How large and diverse are field populations of fungal plant pathogens? The case of Zymoseptoria tritici

Abstract

Pathogen populations differ in the amount of genetic diversity they contain. Populations carrying higher genetic diversity are thought to have a greater evolutionary potential than populations carrying less diversity. We used published studies to estimate the range of values associated with two critical components of genetic diversity, the number of unique pathogen genotypes and the number of spores produced during an epidemic, for the septoria tritici blotch pathogen Zymoseptoria tritici. We found that wheat fields experiencing typical levels of infection are likely to carry between 3.1 and 14.0 million pathogen genotypes per hectare and produce at least 2.1 to 9.9 trillion pycnidiospores per hectare. Given the experimentally derived mutation rate of 3 x 10‐10 substitutions per site per cell division, we estimate that between 27 and 126 million pathogen spores carrying adaptive mutations to counteract fungicides and resistant cultivars will be produced per hectare during a growing season. This suggests that most of the adaptive mutations that have been observed in Z. tritici populations can emerge through local selection from standing genetic variation that already exists within each field. The consequences of these findings for disease management strategies are discussed.

Full text

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Evolutionary Applications. 2022;15:1360–1373.wileyonlinelibrary.com/journal/eva
1 | INTRODUCTION
The evolutionary potential of pathogen populations is affected by
many factors, including the amount of genetic diversity present in
a population and the number of offspring produced during an ep-
idemic. Pathogen population size is known to impact the rates at
which genetic diversity is eroded by genetic drif t or increased by ac-
cumulation of mutations (Charlesworth, 2009; Zwar t et al., 2011) an d
is influenced by the reproductive capacity of the pathogen as quan-
tified by the basic reproductive number R0. While several empirical
studies have documented that natural epidemics are caused by plant
pathogen populations with different degrees of genotypic diversity,
very few sought to estimate the actual number of pathogen gen-
otypes colonizing a crop during a typical epidemic. Many studies
have quantified the number of offspring, usually spores, produced
in vitro by a relatively small number of strains for various species of
plant pathogens, but fewer have quantified the number of spores
produced by these species in planta (mostly in grow th chambers or
greenhouses under highly standardized environmental conditions).
Even fewer studies have estimated the number of pathogen spores
Received: 14 March 2022
|
Revised: 28 May 2022
|
Accepted: 6 June 2022
DOI : 10.1111/eva .13434
ORIGINAL ARTICLE
How large and diverse are field populations of fungal plant
pathogens? The case of Zymoseptoria tritici
Bruce A. McDonald1 | Frederic Suffert2 | Alessio Bernasconi1 |
Alexey Mikaberidze3
This is an op en acces s article unde r the terms of the Creative Commons At tribution License, which permits use, distribution and reproduction in any medium,
provide d the original wor k is properly cited.
© 2022 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.
1Plant Pat holog y Group, Instit ute of
Integrative Biology, ETH Zurich, Zurich,
Switzerland
2Université Paris- Saclay, INR AE, UR
BIOGER, Thiverval- Grignon, France
3School of Agriculture, Policy and
Development, University of Reading,
Reading, UK
Correspondence
Frederic Suffert, Université Paris- Saclay,
INRAE, UR BIOGER, Thiverval- G rignon
78850, France.
Email: frederic.suffert@inrae.fr
Abstract
Pathogen populations differ in the amount of genetic diversity they contain.
Populations carrying higher genetic diversity are thought to have a greater evolu-
tionary potential than populations carrying less diversity. We used published studies
to estimate the range of values associated with two critical components of genetic
diversity, the number of unique pathogen genotypes and the number of spores pro-
duced during an epidemic, for the septoria tritici blotch pathogen Zymoseptoria tritici.
We found that wheat fields experiencing typical levels of infection are likely to carry
between 3.1 and 14.0 million pathogen genotypes per hectare and produce at least
2.1– 9.9 trillion pycnidiospores per hectare. Given the experimentally derived muta-
tion rate of 3 × 10−10 substitutions per site per cell division, we estimate that between
27 and 126 million pathogen spores carrying adaptive mutations to counteract fun-
gicides and resistant cultivars will be produced per hectare during a growing season.
This suggests that most of the adaptive mutations that have been observed in Z. tritici
populations can emerge through local selection from standing genetic variation that
already exists within each field. The consequences of these findings for disease man-
agement strategies are discussed.
KEYWORDS
disease control, evolutionar y potential, genetic diversity, population size

