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.     

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.     

Production of Cyclic Lipopeptides by Pseudomonas fluorescens Strains in Bulk Soil and in the Sugar Beet Rhizosphere

The production of cyclic lipopeptides (CLPs) with antifungal and biosurfactant properties by Pseudomonas fluorescens strains was investigated in bulk soil and in the sugar beet rhizosphere. Purified CLPs (viscosinamide, tensin, and amphisin) were first shown to remain highly stable and extractable (90%) when applied (ca. 5 μg g⁻¹) to sterile soil, whereas all three compounds were degraded over 1 to 3 weeks in nonsterile soil. When a whole-cell inoculum of P. fluorescens strain DR54 containing a cell-bound pool of viscosinamide was added to the nonsterile soil, declining CLP concentrations were observed over a week. By comparison, addition of the strains 96.578 and DSS73 without cell-bound CLP pools did not result in detectable tensin or amphisin in the soil. In contrast, when sugar beet seeds were coated with the CLP-producing strains and subsequently germinated in nonsterile soil, strain DR54 maintained a high and constant viscosinamide level in the young rhizosphere for ~2 days while strains 96.578 and DSS73 exhibited significant production (net accumulation) of tensin or amphisin, reaching a maximum level after 2 days. All three CLPs remained detectable for several days in the rhizosphere. Subsequent tests of five other CLP-producing P. fluorescens strains also demonstrated significant production in the young rhizosphere. The results thus provide evidence that production of different CLPs is a common trait among many P. fluorescens strains in the soil environment, and further, that the production is taking place only in specific habitats like the rhizosphere of germinating sugar beet seeds rather than in the bulk soil.     

Plasmid Stability in Pseudomonas fluorescens in the Rhizosphere

Plasmids belonging to various incompatibility (Inc) groups were introduced into the efficiently root-colonizing strain Pseudomonas fluorescens WCS365, and their stabilities in complex and minimal media and in the rhizospheres of tomato, wheat, and potato plants grown under gnotobiotic conditions without selection pressure were tested. The IncP plasmid was found to be highly unstable under all conditions tested, whereas the IncQ and IncW plasmids showed intermediate stabilities and the plasmids pVSP41 and pWTT2081, for which the Inc group is unknown, both containing the origin of replication (rep) and stability (sta) regions of the Pseudomonas aeruginosa pVS1 replicon, were stably maintained under all conditions tested. Growth experiments in which cells of strain WCS365 carrying the plasmid pWTT2081 were grown in the presence of WCS365 without the plasmid showed that the presence of pWTT2081 acts as a burden. We conclude that pVSP41 and pWTT2081 are valuable as stable vectors for the functional analysis of genes involved in root colonization, provided that control cells carry the empty vector.     

Frequency and Biodiversity of 2,4-Diacetylphloroglucinol-Producing Bacteria Isolated from the Maize Rhizosphere at Different Stages of Plant Growth

A Pseudomonas 2,4-diacetylphloroglucinol (DAPG)-producing population that occurred naturally on the roots, in rhizosphere soil of Zea mays and in the nonrhizosphere soil was investigated in order to assess the microbial diversity at five stages of plant growth. A total of 1,716 isolates were obtained, and 188 of these isolates were able to produce DAPG. DAPG producers were isolated at each stage of plant growth, indicating that the maize rhizosphere is colonized by natural DAPG producers throughout development. The frequency of DAPG producers was very low in the first stage of plant growth and increased over time. An analysis of the level of biodiversity of the DAPG producers at the species level was performed by comparing the AluI restriction patterns of the 16S ribosomal DNAs (rDNAs) amplified by PCR from 167 isolates. This comparison allowed us to cluster the isolates into four amplified rDNA restriction analysis (ARDRA) groups, and the main group (ARDRA group 1) contained 89.8% of the isolates. The diversity of the 150 isolates belonging to ARDRA group 1 was analyzed by the random amplified polymorphic DNA (RAPD) technique. An analysis of RAPD patterns by a molecular variance method revealed that there was a high level of genetic diversity in this population and that the genetic diversity was related to plant age. Finally, we found that some of the DAPG producers, which originated from all stages of plant growth, had the same genotype. These DAPG producers could be exploited in future screening programs for biocontrol agents.     

Uptake of 2,4-Dichlorophenoxyacetic Acid by Pseudomonas fluorescens

Factors influencing the uptake of the sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D), under conditions in which no net metabolism occurred, were investigated in an effort to determine both the significance of "non-metabolic" uptake as a potential agent in reducing pesticide levels and the mechanisms involved. Uptake of 2,4-D was affected by pH, temperature, and the presence of other organic and inorganic compounds. Uptake was more pronounced at pH values less than 6, which implies that there may be some interaction between charged groups on the cell and the ionized carboxyl group of 2,4-D. Active transport, carrier-mediated diffusion, passive diffusion, and adsorption were considered as possible mechanisms. Though uptake was inhibited by glucose, sodium azide, and fluorodinitrobenzene (but not by uranyl ion), 2,4-D was not accumulated against a concentration gradient, a necessary consequence of an active transport system, nor was isotope counterflow found to occur. Thus, carrier-mediated diffusion was finally precluded, implying that uptake probably occurs by a two-step process: sorption onto the cell wall followed by passive diffusion into the cytoplasm.     

