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Title: Multiple scenarios for sexual crosses in the fungal pathogen Zymoseptoria tritici on
wheat residues: potential consequences for virulence gene transmission
Authors: Carolina Orellana-Torrejon
1,2, Tiphaine Vidal
1, Gwilherm Gazeau
1, Anne-Lise
Boixel 1, Sandrine Gélisse1, Jérôme Lageyre 2, Sébastien Saint-Jean 2, Frédéric Suffert 1*
1 Université Paris-Saclay, INRAE, UR BIOGER, 78850 Thiverval-Grignon, France
2 Université Paris-Saclay, INRAE, AgroParisTech, UMR ECOSYS, 78850 Thiverval-
Grignon, France
* corresponding author: frederic.suffert@inrae.fr
Abstract:
Little is known about the impact of host immunity on sexual reproduction in fungal
pathogens. In particular, it is unclear whether crossing requires both sexual partners to infect
living plant tissues. We addressed this issue in a three-year experiment investigating different
scenarios of Zymoseptoria tritici crosses on wheat according to the virulence (‘vir’) or
avirulence (‘avr’) of the parents against a qualitative resistance gene. Co-inoculations (‘vir ×
vir’, ‘avr × vir’, ‘avr × avr’) and single inoculations were performed on a cultivar carrying the
resistance gene (Cellule) and a susceptible cultivar (Apache), in the greenhouse. We assessed
the intensity of asexual multiplication by scoring disease severity, and the intensity of sexual
reproduction by counting the ascospores discharged from wheat residues. As expected,
disease severity was more intense on Cellule for ‘vir × vir’ co-inoculations than for ‘avr × vir’
co-inoculations, with no disease for ‘avr × avr’. However, all types of co-inoculation yielded
sexual offspring, whether or not the parental strains caused plant symptoms. Parenthood was
confirmed by genotyping (SSR markers), and the occurrence of crosses between (co-
)inoculated and exogenous strains (other strains from the experiment, or from far away) was
determined. We found that symptomatic asexual infection was not required for a strain to
participate in sexual reproduction, and that avirulent strains could be maintained
asymptomatically “on” or “in” leaf tissues of plants carrying the corresponding resistant gene
for long enough to reproduce sexually. In two of the three years, the intensity of sexual
reproduction did not differ significantly between the three types of co-inoculation in Cellule,
suggesting that crosses involving avirulent strains
are not anecdotal. We discuss the possible
mechanisms explaining the maintenance of avirulence in Z. tritici populations and supporting
the potential efficacy of cultivar mixtures for limiting resistance gene breakdown.
Key words: asexual multiplication, avirulence, infection, plant disease epidemiology, sexual
reproduction, resistance breakdown
Highlights:
• Avirulent Zymoseptoria tritici strains can reproduce sexually in wheat plants carrying
the corresponding resistant gene.
• Symptomatic infection of plant tissues is not essential for a strain to reproduce
sexually.
• Avirulent strains can be maintained asymptomatically “on” or “in” leaf tissues of
plants carrying the corresponding resistant gene for long enough to reproduce
sexually.
• Crosses of virulent strains with virulent and avirulent strains in a plant host carrying
the corresponding resistance gene can produce offspring with similar population sizes.
• Several possible scenarios for sexual crosses can explain the maintenance of
avirulence in Zymoseptoria tritici populations evolving in a wheat canopy, particular
in cultivar mixtures.
1. Introduction
Sexual reproduction drives genetic recombination in many phytopathogenic fungal
populations (Ni et al., 2011). It also plays an essential role in their interseason survival and,
thus, in the multiannual recurrence of many plant disease epidemics (Burdon and Laine,
2019). It is widely believed that sexual reproduction in foliar pathogens requires “infection”
— penetration and growth in the organs of a living host plant, including the appearance of
symptoms — by both individual partners during an epidemic. Some host plants carry
qualitative resistance genes that prevent or stop infection by pathogenic strains that are
“avirulent” (‘avr’), according to the “gene-for-gene” model (Dangl and Jones, 2001).
Pathogenic “virulent” strains (‘vir’) able to infect these “resistant” plants can then reproduce
sexually in host tissues, whereas avirulent strains, having been prevented from infecting the
host plants, are presumed not to be able to engage in sexual reproduction. Avirulent strains
are, therefore, thought to be progressively eliminated from host plans populations carrying the
corresponding resistance gene. However, this assumption is simplistic, because the absence of
infection (and, thus, of symptoms) does not necessarily prevent a physical encounter between
sexual partners. Indeed, many fungal species reported to act as plant pathogens can also
develop epiphytically, endophytically (causing an “asymptomatic infection”), or as
saprotrophs on plant residues (Kerdraon et al., 2019; Selosse et al., 2018; Wheeler et al.,
2019).
As pathogens can complete their life cycle without necessarily causing symptoms, it
may be possible for avirulent strains to reproduce sexually on a host that they failed to infect.
Clarification of this point is essential, not only from the standpoint of fungal genetics and
biology, but also for epidemiology, as ‘unexpected’ sexual reproduction events may affect the
ratio of virulent to avirulent strains in the offspring population developing on a cultivar
considered resistant. Such events could affect the efficacy of the resistance gene and the
durability of resistance deployment strategies.
There are still many gaps in our knowledge of the interactions between living plants and fungi
during sexual reproduction in natural conditions, because of experimental difficulties
promoting sexual reproduction in some of the organisms studied, and because of their ability
to switch between lifestyles — epiphytic, endophytic and pathogenic — under changing
environmental conditions (e.g., Schulz and Boyle, 2005). Sexual reproduction in several fungi
can be achieved under controlled conditions in planta, but also sometimes in dead plant
tissues colonised in a saprophytic way, or in axenic conditions in vitro. The ease of crossing is
heterogeneous in heterothallic species, which require two sexually compatible parental
strains. For example, crosses were obtained for Phaeosphaeria nodorum on wheat residues
(straw) by Halama & Lacoste (1992), but the same authors were unable to obtain crosses of
Zymoseptoria tritici, the causal agent of Septoria tritici blotch (STB) in wheat under similar
conditions. These difficulties do not necessarily reflect what happens in natural conditions. In
several ascomycetes, the sexual form is cryptic or facultative, and therefore difficult to detect.
For instance, the sexual form has never been identified for the rice pathogen Magnaporthe
oryzae in natural conditions, whereas it can be obtained in vitro (Saleh et al., 2012a, 2012b).
Controlled crosses of Venturia inaequalis were first obtained on apple trees in the greenhouse
by Keitt & Palmiter (1937), then on detached apple leaf discs by Ross & Hamlin (1962) and
Martin et al. (1981), and, later, on artificial medium by Sierotzki & Gessler (1998). The
causal agent of oilseed rape stem canker, Leptosphaeria maculans, was successfully crossed
on artificial media (Shoemaker and Brun, 2011), but sexual reproduction in planta under
controlled conditions was not achieved until recently (Bousset et al., 2018). The physiological
and cellular mechanisms occurring during sexual reproduction have been described in some
model ascomycetes (e.g. Neurospora crassa; Brun et al., 2021), but the interaction with the
host plant remains poorly understood for biotrophic and hemibiotrophic plant pathogens.
Sexual reproduction helps maintain pathogen populations between epidemics and is involved
in the interseason transmission of primary inoculum. It is, thus, essential to understand how
plant host immunity affects this process and its consequences for a local pathogen population.
The efficacy of strategies for controlling fungal plant pathogens through cultivar resistance is
generally assessed at cropping-season scale. For this reason, the impact of host “immunity” on
the sexual reproduction of the pathogen, which mainly occurs between cropping seasons, has
been much less studied. Host-plant resistance could be considered as a form of stress, in
which case it would be expected to increase sexual reproduction rates. However, contrasting
results have been obtained for pathosystems in which this question has been addressed. V.
inaequalis has been reported to grow and produce sexual fruiting bodies (pseudothecia) on
pear and apple leaves, but its growth on different apple cultivars was not related to the
susceptibility of these cultivars to apple scab (Ross and Hamlin, 1962). Conversely, Marcroft
et al. (2004), Bousset et al. (2021) and Fortune et al. (2021) highlighted a significant effect of
oilseed rape genotype on the density of L. maculans pseudothecia produced on crop residues.
The intensity of sexual reproduction was shown to depend on disease severity at harvest, with
higher stem canker severities leading to the production of more pseudothecia. A similar
finding was reported for the hemibiotrophic pathogen Z. tritici: a positive correlation was
found between the susceptibility of wheat cultivars, expressed as disease severity during the
epidemic stage, and the intensity of sexual reproduction on wheat residues (Cowger et al.,
2002). Such a correlation is not surprising, given that this fungus is heterothallic, the opposite
mating types must come into contact for sex to occur (Kema et al., 1996), and the two mating-
type idiomorphs are consistently found to be evenly distributed, even at a fine scale (plant and
field; Zhan et al., 2002; Siah et al., 2010; Morais et al., 2019). However, data are lacking as to
whether there is systematically a good match between the levels of host susceptibility at the
asexual and sexual stages (Suffert et al., 2016), apart from the empirical characterisation of
density-dependent processes established by Suffert et al. (2018a). Moreover, it has been
shown that a few days’ difference in the latent periods of the strains, with one progressing
faster than the other in wheat tissues, is slightly beneficial to ascosporogenesis, whereas a
two- to three-week interval between infections with the two strains is detrimental (Suffert et
al., 2016). More generally, the conditions of asexual infection by two parental strains of Z.
tritici affect the dynamics of sexual reproduction, but it remains challenging to identify these
conditions and to decipher the mechanisms involved.
