Abstract
Two field trials were undertaken to determine the influence of fall fertilization using two commercially available, calcium-based fertilizers (calcium nitrate, calcium nitrate borate) and a high-nitrogen fertilizer (N:P:K = 24:7:7), at a range of concentrations, on the freezing and salinity tolerance of two urban tree species, evergreen oak (Quercus ilex) and holly (Ilex aquifolium). In both the 2001 and 2002 field trials, fertilization with calcium nitrate and calcium nitrate borate at a concentration of 40 g/m2 (0.12 oz/ft2) increased the freezing and salinity tolerance of both species as measured by leaf chlorophyll fluorescence, electrolyte leakage, and chlorophyll content. In addition, calcium fertilization at this concentration significantly increased total plant dry weight recorded at the cessation of the experiment. Application of both calcium fertilizers at concentrations of less than 40 g/m2 provided no significant protectant properties. Applications of more than 40 g/m2 proved phytotoxic to the two test species. Irrespective of concentration, applications of N:P:K (24:7:7) fertilizer did not enhance or increase susceptibility to freezing and salinity damage compared to nonfertilized controls. However, N:P:K (24:7:7) fertilization significantly increased leaf chlorophyll content and total plant dry weight. Results indicate that fall applications of calcium nitrate and calcium nitrate borate at 40 g/m2 can increase the freezing and salinity tolerance of evergreen oak and holly.
- Evergreen oak
- holly
- chlorophyll fluorescence
- electrolyte leakage
- SPAD values
- chlorophyll content
- fertilizer
Application of road de-icing salts in combination with direct freezing damage to urban trees following subzero temperatures during winter can result in significant tree mortalities (Dobson 1991; Percival and Fraser 2001). Likewise, late spring frosts can be devastating to newly emerged leaf tissue, resulting in reduced plant quality and often death (Cannel and Smith 1986; Cameron and Dixon 1997). Indeed, it has been estimated that freezing damage is indirectly and directly responsible for the deaths of more than 1 million urban trees annually on a global basis (Sakai and Larcher 1987). Protection against freezing and salt damage will become more important as climatic change may increase the unpredictability of weather patterns, resulting in progressively later frosts on an annual basis (Biggs 1996), and increased traffic volume and road network expansion may increase the quantity of salt used for de-icing operations (Percival and Fraser 2001).
Attempts to improve hardiness, through plant breeding, are limited by the nature of freezing and salt tolerance, which are quantitatively inherited traits controlled by many genes. Consequently, there is a demand for protectant compounds that are both inexpensive and can be applied at relatively short notice. It is now well recognized that applications of calcium (Ca) can reduce stress injury in plants by increasing cell wall strength (Ca is a major nutrient responsible for cell wall rigidity), maintaining plasma membrane integrity and transport function (a primary site of tissue injury during stress), and inducing formation of new proteins with stress-protective characteristics (Palta 1996). Previous research has shown that applications of calcium sprays can improve winter hardiness of fruit trees and increase time spent in cold store of fruit and vegetables such as avocados, mangoes, cherries, and apples (Anderson and Campbell 1995; Raese 1996). Little investigation exists into whether applications of calcium can improve the freezing and salt tolerance of urban trees.
Objectives of this investigation were to (1) determine the effectiveness and feasibility of two commercially available Ca fertilizers (calcium nitrate and calcium nitrate borate) to improve the freezing and salt tolerance of two tree species commonly planted in urban environments; (2) determine the concentration conferring maximal resistance to freezing and salt damage; and (3) comparatively evaluate both Ca fertilizers against a conventionally used, high-nitrogen fertilizer (N:P:K = 24:7:7).
METHODS
Plant Material and Experimental Design
Cell grown, 3-year-old, 45 cm (18 in.) high stock of evergreen oak (Quercus ilex L.) and holly (Ilex aquifolium L.) were obtained from a commercial supplier on 13 September 2001 and stored outdoors on a free-draining gravel surface. To ensure uniformity of stock for experimental purpose, trees were graded, and only those confirming to the characteristics specified in Table 1 were used. The experimental design consisted of three randomized, complete block designs at the University of Reading, Shinfield Field Experimental Station, Reading, United Kingdom. Within each block were eighteen 3 × 2 m (9.9 × 6.6 ft) plots separated by rows of guard trees at 0.5 m (1.65 ft) spacings. Within each plot, one of nine fertilizer treatments was used per species, along with a nonfertilized control:
Physical characteristics of evergreen oak (Quercus ilex) and holly (Ilex aquifolium) after grading.
