Ralstonia solanacearum ΔPGI-1 strain KZR-5 is affected in growth, response to cold stress and invasion of tomato

Abstract

The survival and persistence of Ralstonia solanacearum biovar 2 in temperate climates is still poorly understood. To assess whether genomic variants of the organism show adaptation to local conditions, we compared the behaviour of environmental strain KZR-5, which underwent a deletion of the 17.6 kb genomic island PGI-1, with that of environmental strain KZR-1 and potato-derived strains 1609 and 715. PGI-1 harbours two genes of potential ecological relevance, i.e. one encoding a hypothetical protein with a RelA/SpoT domain and one a putative cellobiohydrolase. We thus assessed bacterial fate under conditions of amino acid starvation, during growth, upon incubation at low temperature and invasion of tomato plants. In contrast to the other strains, environmental strain KZR-5 did not grow on media that induce amino acid starvation. In addition, its maximum growth rate at 28°C in rich medium was significantly reduced. On the other hand, long-term survival at 4°C was significantly enhanced as compared to that of strains 1609, 715 and KZR-1. Although strain KZR-5 showed growth rates (at 28°C) in two different media, which were similar to those of strains 1609 and 715, its ability to compete with these strains under these conditions was reduced. In singly inoculated tomato plants, no significant differences in invasiveness were observed among strains KZR-5, KZR-1, 1609 and 715. However, reduced competitiveness of strain KZR-5 was found in experiments on tomato plant colonisation and wilting when using 1:1 or 5:1 mixtures of strains. The potential role of PGI-1 in plant invasion, response to stress and growth in competition at high and moderate temperatures is discussed.

Figure 1

Comparison between SpoT, RelA and RelA/SpoT domain containing proteins. a Comparison of conserved domains found in SpoT, RelA and RelA/SpoT domain containing proteins. HD domain (possessing a conserved histidine [H] and aspartate [D] residue): possibly involved in (p)ppGpp degradation, only present in SpoT. TGS and ACT domains: conserved ATP/GTP-binding and GTP-binding domains, respectively [6]; found in SpoT as well as RelA. The RelA/SpoT domain is shared by all three types of the RelA/SpoT-like proteins. b Alignment of the RelA/SpoT domain of SpoT and RelA of E. coli, R. solanacearum biovar 2, PGI-1 encoded RSIPO_04909, and eight related RelA/SpoT domain containing proteins using ClustalW. The position of the first amino acid of the four partial protein sequences (I–IV) is indicated and corresponds to the position of the first amino acid along the full-length protein. Stars Residues that are fully conserved among all sequences; triangles residues conserved among the SAS proteins of B. subtilis and S. mutans, the R. solanacearum RelA/SpoT domain protein and its closest homologues, but not with the RelA and SpoT proteins of E. coli and R. solanacearum; squares residues that were experimentally shown to be functionally significant in Streptococcus dysgalactiae [12]. E. coli, Eschericha coli; R. sol, Ralstonia solanacearum biovar 2; B. sub, Bacillus subtilis; S. mut, Streptococcus mutans; R. etli, Rhizobium etli; S. vir, Streptomyces viridochromogenes; Exi, Exiguobacterium; B.cer, Bacillus cereus. Asterisk For R. etli, a partial protein sequence was used due to unavailability of the full-length sequence. c Phylogenetic analysis of SpoT and RelA of E. coli and R. solanacearum biovar 2, together with the PGI-1-encoded RelA/SpoT domain protein, RSIPO_04909 and related RelA/SpoT domain containing proteins. The tree was conducted using Mega4 software [28], option neighbor joining, bootstrap 1,000 replicates and Poisson correction)

Figure 2

Maximum growth rate (μ _max; h⁻¹) of strains KZR-5, 1609 and 715 at 28°C in BG, 0.1× TSBS and M63 and at 16°C in 0.1× TSBS. Maximum growth rate was determined for exponentially growing cells. Values are the average of at least two independent experiments. Statistical classes (a, b, c, d and e) are indicated (P < 0.05.

Figure 3

Competitiveness of R. solanacearum strain KZR-5 against strains 1609 and 715 in liquid 0.1× TSBS. The ratio between ΔPGI-1 strain (KZR-5) and the comparator strains (1609 or 715) is shown at the beginning of the experiment (1:1) and after growth in mixed cultures. For competition at 28°C, the ratio between strains was determined from duplicate overnight cultures. For competition at 16°C, the ratio between the strains was determined from (duplicate) batch cultures after four serial transfers. Statistical classes (a and b) are indicated; P < 0.05

Figure 4

Survival of R. solanacearum strains 1609, 715, KZR-5 and KZR-1 at 4°C. The graph shows the colony forming unit enumerations on BG plates. Points represent the means of three replicate experiments. The limit of detection (LOD), 10 CFU/mL, is indicated

Figure 5

Competitiveness of R. solanacearum strain KZR-5 against strains 1609, 715 and KZR-1 upon invasion of tomato plants. The ratio between ΔPGI-1 strain KZR-5 and the comparator strains (1609, 715 and KZR-1) was determined by dilution plating on agar plates (BG or SMSA) at four time points. First, the ratio between strains was determined for the cell mixture used for inoculation of the plants on BG agar plates. Second, the ratio between strains in the soil was determined from CFUs on SMSA plates at day 2 (soil). Extraction of R. solanacearum from the tomato stems was done 10 and 21 days post-inoculation, and the ratio between strains was determined from CFU counts on SMSA plates (stems)