ORIGINAL RESEARCH published: 10 November 2021 doi: 10.3389/fpls.2021.755708 The Bacterial Wilt Reservoir Host Solanum dulcamara Shows Resistance to Ralstonia solanacearum Infection Pau Sebastià 1† , Roger de Pedro-Jové 1,2† , Benoit Daubech 1 , Anurag Kashyap 1 , Núria S. Coll 1 and Marc Valls 1,2* 1 Centre for Research in Agricultural Genomics (CSIC-IRTA-UAB-UB), Bellaterra, Spain, 2 Department of Genetics, University of Barcelona, Barcelona, Spain Ralstonia solanacearum causes bacterial wilt, a devastating plant disease, responsible for serious losses on many crop plants. R. solanacearum phylotype II-B1 strains have caused important outbreaks in temperate regions, where the pathogen has been identified inside asymptomatic bittersweet (Solanum dulcamara) plants near rivers and in potato fields. S. dulcamara is a perennial species described as a reservoir host where Edited by: R. solanacearum can overwinter, but their interaction remains uncharacterised. In this Adi Avni, Tel Aviv University, Israel study, we have systematically analysed R. solanacearum infection in S. dulcamara, Reviewed by: dissecting the behaviour of this plant compared with susceptible hosts such as Guido Sessa, tomato cv. Marmande, for which the interaction is well described. Compared with Tel Aviv University, Israel susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and Antonio Cellini, University of Bologna, Italy bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, *Correspondence: (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the Marc Valls stem. Taken together, these results demonstrate that S. dulcamara behaves as partially marcvalls@ub.edu resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study † These authors have contributed equally to this work proves that tolerance (i.e., the capacity to reduce the negative effects of infection) is not required for a wild plant to act as a reservoir host. We propose that inherent resistance Specialty section: (impediment to colonisation) and a perennial habit enable bittersweet plants to behave This article was submitted to Plant Pathogen Interactions, as reservoirs for R. solanacearum. a section of the journal Keywords: bacterial wilt, Ralstonia solanacearum, disease resistance, reservoir host plants, vascular Frontiers in Plant Science reinforcements, overwintering Received: 09 August 2021 Accepted: 04 October 2021 Published: 10 November 2021 INTRODUCTION Citation: Sebastià P, de Pedro-Jové R, Alternate or reservoir hosts are non-target organisms that can harbour high amounts of pathogens Daubech B, Kashyap A, Coll NS and for long periods of time and serve as an inoculum source for further infections on the primary Valls M (2021) The Bacterial Wilt host (Haydon et al., 2002; Morris et al., 2009). However, the term “reservoir host” has been also Reservoir Host Solanum dulcamara applied to natural or economically unimportant hosts or to hosts where infections are always non- Shows Resistance to Ralstonia pathogenic (Haydon et al., 2002). In many important crop diseases, non-agricultural reservoirs solanacearum Infection. Front. Plant Sci. 12:755708. have also been proposed to enhance the adaptive potential of pathogens and influence disease doi: 10.3389/fpls.2021.755708 epidemiology (Mueller et al., 2012; Monteil et al., 2013; Thinakaran et al., 2015; McCann, 2020). Frontiers in Plant Science | www.frontiersin.org 1 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction For instance, Pseudomonas syringae isolated from wild species wilt because colonisation was only apparent in 25% of the was shown to potentially develop into novel crop pathovars in plants, in which the bacterium occupied a few xylem vessels a few evolutionary steps (Monteil et al., 2013; Bartoli et al., or occasionally all xylem bundles (Álvarez et al., 2008b). In 2015), and experimental evolution experiments with Ralstonia another report, wilting was more apparent after soil drench solanacearum demonstrated high fitness gains when this inoculations, and S. dulcamara plants showed intermediate pathogen was inoculated on distant hosts (Guidot et al., 2014). symptomatology compared with susceptible (Bonny Best) and Ralstonia solanacearum is the agent causing the devastating resistant (Hawaii 7996) tomato plants (Jacobs et al., 2013). bacterial wilt disease in over 200 plant species, including In a third study, all plants became infected and 97% showed economically important crops such as potato, tomato, peanut, symptoms when the bacterium was directly inoculated in the eggplant, and banana (Hayward, 1994; Mansfield et al., 2012; Coll stem. However, symptomatology and pathogen presence was and Valls, 2013). R. solanacearum can survive in the soil and restricted to inoculated shoots, indicating slow or no spreading waterways (Van Elsas et al., 2000; Álvarez et al., 2008a), from of the bacterium throughout the plant (Wenneker et al., 1999). where it infects plants through the roots and colonises the xylem In this same study, only 13–19% of the plants were infected and tissue, blocking water flow and causing plant wilting (Hayward, 9% showed symptoms when plants were soil-drench inoculated 1991; Schell, 2000). The disease is endemic in tropical and (Wenneker et al., 1999). In summary, S. dulcamara presents subtropical areas, but R. solanacearum phylotype II-B1 (formerly highly variable symptomatology in response to R. solanacearum race 3 biovar 2) strains are adapted to cooler temperatures depending on the inoculation method, although it usually shows and have caused important outbreaks in temperate regions an intermediate behaviour between a susceptible and a resistant (Elphinstone, 1996; Janse et al., 2004; Champoiseau et al., 2009). host. The mechanisms responsible for this partial restriction of Survival and overwintering of R. solanacearum in temperate colonisation by R. solanacearum have not yet been described. regions appears to rely on infection of perennial reservoir host In this study, we have undertaken a thorough characterisation plants because its persistence in the soil is limited (Olsson, of the interaction between R. solanacearum and its wild host 1976; Shamsuddin et al., 1978; Elphinstone, 1996). Bittersweet S. dulcamara. We describe the localisation of the pathogen during (Solanum dulcamara) is a common holarctic perennial weed that the infection process and the symptomatology