Arboriculture & Urban Forestry 33(5): September 2007 311 were obtained by isolation in California forests from infected oaks or tanoaks. Isolates were first inoculated on bay leaves, and then mar- gins of the resulting lesions were subcultured first on corn meal-based P10ARP followed by V8 agar. Plugs measuring 8 mm(0.32 in) in diameter were then taken from the margins of the resulting colonies and placed in the outer xylem of oak trees after removal of the bark using a sterile 10 mm (0.4 in) diameter cork borer. The bark was then replaced on top of the inoculum plug and the inoculation area sealed with grafting wax, Parafilm™ (Alcan Inc., Neenah, WI), and aluminum tape. Inoculations were performed on the stem 1 m (3.3 ft) from the root collar. Lesion Evaluation At the end of each trial (12 to 33 weeks long; see Table 1), the bark of all trees was carefully peeled off and the size of lesions was measured. Longitudinal growth along the stem was determined by overlaying a flexible, graduated ruler on top of the lesion. Care was taken to measure the entire length of the lesion by progressively peeling deeper into the bark and the outer xylem of trees in the trial. This was a necessary requirement, because P. ramorum tends to grow in a serpen- tine fashion in and out between the cambium and the outer xylem. The length of the longitudinal lesion acropetally to- ward the tree top was measured independently of the basip- etal lesion toward the root collar. The extremities of each lesion were plated on PARP media to ensure lesions were caused by the inoculated pathogen. Each culture resulted in the growth of a P. ramorum colony. In each case, the patho- gen was identified by microscopic observation of the growing colonies. Treatments Seven different experiments were performed to evaluate the therapeutic and preventive efficacy of phosphite treatments. Table 1 summarizes treatment types and products used. The following products were tested: NutriPhyte (Chemical Dy- namics Inc., Plant City, FL), Agrifos (Agrichem Manufactur- ing Industries Pty. Ltd., Loganholme, Queensland, Australia), Phostrol (Nufarm Americas Inc., Burr Ridge, IL), Aliette (wettable powder from Rhone-Poulenc for foliar and soil ap- plications, TreeTech for injections), Vital (Luxembourg- Pamol Inc., Memphis, TN), and SuperSODAway (University of California Berkeley, not commercially available). Concen- tration of active ingredients was 6% to 9.5% for injections (6% for Phostrol and Phytoguard; 8% for Nutriphyte, Agri- fos, and SuperSODAway; 9.5% for Aliette), 0.5% for foliar or soil drenches, and 18% for bark application of Agrifos + Pentrabark™ (Agrichem, Medina, OH). Surfactants were added to phosphites as described by label or as recommended by the manufacturer. Breakthru (Western Farm Services Inc, Fresno, CA) was mixed to the foliar spray at a final concentration of 0.05%; Pentrabark™ was tested at con- centrations of 7.5% and 2.5%. Tree plots were divided in blocks, each containing one replicate of each treatment. Po- sitioning of treatment within the block was randomly se- lected. Analyses were done as for a completely randomized design, because there was no treatment replication within the block. Treatments were avoided in the period between late December and the end of January, when temperatures are relatively cold. When possible, they were administered in October or November or between March and May when trees are physiologically active and can translocate and process phosphites. Therapeutic treatments were administered 64 to 250 hr after the inoculum had been placed in the stems. Preventive treatments were always administered 1 week be- fore inoculation. A single injection per tree, 10 mL (0.3 fl oz) in volume, was administered by drilling a hole above the root collar using a bit slightly smaller than the tip of the tree injector. Depth of injection was variable depending on tree size, but all injections remained in the outer layer of rings of the xylem. The tip of the plastic injector (Marley injector, Quest Prod- ucts, Louisburg, KS) is conical and a seal is formed between the injector and the tree simply by slightly pressing the in- jector into the drill hole. Holes are normally drilled, not per- pendicular to the stem, but at a slight angle to ease flow of the solution. Positive pressure by means of a piston or spring mechanism is constantly applied until the product is com- pletely absorbed. Foliar treatments were applied by either mixing a wettable powder (weight:volume dilution) or by diluting a concen- trated product to the desired concentration. The solution was then sprayed using a backpack sprayer until runoff (approxi- mately 500 mL [15 fl oz] per tree). Soil drenches are applied by mixing the solution as indicated and by watering each tree with 2 L (0.52 gal) of solution. Topical bark applications were performed by spraying the same solution as in the foliar treatments on the bark of trees. Bark applications of Pentrabark™ + Agrifos were per- formed by mixing different percentages of Pentrabark™with a 1:1 solution of AgriFos and water. Statistical Analyses All statistical analyses were performed using the program JMP (SAS Institute Inc., Cary, NC). The variable measured to assess efficacy of treatments was the longitudinal growth of the pathogen along the stem in both directions from the in- oculation point. Data sets for each trial were independently tested for lack of normality and unequal variances among treatments using the Shapiro-Wilk and the O’Brien tests, re- spectively. When the data from a trial violated either one of ©2007 International Society of Arboriculture
September 2007
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