DAPG Production Research Articles

Evaluation of antagonistic activity of DAPG‐producing fluorescent pseudomonads from rhizospheres of Solanaceous crops for biocontrol of bacterial wilt

AbstractPseudomonads produce a wide array of antimicrobial compounds including 2,4‐diacetylphloroglucinol (DAPG), which possess a broad‐spectrum antagonistic effect against phytopathogens. Rhizospheric soil samples were collected from the Solanaceous crops for isolation of fluorescent pseudomonads (FPs). Out of the 32 fluorescent bacterial isolates, 21 showed antagonistic activity against Ralstonia solanacearum (NAIMCC‐B‐01630) with the highest zone of inhibition at 38.33 mm. Among them, 11 bacterial isolates depicted the production of DAPG in Kings' B medium. DAPG production was detected by TLC and quantified through HPLC analysis. Moreover, the bacterial isolate OD13 exhibited higher DAPG production. Further, the presence of the DAPG‐encoding phlD gene within the size of 629 bp and 725 bp was also validated through amplification. These bacterial isolates belonged to the genera Pseudomonas as confirmed by morpho‐physiological characterization using the VITEK 2 system. The higher DAPG‐producing bacterial isolate was identified as Pseudomonas aeruginosa by polyphasic approach with GenBank accession number OQ781265. Thus, the DAPG‐producing rhizospheric Pseudomonas strains can be used as biocontrol agents for bacterial wilt. However, field study is highly essential before its successful application.

Resistance towards and biotransformation of a Pseudomonas-produced secondary metabolite during community invasion.

The role of antagonistic secondary metabolites produced by Pseudomonas protegens in suppression of soil-borne phytopathogens has been clearly documented. However, their contribution to the ability of P. protegens to establish in soil and rhizosphere microbiomes remains less clear. Here, we use a four-species synthetic community (SynCom) in which individual members are sensitive towards key P. protegens antimicrobial metabolites (DAPG, pyoluteorin, and orfamide A) to determine how antibiotic production contributes to P. protegens community invasion and to identify community traits that counteract the antimicrobial effects. We show that P. protegens readily invades and alters the SynCom composition over time, and that P. protegens establishment requires production of DAPG and pyoluteorin. An orfamide A-deficient mutant of P. protegens invades the community as efficiently as wildtype, and both cause similar perturbations to community composition. Here, we identify the microbial interactions underlying the absence of an orfamide A mediated impact on the otherwise antibiotic-sensitive SynCom member, and show that the cyclic lipopeptide is inactivated and degraded by the combined action of Rhodococcus globerulus D757 and Stenotrophomonas indicatrix D763. Altogether, the demonstration that the synthetic community constrains P. protegens invasion by detoxifying its antibiotics may provide a mechanistic explanation to inconsistencies in biocontrol effectiveness in situ.

Sequential interspecies interactions affect production of antimicrobial secondary metabolites in Pseudomonas protegens DTU9.1.

Soil and rhizosphere microbiomes play important roles in suppression of plant pathogens through production of antagonistic secondary metabolites, yet mechanisms that determine the strength of pathogen control are not well understood. Many Pseudomonas species are associated with soil and rhizosphere microbiomes, and their ability to suppress pathogens is well documented. Here, we investigate how interactions within the Pseudomonas genus affect their production of antimicrobial metabolites. From a biosensor-based screen, we identify P. capeferrum species as capable of modulating secondary metabolite production in P. protegens. We show that P. capeferrum alters production of pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) in P. protegens via two distinct and sequential mechanisms that depends on spatial proximity of the two species. Specifically, P. capeferrum secretes a diffusible signal that induce pyoluteorin production up to 100-fold in neighboring P. protegens colonies. In contrast, the interaction results in reduced DAPG production, but only within mixed-species colonies. Additionally, we found that increased pyoluteorin production and cell lysis of P. capeferrum is required for inhibition of DAPG production, suggesting that pyoluteorin-facilitated antibiosis of P. protegens on P. capeferrum leads to release of cell-associated metabolites and subsequent inhibition of DAPG production in P. protegens. As the interaction modulates in vitro bioactivity of the species, genus-specific interactions may assist in improving efficacy of biocontrol strains and consortia.

