Quantification of 2,4-diacetylphloroglucinol-producing Pseudomonas fluorescens strains in the plant rhizosphere by real-time PCR.

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.