A GacS deficiency does not affect Pseudomonas chlororaphis PA23 fitness when growing on canola, in aged batch culture or as a biofilm

Pseudomonas chlororaphis PA23 is a biocontrol agent that protects against the fungal pathogen Sclerotinia sclerotiorum. Employing transposon mutagenesis, we isolated a gacS mutant that no longer exhibited antifungal activity. Pseudomonas chlororaphis PA23 was previously reported to produce the nonvolatile antibiotics phenazine 1-carboxylic acid and 2-hydroxyphenazine. We report here that PA23 produces additional compounds, including protease, lipase, hydrogen cyanide, and siderophores, that may contribute to its biocontrol ability. In the gacS mutant background, generation of these products was markedly reduced or delayed with the exception of siderophores, which were elevated. Not surprisingly, this mutant was unable to protect canola from disease incited by S. sclerotiorum. The gacS mutant was able to sustain itself in the canola phyllosphere, therefore, the loss of biocontrol activity can be attributed to a reduced production of antifungal compounds and not a declining population size. Competition assays between the mutant and wild type revealed equivalent fitness in aged batch culture; consequently, the gacS mutation did not impart a growth advantage in the stationary phase phenotype. Under minimal nutrient conditions, the gacS-deficient strain produced a tenfold less biofilm than the wild type. However, no difference was observed in the ability of the mutant biofilm to protect cells from lethal antibiotic challenge.     

Nematicidal activity and chemical component of Poria cocos

Poria cocos, a famous traditional Chinese medicine, was found to have nematicidal activity in experiments searching for nematicidal fungi. The experiment showed it could kill 94.9% of the saprophytic nematode, Panagrellus redivivue, 92.6% of the root-knot nematode, Meloidogyne arenaria, and 93.5% of the pine nematode, Bursaphelenchus xylophilus, on PDA plate within 12 hours. According to the nematicidal activity, three new compounds, 2, 4, 6-triacetylenic octane diacid, 2, 4, 5, 6-tetrahydroxyhexanoic acid and 3, 4-dihydroxy-2-keto-n-butyl 2,4,5,6-tetrahydroxyhexanate, were isolated from submerged cultures of Poria cocos. Of these, 2, 4, 6-triacetylenic octane diacid could kill 83.9% Meloidogyne arenaria and 73.4% Panagrellus redivivus at 500 ppm within 12 hours. Here, it is reported for the first time that Poria cocos has nematicidal activity.     

Inactivation of gacS does not affect the competitiveness of Pseudomonas chlororaphis in the Arabidopsis thaliana rhizosphere

Quorum-sensing-controlled processes are considered to be important for the competitiveness of microorganisms in the rhizosphere. They affect cell-cell communication, biofilm formation, and antibiotic production, and the GacS-GacA two-component system plays a role as a key regulator. In spite of the importance of this system for the regulation of various processes, strains with a Gac(-) phenotype are readily recovered from natural habitats. To analyze the influence of quorum sensing and the influence of the production of the antibiotic phenazine-1-carboxamide on rhizosphere colonization by Pseudomonas chlororaphis, a gnotobiotic system based on Arabidopsis thaliana seedlings in soil was investigated. Transposon insertion mutants of P. chlororaphis isolate SPR044 carrying insertions in different genes required for the production of N-acyl-homoserine lactones and phenazine-1-carboxamide were generated. Analysis of solitary rhizosphere colonization revealed that after prolonged growth, the population of the wild type was significantly larger than that of the homoserine lactone-negative gacS mutant and that of a phenazine-1-carboxamide-overproducing strain. In cocultivation experiments, however, the population size of the gacS mutant was similar to that of the wild type after extended growth in the rhizosphere. A detailed analysis of growth kinetics was performed to explain this phenomenon. After cells grown to the stationary phase were transferred to fresh medium, the gacS mutant had a reduced lag phase, and production of the stationary-phase-specific sigma factor RpoS was strongly reduced. This may provide a relative competitive advantage in cocultures with other bacteria, because it permits faster reinitiation of growth after a change to nutrient-rich conditions. In addition, delayed entry into the stationary phase may allow more efficient nutrient utilization. Thus, GacS-GacA-regulated processes are not absolutely required for efficient rhizosphere colonization in populations containing the wild type and Gac(-) mutants.     

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.     

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.     

