The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta

The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (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];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results The EPS-I exopolysaccharide transforms Ralstonia wilt pathogen biofilms into viscoelastic fluids for rapid dissemination in planta View ORCID Profile Matthew Cope-Arguello , View ORCID Profile Jiayu Li , View ORCID Profile Zachary Konkel , View ORCID Profile Nathalie Aoun , View ORCID Profile Tabitha Cowell , View ORCID Profile Nicholas Wagner , View ORCID Profile A. Li Han Chan , View ORCID Profile Lan Thanh Chu , View ORCID Profile Samantha Wang , View ORCID Profile Mariama D. Carter , Caitilyn Allen , View ORCID Profile Lindsay J. Caverly , View ORCID Profile Loan Bui , View ORCID Profile Kristen DeAngelis , View ORCID Profile Matthew J. Wargo , View ORCID Profile Tuan M. Tran , View ORCID Profile Jonathan M. Jacobs , View ORCID Profile Harishankar Manikantan , View ORCID Profile Tiffany M. Lowe-Power doi: https://doi.org/10.1101/2025.05.22.655385 Matthew Cope-Arguello 1 Department of Plant Pathology, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthew Cope-Arguello Jiayu Li 2 Department of Chemical Engineering, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jiayu Li Zachary Konkel 3 Infectious Diseases Institute, The Ohio State University , Columbus, OH, 43210, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Zachary Konkel Nathalie Aoun 1 Department of Plant Pathology, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nathalie Aoun Tabitha Cowell 1 Department of Plant Pathology, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tabitha Cowell Nicholas Wagner 5 Department of Biology, University of South Alabama , Mobile, AL 36688 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nicholas Wagner A. Li Han Chan 6 Department of Microbiology, University of Massachusetts , Amherst, MA, USA 01003 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for A. Li Han Chan Lan Thanh Chu 7 Department of Biology, University of Dayton , Dayton, OH 45469 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lan Thanh Chu Samantha Wang 2 Department of Chemical Engineering, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samantha Wang Mariama D. Carter 8 Department of Plant Pathology, University of Wisconsin-Madison , Madison, WI 53706, U.S.A. 9 Department of Crop Genetics, John Innes Centre , Norwich Research Park, Norwich NR4 7UH, United Kingdom Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mariama D. Carter Caitilyn Allen 8 Department of Plant Pathology, University of Wisconsin-Madison , Madison, WI 53706, U.S.A. Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lindsay J. Caverly 10 Department of Pediatrics, University of Michigan Medical School , Ann Arbor, MI 48109 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Lindsay J. Caverly Loan Bui 7 Department of Biology, University of Dayton , Dayton, OH 45469 USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Loan Bui Kristen DeAngelis 6 Department of Microbiology, University of Massachusetts , Amherst, MA, USA 01003 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Kristen DeAngelis Matthew J. Wargo 11 Department of Microbiology & Molecular Genetics, University of Vermont Larner College of Medicine , Burlington, VT 05405 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Matthew J. Wargo Tuan M. Tran 5 Department of Biology, University of South Alabama , Mobile, AL 36688 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tuan M. Tran Jonathan M. Jacobs 3 Infectious Diseases Institute, The Ohio State University , Columbus, OH, 43210, USA 4 Department of Plant Pathology, The Ohio State University , Columbus, OH, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonathan M. Jacobs Harishankar Manikantan 2 Department of Chemical Engineering, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Harishankar Manikantan For correspondence: hmanikantan{at}ucdavis.edu tlowepower{at}ucdavis.edu Tiffany M. Lowe-Power 1 Department of Plant Pathology, University of California , Davis, Davis, CA, USA 95616 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tiffany M. Lowe-Power For correspondence: hmanikantan{at}ucdavis.edu tlowepower{at}ucdavis.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Ralstonia solanacearum species complex (RSSC) pathogens cause destructive plant wilt diseases of a wide variety of crops, leading to significant agricultural losses worldwide. These bacteria rapidly spread through the water-transporting xylem where they grow prolifically and produce abundant biofilm that clogs xylem vessels. To understand RSSC biofilm behavior in planta , we examined their complex fluid mechanics. Rheological analyses revealed that unlike all previously analyzed microbial biofilms, RSSC biofilms are shear-thinning, viscoelastic fluids at physiologically relevant shear forces. To determine which factors confer these unique mechanics, we analyzed biofilms of bacterial mutants with altered biofilm components. Genetic analysis demonstrated that development of the viscous-dominant biofilms required production of EPS-I, an amphiphilic exopolysaccharide that is a major virulence factor for all RSSC pathogens. We show that EPS-I confers “biofilm mobility”, which allows wildtype RSSC colonies to passively expand when deformed. Despite its high metabolic cost, bioassays demonstrated that EPS-I production conferred a net fitness benefit where biofilm mobility allowed the pathogen to spread and access more nutrients in complex environments like xylem vessels. The RSSC are a monophyletic lineage of aggressive plant wilt pathogens, and our evolutionary hypothesis testing suggests the origin of the eps biosynthetic gene cluster coincides with the emergence of wilt pathogenesis in the RSSC ancestor. Furthermore, comparative physiological assays demonstrated that biofilm mobility is unique to the RSSC within the genus Ralstonia . In summary, EPS-I production is a key evolutionary innovation that enables RSSC dispersal and virulence by conferring unique biofilm mechanics. Significance Statement Ralstonia solanacearum species complex (RSSC) pathogens threaten global food security by fatally wilting plants. A soft matter physics lens demystified the cryptic role of a major virulence factor, the EPS-I exopolysaccharide. EPS-I transforms RSSC biofilms into viscoelastic fluids, a mechanical behavior not previously described for other microbial biofilms that are almost always viscoelastic solids. We demonstrate that the development of fluid biofilms was a key evolutionary innovation that enabled pathogenic success of these aggressive pathogens that rapidly wilt plants. Introduction Approximately 80% of bacteria on earth live in complex, three-dimensional aggregates called biofilms ( 1 ). Biofilms are assemblages of one-or-more microbial species embedded in a self-produced matrix of extracellular DNA (exDNA), exopolysaccharides, lipids, and proteins. By