Phloroglucinol mediates cross-talk between the pyoluteorin and 2,4-diacetylphloroglucinol biosynthetic pathways in Pseudomonas fluorescens Pf-5

The antibiotics pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) contribute to the biological control of soilborne plant diseases by some strains of Pseudomonas fluorescens, including Pf-5. These secondary metabolites also have signalling functions with each compound reported to induce its own production and repress the other's production. The first step in DAPG biosynthesis is production of phloroglucinol (PG) by PhlD. In this study, we show that PG is required at nanomolar concentrations for pyoluteorin production in Pf-5. At higher concentrations, PG is responsible for the inhibition of pyoluteorin production previously attributed to DAPG. DAPG had no effect on pyoluteorin production, and monoacetylphloroglucinol showed both stimulatory and inhibitory activities but at concentrations 100-fold greater than the levels of PG required for similar effects. We also demonstrate that PG regulates pyoluteorin production in P. aeruginosa and that a phlD gene adjacent to the pyoluteorin biosynthetic gene cluster in P. aeruginosa strain LESB58 can restore pyoluteorin biosynthesis to a ΔphlD mutant of Pf-5. Bioinformatic analyses show that the dual role of PhlD in the biosynthesis of DAPG and the regulation of pyoluteorin production could have arisen within the pseudomonads during the assembly of these biosynthetic gene clusters from genes and gene subclusters of diverse origins.     

RpoN (sigma54) controls production of antifungal compounds and biocontrol activity in Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 is an effective biocontrol agent of root diseases caused by fungal pathogens. The strain produces the antibiotics 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) that make essential contributions to pathogen suppression. This study focused on the role of the sigma factor RpoN (sigma54) in regulation of antibiotic production and biocontrol activity in P. fluorescens. An rpoN in-frame-deletion mutant of CHAO had a delayed growth, was impaired in the utilization of several carbon and nitrogen sources, and was more sensitive to salt stress. The rpoN mutant was defective for flagella and displayed drastically reduced swimming and swarming motilities. Interestingly, the rpoN mutant showed a severalfold enhanced production of DAPG and expression of the biosynthetic gene phlA compared with the wild type and the mutant complemented with monocopy rpoN+. By contrast, loss of RpoN function resulted in markedly lowered PLT production and plt gene expression, suggesting that RpoN controls the balance of the two antibiotics in strain CHA0. In natural soil microcosms, the rpoN mutant was less effective in protecting cucumber from a root rot caused by Pythium ultimum. Remarkably, the mutant was not significantly impaired in its root colonization capacity, even at early stages of root infection by Pythium spp. Taken together, our results establish RpoN for the first time as a major regulator of biocontrol activity in Pseudomonas fluorescens.     

Assessment of genotypic diversity of antibiotic-producing pseudomonas species in the rhizosphere by denaturing gradient gel electrophoresis

The genotypic diversity of antibiotic-producing Pseudomonas spp. provides an enormous resource for identifying strains that are highly rhizosphere competent and superior for biological control of plant diseases. In this study, a simple and rapid method was developed to determine the presence and genotypic diversity of 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas strains in rhizosphere samples. Denaturing gradient gel electrophoresis (DGGE) of 350-bp fragments of phlD, a key gene involved in DAPG biosynthesis, allowed discrimination between genotypically different phlD(+) reference strains and indigenous isolates. DGGE analysis of the phlD fragments provided a level of discrimination between phlD(+) genotypes that was higher than the level obtained by currently used techniques and enabled detection of specific phlD(+) genotypes directly in rhizosphere samples with a detection limit of approximately 5 x 10(3) CFU/g of root. DGGE also allowed simultaneous detection of multiple phlD(+) genotypes present in mixtures in rhizosphere samples. DGGE analysis of 184 indigenous phlD(+) isolates obtained from the rhizospheres of wheat, sugar beet, and potato plants resulted in the identification of seven phlD(+) genotypes, five of which were not described previously based on sequence and phylogenetic analyses. Subsequent bioassays demonstrated that eight genotypically different phlD(+) genotypes differed substantially in the ability to colonize the rhizosphere of sugar beet seedlings. Collectively, these results demonstrated that DGGE analysis of the phlD gene allows identification of new genotypic groups of specific antibiotic-producing Pseudomonas with different abilities to colonize the rhizosphere of sugar beet seedlings.     

