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

Article https://doi.org/10.1038/s41467-023-43926-4 Crop rotation and native microbiome inoculation restore soil capacity to suppress arootdisease Yanyan Zhou 1,12 ,ZhenYang 2,3,12 ,JinguangLiu 2 ,XudongLi 1 , Xingxiang Wang 2,4 , Chuanchao Dai 3 , Taolin Zhang 2,4 ,VíctorJ.Carrión 5,6,7,8 , Zhong Wei 9 , Fuliang Cao 10 , Manuel Delgado-Baquerizo 11 &XiaogangLi 1,2,10 It is widely known that some soils have strong levels of disease suppression and prevent the establishment of pathogens in the rhizosphere of plants. However, what soils are better suppressing disease, and how management can help us to boost disease suppressionremainunclear.Here,weusedfield, greenhouse and laboratory experiments to investigate the effect of management (monocropping and rotation) on the capacity of rhizosphere microbiomes in suppressing peanut root rot disease. Compared with crop rotations, monocropping resulted in microbial assemblies that were less effective in suppressing root rot diseases. Further, the depletion of key rhizosphere taxa in monocropping, which were at a disadvantage in the competition for limited exudates resources, reduced capacity to protect plants against pathogen invasion. However, the supplementation of depleted strains restored rhizosphere resistance to pathogen. Taken together, our findings highlight the role of native soil microbes in fighting disease and supporting plant health, and indicate the potential of using microbial inocula to regenerate the natural capacity of soil to fight disease. Some soils have a larger capacity to support disease suppression than others helping to prevent the establishment of pathogens in the rhizosphere of plants1,2. However, the characteristics defining these soils are largely unknown. The rhizosphere refers to the soil directly associated with the roots. This complex environment, enriched with carbon and nutrients, is home to a diverse microbial community that plays an essential role in promoting plant growth and health3,4. This rhizosphere microbiome serves as the first line of defense against plant pathogens, with direct consequences for plant disease outcomes5–7. Because of this, advancing our mechanistic understanding on how variations in the assembly of rhizosphere microbiomes influence plant disease is of paramount important to provide innovative strategies for improving plant health and productivity. Received: 2 November 2022 Accepted: 24 November 2023 Check for updates 1 State Key Laboratory of Tree Genetics and Breeding, College of Ecology and Environment, Nanjing Forestry University, Nanjing 210037, China. 2 State Key Laboratory of Soil & Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China. 3 Jiangsu Key Laboratory for Microbes and Functional Genomics, College of Life Sciences, Nanjing Normal University, Nanjing 210023 Jiangsu, China. 4 Ecological Experimental Station of Red Soil, Chinese Academy of Sciences, Yingtan 335211, China. 5 Departamento de Microbiología, Facultad de Ciencias, Campus Universitario de Teatinos s/n, Universidad de Málaga, 29010 Málaga, Spain. 6 Instituto de Hortofruticultura Subtropical y Mediterránea La Mayora (IHSM) UMA-CSIC, 29010 Málaga, Spain. 7 Institute of Biology, Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands. 8 Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Droevendaalsesteeg 10, 6708 PB Wageningen, The Netherlands. 9 College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing 210095, China. 10 Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China. 11 Laboratoriode Biodiversidad y Funcionamiento Ecosistémico, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Sevilla, Spain. 12 These authors contributed equally: Yanyan Zhou, Zhen Yang. e-mail: [email protected] Nature Communications | (2023) 14:8126 1 1234567890():,; 1234567890():,; Crop management is known to have a critical role in regulating rhizosphere microbiomes. In China, peanut represents one of the most profitable crops. However, the yield and quality of this product have been reported to be greatly compromised by soil-borne diseases, especially under intensive monocropping managements. In subtropical China, 10–40% of crop yield is lost due to increasing disease pressure, with peanut root rot caused by fungal pathogens being a major constraint8. Unfortunately, few effective control measures are available, and the use of chemical pesticides is limited by environmental concerns9. Crop rotations, including different crop varieties, have been proposed to mitigate the negative impacts of pathogens on crop production, by breaking the link between plant host and pathogens, becoming a non-expensive alternative for disease control. Yet, how management by regulating the impact of rhizosphere microbial communities on peanut root rot diseases remains largely unknown. Microbial and plant-based tools, such as synthetic microbial communities (SynComs) and root-derived metabolites, are promising strategies to fight against soil-borne pathogens and plant disease, and can help to promote plant health. However, what strategies work best under contrasting management remains poorly understood. Recent studies have shown that rhizosphere microbiome assembly is significantly influenced by the selection effects of plant root metabolisms. As a result, different outcomes of plant health can be fostered depending on which microbial populations are able to take advantage of the root metabolites10,11. Thus, agricultural practices could potentially exploit the differences in root metabolisms of various crops to disrupt the directional selection for maintaining the stability of agricultural ecosystem12,13. Similarly, SynComs are currently being developed as bio-products to control soil-borne disease. Yet, there are needs for understanding the impacts of these SynComs on crop rhizosphere and as management tools for plant health. Here, we conducted multiple experiments to unravel the influence of crop management on the capacity of the rhizosphere microbiome to support disease suppression. The findings revealed that under peanut monocropping, there was a selective impoverishment of key bacteria, with weaker responses to root exudates, but that were associated with the capacity of rhizosphere to suppress pathogen invasion. The restoration of