Phylogenetic diversity of fluorescent pseudomonads in agricultural soils from Korea

AIMS: To identify and compare the relative diversity and distribution of genotypes of culturable fluorescent pseudomonads from soils. METHODS AND RESULTS: Analysis of 160 isolates from seven soil samples using randomly amplified polymorphism DNA methods revealed 53 genotypes, which were subsequently identified by their 16S ribosomal DNA sequences. Phylogenetic analyses of the 53 genotypes along with 43 fluorescent pseudomonad type strains separated the genotypes into 10 distinct clusters that included two phylogenetic groups that were not represented by previously described type strains. CONCLUSIONS: The diversity of genotypes that was obtained from the soil samples was highly variable among the different soils and appeared to be associated with different soil management practices that also influence plant yields. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification and phylogenetic analysis of these genotypes offers opportunities for study of phenotypic traits that may be associated within taxonomically related groups of fluorescent pseudomonad species and how these groups vary in relation to soil management practices.     

Genetic diversity of phenazine- and pyoluteorin-producing pseudomonads isolated from green pepper rhizosphere

The genetic diversity among indigenous phenazine-1-carboxylic acid (PCA)-producing and pyoluteorin (Plt)-producing isolates of pseudomonads screened from green pepper rhizosphere was exploited in this study. A total of 48 bacterium isolates producing one or both of these antibiotics were screened from green pepper rhizosphere in diverse regions in China. Among these isolates, 45 could produce PCA, 3 could produce both PCA and Plt, and none could produce Plt only. Based on the restriction patterns of partial 16S and 16S-23S internal transcribed spacer (ITS) PCR fragments generated by enzyme HaeIII or HinfI, these isolates fell into 19 or 17 distinct groups respectively, indicating that there was a significant diversity among them. Polygenetic analysis of the partial 16S rDNA and 16S-23S ITS sequence from the representative in each group in the context of similar sequence from previously described bacterial species indicated that most isolates were closely related to the species of Pseudomonas fluorescens, P. putida, and Stenotrophomonas maltophilia. Some of these representatives of these isolates, then, are likely to be novel strains or species in these two genera. The GenBank accession numbers for DNA sequences of the partial 16S rDNA with ITS region in each isolate determined in this study were: GP30 DQ003219; GP127 DQ003220; GP83 DQ003221; GP42, DQ003222; GP59 DQ003223; GP50 DQ003224; GP36 DQ003225; GP110 DQ003226; GP26 DQ003227; GP37 DQ003228; GP60 DQ003229; GP31 DQ003230; GP57 DQ003231; GP75 DQ003232; GP115 DQ003233; GP65 DQ003234; GP32 DQ003235; GP76 DQ003236; GP78 DQ003237.     

Field performance in northern Spain of Douglas-fir seedlings Inoculated with ectomycorrhizal fungi

 Experimental plantations were established in northern Spain to determine the effects of different ectomycorrhizal fungi on growth and survival of Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) under field conditions. Douglas-fir seedlings were inoculated with Laccaria bicolor S238 mycelia in two bareroot nurseries in central France or with spore suspensions of three hypogeous ectomycorrhizal species: Melanogaster ambiguus, Rhizopogon colossus and R. subareolatus, in a Spanish containerised nursery. The effects of ectomycorrhizal inoculation on plant survival after outplanting were limited, being only significant at the Guipuzkoan (Spain) site, when plants inoculated with L. bicolor S238 were compared to non-inoculated plants grown in non-fumigated soil. L. bicolor S238 had a significant effect on plant growth during the phase of bareroot nursery growth and this difference was maintained after field outplanting. Nursery inoculations with M. ambiguus, R. colossus and R. subareolatus improved plant growth during the first 2 and 3 years after field outplanting. The positive effects of the inoculation treatment on seedling height, root collar diameter and stem volume persisted after 5 years of field growth. Inoculation with these ectomycorrhizal fungi may improve the field performance of Douglas-fir seedlings in northern Spain. Accepted: 12 February 1999     

Effect of an introduced inoculum on soil microbial diversity

Abstract A laboratory study was carried out to evaluate the impact of the introduction of genetically modified microorganisms into soil, in terms of effect on the diversity of the indigenous microflora, and at the process level. The impact on microbial phenotypic diversity, and on soil denitrification of an inoculum of a lux -modified denitrifier, Pseudomonas fluorescens , was examined using two different soil types in re-packed soil microcosms. The effect on diversity was found to depend on the soil pore size class into which the modified inoculum was introduced. The introduction of lux -modified cells into the 15–30 μm pore neck size class caused a short-term reduction in the overall microbial diversity. There was no significant change in the diversity of the indigenous microbial community, however, when cells were introduced into the 40–60 μm pore class. Partial chloroform fumigation proved useful in differentiating cell populations with respect to pore location. No change in diversity was observed when dead cells (either heat killed or glutaraldehyde fixed) were introduced into either pore size class. At the process level, the effect on soil denitrification of introduction of lux -modified P. fluorescens was not significantly different from introduction of the equivalent inoculum of the parental wild-type, although denitrification was found to be dependent upon both soil structure and pore size location of the introduced inoculum.     

