Figure 1.
Lemna minor provides both quantitative and qualitative assessments of bacterial strain lethality.
A. 50% lethal dose can be determined by assessing plant survival after a given timepoint. An overnight culture of a given strain was washed and inoculated into each well of column 1, then 10-fold serially diluted into each subsequent column using a multichannel pipette. Bacterial counts were generated using a multichannel pipette by spotting 10 μl from each well on agar. For Bcc infections, surviving plants were counted after 96 h. B. Supernatants of wild type and shvR-deficient B. cenocepacia affect duckweed at different dilutions. Two 5-day cultures were lyophilized, resuspended in deionized water and 4-fold serially diluted into duckweed-containing wells for each strain: WT, wild type B. cenocepacia K56-2; S, K56-2 harbouring a TpR cassette in gene bcas0225, also known as shvR; Ø, blank SHS media control. Results are shown at 72 h post-inoculation. C. Heat-killed B. cenocepacia K56-2 has no effect on duckweed. A dense (~ 1x109 cfu/ml) K56-2 suspension was incubated at 65°C for different lengths of time (shown in min beside each well) and then inoculated into a plant-containing well. A lack of viable cells from a 5-min incubation onward was shown by spotting the suspensions on LB agar.
Figure 2.
The virulence of Bcc strains in duckweed correlates with their virulence in wax moth larvae.
A. Each point represents the LD50 of a Bcc strain in duckweed determined from the compiled data of 2-6 independent trials plotted against LD50 values determined in wax moth larvae [13]. B. LD50 values for all strains in both infection models placed in order of rank. The large point represents four B. multivorans strains (C7322, C3430, C5393, and PC249) and one B. dolosa strain (AU0645) for which bacterial loads high enough for killing were not attained. NB. all strains identified by Seed and Dennis [13] to be avirulent in the larva model were also avirulent against duckweed, except for B. multivorans C5568 with an LD50 of 1.06x108 cfu/ml.
Figure 3.
The virulence of EPEC strains in duckweed correlates with their virulence in wax moth larvae.
Wild type EPEC and five mutant strains were inoculated into wells containing duckweed and left to incubate at 30°C for 7 days. Each point represents the LD50 determined in duckweed from the compiled data of 2-4 independent trials plotted against values determined in wax moth larvae by Leuko and Raivio [37].
Figure 4.
Complementation of mutant 46B2 with bcal1124, but not bcal1124 with point mutations at R204H and E336G, results in restoration of plant killing.
Infection experiments were performed as described above following overnight growth of all strains (K56-2/pSCRhaTc, 46B2/pSCRhaTc, 46B2/p1124, 46B2/p1124”) in ½ LB + Tc100 + 0.02% w/v rhamnose. Infections omitted antibiotics but included 0.02% rhamnose to continue induction of the pSCRha promoter.
Figure 5.
B. cenocepacia infection of duckweed is alleviated by bacteriophage treatment up to 12 h but not after 18 h.
A. B. cenocepacia K56-2 was inoculated into plant-containing wells at 2x106 cfu/ml, with 4x108 pfu/ml of phage KS12 (i.e. MOI = 200) applied at 0, 6, 12, 18, and 24 h. Results shown are averages of 3 independent trials using 6 plants per trial +/- SE. “Untreated” refers to both phage-treated and mock-treated plants, as both showed 100% survival. B. Bacterial counts from media surrounding plants during the infections (not phage-treated). Results shown are averages of 3 independent trials performed in triplicate +/- SE. C. Bacterial counts from crushed surface-sterilized plants. Results shown are averages of 3 independent trials +/- SE, with four replicates of each sample per trial.