Quantitative Selective PCR of 16S Ribosomal DNA Correlates Well with Selective Agar Plating in Describing Population Dynamics of Indigenous Pseudomonas spp. in Soil Hot Spots

We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous Pseudomonas DNA in soil hot spots. The amount of Pseudomonas DNA corresponded to the number of culturable Pseudomonas bacteria on Gould’s S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.     

Polymerase chain reaction amplification of naphthalene-catabolic and 16S rRNA gene sequences from indigenous sediment bacteria.

We report the amplification of bacterial genes from uninoculated surface and subsurface sediments by the polymerase chain reaction (PCR). PCR amplification of indigenous bacterial 16S ribosomal DNA genes was unsuccessful when subsurface sediment containing approximately 10(7) cells.g-1 was added directly to a PCR mixture. However, when 10 mg of sediment was inoculated with approximately 10(5) cells of Pseudomonas putida G7, the nahAc naphthalene dioxygenase gene characteristic of the P. putida G7 NAH7 plasmid was detected by PCR amplification. Southern blotting of the PCR amplification product improved sensitivity to 10(3) to 10(4) cells from samples inoculated with P. putida G7, but controls with no sediment added showed that the PCR was partially inhibited by the sediments. Lysozyme-sodium dodecyl sulfate-freeze-thaw DNA extraction was combined with gel electrophoretic partial purification in the presence of polyvinylpyrrolidone to render DNA from indigenous bacteria in surface or subsurface sediment samples amplifiable by PCR using eubacterial 16S ribosomal DNA primers. The nahAc gene could also be amplified from indigenous bacteria by using nahAc-specific primers when PCR conditions were modified by increasing Taq and primer concentrations. Restriction digests of the nahAc amplification products from surface and subsurface sediments revealed polymorphism relative to P. putida G7. The procedures for DNA extraction, purification, and PCR amplification described here demonstrate that the PCR is a potentially useful tool in studies of function- and taxon-specific DNA from indigenous microbial communities in sediment and groundwater environments.     

Succession of indigenous Pseudomonas spp. and actinomycetes on barley roots affected by the antagonistic strain Pseudomonas fluorescens DR54 and the fungicide imazalil

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.     

Manure Enhances Plasmid Mobilization and Survival of Pseudomonas putida Introduced into Field Soil

We report a field study on plasmid mobilization in an agricultural soil. The influence of pig manure on the mobilization of the IncQ plasmid pIE723 by indigenous plasmids or by the IncP(alpha) plasmid pGP527 into the recipient Pseudomonas putida UWC1 (Rif(supr) Nal(supr)) was studied in field soil. Six plots were prepared in duplicate, three of which were treated with manure prior to inoculation of the donor and recipient strains. As a donor strain, either Escherichia coli J53(pIE723) or E. coli 600(pIE723, pGP527) was used. Putative transconjugants obtained on a selective medium were confirmed by DNA hybridization and PCR. Plasmid mobilization by indigenous mobilizing plasmids was observed on two occasions in manured soil. Manuring of soil significantly enhanced the frequency of pIE723 mobilization by pGP527, since mobilization frequencies into P. putida UWC1 were at least 10-fold higher in manured soil than in nonmanured soil. Enhanced numbers of P. putida UWC1 transconjugant and recipient colonies could be observed in manured soil throughout the 79-day field test. Transfer of pIE723 or pG527 into indigenous soil or rhizosphere bacteria could not be detected when indigenous bacteria isolated by selective cultivation were screened for the presence of these plasmids by DNA hybridization. Furthermore, the presence of IncN-, IncP-, or IncQ-specific sequences was confirmed in total community DNA extracted directly from the manured or nonmanured soil by PCR. IncW plasmids were detectable only in manured soil, indicating entry of these plasmids into soil via manure.     

Bacterial origin and community composition in the barley phytosphere as a function of habitat and presowing conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Interaction of higher plant (jute), electrofused bacteria and mycorrhiza on anthracene biodegradation

The interaction of bacteria, mycorrhiza and jute (Corchotus capsulari, a higher plant) to reduce anthracene in different concentrations of spiked soils was investigated. Dominant indigenous bacterium (Pseudomonas sp.) isolated in the rhizosphere of jute was electrofused with anthracene degraders (Sphingomonas paucimobilis and Pseudomonas aeruginosa) which were able to produce different types of biosurfactants. The highest population (56 x 10(5)CFU/g) was found in the planted soil with the inoculation of mixtures of electrofused anthracene degraders after 7 days. The growth of anthracene degraders in the spiked soil was improved by gene transfer from indigenous bacteria. After 35 days, enhanced anthracene removals were observed in inoculated soils planted with jute (65.5-75.2%) compared with unplanted soil without inoculation (12.5%). The interaction of jute and electrofused S. paucimobilis enabled the greatest reduction of soil anthracene with or without the addition of P. aeruginosa. Mycorrhizal colonization was not significantly inhibited by anthracene in soils up to 150 mg/kg. Inoculation of jute with Glomus mosseae and Glomus intraradices improved plant growth and enhanced anthracene removal in the presence of electrofused S. paucimobilis.     