Ca nitrate (Ca(NO3)2, trade name Tropicote™ (Yara UK Ltd, Immingham, N.E, Lincolnshire, UK), applied at 20, 40, and 80 g/m2 (0.07, 1.4, and 2.8 oz/ft2)
Ca nitrate borate (Ca(NO3)2H3BO3, trade name Nitrabor™ (Yara UK Ltd, Immingham, N.E, Lincolnshire, UK), applied at 20, 40, and 80 g/m2
high-nitrogen fertilizer (N:P:K = 24:7:7, trade name Bartlett BOOST, The Doggett Corporation, Lebanon, NJ) applied at 20, 40, and 80 g/m2
Thirty evergreen oak and holly were used per fertilizer treatment (i.e., 2 × 9 × 10 = 180 trees per block, 10 trees per plot [90 evergreen oak, 90 holly]; 3 × 180 = 540 trees total).
Fertilizers were applied uniformly to all plots by broadcast on the soil surface. All fertilizers were applied in the fall (4 October 2001) and trees planted by hand at 0.5 m (1.65 ft) spacings 1 week later. The soil was a sandy loam containing 4% to 6% organic matter and had a pH of 6.2. Available P, K, Mg, Na, and Ca were 52, 659.1, 175.2, 49.4, and 2,188 mg/L, respectively. Weeds were controlled chemically using glyphosate (Roundup, Green-Tech, Sweethills Park, Nun Monkton, York, UK) prior to planting and by hand throughout the experiment. No watering was required during the experimental period. Based on results of the 2001 trial, the experiment was repeated in 2002 on the same dates using only four of the nine treatments: Ca(NO3)2, Ca(NO3)2H3BO3, N:P:K (24:7:7) applied at 40 g/m2 (1.4 oz/ft2), and a nonfertilized control.
Freezing Treatments
At months 1, 2, 4, and 8 post-fertilization, two fully expanded, nonsenescing leaves per tree, 15 trees per fertilizer treatment (5 trees per block) were excised at the base of the petiole using a razor blade and placed abaxial surface down, in a Petri dish on moist Watman’s filter paper and sealed with a thin polythene film permeable to air but not water, then placed in darkness in a Merck environmental freezing chamber, where the temperature was reduced by 2°C (3.6°F) per h from 10°C (50°F) to −8°C (18°F). This temperature was maintained for 4 h, after which the temperature was raised by 2°C per h to 10°C, a cooling regime representative of a naturally occurring severe frost (Sakai and Larcher 1987). All leaf material was prepared within 2 h of collection from the field.
Salt Treatments
At months 1, 2, 4, and 8 post-fertilization two fully expanded, nonsenescing leaves per tree, 15 trees per fertilizer treatment (5 trees per block) were excised at the base of the petiole using a razor blade. Upon arrival at the laboratory (< 2 h after collection), leaves were immersed in an 8% salt (NaCl) solution for 2 min. After salt immersion, leaves were placed, abaxial surface down, in a Petri dish on moist Watman’s filter paper sealed with a thin polythene film permeable to air but not water. Following all treatments, leaf samples were placed in a Merck environmental growth chamber in darkness at 22°C (72°F) for 72 h, a time after which detrimental effects on chlorophyll fluorescence values could be detected (Greaves and Wilson 1987).
Because of the potential influence of leaf removal on total plant dry weights measured at the cessation of the experiment, leaves were taken only from the same 15 trees at months 1, 2, 4, and 8. The remaining 15 undamaged trees were used to obtain dry weight measurements recorded at month 8.
Physiological Tests
Because leaf chlorophyll fluorescence and SPAD measurements are noninvasive and nondestructive, following each measurement the same evergreen oak and holly leaves were used to obtain electrolyte leakage values. All physiological measurements taken at months 1, 2, 4, and 8 were obtained on leaf material present on the plant at the initiation of the experiment (existing leaves). In addition, physiological measurements at month 8 only were also taken from newly formed spring leaf tissue (i.e., new leaves not present at the time of fertilizer application).
Chlorophyll Fluorescence
Immediately after the freezing treatment and 72 h post-salinity treatments, leaves were adapted to darkness for 30 min by attaching light exclusion clips to the leaf surface. Chlorophyll fluorescence was measured using a HandyPEA portable fluorescence spectrometer (Hansatech Instruments Ltd., King’s Lynn, UK). Measurements were recorded up to 1 s with a data acquisition rate of 10 μs 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 μmol m2/s photosynthetically active radiation (PAR) intensity provided by an array of six light-emitting diodes. The ratio of variable (Fv = Fm – Fo) to maximal (Fm) fluorescence (i.e., Fv/Fm, where Fo = minimal fluorescence) of dark adapted leaves and Fo values alone were used to quantify the detrimental effects of freezing on leaf tissue. An increase in Fo is characteristic of destruction of photosystem II reaction centers (Yamada et al. 1996). Fv/Fm is considered a quantitative measure of the maximal or potential photochemical efficiency or 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 (Meinander et al. 1996).