developing biofilms, bacteria remodel their immediate physical, chemical, and biological environments. The biofilm environment confers resilience to environmental stresses, including antimicrobial molecules, desiccation, predation by phages, and attacks from microbial competitors. Biofilms have adhesive, viscous, and elastic mechanical properties that enable many bacteria to form stable and persistent colonies in dynamic environments, including in plant and animal hosts ( 2 ). The mechanical behavior of diverse microbial biofilms has been extensively studied using rheometry ( Table S1 ) ( 3 – 23 ). Although biofilm mechanics differ based on genetic and environmental variation, biofilms typically behave as viscoelastic solids, meaning they have a larger elastic component (termed G` , or elastic modulus) compared to the viscous component (termed G ``, or viscous modulus). Conversely, viscoelastic fluids would have a greater viscous component than elastic. For viscoelastic materials such as a biofilm, acute, fast forces impose short-lived stresses, causing the biofilm to spring back like an elastic band ( 2 ). In contrast, slow, persistent forces relax polymeric molecular springs, causing the biofilm to flow like a fluid, enabling passive expansion of the biofilm. Thus, the mechanical properties of biofilms influence how bacteria colonize complex, dynamic environments, like those with flowing conditions. Bacterial plant pathogens within the Ralstonia genus produce notoriously fluid biofilms in planta and on agar ( 24 ). Collectively, these causal agents of bacterial wilt disease are known as the Ralstonia solanacearum species complex (RSSC), a monophyletic lineage of three species that colonize the water-transporting xylem vessels and disrupt xylem sap flow across a broad range of hosts ( 25 ). Microscopy of RSSC-infected xylem tissues usually shows substantial biofilms of RSSC cells and extracellular matrix ( 26 – 29 ). These in planta biofilms allow diagnosticians to identify the tell-tale sign known as bacterial streaming ( 30 ), where visible plumes of pathogen biofilms “stream” out of xylem vessels when the cut stem of a wilted plant is submerged in water. RSSC pathogens develop biofilms by producing a matrix of extracellular DNA, proteins, and a unique amphiphilic, exopolysaccharide called “EPS-I” ( 29 , 31 , 32 ). These components contribute to the structure and function of RSSC biofilms. RSSC secrete two nucleases, NucA and NucB, which modulate the amount of extracellular DNA ( 29 ). Δ nucAB mutants form thicker biofilms and dome-shaped colonies, suggesting that DNA influences the mechanical behavior of RSSC biofilms. RSSC secrete multiple sugar-binding lectin proteins ( 32 ), and mutants lacking the LecF and LecX lectins develop expansive colonies with apparent zones of collapse in the center ( 32 ). However, the most important biofilm component is the major virulence factor EPS-I ( 29 , 30 , 33 – 35 ). RSSC pathogens secrete abundant EPS-I when growing at high cell densities in vitro and in plant hosts because the expression of the corresponding eps biosynthetic gene cluster is positively regulated by quorum sensing ( 36 – 40 ). Exactly how EPS-I contributes to bacterial wilt disease has perplexed researchers ( 24 ). A potential model is that EPS-I enables pathogen dissemination throughout the host ( 33 , 41 , 42 ). Here, we hypothesize that RSSC pathogens have evolved to produce biofilms with unique mechanical properties, which contribute to pathogenic success by rapidly spreading the pathogens throughout the flowing xylem environment. To test this hypothesis, we employed rotational rheometry to quantify the bulk mechanical behavior of colony biofilms from diverse wildtype RSSC isolates, from RSSC mutants with altered biofilm composition, and from an unrelated plant pathogen. In striking contrast to all previously investigated bacterial biofilms that are viscoelastic solids , we show that EPS-I production transforms RSSC biofilms into viscoelastic fluids . This mechanical behavior enhances RSSC fitness by promoting colony expansion in both in vitro and in planta environments, highlighting the adaptive advantage conferred by fluidal biofilms. Our evolutionary genomic, physiological, and rheological analyses suggest that the emergence of the RSSC as rapid wilt pathogens coincided with the evolutionary invention of fluidal biofilms, offering new insight into the ancient emergence of these globally impactful pathogens. Results and Discussion RSSC wilt pathogens form biofilms that are shear-thinning, viscoelastic fluids Peculiarly, cultures of RSSC isolates appear to be more liquid than solid ( Fig. 1A-B ; Movie S1 ), suggesting that their biofilms are not typical viscoelastic solids. We investigated the rheological properties of RSSC biofilms with selected representatives of each of the three RSSC species ( R. solanacearum “ Rsol ” IBSBF1503, R. pseudosolanacearum “Rpseu ” GMI1000, and R. syzygii “ Rsyz ” PSI07) ( Fig. 1C-D , Fig. 2 , Fig. S1 , and Fig. S2 ). These isolates have subtle natural diversity in their colony morphology ( Fig. S3 ). Rpseu GMI1000 and Rsyz PSI07 produce typical colonies that are wide, fluidal, and have irregular shape whereas Rsol IBSBF1503 produces smaller dome-like colonies characteristic of the “small, fluidal, round (SFR)” morphology ( 43 , 44 ). These data revealed an inverse relationship between colony size and viscosity ( Fig. 1D and Fig. S3 ). At a shear rate of 0.11 s -1 , the RSSC biofilms had mean viscosities of 2.19 ± 0.639 Pa*s ( Rpseu GMI1000), 6.47 ± 0.629 Pa*s ( Rsol IBSBF1503), and 1.39 ± 0.160 Pa*s ( Rsyz PSI07) (± standard deviation [SD]; Fig. 1D ). To further understand the implications of these viscosities, we determined that this shear rate is relevant to the shear rates of xylem flow ( SI “Results and Discussion” ) ( 45 ). Download figure Open in new tab Fig. 1. Ralstonia solanacearum species complex (RSSC) wilt pathogens produce biomass with viscous flow mechanical behavior. Photographs of ( A ) RSSC biomass ( Rpseu GMI1000) streaming from a submerged, cut stem of a wilt symptomatic tomato plant and (B) RSSC colony biofilms ( Rpseu GMI1000) that have coalesced and dripped upon inversion of petri dishes. The full video is available as Video S1. (C) Overview of biofilm rheology experiments. (D) Viscosity of colony biofilms of RSSC pathogens ( Rsol IBSBF1503, Rpseu GMI1000, and Rsyz PSI07) and Xanthomonas XCC8004 at varying shear rates. The dashed line represents an estimated maximum shear rate within the xylem vessels of healthy plants (SI “Results and Discussion” ) ( 45 ). Symbols show mean (N=3); error bars show SD. Download figure Open in new tab Fig. 2. EPS-I confers unique viscoelastic mechanics to colony biofilms of RSSC wilt pathogens. Elastic (G`) and viscous (G``) moduli were measured in frequency sweep experiments and used to calculate the Loss factor for ( A ) wildtype RSSC wilt pathogens (GMI1000, IBSBF1503, and PSI07) and Xanthomonas (XCC8004) and ( B ) RSSC mutants with altered biofilm