Arboriculture & Urban Forestry 34(3): May 2008 a series of minimum temperatures to bracket the temperature causing 50% electrolyte leakage. Consequently, root samples were placed in darkness in a programmable Merck Environmen- tal Freezing Chamber and the temperature was reduced by 2°C (3.6°F) per hr from 10°C (50°F) to –5°C (23°F) (apple) and –6.5°C (20°F) (evergreen oak) and maintained for 4 hr after which the temperature was raised by 2°C (3.6°F) per hr to 10°C (50°F). These temperature treatments have been shown to cor- respond to approximately 50% electrolyte leakage from root tis- sue of each species (Percival unpublished data; Kozlowski et al. 1991). Root freezing treatments were carried out on 18 January 2005 only. Salt Treatments Treatments were performed on 17 December 2004, 17 January 2005, and 8 February 2005. Six fully expanded nonsenescing leaves per tree selected at random from throughout the canopy were excised at the base of the petiole using a razor blade. On arrival at the laboratory (less than 2 hr after collection), leaves were immersed in a series of percentage salt (NaCl) solution (0% to 12%) for 5 min to select a concentration resulting in 50% damage to the leaf photosynthetic system as measured by leaf chlorophyll fluorescence Fv/Fm values. After salt immersion, leaves were placed, abaxial surface down, in a Petri dish on moist Watmans filter paper sealed with a thin polythene film permeable to air but not water. After all treatments, leaf samples were placed in a Merck Environmental Growth Chamber in darkness at 22°C (72°F) for 72 hr, a time after which salt- induced detrimental effects on chlorophyll fluorescence, values could be detected (Greaves and Wilson 1987). In the case of evergreen oak, each salt treatment (0%, 2%, 4%, 6%, 8%, 10%, 12%) required six leaves per tree, eight trees per calcium treat- ment, 48 leaves in total, i.e., 48 leaves were immersed in a zero salt solution, a separate 48 leaves immersed in a 2% salt solution, and so on until a 12% salt solution was reached. Physiological Tests All physiological measurements taken during the trial were ob- tained from existing leaf material on the plant at the initiation of the experiment. Because leaf chlorophyll fluorescence is nonin- vasive and nondestructive, after each measurement, the same evergreen oak leaves were used to obtain electrolyte leakage values. Electrolyte leakage tests were not conducted on NaCl- treated plants. The principle of electrolyte leakage is well known. When healthy plant tissue is immersed in ion-free water, there is a slight leakage into the surrounding water that can be detected and quantified using a conductivity meter. If the cell membrane is ruptured because of adverse environmental stress, the cell ion contents leak at a greater rate and therefore conduc- tivity readings are higher. However, electrolyte leakage is not recommended when quantifying tolerance to salt stress because contamination by NaCl ions within leaf tissue or on the leaf surface can influence these values. Consequently, electrolyte leakage is not considered a good measure of NaCl damage and therefore not used in this study. Chlorophyll Fluorescence Immediately after the freezing treatment and 72 hr postsalinity treatments, leaves were adapted to darkness for 30 min by at- taching light exclusion clips to the leaf surface and chlorophyll 193 fluorescence was measured using a HandyPEA portable fluores- cence spectrometer (Hansatech Instruments Ltd., King’s Lynn, U.K.). Measurements were recorded up to 1 sec with a data acquisition rate of 10 s for the first 2 ms and of 1 ms thereafter. The fluorescence responses were induced by a red (peak at 650 nm) light of 1500 mol/m−2/Hz–1 photosynthetically active ra- diation intensity provided by an array of six light-emitting di- odes. The ratio of variable (Fv Fm-Fo) to maximal (Fm) fluorescence, i.e., Fv/Fm where Fo minimal fluorescence, of dark-adapted leaves were used to quantify the detrimental effects of freezing and salinity on leaf tissue. Fv/Fm is considered a quantitative measure of the maximal or potential photochemical efficiency and optimal quantum yield of photosystem II (Willits and Peet 2001). Likewise, Fv/Fm values are the most popular index used as a measure of plant vitality and early diagnostic of stress in plants (Meinander et al. 1996). Leaf Electrolyte Leakage After freezing, samples were stored at 22°C (72°F) for 24 hr in darkness before conductivity measurements using a Jenway con- ductivity probe and M4070 m (BDH, Leicestershire, Loughbor- ough, U.K.). Total solute leakage was obtained by autoclaving for 1 hr at 121°C (249°F) and 0.103 MPa. Results are presented as percent solute leakage after 24 hr (McKay 1992). Twig and Leaf Calcium Analysis Ten leaf and six twig samples per tree were selected at random from throughout the canopy and pooled to form sufficient ma- terial for calcium analysis. Samples were then thoroughly washed and dried in a convection oven at 85°C (185°F) for 48 hr before grinding through a 0.5 mm (0.02 in) cyclone mill (Retsch, Middlesborough, U.K.). Each separate sample (0.5 g [0.02 oz]) was placed into 150 mL (4.5 fl oz) volumetric flasks and di- gested in 20 mL (0.6 fl oz) of 7:1 nitric/perchloric acid. After cooling, the solutions were brought to volume with deionized water and analyzed by inductively coupled plasma–emission spectroscopy elemental analysis. Nutrient values were expressed as percent tissue dry weight. Measurements were made on twig and leaf tissue taken at the third sampling date only (9 February 2005). The foliar and twig calcium concentration of each tree was correlated with the freezing temperature causing 50% elec- trolyte leakage (LT50) from stem tissue of apple and leaf tissue of evergreen oak derived from the same tree. Data Analysis Freezing and salt resistance was calculated for each calcium treatment as follows. Electrolyte leakage (percent values) and chlorophyll fluorescence Fv/Fm values were plotted against the treatment temperature and a classic logistic function was fitted to the viability data using slide write software. The temperature at 50% damage (LT50) was calculated for each tree as a parameter of the logistic function that was read from the curve fitting protocol. Each LT50 value per tree (eight trees per calcium treat- ment) was subjected to a one-way analysis of variance and when significant differences were found (P < 0.05), means were com- pared using the Duncan multiple range test. Levene’s (1960) test was used to determine the homogeneity of variances, and per- centage data were transformed using arcsine (sqrt [y/100]). Each species was independently statistically analyzed. The influence of sampling date, i.e., time, was initially analyzed as a posttreat- ment variable. In only one instance (leaf salinity of evergreen oak) was time shown to have a significant influence. Conse- ©2008 International Society of Arboriculture
May 2008
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