Rigorous mathematical modeling and physiological experimentation reveal that Ralstonia wilt pathogens consume an in planta diet of amino acids with a dash of sugar

Abstract

How does Ralstonia acquire sufficient carbon for its massive in planta reproduction? To answer questions such as this, bacteriologists typically turn to our favorite approaches, bioassays with metabolic mutants, comparative genomics, gene expression analysis, and occasionally, chemistry, if we must. Molecular genetics provide evidence that sugars (primarily sucrose and glucose) partially support Ralstonia's growth in planta (Jacobs et al., 2012; Lowe-Power et al., 2018a; Gerlin et al., 2021; Hamilton et al., 2021). However, these mutants still wilt plants at nearly wild-type rates. Could amino acids and organic acids be the missing factor? Genetically blocking amino acid and organic acid assimilation is more complex than sugar assimilation, leaving the role of alternative carbon sources untested.

To solve this mystery, Caroline Baroukh et al. at the Laboratoire des Interactions Plantes Microorganismes (LIPME) part of the National Research Institute for Agriculture, Food and the Environment (INRAE), Toulouse, France, have adopted a strategic, stepwise approach integrating quantitative physiology and modeling (Fig. 1). Gerlin et al. (2021) precisely quantified the physiological variables of bacterial wilt disease, measuring concentrations of dozens of metabolites over an 8-day infection course using nuclear magnetic resonance (NMR) metabolomics. They found that tomato xylem sap contains c. 7–11 mM carbon and 2–4 mM organic nitrogen, with many amino acids more abundant than sugars. Notably, glutamine is present at c. 3.3 mM in planta and decreases during infection. Baroukh et al. then investigated Ralstonia's trophic preferences for the putative xylem sap carbon sources (Baroukh et al., 2022). Unlike typical bacteriologists who investigate nutrient utilization solely with bacterial growth assays in minimal media, Baroukh et al. continued using NMR metabolomics to analyze the rate at which Ralstonia assimilated each nutrient from culture media, both individually and in pairs. Here, they discovered that Ralstonia simultaneously assimilates distinct carbon sources, lacking the ‘catabolite repression’ mechanism sometimes seen in bacteria. Thus, Ralstonia are flexible heterotrophs – not picky eaters.

A key innovation of the 2024 study in New Phytologist is the development of a synthetic xylem-mimicking medium (XMM) that mirrors the carbon composition of tomato xylem sap (Baroukh et al., 2025). XMM allows for highly controlled studies of bacterial behavior in conditions similar to those in the xylem. Remarkably, the authors show that the complexity of tomato xylem sap can be simulated with just 13 substrates, including glutamine, asparagine, sucrose, and glucose. XMM supported bacterial growth similarly to the in planta environment. XMM addresses challenges in researching plant pathogens in vivo, such as laboriously harvesting sufficient xylem sap for experiments, especially during early infection stages. Using XMM, researchers tracked bacterial growth and nutrient consumption precisely, reinforcing Ralstonia's omnivorous ability to simultaneously catabolize multiple substrates without diauxic shifts. This insight underscores the pathogen's metabolic adaptability in nutrient-variable environments and offers a valuable tool for studying bacterial behavior under such conditions.

This study examined the enrichment of putrescine and acetate during infection. While acetate is a well-known by-product of overflow metabolism, the biological role of the polyamine putrescine is complex. Recent evidence shows exogenous application of putrescine increases plant susceptibility to Ralstonia through unknown mechanisms (Lowe-Power et al., 2018a). Ralstonia produce copious putrescine in media (Peyraud et al., 2016; Lowe-Power et al., 2018a; Baroukh et al., 2022). Although putrescine is not a carbon or nitrogen source, putrescine is essential for the pathogen's growth (Lowe-Power et al., 2018a). Ralstonia seems to increase putrescine levels in planta through two mechanisms. Similar to culture conditions, Ralstonia exudes copious amounts of putrescine into xylem sap while growing in planta (Lowe-Power et al., 2018a; Gerlin et al., 2021). Moreover, Ralstonia also injects living plant cells with a transcriptional activator-like effector protein that increases plant expression of a key putrescine biosynthesis gene (Wu et al., 2019). This paper sheds light on the fate of the putrescine that Ralstonia exudes into xylem sap: the Monod-like model indicates that the plant host absorbs putrescine. Nevertheless, putrescine's impact on host susceptibility remains to be solved.

