Figure legends
Figure 1 Symptoms caused by Dickeya pathogens. Symptoms
(white arrows) caused by Dickeya solani and Dickeya
dianthicola on potato stems (a) and hyacinths (b).
The five symptom classes (0 to 4) used to compare the aggressiveness ofDickeya solani and Dickeya dianthicola on potato tubers
(c).
Figure 2 Dickeya and Pectobacterium prevalence
from potato fields exhibiting blackleg symptoms. Percentage ofPectobacterium-containing and Dickeya-containing fields
(a) and that of D. solani-containing fields and D.
dianthicola-containing fields (b) were calculated each year
from 2004 to 2015 (with the exception of 2006). (c) Number and
relative abundance of the Pectobacterium, D. dianthicolaand D. solani isolates collected in 19 symptomatic fields sampled
from 2013 to 2016. A hierarchical clustering paired group method
delineated four pathogen population groups, i.e. Pectobacteriumonly (I: 3 fields), D. solani and Pectobacterium (II: 9
fields), D. dianthicola and Pectobacterium (III: 3
fields), and D. dianthicola, D. solani and Pectobacterium(IV: 4 fields).
Figure 3 Symptom incidence and fitness of Dickeya
dianthicola and D. solani in potato plants. In a, mean
value and standard error (SE) of the percentage (%) values of plants
exhibiting blackleg symptoms, which were measured after inoculation of
each of the five D. solani strains (3337, IPO2222, RNS05-1-2A,
Ds0432.1, PPO9019) and five D. dianthicola strains
(RNS11-47-1-1A, CFBP1888, CFBP2982, CFBP2015, MIE34) on 15 plants. The
p-value of the Kruskal-Wallis test comparing of the symptomatic classes
between the two species is indicated below the graph. In b,
mean value and SE between two replicates of the percentages (%) of
plants exhibiting blackleg symptoms which was measured on 15 plants
inoculated by D. dianthicola and D. solani populations and
their mix. The p-values of the pairwise comparisons (Post-hoc Tukey
tests) of the symptomatic classes are indicated below the graph, when p
≤ 0.1. In c, competitive index (CI) values between D.
solani and D. dianthicola populations were calculated in eight
emerging lesions of co-infected plants and revealed a competitive
advantage of D. dianthicola: the CI median (= 2.4
10-5) is represented by a thick bar; p-values
resulting from Kruskal-Wallis tests testing difference from one are
indicated. Legend: * for 0.05<p≤0.1; ** for
0.01<p≤0.05 and *** for p ≤0.01.
Figure 4 Aggressiveness and fitness of Dickeya
dianthicola and D. solani in potato tubers. Rotting assays were
conducted by inoculating each tuber by either 107colony-forming units (CFU) (in a, b and c) or
105 CFU of pathogens (in d, e andf). In a, b, d and e, each
disease severity index (DSI) value was calculated using symptom classes
observed on 10 tubers. In a and d, mean values and
standard errors (SE) of DSI values were measured for each of the fiveD. solani strains (3337, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019)
and five D. dianthicola strains (RNS11-47-1-1A, CFBP1888,
CFBP2982, CFBP2015, MIE34) using five (a) and two (d)
independent experiments. The p-values of the Kruskal-Wallis tests
comparing symptom classes are indicated below the graphs. In band e, mean values and standard errors of DSI values were
measured twice for each of the experimental populations of D.
dianthicola, D. solani and the mixture of the two. The p-values of the
pairwise Tukey tests comparing the symptomatic classes are indicated,
when p ≤ 0.1. In c and f, the competitive index (CI)
between D. solani and D. dianthicola populations was
calculated in 10 co-infected symptomatic tubers: the median values
(indicated by a thick bar) reached 5.7 (c) and 5.8 (f)
and the CI values were statistically different from one (Kruskal-Wallis
test; p = 5 10-5 and p = 0.05 respectively), revealing
a competitive advantage of D. solani. Legend: * for
0.05<p≤0.1; ** for 0.01<p≤0.05 and *** for p ≤0.01.