Chlorophyll Measurements
A Minolta chlorophyll meter SPAD-502 was used. Chlorophyll was measured 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 Unicam) after extraction with 80% v/v aqueous acetone (regression equation = 5.80 + 0.057x; r2 adj = 0.82, P = < 0.01) (Lichtenthaler and Wellburn 1983).
Leaf Electrolyte Leakage
Quantitative damage to leaf tissue (freezing treatment only) was assessed by measuring electrolyte leakage of entire leaves post-freezing. Excised leaves were placed in 50 mL (1.5 oz) Universal bottles containing 30 mL (0.9 oz) distilled water and gently shaken by hand. Samples were stored at 22°C (72°F) for 24 h in darkness prior to conductivity measurements taken with a Jenway conductivity probe and M4070 meter (BDH, Leicestershire, Loughborough, UK). Total solute leakage was obtained by autoclaving for 1 h at 121°C (249°F) and 0.103 MPa. Results are presented as percentage of solute leakage after 24 h (McKay 1992).
Dry Weights and Leaf Area
At the conclusion of the experiment, trees were destructively harvested. Leaf, shoot, and root dry weight were recorded after oven drying at 85°C (184°F) for 48 h. Root areas were quantified using a Delta-T area meter. Stem diameter was quantified using Manta blue precision calipers (Langsele, Haglof AB, Sweden) at one-third of the height of the stem and girth calculated using the equation C = ρD where C = circumference (girth), ρ = 3.14, and D = diameter. Height was recorded by measuring the distance from the tip of the leading apical shoot to the soil surface.
Data Analysis
Because measurements at months 1, 2, 4, and 8 were obtained from the same plant, the relationship (β1) between physiological measurements over time following freezing and salinity treatments was quantified using repeated measures analysis (regression). Significant effects, salient interactions, and whether β1 was significantly different from nonfertilized controls were determined by both two- and one-way analyses of variance (ANOVA) after checks for normality and equal variance distributions (Anderson-Darling test) were conducted. Differences between treatment means were separated by the least significance difference (LSD) at the 95% confidence level (P < 0.05) using the Genstat for Windows® program. The 2001 and 2002 data sets were not different when compared using a t-test; therefore, values presented represent data for 2001 trials only.
RESULTS AND DISCUSSION
A significant effect (P < 0.05) of species was recorded (Table 2). Following freezing, Fo regression values as a measure of damage to photosystem II reaction centers were 10% to 20% higher than following salt damage in both test species. Likewise, Fv/Fm regression values as a measure of photochemical efficiency and SPAD regression values as a measure of leaf chlorophyll content were 10% to 20% lower following freezing compared to salt damage for both species (Tables 3 and 4*). This finding indicates that a freezing temperature of −8°C (18°F) is more detrimental to leaf tissue than salt stress at a concentration of 8%. Similar results were obtained by Percival and Fraser (2001) when evaluating a range of Crataegus genotypes for their salinity and freezing tolerance. Higher Fo and leaf electrolyte leakage (freezing only) regression values (10% to 30%) and lower Fv/Fm and SPAD regression values of 10% to 30% following both salt and freezing stress in holly compared to evergreen oak (Tables 3 and 4) indicate that when selecting species for planting in areas where salinity and freezing temperatures are prevalent, evergreen oak should be chosen to help avoid replacement costs. Previous research investigating the freezing tolerance of a range of trees commonly planted in UK urban environments supports the conclusion that evergreen oak is a more freezing-tolerant species compared to holly (Percival and Henderson 2003).
Statistical analysis of variance for examination of salient effects and interactions on leaf chlorophyll fluorescence (Fo, Fv/Fm), chlorophyll concentration (SPAD), and electrolyte leakage.
The influence of Ca(N03)2, Ca(N03)2H3B03, and N:P:K (24:7:7) fertilization on the freezing tolerance of evergreen oak and holly. Leaves were detached at months 1,2,4, and 8 post-fertilization from trees growing under field conditions and subjected to temperatures of −8°C (18°F) under laboratory conditions. Improvements in tolerance as measured by chlorophyll fluorescence (Fo, Fv/Fm), leaf chlorophyll content (SPAD), and leaf electrolyte leakage values were quantified by repeated measures analysis (regression analysis; the slope value represented by β1).
