Influence of soil type on the transfer of plasmid RP4p from Pseudomonas fluorescens to introduced recipient and to indigenous bacteria

Abstract Transfer of plasmid RP4p from introduced Pseudomonas fluorescens to a co-introduced recipient strain or to members of the indigenous bacterial population was studied in four different soils of varying texture planted with wheat. Donor and recipient strains showed good survival in the four soils throughout the experiment. The numbers of transconjugants found in donor and recipient experiments in two soils, Ede loamy sand and Löss silt loam were significantly higher in the rhizosphere than in corresponding bulk soil. In the remaining two soils, Montrond and Flevo silt loam, transconjugant numbers were not significantly higher in the rhizosphere than in the bulk soil.
The combined utilization of a specific bacteriophage eliminate the donor strain and the pat sequence as a specific marker to detect RP4p was found to be very efficient in detecting indigenous transconjugants under various environmental conditions. The numbers of indigenous transconjugants were consistently higher in rhizosphere than bull soil. A significant rhizosphere effect on transconjugant numbers of transconjugants were recovered from Flevo and Montrond silt loam; these soils possess characteristics such as clay or organic matter contents which may be favorable to conjugation.     

Prevalence of nptII and Tn5 in kanamycin-resistant bacteria from different environments

Abstract Kanamycin (Km)-resistant bacterial populations in different soil, river water, sewage and pig manure slurry samples were enumerated and their prevalence in the total populations determined. About 350 Km-resistant Gram-negative colonies grown in the presence of kanamycin were identified using a rapid presumptive identification scheme. They were then screened for the presence of Tn5 and npt II sequences using hybridization of cells in dot blots, of Southern-blotted genomic DNA extracts and of PCR amplification products. Colonies reacting positively with a 2.7 kb probe of the central region of Tn5, or with a 925 bp npt II specific probe were primarily obtained from sewage samples, whereas fewer were obtained from pig manure slurry, river water and soil. However, in soil samples bacteria containing Tn5 or npt II were not found. Transposon Tn5 carrying the npt II gene could be unequivocally demonstrated in 3 isolates from sewage, identified as Aeromonas spp. (2x) and Escherichia coli . Hin dIII digests of chromosomal DNA obtained from these strains were cloned and shown to confer Km resistance to a sensitive E. coli strain. Further, various strains revealed the presence of npt II homologous sequences in a non-Tn5 background. The occurence of Tn5 and npt II in the samples was also assessed via PCR analysis of total community DNA extracts obtained from the aforementioned environmental samples. Evidence for the occurence of npt II was obtained for sewage, pig manure slurry, for 2 (out of 3) river water (Avon, Rhine) and 3 (out of 6) soil (Flevo silt loam, Westmaas silt loam, Ahlum rhizosphere) samples. Tn5 was not detectable via PCR in any of these environmental DNA extracts but it was found in Ede loamy sand and Flevo silt loam samples taken from a field microplot 2 and 4 weeks after release of a Tn5-containing genetically modified organism.     

Survival of, and induced stress resistance in, carbon-starved Pseudomonas fluorescens cells residing in soil.

We investigated the survival, cell length, and development of general stress resistance in populations of Pseudomonas fluorescens R2f and its rifampin-resistant mutant, R2f Rpr, following exposure to carbon starvation conditions in liquid cultures and residence in two different soils, Flevo silt loam (FSL) and Ede loamy sand (ELS). In much the same way as was recently shown for P. putida KT2442, carbon-starved P. fluorescens R2f populations revealed enhanced resistance to otherwise lethal treatments, such as exposure to ethanol, high temperature, osmotic tension, and oxidative stress. A large population of nonculturable P. fluorescens R2f Rpr cells arose shortly after their introduction into ELS soil, whereas the formation of nonculturable cells was not observed in FSL soil. Also, the inoculant cell (based on immunofluorescence) and CFU counts decreased faster in ELS soil than in FSL soil. Introduction of carbon-starved instead of exponential-growth-phase R2f Rpr cells into ELS soil did not affect bacterial survival. The inoculant cell length decreased in soil, and no large differences in cell length in the two soil types were observed. Addition of glucose to ELS soil resulted in a stable cell length of R2f Rpr cells, whereas carbon-starved cells introduced into ELS soil remained small. Exponentially growing R2f Rpr cells developed enhanced resistance to ethanol, high temperature, osmotic tension, and oxidative stress within 1 day in both soils, whereas cells introduced into ELS soil amended with glucose showed decreased resistance. Cells that were carbon starved prior to introduction into ELS soil showed unchanged stress resistance levels upon residence in soil.     

