The production of antifungal metabolites by Pseudomonas chlororaphis grown on different nutrient sources

It was found that the antifungal activity of Pseudomonas chlororaphis SPB1217 is due to phenazine-1-carboxylic acid, phenazine-1-carboxamide, and two unidentified exometabolites. The carbon source used for the growth of this bacterial strain and iron ions present in the medium considerably influenced the proportion between the antifungal metabolites. The maximum production of phenazines was observed in the media enriched in amino acids and iron ions. The absence of correlation between the production of phenazines and antifungal activity indicates that phenazines are not the only antifungal metabolites of the strain. Organic acids as nutrient sources provide for more intense production of exometabolites and for a higher level of antifungal activity than do sugars.     

The Production of Antifungal Metabolites by Pseudomonas chlororaphis Grown on Different Nutrient Sources

It was found that the antifungal activity of Pseudomonas chlororaphis SPB1217 is due to phenazine-1-carboxylic acid, phenazine-1-carboxamide, and two unidentified exometabolites. The carbon source used for the growth of this bacterial strain and iron ions present in the medium considerably influenced the proportion between the antifungal metabolites. The maximum production of phenazines was observed in the media enriched in amino acids and iron ions. The absence of correlation between the production of phenazines and antifungal activity indicates that phenazines are not the only antifungal metabolites of the strain. Organic acids as nutrient sources provide for more intense production of exometabolites and for a higher level of antifungal activity than sugars.     

A system for screening natural immunosuppressors

Criteria for directed screening of antibiotics with immunosuppressive action were defined. The first stage included screening of cultures producing antiaspergillous antibiotics. At the second stage, the antibiotics whose antifungal activity decreases in the presence of insulin (at the background of calcium salts) and erythromycin and increases in the presence of verapamil were selected. The screening of antibiotic-producing cultures among 123 strains of mycelial fungi and 181 strains of actinomycetes resulted in isolation of 3 fungal cultures and 2 actinomycetes which produced antibiotics corresponding to cyclosporine A as evidenced by thin-layer and high performance liquid chromatographies.     

Taxonomic characteristics of soil Mycobacteria and their antibiotic properties]

Morphological, cultural and chemotaxonomic properties of 12 gram-positive soil cultures isolated were studied by using a test system developed for screening the organisms producing broad-spectrum antibiotics among Nocardiaforms (Coryneforms). The cultures were found to belong to Actinomycetales, Nocardioforms, Mycobacteriaceae and Mycobacterium. The saprophytic rapidly growing soil mycobacteria showed antibiotic activity against a large number of gram-positive and gram-negative test microbes including those belonging to Pseudomonas and Proteus resistant to the majority of the antibiotics currently used in medicine.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria.

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa+), siderophore (Sid+), HCN (HCN+) and fluorescent pigments (Flu+) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid+), antibiotics (Afa+) and antifungal volatiles (Afv+), MRF exhibited the production of antifungal antibiotics (Afa+) and siderophores (Sid+). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lacZ induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lacZ mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lacZ marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

Antifungal characteristics of a fluorescent Pseudomonas strain involved in the biological control of Rhizoctonia solani

A plant growth-promoting isolate of a fluorescent Pseudomonas spp. EM85 was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of cotton. The isolate produced HCN (HCN+), siderophore (Sid+), fluorescent pigments (Flu+) and antifungal antibiotics (Afa+). Tn5::lacZ mutagenesis of isolate EM85 resulted in the production of a series of mutants with altered production of HCN, siderophore, fluorescent pigments and antifungal antibiotics. Characterisation of these mutants revealed that the fluorescent pigment produced in PDA and the siderophore produced in CAS agar were not the same. Afa- and Flu- mutants had a smaller inhibition zone when grown with Rhizoctonia solani than the EM85 wild type. Sid- and HCN mutants failed to inhibit the pathogen in vitro. In a pot experiment, mutants deficient in HCN and siderophore production could suppress the damping-off disease by 52%. However, mutants deficient in fluorescent pigments and antifungal antibiotics failed to reduce the disease severity. Treatments with mutants that produced enhanced amounts of fluorescent pigments and antibiotics compared with EM85 wild type, exhibited an increase in biocontrol efficiency. Monitoring of the mutants in the rhizosphere using the lacZ marker showed identical proliferation of mutants and wild type. Purified antifungal compounds (fluorescent pigment and antibiotic) also inhibited the fungus appreciably in a TLC bioassay. Thus, the results indicate that fluorescent pigment and antifungal antibiotic of the fluorescent Pseudomonas spp. EM85 might be involved in the biological suppression of Rhizoctonia-induced damping-off of cotton.     

Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.     

Steroid interference with antifungal activity of polyene antibiotics

Wide differences exist among the polyene antibiotics, nystatin, rimocidin, filipin, pimaricin, and amphotericin B, with reference to steroid interference with their antifungal activities against Candida albicans. Of the numerous steroids tested, ergosterol was the only one which effectively antagonized the antifungal activity of all five polyene antibiotics. The antifungal activities of nystatin and amphotericin B were the least subject to vitiation by the addition of steroids other than ergosterol, and those of filipin, rimocidin, and pimaricin were the most sensitive to interference. Attempts to delineate the structural requirements of steroids possessing polyene-neutralizing activity in growing cultures of C. albicans are discussed. The ultraviolet absorbance of certain antibiotic steroid combinations was also studied.     

Physiological factors affecting carbon tetrachloride dehalogenation by the denitrifying bacterium Pseudomonas sp. strain KC.

Pseudomonas sp. strain KC was grown on a medium with a low content of transition metals in order to examine the conditions for carbon tetrachloride (CT) transformation. Several carbon sources, including acetate, glucose, glycerol, and glutamate, were able to support CT transformation. The chelators 2,2'-dipyridyl and 1,10-phenanthroline stimulated CT transformation in a rich medium that otherwise did not support this activity. Low (< 10 microM) additions of dissolved iron(II), iron(III), and cobalt(II), as well as an insoluble iron(III) compound, ferric oxyhydroxide, inhibited CT transformation. The addition of 50 microM iron to actively growing cultures resulted in delayed inhibition of CT transformation. CT transformation was seen in aerobic cultures of KC, but with reduced efficiency compared with denitrifying cultures. Inhibition of CT transformation by iron was also seen in aerobically grown cultures. Optimal conditions were used in searching for effective CT transformation activity among denitrifying enrichments grown from samples of aquifer material. No activity comparable to that of Pseudomonas sp. strain KC was found among 16 samples tested.     

The role of antibiotic production by a strain of Pseudomonas fluorescens in the suppression of Pseudocercosporella herpotrichoides, the causal agent of eyespot disease of cereals

Pseudomonas fluorescens strain 220 is an effective antagonist of Pseudocercosporella herpotrichoides , the eyespot pathogen of cereals. Culture filtrates of Ps. fluorescens 220 were inhibitory to spore germination and hyphal growth of P. herpotrichoides and at least two compounds with antifungal and antibacterial activity were identified in cultures grown in nutrient broth. In plant tests, both a culture broth of Ps. fluorescens 220 and a crude antibiotic extract reduced eyespot disease, whereas a mutant strain of 220 deficient in antibiotic production had no effect. Production of antibiotics would therefore appear to be a major factor in the suppression of P. herpotrichoides infection. A loss of disease control when Ps. fluorescens 220 was applied to plants in water was not due to lack of survival, as populations of a marked strain of Ps. fluorescens 220 applied to the stem base of wheat plants were similar whether applied in water or culture broth.     