The influence of Ca(NO3)2, Ca(NO3)2H3BO3, and N:P:K (24:7:7) fertilization on the salinity tolerance of evergreen oak and holly. Leaves were detached at months 1, 2, 4, and 8 post-fertilization from trees growing under field conditions and subjected to 8% salt (NaCl) exposure under laboratory conditions. Improvements in tolerance as measured by chlorophyll fluorescence (Fo, Fv/Fm) and leaf chlorophyll content (SPAD) values were quantified by repeated measures analysis (regression analysis; the slope value represented by β3).
Measurements of Fo and leaf electrolyte leakage were 50% higher, while measurements of Fv/Fm and SPAD values were 50% lower in newly formed spring leaf tissue (new leaves; Tables 5 and 6) sampled at month 8 compared to leaf tissue sampled at month 8 that was present at the time of fertilizer application (existing leaves; Tables 3 and 4), irrespective of form of stress applied and tree species (Tables 4 through 7). Previous research has shown that newly formed leaves produced in spring are highly susceptible to a range of environmental stresses such as freezing, heat, and salinity compared to older, more mature leaf tissue due to the new leaves’ inability to acclimate (Cannel and Smith 1986; Cameron and Dixon 1997).
The influence of Ca(NO3)2, Ca(NO3)2H3BO3, and N:P:K (24:7:7) fertilization on the freezing tolerance of spring foliage of evergreen oak and holly. Newly emerged leaves were detached at month 8 post-fertilization from trees growing under field conditions and subjected to −8°C (18°F) under laboratory conditions. Improvements in tolerance were measured by chlorophyll fluorescence (Fo, Fv/Fm), leaf chlorophyll content (SPAD), and leaf electrolyte leakage.
The influence of Ca(NO3)2, Ca(NO3)2H3BO3, and N:P:K (24:7:7) fertilization on the salt tolerance of spring foliage of evergreen oak and holly. Newly emerged leaves were detached at month 8 post-fertilization from trees growing under field conditions and subjected to 8% salt (NaCl) exposure under laboratory conditions. Improvements in tolerance were measured by chlorophyll fluorescence (Fo, Fv/Fm) and leaf chlorophyll content (SPAD).
The influence of Ca(N03)2, Ca(N03)2H3B03, and N:P:K (24:7:7) fertilization on growth of evergreen oak and holly at month 8 post-fertilization.
A significant effect of fertilizer and concentration applied was recorded (Table 1). Such a response is reflected in the results where, compared to nonfertilized controls, significantly lower (P < 0.05) Fo and electrolyte leakage regression values (20% to 40%), coupled with significantly higher (P < 0.05) Fv/Fm and SPAD regression values (30% to 40%), indicate that the freezing and salinity tolerance of evergreen oak and holly can be increased by fall application of Ca(NO3)2 and Ca(NO3)2H3BO3 at a concentration of 40 g/m2 (1.4 oz/ft2) (Tables 3 and 4). Results also show that increases in freezing and salinity tolerance were apparent in newly formed spring leaf tissue measured at month 8 (Tables 5 and 6). This finding indicates that fall fertilization with Ca(NO3)2 and Ca(NO3)2H3BO3 at 40 g/m2 can improve not only the freezing and salinity tolerance of existing leaf material through the winter months but also that of newly formed leaf tissue in spring.
Improvements in freezing and salinity hardiness of plants following calcium fertilization have been shown to be achieved via alterations to a number of plant physiological processes. The structural stability of cell walls and plasma membranes results from calcium links between phosphate and plasma lipids. Consequently, applications of calcium can physically increase the strength of the plant cell wall (Legge et al. 1982).
Calcium has also been implicated in controlling enzyme activity, important in improving freezing resistance. For example, cold temperatures increase levels of a calcium-dependent NAD kinase, which, in turn, is responsible for activating enzymes that cause the production of proteins necessary to alter the transcription of genes specific to cold acclimation. The progress of freezing injury can be also be halted by bathing or washing freeze-thaw–injured tissue in a calcium-based solution. (Monroy et al. 1993; Berbezy et al. 1996). Electrolyte leakage is widely used to measure freezing damage via alterations in membrane structural integrity (McKay 1992; Percival and Galloway 1999). Reduced electrolyte leakage values in trees fertilized with Ca(NO3)2 and Ca(NO3)2H3BO3 at a concentration of 40 g/m2 (1.4 oz/ft2) following freezing damage indicate increased membrane structural stability and cell wall strength caused by calcium fertilization in both test species. Importantly, improvements in freezing and salinity tolerance were recorded by month 1 (Tables 3 and 4), indicating calcium fertilization works rapidly to induce stress resistance in both evergreen oak and holly. Increased tolerance to salinity and freezing stress as a result of calcium fertilization at 40 g/m2 may also have contributed toward the significantly increased (P < 0.05) plant dry weights recorded at the cessation of the experiment (Table 7).
