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.     

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.     

Colonizing ability of Pseudomonas fluorescens 2112, among collections of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens spp. in pea rhizosphere

Pseudomonas fluorescens 2112, isolated in Korea as an indigenous antagonistic bacteria, can produce 2,4- diacetylphloroglucinol (2,4-DAPG) and the siderophore pyoveridin2112 for the control of phytophthora blight of red-pepper. P. fluorescens 2112 was classified into a new genotype C among the 17 genotypes of 2,4-DAPG producers, by phlD restriction fragment length polymorphism (RFLP). The colonizing ability of P. fluorescens 2112 in pea rhizosphere was equal to the well-known pea colonizers, P. fluorescens Q8r1 (genotype D) and MVP1-4 (genotype P), after 6 cycling cultivations for 18 weeks. Four tested 2,4- DAPG-producing Pseudomonas spp. could colonize with about a 96% dominance ratio against total bacteria in pea rhizosphere. The strain P. fluorescens 2112 was as good a colonizer as other Pseudomonas spp. genotypes in pea plant growth-promoting rhizobacteria.     

Role of ptsP, orfT, and sss recombinase genes in root colonization by Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.     

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。     

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.     

Wheat Cultivar-Specific Selection of 2,4-Diacetylphloroglucinol-Producing Fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> Species from Resident Soil Populations

An emerging body of evidence indicates a role for plant genotype as a determinant of the species and genetic composition of the saprophytic microbial community resident to the rhizosphere. In this study, experiments were conducted to determine the capacity of five different wheat cultivars to enhance resident populations and support introduced strains of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing fluorescent pseudomonads, a group of bacteria known to provide biological control of several soilborne diseases. When soils were cropped with three successive 28-day growth cycles of wheat, the 2,4-DAPG-producing strains were consistently recovered from the rhizosphere of the cultivar Lewjain, and commonly were present at populations higher than those recovered from other wheat cultivars. Based on restriction fragment length polymorphism and sequence analyses of phlD, a key gene involved in 2,4-DAPG production, two previously undefined phlD+ genotypes, referred to as genotypes PfZ and PfY, were discovered. Wheat cultivar Lewjain was the primary source of genotype PfY while cultivar Penawawa yielded the majority of genotype PfZ. Based on 16S rDNA sequence analysis, both new phlD genotypes were classified as P. fluorescens. Comparison of the rhizosphere competence of 2,4-DAPG-producing P. fluorescens Q2-87 (genotype B) and P. fluorescens LR3-A28 (genotype PfY) showed that both strains persisted at similar populations in the rhizosphere of all cultivars tested over a 30 day period when introduced as a seed inoculant. However, when strain LR3-A28 was applied as a soil inoculant, this strain was recovered at higher populations from the rhizosphere of wheat cultivar Lewjain than from the rhizospheres of two other cultivars. No cultivar effects were shown for strain Q2-87. Collectively, these results add further to evidence indicating a degree of specificity in interactions between plant cultivars and specific members of the saprophytic microbial community. Furthermore, as 2,4-DAPG-producing fluorescent Pseudomonas spp. have a central role in the spontaneous reduction in severity of take-all disease of wheat in response to continuous wheat monoculture, we postulate that the use of specific cultivars, such as Lewjain, which possess a superior capacity to enhance resident soil populations of these bacteria may have potential to reduce the length of the monoculture period required to induce natural suppressiveness of soils toward this disease.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Role of ptsP, orfT, and sss Recombinase Genes in Root Colonization by Pseudomonas fluorescens Q8r1-96

