Green manure-driven suppression of Fusarium solani through regulating rhizosphere microorganisms to reduce tobacco root rot incidence

Green manure-driven suppression of Fusarium solani through regulating rhizosphere microorganisms to reduce tobacco root rot incidence | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL Land Degradation & Development This is a preprint and has not been peer reviewed. Data may be preliminary. 27 April 2025 V1 Latest version Share on Green manure-driven suppression of Fusarium solani through regulating rhizosphere microorganisms to reduce tobacco root rot incidence Authors : Zhao Wenjun , Wang Zhengxu , Zhao Wenjia , Chen Hua , Liu Kui , Qian Yingying , Lu Junping , … Show All … , Libo Fu , Wang Jiansong , Yang Jizhou , Cao Jing , Feng Yu [email protected] , and Weidong Cao 0000-0003-1787-9165 Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.174576820.06958313/v1 370 views 188 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Green manure-tobacco rotation system has proven to be an effective strategy for improving soil nutrients and alleviating soil-borne fungal diseases. However, the differential efficacy of various green manures against tobacco root rot and their underlying microbial regulatory mechanisms remains unclear. Through pot-based experiments, this study systematically evaluates the disease-suppressive effects of two green manure, smooth vetch ( Vicia villosa ) and rape ( Brassica campestris ), applied at two incorporation rates on tobacco root rot (caused by Fusarium solani ). Results indicated that green manure incorporation significantly reduced pathogen abundance by 36.1-64.7%, decreased root rot disease incidence by 10-20%, with smooth vetch exhibiting superior disease suppression compared to rape. Smooth vetch incorporation enriched a higher abundance of Bacillus spp. (Bacillus niacini and Bacillus megaterium) in rhizosphere soil, increasing by more than 2 times compared to no incorporation . Co-occurrence network analysis identified four microbial modules, among which Module 0 exhibited a significant negative correlation with pathogen related abundance. Within Module 0, bacterial taxa, particularly Bacillus spp. , occupied central positions with extensive node interactions, while fungi maintained higher relative abundance. This module also contained other disease-resistant communities, including Paenibacillus, Lysobacter soli , Chaetomium sphaerale and Rhizopus arrhizus . Notably, smooth vetch treatment enhanced soil available nutrients, (especially alkaline nitrogen content) more effectively than rape treatment, favoring the increase of these disease-resistant communities. Collectively, smooth vetch demonstrates superior capacity in enhancing resistance to tobacco root rot and reducing disease incidence, offering an effective solution for tobacco soil-borne disease prevention and control. Green manure-driven suppression of Fusarium solani through regulating rhizosphere microorganisms to reduce tobacco root rot incidence Zhao Wenjun 1,2 , Wang Zhengxu 1 , Zhao Wenjia 3 , Chen Hua 4 , Liu Kui 1 , Qian Yingying 2 , Lu Junping 2 , Fu Libo 4 , Wang Jiansong 1 , Yang Jizhou 1 , Cao Jing 1 , Feng Yu 4* , Cao Weidong 5 , 1. Hongta Tobacco (Group) Co., Ltd, Yuxi, Yunnan, 653100, China; 2. Raw Material Center of China Tobacco Yunnan Industrial Co., Ltd, Kunming, Yunnan, 650000, China; 3. College of Plant Protection, Yunnan Agricultural University, Kunming, Yunnan, 650500, China; 4. Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences, Kunming, Yunnan, 650000, China; 5. State Key Laboratory of Efficient Utilization of Arable Land in China, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Abstract Green manure-tobacco rotation system has proven to be an effective strategy for improving soil nutrients and alleviating soil-borne fungal diseases. However, the differential efficacy of various green manures against tobacco root rot and their underlying microbial regulatory mechanisms remains unclear. Through pot-based experiments, this study systematically evaluates the disease-suppressive effects of two green manure, smooth vetch ( Vicia villosa ) and rape ( Brassica campestris ), applied at two incorporation rates on tobacco root rot (caused by Fusarium solani ). Results indicated that green manure incorporation significantly reduced pathogen abundance by 36.1-64.7%, decreased root rot disease incidence by 10-20%, with smooth vetch exhibiting superior disease suppression compared to rape. Smooth vetch incorporation enriched a higher abundance of Bacillus spp. (Bacillus niacini and Bacillus megaterium) in rhizosphere soil, increasing by more than 2 times compared to no incorporation . Co-occurrence network analysis identified four microbial modules, among which Module 0 exhibited a significant negative correlation with pathogen related abundance. Within Module 0, bacterial taxa, particularly Bacillus spp. , occupied central positions with extensive node interactions, while fungi maintained higher relative abundance. This module also contained other disease-resistant communities, including Paenibacillus, Lysobacter soli , Chaetomium sphaerale and Rhizopus arrhizus . Notably, smooth vetch treatment enhanced soil available nutrients, (especially alkaline nitrogen content) more effectively than rape treatment, favoring the increase of these disease-resistant communities. Collectively, smooth vetch demonstrates superior capacity in