Fatty Acid Competition as a Mechanism by Which Enterobacter cloacae Suppresses Pythium ultimum Sporangium Germination and Damping-Off

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 β-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.     

Differential Inactivation of Seed Exudate Stimulation of Pythium ultimum Sporangium Germination by Enterobacter cloacae Influences Biological Control Efficacy on Different Plant Species

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.     

Epicoccum Nigrum as biocontrol agent of Pythium damping-off and root-rot of cotton seedlings

Efficacy of Epicoccum nigrum and its exudate was tested in control the pre- and post-emergence damping-off and root-rot of Egyptian cotton (cv. Giza 83) in vitro and in vivo. Different isolates of Epicoccum nigrum reduced the radial growth of both Pythium debaryanum and P. ultimum significantly, by production considerable inhibition zones. In liquid cultures E. nigrum exudate showed a high fungicidal effect resulting in a significant reduction of the mycelial dry weight of the two investigated Pythium spp. Also E. nigrum exudate inhibited cellulase and pectinase activity by P. debaryanum and P. ultimum. Soaking of cotton seeds in E. nigrum exudate for different intervals resulted in significant reduction of root-rot severity of seedlings as well as the contamination of seeds and seedlings by fungi during and after germination. These treatments also stimulated germination of cotton seeds and enhanced the seedlings vigour significantly. In pot experiments, the use of E. nigrum as a soil mixture or seed dressing significantly alleviated the hazard effect of P. debaryanum. Pythium ultimum seemed to be weak or non pathogen to the used cotton cultivar (Giza 83). Application of E. nigrum or its exudate not only involved in protection of cotton seedlings against Pythium damping-off and root-rot but also enhanced their vigour and growth characteristics. The main conclusion of this study is that E. nigrum could be used successfully as environmentally safe and economic biological control agent to protect cotton (cv. Giza 83) from damping-off and root-rot diseases caused by P. debaryanum.     

Effect of seed exudates ofPinus resinosa on the germination of sporangia and on the population ofPythium irregulare in the soil

Summary Exudates from germinating seed ofP. resinosa stimulated the germination of sporangia and increased thePythium populations in soil. Sporangia ofP. irregulare did not germinate in natural soil and needed exogenous nutrition for their germination. Different components of the exudate, particularly glucose and asparagine, effectively stimulated sporangial germination. This is in agreement with an earlier finding withPythium ultimum ³.     

Mutation in <Emphasis Type="Italic">cyaA</Emphasis> in <Emphasis Type="Italic">Enterobacter cloacae</Emphasis> decreases cucumber root colonization

Strains of Enterobacter cloacae show promise as biological control agents for Pythium ultimum-induced damping-off on cucumber and other crops. Enterobacter cloacae M59 is a mini-Tn5 Km transposon mutant of strain 501R3. Populations of M59 were significantly lower on cucumber roots and decreased much more rapidly than those of strain 501R3 with increasing distance from the soil line. Strain M59 was decreased or deficient in growth and chemotaxis on most individual compounds detected in cucumber root exudate and on a synthetic cucumber root exudate medium. Strain M59 was also slightly less acid resistant than strain 501R3. Molecular characterization of strain M59 demonstrated that mini-Tn5 Km was inserted in cyaA, which encodes adenylate cyclase. Adenylate cyclase catalyzes the formation of cAMP and cAMP levels in cell lysates from strain M59 were approximately 2% those of strain 501R3. Addition of exogenous, nonphysiological concentrations of cAMP to strain M59 restored growth (1 mM) and chemotaxis (5 mM) on synthetic cucumber root exudate and increased cucumber seedling colonization (5 mM) by this strain without serving as a source of reduced carbon, nitrogen, or phosphorous. These results demonstrate a role for cyaA in colonization of cucumber roots by Enterobacter cloacae.     

Effect ofAgrobacterium radiobacter and its polysaccharide on emergence and damping-off of tomato plants

Agrobacterium radiobacter B6 and agrobacteran (an exopolysaccharide of the succinoglycan group) stimulated seed germination and tomato plant emergence. The germination was most stimulated by dipping the seeds in 0.1 % agrobacteran for 30 min whereas plant emergence in garden soil was best with 0.4 % agrobacteran at 10–20°C. Treatment of the plants withA.radiobacter cells and agrobacteran solution at 30–35°C. had a lower effect.A.radiobacter cells applied on seed surface protected the plants against damping off in garden soil artificially inoculated with the fungiRhizoctonia solani andPythium ultimum; in soils contaminated withFusarium solani 0.1 to 0.2% agrobacteran had a higher protective effect than the bacterization. The difference can be attributed to the varying density ofA.radiobacter population in plant rhizosphere in the presence of different plantpathogenic fungi, different interactions of microorganisms in the rhizosphere and different mode of penetration of the pathogens into plant roots.     