properties. Symbols show mean (N=3); error bars show SD. The dotted horizontal line represents the transition from elastic to viscous dominant behavior, corresponding to G`` / G`=1. Data from Wildtype GMI1000 is repeated across panel A and B plots for consistent comparisons As a biologically informative comparison, we analyzed biofilms of a leaf-infecting Xanthomonas pathogen ( Xanthomonas campestris pv. campestris 8004, “XCC8004”). Xanthomonads secrete xanthan gum, a model viscoelastic polymer in rheology ( 46 , 47 ). As expected, the Xanthomonas biofilms were substantially more viscous than RSSC biofilms with a mean viscosity of 319 ± 3.22 Pa*s (± SD) at a shear rate of 0.11 s -1 ( Fig. 1D ). Additionally, all wildtype RSSC and Xanthomonas biofilms were shear-thinning ( Fig. 1D ), meaning the viscosity decreased with increasing shear rate. Shear thinning is a typical trait for biofilms and can be contrasted with a shear thickening fluid such as a corn starch solution that resist deformation from strong forces, but readily flow when perturbed with weak forces ( 48 ). The shear-thinning feature of biofilms assists their ability to deform when external forces are high enough, flowing more easily with increasing flow rate in the xylem. We next quantified the elastic ( G` ) and viscous ( G`` ) moduli of RSSC and Xanthomonas biofilms in the linear viscoelastic region (see methods, SI “ Materials and Methods” and Fig. S1 , for details of rotational rheology). All strains exhibited a direct relationship between rotational frequency and the moduli, and Xanthomonas biofilms had higher elastic and viscous moduli than the RSSC biofilms at all frequencies ( Fig. 2A ). Moreover, the rate at which the viscous and elastic moduli increased as frequency increased was greater for RSSC biofilms than for Xanthomonas biofilms. For example, the elastic moduli of Rsol IBSBF1503 increased across frequencies about 5000-fold from a mean of 0.0137 Pa to 74.2 Pa. By contrast, Xanthomonas biofilms increased about 4.5-fold, from a mean of 89.1 Pa to 404 Pa. To compare the viscous and elastic moduli and characterize the biofilms as either viscoelastic solids or viscoelastic fluids, we determined the “loss factor” of the biofilms by calculating the ratio of G `` to G ` . Materials are elastic dominant (viscoelastic solids) if their loss factor is less than 1 and viscous dominant (viscoelastic fluids) if their loss factor is greater than 1. Xanthomonas biofilms were predominantly elastic at all driving frequencies ( Fig. 2A ). RSSC colony biofilms, on the other hand, were uniquely viscous-dominant with loss factors between 1.51 and 22.5, depending on the RSSC strain and measurement frequency ( Fig. 2A ). Overall, Xanthomonas biofilms are viscoelastic solids like all previously analyzed microbial biofilms ( Table S1 ) ( 3 – 23 ). RSSC biofilms have viscoelastic fluid behavior where the mechanical work performed in deforming RSSC biofilms is lost as they flow rather than being stored and so they are unable to rebound like in an elastic material. The unique exopolysaccharide, EPS-I, is responsible for the viscous-dominant biomechanics of RSSC biofilms The revelation that RSSC biofilms are viscoelastic fluids led us to ask, which matrix components confer these unique mechanics? We repeated the rheological assays with RSSC mutants that have altered biofilm composition: Δ nucAB , Δ lecF , Δ lecX , Δ lecFX , and Δ epsB mutants in the Rpseu GMI1000 background. The Δ nucAB mutant has elevated exDNA due to the absence of secreted DNases ( 29 ). The Δ lecF , Δ lecX , and Δ lecFX mutants lack lectin(s) that contribute structural stability to RSSC colonies ( 32 ). The Δ epsB mutant does not produce EPS-I because it lacks a Wzc family integral inner membrane protein that is required for EPS-I export ( 49 ). All mutant biofilms retained shear-thinning mechanical behavior ( Fig. S2 ) ( 32 ). The DNase and lectin mutant biofilms exhibited moderately altered elastic ( G` ) and viscous ( G`` ) moduli ( Fig. 2B ). Consistent with the Δ nucAB mutant’s development of dome-shaped colonies ( 29 ), Δ nucAB biofilm viscous moduli were up to 3-fold higher and elastic moduli up to 7-fold higher than wildtype GMI1000. In addition to their known increase in colony area and the reduction in biofilm viscosity ( 32 ), lectin mutant biofilms also had reduced elasticity. While these biofilm components clearly contribute to the viscoelastic behavior of RSSC biofilms, mutants lacking these components all still produced viscous dominant biofilms. Notably, the Δ epsB mutant was the only mutant that lacked the characteristic viscous-dominant mechanics of wildtype RSSC biofilms with a loss factor between 0.308 and 1.53, depending on the measurement frequency ( Fig. 2B ). Additionally, the biofilms of the EPS-deficient mutant were dramatically more viscous and elastic than wildtype biofilms across all frequencies. At the median measured frequency (0.156 Hz), biofilm G` was 1,030 ± 199 Pa and 0.173 ± 0.0772 Pa for the Δ epsB mutant vs wild type, respectively (mean ± SD). Similarly, the biofilm G`` was 1,260 ± 91.0 Pa and 1.43 ± 0.177 Pa for the Δ epsB mutant vs wild type, respectively. These data reveal that EPS-I contributes to the viscous-dominant nature of RSSC biofilms, reducing the crossover strain required for RSSC biofilms to flow. EPS-I exopolysaccharide confers collective biofilm mobility of plant pathogenic RSSC biofilms in diverse environments Our discovery that EPS-I confers unique rheological properties to RSSC biofilms motivated us to explore the mechanistic understanding of this major virulence factor because the precise pathogenic function of EPS-I remains cryptic. The SI Results and Discussion summarizes gaps in knowledge from prior in planta studies of EPS-I ( 24 , 33 , 42 ). Briefly, the literature was unclear about whether EPS-I contributes to pathogen growth in planta . Thus, we tested the contribution of EPS-I to pathogen growth and dissemination. Using cut petiole inoculations, we directly inoculated plant xylem with either wildtype GMI1000 or the Δ epsB mutant. After 3 days, population sizes were quantified at the site of inoculation and at distal sites 2-cm and 4-cm above and below the site of inoculation ( Fig. 3A ). At the site of inoculation, Δ epsB mutant had the same incidence as wild type (95%). However, at distal sites below and above the site of inoculation, there was lower incidence of the Δ epsB mutant: 69-81% wild type and 25-50% for the Δ epsB mutant. Overall, our results are consistent with a role of EPS-I as a dissemination factor. Download figure Open in new tab Fig. 3. EPS-I confers biofilm mobility, which enhances RSSC fitness. (A) Dispersal of wildtype and Δ epsB Rpseu GMI1000 in tomato stems after cut-petiole inoculation (4 trials of n=8 plants per strain for a total of n=32 plants per strain; * represents P <0.05, Mann-Whitney test). (B) Biofilm occupancy of CMC-coated microfluidic channels by wildtype and Δ epsB Rpseu GMI1000, following 72 hr of rich media flowing at