Fusaric Acid-Producing Strains of Fusarium oxysporum Alter 2,4-Diacetylphloroglucinol Biosynthetic Gene Expression in Pseudomonas fluorescens CHA0 In Vitro and in the Rhizosphere of Wheat

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA′-′lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA′-′lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA⁺) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA⁻) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.     

Quantification of 2,4-Diacetylphloroglucinol Produced by Fluorescent Pseudomonas spp. In Vitro and in the Rhizosphere of Wheat

The broad-spectrum antibiotic 2,4-diacetylphloroglucinol (Phl) is a major determinant in the biological control of a wide range of plant diseases by fluorescent Pseudomonas spp. A protocol was developed to readily isolate and quantify Phl from broth and agar cultures and from the rhizosphere environment of plants. Extraction with ethyl acetate at an acidic pH was suitable for both in vitro and in situ sources of Phl. For soil samples, the addition of an initial extraction step with 80% acetone at an acidic pH was highly effective in eliminating polar organic soil components, such as humic and fulvic acids, which can interfere with Phl detection by high-performance liquid chromotography. The efficiency of Phl recovery from soil by a single extraction averaged 54.6%, and a second extraction added another 6.1%. These yields were substantially greater than those achieved by several standard protocols commonly used to extract polar phenolic compounds from soil. For the first time Phl was isolated from the rhizosphere environment in raw soil. Following application of Pseudomonas fluorescens Q2-87 and the Phl-overproducing strain Q2-87(pPHL5122) to the seeds of wheat, 2.1 and 2.4 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Ritzville silt loam; 0.47 and 1.3 (mu)g of Phl/g of root plus rhizosphere soil, respectively, were isolated from wheat grown in a Shano silt loam. However, when the amount of Phl was calculated on the basis of cell density, Q2-87(pPHL5122) produced seven and six times more antibiotic than Q2-87 in Ritzville silt loam, and Shano silt loam, respectively.     

Effect of 2,4-Diacetylphloroglucinol Producing,Overproducing, and Nonproducing Pseudomonas fluorescens F113 in the Rhizosphere of Pea

Pseudomonas fluorescens F113lacZY and modified strains carrying different function modifications were assessed for their impact in the rhizosphere of pea. Strain F113lacZY naturally produces the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl) useful in plant disease control. The first modified strain of F113 was repressed in production of Phl, creating the Phl negative strain F113G22. The second was a plasmid based overproducer of Phl (F113Rif (pCUGP)). Both the F113lacZY and the F113Rif (pCUGP) strains increased the rhizoplane fungal populations, whereas the same strains reduced the rhizosphere soil fungal populations with respect to the control. Similar results were found with the rhizoplane and rhizosphere soil bacterial populations. The F113G22 treatment resulted in a significantly greater indigenous fluorescent Pseudomonas population than the F113lacZY and F113Rif (pCUGP) treatments and a greater total Pseudomonas population than the control, F113lacZY, and F113Rif (pCUGP) treatments. Overproduction of Phl did not affect the establishment of the introduced Pseudomonas population. None of the inocula displaced the indigenous populations, but the F113G22 inocula had an additive effect on the total Pseudomonas population. P (phosphatase), S (sulphatase), and N (urease) cycle enzyme activities were increased while C (glucosidase, NAGase) cycle activities were decreased by the F113lacZY and F113Rif (pCUGP) treatments, suggesting C leakage from the roots. Overall, most effects of inoculation compared to the wild type were found with the non-Phl-producing strain. Overproduction of Phl had little environmental effect in relation to wild-type inocula.     

Effect of Introduced Pseudomonas fluorescens Strains on Soil Nematode and Protozoan Populations in the Rhizosphere of Wheat and Pea

