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Review
Evolutionary and
Epidemiological Implications
of Multiple Infection in Plants
Charlotte Tollenaere,
1,2
Hanna Susi,
3
and Anna-Liisa Laine
3,
*
Recent methodological advances have uncovered tremendous microbial
diversity cohabiting in the same host plant, and many of these microbes cause
disease. In this review we highlight how the presence of other pathogen
species, or other pathogen genotypes, within a plant can affect key compo-
nents of host–pathogen interactions: (i) within-plant virulence and pathogen
accumulation, through direct and host-mediated mechanisms; (ii) evolutionary
trajectories of pathogen populations, through virulence evolution, generation
of novel genetic combinations, and maintenance of genetic diversity; and (iii)
disease dynamics, with multiple infection likely to render epidemics more
devastating. The major future challenges are to couple a community ecology
approach with a molecular investigation of the mechanisms operating under
coinfection and to evaluate the evolution and effectiveness of resistance within
a coinfection framework.
Why Does Coinfection Matter?
During the growing season, plants in both agro- and natural ecosystems are likely to
encounter a myriad of microbes, many of which are pathogenic. As a consequence, a
pathogen strain entering a host plant will encounter not only the host's defenses but also
a number of other microbial species or genotypes within the plant[5_TD$DIFF] phytobiome (the entire
microbial community associated with the various plant compartments including the rhizo-
sphere, phyllosphere, and endophytic compartments, following the American Phytopatho-
logical Society). This diversity can fundamentally change the ability of a parasite strain to
establish and grow on its host and dynamics under multiple infection have been suggested to
be a major force driving pathogen evolution [1]. This has sparked a growing interest in the
epidemiological and evolutionary consequences of coinfection in humans [2,3] and wild
animal populations (e.g., [4,5]). By contrast, the impact of coinfection on disease dynamics
and pathogen evolution in plant pathosystems has received comparatively less attention, with
the significant exception of virus–virus interactions (see, for example, [6,7] and [8,9] for
reviews). Plant pathologists have traditionally focused on two-way interactions within the
‘single host–single pathogen’framework [10] and plant resistance is mostly considered in a
‘unique pathogen genotype’framework. However, we are just beginning to understand the
far-reaching consequences of the microbial diversity associated with plants. In this review we
show how sensitive epidemiological and evolutionary dynamics of pathogens are to coinfec-
tion. We consider the methodology and current knowledge regarding coinfection levels in
natural and agricultural plant systems and review the within-host mechanisms that mediate
interactions between pathogen strains or species. We also consider the consequences, in
terms of both the epidemiology and the evolution of the pathogen populations, highlighting
the current challenges in [17_TD$DIFF]this field.
Trends
Molecular tools are becoming readily
available for the study of parasites.
These technical advances have shown
that multiple infection is common in the
wild and in agriculture, with the same
host individual simultaneously infected
by several pathogen genotypes or spe-
cies (i.e., coinfection).
Under coinfection, pathogens may
interact either directly (mechanical or
chemical interactions) or indirectly
through host resources or defenses.
These direct or host-mediated interac-
tions under coinfection can change
virulence, within-host pathogen accu-
mulation, and transmission.
Such plant-leveleffects can also be seen
at the population level, with coinfection
rendering epidemics more devastating.
Coinfection may drive the evolutionary
trajectories of pathogen populations
through its effects on virulence evolu-
tion and on the generation and main-
tenance of genetic diversity in
pathogen populations.
1
[1_TD$DIFF]Interactions Plantes–Microorganismes
et Environnement (IPME), Institut de
Recherches pour le Développement
(IRD) –Cirad –Université de
Montpellier,[2_TD$DIFF] 34394 Montpellier, France
2
Laboratoire Mixte International[4_TD$DIFF] Patho-
Bios, IRD-INERA (Institut de
l’Environnement et de Recherches
Agricoles), BP171, Bobo-Dioulasso,
Burkina Faso
3
Metapopulation Research Centre,
Department of Biosciences, PO Box
65 (Viikinkaari 1), FI-00014 University
of Helsinki, Helsinki, Finland
*Correspondence:
anna-liisa.laine@helsinki.fi (A.-L. Laine).