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produced during natural field epidemics under naturally fluctuating
environmental conditions. Epidemics unfold across different spatial
scales, star ting from individual leaves and groups of nearby plants
to encompass entire crop canopies in affected fields and eventually
spreading across regional cultivated landscapes. Though a long- term
goal will be to integrate evolutionary processes occurring at each
spatial scale during epidemic development, in this work we con-
sider the scale of a crop canopy or an entire field to infer the conse-
quences for the population dynamics and evolutionary potential of
crop pathogens.
Spore production has been quantified for many plant pathogens,
sometimes by combining experimental and modeling approaches
(e.g., Leclerc et al., 2 019; Sache & de Vallavieille- Pope, 1995). Spore
production is a major factor that contributes to the pathogen's basic
reproduction number (R0), defined as the total number of infec-
tions arising from one newly infected individual introduced into a
healthy (disease- free) host population (van den Bosch et al., 2008),
and thereby influences the rate of epidemic increase. In some pa-
thosystems, estimates of R0 are of the order of tens (<35 in Puccinia
striiformis f. sp. tritici; Mikaberidze et al., 2016) or even hundreds
(>300 in Puccinia lagenophorae; Frantzen & van den Bosch, 2000),
highlighting the potential for rapid multiplication to form mas-
sive populations, whereas more moderate estimates are found
for the Zymoseptoria tritici– wheat pathosystem (<10; Mikaberidze
et al., 20 17). Sporulation capacity, often estimated by the number of
spores produced by a fruiting body at a single point in time, is con-
sidered to be a crucial life- history trait of a pathogen (e.g., Delmas
et al., 2016; Pariaud et al., 2009; Suffert et al., 2013). More accurate
assessments of this component take into account the depletion dy-
namics of the sporulating structures, such as the pustules of rusts
or the pycnidia of Ascomycetes, over several points in time (e.g.,
Eyal, 1971; Sache, 1997).
The heterothallic ascomycete fungus Z. tritici causing septoria
tritici blotch (STB) on wheat is a particularly relevant pathogen to
answer the question posed in our title. The development of STB
epidemics is driven by a mixed reproductive system, characterized
by both splash- dispersed asexual pycnidiospores released from
pycnidia during the wheat growing season and wind- dispersed as-
cospores released from pseudothecia, the sexual fruiting bodies
that are produced on senescent tissues and wheat residues (Singh
et al., 2021; Suffert et al., 2011). Lesions can be found in high num-
bers on living leaf tissues in wheat fields over the majority of the
growing season. Asexual fruiting bodies (pycnidia containing pycn-
idiospores) are numerous within lesions and the capacity of sporu-
lation for each fruiting body is high. The intensity of an epidemic is
determined mainly by the number of secondary infections and thus
by the total number of pycnidiospores produced. The genetic diver-
sity of a local pathogen population is high due to recurring cycles of
sexual reproduction that occurs mainly between the end of an epi-
demic season and the beginning of the subsequent growing season,
but also during the growing season (Zhan et al., 1998). The recent
discovery of thick- walled resting spores called chlamydospores in
Z. tritici (Sardinha- Francisco et al., 2 019) adds a new element to the
pathogen life cycle that can also contribute to the effective pop-
ulation size and maintenance of genetic diversity by enabling the
buildup of a chlamydospore bank in soils planted to wheat and long-
term persistence of the pathogen between crop rotations.
The population genetics and population genomics of Z. trit-
ici have been well- characterized by using transect sampling to
obtain well- annotated collections of strains coming from natu-
rally infected wheat fields distributed around the world (Boixel
et al., 2022; McDonald & Mundt, 2016), enabling comparisons
of population structure across different geographical scales.
In some cases, the same field site was sampled over several
years (El Chartouni et al., 2012; Morais et al., 2019; Oggenfuss
et al., 2021; Zhan et al., 2001), enabling comparisons of popula-
tion structure over time. The field populations were characterized
using both neutral genetic markers such as restriction fragment
length polymorphisms (RFLPs) and SSRs (Drabešová et al., 2013;
Linde et al., 2002; McDonald & Martinez, 1990) and DNA se-
quences of strongly selected genes encoding fungicide resistance
and avirulence effectors (Boukef et al., 2012; Brunner et al., 2008;
Brunner & McDonald, 2018). The main outcome of these analy-
ses was to show that field populations around the world typically
contain high levels of both gene and genotype diversity. The gene
diversit y reflects the high effective population size and the gen-
otype diversity reflects recurring cycles of sexual recombination.
Comparisons of neutral allele frequencies over different spatial
scales indicated that gene flow is high across regional scales of
hundreds of km and that populations from different continents are
very similar to each other, consistent with significant gene flow oc-
curring among continents, with the notable exception of Australia
(Jürgens et al., 2006). Measurements of changes in neutral allele
frequencies over time combined with field experiments using
mark- release- recapture experimental designs provided rough es-
timates of ef fective population size and genotype diversity at the
field scale (Zhan et al., 2001; Zhan & McDonald, 2004). But no
attempt was made until now to methodically calculate the amount
of genotype diversity and spore production occurring in naturally
infected fields.
Here we focus on the evolutionar y potential of Z. tritici popula-
tions during the mainly asexual stage of STB epidemics. We bring
together a wide variety of results from the literature to generate
estimates of the number of genot ypes found in a hec tare of wheat
experiencing a natural STB epidemic and also estimate the number
of pycnidiospores produced per hectare in fields with different lev-
els of infection. These results combine concepts and results coming
from two disciplinary fields: “Phy topathometr y” (Bock et al., 2021)
and “Population genetics” (Burdon & Laine, 2019) that are not often
brought together in plant pathology. Based on our analyses, we esti-
mate that wheat fields showing typical levels of STB infection carry
at least 3.1 million Z. tritici genotypes per hectare and produce at
least 2.1 trillion pycnidiospores that can result in 27 million adaptive
mutations per hectare. These numbers underline the tremendous
evolutionary potential of local pathogen populations and the chal-
lenges faced by breeders aiming to create durably resistant wheat

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cultivars and pesticide manufacturers aiming to avoid the emer-
gence of fungicide resistance.
2 | METHODS
In order to estimate the number of pathogen genotypes and the
number of pathogen spores existing in a field, we first need to know
the number of infected host units existing in a well- defined spatial
context. For STB, the most relevant host unit is an infected leaf and
we chose 1 ha as the most appropriate spatial scale. To estimate
the number of wheat leaves found in a square meter in a typical
wheat field, we used information provided in Lloveras et al. (2004).
According to that source, northern central European wheat farm-
ers typically aim for a planting density of approx. 200 plants per
square meter that provide between 475– 500 fertile tillers produc-
ing ears (spikes) of grain per square meter. The recommendation in
North America is also to aim for 200 plants per square meter, with
an increase of up to 300 plants per square meter if the fields are
irrigated, with a goal of achieving between 500 and 600 fertile till-
ers per square meter. In the drier Mediterranean climate, Lloveras
et al. (2004) showed that 4 00– 500 plants were an optimum plant-
ing density that would provide between 380– 500 fertile tillers per
square meter. Based on these repor ted average values, our calcula-
tions assume that a typical wheat field will contain 450 fertile tillers
per square meter. If we assume that only the top four leaves on each
tiller can be infected during the most active phase of an STB epi-
demic (we recognize that this likely underestimates the actual num-
ber of leaves), this would provide an average leaf density of 1800
per square meter, or 18 million leaves per hectare available to be
infected by STB. We will use this estimate in our general calculations
of genotypic diversity and spore production. Other estimates could
be prepared for specific cases where there are lower or higher plant-
ing densities according to the local agricultural practices.
To estimate the number of unique Z. tritici genotypes found in
a field, we used published results coming from naturally infected
wheat fields at 31 field sites in nine countries on four continents
collected over more than 30 years (Table 1) (Boukef et al., 2012;
Drabešová et al., 2013; Jürgens et al., 2006; Linde et al., 2002;
McDonald & Martinez, 199 0; Morais et al., 2019). Pathogen geno-
types were identified using neutral genetic markers, including RFLPs
and microsatellites (SSRs). In these studies, diseased leaves were
sampled from the four upper leaf layers and, in total, more than 2400
pathogen isolates were genotyped (Table 1). To estimate the average
number of unique Z. tritici genot ypes found per leaf in a naturally
infected wheat field, we investigated the relationship between the
total number of leaves and the total number of unique genotypes
found across all these studies (Figure 1). We used linear regression
with a zero intercept to estimate the slope of the line and found an
average of 0.97 ± 0.07 unique Z. tritici genotypes per leaf across the
field populations. The uncertainty represents the standard error of
the regression slope. This estimate is, however, strongly affec ted by
the sampling procedure. In most field collections, only single isola-
tion of Z. tritici was made from each leaf. But field collections made
in California, Switzerland, Israel, and Uruguay often included isola-
tions made from more than one discrete lesion on the same leaf or
from more than one pycnidium in the same lesion. In these cases,
more than one genotype per leaf was commonly found. For example,
in the collections from Uruguay and Israel, where an average of 2 or
3 lesions were sampled per leaf, respectively, there was an average
of 2 or 3 genotypes found per leaf, respectively. In one naturally
infected Swiss field, microtransects were made through 5 lesions,
with one lesion investigated per leaf, and each leaf coming from a
different tiller growing in the same 1 m2 area. In this case, 15 dif fer-
ent Z. tritici genotypes were identified among 158 isolates sampled
from the 5 lesions, giving an average of three different genotypes
per lesion (Linde et al., 2002). Our estimates of genotype diver-
sity, focused on the most active phase of disease development and
TABLE 1 Number of unique Zymoseptoria tritici genotypes found on a determined number of leaves and lesions in 30 naturally infected
wheat fields located on four different continents
Location (number of fields
sampled) Number of leaves Number of lesions Number of genotypes Reference
USA, California, Davis 19 35 22 McDonald and Martinez (1990)
USA, Oregon, Corvallis 544 544 497 Linde et al. (2002)
Switzerland (4) 280 280 3 41 Linde et al. (2002)
Israel, Nahal Oz 47 135 158 Linde et al. (2002)
USA, Texas (2) 75 75 73 Linde et al. (2002)
Argentina (3) 193 193 154 Jürgens et al. (2006)
Uruguay, Colonia 23 41 41 Jürgens et al. (2006)
Australia (4) 193 193 176 Jürgens et al. (2006)
Tunisia (5) 218 218 216 Boukef et al. (2012)
Czech Republic (7 ) 184 184 158 Drabešová et al. (2013)
France, Grignon (2) 680 680 656 Morais et al. (2019)
Tot a l 2456 2578 2492

