Ecology of a plant growth-promoting strain of Pseudomonas fluorescens colonizing the maize endorhizosphere in tropical soil

Pseudomonas fluorescens strain BR-5 stimulated the growth of maize in a natural soil and inhibited fungal root pathogens in vitro. Strain BR-5 was detected inside plant cells, indicating that it is able to colonize the endorhizosphere. No significant effect was detected on soil or ectorhizosphere microbial population after inoculation of strain BR-5 onto seeds.     

Phosphate solubilization and growth promotion by <Emphasis Type="Italic">Pseudomonas fragi</Emphasis> CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude Himalayan rhizosphere

Phosphate solubilization and growth promotion by Pseudomonas fragi CS11RH1 (MTCC 8984), a psychrotolerant bacterium isolated from a high altitude garlic rhizosphere from the Indian Himalayas, are reported here. The identity of the isolate was arrived on the basis of its biochemical features and sequencing of the 16S rRNA gene. The isolate grew and solubilized phosphate at temperatures ranging from 4 to 30°C. Besides solubilizing P it produced indole acetic acid (IAA) and hydrogen cyanide (HCN). Seed bacterization with the isolate significantly increased the percent germination, rate of germination, plant biomass and nutrient uptake of wheat seedlings. While Pseudomonas fragi is normally associated with the spoilage of dairy products stored at cold temperatures, this is an early report on the plant growth promoting ability of the bacterium.     

Genetics of phosphate solubilization and its potential applications for improving plant growth-promoting bacteria

Plant growth-promoting bacteria (PGPB) are soil and rhizosphere bacteria that can benefit plant growth by different mechanisms. The ability of some microorganisms to convert insoluble phosphorus (P) to an accessible form, like orthophosphate, is an important trait in a PGPB for increasing plant yields. In this mini-review, the isolation and characterization of genes involved in mineralization of organic P sources (by the action of enzymes acid phosphatases and phytases), as well as mineral phosphate solubilization, is reviewed. Preliminary results achieved in the engineering of bacterial strains for improving capacity for phosphate solubilization are presented, and application of this knowledge to improving agricultural inoculants is discussed.     

荧光假单胞菌生防机理的研究进展

荧光假单胞菌是植物根际促生细菌(Plant Growth Promoting Rhizobacteria,PGPR)具有分布广、数量多、营养需要简单、繁殖快、竞争定殖力强的特点。它们能通过产生多种次生代谢物及有效的根际定殖防治植物病害,成为植物生防控制的重要研究对象。主要论述了荧光假单胞菌对植物病害生物防治机理的研究进展。     

Factors determining rock phosphate solubilization by microorganisms isolated from soil

Forty two soil isolates (31 bacteria and 11 fungi) were studied for their ability to solubilize rock phosphate and calcium phosphate in culture medium. Eight bacteria and 8 fungi possessed solubilizing ability. Pseudomonas cepacia and Penicillium purpurogenum showed the highest activity. There was a correlation between final pH value and titratable acidity (r=–0.29 to –0.87) and between titratable acidity and soluble phosphate (r=0.22 to 0.99). Correlation values were functions of insoluble phosphate and of the group of microorganisms considered. A high correlation was observed between final pH and soluble phosphate only for the rock phosphates inoculated with the highest concentration of solubilizing bacteria (r=–0.73 to –0.98).     

Cyanide oxygenase and cyanase activities of Pseudomonas fluorescens NCIMB 11764

Abstract Pseudomonas fluorescens NCIMB 11764 is able to utilise cyanide (both KCN and Ni(CN)₄²⁻) as a nitrogen source for growth. Under such conditions cyanide oxygenase activity is induced. When potassium cyanate (KOCN) is supplied as the sole nitrogen source for growth, cyanase activity is induced. It has been demonstrated that these two enzymic activities are physiologically distinct, and are not co-induced under any of the growth conditions tested.     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

Microbial solubilization of rock phosphate on media containing agro-industrial wastes and effect of the resulting products on plant growth and P uptake