Isolation and characterization of a novel banana rhizosphere bacterium as fungal antagonist and microbial adjuvant in micropropagation of banana

AIM: Isolation and characterization of a bacterial isolate (strain FP10) from banana rhizosphere with innate potential as fungal antagonist and microbial adjuvant in micropropagation of banana. METHODS AND RESULTS: Bacterium FP10 was isolated from the banana rhizosphere and identified as Pseudomonas aeruginosa based on phenotypic, biochemical traits and sequence homology of partial 622-bp fragment of 16S ribosomal DNA (rDNA) amplicon, with the ribosomal database sequences. Strain FP10 displayed antibiosis towards fungi causing wilt and root necrosis diseases of banana. Production of plant growth hormone, indole-3-acetic acid (IAA), siderophores and phosphate-solubilizing enzyme in FP10 was determined. Strain FP10 tested negative for hydrogen cyanide, cellulase and pectinase, the deleterious traits for plant growth. Screening of antibiotic genes was carried out by polymerase chain reaction using gene-specific primers. Amplification of a 745-bp DNA fragment confirmed the presence of phlD, which is a key gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG) in FP10. The antibiotic produced by FP10 was confirmed as DAPG using thin layer chromatography, high performance liquid chromatography and Fourier transform infrared and tested for fungal antibiosis towards banana pathogens. Procedures for encapsulation of banana shoot tips with FP10 are described. CONCLUSIONS: Strain FP10 exhibited broad-spectrum antibiosis towards banana fungi causing wilt and root necrosis. DAPG by FP10 induced bulb formation and lysis of fungal mycelia. Encapsulation of banana shoot tips with FP10 induced higher frequency of germination (plantlet development) than nontreated controls on Murashige and Skoog basal medium. Treatment of banana plants with FP10 enhanced plant height and reduced the vascular discolouration as a result of Fusarium oxysporum f. sp. cubense FOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of the innate potential of fungal antibiosis by DAPG antibiotic and production of siderophore, plant-growth-promoting IAA and phosphatase, the strain FP10 can be used as biofertilizer as well as a biocontrol agent.     

Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection

Multitrophic interactions mediate the ability of fungal pathogens to cause plant disease and the ability of bacterial antagonists to suppress disease. Antibiotic production by antagonists, which contributes to disease suppression, is known to be modulated by abiotic and host plant environmental conditions. Here, we demonstrate that a pathogen metabolite functions as a negative signal for bacterial antibiotic biosynthesis, which can determine the relative importance of biological control mechanisms available to antagonists and which may also influence fungus-bacterium ecological interactions. We found that production of the polyketide antibiotic 2,4-diacetylphloroglucinol (DAPG) was the primary biocontrol mechanism of Pseudomonas fluorescens strain Q2-87 against Fusarium oxysporum f. sp. radicis-lycopersici on the tomato as determined with mutational analysis. In contrast, DAPG was not important for the less-disease-suppressive strain CHA0. This was explained by differential sensitivity of the bacteria to fusaric acid, a pathogen phyto- and mycotoxin that specifically blocked DAPG biosynthesis in strain CHA0 but not in strain Q2-87. In CHA0, hydrogen cyanide, a biocide not repressed by fusaric acid, played a more important role in disease suppression.     

Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0

Pseudomonas fluorescens CHA0 protects various crop plants against root diseases caused by pathogenic fungi. Among a range of exoproducts excreted by strain CHA0, the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are particularly relevant to the strain's biocontrol potential. Here, we report on the characterization of MvaT and MvaV as novel regulators of biocontrol activity in strain CHA0. We establish the two proteins as further members of an emerging family of MvaT-like regulators in pseudomonads that are structurally and functionally related to the DNA-binding protein H-NS. In mvaT and mvaV in frame-deletion mutants of strain CHA0, PLT production was enhanced about four- and 1.5-fold, respectively, whereas DAPG production remained at wild-type levels. Remarkably, PLT production was increased up to 20-fold in an mvaT mvaV double mutant. DAPG biosynthesis was almost completely repressed in this mutant. The effects on antibiotic production could be confirmed by following expression of gfp-based reporter fusions to the corresponding biosynthetic genes. MvaT and MvaV also influenced levels of other exoproducts, motility, and physicochemical cell-surface properties to various extents. Compared with the wild type, mvaT and mvaV mutants had an about 20% reduced capacity (in terms of plant fresh weight) to protect cucumber from a root rot caused by Pythium ultimum. Biocontrol activity was nearly completely abolished in the double mutant Our findings indicate that MvaT and MvaV act together as further global regulatory elements in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads.     

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.     

