The Bacterial Wilt Reservoir Host Solanum dulcamara Shows Resistance to Ralstonia solanacearum Infection

Abstract

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 R. solanacearum can overwinter, but their interaction remains uncharacterised. In this study, we have systematically analysed R. solanacearum infection in S. dulcamara, dissecting the behaviour of this plant compared with susceptible hosts such as tomato cv. Marmande, for which the interaction is well described. Compared with susceptible tomatoes, S. dulcamara plants (i) show delayed symptomatology and bacterial progression, (ii) restrict bacterial movement inside and between xylem vessels, (iii) limit bacterial root colonisation, and (iv) show constitutively higher lignification in the stem. Taken together, these results demonstrate that S. dulcamara behaves as partially resistant to bacterial wilt, a property that is enhanced at lower temperatures. This study 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 (impediment to colonisation) and a perennial habit enable bittersweet plants to behave as reservoirs for R. solanacearum.

Keywords: Ralstonia solanacearum; bacterial wilt; disease resistance; overwintering; reservoir host plants; vascular reinforcements.

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.

FIGURE 2

Bacterial colonisation and multiplication in stem-inoculated S. dulcamara and tomato cv. Marmande plants. (A) Non-destructive live luminescence imaging of four representatives S. dulcamara plants throughout a 30-day period after root inoculation with a luminescent R. solanacearum strain. Two symptomatic (A & F) and two asymptomatic (K & Q) plants are shown. Luminescent bacteria are detected in darker areas. Wilting symptoms (Disease index = 0–4) are indicated next to each plant inside the images. (B) R. solanacearum concentrations measured at the root, taproot, internode 1 (2–3 cm above the inoculation point) and 2 (6–9 cm above the inoculation point) in S. dulcamara plants uprooted 30 days post-inoculation (dpi) with the luminescent reporter strain. The results from asymptomatic (disease index 0) and symptomatic (disease index 1–4) plants are shown separately. Bacterial counts were calculated from tissue luminescence and are expressed as log CFUs⋅g^–1 tissue. ^∗indicates statistical differences (p-value < 0.05, T-student significant test).

FIGURE 3

R. solanacearum distribution in stems of inoculated S. dulcamara and tomato cv. Marmande plants. (A) Representative luminescence imaging photographs at different wilting stages (Disease index 0–4) of stem sections from S. dulcamara (top panel) and tomato (bottom panel) plants stem-inoculated with luminescent R. solanacearum. Luminescent bacteria are detected as dark areas in transversal and longitudinal sections of plant internodes 1–4 organised bottom to top. Inoculation points are indicated by an arrow. (B) Representative fluorescence microscopy images of stem sections from S. dulcamara (top panel) and tomato (bottom panel) plants stem-inoculated with an R. solanacearum strain constitutively expressing GFP. Inoculations were performed at the base of the first true leaf and transversal sections obtained in the first internode, 2 cm above the inoculation point. Scale bars indicate 0.5 mm.

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).

FIGURE 5

Lignification of S. dulcamara, Tomato cv. Marmande and S. tuberosum cv. Desirée tissues upon R. solanacearum infection. (A) Representative composed images of S. dulcamara, tomato, and potato taproot transversal sections obtained 9 days after inoculation with R. solanacearum-GFP or mock treatment. First and second row: microscope bright-field images after lignin staining with phloroglucinol HCl (magenta colouration). Third row: fluorescence microscopy images after inoculation to assess the extent of bacterial colonisation. Images were obtained using a Leica DM6 microscope. Scale bars indicate 0.5 mm. (B) Quantification of the phloroglucinol HCL stain -indicative of lignin content- in the vascular area from the images shown in A performed with the ImageJ software. ^∗indicates statistical differences (p-value < 0.05; T-student significant test α = 0.05).