The effects of exotoxin (Thuringiensin) from Bacillus thuringiensis on Meloidogyne incognita and Caenorhabditis elegans

A partially purified preparation as well as two formulations of exotoxin from Bacillus thuringiensis (thuringiensin) were evaluated for nematicidal activity. The methods used in our evaluations included direct contact nematicidal assays, hatching tests, infection tests in seed pouches using the cucumber/root-knot nematode (Meloidogyne incognita) system, and greenhouse test using the root-knot nematode. While contact nematicidal activity was not observed against juveniles of M. incognita, 100% mortality occurred when the free-living nematode, Caenorhabditis elegans, was used as the test organism. Nematode infection evaluations in the seed pouch assay showed reduced root galling at relatively high concentrations (>10 mg kg^-1). Greenhouse assays indicated significant reduction in the soil population. However, the degree of control in relation to the amount of material applied, as measured by the gall numbers, larvae from soil/roots, and plant growth parameters, was not considered adequate. Data on the plant response in relation to treatment with different formulations of the toxin are presented.     

Nematicidal prenylated flavanones from Phyllanthus niruri

Two prenylated flavanones have been isolated from the hexane extract of Phyllanthus niruri plant. The structure of these flavanones were established as 8-(3-Methyl-but-2-enyl)-2-phenyl chroman-4-one (1) and 2-(4-hydroxyphenyl)-8-(3-methyl-but-2-enyl)-chroman-4-one (2) on the basis of spectral analysis. These were evaluated for nematicidal activity against root-knot, Meloidogyne incognita, and reniform, Rotylenchulus reniformis, nematodes. Compound 2 exhibited nematicidal activity at par with the standard carbofuran (LC50 3.3 and 3.1ppm, respectively) when tested against reniform nematode. The LC50 value against root-knot nematode was found to be 14.5ppm. Compound 1 however, showed moderate activity against both the test nematodes.     

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.     

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.     

Process development of oxalic acid production in submerged culture of Aspergillus niger F22 and its biocontrol efficacy against the root-knot nematode Meloidogyne incognita

Bioprocess and Biosystems Engineering - Oxalic acid has potent nematicidal activity against the root-knot nematode Meloidogyne incognita. In this study, fermentation parameters for oxalic acid...     

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.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Nematicidal activity of culture filtrate of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> against <Emphasis Type="Italic">Meloidogyne hapla</Emphasis>

Culture filtrates of Beauveria bassiana at different concentrations were evaluated for nematicidal activity against the northern root knot nematode (Meloidogyne hapla); bioassays included egg hatching, mortality and infectivity on tomato plants in pots under glasshouse conditions. The percentage mortality and inhibition of hatching of root-knot nematode were directly proportional to the concentration of culture filtrates of B. bassiana. Soil drenching with culture filtrate of B. bassiana significantly reduced nematode population densities in soil and in the roots and subsequent gall formation and egg-mass production by M. hapla under glasshouse conditions.     

Observations on the suppression of root-knot nematode (Meloidogyne arenaria) on tomato by incorporation of cyanobacterial powder (Oscillatoria chlorina) into potting field soil

Experiments were carried out to investigate the nematicidal potential of a cyanobacterium, Oscillatoria chlorina, against the root-knot nematode, Meloidogyne arenaria on tomato plants grown in pots filled with 500 cm3 of field soil infested with 12-s stage juveniles (J2)/cm3 soil. Incorporation of freeze-dried cyanobacterial powder into potted field soil at the rate of 0.2%, 0.4%, 0.6%, 0.8% and 1.0% (w/w) 5 days prior to tomato planting, reduced root galling, final population of M. arenaria and increased vegetative growth of tomato plants and root-mass production, compared with untreated control (P > or = 0.05). The beneficial effect of adding cyanobacterial powder into infested potted field soil increased exponentially with concentration up to 0.8%. Root galling and nematode population decreased by 68.9% and 97.6%, respectively at the highest dose (1%) of cyanobacterial powder compared with the untreated control. Addition of cyanobacterial powder into infested potted field soil at 5 days before planting was the most effective followed by 2 days before and at the time of tomato planting. We conclude that application rate and timing are important factors in the control of root-knot nematodes with O. chlorina.     

Coprinus comatus: A basidiomycete fungus forms novel spiny structures and infects nematode

Nematophagous basidiomycete fungi kill nematodes by trapping, endoparasitizing and producing toxin. In our studies Coprinus comatus (O.F.Müll. : Fr.) Pers. is found to be a nematode-destroying fungus; this fungus immobilizes, kills and uses free-living nematode Panagrellus redivivus Goodey and root-knot nematode Meloidogyne arenaria Neal. C. comatus produces an unusual structure designated spiny ball. Set on a sporophore-like branch, the spiny ball is a burr-like structure assembled with a large number of tiny tubes. Purified spiny balls exhibit moderate nematicidal activity. Experiments show that spiny balls are not chlamydospores because of the absence of nuclei in the structures and quick formation within 3 d in a young colony. Nematodes added to C. comatus cultures on potato-dextrose agar (PDA) and cornmeal agar (CMA) become inactive in hours. Infection of nematodes by the fungus occurs only after the nematodes are immobilized (feeble or dead), probably by a toxin. Electron micrographs illustrate that C. comatus infect P. redivivus by producing penetration pegs with which hyphae colonize nematode bodies. An infected nematode is digested and consumed within days and hyphae grow out of the nematode.     

