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Applied and Environmental Microbiology, September 2007, p. 5531-5538, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00925-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Quantification of 2,4-Diacetylphloroglucinol-Producing Pseudomonas fluorescens Strains in the Plant Rhizosphere by Real-Time PCR{triangledown}

Olga V. Mavrodi,1 Dmitri V. Mavrodi,1 Linda S. Thomashow,2 and David M. Weller2*

Department of Plant Pathology, Washington State University, Pullman, Washington,1 USDA-ARS, Root Disease and Biological Control Research Unit, Pullman, Washington2

Received 24 April 2007/ Accepted 3 July 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A real-time PCR SYBR green assay was developed to quantify populations of 2,4-diacetylphloroglucinol (2,4-DAPG)-producing (phlD+) strains of Pseudomonas fluorescens in soil and the rhizosphere. Primers were designed and PCR conditions were optimized to specifically amplify the phlD gene from four different genotypes of phlD+ P. fluorescens. Using purified genomic DNA and genomic DNA extracted from washes of wheat roots spiked with bacteria, standard curves relating the threshold cycles (CTs) and copies of the phlD gene were generated for P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36). The detection limits of the optimized real-time PCR assay were 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures of P. fluorescens and 600 fg to 6.0 pg (80 to 800 CFU, corresponding to log 4 to 5 phlD+ strain CFU/rhizosphere) for bacterial DNA extracted from plant root washes. The real-time PCR assay was utilized to quantify phlD+ pseudomonads in the wheat rhizosphere. Regression analysis of population densities detected by real-time PCR and by a previously described phlD-specific PCR-based dilution endpoint assay indicated a significant linear relationship (P = 0.0016, r2 = 0.2). Validation of real-time PCR assays with environmental samples was performed with two different soils and demonstrated the detection of more than one genotype in Quincy take-all decline soil. The greatest advantage of the developed real-time PCR is culture independence, which allows determination of population densities and the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Strains of Pseudomonas fluorescens that produce the antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are responsible for the natural suppression of take-all diseases of wheat (Gaeumannomyces graminis var. tritici), known as take-all decline (TAD) (34, 42, 43). These bacteria also provide biological control of many root and seedling diseases on a variety of crops (6, 13, 32, 39). Because 2,4-DAPG producers occur in soils worldwide (5, 14, 25) and have such an important role in plant defense, the genotypic and phenotypic diversity of these strains has been studied extensively. Phylogenetic comparisons based on amplified rRNA gene restriction analysis of 16S rRNA genes revealed three distinct lineages among 2,4-DAPG-producing Pseudomonas fluorescens strains (14, 25). A finer degree of variation was defined by genomic fingerprinting utilizing randomly amplified polymorphic DNA analysis (14, 21, 31, 33) or repetitive sequence-based PCR (18, 25) and by restriction fragment length polymorphism (RFLP) (18, 19, 21, 25), DNA sequence (5, 36), and denaturing gradient gel electrophoresis (2) analyses of phlD, a key gene in the biosynthesis of 2,4-DAPG. The terms "phlD+ fluorescent Pseudomonas spp." and "2,4-DAPG producer" have been used synonymously because the detection of phlD correlates with the production of 2,4-DAPG by Pseudomonas spp. (42).

Genotypes defined by RFLP and sequence analysis of phlD correlated closely with clusters defined by BOX-PCR of total genomic DNA, validating the utility of phlD as a marker of genetic diversity and population structure among 2,4-DAPG producers (5, 21). To date, these techniques have distinguished at least 22 different genotypes, designated as A to T, PfY, and PfZ (19, 22, 23, 42). Although most strains from different genotypes are phenotypically similar (25, 33), they differ considerably in their rhizosphere competence (4, 17-19, 33), and it appears that certain genotypes and crop species have a mutual preference or "affinity" for each other (19, 42). For example, genotype D strains have a preference for wheat (17, 33) and pea (18), which accounts for their dominance in Washington State soils that contain multiple genotypes but have undergone continuous wheat or pea monoculture. By understanding the population size and genotype composition of 2,4-DAPG-producing strains of P. fluorescens in a soil, it is possible to predict the soil's suppressiveness to certain soilborne plant pathogens (42).

