Long-term rice-crayfish-turtle co-culture maintains high crop yields by improving soil health and increasing soil microbial community stability

Crop and aquatic animal co-culture systems have been used for over 1200 years and can very efficiently utilize nutrients and materials available in agroecology settings. These co-culture systems are sustainable forms of agriculture, and have been extensively practiced worldwide. Microorganisms play critical roles in promoting ecosystem nutrient transformations and material circulations. However, the long-term effects of this farming system on soil health and microbial community stability, in addition to their relationships with rice yields, remain unclear. Here, 7-and 12-year farming experiments with rice-crayfish-turtle (RCT) co-culture systems were conducted and compared against rice monoculture (RM) in a suburb of Shanghai, China. Increased years of RCT farming led to higher contents of soil organic carbon (SOC), total nitrogen (TN), and available nitrogen (Avail_N), but less total phosphorus (TP) when the same soil layers were compared against the RM treatment. Thus, RCT farming generally had positive effects on soil physiochemical properties across all soil layers. qPCR analyses indicated that RCT farming led to a higher abundance ratio of fungi to bacteria (F:B) in soils than in RM soils, suggesting an improvement in metabolic efficiency and sustainability of these agroecosystems. High throughput sequencing demonstrated that 12-year RCT farming led to much higher soil microbial community diversity. Network analysis of 12-year RCT revealed more abundant microbial network modules and a larger proportion of negative correlations compared against 7-year RCT and RM networks. Overall, the community compositional analyses and network analyses indicated that RCT farming promoted soil health and increased microbial community stability. Stochastic processes dominated the bacterial and fungal community assembly, while random distributions were evident in the soil networks for 12-year RCT communities. Thus, all interactions between amplicon sequence variants (ASVs) were equivalent and these structures promoted network structure stability. Structural equation models (SEM) revealed that RCT farming had positive and indirect effects on rice yields by influencing soil properties or fungal diversity. Altogether, the improvement of soil health by RCT farming revealed the promotion of more suitable environments for soil microbial communities, which could contribute to nutrient cycling and further increase crop production yields. These results provide new insights into the co-associations and shifts in soil fertility quality with microbial community diversity and crop production, thereby helping to inform the ecological sustainability of RCT co-culture systems.