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.     

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.     

Enhancing the biocontrol efficacy of Pseudomonas fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol

Pseudomonas fluorescens F113 is an effective biocontrol agent against Pythium ultimum, the causative agent of damping-off of sugarbeet seedlings. Biocontrol is mediated via the production of the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl). A genetic approach was used to further enhance the biocontrol ability of F113. Two genetically modified (GM) strains, P. fluorescens F113Rif (pCU8.3) and P. fluorescens F113Rif (pCUP9), were developed for enhanced Phl production and assessed for biocontrol efficacy and impact on sugarbeet in microcosm experiments. The multicopy plasmid pCU8.3 contains the biosynthetic genes (phlA, C, B and D) and the putative permease gene (phlE) of F113 cloned into the rhizosphere stable plasmid pME6010, independent of external promoters. The plasmid pCUP9 consists of the Phl biosynthetic genes cloned downstream of the constitutive Plac promoter in pBBR1MCS. Introduction of pCU8.3 and pCUP9 into P. fluorescens F113 significantly altered the kinetics of Phl biosynthesis when grown in SA medium. A significant and substantial increase in Phl production by the GM strains was observed in the early logarithmic phase and stationary phase of growth compared with the wild-type strain. In microcosm, the two Phl overproducing strains proved to be as effective at controlling damping-off disease as the proprietary fungicide treatment, indicating the potential of genetic modification for plant disease control.     

抗生素2,4-二乙酰基间苯三酚作为荧光假单胞菌2P24菌株生防功能因子的实证分析

荧光假单胞杆菌2P24菌株分离自小麦全蚀病自然衰退土壤,它是酚类抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)的高产菌,对多种土传病害具有较好的防治能力。利用同源重组构建2,4-DAPG合成基因的定位突变体,并对突变体进行基因互补,通过检测突变菌株和恢复突变菌株抗生素产量和生防效果确定2,4-DAPG在菌株2P24生防功能中的作用。实验中,定位突变体丧失产生抗生素和拮抗病原菌的能力,而恢复突变体的抗生素产量和拮抗能力均恢复至野生菌水平。在对番茄青枯病的防病试验中,2,4-DAPG突变体的防效低且下降快,而恢复突变体的生防能力与野生菌相当,且效果稳定。由此可确定2,4-DAPG是菌株2P24防治番茄青枯病的主要因子,在防效上起关键作用。     

Novel T-RFLP method to investigate six main groups of 2,4-diacetylphloroglucinol-producing pseudomonads in environmental samples

Strains of fluorescent pseudomonads producing 2,4-diacetylphloroglucinol (DAPG) are involved in the protection of plant roots against soil-borne plant pathogens. Recently, a multilocus sequence analysis of a world wide collection of DAPG-producers led to the identification of six main groups (A-F). In this study a T-RFLP method based on the phlD gene was developed to efficiently identify the members of these six groups in environmental samples. A combination of six restriction enzymes was identified which leads to group specific terminal fragments (T-RF). The detection limit of the phlD-T-RFLP method was determined for the two P. fluorescens strains F113 (group B) and CHA0 (group F) in rhizosphere samples and was found to be 5 × 10³ CFU/g and 5 × 10⁴ CFU/g respectively. PhlD-T-RFLP and phlD-DGGE analysis of wheat and maize root samples from greenhouse and field revealed similarly the presence of multilocus groups A, B and D. However, they were more frequently detected with phlD-T-RFLP. Additionally, groups C and F were detected in greenhouse samples but only by phlD-T-RFLP and not by phlD-DGGE. In conclusion, the new phlD-T-RFLP method proved to be a fast and reliable method to detect strains of the six main groups of DAPG-producers in environmental samples with an improved detection limit compared to phlD-DGGE.     