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.     

Transport of bacterial inoculants through intact cores of two different soils as affected by water percolation and the presence of wheat plants

Abstract Water flow-innduced transport of Burkholderia cepacia strain P2 and Pseudomonas fluorescens strain R2f cells through intact cores of loamy sand and silt loam field soils was measured for two percolation regimes, 0.9 and 4.4 mm h⁻¹, applied daily during 1 hour. For each strain, transport was generally similar between the two water regimes. Translocation of B. cepacia , with 4.4 mm h⁻¹, did occur initially in both soils. In the loamy sand soil, no change in the bacterial distribution occurred during the experiment (51 days). In the silt loam, B. cepacia cell numbers in the lower soil layers were significantly reduced, to levels at or below the limit of detection. Transport of P. fluorescens in both soils also occurred initially and was comparable to that of B. cepacia . Later in the experiment, P. fluorescens was not detectable in the lower soil layers of the loamy sand cores, due to a large decrease in surviving cell numbers. In the silt loam, the inoculant cell distribution did not change with time. Pre-incubation of the inoculated cores before starting percolation reduced B. cepacia inoculant transport in the loamy sand soil measured after 5 days, but not that determined after 54 days. Delayed percolation in the silt loam soil affected bacterial transport only after 54 days. The presence of growing wheat plants overall enhanced bacterial translocation as compared to that in unplanted soil cores, but only with percolating water. Percolation water from silt loam cores appeared the day after the onset of percolation and often contained inoculant bacteria. With loamy sand, percolation water appeared only 5 days after the start of percolation, and no inoculant bacteria were found. The results presented aid in predicting the fate of genetically manipulated bacteria in a field experiment.     

Survival of Pseudomonas fluorescens and Bacillus subtilis introduced into two soils of different texture in field microplots

Abstract Antibiotic-resistant strains of Pseudomonas fluorescens and Bacillus subtilis , produced by transposon Tn5 mutagenesis and transformation with plasmid pFT30, respectively, were characterized. Both strains grew at a rate comparable to that of the wild-type strains, and the antibiotic resistance remained stable for over 50 generations without selective pressure. During the growing season, the survival of these strains was studied in two soils of different texture cropped with wheat. The B. subtilis populations declined rapidly in both soils and then stabilized at the levels of added spores. P. fluorescens showed a slow, steady decline in both soils; survival was better in the finer-textured soil, a silt loam, than in the coarser loamy sand. For both bacteria, some translocation to deeper soil layers was observed. No significant rhizosphere effects were detected in either of the two soils.     

Effect of Burkholderia (Pseudomonas) cepacia and soil type on the control of crown rot in wheat

A rifampicin-resistant isolate of Burkholderia (Pseudomonas) cepacia (A3R) reduced crown rot (Fusarium graminearum Group 1) symptoms significantly (P 0.05) in wheat in glasshouse and field experiments and increased grain yield significantly (P 0.05) in one of two field experiments. In glasshouse experiments, applying the bacteria as a soil drench (2.5 × 109 cfu/g soil) was more effective than coating the bacteria on wheat seed (3.4 × 107cfu/seed). In field experiments, the bacteria were applied as a soil drench at the rate of 1.8 x 10¹⁰ cfu/m row. In both the glasshouse and the field, disease severity in the bacteria-inoculated treatments was significantly less in a silt loam than in a sandy loam. The silt loam had a large proportion of fine clay and silt particles (51.7%), which may have favoured the biocontrol activity and survival of the introduced B. cepacia. In a glasshouse experiment, control by B. cepacia was significantly greater in the silt loam than in the sandy loam, which in turn was greater than in a loamy sand. The loamy sand appeared to favour crown rot development but not the activity or survival of the bacterial antagonist. The latter was reflected by the relative populations of the rifampicin-resistant bacteria re-isolated from the various soils during a 5-week period after application of the bacteria (silt loam > sandy loam > loamy sand). This study further confirms that soil type can influence the populations and the level of biocontrol activity of some bacterial antagonists.     