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involving the four upper leaf layers, assumed that only one genot ype
is found on a leaf. We recognize that this is likely to underestimate
the true number of genotypes because it is common to find more
than one discrete STB lesion on a leaf and more than one genotype
within a lesion, especially in fields experiencing moderate to high
levels of infection.
To estimate the number of spores produced in a single Z. trit-
ici pycnidium, we used published results coming from different re-
search groups over many years. All calculations were obtained from
leaves infected under greenhouse conditions, in some cases from
pycnidia loc ated on the second or third leaves of seedling plants (e.g.,
Stewart et al., 2018), and in other cases from pycnidia located on
the top leaves of adult plants (e.g., Suffert et al., 2013). The earliest
work (Eyal, 1971) marked individual pycnidia and carefully pipetted
the entire mass of spores emerging as a cirrhus from each pycnid-
ium over several wetting and drying periods to estimate the total
number of spores coming from individual pycnidia over time. Most
methods relied on counting the number of pycnidia in a group of leaf
lesions (distinct or coalescing, depending on the STB severity) found
on a whole leaf either manually (Gough, 1978; Stewart et al., 2018)
or by using automated image analysis (e.g., Yates et al., 2 019), and
then washing all spores off of that leaf after placing it into a humid
environment that led to extrusion of cirri from all mature pycnidia.
Another approach took into account the overall capacity of all
pycnidia on a leaf to produce spores over a defined period of time,
by washing spores from a leaf over several cycles of spore produc-
tion (Boixel, 2020, p. 229; Suffert et al., 2013). Individual studies
found differences in spore production among different Z. tritici
strains (Gough, 1978; Stewart et al., 2018; Suffert et al., 2013) and
among dif ferent wheat cultivars (Eyal, 1971; Gough, 1978; Hess &
Shaner, 1987; Suffert et al., 2013). The total number of measure-
ments made and the total numbers of pycnidia included in the mea-
surements also differed widely among studies (Table 2). All told, data
were obtained from 16 different cultivars that differed in their de-
gree of STB resistance and included at least 45 strains of Z. tritici that
differed in their overall reproductive capacity. The average values
reported in different studies ranged from 830 to 11,500 spores per
pycnidium. The mean value over all studies, weighted according to
the number of measurements made (but excluding the values from
Hess & Shaner, 1987 because they did not report the number of
measurements) was 5322 spores per pycnidium.
Estimates of the number of pycnidia found on a leaf came from
only one research group that relied mainly on automated image
analysis to identify and count pycnidia (e.g., Stewart et al., 2016;
Stewart & McDonald, 2014). These data were obtained from nat-
urally infected leaves, with many coalescing lesions in cases of high
STB severity, which made it impractical to estimate the number of
pycnidia per lesion induced by a single genotype. Automated image
analysis has been used in this research group for 10 years to identify
and count Z. tritici pycnidia in the framework of experiments seeking
to quantif y differences in virulence and reproduction among strains
of Z. tritici (Dut ta, Croll, et al., 2021; Dutta, Hartmann, et al., 2021;
Stewart et al., 2018) and differences in quantitative STB resis-
tance among wheat cultivars (Karisto et al., 2018; Mikaberidze &
McDonald, 2020; Stewart et al., 2016; Yates et al., 2 019). These ex-
periments were conducted mainly using seedling plants grown under
controlled greenhouse conditions (e.g., Stewart & McDonald, 2014),
but several more recent experiments collected large datasets from
naturally infected leaves of adult plant s grown under field condi-
tions (Karisto et al., 2018; Mikaberidze & McDonald, 2020; Stewart
et al., 2016). Estimates of pycnidia found within isolated lesions (a
condition closer to what is observed in the field when STB sever-
ity is low) obtained by Morais et al. (2015) from flag leaves of adult
plants inoculated with a low concentration of ascospores, showed
that a lesion, whose size ranged from 10 to 20 mm2 (typically 1– 2 mm
wide × 10– 20 mm long) contains approx. 20 to 40 pycnidia. However,
we used here only the numbers of pycnidia found on naturally in-
fected leaves taken from adult plants grown under natural field
conditions. The numbers shown in Table 3 used automated image
analysis to count a total of 2.95 million pycnidia on a total of 22,395
naturally infected leaves growing under field conditions over 2 years
and in two locations (Karisto et al., 2018; Stewart et al., 2016). The
weighted mean of 132 pycnidia per leaf was used for the calculations
of spore production.
The leaf area exhibiting disease symptoms, measured at differ-
ent spatial scales that can range from individual leaves or plants to
entire crop canopies, is often used to estimate the intensit y of a
field epidemic, typically producing measures of disease incidence
and/or severity. We estimated the percentage of diseased wheat
leaves in natural STB epidemics characterized by low, moderate,
FIGURE 1 The number of unique Zymoseptoria tritici genotypes
identified in naturally infected wheat fields versus the total number
of wheat leaves sampled in each of the studies (reported in Table 1).
Red points show the outcomes of individual locations. The blue
line represents the linear regression with zero intercept, where we
estimated the slope as 0.97 ± 0.07

