Phosphorus availability enriches Massilia in the root microbiome to enhance resistance against Sclerotinia sclerotiorum in rapeseed

Phosphorus availability enriches Massilia in the root microbiome to enhance resistance against Sclerotinia sclerotiorum in rapeseed | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Phosphorus availability enriches Massilia in the root microbiome to enhance resistance against Sclerotinia sclerotiorum in rapeseed Xiaoxiao Dong, Liang Guo, Hanchen Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9380607/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Phosphorus is an essential macronutrient for plant growth and development, especially in P-sensitive crops such as rapeseed ( Brassica napus ). However, the mechanism by which P availability modulates plant disease resistance by shaping the root‑associated microbiome remains poorly understood. Here, we investigated how P homeostasis regulates rapeseed resistance to Sclerotinia sclerotiorum through modulation of the root-associated microbiome. P deficiency significantly inhibited plant growth and increased susceptibility to S. sclerotiorum in multiple rapeseed ecotypes, including spring, semi‑winter, and winter types. Microbiome profiling revealed that Massilia was a key P-responsive biomarker genus significantly enriched under P-sufficient conditions. Both foliar application and root inoculation with Massilia effectively suppressed S. sclerotiorum infection in rapeseed. Mechanistically, Massilia colonization strongly activated the expression of pathogenesis‑related (PR) genes, antioxidant genes, and jasmonic acid (JA) signaling genes. Overall, this study establishes a P-mediated tripartite interaction linking root microbiota assembly and plant immunity. These results highlight that optimizing P supply to enrich beneficial microbes such as Massilia can enhance rapeseed resistance to S. sclerotiorum , providing a sustainable strategy for disease management. rapeseed root microbiome Massilia phosphorus availability plant immunity S. sclerotiorum Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Rapeseed is one of the most economically important oilseed crops worldwide and can be classified into spring, semi-winter, and winter ecotypes based on their vernalization requirements (Calderwood et al., 2021 ; Sun et al., 2025 ; Wang et al., 2023 ). Spring types exhibit little or no requirement for vernalization, whereas winter types require prolonged exposure to low temperatures, and semi-winter types display an intermediate requirement (Wu et al., 2019 ; Yin et al., 2020 ). As a phosphorus (P)-sensitive crop, rapeseed growth, yield, and stress tolerance are strongly dependent on sufficient P supply (Plaxton and Tran, 2011 ; Vance et al., 2003 ; Zhang et al., 2011 ). Sclerotinia stem rot (SR), caused by the necrotrophic fungal pathogen Sclerotinia sclerotiorum , is one of the most devastating diseases limiting rapeseed production globally (Hossain et al., 2023 ; Shahoveisi and Del Río Mendoza, 2020). Disease incidence typically ranges from 10% to 30% annually, and yield losses can reach up to 80% during severe epidemics (Wu et al., 2022 ). Current management relies heavily on fungicides and cultural practices, but long‑term fungicide use has led to the emergence of resistant pathogen populations (Shahoveisi and Del Río Mendoza, 2020). Although host resistance breeding has made progress, highly resistant cultivars remain limited (Chen et al., 2023 ; Lin et al., 2022 ; Mei et al., 2025 ). There is an urgent need to develop eco-friendly and sustainable strategies for controlling sclerotinia rot. Increasing evidence indicates that root-associated microbes play important roles in suppressing soil‑borne and foliar pathogens. Beneficial bacteria can inhibit pathogens directly via antimicrobial metabolites or indirectly by triggering induced systemic resistance (ISR) in the host (Wang et al., 2024 ). Several Bacillus species, including Bacillus macauensis , Bacillus amyloliquefaciens , and Bacillus pumilus exhibit antagonistic activity against S. sclerotiorum (Cavalcanti et al., 2020 ; Chen et al., 2014 ). Similarly, species of Streptomyces griseus , Streptomyces rochei and Streptomyces sampsonii , have been shown to inhibit the growth of S. sclerotiorum (Gebily et al., 2021 ; Selin et al., 2010 ). In addition, the strain Pseudomonas fluorescens P13, isolated from rapeseed field soil, significantly suppresses mycelial growth and sclerotial germination of S. sclerotiorum and reduces disease incidence under field conditions (Jain et al., 2015 ). Notably, the structure and function of the root microbiome are strongly modulated by nutrient availability, especially P. P nutrition, root exudation, root microbes, and plant immunity form a dynamic regulatory network that determines plant health and disease outcomes(Cao et al., 2024 ; Liu et al., 2025a ; Liu et al., 2026 ; Schmidt et al., 2025 ; Zhang et al., 2026 ). P deficiency triggers the phosphate starvation response (PSR), which often prioritizes nutrient acquisition over defense activation, thereby increasing disease susceptibility (Finkel et al., 2019 ; McCombe et al., 2025). In contrast, sufficient P supply can enhance the recruitment of beneficial microbes and strengthen plant defense capacity (Castrillo et al., 2017; Tang et al., 2022). However, how P availability shapes the root microbiome to confer resistance against S. sclerotiorum in rapeseed remains largely unknown. In this study, we hypothesized that P availability modulates root microbiome assembly, particularly enriching beneficial taxa such as Massilia , which in turn activates host immune pathways and enhances resistance to S. sclerotiorum . Using multiple rapeseed ecotypes, 16S rRNA gene amplicon sequencing, and functional validation assays, we demonstrate that adequate P supply enriches Massilia in the root microbiome. Inoculation with Massilia significantly suppresses S. sclerotiorum infection by upregulating defense‑related gene expression. Our findings reveal a link between P nutrition, root microbiome, and plant immunity, providing a sustainable approach for managing sclerotinia rot in rapeseed. Materials and methods Plant growth Low phosphorus (LP) soil was collected from a no n- cultivated field in Yunnan Province, China. After removing the upper 3 cm of surface soil, samples were taken from a depth of 10 cm. The collected soil was sieved through a 3 mm mesh and mixed with vermiculite and perlite at a volumetric ratio of 5:3:2. Rapeseed seeds were surface-sterilized prior to germination and placed in germination boxes containing sufficient water to fully immerse the seeds. Germinated seeds were transferred to sterile gauze for 5–7 days until primary roots and cotyledons emerged. Uniform seedlings were transplanted into pots filled with the prepared soil matrix. Plants were grown in a controlled-environment chamber at 21°C, with a 16 h light/8 h dark photoperiod, a light intensity of 300–320 µmol m⁻² s⁻¹, and 60% relative humidity. For the P-sufficient control (CK), seedlings were treated weekly with 5 mL of ½ MS‑P liquid medium (pH 5.8) supplemented with 4.9 g L⁻¹ KH₂PO₄. For low‑P (LP) treatment, plants received 5 mL per week of the same medium containing only 0.049 g L⁻¹ KH₂PO₄ (1% of the CK level) After 28 days of growth, shoot and root phenotypes were recorded. Root samples were collected for microbial sequencing with four biological replicates per sample. Sample collection and DNA Extraction Root samples were collected from four-week-old rapeseed plants. Total microbial genomic DNA was extracted using the E.Z.N.A.® Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer’s protocol. The concentration and purity of the extracted DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and DNA integrity was verified by 1% agarose gel electrophoresis. Library construction and high-throughput sequencing The V5-V7 hypervariable region of the bacterial 16S rRNA gene was amplified using the primer pair 799F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC), each labeled with a unique barcode. PCR amplification was performed in a T100 Thermal Cycler (BIO-RAD, USA) under the following conditions: 95°C for 3 min; 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. Each 20 µL reaction contained 10 µL of 2 × Phanta Flash Master Mix, 1 µL each of the forward and reverse primers (10 µM) and 10 ng of template DNA and nuclease-free water. Amplicons were separated on 2% agarose gel, purified using a Gel Extraction Kit (YuHua, China), and quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Sequencing libraries were prepared using the NEXTFLEX Rapid DNA‑Seq Kit. Paired‑end sequencing (2 × 250 bp or 2 × 300 bp) was performed on an Illumina MiSeq platform at Shanghai Majorbio Bio‑Pharm Technology Co., Ltd. Bioinformatics analysis ​ Raw paired-end reads were quality-filtered using fastp (Chen et al., 2018 ) and merged using FLASH (Magoč and Salzberg, 2011 ). The resulting high-quality sequences were denoised using the DADA2 plugin (Callahan et al., 2016 ) within the QIIME2 pipeline (Bolyen et al., 2019 ) under default parameters. The sequences obtained after DADA2 denoising are referred to as amplicon sequence variants (ASVs). Sequences annotated as chloroplast or mitochondrial in origin were removed from all samples. Taxonomic assignment of ASVs was performed using the Naive Bayes classifier implemented in QIIME2, with reference to the SILVA 16S rRNA gene database. In vitro antagonism assay sclerotiorum was activated by two successive subcultures on PDA medium (4g potato starch, 20g glucose, 15g agar per liter of water). A flame-sterilized cork borer was used to obtain mycelial plugs (4 mm) from the actively growing edge of the fungal colony, which were then placed at the center of fresh PDA plates. Massilia from overnight cultures were harvested by centrifugation 6000 × g for 5min and resuspended in sterile water (ddH 2 O). A 50 µL aliquot of bacterial suspension was spotted at four symmetrical positions around the central fungal plug on each plate, while equal volumes of sterile water