Alternative host shapes transmission and life-history trait correlations in a multi-host plant pathogen.

Most pathogens are generalists capable of infecting multiple host species or strains. Trade-offs in performance among different hosts are expected to limit the evolution of generalism. Despite the commonness of generalism, the variation in infectivity, transmission, and trade-offs in performance among host species have rarely been studied in the wild. To understand the ecological and evolutionary drivers of multi-host pathogen infectivity and transmission potential, I studied disease severity, transmission dynamics, and infectivity variation of downy mildew pathogen Peronospora sparsa on its three host plants Rubus arcticus, R. chamaemorus, and R. saxatilis. In a survey of 20 wild and cultivated sites of the three host species, disease severity varied by host species and by host population size but not among wild and cultivated sites. To understand how alternative host presence and plant diversity affect transmission of the pathogen, I conducted a transmission experiment. In this experiment, alternative host abundance and plant diversity together modified P. sparsa transmission to trap plants. To understand how resistance to P. sparsa varies among host species and genotypes, I conducted an inoculation experiment using 10 P. sparsa strains from different locations and 20 genotypes of the three host species. Significant variation in infectivity was found among host genotypes but not among host species. When trade-offs for infectivity were tested, high infectivity in one host species correlated with high infectivity in another host species. However, when pathogen transmission-related life-history correlations were tested, a positive correlation was found in R. arcticus but not in R. saxatilis. The results suggest that host resistance may shape pathogen life-history evolution with epidemiological consequences in a multi-host pathogen.

The first arriving virus shapes within-host viral diversity during natural epidemics.

Viral diversity has been discovered across scales from host individuals to populations. However, the drivers of viral community assembly are still largely unknown. Within-host viral communities are formed through co-infections, where the interval between the arrival times of viruses may vary. Priority effects describe the timing and order in which species arrive in an environment, and how early colonizers impact subsequent community assembly. To study the effect of the first-arriving virus on subsequent infection patterns of five focal viruses, we set up a field experiment using naïve Plantago lanceolata plants as sentinels during a seasonal virus epidemic. Using joint species distribution modelling, we find both positive and negative effects of early season viral infection on late season viral colonization patterns. The direction of the effect depends on both the host genotype and which virus colonized the host early in the season. It is well established that co-occurring viruses may change the virulence and transmission of viral infections. However, our results show that priority effects may also play an important, previously unquantified role in viral community assembly. The assessment of these temporal dynamics within a community ecological framework will improve our ability to understand and predict viral diversity in natural systems.

Direct and indirect viral associations predict coexistence in wild plant virus communities.

Viruses are a vastly underestimated component of biodiversity that occur as diverse communities across hierarchical scales from the landscape level to individual hosts. The integration of community ecology with disease biology is a powerful, novel approach that can yield unprecedented insights into the abiotic and biotic drivers of pathogen community assembly. Here, we sampled wild plant populations to characterize and analyze the diversity and co-occurrence structure of within-host virus communities and their predictors. Our results show that these virus communities are characterized by diverse, non-random coinfections. Using a novel graphical network modeling framework, we demonstrate how environmental heterogeneity influences the network of virus taxa and how the virus co-occurrence patterns can be attributed to non-random, direct statistical virus-virus associations. Moreover, we show that environmental heterogeneity changed virus association networks, especially through their indirect effects. Our results highlight a previously underestimated mechanism of how environmental variability can influence disease risks by changing associations between viruses that are conditional on their environment.

Altered within- and between-host transmission under coinfection underpin parasite co-occurrence patterns in the wild.

