Arboriculture & Urban Forestry 42(2): March 2016 when fungi occurred in unison, as they would in the natural environment. Inoculations were carried out by first cleaning the outer bark with cotton rolls soaked in Clean-jet (25% isopropa- nol, 0.5% polyalkoxylated alcohol, Arborjet Inc., Woburn, Massachusetts, U.S.). A sterile razor blade was then used to make one slanting incision (~0.5 cm) into the stem. One full loop of inocu- lum was transferred from a one-week old fungal colony to each treated seedling using a flame- sterilized microbiological loop. After inocula- tion, the incision was closed and tightly sealed with Parafilm and aluminum foil to prevent contamination and desiccation and to immobi- lize the stem at the point of incision. For sterile agar and water controls, one loop full of sterile MEA or 100 µL of sterile water, respectively, were placed into the stem incision and similarly sealed. The seedlings were kept in a greenhouse under 400 watt metal halide lamps (10-hour light cycle) and watered weekly. Assignment of seed- lings to treatments and location of seedlings in the greenhouse was according to a completely randomized design. Study seedlings were as- sessed weekly for foliar necrosis and destructively autopsied by dissection after 120 days to docu- ment lesion development and to re-isolate fungal species from cross sections of seedling stems. Phytotoxicity of fungicides Another 40 seedlings were randomly assigned to four treatments to test the phytotoxicity of injected TREE-äge, Alamo, and Arbotect 20-S, relative to an untreated control (UTC). A Micro I.V. designed for this experiment (Arborjet, Inc., Woburn, Massachusetts, U.S.), based on a 10 cc capacity syringe fitted with 18 ga × 2.5 cm syringe needle, administered TREE-äge, Alamo, or Arbotect by gravity feed into P. monticola seedlings. All treat- ments were infused by drip, where each seedling received 5 mL of a 10% solution. Injections were conducted by first cleaning the injection site using cotton rolls soaked in Clean-jet. A sterile razor blade was used to make a thin incision into the sapwood of the seedling, just large enough to al- low a tight fit of the syringe tip. The syringe was then placed into the incision, while elastic horti- cultural tape was used to secure the site. The valve on the Micro I.V. was opened to allow the pesti- 87 cide to be administered by slow drip. The Micro I.V. was not removed until all of the solution had drained. The seedlings were arranged randomly in a separate section of the greenhouse from that which was occupied by the pathogenicity study, and so were held under the same environmental conditions previously described. Seedlings were visually inspected weekly for foliar phytotoxicity (e.g., foliar necrosis) and destructively autop- sied aſter 120 days to document vascular health. Autopsies and statistics Seedlings were observed weekly to note symp- toms of foliar necrosis and autopsies were con- ducted at 120 days post-treatment, earlier if all needles were dead, to assess cause of death (Table 1). Where oxidized or necrotic phloem completely encircled the stem, the seedling was categorized as dead. The earliest mortality was observed at 40 days in seedlings inoculated with G. clavigera. After 120 days, the percent circum- ferential lesion was measured in all remaining treatments. Circumferential lesion formation reflects the extent of stem girdling, a critical met- ric to tree survival. Autopsies were conducted using the following steps: 1) the bark was re- moved at the base of each seedling, up to the first node (first year stem growth); lesion circum- ference and height were measured; and 2) thin transverse sections of treatments were taken for microscopic examination of discoloration (oxi- dation) and occlusions of the vascular tissues. Statistical analyses were carried out separately for each experiment. For each experiment, the null hypothesis that mortality was the same for all treatments was tested using Fisher’s exact test (FET) on an Rx2 frequency table with seedlings classified as living or dead for each of R treat- ments (R = 5 or 4 for the pathogenicity and phy- totoxicity studies, respectively). Rejection of the null hypothesis at significance level 0.05 was followed by pairwise comparisons of treatments using FET with Bonferroni-adjusted P-values on the corresponding 2x2 tables. For the patho- genicity study, the two control treatments were compared first and, when a non-significant out- come (P > 0.10) was observed; they were pooled for comparisons with other treatments (Table 1). One-way analysis of variance (ANOVA) was used ©2016 International Society of Arboriculture
March 2016
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