Cultivar-dependent responses of tomato rhizosphere bacterial and protistan communities under Ralstonia solanacearum invasion

Abstract

Bacterial wilt, caused by Ralstonia solanacearum (RS), is a major constraint on tomato production worldwide. This study investigated how multi-trophic rhizosphere communities associated with five tomato cultivars (AM, DF, MF, NP, and ZY) respond to RS invasion using sterile plate and greenhouse pot assays. Rhizosphere bacterial and protistan communities were characterized by 16S and 18S rRNA gene amplicon sequencing under control and RS-inoculated conditions. Results showed that both cultivar identity and RS invasion significantly shaped microbial structures and compositions. RS infection exerted a stronger influence on bacterial communities than protists, whereas cultivar identity was the primary driver for protistan shifts. Random forest classification and differential abundance analysis identified specific microbial taxa that effectively discriminated cultivars and infection status. Community assembly analysis further revealed that bacterial assembly shifted from predominantly deterministic to more stochastic processes under pathogen pressure, while protistan assembly remained largely stochastic. Collectively, these results demonstrate that tomato cultivars harbor distinct multi-trophic rhizosphere microbial configurations under pathogen invasion, and highlight the importance of incorporating protists into rhizosphere community frameworks for understanding and potentially enhancing bacterial wilt suppression.

Graphical abstract

Keywords

bacterial wilt / rhizosphere microbiome / microbial community assembly / pathogen invasion / protists

Highlight

	● Both tomato cultivar identity and Ralstonia solanacearum pathogen invasion distinctly reshape rhizosphere bacterial and protistan communities.
	● R. solanacearum pathogen infection primarily alters bacterial community composition, while tomato cultivar identity exerts a stronger effect on protists.
	● R. solanacearum pathogen invasion shifts bacterial community assembly from deterministic toward stochastic processes while protistan assembly remains predominantly stochastic.

References

[1] Aktar, S.N., Shah, M.H., Mohanty, B., Pramanik, K., Hosen, S., Ddoza, R.M.B., Kumawat, S.M., Sharma, N.K., Sayyed, R.Z., 2024. Beneficial microbes in plant health, immunity and resistance. In: Mathur, P., Roy S., eds. Plant Microbiome and Biological Control: Emerging Trends and Applications. Cham: Springer, 1–17.

[2] Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology26, 32–46.

[3] Asghar, W., Craven, K.D., Asghar, N., Saasan, N.K., Kataoka, R., Mahmood, A., Ikhlas, I., 2025. Rhizosphere interactions in relation to resilience of agroforestry system. In: Jatav, H.S., Raiput, V.D., Minkina, T., eds. Ecologically Mediated Development: Promoting Biodiversity Conservation and Food Security. Singapore: Springer, 445–457.

[4] Carrión, V.J., Perez-Jaramillo, J., Cordovez, V., Tracanna, V., de Hollander, M., Ruiz-Buck, D., Mendes, L.W., Van Ijcken, W.F.J., Gomez-Exposito, R., Elsayed, S.S., Mohanraju, P., Arifah, A., Van Der Oost, J., Paulson, J.N., Mendes, R., Van Wezel, G.P., Medema, M.H., Raaijmakers, J.M., 2019. Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome. Science366, 606–612.

[5] Crotty, F.V., Adl, S.M., Blackshaw, R.P., Murray, P.J., 2012. Protozoan pulses unveil their pivotal position within the soil food web. Microbial Ecology63, 905–918.

[6] Das, P.P., Singh, K.R., Nagpure, G., Mansoori, A., Singh, R.P., Ghazi, I.A., Kumar, A., Singh, J., 2022. Plant-soil-microbes: a tripartite interaction for nutrient acquisition and better plant growth for sustainable agricultural practices. Environmental Research214, 113821.