Interactions among parasite species coinfecting the same host individual can have far reaching consequences for parasite ecology and evolution. How these within-host interactions affect epidemics may depend on two non-exclusive mechanisms: parasite growth and reproduction within hosts, and parasite transmission between hosts. Yet, how these two mechanisms operate under coinfection, and how sensitive they are to the composition of the coinfecting parasite community, remains poorly understood. Here, we test the hypothesis that the relationship between within- and between-host transmission of the fungal pathogen, Phomopsis subordinaria, is affected by co-occurring parasites infecting the host plant, Plantago lanceolata. We conducted a field experiment manipulating the parasite community of transmission source plants, then tracked P. subordinaria within-host transmission, as well as between-host transmission to naïve recipient plants. We find that coinfection with the powdery mildew pathogen, Podosphaera plantaginis, causes increased between-host transmission of P. subordinaria by affecting the number of infected flower stalks in the source plants, resulting from altered auto-infection. In contrast, coinfection with viruses did not have an effect on either within- or between-host transmission. We then analyzed data on the occurrence of P. subordinaria in 2018 and the powdery mildew in a multi-year survey data set from natural host populations to test whether the positive association predicted by our experimental results is evident in field epidemiological data. Consistent with our experimental findings, we observed a positive association in the occurrence of P. subordinaria and historical powdery mildew persistence. Jointly, our experimental and epidemiological results suggest that within- and between-host transmission of P. subordinaria depends on the identity of coinfecting parasites, with potentially far-reaching effects on disease dynamics and parasite co-occurrence patterns in wild populations. Supplementary Information: The online version contains supplementary material available at 10.1007/s10682-022-10182-9.

Coinfection with a virus constrains within-host infection load but increases transmission potential of a highly virulent fungal plant pathogen.

The trade-off between within-host infection rate and transmission to new hosts is predicted to constrain pathogen evolution, and to maintain polymorphism in pathogen populations. Pathogen life-history stages and their correlations that underpin infection development may change under coinfection with other parasites as they compete for the same limited host resources. Cross-kingdom interactions are common among pathogens in both natural and cultivated systems, yet their impacts on disease ecology and evolution are rarely studied. The host plant Plantago lanceolata is naturally infected by both Phomopsis subordinaria, a seed killing fungus, as well as Plantago lanceolata latent virus (PlLV) in the Åland Islands, SW Finland. We performed an inoculation assay to test whether coinfection with PlLV affects performance of two P. subordinaria strains, and the correlation between within-host infection rate and transmission potential. The strains differed in the measured life-history traits and their correlations. Moreover, we found that under virus coinfection, within-host infection rate of P. subordinaria was smaller but transmission potential was higher compared to strains under single infection. The negative correlation between within-host infection rate and transmission potential detected under single infection became positive under coinfection with PlLV. To understand whether within-host and between-host dynamics are correlated in wild populations, we surveyed 260 natural populations of P. lanceolata for P. subordinaria infection occurrence. When infections were found, we estimated between-hosts dynamics by determining pathogen population size as the proportion of infected individuals, and within-host dynamics by counting the proportion of infected flower stalks in 10 infected plants. In wild populations, the proportion of infected flower stalks was positively associated with pathogen population size. Jointly, our results suggest that the trade-off between within-host infection load and transmission may be strain specific, and that the pathogen life-history that underpin epidemics may change depending on the diversity of infection, generating variation in disease dynamics.

Effect of maternal infection on progeny growth and resistance mediated by maternal genotype and nutrient availability.

Maternal effects of pathogen infection on progeny development and disease resistance may be adaptive and have important consequences for population dynamics. However, these effects are often context-dependent and examples of adaptive transgenerational responses from perennials are scarce, although they may be a particularly important mechanism generating variation in the offspring of long-lived species.Here, we studied the effect of maternal infection of Plantago lanceolata by Podosphaera plantaginis, a fungal parasite, on the growth, flower production and resistance of the progeny of six maternal genotypes in nutrient-rich and nutrient-poor environments. For this purpose, we combined a common garden study with automated phenotyping measurements of early life stages, and an inoculation experiment.Our results show that the effects of infection on the mother plants transcend to impact their progeny. Although maternal infection decreased total leaf and flower production of the progeny by the end of the growing season, it accelerated early growth and enhanced resistance to the pathogen P. plantaginis.We also discovered that the effects of maternal infection affected progeny development and resistance through a three way-interaction between maternal genotype, maternal infection status and nutrient availability. Synthesis. Our results emphasize the importance of maternal effects mediated through genotypic and environmental factors in long-living perennials and suggest that maternal infection can create a layer of phenotypic diversity in resistance. These results may have important implications for both epidemiological and evolutionary dynamics of host-parasite interactions in the wild.

Agricultural land use disrupts biodiversity mediation of virus infections in wild plant populations.