Genes conferring partial or complete resistance to an ascomycete pathogen on the host plant
may also influence the intensity of the pathogen’s sexual reproduction, thereby limiting the
density of sexual fruiting bodies and/or ascosporogenesis. The production and release of
smaller numbers of ascospores may be another immunity trait lowering disease severity
during the early stages of the subsequent epidemic at least (e.g. Suffert and Sache, 2011), and
ensuring better crop yields. This is an important point consider, although not the focus of this
study. It would have epidemiological consequences, as highlighted by the trade-offs identified
between crop infection and survival or the interseason transmission of primary inoculum in
Phytophthora infestans (Pasco et al., 2016), Plasmopara viticola (Delbac et al., 2019) and Z.
tritici (Suffert et al., 2018b).
STB, one of the most important wheat diseases worldwide, is a suitable biological model for
the investigation of these issues. The presence of Stb genes conferring resistance in wheat
cultivars (Brown et al., 2015) is known to determine the ability of virulent and avirulent
strains to infect them. If we assume that only virulent strains can cross on a cultivar carrying a
resistance gene, then the offspring would be exclusively virulent, resulting in the fixation of
this virulence at a very high frequency, especially when the Stb gene is present in most, if not
all of the cultivars widely deployed in a wheat production area. However, the epidemiological
situation is more complex in the case of Z. tritici. For instance, the frequency of strains
avirulent against Stb6 remains at about 0.1, despite the widespread use of this resistance gene
in wheat cultivars deployed in France (Saintenac et al., 2018; Stephens et al., 2021). Various
processes, such as the possibility of ‘avr × vir’ crosses on a cultivar carrying Stb6, could
counter the generalisation of virulence. Indeed, Kema et al. (2018) established experimentally
that such a cross can occur between a Z. tritici virulent strain successfully infecting host
seedlings carrying the corresponding resistance gene and an avirulent strain unable to infect
the host. This result should be interpreted in light of the ‘epiphytic’ growth of Z. tritici, which
has recently been shown to occur for at least 10 days in specific humidity conditions (Fones et
al., 2017). A second potential mechanism should also be considered: the facilitation between
co-infecting fungal strains demonstrated by Dutt et al. (2021), after a ‘systemic induced
susceptibility’ (SIS) reaction (Seybold et al., 2020) or ‘neighbour-modulated susceptibility’
(NMS) reaction (Pélissier et al., 2021) for instance, in Z. tritici, with consequences modelled
by Sofonea et al. (2017). These mechanisms, explaining the possible simultaneous co-
infection of a plant by an avirulent and a virulent strain, are consistent with the presence of
avirulent strains in STB lesions sampled from resistant cultivars (e.g. Orellana-Torrejon et al.,
2022a). Some authors have also suggested the possible induction of ‘systemic acquired
resistance’ (SAR) or ‘localised acquired resistance’ (LAR) in other pathosystems (Costet et
al., 1999), although their studies focused on levels of aggressiveness (quantitative response)
rather than virulence (qualitative response). Microscopy studies showed, for example, that the
Stb16q gene stops the progression of avirulent Z. tritici strains 10 day after inoculation, either
before their penetration through the stomata or in the substomatal cavities (Saintenac et al.,
2021). The physiological processes induced in such incompatible reactions merit exploration,
as they may also explain the phenomenon of partial resistance against co-inoculation with
virulent and avirulent strains. They could potentially decrease the severity of STB severity
observed in co-infected wheat plants (Barrett et al., 2021; Schürch and Roy, 2004). Other
mechanisms should also be considered, such as crosses between non-infecting strains
developing saprophytically on (or in) wheat residues (i.e. dead tissues). Crossing in such
conditions has been reported for P. nodorum but has never been observed for Z. tritici
(Halama & Lacoste, 1992).
In this experimental study, we investigated the diversity of scenarios in which Z. tritici sexual
reproduction could occur in a wheat cultivar carrying an Stb resistance gene. Our goal was to
determine whether: (i) strains that fail to infect wheat tissues during the asexual phase can
reproduce sexually (“Who?”); (ii) sex can occur if one of the parental strains arrives on plant
tissues later than the other, particularly after the senescence of these tissues (“When?”); (iii)
the presence of parental strains in different proportions affects the intensity of sexual
reproduction (“How?”). As a means of addressing the question “Do these situations actually
lead to sexual crosses?”, we designed an experiment based on different crossing situations,
with three types of co-inoculation (‘vir × vir’, ‘avr × vir’ and ‘avr × avr’) and the
corresponding single inoculations (virulent, avirulent), involving multiple epidemiological
processes (spore transfers between different types of wheat tissues and fungal growth
conditions). Obtaining an answer to this question implied finding a balance between natural
conditions allowing the processes to be expressed and controlled conditions managing and
dissociating the processes considered, at least partially. We, thus, simplified the host-pathogen
interactions, using only pairs of parental strains and two wheat cultivars, for which virulence
(vir, avr) and resistance (R, S) status, respectively, were known.
2. Materials & methods
2.1. Overall strategy
In this experiment, we inoculated adult wheat plants with and without a resistance gene with
several virulent and avirulent Z. tritici strains in the greenhouse, to estimate the intensity of
sexual reproduction by quantifying the offspring. We performed ‘co-inoculations’ with pairs
of compatible parental strains and ‘single inoculations’ of each parent, to estimate the
possibility of a strain crossing with an exogenous strain (used for another cross during the
same experiment or from outside) in the various crossing situations. The experiment was
replicated over three years (2018, 2019 and 2020). Each year, we performed 12 co-
inoculations with eight parental strains, and eight single inoculations (Figure 1). We tested
three types of cross, defined in terms of the virulence status of the parental strains: both
virulent (‘vir × vir’), both avirulent (‘avr × avr’), and one virulent and the other avirulent (‘vir
× avr’). All co-inoculations were performed with suspensions of equal proportions of
blastospores from the two parental strains sprayed on adult wheat plants. Each year, additional
co-inoculations were performed for one of the ‘avr × vir’ treatments, with unbalanced
proportions for the biparental suspensions. Disease severity was assessed to estimate the
intensity of asexual multiplication. Plants were left in the greenhouse and allowed to follow
their natural development cycle until they had completely dried out. The resulting senescent
plants, which were considered to be ‘residues’, were placed outdoors for several months, and
the intensity of sexual reproduction was then estimated by quantifying ascospore discharge, as
described by Suffert et al. (2016), for each crossing situation. Several offspring strains were
sampled from some sets of residues and genotyped with 12 SSR markers (Gautier et al., 2014)
to check their ‘parenthood’: the genotypic profiles of the offspring were compared with those
of the (co-)inoculated strains to determine which strains had actually crossed, their status
(virulent and/or avirulent) and their origin (inoculated and/or exogenous).
2.2. Plant material
We used cv. Cellule
(
Florimond Desprez, France) carrying the Stb16q resistance gene
(Ghaffary et al., 2012) for its resistance to STB and Apache (Limagrain Europe, France) for
its susceptibility. Both cultivars have been widely grown in France in the last decade. A few
years after the deployment of Cellule (2012), strains capable of overcoming Stb16q were
detected. These strains now comprise a substantial proportion of Z. tritici populations in
Europe (Kildea et al., 2020; Orellana-Torrejon et al., 2022a). Seeds of cv. Cellule and Apache
were sown on 11th December 2018, 20th December 2019, and 9th December 2020, in peat pots
(Jiffy). Seedlings were kept in greenhouse conditions for two weeks and were then vernalised
in a growth chamber for 8 weeks at 8°C, with a 10-h light/14-h dark photoperiod. The plants
were then returned to the greenhouse and left to acclimate for one week before transplantation
into individual pots filled with 1 litre of commercial compost (Klasmann Peat Substrate 4;
Klasmann France SARL, France) supplemented with 4 g of slow-release fertiliser (Osmocote
Exact 16-11-11N-P-K 3MgO Te) per pot. The plants were also watered with Hydrokani C2
fertiliser (Hydro Agri Spécialités, France) diluted 1:100 and poured into the saucers under the
pots. During the growth period, the plants were illuminated with natural daylight
supplemented with 400 W sodium vapour lamps. The air temperature was kept below 20°C
during the 15-hour light period and above 15°C during the 9-hour dark period. Plants were
thinned to three stems per pot during the growth period.
2.3. Fungal material
Each year, we selected eight strains (Figure 1 and Table S1) from a Z. tritici population
sampled from single pycnidium on cv. Cellule and Apache in pure-stand field trials in
Grignon (France) in July 2017, July 2018, and June 2019 (Orellana-Torrejon et al., 2022a).
The virulence status of these strains with respect to
Stb16q was determined by phenotyping on
wheat cv. Cellule seedlings. The sexual compatibility of the different pairs of strains was
determined by PCR amplification of the two mating-type idiomorphs (Waalwijk et al., 2002).