were used as a negative control. After the droplets had dried, the plates were sealed with parafilm and incubated at 22°C. The growth of S. sclerotiorum was monitored and recorded. Disease resistance assays The third and fourth true leaves from the apical meristem of one-month-old rapeseed seedlings were used for the experiments. Massilia ( M.s ) was pre-cultured on R2A solid agar plates. A single colony was then inoculated into liquid medium and cultured overnight at 28°C. Bacterial cells were harvested by centrifugation at 6,000 × g for 5 minutes, resuspended in ddH₂O, and adjusted to OD₆₀₀ = 0.5. A 0.025% (v/v) solution of Silwet-77 was added to the bacterial suspension. The suspension was applied to the surface of rapeseed leaves using a pipette. As a negative control, leaves were treated with sterile water containing 0.025% Silwet-77. In parallel, another group of seedlings was subjected to root drenching with M.s . S. sclerotiorum was cultured at the same time. One day after the initial bacterial treatment, the inoculation with M.s was repeated. On the third day, treated leaves were excised and placed in 25 cm × 25 cm plates lined with moistened sterile filter paper. Mycelial plugs (approximately 4 mm in diameter) were taken from the edge of actively growing PDA cultures of S. sclerotiorum and placed on both sides of the midvein of the detached leaves, avoiding the central vein. The plates were incubated in darkness for 24 h. Lesion development was then recorded and photographed. Lesion areas were quantified using ImageJ software. Each treatment included at least 10 biological replicates. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from tissues around inoculation sites using the RNA simple Total RNA Extraction Kit (DP419, TIANGEN). First‑strand cDNA was synthesized using a Vazyme reverse transcription kit (R333-01). Quantitative real-time PCR was preformed using the SYBR Green method. The reaction conditions were as follows: 95°C for 2 minutes, followed by 40 cycles of 95°C for 5 seconds, 60°C for 10 seconds, and 72°C for 15 seconds. BnACTIN was used as the internal reference, and relative gene expression levels were calculated using the 2 −ΔΔCt method. Each experiment included three independent biological replicates. All Primers are listed in Table S3. Results Adequate phosphorus promotes rapeseed growth and resistance to S. sclerotiorum . To determine the effects of P availability on rapeseed growth and disease resistance, four ecotypes representing spring (Westar), semi‑winter (ZS11), and winter (4474, 4180) ecotypes were grown under P‑sufficient (CK) and low‑P (LP) conditions. After 28 days, LP significantly reduced both shoot and root biomass in all tested ecotypes (Figs. 1 a, b). Shoot fresh weight decreased by 72%, 60%, 66.2%, and 54.4% in Westar, ZS11, 4474, and 4180, respectively (Fig. 1 a). Root fresh weight was similarly decreased by 70%, 81%, 74%, and 54% (Fig. 1 b). Detached leaf inoculation assays showed that plants grown under LP conditions developed significantly larger lesions than CK plants (Figs. 1 c, d). Lesion area increased by 56.1% under LP relative to CK. Quantification of S. sclerotiorum biomass further confirmed that LP plants were more susceptible to infection (Fig. 1 e). These results demonstrate that P deficiency not only restricts rapeseed growth but also compromises resistance to S. sclerotiorum . Phosphorus availability shapes root microbiome diversity in rapeseed Root microorganisms play an important role in regulating plant growth and immunity (Tang et al., 2025 ). To investigate the effects of P availability on the root microbiome of different rapeseed ecotypes, 16S rRNA gene amplicon sequencing was performed on plants grown under LP and CK conditions. After quality filtering and denoising, a total of 2,250,424 high‑quality sequences and 17,448 ASVs were obtained (Tables S1, S2). Rarefaction curves plateaued, indicating sufficient sequencing depth (Fig. S1 ). Alpha diversity analysis revealed that LP significantly increased bacterial richness and diversity compared with CK (Figs. 2 a, b). Specifically, the Chao1 and Shannon indices under LP were 1.35- and 1.2-fold higher than those under CK, respectively. At the ecotype level, a similar trend was observed in all genotypes except 4474. In the spring-type rapeseed (Westar), these indices were 3.04- and 1.36-fold higher under LP than CK conditions. In the semi-winter type (ZS11), the corresponding increases were 2.54- and 1.28-fold, while in the winter-type genotype 4180, they were 3.0- and 1.3-fold, respectively (Figs. 2 a–c). Beta diversity analysis further demonstrated a clear separation between samples under different P conditions (Fig. 2 d), whereas no distinct clustering was observed among ecotypes (Fig. 2 e). These results suggest that P availability, rather than ecotype, is the dominant factor shaping the root microbiome in rapeseed. Taxonomic shifts in the root microbiome To further investigate the effects of P availability on microbial community composition, we analyzed taxonomic profiles under different P conditions. P availability was associated with shifts in the taxonomic composition of the rapeseed root microbiome at both the family and genus levels (Fig. 3 ). At the family level, several dominant taxa, including Burkholderiaceae, Micrococcaceae, Nocardioidaceae, and Xanthomonadaceae, exhibited notable changes in relative abundance between LP and CK conditions (Fig. 3 a). Similar patterns were observed at the genus level, where taxa such as Massilia , Sphingomonas , Pseudomonas , Bradyrhizobium , and Allorhizobium varied in relative abundance across treatments (Fig. 3 b). Massilia is a key P‑responsive biomarker To identify key microbial taxa associated with different P conditions, LEfSe analysis was performed to detect differentially enriched genera between LP and CK treatments (Fig. 4 a). Genera including Paenibacillus , Azospirillum , Bacillus , Actinophytocola , Ochrobactrum , and Noviherspirillum were enriched under LP. In contrast, Massilia , Sphingomonas , Pseudomonas , Shinella , and Flavobacterium were significantly more abundant under P‑sufficient conditions. Statistical analysis confirmed that the relative abundance of Massilia was significantly higher in P-sufficient conditions, identifying it as a core P-responsive beneficial genus (Fig. 4 b). a Biomarker genera under different P conditions identified by LEfSe (Linear Discriminant Analysis Effect Size). The bar plot shows LDA scores, reflecting the effect size and contribution of each genus to differences between treatments. differential taxa were determined using the Kruskal–Wallis test followed by pairwise Wilcoxon rank-sum tests. b Mean relative abundance of selected microbial genera across different groups. Error bars represent 95% confidence intervals. Statistical significance was assessed using the Wilcoxon rank-sum test, * P < 0.05, ** P < 0.01, *** P < 0.001. Massilia directly antagonizes S. sclerotiorum and enhances plant resistance To evaluate whether Massilia exhibits antagonistic activity against S. sclerotiorum , dual culture assays were performed using a Massilia strain ( M.s ) maintained in our laboratory. The results showed that M.s significantly inhibited the growth of S. sclerotiorum (Fig. 5 a), with relative hyphal extension distance reduced by 17% compared to the control (Fig. 5 b). To further assess the protective effect of M.s against S. sclerotiorum in rapeseed, M.s was applied either by droplet inoculation onto leaves or by root drenching. Detached leaves were subsequently challenged with S. sclerotiorum , and disease progression was evaluated. Both application methods significantly reduced S. sclerotiorum infection (Figs. 5 c, f). Specifically, foliar application reduced lesion area by 53%, while root drenching reduced it by 29.5%. (Figs. 5 d, g). Consistently, quantification of S. sclerotiorum biomass confirmed a significant reduction in pathogen levels in both treatments (Figs. 5 e, h). These results demonstrate that Massilia effectively enhances rapeseed resistance to S. sclerotiorum. Massilia activates plant defense-related gene expression To explore the molecular mechanism underlying Massilia ‑induced resistance, we analyzed the expression of defense‑related genes by qRT‑PCR (Fig. 6 ). Compared with the mock control, both Massilia _Foliar ( M.s _F) and Massilia _Root ( M.s _R) treatments strongly upregulated the expression of PR genes ( PR1 , PR2 , PAL ), antioxidant genes ( SOD , CAT, POD ), and JA signaling genes ( AOS , AOC , MYC2 ). Foliar application generally triggered stronger induction, whereas root inoculation preferentially enhanced CAT , AOS , and AOC expression. These results indicate that Massilia enhances disease resistance by activating multiple layers of plant immune responses, including PR proteins, ROS scavenging systems, and JA‑mediated signaling. Discussion In this study, we demonstrate that P availability strongly influences rapeseed growth and resistance to S. sclerotiorum by modulating the root microbiome. Low phosphorus stress repressed plant growth and increased disease susceptibility in all tested rapeseed ecotypes. Microbiome profiling revealed that P status, rather than host ecotype, predominantly determines root microbial community structure. Importantly, we identified Massilia as a key beneficial genus enriched under P‑sufficient conditions. Inoculation with Massilia suppressed S. sclerotiorum both in vitro and in planta. These findings establish a mechanistic link among P nutrition, root microbiome assembly, and plant immunity. Consistent with previous studies, P deficiency impaired plant growth and compromised defense capacity (Finkel et al., 2019 ; He et al., 2022 ; Vance et al., 2003 ). Under nutrient limitation, plants often prioritize resource allocation for survival and nutrient acquisition over immune activation, leading to a growth–defense trade‑off that increases disease susceptibility. Our results extend this concept by showing that P availability modulates disease resistance not only directly through plant physiological status