these bacteria brought back the capacity of soils to fight disease suppression. Our study advances our knowledge on how to develop innovative strategies to provide favorable support for sustainable agriculture. Results Monocropping aggravates peanut root rot severity To determine the effects of different agricultural regimes on peanut health, we conducted a field experiment from 2012 to 2016 aiming to quantify the occurrence of peanut (Arachis hypogaea L.) root rot under monocropping and rotation regimes (Fig. 1a). Results indicated that disease index (DI) rapidly increased from 2012 to 2016 in monocropping peanuts that were examined at flowering stage, compared with crops subjected to rotations (DI 2012 = 2.1, DI 2016 =8.0,Fig.2a, b). In fact, we showed that the DI of peanuts in rotation systems remained stable (DI 2012 =1.6, DI 2016 = 3.1, Fig. 2b). We further showed that the DI of monocropping and rotation began to diverge in 2014, with a significantly higher DI in monocropping compared with crops subjected to rotation (Student’st-test, t=8.276,df =16,P< 0.001, Fig. 2b). These results indicated that long-term monocropping can aggravate peanut root rot disease. Based on the difference of DI between monocropped and rotation peanuts at flowering stage, we specifically tracked the changes in DI at different peanut growth stages in 2018. At peanut seedling stage, the DI of peanuts grown on monocropped and rotation plots were very low (DI rotation =3.87, DI monocropping =4.38), and we did not find significant differences in DI for the two regimes at this particular stage (Student’st-test, t=−0.660, df =16,P=0.519,Fig.2c). However, from flowering stage, the DI in peanuts under monocropping regime dramatically increased, and was 2.6 times higher than that of peanuts sown under the rotation regime at pod-bearing stage (Student’st-test, t=−7.901, df =16,P< 0.001, Fig. 2c), indicating the amplified severity of root rot during developmental stage. To identify the pathogen associated with this root rot disease, we characterized the community composition of fungi in healthy and diseased peanut roots using Illumina sequencing (Fig. 1b). A total of 347,214 internal transcribed spacer 1 (ITS1) reads were obtained (range, 31,995–44,592 reads per sample), clustering into 183 fungal OTUs at ≥97% sequence identity. Several taxa from Fusarium sp. have been reported with ability to cause wilting symptoms in peanuts, but specific pathogen lying behind peanut root rots remains to be discovered14,15. Results indicated OTU177 (taxonomically assigned to F. oxysporum)andOTU90(taxonomicallyassignedtoF. solani)were significantly enriched in diseased peanut root. The relative abundance ofthese two fungal species was 34.1 and 2712.7times higher in diseased than in healthy peanut roots respectively (Fig. 2d). In order to obtain cultures associated with the potential pathogens, we isolated 38 fungi from the diseased peanut roots. Based on growth morphology, 20 isolates were selected for 18S rRNA sequencing and subsequently were identified as F. oxysporum (5), Penicillium sp. 196F (1), F. solani (3), Talaromyces pinophilus (2), Talaromyces verruculosus (4), and Neocosmospora striata (5) (Supplementary Fig. 1). Next, we confirmed the pathogenicity of the two isolates linked with F. oxysporum and F. solani. F. oxysporum was highly pathogenic (51 ± 11%), resulting in significantly higher disease incidence than that observed upon inoculation with F. solani (35 ± 9%) (Student’st-test, t= 5.580, df =58,P< 0.001) (Fig. 2e). Since other Fusarium were not enriched and cultivable, we identified our isolated strain (F. oxysporum) as the most likely organism behind the root rot disease observed in the studied peanut fields. We conducted further quantitative real-time PCR (qPCR) analyses to gain deeper insights into the abundance and dynamics of F. oxysporum in the peanut rhizosphere at different growth stages. Consistent with the results of the disease index, there was no significant differences between monocropping and rotation regimes in F. oxysporum abundance at the peanut seedling stage (Student’st-test, t=−1.525, df =10,P= 0.158, Fig. 2f). However, F. oxysporum abundance in the rhizosphere of monocropped peanut was significantly higher than that of rotation during the next developmental stages (Student’s t-test, t=2.980,df =10,P<0.05,Fig.2f). Dynamics in pathogen accumulation after seedling stage in monocropped peanut showed a parallel pattern to that in peanut root rot disease, significantly affecting plant growth and reducing plant yield (Student’st-test, P<0.05,Supplementary Fig. 2). Taken together, our results suggested that the effective colonization of F. oxysporum in the peanut rhizosphere has critical consequences for plant health in crops subjected to monocropping. Crop managements have significant effects on the peanut rhizosphere microbiome The bacterial microbiome plays a crucial role in influencing the severity of soil-borne diseases compared to fungi. Plant roots can effectively recruit a higher diversity and richness of the rhizosphere bacterial community to suppress the invasion of pathogens, particularly those originating from fungal sources16–18. Therefore, we investigated the impacts of crop managements on the bacterial microbiome of peanut rhizosphere at different plant growth stages and bulk soil in our 2018 field experiment. In total, we obtained 1,369,637 sequences from 48 soil samples. Nonmetric multidimensional scaling (NMDS) based on the Bray–Curtis dissimilarity matrix revealed significant differences in bacterial community of monocropping and rotation at all development stages (ANOSIM, P< 0.001, Fig. 3a). Sequentially, we compared the variation of rhizosphere bacterial community between monocropping and rotation at seedling stage. Results indicated that Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 2 183 OTUs were enriched and 156 OTUs were depleted by monocropping (P adjusted <0.05,Fig.3b). Discriminating OTUs mainly clustered in Proteobacteria and Actinobacteriota (Fig. 3c). Further, the average relative abundance of 122 depleted OTUs (account for 82.4% of the discriminating OTUs), and 148 enriched OTUs (account for 83.1% of the discriminating OTUs) in monocropping was <0.1% (Fig. 3c, d). Rhizosphere microbiome of crops under rotation