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The aim of this study was to determine the temporal release of fatty acids and sugars from corn and cucumber seeds during the early stages of seed germination in order to establish whether sugars found in exudate can prevent exudate fatty acid degradation by Enterobacter cloacae. Both saturated (long-chain saturated fatty acids [LCSFA]) and unsaturated (long-chain unsaturated fatty acids [LCUFA]) fatty acids were detected in corn and cucumber seed exudates within 15 min after seed sowing. LCSFA and LCUFA were released at a rate of 26.1 and 6.44 ng/min/seed by corn and cucumber seeds, respectively. The unsaturated portion of the total fatty acid pool from both plant species contained primarily oleic and linoleic acids, and these fatty acids were released at a combined rate of 6.6 and 0.67 ng/min/seed from corn and cucumber, respectively. In the absence of seed exudate sugars, E. cloacae degraded linoleic acid at rates of 29 to 39 ng/min, exceeding the rate of total fatty acid release from seeds. Sugars constituted a significant percentage of corn seed exudate, accounting for 41% of the total dry seed weight. Only 5% of cucumber seed exudate was comprised of sugars. Glucose, fructose, and sucrose were the most abundant sugars present in seed exudate from both plant species. Corn seeds released a total of 137 microg/seed of these three sugars within 30 min of sowing, whereas cucumber seeds released 0.83 microg/seed within the same time frame. Levels of glucose, fructose, and sucrose found in corn seed exudate (90 to 342 microg) reduced the rate of linoleic acid degradation by E. cloacae to 7.5 to 8.8 ng/min in the presence of either sugar, leaving sufficient concentrations of linoleic acid to activate Pythium ultimum sporangia Our results demonstrate that elevated levels of sugars in the corn spermosphere can prevent the degradation of LCUFA by E. cloacae, leading to its failure to suppress P. ultimum sporangial activation, germination, and subsequent disease development.

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.

This study was initiated to understand whether differential biological control efficacy of Enterobacter cloacae on various plant species is due to differences in the ability of E. cloacae to inactivate the stimulatory activity of seed exudates to Pythium ultimum sporangium germination. In biological control assays, E. cloacae was effective in controlling Pythium damping-off when placed on the seeds of carrot, cotton, cucumber, lettuce, radish, tomato, and wheat but failed to protect corn and pea from damping-off. Seeds from plants such as corn and pea had high rates of exudation, whereas cotton and cucumber seeds had much lower rates of exudation. Patterns of seed exudation and the release of P. ultimum sporangium germination stimulants varied among the plants tested. Seed exudates of plants such as carrot, corn, lettuce, pea, radish, and wheat were generally more stimulatory to P. ultimum than were the exudates of cotton, cucumber, sunflower, and tomato. However, this was not directly related to the ability of E. cloacae to inactivate the stimulatory activity of the exudate and reduce P. ultimum sporangium germination. In the spermosphere, E. cloacae readily reduced the stimulatory activity of seed exudates from all plant species except corn and pea. Our data have shown that the inability of E. cloacae to protect corn and pea seeds from Pythium damping-off is directly related to its ability to inactivate the stimulatory activity of seed exudates. On all other plants tested, E. cloacae was effective in suppressing damping-off and inactivating the stimulatory activity of seed exudates.

Leaf composts were studied for their suppressive effects on Pythium ultimum sporangium germination, cottonseed colonization, and the severity of Pythium damping-off of cotton. A focus of the work was to assess the role of fatty-acid-metabolizing microbial communities in disease suppression. Suppressiveness was expressed within the first few hours of seed germination as revealed by reduced P. ultimum sporangium germination, reduced seed colonization, and reduced damping-off in transplant experiments. These reductions were not observed when cottonseeds were sown in a conducive leaf compost. Microbial consortia recovered from the surface of cottonseeds during the first few hours of germination in suppressive compost (suppressive consortia) induced significant levels of damping-off suppression, whereas no suppression was induced by microbial consortia recovered from cottonseeds germinated in conducive compost (conducive consortia). Suppressive consortia rapidly metabolized linoleic acid, whereas conducive consortia did not. Furthermore, populations of fatty-acid-metabolizing bacteria and actinobacteria were higher in suppressive consortia than in conducive consortia. Individual bacterial isolates varied in their ability to metabolize linoleic acid and protect seedlings from damping-off. Results indicate that communities of compost-inhabiting microorganisms colonizing cottonseeds within the first few hours after sowing in a Pythium-suppressive compost play a major role in the suppression of P. ultimum sporangium germination, seed colonization, and damping-off. Results further indicate that fatty acid metabolism by these seed-colonizing bacterial consortia can explain the Pythium suppression observed.

