Seedlings of the mangrove, Avicennia marina, were used in two nitrogen nutrition experiments, carried out at the Australian Institute of Marine Science in a shadehouse, where incident light was reduced to about one-third of full sunlight. Propagules were placed in wet sand and when at least 4 leaves and a healthy root system had developed, seedlings of similar initial fresh weights were chosen for experiments.In the first experiment, seedlings were grown in anaerobic soil collected from the intertidal mangrove zone at Hinchinbrook Island. The soil was stored wet and thoroughly blended before use. Four soil samples were tested initially to measure the mean extractable ammonium-N level and to determine if it was evenly distributed throughout the soil. Seedlings were placed in 12 of 20 PVC pots (8 cm diameter x 14 cm high) and approximately 400 ml of soil slurry, with a 75% water content was added to all pots, which was sufficient to cover the roots of each plant. After allowing the soil to dry sufficiently to absorb treatment solutions, a total of 10 ml of 15N enriched ammonium sulphate (77.6 atom% 15N) solution containing 2.50 mg of ammonium-N was injected into the soil in each pot, at several different positions to ensure uniformity and to prevent leakage to the soil surface. A nitrification inhibitor, N-serve (2-Chloro-6-trichloromethyl pyridine was also added to 10 of the pots (6 with plants), the amount added corresponding to a final concentration of 5 ppm in the soil water. Similar quantities of N-serve were added at 14 to 18 d intervals to maintain efficient nitrification inhibition. After all injections, the soil surface was tamped down and distilled water was added at frequent intervals to maintain a level of 3 to 5 mm above the soil surface. Redox probes were placed in 4 of the pots (1 in each treatment type: plant + soil, soil only and with/without N-serve) to measure Eh throughout the experiment.The experiment was terminated 63 days after 15N additions. The aboveground plant portions were removed and dried to constant weight at 60°C. The soil was washed away from the roots, which were added to the corresponding upper plant portions. Following the addition of 300 ml of 4 M KCl, the soils were shaken for 1 hour and the extract solution cleared by filtration. The dried, ground soil residue was analysed for total N and 15N and the extract solutions analysed for ammonium, nitrate and nitrite and 15N. The dried, ground plant samples were analysed for total N and 15N. A Micromass 622 isotope-ratio mass spectrometer was used to determine the 15N enrichment of the nitrogen gas produced by reaction of the dried ammonium salts with lithium hypobromite.In the second experiment, seedlings were grown in continuously aerated solutions containing 25% seawater and all essential primary and trace nutrients except nitrogen. Nitrogen was supplied to the plants as ammonium sulphate or potassium nitrate, each at 4 different levels: 5, 10, 30 and 50 ppm N. Each treatment group contained 5 replicate seedlings giving a total of 40 seedlings. The nitrification inhibitor, N-serve was added to all solutions at the 5 ppm level. Solutions were changed every 2 weeks and between changes were monitored for ammonium, nitrate and nitrite content at 2 to 3 day intervals. After 158 days of treatment, the plants were harvested for fresh and dry weight determinations of leaves, stems and roots. Total leaf area per plant was measured using a Licor leaf area meter. The dried plant components were finely ground in an agate mill and analysed for total N using a LECO CHN analyser.
Laboratory experiments were undertaken to investigate the role of nitrate uptake in the nitrogen nutrition of plants growing in anaerobic soils. The mangrove Avicennia marina was chosen due to its widespread occurrence in Australian mangrove systems, its ease of propagation and its tolerance of highly anaerobic, perpetually flooded, soils.