enhancing resistance to tobacco root rot and reducing disease incidence, offering an effective solution for tobacco soil-borne disease prevention and control. Key words green manure, smooth vetch, Fusarium, Bacillus, green manure-tobacco rotation 1 Introduction In recent years, due to continuous cropping and unreasonable application of chemical fertilizers, the incidence of tobacco root rot has expanded and intensified in tobacco-growing regions across China. In severely affected areas, the disease incidence can reach 30% (Gai et al., 2022), causing significant economic losses to tobacco production (Gai et al., 2023). Tobacco root rot is primarily caused by fungal pathogens of the genus Fusarium ( Fusarium spp. ), which infect the vascular bundles of tobacco roots, leading to rot, obstruction of water transport and nutrient absorption, and ultimately plant death (Qian et al., 2017). The disease development is influenced by multiple factors, including soil pathogen concentration, pathogens in plant residue, disease resistance of tobacco varieties, and environmental conditions (Liu et al., 1993). Current strategies for controlling tobacco root rot include breeding resistant cultivars (Qiu et al., 2019; Xie et al, 2024), biological control (Wang et al., 2020; Guo et al., 2020), chemical control (Liu et al., 2021; Huang et al., 2014), and agricultural practices such as crop rotation (Chuan et al., 2016; Xue et al., 2015). Among these, crop rotation, as a traditional and cost-effective agricultural practice, has long been utilized for disease management (Zhou et al., 2023). As a traditional agricultural practice, the incorporation of green manure into tobacco rotation systems demonstrates significant agronomic benefits, particularly in enhancing soil fertility, improving soil quality, and regulating microbial community dynamics. Studies indicate that green manure treatments can influence functional microbial communities antagonistic to pathogenic fungi. For instance, Bacillus spp., enriched in soils treated with smooth vetch ( Vicia villosa Roth var. glabrescens ), exhibit well-documented biocontrol and growth-promoting effects (Pieterse CM et al., 2024; Ryan R P et al., 2008). Recent research (Ruan et al., 2024) further demonstrates that endophytic bacteria from smooth vetch inhibit banana fusarium wilt. Additionally, rape ( Brassica campestris ) produces isothiocyanates with antifungal activity against tobacco root-knot nematodes (Fourie et al., 2016; Soheili et al., 2017). However, the regulatory effects and microbial mechanisms of these green manures on tobacco root rot remain unclear. Therefore, it is necessary to investigates the potential roles of green manure such as smooth vetch and rape in suppressing soil-borne diseases. In present study, a pot experiment was conducted to evaluate the efficacy of smooth vetch and rape incorporation as green manures in suppressing tobacco root rot following pathogen inoculation. Rhizosphere microbial community dynamics were analyzed temporally post-inoculation, while co-occurrence network analysis identified key pathogen-antagonistic taxa. This investigation elucidates the microbiological mechanisms underlying green manure-mediated disease suppression, providing theoretical foundations for integrated management strategies against soil-borne pathogens. 2. Materials and Methods 2.1 Experimental Materials The soil used for potting was collected from the 0-20 cm tillage layer of a tobacco field in Longjie Town (102°53′29.418′′E, 24°39′29.654′′N), Chengjiang City, Yunnan Province. Basic soil properties were: pH 7.28, organic matter 31.8 g kg⁻¹, total nitrogen 2.5 g kg⁻¹, total phosphorus 2.6 g kg⁻¹, total potassium 16.0 g kg⁻¹, available nitrogen 210 mg kg⁻¹, available phosphorus 115 mg kg⁻¹, available potassium 346 mg kg⁻¹, chloride ion 55.0 mg kg⁻¹, and bulk density 1.16 g cm⁻³. Green manures included smooth vetch ( Vicia villosa Roth var. glabrescens , cv. Yunguangzaoshao) and rape ( Brassica campestris , cv. Yunyouza 28). The nutrient content of smooth vetch was 34.2 g/kg N, 3.5 g/kg P, and 21.8 g/kg K, while rape contained 20.1 g/kg N, 2.7 g/kg P, and 25.3 g/kg K. Tobacco seedlings (cv. K326) were provided by the Yuxi Tobacco Company. Pots (15 cm height, 10 cm upper diameter) were used for cultivation. The pathogen Fusarium solani was obtained from the Yunnan Academy of Agricultural Sciences. Potato Dextrose Agar (PDA) medium was prepared as follows: 200 g of peeled and diced potatoes were boiled in distilled water for 20 minutes, filtered through gauze, mixed with 20 g glucose, and adjusted to 1,000 mL. Half of the solution was autoclaved at 121°C for 20 minutes, while the other half was supplemented with 8 g agar, sterilized, and poured into petri dishes. 