Plant growth promoting pseudomonads play an important role in disease suppression and there is considerable interest in development of bio-marker genes that can be used to monitor these bacteria in agricultural soils. Here, we report the application ofa PCR primer sets targeting genes encoding the main antibiotic groups. Distribution of the genes was variably distributed across type strains of 28 species with no phylogenetic groupingfor the detected antibioticsgenes, phlD for 2,4-diacetylphloroglucinol (2,4-DAPG) and phzCD for phenazine-1-carboxylic acid or hcnBC for hydrogen cyanide production. Analysis of field soils showed that primer sets for phlD and phzCD detected these genes in a fallowed neutral pH soil following wheat production, but that the copy numbers were below the detection limits in bulk soils having an acidic pH. In contrast, PCR products for the phzCD, pltc and hcnBc genes were detectable in mature root zones following plantingwith wheat. The ability to rapidly characterize populations of antibiotics producers using specific primer sets will improve our ability to assess the impacts of management practices on the functional traits of Pseudomonas spp. populations in agricultural soils.
Plant growth promoting rhizosphere bacteria improve plant growth through the production of hormones and by suppression of root disease through the production of antibiotics and siderophores. Using culture based methods to identify these bacteria, PGPR have been isolated from plants in soils from diverse geographical regions [1-5]. One of the most important groups of PGPR are strains of antibiotic-producing fluorescent Pseudomonas spp. that contribute to suppression of fungal diseases including take-all of wheat, black root rot of tobacco, or fusarium wilt of tomato [6-9]. The genes that are responsible for production of antibiotics are variably dispersed among different genotypes, which vary in their efficacy for disease suppression [5,8,10-12]. Thus there is interest in development of PCR primer sets that can be used to detect the presence of these genes in environmental samples and to monitor the populations of bacteria that carry these genes [6,7,13-15].
Genes encoding the antibiotics pyoluteorin (pltC) [
Molecular methods for the detection of these genes have been described, but are often utilized in combination with culture based methods to quantify the population sizes of Pseudomonas strains carrying specific genes [
With the advent of rapid screening methods for detection of marker genes that are associated with plant health and growth in agricultural soils, we were interested in determining the utility of PCR based methods to detect a suite of genes for plant growth promoting pseudomonads in wheat soils in S.E. Australia where there is still little information on the ecology and importance of these bacteria in soil health. The research reported here examined selected PCR primer sets from the literature for detection of specific marker genes, and the association of antibiotic genes among different type strains of Pseudomonas spp. The primer sets were evaluated for three different soil classes representing the major soils used for wheat cultivation in Victoria, Australia. We examined both the field soil and adjacent remnant areas that had not been cultivated and further compared soils that were collected during the fallow period and in the rhizosphere after cultivation with wheat. The results demonstrate the utility of molecular methods to compare soils, but also illustrate the highly dynamic changes that occur in managed soils.
Soil samples were collected in autumn from three soil classes according to the Australian soil classification system (Isbell, R.F., 2002. The Australian Soil Classification. CSIRO Publishing, Melbourne), including a dermosol (NE Victoria, Rutherglen), a calcarosol (NW Victoria, Birchip) and a chromosol (SW Victoria, Hamilton) collected in southeastern Australia. Within each of these regions, soils were collected from paired sites representing two contrasting land-uses, a “remnant” uncultivated site where indigenous plant species were present and a ‘managed’ site where the soils were subjected to normal agricultural practices with fertilizer and herbicide input regimes, grazing and cropping activities. Soil chemical and physical characteristics for the six sites are summarized in
the soil fractions, after which the soil and root were processed for DNA extraction.
Soil pH and salinity (electrical conductivity, EC) were measured in soil extracts using 1:5 soil water and 1:1 soil water: 1 mM CaCl2 extraction solutions. Exchangeable cations, and heavy metals (iron, zinc, copper, and manganese) were quantified by atomic absorption spectrophotometer (Perkin-Elmer AAnalyst 800). Carbon and nitrogen was analyzed using a C-N-S analyzer (NA 1500 series 2, Carlo Erba). The soil particle size was analyzed using the hydrometer method. Nitrate, nitrite, ammonia and soluble reactive phosphorus were quantified with a Technicon TRAACS 800 Autoanalyzer (Tarrytown, NY).
DNA was extracted from 0.5 g samples of each soil and root using the FastPrepTM Soil DNA Extraction kit (Qbiogene, Carlsbad, CA) according to the manufacturers’ recommendations. Five replicates for each soil and root sample were prepared from each soil and were stored at −80˚C until use for subsequent analyses.
PCR was conducted with 10 μl reaction volumes containing: 1 μl of DNA, 2 μl of Terminator Ready Reaction Mix, and 200 nM each of T7 and SP6 primers. The reagents were combined and heated to 96˚C for 1 min. Twenty-five cycles of PCR were run at 96˚C for 10 s, 50˚C for 10 s, and 60˚C for 4 min. Approximately 1 kb (position 289-1258 [
16S rDNA sequences were aligned with greengenes [25,26]. Evolutionary distances were calculated by the method of Kimura 2-parameter and a phylogenetic tree was constructed by the neighbor-joining method [
The nucleotide sequence data reported in this paper will appear in the GenBank/EMBL/DDBJ nucleotide sequence databases with the accession number(s) AY860450-AY860454.