The rhizosphere microflora of wheat grown under controlled conditions II. Influence of the stage of growth of the plant,soil fertility and leaf treatment with urea on the rhizosphere soil microflora

Summary The rhizosphere effect of seminal roots of seedlings and of nodal roots of tillering plants of spring wheat ‘Kaspar’ was investigated under controlled conditions. The total number of micro-organisms recorded in the rhizosphere soil were significantly higher than for the non-rooted soil when investigated with the soil dilution plate method, but lower when fluorescence microscopy was used. Additions of inorganic fertilizer (NPK) decreased their numbers especially in rhizosphere soil of seminal roots and in non-rhizosphere soil, but did not change the ratio between bacteria and actinomycetes (B/A). In the rhizosphere soil the B/A ratio was higher than in the non-rhizosphere soil. An effect of urea leaf treatment was found with the dilution-plate method only in the rhizosphere soil of nodal roots, 3 to 7 days after the first treatment. Increased numbers of actinomycetes were found in this period in NPK fertilized soil, whereas increased numbers of bacteria were found at both fertility levels.     

Effects of antibiotics in soil on the population dynamics of transposon Tn5 carrying Pseudomonas fluorescens

The effects of kanamycin and streptomycin added to soil on the survival of transposon Tn5 modified Pseudomonas fluorescens strain R2f were investigated. Kanamycin in high (180 g g^-1 dry soil) or low (18 g g^-1) concentration or streptomycin in low concentration in Ede loamy sand soil had no noticeable effect on inoculant population dynamics in soil and wheat rhizosphere, whereas streptomycin in high concentration had a consistent significant stimulatory effect, in particular in the wheat rhizosphere. Streptomycin exerted its effect by selecting P. fluorescens with Tn5 insertion whilst suppressing the unmodified sensitive parent strain, as evidenced by comparing the behaviour of these two strains in separate and mixed inoculation studies.Soil textural type influenced the effect of streptomycin on the Tn5 carrying inoculant; the effect was consistently detected in rhizosphere and rhizoplane samples of wheat grown in Ede loamy sand after 7 and 14 days incubation, whereas it was only apparent after 7 days in rhizoplane or rhizosphere (and bulk soil) samples of wheat grown in two silt loam soils. Modification of soil pH by the addition of CaCO₃ or bentonite clay resulted in an enhancement of the selective effect of streptomycin by CaCO₃ and its abolishment by bentonite clay.The addition to soil of malic acid or wheat root exudate, but not of glucose, enhanced the streptomycin selective effect on the Tn5-modified P. fluorescens strain. Neither the streptomycin producer Streptomyces griseus nor two non-inhibiting mutants obtained following UV irradiation affected the dynamics of P. fluorescens (chr::Tn5) in soil and wheat rhizosphere.The effect of streptomycin in soil on inoculant Tn5 carrying bacteria depends on conditions such as soil type, the presence of (wheat) root exudates and the type of available substrate.     

Fluorescent pseudomonads in the rhizosphere of plants and their relation to root exudates

Fluorescent pseudomonads were present in chernozem soil not influenced by plant roots (10(3)-10(4) per g dry soil) in the rhizosphere soil of various plants (10(4)-10(5) per g soil) and on roots (10(3) to 10(7) per g fresh roots), depending on the species and age of the plant. Relative species representation of fluorescent pseudomonads changed on the roots and in the plant rhizosphere as compared with free soil. Pseudomonas fluorescens, representing 60-93% of the population of fluorescent pseudomonads predominated on the roots of all plants investigated. Somewhat different results were obtained in rhizosphere soil. Relatively higher numbers of P. fluorescens were detected in the rhizosphere soil of cucumber and maize, numbers in the rhizosphere soil of wheat were practically the same as in free soil and higher numbers of P. putida were found in the rhizosphere soil of barley. Almost all components contained in the root exudates of the plants studied, including beta-pyrazolylalanine from the root exudates of cucumbers were utilized as carbon and energy sources. Root exudates of wheat and maize were utilized by the strain P. putida K2 with an efficiency of 73-91%, depending on species and age of the plant.     

