Expanding the toolbox of nutrient limitation studies: novel method of soil microbial in-growth bags to evaluate nutrient demands in tropical forests

1.Ecosystem processes and the organisms involved are generally limited by the availability of one or more element in soil, an important phenomenon to consider for our understanding of ecosystem functioning and future changes. Especially in tropical forests, typically growing on nutrient depleted soils, nitrogen (N), phosphorus (P) or other limitations are assumed. However, large‐scale nutrient manipulation experiments revealed complex site‐specific patterns and several authors raised the need for novel approaches to reveal deeper mechanistic insights on limitation patterns, especially concerning soil microbial activity.

2.In order to gain such deeper knowledge, based on a short review of previous small‐scale studies focusing on soil microorganisms, we developed an experimental approach which controls for common biases, including indirect treatment effects, addition of co‐elements or nutrient pulses. Using this technique, we tested the hypotheses that fungi growing in tropical forest soils are mainly limited by P, and that N versus P limitations shift along an altitudinal gradient.

3.Mesh bags of 38 μm filled with sand were amended with weak ion exchange resins loaded with N, P or potassium (K) and buried underneath the litter layer at three altitudinal sites in Southern Ecuador. After a period of four months, the in‐growth of fungal hyphae was quantified, phospholipid fatty acids were analyzed for a subset of samples and chemical properties were determined.

4.In line with the first hypothesis, hyphal abundance was increased in P amended mesh bags, indicating P limitation. However, this pattern was not significantly shifted along the altitudinal gradient. By contrast, N addition increased hyphal abundance at the lowest site, compared to significant reductions at 2000 and 3000 m – not only in fungi but also in bacterial abundance as indicated by PLFA analyses, contradicting common soil‐age hypotheses. Decreased nutrient immobilization and fungal in‐growth at higher elevations suggest slow microbial activity, including nitrification, which may have caused toxic ammonium accumulation.

5.The experimental design offers a promising tool to provide more mechanistic and soil focused analyses specifically targeting microbes, which in this system strongly supported the hypothesis of primary fungal P limitation in tropical forest soils.

1. 生态系统过程及其相关生物体通常受土壤中一种或多种元素的有效性限制，这一重要现象对于理解生态系统功能与未来变化至关重要。尤其是在通常生长于养分贫瘠土壤的热带森林中，学界普遍认为其存在氮（N）、磷（P）或其他元素的限制。然而，大规模养分操控实验揭示了复杂的位点特异性格局，多位学者提出需要开发新方法，以深入解析养分限制格局的机制，尤其是针对土壤微生物活动相关的机制。
2. 为获取此类深层认知，本研究在简要综述既往聚焦土壤微生物的小型研究基础上，开发了一种可控制常见实验偏倚的实验方法——此类偏倚包括间接处理效应、协同元素添加或养分脉冲输入。依托该方法，我们验证了两项假说：一是热带森林土壤中的真菌主要受磷限制；二是氮与磷的限制格局沿海拔梯度发生变化。
3. 我们将装填沙土的38微米孔径网袋，经负载氮、磷或钾（K）的弱离子交换树脂处理后，埋藏于厄瓜多尔南部三个海拔位点的枯落物层下方。历经4个月培育后，对真菌菌丝的入侵生长量进行定量测定，对部分样品开展磷脂脂肪酸（phospholipid fatty acids，PLFA）分析，并测定相关土壤化学性质。
4. 与第一项假说相符，经磷处理的网袋中菌丝丰度显著升高，表明真菌受磷限制。但该格局并未沿海拔梯度发生显著变化。与之相反，氮添加在最低海拔位点提升了菌丝丰度，而在2000米与3000米海拔位点则导致菌丝丰度显著下降——这一变化不仅见于真菌，磷脂脂肪酸分析结果亦显示细菌丰度呈现相同趋势，与主流的土壤年龄假说相悖。高海拔区域的养分固持能力与真菌入侵生长均有所降低，表明微生物活动（包括硝化作用）速率缓慢，这可能引发有毒铵态氮的积累。
5. 本实验设计为专门针对微生物的机制性、土壤导向型分析提供了极具潜力的工具，在本研究体系中，该设计有力验证了热带森林土壤中真菌主要受磷限制的假说。