suppress the invasion of F. oxysporum To examine the role of rhizosphere community in the occurrence of peanut root rot, we further collected soils from field plots of both monocropping and rotation to grow peanut at a greenhouse experiment (Fig. 1c). The rhizosphere microbiome of peanuts at seedlings was harvested to determine the ability of F. oxysporum inhibition. Antifungal activity against F. oxysporum was tested in antagonism assay of both volatile organic compounds (VOCs)-mediated and directed microcosms. The results showed that the rhizosphere microbiomes of peanuts grown under rotation regime were able to suppress the growth of F. oxysporum,by45–56% higher than monocropped regime (Fig. 4a, b, Student’st-test, P< 0.01). This highlighted the significance of antagonism in rhizosphere community against pathogen invasion. We then investigated the potential role of VOCs in mediating these results. We knew that VOCs produced by the rhizosphere bacterial community can play an important role in preventing fungal pathogen infection19,20. Thus, we used gas chromatography–mass spectrometry (GC-MS) to determine the composition of VOCs produced by the rhizosphere microbiome of monocropped and rotation peanut. Of the VOCs detected, dimethyl sulfide, 2,5-dimethylcyclohexanone, and 6-methyl-3,5-pentadien-2-one produced by the Monocropping Rotation a Field experiment ×3 plots ×3 plots Cultivable potential pathogens Flowering Isolation and ITS identification Pathogenicity surveillance Community analysis Healthy Diseased DNA extraction Analysis of pathogenic fungal community Sampling time ● ● ● ● ● ● ● ● ● ● ● ● −0.4 −0.2 0.0 0.2 −0.5 0.0 0.5 PCoA1 (53.43%) PCoA2 (19.51%) OTU90 OTU177 Developmental period Disease index Disease dynamic curve Monocropping roots b Pathogen determination 2012 2018 Peanuts Maize Potato Soybean Peanuts Peanuts Peanuts Peanuts 2014 Peanuts Peanuts 2016 Peanuts Peanuts Peanuts Peanuts Flowering Seedling Pod-bearing Monocropping Rotation c Pot experiment Rhizosphere soil Cultivable microbiome ×10 pots Community-pathogen interaction Antagonistic VOCs analysis Isolation and 16S rRNA identification ATCG + Community analysis + Functional analysis Monocropping Rotation ● ● ● ● ● ● ● ● Quorum sensing Staphylococcus aureus infection Two−component system ABC transporters Phosphotransferase system (PTS) Folate biosynthesis beta−Alanine metabolism Taurine and hypotaurine metabolism Galactose metabolism Alanine, aspartate and glutamate metabolism 0.1 0.2 0.3 0.4 ko00250 ko00052 ko00430 ko00410 ko00790 ko02060 ko02010 ko02020 ko05150 ko02024 Directed VOCs-mediated 2018 2012 2014 2016 Rhizosphere soil Fig. 1 | Flow charts of field experiments for peanut disease investigation, identification of potential pathogens and pot experiment. a From 2012, experimental plots were managed under two cropping regimes (treatments): peanut monocropping and rotation. For monocropping, peanuts were continuously planted from 2012 to 2018. For rotation, peanut was grown first (2012), and then maize (Zea mays L.), potato (Solanum tuberosum), and soybean (Glycine max) were ordinally planted in every other peanut planting year. The disease index of peanut was investigated at the flowering stage in 2012, 2014, and 2016. At 2018, peanut disease was investigated at seedling, flowering, and pod-bearing stage, and the rhizosphere soil was sampled for community analysis. bRoots from mature monocropped plants with healthy and diseased were used for pathogenic community analysis and potential pathogen identification. cDuring the end of 2018 planting season, soil samples were collected from six plots and used for pot experiments. Therhizosphere soil was collected at the seedling stage for culturable microbiome–pathogen interaction, community analysis, and functional analysis. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 3 rhizosphere microbiome from rotation-grown peanut were not detected in the rhizosphere microbiome from monocropped peanut (Supplementary Fig. 3). Further, the relative contents of α-acorenol, dimethyl disulfide, and 1,3-xylene were significantly reduced under the monocropping regime compared with rotation (Student’st-test, P< 0.05). Further in vitro experiments of pathogen suppression using standard VOCs (dimethyl sulfide, 2,5-dimethylcyclohexanone, 6methyl-3,5-pentadien-2-one and 1,3-xylene), verified that these specific VOCs detected in peanut rhizosphere of rotation regime significantly inhibited pathogen growth, even at low concentrations (0.5–5.0 μg/ mL; Supplementary Fig. 4). In order to provide a more detailed understanding on the mechanisms behind these results, we further performed transcriptome analysis of cultivable microbiome from monocropping and rotation rhizosphere to understand the associated functional variation for F. oxysporum inhibition (Fig. 1c). We extracted mRNA of cultivable microbiome from monocropping and rotation rhizosphere after the VOCs-mediated antagonism assay, and transcribed it into cDNA for sequencing. Pathways enriched in different Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology functional categories in cultivable rhizosphere microbiomes from monocropping and rotation were analyzed. The metabolic pathways that were primarily responsible for the differences in KO functional categories in the monocropping and rotation included ABC transporters and Two-component system (Fig. 4c). To be specific, the expression of genes associated with pathogen inhibition, including Isopentenyldiphosphate Delta-isomerase (K01823), Glutamate decarboxylase (K01580), and 1-pyrroline-5-carboxylate dehydrogenase (K00294) were significantly higher in rotation than that in monocropping (Fig. 4d, Student’st-test, P< 0.05). Cultivable rhizosphere microbiome under monocropping and rotation To understand whether immature microflora was associated with decreased rhizosphere resistance in monocropping seedlings, we first collected cultivable rhizosphere microbiome from the above antagonistic experiments. Considering the difference in inhibition ability of the rhizosphere microbiome against F. oxysporum on agar plates between monocropping and rotation, we first compared the characteristics of the whole cultivable microbiome by Illumina sequencing. The number of sequences of the cultivable microbiome ranged from 14,804 to 22,748, with