A Single, Plastic Population of Mycosphaerella pinodes Causes

Ascochyta Blight on Winter and Spring Peas (Pisum sativum) in

France
Christophe Le May,a,b Michèle Guibert,c Aurélie Leclerc,a,b Didier Andrivon,c and Bernard Tivolic
Agrocampus Ouest, UMR1349 IGEPP, Rennes, Francea; Université Européenne de Bretagne, Rennes, Franceb; and INRA, UMR1349 IGEPP, Le Rheu, Francec

Plant diseases are caused by pathogen populations continuously subjected to evolutionary forces (genetic flow, selection, and
recombination). Ascochyta blight, caused by Mycosphaerella pinodes, is one of the most damaging necrotrophic pathogens of
field peas worldwide. In France, both winter and spring peas are cultivated. Although these crops overlap by about 4 months
(March to June), primary Ascochyta blight infections are not synchronous on the two crops. This suggests that the disease could
be due to two different M. pinodes populations, specialized on either winter or spring pea. To test this hypothesis, 144 pathogen
isolates were collected in the field during the winter and spring growing seasons in Rennes (western France), and all the isolates
were genotyped using amplified fragment length polymorphism (AFLP) markers. Furthermore, the pathogenicities of 33 isolates
randomly chosen within the collection were tested on four pea genotypes (2 winter and 2 spring types) grown under three cli-
matic regimes, simulating winter, late winter, and spring conditions. M. pinodes isolates from winter and spring peas were ge-
netically polymorphic but not differentiated according to the type of cultivars. Isolates from winter pea were more pathogenic
than isolates from spring pea on hosts raised under winter conditions, while isolates from spring pea were more pathogenic than
those from winter pea on plants raised under spring conditions. These results show that disease developed on winter and spring
peas was initiated by a single population of M. pinodes whose pathogenicity is a plastic trait modulated by the physiological sta-
tus of the host plant.

P lant parasites can quickly adapt to their hosts and overcome

resistance genes used in crop protection. This process occurs
both in agroecosystems, where hosts (cultivars) are typically quite
uniform genetically, and in wild pathosystems, where hosts and
parasites show high levels of diversity and are structured as meta-
populations (12). However, a number of crop plants are grown in
complex ecosystems rather than as the monocultures which char-
acterize modern agriculture; this is particularly the case for species
grown as both winter and spring cultivars and for which wild
lengthens the crop life span and can result in higher, more stable
yields (30, 31). As a result, spring and winter pea crops are now
grown simultaneously in many parts of Europe, particularly in
France. In these areas, winter and spring peas constitute two
sympatric niches overlapping temporally for about 4 months
(March to June) and absent for about 5 months (July to No-
vember). Both crops are vulnerable to Ascochyta blight, a seri-
ous disease affecting field peas in most pea-growing regions of
the world, particularly in the temperate areas of Europe, North

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relatives occur as weeds or in field margins.
From a plant pathology point of view, such complex ecosys-
tems are usually characterized by one or more of the following
parameters: (i) spatial and temporal heterogeneity of hosts, which
creates patchiness and an often complex age structure, (ii) intra-
and interspecific diversity, and (iii) multiple host-pathogen inter-
actions. These features favor niche partitioning, as they allow the
coexistence on the same host species of different pathogen popu-
lations separated in space and/or time. Indeed, ecological differ-
ences that lead to niche partitioning can occur in three basic ways:
resource partitioning, temporal niche partitioning, and spatial
niche partitioning (4, 14, 36). For plant-pathogenic species able to
exploit the same host, separation in space and/or time might best
explain niche partitioning (18). For instance, Montarry et al. (36)
showed that temporal isolation of genetic groups of Erysiphe ne-
cator is a mechanism which, by preventing recombination, can
explain the sympatric persistence of two highly differentiated ge-
netic populations of the grapevine powdery mildew pathogen.
This process can eventually lead to sympatric speciation within
pest populations (39).
Dry peas have been grown in Europe for over 30 years, mainly
as a spring crop. However, recent emphasis has been put on breed-
ing and growing winter types, since sowing in autumn or winter
America, Australia, and New Zealand (10, 50). The disease is
caused by three related fungi: Ascochyta pisi Lib., Mycosphaer-
ella pinodes (Berk. et Bloxam) Vestergren (anamorph: Asco-
chyta pinodes Jones), and Phoma medicaginis var. pinodella
(Jones) Boerema. Mycosphaerella pinodes is the main pathogen
infecting winter and spring peas in France. The extent and sever-
ity of disease depend on the cropping system and weather conditions.
The most favorable conditions for the pathogen are frequent rainfall,
high relative humidity, and leaf wetness duration (44, 50). Pea plants
may suffer frost damage during the winter, which can enhance asco-
spore germination and spore penetration into healthy tissue for M.
pinodes (5, 24, 25).
Ascochyta blight symptoms do not appear at the same period
and the same weather conditions in winter and spring crops. On
winter peas, the disease starts in December, when the temperature
Received 24 May 2012 Accepted 19 September 2012
Published ahead of print 28 September 2012
Address correspondence to Christophe Le May, lemay@agrocampus-ouest.fr.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.01543-12

December 2012 Volume 78 Number 23 Applied and Environmental Microbiology p. 8431– 8440 aem.asm.org 8431
Le May et al.