Impact of biocontrol agents Pseudomonas fluorescens CHA0 and its genetically modified derivatives on the diversity of culturable fungi in the rhizosphere of mungbean

AIMS: To assess whether Pseudomonas fluorescens strain CHA0 and its genetically modified derivatives, CHA0/pME3424 (antibiotic over-producer) and CHA89 (antibiotic-deficient) could have an impact on the fungal community structure and composition in the rhizosphere of mungbean. METHODS AND RESULTS: Under glasshouse conditions, mungbean was grown repeatedly in the same soil, which was inoculated with CHA0, CHA0/pME3424, CHA89 or was left untreated. Treatments were applied to soil at the start of each 36-day mungbean growth cycle, and their effects on the diversity of the rhizosphere populations of culturable fungi were assessed at the end of the first, second and third cycles. The effects of CHA0 and CHA0/pME3424 did differ from the controls while CHA89 did not. Whereas all major fungal species were frequently isolated from both bacterized and nonbacterized rhizospheres, certain fungal species were exclusively promoted or specifically suppressed from Pseudomonas-treated soils. In general, fungal diversity and equitability tended to decrease with time while species richness slightly increased. Whilst a total of 29 fungal species were isolated from the mungbean rhizosphere, only eight species colonized the root tissues. CONCLUSIONS: Soil inoculation with Ps. fluorescens CHA0 or CHA0/pME3424 altered fungal community structure in mungbean rhizosphere but strain CHA89 failed to produce such effect. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas fluorescens-mediated alteration in the composition and structure of fungal communities might have acute or lasting effects on ecosystem functioning. Furthermore, the study provides useful data pertinent to characterization of the fate of genetically modified inoculants (e.g. antibiotic-overproducing Pseudomonas strains) released into the environment.     

Enrichment and genotypic diversity of phlD-containing fluorescent Pseudomonas spp. in two soils after a century of wheat and flax monoculture

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) play a key role in the suppressiveness of some soils to take-all of wheat and other diseases caused by soilborne pathogens. Soils from side-by-side fields on the campus of North Dakota State University, Fargo, USA, which have undergone continuous wheat, continuous flax or crop rotation for over 100 years, were assayed for the presence of 2,4-DAPG producers. Flax and wheat monoculture, but not crop rotation, enriched for 2,4-DAPG producers, and population sizes of log 5.0 CFU g root(-1) or higher were detected in the rhizospheres of wheat and flax grown in the two monoculture soils. The composition of the genotypes enriched by the two crops differed. Four BOX-PCR genotypes (D, F, G, and J) and a new genotype (T) were detected among the 2,4-DAPG producers in the continuous flax soil, with F- and J-genotype isolates dominating (41 and 39% of the total, respectively). In contrast, two genotypes (D and I) were detected in the soil with continuous wheat, with D-genotype isolates comprising 77% of the total. In the crop-rotation soil, populations of 2,4-DAPG producers generally were below the detection limit, and only one genotype (J) was detected. Under growth-chamber and field conditions, D and I genotypes (enriched by wheat monoculture) colonized the wheat rhizosphere significantly better than isolates of other genotypes, while a J-genotype isolate colonized wheat and flax rhizospheres to the same extent. This study suggests that, over many years of monoculture, the crop species grown in a field enriches for genotypes of 2,4-DAPG producers from the reservoir of genotypes naturally present in the soil that are especially adapted to colonizing the rhizosphere of the crop grown.     

Differential ability of genotypes of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains to colonize the roots of pea plants

Indigenous populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent Pseudomonas spp. that occur naturally in suppressive soils are an enormous resource for improving biological control of plant diseases. Over 300 isolates of 2,4-DAPG-producing fluorescent Pseudomonas spp. were isolated from the rhizosphere of pea plants grown in soils that had undergone pea or wheat monoculture and were suppressive to Fusarium wilt or take-all, respectively. Representatives of seven genotypes, A, D, E, L, O, P, and Q, were isolated from both soils and identified by whole-cell repetitive sequence-based PCR (rep-PCR) with the BOXA1R primer, increasing by three (O, P, and Q) the number of genotypes identified previously among a worldwide collection of 2,4-DAPG producers. Fourteen isolates representing eight different genotypes were tested for their ability to colonize the rhizosphere of pea plants. Population densities of strains belonging to genotypes D and P were significantly greater than the densities of other genotypes and remained above log 6.0 CFU (g of root)(-1) over the entire 15-week experiment. Genetic profiles generated by rep-PCR or restriction fragment length polymorphism analysis of the 2,4-DAPG biosynthetic gene phlD were predictive of the rhizosphere competence of the introduced 2,4-DAPG-producing strains.     