Development of a novel technique for axenic isolation and culture of thraustochytrids from New Zealand marine environments

Aims: To maintain axenic cultures of commercially important thraustochytrids, a novel procedure was developed for the isolation of zoospores and sporangium from heterotrophic seawater samples and axenic culture on solid media. Methods and Results: Thraustochytrid cultures were isolated from Whangapoua Harbour in North East New Zealand and subjected to two antibiotic and antifungal treatment regimes designed to eliminate bacteria and fungi. Antibiotic trial 1 was designed to determine the appropriate combination of antibiotics (including streptomycin/penicillin, ampicillin, rifampicin, nalidixic acid, tetracycline, gentamicin and the antifungal agent nystatin). Antibiotic trial 2 determined the optimal dosing frequency and concentration of the antibiotics, and antifungal found to be the most promising in trial 1. Axenic cultures were then spread plated onto nutrient agar containing the optimal antibiotic cocktail, and pure thraustochytrid colonies were purified on solid media using standard microbiological techniques. Conclusions: Removal of bacteria and fungi was best accomplished using a mixture of three antibiotics and one antifungal; rifampicin (300 mg l^?1), streptomycin/penicillin (25 mg l^?1) and nystatin (10 mg l^?1) were incorporated in seawater samples and incorporated into cultures every 24 h for a minimum of 2 days. Significance and Impact of the Study: The axenic isolation and culture of marine thraustochytrids from a marine habitat in New Zealand have significant implications for the biotechnological development of these potentially valuable protists. This method has global significance as it is reasonable to assume it could be used throughout the world to obtain axenic thraustochytrid cultures.     

Antibiotic activity and siderophores of Pseudomonas cepacia

The ability of 46 strains of Pseudomonas cepacia to inhibit phytopathogenic fungi and the effect of iron on their antifungal activity were studied. The antifungal effect of the bacteria and the antimicrobial activity of their crude yellow and violet pigments showed a 4-5-fold decrease in the presence of Fe(III). The addition of 100 micrograms/ml of FeCl3 to the medium decreased the biosynthesis of violet and yellow pigments; the complex of the yellow pigment with Fe(III) promoted the growth of the P. cepacia producing strain under iron-deficient conditions. The data obtained suggest a participation of some P. cepacia pigments in iron transport. The resistance of the P. cepacia strains to the synthetic chelating agents hydroxyethylenediphosphonic and diethylenediaminepentaacetic acids was demonstrated, which may indicate a high Fe(III)-binding constant of P. cepacia siderophores.     

New synthetic siderophores and their beta-lactam conjugates based on diamino acids and dipeptides

Linking of siderophores to antibiotics improves the penetration and therefore increases the antibacterial activity of the antibiotics. We synthesized the acylated catecholates and hydroxamates as siderophore components for antibiotic conjugates to reduce side effects of unprotected catecholate and hydroxamate moieties. In this paper, we report on bis- and tris-catecholates and mixed catecholate hydroxamates based on diamino acids or dipeptides. These compounds were active as siderophores in a growth promotion assay under iron limitation. Most of the conjugates with beta-lactams showed high in vitro activity against Gram-negative bacteria especially Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Serratia marcescens and Stenotrophomonas maltophilia. The compounds with enhanced antibacterial activity use active iron uptake routes to penetrate the bacterial outer membrane barrier, demonstrated by assays with mutants deficient in components of the iron transport system. Correlation between chemical structure and biological activity was studied.     

Experience with epidemiologic studies of pyocyaneus infections. I. Feasibility of using serotypes and antibiotic resistance as a epidemiologic label for clinical strains]

The authors carried out serological typing of 98 Pseudomonas aeruginosa strains, isolated from patients of burn department of the Sklifosovsky First Aid Institute in January-July, 1974, and of 215 strains obtained from other sources; their sensitivity to 13 antibiotics was determined. Pseudomonas aeruginosa cultures isolated from the patients were typed with O-sera of 10 serological types. The presence of several hospital strains of Pseudomonas aeruginosa was found by means of serological typing; along with these there were revealed cultures of this causative agent sporadically appearing in the department. Sensitivity to some antibiotics could serve as an additional criterion for differentiation of Pseudomonas aeruginosa strains of the same serological type.     