TABLE 1 Isolates of Mycosphaerella pinodes sampled from winter (WP) TABLE 2 Temperatures used for plant growth and disease incubation in
and spring (SP) pea fields used for individual pathogenicity assessments the aggressiveness test of Mycosphaerella pinodes isolatesa
Source Isolation date (day/mo/yr) Isolate Temp (°C)
Winter pea 20/12/2004 WP-1 Late winter
20/12/2004 WP-2 Parameter Winter condition condition Spring condition
20/12/2004 WP-3 Plant growth 8–10 8–10 18–20
20/12/2004 WP-4 Disease incubation 8–10 18–20 18–20
03/01/2005 WP-5 a
For the winter conditions, plants were grown for 5 weeks, whereas for spring
03/01/2005 WP-6 conditions, plants were grown for 3 weeks, until they reached the 5- to 6-leaf stage.
03/01/2005 WP-7
03/01/2005 WP-8
03/01/2005 WP-9
MATERIALS AND METHODS
17/01/2005 WP-10
17/01/2005 WP-11 Fungal isolates and DNA extraction. A collection of 144 isolates of M.
pinodes sampled in 2004-2005 from two naturally infected fields on the
17/01/2005 WP-12
INRA experimental station of le Rheu (France; 48.06°N, 1.41°W) was
17/01/2005 WP-13
established (Table 1). These fields were separated by more than 3 km and
17/01/2005 WP-14
sown with a winter (Cheyenne, GAE-Recherche, France) and a spring
17/01/2005 WP-15 (Baccara, Florimond-Desprez, France) pea cultivar. One hundred two
17/01/2005 WP-16 isolates were sampled from winter peas (WP) during the winter (Decem-
17/01/2005 WP-17 ber to March) and during the spring (March to June). Forty-two isolates
were sampled from spring peas (SP) during the spring (May to June) of
Spring pea 30/05/2005 SP-1 2005. All isolates were cultured and single spored before being tested. For
13/06/2005 SP-2 isolation, approximately 5 mm2 of diseased leaf tissue was surface steril-
13/06/2005 SP-3 ized for 1 min in 70% ethanol, rinsed three times in sterile water, placed on
13/06/2005 SP-4 sterile filter paper to remove excess water, and cultured on V8 medium (99
13/06/2005 SP-5 ml V8 vegetable juice [Campbell, France], 35 g agar, and 801 ml distilled
13/06/2005 SP-6 water, autoclaved at 105°C for 30 min) in petri dishes for 14 days. Pycni-
13/06/2005 SP-7 diospores from resulting cultures were spread on 2% malt agar and incu-
13/06/2005 SP-8 bated for 12 h as described by Onfroy et al. (37). Single germinating
13/06/2005 SP-9 pycnidiospores were transferred to fresh V8 plates under a dissecting mi-
27/06/2005 SP-10 croscope, and cultures were incubated at 20°C with a 12-h photoperiod
27/06/2005 SP-11 under cool white fluorescent lamps. Single-spore cultures were then
27/06/2005 SP-12 maintained on malt slants and stored in the dark at 4°C.
27/06/2005 SP-13 AFLP typing. Each isolate was grown in 75 ml of LP liquid medium
27/06/2005 SP-14
(10 g tryptone, 5 g extract of yeast powder, 5 g NaCl, 1 liter distilled water;
autoclaved at 115°C for 20 min) supplemented with streptomycin (1.5 g)
27/06/2005 SP-15
and penicillin (0.75 g). Each culture was raised from four pieces (approx-
27/06/2005 SP-16
imately 1 cm2 each) cut from the margin of an actively growing culture on