Bacterial Origin and Community Composition in the Barley Phytosphere as a Function of Habitat and Presowing Conditions

An understanding of the factors influencing colonization of the rhizosphere is essential for improved establishment of biocontrol agents. The aim of this study was to determine the origin and composition of bacterial communities in the developing barley (Hordeum vulgare) phytosphere, using denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes amplified from extracted DNA. Discrete community compositions were identified in the endorhizosphere, rhizoplane, and rhizosphere soil of plants grown in an agricultural soil for up to 36 days. Cluster analysis revealed that DGGE profiles of the rhizoplane more closely resembled those in the soil than the profiles found in the root tissue or on the seed, suggesting that rhizoplane bacteria primarily originated from the surrounding soil. No change in bacterial community composition was observed in relation to plant age. Pregermination of the seeds for up to 6 days improved the survival of seed-associated bacteria on roots grown in soil, but only in the upper, nongrowing part of the rhizoplane. The potential occurrence of skewed PCR amplification was examined, and only minor cases of PCR bias for mixtures of two different DNA samples were observed, even when one of the samples contained plant DNA. The results demonstrate the application of culture-independent, molecular techniques in assessment of rhizosphere bacterial populations and the importance of the indigenous soil population in colonization of the rhizosphere.     

Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization,direct quantification,and in situ location of transconjugant cells

The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied. Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids. The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts. The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively. Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas. Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing. This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts. Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria. In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P. putida in the absence of selective pressure that could favor the presence of the investigated plasmids.     

Fate of the biological control agent Pseudomonas aureofaciens TX-1 after application to turfgrass

The fate and impact of Pseudomonas aureofaciens TX-1 following application as a biocontrol agent for fungi in turfgrass were studied. The organism was applied with a modified irrigation system by using a preparation containing 1 x 10(6) P. aureofaciens TX-1 CFU ml(-1) about 100 times between May and August. We examined the impact of this repeated introduction of P. aureofaciens TX-1 (which is known to produce the antimicrobial compound phenazine-1-carboxylic acid) on the indigenous microbial community of the turfgrass system and on establishment of introduced bacteria in the soil system. A PCR primer-DNA hybridization probe combination was developed to accurately monitor the fate of P. aureofaciens TX-1 following application in irrigation water. To assess the impact of frequent P. aureofaciens TX-1 applications on the indigenous bacterial community, turfgrass canopy, thatch, and rhizosphere samples were obtained during the growing season from control and treated plots and subjected to DNA extraction procedures and denaturing gradient gel electrophoresis (DGGE). PCR amplification and hybridization of extracted DNA with the P. aureofaciens TX-1-specific primer-probe combination revealed that P. aureofaciens TX-1 not only became established in the rhizosphere and thatch but also was capable of overwintering. Separation of PCR-amplified partial 16S rRNA genes by DGGE showed that the repeated application of P. aureofaciens TX-1 in irrigation water resulted in transient displacement of a leaf surface bacterial community member. There was no obvious alteration of any dominant members of the thatch and rhizosphere microbial communities.     

Monitoring of accelerated naphthalene-biodegradation in a bioaugmented soil slurry

The effect of microbial inoculation on the mineralization of naphthalene in a bioslurry treatment was evaluated in soil slurry microcosms. Inoculation by Pseudomonas putida G7 carrying the naphthalene dioxygenase (nahA) gene resulted in rapid mineralization of naphthalene, whereas indigenous microorganisms in the PAH-contaminated soil required a 28 h adaptation period before significant mineralization occurred. The number of nahA-like gene copies increased in both the inoculated and non-inoculated soil as mineralization proceeded, indicating selection towards naphthalene dioxygenase producing bacteria in the microbial community. In addition, 16S rRNA analysis by denaturing gradient gel electrophoresis (DGGE) analysis showed that significant selection occurred in the microbial community as a result of biodegradation. However, the indigenous soil bacteria were not able to compete with the P. putida G7 inoculum adapted to naphthalene biodegradation, even though the soil microbial community slightly suppressed naphthalene mineralization by P. putida G7.     

Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction.