TRPLSC 1360 No. of Pages 11
Trends in Plant Science, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.tplants.2015.10.014 1
© 2015 Elsevier Ltd. All rights reserved.
TRPLSC 1360 No. of Pages 11
Levels of Coinfection in Natural and Agricultural Plant Systems
The study of coinfection was for a long time hindered by methodological constraints, as
symptom-based detection is rarely a reliable means of identifying coinfection. Hence, molecular
or serological markers are generally a mandatory tool for the study of multiple infection (Box 1).
As these tools are becoming available for a wider range of pathogens and next-generation
sequencing opens new possibilities for the characterization of microbial communities, it is
becoming increasingly clear that coinfection is common in both wild plants and agricultural
crops and for both systemic and local diseases [11–14]. Levels of coinfection can be very high in
some pathosystems. For example, multiple infection was found in a majority (16 of 21) of weed
species tested for five viruses [15]. Up to 76% of plants were found to be infected by more than
one pathogen genotype of the barley scald pathogen Rhynchosporium secalis [16] and up to 16
distinct genotypes were detected within a single lesion [18_TD$DIFF]of a Eucalyptus leaf-infecting fungus,
Teratosphaeria nubilosa [12]. In the Central African Republic, cassava was frequently affected by
cassava mosaic disease (CMD) (85% incidence), with up to 58% of diseased plants infected by
various virulent geminiviruses [13]. In addition, high spatiotemporal variability in coinfection levels
was found when investigated, with, for example, 20–100% (average 35.3%) of infected plants
harboring more than one of the six tested viruses within six Arabidopsis thaliana Spanish wild
populations followed over 4 years [17].
Both plant genetics and environmental variation can shape the prevalence of coinfection in
space and time. Host population resistance is a key determinant of pathogen population and
community structure, with plant resistance genes determining whether a pathogen is capable of
infecting a given host genotype (at one extreme gene-for-gene interactions, but also true for
resistance controlled by multiple loci). As a consequence, a mismatch between host resistance
and pathogen infectivity profiles may prevent coexistence. Host community structure is also
likely to impact coinfection, as revealed by the negative relationship between [19_TD$DIFF]plant [20_TD$DIFF]diversity and
[21_TD$DIFF]coinfection [22_TD$DIFF]levels found in grassland experimental plots [18]. In wild Plantago populations in
the Aland archipelago, coinfection by multiple genotypes was found in approximately half of the
populations infected by the powdery mildew Podosphaera plantaginis [11]. In this system, the
prevalence of coinfection was higher in well-connected pathogen populations [19], suggesting
that the dispersal rate among pathogen populations may be an important determinant of the
multiplicity of infections. Moreover, host genotype was a key determinant of coinfection in
common garden populations of Plantago lanceolata, with the lowest levels of coinfection
Box 1. Detecting and Quantifying Pathogens for the Study of Coinfection
The study of coinfection requires: (i) distinguishing coinfection from single infection; (ii) distinguishing coinfecting
[15_TD$DIFF]genotypes/species from each other; and (iii) quantifying each coinfecting pathogen. Given that pathogens are typically
small and clonally reproducing, morphological identification is often impossible and symptom expression can be different
under coinfection than under single infection (see, for example, [85], where unexpected symptoms are observed in dual
viral infection), so that molecular or serological tools are required in most cases (but see [86]). One commonly used
method to detect coinfection in a field sample comprises [16_TD$DIFF]purification of several pathogen strains/spec ies from a single
plant (e.g., [77]). Genetic characterization can then be performed using various molecular markers, such as micro-
satellites [12,22], or restriction length polymorphisms [77].
Alternatively, when researchers are interested only in the presence/absence of coinfection, molecular work can be
performed directly on the sample without the purification step. Multipathogen molecular detection methods that rely on
species- or genotype-specific primers have been developed in a few pathosystems (e.g., [87]). For haploid species,
coinfection may be inferred from SNP genotyping [11]. In addition, recent next-generation sequencing technology allows
the characterization of entire microbial communities [88,89] and consequently the determination of coinfection levels
within each pathogen group (viral, bacterial, or fungal).
