Elec- tronics Corporation, New Taipei City, Taiwan). Xylem tissues examined using CLSM were illu- minated with argon (λ = 543 nm) and helium- neon (λ = 488 nm) lasers. Single channel z-scan 393 collections were recorded (Hutzler et al. 1998). A 10× dry objective was used to magnify trans- verse samples removed from the discolored- unstained interface of trees trunk-injected with TREE-äge. Total slice depth ranged from 10.2 to 14.5 µm; 7 to 21 slices per sample were obtained, depending on optimal image acquisition param- eters. Xylem samples assessed with the hand- held polarizing microscope were examined under 10× and 50× magnification. Polarization was adjusted as necessary to reduce image glare. Statistical Analysis Data were analyzed using SAS statistical soft- ware (SAS Institute, Inc. 2008). Assumption of normality was tested with residual plots and the Shapiro-Wilk test (Shapiro and Wilk 1965). Width and length of discolored tissue on cross sections from treated trees of the two- year study were normalized using log transfor- mations (Ott and Longnecker 2001), while all other variables met assumptions of normality. Three-way ANOVA was performed to assess effects of ash species (green or white), EB rate (EB-low or EB-high), and number of applica- tions (2008 only, or 2008 and 2009) on the pres- ence of xylem laid over injection sites, bark cracks or internal tissue necrosis, and discol- oration length, width, and height (stumps from the two-year study). When ANOVA results were significant (α ≤ 0.05), Fisher’s protected least significant difference (LSD) multi-comparison test was applied with Tukey’s adjustment for unbalanced data sets (Ott and Longnecker 2001) to determine differences between means. RESULTS Two-Year Study Green ash and white ash tree size did not vary among treatments (EB-low or EB-high trees treat- ed in 2008 or in 2008 and 2009) (P = 0.65). The number of injection sites on EB-high and EB-low trees treated in 2008 only was similar (P = 0.36), averaging 4.6 ± 0.53 and 3.3 ± 0.24 injections per tree, respectively (Table 1). On average, EB-low and EB-high trees treated in 2008 and 2009 had 7.4 ± 0.54 and 5.8 ± 0.54 injection sites per tree, respectively, but differences between the EB-low ©2016 International Society of Arboriculture
November 2016
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