Two approaches have been used extensively for the quantification of 2,4-DAPG producers in situ: colony hybridization followed by confirmatory PCR with phlD-specific probes and primers (2, 35) and the phlD-specific PCR-based dilution endpoint assay (dilution endpoint assay) (19, 24). Landa et al. (16) employed these techniques and traditional dilution plating to quantify population densities of 10 strains from five genotypes introduced into the wheat rhizosphere and demonstrated significant linear relationships among the population sizes detected by the three methods. Each technique has strengths and weaknesses. For example, colony hybridization followed by phlD-specific PCR has a detection limit of log 4 phlD+ strain CFU/g of fresh root weight but is labor-intensive and time-consuming and does not discriminate among genotypes (16). In contrast, the phlD-specific PCR-based dilution endpoint technique with the enrichment step developed by McSpadden Gardener et al. (24) has a detection limit of log 3.1 phlD+ strain CFU/rhizosphere and allows the identification of the dominant genotype by RFLP analysis, but detection of subdominant genotypes is limited (5). To overcome this limitation, De La Fuente et al. (5) developed allele-specific PCR primers for genotypes A, B, D, K, and L for use with the dilution endpoint assay, enabling detection of multiple genotypes in a single sample. However, all of these approaches are culture dependent and require incubation of serially diluted root washes in medium selective for fluorescent pseudomonads (24), potentially altering the proportion of each genotype present in a sample (5). In addition, these approaches are not ideal for large-scale high-throughput studies of the biogeography of 2,4-DAPG producers in managed and unmanaged ecosystems.

Culture-independent methods based on extraction and analysis of DNA from environmental samples are becoming more popular for assessing the population structure of indigenous or introduced bacterial communities. One such technique, real-time PCR, is based on the quantification of the amplified PCR product, which in turn is proportional to the concentration of the template DNA. Various real-time systems demonstrating specificity, sensitivity, and speed have been developed to detect and enumerate bacteria, fungi, viruses, and yeasts (8, 10-12, 20, 26, 28, 41).

The objectives of this study were (i) to design real-time PCR primers for specific amplification of phlD from genotypes A, B, D, and I of P. fluorescens; (ii) to develop and optimize real-time PCR assays for the enumeration of 2,4-DAPG producers in bacterial cultures and rhizosphere samples; and (iii) to compare quantification of 2,4-DAPG-producing P. fluorescens strains by real-time PCR with that by phlD-specific dilution endpoint assay and traditional dilution plating.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and plasmids.
All strains used in this study are listed in Table 1 and were grown at 28°C in Luria-Bertani medium (1), Pseudomonas agar F (Difco Laboratories, Detroit, MI), or one-third-strength King's medium B (KMB) (15). Antibiotics were used at the indicated concentrations: rifampin, 100 µg/ml; cycloheximide, 100 µg/ml; chloramphenicol, 13 µg/ml; ampicillin, 40 µg/ml; kanamycin, 25 µg/ml, and gentamicin, 2 µg/ml.


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  		TABLE 1. Bacterial strains and plasmids used in this study

 
DNA preparation.
Two methods were used to obtain genomic DNA. For standard curves from bacteria grown in vitro and optimization of real-time PCR conditions, genomic DNA was isolated from overnight cultures of P. fluorescens by using a cetyltrimethylammonium bromide-based protocol (1). The DNA was resuspended in 100 µl 10 mM Tris buffer, pH 8.0, and quantified by fluorometry with Hoechst 33258 stain utilizing a fluorescent DNA quantitation kit (Bio-Rad, Hercules, CA). For standard curves from rhizosphere samples and for enumeration of phlD+ strain populations on wheat roots, total DNA from root washes was extracted with an UltraClean Soil DNA kit (MO BIO Laboratories, Solana Beach, CA) by using the alternative protocol for wet soil samples. Briefly, the entire root system with adhering rhizosphere soil from an individual 2-week-old wheat seedling grown in natural Shano sandy loam (Quincy Virgin soil) from a noncropped site near Quincy, WA, was placed in 10 ml of sterile distilled water, vortexed, and sonicated, and then 2 ml of the root wash was used for DNA extraction according to the manufacturer's protocol.

