Yeasts associated with nectarines and their potential for biological control of brown rot.

Resident fruit microflora has been the source of biocontrol agents for the control of postharvest decay of fruits and the active ingredient in commercialized biocontrol products. With the exception of grapes and apples, information on the resident microflora of other fruits is only fragmentary, but greater knowledge in this area can be very helpful in developing biocontrol strategies. We characterized the yeast microflora of nectarines ('Croce del Sud') from the early stages of fruit development until harvest. The fruit samples were collected from trees in an unmanaged orchard. The resident fruit microflora was separated from the occasionally deposited microorganisms by discarding initial fruit washings before the final wash, followed by sonication and plating on NYDA medium. The isolated yeasts were identified by BIOLOG and by sequencing the D1/D2 domain of a large subunit of the rRNA gene and, where available, the ITS sequence. BIOLOG identified 19 and the genetic analysis 23 species of yeasts. Although the identification by these two systems was not always the same, the predominant yeasts were Rhodotorula spp., Sporodiobolus spp., Cryptococcus spp., Pichia spp., Candida spp. and yeast-like Aureobasidium pullulans. Several of the taxa appear to represent new species. The preliminary biocontrol tests against brown rot of nectarine fruit caused by Monilinia fructicola indicates significant decay control potential of some of the identified yeast species, namely Cryptococcus magnus, Cryptococcus sp. nov., Sporidiobolus pararoseus, A. pullulans and Rhodotorula sp. nov.

Analysis of fungal communities by sole carbon source utilization profiles.

A simple method for characterization of fungal communities in environmental samples was developed. Dilute suspensions of samples in 0.2% agar containing three different antibiotics were pipetted into 96-well plates (Biolog SF-N) containing a diverse collection of 95 different carbon sources. The plates were incubated for 4-12 days at 22 degrees C and the absorbance measured at 650 nm. Canonical variates analysis was then used to analyze the multivariate data. This method allowed fungal communities in rhizosphere soil of corn and soybean to be distinguished according to soil and plant type. Data taken at a single time-point, which varied greatly in total absorbance of the plate, separated rhizosphere samples primarily by soil type. When multiple time-points were combined to keep the total absorbance constant, differences in substrate utilization patterns due to different plant types could be distinguished. The method was also applicable to analysis of phylloplane and compost fungal communities. This method is readily applied to large numbers of samples and should be useful for community analysis in a variety of agricultural and ecological studies.

4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane.

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.

Importance of pfkA for rapid growth of Enterobacter cloacae during colonization of crop seeds.

Enterobacter cloacae A-11 is a prototrophic, glycolytic mutant of strain 501R3 with a single transposon insertion in pfkA. The populations of strain A-11 on cucumber and radish seeds were smaller than the populations of strain 501R3 in natural soil, but the populations of these two strains on pea, soybean, sunflower, and sweet corn seeds were similar (D. P. Roberts, P. D. Dery, I. Yucel, J. Buyer, M. A. Holtman, and D. Y. Kobayashi, Appl. Environ. Microbiol. 65:2513-2519, 1999). The net effect of the mutation in pfkA in vitro was a shift from rapid growth on certain carbohydrates detected in seed exudates to much slower growth on other carbohydrates, amino acids, and organic acids. The impact of the mutation in pfkA was greatest on the growth rate of E. cloacae on the seeds that released the smallest quantities of fructose, other carbohydrates, and amino acids. Corn, pea, soybean, and sunflower seeds released total amounts of carbohydrates and amino acids at rates that were approximately 10- to 100-fold greater than the rates observed with cucumber and radish seeds for the first 24 h after inhibition began. The growth rate of strain A-11 was significantly less (50% less) than the growth rate of strain 501R3 on radish seeds, and the growth rate of strain A-11 was too low to estimate on cucumber seeds in sterile sand for the first 24 h after inhibition began. The growth rate of strain A-11 was also significantly lower on soybean seeds, but it was only 17% lower than the growth rate of strain 501R3. The growth rates of strains 501R3 and A-11 were similar on pea, sunflower, and corn seeds in sterile sand for the first 30 h after imbibition began. Large reductions in the growth rates of strain A-11 on seeds were correlated with subsequent decreased levels of colonization of seeds compared to the levels of colonization of strain 501R3. The strain A-11 populations were significantly smaller than the strain 501R3 populations only on radish and cucumber seeds. The mutation in pfkA appears to decrease the level of colonization by E. cloacae for seeds that release small quantities of reduced carbon compounds by decreasing the size of the pool of compounds that support rapid growth by this bacterium.

Synthesis of optically pure chrysobactin and immunoassay development.

Chrysobactin (alpha-N-(2,3-dihydroxybenzoyl)-D-lysyl-L-serine), a siderophore that is essential for systemic virulence by plant pathogenic Erwinia chrysanthemi, was synthesized with high diastereomeric purity. Chrysobactin was prepared by coupling the N-hydroxysuccinimide ester of alpha-N-(2,3-dibenzyloxybenzoyl)-epsilon-N-Cbz-D-lysine with L-serine benzyl ester followed by deprotection via hydrogenolysis. Optically pure chrysobactin was obtained with 98% overall yield. A monoclonal antibody to ferric chrysobactin was developed and characterized as IgM. The antibody reacts with chrysobactin, ferric chrysobactin and less strongly with ferric dihydroxybenzoic acid. The antibody reacts weakly with the siderophores ferrichrome, A, ferric pseudobactin and ferric rhodotorulic acid. This antibody was used in a competitive immunoassay to detect ferric chrysobactin at 10(-8) to 10(-10) mol. This immunoassay may provide a useful method for the detection of chrysobactin in plant samples.

