Zinc and glycerol enhance the production of nematicidal compounds in vitro and improve the biocontrol of Meloidogyne javanica in tomato by fluorescent pseudomonads

AIMS: To assess the effects of various carbon and mineral sources on the nematicidal potential of biocontrol inoculants of Pseudomonas aeruginosa IE-6S+ and Ps. fluorescens CHA0 under laboratory and glasshouse conditions. METHODS AND RESULTS: Culture filtrates of strains IE-6S+ and CHA0, cultured in nutrient yeast extract broth, caused substantial mortality of the juveniles of Meloidogyne javanica. The nematicidal activities of the culture filtrates were altered after amendment with various carbon and mineral sources. Soil amendment with zinc alone or in combination with glycerol improved the biocontrol efficacy against root-knot nematode, promoted tomato plant growth and enhanced bacterial rhizosphere and endophytic colonization. CONCLUSIONS: Appropriate quantities of glycerol and zinc alone or in combination enhance the nematicidal activity of Ps. aeruginosa and Ps. fluorescens. Glucose reduces the activity of these bacteria against nematodes. SIGNIFICANCE AND IMPACT OF THE STUDY: Minerals and carbon sources are appealing because they are easy and economical to provide during liquid fermentation of inoculants or as fertilizer amendments to improve the biocontrol activity of indigenous and introduced bacteria.     

Non-pathogenic Fusarium solani represses the biosynthesis of nematicidal compounds in vitro and reduces the biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: The aim of the present investigation was to determine the influence of various Fusarium solani strains on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424. METHODS AND RESULTS: Culture filtrates (CF) of P. fluorescens strain CHA0 and its diacetylphloroglucinol-overproducing derivative CHA0/pME3424 caused substantial mortality of M. javanica juveniles in vitro. Bacterial growth medium amended with the growth medium of F. solani repressed the nematicidal activity of the bacteria. Methanol extract of F. solani CF resulting from Czapek's Dox liquid (CDL) medium without zinc amendment repressed the nematicidal activity of the bacteria while the CF obtained from CDL medium amended with zinc did not. Conidial suspension of F. solani strain Fs5 (repressor strain for the biosynthesis of nematicidal compounds in P. fluorescens) reduced biocontrol potential of the bacterial inoculants against M. javanica in tomato while strain Fs3 (non-repressor) did not. CONCLUSIONS: Fusarium solani strains with increased nematicidal activity repress the biosynthesis of nematicidal compounds by P. fluorescens strains in vitro and greatly alter its biocontrol efficacy against root-knot nematode under natural conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Fusarium solani strains are distributed worldwide and found in almost all the agricultural fields which suggest that some mycotoxin-producing strains will also be found in almost any soil sample taken. Besides the suppressive effect of these metabolite-producing strains on the production of nematicidal compound(s) critical in biocontrol, F. solani strains may also affect the performance of mycotoxin-sensitive biocontrol bacteria effective against plant-parasitic nematodes.     

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.     

Differential impact of some Aspergillus species on Meloidogyne javanica biocontrol by Pseudomonas fluorescens strain CHA0

