The Influence of Sodium Chloride and Waterlogging Stresses on Alnus cordata

Plant material

The experiment used 4-year-old Italian alder, 1.5 to 1.7 m high, which had been grown for 2 years in a seed bed and then transferred into plastic pots (26 cm diameter by 23 cm deep) filled with compost. The soil was loamy in texture (24% clay, 45% silt, 31% sand), 3.1% organic carbon, pH 6.6, and supplemented with the controlled release nitrogen-based fertilizer Enmag (Salisbury House, Weyside Park, Goldmar, Surrey, UK) at a rate of 1 g/kg compost. The experiment started 6 weeks after leaf fall, when trees were deemed to be dormant, on January 19,1996 (week 0). Trees were placed in an unheated transparent polyethylene tunnel at the Department of Horticulture, SAC Auchincruive, Ayr, Scotland, continuing until March 1 (week 6). Thereafter the trees were placed under glass and flushed with tap water to remove excess salt that may have accumulated in the compost. Growing conditions under glass were 22°C ± 2°C (71.6°F ± 35.6°F)supplemented with 400-W high-pressure sodium lamps (SON/T) providing a photoperiod of 16 h light and 8 h dark and 250 μmol m^-2 s^-1 photosynthetically active radiation (PAR) at the tree crown. The experimental design used was a completely randomised block with 12 replications per treatment.

During the 6-week period in the tunnel, the treatments were 1) distilled water, 2%, 4.5%, and 7% sodium chloride solutions applied as weekly drenches to trees growing in freely drained compost until solutions were observed emerging from drainage holes; and 2) waterlogging in distilled water, 2%, 4.5%, and 7% sodium chloride solutions for the 6 weeks’ duration. In the case of treatment no. 1, the compost water content was adjusted daily to maintain it between 75% and 100% of pot capacity by weighing the freely drained pots and flushing with tap water to prevent salt accumulation in the compost. Waterlogging was performed by placing the 26-cm-diameter pots inside larger 28-cm-deep plastic buckets and flooding until 2 cm of water overlaid the compost surface. This depth was maintained by inspecting daily and watering when necessary. Trees growing in freely drained pots and drenched with distilled water were used as controls. During the 6-week period, the minimum and maximum air temperatures were –16.2°C and 8.4°C (2.8°F to 47.1°F), respectively, while minimum and maximum daily relative humidity varied between 24.3% and 64.6%. Irradiance was measured daily at 1300 h using infrared gas analysis and averaged 139 μmol/m²/s PAR. All pots were widely spaced, thereby reducing competition for light.

Analytical techniques: Bud burst

The time to bud burst following treatments (i.e., from March 1 onwards), defined as green leaves emerging from the scales (Cannell and Smith 1983), was recorded visually.

Chlorophyll fluorescence

Before measurements, leaves were dark adapted for 40 min by attaching light exclusion clips to leaf surfaces in situ. Chlorophyll fluorescence measurements were obtained with a portable fluorescence spectrometer (Hansatech Instruments Ltd, Kings’ Lynn, UK). Eight leaves were randomly selected per tree and each leaf tagged, ensuring that measurements came from the same leaf throughout the experiment. Readings were obtained at 3-day intervals at 1300 h until day 15. In all cases, chlorophyll fluorescence measurements refer to the Fm/Fv ratios, which represent the maximum quantum yield of Photosystem II, which in turn is highly correlated to the quantum yield of net photosynthesis—providing a rapid quantitative measurement to determine plant response to stress (Demmig and Bjorkman 1987; Bolhar-Nordenkampf et al. 1989; Adams et al. 1995).

Leaf necrosis

Assessments of sodium chloride and waterlogging damage to leaves were estimated visually on a 1 to 6 scale (0 = no necrosis; 1 = < 1% necrosis; 2 = 1–10% necrosis; 3 = 10–25% necrosis; 4 = 25–50% necrosis; 5 = 50–75% necrosis; 6 = > 75% necrosis). Assessments were made at 3-day intervals until day 15.