Subcultures were grown for five days in Petri dishes containing PDA (potato dextrose agar,
39 g·L−1) at 18 °C in the dark. Blastospore suspensions of each strain were prepared as
described by Suffert et al. (2013). The inoculum concentration was adjusted to 5 × 105
conidia·mL-1 with a Malassez counting chamber, and two drops of surfactant (Tween 20;
Sigma, France) were added. Biparental suspensions containing a Mat1.1 strain and a Mat1.2
strain were prepared by mixing 15 mL of each of the monoparental suspensions for the
equiproportional combination (0.5-0.5). For the non-equiproportional combinations
(unbalanced proportions: 0.1-0.9, 0.25-0.75, 0.75-0.25, and 0.9-0.1), the appropriate volumes
of each suspension were combined to achieve a total volume of 30 mL.
2.4. Inoculation procedure
An atomiser (Ecospray, VWR, France) was used to apply each blastospore suspension onto
three adult plants (nine stems) for biparental suspensions and two adult plants (six stems) for
monoparental suspensions, after the wheat heads had fully emerged (on 27th April 2018, 30th
April 2019, and 19th April 2020), as described by Suffert et al. (2013). Plants were turned
during the 10-second spraying event, to ensure even coverage with inoculum. Infection was
promoted by enclosing each duo or trio of plants in a transparent polyethylene bag containing
a small amount of distilled water for 72 h. The wheat plants were kept in a greenhouse for
about 11-12 weeks until they had completely dried out (from 27th April to 12th July 2018, 30th
April to 9th July 2019, and 19th April to 8th July 2020). Air temperature was kept above 24 °C
during this period. In 2020, three adult plants each of Apache and Cellule were used as non-
inoculated control plants.
2.5. Assessment of the intensity of asexual multiplication
We estimated the intensity of asexual multiplication, by determining the percentage of the leaf
area covered by pycnidia (1, 2, 3 and 5%, and increments of 5% thereafter up to 100%;
Suffert et al., 2013) for the two uppermost leaves of each stem of the inoculated plants,
between five and six weeks after inoculation (on 15th June 2018, 5th June 2019, and 29th May
2020). Mean disease severity was, thus, calculated with 18 replicates for co-inoculations and
12 replicates for single inoculations. In the third year, an additional assessment was performed
eight weeks after inoculation (on 8th June 2020) on some of the leaves of cv. Cellule (co-
)inoculated with avirulent strains, on which some sporulating lesions appeared very late (a
few days before leaf senescence). The 17 strains collected from pycnidia in these lesions were
genotyped, for comparison with the parental strains used in the experiment.
2.6. Promotion of sexual reproduction
After the stems and leaves had completely dried out, the wheat plants were placed outdoors
during the summer and autumn, to induce the formation of pseudothecia and promote
ascosporogenesis. In 2018, each duo or trio of plants was tied up with stiff wire, with the
stems and leaves clamped, and placed about 10 cm away from the nearest neighbouring duo
or trio, to reduce contact with these other groups of plants (from 12th July 2018 to 16th January
2019; ‘arrangement A’ in Figure 2A). In 2019 and 2020, the plants of each duo or trio were
cut and grouped into small bundles, hung on a fence and spaced about 30 cm apart to ensure
that contact between bundles was totally prevented (from 9th July 2019 to 21st January 2020
and from 22nd July 2020 to 21st December 2021; ‘arrangement B’ in Figure 2B). Each duo or
trio of plants was considered to constitute a ‘set of residues’. After several weeks of exposure
to outdoor weather conditions, the senescent leaves and stems from each set of residues were
cut into 2 cm segments and left to dry in a laboratory atmosphere at 18 °C for one week.
2.7. Assessment of the intensity of sexual reproduction
We estimated the intensity of sexual reproduction by evaluating ascospore discharge events in
residue samples split in two series (16th and 17th January 2019, 22nd and 23rd January 2020,
and 8th and 14th January 2021). Each set of residues was weighed, soaked in water for 20 min
and spread on dry filter paper in a box (24 × 36 cm), the lid of which was left half-open to
decrease the relative humidity of the air around the residues. Eight Petri dishes (90 mm in
diameter) containing PDA medium were placed upside down 1 cm above the residues and left
in the dark at 18 °C for 18 h (Figure 2C). The dishes were then closed and incubated in the
same conditions for five days. The number of ascospore-derived colonies (Figure 2D and S1)
was estimated by eye to calculate a cumulative ascospore discharge index (ADI;
ascospores·g-
1), defined as the total number of ascospores discharged per gramme of wheat residues, which
we used as a proxy for sexual reproduction intensity (Suffert et al., 2018a, 2016, 2011).
2.8. Genotyping
We sampled 278 offspring strains from different ascospore-derived colonies obtained from
nine single inoculations (±10 strains sampled for each) and 11 co-inoculations (±20 strains
sampled for each) in 2018, 2019 and 2020. The parenthood of the offspring strains was
established by genotyping the 278 offspring strains and the 24 strains used for (co-
)inoculation and assumed to be their parents (Table S1). We used 12 SSR markers (AC0002-
ST4, AG0003-ST3A, CT0004-ST9, TCC0009-ST6, AC0001-ST7, GCA0003-ST3C, chr_02-
140-ST2, GAC0002-ST1, GGC0001-ST5, CAA0003-ST10, TCC0002-ST12, CAA0005-
ST3B) from the panel developed by Gautier et al., (2014). The genotyping profiles of the
offspring and parental strains were analysed with Peak Scanner software v2.0 (Applied
Biosystems). We then compared these profiles, using a decision tree designed specifically for
this purpose (Figure S2). The actual parental strains could be the strains used to inoculate the
corresponding plants, other strains used to inoculate other plants during the experiment
(evidence of a cross with exogenous strains from neighbouring sets of residues; ‘exo-IN’ in
Table 2), or other strains not used in the experiment (evidence of a cross with exogenous
strains from outside the experiment; ‘exo-OUT’ in Table 2). If all 12 SSR markers for a strain
matched those of the strains used for inoculation, we considered those strains to be the true
parents. However, as several of the 12 alleles are very common in natural Z. tritici
populations, the probability of one of the parents actually being exogenous was extremely low
but not zero. By contrast, if at least one of the markers in an offspring strain did not match the
strains used for inoculation, we could be sure that one of the actual parents was exogenous.
The ‘degree of exogeneity’ of the actual parental strains was estimated by considering the
status of all genotyped offspring sampled for a given (co-)inoculation. This indicator is
expressed as the percentage of offspring strains with at least one parent different from the
strains used for inoculation.
We also genotyped 11 strains sampled from late asexual lesions observed on Cellule
(pycnidiospores) after ‘avr × avr’ co-inoculations and six observed after single inoculations
with different avirulent strains, to identify the strains responsible for these lesions.
2.9. Statistical analysis
All data analyses were performed with R software (v4.0.2 R Core Team 2012). The variables
used to assess the intensity of asexual and sexual reproduction were non-normally distributed,
as revealed by Shapiro-Wilk tests (‘shapiro.test’ function). We analysed the results of co-
inoculations separately from those of single inoculations, due to a difference in the number of
replicates. We, therefore, performed Kruskal-Wallis tests (agricolae R package, ‘kruskal’
function)
for all statistical analyses concerning co-inoculations, and Wilcoxon tests (rstatix R
package, ‘wilcox_test’ function) for all statistical analyses concerning single inoculations.
Both tests were followed, when necessary, by Bonferroni corrections for pairwise
comparisons, due to the high stringency of this method (P value threshold at 0.05). We
estimated the impact on disease severity and ADI of the type of co-inoculation (‘vir × vir’,
‘avr × avr’ and ‘avr × vir’) and virulence status, for single inoculations. To this end, we first
separately assessed the effects of year (2018, 2019, 2020) and cultivar (Apache, Cellule) on
the two aforementioned variables, and checked for an absence of potential two-way
interactions between factors. We used Benjamini & Hochberg correction for pairwise
comparisons to prevent the overestimation of mean differences. For ADI, we split the data
into two groups (‘arrangement A’ in 2018; ‘arrangement B’ in 2019 and 2020). We also
compared the effects of single and co-inoculations on ADI and assessed the effect of the
proportion of the virulent strain in the biparental ‘avr × vir’ suspensions (0.1, 0.25, 0.5, 0.75
and 0.9) on ADI and disease severity.
We used a linear model to analyse the potential correlation between the intensity of asexual
multiplication and the intensity of sexual reproduction. The variance of disease severity was
partitioned into sources attributable to the following factors and their interactions: ADI, the
cultivar inoculated (Apache, Cellule), the type of co-inoculation and year. The model was
fitted by removing non-significant interactions and we used the adjusted R² to evaluate the
state of the correlation.
3. Results
3.1. Intensity of asexual multiplication resulting from single inoculations and co-
inoculations
On Apache, all single inoculations caused symptoms (Figure S3), with significant differences
in mean disease severity between strains (p < 0.001, Kruskall-Wallis test, data not shown).
Disease severity per type of co-inoculation was scored 35 to 80% in 2018 and 2019 and 30 to
50% in 2020 (Figure 3 and Table S2).