Interactions between plant-associated microorganisms play important roles in suppressing plant diseases and enhancing plant growth and development. While competition between plant-associated bacteria and plant pathogens has long been thought to be an important means of suppressing plant diseases microbiologically, unequivocal evidence supporting such a mechanism has been lacking. We present evidence here that competition for plant-derived unsaturated long-chain fatty acids between the biological control bacterium Enterobacter cloacae and the seed-rotting oomycete, Pythium ultimum, results in disease suppression. Since fatty acids from seeds and roots are required to elicit germination responses of P. ultimum, we generated mutants of E. cloacae to evaluate the role of E. cloacae fatty acid metabolism on the suppression of Pythium sporangium germination and subsequent plant infection. Two mutants of E. cloacae EcCT-501R3, Ec31 (fadB) and EcL1 (fadL), were reduced in beta-oxidation and fatty acid uptake, respectively. Both strains failed to metabolize linoleic acid, to inactivate the germination-stimulating activity of cottonseed exudate and linoleic acid, and to suppress Pythium seed rot in cotton seedling bioassays. Subclones containing fadBA or fadL complemented each of these phenotypes in Ec31 and EcL1, respectively. These data provide strong evidence for a competitive exclusion mechanism for the biological control of P. ultimum-incited seed infections by E. cloacae where E. cloacae prevents the germination of P. ultimum sporangia by the efficient metabolism of fatty acid components of seed exudate and thus prevents seed infections.

Composts prepared from a variety of feedstocks were tested for their ability to suppress seedling and root diseases of creeping bentgrass caused by Pythium graminicola. Among the most suppressive materials in laboratory experiments were different batches of a brewery sludge compost and a biosolids compost from Endicott, N.Y. Batches of these composts that were initially not suppressive to Pythium damping-off became more suppressive with increasing compost age. Leaf, yard waste, food, and spent mushroom composts as well as certain biosolids, cow manure, chicken-cow manure, and leaf-chicken manure composts were not suppressive to Pythium damping-off. In some cases, turkey litter, chicken manure, chicken-leaf, and food waste composts were inhibitory to creeping bentgrass seed germination in laboratory experiments. Microbial populations varied among all of the composts tested. Bacterial populations were high in all composts except the turkey litter compost, in which populations were 1,000- to 10,000-fold lower than in the other composts tested. Among the highest populations of heterotrophic fungi and antibiotic-producing actinomycetes were those found in all batches of the brewery sludge compost, whereas the lowest populations were found in turkey litter, chicken manure, and food waste composts. Heat treatment of suppressive composts reduced populations of bacteria, fungi, and actinomycetes in all composts tested. Disease suppressiveness was also reduced or eliminated in heated composts. Amending heated composts with small amounts of nonheated compost restored suppressive properties and partially restored microbial populations to wild-type levels. A strong negative relationship between compost microbial activity (as measured by the hydrolysis of fluorescein diacetate) and Pythium damping-off severity was observed. When composts were applied to creeping bentgrass in field experiments, a significant level of suppressiveness was evident with some composts when disease pressure was high (i.e., disease ratings high in uninoculated plots). A 1991 batch of turkey litter compost and the 1990 batch of Endicott biosolids were consistently suppressive to foliar symptoms of Pythium root rot on creeping bentgrass. This study indicates that suppression of Pythium diseases of creeping bentgrass in batches of brewery sludge and Endicott biosolids composts, and possibly in other suppressive composts examined in less detail in this study, is related directly to the microbial activities in the composts. On the other hand, the mechanisms of Pythium suppression in turkey litter and perhaps other poultry-based composts is not related directly to the compost microbial activity. Although turkey litter showed a lack of suppressiveness in laboratory bioassays and low microbial populations and activity, it resulted in a significant and consistent level of suppressiveness in field experiments. Therefore, the microbiological properties of Pythium-suppressive composts may differ substantially, and measurements of microbial populations and activity may not be predictive of the level of disease suppression in all composts.