Finally, the prevalence of the different strains forming the coinfection can be quantified using quantitative PCR methods.
These have been developed for a few pathogens, such as Phytophthora infestans [75], as well as some viruses (e.g.,
[90,91]). High-throughput sequencing has been used to monitor viruses [92], while genetic engineering and the use of
fluorescent proteins allow the visualization of competition of genotypes in real time [93].
2Trends in Plant Science, Month Year, Vol. xx, No. yy
TRPLSC 1360 No. of Pages 11
detected in experimental populations supporting high accumulation of resistance genes [20].
Finally, environmental conditions may also play a key role in determining coinfection levels, as
suggested by the relationship between latitude and the prevalence of coinfection of four barley
and cereal yellow dwarf viruses (B/CYDVs) in three host grass species across 26 natural
grasslands [21].
Measuring nonrandom associations between pathogen species/strains from field data has been
useful for assessing species interactions in animal parasitology and will also prove useful for plant
pathologists in understanding how coinfections are formed. There are statistical tools available
for estimating the distribution of single and multiple infection compared with random expecta-
tions (see for example [15,22]). Investigating three clover-infecting viral species revealed non-
independent distributions within host populations, with alfalfa mosaic virus (AMV) occurring most
often together with either clover yellow vein virus (ClYVV) or clover mosaic virus (ClMV), while the
two latter species did not co-occur [23]. The probability of contracting bacterial infection has
been shown to be lower in virus-infected than in healthy gourds (Cucurbita) in central Penn-
sylvania [24,25]. In this pathosystem, lower coinfection than expected by chance was generated
by the bacterial beetle vector that preferred healthy plants [26]. Various studies have revealed
positive associations between various viruses [15,27] and vector preference and abundance
have been invoked as underlying mechanisms [27].
Together the available data reveal that levels of coinfection are variable in space and time and
that levels of coinfection can be high. Additional robust field data across different spatiotemporal
scales are required to understand the factors determining levels of coinfection in
agroecosystems.
Overall Virulence and Pathogen Accumulation Driven by the Multiplicity of
Infection
Diseaseimpactontheplant(ordecreaseinhostfitness due to the infection, referred to here as
virulence) may be affected by the number of pathogens present, so that the damage caused
by a single pathogen often differs when the genotype/species is alone compared with multiple
infection [1]. In humans, the health consequences of coinfection are generally expected to be
more negative than those generated by single infections [28]. Mixed results have been
obtained in plants, with studies reporting both milder and more severe symptoms under
coinfection in an unpredictable manner. For plant viruses, stronger symptoms under coinfec-
tion (higher overall virulence) have frequently been observed, with the classic example of
experimental evidence of synergism between the potyviruses potato virus Y (PVY) and potato
virus X (PVX) [6] or the more severe symptoms observed for cassava plants coinfected by
various geminiviruses causing CMD in the field [13], but antagonisms have also been reported
(for a review see [8]). By contrast, milder symptoms (lower overall virulence) have been
obtained when two genotypes of Mycosphaerella graminicola simultaneously infect wheat
compared with single-genotype infection [29]. Similarly, a reduction in disease severity was
found for stem rust (Puccinia graminis) of barley when coinfected with powdery mildew
(Erysiphe graminis)[30] and viral infection delays the progression of a fatal bacterial wilt
infection in gourd plants [31].
Such a modification of overall virulence is often associated with a change in terms of pathogen
accumulation (i.e., within-host pathogen load) and various examples show the drastic impact the
presence of a second pathogen may have on pathogen replication rate (up to ten-times increase
in the PVX concentration when coinfected with PVY compared with a single infection [6]).