On Cellule, only single inoculations of virulent strains caused symptoms (Figure S3). We
observed only rare lesions caused by single inoculations of avirulent strains (Table S2).
Disease severity measured after ‘vir × vir’ and ‘vir × avr’ co-inoculations on Cellule was
rated from 9% to 64% (Table S2) and was, on average, lower than that on Apache (p < 0.001,
Wilcoxon test). As expected, no disease was observed on Cellule after ‘avr × avr’ co-
inoculations (Figure 3 and Figure S3).
Mean disease severity on Apache and Cellule differed significantly between years (p < 0.01
for both single inoculations and co-inoculations, Kruskal-Wallis test), which can be explained
by the difference in disease-scoring dates between years.
Unexpectedly, some rare lesions (isolated sporulating lesions) were observed late on Cellule
in 2020, eight weeks after inoculation, i.e.,
long after the main disease assessment (Figure 4).
These lesions appeared in single inoculations with three different avirulent strains (FS6196,
FS6503 and FS6191) and after ‘avr × avr’ co-inoculation. The genotyping of strains sampled
from these asexual lesions (pycnidiospores) showed that almost all (16/17) had the same
genotyping profile as one of the strains used to inoculate the plant (Table 1 and S3). The
remaining strain had the same genotyping profile as another strain used in the experiment
(FS6181) and might have resulted from of a unique contamination event in the greenhouse.
3.2. Intensity of sexual reproduction and genotyping of offspring resulting from co-
inoculations
Overall, the intensity of sexual reproduction depended on cultivar in 2018 and 2019 (p =
0.002, Wilcoxon), on year for Cellule (p < 0.001, Kruskal-Wallis test; Figure 5), and on the
type of co-inoculation, as described below for each cultivar.
On Apache, we obtained offspring strains for all type of co-inoculations (‘vir × vir’, ‘avr ×
avr’ and ‘avr × vir’) as expected, regardless of the virulence or avirulence status of parental
strains (Figure 5 and Table S4). The intensity of sexual reproduction on this cultivar was not
dependent on the virulence status of the parental strains: there were no significant differences
in mean ascospore discharge index (ADI) between the three types of co-inoculation (Figure 5
and Table S4). This is not surprising, as the virulence status of strains is relevant only for
Cellule, the cultivar carrying the corresponding resistance gene.
On Cellule, we obtained offspring strains for ‘vir × vir’ co-inoculations, as expected, but also
for ‘vir × avr’ and even for ‘avr × avr’ co-inoculations (Figure 5). The intensity of sexual
reproduction depended on the virulence status of the parental strains: ADI was higher for ‘vir
× vir’ than for ‘vir ×x avr’ and ‘avr ×x avr’ inoculations, with significant differences only in
2018 (p = 0.031, Kruskal-Wallis; Figure 5 and Table S4).
The analysis of genotyping data for offspring strains sampled from Cellule residues confirmed
that the parental strains actually involved in sexual reproduction were very probably the
strains used for inoculation (Table 2, Table S3 and Figure S2A). Few cases were identified
in which at least one of the actual parental strains was not among those used for inoculation
(mean degree of exogeneity of the parental strains: 5% for all co-inoculations). The
genotyping results demonstrate that an avirulent strain present asymptomatically on a plant
(without causing sporulating lesions five weeks after inoculation) could cross with a virulent
strain (infecting). For instance, two ‘vir × avr’ co-inoculations, FS2883 × FS2847 in 2018 and
FS6181 × FS6196 in 2020, produced offspring for which we genotyped a sample (20 and 25
strains, respectively): all alleles of the 12 SSR markers for all offspring strains matched those
of the parental strains used for co-inoculation (no exogeneity of the parental strains; Table 2).
Moreover, genotyping demonstrated that two avirulent strains present asymptomatically on a
plant — at least five to six weeks after inoculation — were also able to cross. This is the most
striking finding of this study. The ‘avr × avr’ co-inoculations, FS2851 × FS2855 and FS2849
× FS2847 in 2019 and FS6503 × FS6196 and FS6522 × FS6196 in 2020 all produced
offspring strains in which all alleles of the 12 SSR markers matched those of the parental
strains used for co-inoculation (no exogeneity of the parental strains; Table 2). The
genotyping results for the offspring strains from the ‘vir × avr’ co-inoculations FS1492 ×
FS1721 and FS1425 × FS1754 in 2018 and the ‘avr × avr’ co-inoculation FS1721 × FS1754
are consistent with the results reported above, even though the degree of exogeneity of the
parental strains was non-null (mean value of 16%; Table 2). Four of the 51 offspring strains
genotyped resulted from a cross between one of the parents used for co-inoculation and
another parental strain used in the experiment (‘exo-IN’), and three resulted from a cross
between one of the parental strains used for co-inoculation and a strain from outside the
experiment (‘exo-OUT’; Table 2). The parental strains displayed a higher degree of
exogeneity in 2018 (Table 2), probably due to the difference in the arrangement of the
residues between this and the other two years (Figure 2A and 2B).
3.3. Intensity of sexual reproduction and genotyping of offspring resulting from single
inoculations
Another key finding of this study was that offspring strains could be obtained following single
inoculations, whereas plants not inoculated in 2020 (controls) produced no offspring. At first
sight, this result is surprising, as it shows that sexual reproduction does not necessarily require
inoculation with the two parental strains such that they are present in or on (growing
epiphytically) the host tissues whilst the plant is growing. Surprising as it may seem, this
result was obtained in each of the three years (Table 2, Figure 5). The “unexpected” crosses
can be explained by the arrival of “exogenous” strains on residues (after the senescent plants
were placed outside) from neighbouring sets of residues for the strains used in the experiment
(‘exo-IN’) and from residues present in fields away from the experimental site (‘exo-OUT’).
In 2018, most of the offspring strains obtained after the inoculation of Cellule with a single
avirulent strain (FS1754) were considered to result from a cross between the strain used for
inoculation and another parental strain used in the experiment (Table S3 and Figure S2A).
This can be explained by the difference in the arrangement of the residues (A vs. B; Figure
2). This result is particularly surprising given that the avirulent parental strain used for
inoculation produced no symptoms on the plant during the asexual stage. In 2019 and 2020,
despite the distancing of residues, offspring strains were obtained after single inoculations,
whatever the cultivar or the virulence status of the strain used for inoculation. We genotyped
42 and 21 offspring strains sampled from residues of Apache after single inoculation with a
virulent or avirulent strain, respectively. We found that 36 (86%) and 18 (86%) offspring
strains, respectively, could be considered to result from a cross between the strain used for
inoculation and another parental strain used in the experiment (‘exo-IN’), and that 4 (10%)
and 2 (10%), respectively, resulted from a cross between the inoculated strain and strain from
outside the experiment (‘exo-OUT’; Table 2, Figure S2B). For the remaining cases (two and
one, respectively), the genotyping profiles of the offspring strains matched those of the strains
used for inoculation, and we cannot, therefore, conclude on the origin of the exogenous parent
(‘exo-IN’ or ‘exo-OUT’). On Cellule, 29 of the 33 offspring strains sampled after single
inoculation with a virulent strain (88%) were considered to result from a cross between the
inoculated strain and another of the parental strains used in the experiment. The remaining
four strains (12%) had the same genotyping profile as the strain inoculated, and it was not,
therefore, possible to draw any conclusions about the origin of the exogenous parent (Table 2,
Figure S2B).
The intensity of sexual reproduction was lower after single inoculations than after co-
inoculations in 2019 and 2020 (Figure 5). Support for the role of non-inoculated strains
(arriving on host tissues after the plant was fully senescent) in successful crosses is provided
by the observation that the intensity of sexual reproduction in single inoculations was
dependent on year (p = 0.056 for avirulent strains in Apache and p = 0.002 in Cellule,
Kruskal-Wallis test).
3.4. Impact of diverse factors on the intensity of sexual reproduction
Impact of environmental conditions — As highlighted above, the intensity of sexual
reproduction was significantly dependent on year, with higher values for ‘avr × avr’ co-
inoculations in Apache in 2018 (p = 0.023, Kruskal-Wallis) and for ‘vir × vir’ and ‘avr × avr’
co-inoculations in Cellule in 2018 than for the corresponding co-inoculations in 2019 and
2020 (p = 0.03, p = 0.02, respectively, Kruskal-Wallis; Table S4). This was also the case for
single inoculations in Apache and Cellule in 2018 (p = 0.011 and p = 0.002, respectively,
Kruskal-Wallis). The impact of year is consistent with the difference in the arrangement of the
sets of residues (A vs. B): “cross-contaminations” between sets of residues were frequent in
2018, when the arrangement of residues made physical contact possible, but were more
limited in 2019 and 2020, when the sets of residues were placed further apart. The intensity
of sexual reproduction was higher in field conditions in 2018 than in 2019, and this may be
attributed to differences in climatic conditions, as highlighted in a previous study (Orellana-
Torrejon et al., 2022a).