Pseudomonas fluorescens Q8r1-96 produces 2,4-diacetylphloroglucinol (2,4-DAPG), a polyketide antibiotic that suppresses a wide variety of soilborne fungal pathogens, including Gaeumannomyces graminis var. tritici, which causes take-all disease of wheat. Strain Q8r1-96 is representative of the D-genotype of 2,4-DAPG producers, which are exceptional because of their ability to aggressively colonize and maintain large populations on the roots of host plants, including wheat, pea, and sugar beet. In this study, three genes, an sss recombinase gene, ptsP, and orfT, which are important in the interaction of Pseudomonas spp. with various hosts, were investigated to determine their contributions to the unusual colonization properties of strain Q8r1-96. The sss recombinase and ptsP genes influence global processes, including phenotypic plasticity and organic nitrogen utilization, respectively. The orfT gene contributes to the pathogenicity of Pseudomonas aeruginosa in plants and animals and is conserved among saprophytic rhizosphere pseudomonads, but its function is unknown. Clones containing these genes were identified in a Q8r1-96 genomic library, sequenced, and used to construct gene replacement mutants of Q8r1-96. Mutants were characterized to determine their 2,4-DAPG production, motility, fluorescence, colony morphology, exoprotease and hydrogen cyanide (HCN) production, carbon and nitrogen utilization, and ability to colonize the rhizosphere of wheat grown in natural soil. The ptsP mutant was impaired in wheat root colonization, whereas mutants with mutations in the sss recombinase gene and orfT were not. However, all three mutants were less competitive than wild-type P. fluorescens Q8r1-96 in the wheat rhizosphere when they were introduced into the soil by paired inoculation with the parental strain.     

Exploiting genotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization of superior root-colonizing P. fluorescens strain Q8r1-96

The genotypic diversity that occurs in natural populations of antagonistic microorganisms provides an enormous resource for improving biological control of plant diseases. In this study, we determined the diversity of indigenous 2,4-diacetylphloroglucinol (DAPG)-producing Pseudomonas spp. occurring on roots of wheat grown in a soil naturally suppressive to take-all disease of wheat. Among 101 isolates, 16 different groups were identified by random amplified polymorphic DNA (RAPD) analysis. One RAPD group made up 50% of the total population of DAPG-producing Pseudomonas spp. Both short- and long-term studies indicated that this dominant genotype, exemplified by P. fluorescens Q8r1-96, is highly adapted to the wheat rhizosphere. Q8r1-96 requires a much lower dose (only 10 to 100 CFU seed(-1) or soil(-1)) to establish high rhizosphere population densities (10(7) CFU g of root(-1)) than Q2-87 and 1M1-96, two genotypically different, DAPG-producing P. fluorescens strains. Q8r1-96 maintained a rhizosphere population density of approximately 10(5) CFU g of root(-1) after eight successive growth cycles of wheat in three different, raw virgin soils, whereas populations of Q2-87 and 1M1-96 dropped relatively quickly after five cycles and were not detectable after seven cycles. In short-term studies, strains Q8r1-96, Q2-87, and 1M1-96 did not differ in their ability to suppress take-all. After eight successive growth cycles, however, Q8r1-96 still provided control of take-all to the same level as obtained in the take-all suppressive soil, whereas Q2-87 and 1M1-96 gave no control anymore. Biochemical analyses indicated that the superior rhizosphere competence of Q8r1-96 is not related to in situ DAPG production levels. We postulate that certain rhizobacterial genotypes have evolved a preference for colonization of specific crops. By exploiting diversity of antagonistic rhizobacteria that share a common trait, biological control can be improved significantly.     

Identification and Characterization of a Gene Cluster for Synthesis of the Polyketide Antibiotic 2,4-Diacetylphloroglucinol from Pseudomonas fluorescens Q2-87

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) is produced by many strains of fluorescent Pseudomonas spp. with biocontrol activity against soilborne fungal plant pathogens. Genes required for 2,4-DAPG synthesis by P. fluorescens Q2-87 are encoded by a 6.5-kb fragment of genomic DNA that can transfer production of 2,4-DAPG to 2,4-DAPG-nonproducing recipient Pseudomonas strains. In this study the nucleotide sequence was determined for the 6.5-kb fragment and flanking regions of genomic DNA from strain Q2-87. Six open reading frames were identified, four of which (phlACBD) comprise an operon that includes a set of three genes (phlACB) conserved between eubacteria and archaebacteria and a gene (phlD) encoding a polyketide synthase with homology to chalcone and stilbene synthases from plants. The biosynthetic operon is flanked on either side by phlE and phlF, which code respectively for putative efflux and regulatory (repressor) proteins. Expression in Escherichia coli of phlA, phlC, phlB, and phlD, individually or in combination, identified a novel polyketide biosynthetic pathway in which PhlD is responsible for the production of monoacetylphloroglucinol (MAPG). PhlA, PhlC, and PhlB are necessary to convert MAPG to 2,4-DAPG, and they also may function in the synthesis of MAPG.     