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and high levels of disease intensity in order to cover a wide array
of epidemic situations. We used datasets collected during 10 years
(2008– 2017) of field experiments conducted in Grignon, France.
The experiments had three treatments corresponding to three
different tillage and crop residue management practices repre-
sentative of those practiced in the Paris basin, one of the main
wheat- producing areas in Europe (Suffert et al., 2018; Suffert &
Sache, 2011). The datasets included measurements of disease in-
cidence (the percentage of leaves carrying at least one STB lesion)
and severity (the percentage of leaf area covered by STB lesions)
for the three or four top leaf layers of wheat cv. Soissons (moder-
ately susceptible to STB and among the most popular wheat culti-
vars in France from 1990 to 2015). These assessments were made
several times during each growing season. The measurements
were conducted visually on whole plant s sampled in the field (25
plants per plot from 2008 to 2011 and 15 plants per plot from
2012 to 2017). For the analyses in the present study, we selected
one date per year during the most active phase of disease devel-
opment (centered around May: from late April to early June; see
Table S1). Selecting the final dates for assessment in each season
(i.e., late June to early July) would lead to higher estimates of the
final incidence and severity, but the pathogen population size at
that stage would no longer be relevant for the further epidemic
development during the same season. At earlier dates the epi-
demic development was more vulnerable to unfavorable weather
conditions and measurements may not accurately represent the
epidemiologically relevant pathogen population sizes. Mean val-
ues of incidence and severity were calculated with measurements
averaged over the three different treatments and leaf layers.
These values are shown in Figure 2. By conducting K- mean clus-
tering on these data, we found 18%, 44%, and 83% STB incidence
to represent the percentage of diseased leaves found in fields
with low, moderate, and high STB epidemic severity, respectively.
These measures of disease incidence were used in the calculations
of genotype diversity and spore production.
To estimate the number of pycnidiospores per pycnidium, we
used several datasets (Table 2). First, we computed the weighted
global mean:
where
ai
is the mean value in experiment i,
ni
is the number of mea-
surements conducted in experiment i,
ne
is the number of experiments,
and N is the total number of measurements in all experiments. This
way of computing global means incorporates the fact that different ex-
periments had different numbers of measurements, and hence exper-
iments with higher numbers of measurements should contribute more
to the global mean values. However, we could not characterize the un-
certainty of this estimate, because measures of uncer tainty were not
available for all studies summarized in Table 2.
To characterize the uncer tainty of the estimate of the number of
pycnidiospores per pycnidium, we used the two most comprehensive
studies (Bernasconi and Zala; Suffert and Riahi El Kamel; Table 2), for
(1)
a
=
1
N∑n
e
i=1niai
,
TABLE 2 Average number of pycnidiospores produced within a Zymoseptoria tritici pycnidium
Number of Z. tritici
strains tested
Number of wheat
cultivars tested
Number of pycnidia
tested
Range of spore outputs per
pycnidium
Average spores per pycnidium
(number of measurements) Reference
UNK 2~60 – 75 2956– 4092 ~3600 (60– 75) Eyal (1971)
2 2 23,502 4000– 22,000 ~11,50 0 (12) Gough (1978)
UNK 4UNK 6 0 – 14 0 0 ~830 (UNK ) Hess and Shaner (1987 )
17 110, 742 24– 26,000 ~2600 (90) Stewart et al. (2018)
4 2 76, 509 1020– 27,000 6780 (192) Bernasconi and Zala, this paper (Table S2)
4 4 ~60,000 2363– 9 216a5450 (128) Suffert et al. (2013)
18 1142, 016 2 8 0 7– 9 3 5 9 b527 7 (90 ) Suffert and Riahi El Kamel, this paper (Table S2)
Weighted meanc5322
Regression sloped5020 ± 440
Abbreviation: UNK, unknown.
aObtained from flag leaves of adult wheat plants under controlled conditions; spores were collected on a weekly basis over 7 weeks, and the cumulative number of spores was divided by the final number
of pycnidia.
bObtained from flag leaves of adult wheat plants in controlled conditions; spores were collected once, and pycnidia were counted 6 weeks after inoculation.
cWeighted by the number of measurements, computed using Equation (1).
dCalculated via linear regression (R2 = 0.61) based on raw data from the two most comprehensive studies (Bernasconi and Zala; Suf fert and Riahi El Kamel; data in Table S2). The uncer taint y represents the
standard error of regression slope.

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McDONAL D et AL.
which the raw data is also repor ted (Table S2). Based on these raw
data, we conducted linear regression with a zero intercept using the
number of pycnidia per leaf as an independent variable and the num-
ber of spores per leaf as a dependent variable. As a result, we esti-
mated the number of pycnidiospores per pycnidium as the regression
slope and we characterized it s uncer taint y using the standard error
of regression slope.
We computed global means over a number of different ex-
periments to estimate the number of pycnidia per leaf (Table 3) as
weighted means according to Equation (1). Next, we computed the
standard error of this estimate as:
where
𝜎i
is the standard error of the mean in the experiment i.
Based on our estimates of numbers of spores, we also esti-
mated the numbers of adapted Z. tritici mutants produced during
low, moderate and high epidemic years per hectare of the wheat
field. For Z. tritici, a generation begins when a pycnidiospore lands
on a plant, continues when the lesion resulting from successful
infection by that spore has produced oozing cirri filled with new
pycnidiospores and ends when a newly produced pycnidiospore
has landed on a new plant. To obtain the estimates, we thus con-
sidered that the pathogen population undergoes k nonoverlapping
rounds of asexual reproduction (or generations) and estimated the
number of spores nk produced after k generations using the geo-
metric growth model:
where n0 is the initial number of spores, R0 is the basic reproduction
number of the pathoge n, and nk is the number of sp ores produced af ter
k generations.
The number of adapted mutant s generated at kth round of re-
production is then
where u is the mutation rate per cell division per site, nsite s is the num-
ber of sites at which adaptive mutations can occur, and ncd is the num-
ber of cell divisions per generation. To calculate the total number of
adapted mutant spores Nmut,k produced in the course of k generations,
we compute the sum of nmut,k over k
To estimate the number of adapted mutant spores we set the
number of generations to k = 3 (at the lower boundary of a typical
epidemic year that can include up to six asexual generations), use a
conservative estimate of the basic reproduction number of Z. tritici
R0 = 4 (Mikaberidze et al., 2 017), a mutation rate per cell division
per site of u = 3 × 10−10 (Habig et al., 2021), and assume that there
are 100 cell divisions per generation (ncd = 100). To characterize the
uncertainty in the Nmut,k estimate, we propagated the standard error
associated with the estimate of the number of spores n0.
Data analyses were conducted using the Python programming
language (v. 3.8.5). We conducted linear regressions using “linear_
model.LinearRegression” class in the sklearn package (v. 0.23.2). We
quantified the correlation between the STB severity and incidence
using Spearman's correlation coefficient (“spearmanr” function in
the scipy.stats package v. 1.5.2). We conducted K- mean clustering
of the severity/incidence dataset to characterize different degrees
(2)
𝜎
=
1
N√∑n
e
i=1ni𝜎2
i
,
(3)
nk
=n
0
R
k
0,
nmut,
k=un
sites
n
cd
nk=un
sites
n
cd
n
0
R
k
0,
(4)
N
mut,k=∑
k
i=0
nmut,k=unsitesncd n0∑
k
i=0
R
k
0
TABLE 3 Number of Zymoseptoria tritici pycnidia found per leaf in natural field infections using automated image analysis
Location, year
Number of wheat
lines analyzed
Number of
leaves analyzed
Range in pycnidia
number per leaf
Mean number of pycnidia
per leaf (±standard error) Reference
Switzerland, 2015 39 733 40– 326 148 ± 11 Stewart et al. (2016)
Oregon, 2015 9240 5 2– 147 1 6 41 ± 172 Stewart et al. (2016)
Switzerland, 2016 t1334 10,269 3– 30 4 62 ± 3 Karisto et al. (2018)
Switzerland, 2016 t2335 11,1 53 17– 840 185 ± 7 Kar isto et al. (2018)
Weighted meana132 ± 4
aWeighted by the number of leaves in each experiment, compute d using Equation (1), its st andard error was computed using Equation (2).
FIGURE 2 STB severity versus incidence measured in the field
over 10 consecutive years (2008– 2017; Suffert & Sache, 2011;
Suffert et al., 2018). Mean values over field assessments in each
year are shown as small circles (for dates and values, see Table S1).
Different colors correspond to three clusters obtained using K-
mean clustering: Low epidemics (blue), moderate epidemics (green),
and high epidemics (orange). Large circles show mean values within
each cluster