Survival of Azospirillum brasilense in the Bulk Soil and Rhizosphere of 23 Soil Types

The survival of Azospirillum brasilense Cd and Sp-245 in the rhizosphere of wheat and tomato plants and in 23 types of plant-free sterilized soils obtained from a wide range of environments in Israel and Mexico was evaluated. Large numbers of A. brasilense cells were detected in all the rhizospheres tested, regardless of soil type, bacterial strain, the origin of the soil, or the amount of rainfall each soil type received prior to sampling. Survival of A. brasilense in soils without plants differed from that in the rhizosphere and was mainly related to the geographical origin of the soil. In Israeli soils from arid, semiarid, or mountain regions, viability of A. brasilense rapidly declined or populations completely disappeared below detectable levels within 35 days after inoculation. In contrast, populations in the arid soils of Baja California Sur, Mexico, remained stable or even increased during the 45-day period after inoculation. In soils from Central Mexico, viability slowly decreased with time. In all soils, percentages of clay, nitrogen, organic matter, and water-holding capacity were positively correlated with bacterial viability. High percentages of CaCO(inf3) and fine or rough sand had a highly negative effect on viability. The percentage of silt, pH, the percentage of phosphorus or potassium, electrical conductivity, and C/N ratio had no apparent effect on bacterial viability in the soil. Fifteen days after removal of inoculated plants, the remaining bacterial population in the three soil types tested began to decline sharply, reaching undetectable levels 90 days after inoculation. After plant removal, percolating the soils with water almost eliminated the A. brasilense population. Viability of A. brasilense in two artificial soils containing the same major soil components as the natural soils from Israel did was almost identical to that in the natural soils. We conclude that A. brasilense is a rhizosphere colonizer which survives poorly in most soils for prolonged periods of time; that outside the rhizosphere, seven abiotic parameters control the survival of this bacterium in the soil; and that disturbance of the soil (percolation with water or plant removal) directly and rapidly affects the population levels.     

Induced Reporter Gene Activity, Enhanced Stress Resistance, and Competitive Ability of a Genetically Modified Pseudomonas fluorescens Strain Released into a Field Plot Planted with Wheat

The fates of Pseudomonas fluorescens R2fR and its mutant derivative RIWE8, which contains a lacZ reporter gene responsive to wheat root exudate, were compared in a field microplot. Inoculant survival, root colonization, translocation, resistance to stress factors, and reporter gene activity were assessed in bulk and wheat rhizosphere soils. Populations of both strains declined gradually in bulk and wheat rhizosphere soils and on the wheat rhizoplane as determined by specific CFU and immunofluorescence (IF). In samples from both bulk soil and wheat rhizosphere, IF cell counts were up to 3 orders of magnitude greater than the corresponding numbers of CFU after 120 days, indicating the presence of nonculturable inoculant cells. Estimates of RIWE8-specific target DNA molecule numbers in bulk soil samples 3 and 120 days after inoculation by most-probable-number PCR coincided with the corresponding CFU values. Transport of both strains to deeper soil layers was observed by 3 days after introduction into the microplot. Both strains colonized wheat roots similarly, and cells were seen scattered on the surface of 1-month-old wheat seedling roots by immunogold labelling-scanning electron microscopy. On average, reporter gene activity was significantly higher in wheat rhizosphere soil containing RIWE8 cells than in bulk soil or in soils containing R2fR cells. For both strains, resistance to the four stress factors ethanol, high temperature, high osmotic tension, and oxidative stress increased progressively with residence in soil. Cells from the rhizosphere of 11-day-old seedlings showed similar levels of resistance to osmotic and oxidative stresses and enhanced resistance to ethanol and heat as compared to cells from bulk soil. By 37 days, populations of R2fR and RIWE8 in the rhizosphere were significantly more sensitive to osmotic stress than were populations in bulk soil, whereas differences in response to the other stress factors were less evident. Hence, except for the induction of reporter gene expression in strain RIWE8 in the wheat rhizosphere, the data indicated that there were no great differences in the ecological properties in soil between the lacZ-modified and parental strains.     

Influence of soil pH on the nitrate-reducing microbial populations and their potential to reduce nitrate to NO and N2O

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into natural loamy sand and silt loam, bacterial numbers increased only directly after inoculation. Thereafter, bacterial numbers decreased until an equilibrium was reached. This decrease was exponential on a log scale and could be described by the function Y = A + B − R ', where Y is the log number of rhizobial cells at time: T ; A represents the lgo of the final population size; B is the difference between the log (initial number of bacteria) and A ; R is the daily reduction factor of Y−A and t is time in days after inoculation. The final population sizes increased with increasing inoculum densities (10⁴−10⁸ bacteria/g soil). In sterilized soil, however, the populations increased up to an equilibrium, which was not affected by the inoculum density.
The final population sizes were higher in silt loam than in loamy sand in natural, as well as in sterilized soil. The final population size was reached earlier in natural silt loam than in loamy sand. Also the growth rate in sterilized soil was higher in silt loam than in loamy sand. The growth rate of low inoculum densities in silt loam was exponential and approximately the same as in yeast extract mannitol broth. The growth rate in loamy sand could be improved by incresing the bulk density of the soil from 1.0 to 1.4 g/cm³.     

