Chemotactic disruption as a method to control bacterial wilt caused by Ralstonia pseudosolanacearum.

We have demonstrated that chemotaxis to l-malate facilitated motility of Ralstonia pseudosolanacearum MAFF 106611, a causative agent of bacterial wilt, to plant roots. Here, we evaluated the assumption that the disruption of chemotaxis to l-malate leads to inhibition of plant infection by R. pseudosolanacearum MAFF 106611. Chemotactic assays revealed that chemotaxis to l-malate was completely or partially inhibited in the presence of l-, d-, and dl-malate, respectively. Moreover, l-malate served as a carbon and energy source for R. pseudosolanacearum MAFF 106611, while d-malate inhibited the growth of this bacterium. In the sand-soak inoculation virulence assay for tomato plants, the addition of l-, d-, and dl-malate to sand suppressed the plant infection. We concluded that supplementation of l- and dl-malate suppresses tomato plant infection with R. pseudosolanacearum MAFF 106611 by disrupting its chemotaxis to l-malate, while d-malate suppresses it by both the disruption of l-malate chemotaxis and inhibition of growth.

Negative chemotaxis of Ralstonia pseudosolanacearum to maleate and identification of the maleate chemosensory protein.

Ralstonia pseudosolanacearum Ps29 was repelled by maleate. Screening of a complete collection of Ps29 single-methyl-accepting chemotaxis protein (mcp) gene mutants identified the RSp0303 homolog (McpP) as a chemotaxis sensor mediating negative chemotaxis to maleate. Interestingly, the mcpP-deletion mutant was attracted to maleate, indicating that this bacterium expresses a MCP(s) for both positive and negative chemotaxis to maleate. We constructed a Ps29 derivative (designated POC14) harboring deletions in 14 individual mcp genes, including mcpP, to characterize McpP. Introduction of a plasmid harboring the mcpP gene (pPS16) restored the ability to negatively respond to maleate, confirming that McpP is a MCP for negative chemotaxis to maleate. We thought that maleate might be applied to controlling plant infection by R. pseudosolanacearum. To evaluate this possibility, we measured chemotactic responses of seven other virulent R. pseudosolanacearum strains to maleate. We confirmed that they harbored functional mcpP orthologues, but they showed no chemotactic responses to maleate. Quantitative RT-PCR analysis revealed that these seven R. pseudosolanacearum strains did not show negative chemotaxis to maleate because of negligible transcription of the mcpP genes. We compared the chemotactic responses of POC14 and POC14[pPS16] toward various chemicals and found that McpP senses inorganic phosphate as a chemoattractant.

Identification and characterization of chemosensors for d-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum.

Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.