Abstract Previous studies have shown that inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 increased mineralization of organic nitrogen in the rhizosphere. In contrast, inoculation of the same strains onto wheat seeds reduced mineralization of N from organic residues incorporated into soil. In the present study, we report on a likely explanation of this phenomenon, which appears to be governed by the effect of plant-microbe interactions on bacterial-feeding nematodes and protozoa. In soil microcosm tests, inoculation of pea seeds with Pseudomonas fluorescens strains F113lacZY or F113G22 resulted in an increase in the number of nematodes and protozoa in the rhizosphere as compared to noninoculated controls. This trend was repeated using a model sand system into which the bacteriophagous nematode Caenorhabditis elegans was introduced. It was subsequently found that non-inoculated germinating pea seeds exerted a nematicidal effect on C. elegans, which was remedied by inoculation with either strain F113lacZY or F113G22. This suggests that nematicidal compounds released by the germinating pea seeds were metabolized by the microbial inoculants before they affected nematode populations in the spermosphere or rhizosphere of pea. In contrast, inoculation of wheat plants resulted in significantly lower nematode populations in the rhizosphere, whereas protozoan numbers were unaffected. No nematicidal effects of inoculated or noninoculated wheat seeds could be found, suggesting that microfaunal populations were affected at a later stage during plant growth. Because of their key roles in accelerating the turnover of microbially immobilized N and organic matter, plants that support a larger microfaunal population are likely to benefit from a higher availability of inorganic nitrogen. Therefore, an understanding of plant-microbe interactions and their effects on soil microfaunal populations is essential in order to assess the effects of microbial inocula on plant mineral nutrition. Received: 27 May 1999; Accepted: 15 July 1999; Online Publication: 17 December 1999     

Real-Time PCR Assays for Quantification and Differentiation of Vibrio vulnificus Strains in Oysters and Water

Vibrio vulnificus is an autochthonous estuarine bacterium and a pathogen that is frequently transmitted via raw shellfish. Septicemia can occur within 24 h; however, isolation and confirmation from water and oysters require days. Real-time PCR assays were developed to detect and differentiate two 16S rRNA variants, types A and B, which were previously associated with environmental sources and clinical fatalities, respectively. Both assays could detect 10² to 10³ V. vulnificus total cells in seeded estuarine water and in oyster homogenates. PCR assays on 11 reference V. vulnificus strains and 22 nontarget species gave expected results (type A or B for V. vulnificus and negative for nontarget species). The relationship between cell number and cycle threshold for the assays was linear (R² = >0.93). The type A/B ratio of Florida clinical isolates was compared to that of isolates from oysters harvested in Florida waters. This ratio was 19:17 in clinical isolates and 5:8 (n = 26) in oysters harvested from restricted sites with poor water quality but was 10:1 (n = 22) in oysters from permitted sites with good water quality. A substantial percentage of isolates from oysters (19.4%) were type AB (both primer sets amplified), but no isolates from overlying waters were type AB. The real-time PCR assays were sensitive, specific, and quantitative in water samples and could also differentiate the strains in oysters without requiring isolation of V. vulnificus and may therefore be useful for rapid detection of the pathogen in shellfish and water, as well as further investigation of its population dynamics.     

同步PCR技术及其在植物核酸分子定量中的应用

同步PCR是一种集生化、光电和计算机技术于一体的封闭式DNA扩增系统,采用荧光染料将扩增与检测过程结合在一起,实现了在PCR过程中在线显示PCR反应,通过检测荧光强度来绝对定量起始模板的拷贝数。该技术大大简化和加速了核酸分子的定量过程,不仅快速、灵敏、准确、重复性好,而且很容易计算出待测样品中核酸分子的绝对起始拷贝数。同微阵列等分子生物技术一起,同步PcR技术将会在功能基因解析和病害分子诊断等方面发挥重要作用。本综述除了介绍同步．PCR技术的原理和应用外,还介绍了定量拟南芥,Aux／正4,4基因的转录水平的实验,并就同步PCR操作过程中的问题进行了讨论。     

Engineering Pseudomonas fluorescens for Biodegradation of 2,4-Dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10°C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

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 (10⁷ 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 10⁵ 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.     

Quantification of Tetracycline Resistance Genes in Feedlot Lagoons by Real-Time PCR

A new real-time PCR method is presented that detects and quantifies three tetracycline resistance (Tc^r) genes [tet(O), tet(W), and tet(Q)] in mixed microbial communities resident in feedlot lagoon wastewater. Tc^r gene real-time TaqMan primer-probe sets were developed and optimized to quantify the Tc^r genes present in seven different cattle feedlot lagoons, to validate the method, and to assess whether resistance gene concentrations correlate with free-tetracycline levels in lagoon waters. The method proved to be sensitive across a wide range of gene concentrations and provided consistent and reproducible results from complex lagoon water samples. The log₁₀ of the sum of the three resistance gene concentrations was correlated with free-tetracycline levels (r² = 0.50, P < 0.001; n = 18), with the geometric means of individual resistance concentrations ranging from 4- to 8.3-fold greater in lagoon samples with above-median tetracycline levels (>1.95 μg/liter by enzyme-linked immunosorbent assay techniques) than in below-median lagoon samples. Of the three Tc^r genes tested, tet(W) and tet(Q) were more commonly found in lagoon water samples. Successful development of this real-time PCR assay will permit other studies quantifying Tc^r gene numbers in environmental and other samples.     

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.