Human alteration of natural habitats may change the processes governing species interactions in wild communities. Wild populations are increasingly impacted by agricultural intensification, yet it is unknown whether this alters biodiversity mediation of disease dynamics. We investigated the association between plant diversity (species richness, diversity) and infection risk (virus richness, prevalence) in populations of Plantago lanceolata in natural landscapes as well as those occurring at the edges of cultivated fields. Altogether, 27 P. lanceolata populations were surveyed for population characteristics and sampled for PCR detection of five recently characterized viruses. We find that plant species richness and diversity correlated negatively with virus infection prevalence. Virus species richness declined with increasing plant diversity and richness in natural populations while in agricultural edge populations species richness was moderately higher, and not associated with plant richness. This difference was not explained by changes in host richness between these two habitats, suggesting potential pathogen spill-over and increased transmission of viruses across the agro-ecological interface. Host population connectivity significantly decreased virus infection prevalence. We conclude that human use of landscapes may change the ecological laws by which natural communities are formed with far reaching implications for ecosystem functioning and disease.

Intraspecific host variation plays a key role in virus community assembly.

Infection by multiple pathogens of the same host is ubiquitous in both natural and managed habitats. While intraspecific variation in disease resistance is known to affect pathogen occurrence, how differences among host genotypes affect the assembly of pathogen communities remains untested. In our experiment using cloned replicates of naive Plantago lanceolata plants as sentinels during a seasonal virus epidemic, we find non-random co-occurrence patterns of five focal viruses. Using joint species distribution modelling, we attribute the non-random virus occurrence patterns primarily to differences among host genotypes and local population context. Our results show that intraspecific variation among host genotypes may play a large, previously unquantified role in pathogen community structure.

Genetic analysis reveals long-standing population differentiation and high diversity in the rust pathogen Melampsora lini.

A priority for research on infectious disease is to understand how epidemiological and evolutionary processes interact to influence pathogen population dynamics and disease outcomes. However, little is understood about how population adaptation changes across time, how sexual vs. asexual reproduction contribute to the spread of pathogens in wild populations and how diversity measured with neutral and selectively important markers correlates across years. Here, we report results from a long-term study of epidemiological and genetic dynamics within several natural populations of the Linum marginale-Melampsora lini plant-pathogen interaction. Using pathogen isolates collected from three populations of wild flax (L. marginale) spanning 16 annual epidemics, we probe links between pathogen population dynamics, phenotypic variation for infectivity and genomic polymorphism. Pathogen genotyping was performed using 1567 genome-wide SNP loci and sequence data from two infectivity loci (AvrP123, AvrP4). Pathogen isolates were phenotyped for infectivity using a differential set. Patterns of epidemic development were assessed by conducting surveys of infection prevalence in one population (Kiandra) annually. Bayesian clustering analyses revealed host population and ecotype as key predictors of pathogen genetic structure. Despite strong fluctuations in pathogen population size and severe annual bottlenecks, analysis of molecular variance revealed that pathogen population differentiation was relatively stable over time. Annually, varying levels of clonal spread (0-44.8%) contributed to epidemics. However, within populations, temporal genetic composition was dynamic with rapid turnover of pathogen genotypes, despite the dominance of only four infectivity phenotypes across the entire study period. Furthermore, in the presence of strong fluctuations in population size and migration, spatial selection may maintain pathogen populations that, despite being phenotypically stable, are genetically highly dynamic.

Diverse and variable virus communities in wild plant populations revealed by metagenomic tools.

