Biological control of Tiarosporella phaseolina the causal agent of charcoal rot of soybean

Charcoal rot caused by Tiarosporella phaseolina (Tassi) Van der Aa is an important disease of soybean in Gorgan province of Iran. Experiments were carried out with 95 bactenal isolates that were collected from the rhizosphere of soybean plant. Among these bacteria only 50 isolates showed antagonistic effect on Tiarosporella phaseolina using dual culture test. Six highly effective bacteria were selected for subsequent studies. Based on biochemical physiological and morphological tests, isolates Pf-12 and Pf-63 were identified as Pseudomonas fluorescens, isolates B-13, B-42,B-126 and B-84 as Bacillus subtilis. The isolates of P. fluorescens produced antibiotics as well as volatile metabolites that inhibited mycelial growth of fungus. Bacillus subtilis isolates inhibited the fungal growth through volatile and non-volatile metabolites production. Only P. fluorescens isolates produced hydrogen cyanide. In greenhouse studies, the isolates B-13 and B-126 reduced 59% and 66% the intensity of charcoal rot of soybean respectively. The combinations of isolates B-13 and B-126 were also effective on reducing the intensity of disease.     

Isolation of fluorescent pseudomonads from the rhizosphere of banana plants antagonistic towards root necrosing fungi

Total aerobic bacteria and fluorescent pseudomonads were counted in bulk and rhizospheric soils of banana plants of 14 plantations in Martinique (French West Indies). Fluorescent Pseudomonas isolates were then identified and investigated for in vitro antagonism towards Cylindrocladium sp., a fungal pathogen of banana roots. Total aerobic bacteria and fluorescent pseudomonads were significantly more abundant in rhizospheric soils than in bulk soils. Among 58 fluorescent Pseudomonas isolates, 41 were identified as Pseudomonas fluorescens biovar V and 17 as Ps. putida biovar A. Six strains exhibited an antagonism towards Cylindrocladium isolates. Among them, Ps. putida strain 93.1 totally blocked fungal growth. No relationship was established between the antifungal effect and enzyme or hydrogen cyanide production by bacteria, suggesting that siderophores and other compounds were involved in fungal inhibition. Antagonistic fluorescent pseudomonads represent a potential for the biological control of banana root infections by Cylindrocladium sp.     

Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth

Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.     

A conserved mechanism for nitrile metabolism in bacteria and plants

Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that efficiently colonises the leaf surfaces and rhizosphere of a range of plants. Previous studies have identified a putative plant-induced nitrilase gene ( pinA ) in P. fluorescens SBW25 that is expressed in the rhizosphere of sugar beet plants. Nitrilase enzymes have been characterised in plants, bacteria and fungi and are thought to be important in detoxification of nitriles, utilisation of nitrogen and synthesis of plant hormones. We reveal that pinA is a NIT4-type nitrilase that catalyses the hydrolysis of β-cyano- l -alanine, a nitrile common in the plant environment and an intermediate in the cyanide detoxification pathway in plants. In plants cyanide is converted to β-cyano- l -alanine, which is subsequently detoxified to aspartic acid and ammonia by NIT4. In P. fluorescens SBW25 pinA is induced in the presence of β-cyano- l -alanine, and the β-cyano- l -alanine precursors cyanide and cysteine. pinA allows P. fluorescens SBW25 to use β-cyano- l -alanine as a nitrogen source and to tolerate toxic concentrations of this nitrile. In addition, pinA is shown to complement a NIT4 mutation in Arabidopsis thaliana , enabling plants to grow in concentrations of β-cyano- l -alanine that would otherwise prove lethal. Interestingly, over-expression of pinA in wild-type A. thaliana not only resulted in increased growth in high concentrations of β-cyano- l -alanine, but also resulted in increased root elongation in the absence of exogenous β-cyano- l -alanine, demonstrating that β-cyano- l -alanine nitrilase activity can have a significant effect on root physiology and root development.     

Effect of Respiratory Inhibitors on the Motility of Pseudomonas fluorescens

The translational motility of Pseudomonas fluorescens was weakly inhibited by oligomycin, Dicumarol, 2,4-dinitrophenol, 2n-heptyl-4-hydroxyquinoline N-oxide, and potassium cyanide. Atabrine and antimycin A together with potassium cyanide immediately immobilized this bacterium, but antimycin A alone was without effect. Gramicidin D also immobilized P. fluorescens, but its action was inhibited by K(+) and NH(4) (+) ions. In like manner, the effect of p-chloromercuribenzoate could be counteracted with cysteine, thereby suggesting the involvement of -SH groups in flagellar motility processes. It appears that the energy required for motility of P. fluorescens is generated by oxidative phosphorylation mediated by the cytochrome system.     

Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid

The arylacetonitrilase of Pseudomonas fluorescens EBC191 catalyzes the conversion of (S)-mandelonitrile to (S)-mandelic acid and (S)-mandeloamide. This biotransformation is optimally performed under acidic pH values because (S)-mandelonitrile rapidly decomposes under neutral conditions. Therefore, the gene encoding the arylacetonitrilase of P. fluorescens EBC191 was integrated and expressed under the control of the AOX1 promoter in the methylotrophic yeast Pichia pastoris which was supposed to act as an acidotolerant expression system. These recombinant strains hydrolyzed (R,S)-mandelonitrile at pH values >or=3 to mandelic acid and mandeloamide and were more acidotolerant than previously constructed Escherichia coli whole cell catalysts synthesizing the same nitrilase activity. Subsequently, recombinant P. pastoris strains were constructed which simultaneously expressed the (S)-oxynitrilase of Manihot esculenta and the arylacetonitrilase of P. fluorescens EBC191 each under the control of individual AOX1 promoters in order to obtain a whole cell catalyst for the synthesis of (S)-mandelic acid from benzaldehyde and cyanide. Resting cells of the recombinant strains converted under acidic conditions benzaldehyde and cyanide initially to mandelonitrile which was immediately converted to mandelic acid and mandeloamide. The chiral analysis of the products formed revealed a high enantiomeric excess for the (S)-enantiomers.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa.

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Cyanide production by Pseudomonas fluorescens and Pseudomonas aeruginosa

Of 200 water isolates screened, five strains of Pseudomonas fluorescens and one strain of Pseudomonas aeruginosa were cyanogenic. Maximum cyanogenesis by two strains of P. fluorescens in a defined growth medium occurred at 25 to 30 degrees C over a pH range of 6.6 to 8.9. Cyanide production per cell was optimum at 300 mM phosphate. A linear relationship was observed between cyanogenesis and the log of iron concentration over a range of 3 to 300 microM. The maximum rate of cyanide production occurred during the transition from exponential to stationary growth phase. Radioactive tracer experiments with [1-14C]glycine and [2-14C]glycine demonstrated that the cyanide carbon originates from the number 2 carbon of glycine for both P. fluorescens and P. aeruginosa. Cyanide production was not observed in raw industrial wastewater or in sterile wastewater inoculated with pure cultures of cyanogenic Pseudomonas strains. Cyanide was produced when wastewater was amended by the addition of components of the defined growth medium.     

Uptake and active efflux of polycyclic aromatic hydrocarbons by Pseudomonas fluorescens LP6a

The mechanism of transport of polycyclic aromatic hydrocarbons (PAHs) by Pseudomonas fluorescens LP6a, a PAH-degrading bacterium, was studied by inhibiting membrane transport and measuring the resulting change in cellular uptake. Three cultures were used: wild-type LP6a which carried a plasmid for PAH degradation, a transposon mutant lacking the first enzyme in the pathway for PAH degradation, and a cured strain without the plasmid. Washed cells were mixed with aqueous solutions of radiolabelled PAH; then the cells were removed by centrifugation, and the concentrations of PAH in the supernatant and the cell pellet were measured. The change in the pellet and supernatant concentrations after inhibitors of membrane transport (azide, cyanide, or carbonyl cyanide m-chlorophenyl hydrazone) were added indicated the role of active transport. The data were consistent with the presence of two conflicting transport mechanisms: uptake by passive diffusion and an energy-driven efflux system to transport PAHs out of the cell. The efflux mechanism was chromosomally encoded. Under the test conditions used, neither uptake nor efflux of phenanthrene by P. fluorescens LP6a was saturated. The efflux mechanism showed selectivity since phenanthrene, anthracene, and fluoranthene were transported out of the cell but naphthalene was not.     

Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion.

The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion.

