The intensification of agriculture has resulted in significant soil carbon loss, a critical issue in mitigating climate change. Agroecosystems cover over 40% of the Earth's surface and play a vital role in carbon cycling. Improving management practices to retain soil carbon is crucial but is hampered by a limited understanding of plant-microbe interactions belowground. Plants contribute carbon to the soil, while microbes determine its fate through decomposition and mineralization. This research utilizes a field trial to examine the impact of plant diversity on rhizosphere microbiota and CUE. The overarching hypothesis is that increased plant diversity fosters positive associations within the belowground microbial community, influencing nutrient cycling dynamics and soil carbon storage. Specific hypotheses include a positive influence of plant diversity on rhizosphere microbial structure, an impact of microbial community structure on rhizosphere CUE, and a positive relationship between plant diversity and biomass. The TwinWin platform experiment, funded by the Food and Agriculture Organization of the United Nations, provides the data for this analysis. The study aims to provide empirical evidence for the link between plant diversity and microbial CUE in agricultural soils. Previous research indicated that plant diversity enhances carbon uptake within the rhizosphere microbial community, forming the basis for this investigation.

Existing literature demonstrates that biodiversity loss negatively impacts ecosystem processes due to altered energy and matter fluxes. Long-term ecological experiments have highlighted the positive effects of biodiversity on ecosystem productivity, stability, and resilience to climate extremes. While the relationship between plant diversity and aboveground productivity is well-studied, the belowground mechanisms driving these positive biodiversity-ecosystem functioning relationships are increasingly recognized. The potential of plant diversity to influence soil carbon cycling is acknowledged, given the significant role of soil carbon in climate regulation. While plant biomass and root exudates are primary carbon sources for soil, microbial activity, influenced by microbial biodiversity, dictates carbon decomposition and mineralization. Research suggests that complex microbial-derived soil organic matter (SOM) compounds lead to longer residence times in the soil. Studies have linked microbial community composition to SOM chemical signatures and thermal stability, impacting decomposition rates. The influence of plant carbon and other species on microbial communities has also been noted. Diverse microbial communities show increased growth relative to respiration compared to species-poor communities, possibly due to greater functional diversity and complementarity through niche differentiation and facilitation. This study builds on existing knowledge to investigate the effects of plant diversity on belowground microbial community dynamics, particularly within the rhizosphere, which is significantly impacted by root secretions.

This study utilized data from the TwinWin platform experiment, a field trial designed to assess the impacts of ecological intensification in agroecosystems. The experiment involved barley as the main crop, with varying levels of undersown species diversity (monoculture, +1, +4, +8 species). Soil samples were collected from the rhizosphere of barley in August 2020. The sampling design aimed for 24 replicates per treatment, totaling 168 rhizosphere samples. Aboveground biomass measurements were conducted before barley harvest and before tilling in the spring of each year. Barley yield was assessed at harvest. Microbial community analysis involved measuring microbial biomass using the 20-water method, which assesses microbial CUE based on 18O-DNA enrichment. DNA was extracted, and δ15N values were measured to calculate CUE. Soil organic matter quality and quantity were assessed using Rock-Eval pyrolysis to determine total organic carbon (TOC), labile and recalcitrant carbon fractions. Bacterial and fungal communities were analyzed using amplicon sequencing, and diversity metrics were calculated. Network analysis was performed to investigate microbial associations, focusing on bacterial networks. Statistical analysis included linear mixed models, ANOVA, and structural equation modeling (SEM) to determine direct and indirect effects of plant diversity on CUE and other variables. The SEM incorporated plant diversity, plant biomass, soil properties, microbial community composition, network structure, respiration, and growth to assess their influence on CUE.

The study revealed a positive relationship between plant diversity and soil organic carbon content in the rhizosphere. Increased plant diversity led to greater productivity and biomass, especially in spring when undersown species contributed significantly. The 20-water method revealed that increasing plant diversity enhanced microbial growth in the rhizosphere, while respiration remained unchanged. Consequently, microbial community CUE increased with plant diversity. Network analysis showed that plant diversity positively affected the connectivity of bacterial networks, with an increased ratio of positive to negative associations. This suggests enhanced cross-feeding and facilitation among microbial communities under higher plant diversity. Specific bacterial phyla (Acidobacteria, Gemmatimonadetes, Pseudomonadota) showed stronger positive relationships with network connectivity, while others (Actinobacteria, Chloroflexi, Verucrombacteria) exhibited negative relationships. Structural equation modeling (SEM) demonstrated that plant diversity directly and indirectly (through its impact on microbial associations) influenced CUE. Soil properties indirectly influenced CUE by affecting microbial community composition and growth. The SEM highlighted the importance of positive network connectivity in driving CUE. Fungal networks did not respond to undersown plant diversity, which the authors suggest may be due to the distinct responses of fungi and bacteria to plant-derived carbon and the relatively short duration of the experiment. While the experiment is limited in answering the question of how distinct plant diversity affects microbial community dynamics, the authors observed that at the first level of diversity (barley plus 1), both microbial communities influenced barely phosphorus and improved microbial community associations.

The findings strongly support the hypothesis that increased plant diversity enhances soil carbon cycling in agricultural systems. The positive effects of plant diversity on microbial CUE are mediated by changes in the structure and interactions within the rhizosphere microbial community. Increased plant diversity leads to higher plant biomass, which provides more carbon substrates for microbial growth and activity. The enhanced positive associations within the microbial community likely reflect facilitation and cross-feeding mechanisms, resulting in more efficient resource use and carbon storage. The differential responses of bacterial and fungal networks to plant diversity suggest that the mechanisms driving these responses are complex and might involve distinct functional roles played by different microbial groups. The SEM analysis provides further support for the direct and indirect effects of plant diversity on CUE, highlighting the intricate interactions between biotic and abiotic factors. These results have important implications for sustainable agricultural practices. The incorporation of diverse plant species into agricultural systems could be a key strategy for enhancing soil carbon sequestration and improving soil health.

This study provides compelling evidence for the positive impact of plant diversity on soil carbon sequestration through enhanced microbial CUE in agricultural soils. The findings highlight the importance of considering belowground interactions in managing agroecosystems for increased sustainability. Future research should focus on identifying specific plant functional traits that maximize positive microbial interactions and explore the long-term effects of plant diversity on soil carbon dynamics under varying environmental conditions. Further investigations into the functional roles of different microbial groups and the mechanisms driving the observed interactions are needed to fully understand the complexities of belowground biodiversity and its contribution to soil health.

The study is limited by its relatively short duration (one year of soil sampling), which may not fully capture the long-term dynamics of microbial communities and soil carbon cycling. The focus on bacterial networks, while providing valuable insights, overlooks the potential roles of other microbial groups in these processes. While controlling for plant diversity, the impact of other factors such as root biomass could not be fully disentangled. Finally, the study is limited to a specific geographic location and may not fully generalize to other agricultural systems.