The role of nitrate in nitrogen nutrition of the mangrove Avicennia marina in North Queensland

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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.

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Statement: Statement: The soil used in the first nutrition experiment is fully described in:Boto KG and Wellington JT (1984) Soil characteristics and nutrient status in a northern Australian mangrove forest. Estuaries 7: 61-69.The soil is a fine silt containing about 20 % organic matter, 0.3 % total nitrogen and 0.03 % total phosphorus on a soil dry weight basis. Previous field experiments have shown that the mangroves at this site responded positively to soil ammonium enrichment but not to superphosphate addition. Boto KG and Wellington JT (1983) Phosphorus and nitrogen nutritional status of a northern Australian mangrove forest. Mar Ecol. Prog. Ser. 11: 63-69.The procedures used to inhibit nitrification are described in:Sahrawat KL and Mukerjee SK (1977) Nitrification inhibitors. I. Studies with Karanjin, a furanoflavonoid from Karanja (Pongamia glabra) seeds. Plant Soil 47: 27-36.The Kjeldahl digestion and steam distillation methods used for analyses are described in:Bremmer JM (1965) Total nitrogen. In: Black CA (ed.) Methods of soil analysis. Agron. 9. American Society of Agronomy, Madison, Wisconsin, USA, p 1149-1178.Bremmer JM and Keeney DR (1966) Determination and isotope ratio analysis of different forms of nitrogen in soils. 3: Exchangeable ammonium, nitrate and nitrite by extraction-distillation methods. Soil Sci. Soc. Am. Proc. 30: 577 582.Saffigna PG and Waring SA (1977) Prevention of 15N cross contamination during distillation and potentiometric titration of 15N-labelled samples. Analylica chim. Acta 89: 203-207.The procedure used to determine the 15N enrichment of the nitrogen gas produced by reaction of the dried ammonium salts with lithium hypobromite is described in:Ross PJ and Martin AE (1970) A rapid procedure for preparing gas samples for nitrogen-15 determination. Analyst (Lond.) 95: 817-822.Experiment 2:Despite constant aeration, nitrate losses were relatively severe, equivalent to about 5 ppm every 2 to 3d. Ammonium loss was less severe, although occasional additions to those solutions were also required to maintain the nominal treatment levels. The nitrate and nitrite levels in all ammonium solutions was consistently less than 0.1 ppm N.

Notes

Credit
Boto, Kevin G, Dr (Principal Investigator)