In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR). Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized. To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks. Purification of nucleic acids was achieved by passage through up to three Elutip d columns. Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide. Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used. Specific primers were characterized either in the plasmid-borne vir genes for A. tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp. Specific detection of the inoculated A. tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells. Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample. This allowed us to estimate the indigenous population of Frankia spp. at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.     

Decreased abundance and diversity of culturable Pseudomonas spp. populations with increasing copper exposure in the sugar beet rhizosphere

Recent studies have indicated that culturable bacteria constitute highly sensitive bioindicators of metal-induced stress in soil. We report the impact of different copper exposure levels characteristic of contaminated agricultural soils on culturable Pseudomonas spp. in the rhizosphere of sugar beet. We observed that the abundance of Pseudomonas spp. was much more severely affected than that of the general population of culturable heterotrophic bacteria by copper. For diversity assessment, Pseudomonas isolates were divided into operational taxonomic units based on amplified ribosomal DNA restriction analysis and genomic PCR fingerprinting by universally primed PCR. Copper significantly decreased the diversity of Pseudomonas spp. in the rhizosphere and significantly increased the frequency of copper-resistant isolates. Concomitant chemical and biological analysis of copper in the rhizosphere and in bulk soil extracts indicated no rhizosphere effect and a relatively low copper bioavailability in the studied soil, suggesting that the observed effects of copper may occur at lower total concentrations in other soils. We conclude that culturable Pseudomonas sensu stricto constitutes a highly sensitive and relevant bioindicator group for the impact of copper in the rhizosphere habitat, and suggest that continued application of copper to agricultural soils poses a significant risk to successful rhizosphere colonization by Pseudomonas spp.     

Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene target

Reliability of the most widely used PCR screenings for the human opportunistic pathogen Pseudomonas aeruginosa was evaluated. Specificity analyses showed the gyrB, toxA, and 16S-23S rDNA internal transcribed spacer (ITS) but not the 16S rDNA, oprI, oprL, and fliC PCR screenings to discriminate P. aeruginosa cells from a collection of fifteen Pseudomonas species. Sensitivity analyses showed all these PCR except the toxA one to be reliable for 100% of the P. aeruginosa strains tested in this study. Specificity of the ITS and gyrB PCR screenings were further investigated on 9 soils and 29 freshwater DNA extracts of different origins, and on DNA extracted from 3 horse manures. The ITS PCR showed the highest efficacy on water and soil DNA extracts but only the gyrB one detected P. aeruginosa DNA in horse manure. DNA sequence analyses of ITS and gyrB PCR products revealed uncertainties and false positive results in these P. aeruginosa identification schemes. A novel PCR screening, targeting the ecfX gene, was thus developed. ecfX encodes an ECF (extracytoplasmic function) sigma factor which is restricted to P. aeruginosa, and might play a role in haem-uptake and virulence. Specificity and sensitivity analyses showed the ecfX PCR screening to be highly reliable, giving PCR products of the expected size for all P. aeruginosa strains tested and not amplifying DNA from any of the other Pseudomonas species tested. The ecfX PCR screening was validated on environmental DNA extracts. DNA sequence analyses of the ecfX PCR products confirmed their identity and allocation to P. aeruginosa. These investigations suggest a preferential colonization of water rather than soil environments by P. aeruginosa. Detection limits of P. aeruginosa in environmental samples were improved by the ecfX PCR screening.     

Detection and quantification of degradative genes in soils contaminated by toluene

Abstract: A method based on the polymerase chain reaction (PCR) was developed for a rapid and specific detection of toluene degradative genes in soil. The xylE gene coding for catechol 2,3-dioxygenase was chosen as a target gene. The detection threshold was evaluated in microcosms using a sterilized standard soil inoculated with various amounts of a degradative strain of Pseudomonas putida (mX). The extracted DNA was used as a template to amplify the xylE gene. PCR followed by hybridization with an internal probe allowed us to detect 10² bacteria per g of soil. In polluted soils, quantification of target DNA by competitive PCR was compared with enumeration of degradative microflora. This molecular method appeared to be rapid, sensitive and more suitable than the microbiological approach to estimate the biodegradative potential of a polluted soil.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Use of a biotinylated DNA probe to detect bacteria transduced by bacteriophage P1 in soil.

Presumptive bacteriophage P1 transductants of Escherichia coli, isolated from soil inoculated with lysates of transducing phage P1 and E. coli, were confirmed to be lysogenic for phage P1 by hybridization with a biotinylated DNA probe prepared from the 1.2-kilobase-pair HindIII 3 fragment of bacteriophage P1. No P1 lysogens of indigenous soil bacteria were detected with the DNA probe. The sensitivity and specificity of the DNA probe were assessed with purified and dot blot DNA, respectively. In addition, two techniques for the lysis and deproteinization of bacteria and bacteriophages on nitrocellulose filters were compared. These studies indicated that biotinylated DNA probes may be an effective alternative to conventional radiolabeled DNA probes for detecting specific gene sequences in bacteria indigenous to or introduced into soil.     

