Competition mode and soil nutrient status shape the role of soil microbes in the diversity–invasibility relationship

Understanding the relationship between plant diversity and invasibility is essential in invasion ecology. Species-rich communities are hypothesized to be more resistant to invasions than species-poor communities. However, while soil microorganisms play a crucial role in regulating this diversity–invasibility relationship, the effects of plant competition mode and soil nutrient status on their role remain unclear. To address this, we conducted a two-stage greenhouse experiment. Soils were first conditioned by growing nine native species separately in them for 1 year, then mixed in various configurations with soils conditioned using one, three, or six species, respectively. Next, we inoculated the mixed soil into sterilized substrate soil and planted the alien species Rhus typhina and native species Ailanthus altissima as test plants. We set up two competition modes (intraspecific and interspecific) and two nutrient levels (fertilization using slow-release fertilizer and non-fertilization). Under intraspecific competition, regardless of fertilization, the biomass of the alien species was higher in soil conditioned by six native species. By contrast, under interspecific competition, the biomass increased without fertilization but remained stable with fertilization in soil conditioned by six native species. Analysis of soil microbes suggests that pathogens and symbiotic fungi in diverse plant communities influenced R. typhina growth, which varied with competition mode and nutrient status. Our findings suggest that the soil microbiome is pivotal in mediating the diversity–invasibility relationship, and this influence varies according to competition mode and nutrient status.

Methods
At 8 weeks after the start of the test phase, all aboveground and belowground parts of the test plants were harvested and rinsed with water. Owing to the death of some seedlings during the growth period, 502 R. typhina individuals (interspecific: 239, intraspecific: 263) and 259 A. altissima individuals were harvested. The collected plant tissue was placed in a 70 °C oven to dry for a week and was weighed. The biomass of two R. typhina plants was measured together under intraspecific competition, and the biomass of R. typhina and A. altissima was measured separately under interspecific competition.

Soil sampling, DNA extraction, amplicon sequencing, and bioinformatics analysis

The 27 mixed fresh soil samples, collected from various diversity treatments and subsequently stored in a refrigerator at −80 °C, were analyzed for soil fungal community composition. The total microbial community DNA was extracted according to the instructions of the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA). The quality of the extracted DNA was verified using 1% agarose gel electrophoresis, and DNA concentration and purity were measured using a NanoDrop 2000 device (Thermo Fisher Scientific, Waltham, MA). The fungal rRNA internal transcribed spacer (ITS) region was amplified using the primer set ITS1F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′-GCTGCGTTCTTCATCGATGC-3′). Sequencing was performed using a MiSeq PE300/NovaSeq PE250 platform (Illumina, San Diego, CA). The Illumina MiSeq platform has a higher throughput and lower error rate than other high-throughput sequencers (Loman et al., 2012; Frey et al., 2014).

Fungal sequences were classified using the UNITE database (version 8.0) and using the USEARCH11-uparse algorithm for clustering. Operational taxonomic unit (OTU) sequence similarity was 0.97. Species classification was performed using the unite8.0/its fungi database, and classification confidence was 0.7. As fungal DNA extraction failed for one sample, we obtained 26 samples of ITS rDNA.

解析植物多样性与可入侵性（invasibility）之间的关联是入侵生态学的核心研究方向。学界普遍假说认为，物种丰富的群落相较于物种贫乏群落，对生物入侵的抗性更强。然而，尽管土壤微生物（soil microorganisms）在调控这一多样性-可入侵性关联中发挥关键作用，但植物竞争模式与土壤养分状况对其调控效应的影响仍未明确。
为解答这一科学问题，我们开展了两阶段温室实验。首先，将9种本地物种分别接种于土壤中培育1年，完成土壤预培养；随后将这些预培养土壤按不同配置混合，分别搭配经1种、3种或6种物种预培养的土壤。之后，将混合土壤接种至灭菌的基质土壤中，并种植外来种火炬树（Rhus typhina）与本地种臭椿（Ailanthus altissima）作为受试植物。实验设置两种竞争模式：种内竞争（intraspecific competition）与种间竞争（interspecific competition），以及两种养分水平：施加缓释肥与不施肥。
在种内竞争条件下，无论是否施加肥料，经6种本地物种预培养的土壤中，外来受试植物的生物量均更高。与之相对，在种间竞争条件下，未施肥时该生物量随预培养物种丰富度提升而升高，而施肥条件下则保持稳定。土壤微生物分析结果显示，多样植物群落中的病原菌与共生真菌会影响火炬树的生长，且这种影响随竞争模式与养分状态发生变化。
本研究结果表明，土壤微生物组（soil microbiome）在介导多样性-可入侵性关联中发挥关键作用，且其调控效应随竞争模式与土壤养分状况而异。
实验方法
实验阶段开始8周后，收获受试植物的全部地上与地下部分，并用清水冲洗干净。由于生长期间部分幼苗死亡，最终收获了502株火炬树个体（种间竞争组239株，种内竞争组263株）与259株臭椿个体。将采集的植物组织置于70℃烘箱中烘干1周后称重。种内竞争条件下，将2株火炬树的生物量合并测量；种间竞争条件下，则分别测量火炬树与臭椿的生物量。
土壤采样、DNA提取、扩增子测序及生物信息学分析
从不同多样性处理组中采集的27份新鲜混合土壤样品，保存于-80℃冰箱中，用于分析土壤真菌群落组成。按照E.Z.N.A.土壤DNA试剂盒（Omega Bio-tek，美国佐治亚州诺克罗斯市）的操作说明提取总微生物群落DNA。采用1%琼脂糖凝胶电泳验证提取DNA的质量，使用NanoDrop 2000仪器（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）测定DNA浓度与纯度。采用引物对ITS1F（5′-CTTGGTCATTTAGAGGAAGTAA-3′）与ITS2R（5′-GCTGCGTTCTTCATCGATGC-3′）扩增真菌rRNA内转录间隔区（internal transcribed spacer, ITS）。测序工作依托MiSeq PE300/NovaSeq PE250平台完成（Illumina，美国加利福尼亚州圣地亚哥市）。相较于其他高通量测序仪，Illumina MiSeq平台具有更高的测序通量与更低的错误率（Loman等，2012；Frey等，2014）。
真菌序列采用UNITE数据库（版本8.0）进行分类，并通过USEARCH11-uparse算法进行聚类，操作分类单元（operational taxonomic unit, OTU）的序列相似性阈值设为0.97。物种分类基于unite8.0/its fungi数据库完成，分类置信度阈值为0.7。由于1份样品的真菌DNA提取失败，最终共获得26份ITS rDNA测序样本。