Impact of the proportions of the parental strains — The intensity of sexual reproduction
(ADI) was not significantly affected by the proportion (0.1, 0.25, 0.5, 0.75, and 0.9) of the
virulent parental strain in the three pairs of ‘avr × vir’ strains tested, whatever the wheat
cultivar, probably because of the small number of replicates and the high variability of ADI
(Table S5). ADI was higher on Apache than on Cellule (p = 0.025 Wilcoxon test), with no
significant difference between years in any cultivar, and it tended to be higher for Cellule in
2018 (p = 0.086, Kruskal-Wallis test), when ‘arrangement A’ was applied. By grouping
similar unbalanced proportions (group 1/10, combining the 0.1-0.9 and 0.9-0.1; group 1/4
combining 0.25-0.75 and 0.75-0.25), we found that the intensity of sexual reproduction after
co-inoculation in unbalanced proportions was higher than that when parental strains were
used in equal proportions for co-inoculation (0.5-0.5), whatever the cultivar. As expected,
disease severity on Apache did not differ with the proportion of the virulent parental strain
(Table S6), whereas disease severity increased significantly with the proportion of the
virulent strain on Cellule.
4. Discussion
4.1. “Unexpected” crosses of avirulent strains in plants carrying Stb16q: from
experimental findings to explanatory hypotheses
We showed that sexual reproduction between virulent (‘vir’) and avirulent (‘avr’) strains is
possible in a wheat cultivar carrying the corresponding resistance gene, here Stb16q (process
in Figure 6; see also Figure 5 and S3, Table 2). This finding is consistent with the results
obtained by Kema et al. (2018) in a similar experiment in which all Z. tritici crosses on
seedlings of a cultivar carrying Stb6 were successful even if one of the parental strains was
avirulent, with no pathogenic relationship to the wheat cultivar used. More unexpectedly, we
found that two avirulent strains could cross without causing symptoms in plants, five weeks
after their use for co-inoculation (see below). For several crossing situations (Table 2 and
Figure S2), we established by genotyping that the strains used for co-inoculation could be
considered to be the actual parents, even if the host-pathogen interaction during the asexual
stage was “incompatible”. As a means of understanding the mechanisms underlying the ‘vir’×
‘avr’ and ‘avr × avr’ crosses, we focused on the rare symptoms (Figure 4B) observed on
Cellule late after inoculation in 2020, with an origin — the strains used for inoculation —
confirmed by genotyping (Table 1). The immunity conferred by Stb16q throughout growth of
the wheat plants prevented complete infection until the appearance of lesions by limiting
pathogen progression in the substomatal cavities or the apoplast (Saintenac et al., 2021). We
hypothesize that this type of immunity ceased to operate just before plant senescence,
allowing the pathogen to colonise the host tissues more deeply in some areas of the leaf and
leading to the appearance of a few isolated lesions (Figure 4D). This observation of late
symptoms is also consistent with the epiphytic development and/or survival of Z. tritici on the
surface of leaves, as reported by Fones et al. (2017). This epiphytic development may be
explained by specific experimental conditions in the greenhouse: application of a high
concentration of inoculum on the leaf surface and favourable conditions for fungal growth
over an extended period of time. This phenomenon could however happen in the field: 3.3%
of the strains sampled on Cellule grown in mixture with Apache, from STB lesions, were
found to be avirulent against Stb16q (Orellana-Torrejon et al., 2022a). The success of ‘vir ×
avr’ crosses (process
in Figure 6) can be also explained by the hypothesis that penetration
of the avirulent strain was favoured by a virulent strain that had previously infected the host
tissues (Tollenaere et al., 2016). Facilitation mechanisms of this type have been reported in
multiple infections in some pathosystems. For example, the initial arrival of a virulent strain
of Blumeria graminis on a host plant can suppress resistance, enabling subsequently arriving
avirulent strains to penetrate host tissues (Lyngkjær et al., 2001). The possibility of infection
by “stowaway” strains through a ‘systemic induced susceptibility’ reaction was
experimentally proposed for Z. tritici by Seybold et al. (2020) and recently discussed by
Barrett et al. (2021). This hypothesis is consistent with the intensity of sexual reproduction
tending to be higher for unbalanced proportions of the two parental strains, as observed in our
study (see 4.4), which could promote the penetration of the avirulent strain. However, this
finding requires experimental confirmation and should, therefore, be interpreted with caution.
4.2. Parental strains do not necessarily need to infect their living host to reproduce
sexually: the epidemiological processes potentially involved
Debuchy et al. (2010) suggested that the physical proximity of parental strains might be
sufficient to induce the development of reproductive organs when abiotic conditions are
optimal, in a manner dependent on the pathogenic fungi concerned. The recognition of
sexually compatible strains is based on the reception of pheromones for heterothallic fungi
(Ni et al., 2011). There is, therefore, no evidence that sexual reproduction consistently
requires at least one of the two parents to infect the living host tissues until the appearance of
symptoms. The “unexpected” crossing situations highlighted in this study support this
assumption and suggest that avirulent strains may have developed on leaves, possibly
epiphytically or may have penetrated the host tissues only superficially (Fones et al., 2017;
Fantozzi et al., 2021). Microscopy studies showed that avirulent strains are able to germinate
and penetrate into resistant plants tissues (Cohen & Eyal, 1993; Shetty et al., 2003) but that
the infection process is then blocked: pathogen can growth in the apoplast and plant cells are
not penetrated
.
The resistance based on major resistance genes active against apoplastic fungal
pathogens is not actually associated with fungal death (Stotz et al., 2014), supporting the idea
that avirulent strains may survive in resistant host tissues. Saintenac et al. (2021) provided
experimental evidence of this for Stb16q: they detected fungal biomass 21 days after the
inoculation of a resistant cultivar with an avirulent strain of Z. tritici, despite the absence of
symptoms due to host-pathogen incompatibility. The results obtained by Kema et al. (2018)
for Z. tritici, focusing on Stb6, suggest that the “female” parental strain must infect the host
tissues to form maternal ascogonia: the skew in the segregation of mitochondrial SSRs (and,
thus, an unbalanced ratio for maternal or paternal parenthood) obtained after co-inoculations
showed that the avirulent parent was almost exclusively the paternal parent. This finding is
consistent with hypothetical process
in Figure 6. However, as we obtained crosses on
Cellule after single inoculations with avirulent strains (not only in 2018, but also, to a lesser
extent, in 2019 and 2020; Figure 5 and Table S4), it remains possible that ascogonia were
also formed on senescent tissue after the maternal strain had grown saprophytically (process
in Figure 6).
Our results also suggest that the second exogenous parental (probably paternal) strain arrived
later, on senescent tissues, when the set of residues had been placed outdoors, and that it grew
saprophytically before crossing. Sexual reproduction may, therefore, have been initiated
relatively late, at a stage equivalent to the interseason period in field conditions. Furthermore,
this scenario is the only one capable of explaining the crosses observed following inoculation
with a single virulent or avirulent strain (processes
and
, respectively, in Figure 6;
Figure 5 and Table 2). The exogenous parental strain may have come from: (i)
pycnidiospores transferred from neighbouring residues by rainsplash or direct physical
contact (which was possible in the 2018 arrangement), or (ii) ascospores dispersed by the
wind from field residues farther away in 2019 and 2020. Further experiments are required to
validate processes
in addition to the classical process
for each crossing situation.
For instance, the direct inoculation of residues with the second parental strain (or both
parental strains) might make it possible to demonstrate the ability of a strain to undergo sexual
reproduction after growing saprophytically, as for Phaeosphaeria nodorum (Halama &
Lacoste, 1992). Cytological analyses would be relevant, but complex to perform on senescent
tissues. The current state of knowledge and hypotheses about sexual reproduction are
consistent with the hemibiotrophic status of Z. tritici and a necrotrophic status for P.
nodorum, although the lifestyles of these function have been defined to date on the sole basis
of analyses of the asexual stage (Sanchez-Vallet et al., 2015).
4.3. Relationship between the intensity of asexual multiplication and the intensity of
sexual reproduction
We found no correlation (R2 = 0.38) between the intensity of asexual multiplication and the
intensity of sexual reproduction (Figure S3). Suffert et al. (2018a) highlighted a significant
positive correlation for disease severity scores that were lower (from 10% to 40%) than those
reported here (35% to 80% on Apache), despite the similar experimental conditions. In this
study, disease severity levels were high enough to maximize physical contact between the two
parental strains, but also to induce a decrease in the intensity of sexual reproduction (‘density-
dependent’ processes), as previously shown by Suffert et al. (2018a).
4.4. Impact of the proportions of parental strains: a trend requiring confirmation
We found no significant impact of the proportions of parental strains on the intensity of sexual
reproduction (Table S4). However, we showed, by grouping similar parental strain ratios
(1/10 and 1/4), that the intensity of sexual reproduction tended to be higher for unbalanced
proportions of parental strains. This trend, consistent with the increase in the intensity of
sexual reproduction highlighted by Suffert et al. (2016) when parental strains had latent
periods differing by a few days, requires confirmation in future experiments.
4.5. Epidemiological consequences for avirulent strains reproducing sexually on
resistant cultivars: a possibility that could be exploited to limit the breakdown of a
resistance gene?
By contrast to our findings, all crosses involving two avirulent parental strains failed in the
study by Kema et al. (2018). These divergent results may be explained by differences in the
plant stages considered: adult plants treated until natural senescence and with a long crossing
time in our study, versus seedlings and a short crossing time in the study by Kema et al.