A review of isothiocyanates biofumigation activity on plant parasitic nematodes

Natural isothiocyanates (ITCs) are toxic to a range of soil-borne pest and pathogens, including nematodes and fungi, and can thus be used as natural fumigants called biofumigants. Glucosinolates, β-thioglucoside N-hydroxysulfates, are secondary metabolites of Brassicales plants, stored in the S-cells vacuoles. Upon plant tissue damage myrosinase (thioglucoside glycohydrolase, EC 3.2.3.1), stored in contiguous cells, hydrolyses glucosinalates to an unstable aglycone that eventually eliminates sulfate group producing a wide range of different volatile isothiocyanates that are extremely toxic to root-knot nematodes. In fact, among synthetic commercial nematicidal formulates we can find isothiocyanates as active ingredients. Conventional nematode control practices have included soil sterilants of great environmental impact, most of which are now banned making mandatory the development of eco-sustainable alternative tools. We reviewed the nematicidal activity of isothiocyanates as components of botanical matrixes in the frame of a holistic nematode control approach encompassing secondary beneficial effects on soil structure and microbiology, beneficial preservation, enhanced residual life of biological activity and plant growth.     

Genetic and Proteomic Characterization of rpoB Mutations and Their Effect on Nematicidal Activity in Photorhabdus luminescens LN2

Rifampin resistant (Rif(R)) mutants of the insect pathogenic bacterium Photorhabdus luminescens LN2 from entomopathogenic nematode Heterorhabditis indica LN2 were genetically and proteomically characterized. The Rif(R) mutants showed typical phase one characters of Photorhabdus bacteria, and insecticidal activity against Galleria mellonella larvae, but surprisingly influenced their nematicidal activity against axenic infective juveniles (IJs) of H. bacteriophora H06, an incompatible nematode host. 13 out of 34 Rif(R) mutants lost their nematicidal activity against H06 IJs but supported the reproduction of H06 nematodes. 7 nematicidal-producing and 7 non-nematicidal-producing Rif(R) mutants were respectively selected for rpoB sequence analysis. rpoB mutations were found in all 14 Rif(R) mutants. The rpoB (P564L) mutation was found in all 7 mutants which produced nematicidal activity against H06 nematodes, but not in the mutants which supported H06 nematode production. Allelic exchange assays confirmed that the Rif-resistance and the impact on nematicidal activity of LN2 bacteria were conferred by rpoB mutation(s). The non-nematicidal-producing Rif(R) mutant was unable to colonize in the intestines of H06 IJs, but able to colonize in the intestines of its indigenous LN2 IJs. Proteomic analysis revealed different protein expression between wild-type strain and Rif(R) mutants, or between nematicidal-producing and non nematicidal-producing mutants. At least 7 putative proteins including DsbA, HlpA, RhlE, RplC, NamB (a protein from T3SS), and 2 hypothetical proteins (similar to unknown protein YgdH and YggE of Escherichia coli respectively) were probably involved in the nematicidal activity of LN2 bacteria against H06 nematodes. This hypothesis was further confirmed by creating insertion-deletion mutants of three selected corresponding genes (the downregulated rhlE and namB, and upregualted dsbA). These results indicate that the rpoB mutations greatly influence the symbiotic association between the symbionts and their entomopathogenic nematode hosts.     

Quorum-sensing regulation in soil pseudomonads

228 strains of soil and rhizosphere pseudomonads isolated in different geographic zones were screened, with the use of two tester systems, for the capacity to produce N-acetyl-homoserine lactones (AHLs), which are autoinducers involved in quorum-sensing (QS) regulation. AHL production was found in 11.4% of the strains investigated. In five Pseudomonas chlororaphis strains shown to be active AHL producers and chosen for further study, PCR identified two QS systems that involved the phzI, phzR, csaI, and csaR genes; this finding suggests the conservative nature of these regulation systems in P. chlororaphis. Strain P. chlororaphis 449, chosen as a model object and studied in greater detail, produced three AHL species including N-butanoyl-homoserine lactone and N-hexanoyl-homoserine lactone. This strain produced three types of phenazine antibiotics, as well as siderophores and cyanide; it also exhibited antagonistic properties toward a wide spectrum of phytopathogenic fungi. The phzI and csaI genes, coding for synthases of AHLs of two types, were cloned and sequenced; mutants with knocked-out phzI and csal genes were obtained. With the use of transposon mutagenesis and the gene substitution method, mutations were obtained in the global expression regulator genes gacS, coding for the GacA-GacS regulation system kinase, and rpoS, coding for the sigma S subunit of RNA polymerase. The effect of these mutations on the AHL synthesis and on the regulation of various metabolic processes in P. chlororaphis was studied.