Wild plant populations may harbour a myriad of unknown viruses. As the majority of research efforts have targeted economically important plant species, the diversity and prevalence of viruses in the wild has remained largely unknown. However, the recent shift towards metagenomics-based sequencing methodologies, especially those targeting small RNAs, is finally enabling virus discovery from wild hosts. Understanding this diversity of potentially pathogenic microbes in the wild can offer insights into the components of natural biodiversity that promotes long-term coexistence between hosts and parasites in nature, and help predict when and where risks of disease emergence are highest. Here, we used small RNA deep sequencing to identify viruses in Plantago lanceolata populations, and to understand the variation in their prevalence and distribution across the Åland Islands, South-West Finland. By subsequent design of PCR primers, we screened the five most common viruses from two sets of P. lanceolata plants: 164 plants collected from 12 populations irrespective of symptoms, and 90 plants collected from five populations showing conspicuous viral symptoms. In addition to the previously reported species Plantago lanceolata latent virus (PlLV), we found four potentially novel virus species belonging to Caulimovirus, Betapartitivirus, Enamovirus, and Closterovirus genera. Our results show that virus prevalence and diversity varied among the sampled host populations. In six of the virus infected populations only a single virus species was detected, while five of the populations supported between two to five of the studied virus species. In 20% of the infected plants, viruses occurred as coinfections. When the relationship between conspicuous viral symptoms and virus infection was investigated, we found that plants showing symptoms were usually infected (84%), but virus infections were also detected from asymptomatic plants (44%). Jointly, these results reveal a diverse virus community with newly developed tools and protocols that offer exciting opportunities for future studies on the eco-evolutionary dynamics of viruses infecting plants in the wild.

Host resistance and pathogen aggressiveness are key determinants of coinfection in the wild.

Coinfection, whereby the same host is infected by more than one pathogen strain, may favor faster host exploitation rates as strains compete for the same limited resources. Hence, coinfection is expected to have major consequences for pathogen evolution, virulence, and epidemiology. Theory predicts genetic variation in host resistance and pathogen infectivity to play a key role in how coinfections are formed. The limited number of studies available has demonstrated coinfection to be a common phenomenon, but little is known about how coinfection varies in space, and what its determinants are. Our aim is to understand how variation in host resistance and pathogen infectivity and aggressiveness contribute to how coinfections are formed in the interaction between fungal pathogen Podosphaera plantaginis and Plantago lanceolata. Our phenotyping study reveals that more aggressive strains are more likely to form coinfections than less aggressive strains in the natural populations. In the natural populations most of the variation in coinfection is found at the individual plant level, and results from a common garden study confirm the prevalence of coinfection to vary significantly among host genotypes. These results show that genetic variation in both the host and pathogen populations are key determinants of coinfection in the wild.

Genome sequences of a capulavirus infecting Plantago lanceolata in the Åland archipelago of Finland.

The discovery and full-genome sequences of two isolates of a fourth capulavirus species are reported. The viruses were discovered during a viral metagenomics survey of uncultivated Plantago lanceolata plants in the Åland archipelago of south western Finland. The newly discovered viruses apparently produce no symptoms in P. lanceolata. They have a genome organization that is very similar to that of the three known capulavirus species and additionally share between 62.9 and 67.1% genome-wide sequence identity with the isolates of these species. It is therefore proposed that these viruses be assigned to a new capulavirus species named "Plantago lanceolata latent virus".

Evolutionary and Epidemiological Implications of Multiple Infection in Plants.

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 components 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.

Host Genotype and Coinfection Modify the Relationship of within and between Host Transmission.

Variation in individual-level disease transmission is well documented, but the underlying causes of this variation are challenging to disentangle in natural epidemics. In general, within-host replication is critical in determining the extent to which infected hosts shed transmission propagules, but which factors cause variation in this relationship are poorly understood. Here, using a plant host, Plantago lanceolata, and the powdery mildew fungus Podosphaera plantaginis, we quantify how the distinct stages of within-host spread (autoinfection), spore release, and successful transmission to new hosts (alloinfection) are influenced by host genotype, pathogen genotype, and the coinfection status of the host. We find that within-host spread alone fails to predict transmission rates, as this relationship is modified by genetic variation in hosts and pathogens. Their contributions change throughout the course of the epidemic. Host genotype and coinfection had particularly pronounced effects on the dynamics of spore release from infected hosts. Confidently predicting disease spread from local levels of individual transmission, therefore, requires a more nuanced understanding of genotype-specific infection outcomes. This knowledge is key to better understanding the drivers of epidemiological dynamics and the resulting evolutionary trajectories of infectious disease.

Co-infection alters population dynamics of infectious disease.