Heterozygosis drives maize hybrids to select elite 2,4-diacethylphloroglucinol-producing Pseudomonas strains among resident soil populations

By comparing the distribution of two genomic markers among Pseudomonas strains recovered from the rhizosphere of two maize hybrids with those of strains recovered from the rhizosphere of their four respective parental lines, we showed that both hybrids supported more elite probiotic strains than the parents. Elite Pseudomonas strains showed genomic potential for both an appropriate in vitro 2,4-diacetylphloroglucinol (DAPG) productivity, and a superior root-colonization ability. The actual biocontrol and root-colonization abilities of these strains were confirmed by bioassays on five fungal strains and on axenic maize plants. Furthermore, results on the abundance and genetic diversity of resident DAPG+ Pseudomonas strains indicated that each hybrid was able to select its own specific DAPG+ population, whereas the four parental lines were not. The evidence that heterozygosis can drive maize plants to select elite probiotic rhizospheric DAPG+ Pseudomonas strains opens the way to a new strategy in the set up of plant breeding for low-input and organic agriculture.     

Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin

Fluorescent Pseudomonas strains producing the antimicrobial secondary metabolite 2,4-diacetylphloroglucinol (Phl) play a prominent role in the biocontrol of plant diseases. A subset of Phl-producing fluorescent Pseudomonas strains, which can additionally synthesize the antimicrobial compound pyoluteorin (Plt), appears to cluster separately from other fluorescent Pseudomonas spp. based on 16S rRNA gene analysis and shares at most 98.4% 16S rRNA gene sequence identity with any other Pseudomonas species. In this study, a polyphasic approach based on molecular and phenotypic methods was used to clarify the taxonomy of representative Phl⁺ Plt⁺ strains isolated from tobacco, cotton or wheat on different continents. Phl⁺ Plt⁺ strains clustered separately from their nearest phylogenetic neighbors (i.e. species from the ‘P. syringae’, ‘P. fluorescens’ and ‘P. chlororaphis’ species complexes) based on rpoB, rpoD or gyrB phylogenies. DNA-DNA hybridization experiments clarified that Phl⁺ Plt⁺ strains formed a tight genomospecies that was distinct from P. syringae, P. fluorescens, or P. chlororaphis type strains. Within Phl⁺ strains, the Phl⁺ Plt⁺ strains were differentiated from other biocontrol fluorescent Pseudomonas strains that produced Phl but not Plt, based on phenotypic and molecular data. Discriminative phenotypic characters were also identified by numerical taxonomic analysis and siderotyping. Altogether, this polyphasic approach supported the conclusion that Phl⁺ Plt⁺ fluorescent Pseudomonas strains belonged to a novel species for which the name Pseudomonas protegens is proposed, with CHA0^T (=CFBP 6595^T, =DSM 19095^T) as the type strain.     

Effect of long-term vineyard monoculture on rhizosphere populations of pseudomonads carrying the antimicrobial biosynthetic genes phlD and/or hcnAB

The impact of repeated culture of perennial plants (i.e. in long-term monoculture) on the ecology of plant-beneficial bacteria is unknown. Here, the influence of extremely long-term monocultures of grapevine (up to 1603 years) on rhizosphere populations of fluorescent pseudomonads carrying the biosynthetic genes phlD for 2,4-diacetylphloroglucinol and/or hcnAB for hydrogen cyanide was determined. Soils from long-term and adjacent short-term monoculture vineyards (or brushland) in four regions of Switzerland were baited with grapevine or tobacco plantlets, and rhizosphere pseudomonads were studied by most probable number (MPN)-PCR. Higher numbers and percentages of phlD ⁺ and of hcnAB ⁺ rhizosphere pseudomonads were detected on using soil from long-term vineyards. On focusing on phlD , restriction fragment length polymorphism profiling of the last phlD -positive MPN wells revealed seven phlD alleles (three exclusively on tobacco, thereof two new ones). Higher numbers of phlD alleles coincided with a lower prevalence of the allele displayed by the well-studied biocontrol strain Pseudomonas fluorescens F113. The prevalence of this allele was 35% for tobacco in long-term monoculture soils vs. >60% in the other three cases. We conclude that soils from long-term grapevine monocultures represent an untapped resource for isolating novel biocontrol Pseudomonas strains when tobacco is used as bait.     