However, overall virulence and pathogen accumulation are not always coupled [32], with, for
example, cucurbit yellow stunting disorder virus (CYSDV) [23_TD$DIFF]negatively [24_TD$DIFF]affects [25_TD$DIFF]zucchini yellow[26_TD$DIFF]
mosaic virus ([27_TD$DIFF]ZYMV) [28_TD$DIFF]titer and[8_TD$DIFF] symptoms [29_TD$DIFF]while [30_TD$DIFF]the presence of [31_TD$DIFF]cucumber [32_TD$DIFF]vein [33_TD$DIFF]yellow virus
Trends in Plant Science, Month Year, Vol. xx, No. yy 3
TRPLSC 1360 No. of Pages 11
([34_TD$DIFF]CVYV) [35_TD$DIFF]positively [36_TD$DIFF]affects [37_TD$DIFF]CYSDV [38_TD$DIFF]titer [17_TD$DIFF]with [39_TD$DIFF]no [40_TD$DIFF]effect on cucumber [41_TD$DIFF]symptoms [33].In planta
quantification of pathogen accumulation is required for a comprehensive picture of the effect one
pathogen may have on another (see Box 1 for methodological considerations).
Mechanisms of Within-Plant Interactions between Pathogen Strains or
Species
Pathogen species or genotypes sharing a common host may interact in a direct or indirect
manner or both simultaneously [34] (Figure 1). Direct pathogen–pathogen interactions may be
detected through in vitro assays and have revealed that hyphae from the fungus Didymella are
able to transport bacterial isolates from four different species coinfecting the Styrian oil pumpkin
[35]. Frequent co-occurrence of the fungus and bacteria in the field suggest that such
mechanical facilitation observed in vitro contributes to this emerging multipathogen disease
[35]. Positive or negative direct interactions may also be due to: (i) the production of molecules
affecting other pathogens positively (e.g., siderophores facilitating host exploitation [36])or
negatively (e.g., interference competition through the secretion of toxins that can be targeted to
their non-related competitors [37]); or (ii) interactions between respective proteins of the two
protagonists [38].
Species occupying different host tissues or organs may also interact, with synergism between a
phloem-limited virus and a virus invading non-phloem tissue [39] or a decrease in root-knot
nematode multiplication with leaf inoculation by soybean mosaic virus [40]. Such indirect
interactions may be mediated either by the host immune system (top down) or via the attribution
of host resources (bottom up) (Figure 1). Resource-mediated competition negatively affects the
cohabiting pathogens, especially when resources are highly limiting [41], while host immune
Direct interacon
Facilitaon
Trade-offs between
antagonisc
defense
pathways
Systemic
Acquired
Resistance
(SAR)
Inducon
of local
response
Interference
compeon
+
–
Pathogen counter-aack
strategy
Host-mediated interacon
+
+
–
–
–Exploitaon
compeon for
host resources
Figure 1. Mechanisms of Within-Plant Interactions between Pathogen Strains or Species. Pathogen–pathogen
interactions within a shared plant may be direct (A), host mediated (B), or a combination of the two. The interactions can
either positively (+) or negatively () affect interacting species/genotyp es. Direct pathogen–pathogen interactions (A) include
facilitation and interference competition, whereby the growth, reproduction, or transmission of competitors is modified
either chemically or mechanically. Indirect interactions (B) are mediated by the shared host plant when the pathogens
compete for the same limited resources (exploitation competition, ‘bottom-up’interaction) or when the competitor is
affected by the induction or inhibition of host defense responses by the other pathogen (‘top-down’interaction). In direct
interactions, the strains are in physical contact (local interactions), while host-mediated interactions may occur when the
strains are in contact or occupy different parts of the host plant (systemic and local interactions).
4Trends in Plant Science, Month Year, Vol. xx, No. yy
TRPLSC 1360 No. of Pages 11
system-mediated interactions may be either positive or negative, with, for example, the effect of
microbial induction of the hypersensitive response (HR) on subsequent infections [42]. The
disease synergism involving potyviruses requires suppression of adaptive defense responses
against viruses (gene-silencing mechanisms [43,44]). Trade-offs between plant-defense antag-
onistic pathways may also mediate pathogen–pathogen interactions [10]. For example, the
biotrophic Pseudomonas syringae, which induces salicylic acid (SA)-mediated defense, ren-
dered plants more susceptible to the necrotrophic pathogen Alternaria brassicicola by sup-
pression of the jasmonic acid (JA) signaling pathway [45]. Initial infection by P. syringae also
induces the production of a structural mimic of JA in systemic tissues that counteracts SA
induction and consequently facilitates further infections with the bacterium (systemic induced
susceptibility [46]). Induction of SA can mediate systemic acquired resistance (SAR) following
primary pathogen infection, conferring long-lasting resistance to subsequent attacks [47].A
complex crosstalk between phytohormones induced by pathogens with different lifestyles [48] is
thus involved in the context of multiple attack and the impact of belowground microbes on
aboveground resistance [40,49] is likely to be mediated by both shared resources and hormonal
response.