2.2 Experimental Design and Procedures The experiment included two green manure types (smooth vetch and rape), two incorporation rates (1× and 1.5× field rates), two inoculation methods (with or without F. solani ), and a control without green manure, totaling 10 treatments (Table 1) with 10 pots each. Table 1 Experimental treatments and descriptions CK Control: no green manure; no pathogen inoculation S1 Smooth vetch, 1× field rate; no pathogen S2 Smooth vetch, 1.5× field rate; no pathogen R1 Rape, 1× field rate; no pathogen R2 Rape, 1.5× field rate; no pathogen F No green manure; inoculated with pathogen S1F Smooth vetch, 1× field rate; pathogen S2F Smooth vetch, 1.5× field rate; pathogen R1F Rape, 1× field rate; pathogen R2F Rape, 1.5× field rate; pathogen During pot preparation, the soil matrix underwent sequential processing: initial sieving to remove gravel (>2 mm), plant residues, and organic debris, followed by precision amendment with green manure. Two application rates were established—the conventional field rate (22,500 kg ha⁻¹) and 1.5× conventional rate—calculated based on a soil mass of 2,250 tones ha⁻¹. This protocol generated defined green manure-to-soil mass ratios: 1:100 (10 g green manure + 1 kg soil) and 1.5:100 (15 g green manure + 1 kg soil). Green manure was sheared into 1 cm segments using sterilized scissors, thoroughly homogenized with the processed soil matrix, and subsequently loaded into pots. Fertilizers were applied at twice the field rate: N (180 kg ha⁻¹), P₂O₅ (90 kg ha⁻¹), and K₂O (540 kg ha⁻¹). Healthy tobacco seedlings (4-6 true leaves) were transplanted into pots and placed outdoors with 50 cm spacing between treatments. Fungal hyphae from stock cultures were transferred to fresh PDA plates and incubated at 28°C for 4 days. Ten 5-mm mycelial discs were inoculated into 200 mL PDA broth and shaken at 180 rpm (28°C) for 5 days. Spore suspensions were adjusted to 1×10⁶–10⁷ CFU mL⁻¹ using a hemocytometer. Seven days post-transplanting, 50 mL spore suspension was applied to the stem base. Control plants received sterile water. A second inoculation was performed after 7 days. 2.3 Disease Incidence, Severity, and Control Efficacy Disease incidence and severity were assessed at the onset of symptoms in the F treatment (no green manure, inoculated). Disease severity was graded according to ”GB/T 23222-2008 Tobacco disease severity classification and survey methods.” \(\text{Incidence\ }\left(\%\right)=\frac{\text{ni}}{N}\times 100\)(1-1)\(\text{Disease\ index}=\frac{\sum ni*vi}{N*4}\ \times 100\) (1-2)\(\text{Relative\ control\ efficacy}\mathbf{\ (\%)}=\frac{F-T}{F}\times 100\)(1-3) In the formula, ni represents number of infected plants, N represents total plants surveyed; vi represents the disease level (0, 1, 2, 3, 4); F represents the disease index of the control group, and T represents the disease index of the treatment group. The disease severity classification (surveyed by plant unit): Level 0, no disease on the entire plant; Level 1, 0-25% of leaves wilted; Level 2, 26%-50% of leaves wilted; Level 3, 51%-75% of leaves wilted; Level 4, 76%-100% of leaves wilted, with the diseased plant essentially dead. 2.4 Soil Sampling Rhizosphere soil samples were collected from fully diseased plants (highest severity index) in treatment F. For CK and green manure-only treatments, four healthy plants per group were uprooted with all pot soil. Rhizosphere soil was obtained by gently shaking roots and brushing tightly adhered soil (Fan et al., 2019; Li Guilong et al., 2022). Samples were sieved (2 mm) and stored at −80°C for DNA extraction. Bulk soil samples were air-dried and sieved to 0.149 mm for physicochemical analysis. The same protocol was applied to pathogen-inoculated treatments (with/without green manure), sampling four plants per group for rhizosphere and non-rhizosphere soils. 2.5 Soil Physicochemical Analysis The measurement indices and methods were as follows: soil organic matter (SOM) content was determined by potassium dichromate external heating method; alkaline hydrolyzable nitrogen (AN) by alkaline diffusion method; available phosphorus (AP) by 0.5 mol/L NaHCO 3 and measured by molybdenum-blue colorimetry; available potassium (AK) by 1 mol/L ammonium acetate extraction-flame photometry; soil pH by potentiometric method using a pH meter with a water-to-soil ratio (v/m) of 2.5(Bao, 2000). 2.6 Microbiological analysis Total DNA was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek). The V3–V4 region of bacterial 16S rRNA and fungal ITS regions were amplified using primers 338F/806R and ITS1F/ITS2R, respectively. PCR products were purified and sequenced on an Illumina platform. Fusarium solani abundance was quantified using specific primers (F: 5′-GCAGCGAAATGCGATAAG-3′; R: 5′-TCTCCAGTTGCGAGGTGT-3′) on an ABI7300 system. Standard curves were generated using serial dilutions of recombinant plasmids. Raw sequences were quality-controlled using fastp (v0.20.0; Chen et al., 2018), which trimmed reads with terminal bases <Q20 and removed reads with 50-bp window average quality <20 or length merged paired-end reads (min. overlap: 10 bp, max. mismatch: 0.2), after which samples were demultiplexed via barcodes (0 mismatch) and primers (≤2 mismatches). UPARSE (v7.1; Edgar et al., 2013) clustered sequences into ASVs (97% similarity; Stackebrandt et al., 1994) and removed chimeras. Taxonomic annotation was performed using RDP classifier (v2.2; Wang et al., 2007) against Silva 16S rRNA (v138) and UNITE databases (70% threshold). Raw data were deposited in NCBI SRA (Accession: PRJNA1253315). A weighted gene co-expression network analysis (WGCNA) was performed using the R package WGCNA (v1.70-3; Langfelder & Horvath, 2008) on microbial taxa. OTUs present in ≥80% of soil samples were retained, resulting in merged bacterial (1,703) and fungal (232) OTU tables. Treatments were categorized into two groups: ”Healthy” (CK, S1, S2, R1, R2; control and green manure-only) and ”Diseased” (F, S1F, S2F, R1F, R2F; pathogen-inoculated). Each treatment had 4 replicates, resulting in 20 samples each for the Healthy group and the Diseased group. Spearman correlation matrices were generated with FDR-corrected P -values, retaining robust interactions (|r| ≥0.7, P < 0.01). Separate bacterial-fungal networks were constructed for Healthy and Diseased groups, and a combined cross-kingdom network was analyzed using Gephi (v0.10.0) to identify top four modules by abundance. Network parameters (average degree, connectivity, path length) and node topological properties (degree, closeness centrality, eigenvector centrality) were computed via igraph (v1.2.9; Csárdi & Nepusz, 2006) using its degree , edge_connectivity, average.path.length , closeness , and evcent functions. 