on the plant at has been proposed to play a reservoir role in the persistence different temperatures and compare this interaction with that and spread of R. solanacearum based on several observations. established on susceptible tomato (cv. Marmande) and potato Firstly, common incidences of R. solanacearum in S. dulcamara (cv. Desirée) plants. have been described along waterways (Kempenaar et al., 1998), and most disease outbreaks were related to watercourses in which infected S. dulcamara plants were present (Olsson, 1976; MATERIALS AND METHODS Elphinstone, 1996; Janse et al., 1998). Secondly, winter persistence of R. solanacearum in waterways correlated with the presence of Plant and Bacterial Materials and Growth the pathogen in S. dulcamara plants growing near them (Olsson, Conditions 1976; Elphinstone et al., 1998; Caruso et al., 2005). Thirdly, Bittersweet (S. dulcamara) plants were grown from seeds R. solanacearum was shown to colonise asymptomatically the harvested from wild specimens in Vidrà (NE Catalonia, Spain). roots and vascular tissue of S. dulcamara plants in the wild, The susceptible tomato (Solanum lycopersicum cv. Marmande) and infected plants were shown to release the bacterium onto and susceptible potato (Solanum tuberosum cv. Desirée) plants surface water via aquatic roots (Olsson, 1976; Elphinstone, 1996; used in this study are commercially available. Janse, 1996). Besides S. dulcamara, R. solanacearum phylotype For pot experiments, S. dulcamara and S. lycopersicum cv. IIB sequevar 1 strain have been found growing asymptomatically Marmande seeds were surface-sterilised in 35% bleach and in the wild with other weeds that act as sources of inoculum 0.02% Triton-X 100 for 10 min and then rinsed with sterile to infect potato fields. These include Solanum nigrum (Olsson, distilled water five times before sowing them in soil (Substrate 2, 1976), Solanum cinereum (Graham and Lloyd, 1978), Urtica Klasmann-Deilmann GmbH) mixed with perlite and vermiculite dioica in Europe (Wenneker et al., 1999), and a number of wild (30:1:1) and grown under controlled conditions for 3 weeks species from the Ugandan highlands (Tusiime et al., 1997). In under a long-day photoperiod (16 h light/8 h dark) and under China, tropical R. solanacearum strains were also identified in the a light intensity of 120–150 µmol·m−2 · s−1 at 22◦ C and 60% weed Ageratum conyzoides L., often showing wilting symptoms humidity. For optimal germination, S. dulcamara seeds were (She et al., 2013). stratified at 4◦ C for 2 weeks before transferring them to 22◦ C. The interactions between R. solanacearum and its cultivated S. tuberosum cv. Desirée potato plants were propagated in vitro hosts have been well-characterised, but little is known about the (Puigvert et al., 2017) and 2-week old apex was sown in the same behaviour of this pathogen inside wild hosts. R. solanacearum soil mixture described above and grown in the same conditions. inoculation on S. dulcamara in laboratory conditions has been All infection assays were performed using the R. solanacearum previously reported (Wenneker et al., 1999; Álvarez et al., 2008b; strain UY031 (phylotype IIB, sequevar 1) isolated from potato Jacobs et al., 2013). A first assay screening a large number of tubers in Uruguay (Siri et al., 2011), carrying either the synthetic plant species found that 66% of S. dulcamara plants inoculated luxCDABE operon or the GFPuv gene, both under the control of through soil drenching became infected (Álvarez et al., 2008b). the constitutive psbA promoter (Monteiro et al., 2012). Bacteria However, the authors classified this plant as tolerant to bacterial were routinely grown at 28–30◦ C in rich B medium in liquid Frontiers in Plant Science | www.frontiersin.org 2 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction cultures supplemented with gentamicin (10 µg/ml) and the with the luminescent strain were photographed by placing the same medium, supplemented with 0.5% glucose and 50 mg/l whole plant in a Fuji Film LAS4000 light imager system with a of triphenyl tetrazolium chloride for growth on semi-solid agar 15-min exposure time. plates (Monteiro et al., 2012). Stem-inoculated plants with the fluorescent strain were dissected as described before and photographed using binocular Plant Inoculation and Pathogenicity microscopy equipped with a UV fluorescent lamp (BP330-385 BA420 filter) and an SZX16 stereomicroscope equipped with a Assays DP71 camera system (Olympus) using the following settings: For soil-soaking and stem inoculation assays, plants were grown GFP filter, 10 s exposure time, ISO 1/800. Soil-soaked plants for 3–4 weeks. Soil-soaking root inoculations were performed with the fluorescent strain were photographed with a Leica DM6 by pouring 40 ml of 108 colony-forming units (CFU)·ml−1 microscope. Bright field or fluorescence images merging the UV (OD600 = 0.1) of bacterial suspension on every plant pot without channel for plant structures (blue) and the GFP channel for disturbing the roots. Infected plants were kept in a growth bacteria (green) were automatically assembled by the microscope chamber set at 27◦ C (exceptionally 20◦ C when indicated) and software to obtain single images including the whole root section. scored for wilting symptoms using a scale from 0 to 4, 0 = healthy Quantification of the black signal (luminescence) or the green plant with no wilt, 1 = 25%, 2 = 50%, 3 = 75% of the leaves channel (fluorescence) in the pictures was quantified using the wilted, and 4 = total wilting. Disease indexes were calculated Fiji software (United States National Institutes of Health). by averaging the disease score of each plant of the experiment (n > 15) as indicated in previous publications with S. dulcamara, tomato, and potato (Álvarez et al., 2008b; Siri et al., 2011; Planas- Tissue Stainings After producing root wounds with a 1 ml pipette tip, the Marquès et al., 2020). Stem-inoculation assays were performed plants were soil-soaked with a bacterial solution of 107 CFU·g−1 by applying a 5 µl droplet of a 106 CFU·ml−1 (OD600 = 0.001) (OD600 = 0.01). The day the plants showed an adequate disease bacterial solution twice with a sterile 0.3 × 13 mm needle (30GX index, the taproots were transversally sliced. Then four to five 1/200 , BD Microlance, Becton Dickinson) to the wounds caused slices per plant were placed in a 1.5-ml