AIMS: The aim was to determine the influence of some Aspergillus species on the production of nematicidal agent(s) in vitro and biocontrol of Meloidogyne javanica in tomato by Pseudomonas fluorescens strains CHA0 and CHA0/pME3424. METHODS AND RESULTS: Six species of Aspergillus, isolated from the rhizosphere of certain crops, produced a variety of secondary metabolites in vitro. Culture filtrate (CF) obtained from Ps. fluorescens strain CHA0 and its2,4-diacetylphloroglucinol overproducing mutant CHA0/pME3424 grown in King's B liquid medium caused significant mortality of M. javanica juveniles in vitro. Bacterial growth medium amended with CF of A. niger enhanced nematicidal and beta-galactosidase activities of fluorescent pseudomonads while A. quadrilineatus repressed such activities. Methanol or ethyl acetate extracts of the CF of A. niger markedly optimized bacterial efficacy to cause nematode deaths while hexane extract of the fungus had no influence on the nematicidal activity of the bacterial strains. A. niger applied alone or in conjunction with the bacterial inoculants inhibited root-knot nematode galling in tomato. On the other hand, A. quadrilineatus used alone or together with CHA0 did not inhibit nematode galling but when used in combination with strain CHA0/pME3424 did reduce galling intensity. CONCLUSIONS: Aspergillus niger enhances the production of nematicidal compounds by Ps. fluorescensin vitro and improves biocontrol potential of the bacterial inoculants in tomato while A. quadrilineatus reduces bacterial performance to suppress root-knot nematodes. SIGNIFICANCE AND IMPACT OF THE STUDY: Rhizosphere harbours a variety of micro-organisms including bacteria, fungi and viruses. Aspergillus species are ubiquitous in most agricultural soils and generally produce a variety of secondary metabolites. Such metabolites synthesized by Aspergillus species may influence the production of nematicidal agents and subsequent biocontrol performance of the bacterial inoculants against plant-parasitic nematodes. This fact needs to be taken into consideration when using biocontrol strains in an agriculture system.     

Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato

AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica. METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T. harzianum strain Th6 to P. fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro. In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T. harzianum. Pseudomonas fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots. CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P. fluorescens both in vitro and under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T. harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes. Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T. harzianum and P. fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.     

Phenylacetic acid-producing Rhizoctonia solani represses the biosynthesis of nematicidal compounds in vitro and influences biocontrol of Meloidogyne incognita in tomato by Pseudomonas fluorescens strain CHA0 and its GM derivatives

AIMS: The aim of the present investigation was to determine the influence of Rhizoctonia solani and its pathogenicity factor on the production of nematicidal agent(s) by Pseudomonas fluorescens strain CHA0 and its GM derivatives in vitro and nematode biocontrol potential by bacterial inoculants in tomato. METHODS AND RESULTS: One (Rs7) of the nine R. solani isolates from infected tomato roots inhibited seedling emergence and caused root rot in tomato. Thin layer chromatography revealed that culture filtrates of two isolates (Rs3 and Rs7) produced brown spots at Rf-values closely similar to synthetic phenylacetic acid (PAA), a phytotoxic factor. Filtrates from isolate Rs7, amended with the growth medium of P. fluorescens, markedly repressed nematicidal activity and PhlA'-'LacZ reporter gene expression of the bacteria in vitro. On the contrary, isolate Rs4 enhanced nematicidal potential of a 2,4-diacetylphloroglucinol overproducing mutant, CHA0/pME3424, of P. fluorescens strain CHA0 in vitro. Therefore, R. solani isolates Rs4 and Rs7 were tested more rigorously for their potential to influence biocontrol effectiveness of the bacterial agents. Methanol extract of the culture filtrates of PAA-producing isolate Rs7 resulting from medium amended with phenylalanine enhanced fungal repression of the production of nematicidal agents by bacteria, while amendments with zinc or molybdenum eliminated such fungal repression, thereby restoring bacterial potential to cause nematode mortality in vitro. A pot experiment was carried out, 3-week-old tomato seedlings were infested with R. solani isolates Rs4 or Rs7 and/or inoculated with Meloidogyne incognita, the root-knot nematode. The infested soil was treated with aqueous cell suspensions (10(8) CFU) of P. fluorescens strain CHA0 or its GM derivatives or left untreated (as a control). Observations taken 45 days after nematode inoculation revealed that, irrespective of the bacterial treatments, galling intensity per gram of fresh tomato roots was markedly higher in soil amended with isolate Rs4 than in Rs7-amended soils. Soil amendments with R. solani and the bacterial antagonists resulted in substantial reductions of the number of galls per gram of root. These results are contradictory to those obtained under in vitro conditions where culture filtrates of PAA-positive Rs7 repressed the production of nematicidal compounds. Plants grown in Rs7-amended soils, with or without bacterial inoculants, had lesser shoot and root weights than plants grown in nonamended or Rs4-amended soils. Moreover, amendments with Rs7 substantially retarded root growth and produced necrotic lesions that reduced the number of entry sites for invasion and subsequent infection by nematodes. Populations of P. fluorescens in the tomato rhizosphere were markedly higher in Rs7-amended soils. CONCLUSIONS: PAA-producing virulent R. solani drastically affects the potential of P. fluorescens to cause death of M. incognita juveniles in vitro and influences bacterial effectiveness to suppress nematodes in tomato roots. SIGNIFICANCE AND IMPACT OF THE STUDY: As most agricultural soils are infested with root-infecting fungi, including R. solani, it is likely that some PAA-producing isolates of the fungus may also be isolated from such soils. The inhibitory effect of PAA-producing R. solani on the biosynthesis of nematicidal agent(s) critical in biocontrol may reduce or even eliminate the effectiveness of fluorescent pseudomonads against root-knot nematodes, both in nursery beds and in field conditions. Introduction of bacterial inoculants, for the control of any plant pathogen, should be avoided in soils infested with PAA-producing R. solani. Alternatively, the agents could be applied together with an appropriate quantity of fungicide or chemicals such as zinc to create an environment more favourable for bacterial biocontrol action.     