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is generally below 10°C and climatic conditions are conducive
(rainfall period, leaf wetness durations, and winter frost periods)
(46). On spring pea, the disease starts at the end of May, when the
temperature is about 18°C and rainfall periods are shorter than in
winter. The two crops thus provide two distinct niches regarding
temperature and moisture regimes, and it is possible that M. pi-
nodes populations adjust their ecological requirements to exploit
one niche or the other. Such adaptations were reported by
Harikrishnan and Yang (23) for Rhizoctonia solani and by Frenkel
et al. (19) for Didymella rabiei. Furthermore, winter crops gener-
ally present phenotypic structures different from those of spring
crops (30), which could limit the resource exploitation for plant
pathogens.
To test this hypothesis, we sampled M. pinodes isolates from
winter (WP) and spring (SP) peas during the 2004-2005 growing
season from two separate fields. These isolates were typed to an-
swer the following questions: (i) Are M. pinodes populations from
winter and spring peas genetically different? (ii) Do isolates from
winter and spring peas have different temperature requirements
for growth? (iii) Does their aggressiveness depend on ecological
conditions and host status?
malt agar. Inoculated vials were incubated, under agitation, for 14 days at
20°C with a 12-h photoperiod under cool white fluorescent lamps. Myce-
lia were harvested by vacuum filtration through two layers of sterilized
Miracloth (Calbiochem CN Biosciences, Inc., La Jolla, CA), rinsed twice
in sterile water, and stored at ⫺80°C until lyophilized. DNA was extracted
from lyophilized mycelium as described by Lodhi et al. (33), quantified by
measuring the optical density of extracts at 260 and 280 nm with a Nano-
drop 1000 (Labtech) spectrophotometer, and adjusted to a final concen-
tration of 5 ng/␮l for amplified fragment length polymorphism (AFLP)
analysis.
For AFLP, DNA was digested with restriction endonucleases EcoRI
and MseI, ligated to EcoRI and MseI adapters, and amplified in a PCR
using primers that contain the common sequences of the adapters and one
or two arbitrary supplementary nucleotides as selective sequences. Pri-
mary template DNA was prepared in a one-step restriction ligation reac-
tion. Fungal genomic DNA (250 ng) was digested with EcoRI and MseI at
37°C for 2 h and heated to 70°C for 15 min to inactivate enzymes. The
DNA fragments were ligated to EcoRI and MseI adapters for 2 h at 20°C.
After termination of the reaction, the ligation mixture was stored at
⫺20°C. Ligated fragments served as templates in the preamplification
reaction with EcoRI (5=-AGACTGCGTACCAATTC-3=) and MseI⫹C
(5=-GACGATGAGTCCTGAGTAA/C-3=) primers. The preamplification
reaction was performed in a 25-␮l reaction mixture containing 7.2 ␮l of
template DNA, 30 ng of each primer, 1 U of Taq polymerase (TaqG Pro-
mega, Madison WI), 100 mM Tris-HCl (pH 8.0), 500 mM KCl, 1.5 mM
MgCl2, and 0.2 mM (each) deoxynucleoside triphosphate (dNTP). The