tube with 70% ethanol at the base of the petiole after removal of the first true leaf. After for at least 7 days to remove the chlorophyll. For lignin staining, inoculation, plants were kept in a growth chamber set at 27◦ C individual taproot slices were placed on a microscope slide and unless otherwise specified and scored for wilting symptoms as incubated with two drops of phloroglucinol HCl for about 1 min, described before (Monteiro et al., 2012). then rinsed with 70% ethanol, and a cover slide was placed on top To quantify the bacterial content inside the shoots, 2 cm for visualisation in the upright microscope (Leica DM6) bright- sections were excised from above the taproot (soil-soaked plants) field (Pomar et al., 2004). Mock-infected plants were inoculated or above the inoculation point (stem-inoculated plants), weighed, with water. Lignin quantification was performed by selecting the and incubated for at least 30 min in a sterile 2-ml Eppendorf vascular area in tomato and potato plants and comparing it with tube with 300 µl of sterile distilled water to let the bacterium the same area in S. dulcamara plants using ImageJ software. ooze from the tissue. Luminescence was measured from the tubes Images were converted to a greyscale (eight-bit image) and the containing excised tissue with a luminometer (FB 12, Berthold mean grey value was calculated. Detection Systems) to determine the bacterial concentrations For suberin staining, individual S. dulcamara, tomato cv. since luminescence was proven to strongly correlate with Marmande and potato cv. Desirée taproot slices were placed in bacterial density (Planas-Marquès et al., 2020). To measure a well containing a Sudan IV solution for 5 min and then rinsed bacterial counts in the root, plants inoculated as described were in another well with 70% ethanol as described (Kashyap et al., uprooted from day 1 to day 4 post-inoculation, and the roots were 2021). Clean slices were placed on a slide and visualised with the rinsed with distilled water. Approximately 1–2 cm of root below UV filter on a Leica DM6 microscope. the tap root were cut and ground. Tissue was weighed and CFUs were counted as described above. Dilution plating of samples on rich B medium and CFU counting 24 h later was performed in Statistical Analyses some cases to verify luminescence results. Statistical analyses were performed using Statgraphics software. All statistical tests are indicated in the respective figure legends. Assessment of Bacterial Colonisation Plant colonisation by R. solanacearum was assessed using the RESULTS luminescent and fluorescent strains described above. Plant stems inoculated with the luminescent strain were sliced using a sterile Solanum dulcamara Shows an Enhanced razor blade obtaining internode sections just below and above the petiole where inoculation had been carried out. One millimetre Capacity to Withstand R. solanacearum thick transversal cuts and 1 cm long longitudinal cuts were Infection in Comparison With Tomato cv. placed flat on a square plate and visualised using a live imaging Marmande system (ChemiDoc Touch Imaging System, Bio-Rad) using a 5- To analyse the symptomatology caused by R. solanacearum in min exposure time with 3 × 3 sensitivity. Images were processed S. dulcamara and compare its behaviour with that of tomato, using Image Lab software (Bio-Rad). Soil-soak inoculated plants we inoculated plants in controlled conditions using two different Frontiers in Plant Science | www.frontiersin.org 3 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction methods. First, we used a more naturalistic root inoculation that skips the first infection steps (root entry and vascular method by soaking the soil with a bacterial solution without colonisation). As expected, disease progression was faster after causing any wounding to plants, after which the plants were kept stem inoculation and all the tomato plants were completely at 27◦ C, and then the wilting symptoms were recorded over time. wilted at 8 dpi. S. dulcamara plants still showed a clear delay in All susceptible tomato Marmande plants were completely wilted disease progression after stem inoculation (p-value < 0.0001): 14 days post-inoculation (dpi), while the symptoms just started first symptoms were apparent only by day 8, although most plants to appear in S. dulcamara (Figure 1A). By 28 dpi, less than half were completely wilted by 14 dpi (Figure 1B). Interestingly, of the S. dulcamara plants had completely wilted, showing a clear an important proportion of S. dulcamara plants remained delay in the development of the disease with respect to tomato asymptomatic at the end of our experiments, especially when plants (p-value < 0.0001; Figure 1A). soil inoculation was performed. Quantification of bacterial The second method used was direct bacterial inoculation loads in the stem over time showed an overall correlation inside the plant stem vasculature, a more aggressive procedure with disease symptoms (Figures 1C,D). In soil-drench FIGURE 1 | Bacterial wilt evaluation in S. dulcamara and tomato cv. Marmande plants. Plants of the wild reservoir host S. dulcamara and tomato susceptible to bacterial wilt were root inoculated by soil soaking (A,C,E) or stem inoculated (B,D,F) with R. solanacearum UY031 carrying a luminescent reporter. (A,B) Wilting symptoms were recorded over time using a scale from 0 (no wilting) to 4 (completely wilted). n = 30–35 plants per plant species. (C,D) Bacterial concentrations in the stem at different time points from plants in panels (A,B), respectively. n = 4–8 plants per sampling day. (E,F) Bacterial content in relation to wilting symptoms for each plant individual analysed of the two species. n = 30 plant samples analysed for each species. Bacterial counts are expressed as log CFUs·g−1 tissue. ∗ indicates statistical differences (p value < 0.05, T-student significant test). The experiments in panels (A,C) were repeated three times with similar results. The experiment in panels (B,D) was repeated twice with similar results. Frontiers in Plant Science | www.frontiersin.org 4 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction inoculations, when tomato plants were almost completely wilted (12 dpi), the bacterial concentrations in their stems were ∼109 CFU·g−1 , significantly higher than the ∼107 CFU·g−1 found in S. dulcamara, which only showed minor