Primer design and real-time PCR conditions.
The oligonucleotide primers listed in Table 2 were developed with Oligo 6.65 software (Molecular Biology Insights, West Cascade, CO), and melting temperatures (Tms) of the primers were calculated by Oligo 6.65 using the nearest-neighbor thermodynamic method. Primers targeting the phlD gene were designed from phlD sequences of P. fluorescens Pf-5 (GenBank accession no. AF214457; genotype A), Q2-87 (U41818; genotype B), Q8r1-96 (AF207693; genotype D), FTAD1R34 (AY928638; genotype D), and FTAD1R36 (AY928647; genotype I). The sequences of these strains were aligned with ClustalW (3), and the alignment was used to design primers specific for each genotype. The following criteria were used to design the oligonucleotide primers: (i) an amplification product size of 50 to 200 bp; (ii) an oligonucleotide length of 20 to 30 bp; and (iii) an absence of predicted hairpin loops, duplexes, or primer-dimer formations (Tables 2 and 3). Primer specificity and optimization of PCR conditions were assessed by using 30 pg of genomic DNA of genotypes A, B, D, and I as a template (Table 3).


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  		TABLE 2. Genotype-specific phlD primers for real-time PCR

 

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  		TABLE 3. Optimal conditions for real-time PCR with genotype-specific phlD primers

 
Real-time PCR experiments were conducted in a Roche LightCycler (Roche Applied Science, Indianapolis, IN). PCRs were performed in borosilicate glass capillaries in 20-µl PCR volumes that contained 2.0 µl LightCycler FastStart DNA Master SYBR green I (Roche Applied Science, Indianapolis, IN), 0.5 µM of each primer, 2 µl of template DNA, and, depending on the strain, 2.5 to 4 mM MgCl2 (Table 3). The cycling program included a 10-min initial preincubation at 95°C followed by 50 cycles of 95°C for 10 s, 51 to 58°C for 15 s, and 72°C for 5 to 8 s, with the annealing temperature and extension time adjusted for each strain and primer combination (Table 3). The specificity of amplification was checked by melting curve analysis of the PCR products performed by ramping the temperature to 95°C for 0 s and back to 65°C for 15 s followed by incremental increases of 0.1°C/s up to 95°C. The melting curve calculation and determination of the Tms of the amplified products were performed using the polynomial algorithm function of LightCycler Software v.3 (Roche Applied Science, Indianapolis, IN).

Generation of standard curves from genomic DNA isolated from pure bacterial cultures.
Total DNA isolated from cultures of each strain was diluted to 30 ng/ml and used to prepare dilution series containing 3 x 105, 3 x 104, 3 x 103, 3 x 102, or 3 x 101 fg of DNA ml–1. Sterile deionized water (2 µl) was used as a negative control. Cycle threshold (CT) for the quantification of individual samples was automatically determined by the second derivative maximum method, and real-time PCR data were analyzed with LightCycler Software v.3 (Roche Applied Science, Indianapolis, IN). A standard curve for each strain was generated by plotting the CT number versus the logarithm of bacterial DNA concentration (converted to CFU) from three independent replications of each DNA concentration. The amount of DNA in unknown samples was interpolated via CT from a linear regression line through the standard data points, and the amplification efficiency (E) was calculated from the slope of the standard curve by using the formula E = 10–1/slope – 1. Standard curves were generated by using Sigma Plot, version 8.0 (SYSTAT Software Inc., Richmond, CA). The calculation of phlD gene copy number in a sample was based on the size of the genome of P. fluorescens Pf-5 (7.1 Mbp) (30) and the knowledge that only one copy of the phlD gene is present per genome of P. fluorescens.