Pyoluteorin and 2,4-diacetylphloroglucinol are major contributors to Pseudomonas protegens Pf-5 biocontrol against Botrytis cinerea in cannabis.

Pseudomonas protegens Pf-5 is an effective biocontrol agent that protects many crops against pathogens, including the fungal pathogen Botrytis cinerea causing gray mold disease in Cannabis sativa crops. Previous studies have demonstrated the important role of antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (DAPG) in Pf-5-mediated biocontrol. To assess the potential involvement of PLT and DAPG in the biocontrol exerted by Pf-5 against B. cinerea in the phyllosphere of C. sativa, two knockout Pf-5 mutants were generated by in-frame deletion of genes pltD or phlA, required for the synthesis of PLT or DAPG respectively, using a two-step allelic exchange method. Additionally, two complemented mutants were constructed by introducing a multicopy plasmid carrying the deleted gene into each deletion mutant. In vitro confrontation assays revealed that deletion mutant ∆pltD inhibited B. cinerea growth significantly less than wild-type Pf-5, supporting antifungal activity of PLT. However, deletion mutant ∆phlA inhibited mycelial growth significantly more than the wild-type, hypothetically due to a co-regulation of PLT and DAPG biosynthesis pathways. Both complemented mutants recovered in vitro inhibition levels similar to that of the wild-type. In subsequent growth chamber inoculation trials, characterization of gray mold disease symptoms on infected cannabis plants revealed that both ∆pltD and ∆phlA significantly lost a part of their biocontrol capabilities, achieving only 10 and 19% disease reduction respectively, compared to 40% achieved by inoculation with the wild-type. Finally, both complemented mutants recovered biocontrol capabilities in planta similar to that of the wild-type. These results indicate that intact biosynthesis pathways for production of PLT and DAPG are required for the optimal antagonistic activity of P. protegens Pf-5 against B. cinerea in the cannabis phyllosphere.

Phytobeneficial traits of rhizobacteria under the control of multiple molecular dialogues.

SummaryPseudomonads play crucial roles in plant growth promotion and control of plant diseases. However, under natural conditions, other microorganisms competing for the same nutrient resources in the rhizosphere may exert negative control over their phytobeneficial characteristics. We assessed the expression of phytobeneficial genes involved in biocontrol, biostimulation and iron regulation such as, phlD, hcnA, acdS, and iron‐small regulatory RNAs prrF1 and prrF2 in Pseudomonas brassicacearum co‐cultivated with three phytopathogenic fungi, and two rhizobacteria in the presence or absence of Brassica napus, and in relation to iron availability. We found that the antifungal activity of P. brassicacearum depends mostly on the production of DAPG and not on HCN whose production is suppressed by fungi. We have also shown that the two‐competing bacterial strains modulate the plant growth promotion activity of P. brassicacearum by modifying the expression of phlD, hcnA and acdS according to iron availability. Overall, it allows us to better understand the complexity of the multiple molecular dialogues that take place underground between microorganisms and between plants and its rhizosphere microbiota and to show that synergy in favour of phytobeneficial gene expression may exist between different bacterial species.