symptoms at this time point (Figure 1C). Similar results with bacterial concentrations were obtained in stem inoculation experiments although these plants displayed higher bacterial contents, especially at late disease stages, since structural barriers present in roots are circumvented in this inoculation method (Figure 1D). To precisely determine the bacterial concentrations that the two plant species could withstand inside their tissues, we took the data from all biological replicas and plotted bacterial content in relation to wilting symptoms for each plant analysed. This representation clearly showed that, irrespective of the inoculation method, S. dulcamara and susceptible tomato bore similar bacterial concentrations at intermediate wilting stages (Figures 1E,F). However, two clear differences were observed: (i) as hinted before, at early disease stages (disease index = 1) S. dulcamara plants showed lower bacterial colonisation, and (ii) bacterial loads rarely exceeded 109 CFU·g−1 in S. dulcamara, whereas they often overcame these levels in tomato plants, leading to statistical differences when plants were completely wilted. The differences in the late disease stages were more apparent in stem inoculation experiments because this more FIGURE 2 | Bacterial colonisation and multiplication in stem-inoculated aggressive inoculation method resulted in a higher proportion S. dulcamara and tomato cv. Marmande plants. (A) Non-destructive live of plants showing symptoms and becoming totally wilted luminescence imaging of four representatives S. dulcamara plants throughout (disease index 4) compared with natural root inoculations a 30-day period after root inoculation with a luminescent R. solanacearum by soil drenching (Figures 1E,F). In summary, S. dulcamara strain. Two symptomatic (A & F) and two asymptomatic (K & Q) plants are shown. Luminescent bacteria are detected in darker areas. Wilting symptoms plants displayed delayed bacterial wilt symptom development (Disease index = 0–4) are indicated next to each plant inside the images. compared with susceptible tomato plants, with most of (B) R. solanacearum concentrations measured at the root, taproot, internode the individuals surviving infection in the timeframe of our 1 (2–3 cm above the inoculation point) and 2 (6–9 cm above the inoculation experiments. In addition, lower R. solanacearum concentrations point) in S. dulcamara plants uprooted 30 days post-inoculation (dpi) with the were observed in the stems of S. dulcamara at early and late luminescent reporter strain. The results from asymptomatic (disease index 0) and symptomatic (disease index 1–4) plants are shown separately. Bacterial disease stages, suggesting delayed colonisation and restriction to counts were calculated from tissue luminescence and are expressed as log bacterial growth. CFUs·g−1 tissue. ∗ indicates statistical differences (p-value < 0.05, T-student significant test). A High Proportion of S. dulcamara Plants Show Long-Lasting Latent Infections Ralstonia solanacearum infection heterogeneity amongst panel). Interestingly, R. solanacearum latent infections were different plant individuals is common. To analyse the progression detected in the most asymptomatic plants (four out of six of bacterial colonisation and disease symptoms in single plants plants Supplementary Figure 1), which displayed detectable over time, we took advantage of the luminescent R. solanacearum luminescence in the root at 30 dpi (Figure 2A bottom panel strain used in this work, which could be visualised non- and Supplementary Figure 1). Quantification of the black destructively inside plant tissues (Cruz et al., 2014). Stem signal from the pictures in S1 showed that the area colonised by inoculations were used in these experiments to reduce the high bacteria positively correlates with the disease symptoms, except stochasticity of root inoculations and to facilitate infection so in totally wilted plants where tissue collapse and drying causes that a significant proportion of plants become completely wilted. bacterial death (Supplementary Figure 2). For a more sensitive Live imaging and symptom recording of whole plants were and quantitative analysis, the bacterial contents of root and stem carried out along a 30-day period, after which the plants were sections of plants uprooted at 30 dpi were calculated. The results uprooted to visualise bacterial content in the roots. As observed proved that R. solanacearum was present in all tissues analysed before, in this experiment, half of the S. dulcamara plants showed from asymptomatic S. dulcamara plants, although bacterial symptoms, and half of them remained asymptomatic at the end concentrations were in almost all cases four orders of magnitude of the assay (Figure 2A and Supplementary Figure 1). Bacterial lower than that in symptomatic plants (Figure 2B). colonisation paralleled the onset of disease symptoms in wilting In summary, long-term challenging of S. dulcamara with plants (Figure 2A top panel) and was always undetectable in R. solanacearum always resulted in two distinct behaviours: the aerial tissues of asymptomatic plants (Figure 2A bottom plants with apparent bacterial colonisation and disease symptoms Frontiers in Plant Science | www.frontiersin.org 5 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction and plants that remained symptomless even after direct stem inoculation, but which always carried latent bacterial infections. Ralstonia solanacearum Movement Is Restricted in S. dulcamara Tissues Compared With Susceptible Tomato cv. Marmande We have previously demonstrated that resistant tomato varieties can restrict R. solanacearum root colonisation and hamper bacterial vertical and horizontal movements in the stem (Planas- Marquès et al., 2020). Thus, we hypothesised that this mechanism could be also active in S. dulcamara and cause the observed delay in symptom appearance and infection latency. Next, we evaluated if S. dulcamara plants restricted bacterial movement in the stems compared with susceptible tomatoes. To better compare bacterial behaviour in the two hosts, we stem-inoculated a large number of plants with the luminescent reporter strain and observed bacterial distribution in their stems by grouping the plants according to disease stage. The whole 4-to-5-week-old plants could not be imaged because of size limitations and reduced