Generation of standard curves with DNA purified from rhizospheres spiked with bacteria and enumeration of 2,4-DAPG producers.
Roots with attached rhizosphere soil from individual 2-week-old wheat seedlings were cut from the shoot, placed in 50-ml Falcon tubes containing 10 ml of sterile distilled water, and spiked with cells of each strain to achieve 103, 104, 105, 106, 107, or 108 CFU per tube. The tubes were vortexed and sonicated for 1 min, and 2 ml of the root wash was used for DNA extraction with an UltraClean Soil DNA kit as described above. Subsequently, 2 µl of template DNA was used for real-time PCRs with genotype-specific primers following the protocols described above. No bacteria were added to the control samples. A standard curve for each strain was generated by plotting the CT number versus the logarithm of bacterial DNA concentration (converted to CFU) from the four independent repetitions for each spiked bacterial amount.

The same spiked wheat root samples were used to enumerate bacteria by the dilution endpoint assay and traditional dilution plating assays described previously (16). For the dilution endpoint assay (24), 100 µl of root wash was serially diluted in 96-well microtiter plates containing one-third-strength KMB broth supplemented with rifampin, cycloheximide, ampicillin, and chloramphenicol (1/3 KMB+++rif) and incubated for 72 h at room temperature. An optical density at 600 nm of ≥0.1 was scored as positive (24). Another 100-µl aliquot of the wash solution was serially diluted and plated on 1/3 KMB+++rif agar; plates were incubated at room temperature, and colonies were counted after 72 h.

Rhizosphere colonization assay and comparison of methods for enumeration of 2,4-DAPG producers.
Rhizosphere colonization assays were performed using P. fluorescens strains tagged with mini-Tn7-gfp2 or mini-Tn7-gfp1 (40) (Table 1). Bacterial inocula were prepared and added to natural Shano sandy loam (Quincy Virgin soil) as previously described (16) to give ~1 x 104 CFU g of soil–1 or ~0.5 x 104 CFU g of soil–1 for each strain for single and mixed inoculations (1:1 ratio), respectively. The actual density of each strain was determined by assaying 0.5 g of inoculated soil as described by Landa et al. (16). Control treatments consisted of soil amended with a 1% methylcellulose suspension. Experiments were repeated twice with six replicates per treatment. Spring wheat (Triticum aestivum L.) seeds (cv. Penawawa) were pregerminated on moistened sterile filter paper in petri dishes for 24 h in the dark and were sown in square pots (6.5 cm high by 7 cm wide) containing 200 g of inoculated or noninoculated soil (16). Wheat was grown for six successive cycles, 2 weeks each, in a controlled-environment chamber at 15°C with a 12-h photoperiod, and populations of bacteria were determined after each growth cycle.

Rhizosphere population densities were monitored on root systems from single seedlings that were analyzed by both real-time PCR and the modified dilution endpoint assay (40). Real-time PCR analyses included four independent repetitions per bacterial treatment. To enumerate individual strains in mixed-inoculation treatments by the modified dilution endpoint method (40), dilutions of each root wash sample were first cultured in 96-well microtiter plates containing 1/3 KMB+++rif and incubated for 72 h at room temperature (optical density at 600 nm of ≥0.07 was scored as positive) (40). The cultures then were transferred with a 96-pin replicator as previously described (40) into fresh plates containing KMB broth amended with kanamycin or gentamicin to distinguish each inoculant strain separately.