Improvement of Pseudomonas fluorescens CHA0 biocontrol activity against root-knot nematode by the addition of ammonium molybdate

AIMS: To improve the efficacy of Pseudomonas fluorescens CHA0 and its genetically modified (GM) derivatives by adding ammonium molybdate to control Meloidogyne javanica, the root-knot nematode in mungbean. METHODS AND RESULTS: Culture filtrate of P. fluorescens CHA0 and its GM derivative (antibiotic overproducing strain CHA0/pME3424 and antibiotic-deficient CHA89) obtained from nutrient broth yeast extract medium amended with 1, 2 or 4 mm of ammonium molybdate (NH4-Mo) caused substantial mortality of M. javanica juveniles in vitro. Pseudomonas fluorescens CHA0 or CHA0/pME3424 applied in conjunction with NH4-Mo caused greater reduction of nematode penetration in mungbean roots compared with the bacterial application alone. Ammonium molybdate at 4 mg kg-1 of soil along with CHA0 also enhanced plant height while shoot weight remained unaffected. Either used alone or in conjunction with NH4-Mo, strain CHA89 did not reduce nematode invasion compared with the controls. Bacterial strains did not differ significantly in their colonization potential in the mungbean rhizosphere. Efficacy of the biocontrol bacteria to control root-knot nematode was accentuated when soil was treated with NH4-Mo and zinc (both at 1 mg kg-1 of soil). CONCLUSION: The addition of ammonium molybdate enhances the production of nematicidal compounds by P. fluorescensin vitro and improves bacterial efficacy against root-knot nematode under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Application of minerals such as ammonium molybdate is appealing because they are cheap and can easily be applied under field conditions to improve biocontrol potential of the bacterial inoculants. They also significantly reduce the amount of biocontrol inoculant biomass required to achieve root-knot disease control, with a consequent reduction in cost.     

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.     

Construction of a Pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.

A bacterium, Pseudomonas sp. strain C1S1, able to grow on 2,4,6-trinitrotoluene (TNT), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as N sources, was isolated. The bacterium grew at 30 degrees C with fructose as a C source and accumulated nitrite. Through batch culture enrichment, we isolated a derivative strain, called Pseudomonas sp. clone A, which grew faster on TNT and did not accumulate nitrite in the culture medium. Use of TNT by these two strains as an N source involved the successive removal of nitro groups to yield 2,4- and 2,6-dinitrotoluene, 2-nitrotoluene, and toluene. Transfer of the Pseudomonas putida TOL plasmid pWW0-Km to Pseudomonas sp. clone A allowed the transconjugant bacteria to grow on TNT as the sole C and N source. All bacteria in this study, in addition to removing nitro groups from TNT, reduced nitro groups on the aromatic ring via hydroxylamine to amino derivatives. Azoxy dimers probably resulting from the condensation of partially reduced TNT derivatives were also found.     