but also indirectly by shaping the root microbiome. Root microbiome analysis showed that low P increased α diversity, which may reflect a stress‑induced generalist community. Beta diversity clearly separated CK and LP groups, confirming that P availability is a major driver of root microbiome structure in rapeseed. Similar observations have been reported in other plant systems (Liu et al., 2025b ). LEfSe and differential abundance analysis highlighted Massilia as the most prominent biomarker enriched by sufficient P. Massilia has emerged as a beneficial genus associated with disease suppression and growth promotion in multiple crops (Huang et al., 2025 ). Our dual‑culture assays confirmed direct antagonism against S. sclerotiorum . Furthermore, foliar and root inoculation of Massilia significantly reduced disease severity in rapeseed. The observed reductions in lesion area (up to 53%) highlight the biocontrol potential of this bacterium. Gene expression analysis revealed that Massilia activates a broad‑spectrum defense response including PR genes, antioxidant systems, and JA signaling pathways. These pathways are central to plant resistance against necrotrophic pathogens (Chen et al., 2024 ; Liu et al., 2025b ; Xiao et al., 2025 ). The stronger induction by foliar application suggests tissue‑specific immune modulation, while root inoculation preferentially induced certain stress‑responsive genes, indicating systemic immune priming. Together, our results support a model in which adequate phosphorus supply enriches Massilia in the root microbiome, which in turn triggers systemic immune activation and enhances resistance to S. sclerotiorum in rapeseed. This study provides a sustainable strategy to improve disease management by optimizing phosphorus nutrition to recruit beneficial microbes. Conclusion Our findings reveal that P availability shapes the root microbiome of rapeseed and regulates resistance to S. sclerotiorum . LP stress reduces plant growth, increases disease susceptibility, and alters microbial community structure. CK conditions specifically enrich the beneficial bacterium Massilia , which suppresses pathogen growth and activates plant defense pathways including PR genes, antioxidant systems, and JA signaling. This work establishes a P-microbiome-immunity axis that determines disease outcome in rapeseed. Optimizing P fertilization to promote beneficial microbial colonization represents a promising and sustainable approach for managing sclerotinia stem rot in agricultural production. Declarations Competing interests The authors declare no competing interests. Author Contribution L.G. and H.C. designed and supervised the study. X.D. performed the experiments. X.D. analyzed the data. X.D. wrote the manuscript. L.G. and H.C. revised the manuscript. All authors read and approved the manuscript. Acknowledgements The research was supported by National Key Research and Development Program of China (grant no. 2023YFF1000700 to L.G.) and Basic Research Project of Yazhouwan National Laboratory (2310GL01). Data Availability Data will be made available on request. References Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodríguez AM, Chase J, Cope EK, Da Silva R, von Hippel M, Walters W et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852–857 Calderwood A, Lloyd A, Hepworth J, Tudor EH, Jones DM, Woodhouse S, Bilham L, Chinoy C, Williams K, Corke F, Doonan JH, Ostergaard L, Irwin JA, Wells R, Morris RJ (2021) Total FLC transcript dynamics from divergent paralogue expression explains flowering diversity in Brassica napus. New Phytol 229:3534–3548 Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583 Cao Y, Shen Z, Zhang N, Deng X, Thomashow LS, Lidbury I, Liu H, Li R, Shen Q, Kowalchuk GA (2024) Phosphorus availability influences disease-suppressive soil microbiome through plant-microbe interactions. Microbiome 12:185 Cavalcanti VP, Araújo NAF, Machado NB, Júnior C, Pasqual PSP, Alves M, Schwan-Estrada E, K. R. F., and, Dória J (2020) Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic. Scientia Horticulturae 261 Chen RS, Wang JY, Sarwar R, Tan XL (2023) Genetic breakthroughs in the Brassica napus-Sclerotinia sclerotiorum interactions. Front Plant Sci 14:1276055 Chen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884–i890 Chen Y, Gao X, Chen Y, Qin H, Huang L, Han Q (2014) Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed. Biol Control 78:67–76 Chen Y, Jin G, Liu M, Wang L, Lou Y, Baldwin I, Li R (2024) Multiomic analyses reveal key sectors of jasmonate-mediated defense responses in rice. Plant Cell 36:3362–3377 Finkel OM, Salas-González I, Castrillo G, Spaepen S, Law TF, Teixeira P, Jones CD, Dangl JL (2019) The effects of soil phosphorus content on plant microbiota are driven by the plant phosphate starvation response. PLoS Biol 17:e3000534 Gebily DAS, Ghanem GAM, Ragab MM, Ali AM, Soliman NE-d. K., and, El-Moity A (2021) T. H. Characterization and potential antifungal activities of three Streptomyces spp. as biocontrol agents against Sclerotinia sclerotiorum (Lib.) de Bary infecting green bean. Egyptian Journal of Biological Pest Control 31 He Z, Webster S, He SY (2022) Growth-defense trade-offs in plants. Curr Biol 32:R634–R639 Hossain MM, Sultana F, Li W, Tran LP, Mostofa MG (2023) Sclerotinia sclerotiorum (Lib.) de Bary: Insights into the Pathogenomic Features of a Global Pathogen. Cells 12 Huang Q, Wang R, Ding Q, Liao F, Zhu L, Huang M, Li J, Zeng J, Shen Q, Wang M, Guo S (2025) Low-nitrogen input enriches Massilia bacteria in the phyllosphere to improve blast resistance in rice. New Phytol 248:3151–3167 Jain A, Singh A, Singh S, Singh HB (2015) Biological management of Sclerotinia sclerotiorum in pea using plant growth promoting microbial consortium. J Basic Microbiol 55:961–972 Lin L, Fan J, Li P, Liu D, Ren S, Lin K, Fang Y, Lin C, Wang Y, Wu J (2022) The Sclerotinia sclerotiorum-inducible promoter pBnGH17D7 in Brassica napus: isolation, characterization, and application in host-induced gene silencing. J Exp Bot 73:6663–6677 Liu C, Pan Y, Fei Y, Shen R, Lan P (2025a) Hijacking phosphate signaling: A novel strategy of fungal pathogens in plant disease. J Integr Plant Biol 67:1988–1990 Liu W, Wang J, Zhu D, Yin X, Du G, Qin Y, Zhang Z, Liu Z (2025b) Jasmonic Acid-Mediated Antioxidant Defense Confers Chilling Tolerance in Okra (Abelmoschus esculentus L). Plants (Basel) 14 Liu X, Yang S, Xie P, Niu G, Shen Q, Yuan J (2026) Phosphorus availability drives rhizosphere metabolite-microbial community interactions to modulate cucumber susceptibility to Fusarium wilt. Microbiome Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963 Mei J, Yang S, Linghu Y, Gao Y, Hu Y, Nie W, Zhang Y, Peng L, Wu Y, Ding Y, Luo R, Liao J, Qian W (2025) Unveiling the role of microRNAs in nonhost resistance to Sclerotinia sclerotiorum: Rice-specific microRNAs attack the pathogen via cross-kingdom RNAi. J Integr Plant Biol 67:1179–1195 Plaxton WC, Tran HT (2011) Metabolic adaptations of phosphate-starved plants. Plant Physiol 156:1006–1015 Schmidt M, Raj K, Salas-Oropeza J, Valdés-López O, Ried-Lasi MK (2025) Starve or share? Phosphate availability shapes plant-microbe interactions. PLoS Pathog 21:e1013601 Selin C, Habibian R, Poritsanos N, Athukorala SN, Fernando D, de Kievit TR (2010) Phenazines are not essential for Pseudomonas chlororaphis PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm formation. FEMS Microbiol Ecol 71:73–83 Shahoveisi F, Río Mendoza D, L. E (2020) Effect of Wetness Duration and Incubation Temperature on Development of Ascosporic Infections by Sclerotinia sclerotiorum. Plant Dis 104:1817–1823 Sun C, Zhou X, Wang C, Chen F, Zhang W, Peng Q, Guo Y, Gao J, Wang X, Hu M, Zhang J, Zhao H, Fu S (2025) Ningza 182: A rapeseed variety with a moderately compact plant type, bred for high yield and high oil content. Mol Breed 45:62 Tang Z, Tan W, Li R, Weng L, Chen X, Xi B, Lv D (2025) Advances in Rhizosphere Microbiome and Rhizosphere Immunity Effect: A Review. J Agric Food Chem 73:14707–14721 Vance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423–447 Wang SY, Zhang YJ, Chen X, Shi XC, Herrera-Balandrano DD, Liu FQ, Laborda P (2024) Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review. Phytopathology 114:1447–1457 Wang T, van Dijk ADJ, Bucher J, Liang J, Wu J, Bonnema G, Wang X (2023) Interploidy Introgression Shaped Adaptation during the Origin and Domestication History of Brassica napus. Mol Biol Evol 40 Wu D, Liang Z, Yan T, Xu Y, Xuan L, Tang J, Zhou G, Lohwasser U, Hua S, Wang H, Chen X, Wang Q, Zhu L, Maodzeka A, Hussain N, Li Z, Li X, Shamsi IH, Jilani G, Wu L, Zheng H, Zhang G, Chalhoub B, Shen L, Yu H, Jiang L (2019) Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence. Mol Plant 12, 30–43 Wu J, Yin S, Lin L, Liu D, Ren S, Zhang W, Meng W, Chen P, Sun Q, Fang Y, Wei C, Wang Y (2022) Host-induced gene silencing of multiple pathogenic factors of Sclerotinia sclerotiorum confers resistance to Sclerotinia rot in Brassica napus. Crop J 10:661–671 Xiao J, Nakamura Y, Wu Z, Fu W, Chen Y, Lou Y, Baldwin IT, Boland W, Li R (2025) A synthetic jasmonate receptor agonist uncouples the growth-defense trade-off in rice. Proc Natl Acad Sci U S A 122:e2505675122 Yin S, Wan M, Guo C, Wang B, Li H, Li G, Tian Y, Ge X, King GJ, Liu K, Li Z, Wang J (2020) Transposon insertions within alleles of BnaFLC.A10 and BnaFLC.A2 are associated with seasonal crop type in rapeseed. J Exp Bot 71:4729–4741 Zhang H, Huang Y, Ye X, Xu F (2011) Genotypic variation in phosphorus acquisition from sparingly soluble P sources is related to root morphology and root exudates in Brassica napus. Sci China Life Sci 54:1134–1142 Zhang L, Liu J, Zhou Z, Wang W (2026) Integrated transcriptome and translatome analyses reveal the early regulatory network of Brassica napus roots in response to the growth-promoting rhizobacterium Pseudomonas simiae WCS417. Mol Breed 46:8 Additional Declarations No competing interests reported. Supplementary Files supplymentaryFigure.docx SupplymentaryTables.