Data analysis.
Statistical analyses were performed by using appropriate parametric and nonparametric procedures with STATISTIX 8.0 software (Analytical Software, St. Paul, MN). All treatments in competitive colonization experiments were arranged in a completely randomized design. All population data were converted to log CFU per rhizosphere or root (fresh weight). Differences in population densities among treatments were determined by standard analysis of variance, and mean comparisons among treatments were performed by Fisher's protected least significant difference test (P = 0.05) or by the Kruskal-Wallis all-pairwise comparisons (P = 0.05). Regression analyses were performed on population densities of 2,4-DAPG-producing P. fluorescens strains enumerated by the real-time PCR, dilution endpoint assay, and dilution plating.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Primer design and evaluation of PCR specificity.
Primers specific for the phlD alleles from P. fluorescens strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36) were developed after comparison of the respective phlD DNA sequences (Table 2). The sites for primer annealing were chosen such that the 3' nucleotide and, whenever possible, the penultimate nucleotide would efficiently discriminate between the genotype-specific target allele and closely related sequences during amplification with Taq DNA polymerase. Primer specificity for each genotype was tested by real-time PCR with genomic DNA extracted from pure cultures of each strain. To validate the specificity of the primers and to optimize assay conditions, the real-time PCR products also were analyzed by agarose gel electrophoresis (data not shown). As a result, optimal amplification conditions were identified for each strain and each set of primers (summarized in Table 3).

Standard curves for genomic DNA from bacterial cultures.
Standard curves for each strain were generated with purified genomic DNA added to PCR mixtures in quantities ranging from 60 fg to 6 x 105 fg, corresponding to 8 copies to 8 x 104 copies of phlD (or CFU) of P. fluorescens. The limit of detection by real-time PCR for genomic DNA from cultures of genotypes A (Pf-5) and B (Q2-87) was 60 fg (8 CFU) per reaction or 600 fg (80 CFU) per reaction for genotypes D (Q8r1-96 and FTAD1R34) and I (FTAD1R36). For all five strains, linear relations (r2 = 0.99) were observed between log CFU per reaction (derived from the number of phlD gene copies) and real-time PCR CTs (Table 4).


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  		TABLE 4. Standard curve parameters and real-time PCR amplification efficiencies of genotype-specific primer sets used in the study

 
Variables such as primer composition and secondary structure and amplicon length can affect the efficiency (E) of PCR, which ideally should be 100%, resulting in a standard curve with a slope of –3.3. Standard curves generated with DNA from genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36) had slopes from –3.4 to –3.7 (Table 4), and amplification efficiencies with genotype-specific primers differed for each phlD allele from 85% to 98% (Table 4).

Standard curves for DNA purified from the wheat rhizosphere.
Preliminary experiments were performed to optimize the DNA extraction efficiencies of phlD+ Pseudomonas strains in situ. Among the tested methods for bacterial cell disruption, vortexing of the samples at maximum speed for 10 min was preferred over using a FastPrep FP120 instrument at a speed of 5.0 for 45 s. Because our research is focused on rhizosphere bacteria, we modified the UltraClean Soil DNA protocol and extracted DNA from root washes containing bacteria and soil particles instead of bulk soil. In addition, the use of 2 ml of root washes containing soil particles for DNA isolation was chosen over 10 ml. To precisely calculate the population size in the rhizosphere, we generated new standard curves with DNA extracted from root washes that had been spiked with known quantities (103 to 108 CFU) of each target strain. The calculated extraction efficiencies for this protocol varied by strain and ranged from 5.5% to 9.2%.

For the four genotypes tested, linear relations (r2 = 0.95 or r2 = 0.99) were observed between the log values of bacterial genomic DNA (converted to log CFU/reaction) and CT over a range of 4 or 5 orders of magnitude (Table 4). Slopes of these standard curves varied from –2.9 to –3.7, corresponding to amplification efficiencies (E) of 80% to 98% (Table 4). Limits of detection by real-time PCR were 600 fg (80 CFU) per reaction or log 4 phlD+ strain CFU/rhizosphere for genotypes D (Q8r1-96 and FTAD1R34) and B (Q2-87) and 6.0 pg (800 CFU) per PCR or log 5 phlD+ strain CFU/rhizosphere for genotypes A (Pf-5) and I (FTAD1R36).

