Pseudomonas orientalis F9 Pyoverdine, Safracin, and Phenazine Mutants Remain Effective Antagonists against Erwinia amylovora in Apple Flowers

Cultivation of bacterial strains used.Bacterial overnight cultures were grown at 26°C in tryptic soy broth (TSB; Oxoid, Karlsruhe, Germany) or King’s B (KB) medium (36). Partial stigma-based medium (PSBM) (14) was also used. Microorganisms used in the study are listed in Table 1. Where appropriate, medium was supplemented with kanamycin or rifampin at a concentration of 40 mg liter⁻¹ or 100 mg liter⁻¹, respectively.

Transposon mutagenesis.Random insertion Tn10 transposon mutagenesis was performed using pJA1, an oriR6K-based suicide vector. The plasmid contains the Tn10 transposase and confers kanamycin resistance (37). Donor Escherichia coli SM10 λpir(pJA1) and recipient P. orientalis F9 were grown overnight in either LB medium (10 g liter⁻¹ tryptone, 5 g liter⁻¹ yeast extract, 10 g liter⁻¹ NaCl, pH 7.0 ± 0.2 [Carl Roth]) supplemented with kanamycin or KB medium, respectively. The overnight culture of E. coli SM10 λpir(pJA1) was inoculated into fresh LB medium containing kanamycin and grown to mid-exponential phase. One milliliter of the donor and the recipient in stationary phase was mixed and centrifuged for 30 s at 10,000 × g. The bacterial pellet was washed twice with 1× phosphate-buffered saline (PBS) (2.5 g liter⁻¹ K₂HPO₄, 1.2 g liter⁻¹ KH₂PO₄) and subsequently spotted onto the center of an isopropyl-β-d-thiogalactopyranoside (IPTG)-containing LB plate (10 μl of a 100 mM IPTG solution, spotted onto the center of the plate). The conjugation plate was incubated at 37°C for 3 to 5 h. A dilution series was plated onto MM2-agar plates (4 g liter⁻¹ l-asparagine, 2 g liter⁻¹ K₂HPO₄, 0.2 g liter⁻¹ MgSO₄, 3 g liter⁻¹ NaCl, 10 g liter⁻¹ sorbitol, 15 g liter⁻¹ agar) supplemented with kanamycin. After 2 to 3 days of growth at 26°C, single colonies were picked and inoculated in 96-well plates containing KB medium and grown for 2 days at 26°C. P. orientalis F9 mutants were screened for two phenotypes: (i) a lack of fluorescence in the iron-limited KB agar and (ii) a lack of growth deficiency halos on KB plates overlaid with E. amylovora^Rif in the double-layer assay (see below).

Identification of transposon insertion sides.Transposon insertion sites were identified according to Holenstein (38). As previously described (39), transposon insertion-specific primers arPCR-T7 (5′-GCACCTAACCGCTAGCACGTATACGACTC-3′) and ARB6 (5′-GGCCACGCGTCGACTAGTACNNNNNNNNNNACGCC-3′) were used for a primary PCR. In a second, nested PCR, primers arPCR-T7 inner (5′-TGAACGGTAGCATCTTGACGAC-3′) and ARB2 (5′-GGCCACGCGTCGACTAGTAC-3′) were used. A crude DNA extract of selected transposon mutants was prepared by suspending 24- to 48-h-old bacterial colonies in 300 μl of double-distilled H₂O and incubating them at 95°C for 30 min, followed by centrifugation at 12,000 × g for 1 min. The supernatant was diluted 1:10 with double-distilled H₂O and used as a PCR template. Amplifications were performed using Hotstar Taq polymerase (Qiagen). PCR products were sequenced using an ABI Prism BigDye Terminator, version 1.1, cycle sequencing kit (Applied Biosystems) and analyzed using nucleotide NCBI (National Center for Biotechnology Information) BLAST against the P. orientalis F9 whole-genome sequence (1) and protein BLAST using the UniProt database with default settings (40).

