196 James et al.: Prevention of Pine Wilt of Scots Pine (0.03 fl oz) water. The final suspension contained a mixture of life stages of PWN, a few other nematode species, and organic debris. Two mL (0.02 fl oz) of the nematode suspen- sion (approximately 5,000 nematodes) were pipetted into small glass Petri plates. A solution of Avid was added to obtain final concentrations of 0, 0.01, 0.1, 1.0, and 10.0 L a.i. abamectin per liter (ppm) water. Nematodes were soaked in the solution for 48 hr at 20°C (68°F). The solutions con- taining nematodes were then poured over Kleenex-brand tis- sue suspended by a coarse screen in contact with the surface of tapwater contained in 10 cm (4 in) diameter pots (Alby 1975). Tissue margins were folded to prevent nematode pas- sage into suspension without active movement through the tissue. After 24 hr, tissue and screens were removed and water in pots decanted to approximately 80 mL (2.7 fl oz). Nematodes in the suspension were then counted as previously described. There were four replicates of each nematode/ abamectin concentration and the experiment was repeated. Data from the two experiments were combined for analysis. The number of active nematodes (i.e., those capable of mi- grating through the tissue) was standardized to the predicted y value at concentration 0 and modeled as an exponential function of abamectin concentration using Proc NLIN in SAS (SAS Institute 1999). Tree Injections Followed by Inoculations with Pinewood Nematode The efficacy of abamectin injections to suppress pine wilt was studied in an abandoned Scots pine Christmas tree farm near Lansing, Kansas. The experimental design was a ran- domized complete block with four treatments and 20 trees per treatment. Trees were approximately 4 to 6m(13.2 to 19.8 ft) in height with dbh of 7 to 15 cm (2.8 to 6 in). Injection methods included the Wedgle injector (Arbor Systems Inc., Omaha, NE) and systemic tree injection tubes (STIT) (Helson et al. 2001). Briefly, the Wedgle injector is a system consist- ing of a modified livestock syringe with a flattened, beveled needle that is inserted through the bark and to the cambium. A small amount of liquid (0.5 or 1 mL) is deposited in the xylem/bark interface by squeezing two handles simulta- neously to create pressure for injection. The STIT system consists of tubing (Nalgene™or Tygon™) approximately 60 to 90 cm (2 to 3 ft) long, 1.7 cm (0.68 in) outside diameter, and 1.0 cm (0.4 in) inside diameter. One end of the tube is connected to a maple sap spile (Atkinson Supply, Oro Sta- tion, Ontario, Canada) and the other to a tubeless automobile tire stem by automobile hose clamps. Trees are injected by drilling 8 mm (0.32 in) diameter holes at a slight angle into the trunk to a depth of 3 to 4 cm (1.2 to 1.6 in). The maple spile with attached hose (without the tire stem) is inserted into the hole and tapped with a hammer to seat into place. Mate- rial for injection is loaded into the attached hose by a syringe, the tire stem inserted and then secured with a hose clamp. ©2006 International Society of Arboriculture A bicycle pump is used to pressurize the tube to ≈275 kPa (40 psi). Injections were performed 18 May 2002. A 2% a.i. formu- lation of abamectin (Avid™; Syngenta Corporation) or water was injected with the Wedgle at a rate of 1 mL per 3-cm diameter (dbh) (0.03 fl oz per 1.2 in). Thus, a total of 5 mL (0.15 fl oz) of liquid was injected into trees at five injection points equally spaced around the tree circumference at ground level. Avid or water was injected with the STIT sys- tem at 30 mL (0.9 fl oz) per 15 cm (6 in) trunk diameter with an additional 30 mL (0.9 fl oz) for each 5 cm (2 in) diameter. Because all trees in the planting were 15 cm (6 in) dbh or smaller, all trees were injected with 30 mL (0.9 fl oz) of liquid. Two STIT tubes at 15 mL (0.5 fl oz) per tube were inserted at ground level and on opposite sides of the trunk. All trees were inoculated with PWN on 20 June 2002. Inocu- lum from a single dead Scots pine was prepared by sectioning the trunk into 2 cm (0.8 in) thick wafers. The wafers were debarked and chipped into smaller pieces and put in ≈18 L (4.68 gal) buckets filled with water and agitated with com- pressed air for 48 hr. Nematodes were processed as previ- ously described to produce a suspension containing 4,733 ± 159 nematodes per mL (0.03 fl oz) water. Inoculation in- volved cutting the end of two 1 to 1.5 cm (0.4 to 0.6 in) diameter healthy branches at a height of ≈2 m (6.6 ft) on opposite sides of each tree and drillinga5mm (0.2 in) diameter hole to a depth of 4 to 5 cm (1.6 to 2 in). One-half milliliter (0.015 fl oz) of the nematode suspension was pipet- ted into the hole. The wound was then capped with a plastic or rubber bung. Additionally, a third branch on each tree in 10 replicates was wounded in the manner previously described and capped but was not inoculated with the nematode sus- pension. Visual symptom ratings for the entire tree were assigned on 6 December 2002 per the following rating scale: 1healthy, 2 <25% dieback, 3 25% to 50% dieback, 4 50% to 75% dieback, and 5 complete mortality. Ratings for each treatment were separated (P 0.05) by the Kruskal-Wallis K-sample test, a one-way analysis of ranks using Proc Rank and Proc GLM in SAS. Branch samples 12 to 18 cm (4.8 to 7.2 in) in length were removed from all trees on 6 December 2002. Three samples were collected at the tip and base of an inoculated branch and one from the middle of a noninoculated branch on the oppo- site side from trees with damage ratings of 1 to 4 (no symp- toms to major dieback). Two samples, including one from the base of the inoculated branch and one from middle of a noninoculated branch on the opposite side, were taken from trees with a rating of five (dead). Samples were placed in plastic bags and kept in cold storage at 4°C (39.2°F) before nematode extraction. Extraction and counting procedures were conducted as pre- viously described. After extraction, wood samples were oven-
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