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.

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.