2.7 Data statistical analysis One-way ANOVA with Fisher’s LSD post hoc test (P < 0.05) for multi-group comparisons. Two-way ANOVA to assess effects of sampling time and green manure treatments on microbial diversity. The α-diversity indices and PCoA (Bray-Curtis distance) calculated via the R package vegan (v2.5-7; Oksanen et al., 2007), ANOSIM and PERMANOVA to evaluate treatment impacts on community structure. Wilcoxon rank-sum test for differential taxa identification (threshold: |log2(fold change)| ≥1, P < 0.01), with results visualized through volcano plots. 3. Results 3.1 Disease incidence, severity, and control efficacy of tobacco root rot Compared with F treatment, all green manuring treatments (S1F, S2F, R1F, R2F) decreased the tobacco root rot disease incidence 10-20% (Table 2). At 20 days after pathogen inoculation, the F treatment reached 100% disease incidence, whereas 1–2 plants remained asymptomatic in green manure incorporation + pathogen inoculation treatments. Smooth vetch treatments (S1F, S2F) showed lower disease incidence than rape treatments (R1F, R2F). Disease indices increased across all treatments over time, but rose more slowly in green manure incorporation + inoculation groups compared to F. By 20 days after inoculation, smooth vetch incorporation + inoculation treatments (S1F, S2F) exhibited lower disease indices than rape incorporation groups. Regarding relative control efficacy, smooth vetch incorporation + inoculation demonstrated superior suppression of Fusarium solani compared to rape incorporation + inoculation. Additionally, 1.5 times incorporation rates of smooth vetch (S2F) outperformed conventional rates (S1F). Table 2 Disease incidence of tobacco after inoculation with the pathogen under different treatments 4rd d 13rd d 20rd d 4rd d 13rd d 20rd d 4rd d 13rd d 20rd d F 50 90 100 8.05 13.71 40.51 – – – S1F 30 60 80 2.27 6.86 16.27 71.80 49.97 59.84 S2F 30 50 80 2.20 6.19 13.01 72.67 54.85 67.88 R1F 40 60 90 4.35 7.55 23.61 45.96 44.93 41.72 R2F 30 70 90 4.40 6.03 24.83 45.34 56.02 38.71 3.2 Soil Physicochemical properties and absolute pathogen abundance in soil Smooth vetch incorporation significantly decreased soil pH compared to the control (CK), whereas other treatments showed minimal changes. All green manure treatments increased soil organic matter (SOM), with smooth vetch exhibiting higher SOM accumulation than rape. Alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), and available potassium (AK) followed similar trends but with smaller inter-treatment differences. Especially in terms of AN content, before and after inoculation with pathogen, the increase in smooth vetch was more significant than that in rape (Table 3). Quantitative PCR revealed significantly reduced F. solani abundance in S1F, S2F, R1F, and R2F compared to F (Figure 1). No significant differences were observed between incorporation rates. Table 3 Soil physical and chemical properties of different treatments CK 7.61±0.03ab 34.8±0.36e 136.6±1.47g 47.3±1.34e 179.2±13.08b S1 7.51±0.03cd 38.8±0.6ab 157.3±2.04cd 60±0.76ab 227.7±7.65a S2 7.46±0.02d 38.4±0.51abc 151.7±2.02de 58.1±1.96abcd 245.3±15.3a R1 7.53±0.01bcd 37.4±0.2bcd 147.8±3.94ef 54.1±1.26d 214±5.42ab R2 7.58±0.01abc 37±0.81cd 145.9±0.52ef 56.1±1.5bcd 214.2±6.49ab F 7.66±0.06a 36.3±0.48de 141.1±2.88fg 44.2±0.83e 181.1±6.09b S1F 7.58±0.02abc 39.2±0.61a 169.8±5.92ab 59.5±2.37abc 252.2±14.03a S2F 7.6±0.03ab 38.1±0.86abc 172.2±2a 60.1±0.72ab 247.9±24.23a R1F 7.6±0.03ab 37.9±0.28abcd 161.3±3bc 55.6±1.57cd 222.5±21.06a R2F 7.62±0.01ab 38.8±0.76ab 153.3±4.23cde 60.6±0.76a 243.2±12.34a Figure 1 Quantity of the pathogen in different treatments 3.3 Bacterial communities of rhizosphere soil Bacterial Shannon index showed no significant changes, while Chao1 index decreased ( P < 0.05) in green manure treatments. S2F significantly reduced microbial richness compared to S1, S2, and F (Figure S1). Principal Coordinates Analysis (PCoA) based on Bray-Curtis distances revealed distinct microbial community structures (Figure 2). Smooth vetch incorporation treatments exhibited greater divergence from CK than rape treatments in the healthy group. Diseased group samples displayed higher dispersion and reduced P -values, indicating increased inter-treatment variability post-inoculation. Figure 2 PCoA analysis of rhizosphere soil bacterial communities in healthy group (a) and diseased group (b) Dominant bacterial phyla included Actinobacteriota (29.9%), Proteobacteria (21.4%), Acidobacteriota (12.2%), and Chloroflexi (12.2%). At the genus level, Intrasporangium (6.8%), Sphingomonas (3.8%), and Bacillus (3.2%) were predominant (Figure S2). We analyzed the differential species in Healthy and Diseased groups from two green manure treatments. In the smooth vetch incorporation treatment (Figure 3a) , 28 OTUs were significantly upregulated (compared