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 24 Apr, 2026 Reviews received at journal 24 Apr, 2026 Reviews received at journal 20 Apr, 2026 Reviewers agreed at journal 17 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 12 Apr, 2026 Submission checks completed at journal 12 Apr, 2026 First submitted to journal 10 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9380607","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626458793,"identity":"3a115053-bce3-4d80-9435-3abbeaa2b348","order_by":0,"name":"Xiaoxiao Dong","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxiao","middleName":"","lastName":"Dong","suffix":""},{"id":626458794,"identity":"028f067b-eca0-48cd-956a-09b8f2c3c78e","order_by":1,"name":"Liang Guo","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Guo","suffix":""},{"id":626458795,"identity":"4f14a2d3-f5ba-4c67-8d74-aa37fa6a654e","order_by":2,"name":"Hanchen Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCQYGZhBtwMDA+ABIy4BEiNbCDMQMPCRpYZMgSgv/7OZjjwtq7sibs/ceq+b5c5iHf3YD4+cCfJbcOZZuPOPYM8OdPefSbvPwHOaRuHOAWXoGHi0GEjlm0jxshxMMbuSY3eaRuM3DcCOBjZkHr5b8b9I8/4Ba7r8xK+YxuM0jT1hLDps0bxvIFh4zZp6E2zwGhLRI3Egzk+btO2y44UyOseScA/95DO8cbJbGp4V/RvIzaZ5vh+UNjp8x/PDmT5qc3O3mg5/xaUEBTBCVjA3EagCq/UG82lEwCkbBKBhBAABYokhfPnew/wAAAABJRU5ErkJggg==","orcid":"","institution":"Yazhouwan National Laboratory","correspondingAuthor":true,"prefix":"","firstName":"Hanchen","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2026-04-10 14:23:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9380607/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9380607/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107705688,"identity":"f3537481-54bb-4cf0-8f85-6adc31d2ba2b","added_by":"auto","created_at":"2026-04-24 09:14:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":669875,"visible":true,"origin":"","legend":"\u003cp\u003eLow phosphorus inhibits rapeseed growth and resistance to \u003cem\u003eSclerotinia sclerotiorum.\u003c/em\u003e \u003cstrong\u003ea\u003c/strong\u003eStatistical analysis of shoot fresh weight in different rapeseed ecotypes grown under CK and LP conditions for one month, respectively; \u003cstrong\u003eb\u003c/strong\u003e Statistical analysis of root fresh weight in different rapeseed ecotypes grown under CK and LP conditions for one month, respectively. Westar, ZS11, 4474, 4180 indicate distinct rapeseed ecotypes in (\u003cstrong\u003ea\u003c/strong\u003e) and (\u003cstrong\u003eb\u003c/strong\u003e). \u003cstrong\u003ec\u003c/strong\u003e Disease phenotypes of rapeseed leaves inoculated with \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e. \u003cstrong\u003ed\u003c/strong\u003eQuantification of lesion area. \u003cstrong\u003ee \u003c/strong\u003eQuantification of \u003cem\u003eS. sclerotiorum\u003c/em\u003ebiomass. CK and LP represent normal phosphorus and low phosphorus conditions, respectively. Statistical significance was determined using a Student's \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Bar = 1 cm. The experiments were repeated 3 times with similar results.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/66a26e45b9f019f6b599c156.png"},{"id":107705400,"identity":"d9996e85-5e30-4748-be8d-745548951ee1","added_by":"auto","created_at":"2026-04-24 09:12:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":802646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLow phosphorus significantly affects root microbial diversity across rapeseed ecotypes. a–b\u003c/strong\u003e α-diversity of the root microbiome across all samples under different P conditions and among rapeseed ecotypes, including the Chao1 (\u003cstrong\u003ea\u003c/strong\u003e) and Shannon (\u003cstrong\u003eb\u003c/strong\u003e) indices. \u003cstrong\u003ec–d\u003c/strong\u003eβ-diversity of the root microbiome: \u003cstrong\u003ec \u003c/strong\u003eβ-diversity among all samples under different P conditions; \u003cstrong\u003ed\u003c/strong\u003e β-diversity among different rapeseed ecotypes. CK and LP represent normal phosphorus and low phosphorus conditions, respectively. Westar, ZS11, 4474, 4180 indicate distinct rapeseed ecotypes. Statistical significance was determined using a Student's \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/2668a4cdfc831b364959296f.png"},{"id":107579418,"identity":"47488391-1f11-4b20-ba2b-93fbe51c7fce","added_by":"auto","created_at":"2026-04-22 21:56:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":622523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of root microbial taxonomic composition under different phosphorus levels. \u003c/strong\u003eRelative abundance of microbial communities under LP and CK conditions at different taxonomic levels: \u003cstrong\u003ea \u003c/strong\u003ecommunity composition at the family level; \u003cstrong\u003eb\u003c/strong\u003e community composition at the genus level. Each bar represents the mean relative abundance of taxa in each treatment group; different colors indicate different taxa, and taxa with relative abundance \u0026lt; 0.01 are grouped as “others”.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/1ae77fca2ba9a366b34493f3.png"},{"id":107707014,"identity":"c21bf619-7763-409e-a71c-21b1dc92796f","added_by":"auto","created_at":"2026-04-24 09:19:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":945372,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of P-responsive biomarker genera and their relative abundance.\u003c/strong\u003e\u003cbr\u003e\n \u003cstrong\u003ea\u003c/strong\u003e Biomarker genera under different P conditions identified by LEfSe (Linear Discriminant Analysis Effect Size). The bar plot shows LDA scores, reflecting the effect size and contribution of each genus to differences between treatments. differential taxa were determined using the Kruskal–Wallis test followed by pairwise Wilcoxon rank-sum tests. \u003cstrong\u003eb\u003c/strong\u003e Mean relative abundance of selected microbial genera across different groups. Error bars represent 95% confidence intervals. Statistical significance was assessed using the Wilcoxon rank-sum test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/a06e4f68f43b816d12a1362b.png"},{"id":107579420,"identity":"e8dc8ddd-51ae-447c-a107-6801e32abef5","added_by":"auto","created_at":"2026-04-22 21:56:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":884027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eM.s\u003c/em\u003einhibits \u003cem\u003eS. sclerotiorum\u003c/em\u003e growth and promotes resistance in rapeseed. \u003cstrong\u003ea \u003c/strong\u003eMycelial growth phenotypes of\u003cem\u003e S. sclerotiorum\u003c/em\u003e in single culture and in co-culture with \u003cem\u003eM.s.\u003c/em\u003e \u003cstrong\u003eb\u003c/strong\u003e Statistical analysis of the relative expansion distance of \u003cem\u003eS. sclerotiorum\u003c/em\u003e mycelium. \u003cstrong\u003ec\u003c/strong\u003e Phenotypes of detached leaves inoculated with \u003cem\u003eS. sclerotiorum\u003c/em\u003e following foliar spray treatment with \u003cem\u003eM.s \u003c/em\u003e(\u003cem\u003eM.s_F\u003c/em\u003e), with water-treated (Mock) plants as controls. \u003cstrong\u003ed \u003c/strong\u003eStatistical analysis of lesion area in (c). \u003cstrong\u003ee\u003c/strong\u003e Quantification of \u003cem\u003eS. sclerotiorum\u003c/em\u003e biomass in (c). \u003cstrong\u003ef\u003c/strong\u003e Phenotypes of detached leaves inoculated with \u003cem\u003eS. sclerotiorum\u003c/em\u003e after root irrigation with \u003cem\u003eM.s \u003c/em\u003e(\u003cem\u003eM.s_R\u003c/em\u003e). \u003cstrong\u003eg\u003c/strong\u003e Statistical analysis of lesion area in (f). \u003cstrong\u003eh\u003c/strong\u003eQuantification of \u003cem\u003eS. sclerotiorum\u003c/em\u003e biomass in (f). Statistical significance was determined using a Student's \u003cem\u003et\u003c/em\u003e-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Bar = 1 cm. The experiments were repeated 3 times with similar results.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/1e59e77bdeb4090e49912473.png"},{"id":107579421,"identity":"2c7dac05-8dd9-4af1-ae43-6b95f4d1bf72","added_by":"auto","created_at":"2026-04-22 21:56:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":857806,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eM.s\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e significantly activates the expression of plant defense-related genes. \u003c/strong\u003eGene expression levels of pathogenesis-related (PR) genes (a), reactive oxygen species (ROS) scavenging–related genes (b), and jasmonic acid (JA) signaling–related genes (c) were analyzed in leaves surrounding the infection sites after \u003cem\u003eS. sclerotiorum\u003c/em\u003e inoculation. Rapeseed plants were subjected to three treatments: water-treated control (Mock), foliar spray with \u003cem\u003eMassilia\u003c/em\u003e (\u003cem\u003eM.s\u003c/em\u003e_F), and root irrigation with \u003cem\u003eMassilia\u003c/em\u003e (\u003cem\u003eM.s\u003c/em\u003e_R). Statistical significance was determined using a Student's t-test, *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.001. Bar = 1 cm. The experiments were repeated 3 times with similar results.