clustering as 714 OTUs at 97% similarity. Since the cultivable microbiome sequence came from all bacterial colonies growing on agar plates, we then determined OTUs depleted from monocropping or rotation rhizosphere cultures. Results indicated 362 OTUs, most belonging to Bacillus,Enterobacter,Escherichia−Shigella,Pantoea, Enterococcus,Pseudomonas,Lysinibacillus,Paenibacillus,Kluyvera,and Jeotgalibacillus, were not detected in the rhizosphere of monocropped peanut (Supplementary Fig. 5). By contrast, 257 OTUs, most belonging to Bacillus,Pseudomonas,Virgibacillus,Burkholderia−Paraburkholderia, Ralstonia,Escherichia−Shigella,Klebsiella,Gemmatimonas,Acinetobacter,Paenibacillus,andLysinibacillus, were not detected in the rhizosphere of rotation-regime peanut (Supplementary Fig. 5). In order to obtain the depleted and enriched strains in monocropping rhizosphere, we isolated 173 bacterial strains from agar plates of rhizosphere cultures for 16S rRNA sequencing. These Fig. 2 | Investigation of peanut disease and identification of pathogen in longterm field experiment. a Symptoms of healthy and diseased peanuts in the field. bDisease index of peanut root rot from 2012 to 2016 at the flowering period (P<0.001,n= 9 biologically independent samples). cDisease index of peanut root rot in monocropping and rotation regimes at 2018 across different growing periods (P<0.001,n= 9 biologically independent samples). dSignificantly increased OTUs (Fusarium sp.) in diseased roots compared with healthy roots. −1.5 to 1.5 represents the relative abundance of normalized OTUs. eTest for pathogenicity of the potential pathogens Fusarium spp. (P< 0.001, n= 30 biologically independent samples). Horizontal bars within boxes represent the median. The tops and bottoms of boxes represent 75th and 25th quartiles, respectively. The upper and lower whiskers represent the range of non-outlier data values. fThe abundance of F. oxysporum in peanut rhizosphere of monocropping and rotation regimes in 2018 field experiment (P<0.05,n= 6 biologically independent samples). Asterisks above the bars indicate statistically significant differences between the treatments based on two-sided tests by Student’st-test (*P< 0.05, **P< 0.01, ***P<0.001).Each bars represents the mean ± SD. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 4 sequenced strains were mainly belonged to Paenibacillus sp., Pantoea sp., Bacillus sp., Fictibacillus sp., Lysinibacillus sp., Enterobacter sp., Sporosarcina sp., Pseudomonas sp., Burkholderia sp., Stenotrophomonas sp., Serratia sp., Arthrobacter sp., Buttiauxella sp., and Brevundimons sp. (Supplementary Fig. 6). Further, in order to determine the consistency of depleted or enriched OTUs with the above strains, we compared the similarity between the 16 S rRNA sequences of the isolated strains and the OTUs that were depleted or enriched in the cultivable microbiome of monocropping rhizosphere (Fig. 4e). When the sequence similarity between isolated strains and depleted or enriched OTUs exceeded 97%, the strain was considered representative of the corresponding OTU. We excluded the 16S rRNA sequence of Bacillus sp., a highly abundant genus widely present in the peanut rhizosphere, from the analysis. Among them, strains isolated from the rhizosphere of rotation-grown peanuts, i.e., Paenibacillus sp. (R60), Pantoea sp. (R05), Lysinibacillus sp. (R06), Enterobacter sp. (R09), Sporosarcina sp. (R07), Fictibacillus sp. (R37), and Pseudomonas sp. (R26), were 97% similar to OTUs depleted from the rhizosphere of monocropped peanuts, while strains belonging to Stenotrophomonas sp. (C20) and Burkholderia sp. (C63) were 97% similar to OTUs enriched in the rhizosphere of monocropped peanuts (Fig. 4e). Indeed, the abundance of the OTUs associated with the depleted strains was very low in the rotation plant rhizosphere (Fig. 4e). Characteristics of depleted strains responding to peanut root exudates Plant root exudates are one of the sources that regulate rhizosphere microbial assembly21. To understand whether the depletion of low abundance of taxa was associated with responsiveness to plant root exudates, we collected root exudates of peanuts grown on monocropping and rotation soil, and evaluated the effects of root exudates on bacterial growth in vitro. In this regard, all depleted bacterial strains had less sensitive response to root exudates of monocropped peanut as compared to that from rotation peanut (Student’st-test, P< 0.001, Fig. 4f). However, monocropping root exudates promoted the growth of Burkholderia and Stenotrophomonas that were enriched in monocropping rhizosphere (Student’st-test, P< 0.001, Fig. 4f). Compared to the enriched strains, the growth of depleted strains increased less in responding to root exudates from monocropping peanut. Next, we determined potential plant growth-promoting properties of the above strains in vitro. In total, depleted strains of monocropping had potential growth-promoting properties. Of these, Enterobacter and Pantoea produced siderophores, and Bacillus and Sporosarcina solubilized organic phosphorus (Supplementary Table 1). Accordingly, Paenibacillus and Lysinibacillus produced more than 25 mg indoleacetic acid (IAA) per L. Except Pantoea and Lysinibacillus, all the other depleted strains inhibited the growth of F. oxysporum (Supplementary Table 1). Nevertheless, Burkholderia, a monocropping enriched strain, solubilized phosphorus and produced siderophores and IAA, but did not inhibit the fungal growth. Finally, we determined the interaction between any two depleted bacteria. No significant enhancement or inhibition was observed between any two strains (Supplementary Fig. 7), indicating independent growth and stable coexistence among depleted strains. ● ● ● ● ● ● ● ● Bulk soil Pod-bearing Seedling Flowering ●Monocropping Rotation ●●●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ●● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● −0.4 −0.2 0.0 0.2 0.4 −0.4 0.0 0.4 NMDS1 NMDS2 R=0.9499 P<0.001 Stress=0.10 0 10 20 30 40 −10 0 10 log2(FC) −log10(pVal) Down(156) Up (183) Monocropping enriched(183) Monocropping depleted(156) Acidobacteriota Actinobacteriota Unassigned Bacteroidota Chloroflexi Firmicutes Proteobacteria 0 0 - 0.1% 0.1% - 1% > 1% OTU abundance Monocropping Rotation Monocropping Rotation d ab c Fig. 3 | Changes of rhizosphere microbial community in 2018 peanut season. aNon-metric multidimensional scaling (NMDS) of microbial communities in rhizosphere of monocropping and rotation at seedling, flowering and pod-bearing stages. bCompared with rotation, enriched or depleted OTUs in monocropping seedlings rhizosphere (DESeq2, P< 0.05, FDR adjustment). c,dPhylogenetic trees constructed by enriched/depleted OTUs in monocropping seedlings rhizosphere and the relative abundance. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 5 Depleted strains associated with pathogen suppression by rhizosphere microbiome We next asked why the depletion of specificstrainsdepressedthe ability of monocropped peanut rhizosphere to suppress F. oxysporum. We first analyzed the ability of different SynComs containing 7, 4, and 2 depleted strains to impact F. oxysporum pathogenicity (Fig. 5aand Supplementary Table 2). Overall, SynCom containing the 7, 4, or 2 depleted strains significantly suppressed mycelial development of F. oxysporum (ANOVA, F= 38.483, P< 0.001, Fig. 5b). When the strain was inactivated, the inhibition disappeared. Moreover, the inhibition effect was positively correlated with the diversity of the depleted strains. When there were 7 depleted strains, SynCom showed the best inhibition of F. oxysporum. This indicated thatall depleted strains participate in the suppression of fungal pathogens via synergistic interactions22. Next, we supplemented suspension of SynComs with different depleted strains to rhizosphere microbiome from monocropped ab R07 Sporosarcina R06 Lysinibacillus R37 Fictibacillus R60 Paenibacillus R09 Enterobacter R05 Pantoea R26 Pseudomonas C20 Stenotrophomonas C63 Burkholderia 0 5 10 15 Relative abundance (× 10 -4 %) OTU6966 OTU12223 OTU9456 OTU8675 OTU9041 OTU9439 OTU8770 OTU9294 OTU10342 cd ef Monocropping Rotation ● ● ● ● ● ● ● beta−Alanine metabolism Two−component system Quorum sensing Galactose metabolism ABC transporters Taurine and hypotaurine metabolism Phosphotransferase system (PTS) Alanine, aspartate and glutamate metabolism 0.00 0.04 0.08 0.12 0.16 Rich Factor Pathway Name Gene Number ● 5 10 ● ● ● ● ● ● Monocropping Rotation Rhizosphere bacterial community F. oxysporum Isopentenyldiphosphate Delta-isomerase K01823 0.0 0.1 0.2 0.3 0.4 0.5 TPM K01580 K00294 Glutamate decarboxylase 1-pyrroline5-carboxylate dehydrogenase Rotation Monocropping Pseudomonas Enterobacter Sporosarcina Paenibacillus Lysinibacillus Pantoea Fictibacillus Burkholderia Stenotrophomonas 0.0 0.1 0.2 0.3 0.4 0.5 OD 600nm *** *** *** *** *** *** *** *** *** 0 20 40 60 80 100 Inhibition (%) Directed RotationMonocropping RotationMonocropping 0 20 40 60 80 100 Inhibition (%) VOCs-mediated Rotation Monocropping Rotation Monocropping *** * *** *** *** Fig. 4 | Inhibition of pathogens by rhizosphere microorganisms in monocropping and rotation and determination of depleted bacteria in monocropping rhizosphere. a Directed inhibitory effect of rhizosphere microbial community on F. oxysporum (P=0.006,n= 3 biologically independent samples). bVOCs-mediated inhibitory effect of rhizosphere microbial com=munity on F. oxysporum (P=0.006,n= 3 biologically independent samples). cEnrichment analysis of differential genes of culturable microorganisms in rhizosphere of monocropping and rotation. Rich Factor, the ratio of the number of differential genes in the metabolic pathway to the number of all genes annotated to the pathway (n=3 biologically independent samples). dAbundance of representative genes associated with pathogen inhibition in cultivable microorganisms of monocropping and rotation. TPM, transcripts Per million (P< 0.001, n= 3 biologically independent samples). Each bars represents the mean ± SD. eThe relative abundances of OTUs in the monocropping and rotation samples. The blue bars show the relative abundance of monocropping-enriched OTUs in monocropping samples (<0.002% in rotation samples). The red bars show the relative abundance of monocroppingdepleted OTUs in rotation samples (<0.002% in monocropping samples). The nine depleted or enriched OTUs with >97% similarity to isolates are shown (n= 3 biologically independent samples). Each bars represents the mean ± SEM. fEffects of peanut root exudates from monocropping and rotation on growth of monocropping-depleted and enriched bacteria (P<0.001,n= 12 biologically independent samples). OD, optical density. Asterisks above the bars indicate statistically significant differences between treatments based on two-sided tests by Student’st-test (*P< 0.05, **P< 0.01, ***P< 0.001). Each bars represents the mean ± SD. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 6 peanuts and co-cultured on a plate with F. oxysporum (Fig. 5c). When the monocropping rhizosphere suspensions were supplemented with SynComs, the pathogen growth was inhibited by 64.4%, with the inhibition 4-fold higher than that of the control without rhizosphere suspension (ANOVA, F= 33.501, P< 0.001, Fig. 5d). By contrary, an inactivated SynCom did not make up for the suppression ability of monocropping rhizosphere microbiome (Fig. 5d). The suppression ability on pathogen increased with the supplementation of SynCom diversity, indicating depleted strains compensate the ability of monocropping rhizosphere microbiome to resist pathogen invasion. Further, we tested the compensatory effect of the depleted strains in experiments with sterile peanut seedlings (Fig. 5e). Compared with seedling inoculation with the monocropping rhizosphere suspension, supplementation with the depleted strains significantly improved rhizosphere resistance to F. oxysporum invasion (ANOVA, F=71.755, P< 0.001, Fig. 5f). Consistent with in vitro antagonism, the best inhibitory effect was obtained when the SynCom composed of 7 strains was supplemented. In the treatment with no SynCom inoculation, the root rot protection collapsed. However, compared with the treatment with rhizosphere community, supplementation of the depleted strains did not affect plant growth (ANOVA, P> 0.05, Supplementary Fig. 8). h 7 strains 4 strains 2 strains d f 7 strains4 strains2 strains Antagonism ab e g SynCom 0/2/4/7 strains RhiCom + Antagonism F. oxysporum RhiCom + SynCom RhiCom Control 0/2/4/7 strains Replenishment experiment in vitro Field experiment c Antagonism of SynCom in vitro