Influence of tomato genotype on growth of inoculated and indigenous bacteria in the spermosphere

We previously demonstrated a genetic basis in tomato for support of the growth of a biological control agent, Bacillus cereus UW85, in the spermosphere after seed inoculation (K. P. Smith, J. Handelsman, and R. M. Goodman, Proc. Natl. Acad. Sci. USA 96:4786-4790, 1999). Here we report results of studies examining the host effect on the support of growth of Bacillus and Pseudomonas strains, both inoculated on seeds and recruited from soil, using selected inbred tomato lines from the recombinant inbred line (RIL) population used in our previous study. Two tomato lines, one previously found to support high and the other low growth of B. cereus UW85 in the spermosphere, had similar effects on growth of each of a diverse, worldwide collection of 24 B. cereus strains that were inoculated on seeds and planted in sterilized vermiculite. In contrast, among RILs that differed for support of B. cereus UW85 growth in the spermosphere, we found no difference for support of growth of the biocontrol strains Pseudomonas fluorescens 2-79 or Pseudomonas aureofaciens AB254. Thus, while the host effect on growth extended to all strains of B. cereus examined, it was not exerted on other bacterial species tested. When seeds were inoculated with a marked mutant of B. cereus UW85 and planted in soil, RIL-dependent high and low support of bacterial growth was observed that was similar to results from experiments conducted in sterilized vermiculite. When uninoculated seeds from two of these RILs were planted in soil, changes in population levels of indigenous Bacillus and fluorescent Pseudomonas bacteria differed, as measured over time by culturing and direct microscopy, from growth patterns observed in the inoculation experiments. Neither RIL supported detectable levels of growth of indigenous Bacillus soil bacteria, while the line that supported growth of inoculated B. cereus UW85 supported higher growth of indigenous fluorescent pseudomonads and total bacteria. The vermiculite system used in these experiments was predictive for growth of B. cereus UW85 inoculated on seeds and grown in soil, but the patterns of growth of inoculated strains-both Bacillus and Pseudomonas spp.-did not reflect host genotype effects on indigenous microflora recruited from soil to the spermosphere.     

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).     

Biodiversity of denitrifying and dinitrogen-fixing bacteria in an acid forest soil

Isolated soil DNA from an oak-hornbeam forest close to Cologne, Germany, was suitable for PCR amplification of gene segments coding for the 16S rRNA and nitrogenase reductase (NifH), nitrous oxide reductase (NosZ), cytochrome cd(1)-containing nitrite reductase (NirS), and Cu-containing nitrite reductase (NirK) of denitrification. For each gene segment, diverse PCR products were characterized by cloning and sequencing. None of the 16S rRNA gene sequences was identical to any deposited in the data banks, and therefore each of them belonged to a noncharacterized bacterium. In contrast, the analyzed clones of nifH gave only a few different sequences, which occurred many times, indicating a low level of species richness in the N2-fixing bacterial population in this soil. Identical nifH sequences were also detected in PCR amplification products of DNA of a soil approximately 600 km distant from the Cologne area. Whereas biodiversity was high in the case of nosZ, only a few different sequences were obtained with nirK. With respect to nirS, cloning and sequencing of the PCR products revealed that many false gene segments had been amplified with DNA from soil but not from cultured bacteria. With the 16S rRNA gene data, many sequences of uncultured bacteria belonging to the Acidobacterium phylum and actinomycetes showed up in the PCR products when isolated DNA was used as the template, whereas sequences obtained for nifH and for the denitrification genes were closely related to those of the proteobacteria. Although in such an experimental approach one has to cope with the enormous biodiversity in soils and only a few PCR products can be selected at random, the data suggest that denitrification and N2 fixation are not genetic traits of most of the uncultured bacteria.     

Succession of Indigenous Pseudomonas spp. and Actinomycetes on Barley Roots Affected by the Antagonistic Strain Pseudomonas fluorescens DR54 and the Fungicide Imazalil

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain Pseudomonas fluorescens DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous Pseudomonas organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (Pseudomonas) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting Streptomyces and closely related genera. DR54 constituted more than 75% of the Pseudomonas population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous Pseudomonas organisms negatively, whereas imazalil affected Pseudomonas numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for Pseudomonas during plant growth, but after plant harvest Pseudomonas-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence, Pseudomonas was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.