Dissection of molecular mechanisms involved in pathogen–pathogen interactions is a challeng-
ing task as the molecular dialog between one host and one pathogen is already hard to
elucidate. It should be noted, however, that understanding within-host interactions may also
contribute new insights into the molecular mechanisms of single host–single pathogen inter-
actions. For example, the spatiotemporal pattern of pathogen attack may affect the outcome of
pathogen–pathogen interactions (Box 2) in a manner that can strengthen our understanding of
the underlying mechanisms. In humans, a summary network of parasite interactions revealed
that bottom-up (shared resources) interactions are the most important form of interaction
determining the outcome of coinfection [28]. Also, diet was found to affect parasite interactions
in animals [50,51]. Few investigations manipulating nutrient supply in coinfection experiments
have been conducted in plants to date, but one study found that nitrogen supply decreased the
strength of antagonistic interaction between two viruses in Avena [41]. As not only parasites but
also the immune system depend on host resources, these three components of the pathos-
ystem interact to drive the outcome under multiple infection [51].
Box 2. The Effect of Arrival Sequence on the Outcome of Multiple Infection
Studies following the dynamics of pathogen interactions resulting from sequential or simultaneous coinoculation have
revealed that the outcome critically depends on spatiotemporal patterns of infection ([94–97], but see [98]). This is also
the case for one of the first described synergisms between two phytoviruses: the accumulation of potato virus X (PVX)
and potato virus Y (PVY) was greater when they arrived simultaneously on their host but when the arrival of the other virus
was delayed by more than 24 h the synergistic effect on accumulation was lost [99]. Another example comes from an
experimental study of sequential and simultaneous coinfection by two fungal wheat pathogens, Puccinia triticina and
Pyrenophora tritici-repentis. Compared with single infection, Pyrenophora was stronger and Puccinia produced fewer
spores in coinfection but, interestingly Pyrenophora produced even more spores if Puccinia was inoculated after
Pyrenophora [97].
The mechanisms underlying pathogen–pathogen interactions may explain such effects of arrival sequence on the
outcome of infection. Host resources may be more easily exploited by the first-arriving pathogen, so that the ‘first-come,
first-served’principle may occur in sequential inoculations of pathogens competing for host resources [86,97]. Also,
induced resistance, whereby a first pathogen stimulates host defenses negatively impacting subsequent pathogen
attacks, is highly species specific in terms of both duration and spatial scale. For example, the reaction induced by the
oomycete pathogen Albugo candida is not only local but systemic and was also proved to be durable in the later-
emerging leaves [100]. By contrast, in sequential inoculations with different powdery mildew genotypes on a very fine
spatial scale of single cells, an avirulent genotype of Oidium neolycopersici arriving first may trigger a hypersensitive
reaction against later-arriving virulent genotypes [96], while a virulent genotype of Blumeria graminis arriving first can
suppress resistance and allow the penetration of avirulent genotypes [101]. Finally, in viruses, this phenome non has been
called ‘cross-protection’[8] and historically was discovered in tobacco mosaic virus where prior infection by a mild strain
delayed the multiplication of a severe strain inoculated afterward in a vaccine-like manner [102].
Trends in Plant Science, Month Year, Vol. xx, No. yy 5
TRPLSC 1360 No. of Pages 11
How Coinfection Changes Epidemiological Dynamics
Transmission is often directly or indirectly related to within-host accumulation, so that the
previously described effect of coinfection on within-host multiplication is also expected to
impact between-host transmission [52]. A drastic example of such an effect of coinfection
on phytopathogen transmission comes from umbraviruses, which lack genetic information for
the capsid protein required for vector transmission and can be transmitted only in coinfection
with a suitable virus from the family Luteoviridae [53]. More generally, some viruses facilitate the
transmission of others, in a phenomenon called helper dependence [8].
