Alginate beads as a storage, delivery and containment system for genetically modified PCB degrader and PCB biosensor derivatives of Pseudomonas fluorescens F113

Aims: Pseudomonas fluorescens F113Rifpcb is a genetically engineered rhizosphere bacterium with the potential to degrade polychlorinated biphenyls (PCBs). F113Rifpcbgfp and F113L::1180gfp are biosensor strains capable of detecting PCB bioavailability and biodegradation. The aim of this paper is to evaluate the use of alginate beads as a storage, delivery and containment system for use of these strains in PCB contaminated soils. Methods and Results: The survival and release of Ps. fluorescens F113Rifpcb from alginate beads were evaluated. Two Ps. fluorescens F113‐based biosensor strains were encapsulated, and their ability to detect 3‐chlorobenzoate (3‐CBA) and 3‐chlorobiphenyl (3‐CBP) degradation in soil was assessed. After 250 days of storage, 100% recovery of viable F113Rifpcb cells was possible. Amendments to the alginate formulation allowed for the timed release of the inoculant. Encapsulation of the F113Rifpcb cells provided a more targeted approach for the inoculation of plants and resulted in lower inoculum populations in the bulk soil, which may reduce the risk of unintentional spread of these genetically modified micro‐organisms in the environment. Encapsulation of the biosensor strains in alginate beads did not interfere with their ability to detect either 3‐CBA or 3‐CBP degradation. In fact, detection of 3‐CBP degradation was enhanced in encapsulated biosensors. Conclusions: Alginate beads are an effective storage and delivery system for PCB degrading inocula and biosensors. Significance and Impact of the Study: Pseudomonas fluorescens F113Rifpcb and the F113 derivative PCB biosensor strains have excellent potential for detecting and bioremediation of PCB contaminated soils. The alginate bead delivery system could facilitate the application of these strains as biosensors.     

Effects of the interactions between glutaraldehyde and the polymeric matrix on the efficacy of the biocide against pseudomonas fluorescens biofilms

Glutaraldehyde (GTA) is a widely used biocide due to its high effectiveness. The experimental work reported here was carried out to assess the effectiveness of GTA in controlling biofilms formed by Pseudomonas fluorescens on stainless steel slides, and to compare efficacy against both planktonic and sessile micro‐rganisms. The tests were performed using two concentrations of GTA (50 and 100mg 1^‐1), biofilms of two ages (7 and 15 d), several pH values (5,7 and 9) and a range of exposure times (from 0 (control) to 1,3,7 and 24 h). The action of GTA on biofilm and planktonic populations was assessed by means of activity tests, zeta potential, and the wet weight of the biofilms. Biofilms were not completely removed after treatment with GTA in any of the conditions studied. The higher GTA concentration was more effective in reducing the bacterial activity of the biofilm. The biocide proved to be more effective for longer exposure times. GTA showed good antimicrobial activity against P. fluorescens in suspension, with higher activity at pH 9. The findings of this study suggest that when GTA is used to control biofilms, it reacts with one of the components of the matrix, the proteins, thereby reducing its antimicrobial action.     

耐低温荧光假单胞菌筛选体系建立及其植物促生作用评价

【目的】以前期研究小麦生境分离细菌为菌株库,从中筛选耐低温菌株,评价其植物促生效果。【方法】通过4°C低温培养筛选耐低温菌株。以产吲哚乙酸、1-氨基环丙烷羧酸(ACC)脱氨酶、嗜铁素以及无机、有机磷溶解能力为评价指标,测定15°C和28°C环境中耐低温菌株的植物促生作用潜力,利用赋分评估系统选定潜力菌株进行温室和田间试验验证其植物促生作用。【结果】筛选到34株耐低温菌株,从中选定的8株植物促生潜力细菌温室试验促生效果和评估分值之间的相关性在0.62以上,菌株1b YB22和3b JN2在28°C环境中对黄瓜的促生效果均在28%以上,二者在15°C环境中对青菜种子根长的促生效果分别为116.76%和46.82%,菌株1b YB22在15°C环境中对青菜的促生效果为25.11%。菌株1b YB22在田间应用对小麦生长同样具有促生作用,增产效果达0.58%。【结论】研究建立的筛选体系从小麦生境分离菌株中获得了耐低温荧光假单胞菌株1b YB22和3b JN2,两株细菌在15°C和28°C均具有植物促生作用,同时可以在田间促进小麦生长。     

Methods for assaying cyanide in bacterial culture supernatant

AIMS: To find an easy, rapid and direct method for the quantitation of cyanide in a moderate number of bacterial culture supernatants. METHODS AND RESULTS: Culture supernatant from stationary phase cultures of Pseudomonas aeruginosa, grown in LB media, were analysed for cyanide content using the Merckoquant and Spectroquant cyanide detection kits as well as a cyanide ion-selective electrode (ISE) and a cyanide micro-ISE. The Merckoquant kit, designed for detection of low quantities of cyanide in water systems, proved not to be sufficiently reliable, providing poor comparison with previous assessments of cyanide levels in Ps. aeruginosa. The Spectroquant kit, and the two ISEs all provided very similar results, in agreement with previous data; however, it was the ISEs that fulfilled all the criteria for a rapid, direct test in a moderate number of samples. CONCLUSIONS: Cyanide ISEs can be used for easy assessment of the cyanide quantity in cultures grown in LB medium. Significance and Impact of the Study: The use of a cyanide ISE allows for an easy, direct and reproducible method for assaying cyanide in bacterial culture supernatant, which is of significant advantage over the currently accepted methods. This is especially important in an era of high-output genomic studies for assessing the phenotypic significance of data relating to the cyanide synthetic genes.     