Effect of substrate particle size and additional nitrogen source on production of lignocellulolytic enzymes by Pleurotus ostreatus strains

Two strains of Pleurotus ostreatus (IE-8 and CP-50) were grown on defined medium added with wheat straw extract (WSE). Mycelia from these cultures were used as an inoculum for solid fermentation using sugar cane bagasse (C:N=142). This substrate was used separately either as a mixture of heterogeneous particle sizes (average size 2.9 mm) or as batches with two different particle sizes (0.92 mm and 1.68 mm). Protein enrichment and production of lignocellulolytic enzymes on each particle size was compared. The effect of ammonium sulphate (AS) addition was also analyzed (modified C:N=20), this compound favored higher levels of protein content. Strain CP-50 showed the highest increase of protein content (48% on particle size of 1.68 mm) when compared to media with no additional N source. However, strain IE-8 produced the highest levels of all enzymes: xylanases (5.79 IU/g dry wt on heterogeneous particles) and cellulases (0.18 IU/g dry wt on smallest particles), both without the addition of AS. The highest laccase activity (0.040 IU/g dry wt) was obtained on particles of 1.68 mm in the presence of AS. Since effect of particle size and addition AS was different for each strain, these criteria should be considered for diverse biotechnological applications.     

Role of cyanide production by Pseudomonas fluorescens CHA0 in the suppression of root-knot nematode, Meloidogyne javanica in tomato

Summary Pseudomonas fluorescens strain CHA0 produces hydrogen cyanide (HCN), a secondary metabolite that accounts largely for the biocontrol ability of this strain. In this study, we examined the role of HCN production by CHA0 as an antagonistic factor that contributes to biocontrol of Meloidogyne javanica, the root-knot nematode, in situ. Culture filtrate of CHA0, resulting from 1/10-strength nutrient broth yeast extract medium amended with glycine, inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. The bacterium cultured under high oxygen-tension conditions exhibited better inhibitory effects towards nematodes, compared to its cultivation under excess oxygen situation. Growth medium amended with 0.50 or 1.0 mM FeEDDHA further improved hatch inhibition and nematicidal activity of the strain CHA0. Strain CHA77, an HCN-negative mutant, failed to exert such toxic effects, and in this strain, antinematode activity was not influenced by culture conditions. Exogenous cyanide also inhibited egg hatch and caused mortality of M. javanica juveniles in vitro. Strains CHA0 or CHA77 applied in unsterilized sandy-loam soil as drench, caused marked suppression of root-knot disease development incited by M. javanica in tomato seedlings. However, efficacy of CHA77 was noticeably lower compared to its wild type counterpart CHA0. An increased bioavailability of iron following EDTA application in soil substantially improved nematode biocontrol potential of CHA0 but not that of CHA77. Soil infestation with M. javanica eggs resulted in significantly lower nematode population densities and root-knot disease compared to the juveniles used as root-knot disease-inducing agents. Strain CHA0 significantly suppressed nematode populations and inhibited galling in tomato roots grown in soil inoculated with eggs or juveniles and treated with or without EDTA. Strain CHA0 exhibited greater biocontrol potential in soil inoculated with eggs and treated with EDTA. To demonstrate that HCN synthesis by the strain CHA0 acts as the inducing agent of systemic resistance in tomato, efficacy of the strain CHA0 was compared with CHA77 in a split root trial. The split-root experiment, guaranteeing a spatial separation of the inducing agent and the challenging pathogen, showed that HCN production by CHA0 is not crucial in the induction of systemic resistance in tomato against M. javanica, because the HCN-negative-mutant CHA77 induced the same level of resistance as the wild type but exogenous cyanide in the form of KCN failed to trigger the resistance reaction. In the root section where both nematode and the bacterium were present, strain CHA0 reduced nematode penetration to a greater extent than CHA77, suggesting that for effective control of M. javanica, a direct contact between HCN-producing CHA0 and the nematode is essential.     