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/f761b449f108e4b59ac4a681.png"},{"id":107709130,"identity":"7a1e7bea-6960-4161-8ada-88567390e2ca","added_by":"auto","created_at":"2026-04-24 09:34:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5104069,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/8c01292c-a2b6-4a44-9581-dd877735117e.pdf"},{"id":107579414,"identity":"353ff2a9-360f-4e4c-b500-f01b61db4c0c","added_by":"auto","created_at":"2026-04-22 21:56:00","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":70697,"visible":true,"origin":"","legend":"","description":"","filename":"supplymentaryFigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/a39c0d08bab93bdec38edb73.docx"},{"id":107579416,"identity":"2e5758f1-d4f7-4184-8c71-44b200944cc0","added_by":"auto","created_at":"2026-04-22 21:56:00","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1077073,"visible":true,"origin":"","legend":"","description":"","filename":"SupplymentaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9380607/v1/fe676f5fa5fd51649a6ec73c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phosphorus availability enriches Massilia in the root microbiome to enhance resistance against Sclerotinia sclerotiorum in rapeseed","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRapeseed is one of the most economically important oilseed crops worldwide and can be classified into spring, semi-winter, and winter ecotypes based on their vernalization requirements (Calderwood et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Spring types exhibit little or no requirement for vernalization, whereas winter types require prolonged exposure to low temperatures, and semi-winter types display an intermediate requirement (Wu et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yin et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As a phosphorus (P)-sensitive crop, rapeseed growth, yield, and stress tolerance are strongly dependent on sufficient P supply (Plaxton and Tran, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Vance et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSclerotinia stem rot (SR), caused by the necrotrophic fungal pathogen \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e, is one of the most devastating diseases limiting rapeseed production globally (Hossain et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shahoveisi and Del R\u0026iacute;o Mendoza, 2020). Disease incidence typically ranges from 10% to 30% annually, and yield losses can reach up to 80% during severe epidemics (Wu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Current management relies heavily on fungicides and cultural practices, but long‑term fungicide use has led to the emergence of resistant pathogen populations (Shahoveisi and Del R\u0026iacute;o Mendoza, 2020). Although host resistance breeding has made progress, highly resistant cultivars remain limited (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mei et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). There is an urgent need to develop eco-friendly and sustainable strategies for controlling sclerotinia rot.\u003c/p\u003e \u003cp\u003eIncreasing evidence indicates that root-associated microbes play important roles in suppressing soil‑borne and foliar pathogens. Beneficial bacteria can inhibit pathogens directly via antimicrobial metabolites or indirectly by triggering induced systemic resistance (ISR) in the host (Wang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Several Bacillus species, including \u003cem\u003eBacillus macauensis\u003c/em\u003e, \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e, and \u003cem\u003eBacillus pumilus\u003c/em\u003e exhibit antagonistic activity against \u003cem\u003eS. sclerotiorum\u003c/em\u003e (Cavalcanti et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarly, species of \u003cem\u003eStreptomyces griseus\u003c/em\u003e, \u003cem\u003eStreptomyces rochei\u003c/em\u003e and \u003cem\u003eStreptomyces sampsonii\u003c/em\u003e, have been shown to inhibit the growth of \u003cem\u003eS. sclerotiorum\u003c/em\u003e (Gebily et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Selin et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, the strain \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e P13, isolated from rapeseed field soil, significantly suppresses mycelial growth and sclerotial germination of \u003cem\u003eS. sclerotiorum\u003c/em\u003e and reduces disease incidence under field conditions (Jain et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, the structure and function of the root microbiome are strongly modulated by nutrient availability, especially P. P nutrition, root exudation, root microbes, and plant immunity form a dynamic regulatory network that determines plant health and disease outcomes(Cao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2025a\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2026\u003c/span\u003e; Schmidt et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2026\u003c/span\u003e). P deficiency triggers the phosphate starvation response (PSR), which often prioritizes nutrient acquisition over defense activation, thereby increasing disease susceptibility (Finkel et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; McCombe et al., 2025). In contrast, sufficient P supply can enhance the recruitment of beneficial microbes and strengthen plant defense capacity (Castrillo et al., 2017; Tang et al., 2022). However, how P availability shapes the root microbiome to confer resistance against \u003cem\u003eS. sclerotiorum\u003c/em\u003e in rapeseed remains largely unknown.\u003c/p\u003e \u003cp\u003eIn this study, we hypothesized that P availability modulates root microbiome assembly, particularly enriching beneficial taxa such as \u003cem\u003eMassilia\u003c/em\u003e, which in turn activates host immune pathways and enhances resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e. Using multiple rapeseed ecotypes, 16S rRNA gene amplicon sequencing, and functional validation assays, we demonstrate that adequate P supply enriches \u003cem\u003eMassilia\u003c/em\u003e in the root microbiome. Inoculation with \u003cem\u003eMassilia\u003c/em\u003e significantly suppresses \u003cem\u003eS. sclerotiorum\u003c/em\u003e infection by upregulating defense‑related gene expression. Our findings reveal a link between P nutrition, root microbiome, and plant immunity, providing a sustainable approach for managing sclerotinia rot in rapeseed.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant growth\u003c/h2\u003e \u003cp\u003eLow phosphorus (LP) soil was collected from a no\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003en-\u003c/span\u003ecultivated field in Yunnan Province, China. After removing the upper 3 cm of surface soil, samples were taken from a depth of 10 cm. The collected soil was sieved through a 3 mm mesh and mixed with vermiculite and perlite at a volumetric ratio of 5:3:2. Rapeseed seeds were surface-sterilized prior to germination and placed in germination boxes containing sufficient water to fully immerse the seeds. Germinated seeds were transferred to sterile gauze for 5\u0026ndash;7 days until primary roots and cotyledons emerged. Uniform seedlings were transplanted into pots filled with the prepared soil matrix. Plants were grown in a controlled-environment chamber at 21\u0026deg;C, with a 16 h light/8 h dark photoperiod, a light intensity of 300\u0026ndash;320 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;, and 60% relative humidity. For the P-sufficient control (CK), seedlings were treated weekly with 5 mL of \u0026frac12; MS‑P liquid medium (pH 5.8) supplemented with 4.9 g L⁻\u0026sup1; KH₂PO₄. For low‑P (LP) treatment, plants received 5 mL per week of the same medium containing only 0.049 g L⁻\u0026sup1; KH₂PO₄ (1% of the CK level) After 28 days of growth, shoot and root phenotypes were recorded. Root samples were collected for microbial sequencing with four biological replicates per sample.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSample collection and DNA Extraction\u003c/h3\u003e\n\u003cp\u003eRoot samples were collected from four-week-old rapeseed plants. Total microbial genomic DNA was extracted using the E.Z.N.A.\u0026reg; Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according to the manufacturer\u0026rsquo;s protocol. The concentration and purity of the extracted DNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and DNA integrity was verified by 1% agarose gel electrophoresis.\u003c/p\u003e\n\u003ch3\u003eLibrary construction and high-throughput sequencing\u003c/h3\u003e\n\u003cp\u003eThe V5-V7 hypervariable region of the bacterial 16S rRNA gene was amplified using the primer pair 799F (AACMGGATTAGATACCCKG) and 1193R (ACGTCATCCCCACCTTCC), each labeled with a unique barcode. PCR amplification was performed in a T100 Thermal Cycler (BIO-RAD, USA) under the following conditions: 95\u0026deg;C for 3 min; 27 cycles of 95\u0026deg;C for 30 s, 55\u0026deg;C for 30 s, and 72\u0026deg;C for 30 s; and a final extension at 72\u0026deg;C for 10 min. Each 20 \u0026micro;L reaction contained 10 \u0026micro;L of 2 \u0026times; Phanta Flash Master Mix, 1 \u0026micro;L each of the forward and reverse primers (10 \u0026micro;M) and 10 ng of template DNA and nuclease-free water. Amplicons were separated on 2% agarose gel, purified using a Gel Extraction Kit (YuHua, China), and quantified with a Qubit 4.0 fluorometer (Thermo Fisher Scientific, USA). Sequencing libraries were prepared using the NEXTFLEX Rapid DNA‑Seq Kit. Paired‑end sequencing (2 \u0026times; 250 bp or 2 \u0026times; 300 bp) was performed on an Illumina MiSeq platform at Shanghai Majorbio Bio‑Pharm Technology Co., Ltd.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatics analysis\u003c/b\u003e​\u003c/p\u003e \u003cp\u003eRaw paired-end reads were quality-filtered using fastp (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and merged using FLASH (Magoč and Salzberg, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The resulting high-quality sequences were denoised using the DADA2 plugin (Callahan et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) within the QIIME2 pipeline (Bolyen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) under default parameters. The sequences obtained after DADA2 denoising are referred to as amplicon sequence variants (ASVs). Sequences annotated as chloroplast or mitochondrial in origin were removed from all samples. Taxonomic assignment of ASVs was performed using the Naive Bayes classifier implemented in QIIME2, with reference to the SILVA 16S rRNA gene database.