Stable phylloplane colonization by entomopathogenic bacterium Pseudomonas fluorescens KPM-018P and biological control of Phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae)

An entomopathogenic bacterium was isolated from tomato leaves and used as a microbial agent to control larvae of phytophagous ladybird beetles Epilachna vigintioctopunctata. The isolate was identified as Pseudomonas fluorescens KPM-018P on the basis of its bacteriological characteristics. KPM-018P produced extracellular chitinase to form a transparent zone around their colonies by hydrolyzing chitin in a minimal medium. Pale-yellow colonies turned red after a change of incubation temperature. These characteristics were availed as markers for tracking KPM-018P. The bacteria produced biosurfactants that enabled the bacteria to stably colonize the hydrophobic leaf surface; they were recovered without any considerable decrease even after a suspension of KPM-018P was sprayed onto leaves. KPM-018P, transformed with the gfp gene and observed with fluorescence microscopy, stably dwelled in the junctions of epidermal cells of bacteria-sprayed leaves. Ingestion of KPM-018P-sprayed leaves by the larvae caused prompt death of these insects to eventually suppress their pupation. This method is thus effective for decreasing the population of larvae and adult insect pests in the subsequent generation. The study provides an experimental basis for the biocontrol of herbivorous insect pests using a leaf-inhabiting, entomopathogenic strain of P. fluorescens.     

Use of Pseudomonas fluorescens-based formulations for management of tomato spotted wilt virus (TSWV) and enhanced yield in tomato

Strains of Pseudomonas fluorescens were investigated for biocontrol efficacy against tomato spotted wilt virus (TSWV) in tomato both alone and in mixtures. P. fluorescens strains applied to seed, soil and foliage or as a seedling dip significantly reduced TSWV, with a concomitant increase in growth promotion in both the glasshouse and field. Two native strains (CoP-1 and CoT-1) and one foreign strain (CHAO) reduced TSWV. In P. fluorescens-treated tomato plants, increased activity of polyphenol oxidase, β-1,3-glucanase and chitinase was observed, and induction of chitinase was confirmed by western blot analysis. Induction of new protein (18 kDa) detected by SDS-PAGE in P. fluorescens-treated tomato plants was not found in healthy and P. fluorescens-untreated virus inoculated control plants. Indirect ELISA clearly showed a reduction in viral antigen concentration in P. fluorescens-treated tomato plants corresponding to reduced disease ratings. All the P. fluorescens-treated tomato plants also showed enhanced growth and yield compared to control plants. Hence, plant growth promoting rhizobacteria (PGPR) could play a major role in reducing TSWV and increasing yield in tomato plants.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Verringerung von frostschaden an obstblüten und maispflanzen durch bakterizide

Blüten von Pfirsich, Pflaume, Sauerkirsche, Apfel und Birne sowie im Freiland herangezogene Maispflanzen wurden nach Inokulation mit Ice ⁺ Pseudomonas fluorescens, Isolat 533, mit Antinukleationsagenzien unterschiedlicher chemischer Zusammensetzung tropfnaß besprüht und künstlichen Frostbelastungen bis ‐3,5 °C ausgesetzt. Der Anteil der überlebenden Blüten bzw. Maisblätter von jeder Variante wurde ausgezählt und in Prozent von der gesamten Zahl getesteter Blüten bzw. Maisblätter als Frostschaden dargestellt.

Die Antinukleationsagenzien verringerten an Maispflanzen den Frostschaden signifikant im Vergleich zu den inokulierten Pflanzen. Kocide 101 sowie das quartäre Ammoniumsalz 6151 zeigten sogar einen geringeren Schaden gegenüber der unbehandelten Kontrolle.

An den Obstblüten ergaben die Mittelapplikationen widersprüchliche Resultate. Der Anteil erfrorener Blüten variierte sowohl innerhalb der Versuchsvarianten als auch zwischen den Obstarten.

Die Annahme vieler Autoren, daß die primäre Quelle der Eisbildung in Obstgehölzen, einschließlich der anhaftenden Blüten, innere mit dem Holz assoziierte, jedoch abiotische Eiskeime sind, wurde diskutiert. An krautigen Pflanzen, wie Mais, kann dagegen der Frostschaden durch die Anwendung von Bakeriziden verringert werden.     

The influence of mineral and carbon sources on biological control of charcoal rot fungus,Macrophomina phaseolina by fluorescent pseudomonads in tomato

AIMS: To determine the influence of various trace minerals and carbon source on the biocontrol performance of Pseudomonas aeruginosa strain IE-6S+ and P. fluorescens strain CHA0 against Macrophomina phaseolina. METHODS AND RESULTS: In dual culture plate assay, P. aeruginosa IE-6S+ and P. fluorescens CHA0 inhibited radial growth of M. phaseolina producing zones of inhibition. Czapek's dox agar medium amended with both zinc and glucose remarkably improved antifungal activities of the bacterial inoculants. Under glasshouse conditions, soil amendment with zinc and/or glucose alone did not reduce M. phaseolina infection in tomato roots but did reduce significantly when used in combination with IE-6S+ or CHA0. Soil amendments with zinc and/or glucose increased fresh shoot weights but zinc amendment greatly reduced bacterial populations in the rhizosphere. CONCLUSIONS: Mineral and carbon amendments enhance the biocontrol performance of fluorescent pseudomonads against M. phaseolina. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of mineral and carbon amendments that favour biocontrol of certain bacterial strains may provide clues to soil factors or components of nutrient solutions in hydroponic culture that will improve the level and reliability of control.     

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.