Differential Contribution of Plant-Beneficial Functions from Pseudomonas kilonensis F113 to Root System Architecture Alterations in Arabidopsis thaliana and Zea mays

Fluorescent pseudomonads are playing key roles in plant-bacteria symbiotic interactions due to the multiple plant-beneficial functions (PBFs) they are harboring. The relative contributions of PBFs to plant-stimulatory effects of the well-known plant growth-promoting rhizobacteria Pseudomonas kilonensis F113 (formerly P. fluorescens F113) were investigated using a genetic approach. To this end, several deletion mutants were constructed, simple mutants ΔphlD (impaired in the biosynthesis of 2,4-diacetylphloroglucinol [DAPG]), ΔacdS (deficient in 1-aminocyclopropane-1-carboxylate deaminase activity), Δgcd (glucose dehydrogenase deficient, impaired in phosphate solubilization), and ΔnirS (nitrite reductase deficient), and a quadruple mutant (deficient in the four PBFs mentioned above). Every PBF activity was quantified in the wild-type strain and the five deletion mutants. This approach revealed few functional interactions between PBFs in vitro. In particular, biosynthesis of glucose dehydrogenase severely reduced the production of DAPG. Contrariwise, the DAPG production impacted positively, but to a lesser extent, phosphate solubilization. Inoculation of the F113 wild-type strain on Arabidopsis thaliana Col-0 and maize seedlings modified the root architecture of both plants. Mutant strain inoculations revealed that the relative contribution of each PBF differed according to the measured plant traits and that F113 plant-stimulatory effects did not correspond to the sum of each PBF relative contribution. Indeed, two PBF genes (ΔacdS and ΔnirS) had a significant impact on root-system architecture from both model plants, in in vitro and in vivo conditions. The current work underscored that few F113 PBFs seem to interact between each other in the free-living bacterial cells, whereas they control in concert Arabidopsis thaliana and maize growth and development.

Distribution of 2,4-Diacetylphloroglucinol Biosynthetic Genes among the Pseudomonas spp. Reveals Unexpected Polyphyletism.

Fluorescent pseudomonads protecting plant roots from phytopathogens by producing 2,4-diacetylphloroglucinol (DAPG) are considered to form a monophyletic lineage comprised of DAPG+ Pseudomonas strains in the “P. corrugata” and “P. protegens” subgroups of the “Pseudomonas fluorescens” group. However, DAPG production ability has not been investigated for many species of these two subgroups, and whether or not the DAPG+ Pseudomonas are truly monophyletic remained to be verified. Thus, the distribution of the DAPG biosynthetic operon (phlACBD genes) in the Pseudomonas spp. was investigated in sequenced genomes and type strains. Results showed that the DAPG+ Pseudomonas include species of the “P. fluorescens” group, i.e., P. protegens, P. brassicacearum, P. kilonensis, and P. thivervalensis, as expected, as well as P. gingeri in which it had not been documented. Surprisingly, they also include bacteria outside the “P. fluorescens” group, as exemplified by Pseudomonas sp. OT69, and even two Betaproteobacteria genera. The phl operon-based phylogenetic tree was substantially congruent with the one inferred from concatenated housekeeping genes rpoB, gyrB, and rrs. Contrariwise to current supposition, ancestral character reconstructions favored multiple independent acquisitions rather that one ancestral event followed by vertical inheritance. Indeed, based on synteny analyses, these acquisitions appeared to vary according to the Pseudomonas subgroup and even the phylogenetic groups within the subgroups. In conclusion, our study shows that the phl+ Pseudomonas populations form a polyphyletic group and suggests that DAPG biosynthesis might not be restricted to this genus. This is important to consider when assessing the ecological significance of phl+ bacterial populations in rhizosphere ecosystems.

Interaction between 2,4-Diacetylphloroglucinol- and Hydrogen Cyanide-Producing Pseudomonas brassicacearum LBUM300 and Clavibacter michiganensis subsp. michiganensis in the Tomato Rhizosphere.