Site-directed mutagenesis of P. orientalis F9.A phenazine mutant of P. orientalis F9 (Δphen::Kan^r) was generated by site-directed mutagenesis. For this purpose, the BglII, EcoRI, and EcoRV cut sites within the phenazine gene cluster (Fig. 1) were used. Two fragments of ca. 1.4 kb (primer PhenaF, 5′-GTCGTGGAAGCTGGACAGTG-3′; primer PhenaEVr, 5′-GACTCGGCGATCCTGATTCG-3′; annealing temperature of 58°C) and 1.8 kb (primer PhenaEVf, 5′-TGGTGTTGCCGTGCATCGGG-3′; primer PhenaR, 5′-AACCGGTGAACCCCTTGTTT-3′; annealing temperature of 58°C) of a 5.2-kb BglII/EcoRI fragment of the phenazine gene cluster were amplified by PCR. The 1.4-kb PCR product was digested with BglII/EcoRV, and the 1.8-kb product was digested with EcoRV/EcoRI. The BglII/EcoRV product was ligated in a first step into the similarly digested suicide vector pKAS32 (41), followed by ligation with the EcoRV/EcoRI-digested 1.8-kb fragment and the appropriate cut vector of the first ligation step. The resulting plasmid pSVI1 + 2 harbored the 5′ and 3′ end flanking regions of the BglII/EcoRI phenazine fragment with a 2.2-kb deletion in between. pSVI1 + 2 was EcoRV digested and ligated with a HindII-cut kanamycin cassette from pSB315 (42), resulting in pSVKan. When pKAS32 derivatives are used for the positive selection of a double-allelic exchange, a streptomycin-resistant parental strain is required (41). Spontaneous streptomycin-resistant colonies of P. orientalis F9 were isolated by increasing streptomycin concentrations (starting concentration of 10 μg/ml; final concentration of 100 μg/ml) in KB medium. pSVKan was conjugated into P. orientalis F9 (Sm^r) using two-parental mating employing E. coli S17-1 λpir (43). Kanamycin- and streptomycin-resistant mutants were selected on MM2 medium containing 40 μg/ml kanamycin and 500 μg/ml streptomycin. Strain P. orientalis F9 (Δphen::Kan^r Sm^r) was tested via PCR using primers for the presence of the kanamycin resistance gene (aph157, 5′-GTCACCGAGGCAGTTCCA-3′; aph606, 5′-CGACCATCAAGCATTTTATC-3′; annealing temperature of 58°C) and primers set before the EcoRV restriction site (DelF, 5′-AGGTGAACGTGTCTTCGGCG-3′; DelR, 5′-CTCCCGATCATGTGATCCGC-3′). DelF and DelR amplified a ca. 700-bp fragment and the integrated kanamycin resistance cassette. The position of the kanamycin cassette within the former EcoRV cutting site was confirmed by sequencing.

Bacterial growth rate analysis.For growth rate analysis, a Bioscreen C (Oy Growth Curves Ab, Ltd., Helsinki, Finland) automatic microbiology growth curve analysis system was used. Nine hundred millimeters of medium (KB or PSBM) was pipetted into a reaction mixture, 200 μl of which was not inoculated but used as a negative control for the corresponding growth curves. The remaining 700 μl was inoculated with 3 μl of overnight culture of the tested strain. Three replicates of the inoculated medium, each 200 μl, were loaded in wells of a Bioscreen C honeycomb plate. The plates were incubated at 26°C for 24 h and were shaken every 20 min for 10 s before the absorbance at an optical density of 600 nm (OD₆₀₀) was determined. Growth experiments were performed in two independent trials.

Siderophore assay.Siderophore production was tested using chrome azurol S (CAS) agar (44). Bacteria were grown overnight on TSB plates, harvested, and resuspended in PBS to an OD₆₀₀ of 1. Five microliters of the bacterial suspension was spotted onto chrome azurol S agar and incubated for 3 days at 26°C. Siderophore-producing strains induce a color change from a green-blue CAS-iron complex to orange desferrated CAS.

Phytotoxicity test of P. orientalis F9 and mutant strains.For the phytotoxicity test, P. orientalis F9 and mutant strains were cultivated overnight on TSB plates. Colonies were resuspended in 1× PBS and adjusted to an OD₆₀₀ of 1. Twenty microliters of a 10⁻² dilution of the suspension was applied onto the hypanthium of apple flowers. Inoculated flowers were incubated at 26°C in closed boxes with water-saturated paper towels, and necrosis was evaluated 4 days postinfection (dpi).

Growth inhibition test using a double-layer assay.For a double-layer assay, the strains were cultivated overnight on KB plates. Colonies were resuspended in 1× PBS and adjusted to an OD₆₀₀ of 1. For bacteria seeded in the top layer, approximately 5 × 10⁸ bacteria were added to 10 ml of 0.75% KB top agar (cooled to 45°C). Fifteen milliliters of a top agar was poured on top of a 12 cm by 12 cm KB agar plate. Ten microliters of each P. orientalis F9 mutant strain tested as an antagonist was spotted onto the solidified top layer surfaces. Growth halos were detected after 2 days of incubation at 26°C.