various rates. The image shows a representative result at 10 µL medium /hr from the middle portion of the microchannel, and images from all flow rates are shown in Fig. S5 (Scale bar = 50 μm; * represents adjusted P <0.05, ANOVA with Šídák’s multiple comparisons test). (C) Biofilm mobility of wildtype and Δ epsB Rpseu GMI1000 when grown in static flat vs. tilted conditions. (Scale bar = 1 cm; letters indicate P <0.0001 by Welch’s ANOVA with Dunnett’s multiple comparisons) full data set in Fig S6. (D) Mobility of individual colonies grown rich agar. Colony biofilms grew at a normalized density of 125 CFU/spot for two days on either a flat or tilted angle. A full image set for all RSSC strains and Xanthomonas XCC8004 can be found in Fig. S7. (E) EPS-I enables colony expansion, which increases population sizes per colony (Scale bar: 1 cm; middle panel: shows a superplot of individual colonies (grey) and the median for each petri dish (blue or red symbols) where * indicates P <0.0001 by Welch’s ANOVA with Brown-Forsythe multiple comparisons; right panel: * indicates P <0.0001 by Mann-Whitney test). Nevertheless, our experiments demonstrate that the Δ epsB mutant had a general growth defect at the site of inoculation ( Fig. 3A and Fig. S4 ). Wildtype populations reached a geometric mean 4.8 x10 6 CFU/g at the inoculation site, compared to Δ epsB population sizes of 2.0 x10 5 CFU/g. This growth defect at the inoculation site demonstrates that movement is unlikely the sole virulence function of EPS-I. The growth defect suggests that biofilm viscoelasticity also protects the pathogens from the external chemical stresses imposed by the plant host. Thus, to directly test the contribution of EPS-I to movement of RSSC populations, we sought to investigate RSSC biofilm mobility in the absence of plant immune defenses. Microfluidic devices simulate the highly confined hydrodynamic environment that RSSC pathogens experience in xylem vessels. To investigate the contribution of EPS-I to the colonization of xylem-like environments in a flow-dependent manner, we cultured wildtype GMI1000 and the Δ epsB mutant in microfluidic devices coated with carboxymethyl cellulose-dopamine (CMC) to further replicate the environment of xylem vessels ( Fig. S5 ) ( 50 ). We found that total coverage of wildtype biofilms was significantly greater than Δ epsB biofilms at 10 and 40 µL/hr flow rates ( Fig. 3B ) but not significantly different at higher and lower flow rates. Wildtype GMI1000 coverage of the microfluidic channels had a geometric mean of 20.3% and 12.3%, respectively. By comparison, the Δ epsB mutant covered the microfluidic channels to 10.7% and 5.04% for the respective flow rates. While we recently demonstrated that EPS-I was necessary for biofilm development in these microfluidic channels, our analysis of the rheological contributions of EPS-I led us to question whether the contribution of EPS-I depends on flow. Interestingly, the 10 and 40 µL/hr flow rates translate to dynamic processes in the 1-10 s -1 frequency range, where the Δ epsB mutant biofilm is elastic-dominant ( Fig. 2B ). It is known that flow in microchannels creates shear forces that can cause biofilms to grow, restructure, or detach from walls. Even very small fluid shear stresses can initiate biofilm formation in some species by triggering the secretion of polysaccharides that aid biofilm formation ( 51 , 52 ). Conversely, strong shear associated with high flow rates can suppress biofilm formation. Fast bulk flow can directly inhibit biofilm development by carrying cells at much faster rates than the cells can attach to the surface or indirectly by carrying nutrients away faster than the nutrients can diffuse to surface-attached cells. Thus, the effect of biofilm viscoelasticity on biofilm structure and behavior is most evident in intermediate, physiologically relevant shear stresses, which are approximately up to ∼10 -2 Pa in the case of xylem flow ( SI “Results and Discussion” ). At these stresses, typical viscoelastic solid biofilms would undergo restructuring due to their solid-like mechanical behavior, which is characterized by very slow relaxation and can result in stiffening, softening, or yielding, depending on the species ( 51 ). Our findings that EPS-I contributes to colonization of microfluidic channels in a flow-dependent manner suggests that the unique viscous-dominant rheology of RSSC biofilms contributes to the enhanced surface coverage at physiologically relevant shear stresses. Knowing that wildtype RSSC colony biofilms can drip when deformed by gravity ( Video S1 ), we developed simple assays to compare mobility of RSSC biofilms. In one assay, cell suspensions were spotted on agar and colony biofilms developed while the plates were incubated at constant fixed angles of 0° (flat) or 39° (tilted). As expected, the Δ epsB mutant biofilms remained in place while gravity caused the wildtype biofilms on the tilted plates to deform and disseminate up to 2 cm ( Fig. 3C and Fig S6 ). Thus, EPS-I production enables wildtype RSSC to flow as a viscoelastic fluid, a trait we call “biofilm mobility” hereafter. We repeated the tilt plate assay with plates inoculated at low cell density so that individual cells would grow into spatially separated colonies. Once again, wildtype biofilms were mobile and dripped when grown on tilted agar while the Δ epsB biofilms remained in place ( Fig. 3D and Fig. S7 ). Only the Δ epsB mutant and Xanthomonas XCC8004 biofilms lacked mobility ( Fig. 3D and Fig. S7 ). Intriguingly, the images suggest that when multiple wildtype RSSC biofilms converged, the collective mass traveled longer distances as a confluence. This visualization of RSSC biofilm mobility underscores the demonstrated fact that EPS-I production is a collective good that is produced in a quorum sensing-dependent manner ( 38 – 40 ). Biofilm mobility improves RSSC fitness by enabling the pathogen to passively expand its niche Although EPS-I production is a net benefit to RSSC fitness in planta ( Fig. 3A ), this outcome is context-dependent. When grown in liquid media, EPS-I production reduces RSSC’s maximum growth rate ( Fig. S8 ) ( 53 ), almost certainly due to the metabolic burden of synthesizing and exporting the EPS-I polymer. We hypothesized that EPS-I is a net benefit to RSSC fitness in conditions where the viscoelasticity allows RSSC biofilms to expand their physical niches and gain access to more nutrients. We tested this hypothesis on agar plates, quantifying colony area and population sizes. Even when growing flat, gravity deforms wildtype biofilms, causing colony expansion ( Fig. 3E ). We used CellProfiler to quantify the area of individual colonies ( 54 ), which demonstrated that wildtype RSSC colonies occupy roughly double the area compared to Δ epsB colonies ( Fig. 3E ). Wildtype GMI1000 colonies grew to a geometric mean of 2.48 mm 2 , and the Δ epsB colonies grew to 1.11 mm 2 . However, a larger colony does not necessarily mean that wildtype cells had higher reproduction. In addition to