Grass-legume mixtures maintain forage biomass under microbial diversity loss via gathering Pseudomonas in root zone soil

Abstract

As the most common approach for the restoration of degraded grasslands, the use of grass-legume mixtures has long been recognized for its role in increasing aboveground biomass and resisting grassland degradation. However, whether the legumes in these mixtures can help neighboring plants resist the decline in biomass caused by the loss of soil microbial diversity remains a question worthy of investigation. To address this, we employed a dilution method to create a gradient of decreasing microbial diversity in soil and utilized full-factorial combinations of legumes and two grasses to investigate the crucial role of legumes in the mixture. The results showed that compared to monoculture, the mixture of Medicago sativa L. and Elymus dahuricus Turcz. enhanced the biomass of grass species under conditions of soil microbial diversity loss. We then discovered that a significantly enriched Pseudomonas (ASV53), in the grass-legume mixtures under conditions of microbial diversity loss, was positively correlated with plant biomass and nitrogen-fixing ( nifH ) gene abundance, implying that it could be a keystone species. In addition, the grass-legume mixture increased the deterministic processes of microbial community enrichment in the root zone soil by enhancing the process of homogeneous selection. Functional predictions revealed that grass-legume mixtures increased the potential abundance of N-related and phototrophy-related microbial communities in the root zone soil. This study provides an important insight into the mechanism underlying the role of legumes in increasing and maintaining grass biomass despite soil microbial diversity loss. IMPORTANCE Grass-legume mixtures are a common practice for establishing artificial grasslands, directly or indirectly contributing to the improvement of yield. In addition, this method helps maintain soil and plant health by reducing the use of chemical fertilizers. The impact of grass-legume mixtures on yield and its underlying microbial mechanisms have been a focus of scientific investigation. However, the benefits of mixtures in the context of soil microbial diversity loss remain a problem worthy of exploration. In this study, we examined different aboveground and belowground diversity combinations to elucidate the mechanisms by which grass-legume mixtures help maintain stable yields in the face of diversity loss. We identified the significantly enriched Pseudomonas genus microbial ASV53, which was gathered through homogeneous selection and served as a keystone in the co-occurrence network. ASV53 showed a strong positive correlation with biomass and the abundance of nitrogen-fixing genes. These findings provide a new theoretical foundation for utilizing grass-legume mixtures to enhance grass yields and address the challenges posed by diversity loss.