Four agro-industrial wastes were assayed as substrates for microbial solubilization of rock phosphate (RP). Sugar beet wastes (SB), olive cake (OC) and olive mill wastewaters (OMWW) were treated by Aspergillus niger, and dry olive cake (DOC) was treated by Phanerochaete chrysosporium. In conditions of solid-state fermentation 46% of SB and 21% of OC were mineralized by A. niger while 16% of DOC was mineralized by P. chrysosporium. Repeated-batch mode of fermentation was employed for treatment of OMWW by immobilized A. niger, which resulted in conversion of 80% of the fermentable sugars. Acidification of all media treated by A. niger was registered with a simultaneous solubilization of 59.7% (SB), 42.6% (OC), and 36.4% (OMWW) of the total P present in the RP. The same mechanism of RP solubilization was observed in DOC-based medium inoculated with P. chrysosporium but other mechanisms were probably involved during the process. A series of microcosm experiments were then performed in the greenhouse to evaluate the effectiveness of the resulting fermented products. All amendments improved plant growth and P acquisition, which were further enhanced by mycorrhizal inoculation. The level of all studied parameters including the root mycorrhizal colonization depended on the substrate characteristics. The reported biotechnological schemes offer a potential application particularly for degraded soils.     

Root colonization and iron nutritional status of a Pseudomonas fluorescens in different plant species

Root colonization and induction of an iron stress regulated promoter for siderophore production by Pseudomonas fluorescens 2-79RLI was studied in vitro and in the rhizosphere of different plant species. P. fluorescens 2-79RLI was previously genetically modified with an iron regulated ice nucleation reporter, which allowed calibration of ice nucleation activity with siderophore production. Initial experiments examined ice nucleation activity and siderophore production under different growth conditions in vitro. These studies demonstrated that P. fluorescens 2-79RLI could utilize both Fe-citrate and Fe-phytosiderophore as iron sources, suggesting that production of these compounds by plants would increase iron availability for P. fluorescens 2-79RLI in the rhizosphere. Fe demand and Fe stress were further shown to be a function of nutrient availability and were reduced when carbon was limiting for growth. Subsequent experiments extended these observations to rhizosphere cells. Cells were sampled from the rhizosphere and the rhizoplane. Results of a soil microcosm experiment showed that Fe stress was reduced for P. fluorescens 2-79RLI in the barley rhizosphere as compared to the cells in the rhizosphere.of lupin. In lupin, relative Fe stress of P. fluorescens 2-79RLI was greater at the root tip than in the lateral root zone. In a second experiment comparing zucchini and bean, iron stress was greater for P. fluorescens 2-79RLI associated with zucchini than with bean. In a third experiment with rape plants under P deficient conditions, addition of soluble P was shown to increase Fe stress for P. fluorescens 2-79RLI located at the root tip, but not in the lateral root zone. This study showed that Fe stress of P. fluorescens 2-79RLI in the rhizosphere may be influenced by plant species, P source, root zone and localization of the cells within the rhizosphere.     

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.     

Effect of introduced Pseudomonas fluorescens strains on the uptake of nitrogen by wheat from 15N-enriched organic residues

The effects of an antibiotic-producing Pseudomonas fluorescens strain (F113) carrying the marker gene cassette lacZY and a marked, non-producing strain (F113G22) on the uptake of nitrogen from ¹⁵N-enriched organic residues incorporated into a sandy soil were investigated in microcosm studies. Strain F113 produces the antibiotic 2,4-diacetylphloroglucinol (DAPG), whilst its modified derivative strain F113G22 has DAPG production deleted by Tn5 mutagenesis. Uptake of nitrogen by wheat (Triticum aestivum) from ¹⁵N-enriched organic residues was estimated using stable isotope-ratio mass spectrometry of shoot and root material of 17-day-old plants. In addition, plant growth and active microbial biomass in soil were monitored. In contrast to results obtained in our previous study on pea (Pisum sativum), it was found that in wheat, inoculation with either strain F113 or F113G22 decreased the proportion of nitrogen derived from ¹⁵N-labelled organic residues incorporated into soil as compared to non-inoculated controls. It is therefore suggested that these strains decreased mineralization of organic residues in the rhizosphere of wheat, making less inorganic N (¹⁵N) available for plant uptake. The results of this study indicate that the effects of introduced Pseudomonas fluorescens strains on nitrogen mineralization in the rhizosphere are plant-species dependent, and highlight the importance of testing microbial inocula on a range of plant species.     