Replenishment experiment in vivo Control Control RhiCom 0 2 4 7 0 1 2 3 4 Mycelial diameter (cm) (strains) abbccdde Control RhiCom 0 2 4 7 0 20 40 60 80 100 120 Incidence (%) (strains) abb c cd abbc 247 0 20 40 60 80 100 120 Incidence (%) Control (strains) inactivated 7 strains 21 35 combinations 0 strain Mycelial diameter (cm) (strains) Control 0 2 4 7 0 1 2 3 4 5 aa b b c 1 combination, 4 plots 35 combination, 35 plots 21 combination, 21 plots 1 combination, 4 plots randomized block Fig. 5 | Assessment of suppression ability of monocropping-depleted strains on pathogen development and peanut root rot. a Overview of experiments performed to determine the supplemental effect of monocropping-depleted strains on pathogen invasion. bEffects of re-inoculation of different strain combinations on the growth of F. oxysporum (P<0.001,n control =8,n 7 =8,n 4 =35×8,n 2 =21×8, n 0 = 8; biologically independent samples). cOverview of a replenishment experiment performed to determine the inhibitory effect of the addition of depleted strains on F. oxysporum in vitro. dInhibition of F. oxysporum by monocropping rhizosphere community supplemented with different strain combinations (P<0.001, control =6,n 7 =6,n 4 =35×6,n 2 =21×6,n 0 =6).eOverview of a replenishment experiment performed to determine the disease index of peanuts in vivo. fEffect of monocropping rhizosphere community supplemented with different strain combinations on root rot occurrence (P<0.001, control =5,n 7 =5,n 4 =35×5, n 2 =21×5,n 0 =5).gRandom block design of a field trial plot. hEffects of reinoculation of different strain combinations on root rot occurrence in the field (P<0.001,n control =4,n 7 =4,n 4 =35×4,n 2 = 21 × 4). In figures b, d, f and h, different letters above the bars indicate statistically significant differences between treatments. Pvalues were calculated using ANOVA’s test based on two-sided. RhiCom, treatment with monocropping rhizosphere community; SynCom, supplemented synthetic community composed of monocropping-depleted strains to monocropping rhizosphere community; 0, a SynCom composed of 7 inactivated depleted strains;2, SynComs composed of 2 depleted strains; 4, SynComs composed of 4 depleted strains; 7, a SynCom composed of 7 depleted strains. Eachbarsrepresents the mean ± SD. Horizontal bars within boxes represent the median. The tops and bottoms of boxes represent 75th and 25th quartiles, respectively. The upper and lower whiskers represent the range of non-outlier data values. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 7 Finally, we conducted field experiments to verify the compensatory effect of the depleted strains on the occurrence of root rot in monocropped peanuts (Fig. 5g). Peanut seeds were inoculated with SynComs composed of 7, 4, or 2 strains by soaking. Results indicated compared with the control without their inoculation, SynComs significantly reduced the incidence of peanut root rot (ANOVA, F= 16.169, P< 0.001, Fig. 5h). SynComs composed of 7 bacterial strains were most effective (Fig. 5h). Similarly, no SynCom tested significantly promoted peanut growth compared to the control (ANOVA, P> 0.05), indicating their biocontrol effect in the plant rhizosphere rather than a direct promotion of plant resistance (Supplementary Fig. 9). Discussion In the current study, we investigated the influence of agricultural practices on soil disease suppression. We further manipulated the microbiome of our soils aiming to bring back the capacity of rhizosphere to fight pathogens and disease. We found that the depletion of rhizosphere specific bacteria in response to monocultures of peanut and its associated root exudates can reduce the capacity of soils to suppress disease and further determined the future responses of plant to disease outcomes. On the contrary, crop rotations supported soils with a greater capacity to suppress disease, and we were ableto restore rhizosphere communities to protect soils from pathogens using microbial innocula. Our study adds to the consensus that monocultures have a reduced soildisease suppression capacity compared to crop rotations. Our study revealed that monocultures had a reduced soil disease suppression capacity. In particular, our five-year field peanut experiment, monocropping significantly increased the incidence of root rot disease, compared with fields subjected to rotation-grown peanut. Thus, these contrasting agricultural managements supported opposite capacity to control disease occurrence by preventing soil-borne pathogen invasion. We then tracked root rot occurrence over an entire peanut growing period in 2018. The results showed that disease indices and pathogen abundances were not significantly different under monocropping and rotation regimes at the seedling stage. However, there was a dramatic increase in disease incidence at later growth stages in monocropped peanut compared with rotation-grown peanut. Interestingly, we found significant differences in the rhizosphere microbiome of peanuts in monoculture and crop rotation from seedling stage to the entire plant development. These differences were associated with different disease outcomes, indicating the importance of rhizosphere microbiome for the development of these plants and their disease. Recent studies have shown that differences in rhizosphere microbial composition of seedlings lead to changes in microbial function, and eventually differentiate into two different results (diseased or healthy)23. Therefore, our work suggests that strengthening seedling management by focusing on rhizosphere microbiome could contribute to maintaining plant health. We then sought to understand the mechanisms behind the ability of the rhizosphere microbiome to support pathogen suppression during the seedling stage. As explained above, the ability of the rhizosphere microbiome from monocropped peanuts to inhibit pathogen was significantly lower than that from rotation-grown peanuts. These results indicated that the microflora at seedling stage could determine the suppression ability of plant rhizosphere to pathogen invasion. The underlying mechanisms behind inhibition would associate with the production of volatile organic compounds and antibiotics24,25. For instance, dimethyl sulfide, 2,5-dimethylcyclohexanone, and 6-methyl-3,5-pentadien-2-one that were not detected