Pseudomonas fluorescens and closely-related fluorescent pseudomonads as biocontrol agents of soil-borne phytopathogens

Many strains of Pseudomonas fluorescens show potential for biological control of phytopathogens especially root pathogens. In taxonomic terms, several of them are indeed P. fluorescens sensu stricto , while others belong in fact to neighbouring species of the ' P. fluorescens ' complex or to ill-defined related species within the fluorescent Pseudomonas spp. These bacteria have become prominent models for rhizosphere ecological studies and analysis of bacterial secondary metabolism, and in recent years knowledge on their plant-beneficial traits has been considerably enhanced by widening the focus beyond the case of phytopathogen-directed antagonism. Current genomic analyses of rhizosphere competence and biocontrol traits will likely lead to the development of novel tools for effective management of indigenous and inoculated P. fluorescens biocontrol agents and a better exploitation of their plant-beneficial properties for sustainable agriculture.     

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.     

The influence of mineral and carbon sources on biological control of charcoal rot fungus,Macrophomina phaseolina by fluorescent pseudomonads in tomato

AIMS: To determine the influence of various trace minerals and carbon source on the biocontrol performance of Pseudomonas aeruginosa strain IE-6S+ and P. fluorescens strain CHA0 against Macrophomina phaseolina. METHODS AND RESULTS: In dual culture plate assay, P. aeruginosa IE-6S+ and P. fluorescens CHA0 inhibited radial growth of M. phaseolina producing zones of inhibition. Czapek's dox agar medium amended with both zinc and glucose remarkably improved antifungal activities of the bacterial inoculants. Under glasshouse conditions, soil amendment with zinc and/or glucose alone did not reduce M. phaseolina infection in tomato roots but did reduce significantly when used in combination with IE-6S+ or CHA0. Soil amendments with zinc and/or glucose increased fresh shoot weights but zinc amendment greatly reduced bacterial populations in the rhizosphere. CONCLUSIONS: Mineral and carbon amendments enhance the biocontrol performance of fluorescent pseudomonads against M. phaseolina. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of mineral and carbon amendments that favour biocontrol of certain bacterial strains may provide clues to soil factors or components of nutrient solutions in hydroponic culture that will improve the level and reliability of control.     

Microbial community composition and functional diversity in the rhizosphere of maize

This study investigates the small-scale stratification of bacterial community composition and functional diversity in the rhizosphere of maize. Maize seedlings were grown in a microcosm with a horizontal mesh (53 M) creating a planar root mat and rhizosphere soil. An unplanted microcosm served as control. Thin slices of soil were cut at different distances from the mesh surface (0.2–5.0 mm) and analysed for bacterial community composition by PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) of 16S rDNA and tested for activities of different enzymes involved in C, N, P and S cycling. Bacterial community composition and microbial functional diversity were affected by the presence of the maize roots. The bacterial composition showed a clear gradient up to 2.2 mm from the root surface, while no such gradient was observed in the unplanted pot. Invertase and phosphatase activities were higher in the close vicinity of maize roots (0.2–0.8 mm), whereas xylanase activity was unaffected. This study shows that the changes in bacterial community composition and functional diversity induced by roots may extend several millimetres into the soil.     

Short-term rates of parasite evolution predict the evolution of host diversity

Coevolution with parasites has been implicated as an important factor driving the evolution of host diversity. Studies to date have focussed on gross effects of parasites: how host diversity differs in the presence vs. absence of parasites. But parasite-imposed selection is likely to show rapid variation through time. It is unclear whether short-term fluctuations in the strength of parasite-imposed selection tend to affect host diversity, because increases in host diversity are likely to be constrained by both the supply of genetic variation and ecological processes. We followed replicate populations of coevolving, initially isogenic, bacteria and phages through time, measuring host diversity (with respect to bacterial colony morphologies), host density and rates of parasite evolution. Both host density and time-lagged rates of parasite evolution were good independent predictors of the magnitude of bacterial within- and between-population diversities. Rapid parasite evolution and low host density decreased host within-population diversity, but increased between-population diversity. This study demonstrates that short-term changes in the rate of parasite evolution can predictably drive patterns of host diversity.     