We have previously demonstrated that inoculation of tomato plants with 2,4-diacetylphloroglucinol (DAPG)- and hydrogen cyanide (HCN)-producing Pseudomonas brassicacearum LBUM300 could significantly reduce bacterial canker symptoms caused by Clavibacter michiganensis subsp. michiganensis In this study, in order to better characterize the population dynamics of LBUM300 in the rhizosphere of tomato plants, we characterized the role played by DAPG and HCN production by LBUM300 on rhizosphere colonization of healthy and C. michiganensis subsp. michiganensis-infected tomato plants. The impact of C. michiganensis subsp. michiganensis presence on the expression of DAPG and HCN biosynthetic genes in the rhizosphere was also examined. In planta assays were performed using combinations of C. michiganensis subsp. michiganensis and wild-type LBUM300 or DAPG (LBUM300ΔphlD) or HCN (LBUM300ΔhcnC) isogenic mutant strains. Populations of LBUM300 and phlD and hcnC gene expression levels were quantified in rhizosphere soil at several time points up to 264 h postinoculation using culture-independent quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR) TaqMan assays, respectively. The presence of C. michiganensis subsp. michiganensis significantly increased rhizospheric populations of LBUM300. In C. michiganensis subsp. michiganensis-infected tomato rhizospheres, the populations of wild-type LBUM300 and strain LBUM300ΔhcnC, both producing DAPG, were significantly higher than the population of strain LBUM300ΔphlD A significant upregulation of phlD expression was observed in the presence of C. michiganensis subsp. michiganensis, while hcnC expression was only slightly increased in the mutant strain LBUM300ΔphlD when C. michiganensis subsp. michiganensis was present. Additionally, biofilm production was found to be significantly reduced in strain LBUM300ΔphlD compared to the wild-type and LBUM300ΔhcnC strains.IMPORTANCE The results of this study suggest that C. michiganensis subsp. michiganensis infection of tomato plants contributes to increasing rhizospheric populations of LBUM300, a biocontrol agent, as well as the overexpression of the DAPG biosynthetic operon in this bacterium. The increasing rhizospheric populations of LBUM300 represent one of the key factors in controlling C. michiganensis subsp. michiganensis in tomato plants, as DAPG-producing bacteria have shown the ability to decrease bacterial canker symptoms in tomato plants.

An Interspecies Signaling System Mediated by Fusaric Acid Has Parallel Effects on Antifungal Metabolite Production by Pseudomonas protegens Strain Pf-5 and Antibiosis of Fusarium spp.

Pseudomonas protegens strain Pf-5 is a rhizosphere bacterium that suppresses soilborne plant diseases and produces at least seven different secondary metabolites with antifungal properties. We derived mutants of Pf-5 with single and multiple mutations in biosynthesis genes for seven antifungal metabolites: 2,4-diacetylphoroglucinol (DAPG), pyrrolnitrin, pyoluteorin, hydrogen cyanide, rhizoxin, orfamide A, and toxoflavin. These mutants were tested for inhibition of the pathogens Fusarium verticillioides and Fusarium oxysporum f. sp. pisi. Rhizoxin, pyrrolnitrin, and DAPG were found to be primarily responsible for fungal antagonism by Pf-5. Previously, other workers showed that the mycotoxin fusaric acid, which is produced by many Fusarium species, including F. verticillioides, inhibited the production of DAPG by Pseudomonas spp. In this study, amendment of culture media with fusaric acid decreased DAPG production, increased pyoluteorin production, and had no consistent influence on pyrrolnitrin or orfamide A production by Pf-5. Fusaric acid also altered the transcription of biosynthetic genes, indicating that the mycotoxin influenced antibiotic production by Pf-5 at the transcriptional level. Addition of fusaric acid to the culture medium reduced antibiosis of F. verticillioides by Pf-5 and derivative strains that produce DAPG but had no effect on antibiosis by Pf-5 derivatives that suppressed F. verticillioides due to pyrrolnitrin or rhizoxin production. Our results demonstrated the importance of three compounds, rhizoxin, pyrrolnitrin, and DAPG, in suppression of Fusarium spp. by Pf-5 and confirmed that an interspecies signaling system mediated by fusaric acid had parallel effects on antifungal metabolite production and antibiosis by the bacterial biological control organism.