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of STB epidemics (“Kmeans” function with three clusters in sklearn.
cluster package v. 0.23.2).
3 | RESULTS
3.1 | How many unique pathogen genotypes exist
per hectare?
The STB assessments made during the most active phase of disease
development from each of 10 years of field data from INRA Grignon
allowed us to characterize the relationship between STB disease
severity and the incidence of diseased leaves in natural epidemics.
This relationship was close to linear and the two quantities exhibited
a strong correlation (rSp = 0.84, p = 2.2 × 10−3; Figure 2). Based on
these new experimental findings, we assume the following typical
values of STB severity and incidence corresponding to three classes
of epidemics: low, moderate, and high (with values corresponding to
the large circles in Figure 2 showing means within the three clusters).
In a low epidemic year (on average 2% of the leaf area covered by
lesions; blue points), 18% of the leaves in an infected field had at
least one STB lesion, while in a moderate epidemic year (12% of the
leaf area covered by lesions; green points) 4 4% of the leaves were
diseased, and in a high epidemic year (20% of the leaf area covered
by lesions; orange points) 83% of the leaves were diseased.
Given the new finding that each leaf is infected by a minimum
average of one unique Z. tritici genot ype in a naturally infected field
(Table 1, Figure 1), and assuming that there are 18 million leaves
per hectare, these rates of infection predict 3.1 ± 0.2, 7.6 ± 0.6 and
14.0 ± 1.0 million Z. tritici genotypes per hectare will be found in
fields with low, moderate and high levels of infection, respectively.
The uncer tainties represent standard errors that stem from the
standard error of the regression slope in Figure 1. We consider it
worthwhile to mention some caveats at this point. On the one hand,
these numbers may over- estimate the true number of genotypes be-
cause the lesions present on the four upper leaves result from multi-
ple cycles of asexual reproduction. The number of cycles of asexual
reproduction occurring during an epidemic is estimated to range
from 3– 6 according to local climatic conditions (Karisto et al., 2018;
Lovell et al., 2004). On the other hand, these numbers may under-
estimate the true number of genotypes because there can be more
than four leaves per tiller, an infected leaf often contains more than
one discrete lesion and the majority of lesions contain more than one
genotype (Table 1).
3.2 | How many spores are produced per hectare?
The number of pycnidia forming on a leaf will be affected by the
degree of host resistance, the reproductive capacity of the strain
infecting each leaf, and the microenvironment in the leaf during
the infection process. Using automated image analysis, it was pos-
sible to count large numbers of pycnidia on individual leaves of
seedlings infected by particular pathogen strains under standard-
ized greenhouse conditions. A previous experiment found that the
average numbers of pycnidia per leaf following inoculation of 12
host genot ypes by 145 global strains of Z. tritici (encompassing
approx. 11,000 leaves) ranged from 81 to 377 on the three most
susceptible cultivars and from 7 to 10 on the three most resist-
ant cultivars tested under highly controlled greenhouse conditions
(Dutta, Croll, et al., 2021; Dutta, Har tmann, et al., 2021). It is more
challenging to count pycnidia on naturally infected leaves growing
under variable field conditions, but these data are available from
field sites in Switzerland and Oregon, where measurements were
made from 335 and 9 host genotypes, respectively (Table 3). In
the Oregon field experiment, no fungicides were used and con-
ducive environmental conditions enabled a high intensity of STB
to develop, leading to an average of 641 pycnidia detected per
leaf across 240 leaves. In the 2 years of the Swiss experiments,
environmental conditions favored the development of STB, but
heavy fungicide applications suppressed the overall levels of STB,
leading to a low- moderate level of infection overall, with an aver-
age disease rating at the final time point (approximately GS80) of
only 2.5 on a 1– 9 assessment scale. In these three leaf collections
(22,155 leaves total), an average of 132 ± 4 pycnidia were identi-
fied per leaf. The uncer tainty represents the standard error of the
mean according to Equation (2).
If we assume that only 18% of the upper four leaves are dis-
eased in a low epidemic year, and that only 132 ± 4 pycnidia form,
on average, on these leaves, then we expect to find about 0.4 bil-
lion pycnidia per hectare. In a moderate epidemic year with 44% of
leaves diseased, we expect to find approx. 1.0 billion pycnidia, and in
a high epidemic year with 83% of leaves diseased we expect to find
approx. 2.0 billion pycnidia per hectare. We anticipate that in some
high epidemic years, more pycnidia will form on average on each leaf,
as indicated by the 641 pycnidia found per leaf in the Oregon field
trial. If we combine a high incidence of disease (83%) with a high
number of pycnidia per leaf (641), then we could expect to find up to
9.6 billion pycnidia forming per hectare.
Under the assumption that an average of 5020 ± 440 spores/
pycnidium spores are produced per pycnidium, we estimate that
approx. 2.1 ± 0.3 × 1012 spores per hectare are produced in a low
infection year (2% severity, 18% incidence), approx. 5.3 ± 0.6 × 1012
spores per hectare are produced in a moderate infection year (12%
severity, 44% incidence), and 9.9 ± 1.0 × 1012 spores per hectare are
produced in a high infection year (20% severity, 83% incidence). The
uncertainties represent standard errors that stem from the standard
error of the regression slopes of the number of spores versus the
number of leaves and the standard error in the number of pycnidia
per leaf. We note here that these should be considered average val-
ues of spore production that could be expected across a wide range
of host cultivars, pathogen strains, and environmental conditions.
If we combine the higher number of pycnidia found per leaf in
Oregon (641) with the highest average number of spores found per
pycnidium (11,500, Gough, 1978) and assume that 83% of leaves
are diseased, we arrive at 1014 spores per hectare. If we use the