Molecular diversity in the bacterial community and the fluorescent pseudomonads group in natural and chlorobenzoate-stressed peat-forest soil

Bacterial community shifts in a soil microcosm spiked with 3-chlorobenzoate or 2,5-dichlorobenzoate were monitored. The V6-V8 variable regions of soil bacterial 16S rRNA and rDNA were amplified and separated by temperature gradient gel electrophoresis (TGGE) profiling. Culturing in the presence of 2.5 mM chlorinated benzoates suppressed 10 to 100 fold the total aerobic bacterial community but had no effect on the diversity within the group of fluorescent pseudomonads. In contrast, the uncultured bacterial community showed a decrease in the number of bands in the TGGE profiles of the chlorobenzoate-spiked treatments. Accordingly, the Shannon's diversity and equitability indices of these treatments reflected a decreasing trend in time. The approach allowed a direct assessment of community shifts upon contamination of soil.     

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

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

Molecular and functional diversity in soil micro-organisms

Traditional approaches to the study of microbial diversity have relied on laboratory cultivation of isolates from natural environments and identification by classical techniques, including analysis of morphology, physiological characteristics and biochemical properties. These approaches provide information on fine-scale diversity but suffer from bias, resulting from the media and cultivation conditions employed, and from the inability to grow and isolate significant proportions of natural communities in the laboratory. An alternative approach is the amplification of ribosomal RNA and functional genes from nucleic acids extracted directly from environmental samples, with subsequent analysis by `fingerprinting' methods or by sequencing and phylogenetic analysis. This approach avoids the need for laboratory cultivation and has provided major insights into species and functional diversity of bacterial and archaeal populations. This article reviews molecular approaches to the characterisation of prokaryote diversity in natural environments, their more recent application to fungal diversity and the advantages and limitations of molecular analyses.     

Impact of wild-type and genetically modified Pseudomonas fluorescens on soil enzyme activities and microbial population structure in the rhizosphere of pea

The aim of this study was to determine the impact of wild-type along with functionally and nonfunctionally modified Pseudomonas fluorescens strains in the rhizosphere. The wild-type F113 strain carried a gene encoding the production of the antibiotic 2,4-diacetylphloroglucinol (DAPG) useful in plant disease control, and was marked with a lacZY gene cassette. The first modified strain was a functional modification of strain F113 with repressed production of DAPG, creating the DAPG-negative strain F113 G22. The second paired comparison was a nonfunctional modification of wild-type (unmarked) strain SBW25, constructed to carry marker genes only, creating strain SBW25 EeZY-6KX. Significant perturbations were found in the indigenous bacterial population structure, with the F113 (DAPG+) strain causing a shift towards slower growing colonies (K strategists) compared with the nonantibiotic-producing derivative (F113 G22) and the SBW25 strains. The DAPG+ strain also significantly reduced, in comparison with the other inocula, the total Pseudomonas populations but did not affect the total microbial populations. The survival of F113 and F113 G22 were an order of magnitude lower than the SBW 25 strains. The DAPG+ strain caused a significant decrease in the shoot-to-root ratio in comparison to the control and other inoculants, indicating plant stress. F113 increased soil alkaline phosphatase, phosphodiesterase and aryl sulphatase activities compared to the other inocula, which themselves reduced the same enzyme activities compared to the control. In contrast to this, the β-glucosidase, β-galactosidase and N -acetyl glucosaminidase activities decreased with the inoculation of the DAPG+ strain. These results indicate that soil enzymes are sensitive to the impact of inoculation with genetically modified microorganisms (GMMs).     

Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches

The regional variability of Taxus rhizosphere bacterial community composition and diversity was studied by comparative analysis of three large 16S rRNA gene clone libraries from the Taxus rhizosphere in different regions of China (subtropical and temperate regions). One hundred and forty-six clones were screened for three libraries. Phylogenetic analysis of 16S rRNA gene sequences demonstrated that the abundance of sequences affiliated with Gammaproteobacteria, Betaproteobacteria, and Actinobacteria was higher in the library from the T. xmedia rhizosphere of the temperate region compared with the subtropical Taxus mairei rhizosphere. On the other hand, Acidobacteria was more abundant in libraries from the subtropical Taxus mairei rhizosphere. Richness estimates and diversity indices of three libraries revealed major differences, indicating a higher richness in the Taxus rhizosphere bacterial communities of the subtropical region and considerable variability in the bacterial community composition within this region. By enrichment culture, a novel Actinobacteria strain DICP16 was isolated from the T. xmedia rhizosphere of the temperate region and was identified as Leifsonia shinshuensis sp. via 16S rRNA gene and gyrase B sequence analyses. DICP16 was able to remove the xylosyl group from 7-xylosyl-10-deacetylbaccatin III and 7-xylosyl-10-deacetylpaclitaxel, thereby making the xylosyltaxanes available as sources of 10-deacetylbaccatin III and the anticancer drug paclitaxel. Taken together, the present studies provide, for the first time, the knowledge of the biodiversity of microorganisms populating Taxus rhizospheres.     

First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA

Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO(2)(-) to NO(3)(-) in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrA-like sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.     

Microarray-based analysis of microbial functional diversity along an oil contamination gradient in oil field

To understand better the in situ microbial functional diversity under oil contamination stress, soils were sampled along a contamination gradient at an oil field in north-east China. Microbial community functional structure was examined with a functional gene array, termed GeoChip. Multivariate statistical analysis and meta-analysis were conducted to study the functional gene responses to oil concentrations. The total functional gene abundance and diversity decreased along the gradient of increasing contamination. The overall abundance of soil bacteria, archaea and fungi decreased to 10%, 40% and 80% of those in the pristine soil. Several functional genes in the families pgl, rbcL, nifH and nor and those encoding cellulase, laccase, chitinase, urease and key enzymes in metabolizing organic compounds were significantly decreased with oil contamination, especially under high contamination stress. However, a few genes encoding key enzymes for catechol, protocatechuate, and biphenyl degradation and in the gene families of nir, rbcL and pgl showed a significant increase at a medium level of oil contamination. Oil content and soil available nitrogen were found to be important factors influencing the microbial community structure. The results provide an insight into microbial functional diversity in oil-contaminated soils, providing potential information for on-site management and remediation measures.     

Structural and functional study in the rhizosphere of Oryza sativa L. plants growing under biotic and abiotic stress

Aims

A structural and functional study has been carried out in the rice production area of the Guadalquivir marshes in southern Spain aiming to increase knowledge of rice rhizosphere structure and function for further application on integrated management practices.

Methods and Results

Rhizosphere bacterial structure (analysis of 16S rRNA partial sequences from total soil DNA), metabolic diversity (analysed by Biolog FF for fungal community and GN for microbial community) and a screening for putative plant growth–promoting rhizobacteria (PGPR) to identify potential isolates for development of local biofertilizers, and biodiversity of culturable micro‐organisms (analysis of 16S rRNA partial sequences) from four areas differing in salinity and Magnaporthe oryzae incidence in two moments of the crop cycle were studied. Results indicate that the dominant taxon in libraries from the four areas was Proteobacteria. Metabolic diversity was higher in areas affected only by salinity or incidence of Magnaporthe than in the control or area affected by both stresses. It seems that rice plants selected, in their rhizosphere, micro‐organisms able to affect plant hormonal balance under all conditions, and this activity relied in different bacterial genera depending on the environmental stress.

Conclusions

Bacterial genera for each stress, as well as generalist strains, were found present in all the studied areas. Potential molecular markers and taxonomic markers (Sphingobacteria for salt and Thermococci for Magnaporthe) of the different stress situations have been highlighted, and Class Verrucomicrobiae could be a marker for nonstressed areas. In addition, putative PGPR strains isolated in this study could be used as biofertilizers.

Significance and Impact of the Study

Rice paddies are great ecologically important ecosystems. The results are very relevant as they may be included in the process of rice production, improving crop conditions with less environmental impact.     

Inferring evolutionarily significant units of bacterial diversity from broad environmental surveys of single-locus data

By far the greatest challenge for diversity studies is to characterize the diversity of prokaryotes, which probably encompasses billions of species, most of which are unculturable. Recent advances in theory and analysis have focused on multi-locus approaches and on combined analysis of molecular and ecological data. However, broad environmental surveys of bacterial diversity still rely on single-locus data, notably 16S ribosomal DNA, and little other detailed information. Evolutionary methods of delimiting species from single-locus data alone need to consider population genetic and macroevolutionary theories for the expected levels of interspecific and intraspecific variation. We discuss the use of a recent evolutionary method, based on the theory of coalescence within independently evolving populations, compared with a traditional approach that uses a fixed threshold divergence to delimit species.