Biological control of take-all disease by isolates of Pseudomonas fluorescens and biosynthesis of silver nanoparticles by the culture supernatant of Pseudomonas fluorescens CHA0

Plant diseases are among the main constraints affecting the production and productivity of crops both in terms of quality and quantity. Use of chemicals continues to be the major tactic to mitigate the menace of crop diseases. However, because of the environmental concerns, health conscious attitude of human beings and other hazards associated with the use of chemicals, use of bio agents to suppress the disease-causing activity of plant pathogens is gaining importance. With the emergence and increase of microbial organisms resistant to multiple antibiotics, and the continuing emphasis on health-care costs, many researchers have tried to develop new and effective antimicrobial reagents that do not stimulate resistance and are less expensive. Nanoscale materials have emerged as novel antimicrobial agents owing to their high surface area to volume ratio and the unique chemical and physical properties, which increases their contact with microbes and their ability to permeate cells. Since silver displays multiple modes of inhibitory action to micro-organisms, it may be used for controlling various plant pathogens in a relatively safer way compared to synthetic fungicides. Development of reliable and eco-friendly processes for synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. One of the options to achieve this objective is to use synthesis of nanoparticles of silver by reduction of aqueous Ag⁺ ions with the culture supernatant of Pseudomonas fluorescens CHA0. In this study, P. fluorescens CHA0 that has a medium impact on Gaeumannomyces graminis var. tritici was selected. Then, P. fluorescens CHA0 was used for the synthesis of silver nanoparticles. The morphology of the nanoparticles was characterised by Transmission Electron Microscopy and UV–vis spectroscopy. The silver nanoparticles of approximate size 50 nm were observed. The process of reduction is extracellular which makes it an easier method for the synthesis of silver nanoparticles.     

In vitro analyses are not reliable predictors of the plant growth promotion capability of bacteria; a Pseudomonas fluorescens strain that promotes the growth and yield of wheat

Aims: In this study, we set out to identify bacteria that can be used to promote the growth of cereals, while concurrently investigating the merits of using a range of such tests to preselect bacteria for glasshouse studies. Methods and Results: A panel of 15 strains isolated from the rhizosphere and phyllosphere of cereals was tested for the ability to improve the germination of wheat seeds and for production of a range of factors associated with plant growth promotion. In parallel, all bacteria were tested for their ability to improve biomass and grain yield when applied as a soil amendment in glasshouse trials. Conclusions: There was no significant correlation between growth promotion potential in the glasshouse and the results of either the phenotypic or the germination tests. Glasshouse tests identified that only one strain, Pseudomonas fluorescens strain MKB37, gave a significant increase in head weight and grain yield. Significance and Impact of the Study: While this study has identified a candidate for further field tests, it has also highlighted the fact that the modes of action for plant growth‐promoting bacteria (PGPB) are still not fully understood, and that there is no efficient and effective screening method for identifying PGPB by laboratory tests.     

Plasmid-mediated degradation of o- and p-phthalate by Pseudomonas fluorescens

A Pseudomonas fluorescens strain SKP3 capable of utilizing both phthalic acid and terephthalic acid as sole source of carbon and energy was isolated by enrichment technique. Phthalic acid, terephthalic acid and protocatechuic acid were easily oxidized by both phthalate-grown and glucose-grown cells without a lag period. Phthalic acid is metabolized through the ortho cleavage pathway and terephthalic acid through the meta cleavage pathway and the enzymes of the two pathways are constitutive in nature. A large plasmid of approximately 140kb in size was found to be involved in the degradation of phthalates. The catabolic plasmid pSKL was transferable to different hosts.     

Isolation and Characterization of a Stilbene-degrading Strain of Pseudomonas fluorescens,and Production of Antioxidant Compounds by Stilbene Metabolism

In this study, we consider the use of hydrocarbon-degrading bacteria that degrade trans-stilbene as a novel approach for synthesizing potentially bioactive hydroxylated stilbenes. A trans-stilbene-degrading bacterium, MN2, was isolated from activated sludge through enrichment culture, and identified as Pseudomonas fluorescens using conventional techniques. Degradation of trans-stilbene by this strain yielded two metabolites that had significant antioxidant activity.     

