290 Alamo, PHOSPHO-jet, and Arbotect, respectively) at the time of application. Tree diameters at breast height averaged 14.2 ± 0.2 cm, which led to an average injected volume (for low and high doses, respectively) of: 140 and 280 mLs of PHOSPHO-jet, 224 and 448 mL of Alamo, and 577 and 594 mL of Arbotect 20-S. Thus 28 to 56 mL of PHOSPHO-jet, 56 to 112 mL of Alamo, and 17 to 34 mL of Arbotect for- mulations were applied for low and high doses, respectively. Trees were inoculated with O. minus four times: 30, 112, 391, and 738 days after treatment with fungicides (Figure 1). Lesion areas at the points of inoculation were determined ~30 days af- ter inoculation. At each sampling period, two subsamples were measured per tree, except for 112 days when four subsamples were measured per tree. Each inoculation point of O. minus pro- vided one subsample; tree means were used for all evaluations. 2009 Experiment Results from the 2008 screening experiment suggested that Alamo was the most effective formulation (Figure 1), so 2009 treatments emphasized this product. Arbotect 20-S was used at a rate below the maximum label rate (72 mL per 2.5 cm dbh) in the first exper- iment. Researchers chose to combine the two formulations in this experiment in an effort to improve systemic activity and to reduce the viscosity [of 56 mPas (millipascal seconds) @22C] of the Alamo formulation for easier application (Alamo MSDS). These objectives required that products be mixed, necessitating some applications that were off-label. Five treatments were assigned to an experimental population of 50 loblolly pines (N31°49’08.9”, W092°32’01.7”), providing 10 replicate trees per treatment. Treatments were applied over a four-day period (May 4–7, 2009). In 2009, treatments were applied at 100 mL total volume; the amount of each ingredient varied by the ratio of the treatment. Trees averaged 18.5 ± 0.25 cm dbh. The Alamo-only dose per tree was 100 mL, which was ~13 mL per 2.5 cm dbh, 3 mL high- er than applied in 2008 and closer to the therapeutic label rate (10–20 mL per 2.5 cm). The Arbotect 20-S-only treatment was also 100 mL, or ~13 mL, per 2.5 cm dbh. This was 8 mL more than in 2008 and closer to its therapeutic label rate (12–24 mL per 2.5 cm dbh). To determine if a mixture of these two products was more effective than each fungicide individually, two ratios (2:1 and 3:1, Arbotect to Alamo) were compared. The 2:1 ratio treat- ment was prepared by mixing 67% Arbotect to 33% Alamo v/v, and the 3:1 ratio treatment was prepared by mixing 75% Arbotect to 25% Alamo v/v. In both cases, trees injected with a ratio treat- ment received ~13 mL per 2.5 cm dbh for a 100 mL total per tree. Inoculations with O. minus were made 28 days and 373 days post treatment, and lesion areas determined about 30 days later following the procedures from 2008. Statistical Analysis Although individual inoculations were evaluated only once due to the destructive nature of the sampling procedure, it was ex- pected that responses (lesion areas) on a given tree may be cor- related in time. This led to a repeated measures analysis for evalu- ating impact of the fungicides on lesion area for the 2008 data, where evaluations were made over four periods. In this analysis, fungicide treatment (with seven categories) was regarded as the between-subject factor, and time period (with four levels) was the within-subject factor. Following a non-significant test result ©2011 International Society of Arboriculture Doccola et al.: Systemic Fungicides Impact on Bluestain Fungi for sphericity (P = 0.10), split-plot F-tests (univariate repeated measures) were reported for fungicide treatment and time period main effects, and treatment × time period interaction. Because the goal was to identify the best fungicide treatment(s), and Alamo strongly met this criterion (Figure 1), detailed examination of the treatment × time period interactions provided little information of practical importance and was not pursued in the 2008 data. Individual contrasts were performed on treatment means (aver- aged over periods) to test for rate and rate × fungicide effects, and to carry out pairwise comparisons among fungicides aver- aged over rates. To maintain an experiment-wide alpha level of 0.05, a Bonferroni correction was applied to the pairwise com- parisons among fungicide treatments (including the control, there were six comparisons resulting in a critical P-value of 0.0083). Trees injected with fungicides in 2009 were evaluated for lesion areas twice, and a split-plot analysis was performed to test for fungicide treatment, time period, and treatment × time period effects. A strong treatment × time period interaction led to separate comparisons among treatments at each time. The ef- fect of fungicide treatment was significant at each time period; Tukey’s Honestly Significant Difference (HSD) range test was applied to evaluate relationships among treatment means. Prior to all analyses, lesion areas were square root transformed, then averaged for each tree at each sampling period to better meet the assumptions of parametric statistics for distribution and variance. Statistical analyses were conducted using a combi- nation of SAS (V. 9.2) and JMP (V. 9) (SAS, Inc., Cary, North Carolina, U.S.) with significance being accepted when P < 0.05. RESULTS 2008 Experiment Trees were evaluated for crown fade (phytotoxicity) on June 5, 2008; June 20, 2008; and July 28, 2008, which were 30, 45, and 83 days post-treatment. At 30 days, all four trees treated with PHOS- PHO-jet high and two of the four trees treated with PHOSPHO-jet low exhibited reddish or browning crowns. After 45 days, there was little change but by day 83 post-injection tree canopies were mostly recovered with only two (both PHOSPHO-jet high) having any noticeable crown discoloration. Trees treated with Alamo and Arbotect did not exhibit any noticeable crown discoloration. The study’s researchers did not observe any mortality of treated trees. The repeated measures analysis produced significant treat- ment (= fungicide product) and time period effects (F = 10.21, DF = 6, 21, P < 0.001 and F = 7.38, DF = 3, 63, P < 0.001, respectively) and a non-significant treatment × time period in- teraction (F = 1.57, DF = 18, 63, P = 0.096). Rate and rate × fungicide (averaged over period were non-significant (F = 3.13, DF = 1, 21, P = 0.091 and F = 0.01, DF = 2, 21, P = 0.994, respectively). Contrasts among fungicides, averaging over time periods, revealed that treatment with Alamo or Arbotect 20-S significantly reduced lesion areas produced in response to in- oculation with O. minus (adjusted critical P-value < 0.0083; six pairwise comparisons were made among the untreated and the three products averaged across rates). Alamo was also signifi- cantly more effective than both Arbotect 20-S and PHOSPHO- jet (P < 0.0001 in each case), which did not differ from each other (P = 0.51). Mean lesion size in trees treated with PHOS- PHO-jet was not different than in untreated trees (P = 0.02).
November 2011
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