Arboriculture & Urban Forestry 33(6): November 2007 399 measurements were taken on any newly formed leaf tissue, i.e., new leaves not present at the time of PBZ application. At each sampling date, five leaves per tree were used for chlo- rophyll fluorescence and chlorophyll content measurements randomly selected throughout the crown. Leaves were then tagged to ensure the same leaf was measured throughout the study. Chlorophyll Fluorescence Chlorophyll fluorescence was used as a measure of damage to the leaf photosynthetic system. Leaves were adapted to dark- ness for 30 min by attaching light exclusion clips to the leaf surface and chlorophyll fluorescence was measured using a HandyPEA portable fluorescence spectrometer (Hansatech Instruments Ltd., King’s Lynn, UK). Measurements were re- corded up to 1 sec with a data acquisition rate of 10 s for the first 2 ms and 1 ms thereafter. The fluorescence responses were induced by a red (peak at 650 nm) light of 1500 mol/ m−2/s−1 PAR intensity provided by an array of six light- emitting diodes. A performance index (PI) based on an equa- tion that combines the relationship of calculated relative num- ber of reaction centers (RC) per energy absorbed (ABS) and then multiplied by two expressions describing the yields of light trapping (po) and subsequent electron transport (0), i.e., RC/ABS × po/(1 − po) × 0/(1 − 0; Clark et al. 1998, 2000; Percival and Fraser 2001) was used to quantify any effects on leaf tissue. PI values have been shown to be a highly sensitive measure of leaf photosynthetic activities and provide an indirect measure of plant vitality. PI values were automatically calculated by the HandyPEA. Leaf Chlorophyll Content A Minolta chlorophyll meter SPAD-502 (Spectrum Labora- tories, Inc., Plainfield, IL) was used. Chlorophyll was mea- sured at the midpoint of the leaf next to the main leaf vein. Calibration was obtained by measurement of absorbance at 663 and 645 nm in a spectrophotometer (PU8800 Pye Uni- cam, Cambridge, UK) after extraction with 80% v/v aqueous acetone (regression equation6.01 + 0.055x; r2 adjusted 0.90, P < 0.01) (Lichtenthaler and Wellburn 1983). Photosynthetic CO2 Fixation Light-induced CO2 fixation (Pn) was measured in two pre- darkened (20 min) fully expanded leaves per tree near the top of the canopy (generally approximately the fourth leaf from the apex) using an Infra Red Gas Analyser (LCA-2 ADC; ADC Ltd, Hoddesdon, Herts, UK). The irradiance on the leaves was 700 to 800 mol/m−2 PAR saturating with respect to Pn; the velocity of the airflow was 1 mL/s−1/cm−2 leaf area. Calculation of the photosynthetic rates was carried out ac- cording to Von Caemmerer and Farquhar (1981). All photo- synthetic measurements were taken in the early morning be- tween 8:00 A.M. and 10:00 A.M. on clear or partly cloudy days. Leaf Necrosis Leaf necrosis was assessed visually. Each tree was rated on a 0 to 5 rating scale using a visual indexing technique and ratings on the scale: 0no necrosis observed; 1less than 5% of leaves affected and no aesthetic impact; 2 5% to 20% of leaves affected with some yellowing but little or no defoliation; 3 21% to 50% of leaves affected, significant defoliation and/or leaf yellowing; 451% to 80% of leaves affected, severe foliar discoloration; and 5 81% to 100% of foliage affected with 90% to 100% defoliation. Leaf Electrolyte Leakage Quantitative drought damage to leaf tissue was assessed by measuring electrolyte leakage of entire leaves (two leaves per tree) at each sampling date. Excised leaves were placed in 50 mL (1.5 fl oz) universal bottles containing 30 mL (0.9 fl oz) distilled water and gently shaken by hand. Samples were stored at 22°C (71.6°F) for 24 hr in darkness before conduc- tivity measurements using a Jenway conductivity probe and M4070 m (BDH, Leicestershire, Loughborough, UK). Total solute leakage was obtained by autoclaving for 1 hr at 121°C (249.8°F) and 0.103 MPa. Results are presented as percent solute leakage after 24 hr (McKay 1992) Carotenoids Carotenoids (lutein, -carotene, neoxanthin, -carotene, zea- xanthin, antheraxanthin, violaxanthin) were analyzed using a high-performance liquid chromatography (HPLC) system (Schindler and Lichtenthaler 1994). Leaf samples were ex- cised from trees and immediately placed in liquid nitrogen. Fifty leaf samples per treatment, i.e., five leaves per tree, were taken. Pigment analysis was performed the next day. Pigments were extracted from leaves with 100% aqueous acetone (quartz sand mortar) and separated and quantitatively determined by reverse-phase HPLC applying a Nucleosil C18 column (particle size 4 m). The solvent systems for the combined isocratic and gradient separation and quantification were acetonitrile–methanol–0.2 M tris/HCL buffer and methanol–hexane (Lichtenthaler et al. 1992). Superoxide Dismutase Activity Superoxide dismutase (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 three leaves per plant) in 4 mL (0.12 fl oz) of 0.1MNa2HPO4/NaH2PO4 buffer (pH 7) with a mortar and pestle and centrifuged at 16,000 × g for 10 min. The supernatant was filtered through Watman paper (No. 1) (Sigma-Aldrich, Poole, UK) and 1 mL (0.03 fl oz) was applied to a Pharmacia PD-10 chromatography column (Waters HPLC Solutions, Centennial Park, Elstree, Hertford- shire, UK) containing Sephadex G-250 (Waters HPLC Solu- ©2007 International Society of Arboriculture
November 2007
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