lacking EPS-I, Δ epsB colony biofilms had a significant decrease in moisture content ( Fig. S9 ). Wildtype GMI1000 colonies had a moisture content of 90.3% ± 0.251% (mean ± SD), while Δ epsB colony biofilms had a mean moisture content of 64.6% ± 0.286%, suggesting the difference in colony size might be confounded to a difference in water. Thus, we directly measured the population size of individual colonies by excising colonies with a blade, homogenizing the colony, and dilution plating to enumerate colony forming units (CFUs). This direct test of bacterial fitness demonstrated that individual wildtype colonies contained approximately 4-fold more cells than Δ epsB colonies ( Fig. 3E ). Wildtype GMI1000 colony populations contained a geometric mean of 1.6x10 8 CFU/colony, whereas the Δ epsB colonies contained 6.0x10 7 CFU/colony. We assume that EPS-I production had the same metabolic cost whether the cells grow in liquid broth or on agar. Nevertheless, wildtype cells on agar were able to reproduce more than the Δ epsB mutants. Why? Although we cannot rule out the hypothesis that the Δ epsB cells were constrained by water stress in the absence of hygroscopic EPS-I, we speculate that production of EPS-I allowed the colonies to increase the cells’ access to nutrients via a microstructural change closely linked to biofilm rheology. The resulting flowability increased the area of a colony as it spreads due to gravity, enhancing nutrient flux across the agar-colony interface. Additionally, we speculate that the elastic modulus (G`) roughly indicates the amount of thermal energy stored per unit volume of biofilm matrix. The relevant volume in this case is set by the typical mesh size within the scaffolded structure of the entangled extracellular polymer matrix ( 55 ). G` is then inversely related to the volume of the space between polymeric entanglements. A reduction in the elastic modulus due to EPS-I then indicates an increase in the volume available for nutrients to reside in and diffuse freely into without the close confinements of a tight polymeric mesh. Thus, the increased opportunity to acquire nutrients exceeds the cost of EPS-I production. Viscoelastic fluid biofilms are a key evolutionary event that coincides with the emergence of wilt pathogenesis within the Ralstonia genus RSSC wilt pathogens are a monophyletic lineage within the Ralstonia genus. Other species of Ralstonia are found in habitats such as soil, the plant rhizosphere, surface water, industrialized water systems such as the water recycling system on the International Space Station, and opportunistic infections of humans. To explore the evolutionary history of the RSSC wilt pathogens’ EPS-I dependent, viscoelastic liquid biofilms, we explored publicly available genomic data and biofilm mobility phenotypes of diverse isolates across the Ralstonia genus. RSSC isolates produce and export EPS-I via the products of the 17-gene eps cluster that is encoded on a megaplasmid ( 49 , 56 , 57 ). We used BLASTp to search 399 RSSC genomes and 72 non-RSSC Ralstonia genomes for homologs of the eps cluster genes and the neighboring xpsR gene whose product activates expression of the eps genes ( 38 ). Within the plant pathogenic RSSC, the eps genes are syntenic and nearly universally conserved ( Fig. 4A-B and Fig S10 ), except for a single genome ( Fig. S11) . Most genes had high amino acid sequence identity with a minimum identity of 84.5%-96.5% to the query, but RSp1005 had higher variation with identity as low as 65.1%. None of the non-RSSC Ralstonia genomes encoded the eps gene cluster although low identity homologs were occasionally detected ( Fig. 4B ). A similar analysis suggests that the nucA nuclease is also specifically present in RSSC genomes while the nucB nuclease is conserved across the entire genus ( Fig. S12 ). Download figure Open in new tab Fig. 4. EPS-I production and biofilm mobility is an evolutionary innovation of RSSC wilt pathogens. (A) The eps gene cluster synteny of strains from the three RSSC species was visualized with Clinker. (B) Presence of eps genes in genomes of the RSSC pathogens and non-RSSC Ralstonia species that do not wilt plants. Rectangles show percent amino acid identity of the best BLASTp hit for queries with Rsol K60 proteins. The approximate maximum likelihood phylogenetic tree was built from 49 conserved bacterial COGs using the KBase Insert Genomes into SpeciesTree v2.2.0 application and visualized in iToL. (C-D) Results of biofilm mobility assay non-wilt pathogenic Ralstonia . Full results are in Fig. S15 ( C ) Representative results showing the soil isolate R. thomasii GP103 (D) Summary of colony biofilm phenotypes of non-wilt-pathogenic Ralstonia isolates. The pattern of eps gene presence suggests that either 1.) the eps genes were acquired or evolved in the common ancestor of the RSSC when this lineage diverged from the rest of the genus, or 2.) the ancestor of the entire Ralstonia genus encoded the eps gene cluster, and the non-RSSC lineage lost the eps genes. To investigate these evolutionary hypotheses, we used a cluster reconstruction and phylogenetic analysis pipeline ( 58 , 59 ) to reconstruct phylogenies for the 17 RSSC eps cluster genes that encode the proteins for synthesis and export of EPS-I. Each EPS protein was BLASTp searched against a database of 16,355 betaproteobacterial genomes, including 508 RSSC genomes, and phylogenies of each gene were constructed after the BLAST datasets were truncated through sequence similarity clustering ( 58 ). This analysis identified 1,563 non-RSSC betaproteobacterial genomes that had 2 or more eps genes within 20 kb, but only the RSSC pathogen genomes encoded the full eps gene cluster. Several eps genes were rarely detected in the betaproteobacterial genomes: epsE, epsF , RSp1013, RSp1015, and RSp1003 ( Fig. S13 ). Several non-RSSC genomes had as many as 13 homologs to the 17 eps genes ( Fig. S13 ), but those gene clusters contained additional non-homologous genes and often varied in gene organization ( Fig. S14 ), suggesting the gene products would produce a different exopolysaccharide than EPS-I. The gene phylogenies provided a second line of evidence that the eps cluster is conserved across the RSSC and shares a recent origin within the clade of RSSC wilt pathogens. The outgroups of the RSSC eps genes were commonly identified in Cupriavidus , the sister genus of Ralstonia . There was also evidence of recombination of partial eps -like clusters in sporadic genomes of R. pickettii and R. mannitolilytica and throughout the Alcaligenaceae and Burkholderiaceae. As a third approach, we used cblaster to query the 17 eps genes against public genomes in NCBI’s nr database. Once again, the eps gene cluster was only identified in RSSC genomes. Overall, the gene cluster responsible for EPS-I production is unique to the RSSC. This result aligns with the effectiveness of antibody-based diagnostics to accurately detect bacterial wilt disease through the identification of EPS-I ( 60 ) . Together, the evolutionary