to no green manure incorporation), and after pathogen inoculation (Figure 3b) , 41 OTUs were significantly upregulated ( P < 0.01). Meanwhile, downregulated OTUs decreased from 7 to 3 before and after inoculation (Figure 3a, b) . The most significantly different OTUs were primarily upregulated ones. Among these upregulated OTUs, Bacillus (bac_OTU20490) was present among the top 8 OTUs with the greatest differences both before and after inoculation, increasing by 2.7 and 2.3 times, respectively (Table S2). After inoculation, the OTUs with the most significant differences were mainly concentrated in the upregulated bacterial communities, including not only Bacillus (bac_OTU20490) but also Massilia (bac_OTU19123), Rhizobium (bac_OTU20430), Methylotenera (bac_OTU18621), and others. In the rape treatment (Figure 3c) , 10 OTUs were significantly upregulated (compared to no green manure incorporation), and after pathogen inoculation (Figure 3d) , 11 OTUs were significantly upregulated ( P < 0.01) (compared to pathogen-only treatment). The downregulated OTUs were 3 and 2, respectively. After inoculation, the significantly different OTUs were mainly concentrated in the upregulated bacterial communities. Figure 3 Volcano plots illustrating significant changes in bacterial communities under different green manure treatments in healthy and diseased groups. The top 8 statistically significant ( P < 0.01) up-regulated or down-regulated OTUs are specifically labeled. a and c show significant differences in OTUs for smooth vetch and rape treatments, respectively, compared to the CK. b and d illustrate significant differences in OTUs for incorporated smooth vetch and rape treatments after pathogen inoculation, respectively, compared to the F. 3.4 Fungal Communities of Rhizosphere Soil Fungal diversity (Shannon index) and richness (Chao1 index) increased significantly ( P < 0.05) with smooth vetch incorporation but declined post-inoculation (Figure S3). PCoA indicated greater structural divergence in smooth vetch treatments compared to rape (Figure 4). Figure 4 PCoA analysis of the rhizosphere soil fungal community Dominant fungal phyla included Ascomycota (63–87%), Chytridiomycota (2.5–15.8%), and Basidiomycota (1.1–10.6%). At the genus level, Chaetomium , Fusarium , and unclassified_f__Didymellaceae had relatively high proportions (Figure S4). Analysis of differential species in fungal communities revealed distinctly different patterns of change compared to bacteria. In the smooth vetch treatment’s healthy group (Figure 5a), significantly upregulated fungal taxa included Chaetomium (fungi_OTU1131, fungi_OTU3411), Podospora (fungi_OTU3256), Aspergillus (fungi_OTU3264), and unclassified Fungi (fungi_OTU3227). In the diseased group (Figure 5b), only 4 OTUs were upregulated in smooth vetch, marking a notable decrease compared to the healthy group, included Chaetomium (fungi_OTU1131), Rhizopus (fungi_OTU3192), and unclassified Fungi (fungi_OTU3227, fungi_OTU3496). Notably, Chaetomium (fungi_OTU1131) remained consistently upregulated before and after inoculation.Significantly downregulated species increased from 2 before inoculation to 12 after inoculation, represented by Penicillium (fungi_OTU173, fungi_OTU3644), Mortierella_alpina (fungi_OTU3408), and unclassified_f__Microascaceae (fungi_OTU3184). In the rape treatment’s healthy group (Figure 5c), 5 OTUs were upregulated: Chaetomium (fungi_OTU1131), Penicillium (fungi_OTU3501, fungi_OTU3644), Aspergillus (fungi_OTU4896), and Lycoperdon (fungi_OTU3427). After inoculation (Figure 5d), only Apiotrichum (fungi_OTU3583) was upregulated, with Papulaspora (fungi_OTU3566) downregulated. Figure 5 Volcano plots illustrating significant changes in fungal communities under different green manure treatments in healthy and diseased groups. The top 8 statistically significant ( P < 0.01) up-regulated or down-regulated OTUs are specifically labeled. a and c show significant differences in OTUs for smooth vetch and rape treatments, respectively, compared to the CK. b and d illustrate significant differences in OTUs for incorporated smooth vetch and rape treatments after pathogen inoculation, respectively, compared to the F. 3.5 Comparative analysis of rhizosphere microbial co-occurrence patterns To elucidate microbial interaction dynamics under healthy and pathogen-inoculated conditions, weighted gene co-expression network analysis (WGCNA) was applied to construct bacterial and fungal networks for Healthy and Diseased groups (Figure 6a). Bacterial networks of healthy group exhibited higher complexity (Figure 10b) (1,417 nodes, 4,913 edges; average degree = 6.93) compared to fungal networks (81 nodes, 59 edges，degree = 1.46) (Table S1), characterized by dense connectivity and positive correlations dominating interactions (Figure 6e). Elevated clustering coefficients and assortativity indicated modular structures and homophilic node connections, suggesting robust cooperative interactions that stabilize community functionality. In contrast, fungal networks displayed sparser connectivity but higher edge density, reflecting compact yet centralized interaction patterns. The diseased group’s bacterial networks had lower edge density and connectivity (average degree = 5.48) (Table S1), with diminished clustering coefficients, signifying disrupted cooperative interactions and structural fragmentation under pathogen stress. Conversely, fungal networks exhibited enhanced