sensitivity due to stem thickness. Thus, we obtained stem sections of internodes one to four from plants and imaged the top and bottom slices of each section and the remaining stem longitudinally divided in two. Representative pictures presented in Figure 3A show that luminescence matched the location of xylem bundles and was FIGURE 3 | R. solanacearum distribution in stems of inoculated S. dulcamara less intense in S. dulcamara plants compared with tomatoes at and tomato cv. Marmande plants. (A) Representative luminescence imaging early disease stages. Quantification of the luminescence signal photographs at different wilting stages (Disease index 0–4) of stem sections from S. dulcamara (top panel) and tomato (bottom panel) plants (Supplementary Figure 3A) corroborated this result, supporting stem-inoculated with luminescent R. solanacearum. Luminescent bacteria are the lower bacterial loads previously observed in asymptomatic detected as dark areas in transversal and longitudinal sections of plant S. dulcamara (Figures 1E,F). In addition, the luminescence of internodes 1–4 organised bottom to top. Inoculation points are indicated by xylem bundles tended to decrease with height in S. dulcamara, an arrow. (B) Representative fluorescence microscopy images of stem sections from S. dulcamara (top panel) and tomato (bottom panel) plants while it remained constant in the susceptible tomato plants stem-inoculated with an R. solanacearum strain constitutively expressing GFP. (Figure 3A), suggesting stronger restriction to vertical bacterial Inoculations were performed at the base of the first true leaf and transversal movement along the vessels in S. dulcamara. sections obtained in the first internode, 2 cm above the inoculation point. To further analyse if S. dulcamara restricts the horizontal Scale bars indicate 0.5 mm. spread of R. solanacearum to neighbouring xylem and parenchyma tissues, we observed shoot sections of plants stem- inoculated with a GFP-tagged strain (Cruz et al., 2014) using points, demonstrating no difference in root entry. However, fluorescence microscopy. Representative images of internode statistically lower bacterial loads were observed in S. dulcamara cuts above the inoculation point showed that the bacterium was roots at 4 dpi, proving that the root tissues of S. dulcamara also slightly more confined to the vasculature in S. dulcamara, and limit R. solanacearum colonisation. that a lower number of xylem vessels appeared fluorescent in this Taken together, the assessment of bacterial colonisation in species with respect to tomato plants at comparable disease stages shoots and roots of both hosts suggests that S. dulcamara (Figure 3B). Despite the differences observed in asymptomatic plants cope better with bacterial wilt because they have the plants, quantification of the fluorescence intensity in diseased ability to effectively restrict pathogen movement and colonisation plants (Supplementary Figure 3B) showed that colonisation inside their tissues. was comparable in tomato and S. dulacamara plants displaying similar symptomatology. Solanum dulcamara Displays Since stem inoculation skips the initial stages of infection, and to determine whether R. solanacearum root entry and Dramatically Reduced Bacterial Wilt colonisation were also restricted in S. dulcamara plants, we Symptoms and Bacterial Colonisation at carried out a root inoculation experiment. Briefly, plants were 20◦ C inoculated with the luminescent reporter strain by soil drenching Solanum dulcamara has been demonstrated to be a reservoir and the bacterial counts were measured at short times after plant host in which R. solanacearum can overwinter (Olsson, inoculation (1–4 dpi). As can be observed in Supplementary 1976; Elphinstone et al., 1998; Janse et al., 1998; Wenneker Figure 4, bacterial concentrations were comparable at early time et al., 1999; Caruso et al., 2005). To test the plant behaviour at Frontiers in Plant Science | www.frontiersin.org 6 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction FIGURE 4 | Influence of temperature on bacterial wilt symptomatology in S. dulcamara and susceptible tomato and potato varieties. (A) Bacterial wilt symptom development (0 = asymptomatic to 4 = completely wilted) after soil-drench inoculation in S. dulcamara, tomato cv. Marmande and potato cv. Desirée plants inoculated with the R. solanacearum luminescent reporter and kept at 20◦ C. n = 30–35 plants per species. (B) Bacterial concentrations at the taproot level quantified by measuring the luminescence at 10, 20 and 28 dpi from S. dulcamara, susceptible tomato cv. Marmande, and susceptible potato cv. Desirée inoculated as in panel (A). Results are grouped according to disease symptoms: asymptomatic (grey, disease index 0) and symptomatic (white, disease indices 1–4). n = 4–8 plants per sampling day and condition. ∗ indicates statistical differences (p value < 0.05, T-student significant test). lower temperatures that mimic those encountered in temperate throughout the experimental period, bacterial concentrations environments, S. dulcamara and tomato plants kept at 20◦ C were calculated separately for asymptomatic and symptomatic were soil-soak inoculated with luminescent R. solanacearum, plants. Symptomatic plants carried bacterial counts above 107 and the symptoms and bacterial loads in the stems were CFUs·g−1 in all species, concentrations being the lowest in evaluated over time. A temperature of 20◦ C was chosen as S. dulcamara because disease symptoms were less developed the lower temperature, compared with 27◦ C to avoid strong in this species compared with the two susceptible crops. For effects on plant or pathogen growth. To rule out specific instance, 30 days after inoculation R. solanacearum counts effects of lower temperatures on the tomato-control plants, reached a maximum of 5 × 108 CFUs·g−1 in S. dulcamara, susceptible potato plants (cv. Desirée), which are adapted to whereas wilted potato and tomato plants harboured up to 1010 cooler conditions than tomatoes (Ingram and McCloud, 1984), CFUs·g−1 (Figure 4B). were also included in this experiment. Few days after inoculation, In conclusion, cooler temperatures slowed down disease the first tomato plants started to wilt, followed by the first development in all species, but this effect was more pronounced potato plants 2 weeks after inoculation. By 