Comparison of techniques for quantification of phlD+ strains in the rhizosphere.
In order to compare the population sizes determined by the real-time PCR assay to those detected by the dilution endpoint assay, strains belonging to genotypes A (Pf-5), B (Q2-87), D (Q8r1-96 and FTAD1R34), and I (FTAD1R36) were introduced, either individually or in mixtures, at 104 CFU g of soil–1 into pots containing natural Quincy Virgin soil sown with wheat cultivar Penawawa. After 2 weeks of incubation in a growth chamber, population densities of the introduced bacteria in the wheat rhizosphere were determined by both methods. The data from single and mixed inoculations of each strain were combined and used to compare the performance of the two techniques. The results indicated that for genotypes D (Q8r1-96 and FTAD1R34) and I (FTAD1R36), the population sizes estimated by real-time PCR were significantly greater than those detected by the dilution endpoint method (Fig. 1). In contrast, the population density of genotype B (Q2-87) as estimated by the dilution endpoint assay was significantly higher than that detected by real-time PCR (7.1 versus 6.3 log CFU/g root), while similar populations of genotype A (Pf-5) were detected by the two assays (Fig. 1). Correlation and regression analyses of pooled data across all strains revealed a significant linear relationship (P = 0.0016, r2 = 0.2) between population levels detected by the two assays (Fig. 2).


Figure 1
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  		FIG. 1. Comparison of population levels detected by the phlD-specific PCR-based dilution endpoint (black bars) and real-time PCR (white bars) assays. Bacteria were introduced into natural Shano sandy loam (Quincy Virgin) at 104 CFU/g of soil, and wheat was grown for 2 weeks. Bars above the same strain with the same letter are not significantly different (P = 0.05).

 

Figure 2
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  		FIG. 2. Enumeration of 2,4-DAPG-producing Pseudomonas fluorescens strains in the rhizosphere of wheat by the endpoint dilution and real-time PCR assays. Bacteria were introduced into natural Shano sandy loam (Quincy Virgin) at 104 CFU/g of soil, and wheat was grown for 2 weeks. A significant (P = 0.0016) linear relationship was found between population densities of different bacteria detected by the two techniques. Each symbol represents the population of bacteria detected on roots plus adhering rhizosphere soil from a single seedling.

 
Previously, population sizes of phlD+ pseudomonads detected by the dilution endpoint technique were compared and found to correlate with traditional dilution plating and colony hybridization followed by PCR (16). Similarly, regression analysis of our data demonstrated a significant linear relationship (P < 0.0001, r2 = 0.91) between population densities of the tested strains estimated in the rhizosphere of wheat by the dilution endpoint assay and dilution plating onto 1/3 KMB+++rif (Fig. 3).


Figure 3
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  		FIG. 3. Enumeration of 2,4-DAPG-producing Pseudomonas fluorescens strains by the endpoint dilution assay in 1/3 KMB+++rif broth and dilution plating on 1/3 KMB+++rif plates. Root washes of 2-week-old wheat seedlings were spiked with individual strains to achieve 103, 104, 105, 106, 107, and 108 CFU in 10 ml of water and serially diluted, and bacteria were enumerated after incubation for 72 h at room temperature. A significant (P < 0.0001) linear relationship was found between population densities of different bacteria detected by the two techniques. Each symbol represents the population of bacteria detected on a single wheat root system.

 
Validation of real-time PCR assays with environmental samples.
Soils from two sites near Quincy, WA (Quincy Virgin soil and Quincy TAD soil) (34), were checked for the presence of phlD+ pseudomonads. Wheat was grown in the soils for 3 weeks, and then population densities of indigenous phlD+ pseudomonads in the wheat rhizosphere were determined by real-time PCR and by the dilution endpoint assay. Both methods showed that Quincy Virgin soil did not contain indigenous phlD+ pseudomonads. From the Quincy TAD soil, the dilution endpoint assay detected log 8.4 CFU of phlD+ pseudomonads/g root and real-time PCR detected log 7.0 and log 8.1 CFU/g root of D-genotype and B-genotype indigenous phlD+ pseudomonads, respectively.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It is well known that only a small fraction of environmental bacterial communities are culturable in vitro. Methods based on analysis of DNA extracted directly from environmental samples are greatly enhancing our knowledge of the viable but unculturable fraction of indigenous and introduced bacterial communities. Among such methods is quantitative PCR, which allows quantification of the target DNA and subsequently an estimation of the number of the corresponding cells. The early version of quantitative PCR, called quantitative competitive PCR (QC-PCR), has been used successfully to detect indigenous Pseudomonas DNA in soil hot spots (12), with a significant linear relationship between population densities of bacteria detected by QC-PCR and dilution plating on a Pseudomonas selective medium (12). QC-PCR is based on coamplification of the target DNA and a known amount of competitor DNA with the same set of primers. The amplification product of the competitor DNA (internal standard) differs slightly in size from the target DNA and can be separated on a gel.