Production of DAPG and HCN by Pseudomonas sp. LBUM300 Contributes to the Biological Control of Bacterial Canker of Tomato

Bacterial canker caused by Clavibacter michiganensis subsp. michiganensis is known to cause significant economic losses to tomato production worldwide. Biological control has been proposed as an alternative to current chemical containment methods, which are often inefficient and may leave adverse effects on the environment. However, only little headway has so far been made in developing biocontrol strategies against C. michiganensis subsp. michiganensis. To address this knowledge gap, we investigated the antagonistic capacity of PCA, produced by Pseudomonas sp. LBUM223, and DAPG and HCN, both produced by Pseudomonas sp. LBUM300, on C. michiganensis subsp. michiganensis under in vitro and in planta conditions. Nonsynthesizing isogenic mutants of the producer strains were also developed to further dissect the role of each individual metabolite on C. michiganensis subsp. michiganensis biological control. Novel specific quantitative polymerase chain reaction TaqMan assays allowed quantification of C. michiganensis subsp. michiganensis in tomato plants and rhizospheric soil. Pseudomonas spp. LBUM223 and LBUM300 significantly repressed C. michiganensis subsp. michiganensis growth in vitro, while their respective nonproducing mutants showed less or no significant antagonistic activity. In planta, only Pseudomonas sp. LBUM300 was capable of significantly reducing disease development and C. michiganensis subsp. michiganensis rhizospheric population, suggesting that the production of both DAPG and HCN was involved. In summary, simultaneous DAPG/HCN production by Pseudomonas sp. LBUM300 shows great potential for controlling bacterial canker of tomato.

Cloning and Expression of Genes Encoding Enzymes Involved in the Synthesis of Phenolic Antibiotic in Recombinant E. Coli

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) causes serious public health problems throughout the world. In order to find an appropriate antibiotic against S. aureus scientists have focused on 2,4-diacetylphloroglucinol (DAPG) antibiotic produced by certain Pseudomonas spp. strains. Pseudomonas sp. strains residing in the rhizosphere have been considered due to their ability to produce antimicrobial metabolites which protect plants against different types of pathogens. In this study, Pseudomonas fluorescens UTPf100 was selected among 103 different Iranian isolates as an overproducer of DAPG on the basis of biological control and DAPG production. From this strain we isolated four genes that directly affect DAPG biosynthesis. The isolation was then confirmed by nested PCR and digestion. The encoding sequences responsible for DAPG biosynthesis were sub-cloned to pET28a (+) as an expression vector and transformed in E. coli BL21. To the best of our knowledge this is the first report for expression of the operon responsible for DAPG synthesys in recombinant E. coli.

Mannitol Amendment as a Carbon Source in a Bean-based Formulation Enhances Biocontrol Efficacy of a 2,4-diacetylphloroglucinol-producing Pseudomonas sp. NJ134 Against Tomato Fusarium Wilt

Fusarium wilt caused by Fusarium oxysporum has become a serious problem world-wide and relies heavily on chemical fungicides. We selected Pseudomonas sp. NJ134 to develop an effective biocontrol strategy. This strain shows strong antagonistic activity against F. oxysporum. Biochemical analyses of ethyl-acetate extracts of NJ134 culture filtrates showed that 2,4-diacetylphloroglucinol (DAPG) was the major compound inhibiting in vitro growth of F. oxysporum. DAPG production was greatly enhanced in the NJ134 strain by adding mannitol to the growth media, and in vitro antagonistic activity against F. oxysporum increased. Bioformulations developed from growth of NJ134 in sterile bean media with mannitol as the carbon source under plastic bags resulted in effective biocontrol efficacy against Fusarium wilt. The efficacy of the bioformulated product depended on the carbon source and dose. Mannitol amendment in the bean-based formulation showed strong effective biocontrol against tomato Fusarium wilt through increased DAPG levels and a higher cell density compared to that in a glucose-amended formulation. These results suggest that this bioformulated product could be a new effective biocontrol system to control Fusarium wilt in the field.