complexity (122 nodes, 188 edges; average degree = 3.08) and increased modularity, indicative of intensified interactions and cluster formation (Figure 6b). We analyzed the top ten nodes with the highest connectivity in each network and found that in diseased bacterial networks (Figure 6c), highly connected OTUs (e.g., Bacillus OTU20490, Rhizobium OTU20430, Lysobacter OTU18612) were significantly enriched in smooth vetch treatments (S1F/S2F), suggesting their pivotal roles in network resilience and pathogen suppression. Notably, these hub taxa aligned with differentially abundant OTUs identified in prior analyses, underscoring their dual roles as topological keystones and functional defenders. In fungal networks, only Rhizopus OTU3192 was upregulated post-inoculation, while other central OTUs (e.g., Penicillium OTU173, Mortierella OTU3408) were downregulated, implying that fungal suppression mechanisms may rely on alternative pathways rather than direct hub-mediated interactions. Figure 6 a. Rhizosphere soil microbial ecological networks; b. Comparison of topological features (degree and closeness centrality) of nodes in the networks; c. Types of interactions among the top 10 hub nodes with the highest degree in the networks; Comparison of d. nodes and e. edges among the four networks, with significance of differences tested using Fisher’s LSD test. 3.6 Ecological Network Analysis of Rhizosphere Microbiota To explore the correlation between rhizosphere microbial community ecological network structure and pathogen abundance, an overall ecological network of rhizosphere microbial communities was constructed based on all treatment samples (Figure. 7a). The network comprised 299 nodes and 665 edges, with positive correlations dominating the network. The network was divided into four ecological modules (Modules 0-3), among which only Module 0’s relative abundance showed a significant negative correlation with the abundance of Fusarium solani (R²=0.39, P < 0.05; Figure. 7b). This module was significantly enriched in smooth vetch treatments, both in single application (S1/S2) and in inoculated treatments (S1F/S2F) (Figure. 7c), while no significant changes were observed in the rape treatments. The discovery of key ecological cluster Module 0 is significantly correlated with the increase in SOM, AN, AP, and AK content, as well as the decrease in soil pH (Figure. S5). At the phylum level (Figure. 7d), Module 0 was primarily composed of Ascomycota (57.2%), Firmicutes (16.6%), and Proteobacteria (9.9%). The core taxa at the genus level (Figure. 7e) included fungi such as Chaetomium (OTU1131/3411) and Rhizopus (OTU3192), as well as bacteria including Bacillus (OTU20490/20147) and Microlunatus (OTU18714). Figure 7 a Co-occurrence network of bacterial-fungal based on all treatments; b linear regression analysis of the abundance of key ecological module and pathogen quantity; c the relative abundance of key ecological module in each treatment and the community composition of ecological clusters d phylum level e genus level. Within Module 0, positive interactions were dominant (101 edges, 89% positive correlations), with high-connectivity nodes concentrated in Firmicutes (e.g., Bacillus_niacini OTU20490) and Proteobacteria (e.g., Ramlibacter OTU19452) (Figure. 8a-b). These hub taxa were significantly enriched in S1F and S2F treatments under pathogen stress, supporting the hypothesis that they inhibit pathogens through a dual strategy of ”network centrality + functional enrichment.” Fungal taxa (such as Chaetomium ), though having lower connectivity, exhibited higher abundance (>50% proportion), suggesting that fungi may inhibit pathogen proliferation by competing for ecological niches through numerical advantage (Figure. 7e). Collectively, our results identified Bacillus (Bacillus_niacini, Bacillus_megaterium), Paenibacillus, and Lysobacter soli among bacteria, and Rhizopus_arrhizus and Chaetomium_sphaerale among fungi, as key microbial communities in the suppression of tobacco root rot pathogens (Table 4). Figure 8 a Network of key ecological module and b topological structure characteristics of node degree and closeness centrality Table 4 Classification information of core microorganisms Bacteria Firmicutes Bacilli Bacillales Bacillaceae Bacillus s_Bacillus_niacini OTU20490 Activate plant immunity, antagonize the pathogen of bacterial wilt （Lee, 2021）， produce plant hormone IAA（Spaepen S, 2007） s_Bacillus_megaterium_NBRC_15308_ATCC_14581 OTU20147 Inducing systemic resistance in plants （ISR）（Ryan R P, 2008）（Bai，2024） Paenibacillales Paenibacillaceae Paenibacillus s_unclassified_g_Paenibacillus OTU19419 Promote plant growth, degrade pollutants, produce industrial enzymes and antimicrobial peptides （Yuan, P. 2023） Proteobacteria Alphaproteobacteria Rhizobiales Beijerinckiaceae Microvirga s_uncultured_bacterium_g__Microvirga OTU19945 Rhizobium; Enriching carbon decomposition genes （Zhang, Y., 2024; Ye, X., 2020） Related to sugars and organic acids （Liu, K., 2020）, Helps with plant growth （Wan, P., 2024） Gammaproteobacteria Burkholderiales Comamonadaceae Ramlibacter s_unclassified_g_Ramlibacter OTU19452 Coordinate multiple nitrogen metabolism pathways to promote the generation of ammonium nitrogen （Hu X, 2023） Xanthomonadales Rhodanobacteraceae g_norank_f_Rhodanobacteraceae s_uncultured_bacterium_g_norank_f_Rhodanobacteraceae OTU19754 Ammonia oxidizing denitrifying bacteria, adapted to a wide range of pH and high nitrogen environments, participate in nitrogen cycle regulation （Ramírez-Fernández L, 2021）。 