Crop rotation and native microbiome inoculation restore soil capacity to suppress a root disease

Soil-borne diseases significantly impact crop yields, particularly in intensive monoculture systems. Peanut root rot, caused by fungal pathogens, is a major constraint in subtropical China, resulting in substantial yield losses. While some soils exhibit inherent disease suppression, the underlying mechanisms and management strategies for enhancing this capacity remain unclear. The rhizosphere, the soil region surrounding plant roots, harbors a diverse microbial community crucial for plant health and disease resistance. This microbiome acts as a first line of defense against pathogens, influencing disease outcomes. Understanding how rhizosphere microbiome assembly affects plant disease is crucial for developing innovative strategies to improve plant health and productivity. Crop rotations offer a cost-effective alternative to chemical pesticides for disease control by breaking the host-pathogen cycle. However, the precise mechanisms by which crop rotations regulate rhizosphere microbial communities and their impact on peanut root rot remain largely unknown. This research aimed to elucidate the influence of crop management on rhizosphere microbiome function in disease suppression, focusing on the impact of monocropping versus crop rotation on peanut root rot caused by *Fusarium oxysporum*. The study hypothesized that monocropping would lead to a less diverse and less effective rhizosphere microbiome in suppressing root rot compared to crop rotation, and that restoration of depleted microbial taxa could enhance disease suppression.

Extensive research highlights the importance of soil microbial communities in disease suppression. Studies have shown that diverse and well-balanced rhizosphere microbiomes can effectively inhibit the growth and spread of plant pathogens. However, the specific microbial taxa involved and their mechanisms of action often remain unclear. The role of plant root exudates in shaping the rhizosphere microbiome is increasingly recognized; different plants release different exudates, which selectively promote the growth of certain microbial communities. This suggests that agricultural practices, such as crop rotation, can manipulate root exudates to modulate the composition and function of the rhizosphere microbiome. The use of synthetic microbial communities (SynComs) is emerging as a potential biocontrol strategy, but the effectiveness of SynComs under diverse agricultural management practices needs further investigation. The literature demonstrates that monocultures can deplete beneficial microbial populations compared to diverse cropping systems, leading to reduced soil health and increased susceptibility to diseases. Previous studies have shown the effectiveness of crop rotation in managing soil-borne diseases, but the underlying microbial mechanisms are not fully understood. This research builds upon this existing literature by investigating the specific microbial taxa involved in disease suppression, their responses to different crop management practices and the potential of microbial inoculants to restore soil health and disease resistance.

The study employed a multifaceted approach involving field experiments, greenhouse experiments, and laboratory assays. A long-term (2012-2016) field experiment compared peanut root rot severity under monocropping and rotation regimes. In 2018, a more detailed investigation was conducted, examining disease incidence at various growth stages (seedling, flowering, pod-bearing). Illumina sequencing was used to characterize fungal communities in healthy and diseased peanut roots to identify the primary pathogen. The pathogenicity of identified isolates was confirmed through pathogenicity tests. Quantitative real-time PCR (qPCR) quantified *F. oxysporum* abundance in the rhizosphere at different growth stages. To assess the effect of crop management on the rhizosphere bacterial microbiome, 16S rRNA gene sequencing was performed on rhizosphere and bulk soil samples collected at different growth stages. Greenhouse experiments evaluated the ability of rhizosphere microbiomes from monocropped and rotation-grown peanuts to suppress *F. oxysporum* growth using both volatile organic compound (VOC)-mediated and direct microcosm antagonism assays. Gas chromatography-mass spectrometry (GC-MS) analyzed VOC profiles of rhizosphere microbiomes. Metatranscriptomic analysis investigated functional differences in cultivable rhizosphere microbiomes under monocropping and rotation. Cultivable rhizosphere microbiomes were characterized using 16S rRNA gene sequencing to identify depleted and enriched strains in monocropping. The response of depleted strains to root exudates was examined in vitro. The plant growth-promoting properties (siderophore and IAA production, phosphate solubilization, and *F. oxysporum* inhibition) of selected strains were evaluated. Finally, laboratory and field experiments tested the ability of SynComs composed of different combinations of depleted strains to suppress *F. oxysporum* growth and reduce root rot disease incidence.

