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 .