Evaluation of Physiological Tests as Predictors of Young Tree Establishment and Growth

Plant Material and Experimental Design

Four-year-old bare-root birch (Betula pendula), evergreen oak (Quercus ilex), and holly (Ilex aquifolium) were obtained from a commercial supplier on 10 December 2001. Immediately upon arrival and to ensure uniformity of stock for experimental purposes, trees were graded, and only those confirming to the physical characteristics specified in Table 1* were used. Trees were then sorted into bundles of 40 (Q. ilex, I. aquifolium) and 55 (B. pendula). Forty bundled trees for Q. ilex and I. aquifolium [25 for physiological tests (10 for stem electrolyte leakage and 15 for RGP and chlorophyll fluorescence) and 15 for a field growth trial] and 55 for B. pendula (15 extra for chlorophyll fluorescence analysis) were sealed in plastic bags, placed inside larger paper bags, and stored at 6°C ± 0.5°C (43°F ± 1°F), a standard storage temperature for trees in the United Kingdom, in a refrigerated cold store in darkness.

Freezing Treatments

Following 6 weeks at 6°C ± 0.5°C (43°F ± 1°C), darkstorage trees were removed from cold store (21 January 2002) and placed in a Merck environmental freezing chamber and the temperature reduced by 2°C h⁻¹ (3.8°F h⁻¹) from 10°C to 0°C, −2°C, −4°C, −6°C, −8°C, and −10°C (50°F to 32°F, 28°F, 25°F, 21°F, 18°F, and 14°F). Each respective temperature was maintained for 4 hours, after which the temperature was raised by 2°C h⁻¹ to 10°C. There was one bag of 40 (Q. ilex, I. aquifolium) and 55 (B. pendula) young trees per species for each respective temperature treatment. Trees stored at 6°C served as controls.

Physiological Tests

Statistical Analysis

Stem electrolyte leakage (10 replications), chlorophyll fluorescence (45 replications, Q. ilex and I. aquifolium; 30 replications, B. pendula), and RGP (15 replications), as well as significant interactions between freezing and species on each parameter, were determined by both two- and one-way analyses of variance (ANOVA) following checks for normality and equal variance distributions. Dunn’s or Dunnett’s tests (for unequal and equal sample sizes, respectively) were then used to compare the six freezing treatments to the control values using the Genstat V program.

Tolerance of Young Trees to Freezing

Post-freezing Fv/Fm was reduced to between 62.5% and 91.9% of the control values depending on species (Table 2). Significant differences (P < 0.05) in the reduction of Fv/Fm were recorded between species, with birch appearing to be the most resistant to freezing of the three species tested and holly the most freezing sensitive. Freezing also had a significant effect (P < 0.05) on stem electrolyte leakage, with values reduced from 88% to 95.7% of controls (Table 2). Reductions in stem electrolyte leakage reflected species tolerance based on Fv/Fm values, with birch again appearing to be the most freezing resistant and holly the most sensitive. Irrespective of species, RGP was markedly reduced 28 days post-freezing under glasshouse conditions. The greatest reduction in RGP, compared to the control values, was recorded in evergreen oak (84.3%), followed by birch (78.9%) and holly (77.4%). Irrespective of species, little variation existed between stem electrolyte leakage values of controls (ranging from 2.16% to 2.79%). Likewise, pre-treatment Fv/Fm values of evergreen oak and holly were 0.795 and 0.790, respectively. However, pre-treatment values for birch were markedly lower (0.681), possibly as a result of differences in Fv/Fm values between leaf and periderm tissue used for chlorophyll fluorescence measurements. Large variability in pre-treatment RGP values between species was recorded. Values ranged from 10.13 (birch) to 7.07 (holly) to 4.27 (evergreen oak). Irrespective of species, significant detrimental effects (P < 0.05) on Fv/Fm, RGP, and stem electrolyte were recorded following freezing at ≥ −2°C (28°F). A significant influence (P < 0.001) of species, freezing, and species × freezing interaction on the variates Fv/Fm, RGP, and stem electrolyte was recorded in this investigation (Table 2).

Physiological responses and growth attributes of freezing on birch (Betula pendula), holly (Ilex aquifolium), and evergreen oak (Quercus ilex) trees.

Seventeen weeks post-freezing under field conditions, height increment was reduced by 55.1%, 85.2%, and 85.1% of the control values in birch, holly, and evergreen oak, respectively (Table 2). Likewise, foliar damage 17 weeks post-freezing was increased to between 92.3% and 95.2% of the control values, depending on species (Table 2). Significant effects on height increment and foliar damage were manifest post-freezing at ≥ −2°C (28°F) (Table 2). Effects on survival were recorded post-freezing at −4°C (25°F) for birch and evergreen oak, and at −6°C (21°F) for holly. Freezing tolerance based on survival 17 weeks post-freezing at −10°C (14°F), however, was in the order of birch (47.7), evergreen oak (27.0), and holly (6.7). Irrespective of species, little variation existed between foliar damage values of controls (ranging from 2.4% to 6.7%). Likewise, survival rates in control trees of all three species tested were 100%. Large variability in height increment between controls of each species was recorded. Values ranged from 182.13 (birch) to 83.84 (evergreen oak) to 73.23 (holly).