A more recent version of quantitative PCR, called real-time PCR, is based on the measurement of fluorescence generated by a fluorochrome or hybridization probe that binds to double-stranded DNA after each cycle and does not require post-PCR manipulation. The cycle at which the fluorescent signal crosses the threshold line, known as CT, is directly proportional to the amount of target DNA present in the sample. Real-time PCR has a dynamic range of 4 to 7 orders of magnitude of target DNA depending on the model organism and has been applied for the direct quantification of bacterial populations in a number of different environments: Pseudomonas stutzeri in lake sediments and groundwater (8); ammonia-oxidizing bacteria in soil (28); Escherichia coli O157:H7 in soil, manure, feces, and dairy waste wash water (11); and Streptococcus pneumoniae in nasopharyngeal secretions (7). Recently, real-time PCR was also successfully employed for the detection and quantification of soilborne plant pathogens in soil and plant samples (29).

In the current work, we describe the development of a SYBR green real-time PCR assay targeting the phlD gene in several 2,4-DAPG-producing strains of P. fluorescens belonging to four different genotypes (A, B, D, and I). The assay proved to be quantitative over 4 to 5 orders of magnitude, and because the genome size of P. fluorescens (30) and the copy number of phlD are known, the method can also be employed for direct estimation of cell counts of target strains. The PCR assay was first optimized for quantification of phlD in bacterial DNA isolated from pure cultures, but the use of standard curves generated from bacterial DNA extracted from the plant rhizosphere allowed for the enumeration of the population densities of each strain directly on wheat roots. The detection limit of the optimized real-time PCR assay was 60 to 600 fg (8 to 80 CFU) for genomic DNA isolated from pure cultures and 600 fg to 6.0 pg (80 to 800 CFU) for bacterial DNA extracted from plant root washes. It may be possible to improve the sensitivity of the assay by further concentrating the sample and/or by improving the efficiency of extraction of bacterial DNA from plant rhizospheres. It is notable that the amplification efficiencies with D-genotype-specific primers and the detection limits of the real-time PCR were the same for genotype D strains Q8r1-96 and FTAD1R34, which were originally isolated in Washington State and North Dakota, respectively. This indicates the validity of the real-time assay as a genotype-distinguishing method and not just as a strain-specific method.

We also compared the performance of the real-time PCR assay to that of the dilution endpoint assay, a technique that is currently used by our group to estimate population densities of 2,4-DAPG-producing Pseudomonas fluorescens strains in the rhizosphere of wheat. The regression analysis revealed a significant linear relationship (P = 0.0016, r2 = 0.2) between the population densities of all of the strains of 2,4-DAPG producers in the wheat rhizosphere as detected by the two methods (Fig. 2). The significantly higher population densities of genotypes D (Q8r1-96 and FTAD1R34) and I (FTAD1R36) detected by real-time PCR than of those estimated by the dilution endpoint method (Fig. 1) suggest that the real-time PCR assay may detect nonculturable but viable cells. Interestingly, in a study by Rezzonico et al. (37), the authors used QC-PCR targeting phlA to enumerate P. fluorescens CHA0 bacteria in vitro and found a significant correlation between populations of CHA0 detected by QC-PCR and dilution plating only when cells were in logarithmic growth or stationary phase (37). On the other hand, when the cells were stressed, this correlation disappeared owing to DNA from nonculturable or dead cells being detected by QC-PCR (37). We speculate that a similar phenomenon may explain the fact that real-time PCR estimated a lower population density for genotype B (Q2-87) than did the dilution endpoint assay.