Contribution of phlA and some metabolites of fluorescent pseudomonads to antifungal activity

Fluorescent Pseudomonas species are an important group of PGPR that suppress fungal root and seedling disease by production of antifungal metabolites such as 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, pyrolinitrin, siderophores and HCN. The compound 2,4-DAPG is a major determinant in biocontrol of plant pathogens. A 7.2 kbp chromosomal DNA region, carrying DAPG biosynthetic genes (phlA, phlC, phlB, phlD, phIE and phlF). Detecting the ph1 genes make them an ideal marker gene for 2,4-DAPG-producing fluorescent pseudomonad's. In this study we detected ph1A gene (that convert MAPG to 2,4-DAPG) using PCR assay with primers phlA-1r and phlA- f that enabled amplification of phlA sequences from fluorescent pseudomonad's from ARDRA group 1 and 3. We could detect phlA gene in P. fluorescens strains CHAO, Pf-44, Pf-1, Pf-2, Pf-3, Pf-17, Pf-62 and Pf-64, native isolates of Iran. The efficacy of this method for rapid assay characterizing rhizosphere population of 2,4-DAPG producing bacteria from soil of different area of Iran is in progress. We used a collection of 48 fluorescent pseudomonas strains in vitro, with known biological control activity against some soil born phytopathogenic fungi such as, Macrophomina phaseoli, Rhizoctonia solani Vericillium dahlia, Phytophthora nicotiana, Pythium spp. and Fusarium spp. and the potential to produce known secondary metabolites such as protease. Strains Pf-1, Pf-2, Pf-3, Pf-17, Pf-33 and Pf-44 showed the best antifungal activity against all fungi used in this study. Thirty-eight of 48 strains produced protease. The ability to rapidly characterize populations of 2,4-DAPG producers will greatly enhance our understanding of their role in the suppression of root disease.     

Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Genotypic and Phenotypic Diversity of phlD-Containing Pseudomonas Strains Isolated from the Rhizosphere of Wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2,4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

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.     

Comparison of Barley Succession and Take-All Disease as Environmental Factors Shaping the Rhizobacterial Community during Take-All Decline

The root disease take-all, caused by Gaeumannomyces graminis var. tritici, can be managed by monoculture-induced take-all decline (TAD). This natural biocontrol mechanism typically occurs after a take-all outbreak and is believed to arise from an enrichment of antagonistic populations in the rhizosphere. However, it is not known whether these changes are induced by the monoculture or by ecological rhizosphere conditions due to a disease outbreak and subsequent attenuation. This question was addressed by comparing the rhizosphere microflora of barley, either inoculated with the pathogen or noninoculated, in a microcosm experiment in five consecutive vegetation cycles. TAD occurred in soil inoculated with the pathogen but not in noninoculated soil. Bacterial community analysis using terminal restriction fragment length polymorphism of 16S rRNA showed pronounced population shifts in the successive vegetation cycles, but pathogen inoculation had little effect. To elucidate rhizobacterial dynamics during TAD development, a 16S rRNA-based taxonomic microarray was used. Actinobacteria were the prevailing indicators in the first vegetation cycle, whereas the third cycle—affected most severely by take-all—was characterized by Proteobacteria, Bacteroidetes, Chloroflexi, Planctomycetes, and Acidobacteria. Indicator taxa for the last cycle (TAD) belonged exclusively to Proteobacteria, including several genera with known biocontrol traits. Our results suggest that TAD involves monoculture-induced enrichment of plant-beneficial taxa.The root disease take-all, caused by the soilborne ascomycete fungus Gaeumannomyces graminis var. tritici, has a significant impact on the global production of wheat and barley (23). Usually, take-all is managed by crop rotation, but low disease levels may also be achieved by monoculture-induced disease suppressiveness (13, 50, 61). Indeed, a spontaneous reduction of disease severity can be observed during wheat or barley monoculture after at least one severe outbreak of the disease, a phenomenon called take-all decline (TAD). Disease symptoms then remain at a low level as long as monoculture continues. TAD is related to the enrichment of certain rhizosphere populations antagonistic to the pathogen (13) and parallel changes in G. graminis var. tritici populations (28).Though TAD is the best-studied example of induced disease suppressiveness (61), knowledge of the mechanisms involved in TAD is fragmentary. Take-all research so far has mainly focused on the role of fluorescent pseudomonads, as they are easy to cultivate and known to be antagonistic to various phytopathogens (21, 35, 62, 63). Several studies have indicated the involvement of other microorganisms in TAD, e.g., Bacillus species and various actinomycetes (3, 26, 45), but none of them has considered the whole bacterial community. Yet, it has been shown that community-based approaches are important in identifying microbial populations potentially involved in suppressiveness (10, 27).The first community-based assessment was carried out based on molecular fingerprinting and sequencing, and it showed that take-all disease of wheat induced changes in several rhizosphere bacterial populations (39). However, disease-suppressive stages were not included. More recently, the taxonomic microarray-based study of Sanguin et al. (48) revealed multiple changes in the rhizobacterial community of wheat when comparing take-all disease and suppressive stages, which paralleled changes in Pseudomonas populations (46). These differences were attributed to TAD, but it is not known whether they occurred as a result of repeated wheat cropping (leading to enrichment of particular bacterial taxa), a disease outbreak and subsequent attenuation (modifying nutrient conditions on roots), or both.Therefore, the objective of this study was to compare the significance of plant succession and take-all disease as environmental factors shaping the rhizobacterial community during TAD. Repeated barley cropping was carried out in microcosms inoculated with G. graminis var. tritici or not inoculated, and the physiologically active bacterial rhizosphere community was monitored using terminal restriction fragment length polymorphism (t-RFLP) (32), as well as microarray analysis of 16S rRNA.     

