Mixedwood conifer trial to compare ectomycorrhizal and arbuscular fungal effects on soil organic matter decay

The purportedly enhanced decay of soil organic matter (SOM) by ectomycorrhizal fungi (EMF), in contrast to arbuscular mycorrhiza (AMF), was tested in a 30-year old mixedwood trial. Total peroxidase activity, including manganese-peroxidase (MnP), and mass of the humus layer were compared between Pseudotsuga menziesii (EMF) and Thuja plicata (AMF) across a natural productivity gradient. We found total peroxidase and MnP activity diverged between hosts as humus C:N increased, culminating in 3- to 4-fold greater enzyme activity for EMF on low fertility soils. This edaphic effect also correlated significantly with exchangeable Mn, highlighting a possible further restriction on SOM turnover by EMF. Peroxidase activity was well aligned with EMF species other than Cortinariaceae, notably Piloderma olivaceum and Piloderma sphaerosporum. The 3-fold decline in humus mass with decreasing soil fertility under P. menziesii, unlike T. plicata, was strong affirmation that EMF engaged in organic N liberation can lead to reductions in SOMcarbon sequestration

Methods
The assay method for total and Mn-dependent peroxidase activity was based on oxidative coupling of DMAB (3-dimethylaminobenzoic acid) and MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) in the presence of Mn2+ and H2O2. The residual peroxidase activity not dependent on Mn as a cofactor was subtracted based on reactions in which MnSO4 was replaced by a chelator (2 mM Na2-EDTA-2H2O). Substrate controls for background absorption unrelated to enzyme activity were supernatants plus reaction solutions without H2O2, while boiled samples were used as negative controls to confirm enzyme involvement in the colour change. We assayed 2 subsamples per composite for peroxidase activity (= 6 readings per plot). Enzyme activity was expressed as μmol h-1 g-1 dry soil.

Humus samples were freeze-dried and ground with a ring mill before DNA extraction. Briefly, the full length ITS region was amplified in duplicate from template DNA using high-fidelity Phusion polymerase and fusion primers and sequenced on a Sequel II instrument at the Integrated Microbiome Resource facility (Halifax, Canada). Fastq files were quality filtered and binned into amplicon sequence variants (ASVs) using the DADA2 software package in R. Taxonomy was assigned using DADA2’s assignTaxonomy() function and the UNITE database. Species were further assigned to functional guild using FUNguild and published literature.

本研究在一项开展30年的混交林定位试验中，验证了外生菌根真菌 (ectomycorrhizal fungi, EMF) 相较于丛枝菌根真菌 (arbuscular mycorrhiza, AMF) 据称可增强土壤有机质 (soil organic matter, SOM) 降解的假说。研究沿自然生产力梯度，比较了北美黄杉 (*Pseudotsuga menziesii*，EMF宿主) 与北美乔柏 (*Thuja plicata*，AMF宿主) 林下的总过氧化物酶活性（含锰过氧化物酶 (manganese-peroxidase, MnP)）以及腐殖质层质量。

研究结果显示，随着腐殖质碳氮比升高，两种宿主的总过氧化物酶与MnP活性出现显著分化；在低肥力土壤中，EMF宿主的酶活性较对照提升3至4倍。这一土壤介导的效应还与可交换态锰含量呈显著相关，提示EMF可能通过额外途径进一步限制土壤有机质周转。除丝膜菌科 (Cortinariaceae) 类群外，过氧化物酶活性与其余EMF物种的匹配度较高，尤以橄榄色被丝膜菌 (*Piloderma olivaceum*) 和球孢被丝膜菌 (*Piloderma sphaerosporum*) 最为典型。与北美乔柏林下不同，北美黄杉林下的腐殖质质量随土壤肥力降低下降3倍，这一结果有力证实：参与有机氮释放过程的EMF可减少土壤有机质的碳固存。

## 材料与方法
### 过氧化物酶活性测定方法
总过氧化物酶与锰依赖型过氧化物酶活性的测定基于Mn²+与H₂O₂存在条件下，DMAB (3-dimethylaminobenzoic acid) 与MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) 的氧化偶联反应。以MnSO₄被螯合剂（2 mM Na₂-EDTA·2H₂O）替代的反应体系，扣除不依赖锰作为辅因子的残余过氧化物酶活性。以不含H₂O₂的上清液与反应混合液作为非酶促本底吸收底物对照，以煮沸灭活的样品作为阴性对照，验证酶促反应参与颜色变化过程。每份混合样品设置2份子样本进行过氧化物酶活性测定（即每个样地共6次读数），酶活性单位以μmol·h⁻¹·g⁻¹干土表示。

### 微生物组测序与分析方法
腐殖质样品经冷冻干燥后，使用环磨粉碎，随后进行DNA提取。简要流程如下：以高保真Phusion聚合酶与融合引物，从模板DNA中对全长ITS区域进行重复扩增，随后在加拿大哈利法克斯的整合微生物组资源中心 (Integrated Microbiome Resource facility) 使用Sequel II测序仪完成测序。Fastq格式文件经质量过滤后，通过R语言中的DADA2软件包聚类为扩增子序列变异 (amplicon sequence variants, ASVs)。分类学注释采用DADA2的assignTaxonomy()函数与UNITE数据库完成。物种功能群通过FUNguild工具与已发表文献进一步判定。