Differential interference with Pythium ultimum sporangial activation and germination by Enterobacter cloacae in the corn and cucumber spermospheres

Abstract

Differential protection of plants by Enterobacter cloacae was studied by investigating early sensing and response behavior of Pythium ultimum sporangia toward seeds in the presence or absence of E. cloacae. Ten percent of P. ultimum sporangia were activated within the first 30 min of exposure to cucumber seeds. In contrast, 44% of the sporangia were activated as early as 15 min after exposure to corn seeds with over 80% sporangial activation by 30 min. Germ tubes emerged from sporangia after 2.5 and 1.0 h in the cucumber and corn spermospheres, respectively. Seed application of the wild-type strain of E. cloacae (EcCT-501R3) reduced sporangial activation by 45% in the cucumber spermosphere, whereas no reduction was observed in the corn spermosphere. Fatty acid transport and degradation mutants of E. cloacae (strains EcL1 and Ec31, respectively) did not reduce sporangial activation in either of the spermospheres. Although wild-type or mutant strains of E. cloacae failed to reduce seed colonization incidence, pathogen biomass on cucumber seeds was reduced in the presence of E. cloacae strains EcCT-501R3 and Ec31 by 4 and 8 h after sowing, respectively. By 12 h, levels of P. ultimum on cucumber seeds treated with E. cloacae EcCT-501R3 did not differ from levels on noninoculated seeds. On corn seeds, P. ultimum biomass was not affected by the presence of any E. cloacae strain. When introduced after sporangial activation had occurred, E. cloacae failed to reduce P. ultimum biomass on cucumber seeds compared with that on nontreated seeds. Also, increasing numbers of sporangia used to inoculate seeds yielded increased pathogen biomass at each sampling time. This indicates a direct link between the level of seed-colonizing biomass of P. ultimum and the number of activated and germinated sporangia in the spermosphere, suggesting that E. cloacae suppresses P. ultimum seed infections by reducing sporangial activation and germination within the first 30 to 90 min after sowing.

FIG. 1.

Activation (A) and germ tube emergence (B) of Pythium sporangia in the corn (•) and cucumber (□) spermospheres and in the absence of seeds (○) during the first 4 and 6 h of seed germination, respectively. Activation was assessed as percent sporangia with visible germ tubes after sporangia were exposed to seeds for various periods of time and then incubated in the absence of seeds, allowing germ tubes to emerge. Each point with the error bars represents the mean ± standard deviation of 15 observations from three repeated experiments.

FIG. 2.

Activation of Pythium ultimum sporangia in the spermospheres of cucumber and corn in the presence of E. cloacae EcCT-501R3 and fatty acid degradation mutant derivative strains EcL1 and Ec31 during the first 4 (corn) to 6 (cucumber) h of seed germination. (A and B) Sporangial activation in the cucumber (A) and corn (B) spermospheres in response to seeds treated with 0 (▪), 10⁴ (○), 10⁶ (•), or 10⁸ (▵) cells of E. cloacae EcCT-501R3 per cm³ of sand. (C and D) Sporangial activation in the cucumber (C) and corn (D) spermospheres in response to nontested seeds (▪) or seeds treated with 10⁸ cells of E. cloacae EcL1 (○), Ec31 (•), or EcCT-501R3 (▵) per cm³ substrate. Each point represents the mean of nine observations from three repeated experiments. No-seed treatments (□) served as negative controls.

FIG. 3.

Frequency of cucumber (A) and corn (B) seeds colonized by Pythium ultimum in the presence of E. cloacae strain EcL1 (○), Ec31 (•), or EcCT-501R3 (▵). Controls consisted of noninoculated seeds (□) as well as inoculated seeds not treated with bacteria (▪). Each point represents the mean of three observations (six replicates each) from three repeated experiments.

FIG. 4.

Pythium ultimum biomass development on cucumber (A) and corn (B) seeds in the presence of E. cloacae strain Ec31 (○) or EcCT-501R3 (•). Seeds were exposed to Pythium for 4, 8, and 12 h, after which amounts of P. ultimum DNA were determined using qPCR. Seeds that were inoculated but not treated with E. cloacae (▪) and noninoculated seeds (□) served as positive and negative controls, respectively. Each point with the error bars represents the mean ± standard deviation of six seeds from two repeated experiments. Note the different y axes.