8432 aem.asm.org Applied and Environmental Microbiology


TABLE 3 Origin, sample sizes, and genotypic diversity descriptors assessed in populations of M. pinodes collected from winter (WP) and spring (SP)
peas
No. of genotypes
Population No. of isolates observed
WP 102 102
SP 42 42
Total collection 144 42
a
Fifteen loci in total.
b
Multilocus genotypic diversity based on Stoddard and Taylor’s index (48).
c
G/g is an estimate of evenness (Grünwald et al. [22]).
d
Index of association (12a). **, P ⬍ 0.01 based on 1,000 artificially recombined data sets.
fragments were preamplified in a 51000 thermal cycler (Bio-Rad) for 20
PCR cycles (94°C for 30 s, 56°C for 60 s, 72°C for 60 s), followed by 5 min
at 72°C and 10 min at 10°C, as described by Vos et al. (51). The samples of
the preamplified fragments were diluted 30-fold in sterile distilled water
to be used as DNA templates in selective amplification with three EcoRI
selective primers with two selective nucleotides (EcoRI ⫹AA, ⫹AC, or
⫹AT), developed by Zhang et al. (55), combined with selective primer
MseI⫹C. The selective amplification was performed in a 20-␮l reaction
mixture containing 2 ␮l of diluted product of the preamplification reac-
tion, 5 ng of EcoRI and MseI primers, 1 U of Taq polymerase (Fisher-
Brand), 100 mM Tris-HCl (pH 8.0), 500 mM KCl, 1.5 mM MgCl2, and 0.2
mM each dNTP. It started with one cycle at 94°C for 30 s, 65°C for 30 s,
and 72°C for 60 s, and then the annealing temperature was lowered during
each cycle by 0.7°C for 3 cycles, followed by 30 cycles at 94°C for 30 s, 56°C
for 30 s, and 72°C for 60 s. The reaction was terminated by incubation at
72°C for 5 min and then at 10°C for 10 min. Four-microliter aliquots of
the selective amplification products were then sequenced in an ABI Prism
3130xl sequencer (AB Applied Biosystems, Hitachi). PCR repetitions us-
ing the same set of primers and isolates and different DNA preparations of
the same isolates were conducted to check the repeatability of results.
Analysis of AFLP data. The presence and absence of all fragments
between 100 and 500 bp were scored in each of the 144 isolates. Bands with
molecular sizes exceeding 500 bp were not scored because of insufficient
resolution. The data matrix was analyzed using GeneMapper software 3.5
(ABI Prism AB Applied Biosystem), based on the assumption that bands
of the same molecular weight were identical.
A phylogenetic tree was built using the neighbor-joining (NJ) method
proposed by Saitou and Nei (45), implemented in the DARwin5 software
(41, 42) (http://Darwin.cirad.fr/darwin). To determine the robustness of
the dendrogram, the presence or absence of data was resampled by re-
placement with 1,000 bootstrap replicates. The analysis of molecular vari-
ance (AMOVA; online as Arlequin version 3.1 software, hosted by the
Department of Anthropology, University of Geneva, Switzerland [17])
was used to partition molecular variance between and within populations.
A principal component analysis (PCA) was also performed using the
procedure available in the package adegenet (27) for the statistical free-
ware R version 2.7.2 (The R Foundation for Statistical Computing, 2008).
PCA has an important advantage over other methods, such as the Bayes-
ian clustering algorithm implemented in Structure 2.2 (43): it does not
require strong assumptions about an underlying genetic model, such as
the Hardy-Weinberg equilibrium or the absence of linkage disequilibrium
between loci (27).
Structure software version 2.2 (43) was used without any assumptions
about population structure or assigning of individuals to populations.
The analysis was performed using 3 ⫻ 104 burn-in replicates and a run
length of 2 ⫻ 104 Markov chain Monte Carlo (MCMC) replicates, adopt-
ing the admixed model and the correlated allele frequency option. The
number of genetic groups (K value) was estimated using the model devel-
oped by Evanno et al. (16), which provides an estimate of the posterior
probability of the data for a given K value, Pr(X/K) (42). We used the
height of the modal value of the distribution as an indicator of the strength
of the signal detected by Structure.
December 2012 Volume 78 Number 23
Plasticity of Ascochyta Blight Populations on Pea
% of polymorphic
locia Gb G/gc Iad
97.1 1.509 0.036 0.064**
99.6 1.502 0.014 0.064**
100.0 1.523 0.010 35.3587
Standard population genetic statistics were calculated using the Pop-
Gene software, version 1.32 (http://www.ualberta.ca/⬃fyeh/popgene
n
.html). The genotypic diversity (G) was calculated as G ⫽ 1⁄ ⌺ gj2, where gj
k⫽0
is the frequency of the jth genotype and n is the total number of strains
(48). Following Grünwald et al. (22), we also used a weighting of G by g,
the total number of genotypes observed, giving an estimate of evenness.
Genotypic differentiation between populations (complete population)
and subpopulations (WP and SP) was estimated as a ␪ statistic (52, 53), anal-
ogous to Wright’s Fst, using the multiLocus 1.3b software (2, 3). Clonality was
assessed with the index of association (Ia), a measure of the multilocus linkage
disequilibrium (3, 11, 35), also calculated in multiLocus 1.3b. The distance
between all pairs of individuals (number of loci by which they differ) is cal-
culated, and the variance of these distances is compared to that of the “no
linkage disequilibrium” case. The higher the Ia, the more clonal is the popu-
lation structure. In these analyses, the null hypothesis of complete panmixia
was tested by comparing the Ia of the observed data set to those of artificially
recombined data sets (alleles were mixed at random between individuals,
independently for each locus). Five hundred recombined data sets were used
here.
Effect of temperature on in vitro colony hyphal growth. The hyphal
growth rate of 17 WP and 16 SP isolates (randomly chosen within the M.
pinodes collection) was determined in vitro at 5, 10, 15, 20, 25, and 30°C by
depositing a piece of malt agar (approximately 1 cm2) containing the
isolate on the center of a 90-mm (medium) plate. Each treatment in-
cluded three replicates per isolate, and the whole experiment was done
twice. Colony diameter (mm) was measured every second day for 14 days
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for each plate. To consider the whole growth period, the area under the
hyphal growth curve was then calculated for each isolate and used for the
statistical analysis. Statistical analysis of the data was performed with R
statistical software, version 2.12.0 (26), by using a mixed model in which
temperature and isolate origin (WP or SP) were considered fixed factors
and isolates were considered a random effect. Finally, our data showed
that isolates presented optimum hyphal growth at 20 and 25°C. A ␹2 test
was done to see if isolates from SP and WP crops showed similar distri-
butions.
FIG 1 Genetic assignation of the 144 individual M. pinodes isolates, using
Structure. Each isolate is represented by a single vertical bar divided into two
colored segments (red and green) corresponding to the inferred membership
fraction in groups 1 and 2, respectively. Isolates are classified according to their
original host.
aem.asm.org 8433
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FIG 2 Consensus unrooted NJ tree (1,000 bootstraps) based on Nei’s genetic distance of the 144 M. pinodes isolates sampled from winter (WP; black lines) and
spring (SP; red lines) pea crops.