[7] Deng, Y., Kong, W.Y., Zhang, X.M., Zhu, Y., Xie, T., Chen, M., Zhu, L., Sun, J.Z., Zhang, Z.H., Chen, C.Y., Zhu, C.W., Yin, H.Q., Huang, S.Q., Gu, Y.B., 2024. Rhizosphere microbial community enrichment processes in healthy and diseased plants: implications of soil properties on biomarkers. Frontiers in Microbiology15, 1333076.

[8] Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST. Bioinformatics26, 2460–2461.

[9] Fan, X.Y., Ge, A.H., Qi, S.S., Guan, Y.F., Wang, R., Yu, N., Wang, E.T., 2025. Root exudates and microbial metabolites: signals and nutrients in plant-microbe interactions. Science China Life Sciences,68, 2290–2302.

[10] Gao, M., Xiong, C., Gao, C., Tsui, C.K.M., Wang, M.M., Zhou, X., Zhang, A.M., Cai, L., 2021. Disease-induced changes in plant microbiome assembly and functional adaptation. Microbiome9, 187.

[11] Gao, Z.L., Karlsson, I., Geisen, S., Kowalchuk, G., Jousset, A., 2019. Protists: puppet masters of the rhizosphere microbiome. Trends in Plant Science24, 165–176.

[12] Geisen, S., Mitchell, E.A.D., Adl, S., Bonkowski, M., Dunthorn, M., Ekelund, F., Fernández, L.D., Jousset, A., Krashevska, V., Singer, D., Spiegel, F.W., Walochnik, J., Lara, E., 2018. Soil protists: a fertile frontier in soil biology research. FEMS Microbiology Reviews42, 293–323.

[13] Geisen, S., Tveit, A.T., Clark, I.M., Richter, A., Svenning, M.M., Bonkowski, M., Urich, T., 2015. Metatranscriptomic census of active protists in soils. The ISME Journal9, 2178–2190.

[14] Guillou, L., Bachar, D., Audic, S., Bass, D., Berney, C., Bittner, L., Boutte, C., Burgaud, G., De Vargas, C., Decelle, J., Del Campo, J., Dolan, J.R., Dunthorn, M., Edvardsen, B., Holzmann, M., Kooistra, W.H.C.F., Lara, E., Le Bescot, N., Logares, R., Mahé, F., Massana, R., Montresor, M., Morard, R., Not, F., Pawlowski, J., Probert, I., Sauvadet, A.L., Siano, R., Stoeck, T., Vaulot, D., Zimmermann, P., Christen, R., 2013. The Protist Ribosomal Reference database (PR²): a catalog of unicellular eukaryote Small Sub-Unit rRNA sequences with curated taxonomy. Nucleic Acids Research41, D597–D604.

[15] Guo, S., Geisen, S., Mo, Y.N., Yan, X.Y., Huang, R.L., Liu, H.J., Gao, Z.L., Tao, C.Y., Deng, X.H., Xiong, W., Shen, Q.R., Kowalchuk, G.A., Li, R., 2024a. Predatory protists impact plant performance by promoting plant growth-promoting rhizobacterial consortia. The ISME Journal18, wrae180.

[16] Guo, S., Jiao, Z.X., Yan, Z.G., Yan, X.Y., Deng, X.H., Xiong, W., Tao, C.Y., Liu, H.J., Li, R., Shen, Q.R., Kowalchuk, G.A., Geisen, S., 2024b. Predatory protists reduce bacteria wilt disease incidence in tomato plants. Nature Communications15, 829.

[17] Guo, S., Xiong, W., Hang, X.N., Gao, Z.L., Jiao, Z.X., Liu, H.J., Mo, Y.N., Zhang, N., Kowalchuk, G.A., Li, R., Shen, Q.R., Geisen, S., 2021. Protists as main indicators and determinants of plant performance. Microbiome9, 64.

[18] Hinsinger, P., Gobran, G.R., Gregory, P.J., Wenzel, W.W., 2005. Rhizosphere geometry and heterogeneity arising from root-mediated physical and chemical processes. New Phytologist168, 293–303.