(2018). Moreover, we found that the most classical scenario of ‘vir × vir’ crosses (process
in Figure 6) and the “unexpected” ‘vir × avr’ and ‘avr × avr’ scenarios on plants carrying a
resistance gene generated offspring populations of similar sizes. Thus, not only can avirulent
strains reproduce sexually, but they can also produce substantial numbers of ascospores. This
finding conflicts with the paradigm that avirulent strains tend to be eliminated from a
population because they do not infect the host, and cannot therefore reproduce and contribute
to subsequent epidemics, at least in agrosystems characterised by monovarietal fields. In fact,
avirulence genes can be transmitted to the next generation even in a wheat canopy considered
resistant. This can temporally affect the local evolution of the pathogen population and,
hence, the dynamics of resistance breakdown. The extent of resistance breakdown should
depend: (i) on the number of avirulent strains arriving on the cultivar carrying the resistance
gene, and (ii) the available opportunities for encountering and crossing with other strains. ‘Vir
× avr’ and ‘avr × avr’ crosses are probably rare in pure stands (the lower the frequency of
avirulent strains in the pathogen population, the rarer), but their frequency could be enhanced
by certain resistance deployment strategies. For instance, the use of cultivar mixtures would
increase the exposure of plants carrying a resistance gene to the corresponding avirulent
strains, due to the presence of neighbouring susceptible plants on which these strains would
be then able to multiply asexually. Orellana-Torrejon et al. (2022a, 2022b) highlighted a
consequence of this phenomenon in the field: the frequency of avirulent Z. tritici strains was
higher after sexual reproduction on the residues of a resistant wheat cultivar grown in a
mixture than after sexual reproduction on the residues of a pure stand, providing experimental
evidence of an “overtransmission” of avirulence to the next epidemic season. Cultivar
mixtures, therefore, appear to be a promising way to exploit the epidemiological
consequences of avirulent strains crossing on resistant cultivars for limiting the breakdown of
resistance genes.
5. Conclusion
The selection pressure exerted on pathogen populations by the deployment of a resistance
gene in fields planted with a single cultivar promotes the generalisation of virulent strains
(Brown and Tellier, 2011). Virulence is, theoretically, accompanied by a fitness cost (Burdon
and Laine, 2019), but it was thought that this advantage for avirulent strains (rarely
demonstrated; see, for instance, Orellana-Torrejon et al., 2022a) was compensated by their
inability to persist in pathogen populations over a long period of time. The experimental
findings presented here challenge partly this assumption. The hypothetical epidemiological
processes we propose can explain the diversity of possible crossing scenarios (detailed in
Figure 6) contributing to the maintenance of avirulence in Z. tritici populations. Epi-
evolutionary models incorporating sexual reproduction should take the diversity of these
scenarios into account, to improve our understanding of their impact on the transmission of
avirulence in pathogen populations (Fabre et al., 2022; Rimbaud et al., 2021), particularly
when the aim is to compare different deployment strategies for cultivar resistances at large
spatiotemporal scales.
Funding
This research was supported by a grant from the Fonds de Soutien à l'Obtention Végétale
(FSOV PERSIST project; 2019-2022) and by a PhD fellowship from the French Ministry of
Education and Research (MESR) awarded to Carolina Orellana-Torrejon for the 2018-2022
period. The BIOGER laboratory also receives support from Saclay Plant Sciences-SPS (ANR-
17-EUR-0007).
Acknowledgements
We thank Marie-Pierre Guillot (AgroParisTech BIOGER, Thiverval-Grignon, FR), Nathalie
Retout and Laurent Gerard (INRAE BIOGER, Thiverval-Grignon, FR) for technical
assistance, Dr. Cyrille Saintenac (INRAE GDEC, Clermont-Ferrand, FR), Dr. Romain Valade
(ARVALIS-Institut du Végétal, Boigneville, FR) and Dr. Marc-Henri Lebrun (INRAE
BIOGER, Thiverval-Grignon, FR) for sharing information and relevant suggestions
throughout this project. We also thank Dr. Julie Sappa for her help correcting our English.
Conflict of Interest Statement
The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.
Data Availability Statement
The raw data used for the analysis of the intensity of asexual multiplication and the intensity
of sexual reproduction of Z. tritici are available from the INRAE Dataverse online data
repository (https://data.inrae.fr/) at https://doi.org/10.15454/IPMIOX.
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Table 1. Identification of 17 Zymoseptoria tritici strains causing late lesions on wheat plants
of cv. Cellule, 8 weeks after (co-)inoculation (June 15th, 2020), based on the comparison of
their genotyping profiles with those of the 8 strains used for co-inoculation. The genotyping
profiles, established with 12 SSR markers from the panel developed by Gautier et al. (2014),
are presented in Table S3. Sampled strains that did not match the strains used for (co-
)inoculation are indicated by *.
Cv. Strains used for
(co-)inoculation
Number of
strains sampled Identity of strains
sampled
Cellule FS6503
(avr1) - 1 FS6181 (× 1) *
Cellule
FS6191 (avr)
-
4 FS6191 (× 4)
Cellule
FS6196 (avr)
-
1 FS6196 (× 1)
Cellule
FS6503 (avr)
FS6191 (avr)
3 FS6191 (× 3)
Cellule
FS6503 (avr)
FS6196 (avr)
3 FS6196 (× 3)
Cellule
FS6522 (avr)
FS6191 (avr)
2
FS6191 (× 2)
Cellule
FS6522 (avr)
FS6196 (avr)
3 FS6196 (× 3)
1 ‘avr’ for avirulent against Stb16q
Table 2. Parenthood of 278 Zymoseptoria tritici offspring strains sampled from ascospore-derived colonies obtained after nine single
inoculations and 11 co-inoculations (a subset of all the inoculations performed in the three-year experiment). Parenthood was established by
comparing the genotypic profiles of the 278 strains with those of the three sets of eight strains used for (co-)inoculation, based on the decision
tree presented in Figure S2. The genotypic profiles, established with 12 SSR markers from the panel developed by Gautier et al. (2014), are
presented in Table S3.
Type of
inoculation
Year Cv.
1 (Co-)inoculated strain(s)
2 Intensity of
asexual
multiplication 3
Intensity of
sexual
reproduction 4
Number of
genotyped
offspring
strains
Number of offspring strains per
parental origin 5 ‘Degree of
exogeneity’ of
the parental
strain(s) 6
‘inoc’ ‘exo-IN’ ‘exo-OUT’ NA
Co-
inoculation
2018
CEL FS1721 (avr) FS1754 (avr) 0.1 1034 10 7 1 2 0 30%
CEL FS1492 (vir) FS1721 (avr) 10.6 663 21 18 3 0 0 14%
CEL FS1425 (vir) FS1754 (avr) 14.7 220 20 19 0 1 0 5%
2019
CEL FS3250 (vir) FS2883 (vir) 27.8 551 20 20 0 0 0 0%
CEL FS2883 (vir) FS2847 (avr) 34.7 98 20 20 0 0 0 0%
CEL FS2851 (avr) FS2855 (avr) 0.3 224 20 20 0 0 0 0%
CEL FS2849 (avr) FS2847 (avr) 0.6 89 20 20 0 0 0 0%
2020
CEL FS6181 (vir) FS6141 (vir) 32.8 740 26 26 0 0 0 0%
CEL FS6181 (vir) FS6196 (avr) 19.4 868 25 25 0 0 0 0%
CEL FS6503 (avr) FS6196 (avr) 0.1 442 22 20 0 0 2 (0%)
CEL FS6522 (avr) FS6196 (avr) 0.0 212 24 24 0 0 0 0%
Single
inoculation
2018
CEL FS1754 (avr) - 0.0 454 10 1 9 0 0 -
2019
APA FS3098 (vir) - 21.5 407 10 0 10 0 0 -
APA FS3250 (vir) - 23.8 312 10 1 9 0 0 -
APA FS2851 (avr) - 67.1 177 10 0 8 2 0 -
CEL FS3250 (vir) - 11.5 28 10 4 6 0 0 -
2020
APA FS6181 (vir) - 44.5 613 11 0 11 0 0 -
APA FS6141 (vir) - 33.1 52 11 1 6 4 0 -
APA FS6196 (avr) - 40.4 128 11 0 10 0 1 -
CEL FS6181 (vir) - 21.7 112 23 0 23 0 0 -
1 CEL for Cellule, APA for Apache.
2 ‘vir’ for virulent against Stb16q and ‘avr’ for avirulent against Stb16q.
3 disease severity, expressed as the mean percentage of the leaf area displaying sporulation on the two uppermost leaves of adult wheat plants five weeks after
inoculation.
4 number of ascospores discharged per gramme of residues (ADI).
5 number of offspring strains for which it is certain that at least one parental strain was not among those used to inoculate the adult wheat plants in the
greenhouse, i.e. has an exogenous origin: ‘inoc’ when we considered the two parental strains to be those used for co-inoculation; ‘exo-IN’ when we considered
that at least one parental strain was another strain used in the experiment (evidence of a cross with an exogenous strain from a neighbouring sets of residues);
‘exo-OUT’ when we considered that at least one parental strain was not a strain used in the experiment (evidence of a cross with an exogenous strain from
outside the experiment); ‘NA’ for non-assigned, when it was not possible to conclude (see decision trees in Figure S2).