\u003c/p\u003e\n\u003ch3\u003eIn vitro antagonism assay\u003c/h3\u003e\n\u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003e \u003cem\u003esclerotiorum\u003c/em\u003e was activated by two successive subcultures on PDA medium (4g potato starch, 20g glucose, 15g agar per liter of water). A flame-sterilized cork borer was used to obtain mycelial plugs (4 mm) from the actively growing edge of the fungal colony, which were then placed at the center of fresh PDA plates. \u003cem\u003eMassilia\u003c/em\u003e from overnight cultures were harvested by centrifugation 6000 \u0026times; g for 5min and resuspended in sterile water (ddH\u003csub\u003e2\u003c/sub\u003eO). A 50 \u0026micro;L aliquot of bacterial suspension was spotted at four symmetrical positions around the central fungal plug on each plate, while equal volumes of sterile water were used as a negative control. After the droplets had dried, the plates were sealed with parafilm and incubated at 22\u0026deg;C. The growth of \u003cem\u003eS. sclerotiorum\u003c/em\u003e was monitored and recorded.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e\n\u003ch3\u003eDisease resistance assays\u003c/h3\u003e\n\u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eThe third and fourth true leaves from the apical meristem of one-month-old rapeseed seedlings were used for the experiments. \u003cem\u003eMassilia\u003c/em\u003e (\u003cem\u003eM.s\u003c/em\u003e) was pre-cultured on R2A solid agar plates. A single colony was then inoculated into liquid medium and cultured overnight at 28\u0026deg;C. Bacterial cells were harvested by centrifugation at 6,000 \u0026times; g for 5 minutes, resuspended in ddH₂O, and adjusted to OD₆₀₀ = 0.5. A 0.025% (v/v) solution of Silwet-77 was added to the bacterial suspension. The suspension was applied to the surface of rapeseed leaves using a pipette. As a negative control, leaves were treated with sterile water containing 0.025% Silwet-77. In parallel, another group of seedlings was subjected to root drenching with \u003cem\u003eM.s\u003c/em\u003e. \u003cem\u003eS. sclerotiorum\u003c/em\u003e was cultured at the same time. One day after the initial bacterial treatment, the inoculation with \u003cem\u003eM.s\u003c/em\u003e was repeated. On the third day, treated leaves were excised and placed in 25 cm \u0026times; 25 cm plates lined with moistened sterile filter paper. Mycelial plugs (approximately 4 mm in diameter) were taken from the edge of actively growing PDA cultures of \u003cem\u003eS. sclerotiorum\u003c/em\u003e and placed on both sides of the midvein of the detached leaves, avoiding the central vein. The plates were incubated in darkness for 24 h. Lesion development was then recorded and photographed. Lesion areas were quantified using ImageJ software. Each treatment included at least 10 biological replicates.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eTotal RNA was extracted from tissues around inoculation sites using the RNA simple Total RNA Extraction Kit (DP419, TIANGEN). First‑strand cDNA was synthesized using a Vazyme reverse transcription kit (R333-01). Quantitative real-time PCR was preformed using the SYBR Green method. The reaction conditions were as follows: 95\u0026deg;C for 2 minutes, followed by 40 cycles of 95\u0026deg;C for 5 seconds, 60\u0026deg;C for 10 seconds, and 72\u0026deg;C for 15 seconds. \u003cem\u003eBnACTIN\u003c/em\u003e was used as the internal reference, and relative gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. Each experiment included three independent biological replicates. All Primers are listed in Table S3.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eAdequate phosphorus promotes rapeseed growth and resistance to\u003c/b\u003e \u003cb\u003eS. sclerotiorum\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTo determine the effects of P availability on rapeseed growth and disease resistance, four ecotypes representing spring (Westar), semi‑winter (ZS11), and winter (4474, 4180) ecotypes were grown under P‑sufficient (CK) and low‑P (LP) conditions. After 28 days, LP significantly reduced both shoot and root biomass in all tested ecotypes (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Shoot fresh weight decreased by 72%, 60%, 66.2%, and 54.4% in Westar, ZS11, 4474, and 4180, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Root fresh weight was similarly decreased by 70%, 81%, 74%, and 54% (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). Detached leaf inoculation assays showed that plants grown under LP conditions developed significantly larger lesions than CK plants (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Lesion area increased by 56.1% under LP relative to CK. Quantification of \u003cem\u003eS. sclerotiorum\u003c/em\u003e biomass further confirmed that LP plants were more susceptible to infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee). These results demonstrate that P deficiency not only restricts rapeseed growth but also compromises resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePhosphorus availability shapes root microbiome diversity in rapeseed\u003c/h3\u003e\n\u003cp\u003eRoot microorganisms play an important role in regulating plant growth and immunity (Tang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). To investigate the effects of P availability on the root microbiome of different rapeseed ecotypes, 16S rRNA gene amplicon sequencing was performed on plants grown under LP and CK conditions. After quality filtering and denoising, a total of 2,250,424 high‑quality sequences and 17,448 ASVs were obtained (Tables S1, S2). Rarefaction curves plateaued, indicating sufficient sequencing depth (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlpha diversity analysis revealed that LP significantly increased bacterial richness and diversity compared with CK (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Specifically, the Chao1 and Shannon indices under LP were 1.35- and 1.2-fold higher than those under CK, respectively. At the ecotype level, a similar trend was observed in all genotypes except 4474. In the spring-type rapeseed (Westar), these indices were 3.04- and 1.36-fold higher under LP than CK conditions. In the semi-winter type (ZS11), the corresponding increases were 2.54- and 1.28-fold, while in the winter-type genotype 4180, they were 3.0- and 1.3-fold, respectively (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026ndash;c). Beta diversity analysis further demonstrated a clear separation between samples under different P conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), whereas no distinct clustering was observed among ecotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). These results suggest that P availability, rather than ecotype, is the dominant factor shaping the root microbiome in rapeseed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTaxonomic shifts in the root microbiome\u003c/h2\u003e \u003cp\u003eTo further investigate the effects of P availability on microbial community composition, we analyzed taxonomic profiles under different P conditions. P availability was associated with shifts in the taxonomic composition of the rapeseed root microbiome at both the family and genus levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). At the family level, several dominant taxa, including Burkholderiaceae, Micrococcaceae, Nocardioidaceae, and Xanthomonadaceae, exhibited notable changes in relative abundance between LP and CK conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Similar patterns were observed at the genus level, where taxa such as \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eBradyrhizobium\u003c/em\u003e, and \u003cem\u003eAllorhizobium\u003c/em\u003e varied in relative abundance across treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMassilia\u003c/b\u003e \u003cb\u003eis a key P‑responsive biomarker\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo identify key microbial taxa associated with different P conditions, LEfSe analysis was performed to detect differentially enriched genera between LP and CK treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). Genera including \u003cem\u003ePaenibacillus\u003c/em\u003e, \u003cem\u003eAzospirillum\u003c/em\u003e, \u003cem\u003eBacillus\u003c/em\u003e, \u003cem\u003eActinophytocola\u003c/em\u003e, \u003cem\u003eOchrobactrum\u003c/em\u003e, and \u003cem\u003eNoviherspirillum\u003c/em\u003e were enriched under LP. In contrast, \u003cem\u003eMassilia\u003c/em\u003e, \u003cem\u003eSphingomonas\u003c/em\u003e, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eShinella\u003c/em\u003e, and \u003cem\u003eFlavobacterium\u003c/em\u003e were significantly more abundant under P‑sufficient conditions. Statistical analysis confirmed that the relative abundance of \u003cem\u003eMassilia\u003c/em\u003e was significantly higher in P-sufficient conditions, identifying it as a core P-responsive beneficial genus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ea\u003c/b\u003e Biomarker genera under different P conditions identified by LEfSe (Linear Discriminant Analysis Effect Size). The bar plot shows LDA scores, reflecting the effect size and contribution of each genus to differences between treatments. differential taxa were determined using the Kruskal\u0026ndash;Wallis test followed by pairwise Wilcoxon rank-sum tests. \u003cb\u003eb\u003c/b\u003e Mean relative abundance of selected microbial genera across different groups. Error bars represent 95% confidence intervals. Statistical significance was assessed using the Wilcoxon rank-sum test, *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eMassilia\u003c/em\u003e directly antagonizes \u003cem\u003eS. sclerotiorum\u003c/em\u003e and enhances plant resistance\u003c/h2\u003e \u003cp\u003eTo evaluate whether \u003cem\u003eMassilia\u003c/em\u003e exhibits antagonistic activity against \u003cem\u003eS. sclerotiorum\u003c/em\u003e, dual culture assays were performed using a \u003cem\u003eMassilia\u003c/em\u003e strain (\u003cem\u003eM.s\u003c/em\u003e) maintained in our laboratory. The results showed that \u003cem\u003eM.s\u003c/em\u003e significantly inhibited the growth of \u003cem\u003eS. sclerotiorum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea), with relative hyphal extension distance reduced by 17% compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). To further assess the protective effect of \u003cem\u003eM.s\u003c/em\u003e against \u003cem\u003eS. sclerotiorum\u003c/em\u003e in