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett–Burman design (PBD). Only Zn 2+, Mn 2+ and MoO 4 2− were the most significant components ( p < 0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn 2+ (ZnSO 4·7H 2O), Mn 2+ (MnCl 2·4H 2O), and MoO 4 2− (Na 2MoO 4·2H 2O) were 83, 42 and 135 μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14 L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25 μg DAPG/g carrier) when stored at 28 °C for 6 months. The structure of the purified DAPG was confirmed using 1H NMR and mass spectrometry.

Phenazine antibiotics produced by fluorescent pseudomonads contribute to natural soil suppressiveness to Fusarium wilt

Natural disease-suppressive soils provide an untapped resource for the discovery of novel beneficial microorganisms and traits. For most suppressive soils, however, the consortia of microorganisms and mechanisms involved in pathogen control are unknown. To date, soil suppressiveness to Fusarium wilt disease has been ascribed to carbon and iron competition between pathogenic Fusarium oxysporum and resident non-pathogenic F. oxysporum and fluorescent pseudomonads. In this study, the role of bacterial antibiosis in Fusarium wilt suppressiveness was assessed by comparing the densities, diversity and activity of fluorescent Pseudomonas species producing 2,4-diacetylphloroglucinol (DAPG) (phlD+) or phenazine (phzC+) antibiotics. The frequencies of phlD+ populations were similar in the suppressive and conducive soils but their genotypic diversity differed significantly. However, phlD genotypes from the two soils were equally effective in suppressing Fusarium wilt, either alone or in combination with non-pathogenic F. oxysporum strain Fo47. A mutant deficient in DAPG production provided a similar level of control as its parental strain, suggesting that this antibiotic does not play a major role. In contrast, phzC+ pseudomonads were only detected in the suppressive soil. Representative phzC+ isolates of five distinct genotypes did not suppress Fusarium wilt on their own, but acted synergistically in combination with strain Fo47. This increased level of disease suppression was ascribed to phenazine production as the phenazine-deficient mutant was not effective. These results suggest, for the first time, that redox-active phenazines produced by fluorescent pseudomonads contribute to the natural soil suppressiveness to Fusarium wilt disease and may act in synergy with carbon competition by resident non-pathogenic F. oxysporum.

Evaluation of fluorescent pseudomonads for plant growth promotion, antifungal activity against Rhizoctonia solani on common bean, and biocontrol potential

Fluorescent pseudomonads are ubiquitous bacteria that are common inhabitants of the rhizosphere and are the most studied group within the genus Pseudomonas. Bacterial isolates ( n = 103) from the rhizosphere of wheat and common bean were assessed as potential biocontrol agents in this study. Fungal inhibition tests were performed by a plate assay in which each isolate was tested directly for the production of hydrogen cyanide, protease, siderophore and cellulase. Production of DAPG was verified by using an analytical high performance liquid chromatography assay (HPLC). Plant growth promotion was assessed in phytochamber trials and biocontrol activity was evaluated in greenhouse trials. In all, 52 bacterial isolates with antifungal activity against Rhizoctonia solani were found. Of the 52 isolates, 41 were selected according to their high efficiency in in vitro antagonism, which was shown as inhibition zones in the dual-culture assay. Six of the 41 rhizobacteria, including isolates UTPF7, UTPF13, UTPF18, UTPF22, UTPF27 and strain CHA0 produced HCN. Production of protease enzyme was detected for all isolates excluding UTPF30 isolate. Although some stains appeared not to produce any compound with affinity for ferric iron, other isolates produced prolific amounts, creating a large zone of orange (up to 160 mm 2, i.e., UTPF16). Seventeen of 41 isolates of fluorescent pseudomonads including strain CHAO produced different amounts of DAPG ranging from 0.6 to 11.4 ng/10 8 cfu. A total of 39 isolates induced statistically significant effects on plant growth compared with the non-treated control for at least one parameter. The predominant influence observed was increased root length. No bacteria could completely protect the plant against R. solani, although all isolates significantly increased fresh weight as compared to the infested control in greenhouse trials. Pseudomonas fluorescens isolates UTPF16 and UTPF26 significantly ( P < 0.05) decreased the number of seedlings with damping-off symptoms in the means of the experiments.

