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

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.

本试验以白骨壤（Avicennia marina）幼苗为材料，于澳大利亚海洋科学研究所（Australian Institute of Marine Science）的荫棚内开展两项氮营养相关实验，实验环境中将入射光强度降至全光照的约1/3。红树繁殖体（propagules）被置于湿沙中培养，待其长出至少4片叶片并形成健康根系后，选取初始鲜重相近的幼苗用于试验。在第一项试验中，幼苗种植于从欣钦布鲁克岛（Hinchinbrook Island）潮间带红树带采集的厌氧土壤（anaerobic soil）中，土壤在使用前保持湿润并充分混匀。首先对4份土壤样品开展初始检测，以测定平均可提取态铵态氮（ammonium-N）水平，并确认其在土壤中分布均匀性。将幼苗栽种于20个PVC盆（直径8 cm × 高14 cm）中的12个内，向每个盆中加入约400 ml含水量75%的土壤泥浆，该泥浆足以覆盖各植株的根系。待土壤干燥至可吸附处理液后，向每个盆内的土壤多个位置注入总计10 ml的15N标记硫酸铵（15N enriched ammonium sulphate，丰度为77.6 atom% 15N）溶液，该溶液含2.50 mg铵态氮，以保证养分分布均匀并防止溶液渗漏至土壤表面。向其中10个盆（其中6个种植有幼苗）加入硝化抑制剂（nitrification inhibitor）N-serve（2-Chloro-6-trichloromethyl pyridine），添加量使土壤水中的最终浓度达到5 ppm。后续每隔14至18天添加等量的N-serve，以维持高效的硝化抑制效果。所有注入操作完成后，将土壤表面压实，并定期添加蒸馏水以维持土壤表面上方3至5 mm的水层。在4个盆中各放置1个氧化还原探针，分别对应植物+土壤、仅土壤、有/无N-serve四种处理类型，以在整个试验过程中监测氧化还原电位（Eh）。试验在15N标记液添加63天后终止。移除植株地上部分，于60℃下烘干至恒重。冲洗掉根系上附着的土壤，并将根系与对应的地上植株部分合并。向土壤样品中加入300 ml 4 M氯化钾（KCl）溶液，振荡1小时后通过过滤得到澄清的提取液。对烘干、研磨后的土壤残渣进行总氮（total N）和15N丰度分析，对提取液则分别测定铵态氮、硝态氮、亚硝态氮含量及15N丰度。对烘干、研磨后的植株样品进行总氮和15N丰度分析。采用Micromass 622同位素比值质谱仪（isotope-ratio mass spectrometer），通过烘干铵盐与次溴酸锂（lithium hypobromite）的反应生成氮气，以此测定其15N富集度。在第二项试验中，幼苗种植于持续曝气的培养液中，培养液包含25%的海水（seawater）以及除氮以外的所有必需大量和微量营养元素。试验以硫酸铵（ammonium sulphate）或硝酸钾（potassium nitrate）作为氮源，两种氮源各设置4个浓度梯度：5、10、30和50 ppm N。每个处理组包含5株重复幼苗，总计40株幼苗。向所有培养液中添加浓度为5 ppm的硝化抑制剂N-serve。每两周更换一次培养液，更换间隔期间每隔2至3天监测一次培养液中的铵态氮、硝态氮和亚硝态氮含量。处理158天后，收获植株，测定其叶片、茎秆和根系的鲜重与干重。采用Licor叶面积仪（Licor leaf area meter）测定单株总叶面积。将烘干后的植株各组分在玛瑙球磨机（agate mill）中充分研磨，使用LECO CHN元素分析仪（LECO CHN analyser）测定总氮含量。本研究还开展了室内试验，以探究硝酸盐吸收在厌氧土壤中生长植物的氮营养中的作用。选择白骨壤（Avicennia marina）作为试验材料，因其广泛分布于澳大利亚红树生态系统中，且易于繁殖，同时能够耐受高度厌氧且长期淹水的土壤环境。