Impact of Intercropping on Microbial Dynamics and Functionality in Bulk Soil and Rhizosphere

As a result of the impacts of global warming and anthropogenic activities, agroecosystems are currently experiencing an unprecedented crisis of soil degradation, characterized by nutrient depletion, biodiversity loss, and decline of their services. Intercropping, practiced for thousands of years in agriculture, has recently emerged as a promising strategy to traditional farming methods to enhance soil health and productivity. However, the effects of intercropping on soil microbial ecology remain underexplored. This study assessed soil enzymatic activities, microbial biomass C and N, and microbial community’s composition (determined by PLFAs analyses) in bulk soil and rhizosphere from a randomized field experiment in semiarid central Spain established in 2022. The treatments included monocropping of alfalfa, monocropping of barley, and alfalfa-barley intercropping. Soil samples were collected in May 2023, one month before harvest, at the peak of microbial activity.

The results revealed distinct patterns in soil microbial dynamics depending on the cropping system. Enzyme activities in the rhizosphere were significantly higher under alfalfa monocropping, likely due to enhanced microbial diversity and availability of root-derived exudates metabolites, while they were lower under barley monocropping, reflecting a higher nutrient demand or a lower substrate availability that stimulate the synthesis of those enzymes. In intercropping, C-cycling enzymes (β-glucosidase and cellulose) were mainly influenced by barley while N-cycling enzyme (leucine aminopeptidase) was by alfalfa, and P-cycling enzyme (acid phosphatase) showed intermediate activity, indicating balanced contributions from both crops. In bulk soil, enzymatic activities exhibited minimal differences across treatments. For all treatments, microbial biomass C and N were consistently higher in the rhizosphere compared to bulk soil, emphasizing the influence of root exudates in stimulating microbial growth and activity. The intercropping system showed the highest microbial biomass values within each soil compartment, suggesting an enhanced biological activity and nutrient cycling efficiency. Regarding microbial composition, mycorrhizal abundance peaked under intercropping in both the bulk soil and rhizosphere, highlighting the synergistic effect of the crop mixture on mycorrhizal communities. Fungal abundance also increased under intercropping but only in the rhizosphere. Gram-positive and gram-negative bacteria were most abundant in the rhizosphere under alfalfa monocropping, while in bulk soil, their levels showed no significant differences across treatments.

The alfalfa-barley intercropping system demonstrated strong potential to enhance soil microbial communities’ composition, particularly mycorrhizal associations, which are essential for nutrient cycling and soil functionality. Intercropping fosters beneficial plant-microbe interactions, especially in the rhizosphere, where microbial activity is most dynamic. These findings highlight intercropping as a sustainable agricultural practice that optimizes soil microbial processes and supports soil ecosystem services. Future studies should investigate the mechanisms driving these responses and the long-term sustainability of intercropping in diverse agroecological contexts.

Acknowledgments: This research was supported by the LEGUMINOSE project (grant ref. 101082289). M.J.C thanks to MINECO for her “Juan de la Cierva-Formación” postdoctoral contract.