PCR primer sets for conserved sequences of genes involved in expression and regulation of six antibiotics were targeted against 28 type strains of Pseudomonas spp. Of the six sequences evaluated, antibiotic gene sequences for 2,4-DAPG (phlD) and phenazine (phzCD) were detected in 3 and 5 of the type strains, respectively (
PCR products were obtained in all of the type strains with primers for 16S rRNA sequences, and in 25 and 23 of the strains using primers for the proposed phylogenetic classification markers gyrB and rpoD. The hcnBC genes for cyanide production and gacA for global activator cyanide, which are involved in a regulatory circuit affecting antibiotic production, were widely dispersed in 8 and 14 of the 28 type strains. In this case, the gacA gene was detected in 5 of the 8 type strains having positive signals for hcnBC. Marker gene sequences for aminocyclopropane deaminase (acc) and indole acetic acid production (ipdc) were not detected in any of the type strains tested here.
The fully set of gene targets for Pseudomonas sp. and functional genes were tested in bulk soils and in root samples from wheat for managed and remnant soils
1fluoresecent Pseudomonas; 2global activator of cyanide and antibiotic production; 31-aminocyclopropane-1-carboxylic acid.
represented 3 soil geomorphic classes in Victoria, Australia. These soils were of similar texture, but varied in pH and organic carbon (
The hcnBC gene was detected in two of the six bulk soil samples, and in mature root zones in all three soil classes. The gacA gene was not detected in any of the bulk or root samples, nor were the two growth regulator genes, the ipdc and acc genes were not detected in any of the root samples. The phylogenetic group marker 16S rRNA gene target for Pseudomonas was detected in all soil samples, but the gyrB and rpoD genes were not detected in any of the soil samples.
The objective of this study was to evaluate a selected set of gene targets for detection of Pseudomonas sp. and functional genes for antibiotic production, regulation of cyanide and antibiotic production, and plant growth hormone production across different soil types representing a range of important soils used for wheat production in Australia. There is considerable interest in developing molecular methods to monitor these and other species of plant growth promoting bacteria as part of a strategy to monitor soil health and the impacts of various management practices on the population densities of PGPR bacteria. Previous research has identified a number of candidate genes that are potential markers for antibiotic-producing Pseudomonas spp. It has also been shown that there is considerable variation in genotypes of specific antibiotic-producing Pseudomonas that vary in their ability to colonize the rhizosphere (Bergsma-Vlami et al., 2005). In general, population densities of pseudomonads are highly dynamic in soils and are highest in the plant rhizosphere, but are also influence by the host plant species [
tion that may be preferable, allowing uniform conditions for establishment of a stable population size for comparative analysis of soils over time and across different soil types and management regimes. This is analogous to monitoring of chemical and physical variables used to describe soil quality, where specific reference locations, or “benchmarks”, are used for comparison of changes in chemical and physical variables such as bulk density and hydraulic conductivity. In the case of soil biological variables, the benchmark conditions employ a standardized test plant species, plant age, and rhizosphere location.
The gacA sequence was of particular interest here for its role in regulation of both cyanide and antibiotic production. However, our study of the type strains for 28 species shows that the hcnBC and gacA genes were coassociated only in 30% of the strains. This could be due to variations in the sequence that was targeted. The gacA gene, which was detected in 3 of the 14 strains was consistently co-associated with the phlD gene for 2,4-DAPG production, but was inconsistently associated with phenazine production for 3 of the 5 phzCD containing type strains.
Several species of soil bacteria contain the acc gene [17,18]. The gene appears to undergo horizontal transfer and has been found in various Pseudomonas sp. The sequence used here was not detected in any of the type strains, or in the soils that were screened. This was unexpected as the target sequence we used is conserved. It is possible of course that our inability to detect this gene and others in our primer set such as the pupA gene for siderophores production is due to low copy numbers or interferences from PCR inhibitors in the soil extracts that lowered the detection efficiency.
Molecular methods for detection of specific gene sequences for monitoring bacterial species population densities and copy numbers of functional genes are advanceing rapidly. Results of this research demonstrate that it will be necessary to empirically test many of the sequences that have been identified in the laboratory for their efficacy in actual field samples, and standardize the conditions that are to be used for screening soils. This may be best achieved by bringing soils to a standard reference state that reflects the conditions under which the PGPR perform their ecosystem function; in this case the presence of a plant rhizosphere of a particular plant species. The ability to rapidly characterize populations of antibiotics producers will greatly enhance our understanding of their role in enhancing plant health and yields, and the impacts of management practices on PGPR bacteria.
The authors gratefully acknowledge the support of the Department of Primary Industries Victoria as part of the ‘Our Rural Landscape’ initiative (sub-project 1.2 Our Soils: Understanding and Protecting the Soils of our Rural Landscapes) funded by the Victorian State Government (Department of Innovation, Industry and Regional Development).