genomic analyses strongly suggest that production of viscous-dominant EPS-I is unique to the RSSC. But what about the physiology? To determine whether the non-RSSC Ralstonia shared the trait of forming viscoelastic fluid colony biofilms, we investigated the biofilm mobility phenotype of other Ralstonia spp. isolated from the International Space Station, soil, and human patients. All 23 of these isolates lacked biofilm mobility ( Fig. 4C-D and Fig. S15 ), which demonstrates that the mobile, fluid biofilm trait is specific to the RSSC wilt pathogens. An improved model of the role of EPS-I in RSSC virulence We propose that EPS-I is a key in planta movement factor that transforms RSSC biofilms into viscoelastic fluids. Based on the measured viscosities of wildtype RSSC biofilms ( Fig. 1D ) and estimated shear rates in xylem vessels ( 45 ), we calculate that viscous flow could passively disseminate RSSC biofilms in the range of centimeters per day while the Δ epsB mutant is practically immobile against flow ( SI “Results and Discussion” ). The chemical structure of EPS-I sheds light on the unique mechanics. Typical polysaccharides are composed of polar sugars. However, EPS-I is an amphiphilic polysaccharide composed of trimeric repeats of N-acylated derivatives of galactosamine, deoxygalacturonic acid, and the reduced sugar bacillosamine ( Fig. S16 ) ( 31 ). We suspect that when in the extracellular matrix, this polymer has minimal inter- and intra-molecular hydrogen bonding when compared to canonical polysaccharides, leading to a decrease in biofilm elasticity and viscosity. As an amphiphilic polymer, EPS-I appears to play an analogous role in bacterial movement as rhamnolipids and other small molecule biosurfactants that coat surfaces and enable cells to slide ( 61 ). Our results suggest that EPS-I is analogous to a biosurfactant that enables biofilms to expand by flow when deformed by the modest shear forces within xylem vessels. Thus, RSSC pathogens have three movement mechanisms: flagellar swimming motility, type IV pilus-driven twitching motility, and EPS-I dependent biofilm mobility. RSSC predominantly use swimming motility to invade roots ( 62 ). Swimming motility is repressed in the xylem ( 62 ), and non-flagellated mutants have wildtype virulence and normal in planta dissemination ( 62 , 63 ). Multiple lines of evidence suggest that pilus-mediated motility and EPS-mediated biofilm mobility have complimentary roles in RSSC dissemination through plant hosts. Even when direct inoculation into plant stems allows the pathogen to bypass root invasion, type IV pili and EPS-I are both strongly required for virulence ( 33 , 64 ) and dissemination ( Fig. 3 ) ( 42 , 63 ). The evolutionary conservation of EPS-I biosynthesis across the three RSSC species suggests that biofilm fluidity contributes to these pathogens’ success. Biofilm mobility has also been demonstrated for the distantly-related xylem pathogen Erwinia amylovora although the rheological properties for biofilms of this pathogen have not been analyzed ( 65 ). Not all vascular plant pathogens have mobile biofilms. Here, we demonstrated that the vascular black rot pathogen Xanthomonas XCC8004 produces highly elastic, viscoelastic solid biofilms, similar to all previously characterized bacterial and fungal biofilms. These Xanthomonas biofilms were non-mobile under the force of gravity. However, a key principle of rheology is “everything flows;” with sufficient strain, viscoelastic solid biofilms will migrate by viscous flow ( 2 ). Nevertheless, RSSC pathogens can fully wilt a plant host within days, which is dramatically faster than Xanthomonas and all other bacterial or fungal wilt pathogens. Thus, viscoelastic fluid biofilms are key factors for systemic in planta spread of RSSC pathogens. Materials and Methods Detailed methods for bacterial strains and culture media and experiments on rheometry of colony biofilms, bacterial growth and dispersal in tomato stems, biofilms in CMC-coated microfluidic, biofilm mobility on agar, image analysis with CellProfiler, growth curves, and bioinformatic analyses are present in SI Appendix, Supplementary Methods . Data availability All data in this publication can be found on Zenodo ( https://zenodo.org/ ) with the DOI: 10.5281/zenodo.14767251 Competing Interest Statement The authors declare no competing interests. Author Contributions MCA: Conceptualization, Analysis, Experiments / Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing; JL: Conceptualization, Analysis, Experiments / Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing; TML: Conceptualization, Data curation, Analysis, Funding acquisition, Methodology, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing; HM: Conceptualization, Analysis, Funding acquisition, Methodology, Resources, Supervision, Writing – original draft, Writing – review & editing; NA: Analysis, Experiments / Investigation, Methodology, Visualization, Writing – review & editing; TMT: Conceptualization, Data curation, Analysis, Funding acquisition, Experiments / Investigation, Methodology, Resources, Supervision, Validation, Writing – review & editing; TC: Analysis, Experiments / Investigation, Visualization, Writing – review & editing; MDC: Conceptualization, Resources, Writing – review & editing; CA: Conceptualization, Resources, Supervision, Writing – review & editing; ZK: Data curation, Analysis, Experiments / Investigation, Methodology, Validation, Visualization, Writing – review & editing; JJ: Funding acquisition, Supervision, Writing – review & editing; AC: Experiments / Investigation, Resources, Visualization, Writing – review & editing; KMD: Funding acquisition, Resources, Supervision, Writing – review & editing; SW: Experiments / Investigation, Writing – review & editing; MJW: Experiments / Investigation, Resources, Writing review & editing; LJC: Resources, Writing – review & editing; NW: Data curation, Analysis, Experiments / Investigation, Methodology, Validation, Visualization, Writing – review & editing; LB: Experiments / Investigation, Writing – review & editing; LTC: Experiments / Investigation, Writing – review & editing Funding Information None of the funders had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. View this table: View inline View popup Download powerpoint Acknowledgments We thank Prof. Cooper Battle (Willamette University) and Clay Fuqua (Univ. Indiana) for valuable discussion. We also thank Prof. Pamela Ronald (University of California, Davis) for the contribution of the Xanthomonas campestris pv. campestris 8004 isolate. Funder Information Declared United States Department of Agriculture (USDA), Washington, DC (Hatch Grant) , #1023861 , #7004345 USDA National Institute of Food and Agriculture , #2024-67011-42914 , #2024-67012-42635 , #2022-67013-36272 University of California, Davis, Davis, US (Academic Senate) U. of South Alabama Office of Research & Economic Development , #A24-0097-001 USDA Specialty Crop Block Grant , #23SCBGPAL 1173-00 Cystic Fibrosis Foundation Funding , CAVERL22AB0 National Science Foundation, https://ror.org/021nxhr62 , DGE-1747503 References 1. ↵ H.