Genetic diversity of phlD gene from 2,4-diacetylphloroglucinol-producing Pseudomonas spp. strains from the maize rhizosphere

In biocontrol Pseudomonads, phlD is an essential gene involved in the biosynthesis of 2,4-diacetylphloroglucinol (DAPG). HaeIII restriction of amplified phlD gene, previously proposed as the most discriminant analysis, showed no polymorphism among 144 Pseudomonas strains isolated from maize roots. However, these strains fell into three statistically significant DAPG production level groups. phlD sequences of 13 strains belonging to the three DAPG groups revealed a KspI restriction site only in good DAPG-producing strains. This result was confirmed on the 144 strains, 82 of which were identified as good-DAPG producers by both biochemical and amplified phlD KspI restriction analysis. They are candidates as potential biocontrol agents.     

phlD-based genetic diversity and detection of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens

Diversity within a worldwide collection of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains was assessed by sequencing the phlD gene. Phylogenetic analyses based on the phlD sequences of 70 isolates supported the previous classification into 18 BOX-PCR genotypes (A-Q and T). Exploiting polymorphisms within the sequence of phlD, we designed and used allele-specific PCR primers with a PCR-based dilution endpoint assay to quantify the population sizes of A-, B-, D-, K-, L- and P-genotype strains grown individually or in pairs in vitro, in the rhizosphere of wheat and in bulk soil. Except for P. fluorescens Q8r1-96, which strongly inhibited the growth of P. fluorescens Q2-87, inhibition between pairs of strains grown in vitro did not affect the accuracy of the method. The allele-specific primer-based technique is a rapid method for studies of the interactions between genotypes of 2,4-diacetylphloroglucinol producers in natural environments.     

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.     

The Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion

During evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-β-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P. fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR.     

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.     

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.     

Influence of plant species on population dynamics, genotypic diversity and antibiotic production in the rhizosphere by indigenous Pseudomonas spp

The population dynamics, genotypic diversity and activity of naturally-occurring 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. was investigated for four plant species (wheat, sugar beet, potato, lily) grown in two different soils. All four plant species tested, except lily and in some cases wheat, supported relatively high rhizosphere populations (5 x 10(4) to 1 x 10(6) CFU/g root) of indigenous DAPG-producing Pseudomonas spp. during successive cultivation in both a take-all suppressive and a take-all conducive soil. Although lily supported on average the highest population densities of fluorescent Pseudomonas spp., it was the least supportive of DAPG-producing Pseudomonas spp. of all four plant species. The genotypic diversity of 492 DAPG-producing Pseudomonas isolates, assessed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the phlD gene, revealed a total of 7 genotypes. Some of the genotypes were found only in the rhizosphere of a specific plant, whereas the predominant genotypes were found at significantly higher frequencies in the rhizosphere of three plant species (wheat, sugar beet and potato). Statistical analysis of the phlD(+) genotype frequencies showed that the diversity of the phlD(+) isolates from lily was significantly lower than the diversity of phlD(+) isolates found on wheat, sugar beet or potato. Additionally, soil type had a significant effect on both the phlD(+) population density and the phlD(+) genotype frequencies, with the take-all suppressive soil being the most supportive. HPLC analysis further showed that the plant species had a significant effect on DAPG-production by the indigenous phlD(+) population: the wheat and potato rhizospheres supported significantly higher amounts of DAPG produced per cell basis than the rhizospheres of sugar beet and lily. Collectively, the results of this study showed that the host plant species has a significant influence on the dynamics, composition and activity of specific indigenous antagonistic Pseudomonas spp.     

Carbon fractions in the rhizosphere of pea inoculated with 2,4 diacetylphloroglucinol producing and non-producing Pseudomonas fluorescens F113

The aim of this work was to determine the effect of wild type and functionally modified Pseudomonas fluorescens strains on C fractions in the rhizosphere of pea. The lac ZY marked F113 strain produces the antibiotic 2,4 diacetylphloroglucinol (DAPG) useful in plant disease control. The modified strain of F113 was represented in production of DAPG, creating the DAPG negative strain F113 G22. The F113 treatment resulted in a significantly lower shoot/root ratio. The F113 G22 treatment had a significantly greater indigenous and total fluorescent Pseudomonas population than the control and F113 (DAPG₊) treatment. Both strains significantly increased the water soluble carbohydrates and the total water soluble carbon in the pea rhizosphere soil. Strain F113 significantly increased the soil protein content relative to the control, but not in relation to the F113 G22 treatment. The F113 treatment had a significantly greater organic acid content than the control and F113 G22 treatments, whilst the F113 G22 treatment was also significantly greater than the control. Both inocula resulted in significantly lower phosphate contents than the control. The F113 inocula significantly increased alkaline phosphatase, sulphatase and urease activities, and reduced β glucosidase activities indicating increased carbon availability. Both inocula increased C availability ; however, antibiotic production by strain F113 reduced the utilisation of organic acids released from the plant resulting in differing effects of the two strains on nutrient availability, plant growth, soil enzyme activities and Pseudomonas populations.     

Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR

A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (C(T)s) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.     