荧光假单胞杆菌P13菌株对油菜化感作用的研究

通过化感试验研究荧光假单胞杆菌P13菌株对油菜种子萌发、幼苗生长的影响及其在油菜根际土壤和根部定植的能力。结果表明,P13菌株发酵液对油菜种子萌发没有明显的促进作用,稀释10倍的菌体发酵液处理种子与对照组无显著差异,而低浓度和高浓度都抑制种子萌发;田间试验发现P13菌株能促进植物幼苗生长,根长、苗高、干重和长1片叶子的株数均与对照组差异显著;1周内P13菌株在油菜根际土壤和根部定植良好,定植数量均达到107cfu/g以上。说明P13菌株可被开发为微生物菌剂,但在施用时不宜用作种子处理剂。     

A new DGGE protocol targeting 2,4-diacetylphloroglucinol biosynthetic gene phlD from phylogenetically contrasted biocontrol pseudomonads for assessment of disease-suppressive soils

In the rhizosphere, biocontrol pseudomonads producing 2,4-diacetylphloroglucinol (Phl) can protect plants from soil-borne pathogens. DGGE of phlD has been proposed to monitor these bacteria, but two distinct protocols were needed for analysis of both the 'Pseudomonas fluorescens' species complex and the strains from rrs restriction group ARDRA-1. Here, a single DGGE protocol performed on 668-bp GC-clamp-containing phlD amplicons was effective with both types of pseudomonads, and 36 reference biocontrol strains from the 'P. fluorescens' complex or group ARDRA-1 gave a total of 11 distinct DGGE bands. phlD amplicons with at least two to seven nucleotidic differences could be discriminated, and the discrimination level was similar to that of phlD restriction analysis with four enzymes. Multiple phlD-DGGE bands were obtained when studying rhizosphere soil containing indigenous phlD+ pseudomonads, and phlD diversity was higher when DGGE was implemented after incubation of tobacco rhizosphere extracts in semi-selective medium (MPN approach) in comparison with approaches based on direct analysis of rhizosphere DNA extracts or assessment of phlD+colonies. phlD-DGGE profiles differed for a soil suppressive and a soil conducive to black root rot of tobacco, and each soil yielded new phlD sequences. In conclusion, this DGGE protocol was useful for monitoring indigenous rhizosphere consortia of phlD+ pseudomonads.     

Biological control of soil-pathogenic nematodes infecting mungbean using Pseudomonas fluorescens

The addition of organic matters to soil has been explored as an alternative means of nematode control under field conditions. Several oil-seed cakes of neem (Azadirachta indica), castor (Ricinus communis), groundnut (Arachis hypogeae), linseed (Linum usitatissimum) and sunflower (Helianthus annuus) were found to be highly effective in reducing the multiplication of soil-pathogenic nematodes Meloidogyne incognita, Rotylenchulus reniformis, Tylenchorhynchus brassicae, etc. The plant growth parameters such as plant weight, per cent pollen fertility, number of pods per plant, root-nodulation and chlorophyll content of mungbean increased significantly. The multiplication rate of nematodes and number of root-galls were less in the presence of Pseudomonas fluorescens as compared to its absence. Damage caused by the nematodes was further reduced when P. fluorescens was added along with the oil-seed cakes. Neem cake was found most effective in combination with P. fluorescens.     

Bacterial endophyte-enhanced phytoremediation of the organochlorine herbicide 2,4-dichlorophenoxyacetic acid

2,4-Dichlorophenoxyacetic acid is a selective systemic herbicide for the control of broad-leaved weeds, which is widely used throughout the world. The persistence of its residues and its potential to migrate in the soil make it necessary to reduce its concentrations in contaminated soil and groundwater. The nature of this compound makes it particularly toxic to the broad-leaved plants, such as the poplar (Populus) and willow (Salix), which are often used in phytoremediation projects. We describe the inoculation of a model plant, the pea (Pisum sativum), with a genetically tagged bacterial endophyte that naturally possesses the ability to degrade 2,4-dichlorophenoxyacetic acid. The results showed that this strain actively colonized inoculated plants internally (and in the rhizosphere). Inoculated plants showed a higher capacity for 2,4-dichlorophenoxyacetic acid removal from soil and showed no 2,4-dichlorophenoxyacetic acid accumulation in their aerial tissues. This demonstrates the usefulness of bacterial endophytes to enhance the phytoremediation of herbicide-contaminated substrates and reduce levels of toxic herbicide residues in crop plants.     