-C. Flemming , S. Wuertz , Bacteria and archaea on Earth and their abundance in biofilms . Nat. Rev. Microbiol . 17 , 247 – 260 ( 2019 ). OpenUrl CrossRef PubMed 2. ↵ E. S. Gloag , S. Fabbri , D. J. Wozniak , P. Stoodley , Biofilm mechanics: Implications in infection and survival . Biofilms 2 , 100017 ( 2020 ). OpenUrl CrossRef 3. ↵ J. K. Beckwith , M. Ganesan , J. S. VanEpps , A. Kumar , M. J. Solomon , Rheology of Candida albicans fungal biofilms . Journal of Rheology 66 , 683 – 697 ( 2022 ). OpenUrl CrossRef 4. L. I. Brugnoni , M. C. Tarifa , J. E. Lozano , D. Genovese , In situ rheology of yeast biofilms . Biofouling 30 , 1269 – 1279 ( 2014 ). OpenUrl CrossRef PubMed 5. E. S. Gloag , D. J. Wozniak , P. Stoodley , L. Hall-Stoodley , Mycobacterium abscessus biofilms have viscoelastic properties which may contribute to their recalcitrance in chronic pulmonary infections . Sci. Rep . 11 , 5020 ( 2021 ). OpenUrl CrossRef PubMed 6. M. Horvat , A. Pannuri , T. Romeo , I. Dogsa , D. Stopar , Viscoelastic response of Escherichia coli biofilms to genetically altered expression of extracellular matrix components . Soft Matter 15 , 5042 – 5051 ( 2019 ). OpenUrl CrossRef PubMed 7. A. Houari , et al. , Rheology of biofilms formed at the surface of NF membranes in a drinking water production unit . Biofouling 24 , 235 – 240 ( 2008 ). OpenUrl CrossRef PubMed 8. S. Jana , et al. , Nonlinear rheological characteristics of single species bacterial biofilms . NPJ Biofilms Microbiomes 6 , 19 ( 2020 ). OpenUrl CrossRef PubMed 9. F. Kaniz , W. Zheng , H. Bais , Y. Jin , Plant growth-promoting rhizobacteria mediate soil hydro-physical properties: An investigation with Bacillus subtilis and its mutants . Vadose Zone J . 22 ( 2023 ). 10. J. A. Kochanowski , B. Carroll , M. E. Asp , E. C. Kaputa , A. E. Patteson , Bacteria colonies modify their shear and compressive mechanical properties in response to different growth substrates . ACS Appl. Bio Mater . ( 2024 ). doi: 10.1021/acsabm.3c00907 . OpenUrl CrossRef 11. K. Kovach , et al. , Evolutionary adaptations of biofilms infecting cystic fibrosis lungs promote mechanical toughness by adjusting polysaccharide production . NPJ Biofilms Microbiomes 3 , 1 ( 2017 ). OpenUrl CrossRef PubMed 12. M. Kretschmer , O. Lieleg , Chelate chemistry governs ion-specific stiffening of Bacillus subtilis B-1 and Azotobacter vinelandii biofilms . Biomater. Sci . ( 2020 ). doi: 10.1039/c9bm01763a . OpenUrl CrossRef PubMed 13. O. Lieleg , M. Caldara , R. Baumgrtel , K. Ribbeck , Mechanical robustness of Pseudomonas aeruginosa biofilms . Soft Matter 7 , 3307 – 3314 ( 2011 ). OpenUrl CrossRef PubMed Web of Science 14. I. M. Oliveira , et al. , The impact of potassium peroxymonosulphate and chlorinated cyanurates on biofilms of Stenotrophomonas maltophilia: effects on biofilm control, regrowth, and mechanical properties . Biofouling 39 , 691 – 705 ( 2023 ). OpenUrl CrossRef PubMed 15. L. Pavlovsky , J. G. Younger , M. J. Solomon , In situ rheology of Staphylococcus epidermidis bacterial biofilms . Soft Matter 9 , 122 – 131 ( 2013 ). OpenUrl CrossRef PubMed 16. L. Qi , G. F. Christopher , Rheological variability of Pseudomonas aeruginosa biofilms . Rheol. Acta 60 , 219 – 230 ( 2021 ). OpenUrl CrossRef 17. A. Sarlet , V. Ruffine , K. G. Blank , C. M. Bidan , Influence of metal cations on the viscoelastic properties of Escherichia coli biofilms . ACS Omega 8 , 4667 – 4676 ( 2023 ). OpenUrl CrossRef PubMed 18. A. A. Snowdon , S. P. Dennington , J. E. Longyear , J. A. Wharton , P. Stoodley , Surface properties influence marine biofilm rheology, with implications for ship drag . Soft Matter 19 , 3675 – 3687 ( 2023 ). OpenUrl CrossRef PubMed 19. V. Souza-Egipsy , J. F. Vega , E. González-Toril , Á. Aguilera , Biofilm mechanics in an extremely acidic environment: microbiological significance . Soft Matter 17 , 3672 – 3680 ( 2021 ). OpenUrl CrossRef PubMed 20. B. W. Towler , C. J. Rupp , A. L. B. Cunningham , P. Stoodley , Biscoelastic properties of a mixed culture biofilm from rheometer creep analysis . Biofouling 19 , 279 – 285 ( 2003 ). OpenUrl CrossRef PubMed Web of Science 21. A. M. Vinogradov , M. Winston , C. J. Rupp , P. Stoodley , Rheology of biofilms formed from the dental plaque pathogen Streptococcus mutans . Biofilms 1 , 49 – 56 ( 2004 ). OpenUrl CrossRef 22. M. S. Waters , S. Kundu , N. J. Lin , S. Lin-Gibson , Microstructure and mechanical properties of in situ Streptococcus mutans biofilms . ACS Appl. Mater. Interfaces 6 , 327 – 332 ( 2014 ). OpenUrl CrossRef PubMed 23. ↵ S. G. V. Charlton , et al. , Microstructural and rheological transitions in bacterial biofilms . Adv. Sci. (Weinh .) 10 , e2207373 ( 2023 ). OpenUrl CrossRef 24. ↵ T. P. Denny , Involvement of bacterial polysaccharides in plant pathogenesis . Annu. Rev. Phytopathol . 33 , 173 – 197 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 25. ↵ P. Prior , et al. , Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species . BMC Genomics 17 , 90 ( 2016 ). OpenUrl CrossRef PubMed 26. ↵ D. Caldwell , B.-S. Kim , A. S. Iyer-Pascuzzi , Ralstonia solanacearum differentially colonizes roots of resistant and susceptible tomato plants . Phytopathology 107 , 528 – 536 ( 2017 ). OpenUrl CrossRef PubMed 27. C. Digonnet , et al. , Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall . Planta 236 , 1419 – 1431 ( 2012 ). OpenUrl CrossRef PubMed Web of Science 28. D. Khokhani , T. M. Lowe-Power , T. M. Tran , C. Allen , A single regulator mediates strategic switching between attachment/spread and growth/virulence in the plant pathogen Ralstonia solanacearum . mBio 8 ( 2017 ). 29. ↵ T. M. Tran , A. MacIntyre , D. Khokhani , M. Hawes , C. Allen , Extracellular DNases of Ralstonia solanacearum modulate biofilms and facilitate bacterial wilt virulence . Environ. Microbiol . 18 , 4103 – 4117 ( 2016 ). OpenUrl CrossRef 30. ↵ R. O. García , J. P. Kerns , L. Thiessen , Ralstonia solanacearum species complex: a quick diagnostic guide . Plant Health Prog . 20 , 7 – 13 ( 2019 ). OpenUrl CrossRef 31. ↵ G. Orgambide , et al. , High heterogeneity of the exopolysaccharides of Pseudomonas solanacearum strain GMI1000 and the complete structure of the major polysaccharide . J. Biol. Chem . 266 , 8312 – 8321 ( 1991 ). OpenUrl Abstract / FREE Full Text 32. ↵ M. D. Carter , et al. , Lectins and polysaccharide EPS I have flow-responsive roles in the attachment and biofilm mechanics of plant pathogenic Ralstonia . PLOS Pathogens 20 , e1012358 ( 2024 ). OpenUrl CrossRef PubMed 33. ↵ Denny T. P. and Baek S. R. , Genetic evidence that extracellular polysaccharide is a virulence factor of Pseudomonas solanacearum . Molecular Plant-Microbe Interactions 4 , 198 – 206 ( 1991 ). OpenUrl CrossRef Web of Science 34. C. C. Kao , E. Barlow , L. Sequeira , Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum . J. Bacteriol . 