rapeseed, \u003cem\u003eM.s\u003c/em\u003e was applied either by droplet inoculation onto leaves or by root drenching. Detached leaves were subsequently challenged with \u003cem\u003eS. sclerotiorum\u003c/em\u003e, and disease progression was evaluated. Both application methods significantly reduced \u003cem\u003eS. sclerotiorum\u003c/em\u003e infection (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, f). Specifically, foliar application reduced lesion area by 53%, while root drenching reduced it by 29.5%. (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed, g). Consistently, quantification of \u003cem\u003eS. sclerotiorum\u003c/em\u003e biomass confirmed a significant reduction in pathogen levels in both treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, h). These results demonstrate that \u003cem\u003eMassilia\u003c/em\u003e effectively enhances rapeseed resistance to \u003cem\u003eS. sclerotiorum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMassilia\u003c/b\u003e \u003cb\u003eactivates plant defense-related gene expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the molecular mechanism underlying \u003cem\u003eMassilia\u003c/em\u003e‑induced resistance, we analyzed the expression of defense‑related genes by qRT‑PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Compared with the mock control, both \u003cem\u003eMassilia\u003c/em\u003e_Foliar (\u003cem\u003eM.s\u003c/em\u003e_F) and \u003cem\u003eMassilia\u003c/em\u003e_Root (\u003cem\u003eM.s\u003c/em\u003e_R) treatments strongly upregulated the expression of PR genes (\u003cem\u003ePR1\u003c/em\u003e, \u003cem\u003ePR2\u003c/em\u003e, \u003cem\u003ePAL\u003c/em\u003e), antioxidant genes (\u003cem\u003eSOD\u003c/em\u003e, \u003cem\u003eCAT, POD\u003c/em\u003e), and JA signaling genes (\u003cem\u003eAOS\u003c/em\u003e, \u003cem\u003eAOC\u003c/em\u003e, \u003cem\u003eMYC2\u003c/em\u003e). Foliar application generally triggered stronger induction, whereas root inoculation preferentially enhanced \u003cem\u003eCAT\u003c/em\u003e, \u003cem\u003eAOS\u003c/em\u003e, and \u003cem\u003eAOC\u003c/em\u003e expression. These results indicate that \u003cem\u003eMassilia\u003c/em\u003e enhances disease resistance by activating multiple layers of plant immune responses, including PR proteins, ROS scavenging systems, and JA‑mediated signaling.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we demonstrate that P availability strongly influences rapeseed growth and resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e by modulating the root microbiome. Low phosphorus stress repressed plant growth and increased disease susceptibility in all tested rapeseed ecotypes. Microbiome profiling revealed that P status, rather than host ecotype, predominantly determines root microbial community structure. Importantly, we identified \u003cem\u003eMassilia\u003c/em\u003e as a key beneficial genus enriched under P‑sufficient conditions. Inoculation with \u003cem\u003eMassilia\u003c/em\u003e suppressed \u003cem\u003eS. sclerotiorum\u003c/em\u003e both in vitro and in planta. These findings establish a mechanistic link among P nutrition, root microbiome assembly, and plant immunity.\u003c/p\u003e \u003cp\u003eConsistent with previous studies, P deficiency impaired plant growth and compromised defense capacity (Finkel et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vance et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Under nutrient limitation, plants often prioritize resource allocation for survival and nutrient acquisition over immune activation, leading to a growth\u0026ndash;defense trade‑off that increases disease susceptibility. Our results extend this concept by showing that P availability modulates disease resistance not only directly through plant physiological status but also indirectly by shaping the root microbiome.\u003c/p\u003e \u003cp\u003eRoot microbiome analysis showed that low P increased α diversity, which may reflect a stress‑induced generalist community. Beta diversity clearly separated CK and LP groups, confirming that P availability is a major driver of root microbiome structure in rapeseed. Similar observations have been reported in other plant systems (Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e). LEfSe and differential abundance analysis highlighted \u003cem\u003eMassilia\u003c/em\u003e as the most prominent biomarker enriched by sufficient P.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMassilia\u003c/em\u003e has emerged as a beneficial genus associated with disease suppression and growth promotion in multiple crops (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Our dual‑culture assays confirmed direct antagonism against \u003cem\u003eS. sclerotiorum\u003c/em\u003e. Furthermore, foliar and root inoculation of \u003cem\u003eMassilia\u003c/em\u003e significantly reduced disease severity in rapeseed. The observed reductions in lesion area (up to 53%) highlight the biocontrol potential of this bacterium.\u003c/p\u003e \u003cp\u003eGene expression analysis revealed that \u003cem\u003eMassilia\u003c/em\u003e activates a broad‑spectrum defense response including PR genes, antioxidant systems, and JA signaling pathways. These pathways are central to plant resistance against necrotrophic pathogens (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025b\u003c/span\u003e; Xiao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The stronger induction by foliar application suggests tissue‑specific immune modulation, while root inoculation preferentially induced certain stress‑responsive genes, indicating systemic immune priming.\u003c/p\u003e \u003cp\u003eTogether, our results support a model in which adequate phosphorus supply enriches \u003cem\u003eMassilia\u003c/em\u003e in the root microbiome, which in turn triggers systemic immune activation and enhances resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e in rapeseed. This study provides a sustainable strategy to improve disease management by optimizing phosphorus nutrition to recruit beneficial microbes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings reveal that P availability shapes the root microbiome of rapeseed and regulates resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e. LP stress reduces plant growth, increases disease susceptibility, and alters microbial community structure. CK conditions specifically enrich the beneficial bacterium \u003cem\u003eMassilia\u003c/em\u003e, which suppresses pathogen growth and activates plant defense pathways including PR genes, antioxidant systems, and JA signaling. This work establishes a P-microbiome-immunity axis that determines disease outcome in rapeseed. Optimizing P fertilization to promote beneficial microbial colonization represents a promising and sustainable approach for managing sclerotinia stem rot in agricultural production.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.G. and H.C. designed and supervised the study. X.D. performed the experiments. X.D. analyzed the data. X.D. wrote the manuscript. L.G. and H.C. revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe research was supported by National Key Research and Development Program of China (grant no. 2023YFF1000700 to L.G.) and Basic Research Project of Yazhouwan National Laboratory (2310GL01).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, Alexander H, Alm EJ, Arumugam M, Asnicar F, Bai Y, Bisanz JE, Bittinger K, Brejnrod A, Brislawn CJ, Brown CT, Callahan BJ, Caraballo-Rodr\u0026iacute;guez AM, Chase J, Cope EK, Da Silva R, von Hippel M, Walters W et al (2019) Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol 37:852\u0026ndash;857\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalderwood A, Lloyd A, Hepworth J, Tudor EH, Jones DM, Woodhouse S, Bilham L, Chinoy C, Williams K, Corke F, Doonan JH, Ostergaard L, Irwin JA, Wells R, Morris RJ (2021) Total FLC transcript dynamics from divergent paralogue expression explains flowering diversity in Brassica napus. New Phytol 229:3534\u0026ndash;3548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581\u0026ndash;583\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao Y, Shen Z, Zhang N, Deng X, Thomashow LS, Lidbury I, Liu H, Li R, Shen Q, Kowalchuk GA (2024) Phosphorus availability influences disease-suppressive soil microbiome through plant-microbe interactions. Microbiome 12:185\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCavalcanti VP, Ara\u0026uacute;jo NAF, Machado NB, J\u0026uacute;nior C, Pasqual PSP, Alves M, Schwan-Estrada E, K. R. F., and, D\u0026oacute;ria J (2020) Yeasts and Bacillus spp. as potential biocontrol agents of Sclerotinia sclerotiorum in garlic. \u003cem\u003eScientia Horticulturae\u003c/em\u003e 261\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen RS, Wang JY, Sarwar R, Tan XL (2023) Genetic breakthroughs in the Brassica napus-Sclerotinia sclerotiorum interactions. Front Plant Sci 14:1276055\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Zhou Y, Chen Y, Gu J (2018) fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34:i884\u0026ndash;i890\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Gao X, Chen Y, Qin H, Huang L, Han Q (2014) Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed. Biol Control 78:67\u0026ndash;76\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Jin G, Liu M, Wang L, Lou Y, Baldwin I, Li R (2024) Multiomic analyses reveal key sectors of jasmonate-mediated defense responses in rice. Plant Cell 36:3362\u0026ndash;3377\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinkel OM, Salas-Gonz\u0026aacute;lez I, Castrillo G, Spaepen S, Law TF, Teixeira P, Jones CD, Dangl JL (2019) The effects of soil phosphorus content on plant microbiota are driven by the plant phosphate starvation response. PLoS Biol 17:e3000534\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGebily DAS, Ghanem GAM, Ragab MM, Ali AM, Soliman NE-d. K., and, El-Moity A (2021) T. H. Characterization and potential antifungal activities of three Streptomyces spp. as biocontrol agents against Sclerotinia sclerotiorum (Lib.) de Bary infecting green