8434 aem.asm.org Applied and Environmental Microbiology


FIG 3 Distribution of the M. pinodes isolates collected on winter pea (WP; solid dots) and spring pea (SP; open dots) according to a principal component analysis
(PCA) on AFLP alleles.
Aggressiveness of M. pinodes isolates. The pathogenicity of the 33 M.
pinodes isolates tested in the colony growth assays was evaluated on four
pea genotypes: two winter cultivars (Cheyenne [GAE-Recherche, France]
and Dove [Agri-Obtentions, France]), one spring cultivar (Baccara [Flo-
rimond-Desprez, France]), and one spring breeding line (DP; Baranger et
al. [6]). To mimic winter and spring conditions, plants were grown at
either 8 to 10°C or 18 to 20°C for 5 and 3 weeks, respectively, until they
reached the 5- to 6-leaf stage, before inoculation. Plant preparation and
experimental design were as described by Onfroy et al. (37).
The inoculation method used was based on that proposed by Onfroy
et al. (38), consisting of depositing a drop of spore suspension on detached
leaflets. Isolates were grown for 10 days on V8 medium under white light
with a 12-h photoperiod at 20°C (wavelengths between 350 and 750 nm)
before pycnidiospore suspensions were prepared by flooding the surface
of cultures with sterile distilled water, gently scraping with a glass rod, and
filtering the suspension through two layers of sterile cheesecloth. The
concentration of spores was determined with a hemocytometer and ad-
justed to 5 ⫻ 104 spores ml⫺1. Tween 20 (VWR International SAS, Stras-
bourg, France) was added as a wetting agent (2 drops/500 ml spore sus-
pension).
Inoculation consisted of depositing a 10-␮l drop of the spore suspen-
sion on the upper surface of freshly detached stipules floated, lower sur-
face down, on tap water in a compartmentalized square petri dish (12-cm
side; Gosselin, France). Drops were deposited away from the main veins
(38). To avoid drop evaporation, petri dishes were placed into large trans-
parent plastic boxes immediately after inoculum deposition and incu-
bated in a climate chamber for 7 days with a continuous cycle of 14 h of
light and 10 h of darkness at either 8 to 10°C or 18 to 20°C (Table 2).
Symptom development was assessed 2, 3, and 7 days after inoculation, as
described by Onfroy et al. (38). A 0-to-3 semiquantitative scale (0 ⫽
symptom free, 1 ⫽ flecks appearing, 2 ⫽ flecks covering half of the area of
drop deposition, 3 ⫽ coalescence of the flecks within the area of drop
deposition) was used to score symptoms not extending past the inocula-
tion droplet. For stipules with necrosis extending beyond the borders of
inoculum drops, lesion diameter (mm) was measured with a graduated
ruler. A visual assessment using a 0-to-7 scale adapted from Wroth (54),
where 0 ⫽ symptom free, 1 ⫽ flecks appearing, 2 ⫽ flecks covering half of
the drop deposit, 3 ⫽ coalescence of the flecks in the area of the drop
deposit, 4 ⫽ 3- to 6-mm lesion diameter, 5 ⫽ 6- to 9-mm lesion diameter,
6 ⫽ 9- to 12-mm lesion diameter, 7 ⫽ ⬎12-mm diameter, was also per-
formed. For each treatment (isolate, plant growth condition, incubation
December 2012 Volume 78 Number 23
Plasticity of Ascochyta Blight Populations on Pea
condition), two stipules from each of seven different plants per genotype
were inoculated. For each genotype, the area under the disease progres-
sion curve (AUDPC) was calculated as follows:
m (Di,j ⫹ Di,j⫹1)
AUDPCi ⫽ 兺
j⫽1 2
⫻ (tj⫹1 ⫺ tj)
where Di,j and Di,j⫹1 correspond to disease scores at two consecutive
dates, tj and tj⫹1 (47).
Statistical analysis of the data was performed with R statistical software,
version 2.12.0 (26), by using a mixed model with a random effect. Incubation
temperature, cultivar, and origin of the isolates (WP or SP) were considered
fixed factors, and isolates were considered a random effect.
RESULTS
Allelic variation and genetic structure within populations. The
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three AFLP primer sets detected a total of 561 loci among the 144
M. pinodes isolates, of which 548 were polymorphic in each pop-
ulation. The largest AFLP variation occurred within populations,
with a total estimated genetic diversity of 0.674 and 0.670 for M.
pinodes from WP and SP populations, respectively. Gene diversi-
ties ranged from 97 to 99% (Table 3). Indices of association indi-
cated that all populations had a clonal structure (Table 3).
The ␪ statistic between the two subpopulations of M. pinodes
isolates was significant (␪WP-SP ⫽ 0.0789, P ⬍ 0.001), suggesting
genetic differentiation. However, Nei’s genetic identity showed
strong similarities between WP and SP populations (Ia ⫽
35.3587). Furthermore, only 1.5% of AFLP allelic diversity was
due to differences between WP and SP populations, with most of
the variation (H=j ⫽ 0.680) being observed within populations
(Table 3). These results were confirmed by the Structure analysis,
which showed the highest posterior probability (P ⫽ 0.9989) for
the existence of two groups, including both WP and SP isolates
(Fig. 1). A consensus unrooted NJ tree with 1,000 bootstraps based
on Nei’s distance (36a) also did not separate populations from WP
and SP into 2 distinct groups (Fig. 2). A principal component
analysis (PCA) also failed to separate the WP and SP isolates into
different groups. The first axis of the PCA explained 22.7% of the
structuration, whereas the second explained only 9.3% (Fig. 3).
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Le May et al.

