Development of qPCR Methods for Quantification of Pseudomonads Harboring DAPG, PHZ, and PRN Biosynthetic Genes

To quantify the abundance of DAPG and PHZ producing bacteria in soil, we developed quantitative real-time polymerase chain reaction (qPCR) assays targeting phlD and phzF genes. These genes encode, respectively, a polyketide synthase involved in the synthesis of phloroglucinols from malonyl-CoA (Bangera and Thomashow, 1996; Achkar et al., 2005) and an isomerase involved in the synthesis of phenazine-1-carboxylic acid (Mavrodi et al., 1998; Blankenfeldt et al., 2004). Alignments were created with publicly available phlD and phzF sequences from GenBank² and conserved regions were chosen for the design of primers and probes (Table ​Table22), which was carried out with the Primer 3 Plus software (Untergasser et al., 2007). The parameters were amplicon length between 100 and 200 bp, melting temperature (TM) between 50 and 70°C, TM of probe 5°C higher than TM of primers, and the default setting of the program for self-complementarity and 3′-end stability. Partial sequences of phlD (GenBank accession {"type":"entrez-nucleotide","attrs":{"text":"CP003190.1","term_id":"500239649","term_text":"CP003190.1"}}CP003190.1| :6563260-6563937) of strain P. protegens CHA0 (Jousset et al., 2014) and phzF (locus tag, PFLU3_RS28075) of P. synxantha 2–79 (Nesemann et al., 2015) were used for primer design. The specificity of the primers was tested in silico with Primer-Blast (Ye et al., 2012) and in vitro with genomic DNA from 28 DAPG-producing strains and 38 PHZ-producing strains of the P. fluorescens group and nine additional PHZ-producing strains (Supplementary Table S1). Results of these tests revealed that our qPCR assays amplify phlD and phzF genes exclusively from DAPG and PHZ producing species of the P. fluorescens lineage. The PRN biosynthetic genes were quantified on wheat roots by the qPCR method of Garbeva et al. (2004). That assay targets a gene for the class IA oxygenase PrnD that is involved in the final step of PRN biosynthesis (Kirner et al., 1998). In contrast to our phlD and phzF primers, the primers of Garbeva et al. (2004) have broader specificity and, in addition to Pseudomonas, amplify prnD from PRN-producing strains of Burkholderia and Serratia.

Primers and probes used to quantify antimicrobial genes with qPCR.

The efficiency of phlD and phzF primers at low gene copy numbers was evaluated using in vitro standard curves prepared by serially diluting genomic DNA of P. protegens CHA0 and P. synxantha 2–79. The genomic DNA was prepared by growing both strains in lysogeny broth (LB) (Bertani, 1951) overnight at 24°C on a rotary shaker at 180 rpm and extracting DNA with the Wizard Genomic DNA Purification Kit (Promega AG, Dübendorf, Switzerland). The concentration of purified DNA was quantified by fluorimetry with Qbit (Thermo Fisher Scientific). We also generated an in vivo standard curve for each qPCR assay to quantify the corresponding target genes on wheat roots. To this end, aliquots of 1 g of 21-days-old roots of spring wheat cv. Rubli grown in autoclaved soil were inoculated with decreasing concentrations of a mixture of bacterial cells belonging to different strains carrying the respective target gene. Strains used for in vivo standard curves are listed in Supplementary Table S1. Bacterial cells were harvested from overnight cultures in LB, washed and suspended in sterile 0.9% NaCl solution. Cell suspensions from each strain were set to the same optical density at 600 nm (OD₆₀₀) and then mixed together at equal proportions. The mixed suspensions were adjusted to an OD₆₀₀ of 0.125, corresponding to approximately 10⁸ CFU mL^-1, serially diluted and inoculated at 10¹, 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷ and 10⁸ CFU g^-1 roots for the preparation of the standard curve. For each concentration and for the control without bacteria, three replicates were performed. The inoculated root samples used for standard curves were processed with the same method as the samples from pot experiments with the different soils (see following chapter). In vivo standard curves were prepared as described above for the phlD, phzF and prnD qPCR, using strains listed in Supplementary Table S1. Since all in vivo standard curves were prepared with bacterial cells recovered from wheat roots, the CT values can be directly converted to numbers of bacteria harboring phlD, phzF, or prnD per g root. Our qPCR data also directly reflect the abundance of the antimicrobial biosynthesis genes because phlD, phzF, or prnD are present in single copy in genomes of the P. fluorescens group (Flury et al., 2016). A survey of published bacterial genomes revealed that phzF and prnD are also found as a single copy in other bacterial species such as Burkholderia (phzF and prnD), Pectobacterium (phzF) or Serratia (prnD).