Predictive Ability of Physiological Tests

All physiological tests of young trees measured after freezing were highly correlated to height increment, foliar damage, and survival under field conditions for 17 weeks (Table 3 and Figures 1–3). Stem electrolyte leakage proved to be the best predictor of foliar damage for all three species (r² = 0.96), whereas relationships between chlorophyll fluorescence and survival were strongest for all three species, with a mean r² of 0.94 (Table 3). RGP proved to be the best predictor of height increment for all three species (r² = 0.85). Of the three physiological tests considered, (chlorophyll fluorescence, RGP, and stem electrolyte leakage), correlations of field performance with stem electrolyte leakage had the highest mean r² values (0.93 to 0.63; Table 3).

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Figure 1.

Relationship of chlorophyll fluorescence (Fv/Fm) with survival, foliar damage, and height increment of birch (Betula pendula), holly (Ilex aquifolium), and evergreen oak (Quercus ilex) trees at week 17 post-freezing under field conditions.

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Figure 2.

Relationship of root growth potential (RGP) with survival, foliar damage, and height increment of birch (Betula pendula), holly (Ilex aquifolium), and evergreen oak (Quercus ilex) trees at week 17 post-freezing under field conditions.

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Figure 3.

Relationship of stem electrolyte leakage with survival, foliar damage, and height increment of birch (Betula pendula), holly (Ilex aquifolium), and evergreen oak (Quercus ilex) trees at week 17 post-freezing under field conditions.

Chlorophyll fluorescence was highly correlated to foliar damage, height, and survival in all species (Figure 1, Table 3). Values for r² ranged from 0.94 (holly) to 0.98 (birch and evergreen oak) for survival; 0.98 (birch) to 0.99 (holly and evergreen oak) for foliar damage, and 0.96 (holly) to 0.99 (birch and evergreen oak) for height increment (Figure 1). Highly significant relationships between chlorophyll fluorescence and all three performance attributes were obtained using quadratic polynomial and linear regression models (Figure 1). For the quadratic polynomial curves of RGP versus survival, r² ranged from 0.93 for evergreen oak to 0.98 for birch and holly (Figure 2). Likewise, for the quadratic polynomial curves of RGP versus foliar damage, r² ranged from 0.96 for evergreen oak to 0.99 for holly. Linear and quadratic polynomial curves were used to describe the relationship of height increment to RGP; in these cases, r² ranged from 0.94 (evergreen oak) to 0.99 (holly; Figure 2). Stem electrolyte leakage was highly correlated to foliar damage, height, and survival in all species using quadratic polynomial and linear regression models (Figure 3, Table 3). Values for r² ranged from 0.95 (birch) to 0.99 (evergreen oak) for survival; 0.95 (birch) to 0.99 (holly and evergreen oak) for foliar damage, and 0.94 (birch) to 0.99 (holly) for height increment (Figure 3).

Tolerance of Young Trees to Freezing

Current nursery practices in Europe and North America rely on the rapid production of immature woody plants grown in small containers. Such intensive husbandry renders the plants very susceptible to stress-induced injury. Freezing temperatures, especially late spring frosts, can be devastating to leaf tissue of woody plants and are a major source of injury resulting in reduced plant quality and often death (Cannell and Smith 1983; 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). Selection criteria against freezing damage will become more important because climatic change may increase the unpredictability of weather patterns, resulting in progressively later frosts on an annual basis (Biggs 1996). Survival rates among the three test species were not affected until a freezing stress of ≥ −4°C (25°F) (birch and evergreen oak) and −6°C (21°F) (holly) was imposed (Table 2). Freezing tolerance based on survival 17 weeks post-freezing at −10°C (14°F), however, was in the order birch > evergreen oak > holly. This finding indicates that holly is better able to withstand moderate freezing stress compared with birch and evergreen oak, yet following severe freezing, damage birch and evergreen oak are more ideally suited.

Predictive Ability of Physiological Tests

All physiological tests of young trees used in this investigation were highly correlated to height increment, foliar damage, and survival under field conditions at week 17 post-freezing (Table 3 and Figures 1–3). Of the three physiological tests considered, correlations of field performance with stem electrolyte leakage had the highest mean r² values (Table 3) and, therefore, based on results of this investigation, were marginally better as a predictor of future plant performance compared to chlorophyll fluorescence and RGP. This trial found a strong relationship between growth and RGP, stem electrolyte leakage, and chlorophyll fluorescence, and is one of few investigations to use these measures as predictors of plantation establishment success in a field scenario that is more representative of landscape conditions. Likewise, young 2 + 2 transplants are generally used in preference to seedling material when establishing amenity plantings; therefore, they were used in this trial instead of seedling material as commonly used in other investigations (McCreary and Duryea 1987; McKay 1992, 1994; Simpson and Ritchie 1997).

