# Changes in Soil Nutrients and Bacterial Communities in Perennial Grass Mixtures in Alpine Ecological Zones After 20 Years of Establishment

## TL;DR

Mixing different grass species in alpine meadows improves soil quality and bacterial diversity over 20 years, with the best results from a four-species mix.

## Contribution

This study demonstrates that perennial grass mixtures significantly enhance soil properties and bacterial community diversity in alpine grasslands over two decades.

## Key findings

- Mixed-sowing treatments improved soil physicochemical properties and plant community characteristics compared to monocultures.
- The P4 mixed-sowing treatment had the highest bacterial diversity indices and OTU richness compared to monoculture treatments.
- Soil organic matter was identified as a key driver shaping bacterial community assembly in alpine grasslands.

## Abstract

Monoculture and mixed sowing are common practices for restoring degraded alpine meadow grasslands. To investigate the effects of different sowing patterns on soil bacterial community characteristics in alpine artificial grasslands, this study examined a 20-year-old established artificial grassland, systematically analyzing plant community attributes, soil physicochemical properties, and the diversity and functional structure of soil bacterial communities under various monoculture and mixed-sowing treatments. The results showed that: (1) Mixed-sowing treatments significantly improved soil physicochemical properties and plant community characteristics. The P4 (Elymus nutans + Poa pratensis + Festuca sinensis + Poa crymophila) mixed-sowing treatment notably enhanced vegetation performance and soil conditions. Compared with the monoculture P1 (Elymus nutans) treatment, aboveground biomass (AGB) and soil organic matter (SOM) content increased by 57.23% and 68.25%, respectively, indicating that perennial grass mixtures improve soil water and nutrient retention, thereby promoting plant growth. (2) Microbiome analysis revealed that mixed sowing significantly optimized the structure of rhizosphere bacterial communities. Operational Taxonomic Units (OTUs), which represent sequence-based taxonomic units and their abundance information, were most abundant in the P4 mixed-sowing treatment, reaching a total of 5685 OTUs. In terms of bacterial diversity indices, the OTU richness, Ace index, and Chao1 index in the P4 mixed-sowing treatment were 26.12%, 25.81%, and 24.34% higher, respectively, than those in the monoculture P1 treatment, with all differences being statistically significant (p < 0.05). (3) Mantel test and redundancy analysis (RDA) revealed that soil electrical conductivity (SEC) and pH were negatively correlated with bacterial diversity indices, while soil organic matter (SOM) was identified as the key environmental driver shaping bacterial community assembly. In summary, appropriate grass mixtures effectively enhance “plant–soil–microbe” interactions, leading to improved soil fertility and optimized bacterial communities, representing a viable strategy for long-term ecological restoration and sustainability of alpine artificial grassland ecosystems. The P4 treatment—comprising a four-species mixture of Elymus nutans, Poa pratensis, Poa crymophila, and Festuca sinensis—achieved the best overall performance.