Although Fo and electrolyte leakage regression values were generally lower in both test species supplemented with Ca(NO3)2 and Ca(NO3)2H3BO3 at a concentration of 20 g/m2 (0.7 oz/ft2) and Fv/Fm, SPAD regression values and plant dry weights were generally higher compared to controls; in few instances were they significantly so (Tables 3 through 7). This finding indicates that applications of calcium-based fertilizers at 20 g/m2 conferred no significant protectant properties in the two test species. Likewise, applications of both calcium fertilizers at a concentration of 80 g/m2 (2.8 oz/ft2) had no significant effect on freezing and salinity tolerance or plant dry weights (Tables 3 through 7). Symptoms of fertilizer burn were observed in both tree species at this concentration (marginal necrosis of the leaf peripheral edges) indicating phytotoxicity of both calcium fertilizers at 80 g/m2, which may account for this result.
Irrespective of species, there was no marked difference in freezing and salinity tolerance and dry weight values between trees treated with Ca(NO3)2 and Ca(NO3)2H3BO3 at a concentration of 40 g/m2 (Tables 3 through 7). Both fertilizers are commercially available at a cost of US$288 (£160) for Ca(NO3)2 and $US304 (£169) for Ca(NO3)2H3BO3 per metric ton at farm gate prices. Such knowledge may prove of importance to those involved in urban tree care, when the cost–benefit ratio involved with calcium fertilization needs to be considered.
Nitrogen fertilization has been shown to reduce winter hardiness in apple (Way 1953; Edgerton 1957), pear (Raese 1997), and peach (Proebstring 1961). Contrary to these findings, work elsewhere suggests nitrogen fertilization can enhance winter hardiness in Picea ruben (Dehaynes et al. 1998) and Juniperus chinensis (Pellet and White 1969), while Smiley and Shirazi (2000) suggest nitrogen fertilization has no significant effect on winter hardiness in Forsythia, maple (Acer spp.), Leyland cypress (× Cupressocyparis leylandii), and oak (Quercus spp.).
Irrespective of species, chlorophyll fluorescence (Fo, Fv/Fm) and electrolyte leakage regression values did not significantly differ from controls following freezing and salinity stresses following fall fertilization with a high-nitrogen (N:P:K 24:7:7) fertilizer (Tables 3-7). This result indicates that fertilization of evergreen oak and holly with a N:P:K (24:7:7) fertilizer did not enhance or increase susceptibility to freezing and salinity damage. However, significantly higher SPAD regression values as a measure of leaf chlorophyll concentration and overall plant dry weights were recorded (Tables 3 through 7).
Leaf nitrogen status has been shown to be highly correlated with leaf chlorophyll concentration (i.e., “greenness”) and yield in a range of plants (Evans 1983; Seemann et al. 1987; Hoel 1998). Higher leaf nitrogen content as a result of N:P:K (24:7:7) fertilization in both evergreen oak and holly may account for the improved leaf color and total plant dry weights recorded. Consequently, results of this investigation indicate that although application of a N:P:K (24:7:7) fertilizer induced no significant freezing and salinity protectant properties in evergreen oak and holly, leaf color and overall growth were significantly (P < 0.05) better in both species compared to nonfertilized controls. Similar results have been obtained elsewhere using pear (Pyrus spp.) as a test species (Raese 1997).
In conclusion, previous investigations have shown that calcium fertilization can improve the salinity and freezing tolerance of apple and pear trees cultivated under orchard conditions (Raese 1996); trees used in urban landscapes such as hornbeam (Carpinus spp.), and white poplar (Populus alba) (Percival et al. 1999); fruit and vegetables such as beans, potatoes, avocados, mangoes, cherries; and various grass species (Akhavankharazian et al. 1991; Anderson and Campbell 1995; Palta 1996). The present study offers further evidence in this respect.
Acknowledgments
The authors are grateful for funding from the TREE Fund (John Z. Duling grant program).
- © 2005, International Society of Arboriculture. All rights reserved.
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