The two-component colR/S system of Pseudomonas fluorescens WCS365 plays a role in rhizosphere competence through maintaining the structure and function of the outer membrane

Pseudomonas fluorescens strain PCL1210, a competitive tomato root tip colonization mutant of the efficient root colonizing wild type strain WCS365, is impaired in the two-component sensor-response regulator system ColR/ColS. Here we show that a putative methyltransferase/wapQ operon is located downstream of colR/colS and that this operon is regulated by ColR/ColS. Since wapQ encodes a putative lipopolysaccharide (LPS) phosphatase, the possibility was studied that the integrity of the outer membrane of PCL1210 was altered. Indeed, it was shown that mutant PCL1210 is more resistant to various chemically unrelated antibiotics which have to pass the outer membrane for their action. In contrast, the mutant is more sensitive to the LPS-binding antibiotic polymyxin B. Mutant PCL1210 loses growth in competition with its wild type when grown in tomato root exudate. Mutants in the methyltransferase/wapQ operon are also altered in their outer membrane permeability and are defective in competitive tomato root tip colonization. A model for the altered outer membrane of PCL1210 is discussed.     

Biological control of rice bacterial blight by plant-associated bacteria producing 2,4-diacetylphloroglucinol

Certain plant-associated strains of fluorescent Pseudomonas spp. are known to produce the antimicrobial antibiotic 2,4-diacetylphloroglucinol (DAPG). It has antibacterial, antifungal, antiviral, and antihelminthic properties and has played a significant role in the biological control of tobacco, wheat, and sugar beet diseases. It has never been reported from India and has not been implicated in the biological suppression of a major disease of the rice crop. Here, we report that a subpopulation of 27 strains of plant-associated Pseudomonas fluorescens screened in a batch of 278 strains of fluorescent pseudomonads produced DAPG. The DAPG production was detected by a PCR-based screening method that used primers Phl2a and Phl2b and amplified a 745-bp fragment characteristic of DAPG. HPLC, 1H NMR, and IR analyses provided further evidence for its production. We report also that this compound inhibited the growth of the devastating rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae in laboratory assays and suppressed rice bacterial blight up to 59%-64% in net-house and field experiments. Tn5 mutants defective in DAPG production (Phl-) of P. fluorescens PTB 9 were much less effective in their suppression of rice bacterial blight.     

Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0

AIMS: To develop reporter constructs based on stable and unstable variants of the green fluorescent protein (GFP) for monitoring balanced production of antifungal compounds that are crucial for the capacity of the root-colonizing Pseudomonas fluorescens strain CHA0 to control plant diseases caused by soil-borne pathogenic fungi. METHODS AND RESULTS: Pseudomonas fluorescens CHA0 produces the three antifungal metabolites 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT) and pyrrolnitrin (PRN). The gfp[mut3] and gfp[AAV] reporter genes were fused to the promoter regions of the DAPG, PLT and PRN biosynthetic genes. The reporter fusions were then used to follow the kinetics of expression of the three antifungal metabolites in a microplate assay. DAPG and PLT were found to display an inverse relationship in which each metabolite activates its own biosynthesis while repressing the synthesis of the other metabolite. PRN appears not to be involved in this balance. However, the microbial and plant phenolic metabolite salicylate was found to interfere with the expression of both DAPG and PLT. CONCLUSIONS: The results obtained provide evidence that P. fluorescens CHA0 may keep the antifungal compounds DAPG and PLT at a fine-tuned balance that can be affected by certain microbial and plant phenolics. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, the present study is the first to use stable and unstable GFP variants to study antibiotic gene expression in a biocontrol pseudomonad. The developed reporter fusions will be a highly valuable tool to study in situ expression of this bacterial biocontrol trait on plant roots, i.e. at the site of pathogen suppression.     

Impact on Arbuscular Mycorrhiza Formation of Pseudomonas Strains Used as Inoculants for Biocontrol of Soil-Borne Fungal Plant Pathogens

The arbuscular mycorrhizal symbiosis, a key component of agroecosystems, was assayed as a rhizosphere biosensor for evaluation of the impact of certain antifungal Pseudomonas inoculants used to control soil-borne plant pathogens. The following three Pseudomonas strains were tested: wild-type strain F113, which produces the antifungal compound 2,4-diacetylphloroglucinol (DAPG); strain F113G22, a DAPG-negative mutant of F113; and strain F113(pCU203), a DAPG overproducer. Wild-type strain F113 and mutant strain F113G22 stimulated both mycelial development from Glomus mosseae spores germinating in soil and tomato root colonization. Strain F113(pCU203) did not adversely affect G. mosseae performance. Mycelial development, but not spore germination, is sensitive to 10 μM DAPG, a concentration that might be present in the rhizosphere. The results of scanning electron and confocal microscopy demonstrated that strain F113 and its derivatives adhered to G. mosseae spores independent of the ability to produce DAPG.