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McDONAL D et AL.
highest reported values for each factor, with 97% of leaves in-
fected (i.e., 17.5 × 106 leaves per hectare), 1471 pycnidia per leaf,
and 27,000 spores produced per pycnidium, we arrive at an upper
estimate of approx. 7 × 1014 spores per hectare. Given these last
two calculations, we consider it possible that 1– 7 × 1014 spores per
hectare may be produced on a susceptible cultivar grown without
fungicide treatments in an STB- conducive environment (i.e., in a
very wet year).
3.3 | How many spores carry adaptive mutations
per hectare?
We use our estimates of the overall number of spores produced
per hectare of wheat to estimate the number of Z. tritici spores that
carry mutations potentially conferring adaptation to control meas-
ures such as fungicides or disease- resistant cultivars. We consider
pathogen adaptation to azole fungicides targeting the sterol 14α-
demethylase cytochrome P450 encoded by the Cyp 51 gene (Cools
& Fraaije, 2013) and to STB resistance encoded by the Stb6 resist-
ance gene (Saintenac et al., 2018) in wheat as prime examples in our
estimates. A number of mutations in the Cyp51 gene of Z. tritici are
known to confer varying degrees of resistance to azole fungicides
(Cools et al., 2011). At least 130 different haplotypes of the C yp51
gene were identified encoding various combinations of 30 amino
acid substitutions (Huf et al., 2018). Similarly, a number of muta-
tions in Avr Stb6 allow the pathogen to avoid recognition by the Stb6
gene product, thereby conferring an adaptive advantage (Brunner &
McDonald, 2018; Stephens et al., 2021). At least 44 different AvrStb6
haplotypes were identified that encode various combinations of >60
amino acid substitutions (Stephens et al., 2021). For both genes,
however, different mutations are likely to confer different quantit a-
tive phenotypes due to the complex shape of the associated fitness
landscapes (Cools et al., 2013). To take this into account, we assume
that adaptive point mutations can occur at only five nucleotide posi-
tions in each of the two genes.
The number of adapted mutant spores was estimated using the
geometric growth model (Equation 3). According to Equation (4), we
estimated the total number of adapted mutant spores of Z. tritici pro-
duced during three generations that correspond to the decisive, final
period of epidemic development as 2.7 ± 0.3 × 107 (low epidemic
year), 6.7 ± 0.8 × 107 (moderate epidemic year) and 12.6 ± 1.5 × 107
(high epidemic year). The uncertainties represent standard errors
that stem from the st andard error associated with the estimate of
the number of pycnidiospores per hectare obtained above. These
numbers should be considered conservative estimates because
Z. tritici is a multicellular fungus and the number of mitotic cell divi-
sions occurring during its development in planta that contribute to
mutations in spores can be much higher than 100, but it is difficult to
estimate the total number of cell divisions occurring from the time a
germinating spore successfully infects a leaf until a resulting pycnid-
ium produces a new generation of spores. Hence the ac tual number
of mutant spores may be considerably higher.
4 | DISCUSSION
According to our estimates, a typical wheat field infected by Z. tritici
contains between 3– 14 million unique Z. tritici genotypes per hec-
tare, with an average of at least one unique pathogen genotype per
diseased leaf. We estimate that this population will produce a mini-
mum of between 2– 10 trillion asexual spores per hectare. It is widely
accepted that many plant pathogens produce large numbers of prop-
agules during an epidemic and that many pathogen populations can
carry a large amount of genetic diversity, but we are not aware of
any other robust quantitative estimates for these parameters in the
peer- reviewed literature. To obtain these estimates (summarized in
Table 4), we brought together a number of datasets collected over
many years across a range of geographically diverse locations. These
numbers highlight the remarkably high degree of genotype diversity
and spore production associated with natural STB epidemics, and
can be used to inform our understanding of pathogen adaptation
to fungicides, disease resistance genes, and changing environments.
4.1 | The range of estimates indicates very high
values for each parameter
We sought to obtain “average” values for each parameter used in our
calculations, though we recognize that a wide range of values can
be ascribed to each parameter due to differences in local conditions
(e.g., in degrees of host susceptibility, pathogen virulence, and con-
duciveness of environmental conditions). We believe that hectares
and annual cropping seasons are the most reasonable spatial and
temporal units for measurements of genetic diversity and spore pro-
duction in an agricultural pathogen because wheat is typically grown
as annual monocultures in fields that cover a large sur face area.
Wheat is grown globally, with different densities of leaves re-
flecting differences in local growing conditions. We chose an aver-
age value of 18 million leaves per hectare for all of our calculations,
but our approach can readily accommodate higher or lower planting
densities to prepare locally relevant estimates. Our assumptions of
the proportion of diseased leaves in a field (incidence) came from
10 years of field data collected from only one cultivar growing in only
one location. We recognize the added value of obtaining comparable
estimates of incidence from a wider array of cultivars and growing
environments and hope that these data will become available in fu-
ture. If they already are available for some locations, our approach
can easily accommodate differences in disease incidence. Our esti-
mates of the number of pycnidia formed on naturally infected leaves
were obtained using a single technology (automated image analysis)
and included leaves coming from only two locations and 2 years.
Though we recognize the limitations inherent in these datasets, we
think the estimates are reasonable given that they included counts
of nearly 3 million pycnidia coming from a wide array of host and
pathogen genotypes. The average value used in our calculations also
fell within the range of estimates coming from greenhouse experi-
ments conducted under highly controlled conditions using 12 host

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lines and 145 global pathogen genotypes (Dutta, Croll, et al., 2021;
Dutta, Hartmann, et al., 2021), increasing our confidence in using
this value. Our estimates of the number of spores produced per
pycnidium were based on several dif ferent approaches and were ob-
tained from different research groups over many years. Though the
range of reported values was quite wide, we feel reasonably confi-
dent that our estimate of 5020 ± 440 reflects the typical number of
pycnidiospores per pycnidium likely to be found across a wide vari-
ety of cultivars, pathogen strains, and field environments.
We brought together many studies of genotype diversit y based
on >2400 genotyped Z. tritici individuals collected in naturally in-
fected wheat fields from around the world (Table 1). Based on these
analyses, we estimated that a t ypical field infected by Z. tritici car-
ries between 3.1– 14.0 million unique Z. tritici strains per hectare. We
consider these to be conservative estimates. Actual numbers could
be 2– 3 times higher given that most studies of genotype diversity
included only one strain per leaf, while studies that included more
than one strain per leaf typically reported more than one unique
genotype per diseased leaf (Jürgens et al., 2006; Linde et al., 2002).
Bringing together the average values of leaf density, STB inci-
dence, pycnidia per leaf, and spores per pycnidium allowed us to
estimate an average number of Z. tritici pycnidiospores likely to be
produced during an epidemic under low, moderate, and high disease
intensities. Our calculations suggest that a typical field infected by
STB will produce between 2.1 and 9.9 trillion pycnidiospores per
hectare, though a heavily infected field may produce as many as
700 trillion spores per hectare. It is important to note here that only
a small fraction of these spores will ac tually contribute to each infec-
tion cycle because a great majority of the generated propagules fail
to cause new infections for a variety of reasons, e.g., the spores do
not land on the host tissues, are degraded or nonviable, fail to pen-
etrate the epidermis, etc. But these spores contain a vast reservoir
of mutations on which selection may operate as described below.
4.2 | What is the source of the high genotype
diversity found in Z. tritici populations?
To answer this question we can draw on recent, det ailed analyses of
whole- genome sequences that were conducted in Z. tritici (Hartmann
et al., 2017; Plissonneau et al., 2018; Singh et al., 2021). Among 107
strains from a global collection, about 0.78 million high- confidence
single- nucleotide polymorphisms (SNPs) were detected with an
average of 20 SNPs per kilobase (Hartmann et al., 2017). Among 177
isolates collected from the same wheat field in Switzerland, about
1.5 million high- confidence SNPs were detected with an average of
37 SNPs per kilobase (Singh et al., 2021). While SNPs seem to domi-
nate the genetic variation in Z. tritici populations, structural poly-
morphisms resulting from chromosomal rearrangements (Croll et al.,
2013; Goodwin et al., 2011) and gene gain/loss (Badet et al., 2020;
Hartmann & Croll, 2017; Plissonneau et al., 2018) also make substan-
tial contributions to genotype diversity. Considered together, these
studies suggest that a substantial portion of the genetic diversity
observed in Z. tritici populations results from random mutations that
produce polymorphisms within local populations. These mutations
can persist within local populations as a result of the large population
sizes associated with the estimates of spore production presented
here and the high effective population sizes estimated in earlier stud-
ies (Singh et al., 2021; Zhan et al., 2001; Zhan & McDonald, 2004).
Sexual recombination does not influence nucleotide diversity,
but it does increase genotype diversity via the formation of new
combinations of alleles of different genes and through intragenic
recombination. Z. tritici has a mixed reproductive system (Chen &
McDonald, 1996; Zhan et al., 1998): within- season epidemics are
driven by multiple rounds of asexual reproduction via pycnidio-
spores, while the initial inoculum in the following season is domi-
nated by sexual ascospores (Shaw & Royle, 1989; Suffert et al., 2011,
2018; Suffert & Sache, 2011). Despite a likely bottleneck in patho-
gen population size in the absence of living wheat plants between
wheat growing seasons, the transition from one season to the next
is expected to re- establish high levels of genotype diversity because
ascospores resulting from sexual recombination initiate the STB
epidemic each season. Additional factors that contribute to high
genoty pe diversity are new cycles of ascospore p roduction with in in-
fected fields during the growing season (Zhan et al., 1998), high gene
flow among fields (Boeger et al., 1993 ) enabled by long- distance dis-
persal of the sexual ascospores (Shaw & Royle, 1989), and the patho-
gen's ability to persist on infected wheat debris between growing
seasons (McDonald & Mundt, 2016; Morais et al., 2016; Suffert &
Sache, 2011). The recent discovery that Z. tritici forms chlamydo-
spores suggests an additional contribution from a long- lived spore
bank in the soil that may serve as a long- term reser voir of genetic
diversity (Sardinha- Francisco et al., 20 19). Thus, the high genotype
diversit y found in Z. tritici populations is consistent with the current
knowledge of the population biology and population genomics of
this pathogen.
Levels of disease intensity
Low Moderate High
Number of unique Z. tritici genotypes per
hectare (in millions)
3.1 ± 0.2 7.6 ± 0.6 14.0 ± 1.0
Number of Z. tritici pycnidiospores per hectare
(in trillions)
2.1 ± 0.3 5.3 ± 0.6 9. 9 ± 1.0
Number of adapted mutant pycnidiospores of
Z. tritici per hectare (in millions)
27 ± 3 67 ± 8 126 ± 15
TABLE 4 Summary of estimates
characterizing population biology of
Zymoseptoria tritici. All numbers represent
values per hectare of wheat field. The
uncertainties represent associated
standard errors