[19] Hu, Y.X., Jiang, K.Y., Xia, S.Q., Zhang, W.X., Guo, J.H., Wang, H., 2025. Amoeba community dynamics and assembly mechanisms in full-scale drinking water distribution networks under various disinfectant regimens. Water Research271, 122861.

[20] Jiang, G.F., Wei, Z., Xu, J., Chen, H.L., Zhang, Y., She, X.M., Macho, A.P., Ding, W., Liao, B.S., 2017. Bacterial wilt in China: history, current status, and future perspectives. Frontiers in Plant Science8, 1549.

[21] Kranawetter, C., Sumner, L.W., 2025. Differential root zone secretions and the role of root border cells in rhizosphere manipulation. Phytochemistry Reviews24, 5639–5658.

[22] Kwak, M.J., Kong, H.G., Choi, K., Kwon, S.K., Song, J.Y., Lee, J., Lee, P.A., Choi, S.Y., Seo, M., Lee, H.J., Jung, E.J., Park, H., Roy, N., Kim, H., Lee, M.M., Rubin, E.M., Lee, S.W., Kim, J.F., 2018. Rhizosphere microbiome structure alters to enable wilt resistance in tomato. Nature Biotechnology36, 1100–1109.

[23] Li, C.H., Jiao, N., Liu, S.Y., Qu, L.Y., Zhang, Y., Zhang, N.L., 2025. Patterns of rhizosphere microbial community assembly affect blueberry growth: environmental and host effects. Rhizosphere36, 101228.

[24] Liang, J.C., Wei, C.J., Song, X.R., Wang, R., Shi, H.L., Tan, J., Cheng, D.J., Wang, W.J., Wang, X.Q., 2024. Bacterial wilt affects the structure and assembly of microbial communities along the soil-root continuum. Environmental Microbiome19, 6.

[25] Lin, C.S., Li, W.J., Li, L.J., Neilson, R., An, X.L., Zhu, Y.G., 2023. Movement of protistan trophic groups in soil–plant continuums. Environmental Microbiology25, 2641–2652.

[26] Lin, H., Lai, C.X., Yu, G., Sunahara, G.I., Liu, L.H., Ullah, H., Liu, J., 2025. Root exudate-driven rhizospheric recruitment of plant growth-promoting rhizobacteria. Pedosphere35, 216–228.

[27] Liu, C., Wang, Y.J., Zhou, Z.Y., Wang, S.M., Wei, Z., Ravanbakhsh, M., Shen, Q.R., Xiong, W., Kowalchuk, G.A., Jousset, A., 2024a. Protist predation promotes antimicrobial resistance spread through antagonistic microbiome interactions. The ISME Journal18, wrae169.

[28] Liu, C., Zhou, Z.Y., Sun, S., Zhang, Q., Sun, S.Q., Hang, X.N., Ravanbakhsh, M., Wei, Z., Li, R., Wang, S.M., Xiong, W., Kowalchuk, G.A., Shen, Q.R., 2024b. Investigating protistan predators and bacteria within soil microbiomes in agricultural ecosystems under organic and chemical fertilizer applications. Biology and Fertility of Soils60, 1009–1024.

[29] Love, M.I., Huber, W., Anders, S., 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology15, 550.

[30] Malla, M.A., Dubey, A., Yadav, S., Kumar, A., Hashem, A., Abd_Allah, E.F., 2018. Understanding and designing the strategies for the microbe-mediated remediation of environmental contaminants using omics approaches. Frontiers in Microbiology9, 1132.

[31] Mashabela, M.D., Piater, L.A., Dubery, I.A., Tugizimana, F., Mhlongo, M.I., 2022. Rhizosphere tripartite interactions and PGPR-mediated metabolic reprogramming towards ISR and plant priming: a metabolomics review. Biology11, 346.

[32] Mendes, R., Garbeva, P., Raaijmakers, J.M., 2013. The rhizosphere microbiome: significance of plant beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiology Reviews37, 634–663.

[33] Munoz-Ucros, J., Zwetsloot, M.J., Cuellar-Gempeler, C., Bauerle, T.L., 2021. Spatiotemporal patterns of rhizosphere microbiome assembly: from ecological theory to agricultural application. Journal of Applied Ecology58, 894–904.