Influence of Sugar Beet Seed Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality

The effect of sugar beet seed inoculation with the bacterium Pseudomonas fluorescens and treatment with the fungicides Thiram 42‐S and Dithane S‐60 with and without seed inoculation aiming to control the root decay agents Pythium ultimum and Pythium debarianum was studied during a 2‐year trial on two soil types (Mollic Gleysols and Eutric Cambisols). The influence of the treatments on parameters of sugar beet yield and quality such as root yield, sugar content, sugar in molasses, sugar yield as well as percentage of the infected and decayed plants as a consequence of parasitic oomycete infestation will be described.     

Production and Application of Syringomycin E as an Organic Fungicide Seed Protectant against Pythium Damping‐off

Syringomycin E (SRE) is a cyclic lipodepsinonapeptide with potent antifungal activity and is produced by certain strains of Pseudomonas syringae pv. syringae. In this study, its potential as an organic‐compatible agrofungicide and vegetable seed treatment against the soilborne pathogen Pythium ultimum var. ultimum was examined. A variant of P. syringae pv. syringae strain B301D with enhanced SRE‐producing capabilities was isolated and grown in a bioreactor with SRE yields averaging 50 mg/l in 40 h. SRE was extracted and purified through a large‐scale chromatography system using organic‐compatible processes and reagents. The minimum concentrations of the purified product required to inhibit 50 and 90% of P. ultimum oospore germination were determined as 31.3 and 250 μg/ml, respectively. Drench treatment of cucumber seeds in P. ultimum‐infested potting medium (500 oospores/g) with 50 μg/ml SRE or water with no SRE resulted in 90.2 ± 4.5% and 65.7 ± 4.6% germination rates, respectively. Seed coating with 0.03% (w/w) SRE allowed 65.7 ± 4.6% seedlings to germinate on naturally infested soil while 100.0 ± 0.0% of non‐coated seeds were unable to germinate due to Pythium infection. Organic‐compatible and scalably produced SRE is potentially a novel organic fungicide seed protectant.     

Enterobacter cloacae is an endophytic symbiont of corn

The bacteriumEnterobacter cloacae is presently used for biocontrol of postharvest diseases of fruits and vegetables and as a preplant seed treatment for suppression of damping-off. This bacterium has apparent affinities for several grass species, but it is not considered to be an endophyte. While screening corn for fungi and bacteria with potential for biocontrol of various corn diseases, the surface-sterilized kernels of one unknown Italian corn cultivar produced fungus-free corn seedlings with roots endophytically infected byE. cloacae. This paper describes the microscopic nature ofE. cloacae RRC 101 with corn, and the in vitro control ofFusarium moniliforme and other fungi with this bacterium. Light and electron microscopy determined that this isolate ofE. cloacae was biologically associated with corn seedling roots, where it was distributed intercellularly within the cortex and stele. This is a first report of a strain of this bacterium as an endophytic symbiont of roots. Following a topical application ofE. cloacae to kernels, and upon germination this bacterium readily infected roots of two other corn cultivars. The bacterium was observed within the endosperm of germinating corn seedling, but germination was not affected. Further, the bacterium was isolated from leaves and stems of 3- to 6-week-old seedlings indicating that the above ground portions of corn were also colonized. There was no evidence of damage to cells of the root during a three to four week observation period. This bacterium was antagonistic to several isolates of the corn pathogenFusarium moniliforme, and to two other species of fungi, all of which produce mycotoxins on corn.     

Functional aspects of mature seed coat of theCannaceae

Cannaceae seeds have been analysed regarding seed coat structure, germination and macromolecular composition of the seed coats. Data of several mass spectrometric techniques were combined with those of microscopic and histochemical techniques to acquire insight into the functions of the seed coat.Cannaceae seeds have an exotestal layer of Malpighian cells with a hydrophobic and a hydrophilic part. The hydrophobic part is mainly responsible for the impermeability of the seed and contains silica, callose, lignin as water repellent substances. Water can only enter the seed after a certain temperature-induced opening of an imbibition lid. During imbibition the hydrophilic part of the Malpighian cells swells and the seed coat ruptures due to differences in pressure in the upper and lower part of the Malpighian cells.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

Testa is involved in the control of storage mobilisation in seeds of <Emphasis Type="Italic">Sesbania virgata</Emphasis> (Cav.) Pers., a tropical legume tree from of the Atlantic Forest

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) contain galactomannan as a cell wall storage polysaccharide in the endosperm. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the role of the testa (seed coat) on galactomannan degradation during and after germination. Seeds were imbibed in water, with and without the testa, and used to evaluate the effect of this tissue on storage mobilisation, as well as its possible role in the galactomannan hydrolases activities. Immunocytochemistry and immunodotblots were used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. Endo-β-mannanase and α-galactosidase activities were found in the testa and latter in the endosperm during galactomannan degradation. The former enzyme was immunologically detected in the testa, mainly during germination. The absence of the testa during imbibition led to the anticipation of protein mobilisation and increased of the α-galactosidase activity and galactomannan degradation. Thus, the testa appears to play a role during storage mobilisation in the legume seed of S. virgata probably by participating in the control of the production, modification and/or storage of the hydrolases.     

