2.3 Plant and tuber inoculations by D. solani andD. dianthicola strains and experimental populations
In our plant and tuber assays, we use the term virulence as the capacity of the pathogen to parasitize a host (symptom incidence) and the term aggressiveness as the degree of damage caused by a pathogen on a host (symptom or disease severity). The terms used to describe pathogens behaviors (virulence, symptom incidence, aggressiveness, disease severity index, fitness and competitive index) are defined more extensively in Supplementary Methods 1 (SM1) . We designed the whole plant experiments based on our knowledge of the pathosystem, to investigate contrasting conditions relevant for the disease expression in fields. Each bacterial strain was cultivated individually before being used for plant and tuber inoculations, either separately or assembled in populations. To constitute experimental populations ofD. solani, D. dianthicola, or mixtures of the two species, cell suspensions of different strains were assembled just before plant inoculation. Potato plants (S. tuberosum var. Bintje) and hyacinths (Hyacinthus orientalis var. Delft blue) were cultivated in horticultural compost in individual pots (2 L) in a greenhouse at around 23°C (minimum 20°C maximum 28°C) with a 12-hour photoperiod. For mimicking plant infection by soil pathogens, potato plants (three weeks post-tuber plantation) and hyacinths (four weeks post-bulb plantation) were inoculated by watering the substrate with a pathogen cell suspension at 109 colony-forming units (CFU) per pot. This inoculation load was calibrated to obtain a large range (20 to 60%) of symptom incidence in the unwounded potato plant condition (Raoul des Essarts et al., 2015). In a wounded condition, the potato roots were wounded with a sterile knife before infection. The symptom monitoring was adapted from Raoul des Essarts et al. (2015). The number of symptomatic potato plants (blackleg disease) and hyacinths (soft-rot disease) was recorded twice a week (see examples of symptoms inFigure 1a-b ). For each strain and experimental population and unwounded and wounded conditions, the number of symptomatic and asymptomatic plants (hence two symptom classes that were used in statistical comparisons) was counted at 61 days post infection (dpi) using 15 potato plants (up to 25 potato plants when indicated) and 8 hyacinths. A set of non-inoculated plants was used as an asymptomatic control. In order to facilitate visualization of the virulence data in the figures, they are presented as a percentage of symptomatic plants (symptom incidence).
In tuber assays, potato tubers (S. tuberosum var Bintje) were inoculated using a tip to inject 10 µL of calibrated pathogen suspensions. Based on previous results (Raoul des Essarts et al., 2019), we chose two different inoculum loads of strains and experimental populations (107 CFU and 105 CFU per tuber) to obtain a large range of symptom severity, to compare the capacity to initiate maceration at different population sizes, to investigate fitness of the two pathogens in two contrasting ecological conditions. After 5 days of incubation at 24°C, the tubers were cut in half and scored based on five aggressiveness classes according to the symptom severity in each tuber (from no symptom to the most severe symptoms; Figure 1c ). For each strain and experimental population, at least 10 tubers were inoculated. A set of non-inoculated tubers was used as an asymptomatic control. In order to facilitate the presentation of the aggressiveness data in the figures, results are presented as normalized disease severity index (DSI) of which values, ranging from 0 to 100 (arbitrary unit), increase with symptom severity. DSI was calculated using the five aggressiveness classes assigned to 10 tubers, see formula in Supplementary Methods 1 (SM1).
Emerging lesions from potato stems and hyacinths and rotting tissue recovered from tubers at 5 dpi were collected and frozen at -80°C, and then used to quantify the abundance D. solani and D. dianthicola pathogens within hosts by Taq Man qPCR as described inSupplementary Methods 2 (SM2) . Assessing D. dianthicolaand D. solani in inoculum and plant tissues (potato stems and tubers, and hyacinths) permitted the calculation of competitive index (CI) values following the formula presented in SM1 . A CI value equal to one indicated an equal fitness between D. solani andD. dianthicola , CI value greater than one indicated a fitness advantage to D. solani , and CI value below one indicated a fitness advantage to D. dianthicola .