3.2 Dickeya dianthicola exhibited a fitness advantage over D. solani in potato plants.
Plant assays were performed in the greenhouse to compare the incidence of blackleg symptoms after inoculation by D. dianthicola orD. solani on non-wounded potato plants. FiveD. dianthicola strains (RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34) and five D. solani strains (3337 = RNS08-23-3-1A, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019) were used in these assays.
In a first assay, each strain was inoculated individually on 15 plants and the number of asymptomatic and symptomatic plants were counted at 61 days post inoculation (dpi). A Kruskal-Wallis test revealed differences between D. solani and D. dianthicola in terms of symptom incidence (k=7; DF=1; p=8 x 10-3): D. dianthicola was found more virulent than D. solani (percentages of symptomatic plants of 61% ± 10 versus 15% ± 11; Figure 3a).
In a second plant assay, the five bacterial cultures (i.e., the five strains) of each species were assembled to constitute two experimental populations and each was inoculated on 15 plants. A mix of the two species (10 strains) was also inoculated on 15 plants. The assay was duplicated (2 x 15 plants per treatment). We compared symptom incidence at 61 dpi. Kruskal-Wallis test revealed differences between the three treatments, i.e., D. solani strain mixture, D. dianthicolastrain mixture and species mixture (k=6.1; DF=2; p=0.04). Pairwise comparison (Post-hoc Tukey test) showed that D. solani was less virulent than D. dianthicola (F=-2.5; p= 0.03) and the species mixture (F=1.9; p= 0.10). However, symptom incidence caused by the species mixture did not differ from those of the D. dianthicolamixture (F=-0.5; p= 0.86). Post-hoc Tukey p-values and mean value (± SE) of the percentage of symptomatic plants inoculated by the D. dianthicola (53% ± 7), D. solani (23% ± 3) and mixed (47% ± 0) populations are presented in the figure 3b . To assess the dynamics of the appearance of symptomatic plants, we also drew a disease progress curve for each replicate (Figure S3a ) and compared the areas under disease progress curve (Figure S3b ). Pairwise comparisons (Post-hoc Tukey tests) confirmed that D. solani was less virulent than D. dianthicola (F=-336; p<10-11) and the species mixture (F=386; p<10-11).
To evaluate which pathogen had emerged at the beginning of the infection process, hence to identify the most probable causative agent of the observed symptoms, we used qPCR to quantify pathogen loads. When the two species mixtures were inoculated separately, D. dianthicola andD. solani reached the mean values ± SE of 2 x 1011 ± 1 x 104 cells and 3 x 109 ± 1 x 102 cells per gram of symptomatic stem tissues, respectively. In eight co-infected symptomatic plants, quantifications of the pathogen abundance revealed an excess ofD. dianthicola with a CI median value of 10-5(Figure 3c ). The eight CI values differed from one (Kruskal-Wallis test, k=9.5; DF=1; p= 2 x 10-3), meaning that D. dianthicola outcompeted D. solani in blackleg tissues. Altogether, these data revealed a fitness advantage ofD. dianthicola in terms of multiplication within lesions in the aerial parts of the host in either the absence or presence of theD. solani invader.