INTERFACES DEL SISTEMA

Pectobacterium atrosepticum SCRI1043 tagged with an antibiotic resistance

gene retain the wild type phenotypes relating to antibiotic production and

sensitivity in vitro

To study the interactions between P. c. subsp. brasiliensis ICMP 19477 and P. atrosepticum SCRI1043 further using in vitro and in planta competition assays, antibiotic resistant strains were produced (as described in Section 2.1.4, Table 2.2). Firstly, spontaneous Rif mutants were created for both P. c. subsp. brasiliensis ICMP 19477 (P. c. subsp. brasiliensis R) and P. atrosepticum SCRI1043 (P. atrosepticum R). Rifampicin resistance is conferred by a mutation in rpoB, the gene encoding the β subunit of the RNA polymerase (Heep et al., 2000; Vogler et al., 2002). SpontaneousRif resistant mutants of a P. atrosepticum strain have successfully been used in in vitro and in planta studies without any observed difference in phenotype compared to the wild-type (WT) (Cronin et al., 1997; Vanga et al., 2012). Genetic mutants of P. c. subsp. brasiliensis ICMP 19477 and P. atrosepticum

SCRI1043, resistant to Km (P. c. subsp. brasiliensis Kand P. atrosepticum K) were also generated for co-inoculations. Similar Km resistant-tagged strains have previously been used successfully in in vitro

and in planta assays (Vanga et al., 2012). Both the P. c. subsp. brasiliensis ICMP 19477 Kand P. c. subsp. brasiliensis ICMP 19477 R strains retained the producer phenotype, whilst the P. atrosepticum

SCRI1043 Kand P. atrosepticum SCRI1043 R strains remained sensitive to the producer (Figure 3.2). Similar sized zones of inhibition were generated using these strains, indicating that they were suitable for use in future antagonism assays.

Figure 3.2. In vitro growth inhibition of P. atrosepticum (Pba) SCRI1043 by P. c. subsp. brasiliensis

(Pbr)ICMP 19477 was unaffected by tagging the strains with antibiotic resistance.

Antagonism assays were conducted using P. c. subsp. brasiliensis ICMP 19477 R or P. c. subsp. brasiliensis ICMP 19477 Kas the producer strain andP. atrosepticum SCRI1043 K or P. atrosepticum

SCRI1043 R as the sensitive strains in assays described in Section 2.1.21. Assay plates were incubated at 28°C for 24 h, with zones of inhibition around the P. c. subsp. brasiliensis ICMP 19477 colonies indicative of antimicrobial production.

The growth of the antibiotic resistant mutants was also compared to the WTs on solid agar plates (Section 2.1.22) and in liquid cultures (Section 2.1.23), to confirm that the tagged strains were representative of the WTs in vitro. On solid agar there were no significant differences in growth between the P. c. subsp. brasiliensis ICMP 19477 mutants and the WT (p > 0.22, F-test), with the exception of 24, 32 and 72 h (p = 0.073, 0.007 and 0.001 respectively, F-test) (Figure 3.3 A). There were similar results for the P. atrosepticum SCRI1043 strains, as the CFUs did not vary significantly between the strains at most time points (p > 0.4, F-test). There was, however, a significant variation at 48 h (p =0.032, F-test) (Figure 3.3 B). Regardless of the statistical differentiation between the strains at these few time points on solid media, the overall differences did not appear to be biologically relevant, confirming that the tagged strains were suitable for use in future competition assays performed on solid plates. Furthermore, the growth curves for both species showed that both bacterial species reached mid-stationary phase after 48 h and there was little increase in CFUs after

Pba SCRI1043 RifR

Pbr ICMP19477

KanR

Pbr ICMP

19477

Pba SCRI1043 KanR

Pbr ICMP

19477

Pbr ICMP19477

this time. Therefore, subsequent growth assays were terminated at 48 h. A second independent experiment confirmed the results above.

Figure 3.3. Growth of P. c. subsp. brasiliensis (Pbr) ICMP 19477 (A) and P. atrosepticum (Pba)

SCRI1043 (B) on solid agar plates was not affected by tagging them with Km and Rif resistance.

Each graph shows the growth (mean CFUs) of the WT as well as the antibiotic tagged strains over 72 h post inoculation with 104 _{CFUs/ml of each bacteria (as described in section 2.1.22). Pectobacterium}

caratovorum subsp. brasiliensis ICMP 19477 Km resistant (Pbr K); P. c. subsp. brasiliensis ICMP 19477 Rif resistant (Pba R); P. atrosepticum SCRI1043 Km resistant (Pba K); P. atrosepticum SCRI1043 Rif resistant (Pba R). The mean CFUs represent the mean over three test plates. Error bars are 95% confidence limits.

In liquid cultures, the CFUs for the marked P. c. subsp. brasiliensis ICMP 19477 strains did not differ significantly from those of the WT at 6, 12 and 16 h (p = 0.093, 0.118 for K and R, F-test). However at 24 h, the CFUs for both marked strains were significantly lower than for the WT (p = 0.013 and 0.021 for the Km and Rif resistant strains respectively, F-test). At 32 and 48 h, numbers for P. c. subsp. brasiliensis K were again similar to the P. c. subsp. brasiliensis ICMP19477 WT (p = 0.113 and 0.553, F- test), but those for P. c. subsp. brasiliensis Rwere statistically significantly lower (p < 0.001 and 0.007, F-test) (Figure 3.4 A). For P. atrosepticum SCRI1043, significant differences were only observed between the strains at 12 and 32 h. The CFUs for the two marked strains were significantly lower than for the P. atrosepticum WT strain (p < 0.001 at 12 h, and p = 0.010 for P. atrosepticum K, p = 0.002 for

P. atrosepticum R F-test) (Figures 3.4 B). As for the growth experiments on solid media, although

2D Graph 10

Time (hours post inoculation)

0 10 20 30 40 50 60 70 80 P br cel l cou nt (m ea n CFU) 103 104 105 106 107 108 109 Pbr Pbr K Pbr R A. 2D Graph 12

Time (hours post inoculation)

0 10 20 30 40 50 60 70 80 P br cel l cou nt (m ea n CFU) 103 104 105 106 107 108 109 Pba Pba K Pba R B.

statistically significant differences were observed at a couple of time points, overall there appeared to be no major biological differences in the growth of the WTs and the relevant tagged strains.

Figure 3.4. Growth of P. c. subsp. brasiliensis (Pbr) ICMP 19477 (A) and P. atrosepticum (Pba)

SCRI1043 (B) and in liquid cultures was not affected by tagging them with Km and Rif resistance.

Each graph shows the growth (mean CFUs) of the WT as well as the antibiotic tagged strains over 48 h post inoculation with 104 _{CFUs/ml of each bacteria (as described in section 2.1.23). P. c. subsp.}

brasiliensis ICMP 19477 Km resistant (Pbr K); P. c. subsp. brasiliensis ICMP 19477 Rifresistant (Pbr R);

P. atrosepticum SCRI1043 Km resistant (Pbr K); P. atrosepticum SCRI1043 Rifresistant (Pba R). The mean CFUs represent the mean over three test plates. Error bars are 95% confidence limits.

3.2.4

Pectobacterium carotovorum subsp. brasiliensis

ICMP 19477 reduces the