Xanthomonadaceae Lysobacter s_Lysobacter_soli OTU18612 Plant fungal disease biocontrol agents （Tang, B., 2019）， Regulating various stress responses in microbial communities （Zhu, Y., 2022）， Activate antifungal antibiotic 2,4-DAPG （Wang, B. 2023） Fungi Ascomycota Sordariomycetes Sordariales Chaetomiaceae Chaetomium s_Chaetomium_sphaerale OTU3411 Degradation of lignocellulose （Feng, J., 2021）, Phosphorus solubility （Li, Q., 2022） Mucoromycota Mucoromycetes Mucorales Rhizopodaceae Rhizopus s_Rhizopus_arrhizus OTU3192 Production of fumaric acid （Gu, C., 2013） 4 Discussion 4.1 Regulation of microbial communities by green manures incorporation The experimental results demonstrated that smooth vetch exhibited significantly superior efficacy in suppressing tobacco root rot compared to rape, primarily attributable to its faster decomposition rate and nutrient release dynamics. Specifically, smooth vetch exhibited a lower carbon-to-nitrogen (C/N) ratio (12.3) than rape (20), a trait strongly correlated with accelerated decomposition and rapid nutrient mineralization (Ning et al., 2024). Green manures with low C/N ratios are rapidly converted into microbially available forms during decomposition, particularly facilitating nitrogen release, which enriches soil microbial communities with bioavailable nutrients (Liu et al., 2016; Duan et al., 2024). This rapid nutrient flux likely stimulates microbial activity and diversity, fostering a rhizosphere microenvironment antagonistic to fungal pathogens and enhancing plant resistance against soil-borne diseases. The optimal C/N ratio of smooth vetch supports balanced decomposition, maintaining microbial community equilibrium and promoting beneficial taxa proliferation. This equilibrium creates a pathogen-suppressive soil environment by limiting resource availability to pathogens. In contrast, rape’s higher C/N ratio delays decomposition (Su et al., 2022), resulting in slower nitrogen mineralization and transient nutrient limitations that constrain microbial activity and diversity, thereby reducing short-term disease suppression efficacy. This study demonstrated that the decomposition of smooth vetch promotes the enrichment of antagonistic microorganisms, including Bacillus spp ., aligning with previous findings (Liang et al., 2024; Ruan et al., 2024; Zu et al., 2022). These microbes competitively exclude pathogens via antimicrobial metabolite production (e.g., lipopeptides) or induction of plant systemic resistance (Sennett et al., 2021). Future investigations should prioritize characterizing secondary metabolites derived from smooth vetch-recruited beneficial microbes during decomposition to elucidate their roles in disease suppression. While rape releases glucosinolates, whose hydrolyzed products (e.g., isothiocyanates) exhibit pathogen-suppressive properties (Gardiner et al., 1999), its high C/N ratio likely slows the release kinetics of these compounds, preventing rapid accumulation to effective inhibitory concentrations. 4.2 Mechanism of green manure incorporation in suppressing fungal soil-borne diseases through microecological network reconstruction This study revealed that the incorporation of smooth vetch drives the reconstruction of soil microbial communities by specifically enriching core functional microorganisms, including bacteria such as Bacillus niacini , Bacillus megaterium , Paenibacillus , and Lysobacter soli , as well as fungi like Chaetomium sphaerale and Rhizopus arrhizus . These microorganisms collectively establish a bacterial network regulation and fungal resource competition dual-track disease suppression mechanism. Our study demonstrates that green manure incorporation significantly enhances soil available nutrient pools, with smooth vetch exhibiting particularly pronounced effects on AN elevation. This nitrogen enrichment establishes critical biochemical foundations for microbial-mediated disease suppression (Liang et al., 2023; Sun et al., 2020). Mechanistically, the increased AN availability strengthens bacterial community interactions while promoting rhizosphere-beneficial microbial proliferation, thereby achieving dual pathogen suppression and plant systemic resistance induction. Within the smooth vetch-induced disease-suppressive module, phylogenetically distinct yet functionally complementary bacteria—including Bacillus spp ., Paenibacillus sp ., and Lysobacter sp .—occupy central network nodes (Figure 8). These microorganisms exhibit remarkable metabolic diversity (Bertin et al., 2011), enabling direct antagonism against fungal pathogens. For instance, Bacillus niacin produces peptide or non-peptide antibiotic compounds (Stein et al., 2005), which directly inhibit pathogens and modulate microbial community structure, while Lysobacter sp. secretes chitinases to degrade fungal cell walls. Additionally, Bacillus niacin and Bacillus megaterium trigger Induced Systemic Resistance (ISR) in plants, and Paenibacillus sp. synthesizes auxins to promote growth, with these plant-microbe interactions being further optimized under nitrogen-enhanced conditions (Liang et al., 2024). Microvirga sp.’s nitrogen-fixing activity, collectively enhancing plant fitness. Bacillus spp. physically block pathogen colonization sites through biofilm formation (Arnaouteli et al., 2021), whereas Lysobacter sp. indirectly improves plant stress tolerance