Multispecies pasture reshapes subsoil bacterial and fungal communities compared to ryegrass monoculture

Perennial ryegrass (Lolium perenne L.) underpins the pasture-based dairy industry in Australia, yet its shallow root system limits adaptation to drought conditions. Multispecies pastures, consisting of grasses, legumes and herbs, are increasingly adopted to improve ecosystem resilience, but their interactions with soil microorganisms, particularly in deeper soil layers, remain poorly understood. This study investigated bacterial and fungal communities in both topsoils and subsoils across monoculture (perennial ryegrass) and multispecies (ryegrass, chicory, clovers and other plants) pastures at eight dairy farms in Victoria, Australia, using quantitative PCR and 16S and ITS rRNA gene amplicon sequencing. We found that microbial abundance and diversity declined with soil depth in both pastures, and were broadly comparable between monoculture and multispecies pasture systems. Microbial community compositions differed significantly between monoculture and multispecies pasture systems in both the topsoil and subsoil, across all sampling sites. Co-occurrence network analysis indicated that at three of the farm sites, multispecies pastures exhibited reduced bacterial and fungal network size in the subsoil compared to monoculture ryegrass at three of the sample locations. Overall, our findings suggest that multispecies pastures exert a different interaction with subsoil microbial communities than that of monocultures, which potentially benefit pasture drought resilience in sustainable agriculture.