Antagonistic activity among 2,4-diacetylphloroglucinol-producing fluorescent Pseudomonas spp

Strains of fluorescent Pseudomonas spp. that produce 2,4-diacetylphloroglucinol (2,4-DAPG) differ in their ability to colonize roots. In this study, we screened 47 2,4-DAPG-producing strains representing17 distinct genotypes for antagonistic activity associated with the production of bacteriocins. Upon induction, over 70% of the strains inhibited the growth of other isolates in vitro. Greenhouse assays indicated that populations of sensitive strains in wheat rhizosphere soil declined more rapidly in the presence of antagonists than when introduced alone. Antagonism can influence the ability of biocontrol agents to establish and maintain effective population densities in situ.     

生防假单胞菌2P24中mvaT和mvaV基因对PcoI/PcoR群体感应系统的调控作用

【目的】自小麦全蚀病自然衰退土壤分离得到的荧光假单胞菌(Pseudomonas fluorescens)2P24,可防治多种由植物病原菌引起的土传病害。菌株2P24具有群体感应(quorum-sensing,QS)系统PcoI/PcoR,该系统影响生防菌2P24生物膜的形成以及其在小麦根围的定殖能力,从而影响2P24的生防能力。本文利用遗传学方法进一步研究了2P24中QS系统的调控途径。【方法】将QS系统信号合成基因pcoI的转录报告质粒p970Gm-pcoIp转入gacA基因突变菌株PM201中,再利用Tn5转座子对该菌株进行随机突变,筛选影响pcoI基因表达的调控因子。【结果】根据菌落颜色的变化筛选到2株突变菌株。Tn5插入位点和基因序列分析表明这2个突变体中Tn5破坏了同一个基因mvaT;设计引物利用PCR方法从2P24基因组中获得mvaT基因及其同源基因mvaV。转录融合报告实验表明:与野生菌株2P24相比,mvaT及mvaV突变体中pcoI基因的表达和N-乙酰高丝氨酸内酯的产量显著提高;HPLC试验表明mvaT和mvaV基因影响抗生素2,4-二乙酰基间苯三酚的合成。细菌双杂交试验证实,MvaT蛋白和MvaV蛋白在体内发生自身互作,这两个蛋白也可相互作用。【结论】以上结果表明mvaT和mvaV参与调控生防假单胞菌2P24的PcoI/PcoR群体感应系统,并可能影响其生防功能基因的表达。