Cell electrolyte leakage

At 3, 6, 9, 12, and 15 days after bud burst, quantitative damage to leaf cell tissue was assessed by measuring electrolyte leakage. Leaves were excised and placed in 30 mL Universal bottles containing 20 mL distilled water. Samples were stored at 22°C for 24 h in darkness before conductivity measurements were taken with a Jenway conductivity probe and M4070 meter (BDH, Loughborough, UK). Total solute leakage was obtained by autoclaving for 1 h at 121 °C (249.8°F) and 0.103 MPa. Results are presented as percentage of solute leakage after 24 h.

Inductively coupled plasma-emission spectroscopy (ICP) elemental analysis

Root, shoot, and leaf samples were thoroughly washed, then dried in a convection oven at 85°C for 48 h before grinding through a 0.5 mm cyclone mill (Retsch, Middlesborough, UK). Samples (0.5 g) were placed into 150 mL volumetric flasks and digested in 20 mL of 7:1 nitric/perchloric acid. After cooling, the solutions were brought to volume with deionized water and analysed by ICP. Nutrient values were expressed as g/kg dry weight.

Statistical analysis

Treatment effects were determined by analysis of variance (ANOVA) using the Genstat 5 program (Lawes Agricultural Trust 1990). Differences among treatment means were separated by the least significance difference (LSD) at the 0.05 level of probability.

Time to bud burst

Applications of sodium chloride at higher concentrations tended to increase time to bud burst. These effects, however, were not significant compared to controls. Mean bud burst ranged from 57.2 to 68 days for trees in freely drained substrates and from 57.7 to 70 days in waterlogged compost.

Mortality

By day 15 after bud burst, all trees watered with sodium chloride solutions ≥ 4.5% or waterlogged in sodium chloride solutions ≥ 2% were dead. Watering or waterlogging with distilled water resulted in no mortality, while 2/3 of trees watered with a 2% NaCI solution died.

Chlorophyll fluorescence, cell electrolyte leakage, leaf necrosis

Irrespective of whether trees were grown in freely drained soil or waterlogged in salt-amended solutions, exposure of the roots to sodium and chloride ions decreased leaf chlorophyll fluorescence (Figure 1) and increased cell electrolyte leakage (Figure 2) and leaf necrosis (Figure 3), from days 0 to 15. Significant reductions in chlorophyll fluorescence were detected by day 3 (P < 0.05), with significant increases in cell electrolyte leakage and leaf necrosis occurring by day 6. The greatest severity of effects on chlorophyll fluorescence, cell electrolyte leakage, and leaf necrosis resulted from drenching trees grown in freely drained compost with 4.5%, 7%, and 7% NaCI, respectively (P < 0.01).

Root and shoot macronutrient concentrations

Applications of sodium chloride significantly increased (P< 0.01) sodium and chloride concentrations in root (Table 1) and shoot tissue (Table 2) of trees grown in freely drained soil or waterlogged in salt-amended solutions. Higher root and shoot sodium and chloride concentrations reflected applications of stronger NaCI solutions.

Greater amounts of sodium were found in the shoots of trees waterlogged in a 7% NaCI solution and in the roots of trees watered with a 7% NaCI solution grown in freely drained compost, respectively. Maximal concentrations of chloride were recorded in the shoots of trees watered with a 7% NaCI and in roots of trees waterlogged in a 7% NaCI solution, respectively. Minimal foliar and root concentrations of each element were apparent in trees not subjected to salinity.

No treatment effects upon shoot and root calcium, phosphorous, magnesium, and potassium concentrations were identified (data not shown). Irrespective of treatment, no significant effects upon copper concentrations in root tissue were recorded. Significantly reduced concentrations (P < 0.01) of copper were, however, recorded in shoot tissue with greater reductions reflected by stronger NaCI applications. Shoot concentrations of zinc and iron were significantly lower (P < 0.05) in trees waterlogged in NaCI solutions ≥ 4.5% only. Irrespective of treatment, no significant effects upon manganese concentrations in shoot tissue were recorded. Significantly reduced concentrations (P < 0.01) of manganese were, however, recorded in roots with greater reductions generally reflected by stronger NaCI applications.