‘All models are wrong’ (Box, 1976), but the models in Baroukh et al. (2025) provide useful insights into bacterial wilt disease. This study used macroscopic modeling to link xylem sap flow, metabolite abundance, and the pathogen's biomass growth. The macroscopic modeling was based on the Monod equation, an empirical equation that quantifies microbial growth based on the concentration of the rate-limiting nutrients. This new modeling further emphasizes the importance of xylem flow for Ralstonia's population growth in planta (Lowe-Power et al., 2018b; Baroukh et al., 2022). For Ralstonia, xylem sap flow delivers 8 M of carbon over 3 days, which this paper demonstrates is sufficient to support a population size of 10⁹ pathogen cells g⁻¹ tomato stem (Baroukh et al., 2022). Modeling the plant environment as a continuous nutrient flow, rather than static, showed that Ralstonia growth is limited by how wilt symptoms restrict nutrient availability. Bacterial occlusion of xylem vessels reduces nutrient flow, creating a negative feedback loop that limits pathogen proliferation. This balance between pathogen growth and pathogen damage to host physiology has broad implications for understanding disease progression.

Like all organisms, Ralstonia's population size is determined by reproduction, death, migration, and emigration rates. How many Ralstonia bacteria must migrate into a plant's roots to successfully initiate an infection? This simple question is surprisingly difficult to test due to the complexity of soil, plant root systems, and Ralstonia movement in soil. The integration of mathematical modeling and experimental data allows researchers to investigate infection conditions that are difficult to achieve in reality. This paper uses a playfully clever approach to extend the Monod-type model to simulate alternative realities of Ralstonia's early population dynamics. Thus, the paper develops testable hypotheses about the founding population size and the time required for the pathogen to enter the xylem.

The current model represents the plant as a simple chemostat rather than a dynamic organism. We anticipate future work that integrates Ralstonia and tomato physiological data into the authors' Virtual Young TOmato Plant (VYTOP; Gerlin et al., 2022). VYTOP integrates flux exchanges between multiple tomato organs, including the xylem, setting the groundwork for understanding the metabolic interface of pathogenesis. For example, VYTOP could explore how pathogen-produced putrescine impacts the tomato host's polyamine and other interconnected metabolites. VYTOP may also reveal how the diurnal shifts in nutrient concentrations and xylem sap flow rate (Windt et al., 2006) impact pathogen physiology during the day vs the night.

Refining the mathematical model into a compartmental model might allow analysis of pathogen biomass within a population of xylem vessels with varying flow rates. Decreased whole-plant transpiration rates result from the stepwise clogging of vessels. X-ray microcomputed tomography shows that wilt symptoms occur in young tomato plants when the pathogen occludes 50% of the xylem vessels (Ingel et al., 2023). How does the bacterial biomass migrate through the host? Future studies should examine how pathogen biomass migrates within the xylem under varying flow conditions. Recent technological breakthroughs enable visualization of Ralstonia's population growth in laboratory conditions mimicking xylem flow (Chu et al., 2024). Adapting live-cell imaging to these cellulose-coated microfluidic devices could enable cell-level quantification of bacterial reproduction and death in a xylem-like environment, providing new insights.

In summary, Baroukh et al. (2025) demonstrate the value of combining computational models and experimental quantification. By integrating data from the XMM, the authors developed a model that predicts bacterial density, sap composition, and vascular clogging conditions. This framework allows researchers to explore how metabolic activities drive pathogen colonization and disease onset. By identifying metabolic and population dynamics, the study provides insights into how nutrient fluxes correlate with disease severity. This approach opens new avenues for understanding Ralstonia dynamics and offers a framework adaptable for diverse plant–pathogen studies.

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