The effects of coinfection on epidemiology have been studied in human and animal diseases,
with intuitive expectations formalized by modeling [54] and investigated through field surveys
[4,5,55], but such investigations in plants remain scarce. However, such studies are needed
because the effect of coinfection on virulence (plant symptoms) and pathogen accumulation
(and consequently transmission) may have opposite consequences for the coexistence of
obligate pathogens. Under certain conditions, increased virulence decreases the probability
of coexistence[11_TD$DIFF] ([12_TD$DIFF]1[42_TD$DIFF]), whereas increased transmission increases this opportunity. The net effect of
coinfection on disease epidemiology would thus depend on the relative effect on overall virulence
and transmission of each protagonist (as the effect on each species/genotype is not necessarily
the same) as well as on the initial state of the pathosystem [7]. Consequently, modeling each
particular case is required, even for simple cases where transmission is linked to within-host
accumulation because these two components are likely to have nonlinear and different propor-
tional effects on virulence and disease dynamics. Detailed experimental data comparing co-
infection with single infections is thus required for each considered pathosystem, but such data
remain scarce.
In the fungal [43_TD$DIFF]endophyte [44_TD$DIFF]Epichloë bromicola, simultaneous inoculation with two genotypes
[45_TD$DIFF]slightly [46_TD$DIFF]increased [47_TD$DIFF]overall infection success [48_TD$DIFF]compared [49_TD$DIFF]to single infection; however, it did not
lead to coexistence of pathogen strains but rather to a superinfection where only one pathogen
persists [56]. In the powdery mildew P. plantaginis the relationship between within-host disease
accumulation and transmission varied between single infection and coinfection [57]. Coinfected
host plants shed more spores, resulting in higher disease prevalence in a common garden
experiment. Also, more devastating epidemics were observed in the natural host populations
with higher levels of coinfection [19].
Coinfection may thus be an important factor driving plant epidemiological dynamics and such
complexity has been identified as one of the major challenges for modeling plant diseases [58].
Detailed experimental work coupled with field surveys are required to unravel how possible
trade-offs between within-host multiplication and between-host transmission under coinfection
impact epidemiological dynamics in phytopathogens, offering an exciting avenue of future
research.
Coinfection Driving Virulence Evolution in Pathogen Populations
Coinfection may have a drastic impact on the evolution of pathogen populations, from the
generation of novel genetic combinations to its potential role in driving evolution through natural
selection. Coinfection has been traditionally considered to help us understand the classic
dilemma of virulence. This harm caused by parasites to their host is an important parasite trait
from both a fundamental and an applied perspective and its evolution has long puzzled
evolutionary biologists because parasite virulence has negative impacts on the host they depend
on for their survival. Among-host transmission is expected to require a certain level of virulence
and hence virulence evolution is considered to be driven by transmission–virulence trade-offs
[59]. However, understanding virulence evolution requires knowledge not only of between-host
transmission but also of within-host interactions between different strains, as higher host
6Trends in Plant Science, Month Year, Vol. xx, No. yy
TRPLSC 1360 No. of Pages 11
exploitation rates may be favored under coinfection rather than under single infection [1]. This
was probably the case in the Spanish epidemic of tomato necrosis caused by cucumber mosaic
virus (CMV); taking coinfection into account in the model was needed to understand the onset of
a highly virulent strain during the epidemic, while a decrease in damage [50_TD$DIFF](corresponding to the
evolution of decreased virulence[51_TD$DIFF]) was observed afterward [60,61].
The effect within-host competition may have on the evolution of pathogen populations would
depend on the mechanisms of interaction (see above). Competition for host resources between
unrelated pathogens would lead to increased host exploitation and favor increased virulence [1],
with one pathogen outcompeting the other [9]. However, in the case of pathogens producing
public goods (facilitation) a nonvirulent cheater genotype would be favored by natural selection
and evolution may lead to lower virulence for the host [1] with pathogens able to coexist for a long
time [9]. Finally, transmission processes may also be affected by the presence of other strains or
species (with, for example, helper dependence in viruses; see above). Notably, cotransmission
of various pathogen strains was shown to select for less-virulent variants in a theoretical model of
parasites competing for host resources [62].