6 number of genotyped offspring strains / (‘exo-IN’ + ‘exo-OUT’) × 100, in parenthesis when few strains were non-assigned.
Figure 1. Design of the three-year experiment (2018, 2019, 2020) involving Zymoseptoria
tritici strains virulent (‘vir’) and avirulent (‘avr’) against Stb16q. Each adult wheat plant of cv.
Apache or Cellule received the same number of blastospores, whether inoculated with one
(eight single inoculations) or two strains (12 co-inoculations). All co-inoculations were
performed with equiproportional biparental inoculum suspensions. For one of the ‘avr × vir’
co-inoculations (FS1425 × FS1754 in 2018, FS2883 × FS2847 in 2019, FS6176 × FS6191 in
2020), four additional co-inoculations were performed with biparental suspensions in
unbalanced proportions (0.1-0.9, 0.25-0.75, 0.75-0.25, and 0.9-0.1).
FS-1441
FS-3098
FS-6181
FS-1477
FS-2881
FS-6141
FS-1492
FS-3250
FS-6152
Strains ‘vir’ (
avrStb16q
)
Single inoculations Co-inoculations
FS-1425
FS-2883
FS-6176
FS-1441
FS-3098
FS-6181
FS-1477
FS-2881
FS-6141
FS-1492
FS-3250
FS-6152
FS-1721
FS-2849
FS-6503
FS-1738
FS-2855
FS-6522
FS-1775
FS-2851
FS-6196
FS-1425
FS-2883
FS-6181
FS-1754
FS-2847
FS-6191
FS-1721
FS-2849
FS-6503
FS-1738
FS-2855
FS-6522
FS-1775
FS-2851
FS-6196
FS-1754
FS-2847
FS-6191
Strains ‘avr’ (
AvrStb16q
)
Single inoculations
Mat1.2
Mat1.1
2018
2019
2020
Figure 2. Management of sets of wheat residues, from the induction of Zymoseptoria tritici
pseudothecia to the assessment of sexual reproduction intensity. A. Dry wheat plants placed
outdoors (2018; ‘arrangement A’), which led to the residues partially falling on each other. B.
Bundles of dry wheat plants hung on a fence (2019, 2020; ‘arrangement B’), with no
possibility of direct contact. C. Petri dishes containing PDA medium placed upside down
above wheat residues to collect Z. tritici ascospores. D. Ascospore-derived colonies on PDA
at the time of counting, i.e., four days after discharge.
Figure 3. Impact of virulence status with respect to Stb16q of the Zymoseptoria tritici strains
used for co-inoculation (‘avr × avr’, ‘avr × vir’, ‘vir × vir’) on the intensity of asexual
multiplication (expressed as disease severity) and the intensity of sexual reproduction
(expressed as the number of ascospores discharged per gramme of residues; ADI) by cultivar
(Apache, Cellule) and the arrangement of the different sets of residues (‘arrangement A’ in
Figure 2A; ‘arrangement B’ in Figure 2B) during the three-year experiment. Kruskal-Wallis
tests, with Bonferroni correction for pairwise comparisons, were performed to compare the
types of co-inoculation by cultivar and arrangement.
Figure 4. Contrasted timing of lesion appearance on adult wheat plants of cv. Cellule in 2020:
five vs. eight weeks after co-inoculation. Flag leaves of Cellule were co-inoculated with either
two virulent strains (Leaf A, FS6503 × FS619) or with two avirulent strains (Leaf B, FS6176
× FS6191).
Figure 5. Impact of the type of inoculation (single inoculations, co-inoculations) on the
intensity of Zymoseptoria tritici sexual reproduction (expressed as the number of ascospores
discharged per gramme of residues; ADI), by cultivar (Apache, Cellule) and the arrangement
of the different sets of residues (‘arrangement A’ in Figure 2A; ‘arrangement B’ in Figure
2B) during the three-year experiment. Kruskal-Wallis tests, with Bonferroni correction for
pairwise comparisons, were performed to compare the types of co-inoculation by cultivar and
arrangement.
Figure 6. Epidemiological processes, including spore transfers, leading to sexual crosses on
wheat residues according to the origin of the Zymoseptoria tritici parental strains and their
virulence status, (A, B) in the conditions of the three-year experiment and (C) in field
conditions. Each crossing event is characterised by cultivar — with or without a resistant gene
R (Cellule R, Apache R) — where the cross occurs, the virulence status (‘vir’, ‘avr’) of the
parental strains, and the status of the offspring. Four crossing scenarios are proposed
according to the origin and type of growth of the parental strains:
both parents (virulent
strains) infect independently living tissues;
one parent (virulent strain, acting as the
maternal parent) infects living tissues whereas the other parent (avirulent strain) grows
asymptomatically, possibly epiphytically, penetrates living tissues superficially and growth
only in the apoplast (asymptomatic infection), or co-infects as a ‘stowaway’ taking advantage
of the infection with the other strain;
one parent (virulent strain) infects living tissues
whereas the other parent (virulent or avirulent strain) arrives later on residues from a
neighbouring set of residues (pycnidiospores; possible in A and C, but not possible in B) or
after covering a longer distance (ascospores; possible in A, B, C) and then grows
saprophytically;
one parent (avirulent strain) grows asymptomatically, possibly
epiphytically, whereas the other parent (virulent or avirulent strain) arrives later on residues
from a neighbouring set of residues (pycnidiospores; possible in A and C, but not possible in
B) or after covering a longer distance (ascospores; possible in A, B, C) and then grows
saprophytically.
parental strain#1
(vir)
parental strain#2
(avr)
offspring strains
(mixture vir + avr)
Strain that infectedliving host tissues (symptomaticasexual multiplication)
Strain that grew possibly epiphytically, penetrates living tissue s superficiall y and
growth only in the apoplast (asymptomatic inf ection), or co- infects as a ‘stowaway’
Strain that landedon host residues, i.e. alread ydead tissues (pycnidiospore
splash-dispersedfrom neighbouring residues or long-distanc e wind- di spersed
ascospore), and grew saprophytic ally
Origin of the parental strains
Example of crossing
A
R
1 avr
1 vir
R
C
R
R
?
?
?
n avr
n vir
B
R
RR
co-inoculation
natural spores
transfer
R
R
1 avr
single inoculation
R
1 avr
1 vir
co-inoculation
1 avr
single inoculation
?
Table S1. List of the 24 Zymoseptoria tritici strains used as parental strains in the three-year
experiment. Strains were sampled on wheat cv. Cellule and Apache in pure-stand field trials
located in Grignon (France) in July 2017, July 2018, and June 2019 (Orellana-Torrejon et al.,
2022a).
Year Code Mating type
1 Wheat cv.
of origin
Virulence status 2
2018 (INRA17-)FS1425 Mat1.2 Cellule vir
(INRA17-)FS1441 Mat1.2 Cellule vir
(INRA17
-
)FS1477
Mat1.1
Cellule
vir
(INRA17
-
)FS1492
Mat1.1
Cellule
vir
(INRA18-)FS1721 Mat1.2 Apache avr
(INRA18-)FS1738 Mat1.2 Apache avr
(INRA18-)FS1754 Mat1.1 Apache avr
(INRA18-)FS1775 Mat1.1 Apache avr
2019 (INRA18-)FS2883 Mat1.2 Cellule vir
(INRA18-)FS3098 Mat1.2 Cellule vir
(INRA18
-
)FS2881
Mat1.1
Cellule
vir
(INRA18
-
)FS3250
Mat1.1
Cellule
vir
(INRA18-)FS2849 Mat1.2 Apache avr
(INRA18-)FS2855 Mat1.2 Apache avr
(INRA18-)FS2847 Mat1.1 Apache avr
(INRA18-)FS2851 Mat1.1 Apache avr
2020 (INRA19-)FS6176 Mat1.2 Cellule vir
(INRA19-)FS6181 Mat1.2 Cellule vir
(INRA19
-
)FS6141
Mat1.1
Cellule
vir
(INRA19-)FS6152 Mat1.1 Cellule vir
(INRA19-)FS6503 Mat1.2 Apache avr
(INRA19-)FS6522 Mat1.2 Apache avr
(INRA19-)FS6191 Mat1.1 Apache avr
(INRA19-)FS6196 Mat1.1 Apache avr
1 determined by PCR amplification of the two mating type idiomorphs (Waalwijk et al., 2002).
2 based on the results of pathotyping on wheat seedlings, using cv. Cellule and Apache; ‘vir’ for
virulent against Stb16q and ‘avr’ for avirulent against Stb16q.
Table S2. Intensity of asexual multiplication (disease severity) assessed for each type of co-
inoculation and single inoculation in the three-year experiment. Disease severity is expressed
as the mean percent sporulating area on the two uppermost leaves of wheat plants five weeks
after inoculation. The impact of the type of co-inoculation on the intensity of asexual
multiplication, by cultivar and year (with subsequent pooling of the three years) was assessed
in Kruskal-Wallis tests with Bonferroni correction for pairwise comparisons. For single
inoculations, the impact of strain status was assessed with Wilcoxon tests. Different letters
indicate significant differences in means (p < 0.05) between statuses (columns) for a specific
year × cultivar (rows) combination.