in the rhizosphere microbiome of monocropping had strong inhibition on pathogen growth even at low concentrations. In terms of rhizosphere microbiome function, the expression of genes associated with pathogen inhibition, including Isopentenyl-diphosphate Delta-isomerase, Glutamate decarboxylase, and 1-pyrroline-5-carboxylate dehydrogenase were significantly higher in rotation compared with monocropping26. Our results revealed the potential mechanisms behind the greater disease suppression capacity in croplands under rotation. The negative influence of root exudates in monoculture peanut field on key bacteria supporting suppression may also explain the reduced capacity to protect soils against pathogens and disease. This highlights the importance of low-abundance microbial species regulating plant rhizosphere resistance, as the ability of the community assembly without them in the monocropped peanut rhizosphere to suppress pathogen growth was reduced. In our study, the growthpromoting effect of monocropping root exudates on Burkholderia sp. and Stenotrophomonas sp. may lead to the occupation of ecological niche, and thus affect the rhizosphere depletion of bacteria with a weak response to monocropping root exudates such as Paenibacillus sp., Pantoea sp., Lysinibacillus sp., Enterobacter sp., Sporosarcina sp., Fictibacillus sp., and Pseudomonas sp. Accordingly, the enriched strains preferentially occupy root resources, and the depleted taxa are thereby unable to effectively participate in community assembly in the monocropped rhizosphere. We then investigated whether reintroducing the depleted microbial taxa could bring back the capacity of soils to resist pathogen invasions and avoid disease. Despite the growing interest in SynComs, the detailed mechanisms by which they operate remain largely unknown. We first determined the inhibitory capacity of microbial communities composed of random combinations of the deleted strains in monoculture compared with rotation croplands. Our result revealed that microbes that were lost under monoculture, participated in the suppression of fungal pathogens via synergistic interactions. We secondly reintroduced these strains into the rhizosphere of monocropped peanuts. The supplementation significantly alleviated pathogen attack of peanut root, strongly supporting the notion that the addition of low abundance microbes during community succession can improve the overall function of the rhizosphere community27. The inhibitory capacity of microbial communities composed of random combinations of strains was consistent with an increased phylogenetic diversity, suggesting that all depleted strains participate in the suppression of fungal pathogens via synergistic interactions22.Indeed, our microbial inoculation experiments revealed that the reintroduction of depleted strains strongly protected the plant root against pathogen infection, while failing to promote plant growth. Data obtained by different approaches suggests that the substantial participation of the depleted strains in the resident community assembly could determine their success in fulfilling functional services in the rhizosphere4,28. Applications of single-function microbes or SynComs composed of multi-functional microbes generally have satisfactory outcomes in the laboratory setting but not in practice. Indeed, lack of consideration of the appropriate ecological niches for functional strains, factors driving the variation in resident microbial interactions, and the impact of environmental factors on microbial colonization ability can have undesirable effects29.Inthefield experiments presented in the current study, SynCom composed of 7 depleted strains showed an excellent ability to prevent root rot. SynComs composed of some depleted strains also controlled root rot, suggesting the stability of their application. Recently, a simplified SynCom, developed based on hostmediated microbial community selection, had effectively captured the dominant members of the maize root microbiota, inhibiting pathogen colonization30. However, it is unlikely that the such simplified SynComs would take over all the functions of rhizosphere microbiota31.Conversely, specific microbial taxa that drive resident microbial community assembly can amplify rhizosphere community functions. For instance, the addition of biological agents containing Bacillus amyloliquefaciens W19 protected banana from fungal pathogen infection by activating specificplant-beneficial bacterial genera (e.g. Pseudomonas) Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 8 to form a plant-benefitting consortium27. Consequently, the current study seeks to rationally control plant disease from a holistic microbial perspective, in that SymComs should switch core from basic functions to activate community functionality. Taken together, our results indicate that management and rhizosphere microbiomes play a role in supporting soil pathogen suppression against important soil disease. Our work shows that, compared with rotation managements, intensive monocropping strategies weakened the capacity of soils to prevent the entrance of pathogens to the rhizosphere, and promoted the development of soilborne diseases. Importantly, we provide experimental evidence that pathogen resistance in weakened rhizospheres may be restored by applying targeted microbial inocula. Therefore, our study highlights the role of management to fight against soil crop disease and suggests the active restoration of rhizosphere communities to promote soil disease suppression. Methods Field site description and disease assessment From April 2012, we conducted a field experiment at Yujiang County, Jiangxi Province, China. The soil at the study site is classified as Udic Ferrosol (FAO classification), covering an area of ~1 × 108ha in southern China. Because of low soil organic matter content and fertility, and climatic suitability, peanut (Arachis hypogaea L.) is a particularly popular crop in the region. The field experiment included two cropping regimes (treatments): (1) monocropping with peanut, the same peanut cultivar (Ganhua-5) was consecutively planted for the growing season (April–August) of each year; and (2) rotation, peanut was grown first (2012), and then maize (Zea mays L.), potato (Solanum tuberosum), and soybean (Glycine