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae

S.J. HIOM, J.R. FURR, A.D. RUSSELL AND J.R. DICKINSON, 1992. The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 μg/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 μg/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

北部湾徐闻海域红树内生细菌物种多样性及其杀线虫活性研究

为发掘红树植物内生细菌资源和寻找新型微生物杀虫剂,该研究从北部湾徐闻海域采集7种红树植物共16份样品,设计10种分离培养基,使用稀释涂布法分析红树植物内生细菌的分布特征,通过16S rRNA分子生物学方法对内生细菌进行多样性分析,并利用秀丽隐杆线虫模型,通过杀线虫活性实验测试内生细菌乙酸乙酯提取物的杀线虫活性。结果表明:(1)从16份红树植物各组织器官中获得33株内生细菌,分布于19个科23个属。其中,芽孢杆菌属(Bacillus)为优势菌属,并发现了10株潜在的新种或新属。(2)筛选到具有显著杀线虫活性的菌株IMDGX 4725和IMDGX 4744,半数致死浓度(LC_(50))分别为61.58、100.89mg·mL~(-1)。研究结果证实了徐闻海域红树具有多样性丰富的内生细菌,同时部分细菌具有较强的杀线虫活性,具有发现新型微生物杀虫剂的潜力。     

Sulfobacillus sibiricus sp. nov., a new moderately thermophilic bacterium

In the course of pilot industrial testing of a biohydrometallurgical technology for processing goldarsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha, Yakutiya), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe2+, S0, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55 degrees C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow, in the absence of an inorganic energy source, on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G + C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA-DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis) which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9-98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.     

Secondary metabolites help biocontrol strain Pseudomonas fluorescens CHA0 to escape protozoan grazing

In soil ecosystems, bacteria must cope with predation activity, which is attributed mainly to protists. The development of antipredation strategies may help bacteria maintain higher populations and persist longer in the soil. We analyzed the interaction between the root-colonizing and biocontrol strain Pseudomonas fluorescens CHA0 and three different protist isolates (an amoeba, a flagellate, and a ciliate). CHA0 produces a set of antibiotics, HCN, and an exoprotease. We observed that protists cannot grow on CHA0 but can multiply on isogenic regulatory mutants that do not produce the extracellular metabolites. The in vitro responses to CHA0 cells and its exoproducts included growth inhibition, encystation, paralysis, and cell lysis. By analyzing the responses of protists to bacterial supernatants obtained from different isogenic mutants whose production of one or more exometabolites was affected and also to culture extracts with antibiotic enrichment, we observed different contributions of the phenolic antifungal compound 2,4-diacetylphloroglucinol (DAPG) and the extracellular protease AprA to CHA0 toxicity for protists and to the encystation-reactivation cycle. The grazing pressure artificially produced by a mixture of the three protists in a microcosm system resulted in reduced colonization of cucumber roots by a regulatory isogenic CHA0 mutant unable to produce toxins. These results suggest that exometabolite production in biocontrol strain CHA0 may contribute to avoidance of protist grazing and help sustain higher populations in the rhizosphere, which may be a desirable and advantageous trait for competition with other bacteria for available resources.     