174 , 1068 – 1071 ( 1992 ). OpenUrl Abstract / FREE Full Text 35. ↵ A. Husain , A. Kelman , Relation of slime production to mechanism of wilting and pathogenicity of Pseudomonas solanacearum . Phytopathology 48 , 155 – 165 ( 1958 ). OpenUrl 36. ↵ Y. Kang , E. Saile , M. A. Schell , T. P. Denny , Quantitative immunofluorescence of regulated eps gene expression in single cells of Ralstonia solanacearum . Appl. Environ. Microbiol . 65 , 2356 – 2362 ( 1999 ). OpenUrl Abstract / FREE Full Text 37. M. A. Schell , Control of virulence and pathogenicity genes of Ralstonia solanacearum by an elaborate sensory network . Annu. Rev. Phytopathol . 38 , 263 – 292 ( 2000 ). OpenUrl CrossRef PubMed Web of Science 38. ↵ J. Huang , B. F. Carney , T. P. Denny , A. K. Weissinger , M. A. Schell , A complex network regulates expression of eps and other virulence genes of Pseudomonas solanacearum . J. Bacteriol . 177 , 1259 – 1267 ( 1995 ). OpenUrl Abstract / FREE Full Text 39. J. Huang , W. Yindeeyoungyeon , R. P. Garg , T. P. Denny , M. A. Schell , Joint transcriptional control of xpsR, the unusual signal integrator of the Ralstonia solanacearum virulence gene regulatory network, by a response regulator and a LysR-type transcriptional activator . J. Bacteriol . 180 , 2736 – 2743 ( 1998 ). OpenUrl Abstract / FREE Full Text 40. ↵ S. J. Clough , A. B. Flavier , M. A. Schell , T. P. Denny , differential expression of virulence genes and motility in Ralstonia (Pseudomonas) solanacearum during exponential growth . Appl. Environ. Microbiol . 63 , 844 – 850 ( 1997 ). OpenUrl Abstract / FREE Full Text 41. ↵ S. S. Gnanamanickam T. Denny , “ Plant pathogenic Ralstonia species ” in Plant-Associated Bacteria , S. S. Gnanamanickam , Ed. ( Springer Netherlands , 2006 ), pp. 573 – 644 . 42. ↵ E. Saile , J. A. McGarvey , M. A. Schell , T. P. Denny , Role of extracellular polysaccharide and endoglucanase in root invasion and colonization of tomato plants by Ralstonia solanacearum . Phytopathology 87 , 1264 – 1271 ( 1997 ). OpenUrl CrossRef PubMed Web of Science 43. ↵ L. Sequeira , Bacterial wilt of bananas: dissemination of the pathogen and control of the disease . Phytopathology 48 , 64 – 69 ( 1958 ). OpenUrl 44. ↵ A. C. Woods , Moko disease: Atypical symptoms induced by afluidal variants . Phytopathology 74 , 972 – 976 ( 1984 ). OpenUrl CrossRef 45. ↵ M. Bouda , C. W. Windt , A. J. McElrone , C. R. Brodersen , In vivo pressure gradient heterogeneity increases flow contribution of small diameter vessels in grapevine . Nat. Commun . 10 , 5645 ( 2019 ). OpenUrl CrossRef PubMed 46. ↵ W. E. Rochefort , S. Middleman , Rheology of xanthan gum: Salt, temperature, and strain effects in oscillatory and steady shear experiments . Journal of Rheology 31 , 337 – 369 ( 1987 ). OpenUrl CrossRef 47. ↵ R. H. Ewoldt , P. Winter , J. Maxey , G. H. McKinley , Large amplitude oscillatory shear of pseudoplastic and elastoviscoplastic materials . Rheol. Acta 49 , 191 – 212 ( 2010 ). OpenUrl CrossRef 48. ↵ A. Fall , F. Bertrand , G. Ovarlez , D. Bonn , Shear thickening of cornstarch suspensions . Journal of Rheology 56 , 575 – 591 ( 2012 ). OpenUrl CrossRef Web of Science 49. ↵ J. Huang , M. Schell , Molecular characterization of the eps gene cluster of Pseudomonas solanacearum and its transcriptional regulation at a single promoter . Mol. Microbiol . 16 , 977 – 989 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 50. ↵ L. T. Chu , et al. , Development of a tomato xylem-mimicking microfluidic system to study Ralstonia pseudosolanacearum biofilm formation . Front. Bioeng. Biotechnol . 12 , 1395959 ( 2024 ). OpenUrl CrossRef PubMed 51. ↵ J. C. Conrad , R. Poling-Skutvik , Confined flow: Consequences and implications for bacteria and biofilms . Annu. Rev. Chem. Biomol. Eng . 9 , 175 – 200 ( 2018 ). OpenUrl CrossRef PubMed 52. ↵ W. M. Weaver , V. Milisavljevic , J. F. Miller , D. Di Carlo , Fluid flow induces biofilm formation in Staphylococcus epidermidis polysaccharide intracellular adhesin-positive clinical isolates . Appl. Environ. Microbiol . 78 , 5890 – 5896 ( 2012 ). OpenUrl Abstract / FREE Full Text 53. ↵ R. Peyraud , L. Cottret , L. Marmiesse , J. Gouzy , S. Genin , A resource allocation trade-off between virulence and proliferation drives metabolic versatility in the plant pathogen Ralstonia solanacearum . PLoS Pathog . 12 , e1005939 ( 2016 ). OpenUrl CrossRef PubMed 54. ↵ A. E. Carpenter , et al. , CellProfiler: image analysis software for identifying and quantifying cell phenotypes . Genome Biol . 7 , R100 ( 2006 ). OpenUrl CrossRef PubMed 55. ↵ J. N. Wilking , T. E. Angelini , A. Seminara , M. P. Brenner , D. A. Weitz , Biofilms as complex fluids . MRS Bull . 36 , 385 – 391 ( 2011 ). OpenUrl CrossRef Web of Science 56. ↵ H. J. Lee , S.-M. Lee , M. Choi , J. H. Kwon , S.-W. Lee , A mutation of a putative NDP-sugar epimerase gene in Ralstonia pseudosolanacearum attenuates exopolysaccharide production and bacterial virulence in tomato plant . Plant Pathol. J . 39 , 417 – 429 ( 2023 ). OpenUrl CrossRef PubMed 57. ↵ C. Tsumori , S. Matsuo , Y. Murai , K. Kai , Quorum sensing-dependent invasion of Ralstonia solanacearum into Fusarium oxysporum chlamydospores . Microbiol Spectr 11 , e0003623 ( 2023 ). OpenUrl 58. ↵ Z. Konkel , J. C. Slot , Mycotools: An automated and scalable platform for comparative genomics . bioRxiv ( 2023 ). doi: 10.1101/2023.09.08.556886 . OpenUrl Abstract / FREE Full Text 59. ↵ J. C. Slot , A. Rokas , Horizontal transfer of a large and highly toxic secondary metabolic gene cluster between fungi . Current Biology 21 , 134 – 139 ( 2011 ). OpenUrl CrossRef PubMed 60. ↵ G. L. Hartman , A. C. Hayward A. M. Alvarez , J. Berestecky , J. I. Stiles , S. A. Ferreira , A. A. Benedict , Serological and molecular approaches to identification of Pseudomonas solanacearum strains from Heliconia . in Bacterial Wilt , G. L. Hartman , A. C. Hayward , Eds. ( Australian Center for International Agricultural Research , 1993 ), pp. 62 – 69 . 61. ↵ T. Hölscher , Á. T. Kovács , Sliding on the surface: bacterial spreading without an active motor . Environ. Microbiol . 19 , 2537 – 2545 ( 2017 ). OpenUrl CrossRef 62. ↵ J. Tans-Kersten , H. Huang , C. Allen , Ralstonia solanacearum needs motility for invasive virulence on tomato . J. Bacteriol . 183 , 3597 – 3605 ( 2001 ). OpenUrl Abstract / FREE Full Text 63. ↵ J. Corral , et al. , Twitching and swimming motility play a role in Ralstonia solanacearum pathogenicity . mSphere 5 ( 2020 ). 64. ↵ Y. Kang , H. Liu , S. Genin , M. A. Schell , T. P. Denny , Ralstonia solanacearum requires type 4 pili to adhere to multiple surfaces and for natural transformation and virulence . Mol. Microbiol . 46 , 427 – 437 ( 2002 ). OpenUrl CrossRef PubMed Web of Science 65. ↵ X. Yuan , L. I. Eldred , G. W. Sundin , Exopolysaccharides amylovoran and levan contribute to sliding motility in the fire blight pathogen Erwinia amylovora . Environ. Microbiol . 24 , 4738 – 4754 ( 2022 ). OpenUrl CrossRef View the discussion thread. Back to top Previous Next Posted May 22, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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