30 dpi, around in S. dulcamara, which always survived the disease in the 50% of the potatoes and over 25% of the tomatoes were tested period while holding mostly asymptomatic (latent) completely wilted (Figure 4A), in accordance with previous bacterial infections. results in tomatoes inoculated at these temperatures with a closely related II-B1 strain (Milling et al., 2009). On the contrary, all S. dulcamara plants survived the infection at Solanum dulcamara Contains a 30 dpi with only a few of them (six out of 25) showing mild Constitutively and Highly Lignified Xylem wilting symptoms in individual leaves (disease index <0.5, The colonisation pattern of R. solanacearum in S. dulcamara Figure 4A). Quantification of bacterial levels in the stem over compared with tomato cv. Marmande suggested that the time correlated with wilting, showing overall lower bacterial former may contain vascular structures or components that titres in S. dulcamara than in susceptible tomato or potato make bacterial movement difficult. Lignin is one of the main plants (Figure 4B). Since most plants remained asymptomatic components of the secondary plant cell wall, and it has been Frontiers in Plant Science | www.frontiersin.org 7 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction described to play an important role as a structural defence (Kashyap et al., 2021). To avoid the effect of lower bacterial mechanism in resistant tomato varieties against R. solanacearum concentrations usually found in S. dulcamara tissues, plants (Nakaho et al., 2000; Ishihara et al., 2012; Kashyap et al., that contained comparable bacterial colonisation, as assessed by 2021). Therefore, we tested whether S. dulcamara xylem bacterial GFP fluorescence, were used for staining (Figure 5A vessels presented differential lignin accumulation in their cell lower panel). Quantification of the lignin stain intensity in mock walls compared with susceptible tomato and potato plants. and infected plants clearly confirmed a decrease in inoculated Taproot sections obtained 9 days after mock or soil inoculation tomato and potato that was not observed in S. dulcamara plants with the R. solanacearum GFP reporter strain were stained (Figure 5B). The same results were observed after lignin staining with phloroglucinol HCl to identify lignified structures. This from samples obtained at 6 dpi (Supplementary Figure 3), revealed constitutive and conspicuous lignification of the when bacterial colonisation was still low (Supplementary S. dulcamara vasculature, whereas, in susceptible tomato and Figure 3A lower panel). potato plants, the parenchyma cells surrounding the vascular We have recently described (Kashyap et al., 2021) that cylinder did not appear lignified (Figure 5A). In addition, suberin plays an important role in tomato resistance to bacterial while lignification remained stable in S. dulcamara after wilt. To evaluate if this compound had an effect on the R. solanacearum infection, both tomato and Desirée plants S. dulcamara restriction to R. solanacearum colonisation, we also showed a significant decrease in lignin accumulation upon stained inoculated or mock-treated stem sections with Sudan R. solanacearum infection (Figure 5A), as previously described IV, which binds to the aliphatic domain of suberin to produce a reddish-brown colouration. No detectable increase in the accumulation of suberin was observed in S. dulcamara or in the susceptible plants after infection or mock treatment, as shown in Supplementary Figure 6. In sum, S. dulcamara presented a constitutive accumulation of lignin in the xylem vessels and surrounding parenchyma that was not reduced upon pathogen infection as observed in susceptible tomato and potato, which may explain a higher restriction of bacterial colonisation in this species. DISCUSSION Solanum dulcamara Shows Partial Resistance to Bacterial Wilt It has been established that plants display two main types of defence against pathogens: resistance, which is the ability of the host to limit pathogen multiplication; and tolerance, defined as the ability of the host to reduce the negative effects of infection (Clarke, 1986; Pagán and García-Arenal, 2020). However, the term tolerance has often been used incorrectly to describe partial levels of plant resistance. To differentiate bona fide tolerance from partial resistance a key aspect is that tolerance implies that the plant shows less symptoms or yield effects at equivalent levels of pathogen loads (Pagán and García-Arenal, 2020). According to these definitions, S. dulcamara shows some degree of resistance to bacterial wilt and no tolerance to the pathogen. A clear proof that tolerance does not play a role in the response of S. dulcamara to R. solanacearum is that all direct and indirect FIGURE 5 | Lignification of S. dulcamara, Tomato cv. Marmande and quantifications R. solanacearum inside S. dulacamara plants are S. tuberosum cv. Desirée tissues upon R. solanacearum infection. comparable with those observed in tomato plants showing similar (A) Representative composed images of S. dulcamara, tomato, and potato symptoms (Figures 1E,F, 2 and Supplementary Figures 2, 3, 6). taproot transversal sections obtained 9 days after inoculation with The only exception to this is totally wilted plants, where bacterial R. solanacearum-GFP or mock treatment. First and second row: microscope bright-field images after lignin staining with phloroglucinol HCl (magenta populations declined, likely due to the lack of humidity in dry colouration). Third row: fluorescence microscopy images after inoculation to dead tissues (e.g., plant F, Figure 2A). assess the extent of bacterial colonisation. Images were obtained using a Several observations support that S. dulcamara plants Leica DM6 microscope. Scale bars indicate 0.5 mm. (B) Quantification of the display partial resistance to bacterial wilt (Table 1). First, phloroglucinol HCL stain -indicative of lignin content- in the vascular area from irrespective of the inoculation method used, S. dulcamara the images shown in A performed with the ImageJ software. ∗ indicates statistical differences (p-value < 0.05; T-student significant test α = 0.05). showed delayed symptomatology (Figures 1A,B), delayed stem colonisation (Figures 1C,D), and slightly delayed root Frontiers in Plant Science | www.frontiersin.org 8 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction TABLE 1 | Schematic comparison of the interaction at different levels between We thus explored the behaviour of S. dulcamara resistance to R. solanacearum and a susceptible tomato, S. dulcamara, and a resistant tomato. bacterial wilt when plants are grown and inoculated at lower Susceptible S. dulcamara Resistant temperatures. A decrease in temperature resulted in delayed tomato tomato symptom appearance and bacterial colonisation in both the susceptible hosts and in S. dulcamara (Figure 4) and the Disease symptoms +++ ++ ± difference in resistance between them was maintained. This Bacterial levels in roots +++ ++ + indicates that the ability of a pathogen to cause disease is Bacterial levels in stems +++ ++ + compromised at a lower temperature, as has been described for Bacterial vertical spread +++ ++ + many pathosystems. Thus, in these conditions, S. dulcamara Bacterial horizontal spread +++ ++ + plants displayed a stronger resistance to the disease, as all Symptoms at lower temp. ++ ± NT plants survived a month after inoculation and only very Structural reinforcements ± ++ +++ few of them showed minor wilting symptoms (Figure 4A ±: few or inexistent; +: low levels; ++: intermediate levels; +++: high and Table 1), but they all carried asymptomatic (latent) levels; NT: not tested. infections (Figure 4B). Tolerance to disease, i.e., the ability to keep high bacterial colonisation (Supplementary Figure 4). These phenotypes are levels without showing symptoms, has been proposed as a key similar but less pronounced than those observed in resistant trait for plants to act as reservoir hosts, providing a source of tomato cv. Hawaii 7996 (Table 1 and Planas-Marquès et al., pathogen inoculum that spreads when environmental conditions 2020). Further proofs of this are that an important proportion of become appropriate (Roberts and Heesterbeek, 2020). Based S. dulcamara plants remained asymptomatic when tomatoes were on our findings with S. dulcamara (Table 1), we propose that completely wilted and that stem inoculation and large numbers resistance, i.e., limiting pathogen colonisation, could also enable had to be used to obtain enough plants at advanced disease stages plants to act as reservoirs. Intermediate resistance would be to compare with susceptible tomatoes. required in this case for two reasons: first, it would allow a limited Second, S. dulcamara restricted R. solanacearum vertical amount of pathogen to colonise and survive under unfavourable movement in the stem. A luminescent R. solanacearum reporter environmental conditions, such as winter temperatures, as latent strain was able to entirely colonise susceptible tomato, while infections inside the plant, and second, when environmental in S. dulcamara plants the upper stem displayed less pathogen conditions favour disease (high temperature in our case), the colonisation (Figures 2, 3A). In accordance with this, it has been pathogen could overcome resistance in some plants, multiplying described in S. dulcamara that symptomatology and pathogen to high numbers and spreading to other hosts. These two presence was restricted only to shoots directly inoculated, conditions could not take place if plants were either fully resistant indicating slow or no spreading of the bacterium throughout the or tolerant. This theory is supported by the original description plant (Wenneker et al., 1999). We previously reported similar of S. dulcamara as a symptomless R. solanacearum carrier in behaviour, although clearly more apparent (Table 1) in resistant the wild (Olsson, 1976; Hayward, 1991), and studies show that tomatoes (Planas-Marquès et al., 2020). environmental conditions can break resistance to the disease. Third, bacterial movement between xylem vessels was For instance, in eucalypt, R. solanacearum usually behaves as also limited in S. dulcamara compared with susceptible a latent colonist, and only in the presence of other stressing tomatoes (Figure 3B). This could explain the stem colonisation factors the pathogen is able to proliferate and cause disease delay observed, as S. dulcamara restricts R. solanacearum (Coutinho and Wingfield, 2017). to specific xylem vessels, while others remain pathogen- free, a behaviour also reported for the resistant tomato Constitutive Xylem Lignification in an (Planas-Marquès et al., 2020). S. dulcamara Is Likely Responsible for Taken together, our results confirm previous studies that reported S. dulcamara as partially resistant to bacterial Its Resistance to R. solanacearum wilt, although it was misleadingly described as tolerance. Observation of S. dulcamara stem transversal sections indicated Discrepancies amongst previous reports where infection rates a highly lignified xylem compared with susceptible tomato and varied from 100 to 66% and 13 to 19% (Wenneker et al., 1999; potato varieties (Figure 5 and Supplementary Figure 5). This Álvarez et al., 2008b; Jacobs et al., 2013) can be explained by the is in accordance with previous reports that lignin biosynthesis different inoculation methods used, by different assay conditions genes were upregulated in the bacterial wilt resistant tomato (e.g., temperature and inoculum), and/or by genetic differences variety LS-89 upon R. solanacearum infection (Ishihara et al., amongst the plant accessions used. 2012). Furthermore, we have recently shown striking differences in lignin composition between susceptible (Marmande) and resistant (Hawaii 7996) tomatoes, which indicate that the Solanum dulcamara Carries Latent properties of paravascular lignin may be key for resistance to R. solanacearum Infections at 20◦ C bacterial wilt (Kashyap et al., 2021). Three conditions are required for the establishment and Interestingly, S. dulcamara lignification was already high in development of plant diseases: a virulent pathogen, a susceptible mock-treated plants and was not affected by infection (Figure 5 host, and permissive environmental conditions (McNew, 1960). and Supplementary Figure 5), whereas susceptible tomato and Frontiers in Plant Science | www.frontiersin.org 9 November 2021 | Volume 12 | Article 755708 Sebastià et al. Solanum dulcamara–Ralstonia solanacearum Interaction potato plants reduced their lignin content significantly both at 6 AUTHOR CONTRIBUTIONS and 9 dpi upon R. solanacearum inoculation. This constitutive lignification and the irrelevance