bean. \u003cem\u003eEgyptian Journal of Biological Pest Control\u003c/em\u003e 31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe Z, Webster S, He SY (2022) Growth-defense trade-offs in plants. Curr Biol 32:R634\u0026ndash;R639\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHossain MM, Sultana F, Li W, Tran LP, Mostofa MG (2023) Sclerotinia sclerotiorum (Lib.) de Bary: Insights into the Pathogenomic Features of a Global Pathogen. \u003cem\u003eCells\u003c/em\u003e 12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang Q, Wang R, Ding Q, Liao F, Zhu L, Huang M, Li J, Zeng J, Shen Q, Wang M, Guo S (2025) Low-nitrogen input enriches Massilia bacteria in the phyllosphere to improve blast resistance in rice. New Phytol 248:3151\u0026ndash;3167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJain A, Singh A, Singh S, Singh HB (2015) Biological management of Sclerotinia sclerotiorum in pea using plant growth promoting microbial consortium. J Basic Microbiol 55:961\u0026ndash;972\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin L, Fan J, Li P, Liu D, Ren S, Lin K, Fang Y, Lin C, Wang Y, Wu J (2022) The Sclerotinia sclerotiorum-inducible promoter pBnGH17D7 in Brassica napus: isolation, characterization, and application in host-induced gene silencing. J Exp Bot 73:6663\u0026ndash;6677\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu C, Pan Y, Fei Y, Shen R, Lan P (2025a) Hijacking phosphate signaling: A novel strategy of fungal pathogens in plant disease. J Integr Plant Biol 67:1988\u0026ndash;1990\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu W, Wang J, Zhu D, Yin X, Du G, Qin Y, Zhang Z, Liu Z (2025b) Jasmonic Acid-Mediated Antioxidant Defense Confers Chilling Tolerance in Okra (Abelmoschus esculentus L). Plants (Basel) 14\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Yang S, Xie P, Niu G, Shen Q, Yuan J (2026) Phosphorus availability drives rhizosphere metabolite-microbial community interactions to modulate cucumber susceptibility to Fusarium wilt. \u003cem\u003eMicrobiome\u003c/em\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957\u0026ndash;2963\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei J, Yang S, Linghu Y, Gao Y, Hu Y, Nie W, Zhang Y, Peng L, Wu Y, Ding Y, Luo R, Liao J, Qian W (2025) Unveiling the role of microRNAs in nonhost resistance to Sclerotinia sclerotiorum: Rice-specific microRNAs attack the pathogen via cross-kingdom RNAi. J Integr Plant Biol 67:1179\u0026ndash;1195\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlaxton WC, Tran HT (2011) Metabolic adaptations of phosphate-starved plants. Plant Physiol 156:1006\u0026ndash;1015\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmidt M, Raj K, Salas-Oropeza J, Vald\u0026eacute;s-L\u0026oacute;pez O, Ried-Lasi MK (2025) Starve or share? Phosphate availability shapes plant-microbe interactions. PLoS Pathog 21:e1013601\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSelin C, Habibian R, Poritsanos N, Athukorala SN, Fernando D, de Kievit TR (2010) Phenazines are not essential for Pseudomonas chlororaphis PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm formation. FEMS Microbiol Ecol 71:73\u0026ndash;83\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShahoveisi F, R\u0026iacute;o Mendoza D, L. E (2020) Effect of Wetness Duration and Incubation Temperature on Development of Ascosporic Infections by Sclerotinia sclerotiorum. Plant Dis 104:1817\u0026ndash;1823\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun C, Zhou X, Wang C, Chen F, Zhang W, Peng Q, Guo Y, Gao J, Wang X, Hu M, Zhang J, Zhao H, Fu S (2025) Ningza 182: A rapeseed variety with a moderately compact plant type, bred for high yield and high oil content. Mol Breed 45:62\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Z, Tan W, Li R, Weng L, Chen X, Xi B, Lv D (2025) Advances in Rhizosphere Microbiome and Rhizosphere Immunity Effect: A Review. J Agric Food Chem 73:14707\u0026ndash;14721\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVance CP, Uhde-Stone C, Allan DL (2003) Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 157:423\u0026ndash;447\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang SY, Zhang YJ, Chen X, Shi XC, Herrera-Balandrano DD, Liu FQ, Laborda P (2024) Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review. Phytopathology 114:1447\u0026ndash;1457\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, van Dijk ADJ, Bucher J, Liang J, Wu J, Bonnema G, Wang X (2023) Interploidy Introgression Shaped Adaptation during the Origin and Domestication History of Brassica napus. Mol Biol Evol 40\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu D, Liang Z, Yan T, Xu Y, Xuan L, Tang J, Zhou G, Lohwasser U, Hua S, Wang H, Chen X, Wang Q, Zhu L, Maodzeka A, Hussain N, Li Z, Li X, Shamsi IH, Jilani G, Wu L, Zheng H, Zhang G, Chalhoub B, Shen L, Yu H, Jiang L (2019) Whole-Genome Resequencing of a Worldwide Collection of Rapeseed Accessions Reveals the Genetic Basis of Ecotype Divergence. \u003cem\u003eMol Plant\u003c/em\u003e 12, 30\u0026ndash;43\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu J, Yin S, Lin L, Liu D, Ren S, Zhang W, Meng W, Chen P, Sun Q, Fang Y, Wei C, Wang Y (2022) Host-induced gene silencing of multiple pathogenic factors of Sclerotinia sclerotiorum confers resistance to Sclerotinia rot in Brassica napus. Crop J 10:661\u0026ndash;671\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao J, Nakamura Y, Wu Z, Fu W, Chen Y, Lou Y, Baldwin IT, Boland W, Li R (2025) A synthetic jasmonate receptor agonist uncouples the growth-defense trade-off in rice. Proc Natl Acad Sci U S A 122:e2505675122\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYin S, Wan M, Guo C, Wang B, Li H, Li G, Tian Y, Ge X, King GJ, Liu K, Li Z, Wang J (2020) Transposon insertions within alleles of BnaFLC.A10 and BnaFLC.A2 are associated with seasonal crop type in rapeseed. J Exp Bot 71:4729\u0026ndash;4741\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Huang Y, Ye X, Xu F (2011) Genotypic variation in phosphorus acquisition from sparingly soluble P sources is related to root morphology and root exudates in Brassica napus. Sci China Life Sci 54:1134\u0026ndash;1142\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Liu J, Zhou Z, Wang W (2026) Integrated transcriptome and translatome analyses reveal the early regulatory network of Brassica napus roots in response to the growth-promoting rhizobacterium Pseudomonas simiae WCS417. Mol Breed 46:8\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"rapeseed, root microbiome, Massilia, phosphorus availability, plant immunity, S. sclerotiorum","lastPublishedDoi":"10.21203/rs.3.rs-9380607/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9380607/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhosphorus is an essential macronutrient for plant growth and development, especially in P-sensitive crops such as rapeseed (\u003cem\u003eBrassica napus\u003c/em\u003e). However, the mechanism by which P availability modulates plant disease resistance by shaping the root‑associated microbiome remains poorly understood. Here, we investigated how P homeostasis regulates rapeseed resistance to \u003cem\u003eSclerotinia sclerotiorum\u003c/em\u003e through modulation of the root-associated microbiome. P deficiency significantly inhibited plant growth and increased susceptibility to \u003cem\u003eS. sclerotiorum\u003c/em\u003e in multiple rapeseed ecotypes, including spring, semi‑winter, and winter types. Microbiome profiling revealed that \u003cem\u003eMassilia\u003c/em\u003e was a key P-responsive biomarker genus significantly enriched under P-sufficient conditions. Both foliar application and root inoculation with \u003cem\u003eMassilia\u003c/em\u003e effectively suppressed \u003cem\u003eS. sclerotiorum\u003c/em\u003e infection in rapeseed. Mechanistically, \u003cem\u003eMassilia\u003c/em\u003e colonization strongly activated the expression of pathogenesis‑related (PR) genes, antioxidant genes, and jasmonic acid (JA) signaling genes. Overall, this study establishes a P-mediated tripartite interaction linking root microbiota assembly and plant immunity. These results highlight that optimizing P supply to enrich beneficial microbes such as \u003cem\u003eMassilia\u003c/em\u003e can enhance rapeseed resistance to \u003cem\u003eS. sclerotiorum\u003c/em\u003e, providing a sustainable strategy for disease management.\u003c/p\u003e","manuscriptTitle":"Phosphorus availability enriches Massilia in the root microbiome to enhance resistance against Sclerotinia sclerotiorum in rapeseed","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 21:55:55","doi":"10.21203/rs.3.rs-9380607/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-24T23:55:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-24T08:03:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-20T15:47:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65978741432841659188418285263463500596","date":"2026-04-17T12:38:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170027979052994495088549863809966094100","date":"2026-04-14T23:26:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-13T16:29:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-13T00:39:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-12T22:40:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Breeding","date":"2026-04-10T14:17:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-breeding","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"molb","sideBox":"Learn more about [Molecular Breeding](https://www.springer.com/journal/11032)","snPcode":"11032","submissionUrl":"https://submission.nature.com/new-submission/11032/3","title":"Molecular Breeding","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"02e6c322-91ce-4383-816b-b1af0c733aab","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-28T05:56:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 21:55:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9380607","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9380607","identity":"rs-9380607","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}