Effect of genetically modified Pseudomonas putida WCS358r on the fungal rhizosphere microflora of field-grown wheat

We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the antifungal compound phenazine-1-carboxylic acid (PCA). In the springs of 1997 and 1998 we sowed wheat seeds treated with either GMM 2, GMM 8, or WCS358r (approximately 10(7) CFU per seed), and measured the numbers, composition, and activities of the rhizosphere microbial populations. During both growing seasons, all three bacterial strains decreased from 10(7) CFU per g of rhizosphere sample to below the limit of detection (10(2) CFU per g) 1 month after harvest of the wheat plants. The phz genes were stably maintained, and PCA was detected in rhizosphere extracts of GMM-treated plants. In 1997, but not in 1998, fungal numbers in the rhizosphere, quantified on 2% malt extract agar (total filamentous fungi) and on Komada's medium (mainly Fusarium spp.), were transiently suppressed in GMM 8-treated plants. We also analyzed the effects of the GMMs on the rhizosphere fungi by using amplified ribosomal DNA restriction analysis. Introduction of any of the three bacterial strains transiently changed the composition of the rhizosphere fungal microflora. However, in both 1997 and 1998, GMM-induced effects were distinct from those of WCS358r and lasted for 40 days in 1997 and for 89 days after sowing in 1998, whereas effects induced by WCS358r were detectable for 12 (1997) or 40 (1998) days. None of the strains affected the metabolic activity of the soil microbial population (substrate-induced respiration), soil nitrification potential, cellulose decomposition, plant height, or plant yield. The results indicate that application of GMMs engineered to have improved antifungal activity can exert nontarget effects on the natural fungal microflora.     

Population Increase of Pratylenchus hexincisus on Corn as Related to Soil Temperature and Type

Population increase of Pratylenchus hexincisus on corn was tested over 3 months at 15, 20, 25, and 30 C in Marshall silt loam, Clarion silt loam, Buckner coarse sand, and Haig silty clay loam soils. The optimum temperature for increase was 30 C in all soils. The nematode population was significantly larger in Buckner coarse sand than in other soil types at 50 C. The recovered P. hexincisus populations equaled or exceeded initial inoculum levels at the two higher temperatures in Marshall silt loam and Haig silty clay loam and at 30 C in Clarion silt loam and Buckner coarse sand. P. hexincisus required 32,400 heat units in Haig silty clay loam and more than 40,000 heat units in the three other soil types to reach a level that is known to cause significant height and biomass reduction in corn under controlled condition.     

Quantitative detection of Sphingomonaschlorophenolica in soil via competitive polymerase chain reaction

The 16S ribosomal RNA gene sequence of the pentachlorophenol degrader Sphingomonas chlorophenolica strain RA2 was used to generate specific polymerase chain reaction (PCR) primers for the detection of this strain in soil, whereas a region internal to the two primers was used to provide an S. chlorophenolica strain RA2-specific oligonucleotide probe. The PCR detection system resulted in a 727 bp product detectable via gel electrophoresis and hybridization. It was specific for strain RA2 and its close relative, S. chlorophenolica ATCC 39723, as evidenced by PCR amplifications of a range of bacterial genomic DNAs. Tests of total microbial community DNA obtained from five uninoculated and two RA2-inoculated soils confirmed this specificity for introduced S. chlorophenolica RA2. Strain RA2 could be detected in soil down to a level of 10³ cfu g⁻¹ soil. Two strategies were followed to generate internal standard DNA for competitive PCR. First, a 479 bp MIMICS fragment was obtained based on a previously constructed gene cassette; however, this standard did not reliably quantify RA2 targets. Low stringency PCR performed with a range of bacterial genomic DNAs resulted in the generation of an amplicon with a Paenibacillus azotofixans strain that was slightly smaller than the RA2-derived product. Both products were easily separable via conventional gel electrophoresis. The use of this competitor in a threefold dilution scheme applied to the target DNA allowed for the quantitative detection of RA2-specific target DNA molecules from pure culture and from soil. The fate of strain RA2 in pentachlorophenol-contaminated soil was described using this competitive PCR approach, and the organism was shown to persist at two inoculum levels over prolonged periods of time.     