Secondary Metabolites Help Biocontrol Strain Pseudomonas fluorescens CHA0 To Escape Protozoan Grazing

In soil ecosystems, bacteria must cope with predation activity, which is attributed mainly to protists. The development of antipredation strategies may help bacteria maintain higher populations and persist longer in the soil. We analyzed the interaction between the root-colonizing and biocontrol strain Pseudomonas fluorescens CHA0 and three different protist isolates (an amoeba, a flagellate, and a ciliate). CHA0 produces a set of antibiotics, HCN, and an exoprotease. We observed that protists cannot grow on CHA0 but can multiply on isogenic regulatory mutants that do not produce the extracellular metabolites. The in vitro responses to CHA0 cells and its exoproducts included growth inhibition, encystation, paralysis, and cell lysis. By analyzing the responses of protists to bacterial supernatants obtained from different isogenic mutants whose production of one or more exometabolites was affected and also to culture extracts with antibiotic enrichment, we observed different contributions of the phenolic antifungal compound 2,4-diacetylphloroglucinol (DAPG) and the extracellular protease AprA to CHA0 toxicity for protists and to the encystation-reactivation cycle. The grazing pressure artificially produced by a mixture of the three protists in a microcosm system resulted in reduced colonization of cucumber roots by a regulatory isogenic CHA0 mutant unable to produce toxins. These results suggest that exometabolite production in biocontrol strain CHA0 may contribute to avoidance of protist grazing and help sustain higher populations in the rhizosphere, which may be a desirable and advantageous trait for competition with other bacteria for available resources.     

Caldimonas taiwanensis sp. nov., a amylase producing bacterium isolated from a hot spring

During screening for amylase-producing bacteria, a strain designated OnlT was isolated from a hot spring located in Pingtung area, which is near the very southern part of Taiwan. Cells of this organism were Gram-negative rods motile by means of a single polar flagellum. Optimum conditions for growth were 55 degrees C and pH 7. Strain On1(T) grew well in minimal medium containing starch as the sole carbon source, and its extracellular products expressed amylase activity. The 16S rRNA gene sequence analysis indicates that strain On1(T) is a member of beta-Proteobacteria. On the basis of a phylogenetic analysis of 16S rDNA sequences, DNA-DNA similarity data, physiological and biochemical characteristics, as well as fatty acid compositions, the organism belonged to the genus Caldimonas and represented a novel species within this genus. The predominant cellular fatty acids of strain On1(T) were 16:0 (about 30%), 18:1 omega 7c (about 20%) and summed feature 3 (16:1 omega 7c or 15:0 iso 2OH or both [about 31%]). Its DNA base ratio was 65.9 mol% G + C. We propose to classify strain On1(T) (= BCRC 17405T = LMG 22827T) as Caldimonas taiwanensis sp. nov.     

Effect of nitrogen limitation and nature of the feed upon Oenococcus oeni metabolism and extracellular protein production

AIMS: The aim of the study was to characterize the effect of various nitrogen sources on Oenococcus oeni growth, carbon source utilization, extracellular protease activity and extracellular proteins. More generally, the goal is to understand how nitrogen-based additives might act to enhance malolactic fermentation in wine. METHODS AND RESULTS: Five yeast extracts were used. As the amino acid and nitrogen analyses revealed, they were similar in global amino acid composition, except for arginine level. Nevertheless the ratio of amino acids between free/bound, and low/high molecular weight fractions were highly different. One of the yeast extracts led to a significant protease activity in the supernatant and to a poor final biomass of the IOB84.13 strain compared to the other ones. For the IOB84.13 strain specifically, arginine addition to the arginine poor yeast extract did not restore growth. 35S-methionine-labelled extracellular proteins were separated by SDS-PAGE. Signals were detected in all media early in the growth phase and were maintained during 48 h of culture. CONCLUSIONS: A significant protease activity was detected for O. oeni supernatants during growth under nitrogen limitation but only for certain nitrogen sources. Moreover, the activity was strain dependent. Peptides (0.5-10 kDa) seemed to be more favourable for growth of wine bacteria than <0.5 kDa nitrogen sources. The extracellular protein signal patterns differed more greatly between the bacterial strains tested than between the nitrogen molecules in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study extensively considering the role of the nitrogen source composition and level upon O. oeni growth and metabolism.     

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.