[34] Nguyen, T.B.A., Bonkowski, M., Dumack, K., Chen, Q.L., He, J.Z., Hu, H.W., 2023. Protistan predation selects for antibiotic resistance in soil bacterial communities. The ISME Journal17, 2182–2189.

[35] Nilsen, T., Snipen, L.G., Angell, I.L., Keeley, N.B., Majaneva, S., Pettersen, R., Rudi, K., 2024. Swarm and UNOISE outperform DADA2 and Deblur for denoising high-diversity marine seafloor samples. ISME Communications4, ycae071.

[36] Pajdak-Stós, A., Sobczyk, M., Fiałkowska, E., Kocerba-Soroka, W., Fyda, J., 2017. The effect of three different predatory ciliate species on activated sludge microfauna. European Journal of Protistology58, 87–93.

[37] Ping, X.X., Khan, R.A.A., Chen, S.M., Jiao, Y., Zhuang, X., Jiang, L.J., Song, L.Q., Yang, Y.H., Zhao, J.L., Li, Y., Mao, Z.C., 2024. Deciphering the role of rhizosphere microbiota in modulating disease resistance in cabbage varieties. Microbiome12, 160.

[38] Quast, C., Pruesse, E., Yilmaz, P., Gerken, J., Schweer, T., Yarza, P., Peplies, J., Glöckner, F.O., 2013. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Research41, D590–D596.

[39] Riseh, R.S., Fathi, F., Vazvani, M.G., Tarkka, M.T., 2025. Plant colonization by biocontrol bacteria and improved plant health: a review. Frontiers in Bioscience-Landmark30, 23223.

[40] Sahu, P.K., Tilgam, J., Mishra, S., Hamid, S., Gupta, A., K ., J ., Verma, S.K., Kharwar, R.N., 2022. Surface sterilization for isolation of endophytes: ensuring what (not) to grow. Journal of Basic Microbiology62, 647–668.

[41] Santoyo, G., 2022. How plants recruit their microbiome? New insights into beneficial interactions. Journal of Advanced Research40, 45–58.

[42] Song, Y.Q., Liu, C., Yang, K.M., Sun, S.Q., Wang, L., Yuan, C.S., Xu, Y.C., Xiong, W., Shen, Q.R., Wei, Z., 2025. Predatory protist promotes disease suppression against bacterial wilt through enriching plant beneficial microbes at the early stage of plant growth. Plant and Soil511, 1241–1252.

[43] Tagyan, A.I., Elghoul, O., Hozzein, W.N., Rabie, W., Alkhalifah, D.H.M., El-Far, N.A., 2025. Implications for agricultural sustainability: predicting the global distribution of Ralstonia solanacearum under current and future climate scenarios. Frontiers in Plant Science16, 1548640.

[44] Trivedi, P., Leach, J.E., Tringe, S.G., Sa, T., Singh, B.K., 2020. Plant–microbiome interactions: from community assembly to plant health. Nature Reviews Microbiology18, 607–621.

[45] Wagner, M.R., 2021. Prioritizing host phenotype to understand microbiome heritability in plants. New Phytologist232, 502–509.

[46] Wang, Z.J., Luo, W.B., Cheng, S.J., Zhang, H.J., Zong, J., Zhang, Z., 2023. Ralstonia solanacearum – A soil borne hidden enemy of plants: research development in management strategies, their action mechanism and challenges. Frontiers in Plant Science14, 1141902.

[47] Wei, Z., Friman, V.P., Pommier, T., Geisen, S., Jousset, A., Shen, Q., 2020. Rhizosphere immunity: targeting the underground for sustainable plant health management. Frontiers of Agricultural Science and Engineering7, 317–328.

[48] Wei, Z., Gu, Y.A., Friman, V.P., Kowalchuk, G.A., Xu, Y.C., Shen, Q.R., Jousset, A., 2019. Initial soil microbiome composition and functioning predetermine future plant health. Science Advances5, eaaw0759.