Factors affecting catalase expression in Pseudomonas aeruginosa biofilms and planktonic cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 microM as FeCl(3)) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

PA-5 and PA-7, pentaene and heptaene macrolide antibiotics produced by a new isolate of Streptoverticillium from Spanish soil

Summary Two antifungal antibiotics, named PA-5 and PA-7 were produced by an actinomycete strain, characterized and identified as Streptoverticillium sp 43/16. These antibiotics were extracted from mycelial cake with methanol and purified using different organic solvents and LH-20 Sephadex column chromatography. PA-5 and PA-7 were characterized as, a pentaene and a heptaene macrolide antibiotic, respectively. These antibiotics exhibit strong antifungal activity against pathogen yeasts and molds.     

Antifungal Effects of Bacilysin and Fengymycin from Bacillus subtilis F-29-3 A Comparison with Activities of Other Bacillus Antibiotics

Of the two antifungal antibiotics produced by Bacillus subtilis F-29-3, the dipeptide compound bacilysin inhibits yeasts (and bacteria), whereas the formerly unknown fengymycin, a complex of closely related lipopeptide components, shows antibiotic activity against filamentous fungi. Bacilysin production, formerly known for a few strains only, could be demonstrated for all 12 wild-type cultures of Bacillus subtilis tested during this study. The antibiotic also occurs in some strains of three other Bacillus species considered as closely realted to B. subtilis. Members of the lipopeptide class of antifungal Bacillus metabolites were formed by 8 of 12 Bacillus subtilis-isolates and several other Bacillus strains. The antibiotics of F-29-3 were compared with antifungal metabolites of other Bacillus isolates using TLC, agar-diffusion techniques and tests demonstrating the capacity of six lipopeptide and peptide preparations to protect rice seedlings from phytomycosis due to Rhizoctonia solani. Fengymycin proved to be different from the other compounds tested. It was less toxic to the test plants and protected them better from Rhizoctonia disease than the other antibiotics of the study did.     

Plants respond to pathogen infection by enhancing the antifungal gene expression of root-associated bacteria

Plant health and fitness widely depend on interactions with soil microorganisms. Some bacteria such as pseudomonads can inhibit pathogens by producing antibiotics, and controlling these bacteria could help improve plant fitness. In the present study, we tested whether plants induce changes in the antifungal activity of root-associated bacteria as a response to root pathogens. We grew barley plants in a split-root system with one side of the root system challenged by the pathogen Pythium ultimum and the other side inoculated with the biocontrol strain Pseudomonas fluorescens CHA0. We used reporter genes to follow the expression of ribosomal RNA indicative of the metabolic state and of the gene phlA, required for production of 2,4-diacetylphloroglucinol, a key component of antifungal activity. Infection increased the expression of the antifungal gene phlA. No contact with the pathogen was required, indicating that barley influenced gene expression by the bacteria in a systemic way. This effect relied on increased exudation of diffusible molecules increasing phlA expression, suggesting that communication with rhizosphere bacteria is part of the pathogen response of plants. Tripartite interactions among plants, pathogens, and bacteria appear as a novel determinant of plant response to root pathogens.     

Factors Affecting Catalase Expression in Pseudomonas aeruginosa Biofilms and Planktonic Cells

Previous work with Pseudomonas aeruginosa showed that catalase activity in biofilms was significantly reduced relative to that in planktonic cells. To better understand biofilm physiology, we examined possible explanations for the differential expression of catalase in cells cultured in these two different conditions. For maximal catalase activity, biofilm cells required significantly more iron (25 μM as FeCl₃) in the medium, whereas planktonic cultures required no addition of iron. However, iron-stimulated catalase activity in biofilms was still only about one-third that in planktonic cells. Oxygen effects on catalase activity were also investigated. Nitrate-respiring planktonic cultures produced approximately twice as much catalase activity as aerobic cultures grown in the presence of nitrate; the nitrate stimulation effect could also be demonstrated in biofilms. Cultures fermenting arginine had reduced catalase levels; however, catalase repression was also observed in aerobic cultures grown in the presence of arginine. It was concluded that iron availability, but not oxygen availability, is a major factor affecting catalase expression in biofilms.