The five-year field experiment showed significantly higher peanut root rot disease incidence under monocropping compared to rotation (P<0.001). The disease index increased dramatically in monocropped peanuts from the flowering stage onward (P<0.001). *F. oxysporum* was identified as the primary pathogen, with significantly higher abundance in diseased roots (P<0.001). Analysis of rhizosphere bacterial communities revealed significant differences between monocropping and rotation at all growth stages (P<0.001). Monocropping depleted 183 OTUs and enriched 156 OTUs compared to rotation (Padjusted <0.05). Rhizosphere microbiomes from rotation-grown peanuts exhibited significantly greater *F. oxysporum* suppression (45-56% higher than monocropped, P<0.01) in both VOC-mediated and direct antagonism assays. GC-MS analysis revealed that specific VOCs (dimethyl sulfide, 2,5-dimethylcyclohexanone, 6-methyl-3,5-pentadien-2-one) produced by the rotation rhizosphere microbiome were absent in the monocropping microbiome and exhibited strong antifungal activity (Supplementary Fig. 3, 4). Metatranscriptomic analysis revealed significant differences in pathways involved in pathogen inhibition between monocropping and rotation (Fig. 4c,d). 362 OTUs were depleted from the monocropping rhizosphere, most belonging to genera known for plant growth promotion and pathogen inhibition. In vitro experiments showed that monocropping root exudates suppressed the growth of these depleted strains, whereas they promoted growth of enriched strains (P<0.001). SynComs containing the depleted strains significantly suppressed *F. oxysporum* growth in vitro (P<0.001) and in vivo (P<0.001), and reduced root rot incidence in field experiments (P<0.001). Supplementation of depleted strains improved disease resistance without affecting plant growth.

The study's findings strongly support the hypothesis that monocropping weakens the soil's capacity to suppress root rot disease, while crop rotation enhances this capacity. The observed differences in rhizosphere microbiome composition and function between monocropping and rotation explain these contrasting disease outcomes. The depletion of key bacterial taxa in monocropping, due to their less competitive response to monoculture root exudates, compromises the rhizosphere's ability to inhibit pathogen invasion. The successful restoration of disease suppression by reintroducing the depleted strains underscores the importance of these low-abundance taxa and their synergistic interactions in pathogen control. The study's results highlight the importance of considering rhizosphere microbiome dynamics in agricultural management practices and demonstrate the potential of targeted microbial inoculation as a sustainable approach for disease management. The observation that VOCs from the rotation rhizosphere microbiome showed strong antifungal activity offers additional insights into the mechanisms of disease suppression. Further research could focus on identifying and characterizing the specific genes and metabolic pathways responsible for the production of these VOCs, as well as exploring the role of other microbial interactions in disease suppression. The use of SynComs holds promise as a biocontrol tool, but the complexity of microbial community interactions requires further investigation.

This study demonstrates that long-term monocropping of peanuts significantly increases the incidence of root rot disease, while crop rotation enhances soil disease suppression. The depletion of key rhizosphere bacteria under monoculture reduces disease resistance, while targeted inoculation with these depleted strains restores this capacity. The findings highlight the crucial role of rhizosphere microbiome diversity and function in disease suppression and advocate for the use of microbial inoculants as a sustainable biocontrol strategy for managing soil-borne diseases. Future research could explore the optimization of SynComs, investigate the long-term effects of microbial inoculation and investigate the role of other environmental factors in shaping the rhizosphere microbiome and disease suppression.

The study focused on a specific geographic region and soil type, limiting the generalizability of the findings to other environments. The field experiments were conducted over a relatively short period, potentially overlooking long-term effects of crop management on soil microbial communities. The SynCom experiments used a limited number of bacterial strains and combinations, and further research could explore the effects of more diverse and complex communities. While plant growth was not significantly affected by the inoculation of depleted strains, it is important to note the potential for long-term effects that may not be evident within the timeframe of the study.