Relatedness among coinfecting pathogen genotypes is generally assumed to favor less
competitive interactions (kin selection), but the relationship between relatedness and virulence
is crucially dependent on the form of the interaction [63]. To date, the best examples in plant
pathogens of how relatedness under coinfection impacts virulence come from Microbotryum
species causing anther-smut disease, which sterilizes the host. First, it has been shown that the
probability of multiple infection is higher when strains are more closely related, suggesting
mechanisms of competitive exclusion that are conditional on the genotypic characteristics of
the strains involved [64]. Second, virulence increased in cases of multiple infection compared
with single infection: both spore production and degree of plant sterilization were higher under
multiple infection [14,65]. Finally, Microbotryum also appeared able to interfere with compet-
itors, reducing their ability to colonize the host, and this effect was smaller between close
relatives [14].
Coinfection may thus affect the evolution of virulence in pathogen populations, with precise
mechanisms of interaction (Figure 1), virulence/transmission trade-offs, and relatedness among
strains affecting the direction and intensity of selective pressures. Experimental evolution work
involving serial passages with or without coinfecting strain or species, and comparison of
virulence between initial and passaged strains, is required to investigate properly the effect
of multiple infection on virulence.
Coinfection and the Genetic Diversity of Pathogen Populations
Coinfection is also expected to play a major role in how genetic diversity is generated and
maintained in pathogen populations. Coinfection is the prerequisite for sexual recombination in
many parasite species and the close contact between pathogen species or genotypes in the
context of coinfection allows the generation of novel genetic combinations. For example, in fungi
the reshuffling of alleles, a fundamentally important process for the evolutionary potential of
fungal pathogen populations, is due to sexual reproduction between different pathogen gen-
otypes [66]. Coinfection by multiple viruses has been shown to lead to frequent events of
genomic recombination (e.g., [67]). Coinfection also offers opportunities for hybridization
between different species [68] and may lead to changes in pathogen host range [69] as well
as the emergence of new pathogen species [70]. A recent study reported that suppression of
immunity by a race of Albugo candida allowed the subsequent infection of a normally non-host
species by another race, leading to genomic introgression between the two races [71].By
contrast, the absence of coinfection promotes reproductive isolation of obligate pathogens and
may ultimately promote sympatric speciation [72].
Trends in Plant Science, Month Year, Vol. xx, No. yy 7
TRPLSC 1360 No. of Pages 11
Diversity is the raw material for evolution and pathogen–pathogen interactions may contribute to
the maintenance of genetic diversity within pathogen populations [73]. In natural populations of
P. syringae, maintenance of nonvirulent strains is partly attributed to growth enhancement [52_TD$DIFF]when
[53_TD$DIFF]coinfected with virulent strains [74]. More generally, the fitness of one genotype measured alone
is a poor predictor of the fitness observed in coinfection, with, for example, some genotypes of
Phytophthora infestans reproducing more spores in competition while others reproduce more
when in single-genotype infection [75]. Interestingly, relative fitness differences between single
infection and coinfection for two strains of tomato yellow leaf curl virus allowed an explanation of
the patterns observed in the field: the genotype more recently introduced on the island of
Réunion was found to be fitter in single infection and consequently invaded the population, while
the resident strain was allowed to persist in the long term, mostly in coinfection with the other
genotype [76]. By contrast, competitive exclusion may occur between pathogen species or
genotypes. For example, although M. graminicola lesions on wheat leaves can contain up to six
pathogen genotypes, one or two of them occupied larger areas than others, which can be
interpreted as higher competitive ability of the most common genotype [77]. These studies
illustrate how dynamics under coinfection may change the frequency of strains in natural
populations. The picture may be even more complicated, with data from animals showing
interactions to be genotype specific in coinfecting pathogen species where the relative fitness of
different strains within populations differs under single-infection dynamics versus coinfection and
furthermore is affected by the particular genotypes that form the coinfection (i.e., specific
pathogen genotype-by-genotype interactions determine the outcome of multiple infection
[78]). These results suggest that the evolutionary trajectory of the pathogen population could
be affected by the genetic structure of other pathogen species sharing a host and by coinfection
levels in this pathosystem, but to our knowledge this has never been assessed in plant
pathogens.