[49] Wei, Z., Huang, J.F., Hu, J., Gu, Y.A., Yang, C.L., Mei, X.L., Shen, Q.R., Xu, Y.C., Friman, V.P., 2015. Altering transplantation time to avoid periods of high temperature can efficiently reduce bacterial wilt disease incidence with tomato. PLoS One10, e0139313.

[50] Wen, T., Xie, P.H., Penton, C.R., Hale, L., Thomashow, L.S., Yang, S.D., Ding, Z.X., Su, Y.Q., Yuan, J., Shen, Q.R., 2022. Specific metabolites drive the deterministic assembly of diseased rhizosphere microbiome through weakening microbial degradation of autotoxin. Microbiome10, 177.

[51] Wu, J.L., Wu, C.F., Zhang, Q., Zhang, H.Q., Wang, S., Wang, F., Jin, S.Q., Kuzyakov, Y., Chen, J.Q., Ge, T.D., 2024. Microbial diversity loss and wheat genotype-triggered rhizosphere bacterial and protistan diversity constrain soil multifunctionality: evidence from greenhouse experiment. Plant and Soil504, 475–491.

[52] Xiong, W., Jousset, A., Guo, S., Karlsson, I., Zhao, Q.Y., Wu, H.S., Kowalchuk, G.A., Shen, Q.R., Li, R., Geisen, S., 2018. Soil protist communities form a dynamic hub in the soil microbiome. The ISME Journal12, 634–638.

[53] Xiong, W., Jousset, A., Li, R., Delgado-Baquerizo, M., Bahram, M., Logares, R., Wilden, B., De Groot, G.A., Amacker, N., Kowalchuk, G.A., Shen, Q.R., Geisen, S., 2021. A global overview of the trophic structure within microbiomes across ecosystems. Environment International151, 106438.

[54] Xiong, W., Song, Y.Q., Yang, K.M., Gu, Y.A., Wei, Z., Kowalchuk, G.A., Xu, Y.C., Jousset, A., Shen, Q.R., Geisen, S., 2020. Rhizosphere protists are key determinants of plant health. Microbiome8, 27.

[55] Xun, W.B., Li, W., Xiong, W., Ren, Y., Liu, Y.P., Miao, Y.Z., Xu, Z.H., Zhang, N., Shen, Q.R., Zhang, R.F., 2019. Diversity-triggered deterministic bacterial assembly constrains community functions. Nature Communications10, 3833.

[56] Yue, Y., Liu, C., Xu, B.T., Wang, Y.J., Lv, Q.H., Zhou, Z.Y., Li, R., Kowalchuk, G.A., Jousset, A., Shen, Q.R., Xiong, W., 2023. Rhizosphere shapes the associations between protistan predators and bacteria within microbiomes through the deterministic selection on bacterial communities. Environmental Microbiology25, 3623–3629.

[57] Yue, Y., Xu, Z.H., Wang, Y.J., Liu, C., Sun, S., Ren, X.Y., Lv, Q.H., Liu, J.C., Dini-Andreote, F., Xiong, W., Shen, Q.R., 2025. Soil-dwelling Naegleria enhances plant performance by stimulating beneficial bacterial functions in the rhizosphere. Nature Communications16, 9079.

[58] Zhao, J., Rodriguez, J., Martens-Habbena, W., 2023b. Fine-scale evaluation of two standard 16S rRNA gene amplicon primer pairs for analysis of total prokaryotes and archaeal nitrifiers in differently managed soils. Frontiers in Microbiology14, 1140487.

[59] Zhao, X.W., Yuan, X.M., Xing, Y.J., Dao, J.C., Zhao, D.Q., Li, Y.Z., Li, W.W., Wang, Z.T., 2023a. A meta-analysis on morphological, physiological and biochemical responses of plants with PGPR inoculation under drought stress. Plant, Cell & Environment46, 199–214.

RIGHTS & PERMISSIONS

Higher Education Press