The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of rhizosphere bacteria with potential for biological control of weeds in vineyards

AIMS: Deleterious rhizosphere inhabiting bacteria (DRB) have potential to suppress plant growth. This project focuses on the isolation of DRB with potential for development as commercial products for weed control. METHODS AND RESULTS: Bacteria were isolated from the rhizosphere, rhizoplane, and endorhizosphere of seedlings and mature plants of wild radish (Raphanus raphanistrum), annual ryegrass (Lolium rigidum) and capeweed (Arctotheca calendula) growing in vineyards in the Swan Valley, Western Australia. A majority (81.5%) of the 442 strains was obtained from either rhizospheres or rhizoplanes. Rapid screening techniques were developed to evaluate in the laboratory and glasshouse the effects of bacteria on plants. Strains were screened in the glasshouse for deleterious effects on annual ryegrass, wild radish, grapevine rootlings (Vitis vinifera) and the legume cover crop subterranean clover (Trifolium subterraneum). Three strains were identified using the Biolog system and 16S rRNA gene sequencing as two strains of Pseudomonas fluorescens (WSM3455 and WSM3456) and one strain of Alcaligenes xylosoxidans (WSM3457). One of the P. fluorescens (WSM3455) strain produced hydrogen cyanide, an inhibitor of plant roots and a broad-spectrum antimicrobial compound. CONCLUSIONS: Three strains specifically inhibited wild radish but had no significant deleterious effects on either grapevine rootlings or subterranean clover. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggested manipulation of the weed seedling rhizosphere using identified DRB as a potential biocontrol agent for wild radish.     

Assessment of the role of a terrestrial isopod in the survival of a genetically modified pseudomonad and its detection using the polymerase chain reaction

Abstract The effects of a terrestrial isopod, Porcellio scaber , on the survival of a genetically modified pseudomonad were studied. Pseudomonas fluorescens KTG was inoculated onto ash leaf litter and supplied to populations of P. scaber . Plate counts were lower in fresh faeces than the ash leaf litter for P. fluorescens KTG, and higher counts were detected in the faeces for the total bacterial population. When faeces were aged by incubation for up to 7 days at 15–17°C, plate counts for P. fluorescens KTG increased during the first day to a level similar to those in the corresponding ash leaf litter, and remained relatively constant thereafter. The total bacterial population in the faeces continued to increase steadily over the 7 days, whilst remaining at a constant level in the ash leaf litter during the same period. Counts of bacteria in faecal material showed that P. fluorescens KTG was present for 6 days after the isopods had fed on inoculated litter although transit times of food through the gut were as little as 5 h. The implications for GEMMO dispersal of bacterial retention in the gut is considered. The polymerase chain reaction was utilised in the detection of the inserted DNA. Positive amplification of the inserted DNA sequence of P. fluorescens KTG was achieved in ash leaf litter, fresh faeces, and faeces from animal which were supplied uninoculated litter for one day after feeding on the inoculated litter. However, plate counts were more sensitive than the polymerase chain reaction in detecting P. fluorescens KTG in the faeces. Our findings suggest that when the GEMMO is ingested by the woodlouse it can survive within the guts and faeces. This has implications for risk assessment of genetically modified bacteria in terrestrial environments.     

Evidence that bacterial cyanide oxygenase is a pterin-dependent hydroxylase

The soluble cell-free fraction (150,000g high-speed supernatants [HSS]) of Pseudomonas fluorescens NCIMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O(2) or NADH uptake, or by conversion of radioactive K(14)CN to (14)CO(2), was detected at micromolar concentrations (apparent half-saturation constant, 4 microM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (<500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.     

Production of Hydrogen Cyanide by Pseudomonas flurescens

Two Pseudomonas fluorescens isolates were found to produce hydrogen cyanide when cultured on either Trypticase soy agar supplemented with 0.5% yeast extract or on irradiation-sterilized chicken.     

Colonizing ability of Pseudomonas fluorescens 2112, among collections of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens spp. in pea rhizosphere

Pseudomonas fluorescens 2112, isolated in Korea as an indigenous antagonistic bacteria, can produce 2,4- diacetylphloroglucinol (2,4-DAPG) and the siderophore pyoveridin2112 for the control of phytophthora blight of red-pepper. P. fluorescens 2112 was classified into a new genotype C among the 17 genotypes of 2,4-DAPG producers, by phlD restriction fragment length polymorphism (RFLP). The colonizing ability of P. fluorescens 2112 in pea rhizosphere was equal to the well-known pea colonizers, P. fluorescens Q8r1 (genotype D) and MVP1-4 (genotype P), after 6 cycling cultivations for 18 weeks. Four tested 2,4- DAPG-producing Pseudomonas spp. could colonize with about a 96% dominance ratio against total bacteria in pea rhizosphere. The strain P. fluorescens 2112 was as good a colonizer as other Pseudomonas spp. genotypes in pea plant growth-promoting rhizobacteria.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.