Figure 5 Expression of the pectate lyase genes pelA,pelD and pelE. The expression level of the pelA,pelD and pelE genes was evaluated in each of the fiveD. solani strains (3337, IPO2222, RNS05-1-2A, Ds0432.1, PPO9019)
and five D. dianthicola strains (RNS11-47-1-1A, CFBP1888,
CFBP2982, CFBP2015, MIE34), grown in three conditions: a rich culture
medium in the absence of pectin (exponential growth phase) and
symptomatic tubers (at 5 days post infection) and emerging lesions in
stems. Relative expression was measured four times and normalized using
the rpoB and yafS gene expression. In the graphs, the mean
values and standard error (SE) of gene expression from all strains of a
given species are indicated, as well as p-values of pairwise comparisons
by Tukey tests. Legend: * for 0.05<p≤0.1; ** for
0.01<p≤0.05 and *** for p ≤0.01.
Figure 6 Population genomics of Dickeya solani. Ina, scan of 67 D. solani genomes revealed that the genome
position 2,930,940, (according the D. solani 3337 genome), in thevfmB gene, was the most balanced non-synonymous variation, with
alternated VfmBPro (71%) and VfmBSer(29%) alleles. In b, single nucleotide polymorphism
(SNP)-based tree of D. solani strains using PHYLOViZ; the name of
the French isolates is indicated in blue font; the
VfmBSer and VfmBPro strains used in the
plant assays are underlined. In c, dynamics of the
VfmBSer allele (%) among the D. solani isolates
along the sampling period 2005-2015 in France.
Figure 7 Characterization of the VfmB alleles. In a,
using the Escherichia coli AidB protein (the PDB accession is
3DJL) as a model, the Phyre2-predicted structure of the VfmB protein
showed conformational differences in the beta-sheet associated with the
VfmBPro and VfmBSer alleles; the Pro55
and Ser55 positions are in red color in the VfmB representations
obtained using the EzMol web server. In b, mean values and
standard error (SE) of disease severity index (DSI) values, which were
calculated by recording symptom classes on 10 tubers infected by
107 CFU of each of the D. solani strains
carrying either VfmBPro (IPO2222, MIE35, AM3a and 3337)
or VfmBSer (Ds0432.1, RNS10-27-2A, Sp1a and M21a); the
p-value of the Kruskal-Wallis test comparing the four
VfmBPro and four VfmBSer strains using
symptom classes is indicated below the graph. In c, comparative
transcriptome of D. solani 3337 (VfmBPro) and
Ds0432.1 (VfmBSer) recovered from soft-rot lesions in
potato tubers; upregulated virulence genes were enriched in Ds0432.1
(VfmBSer) as illustrated by the pelB, pelC, pelC,
hcpA and prtA genes (closed circles).
Figure 8 Aggressiveness, symptom incidence and fitness assays of
two Dickeya solani experimental populations expressing the VfmB
alleles. Soft-rot assays were conducted by inoculating each tuber by
either 107 colony-forming units (CFU) (a) or
105 CFU (b) of the VfmBProand VfmBSer D. solani populations and a mixture
of the two. In a and b, each disease severity index
(DSI) value (measuring aggressiveness) was calculated using symptomatic
classes observed on 10 tubers; the assays were performed in triplicate.
The p-values of the pairwise Tukey tests comparing the symptom classes
are indicated, when p ≤ 0.1. In c, percentage (%) of plants
exhibiting blackleg symptoms was measured on plants inoculated by the
VfmBPro and VfmBSer D. solanipopulations and a mixture of the two. These assays measuring symptom
incidence were performed in triplicate. Kruskal-Wallis test revealed no
difference between the three treatments using symptom classes (p =
0.86). In a, b and c, competitive index (CI) values of
VfmBPro and VfmBSer populations were
calculated using allele counts based on shotgun sequencing of bacterial
populations recovered from co-infected tissues. Median values of CI are
represented as thick lines and reached 1.7 (8 repeats in a),
2.0 (3 repeats in b) and 14.0 (16 repeats in c) in the
three treatments. Statistical differences between CI values and 1
(Kruskal-Wallis test) revealed that neither D. solaniVfmBPro or D. solani VfmBSer had
significant advantage in tubers inoculated with a high load (p = 0.3 ina), but D. solani VfmBPro was more
competitive in tubers inoculated with a low load (p = 0.04 inb) and in stems (p = 9 x 10-3 in c).
Legend: * for 0.05<p≤0.1; ** for 0.01<p≤0.05 and ***
for p ≤0.01.