Root colonization and iron nutritional status of a Pseudomonas fluorescens in different plant species

Root colonization and induction of an iron stress regulated promoter for siderophore production by Pseudomonas fluorescens 2-79RLI was studied in vitro and in the rhizosphere of different plant species. P. fluorescens 2-79RLI was previously genetically modified with an iron regulated ice nucleation reporter, which allowed calibration of ice nucleation activity with siderophore production. Initial experiments examined ice nucleation activity and siderophore production under different growth conditions in vitro. These studies demonstrated that P. fluorescens 2-79RLI could utilize both Fe-citrate and Fe-phytosiderophore as iron sources, suggesting that production of these compounds by plants would increase iron availability for P. fluorescens 2-79RLI in the rhizosphere. Fe demand and Fe stress were further shown to be a function of nutrient availability and were reduced when carbon was limiting for growth. Subsequent experiments extended these observations to rhizosphere cells. Cells were sampled from the rhizosphere and the rhizoplane. Results of a soil microcosm experiment showed that Fe stress was reduced for P. fluorescens 2-79RLI in the barley rhizosphere as compared to the cells in the rhizosphere.of lupin. In lupin, relative Fe stress of P. fluorescens 2-79RLI was greater at the root tip than in the lateral root zone. In a second experiment comparing zucchini and bean, iron stress was greater for P. fluorescens 2-79RLI associated with zucchini than with bean. In a third experiment with rape plants under P deficient conditions, addition of soluble P was shown to increase Fe stress for P. fluorescens 2-79RLI located at the root tip, but not in the lateral root zone. This study showed that Fe stress of P. fluorescens 2-79RLI in the rhizosphere may be influenced by plant species, P source, root zone and localization of the cells within the rhizosphere.     

Ecology of a plant growth-promoting strain of Pseudomonas fluorescens colonizing the maize endorhizosphere in tropical soil

Pseudomonas fluorescens strain BR-5 stimulated the growth of maize in a natural soil and inhibited fungal root pathogens in vitro. Strain BR-5 was detected inside plant cells, indicating that it is able to colonize the endorhizosphere. No significant effect was detected on soil or ectorhizosphere microbial population after inoculation of strain BR-5 onto seeds.     

Survival of genetically modified Pseudomonas fluorescens introduced into subtropical soil microcosms

Abstract A genetically modified strain of Pseudomonas fluorescens and its parent showed grossly similar decline rates following introduction into subtropical clay and sandy soils. In unplanted clay soit at pH 6.9 and 25°C, population densities declined progressively from about 10⁸ to 10³ colony forming units (cfu) g⁻¹ dry soil over 75 days, but in unplanted sandy soil the introduced populations could not be detected after 25 days. In clay soil at pH 8.7 or 4.7, or at environmental temperature, decay rates were enhanced as compared to those at pH 6.9 and 25°C. Counts of introduced strains in clay bulk soil and in rhizosphere and rhizoplane of maize suggested that the introduced bacteria competed well with the native bacteria, and colonized the roots at about 10⁶ cfu g⁻¹ dry root at 25°C, over 20 days. However, rhizoplane colonization was lower at environmental temperature. The decay rate of both strains was slower in planted than in unplanted sandy soil. The population densities in the rhizosphere and rhizoplane in the sandy soil were significantly lower than those in the clay soil. Both introduced strains colonized the maize roots in both soils, using seeds coated with bacteria in 1% carboxymethyl cellulose. Introduced cells were localized at different sites along the roots of plants developing in clay soil, with higher densities in the original (near the seeds) and root hair zones as compared to the intermediate zones. No significant difference was observed between the extent of root colonization of the genetically modified strain and its parent.     

Degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9

Abstract The aerobic degradation of 2,4-dihydroxybenzoate by Pseudomonas sp. BN9 was studied. Intact cells of Pseudomonas sp. BN9 grown with 2,4-dihydroxybenzoate oxidized 2,4-dihydroxybenzoate but not salicylate. Cell-free extracts of Pseudomonas sp. BN9 converted 2,4-dihydroxybenzoate after the addition of NAD(P)H. A partially purified protein fraction converted 2,4-dihydroxybenzoate with NADH to 1,2,4-trihydroxybenzene. 1,2,4-Trihydroxybenzene was converted by a 1,2-dioxygenase to maleylpyruvate, which was reduced by a NADH-dependent enzyme to 3-oxoadipate. 2,4-Dihydroxybenzoate 1-monooxygenase, 1,2,4-trihydroxybenzene 1,2-dioxygenase and maleylpyruvate reductase were induced in Pseudomonas sp. BN9 after growth with 2,4-dihydroxybenzoate.