Role of sdhA and pfkA and catabolism of reduced carbon during colonization of cucumber roots by Enterobacter cloacae

Authors: LIU, SHENGYI - Chinese Academy Of Agricultural Sciences; HU, XIAOJIA - Chinese Academy Of Agricultural Sciences; Lohrke, Scott; Baker, Con; Buyer, Jeffrey; DE SOUZA, JORGE - Chinese Academy Of Agricultural Sciences; Roberts, Daniel

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/20/2007
Publication Date: 9/1/2007
Citation: Liu, S., Hu, X., Lohrke, S.M., Baker, C.J., Buyer, J.S., De Souza, J.T., Roberts, D.P. 2007. Role of sdhA and pfkA and catabolism of reduced carbon during colonization of cucumber roots by Enterobacter cloacae. Microbiology. 153:3197-3210.

Interpretive Summary: Soilborne plant pathogenic fungi cause diseases that result in major economic losses to farmers in the United States. Biological control measures for these diseases need to be developed due to environmental problems associated with existing chemical controls. However, little is known regarding the genetic and biochemical means by which biological control agents grow on seeds and roots of plants and suppress pathogens that infect these seeds and roots. Growth on plant parts and suppression of pathogen infection of plants are thought to be important for biological control. In this study we determined the role of the pfkA gene of the biocontrol bacterium Enterobacter cloacae in growth on cucumber seedlings and roots and in suppression of infection of the important seed rot pathogen Pythium ultimum. This gene was important for growth on cucumber but the importance of this gene varied with population density of E. cloacae on cucumber. This gene was not required for suppression of cucumber seed rot or of pea seed rot caused by Pythium ultimum. This information will be useful to scientists devising strategies to improve biological control through enhancement of growth on seeds or of disease suppression.

Technical Abstract: Strain A-11 of the plant-beneficial bacterium Enterobacter cloacae was used as a tool to determine the importance of pfkA and catabolism of carbohydrates in exudate to the plant-associated activities of root colonization and suppression of damping-off. E . cloacae A-11 is a near-isogenic mutant of strain 501R3 with a single transposon insertion in pfkA. pfkA encodes phosphofructose kinase, a key enzyme in glycolysis. Consequently strain A-11 is reduced in catabolism of carbohydrates in seed and root exudate with the exception of fructose (D.P. Roberts, P.D. Dery, I. Yucel, J.S. Buyer, M.A. Holtman, and D.Y. Kobayashi, Appl. Environ. Microbiol. 65:2513-2519, 1999). In situ competitiveness experiments with strain A-11 were used to determine the importance of pfkA and catabolism of carbohydrate in exudate to plant-associated activities of E. cloacae at various population densities on cucumber seedlings. Strain A-11 was equally competitive with strain 501R3 in De Wit replacement series experiments at 9.49 + 0.02 log10 CFU per seed but became increasingly less competitive with strain 501R3 as population densities in the initial inoculum were decreased to 7.71 + 0.02 log10 CFU per seed and to 3.65 + 0.08 log10 CFU per seed. A similar relationship between metabolic activity of strain A-11 and initial population density was found. Competitiveness (tested at 7.71 + 0.02 log10 CFU per seed) by strain A-11 relative to strain 501R3 was restored on cucumber seedlings supplemented with fructose indicating that reduced competitiveness was due to decreased carbohydrate utilization capabilities of strain A-11. Together this data indicates that the importance of pfkA and catabolism of carbohydrate in exudate is dependent on, and negatively correlated with, population densities of this bacterium on cucumber. pfkA and catabolism of carbohydrate in exudate were important for colonization of cucumber roots by E . cloacae in natural soil and in soil-less mix. Differences in populations between strains A-11 and 501R3 on cucumber roots were maximized when population densities of these strains decreased to within the range where the impact of pfkA on competitiveness was maximized. In contrast, pfkA and carbohydrate catabolism were not required for suppression of damping-off of cucumber caused by Pythium ultimum. Strain A-11 provided similar or greater suppression of damping-off than strain 501R3 at population densities of approximately 10.00 log10 CFU per seed and 8.00 log10 CFU per seed in seed treatments. Strain A-11 was reduced in competitiveness at approximately 8.00 log10 CFU on cucumber seedlings, however, neither strain was effective in disease suppression at lower population densities where the impact of pfkA on competitiveness was maximized. As the window of vulnerability of cucumber to damping-off caused by P. ultimum is short, populations of E. cloacae did not decrease to levels where the impact of pfkA was maximized. This is the first report demonstrating a differential role for a gene in root colonization and in disease suppression. We propose that this differential role for pfkA is due to the differing population densities that occur on cucumber during these two plant-associated activities. These experiments illustrate the complex relationship between catabolic capabilities and plant-associated activities of beneficial bacteria in the spermosphere and rhizosphere.