Influence of Nitrogen Source on 2,4-diacetylphloroglucinol Production by the Biocontrol Strain Pf-5

The production of 2,4-diacetylphloroglucinol (DAPG) by the biocontrol agent Pseudomonas fluorescens Pf-5 was studied in nutrient-solution based media with varying nitrogen content. No production of DAPG was observed when organic nitrogen was omitted from the media, regardless of the inorganic nitrogen source used. Furthermore, a micromolar concentration range of organic nitrogen was insufficient to sustain production. When a millimolar concentration range of organic nitrogen was used, DAPG production was observed in the medium with ammonium as the inorganic nitrogen source. No production was observed in the treatments with ammonium and nitrate or nitrate only, despite growth of the bacterial strain being the same for all treatments. These results suggest that it is possible to manipulate the nutrient solution to increase the reliability and efficacy of biological control agents.

Novel Screen for Investigating In Situ Rhizosphere Production of the Antibiotic 2,4‐Diacetylphloroglucinol by Bacterial Inocula

The rhizosphere is a complex zone of multitrophic interactions comprising plant roots, associated bacteria, fungi, and micro‐, meso‐, and macro‐fauna. Of considerable importance in this system is the production of antibiotics by root‐associated or “rhizo” bacteria. This is a widespread phenomenon of which a much‐studied exemplar is the production by pseudomonads of 2,4‐diacetylphloroglucinol (DAPG), known to be effective in the suppression of soil‐borne fungal pathogens. Rapid advances in understanding the molecular and biochemical bases of antibiotic (particularly DAPG) production have been made. However, our understanding of in situ antibiotic production currently lags behind this. There is therefore a need for a rapid soil‐based screen with which to identify antibiotic producers under rhizosphere C‐flow conditions. Here, a novel “rhizocosm,” comprising porous pipe and Rhizon sampler®, was superior to a simple, nonperfusing incubation‐type microcosm with respect to supporting a rhizobacterial inoculum. Its use as a screening tool is illustrated by screening known DAPG‐producing inocula in soil continuously supplied with simulated rhizosphere carbon flow. The DAPG was then extracted from soil using acetone and quantified by high‐performance liquid chromatography (HPLC). Simple sugars (glucose and fructose) stimulated the greatest DAPG production, while glucose with an additional organic or amino acid, or with a signal molecule, resulted in strongly variable DAPG expression.

Influence of mineral amendment on disease suppressive activity of Pseudomonas fluorescens to Fusarium wilt of chickpea

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceri causes considerable yield loss of chickpea. Pseudomonas fluorescens4-92 (Pf4-92) strain can suppress the disease. Amendment of zinc EDTA and copper EDTA could not suppress the disease significantly when used alone; however, they significantly suppressed the disease in presence of Pf4-92. In vitro observation showed that at 40, 30 and 20microgml(-1) concentrations of these minerals, i.e. Zn, Cu and Zn plus Cu, respectively, completely repressed the production of the phytotoxin, fusaric acid (FA). FA concentration (0.5microgml(-1)) has been shown to suppress the production of 2,4-diacetylphloroglucinol (DAPG) by Pf4-92, and DAPG, salicylic acid, pyochelin and pyoluteorin production was enhanced by these mineral amendments. In rockwool bioassays, Zn, Cu and Zn plus Cu amendments reduced FA production and enhanced DAPG production. This study demonstrates that Zn and Cu enhance biocontrol activity by reducing FA produced by the pathogen, F. oxysporum f. sp. ciceri.

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

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. 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. 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. 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.

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.