2.3 Plant inoculations by D. solani and D.
dianthicola strains and experimental populations
Each bacterial strain was cultivated individually before being used for
plant and tuber inoculations, either separately or assembled in
populations. To constitute experimental populations of D. 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. 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. 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 biweekly (see examples of symptoms in Figure 1a-b ).
For each strain and experimental population and unwounded and wounded
conditions, the number of symptomatic and asymptomatic plants (hence two
symptom classes) was counted at 60 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 aggressiveness data in the
figures, they are presented as a percentage of symptomatic plants.
Potato tubers (S. tuberosum var Bintje) were inoculated using a
tip to inject 10 µL of pathogen strains and experimental populations at
107 (or 105 when indicated) CFU per
tuber. After 5 days of incubation at 24°C, the tubers were cut in half
and scored in five aggressiveness classes according to the symptom
gravity 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).
Symptomatic tissues from potato stems and tubers, as well as from
hyacinths, were also used to quantify D. solani and D.
dianthicola pathogens 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 .