Pseudomonas fluorescens enhances resistance and natural enemy population in rice plants against leaffolder pest

Plant growth promoting fluorescent pseudomonad strains Pf1, TDK1 and PY15 were evaluated for their efficacy against leaffolder ( Cnaphalocrocis medinalis ) pest in rice plants under field conditions individually and in combinations. Application of mixtures of Pseudomonas fluorescens strains Pf1, TDK1 and PY15 significantly reduced the leaffolder damage in rice plants compared with untreated control. Interestingly, natural enemy population in plots treated with P. fluorescens was greater than the chemical and untreated controls. Further, support for these results was gathered by assaying activities of polyphenol oxidase (PPO) and lipoxygenase (LOX) under glasshouse conditions. The results showed the higher activity of PPO and LOX in plants treated with P. fluorescens mixtures (Pf1 + TDK1 + PY15) than the plants treated with individual strains, chemical and untreated controls. Further, fluorescent pseudomonad mixtures increased the rice yield compared with individual strain and non-bacterized treatments. The present study reveals that in addition to plant growth promotion, plant growth-promoting rhizobacterial (PGPR) strains-mediated induction of PPO and LOX in rice plants could be involved in enhanced natural enemy populations and resistance mechanisms against leaffolder attack.     

Rapid solubilization of insoluble phosphate by a novel environmental stress-tolerant Burkholderia vietnamiensis M6 isolated from ginseng rhizospheric soil

We isolated and characterized novel insoluble phosphate (P)-solubilizing bacteria tolerant to environmental factors like high salt, low and high pHs, and low temperature. A bacterium M6 was isolated from a ginseng rhizospheric soil and confirmed to belong to Burkholderia vietnamiensis by BIOLOG system and 16S rRNA gene analysis. The optimal cultural conditions for the solubilization of P were 2.5% (w/v) glucose, 0.015% (w/v) urea, and 0.4% (w/v) MgCl₂·6H₂O along with initial pH 7.0 at 35°C. High-performance liquid chromatography analysis showed that B. vietnamiensis M6 produced gluconic and 2-ketogluconic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with P solubilization. Insoluble P solubilization in the optimal medium was about 902 mg l⁻¹, which was approximately 1.6-fold higher than the yield in NBRIP medium (580 mg l⁻¹). B. vietnamiensis M6 showed resistance against different environmental stresses like 10–45°C, 1–5% (w/v) salt, and 2–11 pH range. The maximal concentration of soluble P produced by B. vietnamiensis M6 from Ca₃(PO₄)₂, CaHPO₄, and hydroxyapatite was 1,039, 2,132, and 1,754 mg l⁻¹, respectively. However, the strain M6 produced soluble P with 20 mg l⁻¹ from FePO₄ after 2 days and 100 mg l⁻¹ from AlPO₄ after 6 days, respectively. Our results indicate that B. vietnamiensis M6 could be a potential candidate for the development of biofertilizer applicable to environmentally stressed soil.     

Exocellular and intracellular accumulation of lead in Pseudomonas fluorescens ATCC 13525 is mediated by the phosphate content of the growth medium

Pseudomonas fluorescens appears to elicit disparate lead detoxification mechanisms in phosphate-rich and phosphate-deficient media. When grown in the presence of 0.1 mM Pb2+ complexed to citrate, the sole source of carbon, only a slight diminution in cellular yield was observed in the former medium. However, in a phosphate-deficient milieu, lead imposed approximately a 30% reduction in bacterial multiplication. At stationary phase of growth, 72% of the metal was found in the bacterial cells from the phosphate-deficient medium, while that from phosphate-rich broth contained only 12.5%. The latter medium was characterized by an insoluble pellet that accounted for 73.5% of the lead. Although no citrate was detected in the phosphate-rich media after 40 h of incubation, only 72% of citrate was consumed even after 70 h of growth in the phosphate-deficient cultures. The inclusion of lead did not appear to enhance the production of either extracellular proteins or carbohydrates.