Concluding Remarks
We conclude that coinfection is common in both natural and agricultural environments and has
the potential to change pathogen accumulation[54_TD$DIFF], transmission, and [55_TD$DIFF]virulence, the key compo-
nents of host–pathogen interactions. As a result, epidemiological dynamics are altered, with
fitness consequences of infection for the host that also depend on the multiplicity of infection. We
further conclude that it is crucial to combine field surveys and experimental approaches for the
study of multiple infection (see, for example, in animals [79]). Currently, integrated studies of
different pathogen genotypes or species aimed at a better understanding of coinfection impact
on epidemiology and evolution remain scarce in plant pathosystems (but see [19,27,31]). In
addition, most studies conducted on infection by multiple species to date involve a couple of
related species (at least within the wide groups of viruses, bacteria, or fungi). Multiple infection by
unrelated species should be more studied, as it represents the true diversity of pathogens
associated with any given host, with strong effects reported in the human and animal literature
[5,80] as well as the few studies performed to date in plants [31,40,45]. Also, we should aim for a
better understanding of the interactions between belowground and aboveground communities
that are likely to be mediated by shared resources and hormonal pathways. Belowground
microbial communities may play a key role plant health protection, analogous to the impact of the
intestinal microbiome on human health [49]. Researchers would benefit from genomics advan-
ces allowing us to appreciate and understand the range of pathogens that any given host is
associated with (Box 1) and new questions are raised within the pathobiome framework (i.e.,
integrating the pathogenic agent within its biotic environment; that is, not only its host and vector,
but also various other organisms susceptible to interaction [81] –see Outstanding Questions).
Recent methodological advances now allow the fascinating characterization of entire within-host
communities, but we are facing the nontrivial challenge of quantifying the ecological, physiologi-
cal, and evolutionary consequences of this diversity for both the host and the pathogens.
Tremendous levels of complexity could be reached in this context, with coinfection patterns
Outstanding Questions
How sensitive are infection dynamics to
the assemblage of coinfecting patho-
gens, including a broad array of facili-
tative, neutral, and antagonistic
interactions?
How do nonpathogenic species (com-
mensal, mutualistic) affect infection and
coinfection outcome?
How does the environment shape the
outcome of multiple infection?
What is the role of host resistance in
mediating the outcome of multiple
infection?
How effective and durable is resistance
under multiple infection?
What are the ecological and evolution-
ary consequences of coinfection for
plant resistance?
Is the evolution of resistance toward
one pathogen constrained by the need
to resist others?
8Trends in Plant Science, Month Year, Vol. xx, No. yy
TRPLSC 1360 No. of Pages 11
potentially differing among host species [52,82], host genotypes [19,83], or developmental stage
[39], and tools developed within community ecology framework are required to disentangle the
major processes [84].
Overall, establishing direct links between the molecular mechanisms of dynamics under coin-
fection and the formation of pathogen communities with the ecoevolutionary aspects of host
resistance and epidemiological dynamics in single infection versus coinfection scenarios offers
an exciting future avenue of research (see Outstanding Questions). This is needed to truly
account for the role of coinfection when designing epidemiological interventions or virulence
management efforts.
Acknowledgments
The manuscript benefited from discussions with and comments from Aurélien Tellier, Philippe Roumagnac, Samuel Alizon,
and Christophe Brugidou[14_TD$DIFF]. The [56_TD$DIFF]study [57_TD$DIFF]was [58_TD$DIFF]supported by funding from Labex Agro (ANR-10-LABX-001-01, project ID
1403-041) to C.T. and from the Academy of Finland (Center of Excellence in Metapopulation Biology 2015–2017; 284601)
and the European Research Council (Independent Starting Grant PATHEVOL; 281517) to A-L.L.
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