Polyamine contents, ethylene synthesis, and BrACO2 expression during turnip germination

Contents of total free [PA(S)] and conjugated polyamines [PA(SH), PA(PH)] were higher in turnip (Brassica rapa L. cv. Rapa) seeds during imbibition (0–36 h) and radicle protrusion (36–48 h) than during the further growth (10 d). Ethylene production was activated with the protrusion, reaching a maximum at the second day of germination and dropping afterwards. The application of ethrel accelerated radicle emergence but the direct intervention of ethylene in the breaking of the seed coat was not clear from the use of ethylene-biosynthesis inhibitors (CoCl₂ and AVG). Finally, in this work the gene BrACO2 was characterized. Although its expression was not detected in seeds through zygotic embryogenesis, it increased concomitantly with the germination process.     

SEEDS OF KOSTELETZKYA VIRGINICA (MALVACEAE): THEIR STRUCTURE,GERMINATION, AND SALT TOLERANCE. I. SEED STRUCTURE AND GERMINATION

Dormancy of Kosteletzkya virginica (L.) Presl. seeds is primarily due to the impermeability of the seed coat to water. The impermeable structure is assumed to be, in other Malvaceae, the palisade layer of the seed coat. The percentage of seeds capable of imbibition and germination increased with increasing time of storage at low temperatures, but the release from dormancy was not accompanied by decreased seed coat resistance to pressure. Under natural conditions, mechanical damage to the seed coat due to changes in temperature and/or abrasion may render the seeds water permeable. It is not clear what causes water permeability during storage under laboratory conditions. During seed maturation and drying, the inner epidermis of the tegmen partly separates from the rest of the seed coat and an air space, which makes the seed buoyant, is formed around the region of the chalazal cleft. The optimal temperature for germination of K. virginica seeds is between 28 and 30 C in light or darkness.     

Compost-Induced Suppression of Pythium Damping-Off Is Mediated by Fatty-Acid-Metabolizing Seed-Colonizing Microbial Communities

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.     

Pepper seed germination assessed by combined X-radiography and computer-aided imaging analysis

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97–100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52–56 h and 64–72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.     

Conversion of crude chitosan to an anti-fungal protease by <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Bacillus cereus AU004, isolated from soil samples, secreted a complex of hydrolytic enzymes into the culture broth when it was grown aerobically in a medium containing crude chitosan flakes. The presence of the AU004 culture supernatant substantially influenced the growths of the plant-pathogenic fungi Fusarium oxysporum, F. solani and Pythium ultimum in terms of dry weight. AU004 excreted a protease when cultivated in a medium that contained 4% (w/v) chitosan as the major nutritional source. The protease was purified by sequential chromatography and characterized as a novel extracellularly neutral protease. The protease had an Mr of 28.8 kDa. The optimal pH and temperature for protease activity were 7 and 50°C, respectively. Antifungal activity of the protease was observed using an assay based on the inhibition of spore germination and hyphal extension of the fungal Pythium ultimum. This investigation is the first report of the production of an anti-fungal protease from Bacillus spp.     

An image analysis method for determination of spatial colonization patterns of bacteria in plant rhizosphere

A method that allows the rapid visualization of bacterial spatial colonization patterns on roots for the determination of general colonization trends was developed. This method, which analyzes images of roots, and bioluminescence-enhanced images of bacterial colonization patterns on these roots, was used to study the colonization patterns of seed-applied Enterobacter cloacae strain E6 on 3-day-old cucumber plants. Conventional dilution-plating methods indicated that E6 colonized cucumber tap roots in high populations and that these populations significantly decreased as the distance from the seed increased. In addition to confirming these observations, image analysis indicated that colonization by E6 significantly decreased on lateral roots as the distance increased horizontally away from the tap root, and that this bacterium did not evenly cover the most densely colonized regions of the cucumber root system. Results from these experiments indicate that the majority of E6 populations on cucumber roots after seed application are limited to the upper regions of the tap root and that E6 does not effectively colonize other regions of the root system. Received: 15 June 1988 / Received revision: 19 November 1998 / Accepted: 29 November 1998     

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin. Received: 16 March 1999 / Accepted: 21 August 1999     

Electrophysiological membrane characteristics of the salt tolerant Plantago maritima and the salt sensitive Plantago media

Plasmid pUCD607 was mobilized into the biocontrol agent Enterobacter cloacae strain E6 by conjugation and the resultant strain, E6(pUCD607), was bioluminescent. Biocontrol of Pythium ultimum by E6(pUCD607) was similar to that of the parent strain, E6. The location of E6(pUCD607) in the soil and in the rhizosphere of lettuce was readily determined by pressing agar medium against plant roots in a root box, allowing the bacteria to grow overnight on the medium, and detecting the presence of bioluminescence by autophotography. There was a positive, linear correlation between population sizes determined by dilution plating and the quantity of light emitted due to bioluminescence. However, both the intercept and slope of this line varied among experiments possibly due to the differing physiological states of cells recovered from soils. The amount of light emitted by the bioluminescent strain E6(pUCD607) was not quantitative. This technique is useful for qualitative determinations of populations and for photographically locating bacteria.