Arboriculture & Urban Forestry 46(5): September 2020 337 Table 3. Evaluation of TREE-äge-treated and untreated western white pine (Pinus monticola) use by Dendroctonus ponderosae in three experiments in the Warner Mountains of California (mean ± SEM). Bark samples were collected at breast height (1.4 m) and were approximately 210 cm2 . Data are based on 10 trees per treatment, however, actual sample size varied due to the number of trees killed in each treatment and the adequacy of bark samples for measuring response variables. Larval data from experiment 2 were determined from 7 untreated trees; 3 were discarded due to a lack of discernible MPB activity caused by secondary agents in the phloem. Trees in experiment 1 were treated on 17 to 18 September 2007, baited with attractant semiochemicals to promote attack by MPB from 1 July to 4 September 2008, and had bark samples removed on 11 to 12 August 2009. Trees in experiment 2 were treated on 5 November 2009, baited from 7 July to 4 November 2010, and had bark samples removed on 27 October 2011. Trees in experiment 3 were treated on 11 June 2013, baited from 11 July 2013 to 21 May 2014, and sampled 15 October 2014. Experiment Treatment 1 2 3 Control TREE-äge Control TREE-äge Control No. of attacks 3.4 ± 0.5 4.9 ± 0.7 4.8 ± 0.4* 8.3 ± 0.7 TREE-äge/V/GLV 0.6 ± 0.49 TREE-äge/PPZ 1.3 ± 0.26 1.9 ± 0.47 Exit holes 8.5 ± 1.1* 1.1 ± 0.4 0.00 5.6 ± 1.01* 0.00 0.00 Adult gallery (no.) 11.5 ± 1.2* 6.7 ± 0.5 17.8 ± 2.3* 8.7 ± 1.4 4.9 ± 0.38* 0.03 ± 0.03 0.63 ± 0.42 Adult gallery (cm) 74.9 ± 7.5* 21.8 ± 3.9 120.6 ± 13.4* 10.1 ± 2.1 38.7 ± 3.07* 0.08 ± 0.08 0.66 ± 0.44 * Indicates significant difference between means within a year (t-test or Welch’s approximation; P < 0.05). length) or not, and the circumferential distance of cat- egory types was recorded using a flexible measuring tape. For each tree, two sums were determined, i.e., the circumferential distances colonized and un-colonized by MPB. In experiment 3, a subset of 21 trees (7 from each treatment) were felled for autopsy. Insecticide Residues Residues of EB in phloem tissue were determined using enzyme-linked immunosorbent assay (ELISA) kits (HORIBA Biotechnology Co., Ltd., Kyoto, Japan). Phloem samples were field collected using a 5-cm hole saw and kept frozen until processing. Sam- ples were collected from two compass directions (North and South) at each sampling period. In exper- iment 1, trees were sampled on 1 July 2008 and 7 July 2009. In experiment 2, trees were sampled on 7 July 2010, 4 November 2010, and 27 July 2011. In exper- iment 3, trees were sampled on 11 July 2013, 18 June 2014, and 15 October 2014. To allow the evaluation of height effects on movement of EB, the first sample collection (1 July 2008) included height (1.2 and 2.4 m) as a factor in addition to direction. All other samples were removed at breast height while avoiding previ- ously sampled areas. Phloem samples were kept frozen (−19 °C) until preparation for residue determinations. They were oven- dried at 38 °C for at least 48 hours, then ground in a Wiley mill (Thomas Scientific, Swedesboro, NJ, USA, mesh size 20). A 0.5-g portion of each ground sample was extracted in 10 mL of chromatography grade meth- anol (Burdick and Jackson, Muskegon, MI, USA) by overnight horizontal agitation. After removal from the agitator, sediment was allowed to settle, and 1.5 mL from each sample was transferred to a 12 × 32 mm auto-sampler vial with a Teflon-lined screw cap (SUN SRI, Rockwood, TN, USA). Samples were refriger- ated (4 °C) until determinations of residue concentrations were made. For ELISA kits, all manufacturer instructions were followed. Duplicate wells were used for each sample and their mean recorded. The useable range of the Horiba EB kits is 0.3 to 3.0 ng/mL, so sample extracts were diluted serially with 10% (v/v) methanol/water until a result within this range was achieved. A Biotek ELx808 plate reader (Biotek, Inc., Winooski, VT, USA) was used to measure absorbance at 450 nm and results were calculated using 4-parameter curve fitting software (Biotek Gen5, Biotek, Inc.). Both matrix- only blanks and EB spikes (0.6 μg/g EB) were included with each plate to provide an estimate of matrix interferences and to ensure kit performance; the mean value of the tray blanks in the same run was subtracted from each treated sample value to provide the data used for presentation and analysis. STATISTICAL ANALYSES The response variable of primary interest was tree mortality as indicated by crown fade at the conclusion of the experiment. We used the definition of treatment ©2020 International Society of Arboriculture Larval gallery (no.) 41.6 ± 8.3 21.6 ± 5.1 47.1 ± 10.6* 0.00 35.8 ± 4.12* 0.00 0.00 Larval gallery (cm) 26.5 ± 5.2 13.8 ± 4.0 50.3 ± 11.2* 0.00 43.2 ± 4.93* 0.00 0.00
September 2020
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