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McDONAL D et AL.
4.3 | How do Z. tritici populations maintain high
genotype diversity under strong selection?
In wild plant pathosystems, local genetic and environmental hetero-
geneity of host plant populations contributes to the maintenance of
genetic diversity in the corresponding pathogen populations (Laine
et al., 2011). In contrast, agricultural ecosystems such as those
planted to wheat are typically grown in large, environmentally uni-
form fields consisting of genetically identical hosts. These wheat
fields are often planted with disease- resistant cultivars and sprayed
with fungicides to control diseases. Hence, we might expect that the
uniform environment coupled with strong selective pressures will
rapidly diminish the degree of genetic diversity found in Z. tritici pop-
ulations. How do Z. tritici populations maintain such a high degree of
genotype diversity over time and space under such highly selective
conditions?
Fungicides and disease- resistant wheat cultivars are not always
expected to impose directional selection that will diminish genetic
diversit y in pathogen populations. In cases when a large and con-
stant selective advantage is conferred by a single mutation, the
selection is indeed direc tional (e.g., resistance to quinone outside
inhibitor fungicides; Estep et al., 2015; Torriani et al., 2009). In con-
trast, when pathogen adaptation occurs via the accumulation of
several mutations, with each having a moderate and/or additive
effect, selection can generate more complex and variable fitness
landscapes associated with individual mutations and their combi-
nations. E xamples include the adaptation of Z. tritici to azole fungi-
cides (Estep et al., 2015) and to the disease resistance gene Stb6 in
wheat (Brunner & McDonald, 2018): both the azole target site gene
Cy p51 and the avirulence gene AvrStb6 exhibit substantial polymor-
phisms in Z. tritici, with population genetic analyses providing strong
evidence of intragenic recombination and diversifying selection
(Brunner & McDonald, 2018; Estep et al., 2015). An analysis that
included only recently collected Z. tritici populations found evidence
for directional selection favoring a particular AvrStb6 allele with a
global distribution that likely emerged through convergent evolution
in different populations. The same study showed that high levels of
nucleotide and amino acid diversity were maintained for this gene in
most sampled populations (Stephens et al., 2021). Additional selec-
tive factors that may maintain genotype diversity in Z. tritici include
tradeoffs among life- history traits (Dutta, Croll, et al., 2021; Dutta,
Hartmann, et al., 2021) and differential fitness of strains when they
co- infect and co- exist within the same leaf (Barrett et al., 2021;
Linde et al., 2002).
4.4 | Overall implications for pathogen adaptation
While the total number of spores produced during an epidemic
has significant consequences for epidemic development, more
important for long- term evolutionary processes is the effective
size of a pathogen population and the ability of this population
to generate genotypes that are especially well- adapted to the
local environment, which may include resistant hosts and fungi-
cides. Our estimates of the number of different genotypes include
those resulting from sexual reproduction occurring between and
within growing seasons within regions and those resulting from
mutations that occur within fields. As the number of genotypes
in a field increases, the probability that a genotype occurs that
is able to overcome an STB control strategy implemented in that
field (e.g., deployment of fungicides or resistant cultivars) also
increases. Paradoxically, as the total number of genotypes in-
creases, it becomes more difficult for a particular genotype to
become dominant. The consequences of the pathogen population
diversit y for local adapt ation also depend on the ability of adapted
genotypes to persist over time (i.e., across asexual and/or sexual
generations) and to be transmit ted over larger spatial scales (i.e.,
acro ss an en tire fie ld and/or be twe en fie lds). A well- ada pted gen o-
type must first multiply locally and then be transmitted over a long
distance in order to make a significant contribution to an epidemic.
Then, it must persist between growing seasons to contribute to fu-
ture epidemics. These processes are considered in the remainder
of this section.
The high genetic diversity found in wheat fields infected by
Z. tritici is expected to drive a field population's adaptation to local
control measures and environmental conditions. Using the example
of population adaptation to azole fungicides and the Stb6 resistance
gene in wheat, if we assume three generations of pathogen repro-
duction during the final phase of a cropping season, we estimate that
there will be 27, 67, and 126 million adapted mutant spores appear-
ing in every hectare experiencing low, moderate, and high epidemic
intensity, respectively. Can these adapted mutants be maintained
in a field population as standing genetic variation even in the ab-
sence of a specific selection pressure (i.e., deployment of an azole
fungicide or a wheat cultivar with the Stb6 resistance gene), or are
they more likely to emerge de novo following the deployment of the
selection pressure? To address this question, we would need to es-
timate the proportion of mut ant spores that land on wheat leaves
and are able to cause sporulating lesions. These estimates can then
be incorporated into a population dynamical mathematical model
that takes into account demographic stochasticity and the associ-
ated probability of stochastic extinction. However, even before such
models are developed, these numbers (between 27 and 126 million
adapted mutants per hectare per season) appear to be high enough
to allow the adapted mutants to escape stochastic extinction and
to be maintained in any field population even in the absence of the
specific control measure. The mechanism of such maintenance can
be mutation- drift equilibrium if the mutants are neutral or mutation-
selection equilibrium in case the mutations are slightly deleterious
in the absence of the control measure. These considerations lead
us to conclude that resistance to fungicides and virulence to major
resistance genes are likely to emerge from standing genetic variation
in the majority of wheat fields.
We expect that any mutants conferring fungicide resistance or
virulence against a given resistance gene will first be selected during
the growing season and increase in frequency in treated fields via

