Arboriculture & Urban Forestry 34(5): September 2008 technique as follows: 0no powdery mildew observed; 1less than 5% of leaf surface covered with hyphal growth; 2 5% to 20% of leaf surface covered; 3 21% to 50% of leaf surface covered; 451% to 80% of leaf surface covered; and 581% to 100% of leaf surface covered. Superoxide Dismutase Activity Superoxide dismutase activity (SOD) activity was based on the method described by Kraus and Fletcher (1994). In summary, extracts were prepared at 4°C (39.2°F) by homogenizing 0.2 g (0.07 oz) of leaf tissue (generally six leaves per tree in 4 mL [0.12 fl oz] of 0.1 M Na2HPO4/NaH2PO4 buffer [pH 7] with a mortar and pestle and centrifuged at 16,000 × g for 10 min). The supernatant was filtered through Whatman paper (No. 1) and 1 mL (0.03 fl oz) was applied to a Pharmacia PD-10 chromatog- raphy column containing Sephadex G-250 equilibrated with 50 mM Na2CO3 buffer (pH 10.2) to remove low-molecular-weight components and exchange the buffer. The assay was performed using 0.1 mM diethylenetriaminepentaacetic acid in the reaction mixture and the subsequent increase in absorbance at 550 nm followed on a spectrophotometer (PU8800 Pye Unicam, Ports- mouth, U.K.) equipped with a kinetics module for 6 min at 24°C (75.2°F). SOD activity was assessed at days 10, 20, and 60 after application of the SIR-inducing chemical to determine the short- and long-term effects of each product on enzymatic activity. Peroxidase Activity A total of 0.3 g (0.01 oz) of foliar tissue (generally six leaves per tree) were homogenized in 5 mL (0.15 fl oz) of 0.2 M Tris-Cl− (buffer pH 7.8) containing 0.13 mM EDTA and 80 M soluble polyvinylpyrrolidone with a Polytron homogenizer (Glen Mills Inc., Clinton, NJ) for 30 sec on ice and centrifuged at 3,000 × g for 10 min. Catalase activity was determined by following the consumption of hydrogen peroxide (extinction coefficient 39.4 mm−1/cm−1) at 240 nm for 2 min. The reaction mixture con- tained 2 mL (0.06 fl oz) of 100 mM Na2HPO4/NaH2PO4 buffer (pH 6.5) and 50 L of plant extract, and the reaction was initi- ated by adding 10 of 30% (w/v) hydrogen peroxide. Leaf peroxidase activity was assessed at days 10, 20, and 60 after application of the SIR-inducing chemical to determine the short- and long-term effects of each product on enzymatic activity. Statistical Methods Before the analysis, data were examined to ensure normality and homogeneity of variance (Anderson-Darling test) were met to meet the assumptions for the analysis of variance. Data were then analyzed as a randomized complete block using an analysis of variance (ANOVA). If trial data violated the basic assumptions required by ANOVA, data were log-transformed and then back- transformed for presentation in tables. Differences in levels of disease severity, leaf superoxidase dismutase, and peroxidase- specific activity from control values were determined and means were separated at the P < 0.05 level of significance using the Genstat for Windows program (VSN International Ltd., Hemel Hempstead, U.K.). A simple linear mathematical model was used to record relationships between leaf superoxidase dismutase and peroxidase-specific activity versus leaf disease severity us- ing goodness-of-fit R2 values. RESULTS In both the 2005 and 2006 trials, damaging outbreaks of powdery mildew were recorded on control trees as indicated by disease 273 severity ratings of 5.0 to 5.5, respectively, on leaves of English oak (Figure 1). During the study, none of the treated or control trees died as a result of pathogen attack. None of the SIR agents or fungicide evaluated was phytotoxic to the test trees. The SIR- inducing compound RESISTIM (a.i. betaine) and a single spray treatment of penconazole had no significant influence on disease severity and specific activity of peroxidase and superoxide dis- mutase in both the 2005 and 2006 trials (Figures 1 to 4). Con- sequently, results for RESISTIM are shown that reflect those of a single penconazole spray treatment. Results of this study in- dicate that the commercially available compound RESISTIM and a single spray of penconazole have no significant role in the control of powdery mildew when applied as a therapeutic treat- ment (Figure 1). Disease Severity In both the 2005 and 2006 trials, salicylic acid and potassium phosphite had no significant effect on disease severity at the cessation of the growing season (Figure 1). Consequently, results of this study indicate no beneficial long-term effects of salicylic acid and potassium phosphite on reducing powdery mildew se- verity when used as a single therapeutic spray treatment. Applica- tion of the SIR-inducing agent harpin protein significantly (P < 0.05) reduced disease severity of powdery mildew from 5.5 to 2.1 in the 2005 trial. No significant effects, however, on disease severity were recorded in the 2006 trial. The fungicide Topas (penconazole) applied at three weekly intervals after the visible observation of powdery mildew resulted in a significant (P < 0.05) reduction in disease severity (Figure 1). Disease severity rates in this instance were reduced from 5.5 to 1.9 (2005 trial) and 5.0 to 1.8 (2006) trial. Leaf Superoxidase Dismutase, Peroxidase-Specific Activity, and Disease Severity The SIR-inducing agents harpin protein, salicylic acid, and po- tassium phosphite increased leaf-specific activity of superoxidase dismutase and peroxidase by 16% to 28% at days 10 and 20 post- application compared and nontreated controls (Figures 2 and 3). In all cases, such an increase in activity was significant at P < 0.05. By day 60, however, specific activity of both enzymes in leaves was nonsignificant when compared with controls (Figures 2 and 3). Such a result indicates a single spray of harpin protein, salicylic acid, and potassium phosphite induces superoxidase dismutase and peroxidase activity 20 days or more but less than 60 days. Concomitant with an increase in superoxidase dismutase and peroxidase activity at days 10 and 20, a significant (P < 0.05) reduction in disease severity was recorded at these time intervals. Such a result indicates an association between enhanced super- oxidase dismutase and peroxidase activity and resistance against oak powdery mildew. Applications of penconazole had no sig- nificant effect on specific leaf activity of superoxidase dismutase at days 10, 20, and 60, although the level of activity was always higher than controls (Figure 3). However, a significant increase (P < 0.05) in leaf peroxidase activity was recorded at days 10 and 20 (Figure 3). At day 60, levels were higher than controls, only not significantly so. At days 10, 20, and 60, penconazole appli- cation significantly (P < 0.05) reduced disease severity com- pared with nontreated controls (Figure 4). ©2008 International Society of Arboriculture
September 2008
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