TABLE 4 Mean colony diameters of M. pinodes isolates sampled on winter peas (WP) and spring peas (SP) for the temperatures 5, 10, 15, 20, 25,
and 30°C after 14 days on malt medium
Mean colony diam (mm) by incubation tempa
Source Isolate 5°C 10°C 15°C 20°C 25°C 30°C
WP WP-1 1 (0) 0 (0) 27.4 (0.32) 29.0 (0.10) 26.8 (0.16) 0 (0)
WP-2 0 (0) 0 (0) 5.0 (0.19) 7.4 (0.26) 18.8 (0.25) 0 (0)
WP-3 0 (0) 0 (0) 5.9 (0.52) 14.0 (0.42) 20.4 (0.42) 0 (0)
WP-4 0 (0) 0 (0) 12.0 (1.36) 22.8 (0.49) 28.8 (0.37) 0 (0)
WP-5 0 (0) 0 (0) 8.1 (0.52) 11.8 (0.80) 22.8 (0.41) 0 (0)
WP-6 0 (0) 0 (0) 6.4 (0.18) 14.2 (0.69) 23.3 (0.31) 0 (0)
WP-7 0 (0) 0 (0) 10.6 (0.80) 25.6 (0.96) 20.3 (0.49) 0 (0)
WP-8 0 (0) 0 (0) 8.6 (0.26) 23.5 (0.57) 21.9 (0.52) 0 (0)
WP-9 0 (0) 0 (0) 14.2 (0.49) 23.6 (0.65) 18.0 (0.38) 0 (0)
WP-10 0 (0) 0 (0) 8.8 (0.49) 25.5 (0.98) 24.6 (0.26) 0 (0)
WP-11 0 (0) 0 (0) 9.2 (0.35) 12.6 (0.18) 25.4 (0.18) 0 (0)
WP-12 0 (0) 0 (0) 24.6 (0.82) 31.3 (0.37) 21.7 (0.18) 0 (0)
WP-13 0 (0) 0 (0) 9.2 (0.44) 14.9 (0.81) 17.2 (0.13) 0 (0)
WP-14 0 (0) 0 (0) 7.3 (0.31) 15.4 (0.71) 24.8 (0.31) 0 (0)
WP-15 0 (0) 0 (0) 6.5 (0.19) 15.3 (0.59) 26.9 (0.52) 0 (0)
WP-16 0 (0) 0 (0) 8.8 (0.25) 25.7 (0.46) 20.4 (0.38) 0 (0)
WP-17 0 (0) 0 (0) 8.9 (0.13) 15.0 (0.73) 22.8 (0.25) 0 (0)
Mean 0.06 (0.05) 0 (0) 10.7 (1.50) 19.3 (1.68) 22.6 (0.79) 0 (0)

SP SP-1 0 (0) 0 (0) 9.6 (0.38) 18.6 (1.52) 17.6 (0.94) 0 (0)
SP-2 0 (0) 0 (0) 7.4 (0.26) 12.6 (0.32) 23.3 (0.16) 0 (0)
SP-3 0 (0) 0 (0) 27.0 (0.91) 31.3 (0.25) 34.0 (0.1) 13.9 (0.40)
SP-4 0 (0) 0 (0) 4.8 (0.16) 12.5 (0.91) 21.6 (0.32) 0.0 (0)
SP-5 0 (0) 0 (0) 5.2 (0.23) 15.9 (0.52) 19.8 (0.70) 0 (0)
SP-6 0 (0) 0 (0) 14.4 (1.50) 30.8 (0.16) 29.9 (1.81) 0 (0)
SP-7 0 (0) 0 (0) 9.3 (0.25) 14.0 (0.38) 16.9 (0.30) 0 (0)
SP-8 0 (0) 0 (0) 6.9 (0.88) 12.0 (0.53) 20.4 (0.65) 0 (0)
SP-9 0 (0) 0 (0) 8.2 (0.35) 14.0 (0.50) 21.5 (0.60) 0 (0)
SP-10 0 (0) 0 (0) 7.3 (0.25) 12.9 (0.44) 21.8 (0.31) 0 (0)
SP-11 0 (0) 0 (0) 6.0 (0.27) 13.3 (0.25) 16.8 (0.25) 0 (0)
SP-12 0 (0) 0 (0) 10.3 (0.25) 14.3 (0.59) 16.0 (0.46) 0 (0)
SP-13 0 (0) 0 (0) 11.9 (0.48) 20.0 (0.33) 20.8 (0.49) 0 (0)
SP-14 0 (0) 0 (0) 8.4 (0.60) 20.0 (0.82) 18.0 (0.33) 0 (0)
SP-15 0 (0) 0 (0) 2.3 (0.31) 12.4 (0.32) 17.3 (0.67) 0 (0)
SP-16 0 (0) 0 (0) 4.7 (0.56) 10.9 (0.44) 16.3 (0.53) 0 (0)

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Mean 0 (0) 0 (0) 8.9 (1.41) 16.6 (1.57) 20.7 (1.244) 0.87 (0.86)
a
Values in parentheses are standard deviations.