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mostly asexual reproduction. At the next stage, the selected strains
will undergo mating and sexual recombination with a diverse pool of
wild- type or mutated pathogen individuals and thereby embed the
selected mutant alleles into diverse genetic backgrounds. These reg-
ular cycles of recombination make the adapted pathogen subpopu-
lation carrying the mutation more robust with respect to changing
environmental conditions and enable the long- term persistence of
the adapted mutation. In a final step, any fitness penalties associated
with the mutant alleles are likely to be ameliorated through compen-
satory mutations occurring either in the same gene or in unlinked
genes that c an be brought together by recombination.
4.5 | Implications for field- scale strategies of
disease management
Current epidemiological/evolutionary models that inform the man-
agement of fungal diseases of crops, including STB, typically consider
only two pathogen strains, a wild- type and an adapted strain (e.g.,
a fungicide- resistant or a virulent strain) that reproduce asexually
(e.g., Lo Iacono et al., 2013; Mikaberidze et al., 2014; van den Bosch
& Gilligan, 2008). One conclusion from these models that is relevant
to STB management is that in cultivar mixtures consisting of disease-
resistant and disease- susceptible cultivars, higher proportions of a
resistant cultivar make the mixture more durable with respect to the
breakdown of host resistance genes (Lo Iacono et al., 2013; Stam &
McDonald, 2018). Furthermore, studies that specifically parameter-
ized these modeling approaches to Z. tritici populations concluded
that lower fungicide doses slow down the emergence of fungicide
resistance (Hobbelen et al., 2014; Mikaberidze et al., 2017 ), and
that fungicide mixtures are typically more durable with respect to
pathogen evolution compared with fungicide alternations when a
high- risk fungicide is mixed with a low- risk fungicide (Dooley et al.,
2015; Elderfield et al., 2018). However, as we argue above, the high
standing genetic diversity in Z. tritici field populations combined
with large local populations and abundant sexual recombination
represents key unconsidered factors that are likely to affect the
emergence and persistence of adapted pathogen strains. If adapted
mutants indeed pre- exist in local populations as par t of standing ge-
netic variation, this would alleviate the detrimental role of stochastic
extinctions in the emergence of adapted pathogen strains (Lo Iacono
et al., 2013; Mikaberidze et al., 2017). Moreover, as we explained
above, reshuffling of alleles via sexual recombination would embed
the adapted mutants into diverse genetic backgrounds, making them
more robust with respect to environmental stochasticity, which may
constitute a key factor in their long- term persistence. These insights
call for a reconsideration of mathematic al modeling approaches that
currently inform management of STB, because incorporating the
above factors may put the current conclusions into question.
Our findings also raise questions regarding the strategy of plant-
ing mixtures of wheat cultivars carrying different Stb resistance
genes (Kristoffersen et al., 2020) to manage STB. We suggest that
increasing the diversity of host populations at the field scale can
have both beneficial and deleterious impacts in the presence of such
highly diverse pathogen populations. For example, consider a field
planted with a mixture of a susceptible wheat cultivar and a resistant
cultivar carrying a fully effective Stb resistance gene with properties
like Stb6. A mutation occurring in the larger pathogen population ex-
isting on the susceptible cultivar could confer virulence on the Stb
gene found in the resistant cultivar. In a homogeneous population of
susceptible cultivars, this mutant virulence allele exists as a stand-
ing genetic variation but would be unlikely to increase in frequency
because it would not provide a competitive advantage. But in a mix-
ture with the resistant cultivar, spores carrying this mutant allele are
more likely to land on the nearby resistant cultivar where they can
cause infection and multiply in the absence of competition from the
more common avirulent strains found on the susceptible cultivar.
In this way, cultivar mixtures can drive the emergence of new viru-
lence alleles and facilitate the process of breaking resistance genes.
In contrast, in a resistant pure stand, the number of new virulent
mutant strains is expected to be lower because the total number of
spores produced will be lower, but when virulent mutants emerge
or are introduced into the field through gene flow, they will undergo
stronger selection than in a mixture and will likely increase in fre-
quency more rapidly in the pure stand.
However, cultivar mixtures can still effectively control STB and
limit the loss in effectiveness of a resistance gene— in opposition to
what we just described— when these mixtures are composed of cul-
tivars carrying already broken Stb resistance genes (i.e., Stb genes
whose corresponding virulence mutations are already present but
do not yet occur at a high frequency in the pathogen population). A
recent field experiment suggested that a mixture can maintain the
efficacy of the resistance encoded by St b16 q through a decrease in
the frequency of virulent strains infecting the susceptible cultivar
and an increase in the frequency of avirulent strains occurring on the
cultivar carrying S tb16 q in the mixtures compared to pure stands.
The observed changes resulted (i) on the one hand from virulence
selection/counter- selection driven by exchanges of splash- dispersed
asexual spores between cultivars depending on their respective pro-
portions in the mixture (Orellana- Torrejon, Vidal, Boixel, et al., 2022),
and (ii) on the other hand from sexual reproduction between virulent
strains and avirulent strains that land on the cultivar carrying Stb16 q
and then recombine with virulent strains without the need to infect
host tissues (Orellana- Torrejon, Vidal, Saint- Jean, & Suffert, 2022).
This mechanism that explains the persistence (or even a slight in-
crease) of avirulent strains in mixtures was experimentally estab-
lished by Orellana- Tor re jon, Vidal, Gaze au, et al . (2022), who showed
that symptomatic asexual infection is not required for a strain to
engage in sexual reproduction [a similar finding was also reported
for the Stb6- AvrStb6 interaction (Kema et al., 2018)]. While cultivar
mixtures can thus be an effective strategy to extend the useful life
of already- defeated resistance genes or to enable the recycling of
defeated resistance genes, we should be aware, as previously stated,
that cultivar mixtures including fully susceptible cultivars may also
facilitate the emergence of virulent mutants that can overcome
newly- deployed resistance genes. Following this line of reasoning,

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McDONAL D et AL.
the benefit of mixtures versus pure stands would depend on the
frequency of virulent strains in a local population, which in turn re-
flect s the commercial life cycle of the corresponding varieties. The
optimum strategy to formulate cultivar mixtures may be to mix vari-
eties carrying quantitative resistance with varieties containing bro-
ken qualitative resistance (e.g., Stb6) while avoiding the use of fully
susceptible varieties (to improve effic acy) and the introduction of
new varieties containing unbroken qualitative resistance (to improve
durability).
ACKNOWLEDGMENTS
Marcello Zala and Ons Riahi El Kamel assisted AB and FS, respec-
tively, with generating the two previously unpublished spore count
dataset s shown in Table S2. AM and FS thanks Daniel Croll and
Carolina Orellana- Torrejon, respectively, for fruitful discussions.
CONFLICT OF INTEREST
The authors have no competing interest s to declare.
DATA AVAIL ABI LIT Y STAT EME NT
The data that support the findings of this study are available in
Supporting Information.
ORCID
Bruce A. McDonald https://orcid.org/0000-0002-5332-2172
Frederic Suffert https://orcid.org/0000-0001-6969-3878
Alessio Bernasconi https://orcid.org/0000-0003-3833-288X
Alexey Mikaberidze https://orcid.org/0000-0003-2278-2288
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SUPPORTING INFORMATION
Additional supporting information can be found online in the
Supporting Information section at the end of this article.
How to cite this article: McDonald, B. A., Suffert, F.,
Bernasconi, A., & Mikaberidze, A. (2022). How large and
diverse are field populations of fungal plant pathogens? The
case of Zymoseptoria tritici. Evolutionary Applications, 15,
1360–1373. https://doi. or g/10.1111/eva.134 34