Discussion

Applications of sodium chloride to roots resulted in high mortality rates by day 15 after bud burst. These results highlight the importance of a more rigorous approach to quantifying salt tolerance of species through the use of NaCI applications to both foliage and roots. Previous experimentation demonstrated A. cordata to be highly tolerant to foliar salt spray even under waterlogged conditions (Percival and Dixon 1997). Similar phenomena have been recorded with Acer platanoides, which was classified as salt tolerant by Lumis et al. (1973) and Sucoff (1975) but as salt sensitive by Schiechtl (1978) and Kreutzer (1977), Similarly, Thuja occidentalis was reported as tolerant of salt applied to roots but sensitive to foliar salts (Lumis et al. 1973; Sucoff 1975), and Juglans nigra is reported to be sensitive to salt in the soil but reasonably tolerant of sprays (Monke and Weibe 1961; Monk and Peterson 1962; Leh 1975).

Data for a range of trees (Dobson 1991) suggest that salt toxicity resulting in visual leaf necrosis develops at chlorine contents greater than 1% oven dry weight for broadleaves and circa 0.5% for conifers. There are few reports that correlate such treatments with tree death. Because 2/3 of trees watered with a 2% sodium chloride solution in this investigation died, the tolerance threshold for sodium and chloride ions in roots and shoots of A. cordata was 5.77 and 5.39, and 13.0 and 16.1 g/kg dry weight, respectively.

Regardless of drainage, applications of sodium and chloride ions to roots decreased leaf chlorophyll fluorescence and increased cell electrolyte leakage and leaf necrosis from days 3 and 6, respectively. Chlorophyll fluorescence identifies stress injury before it becomes visible and thus provides a rapid method for screening plants in breeding programmes (Greaves and Wilson 1987). The technique has been used to evaluate responses of crop species to detrimental influences such as freezing, salinity, and drought. It can provide rapid quantitative assessment with which to rank species depending on their sensitivity. Chlorophyll fluorescence may also have implications for arboriculturists (Percival and Dixon 1997). Although established techniques are available to detect tree decline due to biotic causes, there are few that detect abiotic stress in situ. Early detection of stress, before obvious physical deterioration, may allow effective remedial action. However, it is debatable whether detection of stress by chlorophyll fluorescence would occur early enough for remedial action to be effective. Results do highlight a potential use of chlorophyll fluorescence as a diagnostic system for the utility industry with which to detect stress in trees (Percival and Dixon 1997).

Sodium and chloride ions influence the uptake and internal distribution of other nutrients. Results elsewhere suggest that they decrease the levels of tissue calcium, magnesium, potassium, and phosphorous (Stavarek and Rains 1983). Other studies indicate that waterlogging alone reduced the uptake and transport of nitrogen, phosphorous, potassium, calcium, and iron (Mclean 1993). Townsend (1984), however, demonstrated that essential elements present in stem and leaf tissue of 6 tree species increased in response to applications of sodium chloride. Few studies have analysed the combined effects of salinity and waterlogging on uptake and translocation of nutrients (Farrell et al. 1996).

Results presented here demonstrate an increase in the root concentration of iron and manganese for trees waterlogged with distilled water only. An adaptation to waterlogging involves the internal transport of oxygen within the plant. Oxygen is absorbed by aerial tissue and translocated via the phloem to the roots. There it diffuses into the rhizosphere, preventing an accumulation of toxic compounds such as hydrogen sulphide (Kozlowski et al. 1991). Trees reported to possess this ability include Populus x petrowskiana, Salix alba, S.fragilis, and S.atrocinereal (Kozlowski et al. 1991). Following this adaptation, the roots became coated by minerals (Fisher and Stone 1991), primarily iron and manganese oxides (Ding and Musgrave 1995). Results indicated such a metabolic adaptation in response to waterlogging by A. cordata. An increase in these elements was not observed, however, in the presence of sodium and chloride ions. Indeed iron, manganese copper, and zinc concentrations were, on occasion, reduced compared to controls, suggesting salt inhibited such a metabolic adaptation.