Co-infections by multiple pathogen strains are common in the wild. Theory predicts co-infections to have major consequences for both within- and between-host disease dynamics, but data are currently scarce. Here, using common garden populations of Plantago lanceolata infected by two strains of the pathogen Podosphaera plantaginis, either singly or under co-infection, we find the highest disease prevalence in co-infected treatments both at the host genotype and population levels. A spore-trapping experiment demonstrates that co-infected hosts shed more transmission propagules than singly infected hosts, thereby explaining the observed change in epidemiological dynamics. Our experimental findings are confirmed in natural pathogen populations-more devastating epidemics were measured in populations with higher levels of co-infection. Jointly, our results confirm the predictions made by theoretical and experimental studies for the potential of co-infection to alter disease dynamics across a large host-pathogen metapopulation.

Disease ecology. Ecological and evolutionary effects of fragmentation on infectious disease dynamics.

Ecological theory predicts that disease incidence increases with increasing density of host networks, yet evolutionary theory suggests that host resistance increases accordingly. To test the combined effects of ecological and evolutionary forces on host-pathogen systems, we analyzed the spatiotemporal dynamics of a plant (Plantago lanceolata)-fungal pathogen (Podosphaera plantaginis)relationship for 12 years in over 4000 host populations. Disease prevalence at the metapopulation level was low, with high annual pathogen extinction rates balanced by frequent (re-)colonizations. Highly connected host populations experienced less pathogen colonization and higher pathogen extinction rates than expected; a laboratory assay confirmed that this phenomenon was caused by higher levels of disease resistance in highly connected host populations.

Pathogen life-history trade-offs revealed in allopatry.

Trade-offs in life-history traits is a central tenet in evolutionary biology, yet their ubiquity and relevance to realized fitness in natural populations remains questioned. Trade-offs in pathogens are of particular interest because they may constrain the evolution and epidemiology of diseases. Here, we studied life-history traits determining transmission in the obligate fungal pathogen, Podosphaera plantaginis, infecting Plantago lanceolata. We find that although traits are positively associated on sympatric host genotypes, on allopatric host genotypes relationships between infectivity and subsequent transmission traits change shape, becoming even negative. The epidemiological prediction of this change in life-history relationships in allopatry is lower disease prevalence in newly established pathogen populations. An analysis of the natural pathogen metapopulation confirms that disease prevalence is lower in newly established pathogen populations and they are more prone to go extinct during winter than older pathogen populations. Hence, life-history trade-offs mediated by pathogen local adaptation may influence epidemiological dynamics at both population and metapopulation levels.

SNP design from 454 sequencing of Podosphaera plantaginis transcriptome reveals a genetically diverse pathogen metapopulation with high levels of mixed-genotype infection.

BACKGROUND: Molecular tools may greatly improve our understanding of pathogen evolution and epidemiology but technical constraints have hindered the development of genetic resources for parasites compared to free-living organisms. This study aims at developing molecular tools for Podosphaera plantaginis, an obligate fungal pathogen of Plantago lanceolata. This interaction has been intensively studied in the Åland archipelago of Finland with epidemiological data collected from over 4,000 host populations annually since year 2001. PRINCIPAL FINDINGS: A cDNA library of a pooled sample of fungal conidia was sequenced on the 454 GS-FLX platform. Over 549,411 reads were obtained and annotated into 45,245 contigs. Annotation data was acquired for 65.2% of the assembled sequences. The transcriptome assembly was screened for SNP loci, as well as for functionally important genes (mating-type genes and potential effector proteins). A genotyping assay of 27 SNP loci was designed and tested on 380 infected leaf samples from 80 populations within the Åland archipelago. With this panel we identified 85 multilocus genotypes (MLG) with uneven frequencies across the pathogen metapopulation. Approximately half of the sampled populations contain polymorphism. Our genotyping protocol revealed mixed-genotype infection within a single host leaf to be common. Mixed infection has been proposed as one of the main drivers of pathogen evolution, and hence may be an important process in this pathosystem. SIGNIFICANCE: The developed SNP panel offers exciting research perspectives for future studies in this well-characterized pathosystem. Also, the transcriptome provides an invaluable novel genomic resource for powdery mildews, which cause significant yield losses on commercially important crops annually. Furthermore, the features that render genetic studies in this system a challenge are shared with the majority of obligate parasitic species, and hence our results provide methodological insights from SNP calling to field sampling protocols for a wide range of biological systems.