by regulating microbial community responses to environmental stressors (Nitzan et al., 2015). The increase of effective nitrogen is also beneficial to the competition mechanism of fungal resources. Building on established evidence that nitrogen enrichment reinforces rhizosphere fungal community stability and ecological network complexity—effectively constraining Fusarium pathogen proliferation (Gu et al., 2020). Our research findings are the resource competition strategy is dominated by functional taxa such as Chaetomium sphaerale , a critical biocontrol agent against plant pathogens and one of the most abundant saprophytic ascomycetes. Chaetomium efficiently degrades cellulose and organic matter while antagonizing diverse microorganisms (Feng et al., 2021). In the key disease-suppressive Module 0, Chaetomium and Rhizopus account for over 50% of the fungal biomass, enabling spatial dominance and nutrient sequestration (Finlay et al., 2002). Their extensive hyphal networks (Thormann et al., 2006) facilitate efficient nutrient absorption, with nitrogen enrichment particularly enhancing their carbon-degrading enzymatic systems’ competitiveness against pathogens (Gu Z et al.,2020). Equipped with diverse enzymatic systems, these fungi enhance resource acquisition competitiveness. Chaetomium ’s cellulose-degrading system efficiently converts cellulose into glucose, providing energy and nutrients for microbial consortia, while non-enzymatic byproducts (e.g., oligosaccharides) may act as elicitors to prime plant immune responses (Xu et al., 2019). These findings reflect the distinct ecological roles and adaptive strategies of bacteria and fungi. Bacteria rely on rapid responses and network-level regulation, whereas fungi prioritize resource acquisition and utilization to establish competitive dominance. The integration of these complementary mechanisms underpins the sustainable suppression of soil-borne fungal pathogens through green manure-mediated microecological network reconstruction. 5 Conclusions Green manure incorporation can alleviate the occurrence of tobacco root rot. Our pot experiment demonstrated that incorporating smooth vetch had a better inhibitory effect on tobacco root rot pathogens compared to rape. The improvement in available soil nutrients due to smooth vetch incorporation promoted the enrichment of beneficial functional microbial community such as Bacillus, Paenibacillus, Lysobacter , and Chaetomium . Through ecological network analysis based on bacteria and fungi, we found that incorporating smooth vetch could strengthen the hub position and synergistic effects of bacterial communities in key suppressive modules, and promote fungal communities’ participation in resource competition, thereby limiting pathogen growth. This represents a potential approach for controlling soil-borne diseases in tobacco. ACKNOWLEDGEMENTS This research was supported by the Hongta Group Science and Technology Project ”Research on the Technical System for High-Quality Development of Premium Tobacco Leaf Production in Highland Lake Basin Areas” (2022YL02), the National Green Manure Industry Technology System Project (CARS-22), and the National Key Research and Development Program of China (2021YFD1700204). The authors gratefully acknowledge the support provided by the team led by Ms. Fu from the Institute of Agricultural Environment and Resources, Yunnan Academy of Agricultural Sciences. REFERENCES Arnaouteli, S.,Bamford, N.C,Stanley-Wall, N.R, & Kovács, Á.T (2021). Bacillus subtilis biofilm formation and social interactions. Nature reviews. Microbiology, 19 (9), 600-614. Bao S D. (2000). Soil and agriculture chemistry analysis. Beijing: China Agriculture Press. Bertin, P.N, Heinrich-Salmeron, A., Pelletier, et al (2011). 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Collection Land Degradation & Development Keywords bacillus fusarium green manure green manure-tobacco rotation smooth vetch Authors Affiliations Zhao Wenjun Hongta Group View all articles by this author Wang Zhengxu Hongta Group View all articles by this author Zhao Wenjia Yunnan Agricultural University View all articles by this author Chen Hua Yunnan Academy of Agricultural Sciences View all articles by this author Liu Kui Hongta Group View all articles by this author Qian Yingying China Tobacco Yunnan Industrial Corporation View all articles by this author Lu Junping China Tobacco Yunnan Industrial Corporation View all articles by this author Libo Fu Yunnan Academy of Agricultural Sciences View all articles by this author Wang Jiansong Hongta Group View all articles by this author Yang Jizhou Hongta Group View all articles by this author Cao Jing Hongta Group View all articles by this author Feng Yu [email protected] Yunnan Academy of Agricultural Sciences View all articles by this author Weidong Cao 0000-0003-1787-9165 Chinese Academy of Agricultural Sciences Institute of Agricultural Resources and Regional Planning View all articles by this author Metrics & Citations Metrics Article Usage 370 views 188 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Zhao Wenjun, Wang Zhengxu, Zhao Wenjia, et al. Green manure-driven suppression of Fusarium solani through regulating rhizosphere microorganisms to reduce tobacco root rot incidence. Authorea . 27 April 2025. DOI: https://doi.org/10.22541/au.174576820.06958313/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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