Temperature effect on colony hyphal growth. Temperature
had a highly significant effect on hyphal growth (P ⬍ 0.0001). All
SP and WP strains showed only hyphal growth for temperatures of
15, 20, and 25°C. No hyphal growth was observed at low temper-
atures (5 and 10°C), and only one SP strain showed some hyphal
growth at 30°C. Seven of the 17 WP isolates (41%), but only 3 of
the 16 SP isolates (12%), had the largest colony diameters at 20°C
(Table 4); however, these distributions were not statistically dif-
ferent according to a ␹2 test (df ⫽ 1, P ⫽ 0.109). Similar results
were obtained for the areas under mycelium growth curves (data
not shown).
Winter and spring pea Mycosphaerella pinodes isolates do
not present any specialization according to host genotypes and
the growing conditions. Frost hardening of pea plants de-
creased the disease level for all hosts, as disease severity was
lower on all genotypes in winter than in spring conditions (P ⬍
0.001; Fig. 4A and B). For the late-winter condition, an inter-
mediate level of disease was observed with both groups of iso-
lates. Significant differences between host genotypes were ob-
served only for spring conditions, where DP showed a lower
disease level with both WP and SP isolates (P ⫽ 0.0044). For the
late-winter condition, DP showed a significant difference only
with the other spring host, Baccara (Fig. 4A and B). In addition,
results showed that pea genotypes do not distinguish between
WP and SP isolates.
Isolates did not present any specialization to either the host
type (winter or spring pea) or the host growing conditions (Fig.
4A and B). The origin of the isolates (WP or SP) did not have
any significant effect on the aggressiveness level measured on
the genotypes (P ⫽ 0.7401). However, significant aggressive-
ness differences were observed between individual isolates for
each experimental condition (P ⬍ 0.001). They were mostly
evident in the suboptimal winter condition (Fig. 5). AUDPC
values ranged from 0.57 to 6.18 for the WP isolates and from
0.57 to 6.27 for the SP isolates in winter conditions (Fig. 5A and
D). These differences were less important for WP than for SP
isolates in the late winter condition. The aggressiveness level
ranged from 4.31 to 11.16 for the WP isolates and from 3.68 to

8436 aem.asm.org Applied and Environmental Microbiology


FIG 4 Mean aggressiveness (AUDPC) on four different hosts (Baccara, DP,
Cheyenne, and Dove) and three growing conditions (winter, late winter, and
spring) of M. pinodes isolates sampled from winter (A) and spring (B) pea
crops.
12.48 for the SP isolates (Fig. 5B and E). Finally, for the spring
condition, WP and SP isolates displayed similar ranges of ag-
gressiveness. AUDPC values ranged from 8.40 to 17.97 for the
WP isolates and from 11.79 to 18.50 for the SP isolates (Fig. 5C
and F).
DISCUSSION
Plant pathogens can emerge either sympatrically or allopatri-
cally in agricultural ecosystems through several mechanisms,
including host tracking, host jumps, hybridization, and hori-
zontal gene transfer (34, 49). In this study, M. pinodes isolates
from sympatric winter (WP) and spring (SP) peas were studied
to see whether or not the population of M. pinodes was special-
ized to the crop. The host agroecology in the pea-M. pinodes
pathosystem indeed warranted a direct investigation of two
factors liable to lead to niche partitioning and eventually to
genetic differentiation of specialized pathogen populations.
First, M. pinodes isolates infecting winter pea may be adapted to
survive and develop at lower temperatures than those infecting
spring pea. Second, we expected differences in the aggressive-
ness level of M. pinodes isolates according to the ecophysiologi-
cal status of the host, influenced by the climatic conditions
present during its own growth.
The first important finding in this work is that, contrary to
earlier results obtained by Onfroy et al. (37) using randomly
amplified polymorphic DNA (RAPD) markers, we observed
substantial molecular diversity within the French M. pinodes
population using three of the six AFLP primers developed by
Zhang et al. (55). Despite this genetic polymorphism, the mo-
December 2012 Volume 78 Number 23
Plasticity of Ascochyta Blight Populations on Pea
lecular analysis (H=j ⫽ 0.68 and multilocus genotypic diversity
[Gst] ⫽ 0.015) showed that M. pinodes populations were not
structured genetically through parasitic specialization accord-
ing to host types (winter or spring pea). Indeed, clustering (NJ
consensus tree) and assignation methods (Structure and PCA
analyses) did not place isolates collected from winter and
spring peas into distinct groups. This conclusion is also consis-
tent with the fact that the host type accounted for only 1.2% of
the total genetic variation within the collection of 144 isolates
tested.
Because temperature is an important ecological determinant,
the seasonal differences in temperature profiles prevailing during
the winter and spring pea cropping seasons were expected to be
reflected in different temperature optima for the isolates collected
on each host type. Such observations have been reported previ-
ously in other fungal pathogens, such as R. solani (23) or D. rabiei
(19), a close relative of M. pinodes. In contrast, in our work, the
extensive variation for growth speed and thermal optima existing
among isolates could not be shown to affect the distribution of
isolates according to host types. Although the proportion of iso-
lates with a lower thermal optimum (20°C) seemed to be higher
among those collected from winter pea than among those from
spring pea, the difference was not statistically significant, perhaps
owing to the relatively small sample sizes in both groups (16 to 17
isolates).
Despite the lack of differential thermal adaptation between SP
and WP populations of M. pinodes, all isolates displayed a pheno-
typic plasticity for the physiological adaptation to their hosts. We
indeed showed an increase in aggressiveness on plants grown be-
fore inoculation under spring conditions relative to that on plants
of the same host genotype grown under winter conditions, with no
particular distinction between the isolates. Winter and spring pea
genotypes are usually characterized by different leaf structures (7,
30). Due to the low temperature during the first stage of plant
growth, winter genotype peas develop smaller and thicker leaves
than spring genotype peas, as well as cold tolerance. In our exper-
iment, we did not see visual differences in leaf structure between
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winter and spring genotype peas in a given growing condition.
This suggests that leaf structure depends more upon the climatic
conditions during growth than upon genetic differences between
host genotypes, which may in turn explain the absence of struc-
turation of M. pinodes populations by host types. The low temper-
atures during plant development used in the winter and interme-
diate conditions are known to alter the development of the
different genotypes and therefore possibly also affect the expres-
sion of resistance genes (15, 20, 31). Indeed, as suggested by Ergon
et al. (15) and Gaudet et al. (20), cold hardening is known to
strongly increase the resistance of plants to disease by increasing
the PR gene expression. This could explain the similar levels of
resistance of all four pea genotypes in the winter conditions and
hence the fact that the higher level of resistance of DP than that of
the other three hosts was manifest only in spring conditions, con-
firming earlier observations (38).
Our results clearly support the idea of synchronic and sympa-
tric M. pinodes populations on winter and spring peas within a
cropping season, allowing their transfer between winter and
spring pea crops. As shown by Schoeny et al. (46) and Le May et al.
(32), significant gene flow can be observed between these two
crops. The persistence of the disease is favored by the connection
between host populations, which probably restrict the chance of
aem.asm.org 8437
Le May et al.