of suberin components, whose MV, PS, NC, and AK conceived and designed the work. PS, AK, levels are comparable with susceptible plants (Supplementary RP-J, and BD performed the experiments and statistical analyses. Figure 6), are key differences in the factors controlling MV and NC provided reagents and materials. PS, RP-J, and S. dulcamara resistance compared with tomato H7996, where MV analysed the results and edited the figures. MV, PS, and suberin components play a major role (Kashyap et al., 2021). The NC wrote the manuscript. All authors have made a substantial, fact that S. dulcamara is a perennial, with the ensuing secondary direct and intellectual contribution to the work, and approved it growth present in these plants (Caldwell, 2016), may explain for publication. the high lignification of its tissues, which must be even more pronounced in wild plants- and that this phenomenon is not inducible like in the annual tomato plants. FUNDING The correlation observed between resistance to infection and the presence of cell wall reinforcements both in resistant This work was supported by the Spanish Ministry of tomato and in the wild S. dulcamara plants indicates that Economy and Competitiveness (AGL2016-78002-R) and the lignification hinders R. solanacearum movement throughout Ministry of Science and Innovation PID2019-108595RB- the plant tissues and entry in the xylem vessels. This would I00/AEI/10.13039/501100011033. We also acknowledge financial explain the delay in symptom appearance compared with support from the “Severo Ochoa Program for Centers of susceptible tomatoes (Figures 1A,B) and also account for Excellence in R&D” (SEV-2015-0533 and CEX2019-000902- the low bacterial content in inoculated plants that remained S), and the CERCA Program from the Catalan Government healthy (Figures 1C–F). Microscopically, cell wall reinforcements (Generalitat de Catalunya). The funders had no role in study could have a major contribution to the stronger bacterial design, data collection and analysis, decision to publish, restriction in specific xylem vessels and decreased spread to or preparation of the manuscript. PS held an INPHINIT neighbouring parenchyma cells, as observed in S. dulcamara Programme fellowship from Fundació “La Caixa” and AK was compared with tomato cv. Marmande (Figure 3). Restriction a recipient of a Netaji Subhas - Indian Council of Agricultural of R. solanacearum infection to primary xylem vessels while Research (ICAR) International Fellowship. RP-J received FI secondary xylem vessels remain functional (Esau, 1977) could and FPU fellowships from Generalitat de Catalunya and the explain why S. dulcamara better survives the infection. Ministry of Education and Professional Formation, respectively. Restricting pathogen movement is an important mechanism for BD have received funding from the European Union’s Horizon resistance against R. solanacearum in tomato (Caldwell et al., 2020 Research and Innovation Programme under the Marie 2017; Planas-Marquès et al., 2020) and potato (Cruz et al., 2014) Skłodowska-Curie Grant Agreement No. 945043. that is also conserved in grapevine against Xylella fastidiosa (Chatterjee et al., 2008). In summary, strong preexisting lignified xylem vessels present ACKNOWLEDGMENTS in S. dulcamara are likely the factor that supports its resistance to R. solanacearum and allows it to behave like a reservoir host. The The authors are grateful to all the members of the Bacterial generation of S. dulcamara mutants in lignin biosynthesis genes Pathogens and Plant Cell Death Team (Center for Research in would be extremely useful to confirm this hypothesis. Agricultural Genomics, CRAG), especially to Marc Planas for their technical assistance and critical comments. DATA AVAILABILITY STATEMENT SUPPLEMENTARY MATERIAL The original contributions presented in the study are included The Supplementary Material for this article can be found in the article/Supplementary Material, further inquiries can be online at: https://www.frontiersin.org/articles/10.3389/fpls.2021. directed to the corresponding author. 755708/full#supplementary-material REFERENCES Bartoli, C., Lamichhane, J. 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Silverleaf nightshade (Solanum elaeagnifolium), a reservoir host absence of any commercial or financial relationships that could be construed as a for ‘Candidatus Liberibacter solanacearum’, the putative causal agent of zebra potential conflict of interest. chip disease of potato. Plant Dis. 99, 910–915. doi: 10.1094/PDIS-12-14-1254- RE Publisher’s Note: All claims expressed in this article are solely those of the authors Tusiime, G., Adipala, E., Opio, F., and Bhagsari, A. S. (1997). “Weeds as and do not necessarily represent those of their affiliated organizations, or those of latent hosts of Ralstonia solanacearum in highland Uganda: implications to the publisher, the editors and the reviewers. Any product that may be evaluated in development of an integrated control package for bacterial wilt,” in Bacterial this article, or claim that may be made by its manufacturer, is not guaranteed or Wilt Disease: Molecular and Ecological Aspects, eds P. Prior, C. Allen, and endorsed by the publisher. J. Elphinstone (Berlin: Springer-Verlag), 413–419. doi: 10.1007/978-3-662-035 92-4_63 Copyright © 2021 Sebastià, de Pedro-Jové, Daubech, Kashyap, Coll and Valls. This Van Elsas, J. D., Kastelein, P., Van Bekkum, P., Van der Wolf, J. M., De Vries, P. M., is an open-access article distributed under the terms of the Creative Commons and Van Overbeek, L. S. (2000). Survival of Ralstonia solanacearum biovar 2, the Attribution License (CC BY). The use, distribution or reproduction in other forums causative agent of potato brown rot, in field and microcosm soils in temperate is permitted, provided the original author(s) and the copyright owner(s) are credited climates. Phytopathology 90, 1358–1366. doi: 10.1094/PHYTO.2000.90.12.1358 and that the original publication in this journal is cited, in accordance with accepted Wenneker, M., Verdel, M. S. W., Groeneveld, R. M. W., Kempenaar, C., academic practice. No use, distribution or reproduction is permitted which does not Van Beuningen, A. R., and Janse, J. D. (1999). Ralstonia (Pseudomonas) comply with these terms. Frontiers in Plant Science | www.frontiersin.org 12 November 2021 | Volume 12 | Article 755708