A Soil and Rhizosphere Microorganism Isolation and Enumeration Medium That Inhibits Bacillus mycoides.

A new solid medium has been developed for the enumeration and isolation of soil and rhizosphere microorganisms. This medium, named rhizosphere isolation medium, contains glucose and 15 of the 20 common amino acids. The absence of five other amino acids, namely, aspartic acid, asparagine, cysteine, proline, and threonine, inhibits the growth of Bacillus mycoides, a commonly encountered bacterium that rapidly spreads on agar media and complicates the isolation and enumeration of other microorganisms. Compared with a similar medium containing Casamino Acids, rhizosphere isolation medium had half as many colonies of B. mycoides, with each colony approximately half the diameter. The two media had similar total numbers of bacterial colonies. Isolates were divided into taxononomic groups, roughly corresponding to species and genus, by fatty acid methyl ester analysis and numerical methods. There were 24 genera and 41 species found in the isolates from rhizosphere isolation medium, while 19 genera and 35 species were found in the isolates from the medium prepared with Casamino Acids. No major group of bacteria was found to occur only on one medium or on the other, indicating that the five missing amino acids had no great effect on organisms other than B. mycoides. This medium may prove useful in soil and rhizosphere studies in which the growth of B. mycoides is undesirable.

A method for detection of pseudobactin, the siderophore produced by a plant-growth-promoting pseudomonas strain, in the barley rhizosphere.

Detection in the rhizosphere of the siderophore produced by an inoculated microorganism is critical to determining the role of microbial iron chelators on plant growth promotion. We previously reported the development of monoclonal antibodies (MAb) to ferric pseudobactin, the siderophore of plant-growth-promoting Pseudomonas strain B10. One of these MAb reacted less strongly to pseudobactin than to ferric pseudobactin. The MAb reacted to Al(III), Cr(III), Cu(II), and Mn(II) complexes of pseudobactin at a level similar to the level at which it reacted to ferric pseudobactin and reacted less to the Zn(II) complex, but these metals would make up only a small fraction of chelated pseudobactin in soil on the basis of relative abundance of metals and relative binding constants. Fourteen-day-old barley plants grown in limed and autoclaved soil were inoculated with 10 CFU of Pseudomonas strain Sm1-3, a strain of Pseudomonas B10 Rif Nal selected for enhanced colonization, and sampled 3 days later. Extraction and analysis of the roots and surrounding soil using the MAb in an immunoassay indicated a concentration of 3.5 x 10 mol of ferric pseudobacting g (wet weight). This is the first direct measurement of a pseudobactin siderophore in soil or rhizosphere samples.

Production of the siderophore aerobactin by a halophilic pseudomonad.

A bacterial strain, isolated from a cyanobacterial culture, was identified as Pseudomonas sp. strain X40. Under iron-limiting conditions, the Pseudomonas sp. produced aerobactin, a dihydroxamate siderophore previously found only in the family Enterobacteriaceae. Aerobactin was identified by electrophoretic mobility, spectrophotometric titration, proton nuclear magnetic resonance spectroscopy, mass spectrometry, acid hydrolysis, and biological activity. Aerobactin was used as a siderophore in the Pseudomonas sp. and Escherichia coli. Two iron-repressed outer membrane proteins were observed in the Pseudomonas sp., neither of which had electrophoretic mobility identical to that of the aerobactin outer membrane receptor protein from E. coli. DNA hybridization assays showed no hybridization to the aerobactin genes from the E. coli plasmid pColV, indicating that the genetic determinants for aerobactin production by Pseudomonas strain X40 differ substantially from those found in the archetypic enteric plasmid pColV-K30.

Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10.

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 x 10 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores.

Structure of pseudobactin A214, a siderophore from a bean-deleterious Pseudomonas.

Bean-deleterious Pseudomonas A214 produced the extracellular yellow-green, fluorescent siderophore [microbial iron(III) transport agent] pseudobactin A214 under iron-limiting conditions. Pseudobactin A214 has a molecular formula of C46H64N13O22 and a molecular mass of 1151 g/mol. Pseudobactin A214 contained an N-blocked linear octapeptide with the amino acid sequence Ser-Ala-Gly-Ser-Ala-threo-beta-OH-Asp-L-allo-Thr-N delta-OH-Orn with a yellow-green, fluorescent quinoline derivative attached via an amide bond to the amino terminus. A succinamide group was linked to carbon 3 of the quinoline derivative. Sequencing was accomplished by two-dimensional NMR spectroscopy and by Edman degradation of smaller peptides obtained from partial acid hydrolysis. Since pseudobactin A214 was not affected by nonspecific proteolytic enzymes, it might contain D-amino acids. The three bidentate iron-(III)-chelating groups consisted of a 1,2-dihydroxy aromatic group in the quinoline chromophore, an alpha-hydroxy acid group present as beta-hydroxyaspartic acid, and a hydroxamate group derived from N delta-acetyl-N delta-hydroxyornithine. The chemical structure of pseudobactin A214 is remarkably similar to those of pseudobactin and pseudobactin 7SR1, the siderophores of plant growth promoting and plant-deleterious Pseudomonas B10 and Pseudomonas 7SR1, respectively.

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains.

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.