max) were ordinally planted in every other peanut planting year (Fig. 1a). Three plots of the two cropping regimes were laid out in a randomized block design, and lay fallow after harvest until the following sowing period. Commonly used management practices, including tillage, fertilizer application, and weed control, were applied manually32. In the 2012, 2014, and 2016, all plots were planted with peanut, and the severity of root rots was persistently evaluated at flowering stage. In 2018 peanut planting season, we further examined peanut root rots at seedling, flowering and pod-bearing stages (Fig. 1a). For each examination, 30 plants from each plot were carefully removed from the soil, and peanut root rot was evaluated using a five-class rating scale (0 = no lesions, 1 = small root lesions, 2 = central root lesions, 3 = large root lesions, 4 = dead plant)15. Overall, 540 plants (6 plots, 3 time points) were removed from the plots for examination. Following a gentle wash with tap water, the plant height, shoot and root fresh weight, root length, and nodule number were determined at each sampling. Sample collection and processing Bulk soil and rhizosphere soil samples were collected before the peanuts harvest in 2018. Before peanut planting, 10 soil cores with a depth of 20 cm were randomly selected from each plot. Every 5 soil cores were fully mixed into a singlecomposite soil sample as bulk soil. Excess soil on the roots was discarded by gently shaking the plants, and the remaining soil particles attached to the root surface were collected as rhizosphere soil. At seedling, flowering, and pod-bearing stage, 10 peanuts were randomly selected from each plot, and rhizosphere soil of every 5 peanut plants was fully mixed into a single composite soil sample. In total, 36 soil samples (6 plots, 3 time points) were collected. Soil DNA of rhizosphere soil collected from rhizosphere and bulk soil ware extracted for microbial community analysis. Roots from healthy or severe disease peanuts at pod-bearing stages were surface-sterilized in 3% hydrogen peroxide, and washed with sterile water and 70% ethanol. Excess fluid on the sterilized root surface was wiped off using sterilized filter papers and root samples were cut into pieces. All the root samples were stored at −80 °C before DNA extraction. In addition, a portion of diseased peanut roots were used to isolate potential pathogens. Sequencing analysis of endophytic fungal community Root samples were ground in liquid nitrogen and DNA was extraction using the MiniBEST Plant Genomic DNA Extraction Kit (Takara, Japan) (Fig. 1b). Specific sequences were amplified using the primer pairs ITS1F(5’-CTTGGTCATTTAGAGGAAGTAA-3’)/ITS2(5’-GCTGCGTTCTTC ATCGATGC-3’). The samples were initially denatured for 3 min at 95 °C; this was followed by 27 cycles of denaturation (95 °C, 30 s), annealing (55 °C, 30 s), and elongation (72 °C, 45 s). The PCR program ended with a 10-min incubation at 72 °C. The PCR products were separated by electrophoresis on 1% agarose gel. All samples were pooled in equimolar concentrations and then sequenced with a paired-end protocol at Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China) using the Illumina MiSeq platform, according to the manufacturer’s instructions. Raw fastq files were quality-filtered using QIIME33.Lowquality sequences (<150 bp long, with an average quality score <25) were removed. The reads were trimmed and assigned based on unique 7-base barcodes. The barcode and primer sequences were then removed. The forward and reverse reads were incorporated into fulllength sequences based on the thresholds: overlap length >10 bp and mismatch ratio <0.2. After discarding unqualified reads, the OTUs were assigned at 97% identity similarity level using UPARSE34. Chimeric sequences were identified and removed using UCHIME35. Taxonomic assignment was performed using UNITE database (v7.0) for fungi36. Isolation and identification of potential pathogens Potential fungal pathogens were isolated from peanut roots that displayed disease symptom37.Briefly, a clean knife was used to cut the pathogen-infected roots into sections. The root sections were surfacesterilized (submerged in 4 v/v sodium hypochlorite for 5 min), washed (two times, in sterile distilled water), and placed on a PDA plate containing streptomycin and penicillin (20 μg/mL) to obtain fungal isolates38,39. DNA was extracted from each fungal culture by using the FastDNA Spin Kit (MP Biomedicals, Santa Ana, CA), according to the manufacturer’s instructions. DNA sequences from the fungal 18S rRNA region were amplified using primers NS1(5′- GTAGTCATATGCT TGTCTC-3′)andNS8(5′-TCCG-CAGGTTCACCTACGGA-3′)40.ThePCR mixture contained (per 50 μL) 1.5 U of Taq polymerase (Red Taq, Sigma Chemical Co.) and the following reagents: 1 × Sigma PCR buffer, 0.20 mM PCR nucleotide mix (Promega), 4.0mM MgCl 2 ,6.25mg bovine serum albumin (Roche Diagnostics), and 25 pmol of each primer. For the amplification reaction, the DNA samples were initially denatured for 3 min at 95 °C. This was followed by 35 cycles of denaturation (94 °C, 30 s), annealing (57 °C, 30 s), and elongation (72 °C, 105 s).The PCR program ended with a 2-min incubation at 72 °C40.Each fragment was compared phylogenetically to sequences of known species in the GenBank database of the National Center for Biotechnology Information (NCBI) by using BLAST. Phylogenetic trees were analyzed using MEGA v5. Phylogenetic trees were constructed by using the neighbor-joining (NJ) method. Determination of pathogenicity of potential pathogens Potential pathogens were highly enriched isolates from the diseased roots, including F. oxysporum and F. solanum. A fungal plug (6-mm diameter) was transferred to PDA medium and cultured at 28 °C for 7 days. Obtained fermentation solution was filtered to remove mycelia, and centrifuged for 10 min (3000 × g) to retain precipitation. The precipitated spores were re-suspended with sterile water, and the concentration of spore suspension was adjusted to 109CFU/ mL. To confirm pathogenicity of potential pathogens, peanuts were planted in glasshouse (30 °C, 70% relative humidity, light intensity 500 μMm −2s−1). 10 mL spore suspensions of F. oxysporum or F. Article https://doi.org/10.1038/s41467-023-43926-4 Nature Communications | (2023) 14:8126 9