Evaluation of the Effect of Surfactants on the Movement of Pesticides in Soils Using a Soil Thin-Layer Chromatography Technique

In this study, the effect of concentration (1/2 CMC, at CMC and 2 x CMC) of surfactants, cetyl trimethyl ammonium bromide (cationic), sodium dodecyl sulfate (anionic), and tween ‘20’ (non-ionic) on the movement of carbofuran, chlorpyrifos and en-dosulfan in soils was evaluated by using a soil thin-layer chromatographic technique. The movement of pesticides was detected by spray reagents and expressed in terms of Rf values. The penetrability K was found to increase by decreasing the plate angle and followed the order as: sandy loam > loam > silt loam soils. The penetrability K also decreases in surfactant-free and surfactant-amended soils when developed in distilled water and aqueous surfactant solutions of different CMCs, respectively. The higher movement of pesticides was observed in sandy loam soil followed by loam and silt loam soils. On the basis of Rf values, the movement of pesticides follows the order as: carbofuran > chlorpyrifos > endosulfan, both in surfactant-amended and surfactant-free soils when developed in distilled water and aqueous surfactant solutions of different CMCs. The movement is directly proportional to the aqueous solubilities, polarities, and carbon numbers and inversely related to the molecular weights of pesticides. A significant increase or decrease of pesticides movement in soils was discussed on the basis of adsorption of pesticides on soils, chemical nature of the surfactants, and its concentrations in terms of critical micelle concentrations (CMCs) in soils and eluents. Results obtained may provide insights pertaining to the use of surfactants for solving soil pollution problems posed by pesticides.     

The dynamics of Rhizobium leguminosarum biovar trifolii introduced into soil as determined by immunofluorescence and selective plating techniques

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into a loamy sand and a silt loam, high recovery percentages were determined using quantitative immunofluorescence. Soil type, but not inoculum density between 10⁴ and 10⁸ cells per gram of soil, significantly influenced the recovery percentage of the immunofluorescence technique. Recovery percentages determined using selective plating were independent of either soil type or inoculum density and exceeded those determined by immunofluorescence.
The serological and genetic markers used for detection were stable during 55 days of incubation in phosphate-buffered saline and soil extract solution. After the introduction of R. leguminosarum biovar trifolii into both sterilized soil types, the population increased to 0.5–1×10⁹ cells per gram of soil, but a decline was demonstrated in non-sterile loamy sand and silt loam during incubation of 90 days at 15°C. Starvation of rhizobial cells in the phosphate-buffered saline and soil extract solution, as well as incubation in both soil types, resulted in a significant decrease in mean cell size.     

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.     

Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats.

Phenazine antibiotics produced by Pseudomonas fluorescens 2-79 and Pseudomonas aureofaciens 30-84, previously shown to be the principal factors enabling these bacteria to suppress take-all of wheat caused by Gaeumannomyces graminis var. tritici, also contribute to the ecological competence of these strains in soil and in the rhizosphere of wheat. Strains 2-79 and 30-84, their Tn5 mutants defective in phenazine production (Phz-), or the mutant strains genetically restored for phenazine production (Phz+) were introduced into Thatuna silt loam (TSL) or TSL amended with G. graminis var. tritici. Soils were planted with three or five successive 20-day plant-harvest cycles of wheat. Population sizes of Phz- derivatives declined more rapidly than did population sizes of the corresponding parental or restored Phz+ strains. Antibiotic biosynthesis was particularly critical to survival of these strains during the fourth and fifth cycles of wheat in the presence of G. graminis var. tritici and during all five cycles of wheat in the absence of take-all. In pasteurized TSL, a Phz- derivative of strain 30-84 colonized the rhizosphere of wheat to the same extent that the parental strain did. The results indicate that production of phenazine antibiotics by strains 2-79 and 30-84 can contribute to the ecological competence of these strains and that the reduced survival of the Phz- strains is due to a diminished ability to compete with the resident microflora.