Stem electrolyte leakage indicates tissue damage resulting in the loss of cell membrane semi-permeability (Cameron and Dixon 1997). Advantages of stem electrolyte leakage as a system to measure plant vitality include the simple nature of the task along with a requirement of only 48 hours to complete (Ketchie et al. 1972). Practical disadvantages of stem electrolyte leakage measurements include a requirement for laboratory facilities and the destructive nature of the assay (Figueiredo 1985). Although measurements of stem electrolyte leakage were used as predictors of future growth responses in this investigation, fine root electrolyte leakage is the more commonly used test to determine plant vitality (McKay and Mason 1991; McKay 1992, 1994). Root electrolyte leakage was not considered in this investigation due to the practical difficulty of collecting root samples from trees in urban sites where roots frequently grow beneath roads and pavements. The fact that stem tissue can be obtained at any time means estimates of vitality can be determined through out the year if required.

RGP is probably the most widely used method to assess seedling viability and vigor (McCreary and Duryea 1987; Dunsworth 1997). The test is inexpensive to conduct if a controlled growth environment is available. However, RGP testing is destructive and, as in this study, requires 28 days to obtain results, although a 14-day test has provided meaningful results elsewhere (Maki and Columbo 2001). In addition, RGP varies by species, so that a database of acceptable levels of performance is usually a prerequisite to using this test operationally to assess young tree condition. Indeed, in the current study, the highest mean RGP for birch was 10.1 (new roots), while for evergreen oak it was 4.7. Despite these differences in magnitude among the species tested, RGP had the highest predictive ability for height increment of the three physiological tests evaluated (Table 3 and Figures 1–3). Consequently, this gives cause to consider the operational use of RGP to grade stock according to performance potential prior to leaving a nursery and planting in situ. Numerous reports exist of positive relationships between RGP and seedling growth and survival after planting (Simpson and Ritchie 1997), and the present study offers further evidence in this regard. Nevertheless, the view has been stated that tests such as RGP should not be used to predict field performance because correlations with field performance are at times poor or absent (Binder et al. 1988; Simpson and Ritchie 1997).

Chlorophyll fluorescence is a method for studying how environmental factors influence photosynthetic efficiency (Lichtenthaler and Rinderle 1988). The chlorophyll fluorescence measurement Fv/Fm has been shown to be a useful estimator of photosystem II quantum efficiency and an indirect measurement of plant physiological status (Maxwell and Johnson 2000). In this investigation, relationships between chlorophyll fluorescence and survival were strongest for all three test species (Figure 1, Table 3), a result supported elsewhere (Maki and Columbo 2001). Fv/Fm measurements of healthy, nonstressed plants are associated with values ranging from 0.78 to 0.85 (Demmig and Björkman 1987). However, results here and by Maki and Columbo (2001) indicated a threshold fluorescence value of about 0.6 above which trees were unaffected in survival, height growth, and foliar damage (Table 2, Figures 1–3). Although the physiological principles of chlorophyll fluorescence are complex, the operation of the fluorimeter used in this trial is simple. The practical advantages of using chlorophyll fluorescence include the fact that fluorescence measurements use a light, portable piece of equipment; measurements are nondestructive and noninvasive; and readings are obtained in less than 1 second, allowing for many trees to be evaluated in a single day. The fluorescence value Fv/Fm has been shown to be a sensitive and rapid method of assessing woody tissue viability in birch following freezing stress and can be used as a reliable predictor of foliar damage, growth, and survival under field conditions. However, this work was conducted on one species of one genus and, although preliminary results are promising, further work is needed to assess the applicability and limits of this method for other ornamental tree species. One of the major limitations of fluorescence analysis at present is the initial cost of the fluorimeter, which can retail at more than US$5,000.

The strong relationship between two of the physiological tests (stem electrolyte leakage and chlorophyll fluorescence) and survival of all three test species in this investigation and conifer species elsewhere (Maki and Columbo 2001) may have other implications for professionals involved in urban tree management. Stress detection in woody plants relies heavily on visual observations of, for example, leaf yellowing, crown dieback, and other factors. Such indicators can be subjective because they are based on human knowledge and interpretation, which can differ among individuals. Consequently, tree health evaluations can differ markedly among assessors. Use of stem electrolyte leakage and/or chlorophyll fluorescence may aid in the decision-making process by quantifying the odds of survival following a prolonged period of stress such as drought or waterlogging, in turn determining the economic feasibility of applying remedial measures or tree removal. Further research in this area is warranted using a wider range of species at different stages of maturity so that “thresholds of concern” can be identified before symptoms of physical deterioration are observed, which, if ignored may, with time, make the tree a potential danger to people and property.

In conclusion, RGP, chlorophyll fluorescence, and shoot tip electrolyte leakage were all able to predict when freezing temperatures caused damage and reduced tree vigor. A high correlation between chlorophyll fluorescence obtained from woody tissue and foliar damage, height, and survival indicates a useful potential of this system to evaluate the vitality and predict the future performance of deciduous ornamental stock that has been subjected to freezing stress. The ability of all three physiological tests to predict damage and subsequent growth supports the continued use of these measures as operational tools to measure plant viability.