In addition, we compared the performance of the real-time PCR assay to that of the dilution endpoint assay in detecting indigenous phlD+ pseudomonads in wheat rhizospheres from Quincy Virgin and Quincy TAD soils. Enumerations of bacteria by the two methods were very similar, but the advantage of real-time PCR was the detection of dominant and subdominant genotypes in Quincy TAD soil.

The average detection limit of the dilution endpoint assay for 10 strains of 2,4-DAPG producers is log 3.26 CFU/g of fresh root weight (16), but without the culture enrichment step in the assay the detection limit drops to log 5.6 cells per rhizosphere (24). Reliance on pregrowth of bacteria in selective medium increases the possibility that the proportion and composition of genotypes detected in a sample do not precisely reflect those which occur in situ because of inhibition among strains of certain genotypes in vitro (5). For example, it was reported that genotype D (Q8r1-96) inhibited the growth of genotype B (Q2-87) in vitro, thus making detection of genotype B (Q2-87) difficult when the two strains were cultured together (5). Our study demonstrated a detection limit of log 4 to 5 phlD+ strain CFU/rhizosphere for the real-time PCR assay. This detection limit is comparable to that of other real-time PCR systems for quantification of bacterial populations. For example, real-time PCR with SYBR green was used to detect 16S rRNA genes of the gram-positive sulfate-reducing Desulfotomaculum lineage 1 with a detection limit of 106 targets per gram (dry weight) of rice bulk soil and rice roots (38). This detection limit reflected the dilution of the DNA extracts that was necessary to overcome PCR inhibition by humic substances (38). Likewise, the limit of detection of Corynebacterium casei, a major species used for smear-ripened cheeses, by SYBR green real-time PCR targeting 16S rRNA was about log 5 CFU/g (26), and a TaqMan real-time PCR assay targeting part of the ammonia-monooxygenase gene (amoA) for estimation of populations of ammonia-oxidizing bacteria in soil had a detection limit of 1.3 x 105 cells g of dry soil–1 (28). Similarly, the detection limit for Escherichia coli O157:H7 in environmental samples was 2.6 x 104 CFU g of soil–1 with SYBR green real-time PCR (11).

In the Pacific Northwest, the enrichment of 2,4-DAPG producers by intensive cropping of wheat or barley is responsible for the natural suppression of take-all in almost 0.8 million hectares (42). Our real-time PCR assay is the first step toward rapid screening of soils for their suppressiveness to take-all, the most important root disease of wheat worldwide. The turnaround time for the real-time PCR assay is more favorable: 1 to 2 days versus 5 to 6 days for the phlD-specific PCR-based dilution endpoint assay. The greatest advantage of real-time PCR is culture independence, which allows determination of population densities and, more importantly, the genotype composition of 2,4-DAPG producers directly from the plant rhizospheres and soil. Knowing the genotype is essential because in the Pacific Northwest and elsewhere, genotype D isolates are primarily responsible for the natural suppressiveness of take-all. In summary, this study demonstrates the potential of real-time PCR for direct quantification and characterization of genotypes of 2,4-DAPG producers present in plant rhizosphere samples. The method will enhance our understanding of the ecology, biogeography, and interactions among and contributions to root defense by 2,4-DAPG-producing P. fluorescens strains.


   ACKNOWLEDGMENTS
 
We thank Patricia Okubara and Kurtis Schroder for their help with initial quantitative PCR work, Amanda Park and Jennifer Kleene for technical assistance, and Nathalie Walter for comments on the manuscript. Also, we thank M. Evans from the Department of Statistics at Washington State University for consultation on statistical analyses of our data.


   FOOTNOTES
 
* Corresponding author. Mailing address: USDA-ARS, Root Disease and Biological Control Research Unit, Washington State University, Pullman, WA 99164-6430. Phone: (509) 335-6210. Fax: (509) 335-7674. E-mail: wellerd{at}mail.wsu.edu Back

{triangledown} Published ahead of print on 13 July 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Applied and Environmental Microbiology, September 2007, p. 5531-5538, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00925-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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