Year Cv. Virulence status of the (co-) inoculated parental strains
vir × vir avr × vir avr × avr vir 1 avr
1
2018 Apache 79.9 a 77.1 a 61.0 b 59.8 α 72.5 α
Cellule 30.5 a 8.9 b 0.1 c 17.2 α 0.1 β
2019 Apache 56.4 a 58.4 a 64.1 a 33.8 α 48.2 β
Cellule 64.1 a 24.1 b 0.5 c 17.4 α 0.7 β
2020 Apache 35.2 a 41.4 ab 50.6 b 48.3 α 41.2 α
Cellule 38.7 a 22.9 b 0.1 c 37.2 α 0.0 β
Means
2018-2019-2020 Apache 57.2 a 59.0 a 58.6 a 47.3 α 2 54.0 α
Cellule 44.4 a 18.6 b 0.2 c 23.9 α 0.3 β
1 single inoculation
2 p = 0.06
Table S3. Genotyping data set (Excel file).
Table S4. Intensity of sexual reproduction (ADI) assessed for each type of co-inoculation and
single inoculation in the three-year experiment. ADI is expressed as the number of ascospores
discharged per gramme of residues. The impact of the type of co-inoculation on the intensity
of sexual reproduction for each cultivar and year (with subsequent pooling of the three years)
was assessed in Kruskal-Wallis tests followed by Bonferroni correction for pairwise
comparisons. For single inoculations, the impact of strain status was assessed with Wilcoxon
tests. Different letters indicate significant differences in means (p < 0.05) between statuses
(columns) for a specific year × cultivar (rows) combination.
Year Cv. Virulence status of the (co)inoculated parental strains
vir × vir avr × vir avr × avr vir 1 avr
1
2018 Apache 529 a 661 a 339 a 2292 α 864 α
Cellule 2065 a 680 b 1564 ab 1980 α 695 α
2019 Apache 493 a 491 a 1140 a 320 α 172 α
Cellule 276 a 103 a 187 a 15 α 4 α
2020 Apache 389 a 493 a 426 a 303 α 62 α
Cellule 654 a 406 a 368 a 51 α 6 α
Mean
2019-2020 Apache 441 a 492 a 783 a 312 α 117 α
Cellule 465 a 254 a 278 a 33 α 5 α
1 single inoculation
Table S5. Intensity of sexual reproduction (ADI) according to the proportion of the
Zymoseptoria tritici virulent strain in the biparental inoculum suspension (‘avr × vir’). ADI is
expressed as the number of ascospores discharged per gramme of residues of each cultivar
(Apache, Cellule). Data were obtained in 2018, 2019 and 2020 for the FS1754 × FS1425,
FS2883 × FS2847 and FS6176 × FS6191 co-inoculations, respectively. Differences in the
mean intensity of sexual reproduction (pooling of the data for the three years) in a specific
cultivar according to the proportion of the virulent parental strain were not significant (p >
0.05, Kruskal-Wallis test).
Proportion of the virulent parental strain
Cv. year 0.10 0.25 0.5 0.75 0.90
Apache 2018 638 1367 238 724 497
2019 302 59 665 318 1087
2020 1237 1340 398 273 1545
mean 726 922 434 438 1043
Cellule 2018 2043 883 375 48 1461
2019 82 3 167 111 0
2020 685 86 0 14 95
mean 937 324 181 57 518
Unbalanced ratio of parental strains
Cv. 1/10
1 1/4
2 1/2
3
Apache mean 884 680 434
Cellule mean 728 191 181
1 0.10-0.90 and 0.90-0.10
2 0.25-0.75 and 0.75-0.25
3 0.50-0.50
Table S6. Intensity of asexual multiplication (disease severity) according to the proportion of
the Zymoseptoria tritici virulent strain in the biparental suspension (‘avr × vir’). Disease
severity is expressed as the mean percentage sporulating area on the two uppermost leaves of
wheat plants five weeks after the inoculation of each cultivar (Apache, Cellule). Data were
obtained in 2018, 2019 and 2020 for the FS1754 × FS1425, FS2883 × FS2847 and FS6176 ×
FS6191 co-inoculations, respectively. Different letters indicate significant differences in the
mean intensity of asexual multiplication (pooling of the data for the three years) in a specific
cultivar according to the proportion of the virulent parental strain (p < 0.01, Kruskal-Wallis
test with Bonferroni correction for pairwise comparisons).
Proportions of virulent parental strains
Cv. year 0.10 0.25 0.5 0.75 0.90
Apache 2018 92.9 87.1 86.1 80.4 88.3
2019 34.0 25.5 45.0 43.9 35.0
2020 48.8 60.0 56.4 63.1 60.0
mean 58.6 57.5 62.5 62.5 56.2
Cellule 2018 7.5 13.8 14.7 30.8 16.7
2019 13.2 6.2 34.7 42.5 19.2
2020 19.1 30.8 36.5 31.7 45.4
mean 13.3 a 16.9 a 28.6 b 35.0 b 27.1 b
Figure S1. Clusters of Zymoseptoria tritici ascospore-derived colonies photographed seven
days after ascospore ejection. (A) Little or no contamination; a few, distant, non-coalescent
Z. tritici colonies of similar size, easy to identify and count. (B) Possible confusion between
Z. tritici colonies (see F) and white bacterial or yeast colonies (see G). (C) Invasion of the
plate by fast-growing fungi or bacteria covering all or part of the cluster of Z. tritici colonies.
(D) Coalescence of Z. tritici colonies when cluster density is very high, precluding automated
counting approaches based on image analysis. (E) High-density effect leading to different
colony diameters, hindering automated area measurement approaches based on image analysis
with software.
Figure S2. Decision trees used for investigation of the parenthood of Zymoseptoria tritici
offspring strains based on the comparison of alleles for 12 SSR markers (mi) in the cases of
(A) co-inoculation and (B) single inoculation. The color code is the one used for the
genotyping data set in Table S3.
marker
Mixture of alleles
in the well
no yes
The allele matches
with one of the two
inoculated strains
no
yes
One of the parents
may be the
inoculated strain
yes
The allele matches with one
of the otherstrainsused in
the experiment
no
Both alleles match with the
two inoculated strains
yes no
At least one allele matches
with one of the inoculated
strains
One of the parents may be
another strain used in the
experiment (‘exo-IN’ transfer)
Both alleles match
with two of the other
strains used in the
experiment
At least one allele
matches with one of
the other strains used
in the experiment
no yes
yes no
yes
no
One of the parents is not a strain used in the
experiment (‘exo-OUT’ transfer) Not relevant
indication (NA)
A
marker
Mixture of alleles
in the well
no yes
The allele matches with
the inoculated strain
yes
One of the parents
may be the
inoculated strain
The allele matches with one
of the otherstrainsused in
the experiment
no
One allele matches with
the inoculated strains
yes no
At least one allele
matches withone of
the other strains used
in the experiment
One of the parents may be
another strain used in the
experiment (‘exo-IN’
transfer)
no yes
Neither parent is one of the strains used
in the experiment (‘exo-OUT’ transfer)
Not relevant
indication (NA)
yes
yes
no
B
Figure S3. Intensity of Zymoseptoria tritici sexual reproduction (expressed as the number of
ascospores discharged per gramme of residues of the same plant; ADI) and asexual
multiplication (expressed as disease severity), by cultivar (Apache, Cellule) and type of
inoculation during the three-year crossing experiment. The number of isolates used for
inoculation (+ for single inoculations, • for co-inoculations), their virulence status with
respect to Stb16q (‘v’ for a virulent strain, ‘a’ for an avirulent strain) and the wheat cultivar
(red for Cellule, blue for Apache) are indicated for each point, with the data averaged for nine
stems (three plants). There was no correlation between the intensity of sexual reproduction
and the intensity of asexual multiplication (R² =
0.38).
avr x vir
avr x vir
avr x vir
avr x vir
avr x vir vir x vir
vir x vir
vir x vir
vir x vir
avr x vir
avr x vir
avr x vir
avr x avr
avr x avr
avr x avr
avr x avr
avr x vir
avr x vir
vir x vir
vir x vir
vir x vir
avr x vir
vir x vir
avr x vir
0
500
1000
1500
2000
2500
3000
3500
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4500
0 20406080100
A
D
I
Disease severity (%)
Cellule
Apache
2018
avr x vir
vir x vir
vir x vir
vir x vir
vir x vir
avr x vir
avr x vir
avr x vir
avr x avr
avr x avr
avr x avr
avr x avr
avr x vir
vir x vir
vir x vir
vir x vir
vir x vir
avr x vir
avr x vir avr x vir
avr x avr
avr x avr
avr x avr
avr x avr
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0 20406080100
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Disease severity (%)
2020
avr x vir
avr x avr
vir x vir
avr x avr
vir x vir
avr x vir
avr x avr
avr x vir
vir x vir
avr x vir
vir x vir
avr x avr
vir x vir vir x vir
avr x avr
avr x vir
avr x vir
avr x vir
avr x avr
vir x vir
avr x vir
avr x avr
avr x avr
vir x vir
0
500
1000
1500
0 20406080100
A
D
I
Disease severity (%)
2019