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FIG 5 Mean aggressiveness (AUDPC) of individual M. pinodes isolates sampled on winter pea (A, B, C) or spring pea (D, E, F) according to three climatic
conditions (winter condition, panels A and D; late winter, panels B and E; and spring, panels C and F).

niche adaptation. However, the connection between hosts is lost
during the intercrop period, i.e., from midsummer (harvest of
spring peas) to early autumn (emergence of the next winter pea
crop). M. pinodes produces its pseudothecia on the senescent or-
gans during the second part of the cropping season (50), pending
specific environmental and/or nutritional conditions. The high
levels of infection that can be observed early in the winter crops
probably owe more to longer periods of exposure to leaf wetness
than to the presence of high levels of M. pinodes primary inoculum
early in crop growth. Indeed, as shown by Schoeny et al. (46) in an
investigation of the seasonal dynamics of airborne inoculum
through trap plants, the availability of airborne primary inoculum
is extremely low during autumn and winter (0 to 8 lesions per
plant). Thus, the sexual fruiting structures allow only the fungus
to oversummer and probably play an important role in generating
pathogen variability (13, 28, 29).
Since M. pinodes is not specific to pea crops, we wonder
whether an alternative host could select for aggressiveness and
genetic variability. Indeed, M. pinodes is a polyphagous fungus
that can infect different wild legumes (9, 40, 50). Wild legumes
have sometimes been assumed or shown to be involved in the
transmission of inoculum from field borders into fields. More-
over, as discussed by Abbo et al. (1), wild hosts can also act as a
selection pressure which could affect the fitness of M. pinodes iso-
lates. Gerard et al. (21) and Frenkel et al. (19) showed that an
exchange between wild and domesticated plants could exist in
nature. Moreover, Peever (40) showed that as wild host popula-
tions are generally more diverse, they could influence the genetic

8438 aem.asm.org Applied and Environmental Microbiology


structure of the plant pathogen populations. Although primary
inoculum can also be generated by volunteers in neighboring
fields (8), the initiation of disease in the winter crops could there-
fore be due mainly to wild hosts acting as reservoirs. If this is the
case, the populations present on those wild hosts would not be
genetically differentiated from those present on pea. While we
restricted the present study to the pea isolates, cross-inoculations
and genotyping experiments are under way to test this hypothesis.
Overall, our results indicate that M. pinodes populations on pea
crops in France do not show any evidence for niche adaptation
either to host type or to temperature but do show a high plasticity
for adaptation to host physiology. This has important conse-
quences for the management of disease, since the connection be-
tween hosts plays a main role in the persistence and spread of the
pathogen throughout the growing season. The development of
winter pea crops, fully warranted for agronomic reasons (better
yield stability, ground coverage in winter, etc.), provides an in-
creased opportunity for the pathogen to bridge cropping seasons
and to persist in the environment. This might be further enhanced
if the pea-infecting populations are not genetically and ecologi-
cally separated from those infecting wild legume hosts present in
field margins. The lack of highly resistant cultivars and the pres-
ence of extensive genetic and phenotypic variation within M. pi-
nodes populations make the sustainable management of disease a
challenging goal; meeting it will require limiting the connectivity
between susceptible host fields and decreasing the inoculum dy-
namics (primary and secondary infections) within winter crops.
ACKNOWLEDGMENTS
We thank Lionel Lebreton (INRA, UMR1349 IGEPP) and Caroline On-
froy (UNIP) for their valuable advice and Emmanuel Wicker (CIRAD, La
Réunion) and Yannick Outreman (INRA AGROCAMPUS Ouest UMR
1349 IGEPP) for their valuable advice on the phylogenetic analyses.
This work was supported by Union Nationale Interprofessionnelle des
Plantes Riches en Protéines (UNIP; Paris), INRA Institute, and Agrocam-
pus Ouest.
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