Stigma-based medium in vitro competition assay.E. amylovora^Rif, P. orientalis F9, and mutants were cultivated on KB plates overnight. Colonies were harvested and resuspended in 1× PBS to an OD₆₀₀ of 1 before being serially diluted up to 10⁻⁴. Twenty microliters of this bacterial suspension was then added to 980 μl of 1× PBS, or, in the case of a coinoculation, 20 μl of each strain was added to 960 μl of 1× PBS. Sixty microliters of the bacterial suspension was directly pipetted into 1,440 μl of PSBM, vortexed, and then aliquoted to 400 μl in three columns of a 96-deep-well plate. After 3 days at 26°C, the CFU counts of E. amylovora^Rif were determined. A serial dilution of the inoculated medium was performed up to 10⁻⁷. Three microliters of each dilution was spotted onto TSB plates supplemented with rifampin (100 μg/ml). Experiments were repeated three times independently.

Cress assay.For the cress assay (45), two 1-cm-diameter discs of Pythium ultimum culture grown on malt extract agar (Oxoid) were punched out, laid on the bottom of a 9-cm-diameter petri dish, and carefully overlaid with 14 g of doubly autoclaved soil. P. orientalis F9 and F9 mutants were grown on KB plates overnight and resuspended in 1× PBS to an OD₆₀₀ of 0.1. Ten milliliters of the bacterial suspensions or mock control (PBS only) was evenly spread over the soil surface, followed by 0.4 g of cress seeds (Lepidium sativum). Petri dishes were incubated at 22°C and 65% humidity. After 2, 4, and 6 days of incubation, 20 ml of autoclaved water was added. After 7 days of incubation, the complete above-ground cress biomass was harvested, and its wet weight was determined. Each treatment was performed in duplicates for inoculation of cress only with P. orientalis F9, TM16, TM18, P. orientalis F9 (Δphen::Kan^r), and P. protegens CHA0 or in quadruplicates for P. ultimum controls and coinoculations of P. orientalis F9, TM16, TM18, P. orientalis F9 (Δphen::Kan^r), and P. protegens CHA0 with P. ultimum. The experiments were repeated two to three times independently.

Detached-flower assay.For the detached-flower assay (46), freshly opened flowers of 2-year-old potted Malus domestica Golden Delicious in the greenhouse were used. E. amylovora^Rif, P. orientalis F9, and mutants were cultivated and resuspended to an OD₆₀₀ of 1 as described above. A serial dilution of each bacterial suspension was performed. For the E. amylovora control, 20 μl of the 10⁻⁴ dilution was transferred into 980 μl of PBS; for coinoculations, 20 μl of each strain was transferred into 960 μl of PBS. Twenty microliters of the bacterial suspensions was directly pipetted onto the hypanthium of individual flowers. Mock treatments were performed with 1× PBS. After inoculation, flowers were incubated at 26°C in closed boxes with water-saturated paper towels on the bottom. Two and four days postinfection (dpi), the CFU counts of E. amylovora^Rif in eight individual flowers of each treatment were determined as described previously (1). Briefly, petals, pedestals, stamens, and stigmas of the flowers were removed, and the remaining flowers were shaken in 1 ml of 1× PBS for 30 min at 1,400 rpm. Afterwards, tubes were vortexed for 30 s. A serial dilution of the supernatant suspension was performed up to 10⁻⁷, and 3 μl of each dilution was spotted onto TSB plates supplemented with rifampin (100 μg/ml). Detached-flower assays for transposon mutants TM16 and TM18 (and alternative mutants TM10, TM13, and TM19) were performed during the spring and summer of 2018, and for the phenazine mutant P. orientalis F9 (Δphen::Kan^r), they were performed in the spring of 2019. Assays were repeated at least three times independently.

Genome analysis of P. orientalis F9.The genome of P. orientalis F9 (CP018049.1) was analyzed for additional metabolites and factors with potential antagonistic activity using the Virulence Factors Database (VFDB; virulence factors of bacterial pathogens) and antiSMASH. In addition, blastn/tblastn (NCBI) comparison of the P. orientalis F9 genome and synthesis genes for additional secondary siderophores of pseudomonads was also performed using the sequences for enantio-pyochelin (47), pseudomonine (48), pyridine-2,6-bis(monothiocarboxylic acid) (PDTC) (49), and thioquinolobactin (50) synthesis genes.